Thumbnailjpg

The ReTinoids

The ReTinoidsBiology Biochemistry

and diseaseediTed By

Pascal Dolleacute

and

Karen Niederreither

Copyright copy 2015 by Wiley-Blackwell All rights reserved

Published by John Wiley amp Sons Inc Hoboken New JerseyPublished simultaneously in Canada

No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording scanning or otherwise except as permitted under Section 107 or 108 of the 1976 United States Copyright Act without either the prior written permission of the Publisher or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center Inc 222 Rosewood Drive Danvers MA 01923 (978) 750-8400 fax (978) 750-4470 or on the web at wwwcopyrightcom Requests to the Publisher for permission should be addressed to the Permissions Department John Wiley amp Sons Inc 111 River Street Hoboken NJ 07030 (201) 748-6011 fax (201) 748-6008 or online at httpwwwwileycomgopermission

Limit of LiabilityDisclaimer of Warranty While the publisher and author have used their best efforts in preparing this book they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose No warranty may be created or extended by sales representatives or written sales materials The advice and strategies contained herein may not be suitable for your situation You should consult with a professional where appropriate Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages including but not limited to special incidental consequential or other damages

For general information on our other products and services or for technical support please contact our Customer Care Department within the United States at (800) 762-2974 outside the United States at (317) 572-3993 or fax (317) 572-4002

Wiley also publishes its books in a variety of electronic formats Some content that appears in print may not be available in electronic formats For more information about Wiley products visit our web site at wwwwileycom

Library of Congress Cataloging-in-Publication Data

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

The ReTinoids

The ReTinoidsBiology Biochemistry

and diseaseediTed By

Pascal Dolleacute

and

Karen Niederreither

Copyright copy 2015 by Wiley-Blackwell All rights reserved

Published by John Wiley amp Sons Inc Hoboken New JerseyPublished simultaneously in Canada

No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording scanning or otherwise except as permitted under Section 107 or 108 of the 1976 United States Copyright Act without either the prior written permission of the Publisher or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center Inc 222 Rosewood Drive Danvers MA 01923 (978) 750-8400 fax (978) 750-4470 or on the web at wwwcopyrightcom Requests to the Publisher for permission should be addressed to the Permissions Department John Wiley amp Sons Inc 111 River Street Hoboken NJ 07030 (201) 748-6011 fax (201) 748-6008 or online at httpwwwwileycomgopermission

Limit of LiabilityDisclaimer of Warranty While the publisher and author have used their best efforts in preparing this book they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose No warranty may be created or extended by sales representatives or written sales materials The advice and strategies contained herein may not be suitable for your situation You should consult with a professional where appropriate Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages including but not limited to special incidental consequential or other damages

For general information on our other products and services or for technical support please contact our Customer Care Department within the United States at (800) 762-2974 outside the United States at (317) 572-3993 or fax (317) 572-4002

Wiley also publishes its books in a variety of electronic formats Some content that appears in print may not be available in electronic formats For more information about Wiley products visit our web site at wwwwileycom

Library of Congress Cataloging-in-Publication Data

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

The ReTinoidsBiology Biochemistry

and diseaseediTed By

Pascal Dolleacute

and

Karen Niederreither

Copyright copy 2015 by Wiley-Blackwell All rights reserved

Published by John Wiley amp Sons Inc Hoboken New JerseyPublished simultaneously in Canada

No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording scanning or otherwise except as permitted under Section 107 or 108 of the 1976 United States Copyright Act without either the prior written permission of the Publisher or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center Inc 222 Rosewood Drive Danvers MA 01923 (978) 750-8400 fax (978) 750-4470 or on the web at wwwcopyrightcom Requests to the Publisher for permission should be addressed to the Permissions Department John Wiley amp Sons Inc 111 River Street Hoboken NJ 07030 (201) 748-6011 fax (201) 748-6008 or online at httpwwwwileycomgopermission

Limit of LiabilityDisclaimer of Warranty While the publisher and author have used their best efforts in preparing this book they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose No warranty may be created or extended by sales representatives or written sales materials The advice and strategies contained herein may not be suitable for your situation You should consult with a professional where appropriate Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages including but not limited to special incidental consequential or other damages

For general information on our other products and services or for technical support please contact our Customer Care Department within the United States at (800) 762-2974 outside the United States at (317) 572-3993 or fax (317) 572-4002

Wiley also publishes its books in a variety of electronic formats Some content that appears in print may not be available in electronic formats For more information about Wiley products visit our web site at wwwwileycom

Library of Congress Cataloging-in-Publication Data

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

Copyright copy 2015 by Wiley-Blackwell All rights reserved

Published by John Wiley amp Sons Inc Hoboken New JerseyPublished simultaneously in Canada

No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording scanning or otherwise except as permitted under Section 107 or 108 of the 1976 United States Copyright Act without either the prior written permission of the Publisher or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center Inc 222 Rosewood Drive Danvers MA 01923 (978) 750-8400 fax (978) 750-4470 or on the web at wwwcopyrightcom Requests to the Publisher for permission should be addressed to the Permissions Department John Wiley amp Sons Inc 111 River Street Hoboken NJ 07030 (201) 748-6011 fax (201) 748-6008 or online at httpwwwwileycomgopermission

Limit of LiabilityDisclaimer of Warranty While the publisher and author have used their best efforts in preparing this book they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose No warranty may be created or extended by sales representatives or written sales materials The advice and strategies contained herein may not be suitable for your situation You should consult with a professional where appropriate Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages including but not limited to special incidental consequential or other damages

For general information on our other products and services or for technical support please contact our Customer Care Department within the United States at (800) 762-2974 outside the United States at (317) 572-3993 or fax (317) 572-4002

Wiley also publishes its books in a variety of electronic formats Some content that appears in print may not be available in electronic formats For more information about Wiley products visit our web site at wwwwileycom

Library of Congress Cataloging-in-Publication Data

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

v

Contents

Contributors ix

Preface xiii

Part I VItamIn a metabolIC and enzymatIC Pathways 1

1 VItamIn a metabolIsm storage and tIssue delIVery meChanIsms 3William S Blaner and Yang Li

2 assImIlatIon and ConVersIon of dIetary VItamIn a Into bIoaCtIVe retInoIds 35Earl H Harrison and Carlo dela Senarsquos

3 IntraCellular storage and metabolIC aCtIVatIon of retInoIds lIPId droPlets 57Joseph L Napoli and Charles R Krois

4 eVolutIon of the retInoIC aCId sIgnalIng Pathway 75Vincent Laudet Elisabeth Zieger and Michael Schubert

Part II bIoChemIstry and Cellular bIology of retInoIC aCId sIgnalIng 91

5 Control of gene exPressIon by nuClear retInoIC aCId reCePtors Post-translatIonal and ePIgenetIC regulatory meChanIsms 93Marilyn Carrier and Ceacutecile Rochette-Egly

6 retInoIC aCId reCePtor Coregulators In ePIgenetIC regulatIon of target genes 117Li-Na Wei

7 retInoId reCePtors ProteIn struCture dna reCognItIon and struCturendashfunCtIon relatIonshIPs 131William Bourguet and Dino Moras

8 how the rarndashrxr heterodImer reCognIzes the genome 151Sylvia Urban Tao Ye and Irwin Davidson

vi Contents

9 retInoId reCePtor-seleCtIVe modulators ChemIstry 3d struCtures and systems bIology 165Marco-Antonio Mendoza-Parra William Bourguet Angel R de Lera and Hinrich Gronemeyer

10 use of retInoId reCePtor lIgands to IdentIfy other nuClear reCePtor lIgands retInoId-related moleCules are lIgands for the small heterodImer Partner (shP) ldquoorPhanrdquo reCePtor 193Marcia I Dawson and Zebin Xia

11 the dual transCrIPtIonal aCtIVIty of retInoIC aCId 273Noa Noy

12 retInoIds ePIgenetIC Changes durIng stem Cell dIfferentIatIon and Cell lIneage ChoICe 291Lorraine J Gudas

Part III retInoIC aCId sIgnalIng In deVeloPment 307

13 retInoIC aCId sIgnalIng and Central nerVous system deVeloPment 309Malcolm Maden

14 the role of retInoIC aCId In lImb deVeloPment 339Gregg Duester

15 retInoIC aCId sIgnalIng and heart deVeloPment 353Steacutephane Zaffran and Karen Niederreither

16 retInoIC aCId In the deVeloPIng lung and other foregut derIVatIVes 371Wellington V Cardoso and Felicia Chen

17 retInoIC aCId and the Control of meIotIC InItIatIon 383Josephine Bowles and Peter Koopman

Part IV retInoIds and PhysIologICal funCtIons 401

18 retInoIds and the VIsual CyCle new aCtors for an ldquooldrdquo funCtIon 403Darwin Babino and Johannes von Lintig

19 retInoId sIgnalIng In the Central nerVous system 421Peter McCaffery and Wojciech Krezel

20 retInoId turnoVer and CatabolIsm InfluenCes of dIet and InflammatIon 449A Catharine Ross and Reza Zolfaghari

Contents vii

21 retInoIds and the Immune system 465J Rodrigo Mora and Makoto Iwata

22 retInoIC aCId reCePtor sIgnalIng In Post-natal male germ Cell dIfferentIatIon 485Manuel Mark and Norbert B Ghyselinck

Part V retInoIds dIsease and theraPy 505

23 ePIdemIology and PreVentIon of VItamIn a defICIenCy dIsorders 507Keith P West Jr

24 retInoId Pathway gene mutatIons and the PathoPhysIology of related VIsual dIseases 529Yaroslav Tsybovsky and Krzysztof Palczewski

25 retInoIC aCId In aCute myeloId leukemIas 543Hugues de Theacute and Pierre Fenaux

26 adVanCes In the use of retInoIds In CanCer theraPy and PreVentIon 557Michael J Spinella Sarah J Freemantle and Ethan Dmitrovsky

Index 575

ix

Contributors

Darwin Babino Department of Pharmacology School of Medicine Case Western Reserve University Cleveland OH USA

William S Blaner Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

William Bourguet Centre de Biochimie Structurale Montpellier France Universiteacutes Montpellier 1 amp 2 Montpellier France

Josephine Bowles Institute for Molecular Bioscience The University of Queensland Brisbane QLD Australia

Wellington V Cardoso Columbia Center for Human Development Department of Medicine Columbia University Medical Center New York NY USA

Marilyn Carrier Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Felicia Chen Pulmonary Center Boston University School of Medicine Boston MA USA

Irwin Davidson Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Marcia I Dawson Cancer Center Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Ethan Dmitrovsky Department of Pharmacology and Toxicology Department of Medicine The Norris Cotton Cancer Center Geisel School of Medicine Dartmouth Hanover NH USA

Gregg Duester Development Aging and Regeneration Program Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Pierre Fenaux Universiteacute Paris Diderot Sorbonne Paris Citeacute Assistance PubliquendashHocircpitaux de Paris Service drsquoHeacutematologie Hocircpital St Louis Paris France

Sarah J Freemantle Department of Pharmacology and Toxicology Geisel School of Medicine Dartmouth Hanover NH USA

Norbert B Ghyselinck Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Hinrich Gronemeyer Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

x Contributors

Lorraine J Gudas Pharmacology Department Weill Cornell Medical College Cornell University New York NY USA

Earl H Harrison Department of Human Nutrition and Ohio State Biochemistry Program Ohio State University Columbus OH USA

Makoto Iwata Kagawa School of Pharmaceutical Sciences Tokushima Bunri University amp JST CREST Kagawa Japan

Peter Koopman Institute for Molecular Bioscience The University of Queensland Brisbane QLD Australia

Wojciech Krezel Development and Stem Cells Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Charles R Krois Graduate Program in Metabolic Biology Nutritional Sciences and Toxicology University of California-Berkeley CA USA

Vincent Laudet Molecular Zoology Team Institut de Geacutenomique Fonctionnelle de Lyon (IGFL) Ecole Normale Supeacuterieure de Lyon Lyon France

Angel R de Lera Departamento de Quiacutemica Orgaacutenica Facultad de Quiacutemica Universidad de Vigo Vigo Spain

Yang Li Columbia College Columbia University New York NY USA

Johannes von Lintig Department of Pharmacology School of Medicine Case Western Reserve University Cleveland OH USA

Malcolm Maden Department of Biology and University of Florida Genetics Institute University of Florida Gainesville FL USA

Manuel Mark Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France Hocircpitaux Universitaires de Strasbourg Strasbourg France

Peter McCaffery Institute of Medical Sciences School of Medical Sciences University of Aberdeen Aberdeen UK

Marco-Antonio Mendoza-Parra Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

J Rodrigo Mora Massachusetts General Hospital and Harvard Medical School Boston MA USA

Dino Moras Integrated Structural Biology Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Joseph L Napoli Graduate Program in Metabolic Biology Nutritional Sciences and Toxicology University of California-Berkeley CA USA

Karen Niederreither Development and Stem Cells Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Noa Noy Department of Cellular and Molecular Medicine Lerner Research Institute Cleveland Clinic Foundation Cleveland OH USA

Contributors xi

Krzysztof Palczewski Department of Pharmacology Case Western Reserve University Cleveland OH USA

Ceacutecile Rochette-Egly Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

A Catharine Ross Department of Nutritional Sciences Pennsylvania State University University Park PA USA

Michael Schubert Laboratoire de Biologie du Deacuteveloppement de Villefranche-sur-Mer Sorbonne Universiteacutes UPMC Universiteacute Paris 06 Observatoire Oceacuteanologique de Villefranche-sur-Mer Villefranche-sur-Mer France

Carlo dela Sentildearsquos Department of Human Nutrition and Ohio State Biochemistry Program Ohio State University Columbus OH USA

Michael J Spinella Department of Pharmacology and Toxicology The Norris Cotton Cancer Center Geisel School of Medicine Dartmouth College Hanover NH USA

Hugues de Theacute Universiteacute Paris Diderot Sorbonne Paris Citeacute Institut Universitaire drsquoHeacutematologie Assistance PubliquendashHocircpitaux de Paris Service de Biochimie Hocircpital St Louis Paris France

Yaroslav Tsybovsky Department of Pharmacology Case Western Reserve University Cleveland OH USA

Sylvia Urban Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Li-Na Wei Department of Pharmacology University of Minnesota Medical School Minneapolis MN USA

Keith P West Jr Center and Program in Human Nutrition Department of International Health Johns Hopkins Bloomberg School of Public Health Baltimore MD USA

Zebin Xia Cancer Center Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Tao Ye Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Steacutephane Zaffran Aix-Marseille Universiteacute Uniteacute de Geacuteneacutetique Meacutedicale et Geacutenomique Fonctionnelle (GMGF) Faculteacute de Meacutedecine Marseille France

Elisabeth Zieger Laboratoire de Biologie du Deacuteveloppement de Villefranche-sur-Mer Sorbonne Universiteacutes UPMC Universiteacute Paris 06 Observatoire Oceacuteanologique de Villefranche-sur-Mer Villefranche-sur-Mer France

Reza Zolfaghari Department of Nutritional Sciences Pennsylvania State University University Park PA USA

xiii

Preface

VITAMIN A AND RETINOIDS AN AMAZING HISTORY AND HOPES FOR DISEASE PREVENTION

Historically the vitamin A precursor carotenoids had been long suspected of having medicinal properties For thousands of years humans and animals suffered from vitamin A deficiency typified by night blindness and xerophthalmiamdasha failure of tear production which if untreated would result in blindness While the underlying causes of such maledictions were a mystery in 1500 BC the ancient Egyptians rec-ommended eating animal or fish liver for their curative powers The native people of the Arctic also long knew to avoid eating the liver of polar bears because they became sick (unknowingly by vitamin A) Western explorers not savvy to the vitamin A rich properties of polar bear liver as early as 1596 accounted a horrible illnessndashsluggish-ness blurred vision nausea headache and skin loss resulting in coma and even deathmdashall signs of acute hypervitaminosis A or vitamin A toxicity

Not until the twentieth century was vitamin A actually isolated In 1909 Hopkins and Steep extracted a lipidfat substance that mice and rats absolutely required for their growth Elmer McCollum performed a careful analysis of the growth-promoting factors in protein-free milk leading to the isolation of the first known fat-soluble vitamin This essential growth promoting compound was named ldquofat soluble Ardquo a terminology distinguishing it from other water-soluble vitamins such as the recently discovered anti-scurvey factormdashvitamin C In 1920 Jack Drummond suggested that the ldquovital sub-stancerdquo be given the name vitamin A a substance later associated with a pigmented yellow color In 1935 George Wald found vitamin A was a component of the retina When the rhodopsin pigment was exposed to light it yielded opsin and a vitamin A-containing compound (the chromophore) indicating that vitamin A was essential in retinal function While the nature of the chromophore and the reactions occurring dur-ing the visual cycle were characterized long ago there have been recent developments in the characterization of the enzymes and carrier proteins involved in this cycle with novel findings indicating that an alternative pathway for chromophore regeneration has evolved in cones (the photoreceptors of the retina that operate in bright daylight and which are responsible visual acuity and color discrimination) Two chapters in this book (Chapters 18 and 24) review these findings the latter chapter describing gene mutations leading to visual diseases and discussing therapeutic strategies

In the late 1970s researchers suggested that the physiological activity of vitamin A may be occurring through ligand binding to nuclear receptors In the 1980s the goal of many researchers became to elucidate how these signaling molecules prodded gene expression In 1986 the groups of Pierre Chambon and Ronald Evans independently

xiv Preface

cloned and characterized the first retinoic acid receptormdasha molecule providing an entry point for detailing vitamin A biology In 2004 the two scientists were awarded the Albert Lasker Basic Medical Research Award for their discoveries that retinoids and other pathways including steroids vitamin D thyroid hormone and many other lipid based drugs transmit signals through similar pathways These discoveries advanced the mechanisms of vitamin A and retinoid signaling Several authors at the forefront of research aiming to solve structurendashfunction relationships of retinoid receptors their genomic target loci and the post-translational and epigenetic regulatory mechanisms that fine-tune their activity according to the cellular context provide a comprehensive overview of these complex phenomena in Part II of this book (Chapters 5ndash8) These are followed by two chapters on the chemistry of retinoid-related synthetic molecules an important research field for drug design of selective receptor agnonistsantagonists with Chapter 10 emphasizing how research on retinoid-related molecules led to the identification of modulators of another nuclear receptor the small heterodi-mer partner ldquoorphanrdquo receptor

Worldwide problems of nutritional deficiencies appear insurmountable as the planetrsquos population tops seven billion Vitamin A deficiency and its associated dis-eases afflict impoverished populations and are endemic in sub-Saharan Africa and southern Asia These deficiencies preferentially target infants preschool children and pregnant women Vitamin A deficiency is the leading preventable cause of vision loss Over 200 million are estimated as lacking sufficient serum retinol levels Vitamin A deficiency diseases frequently include rod photoreceptor dysfunction and night blindness and in more severe cases blinding ulcerations and necrosis of the cornea The World Health Organization estimates that 14 million children have pre-ventable irreversible blindness with half dying within a year of losing sight Also because inflammation reduces retinol-binding protein levels compromised immune function increases death rate following bacterial-induced diarrhea malaria or HIV infection Lower food intake during disease results in a synergistic downward pro-gression of malnutrition and disease which can be lethal Providing a small concen-trated dose of retinol has proven effects increasing infant survival by over 30 Retinol treatments are extremely low cost with consequent saving of visionmdashwith sometimes only two days of treatment restoring night blindness One chapter of this book (Chapter 23) describes the consequences to world health of retinoid defi-ciencies while others provide updated reviews on the relationship between the reti-noid pathway and inflammatory processes (Chapter 20) and its functions in the immune system (Chapter 21)

Nutritional deficiencies are a common survival challenge and for the fat-soluble vitamin A our organism has developed exquisite mechanisms to enzymatically con-vert and maintain adequate stores of retinoids (mainly in the liver) allowing adults to survive under vitamin A deficiency for months or even years The first part of this book provides detailed accounts on the mechanisms of vitamin A and carotenoid absorption liver storage and tissue-targeted delivery through the blood circulation with one chapter (Chapter 4) discussing current knowledge on the ancestry of reti-noid receptors in multicellular organisms and the molecular evolution of the retinoic acid signaling pathway

In the 1970s Michael Sporn first used the term ldquochemopreventionrdquo for clinical trials using retinoids to prevent or delay the occurrence of certain forms of cancer

Preface xv

Unfortunately large-scale clinical trials providing retinol to smokers with premalig-nant oral lesions had disappointing results and in some cases had to be interrupted Potentially these high dose treatments decreased cellular retinoid signalingmdashperhaps by inducing CYP26 enzymatic activity leading to retinoic acid (RA) degradation Currently compounds inhibiting these enzymes are being tested for cancer treatment efficacy Clinical pharmacology employing the synthetic retinoid fenretinide indicates retinoid derivatives are plausible cancer treatment strategies Phase III clinical trial data suggested that fenretinide reduces breast cancer relapse inducing tumor cell death and necrosis The retinoid X receptor (RXR) agonist bexarotene is being tested for treatment of T-cell lymphoma and Kaposi sarcoma These data are reviewed in the last chapter of this book

How can we better understand intracellular functions related to cancer As detailed in Chapter 11 there are key examples of how the biological activity of RA critically relies on a balance of several lipid-binding proteins Reductions in one of these proteins cellular retinoic acid-binding protein 2 (CRABP2) and augmentations in fatty acid-binding protein 5 (FABP5) change RA signaling from normal phy-siological growth inhibition to pathological states of neoplastic breast cancer growth There are also antagonistic effects of RA and estrogenmdashanother hormone acting through nuclear receptorsmdashin breast cancer cells (Chapter 8) Selective gene targets are either induced or inhibited within cell-specific contexts indicating some of the selective parameters in which RA can be capable of arresting neoplastic growth

