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Department of Endocrinology (Q.H., Z.G., L.Y., B.L., Z.F.), Changhai Hospital; Department of Physiology (S.B.), Basic Medicine College; Department of Pathology (Y.Y.), Changhai Hospital; and Department of Histology and Embryology (S.L., F.W.), Basic Medicine College, Second Military Medical University, Shanghai, 200433, People’s Republic of China; and Department of Cell Biology and Anatomy (G.G., M.B.), Medical University of South Carolina, Charleston, South Carolina 29425
Address all correspondence and requests for reprints to: Qin Huang, Department of Endocrinology, Changhai Hospital, 174 Changhai Road, Shanghai 200433, People’s Republic of China. E-mail: qinhuang20{at}yahoo.com.cn or qinhuang{at}smmu.edu.cn.
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Abstract |
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Introduction |
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A number of potential mechanisms for increased ß-cell apoptosis in type 2 diabetes have been postulated; an important one is the chronically stimulating action of hyperglycemia (5, 6). Increase in glucose levels is a principal regulator of pancreatic ß-cell function. Moreover, sulfonylurea tolbutamide, a current drug for treating type 2 diabetes, triggers apoptosis in pancreatic ß-cells (7). Although these changes result from chronic hyperglycemia and the exact mechanism behind this process is not understood, the progressive increase in ß-cell apoptosis, which is due to ß-cell dysfunction and/or a reduction in ß-cell mass, is a prerequisite for development of type 2 diabetes. Thus, elucidating the mechanisms of apoptosis and its modulation are important in understanding the molecular mechanisms underlying diabetes.
Interestingly, high glucose-induced rat blastocyst apoptosis is characterized by a decrease of Bcl-2 expression (8). The Bcl-2 gene family is widely accepted as regulators of cell death, and Bax and Bcl-2 are considered dominant regulators of apoptosis. It has been shown that the Bcl-2 proteins promote cell survival by inhibiting the process of apoptosis, and the ratio of Bcl-2 to Bax is critical in determining susceptibility to apoptosis (9, 10). Bcl-2 overexpression is associated with resistance to apoptosis induced by toxic stimuli such as chemotherapy growth factor withdrawal (11, 12). p38 MAPK is also important for programed cell death (13, 14). Studies have demonstrated that p38 MAPK can be activated by growth factors, leading to the induction of growth-promoting genes (14, 15). In cardiomyocytes, p38ß isozyme was shown to exert hypertrophic action and mediate IGF-I induction of Bcl-2 promoter (16), whereas p38
induced apoptosis (17).
Diazoxide, a known opener of KATP channels, has been shown to be associated with preventing the development of diabetes (2, 18) and could inhibit the pancreatic ß-cell death triggered by high glucose or tolbutamide in vitro (7). However, the in vivo mechanisms and antiapoptotic effects of diazoxide have not been well studied. Theoretically, if decreased apoptosis could be achieved without inducing hyperglycemia, the onset and development of diabetes would be prevented. To test this hypothesis, diazoxide was administered to Otsuka Long Evans Tokushima Fatty (OLETF) rats, an established animal model of human type 2 diabetes. OLETF rats exhibit a late onset of chronic and slowly progressive hyperglycemia. Overt diabetes develops at 20 to 30 wk of age (19, 20). This study, including in vivo and in vitro parts, examined the effect of diazoxide administration at continuous and discontinuous dosing protocols on pancreatic ß-cell apoptosis and its mechanism, glucose tolerance, and insulin secretion function in OLETF rats from an early age to prediabetic and diabetic stages.
