THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2002 by The by Ilhamjan Sabit

Vol. 277, No. 28, Issue of July 12, pp. 25277–25282, 2002 Printed in U.S.A.

Normalization of Intracellular Ca2ⴙ Induces a Glucose-responsive State in Glucose-unresponsive ␤-Cells* Received for publication, April 24, 2002 Published, JBC Papers in Press, May 6, 2002, DOI 10.1074/jbc.M203988200

Kohtaro Minami‡§, Masaaki Yokokura‡, Nobuko Ishizuka‡, and Susumu Seino‡¶ From the ‡Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan and the §Department of Medical Genetics (Novo Nordisk Pharma), School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan

Because intracellular Ca2⫹ is involved in a variety of cellular processes such as signal transduction, gene expression, and hormone release (1– 6), disturbed intracellular Ca2⫹ homeostasis readily induces cell dysfunction (7, 8). In pancreatic ␤-cells, a rise in the intracellular Ca2⫹ concentration ([Ca2⫹]i) is the trigger for insulin secretion. As the extracellular glucose concentration increases, intracellular ATP is increased and the ATP-sensitive K⫹ (KATP) channels are closed, depolarizing the plasma membrane and opening the voltage-dependent Ca2⫹ channels (VDCCs),1 which allows Ca2⫹ influx. The rise in

[Ca2⫹]i in the ␤-cells triggers exocytosis of the insulin granules (9). When the blood glucose level falls, [Ca2⫹]i returns to basal level. In this context, persistent hyperglycemia might well cause sustained elevated [Ca2⫹]i and abnormalities in glucoseinduced insulin secretion. It has been reported that human pancreatic islets cultured with high glucose show elevated basal [Ca2⫹]i together with loss of the glucose-induced rise in [Ca2⫹]i and glucose-induced insulin secretion (10). Normal pancreatic ␤-cells exposed to high glucose exhibit an abnormal response of intracellular Ca2⫹ and impaired insulin secretion (11), impairments which also are observed in the ␤-cells of diabetic animals (12–14). However, the molecular basis of the effect of chronic elevation of [Ca2⫹]i on insulin secretion has not been examined in detail, primarily because an appropriate in vitro model has not been available. We recently established two pancreatic ␤-cell lines with contrary features, glucose-responsive (MIN6-m9) and glucose-unresponsive (MIN6-m14), and have shown these cell lines to be useful in ␤-cell studies (15). MIN6-m9 exhibit glucose metabolism and insulin secretion similar to normal pancreatic ␤-cells, while MIN6-m14 exhibit abnormalities in glucose metabolism, KATP channel activity, VDCC activity, and glucose-induced insulin secretion (15). In the present study we have determined the factors responsible for the glucose-unresponsiveness in MIN6-m14. [Ca2⫹]i in MIN6-m14 is significantly higher than in MIN6-m9. When the [Ca2⫹]i level was normalized by nifedipine, a Ca2⫹ channel blocker, the glucose-induced insulin secretion was increased dramatically in MIN6-m14 with a concomitant improvement of KATP channel and VDCC activities. Accordingly, chronically elevated [Ca2⫹]i is the major factor contributing to the defect in glucose responsiveness of MIN6-m14, and normalization of [Ca2⫹]i restores the glucose-responsive state of the cells. Because abnormalities of [Ca2⫹]i in pancreatic ␤-cells are associated with chronic exposure to high glucose in both normal and diabetic animals (8, 10 –14), the present study suggests normalization of [Ca2⫹]i as a therapeutic strategy for the glucose unresponsiveness of ␤-cells in type 2 diabetic patients. EXPERIMENTAL PROCEDURES

* This work was supported by Grants-in-Aid for Creative Scientific Research 10NP0201 and for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology; by a scientific research grant from the Ministry of Health, Labour, and Welfare, Japan; and by grants from Novo Nordisk Pharma Ltd., from Takeda Chemical Industries Ltd., and from the Yamanouchi Foundation for Research on Metabolic Disorders. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ To whom correspondence should be addressed. Tel.: 81-43-2262187; Fax: 81-43-221-7803; E-mail: seino@med.m.chiba-u.ac.jp. 1 The abbreviations used are: VDCC, voltage-dependent Ca2⫹ chanThis paper is available on line at http://www.jbc.org

