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Phosphatidylinositol 3-Kinase Activation Is Required for Sulfonylurea Stimulation of Glucose Transport in Rat Skeletal Muscle

Department of Endocrinology, Fundación Jiménez Díaz, Universidad Autónoma de Madrid, 28040 Madrid, Spain

Address all correspondence and requests for reprints to: Adela Rovira, M.D., Department of Endocrinology, Fundación Jiménez Díaz, Avda. Reyes Católicos, 2, 28040 Madrid, Spain. E-mail: arovira{at}fjd.es.

	Abstract

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Sulfonylureas are drugs widely used in the treatment of patients with type 2 diabetes mellitus. In addition to their pancreatic effect of stimulating insulin secretion, many studies suggest that sulfonylureas also have extrapancreatic actions. We have previously reported that gliclazide, a second-generation sulfonylurea, stimulates the glucose uptake by rat hindquarter skeletal muscle directly and immediately by promoting the translocation of glucose transporter 4 to the plasma membrane. The aim of our study was to approach the gliclazide intracellular signaling pathway. For this purpose, we incubated clamped and isolated soleus muscle from rat with gliclazide. The following results were obtained: 1) gliclazide stimulates insulin receptor substrate (IRS)-1-phosphatidylinositol 3 (PI3)-kinase-associated activity, and this activity is necessary for gliclazide-stimulated glucose transport; 2) gliclazide treatment produces a gradual translocation of the diacylglycerol (DAG)-dependent isoforms protein kinase C (PKC) , , and from cytosolic to membrane fraction that is dependent on PI3-kinase and phospholipase C (PLC)- activation; and 3) PKC and PLC- activation is necessary for gliclazide-stimulated glucose transport. We propose a hypothetical signaling pathway by which gliclazide could stimulate IRS-1 that would allow its association with PI3-kinase, promoting its activation. PI3-kinase products could induce PLC- activation, whose hydrolytic activity could activate the DAG-dependent isoforms PKC , , and .

	Introduction

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THE HYPOGLYCEMIC POTENCY of sulfonylurea drugs has been attributed primarily to an acute stimulation of the rate of insulin secretion by inhibiting ATP-sensitive K⁺ channels (K_ATP) in pancreatic ß-cells, after its binding to the sulfonylurea receptor (SUR) subunit of the channel. However, the long-term efficacy of these drugs in type 2 diabetic patients also seems to involve extrapancreatic effects because it has been reported that these patients show a decrease in basal hepatic glucose production and an increase in insulin-mediated glucose disposal after a period of sulfonylurea treatment (1, 2). It has been reported that sulfonylureas enhance insulin-mediated glucose utilization by muscle tissue and cultured muscle cells (3, 4) by a mechanism distal to the insulin receptor (5, 6). In poststreptozotocin diabetic rats, treatment with gliclazide increases the glucose uptake by hindquarters and also has an additive effect to insulin (7). In addition, our group and others have reported a dose-dependent, direct, and rapid effect of sulfonylureas on glucose uptake by rat skeletal muscle (8, 9).

Sulfonylureas promote the movement of glucose transporters to the plasma membrane in adipocytes and in cultured myocytes (10, 11). In fact, we have showed that gliclazide, a second-generation sulfonylurea, promotes the movement of glucose transporter (GLUT) 4 to the plasma membrane in rat gastrocnemius muscle (12). The effect of gliclazide and insulin on both glucose uptake and GLUT4 translocation in rat skeletal muscle is additive, suggesting that these two stimuli act through different mechanisms. The work of Muller et al. (13), who examined the mechanism of glucose transport induced by the sulfonylurea glimepiride in adipocytes, has shown an insulin receptor-independent signaling pathway that includes insulin receptor substrate (IRS)-1/2 tyrosine phosphorylation and phosphatidylinositol 3 (PI3)-kinase activation. Protein kinase C (PKC) activation has been implicated in the sulfonylurea-stimulating glucose uptake by skeletal muscle (14, 15), but there is no information on the activation of other signaling pathways that could plausibly be enrolled in glucose transport in this tissue, mainly PI3-kinase and phospholipase C (PLC).

The aim of this study was to know whether sulfonylurea activates some intracellular enzymes that currently are implicated in glucose transport in skeletal muscle and to define the signaling pathway.

