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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR, CELLULAR, AND REGULATORY ASPECTS OF OBESITY DEVELOPMENT IN CHILDREN

Location: Children's Nutrition Research Center

2012 Annual Report


1a.Objectives (from AD-416):
1) determine role of circadian clock in regulation of food intake and interaction between diet composition and circadian rhythms of food intake on body weight control during post-weaning and adult life; determine specific role of central and peripheral clocks, as well as circadian output pathways in maintaining homeostasis of food intake; (2-3 removed; SYs left);.
4)investigate impact of prematurity on GI and metabolic response to perinatal nutrition;.
5)compare impact of continuous vs. intermittent bolus delivery of nutrients provided enterally or parenterally on protein synthesis and accretion in neonatal models and identify intracellular signaling mechanism involved;.
8)investigate changes of SIRT3 gene expression in the liver, and study effects of SIRT3 expression on hepatic metabolism, oxidative stress, and fat deposition;.
9)determine role of protein kinase C interacting cousin of thioredoxin in insulin-mediated growth, macronutrient metabolism, and insulin resistance in the liver; 10) define action of glucagon-like peptide-2 (GLP-2) receptor on food intake and inter-organ macronutrient flux; 11) study ghrelin peptide expression profile under different diet regimes; 12) conduct mechanistic analyses of differences in metabolic profile between WT and null mice; 13) confirm predicted lipotropic effects of lecithin, choline and betaine in high-fat-fed mouse models of the metabolic syndrome; 14) test impact of liver specific LRH-1 knockout on the lipotropic effects of lecithin, choline and betaine in high-fat-fed mouse models of the metabolic syndrome; 15)identify genes that show epigenetic dysregulation in obesity; 16) 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; 17) determine if maternal obesity and/or nutrition before and during pregnancy persistently alters epigenetic regulation in offspring hypothalamus or adipose tissue; 18) identify placental epigenetic mechanisms that affect fetal nutrition, growth and development; 19) determine how programming of glucose intolerance, obesity, and the epigenetic dysregulation of skeletal muscle-growth in mice is affected by maternal diet during development; 20) determine if epigenetic programming and reprogramming contribute to lineage-specific patterns of gene expression; 21) develop targested knock-in mouse model to determine if nutrients can modulate hypermethylation, epigenetic silencing and increase susceptibility to disease; 22) evaluate leukocyte patterns, gene expression profiles, and inflammatory mediators in adipose tissue under influence of diatary manipulation that leads to obesity.


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. Additionally we will investigate the mechanisms regulating DNA methylation during development, and characterize their involvement in nutritional programming during critical ontogenic periods. We will characterize the role of ghrelin and its receptor in nutritional regulation of energy and glucose homeostasis.


3.Progress Report:
Significant research progress was accomplished during the year. To review the progress, please refer to project 6250-51000-055-10S (Project 1), 6250-51000-055-20S (Project 2), 6250-51000-055-30S (Project 3), 6250-51000-055-40S (Project 4), and 6250-51000-055-50S (Project 5).


4.Accomplishments
1. Continuous feeding induces metabolic dysfunction in neonatal pigs. Thousands of premature infants born in the United States every year are unable to handle normal oral or enteral feeding, and instead receive parenteral or intravenous nutrition (PN). Previous studies conducted at the Children's Nutrition Research Center in Houston, Texas, using the neonatal piglet as a model of human premature infants showed that PN induces metabolic dysfunction which was marked by an accumulation of fat in the liver and a condition of glucose intolerance known as insulin resistance, which is similar to type 2 diabetes. In this study CNRC researchers used neonatal piglets to further test if feeding patterns contribute to these metabolic dysfunctions. Our results showed that the intermittent feeding pattern produced the optimum metabolic function and this was more important than whether feeding occurred by enteral vs. PN route. These findings are important and suggest that hospitalized premature infants should be given intermittent or bolus feedings during the early period after birth to maintain optimum metabolic function.

2. Intermittent feeding has a greater effect on protein deposition in muscle of neonates. More than 10% of newborns are of low birth-weight and many exhibit adverse long-term health problems. Orogastric tube feeding (passing a stomach tube through the mouth) by continuous infusion or intermittent bolus delivery is necessary for newborns who are unable to coordinate food ingestion. However, no study had examined the effect of these feeding modalities on protein deposition in newborns. Scientists at the Children's Nutrition Research Center in Houston, Texas, conducted studies in models that demonstrated that the intermittent bolus pattern of feeding increases the synthesis of proteins in skeletal muscle to a greater extent than continuous feeding, leading to an increase in protein deposition in muscle. These findings are important for pediatric nutrition as they suggest that the intermittent bolus pattern of feeding has the potential to enhance lean body mass and improve clinically important outcomes, such as weight gain, compared to continuous feeding, in newborns.

