Jennifer Wood, Ph.D. Dept. of Animal Science, UNL

Jennifer Wood, Ph.D.Dept. of Animal Science, UNL

Nebraska Center for the Prevention of Obesity Diseases through Dietary

Molecules (NPOD):

Abnormalities in Regulation of Myocyte versus Adipocyte Differentiation by Maternal Obesity

Significance

The economic cost of obesity is estimated at 147 billion dollars which is > 9% of national health expenditures and is comparable to health care costs associated with smoking

Finkelstein et al. (2009) Health Aff; NIH (1998) Obesity Research

Visceral Adipose

FreeFatty Acids

Pro-InflammatoryCytokines

InsulinResistance

DyslipidemiaHypertension

Type 2Diabetes

CardiovascularDisease

MetabolicSyndrome

SedentaryLifestyle

Nutrient-DenseFood

Genetic Predisposition

Metabolic Syndrome

Criteria (3 of 5)• Elevated Waist Circumference• Elevated Triglycerides• Reduced HDL-C• Elevated Blood Pressure• Elevated Fasting Glucose

Significance

Children of obese mothers are at increased risk of becoming obese and developing metabolic syndrome

Bruce et al. (2010) J Nutr.; Power et al. (2011) Physiol Behav; Heerwagen et al. (2010) Am J Physiol Regul Integr Comp Physiol

Approximately 22% of obstetric patients are obeseKim et al. (2007) Obesity

SignificanceIn a sheep model of maternal obesity, fetal muscle mass is decreased due to increased signaling of AMP kinase and activation of FOXO3a and NFkB p65 transcription factors

Zhu et al (2008) J Physiol; Tong et al (2009) Am J Physiol Endocrinol Metab; Du et al (2010) Biol Reprod

Sarcopenic obesity is the loss of muscle mass in combination with increases in visceral fat resulting in the development of metabolic syndrome in 1/3 of the female population and 2/3 of the male population >60 years old in the US

Stenholm et al. (2008) Curr Opin Clin Nutr Metab Care; Jensen (2008) J Parenter Enteral Nutr

Epigenetic modifications of the embryonic genome determine cell fate during normal development and therefore changes in these modifications due to maternal obesity may contribute to the “programming” of decreased fetal muscle mass and the development of sarcopenic obesityHeerwagen et al. (2010) Am J Physiol Regul Integr Comp Physiol; Gluckman (2009)

Nat Rev Endocrinol; Campion (2009) Obes Rev

Developmental Trajectory of Mesenchymal Stem Cells

Gesta et al (2007) Cell

Pref-1CEBPb

Mesodermal Cell

ROSPlacenta

Maternal Obesity

TNFa

BMPTNFa

TNFR1Wnt

SMAD 2/3

SMAD 4

NFkBp65/p50

Myf5, Pax3, Pax7;

JNK

b-catenin

FOXOTCF

TrxPrx

Pref-1, CEBPb

Adipoblasts

Myoblasts

Omega-3Fatty Acids

Central Hypothesis

Curcumin

InnovationBiochemical studies → altered insulin signaling

Genetic studies → insulin resistance is highly heritable (above 40%)

However, genetic loci only account for ~10% of the obese population and the success of current therapeutic interventions based on biochemical studies are limited.

Novel hypothesis → children of obese mothers develop sarcopenic obesity at an early age due to decreased programming of myoblasts and increased programming of adipoblasts.

Novel Experimental Model → obese mouse model which expresses a phenotype consistent with human metabolic syndrome.

B6

LY

Lethal Yellow (LY) mice Mimics human obesity

Merc

1Ay 1A 1A’ 1B 1C 2

AgoutiAgouti

Merc/agouti fusion

1Ay 1A 1A’ 1B 1C 2

Agouti

( 120 kb Ay deletion )

Blood Hypothalamus

ME Arcuate

ααα-MSHCART Satiety

Insulin

Leptin-

Agouti (Ay)

+ MC4R

Yang et al. 2011, Mol Reprod Dev

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Maternal Obesity Decreases Growth and Development of Embryos

Norwood et al., unpublished data

Specific Aim 1: Identify TNFa-dependent mechanisms of decreased myogenic and increased adiopogenic regulatory factor expression.

