The Role of Arginine Transport on Pancreatic Alpha Cell Proliferation and Function

项目摘要 本文档中展示的培训策略将帮助我将职业生涯推向 糖尿病领域的独立研究科学家。我建议评估精氨酸转运在 胰岛细胞增殖和激素分泌的调节。糖尿病的疾病进展是 归因于胰腺β细胞不能充分分泌胰岛素以及联合抑制 胰腺α-细胞分泌的胰升糖素。抑制胰高血糖素信号可降低个体的高血糖 然而，胰升糖素信号的障碍会导致高血糖素血症，高氨基酸血症， 和α细胞增殖。4，5我们的实验室已经确定了一个肝α细胞轴，它通过以下途径促进α细胞的增殖 血液中氨基酸的积累。4我们已经确定了两种主要的氨基酸对α-细胞有贡献 增殖、谷氨酸4和精氨酸(未公布的数据)。然而，精氨酸的潜在机制 α细胞的特异性转运及其对α细胞增殖和分泌的作用还没有很好的定义。 阳离子氨基酸转运蛋白SLC7A2在小鼠和人胰腺α-细胞中高表达。 因此，我们假设高氨基酸血症是由高血糖素信号中断引起的。 有助于增加精氨酸的运输，促进α细胞的增殖和功能障碍。我们的 初步研究表明，SLC7A2是α细胞增殖和分泌胰高血糖素所必需的，即使在 用强膜去极剂挑战当前以阳离子为中心的精氨酸模型 刺激分泌(图2和图4)。使用一种新的α细胞特异性SLC7A2基因敲除小鼠模型，我们将揭开 精氨酸刺激α细胞增殖和胰升糖素分泌的分子机制。评估 α细胞中的SLC7A2是否是氨基酸依赖的α细胞增殖所必需的，SLC7A2基因敲除 永生化的小鼠αTc1-6细胞和可诱导的α细胞特异性SLC7a2基因敲除小鼠模型将用于 评估α-细胞增殖和质量的变化。此外，精氨酸诱导mTORC1的机制 激活的目标是确定精氨酸是否通过使CASTOR1-1失活来激活mTORC1- GATOR2途径(目标1)。此外，为了测试SLC7A2对精氨酸转运的调节能力 我们将把AIM 1中使用的工具与化学和遗传编码的钙离子传感器相结合，以 观察α细胞分泌胰高血糖素的变化。我们还将测量一氧化氮水平，并测试 一氧化氮对胰高血糖素分泌的影响 (目标2)。成功完成这项研究将加强我们目前对氨基酸诱导的α的理解。 细胞增殖和功能，以及扩大糖尿病治疗的可能性。我的 培训将通过执行这项研究来实现，利用提供的极好的资源和设施 范德比尔特和我的高素质导师丹妮尔·迪恩博士和大卫博士的全面指导 雅各布森。

Project Summary The training strategy demonstrated in this document will help me advance my career to be an independent research scientist in the field of diabetes. I propose to assess the role of arginine transport in the regulation of pancreatic islet cell proliferation and hormone secretion. Disease progression of diabetes is attributed to the inability of pancreatic β-cells to sufficiently secrete insulin and the combined failure to suppress pancreatic α-cell secretion of glucagon. Inhibition of glucagon signaling reduces hyperglycemia for individuals with diabetes.3 However, impairment of glucagon signaling leads to hyperglucagonemia, hyperaminoacidemia, and α-cell proliferation.4,5 Our lab has identified a liver-α-cell axis that contributes to α-cell proliferation through the accumulation of amino acids in the blood.4 We have identified two major amino acids that contribute to α-cell proliferation, glutamine4 and arginine (unpublished data). However, the mechanisms underlying arginine transport in the α-cell specifically and its contribution to α-cell proliferation and secretion are not well defined. The cationic amino acid transporter SLC7A2 is highly expressed in mouse and human pancreatic α-cells. Therefore, we hypothesize that hyperaminoacidemia that results from interrupted glucagon signaling contributes to increased arginine transport promoting α-cell proliferation and dysfunction. Our preliminary studies show that SLC7A2 is required for α-cell proliferation and glucagon secretion even when challenged with strong membrane depolarizing agents challenging current cation-centric models of arginine stimulated secretion (Figure 2 and 4). Using a new α-cell specific Slc7a2 knockout mouse model, we will unravel the molecular mechanisms that lead to arginine-stimulated α-cell proliferation and glucagon secretion. To assess whether SLC7A2 in α-cells is necessary for amino acid-dependent α-cell proliferation, Slc7a2 knockout in immortalized mouse αTC1-6 cells and an inducible α-cell specific Slc7a2 knockout mouse model will be used to assess changes in α-cell proliferation and mass. Additionally, the mechanism of arginine-induced mTORC1 activation will be targeted to determine if arginine activates mTORC1 through the inactivation of the CASTOR1- GATOR2 pathway (Aim 1). Furthermore, to test the ability for arginine transport via SLC7A2 to modulate glucagon secretion we will combine tools used in Aim 1 with chemical and genetically encoded Ca2+ sensors to observe changes in α-cell glucagon secretion. We will also measure nitric oxide levels, and test the affect of nitric oxide on glucagon secretion to understand the mechanism behind arginine-induced glucagon secretion (Aim 2). Successfully accomplishing this study will enhance our current understanding of amino acid-induced α- cell proliferation and function, as well as broaden the possibilities of therapeutic treatments for diabetes. My training will be achieved through the execution of this study utilizing the fantastic resources and facilities provided by Vanderbilt and thorough mentorship from my highly qualified mentors, Drs. Danielle Dean and David Jacobsen.