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

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

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  1. Nebraska Center for the Prevention of Obesity Diseases through Dietary Molecules (NPOD): Abnormalities in Regulation of Myocyte versus Adipocyte Differentiation by Maternal Obesity Jennifer Wood, Ph.D. Dept. of Animal Science, UNL

  2. 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

  3. Metabolic Syndrome Genetic Predisposition Sedentary Lifestyle Nutrient-Dense Food Visceral Adipose • Criteria (3 of 5) • Elevated Waist Circumference • Elevated Triglycerides • Reduced HDL-C • Elevated Blood Pressure • Elevated Fasting Glucose Pro-Inflammatory Cytokines Free Fatty Acids Metabolic Syndrome Dyslipidemia Hypertension Insulin Resistance Type 2 Diabetes Cardiovascular Disease

  4. 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 obese Kim et al. (2007) Obesity

  5. Significance In 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 obesity Heerwagen et al. (2010) Am J Physiol Regul Integr Comp Physiol; Gluckman (2009) Nat Rev Endocrinol; Campion (2009) Obes Rev

  6. Developmental Trajectory of Mesenchymal Stem Cells Pref-1 CEBPb Gesta et al (2007) Cell

  7. Central Hypothesis Maternal Obesity Placenta Omega-3 Fatty Acids ROS TNFa Curcumin BMP TNFa Wnt Trx Prx TNFR1 JNK b-catenin NFkB p65/p50 TCF FOXO SMAD 2/3 SMAD 4 Pref-1, CEBPb Myf5, Pax3, Pax7; Mesodermal Cell Myoblasts Adipoblasts

  8. Innovation Biochemical 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.

  9. Lethal Yellow (LY) mice Mimics human obesity 1Ay 1A’ 1A 1B 1C 2 B6 Merc Agouti Agouti 1Ay 1A’ 1A 1B 1C 2 ( 120 kb Ay deletion ) LY Merc/agouti fusion Agouti Arcuate ME αα Agouti (Ay) Insulin - α-MSH CART Satiety Leptin + MC4R Blood Hypothalamus

  10. Yang et al. 2011, Mol Reprod Dev

  11. 50 15 B6 B6 LY LY 40 10 30 Maternal Weight (g) Litter Size (# per dam) 20 5 10 0 0 80 B6 30 100 B6 B6 LY LY LY 80 * 60 * * 20 60 Tail Somites (# per embryo) Embryo Weight (mg) 40 Placental Weight (mg) 40 10 20 20 0 0 0 Maternal Obesity Decreases Growth and Development of Embryos Norwood et al., unpublished data

  12. Placenta Specific Aim 1: Identify TNFa-dependent mechanisms of decreased myogenic and increased adiopogenic regulatory factor expression. Omega-3 Fatty Acids TNFa BMP TNFa TNFR1 NFkB p65/p50 SMAD 2/3 SMAD 4 Myf5, Pax3, Pax7; Pref-1, CEBPb Somite

  13. NFkB-dependent regulation of gene expression

  14. Smad-dependent regulation of gene expression Massagué J et al. Genes Dev. 2005;19:2783-2810

  15. Experimental Design Year 1: • Establish somiteexplant 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 SmadsiRNA 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, Cebpbin TNFa-treated somites OR somites collected from embryos developed in an obese (LY) compared to lean (B6) in uteroenvironment (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

  16. Experimental Design Year 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

  17. Specific Aim 2: Identify ROS-dependent mechanisms of decreased myogenic and increased adiopogenic regulatory factor expression. ROS Curcumin Placenta Wnt Trx Prx JNK b-catenin TCF FOXO Myf5, Pax3, Pax7; Pref-1 Somite

  18. FOXO-dependent regulation of gene expression Huang et al. (2007) J Cell Sci

  19. Experimental Design Year 1: • Establish somiteexplant 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, Cebpbin H2O2 -treated somites OR somites collected from embryos developed in an obese (LY) compared to lean (B6) in uteroenvironment (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

  20. Experimental Design Year 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

  21. 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 Maternal Obesity Omega-3 Fatty Acids Curcumin Myoblasts Adipoblasts Mesenchymal Stem Cell Niche

  22. Experimental Design Year 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 uteroenvironment 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 uteroenvironment 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

  23. Expected Outcomes • Identify signaling pathways and transcription factors activated by the inflammatory response, oxidative stress, or both which regulate the expression of myogenic and adipogenicregulatory 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

  24. 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:

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