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United States Department of Agriculture

Agricultural Research Service

Research Project: METABOLIC REGULATION IN OBESITY DEVELOPMENT

Location: Children's Nutrition Research Center

2013 Annual Report


1a.Objectives (from AD-416):
Objective 1. 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. Sub-objective 1.A. Investigate whether SIRT3 expression in the liver is regulated by various physiological and pathophysiological stimuli. Sub-objective 1.B. Study SIRT3 action in hepatocytes by over-expression or RNAi knockdown of SIRT3. Sub-objective 1.C. Investigate the effect of SIRT3 on liver oxidative stress and steatosis during obesity and diabetes. Objective 2. Determine the role of protein kinase C interacting cousin of thioredoxin (PICOT) in insulin-mediated growth, macronutrient metabolism, and insulin resistance in the liver. Characterize PICOT and link it to redox signaling in the liver. Sub-objective 2.A. Define the regulatory mechanism underlying how a novel redox-regulatory gene, PICOT, responds to nutrient homeostasis. Sub-objective 2.B. Investigate the function of PICOT in liver and dissect the cross talk between PICOT-mediated redox regulation and insulin action and signaling that contributes to insulin resistance. Objective 3. Define the central action of glucagon-like peptide-2 (GLP-2) receptor on food intake and inter-organ macronutrient flux. Characterize the peripheral role of GLP-2 receptor in energy expenditure and glucose homeostasis. Sub-objective 3.A. Define the central action and signaling network of GLP-2 receptor (GLP-2R) on food intake and glucose homeostasis. Sub-objective 3.B. Characterize the peripheral, physiological role of GLP-2 receptor in energy expenditure and glucose homeostasis. Objective 4. Study ghrelin peptide expression profile under different diet regimes. Objective 5. Charactize metabolic profile. Sub-Objective 5.A. Whole body analysis. Sub-Objective 5.B. Functional analysis. Objective 6. Conduct mechanistic analyses of differences in metabolic profile between WT and null mice. Objective 7. Confirm the predicted lipotropic effects of lecithin, choline and betaine in our high fat fed mouse models of the metabolic syndrome. Objective 8. 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. Objective 9: Unravel the complex brain circuits that are physiologically relevant in the control of energy and glucose balance.


1b.Approach (from AD-416):
To elucidate the physiological roles of SIRT3 and PICOT (monothiol glutaredoxin 3, GRX3) genes in liver, the expression of these two genes in cultured hepatocytes and in mice responding to physiological and nutritional stimuli will be investigated by Children's Nutrition Research Center scientists. In addition, SIRT3 or PICOT will be over-expressed or suppressed by RNA interference using adenoviral delivery into cultured hepatocytes or mice liver to study their functions. Knockout mice of SIRT3 or picot genes will also be investigated under nutritional interventions. Furthermore, mice with GLP-2 receptor deficiency specifically in the POMC neurons will be generated, and intracerebroventricular (icv) infusion of GLP-2 in mice will be performed to study the physiological function of GLP-2 receptor and the role in regulation of glucose and energy homeostasis. A primary culture model of hippocampal neurons and an ex vivo model of hypothalamic slices will also be established to explore GLP-2-mediated intracellular action and neuronal signaling using the whole-cell patch clamp technique. Researchers will characterize the role of ghrelin and its receptor in nutritional regulation of energy and glucose homeostasis. Additionally, we will explore the impact of the dietary supplementation on additional pathways thought to link obesity and insulin resistance. The study will further our understanding of nutritional interventions, which may provide new information for dietary guidelines, and lead to novel therapeutic and nutritional approaches for managing obesity and diabetes. Manipulate specific receptors to alter the susceptibility of animal models to binge eat for understanding homeostasis.


3.Progress Report:

