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University of Groningen
Towards novel strategies to improve lipid homeostasis - targeting the intestineWulp, Mariëtte Ymkje Maria van der
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Towards novel strategies to improve lipid homeostasis –
targeting the intestine
Mariëtte van der Wulp
Research on nutrition and health integrates laboratory and clinical studies to identify food The research described in this thesis was conducted at the Department of Pediatrics, Beatrix Children's Hospital, Center for Liver, Digestive and Metabolic Diseases, University of Groningen, University Medical Center Groningen, The Netherlands and was financially supported by Top Institute Food and Nutrition, Programme Nutrition and Health.
The author gratefully acknowledges the financial support for printing of this thesis by:
Top Institute Food and Nutrition (TIFN)
Rijksuniversiteit Groningen (University of Groningen)
University Medical Center Groningen (UMCG)
Groningen University Institute for Drug Exploration (GUIDE)
Nederlandse Vereniging voor Gastroenterologie (NVGE)
Sectie experimentele Gastroenterologie van de NVGE
Restaurant en feestgelegenheid Louis XV, Groningen
Cover design by: Hendrix Grafische Vormgeving (Groningen) en Mariëtte van der WulpLayout by: Nikki Vermeulen, Ridderprint BV, Ridderkerk, the NetherlandsPrinting by: Ridderprint BV, Ridderkerk, the Netherlands
ISBN 978-90-367-5797-3 (printed)978-90-367-5798-0 (digital)
© 2012 M.Y.M. van der WulpAll rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means without permission of the author and the publisher holding the copyrights of the articles.
NVGE
Towards novel strategies to improve lipid homeostasis –
targeting the intestine
Proefschrift
ter verkrijging van het doctoraat in deMedische Wetenschappen
aan de Rijksuniversiteit Groningenop gezag van de
Rector Magnificus, dr. E. Sterken,in het openbaar te verdedigen op
maandag 3 december 2012om 14.30 uur
door
Mariëtte Ymkje Maria van der Wulp
geboren op 14 maart 1981te Deventer
Promotores: Prof. dr. H.J. Verkade Prof. dr. A.K. Groen Prof. dr. E.H.H.M. Rings
Beoordelingscommissie: Prof. dr. R.P.J. Oude Elferink Prof. dr. K.N. Faber Prof. dr. G.P.A. Smit
Voor Nathanja,mijn lieve zusje
I carry your heart
I carry it in my heart
(E.E. Cummings - 95 Poems, 1958)
Paranimfen: Sjoerdtje Johanna van der Veen Marjan Wouthuyzen-Bakker
Table of Contents
Chapter 1 General introduction 9
Chapter 2 Laxative treatment with polyethylene glycol does 39 not affect lipid absorption in rats
Chapter 3 Laxative treatment with polyethylene glycol decreases 55 microbial sterol conversion in the intestine of rats
Chapter 4 Transintestinal cholesterol excretion can be 73 manipulated by dietary fat composition in mice
Chapter 5 Genetic inactivation of the bile salt export pump in 93 mice profoundly increases fecal cholesterol excretion
Chapter 6 Conclusion, discussion and future perspectives 109
Appendices Summary 123
Nederlandse samenvatting 125
Dankwoord 127
Biography/Biografie 132
List of publications 133
Chapter 1
General Introduction
Mariëtte Y.M. van der Wulp, Henkjan J. Verkade, Albert K. Groen
Adapted from: Regulation of cholesterol homeostasis. Mol Cell Endocrinol. 2012 Jun
General Introduction
11
Chapter
1Introduction
The intestinal epithelium forms a unique interface for interactions between the body and food
components. It allows selective passage of nutrients, while offering protection against harmful
substances. Under physiological conditions, the epithelium maintains a peaceful relationship with the
extremely large community of commensal bacteria that live in the intestine and influence our health. 1
The intrinsically dynamic epithelium with its ability to respond to luminal stimuli, provides ample
possibilities to modulate intestinal physiology by nutrition and microbiota.
Cholesterol is of vital importance for vertebrate cell membrane structure and function. 2 Metabolites of
cholesterol, such as bile salts (BS), steroid hormones and oxysterols, fulfill important biological functions. 3
Hypercholesterolemia however represents a major risk factor for cardiovascular disease. 4,5 Cholesterol
homeostasis is tightly regulated by its intestinal absorption, fecal excretion and de novo synthesis. 6
Classically, fecal cholesterol excretion was believed to be primarily driven by cholesterol secreted via
the hepatobiliary pathway. 7 However, it has very recently become apparent that direct secretion of
cholesterol from the blood compartment to the intestine, the process now adopted “TransIntestinal
Cholesterol Excretion” (TICE), plays a major role in fecal cholesterol disposal. 6,8 Reduction of cholesterol
absorption and induction of TICE represent attractive targets to facilitate cholesterol disposal. Ideally,
inhibition of cholesterol absorption and/or induction of TICE would be facilitated by simple dietary
intervention.
BS are important for absorption of cholesterol, fat and fat-soluble vitamins. On the other hand, elimination
of excess cholesterol from the body is partly facilitated by breakdown of cholesterol to BS in the liver and
their subsequent fecal excretion. 9
In this theses we assessed, in a quantitative manner, intestinal function with respect to its capacity to
digest and absorb lipids (dietary fats, cholesterol and BS), and its capacity to secrete cholesterol under
varying intestinal conditions.
In this general introduction, first of all a short overview of BS homeostasis [1] and dietary lipid (dietary fat
and cholesterol) absorption [2] will be provided. Subsequently, the introduction will cover cholesterol
homeostasis including the transport of cholesterol through plasma [3], the characteristics, analytical
possibilities and (pharmacological) inhibition of cholesterol synthesis [4] and absorption [5], and finally
pathways of cholesterol excretion [6].
1. Bile salt homeostasis
Under physiological pH (6-8) biliary and intestinal bile acids are present in the form of sodium salts 10 and
will therefore be referred to as BS. BS are amphiphatic molecules, which are produced by the liver from
cholesterol. BS stimulate bile flow and biliary phospholipid (PL) secretion, regulate their own synthesis,
and are indispensable for efficient lipid absorption (including dietary fats, cholesterol and fat-soluble
vitamins A,D,E and K). Chenodeoxycholate (CDC) and cholate are the major primary (produced by the
liver) BS. The rodent liver converts the majority of CDC to more hydrophilic α- and β-muricholic acid (α-/
β-MC). 11 Intrinsically, rodents show a more hydrophilic BS pool compared with humans. 12
Chapter 1
12
Nearly all BS are conjugated by liver peroxisomes, mainly with taurine in rodents and glycine in humans. 12,13 The liver secretes these BS conjugates across the canalicular membrane into the bile canaliculi.
This canalicular secretion occurs against a high concentration gradient and is facilitated by adenosine-
triphospate (ATP)-dependent transporters, the most important of which is the Bile Salt Export Pump
(BSEP or ABCB11). 14,15 Via the bile canaliculi, BS are finally transported to the duodenum where they
facilitate the solubilization of lipids.
BS can be reabsorbed passively, but are mainly reabsorbed actively by the Apical Sodium-dependent
BS Transporter (ASBT) 16 in the terminal ileum (overall ~95%). At the basolateral enterocyte membrane,
BS leave the cells via the organic solute transporter (OSTα/β). 17 They are transported back to the liver
through the portal system and are mostly taken up via the Na+-dependent Taurocholate Co-transporting
Polypeptide (NTCP) at the basolateral hepatocyte membrane. 18 This process is called enterohepatic
cycling (EHC). 9 Under physiological circumstances BS transport from hepatocyte into the bloodstream
is negligible. However, this may rapidly change under cholestatic conditions, where BS are delivered to
the blood via members of the Multi drug resistance Related Proteins (MRPs), such as MRP4/ABCC4. 19
The BS that escape absorption in the terminal ileum enter the colon. Primary BS can be deconjugated
and converted to secondary BS species by the intestinal microbiota. Bacteria can convert β-MC to
hyodeoxycholate (HDC) 20,21 and ω-MC. In addition, they are able to convert CDC to lithocholate (LC,
mainly in humans) 22 or ursodeoxycholate (UDC) 23, and cholate to deoxycholate (DC) 22. A part of colonic
BS is passively absorbed, while the remaining part is excreted with feces. Excretion of BS with feces
actually represents an important pathway for cholesterol disposal. Under steady state conditions, the
liver compensates for fecal BS loss by de novo BS synthesis.
BS regulate their own secretion and synthesis via an elaborate feedback pathway. 24 Hepatic BS activate
the nuclear receptor (NR) farnesoid X receptor (FXR), which induces BS secretion via BSEP and reduces
basolateral BS uptake via NTCP.
After uptake in the terminal ileum, BS activate intestinal FXR which activates OSTα/β-mediated export
of BS. FXR also induces the short heterodimer partner (SHP), resulting in release of fibroblast growth
factor (Fgf ) 15 (mice) or FGF19 (humans) into the portal blood. 24 Fgf15/FGF19 activates the fibroblast
growth factor receptor 4 (FGFR4) in the liver, which ultimately facilitates transcriptional inhibition of
cholesterol 7 alpha-hydroxylase (Cyp7a1, encoding the rate-limiting enzyme for BS synthesis). 25 Both
SHP and FXR lack a Cyp7a1 DNA binding site. Liver FXR is activated primarily by primary BS (mainly
CDC), which induces SHP. SHP can activate the liver-related-homolog-1 (LRH-1, rodents) or α-fetoprotein
transcription factor (FTF, humans). LRH-1/ FTF can bind the bile acid response element II (BARE-II) in the
Cyp7a1 gene, inhibiting its transcription. SHP blocks the interaction between hepatocyte nuclear factor
4α (HNF4α) and peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α), which also inhibits
Cyp7a1 transcription via BARE-II. Cyp7a1 transcription can in addition be inhibited via steroid hormones,
inflammatory cytokines, insulin, growth factors and NRs that operate independently of FXR. 26 LC is a
ligand for the pregnane X receptor (PXR), constitutive androstane receptor (CAR; indirectly) and vitamin
D receptor (VDR).
General Introduction
13
Chapter
1PXR and VDR bind the BARE-I and block recruitment of PGC1α, inhibiting Cyp7a1 transactivation by
HNF4α. CAR binds the BARE-II and competes with HNF4α for several coactivators, including PGC1α. 26
On the other hand, when the liver X receptor (LXR) is activated by oxysterols, transcription of Cyp7a1 is
induced via the BARE-I in rodents (not in humans due to an alteration in the BARE-I sequence), resulting
in production of BS from cholesterol. 24
It is becoming more and more clear that BS are not just simple detergents necessary for lipid absorption,
but are also involved in the regulation of glucose, lipid homeostasis as well as energy expenditure. 27
Recent data show that removal of BS from the intestine with sequestrants can decrease plasma low
density lipoprotein (LDL) levels and hyperglycemia in patients with type II diabetes. 28 The mechanisms
by which the total pool size and/or profile of BS influence different physiological processes are only
beginning to be understood.
2. Dietary lipid absorption
2.1 Dietary fat absorption
Lipid absorption in general involves emulsification, lipolysis, micellar solubilization, uptake by mucosal
epithelium, re-esterification, chylomicron (CM) formation and lipoprotein metabolism. 29 This paragraph
will focus on dietary fat, whereas specific aspects of cholesterol absorption will be discussed in paragraph
2.2. Triglycerides (also called triacylglycerols; TG) form the major lipid component in the human diet.
Lipids are dispersed by chewing, which increases the surface area. In the stomach, partial hydrolysis by
gastric and lingual lipase takes place (10-30% of TG) and diacylglycerol and free fatty acids (free FA) are
released. 30,31 Pancreatic lipase facilitates further hydrolysis, producing monoacylglycerol and free FA. 32
PL (from diet, bile and sloughed intestinal epithelial cells) are hydrolyzed by phospholipase A2, yielding
lyso-PL and free FA. 33 The products of pancreatic lipolysis have limited solublility and need subsequent
solubilization by BS and/or association with PL. 34,35
Adjacent to the luminal surface of enterocytes, micellar dissociation is promoted by decreased pH (5.3-
6.0) 36 in the so-called unstirred water layer 37, allowing for diffusion of FA across the cellular membrane.
FA can also be taken up by transporters such as FA transporter protein 4 (FATP4) and FA translocase (FAT/
CD36), but none of these transporters were shown to be critical for FA absorption. 38,39
In the enterocyte, FA may bind to the intestinal FA binding protein (IFABP) and diffuse into the
endoplasmatic reticulum. After activation acyl-CoAs are produced and re-esterified into TGs via
several steps. 40 Microsomal TG transfer protein (MTP) assembles TG, PL and cholesterol together with
apolipoprotein B48 (ApoB48) to form a CM particle or with ApoB100 (not in humans) to form a very low
density lipoprotein (VLDL) particle, both of which are secreted to the interstitium via the basolateral
membrane. 41 From the intestitium the particles enter lymphatic capillaries that drain into omental
lymphatic channels and eventually reach the systemic circulation via the thoracic duct. 40
Chapter 1
14
2.2 Cholesterol absorption
Cholesterol present in the intestinal lumen derives from several sources, including diet, bile, intestinal
secretion and desquamated epithelial cells. In humans consuming Western type diets, 300-500 mg
dietary cholesterol enters the intestinal lumen per day, whereas the contribution of biliary cholesterol
has been estimated to be approximately 800-1200 mg per day. 42 Cholesterol is a hydrophobic molecule,
and it intestinal absorption is facilitated via similar steps as described for FA (emulsification, hydrolysis of
dietary esterified cholesterol, micellar solubilization, and uptake within enterocytes). In healthy humans
normally approximately 50% of intestinal cholesterol is absorbed. 43 Micellar solubilization of cholesterol
is essential for cholesterol absorption. 44 The physical chemistry of biliary cholesterol may influence its
absorption in the intestine. Whereas micellar biliary cholesterol is readily available for absorption, dietary
cholesterol first has to be released from food oils or tissue membranes. Elegant isotope infusion studies
in rats, however, showed that only on high cholesterol diet dietary cholesterol is relatively malabsorbed
compared with biliary cholesterol.
On low and moderate cholesterol enriched diets micellized and non-micellized cholesterol appear
in lymph in similar amounts. 45 Biliary cholesterol is unesterified, whereas dietary cholesterol is partly
esterified. Dietary cholesterol esters (CE) thus must be hydrolyzed by pancreatic carboxyl ester lipase
(CEL) before cholesterol can be transported into enterocytes. Howles et al. showed that dietary esterified
cholesterol absorption was reduced by >60% in CEL-null mice, whereas absorption of free cholesterol
was normal. 46 Considering the low daily supply of intestinal CE 47, hydrolysis of CE by CEL may not be
critical for overall cholesterol absorption. CEL may however serve a function similar to phospholipase A2,
in providing sufficient hydrolysis of PL (mainly phosphatidylcholine (PC), which is required for adequate
micelle formation. For example, in rats drained of bile and pancreatic juice, administration of CEL
enhanced lymphatic recovery of cholesterol infused into the duodenum as a micellar, PC containing,
solution. 48 Moreover, CEL itself may be absorbed by enterocytes via endocytosis. 49 and the ceramidase
activity of CEL has been implicated in proper intracellular cholesterol trafficking and CM assembly. 50
In addition to cholesterol, a typical Western diet contains structurally similar phytosterols (including
plant sterols (~95%) and stanols (~5%)), in similar amounts as cholesterol, depending on the diet.
Plant sterols and stanols inhibit the absorption of cholesterol, however the mechanism(s) by which they
do so remain a matter of discussion (see paragraph 5.3.2). Uptake of cholesterol and plant sterols and
stanols is believed to be facilitated by the Niemann-Pick C1 Like 1 (NPC1L1) transporter 51, which is
located in the brush border membrane of enterocytes in the proximal (jejunum), but not the distal
(ileum) small intestine. Npc1l1 null mice showed 70% and 90% reduction in cholesterol and plant sterol/
stanol absorption, respectively, compared with control mice. 52,53
In addition, Npc1l1 null mice showed upregulation of intestinal and hepatic HMG-CoA synthase mRNA
and intestinal cholesterol synthesis. 53 In the proximal small intestine (and apical hepatocyte membrane),
NPC1L1 co-localizes with ABCG5 and ABCG8 (figure 1).
General Introduction
15
Chapter
1HDL
ApoA1
De novo synthesis
CM ApoB48
MTP
CE ACAT2
PS
Chol
LDL ApoB100
NPC1L1 ABCG5/G8
Chol PS Intestinal lumen
Lymph CM ApoB48
ABCA1 SR-‐B1 LDLR
A
LDL ApoB100
HDL ApoA1
De novo synthesis
IDL ApoB100
VLDL ApoB100 VLDL
Chol
ACAT2
MTP
CE
Apical membrane
Basolateral membrane
BSEP
BS
ABCG5/G8
NPC1L1
LDLR
ABCA1
ApoB100
SR-‐B1
LRP1 CR ApoE
B
Figure 1. Cholesterol transporters, converting enzymes and lipoproteins in liver and intestine. A: Cholesterol in enterocytes originates from absorption, synthesis and uptake from the circulation. Cholesterol and plant sterols/ stanols are absorbed from the intestinal lumen via NPC1L1 and secreted back to the lumen via ABCG5/G8. Intracellular cholesterol and plant sterols/ stanols can be esterifi ed by ACAT2, packaged into apoB48 containing CM by MTP, and secreted to the lymph. Alternatively, cholesterol can be secreted via ABCA1 in ApoA1 containing HDL particles. Uptake of cholesterol on the basolateral side occurs via SR-B1 (HDL-c) and LDLR (LDL-c), which recognizes ApoB100. B: The liver takes up HDL-c via SR-B1 and ApoE containing CR-c via LRP1 and the LDLR. In the hepatocyte ACAT2 esterifi es cholesterol and CE are reassembled into VLDL particles together with CR-derived products. VLDL particles contain apoB100 instead of apoB48. ApoB100 binding to MTP results in loading of lipids to the VLDL particle. The liver secretes the VLDL particles into the circulation for delivery of lipids to the periphery. VLDL is in part cleared by the hepatic LDLR, whereas the rest is transformed to IDL and LDL, which still contain apoB100 necessary for reuptake in the hepatocyte via the LDLR. The liver secretes cholesterol to bile via ABCG5/G8 and transforms cholesterol to BS, which are secreted via BSEP. Hepatic NPC1L1 can facilitate reuptake of biliary cholesterol. Abbreviations: ACAT2: acyl CoA:cholesterol acyltransferase 2, CM: chylomicrons, CR: chylomicron remnants, LRP1: LDLR-related protein 1, MTP: microsomal triglyceride transfer protein, CE: cholesteryl esters, BS: bile salts, BSEP: bile salt export pump.
Effl ux of unesterifi ed cholesterol and plant sterols and stanols from the enterocyte back to the intestinal
lumen, as well as biliary cholesterol secretion are facilitated by ABCG5/G8. 54,55 Intracellular cholesterol
that is not effl uxed by ABCG5/G8 travels to the endoplasmatic reticulum, where it is esterifi ed by acyl
CoA:cholesterol acyltransferase 2 (ACAT2). 56 When LXR is activated, it forms a heterodimer with the
Retinoid X receptor (RXR) and the complex induces transcription of target genes encoding proteins
involved in cholesterol disposal, including the two half-transporters ATP-binding cassette sub-family G
member 5 and 8 (Abcg5/ Abcg8). 57 Transcription of Npc1l1 on the other hand is downregulated upon
LXR activation. This likely repesents an indirect eff ect, since LXR is not known as a direct repressor. 24
Intracellular cholesterol is partly re-esterifi ed and fi nally delivered to the bloodstream, mainly as
component of CM, together with TG, PL and apoB48, for transport to the lymph. MTP regulates this
process, and also facilitates VLDL formation after cholesterol esterifi cation by ACAT2 in hepatocytes. 58
Chapter 1
16
In addition, enterocytic cholesterol can be transferred to apoAI high density lipoprotein (HDL) particles
via ABCA1 (basolaterally located in enterocytes; figure 1). 59
Up to now, only NPC1l1 seems to be critical for apical intestinal cholesterol import. 52,53 Studies, in which
proposed alternative candidates, such as scavenger receptor class B member 1 (SR-B1), CD36 and
caveolin-1, were eliminated, showed that none of these proteins was crucial for cholesterol uptake. 60-64
ABCA1 was implicated in cholesterol absorption in the past. However, unchanged fecal sterol excretion
in Abca1-/- mice indicated that ABCA1 plays no role in control of cholesterol absorption. 65
In vitro studies suggested a possible role for aminopeptidase N (CD13) in cholesterol absorption 66 which
has not yet been confirmed in in vivo studies. In contrast to ABCA1, ACAT2 and MTP do affect cholesterol
absorption (paragraph 5.3).
Cholesterol homeostasis
Disturbances in cholesterol homeostasis are associated with potential life-threatening consequences.
Hypercholesterolemia promotes atherosclerosis and thereby represents a major risk factor for
cardiovascular disease. 67,68 Pharmacological inhibition of cholesterol synthesis has been the most potent
treatment option for hypercholesterolemia during the last decades. Cholesterol synthesis inhibition by
itself however, can reduce the risk of cardiovascular disease by only a third. 69 This underlies the ongoing
search for alternative therapeutic modalities.
The liver has been considered the major site of control in maintenance of cholesterol homeostasis. 70 The
liver facilitates clearance of VLDL/ LDL particles and cholesterol-containing CM remnants, synthesizes
cholesterol, synthesizes and secretes (nascent) HDL particles, secretes cholesterol and BS to bile and is
involved in reverse cholesterol transport (RCT). 7
RCT is classically defined as the process by which cholesterol from peripheral tissues is transported to
the liver, followed by excretion via bile to feces in the form of neutral sterols and BS. In recent years,
however, the importance of the intestine in many aspects of cholesterol physiology is increasingly
recognized. The intestine has a major impact on cholesterol homeostasis at the level of cholesterol (re-)
absorption, fecal excretion and de novo synthesis. 71 It has become apparent that, at least in mice, direct
secretion of cholesterol from the blood compartment into the intestine, i.e. TICE, plays a major role in
disposal of cholesterol via the feces. 72
It would be desirable to induce fecal cholesterol loss via the intestine without increasing biliary
cholesterol secretion and thereby the risk of cholesterol gallstones. 73
3. Transport of cholesterol through plasma
A plethora of epidemiological studies have unequivocally shown that increased plasma cholesterol
levels are associated with cardiovascular disease risk. Interestingly this does not necessarily coincide
with increased tissue cholesterol, but is probably caused by changes in rates of secretion and uptake of
cholesterol. 74 Cholesterol is a lipophilic molecule which is transported through blood in lipoproteins.
General Introduction
17
Chapter
1The type of lipoprotein is determined by its buoyant density and apoprotein composition, which act as
emulsifying coating and target their metabolism. 75
There are marked differences in lipoprotein metabolism between humans and rodents. For example,
mice do not possess cholesterol-ester transport protein (CETP) and have an up to 40-fold higher LDL
clearance by the liver compared to humans. 70,76 Mice carry most of their plasma cholesterol in HDL
particles and as such are not a good model for human disease. 70,76
This is the reason why many studies on cholesterol metabolism have been performed in mice with
genetic deficiency of major determinators of plasma cholesterol metabolism such as the Ldl receptor
(Ldlr) 77 or ApoE 78. More recently “humanized” mice have become available in which human CETP 79 is expressed. On an Ldlr null or ApoE3 Leiden background these mice mimic many aspects of the
hyperlipidemic human phenotype. 80,81 Inhibition of CETP increases HDL and should ameliorate
atherosclerosis, but clinical trials with the first CETP inhibitor (torcetrapib) were terminated because of
adverse off-target effects (increased mortality and cardiovascular events).
Recently, a new CETP-related drug (dalcetrapib) showed lack of a clinically meaningful benefit in a clinical
trial, and further testing of the drug has been halted.82 Until now, inhibition of cholesterol synthesis has
remained the first line of therapy for hypercholesterolemia.
4. Cholesterol synthesis
4.1 Control of cholesterol synthesis
Cholesterol is synthesized from its precursor unit acetyl-CoA via a complex metabolic pathway
summarized in figure 2. 83,84 HMG-CoA reductase is the rate-limiting enzyme in cholesterol synthesis 85.
Recently squalene monooxygenase, which catalyzes the first oxygenation step in cholesterol synthesis,
was suggested to represent a possible second control point in cholesterol synthesis beyond HMG-
CoA reductase. 86 Cholesterol and fat biosynthesis are under control of a family of transcription factors
designated Sterol Regulatory Element Binding Proteins (SREBPs). Three isoforms of SREBP have been
described, i.e., SREBP1a, SREBP1c and SREBP2. Genes encoding enzymes and tranporters involved in
cholesterol absorption and efflux are under tight transcriptional control (reviewed recently by 57).
Cholesterol and its biologically active metabolites act as ligands for NR that regulate gene expression.
LXR is a major regulator of cholesterol metabolism. 57 When sterols are present in excess intracellularly,
LXR-mediated activation of SREBP1c transcription leads to induction of oleic acid synthesis. Oleic acid
is the preferred FA for the synthesis of cholesterol esters (CE), which can be stored intercellularly. LXR
activation thus protects cells from accumulation of excess free cholesterol, which is toxic and can result
in cell death.
Chapter 1
18
Figure 2. Cholesterol biosynthesis pathway. Cholesterol is synthesized from its precursor unit acetyl-CoA (Ac-CoA). Two acetyl-CoAs are condensed, forming acetoacetyl-CoA (AcAc-CoA). AcAc-CoA and a third acetyl-CoA are converted to 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) by the action of HMG-CoA synthase. HMG-CoA is converted to mevalonate by HMG-CoA reductase. Mevalonate is subsequently converted to an isoprenoid molecule, isopentenyl pyrophosphate (PP), with the concomitant loss of CO
2. Geranyl-PP and farnesyl-PP are produced from
isopentenyl-PP. Two farnesyl-PP subunits are combined to form squalene. Squalene is converted to lanosterol and subsequently cholesterol via many intermediates, including zymosterol, desmosterol and lathosterol. Solid line: direct step. Dashed line: product is formed via intermediate steps.
Cholesterol synthesis is tightly regulated by SREBPs (mainly type 2). When cellular cholesterol is high,
SREBP2 is located to the ER in a complex with SREBP2 cleavage–activating protein (SCAP). When cells
are depleted of sterols, SCAP escorts SREBP2 from the ER to the Golgi apparatus, where it is cleaved
in order to release part of the protein from the membrane. SREBP2 then can enter the nucleus, bind
to a sterol response element (SRE) in the enhancer/ promoter region of many target genes involved
in cholesterol synthesis, and activate their transcription. 87 The importance of cholesterol synthesis for
survival is illustrated by the fact that defects in the cholesterol synthesis pathway are generally lethal in
mice. Complete loss of function of early cholesterogenic enzymes is rarely described in humans and
deficiencies of these enzymes lead to severe malformations and disease.88
4.2 Measurement of cholesterol synthesis in vivo
The different methods available to determine cholesterol synthesis in vivo include sterol balance,
(plasma) cholesterol precursor measurement and tracer incorporation techniques [such as deuterium
incorporation (DI) and mass isotopomer distribution analysis (MIDA)], reviewed in ref. 89.
General Introduction
19
Chapter
1An early approach to estimate cholesterol synthesis has been cholesterol balance: measurement of
cholesterol and BS excretion in feces followed by subtraction of dietary cholesterol intake yields whole
body synthesis. Surrogate serum markers of cholesterol synthesis (and absorption) will be discussed
in section 3. Here we will briefly discuss the two more recently developed and refined methods:
DI and MIDA. The DI (2H) method has a similar background as the tritiated (3H) water method used
in experimental animals. The theory behind the method is described below. Deuterated water can
equilibrate in total body water and NADPH. Enrichment of deuterium in plasma water, representing
the precursor pool, and deuterium enrichment of cholesterol in either plasma or red blood cells can be
determined sensitively by isotope ratio mass spectrometry.
Eighteen acetyl CoA units containing 36 carbon (C) atoms are utilized to synthesize one molecule of
cholesterol, which contains 27 C atoms and 46 hydrogen (H) atoms. During synthesis of a cholesterol
molecule, 7 H atoms are incorporated originating directly from water, while another 15 H atoms are
inserted from nicotinamide adenine dinucleotide phosphate-oxidase (NADPH).
H atoms from water may also become incorporated into substrates that, later on, are used for the
generation of the cytosolic acetyl CoA pool that is used for cholesterol synthesis. If no labeled H from 2H
water were incorporated into these precursors of the cytosolic acetyl CoA pool, and if the reductive H of
the NADPH were derived entirely from unlabeled sources, only 7 labeled H atoms (those orginating from
the administered 2H water) could be found in each newly synthesized cholesterol molecule (minimal
theoretical labeled H/C incorporation ratio would be 7/27). Alternatively, if NADPH fully equilibrates with 2H labeled water, then a final labeled H/C incorporation ratio of 22/27 would be found. This is considered
the maximal labeled H/C ratio in short term measurements. 90 On the long run, an even greater value
might be obtained when there is significant incorporation of labeled H into an important acetyl-CoA
precursor, which by then has been used for cholesterol synthesis.
On a theoretical basis it is difficult to predict exactly how many labeled H atoms will be incorporated
into each cholesterol molecule. Labeled H/C ratios are not available for all organs. In short term in
vivo studies in mice and rats, 21-25 tritiated H atoms were incorporated into cholesterol molecules of
whole carcass, liver or brain per C atom entering the biosynthetic pathway as acetyl CoA 90 and these
values were subsequently used in other studies to calculate absolute cholesterol synthesis. However,
combining the DI method with MIDA (discussed below) in long term experiments (up to 8 weeks), the
maximum incorporation number in rat liver was found to be 30. 91 This could mean that earlier studies
have overestimated true cholesterol synthesis rates. Many human studies using the DI method have
used H incorporation values obtained from literature 92 and thus provide rough estimates of absolute
cholesterol synthesis rates.
With MIDA the precursor-pool enrichment, fractional synthesis, and absolute (whole body) synthesis
of cholesterol are calculated based on the pattern of excess enrichment among mass isotopomers of
cholesterol present in plasma after administration of stable isotope-labeled 13C-acetate. 93
Chapter 1
20
The distribution of abundances in newly formed cholesterol molecules, measured by gas-
chromatography-mass spectrometry, allows calculation of the abundance in the precursor. MIDA thus
eliminates the need to measure the precursor pool enrichment directly.
Knowing the true precursor-pool enrichment allows calculation of the fractional and absolute cholesterol
synthesis rates. MIDA reveals the weighted mean abundance of the precursor pools contributing to the
product mixture. 94
In the past, human as well as animal studies applying MIDA were performed in relatively short time
frames, which could underestimate whole body synthesis due to circadian effects and/or incomplete
equilibration of the free cholesterol pool. A human study showed that the DI method and MIDA yield
similar rates of fractional and absolute cholesterol synthesis when measured over at least 24h. 95
4.3 The body cholesterol pools
A complication when assessing de novo cholesterol synthesis is the existence of different pools of
cholesterol in the body with a differential rate of exchange. To incorporate this aspect, the turnover
of plasma cholesterol has been modeled by dividing total body cholesterol into 2 or 3 exchangeable
pools. 92,93
In the 3-pool model, pool 1 (rapidly miscible) is considered to consist of cholesterol in fairly rapid
equilibrium with plasma cholesterol (plasma, blood cells, liver and intestines), pool 2 consists of
cholesterol that equilibrates at an intermediate rate (visceral and peripheral tissues) and pool 3
represents the slowest cholesterol turnover compartment (adipose and connective tissue, skeletal
muscle and arterial walls).
The 3-pool model has been used for long term studies (up to 48 weeks). 92 Differentiation between pools
2 and 3 in short term experiments appeared not essential for accurate mathematical modeling. Pool 1 in
the 2-pool model represents the rapidly exchangeable pool similar to that in the 3-pool model, whereas
pool 2 of the 2-pool model represents a combination of pool 2 and 3 of the 3-pool model. 93
The central nervous system contains a major part of total body cholesterol (15% and 23% in mice and
humans, respectively). There is no detectable uptake of plasma cholesterol into the brain via the blood-
brain-barrier. 96 The central nervous system presumably excretes only a very small amount of cholesterol
to pool 3 under physiological conditions. Thus, it must be kept in mind that brain synthesis is not taken
into account when calculating whole body cholesterol synthesis.
In general, whole body cholesterol synthesis rates measured using either the DI method or MIDA were
shown to be comparable with those reported with the classical cholesterol balance (cholesterol intake
+ synthesis = fecal excretion of BS + neutral sterols). 93,97
4.4 Regional and whole body cholesterol synthesis
Virtually every mammalian cell synthesizes cholesterol, in most animals the main part being synthesized
in extrahepatic organs. 70,98,99 Whole body synthesis and the contribution of liver and intestine to whole
body synthesis have been determined for several species.
General Introduction
21
Chapter
1A complication in evaluating the value of these measurements is the circadian rhythm of cholesterol
synthesis, which has not always been taken into account (see below). It seems justified to conclude
that species such as hamsters, guinea pigs, rabbits and squirrel monkeys and humans have much lower
synthesis rates than rats and mice.
