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Originally published In Press as doi:10.1074/jbc.M601228200 on March 16, 2006

J. Biol. Chem., Vol. 281, Issue 19, 13015-13020, May 12, 2006
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Essential Role of Ubiquitin-Proteasome System in Normal Regulation of Insulin Secretion*

Miho Kawaguchi{ddagger}§1, Kohtaro Minami{ddagger}1, Kazuaki Nagashima||, and Susumu Seino{ddagger}§2

From the §Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan, the Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507, Japan, {ddagger}Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, and the ||Department of Diabetes and Clinical Nutrition, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan

Received for publication, February 8, 2006 , and in revised form, March 13, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin secretion from pancreatic beta-cells occurs by sequential cellular processes, including glucose metabolism, electrical activity, Ca2+ entry, and regulated exocytosis. Abnormalities in any of these functions can impair insulin secretion. In the present study, we demonstrate that inhibition of proteasome activity severely reduces insulin secretion in the mouse pancreatic beta-cell line MIN6-m9. Although no significant effects on glucose metabolism including ATP production were found in the presence of proteasome inhibitors, both glucose- and KCl-induced Ca2+ entry were drastically reduced. As Ca2+-ionophore-induced insulin secretion was unaffected by proteasome inhibition, a defect in Ca2+ entry through voltage-dependent calcium channels (VDCCs) is the likely cause of the impaired insulin secretion. We found that the pore-forming {alpha}-subunit of VDCCs undergoes ubiquitination, which does not decrease but slightly increases expression of the {alpha}-subunit protein at the plasma membrane. However, electrophysiological analysis revealed that treatment with proteasome inhibitors results in a severe reduction in VDCC activity in MIN6-m9 cells, indicating that VDCC function is suppressed by proteasome inhibition. Furthermore, insulin secretion in isolated mouse pancreatic islets was also decreased by proteasome inhibition. These results demonstrate that the ubiquitin-proteasome system plays a critical role in insulin secretion by maintaining normal function of VDCCs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin secretion from pancreatic beta-cells is regulated by a variety of extracellular stimuli and intracellular signals (1–3). Regulated exocytosis of insulin-containing secretory granules is triggered by a rise in the intracellular (cytosolic) Ca2+ concentration ([Ca2+]i)3 (3). In pancreatic beta-cells, elevation of [Ca2+]i occurs by release of Ca2+ from intracellular pools such as the endoplasmic reticulum (ER), mitochondria, and secretory granules and/or by influx of extracellular Ca2+ mainly through voltage-dependent calcium channels (VDCCs) (4, 5). However, insulin secretion is induced by many secretagogues including glucose, sulfonylureas, and glucose-dependent potentiators such as glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1, and pituitary adenylate cyclase-activating peptide, all of which depend critically on Ca2+ entering the pancreatic beta-cells through the VDCCs, as these secretions are abolished by specific VDCC channel blockers (4, 6, 7).

We previously established two different mouse pancreatic beta-cell sublines from MIN6 cells, which facilitate study of the mechanisms of impaired insulin secretion (8). One of them is MIN6-m9, a model of normal beta-cells having insulin secretory properties similar to those of pancreatic islets, and the other is MIN6-m14, which exhibits abnormalities in glucose metabolism, ATP-sensitive potassium (KATP) channels, and VDCCs that impair insulin secretion (8). In the course of comparing these sublines, we found that several genes involved in the ubiquitin-proteasome system are down-regulated in MIN6-m14 cells and that inhibition of proteasome activity impairs insulin secretion in MIN6-m9 cells (9). The ubiquitin-proteasome pathway is an energy (ATP)-dependent and well regulated system of proteolysis (10). Misfolded or otherwise unnecessary proteins are subjected to degradation in the ubiquitin-proteasome system. Many proteins and cellular functions are currently known to be regulated by the ubiquitin-proteasome system (10). Thus, it is likely that pathway(s) leading to insulin secretion are affected by the system.

Recently, it was reported that KATP channels undergo ER-associated degradation (ERAD) via the ubiquitin-proteasome system and that proteasome inhibition increases the channel number at the plasma membrane (11). However, that study did not investigate the effect of proteasome inhibition on insulin secretion. Functional expression of KATP channels is essential for normal insulin secretion, as they link glucose metabolism to insulin secretion in pancreatic beta-cells by depolarizing the plasma membrane and opening the VDCCs (12).