Cell Culture and Measurement of [Ca2⫹]i—MIN6 cells were cultured in Dulbecco’s modified Eagle’s medium with 25 mM glucose supplemented with 10% heat-inactivated fetal calf serum under humidified condition of 5% CO2/95% air at 37 °C (15). Cells were loaded with 5 ␮M fura-2 acetoxymethyl ester (Fura-2 AM) (Dojindo, Kumamoto, Japan) for 1 h in culture medium without pH indicator or in HEPES-balanced Krebs-Ringer bicarbonate buffer (KRH: 119 mM NaCl, 4.74 mM KCl, 2.54 mM CaCl2, 1.19 mM MgCl2, 1.19 mM KH2PO4, 25 mM NaHCO3, and

nel; KRH, Krebs-Ringer HEPES; BSA, bovine serum albumin; LDH, lactate dehydrogenase; MOPS, 4-morpholinepropanesulfonic acid; HK, hexokinase; GK, glucokinase; nt, nucleotide; PM, plasma membrane.

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Although intracellular Ca2ⴙ in pancreatic ␤-cells is the principal signal for insulin secretion, the effect of chronic elevation of the intracellular Ca2ⴙ concentration ([Ca2ⴙ]i) on insulin secretion is poorly understood. We recently established two pancreatic ␤-cell MIN6 cell lines that are glucose-responsive (MIN6-m9) and glucose-unresponsive (MIN6-m14). In the present study we have determined the cause of the glucose unresponsiveness in MIN6-m14. Initially, elevated [Ca2ⴙ]i was observed in MIN6-m14, but normalization of the [Ca2ⴙ]i by nifedipine, a Ca2ⴙ channel blocker, markedly improved the intracellular Ca2ⴙ response to glucose and the glucose-induced insulin secretion. The expression of subunits of ATP-sensitive Kⴙ channels and voltage-dependent Ca2ⴙ channels were increased at both mRNA and protein levels in MIN6-m14 treated with nifedipine. As a consequence, the functional expression of these channels at the cell surface, both of which are decreased in MIN6-m14 without nifedipine treatment, were increased significantly. Contrariwise, Bay K8644, a Ca2ⴙ channel agonist, caused severe impairment of glucose-induced insulin secretion in glucose-responsive MIN6-m9 due to decreased expression of the channel subunits. Chronically elevated [Ca2ⴙ]i, therefore, is responsible for the glucose unresponsiveness of MIN6-m14. The present study also suggests normalization of [Ca2ⴙ]i in pancreatic ␤-cells as a therapeutic strategy in treatment of impaired insulin secretion.