The gastrointestinal tract contributes around 15-35% to total cholesterol synthesis in experimental
animals. In rodents, the contribution of the liver to whole body synthesis at night has been shown to
vary from as low as ~15-20% in rabbits and guinea pigs to as high as 50% in rats. 70 In humans the liver
is thought to contribute only around 10% to whole body synthesis 70 (~10 mg/kg/d). Low rates of local
synthesis relative to the rates of uptake of newly synthesized cholesterol from blood in rats were found
in adrenal glands, spleen, lung and kidneys. These organs increase their cholesterol synthesis when
circulating levels of plasma cholesterol are decreased. 99
4.5 Influence of diet and time of day on cholesterol synthesis
Whole body as well as organ specific cholesterol synthesis rates vary depending on the presence of
cholesterol and other lipids in the diet. Dietary cholesterol suppresses hepatic cholesterol synthesis. In
rats fed no dietary fat, liver synthesis rates are decreased when feeding a 2% cholesterol containing diet,
while synthesis rates in the intestine and other extrahepatic tissue remain similar, suggesting that only
the liver senses the increase in cholesterol uptake. 100 Adding cholesterol to either a low or high fat diet
also leads to decreased hepatic cholesterol synthesis in hamsters. 101
It appears that dietary fat alone however does not affect hepatic and whole body cholesterol synthesis,
but can induce intestinal cholesterol synthesis. This was illustrated by several studies. First of all, on a high
fat compared with low fat diet, hepatic cholesterol synthesis is comparable in hamsters. 101 In addition,
it was shown in mice on a 0.2% cholesterol diet that dietary short, medium and long chain fatty acids
did not differentially affect extrahepatic cholesterol synthesis. 102 Infusion of corn oil in non-cholesterol,
non-fat fed rats increases cholesterol synthesis in the intestine, while liver synthesis remains the same. 100
A high fat diet in hamsters also increases cholesterol synthesis in the intestine in the presence of dietary
cholesterol, however whole body synthesis is not affected 103.
In addition to dietary fat and cholesterol content, the type of diet influences cholesterol synthesis
rates. These were shown to be lower on purified diets as compared to non-purified diets. 104,105 Energy
restriction per se seems to have the greatest (lowering) effect on cholesterol synthesis (reviewed by 89).
Both animals and humans display a circadian rhythm of cholesterol synthesis, with a peak in synthesis
several hours after feeding. Most studies on cholesterol turnover in experimental animals have been
performed during the dark phase of the light cycle. 106 It must be kept in mind that cholesterol synthesis
is 2-3 fold higher during the (end of the) dark phase compared to the light phase. 93,107,108 In extrahepatic
tissues the difference between day and night in cholesterol synthesis is much smaller compared with
the liver, at least in mice. 107 Circadian rhythm in the liver is controlled by different molecular mechanisms,
including cAMP-dependent and CLOCK/ BMAL1 regulation, which for example regulate expression of
cholesterol synthesis genes Hmgcr, Hmgcs, Sqs, Fpps and Cyp51. For details, see ref. 109-111.
Chapter 1
22
4.6 Targeting cholesterol synthesis in hypercholesterolemia
HMG-CoA reductase inhibitors (statins) have been used successfully to inhibit cholesterol synthesis and
reduce all-cause mortality since the late 1980’s. 112 Reduced hepatic cholesterol is compensated for by
synthesis of LDLR to draw cholesterol out of the circulation 113 leading to lowering of plasma cholesterol
levels. Some statins (pravastatin) seem to preferentially inhibit synthesis in liver and intestine (90%) 114,115 compared with synthesis in extrahepatic organs, such as kidneys (73%), testes (55%), lungs (53%),
spleen (53%) and adrenals (49%). 114 Other statins (lovastatin) exhibit widespread cholesterol synthesis
inhibition. 116 This may be mainly due to differences in cellular uptake 115, which can be caused by
differences in lipophilicity, pH-sensitivity, plasma protein binding, activity of metabolites and transporter
facilitated uptake and export. For example, simvastatin and lovastatin are administered as very lipophilic
(tissue penetrating) lactone prodrugs, whereas other statins are administered in their active form. 117
Different statins have different affinities for import proteins of hepatocytes such as organic anion
transporting polypeptides (OATP) and NTCP. 118-121 In addition, polymorphisms in these transporters
differentially affect statin uptake and (in vitro) affinity for transporters can differ between human and
experimental animal cells. 121
Although highly effective, statins do not produce the desired health effect in a significant group of
patients and can cause severe side effects such as myalgia and myopathies (rhabdomyolysis), 122 in
particular in case of polypharmacy. Statins decrease farnesyl and geranyl pyrophosphate (isoprenoids)
and dolichol synthesis, reducing essential post-translational prenylation and N-linked glycosylation of
proteins. This may result in myocyte and hepatocyte cell damage and even cell death. 123,124 Newer statins,
such as the recently approved pitavastatin, with a distinctive metabolic profile, may display a reduced
incidence of these adverse effects. 125 In addition, efforts are ongoing to produce new drugs targeting
cholesterol synthesis at different levels of the pathway. Squalene synthase inhibitors for example
increase farnesyl diphosphate while decreasing cholesterol synthesis. They are able to prevent statin
induced changes in protein farnesylation and cell death in vitro. 124 Since these inhibitors do not target
true rate-limiting steps in cholesterol synthesis, they may not effectively reduce cholesterol synthesis as
a monotherapy in tolerable dosages.
Variations in efficacy of statins can be caused by many factors, including race (genetics), body weight
and diet. Humans that inherently have lower cholesterol synthesis rates may respond less adequate to
statin treatment. LDL-cholesterol (LDL-c) response to statins is lower in black compared to white people,
which is associated with lower baseline LDL-c and variant haplotypes of the HMG-CoA reductase gene
in black people. 126 Statins may induce expression of proprotein convertase subtilisin-like kexin type
9 (PCSK9) and SREBP2. PCSK9 is a circulating protein that impairs LDL clearance by promoting LDLR
degradation. PCSK9 thus could diminish statin efficacy, hence PCSK9 inhibitors are currently tested in
clinical trials. 127
In general, cholesterol synthesis inhibition leads to a reciprocal increase in cholesterol absorption, 128
which has led to elaborate research to develop cholesterol absorption inhibitors (discussed below). With
growing knowledge on the determinants of response to cholesterol lowering therapy, in the future it
may become feasible to individually tailor treatment.
General Introduction
23
Chapter
15. Measurement and inhibition of cholesterol absorption
5.1 Measurement of cholesterol absorption in vivo
The available methods to measure cholesterol absorption in humans and mice include (radiolabeled or
stable) isotope based methods (fecal and plasma dual isotope method), intestinal perfusion and blood
levels of surrogate markers such as plant sterols and intermediates of cholesterol synthesis. Details can
be found in several reviews. 129,130 The intestinal perfusion method is the only method that can directly
quantitate absorption of intestinal cholesterol in humans. 131,132 Widespread use of this method is limited
by the need for intubation and radiation exposure.
The dual isotope ratio method has been optimized using stable instead of radioactive isotopes both in
the plasma (ratio of intravenously and orally administered labeled cholesterol) 133 and fecal (ratio of orally
administered labeled cholesterol and sitostanol in feces) dual isotope ratio method. 134 The fecal dual
isotope ratio method is limited by requirement of 72h feces collection. The plasma dual isotope ratio
method has been limited by measurement of single time point absorption, however this method can
nowadays be applied for longer periods, at least in mice. 135
The only approach available to easily estimate (relative) changes in cholesterol absorption in large scale
studies is to measure plasma surrogate markers. These include the ratio of plant sterols (campesterol
and sitosterol) or cholestanol (cholesterol metabolite) to cholesterol in plasma. 136 Surrogate makers
have shown to be valuable to answer questions related to changes in cholesterol absorption during
treatments. However, dietary factors (fat, cholesterol, plant sterols and stanols 137, drugs (statins) 138 and
metabolic diseases (diabetes mellitus) 139,140 can disturb correct interpretation of surrogate markers levels.
For example, the reduction in plasma LDL-c correlates poorly with baseline levels of noncholesterol sterol
markers of absorption (campesterol) and synthesis (lathosterol) in African- and European-American men
treated with ezetimibe (a cholesterol absorption inhibitor, discussed in paragraph 3.3.1), simvastatin or
a combination of the two drugs. 141 Similarly, although ezetimibe + simvastatin treatment significantly
reduces campesterol and sitosterol levels in patients with familial hypercholesterolemia, baseline
cholesterol absorption status does not determine LDL-c lowering response to ezetimibe + simvastatin
therapy. 142 It is currently recommended to use several rather than one serum marker, if possible in
addition to absolute measurements. 143
5.2 Targeting cholesterol absorption in hypercholesterolemia
Statin therapy is not sufficient to prevent cardiovascular disease risk in a substantial proportion
of individuals. 144 To increase treatment efficacy considerable interest has arisen in dietary and
pharmacological interventions that inhibit cholesterol absorption, possibly for combining this with
inhibition of cholesterol synthesis . Most agents are nonspecific and require consumption in high daily
quantities while modestly lowering plasma LDL-c. ACAT2 deficiency resulted in cholesterol malabsorption
in mice, however only in cholesterol fed and not in chow fed mice. 145 MTP inhibition normalized plasma
lipoprotein levels in a rabbit model for human homozygous familial hypercholesterolemia. 146
Chapter 1
24
However, this process is related to disturbed CM formation, not specific for absorption of cholesterol
and not intestine specific as it induces fat accummulation in the liver. 146 We will not discuss inhibition
of these intracellular factors. Ezetimibe represents an exception, as it relatively specifically decreases
cholesterol absorption. The impact of dietary plant sterols and stanols is discussed since it has become
apparent very recently that they might have an unexpected intestinal effect (see section 6). Other
cholesterol lowering food components are discussed elsewhere 147
5.2.1 Ezetimibe
Specific inhibition of cholesterol absorption, either from biliary or dietary origin, is accomplished after
binding of ezetimibe to NPC1L1. 148 The compound is first glucuronidated in the intestine before
travelling to the liver via the portal vein. Glucuronidated ezetimibe blocks the internalization of the
NPC1l1/ cholesterol complex 149 but does not block cholesterol absorption completely.
In the Npc1l1 null mouse there is residual cholesterol absorption, indicating that another pathway of
cholesterol absorption must exist. Ezetimibe does not inhibit this residual cholesterol absorption in
Npc1l1 null mice, indicating that this residual pathway is ezetimibe independent. 52
Studies in experimental animals indicated that ezetimibe may decrease absorption of vitamin E
(α-tocopherol), but not vitamin A and D. 150,151 However, ezetimibe did not seem to affect fat soluble
vitamin status in patients. 152,153 Ezetimibe can decrease plasma TG concentration. This does not seem to
be absorption dependent, but rather a secondary effect of ezetimibe induced hepatic LDLR expression
and subsequent increased clearance of ApoB100 and ApoB48 containing lipoproteins, particularly
during co-treatment with statins. 154
In humans, ezetimibe alone decreases cholesterol absorption by 54% and decreases plasma LDL-c
by ~19%, but increases whole body synthesis by 89%. 43 Combined treatment with statins for
hypercholesterolemia, should therefore be very effective. 155 Recently however, the effect of combination
therapy has become controversial. Some trials reported no additive effect of ezetimibe or even a worse
atherosclerosis outcome when ezetimibe is added to patients previously treated with statins. 156 Others
report increased efficacy of combination therapy over statin monotherapy, however data are limited
to the level of serum lipid profiles. 157-160 Due to the lack of well-executed long term trials it is presently
unclear whether combination therapy is better than increasing statin dosage. In the future, ezetimibe
may be prescribed in patients in whom statin therapy is inadequate or intolerated. As monotherapy
ezetimibe may improve postprandial hyperlipidemia and endothelial dysfunction. 161 In the presence of
pancreatic dysfunction, CEL inhibitors may provide additional inhibition of cholesterol absorption when
co-administered with ezetimibe. 150
5.2.2 Plant sterol/ stanol supplementation
In contrast to cholesterol, plant sterols and stanols can not be synthesized by the body and are poorly
absorbed (plant sterols 0.4-3.5% and plant stanols 0.02-0.3%). 162 Fractional absorption of different plant
sterols and stanols varies depending on their side chain length. 163
General Introduction
25
Chapter
1The role of ABCG5/G8 in intestinal and hepatobiliary efflux of plant sterols and stanols versus cholesterol
is not completely clear. In vitro studies showed comparable effectiveness of plant sterol and cholesterol
transport via ABCG5/G8. 164 However, loss of ABCG5/G8 function in mice results in hyperabsorption of
plant sterols and stanols, but not cholesterol, implying that ABCG5/G8 is more important for efflux of
plant sterols and stanols compared to cholesterol. 165 In contrast to cholesterol, plant sterols and stanols
are poor substrates for ACAT2 166, which seems to be the major factor limiting their absorption. Plant
sterols and stanols effectively lower plasma LDL-c levels in adults as well as children with (familial)
hypercholesterolemia. 167
Plant sterols and stanols naturally occur in foods such as oils, (wheat) cereals, nuts and seeds. There are
large differences in plant sterol/stanol consumption throughout the world, within the Western population
and particularly between vegetarians and meat consumers. 168 Plant sterols/stanols are added to food
products such as margarine, yoghurt and juices. To enable emulsification, added plant sterols/tanols are
often esterified and require hydrolysis before being able to compete with cholesterol for absorption.
It was recently suggested that the hydrolysis products of esterified plant sterols/ stanols (stigmasterol
and especially saturated stearic acid) together may be more effective at lowering cholesterol micellar
solubility than free plant sterols/stanols alone. 169
The mechanism via which plant sterols and stanols lower plasma cholesterol does however not seem
to be confined to decreasing cholesterol solubilization in the intestinal lumen. A recent meta-analysis
showed that despite differences in absorption, plant sterols and plant stanols have similar effects on
plasma lipid profiles. 170 Smaller repeated doses (3 times 0.6 g/d) of plant sterol/ stanol margarine as well
as a single high daily dose (1.8 g/d) are equally effective at lowering cholesterol absorption and plasma
LDL-c. 171
Given that plant sterols and stanols are taken up in enterocytes, it was hypothesized that cholesterol
absorption may be decreased by plant sterols/ stanols via increased cholesterol secretion to the
intestinal lumen, for example via ABCG5/G8 under transcriptional control of the Liver X Receptor (LXR).
However, Abcg5/g8 and Lxr knock-out mice did not reveal any change in cholesterol absorption 172-174,
implying that others factors are involved.
The combination of plant sterols with ezetimibe did not show an additive effect on (surrogate markers
of ) cholesterol absorption and plasma LDL-c in mildly hypercholesterolemic subjects in an open-label
study which was not controlled for dietary plant sterol and stanol content. 175 A recent randomized,
double-blind, placebo-controlled, triple cross-over study on the other hand showed that plant sterols
and stanols further reduced cholesterol absorption (fecal dual isotope method) and further increased
fecal sterol excretion compared with ezetimibe alone in mildly hypercholesterolemic subjects 176,177. This
study showed that the combination of statin and plant sterols and stanols exerted a minor additional
effect on plasma LDL-c levels. Further studies in severe hypercholesterolemic patients may provide
additional information on the usefulness of plant sterol/ stanol-ezetimibe combination therapy.
Chapter 1
26
An adverse effect of plant sterol and stanol consumption could be disturbed micellar incorporation
and absorption of fat-soluble vitamins.178,179 The long term potential adverse effects of plant sterols and
stanols, such as increased atherogenesis and tissue (liver, brain) accumulation of plant sterols and stanols
may seem negligible, but await further study. 168,180
6. Cholesterol excretion
6.1 Classical Reverse Cholesterol Transport (RCT)
The body can dispose itself from cholesterol predominantly in two ways: as neutral sterols (cholesterol
and its intestinal bacterial degradation metabolites) or as acidic sterols (BS). Cholesterol and BS are
excreted mostly with feces and only minimal amounts of cholesterol are additionally lost via the skin. The
classic view of RCT includes the flux of cholesterol from peripheral tissues to the liver mediated mainly
by HDL particles, and the subsequent secretion of this cholesterol by the liver in bile that is transported
to the intestinal lumen, leading to fecal excretion of cholesterol.
Hepatobiliary RCT for decades has been considered the main, if not the only, pathway for cholesterol
elimination. We will briefly discuss this pathway and then turn to other, more recently identified
pathway involved in cholesterol excretion.The liver plays an important role in cholesterol homeostasis
by regulating uptake of lipoproteins, cholesterol synthesis and cholesterol secretion (figure 1). Uptake of
cholesterol is facilitated primarily via basolateral LDLR (LDL and VLDL uptake) and SR-B1 (HDL uptake).
Cholesterol is basolaterally secreted from hepatocytes to the plasma compartment in VLDL and HDL
particles and apically from hepatocytes to bile either directly as free cholesterol (via ABCG5/G8) or after
conversion to BS (via the BS export pump (BSEP or ABCB11) 181 Biliary BS secretion is the main driving
force for secretion of cholesterol and PL. However, studies in mice in which the PL transporter multi-
drug resistance P-glycoprotein (Mdr2 or Abcb4) was eliminated 182, showed that PL secretion itself is also
required for cholesterol secretion.
Cholesterol as well as plant sterols and stanols are secreted into bile mainly via ABCG5/G8.
Recent studies of mice with altered hepatic expression of Niemann-Pick C2 (NPC2, a cholesterol-binding
protein that is involved in intracellular cholesterol trafficking in hepatocytes, but can also be secreted
to bile 183) revealed that NPC2 may positively regulate the biliary secretion of cholesterol, which was
supported by the correlation between levels of NPC2 protein and cholesterol in human bile. Secreted
NPC2 appears to specifically stimulate ABCG5/G8-mediated cholesterol efflux. It was suggested that
NPC2 binds cholesterol and thereby accelerates the transfer of cholesterol to micelles via ABCG5/G8. 183
In recent years it has become clear that the definition of RCT needs revision. 184 Elimination of cholesterol
via feces, at least in mice, mainly occurs not via the classical hepatobiliary route, but via an alternative
pathway, which is adopted Trans Intestinal Cholesterol Excretion (TICE).
General Introduction
27
Chapter
16.2 Transintestinal Cholesterol Excretion
6.2.1 The concept of TICE
The TICE concept has been reviewed recently. 8,73,184,185 Here we will briefly describe the pathway
and latest insights in its dynamics. It was shown in several mouse models with extremely low biliary
cholesterol secretion rates (inactivation of Abcg5, Abcg8 or Abcg5/g8 and Mdr2 or overexpression of
hepatic Npc1l1, see figure 3), that fecal neutral sterol excretion was unchanged or even increased 186-190.
This could not be explained by increased fecal loss of newly synthesized intestinal cholesterol. 191 At a
stable dietary cholesterol intake, this suggested that cholesterol could be excreted directly from blood
to feces via the intestinal mucosa.
Figure 3. Schematic presentation of cholesterol input and excretion in several mouse models. Mice deficient in Abcg8 or Mdr2 have extremely low biliary cholesterol secretion rates, however fecal neutral sterol excretion is relatively similar (Abcg8-/-) or even increased (Mdr2-/-). Similarly, in wildtype mice treated with an LXR-agonist the sum of dietary and biliary cholesterol secretion is outleveled by fecal cholesterol excretion. These models provided the first evidence for the existence of transintestinal cholesterol excretion in mice.
Definitive indications for the existence of TICE were obtained in intestinal perfusion studies in which the
bile duct was ligated and the absence of biliary components was compensated for by infusion of several
(cholesterol free) model bile solutions. Van der Velde et al. demonstrated that TICE occurs in the entire
small intestine, but particularly in the proximal part and plays quantitatively a more prominent role than
the hepatobiliary route in mice. Importantly, intestinal cell shedding and synthesis could not account for
the secreted cholesterol. 187,191 Brown et al. showed that mice with a targeted deletion of hepatic ACAT2
have normal biliary cholesterol secretion rates, in the presence of doubled fecal cholesterol excretion. The
authors determined the fate of newly secreted liver-derived cholesterol. For this purpose, hepatic sterol
pools were radiolabeled and nascent hepatic cholesterol-labeled lipoproteins were collected by isolated
liver perfusion. The liver-derived lipoproteins were then re-injected intravenously into hepatic ACAT2
deficient and control mice to examine the movement of liver-derived radiolabeled cholesterol. It was
found that radiolabeled cholesterol from perfusate of hepatic ACAT2 deficient mice was preferentially
delivered to the proximal small intestine of wild type mice. 192
Chapter 1
28
A similar phenomenon probably occurs in mice deficient in the rate-limiting enzyme for BS synthesis
(cholesterol 7α-hydroxylase, Cyp7a1). Cyp7a1 null mice have reduced BS synthesis and excretion and
a smaller intestinal BS pool, which is associated with a major impairment in cholesterol absorption.
Compared to wildtype mice, they have the same dietary intake and biliary cholesterol level. Nevertheless,
fecal neutral sterol excretion greatly exceeds the sum of total dietary and estimated biliary cholesterol
input to the intestine in these mice. 193
Our lab contributed to the evidence by quantification of in vivo cholesterol fluxes from plasma and bile
to feces by stable isotope methodology. 191 In these experiments, differentially stable isotope labeled
cholesterol was administered to mice intravenously and orally to determine cholesterol absorption and
label distribution over time in blood, bile and feces. In addition, cholesterol synthesis was measured
by MIDA of blood, bile and fecal samples collected over time during administration of stable isotope
labeled cholesterol precursor in drinking water. 191
6.2.2 Transport of cholesterol from blood to feces
The transport of cholesterol from the blood compartment to the enterocyte and the subsequent
excretion of cholesterol is probably facilitated by transport proteins. Sr-b1, being expressed on both
the apical and basolateral side of enterocytes, was considered a possible candidate. However TICE was
shown to be upregulated, rather than downregulated, in Sr-b1 knockout mice. 194 TICE was not altered by
the absence of either Npc1l1 or Ldlr. 192,195 The relation between TICE and cholesterol efflux via Abcg5/g8
is somewhat puzzling 135,185 Compared with wildtype mice, TICE was decreased in Abcg5 knockout mice
during LXR agonist treatment and plant sterol/stanol consumption. 135,191. Strikingly however, TICE was
not decreased by deletion of Abcg8 in mice. 187
By gene expression analyses, so far no apical or basolateral transporters have been identified that
regulate TICE. 8 At present it is unclear which lipoproteins are involved in donating the cholesterol to
the TICE pathway and how it is targeted to the enterocytes. TICE does not seem to be mediated by HDL
particles, based on unchanged fecal cholesterol excretion in mice deficient in HDL (Abca1 knockout
mice). 65 Some evidence points to VLDL as a possible candidate origin for cholesterol involved in TICE. 192
Mice with liver specific depletion of Acat2 showed normal HDL levels, but increased delivery of liver-
derived cholesterol to the lumen of the proximal small intestine as well as increased fecal cholesterol
excretion. 65,192 Alternatively, TICE may not be facilitated via lipoprotein transport, but for example via
increased delivery of erythrocyte cholesterol to the intestinal lumen. Additional research is required to
identify key players in the pathway.
6.2.3 Stimulation of TICE
It is currently unclear whether or not ezetimibe, via inhibition of cholesterol absorption, could stimulate
TICE. One study suggested pharmacological activation of TICE with ezetimibe. 196 However, this was not
confirmed in intestinal perfusion studies 195 indicating that several pathways may co-exist in TICE.
General Introduction
29
Chapter
1TICE can be stimulated with pharmaceuticals such as nuclear receptor agonists of LXR 189,191 and
peroxisome proliferator-activated receptor delta (PPARδ). 195 A high fat (low cholesterol) diet also induces
TICE. 194,197
Dietary plant sterols were shown to inhibit cholesterol absorption in mice. Brufau et al. recently found
that dietary plant sterols/ stanols dose dependently induce fecal neutral sterol excretion, without
affecting biliary cholesterol secretion. These data provide new insight into the cholesterol lowering
mechanism of action of plant sterols/ stanols. 135
A recent study showed that mice with acute biliary diversion are able to secrete labeled cholesterol
derived from intraperitoneally injected cholesterol-loaded macrophages to the intestinal lumen. 198
This study adds to the evidence that TICE may be quantitatively more important than hepatobiliary
cholesterol secretion in the elimination of cholesterol via fecal excretion (RCT).
6.2.4 TICE in humans?
Although the available evidence suggests that TICE may be present in humans 199,200, it is at present
unclear if TICE is present under healthy conditions and whether it can be stimulated pharmacologically
or, preferentially, by dietary means. It has been estimated that TICE may contribute to one-third of fecal
excretion in humans. 187 The possibility of TICE has never been tested in models for predicting plasma
cholesterol. In the future measurement of body cholesterol kinetics by stable isotope studies in humans
may provide new insight into the excretion and regulation of plasma cholesterol, and the dietary and
therapeutic strategies to decrease hypercholesterolemia and associated cardiovascular disease in man.
Scope of this thesis
The specific aim of the research described in this thesis was to determine the effect of manipulation
of intestinal function (by dietary, pharmacological and genetic intervention) on lipid homeostasis,
in particular cholesterol excretion. We hypothesized that acceleration of intestinal transit time could
induce fecal lipid excretion. This could be desirable in case of hypercholesterolemia. On the other hand,
many children nowadays suffer from constipation and are treated with oral laxatives. Administration of
these laxatives could negatively impact on their intestinal absorbtive function and lead to fecal lipid loss.
In chapter 2, we describe the effect of acceleration of intestinal transit on absorption of dietary fat
and cholesterol. In chapter 3, we studied the effect of accelerated intestinal transit on BS homeostasis
and intestinal microbiota. Changes in the composition of dietary fat have been shown to alter fecal fat
excretion. For example, lowering the ratio of polyunsaturated to saturated fatty acids (P/S ratio) was
shown to induce fecal fat excretion.In chapter 4, we studied the effect of two high fat diets with different
P/S ratios on cholesterol homeostasis. We used stable isotope methodologies to determine cholesterol
absorption, synthesis and we modified the method previously used 191 to determine the origin of fecal
sterols. Bsep-/- mice were previously shown to display defective biliary BS secretion. However, this was by
far not as prominent as in their human couterparts. 15 In chapter 5, we studied the effect of absence of
hepatic Bsep in mice on cholesterol homeostasis.
Chapter 1
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164. Wang J, Sun F, Zhang DW, et al. Sterol transfer by ABCG5 and ABCG8: in vitro assay and reconstitution. J Biol Chem. 2006 Sep;281(38):27894-904.
165. Yu L, Hammer RE, Li-Hawkins J, et al. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion. Proc Natl Acad Sci U S A. 2002 Dec;99(25):16237-42.
166. Temel RE, Gebre AK, Parks JS, et al. Compared with Acyl-CoA:cholesterol O-acyltransferase (ACAT) 1 and lecithin:cholesterol acyltransferase, ACAT2 displays the greatest capacity to differentiate cholesterol from sitosterol. J Biol Chem. 2003 Nov;278(48):47594-601.
167. Guardamagna O, Abello F, Baracco V, et al. Primary hyperlipidemias in children: effect of plant sterol supplementation on plasma lipids and markers of cholesterol synthesis and absorption. Acta Diabetol. 2011 Jun;48(2):127-33.
168. Marangoni F, Poli A. Phytosterols and cardiovascular health. Pharmacol Res. 2010 Mar;61(3):193-9.
169. Brown AW, Hang J, Dussault PH, et al. Phytosterol ester constituents affect micellar cholesterol solubility in model bile. Lipids. 2010 Sep;45(9):855-62.
170. Talati R, Sobieraj DM, Makanji SS, et al. The comparative efficacy of plant sterols and stanols on serum lipids: a systematic review and meta-analysis. J Am Diet Assoc. 2010 May;110(5):719-26.
171. AbuMweis SS, Vanstone CA, Lichtenstein AH, et al. Plant sterol consumption frequency affects plasma lipid levels and cholesterol kinetics in humans. Eur J Clin Nutr. 2009 Jun;63(6):747-55.
172. Calpe-Berdiel L, Escola-Gil JC, Blanco-Vaca F. Phytosterol-mediated inhibition of intestinal cholesterol absorption is independent of ATP-binding cassette transporter A1. Br J Nutr. 2006 Mar;95(3):618-22.
173. Calpe-Berdiel L, Escola-Gil JC, Blanco-Vaca F. Are LXR-regulated genes a major molecular target of plant sterols/stanols? Atherosclerosis. 2007 Nov;195(1):210-1.
174. Plosch T, Kruit JK, Bloks VW, et al. Reduction of cholesterol absorption by dietary plant sterols and stanols in mice is independent of the Abcg5/8 transporter. J Nutr. 2006 Aug;136(8):2135-40.
175. Jakulj L, Trip MD, Sudhop T, et al. Inhibition of cholesterol absorption by the combination of dietary plant sterols and ezetimibe: effects on plasma lipid levels. J Lipid Res. 2005 Dec;46(12):2692-8.
176. Lin X, Racette SB, Lefevre M, et al. Combined effects of ezetimibe and phytosterols on cholesterol metabolism: a randomized, controlled feeding study in humans. Circulation. 2011 Aug;124(5):596-601.
General Introduction
37
Chapter
1177. Scholle JM, Baker WL, Talati R, et al. The effect of adding plant sterols or stanols to statin therapy in
hypercholesterolemic patients: systematic review and meta-analysis. J Am Coll Nutr. 2009 Oct;28(5):517-24.
178. Richelle M, Enslen M, Hager C, et al. Both free and esterified plant sterols reduce cholesterol absorption and the bioavailability of beta-carotene and alpha-tocopherol in normocholesterolemic humans. Am J Clin Nutr. 2004 Jul;80(1):171-7.
179. Goncalves A, Gleize B, Bott R, et al. Phytosterols can impair vitamin D intestinal absorption in vitro and in mice. Mol Nutr Food Res. 2011 Sep;55 Suppl 2:S303-11.
180. Kreuzer J. Phytosterols and phytostanols: is it time to rethink that supplemented margarine? Cardiovasc Res. 2011 Jun;90(3):397-8.
181. Dikkers A, Tietge UJ. Biliary cholesterol secretion: more than a simple ABC. World J Gastroenterol. 2010 Dec;16(47):5936-45.
182. Oude Elferink RP, Ottenhoff R, van Wijland M, et al. Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse. J Clin Invest. 1995 Jan;95(1):31-8.
183. Yamanashi Y, Takada T, Yoshikado T, et al. NPC2 regulates biliary cholesterol secretion via stimulation of ABCG5/G8-mediated cholesterol transport. Gastroenterology. 2011 May;140(5):1664-74.
184. Brufau G, Groen AK, Kuipers F. Reverse cholesterol transport revisited: contribution of biliary versus intestinal cholesterol excretion. Arterioscler Thromb Vasc Biol. 2011 Aug;31(8):1726-33.
185. Vrins CL. From blood to gut: direct secretion of cholesterol via transintestinal cholesterol efflux. World J Gastroenterol. 2010 Dec;16(47):5953-7.
186. Plosch T, Bloks VW, Terasawa Y, et al. Sitosterolemia in ABC-transporter G5-deficient mice is aggravated on activation of the liver-X receptor. Gastroenterology. 2004 Jan;126(1):290-300.
187. van der Velde AE, Vrins CL, van den Oever K, et al. Direct intestinal cholesterol secretion contributes significantly to total fecal neutral sterol excretion in mice. Gastroenterology. 2007 Sep;133(3):967-75.
188. Tang W, Ma Y, Jia L, et al. Genetic inactivation of NPC1L1 protects against sitosterolemia in mice lacking ABCG5/ABCG8. J Lipid Res. 2009 Feb;50(2):293-300.
189. Kruit JK, Plosch T, Havinga R, et al. Increased fecal neutral sterol loss upon liver X receptor activation is independent of biliary sterol secretion in mice. Gastroenterology. 2005 Jan;128(1):147-56.
190. Temel RE, Tang W, Ma Y, et al. Hepatic Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe. J Clin Invest. 2007 Jul;117(7):1968-78.
191. van der Veen JN, van Dijk TH, Vrins CL, et al. Activation of the liver X receptor stimulates trans-intestinal excretion of plasma cholesterol. J Biol Chem. 2009 Jul;284:19211-9.
192. Brown JM, Bell TA,3rd, Alger HM, et al. Targeted depletion of hepatic ACAT2-driven cholesterol esterification reveals a non-biliary route for fecal neutral sterol loss. J Biol Chem. 2008 Apr;283(16):10522-34.
193. Schwarz M, Russell DW, Dietschy JM, et al. Marked reduction in bile acid synthesis in cholesterol 7alpha-hydroxylase-deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia. J Lipid Res. 1998 Sep;39(9):1833-43.
194. van der Velde AE, Vrins CL, van den Oever K, et al. Regulation of direct transintestinal cholesterol excretion in mice. Am J Physiol Gastrointest Liver Physiol. 2008 Jul;295(1):G203-8.
195. Vrins CL, van der Velde AE, van den Oever K, et al. Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux. J Lipid Res. 2009 Oct;50(10):2046-54.
196. Jakulj L, Vissers MN, van Roomen CP, et al. Ezetimibe stimulates faecal neutral sterol excretion depending on abcg8 function in mice. FEBS Lett. 2010 Aug;584(16):3625-8.
197. de Vogel-van den Bosch, H.M., de Wit NJ, Hooiveld GJ, et al. A cholesterol-free, high-fat diet suppresses gene expression of cholesterol transporters in murine small intestine. Am J Physiol Gastrointest Liver Physiol. 2008 May;294(5):G1171-80.
198. Temel RE, Sawyer JK, Yu L, et al. Biliary sterol secretion is not required for macrophage reverse cholesterol transport. Cell Metab. 2010 Jul;12(1):96-102.
199. Cheng SH, Stanley MM. Secretion of cholesterol by intestinal mucosa in patients with complete common bile duct obstruction. Proc Soc Exp Biol Med. 1959 Jun;101(2):223-5.
200. DenBesten L, Reyna RH, Connor WE, et al. The different effects on the serum lipids and fecal steroids of high carbohydrate diets given orally or intravenously. J Clin Invest. 1973 Jun;52(6):1384-93.
