1. JACOB E. (JED) FRIEDMAN, PHDJACOB E. (JED) FRIEDMAN, PHD
DEPARTMENT OF PEDIATRICS, BIOCHEMISTRY & MOLECULAR GENETICSDEPARTMENT OF PEDIATRICS, BIOCHEMISTRY & MOLECULAR GENETICS
UNIVERSITY OF COLORADO SCHOOL OF MEDICINEUNIVERSITY OF COLORADO SCHOOL OF MEDICINE
Director, Colorado Program for Nutrition and Healthy DevelopmentDirector, Colorado Program for Nutrition and Healthy Development
Hot Topics in Obesity- The Obesity Society (TOS)Hot Topics in Obesity- The Obesity Society (TOS)
Boston, MA, May 16, 2013Boston, MA, May 16, 2013
Transgenerational Effects of Maternal Nutrition:Transgenerational Effects of Maternal Nutrition:
Molecular Mechanisms, pre-clinical models.Molecular Mechanisms, pre-clinical models.

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*Independent
InvestigatorsInvestigators
It Takes a Village……….It Takes a Village……….

4. Outline of Talk:
1) New Concepts in Fetal Programming
Maternal 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

5. Critical Early Life Factors affect HealthCritical Early Life Factors affect Health
Across the LifespanAcross the Lifespan
GenesGenes Gestational Exposure Post-natal EnvironmentGestational Exposure Post-natal Environment
World-WideWorld-Wide Childhood Obesity EpidemicChildhood Obesity Epidemic
The Childhood Obesity Pipeline is Full and getting worseThe Childhood Obesity Pipeline is Full and getting worse

6. • Obese infants are up to 2-9 times as likelyObese infants are up to 2-9 times as likely
to 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% obesity
risk at age 4 (~3-fold) indep of BWrisk at age 4 (~3-fold) indep of BW Whitaker RCWhitaker RC
Pediatrics 2004;114:29.Pediatrics 2004;114:29.
• 25% of obese children age 4-10 have IGT25% of obese children age 4-10 have IGT
Long Term ImplicationsLong Term Implications
for Infantfor Infant

7. Fetal Programming:
The intrauterine environment can
impact fetal development at both
a morphological and a molecular
level.
An “adverse” environment can
predispose an infant to later life
diseases, such as obesity,
diabetes, and CVD.

8. The Developing World is catching upThe Developing World is catching up
NYTimes, 3-11-2012

9. Yajnik, Proc Nutr Soc; 2004Yajnik, Proc Nutr Soc; 2004
Caucasian, 3500gCaucasian, 3500g Indian, 2700g
THE FAT-THIN INDIAN BABY
8% fat8% fat
16% fat16% fat

10. Insulin resistance **
Hyperglycemia **
Islet dysfunction **
Dyslipidemia **
Endothelial dysfunction
Hypertension **
Prothrombotic State
Cardiac vulnerability **
Increased ANS activity
Appetite dysregulation **
Reduced immune function
Osteoporosis
High Risk Outcomes
In Low or High** Birthweight Adults

11. October 4, 2010
Development origins of
obesity, metabolic and
chronic diseases

12. FETAL ORIGINS HYPOTHESISFETAL ORIGINS HYPOTHESIS
AND METABOLIC MEMORYAND METABOLIC MEMORY
• Excessive metabolic factors in the intrauterine environment (gluc, FFAs, TGs,
inflammatory cytokines, insulin, hormones, growth factors), have a profound effect on
prenatal development and enhances susceptibility to later chronic disease.
• Early exposure: alters embryogenesis and placentation; alters nutrient transport by
placenta 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 oxidative
capacity

13. Heerwagen et.al. AJP Review September 2010
When Obesity and Pregnancy Combine:
Significant evidence here
Not a lot of evidence here

14. What We Don’t Know.What We Don’t Know.
• What are the consequences of exposure to maternal obesity
during pregnancy on development of fetal metabolic systems
and 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 our
modern maternal diet on the childhood obesity epidemic
and what can we do about it?

