Phosphatidylinositol 3-Kinase Activation Is Required for Sulfonylurea ...

0013-7227/04/$15.00/0 Printed in U.S.A.

Endocrinology 145(2):679 – 685 Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2003-0755

Phosphatidylinositol 3-Kinase Activation Is Required for Sulfonylurea Stimulation of Glucose Transport in Rat Skeletal Muscle ESTHER RODRI´GUEZ, NIEVES PULIDO, REMEDIOS ROMERO, FRANCISCO ARRIETA, ARANZAZU PANADERO, AND ADELA ROVIRA Department of Endocrinology, Fundacioń Jimeńez Dıáz, Universidad Autońoma de Madrid, 28040 Madrid, Spain 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 DAGdependent isoforms PKC ␣, ␪, and ⑀. (Endocrinology 145: 679 – 685, 2004)

T

HE 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 (KATP) in pancreatic ␤-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 insulinmediated 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 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

Abbreviations: DAG, Diacylglycerol; GLUT, glucose transporter; IRS, insulin receptor substrate; KATP, ATP-sensitive K⫹ channel; KHB, KrebsHenseleit buffer; PI3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; SUR, sulfonylurea receptor. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

679

680

Endocrinology, February 2004, 145(2):679 – 685

Rodrı´guez et al. • PI3-Kinase Activation by Sulfonylureas

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

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.

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 Fundacioń Jimeńez Dıáz 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 ␮g/ml) was added. After 1 h of preincubation, muscles were transferred into fresh identical medium containing 2-deoxyglucose (2 ␮Ci/ ml, 5 mm) and 14C sorbitol (0.11 ␮Ci/ml, 20 mm) and were incubated for 60 min in the absence and presence of insulin (1 nm) or gliclazide (300 ␮g/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 ␮m), PKC inhibitor, Ro-318220 (20 ␮m), and PLC-␥ inhibitor, U-73122 (5 ␮m), were added in the preincubation period, 15 min before addition of vehicle, insulin (1 nm), or gliclazide (300 ␮g/ml). The glucose uptake was measured as described above.

To determine PKC translocation, soleus muscles were incubated in KHB, as described above, with and without 100 nm insulin or 300 ␮g/ml gliclazide for the indicated times (2, 5, 10, and 15 min). In another set of experiments, wortmannin (1 ␮m) or U-73122 (5 ␮m) 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 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, 174.2 ␮g/ml phenylmethylsulfonyl fluoride, and 20 mm dithiothreitol. The homogenate was centrifuged at 100,000 ⫻ 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 ⫻ 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 ␮g) 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.

Measurement of PI3-kinase activity

Statistical analysis

Soleus muscles were incubated in the absence and presence of 100 nm insulin or 300 ␮g/ml gliclazide for 2, 4, and 8 min. PI3-kinase activity was measured as previously described (17, 18). Briefly, after homogenization, muscle samples (500 ␮g 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 ␮l 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 ␮m at 0.2 ␮Ci/␮l). After 15 min, the reaction was stopped by the addition of 100 ␮l of 1 n HCl. Lipids were extracted from the reaction mixture with 200 ␮l chloroform-methanol (1:1), and 60 ␮l of the lower organic phase were spotted onto a silica-gel, thin-layer chromatography plate and then developed. The plate was analyzed by autoradiography.

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

Determination of IRS-1 tyrosine phosphorylation and IRS1 binding to p85 by immunoprecipitation and immunoblotting A total of 500 ␮g 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 ␮l 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%

PKC studies

Results Effect of gliclazide on glucose uptake

Gliclazide at the concentration assayed of 300 ␮g/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 ␮g/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

TABLE 1. 2-Deoxyglucose uptake by rat soleus muscle

None Insulin (1 nM) Gliclazide (300 ␮g/ml) Results represent mean ⫾ a P ⬍ 0.05 vs. none. b P ⬍ 0.05 vs. insulin. c P ⬍ 0.05 vs. gliclazide.

