Knockout of the α2 but Not α1 5′-AMP-activated Protein Kinase Isoform Abolishes 5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranosidebut Not Contraction-induced Glucose Uptake in Skeletal Muscle

We investigated the importance of the two catalytic α-isoforms of the 5′-AMP-activated protein kinase (AMPK) in 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) and contraction-induced glucose uptake in skeletal muscle. Incubated soleus and EDL muscle from whole-body α2- or α1-AMPK knockout (KO) and wild type (WT) mice were incubated with 2.0 mm AICAR or electrically stimulated to contraction. Both AICAR and contraction increased 2DG uptake in WT muscles. KO of α2, but not α1, abolished AICAR-induced glucose uptake, whereas neither KO affected contraction-induced glucose uptake. AICAR and contraction increased α2- and α1-AMPK activity in wild type (WT) muscles. During AICAR stimulation, the remaining AMPK activity in KO muscles increased to the same level as in WT. During contraction, the remaining AMPK activity in α2-KO muscles was elevated by 100% probably explained by a 2–3-fold increase in α1-protein. In α1-KO muscles, α2-AMPK activity increased to similar levels as in WT. Both interventions increased total AMPK activity, as expressed by AMPK-P and ACCβ-P, in WT muscles. During AICAR stimulation, this was dramatically reduced in α2-KO but not in α1-KO, whereas during contraction, both measurements were essentially similar to WT in both KO-muscles. The results show that α2-AMPK is the main donor of basal and AICAR-stimulated AMPK activity and is responsible for AICAR-induced glucose uptake. In contrast, during contraction, the two α-isoforms seem to substitute for each other in terms of activity, which may explain the normal glucose uptake despite the lack of either α2- or α1-AMPK. Alternatively, neither α-isoform of AMPK is involved in contraction-induced muscle glucose uptake. We investigated the importance of the two catalytic α-isoforms of the 5′-AMP-activated protein kinase (AMPK) in 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) and contraction-induced glucose uptake in skeletal muscle. Incubated soleus and EDL muscle from whole-body α2- or α1-AMPK knockout (KO) and wild type (WT) mice were incubated with 2.0 mm AICAR or electrically stimulated to contraction. Both AICAR and contraction increased 2DG uptake in WT muscles. KO of α2, but not α1, abolished AICAR-induced glucose uptake, whereas neither KO affected contraction-induced glucose uptake. AICAR and contraction increased α2- and α1-AMPK activity in wild type (WT) muscles. During AICAR stimulation, the remaining AMPK activity in KO muscles increased to the same level as in WT. During contraction, the remaining AMPK activity in α2-KO muscles was elevated by 100% probably explained by a 2–3-fold increase in α1-protein. In α1-KO muscles, α2-AMPK activity increased to similar levels as in WT. Both interventions increased total AMPK activity, as expressed by AMPK-P and ACCβ-P, in WT muscles. During AICAR stimulation, this was dramatically reduced in α2-KO but not in α1-KO, whereas during contraction, both measurements were essentially similar to WT in both KO-muscles. The results show that α2-AMPK is the main donor of basal and AICAR-stimulated AMPK activity and is responsible for AICAR-induced glucose uptake. In contrast, during contraction, the two α-isoforms seem to substitute for each other in terms of activity, which may explain the normal glucose uptake despite the lack of either α2- or α1-AMPK. Alternatively, neither α-isoform of AMPK is involved in contraction-induced muscle glucose uptake. The 5′-AMP-activated protein kinase (AMPK) 1The abbreviations used are: AMPK, 5′-AMP-activated protein kinase; AMPK-P, phosphorylated AMPK; ACCβ, acetyl-CoA carboxylase β; ACCβ-P, phosphorylated ACCβ; AICAR, 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside; EDL, extensor digitorum longus; KO, knockout; WT, wild type; 2DG, 2-deoxy-d-glucose. is a multisubstrate serine/threonine protein kinase that is ubiquitously expressed and functions as an intracellular fuel sensor activated by depletion of high energy phosphor compounds (1.Corton J.M. Gillespie J.G. Hardie D.G. Curr. Biol. 1994; 4: 315-324Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 2.Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1276) Google Scholar). 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In to the AMPK catalytic a was with of and a of the catalytic of α1-AMPK from shown in of with the to WT of and of of protein from α1-AMPK knockout and skeletal muscle and a level of with that in from WT and for either the α2- or in lack of of the protein level and In the knockout muscles, a increase in the protein level of the remaining was in EDL and In the α1-KO soleus muscles, a increase in the remaining was whereas was in The protein level of was in the two muscles and was expressed to the same level in both of the two knockout and The protein content was in the muscle and was expressed to the same level in both of the two knockout and α2- and levels in EDL and soleus muscles from or α2-AMPK knockout in a protein level in muscle from or α2-AMPK knockout in a of of the α2-AMPK KO B. F. Jorgensen S.B. C. A. D. Mu J. C. O. M. G. J.F. A. Carling D. Birnbaum M.J. Richter E.A. S. J. 2003; PubMed Scopus Google Scholar). In mice and In contrast, the α1-AMPK KO normal glucose and to and AICAR not glucose and levels α1-KO, normal in α1-AMPK KO mice with WT. both AICAR increased 2DG uptake by in WT soleus and by in WT EDL knockout of the abolished the of AICAR 2DG uptake in both EDL and soleus muscles. In contrast, knockout of the not AICAR-induced 2DG uptake in either of the muscles In both mice muscle contraction increased 2DG uptake by in WT EDL and by in WT soleus knockout of the increased contraction-stimulated glucose uptake by with WT in EDL, whereas the α2-KO not contraction-stimulated glucose uptake in The α1-KO not contraction-stimulated glucose uptake in EDL, but in the α1-KO decreased glucose uptake by during contraction AMPK an to the signaling that explain the of the knockout glucose uptake, of AMPK signaling were and AMPK the WT muscles of both AICAR increased α2-AMPK and α1-AMPK activity in both muscles in the α2-KO the remaining α1-AMPK activity in EDL increased with AICAR to the WT muscles, and in the soleus the AICAR-induced increase in α1-AMPK activity to in the WT muscle AICAR increased α1-AMPK activity in the α2-KO this was not by increased glucose uptake. In α1-KO muscles, AICAR a normal increase in the remaining α2-AMPK activity activated both α1-AMPK and α2-AMPK activity in the WT muscles in the α2-KO muscles, the remaining α1-AMPK activity was increased by during contraction with WT muscles. In the α1-KO muscles, the remaining α2-AMPK activity was similar to the activity in WT muscles of the of AMPK activity by of The used not the two and the the total and In the WT muscles of both AICAR increased AMPK-P by in both muscles in the α2-KO AMPK-P was reduced to and in the soleus AICAR in a increase in in neither of the two α2-KO muscles AICAR in of AMPK-P that the basal in WT muscles. In α1-KO muscles, AICAR similar in AMPK-P as in the WT muscles These that α2-AMPK is the main donor of AMPK activity in both and skeletal muscle in the basal and during with AICAR and probably explain the AICAR glucose uptake is in the α1-KO muscles. increased AMPK-P by in WT muscles from both AMPK-P not increase during contraction in α2-KO EDL, and in the AMPK-P level in the muscle was with WT In contrast, in α2-KO contraction increased AMPK-P to a similar level with In α1-KO muscles, contraction similar in AMPK-P as in the WT muscles These results that the is the main donor of AMPK activity during contraction in EDL but not in the soleus muscle and that α1-KO not total AMPK-P or during contraction. of total AMPK kinase activity and by of the AMPK substrate During AICAR stimulation, in with the AMPK-P in the WT muscles of both AICAR increased and in α2-KO muscles, both the basal and the