AS160 Regulates Insulin- and Contraction-stimulated Glucose Uptake in Mouse Skeletal Muscle

Abstract

Insulin and contraction are potent stimulators of GLUT4 translocation and increase skeletal muscle glucose uptake. We recently identified the Rab GTPase-activating protein (GAP) AS160 as a putative point of convergence linking distinct upstream signaling cascades induced by insulin and contraction in mouse skeletal muscle. Here, we studied the functional implications of these AS160 signaling events by using an in vivo electroporation technique to overexpress wild type and three AS160 mutants in mouse tibialis anterior muscles: 1) AS160 mutated to prevent phosphorylation on four regulatory phospho-Akt-substrate sites (4P); 2) AS160 mutated to abolish Rab GTPase activity (R/K); and 3) double mutant AS160 containing both 4P and R/K mutations (2M). One week following gene injection, protein expression for all AS160 isoforms was elevated over 7-fold. To determine the effects of AS160 on insulin- and contraction-stimulated glucose uptake in transfected muscles, we measured [3H]2-deoxyglucose uptake in vivo following intravenous glucose administration and in situ muscle contraction, respectively. Insulin-stimulated glucose uptake was significantly inhibited in muscles overexpressing 4P mutant AS160. However, this inhibition was completely prevented by concomitant disruption of AS160 Rab GAP activity. Transfection with 4P mutant AS160 also significantly impaired contraction-stimulated glucose uptake, as did overexpression of wild type AS160. In contrast, overexpressing mutant AS160 lacking Rab GAP activity resulted in increases in both sham and contraction-stimulated muscles. These data suggest that AS160 regulates both insulin- and contraction-stimulated glucose metabolism in mouse skeletal muscle in vivo and that the effects of mutant AS160 on the actions of insulin and contraction are not identical. Our findings directly implicate AS160 as a critical convergence factor for independent stimulators of skeletal muscle glucose uptake. Insulin and contraction are potent stimulators of GLUT4 translocation and increase skeletal muscle glucose uptake. We recently identified the Rab GTPase-activating protein (GAP) AS160 as a putative point of convergence linking distinct upstream signaling cascades induced by insulin and contraction in mouse skeletal muscle. Here, we studied the functional implications of these AS160 signaling events by using an in vivo electroporation technique to overexpress wild type and three AS160 mutants in mouse tibialis anterior muscles: 1) AS160 mutated to prevent phosphorylation on four regulatory phospho-Akt-substrate sites (4P); 2) AS160 mutated to abolish Rab GTPase activity (R/K); and 3) double mutant AS160 containing both 4P and R/K mutations (2M). One week following gene injection, protein expression for all AS160 isoforms was elevated over 7-fold. To determine the effects of AS160 on insulin- and contraction-stimulated glucose uptake in transfected muscles, we measured [3H]2-deoxyglucose uptake in vivo following intravenous glucose administration and in situ muscle contraction, respectively. Insulin-stimulated glucose uptake was significantly inhibited in muscles overexpressing 4P mutant AS160. However, this inhibition was completely prevented by concomitant disruption of AS160 Rab GAP activity. Transfection with 4P mutant AS160 also significantly impaired contraction-stimulated glucose uptake, as did overexpression of wild type AS160. In contrast, overexpressing mutant AS160 lacking Rab GAP activity resulted in increases in both sham and contraction-stimulated muscles. These data suggest that AS160 regulates both insulin- and contraction-stimulated glucose metabolism in mouse skeletal muscle in vivo and that the effects of mutant AS160 on the actions of insulin and contraction are not identical. Our findings directly implicate AS160 as a critical convergence factor for independent stimulators of skeletal muscle glucose uptake. Skeletal muscle insulin resistance is a salient feature of type 2 diabetes. In humans and other mammals, skeletal muscle normally accounts for ∼75% of whole body insulin-stimulated glucose transport (1DeFronzo R.A. Gunnarsson R. Bjorkman O. Olsson M. Wahren J. J. Clin. Investig. 