15. Diet and a Plastic Epigenome?
Science, Jan 11, 2013

16. Childhood obesity—methylate now, pay later?
Choudhury, M. & Friedman, J. E. Nat. Rev. Endocrinol. 7, 439–440 (2011).
NEWS & VIEWS
A recent report has found an association between the methylation status of
specific genes in human fetal tissue and the subsequent development of
childhood adiposity in two longitudinal cohorts. Would epigenetic analysis at
birth, therefore, have utility in identifying future risk of obesity?

17. Fruchart Atherosclerosis 2009.
RXRα as a potential candidate for epigenetic modification
- K. Boyle

18. An Epigenetic Hypothesis
Lipid Excess Oxidative Stress
e.g. Methylation during development
- K. Boyle

19. Current Studies: Fetal Stem Cell
Programming
- K. Boyle

20. Reduced Lipid Oxidation in Human Umbilical Cord derived
Stem Cells from Infants born to Obese mothers.
= lack of “Metabolic Flexibility”
Experiment: 21-day myogenic
differentiation in hUC-MSC-s
from offspring of lean (n=2) and
obese (n=2) mothers
+/- lipid treatment
(200 µP Oleate:palmitate [2:1])
Boyle et al. Unpublished Data

21. MOTHER
Exposures during pregnancy
Maternal Obesity/Diet - The Healthy Start Cohort
Metabolome
Epigenome
NEONATE
Metabolome
Epigenome
Imprinted
Genes
Infant Outcomes
Body weight
Fat massImprinted & Non-
imprinted Genes
Measurements:
- Maternal phenotypes
- Maternal Metabolomics
- Maternal DNA methylation at
imprinted genes
- Neonatal Metabolomics
- Neonatal DNA
methylation (imprinted
& non-imprinted genes)
- Cell differentiation &
signaling in MSC
- Birth and 5 months of age

22. May 12, 2013

23. Cell, 2012
The Maternal Microbiome as a source of Inflammation in Human Pregnancy

24. 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 events
appears 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 the
microbiome of the mother-infant pairs
– To establish how maternal characteristics during a critical window of
development (4 months) impacts the infant microbiome community, and
whether it plays a role in infant adiposity.

25. -Recruitment of Myeloid
Cell types.
-Leakage of cytokines,
unknown endotoxins.
-May promote energy
Retention & cross-talk
with key metabolic
tissues.

26. Bacterial vs. host
Transcriptomics ?

27. Outline of Talk:
1) New Concepts in Fetal Programming
Maternal 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

28. METABOLIC PROGRAMMING IN THE
FETUS:
IS IT A MATTER OF FAT?

29. Collaborative ResearchCollaborative Research
Oregon National Primate Research Center,Oregon National Primate Research Center,
University of ColoradoUniversity of Colorado
LONG-TERM GOAL:
• To develop a Non-Human Primate Model to study the effects of
Maternal Diet, Obesity and GDM on the development of metabolic
systems (liver, muscle, fat, heart, brain) in utero and beyond.

30. CTR = 15% Fat Calories
HFD = 35% Fat Calories – Western Style Diet
Diet sensitive (HFD-S) vs resistant (HFD-R)
Diet reversal group (DR) – HFD animals switched
back to CTR diet just prior to pregnancy.
Young adult female Japanese macaques - CTR
or HFD for 2-6 years
MODEL

31. 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- HFS
Non-Preg
Pregnant

32. J. Clin. Invest. 2009J. Clin. Invest. 2009
▪▪Maternal HF diet/obesity leads to an earlyMaternal HF diet/obesity leads to an early
Fetal exposure (day 130) to elevated plasma TGFetal exposure (day 130) to elevated plasma TG