AICAR-stimulated were in WT. in neither of the muscles AICAR in that basal in WT. In α1-KO muscles, AICAR similar in as in the WT muscles These that α2-AMPK is the main donor of kinase activity in the basal and during AICAR increased in WT EDL and soleus from both In to the during AICAR stimulation, contraction increased in both α2-KO and α1-KO muscles to the WT muscles that α2-KO reduced AMPK-P in muscle the total AMPK activity was by both the α2-KO and the α1-KO in the two investigated muscles during contraction. of AICAR, and muscle contraction to glucose uptake is to muscle glycogen content (6.Wojtaszewski J.F. Jorgensen S.B. Hellsten Y. Hardie D.G. Richter E.A. Diabetes. 2002; 51: 284-292Crossref PubMed Scopus (233) Google Scholar, E.A. Galbo H. Kiens B. J. Physiol. 1989; PubMed Scopus Google Scholar, W. S. J. O. Richter E.A. Am. J. Physiol. 1999; 277: Google Scholar). The α2-KO EDL a decreased level of glycogen whereas α2-KO soleus a normal glycogen content The α1-KO not the level of glycogen in either of the two muscles. with WT, glycogen levels were reduced to a similar in both α2-KO and α1-KO muscles contraction glycogen content in EDL and soleus basal and contraction in muscles from or α2-AMPK whole-body knockout in a both for a contraction during of in EDL and in soleus and was not affected by KO of either the or α2-AMPK the of the contraction was reduced with and in EDL and and this was not affected by KO of either the or the In the have shown that knockout of the of AMPK abolished AICAR-induced glucose uptake in skeletal whereas knockout of the in this The of knockout AICAR-induced glucose uptake was of muscle type and was not by a in muscle protein content but was with a in AMPK the the of total AMPK activity in the basal and during AICAR In contrast, contraction-induced glucose uptake was essentially by knockout of either α-isoform of AMPK, and knockout AMPK signaling during contraction. the by Mu J. J.T. O. M. Birnbaum M.J. Full Text Full Text PDF PubMed Scopus Google Scholar), in which a of a of the AMPK catalytic α-isoform abolished AICAR-induced glucose uptake, whereas contraction-induced muscle glucose uptake was reduced by the were to show that the α2-KO is and and whereas was in the α1-KO The glucose and probably to an elevated as B. F. Jorgensen S.B. C. A. D. Mu J. C. O. M. G. J.F. A. Carling D. Birnbaum M.J. Richter E.A. S. J. 2003; PubMed Scopus Google Scholar). AICAR as in the α2-KO was in mice J. J.T. O. M. Birnbaum M.J. Full Text Full Text PDF PubMed Scopus Google Scholar), and it in to a reduced glucose uptake in skeletal muscles. is to that knockout of the to a increase in protein content of the in the soleus and EDL muscles, whereas knockout of the increased α2-AMPK and in the The of α-isoform the other is a to AMPK Therefore, the increase of in α2-KO muscles the that the α2-AMPK activity in is the of AMPK activity in skeletal muscles. is by the that the α2-KO was whereas the α1-KO was the activity of the by of of AMPK by phosphorylated in the two α-isoforms the These in the the to the kinase activity of α2-AMPK and α1-AMPK AMPK and is that α2-KO dramatically decreased basal as as AICAR-induced in both muscles, whereas α1-KO in this α1-AMPK activity increased in response to AICAR in the α2-KO muscles, the increase in in levels in the WT muscles that is the phosphorylated in response to AICAR either as the of a protein or as a of a for by the similar results were AMPK activity by the total AMPK activity, as by was reduced in the α2-KO muscles In with the the knockout of the not in basal or AICAR-stimulated the that AMPK the main of AMPK activity during AICAR in muscles and for glucose uptake during this type of is in the α2-KO muscles, AICAR increased α1-AMPK activity to the same level as in WT muscles, the protein level of the in the α2-KO muscles was increased that AICAR not to the same in α2-KO as in WT muscles or that the increased of is in that for by AMPK kinase during AICAR the of from the incubated muscles measurements of the