Does Nitric Oxide Regulate Skeletal Muscle Glucose Uptake during Exercise?

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1 ARTICLE Does Nitric Oxide Regulate Skeletal Muscle Glucose Uptake during Exercise? Glenn K. McConell 1 and Bronwyn A. Kingwell 2 1 Department of Physiology, The University of Melbourne, Parkville, Australia; and 2 Alfred & Baker Medical Unit, Baker Medical Research Institute, Melbourne, Victoria, Australia MCCONELL, G.K., and B.A. KINGWELL. Does nitric oxide regulate skeletal muscle glucose uptake during exercise? Exerc. Sport Sci. Rev., Vol. 34, No. 1, pp , Although rodent studies are contradictory, there is accumulating evidence in humans suggesting that nitric oxide (NO) is involved in skeletal muscle glucose uptake during exercise. This brief review discusses this controversial area, including potential upstream regulators of skeletal muscle NO synthase (NOS). Key Words: nitric oxide synthase, AMP-activated protein kinase, calcium, peroxynitrite, contraction INTRODUCTION The rate of increase in the prevalence of diabetes in the Western world has been described as an epidemic. Type 2 diabetes is the predominant form of diabetes, accounting for more than 85% of people with diabetes. The uptake and metabolism of glucose by skeletal muscle is a major determinant of whole body glucose homeostasis. People with type 2 diabetes have relatively normal levels of the glucose transporter GLUT-4 in their skeletal muscle, but GLUT-4 translocation from intracellular vesicles to the plasma membrane in response to insulin is reduced. This is a major contributor to the reduced insulin-stimulated skeletal muscle glucose uptake in people with type 2 diabetes. Importantly, however, skeletal muscle GLUT-4 translocation and glucose uptake during exercise are normal in people with type 2 diabetes. As a result, the blood glucose concentration can decrease substantially during strenuous aerobic exercise in people with type 2 diabetes. Exercise is thus an effective preventive and treatment option for diabetes, but unfortunately, many people with diabetes do not (or cannot) exercise regularly. Alternatives therapies therefore are critical to manage diabetes effectively. Understanding the signaling pathways linking exercise and skeletal muscle glucose uptake may provide potential targets for Address for correspondence: Glenn K. McConell, Ph.D., Department of Physiology, The University of Melbourne, Parkville, Victoria, 3010, Australia ( mcconell@unimelb.edu.au). Accepted for publication: September 25, Associate Editor: Mark Hargreaves, Ph.D /3401/36 41 Exercise and Sport Sciences Reviews Copyright 2006 by the American College of Sports Medicine new therapies. The intramuscular signaling pathways associated with exercise-stimulated glucose uptake are not fully understood, but are known to differ from the insulin-mediated pathway. Possible candidates include calcium, calmodulin-dependent protein kinase (CaMK), protein kinase C (PKC), p38 mitogen-activated protein kinase (MAPK), bradykinin, adenosine, AMP-activated protein kinase (AMPK), and nitric oxide (NO). It is likely that more than one regulator is involved in the control of contraction-stimulated skeletal muscle glucose uptake and that some redundancy exists. This paper focuses on the potential role of NO/NO synthase (NOS) in this process. The biogenesis of NO is catalyzed by NOS enzymes, which form L-citrulline and NO from L-arginine, oxygen, and nicotinamide adenine dinucleotide phosphate (NADPH)-derived electrons. Neuronal NOS mu (nnos ) is highly expressed within human skeletal muscle (Fig. 1) and is likely to be the isoform most relevant to glucose uptake. NITRIC OXIDE INCREASES SKELETAL MUSCLE BASAL GLUCOSE DISPOSAL Studies in rats have shown universally that NO donors increase skeletal muscle GLUT-4 translocation (6) and glucose uptake and transport (1,7,14). Although increases in glucose uptake and transport have been observed with 1 mm sodium nitroprusside (SNP) (1), in other studies (6,7), 10 mm SNP was required. This is a pharmacological dose that yields NO levels well above those associated with contraction and exercise. In humans, SNP infusion into the femoral artery increases basal leg glucose uptake in healthy individuals (5). 36