In the present study, we have identified the target molecules responsible for impaired insulin secretion because of proteasome inhibition. We found that both glucose- and high KCl-induced Ca2+ entry are severely suppressed in the presence of proteasome inhibitors and that VDCC activity is diminished by proteasome inhibition. These results demonstrate that the ubiquitin-proteasome system is critical in insulin secretion by maintaining normal function of VDCCs.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Treatment with Proteasome Inhibitors—MIN6-m9 cells were cultured in Dulbecco's modified Eagle's medium with 25 mM glucose supplemented with 10% (v/v) heat-inactivated fetal calf serum under humidified condition of 5% CO2, 95% air at 37 °C (8). Cells were exposed to proteasome inhibitors, MG-132 or epoxomicin (13, 14) (both from Peptide Institute, Osaka, Japan), for 6 h in culture medium. E-64-d (Peptide Institute), an inhibitor of cysteine proteases that does not inhibit proteasome (15), was used as a negative control. Because these inhibitors were dissolved with dimethyl sulfoxide (Me2SO), all culture media including control condition (without inhibitors) contained Me2SO at 0.1% in final concentration.

Measurement of Insulin Secretion—Cells (1 x 105 cells/well) were pre-exposed to proteasome inhibitors for 6 h. The cells then were washed with 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 10 mM HEPES, pH 7.4) containing 0.1% BSA (BSA-KRH) and preincubated for 30 min in the same buffer containing 1 mM glucose. Incubation was performed with various concentrations of glucose or other stimuli as indicated for 1 h at 37°C. A calcium ionophore, A23187 [GenBank] (10 µM; Nacalai Tesque, Kyoto, Japan), was added to the incubation buffer in some experiments. Released and intracellular insulin were quantified as described (8). The amount of insulin secretion was normalized by cellular insulin content or total protein content.

Measurement of Glucose Utilization—Glucose utilization was measured according to the method described by Ishihara et al. (16). In brief, cells were incubated for 1 h at 37°C in 500 µl of BSA-KRH with various concentrations of glucose (1, 10, 25 mM) containing 5 and 20 µCi/ml [5-3H]glucose for the lower (1 mM) and higher (10, 25 mM) glucose concentration, respectively. After incubation, 100 µl of incubation buffer was mixed with 20 µl of 1 N HCl in a microcentrifuge tube. The tubes were placed in 22-ml plastic scintillation vials containing 600 µlof distilled water to allow [3H]H2O in the tubes to equilibrate with the water in the vials. After a 36-h incubation at 37 °C, 10 ml of ACS II scintillation fluid (Amersham Biosciences) was added to the vials. The rate of glucose utilization was calculated as reported (16).

Determination of Cellular ATP Content—For measurement of ATP content, cells were incubated for 1 h in the presence or absence of 25 mM glucose. The cells then were washed twice with ice-cold phosphate-buffered saline and solubilized. The amount of ATP was measured with ATP bioluminescent assay kit (Roche Diagnostics), according to the manufacturer's instruction.

Measurement of [Ca2+]i—Cells were loaded with 5 µM Fura-2 acetoxymethyl ester (Fura-2 AM) (Dojindo, Kumamoto, Japan) for 1 h in BSA-KRH. [Ca2+]i was measured by dual excitation wavelength method (340/380 nm) with a fluorometer (Fluoroskan Ascent CF, Labsystems, Helsinki, Finland) (17). [Ca2+]i was calibrated using solutions containing known Ca2+ concentrations (Molecular Probes, Eugene, OR).

Construction of pCMV-{alpha}1C-FLAG and pCIneo-beta3—cDNA for rabbit {alpha}1C (Cav1.2)-subunit of VDCC (18) was introduced into pCMV-Tag4 plasmid containing C-terminal FLAG epitope tag (Stratagene, La Jolla, CA). pCIneo-beta3 was constructed with pCIneo (Promega, Madison, WI) and the rat beta3-subunit of VDCC (18).

Immunoprecipitation and Immunoblotting—Cells were co-transfected with pCMV-{alpha}1C-FLAG and pCIneo-beta3 using Lipofectamine 2000 (Invitrogen) and cultured for 2 days. After exposure to proteasome inhibitors for 6 h, the cells were harvested and sonicated in a lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100) containing phenylmethylsulfonyl fluoride and protease inhibitor mixture (Nacalai Tesque) and centrifuged at 20,400 x g for 10 min. Protein concentration was determined with Bio-Rad protein assay (Bio-Rad Laboratories). To collect FLAG-tagged protein, the cell lysate (750 µg of protein) was added to 40 µl of anti-FLAG M2-agarose (Sigma) to a total volume of 1 ml, and the reaction was gently rotated overnight at 4 °C. After washing three times with Tris-buffered saline, proteins were eluted in 30 µl of sample buffer for SDS-PAGE. The eluted proteins were subjected to SDS-PAGE (7.5%), and resolved proteins were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA) and probed with anti-ubiquitin antibody FK1 or FK2 (Nippon Bio-Test Laboratories, Tokyo, Japan). Secondary antibodies used were conjugated to horseradish peroxidase, and proteins were visualized by enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Santa Cruz, CA).

Alternatively, ubiquitinated proteins were collected by Ubiquitinated Protein Enrichment Kit (EMD Biosciences, San Diego, CA) and followed by immunoblotting with anti-Cav1.2 antibody (Alomone, Jerusalem, Israel). Expression of the cellular Cav1.2-subunit was detected in both intact and transfected cell lysates.

Subcellular Fractionation—Cells were scraped into a lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, and 1 mM dithiothreitol with protease inhibitors) and sonicated. The 700 x g pellet, which contained nuclei and undisrupted cells, was discarded. The supernatant was centrifuged at 8,000 x g for 15 min. The resultant pellet (plasma membrane-enriched fraction) was resuspended in the same volume of lysis buffer with 1% Triton X-100 (17).

Biotinylation of Cell Surface Proteins—Biotinylation and collection of cell surface proteins was carried out using a cell surface protein biotinylation and purification kit (Pierce). Immunoblot analysis was performed with anti-Cav1.2 antibody (Alomone). Na+/K+-ATPase detected with anti-Na+/K+-ATPase antibody (Upstate%20Biotechnology">Upstate Biotechnology, Lake Placid, NY) was used as a marker for cell surface protein. Anti-p44/42 mitogen-activated protein kinase (MAPK) antibody (Cell Signaling Technology, Danvers, MA) was used to confirm the absence of intracellular proteins in the biotinylated protein fraction.

Electrophysiology—Whole cell VDCC currents were recorded as described previously (8, 18). 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, 110 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, 10 mM MOPS, and 5 mM Mg-ATP, pH 7.2. The holding potential was –70 mV, and test pulses of 400 ms at potentials between –40 and +60 mV in steps of 10 mV were applied every 4 s. Recordings were performed using Axopatch 200B amplifier (Axon Instruments, Foster City, CA) at 37 °C. For normalization, the currents were divided by the membrane capacitance measured for each cell.

Islet Isolation and Batch Incubation—Mouse pancreatic islets were isolated by collagenase digestion followed by Ficoll density gradient centrifugation. Isolated pancreatic islets were exposed to 1 µM MG-132 or 0.1 µM epoxomicin for 24 h in RPMI 1640 medium containing 10% fetal calf serum. Islets were then washed and preincubated at 37 °C for 30 min in BSA-KRH with 2.8 mM glucose. The islets were incubated in the buffer containing various stimuli for 30 min. Cellular insulin was extracted with acid-ethanol.

Statistical Analysis—Values are expressed as means ± S.E. The significance of differences between test groups was evaluated by use of paired Student's t test or multiple analysis of Tukey-Kramer's test. p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Impairment of Glucose-induced Insulin Secretion by Proteasome Inhibition—Our preliminary data indicated that inhibition of proteasome activity impairs insulin secretion in mouse pancreatic beta-cell line MIN6-m9 (9). In the present study, we examined the effects of proteasome inhibition in MIN6-m9 cells in detail. Proteasome activity was blocked by either MG-132 (13) or epoxomicin (14), both of which are well established proteasome inhibitors. Optimum duration of exposure and concentrations of these inhibitors were determined by preliminary experiments (data not shown). When MIN6-m9 cells were exposed to 1 µM MG-132 for 6 h, glucose-induced insulin secretion was markedly reduced (Fig. 1A). The other proteasome inhibitor, epoxomicin at 0.1 µM, exerted similar effects (Fig. 1A). E-64-d, a thiol protease inhibitor that does not suppress proteasome activity (15), showed no significant effects on insulin secretion in MIN6-m9 cells, even when an excessive concentration of the agent (100 µM) was applied (Fig. 1A). These data demonstrate that inhibition of proteasome activity causes impairment of glucose-induced insulin secretion in pancreatic beta-cells.


