Obesogens, Stem Cells and Obesogens, Stem Cells and the Maternal Programming the Maternal Programming

of Obesityof Obesity

Bruce Blumberg, Ph.D.Bruce Blumberg, Ph.D. Department of Developmental and Cell BiologyDepartment of Developmental and Cell Biology Department of Pharmaceutical SciencesDepartment of Pharmaceutical Sciences Developmental Biology CenterDevelopmental Biology Center University of California, IrvineUniversity of California, Irvine

Main Points

• Epigenetics links environment to altered gene expression

• Obesogens exist and contribute to obesity epidemic

• Obesogen action may involve reprogramming of stem cells

• Literally “on top of genetics” – coined by C.H. Waddington in 1957

What is Epigenetics ?

• Epigenetics involves changes in gene expression without changes in the DNA sequence– Heritable, maintained– Reversible – Encoded in chromatin– Cellular memory

• Examples– X inactivation– Genomic imprinting– Cancer – widespread silencing

or overexpression of genes

Goldberg et al. (2007) Cell 128, 635-638

• Methylation/demethylation of DNA, proteins• Acetylation/deacetylation of DNA-binding proteins• Changes can act at very long range, 100s of kb to mb

– from other chromosomes!

Epigenetics acts through chromatin stucture

Epigenetics acts through chromatin stucture

• Chromatin conformation affects accessibility of DNA to transcriptional machinery – epigenetics controls genetics

• Widespread changes in DNA methylation can be associated with diseases, e.g., cancer

Genetics and epigenetics of disease• Some genetic diseases

– Sickle cell anemia– Cystic fibrosis– Hemophilia – Marfan syndrome– Duchenne muscular dystrophy– Huntington’s disease

• Diseases with an epigenetic component (from ID twin studies)– Fragile X syndrome– Prader-Willi syndrome– Scleroderma– Autism – Schizophrenia– Inflammatory bowel disease– Cancer (e.g. melanoma)

Developmental Basis of Disease• Barker Hypothesis - gestational under-nutrition leads to a thrifty phenotype

– reduced fetal growth strongly linked with chronic conditions later in life– Increased susceptibility results from adaptations made by the fetus in an

environment limited in its supply of nutrients

• Developmental Origins of Health and Disease (Mark Hanson) more generally proposes that development is exquisitely sensitive to perturbations that lead to permanent changes in disease susceptibility– Birth defects, low birth weight, premature birth– Functional changes –appears normal but has molecular

abnormalities that persist and lead to increased disease sensitivity later in life

• Diseases with Developmental Origins (from animal models)– Cardiovascular, Pulmonary (asthma)– Neurological (ADHD, Neurodegenerative diseases),– Immune/autoimmune– Endocrine, reproductive/fertility, cancer– Obesity/diabetes

• Developmental programming continues into adolescence– Överkalix studies in Sweden linking nutrition and longevity– Programming of adipocyte number continues into puberty

The Worldwide Obesity Epidemic

BMI = 31.5From Lars Lind

Visceral obesitypathological

Subcutaneous obesityadaptive

BMI ~32BMI ~32

• 34% of the US population are clinically obese (BMI > 30)– Double worldwide average (Flegal et al. JAMA

2010;303:235-241)

• 68% are overweight (BMI > 25) – 86% estimated by 2020

1999

Obesity Trends* Among U.S. AdultsBRFSS, 1990, 1999, 2008

(*BMI 30, or about 30 lbs. overweight for 5’4” person)

2008

1990

No Data <10% 10%–14% 15%–19% 20%–24% 25%–29% ≥30%

~17,000 22,401

30,961

Sources: CDC (map), U.S. Census bureau (numbers)

The Worldwide Obesity Epidemic

• Obesity accounts for 8% of healthcare costs in Western Countries– $75 billion annually in US (2005), $147 billion (2009)

• Obesity is associated with “metabolic syndrome” -> type 2 diabetes and cardiovascular disease– Central (abdominal obesity)– Atherogenic dyslipidemia (high triglycerides, high LDL, low

HDL)– Hypertension– Insulin resistance– Prothrombotic state– Pro-inflammatory state (elevated CRP)

• 34% of the US population are clinically obese (BMI > 30)– Double worldwide average (Flegal et al. JAMA

2010;303:235-241)

• 68% are overweight (BMI > 25 ) – 86% estimated by 2020

• Prevailing wisdom – “couch potato syndrome”– Positive energy balance, i.e., too much food, too little

exercise

How does obesity occur ?

