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Role of the nitric oxide pathway in AMPK-mediated glucose uptake and GLUT4 translocation in heart muscle

¹Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520; ²National Institutes of Health, Bethesda, Maryland 20892; and ³Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom

Submitted 1 June 2004 ; accepted in final form 28 June 2004

	ABSTRACT

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AMP-activated protein kinase (AMPK) is a serine-threonine kinase that regulates cellular metabolism and has an essential role in activating glucose transport during hypoxia and ischemia. The mechanisms responsible for AMPK stimulation of glucose transport are uncertain, but may involve interaction with other signaling pathways or direct effects on GLUT vesicular trafficking. One potential downstream mediator of AMPK signaling is the nitric oxide pathway. The aim of this study was to examine the extent to which AMPK mediates glucose transport through activation of the nitric oxide (NO)-signaling pathway in isolated heart muscles. Incubation with 1 mM 5-amino-4-imidazole-1--carboxamide ribofuranoside (AICAR) activated AMPK (P < 0.01) and stimulated glucose uptake (P < 0.05) and translocation of the cardiomyocyte glucose transporter GLUT4 to the cell surface (P < 0.05). AICAR treatment increased phosphorylation of endothelial NO synthase (eNOS) 1.8-fold (P < 0.05). eNOS, but not neuronal NOS, coimmunoprecipitated with both the ₂ and ₁ AMPK catalytic subunits in heart muscle. NO donors also increased glucose uptake and GLUT4 translocation (P < 0.05). Inhibition of NOS with N-nitro-L-arginine and N-methyl-L-arginine reduced AICAR-stimulated glucose uptake by 21 ± 3% (P < 0.05) and 25 ± 4% (P < 0.05), respectively. Inhibition of guanylate cyclase with ODQ and LY-83583 reduced AICAR-stimulated glucose uptake by 31 ± 4% (P < 0.05) and 22 ± 3% (P < 0.05), respectively, as well as GLUT4 translocation to the cell surface (P < 0.05). Taken together, these results indicate that activation of the NO-guanylate cyclase pathway contributes to, but is not the sole mediator of, AMPK stimulation of glucose uptake and GLUT4 translocation in heart muscle.

nitric oxide synthase; AMP-activated protein kinase; glucose transporter

The mechanisms through which AMPK modulates glucose uptake are only partially understood and may involve both acute and chronic changes in glucose uptake. AMPK increases glucose transport by stimulating translocation of GLUT4 to the sarcolemma in heart (36) and skeletal muscle (25). In Clone 9 cells, AICAR increases GLUT1 activity without a change in content or distribution (1). Long-term AMPK activation with AICAR also increases the expression of GLUT4 in skeletal muscle (17). However, AMPK is not necessary for GLUT4 expression in muscle tissues, as transgenic mice with AMPK deficiency do not have reduced GLUT4 content (29, 35, 45).

The downstream mechanisms through which AMPK mediates the acute activation of glucose transport remain uncertain. However, AMPK may potentially mediate its effect on glucose transport in part through interaction with the nitric oxide pathway. AMPK phosphorylates endothelial nitric oxide synthase (eNOS) on Ser¹¹⁷⁷ (7, 8, 28), leading to NOS activation in a calcium-independent fashion. AMPK also phosphorylates muscle neuronal NOS (nNOS) on Ser¹⁴⁵¹ (7), although the functional significance of this interaction remains uncertain. In addition, NOS inhibition with N-nitro-L-arginine (L-NAME) reduces AICAR stimulation of glucose transport in skeletal muscle (12), and recent findings indicate that L-NAME also partially inhibits AICAR stimulation of muscle deoxyglucose uptake in the rat (40). However, the extent to which NOS modulates glucose uptake and GLUT translocation in heart muscle remains uncertain.

Nitric oxide produced in endothelial cells may have an important paracrine role to modulate myocyte metabolism and function in muscle tissues. Nitric oxide has pleiotropic effects, depending in part on its concentration, and both the metabolic and hemodynamic states of the heart (39). In skeletal muscle from eNOS knockout mice, there is diminished insulin-stimulated glucose uptake, indicating that insulin activation of nitric oxide may contribute to the stimulation of glucose transport (5, 10, 42). At the present time, the role of the nitric oxide pathway in mediating AMPK’s action in the heart is unknown.

The purpose of this study was to determine whether AMPK activation of heart glucose transport and GLUT4 translocation is mediated in part through interaction with and activation of NOS. An isolated rat left ventricular papillary muscle preparation was utilized to assess these effects independently of the hemodynamic and systemic consequences of NOS inhibition. The results indicate that AMPK binds to and phosphorylates eNOS in heart muscle and that nitric oxide and its downstream cGMP pathway modulate in part AMPK activation of glucose uptake and GLUT4 translocation in heart muscle.