There are many additional reasons that populations in affluent well-nourished countries should be concerned about retinoid signaling The most oppressing health problem in the United States (and a growing plague in the entire world) is the alarm-ing increase in obesity This rise in obesity is even found in poorer countries due to food insecurity choices based on high calorie intake The increase in the overweight population and its associated diseases (cardiovascular diabetes and metabolic dis-eases) will have a predicted economic impact of more than $30 trillion over the next 20 years according to a 2011 report by the World Economic Forum and Harvard School of Public Health The collective impact of noncommunicable cardiovascular diabetic and other metabolic diseases linked to obesity impacts large segments of the world population Proteins selectively binding retinoids (such as retinol-binding pro-tein) are increased in obese patients Retinoic acid has a pharmacological side effect of inducing weight loss While this clinical side effect is clearly disadvantageous in retinoid treatments used to reduce cancer growth it has potential actions as an anti-obesity agent if other side effects can be reduced Structural studies on how retinoid receptors bind ligands and coregulators reveal allosteric changes altering their sig-naling properties and provide critical information needed to improve retinoid phar-macology to provide drugs with improved selectivity and reduced side effects (Chapters 7 and 9) Authors also address how retinoid fat-reducing metabolic actions may be controlled by cellular sites of storage namely in lipid droplets (Chapter 3) Pressing questions are if and should retinoids be a widely used treatment strategy to reduce obesity and consequent metabolic disease Since side effects of severe hyper-vitaminosis A or retinoid treatments include skin scaling lethargy and nausea long-term pilot trials appear to be required before the weight loss potential of reti-noids is exploited in large populations Treatment may require pharmacologically selective retinoids specific to retinoid metabolic targets Public health campaigns to

xvi Preface

increasing intake of fruits and vegetables (especially those rich in vitamin A) serve as a more immediate strategy to address increasing obesity

Human nutrition can have a dynamic effect on life processes and disease by influencing epigenetic regulation Gene regulatory systems have evolved for extremely precise and specific regulatory control The retinoid signal acts through epigenetic control of transcriptional coregulatory modulators that can respond to environmental clues Retinoid responsiveness relies on promoter occupancy and organization of the epigenetic landscape at the chromatin level Many genes that are rapidly activated by retinoids have transcriptional response elements with ldquobivalentrdquo promoter chromatin access (Chapter 12) Responding genes are in a poised state and are rapidly activated by increasing the activation to repressive transcriptional complex ratios This chromatin reorganization can be coordinately managed by phosphorylation of several actors of RA signaling integrating and managing cellular decision (Chapter 5) The disease-related examples of aberrant epigenetic landscape contributing to malignancy can be found with retinoic acid treatment of acute promy-elocytic leukemia (Chapter 25) Current evidence supports that compounds inhibit-ing histone deacetylase activity augment retinoic acid receptorndashretinoid X receptor (RARndashRXR) response acting synergistically on condensed chromatin RXRs can be specifically activated by a class of drugs termed rexinoids currently used in the clinical treatment of selective lymphomas These treatments are effective grim reapers bringing about tumor cell apoptosis

The promise of stem cell research has provided public hope in curative ther-apies including neuroregenerative treatment of spinal cord injury or Alzheimerrsquos disease repairing damaged heart tissue subsequent to myocardial infarction or replacing skin in severe burn victims Are these plausible strategies or are realistic expectations being exceeded Retinoids are regulators of a number of stem cell line-ages in a variety of organ progenitor sites (pancreas blood heart central nervous system and gonads) at various stages of development and postnatal life The under-lying concept that RA regulates stemprogenitor cell lineages during developmentmdashand may have later roles in regulating organ regeneration or repairmdashis the subject of several chapters in Part III of this book Various animal models of retinoid deficiency show defects in the developing nervous system heart and lung These defects per-turb organ function disrupting patterning hence inducing fetal or embryonic-stage lethality Interestingly the same genes expressed in adult stem cell populations are often acting in progenitor cell populations at embryonic and fetal stages Could the changes in gene expression observed in such cell populations in murine models of retinoid deficiency be considered as a paradigm for further studies providing clues on how retinoids might modulate stem cell dynamics at later (adult) stages These questions which are touched upon in several chapters of this book will undoubtedly be the subject of many future investigations

In summary the strong interest in vitamin A signaling through its active compo-nents the retinoids is rooted in the biological implications in so many physiological and disease processes Who would want to delve into the complexity of all of these subjects There are currently over 50 000 references when the word ldquoretinoidsrdquo is enteredmdashalthough its coselection with ldquoreviewsrdquo ldquocancerrdquo or ldquoobesityrdquo will reduce this number to thousands This book presents integrated knowledge from many current experts in the field We congratulate authors for their commitmentmdashin spite of their

Preface xvii

other overburdening academic responsibilities And for this opus we as editors wish to give a million ldquothanksrdquo to all of the contributing authors This book is the ldquoRetinoid Toolrdquo that can serve both the debutant and experienced researcher advancing their knowledge and understanding and leading to successful research discoveries

Pascal DolleacuteKaren Niederreither

PART I

Vitamin a metabolic and enzymatic Pathways

The Retinoids Biology Biochemistry and Disease First Edition Edited by Pascal Dolleacute and Karen Niederreither copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

1

Vitamin a metabolism storage and tissue deliVery mechanisms

William s blaner1 and yang li2

1 Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

2 Columbia College Columbia University New York NY USA

I IntroductIon

Vitamin A is an essential micronutrient that must be acquired from the diet either as preformed vitamin A or as provitamin A carotenoid By definition all-trans-retinol is vitamin A although retinol metabolites including retinyl esters and retinoic acid are often referred to collectively as vitamin A Throughout this chapter vitamin A is used solely to refer to all-trans-retinol The term retinoid which was first coined by Sporn in the mid-1970s refers to a family of chemicals both natural and synthetic that bear a structural resemblance to all-trans-retinol with or without the biologic activity of vitamin A (Sporn et al 1976) Thus the retinoid family of compounds comprises both naturally-occurring vitamin A metabolites and synthetic compounds that bear a structural resemblance to vitamin A

Within the body the two most abundant vitamin A species are retinol and retinyl esters Both retinol and retinyl esters are central to the metabolism storage and delivery of vitamin A to tissues The great majority of the vitamin A present in the body is stored as retinyl esters primarily in the liver but also in other tissues Dietary vitamin A is also packaged as retinyl esters in nascent chylomicrons and is thus taken into the body Because of this retinyl esters are a transport form of vitamin A that can

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

vi Contents

9 retInoId reCePtor-seleCtIVe modulators ChemIstry 3d struCtures and systems bIology 165Marco-Antonio Mendoza-Parra William Bourguet Angel R de Lera and Hinrich Gronemeyer

10 use of retInoId reCePtor lIgands to IdentIfy other nuClear reCePtor lIgands retInoId-related moleCules are lIgands for the small heterodImer Partner (shP) ldquoorPhanrdquo reCePtor 193Marcia I Dawson and Zebin Xia

11 the dual transCrIPtIonal aCtIVIty of retInoIC aCId 273Noa Noy

12 retInoIds ePIgenetIC Changes durIng stem Cell dIfferentIatIon and Cell lIneage ChoICe 291Lorraine J Gudas

Part III retInoIC aCId sIgnalIng In deVeloPment 307

13 retInoIC aCId sIgnalIng and Central nerVous system deVeloPment 309Malcolm Maden

14 the role of retInoIC aCId In lImb deVeloPment 339Gregg Duester

15 retInoIC aCId sIgnalIng and heart deVeloPment 353Steacutephane Zaffran and Karen Niederreither

16 retInoIC aCId In the deVeloPIng lung and other foregut derIVatIVes 371Wellington V Cardoso and Felicia Chen

17 retInoIC aCId and the Control of meIotIC InItIatIon 383Josephine Bowles and Peter Koopman

Part IV retInoIds and PhysIologICal funCtIons 401

18 retInoIds and the VIsual CyCle new aCtors for an ldquooldrdquo funCtIon 403Darwin Babino and Johannes von Lintig

19 retInoId sIgnalIng In the Central nerVous system 421Peter McCaffery and Wojciech Krezel

20 retInoId turnoVer and CatabolIsm InfluenCes of dIet and InflammatIon 449A Catharine Ross and Reza Zolfaghari

Contents vii

21 retInoIds and the Immune system 465J Rodrigo Mora and Makoto Iwata

22 retInoIC aCId reCePtor sIgnalIng In Post-natal male germ Cell dIfferentIatIon 485Manuel Mark and Norbert B Ghyselinck

Part V retInoIds dIsease and theraPy 505

23 ePIdemIology and PreVentIon of VItamIn a defICIenCy dIsorders 507Keith P West Jr

24 retInoId Pathway gene mutatIons and the PathoPhysIology of related VIsual dIseases 529Yaroslav Tsybovsky and Krzysztof Palczewski

25 retInoIC aCId In aCute myeloId leukemIas 543Hugues de Theacute and Pierre Fenaux

26 adVanCes In the use of retInoIds In CanCer theraPy and PreVentIon 557Michael J Spinella Sarah J Freemantle and Ethan Dmitrovsky

Index 575

ix

Contributors

Darwin Babino Department of Pharmacology School of Medicine Case Western Reserve University Cleveland OH USA

William S Blaner Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

William Bourguet Centre de Biochimie Structurale Montpellier France Universiteacutes Montpellier 1 amp 2 Montpellier France

Josephine Bowles Institute for Molecular Bioscience The University of Queensland Brisbane QLD Australia

Wellington V Cardoso Columbia Center for Human Development Department of Medicine Columbia University Medical Center New York NY USA

Marilyn Carrier Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Felicia Chen Pulmonary Center Boston University School of Medicine Boston MA USA

Irwin Davidson Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Marcia I Dawson Cancer Center Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Ethan Dmitrovsky Department of Pharmacology and Toxicology Department of Medicine The Norris Cotton Cancer Center Geisel School of Medicine Dartmouth Hanover NH USA

Gregg Duester Development Aging and Regeneration Program Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Pierre Fenaux Universiteacute Paris Diderot Sorbonne Paris Citeacute Assistance PubliquendashHocircpitaux de Paris Service drsquoHeacutematologie Hocircpital St Louis Paris France

Sarah J Freemantle Department of Pharmacology and Toxicology Geisel School of Medicine Dartmouth Hanover NH USA

Norbert B Ghyselinck Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Hinrich Gronemeyer Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

x Contributors

Lorraine J Gudas Pharmacology Department Weill Cornell Medical College Cornell University New York NY USA

Earl H Harrison Department of Human Nutrition and Ohio State Biochemistry Program Ohio State University Columbus OH USA

Makoto Iwata Kagawa School of Pharmaceutical Sciences Tokushima Bunri University amp JST CREST Kagawa Japan

Peter Koopman Institute for Molecular Bioscience The University of Queensland Brisbane QLD Australia

Wojciech Krezel Development and Stem Cells Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Charles R Krois Graduate Program in Metabolic Biology Nutritional Sciences and Toxicology University of California-Berkeley CA USA

Vincent Laudet Molecular Zoology Team Institut de Geacutenomique Fonctionnelle de Lyon (IGFL) Ecole Normale Supeacuterieure de Lyon Lyon France

Angel R de Lera Departamento de Quiacutemica Orgaacutenica Facultad de Quiacutemica Universidad de Vigo Vigo Spain

Yang Li Columbia College Columbia University New York NY USA

Johannes von Lintig Department of Pharmacology School of Medicine Case Western Reserve University Cleveland OH USA

Malcolm Maden Department of Biology and University of Florida Genetics Institute University of Florida Gainesville FL USA

Manuel Mark Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France Hocircpitaux Universitaires de Strasbourg Strasbourg France

Peter McCaffery Institute of Medical Sciences School of Medical Sciences University of Aberdeen Aberdeen UK

Marco-Antonio Mendoza-Parra Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

J Rodrigo Mora Massachusetts General Hospital and Harvard Medical School Boston MA USA

Dino Moras Integrated Structural Biology Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Joseph L Napoli Graduate Program in Metabolic Biology Nutritional Sciences and Toxicology University of California-Berkeley CA USA

Karen Niederreither Development and Stem Cells Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Noa Noy Department of Cellular and Molecular Medicine Lerner Research Institute Cleveland Clinic Foundation Cleveland OH USA

Contributors xi

Krzysztof Palczewski Department of Pharmacology Case Western Reserve University Cleveland OH USA

Ceacutecile Rochette-Egly Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

A Catharine Ross Department of Nutritional Sciences Pennsylvania State University University Park PA USA

Michael Schubert Laboratoire de Biologie du Deacuteveloppement de Villefranche-sur-Mer Sorbonne Universiteacutes UPMC Universiteacute Paris 06 Observatoire Oceacuteanologique de Villefranche-sur-Mer Villefranche-sur-Mer France

Carlo dela Sentildearsquos Department of Human Nutrition and Ohio State Biochemistry Program Ohio State University Columbus OH USA

Michael J Spinella Department of Pharmacology and Toxicology The Norris Cotton Cancer Center Geisel School of Medicine Dartmouth College Hanover NH USA

Hugues de Theacute Universiteacute Paris Diderot Sorbonne Paris Citeacute Institut Universitaire drsquoHeacutematologie Assistance PubliquendashHocircpitaux de Paris Service de Biochimie Hocircpital St Louis Paris France

Yaroslav Tsybovsky Department of Pharmacology Case Western Reserve University Cleveland OH USA

Sylvia Urban Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Li-Na Wei Department of Pharmacology University of Minnesota Medical School Minneapolis MN USA

Keith P West Jr Center and Program in Human Nutrition Department of International Health Johns Hopkins Bloomberg School of Public Health Baltimore MD USA

Zebin Xia Cancer Center Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Tao Ye Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Steacutephane Zaffran Aix-Marseille Universiteacute Uniteacute de Geacuteneacutetique Meacutedicale et Geacutenomique Fonctionnelle (GMGF) Faculteacute de Meacutedecine Marseille France

Elisabeth Zieger Laboratoire de Biologie du Deacuteveloppement de Villefranche-sur-Mer Sorbonne Universiteacutes UPMC Universiteacute Paris 06 Observatoire Oceacuteanologique de Villefranche-sur-Mer Villefranche-sur-Mer France

Reza Zolfaghari Department of Nutritional Sciences Pennsylvania State University University Park PA USA

xiii

Preface

VITAMIN A AND RETINOIDS AN AMAZING HISTORY AND HOPES FOR DISEASE PREVENTION

Historically the vitamin A precursor carotenoids had been long suspected of having medicinal properties For thousands of years humans and animals suffered from vitamin A deficiency typified by night blindness and xerophthalmiamdasha failure of tear production which if untreated would result in blindness While the underlying causes of such maledictions were a mystery in 1500 BC the ancient Egyptians rec-ommended eating animal or fish liver for their curative powers The native people of the Arctic also long knew to avoid eating the liver of polar bears because they became sick (unknowingly by vitamin A) Western explorers not savvy to the vitamin A rich properties of polar bear liver as early as 1596 accounted a horrible illnessndashsluggish-ness blurred vision nausea headache and skin loss resulting in coma and even deathmdashall signs of acute hypervitaminosis A or vitamin A toxicity

Not until the twentieth century was vitamin A actually isolated In 1909 Hopkins and Steep extracted a lipidfat substance that mice and rats absolutely required for their growth Elmer McCollum performed a careful analysis of the growth-promoting factors in protein-free milk leading to the isolation of the first known fat-soluble vitamin This essential growth promoting compound was named ldquofat soluble Ardquo a terminology distinguishing it from other water-soluble vitamins such as the recently discovered anti-scurvey factormdashvitamin C In 1920 Jack Drummond suggested that the ldquovital sub-stancerdquo be given the name vitamin A a substance later associated with a pigmented yellow color In 1935 George Wald found vitamin A was a component of the retina When the rhodopsin pigment was exposed to light it yielded opsin and a vitamin A-containing compound (the chromophore) indicating that vitamin A was essential in retinal function While the nature of the chromophore and the reactions occurring dur-ing the visual cycle were characterized long ago there have been recent developments in the characterization of the enzymes and carrier proteins involved in this cycle with novel findings indicating that an alternative pathway for chromophore regeneration has evolved in cones (the photoreceptors of the retina that operate in bright daylight and which are responsible visual acuity and color discrimination) Two chapters in this book (Chapters 18 and 24) review these findings the latter chapter describing gene mutations leading to visual diseases and discussing therapeutic strategies

In the late 1970s researchers suggested that the physiological activity of vitamin A may be occurring through ligand binding to nuclear receptors In the 1980s the goal of many researchers became to elucidate how these signaling molecules prodded gene expression In 1986 the groups of Pierre Chambon and Ronald Evans independently

xiv Preface

cloned and characterized the first retinoic acid receptormdasha molecule providing an entry point for detailing vitamin A biology In 2004 the two scientists were awarded the Albert Lasker Basic Medical Research Award for their discoveries that retinoids and other pathways including steroids vitamin D thyroid hormone and many other lipid based drugs transmit signals through similar pathways These discoveries advanced the mechanisms of vitamin A and retinoid signaling Several authors at the forefront of research aiming to solve structurendashfunction relationships of retinoid receptors their genomic target loci and the post-translational and epigenetic regulatory mechanisms that fine-tune their activity according to the cellular context provide a comprehensive overview of these complex phenomena in Part II of this book (Chapters 5ndash8) These are followed by two chapters on the chemistry of retinoid-related synthetic molecules an important research field for drug design of selective receptor agnonistsantagonists with Chapter 10 emphasizing how research on retinoid-related molecules led to the identification of modulators of another nuclear receptor the small heterodi-mer partner ldquoorphanrdquo receptor

Worldwide problems of nutritional deficiencies appear insurmountable as the planetrsquos population tops seven billion Vitamin A deficiency and its associated dis-eases afflict impoverished populations and are endemic in sub-Saharan Africa and southern Asia These deficiencies preferentially target infants preschool children and pregnant women Vitamin A deficiency is the leading preventable cause of vision loss Over 200 million are estimated as lacking sufficient serum retinol levels Vitamin A deficiency diseases frequently include rod photoreceptor dysfunction and night blindness and in more severe cases blinding ulcerations and necrosis of the cornea The World Health Organization estimates that 14 million children have pre-ventable irreversible blindness with half dying within a year of losing sight Also because inflammation reduces retinol-binding protein levels compromised immune function increases death rate following bacterial-induced diarrhea malaria or HIV infection Lower food intake during disease results in a synergistic downward pro-gression of malnutrition and disease which can be lethal Providing a small concen-trated dose of retinol has proven effects increasing infant survival by over 30 Retinol treatments are extremely low cost with consequent saving of visionmdashwith sometimes only two days of treatment restoring night blindness One chapter of this book (Chapter 23) describes the consequences to world health of retinoid defi-ciencies while others provide updated reviews on the relationship between the reti-noid pathway and inflammatory processes (Chapter 20) and its functions in the immune system (Chapter 21)

Nutritional deficiencies are a common survival challenge and for the fat-soluble vitamin A our organism has developed exquisite mechanisms to enzymatically con-vert and maintain adequate stores of retinoids (mainly in the liver) allowing adults to survive under vitamin A deficiency for months or even years The first part of this book provides detailed accounts on the mechanisms of vitamin A and carotenoid absorption liver storage and tissue-targeted delivery through the blood circulation with one chapter (Chapter 4) discussing current knowledge on the ancestry of reti-noid receptors in multicellular organisms and the molecular evolution of the retinoic acid signaling pathway

In the 1970s Michael Sporn first used the term ldquochemopreventionrdquo for clinical trials using retinoids to prevent or delay the occurrence of certain forms of cancer

Preface xv

Unfortunately large-scale clinical trials providing retinol to smokers with premalig-nant oral lesions had disappointing results and in some cases had to be interrupted Potentially these high dose treatments decreased cellular retinoid signalingmdashperhaps by inducing CYP26 enzymatic activity leading to retinoic acid (RA) degradation Currently compounds inhibiting these enzymes are being tested for cancer treatment efficacy Clinical pharmacology employing the synthetic retinoid fenretinide indicates retinoid derivatives are plausible cancer treatment strategies Phase III clinical trial data suggested that fenretinide reduces breast cancer relapse inducing tumor cell death and necrosis The retinoid X receptor (RXR) agonist bexarotene is being tested for treatment of T-cell lymphoma and Kaposi sarcoma These data are reviewed in the last chapter of this book

How can we better understand intracellular functions related to cancer As detailed in Chapter 11 there are key examples of how the biological activity of RA critically relies on a balance of several lipid-binding proteins Reductions in one of these proteins cellular retinoic acid-binding protein 2 (CRABP2) and augmentations in fatty acid-binding protein 5 (FABP5) change RA signaling from normal phy-siological growth inhibition to pathological states of neoplastic breast cancer growth There are also antagonistic effects of RA and estrogenmdashanother hormone acting through nuclear receptorsmdashin breast cancer cells (Chapter 8) Selective gene targets are either induced or inhibited within cell-specific contexts indicating some of the selective parameters in which RA can be capable of arresting neoplastic growth

There are many additional reasons that populations in affluent well-nourished countries should be concerned about retinoid signaling The most oppressing health problem in the United States (and a growing plague in the entire world) is the alarm-ing increase in obesity This rise in obesity is even found in poorer countries due to food insecurity choices based on high calorie intake The increase in the overweight population and its associated diseases (cardiovascular diabetes and metabolic dis-eases) will have a predicted economic impact of more than $30 trillion over the next 20 years according to a 2011 report by the World Economic Forum and Harvard School of Public Health The collective impact of noncommunicable cardiovascular diabetic and other metabolic diseases linked to obesity impacts large segments of the world population Proteins selectively binding retinoids (such as retinol-binding pro-tein) are increased in obese patients Retinoic acid has a pharmacological side effect of inducing weight loss While this clinical side effect is clearly disadvantageous in retinoid treatments used to reduce cancer growth it has potential actions as an anti-obesity agent if other side effects can be reduced Structural studies on how retinoid receptors bind ligands and coregulators reveal allosteric changes altering their sig-naling properties and provide critical information needed to improve retinoid phar-macology to provide drugs with improved selectivity and reduced side effects (Chapters 7 and 9) Authors also address how retinoid fat-reducing metabolic actions may be controlled by cellular sites of storage namely in lipid droplets (Chapter 3) Pressing questions are if and should retinoids be a widely used treatment strategy to reduce obesity and consequent metabolic disease Since side effects of severe hyper-vitaminosis A or retinoid treatments include skin scaling lethargy and nausea long-term pilot trials appear to be required before the weight loss potential of reti-noids is exploited in large populations Treatment may require pharmacologically selective retinoids specific to retinoid metabolic targets Public health campaigns to

xvi Preface

increasing intake of fruits and vegetables (especially those rich in vitamin A) serve as a more immediate strategy to address increasing obesity