Figure 1
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  		FIGURE 1.
Effects of proteasome inhibitors on glucose-induced insulin secretion (A) and insulin content (B). Cells were pretreated with 0.1% Me2SO (Con), 1 µM MG-132 (MG), 0.1 µM epoxomicin (Epx), or 100 µM E-64-d for 6 h. A, secreted insulin accumulated in the incubation buffer was measured after a 1-h stimulation of indicated concentrations of glucose. Data are the means ± S.E. (n = 11–12). B, intracellular insulin was extracted by acid-ethanol. Vertical axes represent relative levels of cellular insulin content when the level of control condition is 100. Data are the means ± S.E. (n = 3–6). **, p < 0.01 versus control; NS, not significant.

 
Cellular insulin content also was influenced by proteasome inhibition. When MIN6-m9 cells were exposed to either MG-132 or epoxomicin, the insulin content of the cells was reduced to ~70% of the levels in the control condition (Fig. 1B). However, the drastic suppression of insulin secretion by proteasome inhibitors cannot be explained by the moderate reduction of insulin content. Thus, these results indicate that inhibition of proteasome activity in pancreatic beta-cells impairs cellular functions that lead to exocytosis of insulin-containing secretory granules.

Effects of Proteasome Inhibitors on Glucose Utilization and Intracellular ATP Contents—In pancreatic beta-cells, glucose is taken up by specific transporters and metabolized to generate ATP, leading to closure of the KATP channels and activation of VDCCs (10). As a result, Ca2+ entry through the VDCCs triggers exocytosis of insulin-containing granules. Accordingly, we evaluated the effects of proteasome inhibition on glucose utilization and intracellular ATP contents after glucose loading.

As shown in Fig. 2A, the rate of glucose utilization was unaltered by the presence of MG-132 or epoxomicin at any of the glucose concentrations examined (1, 10, and 25 mM), indicating that proteasome inhibition does not exert a significant effect on glycolytic pathways in MIN6-m9 cells. We then measured intracellular ATP contents after 25 mM glucose stimulation in the presence or absence of proteasome inhibitors. Neither MG-132 nor epoxomicin affected intracellular ATP contents (Fig. 2B), suggesting that proteasome activity is not required for glucose-stimulated ATP production in MIN6-m9 cells. These data indicate that the reduction of glucose-induced insulin secretion by proteasome inhibition in MIN6-m9 cells is not caused by impairment of glucose metabolism and suggest that the major defects lie in steps after glucose metabolism in the insulin secretion signaling pathway.

Defect in Elevation of [Ca2+]i through VDCCs—To bypass glucose metabolism, glibenclamide and a high concentration of KCl (30 mM) were used to stimulate insulin secretion. Glibenclamide is the sulfonylurea widely used in treatment of type 2 diabetes, which binds to SUR1, a regulatory subunit of the KATP channels in pancreatic beta-cells and depolarizes the beta-cell membrane (19). Although insulin secretion was markedly increased by 100 nM glibenclamide in the absence of proteasome inhibitors, pretreatment with MG-132 or epoxomicin nearly abolished the stimulation in MIN6-m9 cells (Fig. 3A). A high concentration of KCl directly depolarizes the beta-cell membrane and activates the VDCCs, which allows Ca2+ influx. Insulin secretion was remarkably stimulated by the depolarizing concentration of KCl under control condition but was significantly reduced in the presence of proteasome inhibitors (Fig. 3A). These results suggest that Ca2+ influx through VDCCs is impaired by proteasome inhibition.