	MATERIALS AND METHODS

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Animal preparationss. Male Sprague-Dawley rats weighing 250–350 g were allowed to have standard chow and water ad libitum before experiments. All procedures were approved by the Yale University Animal Care and Use Committee. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg). Anterior and posterior left ventricular papillary muscles (3–5 mg) were equilibrated in oxygenated phosphate-buffered saline (PBS) containing 1 mM MgCl₂, 1 mM CaCl₂, 5 mM glucose, and 1% BSA, as previously described (36). There was constant oxygen flow through the sealed muscle incubation containers, which were oscillated in a water bath at 37°C. Papillary muscles were then preincubated in buffer containing inhibitor, or inhibitor vehicle as control, for 30 min before the addition of pharmacological activators for 30–60 min, as designated in RESULTS. NOS and guanylate cyclase inhibitors were used in optimally effective concentrations (33).

Glucose transport. In experiments designed to assess glucose transport, papillary muscles were incubated as above, and 2-deoxy-[1-³H]glucose (1 µCi/ml) was added during the final 30 min of incubation to measure the rates of glucose transport and phosphorylation. In addition, [U-¹⁴C]mannitol (0.1 µCi/ml) was added to measure the muscle extracellular space to correct for extracellular deoxyglucose (36). After incubations, muscles were washed in ice-cold PBS, blotted dry, weighed, solubilized, and counted by liquid scintillation (36).

Glucose transporter surface labeling. Cell surface (sarcolemma and T tubule) glucose transporters were photoaffinity labeled with the cell-impermeant compound 4,4'-O-[2-[2-[2-[2-[2-[6 (biotinylamino)hexanoyl]amino]ethoxy]ethoxy]ethoxy]-4-(1-azi-2,2,2,-trifluoroethyl)benzoyl]amino-1,3-propanediyl]bis-D-mannose (Bio-LC-ATB-BGPA), as previously described (14a, 38). In brief, after experimental incubations, muscles were labeled for 15 min in buffer containing 400 µM Bio-LC-ATB-BGPA at 4°C and then irradiated with ultraviolet light (300 nm) twice for 3 min to cross-link the label with glucose transport proteins. Pooled (3–4) labeled muscles (10–20 mg wet wt) were homogenized in buffer containing 250 mM sucrose, 1 mM EDTA, 20 mM HEPES, and 1 µg/ml of the proteinase inhibitors antipain, aprotinin, pepstatin, leupeptin, and 100 µM 4-(2-aminoethyl)benzenesulfonyl fluoride (pH 7.2). Cell membranes were prepared by ultracentrifugation (227,000 g for 50 min at 4°C) and solubilized in PBS containing 2% Thesit and proteinase inhibitors. Membranes underwent centrifugation at 30,000 g for 30 min, and the solubilized membrane proteins were precipitated with streptavidin-agarose (Pierce Chemical). The streptavidin-precipitated surface-labeled proteins were washed several times and then subjected to SDS-PAGE and immunoblotting with GLUT4 or GLUT1 antibody. The surface-labeled GLUT1 and GLUT4 contents were normalized to the amount of these proteins in the total cell membrane fraction.

AMPK activity. In experiments designed to assess AMPK activation, papillary muscles were frozen in liquid nitrogen, stored at –80°C, and subsequently homogenized in buffer containing 125 mM Tris, 1 mM EDTA, 1 mM EGTA, 250 mM mannitol, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM DTT, 1 mM benzamidine, 0.004% trypsin inhibitor, and 3 mM sodium azide, pH 7.5, at 4°C (9, 36). After centrifugation (13,200 g for 30 min), the supernatant underwent polyethylene glycol (PEG) precipitation, and the 2.5–6% fraction was used for measurement of total AMPK activity by use of the SAMS assay, as previously described (9, 36). Protein concentration was determined spectrophotometrically using the Bio-Rad reagent. The AMPK activity associated with specific catalytic subunits was examined following immunoprecipitation with polyclonal ₁ and ₂ antibodies, as previously described (9).

AMPK-eNOS coimmunoprecipitation. After incubation with or without AICAR, heart muscles were homogenized in buffer containing 20 mM HEPES, 50 mM -glycerol phosphate, 2 mM EGTA, 1 mM DTT, 10 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100, 10% glycerol, 0.1% PMSF, 0.1 µM leupeptin, and 10 ng/ml aprotinin at 4°C. After low-speed centrifugation, 400 µg of protein were immunoprecipitated with ₁ or ₂ polyclonal antibodies, or nonimmune IgG as a negative control (9). After extensive washing, the immunoprecipitates were resuspended in Laemmli buffer for immunoblotting with eNOS or nNOS antibodies.