3. The expression and function of leptin follow a circadian rhythm in vivo. Leptin is a hormone secreted from the fat tissue and acts on the brain via blood circulation to suppress appetite and stimulate metabolism. The level of Leptin peaks during the day in response to food-intake (which stimulates energy expenditure to prevent obesity) and troughs at night during sleeping, and is also proportional to the size of fat tissue in the body. Researchers at the Children's Nutrition Research Center in Houston, Texas, have found that Leptin is controlled by the circadian clock. The disruption of circadian rhythm induces and maintains obesity in mice even when they have higher levels of Leptin due to increasing fat storage, a phenomenon called "Leptin resistance", which is commonly observed among obese humans. Since the industrial revolution, frequent disruption of circadian rhythm has become common to all human societies, especially nightshift workers who show a nearly two-fold greater risk of developing metabolic syndromes and obesity. Further study the mechanism of circadian control of the expression and function of Leptin will have a very high impact on the prevention and treatment of obesity in humans.

4. The role of alpha Beta T cells in diet induced inflammation. The alpha Beta T cells, an important class of white blood cells that are known to help protect the body from infection, have been found to increase in fat tissue during obesity, but their importance there has been unknown. Researchers at the Children's Nutrition Research Center in Houston, Texas, studied mice that are deficient in these cells by placing them on a high fat diet for 3 months and analyzing tissue inflammation and diabetic changes that occur as the mice become obese. They found that inflammation and diabetic changes were markedly reduced in these mice. These observations provide strong evidence that these cells in normal mice may be stimulated by the high fat diet and instead of protecting the body, actually cause tissue injury.

5. Estrogen acts in the amygdala region of the brain to prevent obesity. The action sites of estrogen to prevent body weight gain are not fully revealed. Researchers at the Children's Nutrition Research Center in Houston, Texas, successfully generated mice lacking estrogen receptors in the amygdala, a particular structure within the brain, which caused obesity in both female and male mice. These findings indicate that estrogen actions within the amygdala are required to maintain normal body weight in both genders. These results may provide rational targets for the development of novel anti-obesity therapies.

6. Discovery of a link between LRH-1 activity and the unfolded protein response. Liver receptor homolog-1 (LRH-1) is an important human regulatory protein. Previous research results identified a role for LRH-1 counteracting diabetes, but the mechanism of this effect was not clear. Children's Nutrition Research Center researchers in Houston, Texas, have uncovered an unexpected impact of increased LRH-1 activity on a well-studied cellular stress pathway that is called the unfolded protein response. Since activation of the unfolded protein response inhibits insulin action and promotes diabetes, the ability of LRH-1 to counteract this stress pathway could promote insulin sensitivity.

7. The ghrelin receptor (GHS-R) plays a key role in inflammation of fat. Macrophages are a particular type of immune cells and under high-fat feeding conditions, macrophages infiltrate fat tissues to cause inflammation. Macrophages in fat tissues are linked to obesity and insulin resistance (a pre-diabetic condition, in which the body's insulin is less effective). There are two types of macrophages in fat: proinflammatory M1 macrophages are associated with an obese and insulin-resistant state, while anti-inflammatory M2 macrophages are associated with a lean and insulinsensitive state. Researchers at the Children's Nutrition Research Center in Houston, Texas, found that the deletion of the ghrelin receptor (GHS-R) decreases proinflammatory M1 macrophages, but increases anti-inflammatory M2 macrophages in fat. These macrophage changes in GHS-R deleted mice reduce diet-induced obesity, inflammation, and insulin resistance. This finding suggests that suppression of the ghrelin receptor may represent an attractive new approach for combating obesity and insulin resistance.

8. GLP-2 suppresses feeding behavior and glucose production. Glucagon-like peptides (GLP-1/2) are key modulators for maintaining glycemic control and improving insulin sensitivity. However, it is unknown if GLP-2 receptor (Glp2r) in the brain plays any physiological role in the control of feeding behavior and glucose balance. Using Glp2r knockout mice, Children's Nutrition Research Center researchers in Houston, Texas, have demonstrated that GLP-2 suppresses food intake and glucose production in the liver through GLP-2 receptor action on neuronal excitation in the brain. These findings will facilitate the development of food-based approaches for the treatment of intestinal dysfunctions, obesity, and diabetes.

9. A novel protein plays a dual role in scavenging free radicals and repairing DNA damage. Cells constantly monitor and repair damage of genetic materials (DNA) caused by free radicals. However, the underlying mechanisms are not fully understood. Children's Nutrition Research Center researchers in Houston, Texas have identified a novel protein, AtGRXS16, involved in those processes and demonstrated that this protein comprises two regions with distinct functions, in which one half of the protein acts as an enzyme to cut damaged DNA and another half acts as a scavenger to remove excess free radicals from the cells. We further demonstrate that activities of the two parts are regulated accordingly through interacting with each other. Our findings may provide insights into the rational strategy to prevent free radical induced damages in human disorders, including aging, cancer, and chronic diseases.

10. Sirtuin genes regulate tumor suppressor FOXO3 protein stability in cancer cells. The FOXO3 protein can suppress tumor formation and nutritional deprivation, such as starvation, can increase its expression level. Researchers at the Children's Nutrition Research Center in Houston, Texas, found that the enzymes produced by two sirtuin genes (SIRT1 and SIRT2) can bind and modify FOXO3 proteins, which leads to the recruitment of another enzyme Skp2 to add ubiquitin chains to FOXO3 proteins and mark it for degradation. Therefore, in cells with high expression of SIRT1, SIRT2 or Skp2, the levels of FOXO3 are decreased. For example, in prostate cancer cells, the higher the malignancy the higher the expression of SIRT1 and Skp2 and the lower the expression of FOXO3; and suppressing either SIRT1 or Skp2 in these cells bolsters FOXO3 expression and reduces malignancy. This study uncovers a novel mechanism regulating FOXO3 that can be targeted for cancer treatment.

11. The role of the PU.1 gene in obesity-induced fat cell inflammation. It is understood that when compared to subcutaneous obesity, abdominal obesity is associated with a worse metabolic outcome. Obesity induces profound changes in adipose tissue, especially increased production of free radicals and factors that stimulate inflammation, resulting in metabolic imbalance and diseases. The mechanism by which this event occurs is not fully understood. Children's Nutrition Research Center scientists in Houston, Texas found that obesity induces an increased expression of the PU.1 transcription factor in the fat cells of abdominal but not subcutaneous fat; and demonstrated that suppressing PU.1 expression in fat cells results in decreased expression of genes controlled by PU.1, such as enzymes responsible for free radical production and pro-inflammatory factors. As a result of these studies, downregulation of PU.1 in obese fat cells could be a new strategy to combat obesityassociated diseases.


Review Publications
Elbjeirami, W.M., Donnachie, E.M., Burns, A.R., Smith, C.W. 2011. Endothelial-derived GM-CSF influences expression of oncostatin M. American Journal of Physiology - Cell Physiology. 301(4):C947-C953.

Lin, L., Saha, P.K., Ma, X., Henshaw, I.O., Shao, L., Chang, B.J., Buras, E., Tong, Q., Chan, L., Mcguinness, O.P., Sun, Y. 2011. Abalation of ghrelin receptor reduces adiposity and improves insulin sensitivity during aging by regulating fat metabolism in white and borwn adipose tissues. Aging Cell. 10(6):996-1010.

Brand, S., Teich, R., Dicke, T., Harb, H., Yildirim, A.O., Tost, J., Schneider-Stock, R., Waterland, R.A., Bauer, U., Mutius, E., Garn, H., Pfefferle, P.I., Renz, H. 2011. Epigenetic regulation in murine offspring as a novel mechanism for transmaternal asthma protection induced by microbes. Journal of Allergy Clinical Immunology. 128(3):618-625.

Yang, Y., Chen, K., Tong, Q. 2011. Murine Sirt3 protein isoforms have variable half-lives. Islets. 488:46-51.

Albarran-Zeckler, R., Sun, Y., Smith, R.G. 2011. Physiological roles revealed by ghrelin and ghrelin receptor deficient mice. Peptides. 32(11):2229-2235.

Lee, J., Lee, Y., Mamrosh, J.L., Busby, S.A., Griffin, P.R., Pathak, M.C., Ortlund, E.A., Moore, D.D. 2011. A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects. Nature. 474(23):506-510.

Jain, A.K., Stoll, B., Burrin, D.G., Holst, J.J., Moore, D.D. 2011. Enteral bile acid treatment improves parenteral nutrition-related liver disease and intestinal mucosal atrophy in neonatal pigs. American Journal of Physiology - Gastrointestinal and Liver Physiology. 302(2):G218-G224.

Last Modified: 8/19/2014
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