33. Fetal Hepatic Pathology UnderFetal Hepatic Pathology Under
Conditions of Maternal ObesityConditions of Maternal Obesity
Fetallivertriglycerides,
mg/g
0
4.
0
6.0
2.0
8.0
Maternal
Diet
Control High
fat
Revers
al to
control
* p <0.01
*
#
#
p <0.05
J. Clin. Invest. 2009J. Clin. Invest. 2009

34. ▪▪Elevated Fetal Liver TG occurred in all HF Y2-Y4 G130 fetusesElevated Fetal Liver TG occurred in all HF Y2-Y4 G130 fetuses
REGARDLESS of maternal diet responsivenessREGARDLESS of maternal diet responsiveness
J. Clin. Invest. 2009J. Clin. Invest. 2009

35. C-FOSC-FOS p-JNK1p-JNK1 p38 MAPKp38 MAPK
Elevated Stress Response pattern in Fat Y2/Y3
-Elevated Stress Response in Fetal Y2-Y3 Livers-Elevated Stress Response in Fetal Y2-Y3 Livers
in 3in 3rdrd
Trimester- G130Trimester- G130
J. Clin. Invest. 2009J. Clin. Invest. 2009

36. • Hepatic Fat Accumulation
Oxidative Damage
Mitochondrial Dysfunction (Sirtuins)
Kupffer Cell Priming
Epigenetic changes
TG
•Post-natal western, high-fat diet
•Genetic polymorphisms?
•Environmental or dietary factors?
Insulin Resistance
Increased Fuel to Fetus
•Pre-Gravid Obesity
•Excess Weight Gain
•Inflammation
•Excess Lipids/glucose
Other factors
•Maternal Microbiome Transfer to Neonate
•Placental Inflammation
•Oxidative Stress
Childhood “second hit”
• Ongoing Fat Accumulation
De novo Lipogenesis
Reduced FA oxidation
• Hepatocellular Injury
Kupffer Cell Activation
Stellate Cell Activation
Hepatocyte Apoptosis
Oxidative Damage
Endoplasmic Reticulum Stress
Inflammation
Hepatocyte Injury Fibrosis
Fetal Liver – a “first hit?”
Steatohepatitis
TG
DG
DG
SREBP1C

37. Why Does the Fetus Store Excess Lipids inWhy Does the Fetus Store Excess Lipids in
Liver and other organs?Liver and other organs?
• Excess lipid exposure exceeds fetal storage capacityExcess lipid exposure exceeds fetal storage capacity
during 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 promotes
inflammation and causes metabolic re-programming?inflammation and causes metabolic re-programming?

38. Chow
Maternal Fetal Breast Milk
*
*
* *
*
*
The increased n6/n3 ratio in the
chow is made worse in the fetus
N3s are critical for development
Plos One. 2011Plos One. 2011

39. Ragavendra et al., Placenta 2001
Placental function is key to a healthy
pregnancy and normal fetal development
• Hyperinsulinemia andHyperinsulinemia and
hyperglycemia (GDM) causehyperglycemia (GDM) cause
complications in placentalcomplications in placental
function.function.
• What are the potential impactsWhat are the potential impacts
of HFD consumption?of HFD consumption?
– InflammationInflammation
– Vascular dysfunctionVascular dysfunction

40. Placenta HistologyPlacenta Histology
ControlControl HFD-SHFD-S
Frias et al, Endocrinology 2011
Pregnancy complications resulting in fetal death:
CTRs 1 in 5 yrs (3%)
HFD 8 in 5 yrs (24%); 7 in HFD-S animals

41. These are actually decreases in
inflammatory markers.
Sex differences in inflammation
associated with obesity.
These two significant differences
were not observed in fetal offspring
Maternal Circulation Fetal PlacentaControl vs HF diet
Frias et al, Endocrinology 2010Frias et al, Endocrinology 2010

42. Fetal circulating cytokinesFetal circulating cytokines
Includes both HFD-S and HFD-R offspringIncludes both HFD-S and HFD-R offspring
J. Clin. Invest. 2009J. Clin. Invest. 2009

