Protein kinase Cδ but not PKCα is involved in insulin-induced glucose metabolism in hepatocytes.

The liver is a major insulin-responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin-induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin-induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin-induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML-12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin-induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin-induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin-induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin-induced glucose uptake and glycogenesis in hepatocytes.

Insulin stimulation of PKCδ triggers its rapid degradation via the ubiquitin-proteasome pathway.

Insulin rapidly upregulates protein levels of PKCδ in classical insulin target tissues skeletal muscle and liver. Insulin induces both a rapid increase in de novo synthesis of PKCδ protein. In this study we examined the possibility that insulin may also inhibit degradation of PKCδ. Experiments were performed on L6 skeletal muscle myoblasts or myotubes in culture. Phorbol ester (PMA)- and insulin-induced degradation of PKCδ were abrogated by proteasome inhibition. Both PMA and insulin induced ubiquitination of PKCδ, but not of that PKCα or PKCε and increased proteasome activity within 5 min. We examined the role of tyrosine phosphorylation of PKCδ in targeting PKCδ for degradation by the ubiquitin-proteasome pathway. Transfection of cells with PKCδY(311)F, which is not phosphorylated, resulted in abolition of insulin-induced ubiquitination of PKCδ and increase in proteasome activity. We conclude that insulin induces degradation of PKCδ via the ubiquitin-proteasome system, and that this effect requires phosphorylation on specific tyrosine residues for targeting PKCδ for degradation by the ubiquitin-proteasome pathway. These studies provide additional evidence for unique effects of insulin on regulation of PKCδ protein levels.

Protein kinase Cdelta participates in insulin-induced activation of PKB via PDK1.

PKCdelta has been shown to be activated by insulin and to interact with insulin receptor and IRS. PKB(Akt) plays an important role in glucose transport and glycogen synthesis. In this study, we investigated the possibility that PKCdelta may be involved in insulin-induced activation of PKB. Studies were conducted on primary cultures of rat skeletal muscle. PKB was activated by insulin stimulation within 5min and reached a peak by 15-30min. Insulin also increased the physical association between PKCdelta with PKB and with PDK1. The insulin-induced PKCdelta-PKB association was PI3K dependent. PKB-PKCdelta association was accounted for by the involvement of PDK1. Overexpression of dominant negative PKCdelta abrogated insulin-induced association of PKCdelta with both PKB and PDK1. Blockade of PKCdelta also decreased insulin-induced Thr308 PKB phosphorylation and PKB translocation. Moreover, PKCdelta inhibition reduced insulin-induced GSK3 phosphorylation. The results indicate that insulin-activated PKCdelta interacts with PDK1 to regulate PKB.

Insulin rapidly upregulates protein kinase Cdelta gene expression in skeletal muscle.

Recent studies in our laboratories have shown that Protein Kinase C delta (PKCdelta) is essential for insulin-induced glucose transport in skeletal muscle, and that insulin rapidly stimulates PKCdelta activity skeletal muscle. The purpose of this study was to examine mechanisms of regulation of PKCdelta protein availability. Studies were done on several models of mammalian skeletal muscle and utilized whole cell lysates of differentiated myotubes. PKCdelta protein levels were determined by Western blotting techniques, and PKCdelta RNA levels were determined by Northern blotting, RT-PCR and Real-Time RT-PCR. Insulin stimulation increased PKCdelta protein levels in whole cell lysates. This effect was not due to an inhibition by insulin of the rate of PKCdelta protein degradation. Insulin also increased 35S-methionine incorporation into PKCdelta within 5-15 min. Pretreatment of cells with transcription or translation inhibitors abrogated the insulin-induced increase in PKCdelta protein levels. We also found that insulin rapidly increased the level of PKCdelta RNA, an effect abolished by inhibitors of transcription. The insulin-induced increase in PKCdelta expression was not reduced by inhibition of either PI3 Kinase or MAP kinase, indicating that these signaling mechanisms are not involved, consistent with insulin activation of PKCdelta. Studies on cells transfected with the PKCdelta promoter demonstrate that insulin activated the promoter within 5 min. This study indicates that the expression of PKCdelta may be regulated in a rapid manner during the course of insulin action in skeletal muscle and raise the possibility that PKCdelta may be an immediate early response gene activated by insulin.