	Materials and Methods

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Materials
Human insulin, Actrapid, was from Novo (Mainz, Germany). Gliclazide was a kind gift from Institut the Recherches Internationales Servier (Madrid, Spain). 2[³H]deoxy-D-glucose and ¹⁴C sorbitol were obtained from NEN Life Science Products Corp. (Boston, MA). BSA RIA-grade Fraction V was obtained from Sigma (St. Louis, MO). Wortmannin, Ro-318220, and U-73122 were obtained from Calbiochem (La Jolla, CA). L--Phosphatidylinositol (sodium salt) was purchased from Avanti Polar Lipids (Alabaster, AL). Antibodies were purchased from Transduction Laboratories (Lexington, KY). Reagents for polyacrylamide gel electrophoresis and Triton X-100 were obtained from Bio-Rad Laboratories (Richmond, CA). All other chemicals were of analytical grade. Protein determination was performed by the Bradford dye method (Bio-Rad Laboratories).

Measurement of 2-deoxyglucose uptake
Glucose uptake was measured as previously described (16), with minor modifications. Briefly, male Wistar rats (180–200 g) were fasted for 16 h before the experiments. (Note, all research protocols were approved by Fundación Jiménez Díaz Animal Research Committee). Soleus muscles from rats were mounted on Plexi glass clamps to maintain them at the resting position; the soleus muscles were then preincubated at 37 C under 95%O₂-5%CO₂ in glucose-free Krebs-Henseleit buffer (KHB) containing 0.1% BSA and 1 mM pyruvate for 1 h. During the last 10 min of the preincubation period, insulin (1 nM) or gliclazide (300 µg/ml) was added. After 1 h of preincubation, muscles were transferred into fresh identical medium containing 2-deoxyglucose (2 µCi/ml, 5 mM) and ¹⁴C sorbitol (0.11 µCi/ml, 20 mM) and were incubated for 60 min in the absence and presence of insulin (1 nM) or gliclazide (300 µg/ml). Glucose uptake was terminated by washing the muscles in ice-cold KHB. Thereafter, the muscles were dissolved in solubilization buffer containing 0.3 M C₁₉H₄₂NBr and 0.3 M KOH. Sample-associated radioactivity was determined by scintillation counting. In each case, one treated soleus muscle was directly compared with the contralateral control muscle. ¹⁴C sorbitol was used as an extracellular space marker.

In another set of experiments, the PI3-kinase inhibitor, wortmannin (1 µM), PKC inhibitor, Ro-318220 (20 µM), and PLC- inhibitor, U-73122 (5 µM), were added in the preincubation period, 15 min before addition of vehicle, insulin (1 nM), or gliclazide (300 µg/ml). The glucose uptake was measured as described above.

Measurement of PI3-kinase activity
Soleus muscles were incubated in the absence and presence of 100 nM insulin or 300 µg/ml gliclazide for 2, 4, and 8 min. PI3-kinase activity was measured as previously described (17, 18). Briefly, after homogenization, muscle samples (500 µg of protein) were immunoprecipitated with anti-IRS-1 antibody, followed by protein A-Sepharose. PI3-kinase activity was measured directly on the Sepharose beads in 45 µl reaction mixture containing 20 mM HEPES (pH 7.4), 0.2 mg/ml phosphatidylinositol, 7.5 mM LiCl, 10 mM MgCl₂, and ³²P ATP (30 µM at 0.2 µCi/µl). After 15 min, the reaction was stopped by the addition of 100 µl of 1 N HCl. Lipids were extracted from the reaction mixture with 200 µl chloroform-methanol (1:1), and 60 µl of the lower organic phase were spotted onto a silica-gel, thin-layer chromatography plate and then developed. The plate was analyzed by autoradiography.

Determination of IRS-1 tyrosine phosphorylation and IRS-1 binding to p85 by immunoprecipitation and immunoblotting
A total of 500 µg of muscle lysate, obtained as described above, was immunoprecipitated either with antiphosphotyrosine PY20 antibody for measuring IRS-1 tyrosine phosphorylation or with anti-IRS-1 antibody for determining the association of IRS-1 with p85 subunit. After overnight rocking at 4 C, 50 µl of protein A-Sepharose were added to the immunoprecipitates, and incubation was continued for 1 h at 4 C followed by brief centrifugation at 9000 rpm. The Sepharose pellets were then washed three times with ice-cold solubilization buffer. Fifty microliters of Laemmli buffer were added, and the samples were boiled for 5 min at 100 C. Immunoprecipitates were subjected to SDS-PAGE on 8% resolving gel. Proteins were transferred onto nitrocellulose sheets and then incubated with anti-IRS-1 or anti-p85 antibodies, followed by incubation with a secondary antibody bound to horseradish peroxidase. Immunodetection was performed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Uppsala, Sweden). Blots were quantified by scanning densitometry.

