Location: Children's Nutrition Research Center2010 Annual Report
1a. Objectives (from AD-416)
Obesity and related co-morbidities are among the most profound public health problems today. Obesity prevalence has increased dramatically in recent decades and 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 animals and determine if maternal obesity and/or nutrition before and during pregnancy persistently alters epigenetic regulation in offspring; 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
Project 1. We learned that the peripheral clock indirectly controls leptin transcription by interacting with other transcription factors on the leptin promoter. We discovered that the peripheral clock controls bile acid synthesis in the liver by regulating the expression of the FXR and SHP that inhibit the expression of Cyp7A1 for bile acid synthesis. Animal models show a significant decrease in the expression of FXR and SHP in the liver which is correlated with a dramatic increase in Cyp7A1 expression and bile acid levels in serum and the liver. Project 2. We studied whether pretreatment with GLP-2 reduces inflammation and NEC in premature piglets; we may extend the GLP-2 treatment period to test whether this might prevent NEC. We completed studies on the effects of continuous parenteral amino acid infusion on protein synthesis in newborns to identify optimum nutrition support regimens to increase growth of premature infants. We compared how amino acids and insulin separately increase protein synthesis in different tissues of newborn pigs. We showed that both amino acids and insulin increased protein synthesis in a variety of different types of muscles. We studied the effect of prolonged parenteral leucine infusion on protein synthesis in neonatal pigs. We examined the time course of changes in protein synthesis in newborn muscle after a meal. Project 3. We examined livers of male C57BL/6 mice under various diet conditions for detection of fat tissue. The function of the CD11c macrophage surface protein was analyzed by studying mice deficient in CD11c. We studied diet reversal for changes in inflammatory gene expression. We began collaborating on a study on inflammatory bowel disease utilizing our mouse strains with genetically engineered deletions of proteins important in obesity-related inflammation. Project 4. We studied expression of SIRT3 in mouse liver under various conditions; mouse liver SIRT3 protein levels are increased in mice that underwent 12 or 18 months of 40% food restriction, but is down-regulated in mice fed high-fat diet for 16 wks. We analyzed PICOT expression in mouse embryos and adult tissue. We genetically generated a mouse line that lacks the GLP-2 receptor in brain neurons. We found GLP-2 receptor activation is essential for maintaining blood glucose levels by modulating the brain neuron activities. We found deficiency of GLP-2 receptor in brain neurons increases gastric emptying, decreases insulin sensitivity, and has higher rates of glucose synthesis in liver in type 2 diabetes. We began feeding studies of mice lacking the receptor for a stomach-secreted feeding regulating hormone GHS-R. Project 5. We identified over 1000 genomic regions undergoing postnatal DNA methylation changes, and validated over 10. We longitudinally measured body weight in a cohort of Avy/a mice. We performed DNA methylation profiling on Illumina arrays on 6 AnCHM tissues as an alternative to MSAM. We tested several new antibodies to the NLRP7 protein; we found some evidence that NLRP7 may be cleaved by caspase. We isolated RNA from liver at 1 yr, and muscle (soleus) at p21 and 1 yr and performed gene expression profiling.
1. Disruption of circadian rhythm promotes obesity and tumor development. Human studies have indicated that a disruption of the circadian rhythm (our internal 24-hour time clock) increases the risk of obesity and cancer development in individuals that perform shift work during the night. However, the biological mechanisms leading to such risk remains unclear. Researchers at the Children's Nutrition Research Center, Houston, Texas, have recently demonstrated in mice that a disruption of the circadian rhythm induces similar types of tumor development to night-shift human workers. In addition, mice treated with chronic jet-lag also show increased risk of fatty liver development and higher body mass index without changing food choice or amount food-intake, and that uncontrolled activity of the sympathetic nervous system, which aids in the control of most of the body's internal organs, is involved in the increased tumor and obesity development under jet-lag conditions. These findings strongly suggest that educating the public to adopt a healthy life-style is important for both obesity and cancer prevention.
2. The importance of a white blood cell protein. During the development of obesity in children and adults, certain types of white blood cells migrate into fat tissue and cause some of the disease symptoms, such as diabetes, common in obese individuals. Scientists at the Children's Nutrition Research Center, Houston, Texas, are studying a protein called CD11c found on the surface of white blood cells in obese fat tissues. To determine the importance of this protein in obesity-related disease, they studied mice that were genetically engineered to be unable to make this protein. Animals that lacked CD11c became obese when put on a high fat diet, but they showed very little signs of diabetes in contrast to normal mice that developed signs of diabetes within a few weeks of eating a high fat diet. These results show for the first time that the white blood cell protein CD11c contributes to obesity-related disease, and they raise the possibility that a drug blocking CD11c could be used to reduce obesityrelated disease.
