Aims: Protein kinase C epsilon (PKCε) has been shown to play a causative role in the generation of glucose intolerance and insulin resistance. Tissue-specific deletion of PKCε indicates multiple sites of action, highlighting the kinase as a potential target for the treatment of type 2 diabetes. CIDD-0150612 has been developed as a PKCε inhibitor from the Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor Y-27632. Here, we examined the effects of a CIDD-0150612 on insulin action in palmitate-treated HepG2 hepatocytes in vitro and on glucose homeostasis in fat-fed mice in vivo.
Methods: HepG2 cells were treated with palmitate and CIDD-0150612, and stimulated with insulin. Mice were fed a high-fat diet and treated with CIDD-0150612 for 2 weeks. Proteomic analysis was carried out on liver proteins by liquid chromatography with tandem mass spectrometry.
Results: CIDD-0150612 had no effect on insulin-stimulated insulin receptor or IRS-1 tyrosine phosphorylation in lipid-treated hepatocytes. However, the inhibitor promoted Akt phosphorylation in a highly insulin-dependent manner, and reversed the inhibition of insulin-stimulated Akt phosphorylation and glucose incorporation into glycogen by palmitate. Fat-fed mice treated with CIDD-0150612 had reduced body fat and body weight, but not lean mass, compared with vehicle-treated littermates. Mice treated acutely with CIDD-0150612 exhibited elevated fasting blood glucose. However, mice studied 24h after the last dose had lower fasting glucose and improved glucose tolerance with a lower insulin excursion. CIDD-0150612 treatment did not affect insulin signalling in liver or muscle in response to an insulin challenge. Proteomic analysis of liver from CIDD-0150612-treated fat-fed mice indicated altered levels of proteins of lipid metabolism and reduced levels of proteins involved in gluconeogenesis. Phosphoproteomic analysis identified 49 phosphosites that were affected by inhibitor treatment. However, filtering for proteins that also changed in abundance strongly implicated reduced phosphorylation of the transcription factor Foxk1, which modifies gluconeogenic gene expression. Phosphorylation site analysis suggested reduced activity of ERK2 and cyclin-dependent kinases, and altered compartmentalization of GSK-3 activity.
Conclusions: The PKCε inhibitor CIDD-0150612 had beneficial effects on insulin action in hepatocytes and on fat mass and glucose homeostasis in mice. Because certain effects, such as the reduction in fat mass, were not previously observed in studies of PKCε-deficient mice, off-target effects, most likely through residual ROCK inhibition, may be partly responsible. More specific inhibition of PKCε will require approaches independent of the highly conserved catalytic site of the kinase.