Location: Houston, Texas2011 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
In Obj. 1, we have examined the effects of nutritional changes (hypo- or hyperglycemia, serum deprivation or fatty acid loading) or hormones (insulin, thyroid hormone, catecholamine, or glucocorticoids) on SIRT3 expression in cultured hepatocytes, the primary cells of the liver. We have also established methods to express SIRT3 or knockdown SIRT3 in cultured hepatocytes using a particular virus strain called adenovirus. In obj. 2, we have generated transgenic mice targeting the specific gene Grx3, which will be used as a genetic tool to delete the Grx3 gene in specific tissues. Cell and mouse models were established to study the function of Grx3 in tumor development. Clinical data were analyzed to correlate Grx3 expression with human cancer metastasis and survival. In addition, the three-dimensional structure of the functional Grx-domain of mammalian Grx3 was determined. One new member of the glutaredoxin family from the Arabidopsis plant was identified and its function was characterized. In Obj. 3 we conducted studies to determine the physiological role of glucagon-like peptide 2 (GLP-2, a gut hormone) in the central nervous system in the control of blood glucose; we deleted GLP-2 receptor (GLP-2R) in hypothalamic neurons and demonstrated that GLP-2R deletion in these neurons increases glucose production in the liver by decreasing insulin responsiveness, suggesting that brain GLP-2R activation is important for maintaining blood glucose levels. We also demonstrated that GLP-2R activation stimulated protein synthesis in neurons by activating the PI3-kinase-dependent Akt-mTOR signaling pathway. In Obj. 4-6, we analyzed the basic metabolic parameters of mice fed with regular chow, high-fat diet, and high fructose corn syrup (HFCS). As expected, high-fat feeding increases weight gain, inducing obesity. In contrast, HFCS only induced modest weight gain without inducing obesity (the fat and lean mass increased proportionally). Surprisingly, HFCS-fed mice exhibited more severe insulin resistance than that of high fat diet-fed mice. Obj. 7 was initiated this project year, and we demonstrated that potent antidiabetic effects of an unusual form of phosphatidylcholine, termed dilauroyl phosphatidylcholine (DLPC) This was published in Nature. In Obj. 8, we have bred reactivatable 5-HT2CR (serotonin 2C receptor) null mice and their wildtype littermates. We will test if global deletion of 5-HT2CR in mice alters binge eating. Meanwhile, we have generated a new 5-HT2CR floxed mouse model. In parallel, we used mouse models with estrogen receptor-alpha (a receptor that is activated by the sex hormone estrogen) deleted in the central nervous system. We also demonstrated that distinct hypothalamic ERalpha neurons are required to regulate feeding, energy expenditure, fat distribution and fertility. The ADODR monitors project activities by visits, review of purchases of equipment, review of ARS-funded foreign travel, and review of ARS funds provided through the SCA.
1. The brain may be a target for energy balance and reproduction concerns. Estrogens act upon estrogen receptor-alpha (ERa) to regulate body weight and reproduction, but ERa-expressing cells that are critical for these effects have not been identified. Researchers at the Children's Nutrition Research Center, Houston, TX, demonstrated that ERa in the brain is required to mediate estrogenic effects on the regulation of body weight and reproduction. These findings may provide rational targets for the development of new therapeutic interventions that may be used to treat and prevent obesity and infertility in women.
2. A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects. Nuclear hormone receptors are important regulators of metabolism, and their activity is controlled by binding of hormones or other specific ligands. Loss of appropriate metabolic control occurs in obesity and diabetes. Researchers at the Children's Nutrition Research Center, in Houston, TX, have discovered a new ligand for the nuclear receptor LRH-1 (also known as NR5A2), that is an unusual but natural lipid species called dilauroyl phosphatidylcholine (DLPC). DLPC treatment of mice lowers liver fat and improves control of sugar metabolism in mouse diabetes models. These findings identify LRH-1 as a new metabolic regulator and suggest that DLPC may be useful in treating metabolic disorders. The antidiabetic effects of DLPC are currently being examined in a small clinical trial in obese humans.
3. GLP-2 receptor deficiency impairs glucose balance. Hyperglycemia is a common problem with diabetes, and is primarily attributed to glucose production. The brain can sense hormones and nutrients to control energy and glucose homeostasis, but the physiological significance of a particular brain GLP-2 receptor (GLP-2R) has not been defined. Researchers at the Children's Nutrition Research Center in Houston, TX, demonstrated that GLP-2R deficiency in brain neurons impairs glucose homeostasis by decreasing insulin sensitivity, which suggests that the brain GLP-2R plays an important role in the control of glucose homeostasis. GLP-2R signaling in the brain may be a potential target for future interventions for the treatment of diabetes and obesity.
4. An antioxidant protein feels "heat". Global environmental temperature changes (global warming) threaten agriculture practice and food production, as well as human health. How cells feel "heat" and further adapt their metabolism and behaviors to the environmental changes is poorly understood. Scientists at the Children's Nutrition Research Center in Houston, TX ,showed that a type of antioxidant protein, called AtGRXS17, helps protect plants from high temperature. These findings provide unique insights into the biological function of these proteins.
5. Deletion of the ghrelin receptor "turns up the heat" to increase energy expenditure. Energy balance is determined by energy intake and energy expenditures. Non-shivering thermogenesis in brown fat plays a pivotal role in energy balance in rodents, and its significance has recently been recognized in adult humans. Thermogenic function is severely impaired in obese individuals. Children's Nutrition Research Center researchers found that ghrelin receptor ablation elevates thermogenesis in brown fat, and produces higher core body temperatures. This is the first evidence that grhelin signaling has a role in thermogenesis, suggesting that suppression of the ghrelin receptor may represent an attractive novel option for combating obesity and insulin resistance by "turning up the heat".
6. Characterization of three mouse SIRT3 enzyme isoforms. SIRT3 is an important protein modification enzyme (deacetylase) that regulates the function of many metabolic enzymes. It was recently found that three variants can be expressed from the mouse SIRT3 gene. Scientists at the Children's Nutrition Research Center in Houston, TX, characterized the tissue distribution of these three SIRT3 variants and examined the cellular localization and maturation of the three protein isoforms of mouse SIRT3. Since the human SIRT3 gene also has these same transcript variants, our finding sheds light on the possible functional difference of human SIRT3 isoforms. This is important since our finding helps scientists to understand if any SIRT3 isoform plays a role in the regulation of aging or age-related diseases.