Role of hVps34/mTOR Complex1 in Amino Acid-Induced Obesity and Insulin Resistance

hVps34/mTOR Complex1 在氨基酸诱导的肥胖和胰岛素抵抗中的作用

• 批准号: 7657501
• 负责人: GEORGE THOMAS
• 金额: $ 34.22万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-15 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7657501
• 关键词: Ablation; Adult; Affect; Affinity Chromatography; Amino Acids; Binding; Biochemical; Calmodulin; Carbohydrates; Cardiovascular Diseases; Cardiovascular system; Cells; Cessation of life; Chelating Agents; Cloning; Complex; Consumption; Core Facility; Developed Countries; Developing Countries; Development; Diet; Drops; Energy Intake; Epidemic; Epidemiologic Studies; Event; Fatty acid glycerol esters; Figs - dietary; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Food; Goals; Health; Human; Human Activities; Hyperphagia; Immunofluorescence Immunologic; Immunoprecipitation; In Vitro; Infusion procedures; Insulin; Insulin Receptor; Insulin Resistance; Intake; Laboratories; Lead; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methods; Molecular; Molecular Conformation; Muscle Fibers; Non-Insulin-Dependent Diabetes Mellitus; Nutrient; Obesity; Pathology; Pathway interactions; Peripheral; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Production; Proteins; Proteomics; Research; Research Design; Research Project Grants; Ribosomal Protein S6 Kinase; Risk; Risk Factors; Role; Serine/Threonine Phosphorylation; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Sirolimus; Small Interfering RNA; Smoke; Source; Techniques; Testing; Tissues; Vacuolar Protein Sorting; Western World; Work; cost; design; glucose uptake; human morbidity; inhibitor/antagonist; mTOR protein; novel; nutrition; public health relevance; research study; response; ribosomal protein S6 kinase 1; treatment effect; treatment strategy

DESCRIPTION (provided by applicant): Obesity is driven by excessive intake of fats and carbohydrates and is a major contributor to insulin resistance and type 2 diabetes. However, excess protein consumption is also becoming recognized as a potential contributor to insulin resistance, acting through elevated levels of circulating amino acids (AAs). The goal of this research project is to (i) identify the AA(s) responsible for mediating this response, (ii) to determine the role of increased intracellular Ca2+ ([Ca2+]i) in regulating the AA effects and (iii) to elucidate the underlying molecular mechanism by which [Ca2+]i controls this response through the mammalian target of rapamycin (mTOR) Complex1. It is known that AAs promote insulin resistance through serine/threonine phosphorylation of insulin substrate 1 and 2 (IRSI/2). We and others recently showed that, in cases of excess nutrients, this effect on IRSI phosphorylation is mediated in part through the activation of the mTOR Complex1 and its downstream effector, S6 kinase 1 (S6K1). Importantly, we have also shown that the effects of AAs are mediated through class 3 PI3K, or human Vps34, and not through the canonical class I PI3K signal transduction pathway. Furthermore, we found that hVps34 co-immunoprecipitates with mTOR, suggesting that this complex is critical for AA-induced S6K1 activation. Our recent findings implicate an AA- induced increase in [Ca2+]i, as the triggering event leading to increased mTOR Complex1/signaling via hVps34. Our hypothesis is that AA overload triggers hVps34 activation through a distinct form of mTOR Complex1 leading to downstream hyperactivation of S6K1 and insulin resistance. Our research plan in testing this hypothesis in L6 myotubes is to (i) identify, analyze, and localize the protein partners that make up the hVps34-mTOR Complex1 using immunoprecipitation, mass spectrometry, cloning, siRNA ablation, immunofluorescence and FRET; (ii) determine which AA(s) is responsible for eliciting the rise in [Ca2+]i, and identify the source of the [Ca2+]i and (iii) elucidate the mechanism by which AAs and Ca2+ affect the activity of the hVps34-mTOR signaling complex. The major health risks associated with nutrient overload are type 2 diabetes and the onset of cardiovascular disease. Moreover, recent epidemiological studies show that obesity is a major risk factor for cancer. These pathologies have been driven by the worldwide expansion of the obesity epidemic, fueled by a dramatic rise in caloric intake and the increased availability and low cost of food. As this proposal is designed to identify the specific molecules responsible for excess nutrient- induced insulin resistance, it will provide the potential for the development of novel pharmacological therapies. Furthermore, current treatments are largely focused on ameliorating insulin resistance by increasing insulin production or activity. In contrast, the studies described here could contribute to new treatment strategies that directly target insulin resistance in peripheral tissues. PUBLIC HEALTH RELEVANCE: Insulin resistance and type 2 diabetes are becoming exceedingly insidious health problems because of the worldwide obesity epidemic. This research will increase our understanding of the cellular mechanisms that link overeating, specifically excess protein consumption, to insulin resistance. This work could contribute to the development of new and more effective drugs to treat insulin resistance and type 2 diabetes.

