Bruce Blumberg, Ph.D.Department of Developmental and Cell BiologyDepartment of Pharmaceutical SciencesDevelopmental Biology CenterUniversity of California, Irvine

Obesogens, Stem Cells and the Maternal Programming of

Obesity

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 – positive energy balance– “couch potato syndrome” - 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

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

• 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

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 toincreased fat mass together withweight gain in mice, in vivo

Tributyltin-ClSnCl

Grun et al., 2006. Molec Endocrinol 20, 2141-2155

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 home to adipose depots after induction (controversial)

• 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

hMSCs

+ MDII

+ TBT+DMSO, TBT or ROSI

BM WAT

mBMSCs mADSCs

+DMSO, TBT or ROSI

BM WAT

mBMSCs mADSCs

AdipocyteBone

Cartilagedifferentiation

conditions

TBT induces adipogenic genes in MSCs

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Osteogenic capacity of hADSCs

Control (-) Osteo

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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 MSCs– 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– PPARγ antagonists block adipogenic effects of TBT and ROSI

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

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

In vivo assaysto 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

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

• Prenatal TBT exposure inhibits calcium and expression of bone markers while it enhances lipid deposition and adipocyte marker expression

In utero TBT exposure inhibits osteogenesis

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Prenatal TBT exposure increases white fat (WAT) and decreases bone mineral density (BMD)

y = -0.0302x + 0.0877R² = 0.703

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• 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? Ki

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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+

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ADIPOQ

Prenatal TBT exposure reduces promoter methylation in PPARγ target genes with increased expression

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LEPResistinIRS-2

ADIPOQ

How does TBT affect PPARγ regulators?

Zfp423

Ezh2

SIRT1CBP/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)

Conclusions – organotins and nuclear receptors• Organotins are exceptionally potent agonists of RXR and PPARγ

– ~5 nM EC50, 12.5 nM Kd on RXRα– ~20 nM EC50 and Kd on PPARγ

• TBT drives adipocyte differentiation in mouse and human cell cultures

• TBT exposure during development induces adipogenesis in two vertebrate species: mouse and Xenopus– TBT induces expression of expected RXR/PPARγ target genes

involved in adipogenesis, in vivo.

• Induction of adipogenesis is novel and unexpected endocrine disrupting effect of TBT

• 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 PPARγ ligands

Is the environment making us fat?

Obesogens - Just the Tip of the Iceberg ?TBT/TPTPhthalates

PFOA

NicotineBisphenol AGenistein

DES

Organophosphate pesticidesPCBs ?, PBDEs ?

• What don’t we know yet?– How many obesogens are out there?– Body burdens in population?– Molecular targets of action beyond RXR-PPARγ– Critical windows of exposure?– How does prenatal exposure alter adult phenotype ?– Are the results of obesogen exposure reversible?

fructose

BaPAir pollution COX2 inhibitors

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

• Thiazolidinedione anti-diabetic drugs (Actos, Avandia)• 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

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

What do I believe needs to be done?

• Reduce obesogen exposure during sensitive periods (perinatal, early life through puberty)

• Decrease availability of highly palatable, poorly nutritive junk foods that engage pleasure centers in schools

• Encourage increased exercise and eating healthier diet.

• Tax incentives to encourage organic farming practices

• Consider restricting the use of high fructose corn syrup

• UCI - Blumberg LabRachelle AbbeyStephanie CaseyRaquel Chamorro-

GarciaAmanda FreiseAmanda JanesickJhyme LaudeJasmine LiSophia LiuHang PhamNhieu 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 KirchnerLauren MaedaZamaneh Zamanian

• 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