Introduction
Nitric oxide is implicated in a wide array of signalling networks [
1]. In rodents and humans, exercise increases skeletal muscle nitric oxide production, concomitant with increased glucose uptake [
2‐
4]. Nitric oxide biogenesis is catalysed by different isoforms of nitric oxide synthase (NOS) [
5], of which neuronal-type NOS and endothelial-type NOS isoforms are produced in skeletal muscle [
1]. Neuronal-type NOS is produced at higher levels in human skeletal muscle than brain [
6] and therefore is likely to be the major isoform involved in the effect of nitric oxide on glucose metabolism [
1]. Acute administration of the NOS inhibitor NG-monomethyl-
l-arginine during exercise in humans reduces leg muscle glucose uptake [
4], implicating nitric oxide signalling in the mechanism by which exercise controls glucose homeostasis.
The intracellular mechanism by which nitric oxide increases skeletal muscle glucose uptake in humans is incompletely resolved. Intra-femoral artery infusion of a nitric oxide donor increased glucose uptake in healthy participants [
7], as well as in type 2 diabetic patients [
8]. Furthermore, exposure of isolated rat skeletal muscle to the nitric oxide donor sodium nitroprusside increased glucose transport in a dose-dependent manner [
9,
10]. Stimulation of glucose transport by nitric oxide involves the activation of a calcium/contraction- and phosphatidylinositol-3-kinase-independent pathway that acutely increases GLUT4 content at the cell surface [
11]. Nitric oxide seems to exert its action on muscle glucose transport partly via activation of guanylate cyclase, leading to elevation of cyclic GMP (cGMP) levels [
10,
12]. Indeed, the cGMP analogue 8-bromo-cGMP also increases glucose uptake in isolated rat skeletal muscle [
10]. Thus, nitric oxide/cGMP signalling may be part of a novel pathway that regulates skeletal muscle glucose uptake.
The effect of exercise on the acute regulation of skeletal muscle glucose transport has been attributed to several signalling nodes including Ca
2+-calmodulin-dependent protein kinase (CaMK)II, AMP-activated protein kinase (AMPK) and nitric oxide [
13]. In this regard, the interaction between AMPK and nitric oxide signalling pathways is especially intriguing. AMPK is a serine/threonine protein kinase, which acts as a sensor of cellular energy status and regulates a wide variety of gene regulatory and metabolic pathways, including glucose uptake and fatty acid oxidation in skeletal muscle [
14]. AMPK subunits (α1, α2, β1, β2, γ1, γ2, γ3) form a heterotrimeric enzyme consisting of α (catalytic), and β and γ (regulatory) isoforms. In rodent skeletal muscle, sodium nitroprusside increases glucose transport, concomitant with α1-associated AMPK activation [
15]. Moreover, chronic exposure of L6 muscle cells to sodium nitroprusside increases
Glut4 (also known as
SLC2A4) mRNA expression by an AMPK-dependent mechanism [
16], positioning AMPK downstream of nitric oxide signalling. AMPK is also considered to be an upstream kinase for NOS, since it phosphorylates and activates endothelial and neuronal NOS [
17‐
19]. Thus, a positive feedback interaction between AMPK and NOS in the control of skeletal muscle metabolism is implicated [
16].
In the present study, we determined the effect of the nitric oxide donor spermine N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine NONOate on glucose transport and intracellular signalling in isolated human skeletal muscle. Using L6 skeletal muscle cells, we also determined whether spermine NONOate and insulin have additive effects on glucose uptake and intracellular signalling. We hypothesised that pharmacological treatment of human skeletal muscle with a compound that increases cGMP levels may promote glucose uptake.
Discussion
Regular exercise training improves glucose tolerance [
27] and skeletal muscle insulin sensitivity [
28] in type 2 diabetic patients, but the molecular mechanisms are incompletely resolved. Although several strategies designed to enhance compliance with physical activity regimens in patients with insulin resistance have been proposed [
29], many type 2 diabetic patients rely on pharmacological treatments to improve glucose homeostasis. Yet, these current pharmacological treatments to enhance peripheral insulin sensitivity have limited efficacy [
30]. Therefore, insight into novel mechanisms capable of enhancing skeletal muscle glucose uptake could lead to new pharmaceutical strategies to improve treatment and possibly prevent peripheral insulin resistance in patients with type 2 diabetes.
Nitric oxide signalling plays a key role in exercise/contraction-induced metabolic responses in skeletal muscle [
1], partly via an AMPK-dependent mechanism [
15]. During exercise/contraction, increased nitric oxide levels are associated with induction of glucose uptake in skeletal muscle [
2‐
4]. Conversely, NOS inhibition reduces glucose uptake during exercise in type 2 diabetic patients more than in control participants [
31]. Previous reports characterising the direct effects of nitric oxide on glucose uptake are limited to in vitro studies of isolated rodent skeletal muscle [
9‐
11]. Here we provide evidence that pharmacological treatment of human skeletal muscle with the nitric oxide donor spermine NONOate increases glucose transport, concomitant with increased cGMP levels and AMPK-α1-associated activity. Using L6 myotubes (producing only the α1-subunit of AMPK), we provide evidence that spermine NONOate increases phosphorylation of AMPK and ACC, with a concomitant increase in glucose transport and glycogen synthesis. Our findings in human skeletal muscle are compatible with earlier studies in rodent muscle indicating that nitric oxide donors increase cGMP levels and glucose transport [
10]. Similar effects on glucose transport stimulation were also observed in isolated rat skeletal muscle exposed to the cGMP analogues 8-bromo-cGMP [
10] and dibutyryl cGMP [
11]. The mechanism by which cGMP regulates glucose uptake may involve two enzymes, namely guanylate cyclase and phosphodiesterase, but other mechanisms are also likely to be involved. Inhibition of guanylate cyclase prevents the sodium nitroprusside-induced increase in cGMP levels and glucose transport [
10]. Conversely, treatment of isolated skeletal muscle with a phosphodiesterase inhibitor (Zaprinast) increases cGMP levels, with a concomitant increase in glucose uptake [
32]. Collectively, these studies provide evidence that the nitric oxide/cGMP pathway is likely to be important in the regulation of glucose transport.
Nitric oxide signalling has been linked to AMPK activation and glucose uptake [
15,
16]. Here, we provide evidence that in vitro exposure to a nitric oxide donor increases glucose transport, with concomitant increase in AMPK-α1-specific activity and AMPK-α1 phosphorylation in isolated human skeletal muscle and L6 myotubes. Among the 12 possible AMPK heterotrimers, only three (α1β2γ1, α2β2γ1, α2β2γ3) have been identified in human skeletal muscle [
33]. Each individual heterotrimer is activated in a manner dependent on time and intensity [
34,
35], which may elicit signalling specificity in response to a distinct set of stimuli. Our results in human skeletal muscle and L6 myotubes provide evidence that nitric oxide specifically increases AMPK-α1-associated activity and AMPK-α1 phosphorylation respectively. Moreover in L6 myotubes, the guanylate cyclase enzyme inhibitor LY-83583 prevents the nitric oxide donor-induced increase in glycogen synthesis and α1-AMPK phosphorylation, providing evidence for a potential role of the AMPK-α1 subunit in the mechanism by which nitric oxide donors increase glucose transport. These observations are consistent with a previous study in rodent skeletal muscle [
15], which provided evidence that sodium nitroprusside increases AMPK-α1-, but not AMPK-α2-associated activity. Furthermore, in rodent skeletal muscle, sodium nitroprusside-induced increases in AMPK-isoform-specific activity occurred independently of changes in ATP, creatinine phosphate or glycogen levels [
15]. Thus, the nitric oxide–cGMP pathway may be involved in the regulation of AMPK-α1 activity and/or inhibition of the protein phosphatase responsible for AMPK regulation.
