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Am J Physiol Endocrinol Metab 295: E162-E169, 2008. First published May 20, 2008; doi:10.1152/ajpendo.00622.2007
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The nitric oxide-donating derivative of acetylsalicylic acid, NCX 4016, stimulates glucose transport and glucose transporters translocation in 3T3-L1 adipocytes

V. Kaddai,1,* T. Gonzalez,1,* M. Bolla,2 Y. Le Marchand-Brustel,1,3 and M. Cormont1

1Institut National de la Santé et de la Recherche Médicale Unité 895, Cellular and Molecular Physiopathology of Obesity and Diabetes, Faculté de Médecine, University of Nice/Sophia-Antipolis, Nice; 2NicOx, Sophia-Antipolis; and 3Centre Hospitalier Universitaire de Nice, Hôpital l'Archet, Service d'Hépatologie, Nice, France

Submitted 26 September 2007 ; accepted in final form 11 May 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
NCX 4016 is a nitric oxide (NO)-donating derivative of acetylsalicylic acid. NO and salicylate, in vivo metabolites of NCX 4016, were shown to be potential actors in controlling glucose homeostasis. In this study, we evaluated the action of NCX 4016 on the capacity of 3T3-L1 adipocytes to transport glucose in basal and insulin-stimulated conditions. NCX 4016 induced a twofold increase in glucose uptake in parallel with the translocation of the glucose transporters GLUT1 and GLUT4 to the plasma membrane, leaving unaffected their total expression levels. Importantly, NCX 4016 further increased glucose transport induced by a physiological concentration of insulin. The stimulatory effect of NCX 4016 on glucose uptake appears to be mediated by its NO moiety. Indeed, it is inhibited by a NO scavenger and treatment with acetylsalicylic or salicylic acid had no effect. Although NO is involved in the action of NCX 4016, it did not mainly depend on the soluble cGMP cyclase/protein kinase G pathway. Furthermore, NCX 4016-stimulated glucose transport did not involve the insulin-signaling cascade required to stimulate glucose transport. NCX 4016 induces a small activation of the mitogen-activated protein kinases p38 and c-Jun NH2-terminal kinase and no activation of other stress-activated signaling molecules, including extracellular signal-regulated kinase, inhibitory factor {kappa}B, or AMP-activated kinases. Interestingly, NCX 4016 modified the content of S-nitrosylated proteins in adipocytes. Taken together, our results indicate that NCX 4016 induced glucose transport in adipocytes through a novel mechanism possibly involving S-nitrosylation. NCX 4016 thus possesses interesting characteristics to be considered as a candidate molecule for the treatment of patients suffering from metabolic syndrome and type 2 diabetes.

nitric oxide donation; adipocytes; glucose transporter translocation; S-nitrosylation; diabetes

ORIGINALLY CREATED as a nitric oxide (NO)-donating aspirin, NCX 4016 {2-(acetyloxy)benzoic acid 3-[(nitrooxy)methyl] phenyl ester} tested in human as treatment for peripheral artery occlusive disease showed higher clinical efficacy in the subpopulation of patients suffering from Type 2 diabetes. Further investigations showed that NCX 4016 released in vivo salicylates and NO-derived species following oral administration and absorption (8).

Very high doses of salicylate are known to exert a glucose-lowering effect in patients suffering from Type 2 diabetes (3). This improved glycemic control is attributed to an improved insulin sensitivity and could be explained at least by two mechanisms. The first one results in the rescue of insulin signaling in the insulin-sensitive tissues. By inhibiting IKK (47) salicylate indeed decreases phosphorylation of insulin receptor substrate 1 (IRS1) on serine-307, a deleterious phosphorylation event for normal insulin signaling (18, 32). Second, because salicylate inhibits inflammatory responses through inhibition of IKK and cyclooxygenases (44, 47), it could inhibit the secretion of proinflammatory cytokines that alter insulin signaling (1).

The NO moiety can also have anti-inflammatory potential with benefit in insulin action because it was reported to directly inhibit IKKβ (35). NO could also be an important player in glucose and lipid homeostasis. NO may improve glucose disposal through its well-known vasodilatory activity (23) and may contribute to the control of glycemia through regulation of the IKKβ pathway (35). The phenotypic characterization of endothelial nitric oxide synthase (eNOS)-deficient and heterozygous mice clearly unmasks an important role of NO in the control of glucose and lipid homeostasis, on top of its role in the control of arterial pressure (9, 11). Indeed, eNOS expression is required for full insulin-induced glucose transport in isolated muscle (11) and adipocyte (4).