Human nutrition can have a dynamic effect on life processes and disease by influencing epigenetic regulation Gene regulatory systems have evolved for extremely precise and specific regulatory control The retinoid signal acts through epigenetic control of transcriptional coregulatory modulators that can respond to environmental clues Retinoid responsiveness relies on promoter occupancy and organization of the epigenetic landscape at the chromatin level Many genes that are rapidly activated by retinoids have transcriptional response elements with ldquobivalentrdquo promoter chromatin access (Chapter 12) Responding genes are in a poised state and are rapidly activated by increasing the activation to repressive transcriptional complex ratios This chromatin reorganization can be coordinately managed by phosphorylation of several actors of RA signaling integrating and managing cellular decision (Chapter 5) The disease-related examples of aberrant epigenetic landscape contributing to malignancy can be found with retinoic acid treatment of acute promy-elocytic leukemia (Chapter 25) Current evidence supports that compounds inhibit-ing histone deacetylase activity augment retinoic acid receptorndashretinoid X receptor (RARndashRXR) response acting synergistically on condensed chromatin RXRs can be specifically activated by a class of drugs termed rexinoids currently used in the clinical treatment of selective lymphomas These treatments are effective grim reapers bringing about tumor cell apoptosis

The promise of stem cell research has provided public hope in curative ther-apies including neuroregenerative treatment of spinal cord injury or Alzheimerrsquos disease repairing damaged heart tissue subsequent to myocardial infarction or replacing skin in severe burn victims Are these plausible strategies or are realistic expectations being exceeded Retinoids are regulators of a number of stem cell line-ages in a variety of organ progenitor sites (pancreas blood heart central nervous system and gonads) at various stages of development and postnatal life The under-lying concept that RA regulates stemprogenitor cell lineages during developmentmdashand may have later roles in regulating organ regeneration or repairmdashis the subject of several chapters in Part III of this book Various animal models of retinoid deficiency show defects in the developing nervous system heart and lung These defects per-turb organ function disrupting patterning hence inducing fetal or embryonic-stage lethality Interestingly the same genes expressed in adult stem cell populations are often acting in progenitor cell populations at embryonic and fetal stages Could the changes in gene expression observed in such cell populations in murine models of retinoid deficiency be considered as a paradigm for further studies providing clues on how retinoids might modulate stem cell dynamics at later (adult) stages These questions which are touched upon in several chapters of this book will undoubtedly be the subject of many future investigations

In summary the strong interest in vitamin A signaling through its active compo-nents the retinoids is rooted in the biological implications in so many physiological and disease processes Who would want to delve into the complexity of all of these subjects There are currently over 50 000 references when the word ldquoretinoidsrdquo is enteredmdashalthough its coselection with ldquoreviewsrdquo ldquocancerrdquo or ldquoobesityrdquo will reduce this number to thousands This book presents integrated knowledge from many current experts in the field We congratulate authors for their commitmentmdashin spite of their

Preface xvii

other overburdening academic responsibilities And for this opus we as editors wish to give a million ldquothanksrdquo to all of the contributing authors This book is the ldquoRetinoid Toolrdquo that can serve both the debutant and experienced researcher advancing their knowledge and understanding and leading to successful research discoveries

Pascal DolleacuteKaren Niederreither

PART I

Vitamin a metabolic and enzymatic Pathways

The Retinoids Biology Biochemistry and Disease First Edition Edited by Pascal Dolleacute and Karen Niederreither copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

1

Vitamin a metabolism storage and tissue deliVery mechanisms

William s blaner1 and yang li2

1 Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

2 Columbia College Columbia University New York NY USA

I IntroductIon

Vitamin A is an essential micronutrient that must be acquired from the diet either as preformed vitamin A or as provitamin A carotenoid By definition all-trans-retinol is vitamin A although retinol metabolites including retinyl esters and retinoic acid are often referred to collectively as vitamin A Throughout this chapter vitamin A is used solely to refer to all-trans-retinol The term retinoid which was first coined by Sporn in the mid-1970s refers to a family of chemicals both natural and synthetic that bear a structural resemblance to all-trans-retinol with or without the biologic activity of vitamin A (Sporn et al 1976) Thus the retinoid family of compounds comprises both naturally-occurring vitamin A metabolites and synthetic compounds that bear a structural resemblance to vitamin A

Within the body the two most abundant vitamin A species are retinol and retinyl esters Both retinol and retinyl esters are central to the metabolism storage and delivery of vitamin A to tissues The great majority of the vitamin A present in the body is stored as retinyl esters primarily in the liver but also in other tissues Dietary vitamin A is also packaged as retinyl esters in nascent chylomicrons and is thus taken into the body Because of this retinyl esters are a transport form of vitamin A that can

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

Contents vii

21 retInoIds and the Immune system 465J Rodrigo Mora and Makoto Iwata

22 retInoIC aCId reCePtor sIgnalIng In Post-natal male germ Cell dIfferentIatIon 485Manuel Mark and Norbert B Ghyselinck

Part V retInoIds dIsease and theraPy 505

23 ePIdemIology and PreVentIon of VItamIn a defICIenCy dIsorders 507Keith P West Jr

24 retInoId Pathway gene mutatIons and the PathoPhysIology of related VIsual dIseases 529Yaroslav Tsybovsky and Krzysztof Palczewski

25 retInoIC aCId In aCute myeloId leukemIas 543Hugues de Theacute and Pierre Fenaux

26 adVanCes In the use of retInoIds In CanCer theraPy and PreVentIon 557Michael J Spinella Sarah J Freemantle and Ethan Dmitrovsky

Index 575

ix

Contributors

Darwin Babino Department of Pharmacology School of Medicine Case Western Reserve University Cleveland OH USA

William S Blaner Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

William Bourguet Centre de Biochimie Structurale Montpellier France Universiteacutes Montpellier 1 amp 2 Montpellier France

Josephine Bowles Institute for Molecular Bioscience The University of Queensland Brisbane QLD Australia

Wellington V Cardoso Columbia Center for Human Development Department of Medicine Columbia University Medical Center New York NY USA

Marilyn Carrier Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Felicia Chen Pulmonary Center Boston University School of Medicine Boston MA USA

Irwin Davidson Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Marcia I Dawson Cancer Center Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Ethan Dmitrovsky Department of Pharmacology and Toxicology Department of Medicine The Norris Cotton Cancer Center Geisel School of Medicine Dartmouth Hanover NH USA

Gregg Duester Development Aging and Regeneration Program Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Pierre Fenaux Universiteacute Paris Diderot Sorbonne Paris Citeacute Assistance PubliquendashHocircpitaux de Paris Service drsquoHeacutematologie Hocircpital St Louis Paris France

Sarah J Freemantle Department of Pharmacology and Toxicology Geisel School of Medicine Dartmouth Hanover NH USA

Norbert B Ghyselinck Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Hinrich Gronemeyer Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

x Contributors

Lorraine J Gudas Pharmacology Department Weill Cornell Medical College Cornell University New York NY USA

Earl H Harrison Department of Human Nutrition and Ohio State Biochemistry Program Ohio State University Columbus OH USA

Makoto Iwata Kagawa School of Pharmaceutical Sciences Tokushima Bunri University amp JST CREST Kagawa Japan

Peter Koopman Institute for Molecular Bioscience The University of Queensland Brisbane QLD Australia

Wojciech Krezel Development and Stem Cells Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Charles R Krois Graduate Program in Metabolic Biology Nutritional Sciences and Toxicology University of California-Berkeley CA USA

Vincent Laudet Molecular Zoology Team Institut de Geacutenomique Fonctionnelle de Lyon (IGFL) Ecole Normale Supeacuterieure de Lyon Lyon France

Angel R de Lera Departamento de Quiacutemica Orgaacutenica Facultad de Quiacutemica Universidad de Vigo Vigo Spain

Yang Li Columbia College Columbia University New York NY USA

Johannes von Lintig Department of Pharmacology School of Medicine Case Western Reserve University Cleveland OH USA

Malcolm Maden Department of Biology and University of Florida Genetics Institute University of Florida Gainesville FL USA

Manuel Mark Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France Hocircpitaux Universitaires de Strasbourg Strasbourg France

Peter McCaffery Institute of Medical Sciences School of Medical Sciences University of Aberdeen Aberdeen UK

Marco-Antonio Mendoza-Parra Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

J Rodrigo Mora Massachusetts General Hospital and Harvard Medical School Boston MA USA

Dino Moras Integrated Structural Biology Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Joseph L Napoli Graduate Program in Metabolic Biology Nutritional Sciences and Toxicology University of California-Berkeley CA USA

Karen Niederreither Development and Stem Cells Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Noa Noy Department of Cellular and Molecular Medicine Lerner Research Institute Cleveland Clinic Foundation Cleveland OH USA

Contributors xi

Krzysztof Palczewski Department of Pharmacology Case Western Reserve University Cleveland OH USA

Ceacutecile Rochette-Egly Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

A Catharine Ross Department of Nutritional Sciences Pennsylvania State University University Park PA USA

Michael Schubert Laboratoire de Biologie du Deacuteveloppement de Villefranche-sur-Mer Sorbonne Universiteacutes UPMC Universiteacute Paris 06 Observatoire Oceacuteanologique de Villefranche-sur-Mer Villefranche-sur-Mer France

Carlo dela Sentildearsquos Department of Human Nutrition and Ohio State Biochemistry Program Ohio State University Columbus OH USA

Michael J Spinella Department of Pharmacology and Toxicology The Norris Cotton Cancer Center Geisel School of Medicine Dartmouth College Hanover NH USA

Hugues de Theacute Universiteacute Paris Diderot Sorbonne Paris Citeacute Institut Universitaire drsquoHeacutematologie Assistance PubliquendashHocircpitaux de Paris Service de Biochimie Hocircpital St Louis Paris France

Yaroslav Tsybovsky Department of Pharmacology Case Western Reserve University Cleveland OH USA

Sylvia Urban Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Li-Na Wei Department of Pharmacology University of Minnesota Medical School Minneapolis MN USA

Keith P West Jr Center and Program in Human Nutrition Department of International Health Johns Hopkins Bloomberg School of Public Health Baltimore MD USA

Zebin Xia Cancer Center Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Tao Ye Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Steacutephane Zaffran Aix-Marseille Universiteacute Uniteacute de Geacuteneacutetique Meacutedicale et Geacutenomique Fonctionnelle (GMGF) Faculteacute de Meacutedecine Marseille France

Elisabeth Zieger Laboratoire de Biologie du Deacuteveloppement de Villefranche-sur-Mer Sorbonne Universiteacutes UPMC Universiteacute Paris 06 Observatoire Oceacuteanologique de Villefranche-sur-Mer Villefranche-sur-Mer France

Reza Zolfaghari Department of Nutritional Sciences Pennsylvania State University University Park PA USA

xiii

Preface

VITAMIN A AND RETINOIDS AN AMAZING HISTORY AND HOPES FOR DISEASE PREVENTION

Historically the vitamin A precursor carotenoids had been long suspected of having medicinal properties For thousands of years humans and animals suffered from vitamin A deficiency typified by night blindness and xerophthalmiamdasha failure of tear production which if untreated would result in blindness While the underlying causes of such maledictions were a mystery in 1500 BC the ancient Egyptians rec-ommended eating animal or fish liver for their curative powers The native people of the Arctic also long knew to avoid eating the liver of polar bears because they became sick (unknowingly by vitamin A) Western explorers not savvy to the vitamin A rich properties of polar bear liver as early as 1596 accounted a horrible illnessndashsluggish-ness blurred vision nausea headache and skin loss resulting in coma and even deathmdashall signs of acute hypervitaminosis A or vitamin A toxicity

Not until the twentieth century was vitamin A actually isolated In 1909 Hopkins and Steep extracted a lipidfat substance that mice and rats absolutely required for their growth Elmer McCollum performed a careful analysis of the growth-promoting factors in protein-free milk leading to the isolation of the first known fat-soluble vitamin This essential growth promoting compound was named ldquofat soluble Ardquo a terminology distinguishing it from other water-soluble vitamins such as the recently discovered anti-scurvey factormdashvitamin C In 1920 Jack Drummond suggested that the ldquovital sub-stancerdquo be given the name vitamin A a substance later associated with a pigmented yellow color In 1935 George Wald found vitamin A was a component of the retina When the rhodopsin pigment was exposed to light it yielded opsin and a vitamin A-containing compound (the chromophore) indicating that vitamin A was essential in retinal function While the nature of the chromophore and the reactions occurring dur-ing the visual cycle were characterized long ago there have been recent developments in the characterization of the enzymes and carrier proteins involved in this cycle with novel findings indicating that an alternative pathway for chromophore regeneration has evolved in cones (the photoreceptors of the retina that operate in bright daylight and which are responsible visual acuity and color discrimination) Two chapters in this book (Chapters 18 and 24) review these findings the latter chapter describing gene mutations leading to visual diseases and discussing therapeutic strategies

In the late 1970s researchers suggested that the physiological activity of vitamin A may be occurring through ligand binding to nuclear receptors In the 1980s the goal of many researchers became to elucidate how these signaling molecules prodded gene expression In 1986 the groups of Pierre Chambon and Ronald Evans independently

xiv Preface

cloned and characterized the first retinoic acid receptormdasha molecule providing an entry point for detailing vitamin A biology In 2004 the two scientists were awarded the Albert Lasker Basic Medical Research Award for their discoveries that retinoids and other pathways including steroids vitamin D thyroid hormone and many other lipid based drugs transmit signals through similar pathways These discoveries advanced the mechanisms of vitamin A and retinoid signaling Several authors at the forefront of research aiming to solve structurendashfunction relationships of retinoid receptors their genomic target loci and the post-translational and epigenetic regulatory mechanisms that fine-tune their activity according to the cellular context provide a comprehensive overview of these complex phenomena in Part II of this book (Chapters 5ndash8) These are followed by two chapters on the chemistry of retinoid-related synthetic molecules an important research field for drug design of selective receptor agnonistsantagonists with Chapter 10 emphasizing how research on retinoid-related molecules led to the identification of modulators of another nuclear receptor the small heterodi-mer partner ldquoorphanrdquo receptor

Worldwide problems of nutritional deficiencies appear insurmountable as the planetrsquos population tops seven billion Vitamin A deficiency and its associated dis-eases afflict impoverished populations and are endemic in sub-Saharan Africa and southern Asia These deficiencies preferentially target infants preschool children and pregnant women Vitamin A deficiency is the leading preventable cause of vision loss Over 200 million are estimated as lacking sufficient serum retinol levels Vitamin A deficiency diseases frequently include rod photoreceptor dysfunction and night blindness and in more severe cases blinding ulcerations and necrosis of the cornea The World Health Organization estimates that 14 million children have pre-ventable irreversible blindness with half dying within a year of losing sight Also because inflammation reduces retinol-binding protein levels compromised immune function increases death rate following bacterial-induced diarrhea malaria or HIV infection Lower food intake during disease results in a synergistic downward pro-gression of malnutrition and disease which can be lethal Providing a small concen-trated dose of retinol has proven effects increasing infant survival by over 30 Retinol treatments are extremely low cost with consequent saving of visionmdashwith sometimes only two days of treatment restoring night blindness One chapter of this book (Chapter 23) describes the consequences to world health of retinoid defi-ciencies while others provide updated reviews on the relationship between the reti-noid pathway and inflammatory processes (Chapter 20) and its functions in the immune system (Chapter 21)

Nutritional deficiencies are a common survival challenge and for the fat-soluble vitamin A our organism has developed exquisite mechanisms to enzymatically con-vert and maintain adequate stores of retinoids (mainly in the liver) allowing adults to survive under vitamin A deficiency for months or even years The first part of this book provides detailed accounts on the mechanisms of vitamin A and carotenoid absorption liver storage and tissue-targeted delivery through the blood circulation with one chapter (Chapter 4) discussing current knowledge on the ancestry of reti-noid receptors in multicellular organisms and the molecular evolution of the retinoic acid signaling pathway

In the 1970s Michael Sporn first used the term ldquochemopreventionrdquo for clinical trials using retinoids to prevent or delay the occurrence of certain forms of cancer

Preface xv

Unfortunately large-scale clinical trials providing retinol to smokers with premalig-nant oral lesions had disappointing results and in some cases had to be interrupted Potentially these high dose treatments decreased cellular retinoid signalingmdashperhaps by inducing CYP26 enzymatic activity leading to retinoic acid (RA) degradation Currently compounds inhibiting these enzymes are being tested for cancer treatment efficacy Clinical pharmacology employing the synthetic retinoid fenretinide indicates retinoid derivatives are plausible cancer treatment strategies Phase III clinical trial data suggested that fenretinide reduces breast cancer relapse inducing tumor cell death and necrosis The retinoid X receptor (RXR) agonist bexarotene is being tested for treatment of T-cell lymphoma and Kaposi sarcoma These data are reviewed in the last chapter of this book

How can we better understand intracellular functions related to cancer As detailed in Chapter 11 there are key examples of how the biological activity of RA critically relies on a balance of several lipid-binding proteins Reductions in one of these proteins cellular retinoic acid-binding protein 2 (CRABP2) and augmentations in fatty acid-binding protein 5 (FABP5) change RA signaling from normal phy-siological growth inhibition to pathological states of neoplastic breast cancer growth There are also antagonistic effects of RA and estrogenmdashanother hormone acting through nuclear receptorsmdashin breast cancer cells (Chapter 8) Selective gene targets are either induced or inhibited within cell-specific contexts indicating some of the selective parameters in which RA can be capable of arresting neoplastic growth

There are many additional reasons that populations in affluent well-nourished countries should be concerned about retinoid signaling The most oppressing health problem in the United States (and a growing plague in the entire world) is the alarm-ing increase in obesity This rise in obesity is even found in poorer countries due to food insecurity choices based on high calorie intake The increase in the overweight population and its associated diseases (cardiovascular diabetes and metabolic dis-eases) will have a predicted economic impact of more than $30 trillion over the next 20 years according to a 2011 report by the World Economic Forum and Harvard School of Public Health The collective impact of noncommunicable cardiovascular diabetic and other metabolic diseases linked to obesity impacts large segments of the world population Proteins selectively binding retinoids (such as retinol-binding pro-tein) are increased in obese patients Retinoic acid has a pharmacological side effect of inducing weight loss While this clinical side effect is clearly disadvantageous in retinoid treatments used to reduce cancer growth it has potential actions as an anti-obesity agent if other side effects can be reduced Structural studies on how retinoid receptors bind ligands and coregulators reveal allosteric changes altering their sig-naling properties and provide critical information needed to improve retinoid phar-macology to provide drugs with improved selectivity and reduced side effects (Chapters 7 and 9) Authors also address how retinoid fat-reducing metabolic actions may be controlled by cellular sites of storage namely in lipid droplets (Chapter 3) Pressing questions are if and should retinoids be a widely used treatment strategy to reduce obesity and consequent metabolic disease Since side effects of severe hyper-vitaminosis A or retinoid treatments include skin scaling lethargy and nausea long-term pilot trials appear to be required before the weight loss potential of reti-noids is exploited in large populations Treatment may require pharmacologically selective retinoids specific to retinoid metabolic targets Public health campaigns to

xvi Preface

increasing intake of fruits and vegetables (especially those rich in vitamin A) serve as a more immediate strategy to address increasing obesity

Human nutrition can have a dynamic effect on life processes and disease by influencing epigenetic regulation Gene regulatory systems have evolved for extremely precise and specific regulatory control The retinoid signal acts through epigenetic control of transcriptional coregulatory modulators that can respond to environmental clues Retinoid responsiveness relies on promoter occupancy and organization of the epigenetic landscape at the chromatin level Many genes that are rapidly activated by retinoids have transcriptional response elements with ldquobivalentrdquo promoter chromatin access (Chapter 12) Responding genes are in a poised state and are rapidly activated by increasing the activation to repressive transcriptional complex ratios This chromatin reorganization can be coordinately managed by phosphorylation of several actors of RA signaling integrating and managing cellular decision (Chapter 5) The disease-related examples of aberrant epigenetic landscape contributing to malignancy can be found with retinoic acid treatment of acute promy-elocytic leukemia (Chapter 25) Current evidence supports that compounds inhibit-ing histone deacetylase activity augment retinoic acid receptorndashretinoid X receptor (RARndashRXR) response acting synergistically on condensed chromatin RXRs can be specifically activated by a class of drugs termed rexinoids currently used in the clinical treatment of selective lymphomas These treatments are effective grim reapers bringing about tumor cell apoptosis

The promise of stem cell research has provided public hope in curative ther-apies including neuroregenerative treatment of spinal cord injury or Alzheimerrsquos disease repairing damaged heart tissue subsequent to myocardial infarction or replacing skin in severe burn victims Are these plausible strategies or are realistic expectations being exceeded Retinoids are regulators of a number of stem cell line-ages in a variety of organ progenitor sites (pancreas blood heart central nervous system and gonads) at various stages of development and postnatal life The under-lying concept that RA regulates stemprogenitor cell lineages during developmentmdashand may have later roles in regulating organ regeneration or repairmdashis the subject of several chapters in Part III of this book Various animal models of retinoid deficiency show defects in the developing nervous system heart and lung These defects per-turb organ function disrupting patterning hence inducing fetal or embryonic-stage lethality Interestingly the same genes expressed in adult stem cell populations are often acting in progenitor cell populations at embryonic and fetal stages Could the changes in gene expression observed in such cell populations in murine models of retinoid deficiency be considered as a paradigm for further studies providing clues on how retinoids might modulate stem cell dynamics at later (adult) stages These questions which are touched upon in several chapters of this book will undoubtedly be the subject of many future investigations

In summary the strong interest in vitamin A signaling through its active compo-nents the retinoids is rooted in the biological implications in so many physiological and disease processes Who would want to delve into the complexity of all of these subjects There are currently over 50 000 references when the word ldquoretinoidsrdquo is enteredmdashalthough its coselection with ldquoreviewsrdquo ldquocancerrdquo or ldquoobesityrdquo will reduce this number to thousands This book presents integrated knowledge from many current experts in the field We congratulate authors for their commitmentmdashin spite of their

Preface xvii

other overburdening academic responsibilities And for this opus we as editors wish to give a million ldquothanksrdquo to all of the contributing authors This book is the ldquoRetinoid Toolrdquo that can serve both the debutant and experienced researcher advancing their knowledge and understanding and leading to successful research discoveries

Pascal DolleacuteKaren Niederreither

PART I

Vitamin a metabolic and enzymatic Pathways

The Retinoids Biology Biochemistry and Disease First Edition Edited by Pascal Dolleacute and Karen Niederreither copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