3. Studies on fat as a danger signal. Most of the body's cells have proteins on their surface that allow them to activate inflammation when they recognize different danger signals such as foreign biochemicals. Scientists at the Children's Nutrition Research Center, Houston, Texas, are studying one such protein, called TLR2, because this protein may recognize certain types of fat as a danger signal. Mice genetically engineered to be deficient in this protein when placed on a high fat diet developed very little signs of the inflammation that causes obesity-related symptoms, in contrast to normal mice that readily developed obesity and symptoms of disease. These findings show for the first time that TLR2 is involved in high fat diet-induced inflammation, and they indicate that future in-depth study of TLR2 will provide new insights into how the body responds to excess fat as a danger signal.
4. Protein production in the mitochondria. Additional knowledge is needed to better understand the regulation of cellular metabolism. Mitochondria are the structures within cells that produce power for the cells to function. SIRT3 proteins reside in mitochondria and regulate several processes in mitochondria. In collaboration with researchers at Penn State University, scientists at the Children's Nutrition Research Center found MRPL10 proteins (mitochondrial ribosomal protein L10, a part of mitochondrial machinery for the protein production) contain a type of chemical modification, namely acetylation. SIRT3 can remove this chemical modification on MRPL10 and regulate the function of MRPL10 to reduce the production of mitochondrial proteins. This is the first time such kind of control of mitochondrial protein production has been discovered.
5. Structure of an oxidative balance-regulating protein. Free radicals that often cause oxidative stress are harmful to cells and organisms and are involved in the development of some diseases; however, how cells utilize redox (oxidation and reduction balance)-sensitive proteins (enzymes) to remove the free radicals and avoid cellular damage is still largely unknown. Determination of the active elements within a protein molecule is critical toward understanding of the physiological functions of those proteins. In collaboration with The Samuel Roberts Noble Foundation, Children's Nutrition Research Center researchers produced a crystal form of one of the redoxsensitive proteins, called PICOT, and determined some important amino acid residues in this structure that may contribute to different biochemical functions related to oxidative stress. These findings will help other scientists to potentially engineer this protein to enhance cell's capacity of tolerance to oxidative damage.
6. The role of GLP-2 receptor in obesity. Diabetics cannot maintain blood glucose within normal levels, and until now the role of GLP-2 (a nutrient-responsive gut hormone) has not been explored for the potential control of blood glucose. Researchers at the Children's Nutrition Research Center, Houston, Texas, were able to quantify the synthesis of glucose (i.e., gluconeogenesis) in the liver. They have found that the GLP-2 receptor deficiency impairs liver gluconeogenesis, thus resulting in a high level of blood glucose even with high dose of insulin. This research will provide a powerful, genetically-modified mouse model to define the physiological role and signaling network of GLP-2 in the control of glucose level and energy balance, and to advance our understanding at cellular and molecular levels how glucose metabolism is disrupted in diabetes.
7. Improving preterm infant nutrition by getting a grasp on NEC. Necrotizing enterocolitis (NEC) is a serious intestinal disease in premature infants, and there is an urgent need to develop nutritional approaches to prevent NEC. Researchers at the Children's Nutrition Research Center, Houston, Texas, have completed studies testing if the type of dietary carbohydrate, either polycose (an easily digestible source of carbohydrate calories) or lactose-based formula, used in infant formulas induces NEC. Our results showed that the incidence and severity of NEC was higher in preterm animal models fed a formula containing polycose and such findings highlight the potential risk associated with poor digestion of polycose in infant formula. These results have the potential impact of modifying the design of human infant formulas since considerable evidence shows that human milk is protective against NEC and contains mainly lactose, whereas many formulas fed to preterm infants contain substantial amounts of polycose along with lactose.
8. Development of a mouse model to understand how maternal obesity during fetal development promotes obesity in the next generation. Previous studies have shown that maternal obesity promotes obesity in the next generation; but exactly when maternal obesity affects development, however, is not known. Researchers at the Children's Nutrition Research Center in Houston, Texas, have shown using a mouse model that maternal obesity during fetal development promotes excess fat levels in her offspring, whereas maternal obesity during the lactation period actually leads to lower body weight in the offspring. This is an important accomplishment in that it provides a 'cleaner' model in which we can study the effects of maternal obesity on epigenetics (developmental establishment of gene regulatory mechanisms) in the hypothalamus, and focuses the relevant period of development.
9. Gene expression profiling in mouse model liver. Nutritional protein deprivation early in life has an impact on the liver, but the long-term consequences are not known, especially if this may predispose individuals to metabolic disease. Researchers at the Children's Nutrition Research Center in Houston, Texas, have found several up- and down-regulated genes that play a role in DNA organizational structure. This will help researchers understand the mechanisms of long-term altered gene regulation after early exposure to protein deprivation.
Kellermayer, R., Balasa, A., Zhang, W., Lee, S., Mirza, S., Chakravarty, A., Szigeti, R., Laritsky, E., Tatevian, N., Smith, W.C., Shen, L., Waterland, R.A. 2010. Epigenetic maturation in colonic mucosa continues beyond infancy in mice. Human Molecular Genetics. 19(11):2168-2176.