JACOB E. (JED) FRIEDMAN, PHDJACOB E. (JED) FRIEDMAN, PHDDEPARTMENT OF PEDIATRICS, BIOCHEMISTRY & MOLECULAR GENETICSDEPARTM...
 Lynn Barbour, MDLynn Barbour, MD Teri Hernandez, PhD*Teri Hernandez, PhD* Rachael Van Pelt, PhD*Rachael Van Pelt, PhD*...
Outline of Talk:1) New Concepts in Fetal ProgrammingMaternal Obesity and the fetus:- Stem cell programming.- Microbiome in...
Critical Early Life Factors affect HealthCritical Early Life Factors affect HealthAcross the LifespanAcross the LifespanGe...
• 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 a...
Fetal Programming:The intrauterine environment canimpact fetal development at botha morphological and a molecularlevel.An ...
The Developing World is catching upThe Developing World is catching upNYTimes, 3-11-2012
Yajnik, Proc Nutr Soc; 2004Yajnik, Proc Nutr Soc; 2004Caucasian, 3500gCaucasian, 3500g Indian, 2700gTHE FAT-THIN INDIAN BA...
Insulin resistance **Hyperglycemia **Islet dysfunction **Dyslipidemia **Endothelial dysfunctionHypertension **Prothromboti...
October 4, 2010Development origins ofobesity, metabolic andchronic diseases
FETAL ORIGINS HYPOTHESISFETAL ORIGINS HYPOTHESISAND METABOLIC MEMORYAND METABOLIC MEMORY• Excessive metabolic factors in t...
Heerwagen et.al. AJP Review September 2010When Obesity and Pregnancy Combine:Significant evidence hereNot a lot of evidenc...
What We Don’t Know.What We Don’t Know.• What are the consequences of exposure to maternal obesityduring pregnancy on devel...
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 & ...
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 ...
MOTHERExposures during pregnancyMaternal Obesity/Diet - The Healthy Start CohortMetabolomeEpigenomeNEONATEMetabolomeEpigen...
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 primaril...
-Recruitment of MyeloidCell types.-Leakage of cytokines,unknown endotoxins.-May promote energyRetention & cross-talkwith k...
Bacterial vs. hostTranscriptomics ?
Outline of Talk:1) New Concepts in Fetal ProgrammingMaternal Obesity and the fetus:- Stem cell programming.- Microbiome in...
METABOLIC PROGRAMMING IN THEFETUS:IS IT A MATTER OF FAT?
Collaborative ResearchCollaborative ResearchOregon National Primate Research Center,Oregon National Primate Research Cente...
CTR = 15% Fat CaloriesHFD = 35% Fat Calories – Western Style DietDiet sensitive (HFD-S) vs resistant (HFD-R)Diet reversal ...
DIFFERENTIAL SENSITIVITY TO HIGH FAT DIET IN NHPDIFFERENTIAL SENSITIVITY TO HIGH FAT DIET IN NHP::–LOW WEIGHT GAIN = HIGH ...
J. Clin. Invest. 2009J. Clin. Invest. 2009▪▪Maternal HF diet/obesity leads to an earlyMaternal HF diet/obesity leads to an...
Fetal Hepatic Pathology UnderFetal Hepatic Pathology UnderConditions of Maternal ObesityConditions of Maternal ObesityFeta...
▪▪Elevated Fetal Liver TG occurred in all HF Y2-Y4 G130 fetusesElevated Fetal Liver TG occurred in all HF Y2-Y4 G130 fetus...
C-FOSC-FOS p-JNK1p-JNK1 p38 MAPKp38 MAPKElevated Stress Response pattern in Fat Y2/Y3-Elevated Stress Response in Fetal Y2...
• Hepatic Fat AccumulationOxidative DamageMitochondrial Dysfunction (Sirtuins)Kupffer Cell PrimingEpigenetic changesTG•Pos...
Why Does the Fetus Store Excess Lipids inWhy Does the Fetus Store Excess Lipids inLiver and other organs?Liver and other o...
ChowMaternal Fetal Breast Milk*** ***The increased n6/n3 ratio in thechow is made worse in the fetusN3s are critical for d...