• Are there other factors in obesity ?– Stress (elevated glucocorticoids)– Inadequate sleep (stress?)– “Thrifty” genes which evolved to make the most of scarce

calories– Viruses, gut microbes, SNPs

• What about role of prenatal nutrition or in utero experience?– Southampton studies – Maternal smoking decreases birth weight and increases

obesity

• What about the role of industrial chemicals in rise of obesity?– Baillie-Hamilton (2002) postulated a role for chemical

toxins– obesity epidemic roughly correlates with a marked

increase in the use of chemicals (plastics, pesticides, etc.)

• Many chemicals have effects on the endocrine system

Hormonal control of weight

• Hormonal control of appetite and metabolism– Leptin, adiponectin, ghrelin are key

players– Leptin, adiponectin – adipocytes– Grehlin – stomach– Thyroid hormone/receptor

• Sets basal metabolic rate

From Nature Medicine  10, 355 - 361 (2004)

• Hormonal control of fat cell development and lipid balance– Regulated through nuclear

hormone receptors RXR, PPARγ– PPARγ – master regulator of

fat cell development• increased fat cell

differentiation• Increased storage in existing

cells• Increased insulin sensitivity

Endocrine Disrupting Chemicals (EDCs)• Endocrine disrupter - a compound that mimics or blocks the

action of endocrine hormones, either directly or indirectly– Often persistent pollutants or dietary components that

disturb development, physiology and homeostasis

• Frequently act through nuclear hormone receptors– Environmental estrogens– Anti-androgens– Anti-thyroid

• Recent white paper from the Endocrine Society - Diamanti-Kandarakis, et al, Endocrine Reviews 30 (4): 293-342 (2009)– Details scientific support for existence and effects of EDCs– Endorsed by American Medical Association– Led to H.R.4190 - Endocrine Disruption Prevention Act of

2009– Moves responsibility for research from EPA to NIEHS

Endocrine Disrupting Chemicals (EDCs)

• Are EDC-mediated disturbances in endocrine signaling pathways involved in adipogenesis and obesity

The Nuclear Hormone Receptor Superfamily

Known Receptors

Classical receptors (from biochemistry)GR cortisolMR aldosteroneAR testosteronePR progesteroneER α,β estradiolVDR 1,25-(OH)2 vit D3TR α,β triiodothyronineEcR 20-OH ecdysone

Orphan Receptors

Vertebrate DrosophilaTR-2 α,β DHR78NGFI-B α,β,γ DHR38ROR α,β,γ DHR3Rev-erb α,β E75, E78SF-1 α,β FTZ-F1 α,β COUP α,β,γ svpHNF-4 α, β HNF-4Tlx α,β tll

No known homologsERR α,β,γ knirpsDAX-1 knirps-relatedSHP egonGCNF DHR96

C. elegans ~250 nuclear receptorsD. melanogaster ~20 nuclear receptorsH. sapiens ~48 genesArabidopsis no family members

DNA LIGANDA/B C D E F

Adopted (EX) OrphansRAR α,β,γ all-trans retinoic acidRXR α,β,γ 9-cis retinoic acidPPAR α,β,γ fatty acids, eicosanoidsLXR α,β oxy-sterolsFXR α,β bile acidsBXR α,β benzoates

Nearly adopted orphans (natural ligands?)CAR androstanes, xenobioticsSXR/PXR steroids, xenobiotics

EDCs and the obesogen hypothesis • Obesogens - chemicals that inappropriately stimulate adipogenesis

and fat storage, disturb adipose tissue homeostasis, or alter control of appetite/satiety to lead to weight gain and obesity

• several compounds cause adipocyte differentiation in vitro (PPARγ)– phthalates, BPA, aklylphenols, PFOA, organotins

• Pre- and postnatal exposure to EDCs such as environmental estrogens (ER) increases weight– DES, genistein, bisphenol A

• Existence of obesogens is plausible

• Thiazolidinedione anti-diabetic drugs (PPARγ)– Increase fat storage and fat cell number at all ages in

humans

• Urinary phthalates correlate with waist diameter and insulin resistance in humans– Many chemicals linked with obesity in epidemiological

studies

• Organotins -> imposex in mollusks

• Sex reverses genetically femaleflounder and zebrafish -> males

• Which hormone receptors might be organotin targets?