the that α1-AMPK activity in muscles increased with AICAR to WT that α1-AMPK was an in AICAR-stimulated glucose uptake, glucose have increased in the muscles. In to the of AICAR, contraction-induced glucose uptake was by KO of either The is the lack of of either KO glucose during contraction is to of the other α-isoform for the or AMPK activity is not for contraction-induced muscle glucose In to the during AICAR lack of α2-AMPK was for by an increase in α1-AMPK activity during contraction in both muscle in the muscles the was reduced in the EDL but not in the as by the lack of α2-AMPK was not for in the EDL, whereas it was in the glucose was not decreased with WT in either muscle of ACCβ, a of AMPK, a of total AMPK activity, for the lack of the α2-AMPK was essentially during contraction in both muscle In this it in that other D. A. Saha A.K. J. Patel A. Kemp B.E. Witters L.A. Ruderman N.B. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). it that during exercise, a of AMPK activity and J.F. M. T. B. H. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar). it is not is a of total AMPK activity, and it is not with to from AMPK activity was or not during in the two knockout in the AMPK contraction during was reduced in both soleus and EDL J. Birnbaum M.J. Biochem. 2003; PubMed Google Scholar), that total lack of AMPK activity a role for contraction In incubated it shown that a in contraction is with a in glucose uptake J. Ploug T. Hellsten Y. Galbo H. Am. J. Physiol. 1999; 277: E208-E214Crossref PubMed Google Scholar), but it the reduced contraction in the J. Birnbaum M.J. Biochem. 2003; PubMed Google Scholar) is for the reduced glucose uptake. In and α2-KO muscles, contraction was not from the WT muscles, and a similar of was in knockout in glucose uptake not explained by in and AMPK signaling were essentially normal in α1-KO muscles and during was a in contraction-induced glucose in the α1-KO glycogen was not increased in the KO muscles, and the in the contraction-induced glucose in the soleus explained by an of increased muscle glycogen glucose uptake as shown W. Ai H. Ihlemann J. Witters L.A. Kristiansen S. Richter E.A. Ploug T. Diabetes. 2000; 49: 1281-1287Crossref PubMed Scopus (145) Google Scholar, W. S. J. O. Richter E.A. Am. J. Physiol. 1999; 277: Google Scholar, P. Richter E.A. J. Physiol. Scopus Google Scholar). In contrast, in the α2-KO muscles, glycogen was decreased in the EDL, a for the increase in contraction-induced glucose of the α2-KO EDL muscles with WT. Activation of AMPK with AICAR in muscle is by investigators a for of AMPK during muscle contraction. in to contraction, AICAR of muscle was not to increase glucose in α2-KO muscles despite an increase in α1-AMPK AICAR of glucose uptake in muscle is α2-AMPK activity, whereas during muscle contraction, lack of α2-AMPK activity it in that of skeletal muscles to as in the is a causing in the contraction the Therefore, from such may not to in vivo exercise In have shown that knockout of the of AMPK abolished AICAR-induced glucose uptake in skeletal muscle of whereas knockout of the in this We that the the of total AMPK activity in the basal and during AICAR In contrast, contraction-induced glucose was essentially by knockout of either α-isoform of AMPK, explained by essentially signaling of AMPK during contraction. Alternatively, neither α-isoform is involved in contraction-stimulated muscle glucose uptake. In either the that an of signaling to AMPK for the lack of AMPK AICAR-induced muscle glucose used as a substitute for muscle contraction, it is the whereas neither α-isoform of AMPK is for contraction-induced muscle glucose uptake. The that the of AMPK is the glucose uptake both to the importance of this in AICAR-induced glucose uptake and of the in skeletal muscles D. G. J. T. T. Witters L.A. Kemp B.E. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Salt I.P. Davies S.P. Hardie D.G. Carling D. Biochem. J. 2000; PubMed Scopus Google Scholar). We D. Hardie for the of and K. for the of and for