In the present study, we explore whether NCX 4016 could control glucose transport in adipocytes, a cell type playing an important role in regulating body glucose homeostasis (21). The potential effect of NCX 4016 was determined in 3T3-L1 adipocytes in a comparative study with acetylsalicylic acid and sodium nitroprusside (SNP).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Antibodies. Polyclonal antibodies against GLUT4 and GLUT1 were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against AS160 was from Abcam (ab5909; Cambridge, UK), and against phospho-Thr642-AS160 was from Biosource International (Carmarillo, CA). Anti-phospho-Thr172-AMP-activated kinase (AMPK), phospho-Thr308-protein kinase B (PKB), phospho-Ser473-PKB, phospho-Thr180/Tyr182-p38, phospho-Thr202/Tyr204-extracellular signal-regulated kinase (ERK)1/2, and phospho-Thr183/Tyr185-c-Jun NH2-terminal kinase (JNK) were from Cell Signaling Technology (Danvers, MA). Anti-ERK1/2 was from Santa Cruz Biotechnology, whereas anti-PKB and p38 were from Cell Signaling Technology. Anti-JNK and anti cytochrome c were from BD Biosciences (San Jose, CA). Anti-biotin was from Sigma-Aldrich (St. Louis, MO). Fluorescein isothiocyanate (FITC)-coupled and horseradish peroxidase-coupled anti-species antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA).

Chemicals. NCX 4016 and acetylsalicylic acid were provided by NicOx (Sophia-Antipolis, France). Poly-L-lysine, the NO scavenger carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1oxyl (c-PTIO), the NO donor SNP, the inhibitor of NO-dependent guanylyl cyclase [1,2,4]oxadiazolo[4,3-{alpha}]quinoxalin-1-one (ODQ), the cGMP-dependent protein kinase 1a (DT-2), S-methylmethanethiosulfonate (MMTS), neocuproine, and streptavidine-agarose were from Sigma. The inhibitor of NO-dependent guanylyl cyclase carnosine was from Alexis Biochemicals (Lausen, Switzerland). The inhibitor KT-5823 of protein kinases G was from EMD Biosciences (San Diego, CA). Medium and chemicals for cell culture were from Invitrogen. 2-deoxy-D-[3H]glucose (2-DG) was from NEN products (Boston, MA). Other chemicals were from Sigma or from Fisher Scientific Labosi (Elancourt, France). Biotin-BMCC was from Pierce (Rockford, IL).

Cell culture. 3T3-L1 fibroblasts were cultured in DMEM-10% (vol/vol) FCS and induced to differentiate in adipocytes as previously described (30). Briefly, 3 days after confluence, fibroblasts were treated for 2 days with DMEM-10% FCS supplemented with isobutyl methylxanthine (250 nM), dexamethasone (250 nM), and insulin (800 nM) and then for two additional days with DMEM, 10% FCS, and insulin (800 nM). The cells were then fed every 2 days with DMEM and 10% FCS and used between day 2 and 7 after the end of the differentiation protocol when the adipocyte phenotype appeared in >90% of cells.

Measurement 2-DG uptake. After serum starvation, 3T3-L1 adipocytes were incubated or not with the different compounds as specified in the legends for Figs. 1, 3, and 4. The compounds were diluted in Krebs-Ringer phosphate buffer (in mM: 10 sodium phosphate, pH 7.4 containing 1 MgSO4, 1 CaCl2, 136 NaCl, and 4.7 KCl) or, for the 24-h incubation duration, in DMEM containing 0.5% BSA. Before measurement of glucose transport, incubation medium was exchanged for Krebs-Ringer phosphate buffer containing the same compounds. The time of incubation of the cells in Krebs-Ringer phosphate buffer was identical within an experiment, even when the length of incubation with the tested compounds was different. Glucose transport was initiated by adding 2-DG (0.1 mM, 18,500 Bq/ml) to the culture medium as previously described. The reaction was stopped after 3 min by aspiration of the medium, and cells were washed four times with ice-cold PBS (43). Cells were lysed in 20 mM Tris·HCl, pH 7.4, containing 1% Triton X-100 and 1% SDS, cocktails of protease inhibitors (Complete; Roche Diagnostics, Mannheim, Germany), and phosphatase inhibitors (10 mM NaF, 10 mM tetrasodium diphosphate, and 200 µM sodium orthovanadate). Aliquots of homogenates were used to assess glucose uptake by scintillation counting. Results were normalized for protein contents in each sample measured by the BCA Protein Assay Kit (Pierce). Remaining aliquots were used for the quantification of GLUT1 and GLUT4 expression by immunoblotting (31).


Figure 1
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  		Fig. 1. NCX 4016 increases 2-deoxy-D-[3H]glucose (2-DG) uptake by 3T3-L1 adipocytes without modification of the expression of the glucose transporters GLUT4 and GLUT1. A: 3T3-L1 adipocytes were treated during 0.5 h with 0.5 nM insulin or during 3 h with 200 µM NCX 4016, 200 µM acetylsalicylic acid (ASA), 200 µM salicylic acid (SA), 200 µM sodium nitroprusside (SNP), or vehicle [0; 0.25% dimethyl sulfoxide (DMSO)]. 2-DG uptake was then measured. The means ± SE of 5 experiments are shown. The increase induced by NCX 4016 treatment is significant vs. basal (P < 0.02) and ASA conditions (P < 0.005). B: 3T3-L1 adipocytes were treated as in A. Total cell homogenates were prepared and 40 µg of proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes and probed with antibodies against GLUT4 or GLUT1. Shown is a typical immunodetection among 3 independent experiments.

 

Figure 3
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  		Fig. 3. A 24-h treatment with NCX 4016 induces an increase in 2-DG uptake by 3T3-L1 adipocytes. A: 3T3-L1 adipocytes were treated during 24 h with 200 µM of NCX 4016 or 200 µM ASA or vehicle (control, 0.25% DMSO). At the end of the incubation, insulin was added or not at the indicated concentrations for a further 30-min incubation. 2-DG uptake was then measured. Means ± SE of 3 experiments are shown. B: 3T3-L1 adipocytes were treated for 24 h as in A, and the total amount of GLUT4 or GLUT1 was determined as described in B. C: 3T3-L1 adipocytes were treated during 24 h with vehicle alone (0), 200 µM of NCX 4016, or with 200 µM SNP in presence of the indicated concentrations of carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1oxyl (c-PTIO) before 2-DG uptake measurement. Means ± SE of 3 experiments are shown.

 

Figure 4
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  		Fig. 4. NCX 4016-induced 2-DG uptake does not require the soluble cGMP cyclase/protein kinase G pathway. A: 3T3-L1 adipocytes were treated during 24 h with vehicle (0) and with 25 µM of ODQ or 5 mM carnosine in the presence or absence of 200 µM of NCX 4016, before 2-DG uptake measurement. Data represent means ± SE of 3 independent experiments for ODQ and of 2 independent experiments for carnosine. B: 3T3-L1 were treated for 48 h with solvent or NCX 4016 in the presence or absence of 25 µM ODQ. The incubation medium was renewed one time during the experiment. Homogenates were prepared, and the amount of cytochrome c was determined by immunoblotting. p85 immunodetection was used as a loading control. Chemiluminescence was detected using the FUJIFILM Las-3000 apparatus, and numbers correspond to the quantification of the signal performed with Multi Gauge software. C: 3T3-L1 adipocytes were treated during 24 h with vehicle (open bars) or with 1 µM of KT-5823 or 125 nM cGMP-dependent protein kinase 1a (DT-2; filled bars) in the presence or absence of 200 µM of NCX 4016 before 2-DG uptake measurement. Data represents means ± SE of 3 independent experiments for KT-5823 and of 2 experiments for DT-2.

 
Preparation of plasma membrane sheets and immunodetection of GLUT. 3T3-L1 cells were grown on glass cover slips and differentiated as described before. Cells were incubated overnight in DMEM containing 0.5% BSA. They were then treated with the compounds as indicated in the legends to Fig. 2. After rapid washes with PBS, cells were fixed with 0.55 mg/ml poly-L-lysine for 1 min and then swollen in a hypotonic buffer (in mM: 30 HEPES-NaOH, pH 7.5, 70 KCl, 5 MgCl2, and 3 EGTA). Swollen cells were sonicated, and cover slips were washed three times with the hypotonic buffer. Membrane sheets remaining on the cover slips were fixed with 4% paraformaldehyde, blocked with PBS containing 4% BSA, and incubated with goat polyclonal anti-GLUT4 or anti-GLUT1. After washes, cover slips were incubated with FITC-coupled anti goat antibodies and Texas red (TxRed)-coupled wheat germ agglutinin as an internal marker of membrane amount (Molecular Probes, Invitrogen) (31).