Chapter 2
Laxative treatment with polyethylene glycol does not aff ect lipid absorption in rats
Mariëtte Y.M. van der Wulp 1, 2, Frans J.C. Cuperus 2, Frans Stellaard 2, Theo H. van Dijk 2, Jan Dekker 1,
Edmond H.H.M. Rings 1, 2, Albert K. Groen 1,2 and Henkjan J. Verkade 1, 2
1 Top Institute Food and Nutrition, Wageningen, Gelderland, The Netherlands2 Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Beatrix Children’s Hospital,
Groningen University Institute for Drug Exploration, Center for Liver, Digestive and Metabolic Diseases,
University of Groningen, University Medical Center Groningen, Groningen, Groningen, The Netherlands
J Pediatr Gastroenterol Nutr. 2012 Oct;55(4):457-62
Chapter 2
40
Abstract
Objectives Polyethylene glycol (PEG) is a frequently used laxative agent. It is unknown, however, if PEG
aff ects the absorptive capacity of the intestine. Reduced lipid (dietary fat and cholesterol) absorption
induced by long-term PEG treatment could negatively aff ect growth in children. We tested whether
PEG accelerates the gastrointestinal transit and alters lipid absorption and plasma lipid levels.
Methods Wistar rats were administered drinking water with or without PEG (7%) for two weeks. We
studied whole gut transit time by recording the fi rst appearance of red feces after intragastric carmine
red administration. We measured plasma concentrations of cholesterol and triglycerides, dietary fat
absorption by 48h fat balance and by plasma appearance of intragastrically administered stable-
isotope labeled fats, and cholesterol absorption with a dual stable isotope technique.
Results PEG decreased whole gut transit time by 20% (p = 0.028) without causing diarrhea. PEG
treatment did neither aff ect overall dietary fat balance, nor fat uptake kinetics, cholesterol absorption
or plasma lipid concentrations.
Conclusion PEG does not aff ect lipid absorption, nor steady state plasma lipid levels in rats, although
it accelerates the gastrointestinal transit.
Effect of PEG treatment on lipid absorption
41
Chapter
2
Introduction
Polyethylene glycol (PEG) is an inert polymer that originally was used to prepare bowels for endoscopy. 1
In the 1990’s PEG became available as osmotic laxative for the treatment of chronic constipation. 2
Currently it is one of the most widely prescribed laxative agents. Long-term treatment with PEG seems
safe and effective in adults 3 as well as children. 4,5 Recently it was stated that PEG shows better outcomes
than lactulose in terms of stool frequency per week, stool form, relief of abdominal pain and the need
for additional products. 6 However, it is not known whether PEG treatment affects intestinal absorptive
functions, for example by its effect on gastrointestinal transit.
Little attention has been given to the effect of PEG on gastrointestinal transit. Coremans et al. reported
acceleration of oro-cecal transit during PEG treatment as determined by breath tests in healthy
volunteers. 7 In constipated children, both high-dose (1.5 g. kg-1. d-1) and low dose (0.3 g. kg-1. d-1) PEG
were shown to accelerate total and segmental colonic transit after six and fourteen days of treatment,
respectively. 8,9 Only one study, performed in healthy adults, determined fecal excretion of nutrients
during PEG ingestion. 10 In that study, designed to study effects of diarrhea, PEG was shown to increase
fecal loss of fat and carbohydrates. 10 However, it has not been studied in detail in an appropriate animal
model whether long-term, non-diarrhea inducing, PEG dosages affect intestinal lipid (dietary fat and
cholesterol) absorption.
Several steps are required for adequate absorption of dietary lipids in the intestine, which in theory could
be affected by acceleration of intestinal transit, particularly of the small intestine. Triglycerides, the main
components of dietary fat, are partly lipolyzed in the stomach. 11 Emulsification in the stomach further
enhances lipolysis by pancreatic lipase in the intestinal lumen. 12 Lipolytic products and cholesterol are
solubilized by bile salts, providing efficient translocation into enterocytes. 13
The aim of this study was to test whether PEG, by acceleration of whole gut transit time (WGTT), alters
intestinal lipid absorption.
Previous studies on acceleration of intestinal transit were mainly executed with metabolized drugs. 14
Therefore, it is not clear whether the effects observed were directly related to acceleration of
gastrointestinal transit. PEG however, is minimally absorbed and not metabolized in the intestine. 15 We
attempted to accelerate WGTT with PEG without causing side-effects such as diarrhea and dehydration,
similar to its optimal clinical effect in patients.
We determined absorption and plasma levels of dietary fats and cholesterol. We studied fat absorption
with two independent methodologies, namely fat balance and stable isotope techniques. The
quantitative fat balance involves comparing the amount of fat intake and fecal output per time unit.
The fat balance technique can not discriminate between potential causes of fat malabsorption (e.g.
intraluminal versus intracellular processes). 16 We performed a qualitative analysis of fat absorption 16,17
by administering stable isotope labeled triglycerides and measuring plasma appearance of triglyceride-
derived fatty acids. This technique allows to assess differences in the rate of fat digestion and absorption.
We simultaneously studied the absorption of a stable isotope labeled free fatty acid (FFA) and triglyceride.
Chapter 2
42
By comparison of the plasma appearance of the two labels, the intestinal fatty acid absorption per se
can be discriminated from the process of digestion. 16 Cholesterol absorption was determined by a dual
stable isotope test. 18
Materials and Methods
Materials
Colofort® (polyethylene glycol + electrolytes) was obtained from Ipsen Farmaceutica B.V. (Hoofddorp,
The Netherlands). Colofort® contained per sachet (74 g): 64 g PEG, molecular weight 4 kDa, 5.7 g
sodium sulphate (anhydric), 1.68 g sodium bicarbonate, 1.46 g sodium chloride and 0.75 g potassium
chloride. Carmine was obtained from Macro-imPulse Saveur Ltd. (Stadtoldendorf, Germany). Intralipid®
(20%) was obtained from Fresenius Kabi, Den Bosch, The Netherlands. 2,2,4,4,6-Deuterium-cholesterol
(D5-cholesterol) was obtained from Medical Isotopes and 25,26,26,26,27,27,27-Deuterium-cholesterol
(D7-cholesterol) from Cambridge Isotope Laboratories Inc. 1-13C-stearate and tri-1-13C-palmitate were
obtained from Sigma Aldrich (St. Louis, MO). All isotopes were of 98-99% isotopic purity.
Animals
Growing male Wistar Unilever rats (150-174 g) were obtained from Harlan (Horst, The Netherlands). Rats
were housed individually in an environmentally controlled facility with diurnal (12/12h) light cycle. Food
and water were available ad libitum during the entire study period. The experiments were performed
in conformity with Public Health Service policy and in accordance with the national laws. The Ethics
Committee for Animal Experiments of the University Medical Center of Groningen approved the
experimental protocols.
Cholesterol absorption study
Rats were maintained on semisynthetic purified diet (supplementary table 1, code 4063.02, obtained from
Arie Blok, Woerden, The Netherlands). Upon arrival rats were randomly given a number from 1 to 14. After
a three week run-in period on semisynthetic diet, baseline parameters of all rats were obtained, including
WGTT. Subsequently, rats with an even number received PEG treatment (n= 7), whereas rats with an
uneven number were assigned to control group (no treatment; n= 7) for a total period of 16 days. PEG was
continuously available to the rats in their drinking water at a concentration (71g.l-1 PEG4000) previously
found to significantly accelerate intestinal transit. 19 Steady state conditions are expected to be reached
within five days of treatment. 15 Feces were collected during a period of 48h before the start of treatment and
again after one week of PEG treatment. WGTT was measured after intragastric administration of carmine
red inert dye 20 (1 ml 60 mg.ml-1 drinking water 19) under brief isoflurane/ oxygen inhalation anesthesia
(for handling) one day before, and during PEG treatment at day 14 at 9 A.M. during the light phase.
WGTT was defined as the time to appearance of the first red feces after administration of carmine.
Effect of PEG treatment on lipid absorption
43
Chapter
2
Food and fluids were available to the rats during the entire experiment. Food and fluid intake as well as
body weight (BW) were measured daily.
Plasma dual isotope ratio method
At day 10 after the start of PEG treatment, rats received an intravenous injection (tail) of 1.5 mg D7-
cholesterol dissolved in 500 μl intralipid and an oral dose of 3 mg D5-cholesterol dissolved in 1 ml
medium chain triglyceride (MCT) oil. At time points 0, 3, 6, 9, 12, 24, 48, 72, 96, 120, 144 and 168h after
administration, blood samples were obtained from the tail vein under isoflurane anesthesia.
Blood was collected in sodium-heparinized micro-hematocrit tubes (75 μl). Plasma was separated by
centrifugation (10 min, 2000 rpm, 4°C) and stored at -20°C until analyses. Feces were collected from day
three until day six after label administration.
At day 17, rats were anesthetized one by one by intraperitoneal injection of a mixture of Hypnorm
(fentanyl/ fluanisone 1 ml.kg-1) and diazepam (10 mg.kg-1). The common bile duct was cannulated for
bile collection. To ensure that hepatic production was accurately measured, bile produced during the
initial 5 min after cannulation was discarded, and bile was sampled for 30 min thereafter. During the
bile collection period, body temperature was maintained by keeping animals in a humidified incubator.
Blood was obtained by cardiac puncture and divided over lithium-heparin (for determination of renal
parameters) and EDTA-containing tubes. Rats were terminated by cervical dislocation.
Analytical procedures and calculations
Plasma parameters
Plasma cholesterol and triglyceride concentrations were determined using commercially available kits
(Roche Diagnostics, Mannheim, Germany). Renal functions (sodium, potassium, urea and creatinin) were
determined in plasma by routine spectrophotometry on a P800 unit of a modular analytics serum work
area from Roche Diagnostics Ltd. (Basel, Switzerland).
Feces
Fecal calcium and phosphate output were measured as described previously. 21 We measured calcium
concentration in 1.5 g aliquots of freeze-dried feces, and phosphate concentration in 1.0 g aliquots.
Fat balance
Fatty acid concentrations were determined in 50 mg aliquots of feces and of crushed food. 22 Fatty acid
methyl ester derivatives were measured by gas-chromatography (GC) on an HP-Ultra-1 column from
Hewlett-Packard (Palo-Alto, CA), using 100 μl heptadecanoic acid (C17:0, 50 mg.100 ml-1) as internal
standard. 22 Fat absorption was calculated for individual and total fatty acids as (intake - output)/ intake
*100%.
Chapter 2
44
Cholesterol absorption
Cholesterol was extracted from 10 μl plasma and isotope enrichments of sterols were determined in
the cholesterol fraction by gas chromatography-mass spectrometry (GC-MS). 18 Total fecal neutral sterols
(cholesterol plus bacterial metabolites) were separately determined by GC. 23 Biliary lipids were extracted
according to the method of Bligh and Dyer. 24 Total plasma and biliary cholesterol concentrations were
determined as described previously. 18 Fractional cholesterol absorption was determined as previously
described by Van der Veen et al. 18, modified for the influx of labeled cholesterol.
Fat absorption kinetic study
Theoretically, the composition of the diet could affect results. To evaluate this, we also performed
experiments with rats fed a different diet, containing for example less sugars and fiber (supplementary
table 1, AIN-93G, 25 Research Diet Services BV, Wijk bij Duurstede, The Netherlands). Rats served as their
own controls. We analyzed the effect of diet by repeating key measurements in rats before or during
PEG treatment. Body weight, fluid and food intake were determined daily. Feces (48h) and food were
collected. We administered stable isotope labeled saturated fat, in the form of FFA 1-13C-stearate and
triglyceride tri-1-13C-palmitate.
We administered, through oral gavage, a bolus of 500 μl oil (olive oil: MCT oil 1:3) per 300 g BW, which
represents 30% of daily fat intake. This bolus contained 10 mg of each labeled fat. Our rats consumed a
diet containing 7.2% fat. Daily average food intake was 23 g, containing 1.7 g of fat. The daily consumed
stearate and palmitate from food pellets was 4.6 mg (2.8 g. kg-1 in food) and 12.8 mg (7.7 g. kg-1 food)
of stearate and palmitate, respectively. At time points 0, 2, 4, 6, 8 and 10 h after administration blood
was drawn from the tail to determine label appearance. Baseline WGTT was determined two days later.
One day after WGTT measurement, PEG treatment was started as described above and continued until
termination. One week after the start of PEG treatment, all above measurements were repeated.
Analytical procedures
Plasma lipids were hydrolyzed with hydrogen chloride in acetonitrile. α-bromopentafluorotoluene
derivatives of fatty acids were extracted with hexane and analyzed by GC-MS. 26 Enrichment was defined
as the increase in M1/ M
0 fatty acid relative to baseline measurements. The ratio of plasma labeled fatty
acid and administered label (μmol) was calculated to obtain the % enrichment of label in plasma.
Palmitate % enrichment was corrected for molecular weight since we administered triglycerides, which
are detected in plasma as FFA.
Statistical Analyses
Normal distribution was examined by normal probability plots and Shapiro-Wilk tests. Depending on
normality of data, differences between two groups were determined by either Mann Whitney U or
Student’s t-test. The effect of PEG on WGTT was evaluated by a one way ANOVA repeated measurements
test, followed by post-hoc Wilcoxon Signed Rank tests to test within groups.
Effect of PEG treatment on lipid absorption
45
Chapter
2
Results are presented as means ± SD. The level of significance for all statistical analyses was set at
p < 0.05. Analyses were performed using SPSS version 16.0 for Windows (SPSS Inc., Chicago, IL).
Results
Animal characteristics
PEG-treated rats ingested similar amounts of food and water and showed similar body weights and
growth as control rats during the entire study period (table 1). PEG treatment did not affect biochemical
plasma parameters of renal function. PEG increased fecal output (dry weight, +42%; p= 0.001). No overt
diarrhea was noticed during PEG treatment (fecal pellets were formed). Because increased intestinal
calcium and phosphate availability can decrease fat absorption, we measured fecal concentrations and
calculated their fecal excretion. Both calcium and phosphate excretion were decreased in PEG-treated
vs. control rats (calcium -13% on average; p= 0.028; phosphate -19%; p= 0.022).
Table 1: Animal characteristics
Control PEG treated
Body weight (g) 398 ± 32 391 ± 44
Growth (g) 31 ± 4 26 ± 16
Intake and output
Food intake (g. kg-1.day-1) 58.7 ± 2.3 58.4 ± 4.0
Fluid intake (g. kg-1.day-1) 66.9 ± 13.1 79.2 ± 12.0
PEG intake (g. kg-1.day-1) 5.6 ± 0.6
Fecal dry weight (g. kg-1.day-1) 8.0 ± 0.8 11.4 ± 1.1*
Fecal calcium (mmol. kg-1.day-1) 4.8 ± 0.5 4.2 ± 0.4*
Fecal phosphate (mmol. kg-1.day-1) 3.8 ± 0.5 3.1 ± 0.5*
Plasma parameters
Cholesterol (mmol/ L) 2.3 ± 0.5 2.5 ± 0.3
Triglycerides (mmol/ L) 1.0 ± 0.4 0.9 ± 0.4
Creatinin (µmol/ L) 21.7 ± 2.3 20.8 ± 2.4
Ureum (mmol/ L) 8.5 ± 1.3 8.9 ± 1.0
Na (mmol/ L) 142.2 ± 2.1 140.1 ± 4.7
K (mmol/ L) 5.4 ± 0.4 5.3 ± 0.9
Body weight, growth, average daily intake and output, and plasma parameters in control rats and rats treated with 7% PEG in drinking water. Data are represented as mean ± SD, n=7 per group. Data of PEG treated rats were compared with those of control rats by unpaired two-sided Students’ t-tests. *P= <0.05
Chapter 2
46
PEG accelerates WGTT
PEG treatment decreased WGTT by 20% (from 10.1 ± 2.2h to 8.1 ± 2.7h; p= 0.028). Control rats showed
similar WGTT in both episodes (11.2 ± 2.3h and 11.3 ± 3.3h, figure 1).
Whole gut transit time
0 10
5
10
15
20
*
Control
PEG
Transit(h)
Figure 1. Effect of PEG on Whole gut transit time. WGTT was measured in rats fed a semisynthetic diet for 3 weeks. First a baseline measurement (0) was performed in all rats. Subsequently rats were randomly appointed to either control (white dots) or PEG treated (black dots) group and WGTT was measured in both groups (1). To test for differences in WGTT depending on PEG treatment, first a one way ANOVA repeated measurements test was executed. The error variance of the dependent variable (WGTT) was equal across groups (Levene’s test), indicating that it was correct to use this analysis. The repeated measurements test showed significant interaction (group*timepoint) (p<0.05). We used post-hoc Wilcoxon Signed rank tests to compare control WGTT at timepoints 0 and 1 (NS) and PEG treated WGTT (timepoint 1) compared to baseline WGTT in the same animals (timepoint 0; p=0.028). Data are represented as mean ± SD, n=7 per group. *P<0.05
Overall dietary fat balance is not affected by PEG treatment
We studied dietary fat absorption by subtracting fecal fat output from fat intake. Detailed analysis of fat
absorption using GC demonstrated that the absorption of unsaturated fatty acids (oleic, linoleic and
α-linolenic acid) was almost complete in control and PEG-treated animals (~93-100%). As described
previously 27, control rats showed relative malabsorption of the long chain saturated fatty acids palmitic
(~89%) and stearic acid (~66%) (figure 2).
PEG-treated animals absorbed significantly more saturated fatty acids than controls (palmitic acid 94
± 2% vs. 90 ± 2; p= 0.001; and stearic acid 76 ± 8 vs. 66 ± 7%; p= 0.03, respectively.) However, the
absorption of unsaturated fatty acids did not differ between both groups.
Total C16:0 C18:0 C18:1 C18:2 C18:30
25
50
75
100*
**
Fatt
y ac
id a
bsor
ptio
n (%
) Figure 2. 48h Fat balance of control (white bars) and PEG treated rats (black bars). Molar absorption percentage of total and separate dietary fatty acids. Major dietary fatty acids: palmitic acid (C16:0), linoleic acid (C18:2n6) and oleic acid (C18:1n9). Minor dietary fatty acids: stearic acid (C18:0) and α-linolenic acid (C18:3n3). Data are presented as mean + SD, n=7 per group. Data of PEG treated rats were compared with those of control rats by unpaired two-sided Students’ t-tests. *P<0.05, **p<0.01
Effect of PEG treatment on lipid absorption
47
Chapter
2
The overall percentage of lipid absorption did not differ between groups (96 ± 1 vs. 95 ± 2% in PEG-
treated vs. control animals), attributable to the minor relative contribution of the long-chain saturated
fatty acids to dietary fat.
In a separate experiment, we analyzed the effect of diet by repeating key measurements in rats on a
different semisynthetic diet (AIN-93G), again with or without PEG treatment. All relevant effects of PEG
treatment were virtually identical, including acceleration of WGTT by 27% and unchanged fat balance
(data not shown). This indicated that the PEG effect does not (strongly) depend on the type of diet.
0 2 4 6 8 100.00
0.05
0.10
0.15
0.20
Time after administration (h)
Plasma1-13C-palmitate
originating fromlabeled tripalmitin(%adm dose.ml -1)
A
0 2 4 6 8 100.00
0.05
0.10
0.15
Time after administration (h)
Plasma1-13C-stearate
(%adm dose.ml -1)
B
Figure 3. Fat uptake kinetics at baseline (white dots) and during PEG treatment (black dots). Panel A: Palmitate originating from tripalmitate was adequately absorbed during PEG-treatment, suggesting preserved digestion and absorption of saturated fat. Panel B: absorption of free stearate was likewise preserved during PEG-treatment (Area Under Curve). Plasma kinetics were studied during ten hours after administration of stable isotope labeled stearate and tripalmitate by oral gavage. Rats were used as their own control. Data are presented as mean + SD, n= 7 per group. *P(Wilcoxon Signed Rank test) <0.05
Chapter 2
48
PEG treatment does not affect the rate of fat digestion and absorption
Plasma 13C-fatty acid concentrations
Fat balance provides a quantitative analysis of fat absorption. It can not discriminate between potential
causes of differences, such as intraluminal factors (lipolysis of triglycerides, solubilization) or intracellular
factors (chylomicron formation). We used a different approach than fat balance to separately assess
whether PEG affected the rate of triglyceride lipolysis or the absorption of FFA. We administered stable
isotope labeled triglyceride (tri-1-13C-palmitate) and FFA (1-13C-stearate) intragastrically and determined
plasma appearance of 1-13C-palmitate and 1-13C-stearate, respectively (figure 3). Plasma 1-13C-stearate
was higher 2h after administration during PEG treatment (p= 0.028). A similar (non-significant) effect was
observed for 1-13C-palmitate (p= 0.063).
Maximum enrichment values of 1-13C-palmitate were reached after 2h in PEG-treated vs. 6h at baseline
conditions. Maximum values of 1-13C-stearate were reached after 6h in both conditions. The area under
the curves for both 1-13C-stearate and 1-13C-palmitate (0.81 ± 0.16 vs. 0.64 ± 0.11 and 0.91 ± 0.11 vs. 0.78
± 0.08 % of administered dose.ml plasma-1 under PEG treatment versus baseline conditions respectively
(not shown) did not differ, indicating that PEG did neither affect lipolysis nor FFA absorption.
PEG treatment does not affect cholesterol absorption
Similarly to the absorption of long-chain saturated fatty acids, cholesterol absorption is strongly bile
salt dependent. 13 We studied cholesterol absorption by means of a dual stable isotope technique. With
this technique we determine levels of stable isotope labeled cholesterols in plasma after prior i.v. and
oral administration of differentially labeled cholesterols. This well-known methodology 18,28 allows us to
calculate the fractional absorption of cholesterol from the intestine. Similarly to overall fat absorption,
fractional cholesterol absorption did not differ between PEG-treated and control rats (~54 and ~58%,
respectively, figure 4). PEG neither affected biliary cholesterol secretion, nor fecal sterol excretion (data
not shown).
Control PEG0
50
100
Frac
tiona
l cho
lest
erol
abso
rptio
n (%
)
Figure 4. Fractional cholesterol absorption in control rats (white bars) and in rats treated with PEG in drinking water (black bars). Absorption kinetics were calculated from labeled cholesterol in plasma collected after administration of i.v. D
7- and intragastric D
5-cholesterol.
Data are presented as mean + SD. Data were analyzed by unpaired two-sided Student’s t-test, n= 7 per group.
Effect of PEG treatment on lipid absorption
49
Chapter
2
Discussion
In the present study we determined in a rat model whether a frequently applied laxative, PEG, influences
intestinal lipid absorption via acceleration of the WGTT. Our data convincingly show that PEG treatment
does accelerate WGTT, but that it neither affects absorption of triglycerides, fatty acids or cholesterol,
nor plasma lipid levels.
We used PEG with added electrolytes because of its broad applicability with no net exchange of
electrolytes in the intestine. We considered this as a safe method during continuous administration
of PEG in drinking water. To our knowledge, there are no studies that directly compare effects of PEG
with or without added electrolytes in children. However, clinical experience and experimental data of
children taking PEG with or without added electrolytes show that both methods are highly effective and
safe. 29,30 PEG increased dry fecal output, which is mostly, if not entirely, attributable to appearance of PEG
itself in feces. 15 Unexpectedly, we found decreased fecal calcium and phosphate excretion. We cannot
exclude loss of these water soluble ions with fecal water in the bedding of PEG-treated rats.
Although probably underestimated due to evaporation and loss in bedding, we found increased water
content in PEG feces (up to 3-fold, data not shown) as determined by weight of fresh feces and that
after freeze-drying. We did not use metabolic cages to collect feces for our study, since the extra stress
would alter WGTT 31 and influence the effect of PEG. In addition, rats are able to consume feces directly
from their anus, which cannot be prevented in metabolic cages. It is known that coprophagia could
theoretically influence the absolute amounts of fat absorption. However, we do not have any indication
that PEG treatment selectively affects coprophagia and thereby relative fat absorption. We analyzed
the effects of PEG on dietary fat absorption with different, independent methodologies. 16,17 First, we
studied fat balance comparing the amounts of fat ingested over 48h via the food and the amount
excreted via feces. 16 PEG treatment did not affect the overall dietary fat absorption. PEG treatment
did slightly increase saturated fatty acid absorption, compared with control rats. This could be due to
increased intestinal bile salt availability as indicated by decreased fecal bile salt excretion during PEG
treatment. 19 Fat balance cannot discriminate between potential causes of differences in fat absorption,
such as intraluminal factors and intracellular factors. To further investigate fat uptake, we studied plasma
appearance of 13C-labeled fats, derived from intragastrically administered 13C-labeled FFA or triglycerides. 16 We found absorption kinetics to be comparable in PEG-treated versus baseline conditions. The bolus
of stable isotope labeled fat we administered contained 100-200% of the daily consumed saturated
fats by diet. Apparently, the difference in absorption during PEG treatment of specifically the saturated
fats (fat balance), when intake of those fats is minor, is abolished when we administer a single high
dose. Together, these results indicate that there are no relevant changes in fat absorption during PEG
treatment.
We cannot completely exclude that the amount of dietary fat may influence extrapolation of our results
towards the human situation, where a Western high-fat diet is often consumed. However, only one study
by Hammer et al. 10 in humans showed malabsorption of nutrients during PEG intake.
Chapter 2
50
This particular study was designed to study effects of osmotic diarrhea induced by high dose PEG. PEG
is nowadays most frequently used in lower dosages in humans to offer a long-term laxative treatment
by softening of stools and not to induce diarrhea. We mimicked this scenario in our study and did not
observe fat malabsorption.
We did not specifically measure small intestinal transit in this study, since it requires termination of the
rats. However, we previously showed that PEG induces a significant acceleration of small intestinal transit
in rats (+17%), comparable to the acceleration in total WGTT we found. 19
Whereas fat absorption percentage in healthy states is nearly complete 27, cholesterol absorption
is substantially lower. At least in part depending on the method applied 32, cholesterol absorption
percentage has been estimated between 30-70% in humans 33, in rodents 18,34,35 and in our present study
in rats. The substantially lower basal cholesterol absorption may provide increased sensitivity to detect
differences in cholesterol absorption as compared to fat absorption during an intervention. Our finding
that not only fat but also cholesterol absorption was not changed during PEG treatment supports the
concept that PEG treatment does not affect the absorptive capacity of the small intestine.
Together our data indicate that PEG treatment does not lead to relevant changes in lipid absorption
or plasma lipid levels in rats. These data suggest that in terms of lipid absorption, PEG can be safely
administered to children during growth and development.
Acknowledgements
The authors would like to thank Juul Baller and Rick Havinga for their help during animal experiments,
Renze Boverhof and Theo Boer for technical and Vincent Bloks for statistical assistance, respectively.
Conflicts of interest and source of funding
M.Y.M. van der Wulp is currently receiving an unrestricted research grant from Top Institute Food and
Nutrition via the University Medical Center Groningen. We have no conflicts of interest.
Effect of PEG treatment on lipid absorption
51
Chapter
2
References 1. Davis GR, Santa Ana CA, Morawski SG, Fordtran JS. Development of a lavage solution associated with minimal
water and electrolyte absorption or secretion. Gastroenterology. 1980 Feb;78(5 Pt 1):991-5.
2. Andorsky RI, Goldner F. Colonic lavage solution (polyethylene glycol electrolyte lavage solution) as a treatment for chronic constipation: a double-blind, placebo-controlled study. Am J Gastroenterol. 1990 Mar;85(3):261-5..
3. DiPalma JA, Cleveland MV, McGowan J, Herrera JL. A randomized, multicenter, placebo-controlled trial of polyethylene glycol laxative for chronic treatment of chronic constipation. Am J Gastroenterol. 2007 Jul;102(7):1436-41.
4. Pashankar DS, Loening-Baucke V, Bishop WP. Safety of polyethylene glycol 3350 for the treatment of chronic constipation in children. Arch Pediatr Adolesc Med. 2003 Jul;157(7):661-4.
5. Voskuijl W, de Lorijn F, Verwijs W, Hogeman P, Heijmans J, Makel W, et al. PEG 3350 (Transipeg) versus lactulose in the treatment of childhood functional constipation: a double blind, randomised, controlled, multicentre trial. Gut. 2004 Nov;53(11):1590-4.
6. Lee-Robichaud H, Thomas K, Morgan J, Nelson RL. Lactulose versus Polyethylene Glycol for Chronic Constipation. Cochrane Database Syst Rev. 2010 Jul;(7):CD007570.
7. Coremans G, Vos R, Margaritis V, Ghoos Y, Janssens J. Small doses of the unabsorbable substance polyethylene glycol 3350 accelerate oro-caecal transit, but slow gastric emptying in healthy subjects. Dig Liver Dis. 2005 Feb;37(2):97-101.
8. Bekkali NL, van den Berg MM, Dijkgraaf MG, van Wijk MP, Bongers ME, Liem O, et al. Rectal fecal impaction treatment in childhood constipation: enemas versus high doses oral PEG. Pediatrics. 2009 Dec;124(6):e1108-15.
9. Gremse DA, Hixon J, Crutchfield A. Comparison of polyethylene glycol 3350 and lactulose for treatment of chronic constipation in children. Clin Pediatr(Phila). 2002 May;41(4):225-9.
10. Hammer HF, Santa Ana CA, Schiller LR, Fordtran JS. Studies of osmotic diarrhea induced in normal subjects by ingestion of polyethylene glycol and lactulose. J Clin Invest. 1989 Oct;84(4):1056-62.
11. Moreau H, Laugier R, Gargouri Y, Ferrato F, Verger R. Human preduodenal lipase is entirely of gastric fundic origin. Gastroenterology. 1988 Nov;95(5):1221-6.
12. Liu CC, Carlson SE, Rhodes PG, Rao VS, Meydrech EF. Increase in plasma phospholipid docosahexaenoic and eicosapentaenoic acids as a reflection of their intake and mode of administration. Pediatr Res. 1987 Sep;22(3):292-6.
13. Westergaard H, Dietschy JM. The mechanism whereby bile acid micelles increase the rate of fatty acid and cholesterol uptake into the intestinal mucosal cell. J Clin Invest. 1976 Jul;58(1):97-108.
14. Holgate AM, Read NW. Relationship between small bowel transit time and absorption of a solid meal. Influence of metoclopramide, magnesium sulfate, and lactulose. Dig Dis Sci. 1983 Sep;28(9):812-9.
15. Pelham RW, Nix LC, Chavira RE, Cleveland MV, Stetson P. Clinical trial: single- and multiple-dose pharmacokinetics of polyethylene glycol (PEG-3350) in healthy young and elderly subjects. Aliment Pharmacol Ther. 2008 Jul;28(2):256-65.
16. Kalivianakis M, Minich DM, Havinga R, Kuipers F, Stellaard F, Vonk RJ, et al. Detection of impaired intestinal absorption of long-chain fatty acids: validation studies of a novel test in a rat model of fat malabsorption. Am J Clin Nutr. 2000 Jul;72(1):174-80.
17. Rings EH, Minich DM, Vonk RJ, Stellaard F, Fetter WP, Verkade HJ. Functional development of fat absorption in term and preterm neonates strongly correlates with ability to absorb long-chain Fatty acids from intestinal lumen. Pediatr Res. 2002 Jan;51(1):57-63.
18. van der Veen JN, van Dijk TH, Vrins CL, van Meer H, Havinga R, Bijsterveld K, et al. Activation of the liver X receptor stimulates trans-intestinal excretion of plasma cholesterol. J Biol Chem. 2009 Jul;284(29):19211-9.
19. Cuperus FJ, Iemhoff AA, van der Wulp MY, Havinga R, Verkade HJ. Acceleration of the Gastrointestinal Transit by Polyethylene Glycol Effectively Treats Unconjugated Hyperbilirubinemia in Gunn Rats. Gut. 2010 Mar;59(3):373-80.
20. Kotal P, Vitek L, Fevery J. Fasting-related hyperbilirubinemia in rats: the effect of decreased intestinal motility. Gastroenterology. 1996 Jul;111(1):217-23.
21. Cuperus FJ, Hafkamp AM, Havinga R, Vitek L, Zelenka J, Tiribelli C, et al. Effective treatment of unconjugated hyperbilirubinemia with oral bile salts in Gunn rats. Gastroenterology. 2009 Feb;136(2):673-82.
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22. Werner A, Minich DM, Havinga R, Bloks V, van Goor H, Kuipers F, et al. Fat malabsorption in essential fatty acid-deficient mice is not due to impaired bile formation. Am J Physiol Gastrointest Liver Physiol. 2002 Oct;283(4):G900-8.
23. van Meer H, Boehm G, Stellaard F, Vriesema A, Knol J, Havinga R, et al. Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats. Am J Physiol Gastrointest Liver Physiol. 2008 Feb;294(2):G540-7.
24. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911-7.
25. Reeves PG, Nielsen FH, Fahey GC,Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993 Nov;123(11):1939-51.
26. Oosterveer MH, van Dijk TH, Tietge UJ, Boer T, Havinga R, Stellaard F, et al. High fat feeding induces hepatic fatty acid elongation in mice. PLoS One. 2009 Jun;4(6):e6066.
27. Minich DM, Havinga R, Stellaard F, Vonk RJ, Kuipers F, Verkade HJ. Intestinal absorption and postabsorptive metabolism of linoleic acid in rats with short-term bile duct ligation. Am J Physiol Gastrointest Liver Physiol. 2000 Dec;279(6):G1242-8.
28. Turley SD, Herndon MW, Dietschy JM. Reevaluation and application of the dual-isotope plasma ratio method for the measurement of intestinal cholesterol absorption in the hamster. J Lipid Res. 1994 Feb;35(2):328-39.
29. Candy D, Belsey J. Macrogol (polyethylene glycol) laxatives in children with functional constipation and faecal impaction: a systematic review. Arch Dis Child. 2009 Feb;94(2):156-60.
30. Tabbers MM, Boluyt N, Berger MY, Benninga MA. Clinical practice : diagnosis and treatment of functional constipation. Eur J Pediatr. 2011 Aug;170(8):955-63.
31. Enck P, Merlin V, Erckenbrecht JF, Wienbeck M. Stress effects on gastrointestinal transit in the rat. Gut. 1989 Apr;30(4):455-9.