Immunoblotting. Proteins were combined with Laemmli sample buffer prior to SDS-PAGE. After transfer to PDVF membranes, proteins were immunoblotted, detected with enhanced chemiluminence, and quantified by densitometry of autoradiographs, as previously described (9). Immunoblots were performed with rabbit pan- (₁/₂) AMPK antibody at 1:10,000 dilution (kind gift from Dr. M. Birnbaum), sheep anti-₂ AMPK at 1:1,000 dilution (kind gift from Dr. D. G. Hardie), rabbit anti-pThr¹⁷² AMPK antibody at 1:5,000 dilution (Cell Signaling), rabbit anti-pSer¹¹⁷⁷ eNOS (Cell Signaling) at 1:1,000 dilution, mouse anti-eNOS (BD Transduction Laboratory) at 1:2,500 dilution, rabbit anti-pSer¹⁴¹⁶ nNOS (Upstate Biotechnology) at 1:2,000 dilution, and mouse anti-nNOS (Santa Cruz Biotechnology) at 1:1,000 dilution.

Statistical analysis. All data are reported as means ± SE. The number of experiments in each group is presented in the text, table, or figure legend. Data were analyzed by two-tailed, unpaired Student’s t-test. Differences were considered significant at P < 0.05.

	RESULTS

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Effects of AICAR on AMPK activation. We initially examined the effects of AICAR (1 mM) on AMPK activation in the isolated left ventricular papillary muscles. AICAR increased total AMPK activity threefold (P < 0.01) in PEG-precipitated muscle homogenates (Fig. 1A). Immunoblots with anti-pThr¹⁷² AMPK antibody showed that AICAR also increased AMPK catalytic subunit phosphorylation at its key regulatory site, reflecting phosphorylation by the upstream AMPK activating protein kinase (Fig. 1B). Finally, AICAR stimulated AMPK activity in both ₁ (P < 0.05) and ₂ (P < 0.01) immunoprecipitates in the heart muscles (Fig. 1C).

View larger version (21K): [in this window] [in a new window]   Fig. 1. 5-Amino-4-imidazole-1--D-carboxamide ribofuranoside (AICAR) activation of AMP-activated protein kinase (AMPK) A: AMPK activity. Total AMPK activity was assessed using the SAMS kinase assay in polyethylene glycol extracts of heart muscles incubated for 60 min with or without 1 mM AICAR (see MATERIALS AND METHODS). B: AMPK phosphorylation. AMPK catalytic subunit phosphorylation was assessed by immunoblotting with a phospho-Thr172 AMPK antibody. C: AMPK isoform kinase activity. The activity associated with the 1 or 2 catalytic AMPK subunits was measured after immunoprecipitation with isoform-specific antibodies. Values are means ± SE for 3–5 experiments, with 2–4 pooled heart muscles per experiment. P < 0.05, *P < 0.01 vs. control.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Stimulation of glucose transport by AICAR. A: deoxyglucose transport. Isolated heart muscles were preincubated for 15–60 min with or without 1 mM AICAR before addition of 2-deoxy-[1-3H]glucose for an additional 30 min to measure glucose uptake (see MATERIALS AND METHODS). Values are means ± SE for 3 experiments, with 2–4 independently analyzed muscles per experiment. *P < 0.05 vs. control. B: GLUT translocation. After control or AICAR incubations, cell surface GLUT4 (s-GLUT4) and GLUT1 (s-GLUT1) proteins were labeled with a cell-impermeant biotinylated bis-mannose compound (Bio-LC-ATB-BGPA) and cross-linked with UV irradiation (see MATERIALS AND METHODS). Labeled surface proteins were isolated on streptavidin-agarose and immunoblotted with GLUT4 or GLUT1 antibodies. Total cell membrane GLUT4 (t-GLUT4) or GLUT1 (t-GLUT 1) was measured before isolation with streptavidin. Representative immunoblots are shown, and ratios of surface to total GLUT4 and GLUT1 are expressed as means ± SE for 3 experiments, including 2–3 pooled muscles per experiment. *P < 0.05 vs. control.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Activation of glucose transport by nitric oxide donors. A: deoxyglucose transport. Isolated heart muscles were incubated without (control) or with the nitric oxide donors sodium nitroprusside (SNP) or S-nitroso-N-acetyl-DL-penicillamine (SNAP) (0.1–10 µM) for 60 min (see MATERIALS AND METHODS). Results indicate the means ± SE for 3–5 experiments, with 2–4 independently analyzed muscles per experiment. *P < 0.05 vs. control. B: GLUT surface labeling. After control, SNP (1 µM), or SNAP (1 µM) incubations, surface GLUT4 and GLUT1 were labeled with Bio-LC-ATB-BGPA, isolated on streptavidin-agarose, and immunoblotted with specific GLUT4 or GLUT1 antibodies. Representative immunoblots are shown, and ratios of surface to total GLUT4 and GLUT1 are expressed as means ± SE for 3 experiments, including 2–3 pooled papillary muscles per experiment. *P < 0.05 vs. control. C: bromo (Br)-cGMP and deoxyglucose transport. Heart muscles were incubated for 60 min with 10 µM Br-cGMP, 1 µM SNP, or 1 µM SNAP (see MATERIALS AND METHODS). Results indicate means ± SE for 3 experiments, including 2–4 independently analyzed muscles per experiment. *P < 0.05 vs. control.