Ragavendra et al., Placenta 2001Placental function is key to a healthypregnancy and normal fetal development• Hyperinsulin...
Placenta HistologyPlacenta HistologyControlControl HFD-SHFD-SFrias et al, Endocrinology 2011Pregnancy complications result...
These are actually decreases ininflammatory markers.Sex differences in inflammationassociated with obesity.These two signi...
Fetal circulating cytokinesFetal circulating cytokinesIncludes both HFD-S and HFD-R offspringIncludes both HFD-S and HFD-R...
Summary - PlacentaSummary - PlacentaDietary LipidsPostprandial sat. FFAN3-FFAN6/N3 ratioPlacentaPostprandial sat. FFAN3-FF...
What Persistsvs. gets betterin Juvenileanimals on ahealthy diet?
Study DesignStudy DesignConception0 mos 8 mos 12 mos 14 mosBirth WeaningAge:Maternal DietBody WeightDEXANecropsyHormonesMe...
Figure 1
Maternal Control-fed Mother-HFD-sensitiveMother-HFD-sensitive
Persistent liver TG in Juveniles exposed to HFD-SensitivePersistent liver TG in Juveniles exposed to HFD-Sensitivemothers,...
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 laterSimil...
Liver macrophage multiply in HFDLiver macrophage multiply in HFDexposed animals even on healthy dietexposed animals even o...
Increased Inflammatory and Lipogenic Genes inIncreased Inflammatory and Lipogenic Genes inHepatocytes from HFD-Sensitive M...
Summary: Maternal HFD:• Significant impact on placental function & development– Cytokine production – n6/n3 increased in d...
Excess FFA/TG DeliveryPlacental InflammationPlacental Nutrient TransferFetal Hepatic Fat AccumulationOxidative StressInfla...
Our Approach in Moms & Infants–Work in ProgressSubcutaneousFatHepatic FatVisceral FatCan we observe physiologic difference...
• Visceral fat is associated with severe insulinresistance; hepatic fat is associated with non-alcoholicfatty liver diseas...
18 obese mothers w/ GDM17 lean mothersExclusion Criteria:Pre-Pregnancy DiabetesIUGRPremature deliveryNICU admissionbirth S...
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 ...
Variable ß - coefficient P-valueMaternal Pre-pregnancy BMI 0.03170 0.0456Infant Sex 0.20682 0.4877Infant Age at Study -0.0...
Top Intrauterine Contributors to Neonatal AdiposityTop Intrauterine Contributors to Neonatal Adiposity Maternal Insulin R...
How do we modify this?Pregnant population limits drug options, especially innon-diabetic patients…• Pre-pregnancy lifestyl...
Omega-3 Fatty Acids and Pregnancy:Current Research:Focus on DHA forCognitive developmentReports of impairedOmega-3 transfe...
2 views of Inflammation
Figure 3 Cardiovascular effects of resveratrol. Resveratrol and/or Sirt1/AMP-activated kinase (AMPK) activation and/orphos...
Supplementary Figure 1-1 0 G120Necropsyand SampleCollectionNecropsyand SampleCollectionPre-pregMetabolicAnalysisPre-pregMe...
Figure 2 A B CD E FG H IGlucoseAUCPre-Preg 3rd Trim050001000015000 ∆WSD/RESV v CTR p<0.01Pre-Preg 3rd Trim0123Triglyceride...
Figure 3 A B CD E FG H I
Supplementary Table 2Maternal fatty acidsCTR (n=10) WSD (n=11) WSD/RESV (n=6) StatisticsSaturated FA 2987 ± 727 3329 ± 460...
Figure 4 Liver TriglyceridesA BCTR WSD WSD/RESV
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 (...
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/RESV02468...
Resveratrol SummaryResveratrol Summary• Resv caused weight loss prior to gestation despite noResv caused weight loss prior...
Figure 1. Complex Pathogenesis of Type 2 Diabetes.Genetic and environmental factors may influence the risk of diabetes thr...
Acknowledgement-Funding Sources:
Friedman o&p2013
Friedman o&p2013
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  • But how to tease apart question of genes vs. gestational exposure vs. post-natal environment?????