1

Vitamin a metabolism storage and tissue deliVery mechanisms

William s blaner1 and yang li2

1 Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

2 Columbia College Columbia University New York NY USA

I IntroductIon

Vitamin A is an essential micronutrient that must be acquired from the diet either as preformed vitamin A or as provitamin A carotenoid By definition all-trans-retinol is vitamin A although retinol metabolites including retinyl esters and retinoic acid are often referred to collectively as vitamin A Throughout this chapter vitamin A is used solely to refer to all-trans-retinol The term retinoid which was first coined by Sporn in the mid-1970s refers to a family of chemicals both natural and synthetic that bear a structural resemblance to all-trans-retinol with or without the biologic activity of vitamin A (Sporn et al 1976) Thus the retinoid family of compounds comprises both naturally-occurring vitamin A metabolites and synthetic compounds that bear a structural resemblance to vitamin A

Within the body the two most abundant vitamin A species are retinol and retinyl esters Both retinol and retinyl esters are central to the metabolism storage and delivery of vitamin A to tissues The great majority of the vitamin A present in the body is stored as retinyl esters primarily in the liver but also in other tissues Dietary vitamin A is also packaged as retinyl esters in nascent chylomicrons and is thus taken into the body Because of this retinyl esters are a transport form of vitamin A that can

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

ix

Contributors

Darwin Babino Department of Pharmacology School of Medicine Case Western Reserve University Cleveland OH USA

William S Blaner Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

William Bourguet Centre de Biochimie Structurale Montpellier France Universiteacutes Montpellier 1 amp 2 Montpellier France

Josephine Bowles Institute for Molecular Bioscience The University of Queensland Brisbane QLD Australia

Wellington V Cardoso Columbia Center for Human Development Department of Medicine Columbia University Medical Center New York NY USA

Marilyn Carrier Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Felicia Chen Pulmonary Center Boston University School of Medicine Boston MA USA

Irwin Davidson Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Marcia I Dawson Cancer Center Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Ethan Dmitrovsky Department of Pharmacology and Toxicology Department of Medicine The Norris Cotton Cancer Center Geisel School of Medicine Dartmouth Hanover NH USA

Gregg Duester Development Aging and Regeneration Program Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Pierre Fenaux Universiteacute Paris Diderot Sorbonne Paris Citeacute Assistance PubliquendashHocircpitaux de Paris Service drsquoHeacutematologie Hocircpital St Louis Paris France

Sarah J Freemantle Department of Pharmacology and Toxicology Geisel School of Medicine Dartmouth Hanover NH USA

Norbert B Ghyselinck Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Hinrich Gronemeyer Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

x Contributors

Lorraine J Gudas Pharmacology Department Weill Cornell Medical College Cornell University New York NY USA

Earl H Harrison Department of Human Nutrition and Ohio State Biochemistry Program Ohio State University Columbus OH USA

Makoto Iwata Kagawa School of Pharmaceutical Sciences Tokushima Bunri University amp JST CREST Kagawa Japan

Peter Koopman Institute for Molecular Bioscience The University of Queensland Brisbane QLD Australia

Wojciech Krezel Development and Stem Cells Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Charles R Krois Graduate Program in Metabolic Biology Nutritional Sciences and Toxicology University of California-Berkeley CA USA

Vincent Laudet Molecular Zoology Team Institut de Geacutenomique Fonctionnelle de Lyon (IGFL) Ecole Normale Supeacuterieure de Lyon Lyon France

Angel R de Lera Departamento de Quiacutemica Orgaacutenica Facultad de Quiacutemica Universidad de Vigo Vigo Spain

Yang Li Columbia College Columbia University New York NY USA

Johannes von Lintig Department of Pharmacology School of Medicine Case Western Reserve University Cleveland OH USA

Malcolm Maden Department of Biology and University of Florida Genetics Institute University of Florida Gainesville FL USA

Manuel Mark Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France Hocircpitaux Universitaires de Strasbourg Strasbourg France

Peter McCaffery Institute of Medical Sciences School of Medical Sciences University of Aberdeen Aberdeen UK

Marco-Antonio Mendoza-Parra Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

J Rodrigo Mora Massachusetts General Hospital and Harvard Medical School Boston MA USA

Dino Moras Integrated Structural Biology Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Joseph L Napoli Graduate Program in Metabolic Biology Nutritional Sciences and Toxicology University of California-Berkeley CA USA

Karen Niederreither Development and Stem Cells Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Noa Noy Department of Cellular and Molecular Medicine Lerner Research Institute Cleveland Clinic Foundation Cleveland OH USA

Contributors xi

Krzysztof Palczewski Department of Pharmacology Case Western Reserve University Cleveland OH USA

Ceacutecile Rochette-Egly Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

A Catharine Ross Department of Nutritional Sciences Pennsylvania State University University Park PA USA

Michael Schubert Laboratoire de Biologie du Deacuteveloppement de Villefranche-sur-Mer Sorbonne Universiteacutes UPMC Universiteacute Paris 06 Observatoire Oceacuteanologique de Villefranche-sur-Mer Villefranche-sur-Mer France

Carlo dela Sentildearsquos Department of Human Nutrition and Ohio State Biochemistry Program Ohio State University Columbus OH USA

Michael J Spinella Department of Pharmacology and Toxicology The Norris Cotton Cancer Center Geisel School of Medicine Dartmouth College Hanover NH USA

Hugues de Theacute Universiteacute Paris Diderot Sorbonne Paris Citeacute Institut Universitaire drsquoHeacutematologie Assistance PubliquendashHocircpitaux de Paris Service de Biochimie Hocircpital St Louis Paris France

Yaroslav Tsybovsky Department of Pharmacology Case Western Reserve University Cleveland OH USA

Sylvia Urban Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Li-Na Wei Department of Pharmacology University of Minnesota Medical School Minneapolis MN USA

Keith P West Jr Center and Program in Human Nutrition Department of International Health Johns Hopkins Bloomberg School of Public Health Baltimore MD USA

Zebin Xia Cancer Center Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Tao Ye Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Steacutephane Zaffran Aix-Marseille Universiteacute Uniteacute de Geacuteneacutetique Meacutedicale et Geacutenomique Fonctionnelle (GMGF) Faculteacute de Meacutedecine Marseille France

Elisabeth Zieger Laboratoire de Biologie du Deacuteveloppement de Villefranche-sur-Mer Sorbonne Universiteacutes UPMC Universiteacute Paris 06 Observatoire Oceacuteanologique de Villefranche-sur-Mer Villefranche-sur-Mer France

Reza Zolfaghari Department of Nutritional Sciences Pennsylvania State University University Park PA USA

xiii

Preface

VITAMIN A AND RETINOIDS AN AMAZING HISTORY AND HOPES FOR DISEASE PREVENTION

Historically the vitamin A precursor carotenoids had been long suspected of having medicinal properties For thousands of years humans and animals suffered from vitamin A deficiency typified by night blindness and xerophthalmiamdasha failure of tear production which if untreated would result in blindness While the underlying causes of such maledictions were a mystery in 1500 BC the ancient Egyptians rec-ommended eating animal or fish liver for their curative powers The native people of the Arctic also long knew to avoid eating the liver of polar bears because they became sick (unknowingly by vitamin A) Western explorers not savvy to the vitamin A rich properties of polar bear liver as early as 1596 accounted a horrible illnessndashsluggish-ness blurred vision nausea headache and skin loss resulting in coma and even deathmdashall signs of acute hypervitaminosis A or vitamin A toxicity

Not until the twentieth century was vitamin A actually isolated In 1909 Hopkins and Steep extracted a lipidfat substance that mice and rats absolutely required for their growth Elmer McCollum performed a careful analysis of the growth-promoting factors in protein-free milk leading to the isolation of the first known fat-soluble vitamin This essential growth promoting compound was named ldquofat soluble Ardquo a terminology distinguishing it from other water-soluble vitamins such as the recently discovered anti-scurvey factormdashvitamin C In 1920 Jack Drummond suggested that the ldquovital sub-stancerdquo be given the name vitamin A a substance later associated with a pigmented yellow color In 1935 George Wald found vitamin A was a component of the retina When the rhodopsin pigment was exposed to light it yielded opsin and a vitamin A-containing compound (the chromophore) indicating that vitamin A was essential in retinal function While the nature of the chromophore and the reactions occurring dur-ing the visual cycle were characterized long ago there have been recent developments in the characterization of the enzymes and carrier proteins involved in this cycle with novel findings indicating that an alternative pathway for chromophore regeneration has evolved in cones (the photoreceptors of the retina that operate in bright daylight and which are responsible visual acuity and color discrimination) Two chapters in this book (Chapters 18 and 24) review these findings the latter chapter describing gene mutations leading to visual diseases and discussing therapeutic strategies

In the late 1970s researchers suggested that the physiological activity of vitamin A may be occurring through ligand binding to nuclear receptors In the 1980s the goal of many researchers became to elucidate how these signaling molecules prodded gene expression In 1986 the groups of Pierre Chambon and Ronald Evans independently

xiv Preface

cloned and characterized the first retinoic acid receptormdasha molecule providing an entry point for detailing vitamin A biology In 2004 the two scientists were awarded the Albert Lasker Basic Medical Research Award for their discoveries that retinoids and other pathways including steroids vitamin D thyroid hormone and many other lipid based drugs transmit signals through similar pathways These discoveries advanced the mechanisms of vitamin A and retinoid signaling Several authors at the forefront of research aiming to solve structurendashfunction relationships of retinoid receptors their genomic target loci and the post-translational and epigenetic regulatory mechanisms that fine-tune their activity according to the cellular context provide a comprehensive overview of these complex phenomena in Part II of this book (Chapters 5ndash8) These are followed by two chapters on the chemistry of retinoid-related synthetic molecules an important research field for drug design of selective receptor agnonistsantagonists with Chapter 10 emphasizing how research on retinoid-related molecules led to the identification of modulators of another nuclear receptor the small heterodi-mer partner ldquoorphanrdquo receptor

Worldwide problems of nutritional deficiencies appear insurmountable as the planetrsquos population tops seven billion Vitamin A deficiency and its associated dis-eases afflict impoverished populations and are endemic in sub-Saharan Africa and southern Asia These deficiencies preferentially target infants preschool children and pregnant women Vitamin A deficiency is the leading preventable cause of vision loss Over 200 million are estimated as lacking sufficient serum retinol levels Vitamin A deficiency diseases frequently include rod photoreceptor dysfunction and night blindness and in more severe cases blinding ulcerations and necrosis of the cornea The World Health Organization estimates that 14 million children have pre-ventable irreversible blindness with half dying within a year of losing sight Also because inflammation reduces retinol-binding protein levels compromised immune function increases death rate following bacterial-induced diarrhea malaria or HIV infection Lower food intake during disease results in a synergistic downward pro-gression of malnutrition and disease which can be lethal Providing a small concen-trated dose of retinol has proven effects increasing infant survival by over 30 Retinol treatments are extremely low cost with consequent saving of visionmdashwith sometimes only two days of treatment restoring night blindness One chapter of this book (Chapter 23) describes the consequences to world health of retinoid defi-ciencies while others provide updated reviews on the relationship between the reti-noid pathway and inflammatory processes (Chapter 20) and its functions in the immune system (Chapter 21)

Nutritional deficiencies are a common survival challenge and for the fat-soluble vitamin A our organism has developed exquisite mechanisms to enzymatically con-vert and maintain adequate stores of retinoids (mainly in the liver) allowing adults to survive under vitamin A deficiency for months or even years The first part of this book provides detailed accounts on the mechanisms of vitamin A and carotenoid absorption liver storage and tissue-targeted delivery through the blood circulation with one chapter (Chapter 4) discussing current knowledge on the ancestry of reti-noid receptors in multicellular organisms and the molecular evolution of the retinoic acid signaling pathway

In the 1970s Michael Sporn first used the term ldquochemopreventionrdquo for clinical trials using retinoids to prevent or delay the occurrence of certain forms of cancer

Preface xv

Unfortunately large-scale clinical trials providing retinol to smokers with premalig-nant oral lesions had disappointing results and in some cases had to be interrupted Potentially these high dose treatments decreased cellular retinoid signalingmdashperhaps by inducing CYP26 enzymatic activity leading to retinoic acid (RA) degradation Currently compounds inhibiting these enzymes are being tested for cancer treatment efficacy Clinical pharmacology employing the synthetic retinoid fenretinide indicates retinoid derivatives are plausible cancer treatment strategies Phase III clinical trial data suggested that fenretinide reduces breast cancer relapse inducing tumor cell death and necrosis The retinoid X receptor (RXR) agonist bexarotene is being tested for treatment of T-cell lymphoma and Kaposi sarcoma These data are reviewed in the last chapter of this book

How can we better understand intracellular functions related to cancer As detailed in Chapter 11 there are key examples of how the biological activity of RA critically relies on a balance of several lipid-binding proteins Reductions in one of these proteins cellular retinoic acid-binding protein 2 (CRABP2) and augmentations in fatty acid-binding protein 5 (FABP5) change RA signaling from normal phy-siological growth inhibition to pathological states of neoplastic breast cancer growth There are also antagonistic effects of RA and estrogenmdashanother hormone acting through nuclear receptorsmdashin breast cancer cells (Chapter 8) Selective gene targets are either induced or inhibited within cell-specific contexts indicating some of the selective parameters in which RA can be capable of arresting neoplastic growth

There are many additional reasons that populations in affluent well-nourished countries should be concerned about retinoid signaling The most oppressing health problem in the United States (and a growing plague in the entire world) is the alarm-ing increase in obesity This rise in obesity is even found in poorer countries due to food insecurity choices based on high calorie intake The increase in the overweight population and its associated diseases (cardiovascular diabetes and metabolic dis-eases) will have a predicted economic impact of more than $30 trillion over the next 20 years according to a 2011 report by the World Economic Forum and Harvard School of Public Health The collective impact of noncommunicable cardiovascular diabetic and other metabolic diseases linked to obesity impacts large segments of the world population Proteins selectively binding retinoids (such as retinol-binding pro-tein) are increased in obese patients Retinoic acid has a pharmacological side effect of inducing weight loss While this clinical side effect is clearly disadvantageous in retinoid treatments used to reduce cancer growth it has potential actions as an anti-obesity agent if other side effects can be reduced Structural studies on how retinoid receptors bind ligands and coregulators reveal allosteric changes altering their sig-naling properties and provide critical information needed to improve retinoid phar-macology to provide drugs with improved selectivity and reduced side effects (Chapters 7 and 9) Authors also address how retinoid fat-reducing metabolic actions may be controlled by cellular sites of storage namely in lipid droplets (Chapter 3) Pressing questions are if and should retinoids be a widely used treatment strategy to reduce obesity and consequent metabolic disease Since side effects of severe hyper-vitaminosis A or retinoid treatments include skin scaling lethargy and nausea long-term pilot trials appear to be required before the weight loss potential of reti-noids is exploited in large populations Treatment may require pharmacologically selective retinoids specific to retinoid metabolic targets Public health campaigns to

xvi Preface

increasing intake of fruits and vegetables (especially those rich in vitamin A) serve as a more immediate strategy to address increasing obesity

Human nutrition can have a dynamic effect on life processes and disease by influencing epigenetic regulation Gene regulatory systems have evolved for extremely precise and specific regulatory control The retinoid signal acts through epigenetic control of transcriptional coregulatory modulators that can respond to environmental clues Retinoid responsiveness relies on promoter occupancy and organization of the epigenetic landscape at the chromatin level Many genes that are rapidly activated by retinoids have transcriptional response elements with ldquobivalentrdquo promoter chromatin access (Chapter 12) Responding genes are in a poised state and are rapidly activated by increasing the activation to repressive transcriptional complex ratios This chromatin reorganization can be coordinately managed by phosphorylation of several actors of RA signaling integrating and managing cellular decision (Chapter 5) The disease-related examples of aberrant epigenetic landscape contributing to malignancy can be found with retinoic acid treatment of acute promy-elocytic leukemia (Chapter 25) Current evidence supports that compounds inhibit-ing histone deacetylase activity augment retinoic acid receptorndashretinoid X receptor (RARndashRXR) response acting synergistically on condensed chromatin RXRs can be specifically activated by a class of drugs termed rexinoids currently used in the clinical treatment of selective lymphomas These treatments are effective grim reapers bringing about tumor cell apoptosis

The promise of stem cell research has provided public hope in curative ther-apies including neuroregenerative treatment of spinal cord injury or Alzheimerrsquos disease repairing damaged heart tissue subsequent to myocardial infarction or replacing skin in severe burn victims Are these plausible strategies or are realistic expectations being exceeded Retinoids are regulators of a number of stem cell line-ages in a variety of organ progenitor sites (pancreas blood heart central nervous system and gonads) at various stages of development and postnatal life The under-lying concept that RA regulates stemprogenitor cell lineages during developmentmdashand may have later roles in regulating organ regeneration or repairmdashis the subject of several chapters in Part III of this book Various animal models of retinoid deficiency show defects in the developing nervous system heart and lung These defects per-turb organ function disrupting patterning hence inducing fetal or embryonic-stage lethality Interestingly the same genes expressed in adult stem cell populations are often acting in progenitor cell populations at embryonic and fetal stages Could the changes in gene expression observed in such cell populations in murine models of retinoid deficiency be considered as a paradigm for further studies providing clues on how retinoids might modulate stem cell dynamics at later (adult) stages These questions which are touched upon in several chapters of this book will undoubtedly be the subject of many future investigations

In summary the strong interest in vitamin A signaling through its active compo-nents the retinoids is rooted in the biological implications in so many physiological and disease processes Who would want to delve into the complexity of all of these subjects There are currently over 50 000 references when the word ldquoretinoidsrdquo is enteredmdashalthough its coselection with ldquoreviewsrdquo ldquocancerrdquo or ldquoobesityrdquo will reduce this number to thousands This book presents integrated knowledge from many current experts in the field We congratulate authors for their commitmentmdashin spite of their

Preface xvii

other overburdening academic responsibilities And for this opus we as editors wish to give a million ldquothanksrdquo to all of the contributing authors This book is the ldquoRetinoid Toolrdquo that can serve both the debutant and experienced researcher advancing their knowledge and understanding and leading to successful research discoveries

Pascal DolleacuteKaren Niederreither

PART I

Vitamin a metabolic and enzymatic Pathways

The Retinoids Biology Biochemistry and Disease First Edition Edited by Pascal Dolleacute and Karen Niederreither copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

1

Vitamin a metabolism storage and tissue deliVery mechanisms

William s blaner1 and yang li2

1 Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

2 Columbia College Columbia University New York NY USA

I IntroductIon

Vitamin A is an essential micronutrient that must be acquired from the diet either as preformed vitamin A or as provitamin A carotenoid By definition all-trans-retinol is vitamin A although retinol metabolites including retinyl esters and retinoic acid are often referred to collectively as vitamin A Throughout this chapter vitamin A is used solely to refer to all-trans-retinol The term retinoid which was first coined by Sporn in the mid-1970s refers to a family of chemicals both natural and synthetic that bear a structural resemblance to all-trans-retinol with or without the biologic activity of vitamin A (Sporn et al 1976) Thus the retinoid family of compounds comprises both naturally-occurring vitamin A metabolites and synthetic compounds that bear a structural resemblance to vitamin A

Within the body the two most abundant vitamin A species are retinol and retinyl esters Both retinol and retinyl esters are central to the metabolism storage and delivery of vitamin A to tissues The great majority of the vitamin A present in the body is stored as retinyl esters primarily in the liver but also in other tissues Dietary vitamin A is also packaged as retinyl esters in nascent chylomicrons and is thus taken into the body Because of this retinyl esters are a transport form of vitamin A that can

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

x Contributors

Lorraine J Gudas Pharmacology Department Weill Cornell Medical College Cornell University New York NY USA

Earl H Harrison Department of Human Nutrition and Ohio State Biochemistry Program Ohio State University Columbus OH USA

Makoto Iwata Kagawa School of Pharmaceutical Sciences Tokushima Bunri University amp JST CREST Kagawa Japan

Peter Koopman Institute for Molecular Bioscience The University of Queensland Brisbane QLD Australia

Wojciech Krezel Development and Stem Cells Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Charles R Krois Graduate Program in Metabolic Biology Nutritional Sciences and Toxicology University of California-Berkeley CA USA

Vincent Laudet Molecular Zoology Team Institut de Geacutenomique Fonctionnelle de Lyon (IGFL) Ecole Normale Supeacuterieure de Lyon Lyon France

Angel R de Lera Departamento de Quiacutemica Orgaacutenica Facultad de Quiacutemica Universidad de Vigo Vigo Spain

Yang Li Columbia College Columbia University New York NY USA

Johannes von Lintig Department of Pharmacology School of Medicine Case Western Reserve University Cleveland OH USA

Malcolm Maden Department of Biology and University of Florida Genetics Institute University of Florida Gainesville FL USA

Manuel Mark Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France Hocircpitaux Universitaires de Strasbourg Strasbourg France

Peter McCaffery Institute of Medical Sciences School of Medical Sciences University of Aberdeen Aberdeen UK

Marco-Antonio Mendoza-Parra Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

J Rodrigo Mora Massachusetts General Hospital and Harvard Medical School Boston MA USA

Dino Moras Integrated Structural Biology Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Joseph L Napoli Graduate Program in Metabolic Biology Nutritional Sciences and Toxicology University of California-Berkeley CA USA

Karen Niederreither Development and Stem Cells Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Noa Noy Department of Cellular and Molecular Medicine Lerner Research Institute Cleveland Clinic Foundation Cleveland OH USA

Contributors xi

Krzysztof Palczewski Department of Pharmacology Case Western Reserve University Cleveland OH USA

Ceacutecile Rochette-Egly Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

A Catharine Ross Department of Nutritional Sciences Pennsylvania State University University Park PA USA

Michael Schubert Laboratoire de Biologie du Deacuteveloppement de Villefranche-sur-Mer Sorbonne Universiteacutes UPMC Universiteacute Paris 06 Observatoire Oceacuteanologique de Villefranche-sur-Mer Villefranche-sur-Mer France

Carlo dela Sentildearsquos Department of Human Nutrition and Ohio State Biochemistry Program Ohio State University Columbus OH USA

Michael J Spinella Department of Pharmacology and Toxicology The Norris Cotton Cancer Center Geisel School of Medicine Dartmouth College Hanover NH USA

Hugues de Theacute Universiteacute Paris Diderot Sorbonne Paris Citeacute Institut Universitaire drsquoHeacutematologie Assistance PubliquendashHocircpitaux de Paris Service de Biochimie Hocircpital St Louis Paris France