PKC studies
To determine PKC translocation, soleus muscles were incubated in KHB, as described above, with and without 100 nM insulin or 300 µg/ml gliclazide for the indicated times (2, 5, 10, and 15 min). In another set of experiments, wortmannin (1 µM) or U-73122 (5 µM) were added 15 min before the addition of insulin or gliclazide.

Cytosol and membrane fractions were prepared by the method of Heydrick et al. (19). Briefly, muscles were homogenized in 0.4 ml homogenizing buffer containing 250 mM sucrose, 20 mM Tris (pH 7.5), 2 mM EDTA, 0.5 mM EGTA, 20 µg/ml leupeptin, 10 µg/ml aprotinin, 174.2 µg/ml phenylmethylsulfonyl fluoride, and 20 mM dithiothreitol. The homogenate was centrifuged at 100,000 x g for 1 h at 4 C. The supernatant (cytosolic extract) was transferred to a tube kept on ice, whereas the pellet was resuspended in 0.45 ml homogenizing buffer containing 5% Triton X-100. The resuspended pellet fraction was then centrifuged at 14,000 x g for 5 min at 4 C, and the pellet was discarded. The supernatant from this spin constitutes the membrane extract. An aliquot of cytosol and membrane fractions (150 µg) was subjected to SDS-PAGE on 8% resolving gel. Proteins were transferred onto nitrocellulose sheets and then incubated with PKC , , and monoclonal antibodies. Immunoblots were quantified by scanning densitometry, and treated samples were compared with their corresponding controls.

Statistical analysis
Statistical significance was assessed by the Student’s two-tailed t test, and a P < 0.05 was considered significant.

	Results

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Effect of gliclazide on glucose uptake
Gliclazide at the concentration assayed of 300 µg/ml produced a significant increase in glucose uptake by soleus muscle over the basal value of 64% (95% confidence interval, 45–78%). Gliclazide at a higher concentration (1000 µg/ml) produced a similar increase in glucose uptake over the basal value of 60%. The rate of glucose uptake induced by gliclazide was linear from 10–60 min of incubation periods (data not shown). As indicated in Table 1, insulin (1 nM) induced an increase in glucose uptake by soleus muscle over basal value by approximately 153% (95% confidence interval: 34–246%). Soleus muscles from rats weighing 180–200 g had similar rates of either insulin- or gliclazide-stimulated glucose uptake compared with the rates from smaller rats (100 g). To demonstrate the metabolic viability of this muscle preparation, we measured ATP (20) in soleus muscle incubated as describe above and in soleus muscle frozen in liquid nitrogen immediately after it was removed (fresh muscle). The concentration of ATP in fresh and incubated muscle was similar (2.1 ± 0.3 vs. 2.0 ± 0.2 µmol/g, respectively; n = 5, P = not significant).

Effect of wortmannin on gliclazide-stimulated glucose uptake (Table 1)

Effects of Ro-318220 on gliclazide-stimulated glucose uptake (Table 1)
To examine whether gliclazide-stimulated glucose uptake occurred through a PKC-dependent pathway, we used 20 µM of Ro-318220, an inhibitor of the catalytic domain of PKC. The inhibitor avoided the gliclazide stimulation of glucose uptake. Glucose uptake in the presence of both gliclazide and Ro-318220 was similar to basal glucose uptake. Insulin-stimulated glucose uptake decreased in the presence of Ro-318220, but not significantly. Glucose uptake in presence of both agents (insulin and Ro-318220) achieved values higher than those obtained in basal conditions.

Effects of U-73122 on gliclazide-stimulated glucose uptake (Table 1)
To examine whether gliclazide-stimulated glucose uptake requires PLC activation, we evaluated the effect of a specific PLC- inhibitor, U-73122 (5 µM). The inhibitor blocked the gliclazide stimulation of glucose uptake. Gliclazide-stimulated glucose uptake decreased significantly. In contrast, U-73122 at a concentration of 5 µM did not inhibit insulin-stimulated glucose uptake; a concentration of 10 µM did not inhibit insulin-stimulated glucose uptake either. Basal glucose uptake was not altered by the presence of the inhibitor.