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10 mM HEPES, pH 7.4) containing 0.2% BSA (BSA-KRH) with 0.1 mM glucose. [Ca2⫹]i was measured by a dual-excitation wavelength method (340/380 nm) with a fluorometer (Fluoroskan Ascent CF; Labsystems, Helsinki, Finland). [Ca2⫹]i was calibrated using solutions containing known Ca2⫹ concentrations (Molecular Probes, Eugene, OR). Measurement of Insulin Secretion—Cells (1 ⫻ 105 cells/well, 48-well plate) were exposed to 10 ␮M nifedipine (Sigma), 300 ␮M diazoxide (Sigma), or 1 ␮M Bay K8644 (Sigma) for 24 h (preculture). All media contained 0.1% Me2SO. The cells were then washed with BSA-KRH and preincubated for 30 min in the same buffer containing 1 mM glucose. Incubation was performed with various concentrations of glucose with or without other agents as indicated for 1 h at 37 °C. The drugs used in preculture were omitted throughout the secretion experiments. Released insulin was measured as described previously (15). The amounts of insulin secretion were normalized by the cellular insulin contents determined by acid-ethanol extraction. Assay for Enzyme Activities—For determination of glucose-phosphorylating activity, disrupted cells were centrifuged, and supernatants were incubated in a triethanolamine buffer, pH 7.4, containing 0.5 mM NADP, 5 mM ATP, 1 unit/ml glucose-6-phosphate dehydrogenase, and 0.5 or 50 mM glucose at 30 °C. Velocity of NADPH formation was monitored by reading absorbencies at 340 nm. Enzyme activity was expressed as nmol NADPH/min/mg of protein (15). Activity of lactate dehydrogenase (LDH) was determined as follows: briefly, cell extracts were incubated in a glycylglycine buffer, pH 10.0, with 1 mM lactate, 5 mM NAD, 50 mM glutamate, and 10 unit/ml glutamate-pyruvate transaminase at 25 °C. Velocity of NADH formation was monitored by reading absorbencies at 340 nm. Enzyme activity is expressed as nmol NADH/min/mg of protein (15). Determination of ATP Production—For measurement of ATP production, cells were incubated for 1 h in the presence or absence of 25 mM glucose. The cells then were washed twice with ice-cold PBS and solubilized, and the amount of ATP was measured with an ATP bioluminescent assay kit (Roche Molecular Diagnostics, Mannheim, Germany), according to the manufacturer’s instruction. Electrophysiological Analyses—Whole cell recordings of ATP-sensitive K⫹ current were performed as described previously (15). The extracellular solution contained 135 mM NaCl, 5 mM KCl, 5 mM CaCl2, 2 mM MgSO4, 5 mM HEPES, and 3 mM glucose, pH 7.4. The pipette solution contained 107 mM KCl, 11 mM EGTA, 2 mM MgSO4, 1 mM CaCl2, and 11 mM HEPES, pH 7.2. Whole cell Ba2⫹ currents through the VDCCs were recorded as described (15). Briefly, Ba2⫹ was used as a charged carrier for measurement of VDCC currents. The extracellular solution contained 40 mM Ba(OH)2, 20 mM 4-aminopyridine, 90 mM tetraethylammonium hydroxide, 10 mM tetraethylammonium chloride, 140 mM methanesulfonate, and 10 mM MOPS, pH 7.4. The pipette solution contained 10 mM CsCl, 130 mM cesium aspartate, 10 mM EGTA, 5 mM Mg-ATP, and 10 mM MOPS (pH 7.2). Cells were maintained at a holding potential of ⫺60 mV, and square pulses of 400-msec duration at potentials between ⫺40 and ⫹70 mV in steps of 10-mV were

FIG. 2. Insulin secretory response in MIN6-m14 before and after normalization of [Ca2ⴙ]i by nifedipine. Cells (1 ⫻ 105 cells/ well, 48-well plate) were treated with or without 10 ␮M nifedipine for 24 h. The cells were washed and pre-incubated for 30 min at 37 °C in BSA-KRH containing 1 mM glucose without nifedipine. Incubation was performed in BSA-KRH with indicated concentrations of glucose (A), glibenclamide (B), or Bay K8644 (C) for 1 h at 37 °C. Note that nifedipine was absent throughout the secretion experiment. Values of secreted insulin were normalized by cellular insulin contents. m14, MIN6-m14; m14-Nif, nifedipine-treated MIN6-m14. n ⫽ 4, *, p ⬍ 0.05, and **, p ⬍ 0.01. applied every 4 s. Recordings were made using the EPC-7 amplifier (List Electronics, Darmstadt, Germany). RNA Blotting—Total RNA (10 ␮g) from cells was subjected to formaldehyde-agarose gel electrophoresis. RNA was transferred to a nylon membrane and UV-cross-linked. Membrane was hybridized with ␣-[32P]dCTP-labeled probes corresponding to the cDNA of mouse hexokinase-I (HK-I) (GenBankTM accession no. J05277, nt 1464 –1903), mouse glucokianse (GK) (L38990, nt 98 – 652), mouse lactate dehydrogenase-A (LDH-A) (NM_010699, nt 291–980), mouse Kir6.2 (U73626, nt 753–1427), hamster sulfonylurea receptor 1 (SUR1) (L40623, nt 3126 – 4049), mouse ␣1-subunit of VDCC (NM_009781, nt 4889 –5446), or mouse ␤3-subunit of VDCC (NM_007581, nt 801–1600). Rediprime random primer labeling kit (Amersham Biosciences) was used to label the probes. Blots were exposed to Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY) at ⫺80 °C. Subcellular Fractionation and Immunoblotting—Cells were scraped into a lysis buffer termed buffer A, containing 150 mM NaCl, 50 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, and 1 mM dithiothreitol with protease inhibitors and sonicated (TOMY Sonicator UD201; Tomy, Tokyo, Japan; 1 ⫻ 10-s burst; output power ⫽ 1) and then centrifuged at 700 ⫻ g for 15 min. The pellet, which contained nuclei and undisrupted cells, was discarded. The supernatant was centrifuged at 8000 ⫻ g for 15 min. The 8,000 ⫻ g pellet was referred to as the plasma membrane-enriched fraction. The supernatant was then ultracentrifuged at 100,000 ⫻ g for 1 h. The pellet was enriched in internal membranes. The final supernatant obtained was the cytosolic fraction. All fractions were resuspended in the same volume of buffer A with 1% Triton X-100. Aliquots from each subcellular fraction (volumes equal to PM fractions containing 5 ␮g of protein) were subjected to SDS-PAGE. Resolved proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) and probed with antibodies against Kir6.2 (16), SUR1 (17), the ␣1-subunit of VDCCs (Alomone, Jerusalem, Israel), or the ␤3-subunit of VDCCs (Alomone). Secondary antibodies were conjugated to horseradish peroxidase and visualized by enhanced chemiluminescence reagent (Amersham Biosciences). Kir6.2 was detected as a multimeric complex with SUR1 because it was difficult to denature completely. Mobility of the complex on the gel was determined

