Location: Houston, Texas2010 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.
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.
3. Progress Report
SIRT3 deacetylase is an enzyme that removes a chemical (acetyl) group from proteins to regulate a target protein's function. We studied the expression of SIRT3 in mouse liver under various conditions and found that 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 have set up the culture of rat liver cells and begun to investigate the expression of SIRT3 in these cells under various treatments and conditions. To learn the physiological function of PICOT, a gene regulating oxidative balance, we analyzed its expression in mouse embryos and adult tissue, including liver of mice at different ages and various nutritional interventions (calorie restriction, diet-induced-obesity, and fasting). We checked PICOT expression in fat cells treated with hormones, free fatty acids, anti-diabetic drug, and different growth or differentiation conditions. We found: PICOT expression in liver was significantly increased in aged mice; PICOT expression in liver was significantly increased in mice fasted for 24 h, but not in 12-h fasted or re-fed mice; and PICOT expression was significantly up-regulated during fat cell differentiation. We produced crystal from purified plant PICOT functional domain proteins and determined the structure of this functional domain of PICOT. We have genetically generated a mouse line that lacks the GLP-2 receptor in brain neurons. We found that GLP-2 receptor activation is essential for maintaining blood glucose levels by modulating the brain neuron activities. Infusion of GLP-2 into the brain suppresses food intake and increases glucose utilization by enhancing the expression of appetite-suppressing peptides in the brain. We also found that GLP-2 receptor activation facilitates calcium channel activity with stimulated glucose uptake by brain neurons. Stable isotope tracer techniques were developed to quantify glucose synthesis under high levels of insulin (as in type 2 diabetes), and to assess gastric emptying in conscious mice. We found that deficiency of GLP-2 receptor in brain neurons increases gastric emptying, decreases insulin sensitivity, and has higher rates of glucose synthesis in the liver in type 2 diabetes. We initiated feeding studies of mice lacking the receptor for a stomach-secreted feeding regulating hormone ghrelin (GHS-R). On mice fed with high-fat diet that induced weight gain, we saw a lower body weight in GHS-R null mice compared to controls. A diet containing 60% fructose didn't eliminate obesity in mice, so we have switched to a regimen with drinking water containing 8% high fructose corn syrup, to mimic soft drinks. Since ghrelin is unstable, it is difficult to measure ghrelin levels experimentally. The specificity of the available assays for mouse obestatin, a ghrelin-related hormone, has been questionable. We have validated immune-assays for mouse ghrelin and obestatin. Our pilot study suggests that fasting increased ghrelin but decreased obestatin levels, so those hormones will be studied under fed/fast conditions. The ADODR monitors activities for the project by routine site visits.
1. 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.
2. 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 redox-sensitive 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.
3. 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.