Figure 2
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  		FIGURE 2.
Effects of proteasome inhibitors on glucose utilization (A) and ATP production (B). Cells were pretreated with 0.1% Me2SO (Con), 1 µM MG-132 (MG), or 0.1 µM epoxomicin (Epx) for 6 h. A, glucose utilization was measured by following the conversion of [5-3H]glucose into [3H]H2O. Data are the means ± S.E. (n = 3). B, ATP content was measured after a 1-h stimulation by 25 mM glucose. Data are the means ± S.E. (n = 3–4). NS, difference from control value is not significant.

 


Figure 3
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  		FIGURE 3.
Impairment of Ca2+ entry by proteasome inhibition. Cells were pretreated with 0.1% Me2SO (Con), 1 µM MG-132 (MG), or 0.1 µM epoxomicin (Epx) for 6 h. A, insulin secretion was stimulated by 100 nM glibenclamide (Glib) or 30 mM KCl. Data are the means ± S.E. (n = 12–20). B, the cells were loaded with 5 µM Fura-2 AM, and [Ca2+]i was measured in BSA-KRH. Data are the means ± S.E. (n = 3). C, insulin secretion was induced by an application of a Ca2+-ionophore A23187 [GenBank] (10 µM). Data are the means ± S.E. (n = 5–8). **, p < 0.01 versus control; NS, not significant.

 
To confirm this, we measured changes in [Ca2+]i in response to KCl. In the presence of proteasome inhibitors, the KCl-induced rise in [Ca2+]i was significantly suppressed compared with that in the absence of inhibitors (Fig. 3B). The glucose-induced increase in [Ca2+]i also was reduced by proteasome inhibition (data not shown). These findings demonstrate that Ca2+ entry through VDCCs is impaired by proteasome inhibition in MIN6-m9 cells.

We then investigated to determine whether a rise in [Ca2+]i when VDCCs are closed causes insulin secretion in the presence of proteasome inhibitors. We utilized A23187 [GenBank] , a specific membrane carrier ionophore for divalent cations, which allows Ca2+ entry without the channels being open (20). When MIN6-m9 cells were treated with A23187 [GenBank] , insulin secretion was increased at a low glucose concentration (3 mM) (Fig. 3C). A23187 [GenBank] also increased insulin secretion to the same extent from cells exposed to proteasome inhibitors (Fig. 3C). These results suggest that the Ca2+-responsive exocytotic machinery of insulin-containing granules is not severely impaired, if at all, by proteasome inhibition. Considered together, these findings indicate that the inhibition of proteasome activity in MIN6-m9 cells affects VDCC function.


Figure 4
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  		FIGURE 4.
Ubiquitination of {alpha}-subunit of VDCCs. Cells were transfected with pCMV-{alpha}1C-FLAG and pCIneo-beta3, and cultured for 2 days. The cells then were pretreated with 0.1% Me2SO (Con), 1 µM MG-132 (MG), or 0.1 µM epoxomicin (Epx) for 6 h. Cell lysates were immunoprecipitated (IP) with anti-FLAG M2 antibody or anti-ubiquitin (anti-Ub) antibody and subjected to SDS-PAGE. Immunoblotting (IB) was performed with anti-ubiquitin antibodies (FK1 or FK2) or anti-Cav1.2 antibody.

 
Ubiquitination of VDCCs—Proteasome inhibition can result in accumulation of ubiquitinated proteins. Many of the transporters and channel proteins are ubiquitinated, and, in some cases, subcellular localization and function are regulated by ubiquitination (21). Although the pore-forming subunits of voltage-dependent sodium channels, the structures of which are closely related to VDCCs, are known to undergo ubiquitination (22), ubiquitination of VDCCs has not been reported. Accordingly, we examined the ubiquitination of {alpha}-subunits of VDCCs. MIN6-m9 cells were transfected with FLAG-tagged {alpha}-subunit of VDCC (fCav1.2) together with beta3 subunit (Cabeta3), and cell lysates were subjected to immunoprecipitation by anti-FLAG antibody, followed by immunoblotting with anti-ubiquitin antibodies (FK1 and FK2). FK1 recognizes only polyubiquitinated proteins, whereas FK2 recognizes both mono- and polyubiquitinated proteins (23). As shown in Fig. 4, ubiquitinated forms of fCav1.2 were detected at very low levels in the absence of proteasome inhibitors. In the presence of proteasome inhibitors, the accumulation of ubiquitinated fCav1.2 protein was clearly detected by both antibodies (Fig. 4). To further confirm ubiquitination of Cav1.2, we immunoprecipitated polyubiquitinated proteins of the cell lysates with polyubiquitin affinity beads, followed by immunoblotting with anti-Cav1.2 antibody. Signals of fCav1.2 were detected by proteasome inhibition (Fig. 4). These results demonstrate that the {alpha}-subunits of VDCCs undergo polyubiquitination.

Change in Quantity of VDCCs—The loss of Ca2+ entry might be because of either reduction in the quantity of the VDCCs (total or at plasma membrane) or the quality (intrinsic activity) of the channels. We first evaluated the total amount of {alpha}-subunits of VDCCs. Whole cell lysates were subjected to immunoblotting with anti-Cav1.2 antibody. In both intact and transfected conditions, Cav1.2 protein signal intensity was not reduced but rather slightly increased by proteasome inhibition (Fig. 5A), probably because of a decrease in their degradation.

We then examined the effects of proteasome inhibition on cell surface expression of VDCCs. Two different experiments were conducted to detect VDCCs in the plasma membrane. Subcellular fractionation by serial centrifugation (17) revealed a slight increase rather than a reduction in plasma membrane expression of VDCCs by proteasome inhibitors (Fig. 5A). In addition, we specifically labeled cell surface proteins with biotin, and the biotinylated proteins were collected using streptavidin-conjugated agarose beads. We confirmed that pretreatment with proteasome inhibitors did not cause a reduction but did cause a slight increase in {alpha}-subunit protein levels in the cell surface protein fraction of MIN6-m9 cells (Fig. 5B). These results indicate that the loss of Ca2+ entry is not because of reduction in quantity of VDCCs.


Figure 5
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  		FIGURE 5.
Changes in quantity of VDCCs. A, cells were transfected with or without pCMV-{alpha}1C-FLAG and pCIneo-beta3, and cultured for 2 days. The cells then were pretreated with 0.1% Me2SO (Con), 1 µM MG-132 (MG), or 0.1 µM epoxomicin (Epx) for 6 h. Proteins from total cell lysates were separated by SDS-PAGE and immunoblotted (IB) with anti-Cav1.2 antibody. B, subcellular fractionation was performed by the serial centrifugation method, and the plasma membrane (PM) fraction was subjected to immunoblotting (top panel). Cell surface proteins were biotinylated and collected to perform immunoblotting for Cav1.2 as described under "Experimental Procedures" (bottom panel).

 


Figure 6
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  		FIGURE 6.
Effects of proteasome inhibitors on VDCC activity. A, current-voltage relationships of VDCCs in MIN6-m9 cells are shown. VDCC currents with or without preexposure to proteasome inhibitors were recorded (n = 10–15). The currents were normalized by membrane capacitance measured for each cell. B, relative activities of VDCCs are shown as % of control. The normalized peak VDCC current without proteasome inhibition represents 100%. **p < 0.01 versus control. Con, Me2SO; MG, MG-132; Epx, epoxomicin.

 
Impairment of VDCC Activity by Proteasome Inhibition—We then evaluated activity of the VDCCs using patch clamp technique. The current-voltage relationships of VDCCs were recorded in the presence or absence of proteasome inhibitors. VDCC currents were drastically decreased by proteasome inhibition (Fig. 6A). The peak currents of VDCCs in MIN6-m9 cells exposed to proteasome inhibitors were significantly lower than in control cells (Fig. 6B). These data demonstrate that inhibition of proteasome activity in MIN6-m9 cells results in a loss of VDCC activity that results in impaired insulin secretion.