Endocrine disruption by organotins

• We found that tributyltin (TBT)– Binds and activates at ppb (low

nM) two nuclear receptors, RXR and PPARγ critical for adipogenesis

– TBT induced adipogenesis in cell culture models (nM)

– Prenatal TBT exposure led to weight gain in mice, in vivo

Tributyltin-ClSn

Cl

Structures of RXR and PPARγ-specific agonists

LG268 Kd = 3 nM; EC50 = 3 nM

9-cis-RA Kd = 1 nM EC50 = 15 nM

COOH

N

COOH

NH

S

O

N CH3

N

OO

H

Rosiglitazone Kd = 47 nM; EC50 = 300 nM

Tributyltin-Cl Kd = 12 nM EC50 = 5 nM

Sn

Cl

0.01 0.1 1 10 100 1000 100000

10

20

30

40

50

60

70

80LG268ButyltinDibutyltin

TetrabutyltinButyltin Tris(2-EHA)

Tributyltin

Concentration nM

Organotins show strong SAR on hRXR

EC50

DBT > 2800 nMTBT 5 nM4BT 150 nM

Fold

Act

ivati

on

Grun et al., Molec Endocrinol, 2006

TBT activates PPAR

1 10 100 1000 10000100000None0

1

2

3

4

5

6

7

8

9

10

TroglitazoneTBTLG268AGN203

Concentration (nM)

Fold

Act

ivati

on

toxic

PPAR –regulates lipid metabolism and adipocyte differentiationGrun et al., Molec Endocrinol, 2006

Nuclear receptor activation by organotins

Nuclear Receptor LBD EC50 nM

Ligand hRXRα hRARα hPPARγ

LG268 2-5 na na

AGN203 0.5-2 na na

9-cis RA 15 na

all-trans RA na 8 na

Butyltin chloride na na na

Dibutyltin chloride 3000 na na

Tributyltin chloride 3-8 na 20

Tetrabutyltin chloride 150 ND ND

Di(triphenyltin) oxide 2-10 na 20

Butyltin-tris (2-ethylhexanoate) na ND ND

Troglitazone na na 1000

Organotins are highly potent nuclear receptor agonistsDo they bind to the receptors?

RXRRXR PPARPPAR

Competitive Binding of TBTSpeci

fic

Bound c

pm

00

500

1000

1500

2000

2500

3000

LG268TBT

0.1 1 10 100 103

Concentration nM

his6-hRXR

Kd = 12.5 nM

Kd = 7.5 nM

00

1000

2000

3000

4000

5000

6000

7000

TroglitazoneTBT

1 10 100 103 104

his6-hPPAR

Concentration nM

Kd = 20 nM

Kd = 300 nM

• TBT binds to and activates RXR and PPARγ with high affinity

• How does it behave in adipogenic models?Grun et al., Molec Endocrinol, 2006

TBT

Newborn Liver ± TBT (in utero)

Vehicle (corn oil)

What is the effect of TBT treatment, in vivo?

Grun et al., Molec Endocrinol, 2006

What is the effect of prenatal TBT exposure on adult animals?

TBT increases testis fat pad weightat 10 weeks

Fat depot size increases at the expense of overall body mass

Controln=9

TBTn=10

We

igh

t(g

ram

s)

0.0

0.1

0.2

0.3

0.4

16% increasep = 0.037

Grun et al., Molec Endocrinol, 2006

How does TBT exposure cause weight gain?

• Changes in the hormonal control of appetite and satiety?

• Mesenchymal stem cells (MSCs) (now called multipotent stromal cells) precursors to many lineages including bone, cartilage, and adipose.– MSCs differentiate into adipocytes following rosiglitazone

exposure– MSCs may (or may not) home to adipose depots after

induction

• Hypothesis: TBT induces adipogenesis in MSCs

Hypertrophy

adipocytes• Altered ability of adipocytes to

process and store lipids?Hyperplasia

Commitmentdifferentiation

Preadipocytes

• Increased number of adipocytes

or pre-adipocytes?

