In vitro and in vivo activation of the insulin receptor kinase in control and denervated skeletal muscle.
Skeletal muscle rapidly develops severe insulin resistance following denervation, although insulin binding is unimpaired. Insulin-stimulated receptor tyrosyl kinase activity was studied in intact and 24-h denervated rat hind limb muscles using three preparations: (a) solubilized insulin receptors incubated +/- insulin with gamma-[32P]ATP and histone H2b; (b) soleus muscles prelabeled in vitro with [32P]phosphate with subsequent insulin-stimulated phosphorylation of the receptor in situ; (c) assessment of in vivo activation of muscle receptor tyrosyl kinase by insulin. The latter was achieved by solubilizing muscle insulin receptors in the presence of phosphoprotein phosphatase and kinase inhibitors and measuring receptor-catalyzed histone H2b phosphorylation in the presence of limiting (5 microM) gamma-[32P]ATP. Receptors isolated 5 and 30 min after intravenous insulin injection catalyzed 32P incorporation into histone H2b twice as fast as those from saline-treated controls; insulin stimulated histone H2b labeling exclusively on tyrosine. In vivo activation was demonstrated using solubilized and insulin-agarose-bound receptors. Autophosphorylation of the beta-subunit and receptor tyrosyl kinase activity toward histone H2b was stimulated by insulin in denervated muscles as in controls, although the biological response to insulin, in vitro and in vivo, was markedly impaired after denervation, suggesting a postreceptor defect. The method developed to assess insulin-stimulated receptor activation in vivo seems useful in characterizing mechanisms of insulin resistance.
D000818:Animals; D004789:Enzyme Activation; D006003:Glycogen / Q000096:biosynthesis; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism; D007333:Insulin Resistance; D007700:Kinetics; D008297:Male; D009121:Muscle Denervation; D009132:Muscles / Q000201:enzymology* / Q000378:metabolism; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism
D007328:Insulin; D012265:Ribonucleotides; C008147:insulin, iodo-; D006003:Glycogen; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D006656:Protamine Kinase