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Materials and Methods |
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Experimental protocols
A total of 31 OLETF rats were divided randomly into two groups at 8 wk of age to receive diazoxide (Sigma, St. Louis, MO; n = 19; 25 mg·kg–1·d–1), or saline (n = 12; 2 ml·d–1 for 8 wk) once daily at 1000 h. At the same time, 12 LETO rats received saline (2 ml·d–1) for 8 wk as controls. Diazoxide was administered ip in a dose volume of 2 ml per rat according to the following dosing regimens: 1) continuous dosing group (n = 7), in which rats received diazoxide each day from 8 wk of age until they were killed; and 2) discontinuous dosing group (n = 12), in which rats only received diazoxide each day from 8 to 16 wk of age. If diabetes was diagnosed in the vehicle group, rats were allocated randomly into two experimental groups (n = 3) and received diazoxide or saline, respectively, once daily for 2 wk. Body weight was recorded once weekly. Qualitative glycosuria and ketonuria (Chem strip uG 5000 K; Roche Molecular Biochemicals, Indianapolis, IN) were recorded every week from 20 wk of age.
Oral glucose tolerance test (OGTT)
OGTT was performed every 4 wk from 8 wk of age in all nonglycosuric rats. Rats were fasted overnight and administered 2 g/kg glucose orally by gavage. Blood samples were collected from the retro-orbital sinus using heparinized capillary tubes without anesthesia at identical time points before and after dosing. Blood glucose and insulin were measured using a Surestep monitoring system (LifeScan, Milpitas, CA), and Sensitive Rat Insulin RIA kit (LINCO Research, Inc., St. Charles, MO), respectively. The animals showing a peak blood glucose level of 300 mg/dl or higher and a level of 200 mg/dl or higher at 120 min during OGTT were regarded as diabetic.
Insulin secretion test
A stimulated insulin secretion test was performed by iv administration of the secretagogue, arginine (0.5 g/kg, L-arginine hydrochloride; Sigma) in the lateral tail vein of the rat as previously described (14).
Pancreatic morphological techniques
OLETF rats with or without therapy and LETO rats were killed using an ip injection of pentobarbital sodium (40 mg/kg) at 8, 16, and 24 wk of age or at the onset of diabetes. The remaining rats were killed at 40 wk of age. Pancreatic tissue was collected, paraffin embedded, and sectioned. Sections were stained with hematoxylin and eosin using a standard protocol. To detect insulin, sections were incubated with a guinea pig anti-insulin polyclonal antibody (Zymed Laboratories Inc., South San Francisco, CA) followed by the avidin-biotin peroxidase complex method of visualization. Apoptotic ß-cells in islets were stained using the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) method according to the manufacturer’s instructions (Roche Molecular Biochemicals, Mannheim, Germany) as previously described (21). An average of 50 islet cells were examined for each sample, and the scoring was done in a blinded fashion. The numbers of apoptotic ß-cells were counted and characterized as the apoptotic index (TUNEL positive ß-cells/islet) (21).
Measurement of ß-cell mass
ß-cell mass (in milligrams) was measured using the method previously described by Suzuki et al. (22). The areas of the ß-cells were determined by Cell Slide Audit Diagnostic System (Audit Diagnostics, Carrigtwohill, Ireland). All observations were made by one person (Y.Y.) to ensure consistency.
Islet isolation and cell culture
Pancreatic islets were isolated and cultured as previously described (23, 24). Purity was greater than 95%, as judged by dithizone staining. Islets were cultured in CMRL 1066 (supplemented with 10% fetal calf serum, 5.5 mmol/liter glucose, 100 U/ml penicillin, and 100 µg/ml streptomycin) medium at 37 C (95% air and 5% CO2). Two days after plating, when most islets were attached and began to flatten, the medium was changed to culture medium that contained high (20 mmol/liter) glucose. In some experiments, islets were additionally cultured with 50 µM diazoxide, 1 µM SB 203580 (Alexis Corp., Nottingham, UK), and 100 µM 5-hydroxydecanoate (5-HD) purchased from Research Biochemicals International (Natick, MA), or with diazoxide with SB 203580 or 5-HD.