Somite

Placenta

TNFa

BMPTNFa

TNFR1

SMAD 2/3

SMAD 4

NFkBp65/p50

Myf5, Pax3, Pax7; Pref-1, CEBPb

Omega-3Fatty Acids

NFkB-dependent regulation of gene expression

Massagué J et al. Genes Dev. 2005;19:2783-2810

Smad-dependent regulation of gene expression

Experimental DesignYear 1:

–Establish somite explant cultures from E8.5 CF-1 mouse embryos–Treat with increasing concentration of TNFa, collect protein, and carry out Western blot analysis using antibodies against JNK, NFkB, Smad–Monitor TNFa-dependent increases in Pax3, Pax7, Myf5, Pref1, and Cebpb mRNAs in the absence or presence of JNK inhibitor or Smad siRNA

Year 2: –Carry out ChIP assays using antibodies against NFkB, Smads, HDAC, p300/CBP coupled to QPCR at the promoter of Pax3, Pax7, Myf5, Pref1, Cebpb in TNFa-treated somites OR somites collected from embryos developed in an obese (LY) compared to lean (B6) in utero environment (E10.5)–Alternatively, use Chromosome Conformation Capture assays (3C) to determine long-range regulation of Pax3, Pax7, Myf5, Pref1, or Cebpb by NFkB or Smad transcription factors

Experimental DesignYear 3:

–Supplement dams with omega 3 fatty acid–Collect somites from E10.5 embryos developed in obese (LY) or lean (B6) dams without or with supplement–Collect protein and carry out Western blot analyses using antibodies against JNK, NFkB, Smad–Monitor supplement-dependent changes in Pax3, Pax7, Myf5, Pref1, and Cebpb mRNAs

Somite

ROS

Placenta

Wnt

Myf5, Pax3, Pax7;

JNK

b-catenin

FOXOTCF

TrxPrx

Pref-1

Curcumin

Specific Aim 2: Identify ROS-dependent mechanisms of decreased myogenic and increased adiopogenic regulatory factor expression.

FOXO-dependent regulation of gene expression

Huang et al. (2007) J Cell Sci

Experimental DesignYear 1:

–Establish somite explant cultures from E8.5 CF-1 mouse embryos–Treat with increasing concentration of H2O2, collect protein, and carry out Western blot analysis using antibodies against JNK, b-catenin, TCF, FOXO–Monitor H2O2 -dependent increases in Pax3, Pax7, Myf5, Pref1, and Cebpb mRNAs in the absence or presence of JNK inhibitor or FOXO siRNA

Year 2: –Carry out ChIP assays using antibodies against b-catenin, TCF, FOXO, SIRT1, or p300/CBP coupled to QPCR at the promoter of Pax3, Pax7, Myf5, Pref1, Cebpb in H2O2 -treated somites OR somites collected from embryos developed in an obese (LY) compared to lean (B6) in utero environment (E10.5)–Alternatively, use Chromosome Conformation Capture assays (3C) to determine long-range regulation of Pax3, Pax7, Myf5, Pref1, or Cebpb by FOXO or TCF transcription factors

Experimental DesignYear 3:

–Supplement dams with curcumin–Collect somites from E10.5 embryos developed in obese (LY) or lean (B6) dams without or with supplement–Collect protein and carry out Western blot analyses using antibodies against JNK, b-catenin, TCF, FOXO –Monitor supplement-dependent changes in Pax3, Pax7, Myf5, Pref1, and Cebpb mRNAs

Mesenchymal Stem Cell Niche

Maternal Obesity

Adipoblasts

Myoblasts

Omega-3Fatty Acids

Curcumin

Specific Aim 3: Identify maternal-obesity dependent decreases in myoblast and increases in adipoblast populations in the mesenchymal stem cell niche within adipose tissue, skeletal muscle, and bone marrow

Experimental DesignYear 2 and 3:

–Collect mesenchymal stem cells (adipose, bone marrow, muscle) from 6 and 12 week old B6 males developed in an obese (LY) or lean (B6) in utero environment in the absence or presence of an anti-inflammatory (omega-3 fatty acids) or an anti-oxidant (curcumin)–Carry out QPCR using primers against Pax3, Pax7, Myf5, Pref1, and Cebpb –Carry out ChIP analyses coupled to QPCR in mesenchymal stem cells using antibodies against transcription factors (NFkB, FOXO, Smads) and coactivators (HDAC, SIRT, p300/CBP) associated with the promoter of Pax3, Pax7, Myf5, Pref1, and Cebpb–Determine differences in body fat composition in 6 and 12 week old B6 males developed in an obese (LY) or lean (B6) in utero environment using DEXA. The effect of in utero exposure to an anti-inflammatory (omega-3 fatty acids) or an anti-oxidant (curcumin) as well as the response to a dietary challenge (high fat diet) on body fat composition will also be measured using DEXA

Expected Outcomes• Identify signaling pathways and transcription factors activated by the

inflammatory response, oxidative stress, or both which regulate the expression of myogenic and adipogenic regulatory factors

• Determine the ability of omega-3 fatty acids or curcumin to modify or reverse TNFa- and ROS dependent regulation of mesodermal cell differentiation

• Determine the effect of maternal obesity on the phenotype of mesenchymal stem cell niches in the bone marrow, adipose tissue, and skeletal muscle

Environment and Mentors• Core Facilities:

– Epigenetics– Biostatistics

• Mentors– John Davis: cell signaling, reproductive physiology– Andrea Cupp: growth factor signaling, stem cell biology,

reproductive physiology– Robert Powers: