GLUT2 and glucokinase expression is coordinately regulated by sulfonylurea

Molecular and Cellular Endocrinology 153 (1999) 155 – 161

GLUT2 and glucokinase expression is coordinately regulated by sulfonylurea O. Porzio a, L.N.J.L. Marlier b, M. Federici a, M.L. Hribal a, R. Magnaterra a, D. Lauro a, A. Fusco a, G. Sesti a, P. Borboni a,* a

Department of Internal Medicine, Uni6ersity of Rome ‘Tor Vergata’, Via di Tor Vergata 135, 00133 Rome, Italy b Institute of Experimental Medicine, National Research Council (CNR), Rome, Italy Received 18 November 1998; accepted 1 March 1999

Abstract In the present study we examined the effect of sulfonylurea on the expression of the glucose transporter GLUT2 and the glucose phosphorylating enzyme Glucokinase (GK) in bTC6-F7 cells; furthermore, we studied the modifications induced by sulfonylurea on glucose-responsiveness and -sensitivity. Results demonstrate that sulfonylurea increases GLUT2 and GK mRNA expression after 24 h in a dose–dependent manner. On the contrary, after 48 and 72 h a time – dependent reduction of both GLUT2 and GK mRNA occurs. GLUT2 and GK protein expression follow the same modifications. Therefore, GLUT2 and GK are coordinately regulated by sulfonylurea, probably by a common mechanism. Glucose-induced insulin release is increased by sulfonylurea as well as glucose sensitivity. Our study suggests that short-term effect of sulfonylurea increases while long-term effect reduces the expression of glucose sensing elements. The long-term inhibitory effect on glucose sensing elements would explain the reduced insulin secretion occurring after chronic sulfonylurea treatment. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Glucose transporters; Glucokinase; Pancreatic b cells; Sulfonylurea drugs; Insulin secretion

1. Introduction Sulfonylureas exert their acute hypoglycemic effect by a direct stimulation of insulin release from pancreatic b cells through the interaction with a specific plasma membrane receptor coupled to the ATP – dependent K + channel leading to its closure (Loubatieres, 1969; Ashcroft, 1988; Ashcroft and Ashcroft, 1992; Aguilar-Bryan et al., 1995; Ammala et al., 1996, ). As a consequence of the inhibition of K + efflux b cells depolarize, voltage– dependent Ca2 + channels open causing influx of Ca2 + into cytosol and the increased [Ca2 + ]cyt in turn stimulates extrusion of insulin granules (Ashcroft and Rorsman, 1990; Malaisse and Lebrun, 1990). Chronic sulfonylurea treatment leads to amelioration of glucose tolerance in diabetic patients, which is maintained even though in the absence of elevated insulin levels (Yalow et al., 1960; Kolterman et * Corresponding author. Tel.: + 39-6-72596530; fax: +39-672596538. E-mail address: [email protected] (P. Borboni)

al., 1984). This effect has been attributed to extrapancreatic actions of sulfonylureas such as stimulation of basal and/or insulin stimulated glucose transport and metabolism in muscle and fat cells (Altan et al., 1985; Zuber et al., 1985; Rogers et al., 1987; Hirshman and Horton, 1990; Jacobs et al., 1991). Several studies indicated that sulfonylureas are able to influence glucose transport by a mechanism involving protein kinase C and/or cAMP signaling cascade (Farese et al., 1991; Davidson et al., 1991; Muller et al., 1994); further, a direct effect on the expression of GLUT 1 and GLUT4 has been demonstrated (Muller and Wied, 1993). Finally, Lenzen et al. (1986) demonstrated that the longterm hypoglycemic effect of sulfonylureas is related to the induction of liver and pancreatic glucokinase activity. In the present study we investigated the effect of sulfonylurea treatment on the expression of the pancreatic high Km glucose transporter GLUT2 and the high Km glucose phosphorylating enzyme Glucokinase (GK) in cultured b cells; furthermore we analyzed the modifications induced by sulfonylurea treatment on glucose-

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2. Materials and methods

amount of wild type (w.t.) RNA isolated from bTC6F7 cells, as previously described (Borboni et al., 1996). Briefly, base mutations have been introduced by PCRbased site-directed mutagenesis (one or three bases mutation respectively for GK i.s and GLUT2 i.s) in order to generate a BglII restriction site, allowing to distinguish the amplification products arising from the i.s. RNA from those arising from the w.t. RNA. Known amounts of GLUT2 or GK i.s. cRNA were incubated in the presence of constant amount of w.t. RNA (0.5mg) isolated from bTC6-F7 cells. The i.s./w.t RNA mixtures were reverse transcribed and amplified for 28 cycles (94°C/1 min, 62°C/1 min, 72°C/1 min with a final 72°C/5 min extension) by 2.5U Taq DNA polymerase (Promega Corp. Madison, WI) in a mixture containing 2.1 mM MgCl2, 100 pmol upstream and downstream amplification primers, in the presence of 0.5× 106 cpm of 32P-gATP labeled upstream primer (Amersham Corp., Arlington Heights, IL). The upstream primer was labeled by 10U T4 kinase (Promega Corp. Madison, WI) and purified on Stratagene purification columns (Stratagene, La Jolla, CA).

2.1. Cell cultures and cell treatment

2.4. Analysis of the PCR-amplified products

bTC6-F7 cells (kindly provided from Dr S. Efrat, New York) were cultured in Dulbecco’s modified Eagle’s medium containing 11.1 mmol/l glucose and supplemented with 15% horse serum, 5% foetal calf serum (FCS), 50 IU penicillin and 50 mg/ml streptomycin at 37°C in an atmosphere of 95% humidified air/5% CO2. Cells were subcultured weekly and were used at passage 28 – 35, during which glucose responsiveness is maintained (Efrat et al., 1993). Glimepiride (kindly provided by Hoechst-Roussel Pharm., Somerville, NJ) was added to the cells at increasing concentrations (1 – 10 and 100 mM) for 24 h (dose–response experiments) or at fixed concentration (10mM) for 6–24–48 or 72 h (time course experiments).

PCR products were chloroform/isoamylalcohol (49:1) extracted, aliquots were digested with 20U of BglII (Promega Corp. Madison, WI) overnight at 37°C and successively resolved on 1.5% agarose gel (in triplicates). Because the BglII restriction site was introduced midway to the target sequence, only two bands were obtained in the gel, corresponding respectively to the undigested w.t. (560 bp for GLUT2 or 440 bp for GK) and to the digested i.s. (280 bp for GLUT2 or 220 bp for GK). To quantitate the amount of product arising from the amplified i.s. or w.t. RNA, the corresponding ethidium bromide-stained bands were excised from the gel and the amount of incorporated radioactivity was determined in a b counter (LKB). Gel slices corresponding to lanes containing a control sample (water) were also excised at the size of i.s. or w.t. bands in order to determine the amount of background radioactivity. Data were plotted as the ratio of cpm incorporated into the w.t. amplification products and the cpm incorporated into the i.s. amplification products as a function of the known amount of i.s.. The plot was analysed by logarithmic regression; the extrapolated point of equivalence corresponds to the amount of GLUT2 or GK mRNA present in the unknown sample.

responsiveness and glucose-sensitivity. For this purpose, we have used Glimepiride, a second-generation sulfonylurea with equivalent glucose lowering effect to Glybenclamide and less insulinotropic effect (Geisen, 1988). Pancreatic GLUT2 and GK studies are complicated by technical difficulties in islets isolation and low expression of GK and GLUT2 mRNA in b cells. Various insulinoma cell lines are available, deriving either from b cell tumors induced by irradiation of rats or from insulin promoter-driven T-antigen expression in transgenic mouse, that can be used as a model to study GK activity and mRNA expression (Chick et al., 1977; Efrat et al., 1988; Clark et al., 1990). In the present study we used bTC6-F7 cells as experimental model, an insulinoma cell line arising in transgenic mice that maintains a stable phenotype of insulin secretion in response to physiological glucose concentrations (Efrat et al., 1993; Knaack et al., 1994).

2.2. RNA isolation For RNA studies cells were seeded in culture medium in 25 cm2 Tissue Culture Flask at a density of 2× 105 cells/ml. After Glimepiride treatments (dose – response and time-course) total RNA was extracted by RNAFast II (Molecular System, San Diego, CA), quantified by spectrophotometry UV and stored in diethylpyrocarbonate (DEPC-) treated water at − 70°C until use.

2.3. Analysis of GK and GLUT2 mRNA by competiti6e RT-PCR Competitive RT-PCR assay was performed by coamplification of increasing amounts of internal standard (i.s.) GLUT2 or GK cRNA with a constant

2.5. Western blot analysis of GLUT2 and GK protein expression Cells treated with 10 mM Glimepiride for 24 and 72 h were lysed in a solution containing 1% NP40, 50 mM

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Tris, 150 mM NaCl, 4 mM EDTA, 10 mg/ml leupeptin, 10 mg/ml pepstatin A and 2 mM phenylmethylsulfonyl fluoride. Equal amounts of proteins were separated by 7.5% SDS/polyacrylamide gel and transferred on nitrocellulose membrane in a cooled chamber. Uniform loading of proteins was verified by staining of filters with Ponceau S. Filters were blocked in TBST (10 mM Tris –HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) containing 5% non-fat dried milk. A rabbit anti-mouse antibody directed against the COOH-terminal region of GLUT2 was used at a 1:1000 dilution as primary antibody (kindly provided by Dr B. Thorens, Losanna, Switzerland); a rabbit anti-mouse antibody directed against the COOH-terminal region of GK was used at a 1:500 dilution as primary antibody (kindly provided from Dr M. Magnuson, Nashville, TN). Filters were incubated overnight with the primary antibodies and subsequently washed in TBST and incubated with 1:10000 dilution of peroxidase-conjugated goat antirabbit IgG (Sigma) for 1 h. GLUT2 and GK protein bands were visualised on autoradiographies with a chemiluminiscent reagent detection system (ECL, Amersham, Arlington Heights, IL). Apparent molecular weight was determined by comparison with molecular size markers (Gibco BRL). Densitometric analysis of autoradiographies were performed by an image analyzer (Fluor S, Biorad).

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3.1. Insulin radioimmunoassay Insulin was determined by a dextran–charcoal method as previously described (Herbert et al., 1965), using an anti-insulin antibody raised in guinea pig, porcine insulin standard (Sigma) and 125I-Insulin from Amersham Corp., Arlington Heights, IL.

4. Results

4.1. GLUT2 and GK mRNA expression Fig. 1 illustrates changes in GLUT2 (A) and GK (B) mRNA expression in bTC6-F7 cells after dose-response experiments. Absolute amount of GLUT2 and GK mRNA is respectively 1.479 0.11 pg i.s./mg total RNA and 8.790.56 pg i.s./mg total RNA. Glimepiride determines a dose–dependent increase of GLUT2 and GK mRNA expression after 24 h. Time course experiments (Fig. 2) demonstrate an increase of both GLUT2 (A) and GK (B) mRNA expression after 6 and 24 h exposure to 10 mM Glimepiride. On the contrary, after 48 and 72 h

3. Insulin release experiments For dose–response experiments, bTC6-F7 cells were seeded in culture medium in 24 multiwell plates at a density of 105 cells/ml. After 48 h, the medium was removed and the cells were washed twice at 37°C for 30 min with a glucose-free Krebs-Ringer Buffer (KRB) containing 119 mmol/l NaCl, 4.74 mmol/l KCl, 2.54 mmol/l CaCl2, 1.19 mmol/l MgSO4, 1.19 mmol/l KH2PO4, 25 mmol/l NaHCO3, 10 mM HEPES, 0.1% BSA, pH 7.4). Thereafter, cells were incubated for 2 h in fresh KRB containing 5.6 mM glucose in the absence or in the presence of different Glimepiride concentrations (ranging from 1 to 100 mM). For time course experiments, cells were seeded in culture medium in 6 multiwell plates and Glimepiride (10 mM) was added to the cells for 6, 24, 48 or 72 h and insulin release in response to glucose was evaluated. The effect of 10 mM Glimepiride on insulin release was studied at different glucose concentrations (ranging from 0.1 to 16.7 mM) in order to evaluate modifications of glucose sensitivity. At the end of each experimental protocol, aliquots of the supernatant were collected and stored at −20°C for subsequent insulin radioimmunoassay; cells were extracted overnight at 4°C with a solution of acidified ethanol for intracellular insulin content assay.

Fig. 1. Dose – response effect of Glimepiride on GLUT2 (A) and GK (B) mRNA expression. Cells were treated for 24 h. Data are expressed as a percentage of increment versus control, where the control is the GLUT2 and GK mRNA expression in untreated cells; control values are reported in the text; n = 3; (*=P B0.05; ** PB 0.01).

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Fig. 3. Western blot analysis of GLUT2 protein expression. Data, representing the mean of three independent experiments, are expressed in arbitrary units. A representative blot is shown in the lower panel.

of Glimepiride tested, reaching the maximum response at 100 mM Glimepiride, with an ED50 of 9.5 mM (Fig. 5).

Fig. 2. Time course of GLUT2 (A) and GK (B) mRNA expression in cells treated with 10 mM Glimepiride. Data are expressed as a percentage of increment versus control, where the control is the GLUT2 and GK mRNA expression in untreated cells; control values are reported in the text; n= 3; (*= PB 0.05; ** PB 0.01).

GLUT2 mRNA expression as well as GK mRNA expression are progressively reduced.

4.2. GLUT2 and GK protein expression GLUT2 and GK protein expression are increased after short-term treatment (24 h) with 10 mM Glimepiride. Densitometric analysis indicates an increment of 23.36 and 35.15%, respectively for GLUT2 (Fig. 3) and GK (Fig. 4) compared to untreated cells. On the contrary, after long-term treatment (72 h) both, GLUT2 and GK protein expression are reduced. Densitometric analysis indicates a reduction of 69.80 and 58.18%, respectively for GLUT2 (Fig. 3) and GK (Fig. 4) compared to untreated cells.

4.3.2. Time course experiments Time course experiments were performed in the presence of 11.1 mM glucose since cell detachment occurred with lower glucose concentrations for long-term incubations. Glimepiride 10 mM produces a time–dependent increase of insulin release reaching the plateau at 72 h (Fig. 6). 4.3.3. Glucose sensiti6ity experiments Fig. 7 shows the insulin response of bTC6F7 cells induced by increasing glucose concentrations in the absence or in the presence of 10 mM Glimepiride. An increased insulin response to all the glucose concentrations tested has been observed in Glimepiride treated

4.3. Insulin release experiments 4.3.1. Dose response results Dose response experiments were performed in the presence of 5.6 mM glucose, representing the EC50 of glucose for insulin response in this cell line. In the absence of Glimepiride, basal insulin secretion with 5.6 mM glucose is 7.52 9 0.59% of total intracellular insulin content and it increases significantly in a dose –dependent manner in the presence of all the doses

Fig. 4. Western blot analysis of GK protein expression. Data, representing the mean of five independent experiments, are expressed in arbitrary units. A representative blot is shown in the lower panel.