43. Summary - PlacentaSummary - Placenta
Dietary Lipids
Postprandial sat. FFA
N3-FFA
N6/N3 ratio
Placenta
Postprandial sat. FFA
N3-FFA
N6/N3 ratio
TLR4
MATERNAL Fetal
Cytokines
Cytokines
Oxidative damage
Insulin resistance
Metabolic mal-programming
Hyperglycemia
hyperinsulinemia
Pregnancy complications

44. What Persists
vs. gets better
in Juvenile
animals on a
healthy diet?

45. Study DesignStudy Design
Conception
0 mos 8 mos 12 mos 14 mos
Birth Weaning
Age:
Maternal Diet
Body Weight
DEXA
Necropsy
Hormones
Metabolites
Liver Tissue
Hepatocytes
Kupffer cells
Post-weaning Diet
Control (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)

46. Figure 1

47. Maternal Control-fed Mother-HFD-sensitiveMother-HFD-sensitive

48. Persistent liver TG in Juveniles exposed to HFD-SensitivePersistent liver TG in Juveniles exposed to HFD-Sensitive
mothers, despite weaning to a healthy dietmothers, despite weaning to a healthy diet
Liver

49. No effect on adipose tissue orNo effect on adipose tissue or
circulating cytokines at 1 yoa.circulating cytokines at 1 yoa.

50. Increased inflammatory response
in Liver Kupffer cells (macrophage) from animals
exposed to maternal HFD – 1 year later
Similar results for TNFα and IL-6
Kupffer Cells = 100X ↑ in IL-1β vs Hepatocytes
(N=2-3 per group)

51. Liver macrophage multiply in HFDLiver macrophage multiply in HFD
exposed animals even on healthy dietexposed animals even on healthy diet
15-75-fold enrichment in Kupffer cells
(N=2-3 per group)

52. Increased Inflammatory and Lipogenic Genes inIncreased Inflammatory and Lipogenic Genes in
Hepatocytes from HFD-Sensitive Mothers.Hepatocytes from HFD-Sensitive Mothers.
*
*
*
*
*

53. 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 gene
expression (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 in
offspring from obese mothers.

54. Excess FFA/TG Delivery
Placental Inflammation
Placental Nutrient Transfer
Fetal Hepatic Fat Accumulation
Oxidative Stress
Inflammation
Gluconeogenesis
Recruitment and
Activation of Bone
Marrow WBC
Precursors
Lifelong Increased Risk of a
Proinflammatory Response
to Overnutrition
Consequences of Maternal Overfeeding on Fetal LiverConsequences of Maternal Overfeeding on Fetal Liver
US adults: 20-30%
Obese adults: 60%
US kids 9-19*: 17%
Obese kids: 55%

56. Our Approach in Moms & Infants
–Work in Progress
Subcutaneous
FatHepatic Fat
Visceral Fat
Can we observe physiologic differences in harmful fat deposition that predate
Influence of diet and lifestyle?

57. • Visceral fat is associated with severe insulin
resistance; hepatic fat is associated with non-alcoholic
fatty liver disease; abdominal subcutaneous fat more
lipolytic than peripherally stored fat
• How does fetus store fat in visceral organs or liver if
excess delivery?
• If liver fat is present at birth, what happens?
• Redistribution to subcutaneous adipose tissue?
• “Programming” of liver to favor partitioning of fat towards
storage
• Sequential measures of total fat by PEAPOD and
hepatic and visceral fat by MRI at 2 weeks
Location of Fat May Be Key!

