Depalmitoylation regulates hepatic glucose metabolism

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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.