  • And this brings us to the concept of fetal programming. Morphological – that is the gross structure of tissues, and molecular – subcellular changes at the level of gene expression. It’s known from both human and animal data that an “adverse” environment can predispose the infant to later chronic disease.
  • When obesity and pregnancy combine, these metabolic changes can be exacerbated, creating a sort of perfect storm in terms of fuel exposure to the fetus. Importantly, we are not talking about diabetic pregnancy here, so while glucose may still play a role, we are focusing on other factors such as increased lipid mobilization and inflammatory cytokines. There is good evidence for later childhood disease, but there is less evidence as to why.
  • WE chose G130 because, as I have shown, it is a critical period for hypothalamic development. It is also a critical period for the development of other metabolic systems in peripheral organs like the pancrease
  • For those not familiar with the first &amp; most important formed in primate development – this is a schematic of a human placenta. The placenta is the endocrine organ responsible for nutrient exchange and thus a key determinant of fetal growthn Abnormalities in placentation have been associated with emecvery obstetric complication. Despite this fact, the mechanismsr regulating placental growth / function are poorly understood. From implantation, the uterine vasculature undergoes significant angiogenesis intended to create a low resistance unit capable of maximizing nutrieant exchange. In this schematic….
  • We have previously reported that G130 fetuses have increased circulating cytokines as shown here. The cytokines with a statistically significant increase are highlighted in yellow.   This fetal inflammatory response is independent of maternal obesity. Is this systemic inflammatory response secondary to maternal inflammation?
  • Our study design is as follows- Offspring were used from mothers fed the control (blue) or HFD (red) . The offspring were exposed to the maternal diet throughout fetal life, from conception to birth, and during the nursing period until weaning at 8 mos of age. After weaning, offspring were placed on a control diet, allowing us to test if diet-reversal in HFD improved metabolic outcomes. Other groups of CON and HFD offspring were also exposed to a post-weaning HFD to test if metabolic outcomes are more severe. These groups will be shown with white shading. Studies have been ongoing for 3 years and final 4 th cohort of offspring will be studied this summer. Numerous measurements and data have been collected on these animals. Measurements of interest for today’s talk include bwt and dexa at 12 mos of age. These animals were sacrificed at 14 mos of age and plasma has analyzed for hormones and metabolites; liver tissue was collected for gene expression, protein expression, and lipid analysis. We also isolated hepatocytes and kupffer cells from a small subset of animals to perform more mechanistic in vitro studies.
  • Next, we took a closer look at the composition of FA in TG fraction using Mass Spec. We found a trend for increased TG in maternal HFD on postwean HFD compared CTR-HFD suggesting a programmed effect and is consistent with our fetal data. However, we also found high levels of TG in offspring on postwean CTR. This analysis has been initially performed in a small subset of samples and addition of more samples is planned. Most interestingly, we found increased N6:N3 in CON offsping on postweaning HFD and an even greater increase in offspring exposed to maternal and postweaning HFD. These data suggest increased exposure to potentially inflammatory FA.
  • We will study a total of 35 infants born to both obese and lean mothers. We will exclude… Infants will be studied at a single visit between 1-3 weeks of age. We will obtain the magnetic resonance studies described, a Pea Pod measurement, which is a standard instrument for measuring total body adiposity, skinfold measurements to see if easily obtained anthropometric measures correlate with our MR measures, and a feeding questionnaire to assess for differences between bottle fed and breastfed infants.
  • We will use magnetic resonance spectroscopy to quantify liver fat. MRS is the most sensitive radiographic technique for measuring liver fat, and its validity has been established in comparisons to liver biopsy triglyceride measurements. MRS is based on the fac that each molecule has its own resonance signature within the magnetic field. This square is called the voxel, and it is placed in the area of interest, and you then are given the signatures of molecules in that volume of tissue. The area under the curve then represents the abundance of that particular molecule. On the left is a spectroscopic graph with limited liver fat, as demonstrated in the right sided peak compared to the water peak. On the right is an example of a spectroscopic measure of increased liver fat.
  • To try to condense this talk into a punch line…..
  • One way of answering why this happens, is to modify the environment. So we want to reduce inflammation, IR, and lipids…. Clinician are understandably hesistant to use drugs on patients whose obesity is not overtly pathological Focusing in on dietary modifications – what can we modify?
