Requirement of Atypical Protein Kinase Cλ for Insulin Stimulation of Glucose Uptake but Not for Akt Activation in 3T3-L1 Adipocytes

Abstract

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKC and PKC) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKC in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKC (KD or ⌬NKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKC, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone-or hyperosmolarity-induced glucose uptake, were inhibited by KD or ⌬NKD in a dosedependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKC was ϳ50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKC mutant that lacks the pseudosubstrate domain (⌬PD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of ⌬PD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKC. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKC pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.Phosphoinositide (PI) 3-kinase, a lipid kinase composed of an SRC homology 2 (SH2) domain-containing regulatory subunit and a 110-kDa catalytic subunit, catalyzes phosphorylation of the D3 position of PIs (46,48). This enzyme was first identified complexed with SRC kinase and the middle T antigen of polyomavirus and was later found to associate with various tyrosine-phosphorylated proteins in response to stimulation of cells with growth factors or cytokines (46, 48). Activation of PI 3-kinase, either by targeting of the enzyme to the plasma membrane (27) or as a consequence of direct interaction between the SH2 domain of the regulatory subunit and phosphorylated tyrosine residues present within specific motifs (5), results in the triggering of various important biological actions. Thus, with the use of either a dominant negative protein that blocks the interaction between PI 3-kinase and tyrosine-phosphorylated proteins (21,33,41) or pharmacological inhibitors of the enzyme, such as wortmannin or LY294002 (...

Engagement of the insulin-sensitive pathway in the stimulation of glucose transport by α-lipoic acid in 3T3-L1 adipocytes

“…A recent report showed that vanadium compounds activated Akt1 and this activation was inhibited by wortmannin [54]. This is, however, inconsequential for glucose transport because the stimulation of glucose transport by vanadate is wortmannin-insensitive [55]. In contrast, both the stimulation of glucose transport and activation of Akt1 by R (+) a-lipoic acid in 3T3-L1 adipocytes are sensitive to inhibition of PI3K with wortmannin.…”

Metformin improves atypical protein kinase C activation by insulin and phosphatidylinositol-3,4,5-(PO4)3 in muscle of diabetic subjects

“…Increases in aPKC activity that occur with AMPK activation during AICAR and DNP treatment, and with PI 3-kinase activation during insulin treatment, appear to be required for the activation of GLUT4 translocation/glucose transport during actions of AICAR and DNP [15], as well as insulin [25][26][27][28][29][30]. However, we can only speculate on whether the increases observed in our study in muscle aPKC activity following metformin treatment were effective in stimulating glucose transport into muscles of metformin-treated diabetic subjects, either without or with concurrent acute insulin administration.…”

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

“…Schematic diagram of PKC isoforms expressed in insulin-responsive tissues and potential substrates in the insulin signaling cascade. Insulin resistance, ▲ glucose uptake, no effect on ISGT Cultured cells [12,14,159,164,166,167] …”