O. Porzio et al. / Molecular and Cellular Endocrinology 153 (1999) 155–161

Fig. 5. Dose-response effect of Glimepiride on insulin secretion induced by 5.6 mM glucose in bTC6-F7 cells. Data are expressed as a percentage of increment versus control where the control is the insulin secretion from untreated cells; control values are reported in the text; n =3; (*= P B0.05; ** P B0.01).

cells compared to untreated cells. In particular, Glimepiride treated cells show a leftward shift of glucose-induced insulin release compared to control cells, indicating an increased glucose-sensitivity: EC50 in control cells has been obtained at 8.0 mM glucose while in treated cells has been obtained at 1.5 mM glucose.

5. Discussion Sulfonylureas are widely used drugs for the treatment of non-insulin dependent diabetes mellitus. The mechanism of action of sulfonylureas are principally mediated by their ability to determine the closure of the ATP–dependent K + channels (Sturgess et al., 1985). It is now clear that ATP-dependent K + channels consist of two subunits, named Kir6.2 and SUR1 (Inagaki et al., 1995; Sakura et al., 1995). Kir6.2, an inwardly-rectifying Kchannel subunit, is the pore forming subunit while SUR1 is a regulator subunit, which binds sulfonylureas and may also have K + sensitivity. The two subunits are

Fig. 6. Time course of insulin secretion induced by 11.1 mM glucose in bTC6-F7 cells treated with 10 mM Glimepiride. Data are expressed as a percentage of increment vs control, where the control is the insulin secretion from untreated cells. n= 3; (*= PB 0.05; ** PB 0.01).

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Fig. 7. Glucose sensitivity in bTC6-F7 cells, untreated (squares) or treated with 10 mM Glimepiride (triangles). Data are expressed as a percentage of maximum insulin release obtained in the presence of 16.7 mM glucose; EC50 is indicated by dotted lines. n= 3; (*= PB 0.05; ** P B0.01).

required for functional channel. SUR1 is a member of the ATP Binding Cassette (ABC) transporters family. It is possible that sulfonylureas bind to other ABC transporters, such as cystic fibrosis transmembrane conductance regulator (CFTR) which is known to function as a chloride channel (Higgins, 1992, 1995). In fact sulfonylureas are able to block chloride channels even though at higher concentrations than that required for K-ATP channels activation (Egan et al., 1992; Sheppard and Welsh, 1992). In addition to K-ATP channels, sulfonylureas may act at a more distal site of the b cell secretory machinery. In particular, it has been recently demonstrated that sulfonylureas are also able to potentiate the Ca2 + –dependent exocytosis directly (Eliasson et al., 1996). The possible mechanism of action seems to involve protein kinase C (PKC) activation since the effect is blocked by PKC inhibitors. Ca2 + –dependent exocytosis is regulated by a 145 kD protein which is a substrate for PKC (Nishizaki et al., 1992). The high affinity 140 kD sulfonylurea receptor has a consensus site for PKC phosphorylation suggesting that it may be itself the regulatory protein of Ca2 + –dependent exocytosis (Walent et al., 1992). Moreover, the presence of sulfonylurea receptors in the secretory granules has been hypothesized, suggesting a direct action of sulfonylureas at this level (Thevenod et al., 1992). Finally, K-ATP-sulfonylurea sensitive channels may be involved in the fusion of secretory granules with the plasma membrane, therefore sulfonylureas may affect this process. The possibility that sulfonylureas can interact with the glucose sensing mechanism of b cells has not been previously investigated. Insulin release from b cells is regulated by the high-Km plasma membrane glucose transporter GLUT2 and by the high-Km hexokinase type IV Glucokinase (Matschinsky, 1990; Matschinsky et al., 1993). GLUT2 and GK are key regulators of the