Western blotting assay
Proteins were isolated on iced sodium dodecyl sulfate (SDS), Nonidet P40 lysis buffer containing protease inhibitors (2 µmol/liter complete; Roche Molecular Biochemicals; Indianapolis, IN). Protein content was determined using the Pierce BCA kit (Pierce Chemical Co., Rockford, IL). Equal amounts of protein (50 µg) were fractionated on 10% SDS-polyacrylamide gels (Invitrogen, Carlsbad, CA) and electroblotted to a nitrocellulose membrane (Invitrogen). Membranes were blocked for 2 h at room temperature with 5% nonfat milk in TBS (25 mmol/liter Tris, 137 mmol/liter NaCl, and 2.7 mmol/liter KCl).
Immunoblotting was performed as previously described (25) using monoclonal antiactin clone AC-40 (1:1000 dilution; Sigma); Bax (N-20) and Bcl-2 (N-19) polyclonal antibodies (1:200 dilution each; Santa Cruz Biotechnology, Santa Cruz, CA); phospho-p38, phospho-p38ß, p38, phospho-c-jun N-terminal kinase (JNK), JNK, phospho-ERK and ERK antibodies (1:1000 dilution each; New England Biolabs, Ipswich, MA) at 4 C overnight. After washing, the blots were incubated for 1 h with appropriate horseradish peroxidase-conjugated antibodies, and detected using ECL Plus (Amersham Pharmacia Biotech, Piscataway, NJ).
DNA fragmentation assay
Genomic DNA was extracted from pancreatic tissues and primary culture cells. DNA fragmentation was assayed by agarose gel electrophoresis. Frozen tissues were pulverized in liquid nitrogen and lysed with 300 µl of lysis buffer [50 mmol/liter Tris-HCl (pH 7.8), 100 mmol/liter EDTA, and 0.1% SDS). Lysates were treated with 0.5 mg/ml proteinase K and incubated at 37 C for 1 h. RNase A (1 mg/ml) was added, and the samples were incubated at 65 C for 15 min, while cells were collected by centrifugation and pellet was resuspended in 20 µl of sterile 10 mM EDTA, 50 mM Tris-HCl (pH 8.0), 0.5% (w/v) natrium lauryl sarcosinate, and 0.5 mg/ml proteinase K and incubated at 37 C for 1 h. RNase A (1 mg/ml) was added, and the sample was incubated at 65 C for 15 min. DNA was extracted by phenol-chloroform and electrophoresed on a 1.2% agarose gel in the presence of 0.1 µg/ml ethidium bromide at 80 V for 2–4 h. DNA fragmentation was visualized by UV light, and gels were photographed.
Statistical analysis
Data are shown as means ± SE. Statistical significance of differences between groups was calculated by a Student’s t test or one-way ANOVA when more than two groups were compared. Findings were assumed to be statistically significant at P < 0.05.
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Results |
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Diazoxide improved fasting blood glucose (FBG), fasting plasma insulin (FPI), and area under the curve (AUC) for glucose and insulin during OGTT
Before initiation of treatment, FBG levels were similar in all four groups, but FPI levels and AUC of glucose during OGTT were higher in OLETF rats vs. LETO rats (P < 0.001 by ANOVA). From 8 to 24 wk of age, FBG, FPI, and AUC of glucose all increased gradually in untreated OLETF rats. In contrast, they were significantly lower in treated groups. Moreover, the AUC of glucose for the continuously treated OLETF rats was similar to LETO rats and lower than discontinuously treated and untreated OLETF rats at 24 wk of age (P < 0.0001 by ANOVA). The AUC of insulin was higher in all OLETF rats vs. LETO rats during the experimental period (P < 0.0001 by ANOVA). But the AUC of insulin on wk 16 in treated rats was lower vs. untreated rats (P < 0.005). From 16 to 24 wk of age, the continuously treated group had a smaller AUC increase than the discontinuously treated and untreated group (P < 0.0001 by ANOVA) (Table 1
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). The insulin secretion in response to arginine and the AUC of insulin in treated OLETF rats (67.74 ± 5.59 ng·min·ml–1) were similar to LETO rats (60.68 ± 2.40 ng·min·ml–1) and significantly higher vs. untreated rats (57.37 ± 4.39 ng·min·ml–1; P < 0.05) at 16 wk of age. At 24 wk, the insulin levels after arginine load were higher in continuously treated OLETF rats (P < 0.05 by ANOVA except min 2; P < 0.05 vs. LETO at min 3 and 6; P < 0.05 vs. untreated except min 2; P < 0.05 vs. discontinuously treated only at min 3) (Fig. 1A). After 2 wk, diazoxide treatment also improved the pattern of insulin release after arginine load in diabetic rats (P < 0.05 by ANOVA at min 3 and 4) (Fig. 1B).