TBT induces adipogenic differentiation in MSCs

Kirchner et al., 2010 Molec Endocrinol, 24, 526-539

hMSCshMSCs

+ MDII+ MDII

+ TBT+ TBT+DMSO, TBT or ROSI

BM WAT

mBMSCs mADSCs

+DMSO, TBT or ROSI

BM WAT

mBMSCs mADSCs

AdipocyteBone

Cartilage differentiation

conditions

TBT induces adipogenic genes in MSCs

Kir

chner

et

al.,

2010

Mole

c Endocr

inol, 2

4,

52

6-5

39

Adipogenic effects of TBT and ROSI in MSCs require PPARγ

+0 nM

Induction : MDII

+DMSO

T0

07

090

7

+0 nM +100 nM

+DMSO

+100 nM +100 nM +100 nM

+100 nM TBT +1000 nM ROSI

+100 nM +100 nM

+DMSO

T0

07

090

7

Kirchner et al., 2010 Molec Endocrinol, 24, 526-539

Induction : 100 nM ROSI + MDII

+0 nM +10 nM +100 nM +1000 nM

T0

07

090

7

Induction : 50 nM TBT + MDII

+0 nM +10 nM +100 nM +1000 nM

T0

07

090

7

Osteogenic capacity of hADSCs

Control (-) Osteo

Osteo + Rosi Osteo + TBT

Aliz

arin

Re

d-S

+ S

ud

an

Bla

ck

LEPaP2OPN OSN

x70 x240

0

1

2

3

4

5

6

***

*

****

*

ratio

Ta

rge

t /

Ho

use

kee

pin

g

Contro

l (-)

Osteo

Osteo

+ Ros

i

Osteo

+ TBT

Contro

l (-)

Osteo

Osteo

+ Ros

i

Osteo

+ TBT

Contro

l (-)

Osteo

Osteo

+ TBT

Contro

l (-)

Osteo

Osteo

+ R

osi

Osteo

+ T

BT

Osteo

+ Ros

i

*** ***

TBT overrides the effects of the bone-inducing cocktail, insteadcausing the cells to become adipocytes

Kirchner et al., 2010 Molec Endocrinol, 24, 526-539

Effects of TBT on cultured MSCs • TBT increases the amount of adipocyte differentiation in ADSCs

– Increased number of cells with lipid– Increased amount of lipid stored in cells– Decreased expression of adipgenesis inhibitor Pref-1– Increased expression of pre- and adipocyte markers

• Adipogenic effects of TBT and ROSI require PPARγ– TBT and ROSI rescue effects of PPARγ antagonist– TBT acts through PPARγ

• TBT inhibits ability of osteogenic cocktail to induce ADSCs to become adipocytes

• What is the effect of prenatal exposure on ability of ADSCs to differentiate into adipocytes or other lineages?

In vivo assays

to assessstem cell

commitment

+DMSO, TBT or ROSI

BM WAT

mBMSCs mADSCs

+DMSO, TBT or ROSI

BM WAT

mBMSCs mADSCs

+DMSO, TBT or ROSI

BM WAT

mBMSCs mADSCs

+DMSO, TBT or ROSI

BM WAT

mBMSCs mADSCs

+DMSO, TBT or ROSI

BM WAT

mBMSCs mADSCs

+DMSO, TBT or ROSI

BM WAT

mBMSCs mADSCs

adipocyte differentiationconditions

bone differentiationconditions

cartilage differentiationconditions

C57BLK6 - Pregnant dam

E16 – chemical exposure by gavage

CD-1 unexposed surrogate

CMC TBT ROSI

in utero exposed offspring

-

–

CMC TBT ROSI

in utero exposed offspring

=

Prenatal TBT exposure increases MSC differentiation into adipocytes

Kir

chner

et

al.,

2010

Mole

c Endocr

inol, 2

4,

52

6-5

39

OPNCMC ROSI TBT

In utero gavage treatment

+DMSO +DMSO +DMSO

+TBT +TBT +TBT

In utero CMCIn utero ROSIIn utero TBT

0

0.2

0.4

0.6

0.8

1

+DM

SO

+TBT

+DM

SO

+TBT

+DM

SO

+TBT

0

200

600

1000

+DM

SO

+TBT

+DM

SO

+TBT

+DM

SO

+TBT

Fabp4

ratio

Ta

rge

t /

Ho

use

kee

pin

gra

tio T

arg

et

/ H

ou

seke

ep

ing

0

20

40

60

80

100

sta

inin

g (

%su

rfa

ce)