SEM

Control

Wortmannin, 1 ␮M

Ro-318220, 20 ␮M

U-73122, 5 ␮M

2.4 ⫾ 0.3 5.1 ⫾ 0.6a 4.1 ⫾ 0.3a

2.5 ⫾ 0.2 2.4 ⫾ 0.6b 2.4 ⫾ 0.5c

2.9 ⫾ 0.4 4 ⫾ 0.7 2.6 ⫾ 0.3c

2.5 ⫾ 0.1 5.3 ⫾ 0.2 2.3 ⫾ 0.4c

(nmol/g⫺1䡠min⫺1) of 11 independent experiments each with triplicate determinations.


Endocrinology, February 2004, 145(2):679 – 685 681

similar (2.1 ⫾ 0.3 vs. 2.0 ⫾ 0.2 ␮mol/g, respectively; n ⫽ 5, P ⫽ not significant). Effect of wortmannin on gliclazide-stimulated glucose uptake (Table 1)

To examine the functional role of PI3-kinase in gliclazide action, we used wortmannin, a PI3-kinase inhibitor. Pretreatment of soleus muscles with 1 ␮m wortmannin inhibited the stimulatory effect of gliclazide on glucose uptake. Glucose uptake in the presence of both gliclazide and 1 ␮m wortmannin was not different from the uptake obtained in basal conditions. Wortmannin, as expected, also inhibited the insulin-stimulated glucose uptake. Glucose uptake in the presence of both 1 nm insulin and 1 ␮m wortmannin was no different from the uptake obtained in basal conditions. Wortmannin did not affect the basal glucose uptake. 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 ␮m 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. Insulinstimulated 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 ␮m). The inhibitor blocked the gliclazide stimulation of glucose uptake. Gliclazide-stimulated glucose uptake decreased significantly. In contrast, U-73122 at a concentration of 5 ␮m did not inhibit insulinstimulated glucose uptake; a concentration of 10 ␮m 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). Effects of gliclazide on IRS-1 tyrosine phosphorylation. Tyrosine phosphorylation of IRS-1 has been shown to be critical in the activation of PI3-kinase by insulin (22). Therefore, we tested

FIG. 1. Measurement of PI3-kinase activity in rat soleus muscle. A, Effect of gliclazide (300 ␮g/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.

whether PI3-kinase activation by gliclazide would be accompanied by increased IRS-1 tyrosine phosphorylation. As seen in Fig. 2, gliclazide increased IRS-1 tyrosine phosphorylation 191% over basal situation. Effects of gliclazide on IRS-1-p85 binding. To confirm the results described above, we analyzed the effect of gliclazide on the binding of IRS-1 to the p85 subunit. The enzyme PI3-kinase is composed of two subunits, a regulatory p85 subunit and a catalytic p110 subunit. IRS-1 binding to the regulatory p85 subunit is necessary for the activation of PI3-kinase (22). Muscle extracts were subjected to immunoprecipitation with anti-IRS-1 antibody, and Western blot analysis was performed with an anti-p85 antibody. Gliclazide significantly increased the p85IRS-1 association at 2 min of incubation (139 ⫾ 10% of basal, P ⬍ 0.05, n ⫽ 5) and at 8 min (130 ⫾ 8% of basal, P ⬍ 0.05, n ⫽ 5). Insulin treatment for 2 min increased p85-IRS-1 association (190 ⫾ 25% of basal, P ⬍ 0.05, n ⫽ 5). PKC studies

PKC translocation. To know whether gliclazide stimulates the PKC isoforms ␣, ␪, and ⑀, we studied the PKC levels in

682



Effects of U-73122 on PKC translocation. To analyze the implication of PLC on gliclazide-stimulated PKC ␣, ␪, and ⑀ translocation, we used the PLC inhibitor U-73122. As shown in Table 2, the increase by gliclazide on the translocation of PKC ␣, ␪, and ⑀ to membrane fractions was fully inhibited by U-73122. In contrast, U-73122 had no effect on insulin-stimulated PKC translocation. These results suggest that the activation of PLC seems necessary for gliclazide-stimulated PKC translocation. Discussion

FIG. 2. Effect of 300 ␮g/ml gliclazide and 100 nM insulin on IRS-1 tyrosine phosphorylation. A 500-␮g 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.