For objective 1, we have generated two transgenic mice lines with ubiquitosue expression of SIRT3 in all tissues: one line has high liver SIRT3 expression and another line has low liver SIRT3 expression. We found that transgenic mice with high liver SIRT3 expression have increased glucose production, manifested by elevated circulating glucose levels when mice were administed with a precursor of glucose (pyruvate). We found that factors contributing to liver glucose synthesis, such as the mRNA expression of liver glucogenic genes and insulin signaling, were not changed in SIRT3 transgenic mice. We are exploring whether SIRT3 regulates the activity of liver glucose synthetic enzymes directly. As liver glucose production plays an important role in the high glucose levels in diabetic patients, understading the regulation of this process may lead to novel treatment for diabetes. In objective 2, we determined the structure of a novel protein, called glutaredoxin S16, and demonstrated a unique regulation of this protein that is important for counteracting external stresses caused by free radicals. We discovered that mammalian glutaredoxin 3 (Grx3), also called protein kinase C, an interacting cousin of thioredoxin (PICOT), could translocate to the nucleus from cytoplasm under oxidative stress, and also found that Grx3/PICOT was able to physically interact with p65 subunit under the same condition. We are continuing to explore whether the nuclear Grx3/PICOT is required for NF-KB activation and its signaling pathway in response to oxidative stress. In objective 3, we made significant progress that Glp2r in hypothalamic POMC neurons is required for the physiological, short-term control of feeding behavior and gastric emptying, which may contribute to the homeostatic, long-term control of energy balance and body weight. We also learned that GLP-2 action on POMC neurons is mediated by activating the GLP-2R-PI3K signaling pathway, and engaged in the central melanocortin system, which is a collection of central nervous system (CNS) components involved with body weight regulation. We learned that CNS GLP-2R activation suppresses feeding behavior and hepatic glucose production through directly modulating membrane excitability and nuclear transcription of POMC neurons via the PI3K-Akt-FoxO1 signaling pathway. Thus, GLP-2R activation in POMC neurons is essential for the maintenance of energy balance and glucose homeostasis. In addition, we also found that Glp2r in intestinal epithelium is required for glycemical control after a meal. For objectives 4-6, we have made the following new findings with ghrein receptor GHS-R global knockout mice:.
1)GHS-R deletion reduces the hunger signal but enhances the satiety signal in the brain feeding center;.
2)GHS-R deletion increases heat production in brown fat, but has no effects on total daily food intake or activity;.
3)GHS-R regulates fat metabolism in both central and peripheral tissues. These findings suggest that GHS-R blockers may function as a new class of anti-obesity drugs, which can combat obesity without diet or exercise. In objectives 7-8, progress has focused mainly on the mechanisms of the lipotropic effects of the activation of the nuclear receptor protein LRH-1 (Liver Receptor Homolog-1) activation by the signaling molecule DLPC (DiLauroyl PhosphatidylCholine) and other signals. We have made two signficant advances, the first of which is the demonstration that increased and decreased LRH-1 activity are associated with improved or impaired responses to an evolutionarily conserved, and very basic, cell stress pathway termed endoplasmic reticulum stress, and the cell's response to this stress, which is termed the unfolded protein response. We have also identified the coactivator protein SRC-2, a global regulator of messenger RNA production, as a significant mediator of the transcriptional output of LRH-1 activation. This provides new insights into the basis for fatty liver disease and its potential treatment, and we are continuing to explore both of these new mechanisms. For objective 9, we previously demonstrated that 5-hydroxytryptamine (5-HT) compounds, or also known as serotonin, inhibit binge eating in wild type mice, but have no effects in mice lacking 5-HT 2C receptors (5-HT2CRs). Futher, we found that these 5-HT compounds effectively inhibit binge eating in mice expressing 5-HT2CRs only in dopamine neurons. These support a model that a 5-HT-dopamine circuit in the brain inhibits binge eating. In another project investigating the role of estrogen in the brain on body weight balance, we found that an agonist of estrogen receptor-alph (ERalpha) can activate neurons in the amygdala. These finding further support our previous observations that ERalpha in the amygdala is required to maintain normal body weight.


4.Accomplishments
1. SIRT3 gene increases liver synthesis of glucose. Liver produces glucose and releases it into bloodstream. This process contributes to the high glucose levels in diabetics. Researchers at the Children's Nutrition Research Center in Houston, Texas, have generated genetically engineered mice with elevated expression of the SIRT3 gene in all of their tissues and found that these animals had increased glucose production. However, other factors that play important roles in liver glucose production were not changed in these mice. Evidence suggests that the SIRT3 gene may directly modify liver glucose synthetic enzymes to regulate their activity. These findings shed light on novel mechanisms of liver glucose production and possibly new ways to treat diabetes.

2. Diverse functions of glutaredoxins. A group of proteins, called Glutaredoxins (Grxs), are important for protecting the life of cells from damage caused by free radicals. However, how these proteins act in the cell and whether these proteins are directly involved in the process of damage repair are still largely unknown. Children's Nutrition Research Center researchers in Houston, Texas, have identified a new member of Grxs that comprises two distinct functional regions, in which one region is similar to other members of Grxs, but another one is unique and its function is unknown. They further determined that this unique region had enzymatic activities and could be crucial for repairs of damaged genetic material in the cell. Their findings may provide insights into strategies to prevent free radical induced damages in human disorders, including aging, cancer, and chronic diseases.

3. Generation of GHS-R knockout mice to unravel Ghrelin's unknowns. Ghrelin is a hormone within your body that is responsible for stimulating appetite and satiety. But, despite an enormous body of literature documenting ghrelin's multifaceted functions, the exact molecular mechanisms responsible for its function remain largely elusive. A major obstacle hindering progress of the field is insufficient knowledge of the sites of ghrelin's action, due to the absence of tissue-specific knockout models for ghrelin signaling. Scientists at the Children's Nutrition Research Center in Houston, Texas, have successfully generated the first set of tissue-specific knockout mice for the ghrelin receptor (GHS-R). These novel mouse models will serve as powerful tools for unraveling the tissue-specific roles of GHS-R, thus filling critical knowledge gaps in understanding of biological functions of GHS-R, and providing a foundation for future theraputic developments targeting the ghrelin receptor.

4. GLP-2R action on POMC neurons in food intake and glycemic control. Uncontrolled blood glucose is a hallmark of diabetic patients. However, little is known about whether Glucagon-like peptide-2 (GLP-2) can act like insulin to suppress blood glucose. Using genetic mouse models and pharmacological approaches, scientists at the Children's Nutrition Research Center in Houston, Texas, found that GLP-2 is a key satiety signal for the physiological control of food intake and gastric emptying, and this contributes to the control of energy balance, body weight, and blood glucose. We also learned that GLP-2 directly modulates activities of POMC neurons, enhances the PI3K signaling pathway, and suppresses glucose production in the liver. These findings provided insight into the gut-brain-liver axis in the control of blood glucose and food intake.


Last Modified: 7/23/2014
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