Requirement of atypical protein kinase Cλ for insulin stimulation of glucose uptake but not for Akt activation in 3T3-L1 adipocytes

Phosphoinositide (PI) 3-kinase contributes to a,vide 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 zeta and PKC lambda) have been implicated as downstream effecters of PI 3-kinase, Endogenous or transfected PKC lambda 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 lambda (lambda KD or lambda Delta NKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKC lambda, 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 lambda KD or lambda Delta NKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKC lambda was similar to 50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKC lambda mutant that lacks the pseudosubstrate domain (lambda Delta PD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of lambda Delta 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 lambda. 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 lambda pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.