1985; 76: 149-155Crossref PubMed Scopus (885) Google Scholar, 2Zierath J.R. Krook A. Wallberg-Henriksson H. Diabetologia. 2000; 43: 821-835Crossref PubMed Scopus (289) Google Scholar, 3Bjornholm M. Zierath J.R. Biochem. Soc. Trans. 2005; 33: 354-357Crossref PubMed Scopus (156) Google Scholar). Impaired ability of the muscle to respond to insulin is therefore disruptive to systemic glucose homeostasis. Skeletal muscle also possesses contractile properties that can effectively restore glucose control in an insulin-independent manner, and this element is preserved in individuals with type 2 diabetes (4Goodyear L.J. Kahn B.B. Annu. Rev. Med. 1998; 49: 235-261Crossref PubMed Scopus (800) Google Scholar, 5Kennedy J.W. Hirshman M.F. Gervino E.V. Ocel J.V. Forse R.A. Hoenig S.J. Aronson D. Goodyear L.J. Horton E.S. Diabetes. 1999; 48: 1192-1197Crossref PubMed Scopus (289) Google Scholar). Although the precise mechanisms remain elusive, it is clear that both insulin and contraction signals converge upon GLUT4 vesicles and promote their appearance at the cell membrane (6Koistinen H.A. Zierath J.R. Ann. Med. 2002; 34: 410-418Crossref PubMed Scopus (80) Google Scholar, 7Jessen N. Goodyear L.J. J. Appl. Physiol. 2005; 99: 330-337Crossref PubMed Scopus (228) Google Scholar). AS160 2The abbreviations used are: AS160, Akt substrate of 160 kDa; GAP, GTPase-activating protein; PAS, phospho-Akt substrate; WT, wild type; 4P, AS160 mutated at four PAS motifs; R/K, mutation of arginine to lysine in AS160 GAP domain; 2M, double mutant AS160 containing both 4P and R/K mutations; AMPK, AMP-activated protein kinase. 2The abbreviations used are: AS160, Akt substrate of 160 kDa; GAP, GTPase-activating protein; PAS, phospho-Akt substrate; WT, wild type; 4P, AS160 mutated at four PAS motifs; R/K, mutation of arginine to lysine in AS160 GAP domain; 2M, double mutant AS160 containing both 4P and R/K mutations; AMPK, AMP-activated protein kinase. (Akt substrate of 160 kDa) is a Rab GTPase-activating protein (GAP) shown to regulate GLUT4 translocation in insulin-sensitive 3T3-L1 adipocytes (8Sano H. Kane S. Sano E. Miinea C.P. Asara J.M. Lane W.S. Garner C.W. Lienhard G.E. J. Biol. Chem. 2003; 278: 14599-14602Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar) and L6 myoblasts (9Thong F. Duagani C.B. Klip A. Physiology. 2005; 20: 271-284Crossref PubMed Scopus (177) Google Scholar). In addition to its Rab GAP domain, AS160 also contains two phosphotyrosine-binding domains and multiple putative phosphorylation sites, including six phospho-Akt substrate (PAS) motifs (RXRXX(S*/T*) where asterisks denote phosphorylation residues) targeted by Akt, AMPK, and potentially other upstream kinases (10Kramer H.F. Witczak C.A. Fujii N. Jessen N. Taylor E.B. Arnolds D.E. Sakamoto K. Hirshman M.F. Goodyear L.J. Diabetes. 2006; 55: 2067-2076Crossref PubMed Scopus (270) Google Scholar). Under basal conditions, AS160 has been shown to retain GLUT4 vesicles intracellularly through the activity of its GAP domain in 3T3-L1 cells (11Eguez L. Lee A. Chavez J.A. Miinea C.P. Kane S. Lienhard G.E. McGraw T.E. Cell Metab. 2005; 2: 263-272Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 12Larance M. Ramm G. Stockli J. van Dam E.M. Winata S. Wasinger V. Simpson F. Graham M. Junutula J.R. Guilhaus M. James D.E. J. Biol. Chem. 2005; 280: 37803-37813Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). GLUT4 vesicles are dynamic complexes that constantly migrate/recycle along cytoskeletal elements and are directed by GTP-bound Rab proteins and other molecular chaperones (9Thong F. Duagani C.B. Klip A. Physiology. 2005; 20: 271-284Crossref PubMed Scopus (177) Google Scholar, 13Watson R.T. Pessin J.E. Trends Biochem. Sci. 2006; 31: 215-222Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Thus, AS160 GAP activity could inactivate a critical, still unidentified Rab protein as part of the mechanism for controlling basal GLUT4 trafficking (13Watson R.T. Pessin J.E. Trends Biochem. Sci. 2006; 31: 215-222Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). When cells are treated with insulin, however, AS160 is rapidly phosphorylated at PAS motifs (14Kane S. Sano H. Liu S.C. Asara J.M. Lane W.S. Garner C.C. Lienhard G.E. J. Biol. Chem. 2002; 277: 22115-22118Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar) and dissociates from GLUT4 vesicles (12Larance M. Ramm G. Stockli J. van Dam E.M. Winata S. Wasinger V. Simpson F. Graham M. Junutula J.R. Guilhaus M. James D.E. J. Biol. Chem. 2005; 280: 37803-37813Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). This is associated with accelerated rates of GLUT4 vesicular exocytosis, such that GLUT4 manifests predominantly at the cell surface and enhances glucose transport (15Zeigerer A. McBrayer M.K. McGraw T.E. Mol. Biol. Cell. 2004; 15: 4406-4415Crossref PubMed Scopus (189) Google Scholar). Both 3T3-L1 adipocytes and L6 GLUT4-Myc myoblasts transfected with a constitutively active AS160 incapable of being phosphorylated at four PAS regulatory motifs (4P mutant) exhibit significantly reduced GLUT4 translocation (8Sano H. Kane S. Sano E. Miinea C.P. Asara J.M. Lane W.S. Garner C.W. Lienhard G.E. J. Biol. Chem. 2003; 278: 14599-14602Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar, F. Duagani C.B. Klip A. Physiology. 2005; 20: 271-284Crossref PubMed Scopus (177) Google Scholar). AS160 phosphorylation therefore to in a by the GAP activity of the protein such that of GLUT4 vesicles is this of the Rab GAP domain point mutation of to lysine to restore insulin-stimulated GLUT4 translocation in adipocytes phosphorylation mutations in AS160 (8Sano H. Kane S. Sano E. Miinea C.P. Asara J.M. Lane W.S. Garner C.W. Lienhard G.E. J. Biol. Chem. 2003; 278: 14599-14602Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar). are a regulatory for AS160 on skeletal muscle glucose by (10Kramer H.F. Witczak C.A. Fujii N. Jessen N. Taylor E.B. Arnolds D.E. Sakamoto K. Hirshman M.F. Goodyear L.J. Diabetes. 2006; 55: 2067-2076Crossref PubMed Scopus (270) Google Scholar) and E.B. Lienhard G.E. Diabetes. 2005; PubMed Scopus Google Scholar) that AS160 phosphorylation at PAS motifs following both insulin and contraction in skeletal muscle. these phosphorylation events are in a distinct and potentially by insulin-stimulated and contraction-stimulated (10Kramer H.F. Witczak C.A. Fujii N. Jessen N. Taylor E.B. Arnolds D.E. Sakamoto K. Hirshman M.F. Goodyear L.J. Diabetes. 2006; 55: 2067-2076Crossref PubMed Scopus (270) Google Scholar). is that AS160 is a point of convergence the effects of both insulin and contraction on skeletal muscle glucose uptake. of this was to determine the effects of wild type and mutant AS160 on basal and insulin- and contraction-stimulated glucose uptake in mouse skeletal muscle in we wild type and three mutant AS160 by the tibialis anterior muscle of by in vivo 1) AS160 mutated to prevent phosphorylation on four regulatory phospho-Akt-substrate (PAS) sites (4P); 2) AS160 mutated to Rab GTPase activity and 3) a double mutant AS160 containing both 4P and R/K mutations (2M). of glucose uptake in vivo using suggest that AS160 phosphorylation at PAS motifs is for insulin- and contraction-stimulated glucose uptake in mouse skeletal muscle. inhibition with 4P mutant overexpression was upon the Rab GAP activity of AS160, muscles transfected with mutant AS160 4P and Rab GAP mutations glucose uptake and glucose uptake following skeletal muscle glucose uptake in transfected muscles was also significantly by Rab GAP mutant overexpression and with and wild type AS160 Thus, AS160 directly regulates insulin- and contraction-stimulated uptake in mouse skeletal muscle. the effects of wild type and mutant AS160 on the actions of insulin and contraction are not and suggest distinct of AS160 was using an the mouse (8Sano H. Kane S. Sano E. Miinea C.P. Asara J.M. Lane W.S. Garner C.W. Lienhard G.E. J. Biol. Chem. 2003; 278: 14599-14602Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar, S. Sano H. Liu S.C. Asara J.M. Lane W.S. Garner C.C. Lienhard G.E. J. Biol. Chem. 2002; 277: 22115-22118Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). Akt and AS160 This was and the on AS160 (10Kramer H.F. Witczak C.A. Fujii N. Jessen N. Taylor E.B. Arnolds D.E. Sakamoto K. Hirshman M.F. Goodyear L.J. Diabetes. 2006; 55: 2067-2076Crossref PubMed Scopus (270) Google Scholar). expression was with and and used to and all for and by the and of the and in with of from of the with a and and to to the of the wild type AS160 and three distinct mutant AS160 been in 3T3-L1 cells (8Sano H. Kane S. Sano E. Miinea C.P. Asara J.M. Lane W.S. Garner C.W. Lienhard G.E. J. Biol. Chem. 2003; 278: 14599-14602Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar). mutant AS160 isoforms 1) AS160 mutated at four PAS motifs and these sites incapable of being phosphorylated (4P); 2) AS160 mutated to lysine at its Rab GAP domain, effectively AS160 GAP activity (R/K); and 3) AS160 double mutant containing both the 4P and R/K mutations (2M). the of and in vivo electroporation in mouse skeletal AS160 was from the to a and and This expression a gene the and has been to activity in skeletal muscle N. Sakamoto K. H. S. H. L. M.F. Hirshman M.F. Goodyear L.J. J. Physiol. 2004; PubMed Scopus Google Scholar, O. Fujii N. Hirshman M.F. Goodyear L.J. J. Physiol. 2004; PubMed Scopus Google Scholar). AS160 for using the at and was in cells using an and in at In in Skeletal of AS160 directly mouse tibialis anterior muscles by a N. Sakamoto K. H. S. H. L. M.F. Hirshman M.F. Goodyear L.J. J. Physiol. 2004; PubMed Scopus Google by and H. J. 1998; PubMed Scopus Google Scholar). with an of and of AS160 was the tibialis anterior muscle with a the muscle and at a of using a Our has that with the gene this is and manifests the muscle N. Sakamoto K. H. S. H. L. M.F. Hirshman M.F. Goodyear L.J. J. Physiol. 2004; PubMed Scopus Google Scholar). of muscle was by by J.V. Biochem. PubMed Scopus Google Scholar). was using a In transfected with in tibialis anterior muscle. injection, the with administration of of body from both for Although was the other was to using a for of at transfected with in tibialis anterior muscle. injection, the with and a glucose of of body through the This a insulin to by at data from the and Insulin and not A. J. 2004; PubMed Scopus Google Scholar, G. C.B. Diabetes. 2004; PubMed Scopus Google Scholar). In Skeletal from the to intravenous of [3H]2-deoxyglucose through the This was with the glucose in glucose and with the of in situ in contraction all the from the at and for the of glucose and [3H]2-deoxyglucose activity. of the the and tibialis anterior muscles and in of [3H]2-deoxyglucose in was a from A. L. J. Biochem. J. 1985; PubMed Scopus Google Scholar) using and of the muscle and used for and muscle was with a in 2 and of this was for of glucose uptake was with over at for and at for at and protein as of skeletal muscle proteins by PubMed Scopus Google Scholar) for H. J. Sci. S. A. 76: PubMed Scopus Google Scholar). proteins using protein by and by data are as the using of When of for multiple was of AS160 in Skeletal the expression of wild type AS160 and 4P, R/K, and mutant AS160 isoforms using gene AS160 and in vivo electroporation induced increases in AS160 protein in mouse tibialis anterior muscles the of overexpression was all AS160 was the that the both the AS160 and mouse AS160 Both AS160 protein used in AS160 did not expression of AS160 in muscles. AS160 overexpression was in tibialis anterior muscles, the muscles increases in AS160 not These the of as a of overexpressing wild type gene skeletal muscle of the phosphorylation of AS160 at a critical PAS targeted by Akt and other kinases as as phosphorylation events at all PAS sites the phospho-Akt substrate phosphorylation of and R/K AS160 at was significantly with However, 4P and mutant AS160, point mutations at and three other PAS increases in basal phosphorylation with Both contraction and insulin AS160 phosphorylation and phosphorylation of and R/K AS160. These in with the PAS Here, clear of and AS160 protein This that AS160 is phosphorylated in vivo and that the of the AS160 point mutations (4P and is preserved following gene AS160 Insulin-stimulated AS160 to in GLUT4 translocation in (8Sano H. Kane S. Sano E. Miinea C.P. Asara J.M. Lane W.S. Garner C.W. Lienhard G.E. J. Biol. Chem. 2003; 278: 14599-14602Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar, F. Duagani C.B. Klip A. Physiology. 2005; 20: 271-284Crossref PubMed Scopus (177) Google Scholar, L. Lee A. Chavez J.A. Miinea C.P. Kane S. Lienhard G.E. McGraw T.E. Cell Metab. 2005; 2: 263-272Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, A. McBrayer M.K. McGraw T.E. Mol. Biol. Cell. 2004; 15: 4406-4415Crossref PubMed Scopus (189) Google is data the of AS160 on glucose metabolism in skeletal muscle glucose in to and an associated insulin increase in A. J. 2004; PubMed Scopus Google and G. C.B. Diabetes. 2004; PubMed Scopus Google glucose uptake muscles was measured in vivo using glucose data from all and are as a transfected regulatory effects of and mutant AS160 on insulin-stimulated glucose uptake are shown in overexpressing 4P mutant AS160 in insulin-stimulated glucose uptake with both and AS160 a mutated Rab GAP domain prevented the effects of the 4P mutations and insulin-stimulated glucose uptake. These with insulin-stimulated signaling to Akt all muscles in insulin-stimulated glucose uptake and WT, R/K, and AS160. In in glucose uptake with muscles in as a These data suggest that phosphorylation of AS160 at PAS motifs is for insulin-stimulated glucose uptake in vivo in skeletal muscle. AS160 and muscle AS160 phosphorylation at PAS motifs through a (10Kramer H.F. Witczak C.A. Fujii N. Jessen N. Taylor E.B. Arnolds D.E. Sakamoto K. Hirshman M.F. Goodyear L.J. Diabetes. 2006; 55: 2067-2076Crossref PubMed Scopus (270) Google Scholar). However, it is not these AS160 phosphorylation in regulate contraction-stimulated glucose uptake. we the effects of AS160 and mutant AS160 on glucose uptake following of in situ in transfected mouse tibialis anterior muscles overexpressing 4P mutant AS160 in contraction-stimulated glucose uptake with glucose uptake in vivo was also inhibited in muscles overexpressing wild type AS160, it was still significantly in muscles. In contrast, in vivo glucose uptake contraction was significantly in muscle overexpressing mutant AS160 of Rab GAP activity and for R/K and 2M, is to that contraction-stimulated phosphorylation of Akt and was and not significantly muscles. These suggest that AS160 phosphorylation at PAS motifs is for contraction-stimulated glucose uptake in skeletal muscle. of constitutively active AS160 a on glucose uptake following muscle in overexpression of AS160 lacking Rab GAP activity skeletal muscle glucose uptake. in basal glucose uptake muscles. Under these conditions, muscles overexpressing Rab GAP mutant AS160 increases in basal glucose uptake with and for R/K and AS160, in not WT, and 4P mutant muscles on basal glucose uptake. overexpression of mutant AS160 of Rab GAP activity to regulate both basal and contraction-stimulated glucose uptake in regulatory effects that AS160 on both insulin- and contraction-stimulated glucose uptake with in vivo are with a for AS160 on molecular AS160 of in Skeletal determine in skeletal muscle glucose uptake with AS160 overexpression to effects on regulatory upstream and we for and muscles with of the AS160 gene Thus, gene by AS160 not disruption in the expression of signaling proteins that regulate glucose metabolism in skeletal of and expression and AS160 expression and phosphorylation in tibialis anterior muscles week following of control AS160, 4P mutant AS160, R/K mutant AS160, double mutant AS160 to to to to to to to in a Skeletal muscle is in its ability to promote glucose through both and insulin-independent to GLUT4 of a point of signaling convergence to GLUT4 a for diabetes Our that insulin- and contraction-stimulated signaling cascades AS160 PAS motifs in a distinct and in mouse skeletal muscle (10Kramer H.F. Witczak C.A. Fujii N. Jessen N. Taylor E.B. Arnolds D.E. Sakamoto K. Hirshman M.F. Goodyear L.J. Diabetes. 