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. On the other hand, insulin provoked a biphasic response with an initial increase in membrane content of PKC ␣, ␪, and ⑀ at 2 min and a secondary increase at 10 min. Concomitantly, cytosolic levels of PKC ␣, ␪, and ⑀ decreased at these time points. Effects of wortmannin on PKC translocation. In view of the fact that both wortmannin, an inhibitor of PI3-kinase, and Ro318220, 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.

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 ␮g/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 PI3kinase 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 Ro318220, a potent inhibitor of PKC activity. The stimulatory effect of gliclazide on glucose uptake was inhibited by 20 ␮m of Ro-318220, indicating that PKC activation seems necessary for gliclazide-stimulated glucose transport. Ro-318220, at the



FIG. 3. Time-dependent effects of insulin (100 nM) and gliclazide (300 ␮g/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.

concentration of 20 ␮m, did not affect the basal glucose uptake. Although not significantly, the insulin-stimulated glucose uptake decreased. At a higher concentration (40 ␮m), 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 ␮m) 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 (␣, ␤ ⬎⬎ ␦, ⑀ ⬎⬎ ␨)] (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

684



TABLE 2. Effects of wortmannin (1 ␮M) or U-73122 (5 ␮M) on translocation of PKC ␣, ␪, and ␧ to plasma membranes stimulated by insulin (100 nM) or gliclazide (300 ␮g/ml) PKC ␣ (%) Cytosol

Gliclazide ⫹ Wortmannin ⫹ U-73122 Insulin ⫹ Wortmannin ⫹ U-73122

123 ⫾ 12 134 ⫾ 4b 94 ⫾ 3 114 ⫾ 7

PKC ␪ (%) Membrane

73 ⫾ 7a 54 ⫾ 11a 127 ⫾ 25 101 ⫾ 29

Cytosol

110 ⫾ 23 139 ⫾ 5b 99 ⫾ 11 141 ⫾ 42

PKC ␧ (%) Membrane

71 ⫾ 11a 72 ⫾ 7a 116 ⫾ 7 86 ⫾ 16

Cytosol

127 ⫾ 17 115 ⫾ 4a 112 ⫾ 11 93 ⫾ 28

Results represent percentage over control (absence of wortmannin or U-73122) and are expressed as mean ⫾ experiments. a P ⬍ 0.05. b P ⬍ 0.01.

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 –30), and in BC3H-1 myocytes (30 – 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 Mu¨ ller 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, ␤, ␥, 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 mus-

Membrane

SEM

55 ⫾ 12b 56 ⫾ 13a 104 ⫾ 19 94 ⫾ 24 of five independent

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.

cles 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 KATP opener, suggesting the implication of this channel in the effect of the sulfonylurea (8). The KATP 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 KATP channels in skeletal muscle may be involved in glucose transport. Elimination of muscle KATP channel currents in mice by disrupting SUR2 has been shown to increase insulin responsiveness in skeletal muscle (36). KATP 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 KATP 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 KATP 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).


16. 17. 18.

19.

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

20. 21.

Received June 16, 2003. Accepted October 10, 2003. Address all correspondence and requests for reprints to: Adela Rovira, M.D., Department of Endocrinology, Fundacio´ n Jime´ nez Dıáz, Avda. Reyes Cato´ licos, 2, 28040 Madrid, Spain. E-mail: [email protected]. 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 Fundacio´ n Conchita Ra´ bago.

22. 23. 24.

