Nitric Oxide-dependent Regulation Of Glut4 Expression In L6 Myotubes

Abstract

The upregulation of GLUT4 expression is one of the most important exercise-induced adaptations leading to improved glucose disposal in skeletal muscle. The GLUT4 gene is transcriptionally regulated and AMP-activated protein kinase (AMPK) is involved in this process. Previous studies in rats and humans have shown that AMPK is capable of phosphorylating and activating nitric oxide synthase (NOS). PURPOSE We hypothesized that NOS inhibition would prevent AMPK-induced upregulation in GLUT4 mRNA in L6 myotubes. METHODS Myoblasts were cultured in growth medium (10% FBS) until reaching 80% con.uence, then switched to differentiation medium (2%HoS) until myotubes were 80% confluent. At this point, myotubes were exposed to treatment medium (10%HoS + 5%FBS) for 16 h, with or without the AMPK activator, AICAR (5- aminoimidazole-4-carboxamide riboside, 1mM) or AICAR + the NOS inhibitor, LNAME (N(G)-L-nitro-arginine methyl ester,100μm). RESULTS AICAR induced ∼9-fold increase in GLUT4 mRNA levels in comparison to the control group (n=5–6/group; p = .0012), whereas co-treatment with L-NAME prevented this effect (p = .42). A dose-dependent positive effect of nitric oxide on GLUT4 expression was confirmed by incubation of myotubes with the nitric oxide donor, SNAP. Additionally, treatment with the cell permeable cGMP analog, 8-BrcGMP (1mM) for 16 h increased GLUT4 mRNA levels by 85% (n=6/group; p = .002). CONCLUSIONS These findings suggest that nitric oxide plays a role in the AMPK-induced upregulation of GLUT4 mRNA in skeletal muscle, and may do so by increasing intracellular levels of cGMP. Supported by UF Opportunity Fund.