Location: Houston, Texas2012 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 characterized the differences between the long and the short SIRT3 protein isoforms in protein stability and uncovered skp2-mediated ubiquitination and subsequent proteasome degradation of the short but not the long isoform of SIRT3. We are exploring adenoivirus mediated SIRT3 over expression or shRNA knock down in the liver and the resulting effects on liver gene expression. In objective 2, we created Grx3/PICOT floxed mice and generated live-specific and inducible Grx3/PICOT KO mice. We have made Grx3 variants with Cys residue mutation or domain truncation and tested their functions. We have demonstrated that Grx3/PICOT can physically interact with p65 subunit and regulate NF-kB signaling pathway in response to oxidative stress. We have also determined the structure of GIY-YIG endonuclease domain of AtGRXS16 and the findings provide a nexus between Fe-S cluster biogenesis, redox regulation, and DNA metabolism. In objective 3, we showed that mice lacking GLP2r in POMC neurons enhance feeding behavior and gastric emptying, resulting in late-onset obesity, and display glucose intolerance and hepatic insulin resistance. Central GLP-2 suppresses food intake and gastric emptying through the MC4R signaling pathway, and suppresses hepatic glucose production and enhances insulin sensitivity in PI3K-dependent manner. GLP-2 facilitates PI3K-Akt-dependent FoxO1 nuclear exclusion, and modulates electric activities of POMC neurons in GLP-2R- and PI3K-dependent manners. For objectives 4, 5, and 6, we have completed functional characterization of the mice fed with regular chow, high fat diet (HFD) and high fructose corn syrup (HFCS). While the diet-induced obesity of HFCS-fed mice is not as pronounced as that of higher calorie HFD-fed mice, the HFCS feeding induced more severe insulin resistance in mice than did HFD-feeding. Furthermore, we have found that ablation of ghrelin receptor (GHS-R) improves HFCS-induced insulin resistance by reducing diet-induced adipose inflammation and liver steatosis. In objectives 7 and 8, progress in the current cycle has focused mainly on the mechanisms of the lipotropic effects of nuclear receptor LRH-1 activation by a natural product called DLPC (dilauroyl phosphatidylcholine) and other signals. We have made 2 significant advances, the first of which is the demonstration that increased and decreased LRH-1 activity are associated with improved or impaired responses to a key liver stress pathway termed the endoplasmic reticulum stress response. We have also identified the coactivator SRC-2 as a significant mediator of the transcriptional output of LRH-1 activation. Preliminary results indicate that DLPC activation of LRH-1 target genes is lost in liver-specific SRC-2 knockout mice. For objective 9, we have demonstrated that a number of 5-HT (serotonin) reagents, namely fluoxetine, fenfluramine, and mCPP, can significantly inhibit binge eating in wildtype mice, but these effects were blocked in mice lacking 5-HT 2C receptors. In parallel, we have generated a new mouse model lacking ER alpha only in a subset of brain cells, namely SIM1 neurons. This mutation caused obesity in both female and male mice.
1. 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.
2. 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.
3. 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: pro-inflammatory M1 macrophages are associated with an obese and insulin-resistant state, while anti-inflammatory M2 macrophages are associated with a lean and insulin-sensitive state. Researchers at the Children's Nutrition Research Center in Houston, Texas, found that the deletion of the ghrelin receptor (GHS-R) decreases pro-inflammatory 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.
4. 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.
5. 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.
6. 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.
7. 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, down-regulation of PU.1 in obese fat cells could be a new strategy to combat obesity-associated diseases.