说明(申请人提供)：肥胖是由过量摄入脂肪和碳水化合物引起的，是导致胰岛素抵抗和2型糖尿病的主要因素。然而，过量的蛋白质摄入也被认为是胰岛素抵抗的一个潜在因素，其作用机制是循环氨基酸(AAs)水平升高。这项研究项目的目标是 (I)确定负责调节这一反应的AA(S)，(Ii)确定细胞内升高的钙离子([Ca~(2+)]i)在调节AA效应中的作用，以及(Iii)阐明[Ca~(2+)]i通过哺乳动物靶标雷帕霉素(MTOR)Complex1控制这一反应的潜在分子机制。已知AAs通过胰岛素底物1和2的丝氨酸/苏氨酸磷酸化(IRSI/2)促进胰岛素抵抗。我们和其他人最近发现，在营养过剩的情况下，这种对IRSI磷酸化的影响部分是通过激活mTOR Complex1及其下游效应因子S6激酶1(S6K1)来介导的。重要的是，我们还证明了AAs的作用是通过3类PI3K或人类Vps34介导的，而不是通过典型的I类PI3K信号转导途径。此外，我们还发现hVps34与mTOR共沉淀，这表明该复合体在AA诱导的S6K1激活中起关键作用。我们最近的发现表明，AA诱导的[Ca~(2+)]i增加，作为触发事件导致mTOR Complex1/信号通过hVps34增加。我们的假设是，AA超载通过一种不同形式的mTOR Complex1触发hVps34的激活，导致S6K1下游的过度激活和胰岛素抵抗。我们在L6肌管中验证这一假说的研究计划是：(I)使用免疫沉淀、质谱学、克隆、siRNA消融、免疫荧光和FRET来鉴定、分析和定位组成hVps34-mTor Complex1的蛋白质伙伴； (Ii)确定哪种氨基酸(S)引起[Ca~(2+)]i升高，并确定[Ca~(2+)]i的来源；(3)阐明AA和Ca~(2+)影响hVps34-mTOR信号复合体活性的机制。与营养过载相关的主要健康风险是2型糖尿病和心血管疾病的发病。此外，最近的流行病学研究表明，肥胖是癌症的主要风险因素。这些病症是由肥胖流行病在全球范围内的扩张推动的，卡路里摄入量的急剧增加以及食物供应的增加和低成本的增加助长了肥胖症的蔓延。由于这项提议旨在确定导致过量营养诱导的胰岛素抵抗的特定分子，它将为开发新的药物疗法提供潜力。此外，目前的治疗主要集中在通过增加胰岛素的产生或活性来改善胰岛素抵抗。相反，这里描述的研究可能有助于制定新的治疗策略，直接针对周围组织的胰岛素抵抗。公共卫生相关性：由于全球肥胖症的流行，胰岛素抵抗和2型糖尿病正变得极其隐蔽的健康问题。这项研究将增加我们对过量饮食，特别是过量蛋白质摄入与胰岛素抵抗之间联系的细胞机制的理解。这项工作可能有助于开发治疗胰岛素抵抗和2型糖尿病的新的、更有效的药物。