Although most
Ampk (also known as
Prkaa2) knockout models provide evidence against a critical role for the AMPK-α1 subunit in the regulation of skeletal muscle glucose uptake [
14], AMPK-α1 activation is required for stimulation of glucose uptake in response to twitch contraction [
36]. Based on our results, the promotion of glucose uptake effected by the nitric oxide donor appears to be at least partly mediated via AMPK complexes containing the α1 subunit. However, the role of AMPK in nitric oxide signalling is complex, since AMPK has also been suggested to be an upstream kinase for NOS [
17‐
19,
37]. Direct activation of AMPK, using 5′-aminoimidazole-4-carboxamide ribonucleoside (AICAR), stimulates nitric oxide production in human aortic endothelial cells [
37] and increases NOS activity in H-2K
b cells [
17], implicating a feedback loop between AMPK and NOS [
16]. The question of whether nitric oxide pathways lie upstream or downstream of AMPK remains unresolved. For example, AICAR-induced AMPK-activated glucose transport is unaltered by NOS inhibition in isolated rat skeletal muscle [
38]. The direct interaction between the nitric oxide/cGMP pathway and AMPK warrant further investigation.
To determine the intracellular mechanism by which the nitric oxide donor spermine NONOate increases glucose transport, components of the canonical insulin signalling cascade were assessed. The Akt- and Rev/Rex activation domain-binding protein (Rab) GTPase-activating proteins, TBC1D1 and TBC1D4, are the most distal signalling proteins implicated in GLUT4 translocation [
39,
40]. In human skeletal muscle, insulin exposure led to an expected increase in phosphorylation of Akt, TBC1D1/D4 (detected using a PAS antibody) and GSK3, while exposure to the nitric oxide donor had no effect. These data indicate that nitric oxide-stimulated glucose transport is mediated via an insulin-independent pathway. Nitric oxide has also been linked as a positive [
16] modulator of GLUT4 production. However, GLUT4 protein content was unaltered in response to spermine NONOate (data not shown), presumably because of the shorter incubation time and low concentration of the nitric oxide donor used in this study. CaMKII signalling has been implicated in the mechanism by which muscle contraction increases glucose uptake [
13]. Nevertheless, CaMKII phosphorylation was unaltered in response to insulin or spermine NONOate. In contrast to our results for AMPK signalling, insulin and CaMKII signalling do not appear to play a role in nitric oxide action on skeletal muscle glucose transport. In L6 myotubes, the nitric oxide donor and insulin had an additive effect on glycogen synthesis and glucose uptake without further increase in insulin-induced Akt and TBC1D1/D4 phosphorylation. These results suggest that nitric oxide promotes glucose uptake by an insulin-independent mechanism. Furthermore, the stimulatory effect of the nitric oxide donor on AMPK and ACC phosphorylation under insulin-stimulated conditions was prevented, excluding a role for AMPK signalling in the additive effect on glycogen synthesis and glucose uptake.
In summary, the nitric oxide donor spermine NONOate increases cGMP levels and promotes glucose transport, concomitantly with AMPK-α1-isoform-specific activation in human skeletal muscle. Further study to delineate mechanisms and the therapeutic window is warranted. Spermine NONOate also increased glucose transport in L6 myotubes, concomitantly with an increase in AMPK-α1-isoform-specific phosphorylation. Moreover, these effects were prevented in presence of a guanylate cyclase inhibitor. Further studies on the mechanisms by which AMPK-α1-isoform-specific signalling is directly linked to nitric oxide action are warranted. Taken together with recent evidence showing that sodium nitroprusside increased glucose uptake in human primary myotubes derived from healthy volunteers and patients with type 2 diabetes [
41], our findings have clear clinical implications, since manipulation of the nitric oxide/cGMP signalling cascade could enhance glucose uptake by an insulin-independent mechanism to potentially improve whole-body glucose homeostasis in type 2 diabetic patients.
Acknowledgements
This work was supported by grants from the European Research Council, the Swedish Research Council, the Swedish Diabetes Association, Novo Nordisk Foundation, the Foundation for Scientific Studies of Diabetology, the Swedish Centre for Sports Research, the Strategic Research Foundation, the Commission of the European Communities (Contract numbers LSHM-CT-2004-005272 EXGENESIS and LSHM-CT-2004-512013 EUGENE2). Additional support to H. A. Koistinen was provided by grants from Finnish Academy of Science and Sigrid Juselius Foundation.