Figure 2
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  		Fig. 2. NCX 4016 induces the translocation of both GLUT4 and GLUT1 to the plasma membrane in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated during 0.5 h with the indicated insulin (Ins) concentration or during 3 h with 200 µM of NCX 4016, 200 µM ASA, or vehicle (0; 0.25% DMSO). Plasma membranes were immobilized and fixed on glass cover slips. GLUT4 (A) and GLUT1 (B) were detected by using specific antibodies and secondary antibodies coupled to fluorescein isothiocyanate (FITC). Typical images were shown, and the corresponding signals were quantified by using Metamorph software. The means ± SE of 3–5 experiments are shown.

 
The plasma membrane sheets were analyzed with an Axiovert 200 microscope using a Plan-Neofluar 40 x 1.3 numeral aperture oil objective (Carl Zeiss, Göttingen, Germany). Images were acquired using a cooled digital camera (CoolSnap HQ; Roper Scientific Princeton Instruments, Evry, France), and quantification was made using Metamorph image analysis software with autothreshold detection of pixels (Universal Imaging, Downington, PA) using the adequate filters for FITC and TxRed fluorochromes. Labeling corresponding to the glucose transporters (FITC, green) was normalized to the total amount of membrane corresponding to the red labeling. Five to ten arbitrary chosen fields were quantified for each experimental condition, and the experiment was reproduced as indicated in the legends for Fig. 2.

Detection of S-nitrosylated proteins by the biotin switch assay. The method used, described by Jaffrey et al. (25), allowed the detection of proteins with NO bound to cysteine thiols because it selectively converted nitrosylated cysteines into biotinylated ones. Adipocytes treated or not with the indicated NO donors were lysed for 20 min in HEN buffer (250 mM HEPES, pH 7.7, 1 mM EDTA, and 0.1 mM neocuproine) containing 0.1% SDS and centrifuged at 20,000 g for 15 min. Lysate proteins (800 µg) were incubated with 4 vol HEN containing 2.5% SDS and 20 mM MMTS for 1 h at 50°C under agitation to block free thiols. MMTS was separated from proteins by cold acetone precipitation. Protein pellets were then resuspended in 100 µl HEN containing 1% SDS adjusted to pH 6.8 to prevent the biotinylation of primary amines. S-nitrosothiols were decomposed by adding 5 mM ascorbate followed by incubation with 2 mM biotin-BMCC for 2 h at room temperature. Biotinylated proteins were then purified by using streptavidin-agarose beads, and biotinylated proteins were detected by Western blot with anti-biotin antibodies. When the reaction is performed in the absence of biotin-BMCC, no proteins were detected with the anti-biotin antibodies (data not shown).

Western blot analysis. The amounts of GLUT1 and GLUT4 were determined in total homogenates from 3T3-L1 adipocytes treated or not for 4 or 24 h with NCX 4016 or acetylsalicylic acid by using specific antibodies against each of the glucose transporters in Western blotting experiments (31). Phosphorylated kinases were detected with the indicated phosphospecific kinase antibodies and represent an index of their activation. The total amount of the kinases was determined with the indicated specific antibodies.