32. Matthan NR, Lichtenstein AH. Approaches to measuring cholesterol absorption in humans. Atherosclerosis. 2004 Jun;174(2):197-205.
33. Grundy SM. Absorption and metabolism of dietary cholesterol. Annu Rev Nutr. 1983;3:71-96.
34. Dietschy JM, Turley SD. Control of cholesterol turnover in the mouse. J Biol Chem. 2002 Feb;277(6):3801-4.
35. Voshol PJ, Schwarz M, Rigotti A, Krieger M, Groen AK, Kuipers F. Down-regulation of intestinal scavenger receptor class B, type I (SR-BI) expression in rodents under conditions of deficient bile delivery to the intestine. Biochem J. 2001 Jun;356(Pt 2):317-25.
Effect of PEG treatment on lipid absorption
53
Chapter
2
Supplementary table 1. Composition of semisynthetic diets
% by weight 4063.02 AIN93G
Protein 17.6 17.7
Carbohydrate 53.8 60.1
Fat 5.2 7.2
g.kg-1
Casein 200 200
Corn-starch 100 397
Maltodextrin 132
Sucrose 100
Cerelose/ dextrose 500
Cellulose 50
DICACEL2+4/ cellulose 100
Soybean oil 50 70
Calcium 5 5
Phosphate 4 3
During the cholesterol absorption study, rats were maintained on semisynthetic purified diet code 4063.02. The fat absorption experiment was performed with rats fed AIN-93G diet.
Chapter 3
Laxative treatment with polyethylene glycol decreases microbial sterol conversion
in the intestine of rats
Mariëtte Y.M. van der Wulp 1, 2, Muriel Derrien 1,3, Frans Stellaard 2, Henk Wolters 2, Michiel Kleerebezem 1,3,
Jan Dekker 1, Edmond H.H.M. Rings 1, 2, Albert K. Groen 1,2 and Henkjan J. Verkade 1, 2
1 Top Institute Food and Nutrition, Wageningen, The Netherlands2 Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Beatrix Children’s Hospital,
Groningen University Institute for Drug Exploration, Center for Liver, Digestive and Metabolic Diseases,
University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 3 Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands
Submitted
Chapter 3
56
Abstract
Objectives Polyethylene glycol (PEG) is a frequently used osmotic laxative that accelerates
gastrointestinal transit. It has remained unclear however, if PEG aff ects intestinal functions. We aimed
to determine the eff ect of PEG treatment on intestinal sterol metabolism.
Methods Rats were treated with PEG in drinking water (7%) for two weeks or left untreated (controls).
We studied the enterohepatic circulation of the major bile salt (BS) cholate with a plasma stable isotope
dilution technique and determined BS profi les and concentrations in bile, intestinal lumen contents
and feces. We determined the fecal excretion of cholesterol plus its intestinally formed metabolites.
Finally, we determined the cytolytic activity of fecal water (a surrogate marker of colorectal cancer risk)
and the amount and composition of fecal microbiota.
Results Compared with control rats, PEG treatment increased the pool size (+51%; p<0.01) and
decreased the fractional turnover of cholate (-32%; p<0.01). PEG did not aff ect the cholate synthesis
rate, corresponding with an unaff ected fecal primary BS excretion. PEG reduced fecal excretion of
secondary BS and of cholesterol metabolites (each p<0.01). PEG decreased the cytolytic activity of
fecal water (54 [46-62] vs. 87 [85-92] % erythrocyte potassium release in PEG treated and control rats,
respectively; p<0.01). PEG treatment increased the contribution of Verrucomicrobia (p<0.01) and
decreased that of Firmicutes (p<0.01) in fecal fl ora.
Conclusion PEG treatment increases the pool size of the primary BS cholate and decreases the
bacterial conversion of BS and cholesterol in rats. PEG decreases cytolytic activity of fecal water, which
could improve intestinal health.
PEG treatment affects enterohepatic circulation of bile salts
57
Chapter
3
Introduction
Polyethylene glycol (PEG) currently is one of the most widely prescribed laxatives. Long-term treatment
with PEG is believed to be safe and highly effective. 1,2 However, it is not known whether or not long-term
PEG treatment affects specific intestinal metabolic functions. PEG may change the intestinal milieu by
accelerating the passage of luminal contents and/or by increasing the luminal water content, possibly
leading to a change in the intestinal microflora. 3
We have previously shown that PEG treatment accelerates the transit through the small intestine as
well as the whole gut in rats. 3 PEG decreases fecal excretion of bile salts (BS) in rats. 3 BS facilitate the
solubilization of lipids (dietary fats, cholesterol and fat-soluble vitamins) in the small intestine, which
is required for efficient lipid absorption. 4 It has recently become clear that BS are not only detergents
necessary for lipid absorption, but are also involved in the regulation of glucose and lipid homeostasis,
and energy expenditure. 5,6 This is illustrated for example by the fact that removal of BS from the intestine
with sequestrants improves plasma low density lipoprotein levels and hyperglycemia in patients with
type II diabetes. 7,8 The mechanisms by which the total pool size and/or profile of BS influence different
physiological processes are just beginning to be understood. 5,6
BS are amphiphatic molecules, produced in the liver (primary BS) by catabolism of cholesterol. 9 The
liver conjugates BS with taurine or glycine and secretes BS into bile, which is transported to the small
intestine. 10 Lipid absorption mainly takes place in the proximal small intestine whereas BS are actively
reabsorbed with high efficiency (~95%) in the terminal ileum. 10 From there, they are transported back
to the liver through the portal system, i.e. completing the enterohepatic cycle (EHC). Under steady state
conditions, the liver compensates for fecal BS loss by synthesis of new BS which then are secreted into
bile.
The small percentage of BS that escapes absorption in the terminal ileum enters the colon. Intestinal
microbiota deconjugate (through BS hydrolases) and dehydroxylate (7α-dehydroxylase) primary BS to
so called secondary BS species 10 A part of colonic BS is passively absorbed, while the remaining part
is lost with feces. Secondary BS such as deoxycholate (DC) and litocholate (LC) are highly hydrophobic.
It is generally thought that these hydrophobic BS can damage colonic mucosal cells and play a role in
initiating or propagating the formation of gastrointestinal malignancies. 10,11 In addition, an increased
hydrophobic BS pool size is associated with the formation of cholesterol gallstones. 12-14
Based on our previous studies 3, we hypothesized that PEG disrupts EHC of BS by changing the intestinal
milieu. We determined relevant parameters of the EHC of cholate (quantitatively the major BS in humans
and rodents) during PEG treatment by an isotope dilution technique. 15,16 We determined the effects of
PEG on BS profiles and concentrations in different compartments of the EHC, on the cytotoxic activity
of fecal water, and on fecal microbiota. This study shows that PEG increases the cholate pool size and
changes the microbial composition, decreases secondary BS formation, and finally decreases fecal water
cytotoxicity.
Chapter 3
58
Materials and Methods
Materials
Colofort® (polyethylene glycol + electrolytes) was obtained from Ipsen Farmaceutica B.V. (Hoofddorp,
The Netherlands). Colofort® contained per sachet (74 g): 64 g PEG, molecular weight 4 kDa, 5.7 g sodium
sulphate (anhydric), 1.68 g sodium bicarbonate, 1.46 g sodium chloride and 0.75 g potassium chloride.
24-13C-cholate was obtained from Cambridge Isotope Laboratories Inc and was of 98-99% isotopic purity.
Complete protease inhibitor was obtained from Roche. Deuterium labeled D7-7α-hydroxycholesterol
(7α-hydroxycholesterol-25,26,26,26,27,27,27-D7) was obtained from CDN isotopes inc. (product nr.
D-4064).
Animals
Outbred male Wistar Unilever rats (150-174 g) were obtained from Harlan (Horst, the Netherlands). Rats
were housed individually in an environmentally controlled facility with diurnal (12/ 12 h) light cycle.
They were maintained on semisynthetic purified diet (code 4063.02 low fat normal calcium food, Arie
Blok BV, Woerden, the Netherlands). Before any intervention, rats were accustomed to the diet during a
three week run-in period. Food and water were available ad libitum during the entire study period. The
experiments were performed in conformity with Public Health Service policy and with national laws. The
Ethics Committee for Animal Experiments of the University Medical Center of Groningen approved the
experimental protocols.
Cholate kinetic study
After the run-in period on semisynthetic diet, rats were randomly assigned to either control group (no
treatment; n=7) or intervention group (71 g/l PEG4000 via drinking water; n=7) for a total period of
sixteen days. Feces were collected during a period of 48 h before the start of treatment and again after
one week of PEG treatment. Food and fluid intake and body weight were measured daily.
Cholate kinetics
Intravenous 24-13C-cholate (3 mg per rat in a solution of 250 μl 0.5% NaHCO3 in phosphate-buffered
saline (PBS), pH 7.4) was administered at day 10 after the start of PEG treatment. Blood was drawn from
the tail vein at 0, 12, 24, 36, 48, and 60 h after administration under isoflurane anesthesia. At day 17, rats
were anesthetized by intraperitoneal injection of a mixture of Hypnorm (fentanyl/ fluanisone 1ml/ kg)
and diazepam (10 mg/ kg). The common bile duct was cannulated for bile collection. To ensure that
hepatic production was accurately measured, bile produced during the initial 5 min after cannulation
was discarded, and bile was sampled for 30 min thereafter. During the bile collection period, body
temperature was maintained by keeping animals in a humidified incubator. Blood was obtained by
cardiac puncture and stored (-20°C). Rats were terminated by cervical dislocation.
PEG treatment affects enterohepatic circulation of bile salts
59
Chapter
3
Analytical procedures and calculations
Feces
BS and neutral sterol composition and concentration in feces were determined by gas chromatography
(GC) to calculate daily excretion. 17
Cholate kinetics
Plasma and bile samples were prepared for isotopic analysis of cholate α-bromopentafluorotoluene
(PFB)- trimethylsilyl (TMS) derivatives by GC-mass spectrometry (MS). 15 The ions monitored were 623
and 624 corresponding to cholate (M0) and 24-13C-cholate (M
1). Enrichment (increase in M
1-cholate/ M
0-
cholate relative to baseline measurements) was expressed as the natural logarithm of the atom percent
excess (ln APE) value. The decay of ln APE over time was calculated by linear regression analysis. The
fractional turnover rate (FTR) per day equals the slope of the regression line. Pool size (μmol.100g BW-1)
and cholate synthesis rate (μmol.100 g BW-1.day-1) were calculated as described previously. 15
Enterohepatic Cycling of Cholate
The cholate biliary secretion rate was calculated by multiplying the bile flow (ml.100 g BW-1.h-1) by
the biliary cholate concentration (mM) as determined by GC analysis. 17 Bile flow was determined
gravimetrically, assuming a density of 1 g.ml-1 for bile. The percentage of cholate reabsorbed per day was
calculated as follows: 100% * (biliary secretion – synthesis) / biliary secretion. The percentage of cholate
lost in feces per enterohepatic cycle was calculated by 100% minus the calculated % reabsorption. 16
Hepatic cholesterol 7α-hydroxylase (cytochrome P450 7a1 or cyp7a1) activity
The activity of microsomal cyp7a1 was assayed essentially as described 18 with some modifications. In
short, microsomes (200 µg protein) in 100 mM K-phosphate buffer, pH 7.4 containing 1 mM EDTA ,
1 mM NADPH and complete protease inhibitor were incubated at 370C (total volume 1 ml). After 4
min the enzymatic reaction was stopped by addition of 8 ml of chloroform/ methanol (2:1, v/v). D7-7α-
hydroxycholesterol was added as internal standard (40 ng). After adding 1 ml 0.9% NaCl and vigorous
shaking, the samples were centrifuged. The chloroform phase was removed and evaporated under a
stream of nitrogen. Calibration standards with unlabeled D0-7α-hydroxycholesterol were treated in a
similar way. Samples were converted to TMS ether by overnight treatment with a mixture of N,O-bis
{TMS} trifluoroacetamide, pyridine and trimethylchlorosilane (50:50:1, v/v). Samples were dried under
a stream of nitrogen, resuspended in hexane, and analyzed on GC-MS (Agilent 9575C inert MSD,
Agilent Technologies, Amstelveen). The column used was J+W Scientific DB-17MS ; 20m; 0.18mm; film
0.18 µm. Operating parameters: helium flow 0.7 ml/min; temperature: source 2200C; column: 1500C
for 0.5 min-600C/min up to 300C-3000C for 5 min. The amount of 7α-hydroxycholesterol produced
from the endogenous microsomal cholesterol was calculated from the ratio labeled: unlabeled
7α-hydroxycholesterol using the calibration standards.
Chapter 3
60
Cytotoxic activity of fecal water
Fecal water was prepared by reconstitution of lyophilized feces. 19 Potassium release resulting from lysis
of human erythrocytes incubated in 154 mM NaCl was set as 0% and distilled water represented 100%
(maximal) potassium release. Subsequently, fecal water was incubated with erythrocytes and potassium
release as a measure of cell lysis was expressed as percentage of maximal potassium release. 20
Intestinal bile salt contents
BS in intestinal contents were measured in a separate group of rats, treated and handled identical
to described above: rats were randomly assigned to control group or PEG-treated group (n=7 each
group). At day 16 after the start of PEG treatment, rats were terminated under anesthesia and the entire
intestinal tract from duodenum until anus was removed. Small intestines were divided into proximal,
mid and distal segments of equal length. Cecum was separated from remaining colon and its contents
were collected directly in pre-weighed cups. Small intestine and remaining colon were flushed with 5
ml PBS to collect contents. Samples were lyophilized overnight and weighed again to calculate total
dry content weight. Aliquots of 10 (proximal and middle part of small intestine) to 50 (distal small
intestine, cecum and colon) mg were used to determine BS composition by GC. BS were first isolated
by reversed-phase solid-phase extraction. 17 The eluate was evaporated to dryness under a stream of
nitrogen and dry material was redissolved in hexane. The solution was divided over two glass tubes, after
which samples were dried once more. The first part was converted to TMS derivatives and free cholate
was determined as described previously. 17 The remaining part was mixed in 500 μl DEMI water, 500 μl
sodium acetate (0.2 M; pH 5.6) and 12 U choloyl glycine hydrolase (0.6 U/ μl) to hydrolyze BS for 15 h
at 37°C. Afterwards, BS were isolated and derivatized as described. This way, we obtained the total and
free cholate fractions. Free cholate was subtracted from total cholate to obtain the mass of conjugated
cholate in different compartments.
Bacterial DNA extraction
Fecal bacterial genomic DNA was isolated using the Fast DNA Spin kit (Qbiogene, Inc, Carlsbad, CA, USA)
using 0.1 g of fecal sample and eluted in 100 μl DES. Purity and amount of DNA was measured using
Nanodrop-1000 spectrophotometer (NanoDrop® Technologies, Wilmington, DE).
Quantitative PCR
Quantitative PCR (qPCR) was performed with an IQ5 Cycler apparatus (Bio-Rad, Veenendaal, The
Netherlands). All reactions were performed in triplicate in one run. Samples were analysed in a 25-μl
reaction mix consisting of 12.5 μl Bio-Rad master mix SYBR Green (50 mM KCl, 20 mM Tris-HCl, pH 8.4, 0.2
mM of each dNTP, 0.625 U iTaq DNA polymerase, 3 mM MgCl2, 10 nM fluorescein), 0.1 µM of each primer
Bact-1369F (5’ CGGTGAATACGTTC-3’) and Prok-1492R (5’-GGWTACCTTGTTAC-3’) and 5 μl of template
fecal DNA diluted 1:100 or 1:1000.
PEG treatment affects enterohepatic circulation of bile salts
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Chapter
3
Standard curves of 16S rRNA PCR product from Lactobacillus casei were created using serial 10-
fold dilution of the purified PCR product corresponding to 108 to 100 16S rRNA copies. The following
conditions of qPCR used were: 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 15
sec, annealing temperature of 60°C for 20 sec, extension at 72°C for 30 sec and a final extension step at
72°C for 5 min. A melting curve was performed at the end of each run to verify the specificity of the PCR
amplicons by slowly heating the final reaction mix to 95°C (0.5°C per cycle). Data analysis was performed
using the Bio-Rad software.
Microbial fermentation product analysis
Fresh large bowel content samples were analyzed for short chain fatty acid (SCFA) profiles, including
quantitative detection of acetate, butyrate, propionate and lactate using HPLC (Spectra System, RI-150).
Samples of intestinal content (approximately 0.1 g) were thoroughly mixed with four volumes of distilled
water. Insoluble residue was removed by centrifugation (15 min at 13.000 g, 4°C). The subsequent
supernatant was mixed with the same volume of 1M HCLO4 and mixed organic acid was analyzed by
HPLC as previously described. 21
16S rRNA gene amplicon pyrosequencing
Amplicons from the V1-V3 region of 16S rRNA genes were generated by PCR using 27F-DegS
(5’-GTTYGATYMTGGCTCAG-3’) in combination with 520R-Deg for 14 fecal samples. To facilitate
pyrosequencing using titanium chemistry, each forward primer was appended with the titanium adaptor
A (5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3’) and a ‘NNNN’ barcode sequence on the 5’ end, where
NNNN is a sequence of four nucleotides that was unique for each sample. The reverse primer carried the
titanium adaptor B (5’-BioTEG/CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-3’) on the 5’ end. Sequencing
was performed from adaptor A. Adaptor and barcode sequences were provided by GATC Biotech (www.
GATC-Biotech.com). PCRs were performed in a total volume of 50 μl containing 1× PCR buffer, 1 μl PCR
Grade Nucleotide Mix, 0.4 μl of Faststart Taq DNA polymerase (Roche, Diagnostics GmbH, Mannheim,
Germany), 200 nM of a forward and the reverse primer (Biolegio BV, Nijmegen, The Netherlands), and 20
of template DNA. The amplification program consisted of an initial denaturation step at 95°C for 5 min,
35 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 40 s and elongation at 72°C for 70 s and a
final extension step at 72°C for 10 min. The size of the PCR products was confirmed by gel electrophoresis
using 1 μl of the reaction mixture on a 1% (w/v) agarose gel containing ethidium bromide. DNA yield
from purified PCR products were measured by NanoDrop ND-1000 spectrophotometer. Pooled purified
PCR (final DNA concentration of 100 ng/μl) was subsequently sent to GATC-Biotech for pyrosequencing
using a Genome Sequencer FLX in combination with titanium chemistry.
16S rRNA gene sequence analysis 16S rRNA sequences generated from pyrosequencing were quality
filtered. Sequences were removed if they were shorter than 200 nucleotide, longer than 1,000 nucleotide,
contained primer mismatches or ambiguous bases.
Chapter 3
62
The remaining sequences were analysed using the open source software package Quantitative Insights
Into Microbial Ecology (QIIME). 22 16S rRNA gene sequences were assigned to operational taxonomic
units (OTUs) using UCLUST with a threshold of 97% pair-wise identity, then classified taxonomically using
the Ribosomal Database Project (RDP) classifier 2.0.1. Results were displayed as relative abundance of
bacterial taxa and richness.
Statistical AnalysesNormal distribution was examined by Kolmogorov-Smirnov and Shapiro-Wilk tests and normal
probability plots. Nonparametrically distributed data were tested for significant differences by Mann
Whitney U test (values represent median and interquartile range) and parametrically distibuted data by
Student’s unpaired t-test (values represent mean ± SD). Since some of the data regarding BS kinetics and
sterols in intestinal lumen contents and feces were clearly nonparametrically distributed, we decided
to test all these data nonparametrically to allow for comparison between graphs. Correlations are
expressed as nonparametric Spearman correlation coefficient. Statistical analyses were performed using
SPSS 18.0 for Windows, Chicago, IL and GraphPad Prism. Differences between groups were considered
statistically significant at p< 0.05.
Results
PEG treatment results in major changes of the EHC of cholateTo determine the effects of PEG on the EHC of BS, we studied the kinetics of cholate, representing the
major BS in human as well as rodent BS pools. PEG-treated rats had a similar bile flow and total biliary BS
concentration and secretion rate compared to control rats (table 1).
Table 1. Bile flow and biliary output parameters
Control PEG
Bile flow (ml.day-1.100g BW-1) 6.5 [5.7-6.9] 6.2 [5.5-6.5]
BS concentration (mM) 22.7 [19.3-25.0] 19.8 [16.2-23.3]
Cholate concentration (mM) 8.3 [6.3-12.7] 10.2 [6.9-13.0]
Biliary BS secretion rate (µmol.day-1.100 g BW-1)
133.0 [112.1-172.5] 117.5 [101.7-132.0]
Bile flow was measured during a 30 min. collection period. BS secretion rate was calculated from total biliary BS concentration as determined by GC. Data are presented as median and interquartile range, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by Mann Whitney U tests.
Cholate kinetic parameters are schematically summarized in figure 1A and 1B. PEG did not change biliary
secretion rate or intestinal reabsorption (~96%) of cholate, nor the cholate synthesis rate. The latter
finding corresponded with similar Cyp7a1 (rate-limiting enzyme for BS synthesis) activity in livers of PEG
treated and control rats (figure 1C). PEG treatment increased the cholate pool size (+51%; p=0.008) and
decreased the Fractional Turnover Rate (FTR) by 32% (p=0.003).
PEG treatment affects enterohepatic circulation of bile salts
63
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3
μ
μ
μ
C
Figure 1. Cholate kinetics (A and B). Biliary secretion, intestinal reabsorption, fecal excretion and synthesis of cholate did not differ between control (A) and PEG-treated (B) rats. Cholate pool size on the other hand was increased during PEG-treatment, in the presence of reduced fractional turnover rate (FTR). Plasma cholate kinetics were studied during 60h after intravenous administration of stable isotope labeled 13C-cholate. If not indicated, values are expressed in µmol. 100g BW-1.day-1. Data are presented as median and interquartile range, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by Mann Whitney U tests. **P<0.01. As an indicator of total BS synthesis, Cyp7a1 activity was measured in liver microsomes. (C) Data are presented as mean ± SD, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by unpaired Student’s t-test.
Chapter 3
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PEG did not change fecal primary BS excretion, but decreased fecal secondary BS excretion by 46% (2.0
[1.6-2.6] vs. controls 3.6 [3.1-4.1] µmol.100 g BW-1.day-1, respectively. P=0.001; figure 2A and 2B).
A B
Figure 2. Excretion of individual BS (A) and primary versus secondary BS (B) in feces. Primary BS include chenodeoxycholate (CDC), cholate, allo-cholate (allo-C), α- and β-muricholate (MC). Secondary BS include ursodeoxycholate (UDC), deoxycholate (DCA), δ22-β-MC, hyodeoxycholate (HDC) and ω-MC. Fecal BS excretion was determined in 48h collected feces on day 8 and 9 after the start of PEG treatment. Data are presented as median and interquartile range, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by Mann Whitney U tests. *P<0.05, **p<0.01
PEG treatment alters intestinal BS composition
Considering the kinetics of the enterohepatic circulation, we hypothesized that the increased cholate
pool size would be reflected by higher CA amounts in the intestinal luminal content. Indeed, the total
CA content in the intestinal lumen was similarly increased in PEG-treated rats (figure 3) as was the CA
pool size measured by the stable isotope dilution technique (+60% and +51%, respectively). However,
the variation in the total CA content in the intestinal lumen was considerable and the difference did not
reach statistical significance (figure 3; p=0.051).
Figure 3. Cholate amount in different intestinal compartments. The small intestine (SI) was divided in three segments of equal size, colon was divided in cecum and remaining colon. SIP= Small Intestine Proximal segment, SIM= Small Intestine Middle segment, SID= Small Intestine Distal segment, Cec= cecum, Col= remaining colon. Data are presented as median and interquartile range, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by Mann Whitney U tests. *P<0.05
PEG treatment affects enterohepatic circulation of bile salts
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PEG treatment decreases lipid metabolites
The unaffected primary BS and decreased secondary BS excretion via the feces could be due to altered
bacterial deconjugation and conversion of primary BS. In agreement with this, PEG treated rats had
higher amounts of primary BS in cecum and total intestinal lumen contents (the latter not statistically
significant; figure 4A).
A
B
C
D
E
F
Figure 4. Bile salts (BS), neutral sterols (NS) and short chain fatty acids (SCFA). Distribution of primary (A) and secondary (B) BS in intestinal lumen. Secretion of individual BS (C) and primary versus secondary BS (D) in bile. Fecal NS excretion; Copr= coprostanol, Chol= cholesterol, DiH-Chol= dihydrocholesterol. (E) Fecal SCFA concentration; A (Acetate), P (Propionate), B (Butyrate), L (Lactate). (F) Data are presented as median and interquartile range, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by Mann Whitney U tests. *P<0.05, **p<0.01.
Chapter 3
66
On the other hand, PEG treated rats had lower amounts of secondary BS (figure 4B) in their intestinal
lumen contents. The data corresponded with those found in bile, in which we also found decreased
secondary BS (figure 4C and 4D). To determine whether the decreased microbial conversion upon PEG
treatment was specific for BS metabolism, we analyzed fecal neutral sterols and SCFA. In accordance
with the BS data, we found almost no cholesterol metabolites (figure 4E) or SCFA (figure 4F) in feces.
PEG treatment decreases fecal water cytotoxic activity
The aqueous, rather than the solid, phase of feces is considered to contain the compounds that interact
with (large) intestinal mucosal cells. 23 In our study, the cytotoxic activity induced by fecal water was
significantly decreased in PEG treated compared with control rats (54 [46-62] vs. 87 [85-91] % erythrocyte
K release, respectively. Figure 5A; p=0.001). The fecal water cytotoxicity was related to both the total fecal
BS excretion (figure 5B) and the hydrophobic DC excretion (figure 5C).
A
B
C
Figure 5. (A) Cytotoxic activity of fecal water as determined by potassium (K) release of human erythrocytes after incubation with fecal water. Data are presented as mean ± SD, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by unpaired Student’s t-test. **P<0.01. (B) Correlation between fecal water cytotoxicity and BS excretion. Spearman correlation (r) and p value are indicated. (C) Correlation between fecal water cytotoxicity and DC excretion. Spearman correlation (r) and p value are indicated.
PEG treatment changes intestinal microbiota profile
The PEG-induced differences in intestinal BS and sterol metabolite contents suggested alterations in
the amount and/or activity of the intestinal microflora. Quantitative PCR of 16S amplicon showed that
PEG-fed rats harbored a non-significantly lower bacterial load expressed as Log10 16SrRNA copies.g
wet feces-1 (p=0.053, figure 6A; 11.4 ± 0.1 versus 11.0 ± 0.10 in control and PEG-fed rats, respectively).
PEG treatment affects enterohepatic circulation of bile salts
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Microbiota composition of both groups was investigated by 454-pyrosequencing of the V1-V2 variable
region. A total of 192 158 sequences were obtained.
After quality filtering, the average of sequences size was 314.6 (min: 200, max: 551.0). The number of
reads per sample was on average 13725. PEG treatment increased Verrucomicrobia (p=0.006), but did
not change other phyla significantly (Figure 6B). Looking at lower taxonomical level, multivariate analysis
on relative abundance of bacterial taxa (genus level) showed that microbiota from PEG-treated rats differ
significantly from that of control rats (Monte Carlo permutation test, p=0.004, figure 6C).
Figure 6. Microbiota. (A) Quantification of total bacteria in control and PEG-fed rats, expressed as log10 number of 16S copies.g wet feces-1. Data of PEG treated rats were compared with those of control rats by Mann Whitney U tests. (B) Relative abundance (% of sequences) of bacterial phyla detected in control and PEG-fed rats. (C) Redundancy analysis on relative abundance of bacteria taxa (genus level) in control (grey circles) and PEG-fed rats (black circles). (D) Relative abundance of major bacterial taxa (>5%) that were significantly different between control and PEG-fed rats. (E) Relative abundance of bacterial taxa that were significantly different between control and PEG-fed rats and that account for <5% of the total sequences. Data of PEG treated rats were compared with those of control
rats by Mann Whitney U tests. *P<0.05, **p<0.01.
A
C
E
B
D
Chapter 3
68
Significant changes of genus level included mostly increased relative abundance of known mucus
associated bacteria (Akkermansia, Bacteroides, Ruminococcus), members of Proteobacteria and
concomitant decreased relative abundance of Clostridia (Firmicutes) (Figure 6D and 6E). Moreover, the
total species richness, as estimated by Chao1 index was 1573 ± 209 and 1639 ± 135 OTUs for control and
PEG-fed rats respectively, showed no significant difference between both groups. Overall, microbiota
analysis indicated a trend towards reduced number of fecal bacteria, with no change of richness, but
changes in microbiota composition with a relative increase of mucus-associated bacteria in PEG treated
rats.
Discussion
Our study shows that PEG treatment increases the pool size of the primary bile salt cholate and decreases
its fractional turnover rate in rats. PEG changes the microbiota composition and decreases intestinal
metabolism of bile salts and cholesterol. Finally, PEG treatment decreases the cytotoxicity of fecal water,
a surrogate marker of colon cancer risk.
PEG increased the amount of cholate in the cecum contents of rats. Theoretically, this could be caused
by decreased ileal cholate absorption or decreased bacterial conversion of cholate. The re-absorption of
cholate in quantitative terms did not differ between PEG treated and control rats. Our data do indicate
decreased bacterial conversion of primary BS to secondary BS. PEG also increases the cecum content
weight (+97%, p<0.01). It seems likely that the increased poolsize is at least partly attributable to an
increased volume of luminal contents, rather than to increased BS concentrations per volume unit. In
control rats, we found a sharp decrease in cholate in cecum contents compared with small intestinal
contents, and a concomitant increase in DC. These effects are expected, since most of the cholate will
be actively absorbed in the distal small intestine 24, and the part that is not absorbed will be mostly
converted to DC by the microbiota of the cecum. 10 However, in PEG treated rats cholate remained
higher in the cecum compared to controls and DC appeared minimally (data not shown). Together, these
findings indicate decreased microbial conversion of primary BS in the cecum during PEG treatment. This
was consistent with the significant relative decrease of Clostridia related bacteria, that were previously
identified as major bacteria involved in conversion of primary to secondary BS. 25,26
We have previously shown that PEG accelerates whole gut transit time (WGTT) and small intestinal
transit in rats. 3 In the current study PEG treated rats received the same diet and PEG dosage, and WGTT
was similarly accelerated. Marcus and Heaton have shown significant correlations between acceleration
of WGTT with senna (by 62%) and decreased DC pool (-30%) and between prolongation of WGTT
with loperamide (by 115%) and increased DC pool (+43%). 14 Veysey et al. have also found significant
correlations between prolonged colonic transit and increased DC pool size in humans. 27
Serum DC % was related to microbial BS converting enzyme activity and colonic pH. 13,28 It was found that
cholate pool size was reciprocally decreased during prolonged colonic transit. 27 Berr et al. found that
increased activity of microbial BS converting enzymes in patients with cholesterol gallstones enhances
DC pool size in the absence of changes in WGTT. 12
PEG treatment affects enterohepatic circulation of bile salts
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Together, the data show that changes in BS pool size may be due to alterations in WGTT, but could also
be facilitated via other mechanisms.
Many bacterial species are able to deconjugate BS, whereas the conversion of primary to secondary BS
seems to be restricted to few gram-positive anaerobes. 10 A lower bacterial load, with a shift in intestinal
microbial profile, leading to decreased amount of luminal bacteria, such as Clostridia, that were able
to convert cholate (and HDC) into other BS, seems to have primarily influenced cholate kinetics. The
exact mechanism inducing these microbial changes is currently unknown, but may include increased
intestinal water content and changes in luminal pH induced by PEG.
The cytotoxic effect of fecal water is primarily enhanced by BS and fatty acids. 23 Fatty acids are not
metabolized in the intestinal lumen and we previously showed that the absorption and excretion of fatty
acids is not changed by PEG 29, implying that fatty acids would not differentially influence fecal water
cytotoxicity in PEG treated rats. The decrease in fecal water cytotoxicity during PEG treatment correlated
with decreased (secondary) BS excretion. Previous studies reported decreased tumor development
during PEG treatment in different mouse and rat models of colon cancer. 30-32 A population-based study
in patients undergoing colonoscopy showed a lower prevalence of colorectal tumors in PEG4000 users.33
An increased primary BS (cholate) pool may prevent BS damage in colonocytes. 11 It is tempting to
speculate that PEG positively influences gut health by decreasing the production of secondary BS in
the colon. Moreover, we found a relative increase of mucus associated bacteria (Akkermansia spp. 34),
shown to be important for a healthy mucus layer in the human gut with respect to mucus production
and thickness.
Given the frequent and long-term prescriptions of PEG in clinical practice, we believe that human
studies are warranted to further delineate the effect of PEG on intestinal microbiota and BS metabolism.
AcknowledgementsThe authors would like to thank Juul Baller and Rick Havinga for their help during animal experiments
and Theo van Dijk for contributing to the analyses of cholate kinetics. In addition, we would like to thank
Renze Boverhof and Theo Boer for technical and Vincent Bloks for statistical assistance.
Conflicts of interest and source of funding
M.Y.M. van der Wulp is currently receiving an unrestricted research grant from Top Institute Food and
Nutrition via the University Medical Center Groningen. None of the authors has a conflict of interest to
declare.
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References1. DiPalma JA, Cleveland MV, McGowan J, et al. A randomized, multicenter, placebo-controlled trial of polyethylene
glycol laxative for chronic treatment of chronic constipation. Am J Gastroenterol. 2007 Jul;102(7):1436-41.
2. Pashankar DS, Loening-Baucke V, Bishop WP. Safety of polyethylene glycol 3350 for the treatment of chronic constipation in children. Arch Pediatr Adolesc Med. 2003 Jul;157(7):661-4.
3. Cuperus FJ, Iemhoff AA, van der Wulp MY, et al. Acceleration of the Gastrointestinal Transit by Polyethylene Glycol Effectively Treats Unconjugated Hyperbilirubinemia in Gunn Rats. Gut. 2010 Mar;59(3):373-80.