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rate of glucose metabolism in liver and islets, and in b cells they couple changes in the extracellular glucose concentration to insulin secretion (Efrat et al., 1994; Unger, 1991). In the present study we investigated the hypothesis that sulfonylureas can regulate the expression of GLUT2 and GK in b cells. Results demonstrate that Glimepiride increases the expression of GLUT2 and GK mRNA in a dose – dependent manner; the effect is maximum after 24 h, while after 48 and 72 h a progressive reduction occurs. GLUT2 and GK protein expression shows the same time – dependent modifications, being increased after 24 h and reduced after 72 h. Therefore, GLUT2 and GK are coordinately modified by sulfonylurea treatment, suggesting that the drug affects GLUT2 and GK with the same mechanism. The long-term inhibitory effect of Glimepiride on GLUT2 and GK can be due to a loss of sensitivity of b cells to long-term exposure to sulfonylureas, as already demonstrated in in vivo studies, or alternatively to a negative feed-back mechanism (Karam et al., 1986). A further hypothesis to be considered is that insulin, released from the b cells in response to the sulfonylurea, can secondarily affect GLUT2 and GK mRNA expression. GLUT2 gene expression is known to be modulated by glucose in vitro and in vivo (Chen et al., 1992; Inagaki et al., 1992; Yasuda et al., 1992); in particular signals deriving from glucose metabolism are required for regulation of GLUT2 (Ferrer et al., 1993). On the other hand the role of insulin on GLUT2 gene expression has been extensively investigated. Studies from genetic models of diabetes indicate that Zucker and Wistar Kyoto rats, which are hyperinsulinemic, have reduced GLUT2 levels (Johnson et al., 1990; Orci et al., 1990a); by contrast, neonatal streptozocin and GK diabetic rats, with slightly reduced or normal insulin levels, have reduced GLUT2 expression, whereas female Zucker rats which are hyperinsulinemic but do not become diabetic, have normal GLUT2 levels (Johnson et al., 1990; Orci et al., 1990a,b). Therefore there does not seem to exist a clear correlation between GLUT2 levels and ambient insulinemia. On the contrary, Postic et al. have demonstrated that GLUT2 mRNA expression is decreased by hyperinsulinemia in liver (Postic et al., 1992). GLUT2 gene does not contain alternative tissue-specific promoters or start sites such as GK gene, thus a tissue – specific regulation of GLUT2 gene expression has not been described. Therefore our hypothesis that insulin induced by Glimepiride can secondarily affect GLUT2 expression should be considered. GK gene expression in b cells is mainly regulated by glucose, while in liver it is mainly regulated by insulin. The GK gene and its transcript have been characterized in liver, pancreatic b cells and neurones of the ventromedial hypothalamus (VMH); two different transcription control regions have been described, one specific for the liver and the other specific

for pancreatic b cells and neuroendocrine cells, allowing tissue-specific regulation of the gene (Bedoya et al., 1986; Iynedjian et al., 1989; Magnuson and Shelton, 1989; Jetton et al., 1994). Moreover, it is possible that GLUT2 is regulated by GK as suggested by a previous study in which RIN1046-38 cells transfected with GLUT2 show increased GK activity (Ferber et al., 1994). Taken together these data suggest that there may be physiological circumstances in which GK and GLUT2 expression are coordinately regulated, with consequent specific modulation of glucose metabolism. For instance, a concordant regulation of GLUT2 and GK gene expression in b cells has been evidenced during the fasting/refeeding cycle: fasting reduces while refeeding increases GLUT2 and GK mRNA expression (Iwashima et al., 1994). Glimepiride enhances glucose-induced insulin release in bTC6-F7 cells in a dose–dependent manner with an ED50 of 9.5 mM which is consistent with the blood concentration obtained by therapy. The effect is time– dependent and is in agreement with the time–dependent modifications of GLUT2 and GK expression. Interestingly, glucose sensitivity of b cells is enhanced by Glimepiride treatment as demonstrated by the leftward shift of insulin response to glucose. Our study suggests that sulfonylureas stimulate insulin release by acting both at proximal and distal site of b cells secretory machinery. Short-term effect induces the expression of the glucose sensing elements, while long-term effect determines a reduction of both GLUT2 and GK expression. This long-term inhibitory effect on glucose sensing elements may explain the reduced insulin secretion occurring after chronic sulfonylurea treatment.

Acknowledgements This paper has been supported by Hoechst-Roussel Pharm., NJ, USA.

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