View larger version (50K): [in this window] [in a new window]   Fig. 4. AMPK activation of endothelial nitric oxide synthase (eNOS). A: eNOS phosphorylation. Isolated heart papillary muscles were incubated in the presence or absence of AICAR (1 mM) for 30 min and homogenates immunoblotted with antibodies to pSer1177 eNOS and total eNOS. Representative immunoblots are shown, and ratios of p-eNOS to total eNOS are expressed as means ± SE for 3 experiments, including 2–3 pooled muscles per experiment. *P < 0.05 vs. control. B: AMPK binding to eNOS. Muscles were incubated in the presence or absence of AICAR (1 mM) for 30 min and homogenates immunoprecipitated (IP) with antibodies to 1 or 2 AMPK or nonimmune sheep IgG. Immunoprecipitates were immunoblotted with antibodies to eNOS, as well as with antibodies to 1 and 2 AMPK to determine recovery. C. neuronal (n)NOS phosphorylation. Isolated heart muscles were incubated in the presence or absence of AICAR (1 mM) for 30 min and homogenates immunoblotted with antibodies to pSer1416 nNOS and total nNOS (rat brain homogenate was used as a positive control). D: nNOS binding. Heart 1 or 2 immunoprecipitates were immunoblotted with antibodies to nNOS, as well as with antibodies to 1 and 2 AMPK to determine recovery.

View larger version (19K): [in this window] [in a new window]   Fig. 5. NOS inhibition and AICAR-stimulated deoxyglucose transport. Isolated heart muscles were preincubated for 30 min with 1 mM L-NAME (A) or L-NMMA (B) before incubation with or without AICAR (1 mM) for 60 min. 2-Deoxy-[1-3H]glucose was added during the last 30 min to measure glucose uptake (see MATERIALS AND METHODS). Results indicate means ± SE for 3 experiments, including 2–4 independently analyzed papillary muscles per experiment. *P < 0.01 vs. control, P < 0.05 vs. control, P < 0.05 vs. AICAR.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Guanylate cyclase inhibition and AICAR-stimulated deoxyglucose uptake. Isolated heart muscles were preincubated for 30 min with 25 µM ODQ (A) or 10 µM LY-83583 (C), before incubation with or without AICAR (1 mM) for 60 min (see MATERIALS AND METHODS). Results indicate means ± SE for 3 experiments, including 3–4 independently analyzed muscles per experiment. *P < 0.01 vs. control, P < 0.05 vs. control, P < 0.05 vs. AICAR. B: guanylate cyclase inhibiton and AICAR-stimulated GLUT4 translocation. After control or AICAR incubation with or without ODQ (25 µM), cell surface GLUT4 was labeled with Bio-LC-ATB-BGPA, isolated on streptavidin-agarose, and immunoblotted with specific GLUT4 antibodies. Representative immunoblots are shown, and ratios of surface to total GLUT4 are expressed as means ± SE for 3 experiments, including 2–3 pooled muscles per experiment. *P < 0.05 vs. control, P < 0.05 vs. AICAR.

	DISCUSSION

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These results expand our understanding of the mechanisms through which AMPK stimulates glucose transport in the heart and are of interest in light of recent studies showing that AMPK has an important role in the activation of glucose transport in the ischemic heart. Transgenic mice expressing dominant negative catalytic subunits of AMPK have a reduced glucose uptake response during both ischemia (35) and postischemic reperfusion (35, 45). AMPK deficiency also blocks hypoxia-stimulated glucose transport in skeletal muscle (29). AMPK is also known to be activated during exercise in both heart (9) and skeletal muscle (43), although it remains uncertain how important a role AMPK has in modulating glucose transport during exercise. In isolated skeletal muscle, contraction-stimulated glucose uptake is partially inhibited in AMPK-deficient dominant negative transgenic mice (29), but appears to be normal in muscles from ₂ knockout mice (19). These latter findings suggest that additional redundant signaling pathways may exist that compensate for AMPK deficiency during contraction. Whether such mechanisms are simply recruited in transgenic mice as the result of developmental and/or chronic AMPK deficiency, or would be operative in normal muscle tissue with acute AMPK inhibition, remains uncertain.

The present results support the hypothesis that activation of the nitric oxide-guanylate cyclase pathway plays a role in mediating the effects of AMPK on glucose transport in heart muscle. These experiments were performed in isolated left ventricular papillary muscles, which contain a variety of cell types, including cardiac myocytes and endothelial cells. However, cardiac myocytes are the only cells in the heart which contain GLUT4 (47) and account for the bulk of glucose transport in heart because they also predominate by mass (46). Thus, taken together, the results from GLUT4 surface labeling and glucose uptake experiments indicate that the eNOS-guanylate cyclase pathway impacts on cardiomyocyte glucose uptake during AICAR stimulation.