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FIG. 1. Intracellular Ca2ⴙ concentration ([Ca2ⴙ]i) in MIN6 cells. A, [Ca2⫹]i in standard culture condition. Cells (1 ⫻ 105 cells/well, 96-well plate) were treated with or without 10 ␮M nifedipine. The cells were loaded with 5 ␮M Fura-2 AM, and [Ca2⫹]i was measured in the culture medium without a pH indicator. n ⫽ 8, **, p ⬍ 0.01. m9, MIN6-m9, m14, MIN6-m14, m14-Nif, nifedipine-treated MIN6-m14. B, [Ca2⫹]i change in response to glucose. Cells were treated with or without 10 ␮M nifedipine and loaded with 5 ␮M Fura-2 AM. [Ca2⫹]i was measured in BSA-KRH with 0.1 mM glucose at 30-s intervals, and 25 mM glucose (final concentration) was added to each well at the indicated time points. m14, MIN6-m14, m14-Nif, nifedipine-treated MIN6-m14. n ⫽ 6.

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FIG. 3. Enzyme activities and ATP production. A, glucose phosphorylating activity. Cells were grown on a culture dish (10-cm diameter) with or without 10 ␮M nifedipine for 24 h. The cells were lysed and incubated in a buffer containing 0.5 mM NADP, 5 mM ATP, 1 unit/ml glucose-6-phosphate dehydrogenase, and 0.5 or 50 mM glucose at 30 °C. Enzyme activity is expressed as nmol of NADPH/min/mg of protein. Glucose phosphorylating activity at 0.5 mM glucose represents HK activity, and GK activity is the activity at 50 mM glucose minus that at 0.5 mM glucose. B, LDH activity. Cells (1 ⫻ 105 cells/well, 48-well plate) were treated with or without 10 ␮M nifedipine for 24 h. Cell extracts were incubated in buffer with 1 mM lactate, 5 mM NAD, 50 mM glutamate, and 10 unit/ml glutamate-pyruvate transaminase at 25 °C. Enzyme activity was expressed as nmol of NADH/min/mg of protein. C, cellular ATP contents. Cells treated with or without 10 ␮M nifedipine were incubated for 1 h in the presence or absence of 25 mM glucose. The ATP concentration of the cell lysate was measured with an ATP bioluminescent assay kit. m14, MIN6-m14; m14-Nif, nifedipine-treated MIN6-m14. n ⫽ 4. There was no significant difference between treatments in all experiments.

