Nitric oxide increases GLUT4 expression and regulates AMPK signaling in skeletal muscle

Department of Applied Physiology and Kinesiology, Center for Exercise Science, University of Florida, Gainesville, Florida Submitted 17 January 2007 ; accepted in final form 29 July 2007 Nitric oxide (NO) and 5'-AMP-activated protein kinase (AMPK) are involved in glucose transport and mitochondrial biogenesis in skeletal muscle. Here, we examined whether NO regulates the expression of the major glucose transporter in muscle (GLUT4) and whether it influences AMPK-induced upregulation of GLUT4. At low levels, the NO donor S -nitroso- N -penicillamine (SNAP, 1 and 10 µM) significantly increased GLUT4 mRNA ( 3-fold; P < 0.05) in L6 myotubes, and cotreatment with the AMPK inhibitor compound C ablated this effect. The cGMP analog 8-bromo-cGMP (8-Br-cGMP, 2 mM) increased GLUT4 mRNA by 50% ( P < 0.05). GLUT4 protein expression was elevated 40% by 2 days treatment with 8-Br-cGMP, whereas 6 days treatment with 10 µM SNAP increased GLUT4 expression by 65%. Cotreatment of cultures with the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one prevented the SNAP-induced increase in GLUT4 protein. SNAP (10 µM) also induced significant phosphorylation of -AMPK and acetyl-CoA carboxylase and translocation of phosphorylated -AMPK to the nucleus. Furthermore, L6 myotubes exposed to 5-aminoimidazole-4-carboxamide-1- - D -ribofuranoside (AICAR) for 16 h presented an approximately ninefold increase in GLUT4 mRNA, whereas cotreatment with the non-isoform-specific NOS inhibitor N G -nitro- L -arginine methyl ester, prevented 70% of this effect. In vivo, GLUT4 mRNA was increased 1.8-fold in the rat plantaris muscle 12 h after AICAR injection, and this induction was reduced by 50% in animals cotreated with the neuronal and inducible nitric oxide synthases selective inhibitor 1-(2-trifluoromethyl-phenyl)-imidazole. We conclude that, in skeletal muscle, NO increases GLUT4 expression via a cGMP- and AMPK-dependent mechanism. The data are consistent with a role for NO in the regulation of AMPK, possibly via control of cellular activity of AMPK kinases and/or AMPK phosphatases. 5-aminoimidazole-4-carboxamide-1- - D -ribofuranoside; guanosine 3',5'-cyclic monophosphate; L6 myotubes; compound C; N G -nitro- L -arginine methyl ester; glucose transporter 4; adenosine 5'-monophosphate-activated protein kinase Address for reprint requests and other correspondence: D. Criswell, P.O. Box 118206, Center for Exercise Science, Univ. of Florida, Gainesville, FL 32611 (e-mail: dcr