Original Article Exposure to Chronic High Glucose Induces ␤-Cell Apoptosis Through Decreased Interaction of Glucokinase With Mitochondria Downregulation of Glucokinase in Pancreatic ␤-Cells Won-Ho Kim,1 June Woo Lee,1 Young Ho Suh,1 Shin Hee Hong,2 Joo Sun Choi,1 Joo Hyun Lim,1 Ji Hyun Song,1 Bin Gao,3 and Myeong Ho Jung1

Chronic hyperglycemia is toxic to pancreatic ␤-cells, im- pairing cellular functioning as observed in type 2 diabetes; however, the mechanism underlying ␤-cell dysfunction everal recent studies have demonstrated that and the resulting apoptosis via glucose toxicity are not apoptosis of pancreatic ␤-cells is induced as a fully characterized. Here, using MIN6N8 cells, a mouse consequence of type 2 diabetes, release of cyto- pancreatic ␤-cell line, we show that chronic exposure to kines and free fatty acids from adipocytes, and high glucose increases cell death mediated by Bax oli- S hyperglycemia (1–3). This suggests that type 2 diabetes gomerization, cytochrome C release, and caspase-3 activa- may lead to inadequate functional adaptation of pancreatic tion. During apoptosis, glucokinase (GCK) expression ␤ decreases in high-glucose–treated cells, concomitant with -cell mass in the face of insulin resistance. According to a decrease in cellular ATP production and insulin secre- current models, glucose metabolism in the pancreatic tion. Moreover, exposure to a chronically high dose of ␤-cell leads to ATP generation, closure of ATP-regulated glucose decreases interactions between GCK and mito- Kϩ channels, plasma membrane depolarization, opening of chondria with an increase in Bax binding to mitochondria voltage-dependent Ca2ϩ channels, and an increase in free and cytochrome C release. These events are prevented by cytosolic Ca2ϩ concentration resulting in insulin release GCK overexpression, and phosphorylation of proapo- (4–6). In contrast to the ability of acute glucose to ptotic Bad proteins in GCK-overexpressing cells is pro- stimulate insulin secretion, chronic exposure of ␤-cells to longed compared with Neo-transfected cells. Similar ␤ results are obtained using primary islet cells. Collectively, a hyperglycemic environment causes -cell dysfunction these data demonstrate that ␤-cell apoptosis from expo- and ultimately ␤-cell death, a phenomenon termed gluco- sure to chronic high glucose occurs in relation to lowered toxicity (4,7). Despite convincing evidence of glucotoxic- GCK expression and reduced association with mitochon- ity in pancreatic ␤-cells, the exact mechanisms underlying dria. Our results show that this may be one mechanism by impairment of ␤-cell function and induction of apoptosis which glucose is toxic to ␤-cells and suggests a novel from chronic exposure to elevated glucose are not com- approach to prevent and treat diabetes by manipulating pletely understood. Bax- and GCK-controlled signaling to promote apoptosis Glucokinase (GCK), or hexokinase IV, is a well-known or proliferation. Diabetes 54:2602–2611, 2005 member of the mammalian hexokinase family that cata- lyzes the initial step of glucose metabolism in several metabolic pathways (8,9). Glucose-stimulated insulin se- From the 1Division of Metabolic Disease, Department of Biomedical Science, cretion is regulated by the rate of glucose metabolism National Institutes of Health, Seoul, South Korea; the 2Division of Optical Metrology, Korea Research Institute of Standards and Science, Yuseong, within ␤-cells, and a key event in this process is the South Korea; the 3Section on Liver Biology, Laboratory of Physiologic Studies, phosphorylation of glucose by GCK (10). Moreover, muta- National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland. tions in GCK have been associated with maturity-onset Address correspondence and reprint requests to Dr. Myeong Ho Jung, type 2 diabetes of the young (5,11), a disease characterized Division of Metabolic Disease, Department of Biomedical Science, National by early-onset and persistent hyperglycemia. Similar de- Institutes of Health, #5 Nokbun-dong, Eunpyung-gu, Seoul 122-701, South Korea. E-mail: [email protected]. fects in glucose regulation also have been observed in Received for publication 18 January 2005 and accepted in revised form 16 mice with genetic alterations in the GCK gene, indicating June 2005. that optimal ␤-cell function may be dependent on expres- FITC, fluorescein isothiocyanate; GCK, glucokinase; GFP, green fluorescent protein; GST, glutathione S-transferase; KRBB, Krebs-Ringer bicarbonate sion of genes involved in glucose sensing, such as GCK buffer; PARP, poly(ADP-ribose) polymerase; ROS, reactive oxygen species; and Glut2 as well as the insulin gene, and this has been TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end label- ing; VDAC, voltage-dependent anion channel. confirmed by a study demonstrating that downregulation © 2005 by the American Diabetes Association. of GCK and Glut2 increased blood glucose and prolonged The costs of publication of this article were defrayed in part by the payment of page duration of hyperglycemia in hyperglycemia-induced rat charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. islets (12,13). Conversely, intracellular ATP levels were

2602 DIABETES, VOL. 54, SEPTEMBER 2005 W.-H. KIM AND ASSOCIATES strongly and acutely reduced in GCK- or Glut2-overex- Terminal deoxynucleotidyl transferase-mediated dUTP nick-end label- pressing cells exposed to high glucose, resulting in pro- ing. Apoptotic cells were detected using Apop Tag, an in situ apoptosis detection kit from Oncor (Gaitherburg, MD) as previously described (23). nounced apoptotic cell death (14). Despite studies Immunocytochemistry. Immunocytochemistry for anti-Bax, anti-GCK, anti- demonstrating that Glut2 and GCK are important compo- p53, and anti-inuslin was performed as previously described (23). nents for glucose metabolism in pancreatic ␤-cells, it is not Bax oligomerization assay. The Bax oligomerization assay was adapted as clear whether they are involved in glucotoxicity or how previously described (17,23). Briefly, the mitochondrially enriched fraction ␤ from isolated cells (21) was subjected to protein cross-linking by incubation in -cell apoptosis is mediated. a freshly prepared mixture of 10 mmol/l bismaleimidohexane (Pierce), 16.8% Glucose regulates Bad phosphorylation in hepatocytes, DMSO, and PBS for 30 min at room temperature with occasional mixing. For forming large complexes containing proteins involved in the Bax-VDAC–binding assay, mitochondrial fractions isolated from total regulating its phosphorylation state (15,16). Phosphory- cellular extracts were immunoprecipitated with anti-VDAC antibody, washed lated Bad promotes interactions between hexokinase and twice in lysis buffer, and subjected to Western blotting. Glutathione S-transferase pull-down assay. For in vitro–binding assays, the voltage-dependent anion channel (VDAC) necessary 500 ␮g lysates were incubated with 3.0 ␮g glutathione S-transferase (GST) or for pumping newly synthesized ATP from the mitochon- fusion GST GCK proteins coupled to glutathione sepharose beads in 300 ␮l dria (17). Recent evidence indicates that interactions lysis buffer overnight at 4°C with continuous rocking, as previously described between hexokinase and mitochondrial VDAC inhibit ap- (24). Insulin secretion and ATP content assay. To determine insulin release in optosis by preventing the channel from binding to Bax and response to glucose stimulation, MIN6N8 cells or isolated islet cells stimulated releasing cytochrome C. Hence, as hexokinase is regulated with glucose for 4 days were washed in Krebs-Ringer bicarbonate buffer by the level of glucose metabolism, the interactions be- (KRBB) containing 3.3 mmol/l glucose and preincubated for 30 min in the tween Bax and VDAC may be responsive to glucose levels same buffer. The KRBB was then discarded and replaced by fresh buffer (18–21). containing 3.3 mmol/l glucose for 1 h, followed by an additional 1-h incubation in KRBB containing 16.7 mmol/l glucose. Supernatants were collected and The goal of this study was to determine whether chronic frozen for insulin assays. Thereafter, cells were washed with PBS and exposure of ␤-cells to high glucose induces apoptosis and extracted with 0.18 N HCl in 70% ethanol for 24 h at 4°C. The acid-ethanol to examine the regulatory mechanisms involved in apopto- extracts were collected for determination of insulin content. Insulin was sis. In this investigation, we found that chronic exposure determined by radioimmunoassay using mouse insulin as the standard (14). ATP levels in MIN6N8 cells and islets were determined using a luminometric to high glucose significantly reduces GCK association with assay kit from Promega as previously described (14). mitochondria by downregulating GCK expression, thereby Statistical analysis. For comparing values obtained in three or more groups, increasing interactions between Bax and mitochondria one-factor ANOVA was used followed by Tukey’s post hoc test, and P Ͻ 0.05 and resulting in Bax oligomerization, cytochrome C re- was taken to imply statistical significance. lease, and ␤-cell apoptosis. Changes in pro- and antiapop- totic proteins, as well as impairment of ␤-cell function, were also observed. These findings show that GCK plays RESULTS an important role in ␤-cell apoptosis by glucotoxicity and Chronic exposure to high glucose induces apoptosis is also involved in glucose metabolism and ␤-cell survival. in MIN6N8 cells. To examine the cytotoxic effect of high glucose on a pancreatic insulinoma cell line, MIN6N8 cells were treated with glucose at different concentrations for RESEARCH DESIGN AND METHODS varying time periods. Treatment with 33.3 mmol/l glucose Cell line and reagents. MIN6N8 cells, which are SV40 T-transformed induced marked genomic DNA fragmentation in a time- insulinoma cells derived from NOD mice, were kindly provided by Dr. M.S. and dose-dependent manner and caused a significant Lee (Sungkyunkwan University School of Medicine, Seoul, Korea). These cells were grown in DMEM containing 15% fetal bovine serum, 2 mmol/l glutamine, increase in the number of terminal deoxynucleotidyl trans- 100 IU/ml penicillin, and 100 ␮g/ml streptomycin (Life Technologies, Gaith- ferase-mediated dUTP nick-end labeling (TUNEL)-positive ersburg, MD). All antibodies were obtained from Cell Signaling Technology MIN6N8 cells relative to cells treated with 5.5 mmol/l (Beverly, MA) or Santa Cruz Biotechnology (Santa Cruz, CA), and chemicals glucose, concomitant with cleavage of poly(ADP-ribose) were purchased from Sigma (St. Louis, MO). Bismaleimidohexane was obtained from Pierce Biotechnology (Rockford, IL). polymerase (PARP) similar to caspase-3 cleavage (Fig. Plasmids. Mouse GCK cDNA was amplified by PCR and cloned into pcDNA3 1A). Pretreatment of the cells with a specific caspase-3 and pEGFP C2 vectors (Clontech). The pEGFP GCK plasmid was generated inhibitor, z-DEVD-CHO, completely reduced 33.3 mmol/l using forward 5Ј-gggaagtctgggctacttctg-3Ј and reverse 5Ј-ctagtggactgggagagca glucose–induced PARP cleavage (Fig. 1C) and apoptosis tttg-3Ј primers to produce oligonucleotides that were subcloned within (data not shown). EcoRI/BamH1 sites in the pEGFP vector. The pcDNA GCK plasmid was generated by cutting pEGFP mGCK with HindIII/BamH1 restriction enzymes Exposure to high glucose resulted in a time- and dose- and inserted into the HindIII/BamH1 site of the pcDNA3.1C-V5His vector dependent decrease in Bcl-2 and Bcl-xL expression, (Invitrogen, Carlsbad, CA). whereas expression of Bax significantly increased, thereby Isolation of mouse pancreatic islets. Islets were isolated from overnight- increasing Bax/Bcl-2 or Bcl-xL