Possibility of autocrine beta-adrenergic signaling in C2C12 myotubes.

Levodopa reportedly inhibits insulin action in skeletal muscle. Here we show that C2C12 myotubes produce levodopa and that insulin-stimulated glucose transport is enhanced when endogenous levodopa is depleted. Exogenous levodopa prevented the stimulation of glucose transport by insulin (P < 0.05) and increased cAMP concentrations (P < 0.05). The decrease in insulin-stimulated glucose transport caused by levodopa was attenuated by propranolol (a beta-adrenergic antagonist) and prevented by NSD-1015 (NSD), an inhibitor of DOPA decarboxylase (DDC; converts levodopa to dopamine). Propranolol and NSD both prevented levodopa-related increases in [cAMP]. However, the effects of levodopa were unlikely to be dependent on the conversion of levodopa to catecholamines because we could detect neither DDC in myotubes nor catecholamines in media after incubation of myotubes with levodopa. The data suggest the possibility of novel autocrine beta-adrenergic action in C2C12 myotubes in which levodopa, produced by myotubes, could have hormone-like effects that impinge on glucose metabolism.

Muscle contractions, AICAR, and insulin cause phosphorylation of an AMPK-related kinase.

We hypothesized that AMP-activated protein kinase-related kinase 5 (ARK5)/novel kinase family 1 (NUAK1), an AMP-activated protein kinase (AMPK)-related kinase that has been found to be stimulated by protein kinase B (Akt), would be expressed in rat skeletal muscle and activated by electrically elicited contractions, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), or insulin. We verified expression of ARK5 in muscle through RT-PCR and Western blot. Cross-reactivity of ARK5 immunoprecipitates with antibodies against phospho-AMPK was increased by approximately 30% by muscle contractions and approximately 60% by incubation of muscle with AICAR. AMPK was not detected in the ARK5 immunoprecipitates. Despite the apparent increase in phosphorylation of ARK5 at a site essential to its activation, neither contractions nor AICAR increased ARK5 activity. For muscles from animals injected with saline or insulin, we probed nonimmunoprecipitated samples in sequence for phosphotyrosine (P-Tyr), ARK5, and phosphorylated substrates of Akt (P-AS) and found that the ARK5 band could be precisely superimposed on phosphoprotein bands from the P-Tyr and P-AS blots. In the band corresponding to ARK5, insulin increased P-Tyr content by approximately 45% and cross-reactivity with the antibody against P-AS by approximately threefold. We also detected ARK5 in phosphotyrosine immunoprecipitates. Our data suggest that increased phosphorylation of ARK5 by muscle contractions or exposure to AICAR is insufficient to activate ARK5 in skeletal muscle, suggesting that some other modification (e.g., phosphorylation on tyrosine or by Akt) may be necessary to its activity in muscle.

AICAR and hyperosmotic stress increase insulin-stimulated glucose transport.

Sensitivity of glucose transport to stimulation by insulin has been shown to occur concomitant with activation of the AMP-activated protein kinase (AMPK) in skeletal muscle, suggesting a role of AMPK in regulation of insulin action. The purpose of the present study was to evaluate a possible role of AMPK in potentiation of insulin action in muscle cells. The experimental model involved insulin-responsive C2C12 myotubes that exhibit a twofold increase in glucose transport in the presence of insulin. Treatment of myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), followed by a 2-h recovery, augmented the ability of insulin to stimulate glucose transport. Similarly, incubation in hyperosmotic medium, another AMPK-activating treatment, acted synergistically with insulin to stimulate glucose transport. Furthermore, the increase in insulin action caused by hyperosmotic stress was prevented by inclusion of compound C, an AMPK inhibitor, in hyperosmotic medium. In addition, iodotubercidin, a general kinase inhibitor that is effective against AMPK, also prevented the combined effects of insulin and hyperosmotic stress on glucose transport. The new information provided by these data is that previously reported AICAR effects on insulin action are generalizable to myotubes, hyperosmotic stress and insulin synergistically increase glucose transport, and AMPK appears to mediate potentiation of insulin action.

Creatine feeding increases GLUT4 expression in rat skeletal muscle.

The purpose of this study was to investigate the potential role of creatine in GLUT4 gene expression in rat skeletal muscle. Female Wistar rats were fed normal rat chow (controls) or chow containing 2% creatine monohydrate ad libitum for 3 wk. GLUT4 protein levels of creatine-fed rats were significantly increased in extensor digitorum longus (EDL), triceps, and epitrochlearis muscles compared with muscles from controls (P < 0.05), and triceps GLUT4 mRNA levels were approximately 100% greater in triceps muscles from creatine-fed rats than in muscles from controls (P < 0.05). In epitrochlearis muscles from creatine-fed animals, glycogen content was approximately 40% greater (P < 0.05), and insulin-stimulated glucose transport rates were higher (P < 0.05) than in epitrochlearis muscles from controls. Despite no changes in [ATP], [creatine], [phosphocreatine], or [AMP], creatine feeding increased AMP-activated protein kinase (AMPK) phosphorylation by 50% in rat EDL muscle (P < 0.05). Creatinine content of EDL muscle was almost twofold higher for creatine-fed animals than for controls (P < 0.05). Creatine feeding increased protein levels of myocyte enhancer factor 2 (MEF2) isoforms MEF2A ( approximately 70%, P < 0.05), MEF2C ( approximately 60%, P < 0.05), and MEF2D ( approximately 90%, P < 0.05), which are transcription factors that regulate GLUT4 expression, in creatine-fed rat EDL muscle nuclear extracts. Electrophoretic mobility shift assay showed that DNA binding activity of MEF2 was increased by approximately 40% (P < 0.05) in creatine-fed rat EDL compared with controls. Our data suggest that creatine feeding enhances the nuclear content and DNA binding activity of MEF2 isoforms, which is concomitant with an increase in GLUT4 gene expression.

Levodopa with carbidopa diminishes glycogen concentration, glycogen synthase activity, and insulin-stimulated glucose transport in rat skeletal muscle.

We hypothesized that levodopa with carbidopa, a common therapy for patients with Parkinson's disease, might contribute to the high prevalence of insulin resistance reported in patients with Parkinson's disease. We examined the effects of levodopa-carbidopa on glycogen concentration, glycogen synthase activity, and insulin-stimulated glucose transport in skeletal muscle, the predominant insulin-responsive tissue. In isolated muscle, levodopa-carbidopa completely prevented insulin-stimulated glycogen accumulation and glucose transport. The levodopa-carbidopa effects were blocked by propranolol, a beta-adrenergic antagonist. Levodopa-carbidopa also inhibited the insulin-stimulated increase in glycogen synthase activity, whereas propranolol attenuated this effect. Insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was reduced by levodopa-carbidopa, although Akt phosphorylation was unaffected by levodopa-carbidopa. A single in vivo dose of levodopa-carbidopa increased skeletal muscle cAMP concentrations, diminished glycogen synthase activity, and reduced tyrosine phosphorylation of IRS-1. A separate set of rats was treated intragastrically twice daily for 4 wk with levodopa-carbidopa. After 4 wk of treatment, oral glucose tolerance was reduced in rats treated with drugs compared with control animals. Muscles from drug-treated rats contained at least 15% less glycogen and approximately 50% lower glycogen synthase activity compared with muscles from control rats. The data demonstrate beta-adrenergic-dependent inhibition of insulin action by levodopa-carbidopa and suggest that unrecognized insulin resistance may exist in chronically treated patients with Parkinson's disease.