We observed not only that AICAR increased eNOS Ser¹¹⁷⁷ phosphorylation but also that AMPK coimmunoprecipitated with eNOS, suggesting that AMPK directly activated eNOS. These findings are consistent with previous reports in intact heart and skeletal muscle (8, 41). Experiments in isolated mouse H-2Kb muscle cells (12) raised the possibility that AMPK interacts directly with eNOS in muscle cells. However, the current experiments, as well as those in heart and skeletal muscle (8, 41), do not directly define the extent to which AMPK activation of eNOS occurs in the endothelial cells, endocardial cells, or myocytes in the intact muscle tissue.

Vascular endothelial cell eNOS is known to be activated by AICAR (18), and endothelial cells predominantly express the ₁ isoform of AMPK (28). It is interesting in this regard that we found that AICAR activated the ₁ isoform and bound to eNOS in the heart muscles, suggesting that AICAR activation of endothelial cell AMPK and eNOS may have an important role in the stimulation of cardiomyocyte glucose transport through a paracrine mechanism. However, additional immunoprecipitation experiments demonstrated that eNOS was also associated with ₂ AMPK, the more predominant isoform in cardiac myocytes, which is virtually absent from endothelial cells (28). Although cardiac myocyte expression of eNOS is generally low, there is heterogeneity within the heart with greater eNOS expression in specialized endocardial cells that line the cardiac chambers (6, 30) and epicardial cardiomyocytes (4). Thus these findings suggest that both autocrine and paracrine mechanisms may be involved to some extent in the interaction between the AMPK, eNOS, and glucose transport pathways in heart muscle.

Heart and skeletal muscle cells also express nNOS (7, 12, 30, 39). In skeletal muscle, exercise activates AMPK and increases the phosphorylation of the muscle isoform of nNOS (nNOS-mu) on pSer¹⁴⁵¹ (7). The physiological effect of nNOS phosphorylation in skeletal muscle remains uncertain. We found a small amount of nNOS present in the heart papillary muscles, but there was no evidence that AICAR treatment increased nNOS phosphorylation or that nNOS was bound to either the ₁ or ₂ isoform of AMPK. Thus the results of these experiments suggest that AMPK interaction with the nitric oxide-cGMP pathway involves modulation of eNOS, rather than nNOS, in heart muscle.

Nitric oxide stimulates glucose transport in isolated skeletal muscle (2, 48), where it is thought to have a role in mediating both insulin- (20, 22) and exercise-stimulated glucose uptake (34). In skeletal muscle and other tissues from eNOS knockout mice, there is diminished insulin-stimulated glucose uptake, indicating that insulin activation of NOS may contribute to the stimulation of glucose transport (5, 10, 42). In this study, we observed that relatively low concentrations of nitric oxide donors (0.1 µM) increased glucose uptake and stimulated GLUT4 translocation in isolated heart papillary muscles. The effects of nitric oxide on heart GLUT4 translocation have not been previously examined. Prior studies in working hearts have suggested that nitric oxide may activate fatty acid metabolism and inhibit overall glucose utilization (32), leading to the postulate that deficient eNOS activity in heart failure may reduce fatty acid oxidation and increase glucose metabolism (31). However, nitric oxide modulates cardiac contractility and oxidative metabolism in vivo, which may counterbalance the direct effects of nitric oxide to activate glucose transport. In the present experiments, the utilization of an isolated, quiescent papillary muscle preparation enabled us to examine more directly the role of nitric oxide and NOS inhibition on glucose transport and GLUT4 translocation. Although increased nitric oxide activates GLUT4 translocation, NOS inhibition experiments had no effect on basal glucose uptake, indicating that nitric oxide is not required to maintain basal glucose uptake in isolated heart muscles.

The findings that NOS inhibitors decrease AICAR-stimulated glucose transport in heart muscles are consistent overall with findings in skeletal muscle-derived mouse H-2Kb cells and isolated rat skeletal muscles (12), as well as in rat skeletal muscle in vivo (40). In isolated skeletal muscles, there was virtually complete inhibition of AICAR-stimulated glucose uptake by NOS inhibitors (12). In recent in vivo studies, L-NAME decreased AICAR-stimulated deoxyglucose uptake but also decreased basal uptake (40). There was partial (20–40%) inhibition of AICAR-stimulated glucose transport by NOS inhibitors in the present study in heart muscles. These results indicate that the majority of AMPK-mediated glucose transport in the heart is mediated either through additional downstream signaling pathways or by the direct effects of AMPK on GLUT4 vesicular trafficking. Although additional pathways that might mediate AMPK’s effects on glucose transport in heart are unknown, the phosphatidylinositol-3-kinase pathway, which plays an essential role in insulin signaling, does not appear to be involved (3, 16, 36). In addition, the partial inhibition of glucose transport and GLUT4 translocation seen with guanylate cyclase inhibitors is also consistent with prior studies in skeletal muscle cells (12). These findings indicate that guanylate cyclase is an important downstream mediator of glucose transport activation by nitric oxide, although the mechanism by which cGMP activates glucose transport in muscle remains uncertain.