PI3-kinase studies
PI3-kinase activity.
It has been shown that PI3-kinase activation is one of the steps of the insulin-signaling pathway for glucose uptake (21). As shown above, wortmannin inhibited the effect of gliclazide on glucose uptake by soleus muscle. Therefore, we studied the effect of gliclazide on PI3-kinase activity. Gliclazide treatment for 2, 4, and 8 min increased IRS-1-mediated PI3-kinase activation (Fig. 1). PI3-kinase activity was stimulated at 2 min of exposure to gliclazide; thereafter, it tended to decline at 4 and 8 min (Fig. 1A). The kinetics of the PI3-kinase response was similar to that produced by insulin (Fig. 1B).

View larger version (22K): [in this window] [in a new window]   FIG. 1. Measurement of PI3-kinase activity in rat soleus muscle. A, Effect of gliclazide (300 µg/ml) on IRS-1-mediated PI3-kinase activity. B, Effect of insulin (100 nM) on IRS-1-mediated PI3-kinase activity. PI3-kinase activity is expressed as a percentage of basal activity. Results represent the mean ± SEM of five independent experiments. *, P < 0.05 vs. basal activity. A representative autoradiograph is shown. The position of PI3-phosphate (PI3-P) is indicated.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Effect of 300 µg/ml gliclazide and 100 nM insulin on IRS-1 tyrosine phosphorylation. A 500-µg sample of muscle lysate was immunoprecipitated with anti-PY-20 antibody, followed by SDS-PAGE, and transferred to nitrocellulose. IRS-1 tyrosine phosphorylation was detected by immunoblotting using anti-IRS-1 antibody. IRS-1 tyrosine phosphorylation is expressed as a percentage of basal value. Results represent the mean ± SEM of five independent experiments. *, P < 0.05 vs. basal. A representative autoradiograph is shown.

PKC studies
PKC translocation.
To know whether gliclazide stimulates the PKC isoforms , , and , we studied the PKC levels in membrane and cytosol fractions obtained from soleus muscles incubated at 2, 5, 10, and 15 min. Studies were also performed with insulin.

Gliclazide treatment produced a gradual increase in membrane contents of PKC isoforms (Fig. 3). Scanning densitometry of autoradiography revealed that the highest increment was at 10 min of gliclazide treatment, representing 154% (PKC ), 164% (PKC ), and 157% (PKC ) over basal situation. The increase in the content of PKC in membrane fractions was accompanied by a reduction of PKC in the cytosolic fraction.

View larger version (28K): [in this window] [in a new window]   FIG. 3. Time-dependent effects of insulin (100 nM) and gliclazide (300 µg/ml) on PKC , , and levels in membrane (white squares) and cytosolic (black rhombus) fractions of rat soleus muscles incubated in vitro. Results represent mean ± SEM values of five comparisons of insulin- and gliclazide-treated muscles vs. control muscles at each time point. *, P < 0.05 vs. basal. (A) Representative immunoblot of the PKC , , and subcellular distribution at 10 min of insulin treatment. (B) Representative immunoblot of the PKC , , and subcellular distribution at 10 min of gliclazide treatment.

Effects of wortmannin on PKC translocation.
In view of the fact that both wortmannin, an inhibitor of PI3-kinase, and Ro-318220, an inhibitor of PKC, inhibit gliclazide effects on glucose transport in skeletal muscle, we evaluated the possibility that PI3-kinase and PKC activation may be interrelated events in gliclazide intracellular signaling pathway. We measured gliclazide-stimulated PKC , , and translocation in absence or presence of wortmannin. As shown in Table 2, increases in the translocation of PKC , , and to membrane fractions were fully inhibited by wortmannin. The effect of wortmannin on insulin-induced PKC translocation was also investigated. In contrast with the results seen with gliclazide, wortmannin had no effect on insulin-stimulated PKC translocation. These results suggest that PKC may operate downstream of PI3-kinase during gliclazide action and that gliclazide actives PKC by a signaling pathway that is different from that used by insulin.

