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Obesogens, Stem Cells and the Maternal Programming of Obesity
Bruce Blumberg, Ph.D. Department of Developmental and Cell Biology Department of Pharmaceutical Sciences Developmental Biology Center University of California, Irvine 1
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Main Points Epigenetics links environment to altered gene expression
Obesogens exist and contribute to obesity epidemic Obesogen action may involve reprogramming of stem cells
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What is Epigenetics ? Literally “on top of genetics”
Goldberg et al. (2007) Cell 128, Literally “on top of genetics” coined by C.H. Waddington in 1957 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
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Epigenetics acts through chromatin stucture
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!
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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
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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)
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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 Developmental programming continues into adolescence Överkalix studies in Sweden linking nutrition and longevity Programming of adipocyte number continues into puberty Diseases with Developmental Origins (from animal models) Cardiovascular, Pulmonary (asthma) Neurological (ADHD, Neurodegenerative diseases), Immune/autoimmune Endocrine, reproductive/fertility, cancer Obesity/diabetes
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The Worldwide Obesity Epidemic
34% of the US population are clinically obese (BMI > 30) Double worldwide average (Flegal et al. JAMA 2010;303: ) 68% are overweight (BMI > 25) – 86% estimated by 2020 BMI = 31.5 From Lars Lind Visceral obesity pathological Subcutaneous obesity adaptive BMI ~32
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Obesity Trends* Among U.S. Adults BRFSS, 1990, 1999, 2008
(*BMI 30, or about 30 lbs. overweight for 5’4” person) 1990 1999 ~17,000 22,401 30,961 2008 No Data <10% %–14% %–19% %–24% %–29% ≥30% Sources: CDC (map), U.S. Census bureau (numbers)
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The Worldwide Obesity Epidemic
34% of the US population are clinically obese (BMI > 30) Double worldwide average (Flegal et al. JAMA 2010;303: ) 68% are overweight (BMI > 25 ) – 86% estimated by 2020 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)
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How does obesity occur ? Prevailing wisdom – “couch potato syndrome” Positive energy balance, i.e., too much food, too little exercise 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
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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, (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
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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): (2009) Details scientific support for existence and effects of EDCs Endorsed by American Medical Association Led to H.R Endocrine Disruption Prevention Act of 2009 Moves responsibility for research from EPA to NIEHS
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Endocrine Disrupting Chemicals (EDCs)
Are EDC-mediated disturbances in endocrine signaling pathways involved in adipogenesis and obesity
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The Nuclear Hormone Receptor Superfamily
Known Receptors Classical receptors (from biochemistry) GR cortisol MR aldosterone AR testosterone PR progesterone ER α,β estradiol VDR 1,25-(OH)2 vit D3 TR α,β triiodothyronine EcR 20-OH ecdysone Orphan Receptors Vertebrate Drosophila TR-2 α,β DHR78 NGFI-B α,β,γ DHR38 ROR α,β,γ DHR3 Rev-erb α,β E75, E78 SF-1 α,β FTZ-F1 α,β COUP α,β,γ svp HNF-4 α, β HNF-4 Tlx α,β tll No known homologs ERR α,β,γ knirps DAX-1 knirps-related SHP egon GCNF DHR96 C. elegans ~250 nuclear receptors D. melanogaster ~20 nuclear receptors H. sapiens ~48 genes Arabidopsis no family members Adopted (EX) Orphans RAR α,β,γ all-trans retinoic acid RXR α,β,γ 9-cis retinoic acid PPAR α,β,γ fatty acids, eicosanoids LXR α,β oxy-sterols FXR α,β bile acids BXR α,β benzoates Nearly adopted orphans (natural ligands?) CAR androstanes, xenobiotics SXR/PXR steroids, xenobiotics