Thus these findings indicate that the nitric oxide-guanylate cyclase pathway partially contributes to the AMPK stimulation of glucose transport in heart muscle. The importance of this pathway in mediating the activation of glucose transport in the heart during exercise and ischemia is of interest for future studies.

	GRANTS

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This work was supported by United States Public Health Service (National Heart, Lung, and Blood Institute) Grant RO1-HL-63811 (L. H. Young) and a Medical Student Research fellowship from the Heritage Affiliate of the American Heart Association (P. Selvakumar).

	ACKNOWLEDGMENTS

 
We express appreciation to Dr. William Sessa for helpful suggestions.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. H. Young, Section of Cardiovascular Medicine, TAC S460, Yale Univ. School of Medicine, New Haven, CT 06520 (E-mail: lawrence.young{at}yale.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

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1. Abbud W, Habinowski S, Zhang JZ, Kendrew J, Elkairi FS, Kemp BE, Witters LA, and Ismail-Beigi F. Stimulation of AMP-activated protein kinase (AMPK) is associated with enhancement of Glut1-mediated glucose transport. Arch Biochem Biophys 380: 347–352, 2000.[CrossRef][Web of Science][Medline]
2. Balon TW and Nadler JL. Nitric oxide mediates skeletal glucose transport. Am J Physiol Endocrinol Metab 270: E258–E259, 1996.
3. Bergeron R, Russell RR, Young LH, Ren JM, Marcucci M, Lee A, and Shulman GI. Effect of AMPK activation on muscle glucose metabolism in conscious rats. Am J Physiol Endocrinol Metab 276: E938–E944, 1999.[Abstract/Free Full Text]
4. Brahmajothi MV and Campbell DL. Heterogeneous basal expression of nitric oxide synthase and superoxide dismutase isoforms in mammalian heart : implications for mechanisms governing indirect and direct nitric oxide-related effects. Circ Res 85: 575–587, 1999.[Abstract/Free Full Text]
5. Browne SE, Ayata C, Huang PL, Moskowitz MA, and Beal MF. The cerebral metabolic consequences of nitric oxide synthase deficiency: glucose utilization in endothelial and neuronal nitric oxide synthase null mice. J Cereb Blood Flow Metab 19: 144–148, 1999.[CrossRef][Web of Science][Medline]
6. Brutsaert DL. Cardiac endothelial-myocardial signaling: its role in cardiac growth, contractile performance, and rhythmicity. Physiol Rev 83: 59–115, 2003.[Abstract/Free Full Text]
7. Chen ZP, McConell GK, Michell BJ, Snow RJ, Canny BJ, and Kemp BE. AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation. Am J Physiol Endocrinol Metab 279: E1202–E1206, 2000.[Abstract/Free Full Text]
8. Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, Power DA, Ortiz de Montellano PR, and Kemp BE. AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett 443: 285–289, 1999.[CrossRef][Web of Science][Medline]
9. Coven DL, Hu X, Cong L, Bergeron R, Shulman GI, Hardie DG, and Young LH. Physiological role of AMP-activated protein kinase in the heart: graded activation during exercise. Am J Physiol Endocrinol Metab 285: E629–E636, 2003.[Abstract/Free Full Text]
10. Duplain H, Burcelin R, Sartori C, Cook S, Egli M, Lepori M, Vollenweider P, Pedrazzini T, Nicod P, Thorens B, and Scherrer U. Insulin resistance, hyperlipidemia, and hypertension in mice lacking endothelial nitric oxide synthase. Circulation 104: 342–345, 2001.[Abstract/Free Full Text]
11. Friebe A and Koesling D. Regulation of nitric oxide-sensitive guanylyl cyclase. Circ Res 93: 96–105, 2003.[Abstract/Free Full Text]
12. Fryer LG, Hajduch E, Rencurel F, Salt IP, Hundal HS, Hardie DG, and Carling D. Activation of glucose transport by AMP-activated protein kinase via stimulation of nitric oxide synthase. Diabetes 49: 1978–1985, 2000.[Abstract/Free Full Text]
13. Hallows KR, McCane JE, Kemp BE, Witters LA, and Foskett JK. Regulation of channel gating by AMP-activated protein kinase modulates cystic fibrosis transmembrane conductance regulator activity in lung submucosal cells. J Biol Chem 278: 998–1004, 2003.[Abstract/Free Full Text]