2 Figure 1. nnos and enos protein expression in human skeletal muscle. Representative gel showing (A) nnos and (B) enos protein from muscle biopsy samples from four different humans. Recombinant human nnos and enos were used as positive controls (note that recombinant nnos is smaller than nnos since nnos has 34 extra amino acids). (McConell et al., unpublished observations.) NITRIC OXIDE AND CONTRACTION-STIMULATED GLUCOSE UPTAKE Human Studies We have found that femoral artery infusion of the NOS inhibitor N G -monomethyl L-arginine (L-NMMA) during cycle exercise reduces leg glucose uptake during exercise by 40 50% in young, healthy humans (3). This inhibitory effect seems to be reversed by infusion of the NOS substrate L-arginine (Fig. 2) (3). In addition, in a similar design, we found that NOS inhibition reduced leg glucose uptake during exercise to a greater extent in people with type 2 diabetes than in matched controls (8). In that study, leg glucose uptake was close to identical in the two groups after 10 min of exercise (type 2 group, mmol min 1 ; control group, mmol min 1 ) and then decreased significantly after 5 min of NOS inhibition to mmol min 1 in the people with type 2 diabetes and mmol min 1 in the matched controls (8). In both studies there was no effect of NOS inhibition on leg blood flow, blood pressure, or arterial plasma glucose or insulin concentration. We also recently found that infusion of the NOS precursor L-arginine (0.5 g min 1 ) during prolonged exercise in healthy nondiabetic humans significantly increased glucose disposal during exercise (Fig. 3) (9). L-arginine infusion significantly reduced the plasma glucose concentration and increased the rate of glucose disappearance (glucose Rd) as determined by stable glucose tracer infusion. The glucose clearance rate (glucose Rd/plasma glucose concentration) was higher during L-arginine infusion compared with saline infusion (Fig. 3). Given that we (3) and others have previously shown that L-arginine infusion has no effect on blood flow during exercise in humans, and given that L-arginine infusion had no significant effect on plasma insulin concentration, we hypothesize that L-arginine infusion increased NO production by skeletal muscle NOS, which then increased muscle glucose uptake. It has been shown that L-arginine increases basal NO production by rat skeletal muscle in vitro (2). These results in humans suggest that NO has a role in the regulation of skeletal muscle glucose uptake during exercise and that individuals with type 2 diabetes have a greater reliance on NO for glucose uptake during exercise than healthy controls. Rodent Studies Studies in rodents examining the effect of NOS inhibition on glucose transport and uptake with exercise and contraction have yielded contradictory results (Table 1) (1,6,7,10 12). Only one of these studies can be compared directly with our human studies in that glucose uptake was examined during, rather than after, the exercise bout (11). In this study in mice, ingestion of the NOS inhibitor nitro-l-arginine methyl ester (L-NAME) for 3 d had no effect on 2-deoxyglucose uptake during 30 min of treadmill exercise. It should be kept in mind, however, that ingestion of L-NAME over several days in rodents has been shown to increase blood pressure and causes insulin resistance, which may have impacted on the results. All of the other rodent studies involving NOS inhibition assessed glucose transport and uptake after rather than during the series of contractions or treadmill exercise (1,6,7,10,12). Most of these studies (1,6,7,12) actually measured glucose transport and uptake 20 min or more after contractions and exercise (Table 1), so their relevance to the regulation of glucose uptake during exercise is questionable. In the in vitro studies (6,7,12), muscle contractions were undertaken in the presence of 100 M of L-NMMA, and then glucose uptake (7,12) or transport (6) was measured at least 20 min after the contractions ceased using labeled glucose analogs. In two of the studies, NOS inhibition had no effect on contraction-stimulated glucose uptake and transport (6,7), whereas in the third, glucose uptake was significantly reduced (12). The reason for this discrepancy is unclear but may be related to seemingly minor methodological differences. The study by Higaki et al. (7) involved EDL and soleus muscles, whereas the other two involved epitrochlearis muscle (6,12). In addition, the Etgen et al. (6) study was conducted in female rats, whereas the study by Stephens et al. (12) was conducted in male rats. Volume 34 Number 1 January 2006 Exercise and Muscle Glucose Uptake 37

