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Effects of a protein kinase C epsilon inhibitor on insulin signalling in lipid-treated HepG2 hepatocytes and glucose tolerance in fat-fed mice

ABSTRACT: Protein kinase C epsilon (PKCe) has been shown to play a causative role in the generation of glucose intolerance and insulin resistance. Tissue-specific deletion of PKC 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 PKCe 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. HepG2 cells were treated with palmitate and CIDD-0150612, and stimulated with insulin. Insulin signalling was examined by immunoblotting and glucose incorporation into glycogen was measured using glucose tracer. Mice were fed a high-fat diet for 8 weeks and treated with CIDD-0150612 in the final 2 weeks, prior to glucose tolerance tests, insulin challenge and tissue harvest. Proteomic and phosphoproteomic analysis was carried out on liver proteins by liquid chromatography with tandem mass spectrometry. 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 reduced skeletal muscle triglyceride content, but 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 a reduction in gluconeogenic gene expression and decreased phosphorylation of the transcription factor Foxk1. The PKCe 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 PKCe-deficient mice, off-target effects, most likely through residual ROCK1 inhibition, may be partly responsible.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Liver

SUBMITTER: Lewin Small

LAB HEAD: Carsten Schmitz-Peiffer

PROVIDER: PXD054895 | Pride | 2025-03-11

REPOSITORIES: Pride

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			Action	DRS
	20240603_EP_Liver612_Phospho_CSP_31_241.raw	Raw
	20240603_EP_Liver612_Phospho_CSP_33.raw	Raw
	20240605_EP_Liver612_Phospho_CSP_10_255.raw	Raw
	20240605_EP_Liver612_Phospho_CSP_11_261.raw	Raw
	20240605_EP_Liver612_Phospho_CSP_12_241.raw	Raw

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Effects of a protein kinase C epsilon inhibitor on insulin signalling in lipid-treated HepG2 hepatocytes and glucose tolerance in fat-fed mice.

Agoncillo Miguel L ML Gao Zhongmin Z De Kraker Harmannus C HC McHardy Stanton F SF Messing Robert O RO Small Lewin L Schmitz-Peiffer Carsten C

European journal of pharmacology 20250305

<h4>Aims</h4>Protein kinase C epsilon (PKCε) plays a causative role in the development of glucose intolerance, and is a potential target for the treatment of type 2 diabetes. Here, we examined the effects of the PKCε inhibitor CIDD-0150612 (CP612) on insulin action in palmitate-treated HepG2 hepatocytes in vitro and on glucose homeostasis in fat-fed mice in vivo.<h4>Methods</h4>HepG2 cells were treated with palmitate and CP612 and stimulated with insulin. Insulin signalling was examined by immun ...[more]

PMID: 40054721

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Project description:Obesity-induced insulin resistance of the liver is characterised by increased gluconeogenesis, which contributes to elevated blood glucose levels in individuals with type 2 diabetes. Research into how fatty acids induce insulin resistance has commonly focused on the induction of insulin resistance. We hypothesise that by shifting focus to the reversal of an insulin resistant phenotype, novel insights can be made into the mechanisms by which insulin resistance can be overcome. Using global gene and lipid expression profiling, we aimed to identify biological pathways altered in parallel with restoration of palmitate-induced deregulation of glucose production using metformin and sodium salicylate. FAO hepatoma cells were treated with palmitate (0.075mM, 48h) with or without metformin (0.25mM) and sodium salicylate (2mM) in the final 24h of palmitate treatment, and effects on glucose production were determined. Microarray followed by gene set enrichment analysis was performed to investigate pathway regulation. A lipidomic analysis (HPLC-MS/MS) and measurement of secreted bile acids and cholesterol were performed. Reversal of palmitate-induced impairment of glucose production by metformin and sodium salicylate was characterised by down-regulated expression of metabolic pathways regulating acetyl-CoA to cholesterol and bile acid biosynthesis. Total levels of intracellular and secreted cholesterol and bile acids were not different between impaired and restored glucose production. Total intracellular levels of diacylgycerol, triacylglycerol and cholesterol esters increased with palmitate (impaired glucose production), however, these were not further altered with metformin and sodium salicylate (restored glucose production). Six individual lipid species containing 18:0 and 18:1 side-chains were reduced by metformin and sodium salicylate. Widespread lipid metabolism changes induced by the reversal of palmitate-induced deregulation of glucose production with metformin and sodium salicylate were identified. While cholesterol and bile acid levels remained unchanged, the flux through these pathways may in part explain these findings. The identification of lipid species containing 18:0 and 18:1 side chains being regulated alongside changes to glucose production may indicate potential mediators of glucose production and insulin resistance. Three-condition experiment, Vehicle, Palmitate (PA) and Palmitate (PA) + Metformin (Met) + Sodium Sailcylate (NaS) with biological replicates: 8 Vehicle, 20 PA and 20 PA+Met+NaS , independently grown and harvested. One replicate per array.