Statistical analysis. Paired Student's t-test was performed to determine the significance between the various experimental treatments of the cells. P < 0.05 was considered as significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
NCX 4016 increases glucose uptake in 3T3-L1 adipocytes. We first looked at the effect of a short-term treatment with NCX 4016 on glucose uptake. Because the NCX 4016 is subjected to enzymatic metabolism to produce NO at slow rates, we treated adipocytes for 3 h to allow suitable intracellular delivery of NO (for review, see Ref. 6). 3T3-L1 adipocytes were treated with 200 µM of NCX 4016 or equimolar acetylsalicylic acid, the NO-donor SNP as the positive control, or a physiological concentration of insulin (0.5 nM) before measurement of 2-DG uptake as described in MATERIALS AND METHODS. Compared with control cells, NCX 4016 induced a significant increase in 2-DG uptake into adipocytes (Fig. 1A), which was lower with respect to insulin stimulation. Treatment with an equimolar concentration of acetylsalicylic acid (200 µM) did not induce any change in the amount of 2-DG uptake, whereas 200 µM of the fast NO donor SNP significantly increased 2-DG uptake. Because acetylsalicylic acid must be deacetylated to be active, we also tested the effect of salicylic acid and found that 200 µM of salicylic acid had no effect on 2-DG uptake (Fig. 1A). Expression levels of the two major glucose transporters expressed in adipocytes, GLUT4 and GLUT1 (28), were not modified by this short-term treatment with NCX 4016 or acetylsalicylic acid (Fig. 1B). The effect of NCX 4016 was dose dependent, with a detectable effect at 50 µM and a maximal effect at 200 µM, and time dependent because it increased by 25% glucose uptake after 1 h of incubation (data not shown).

NCX 4016 increases the plasma membrane levels of both GLUT1 and GLUT4 glucose transporters. Because expression levels of GLUT4 and GLUT1 were unchanged by NCX 4016, the increase in 2-DG uptake was likely to be due to an increase in the amount of glucose transporters at the plasma membrane. To measure the amount of GLUT4 and GLUT1 at the plasma membrane, 3T3-L1 adipocytes were treated for 0.5 h with insulin or for 3 h with 200 µM of NCX 4016, 200 µM acetylsalicylic acid, or vehicle (0.25% dimethyl sulfoxide). Plasma membrane sheets immobilized on the glass cover slips were then prepared as indicated in MATERIALS AND METHODS before measuring GLUT4 (Fig. 2A) and GLUT1 (Fig. 2B) amount by quantitative immunofluorescence. NCX 4016 significantly increased the amount of GLUT4 at the plasma membrane, but moderately compared with insulin. By contrast, NCX 4016 was as efficient as 100 nM insulin to increase the amount of GLUT1 at the plasma membrane. Acetylsalicylic acid alone did not modify the amount of either GLUT1 or GLUT4 present at the plasma membrane, suggesting that the observed effect for NCX 4016 was dependent on the release of NO. This series of experiments indicates that NCX 4016 increases basal glucose uptake by inducing translocation of glucose transporters to the plasma membrane of 3T3-L1 adipocytes, with a more potent effect on GLUT1 than on GLUT4.

NCX 4016 increases 2-DG uptake after 24 h exposure in 3T3-L1 adipocytes through a NO-releasing dependent pathway. We next determined whether a longer incubation (24 h) with NCX 4016 could be more efficient in stimulating 2-DG uptake. 3T3-L1 adipocytes were treated for 24 h with 200 µM NCX 4016, acetylsalicylic acid, or vehicle as control and then stimulated or not with insulin. We observed that NCX 4016 induced a threefold increase in 2-DG uptake compared with control and acetylsalicylic acid-treated cells (Fig. 3A), whereas a 24-h treatment with 200 µM SNP increased 2-DG uptake by less than twofold (Fig. 3C). Also of importance, this effect was additive with the effect of a physiological concentration of insulin (0.5 nM) (Fig. 3A). Furthermore, the long-term exposure with NCX 4016 did not affect an acute effect of insulin at a maximally stimulating concentration. The increase in glucose transport activity of the adipocytes treated with NCX 4016 occurred without any change in the levels of expression of the transporters GLUT4 and GLUT1 (Fig. 3B). Because an equimolar amount of acetylsalicylic acid did not induce 2-DG uptake as does NCX 4016, the NO moiety of the compound was likely responsible for the observed effect. To demonstrate this, we used the specific NO scavenger c-PTIO, which abrogated in a concentration-dependent manner the effect of NCX 4016 (Fig. 3C). c-PTIO had no significant effect on basal glucose uptake (data not shown) but also inhibited the effect on 2-DG uptake induced by a 24-h treatment with 200 µM SNP (Fig. 3C). Taken together, our results indicate that the NO moiety of NCX 4016 induces glucose uptake in 3T3-L1 adipocytes and that NCX 4016 did not alter insulin action on 2-DG uptake.

