Hepatic Acyl-Protein Thioesterases Prevent Insulin Resistance

Elevated fatty acids in the context of metabolic disease are associated with insulin resistance. Fatty acids covalently and reversibly modify proteins through a process known as palmitoylation. We hypothesized that depalmitoylation by acyl-protein thioesterases 1 and 2 (APT1 and APT2) in the liver regulates glucose metabolism. The hepatic roles of APT1 and APT2 are unknown. We deleted APT1 and/or APT2 from the livers of mice, followed by high-fat diet (HFD) feeding. We performed acyl resin-assisted capture (acyl-RAC) to isolate palmitoylated proteins from APT KO livers followed by mass spectrometry. APT1 primarily depalmitoylated proteins linked to mitochondrial function and amino acid metabolism, including glutamine and branched-chain amino acid metabolism. To identify direct substrates of APT1 and APT2, we determined the protein-protein proximity network of each APT by fusing each enzyme to the highly efficient biotin ligase, miniTurbo (mT). We expressed these APT-mT constructs in human hepatocellular carcinoma HepG2 cells, followed by purification of biotin-labeled proteins and analysis by mass spectrometry. The APT proximity networks demonstrated that both APT1 and APT2 interact with mitochondrial proteins. APT1 and APT2 were also proximal to several glutamine transporters including Slc1a5 (also known as ASCT2), the only glutamine transporter known to localize to mitochondria. Male mice with dual but not single deletion of APT1 and APT2 from the liver (DLKO mice) had impaired insulin sensitivity independent of p-Akt levels or expression of Pck1 and G6pc. Elevated overnight fasting glucose in DLKO mice was associated with increased glutamine-driven gluconeogenesis and decreased liver mass. These data suggest that APT1 and APT2 regulate hepatic glucose metabolism, insulin signaling, and amino acid flux in a functionally redundant manner. These novel findings are the first to illustrate the hepatic depalmitoylation substrates and protein-protein proximity networks of APT1 and APT2. Disclosure S.L.Speck: None. Q.Zhang: None. D.P.Bhatt: None. S.Adak: None. G.Dong: None. X.Wei: None. C.F.Semenkovich: Consultant; Alnylam Pharmaceuticals. Funding National Institutes of Health (T32GM007200, F30DK131830, R01HL157154, P30DK020579)