2006; 55: 2067-2076Crossref PubMed Scopus (270) Google Scholar). of this was to a functional for AS160 on skeletal muscle glucose uptake. a of protein we that AS160 regulates insulin- and contraction-stimulated glucose uptake in of AS160 at PAS motifs is for insulin- and contraction-stimulated glucose uptake in skeletal muscle. To this is the of a protein upstream of GLUT4 of independent insulin and contraction effects on skeletal muscle glucose uptake. We AS160 on glucose metabolism by mouse tibialis anterior muscles with wild type AS160, and three mutant AS160 of in with in vivo electroporation for and expression of the gene an this to other such as N. J.M. J.M. K. J. PubMed Scopus Google Scholar). it is to the tibialis anterior skeletal muscle in a the of in vivo electroporation in its of this has been by N. Sakamoto K. H. S. H. L. M.F. Hirshman M.F. Goodyear L.J. J. Physiol. 2004; PubMed Scopus Google Scholar, O. Fujii N. Hirshman M.F. Goodyear L.J. J. Physiol. 2004; PubMed Scopus Google Scholar) and H. J. 1998; PubMed Scopus Google Scholar) and for overexpression of wild type and all of the mutant AS160 isoforms in the in the expression activity of signaling proteins upstream of AS160, effects on expression of and of the of AS160 on skeletal muscle glucose uptake. AS160 of insulin-stimulated glucose uptake in mouse skeletal muscles was following intravenous administration of Thus, of a insulin a glucose to a insulin A. J. 2004; PubMed Scopus Google Scholar, G. C.B. Diabetes. 2004; PubMed Scopus Google Scholar). We that 4P mutant AS160 in a insulin-stimulated glucose uptake by over with and AS160 This is with impaired insulin-stimulated of GLUT4 in adipocytes (15Zeigerer A. McBrayer M.K. McGraw T.E. Mol. Biol. Cell. 2004; 15: 4406-4415Crossref PubMed Scopus (189) Google Scholar). overexpression of the double mutant AS160, the 4P and Rab GAP domain effectively insulin-stimulated glucose uptake to and AS160 transfected skeletal muscle. These data suggest that activity of the AS160 Rab GAP domain to glucose uptake, and is in by Sano (8Sano H. Kane S. Sano E. Miinea C.P. Asara J.M. Lane W.S. Garner C.W. Lienhard G.E. J. Biol. Chem. 2003; 278: 14599-14602Abstract Full Text Full Text PDF PubMed Scopus (725) Google the effects of mutant on GLUT4 translocation in 3T3-L1 However, we did not basal glucose transport following overexpression of Rab GAP mutant AS160, is with mechanisms for basal GLUT4 (11Eguez L. Lee A. Chavez J.A. Miinea C.P. Kane S. Lienhard G.E. McGraw T.E. Cell Metab. 2005; 2: 263-272Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). activity of AS160 protein also to basal in glucose findings implicate a for AS160 phosphorylation to Rab GAP activity and the effects of insulin on glucose uptake. These are the data directly a functional for AS160 on insulin-stimulated glucose metabolism in skeletal muscle effects of AS160 on contraction-stimulated glucose uptake are also of AS160 on PAS motifs is by in signaling to glucose however, these motifs are also by and potentially other kinases in insulin-independent signaling to glucose uptake (10Kramer H.F. Witczak C.A. Fujii N. Jessen N. Taylor E.B. Arnolds D.E. Sakamoto K. Hirshman M.F. Goodyear L.J. Diabetes. 2006; 55: 2067-2076Crossref PubMed Scopus (270) Google Scholar). In lacking both AS160 phosphorylation (10Kramer H.F. Witczak C.A. Fujii N. Jessen N. Taylor E.B. Arnolds D.E. Sakamoto K. Hirshman M.F. Goodyear L.J. Diabetes. 2006; 55: 2067-2076Crossref PubMed Scopus (270) Google Scholar) and glucose transport N. Hirshman M.F. Kane E.M. Goodyear L.J. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar) are that AS160 phosphorylation is for glucose uptake. However, these also exhibit significantly contraction-stimulated AS160 phosphorylation contraction-stimulated increases with (10Kramer H.F. Witczak C.A. Fujii N. Jessen N. Taylor E.B. Arnolds D.E. Sakamoto K. Hirshman M.F. Goodyear L.J. Diabetes. 2006; 55: 2067-2076Crossref PubMed Scopus (270) Google glucose uptake following contraction controlling for in contraction N. Hirshman M.F. Kane E.M. Goodyear L.J. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). could for this in N. J.M. J.M. K. J. PubMed Scopus Google we directly the of contraction-stimulated AS160 phosphorylation at PAS motifs using protein overexpression in this Our data that AS160 phosphorylation at four PAS motifs is for the effects of contraction on skeletal muscle glucose uptake in in the insulin-stimulated overexpression of 4P mutant AS160 a contraction-stimulated glucose uptake by over with control muscles and by with muscles overexpressing AS160. These data that phosphorylation events on AS160 are for of glucose uptake. We to that overexpression of wild type AS160 also glucose uptake following contraction, this was still significantly muscles. are at two of these of wild type and AS160 was for contraction-stimulated AS160 and the of phosphorylated AS160 a Our findings this we AS160 overexpression and contraction-stimulated AS160 phosphorylation These suggest that activity not the AS160 in to its therefore for the inhibition of contraction-stimulated glucose uptake by wild type AS160 overexpression of wild type AS160 a by contraction not insulin that glucose uptake. of mutant AS160 of Rab GAP activity R/K and increases in both sham and contraction-stimulated glucose uptake with These in basal glucose uptake in transfected muscles are of to AS160 in 3T3-L1 adipocytes also resulted in basal glucose transport (11Eguez L. Lee A. Chavez J.A. Miinea C.P. Kane S. Lienhard G.E. McGraw T.E. Cell Metab. 2005; 2: 263-272Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 12Larance M. Ramm G. Stockli J. van Dam E.M. Winata S. Wasinger V. Simpson F. Graham M. Junutula J.R. Guilhaus M. James D.E. J. Biol. Chem. 2005; 280: 37803-37813Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). of wild type AS160 basal glucose transport in this expression of R/K mutant AS160 did of these data that AS160 GAP activity is for of GLUT4 in the basal (11Eguez L. Lee A. Chavez J.A. Miinea C.P. Kane S. Lienhard G.E. McGraw T.E. Cell Metab. 2005; 2: 263-272Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). of AS160 in skeletal muscle resulted in of basal glucose uptake, by the of these mutants on GLUT4 To the basal glucose uptake in these from muscles in the of muscles to in We did not in basal glucose uptake in as a control glucose These basal glucose uptake the sham basal uptake in contraction We that sham by not significantly increase AS160 phosphorylation control in vivo (10Kramer H.F. Witczak C.A. Fujii N. Jessen N. Taylor E.B. Arnolds D.E. Sakamoto K. Hirshman M.F. Goodyear L.J. Diabetes. 2006; 55: 2067-2076Crossref PubMed Scopus (270) Google Scholar). are to increases in resistance of the Thus, in of for the and sham in as has been shown in A. J. Physiol. Google Scholar, G. L. Wahren J. J. Appl. Physiol. PubMed Scopus Google Scholar). However, the of sham basal uptake an to in basal glucose uptake the AS160 Transfection of AS160 also resulted in increases in contraction-stimulated glucose uptake in mouse skeletal muscle. In this we a of AS160 lacking (GAP) on GLUT4 AS160 is a protein with multiple phosphorylation and sites for 2003; 31: PubMed Scopus Google Scholar). is that AS160 its activity could as a that glucose uptake. of AS160 as a protein in To we used an in vivo electroporation technique to overexpress wild type and mutant AS160 in skeletal muscle and AS160 on glucose uptake. Our findings a regulatory for AS160 on insulin- and contraction-stimulated glucose uptake. of 4P mutant AS160, incapable of being phosphorylated at PAS significantly inhibited glucose uptake following insulin contractile in transfected muscles. type AS160 overexpression also uptake, the mechanisms for this remain In contrast, both sham and contraction-stimulated glucose uptake significantly by overexpressing mutant AS160 lacking Rab GAP activity. These in the of in the activity expression of putative upstream AS160 kinases expression of AS160 directly regulates insulin- and contraction-stimulated glucose transport in mouse skeletal muscle. We Lienhard for the and AS160 as as We also for the of and for on gene and on in vivo glucose uptake