NCX 4016-induced 2-DG uptake is not efficiently affected by inhibitors of the soluble guanylyl cyclase pathway. Because NCX 4016 effect appears to be linked to the NO moiety, we tested whether the soluble guanylyl cyclase (the canonical physiological target of the NO signaling pathway) was involved in this effect. The incubation of adipocytes with ODQ or carnosine, inhibitors of the soluble form of the guanylyl cyclase, did not affect NCX 4016-induced 2-DG uptake, and ODQ also had no effect on basal glucose uptake (Fig. 4A). ODQ was effective at the concentration used. It indeed inhibited the induction by NCX 4016 of cytochrome c expression, a NO-induced effect that was previously shown to be dependent on the soluble guanylyl cyclase (33) (Fig. 4B). Furthermore, the inhibition of the cGMP-dependent protein kinases, a downstream protein in the signaling cascade, by using KT-5823 slightly inhibited NCX 4016-induced 2-DG uptake without modifying basal glucose uptake, whereas the inhibition of GMP-dependent protein kinase-1a by the inhibitor DT-2 had no effect (Fig. 4C). The action of NCX 4016 is thus probably independent of the activation of the classical NO-activated signaling pathway.

NCX 4016 does not induce IRS phosphorylation and does not activate the PKB pathway. We investigated whether NCX 4016 could induce 2-DG uptake by activating insulin signaling molecules. We observed that NCX 4016 did not increase the tyrosine phosphorylation of the insulin receptor, nor of its substrates IRS, either after a short-term incubation (Fig. 5A) or a long-term incubation (Fig. 5B), the two first events after insulin binding required for insulin-induced glucose transport (45). Furthermore, it also did not induce the activation of insulin receptor downstream pathways, including the PKB pathway, which is involved in insulin-induced glucose uptake (20, 24). Indeed, NCX 4016 did not induce the phosphorylation of PKB and of the PKB substrate AS160 that is required for insulin-induced glucose transport (38) (Fig. 5).


Figure 5
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  		Fig. 5. NCX 4016 does not activate signaling molecules involved in insulin action in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated during the indication period of times with vehicle (0), 100 nM of insulin, 200 µM of ASA, or 200 µM of NCX 4016. Total cell homogenates were then prepared, and 40 µg of proteins from each condition were separated by SDS-PAGE, transferred to PVDF membrane, and probed with the indicated antibodies. Results for short-term incubations and longer incubations with NCX 4016 are shown in A and B, respectively. A typical experiment representative of 3 is shown.

 
Effect of NCX 4016 on stress-activated kinase pathways. Many cell types, including adipocyte and muscle, respond to multiple stresses such as inflammation, nutrient deprivation, or oxidative stress by increasing their capacity to transport glucose (see the following references for some examples: 2, 26, 30, 36, 39). We thus examined whether NCX 4016 is able to activate signaling pathways induced by these various stresses. Among the mitogen-activated protein kinases, NCX 4016 did not activate ERK1/2 at any time of the incubation period, whereas insulin did it transiently (Fig. 6, A and B). We observed an activation of p54 JNK and p38. This activation was detectable at all of the incubation periods, although it declined after 24 h, and it reached the level induced by insulin (Fig. 6, A and B). Thus the activation of these pathways is not robust compared with stress activators of these two kinases. We did not observe any phosphorylation of the AMPK by NCX 4016, a kinase activated by fuel deprivation and in muscle also by exercise (17, 29). NCX 4016 did not decrease either the amount of inhibitory factor {kappa}B, unlike interleukin-1β (Fig. 6C), indicating that it did not activate the inflammatory nuclear factor-{kappa}B pathway in 3T3-L1 adipocytes. Taken together, our results indicate that NCX 4016 are not strong activators of stress kinase pathways.


Figure 6
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  		Fig. 6. Effects of NCX 4016 on various stress-activated kinases. 3T3-L1 adipocytes were incubated during the indicated period of times with vehicle (0), 100 nM of insulin, 200 µM of ASA, or 200 µM of NCX 4016. In B, as a control of AMPK activation, 3T3-L1 adipocytes were treated for 24 h with 100 µM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Results for short-term incubations and longer incubations with NCX 4016 are shown in A and B, respectively. In C, the degradation of inhibitory factor {kappa}B (I{kappa}B) was induced by a treatment with 20 ng/ml interleukin (IL)-1β for 0.5 h. NS, nonspecific band detected with the anti-I{kappa}B antibody. Total cell homogenates were then prepared, and 40 µg of proteins from each condition were separated by SDS-PAGE, transferred to PVDF membrane, and probed with the indicated antibodies.