3 Figure 2. Effect of NOS inhibition during exercise in young, healthy males. Leg blood flow (top panel), leg a-v glucose difference (middle panel), and leg glucose uptake (bottom panel) in six healthy young male subjects (N 7 for a-v glucose difference) during 30 min of supine cycling exercise at 60 2%V O2 peak. N G -monomethyl-larginine (L-NMMA; total dose, 5 mg kg 1 body weight; closed circles) or saline (control; open circles) was infused over the last 20 min of exercise. L-arginine (5 mg kg 1 body weight) was coinfused during the last 5 min of exercise. Values are mean SEM. *P 0.05 between trials. (Reprinted from Bradley, S.J., B.A. Kingwell, and G.K. McConell. Nitric oxide synthase inhibition reduces leg glucose uptake but not blood flow during dynamic exercise in humans. Diabetes 48: , Copyright 1999 The American Diabetes Association. Used with permission.) In other studies (1,7), muscle contractions were undertaken in male rats via sciatic nerve electrical stimulation in situ without NOS inhibition present, and then the EDL muscle was dissected out and exposed to L-NMMA ex vivo. In one study, glucose uptake was reduced to the basal level by NOS inhibition (1), whereas in the other study, there was no effect of the NOS inhibitor (7). The major difference between these studies was that in one study, the muscle was exposed to L-NMMA for 70 min after contractions ceased before the glucose uptake measurements were conducted (1), whereas in the other study, the muscle was exposed to L-NMMA for 20 min (7). Also it seems that younger rats were used by Higaki et al. (7) than Balon and Nadler (1). The fact that there was no NOS inhibition present during contractions in these in situ contraction studies means that they have little relevance to the regulation of glucose uptake during exercise and are more related to the possible role of NO in the higher glucose disposal observed after exercise compared with rest. Two rat studies used in vivo exercise with rats running on a treadmill after ingestion of the NOS inhibitor L-NAME over the 2 d before the exercise (7,10). The L-NAME dose delivered reduces skeletal muscle NOS activity at rest by 75% to 90% (e.g., (7)). Roberts et al. (10) found that NOS inhibition prevented the increase in glucose uptake and GLUT-4 translocation with exercise, whereas Higaki et al. (7) found no effect of NOS inhibition on glucose uptake with exercise. The main difference between these two studies was that in Higaki et al. (7), the muscle was dissected and then glucose uptake was examined longer than 20 min after the exercise ceased, whereas Roberts et al. (10) immediately killed the rats and froze the muscle to examine glucose uptake later in sarcolemmal vesicles. It is likely that the protocol of Roberts et al. (10) more closely reflected exercise metabolism. In addition, Higaki et al. (7) presented results only from slow twitch soleus muscle, whereas Roberts et al. (10) used a mixture of muscles (see Table) which they, perhaps not quite correctly, designated fast muscles. nnos protein and NOS activity are much greater in rat fast-twitch muscle than slow-twitch muscle, so it is perhaps not surprising that NOS inhibition had little effect on soleus muscle in the study by Higaki et al. (7). The reason(s) for the different results observed among some of the rodent studies is difficult to reconcile fully. When considering the disparity between some of the rodent studies and our human studies, it is also worth considering that species differences, including evidence that NOS expression is greater in slow-twitch than fast-twitch muscle fibers in humans, whereas the opposite is the case in rats, may be a consideration. Figure 3. Effect of L-arginine infusion during prolonged exercise in humans. Plasma glucose concentration (A) and glucose clearance rate (Glucose CR) (B) at rest during 120 min of steady-state exercise at 72 1% V O2 peak and at the end of an approximately 15-min performance time-trial (TT) ride ( kj) in CON and L-Arg conditions. Values are mean SEM (N 9). *P 0.05 vs CON. Data from (9). 38 Exercise and Sport Sciences Reviews

