Location: Children's Nutrition Research Center2009 Annual Report
1a. Objectives (from AD-416)
Obesity and related co-morbidities, including type 2 diabetes and cardiovascular disease, are among the most profound public health problems today. Obesity prevalence in the US and other developed countries has increased dramatically in recent decades. This trend is affecting individuals at every age, including women of child-bearing age. Children's Nutrition Research Center researchers will address these issues by targeting these objectives: 1) determine the role of the circadian clock in regulation of food intake and the interaction between diet composition and circadian rhythms of food intake on body weight control during post-weaning and adult life; determine the specific role of central and peripheral clocks, as well as circadian output pathways in maintaining the homeostasis of food intake; 2)determine roles for adipocyte- and skeletal myocyte-specific circadian clocks in high fat feeding-induced alterations of feeding behavior, adiposity, and insulin sensitivity; relate observed changes to circadian patterns in gene expression, protein levels, and metabolic fluxes; 3) determine whether early nutritional intervention-mediated metabolic programming predisposes toward age-onset induced obesity and insulin resistance through modulation of intracellular circadian clocks; 4) establish a model to investigate the impact of prematurity on the gastrointestinal and metabolic response to perinatal nutrition; 5) compare the impact of continuous versus intermittent bolus delivery of nutrients provided enterally or parenterally on protein synthesis and accretion in neonatal pigs and identify the intracellular signaling mechanism involved; 6) define the specific classes of resident and emigrated leukocytes in adipose tissue, their phenotypic changes during development of obesity, and inflammatory mediators involved; 7) characterize leukocyte patterns and gene expression in adipose tissue of animals fed normal and high fat diets; 8) investigate changes of SIRT3 gene expression in the liver by various physiological and pathophysiological stimuli, and study the effects of SIRT3 expression on hepatic metabolism, oxidative stress, and fat deposition; 9) determine the role of protein kinase C interacting cousin of thioredoxin in insulin-mediated growth, macronutrient metabolism, and insulin resistance in the liver; 10) define the central action of glucagon-like peptide-2 (GLP-2) receptor on food intake and inter-organ macronutrient flux; 11) identify genes that show epigenetic dysregulation in obesity using a candidate-gene approach in a mouse model; 12) determine if methylation and expression of specific genes in hypothalamus and/or adipose tissue differ between lean and obese mice models using a genome-wide screen; 13) determine if maternal obesity and/or nutrition before and during pregnancy persistently alters epigenetic regulation in offspring hypothalamus or adipose tissue; 14) identify placental epigenetic mechanisms that affect fetal nutrition, growth and development; and 15) determine how programming of glucose intolerance, obesity, and the epigenetic dysregulation of skeletal muscle-growth in mice is affected by maternal diet during development.
1b. Approach (from AD-416)
The research will be accomplished using a variety of animal models and scientific tools to simulate the human newborn and/or child. Animal models will be used to understand the central and peripheral circadian clock mechanisms that influence eating behavior, metabolism, and energy balance. Newborm animal models will be used to examine the effect of chronic parenteral nutrition during the neonatal period on glucose tolerance, insulin sensitivity, and body composition during late infancy and adolescence. Researchers will investigate the effects of intermittent bolus feeding versus continuous feeding, delivered either enterally or parenterally, on protein synthesis in neonatal animal models. This will allow our team to determine the long-term impact of these feeding modalities on growth and body composition. Various models will be placed on obesigenic diets at 5-6 weeks of age and evaluated at 7 days, 5 weeks, and 6 months to define a blood leukocyte expression profile at these time points. Children's Nutrition Research Center scientists will also characterize the functions of intracellular systems in the liver and their influences on the onset of fatty liver disease and glucose homeostasis. Additional investigation will occur on the intracellular signaling pathways of GLP-2 and their metabolic effects on food intake, energy expenditure, and glucose homeostasis. Various mouse models, and a human model of epigenetic dysregulation compromising placental development, will be used to test if maternal obesity and fetal nutrition during development affects the establishment of gene-specific DNA methylation patterns in the developing fetus, which would cause permanent changes in gene expression, metabolism, food intake regulation, and body weight.
