Presentation - University of Colorado Denver

Maternal Nutrition and Metabolic Programming
in Mouse, Monkey, and Man
JACOB E. (JED) FRIEDMAN, PHD
DEPARTMENT OF PEDIATRICS, BIOCHEMISTRY & MOLECULAR GENETICS
UNIVERSITY OF COLORADO-DENVER SCHOOL OF MEDICINE
Director, Colorado Program for Nutrition and Healthy Development
Developmental Psychobiology Research Retreat
Morrison, Colorado May 07, 2012

Acknowledgements:
Oregon National Primate Research Ctr
Kevin Grove, Ph.D.
Dan Marks, MD, Ph.D.
Antonio Frias, MD, PhD
Diana Takahashi, MS.
Staff and support Oregon National Primate
Research Center, Beaverton Oregon
NIH-RO1 DK060685, RO1 DK074643,
R24 DK90964, P30 DK048520 , ADA
University of Colorado Nutrition & Obesity
Research -Center (NORC); University of Colorado
Maternal-Child Clinical Translational Research
Center
University of Colorado-Denver
Center for Proteomics:
�Karen Jonscher, Ph.D.
�Aga Kendrick, M.S.
Baylor College of Medicine:
Kjersti Agaard-Tillery, M.D. Ph.D.
University of Colorado-Denver
Carrie McCurdy, Ph.D.
Stephanie Thorn, Ph.D.
Lynn Barbour, M.D.
Teri Hernandez, Ph.D.
Margaret Heerwagen, B.S.
Becky De La Houssaye, M.S.
Rachel Van Pelt, Ph.D.
Michael Stewart, M.D.
Sean Newsom, Ph.D.
Kristen Boyle, Ph.D.
David Brumbaugh, M.D.
Rachel Janssen, M.S.
Rebecca Aiken, M.S.

Colorado Program in Nutrition and Health Development
Jed Friedman, Director, Lynn Barbour-Co-director
Goals of the Program:
• To understand basic mechanisms underlying the role of
nutrition and the environment on maternal-infant health in
animal models that can be translated to humans.
• To develop safe nutritional/interventional strategies to
achieve a healthier pregnancy and improve newborn health
during the first 1000 days of life.
• To facilitate collaborative research within Colorado and
beyond.
• To provide education for trainees, medical professionals, and
the lay public.

Colorado Program for Nutrition and Healthy Development

Outline of Talk:
1) The new science of Fetal Programming
- Fetal Programming: What is it?
- Maternal Obesity and the fetus:
Response to an Epidemic
2) Studies in Non-Human Primate
3) Studies in Human pregnancy
4) - Omega-3’s
- Fat-1 mouse model
5) Future Directions

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.

Oct. 6, 2010

How Big Is the Problem?
% Women with BMI > 30
(OBESE) (Overweight)
Catalano OM, Ehrenberg HM. BJOG. 2006 Oct;113(10):1126-33.

March 18, 2010
700,000 children and teens (2-19) in Southern California
1/6 of children are obese (>95%)
7% boys and 5% girls extremely obese
“These kids face 10-20 years shorter life span and will
develop health problems in their 20s that we typically see
in 40-60 yr olds”
Fatty Liver Disease in Obese Youth: 1 in 5.

Trends in child and adolescent overweight
Age 6-11
Age 12-19
Age 2-5
Note: Overweight is defined as BMI>=gender and weight-specific 95th percentile from the 2000 CDC
Growth Charts. Source: National Health Examination Surveys II (ages 6-11) and III (ages 12-17),
National Examination Surveys, I, II, III and 1999-204, NCHS, CDC.

