A Role for Protein Phosphatase 2A–Like Activity, but Not Atypical Protein Kinase Cζ, in the Inhibition of Protein Kinase B/Akt and Glycogen Synthesis by Palmitate

Abstract

We have shown previously that palmitate treatment of C2C12 skeletal muscle myotubes causes inhibition of the protein kinase B (PKB) pathway and hence reduces insulin-stimulated glycogen synthesis through the elevation of intracellular ceramide levels. Ceramide is known to activate both atypical protein kinase C (aPKC) and protein phosphatase (PP) 2A, and each of these effectors has been reported to inhibit PKB. In the present study, palmitate pretreatment was found to elevate PP2A-like activity in myotubes and to prevent its inhibition by insulin. Incubation with the phosphatase inhibitor okadaic acid before insulin stimulation protected against the effect of the fatty acid on PKB phosphorylation. Palmitate was unable to inhibit PKB activity and glycogen synthesis in cells overexpressing the activated PKB mutant (T308D,S473D)-PKB␣, which is unaffected by phosphatase. In contrast, PKB activity and glycogen synthesis were still inhibited by palmitate in cells overexpressing a membrane-targeted and, hence, activated PKB mutant that retains sensitivity to phosphatase. Although aPKC activity was also increased in palmitate-treated cells, overexpression of wild-type or kinase-dead aPKC did not alter the inhibitory effects of the lipid on either stimulation of PKB or glycogen synthesis by insulin. We conclude that palmitate disrupts insulin signaling in C2C12 myotubes by promoting PP2A-like activity and, therefore, the dephosphorylation of PKB, which in turn reduces the stimulation of glycogen synthesis.

The Par6α/aPKC complex regulates Akt1 activity by phosphorylating Thr34 in the PH-domain

“…The role of Akt1-T34 as a target of aPKC has been identified in L6 myotubes stimulated with ceramide (Powell et al, 2003), a sphingolipid enriched in skeletal muscle of insulin-resistant subjects (Adams et al, 2004). In C2C12 cells, ceramide also caused Akt1 down-regulation, but this effect was independent of an increased aPKC activity and relied on a protein phosphatase 2A-like activity dephosphorylating Akt1 (Cazzolli et al, 2001). By contrast, our results indicate that the Par6α/aPKC complex employs Akt1-T34 phosphorylation in C2C12 cells.…”

Nelfinavir-induced insulin resistance is associated with impaired plasma membrane recruitment of the PI 3-kinase effectors Akt/PKB and PKC-?

“…A defect in the transmission of the insulin signal between PI 3-kinase and its downstream effectors Akt/PKB and PKC-ζ was observed in several cellular models of insulin resistance. These include insulin resistance induced by high glucose [28], palmitate and ceramide [26,30], hyperosmolarity [29], actin cytoskeleton disruption [41] and oxidative stress [20]. In the case of palmitate and ceramide, it was proposed that increased PP2A activity underlies this signalling defect.…”

“…In the case of palmitate and ceramide, it was proposed that increased PP2A activity underlies this signalling defect. This proposition was based on either a demonstrated increase in PP2A expression and/or activity, or on the capacity of okadaic acid or calyculin A to reverse the signalling defect [26]. None of these seem to apply in nelfinavir-treated adipocytes (Fig.…”

“…PP2A is a family of Ser/Thr protein phosphatases that are the major phosphatases of Akt/PKB, p70S6 kinase, as well as ERK1/2 [26,27]. Hence, a potential cellular mechanism to explain the results described above is an enhanced rate of dephosphorylation of these kinases by PP2A.…”

“…Hence, a potential cellular mechanism to explain the results described above is an enhanced rate of dephosphorylation of these kinases by PP2A. Moreover, increased PP2A activity was recently implicated in several models of insulin resistance in which normal PI 3-kinase, but reduced Akt/PKB activation were observed [26,28,29,30]. Different complementary ways of assessing a potential role of PP2A in nelfinavir-induced insulin resistance were utilised.…”