Insulin-Stimulated Phosphorylation of the Akt Substrate AS160 Is Impaired in Skeletal Muscle of Type 2 Diabetic Subjects

Abstract

AS160 is a newly described substrate for the protein kinase Akt that links insulin signaling and GLUT4 trafficking. In this study, we determined the expression of and in vivo insulin action on AS160 in human skeletal muscle. In addition, we compared the effect of physiological hyperinsulinemia on AS160 phosphorylation in 10 lean؊to؊moderately obese type 2 diabetic and 9 healthy subjects. Insulin infusion increased the phosphorylation of several proteins reacting with a phosphoAkt substrate antibody. We focused on AS160, as this Akt substrate has been linked to glucose transport. A 160-kDa phosphorylated protein was identified as AS160 by immunoblot analysis with an AS160-specific antibody. Physiological hyperinsulinemia increased AS160 phosphorylation 2.9-fold in skeletal muscle of control subjects (P < 0.001). Insulin-stimulated AS160 phosphorylation was reduced 39% (P < 0.05) in type 2 diabetic patients. AS160 protein expression was similar in type 2 diabetic and control subjects. Impaired AS160 phosphorylation was related to aberrant Akt signaling; insulin action on Akt Ser 473 phosphorylation was not significantly reduced in type 2 diabetic compared with control subjects, whereas Thr 308 phosphorylation was impaired 51% (P < 0.05). In conclusion, physiological hyperinsulinemia increases AS160 phosphorylation in human skeletal muscle. Moreover, defects in insulin action on AS160 may impair GLUT4 trafficking in type 2 diabetes. Diabetes 54:1692-1697, 2005 P eripheral insulin resistance is a major clinical feature of type 2 diabetes. Insulin resistance in skeletal muscle from type 2 diabetic patients is associated with impaired signal transduction at the level of insulin receptor substrate 1 (IRS-1) and phosphatidylinositol (PI) 3-kinase (1-4) and glucose transport (1,2,5). Akt is a downstream effector of PI 3-kinase that is directly linked to the regulation of glucose transport (6). Although defects in IRS-1/PI 3-kinase and glucose transport have been observed in several cohorts of type 2 diabetic subjects, impairments at the level of the Ser/Thr kinase Akt have been less apparent (rev. in 7). The role of Akt in skeletal muscle insulin resistance has been a challenge to resolve due to the presence of multiple isoforms that offer compensatory regulation.Recently, a novel 160-kDa substrate of Akt has been identified in 3T3L1 adipocytes as AS160, a protein containing a Rab GTPase-activating protein (GAP) domain (8). Phosphorylation of AS160 is required for the insulininduced translocation of GLUT4 to the plasma membrane in 3T3L1 adipocytes (9). The expression of a dominant inhibitory mutant AS160 markedly reduces GLUT4 exocytosis, without altering endocytosis (10). We hypothesized that insulin action on AS160, the most proximal step identified thus far in the insulin-signaling cascade to glucose transport, is impaired in skeletal muscle from type 2 diabetic patients. Here we report that physiological hyperinsulinemia increases AS160 phosphorylation in human skeletal muscle. Moreover AS160 phosphorylation is impai...

Partial rescue of in vivo insulin signalling in skeletal muscle by impaired insulin clearance in heterozygous carriers of a mutation in the insulin receptor gene

“…Thus, blunted activation of PI3K has been observed in several insulin-resistant states [14][15][16]27]. Reduced Thr308 phosphorylation of Akt has been reported in obese non-diabetic subjects [28] and in nonobese patients with type 2 diabetes [29], but contrasts with findings in skeletal muscle of obese patients with type 2 diabetes and cultured muscle cells established from such patients [10,15,17]. The lower Thr308 phosphorylation of Akt may cause lower glucose transport in Arg1174Gln carriers, but was not responsible for the impaired insulin activation of glycogen synthase, because insulin-mediated inhibition of GSK-3 and subsequent dephosphorylation of glycogen synthase at sites 3a+3b was normal.…”

Augmenting muscle diacylglycerol and triacylglycerol content by blocking fatty acid oxidation does not impede insulin sensitivity

“…Thus, it has been shown that Akt and Akt substrate of 160 kDa (AS160) are important downstream substrates involved in AMPK-mediated glucose uptake (29), and AMPK activation could thereby overcome fat-induced insulin resistance. However, in our study not only Akt phosphorylation was increased after etomoxir treatment of primary human myotubes and mice, but also the upstream activator of Akt, IRS1 serine 307 phosphorylation, was decreased in etomoxir-treated mice.…”

Are cultured human myotubes far from home?

“…Other components in the insulin-signaling cascade, such as activation of Akt substrate of 160 kDa (AS160), are also impaired in muscles from type 2 diabetic subjects (Karlsson, et al, 2005). Interestingly, AS160 phosphorylation was also reduced after 72 h fasting, and AS160 may be a better intracellular signaling molecule reflecting insulin-stimulated glucose uptake (Vendelbo, et al, 2012).…”