FIG. 4. Electrophysiology of KATP channels and VDCCs. A, normalized peak KATP channel conductance. Cells cultured on collagencoated slide glass were treated with or without 10 ␮M nifedipine for 24 h and then used for recording. Because the membrane area of each cell varies, the ATP-sensitive conductance was normalized by dividing by the membrane capacitance for each cell. m14, MIN6-m14; m14-Nif, nifedipine-treated MIN6-m14. n ⫽ 7–9, *, p ⬍ 0.05. B, current-voltage relationships of VDCCs. Whole-cell Ba2⫹ currents through VDCCs were recorded. m14, MIN6-m14; m14-Nif, nifedipine-treated MIN6-m14. n ⫽ 19 –22. **, p ⬍ 0.01.

by Kir6.2/SUR1-co-transfected Ltk cell extracts. Blots were quantified by scanning densitometry (Amersham Biosciences). To verify subcellular fractionation, Na⫹/K⫹-ATPase (Upstate Biotechnology, Lake Placid, NY) and sarco/endoplasmic reticulum Ca2⫹-ATPase (Santa Cruz Bio-

technology, Santa Cruz, CA), markers for plasma membrane and internal membrane, respectively, were detected by immunoblotting. Statistical Analysis—Values are expressed as means ⫾ S.E. The significance of differences between test groups was evaluated by unpaired Student’s t test or one-way analysis of variance followed by Tukey’s test. p ⬍ 0.05 was considered significant. RESULTS

[Ca2⫹]i of MIN6-m9 and MIN6-m14 —In the standard culture condition (Dulbecco’s modified Eagle’s medium with 10% fetal calf serum and 25 mM glucose), [Ca2⫹]i in MIN6-m14 (317 ⫾ 7 nM, n ⫽ 8) was significantly higher than in MIN6-m9 (227 ⫾ 4 nM, n ⫽ 8) (Fig. 1A). Application of the Ca2⫹ channel blocker nifedipine (10 ␮M for 24 h) lowered [Ca2⫹]i of MIN6m14 to levels similar to those in MIN6-m9 (Fig. 1A). Intracellular Ca2⫹ was poorly responsive to glucose in control MIN6m14 (without nifedipine treatment), but the response was markedly improved in nifedipine-treated MIN6-m14 (Fig. 1B). Insulin Secretion before and after Normalization of [Ca2⫹]i— Although normalization of [Ca2⫹]i by nifedipine did not alter basal levels of insulin secretion in MIN6-m14, glucose-induced insulin secretion was dramatically increased after normalization of [Ca2⫹]i (Fig. 2A). Because the cellular insulin content also was increased by nifedipine (226.2 ⫾ 4.2 versus 505.2 ⫾ 12 pmol/mg of protein, n ⫽ 4; control MIN6-m14 versus nifedipine treated-MIN6-m14), insulin secretion was normalized by the contents. The insulin secretion at 25 mM glucose was 20.8 ⫾ 1.1 and 121.4 ⫾ 10.9 pmol/h/mg of protein in control MIN6-m14 and nifedipine-treated MIN6-m14, respectively. The nifedipine was washed out before the secretion experiments and was omitted thereafter throughout the experiments. These results indicate that the normalization of [Ca2⫹]i restored the glucose responsiveness of MIN6-m14 both in intracellular Ca2⫹ and insulin secretion. Glibenclamide- and Bay K8644-stimulated Insulin Secretion—We then examined the effects of normalization of [Ca2⫹]i on glibenclamide- or Bay K8644-stimulated insulin secretion. Glibenclamide, a sulfonylurea, stimulates insulin secretion by inhibiting the KATP channels (18), while Bay K8644 stimulates insulin secretion by activating the VDCCs of pancreatic ␤-cells (19). Neither glibenclamide (1 ␮M) nor Bay K8644 (1 ␮M) had a significant stimulatory effect on insulin secretion in control MIN6-m14, but effects were observed when [Ca2⫹]i was nor-

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FIG. 5. RNA blotting of molecules involved in glucose metabolism (A) and KATP channels and VDCCs (B). Total RNA was isolated from MIN6-m14 treated with or without 10 ␮M nifedipine for 24 h, and 10 ␮g of aliquot was electrophoresed and transferred onto a nylon membrane. The membrane was probed with 32P-labeled DNA fragments corresponding to cDNA indicated in the figure. Photographs of 18 S RNA are shown to confirm equal loading. m14, MIN6-m14; m14-Nif, nifedipine-treated MIN6-m14.