  • In the context of pregnancy, treatment is focused on proper delivery to the fetus for brain development….. Concern has been raised about improper transport in context of obese pregnancy, but the focus has more been on increasing fetal levels vs. utilization as a maternal treatment.
  • Friedman o&p2013

    1. 1. 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.
    2. 2.  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……….
    3. 3. 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
    4. 4. 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
    5. 5. • 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
    6. 6. 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.
    7. 7. The Developing World is catching upThe Developing World is catching upNYTimes, 3-11-2012
    8. 8. Yajnik, Proc Nutr Soc; 2004Yajnik, Proc Nutr Soc; 2004Caucasian, 3500gCaucasian, 3500g Indian, 2700gTHE FAT-THIN INDIAN BABY8% fat8% fat16% fat16% fat
    9. 9. Insulin resistance **Hyperglycemia **Islet dysfunction **Dyslipidemia **Endothelial dysfunctionHypertension **Prothrombotic StateCardiac vulnerability **Increased ANS activityAppetite dysregulation **Reduced immune functionOsteoporosisHigh Risk OutcomesIn Low or High** Birthweight Adults
    10. 10. October 4, 2010Development origins ofobesity, metabolic andchronic diseases
    11. 11. 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
    12. 12. Heerwagen et.al. AJP Review September 2010When Obesity and Pregnancy Combine:Significant evidence hereNot a lot of evidence here
    13. 13. 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?
    14. 14. Diet and a Plastic Epigenome?Science, Jan 11, 2013
    15. 15. 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?
    16. 16. Fruchart Atherosclerosis 2009.RXRα as a potential candidate for epigenetic modification- K. Boyle
    17. 17. An Epigenetic HypothesisLipid Excess Oxidative Stresse.g. Methylation during development- K. Boyle
    18. 18. Current Studies: Fetal Stem CellProgramming- K. Boyle
    19. 19. 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
    20. 20. 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
    21. 21. May 12, 2013
    22. 22. Cell, 2012The Maternal Microbiome as a source of Inflammation in Human Pregnancy
    23. 23. 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.
    24. 24. -Recruitment of MyeloidCell types.-Leakage of cytokines,unknown endotoxins.-May promote energyRetention & cross-talkwith key metabolictissues.
    25. 25. Bacterial vs. hostTranscriptomics ?
    26. 26. 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
    27. 27. METABOLIC PROGRAMMING IN THEFETUS:IS IT A MATTER OF FAT?
    28. 28. 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.
    29. 29. 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
    30. 30. 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
    31. 31. 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
    32. 32. 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
    33. 33. ▪▪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
    34. 34. C-FOSC-FOS p-JNK1p-JNK1 p38 MAPKp38 MAPKElevated Stress Response pattern in Fat Y2/Y3-Elevated Stress Response in Fetal Y2-Y3 Livers-Elevated Stress Response in Fetal Y2-Y3 Liversin 3in 3rdrdTrimester- G130Trimester- G130J. Clin. Invest. 2009J. Clin. Invest. 2009
    35. 35. • 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
    36. 36. 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?
    37. 37. 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
    38. 38. 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
    39. 39. 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
    40. 40. 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
    41. 41. 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
    42. 42. Summary - PlacentaSummary - PlacentaDietary LipidsPostprandial sat. FFAN3-FFAN6/N3 ratioPlacentaPostprandial sat. FFAN3-FFAN6/N3 ratioTLR4MATERNAL FetalCytokinesCytokinesOxidative damageInsulin resistanceMetabolic mal-programmingHyperglycemiahyperinsulinemiaPregnancy complications
    43. 43. What Persistsvs. gets betterin Juvenileanimals on ahealthy diet?
    44. 44. Study DesignStudy DesignConception0 mos 8 mos 12 mos 14 mosBirth WeaningAge:Maternal DietBody WeightDEXANecropsyHormonesMetabolitesLiver TissueHepatocytesKupffer cellsPost-weaning DietControl (CON)HFD-Resistant (HFD-R)HFD- Sensitive (HFD-S)CON (n = 22)CON (n = 6)CON (n = 8)HFD (n = 9)HFD (n= 11)HFD (n= 10)
    45. 45. Figure 1
    46. 46. Maternal Control-fed Mother-HFD-sensitiveMother-HFD-sensitive
    47. 47. 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
    48. 48. No effect on adipose tissue orNo effect on adipose tissue orcirculating cytokines at 1 yoa.circulating cytokines at 1 yoa.