Yaroslav Tsybovsky Department of Pharmacology Case Western Reserve University Cleveland OH USA

Sylvia Urban Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Li-Na Wei Department of Pharmacology University of Minnesota Medical School Minneapolis MN USA

Keith P West Jr Center and Program in Human Nutrition Department of International Health Johns Hopkins Bloomberg School of Public Health Baltimore MD USA

Zebin Xia Cancer Center Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Tao Ye Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Steacutephane Zaffran Aix-Marseille Universiteacute Uniteacute de Geacuteneacutetique Meacutedicale et Geacutenomique Fonctionnelle (GMGF) Faculteacute de Meacutedecine Marseille France

Elisabeth Zieger Laboratoire de Biologie du Deacuteveloppement de Villefranche-sur-Mer Sorbonne Universiteacutes UPMC Universiteacute Paris 06 Observatoire Oceacuteanologique de Villefranche-sur-Mer Villefranche-sur-Mer France

Reza Zolfaghari Department of Nutritional Sciences Pennsylvania State University University Park PA USA

xiii

Preface

VITAMIN A AND RETINOIDS AN AMAZING HISTORY AND HOPES FOR DISEASE PREVENTION

Historically the vitamin A precursor carotenoids had been long suspected of having medicinal properties For thousands of years humans and animals suffered from vitamin A deficiency typified by night blindness and xerophthalmiamdasha failure of tear production which if untreated would result in blindness While the underlying causes of such maledictions were a mystery in 1500 BC the ancient Egyptians rec-ommended eating animal or fish liver for their curative powers The native people of the Arctic also long knew to avoid eating the liver of polar bears because they became sick (unknowingly by vitamin A) Western explorers not savvy to the vitamin A rich properties of polar bear liver as early as 1596 accounted a horrible illnessndashsluggish-ness blurred vision nausea headache and skin loss resulting in coma and even deathmdashall signs of acute hypervitaminosis A or vitamin A toxicity

Not until the twentieth century was vitamin A actually isolated In 1909 Hopkins and Steep extracted a lipidfat substance that mice and rats absolutely required for their growth Elmer McCollum performed a careful analysis of the growth-promoting factors in protein-free milk leading to the isolation of the first known fat-soluble vitamin This essential growth promoting compound was named ldquofat soluble Ardquo a terminology distinguishing it from other water-soluble vitamins such as the recently discovered anti-scurvey factormdashvitamin C In 1920 Jack Drummond suggested that the ldquovital sub-stancerdquo be given the name vitamin A a substance later associated with a pigmented yellow color In 1935 George Wald found vitamin A was a component of the retina When the rhodopsin pigment was exposed to light it yielded opsin and a vitamin A-containing compound (the chromophore) indicating that vitamin A was essential in retinal function While the nature of the chromophore and the reactions occurring dur-ing the visual cycle were characterized long ago there have been recent developments in the characterization of the enzymes and carrier proteins involved in this cycle with novel findings indicating that an alternative pathway for chromophore regeneration has evolved in cones (the photoreceptors of the retina that operate in bright daylight and which are responsible visual acuity and color discrimination) Two chapters in this book (Chapters 18 and 24) review these findings the latter chapter describing gene mutations leading to visual diseases and discussing therapeutic strategies

In the late 1970s researchers suggested that the physiological activity of vitamin A may be occurring through ligand binding to nuclear receptors In the 1980s the goal of many researchers became to elucidate how these signaling molecules prodded gene expression In 1986 the groups of Pierre Chambon and Ronald Evans independently

xiv Preface

cloned and characterized the first retinoic acid receptormdasha molecule providing an entry point for detailing vitamin A biology In 2004 the two scientists were awarded the Albert Lasker Basic Medical Research Award for their discoveries that retinoids and other pathways including steroids vitamin D thyroid hormone and many other lipid based drugs transmit signals through similar pathways These discoveries advanced the mechanisms of vitamin A and retinoid signaling Several authors at the forefront of research aiming to solve structurendashfunction relationships of retinoid receptors their genomic target loci and the post-translational and epigenetic regulatory mechanisms that fine-tune their activity according to the cellular context provide a comprehensive overview of these complex phenomena in Part II of this book (Chapters 5ndash8) These are followed by two chapters on the chemistry of retinoid-related synthetic molecules an important research field for drug design of selective receptor agnonistsantagonists with Chapter 10 emphasizing how research on retinoid-related molecules led to the identification of modulators of another nuclear receptor the small heterodi-mer partner ldquoorphanrdquo receptor

Worldwide problems of nutritional deficiencies appear insurmountable as the planetrsquos population tops seven billion Vitamin A deficiency and its associated dis-eases afflict impoverished populations and are endemic in sub-Saharan Africa and southern Asia These deficiencies preferentially target infants preschool children and pregnant women Vitamin A deficiency is the leading preventable cause of vision loss Over 200 million are estimated as lacking sufficient serum retinol levels Vitamin A deficiency diseases frequently include rod photoreceptor dysfunction and night blindness and in more severe cases blinding ulcerations and necrosis of the cornea The World Health Organization estimates that 14 million children have pre-ventable irreversible blindness with half dying within a year of losing sight Also because inflammation reduces retinol-binding protein levels compromised immune function increases death rate following bacterial-induced diarrhea malaria or HIV infection Lower food intake during disease results in a synergistic downward pro-gression of malnutrition and disease which can be lethal Providing a small concen-trated dose of retinol has proven effects increasing infant survival by over 30 Retinol treatments are extremely low cost with consequent saving of visionmdashwith sometimes only two days of treatment restoring night blindness One chapter of this book (Chapter 23) describes the consequences to world health of retinoid defi-ciencies while others provide updated reviews on the relationship between the reti-noid pathway and inflammatory processes (Chapter 20) and its functions in the immune system (Chapter 21)

Nutritional deficiencies are a common survival challenge and for the fat-soluble vitamin A our organism has developed exquisite mechanisms to enzymatically con-vert and maintain adequate stores of retinoids (mainly in the liver) allowing adults to survive under vitamin A deficiency for months or even years The first part of this book provides detailed accounts on the mechanisms of vitamin A and carotenoid absorption liver storage and tissue-targeted delivery through the blood circulation with one chapter (Chapter 4) discussing current knowledge on the ancestry of reti-noid receptors in multicellular organisms and the molecular evolution of the retinoic acid signaling pathway

In the 1970s Michael Sporn first used the term ldquochemopreventionrdquo for clinical trials using retinoids to prevent or delay the occurrence of certain forms of cancer

Preface xv

Unfortunately large-scale clinical trials providing retinol to smokers with premalig-nant oral lesions had disappointing results and in some cases had to be interrupted Potentially these high dose treatments decreased cellular retinoid signalingmdashperhaps by inducing CYP26 enzymatic activity leading to retinoic acid (RA) degradation Currently compounds inhibiting these enzymes are being tested for cancer treatment efficacy Clinical pharmacology employing the synthetic retinoid fenretinide indicates retinoid derivatives are plausible cancer treatment strategies Phase III clinical trial data suggested that fenretinide reduces breast cancer relapse inducing tumor cell death and necrosis The retinoid X receptor (RXR) agonist bexarotene is being tested for treatment of T-cell lymphoma and Kaposi sarcoma These data are reviewed in the last chapter of this book

How can we better understand intracellular functions related to cancer As detailed in Chapter 11 there are key examples of how the biological activity of RA critically relies on a balance of several lipid-binding proteins Reductions in one of these proteins cellular retinoic acid-binding protein 2 (CRABP2) and augmentations in fatty acid-binding protein 5 (FABP5) change RA signaling from normal phy-siological growth inhibition to pathological states of neoplastic breast cancer growth There are also antagonistic effects of RA and estrogenmdashanother hormone acting through nuclear receptorsmdashin breast cancer cells (Chapter 8) Selective gene targets are either induced or inhibited within cell-specific contexts indicating some of the selective parameters in which RA can be capable of arresting neoplastic growth

There are many additional reasons that populations in affluent well-nourished countries should be concerned about retinoid signaling The most oppressing health problem in the United States (and a growing plague in the entire world) is the alarm-ing increase in obesity This rise in obesity is even found in poorer countries due to food insecurity choices based on high calorie intake The increase in the overweight population and its associated diseases (cardiovascular diabetes and metabolic dis-eases) will have a predicted economic impact of more than $30 trillion over the next 20 years according to a 2011 report by the World Economic Forum and Harvard School of Public Health The collective impact of noncommunicable cardiovascular diabetic and other metabolic diseases linked to obesity impacts large segments of the world population Proteins selectively binding retinoids (such as retinol-binding pro-tein) are increased in obese patients Retinoic acid has a pharmacological side effect of inducing weight loss While this clinical side effect is clearly disadvantageous in retinoid treatments used to reduce cancer growth it has potential actions as an anti-obesity agent if other side effects can be reduced Structural studies on how retinoid receptors bind ligands and coregulators reveal allosteric changes altering their sig-naling properties and provide critical information needed to improve retinoid phar-macology to provide drugs with improved selectivity and reduced side effects (Chapters 7 and 9) Authors also address how retinoid fat-reducing metabolic actions may be controlled by cellular sites of storage namely in lipid droplets (Chapter 3) Pressing questions are if and should retinoids be a widely used treatment strategy to reduce obesity and consequent metabolic disease Since side effects of severe hyper-vitaminosis A or retinoid treatments include skin scaling lethargy and nausea long-term pilot trials appear to be required before the weight loss potential of reti-noids is exploited in large populations Treatment may require pharmacologically selective retinoids specific to retinoid metabolic targets Public health campaigns to

xvi Preface

increasing intake of fruits and vegetables (especially those rich in vitamin A) serve as a more immediate strategy to address increasing obesity

Human nutrition can have a dynamic effect on life processes and disease by influencing epigenetic regulation Gene regulatory systems have evolved for extremely precise and specific regulatory control The retinoid signal acts through epigenetic control of transcriptional coregulatory modulators that can respond to environmental clues Retinoid responsiveness relies on promoter occupancy and organization of the epigenetic landscape at the chromatin level Many genes that are rapidly activated by retinoids have transcriptional response elements with ldquobivalentrdquo promoter chromatin access (Chapter 12) Responding genes are in a poised state and are rapidly activated by increasing the activation to repressive transcriptional complex ratios This chromatin reorganization can be coordinately managed by phosphorylation of several actors of RA signaling integrating and managing cellular decision (Chapter 5) The disease-related examples of aberrant epigenetic landscape contributing to malignancy can be found with retinoic acid treatment of acute promy-elocytic leukemia (Chapter 25) Current evidence supports that compounds inhibit-ing histone deacetylase activity augment retinoic acid receptorndashretinoid X receptor (RARndashRXR) response acting synergistically on condensed chromatin RXRs can be specifically activated by a class of drugs termed rexinoids currently used in the clinical treatment of selective lymphomas These treatments are effective grim reapers bringing about tumor cell apoptosis

The promise of stem cell research has provided public hope in curative ther-apies including neuroregenerative treatment of spinal cord injury or Alzheimerrsquos disease repairing damaged heart tissue subsequent to myocardial infarction or replacing skin in severe burn victims Are these plausible strategies or are realistic expectations being exceeded Retinoids are regulators of a number of stem cell line-ages in a variety of organ progenitor sites (pancreas blood heart central nervous system and gonads) at various stages of development and postnatal life The under-lying concept that RA regulates stemprogenitor cell lineages during developmentmdashand may have later roles in regulating organ regeneration or repairmdashis the subject of several chapters in Part III of this book Various animal models of retinoid deficiency show defects in the developing nervous system heart and lung These defects per-turb organ function disrupting patterning hence inducing fetal or embryonic-stage lethality Interestingly the same genes expressed in adult stem cell populations are often acting in progenitor cell populations at embryonic and fetal stages Could the changes in gene expression observed in such cell populations in murine models of retinoid deficiency be considered as a paradigm for further studies providing clues on how retinoids might modulate stem cell dynamics at later (adult) stages These questions which are touched upon in several chapters of this book will undoubtedly be the subject of many future investigations

In summary the strong interest in vitamin A signaling through its active compo-nents the retinoids is rooted in the biological implications in so many physiological and disease processes Who would want to delve into the complexity of all of these subjects There are currently over 50 000 references when the word ldquoretinoidsrdquo is enteredmdashalthough its coselection with ldquoreviewsrdquo ldquocancerrdquo or ldquoobesityrdquo will reduce this number to thousands This book presents integrated knowledge from many current experts in the field We congratulate authors for their commitmentmdashin spite of their

Preface xvii

other overburdening academic responsibilities And for this opus we as editors wish to give a million ldquothanksrdquo to all of the contributing authors This book is the ldquoRetinoid Toolrdquo that can serve both the debutant and experienced researcher advancing their knowledge and understanding and leading to successful research discoveries

Pascal DolleacuteKaren Niederreither

PART I

Vitamin a metabolic and enzymatic Pathways

The Retinoids Biology Biochemistry and Disease First Edition Edited by Pascal Dolleacute and Karen Niederreither copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

1

Vitamin a metabolism storage and tissue deliVery mechanisms

William s blaner1 and yang li2

1 Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

2 Columbia College Columbia University New York NY USA

I IntroductIon

Vitamin A is an essential micronutrient that must be acquired from the diet either as preformed vitamin A or as provitamin A carotenoid By definition all-trans-retinol is vitamin A although retinol metabolites including retinyl esters and retinoic acid are often referred to collectively as vitamin A Throughout this chapter vitamin A is used solely to refer to all-trans-retinol The term retinoid which was first coined by Sporn in the mid-1970s refers to a family of chemicals both natural and synthetic that bear a structural resemblance to all-trans-retinol with or without the biologic activity of vitamin A (Sporn et al 1976) Thus the retinoid family of compounds comprises both naturally-occurring vitamin A metabolites and synthetic compounds that bear a structural resemblance to vitamin A

Within the body the two most abundant vitamin A species are retinol and retinyl esters Both retinol and retinyl esters are central to the metabolism storage and delivery of vitamin A to tissues The great majority of the vitamin A present in the body is stored as retinyl esters primarily in the liver but also in other tissues Dietary vitamin A is also packaged as retinyl esters in nascent chylomicrons and is thus taken into the body Because of this retinyl esters are a transport form of vitamin A that can

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

Contributors xi

Krzysztof Palczewski Department of Pharmacology Case Western Reserve University Cleveland OH USA

Ceacutecile Rochette-Egly Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

A Catharine Ross Department of Nutritional Sciences Pennsylvania State University University Park PA USA

Michael Schubert Laboratoire de Biologie du Deacuteveloppement de Villefranche-sur-Mer Sorbonne Universiteacutes UPMC Universiteacute Paris 06 Observatoire Oceacuteanologique de Villefranche-sur-Mer Villefranche-sur-Mer France

Carlo dela Sentildearsquos Department of Human Nutrition and Ohio State Biochemistry Program Ohio State University Columbus OH USA

Michael J Spinella Department of Pharmacology and Toxicology The Norris Cotton Cancer Center Geisel School of Medicine Dartmouth College Hanover NH USA

Hugues de Theacute Universiteacute Paris Diderot Sorbonne Paris Citeacute Institut Universitaire drsquoHeacutematologie Assistance PubliquendashHocircpitaux de Paris Service de Biochimie Hocircpital St Louis Paris France

Yaroslav Tsybovsky Department of Pharmacology Case Western Reserve University Cleveland OH USA

Sylvia Urban Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Li-Na Wei Department of Pharmacology University of Minnesota Medical School Minneapolis MN USA

Keith P West Jr Center and Program in Human Nutrition Department of International Health Johns Hopkins Bloomberg School of Public Health Baltimore MD USA

Zebin Xia Cancer Center Sanford-Burnham Medical Research Institute (SBMRI) La Jolla CA USA

Tao Ye Functional Genomics and Cancer Department Institut de Geacuteneacutetique et de Biologie Moleacuteculaire et Cellulaire (IGBMC) Universiteacute de Strasbourg Illkirch France

Steacutephane Zaffran Aix-Marseille Universiteacute Uniteacute de Geacuteneacutetique Meacutedicale et Geacutenomique Fonctionnelle (GMGF) Faculteacute de Meacutedecine Marseille France

Elisabeth Zieger Laboratoire de Biologie du Deacuteveloppement de Villefranche-sur-Mer Sorbonne Universiteacutes UPMC Universiteacute Paris 06 Observatoire Oceacuteanologique de Villefranche-sur-Mer Villefranche-sur-Mer France

Reza Zolfaghari Department of Nutritional Sciences Pennsylvania State University University Park PA USA

xiii

Preface

VITAMIN A AND RETINOIDS AN AMAZING HISTORY AND HOPES FOR DISEASE PREVENTION

Historically the vitamin A precursor carotenoids had been long suspected of having medicinal properties For thousands of years humans and animals suffered from vitamin A deficiency typified by night blindness and xerophthalmiamdasha failure of tear production which if untreated would result in blindness While the underlying causes of such maledictions were a mystery in 1500 BC the ancient Egyptians rec-ommended eating animal or fish liver for their curative powers The native people of the Arctic also long knew to avoid eating the liver of polar bears because they became sick (unknowingly by vitamin A) Western explorers not savvy to the vitamin A rich properties of polar bear liver as early as 1596 accounted a horrible illnessndashsluggish-ness blurred vision nausea headache and skin loss resulting in coma and even deathmdashall signs of acute hypervitaminosis A or vitamin A toxicity

Not until the twentieth century was vitamin A actually isolated In 1909 Hopkins and Steep extracted a lipidfat substance that mice and rats absolutely required for their growth Elmer McCollum performed a careful analysis of the growth-promoting factors in protein-free milk leading to the isolation of the first known fat-soluble vitamin This essential growth promoting compound was named ldquofat soluble Ardquo a terminology distinguishing it from other water-soluble vitamins such as the recently discovered anti-scurvey factormdashvitamin C In 1920 Jack Drummond suggested that the ldquovital sub-stancerdquo be given the name vitamin A a substance later associated with a pigmented yellow color In 1935 George Wald found vitamin A was a component of the retina When the rhodopsin pigment was exposed to light it yielded opsin and a vitamin A-containing compound (the chromophore) indicating that vitamin A was essential in retinal function While the nature of the chromophore and the reactions occurring dur-ing the visual cycle were characterized long ago there have been recent developments in the characterization of the enzymes and carrier proteins involved in this cycle with novel findings indicating that an alternative pathway for chromophore regeneration has evolved in cones (the photoreceptors of the retina that operate in bright daylight and which are responsible visual acuity and color discrimination) Two chapters in this book (Chapters 18 and 24) review these findings the latter chapter describing gene mutations leading to visual diseases and discussing therapeutic strategies

In the late 1970s researchers suggested that the physiological activity of vitamin A may be occurring through ligand binding to nuclear receptors In the 1980s the goal of many researchers became to elucidate how these signaling molecules prodded gene expression In 1986 the groups of Pierre Chambon and Ronald Evans independently

xiv Preface

cloned and characterized the first retinoic acid receptormdasha molecule providing an entry point for detailing vitamin A biology In 2004 the two scientists were awarded the Albert Lasker Basic Medical Research Award for their discoveries that retinoids and other pathways including steroids vitamin D thyroid hormone and many other lipid based drugs transmit signals through similar pathways These discoveries advanced the mechanisms of vitamin A and retinoid signaling Several authors at the forefront of research aiming to solve structurendashfunction relationships of retinoid receptors their genomic target loci and the post-translational and epigenetic regulatory mechanisms that fine-tune their activity according to the cellular context provide a comprehensive overview of these complex phenomena in Part II of this book (Chapters 5ndash8) These are followed by two chapters on the chemistry of retinoid-related synthetic molecules an important research field for drug design of selective receptor agnonistsantagonists with Chapter 10 emphasizing how research on retinoid-related molecules led to the identification of modulators of another nuclear receptor the small heterodi-mer partner ldquoorphanrdquo receptor

Worldwide problems of nutritional deficiencies appear insurmountable as the planetrsquos population tops seven billion Vitamin A deficiency and its associated dis-eases afflict impoverished populations and are endemic in sub-Saharan Africa and southern Asia These deficiencies preferentially target infants preschool children and pregnant women Vitamin A deficiency is the leading preventable cause of vision loss Over 200 million are estimated as lacking sufficient serum retinol levels Vitamin A deficiency diseases frequently include rod photoreceptor dysfunction and night blindness and in more severe cases blinding ulcerations and necrosis of the cornea The World Health Organization estimates that 14 million children have pre-ventable irreversible blindness with half dying within a year of losing sight Also because inflammation reduces retinol-binding protein levels compromised immune function increases death rate following bacterial-induced diarrhea malaria or HIV infection Lower food intake during disease results in a synergistic downward pro-gression of malnutrition and disease which can be lethal Providing a small concen-trated dose of retinol has proven effects increasing infant survival by over 30 Retinol treatments are extremely low cost with consequent saving of visionmdashwith sometimes only two days of treatment restoring night blindness One chapter of this book (Chapter 23) describes the consequences to world health of retinoid defi-ciencies while others provide updated reviews on the relationship between the reti-noid pathway and inflammatory processes (Chapter 20) and its functions in the immune system (Chapter 21)

Nutritional deficiencies are a common survival challenge and for the fat-soluble vitamin A our organism has developed exquisite mechanisms to enzymatically con-vert and maintain adequate stores of retinoids (mainly in the liver) allowing adults to survive under vitamin A deficiency for months or even years The first part of this book provides detailed accounts on the mechanisms of vitamin A and carotenoid absorption liver storage and tissue-targeted delivery through the blood circulation with one chapter (Chapter 4) discussing current knowledge on the ancestry of reti-noid receptors in multicellular organisms and the molecular evolution of the retinoic acid signaling pathway

In the 1970s Michael Sporn first used the term ldquochemopreventionrdquo for clinical trials using retinoids to prevent or delay the occurrence of certain forms of cancer

Preface xv

Unfortunately large-scale clinical trials providing retinol to smokers with premalig-nant oral lesions had disappointing results and in some cases had to be interrupted Potentially these high dose treatments decreased cellular retinoid signalingmdashperhaps by inducing CYP26 enzymatic activity leading to retinoic acid (RA) degradation Currently compounds inhibiting these enzymes are being tested for cancer treatment efficacy Clinical pharmacology employing the synthetic retinoid fenretinide indicates retinoid derivatives are plausible cancer treatment strategies Phase III clinical trial data suggested that fenretinide reduces breast cancer relapse inducing tumor cell death and necrosis The retinoid X receptor (RXR) agonist bexarotene is being tested for treatment of T-cell lymphoma and Kaposi sarcoma These data are reviewed in the last chapter of this book