4. Woollett LA, Wang Y, Buckley DD, et al. Micellar solubilisation of cholesterol is essential for absorption in humans. Gut. 2006 Feb;55(2):197-204.
5. Li T, Chiang JY. Bile Acid signaling in liver metabolism and diseases. J Lipids. 2012;2012:754067.
6. Out C, Groen AK, Brufau G. Bile acid sequestrants: more than simple resins. Curr Opin Lipidol. 2012 Feb;23(1):43-55.
7. Beysen C, Murphy EJ, Deines K, et al. Effect of bile acid sequestrants on glucose metabolism, hepatic de novo lipogenesis, and cholesterol and bile acid kinetics in type 2 diabetes: a randomised controlled study. Diabetologia. 2012 Feb;55(2):432-42.
8. Brufau G, Stellaard F, Prado K, et al. Improved glycemic control with colesevelam treatment in patients with type 2 diabetes is not directly associated with changes in bile acid metabolism. Hepatology. 2010 Oct;52(4):1455-64.
9. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and functions. Front Biosci. 2009;14:2584-98.
10. Ridlon JM, Kang DJ, Hylemon PB. Bile salt biotransformations by human intestinal bacteria. J Lipid Res. 2006 Feb;47(2):241-59.
11. Bernstein H, Bernstein C, Payne CM, et al. Bile acids as endogenous etiologic agents in gastrointestinal cancer. World J Gastroenterol. 2009 Jul;15(27):3329-40.
12. Berr F, Kullak-Ublick GA, Paumgartner G, et al. 7 Alpha-Dehydroxylating Bacteria Enhance Deoxycholic Acid Input and Cholesterol Saturation of Bile in Patients with Gallstones. Gastroenterology. 1996 Dec;111(6):1611-20.
13. Thomas LA, Veysey MJ, Bathgate T, et al. Mechanism for the transit-induced increase in colonic deoxycholic acid formation in cholesterol cholelithiasis. Gastroenterology. 2000 Sep;119(3):806-15.
14. Marcus SN, Heaton KW. Intestinal transit, deoxycholic acid and the cholesterol saturation of bile--three inter-related factors. Gut. 1986 May;27(5):550-8.
15. Hulzebos CV, Renfurm L, Bandsma RH, et al. Measurement of parameters of cholic acid kinetics in plasma using a microscale stable isotope dilution technique: application to rodents and humans. J Lipid Res. 2001 Nov;42(11):1923-9.
16. Hulzebos CV, Bijleveld CM, Stellaard F, et al. Cyclosporine A-induced reduction of bile salt synthesis associated with increased plasma lipids in children after liver transplantation. Liver Transpl. 2004 Jul;10(7):872-80.
17. van Meer H, Boehm G, Stellaard F, et al. Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats. Am J Physiol Gastrointest Liver Physiol. 2008 Feb;294(2):G540-7.
18. Einarsson K, Angelin B, Ewerth S, et al. Bile acid synthesis in man: assay of hepatic microsomal cholesterol 7 alpha-hydroxylase activity by isotope dilution-mass spectrometry. J Lipid Res. 1986 Jan;27(1):82-8.
19. Sesink AL, Termont DS, Kleibeuker JH, et al. Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme. Cancer Res. 1999 Nov;59(22):5704-9.
20. Govers MJ, Termont DS, Lapre JA, et al. Calcium in milk products precipitates intestinal fatty acids and secondary bile acids and thus inhibits colonic cytotoxicity in humans. Cancer Res. 1996 Jul;56(14):3270-5.
21. Starrenburg MJ, Hugenholtz J. Citrate Fermentation by Lactococcus and Leuconostoc spp. Appl Environ Microbiol. 1991 Dec;57(12):3535-40.
22. Caporaso JG, Kuczynski J, Stombaugh J, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010 May;7(5):335-6.
23. Pearson JR, Gill CI, Rowland IR. Diet, fecal water, and colon cancer--development of a biomarker. Nutr Rev. 2009 Sep;67(9):509-26.
24. Craddock AL, Love MW, Daniel RW, et al. Expression and transport properties of the human ileal and renal sodium-dependent bile acid transporter. Am J Physiol. 1998 Jan;274(1 Pt 1):G157-69.
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25. Ridlon JM, Kang DJ, Hylemon PB. Isolation and characterization of a bile acid inducible 7alpha-dehydroxylating operon in Clostridium hylemonae TN271. Anaerobe. 2010 Apr;16(2):137-46.
26. Ridlon JM, Hylemon PB. Identification and characterization of two bile acid coenzyme A transferases from Clostridium scindens, a bile acid 7alpha-dehydroxylating intestinal bacterium. J Lipid Res. 2012 Jan;53(1):66-76.
27. Veysey MJ, Thomas LA, Mallet AI, et al. Colonic transit influences deoxycholic acid kinetics. Gastroenterology. 2001 Oct;121(4):812-22.
28. Thomas LA, Veysey MJ, Murphy GM, et al. Octreotide induced prolongation of colonic transit increases faecal anaerobic bacteria, bile acid metabolising enzymes, and serum deoxycholic acid in patients with acromegaly. Gut. 2005 May;54(5):630-5.
29. van der Wulp MY, Cuperus FJ, Stellaard F, et al. Laxative treatment with polyethylene glycol does not affect lipid absorption in rats. J Pediatr Gastroenterol Nutr. 2012 Oct;55(4):457-62.
30. Roy HK, Gulizia J, DiBaise JK, et al. Polyethylene glycol inhibits intestinal neoplasia and induces epithelial apoptosis in Apc(min) mice. Cancer Lett. 2004 Nov;215(1):35-42.
31. Do K, Barnard GF. Effect of concomitant polyethylene glycol and celecoxib on colonic aberrant crypt foci and tumors in F344 rats. Dig Dis Sci. 2005 Jul;50(7):1304-11.
32. Corpet DE, Parnaud G, Delverdier M, et al. Consistent and fast inhibition of colon carcinogenesis by polyethylene glycol in mice and rats given various carcinogens. Cancer Res. 2000 Jun;60(12):3160-4.
33. Dorval E, Jankowski JM, Barbieux JP, et al. Polyethylene glycol and prevalence of colorectal adenomas. Gastroenterol Clin Biol. 2006 Oct;30(10):1196-9.
34. Belzer C, de Vos WM. Microbes inside-from diversity to function: the case of Akkermansia. ISME J. 2012 Mar;6:1449-58.
Chapter 4
Transintestinal cholesterol excretioncan be manipulated by dietary fat
composition in mice
Mariëtte Y.M. van der Wulp 1, 2, Sabina Lukovac 2, Theo H. van Dijk 2, Vincent W. Bloks 2,
Mark V. Boekschoten 1,3, Jan Dekker 1,4, Edmond H.H.M. Rings 1, 2, Albert K. Groen 1,2
and Henkjan J. Verkade 1, 2
1 Top Institute Food and Nutrition, Wageningen, The Netherlands2 Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Beatrix Children’s Hospital,
Groningen University Institute for Drug Exploration, University of Groningen, University Medical Center
Groningen, Groningen, The Netherlands
3 Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands4 Department of Animal Sciences, Wageningen University, Wageningen, The Netherlands
Submitted
Chapter 4
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Abstract
Introduction Cholesterol can be excreted from the body with feces via a hepatobiliary or a
transintestinal route. We investigated the quantitative eff ects of the degree of dietary fatty acid
saturation on the two origins of fecal cholesterol.
Methods Mice were fed either a standard high fat diet (controls) or a high fat diet with a low
polyunsaturated to saturated fatty acid (P/S) ratio. We determined parameters of cholesterol
homeostasis and performed RNA-microarray analysis on jejunal mucosa.
Results Intake and absorption of cholesterol were unchanged during low P/S ratio diet feeding,
but fecal cholesterol excretion was increased compared with control diet (+68%; p<0.01). The low
P/S ratio diet did not aff ect biliary cholesterol secretion but increased transintestinal cholesterol
excretion (TICE; +137%; p<0.01). The low P/S ratio diet increased jejunal expression of genes involved
in cholesterol synthesis (Srebf2 and target genes), however, whole body de novo cholesterol synthesis
was quantitatively unaltered. Feeding a low P/S ratio diet resulted in a net negative body cholesterol
balance (p<0.01).
Conclusion A low P/S ratio diet induces TICE and a net negative cholesterol balance. Dietary
manipulation in general could represent an attractive strategy to induce TICE in prevention and
treatment of hypercholesterolemia and cardiovascular disease.
Dietary fat induced fecal cholesterol disposal
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Introduction
Disturbances in cholesterol homeostasis are associated with potential life-threatening consequences.
For example, hypercholesterolemia represents a major risk factor for cardiovascular disease. 1,2 Cholesterol
homeostasis is mainly regulated by intestinal absorption, fecal excretion and de novo synthesis of
cholesterol. 3 Fecal excretion of cholesterol was classically believed to be primarily driven by cholesterol
secreted via the hepatobiliary pathway. Recently, however, it has become apparent that direct secretion
of cholesterol from the blood compartment to the intestine, or so-called “transintestinal cholesterol
excretion” (TICE), plays a major role in disposal of cholesterol in feces, at least in mice. 3,4
Inhibition of cholesterol synthesis has been the most potent therapy for hypercholesterolemia for
decades. 5 However, inhibition of cholesterol synthesis alone reduces risk of cardiovascular disease by
only 30% and alternative therapeutic modalities are needed. 6,7 In recent years, reduction of intestinal
cholesterol absorption has become increasingly important as a target to decrease plasma cholesterol
levels. 8 A new focus in this field may be induction of TICE, which was shown to be regulated by nuclear
receptor agonists and dietary fat content. 9-12 Recently, Lo Sasso et al. demonstrated that selective
upregulation of intestinal Liver X receptor (Nr1h3 or Lxr) increased fecal sterol output and decreased
atherosclerosis in apo E knock-out mice, suggesting that upregulation of TICE may have beneficial effects
in atherosclerosis. 13 The mechanism involved in cholesterol transport from the blood compartment to
the intestinal lumen remains enigmatic and more studies are needed to clarify the conditions under
which TICE can be stimulated.
We previously induced essential fatty acid deficiency (EFAD) in mice to study its effects on intestinal
function. EFAD was induced by replacing dietary polyunsaturated fatty acids with saturated fatty acids
in a high fat diet, thus highly decreasing the polyunsaturated to saturated fatty acid ratio (P/S ratio). We
found that fecal fat excretion was increased in EFAD mice. 14 A detailed analysis of the development of
fecal lipid (dietary fat and cholesterol) excretion indicated that it increased already before mice became
essential fatty acid deficient (EFAD is defined by plasma triene (20:3n-9)-to-tetraene (20:4n-6) ratio >0.2,
see supplementary figure 1 and ref. 14). This observation suggested that the low P/S ratio, rather than
the EFAD itself, increased the lipid excretion.
Recently, it was shown that, compared with a low fat diet, a high fat diet decreased cholesterol absorption
and increased HMG-CoA reductase activity in the small intestine of mice. 15 In hamsters, it was previously
shown that dietary saturated stearic acid can increase fecal cholesterol excretion. 16,17 Schneider et al.
showed decreased cholesterol absorption and suggested increased cholesterol synthesis in hamsters
fed a high stearic acid diet. 16
The aim of the current study was to test whether cholesterol excretion could be manipulated by feeding
mice a high fat diet with a low P/S ratio as compared with a high fat control diet. We hypothesized that
administration of a low P/S ratio diet would affect total body cholesterol homeostasis. We determined
the consequences of a low P/S ratio diet on cholesterol absorption and synthesis, the origin of fecal
sterols, and total body cholesterol balance.
Chapter 4
76
The stable isotope methodology to obtain quantitative information on these relevant parameters of
cholesterol homeostasis had previously been developed and validated in our laboratory. 18 In order to
characterize the effects of the low P/S ratio diet on the metabolic pathways involved in cholesterol
homeostasis in the small intestinal mucosa, we analyzed the transcriptional response to the low P/S ratio
diet in mice. Our data show that the low P/S ratio diet does not significantly affect de novo cholesterol
synthesis rate or cholesterol absorption, but that it increases cholesterol output via increased TICE.
Collectively, the low P/S diet induced a negative cholesterol balance, indicative of net loss of cholesterol
from the body.
Materials and Methods
MaterialsIntralipid® (20%) was obtained from Fresenius Kabi, Den Bosch, The Netherlands. 2,2,4,4,6-Deuterium-
cholesterol (D5-cholesterol) was obtained from Medical Isotopes and 25,26,26,26,27,27,27-Deuterium-
cholesterol (D7-cholesterol) from Cambridge Isotope Laboratories Inc. 1-13C-acetate was obtained from
Sigma Aldrich. All isotopes were of 98-99% isotopic purity.
Mice and dietFriend Virus B (FVB type NHanHsd) male mice of 8 weeks old were purchased from Harlan (Horst, The
Netherlands) and were housed in a light- and temperature-controlled facility. Tap water and food were
allowed ad libitum. Mice were maintained on either control (#4141.07; 0.20 μmol cholesterol.g food-1)
or low P/S ratio (#4141.08; 0.18 μmol cholesterol.g food-1) diet for 8 weeks (n= 6-7 per group), which
were both custom synthesized by Arie Blok BV (Woerden, The Netherlands). Fatty acid composition
and polyunsaturated to saturated fatty acid (P/S) ratio of the diets are indicated in table 1. We studied
cholesterol homeostasis and in a separate experiment, we performed microarray analyses on small
intestinal RNA. The experiments were performed in conformity with Public Health Service policy and
in accordance with the national laws. The Ethics Committee for Animal Experiments of the University
Medical Center of Groningen approved the experimental protocols.
Table 1. Fatty acid composition and P/S ratio of high fat control and intervention diet
Mol% Control diet (4141.07) Low P/S ratio diet (4141.08)
Myristic acid (14:0)Palmitic acid (16:0)Stearic acid (18:0)Oleic acid (18:1n-9)Vaccenic acid (18:1n-7)Linoleic acid (18:2n-6)α-Linolenic acid (18:3n-3)
0.933.54.6
32.51.1
26.90.6
1.460.132.94.30.21.00.0
P/S ratio 0.7 0.01
Concentrations are indicated in mol% of total fatty acid concentrations determined by GC.
Dietary fat induced fecal cholesterol disposal
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Chapter
4
Cholesterol kinetic study
After 8 weeks of diet, baseline bloodspots were collected on filter paper from the tail vein and feces were
collected during a 24h period (day -1). Food pellet weight was determined before and after the 24h
feces collection period and pellets were collected.
Subsequently, mice were switched to tap water containing 2% stable isotope labeled 1-13C-acetate at
9 A.M. (day 1) for 3 days. Bloodspots were collected every morning at 9 o’clock and body weight and
food intake were determined daily for the remainder of the experiment. Day 4-6 represented a wash-
out period for labeled acetate. At day 7 mice received an intravenous (retro-orbital) injection of 1.5
mg D7-cholesterol dissolved in 500 μl intralipid and an oral dose of 3 mg D
5-cholesterol dissolved in 1
ml medium chain triglyceride (MCT) oil. At time points 3, 6, 12, 24, 48, 72, 96, 120, 144 and 168h after
labeled cholesterol administration, bloodspots were obtained. Feces were collected daily from label
administration on until the end of the experiment. At day 14, mice were anesthetized and the common
bile duct was cannulated for bile collection during 30 minutes as previously described. 19 Mice were
sacrificed by cardiac puncture and cervical dislocation. The small intestine was divided into three equal
parts, which were rinsed with phosphate-buffered saline (PBS). Livers were snap frozen in liquid nitrogen
and stored at -80°C.
Analytical methods Indirect cholesterol balance
Biliary lipids were extracted 20 and total plasma and biliary cholesterol concentrations were determined. 18
Food pellets and fecal samples were ground and 50 mg was prepared for neutral sterol (cholesterol plus
bacterial metabolites coprostanol and dehydrocholesterol) and bile salt analysis by gas chromatography
(GC) as described previously. 21 Indirect cholesterol balance was determined by subtraction of dietary
cholesterol intake and hepatobiliary secretion from fecal output of neutral sterols (all calculated in μmol
cholesterol.day-1.100 g of body weight-1). 10
Neutral sterol content in intestinal lumen
Small intestines were divided into three parts of equal size. Cecum was separated from remaining colon.
Small intestine and remaining colon were flushed with 5 ml PBS. Samples were stored at -20°C. Before
analyses, tubes were lyophilized overnight. Aliquots of 10 (proximal and middle part of small intestine)
to 50 (distal small intestine, cecum and colon) mg were used to determine neutral sterols by GC as
described above.
Fractional cholesterol absorption
The procedure for this study, as described in detail by van der Veen et al. 18, was modified for the influx
of labeled cholesterol. Briefly, cholesterol was extracted overnight from bloodspots with 1 ml 100%
ethanol/ acetone (1:1 v/ v). Unesterified cholesterol was derivatized and enrichments of sterols were
determined by GC-mass spectrometry (GC-MS).
Chapter 4
78
The ions monitored were m/z 458-465, corresponding to the M0-M
7 mass isotopomers of cholesterol-
methyl ester/ trimethylsilyl (TMS) derivatives. Fractional cholesterol absorption (F(a)) was calculated as
the ratio between fraction (area under the curve (AUC) of 7 days following label administration) of orally
administered D5-cholesterol and IV administered D
7-cholesterol, after correction for their administered
doses: F(a)= (AUC oral
/ AUCIV) x (Dose
IV / Dose
oral).
Cholesterol synthesis
Fractional cholesterol synthesis was determined by mass isotopomer distribution analysis (MIDA). 22 With
MIDA we could determine the enrichment of acetyl-CoA precursor units (precursor pool enrichment)
that entered newly synthesized cholesterol during 1-13C-acetate administration by analysis of the mass
isotopomer pattern of cholesterol molecules according to a probability model of cholesterolgenesis. In
short, labeled and unlabeled monomeric acetyl-CoA combine in newly formed polymeric cholesterol
molecules. The mass isotopomer pattern of M1 and M
3 cholesterol-TMS derivatives as quantified
by GC-MS was compared to theoretical patterns calculated for cholesterol over a range of precursor
enrichments. 18 A match between experimental and theoretical combinatorial patterns established the
isotopic enrichment of the precursor pool. Knowing the precursor pool enrichment, it was possible
to calculate the expected frequency of M1 and M
3 isotopomers in newly synthesized cholesterol. The
fractional cholesterol synthesis was then determined by comparing the actual M1 and M
3 enrichment
during the experiment with the expected frequencies.
Origin of fecal sterols
In order to determine the origin of fecal sterols, we modified the method described by van der Veen et
al. 18 In physiological terms, fecal sterols can originate from either diet, or de novo synthesis via various
routes, e.g. biliary secretion, transintestinal cholesterol excretion (TICE) or shedding of enterocytes.
Stable isotope studies in combination with mathematical modeling allow quantification of the different
fluxes. Assuming a similar absorption of dietary and biliary derived cholesterol 23 the different fluxes can
be modeled as follows:
Fecal sterols excreted (totalE) were calculated as the average over the time period 24-144h.
The dietary contribution to fecal neutral sterol loss can be calculated as:
ED→F
= (1-F(a)) * D
In which E D→F
is the cholesterol intake calculated in the diet (D) corrected for fractional cholesterol
absorption.
The fecal output of biliary excreted cholesterol (totalEbile) was calculated as bile flow (in µl.100g BW-1.day-1)
times cholesterol concentration in bile, and corrected for cholesterol absorption:totalEbile = flow * [chol] * (1-F(a))
TICE was calculated as: TICE= totalE- totalEbile -ED→F
Dietary fat induced fecal cholesterol disposal
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4
This method does not allow discrimination of blood-derived TICE and fecal cholesterol derived from
the intestinal mucosa itself (i.e. epithelial shedding is included in the TICE fraction). Previous intestinal
perfusion studies showed that epithelial shedding contributes no more than 15% to intestinal cholesterol
secretion. 9,24
Total body cholesterol balance
Total body cholesterol balance was calculated as follows: (cholesterol intake + cholesterol synthesis) –
(fecal neutral sterol + bile salt excretion).
Microarray analyses
Mice were maintained on semisynthetic diets in two groups as described above (6 mice per group).
Body weight and food intake were determined weekly. After 8 weeks of diet, mice were anesthetized
and sacrificed by cervical dislocation. Small intestines were rinsed with PBS, divided into three equal
parts, and the middle parts were sliced and immediately frozen in liquid nitrogen. Subsequently, small
intestinal tissue was stored at -80°C for RNA isolation. Livers were snap frozen in liquid nitrogen and
stored at -80°C.
Analytical methods RNA isolation and measurement of RNA expression levels by microarray analysis and quantitative PCR
Total RNA was prepared from the middle third of the small intestinal tissue and liver using TRIzol reagent
(Invitrogen, Breda, The Netherlands). Subsequently, cDNA synthesis and quantitative PCR (qPCR) analysis
were performed as described by Grefhorst et al. 25 qPCR results were normalized to mRNA expression
of the housekeeping gene 18S. Primer and probe sequences for all tested genes have been deposited
at the RTprimerDB. 26 For microarray analysis, the Affymetrix platform was employed. After RNA
isolation with TRIzol reagent, RNA was used individually and further purified using RNeasy MinElute
micro columns (Qiagen, Venlo, The Netherlands). RNA integrity was checked on an Agilent 2100 bio-
analyzer (Agilent Technologies, Amsterdam, The Netherlands) using 6000 Nano Chips according to
the manufacturer’s instructions. RNA was judged as suitable for array hybridization only if samples
exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no
chromosomal peaks or RNA degradation products (RNA Integrity Number.7.0-8.5). Five hundred ng of
RNA were used for one cycle cRNA synthesis (Affymetrix, Santa Clara, CA). Hybridization, washing and
scanning of Affymetrix Gene chip mouse 1.0 ST arrays were performed according to standard Affymetrix
protocols. Scans of the Affymetrix arrays were processed using packages from the Bioconductor
project. 27 Quality control of microarray data (using simpleaffy and affyplm packages), normalization
and differential expression analysis were performed through the Management and Analysis
Database for MicroArray eXperiments (MADMAX 28) analysis pipeline (Wageningen, The Netherlands).
Expression levels of probe sets were calculated using regular normalization strategies: VSN in small
intestine followed by identification of differentially expressed probe sets using Limma. 29
Chapter 4
80
Comparisons were made between treated (low P/S ratio diet) and untreated (control) groups
(Limma package, applying linear models and moderated t-statistics that implement empirical Bayes
regularization of standard errors. 30 False discovery rate (FDR) of 1% (p-value <0.01) was used as a
threshold for significance of differential expression. Identification of overrepresented functional
categories among responsive genes and their grouping into functionally related clusters (Biological
Processes:BP-4) was performed using DAVID Functional Annotation Clustering tool. 31 All microarray data
reported in the manuscript are described in accordance with MIAME guidelines 32 and are available in
the Gene Expression Omnibus (GEO) databank (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token
=nrkjxeokqomuwxe&acc=GSE37097).
Determination of cholesterol concentration in small intestinal mucosa
Thirty mg of mucosal scrapings of the mid part of the small intestine was homogenized in 200 μl of 0.9%
NaCl. Lipids were extracted from the homogenate 20 and cholesterol concentrations were determined
with a commercially available kit (DiaSys Diagnostic Systems, Holzheim, Germany).
Statistical analysis
Using SPSS version 16 statistical software (Chicago, IL, USA), we calculated significance of differences
between mice on low P/S ratio versus control diet with Mann-Whitney U-tests. P-values <0.05 were
considered statistically significant. Data represent median values and interquartile range.
Results
Mice on a low P/S ratio diet have an increased fecal cholesterol excretion (indirect cholesterol
balance)
In order to investigate the effect of the dietary fat composition on fecal sterol excretion, FVB mice were
fed for 8 weeks with either a control high fat diet (polyunsaturated/saturated fat or P/S ratio 0.7) or a
high fat diet with a very low P/S ratio (P/S ratio 0.01) (table 2). Feeding the low P/S ratio diet nearly
doubled daily fecal cholesterol excretion compared with control diet (table 2). Interestingly, the low P/S
ratio diet did not affect dietary cholesterol intake nor biliary cholesterol secretion. (table 2). Net fecal
cholesterol excretion thus was higher than the sum of cholesterol derived from intake and cholesterol of
hepatobiliary origin (table 2). Note that this contrasts to the control situation where net fecal cholesterol
excretion is lower than the sum of intake and hepatobiliary flux, indicating net (re)absorption.
Dietary fat induced fecal cholesterol disposal
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Chapter
4
Table 2. Indirect cholesterol balance
μmol.day-1.100g body weight-1 Control Low P/S ratio
Dietary cholesterol intake
Biliary cholesterol secretion
Fecal neutral sterol excretion
2.1 [2.1-2.2]
2.5 [1.6-4.1]
4.7 [3.2-5.3]
2.3 [2.1-2.4]
3.5 [2.5-4.0]
7.9 [6.8-9.8]**
Net non-hepatobiliary cholesterol excretion(<0 = net absorption; >0 = net excretion)
~ -0.5 ~ 2.5*
Data represent medians and interquartile ranges of 6-7 mice per group. *p<0.05, **p<0.01 indicate significant differences between low P/S ratio diet and control mice after 8 weeks of diet, respectively.
To gain more insight in the origin of the fecal cholesterol, we determined neutral sterol content in
the intestinal tract. Neutral sterols recovered from the intestinal lumen during low P/S ratio diet were
significantly increased in the middle part of the small intestine (0.08 [0.07-0.10] versus 0.03 [0.02-0.07]
μmol; p= 0.01) and in the total intestinal tract (0.4 [0.4-0.5] versus 0.3 [0.3-0.4] μmol; p<0.05), compared
with controls (figure 1).
Figure 1. Neutral sterol content in intestinal lumen of low P/S ratio fed (black bars) and control (white bars) mice. SIP= small intestine proximal part, M= middle part, D= distal part, Cec= cecum, Col= remaining part of colon. Values represent medians and interquartile ranges for n=6-7 mice per group. *p<0.05 indicates significant difference between the two groups.
Low P/S ratio diet does not change cholesterol absorption or synthesis, but induces transintestinal
cholesterol excretion
To be able to estimate the quantitatively most important cholesterol fluxes in the body we determined,
in addition to dietary intake and biliary flux, cholesterol absorption and whole body de novo synthesis.
Using stable isotope labeled tracers the contribution of dietary, biliary and intestinal cholesterol to
fecal sterols was calculated. 18 Fractional cholesterol absorption was measured by a dual stable isotope
method. Although fractional cholesterol absorption on average was lower in low P/S ratio diet fed
mice versus controls (49 [39-57] versus 65 [46-71]), the difference did not reach statistical significance
(p>0.1). In order to assess whole body cholesterol synthesis, mice were given drinking water containing
1-13C-acetate.
Chapter 4
82
Subsequently cholesterol synthesis was determined by analysis of cholesterol mass-isotopomer
distributions due to incorporation of the precursor 1-13C-acetate in cholesterol in bloodspots over
3 consecutive days. Mice on a low P/S ratio diet tended to have a lower cholesterol synthesis rate
compared to controls (figure 2, table 3) but the difference was not significant (p>0.07). As absorption
and biliary cholesterol secretion were not altered by the low P/S diet, it was not surprising that fecal
cholesterol originating from diet and bile did not differ between dietary groups (figure 3). The low P/S
ratio diet strongly increased the amount of cholesterol derived from non-hepatobiliary origin, i.e. TICE,
compared with control diet (5.0 [3.7-7.0] versus 2.2 [1.5-3.3] μmol.100g BW-1.day-1; p<0.01, figure 3).
Table 3. Total body cholesterol balance
μmol.day-1.100g body weight-1 Control Low P/S ratio
Dietary cholesterol intake
Cholesterol synthesis
Fecal neutral sterol excretion
Fecal bile salt excretion
2.4 [2.3-2.5]
6.1 [3.3-7.4]
4.7 [3.2-5.3]
4.7 [4.4-7.3]
2.6 2.7-2.8]
2.9 [2.5-4.3]
7.9 [6.8-9.8]**
8.0 [6.0-10.8]
Total body cholesterol balance -3.5 [-4.5-0.8] -9.7 [-14.0-(-7.8)]**
Data represent medians and interquartile ranges of 6-7 mice per group. **P<0.01 indicates significant differences between low P/S ratio diet and control mice, respectively.
Figure 2. Cholesterol synthesis in low P/S ratio fed (black bar) and control (white bar) mice. Values represent medians and interquartile ranges for n=6-7 per group.
Figure 3. Origin of fecal sterols in low P/S ratio fed and control mice. Values represent means for n=6-7 per group. **p<0.01 indicates significant difference between the two groups.
Low P/S ratio diet induces a negative total body cholesterol balance
Bile salts are produced in the liver by breakdown of cholesterol and secreted to the intestine with bile.
Fecal bile salt excretion is a major route of disposal of body cholesterol. 33 A total body cholesterol
balance can be calculated by taking the sum of whole body cholesterol synthesis and dietary intake
minus the excreted fecal neutral sterols and bile salts.
Dietary fat induced fecal cholesterol disposal
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Chapter
4
On a low P/S ratio diet the total body cholesterol balance was negative suggesting lack of compensation
of fecal cholesterol loss by synthesis (table 3).
Low P/S ratio diet induces expression of jejunal genes involved in cholesterol metabolism
To investigate the effect of a low P/S ratio diet on gene expression microarray analysis was carried
out on samples from the middle part of the small intestine (jejunum). A large number of in total 962
genes were differentially regulated during low P/S ratio diet in mice, using a false discovery rate (FDR)
of 1% as a threshold for significance of differential expression. 565 genes were upregulated and 397
were downregulated. Based on the Gene Ontology categorization, low P/S ratio diet mainly affected
metabolic processes (table 4). In the top 10 of the processes most significantly enriched during low P/S
ratio diet, steroid biosynthetic processes and processes involved in steroid and fatty acid metabolism
were listed (table 4, indicated in bold font).
Table 4. Biological processes enriched during low P/S ratio diet in murine jejunum – microarray analysis (GOTERM_BP_4). False discovery rate (FDR) <1%.
GOTERM BP 4: Biological processes Number of genes p-value Fold enrichment
Cellular lipid metabolic process Translation
Lipid biosynthetic process Oxoacid metabolic process
Fatty acid metabolic process Sterol biosynthetic processCholesterol metabolic processSteroid biosynthetic processTransmembrane transport
Steroid metabolic process
69
52
44
60
3414181851
26
1.2E-15 3.7E-14
2.9E-113.4E-116.0E-114.2E-102.7E-83.4E-8
7.4E-8
2.4E-7
2.9 3.4
3.22.63.89.65.35.2
2.3
3.3
Top 10-scoring list of processes enriched in jejunum upon low P/S ratio diet feeding in mice.
Low P/S ratio diet alters expression of genes encoding jejunal cholesterol transporters
Over 20 genes involved in sterol metabolic processes were enriched during low P/S ratio diet, including
several genes involved in cholesterol absorption and efflux (table 5). Transcription of the Liver X receptors
(Lxrs) Lxrα (nr1h3) and Lxrβ (nr1h2), important transcriptional regulators of cholesterol metabolism, was
not significantly higher in jejunum during low P/S ratio diet (table 5). Expression of Lxr targets such
as Abcg5/g8 (cholesterol efflux) and ATP-binding cassette (Abc) A1 (table 5) was also unaltered or
even decreased. Verification by qPCR showed no change in expression for either Abca1 or Abcg5/g8.
Expression of Niemann-Pick c1-like 1 gene (Npc1l1, encoding the small intestinal cholesterol importer),
was decreased in jejunum of mice fed the low P/S ratio diet. Gene expression of the Ldl receptor (Ldlr),
implied in basolateral cholesterol uptake into enterocytes, was increased during low P/S ratio diet. qPCR
analysis confirmed the microarray results for Npc1l1 and Ldlr (figure 4).
Chapter 4
84
Table 5. Fold changes of genes involved in intestinal cholesterol transport and synthesis – microarray analysis.
Gene Description and protein IDFold change low P/S ratio fed versus control mice
Cholesterol transport
Lxrα (Nr1h3) Nuclear receptor subfamily 1, group H, member 3 No change
Lxrβ (Nr1h2) Nuclear receptor subfamily 1, group H, member 2 No change
Abcg5/g8 ATP-binding cassette sub-family G (White) member 5/8 No change*
Abca1 ATP-binding cassette, sub-family A (ABC1), member 1 ↓ 1.5 (NS in qPCR)
Npc1l1 Niemann-Pick C1-like protein 1 ↓ 1.8*
Ldlr Low density lipoprotein receptor ↑ 2.0*
Cholesterol synthesis
Mvk Mevalonate kinase ↑ 1.2
Pmvk Phosphomevalonate kinase ↑ 2.3
Mvd Mevalonatediphosphodecarboxylase ↑ 1.6
Fdps Farnesyldiphosphate synthase ↑ 2.0
Fdft1 Farnesyldiphosphate farnesyl transferase 1 (squalene synthase) ↑ 1.6
Sqle Squalene epoxidase (squalene monooxygenase) ↑ 1.8
Lss Lanosterol synthase ↑ 1.9
Cyp51 Cytochrome P450, family 51 (lanosterol 14-alpha demethylase) ↑ 2.4
Dhcr7 7-dehydrocholesterol reductase ↑ 1.4
Srebf1 Sterol regulatory element binding transcription factor 1 No change
Srebf2 Sterol regulatory element binding factor 2 ↑ 1.3*
Hmgcr 3-hydroxy-3-methylglutaryl-Coenzyme A reductase ↑ 1.4*
Hmgcs1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (cytoplasmic) ↑ 1.8*
Fold changes are indicated by the arrows in the direction of up- (↑) or downregulation (↓) of gene expression. False discovery rate (FDR) <1%. mRNA expression of genes indicated with an asterisk (*) has been confirmed by qPCR analysis (p<0.05).
