JACOB E. (JED) FRIEDMAN, PHDJACOB E. (JED) FRIEDMAN, PHDDEPARTMENT OF PEDIATRICS, BIOCHEMISTRY & MOLECULAR GENETICSDEPARTMENT OF PEDIATRICS, BIOCHEMISTRY & MOLECULAR GENETICSUNIVERSITY OF COLORADO SCHOOL OF MEDICINEUNIVERSITY OF COLORADO SCHOOL OF MEDICINEDirector, Colorado Program for Nutrition and Healthy DevelopmentDirector, Colorado Program for Nutrition and Healthy DevelopmentHot Topics in Obesity- The Obesity Society (TOS)Hot Topics in Obesity- The Obesity Society (TOS)Boston, MA, May 16, 2013Boston, MA, May 16, 2013Transgenerational Effects of Maternal Nutrition:Transgenerational Effects of Maternal Nutrition:Molecular Mechanisms, pre-clinical models.Molecular Mechanisms, pre-clinical models.
Lynn Barbour, MDLynn Barbour, MD Teri Hernandez, PhD*Teri Hernandez, PhD* Rachael Van Pelt, PhD*Rachael Van Pelt, PhD* Nancy Krebs MD*Nancy Krebs MD* Bridget Young, PhDBridget Young, PhD Sean Newsom, PhDSean Newsom, PhD Margaret Heerwagen, PhDMargaret Heerwagen, PhD Melanie Reece PhDMelanie Reece PhD David Brumbaugh, MD*David Brumbaugh, MD* Virginia Winn MD PhD*Virginia Winn MD PhD* Gaea Moore MDGaea Moore MD Anita Kramer, MSAnita Kramer, MS Becky DelaHoussaye, MSBecky DelaHoussaye, MS Molly Anderson, MS ,RDMolly Anderson, MS ,RD Catherine Chartiere Logan MSCatherine Chartiere Logan MS Linda Daniels RDLinda Daniels RD CTRC Nutritional support staffCTRC Nutritional support staff Stephanie Thorn, PhD*Stephanie Thorn, PhD* Carrie McCurdy, PhD*Carrie McCurdy, PhD* Kristen Boyle, PhD*Kristen Boyle, PhD* Dana Dabelea, MD, PhD*Dana Dabelea, MD, PhD* Allison Buti, M.S.Allison Buti, M.S. *Independent*IndependentInvestigatorsInvestigatorsIt Takes a Village……….It Takes a Village……….
Outline of Talk:1) New Concepts in Fetal ProgrammingMaternal Obesity and the fetus:- Stem cell programming.- Microbiome in mother/infant.2) Studies in Non-Human Primate:- Long term exposure to HFD and development- In utero fatty liver – the new “first hit”- Follow up studies at 1 year.3) Studies in Human Pregnancy & Obesity- Top 10 causes of infant adiposity- MRI at 2 weeks of life- Infant fat distribution.4) How can we modify the risks for fetal adiposity?-Use of Resveratrol in NHP model
Critical Early Life Factors affect HealthCritical Early Life Factors affect HealthAcross the LifespanAcross the LifespanGenesGenes Gestational Exposure Post-natal EnvironmentGestational Exposure Post-natal EnvironmentWorld-WideWorld-Wide Childhood Obesity EpidemicChildhood Obesity EpidemicThe Childhood Obesity Pipeline is Full and getting worseThe Childhood Obesity Pipeline is Full and getting worse
• Obese infants are up to 2-9 times as likelyObese infants are up to 2-9 times as likelyto be obese as adultsto be obese as adults Baird J, BMJ 2005;331:929.Baird J, BMJ 2005;331:929.• Maternal BMI ≥ 30 conferred 25% obesityMaternal BMI ≥ 30 conferred 25% obesityrisk at age 4 (~3-fold) indep of BWrisk at age 4 (~3-fold) indep of BW Whitaker RCWhitaker RCPediatrics 2004;114:29.Pediatrics 2004;114:29.• 25% of obese children age 4-10 have IGT25% of obese children age 4-10 have IGTLong Term ImplicationsLong Term Implicationsfor Infantfor Infant
Fetal Programming:The intrauterine environment canimpact fetal development at botha morphological and a molecularlevel.An “adverse” environment canpredispose an infant to later lifediseases, such as obesity,diabetes, and CVD.