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and 3A). At 16 wk, the islets were enlarged in untreated OLETF rats (Fig. 2B), and showed a lower number of insulin-positive cells (Fig. 3B). The insulin staining of the ß-cells was at a lesser degree. Infiltration of inflammatory cells was also seen in the expanded islets. At the onset of diabetes in untreated OLETF rats (mean age, 24.78 wk), the enlargement of islets and connective tissue proliferation surrounding the islets became prominent (Fig. 2C). Separated into clusters by connective tissues, the islets show uneven distribution and low numbers of insulin-positive cells (Fig. 3C). The insulin labeling is significantly weak in the ß-cells. By contrast, the islets of diazoxide-treated OLETF rats at 16 and 24 wk all had a normal appearance and were closer to those of LETO rats (Fig. 2, D and E), with similar increases in insulin-positive cells and intensity in insulin labeling (Fig. 3, D and E). Furthermore, diazoxide treatment not only ameliorated the changes of islet morphology mildly, but also increased insulin stores and intensity markedly of insulin labeling in islet ß-cells in diabetic OLETF rats (Figs. 2F and 3F) vs. untreated diabetic OLETF rats (Figs. 2G and 3G). As expected, no histological changes, such as enlargement of islet and connective tissue proliferation or even distribution of insulin-positive cells, were observed in LETO rats throughout the experimental period (Figs. 2H and 3H).
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). In untreated OLETF rats, the increase in ß-cell mass was about 55.25% at the onset of diabetes compared with the LETO rats. In the diazoxide treatment group, ß-cell mass remained relatively stable throughout the study period: it was still significantly lower than untreated OLETF rats at 24 wk of age (P < 0.001), but higher than LETO rats (P < 0.01) (Fig. 4A). Similarly, diazoxide treatment prevented the ß-cell mass from increasing in diabetic OLETF rats at earlier stages (Fig. 4B).
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, A and B). By contrast, the ß-cell death rate in untreated OLETF rats was significantly higher (Fig. 5, C and D). The untreated OLETF rats at 16 wk of age and during the onset of diabetes showed the presence of apoptosis at 0.156 ± 0.007 and 0.327 ± 0.014 ß-cells per islet, respectively (P < 0.05 by ANOVA; P < 0.005 by t test, 16 wk vs. 24 wk). We observed a 2.1-fold increase in ß-cell apoptosis. Furthermore, diazoxide significantly reduced ß-cell apoptosis in diabetic OLETF rats (P < 0.05 by ANOVA) (Figs. 4C
and 5E).
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Linear regression showed a close correlation between the frequency of apoptotic cells and hyperglycemia (with AUC of glucose, r = 0.913; P < 0.0001; Fig. 4E; with FBG, r = 0.634; P < 0.001) suggesting that diazoxide protects ß-cells from hyperglycemia-induced apoptosis.
p38ß MAPK mediated diazoxide modulation of Bcl-2 and Bax expression, protection of ß-cells from apoptosis, and involvement of mitochondrial KATP channels
To evaluate a possible role of Bcl-2 and its binding partner Bax in the protective action of diazoxide, expression of Bcl-2 and Bax was detected using immunoprecipitation followed by Western blot analysis. Bcl-2 was decreased after diabetes (at 24 and 26 wk, respectively) depending on disease term, whereas Bax was increased. Bcl-2 and Bax expression almost returned to normal levels after 2-wk treatment with diazoxide (Fig. 6, A1 and A2). ß-actin was used as an internal loading control.