14. Hardie DG, Scott JW, Pan DA, and Hudson ER. Management of cellular energy by the AMP-activated protein kinase system. FEBS Lett 546: 113–120, 2003.[CrossRef][Web of Science][Medline]
14. Hashimoto M, Hatanaka Y, Yang J, Dhesi J, and Holman GD. Synthesis of biotinylated bis(D-glucose) derivatives for glucose transporter photoaffinity labelling. Carbohydr Res 331: 119–127, 2001.[CrossRef][Web of Science][Medline]
15. Hayashi T, Hirshman MF, Fujii N, Habinowski SA, Witters LA, and Goodyear LJ. Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism. Diabetes 49: 527–531, 2000.[Abstract]
16. Hayashi T, Hirshman MF, Kurth EJ, Winder WW, and Goodyear LJ. Evidence for 5' AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport. Diabetes 47: 1369–1373, 1998.[Abstract]
17. Holmes BF, Kurth-Kraczek EJ, and Winder WW. Chronic activation of 5'-AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle. J Appl Physiol 87: 1990–1995, 1999.[Abstract/Free Full Text]
18. Ido Y, Carling D, and Ruderman N. Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells: inhibition by the AMP-activated protein kinase activation. Diabetes 51: 159–167, 2002.[Abstract/Free Full Text]
19. Jorgensen SB, Viollet B, Andreelli F, Frosig C, Birk JB, Schjerling P, Vaulont S, Richter EA, and Wojtaszewski JF. Knockout of the ₂ but not ₁ 5'-AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4-carboxamide-1--4-ribofuranoside but not contraction-induced glucose uptake in skeletal muscle. J Biol Chem 279: 1070–1079, 2004.[Abstract/Free Full Text]
20. Kapur S, Bédard S, Marcotte B, Côté C, and Marette A. Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action. Diabetes 46: 1691–1700, 1997.[Abstract]
21. Kemp BE, Stapleton D, Campbell DJ, Chen ZP, Murthy S, Walter M, Gupta A, Adams JJ, Katsis F, Van Denderen B, Jennings IG, Iseli T, Michell BJ, and Witters LA. AMP-activated protein kinase, super metabolic regulator. Biochem Soc Trans 31: 162–168, 2003.[Web of Science][Medline]
22. Kingwell BA, Formosa M, Muhlmann M, Bradley SJ, and McConell GK. Nitric oxide synthase inhibition reduces glucose uptake during exercise in individuals with type 2 diabetes more than in control subjects. Diabetes 51: 2572–2580, 2002.[Abstract/Free Full Text]
23. Kudo N, Barr AJ, Barr RL, Desai S, and Lopaschuk GD. High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5'-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J Biol Chem 270: 17513–17520, 1995.[Abstract/Free Full Text]
24. Kudo N, Gillespie JG, Kung L, Witters LA, Schulz R, Clanachan AS, and Lopaschuk GD. Characterization of 5'AMP-activated protein kinase activity in the heart and its role in inhibiting acetyl-CoA carboxylase during reperfusion following ischemia. Biochim Biophys Acta 1301: 67–75, 1996.[Medline]
25. Kurth-Kraczek EJ, Hirshman MF, Goodyear LJ, and Winder WW. 5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle. Diabetes 48: 1667–1671, 1999.[Abstract]
26. Marsin AS, Bertrand L, Rider MH, Deprez J, Beauloye C, Vincent MF, Van den Berghe G, Carling D, and Hue L. Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia. Curr Biol 10: 1247–1255, 2000.[CrossRef][Web of Science][Medline]
27. Merrill GF, Kurth EJ, Hardie DG, and Winder WW. AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. Am J Physiol Endocrinol Metab 273: E1107–E1112, 1997.[Abstract/Free Full Text]
28. Morrow VA, Foufelle F, Connell JM, Petrie JR, Gould GW, and Salt IP. Direct activation of AMP-activated protein kinase stimulates nitric-oxide synthesis in human aortic endothelial cells. J Biol Chem 278: 31629–31639, 2003.[Abstract/Free Full Text]
29. Mu J, Brozinick JT Jr, Valladares O, Bucan M, and Birnbaum MJ. A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle. Mol Cell 7: 1085–1094, 2001.[CrossRef][Web of Science][Medline]
30. Mungrue IN, Husain M, and Stewart DJ. The role of NOS in heart failure: lessons from murine genetic models. Heart Fail Rev 7: 407–422, 2002.[CrossRef][Medline]
31. Recchia FA, McConnell PI, Bernstein RD, Vogel TR, Xu X, and Hintze TH. Reduced nitric oxide production and altered myocardial metabolism during the decompensation of pacing-induced heart failure in the conscious dog. Circ Res 83: 969–979, 1998.[Abstract/Free Full Text]
