Nutritional and Hormonal Control of the Phosphorylation Regulation of GPAT1

描述（由申请人提供）：在细胞水平，肥胖的特征在于脂肪和非脂肪组织中过量的三酰甘油（TAG）积累。甘油-3-磷酸酰基转移酶1（GPAT 1）对于调节肝脏TAG合成和介导胰岛素抵抗的发展至关重要。来自肝细胞中不一致的GPAT 1 mRNA表达、GPAT 1蛋白水平和GPAT 1活性的越来越多的证据强烈地表明存在翻译后调节机制。此外，GPAT 1在体外被蛋白激酶CK 2和AMPK磷酸化，分别伴随着GPAT 1活性的增加和减少。我们假设GPAT 1在肝脏中以磷酸化特异性方式调节，并且这些磷酸化位点与GPATI在胰岛素抵抗发展中的作用生理相关。目前研究GPAT 1磷酸化调控的方法无法提供GPAT 1磷酸化位点特异性证据和相应的功能相关性。由于其在TAG合成中的关键作用，修饰的GPAT 1的功能对于基本理解肝脏中的TAG合成途径和胰岛素抵抗的发展是必要的。质谱法以非凡的灵敏度测量分子的内在性质（质量），使其成为位点特异性磷酸化作图的无与伦比的技术。因此，我们提出了一个跨学科的方法来开发一个强大的，分析方法，使用质谱的GPAT 1的位点特异性磷酸化映射，以评估营养和疾病介导的变化，在TAG合成的调节。该提案的具体目的是1）通过质谱法对磷酸化GPAT 1残基进行位点特异性定位，2）通过测量AMPK被激活或抑制时肝细胞中的GPAT 1活性和磷酸化状态来确定AMPK介导的GPAT 1磷酸化变化的功能相关性，以及3）鉴定由进食状态介导的内源性GPAT 1磷酸化的变化（禁食、禁食/再喂养）和疾病状态（肥胖和2型糖尿病）。我们预期AMPK位点的内源性GPAT 1磷酸化在肥胖状态下以及在禁食/高碳水化合物再喂养野生型小鼠时会降低。或者，AMPK位点的内源性GPAT 1磷酸化将在野生型小鼠中随着禁食而增加，在2型糖尿病小鼠中二甲双胍治疗后（当AMPK被激活时）。公共卫生相关性：血脂异常与许多健康状况有关，包括糖尿病和肥胖症。该研究旨在进一步表征甘油三酯合成的调节以及这种调节在肥胖和糖尿病状态下如何变化。

DESCRIPTION (provided by applicant): At the cellular level, obesity is characterized by excess triacylglycerol (TAG) accumulation in adipose and non-adipose tissues. Glycerol-3-phosphate acyltransferase 1 (GPAT1) is critical for regulating hepatic TAG synthesis and mediating the development of insulin resistance. The mounting evidence from the discordant GPAT1 mRNA expression, GPAT1 protein level, and GPAT1 activity in hepatocytes strongly suggests a post- translational regulation mechanism. Furthermore, GPAT1 is phosphorylated by protein kinase CK2 and AMPK in vitro concomitant with an increase and decrease in GPAT1 activity, respectively. We hypothesize that GPAT1 is regulated in the liver in a phosphorylation-specific manner, and that these phosphorylated sites are physiologically relevant to GPATI's role in the development of insulin resistance. Current methodologies to study the phosphorylation-regulation of GPAT1 are incapable of providing site-specific evidence of GPAT1 phosphorylation and the corresponding function relevance. Due to its critical role in TAG synthesis, the function of modified GPAT1 is requisite for a fundamental understanding of the TAG synthetic pathway in the liver and the development of insulin resistance. Mass spectrometry measures an intrinsic property of molecule (mass) with extraordinary sensitivity, making it an unparalleled technology for site- specific phosphorylation mapping. Therefore, we propose an interdisciplinary approach to develop a robust, analytical method using mass spectrometry for the site-specific phosphorylation mapping of GPAT1 to assess the nutrient- and disease-mediated changes in the regulation of TAG synthesis. The specific aims of the proposal are 1) site-specific mapping of phosphorylated GPAT1 residues by mass spectrometry, 2) determine the functional relevance of AMPK-mediated changes in GPAT1 phosphorylation by measuring GPAT1 activity and phosphorylation status in hepatocytes when AMPK is activated or inhibited, and 3) identify changes in phosphorylation of endogenous GPAT1 mediated by feeding status (fasting, fasting/re- feeding) and disease states (obesity and type 2 diabetes mellitus). We expect endogenous GPAT1 phosphorylation at AMPK sites will decrease in the obese state and with fasting/high-carbohydrate refeeding of wild-type mice. Alternatively, endogenous GPAT1 phosphorylation at AMPK sites will increase with fasting in wild-type mice and upon metformin treatment in type 2 diabetic mice (when AMPK is activated). PUBLIC HEALTH RELEVANCE: Dyslipidemia is linked to a number of health conditions, including diabetes and obesity. The research proposed aims to further characterize the regulation of triglyceride synthesis and how this regulation changes in the obese and diabetic states.