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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 Pre- and postnatal exposure to EDCs such as environmental estrogens (ER) increases weight DES, genistein, bisphenol A 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 several compounds cause adipocyte differentiation in vitro (PPARγ) phthalates, BPA, aklylphenols, PFOA, organotins Existence of obesogens is plausible
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Endocrine disruption by organotins
Organotins -> imposex in mollusks Sex reverses genetically female flounder and zebrafish -> males Which hormone receptors might be organotin targets? 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 Sn Cl Tributyltin-Cl
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Structures of RXR and PPARγ-specific agonists
9-cis-RA Kd = 1 nM EC50 = 15 nM COOH Rosiglitazone Kd = nM; EC50 = 300 nM Tributyltin-Cl Kd = 12 nM EC50 = 5 nM Sn Cl N COOH LG268 Kd = 3 nM; EC50 = 3 nM
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Organotins show strong SAR on hRXR
80 LG268 Butyltin Dibutyltin 70 Tributyltin Tetrabutyltin Butyltin Tris(2-EHA) 60 EC50 DBT > 2800 nM TBT nM 4BT nM 50 Fold Activation 40 30 20 10 0.01 0.1 1 10 100 1000 10000 Concentration nM Grun et al., Molec Endocrinol, 2006
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TBT activates PPAR Fold Activation toxic Concentration (nM)
1 10 100 1000 10000 100000 None 2 3 4 5 6 7 8 9 Troglitazone TBT LG268 AGN203 Concentration (nM) Fold Activation toxic PPARg –regulates lipid metabolism and adipocyte differentiation Grun et al., Molec Endocrinol, 2006
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Nuclear receptor activation by organotins
Nuclear Receptor LBD EC50 nM Ligand hRXRα hRARα hPPARγ LG268 2-5 na AGN203 0.5-2 9-cis RA 15 all-trans RA 8 Butyltin chloride Dibutyltin chloride 3000 Tributyltin chloride 3-8 20 Tetrabutyltin chloride 150 ND Di(triphenyltin) oxide 2-10 Butyltin-tris (2-ethylhexanoate) Troglitazone 1000 Organotins are highly potent nuclear receptor agonists Do they bind to the receptors?
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Competitive Binding of TBT
Specific Bound cpm 500 1000 1500 2000 2500 3000 LG268 TBT 0.1 1 10 100 103 Concentration nM his6-hRXR Kd = 12.5 nM Kd = 7.5 nM 1000 2000 3000 4000 5000 6000 7000 Troglitazone TBT 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
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What is the effect of TBT treatment, in vivo?
Newborn Liver ± TBT (in utero) Vehicle (corn oil) TBT What is the effect of prenatal TBT exposure on adult animals? Grun et al., Molec Endocrinol, 2006
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TBT increases testis fat pad weight at 10 weeks
. 4 16% increase p = 0.037 ) . 3 s m a r g ( t h . 2 g i e W . 1 . C o n t r o l T B T n = 9 n = 1 Fat depot size increases at the expense of overall body mass Grun et al., Molec Endocrinol, 2006
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How does TBT exposure cause weight gain?
Hypertrophy adipocytes Altered ability of adipocytes to process and store lipids? Changes in the hormonal control of appetite and satiety? Hyperplasia Commitment differentiation Preadipocytes Increased number of adipocytes or pre-adipocytes? 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
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TBT induces adipogenic differentiation in MSCs
Adipocyte Bone Cartilage differentiation conditions hMSCs + MDII + TBT Kirchner et al., 2010 Molec Endocrinol, 24,
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TBT induces adipogenic genes in MSCs
Kirchner et al., 2010 Molec Endocrinol, 24,
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Adipogenic effects of TBT and ROSI in MSCs require PPARγ
Induction : MDII +100 nM +100 nM TBT +1000 nM ROSI T T +0 nM +0 nM +100 nM +100 nM +DMSO +DMSO +DMSO Induction : 100 nM ROSI + MDII +0 nM +10 nM +100 nM +1000 nM T Induction : 50 nM TBT + MDII +0 nM +10 nM +100 nM +1000 nM T Kirchner et al., 2010 Molec Endocrinol, 24,
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Osteogenic capacity of hADSCs
x70 x240 *** *** Control (-) Osteo 6 5 4 * Alizarin Red-S + Sudan Black ratio Target / Housekeeping 3 2 * *** * *** 1 Osteo + Rosi Osteo + TBT Osteo Osteo Osteo Osteo Control (-) Control (-) Control (-) Control (-) Osteo + Rosi Osteo + TBT Osteo + Rosi Osteo + TBT Osteo + Rosi Osteo + TBT Osteo + Rosi Osteo + TBT OPN OSN aP2 LEP TBT overrides the effects of the bone-inducing cocktail, instead causing the cells to become adipocytes Kirchner et al., 2010 Molec Endocrinol, 24,
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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?