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malized by nifedipine (Fig. 2, B and C). These results suggest that normalization of [Ca2⫹]i by nifedipine treatment improves the function of the KATP channels and/or the VDCCs, both of which are impaired in control MIN6-m14 (15). Effects of Normalization of [Ca2⫹]i on Glucose Metabolism in MIN6-m14 —To evaluate the effect of normalization of [Ca2⫹]i by nifedipine on glucose metabolism in MIN6-m14, we measured activity of enzymes involved in glucose metabolism and ATP production. Differently than in normal ␤-cells, glucosephosphorylating activity in MIN6-m14 is due mostly to HK, a low Km isoform of the glucose-phosphorylating enzyme (15). Normalization of [Ca2⫹]i by nifedipine did not alter either HK or GK activity (Fig. 3A). Lactate dehydrogenase activity, which is increased in control MIN6-m14 as compared with MIN6-m9 (14), was not altered by nifedipine (Fig. 3B). Furthermore, normalization of [Ca2⫹]i had no influence on ATP production in MIN6-m14 (Fig. 3C). These results demonstrate that normalization of [Ca2⫹]i by nifedipine does not change glucose metabolism in MIN6-m14. Electrophysiological Analyses of KATP Channels and VDCCs—We then performed functional analyses of the KATP channels and the VDCCs by patch clamp technique. Normalization of [Ca2⫹]i by nifedipine significantly increased KATP channel conductance in MIN6-m14 (Fig. 4A). In addition, VDCC currents at the whole cell level also were restored by normalization of [Ca2⫹]i by nifedipine. Peak current was significantly greater in nifedipine-treated MIN6-m14 than in control MIN6-m14

(Fig. 4B). These data show that both KATP channel and VDCC activities are significantly improved in MIN6-m14 after normalization of [Ca2⫹]i. Expressions of Various Genes Important in Glucose-induced Insulin Secretion—Normalization of [Ca2⫹]i by nifedipine did not alter mRNA expression of either GK or HK (Fig. 5A). mRNA levels of LDH were decreased after normalization of [Ca2⫹]i (Fig. 5A). mRNA expression of Kir6.2, the pore-forming subunit of the ␤-cell KATP channel, was markedly increased by treatment with nifedipine (Fig. 5B). SUR1, the regulatory subunit of KATP channels, showed a slight increase in mRNA levels (Fig. 5B). Expression of the ␣1-subunit of the VDCCs was increased strongly in MIN6-m14 after normalization of [Ca2⫹]i, but there was no significant difference in mRNA expression of the ␤3-subunit of VDCCs (Fig. 5B). Subcellular Localization of the Subunits of KATP Channels and VDCCs—We investigated the subcellular localization of subunits of KATP channels and VDCCs before and after normalization of [Ca2⫹]i. Subcellular fractionation was verified by immunoblot analysis of Na⫹/K⫹-ATPase and sarco/endoplasmic reticulum Ca2⫹-ATPase, markers for plasma membrane and internal membrane fractions, respectively. Normalization of [Ca2⫹]i by nifedipine increased the levels of Kir6.2 in both plasma membrane and internal membrane fractions significantly to a similar degree (Fig. 6B). SUR1 was somewhat increased by treatment with nifedipine, but the differences were small (Fig. 6C). Expression level of the ␣1-subunit of VDCCs was increased significantly both in plasma membrane and internal membrane fractions of MIN6-m14 after normalization of [Ca2⫹]i (Fig. 6D), while the expression level of the ␤3-subunit was unaffected by nifedipine (Fig. 6E). These results indicate that the Kir6.2 subunit of KATP channels and the ␣1-subunit of VDCCs are increased at the protein level, while

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FIG. 6. Immunoblotting of the subunits of KATP channels and VDCCs in subcellular fractions. Cells treated with or without 10 ␮M nifedipine were fractionated into plasma membrane-enriched fraction (PM), internal membrane-enriched fraction (IM), and cytosol fraction (Cyt). Aliquots of each fraction (5 ␮g of protein equivalent to PM fraction) were subjected to SDS-PAGE, and blots were probed with antibodies indicated in the figures. A, verification of specificity for subcellular components. Antibodies to Na⫹/K⫹-ATPase to sarco/endoplasmic reticulum Ca2⫹-ATPase were used as markers for plasma and internal membranes, respectively. B–E, quantification of KATP channel (B, Kir6.2; and C, SUR1) and VDCC (D, ␣1- and E, ␤3-) subunit proteins. Data form four separate experiments are illustrated, normalized to the intensity of the signals of PM fractions (arbitrary units; means ⫾ S.E.). Photographs are representative results. m14, MIN6-m14; m14-Nif, nifedipine-treated MIN6-m14. *, p ⬍ 0.05, **, p ⬍ 0.01.