ratios. The expression of fasted ICR mice (weight 20–25 g) by the collagenase digestion technique, as previously described (22,23). Fas and p53, potent proapoptotic proteins in the mito- Transient and stable transfection. MIN6N8 cells were transfected with chondrial apoptotic pathway (25), also significantly in- green fluorescent protein (GFP) GCK or pCDNA wild-type GCK and Neo creased (Fig. 1B). Moreover, a significant time-dependent vector control DNA using a lipofectin reagent (Life Technologies). After a 16-h increase in mitochondrial release of cytochrome C into the incubation period, growth media was replaced and cells were grown for an cytosol was observed after 2 days. Cytochrome C oxidase additional 48 h. Cells were then exposed to a selective concentration of 400 ␮g/ml G418 sulfate (Life Technologies) to isolate stably transfected cells. IV (mitochondrial protein) was used to confirm whether Immunoblots and coimmunoprecipitation. Cells were lysed in RIPA buffer mitochondrial fraction was isolated purely from cell ex- (23) at 4°C and then vortexed and centrifuged at 16,000 rpm for 10 min at 4°C. tract (Fig. 1C). Release of cytochrome C from the mito- The supernatant was mixed in Laemmli loading buffer, boiled for 4 min, and chondria into the cytosol is mediated by Bax translocation then subjected to SDS-PAGE. For endogenous complexes, mitochondrial lysates (1 mg) fractionated from the cells were immunoprecipitated with 2 ␮g to the mitochondrial outer membrane and its subsequent antibody and immunoblotted. For exogenous complexes, whole-cell lysates oligomerization (14,20). Normally residing within the cy- (500 ␮g) of transfected cells were used. tosol, Bax levels were significantly reduced within cytoso-

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FIG. 1. Effects of high glucose on apoptosis. MIN6N8 cells were treated with different glucose concentrations for the indicated times. A: DNA fragmentation (upper left) and TUNEL assay (lower left) were carried out. The cleavage of caspase-3 (upper right) and PARP (lower right) was analyzed. B: Expression of apoptotic proteins. C: Release of cytochrome (Cyto) C and Bax translocation. The blots were reprobed with antibodies to cytochrome C oxidase (COX) IV and VDAC. D: Bax immunocytochemistry. Fluorescent microscopic images taken for Bax (green), MitoTracker CMXRos (red), and the final merged images (localization of Bax at mitochondria) are shown (upper). Fold of cells exhibiting punctuate Bax and percentage of Bax colocalization with mitochondria was determined by counting ϳ20–100 cells for each condition (lower). Results represent the average ؎ SE from three independent experiments (*P < 0.05, **P < 0.01). E: Interaction of Bax with VDAC. F: Bax oligomerization (*nonspecific bands). All data are representative of three independent experiments. lic fractions after 2 days of exposure and increased in the manner but not in hexokinase I (Fig. 2A and B). To confirm mitochondrial fraction of 33.3 mmol/l glucose–treated these results, immunocytochemistry on MIN6N8 cells was cells (Fig. 1C). Translocation of Bax was further confirmed performed using anti-GCK. As shown in Fig. 2C, glucose by immunostaining using fluorescein isothiocyanate reduced GCK expression dose dependently, decreasing (FITC) Bax and Mito-Tracker CMXRos. While FITC Bax significantly by 40% in 33.3 mmol/l glucose–treated cells. was localized primarily in the cytosol of 5.5 mmol/l glu- Consistent with decreased GCK expression, chronic expo- cose–treated cells, treatment with 33.3 mmol/l glucose sure of the cells for 4 days abolished the ability of acute increased Bax translocation to the mitochondria by 4.3- glucose to stimulate insulin content and inhibited ATP fold (Fig. 1D). To confirm that Bax translocates to the production (Fig. 2D). However, in cells treated with 16 mitochondria through binding with VDAC, we examined mmol/l glucose, insulin content and ATP production in- the interaction of Bax with VDAC in isolated mitochon- creased slightly compared with cells treated with 5.5 drial fractions. Bax interaction with VDAC (Fig. 1E) and mmol/l glucose. Furthermore, expression of other major Bax oligomerization (Fig. 1F) substantially increased 2 glucose metabolism proteins, including Glut2, peroxisome days after treatment with 33.3 mmol/l glucose. proliferator–activated receptor ␥ coactivator 1, and sterol Chronic high glucose induces abnormal glucose me- regulatory element–binding protein 1, was also significantly tabolism. To examine whether chronic exposure to high- reduced, whereas considerable changes in the phosphati- glucose–induced apoptosis occurs as a direct result of dylinositol 3-kinase subunit p85, VDAC, and superoxide glucotoxicity, we studied the effects of high glucose on dismutase were not detected. genes associated with glucose metabolism and insulin Chronic high glucose inhibits GCK translocation to content. Chronic exposure to 33.3 mmol/l glucose strongly the mitochondria. The association of hexokinase I and II reduced expression of GCK in a time- and dose-dependent with mitochondria prevents the interaction of Bax with

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FIG. 2. Effects of high glucose on glu- cose metabolism. A and B: Expression of glucose metabolic-related proteins. C: GCK immunocytochemistry. Re- sults represent the average ؎ SE from three independent experiments (*P < 0.005, ** P < 0.05). D: Content of insulin and production of ATP. Re- sults represent the average ؎ SE from three independent experiments (*P < All data are .(5 ؍ P < 0.01; n ** ,0.05 representative of three independent experiments. HKI, hexokinase I; PGC-1, peroxisome proliferator–acti- vated receptor ␥ coactivator 1; PI3K, phosphatidylinositol 3-kinase; SOD, superoxide dismutase; SREBP1, sterol regulatory element–binding protein 1.