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In vivo assays to assess stem cell commitment
– – chemical exposure by gavage CMC CMC TBT TBT ROSI ROSI C57BLK6 - - Pregnant dam CD - 1 unexposed surrogate = in in utero utero exposed offspring exposed offspring adipocyte differentiation conditions bone differentiation conditions cartilage differentiation conditions
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Prenatal TBT exposure increases MSC differentiation into adipocytes
Kirchner et al., 2010 Molec Endocrinol, 24,
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In utero TBT exposure inhibits osteogenesis
In utero gavage treatment OPN CMC ROSI TBT 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 1 +DMSO +DMSO +DMSO 0.8 +TBT +TBT ratio Target / Housekeeping 0.6 *** +TBT 0.4 *** *** O A O A O A 0.2 *** *** Calcification +DMSO +TBT +DMSO +TBT +DMSO +TBT In utero CMC In utero ROSI In utero TBT 100 Fabp4 80 Kirchner et al., 2010 Molec Endocrinol, 24, 1000 ** 60 Lipid accumulation 800 *** staining (%surface) *** ** 40 40 600 *** ratio Target / Housekeeping *** *** 400 *** 20 20 ** 200 ** ** +DMSO +TBT +DMSO +TBT +DMSO +TBT +DMSO +TBT +DMSO +TBT +DMSO +TBT +DMSO +TBT +DMSO +TBT +DMSO +TBT
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Effects of prenatal TBT exposure on WAT and BMD
Males Females 1.6 1.4 Ab Fat wgt (g) 1.2 Males 1.0 0.8 0.6 CMC Rosi TBT CMC Rosi TBT 0.08 Males Females 0.07 BMD BMD Females 0.06 0.05 0.04 o CMC Rosi TBT CMC Rosi TBT Ab Fat wt Prenatal TBT leads to increased WAT and lower BMD What is the mechanism? 34
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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? Kirchner et al., 2010 Molec Endocrinol, 24,
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How does prenatal TBT exposure promote adipocyte differentiation?
Effects of in utero TBT exposure on adipogenic pathway genes LEP Resistin IRS-2 ADIPOQ uninduced + TBT 14D PPARγ2+/- PPARγ2+ Fabp4+ LEP+ Pref1- GyK+ PEPCK+ / LPL+ ADIPOQ+
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Epigenetic effects of prenatal TBT exposure on promoter methylation of PPARγ target genes
Kirchner et al., 2010 Molec Endocrinol, 24,
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How does TBT affect PPARγ regulators?
LEP Resistin IRS-2 ADIPOQ Ezh2 KLF4 Zfp423 BMP SIRT1 SMART NCoR RIP140 CBP/p300 SRC PGC-1α 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)
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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 RXRa ~20 nM EC50 and Kd on PPARg 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?
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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 PPARg ligands Is the environment making us fat?
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Mechanisms that promote adipose development (and where EDCs can potentially act)
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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
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Funding from NIEHS, US-EPA, UC-TSR&TP
UCI - Blumberg Lab Rachelle Abbey Sathya Balanchadr Stephanie Casey Raquel Chamorro-Garcia Connie Chung Amanda Janesick Jasmine Li Hang Pham Peggy Saha Former lab members Connie Chow Felix Grün Tiffany Kieu Séverine Kirchner Sophia Liu Lauren Maeda Michelle Tabb Gina Turco Zamaneh Zamanian Changcheng Zhou NINS – Okazaki, Japan Taisen Iguchi Hajime Watanabe NIHS - Tokyo, Japan Jun Kanno University of Tokyo Satoshi Inoue Kotaro Azuma UCI collaborators Olivier Cinquin David Fruman Matt Janes Ed Nelson Eric Potma Pathik Wadhwa Funding from NIEHS, US-EPA, UC-TSR&TP
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Human Studies Supporting the Obesogen Hypothesis
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)
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