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and ß, ERK, or JNK. Immunoblots of the primary cell culture extracts showed a slight activation of p38ß within 5 min of diazoxide (50 µM) stimulation, and marked activation was observed at 30 min, which returned to the basic level within 4 h (Fig. 6B). By contrast, diazoxide had no effect on p38, JNK, or ERK (Fig. 6B). Cells exposed to 0.7 M NaCl for 20 min or 10 µM of phorbol-12-myristate-13-acetate for 20 min were used as positive controls.
Similar to our in vivo test, high glucose decreased Bcl-2 expression and increased Bax expression (Fig. 6C, line 2). However, diazoxide was able to restore this abnormity (Fig. 6C, line 3). To confirm that diazoxide’s modulation of Bcl-2 and Bax expression in islet ß-cells was mediated by the activation of p38ß, cells were pretreated overnight with 1 µM of SB 203580, a special inhibitor of p38, before treatment with diazoxide. As expected, SB 203580 alone had no effect on expression of Bcl-2 and Bax in high glucose culture medium (Fig. 6C, line 5). Furthermore, diazoxide-induced increased Bcl-2 and decreased Bax (Fig. 6C, line 3) were significantly blocked by SB 203580 (Fig. 6C, line 4).
To demonstrate whether diazoxide activates p38ß MAPK, up-regulates Bcl-2/Bax, and protects cells from apoptosis by opening mitoKATP channels, we used 5-HD, an inhibitor of mitochondrial KATP channels. ß-Cells were pretreated with 100 µM of 5-HD for 30 min before treatment for another 30 min with diazoxide (50 µM). As shown in Fig. 6D (line 3), 5-HD completely blocked diazoxide-activated p38ß but showed no effect on activation of p38ß alone (Fig. 6D, line 4). In another experiment, cells were pretreated with 100 µM of 5-HD for 30 min before treating them for 24 h with diazoxide (50 µM). Western blot was performed to determine the Bcl-2/Bax ratio. 5-HD almost restored Bcl-2 and Bax levels to those seen with high glucose treatment alone (Fig. 6C, lines 6 and 2). And no effect of 5-HD alone was seen on increasing Bcl-2 and decreasing Bax expression (Fig. 6C, line 7). Diazoxide’s protection against islet ß-cells apoptosis was also confirmed by DNA fragmentation assay. DNA laddering appeared in high glucose culture (Fig. 6E, line 3). It disappeared after treatment with diazoxide (Fig 6E, line 4). As expected, SB 203580 or 5-HD alone had no effect on protection of ß-cells against apoptosis (Fig. 6E, lines 6 and 8). But the protective roles of diazoxide were significantly blocked by SB 203580 (Fig. 6E, line 5) or 5-HD (Fig. 6E, line 7).
Thus, combined with other results from this study, these data strongly demonstrate that p38ß MAPK plays a critical role in protecting ß-cells from apoptosis via Bcl-2/Bax by diazoxide, and the mitoKATP channels are involved in this process.
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Discussion |
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Apoptosis, also called programmed cell death, is a physiological and active process whereby cells commit suicide. However, it is not a beneficial phenomenon if the ß-cell apoptosis rate exceeds the physiological rate and when the death of ß-cells is significantly more frequent and massive (9, 26). ß-Cell apoptosis takes place in all type 2 diabetic rats with or without obesity and contributes to the onset and development of type 2 diabetes, although the mechanism behind this process is not clearly understood (4, 5, 27). Interestingly, our in vivo study showed that diazoxide inhibits pancreatic ß-cell apoptosis at both prediabetic and diabetic stages. As per recent literature, the view that the endocrine pancreas belongs to a category of tissues that are finally differentiated and irreplaceable in the adult, has been drastically changed, and nobody disputes today that the endocrine pancreas is a plastic organ especially because of the high ability of the ß-cell mass to change according to the insulin demand (28, 29). Also, our studies found that diazoxide treatment could prevent this compensated increase of ß-cell mass from the prediabetic stage, and marginally restore this change in the early stage of diabetes. Furthermore, diazoxide not only prevents pancreatic morphological changes of ß-cells during prediabetic stage, but also ameliorates the changes of the pancreatic ß-cells in diabetic rats that have already undergone hypertrophy and hyperplasia. However, the most protective effect seems to be at the prediabetic stage.