World-Wide Childhood Obesity Epidemic
Critical Early Life Factors affect Health
Across the Lifespan
Genes Gestational Exposure Post-natal Environment
The Childhood Obesity Pipeline is Full and getting worse

NIH Consensus Conference,
Sept 23-24, 2005 Bethesda MD
The Maternal Intrauterine Environment and
the Origins of Obesity
• When does it Occur?
• How does it Occur?
• What can we do about it?
“Programming” effects on Obesity, CV-disease, and Cancer

Prevalence (%)
Prevalence of Type 2 DM In Pima Indians,
by Mothers Diabetes in Pregnancy
75
60
45
30
15
0
Offspring of non-diabetic mothers
Offspring of pre-diabetic mothers
Offspring of diabetic mothers
5-9 10-14 15-19 20-24 25-29 30-34
Age (years)
Dabelea D, Pettitt DJ, J Mat. Fetal Med. 2000; 9:83.

Development is characterized by early windows of plasticity:
.
Cells in this window are highly sensitive to environmental stimuli that impact:
- differentiation
- death/proliferation
- gene expression programs
Fowden A L et al. Physiology 2006;21:29-37

The “Obese Environment”
• Adipose Tissue Hypertrophy
• Insulin Resistance
• Hyperlipidemia
• Hyperglycemia
• Hyperinsulinemia
• Inflammation
But we see this kind of metabolic profile somewhere else too…

Metabolic changes during pregnancy:
In the mother, in order to mobilize energy stores
and fuel fetal growth:
Hyperlipidemia
Hyperglycemia
Insulin Resistance
Adipose Hypertrophy
What happens when obesity and pregnancy combine?

When the two combine: Maternal Obesity
Significant evidence here
Heerwagen et.al. AJP Review September 2010

Long Term Implications for Fetus
• Obese infants are up to 2-9 times
as likely to be obese as adults Baird J,
BMJ 2005;331:929
• Maternal BMI ≥ 30 conferred 25%
obesity risk at age 4 (~3-fold) indep
of BW Whitaker RC Pediatrics 2004;114:29
• 25% of obese children age 4-10
have IGT

Co-Morbidities of
Childhood Obesity
• Obesity has tripled in last 2 decades:
– 20-25% of girls 11-15 are obese
• Type 2 DM in children/adolescents has ↑ 300%
– Type 2 > Type 1 in children 10-19 in most ethnicities
• Pediatric fatty liver disease 13% children
• 1 in 3 children born in the year 2000 will
develop DM in their lifetime

Diabetes "massive challenge" as cases hit 366 million
LONDON | Tue Sep 13, 2011 7:36am EDT
(Reuters) - The number of people living with diabetes has soared to 366 million, and the
disease kills one person every seven seconds, posing a "massive challenge" to healthcare
systems worldwide, experts said on Tuesday.
The vast majority of those with the disease have Type 2 -- the kind linked to poor diet, obesity
and lack of exercise -- and the problem is spreading as people in the developing world adopt
more Western lifestyles.
Worldwide deaths from the disease are now running at 4.6 million a year.

Global projections for the diabetes epidemic: 2010–2030 –Nature Reviews-
Endocrinology, 2012

World-wide Food Insecurity

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

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

Fetal Origin of Adult Disease
Fetal Growth Restriction:
• Diabetes
• Adiposity
• Cardiovascular disease
• Osteoporosis
Excessive Fetal Growth:
• Diabetes
• Obesity
• Cardiovascular disease
• Some cancers
Is the mechanism
The same?

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

Infant Adiposity vs. BW
Catalano, Am J Clin Nutr 2009:
• 47% of difference in birth weight
related to Fat.
• SGA vs AGA vs LGA have about the
same lean body mass.
• AGA infants of GDM or Obese moms
have higher %fat than AGA infants of
controls moms.
*Fat Accretion by Species:
Rats/Mice 1%
Sheep: 2-3%
Non-human Primates: 3-4%
Guinea Pigs ~10-14%
Humans: 12-15%
Infant DEXA at 2 wks of Age:
Mother: Lean Obese & GDM
BW = 3370g
Fat = 7.7%
BW = 2893g
Fat = 16.8%

Top Intrauterine Contributors to Neonatal Adiposity
� Maternal Insulin Resistance (could explain chunk of
below)
� Maternal BMI
� Hyperglycemia
� Maternal TG and FFA (Lipolysis, CM-TG, VLDL-TG?;
indep of IR?)
� Maternal High Fat Diet (Indep of mat obesity through
changes infant metabolome, appetite regulation,
behaviors, NAFLD, mitochondrial oxidation)
� Excess Gestational Weight Gain
� Maternal Inflammatory Cytokines (change in placental
gene expression and transport)
� What About?
� Oxidative Stress and Increased Lipid Exposure in early life?
� Placental and Fetal Growth Factors (fetal hyperinsulinemia
response to hyperglycemia, placental IGFs)
� Psychological stressors? ↑CRH and IL1β in mom; ↑GR and
insulin in fetus.

