Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Sep 18;115(38):E8996-E9005.
doi: 10.1073/pnas.1804379115. Epub 2018 Sep 4.

PKCε contributes to lipid-induced insulin resistance through cross talk with p70S6K and through previously unknown regulators of insulin signaling

Brandon M Gassaway  1   2 , Max C Petersen  1   3   4 , Yulia V Surovtseva  5 , Karl W Barber  1   2 , Joshua B Sheetz  1 , Hans R Aerni  1   2 , Jane S Merkel  5 , Varman T Samuel  3   6 , Gerald I Shulman  1   3   4 , Jesse Rinehart  7   2
Affiliations

PKCε contributes to lipid-induced insulin resistance through cross talk with p70S6K and through previously unknown regulators of insulin signaling

Brandon M Gassaway et al. Proc Natl Acad Sci U S A. .

Abstract

Insulin resistance drives the development of type 2 diabetes (T2D). In liver, diacylglycerol (DAG) is a key mediator of lipid-induced insulin resistance. DAG activates protein kinase C ε (PKCε), which phosphorylates and inhibits the insulin receptor. In rats, a 3-day high-fat diet produces hepatic insulin resistance through this mechanism, and knockdown of hepatic PKCε protects against high-fat diet-induced hepatic insulin resistance. Here, we employed a systems-level approach to uncover additional signaling pathways involved in high-fat diet-induced hepatic insulin resistance. We used quantitative phosphoproteomics to map global in vivo changes in hepatic protein phosphorylation in chow-fed, high-fat-fed, and high-fat-fed with PKCε knockdown rats to distinguish the impact of lipid- and PKCε-induced protein phosphorylation. This was followed by a functional siRNA-based screen to determine which dynamically regulated phosphoproteins may be involved in canonical insulin signaling. Direct PKCε substrates were identified by motif analysis of phosphoproteomics data and validated using a large-scale in vitro kinase assay. These substrates included the p70S6K substrates RPS6 and IRS1, which suggested cross talk between PKCε and p70S6K in high-fat diet-induced hepatic insulin resistance. These results identify an expanded set of proteins through which PKCε may drive high-fat diet-induced hepatic insulin resistance that may direct new therapeutic approaches for T2D.

