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.

Activation of the STAT family of transcription factors is regulated by cytokines and growth factors. STAT tyrosine and serine phosphorylation are linked to the transcriptional activation and function of STAT. We have previously described a unique pathway inducing keratinocyte proliferation, which is mediated by insulin stimulation and depends on protein kinase C δ (PKCδ). In this study, we assessed STAT3 activation downstream of this pathway and characterized the role of PKCδ activation in STAT3 tyrosine and serine phosphorylation and keratinocyte proliferation. Following insulin stimulation, STAT3 interacted with PKCδ but not with any other PKC isoform expressed in skin. Activated forms of PKCδ and STAT3 were essential for insulin-induced PKCδ-STAT3 activation in keratinocyte proliferation. Abrogation of PKCδ activity inhibited insulin-induced STAT3 phosphorylation, PKCδ-STAT3 association and nuclear translocation. In addition, overexpression of STAT3 tyrosine mutant eliminated insulin-induced PKCδ activation and keratinocyte proliferation. Finally, overexpression of a STAT3 serine mutant abrogated insulin-induced STAT3 serine phosphorylation and STAT3-induced keratinocyte proliferation, whereas STAT3 tyrosine phosphorylation was induced and nuclear localization remained intact. This study indicates that PKCδ activation is a primary regulator of STAT3 serine phosphorylation and that PKCδ is essential in directing insulin-induced signaling in keratinocyte proliferation.

Incubation of rat hepatoma Fao cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins. This is followed by elevation in their P-Ser/Thr content, and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, abolished the increase in the P-Ser/Thr content of IRS-1, its dissociation from the IR, and the decrease in its P-Tyr content following 60 min of insulin treatment, indicating that the Ser kinases that negatively regulate IRS-1 function are downstream effectors of PI3K. PKC fulfills this criterion, being an insulin-activated downstream effector of PI3K. Overexpression of PKC in Fao cells, by infection of the cells with adenovirus-based PKC construct, had no effect on its own, but it accelerated the rate of insulin-stimulated dissociation of IR⅐IRS-1 complexes and the rate of Tyr dephosphorylation of IRS-1. The insulin-stimulated negative regulatory role of PKC was specific and could not be mimic by infecting Fao cells with adenoviral constructs encoding for PKC ␣, ␦, or . Because the reduction in P-Tyr content of IRS-1 was accompanied by a reduced association of IRS-1 with p85, the regulatory subunit of PI3K, it suggests that this negative regulatory process induced by PKC, has a built-in attenuation signal. Hence, insulin triggers a sequential cascade in which PI3K-mediated activation of PKC inhibits IRS-1 functions, reduces complex formation between IRS-1 and PI3K, and inhibits further activation of PKC itself. These findings implicate PKC as a key element in a multistep negative feedback control mechanism of IRS-1 functions.The insulin receptor mediates insulin action through the phosphorylation of substrate proteins on Tyr residues (1-3). The major substrates of the insulin receptor kinase are Shc (4) and the IRS 1 family of proteins, IRS-1 (5), IRS-2 (6), IRS-3 (7), and IRS-4 (8). IRS proteins contain a conserved PH (pleckstrin homology) domain (9, 10) located at the amino terminus, adjacent to a phosphotyrosine binding (PTB) domain. The PTB domain is present in a number of signaling molecules (11) and shares 75% sequence identity between IRS-1 and IRS-2 (12). This domain interacts with the NPXY motif of the juxtamembrane region of IR and promotes IR-IRS-1 interaction (13-15).The carboxyl-terminal region of IRS proteins is poorly conserved. It contains multiple tyrosine phosphorylation motifs that serve as docking sites for SH2 (Src homology-2) domaincontaining proteins like the p85␣ regulatory subunit of PI3K, Grb2, Nck, Crk, Fyn, and SHP-2, which mediate the metabolic and growth-promoting functions of insulin (1-3). IRS proteins contain more than 70 potential Ser/Thr phosphorylation sites for kinases like PKA (cAMP-dependent protein kinase), PKC, and MAPK (5, 6, 16). The phosphorylation of Ser/Thr residues of IRS proteins has a dual function, serving either for a positive or negative modulation of insulin signal transduction. Phosphorylation of Ser residues within the PTB domain of IRS-1 by ...

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.

Insulin and insulin-like growth factor-1 (IGF-1) are members of the family of the insulin family of growth factors, which activate similar cellular downstream pathways. In this study, we analyzed the effects of insulin and IGF-1 on the proliferation of murine skin keratinocytes in an attempt to determine whether these hormones trigger the same signaling pathways. Increasing doses of insulin and IGF-1 promote keratinocyte proliferation in an additive manner. We identified downstream pathways specifically involved in insulin signaling that are known to play a role in skin physiology; these include activation of the Na + /K + pump and protein kinase C (PKC). Insulin, but not IGF-1, stimulated Na + /K + pump activity. Furthermore, ouabain, a specific Na + /K + pump inhibitor, abolished the proliferative effect of insulin but not that of IGF-1. Insulin and IGF-1 also differentially regulated PKC activation. Insulin, but not IGF-1, specifically activated and translocated the PKC␦ isoform to the membrane fraction. There was no effect on PKC isoforms ␣, , , and , which are expressed in skin. PKC␦ overexpression increased keratinocyte proliferation and Na + /K + pump activity to a degree similar to that induced by insulin but had no affect on IGF-1-induced proliferation. Furthermore, a dominant negative form of PKC␦ abolished the effects of insulin on both proliferation and Na + /K + pump activity but did not abrogate induction of keratinocyte proliferation induced by other growth factors. These data indicate that though insulin or IGF-1 stimulation induce keratinocyte proliferation, only insulin action is specifically mediated via PKC␦ and involves activation of the Na + /K + pump. Diabetes 50:255-264, 2001