First take home message:
Its not all about birth weight
Adiposity Matters!

Taveras E, et al. Pediatrics, 2009;123:1177-83.
• Faster weight gain 0-3 months is
positively associated with adiposity
at 21 yo.
Leunissen RW et al. JAMA, 2009; 301(21):2234-42.
• Weight gain 0-9 months is positively
associated with BMI at 17 yo (p=.002)
even after adjustment for parents’
BMI, sex, and birthweight
Larkjaer A et al. Am J Clin Nutr, 2010; 91:1675-83.

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, Cell Metabolism , 2008.

Prevalence (%)
Prevalence of Diabetes in Offspring from
Women with Diabetes -Pima Indians
70
60
50
40
30
20
10
0
Offspring of:
NonDiabetic
PreDiabetic
GDM
5-9 10-14 15-19
Age (yr)
20-24 25-29 30-34
Dabelea, Knowler, Pettitt J MFM 2000:9:83

Obese Pregnancy and Fetal Nutrients:
nd Take home message:It’s not just glucose…
Most Large for Gestational Age (LGA) infants are born to
mothers without GDM.
In multivariate analyses of pregnant women, glucose
accounted for

Epigenetics:
It’s not what you have, but
what you do with it that counts!
Modifications to DNA/chromatin
Effect TF binding and chromatin
structure
Established largely during early
development
Why our body is composed of
different cell types, though same
genetic sequence.
Time Magazine, February 19, 2010

NEWS & VIEWS
Childhood obesity—methylate now, pay later?
Choudhury, M. & Friedman, J. E. Nat. Rev. Endocrinol. 7, 439–440 (2011).
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?

Fetal Biochemistry:
Limited capacity for de novo lipogenesis in the fetus implies
that most precursors for fetal fat accretion are supplied by
transfer of maternal substrates derived from lipid rather than
from glucose sources.
NEFA
TG
lipase
lipase
Placenta Lipid droplet
Triglycerides
Palmitate
phospholipids
PLA2
PLA2
PUFAs
PUFAs
Omega-3
Omega-3
Omega-6
Omega-6
Fatty Acid Oxidation is present in most fetal tissues (liver,
heart, etc) during development. Therefore the concept that
glucose provides the majority of fuel necessary for fetal
energy metabolism may need to be re-examined.

Adult
NAFLD
Insulin Resistance
Obesity
Appetite, behavior
Diabetes

Nature Medicine
Vol:18,
363–374:
(2012)
“Metainflammation”

Maternal diet-induced obesity alters mitochondrial activity and redox
status in mouse oocytes and zygotes. PLoS One. 2010 Apr 9;5(4):e10074.
Igosheva et al.
Abnormal mitochondrial distribution
and increased activity in oocytes and
zygotes from DIO mice
ROS production
Glutathione levels
Maternal DIO increases ROS generation
And depletes glutathione in oocytes & zygotes.

Mitochondrial Defects
Obesity is accompanied by
� Reduced SkM Mitochondrial Function (OXPHOS)
� Incomplete FAO
� Decreased Mitochondrial Biogenesis
� Impaired Insulin Action

Metabolic Programming in the
Fetus:
Is it simply a matter of FAT?

Collaborative Obesity Research
University of Colorado & Oregon National
Primate Research Center, University of Utah
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, px, brain)
in utero, and beyond.

Benefits and Obstacles to
working in NHP model
• Longer gestation period
(175d)
• Similar Placentation
• Organ development
occurs in utero
– pancreas, brain, adipose
• Perform invasive studies
that cannot be done in
humans or fetal mice
• Heterogenity
• Free-living
– Difficult to control
food intake and
exercise
• Studies/data
collection take a lot
longer
• Collaborative
• Expensive
• Heterogenity

STUDY QUESTIONS
1. Does Maternal Diet Influence
Fetal Mitochondrial
Biogenesis and Subsequent
Mitochondrial Function?
2. Does Skeletal Muscle Insulin
Resistance Begin In Utero?

A Primate Model
Japanese
Macaques
@ ONPRC

CTR = 15% Fat Calories
MODEL
Young adult female Japanese macaques - CTR
or HFD for 2-6 years
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

High Fat Diet Animals
High Fat Diet Resistant
(HFD-R)
Triglycerides
Saturated FFA
Frias AE et al., Endocrinology 2011
McCurdy CE et al, JCI, 2009
Grant WF et al., Plos One, 2011
High Fat Diet Sensitive
(HFD-S)
Body Weight
Adiposity
Insulin
Leptin
Triglycerides
Saturated FFA

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

Fetal Hepatic Pathology Under
Conditions of Maternal Obesity
Fetal liver triglycerides, mg/g
8.0
6.0
4.
0
2.0
0
Maternal
Diet
* p

▪Elevated Fetal Liver TG occurred in all HF Y2-Y4 G130 fetuses
REGARDLESS of maternal diet responsiveness
McCurdy, CE et al. J. Clin. Invest. 119(2), 2009.

-Elevated Stress Response in Fetal Y2-Y3 Livers
Elevated Stress in Response 3 pattern in Fat Y2/Y3
Fetal Livers
rd Trimester- G130
C-FOS p-JNK1 p38 MAPK
McCurdy, CE et al. J. Clin. Invest. 119(2), 2009.

Mito Biogenesis in Skeletal Muscle
Copy Number (mtDNA/Nuc DNA)
1000
800
600
400
200
0
N=5-8 for males
*
Males
CON HFD_R HFD_S

Model showing loss of Sirtuin 3 function in fatty liver:
Potential impact on reduced mitochondrial ETC activity
Lys
NADH--->NAD

Why Does the Fetus Store Excess Lipids in
Liver and other organs?
• Excess HFD exposure exceeds fetal storage capacity
during normal development of adipose tissue depots.
• Hormonal factors (such as fetal hyperinsulinemia)
drive lipid storage.
• Exposure to increased dietary n6/n3 ratio promotes
inflammation and causes metabolic re-programming?

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

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

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

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

Fetal circulating cytokines
Includes both HFD-S and HFD-R offspring
McCurdy CE et al, JCI, 2009

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

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 early adiposity and glucose intolerance
– Fatty liver (Inflammation, steatosis), insulin resistance maintained in
Juvenile livers.
– Epigenetics –HSP70, Bmal1 (clock gene family) (FASEB J, 2010).
– Pancreatic islet hyperplasia
– Hyperphagia of palatable diets
– Abnormalities in the melanocortin system (Endocrinology 2010).
• Social Behavior
– Decreased Serotonin (J Neurosci, 2010)
– Female Offspring – increased anxiety
– Male offspring – increased aggressive behavior
– Both sexes display decreases in social behavior
• Is NOT solely dependent on maternal obesity

Juvenile
Hypotheses
1. Maternal high fat diet exposure will result in hepatic
steatosis, inflammation and insulin resistance in
juvenile offspring at 1 yr of age.
2. These metabolic abnormalities will be exacerbated
when offspring are fed a high fat diet after weaning
to 1 yr of age.
3. Mechanism may involve persistent activation of
inflammatory pathways in liver

Consumption During the Food Preference
Testing is Influenced by Current Diet,
Maternal Diet, and Age
*
*
Age/Experience with post-weaning diet:
F 1,11=93.57, p

Temperament Assessment at 4 months of
Age
Human Intruder Test
Acclimation (12 min)
Profile (2 min)
Control period (2 min)
Stare (2 min)
Control period (2 min)
Novel fruit test (kiwi; 5 min)
Novel toy 1 (Mr. potato head; 5 min)
Novel toy 2 (hanging toy; 5 min)
Novel toy 3 (snake; 5 min)
Anxiety:
•Latency to examine novel objects
•Response to Human Intruder
•Fear grimace
•Teeth grind
•Cage bite
•Escape
•Hiding
•Stereotypy
•Scratching
Aggression:
•Threats
•Attacks

Offspring from HFD fed mothers
exhibit increased latency to explore
novel objects suggesting increased
anxiety
*

Maternal and post-weaning HFD increases
early measures of insulin resistance

Maternal HFD increases liver triglycerides

Macrophage/Kupffer Cell TLR Signaling
Macrophage/KC
TLR2/6
Bacteria
LPS
TLR1/6
TLR5
NFκB
TNFα, IL6, TGFβ
Crispe. Annu. Rev. Immunol. 2009;27:147-63
Sat fatty
acids
TLR4
TRIF
ds
RNA
“Apoptosis”
TLR3
IRF3
IFNα
IFNβ
Viral ss
RNA
RIG-I MDA-I
IPS-1

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

Non-human primate Data: Focal Cell Death
G130 fetal livers show increased lipids, oxidative stress and apoptosis with
maternal HFD….
Oil Red-O
4-HNE
CONTROL HFD
McCurdy et.al. J. Clin. Invest. 119:323–335 (2009)
CONTROL HFD
Grant et.al. PLoS One. 2011 Feb 25;6(2):e17261.

Putative Natural History of Fatty Liver Disease
“first hit”
“second hit”
Steatosis
US adults: 20-30%
Obese adults: 60%
US kids 9-19*: 17%
Obese kids: 55%

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

1
Lean
Obese
GDM Hx
Screen:
Consent
H&P
Dietitian
12
2
NIH RO1: Regulation of Maternal Fuel Supply
And Neonatal Adiposity – 9 VISITS
DEXA/
CGMS #1
3 days
Prepared
Diet
Liquid Feed #1
Day 4 of CGMS
Fasting Blood
BMR and RER
Liquid Meal: q20 min
Blood draws x 4 hrs
13 14 15 16 17 18 19 20 21 22 23
3
4
CGMS #2-
Biopsy
AT biopsy
on Day 1
3 days
Prepared
Diet
24
5
Liquid Feed #2
Day 4 of CGMS
Fasting Blood
BMR and RER
Liquid Meal: q20 min
Blood draws x 4 hrs
6 7
Fetal U/S #1
3-hr OGTT
25 26 27 28 29 30 31 32 33 34 35
Obtain placenta
and Cord Blood
9
Fetal
U/S #2
37 38 39
36
8
40
Delivery
Neonatal Blood/
Anthropometrics
2 weeks PP
Maternal DEXA
Neonatal DEXA/
Anthropometrics

Study Groups: 20 each: Normal Weight (NW) (Pre-Pregnant BMI between 19.0 – 24.0)
Overweight/Obese (OW/Ob) (Pre-Pregnant BMI between 26.0 – 39.9)
Gestational Diabetic (GDM) (Pre-Pregnant BMI between 26.0 – 39.9)
Type II Diabetic (T2D) (Pre-Pregnant BMI between 26.0 – 39.9)
Birth
28-38 weeks
Recruitment 48 hours 2 weeks 1 month
1 2 months 3 months
In Hospital:
Infant
• Anthropometry
• Blood Sample 2
Maternal
• Colostrum
Collection
• Weight
• Chart Review
(Pregnancy)
Clinic Visit:
Infant
• Anthropometry
• PeaPod
• Infant Feeding
Questionnaire
• Feeding Practices
Questionnaire
• Stool
• Urine
Maternal
• Weight
• Fasting Milk
Collection
• Fasting Blood
Sample
• 3-day Food Recall
• Physical Activity
Questionnaire
• Health History
• Optional: Stool
Home Visit:
Infant
• Anthropometry
• Infant Feeding
Questionnaire
• Optional: Stool
Maternal
• Weight
• Fasting Milk
Collection
Study Design:
Home Visit:
Infant
• Anthropometry
• Infant Feeding
Questionnaire
• Optional: Stool
Maternal
• Weight
• Fasting Milk
Collection
Home Visit:
Infant
• Anthropometry
• Infant Feeding
Questionnaire
• Optional: Stool
Maternal
• Weight
• Fasting Milk
Collection
1 At recruitment, participants will be provided with lactation support materials and breast pump if desired.
2 Infant blood sample to be collected at the time of the routine 48 hour infant screening blood sample.
4 months
Clinic Visit:
Infant
• Anthropometry
• PeaPod
• Infant Feeding
Questionnaire
• Feeding Practices
Questionnaire
• Blood Sample
(Heel Stick)
• Stool
• Urine
Maternal
• Weight
• Fasting Milk
Collection
• Fasting Blood
Sample
• 3-day Food Recall
• Physical Activity
Questionnaire
• Health History
• Optional: Stool