References 1. Kolterman OG, Gray RS, Shapiro G, Scarlett JA, Griffin J, Olefsky JM 1984 The acute and chronic effects of sulfonylurea therapy in type II diabetic subjects. Diabetes 33:346 –354 2. Simonson DC, Ferrannini E, Bevilacqua S, Smith D, Barrett E, Carlson R, DeFronzo R 1984 Mechanisms of improvement in glucose metabolism after chronic glyburide therapy. Diabetes 33:838 – 845 3. Feldman JM, Lebovitz HE, Durham NC 1969 An insulin dependent effect of chronic tolbutamide administration on the skeletal muscle carbohydrate transport system. Diabetes 18:84 –95 4. Wang PH, Moller D, Flier JS, Nayak RC, Smith RJ 1989 Coordinate regulation of glucose transporter function, number and gene expression by insulin and sulfonylureas in L6 rat skeletal muscle cells. J Clin Invest 84:62– 67 5. Jacobs DB, Hayes GR, Lockwood DH 1987 Effect of chlorpropamide on glucose transport in rat adipocytes in the absence of changes in insulin binding and receptor-associated tyrosine kinase activity. Metabolism 36:548 –554 6. Bak JF, Schmitz O, Sorensen NS, Pedersen O 1989 Postreceptor effects of sulfonylurea on skeletal muscle glycogen synthase activity in type II diabetic patients. Diabetes 38:1343–1350 7. Pulido N, Sua´rez A, Casanova B, Romero R, Rodrı´guez E, Rovira A 1997 Gliclazide treatment of streptozotocin diabetic rats restores GLUT4 protein content and basal glucose uptake in skeletal muscle. Metabolism 46:10 –13 8. Pulido N, Casla A, Suárez A, Casanova B, Arrieta FJ, Rovira A 1996 Sulphonylurea stimulates glucose uptake in rats through an ATP-sensitive K⫹ channel dependent mechanism. Diabetologia 39:22–27 9. Daniels EL, Lewis SB 1982 Acute tolbutamide administration alone or combined with insulin enhances glucose uptake in the perfused rat hindlimb. Endocrinology 110:1840 –1842 10. Muller M, Wied S 1993 The sulfonylurea drug, glimepiride, stimulates glucose transport, glucose transporter translocation, and dephosphorylation in insulinresistant rat adipocytes in vitro. Diabetes 42:1852–1867 11. Rogers BJ, Standaert ML, Pollet RJ 1987 Direct effects of sulfonylurea agents on glucose transport in the BC3–H1 myocyte. Diabetes 36:1292–1296 12. Pulido N, Romero R, Sua´rez A, Rodrı´guez E, Casanova B, Rovira A 1996 Sulfonylureas stimulate glucose uptake through GLUT4 transporter translocation in rat skeletal muscle. Biochem Biophys Res Commun 228:499 –504 13. Mu¨ller G, Jung C, Wied S, Welte S, Frick W 2001 Insulin-mimetic signaling by the sulfonylurea glimepiride and phosphoinositolglycans involves distinct mechanisms for redistribution of lipid raft components. Biochemistry 40:14603–14620 14. Cooper DR, Vila MC, Watson J, Nair G, Pollet RJ, Standaert M, Farese RV 1990 Sulfonylurea-stimulated glucose transport association with diacylglycerollike activation of protein kinase C in BC3H1 myocytes. Diabetes 39:1399 – 1407 15. Davidson MB, Molnar G, Furman A, Yamaguchi D 1991 Glyburide-stimu-

25. 26. 27. 28.

29. 30.

31. 32.

33. 34. 35. 36.

37.

