ALTERATIONS IN THE EXPRESSION AND CELLULAR-LOCALIZATION OF PROTEIN-KINASE-C ISOZYME-EPSILON AND ISOZYME-THETA ARE ASSOCIATED WITH INSULIN-RESISTANCE IN SKELETAL-MUSCLE OF THE HIGH-FAT-FED RAT

We have tested the hypothesis that changes in the levels and cellular location of protein kinase C (PKC) isozymes might be associated with t he development of insulin resistance in skeletal muscles from the high -fat-fed rat. Lipid measurements showed that triglyceride and diacylgl ycerol, an activator of PKC, were elevated four- and twofold, respecti vely. PKC activity assays indicated that the proportion of membrane-as sociated calcium-independent PKC was also increased. As determined by immunoblotting, total (particulate plus cytosolic) PKC alpha, epsilon, and zeta levels were not different between control and fat-fed rats. However, the ratio of particulate to cytosolic PKC epsilon in red musc les from fat-fed rats was increased nearly sixfold, suggesting chronic activation. In contrast, the amount of cytosolic PKC theta was downre gulated to 45% of control, while the ratio of particulate to cytosolic levels increased, suggesting a combination of chronic activation and downregulation. Interestingly, while insulin infusion in glucose-clamp ed rats increased the proportion of PKC theta in the particulate fract ion of red muscle, this was potentiated by fat-feeding, suggesting tha t the translocation is a consequence of altered lipid flux rather than a proximal event in insulin signaling. PKC epsilon and theta measurem ents from individual rats correlated with triglyceride content of red gastrocnemius muscle; they did not correlate with plasma glucose, whic h was not elevated in fat-fed rats, suggesting that they were not simp ly a consequence of hyperglycemia. Our results suggest that these spec ific alterations in PKC epsilon and PKC theta might contribute to the link between increased lipid availability and muscle insulin resistanc e previously described using high-fat-fed rats.