Composition
•Calories
oLactose
oProtein
oFat
•Glucose
•Fatty Acid
Profile*
•Free Fatty Acids
•Triglycerides
Composition
• Glucose
• RBC Membrane Fatty
Acid Composition*
• Free Fatty Acids
• Triglycerides
• Hb-A1C
Breast Milk (6 samples/Participant = 480 total samples)
Cytokines
•IL-6*
•IL-8*
•IL-10*
•TNF-α*
•IFN-γ*
•CRP
Cytokines
•IL-6*
•IL-8*
•IL-10*
•TNF-α*
•IFN-γ*
•CRP
Biochemical Analyses
Hormones &
Adipokines
•Insulin
•Leptin
•Adiponectin
•Ghrelin (total)
•Ghrelin (acylated)
Maternal Blood (2 Samples/Participant = 160 total samples)
Hormones &
Adipokines
•Insulin
•Leptin
•Adiponectin
•Ghrelin (total)
•Ghrelin (acylated)
Antioxidants Microbiota
• Total antioxidant • Probiotic Content*
capacity
• DPH Radical
Scavenging Activity
• Catalase Activity
Oxidative Stress
•F2-Isoprostanes
•Oxidized-LDL
•TBARS
•8-OH-dG
•4-HNE
Infant Urine and Stool (2 Samples/Participant = 160 total samples)
Oxidative Stress (urine)
•F2-Isoprostanes
Oxidative Stress
•F2-Isoprostanes
•Oxidized-LDL
•TBARS
•8-OH-dG
•4-HNE
Microbiota (stool)
• Intestinal Microbiome Profile*
* All cytokines except CRP will be analyzed on a multiplex panel, microbiota analyses, fatty acid profile, and
membrane fatty acid composition will be analyzed in the labs of Dr. Daniel Frank and Dr. Mary Harris, respectively.
All other analyses can be undertaken in our lab, but analytes highlighted in purple are offered by the CTRC core
labs.

Key Outcome: Infant Infant DEXA DEXA at 2 wks at of 2 Age: Weeks of Age
Mother: Obese & GDM Mother: Lean Normal GT
Birth weight = 2.9 kg
body fat = 16.8%
Birth weight = 3.4 kg
body fat 7.7%

Neonatal %Body Fat
Correlation Between Neonatal % Body Fat and
Change in Maternal Fasting TGs
20
18
16
14
12
10
8
6
4
2
R 2 = 0.831
p = 0.001
r=0.890
0 20 40 60 80 100 120
Maternal ∆ Fasting Triglycerides (Late - Early Gestation)
Neonatal % Body Fa
20
18
16
14
12
10
8
6
4
2
0
r =0.110
15 20 25 30 35 40 45
Maternal % Body Fat

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
Pea Pod
MRI
MRS
Anthropometrics
Feeding
Questionnaire

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

SubQ
Fat
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 (cm 3 ) 707 (138) 777 (159) NS
Sum Skin Folds (mm) 9.9 (2.0) 11.7 (1.3)

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
-after adjusting for sex, age, and overall adiposity, the infant’s liver fat increases
over 3% for every 1 kg/m2 increase in maternal pre-pregnancy BMI.

Conclusions – Spectroscopy Data
• Maternal pre-pregnancy BMI is the strongest
predictor of early liver fat deposition
• This appears to be independent of total
adiposity.
• Maternal obesity/GDM will predispose infants
to increased visceral fat storage?

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

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

Western Diet:
High Saturated Fat
High Omega-6
Low Omega-3
Types and sources of dietary fat:
Hunter-Gatherer Diet:
Low Saturated Fat
Omega-3/6 about 1:1
Copyright ©2004 BMJ Publishing Group Ltd.

Why Balance is important:

Substrate competition reduces pro-inflammatory
eicosanoid production:
(Omega-6 PUFA)
(Omega-3 PUFA)
Massaro et.al. Prostaglandins Leukot Essent Fatty Acids. 2008 Oct 22

Omega-3 Fatty Acids
In Non-Pregnant Studies (Humans, Rodents):
Treatment of obesity/HFD subjects with dietary or omega-3
fatty acid supplementation has been shown to:
- Lower Chronic Inflammation (AT, Systemic)
- Lower blood lipid levels and ectopic lipid deposition
- Improve insulin sensitivity
(potentially due to above)

Omega-3 Fatty Acids and Pregnancy:
Maternal supply of PUFA essential for fetal development
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

Hypothesis:
Increasing maternal omega-3 (relative to omega-6)
fatty acids will mitigate the effects of maternal obesity
on adolescent metabolic health by reducing both
placental and fetal exposure to excess maternal lipids
and inflammation.

Transgenic Animal Model: The Fat-1 Mouse
- Endogenously converts omega-6s to omega-3s
- Expressed under actin promoter
- Results in a tissue n-6:n-3 ratio ~1:1, without dietary change
�
fat-1 (C. elegans):
Encodes n-3 desaturase
J.X. Kang. Prostaglandins, Leukotrienes and Essential Fatty Acids: 77 (2007) 263–267.
Kang et.al. Nature. 2004 Feb 19;427(6976):698.

Early evidence of Fat-1 benefits:
- Improved insulin sensitivity on HFD
(White et.al. Diabetes, December 2010)
- Reduced tissue inflammation
(Above and Gravaghi et.al., J Nutr. Biochem, July 2010)
- Increased mitochondrial lipid oxidation and reduced ROS production.
(Hagopian et.al. PLoS One, September 2010)

Experiment Outline:
8 week-old Virgin Females:
WT�CtrlD WT�HFD Fat-1�HFD
E18.5 (= very late gestation)
Maternal/Fetal Harvest:
Maternal, placental & fetal
weights and measures
taken.
Blood and tissue collected.
**Only WT fetuses/placentas analyzed
8 weeks
Cross with WT chow-fed male:
Experimental diet
maintained through
pregnancy
x
OR…. Birth.
Offspring weaned onto HFD or CtrlD

HFD Mothers are significantly heavier prior to
pregnancy, but gain less weight over gestation.
37
35
33
31
29
27
25
23
21
19
17
Maternal Weight Gain (g)
(Litter and placenta weight subtracted)
mated
0 2 4 6 8 p18.5 -
litter
WT-Ctrl
WT-HFD
Fat1-HFD
n=11-13 mothers/group

Fetuses from WT-HFD mothers have significantly larger
placentas relative to fetal size:
n=11-13 mothers/group:
WT-Ctrl WT-HFD Fat1-HFD

20
15
10
5
0
However, lipid deposition within the placenta is
increased in WT-HFD mothers, but not Fat1:
Placenta TG
(ug/mg dry weight)
p

What could be causing placental overgrowth?
What is the placenta seeing?
MATERNAL OBESITY, OVERNUTRITION:
↑ INFLAMMATION
− INSULIN RESISTANCE (Insulin, Glucose)
↑ Il-1, Il-6,
TNF-a, MCP-1
FFA
CM-TG
VLDL-TG
− LYPOLYSIS
− VLDL SECRETION
LPL

Increase in placental lipids also may correspond with
increased placental F480+ Cells:
WT-Ctrl WT-HFD Fat1-HFD
N=4mothers/group
(*p

