Several reports indicate that protein kinase C (PKC) plays a role in insulin-induced glucose transport in certain cells. The precise effects of insulin on specific PKC isoforms are as yet unknown. Utilizing primary cultures of rat skeletal muscle, we investigated the possibility that insulin may influence the activation state of PKC isoenzymes by inducing their translocation and tyrosine phosphorylation. This, in turn, may mediate insulin effects on glucose transport. We identified and determined the glucose transporters and PKC isoforms affected by insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA). Insulin and TPA each caused an increase in glucose uptake. Insulin translocated GLUT3 and GLUT4 without affecting GLUT1. In contrast, TPA translocated GLUT1 and GLUT3 without affecting GLUT4. Insulin translocated and tyrosine phosphorylated and activated PKC-beta2 and -zeta; these effects were blocked by phosphatidylinositol 3-kinase (PI3K) inhibitors. TPA translocated and activated PKC-alpha, -beta2, and -delta; these effects were not noticeably affected by PI3K inhibitors. Furthermore, wortmannin significantly inhibited both insulin and TPA effects on GLUT translocation and glucose uptake. Finally, insulin-induced glucose transport was blocked by the specific PKC-beta2 inhibitor LY379196. These results indicate that specific PKC isoenzymes, when tyrosine-phosphorylated, are implicated in insulin-induced glucose transport in primary cultures of skeletal muscle.

Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKC in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKC to associate specifically with the GLUT4 compartments and that PKC together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKC and GLUT4 recycled independently of one another. To further establish the importance of PKC in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKC (DNPKC) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKC was associated with a marked increase in the activity of this isoform. The overexpressed, active PKC coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKC caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKC induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKC disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKC regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.

Insulin activates certain protein kinase C (PKC) isoforms that are involved in insulin-induced glucose transport. In this study, we investigated the possibility that activation of PKCdelta by insulin participates in the mediation of insulin effects on glucose transport in skeletal muscle. Studies were performed on primary cultures of rat skeletal myotubes. The role of PKCdelta in insulin-induced glucose uptake was evaluated both by selective pharmacological blockade and by over-expression of wild-type and point-mutated inactive PKCdelta isoforms in skeletal myotubes. We found that insulin induces tyrosine phosphorylation and translocation of PKCdelta to the plasma membrane and increases the activity of this isoform. Insulin-induced effects on translocation and phosphorylation of PKCdelta were blocked by a low concentration of rottlerin, whereas the effects of insulin on other PKC isoforms were not. This selective blockade of PKCdelta by rottlerin also inhibited insulin-induced translocation of glucose transporter 4 (GLUT4), but not glucose transporter 3 (GLUT3), and significantly reduced the stimulation of glucose uptake by insulin. When overexpressed in skeletal muscle, PKCdelta and PKCdelta were both active. Overexpression of PKCdelta induced the translocation of GLUT4 to the plasma membrane and increased basal glucose uptake to levels attained by insulin. Moreover, insulin did not increase glucose uptake further in cells overexpressing PKCdelta. Overexpression of PKCdelta did not affect basal glucose uptake or GLUT4 location. Stimulation of glucose uptake by insulin in cells overexpressing PKCdelta was similar to that in untransfected cells. Transfection of skeletal myotubes with dominant negative mutant PKCdelta did not alter basal glucose uptake but blocked insulin-induced GLUT4 translocation and glucose transport. These results demonstrate that insulin activates PKCdelta and that activated PKCdelta is a major signaling molecule in insulin-induced glucose transport.

Certain protein kinase C (PKC) isoforms, in particular PKCs beta II, delta, and zeta, are activated by insulin stimulation. In primary cultures of skeletal muscle, PKCs beta II and zeta, but not PKC delta, are activated via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. The purpose of this study was to investigate the possibility that PKC delta may be activated upstream of PI3K by direct interaction with insulin receptor (IR). Experiments were done on primary cultures of newborn rat skeletal muscle, age 5--6 days in vitro. The time course of insulin-induced activation of PKC delta closely paralleled that of IR. Insulin stimulation caused a selective coprecipitation of PKC delta with IR, and these IR immunoprecipitates from insulin-stimulated cells displayed a striking induction of PKC activity due specifically to PKC delta. To examine the involvement of PKC delta in the IR signaling cascade, we used recombinant adenovirus constructs of wild-type (W.T.) or dominant negative (D.N.) PKC delta. Overexpression of W.T.PKC delta induced PKC delta activity and coassociation of PKC delta and IR without addition of insulin. Overexpression of D.N.PKC delta abrogated insulin- induced coassociation of PKC delta and IR. Insulin-induced tyrosine phosphorylation of IR was greatly attenuated in cells overexpressing W.T.PKC delta, whereas in myotubes overexpressing D.N.PKC delta, tyrosine phosphorylation occurred without addition of insulin and was sustained longer than that in control myotubes. In control myotubes IR displayed a low level of serine phosphorylation, which was increased by insulin stimulation. In cells overexpressing W.T.PKC delta, serine phosphorylation was strikingly high under basal conditions and did not increase after insulin stimulation. In contrast, in cells overexpressing D.N.PKC delta, the level of serine phosphorylation was lower than that in nonoverexpressing cells and did not change notably after addition of insulin. Overexpression of W.T.PKC delta caused IR to localize mainly in the internal membrane fractions, and blockade of PKC delta abrogated insulin-induced IR internalization. We conclude that PKC delta is involved in regulation of IR activity and routing, and this regulation may be important in subsequent steps in the IR signaling cascade.

Tumor necrosis factor-␣ (TNF-␣) is a multifunctional cytokine that interferes with insulin signaling, but the molecular mechanisms of this effect are unclear. Because certain protein kinase C (PKC) isoforms are activated by insulin, we examined the role of PKC in TNF-␣ inhibition of insulin signaling in primary cultures of mouse skeletal muscle. TNF-␣, given 5 min before insulin, inhibited insulin-induced tyrosine phosphorylation of insulin receptor (IR), IR substrate (IRS)-1, insulin-induced association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase (PI3-K), and insulin-induced glucose uptake. Insulin and TNF-␣ each caused tyrosine phosphorylation and activation of PKCs ␦ and ␣, but when TNF-␣ preceded insulin, the effects were less than that produced by each substance alone.