Increased Phosphorylation of Akt Substrate of 160 kDa (AS160) in Rat Skeletal Muscle in Response to Insulin or Contractile Activity

Abstract

T he signaling pathways that mediate insulin's many actions remain incompletely understood, but the following sequence has been well characterized: insulin binds to its receptor, leading to receptor autophosphorylation and activation of receptor tyrosine kinase, which in turn results in tyrosine phosphorylation of endogenous substrates including insulin receptor substrate proteins. These docking proteins engage downstream signaling molecules such as phosphatidylinositol (PI) 3-kinase (1-3). PI 3-kinase catalyzes the phosphorylation of phosphatidylinositol 4,5-bisphosphate on the D3 position of inositol, and the resultant PI 3,4,5-trisphosphate binds and activates more distal signaling proteins, including phosphoinositide-dependent kinase-1 and Akt, a serine/threonine kinase. Akt has been implicated as a key signaling protein for several of insulin's actions, including activation of glycogen synthesis, protein synthesis, and GLUT4 translocation to the cell surface, thereby increasing glucose transport (1-5). Identification of the intermediate signaling steps linking Akt to insulin's diverse actions remains incomplete.Recent research using 3T3-L1 adipocytes demonstrated that insulin leads to the phosphorylation of multiple proteins that contain one or more consensus sequences for phosphorylation by Akt (6). Among these Akt substrates was a 160-kDa protein, named Akt substrate of 160 kDa (AS160), which contained six Akt consensus sequences that become phosphorylated in insulin-treated adipocytes. AS160 also includes a GTPase-activating domain for small G-proteins, known as Rabs, which participate in vesicular trafficking (7). A point mutation of two or more of the consensus phosphorylation sites for Akt resulted in a marked decline in insulin-stimulated GLUT4 redistribution to the cell surface. Thus, in 3T3-L1 adipocytes, phosphorylation of AS160 was strongly implicated as an intermediate step linking insulin's activation of Akt to increased glucose transport. More recently, Zeigerer et al. (8) demonstrated that AS160 is important for insulin's activation of GLUT4 vesicle exocytosis without altering insulin-mediated inhibition of GLUT4 internalization.Insulin also activates Akt in skeletal muscle (9,10), and AS160 is expressed by this tissue (6), which is a major target for insulin action. An important question is this: Does insulin, in skeletal muscle, lead to increased phosphorylation of AS160 and/or other proteins containing the Akt phosphomotif? Accordingly, our first aim was to AICAR, 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside; AS160, Akt substrate of 160 kDa; KHB, Krebs-Henseleit buffer; PAS, phospho-(Ser/Thr) Akt substrate; PI, phosphatidylinositol; TBST, Tris-buffered saline plus Tween.

Prior exercise increases phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle

“…The lack of an effect of in vivo exercise on Akt activation at IPEX is in agreement with most previous research on rats (27,42) and mice (45), although one study of rats (34) found greater Akt activation determined IPEX compared with sedentary values. Electrically stimulated contractions by rat skeletal muscle can induce Akt activation (6,(33)(34)(35), although enhanced Akt activation has not always been detected with in vitro contractions (5,26,37). The IPEX and sedentary groups in the current study had similar levels for phosphorylation of GSK3, an Akt substrate, consistent with the lack of an IPEX effect on Akt phosphorylation.…”

“…Compelling evidence indicates that phosphorylation of a recently identified protein, known as Akt substrate of 160 kDa (AS160), plays an important role in insulin-stimulated GLUT4 vesicle exocytosis in adipocytes (36,48), making AS160 the most distal insulin-signaling event that has been linked to GLUT4 translocation. In light of the distinct signaling pathways for each stimulus, the recognition that AS160 in skeletal muscle becomes phosphorylated in response to either insulin or in vitro contractile activity (6) raised the surprising possibility that this protein may be a point of convergence between the insulin and exercise/contraction pathways for increasing cell surface GLUT4 and glucose transport.…”

“…Although several studies have indicated that in vitro muscle contractions can increase Akt activation (6,(33)(34)(35), many studies that used in vivo exercise have not detected altered Akt phosphorylation or activity (27,42,45). However, there is considerable evidence that AMPactivated protein kinase (AMPK), which is increased during in vitro contractions or in vivo exercise, can also induce AS160 phosphorylation (6,22). Therefore, we evaluated glucose transport and phosphorylation of AMPK, Akt, and AS160 in muscles dissected out of rats immediately after exercise.…”

Akt mediates mitochondrial protection in cardiomyocytes through phosphorylation of mitochondrial hexokinase-II

“…This antibody recognizes the phosphorylated Akt consensus sequence and has been widely utilized to detect proteins phosphorylated by Akt. 30,31 PAS antibody staining revealed increases in HK-II phosphorylation in both the whole cell lysate and mitochondrial fraction following LIF treatment ( Figure 5b). These increases in HK-II phosphorylation were also shown to be blocked by PI3K (LY) or Akt (V) inhibition ( Figure 5b).…”

General aspects of muscle glucose uptake

“…When 3T3-L1 adipocytes and L6 GLUT4-myc myoblasts were transfected with a constitutively active mutant AS160 (incapable of being phosphorylated at these regulatory sites), a significantly reduced insulin-induced GLUT4 translocation was observed (Sano et al 2003, Thong et al 2005). In addition, previous studies have reported increased AS160 phosphorylation with in vitro contractions in rat epitrochlearis muscles (Bruss et al 2005). This suggests that AS160 operates as a common, downstream point of convergence between insulin-mediated and muscle contraction pathways Kramer et al 2006, using AMPK α2-inactive transgenic mice, observed that AICAR-stimulated AS160 phosphorylation was fully inhibited, whereas contraction-stimulated AS160 phosphorylation was partially reduced in the mice and the combined AMPK α2 and Akt inhibition by wortmannin treatment of AMPK α 2 transgenic mice, did not fully ablate contraction-stimulated AS160 phosphorylation.…”