FIG. 7. Effects of diazoxide in MIN6-m14cells (A–C) and Bay K8644 in MIN6-m9 cells (D–F). MIN6-m14 cells (A–C) and MIN6-m9 cells (D–F) were treated with and without 300 ␮M diazoxide (A–C) or 1 ␮M Bay K8644 (D–F) for 24 h. A and D, glucose-induced insulin secretion. n ⫽ 4, *, p ⬍ 0.05, **, p ⬍ 0.01. B and E, [Ca2⫹]i change in response to glucose. C, F, immunoblotting of Kir6.2 and ␣1-subunits in different subcellular fractions. m14, MIN6-m14; m14-Dzx, diazoxide-treated MIN6-m14; and m9. MIN6-m9, m9-Bay K; Bay K8644-treated MIN6-m9.

Importance of [Ca2⫹]i in Glucose Responsiveness of ␤-Cells

DISCUSSION

We used two clonal pancreatic ␤-cell lines, MIN6-m9 and MIN6-m14, in the present study as models of glucose-responsive and glucose-unresponsive ␤-cells, respectively. We previously demonstrated decreased activities of KATP channels and VDCCs in glucose-unresponsive MIN6-m14 (15). KATP channels and VDCCs serve a crucial role in coupling glucose metabolism to exocytosis of the insulin granules in pancreatic ␤-cells (18). Here we show that MIN6-m14 exhibit elevated [Ca2⫹]i compared with glucose-responsive MIN6-m9, and that normalization of [Ca2⫹]i by the Ca2⫹ channel blocker nifedipine restores the activities of both KATP channels and VDCCs to repair glucose-induced insulin secretion in MIN6-m14. It should be noted that normalization of [Ca2⫹]i also was achieved by the KATP channel opener diazoxide with effects similar to those of nifedipine. Moreover, the Ca2⫹ channel agonist Bay K8644 exerted effects contrary to the channel blocker in glucose-responsive MIN6-m9. These findings confirm that chronically elevated [Ca2⫹]i is the major cause of the glucose unresponsiveness in MIN6-m14. The activities of KATP channels and VDCCs can be regulated by various factors, including gene expression, phosphorylation/ dephosphorylation, and trafficking to the plasma membrane (21–36). Our data show that normalization of [Ca2⫹]i increases the expression of both KATP channels and VDCCs. In particular, Kir6.2, the pore-forming subunit of the KATP channels of pancreatic ␤-cells (23), and the ␣1-subunit of the VDCCs, also the pore-forming subunit of the channel (24), were strongly up-regulated at both the mRNA (Fig. 5) and protein levels (Fig. 6). Contrariwise, Bay K8644, a Ca2⫹ channel agonist, decreased the expression of the Kir6.2 subunit of KATP channels and the ␣1-subunit of VDCCs (Fig. 7F). Little is known of the regulation of these channel expressions, but it has been reported that high glucose leads to marked decreases in both Kir6.2 and SUR1 expression in isolated rat pancreatic islets as well as in the INS-1 ␤-cell line (25). A decrease in Kir6.2 expression also has been noted in pancreatic islets of Zucker diabetic fatty rats (26). Furthermore, mRNA expression of the ␣- and ␤-subunits of VDCCs are down-regulated in high glucose-infused rats, but diazoxide restores expression (27, 28).