3. Progress Report
We've shown that disruption of circadian rhythm increases body weight in Period-mutant mice & in jetlagged wt mice. Restricted feeding disrupts the circadian homeostasis of metabolic parameters, especially, the homeostasis of bile acid synthesis & signaling, which leads to increased liver damage that mimics cholestatic disease. We're studying other metabolic parameters in control wt & Period-mutant mice & in jetlagged wt & Period-littermates to investigate the role of the circadian clock in controlling energy homeostasis. We've found that jetlag disrupts the central clock activity as efficient as SCN lesion. (Proj 1) Animal models were fed 1/6 of their daily nutrient requirement & the activation of regulators of protein synthesis were measured over 1/6 of a day. Results showed feeding rapidly increases muscle protein synthesis by activating the mammalian target of rapamycin signaling pathway, but muscle protein synthesis is increased for a limited period; thus, frequent meals are essential for sustaining optimal protein synthesis for muscle growth in the neonate. We have also shown that maldigestion of polycose (infant formula) corresponded with altered assemblages & metabolic activities of the resident bacteria & increased incidence & severity of NEC compared to models fed a lactose-based (colostrum) formula. (Proj 2) We continue to analyze TLR2 knockout mice at the 5-week period of high-fat feeding & have demonstrated significant reductions in systemic & adipose tissue inflammation. Gamma delta T cell knockout mice have been analyzed & demonstrated that these T cells are necessary for CD11c+ macrophage accumulation in adipose tissue in diet-induced obesity. We have developed methods isolating CD11c+ & CD11c negative macrophages from adipose tissue during different stages of diet-induced obesity. (Proj 3) We've demonstrated that MmGrx3/Picot has conserved function in protecting cells against oxidative stress. We found that MmGrx3 was ubiquitously expressed in most cell types & developing organs, with high accumulation of hybridization signals in the brain. We revealed that mouse Grx3 mRNA accumulated predominately in testis & was also found in other tissues (heart, liver, & muscle). We've found that GLP-2 receptor is essential for maintaining glucose (& energy) homeostasis. (Proj 4) We have completed analysis of hypothalamic methylation at P0 & P21 for all 6 genes promoters and found very low levels of methylation at Cart, Mc4r, & Npy, & no changes from P0 to P21. The Agrp promoter showed substantial interindividual variation in DNA methylation at one of the two sites studied. Both Pomc & Lepr promoters showed substantial methylation changes in methylation in P21 vs. P0 hypothalamus. We have completed MSAM studies comparing P21 & P0 hypothalamus which are pilot studies validating our ability to use MSAM to identify genomic regions showing differential methylation in hypothalamic DNA. We performed two P21 vs. P0 MSAM cohybridizations with hypothalamic DNA isolated from female C57BL/6J mice & found 2241 SmaI/XmaI intervals showing a 2-fold gain of methylation, & 904 showing a 50% decrease in methylation from P0 to P21. (Proj 5)
1. Circadian Dysregulation Leads to Loss of Balance in Bile Acid Production: The circadian clock plays an important role in maintaining the balance of bile acid in blood every day. This is important for processing dietary fat. Children's Nutrition Research Center researchers have found that feeding mice during their normal sleep hours (restrict feeding) disrupts the circadian clock activity and results in a loss of stability of liver metabolism. We also found that restricted feeding leads to a dramatic and temporary elevation in liver bile acid levels, which increases liver damage and can lead to cholestatic disease. These studies contribute to a better understanding of how disruption of circadian rhythms increases the risk of metabolic syndromes. (Project 1: The Circadian Clock in Nutritional Metabolism and Obesity)
2. Characterization of redox gene expression and regulation: In order to understand how a redox-sensitive gene, Grx3/Picot, is regulated in animal cells under various growth and environmental conditions, Children's Nutrition Research Center scientists have analyzed Grx3/Picot expression using several cell culture models. We have found that the expression level of Grx3/Picot gene was significantly enhanced under oxidative stress in muscle cells. Furthermore, our results indicated that Grx3/Picot protein levels in the cultured muscle cells were increased when cells were treated with insulin, a small secreted hormone that controls the level of blood sugar in the body. We further demonstrated that Grx3/Picot expression in breast cancer cells was induced by both estrogen and insulin-like growth factor 1 (IGF1), but not epidermal growth factor (EGF), compared with vehicle controls, suggesting Grx3/Picot may be specifically regulated through IGF1 and/or estrogen. These findings will lead to better understanding of the function of Grx3/Picot in both physiological and pathological conditions. (Project 4: Metabolic Regulation in Obesity Development)
3. Generation of liver-specific gene-deletion animal model: Previously, Children's Nutrition Research Center research scientists in Houston, Texas, demonstrated that a redox (reduction-oxidation balance)-sensitive gene, glutaredoxin 3 (Grx3/Picot), had conserved functions in the protection of cells against oxidative stress and animal models not having this gene showed signs of early embryonic growth defects and eventually death. Those findings implicate a critical role of this gene in the early development of embryos in mammals, including human. Yet, little is known how this gene functions in liver development and repair in response to injury caused by disease conditions, like non-alcohol fatty liver. In an attempt towards defining its function in liver, CNRC research scientists have built up a gene delivery vehicle, which can be used to insert a piece of DNA into the mouse chromosome at the specific location and then destroy the expression of gene of interest in the liver. By this means, the liver-specific gene knockout mice can be created. This unique tool will allow researchers to determine the precise function of this gene in liver under various nutritional interventions. (Project 4: Metabolic Regulation in Obesity Development)