***

**

+DM

SO

+TBT

+DM

SO

+TBT

+DM

SO

+TBT

Calcification

0

20

40

***

**

+DM

SO

+TBT

+DM

SO

+TBT

+DM

SO

+TBT

Lipid accumulation

******

***

***

*** ***

***

***

***

**

800

400

O A O A O A

** **

• Prenatal TBT exposure predisposes MSCs to become adipocytes at the expense of their ability to form osteocytes

• Prenatal TBT

exposure inhibits calcium, and enhances lipid deposition

In utero TBT exposure inhibits osteogenesis

Kir

chner

et

al.,

2010

Mole

c Endocr

inol, 2

4,

52

6-5

39

CMC

Rosi

TBT oCM

CRos

iTBT

0.04

0.05

0.06

0.07

0.08

BM

D

Males Females

CMC

Rosi

TBTCM

CRos

iTBT

0.6

0.8

1.0

1.2

1.4

1.6

Ab

Fat

wgt

(g) Males Females

Effects of prenatal TBT exposure on WAT and BMD

FemalesBM

D

Ab Fat wt

Males

• Prenatal TBT leads to increased WAT and lower BMD• What is the mechanism?

Effects of prenatal TBT on MSC pool

• TBT exposure biases the MSC compartment toward adipocytes– 7-15% more pre-adipocytes in TBT-treated than control animals

• Increased expression of adipocyte markers -> more pre-adipocytes– Decreased potential to form osteoblasts

• TBT exposure may have altered setpoint for adipocyte number– Permanent?

Kir

chner

et

al.,

2010

Mole

c Endocr

inol, 2

4,

52

6-5

39

How does prenatal TBT exposure promote adipocyte differentiation?

Effects of in utero TBTexposure on adipogenicpathway genes

uninduced

+ TBT 14D

PPARγ2+/-

PPARγ2+

Fabp4+ Fabp4+

LEP+ LEP+

Pref1- Pref1-

GyK+ GyK+

PEPCK+ /

/ LPL+

/ ADIPOQ+

LEPLEPResistinResistinIRS-2IRS-2

ADIPOQADIPOQ

Epigenetic effects of prenatal TBT exposure on promoter methylation of PPARγ target genes

Kir

chner

et

al.,

2010

Mole

c Endocr

inol, 2

4,

52

6-5

39

LEPResistinIRS-2

ADIPOQ

How does TBT affect PPARγ regulators?

Zfp423

Ezh2

SIRT1

CBP/p300SRCPGC-1α

BMP

SMARTNCoRRIP140

KLF4

• Zfp423 regulates PPARγ expression (Gupta et al. 2010)• BMP4 activates C/EBPβ (Bowers et al. 2007) • Ezh2 represses Wnt during adipogenesis by methylating H3K27

at its promoter (Wang et al. 2010)• Wnt10b represses adipogenesis, repressed by Ezh2 (Wang et al.

2010)• Wnt5b promotes adipogenesis by inhibiting Wnt/ β-catenin

pathway (Christodoulides et al. 2008)• Sox9 represses C/EBPβ/δ activity (Wang et al. 2009)

Obesogen exposure and development• Organotins are exceptionally potent agonists of RXR and PPAR

at environmentally-relevant levels (ppb)– ~5 nM EC50, 12.5 nM Kd on RXR– ~20 nM EC50 and Kd on PPAR

• TBT drives adipocyte differentiation in cell culture models

• TBT exposure during development induces adipogenesis in two vertebrate species: mouse and Xenopus– Inhibits bone formation in culture and in females

• The effects of maternal TBT exposure are multi-generational in females and fully trans-generational in males– Fat depot size, gene expression but little effect on total

weight

• Multiple potential modes of action– PPARγ-RXR– Aromatase expression/function – estradiol levels– Glucocorticoid levels– Other stressors?

Conclusions – organotins and obesity• Is organotin exposure a contributing factor for obesity?