View this table: [in this window] [in a new window]   TABLE 2. Effects of wortmannin (1 µM) or U-73122 (5 µM) on translocation of PKC , , and to plasma membranes stimulated by insulin (100 nM) or gliclazide (300 µg/ml)

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previously we have reported that gliclazide has a direct effect on glucose uptake by rat hindquarter muscle and promotes the translocation of GLUT4 to the plasma membrane in rat gastrocnemius muscle (12). The present study aimed to know the intracellular signal pathways involved in the gliclazide-stimulated glucose transport. We have used rat soleus muscle that shows similar basic parameters of glucose uptake to other in vitro muscle preparations (23). In comparison with previous results obtained in rat hindquarters, rat soleus muscle displayed a less prominent glucose uptake stimulated by 300 µg/ml gliclazide (2.7-fold increase and 1.7-fold increase, respectively) but within a significant narrow range of confidence interval.

First, we demonstrated that gliclazide induced a rapid activation of PI3-kinase associated to IRS-1. The importance of this activation in the effect of gliclazide on glucose uptake was demonstrated by using wortmannin, a well-known PI3-kinase inhibitor. The effect of gliclazide on glucose uptake was totally suppressed by preincubation of soleus muscle with wortmannin, indicating the involvement of PI3-kinase in the metabolic effect of gliclazide.

IRS-1 is a pivotal molecule in the regulation of insulin signal pathway (22). IRS-1 tyrosine phosphorylation causes its binding to the p85 regulatory subunit of PI3-kinase and subsequent activation of the enzyme. Our results indicate that gliclazide stimulates the activity of PI3-kinase associated to IRS-1 through IRS-1 tyrosine phosphorylation in skeletal muscle. The involvement of PI3-kinase activation via IRS1/2 tyrosine phosphorylation has been reported in the glucose transport induced by glimepiride, a third-generation sulfonylurea, in rat adipocytes (13). Sulfonylureas do not seem to act through insulin receptor activation, at least when they are used in a chronic way (5, 6). In poststreptozotocin diabetic rats, gliclazide treatment for 12 d increased the glucose uptake by hindquarter muscle without modifying the kinase activity of the insulin receptor (7). The mechanism by which sulfonylureas induce tyrosine phosphorylation of IRS-1 remains to be elucidated.

PKC is another intracellular signal that has been implicated in glucose transport. Its precise role in the process of glucose uptake mediated by insulin is controversial. Previous studies have reported that sulfonylureas stimulate glucose uptake by skeletal muscle associated to activation of PKC (14, 15). Therefore, we examined the effects of Ro-318220, a potent inhibitor of PKC activity. The stimulatory effect of gliclazide on glucose uptake was inhibited by 20 µM of Ro-318220, indicating that PKC activation seems necessary for gliclazide-stimulated glucose transport. Ro-318220, at the concentration of 20 µM, did not affect the basal glucose uptake. Although not significantly, the insulin-stimulated glucose uptake decreased. At a higher concentration (40 µM), Ro-318220 produced a significant decrease in insulin-stimulated glucose uptake (data not shown). Similar results on the effect of high concentrations of Ro-318220 (40 µM) on insulin-stimulated glucose uptake by rat soleus muscle have also been reported in previous studies, suggesting a more complete inhibition of PKC isoforms [which varies in sensitivity (, ß >> , >> )] (24). It has been reported that the IC₅₀ for Ro-318220 is 10 nM; however, intact tissues need higher concentrations of the inhibitor (24).

It has been suggested that diacylglycerol (DAG)-dependent isoforms of PKC are involved in the mechanism of action of sulfonylureas on glucose transport (11, 15, 25). Therefore, we studied the effect of gliclazide on the subcellular distribution of the skeletal muscle, abundantly expressed, PKC isoforms , , and (26, 27). The sulfonylurea provoked a progressive increase in membrane content of the three PKC isoforms. The fact that both the Ca²⁺-dependent PKC isoform (PKC ) and Ca²⁺-independent PKC isoforms (PKC and PKC ) were activated by gliclazide suggests that this effect was not mediated by changes in cytosolic Ca²⁺.

The maximal increment in membrane PKC isoforms was seen at 10 min of gliclazide treatment, whereas insulin provoked an initial increase in membrane PKC isoforms at 2 min of treatment and a secondary increase at 10–15 min. The biphasic increase in membrane contents of PKC isoforms has already been described in insulin-treated rat solei in vitro (26), in rat adipocytes (28, 29, 30), and in BC³H-1 myocytes (30, 31, 32). This type of response has been attributed to the different sources of DAG generated through the activation of different phospholipase isoforms. The mechanism of PKC activation by gliclazide cannot be explained by our study; however, it is possible that glycosylphosphatidylinositol-specific PLC could be involved, as suggested by Müller et al. in studies performed with glimepiride in rat adipose cells (33).

