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Abstract
The development of type 2 diabetes is reaching epidemic proportions and identifying ways to modulate insulin levels in order to maintain euglycaemia is important in understanding how to better treat this disease. It is now accepted that lipid oversupply can detrimentally affect insulin action and the lipid activated kinase, protein kinase C epsilon (PKCε), has been implicated in the development of insulin resistance and progression to type 2 diabetes. We have previously shown that global PKCε knockout (PKCε KO) mice are protected from high fat diet induced glucose intolerance, in part through reduced hepatic insulin clearance.
We have shown using wild type (WT) and PKCε KO mouse embryonic fibroblasts (MEFs) as a model that ablation of PKCε perturbs insulin uptake and this was associated with a reduction in insulin-stimulated insulin receptor (InsR) redistribution within the cell, by subcellular fractionation. This was associated with a differential localisation of the InsR in the basal state in PKCε KO MEFs, with a greater proportion of the IR localised to cellular lipid microdomains. Insulin-stimulated InsR tyrosine phosphorylation within the kinase domain at sites 1162/1163 was reduced, however this defect in InsR phosphorylation did not translate to defective downstream signalling, with insulin-stimulated PKCε KO MEFs having normal or enhanced Akt/PKB, Erk1/2 and IRS-1 phosphorylation. Potential mechanisms for these differences were investigated by examining key candidates such as expression of the InsR substrate, Ceacam1 and the docking protein Grb14, to deduce the effect upon InsR internalisation and signalling, with Ceacam1 expression found to be greatly reduced in PKCε KO MEFs. Insulin signalling was further investigated in primary hepatocytes and in vivo following stimulation of de novo insulin secretion to examine these findings in more physiological settings.
The role of PKCε in lipid-induced insulin resistance was also investigated as PKCε has been shown to affect lipid metabolism. The incorporation of the fatty acid palmitate into distinct lipid classes was examined, as alterations in cellular lipid composition could contribute to alterations in insulin action, however no major changes were observed. However, following palmitate treatment of MEFs, PKCε KO MEFs displayed a greater preservation of downstream insulin signalling compared to WT MEFs. The data is consistent with a role for PKCε in the generation of insulin resistance through the modulation of lipid metabolism, rather than merely acting downstream of specific lipid intermediates.