The Developing World is catching upThe Developing World is catching upNYTimes, 3-11-2012
October 4, 2010Development origins ofobesity, metabolic andchronic diseases
FETAL ORIGINS HYPOTHESISFETAL ORIGINS HYPOTHESISAND METABOLIC MEMORYAND METABOLIC MEMORY• Excessive metabolic factors in the intrauterine environment (gluc, FFAs, TGs,inflammatory cytokines, insulin, hormones, growth factors), have a profound effect onprenatal development and enhances susceptibility to later chronic disease.• Early exposure: alters embryogenesis and placentation; alters nutrient transport byplacenta and gene expression• Mid Gestation: Alter number, growth, and function of organs after organogenesis(e.g. pancreatic hyperplasia, nephron mass, cardiac hypertrophy)• Late Pregnancy: Key period for regulatory energy set points on brain and neuronal-metabolic pathways for appetite regulation, metabolism, mitochondrial oxidativecapacity
Heerwagen et.al. AJP Review September 2010When Obesity and Pregnancy Combine:Significant evidence hereNot a lot of evidence here
What We Don’t Know.What We Don’t Know.• What are the consequences of exposure to maternal obesityduring pregnancy on development of fetal metabolic systemsand neonatal adiposity?• What are the potential mediators of these effects?Is there a role for metabolic impact on epigenetic factors?• What are the public health consequences of exposure to ourmodern maternal diet on the childhood obesity epidemicand what can we do about it?
Diet and a Plastic Epigenome?Science, Jan 11, 2013
Childhood obesity—methylate now, pay later?Choudhury, M. & Friedman, J. E. Nat. Rev. Endocrinol. 7, 439–440 (2011).NEWS & VIEWSA recent report has found an association between the methylation status ofspecific genes in human fetal tissue and the subsequent development ofchildhood adiposity in two longitudinal cohorts. Would epigenetic analysis atbirth, therefore, have utility in identifying future risk of obesity?
Fruchart Atherosclerosis 2009.RXRα as a potential candidate for epigenetic modification- K. Boyle
An Epigenetic HypothesisLipid Excess Oxidative Stresse.g. Methylation during development- K. Boyle
Current Studies: Fetal Stem CellProgramming- K. Boyle
Reduced Lipid Oxidation in Human Umbilical Cord derivedStem Cells from Infants born to Obese mothers.= lack of “Metabolic Flexibility”Experiment: 21-day myogenicdifferentiation in hUC-MSC-sfrom offspring of lean (n=2) andobese (n=2) mothers+/- lipid treatment(200 µP Oleate:palmitate [2:1])Boyle et al. Unpublished Data
MOTHERExposures during pregnancyMaternal Obesity/Diet - The Healthy Start CohortMetabolomeEpigenomeNEONATEMetabolomeEpigenomeImprintedGenesInfant OutcomesBody weightFat massImprinted & Non-imprinted GenesMeasurements:- Maternal phenotypes- Maternal Metabolomics- Maternal DNA methylation atimprinted genes- Neonatal Metabolomics- Neonatal DNAmethylation (imprinted& non-imprinted genes)- Cell differentiation &signaling in MSC- Birth and 5 months of age
May 12, 2013
Cell, 2012The Maternal Microbiome as a source of Inflammation in Human Pregnancy
Infant Gut Microbiome• Infants are born essentially “sterile” no microbiome.• Infants are colonized by microbes primarily through mom– Mode of delivery (MOD) e.g. C/S especially important (Dominguez-Bello et al. 2010).– Breast feeding vs Formula Fed (Hunt et al. 2011; Cabrera-rubio et al. 2012– Maternal phenotype?• Timing and acquisition of gut microflora during early life eventsappears to play significant role in health & disease (Flint et al. 2012).• ADA/Glaxo-SmithKline Sponsored study:– To determine how maternal obesity and diabetes act to colonize themicrobiome of the mother-infant pairs– To establish how maternal characteristics during a critical window ofdevelopment (4 months) impacts the infant microbiome community, andwhether it plays a role in infant adiposity.