 
NCX 4016 induces protein S-nitrosylation. Because the canonic NO-induced signaling pathway does not seem to play the major role in the effect of NCX 4016 on glucose transport, we assessed whether it could modulate protein S-nitrosylation in adipocytes. 3T3-L1 adipocytes were incubated for 24 h with solvent, 200 µM SNP, or 200 µM NCX 4016. By using the biotin switch assay, we were able to detect significant amounts of S-nitrosylated proteins in adipocytes cultured in control conditions (Fig. 7). Indeed, protein profiles obtained in the presence or absence of MMTS were quantitatively and qualitatively different, an indication of the efficient blockade of the free thiols that could not react any more with biotin-BMCC. Thus the labeled proteins observed in the presence of MMTS correspond to S-nitrosylated proteins. Interestingly, NCX 4016 increased the amount of two S-nitrosylated proteins with calculated apparent molecular weight of 60 and 68 kDa. SNP also affected the S-nitrosylation of these two proteins but to a lower extent.


Figure 7
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  		Fig. 7. Effects of NCX 4016 on protein S-nitrosylation. 3T3-L1 adipocytes were treated for 24 h with solvent (0), 200 µM SNP, or 200 µM NCX 4016. Total cell lysates were subjected to the biotin switch assay as described in MATERIALS AND METHODS. S-nitrosylated proteins [conditions +S-methylmethanethiosulfonate (MMTS)] were detected with anti-biotin antibodies. The intensity profiles of S-nitrosylated proteins in control (0) and NCX 4016 conditions are shown. Chemiluminescence was detected by using the FUJIFILM Las-3000 apparatus, and the time of exposure is 10 times less for the condition –MMTS compared with that with MMTS. The Western blot is representative of 3 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
In the present study, we report that the NO-donating derivative of acetylsalicylic acid (NCX 4016) evokes deoxyglucose uptake in 3T3-L1 adipocytes. We propose that the NO moiety of NCX 4016 is the inducer of glucose uptake in adipocytes. Indeed, an equimolar amount of the acetylsalicylic or salicylic moiety did not show any activity. This is in accordance with previous reports that demonstrate that the NO donor SNP increases glucose uptake in 3T3-L1 adipocytes (42) and isolated muscle (12). Furthermore, we found that the NO scavenger c-PTIO inhibits the action of NCX 4016 as well as that of SNP. Interestingly, NCX 4016 was more effective after 24 h treatment compared with 3 h treatment. This result suggests that slow release of NO from NCX 4016 induces a sustained increase in basal glucose uptake.

The effect of NCX 4016 on glucose uptake occurs without any changes in the expression levels of the glucose transporters expressed in 3T3-L1 adipocyte GLUT1 and GLUT4. NCX 4016 could thus act either by promoting the activity of plasma membrane-inserted transporters or by increasing the amount of glucose transporters at the plasma membrane. We favor this latter hypothesis because more GLUT4 and GLUT1 in the plasma membranes of NCX 4016-treated adipocytes have been found. The fact that GLUT1 is increased to a similar extent by NCX 4016 and insulin, whereas insulin is more potent to increase GLUT4 content in plasma membranes, strongly suggests that NCX 4016 essentially acts on the endosomal pools of glucose transporters, rather than on the GLUT4-specific sequestration compartments (for review on GLUT4 trafficking in adipocytes see Refs. 7 and 34). Furthermore, it also suggests that the action of NCX 4016 on glucose uptake could be extended to other cells and tissues that all expressed GLUT1. In accordance, NCX 4016 was shown to increase glucose uptake without modifying the expression level of GLUT1 in CD3-CD28 costimulated lymphocytes (14). It is thus possible that NCX 4016 could be effective in vivo to increase glucose disposal in resting conditions, an attractive hypothesis that remains to be tested by in vivo administration of the compound to animals.

Although NO was involved in the action of NCX 4016, it seems that it does not mainly require active soluble guanylate cyclase/protein kinase G pathways. Indeed, the use of various inhibitors interfering with this pathway does not efficiently inhibit NCX 4016-induced glucose uptake. NCX 4016 does not trigger the activation of some of the insulin signaling molecules, such as IRS and PKB, involved in the stimulation of glucose uptake, in accordance with the previously mentioned SNP action in 3T3-L1 adipocytes (42).