4 TABLE Effect of NOS inhibition on contraction-stimulated glucose uptake in rodents Model Used and Publication Species/Muscles Studied/Gender Type of Exercise/Contraction Inhibitor and Dose When and How Glucose Uptake Measured Effect on Glucose Uptake In vitro contraction Stephens et al. (12) Rat, epitrochlearis, male Electrical stimulation in vitro Etgen et al. (6) Rat, epitrochlearis, Electrical female stimulation in vitro Higaki et al. (7) Rat, EDL and Electrical soleus, male stimulation in vitro In situ contraction Balon and Nadler (1) Rat, EDL, male Electrical stimulation in situ Higaki et al. (7) Rat, EDL, male Electrical stimulation in situ In vivo exercise Roberts et al. (10) Rat, combined Treadmill running muscles, female 100 M L-NMMA 20 min after contractions, in vitro 100 M L-NMMA 10 min after contractions, in vitro 100 M L-NMMA 20 min after contractions, in vitro 1 M? L-NMMA 70 min after contractions, ex vivo 100 M L-NMMA 20 min after contractions, ex vivo 2 d of 1 mg ml 1 L-NAME ingestion Higaki et al. (7) Rat, soleus, male Treadmill running 2 d of 1 mg ml 1 Rottman et al. (11) Mouse; several muscles, gender not specified Treadmill running L-NAME ingestion 3 d of 1 mg ml 1 L-NAME ingestion Muscle frozen immediately after exercise, sarcolemmal vesicles Muscle dissected, in vitro During exercise, in vivo Contraction effect attenuated Contraction effect abolished (same as rested muscle) Exercise effect abolished (same as rested muscle) HOW DOES NITRIC OXIDE INCREASE SKELETAL MUSCLE GLUCOSE UPTAKE DURING EXERCISE? If NO does indeed play a role in the regulation of skeletal muscle glucose uptake during exercise, it is likely to do so either by increasing GLUT-4 translocation or by shunting blood preferentially into so-called nutritive vascular beds. Although we (3,8), and others, have found that total leg blood flow is unaffected by infusion of a NOS inhibitor during exercise in humans, it is possible that NOS inhibition caused some of the blood within the muscle to be shunted from nutritive (capillary) vessels to nonnutritive vessels (connective tissue, etc.). This would reduce glucose extraction and therefore glucose uptake without altering total blood flow. However, the fact that leg lactate release, oxygen consumption, and femoral artery blood pressure were unchanged with NOS inhibition (3,8), which would be expected to have changed if there had been reduced nutritive blood flow, argues against this possibility. In addition, preliminary evidence recently was presented at the 2005 American Diabetes Association conference by Vincent et al. demonstrating that NOS inhibition has no affect on nutritive flow during in situ muscle contractions in rats. We hypothesize that NOS inhibition in humans decreased glucose uptake during exercise by reducing GLUT-4 translocation to the plasma membrane. To clarify the precise mechanism, human studies investigating the effect of NOS inhibition on both GLUT-4 translocation and capillary nutritive flow during exercise, although technically difficult, are warranted. The signaling events downstream of NO/NOS are unclear. In recent years, nnos has been identified within skeletal muscle fibers of humans and rats. The endothelial isoform of NOS (enos) also is expressed within skeletal muscle, although in humans it seems that enos is confined to the endothelium and is not expressed within the skeletal muscle fibers themselves. We and others have found much higher protein expression of nnos than enos in human skeletal muscle (Fig. 1). We and others also have found that all the nnos expressed in human skeletal muscle (nnos ) contains an additional 102 nucleotides than nnos, the major brain isoform. We also have found that skeletal muscle nnos is lower in people with type 2 diabetes than matched controls and that skeletal muscle nnos is higher in well-trained endurance athletes than in controls (Bradley et al., unpublished observations). As occurs in smooth muscle cells, NO seems to exert its actions, at least in part, by modulating cyclic guanosine monophosphate (cgmp) levels in skeletal muscle. Increases in NO stimulate soluble guanylate cyclase, producing cgmp. In skeletal muscle, NO donors such as SNP raise skeletal muscle cgmp content (13,15) and increase glucose uptake, presumably as a result of activation of guanylate cyclase (7,13,15). Indeed, addition of LY-83583, an inhibitor of soluble guanylate cyclase, decreased skeletal muscle cgmp content and reduced basal 2-deoxyglucose transport in isolated rat muscle to a similar extent as L-NMMA (15). LY also completely abolished SNP augmented glucose transport (15). Finally, the cgmp analog 8-bromo-cGMP increases glucose uptake in isolated rat muscle (14,15). In mice and frogs, contraction increases skeletal muscle cgmp levels. We hypothesize that the increase in cgmp during exercise is in response to NO-mediated activation of guanylate cyclase (see Fig. 4). We further suggest that this increase in cgmp stimulates glucose uptake, possibly via cgmp-dependent protein kinase (14). In incubated rat soleus muscle, the addition of Zaprinast, a specific inhibitor of cgmp type 5 phosphodiesterase, reduced the breakdown of cgmp and increased skeletal Volume 34 Number 1 January 2006 Exercise and Muscle Glucose Uptake 39

5 also that no study has examined skeletal muscle NO release or NOS activity during exercise in humans or the effect of NOS inhibition on NO production during exercise. Increases in muscle cytosolic Ca 2 levels and the activation of muscle AMPK, both of which occur during exercise, may be responsible, at least in part, for the increase in skeletal muscle glucose uptake during exercise (see Fig. 4). Activation of AMPK increases rat skeletal muscle glucose uptake, as do increases in cytosolic Ca 2. Interestingly, these effects are additive and equal to the stimulation of glucose uptake in response to contraction (in muscle comprising predominately fast-twitch fibers; the evidence is less clear in slow muscle). As explained below (and see Fig. 4), we hypothesize that increases in cytosolic Ca 2 and activation of AMPK during exercise may increase glucose uptake, at least partly, through activation of skeletal muscle nnos. In addition, we hypothesize that increases in ROS during exercise, which increase muscle glucose uptake and activate AMPK, may also operate via NOS (Fig. 4). Figure 4. Speculative pathway for nitric oxide regulation of glucose uptake during exercise. AMPK, AMP-activated protein kinase; ONOO, peroxynitrite; H 2 O 2, hydrogen peroxide; cgmp, cyclic guanosine monophosphate. muscle cgmp levels by approximately 90% (13). Zaprinast also substantially increased glucose uptake into the isolated rat muscle (13). This result highlights cgmp as a potential therapeutic target for controlling blood glucose. Indeed, we have preliminary evidence that femoral artery infusion of the 5 phosphodiesterase inhibitor sildenafil tends to increase leg glucose uptake in healthy humans. However, 5 phosphodiesterase inhibitors have only a minor effect on skeletal muscle cgmp levels and glucose uptake in obese insulin-resistant Zucker rats (13). As discussed below, reactive oxygen species (ROS), which are known to increase during exercise, have been shown to increase skeletal muscle glucose uptake. Because some ROS are generated from NO (e.g., peroxynitrite), it is possible that this forms a non-cgmp NO-related signaling mechanism in muscle during exercise. The distal steps regulating GLUT-4 translocation in response to NO are not known. WHAT STIMULATES THE INCREASE IN SKELETAL MUSCLE NITRIC OXIDE DURING EXERCISE? Nitric oxide release is increased by electrical stimulation in isolated rat skeletal muscle (2). Furthermore, in rat skeletal muscle, NOS activity is increased by in vitro muscle contraction as well as by treadmill running. The mechanism(s) responsible for the increase in NO production and the corresponding increase in NOS activity in skeletal muscle with exercise and contraction have not been fully elucidated. It should be noted Calcium It is necessary for calmodulin to bind to nnos in a calcium-dependent manner before nnos can be activated. Therefore, it is logical to hypothesize that in human skeletal muscle, calcium released from the sarcoplasmic reticulum during excitation contraction coupling also stimulates nnos (and also enos in rodent muscle), which then raises skeletal muscle NO levels (see Fig. 4). It has been shown repeatedly that raising cytosolic calcium concentration in rat isolated skeletal muscle increases glucose uptake. The increase in calcium concentration is achieved by either exposure to caffeine, which increases sarcoplasmic reticulum calcium release, or exposure to a calcium ionophore. Although at least some of the effect of calcium to increase skeletal muscle glucose uptake seems to be via CaMK, PKC, or both, it would be useful to examine whether calcium-stimulated skeletal muscle glucose uptake can be attenuated by a NOS inhibitor (Fig. 4). AMPK Recently, there has been enormous interest in the potential role of skeletal muscle AMPK in exercise-stimulated glucose uptake and as a possible target for antidiabetic drugs. AMPK has been described as the energy sensor of the mammalian cell. 5-Aminoimidazole-4-carboxyamide-ribonucleoside (AICAR), a cell-permeable activator of AMPK, increases rat skeletal muscle glucose uptake in vitro and in vivo. Like exercise, AICAR increases the translocation of GLUT-4 to the sarcolemma and seems to act via an insulin-independent mechanism. During exercise in humans, AMPK phosphorylates nnos at Ser1451 in skeletal muscle (4). It has been suggested that the increased phosphorylation of skeletal muscle nnos by AMPK during exercise may account for the increase in glucose uptake during exercise (see Fig. 4). Indeed, a study in rats found that AICAR increased NOS activity and glucose uptake in muscle strips and that these effects could be prevented by NOS inhibition. In addition, the increase in skeletal muscle glucose uptake by AICAR in vivo in rats can be blocked by prior ingestion of a NOS inhibitor (although this could well be largely a blood flow effect). However, we (12) and others have found that NOS inhibition has no effect on AICAR-activated glucose transport 40 Exercise and Sport Sciences Reviews

6 in isolated rat muscle. In humans, we recently found a relationship between AMPK activity, nnos phosphorylation by AMPK, and glucose uptake during exercise of increasing intensities (4). However, this does not prove cause and effect, and it therefore remains unclear whether AMPK activates glucose uptake during exercise by phosphorylating and activating nnos. It should also be noted that we and others have evidence that there may be situations where contraction-stimulated glucose uptake and AMPK activity are uncoupled. Reactive Oxygen Species (Peroxynitrite and Hydrogen Peroxide) Although it is likely that the major reason for the increase in skeletal muscle AMPK activity during exercise is the result of elevation in skeletal muscle-free AMP (either directly or via AMPKK), it is possible that other factors are responsible, at least in part, for this increase in AMPK activity during exercise. Indeed, AMPK is activated in several situations where there is no increase in free AMP, for example during hyperosmotic stress. The ROSs peroxynitrite and hydrogen peroxide (H 2 O 2 ) both increase in skeletal muscle during exercise and both have been shown to activate AMPK without altering the free AMP concentration. Peroxynitrite is formed when superoxide and NO combine. In endothelial cells, the activation of AMPK by peroxynitrite can be inhibited by an NOS inhibitor, and there are indications that metformin, which activates AMPK in skeletal muscle, may do so via interaction with peroxynitrite. Finally, it is also possible that peroxynitrite and hydrogen peroxide may activate glucose uptake independently of AMPK. We therefore are left with a potential situation where, during exercise, AMPK phosphorylates nnos, but also an increase in NO production (either via AMPK or calcium or in other ways) activates AMPK (via production of peroxynitrite). Much work will be required to clarify whether this situation occurs during exercise. FUTURE RESEARCH It is important that other groups examine the effect of NOS inhibition on glucose uptake during exercise in humans, coupled if possible with measures of GLUT-4 translocation and nutritive (capillary) blood flow. It is also necessary to attempt to reconcile more completely the reason for the discrepant findings in rodents concerning the effects of NOS inhibition on glucose uptake associated with exercise and contraction. In addition, it is important to determine whether the increase in skeletal muscle glucose uptake during exercise is attenuated in nnos deficient mice. If NO does indeed regulate, at least in part, GLUT-4 translocation and glucose uptake during exercise, it is necessary to determine the downstream signaling involved. CONCLUSIONS Although rodent studies are conflicting, there is evidence in humans that NO is important for the regulation of skeletal muscle glucose uptake during exercise. Activation of NOS during exercise may be the result of one or more of the following: increases in intracellular calcium, activation of AMPK, increases in ROSs, or a combination thereof. It seems likely that the cgmp second messenger system links NO production to skeletal muscle glucose uptake, although the exact downstream signaling mechanisms remain to be elucidated. References 1. Balon, T.W., and J.L. Nadler. Evidence that nitric oxide increases glucose transport in skeletal muscle. J. Appl. Physiol. 82: , Balon, T.W., and J.L. Nadler. Nitric oxide release is present from incubated skeletal muscle preparations. J. Appl. Physiol. 77: , Bradley, S.J., B.A. Kingwell, and G.K. McConell. Nitric oxide synthase inhibition reduces leg glucose uptake but not blood flow during dynamic exercise in humans. 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