Lipoprotein Lipase (LPL) activity increased in
WT-HFD placentas:
n=4 mothers/group
(p

Fetal serum

20
18
16
14
12
10
8
6
4
2
0
Fetal liver TG content increased in WT-HFD
mothers, not seen in Fat1-HFD mothers:
Fetal Liver TG
(ug/mg dry weight)
p

Preliminary Microarray:
How does this impact the fetal liver?
Is there a potential “program”?
6 maternally-unique fetal livers (all male), pooled into sets of 3
WT-Ctrl – Pool #1, Pool #2
WT-HFD – Pool #1, Pool #2
Fat1-HFD – Pool #1, Pool #2
Key transcription programs up/down with HFD:
Proliferation FA Oxidation/Metabolism
Inflammation (APR) Oxidative Stress
Fibrosis Apoptosis

Transcription expression patterns similar to chronic liver injury:
Tilg H and Day CP (2007) Management strategies in alcoholic liver disease
Nat Clin Pract Gastroenterol Hepatol 4: 24–34 doi:10.1038/ncpgasthep0683

So what might this mean for adolescent offspring?
Aim 3:
WT-Ctrl (n=10)
WT-HFD (n=6)
Fat1-HFD (n=9)
Ctrl
HFD
16wks
ITT, EchoMRI
ITT, EchoMRI
Harvest
Tissue Collection:
RBCs, Serum
Adipose Tissue
Liver
Muscle
(RNA, Protein, Histology, Lipids)
Immediate Analyses:
Muscle and Liver FA oxidation
AT macrophage quant (flow)

Pups from WT-HFD moms are heavier,
and gain more weight on HFD
BUT this is mitigated in Fat1-HFD mothers

Unlike traditional Barker Hypothesis, this is not a case
of nutrient mismatch – but instead more of a
malaptation.
Possible targets:
• Lipid metabolic pathways:
– Oxidation
– Storage
– Synthesis
So… What’s the Program?
• Inflammatory pathways
– Priming of a pro-inflammatory state

Could the “liver injury” pathway suggested by microarray be leading to
“passive” programming?
Death of more oxidative
cells, repopulation by less
oxidative, pro-storage
cells…?

Previous reports in
adolescents from
HFD moms:
↑Lipogenesis
↓Lipid Oxidation
Corresponding with
increased weight gain
and development of
fatty liver
Previous programming studies:
Hepatology Vol.50, 6 Pages: 1796-1808. Copyright © 2009 American Association for the Study of Liver Diseases

Summary:
Maternal obesity/HFD increased
- Maternal fasting blood glucose
- Maternal HOMA-IR score
- Maternal AT Macrophages
- Placental LPL activity
- Placental lipid deposition
- Placental Macrophages
- Fetal hepatic lipid deposition
- Adolescent weight gain, male IR
All were ameliorated (by differing degrees) by maternal
expression of fat-1 transgene.

Next Steps:
- Analysis of maternal frozen tissues: Inflammation, metabolic pathways
- Analysis of maternal lipid trafficking
(oxidation, storage)
- Localize placental LPL and Macs by IHC
- Staining of Fetal liver for apoptosis,
fibrosis, oxidative stress
- Fetal liver FA oxidation analysis
- Continuation of juvenile offspring analyses

Acknowledgments:
Mentor: Jed Friedman CTSI Clinical Mentor: Lynn Barbour
Committee Members:
Jim McManaman (Chair)
Lynn Barbour
Dwight Klemm
Paul MacLean
Pepper Schedin
Virginia Winn
Friedman Lab
Rachel Janssen
Becky DelaHoussaye
Karalee Baquero
Kristen Boyle
Teri Hernandez
Stephanie Thorn
Mahua Choudhury
Mizanoor Rahman
David Brumbaugh
Michael Stewart
Emily Busta
McCurdy Lab
Carrie McCurdy
Julie Houck
Carlos Ainza
Bergman Lab
Bryan Bergman
David Howard
Funding:
American Heart Association
Colorado CTSI

Last Take Home Message:

Thank You !