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推进职业生涯， 糖尿病领域的独立研究科学家。我建议评估精氨酸转运在 调节胰岛细胞增殖和激素分泌。糖尿病的疾病进展是 这归因于胰腺β细胞不能充分分泌胰岛素以及抑制胰岛素分泌的组合失败。 胰腺α细胞分泌胰高血糖素。抑制胰高血糖素信号传导可降低个体的高血糖症 然而，胰高血糖素信号传导的损伤导致高胰高血糖素血症，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脏-α-细胞轴，它通过以下途径促进α-细胞增殖： 氨基酸在血液中的积累。4我们已经确定了两种主要的氨基酸，有助于α-细胞 增殖、谷氨酰胺4和精氨酸（未发表的数据）。然而，精氨酸的潜在机制 α-细胞中的特异性转运及其对α-细胞增殖和分泌的贡献尚未明确。 阳离子氨基酸转运蛋白SLC 7A 2在小鼠和人胰腺α细胞中高度表达。 因此，我们假设胰高血糖素信号传导中断导致的高氨基酸血症 有助于增加精氨酸转运，促进α细胞增殖和功能障碍。我们 初步研究表明，即使当α-细胞增殖和胰高血糖素分泌时， 用强膜去极化剂挑战当前精氨酸的阳离子中心模型 刺激分泌（图2和4）。使用新的α细胞特异性Slc 7a 2敲除小鼠模型，我们将解开 导致精氨酸刺激α细胞增殖和胰高血糖素分泌的分子机制。评估 α细胞中的SLC 7A 2是否是氨基酸依赖性α细胞增殖所必需的， 永生化小鼠α TC 1 -6细胞和诱导型α细胞特异性Slc 7a 2敲除小鼠模型将用于 评估α细胞增殖和质量的变化。此外，马槟榔诱导mTORC 1的机制 激活将被靶向以确定精氨酸是否通过CASTOR 1的失活激活mTORC 1。 GATOR 2通路（Aim 1）。此外，为了测试精氨酸通过SLC 7A 2转运以调节细胞凋亡的能力， 胰高血糖素的分泌，我们将联合收割机的工具，用于目标1与化学和遗传编码的Ca 2+传感器， 观察α细胞胰高血糖素分泌的变化。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，以了解精氨酸诱导胰高血糖素分泌的机制 (Aim 2）。成功完成这项研究将提高我们目前对氨基酸诱导的α- 细胞增殖和功能，以及扩大糖尿病治疗的可能性。我 培训将通过利用所提供的出色资源和设施执行本研究来实现 范德比尔特和我的高素质的导师，博士Danielle院长和大卫彻底的指导 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the blood.4 We have identified two major amino acids that contribute to α-cell proliferation, glutamine4 and arginine (unpublished data).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.