PKCe contributes to lipid-induced insulin resistance through cross talk with p70S6K and through previously unknown regulators of insulin signaling Brandon M. Gassawaya,b, Max C. Petersena,c,d, Yulia V. Surovtsevae, Karl W. Barbera,b, Joshua B. Sheetza, Hans R. Aernia,b,1, Jane S. Merkele, Varman T. Samuelc,f, Gerald I. Shulmana,c,d, and Jesse Rineharta,b,2 aDepartment of Cellular and Molecular Physiology, Yale University, New Haven, CT 06520; bSystems Biology Institute, Yale University, West Haven, CT 06516; cInternal Medicine, Yale University, New Haven, CT 06520; dHoward Hughes Medical Institute, Yale University, New Haven, CT 06519; eYale Center for Molecular Discovery, Yale University, West Haven, CT 06516; and fSection of Endocrinology, Veterans Affairs Medical Center, West Haven, CT 06516 Edited by Morris F. White, Howard Hughes Medical Institute and Children’s Hospital Boston, Boston, MA, and accepted by Editorial Board Member Barbara B. Kahn August 8, 2018 (received for review March 14, 2018) Insulin resistance drives the development of type 2 diabetes (T2D). For these reasons, we sought to describe the PKCe network in In liver, diacylglycerol (DAG) is a key mediator of lipid-induced HFD-induced hepatic insulin resistance. insulin resistance. DAG activates protein kinase C e (PKCe), which To date, PKCe substrates have been investigated in a limited phosphorylates and inhibits the insulin receptor. In rats, a 3-day number of settings, including the tumor-promoting effects of – – high-fat diet produces hepatic insulin resistance through this mecha- phorbol esters (9 13), in protection against cardiac ischemia – – nism, and knockdown of hepatic PKCe protects against high-fat diet- reperfusion injury (14 17), in regulating cell cell junctions (18, induced hepatic insulin resistance. Here, we employed a systems-level 19), in immune cell activation (20, 21), and in channel regulation approach to uncover additional signaling pathways involved in high- (22, 23). However, most of these studies have investigated indi- fat diet-induced hepatic insulin resistance. We used quantitative phos- vidual phosphosites or protein targets and have not evaluated the PKCe signaling network as a whole. In the two studies where phoproteomics to map global in vivo changes in hepatic protein phos- PKCe was investigated on a proteome level, PKCe was impli- phorylation in chow-fed, high-fat–fed, and high-fat–fedwithPKCe e cated in glucose metabolism through modulation of the glycolytic knockdown rats to distinguish theimpactoflipid-andPKC-induced enzymes pyruvate kinase, enolase, and lactate dehydrogenase protein phosphorylation. This was followed by a functional siRNA- (16) as well as in lipid metabolism through modulation of based screen to determine which dynamically regulated phosphopro- HTATIP2 (24) and the epoxyeicosatrienoic acid pathway (25). teins may be involved in canonical insulin signaling. Direct PKCe sub- Another study investigated the PKCe phosphoproteome using strates were identified by motif analysis of phosphoproteomics data 2D gel electrophoresis and Pro-Q Diamond phosphospecific and validated using a large-scale in vitro kinase assay. These substrates staining with a peptide activator of PKCe translocation in included the p70S6K substrates RPS6 and IRS1, which suggested cross e talk between PKC and p70S6K in high-fat diet-induced hepatic insulin Significance resistance. These results identify an expanded set of proteins through which PKCe may drive high-fat diet-induced hepatic insulin resistance We investigated the role of PKCe in driving lipid-induced he- that may direct new therapeutic approaches for T2D. patic insulin resistance beyond direct insulin receptor phos- phorylation/inhibition using an in vivo model of acute hepatic insulin resistance | phosphoproteomics | cross talk | systems biology | PKCe insulin resistance and phosphoproteomic analysis. Many of the phosphoproteins we uncovered have not been previously as- nsulin resistance, or a diminished tissue response to physiologic sociated with insulin signaling; to validate these connections, Iinsulin concentrations, is the primary driving force behind type we developed a functional siRNA-based screen, which con- 2 diabetes (T2D). In liver, insulin resistance impairs insulin- firmed a direct role in regulating insulin signaling. We validated mediated activation of hepatic glycogen synthesis and suppres- direct PKCe–substrate interactions using a recently developed sion of hepatic glucose production leading to increased fasting peptide substrate library, which confirmed the cross talk be- blood glucose (1, 2). Diacylglycerol (DAG) is a key mediator of tween PKCe and p70S6K that our proteomic analysis suggested lipid-induced hepatic