-Recruitment of MyeloidCell types.-Leakage of cytokines,unknown endotoxins.-May promote energyRetention & cross-talkwith key metabolictissues.
Bacterial vs. hostTranscriptomics ?
Outline of Talk:1) New Concepts in Fetal ProgrammingMaternal Obesity and the fetus:- Stem cell programming.- Microbiome in mother/infant.3) Studies in Human Pregnancy & Obesity- Top 10 causes of infant adiposity- MRI at 2 weeks of life- Infant fat distribution.4) How can we modify the risks for fetal adiposity?-Use of Resveratrol in NHP model
METABOLIC PROGRAMMING IN THEFETUS:IS IT A MATTER OF FAT?
Collaborative ResearchCollaborative ResearchOregon National Primate Research Center,Oregon National Primate Research Center,University of ColoradoUniversity of ColoradoLONG-TERM GOAL:• To develop a Non-Human Primate Model to study the effects ofMaternal Diet, Obesity and GDM on the development of metabolicsystems (liver, muscle, fat, heart, brain) in utero and beyond.
CTR = 15% Fat CaloriesHFD = 35% Fat Calories – Western Style DietDiet sensitive (HFD-S) vs resistant (HFD-R)Diet reversal group (DR) – HFD animals switchedback to CTR diet just prior to pregnancy.Young adult female Japanese macaques - CTRor HFD for 2-6 yearsMODEL
DIFFERENTIAL SENSITIVITY TO HIGH FAT DIET IN NHPDIFFERENTIAL SENSITIVITY TO HIGH FAT DIET IN NHP::–LOW WEIGHT GAIN = HIGH FAT RESISTANT- HFR–BIG WEIGHT GAIN = HIGH FAT SENSITIVE- HFS–BIG WEIGHT GAIN = HIGH FAT SENSITIVE- HFSNon-PregPregnant
J. Clin. Invest. 2009J. Clin. Invest. 2009▪▪Maternal HF diet/obesity leads to an earlyMaternal HF diet/obesity leads to an earlyFetal exposure (day 130) to elevated plasma TGFetal exposure (day 130) to elevated plasma TG
Fetal Hepatic Pathology UnderFetal Hepatic Pathology UnderConditions of Maternal ObesityConditions of Maternal ObesityFetallivertriglycerides,mg/g04.06.02.08.0MaternalDietControl HighfatReversal tocontrol* p <0.01*##p <0.05J. Clin. Invest. 2009J. Clin. Invest. 2009
▪▪Elevated Fetal Liver TG occurred in all HF Y2-Y4 G130 fetusesElevated Fetal Liver TG occurred in all HF Y2-Y4 G130 fetusesREGARDLESS of maternal diet responsivenessREGARDLESS of maternal diet responsivenessJ. Clin. Invest. 2009J. Clin. Invest. 2009
• Hepatic Fat AccumulationOxidative DamageMitochondrial Dysfunction (Sirtuins)Kupffer Cell PrimingEpigenetic changesTG•Post-natal western, high-fat diet•Genetic polymorphisms?•Environmental or dietary factors?Insulin ResistanceIncreased Fuel to Fetus•Pre-Gravid Obesity•Excess Weight Gain•Inflammation•Excess Lipids/glucoseOther factors•Maternal Microbiome Transfer to Neonate•Placental Inflammation•Oxidative StressChildhood “second hit”• Ongoing Fat AccumulationDe novo LipogenesisReduced FA oxidation• Hepatocellular InjuryKupffer Cell ActivationStellate Cell ActivationHepatocyte ApoptosisOxidative DamageEndoplasmic Reticulum StressInflammationHepatocyte Injury FibrosisFetal Liver – a “first hit?”SteatohepatitisTGDGDGSREBP1C
Why Does the Fetus Store Excess Lipids inWhy Does the Fetus Store Excess Lipids inLiver and other organs?Liver and other organs?• Excess lipid exposure exceeds fetal storage capacityExcess lipid exposure exceeds fetal storage capacityduring normal development of adipose tissue depots.during normal development of adipose tissue depots.• Hormonal factors (such as fetal hyperinsulinemia)Hormonal factors (such as fetal hyperinsulinemia)drive lipid storage.drive lipid storage.• Exposure to increased dietary n6/n3 ratio promotesExposure to increased dietary n6/n3 ratio promotesinflammation and causes metabolic re-programming?inflammation and causes metabolic re-programming?
