Insulin Induces Specific Interaction between Insulin Receptor and Protein Kinase C  in Primary Cultured Skeletal Muscle

Abstract

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.

Differential Effects of Tumor Necrosis Factor-α on Protein Kinase C Isoforms α and δ Mediate Inhibition of Insulin Receptor Signaling

“…This association, upstream in insulin signal transduction, is consistent with the suggested role for PKC␦ in regulating the function of IR itself (25,26). Our results, showing that an important component of the ability of TNF-␣ to interfere with IR signaling involves disturbance of the insulin-induced physical association between PKC␦ and IR/IRS-1, further strengthen this idea.…”

“…In preliminary studies, we found that cultured mouse skeletal muscle cells express PKC isoforms ␣, ␤2, ␦, ε, , and , in agreement with other studies on mammalian skeletal muscle in vivo and in culture (15,38 -40). In a recent study on rat skeletal muscle in primary culture, we showed that insulin induces a rapid and specific physical association between IR and PKC␦ and that this physical linkage is essential for the continuation of the IR signaling cascade (26). We therefore reasoned that interference of PKC interactions with upstream elements might be involved in TNF-␣ effects on IR signaling.…”

“…The various PKC isoforms are both activated by insulin and can also modulate insulin signaling by regulating the kinase activity of IR. Indeed, PKC␦ was found to upregulate IR tyrosine phosphorylation in rat skeletal muscle cells (26), whereas PKCs ␣, ␦, and were found to inhibit this phosphorylation in HEK293 cells (27). Because various PKC isoforms have been found to interact with both IR and IRS, it is still not clear whether specific PKC isoforms regulate IR directly or through IRS-1.…”

Specific protein kinase C isoforms as transducers and modulators of insulin signaling

“…Moreover, overexpression of WTPKCδ in the primary skeletal muscle cells increased GLUT4 translocation and glucose uptake in the absence of insulin stimulation. In another study, it was reported that insulin induces PKCδ to associate directly with IR and that this isoform plays an essential role in insulin-induced tyrosine phosphorylation and internalization of IR [13]. In all of these and subsequent studies on skeletal muscle cells, interaction of IR occurred exclusively with PKCδ and not with any other PKC isoform (α, βII, ε or ς).…”

“…Activation of these elements may then lead to stimulation of additional enzymes, among which are certain members of the protein kinase C (PKC) family of serine-threonine kinases. Recent studies implicate specific PKC isoforms in the insulin-signaling cascade [2][3][4][5][6][7][8][9][10][11][12][13][14]. Insulin activates PKCs α, βII, δ and ζ in several cell types including cell lines of skeletal muscle [11,12,15].…”