Diabetes is characterized by hyperglycemia and impaired glucose-mediated insulin secretion (30). Deficient ß-cell function plays a dominating role in the establishment of overt diabetes (31). On the other hand, blood glucose levels are tightly controlled by regulation of insulin release from pancreatic ß-cells (32). Impaired insulin secretion in type 2 diabetes is at least in part due to a reversible depletion of insulin stores as a consequence of hyperglycemia (33, 34). Diazoxide has been well known for its ability to improve the pattern of insulin release (34, 35), which was further confirmed by Guldstrand et al. (18), who demonstrated an improvement in the first-phase insulin release in response to an arginine challenge in type 2 diabetic subjects treated with diazoxide. Three-month treatment of recent onset type 1 diabetic subjects with diazoxide also led to a significant improvement of C-peptide in up to 18 months of follow-up. However, some previous publications do not demonstrate a protective effect of diazoxide or could not confirm that diazoxide could induce ß-cell "rest" in studies using rodent islets (36, 37, 38). The different effects of diazoxide treatment in vitro may not be entirely responsible for the in vivo situation. In our studies in OLETF rats that demonstrate spontaneous development of type 2 diabetes, we were able to show protective action with diazoxide. We demonstrated that the administration of diazoxide actually decreased and delayed the onset of diabetes and prevented its development. Our results imply that besides inhibiting apoptosis, diazoxide increases islet insulin content, accompanied with ameliorated glucose tolerance. These properties of diazoxide may be beneficial in the treatment or prevention of diabetes. Moreover, limiting excessive apoptosis could be a newly available therapeutic strategy to prevent and manage type 2 diabetes, because this approach might actually reverse the disease to a greater degree rather than just palliating glycemia, and alleviate subtle defects in ß-cell function preceding the development of hyperglycemia in individuals at high risk of developing type 2 diabetes.
Current studies, including ours, point to the importance of assessing pancreatic ß-cell apoptosis, both for understanding the mechanisms that cause diabetes and for the development of treatment methods. However, the exact mechanism of increased ß-cell apoptosis and protection of ß-cells from damage and apoptosis by diazoxide is largely unknown. Although it has been known for many years that diazoxide blocks glucose-induced insulin secretion and reduces ß-cell workload by opening the K+-ATP channels (39), it is not well studied whether diazoxide has other actions and effects on ß-cells, such as a direct protective effect on the ß-cells through other mechanisms independent of ß-cell rest (40, 41, 42). Recently, several studies have shown that opening of the mitochondrial KATP channels is an obligatory step in cellular signal transduction involved in the cellular protective actions, such as the role of mitochondrial KATP channels in the cardioprotection of ischemic and pharmacological preconditioning of the human myocardium and their sequence of activation (43, 44, 45). However, opening mitochondrial KATP channels during cardioprotection are simply triggers in the signal transduction mechanism rather than the end effectors (43, 46). In addition, a number of studies have shown that diazoxide, a highly selective mitochondrial KATP channel opener, can mimic cardioprotection by preconditioning (44, 45, 47).
Hence, we postulated that opening mitochondrial KATP channels by diazoxide would trigger the signal transduction pathway and, thereby, regulate the activity of the p38 MAPK and Bcl-2. The members of the Bcl-2 family of genes play a crucial role in regulating apoptosis. The ratio of Bcl-2 to Bax within a cell is thought to be critical to how the cell responds to apoptotic stimuli and determines cell death or survival. Bcl-2 itself functions as a repressor of apoptosis, whereas Bax acts as a promoter of cell death (48, 49). We investigated the ability of Bcl-2 and its binding partner Bax in preventing high glucose-induced ß-cell apoptosis. Both our in vivo and in vitro studies confirm that Bcl-2 alone is unable to prevent apoptosis by itself but becomes capable when it is in a complex with Bax. This heterodimerization may remove Bax from homodimeric complexes, thereby uncoupling downstream apoptotic effectors (50).