VDAC and inhibits Bax-induced apoptosis (15,16,18). served in control and cells treated with 33.3 mmol/l Since GCK is a unique hexokinase family member whose glucose for 2 h (Fig. 3D), suggesting that inhibition of Bad expression is largely restricted to the liver and pancreatic phosphorylation and Bax overexpression by chronic high ␤-cells, the ability of GCK to associate with mitochondria glucose may be involved in the reducing GCK-VDAC through VDAC was examined. Therefore, we first exam- interaction. ined the localization of GCK using Mito-Tracker CMXRos To clarify whether GCK endogenously interacts with after transient transfection with GFP GCK. GCK green VDAC on the mitochondrial outer membrane and if inter- fluorescence was coexpressed in both cytosol and mito- actions are reduced by chronic exposure to glucose, chondria of control cells, while 1 day after glucose treat- GCK-VDAC complexes were immunoprecipitated with an- ment, a large amount of the GFP GCK translocated to the ti-GCK. Whereas a basal level of GCK and VDAC interac- mitochondria. However, the colocalization of GFP GCK in tion was observed in control and cells treated acutely with the mitochondria began diffusing into the cytosol after 2 glucose (1 day and 16 mmol/l), GCK-VDAC interactions days, and the GFP GCK appeared in the cytoplasm sepa- were significantly inhibited by chronic high glucose in a rate from the mitochondria 4 days after treatment, con- time- and dose-dependent manner (Fig. 3E). Similar to comitant with decreased GFP GCK expression at the same this, translocation of GCK to the mitochondria was also time (Fig. 3A). Next, to characterize whether GCKs di- inhibited by chronic exposure to glucose in a dose-depen- rectly bind VDAC and whether 33.3 mmol/l glucose affects dent manner (Fig. 3F). GCK expression in both cytosolic GCK binding to VDAC, the GST pull-down assay was and mitochondrial fractions was significantly decreased by conducted using purified GST or GST GCK. As shown in 33.3 mmol/l glucose, whereas no changes were observed in Fig. 3B, VDAC bound to GST GCK but not to control GST hexokinase I expression in both fractions, indicating that beads. Furthermore, the levels of GCK binding to VDAC inhibition of GCK translocation to the mitochondria by decreased slightly in 33.3 mmol/l glucose–treated cells chronic high glucose may be due to downregulation of compared with 5.5 mmol/l glucose, but this decrease was GCK. not due to changes in GST GCK or VDAC expression in Detachment of GCK from mitochondria potentiates total cell lysates. Concomitant with reduced GCK interac- apoptosis. To confirm that the association between GCK tion with VDAC, Bax was overexpressed in 33.3 mmol/l and mitochondria is an important antiapoptotic determi- glucose–treated cells (Fig. 3C). In contrast, chronic high nant, we investigated the effects of clotrimazole, an agent glucose significantly inhibited Bad phosphorylation ob- known to dissociate hexokinase from mitochondria (21),

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FIG. 3. Effects of chronic high glucose on the interaction of GCK with VDAC and GCK translocation to mitochon- dria. A: Localization of GFP GCK. Af- ter incubation with 33.3 mmol/l glucose for the indicated times, the cells were treated with 100 nmol/l Mi- toTracker CMXRos for 30 min to stain mitochondria before analysis of fluo- rescent microscopic images. Percent- age of GFP-GCK translocation to mitochondria was quantified (right). Results shown are means ؎ SE from three independent experiments (*P < 0.05, **P < 0.01). B: In vitro–binding assay. Upper panel: Purified GST GCK was detected with anti-GST antibody. Lower panel: Isolated mitochondrial fractions were incubated with GST or GST GCK. C: High glucose decreases the interaction of GST GCK with VDAC in vitro. The isolated mitochon- drial fractions were subjected to GST pull-down assay. D: Bad phosphoryla- tion. E: Endogenous interaction of GCK with VDAC. F: GCK translocation to mitochondria. All data are repre- sentative of three independent exper- iments. HK I, hexokinase I. on apoptosis and the interaction of Bax with VDAC with 50 ␮mol/l indomethacin, a nonsteroidal anti-inflam- induced by high glucose. Addition of 20 ␮mol/l clotrim- matory agent that induces Bax-dependent apoptosis azole to the cells almost completely inhibited the associ- (21,26). Cells treated for 2 h with either indomethacin or ation of GCK and hexokinase I with mitochondria induced clotrimazole induced little DNA fragmentation, but when by 16 mmol/l glucose in MIN6N8 cells, similar to inhibition combined, DNA fragmentation markedly increased. Fur- of GCK and hexokinase II in HepG2 cells (Fig. 4A). Next, thermore, apoptosis induced by chronic exposure to 33.3 whether detaching hexokinase I or GCK from mitochon- mmol/l glucose for 2 days was potentiated by pretreatment dria by clotrimazole affects the ability of Bax to induce with clotrimazole (Fig. 4B). When 33.3 mmol/l glucose was apoptosis and release cytochrome C in 33.3 mmol/l glu- administered to cells pretreated with clotrimazole, Bax cose–treated cells was examined. Cells were stimulated binding increased significantly with complete release of

FIG. 4. Effects of detachment of GCK or hexokinase (HK) I from mitochondria on apoptosis by chronic high glucose. A: Cells were pretreated with 20 nmol/l clotrimazole for 30 min, and mitochondrial and cytosolic fractions were subjected to Western blotting. B: Cells were pretreated with clotrimazole (CTZ) for 30 min and then additionally treated with or without indomethacin (IND; 50 ␮mol/l) for 3 h. After incubation, 33.3 mmol/l glucose was administered for 2 days in the presence or absence of clotrimazole or indomethacin. C: Clotrimazole and indomethacin increase Bax translocation and cytochrome C release induced by 33.3 mmol/l glucose. All data are representative of three independent experiments. CTL, control.

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FIG. 5. Effects of GCK overexpression on chronic high-glucose–induced apoptosis. MIN6N8 cells were stably transfected with either an empty Neo or wild-type GCK expression vector. A: Three Neo vector– and three wild-type GCK–transfected clones were selected. B: The selected Neo vector no. 2) and wild-type GCK (no. 3) cells were treated with glucose. C: DNA fragmentation and TUNEL assay. Results shown are means ؎ SE from) three independent experiments (*P < 0.05, **P < 0.01). D: Secretion of insulin and production of ATP. Results represent the average ؎ SE from three independent experiments (*P < 0.05, **P < 0.05). E: Colocalization of GCK with mitochondria. F–H: Effects of GCK overexpression on interaction of GCK with VDAC (F), Bax translocation into mitochondria (G), and Bax oligomerization (H) (*nonspecific bands [H]). All data are representative of three independent experiments. cytochrome C compared with 33.3 mmol/l treatment alone GCK-overexpressing cells may be due to the sustenance of (Fig. 4C), similar to the combination of indomethacin and GCK compared with the complete reduction of GCK by clotrimazole. These results demonstrate that detachment chronic exposure to high glucose in Neo control cells (Fig. of GCK, as well as hexokinase I, from mitochondria 5B). We also have obtained the similar tendentious results increase the susceptibility of cells to apoptosis induced by in other Neo- or GCK-overexpressing clones (data not chronic high glucose by increasing Bax binding to mito- shown). chondria in MIN6N8 cells. GCK translocation was further confirmed by immuno- GCK overexpression inhibits chronic high-glucose– cytochemistry in GCK-overexpressing cells (Fig. 5E). The induced apoptosis. To examine the essential role of GCK results show that reduced GCK translocation induced by in chronic high-glucose–induced apoptosis, MIN6N8 cells 33.3 mmol/l glucose in Neo-transfected cells was attenu- were transfected with wild-type GCK cDNA and selected ated in GCK-overexpressing cells. Additionally, GCK was GCK-overexpressed clones (Fig. 5A), followed by treat- still localized in the mitochondria of GCK-overexpressing ment with 33.3 mmol/l glucose. DNA fragmentation in- cells exposed to 33.3 mmol/l glucose, with a stronger duced by chronic exposure of cells to 33.3 mmol/l glucose expression compared with Neo-transfected cells (Fig. 5E). was significantly inhibited by GCK overexpression (Fig. Next, we examined whether the strong localization of GCK 5C, left). Similarly, GCK overexpression significantly, but in the mitochondria induced by GCK overexpression is not completely, prevented TUNEL-positive apoptotic cells mediated by the association of GCK with mitochondrial induced by 33.3 mmol/l glucose (Fig. 5C, right). Further- VDAC. As shown in Fig. 5F, reduced GCK association with more, reductions in insulin content and ATP production VDAC induced by 33.3 mmol/l glucose in cells transfected induced by glucotoxicity were also restored in GCK- with Neo vector was significantly attenuated in GCK- overexpressing cells (Fig. 5D). The recovery observed in overexpressing cells. GCK overexpression almost com-

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FIG. 6. Effects of GCK overexpression on Bad phos- phorylation and expression of apoptotic-related proteins. Neo- or GCK-overexpressed cells were treated with 33.3 mmol/l glucose. After incubation, the lysates were immunoblotted. A–D: Effects of GCK overexpression on Bad phosphorylation (A and B) and apoptotic-related (C) and glucose met- abolic-related (D) proteins. All data are represen- tative of three independent experiments. pletely inhibited the increase of interaction between Bax decreases in glucose metabolism and alterations in apo- and VDAC induced by 33.3 mmol/l glucose in Neo-trans- ptotic proteins are also responsible for apoptosis of pan- fected cells (Fig. 5G). Consistent with these results, Bax creatic islet cells, isolated islet cells were treated with 33.3 oligomerization, which was increased by 33.3 mmol/l mmol/l glucose. Synergistic apoptosis was observed in glucose in Neo-transfected cells, was also completely single-islet cells, which was confirmed by TUNEL assay inhibited in GCK-overexpressing cells (Fig. 5H), indicating and DNA fragmentation (Fig. 7A and B). Similar to that GCK overexpression inhibits apoptosis induced by MIN6N8 cells, GCK significantly decreased in response to high glucose through increased GCK interaction with 33.3 mmol/l glucose (Fig. 7D), which correlated with mitochondria, thereby inhibiting Bax translocation and decreased insulin content and ATP production (Fig. 7C). oligomerization. Most proapoptotic proteins, including p53, p21, and Bax, GCK overexpression prolongs Bad phosphorylation increased in 33.3 mmol/l glucose–treated islet cells, and reduces the Bax–to–Bcl-2 ratio or p53/p21 ex- whereas Bcl-2 was significantly decreased, correlating pression. Recently, it was reported that phosphorylated with increased release of cytochrome C into the cytosol Bad preserves mitochondrial integrity by forming a com- (Fig. 7D). The decrease in GCK and increase in p53 levels plex with GCK and limiting Bax-induced apoptosis in insulin-positive cells were also confirmed by double through prevention of Bax interaction with mitochondria immunostaining of islets with anti-GCK, anti-p53, and and enhancement of glucose metabolism (15,18). There- anti-insulin antibodies (Fig. 7E and F). To clearly define fore, we have characterized the effects of GCK on the the role of GCK on apoptosis induced by 33.3 mmol/l regulation of Bad phosphorylation by chronic high glucose glucose in isolated islet cells, we also examined the in both Neo- and GCK-overexpressing cells. Acute expo- localization and interaction of GCK with mitochondrial sure to 33.3 mmol/l glucose for 2 h increased Bad phos- VDAC using FITC GCK and MitoTracker CMXRos. As phorylation in both cell groups, with more phosphorylated shown in Fig. 8A, GCK green fluorescence was highly Bad found in GCK-overexpressing cells (Fig. 6A and B). expressed and colocalized in mitochondria in 5.5 mmol/l However, chronic exposure for 48 h significantly de- glucose–treated cells, while significantly decreased in 33.3 creased Bad phosphorylation in Neo vector–transfected mmol/l glucose–treated cells. Furthermore, the interaction cells, whereas Bad phosphorylation was significantly pro- of GCK with VDAC decreased significantly more in 33.3 longed in GCK-overexpressing cells. In contrast to Bad mmol/l glucose–treated cells than in 5.5 mmol/l glucose– phosphorylation, the decrease of phosphorylated Akt ob- treated cells (Fig. 8B), which may be due to decreased served at 48 h after treatment in Neo vector–transfected GCK expression, as its expression was decreased in both cells did not recover in GCK-transfected cells (Fig. 6B). mitochondrial and cytosolic fractions (data not shown). In Furthermore, decreases in Bcl-2 and increases in Bax, p53, contrast, Bax binding with mitochondrial VDAC increased and p21 proteins by high glucose in Neo-transfected cells from treatment with 33.3 mmol/l glucose (Fig. 8C), corre- were nearly prevented in GCK-overexpressing cells (Fig. lating with increased Bax translocation to mitochondrial 6C). On the other hand, GCK overexpression also restored fractions (Fig. 8D). Colocalization of Bax with mitochon- decreases in Glut2, peroxisome proliferator–activated re- dria significantly increased in cells treated with 33.3 ceptor ␥ coactivator 1, and sterol regulatory element– mmol/l glucose compared with 5.5 mmol/l, which was binding protein 1 similar to GCK (Fig. 6D). confirmed by immunostaining (Fig. 8E). Similar to these Chronic high glucose induces apoptosis by GCK results, Bax oligomerization also increased highly after downregulation and alterations of apoptotic proteins treatment with 33.3 mmol/l glucose (Fig. 8F), consistent in mouse primary islet cells. To determine whether with results obtained from MIN6N8 cells. These results

2608 DIABETES, VOL. 54, SEPTEMBER 2005 W.-H. KIM AND ASSOCIATES

FIG. 7. Chronic high glucose induces apoptosis through GCK downregulation and alterations of apoptotic-related proteins in islet cells. Isolated islet cells were treated with 5.5 or 33.3 mmol/l glucose for 4 days. A: TUNEL and diamidinophenylindole (DAPI) assays were performed. -Results shown are means ؎ SE of percentage of TUNEL positive islet cells. The mean number of islets scored from each donor was 38 (range 32–48) for each treatment condition. B: DNA fragmentation. C: Insulin content and ATP production. Significant differences from untreated :D .(5 ؍ controls are indicated (*P < 0.05, ** P < 0.01; n Expression of apoptotic-related proteins and cytochrome C release in islet cells. E and F: Double immunostaining for GCK (E) and p53 (F) appears in green and insulin (positive islet cells) in red in islets cultured and exposed for 4 days. Fluorescent microscopic images taken for GCK, p53, insulin, and the final merged images (expres- sion of GCK or p53 in insulin-positive islet cells) are shown. All data are representative of three independent experiments. show that GCK may play an important role in regulating ␤-cells has been widely studied, defining the functional apoptosis induced by chronic exposure of pancreatic ␤ role of GCK and the mechanisms regulated by GCK in islet cells to high glucose. glucotoxicity-induced ␤-cell apoptosis appears to be worthwhile. DISCUSSION GCK is the proximal and rate-limiting step in the utili- Reports suggest that elevated glucose concentrations have zation of glucose and is therefore critical in regulating a dual function on ␤-cell turnover depending on duration insulin secretion by ␤-cells (5,9). Recently, it was sug- of exposure and the genetic background of the islets gested that GCK is involved in apoptosis associated with (7,14). Although the effects of elevated glucose on ␤-cell glucose metabolism (15,16), but the exact mechanisms by proliferation and apoptosis are still controversial, several which GCK is involved in apoptosis are not known. As studies have demonstrated that chronic exposure of with hexokinase I, our data show that GCK also endog- ␤-cells to high glucose results in ␤-cell dysfunction and enously interacts with VDAC on the mitochondrial outer ultimately ␤-cell death (7,27). During progression of type 2 membrane; this was supported by the GST pull down (Fig. diabetes, glucotoxicity is likely an important factor that 3). Our in vitro–binding GST pull-down assay revealed that contributes to advancing ␤-cell failure and development of GST GCK binds to mitochondrial VDAC (Fig. 3B) and that overt diabetes (28). However, the exact molecular mech- the interaction between GST GCK and VDAC was reduced anism involved in glucotoxicity-induced ␤-cell dysfunction by 33.3 mmol/l glucose and correlated with Bax overex- and apoptosis is not clearly understood. In this study, we pression, but the pull-down GST and total VDAC levels demonstrated that chronic exposure to high glucose in- were unaffected by 33.3 mmol/l glucose (Fig. 3C), suggest- duces ␤-cell apoptosis through decreasing GCK protein ing that reduced interaction between GCK and VDAC by expression and interactions with VDAC in the mitochon- 33.3 mmol/l glucose is not due to changes in protein level. drial outer membrane, correlating with decreases in Bad In contrast to GCK, the expression of hexokinase I and its phosphorylation. Decreased binding of GCK with mito- interaction with VDAC or hexokinase I translocation to the chondria promotes the binding of Bax with VDAC and, mitochondria were not affected by chronic high glucose subsequently, Bax oligomerization, cytochrome C release, (Fig. 3F). Based on these results, GCK binding to VDAC and apoptosis along with decreased cellular ATP produc- may play a critical role in chronic high-glucose–induced tion and insulin secretion. Therefore, based on the pivotal apoptosis, although GCK lacks the NH2-terminal domain role of GCK on glucotoxicity-induced apoptosis, we be- necessary for binding of hexokinase I to VDAC. A different lieve that GCK involvement is integral for the relationship NH2-terminal domain of GCK from that of hexokinase I between glucose metabolism and cell apoptosis in pancre- may be involved in its interaction with VDAC. atic ␤-cells. Although the effects of GCK expression in The detachment of GCK or hexokinase I from mitochon-

DIABETES, VOL. 54, SEPTEMBER 2005 2609 GLUCOKINASE IN HIGH-GLUCOSE–INDUCED APOPTOSIS

FIG. 8. Chronic high glucose induces apoptosis through GCK detachment and Bax translocation to the mitochon- dria in islet cells. Isolated islet cells were treated with chronic high glucose for 4 days. A: GCK immunocyto- chemistry. B and C: Mitochondria-enriched fractions were immunoprecipitated with anti-GCK (B) or VDAC (C), and immunoprecipitates were analyzed by Western blotting. D: Bax translocation. E: Bax immunocytochem- istry. F: Bax oligomerization. All data are representative of three independent experiments. dria using clotrimazole significantly potentiated apoptosis cells (Fig. 6). Moreover, hyperglycemia-induced apoptosis in high–glucose– and indomethacin-treated cells and cor- and GCK downregulation were significantly inhibited in related with an increase in Bax translocation and cyto- constitutively phosphorylated Bad-transfected cells but chrome C release from mitochondria (Fig. 4). This potentiated by constitutively nonphosphorylated Bad suggests that GCK or hexokinase I protects against Bax- (W.-H.K., J.W.L., Y.H.S., M.H.J., unpublished data), sug- dependent apoptosis and that the imbalance between gesting that both Bad phosphorylation and GCK activation hexokinases, including GCK and Bax, may regulate cell may be essential for regulating glucose metabolism in survival or death of pancreatic ␤-cells. However, in our pancreatic ␤-cells and may be autoregulated by each other system, GCK, rather than hexokinase I, may play a more rather than have a cause-and-effect relationship. On the prominent role in mediating susceptibility to apoptosis by other hand, some studies demonstrated that phosphoryla- chronic high glucose, since chronic high glucose de- tion of Bcl-2 family proteins, including Bad, is attenuated creases GCK expression and GCK binding with VDAC but by reactive oxygen species (ROS) (29). In our unpublished not hexokinase I (Figs. 2 and 3). In further support of a data, GCK overexpression inhibited ROS production in- relationship between GCK and protection of apoptosis, the duced by hyperglycemia, concordant with an increase in relative expression of GCK in GCK-transfected cells Bad phosphorylation, suggesting that ROS produced under closely corresponded to the ability of GCK overexpression a hyperglycemic condition may regulate Bad phosphory- to resist 33.3 mmol/l glucose–induced apoptosis, reduc- lation. Interestingly, Bad phosphorylation more sustained tions of GCK-VDAC interactions, and Bax binding to in GCK-overexpressed cells than in Neo cells was still mitochondria and its oligomerization (Fig. 5). However, dephosphorylated after 4 days (Fig. 6A), indicating that we cannot exclude the possibile involvement of hexoki- other pathways may possibly be involved in the restora- nase I itself and alternate pathways in regulating apoptosis tion of GCK overexpression in pancreatic ␤-cells. induced by chronic high glucose, since GCK overexpres- Reportedly, the expression of several genes essential for sion did not completely prevent TUNEL-positive apoptotic ␤-cell function, including GCK, insulin, and GLUT2, are cells induced by 33.3 mmol/l glucose in Neo-transfected regulated by oxidative stress and stress-activated protein cells (Fig. 5C). kinase/c-Jun NH2-terminal kinase activation in obesity Reportedly, Bad phosphorylation is needed for the for- (30,31). Similar to this report, oxidative stress triggers p21 mation of a mitochondrially located complex, consisting induction, leading to suppression of GCK and insulin gene of GCK and associated with phosphorylated Bad, that expression in pancreatic cells (32,33), whose expression is enhances glycolysis and prevents apoptosis in liver cells inhibited by GCK overexpression. Furthermore, activation (15,16). Therefore, Bad phosphorylation may also play an of the c-Jun NH2-terminal kinase pathway may also affect important role in glucose metabolism and apoptosis of the activity and expression of GCK through pancreatic pancreatic ␤-cells by regulating the interaction of GCK duodenal homeobox 1, since stress-activated protein ki- with mitochondria. Supporting this hypothesis, Bad phos- nase/c-Jun NH2-terminal kinase activation decreases pan- phorylation was significantly increased and prolonged in creatic duodenal homeobox 1 activity and subsequent GCK-overexpressing cells than in Neo vector–transfected suppression of GCK transcription (34). Finally, induction

2610 DIABETES, VOL. 54, SEPTEMBER 2005 W.-H. KIM AND ASSOCIATES of glycation reactions by hyperglycemia has also been and glucokinase reside in a mitochondrial complex that integrates glycol- shown to suppress GCK gene expression (35,36), and ysis and apoptosis. Nature 424:952–956, 2003 16. Downward J: Cell biology: metabolism meets death. Nature 424:896–897, AMPK, although previously controversial, may be an addi- 2003 tional candidate factor in regulating GCK activity and 17. Majewski N, Nogueira V, Robey RB, Hay N: Akt inhibits apoptosis expression in pancreatic ␤-cells (37,38). Studies on the downstream of BID cleavage via a glucose-dependent mechanism involv- potential involvement of these signaling proteins are cur- ing mitochondrial hexokinases. Mol Cell Biol 24:730–740, 2004 rently under investigation. 18. Vyssokikh V, Zorova L, Zorov D, Heimlich G, Jurgensmeier J, Schreiner D, Finally, we provide novel insight into mechanisms con- Brdiczka D: The intra-mitochondrial cytochrome C distribution varies ␤ correlated to the formation of a complex between VDAC and the adenine tributing to chronic hyperglycemia-induced -cell apopto- nucleotide translocase: this affects Bax-dependent cytochrome C release. sis, showing that GCK plays an important role in regulating Biochimica Biophysica Acta 1644:27–36, 2004 apoptosis of pancreatic ␤-cells via competition with Bax to 19. Antonsson B, Montessuit S, Lauper S, Eskes R, Martinou JC: Bax oligomer- bind with VDAC and suggesting the essential role of GCK ization is required for channel-forming activity in liposomes and to trigger on glucose metabolism and cell apoptosis in ␤-cells. cytochrome c release from mitochondria. Biochem J 345:271–278, 2000 20. Majewski N, Nogueira V, Bhaskar P, Coy PE, Skeen JE, Gottlob K, Chandel NS, Thompson CB, Robey RB, Hay N: Hexokinase-mitochondria interac- ACKNOWLEDGMENTS tion mediated by Akt is required to inhibit apoptosis in the presence or This study was supported by research grants from the absence of Bax and Bak. Mol Cell 16:819–830, 2004 Korean National Institutes of Health (347-6111-211-207). 21. Pastorino JG, Shulga N, Hock JB: Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome C release and apoptosis. J Biol Chem We thank Dr. M.S. Lee for providing insulinoma cell 277:7610–7618, 2002 lines (MIN6N8 cells) and S.S. Kim for technical assistance 22. Chang IK, Cho NJ, Kim SS, Kim JY, Kim E, Woo JE, Nam JH, Kim SJ, Lee with isolated pancreatic islet cells. We also thank Dr. H.J. MS: Role of calcium in pancreatic islet cell death by IFN-gamma/TNF- Kim for providing ICR mice and Dr. Van-Anh Nguyen for alpha. J Immunol 172:7008–7014, 2004 peer reviewing the manuscript. 23. 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