Depalmitoylation regulates hepatic glucose metabolism

项目总结/摘要 每年有数十万美国人被诊断患有2型糖尿病，这增加了他们患糖尿病的风险。 患上心血管疾病和慢性肾病。而肝脏中的胰岛素信号通常 通过抑制新葡萄糖的合成和诱导葡萄糖储存来调节血糖水平， 肝脏胰岛素抵抗可加重高血糖症。胰岛素抵抗与胰岛素抵抗相关。 脂肪酸代谢脂肪酸可以通过一种可逆的过程直接修饰蛋白质， 棕榈酰化去棕榈酰化酰基蛋白硫酯酶（APTs），如APT1和APT2，可以去除这些酶。 翻译后修饰虽然已经显示了参与葡萄糖稳态的多种蛋白质 为了被棕榈酰化，脱棕榈酰酶在代谢中的作用仍在研究中。初步数据 提示APT1肝基因敲除（APT1LKO）雌性小鼠具有胰岛素抵抗，而APT2LKO雌性小鼠 小鼠表现出空腹血糖水平升高。然而，APT1和APT2的冗余在 葡萄糖代谢途径尚不清楚。本提案的目的是阐明机制， 在葡萄糖稳态的背景下，APT1和APT2的功能冗余的后果。整体 假设是APT1和APT2的去棕榈酰化调节肝葡萄糖代谢。目标1 该建议将使用体外邻近标记来确定APT1和APT2的底物冗余。这 目的是产生APT-生物素连接酶融合构建体以标记靠近APT1和APT2的蛋白质。质量 生物素化蛋白的光谱分析将有助于鉴定共有的APT-蛋白相互作用。目标2 本研究旨在探讨小鼠肝脏葡萄糖代谢中APT1和APT2的功能冗余。 将测试具有肝脏APT1和APT2双重缺失的普通饲料和高脂肪喂养的小鼠的葡萄糖、胰岛素， 和丙酮酸耐受性。还将在单肝和双肝KO小鼠中评价肝胰岛素信号传导。的 这项研究的长期目标是在发展中涉及可逆的脂质修饰， 代谢性疾病 在奖学金期间，申请人将发展成为独立调查员的重要技能， 代谢，强调细胞和分子生物学技术。这项工作的发起人克雷博士 Semenkovich在研究脂肪酸和葡萄糖代谢之间的关系方面有着丰富的经验， 机构环境提供了支持，协作专家在肝脏生理学和分子 生物学华盛顿大学医学院在帮助医生科学家建立 成功的职业生涯。拟议的培训计划将有助于申请人过渡到成为 独立的医生科学家，利用研究发现治疗慢性代谢性疾病的新靶点 疾病

PROJECT SUMMARY/ABSTRACT Hundreds of thousands of Americans are diagnosed with type 2 diabetes every year, increasing their risk of developing cardiovascular disease and chronic kidney disease. While insulin signaling in the liver typically modulates blood glucose levels by suppressing the synthesis of new glucose and inducing glucose storage, hepatic insulin resistance can exacerbate hyperglycemia. Insulin resistance is associated with dysregulated fatty acid metabolism. Fatty acids can directly modify proteins through a reversible process known as palmitoylation. Depalmitoylating acyl protein thioesterases (APTs), such as APT1 and APT2, can remove these posttranslational modifications. Although multiple proteins involved in glucose homeostasis have been shown to be palmitoylated, the role of depalmitoylases in metabolism is still under investigation. Preliminary data suggest that APT1 liver knockout (APT1LKO) female mice have insulin resistance, while APT2LKO female mice demonstrate increased fasting plasma glucose levels. However, the redundancy of APT1 and APT2 in the glucose metabolic pathway is unclear. The objective of this proposal is to elucidate the mechanisms and consequences of functional redundancy of APT1 and APT2 in the context of glucose homeostasis. The overall hypothesis is that depalmitoylation by both APT1 and APT2 regulates hepatic glucose metabolism. Aim 1 of this proposal will determine the substrate redundancy of APT1 and APT2 using in vitro proximity labeling. This aim will generate APT-biotin ligase fusion constructs to label proteins proximal to APT1 and APT2. Mass spectrometry of biotinylated proteins will facilitate identification of shared APT-protein interactions. Aim 2 of this proposal will investigate the functional redundancy of APT1 and APT2 in murine hepatic glucose metabolism. Chow- and high fat-fed mice with dual deletion of hepatic APT1 and APT2 will be tested for glucose, insulin, and pyruvate tolerance. Hepatic insulin signaling will also be evaluated in single and double liver-KO mice. The long-term goal of the proposed research is to implicate reversible lipid modifications in the development of metabolic disease. During the fellowship, the applicant will develop important skills for becoming an independent investigator of metabolism, emphasizing cellular and molecular biology techniques. The sponsor of this work, Dr. Clay Semenkovich, has vast experience studying the relationship between fatty acid and glucose metabolism, and the institutional environment provides supportive, collaborative experts in liver physiology and molecular biology. Washington University School of Medicine has a long history of helping physician-scientists build successful careers. The proposed training plan will facilitate the applicant’s transition into becoming an independent physician-scientist, using research to discover novel targets for treating chronic metabolic diseases.