How can we better understand intracellular functions related to cancer As detailed in Chapter 11 there are key examples of how the biological activity of RA critically relies on a balance of several lipid-binding proteins Reductions in one of these proteins cellular retinoic acid-binding protein 2 (CRABP2) and augmentations in fatty acid-binding protein 5 (FABP5) change RA signaling from normal phy-siological growth inhibition to pathological states of neoplastic breast cancer growth There are also antagonistic effects of RA and estrogenmdashanother hormone acting through nuclear receptorsmdashin breast cancer cells (Chapter 8) Selective gene targets are either induced or inhibited within cell-specific contexts indicating some of the selective parameters in which RA can be capable of arresting neoplastic growth

There are many additional reasons that populations in affluent well-nourished countries should be concerned about retinoid signaling The most oppressing health problem in the United States (and a growing plague in the entire world) is the alarm-ing increase in obesity This rise in obesity is even found in poorer countries due to food insecurity choices based on high calorie intake The increase in the overweight population and its associated diseases (cardiovascular diabetes and metabolic dis-eases) will have a predicted economic impact of more than $30 trillion over the next 20 years according to a 2011 report by the World Economic Forum and Harvard School of Public Health The collective impact of noncommunicable cardiovascular diabetic and other metabolic diseases linked to obesity impacts large segments of the world population Proteins selectively binding retinoids (such as retinol-binding pro-tein) are increased in obese patients Retinoic acid has a pharmacological side effect of inducing weight loss While this clinical side effect is clearly disadvantageous in retinoid treatments used to reduce cancer growth it has potential actions as an anti-obesity agent if other side effects can be reduced Structural studies on how retinoid receptors bind ligands and coregulators reveal allosteric changes altering their sig-naling properties and provide critical information needed to improve retinoid phar-macology to provide drugs with improved selectivity and reduced side effects (Chapters 7 and 9) Authors also address how retinoid fat-reducing metabolic actions may be controlled by cellular sites of storage namely in lipid droplets (Chapter 3) Pressing questions are if and should retinoids be a widely used treatment strategy to reduce obesity and consequent metabolic disease Since side effects of severe hyper-vitaminosis A or retinoid treatments include skin scaling lethargy and nausea long-term pilot trials appear to be required before the weight loss potential of reti-noids is exploited in large populations Treatment may require pharmacologically selective retinoids specific to retinoid metabolic targets Public health campaigns to

xvi Preface

increasing intake of fruits and vegetables (especially those rich in vitamin A) serve as a more immediate strategy to address increasing obesity

Human nutrition can have a dynamic effect on life processes and disease by influencing epigenetic regulation Gene regulatory systems have evolved for extremely precise and specific regulatory control The retinoid signal acts through epigenetic control of transcriptional coregulatory modulators that can respond to environmental clues Retinoid responsiveness relies on promoter occupancy and organization of the epigenetic landscape at the chromatin level Many genes that are rapidly activated by retinoids have transcriptional response elements with ldquobivalentrdquo promoter chromatin access (Chapter 12) Responding genes are in a poised state and are rapidly activated by increasing the activation to repressive transcriptional complex ratios This chromatin reorganization can be coordinately managed by phosphorylation of several actors of RA signaling integrating and managing cellular decision (Chapter 5) The disease-related examples of aberrant epigenetic landscape contributing to malignancy can be found with retinoic acid treatment of acute promy-elocytic leukemia (Chapter 25) Current evidence supports that compounds inhibit-ing histone deacetylase activity augment retinoic acid receptorndashretinoid X receptor (RARndashRXR) response acting synergistically on condensed chromatin RXRs can be specifically activated by a class of drugs termed rexinoids currently used in the clinical treatment of selective lymphomas These treatments are effective grim reapers bringing about tumor cell apoptosis

The promise of stem cell research has provided public hope in curative ther-apies including neuroregenerative treatment of spinal cord injury or Alzheimerrsquos disease repairing damaged heart tissue subsequent to myocardial infarction or replacing skin in severe burn victims Are these plausible strategies or are realistic expectations being exceeded Retinoids are regulators of a number of stem cell line-ages in a variety of organ progenitor sites (pancreas blood heart central nervous system and gonads) at various stages of development and postnatal life The under-lying concept that RA regulates stemprogenitor cell lineages during developmentmdashand may have later roles in regulating organ regeneration or repairmdashis the subject of several chapters in Part III of this book Various animal models of retinoid deficiency show defects in the developing nervous system heart and lung These defects per-turb organ function disrupting patterning hence inducing fetal or embryonic-stage lethality Interestingly the same genes expressed in adult stem cell populations are often acting in progenitor cell populations at embryonic and fetal stages Could the changes in gene expression observed in such cell populations in murine models of retinoid deficiency be considered as a paradigm for further studies providing clues on how retinoids might modulate stem cell dynamics at later (adult) stages These questions which are touched upon in several chapters of this book will undoubtedly be the subject of many future investigations

In summary the strong interest in vitamin A signaling through its active compo-nents the retinoids is rooted in the biological implications in so many physiological and disease processes Who would want to delve into the complexity of all of these subjects There are currently over 50 000 references when the word ldquoretinoidsrdquo is enteredmdashalthough its coselection with ldquoreviewsrdquo ldquocancerrdquo or ldquoobesityrdquo will reduce this number to thousands This book presents integrated knowledge from many current experts in the field We congratulate authors for their commitmentmdashin spite of their

Preface xvii

other overburdening academic responsibilities And for this opus we as editors wish to give a million ldquothanksrdquo to all of the contributing authors This book is the ldquoRetinoid Toolrdquo that can serve both the debutant and experienced researcher advancing their knowledge and understanding and leading to successful research discoveries

Pascal DolleacuteKaren Niederreither

PART I

Vitamin a metabolic and enzymatic Pathways

The Retinoids Biology Biochemistry and Disease First Edition Edited by Pascal Dolleacute and Karen Niederreither copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

1

Vitamin a metabolism storage and tissue deliVery mechanisms

William s blaner1 and yang li2

1 Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

2 Columbia College Columbia University New York NY USA

I IntroductIon

Vitamin A is an essential micronutrient that must be acquired from the diet either as preformed vitamin A or as provitamin A carotenoid By definition all-trans-retinol is vitamin A although retinol metabolites including retinyl esters and retinoic acid are often referred to collectively as vitamin A Throughout this chapter vitamin A is used solely to refer to all-trans-retinol The term retinoid which was first coined by Sporn in the mid-1970s refers to a family of chemicals both natural and synthetic that bear a structural resemblance to all-trans-retinol with or without the biologic activity of vitamin A (Sporn et al 1976) Thus the retinoid family of compounds comprises both naturally-occurring vitamin A metabolites and synthetic compounds that bear a structural resemblance to vitamin A

Within the body the two most abundant vitamin A species are retinol and retinyl esters Both retinol and retinyl esters are central to the metabolism storage and delivery of vitamin A to tissues The great majority of the vitamin A present in the body is stored as retinyl esters primarily in the liver but also in other tissues Dietary vitamin A is also packaged as retinyl esters in nascent chylomicrons and is thus taken into the body Because of this retinyl esters are a transport form of vitamin A that can

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

xiii

Preface

VITAMIN A AND RETINOIDS AN AMAZING HISTORY AND HOPES FOR DISEASE PREVENTION

Historically the vitamin A precursor carotenoids had been long suspected of having medicinal properties For thousands of years humans and animals suffered from vitamin A deficiency typified by night blindness and xerophthalmiamdasha failure of tear production which if untreated would result in blindness While the underlying causes of such maledictions were a mystery in 1500 BC the ancient Egyptians rec-ommended eating animal or fish liver for their curative powers The native people of the Arctic also long knew to avoid eating the liver of polar bears because they became sick (unknowingly by vitamin A) Western explorers not savvy to the vitamin A rich properties of polar bear liver as early as 1596 accounted a horrible illnessndashsluggish-ness blurred vision nausea headache and skin loss resulting in coma and even deathmdashall signs of acute hypervitaminosis A or vitamin A toxicity

Not until the twentieth century was vitamin A actually isolated In 1909 Hopkins and Steep extracted a lipidfat substance that mice and rats absolutely required for their growth Elmer McCollum performed a careful analysis of the growth-promoting factors in protein-free milk leading to the isolation of the first known fat-soluble vitamin This essential growth promoting compound was named ldquofat soluble Ardquo a terminology distinguishing it from other water-soluble vitamins such as the recently discovered anti-scurvey factormdashvitamin C In 1920 Jack Drummond suggested that the ldquovital sub-stancerdquo be given the name vitamin A a substance later associated with a pigmented yellow color In 1935 George Wald found vitamin A was a component of the retina When the rhodopsin pigment was exposed to light it yielded opsin and a vitamin A-containing compound (the chromophore) indicating that vitamin A was essential in retinal function While the nature of the chromophore and the reactions occurring dur-ing the visual cycle were characterized long ago there have been recent developments in the characterization of the enzymes and carrier proteins involved in this cycle with novel findings indicating that an alternative pathway for chromophore regeneration has evolved in cones (the photoreceptors of the retina that operate in bright daylight and which are responsible visual acuity and color discrimination) Two chapters in this book (Chapters 18 and 24) review these findings the latter chapter describing gene mutations leading to visual diseases and discussing therapeutic strategies

In the late 1970s researchers suggested that the physiological activity of vitamin A may be occurring through ligand binding to nuclear receptors In the 1980s the goal of many researchers became to elucidate how these signaling molecules prodded gene expression In 1986 the groups of Pierre Chambon and Ronald Evans independently

xiv Preface

cloned and characterized the first retinoic acid receptormdasha molecule providing an entry point for detailing vitamin A biology In 2004 the two scientists were awarded the Albert Lasker Basic Medical Research Award for their discoveries that retinoids and other pathways including steroids vitamin D thyroid hormone and many other lipid based drugs transmit signals through similar pathways These discoveries advanced the mechanisms of vitamin A and retinoid signaling Several authors at the forefront of research aiming to solve structurendashfunction relationships of retinoid receptors their genomic target loci and the post-translational and epigenetic regulatory mechanisms that fine-tune their activity according to the cellular context provide a comprehensive overview of these complex phenomena in Part II of this book (Chapters 5ndash8) These are followed by two chapters on the chemistry of retinoid-related synthetic molecules an important research field for drug design of selective receptor agnonistsantagonists with Chapter 10 emphasizing how research on retinoid-related molecules led to the identification of modulators of another nuclear receptor the small heterodi-mer partner ldquoorphanrdquo receptor

Worldwide problems of nutritional deficiencies appear insurmountable as the planetrsquos population tops seven billion Vitamin A deficiency and its associated dis-eases afflict impoverished populations and are endemic in sub-Saharan Africa and southern Asia These deficiencies preferentially target infants preschool children and pregnant women Vitamin A deficiency is the leading preventable cause of vision loss Over 200 million are estimated as lacking sufficient serum retinol levels Vitamin A deficiency diseases frequently include rod photoreceptor dysfunction and night blindness and in more severe cases blinding ulcerations and necrosis of the cornea The World Health Organization estimates that 14 million children have pre-ventable irreversible blindness with half dying within a year of losing sight Also because inflammation reduces retinol-binding protein levels compromised immune function increases death rate following bacterial-induced diarrhea malaria or HIV infection Lower food intake during disease results in a synergistic downward pro-gression of malnutrition and disease which can be lethal Providing a small concen-trated dose of retinol has proven effects increasing infant survival by over 30 Retinol treatments are extremely low cost with consequent saving of visionmdashwith sometimes only two days of treatment restoring night blindness One chapter of this book (Chapter 23) describes the consequences to world health of retinoid defi-ciencies while others provide updated reviews on the relationship between the reti-noid pathway and inflammatory processes (Chapter 20) and its functions in the immune system (Chapter 21)

Nutritional deficiencies are a common survival challenge and for the fat-soluble vitamin A our organism has developed exquisite mechanisms to enzymatically con-vert and maintain adequate stores of retinoids (mainly in the liver) allowing adults to survive under vitamin A deficiency for months or even years The first part of this book provides detailed accounts on the mechanisms of vitamin A and carotenoid absorption liver storage and tissue-targeted delivery through the blood circulation with one chapter (Chapter 4) discussing current knowledge on the ancestry of reti-noid receptors in multicellular organisms and the molecular evolution of the retinoic acid signaling pathway

In the 1970s Michael Sporn first used the term ldquochemopreventionrdquo for clinical trials using retinoids to prevent or delay the occurrence of certain forms of cancer

Preface xv

Unfortunately large-scale clinical trials providing retinol to smokers with premalig-nant oral lesions had disappointing results and in some cases had to be interrupted Potentially these high dose treatments decreased cellular retinoid signalingmdashperhaps by inducing CYP26 enzymatic activity leading to retinoic acid (RA) degradation Currently compounds inhibiting these enzymes are being tested for cancer treatment efficacy Clinical pharmacology employing the synthetic retinoid fenretinide indicates retinoid derivatives are plausible cancer treatment strategies Phase III clinical trial data suggested that fenretinide reduces breast cancer relapse inducing tumor cell death and necrosis The retinoid X receptor (RXR) agonist bexarotene is being tested for treatment of T-cell lymphoma and Kaposi sarcoma These data are reviewed in the last chapter of this book

How can we better understand intracellular functions related to cancer As detailed in Chapter 11 there are key examples of how the biological activity of RA critically relies on a balance of several lipid-binding proteins Reductions in one of these proteins cellular retinoic acid-binding protein 2 (CRABP2) and augmentations in fatty acid-binding protein 5 (FABP5) change RA signaling from normal phy-siological growth inhibition to pathological states of neoplastic breast cancer growth There are also antagonistic effects of RA and estrogenmdashanother hormone acting through nuclear receptorsmdashin breast cancer cells (Chapter 8) Selective gene targets are either induced or inhibited within cell-specific contexts indicating some of the selective parameters in which RA can be capable of arresting neoplastic growth

There are many additional reasons that populations in affluent well-nourished countries should be concerned about retinoid signaling The most oppressing health problem in the United States (and a growing plague in the entire world) is the alarm-ing increase in obesity This rise in obesity is even found in poorer countries due to food insecurity choices based on high calorie intake The increase in the overweight population and its associated diseases (cardiovascular diabetes and metabolic dis-eases) will have a predicted economic impact of more than $30 trillion over the next 20 years according to a 2011 report by the World Economic Forum and Harvard School of Public Health The collective impact of noncommunicable cardiovascular diabetic and other metabolic diseases linked to obesity impacts large segments of the world population Proteins selectively binding retinoids (such as retinol-binding pro-tein) are increased in obese patients Retinoic acid has a pharmacological side effect of inducing weight loss While this clinical side effect is clearly disadvantageous in retinoid treatments used to reduce cancer growth it has potential actions as an anti-obesity agent if other side effects can be reduced Structural studies on how retinoid receptors bind ligands and coregulators reveal allosteric changes altering their sig-naling properties and provide critical information needed to improve retinoid phar-macology to provide drugs with improved selectivity and reduced side effects (Chapters 7 and 9) Authors also address how retinoid fat-reducing metabolic actions may be controlled by cellular sites of storage namely in lipid droplets (Chapter 3) Pressing questions are if and should retinoids be a widely used treatment strategy to reduce obesity and consequent metabolic disease Since side effects of severe hyper-vitaminosis A or retinoid treatments include skin scaling lethargy and nausea long-term pilot trials appear to be required before the weight loss potential of reti-noids is exploited in large populations Treatment may require pharmacologically selective retinoids specific to retinoid metabolic targets Public health campaigns to

xvi Preface

increasing intake of fruits and vegetables (especially those rich in vitamin A) serve as a more immediate strategy to address increasing obesity

Human nutrition can have a dynamic effect on life processes and disease by influencing epigenetic regulation Gene regulatory systems have evolved for extremely precise and specific regulatory control The retinoid signal acts through epigenetic control of transcriptional coregulatory modulators that can respond to environmental clues Retinoid responsiveness relies on promoter occupancy and organization of the epigenetic landscape at the chromatin level Many genes that are rapidly activated by retinoids have transcriptional response elements with ldquobivalentrdquo promoter chromatin access (Chapter 12) Responding genes are in a poised state and are rapidly activated by increasing the activation to repressive transcriptional complex ratios This chromatin reorganization can be coordinately managed by phosphorylation of several actors of RA signaling integrating and managing cellular decision (Chapter 5) The disease-related examples of aberrant epigenetic landscape contributing to malignancy can be found with retinoic acid treatment of acute promy-elocytic leukemia (Chapter 25) Current evidence supports that compounds inhibit-ing histone deacetylase activity augment retinoic acid receptorndashretinoid X receptor (RARndashRXR) response acting synergistically on condensed chromatin RXRs can be specifically activated by a class of drugs termed rexinoids currently used in the clinical treatment of selective lymphomas These treatments are effective grim reapers bringing about tumor cell apoptosis

The promise of stem cell research has provided public hope in curative ther-apies including neuroregenerative treatment of spinal cord injury or Alzheimerrsquos disease repairing damaged heart tissue subsequent to myocardial infarction or replacing skin in severe burn victims Are these plausible strategies or are realistic expectations being exceeded Retinoids are regulators of a number of stem cell line-ages in a variety of organ progenitor sites (pancreas blood heart central nervous system and gonads) at various stages of development and postnatal life The under-lying concept that RA regulates stemprogenitor cell lineages during developmentmdashand may have later roles in regulating organ regeneration or repairmdashis the subject of several chapters in Part III of this book Various animal models of retinoid deficiency show defects in the developing nervous system heart and lung These defects per-turb organ function disrupting patterning hence inducing fetal or embryonic-stage lethality Interestingly the same genes expressed in adult stem cell populations are often acting in progenitor cell populations at embryonic and fetal stages Could the changes in gene expression observed in such cell populations in murine models of retinoid deficiency be considered as a paradigm for further studies providing clues on how retinoids might modulate stem cell dynamics at later (adult) stages These questions which are touched upon in several chapters of this book will undoubtedly be the subject of many future investigations

In summary the strong interest in vitamin A signaling through its active compo-nents the retinoids is rooted in the biological implications in so many physiological and disease processes Who would want to delve into the complexity of all of these subjects There are currently over 50 000 references when the word ldquoretinoidsrdquo is enteredmdashalthough its coselection with ldquoreviewsrdquo ldquocancerrdquo or ldquoobesityrdquo will reduce this number to thousands This book presents integrated knowledge from many current experts in the field We congratulate authors for their commitmentmdashin spite of their

Preface xvii

other overburdening academic responsibilities And for this opus we as editors wish to give a million ldquothanksrdquo to all of the contributing authors This book is the ldquoRetinoid Toolrdquo that can serve both the debutant and experienced researcher advancing their knowledge and understanding and leading to successful research discoveries

Pascal DolleacuteKaren Niederreither

PART I

Vitamin a metabolic and enzymatic Pathways

The Retinoids Biology Biochemistry and Disease First Edition Edited by Pascal Dolleacute and Karen Niederreither copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

1

Vitamin a metabolism storage and tissue deliVery mechanisms

William s blaner1 and yang li2

1 Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

2 Columbia College Columbia University New York NY USA

I IntroductIon

Vitamin A is an essential micronutrient that must be acquired from the diet either as preformed vitamin A or as provitamin A carotenoid By definition all-trans-retinol is vitamin A although retinol metabolites including retinyl esters and retinoic acid are often referred to collectively as vitamin A Throughout this chapter vitamin A is used solely to refer to all-trans-retinol The term retinoid which was first coined by Sporn in the mid-1970s refers to a family of chemicals both natural and synthetic that bear a structural resemblance to all-trans-retinol with or without the biologic activity of vitamin A (Sporn et al 1976) Thus the retinoid family of compounds comprises both naturally-occurring vitamin A metabolites and synthetic compounds that bear a structural resemblance to vitamin A

Within the body the two most abundant vitamin A species are retinol and retinyl esters Both retinol and retinyl esters are central to the metabolism storage and delivery of vitamin A to tissues The great majority of the vitamin A present in the body is stored as retinyl esters primarily in the liver but also in other tissues Dietary vitamin A is also packaged as retinyl esters in nascent chylomicrons and is thus taken into the body Because of this retinyl esters are a transport form of vitamin A that can

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

xiv Preface

cloned and characterized the first retinoic acid receptormdasha molecule providing an entry point for detailing vitamin A biology In 2004 the two scientists were awarded the Albert Lasker Basic Medical Research Award for their discoveries that retinoids and other pathways including steroids vitamin D thyroid hormone and many other lipid based drugs transmit signals through similar pathways These discoveries advanced the mechanisms of vitamin A and retinoid signaling Several authors at the forefront of research aiming to solve structurendashfunction relationships of retinoid receptors their genomic target loci and the post-translational and epigenetic regulatory mechanisms that fine-tune their activity according to the cellular context provide a comprehensive overview of these complex phenomena in Part II of this book (Chapters 5ndash8) These are followed by two chapters on the chemistry of retinoid-related synthetic molecules an important research field for drug design of selective receptor agnonistsantagonists with Chapter 10 emphasizing how research on retinoid-related molecules led to the identification of modulators of another nuclear receptor the small heterodi-mer partner ldquoorphanrdquo receptor

Worldwide problems of nutritional deficiencies appear insurmountable as the planetrsquos population tops seven billion Vitamin A deficiency and its associated dis-eases afflict impoverished populations and are endemic in sub-Saharan Africa and southern Asia These deficiencies preferentially target infants preschool children and pregnant women Vitamin A deficiency is the leading preventable cause of vision loss Over 200 million are estimated as lacking sufficient serum retinol levels Vitamin A deficiency diseases frequently include rod photoreceptor dysfunction and night blindness and in more severe cases blinding ulcerations and necrosis of the cornea The World Health Organization estimates that 14 million children have pre-ventable irreversible blindness with half dying within a year of losing sight Also because inflammation reduces retinol-binding protein levels compromised immune function increases death rate following bacterial-induced diarrhea malaria or HIV infection Lower food intake during disease results in a synergistic downward pro-gression of malnutrition and disease which can be lethal Providing a small concen-trated dose of retinol has proven effects increasing infant survival by over 30 Retinol treatments are extremely low cost with consequent saving of visionmdashwith sometimes only two days of treatment restoring night blindness One chapter of this book (Chapter 23) describes the consequences to world health of retinoid defi-ciencies while others provide updated reviews on the relationship between the reti-noid pathway and inflammatory processes (Chapter 20) and its functions in the immune system (Chapter 21)

