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

描述（由申请人提供）：肥胖是由过量摄入脂肪和碳水化合物引起的，是胰岛素抵抗和2型糖尿病的主要原因。然而，过量的蛋白质消耗也被认为是胰岛素抵抗的潜在因素，通过升高循环氨基酸（AA）水平起作用。这个研究项目的目标是 (i)鉴定负责介导该应答的AA，（ii）确定增加的细胞内Ca 2+（[Ca 2 +]i）在调节AA效应中的作用，以及（iii）阐明[Ca 2 +]i通过哺乳动物雷帕霉素靶蛋白（mTOR）复合物1控制该应答的潜在分子机制。已知AA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。我们和其他人最近表明，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复合物1及其下游效应子S6激酶1（S6 K1）介导的。重要的是，我们还表明AA的作用是通过3类PI 3 K或人Vps 34介导的，而不是通过经典的I类PI 3 K信号转导途径。此外，我们发现hVps 34与mTOR共免疫沉淀，表明该复合物对AA诱导的S6 K1活化至关重要。我们最近的发现暗示AA诱导的[Ca 2 +]i增加，作为导致mTOR复合物1/通过hVps信号传导增加的触发事件34。我们的假设是AA过载通过一种不同形式的mTOR复合物1触发hVps 34激活，导致下游S6 K1超活化和胰岛素抵抗。我们在L 6肌管中验证这一假设的研究计划是（i）使用免疫沉淀、质谱、克隆、siRNA消融、免疫荧光和FRET来鉴定、分析和定位构成hVps 34-mTOR复合物1的蛋白伴侣; (ii)确定哪种AA负责引起[Ca 2 +]i的升高，并鉴定[Ca 2 +]i的来源，以及（iii）阐明AA和Ca 2+影响hVps 34-mTOR信号传导复合物活性的机制。与营养过剩相关的主要健康风险是2型糖尿病和心血管疾病的发作。此外，最近的流行病学研究表明，肥胖是癌症的一个主要危险因素。这些病理学是由肥胖流行病在世界范围内的扩张所驱动的，这是由卡路里摄入量的急剧增加以及食物的可获得性和低成本所推动的。由于这一建议旨在确定负责过量营养诱导的胰岛素抵抗的特定分子，因此它将为开发新的药理学疗法提供潜力。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直接靶向外周组织胰岛素抵抗的新治疗策略。公共卫生相关性：胰岛素抵抗和2型糖尿病正在成为非常阴险的健康问题，因为世界范围内的肥胖流行。这项研究将增加我们对暴饮暴食，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新的和更有效的药物来治疗胰岛素抵抗和2型糖尿病。

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.