Insulin receptor Thr1160 phosphorylation mediates lipid-induced hepatic insulin resistance
Max C. Petersen, Anila K. Madiraju, Brandon M. Gassaway, Michael Marcel, Ali R. Nasiri, Gina Butrico, Melissa J. Marcucci, Dongyan Zhang, Abudukadier Abulizi, Xian-Man Zhang, William Philbrick, Stevan R. Hubbard, Michael J. Jurczak, Varman T. Samuel, Jesse Rinehart, Gerald I. Shulman
Max C. Petersen, Anila K. Madiraju, Brandon M. Gassaway, Michael Marcel, Ali R. Nasiri, Gina Butrico, Melissa J. Marcucci, Dongyan Zhang, Abudukadier Abulizi, Xian-Man Zhang, William Philbrick, Stevan R. Hubbard, Michael J. Jurczak, Varman T. Samuel, Jesse Rinehart, Gerald I. Shulman
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Research Article Hepatology Metabolism

Insulin receptor Thr1160 phosphorylation mediates lipid-induced hepatic insulin resistance

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ε (PKCε) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCε inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKCε substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr1160 phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCε in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr1150 (InsrT1150A mice) were protected from high fat diet–induced hepatic insulin resistance. InsrT1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr1160 phosphorylation and provide further mechanistic links between PKCε and INSR in mediating NAFLD-induced hepatic insulin resistance.

Authors

Max C. Petersen, Anila K. Madiraju, Brandon M. Gassaway, Michael Marcel, Ali R. Nasiri, Gina Butrico, Melissa J. Marcucci, Dongyan Zhang, Abudukadier Abulizi, Xian-Man Zhang, William Philbrick, Stevan R. Hubbard, Michael J. Jurczak, Varman T. Samuel, Jesse Rinehart, Gerald I. Shulman

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Figure 1

PKCε phosphorylates the INSR at Thr1160.

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PKCε phosphorylates the INSR at Thr1160. (A) IRK activity with and witho...
(A) IRK activity with and without equimolar PKCε preincubation in vitro. (B) Overlaid MS/MS spectra of the T1160-phosphorylated peptide DIYETPDYYRK (identified only in IRK + PKCε samples; unique peaks are shown in blue) and the Y1162-phosphorylated peptide DIYETDYPYRK (identified in samples with IRK alone; unique peaks are shown in red). An Andromeda score of 167.67, a localization probability of 68%, and a 4.3 Delta score between pT1160 assignment and pY1162 assignment supported assignment of phosphorylation to T1160. pT, phosphothreonine. pY, phosphotyrosine. Y and B series MS/MS fragment ions are defined with respect to primary amino acid sequence at upper right. (C) Mechanism by which Thr1160 phosphorylation inhibits IRK activity. The crystal structure of phosphorylated, activated IRK (19) is shown, with the activation loop in green, the catalytic loop in orange, and the rest of IRK in gray (surface representation). The 3 phosphotyrosines in the activation loop are shown as sticks, as is Thr1160 (carbon atoms in green, oxygen atoms in red, nitrogen atoms in blue, and phosphorus atoms in black). The side chain of (unphosphorylated) Thr1160 is hydrogen bonded (black dashed line) to the backbone carbonyl oxygen of phosphorylated Tyr1162 (pTyr1162). If Thr1160 were phosphorylated (hypothetical phosphate group shown semitransparently), the activation loop could not adopt this configuration because of steric clashes and electrostatic repulsion with pTyr1162. (D) In vitro kinase activity of recombinant WT or T1160A IRK at several PKCε concentrations. (E) In vitro kinase activity of recombinant WT or T1160E IRK. Data represent the mean ± SEM. (A) *P < 0.05, by unpaired, 2-tailed Student’s t test for 5 technical replicates per group; (D and E) ***P < 0.0005, by unpaired, 2-tailed Student’s t test for 3 to 4 technical replicates per group.