    49. 49. 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)
    50. 50. 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)
    51. 51. Increased Inflammatory and Lipogenic Genes inIncreased Inflammatory and Lipogenic Genes inHepatocytes from HFD-Sensitive Mothers.Hepatocytes from HFD-Sensitive Mothers.*****
    52. 52. 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.
    53. 53. 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%
    54. 54. Our Approach in Moms & Infants–Work in ProgressSubcutaneousFatHepatic FatVisceral FatCan we observe physiologic differences in harmful fat deposition that predateInfluence of diet and lifestyle?
    55. 55. • 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!
    56. 56. 18 obese mothers w/ GDM17 lean mothersExclusion Criteria:Pre-Pregnancy DiabetesIUGRPremature deliveryNICU admissionbirth Single study visit1-3 weeks of ageMRIMRSPea PodAnthropometricsFeedingQuestionnaire
    57. 57. Roland-Valadez E et al. Ann Hepatol, 2008.
    58. 58. 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
    59. 59. 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
    60. 60. 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.
    61. 61. 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…
    62. 62. 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
    63. 63. 2 views of Inflammation
    64. 64. 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
    65. 65. Supplementary Figure 1-1 0 G120Necropsyand SampleCollectionNecropsyand SampleCollectionPre-pregMetabolicAnalysisPre-pregMetabolicAnalysisAd libitum WSDAd libitum WSD7 Years3Ad libitum WSD/RESVAd libitum WSD/RESV WSDWSDMaternalMetabolicAnalysisMaternalMetabolicAnalysisMaternalMetabolicAnalysisMaternalMetabolicAnalysisG130Time with resveratrol supplementation (months)Post partumMetabolicAnalysisPost partumMetabolicAnalysis9Breeding seasonBreeding seasonWSD = Western Style Diet, 35% fat
    66. 66. Figure 2 A B CD E FG H IGlucoseAUCPre-Preg 3rd Trim050001000015000 ∆WSD/RESV v CTR p<0.01Pre-Preg 3rd Trim0123Triglycerides(mg/dl)∆WSD/RESV v CTR p<0.01
    67. 67. Figure 3 A B CD E FG H I
    68. 68. Supplementary Table 2Maternal fatty acidsCTR (n=10) WSD (n=11) WSD/RESV (n=6) StatisticsSaturated FA 2987 ± 727 3329 ± 460 2973 ± 828 NSMUFA 1041 ± 510 1330 ± 366 1486 ± 616 NSPUFA 2436 ± 746 2173 ± 512 2199 ± 622 NSN-6 2141 ± 684 2034 ± 452 2059 ± 593 NSN-3 295 ± 98 139 ± 74 140 ± 52p<0.001 CTR v WSDp<0.01 CTR v WSD/RESVN6:N3 8.14 ± 4.53 17.26 ± 6.88 15.49 ± 3.72p<0.01 CTR v WSDp<0.05 CTR v WSD/RESVFetal fatty acidsCTR (n=8) WSD (n=13) WSD/RESV (n=6) StatisticsSaturated FA 1465 ± 161 1577 ± 255 1385 ± 253 NSMUFA 317 ± 84 723 ± 285 729 ± 205 p<0.01 CTR v bothPUFA 1047 ± 221 1304 ± 624 1221 ± 468 NSN-6 863 ± 161 1177 ± 571 1084 ± 405 NSN-3 183 ± 64 127 ± 71 137 ± 70 NSN6:N3 4.92 ± 0.83 10.62 ± 4.52 8.63 ± 2.33 p<0.01 CTR v WSD
    69. 69. Figure 4 Liver TriglyceridesA BCTR WSD WSD/RESV
    70. 70. 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
    71. 71. Figure 5ABIns/Ggn/Ki67Ins/Ggn/CK7CTR WSD WSD/RESV
    72. 72. 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
    73. 73. 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?
    74. 74. 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.
    75. 75. Acknowledgement-Funding Sources:

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