Nutritional deficiencies are a common survival challenge and for the fat-soluble vitamin A our organism has developed exquisite mechanisms to enzymatically con-vert and maintain adequate stores of retinoids (mainly in the liver) allowing adults to survive under vitamin A deficiency for months or even years The first part of this book provides detailed accounts on the mechanisms of vitamin A and carotenoid absorption liver storage and tissue-targeted delivery through the blood circulation with one chapter (Chapter 4) discussing current knowledge on the ancestry of reti-noid receptors in multicellular organisms and the molecular evolution of the retinoic acid signaling pathway

In the 1970s Michael Sporn first used the term ldquochemopreventionrdquo for clinical trials using retinoids to prevent or delay the occurrence of certain forms of cancer

Preface xv

Unfortunately large-scale clinical trials providing retinol to smokers with premalig-nant oral lesions had disappointing results and in some cases had to be interrupted Potentially these high dose treatments decreased cellular retinoid signalingmdashperhaps by inducing CYP26 enzymatic activity leading to retinoic acid (RA) degradation Currently compounds inhibiting these enzymes are being tested for cancer treatment efficacy Clinical pharmacology employing the synthetic retinoid fenretinide indicates retinoid derivatives are plausible cancer treatment strategies Phase III clinical trial data suggested that fenretinide reduces breast cancer relapse inducing tumor cell death and necrosis The retinoid X receptor (RXR) agonist bexarotene is being tested for treatment of T-cell lymphoma and Kaposi sarcoma These data are reviewed in the last chapter of this book

How can we better understand intracellular functions related to cancer As detailed in Chapter 11 there are key examples of how the biological activity of RA critically relies on a balance of several lipid-binding proteins Reductions in one of these proteins cellular retinoic acid-binding protein 2 (CRABP2) and augmentations in fatty acid-binding protein 5 (FABP5) change RA signaling from normal phy-siological growth inhibition to pathological states of neoplastic breast cancer growth There are also antagonistic effects of RA and estrogenmdashanother hormone acting through nuclear receptorsmdashin breast cancer cells (Chapter 8) Selective gene targets are either induced or inhibited within cell-specific contexts indicating some of the selective parameters in which RA can be capable of arresting neoplastic growth

There are many additional reasons that populations in affluent well-nourished countries should be concerned about retinoid signaling The most oppressing health problem in the United States (and a growing plague in the entire world) is the alarm-ing increase in obesity This rise in obesity is even found in poorer countries due to food insecurity choices based on high calorie intake The increase in the overweight population and its associated diseases (cardiovascular diabetes and metabolic dis-eases) will have a predicted economic impact of more than $30 trillion over the next 20 years according to a 2011 report by the World Economic Forum and Harvard School of Public Health The collective impact of noncommunicable cardiovascular diabetic and other metabolic diseases linked to obesity impacts large segments of the world population Proteins selectively binding retinoids (such as retinol-binding pro-tein) are increased in obese patients Retinoic acid has a pharmacological side effect of inducing weight loss While this clinical side effect is clearly disadvantageous in retinoid treatments used to reduce cancer growth it has potential actions as an anti-obesity agent if other side effects can be reduced Structural studies on how retinoid receptors bind ligands and coregulators reveal allosteric changes altering their sig-naling properties and provide critical information needed to improve retinoid phar-macology to provide drugs with improved selectivity and reduced side effects (Chapters 7 and 9) Authors also address how retinoid fat-reducing metabolic actions may be controlled by cellular sites of storage namely in lipid droplets (Chapter 3) Pressing questions are if and should retinoids be a widely used treatment strategy to reduce obesity and consequent metabolic disease Since side effects of severe hyper-vitaminosis A or retinoid treatments include skin scaling lethargy and nausea long-term pilot trials appear to be required before the weight loss potential of reti-noids is exploited in large populations Treatment may require pharmacologically selective retinoids specific to retinoid metabolic targets Public health campaigns to

xvi Preface

increasing intake of fruits and vegetables (especially those rich in vitamin A) serve as a more immediate strategy to address increasing obesity

Human nutrition can have a dynamic effect on life processes and disease by influencing epigenetic regulation Gene regulatory systems have evolved for extremely precise and specific regulatory control The retinoid signal acts through epigenetic control of transcriptional coregulatory modulators that can respond to environmental clues Retinoid responsiveness relies on promoter occupancy and organization of the epigenetic landscape at the chromatin level Many genes that are rapidly activated by retinoids have transcriptional response elements with ldquobivalentrdquo promoter chromatin access (Chapter 12) Responding genes are in a poised state and are rapidly activated by increasing the activation to repressive transcriptional complex ratios This chromatin reorganization can be coordinately managed by phosphorylation of several actors of RA signaling integrating and managing cellular decision (Chapter 5) The disease-related examples of aberrant epigenetic landscape contributing to malignancy can be found with retinoic acid treatment of acute promy-elocytic leukemia (Chapter 25) Current evidence supports that compounds inhibit-ing histone deacetylase activity augment retinoic acid receptorndashretinoid X receptor (RARndashRXR) response acting synergistically on condensed chromatin RXRs can be specifically activated by a class of drugs termed rexinoids currently used in the clinical treatment of selective lymphomas These treatments are effective grim reapers bringing about tumor cell apoptosis

The promise of stem cell research has provided public hope in curative ther-apies including neuroregenerative treatment of spinal cord injury or Alzheimerrsquos disease repairing damaged heart tissue subsequent to myocardial infarction or replacing skin in severe burn victims Are these plausible strategies or are realistic expectations being exceeded Retinoids are regulators of a number of stem cell line-ages in a variety of organ progenitor sites (pancreas blood heart central nervous system and gonads) at various stages of development and postnatal life The under-lying concept that RA regulates stemprogenitor cell lineages during developmentmdashand may have later roles in regulating organ regeneration or repairmdashis the subject of several chapters in Part III of this book Various animal models of retinoid deficiency show defects in the developing nervous system heart and lung These defects per-turb organ function disrupting patterning hence inducing fetal or embryonic-stage lethality Interestingly the same genes expressed in adult stem cell populations are often acting in progenitor cell populations at embryonic and fetal stages Could the changes in gene expression observed in such cell populations in murine models of retinoid deficiency be considered as a paradigm for further studies providing clues on how retinoids might modulate stem cell dynamics at later (adult) stages These questions which are touched upon in several chapters of this book will undoubtedly be the subject of many future investigations

In summary the strong interest in vitamin A signaling through its active compo-nents the retinoids is rooted in the biological implications in so many physiological and disease processes Who would want to delve into the complexity of all of these subjects There are currently over 50 000 references when the word ldquoretinoidsrdquo is enteredmdashalthough its coselection with ldquoreviewsrdquo ldquocancerrdquo or ldquoobesityrdquo will reduce this number to thousands This book presents integrated knowledge from many current experts in the field We congratulate authors for their commitmentmdashin spite of their

Preface xvii

other overburdening academic responsibilities And for this opus we as editors wish to give a million ldquothanksrdquo to all of the contributing authors This book is the ldquoRetinoid Toolrdquo that can serve both the debutant and experienced researcher advancing their knowledge and understanding and leading to successful research discoveries

Pascal DolleacuteKaren Niederreither

PART I

Vitamin a metabolic and enzymatic Pathways

The Retinoids Biology Biochemistry and Disease First Edition Edited by Pascal Dolleacute and Karen Niederreither copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

1

Vitamin a metabolism storage and tissue deliVery mechanisms

William s blaner1 and yang li2

1 Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

2 Columbia College Columbia University New York NY USA

I IntroductIon

Vitamin A is an essential micronutrient that must be acquired from the diet either as preformed vitamin A or as provitamin A carotenoid By definition all-trans-retinol is vitamin A although retinol metabolites including retinyl esters and retinoic acid are often referred to collectively as vitamin A Throughout this chapter vitamin A is used solely to refer to all-trans-retinol The term retinoid which was first coined by Sporn in the mid-1970s refers to a family of chemicals both natural and synthetic that bear a structural resemblance to all-trans-retinol with or without the biologic activity of vitamin A (Sporn et al 1976) Thus the retinoid family of compounds comprises both naturally-occurring vitamin A metabolites and synthetic compounds that bear a structural resemblance to vitamin A

Within the body the two most abundant vitamin A species are retinol and retinyl esters Both retinol and retinyl esters are central to the metabolism storage and delivery of vitamin A to tissues The great majority of the vitamin A present in the body is stored as retinyl esters primarily in the liver but also in other tissues Dietary vitamin A is also packaged as retinyl esters in nascent chylomicrons and is thus taken into the body Because of this retinyl esters are a transport form of vitamin A that can

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

Preface xv

Unfortunately large-scale clinical trials providing retinol to smokers with premalig-nant oral lesions had disappointing results and in some cases had to be interrupted Potentially these high dose treatments decreased cellular retinoid signalingmdashperhaps by inducing CYP26 enzymatic activity leading to retinoic acid (RA) degradation Currently compounds inhibiting these enzymes are being tested for cancer treatment efficacy Clinical pharmacology employing the synthetic retinoid fenretinide indicates retinoid derivatives are plausible cancer treatment strategies Phase III clinical trial data suggested that fenretinide reduces breast cancer relapse inducing tumor cell death and necrosis The retinoid X receptor (RXR) agonist bexarotene is being tested for treatment of T-cell lymphoma and Kaposi sarcoma These data are reviewed in the last chapter of this book

How can we better understand intracellular functions related to cancer As detailed in Chapter 11 there are key examples of how the biological activity of RA critically relies on a balance of several lipid-binding proteins Reductions in one of these proteins cellular retinoic acid-binding protein 2 (CRABP2) and augmentations in fatty acid-binding protein 5 (FABP5) change RA signaling from normal phy-siological growth inhibition to pathological states of neoplastic breast cancer growth There are also antagonistic effects of RA and estrogenmdashanother hormone acting through nuclear receptorsmdashin breast cancer cells (Chapter 8) Selective gene targets are either induced or inhibited within cell-specific contexts indicating some of the selective parameters in which RA can be capable of arresting neoplastic growth

There are many additional reasons that populations in affluent well-nourished countries should be concerned about retinoid signaling The most oppressing health problem in the United States (and a growing plague in the entire world) is the alarm-ing increase in obesity This rise in obesity is even found in poorer countries due to food insecurity choices based on high calorie intake The increase in the overweight population and its associated diseases (cardiovascular diabetes and metabolic dis-eases) will have a predicted economic impact of more than $30 trillion over the next 20 years according to a 2011 report by the World Economic Forum and Harvard School of Public Health The collective impact of noncommunicable cardiovascular diabetic and other metabolic diseases linked to obesity impacts large segments of the world population Proteins selectively binding retinoids (such as retinol-binding pro-tein) are increased in obese patients Retinoic acid has a pharmacological side effect of inducing weight loss While this clinical side effect is clearly disadvantageous in retinoid treatments used to reduce cancer growth it has potential actions as an anti-obesity agent if other side effects can be reduced Structural studies on how retinoid receptors bind ligands and coregulators reveal allosteric changes altering their sig-naling properties and provide critical information needed to improve retinoid phar-macology to provide drugs with improved selectivity and reduced side effects (Chapters 7 and 9) Authors also address how retinoid fat-reducing metabolic actions may be controlled by cellular sites of storage namely in lipid droplets (Chapter 3) Pressing questions are if and should retinoids be a widely used treatment strategy to reduce obesity and consequent metabolic disease Since side effects of severe hyper-vitaminosis A or retinoid treatments include skin scaling lethargy and nausea long-term pilot trials appear to be required before the weight loss potential of reti-noids is exploited in large populations Treatment may require pharmacologically selective retinoids specific to retinoid metabolic targets Public health campaigns to

xvi Preface

increasing intake of fruits and vegetables (especially those rich in vitamin A) serve as a more immediate strategy to address increasing obesity

Human nutrition can have a dynamic effect on life processes and disease by influencing epigenetic regulation Gene regulatory systems have evolved for extremely precise and specific regulatory control The retinoid signal acts through epigenetic control of transcriptional coregulatory modulators that can respond to environmental clues Retinoid responsiveness relies on promoter occupancy and organization of the epigenetic landscape at the chromatin level Many genes that are rapidly activated by retinoids have transcriptional response elements with ldquobivalentrdquo promoter chromatin access (Chapter 12) Responding genes are in a poised state and are rapidly activated by increasing the activation to repressive transcriptional complex ratios This chromatin reorganization can be coordinately managed by phosphorylation of several actors of RA signaling integrating and managing cellular decision (Chapter 5) The disease-related examples of aberrant epigenetic landscape contributing to malignancy can be found with retinoic acid treatment of acute promy-elocytic leukemia (Chapter 25) Current evidence supports that compounds inhibit-ing histone deacetylase activity augment retinoic acid receptorndashretinoid X receptor (RARndashRXR) response acting synergistically on condensed chromatin RXRs can be specifically activated by a class of drugs termed rexinoids currently used in the clinical treatment of selective lymphomas These treatments are effective grim reapers bringing about tumor cell apoptosis

The promise of stem cell research has provided public hope in curative ther-apies including neuroregenerative treatment of spinal cord injury or Alzheimerrsquos disease repairing damaged heart tissue subsequent to myocardial infarction or replacing skin in severe burn victims Are these plausible strategies or are realistic expectations being exceeded Retinoids are regulators of a number of stem cell line-ages in a variety of organ progenitor sites (pancreas blood heart central nervous system and gonads) at various stages of development and postnatal life The under-lying concept that RA regulates stemprogenitor cell lineages during developmentmdashand may have later roles in regulating organ regeneration or repairmdashis the subject of several chapters in Part III of this book Various animal models of retinoid deficiency show defects in the developing nervous system heart and lung These defects per-turb organ function disrupting patterning hence inducing fetal or embryonic-stage lethality Interestingly the same genes expressed in adult stem cell populations are often acting in progenitor cell populations at embryonic and fetal stages Could the changes in gene expression observed in such cell populations in murine models of retinoid deficiency be considered as a paradigm for further studies providing clues on how retinoids might modulate stem cell dynamics at later (adult) stages These questions which are touched upon in several chapters of this book will undoubtedly be the subject of many future investigations

In summary the strong interest in vitamin A signaling through its active compo-nents the retinoids is rooted in the biological implications in so many physiological and disease processes Who would want to delve into the complexity of all of these subjects There are currently over 50 000 references when the word ldquoretinoidsrdquo is enteredmdashalthough its coselection with ldquoreviewsrdquo ldquocancerrdquo or ldquoobesityrdquo will reduce this number to thousands This book presents integrated knowledge from many current experts in the field We congratulate authors for their commitmentmdashin spite of their

Preface xvii

other overburdening academic responsibilities And for this opus we as editors wish to give a million ldquothanksrdquo to all of the contributing authors This book is the ldquoRetinoid Toolrdquo that can serve both the debutant and experienced researcher advancing their knowledge and understanding and leading to successful research discoveries

Pascal DolleacuteKaren Niederreither

PART I

Vitamin a metabolic and enzymatic Pathways

The Retinoids Biology Biochemistry and Disease First Edition Edited by Pascal Dolleacute and Karen Niederreither copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

1

Vitamin a metabolism storage and tissue deliVery mechanisms

William s blaner1 and yang li2

1 Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

2 Columbia College Columbia University New York NY USA

I IntroductIon

Vitamin A is an essential micronutrient that must be acquired from the diet either as preformed vitamin A or as provitamin A carotenoid By definition all-trans-retinol is vitamin A although retinol metabolites including retinyl esters and retinoic acid are often referred to collectively as vitamin A Throughout this chapter vitamin A is used solely to refer to all-trans-retinol The term retinoid which was first coined by Sporn in the mid-1970s refers to a family of chemicals both natural and synthetic that bear a structural resemblance to all-trans-retinol with or without the biologic activity of vitamin A (Sporn et al 1976) Thus the retinoid family of compounds comprises both naturally-occurring vitamin A metabolites and synthetic compounds that bear a structural resemblance to vitamin A

Within the body the two most abundant vitamin A species are retinol and retinyl esters Both retinol and retinyl esters are central to the metabolism storage and delivery of vitamin A to tissues The great majority of the vitamin A present in the body is stored as retinyl esters primarily in the liver but also in other tissues Dietary vitamin A is also packaged as retinyl esters in nascent chylomicrons and is thus taken into the body Because of this retinyl esters are a transport form of vitamin A that can

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

xvi Preface

increasing intake of fruits and vegetables (especially those rich in vitamin A) serve as a more immediate strategy to address increasing obesity

Human nutrition can have a dynamic effect on life processes and disease by influencing epigenetic regulation Gene regulatory systems have evolved for extremely precise and specific regulatory control The retinoid signal acts through epigenetic control of transcriptional coregulatory modulators that can respond to environmental clues Retinoid responsiveness relies on promoter occupancy and organization of the epigenetic landscape at the chromatin level Many genes that are rapidly activated by retinoids have transcriptional response elements with ldquobivalentrdquo promoter chromatin access (Chapter 12) Responding genes are in a poised state and are rapidly activated by increasing the activation to repressive transcriptional complex ratios This chromatin reorganization can be coordinately managed by phosphorylation of several actors of RA signaling integrating and managing cellular decision (Chapter 5) The disease-related examples of aberrant epigenetic landscape contributing to malignancy can be found with retinoic acid treatment of acute promy-elocytic leukemia (Chapter 25) Current evidence supports that compounds inhibit-ing histone deacetylase activity augment retinoic acid receptorndashretinoid X receptor (RARndashRXR) response acting synergistically on condensed chromatin RXRs can be specifically activated by a class of drugs termed rexinoids currently used in the clinical treatment of selective lymphomas These treatments are effective grim reapers bringing about tumor cell apoptosis

The promise of stem cell research has provided public hope in curative ther-apies including neuroregenerative treatment of spinal cord injury or Alzheimerrsquos disease repairing damaged heart tissue subsequent to myocardial infarction or replacing skin in severe burn victims Are these plausible strategies or are realistic expectations being exceeded Retinoids are regulators of a number of stem cell line-ages in a variety of organ progenitor sites (pancreas blood heart central nervous system and gonads) at various stages of development and postnatal life The under-lying concept that RA regulates stemprogenitor cell lineages during developmentmdashand may have later roles in regulating organ regeneration or repairmdashis the subject of several chapters in Part III of this book Various animal models of retinoid deficiency show defects in the developing nervous system heart and lung These defects per-turb organ function disrupting patterning hence inducing fetal or embryonic-stage lethality Interestingly the same genes expressed in adult stem cell populations are often acting in progenitor cell populations at embryonic and fetal stages Could the changes in gene expression observed in such cell populations in murine models of retinoid deficiency be considered as a paradigm for further studies providing clues on how retinoids might modulate stem cell dynamics at later (adult) stages These questions which are touched upon in several chapters of this book will undoubtedly be the subject of many future investigations

In summary the strong interest in vitamin A signaling through its active compo-nents the retinoids is rooted in the biological implications in so many physiological and disease processes Who would want to delve into the complexity of all of these subjects There are currently over 50 000 references when the word ldquoretinoidsrdquo is enteredmdashalthough its coselection with ldquoreviewsrdquo ldquocancerrdquo or ldquoobesityrdquo will reduce this number to thousands This book presents integrated knowledge from many current experts in the field We congratulate authors for their commitmentmdashin spite of their

Preface xvii

other overburdening academic responsibilities And for this opus we as editors wish to give a million ldquothanksrdquo to all of the contributing authors This book is the ldquoRetinoid Toolrdquo that can serve both the debutant and experienced researcher advancing their knowledge and understanding and leading to successful research discoveries

Pascal DolleacuteKaren Niederreither

PART I

Vitamin a metabolic and enzymatic Pathways

The Retinoids Biology Biochemistry and Disease First Edition Edited by Pascal Dolleacute and Karen Niederreither copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

1

Vitamin a metabolism storage and tissue deliVery mechanisms

William s blaner1 and yang li2

1 Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

2 Columbia College Columbia University New York NY USA

I IntroductIon

Vitamin A is an essential micronutrient that must be acquired from the diet either as preformed vitamin A or as provitamin A carotenoid By definition all-trans-retinol is vitamin A although retinol metabolites including retinyl esters and retinoic acid are often referred to collectively as vitamin A Throughout this chapter vitamin A is used solely to refer to all-trans-retinol The term retinoid which was first coined by Sporn in the mid-1970s refers to a family of chemicals both natural and synthetic that bear a structural resemblance to all-trans-retinol with or without the biologic activity of vitamin A (Sporn et al 1976) Thus the retinoid family of compounds comprises both naturally-occurring vitamin A metabolites and synthetic compounds that bear a structural resemblance to vitamin A

Within the body the two most abundant vitamin A species are retinol and retinyl esters Both retinol and retinyl esters are central to the metabolism storage and delivery of vitamin A to tissues The great majority of the vitamin A present in the body is stored as retinyl esters primarily in the liver but also in other tissues Dietary vitamin A is also packaged as retinyl esters in nascent chylomicrons and is thus taken into the body Because of this retinyl esters are a transport form of vitamin A that can

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

Preface xvii

other overburdening academic responsibilities And for this opus we as editors wish to give a million ldquothanksrdquo to all of the contributing authors This book is the ldquoRetinoid Toolrdquo that can serve both the debutant and experienced researcher advancing their knowledge and understanding and leading to successful research discoveries

Pascal DolleacuteKaren Niederreither

PART I

Vitamin a metabolic and enzymatic Pathways

The Retinoids Biology Biochemistry and Disease First Edition Edited by Pascal Dolleacute and Karen Niederreither copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

1

Vitamin a metabolism storage and tissue deliVery mechanisms

William s blaner1 and yang li2

1 Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

2 Columbia College Columbia University New York NY USA

I IntroductIon

Vitamin A is an essential micronutrient that must be acquired from the diet either as preformed vitamin A or as provitamin A carotenoid By definition all-trans-retinol is vitamin A although retinol metabolites including retinyl esters and retinoic acid are often referred to collectively as vitamin A Throughout this chapter vitamin A is used solely to refer to all-trans-retinol The term retinoid which was first coined by Sporn in the mid-1970s refers to a family of chemicals both natural and synthetic that bear a structural resemblance to all-trans-retinol with or without the biologic activity of vitamin A (Sporn et al 1976) Thus the retinoid family of compounds comprises both naturally-occurring vitamin A metabolites and synthetic compounds that bear a structural resemblance to vitamin A