lated glucose transport in cultured muscle cells via protein kinase C-mediated pathway requiring new protein synthesis. Diabetes 40:1531–1538 Dohm GL, Tapscott EB, Pories WJ, Dabbs DJ, Flickinger EG, Meelheim D, Fushiki T, Atkinson SM, Elton CW, Caro JF 1988 An in vitro human muscle preparation suitable for metabolic studies. J Clin Invest 82:486 – 494 Folli F, Saad MJA, Backer JM, Kahn CR 1992 Insulin stimulation of phosphatidylinositol 3-kinase activity and association with insulin receptor substrate 1 in liver and muscle of the intact rat. J Biol Chem 267:22171–22177 Elmendorf JS, Damrau-Abney A, Smith TR, David T S, Turinsky J 1995 Insulin-stimulated phosphatidylinositol 3-kinase activity and 2-deoxy-d-glucose uptake in rat skeletal muscles. Biochem Biophys Res Commun 208:1147– 1153 Heydrick SJ, Ruderman NB, Kurowski TG, Adams HB, Chen KS 1991 Enhanced stimulation of diacylglycerol and lipid synthesis by insulin in denervated muscle. Diabetes 40:1707–1711 Williamson JR, Corkey BE 1969 Assays of intermediates of the citric acid cycle and related compounds by fluorometric enzyme methods. Methods Enzymol 13:434 –513 Cheatham B, Kahn CR 1995 Insulin action and insulin signaling network. Endocr Rev 16:117–142 Sun XJ, Crimmins DL, Myers MG, Miralpeix M, White MF 1993 Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1. Mol Cell Biol 13:7418 –7428 Chaudry IH, Gould MK 1969 Kinetics of glucose uptake in isolated soleus muscle. Biochem Biophys Acta 177:527–536 Avignon A, Yamada K, Zhou X, Spencer B, Cardona O, Saba-Siddique S, Galloway L, Standaert ML, Farese RV 1996 Chronic activation of protein kinase C in soleus muscles and other tissues of insulin-resistant type II diabetic Goto-kakizaki (GK), obese/aged, and obese/Zucker rats. A mechanism for inhibiting glycogen synthesis. Diabetes 45:1396 –1404 Farese RV, Ishizuka T, Standaert ML, Cooper DR 1991 Sulfonylureas activate glucose transport and protein kinase C in rat adipocytes. Metabolism 40:196 – 200 Yamada K, Avignon A, Standaert ML, Cooper DR, Spencer B, Farese RV 1995 Effects of insulin on the translocation of protein kinase C-␪ and other protein kinase C isoforms in rat skeletal muscles. Biochem J 308:177–180 Hug H, Sarre TF 1993 Protein kinase C isoenzymes: divergence in signal transduction? Biochem J 291:329 –343 Arnold TP, Standaert ML, Hernańdez H, Watson J, Mischak H, Kazanietz MG, Zhao L, Cooper DR, Farese RV 1993 Effects of insulin and phorbol esters on MARCKS (myristoylated alanine-rich C-kinase substrate) phosphorylation (and other parameters of protein kinase C activation) in rat adipocytes, rat soleus muscle and BC3H-1 myocytes. Biochem J 295:155–164 Farese RV, Standaert ML, Francois AJ, Ways K, Arnold TP, Hernandez H, Cooper DR 1992 Effects of insulin and phorbol esters on subcellular distribution of protein kinase C isoforms in rat adipocytes. Biochem J 288:319 –323 Hoffman JM, Standaert ML, Nair GP, Farese RV 1991 Differential effects of pertussis toxin on insulin-stimulated phosphatidylcholine hydrolysis and glycerolipid synthesis de novo. Studies in BC3H-1 myocytes and rat adipocytes. Biochemistry 30:3315–3322 Yamada Y, Standaert ML, Yu B, Mischak H, Cooper DR, Farese RV 1994 Insulin-like effects of sodium orthovanadate on diacylglycerol-protein kinase C signaling in BC3H-1 myocytes. Arch Biochem Biophys 312:167–172 Standaert ML, Musunruru K, Yamada Y, Cooper DR, Farese RV 1994 Insulinstimulated phosphatidylcholine hydrolysis, diacylglycerol/protein kinase C signaling, and hexose transport in pertussis toxin-treated BC3H-1 myocytes. Cell Signal 6:707–716 Muller G, Dearey EA, Pu¨nter J 1993 The sulfonylurea drug, glimepiride, stimulates release of glycosylphophatidylinositol-anchored plasma-membrane proteins from 3T3 adipocytes. Biochem J 289:509 –521 Sekiya F, Bae YS, Rhee SG 1999 Regulation of phospholipase C isozymes: activation of phospholipase C-␥ in the absence of tyrosine-phosphorylation. Chem Phys Lipids 98:3–11 Bae YS, Cantley LG, Chen CS, Kim SR, Kwon KS, Rhee SG 1998 Activation of phospholipase C-␥ by phosphatidylinositol 3, 4, 5-triphophate. J Biol Chem 273:4465– 4469 Chutkow WA, Samuel V, Hansen PA, Pu J, Valdivia CR, Makielski JC, Burant CF 2001 Disruption of Sur2-containing KATP channels enhances insulin-stimulated glucose uptake in skeletal muscle. Proc Natl Acad Sci USA 98:11760 –11764 Wasada T, Yano T, Ohta M, Yui N, Iwamoto Y 2001 ATP-sensitive potassium channels modulate glucose transport in cultured human skeletal muscle cells. Endocr J 48:369 –375

Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.