Keywords: PKCε; cross talk; insulin resistance; phosphoproteomics; systems biology.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Quantitative phosphoproteomic analysis of acute hepatic insulin resistance and rescue by PKCε knockdown. Rats were fed either a control, chow diet or a HFD and treated with ASO that was either scrambled (control ASO) or targeted to PKCε (PKCε ASO). (A) HFD resulted in increased liver diacylglycerol independent of PKCε ASO. (B) PKCε translocation was quantified as a marker of PKCε activity [membrane-to-cytosol (m/c) ratio]; from immunoblot in SI Appendix, Fig. S1E. (C) HFD feeding increased basal plasma insulin, which was rescued by PKCε ASO. (D) Phosphoproteomics workflow, indicating three experimental groups and two pairwise comparisons. Phospho-enriched samples were separated into 32 fractions by ERLIC to reduce sample complexity and increase proteomic coverage; three technical replicates of each fraction were analyzed by LC-MS/MS using a 120 method (three replicates of 32 fractions = 96 120-min runs). (E) Effect of HFD and PKCε knockdown on the phosphoproteome. Phosphopeptide ratios obtained from two technical replicates of each comparison were averaged and log2 transformed, and peak intensity ratios were plotted against each other. The y axis represents the effect of the PKCε ASO on the phosphoproteome, whereas the x axis represents the effect of the HFD on the phosphoproteome; the lines represent twofold change in the phosphopeptide ratio. The twofold cutoff lines designate 2D enrichment categories of phosphopeptides that are designated by roman numerals. Error bars represent mean ± SEM (A–C).
Fig. 2.
Fig. 2.
siRNA screen uncovers previously unknown activators and inhibitors of insulin signaling. A total of 125 phosphoproteins was targeted with siRNA and stimulated with 1 or 10 nM insulin. Cells were fixed and stained with a phosphospecific antibody against AKT S473 phosphorylation; staining was quantified by single-cell image analysis as described in SI Appendix, Fig. S2. (A) Heatmap of siRNA screen with 1 nM insulin stimulation (three replicates). Heatmap intensity presented as the percentage of AKT S473 phosphorylation signal relative to the RISC-free siRNA control set at 100%. (B) Heatmap of siRNA screen with 10 nM insulin stimulation (three replicates). Heatmap intensities reflect the relative percent effect in AKT S473 phosphorylation of each individual siRNA, compared with the RISC-free control siRNA set as 0% effect and the INSR control siRNA set as 100% effect. (C) Representative images from the 1 nM insulin stimulation (green, FITC-AKT pS473; blue, DAPI). RISC-free and INSR control siRNAs are shown with Grb2 and Neo1 siRNAs, showing increase in AKT pS473 staining with gene knockdown. (D) Representative images from the 10 nM insulin stimulation siRNA screen (green, FITC-AKT pS473; blue, DAPI). (E) Model of the modulation of canonical insulin signaling by the HFD and PKCε. PKCε may influence insulin signaling through phosphoproteins modulated by the PKCε ASO alone (categories II and VIII) or by the PKCε ASO and HFD (categories III and VII). Insulin signaling may also be influenced by proteins whose phosphorylation is up- or down-regulated by the HFD alone (categories IV and VI); however, these proteins are unlikely to play a role in the rescue of insulin sensitivity by PKCε knockdown. CHD7 and DGKD are represented twice [and designated by an asterisk (*)] as we observed multiple phosphorylation events on these proteins that showed different fold-change values upon different treatments. Phosphoproteins that were shown by siRNA screen to activate or inhibit AKT S473 phosphorylation are shown in blue and yellow, respectively.
Fig. 3.
Fig. 3.
Motif analysis and mass spectrometry-based kinase assays identify previously unknown PKCε substrates. (A) Motif analysis of phosphopeptides from category II revealed a PKC-like motif (RxxS/T). Residues shaded in gray are fixed, while the size of the residue correlates with the enrichment of that residue at a position. Residues above the red lines are statistically significant (P < 0.05). (B) Workflow for kinase–substrate relationship determination using a substrate peptide display library. Substrates were selected based on phosphoproteomic data and PKC motif analysis, expressed as a C-terminal fusion to GST in E. coli, purified, and cleaved from the GST. The peptide substrate display library was incubated with PKCε and digested with trypsin; peptide substrates that were phosphorylated were identified by LC-MS/MS followed by database searching. (C) PKCε substrates identified by substrate peptide display library. The amino acid sequence of each substrate peptide is shown with the site of in vivo phosphorylation at position 0 with 7 aa flanking. “On-target” phosphorylation (in green) indicates the site of in vivo and in vitro phosphorylation matched. “Off-target” phosphorylation (in red) indicates phosphorylation by PKCε at unintended sites. PKCε motif elements are shown in blue. References are included for known members of the insulin signaling pathway (INS Sig) and for known kinase–phosphosite relationships (Known).
Fig. 4.
Fig. 4.
PKCε can cross talk with p70S6K substrates. (A) Western blot of p70S6K1/p70S6K2 DKO cells with or without WT PKCε transfection, treated with or without 1 μM MEKi, followed by treatment with or without 1 μM PMA. Densitometry for RPS6 S235/236 phosphorylation (different exposures for −MEKi and +MEKi) (B), IRS1 S302 phosphorylation (C), or PRAS40 T246 phosphorylation (D). n = 3. Bands are normalized to β-actin. Vertical bars separate +MEKi and −MEKi. P values for two-way ANOVA with Tukey’s multiple-comparisons test are as indicated. Full Western blots with selected lanes indicated in SI Appendix, Fig. S6B. (E) Model of PKCε modulation of canonical insulin signaling. In addition to modulating the INSR itself and other known members of the insulin signaling pathway, PKCε may influence insulin signaling through phosphoproteins modulated by the PKCε ASO alone (categories II and VIII) or by the PKCε ASO and HFD (categories III and VII) that were shown by siRNA screen to activate (blue) or inhibit (yellow) AKT S473 phosphorylation; direct targets of PKCε are shown by the solid lines.
See this image and copyright information in PMC

Comment in

  • Role of PKCε in insulin resistance.
    Leong I. Leong I. Nat Rev Endocrinol. 2018 Nov;14(11):627. doi: 10.1038/s41574-018-0098-x. Nat Rev Endocrinol. 2018. PMID: 30242279 No abstract available.

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Perry RJ, Samuel VT, Petersen KF, Shulman GI. The role of hepatic lipids in hepatic insulin resistance and type 2 diabetes. Nature. 2014;510:84–91. - PMC - PubMed
    1. Petersen MC, Vatner DF, Shulman GI. Regulation of hepatic glucose metabolism in health and disease. Nat Rev Endocrinol. 2017;13:572–587. - PMC - PubMed
    1. Kumashiro N, et al. Cellular mechanism of insulin resistance in nonalcoholic fatty liver disease. Proc Natl Acad Sci USA. 2011;108:16381–16385. - PMC - PubMed
    1. Magkos F, et al. Intrahepatic diacylglycerol content is associated with hepatic insulin resistance in obese subjects. Gastroenterology. 2012;142:1444–1446.e2. - PMC - PubMed
    1. Ter Horst KW, et al. Hepatic diacylglycerol-associated protein kinase Cε translocation links hepatic steatosis to hepatic insulin resistance in humans. Cell Rep. 2017;19:1997–2004. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources