THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2002 by The

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THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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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