58. 18 obese mothers w/ GDM
17 lean mothers
Exclusion Criteria:
Pre-Pregnancy Diabetes
IUGR
Premature delivery
NICU admission
birth Single study visit
1-3 weeks of age
MRI
MRS
Pea Pod
Anthropometrics
Feeding
Questionnaire

61. Roland-Valadez E et al. Ann Hepatol, 2008.

62. Adiposity Outcomes
Normal 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) NS
MRI subcutaneous fat (cm3
) 707 (138) 777 (159) NS
Sum Skin Folds (mm) 9.9 (2.0) 11.7 (1.3) <0.05
MRI visceral fat (cm3
) 20.1 (4.6) 22.5 (9.2) NS
Visceral Fat/Length (cm2
) 0.39 (0.09) 0.43 (0.16) NS
Liver Lipid (fat) #
0.017 0.030 <0.05
(76%increase)
#
Back transformation of natural log (lipid peaks liver/lipid peaks Intralipid)
SubQ
Fat
Pediatrics 2013

63. Variable ß - coefficient P-value
Maternal Pre-pregnancy BMI 0.03170 0.0456
Infant Sex 0.20682 0.4877
Infant Age at Study -0.00480 0.9620
Infant Total Adiposity by Peapod 0.03540 0.5510
Pediatrics, 2013

64. 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 infant
metabolome, 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 expression
and 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 hyperinsulinemia
response 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 in
fetus.fetus.

65. How do we modify this?
Pregnant population limits drug options, especially in
non-diabetic patients…
• Pre-pregnancy lifestyle modifications
• Controlling gestational weight gain
• Exercise
• Dietary modifications
Reduce inflammation, insulin resistance, hyperlipidemia…

66. Omega-3 Fatty Acids and Pregnancy:
Current Research:
Focus on DHA for
Cognitive development
Reports of impaired
Omega-3 transfer in obese
pregnancy
No focus on use as an
intervention in obese
pregnancy
Maternal supply of PUFA essential for fetal development

67. 2 views of Inflammation

69. Figure 3 Cardiovascular effects of resveratrol. Resveratrol and/or Sirt1/AMP-activated kinase (AMPK) activation and/or
phosphodiesterase (PDE) inhibition have been shown to improve vascular function and reduce hypertension via increased nitric
oxide (NO) p...
Jay H. Chung , Vincent Manganiello , Jason R.B. Dyck
Resveratrol as a calorie restriction mimetic: therapeutic implications
Trends in Cell Biology Volume 22, Issue 10 2012 546 - 554
http://dx.doi.org/10.1016/j.tcb.2012.07.004

71. Supplementary Figure 1
-1 0 G120
Necropsy
and Sample
Collection
Necropsy
and Sample
Collection
Pre-preg
Metabolic
Analysis
Pre-preg
Metabolic
Analysis
Ad libitum WSDAd libitum WSD
7 Years
3
Ad libitum WSD/RESVAd libitum WSD/RESV WSDWSD
Maternal
Metabolic
Analysis
Maternal
Metabolic
Analysis
Maternal
Metabolic
Analysis
Maternal
Metabolic
Analysis
G130
Time with resveratrol supplementation (months)
Post partum
Metabolic
Analysis
Post partum
Metabolic
Analysis
9Breeding seasonBreeding season
WSD = Western Style Diet, 35% fat

72. Figure 2 A B C
D E F
G H I
GlucoseAUC
Pre-Preg 3rd Trim
0
5000
10000
15000 ∆WSD/RESV v CTR p<0.01
Pre-Preg 3rd Trim
0
1
2
3
Triglycerides(mg/dl)
∆WSD/RESV v CTR p<0.01