Figure 4. Small intestinal expression of genes involved in cholesterol import and efflux in low P/S ratio fed (black bars) versus control (white bars) mice. Data represent mRNA expression relative to the ribosomal RNA expression of the housekeeping gene 18S. Values represent medians and interquartile ranges for n=6 mice per group. *p<0.05 indicates significant difference between the two groups.
Dietary fat induced fecal cholesterol disposal
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Low P/S ratio diet is associated with enhanced expression of genes involved in cholesterol synthesis in jejunum and liver
Microarray analysis of jejunal samples revealed that expression of 32 genes involved in steroid
biosynthesis was enriched during low P/S ratio diet (table 4), including mRNA expression of the rate
limiting enzyme in cholesterol synthesis, HMG-CoA reductase (Hmgcr, table 5). mRNA expression of
other genes relevant in the cholesterol biosynthesis pathway were also increased, namely soluble HMG-
CoA synthase 1 (Hmgcs1, cytoplasmic), mevalonate kinase (Mvk), phosphomevalonate kinase (Pmvk),
mevalonatediphosphodecarboxylase (Mvd), farnesyldiphosphate synthase (Fdps), squalene synthase
(Fdft1), squalene monooxygenase (Sqle), lanosterol synthase (Lss), Cytochrome P450, family 51 (Cyp51)
and 7-dehydrocholesterol reductase (Dhcr7) (table 5). Transcription of all of these genes is regulated by
the sterol regulatory element binding protein 2 (Srebp2). 34 Microarray analysis showed induction of
the gene encoding Srebp2 (Srebf2) in low P/S ratio fed mice (table 5). Results were validated with qPCR
analysis of mRNA expression of Hmgcr, Hmgcs1 and Srebf2 (figure 5A).
A
C D
B
Figure 5. (A) Small intestinal expression of genes involved in cholesterol synthesis in low P/S ratio fed (black bars) versus control (white bars) mice. Data represent mRNA expression relative to the ribosomal RNA expression of the housekeeping gene 18S. (B) Cholesterol concentrations in small intestinal mucosa of low P/S ratio fed (black bars) and control (white bars) mice. (C) Hepatic Hmgcr expression and (D) Total cholesterol concentration in liver. Values represent medians and interquartile ranges for n=6 mice per group. *p<0.05 and **p<0.01 indicate significant difference between the two groups.
Chapter 4
86
Increased expression of Hmgcr was also detected in liver (figure 5C). We did not detect significant
changes in whole body cholesterol synthesis in our stable isotope study. The low P/S ratio diet did not
affect cholesterol concentration in jejunal mucosa (3.2 [2.8-3.5] versus 3.0 [2.1-3.4] μmol.mg mucosa-1),
and only slightly affected it in liver (13.5 [12.9-14.5] versus 11.1 [9.6-12.2] μmol.mg liver-1; p= 0.01,
respectively) (figure 5B and 5D).
Discussion
The major finding in our study is that a high fat diet containing predominantly saturated fatty acids
induces a major increase in net sterol excretion from the body via induction of TICE. The low P/S ratio diet
had no effect on dietary cholesterol intake, biliary cholesterol secretion or overall cholesterol absorption.
Since de novo cholesterol synthesis did not compensate for the sterol loss, the low P/S diet induced a net
negative total body cholesterol balance after 8 weeks of dietary supplementation.
By performing intestinal perfusion experiments in mice, it was previously shown that a high fat diet (P/S
ratio ~4) increased TICE in the proximal part of the intestine compared with a low fat diet. 10 Van der Velde
et al. suggested that stimulation of TICE induced the observed increase in neutral sterol excretion. 10
The experimental set up of van der Velde et al. did not allow actual quantification of the contribution of
TICE to total cholesterol excretion. We modified the method described by van der Veen et al. 18 which
allows quantification of the net contribution of the major body fluxes of cholesterol to feces. In the
present study we found that the degree of fatty acid saturation in a high fat diet influences cholesterol
excretion. A low P/S ratio diet with high amounts of saturated stearic and palmitic acid and low amounts
of unsaturated oleic and linoleic acid compared with a higher P/S ratio diet turned out to be very
efficient in increasing neutral sterol excretion and this effect was mainly accounted for by increased TICE.
Reduced mRNA expression of the apical uptake transporter Npc1l1 35 in the small intestine suggested
that the increase in fecal neutral sterol excretion could, at least in part, be caused by cholesterol
malabsorption. However, we did not find significantly decreased cholesterol absorption during low
P/S ratio diet feeding using a dual stable isotope test. It was shown previously that induction of TICE
by peroxisome proliferator-activated receptor delta (PPARδ, Nr1c2) activation did not correlate with
decreased Npc1l1 mRNA 11. Stimulation of Lxr has been shown to increase TICE in mice. 12,18 In low P/S
ratio fed mice we did not find changed expression levels of Lxr nor its target genes Abcg5 and Abcg8.
In contrast, expression of Cyp27a1, producing 27OH-cholesterol, a major Lxrα ligand in enterocytes 36,
was significantly downregulated during low P/S ratio diet (not shown). These data suggest that TICE is
induced independently of Lxr activation in our model.
The low P/S ratio diet increased mRNA expression of the transcriptional regulator of cholesterol
biosynthesis Srepf2 37 and its target genes in jejunum, whereas cholesterol concentrations in the jejunal
mucosa were similar compared to mice on control diet. It is possible that intestinal cholesterol synthesis
is indeed increased during low P/S ratio diet to compensate for cholesterol loss via TICE. Our method
does not allow measurement of cholesterol synthesis in individual organs.
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Yet, whole body synthesis was not increased by the low P/S diet. Compared with previous studies in mice,
we found relatively low whole body cholesterol synthesis rates. 18,38-40 Several factors can account for the
differences, including the method applied, the time point, time frame and differences in cholesterol
absorption between different mouse strains. Earlier studies were performed with the tritiated water
incorporation method in the dark phase of the light cycle, when synthesis is known to be higher than
during the light phase. In addition, mice were terminated one hour after label administration. 38-40 Based
on previous studies, it is thought that measurement of cholesterol synthesis with MIDA over at least 24h
yields accurate whole body cholesterol synthesis rates. 22,41 Depending on the mouse strain, cholesterol
absorption and thus synthesis differ. 6 Compared with previous studies (in C57BL/6J 18,38, 129Sv mice 39
and mice with mixed backgrounds 40) we found a relatively high cholesterol absorption in FVB mice,
and therefore not surprisingly a relatively low cholesterol synthesis rate. Since fecal sterol excretion
increased, the mice developed a negative total body cholesterol balance on the low P/S diet. Carcass
analyses should be performed in future studies to determine cholesterol concentration and synthesis
in individual organs.
The question arises where exactly the extra fecal cholesterol excretion originated from. Theoretically,
increased fecal cholesterol excretion on the low P/S ratio diet could be due to increased desquamation of
small intestinal enterocytes. During intestinal perfusions studies in mice, cholesterol from desquamated
cells was estimated to contribute ~15% to fecal sterols. 9 Our group recently demonstrated that most
of the TICE flux induced by plant sterols, measured via the stable isotope method, is abrogated in the
absence of Abcg5/g8 suggesting that these transporters play an important role in the pathway. 42 The
dominant role of Abcg5/g8 also confirms the relatively minor contribution of intestinal cell shedding
to TICE. Previous studies showed that there were no significant differences in proliferative capacity of
enterocytes between low P/S ratio and control diet. 19 Taking these considerations into account, we
suggest hat (most of ) the increased neutral sterol output on a low P/S ratio diet originates from the blood
compartment. In early human studies the estimated contribution of cholesterol from desquamated cells
to the intestinal lumen was at most ~10-20%. 43-45 However, the existence of a transport route from
blood to feces via the intestine was not taken into account in these studies. The turnover of intestinal
epithelial cells in humans (3-5 days) is slower than that in mice. It is tempting to speculate that part of the
suggested desquamation-derived fecal cholesterol in humans represents blood derived TICE. Studies
are warranted to clarify the origin of fecal sterols in humans.
Mice on low P/S ratio diet were not ill and their intake and body weights were normal. We calculated
the loss of body cholesterol with the help of literature values 46 of total body cholesterol. In contrast
to control mice that were balanced, mice on a low P/S ratio diet were estimated to lose a small but
significant fraction (0.5%; p<0.01) of their total body cholesterol per day. This indicates that activation of
TICE may actually lead to net cholesterol disposal from the body. Additional studies (of longer duration)
are necessary to investigate this further. Our model allows for quantification of the net TICE derived
cholesterol in feces, however actual TICE flux cannot be measured. Since part of the secreted cholesterol
is likely reabsorbed in the intestine, the actual flux may in reality be much higher.
Chapter 4
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TICE might thus become much more prominent under conditions of impaired cholesterol absorption. 42
Altogether, our data clearly show that a low P/S ratio diet induces cholesterol excretion via the
transintestinal route. Transcriptional activation of cholesterol synthesis fails to compensate for fecal loss of
cholesterol. Induction of TICE by dietary manipulation (for example addition of long chain unabsorbable
fatty acids to avoid the negative side effects of saturated fatty acids) could represent an attractive target
in prevention and treatment of hypercholesterolemia and cardiovascular disease.
Acknowledgements
The authors would like to thank Juul Baller and Rick Havinga for their help during animal experiments,
and Mechteld Grootte Bromhaar, Renze Boverhof and Theo Boer for technical assistance.
Conflicts of interest and source of funding
M.Y.M. van der Wulp is currently receiving an unrestricted research grant from Top Institute Food and
Nutrition via the University Medical Center Groningen. None of the authors has a conflict of interest to
declare.
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11. Vrins CL, van der Velde AE, van den Oever K, et al. Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux. J Lipid Res. 2009 Oct;50(10):2046-54.
12. Kruit JK, Plosch T, Havinga R, et al. Increased fecal neutral sterol loss upon liver X receptor activation is independent of biliary sterol secretion in mice. Gastroenterology. 2005 Jan;128(1):147-56.
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14. Werner A, Minich DM, Havinga R, et al. Fat malabsorption in essential fatty acid-deficient mice is not due to impaired bile formation. Am J Physiol Gastrointest Liver Physiol. 2002 Oct;283:G900-8.
15. de Vogel-van den Bosch, HM, de Wit NJ, Hooiveld GJ, et al. A cholesterol-free, high-fat diet suppresses gene expression of cholesterol transporters in murine small intestine. Am J Physiol Gastrointest Liver Physiol. 2008 May;294(5):G1171-80.
16. Schneider CL, Cowles RL, Stuefer-Powell CL, et al. Dietary stearic acid reduces cholesterol absorption and increases endogenous cholesterol excretion in hamsters fed cereal-based diets. J Nutr. 2000 May;130(5):1232-8.
17. Imaizumi K, Abe K, Kuroiwa C, et al. Fat containing stearic acid increases fecal neutral steroid excretion and catabolism of low density lipoproteins without affecting plasma cholesterol concentration in hamsters fed a cholesterol-containing diet. J Nutr. 1993 Oct;123(10):1693-702.
18. van der Veen JN, van Dijk TH, Vrins CL, et al. Activation of the liver X receptor stimulates trans-intestinal excretion of plasma cholesterol. J Biol Chem. 2009 Jul;284:19211-9.
19. Lukovac S, Los EL, Stellaard F, et al. Essential fatty acid deficiency in mice impairs lactose digestion. Am J Physiol Gastrointest Liver Physiol. 2008 Sep;295(3):G605-13.
20. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37:911-7.
21. van Meer H, Boehm G, Stellaard F, et al. Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats. Am J Physiol Gastrointest Liver Physiol. 2008 Feb;294:G540-7.
22. Neese RA, Faix D, Kletke C, et al. Measurement of endogenous synthesis of plasma cholesterol in rats and humans using MIDA. Am J Physiol. 1993 Jan;264(1 Pt 1):E136-47.
23. Wilson MD, Rudel LL. Review of cholesterol absorption with emphasis on dietary and biliary cholesterol. J Lipid Res. 1994 Jun;35(6):943-55.
24. Ferezou J, Coste T, Chevallier F. Origins of neutral sterols in human feces studied by stable isotope labeling (D and 13C). Existence of an external secretion of cholesterol. Digestion. 1981;21(5):232-43.
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25. Grefhorst A, Elzinga BM, Voshol PJ, et al. Stimulation of lipogenesis by pharmacological activation of the liver X receptor leads to production of large, triglyceride-rich very low density lipoprotein particles. J Biol Chem. 2002 Sep;277(37):34182-90.
26. Lefever S, Hellemans J, Pattyn F, et al. RDML: structured language and reporting guidelines for real-time quantitative PCR data. Nucleic Acids Res. 2009 Apr;37(7):2065-9.
27. Gentleman RC, Carey VJ, Bates DM, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004;5(10):R80.
28. Lin K, Kools H, de Groot PJ, et al. MADMAX - Management and analysis database for multiple ~omics experiments. J Integr Bioinform. 2011 Jul;8(2):160.
29. Irizarry RA, Wu Z, Jaffee HA. Comparison of Affymetrix GeneChip expression measures. Bioinformatics. 2006 Apr;22(7):789-94.
30. Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004;3:Article3.
31. Dennis G,Jr., Sherman BT, Hosack DA, et al. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 2003;4(5):3.
32. Brazma A, Hingamp P, Quackenbush J, et al. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet. 2001 Dec;29(4):365-71.
33. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and functions. Front Biosci. 2009 Jan;14:2584-98.
34. Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest. 2002 May;109(9):1125-31.
35. Altmann SW, Davis HR,Jr., Zhu LJ, et al. Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption. Science. 2004 Feb;303(5661):1201-4.
36. Li T, Chen W, Chiang JY. PXR induces CYP27A1 and regulates cholesterol metabolism in the intestine. J Lipid Res. 2007 Feb;48(2):373-84.
37. Hua X, Yokoyama C, Wu J, et al. SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element. Proc Natl Acad Sci U S A. 1993 Dec;90(24):11603-7.
38. Jolley CD, Dietschy JM, Turley SD. Genetic differences in cholesterol absorption in 129/Sv and C57BL/6 mice: effect on cholesterol responsiveness. Am J Physiol. 1999 May;276(5 Pt 1):G1117-24.
39. Osono Y, Woollett LA, Herz J, et al. Role of the low density lipoprotein receptor in the flux of cholesterol through the plasma and across the tissues of the mouse. J Clin Invest. 1995 Mar;95(3):1124-32.
40. Woollett LA, Osono Y, Herz J, et al. Apolipoprotein E competitively inhibits receptor-dependent low density lipoprotein uptake by the liver but has no effect on cholesterol absorption or synthesis in the mouse. Proc Natl Acad Sci U S A. 1995 Dec;92(26):12500-4.
41. Di Buono M, Jones PJ, Beaumier L, et al. Comparison of deuterium incorporation and mass isotopomer distribution analysis for measurement of human cholesterol biosynthesis. J Lipid Res. 2000 Sep;41(9):1516-23.
42. Brufau G, Kuipers F, Lin Y, et al. A reappraisal of the mechanism by which plant sterols promote neutral sterol loss in mice. PLoS One. 2011;6(6):e21576.
43. DenBesten L, Reyna RH, Connor WE, et al. The different effects on the serum lipids and fecal steroids of high carbohydrate diets given orally or intravenously. J Clin Invest. 1973 Jun;52(6):1384-93.
44. Cheng SH, Stanley MM. Secretion of cholesterol by intestinal mucosa in patients with complete common bile duct obstruction. Proc Soc Exp Biol Med. 1959 Jun;101(2):223-5.
45. Grundy SM, Metzger AL. A physiological method for estimation of hepatic secretion of biliary lipids in man. Gastroenterology. 1972 Jun;62(6):1200-17.
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Supplementary figure 1. Fecal fatty acid (FA) excretion in low P/S ratio fed (black bars) and control (white bars) mice after 4 weeks of diet (A). FA concentrations were determined in feces by gas chromatography and normalized to daily fecal output and body weight. Fecal neutral sterol excretion (B) and erythrocyte triene/ tetraene (TT) ratio (C) in low P/S ratio fed (black bars) and control (white bars) mice after 4 weeks of diet. TT ratio represents the ratio between eicosatrienoic (mead) acid (C20:3n-9) and arachidonic acid (C20:4n-6). Essential fatty acid deficiency is present when the TT ratio is increased above a threshold value of 0.2. Values represent medians and interquartile ranges for n=6 mice per group. **p<0.01 indicates significant difference between the two groups.
Chapter 5
Genetic inactivation of the bile salt export pump in mice profoundly increases fecal
cholesterol excretion
Mariëtte Y.M. van der Wulp 1,2, Theo H. van Dijk 2, Vincent W. Bloks 2,
Albert K. Groen 1,2, Henkjan J. Verkade 1,2
1 Top Institute Food and Nutrition, Wageningen, The Netherlands2 Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Beatrix
Children’s Hospital, Groningen University Institute for Drug Exploration, University of Groningen,
University Medical Center Groningen, Groningen, The Netherlands
In preparation
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Abstract
Objectives The bile salt export pump (Bsep) is the major hepatobiliary bile salt (BS) transporter,
facilitating transfer of BS from hepatocytes into the bile canaliculus. Bsep-/- mice were previously
shown to escape severe cholestasis by increasing biliary BS hydrophilicity, resulting in activation of
alternative transporters. Bsep-/- mice surprisingly displayed increased biliary cholesterol secretion.
The consequence of Bsep defi ciency and increased biliary cholesterol secretion for intestinal sterol
handling is unknown. To determine these downstream eff ects of Bsep defi ciency we determined BS
and whole body cholesterol fl uxes.
Methods Mice were fed either a low or a high fat diet (LFD or HFD, respectively), since it was shown
previously that a HFD can induce cholesterol excretion. We determined cholesterol intake, biliary
secretion, absorption and synthesis, output (feces), and the origin of fecal sterols.
Results Results were essentially the same in Bsep-/- mice fed a LFD or a HFD versus control mice.
Here, only results of LFD are presented. Biliary BS secretion was decreased in Bsep-/- compared
with Bsep+/+ mice (-40%; p<0.01). Biliary cholate secretion was decreased (-85%; p<0.001), whereas
beta-muricholate secretion was increased (+55%; p<0.01) in Bsep-/- mice. Cholesterol intake (+25%;
p<0.001), biliary secretion (4 fold; p<0.001) and synthesis (11 fold; p<0.001) were increased, whereas
cholesterol absorption was decreased (-90%; p<0.001) in Bsep-/- mice. Bsep-/- mice showed increased
fecal cholesterol excretion (8 fold; p<0.001), mostly via the transintestinal pathway (22 fold increase;
p<0.001).
Conclusion Bsep-/- mice show increased (transintestinal) cholesterol excretion, which appears to be
induced by severely impaired cholesterol absorption in the presence of a hydrophilic BS pool. Potential
health benefi ts of an increasingly hydrophilic BS pool in terms of intestinal cholesterol excretion require
further research.
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Introduction
Bile salts (BS) are produced by enzymatic modification of cholesterol in liver hepatocytes. They are
secreted by hepatocytes into bile canaliculi, to end up with bile in the small intestine. In the small
intestine, BS are indispensable for adequate absorption of lipids (dietary fat and cholesterol) and fat-
soluble vitamins. In the terminal ileum, BS are efficiently reclaimed by absorption (~95%) to continue
their enterohepatic circulation. 1
The main transporter facilitating transfer of BS from hepatocyte to bile canaliculus is the adenosine
triphosphate-dependent binding cassette transporter B11 (ABCB11), or bile salt export pump (BSEP). 2,3
Mutations in the Bsep gene can result in several forms of intrahepatic cholestasis. 4,5 Heterozygous Bsep
mutations have been identified in patients with intrahepatic cholestasis of pregnancy and drug-induced
cholestasis. 5 Missense Bsep mutations predominate in a relatively mild form of cholestatic disease
termed benign recurrent intrahepatic cholestasis type 2 (BRIC-2). 6 Frequently, Bsep mutations result in
the absence of canalicular BSEP protein 7, and are associated with biliary BS concentrations of less than
1% of normal. 3 Absence of functional BSEP results in progressive familial intrahepatic cholestasis type
2 (PFIC-2), a disease characterized by early onset of severe intrahepatic cholestasis, often requiring liver
transplantation within the first decade of life. 3,8
Murine Bsep and human BSEP were previously shown to possess comparable affinities for different
BS. 9 Bsep mainly transports conjugated di- and tri-hydroxylated BS, such as cholate (the main BS of
the human and rodent body BS pool) and chenodeoxycholate. 10,11 Bsep-/- mice were created to study
the effects of Bsep deficiency in detail in vivo. 12 An unexpected finding was that these mice displayed
a preserved basal bile flow rate. 12-15 Whereas the secretion of hydrophobic BS (cholate, deoxycholate
and chenodeoxycholate) was severely impaired, secretion of more hydrophilic BS (ursodeoxycholate
and muricholates) remained relatively unchanged. 12,15 Interestingly, Bsep-/- mice secreted substantial
amounts of tetra-hydroxylated BS in their bile, which were not found in Bsep+/+ mice. 12,14-16
It was shown that the multidrug resistance 1 protein could serve as a salvage (low affinity / high
capacity) pathway for BS secretion in Bsep-/- mice. 13,15 Bsep-/- mice showed uncoupling of secretion of
cholesterol and phospholipids with BS. In Bsep-/-, both biliary cholesterol and phospholipid secretion
were significantly increased. 12
Cholesterol homeostasis is mainly regulated by its intestinal absorption, fecal excretion and de novo
synthesis. 17 The influence of increased biliary cholesterol secretion for its intestinal handling are unknown.
The aim of the current study was to determine parameters of total body cholesterol homeostasis in Bsep-/-
mice and their wildtype littermates (Bsep+/+). Our data show that increased hydrophilic BS secretion in
Bsep-/- mice is associated with severely impaired cholesterol absorption and increased fecal cholesterol
output, mainly via the transintestinal pathway.
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Materials and Methods
Materials
Intralipid® (20%) was obtained from Fresenius Kabi, Den Bosch, The Netherlands. 2,2,4,4,6-Deuterium-
cholesterol (D5-cholesterol) was obtained from Medical Isotopes and 25,26,26,26,27,27,27-Deuterium-
cholesterol (D7-cholesterol) from Cambridge Isotope Laboratories Inc. 1-13C-acetate was obtained from
Sigma Aldrich (St. Louis, MO). All isotopes were of 98-99% isotopic purity. Sucrose and Trizma® base
were obtained from Sigma Aldrich (St. Louis, MO). Trimethylchlorosilane was obtained from Thermo
Scientific, Rockford, IL. Hydrochloric acid 37%, methanol, hexane and pyridine were obtained from
Merck, Darmstadt, Germany. Heptane was obtained from Rathburn chemicals ltd, Walkerburn, Scotland
and N,O-bis-(trimethyl)trifluoroacetamide (BSTFA) from Supelco, Bellefonte, PA.
Mice and diet
Mice were housed in a light- and temperature-controlled facility. Tap water and food were available ad
libitum. Mice were maintained on standard low fat diet (LFD) (RMH-B, 5 wt% fat, 0.66 µmol cholesterol.g-1)
or high fat diet (HFD) (#4141.07, 16 wt% (34 energy%) fat, 0.19 µmol cholesterol.g-1), both obtained from
Arie Blok BV (Woerden, The Netherlands). Bsep-/- mice on a mixed background 12 had been backcrossed
with C57BL/6 mice for at least 10 generations. Due to the low birth rate of Bsep-/- mice, it was inevitable
to use mice of different ages. Every mouse in the Bsep-/- group of a particular age was paired with a
wildtype (Bsep+/+) littermate of the same age. For the cholesterol kinetic study, male Bsep-/- and Bsep+/+
mice 4-15 months of age (LFD, n= 6 per group), and male Bsep-/- and Bsep+/+ mice 3-5 months of age
(HFD, n= 5 per group) were used. The experiments were performed in conformity with Public Health
Service policy and in accordance with the national laws. The Ethics Committee for Animal Experiments
of the University Medical Center of Groningen approved the experimental protocols.
Cholesterol kinetic study
The LFD was supplied to the mice from weaning until the experiment. The HFD was fed for 10 weeks
before the start of the experiment. Before any intervention, baseline bloodspots were collected on filter
paper from the tail vein and feces were collected during a 24h period (day -1). Food pellet weight was
determined before and after the 24h feces collection period and pellets were collected for quantification
of cholesterol content.
At day 1 mice received an intravenous (retro-orbital) injection of 1.5 mg D7-cholesterol dissolved in 500
μl intralipid and an oral dose of 3 mg D5-cholesterol dissolved in 1 ml medium chain triglyceride oil. At
time points 3, 6, 12, 24, 48, 72, 96, 120, 144 and 168h (day 8) after labeled cholesterol administration,
bloodspots were obtained. After taking the bloodspot at time point 168h, mice were switched to tap
water containing 2% stable isotope labeled 1-13C-acetate for 72h. Bloodspots were collected 12, 24, 32,
48, 56 and 72h (day 11, low fat diet) and 24, 32, 48, 56, 72, 80, 96h (day 12, HFD experiment) after the
start of 1-13C-acetate.
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Body weight and food intake were determined and feces were collected daily during the entire
experiment. At day 11 or 12 mice were anesthetized and the common bile duct was cannulated for bile
collection during 30 minutes as previously described. 18 Mice were sacrificed by cardiac puncture and
cervical dislocation. The small intestine was divided into three equal segments, which were rinsed with
phosphate-buffered saline (PBS 5 ml, low fat diet) and in addition with 5 ml of DEMI water containing
protease inhibitor (HFD experiments, 1 tablet per 50 ml of DEMI). Livers and intestines per segment were
snap frozen in liquid nitrogen and stored at -80°C.
Analytical methods
BS and indirect cholesterol balance
Biliary lipids were extracted 19 and total plasma cholesterol and biliary cholesterol and BS concentrations
were determined. 20,21 Food pellets and fecal samples were ground and 50 mg was prepared for neutral
sterol (cholesterol plus bacterial metabolites coprostanol and dehydrocholesterol) and BS analysis by
gas chromatography (GC) as described previously. 21 Indirect cholesterol balance was determined by
subtraction of dietary cholesterol intake and hepatobiliary secretion from fecal output of neutral sterols
(all calculated in μmol cholesterol.day-1). 22 Biliary BS hydrophobicity was calculated according to the
method of Heuman. 23
Fractional cholesterol absorption
The procedure for this study 20, was modified for the influx of labeled cholesterol. Fractional cholesterol
absorption (F(a)) was calculated as the ratio between fraction (area under the curve (AUC) of 7 days
following label administration) of orally administered D5-cholesterol and IV administered D
7-cholesterol,
after correction for their administered doses: F(a)= (AUC oral
/ AUCIV) x (Dose
IV / Dose
oral).
Cholesterol synthesis
Fractional cholesterol synthesis was determined by mass isotopomer distribution analysis (MIDA) 24, of
the M1 and M
3 mass isotopomers. 20
Origin of fecal sterols
In order to determine the origin of fecal sterols, we modified the method described by van der Veen et
al. 20
Neutral sterol content in intestinal lumen
Before analyses, tubes containing flushed intestinal lumen contents were lyophilized for 48h. Aliquots of
10 (proximal and middle part of small intestine) to 50 (distal small intestine, cecum and colon) mg were
used to determine neutral sterols by GC as described above.
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98
Preparation of intestinal mucosa homogenates
Intestinal sections (proximal small intestine (SIP), middle segment of small intestine (SIM) and distal
segment of small intestine (SID) were thawed (and kept) on ice and cut open. Mucosa was scraped off
the interior with an object glass, transferred to a pre-weighed potter glass (2 ml) and the potter glass was
weighed. One ml of ice-cold sucrose buffer (250 mM sucrose in 10 mM Trizma® base, pH 7.4) was added
and the solution was homogenized by pottering (10 strokes). The solution was further homogenized
by putting it through 1 ml syringes with needles of 20 and 26 G, respectively, 5-10 times with each
needle. The homogenate was thoroughly vortexed and divided over 3 eppendorf cups (2 ml). Ten µl of
homogenate was transferred to 2 10 ml glass tubes (tube 1 one for GC 21 and tube 2 for GC-MS 20 analyses
of cholesterol (M0-M
7). Glass tubes and remaining homogenate were kept at -20°C until further analyses.
Determination of cholesterol concentration in intestinal mucosa
The glass tubes containing homogenate were thawed and internal standard (5 nmol 5α-cholestane)
for neutral sterol quantification by GC was added to tube 1. Tube 2 was worked up for cholesterol label
analyses (GC-MS). Lipids were extracted from all tubes as described previously. 19 Cholesterol esters
were hydrolyzed with 2 ml of a mixture containing 37% hydrochloric acid (15 ml), DEMI water (10 ml)
and methanol (125 ml) (1h at 90°C). The mixture was evaporated under a stream of nitrogen at 55°C.
After addition of 2 ml DEMI, lipids were extracted (2 times) by adding 3 ml of hexane, vortexing (30
seconds per tube), centrifuging (5 min at 2500 rpm) and transferring the top (hexane) layer to a new
glass tube. The hexane was evaporated under a stream of nitrogen at 55°C. Unesterified cholesterol
was derivatized using BSTFA/ pyridine (1:1 v/v) with 1% trimethylchlorosilane at RT overnight. The
mixture was evaporated under a stream of nitrogen at RT and samples were redissolved in 1 ml heptane
containing 5% BSTFA (GC) or 150 µl heptane containing 5% BSTFA (GC-MS).
Statistical analysis
Using Brightstat, we calculated the significance of differences between groups (Bsep-/- and Bsep+/+ mice
on a LFD and HFD) with Kruskal-Wallis rank tests (and in case this test indicated significant differences
between groups) with Conover multiple comparisons tests. Data that were only generated in HFD fed
Bsep+/+ and Bsep-/- mice were compared with Mann Whitney U tests. P-values <0.05 were considered
statistically significant. Data represent median and interquartile range.
Results
Characteristics of Bsep-/- compared with Bsep+/+ mice
We compared characteristics of Bsep-/- and Bsep+/+ mice on both a LFD and a HFD (table 1). Mice
consumed a slightly smaller amount of food per day on HFD compared with LFD (p<0.05), but Bsep-/-
mice consumed more than Bsep+/+ mice on either diet (p<0.001). Bsep+/+ and Bsep-/- mice had similar
body weights on LFD at the start and end of the experiment (only final body weight is shown).
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Although the body weights on HFD did not significantly differ between genotypes at the start or at the
end of the experiment (due to one relatively small Bsep+/+ mouse), Bsep-/- mice gained less weight on
HFD compared with Bsep+/+ mice (p<0.01). Bsep-/- mice showed a higher fecal output compared with
Bsep+/+ mice on either diet (p<0.001).
Both Bsep+/+ and Bsep-/- mice showed increased plasma cholesterol levels on HFD compared with LFD
(p<0.001). Bsep-/- mice however had significantly lower plasma cholesterol levels than Bsep+/+ mice on
LFD (p<0.01) and HFD (p<0.05).
Table 1. Characteristics of Bsep-/- and Bsep+/+ mice on low and high fat diet
Bsep+/+ Bsep-/- Bsep+/+ Bsep-/-
Diet LFD HFD
Basal body weight (g) 30.8 [23.0-32.0] 32.6 [31.7-34.2]
Body weight (g) at termination 35.8 [28.6-36.9]
31.9 [29.0-34.9]
40.9 [30.7-42.3]
36.8 [36.4-38.5]
Growth (g) 10.2 [7.5-11.2] a 4.4 [4.0-5.0] b
Bile flow 3.1 [2.4-3.6] a 4.3 [4.1-4.6] b 1.7 [1.3-2.5] c 3.6 [3.1-4.1] a
Small intestine (cm) 32.5 [31.0-34.0] a
37.0 [33.5-38.0] b
31.5 [29.5-34.0] c
33.5 [30.0-36.0] ab
Colon (cm) 6.0 [5.5-6.5] 7.0 [6-8] 7.0 [6.5-7] 6.5 [6.5-8]
Cecum (g) 0.6 [0.4-0.6] a 0.6 [0.4-0.8] a 0.2 [0.2-0.3] b 0.3 [0.2-0.3] b
Intake and output
Food intake (g.day-1) 4.0 [3.7-4.2] a 4.6 [4.5-4.9] b 3.5 [3.4-3.7] c 4.3 [4.0-4.6] d
Fecal output (g.day-1) 0.6 [0.6-0.7] a 1.0 [0.9-1.2] b 0.4 [0.4-0.4] c 0.7 [0.6-0.7] a
Plasma parameters
Cholesterol (mmol.L-1) 2.4 [2.1-2.7] a 0.7 [0.7-0.9] b 4.8 [3.4-5.0] c 2.5 [2.4-3.0] a
Data represent median and interquartile range, n= 5-6 mice per group. Different letters indicate significant differences between groups, one or more letters in common indicate nonsignificant differences between groups.
On HFD both Bsep+/+ and Bsep-/- mice showed a lower bile flow rate than on LFD (p<0.001), but Bsep-/-
mice displayed a significantly higher flow rate than Bsep+/+ mice (p<0.01). Biliary BS secretion was 40%
lower in Bsep-/- compared with Bsep+/+ mice on LFD (p<0.001; figure 1A), decreased in both groups on
HFD (p<0.01) and was comparable in Bsep-/- and Bsep+/+ mice on HFD. On either diet, Bsep-/- mice showed
a decreased biliary secretion of cholate (the main BS in Bsep+/+ mice) compared with Bsep+/+ mice (LFD
-87%; p<0.001; HFD -82%; p<0.001; figure 1B). On the other hand Bsep-/- mice showed increased biliary
secretion of β-muricholate (BMC, the main BS in Bsep-/- mice) compared with Bsep+/+ mice (LFD +58%;
p<0.001; HFD +100%; p<0.01; figure 1C). In accordance with these data, Bsep-/- mice secreted a more
hydrophilic bile in general as shown by decreased BS hydrophobicity. (Heuman index on LFD (-0.66
[-0.69 to -0.62] vs. -0.29 [-0.32 to -0.25]; p<0.001 and on HFD (-0.69 [-0.71 to -0.67] vs. -0.36 [-0.42 to -0.24];
p<0.001; data not shown).