Effect of Proteasome Inhibition on Insulin Secretion in Isolated Islets—Isolated mouse pancreatic islets were used to determine whether proteasome activity also is involved in insulin secretion in normal pancreatic beta-cells. Although cellular insulin content was decreased only slightly (Fig. 7A), both glucose- and high K+-induced insulin secretions were significantly suppressed by proteasome inhibition (Fig. 7B). A23187 [GenBank] -induced insulin secretion was not affected by proteasome inhibition (Fig. 7B), as found in MIN6-m9 cells. These results indicate that the proteasome system is essential for normal regulation of insulin secretion in primary beta-cells as well as in the beta-cell line MIN6-m9.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we show that inhibition of proteasome activity causes a marked reduction of insulin secretion in pancreatic beta-cells. Although it was previously found that glucose-stimulated proinsulin biosynthesis is reduced by proteasome inhibition (24), no studies have been reported concerning the roles of the proteasome system in signaling pathways of insulin secretion. As proper insulin secretory response requires multiple cellular functions including glucose-sensing, ATP production, metabolism-electrical activity coupling, and regulated exocytosis (3, 12, 25, 26); dysfunction in any of these processes might impair insulin secretion. It is well known that mutations in glucokinase, an enzyme having a critical role in glucose-sensing and metabolism in pancreatic beta-cells, affects insulin secretion and causes a form of maturity-onset diabetes of the young (MODY2) (27) and severe persistent hyperinsulinemic hypoglycemia (28). Mutations in the subunits of the KATP channels, which couple cell metabolism to electrical activity, also are known to perturb glucose-induced insulin secretion, most severely in persistent hyperinsulinemic hypoglycemia of infancy and permanent neonatal diabetes mellitus (29–31). It recently has been reported that glucose-induced insulin secretion from pancreatic islets is diminished in ashen mice, which have mutations in a molecule that participates in insulin granule exocytosis (32). We attempted to identify the cellular function affected by proteasome inhibition and found no severe abnormalities in glucose metabolism, including ATP production, but did find drastic suppression of Ca2+ influx after stimulation by glucose or high KCl. Because Ca2+-induced insulin secretion of proteasome inhibitor-treated beta-cells by Ca2+-ionophre is similar to that of intact cells, a defect in Ca2+ entry through VDCCs may well be a primary factor in the impairment of insulin secretion by proteasome inhibition.


Figure 7
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  		FIGURE 7.
Effects of proteasome inhibition on insulin content (A) and insulin secretion (B) in isolated mouse pancreatic islets. Isolated pancreatic islets were pretreated with 0.1% Me2SO (Con), 1 µM MG-132 (MG), or 0.1 µM epoxomicin (Epx) for 24 h. A, intracellular insulin was extracted by acid-ethanol. Data are the means ± S.E. (n = 4–8). B, secreted insulin accumulated in the incubation buffer was measured after a 30-min stimulation of indicated concentrations of glucose, KCl, or A23187 [GenBank] . Data are the means ± S.E. (n = 4–14). *, p < 0.05; **, p < 0.01 versus control; NS, not significant.

 
We found that the VDCCs are ubiquitinated in pancreatic beta-cells. The function of a number of receptors, transporters, and channels are known to be affected by ubiquitination (21, 33). In most cases, ubiquitination of these plasma membrane proteins leads to their internalization and degradation (21, 33). Nedd4 family proteins have a critical role in such plasma membrane protein ubiquitination (21). It is well known that cell surface expression of the epithelial sodium channel (ENaC) is regulated by Nedd4-mediated ubiquitination (34). The interaction between Nedd4 and ENaC is mediated by WW domains of the Nedd4 and PY motif of the ENaC (34). It has recently been shown that voltage-dependent sodium channels also contain the PY motif and that their expression at the plasma membrane is regulated by Nedd4-dependent ubiquitination (22). Despite the structural similarity to voltage-dependent sodium channels, VDCCs do not contain the typical PY motif. Accordingly, although the {alpha}-subunits of VDCCs are ubiquitinated, the mechanism may differ from that in voltage-dependent sodium channels.

There are several possible explanations for VDCC dysfunction without loss of its expression at the plasma membrane: 1) ubiquitination of {alpha}-subunits of VDCCs at the plasma membrane directly reduces channel activity, 2) ubiquitination and/or accumulation of certain molecules that interact with VDCCs to regulate their activity diminish channel activity, and 3) escape from ERAD of misfolded or unassembled VDCCs results in accumulation of non-functional channels at the plasma membrane. Protein ubiquitination is accomplished by covalent binding of the C-terminal glycine of ubiquitin to the [cepsilon]-amino group of a lysine residue on a target protein (10). Voltage-dependent ion channels contain a voltage sensor in which positively charged amino acids (lysine or arginine) exert a critical role (35). This raises possibility that ubiquitination of the lysine residues in the voltage sensor impairs sensing of membrane potential, leading to failure in the voltage-dependent activation of VDCCs. However, this is unlikely, because these residues are located in the transmembrane segment in which ubiquitination rarely occurs.