73. Figure 3 A B C
D E F
G H I

74. Supplementary Table 2
Maternal fatty acids
CTR (n=10) WSD (n=11) WSD/RESV (n=6) Statistics
Saturated FA 2987 ± 727 3329 ± 460 2973 ± 828 NS
MUFA 1041 ± 510 1330 ± 366 1486 ± 616 NS
PUFA 2436 ± 746 2173 ± 512 2199 ± 622 NS
N-6 2141 ± 684 2034 ± 452 2059 ± 593 NS
N-3 295 ± 98 139 ± 74 140 ± 52
p<0.001 CTR v WSD
p<0.01 CTR v WSD/RESV
N6:N3 8.14 ± 4.53 17.26 ± 6.88 15.49 ± 3.72
p<0.01 CTR v WSD
p<0.05 CTR v WSD/RESV
Fetal fatty acids
CTR (n=8) WSD (n=13) WSD/RESV (n=6) Statistics
Saturated FA 1465 ± 161 1577 ± 255 1385 ± 253 NS
MUFA 317 ± 84 723 ± 285 729 ± 205 p<0.01 CTR v both
PUFA 1047 ± 221 1304 ± 624 1221 ± 468 NS
N-6 863 ± 161 1177 ± 571 1084 ± 405 NS
N-3 183 ± 64 127 ± 71 137 ± 70 NS
N6:N3 4.92 ± 0.83 10.62 ± 4.52 8.63 ± 2.33 p<0.01 CTR v WSD

75. Figure 4 Liver Triglycerides
A B
CTR WSD WSD/RESV

76. Table 1
CTR WSD WSD/RESV Statistics
Body Mass (g) 345.5±5.1 (29) 347.6±7.4 (34) 317.2±22.3 (6) NS
Brain Mass (g) 45.85±1.05 (16) 46.56±0.81 (21) 43.38±1.55 (6) NS
Heart Mass (g) 2.09±0.08 (15) 2.11±0.07 (24) 2.05±0.20 (6) NS
Liver Mass (g) 9.43±0.19 (26) 9.99±0.21 (29) 9.47±0.60 (6) NS
Pancreas Mass (mg) 249.4±11.80 (27) 246.5±11.95 (31) 349.3±20.14 (6)
p<0.01 CTR v WSD/RESV
p<0.01 WSD v WSD/RESV
Pancreas: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/RESV
p<0.0001 WSD v WSD/RESV
Fetal Tissues –Increase in Pancreas weight

77. Figure 5
A
B
Ins/Ggn/Ki67Ins/Ggn/CK7
CTR WSD WSD/RESV

78. C D E F
G H I
IsletMass(mg)
CTR WSD WSD/RESV
0
5
10
15
20
β-CellMass(mg)
CTR WSD WSD/RESV
0
5
10
15
20
α-CellMass(mg)
CTR WSD WSD/RESV
0
2
4
6
8
a
a,b
b
α-:β-CellRatio
CTR WSD WSD/RESV
0.0
0.5
1.0
1.5
a
a,b
b
CTR WSD WSD/RESV
0.00000
0.00005
0.00010
0.00015
#Ki67+α-Cells/α-CellArea(#/µm2)
CTR WSD WSD/RESV
0.00000
0.00005
0.00010
0.00015
#Ki67+β-Cells/β-CellArea(#/µm2)
a
a
b
Ins+CK7+Area/CK7+Area
CTR WSD WSD/RESV
0.0
0.5
1.0
1.5
Ggn+CK7+Area/CK7+Area
CTR WSD WSD/RESV
0.0
0.5
1.0
1.5
2.0
2.5
a
a
b
J
Figure 5 Islet Cells – α cell loss relative to β cells

79. Resveratrol SummaryResveratrol Summary
• Resv caused weight loss prior to gestation despite noResv caused weight loss prior to gestation despite no
change 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 and
accumulate in the fetus.accumulate in the fetus.
• No change in fetal weight, significant improvement inNo change in fetal weight, significant improvement in
hepatic lipids.hepatic lipids.
• Pancreas- increase in proliferation-yet loss ofPancreas- increase in proliferation-yet loss of αα-cell-cell
mass relative to b cell.mass relative to b cell.
• Long term implications?Long term implications?

80. Figure 1. Complex Pathogenesis of Type 2 Diabetes.
Genetic and environmental factors may influence the risk of diabetes through
the pathways illustrated in the figure or through as-yet-unidentifed mechanisms
affecting insulin sensitivity and/or insulin secretion. Kahn, CR, CellMetabolism
, 2008.

81. Acknowledgement-Funding Sources: