Distinct Signals Regulate AS160 Phosphorylation in Response to Insulin, AICAR, and Contraction in Mouse Skeletal Muscle

Insulin and contraction increase GLUT4 translocation in skeletal muscle via distinct signaling mechanisms. Akt substrate of 160 kDa (AS160) mediates insulin-stimulated GLUT4 translocation in L6 myotubes, presumably through activation of Akt. Using in vivo, in vitro, and in situ methods, insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR all increased AS160 phosphorylation in mouse skeletal muscle. Insulin-stimulated AS160 phosphorylation was fully blunted by wortmannin in vitro and in Akt2 knockout (KO) mice in vivo. In contrast, contraction-stimulated AS160 phosphorylation was only partially decreased by wortmannin and unaffected in Akt2 KO mice, suggesting additional regulatory mechanisms. To determine if AMPK mediates AS160 signaling, we used AMPK α2-inactive (α2i) transgenic mice. AICAR-stimulated AS160 phosphorylation was fully inhibited, whereas contraction-stimulated AS160 phosphorylation was partially reduced in the AMPK α2i transgenic mice. Combined AMPK α2 and Akt inhibition by wortmannin treatment of AMPK α2 transgenic mice did not fully ablate contraction-stimulated AS160 phosphorylation. Maximal insulin, together with either AICAR or contraction, increased AS160 phosphorylation in an additive manner. In conclusion, AS160 may be a point of convergence linking insulin, contraction, and AICAR signaling. While Akt and AMPK α2 activities are essential for AS160 phosphorylation by insulin and AICAR, respectively, neither kinase is indispensable for the entire effects of contraction on AS160 phosphorylation.

[1]  N. Fujii,et al.  AMP-activated Protein Kinase α2 Activity Is Not Essential for Contraction- and Hyperosmolarity-induced Glucose Transport in Skeletal Muscle* , 2005, Journal of Biological Chemistry.

[2]  S. Kane,et al.  AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain. , 2005, The Biochemical journal.

[3]  S. Kane,et al.  Full intracellular retention of GLUT4 requires AS160 Rab GTPase activating protein. , 2005, Cell metabolism.

[4]  S. Kane,et al.  Calmodulin binds to the Rab GTPase activating protein required for insulin-stimulated GLUT4 translocation. , 2005, Biochemical and biophysical research communications.

[5]  F. Thong,et al.  Turning signals on and off: GLUT4 traffic in the insulin-signaling highway. , 2005, Physiology.

[6]  L. Goodyear,et al.  Contraction signaling to glucose transport in skeletal muscle. , 2005, Journal of applied physiology.

[7]  S. Kane,et al.  Insulin-stimulated phosphorylation of the Akt substrate AS160 is impaired in skeletal muscle of type 2 diabetic subjects. , 2005, Diabetes.

[8]  P. Geiger,et al.  Contraction- and hypoxia-stimulated glucose transport is mediated by a Ca2+-dependent mechanism in slow-twitch rat soleus muscle. , 2005, American journal of physiology. Endocrinology and metabolism.

[9]  J. Tavaré,et al.  Role of protein kinase B in insulin-regulated glucose uptake. , 2005, Biochemical Society transactions.

[10]  M. Sajan,et al.  Atypical protein kinase C in insulin action and insulin resistance. , 2005, Biochemical Society transactions.

[11]  M. Birnbaum,et al.  Isoform-specific Regulation of Insulin-dependent Glucose Uptake by Akt/Protein Kinase B* , 2003, Journal of Biological Chemistry.

[12]  M. Sajan,et al.  Activation of protein kinase C-zeta by insulin and phosphatidylinositol-3,4,5-(PO4)3 is defective in muscle in type 2 diabetes and impaired glucose tolerance: amelioration by rosiglitazone and exercise. , 2003, Diabetes.

[13]  M. Peggie,et al.  14‐3‐3s regulate fructose‐2,6‐bisphosphate levels by binding to PKB‐phosphorylated cardiac fructose‐2,6‐bisphosphate kinase/phosphatase , 2003, The EMBO journal.

[14]  D. Hardie,et al.  Management of cellular energy by the AMP‐activated protein kinase system , 2003, FEBS letters.

[15]  Michael B. Yaffe,et al.  Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs , 2003, Nucleic Acids Res..

[16]  Q. Boese,et al.  Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[17]  John M Asara,et al.  Insulin-stimulated Phosphorylation of a Rab GTPase-activating Protein Regulates GLUT4 Translocation* , 2003, The Journal of Biological Chemistry.

[18]  S. Kane,et al.  A Method to Identify Serine Kinase Substrates , 2002, The Journal of Biological Chemistry.

[19]  L. Goodyear,et al.  Contraction Regulation of Akt in Rat Skeletal Muscle* , 2002, The Journal of Biological Chemistry.

[20]  M. Birnbaum,et al.  Akt1/PKBα Is Required for Normal Growth but Dispensable for Maintenance of Glucose Homeostasis in Mice* , 2001, The Journal of Biological Chemistry.

[21]  E. Horton,et al.  Acute exercise induces GLUT4 translocation in skeletal muscle of normal human subjects and subjects with type 2 diabetes. , 1999, Diabetes.

[22]  B. Kemp,et al.  Contraction-induced Changes in Acetyl-CoA Carboxylase and 5′-AMP-activated Kinase in Skeletal Muscle* , 1997, The Journal of Biological Chemistry.

[23]  D. Hardie,et al.  Identification of Raf‐1 Ser621 kinase activity from NIH 3T3 cells as AMP‐activated protein kinase , 1997, FEBS letters.

[24]  Zhou Songyang,et al.  Determination of the Specific Substrate Sequence Motifs of Protein Kinase C Isozymes* , 1997, The Journal of Biological Chemistry.

[25]  B. Kemp,et al.  Isoform-specific Purification and Substrate Specificity of the 5′-AMP-activated Protein Kinase* , 1996, The Journal of Biological Chemistry.

[26]  J. Holloszy,et al.  Wortmannin inhibits insulin‐stimulated but not contraction‐stimulated glucose transport activity in skeletal muscle , 1995, FEBS letters.

[27]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[28]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[29]  G. Lienhard,et al.  Increased phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle in response to insulin or contractile activity. , 2005, Diabetes.

[30]  L. Goodyear,et al.  Exercise, glucose transport, and insulin sensitivity. , 1998, Annual review of medicine.