– Adult exposure rapidly induces adipogenic genes• Drugs that activate PPARγ increase obesity

– Prenatal TBT exposure permanently alters adult phenotype

– Prenatal TBT exposure recruits MSCs to adipocyte lineage and diverts them from bone lineage

• Are humans exposed to sufficient levels of TBT for concern?– PVC is up to 3% w/w (0.1 M) organotins– Prevalent contaminants in dietary sources– Fungicide on high value crops, used in water systems– Average blood level of 27 nM in 32 random people tested– TPT levels from ~0.5–2 nM in Finnish fishermen

• Human exposure to organotins may reach levels sufficient to

activate high affinity receptors– 1000 x lower dose than natural dietary RXR and PPAR

ligands

Is the environment making us fat?

bonebrown

fat

muscle

cartilage

EDCs canpromote

the adipogeniclineage at the

expense ofother lineages

lin-

CD29+

CD34+

Sca1+

CD24+ SMA

PDGFR+

NG2+

PPAR+ Whiteadipose

precursors

Zfp423

pre-adipocyte

BMP

WNT

EDCs can regulate majorsignaling pathways (e.g.,BMP, WNT) that commitMSCs or progenitor cellsto the adipogenic lineage

Upregulationof PPAR

Expressionmatureadipocyte

activation of nuclearreceptor PPAR

EDCs can promote the differentiationof pre-adipocytes via direct activation

of PPAR (e.g., phthalates, organotins)

EDCs can inducelipogenesis andinhibit lipolysis

in the adipocyte,increasing thestorage of fat

PPAR, fatty acidbinding protein 4,lipoprotein lipase

Adipogenic genes ONMe

EDCs can alterthe chromatin

landscape to favorthe transcriptional

activation ofadipogenic genes

(activatingenhancer

mark)

osteopontin,type II collagen,

myosin heavy chainAc

Osterogenic, Chondrogenic,Myogenic genes OFF

Mechanisms that promote adipose development

(and where EDCs can potentially act)

Implications For Human Health• Diet and exercise are insufficient to explain obesity epidemic

particularly in the very young

• Obesogens inappropriately stimulate adipogenesis and fat storage– Prescription drugs

• Thiazolidinediones, atypical antipsychotics, anti-depressants– Environmental contaminants

• organotins, estrogens (BPA, DEHP), PFOS, DDE, POPs

• Prenatal obesogen exposure reprograms exposed animals to be fat– Epigenetic changes alter fate of stem cell compartment -> more

preadipocytes and more adipocyte progenitors– Effects can be trans-generational

• Obesogens shift paradigm from treatment to prevention during pregnancy, childhood and puberty– Reduced exposure to obesogens, optimized nutrition– Obesity is intractable once established

• UCI - Blumberg LabRachelle AbbeySathya BalanchadrStephanie CaseyRaquel Chamorro-

GarciaConnie ChungAmanda JanesickJasmine LiHang PhamPeggy Saha

• NINS – Okazaki, JapanTaisen IguchiHajime Watanabe

• NIHS - Tokyo, JapanJun Kanno

• University of TokyoSatoshi InoueKotaro Azuma

Funding from NIEHS, US-EPA, UC-TSR&TP

• Former lab membersConnie ChowFelix GrünTiffany KieuSéverine KirchnerSophia LiuLauren MaedaMichelle TabbGina TurcoZamaneh

ZamanianChangcheng Zhou

• UCI collaboratorsOlivier CinquinDavid Fruman Matt JanesEd NelsonEric PotmaPathik Wadhwa

• Prenatal & early life exposures to low levels of PCBs and DDE are associated with increased weight in boys and girls at puberty (Gladen et al, J. Pediatr., 2000).

• Childhood obesity is associated with maternal smoking in pregnancy (Toschke et al, Eur J Pediatr 2002)

• Soy-based formula in infancy is a potential risk factor for overweight later in life (Strom et al., JAMA, 2001; Stettler et al., 2005).

• Concentrations of urinary phthalate metabolites are associated with increased waist circumference and insulin resistance in adult US males (Stahlhut et al, EHP, 2007)

• Exposure to HCB during pregnancy increases the risk of overweight in children aged 6 years ( Smink et al, Acta Paediatrica, 2008)

• Intrauterine exposure to environmental pollutants (POPs) increases body mass during the first 3 years of life (Verhulst et al EHP, 2009)

• Prenatal exposure to DDE is associated with rapid weight gain in the first 6 months and elevated BMI later (Mendez et al EHP, 2011)

Human Studies Supporting the Obesogen Hypothesis