We found that p38 is activated by NCX 4016. However, this NCX 4016-induced p38 activation is certainly not responsive of the effect of NCX 4016 on glucose uptake. Indeed, although p38 has been involved in insulin-induced glucose transport (41), it is not involved in the translocation of glucose transporters at the plasma membrane (41), whereas we demonstrate that NCX 4016 controls this process. Furthermore, it is not known whether activation of p38 would induce by itself glucose transport.

The increase in glucose uptake observed after a treatment with NCX 4016 was comparable with the effect of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR; see Refs. 37 and 46). AICAR is a potent activator of AMPK, which has been involved in the activation of eNOS (10). It is thus possible that NCX 4016 and AICAR induce the same signaling pathway to induce glucose uptake. Because NCX 4016 is supposed to act downstream of AMPK, it is normal that NCX 4016 does not phosphorylate and activate AMPK (Fig. 6). However, AICAR has been described to inhibit insulin action on glucose uptake in 3T3-L1 adipocytes (37), whereas NCX 4016 does not. The various stresses known to increase basal glucose uptake in adipocytes (such as tumor necrosis factor-{alpha} or oxidative stress) are also known to be potent troublemakers of insulin metabolic action (5, 40). Therefore, NCX 4016 represents an interesting compound because, although it increases glucose uptake in adipocyte, it has no negative impact on insulin sensitivity, although it could activate the stress kinases p38 and JNK that are implicated in insulin resistance. It is possible that the level of activation of these kinases by NCX 4016 is too small to induce insulin resistance or that NCX 4016 counteracts the action of these deleterious pathways downstream of the activation of the stress kinases.

The action of the NO moiety of NCX 4016 would trigger a noncanonical signaling pathway possibly involving S-nitrosylation of target proteins (27) to promote glucose uptake. S-nitrosylation of N-ethylmaleimide-sensitive factor is involved in the redistribution to the plasma membrane of the postsynaptic {alpha}-amino-3-hydroxy-5-methyl-4-isoxazole-propioic acid subtype of glutamate receptors elicited by N-methyl-D-aspartate receptor agonists (22). A similar mechanism could be envisaged for glucose transporter translocation. Interestingly, we found that NCX 4016 selectively increased the S-nitrosylation of two proteins with an apparent molecular weight of 60 and 68 in adipocytes. The future identification of these proteins could certainly help to understand how NCX 4016 induced glucose uptake in adipocytes.

In cardiovascular models, NCX 4016 showed a better efficacy than aspirin in counteracting thromboembolism, myocardial infarction, and vascular reactivity and remodeling (6, 19), features well related to the mechanism of action of NO donors. Furthermore, NCX 4016 appeared to have little or no gastric toxicity in various animal models (19) and in healthy human volunteers (13) and prevented induced apoptotic events on endothelial (15) or gastric chief cells (16). The ability of NCX 4016 to induce glucose uptake without negatively affecting insulin sensitivity in adipocytes could thus be of therapeutic help in better controlling glycemia in diabetic patients while maintaining a protective vascular effect through NO activity.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
M. Cormont and Y. Le Marchand-Brustel were supported by the Institut National de la Santé et de la Recherche Médicale (INSERM). V. Kaddai was supported by the Fondation pour la Recherche Médicale, the Association pour la Recherche contre le Cancer. This work was supported by INSERM, the University of Nice-Sophia Antipolis, and the Association for Diabetes Research (ARD, Paris, France). Funds for microscopy equipment were provided by the Fondation Bettencourt-Schueller. The "Conseil General des Alpes Maritimes" funded the FUJIFILM Las-3000 detector for chemoluminescence, and the "Conseil Regional de la region PACA" funded the confocal microscopes.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
M. Bolla is an employee of NicOx SA. The work was in part supported by a Grant from NicOx SA.


   ACKNOWLEDGMENTS
 
We thank Dr. J.-F. Tanti for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Cormont, INSERM U895, Bâtiment Archimed, 151 route de St. Antoine de Ginestière, BP 2 3194, 06204 Nice cedex 3 (e-mail: cormont{at}unice.fr)

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.

* V. Kaddai and T. Gonzalez contributed equally to this work. Back


   REFERENCES
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 DISCLOSURES
 REFERENCES
 

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