The function of VDCCs is modulated by a variety of cellular signals, including G proteins (36), protein kinase A (37), and protein kinase C (37). Thus, it is possible that proteasome inhibition, and accumulation of ubiquitinated forms of these regulatory proteins affects VDCC activity. In addition to these well known VDCC regulatory molecules, a recent study by Wei et al. (38) showed inhibitory regulation of VDCCs by cyclin-dependent kinase 5 (Cdk5). Because Cdk5 is activated by interaction with p35 (39), a short lived protein that is degraded in the ubiquitin-proteasome pathway (40), proteasome inhibition might increase the cellular p35 level and lead to sustained activation of Cdk5, resulting in a reduction of VDCC activity. If this is the case, treatment of MIN6-m9 cells with olomoucine, a Cdk5 inhibitor (41), could mask the inhibitory effects of proteasome inhibition on VDCC activity and insulin secretion. However, proteasome inhibition was found not to increase p35 protein in MIN6-m9 cells, and olomoucine treatment was unable to restore the inhibitory effects of proteasome inhibition (data not shown). The contribution of ubiquitination of other regulatory molecules to VDCC activity remains to be investigated.

Membrane-spanning proteins including ion channels are synthesized in the ER, where nascent protein subunits are folded, assembled, and sorted (42). Misfolded or unassembled proteins are targeted for proteolysis by the ERAD, a quality control system for proofreading newly synthesized proteins in which the ubiquitin-proteasome pathway is involved (42). As a considerable portion of newly synthesized proteins fail to mature properly, this system is especially important for the fidelity of cellular functions (42). However, misfolded membrane proteins occasionally escape the ERAD and reach the plasma membrane (43), and this phenomenon is increased by proteasome inhibition. Ideally, in intact beta-cells, misfolded or unassembled VDCCs are subjected to the ERAD, and only correctly folded and assembled functional channels are transported to the plasma membrane. By contrast, in the presence of proteasome inhibitors, misfolded or unassembled channels escape the quality control checkpoint, resulting in an increase in the proportion of nonfunctional VDCCs at the plasma membrane. A recent study by Yan et al. (11) demonstrated that beta-cell KATP channels undergo ERAD via the ubiquitin-proteasome system. Proteasome inhibition was found to increase plasma membrane expression of KATP channels by 30–40%, as in the VDCCs in the present study. However, in contrast to VDCCs, KATP channel activity is increased in proportion to the increment of surface expression. This difference could arise from the presence of the RKR-ER retention motif in both SUR1 and Kir6.2 subunits of KATP channel (44), which prevents surface expression of unassembled channel subunits. Although ER retention determinants also may be present in VDCCs (45), the molecular basis of assembly and trafficking of VDCCs is largely unknown. However, it is possible that ER retention signals in subunit proteins of VDCCs are not strong enough to prevent trafficking of mis-folded or unassembled proteins as defective channels accumulate by proteasome inhibition. In conclusion, the present study demonstrates that the ubiquitin-proteasome system is critical in normal regulation of insulin secretion and that it participates in quality control of the VDCCs in pancreatic beta-cells.


   FOOTNOTES
 
* This work was supported by grant-in-aid for Specially Promoted Research and a grant for 21st Century Center of Excellence program from the Ministry of Education, Culture, Sports, Science and Technology. 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. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan. Tel.: 81-78-382-5360; Fax: 81-78-382-5370; E-mail: seino{at}med.kobe-u.ac.jp.

3 The abbreviations used are: [Ca2+]i, intracellular Ca2+ concentration; ER, endoplasmic reticulum; VDCC, voltage-dependent calcium channel; KATP, ATP-sensitive potassium; ERAD, ER-associated degradation; MOPS, 3-(N-morpholino)propanesulfonic acid; ENaC, epithelial sodium channel; Cdk5, cyclin-dependent kinase 5; BSA, bovine serum albumin. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Tadao Shibasaki and Takashi Miki for helpful suggestions in this study.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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