Within the body the two most abundant vitamin A species are retinol and retinyl esters Both retinol and retinyl esters are central to the metabolism storage and delivery of vitamin A to tissues The great majority of the vitamin A present in the body is stored as retinyl esters primarily in the liver but also in other tissues Dietary vitamin A is also packaged as retinyl esters in nascent chylomicrons and is thus taken into the body Because of this retinyl esters are a transport form of vitamin A that can

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

PART I

Vitamin a metabolic and enzymatic Pathways

The Retinoids Biology Biochemistry and Disease First Edition Edited by Pascal Dolleacute and Karen Niederreither copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

1

Vitamin a metabolism storage and tissue deliVery mechanisms

William s blaner1 and yang li2

1 Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

2 Columbia College Columbia University New York NY USA

I IntroductIon

Vitamin A is an essential micronutrient that must be acquired from the diet either as preformed vitamin A or as provitamin A carotenoid By definition all-trans-retinol is vitamin A although retinol metabolites including retinyl esters and retinoic acid are often referred to collectively as vitamin A Throughout this chapter vitamin A is used solely to refer to all-trans-retinol The term retinoid which was first coined by Sporn in the mid-1970s refers to a family of chemicals both natural and synthetic that bear a structural resemblance to all-trans-retinol with or without the biologic activity of vitamin A (Sporn et al 1976) Thus the retinoid family of compounds comprises both naturally-occurring vitamin A metabolites and synthetic compounds that bear a structural resemblance to vitamin A

Within the body the two most abundant vitamin A species are retinol and retinyl esters Both retinol and retinyl esters are central to the metabolism storage and delivery of vitamin A to tissues The great majority of the vitamin A present in the body is stored as retinyl esters primarily in the liver but also in other tissues Dietary vitamin A is also packaged as retinyl esters in nascent chylomicrons and is thus taken into the body Because of this retinyl esters are a transport form of vitamin A that can

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

The Retinoids Biology Biochemistry and Disease First Edition Edited by Pascal Dolleacute and Karen Niederreither copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

1

Vitamin a metabolism storage and tissue deliVery mechanisms

William s blaner1 and yang li2

1 Department of Medicine College of Physicians and Surgeons Columbia University New York NY USA

2 Columbia College Columbia University New York NY USA

I IntroductIon

Vitamin A is an essential micronutrient that must be acquired from the diet either as preformed vitamin A or as provitamin A carotenoid By definition all-trans-retinol is vitamin A although retinol metabolites including retinyl esters and retinoic acid are often referred to collectively as vitamin A Throughout this chapter vitamin A is used solely to refer to all-trans-retinol The term retinoid which was first coined by Sporn in the mid-1970s refers to a family of chemicals both natural and synthetic that bear a structural resemblance to all-trans-retinol with or without the biologic activity of vitamin A (Sporn et al 1976) Thus the retinoid family of compounds comprises both naturally-occurring vitamin A metabolites and synthetic compounds that bear a structural resemblance to vitamin A

Within the body the two most abundant vitamin A species are retinol and retinyl esters Both retinol and retinyl esters are central to the metabolism storage and delivery of vitamin A to tissues The great majority of the vitamin A present in the body is stored as retinyl esters primarily in the liver but also in other tissues Dietary vitamin A is also packaged as retinyl esters in nascent chylomicrons and is thus taken into the body Because of this retinyl esters are a transport form of vitamin A that can

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

4 Vitamin a metabolism storage and tissue deliVery mechanisms

reach high concentrations in the postprandial circulation Retinol also is a transport form of vitamin A in the circulation where it is found bound to retinol-binding protein (RBP4 hereafter named RBP the intracellular RBP1-3 proteins being commonly referred to as CRBP1 [cellular retinol-binding protein-1] CRBP2 and IRBP [inter-photoreceptor retinoid-binding protein])

In the fasting state retinol bound to RBP is the predominant form of vitamin A present in the circulations of humans and rodents Retinol is also the metabolic precursor for the biologically active vitamin A metabolites 11-cis-retinaldehyde and retinoic acid 11-cis-retinaldehyde is the visual chromophore which as a first event in the visual cycle undergoes photoisomerization to all-trans-retinaldehyde when activated by a photon of light (Wald 1968) (Chapter 18) Retinoic acid both the all-trans- and 9-cis-isomers of retinoic acid are transcriptionally active forms of vitamin A (Chambon 1994 Mangelsdorf et al 1994) The binding of retinoic acid to retinoic acid receptors (RARs) or to retinoid X receptors (RXRs) can affect the transcription of vitamin A-responsive genes (Chambon 1994 Mangelsdorf et al 1994) (Part II in this book)

Most of the knowledge of vitamin A metabolism storage and mobilization has been obtained from studies of humans and rodents this chapter primarily focuses on this information However it should be noted that these processes vary mark-edly across different species For instance retinol is the predominant vitamin A species present in the circulations of fasting humans mice and rats However this is not universally true for other mammals or even primates and may even be the exception Great apes have relatively high fasting plasma retinyl ester levels com-pared to humans (Garciacutea et al 2006) Unlike the fasting human circulation where retinol accounts for 95 or more of the total vitamin A retinol accounts for only approximately 80 of the total vitamin A present in the fasting circulations of chimpanzees and orangutans with most of the remainder being present as retinyl esters (Garciacutea et al 2006) Retinyl esters are the predominant form of vitamin A present in the fasting circulations of dogs (Schweigert 1988 Raila et al 2002b 2004) domestic cats (Raila et al 2001) and ferrets (Ribaya-Mercado et al 1992 Raila et al 2002a)

Similarly retinyl ester concentrations in the lungs of humans and rats are relatively low compared to those of the liver (Schmitz et al 1991 Ross and Li 2007) Yet unlike humans or rats the lungs of mice contain very high concentrations of retinyl esters (OrsquoByrne et al 2005) The physiological significance of these or other species differences are not understood Nevertheless the reader should be aware that there are very pronounced species differences in how vitamin A is transported in the circulation and stored in tissues among different species

II VItamIn a metabolIsm releVant to Its storage

Unlike most other vitamins vitamin A can be accumulated from the diet and stored at relatively high concentrations within the body When adult rodents which are maintained on a conventional vitamin A-sufficient chow diet are placed on a vitamin A-deficient diet it can take 6ndash9 weeks or even longer for the retinyl ester stores of these rodents to become exhausted (Lamb et al 1974 Kato et al 1985 Shankar and De Luca 1988) The ability to accumulate vitamin A stores and the linked ability to

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

Vitamin a metabolism releVant to its storage 5

mobilize these stores in times of dietary vitamin A insufficiency affords the organism a great evolutionary advantage since these stores relieve the organism of the obligate need for regular dietary vitamin A intake

A scheme for the metabolism of vitamin A is provided in Figure 11 Since vitamin A is stored as retinyl ester and mobilized from tissue stores primarily as retinol this chapter focuses primarily on the metabolic events on the left-hand side of the scheme in Figure 11

Only long chain fatty acyl esters of retinol are synthesized within the body The retinyl esters found in humans and rodents consist of retinyl palmitate retinyl oleate retinyl stearate and retinyl linoleate (Goodman et al 1965 Tanumihardjo et al 1990) Within the liver approximately 70-80 of the total retinyl ester is present as retinyl palmitate (Goodman et al 1965 Tanumihardjo et al 1990) Other long chain acyl esters are also found in the body but these usually constitute less than 1ndash2 of all of the retinyl ester Retinyl acetate which is commonly found in food supplements and vitamin formulations is not naturally present in animals The retinyl acetate consumed in food supplements and vitamin formulations is quickly hydrolyzed by gut hydrolases to retinol which is then rapidly taken up by the small intestine and re-esterified as long chain retinyl esters

a Vitamin a esterification to retinyl ester

Two pathways for retinyl ester formation were proposed in the early literature These are summarized in Figure 12 One originally suggested by Huang and Goodman in the mid-1960s proposed that retinyl esters were formed through a

β-Carotene

Retinylesters

Retinol(vitamin A)

Retinaldehyde Retinoic acid Oxidizedretinoids

Elimination

11-cis-retinaldehyde

The chromophore for rhodopsinAll-trans- and 9-cis-retinoic acid

Transcriptionally active retinoidsall-trans-RA 3 retinoic acid receptors (RARs)9-cis-RA 3 retinoid X receptors (RXRs)

gt 500 genes regulated by retinoic acid

Diet

LRATARAT

Retinaldehydereductase

BCMO1

CYPsRALDHsRDHsADHsREHs

Figure 11 generalized scheme for the metabolism of vitamin a retinyl esters retinol

and β-carotene are taken into the body from the diet Vitamin a by definition all-trans-

retinol may be esterified to retinyl esters (via lrat or arat) and then stored in times of

dietary vitamin a insufficiency retinyl ester stores may be hydrolyzed (via rehs) to retinol

both retinol and β-carotene may be converted into the transcriptionally active vitamin a

forms 9-cis- and all-trans-retinoic acid after first being converted to retinaldehyde retinoic

acid (mainly in its all-trans form as most published studies indicate) then regulates transcrip-

tion of vitamin a-responsive genes When retinoic acid is no longer needed it is catabolized

by cytochrome enzymes (cyPs mainly cyP26 enzymes) and then eliminated from the body

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

6 Vitamin a metabolism storage and tissue deliVery mechanisms

transesterification reaction involving the transfer of the sn-1 acyl group from membrane phosphatidylcholine to retinol (Huang and Goodman 1965 Goodman et al 1966) This deduction preceded by about twenty-five years the discovery of a lecithinretinol acyltransferase (LRAT) activity in tissues (Ong et al 1987 1988 Saari and Bredberg 1988 Yost et al 1988) Another proposed by Ross (Ross

Figure 12 two biochemical pathways for retinyl ester formation lrat versus arat (a)

lrat catalyzes a phosphatidylcholine-dependent transesterification reaction transferring

the sn-1 acyl group from membrane phosphatidylcholine to retinol (either nonprotein

bound retinol or retinol bound to crbP1 crbP2 or crbP3) the products formed upon

lrat action are lysophosphatidylcholine and retinyl ester (and apo-crbP when retinol was

bound to a crbP) (b) arat catalyzes the acyl-coa-dependent transfer of the acyl group

from acyl-coa to free retinol forming coa and retinyl ester as products

O

O

R1

Lysophosphatidylcholine

(a)

Retinol (bound to CRBP)

LRAT

PhosphatidylcholineRetinyl ester

OH

O

O OCHR2 O Ondash

O

H

P N

O

O OOR2

R1

O Ondash

O

H

P N

O

O

O

O

R1

R1 CoA

CoA

S

(b)

ARAT

Retinyl ester

Retinol

Acyl-CoA

OH

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

Vitamin a metabolism releVant to its storage 7

1982 Rasmussen et al 1984)) and by Norum and colleagues (Rasmussen et al 1984 Muller and Norum 1986) maintained that retinyl ester formation was fatty acyl-CoA dependent involving an acyl-CoAretinol acyltransferase (ARAT) activity that had been identified in homogenates prepared from some tissues including liver (Ross 1982 Rasmussen et al 1984) kidney (Rasmussen et al 1984) intestine (Rasmussen et al 1984 Muller and Norum 1986) and skin (Torma and Vahlquist 1987 Kurlandsky et al 1996)

It is now clear from investigations of induced mutant mice that the predominant pathway for retinyl ester formation in the body involves a transesterification reaction catalyzed specifically by one enzyme LRAT (Batten et al 2004 Liu and Gudas 2005 OrsquoByrne et al 2005) Batten et al (2004) reported that the targeted disruption of the Lrat gene in all mouse tissues results in no developmental abnormalities however the mutant mice have only trace levels of retinyl esters in the liver lung eye and blood OrsquoByrne et al (2005) identified adipose tissue of Lratndashndash mice as the only substantial tissue site of retinyl ester accumulation reporting a twofold to threefold elevation of retinyl esters compared to age- genetic background- and gender-matched wild-type mice Moreover OrsquoByrne et al reported that some retinyl esters were present in chylomicrons of Lratndashndash mice when these mice were given an oral physiologic dose of vitamin A Liu and Gudas (2005) reported that Lratndashndash mice much more readily develop vitamin A deficiency than wild-type mice when main-tained on a vitamin A-insufficient diet Subsequent studies by the Gudas laboratory demonstrated that Lratndashndash mice when fed a diet high in vitamin A regulate vitamin A homeostasis differently than wild-type mice by enhancing cytochrome (CYP26A1)-catalyzed catabolism and elimination of the dietary retinol (Liu et al 2008) This is unlike wild-type mice which simply accumulate greater concentrations of retinyl esters in tissue stores

Although the published reports focused on Lratndashndash mice have established that LRAT is the key enzyme responsible for retinyl ester synthesis in the body these studies also provide clear evidence that another enzyme(s) acts in the synthesis of retinyl esters Several enzymes that can catalyze the acyl-CoA-dependent formation of retinyl esters have been identified by in vitro studies These include diacylglycerol acyltransferase 1 (DGAT1) (OrsquoByrne et al 2005 Orland et al 2005 Yen et al 2005) multifunctional acyltransferase (MFAT) (Yen et al 2005) and acyl-CoAmonoacylglycerol acyltrans-ferase (MGAT) (Yen et al 2005)

Of these enzymes the only one that has been studied in vivo as an ARAT is DGAT1 DGAT1 is one of two enzymes the other being diacylglycerol acyltransfer-ase 2 (DGAT2) which catalyzes the final step of triglyceride synthesis transferring an acyl group from acyl-CoA to a diglyceride (Yen et al 2008 Ruggles et al 2013) It should be noted based on in vitro studies that DGAT2 does not possess ARAT activity (OrsquoByrne et al 2005 Yen et al 2005) DGAT1 has been extensively studied because of its importance in metabolic disease development (Yen et al 2008 Ruggles et al 2013)

With regards to a role for DGAT1 as a physiologically relevant ARAT two studies have been reported Wongsiriroj et al reported that mice totally lacking expression of both Lrat and Dgat1 (LratndashndashDgat1ndashndash mice) are unable to incorpo-rate any retinyl esters in nascent chylomicrons in response to an oral challenge with a physiological dose of retinol (Wongsiriroj et al 2008) This is unlike

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

8 Vitamin a metabolism storage and tissue deliVery mechanisms

Lratndashndash mice which still synthesize and incorporate some retinyl ester into nascent cylomicrons in response to a physiologic dose of retinol (OrsquoByrne et al 2005) This implies that DGAT1 can act in vivo as an intestinal ARAT Wongsiriroj et al further reported that the LratndashndashDgat1ndashndash mice possess elevated concentrations of reti-nyl esters in adipose tissue identical to those of Lratndashndash mice Thus DGAT1 cannot be responsible for the observed increase in retinyl ester synthesis and accumulation in adipose tissue of Lratndashndash mice In other studies Farese and colleagues studied the actions of DGAT1 in maintaining vitamin A homeostasis in murine skin using Dgat1ndashndash and matched wild-type mice (Shih et al 2009) These investigators con-cluded that DGAT1 is the major ARAT activity present in murine skin Based on these studies it appears that DGAT1 acts as an ARAT in both intestine (Wongsiriroj et al 2008) and skin (Shih et al 2009) However it remains unclear whether MFAT andor MGAT act as ARATs in vivo or whether either of these enzymes is respon-sible for the elevated retinyl ester concentrations observed in adipose tissue of Lratndashndash and LratndashndashDgat1ndashndash mice

b retinyl ester Hydrolysis to Vitamin a

Unlike retinyl ester synthesis in which only a few enzymes have been identified as being able to catalyze retinyl ester formation in vitro and only one of these is responsible for most retinyl ester synthesis within the body many enzymes have been identified which possess retinyl ester hydrolase (REH) activity in vitro Some of these are relatively abundant and well characterized lipases including pancreatic triglyceride lipase (van Bennekum et al 2000) hepatic lipase (Krapp et al 1996) lipoprotein lipase (LpL) (Blaner et al 1994 van Bennekum et al 1999) cholesteryl ester lipase (CEL) (Lindstrom et al 1988) and hormone sensitive lipase (HSL) (Wei et al 1997) Others are less extensively studied these include pancreatic lipase-related protein 2 (Reboul et al 2006) intestinal brush border membrane REH (also identified as a calcium-independent brush border membrane phospholipase B) (Rigtrup and Ong 1992 Rigtrup et al 1994a 1994b) and a number of carboxylesterases (ES-2 ES-4 ES-10 and ES-22) (Mentlein and Heymann 1987 Schindler et al 1998 Linke et al 2005 Schreiber et al 2009)

A few of these enzymes have been established to have roles within the body in facilitating vitamin A homeostasis acting specifically as REHs For instance LpL acts importantly in facilitating uptake of postprandial vitamin A from chylomicrons by extrahepatic tissues (Blaner et al 1994 van Bennekum et al 1999) It is generally accepted that pancreatic triglyceride lipase brush border membrane REH and prob-ably CEL act to hydrolyze newly ingested dietary retinyl ester into retinol which is then taken up by the enterocyte for processing These are discussed in more detail in the next chapter There is consensus that HSL is a physiologically significant REH in adipocytes (Wei et al 1997 Stroumlm et al 2009) HSL was proposed to be the enzyme in adipocytes responsible for triglyceride hydrolysis but in the last decade with the identification and characterization of adipocyte triglyceride lipase (ATGL) it is now clear that both HSL and ATGL can act to hydrolyze adipocyte triglycerides ATGL has a more prominent role in this process (Lampidonis et al 2011 Lass et al 2011 Ruggles et al 2013)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

Vitamin a metabolism releVant to its storage 9

What remains unclear however is which enzyme or enzymes are importantly involved in vivo in the hydrolysis of retinyl esters within the liver where the majority of dietary vitamin A is taken up and where the majority of the bodyrsquos vitamin A stores are found Retinyl esters can be found in many tissues including lung testis skin and eye However as with the liver there is little general con-sensus as to the molecular identities of physiologically relevant REHs present in these tissues

c mobilization of Vitamin a from tissue stores

The ability of the body to alternatively store or mobilize vitamin A in response to its dietary availability is unique amongst the vitamins Healthy well-nourished indi-viduals who have accumulated vitamin A stores throughout life can go many weeks or even months before they succumb to the adverse physiological effects of vitamin A deficiency As depicted in Figure 13 the biology that underlies this selective advantage involves enzymes able to synthesize retinyl esters (LRATs and ARATs) enzymes able to hydrolyze retinyl esters to retinol (REHs) a capacity to facilitate the mobilization of tissue vitamin A stores and a capacity to accumulate retinyl esters in lipid droplets within cells and tissues

Retinyl ester accrual Retinol mobilization

Retinol

Retinylester

LRAT REH

Retinol intake Retinol-RBP

apo-RBP

Dietary vitamin A-insufficiencyDietary vitamin A-sufficiency

Figure 13 scheme depicting the alternative regulation of vitamin a store accumulation

versus the mobilization of these stores Vitamin a is maintained at constant levels within the

blood through two counteracting regulatory mechanisms during times of dietary vitamin

a sufficiency dietary retinol is taken up and then esterified into retinyl ester primarily via

lrat but in some tissues also through an arat activity the resulting retinyl ester is then

stored during times of dietary vitamin a insufficiency retinol is mobilized from retinyl ester

stores retinyl ester is hydrolyzed into retinol via a reh activity retinol then binds apo-rbP

and is released into the circulation for delivery to tissues

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)

10 Vitamin a metabolism storage and tissue deliVery mechanisms

As discussed in more detail later in this chapter (Section IV) vitamin A and several of its metabolites are present in both the fasting and postprandial circula-tions These include retinol retinyl esters retinoic acid and the β-glucuronides of both retinol and retinoic acid However only retinol is thought to be mobilized from tissue retinyl ester stores in response to insufficient dietary vitamin A intake That is to say blood retinol levels are maintained at a constant level or defended in response to extended consumption of a vitamin A-insufficient diet This is accomplished through the interaction of retinol with its specific serum transport protein retinol-binding protein (RBP) (Kanai et al 1968 Quadro et al 1999) As was convincingly demonstrated through the generation and study of Rbp-deficient mice the presence of RBP allows for the mobilization of stored vitamin A which is liberated through hydrolysis of tissue retinyl esters (Quadro et al 1999) Rbp-deficient mice accumulate tissue retinyl ester stores normally from the diet but are unable to mobilize retinol from these stores in response to insufficient dietary vitamin A intake (Quadro et al 1999 2005) Hence Rbp-deficient mice are prone to developing vitamin A deficiency when subjected to insufficient die-tary vitamin A intake

Since retinyl esters are very highly hydrophobic they are thus incorporated into intracellular lipid droplets The formation and degradation of intracellular lipid droplets is a highly regulated process involving lipid synthesizing and degrad-ing enzymes and many different lipid droplet-associated proteins The best studied retinyl ester-containing lipid droplets are the retinosomes found in the retinal pig-mented epithelial (RPE) cells of the eye (Imanishi et al 2004 2008 Orban et al 2011) the lipid droplets of the hepatic stellate cells (HSCs) (Blaner et al 2009) and the adipocyte lipid droplets (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013) As is discussed in more detail later the RPE retinosomes and the HSC lipid droplets are specialized lipid droplets that accumulate relatively high concentrations of retinyl esters In adipocyte lipid droplets concentration of retinyl esters are low compared to concentrations of triglycerides However the biochem-istry of adipocyte lipid droplets is better studied The reader is referred to recent reviews for general information regarding lipid droplet biochemistry (Lass et al 2011 Brasaemle and Wolins 2012 Konige et al 2013)

III VItamIn a storage

More than two decades ago Blomhoff and colleagues (Blomhoff et al 1991) provided a very thoughtful analysis of the literature concerning what proportion of the total vitamin A (retinol plus all of its metabolites) present in the body is found within the liver versus other tissues They concluded that for a vitamin A-sufficient rat probably greater than 90 of whole body total vitamin A is found in the liver They went on to conclude that this percentage is undoubtedly linked to vitamin A nutritional status with a substantially lower percentage present in the livers of ani-mals experiencing insufficient dietary vitamin A intake This conclusion is strongly supported by compartmental modeling studies carried out in rats receiving different quantities of vitamin A in their diets (Green et al 1987 Lewis et al 1990 Cifelli et al 2005)