ChowMaternal Fetal Breast Milk*** ***The increased n6/n3 ratio in thechow is made worse in the fetusN3s are critical for developmentPlos One. 2011Plos One. 2011
Ragavendra et al., Placenta 2001Placental function is key to a healthypregnancy and normal fetal development• Hyperinsulinemia andHyperinsulinemia andhyperglycemia (GDM) causehyperglycemia (GDM) causecomplications in placentalcomplications in placentalfunction.function.• What are the potential impactsWhat are the potential impactsof HFD consumption?of HFD consumption?– InflammationInflammation– Vascular dysfunctionVascular dysfunction
Placenta HistologyPlacenta HistologyControlControl HFD-SHFD-SFrias et al, Endocrinology 2011Pregnancy complications resulting in fetal death:CTRs 1 in 5 yrs (3%)HFD 8 in 5 yrs (24%); 7 in HFD-S animals
These are actually decreases ininflammatory markers.Sex differences in inflammationassociated with obesity.These two significant differenceswere not observed in fetal offspringMaternal Circulation Fetal PlacentaControl vs HF dietFrias et al, Endocrinology 2010Frias et al, Endocrinology 2010
Fetal circulating cytokinesFetal circulating cytokinesIncludes both HFD-S and HFD-R offspringIncludes both HFD-S and HFD-R offspringJ. Clin. Invest. 2009J. Clin. Invest. 2009
Persistent liver TG in Juveniles exposed to HFD-SensitivePersistent liver TG in Juveniles exposed to HFD-Sensitivemothers, despite weaning to a healthy dietmothers, despite weaning to a healthy dietLiver
No effect on adipose tissue orNo effect on adipose tissue orcirculating cytokines at 1 yoa.circulating cytokines at 1 yoa.
Increased inflammatory responsein Liver Kupffer cells (macrophage) from animalsexposed to maternal HFD – 1 year laterSimilar results for TNFα and IL-6Kupffer Cells = 100X ↑ in IL-1β vs Hepatocytes(N=2-3 per group)
Liver macrophage multiply in HFDLiver macrophage multiply in HFDexposed animals even on healthy dietexposed animals even on healthy diet15-75-fold enrichment in Kupffer cells(N=2-3 per group)
Increased Inflammatory and Lipogenic Genes inIncreased Inflammatory and Lipogenic Genes inHepatocytes from HFD-Sensitive Mothers.Hepatocytes from HFD-Sensitive Mothers.*****
Summary: Maternal HFD:• Significant impact on placental function & development– Cytokine production – n6/n3 increased in developing fetus– Decreased placental function – exacerbated in HFD-S mothers• Increased tissue lipid deposition (all tissues) and signs of NAFLD– Fatty liver (Inflammation, steatosis), SIRT1 genes.– Epigenetics –HSP70, Bmal1 (clock gene family) (FASEB J, 2010).– Fetal Pancreatic islet hyperplasia/-loss of fetal thyroid geneexpression (Mol Endo 2012).• Hyperphagia of palatable diets in offspring.• Social Behavior:Abnormalities in the melanocortin system (Endocrinol 2010).– Decreased Serotonin (J Neurosci, 2010); Female Offspring –increased anxiety. Male offspring – increased aggressive behavior– Both sexes display decreases in social behavior.• In Juvenile Animals- Persistant hepatic TGs & Inflammation inoffspring from obese mothers.