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Figure 1. Biliary bile salt secretion rate in Bsep-/-
and Bsep+/+ mice on LFD (white boxes) and HFD (grey boxes). A) Total biliary bile salt secretion rate (BSSR). B) Biliary cholate secretion rate (CA SR). C) Biliary beta-muricholate secretion rate (βMC SR). Data are represented as median and interquartile range, n= 5-6 mice per group. **p<0.01, ***p<0.001 represent significant differences between the indicated two groups.
Figure 2. Fecal bile salt excretion in Bsep-/- and Bsep+/+ mice on LFD (white boxes) and HFD (grey boxes). A) Total daily fecal bile salt excretion. B) Daily fecal cholate excretion. C) Daily β-muricholate excretion. Data are represented as median and interquartile range, n= 4-7 mice per group. *p<0.05, **p<0.01, ***p<0.001 represent significant differences between the indicated two groups.
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Bsep-/- mice excreted less fecal BS compared with Bsep+/+ mice on LFD (-80%; p<0.001) and on HFD
(-84%; p<0.001; figure 2A). On both diets, Bsep-/- excreted less cholate in their feces compared with
Bsep+/+ mice (LFD -86%; p<0.01; HFD -79%; p<0.05; figure 2B). On the other hand, Bsep-/- excreted a
similar amount of β-muricholate compared with Bsep+/+ mice on LFD and HFD; figure 2C).
Bsep-/- mice display increased non-hepatobiliary cholesterol excretion (indirect cholesterol
balance)
Compared with a LFD, a HFD may induce TICE. 22 In order to investigate the effect of Bsep deficiency
on cholesterol homeostasis, we fed mice both a LFD or a HFD for 10 weeks. Attrituble to the lower
cholesterol content of the HFD, both Bsep+/+ and Bsep-/- mice consumed less cholesterol per day on HFD
compared with LFD (p<0.001). Bsep-/- mice consumed 25% (LFD; p<0.001) to 35% (HFD; p<0.001) more
cholesterol than Bsep+/+ mice (table 2). Both Bsep+/+ and Bsep-/- mice secreted less cholesterol in bile
on HFD compared with LFD (p<0.001). In Bsep-/- mice biliary cholesterol secretion was increased ~4-6
fold on both diets compared with Bsep+/+ mice (table 2; p<0.001). The difference in biliary cholesterol
secretion could however not account for the ~8 fold (LFD; p<0.001) and ~15 fold (HFD; p<0.001)
increase in fecal cholesterol excretion that Bsep-/- displayed compared with Bsep+/+ mice. Altogether, the
data indicate that Bsep-/- mice excreted a higher amount of cholesterol in feces via a non-hepatobiliary
pathway than Bsep+/+ mice (table 2; p<0.001).
Table 2. Indirect cholesterol balance
μmol.day-1 Bsep+/+ Bsep-/- Bsep+/+ Bsep-/-
LFD HFD
Dietary cholesterol intake
Biliary cholesterol secretion
Fecal neutral sterol excretion
2.2 [2.2-2.4] a
2.5 [1.8-3.3] a
4.4 [3.5-5.8] a
2.8 [2.5-3.1] b
10.2 [9.0-10.6] b
34.6 [30.9-42.4] b
0.5 [0.5-0.6] c
0.6 [0.4-0.8] c
1.8 [1.8-3.2] c
0.7 [0.7-0.8] d
3.6 [3.2-4.3] d
27.9 [27.3-34.9] b
Net non-hepatobiliary cholesterol excretion(<0 = net absorption; >0 = net excretion)
-0.5 [-1.3-1.2] a 21.4 [19.0-29.1] b 0.9 [0.7-1.8] a 22.9 [22.7-31.0] b
Data represent median and interquartile range, n= 5-6 mice per group. Different letters indicate significant differences between groups (p<0.05).
Cholesterol absorption is impaired and synthesis is upregulated in Bsep-/- mice
We measured crucial parameters of cholesterol homeostasis, cholesterol absorption and synthesis, with
stable isotope methodology. Bsep-/- mice absorbed cholesterol to a much lower extent than Bsep+/+ mice
on a LFD (5 versus 43% (median); p<0.001; figure 3). On HFD, both Bsep-/- and Bsep+/+ mice absorbed
more cholesterol than on LFD (Bsep+/+ LFD vs. HFD p<0.05 and Bsep-/- mice LFD vs. HFD p<0.01), but
Bsep-/- mice again absorbed much less cholesterol than Bsep+/+ mice (18 versus 84% (median); p<0.001;
figure 3).
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Figure 3. Cholesterol absorption in Bsep-/- and Bsep+/+ mice on LFD (white boxes) and HFD (grey boxes). Data are represented as median and interquartile range, n= 5-6 mice per group. ***p<0.001 represents significant difference between the indicated two groups.
Bsep-/- mice synthesized more cholesterol compared with Bsep+/+ mice on both diets (LFD 13 fold;
p<0.001 and HFD 8 fold; p<0.001; figure 4A). In both Bsep-/- and Bsep+/+ mice, whole body cholesterol
synthesis was higher on HFD compared with LFD (Bsep+/+ LFD vs. HFD p<0.001 and Bsep-/- mice LFD
vs. HFD p<0.001). Bsep-/- mice showed a higher fraction of newly synthesized cholesterol in bile and
feces (both diets, table 3), and intestinal mucosa and intestinal lumen contents (measured on HFD, table
3). Similarly, compared with Bsep+/+ mice, Bsep-/- mice had an increased amount of (preformed and)
de novo synthesized cholesterol in bile (all p<0.001; figure 4B) and feces (all p<0.001; figure 4C), and
intestine luminal contents (all p<0.05,); figure 4D, measured on HFD). The amount of de novo synthesized
cholesterol in feces and bile was very small in Bsep+/+ mice. However, both Bsep-/- and Bsep+/+ mice
excreted lower amounts of de novo synthesized fecal (p<0.05) and biliary (p<0.001) cholesterol
on HFD compared with LFD (figure 4B and 4C). Bsep+/+ and Bsep-/- mice showed similar cholesterol
concentrations in small intestinal mucosa (figure 4E).
Table 3. Fractional cholesterol synthesis
% Bsep+/+ Bsep-/- Bsep+/+ Bsep-/-
LFD HFD
Bile 4.8 [3.6-6.1] a 26.6 [25.6-27.8] b 7.1 [5.9-8.7] c 23.2 [19.0-25.1] d
Feces 6.4 [6.1-6.9] a 21.5 [20.1-22.1] b 5.1 [5.1-7.7] a 12.5 [11.1-15.4] c
SIP mucosa 8.8 [7.6-10.7] a 27.4 [21.9-28.4] b
SIM mucosa 8.6 [7.5-10.8] a 23.3 [17.8-24.2] b
SID mucosa 10.3 [8.4-12.2] a 23.2 [17.6-23.7] b
SIP lumen contents 8.7 [7.6-9.7] a 23.6 [19.4-25.5] b
SIM lumen contents 7.8 [9.2-9.6] a 22.7 [18.2-24.2] b
SID lumen contents 9.3 [8.2-11.2] a 23.1 [17.5-23.8] b
Cecum lumen contents 10.6 [8.9-11.4] a 22.2 [17.3-22.9] b
Colon lumen contents 9.9 [8.6-10.6] a 21.9 [17.2-23.4] b
Data represent median and interquartile range, n= 4-6 mice per group. Different letters indicate significant differences (p <0.05) between groups.
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However, Bsep-/- mice displayed increased small intestinal length (table 1) and mucosal weight (data not
shown), which resulted in an increased total amount of cholesterol in small intestinal mucosa compared
with Bsep+/+ mice, in which preformed and de novo synthesized cholesterol were increased (figure 4F).
Figure 4. Cholesterol synthesis in Bsep-/- and Bsep+/+ mice. A) Total body synthesis on LFD (white boxes) and HFD (grey boxes). B) Preformed (black) and de novo synthesized (white) cholesterol in bile. C) Preformed (black) and de novo synthesized (white) neutral sterols in feces. D) Cholesterol concentration in different parts of the small intestine on HFD in Bsep+/+ (white) and Bsep-/- (grey) mice. E) Preformed (black) and de novo synthesized (white) cholesterol in small intestinal mucosa on HFD. F) Preformed (black) and de novo synthesized (white) neutral sterol content in intestine lumenal contents of Bsep-/- and Bsep+/+ mice on HFD. SIP= small intestine proximal segment, SIM= SI middle segment, SID= SI distal segment, Ce= cecum, Co= colon. Data are represented as medians (and interquartile range in E), n= 5-6 mice per group. *p<0.05, **p<0.01, ***p<0.001 represent significant differences between the indicated two groups. For significance of differences shown in figure B, C and D, see text.
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Transintestinal cholesterol excretion is induced in Bsep-/- mice
We used the data obtained during our stable isotope studies to model the net contribution of dietary,
biliary, and intestinal cholesterol to fecal sterols. 20
Bsep-/- mice excreted more dietary cholesterol compared with Bsep+/+ mice on either diet (LFD 2.2 fold;
p<0.001, HFD 4.5 fold; p<0.001; figure 5), due to decreased absorption as described above. Similarly,
Bsep-/- mice excreted more biliary cholesterol on both diets (LFD 4 fold; p<0.001, HFD 29 fold; p<0.001;
figure 5). Finally, Bsep-/- mice showed increased TICE (LFD 22 fold; p<0.001, HFD 14 fold; p<0.001; figure
5).
Figure 5. Origin of fecal sterols in Bsep-/-
and Bsep+/+ mice on LFD and HFD. N= 5-6 mice per group. For significance of differences shown in figure, see text.
Discussion
The major finding in our study is that Bsep-/- mice, that secrete more hydrophilic BS via bile, display
severely impaired cholesterol absorption, increased cholesterol synthesis and greatly induced (trans-
intestinal) cholesterol excretion either on a low or high fat diet.
Previous studies have shown that dietary hydrophilic BS (including β-muricholate) inhibit cholesterol
absorption. 25-27 In hamsters, it was shown that enrichment of bile with very hydrophilic 6-alpha-
hydroxylated BS induced a global hypocholesterolemic effect and enhanced 3-hydroxy-3-methylglutaryl-
Coenzyme A reductase (Hmgcr, the rate-limiting enzyme for cholesterol synthesis) activity and fecal
cholesterol excretion. 26 Cholesterol input into the intestine of Bsep-/- mice is increased, based on
slightly more food ingestion and on a higher biliary cholesterol secretion rate. However, the severely
impaired intestinal capacity to absorb cholesterol only explains ~10-40% of the amount of cholesterol
lost via the feces in Bsep-/- mice. Apparently, in Bsep-/- mice a non-dietary and non-biliary source of
cholesterol contributes to fecal cholesterol loss. Our data indicate that transintestinal cholesterol
excretion is responsible for ~60-90% of fecal cholesterol excretion in Bsep-/- mice. The induction of TICE
in the present model occurs together with the more hydrophilic biliary BS composition. Future studies
would have to be performed to determine whether the coincidence is “merely” an association or indeed
causally related.
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If the latter were the case, then strategies to enhance the hydrophilicity of the BS composition could
become of preventive and therapeutic potential for hypercholesterolemia. Increased biliary cholesterol
secretion does not seem to facilitate gallstone formation in the presence of a very hydrophilic BS
pool 26,28, which would otherwise be a drawback.
Overall, the differences between Bsep+/+ and Bsep-/- mice were highly consistent and mostly independent
of the diet. Bsep-/- mice displayed increased small intestinal length and mucosal weight, possibly as a
compensatory mechanism for the impaired absorption of cholesterol, and likely other nutrients. 29
Our previous studies showed that absorption of dietary fatty acids is decreased in Bsep-/- mice (~85%)
compared with Bsep+/+ mice (~95%, unpublished data). Another indicator of malabsorption is the
increased fecal output we found in Bsep-/- mice.
Bsep-/- showed a decrease in plasma cholesterol compared with Bsep+/+ mice on both diets, which is
likely due to several factors, including profoundly increased biliary cholesterol secretion, cholesterol
malabsorption and ineffective compensatory increased cholesterol synthesis. Cohen-Solal previously
showed increased biliary cholesterol secretion upon oral hydrophilic BS feeding 26, however, Wang et
al. in contrast showed the opposite. 25 In the presence of low intracellular cholesterol levels, activity
of the transcription factors sterol regulatory element-binding proteins (SREBPs) is increased and genes
involved in cholesterol synthesis (Hmgcr) and uptake (Low density lipoprotein receptor (Ldlr)) are induced. 30
Although hepatic Ldlr mRNA expression in Bsep-/- mice is not evidently increased 13,14, its activity
is unknown. On the other hand, the nuclear receptor Liver X receptor (Lxr) is a major transcriptional
regulator of cholesterol homeostasis, particularly in terms of cholesterol disposal from the body. 31 Lxr
activated transcription of the two half-transporters ATP-binding cassette sub-family G member 5 and
8 (Abcg5/g8), which facilitate cholesterol transfer from liver to bile and from enterocyte to intestinal
lumen 32, may play a role in Bsep-/- mice as well. Future studies are warranted to provide more insight
into the mechanism behind the profoundly decreased plasma cholesterol, increased biliary cholesterol
secretion, and increased TICE in Bsep-/- mice.
Altogether, our data clearly show that absence of Bsep in the mouse liver is associated with secretion of
hydrophilic BS and profound transintestinal cholesterol excretion. This provides a rationale for studying
the effect of hydrophilic BS feeding to hypercholesterolemic mice and perhaps humans on fecal disposal
of cholesterol.
AcknowledgementsThe authors would like to thank Angelika Jurdzinski, Juul Baller and Rick Havinga for their help during
animal experiments, and Renze Boverhof, Klaas Bijsterveld and Theo Boer for technical assistance.
Conflicts of interest and source of funding:
M.Y.M. van der Wulp is currently receiving an unrestricted research grant from Top Institute Food and
Nutrition via the University Medical Center Groningen. None of the authors has a conflict of interest to
report.
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2009;14:2584-98.
2. Gerloff T, Stieger B, Hagenbuch B, et al. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem. 1998 Apr;273(16):10046-50.
3. Jansen PL, Strautnieks SS, Jacquemin E, et al. Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis. Gastroenterology. 1999 Dec;117(6):1370-9.
4. Strautnieks SS, Bull LN, Knisely AS, et al. A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis. Nat Genet. 1998 Nov;20(3):233-8.
5. Stieger B. Recent insights into the function and regulation of the bile salt export pump (ABCB11). Curr Opin Lipidol. 2009 Jun;20(3):176-81.
6. van Mil SW, van der Woerd WL, van der Brugge G, et al. Benign recurrent intrahepatic cholestasis type 2 is caused by mutations in ABCB11. Gastroenterology. 2004 Aug;127(2):379-84.
7. Strautnieks SS, Byrne JA, Pawlikowska L, et al. Severe bile salt export pump deficiency: 82 different ABCB11 mutations in 109 families. Gastroenterology. 2008 Apr;134(4):1203-14.
8. Davit-Spraul A, Gonzales E, Baussan C, et al. Progressive familial intrahepatic cholestasis. Orphanet J Rare Dis. 2009 Jan;4:1.
9. Noe J, Stieger B, Meier PJ. Functional expression of the canalicular bile salt export pump of human liver. Gastroenterology. 2002 Nov;123(5):1659-66.
10. Green RM, Hoda F, Ward KL. Molecular cloning and characterization of the murine bile salt export pump. Gene. 2000 Jan;241(1):117-23.
11. Lecureur V, Sun D, Hargrove P, et al. Cloning and expression of murine sister of P-glycoprotein reveals a more discriminating transporter than MDR1/P-glycoprotein. Mol Pharmacol. 2000 Jan;57(1):24-35.
12. Wang R, Salem M, Yousef IM, et al. Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis. Proc Natl Acad Sci U S A. 2001 Feb;98(4):2011-6.
13. Wang R, Chen HL, Liu L, et al. Compensatory role of P-glycoproteins in knockout mice lacking the bile salt export pump. Hepatology. 2009 Sep;50(3):948-56.
14. Wang R, Lam P, Liu L, et al. Severe cholestasis induced by cholic acid feeding in knockout mice of sister of P-glycoprotein. Hepatology. 2003 Dec;38(6):1489-99.
15. Lam P, Wang R, Ling V. Bile acid transport in sister of P-glycoprotein (ABCB11) knockout mice. Biochemistry. 2005 Sep;44(37):12598-605.
16. Perwaiz S, Forrest D, Mignault D, et al. Appearance of atypical 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid in spgp knockout mice. J Lipid Res. 2003 Mar;44(3):494-502.
17. Kruit JK, Groen AK, van Berkel TJ, et al. Emerging roles of the intestine in control of cholesterol metabolism. World J Gastroenterol. 2006 Oct;12(40):6429-39.
18. Lukovac S, Los EL, Stellaard F, et al. Essential fatty acid deficiency in mice impairs lactose digestion. Am J Physiol Gastrointest Liver Physiol. 2008 Sep;295(3):G605-13.
19. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911-7.
20. van der Veen JN, van Dijk TH, Vrins CL, et al. Activation of the liver X receptor stimulates trans-intestinal excretion of plasma cholesterol. J Biol Chem. 2009 Jul;284:19211-9.
21. van Meer H, Boehm G, Stellaard F, et al. Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats. Am J Physiol Gastrointest Liver Physiol. 2008 Feb;294(2):G540-7.
22. van der Velde AE, Vrins CL, van den Oever K, et al. Regulation of direct transintestinal cholesterol excretion in mice. Am J Physiol Gastrointest Liver Physiol. 2008 Jul;295(1):G203-8.
23. Heuman DM. Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions. J Lipid Res. 1989 May;30(5):719-30.
24. Neese RA, Faix D, Kletke C, et al. Measurement of endogenous synthesis of plasma cholesterol in rats and humans using MIDA. Am J Physiol. 1993 Jan;264(1 Pt 1):E136-47.
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25. Wang DQ, Tazuma S, Cohen DE, et al. Feeding natural hydrophilic bile acids inhibits intestinal cholesterol absorption: studies in the gallstone-susceptible mouse. Am J Physiol Gastrointest Liver Physiol. 2003 Sep;285(3):G494-502.
26. Cohen-Solal C, Parquet M, Ferezou J, et al. Effects of hyodeoxycholic acid and alpha-hyocholic acid, two 6 alpha-hydroxylated bile acids, on cholesterol and bile acid metabolism in the hamster. Biochim Biophys Acta. 1995 Jul;1257(2):189-97.
27. Wang DQ, Tazuma S. Effect of beta-muricholic acid on the prevention and dissolution of cholesterol gallstones in C57L/J mice. J Lipid Res. 2002 Nov;43(11):1960-8.
28. Montet JC, Parquet M, Sacquet E, et al. beta-Muricholic acid; potentiometric and cholesterol-dissolving properties. Biochim Biophys Acta. 1987 Mar;918(1):1-10.
29. Ballatori N, Fang F, Christian WV, et al. Ostalpha-Ostbeta is required for bile acid and conjugated steroid disposition in the intestine, kidney, and liver. Am J Physiol Gastrointest Liver Physiol. 2008 Jul;295(1):G179-86.
30. Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest. 2002 May;109(9):1125-31.
31. Calkin AC, Tontonoz P. Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR. Nat Rev Mol Cell Biol. 2012 Mar;13(4):213-24.
32. Yu L, Li-Hawkins J, Hammer RE, et al. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol. J Clin Invest. 2002 Sep;110(5):671-80.
Chapter 6
Conclusion, discussion and future perspectives
Discussion
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Chapter
6
Conclusion, discussion and future perspectives
Intestinal function in terms of its capacity to absorb lipids in general and to secrete acidic and neutrals
steroids under varying conditions is the main focus of this thesis. We showed in chapter 2 that significant
acceleration of whole gut transit with polyethylene glycol (PEG) laxative treatment did not affect the
absorption and excretion of dietary fat and cholesterol. Chapter 3 on the other hand showed that PEG
treatment did decrease conversion of intestinal sterols (i.e. bile salts (BS) and cholesterol) and changed
microbiota composition. Transintestinal cholesterol excretion (TICE) appears to be an important pathway
of reverse cholesterol transport (RCT), at least in mice. In chapter 4 we showed that a high fat diet with
extremely low amounts of polyunsaturated fatty acids (low P/S ratio) doubled total fecal neutral sterol
excretion and induced TICE compared with a high fat diet with a standard P/S ratio. Chapter 5 showed
that absence of the Bile salt export pump (Bsep) in the murine liver, which induces biliary secretion of
highly hydrophilic BS, severely affects cholesterol homeostasis. Bsep-/- mice display severely impaired
cholesterol absorption, increased cholesterol synthesis and greatly induced (trans-intestinal) cholesterol
excretion.
Laxative treatment with polyethylene glycol does not affect lipid absorption, but decreases intestinal sterol conversion in ratsUsing different methodologies, including fat balance and stable isotope techniques, we showed in
chapter 2 that significant acceleration of whole gut transit time (WGTT) with PEG does not affect lipid
absorption and secretion in rats. A reassuring result, since PEG is used worldwide by many constipated
children who need adequate nutrient absorption to maintain growth. On the other hand, if simple oral
treatment with inert PEG would induce lipid malabsorption and/ or TICE, this could provide a promising
strategy to decrease hyperlipidemia. At this point, it is not possible to exclude the possibility that more
pronounced acceleration of WGTT or small intestinal transit would have increased fecal lipid excretion in
rats. Moreover, our results in rats may not (completely) be translatable to the human situation. However,
it must be kept in mind that gastrointestinal transit in humans would have to be accelerated just to
the extent that it induces fecal lipid disposal, but not diarrhea. Likely, humans would not be willingly
to adhere to the drug if it induces diarrhea, and loss of (additional) important nutrients is undesirable.
Interestingly, PEG treatment led to major changes in enterohepatic circulation of BS, an effect which
was independent of its effect on WGTT (chapter 3). Our study showed that during PEG treatment, the
amount of secondary BS, which are produced by the intestinal microbiota, was significantly decreased in
intestinal contents, feces and bile. The pool of primary BS on the other hand was increased during PEG
treatment. Primary BS are cholesterol derivatives produced in the liver that are conjugated before biliary
secretion. Conjugated BS have many functions besides facilitating lipid absorption in the small intestine.
Via their receptors (farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (GPBAR1 or
TGR5) primary BS may positively influence triglyceride, cholesterol and glucose homeostasis. 1,2
Chapter 6
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Moreover, they repress bacterial growth in the small intestine. 3 The human colon harbors a complex high
density microbial community, which can be seen as an ‘exteriorized organ’. 4 Bacteria in all major divisions
are capable of deconjugating BS via BS hydrolases (BSH; als referred to as choloylglycine hydrolase). 5 BSH
appear to promote bacterial survival and were proposed to facilitate colonization and development of
microbiota in the gut. 5,6 It is conceivable that local mucosal defense mechanisms act in synergy with BS
to prevent overgrowth of BSH producing bacteria in the small intestine.
Upon liberation of free primary BS, these are open to a wider pathway of modification (mainly 7α/β-
dehydroxylation) by a limited number of anaerobes (Clostridium) 6,7, producing secondary BS. 7α/β-
dehydroxylation is a multistep pathway that requires the activity of the gene products of the bile acid
inducible (bai) genes that are located to a bai operon identified in several Clostridium species. 6,8-11,11
We observed a decrease in Clostridium during PEG treatment in rats. Although the use of culture-
independent analysis of microbiota (pyrosequencing) allowed us to identify a large number of microbes,
it did not allow differentiation at the species level. Unfortunately, this made it impossible at this point
to direclty link decreased sterol conversion during PEG treatment to decreases in specific bai operon
carrying microbiota. However, PEG may not decrease intestinal sterol conversion by simply reducing
the number of specific microbes, but could also reduce activity of sterol converting enzymes. Future in
vitro studies (testing specific microbiota and their enzyme activities) might shed light on the mechanism
underlying our findings.
Unconjugated secondary BS, such as deoxycholate (DC), are more hydrophobic and have a higher
pKa, which permits their partial recovery via passive absorption in the colon. Whereas rodents are
capable of 7α-hydroxylation of DC in the liver, forming cholate, humans are not equipped to do so.
Thus, humans do not possess a metabolic pathway for removing DC under physiological conditions.
However, accumulation of DC, for example under conditions of slow gastrointestinal transit 12,13, has
been associated with gastrointestinal disease, ranging from cholesterol gallstones to gastrointestinal
malignancies. 14,15 Gut microbiota increase our capacity to harvest energy from the diet.
Germ-free mice fed a Western type diet are protected from obesity, insulin resistance and dyslipidemia. 16,17
Notably, the hypocholesterolemic effect in germfree mice has been attributed to decreased cholesterol
absorption due to decreased BS deconjugation. We did not observe differences in deconjugation of BS
in rats that we have treated with PEG and did not detect differences in cholesterol absorption. However,
we did find potentially beneficial changes in microbiota composition and showed decreased secondary
BS production during PEG treatment. Because of the link between secondary BS and gastrointestinal
disease, attempts are being made to reduce their production and/ or eliminate them from the
gastrointestinal tract. Several possible interventions with secondary BS production are summarized here.
Discussion
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· Probiotics: probiotics have potential health benefits via modulation of BS and cholesterol metabolism.
Orally administered probiotics first have to survive passage through gastric juice and bile. The finding
that microencapsulated BSH-active Lacobacillus plantarum in rats are excreted alive in feces in
this sense is promising. 18 Increased BS deconjugation in the small intestine could lead to BS and
cholesterol malabsorption, lowering plasma cholesterol levels. 19 Jones at al. recently showed that
microencapsulated BSH active Lactobacillus reuteri can decrease plasma cholesterol levels in adults
with hypercholesterolemia. 20 Many Lactobacilli are able to assimilate cholate. 21 Since probiotics do
no produce secondary BS themselves, this could protect BS from being converted to hydrophobic
BS. Thus, if the capacity to assimilate cholate is not sufficient, an increased amount of unconjugated
cholate will become available to the resident colon microbiota 18 during BSH-active probiotic
treatment, which could counteract the positive probiotic effects or even worsening the situation in
terms of increased 7α/β-dehydroxylation. Rigorous human studies would be required to determine
the benefits and possible adverse effects of probiotic treatment.
· Antibiotics: Long term use of antibiotics to decrease secondary BS formation by 7α/β-dehydroxylation
would be impractical since it would induce side effects such as diarrhea and antibiotic resistance.
Furthermore, although antibiotic treatment in mice led to reduction of DC below detection limit, it
disturbed BS signaling in general as evidenced by increased BS synthesis. 22 This option at this point
does not seem favorable.
· Pharmaceuticals that inhibit microbial 7α/β-dehydroxylating enzymes: these compounds would also
be subject to drug resistance, similar to antibiotics.
PEG induced a decrease in the intestinal (microbial) conversion of BS and cholesterol. The decrease in
secondary BS during PEG treatment was related to decreased cytotoxic activity of fecal water, which
represents a surrogate marker of colon cancer risk. PEG induced a (non-significant) decrease in bacterial
load, decreased Clostridium (Firmicutes) and increased mucus-associated bacteria such as Akkermansia
(Verrucomicrobia) and Bacteroides (Bacteroidetes). It was previously shown that the ratio between
Bacteroidetes and Firmicutes is altered in response to dietary changes. High fat diets increase Firmicutes
and decrease Bacteroidetes in mice, a similar effect was shown in one study in which obese subjects
were put on a low calorie diet. 23 Although the value of the Bacteroidetes/ Firmicutes ratio in humans is
at present unclear, our results in PEG treated rats may indicate a positive effect on gut health.
Altogether, our studies provide a rationale to study the effect of PEG treatment on BS metabolism and
microbiota composition in humans. PEG may be beneficial for gastrointestinal health in humans with a
tendency to produce increased amounts of secondary BS.
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114
Transintestinal cholesterol excretion in mice can be manipulated by dietary fat compositionCardiovascular disease represents the second cause of death in our society. Hypercholesterolemia, a
major risk factor for cardiovascular disease, requires adequate monitoring and treatment. Although many
people can benefit from the therapeutic effect of cholesterol synthesis inhibitors (statins), these drug
can have severe side effects and do not produce the necessary decrease in plasma cholesterol levels
in a substantial amount of patients 24,25, which has led to an intense search for alternative therapeutic
modalities. Cholesterol can be excreted with feces via bile (hepatobiliary pathway) or directly via the
intestine (i.e. TICE). 26 Removal of cholesterol via increased secretion into bile seems unpractical, since
it could increase the risk of cholesterol gall stone formation. TICE is an alternative pathway for body
cholesterol removal. Although the steps in the pathway need to be unraveled in detail, TICE may
represent an attractive target for treatment of hypercholesterolemia in humans. Induction of TICE via
simple dietary intervention would be an attractive strategy to decrease plasma cholesterol in a simple
manner with low risk of side effects, increasing compliance.
In chapter 4 and 5, we studied the effects of dietary intervention and defective BS secretion (discussed
below) on TICE and cholesterol homeostasis in general. Chapter 4 shows that it is indeed possible to
induce TICE by dietary means, without an adequate compensatory increase in cholesterol synthesis.
A high fat diet with a very low ratio of polyunsaturated to saturated fatty acids (P/S ratio) doubled the
neutral sterols in feces originating from TICE, compared with a standard high fat diet.
It was previously shown that dietary saturated fatty acids can increase fecal cholesterol excretion in rats 27 and mice 28. Human data on this subject have been conflicting 29; most studies reported increased fecal
neutral sterols by high dietary P/S ratio, whereas one study showed increased fecal neutral sterols by a
low P/S ratio diet (P/S ratio 0.2 vs. 1.9). 30 It has been suggested that the increased fecal neutral sterol
secretion during high P/S ratio diet feeding was transient 27,31 and/or that part of the increase may have
been due to differences in dietary plant sterols that were not adequately separated from the neutral
sterol fraction before the advent of gas-chromotography analysis. 30
The question remains which dietary component was exactly responsible for the induction of fecal
neutral sterol excretion in our experiments. The low P/S diet contained almost no linoleic acid (essential
fatty acid), which was replaced by high amounts of palmitic and stearic acid. Previous studies had shown
that the effect of the low P/S ratio diet on cholesterol excretion was not due to essential fatty acid
deficiency (chapter 4). It is possible that (part of ) the effect we showed of the low P/S ratio diet is due to
the increased dietary stearic acid intake. The possible effects of stearic acid, a long chain fatty acid, are
often overlooked 32 since it belongs to the group of saturated fatty acids, which are generally considered
as plasma (LDL) cholesterol raising. Stearic acid is however considered a biologically neutral fatty acid,
especially with respect to the regulation of LDL-c concentration. 33-35 It is not entirely clear why stearic acid
does not raise plasma cholesterol, but contributing factors may be the lower absorption rate of stearic
acid compared with other (unsaturated) fatty acids 36 and rapid conversion of stearic to oleic acid. 35
Discussion
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6
Schneider et al. showed decreased cholesterol absorption and increased fecal cholesterol excretion in
hamsters fed a high stearic acid diet 28, but no studies have been conducted on the effect of high dietary
stearic acid on fecal cholesterol excretion/ TICE in humans. Dietary stearic acid may however improve
the atherogenic risk factor profile in humans. 32,37,38 In chapter 4 we did not see an effect of the low P/S
ratio diet on cholesterol absorption or plasma cholesterol concentration. However, wildtype mice do not
represent a good model to study effects of different dietary fat compositions on plasma (LDL) cholesterol
levels, since they carry most of their plasma cholesterol in HDL particles. 39 Future studies could include
specific stearic acid enriched diets in other animal models. In addition, it would be interesting to reveal
the effect of non-absorbed long chain fatty acids on fecal cholesterol excretion and TICE.
In addition, dietary omega-3 fatty acids have been suggested to be beneficial in lowering cardiovascular
disease risk. They however mainly lower plasma trigycerides and not cholesterol. 40 Currently, there
is no firm trial evidence that clinical outcomes (in terms of myocardial infarction, stroke or mortality)
improve more with omega-3 fatty acids added to statin therapy as compared to high dose statin
monotherapy. 40,41
The search for compounds that decrease plasma cholesterol levels in humans while inducing
minimal side effects is ongoing. Ezetimibe is the well known pharmaceutical that blocks cholesterol
absorption 42,43 and it may induce TICE. 44 It can be hypothesized that inhibition of cholesterol absorption
in general induces secretion of cholesterol by the intestine. Compounds that create a ‘hydrophobic
sink’ in the intestine, impairing cholesterol absorption, might increase fecal cholesterol excretion by
decreased absorption as well as induced TICE. The indigestible sucrose fatty acid polyester olestra dose-
dependently decreases cholesterol absorption. 45 Olestra does not change fecal BS excretion 46, but
decreases intestinal cholesterol conversion and increases fecal neutral sterol excretion. 47 Caution should
be undertaken however, since olestra decreases plasma fat-soluble vitamins as well. 48 It is unknown
whether cholesterol lowering compounds such as olestra, and perhaps stearic acid, can induce TICE. If
so, these oral compounds, in combination with other cholesterol lowering therapies, may reduce the
need for (high dose) pharmaceuticals such as statins and thereby may reduce treatment burden and
increase compliance.