Interestingly, in this study, we show that the PI3-kinase inhibitor, wortmannin, prevented the gliclazide-stimulated PKC , , and translocation to membranes. This finding suggests that the effect of gliclazide on PKC translocation depends on PI3-kinase activation. In contrast, the effects of insulin on PKC translocation did not appear to be dependent on PI3-kinase. These results suggest two different pathways for insulin and gliclazide to stimulate PKC , , and translocation. However, PKC , , and are DAG-dependent isoforms and are unlikely to serve as direct downstream effectors for PI3-kinase.

As mentioned earlier, previous studies have suggested an implication of glycosylphosphatidylinositol-specific PLC on sulfonylurea signaling pathway (33). PLC is a family of isoenzymes that can be classified into three major subfamilies, ß, , and isoenzymes, according to their structure and mechanism of activation (34). It is known that PLC- may be activated by phosphatidylinositol 3,4,5 triphosphate (34, 35). We evaluated the effects of a PLC- specific inhibitor U-73122 on gliclazide-stimulated PKC translocation.

The PLC- inhibitor suppressed gliclazide-induced PKC translocation, but it did not affect insulin-induced PKC translocation. These results suggest that inhibition of one of the sources of DAG does not affect insulin-induced PKC translocation, whereas the increment of DAG due to PLC activation seems to be necessary for gliclazide-induced PKC translocation. Then, we evaluated the effects of this inhibitor on gliclazide-stimulated glucose uptake. Pretreatment of muscles with U-73122 fully blocked gliclazide-stimulated glucose uptake. In contrast, the presence of the inhibitor did not affect the effects of insulin on glucose uptake.

Previously we have reported that skeletal muscle glucose uptake stimulated by gliclazide was blocked by diazoxide, a K_ATP opener, suggesting the implication of this channel in the effect of the sulfonylurea (8). The K_ATP channels are involved in different physiological functions, including modulation of insulin secretion, protection of myocardium from ischemia, and regulation of vascular tone. The channel is composed of a hetero-octomer of four regulatory SUR subunits and four potassium pore proteins, Kir6.1, or Kir6.2. SUR2 is the primary regulatory subunit expressed in muscle, and it pairs with Kir6.2 in skeletal and cardiac muscle. There are some lines of evidence supporting the view that K_ATP channels in skeletal muscle may be involved in glucose transport. Elimination of muscle K_ATP channel currents in mice by disrupting SUR2 has been shown to increase insulin responsiveness in skeletal muscle (36). K_ATP channel openers, such as nicorandil or PCO-400, have been shown to inhibit both basal and insulin-stimulated glucose transport in cultured human skeletal muscle. These effects were reversed by glibenclamide and gliclazide (37). In the present study, we have not addressed the issue of whether there is a link between K_ATP channels and the activation of the enzymatic cascade by gliclazide in skeletal muscle. Further studies are needed to elucidate the mechanisms that couple the electric activity of the K_ATP channel with the cellular metabolic signals leading to glucose transport in the muscle.

In conclusion, our data suggest that gliclazide promotes glucose transport in skeletal muscle by activating a serial of enzymes, which seems to initiate with IRS-1 tyrosine phosphorylation and its association with PI3-kinase. Thereafter, PLC- is activated and DAG-dependent PKC isoforms , , and translocate to membranes (Fig. 4).

View larger version (12K): [in this window] [in a new window]   FIG. 4. Hypothetical signaling pathway of gliclazide in skeletal muscle. Broken arrows indicate unknown pathway, thin arrows indicate the signaling pathway, and thick arrows indicate enzymatic activities that are necessary for the gliclazide-stimulated glucose transport in skeletal muscle.

	Acknowledgments

 
We are grateful to the Institut the Recherches Internationales Servier for supplying gliclazide.

	Footnotes

 
This work was supported by a grant from Fondo de Investigaciones Sanitarias (FIS) de la Seguridad Social. E.R. and A.P. are recipients of fellowship awards from the Fundación Conchita Rábago.

Abbreviations: DAG, Diacylglycerol; GLUT, glucose transporter; IRS, insulin receptor substrate; K_ATP, ATP-sensitive K⁺ channel; KHB, Krebs-Henseleit buffer; PI3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; SUR, sulfonylurea receptor.

Received June 16, 2003.

Accepted for publication October 10, 2003.

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