Excess FFA/TG DeliveryPlacental InflammationPlacental Nutrient TransferFetal Hepatic Fat AccumulationOxidative StressInflammationGluconeogenesisRecruitment andActivation of BoneMarrow WBCPrecursorsLifelong Increased Risk of aProinflammatory Responseto OvernutritionConsequences of Maternal Overfeeding on Fetal LiverConsequences of Maternal Overfeeding on Fetal LiverUS adults: 20-30%Obese adults: 60%US kids 9-19*: 17%Obese kids: 55%
Our Approach in Moms & Infants–Work in ProgressSubcutaneousFatHepatic FatVisceral FatCan we observe physiologic differences in harmful fat deposition that predateInfluence of diet and lifestyle?
• Visceral fat is associated with severe insulinresistance; hepatic fat is associated with non-alcoholicfatty liver disease; abdominal subcutaneous fat morelipolytic than peripherally stored fat• How does fetus store fat in visceral organs or liver ifexcess delivery?• If liver fat is present at birth, what happens?• Redistribution to subcutaneous adipose tissue?• “Programming” of liver to favor partitioning of fat towardsstorage• Sequential measures of total fat by PEAPOD andhepatic and visceral fat by MRI at 2 weeksLocation of Fat May Be Key!
18 obese mothers w/ GDM17 lean mothersExclusion Criteria:Pre-Pregnancy DiabetesIUGRPremature deliveryNICU admissionbirth Single study visit1-3 weeks of ageMRIMRSPea PodAnthropometricsFeedingQuestionnaire
Roland-Valadez E et al. Ann Hepatol, 2008.
Adiposity OutcomesNormal Weight Mothers (n=13) Obese/GDM Mothers(n=12)Outcome Mean (SD) Mean (SD) p-value*PEA POD (% body fat) 13.1(5.0) 14.7 (3.0) NSMRI subcutaneous fat (cm3) 707 (138) 777 (159) NSSum Skin Folds (mm) 9.9 (2.0) 11.7 (1.3) <0.05MRI visceral fat (cm3) 20.1 (4.6) 22.5 (9.2) NSVisceral Fat/Length (cm2) 0.39 (0.09) 0.43 (0.16) NSLiver Lipid (fat) #0.017 0.030 <0.05(76%increase)#Back transformation of natural log (lipid peaks liver/lipid peaks Intralipid)SubQFatPediatrics 2013
Variable ß - coefficient P-valueMaternal Pre-pregnancy BMI 0.03170 0.0456Infant Sex 0.20682 0.4877Infant Age at Study -0.00480 0.9620Infant Total Adiposity by Peapod 0.03540 0.5510Pediatrics, 2013
Top Intrauterine Contributors to Neonatal AdiposityTop Intrauterine Contributors to Neonatal Adiposity Maternal Insulin Resistance (could explain chunk of below)Maternal Insulin Resistance (could explain chunk of below) Maternal BMIMaternal BMI HyperglycemiaHyperglycemia Maternal TG and FFAMaternal TG and FFA Maternal High Fat Diet (Indep of mat obesity through changes infantMaternal High Fat Diet (Indep of mat obesity through changes infantmetabolome, appetite regulation, behaviors,, mitochondrial oxidation)metabolome, appetite regulation, behaviors,, mitochondrial oxidation) Excess Gestational Weight Gain.Excess Gestational Weight Gain. Maternal Inflammatory Cytokines (change in placental gene expressionMaternal Inflammatory Cytokines (change in placental gene expressionand transport)and transport) What About?What About? Oxidative Stress and Increased Lipid Exposure in early life?Oxidative Stress and Increased Lipid Exposure in early life? Placental and Fetal Growth Factors (fetal hyperinsulinemiaPlacental and Fetal Growth Factors (fetal hyperinsulinemiaresponse to hyperglycemia, placental IGFs)response to hyperglycemia, placental IGFs) Psychological stressors? ↑CRH and IL1Psychological stressors? ↑CRH and IL1ββ in mom; ↑GR and insulin inin mom; ↑GR and insulin infetus.fetus.