Genetic inactivation of the bile salt export pump in mice leads to massive cholesterol excretionThe bile salt export pump (BSEP) is the main transporter facilitating transfer of BS from hepatocyte to bile
canaliculus. 49,50 Mutations in the Bsep gene can result in several forms of intrahepatic cholestasis. 51,52 For
example, heterozygous Bsep mutations have been identified in patients with intrahepatic cholestasis of
pregnancy and drug-induced cholestasis. 51 Missense Bsep mutations on the other hand predominate
in a relatively mild form of cholestatic disease termed benign recurrent intrahepatic cholestasis type 2
(BRIC-2). 53 Frequently however, Bsep mutations result in the absence of canalicular BSEP protein 54, and
are associated with biliary BS concentrations of less than 1% of normal in humans. 50
Chapter 6
116
Absence of functional BSEP results in progressive familial intrahepatic cholestasis type 2 (PFIC-2), a disease
characterized by severe intrahepatic cholestasis in young children, often requiring liver transplantation
within the first decade of life. 50,55 In contrast to these patients, mice in which Bsep is eliminated from
the liver, are able to convert their hepatic BS to more hydrophilic forms and secrete them via alternative
transporters. 56
In chapter 5 we showed that Bsep-/- mice have severely disturbed intestinal function and absorb minimal
amounts of cholesterol. Fecal neutral sterol excretion was increased dramatically, at least in part via
the transintestinal pathway. Previously, Wang et al. fed gallstone-susceptible C57L/J mice with a range
of BS and showed a positive correlation between hydrophobicity indices of the BS pool and percent
cholesterol absorption. 57 In these mice both beta-muricholate (βMC) and ursodeoxycholate (UDC)
reduced cholesterol absorption and the biliary secretion rate of cholesterol. However, βMC was even
more effective in prevention and dissolution of cholesterol gallstones than UDC. 58
Others 59 have shown a global hypocholesterolemic effect of 6-alpha-hydroxylated BS, with concomitant
increased cholesterol synthesis in hamsters. Cholesterol absorption was reduced by 59% and fecal
neutral sterol excretion was increased up to 10-fold. 59 Administration of the 6-alpha-hydroxylated BS led
to increased biliary cholesterol secretion in hamsters 59,60,60, mice 61 and prairie dogs 62. Gallstones were
however not detected; apparently as a result of the weak capacity of very hydrophilic BS (as compared
to UDC and chenodeoxycholate) to form micelles. 63 A defect in biliary micelle formation can also explain
the reduction in cholesterol absorption by hydrophilic BS. 64 UDCA (ursodeoxycholic acid) is used for
treatment of cholesterol gall stones in humans and can decrease cholesterol absorption. 65,66 In patients
with primary biliary cirrhosis, UDCA lowers LDL-c. 67
Potentially, bacterial transformation of UDCA into the highly hydrophobic lithocholate (LC) could be a
drawback for UDCA use in humans. 6,68 However, the evidence is limited (LC is unsolvable) and it was
shown that LC can activate the vitamin D receptor in intestinal epithelial cells 69, which leads to induction
of genes encoding proteins that metabolize LC. 70 This in turn may limit LC toxicity to the intestinal
mucosa. At this point it remains unclear if the therapeutic potential for administration of hydrophilic BS
such as hyodeoxycholate eventually will be greater. Human studies that delineate the effect of feeding
these BS on TICE and cholesterol homeostasis in general are awaited.
Conlusion
This thesis shows that several minor interventions with intestinal function may provide health benefits.
Oral laxative treatment did not affect intestinal lipid absorption, but decreased intestinal acidic and
neutral sterol metabolism. The decrease in secondary bile salt formation observed during treatment
with polyethylene glycol may improve intestinal function and health in humans as well. We showed that
TICE can be induced by dietary means. This provides a rationale for human studies with oral hydrophilic
bile salt ingestion and for example non-absorbable long-chain fatty acids to quantitate the effect of
dietary interventions on TICE and potential health benefits in terms of decreasing hypercholesterolemia
and ultimately cardiovascular disease.
Discussion
117
Chapter
6
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Appendices
Summary
Nederlandse samenvatting
Dankwoord
Biography / Biografi e
List of publications
Appendices
123
Summary
The intestinal epithelium forms a dynamic interface for interactions between the body and food
components. Under physiological conditions, the intestinal epithelium maintains a peaceful equilibrium
with an extremely large community of commensal bacteria that live in the intestinal lumen and regulates
absorption of nutrients and excretion of waste.
Lipids (fat and cholesterol) are of vital importance for vertebrate cell membrane structure and function.
Cholesterol plays a central role in maintaining membrane fluidity, but shows ‘Dr. Jekyll and Mr. Hyde’ type
of characteristics. Its sterol nucleus is so resistant to breakdown that the body can almost not metabolize
cholesterol, leading to accumulation and induction of vascular disease when cholesterol is present in
excess. The only way to dispose of cholesterol is direct excretion with feces or conversion to bile salts
(BS). Besides mediating excretion of the sterol nucleus BS also fulfil important biological functions. BS are
important for efficient absorption of cholesterol, fat and fat-soluble vitamins.
Hypercholesterolemia is common in our Western society and represents a major risk factor for
cardiovascular disease. Classically, fecal cholesterol excretion was believed to be primarily driven by
cholesterol secreted via the hepatobiliary pathway (either in the form of cholesterol, or after breakdown
to BS). However, it has recently become apparent that direct secretion of cholesterol from the blood
compartment to the intestinal lumen, the process now adopted “TransIntestinal Cholesterol Excretion”
(TICE), plays a major role in fecal cholesterol disposal. Reduction of cholesterol absorption and induction
of TICE represent attractive therapeutic targets. Ideally, inhibition of cholesterol absorption and/or
induction of TICE would be facilitated by simple oral (dietary) intervention.
In this thesis we assessed, in a quantitative manner, intestinal function with respect to its capacity to
digest and absorb lipids (dietary fats, cholesterol and BS), and its capacity to secrete cholesterol under
varying intestinal conditions. In chapter 1, we reviewed current insights into the regulation of lipid
homeostasis. In chapter 2 we studied the effect of significant acceleration of whole gut transit (the time
for substances to travel from mouth to anus) with polyethylene glycol (PEG). PEG is a non-absorbed, non-
metabolized drug that is widely used around the world as a laxative agent to treat constipation in adults
and children. We found that PEG treatment did not affect the absorption and excretion of dietary fat
and cholesterol. In chapter 3 on the other hand we showed that PEG treatment did decrease microbial
conversion of intestinal sterols (i.e. BS and cholesterol) and changed fecal microbiota composition
(increased Verrucomicrobia and decreased Firmicutes in PEG treated versus control rats, respectively),
which can possibly be health-promoting. In chapter 4 we showed that a high fat diet with extremely low
amounts of polyunsaturated fatty acids (low polyunsaturated to saturated fatty acid (P/S) ratio) doubled
total fecal neutral sterol (cholesterol and its metabolites formed in the intestinal lumen) excretion and
induced TICE compared with a high fat diet with a standard P/S ratio. This research provides a proof-
of-concept; fecal cholesterol excretion via TICE can be induced by dietary manipulation. Additional
studies are needed to obtain the desired effect (inducing TICE), without inducing well known negative
(hyperlipidemic) side effects of saturated fatty acids.
Appendices
124
The major hepatic transporter facilitating transfer of BS from liver to bile is called the Bile salt export
pump (Bsep). Chapter 5 showed that the absence of Bsep in the murine liver, which induces biliary
secretion of highly hydrophilic BS, severely affects cholesterol homeostasis. Bsep-/- mice display severely
impaired cholesterol absorption, increased cholesterol synthesis and greatly induced (trans-intestinal)
cholesterol excretion.
Together this thesis shows that simple oral interventions, at least in laboratory animals, appear to have
health-promoting effects in terms of lipid homeostasis. The studies described provide a rationale for
human studies to test the effect of for example dietary non-absorbed long chain fatty acids and dietary
hydrophilic BS on TICE. Moreover, a relatively simple human study could provide more insight into
the possible health promoting effects of PEG treatment (via its effect on intestinal microbial flora) of
(apparently) healthy humans and constipated patients.
Appendices
125
Nederlandse samenvatting
Het darmepitheel (binnenste slijmvlieslaag) vormt een dynamische scheidslijn die interactie tussen
voeding en het lichaam toestaat. Onder natuurlijke/ gezonde omstandigheden verkeert het
darmepitheel in evenwicht met een enorme gemeenschap van bacteriën die in de darmholte leeft en
de opname (absorptie) van voedingsstoffen en uitscheiding van afvalstoffen reguleert.
Lipiden (vetten en cholesterol) zijn van vitaal belang voor de structuur en functie van celmembranen.
Cholesterol speelt een centrale rol in het behoud van vloeibaarheid van celmembranen, maar vertoont
‘Dr. Jekyll and Mr. Hyde’ karakteristieken. De zogenaamde sterolenkern van cholesterol is dermate
resistent tegen afbraak dat het lichaam nauwelijks in staat is cholesterol af te breken, wat leidt tot
stapeling en het ontstaan van vaatziekten wanneer er teveel cholesterol aanwezig is. Het lichaam kan
zich alleen van cholesterol ontdoen door het of direct uit te scheiden met ontlasting, of door het om
te zetten in galzouten (GZ) in de lever, waarna deze GZ naar de darm kunnen worden uitgescheiden
met gal en uiteindelijk ook met de ontlasting het lichaam kunnen verlaten. Naast het mediëren van de
uitscheiding van de sterolenkern, vervullen GZ belangrijke biologische functies. De aanwezigheid van
GZ in de dunne darm is essentieel om de opname van cholesterol, (voedings-)vetten en vetoplosbare
vitamines efficiënt te laten verlopen.
Hypercholesterolemie (teveel cholesterol in het bloed) komt zeer veel voor in onze Westerse
samenleving en is een grote risicofactor voor hart- en vaatzieken. In het verleden werd gedacht dat
cholesteroluitscheiding voornamelijk gedreven werd via de lever-gal (hepatobiliaire) route. Echter,
recent is duidelijk geworden dat uitscheiding van cholesterol via de bloedbaan naar de darmholte en
vervolgens de ontlasting, het proces wat “TransIntestinale Cholesterol Excretie” (TICE) gedoopt is, een grote
rol kan spelen in de cholesteroluitscheiding. Het remmen van cholesterolopname en het stimuleren
van TICE kunnen tot twee aantrekkelijke therapeutische doelen verworden. Idealiter zou remming van
cholesterolopname en/of stimulatie van TICE tot stand gebracht worden door simpele dieet (orale)
aanpassingen.
In dit proefschrift hebben wij de darmfunctie bepaald in termen van de capaciteit om lipiden te verteren
en op te nemen, en de capaciteit om cholesterol uit te scheiden onder wisselende omstandigheden in
de darm. Hoofdstuk 1 van dit proefschrift geeft een overzicht van de huidige inzichten in de regulatie
van lipidenhuishouding. In hoofdstuk 2 hebben we het effect bestudeerd van versnelling van de
darmpassage (de zogenaamde “whole gut transit”, ofwel passagetijd van mond tot anus) met het
laxeermiddel polyethyleen glycol (PEG). PEG is een polymeer die in de darm niet opgenomen en niet
omgezet wordt. PEG wordt wereldwijd veel gebruikt bij de behandeling van obstipatie van volwassenen
en kinderen. We zagen dat PEG geen invloed had op de opname en uitscheiding van voedingsvetten en
cholesterol. In hoofdstuk 3 daarentegen, hebben we laten zien dat behandeling van ratten met PEG leidt
tot verminderde bacteriële omzetting van sterolen (GZ en cholesterol) in de darm en een veranderde
samenstelling van de darmbacteriën (zich uitend in een toename in Verrucomicrobia en een afname
in Firmicutes in ontlasting van PEG behandelde versus onbehandelde controle ratten, respectievelijk),
welke mogelijk gezondheidsbevorderend is.
Appendices
126
In hoofdstuk 4 hebben we laten zien dat een hoogvet dieet met extreem lage hoeveelheden
meervoudig onverzadigde vetzuren (lage ‘polyunsaturated to saturated’ vetzuur (P/S) ratio) de
totale neutrale sterolen (cholesterol en stofwisselingsproducten hiervan die door de darmbacteriën
geproduceerd worden) uitscheiding verdubbelt en de TICE stimuleert in vergelijking met een hoogvet
dieet met een standaard P/S ratio. Deze studie geeft een zogenaamd ‘bewijs-van-concept”; namelijk
dat cholesteroluitscheiding wel degelijk gestimuleerd kan worden door een simpele aanpassing van
de dieetsamenstelling. Toekomstige dieetinterventies moeten de focus leggen op stimulatie van
TICE, zonder negatieve effecten (hyperlipidemie; verhoogd vetgehalte in het bloed) van verzadigde
voedingsvetten te veroorzaken.
Het belangrijkste transporteiwit in de lever dat transport van GZ vanuit de levercel naar de gal verzorgt,
wordt de GZ export pomp (Bile salt export pump (Bsep) in het Engels) genoemd. In hoofdstuk 5 hebben
wij laten zien dat afwezigheid (knock-out) van Bsep in de muizenlever, hetgeen uitscheiding van zeer
hydrofiele (‘waterminnende’) GZ veroorzaakt, grote veranderingen in de cholesterolhuishouding teweeg
brengt. Bsep-/- muizen hebben een zeer gestoorde cholesterolopname in de darm, een verhoogde
cholesterolaanmaak, en scheiden zeer veel cholesterol uit in de ontlasting, voornamelijk via TICE.
Samenvattend laat dit proefschrift zien dat simpele (dieet-)aanpassingen, althans in proefdieren,
gezondheidsbevorderende effecten kunnen hebben in termen van GZ- en cholesterolhuishouding
in het lichaam. De beschreven studies bieden perspectief voor vervolgstudies in mensen om te
onderzoeken wat het effect is van de inname van bijvoorbeeld zogenaamde lange keten vetzuren, die
niet door de darm opgenomen kunnen worden, en hydrofiele GZ op TICE. Daarnaast kan een relatief
eenvoudige studie gestart worden om het mogelijk gezondheidsbevorderende effect van PEG (via het
effect op de darmbacteriën) van (ogenschijnlijk) gezonde mensen en mensen met obstipatie is.
Appendices
127
Dankwoord
Elke reis begint met de eerste stap
De afgelopen jaren ben ik volgens mij een echte ‘poepdokter’-maar-dan-anders geworden. Ik heb het
allemaal gezien: (karmijn-) rode poep, bruine (chow) poep, witte (semisynthetisch dieet) poep, droge
poep, natte (PEG) poep, rattenpoep, muizenpoep, mij niks te gek. (Misschien is dit wel de reden dat
ons huis vol Rituals spullen staat?) Laten we het netjes zeggen vanaf nu: geen poep, maar feces. Wie wil
er in mijn Fecesbook? (Concilium Hilaricum Pediatricum 2011.) Darmpassagetijden meten door vijf uur
voor muizenkooien langs te lopen en te noteren wanneer ze eindelijk eens rode feces produceren na
toediening van een rode vloeistofbolus in de maag. En goed opletten, want voor je het weet eten ze
het direct bij het naar buiten komen op, en dan heb jij ‘je moment’ gemist. Met ratten is het ook leuk,
dan mag je tot een uur of drie ‘s nachts door en hoop je dat ze tegen die tijd allemaal aan de beurt
geweest zijn. Gelukkig had ik Sonja, die na het stappen langs kwam om samen chippies te eten en
op de laptop ‘uitzending gemist’ te kijken. Leg maar eens aan je ouders uit: Pap, mam, ik doe nu écht
wetenschappelijk onderzoek! Feces verzamelen, sorteren, wegen, crushen (Doe de poo-jito, met dank
aan Maxi.) en opwerken. Bloed, zweet en tranen, voor het verzamelen van bloed, gal, feces en meer. De
meesten van ons weten het wel: onderzoek doen gaat met pieken en dalen. De dalen meestal vooral
in de eerste helft van het traject, de pieken meestal en gelukkig vooral in de tweede helft (Dankje Karin
L., je had gelijk!). Gelukkig doe je het nooit alleen en staan er veel mensen klaar om je te helpen. Samen
is het zoveel leuker! Ik wil dan ook iedereen ontzettend bedanken die mij op de een of andere manier
bijgestaan heeft in dit traject.
Geachte Prof. dr. H.J. Verkade, beste Henkjan, ik ben heel dankbaar dat ik in jouw groep mocht beginnen
met mijn afstudeeronderzoek voor Biologie, naar leverziekte bij Cystic Fibrosis. Ik had het ontzettend naar
mijn zin en was dan ook vereerd dat je mij halverwege dit project vroeg om ook mijn promotietraject
hier te doen. Jouw scherpe blik en kritische vragen doen mensen soms enigszins sidderen, maar ik weet
zeker dat je mensen gewoon probeert te stimuleren om het beste uit zichzelf te halen. Door alle drukte
(volgens onze e-mail correspondentie slaap je hooguit tussen 01.00 en 06.00 uur) was er soms weinig
tijd voor overleg, maar jouw one-liners (‘Don’t make the reader think’, ‘less is more’, ‘meaner and leaner’
etc.) waren altijd raak en ik zal ze nooit vergeten. De manier waarop jij dingen uitlegt (met prachtige
tekeningen en verhalen over winkelwagentjes in de rij bij de supermarkt) werkt zeer verhelderend en
motiverend. Ik hoop dat onze samenwerking hier niet eindigt.
Geachte Prof. dr. E.H.H.M. Rings, beste Edmond, Henkjan en jij vullen elkaar zo mooi aan. Jij en jouw
zware, kalme stem brengen altijd rust. Je was niet alleen geïnteresseerd in het onderzoek, maar
ook in de persoon erachter en je vroeg altijd even hoe het was en hoe het ervoor stond met mijn
toekomstplannen. Bedankt voor je luisterend oor, je adviezen en al je hulp. Ik hoop dat we elkaar snel
weer zien in het UMCG.
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Geachte Prof. dr. A.K. Groen, beste Bert, bedankt voor de tijd die je in mijn project hebt willen steken en
je aanwezigheid en steun bij de TIFN-meetings. Bedankt dat ik zomaar bij je kon binnenvallen met een
‘acute’ vraag. Cholesterol-onderzoek is leuk, maar soms ook zo frustrerend! Ik hoop dat ‘ons model’ (met
dank aan velen die mij voor gingen, zoals Jelske van der Veen) de komende tijd verder uitgewerkt kan
worden en we nog veel meer informatie uit onze experimenten (o.a. hoofdstuk 4 en 5 van dit boekje)
kunnen halen. Bedankt ook voor je persoonlijke interesse toen plotseling niets meer was zoals het moest
zijn.
Graag wil ik de leden van de leescommissie, bestaande uit Prof. dr. Oude Elferink, Prof. dr. Faber en Prof.
dr. Smit, heel hartelijk bedanken voor het beoordelen (en het goedkeuren!) van mijn proefschrift.
Ook wil ik de leden van ‘mijn’ TIFN project bedanken, met name Jan Dekker, mijn projectleider (Bedankt
voor het meedenken en je vele bezoekjes aan het Verre Groningen.) en Roelof van der Meer (Als niemand
een vraag stelt, doe jij het. En anders ook. Heel motiverend!).
Lieve Els, mijn Groningse mama. Bedankt voor je steun en de goede gesprekken, ik hoop dat je in de
buurt blijft. Astrid, bedankt voor je hulp, je hebt de touwtjes strak in handen! Hilde R. en Gea, bedankt
voor de hulp bij het maken van (bijna onmogelijke) afspraken met alle Profs. tegelijk en bij het faxen naar
het buitenland. ;)
Ik wil graag al mijn collega’s van het lab Kindergeneeskunde bedanken voor de goede samenwerking en
de gezellige buitenschoolse activiteiten. Mijn roomies in de twee kamers naast elkaar op de 3e: Golnar,
Sabina, Annelies, Maxi, Jan Freark (Nu ik ga, moet jij ook! Dat pak stond je goed, dus zet ‘em op. Maar
wel even een van je paranimfen vragen op je fiets en jas te passen ;) ), Krystof, Nienke, Matthijs, Wei-lin,
Marjolein en Shiva. Tim, Agnes, Jolita en Elodi. Margot, Jaana, Wytske, Maurien, Maaike, Jelske, Arne, Niels,
Gijs en Karen. Jurre, Hilde H., Anke (Respect, jij bent zo sterk!), Marijke, Brenda, Carolien, Marije, Jelena
(Jammer dat je niet ’mijn’ co-assistent kon worden, veel succes in Leiden!), Marleen & Gemma (Please
find the key to TICE!) en Harmen (In herinnering). Frans C., Karin G., Marjan, Willemien en Andrea. Janine,
Torsten, Frans S. (’Ik kom even een attachment brengen.’), Mark D., Jaap en Anniek. Dolf, Klary, Barbara,
Rebecca (Zie je in Meerstad?), Hans (Ik zie graag binnenkort je trouwfoto’s! Kunnen we meteen even
bijpraten over het CF onderzoek?), Dirk-Jan, Uwe (Thanks for being my favourite blood-draw-patient
and funniest colleague).
Alle analisten van ons lab wil ik bedanken voor hun inzet en bereidheid tot meedenken: Renze (voor de
hoge tonen) en Henk (voor de lage tonen. Never a dull moment als je tussen jullie in zit op het lab.), Aycha
(Lief!), Juul (Je was overgelopen naar het CDP, maar toch hielp je mij met mijn laatste experiment, dank!),
Angelika (Regel jij nu de Ladies Nights?), Niels, Danny, Nicolette, Trijnie en Wytse. Vincent: dankjewel voor
al je spontane en gevraagde hulp. Jij hebt mijn statistiek significant verbeterd! Dr. Theo, bedankt voor al
je (reken-) hulp, zelfs als je geen tijd had.
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Als we dat TICE model nou maar gewoon uit ons hoofd gezet hadden, was hoofdstuk 4 véél eerder af
geweest en hadden we samen een halfjaar op wereldreis gekund. ;) (Ach, ik zal niet klagen. Ondanks
mijn omzwervingen door de enterohepatische kringloop, heb ik El Fin del Mundo (Argentina!) toch
maar mooi gevonden.) Ik hoop toch dat er nog meer mooie dingen uit ons model voort zullen komen.
P.S.: Nummer 25 nog niet verplaatsen naar de vergane-glorie-map hoor! V. en T.: bedankt voor jullie
heerlijke nuchterheid en flauwe grappen! En snoepjes.
Gerard (Geen Rhesus-aap, maar een Feces-aap!), bedankt voor je hulp, foto’s en humor! Ook dank aan
de collega’s van het metabole lab en LHVG, in het bijzonder Theo B., Ingrid en Klaas. Jullie stonden altijd
open voor vragen en overleg, ik heb dat ontzettend gewaardeerd!
Daarnaast dank aan alle collega’s van de Maag-, Darm en Leverziekten op ons shared lab: Anouk, Golnar,
Mark, Manon, Tjasso, Haukeline, Bojana, Elise, Janette, Martijn, Mariska, Sandra, Floris, Atta, Han en Klaas-
Nico.
Dank aan alle mensen op het dierenlab (CDP) die mijn proefdieren goed verzorgd hebben en mij
geholpen hebben rondom de planning van mijn experimenten, in het bijzonder: Flip, Alex, Hester
(Zonnetje van het CDP), Sylvia, Diana, Ar, Harm, Maurice, Andrea, Angela, Ralph, Ramon, Marcia, Annet,
Arie, Wiebe, Miriam, en Lucas (In herinnering). Rick Havinga (‘Een beetje van ons en een beetje van het
CDP’), bedankt voor alle galcannulaties, zonder gal kom je nergens!
Wim Avis: bij ons kwam de kinderarts gewoon aan huis (en wij bij hem), hoe bijzonder! En hij kwam niet
alleen als dokter, maar ook voor de gezelligheid en voor het klussen met pa. Een posterprijs winnen op
het NVK congres was leuk, maar na al die jaren jou vinden, mijn naambordje omhoog houden en de
blik in je ogen te zien, dat was onbetaalbaar! Bedankt voor de goede zorg voor mijn zus. Je bent een
voorbeeld voor mij. Frank Bodewes, bedankt voor de samenwerking, hopelijk komt er nog een mooi stuk
uit voort en wie weet kunnen we nog eens samenwerken. Gieneke Gonera, Marlon Wilsterman en Linda
Leer: bedankt voor jullie hulp bij het verzamelen van kinder-feces, ik hoop dat we er nog iets moois uit
kunnen halen. Ik wil ook graag de kinderartsen en arts-assistenten van de afdeling Kindergeneeskunde
in het Martini Ziekenhuis bedanken voor alle steun die ik heb ontvangen, ook al voordat ik ‘echt’ begon.
De afgelopen jaren mocht ik deel uitmaken van ‘de Meisjes van Verkade’, a.k.a. Mjan, Mien, Mgot, Mjet
en An. Wat hebben wij genoten van de voorbereiding op het optreden van de ‘Spijsgirls’ (uw zang-
en dansgroep) op het oratie-feest van ‘Dear Prof. Rings’ (You are very smart!). Samen oefenen in het
appartement dat Margot en ik toen deelden. Samen make-uppen voor de start. En toen moesten we
ook nog daadwerkelijk de bühne op...oeps, waar waren we aan begonnen?! Of heb ik juist daardoor een
baan in het Martini Ziekenhuis gekregen? Meiden, bedankt voor alle hysterische lol!
Lieve Karin en Pieter, nu beiden ‘van der Wulp’! Eigenlijk zijn we écht familie. ;)
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Bedankt voor de gezelligheid en goede diners. Lieve Mjannie en Wimpie, bedankt voor jullie steun, jullie
zijn zo lief voor mij geweest.
Willem, dankjewel voor je Meesterlijke Maaltijden, waarvan ik zoveel eet dat ik daarna bijna niet meer
met jullie kan praten. Ik heb zin in een reis met z’n vieren! Lieve Marije (‘Ik ben blij dat jij ook van hard
lachen houdt!’) en Anouk (Almeloooo, brooood), bedankt voor alle gesprekken, etentjes en filmavondjes.
Marije, Anouk en Aycha, bedankt voor jullie steun toen het mis ging met mijn zus, maar ook daarna.
Lieve Anouk en Niek, bedankt voor jullie warmte en gezelligheid, ik wens jullie heel veel geluk straks
met z’n drietjes! Lieve Lisette, ik hoop dat de lijn blijft stijgen, je verdient het! Lieve Margot, vanaf dag
1 van ons promotietraject in hetzelfde schuitje: jonge dokters, samenwonen op afstand, studiootje in
Groningen, vrijdags haasten naar de andere kant van het land, maandagmorgen om 05.00 uur op en
weer terug. (Even kort samengevat.) Alle pieken, dalen, twijfels en successen hebben we gedeeld. Wij
hebben zoveel ‘Weet je nog, die keer...’ momenten. Ik ben heel blij dat ik je heb leren kennen, je bent een
grote steun voor mij geweest de afgelopen jaren, zowel op het werk als privé. Bedankt voor de mooie
tijd in New York (met onze mannen) en veel succes daar de komende tijd, topper! Ondanks de afstand
weten wij elkaar wel te vinden.
Mijn lieve paranimfen, ik ben jullie veel dank verschuldigd. Bedankt dat jullie ook nu voor mij klaarstaan.
Marjan, je bent een schat. Ik begrijp niet hoe jij altijd (ogenschijnlijk?) zo rustig kan blijven. Die rust breng
je over op anderen, een gave. Ik weet zeker dat jou een mooie toekomst als internist-infectioloog te
wachten staat. Lieve Sonja (Sonnie), waar moet ik beginnen? Mijn leven in Groningen zou niet hetzelfde
geweest zijn zonder jou. Samen biologie studeren, allebei geneeskunde studeren, samen eten, sporten
(We DID it! 4 Mijl Groningen 2012), stappen (Antwerpen is niet veilig!), shoppen, vrouwenseries kijken
etc. etc. For better & for worse. Ons leven staat dit jaar op zijn kop. Hopelijk kan ik net zo goed klaar staan
voor jou als jij voor mij. Je hebt een groot hart! Ik hoop dat ik voor altijd jouw vriendinnetje mag zijn.
Home is wherever I’m with you...(Edward Sharpe & The Magnetic Zeros, Home)
Lieve Ilona, Leo en Rico, lieve Remco en papa en mama. Wie zou ik geworden zijn zonder jullie? Ik ben
trots dat ik uit zo’n liefdevol gezin kom. Rico, je gaat al bijna naar school! Tante Mariëtte is heel trots op
je! De wereld ligt aan je voeten.
Papa en mama, mijn helden. Jullie leven heeft altijd in het teken van ons gestaan. Ons verlies, zo kort
geleden, kon niet groter zijn. Ik zal altijd voor jullie klaarstaan, vergeet dat nooit. Als er iets is wat ik van
jullie geleerd heb, is het dat ik ervoor moet gaan als ik iets écht graag wil. Gewoon, op eigen kracht. Ik
hoop dat ik jullie trots maak. Jullie zijn goud.
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Lieve Nathanja, lief zusje. Mijn voorbeeld, mijn geweten, mijn soulmate. Ik heb zo vaak nagedacht over
het moment dat we je zouden verliezen, maar dit scenario had ik nooit kunnen bedenken. Ik kan me
niet herinneren dat ik je 1 keer in mijn leven heb horen klagen. Nooit zei je dat je er genoeg van had,
dat het niet eerlijk was. Waarom jij? Waarom deze rotziekte? Nooit. Je vocht met alles wat je had om de
betere periodes zo goed mogelijk te benutten. Altijd was er jouw lieve, mooie lach. Alle kansen die je
kreeg, greep je met beide handen aan. We hebben gewandeld in Zuid-Europa, gefietst in NL, geskied
in Oostenrijk en gesnorkeld in de Rode Zee. Je vroeg maar 1 ding en dat was een kans op een tweede
kans, met nieuwe longen. Als iemand het verdiende, was jij het wel. Helaas mocht het niet zo zijn. Jou
verliezen was het ergste wat mij kon gebeuren. Ik weet niet hoe ik de rest van mijn leven zonder jou
moet. Ik zou je zo graag zien als mijn paranimf, mijn bruidsmeisje, als tante van mijn kinderen...
Toch weet ik dat het zal lukken. Jouw kracht is mijn kracht en niets komt daar tussen. Ik hou me vast aan
de gedachte dat ik je ooit, ergens, weer zal vinden. Is dat naïef? Laat mij dan maar naïef zijn. Ik hou van
je zusje. Jij was de mooiste.
“Did you ever know that you’re my hero?
You’re everything I wish I could be.
I could fly higher than an eagle,
‘cause you are the wind beneath my wings.”
(Bette Midler, Wind beneath my wings)
Lieve Stef, ondanks alles wat we hebben meegemaakt in slechts een paar jaar tijd, kunnen we nog
steeds samen lachen. Daar gaat het om, toch?
Je weet hoe gek ik op Ilse ben, deze is voor jou:
“You will always be my beautiful distraction
One part sweetness two parts passion
You asked for my heart not perfection
The first one to lead me in the right direction
This crazy world won’t change the same old me
Who loves the same old you”
(Ilse de Lange, Beautiful distraction)
Lieve mensen, vergeet nooit te genieten.
Later is nu!!!
Mariëtte
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132
Biography
Mariëtte van der Wulp was born on March 14th 1981 in Deventer. In 1999 she graduated from high school
(Alexander Hegius Lyceum) in Deventer. After obtaining her diploma, she continued her education at the
University of Groningen where she studied Biology (Master’s degree Medical Biology received in 2008)
and Medicine (MD degree received in 2007). Research projects performed during her studies involved
Pediatric Oncology and Cystic Fibrosis related liver disease. In 2008, Mariëtte started her PhD project
at the Department of Pediatric Gastroenterology and Hepatology at the University Medical Center
Groningen, under supervision of Prof. dr. H.J. Verkade, Prof. dr. A.K. Groen and Prof. E.H.H.M. Rings. Results
obtained from her PhD-studies are described in this dissertation and were part of the Top Institute Food
and Nutrition project ‘Nutrition and Health’. Since August 2012, Mariëtte is employed by the Martini
Hospital in Groningen, where se works as a physician at the Department of Pediatrics.
Biografie
Mariëtte van der Wulp werd op 14 maart 1981 geboren te Deventer. In 1999 behaalde zij haar
Atheneumdiploma aan het Alexander Hegius Lyceum te Deventer. In datzelfde jaar begon zij aan
haar studie Biologie aan de Rijksuniversiteit Groningen en in 2001 startte zij daar tevens haar studie
Geneeskunde. Zij behaalde haar artsdiploma in 2007 en haar doctorandus titel in de Medische Biologie
in 2008. De wetenschappelijke onderzoeken tijdens haar studies richtten zich op kinderoncologie en
leverziekte bij Cystic Fibrosis. In 2008 startte Mariëtte met haar promotieonderzoek bij de afdeling
Kinder-maag-darm-leverziekten in het Universitair Medisch Centrum Groningen onder begeleiding
van Prof. dr. H.J. Verkade, Prof. dr. A.K. Groen en Prof. E.H.H.M. Rings. De resultaten van haar onderzoek,
gefinancieerd door Top Institute Food and Nutrition, project ‘Nutrition and Health’, worden in dit
proefschrift beschreven. Sinds augustus 2012 is Mariëtte werkzaam als arts-assistent Kindergeneeskunde
in het Martini Ziekenhuis Groningen.
Appendices
133
List of publications
van der Wulp MY, Cuperus FJ, Stellaard F, van Dijk TH, Dekker J, Rings EH, Groen AK, Verkade HJ. Laxative
treatment with polyethylene glycol does not affect lipid absorption in rats. J Pediatr Gastroenterol Nutr.
2012 Oct;55(4):457-62.
van der Wulp MY, Verkade HJ, Groen AK. Regulation of cholesterol homeostasis. Mol Cell Endocrinol.
2012 Jun.
Cuperus FJ, Iemhoff AA, van der Wulp M, Havinga R, Verkade HJ. Acceleration of the gastrointestinal
transit by polyethylene glycol effectively treats unconjugated hyperbilirubinaemia in Gunn rats. Gut.
2010 Mar;59(3):373-80.
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