32. Recchia FA, Osorio JC, Chandler MP, Xu X, Panchal AR, Lopaschuk GD, Hintze TH, and Stanley WC. Reduced synthesis of NO causes marked alterations in myocardial substrate metabolism in conscious dogs. Am J Physiol Endocrinol Metab 282: E197–E206, 2002.[Abstract/Free Full Text]
33. Rees DD, Palmer RM, Schulz R, Hodson HF, and Moncada S. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br J Pharmacol 101: 746–752, 1990.[Web of Science][Medline]
34. Roberts CK, Barnard RJ, Scheck SH, and Balon TW. Exercise-stimulated glucose transport in skeletal muscle is nitric oxide dependent. Am J Physiol Endocrinol Metab 273: E220–E225, 1997.[Abstract/Free Full Text]
35. Russell RR, Li J, Coven DL, Pypaert M, Zechner C, Palmieri M, Giordano FJ, Mu J, Birnbaum MJ, and Young LH. AMP-activated protein kinase mediates ischemic glucose uptake and prevents post-ischemic cardiac dysfunction, apoptosis and injury. J Clin Invest 114: 495–503, 2004.[CrossRef][Web of Science][Medline]
36. Russell RR, Bergeron R, Shulman GI, and Young LH. Translocation of myocardial GLUT4 and increased glucose uptake through activation of AMPK by AICAR. Am J Physiol Heart Circ Physiol 277: H643–H649, 1999.[Abstract/Free Full Text]
37. Rutter GA, Da Silva Xavier G., and Leclerc I. Roles of 5'-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis. Biochem J 375: 1–16, 2003.[CrossRef][Web of Science][Medline]
38. Ryder JW, Yang J, Galuska D, Rincon J, Bjornholm M, Krook A, Lund S, Pedersen O, Wallberg-Henriksson H, Zierath JR, and Holman GD. Use of a novel impermeable biotinylated photolabeling reagent to assess insulin- and hypoxia-stimulated cell surface GLUT4 content in skeletal muscle from type 2 diabetic patients. Diabetes 49: 647–654, 2000.[Abstract]
39. Schulz R, Kelm M, and Heusch G. Nitric oxide in myocardial ischemia/reperfusion injury. Cardiovasc Res 61: 402–413, 2004.[Abstract/Free Full Text]
40. Shearer J, Fueger PT, Vorndick B, Bracy DP, Rottman JN, Clanton JA, and Wasserman DH. AMP kinase-induced skeletal muscle glucose but not long-chain fatty acid uptake is dependent on nitric oxide. Diabetes 53: 1429–1435, 2004.[Abstract/Free Full Text]
41. Stephens TJ, Chen ZP, Canny BJ, Michell BJ, Kemp BE, and McConell GK. Progressive increase in human skeletal muscle AMPK₂ activity and ACC phosphorylation during exercise. Am J Physiol Endocrinol Metab 282: E688–E694, 2002.[Abstract/Free Full Text]
42. Vicent D, Ilany J, Kondo T, Naruse K, Fisher SJ, Kisanuki YY, Bursell S, Yanagisawa M, King GL, and Kahn CR. The role of endothelial insulin signaling in the regulation of vascular tone and insulin resistance. J Clin Invest 111: 1373–1380, 2003.[CrossRef][Web of Science][Medline]
43. Winder WW and Hardie DG. Inactivation of acetyl-CoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise. Am J Physiol Endocrinol Metab 270: E299–E304, 1996.[Abstract/Free Full Text]
44. Woods A, Azzout-Marniche D, Foretz M, Stein SC, Lemarchand P, Ferre P, Foufelle F, and Carling D. Characterization of the role of AMP-activated protein kinase in the regulation of glucose-activated gene expression using constitutively active and dominant negative forms of the kinase. Mol Cell Biol 20: 6704–6711, 2000.[Abstract/Free Full Text]
45. Xing Y, Musi N, Fujii N, Zou L, Luptak I, Hirshman MF, Goodyear LJ, and Tian R. Glucose metabolism and energy homeostasis in mouse hearts overexpressing dominant negative alpha2 subunit of AMP-activated protein kinase. J Biol Chem 278: 28372–28377, 2003.[Abstract/Free Full Text]
46. Young LH, Coven DL, and Russell RR III. Cellular and molecular regulation of cardiac glucose transport. J Nucl Cardiol 7: 267–276, 2000.[CrossRef][Web of Science][Medline]
47. Young LH, Renfu Y, Russell RR, Hu X, Caplan MJ, Ren J, Shulman GI, and Sinusas AJ. Low-flow ischemia leads to translocation of canine heart GLUT-4 and GLUT-1 glucose transporters to the sarcolemma in vivo. Circulation 95: 415–422, 1997.[Abstract/Free Full Text]
48. Young ME, Radda GK, and Leighton B. Nitric oxide stimulates glucose transport and metabolism in rat skeletal muscle in vitro. Biochem J 322: 223–228, 1997.[Web of Science][Medline]

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