mRNA levels of the ␣1-subunit of VDCCs also are reduced in pancreatic ␤-cells of Zucker diabetic fatty rats (29). Because persistent hyperglycemia causes sustained elevation of [Ca2⫹]i in the ␤-cells (8, 10), these observations may well be the result of alteration in [Ca2⫹]i. Considering these findings together, [Ca2⫹]i most likely regulates the expression of KATP channels and VDCCs at the transcriptional level. It recently has been shown that intracellular membrane trafficking of ion channel subunits is important in the functional expression of these channels at the cell surface (30 –37). To evaluate the effect of intracellular Ca2⫹ on membrane trafficking of the channels, we measured the protein levels of the channel subunits in different subcellular fractions. However, the relative abundance of the KATP channel subunits and the VDCC subunits in plasma membrane fraction and internal membrane fraction was similar in MIN6-m14 before and after nifedipine treatment, indicating that membrane trafficking of these channel subunits is not affected by [Ca2⫹]i. MIN6-m14 exhibit abnormalities not only in glucose responsiveness but also in glucose sensitivity. As in normal pancreatic islets, half-maximal insulin secretion occurs at 15 mM glucose in MIN6-m9, while the value is below 1 mM in MIN6-m14 cells (15). Although glucose responsiveness (intracellular Ca2⫹ response to glucose and amount of secreted insulin) was dramatically improved by normalization of [Ca2⫹]i, glucose sensitivity remained unchanged (Figs. 2A and 7A). GK is a rate-limiting enzyme in glycolysis in ␤-cells and is thought to be a glucose sensor for glucose-induced insulin secretion (38, 39) because it has a Km value higher than the physiological concentration of glucose. However, MIN6-m14 predominantly expresses HK-I, a low Km isoform of the glucose-phosphorylating enzyme, and ⬎ 90% of the activity occurs at 0.5 mM of glucose (15), which might well lead to abnormal glucose sensitivity. Normalization of [Ca2⫹]i did not alter the expression of two isoforms of the enzyme (Fig. 5) or their activities (Fig. 3A), which might account for the unchanged glucose sensitivity of MIN6-m14 before and after normalization of [Ca2⫹]i by nifedipine. Taken together with the findings on LDH activity and ATP production in MIN6-m14 cells, glucose metabolism apparently is not influenced by changes in [Ca2⫹]i and is regulated independently of KATP channel and VDCC activities. In conclusion, chronically elevated [Ca2⫹]i induces glucose unresponsiveness in MIN6-m14 by decreasing the expression of both KATP channels and VDCCs at the protein level. Normalization of [Ca2⫹]i by nifedipine or diazoxide induces a glucose-responsive state from a glucose-unresponsive state by restoring the functional expressions of these channels. Since abrogation of intracellular Ca2⫹ homeostasis in pancreatic ␤-cells has been shown to be associated with impaired insulin secretion (8, 10 –13), the present study suggests normalization of [Ca2⫹]i in pancreatic ␤-cells as a therapeutic strategy in the treatment of the impaired glucose-induced insulin secretion seen in type 2 diabetes. REFERENCES 1. Ghosh, A., and Greenberg, M. E. (1995) Science 268, 239 –247 2. Berridge, M. J. (1994) Mol. Cell. Endocrinol. 98, 119 –124 3. Bito, H., Deisseroth, K., and Tsien, R. W. (1997) Curr. Opin. Neurobiol. 7, 419 – 429 4. Hardingham, G. E., and Bading, H. (1999) Microsc. Res. Tech. 46, 348 –355 5. Wollheim, C. B., and Sharp, G. W. (1981) Physiol. Rev. 61, 914 –973 6. Artalejo, C. R., Adams, M. E., and Fox, A. P. (1994) Nature 367, 72–76 7. Verkhratsky, A., and Toescu, E. C. (1998) Trends. Neurosci. 21, 2–7 8. Levy, J. (1999) Endocrine 10, 1– 6 9. Prentki, M. (1996) Eur. J. Endocrinol. 134, 272–286 10. Bjorklund, A., Lansner, A., and Grill, V. E. (2000) Diabetes 49, 1840 –1848 11. Okamoto Y., Ishida, H., Taminato T., Tsuji, K., Kurose, T., Tsuura, Y., Kato, S., Imura, H., and Seino, Y. (1992) Diabetes 41, 1555–1561 12. Tsuji, K., Taminato, T., Ishida, H., Okamoto, Y., Tsuura, Y., Kato, S., Kurose, T., Okada, Y., Imura, H., and Seino, Y. (1993) Metabolism 42, 1424 –1428 13. Kato, S., Ishida, H., Tsuura, Y., Tsuji, K., Nishimura, M., Horie, M., Taminato, T., Ikehara, S., Odaka, H., Ikeda, I., Okada, Y., and Seino, Y. (1996) J. Clin.

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