MAPKs transduce signals from the cell membrane to the nucleus in response to a wide range of stimuli and modulate several important biological functions including gene expression, mitosis, proliferation, motility, and apoptosis (51, 52). p38 MAPK belongs to the MAPK superfamily. So far, four isozymes, , ß, 
, and 
, have been identified with several splice variants (52, 53, 54). The apparent discrepancy between these observations can probably be explained by the existence of several p38 MAPK isozymes with distinct functions. According to our and other studies (15, 17), we concluded that p38ß MAPK may be an important mediator of diazoxide-prevented ß-cell apoptosis, whereas SB 203580, a special p38 MAPK inhibitor, not only completely blocked this phenomenon, but also reduced diazoxide-induced Bcl-2/Bax expression changes. These data suggest that p38ß MAPK and Bcl-2/Bax also mediate diazoxide induction of ß-cell apoptosis with p38ß being upstream of Bcl-2/Bax.
Diazoxide depolarizes the mitochondrial membrane potential and causes a reversible oxidation of respiratory chain flavoproteins (43). The stimulation of K+ channel activity could result in p38 MAPK activation via PKC activation (10, 55). We found that pretreatment with 100 µM of 5-HD (44, 45, 56), a specific mitochondrial KATP channel blocker, completely abolished the protective effect of diazoxide in the ß-cells. Although somatostatin (SS) has been shown previously to inhibit insulin secretion (2, 57), SS did not act via p38ß in ß-cells in our studies (data not shown). In accordance with these findings, our data suggests that the KATP-p38ß-Bcl-2/Bax pathway is specific to diazoxide-protected ß-cells, but not SS-protected ß-cells.
In conclusion, the present study demonstrates that diazoxide prevents the onset and development of diabetes in OLETF rats by inhibiting apoptosis directly via opening mitochondrial KATP channels, but that is not the final step. This triggers sequential activation of p38ß MAPK and phosphorylation of the Bcl-2/Bax pathway. We believe that larger and long-term studies are required to evaluate and justify the therapeutic effect of diazoxide in the prevention of diabetes because limiting excessive apoptosis may be an available and novel therapeutic strategy to prevent and manage type 2 diabetes.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to declare.
First Published Online October 19, 2006
1 Q.H. and S.B. contributed equally to this work. 
Abbreviations: AUC, Area under the curve; FBG, fasting blood glucose; FPI, fasting plasma insulin; 5-HD, 5-hydroxydecanoate; JNK, c-jun N-terminal kinase; LETO, Long-Evans Tokushima Otsuka; OGTT, oral glucose tolerance test; OLETF, Otsuka Long Evans Tokushima Fatty; SDS, sodium dodecyl sulfate; SS, somatostatin; TUNEL, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling.
Received June 2, 2006.
Accepted for publication October 12, 2006.
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References |
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-glucosidase inhibitor. Metabolism 48:347–354[CrossRef][Medline]
B. J Biol Chem 279:46748–46754. J Biol Chem 272:30122–30128This article has been cited by other articles:
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  M.-R. Ki, H.-R. Lee, M.-J. Goo, I.-H. Hong, S.-H. Do, D.-H. Jeong, H.-J. Yang, D.-W. Yuan, J.-K. Park, and K.-S. Jeong Differential regulation of ERK1/2 and p38 MAP kinases in VacA-induced apoptosis of gastric epithelial cells Am J Physiol Gastrointest Liver Physiol, March 1, 2008; 294(3): G635 - G647. [Abstract] [Full Text] [PDF]
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, LETO; 
, OLETF-untreated; 
, OLETF-discontinued; 
, OLETF-continued. B, 