How do we modify this?Pregnant population limits drug options, especially innon-diabetic patients…• Pre-pregnancy lifestyle modifications• Controlling gestational weight gain• Exercise• Dietary modificationsReduce inflammation, insulin resistance, hyperlipidemia…
Omega-3 Fatty Acids and Pregnancy:Current Research:Focus on DHA forCognitive developmentReports of impairedOmega-3 transfer in obesepregnancyNo focus on use as anintervention in obesepregnancyMaternal supply of PUFA essential for fetal development
2 views of Inflammation
Figure 3 Cardiovascular effects of resveratrol. Resveratrol and/or Sirt1/AMP-activated kinase (AMPK) activation and/orphosphodiesterase (PDE) inhibition have been shown to improve vascular function and reduce hypertension via increased nitricoxide (NO) p...Jay H. Chung , Vincent Manganiello , Jason R.B. DyckResveratrol as a calorie restriction mimetic: therapeutic implicationsTrends in Cell Biology Volume 22, Issue 10 2012 546 - 554http://dx.doi.org/10.1016/j.tcb.2012.07.004
Table 1CTR WSD WSD/RESV StatisticsBody Mass (g) 345.5±5.1 (29) 347.6±7.4 (34) 317.2±22.3 (6) NSBrain Mass (g) 45.85±1.05 (16) 46.56±0.81 (21) 43.38±1.55 (6) NSHeart Mass (g) 2.09±0.08 (15) 2.11±0.07 (24) 2.05±0.20 (6) NSLiver Mass (g) 9.43±0.19 (26) 9.99±0.21 (29) 9.47±0.60 (6) NSPancreas Mass (mg) 249.4±11.80 (27) 246.5±11.95 (31) 349.3±20.14 (6)p<0.01 CTR v WSD/RESVp<0.01 WSD v WSD/RESVPancreas:Body Mass(mg/g)0.72±0.04 (27) 0.72±0.04 (31) 1.11±0.06 (6)p<0.001 CTR v WSD/RESVp<0.0001 WSD v WSD/RESVFetal Tissues –Increase in Pancreas weight
Figure 5ABIns/Ggn/Ki67Ins/Ggn/CK7CTR WSD WSD/RESV
C D E FG H IIsletMass(mg)CTR WSD WSD/RESV05101520β-CellMass(mg)CTR WSD WSD/RESV05101520α-CellMass(mg)CTR WSD WSD/RESV02468aa,bbα-:β-CellRatioCTR WSD WSD/RESV0.00.51.01.5aa,bbCTR WSD WSD/RESV0.000000.000050.000100.00015#Ki67+α-Cells/α-CellArea(#/µm2)CTR WSD WSD/RESV0.000000.000050.000100.00015#Ki67+β-Cells/β-CellArea(#/µm2)aabIns+CK7+Area/CK7+AreaCTR WSD WSD/RESV0.00.51.01.5Ggn+CK7+Area/CK7+AreaCTR WSD WSD/RESV0.00.51.01.52.02.5aabJFigure 5 Islet Cells – α cell loss relative to β cells
Resveratrol SummaryResveratrol Summary• Resv caused weight loss prior to gestation despite noResv caused weight loss prior to gestation despite nochange in food intake, even on HFD.change in food intake, even on HFD.• Fatty Acid profile significantly improved despite HFD.Fatty Acid profile significantly improved despite HFD.• Resveratrol improved placental function.Resveratrol improved placental function.• Resveratrol appears to cross the placenta andResveratrol appears to cross the placenta andaccumulate in the fetus.accumulate in the fetus.• No change in fetal weight, significant improvement inNo change in fetal weight, significant improvement inhepatic lipids.hepatic lipids.• Pancreas- increase in proliferation-yet loss ofPancreas- increase in proliferation-yet loss of αα-cell-cellmass relative to b cell.mass relative to b cell.• Long term implications?Long term implications?
Figure 1. Complex Pathogenesis of Type 2 Diabetes.Genetic and environmental factors may influence the risk of diabetes throughthe pathways illustrated in the figure or through as-yet-unidentifed mechanismsaffecting insulin sensitivity and/or insulin secretion. Kahn, CR, CellMetabolism, 2008.