Requirement for Activation of the Serine-Threonine Kinase Akt (Protein Kinase B) in Insulin Stimulation of Protein Synthesis but Not of Glucose Transport

Abstract

A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr 308 and Ser 473 ) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by ϳ75% in CHO cells and ϳ30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.Akt is a pleckstrin homology (PH) domain-containing protein serine-threonine kinase whose kinase domain shares structural similarity with protein kinase C (PKC) isozymes and cyclic AMP-dependent protein kinase (PKA) (3). Thus, Akt has also been termed RAC-PK (protein kinase related to A and C kinases) (19) and PKB (protein kinase B) (7). Insulin and various other growth factors activate Akt, and this activation is inhibited by pharmacological blockers of phosphatidylinositol (PI) 3-kinase or by a dominant negative mutant of PI 3-kinase (4,14,25). Furthermore, Akt is activated by overexpression of a constitutively active mutant of PI 3-kinase in quiescent cells (11,23). These observations indicate that Akt is a downstream effector of PI 3-kinase.PI 3-kinase, which consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (5), is implicated in various metabolic effects of insulin (18, 59). A dominant negative mutant of PI 3-kinase as well as various pharmacological inhibitors, such as wortmannin and LY294002, have been used to block specific signaling pathways that include this enzyme (6,16,31,39,61). The metabolic actions of insulin that are sensitive to either a dominant negative mutant or pharmacological inhibitors of PI 3-kinase include stimulation of glucose uptake, antilipolysis, activation of fatty acid synthase ...

Differences in Signaling Properties of the Cytoplasmic Domains of the Insulin Receptor and Insulin-like Growth Factor Receptor in 3T3-L1 Adipocytes

“…On the one hand, glucose uptake was increased in 3T3-L1 cells expressing constitutively active or inducible PKB (44 -47). However, expression of a dominant negative PKB failed to block insulin-stimulated glucose uptake (48,49). We found no difference in levels of PKB activity in isoform-specific immunoprecipitates or in phosphorylation of serine 473 at early and late time points, in TIR and TIGR cells stimulated with NT-3 (Figs.…”

Impaired phosphorylation and insulin-stimulated translocation to the plasma membrane of protein kinase B/Akt in adipocytes from Type II diabetic subjects

“…Furthermore, microinjection of a PKB/Akt substrate peptide or an antibody to PKB/ Akt also inhibited the effect of insulin [19]. In contrast, another PKB/Akt dominant-negative mutant, where the two phosphorylation sites had been mutated, did not prevent insulin-stimulated GLUT-4 translocation although other inhibitory effects were found [20]. The reason for these inconsistent results is not clear but could relate to the cells used, the complex regulation of PKB/Akt activity and cell-specific expression of the PKB/Akt isoforms [11,21].…”

Increased insulin sensitivity in mice lacking p85β subunit of phosphoinositide 3-kinase

“…The lysates from liver (Left) or muscle (Right) were subjected to immunoprecipitation with antip85pan, anti-p85␣, or anti-p85␤ antibody followed by PI3K assay. production (9,10,30,31), although its role in glucose transport is still controversial (32)(33)(34). In the p85␤ Ϫ/Ϫ mice, AKT activity in muscle was significantly up-regulated, whereas AKT activity was unchanged in liver (Fig.…”