insulin resistance; increased DAG content and which may result in aberrant negative feedback upon lipid- in human liver is strongly correlated with hepatic insulin re- induced PKCe activation. Taken together, we expand the poten- sistance (3–5). In rats, a 3-d high-fat diet (HFD) is sufficient to tial landscape of therapeutic targets for the treatment of insulin increase hepatic DAG, which activates protein kinase C e (PKCe) resistance and diabetes. e (6). PKC in turn impairs INSR tyrosine kinase activity, inducing Author contributions: B.M.G., M.C.P., Y.V.S., K.W.B., J.S.M., V.T.S., G.I.S., and J.R. designed hepatic insulin resistance (7). Rats treated with 2′-O-methoxyethyl research; B.M.G., M.C.P., Y.V.S., K.W.B., and J.B.S. performed research; K.W.B. and H.R.A. antisense oligonucleotides (ASOs) to knock down PKCe expres- contributed new reagents/analytic tools; B.M.G., M.C.P., Y.V.S., K.W.B., J.B.S., and J.R. sion were protected from HFD-induced hepatic insulin resistance analyzed data; and B.M.G. and J.R. wrote the paper. (7). Recently, we demonstrated that PKCe inhibits INSR tyrosine The authors declare no conflict of interest. This article is a PNAS Direct Submission. M.F.W. is a guest editor invited by the kinase activity directly through phosphorylation of INSR T1160 Editorial Board. (1150 in mouse, 1161 in rat); a T1150A mutation protected mice Published under the PNAS license. from HFD-induced hepatic insulin resistance (8). Data deposition: The mass spectrometry proteomics data have been deposited to the Although phosphorylation of INSR appears to be a key ProteomeXchange Consortium via the PRIDE partner repository, https://www.ebi.ac.uk/ mechanism for PKCe-induced hepatic insulin resistance, DAG- pride/archive/, (dataset indentifier PXD010209). activated PKCe might also act on additional substrates in an 1Present address: Department of Bioanalytics, Synthorx, Inc., La Jolla, CA 92037. extended network of proteins that may contribute to the insulin- 2To whom correspondence should be addressed. Email: [email protected]. resistant phenotype. Furthermore, identifying PKCe targets in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the HFD-fed liver could shed light not only on the molecular 1073/pnas.1804379115/-/DCSupplemental. basis of insulin resistance but also on PKCe signaling in general. Published online September 4, 2018. E8996–E9005 | PNAS | vol. 115 | no. 38 www.pnas.org/cgi/doi/10.1073/pnas.1804379115 Downloaded by guest on October 2, 2021 cardiomyocytes (17). While this study did identify several addi- Quantitative phosphoproteomic analysis was carried out with tional PKCe targets involved in glucose and lipid metabolism and an established dimethyl labeling strategy to enable direct pair- oxidative phosphorylation that may be relevant to insulin re- wise comparisons between experimental groups (28). We com- sistance, the difference in cell type (cardiac myocytes versus pared control ASO-treated, chow-fed rats to control ASO-treated, liver) and model (peptide activator versus DAG) may have im- HFD-fed rats to determine which phosphorylation events or portant implications for the relevant PKCe network and targets. pathways are altered in the setting of acute hepatic HFD-induced Furthermore, phosphosite localization information was not insulin resistance (Fig. 1D, comparison 1). We also compared obtained for most of these substrates, and candidate site-specific control ASO-treated, HFD-fed rats to PKCe ASO-treated, HFD- kinase–substrate relationships were not directly tested. fed rats to determine which phosphorylation events and signaling To overcome these limitations, we investigated the PKCe networks are affected by PKCe knockdown (Fig. 1D, comparison signaling network using quantitative phosphoproteomics in a 2). Following labeling, phosphopeptides were enriched with TiO2 physiologically relevant model of HFD-induced hepatic insulin and fractionated using electrostatic repulsion liquid chromatography resistance with knockdown of PKCe, evaluated the role of our (ERLIC) into 32 fractions to improve phosphoproteome coverage PKCe network in insulin signaling using an siRNA-based func- (Fig. 1D). In total, 11,224 sites of protein phosphorylation (phos- tional screen, and evaluated direct site-specific kinase–substrate phosites) were reproducibly quantified across all experimental relationships using a recently developed substrate peptide display conditions. In total, 779 phosphosites from 560 distinct proteins library, which were further validated in cells. Our phosphopro- showed a greater than twofold change (Fig. 1E). teomics and kinase–substrate validation suggest that PKCe-mediated HFD-induced hepatic insulin resistance may, in part, be the re- Mapping Phosphorylation