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Endocrinology Vol. 143, No. 1 320-326
Copyright © 2002 by The Endocrine Society

INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS

Specific Expression of Bax-{omega} in Pancreatic ß-Cells Is Down-Regulated by Cytokines before the Onset of Apoptosis

Mark Van de Casteele, Benjamin Ate Kefas, Zhidong Ling, Harry Heimberg and Daniel G. Pipeleers

Diabetes Research Center, Vrije Universiteit Brussel, B-1090 Brussels, Belgium

Address all correspondence and requests for reprints to: Dr. Mark Van de Casteele, Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. E-mail: mvdcaste{at}vub.ac.be


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cytokines have been implicated in the process of pancreatic ß-cell destruction that leads to type 1 diabetes. This study investigates the ß-cell expression of pro- and antiapoptotic proteins from the Bcl-2 family and their variation during cytokine-mediated apoptosis. Exposure of rat ß-cells to the combination of IL-1ß plus interferon-{gamma} causes a time-dependent increase in apoptotic cells starting after 3 d (<10% on d 3 and 28 ± 2% on d 7). This effect was preceded by a marked down-regulation of two antiapoptotic proteins, Bcl-2 and Bax-{omega} (respectively reduced by 60% and 80% after 3 d), whereas no changes occurred in the expression of Bcl-xL and the proapoptotic protein Bax-{alpha}. No apoptosis or down-regulation of Bcl-2 and Bax-{omega} proteins was observed with individual cytokines or in the presence of N-methyl-L-arginine, an inhibitor of nitric oxide synthase. The lowered Bcl-2 protein content was associated with a decrease in Bcl-2 mRNA, which was initiated after 24 h of exposure. In MIN6 cells, the cytokine-induced suppression of Bcl-2- and Bax-{omega}, and apoptosis, occurred within 24 h. Primary rat ß-cells exhibited a higher expression of Bax-{omega} than MIN6 cells or than other rat cell types. These data suggest that suppression of the antiapoptotic proteins Bcl-2 and Bax-{omega} mediates cytokine-induced apoptosis of ß-cells. The ß-cell-specific expression of Bax-{omega} makes this protein a possible effector in the protection of this cell type against apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CYTOKINES HAVE BEEN implicated in the process of ß-cell destruction that leads to type 1 diabetes (1, 2). Their putative role is based on observations that combinations of IL-1ß, interferon-{gamma} (IFN{gamma}), and/or TNF{alpha} are cytotoxic to isolated ß-cell preparations (1, 3, 4, 5). The severity and mode of ß-cell death vary with the experimental condition. In isolated rodent islets, cytokines induce massive necrosis through induction of inducible nitric oxide synthase (iNOS) and subsequent production of toxic nitric oxide (NO) (6, 7, 8, 9). In dispersed rat ß-cells and in human ß-cell preparations, these toxic NO levels are not reached, which explains the low degree of necrosis (10). However, these conditions result in apoptosis, which becomes detectable in a small percentage of ß-cells after 72 h of exposure and progressively increases over subsequent days (10, 11, 12). A similar slow onset and progression of ß-cell apoptosis was noticed when the cells were cultured at low glucose levels (13). These delays in the appearance of apoptotic cells may indicate that time is needed to induce proapoptotic signals or to overcome the cellular protection by antiapoptotic proteins. The latter mechanism certainly seems operative, as the glucose- induced protection against apoptosis depends on the sugar’s stimulation of protein synthesis. We therefore propose that glucose-responsive ß-cells are protected by antiapoptotic proteins. Their nature, specificity, and properties have not yet been characterized. In many cell types, onset of the suicide program is regulated by the interaction, and hence the relative abundance, of members of the Bcl-2 family such as Bax and Bcl-2 (reviewed in Ref. 14). It has been shown that cytokine-induced ß-cell death can be prevented by overexpression of Bcl-2 (15, 16, 17). Furthermore, apoptosis during serum deprivation in pancreatic ß-cell lines is associated with a lower expression of Bcl-2 (18). The present work was undertaken to investigate whether cytokine-mediated apoptosis in ß-cells is mediated by an altered expression of proteins from the Bcl-2 family. During this study we noticed that ß-cells were characterized by a high expression of Bax-{omega}, a recently identified Bcl-2 family member with antiapoptotic activity. This protein was markedly reduced after prolonged exposure of the cells to IL-1ß plus IFN{gamma}, which was then followed by the appearance of apoptotic cells. Bax-{omega} may represent a novel potential target for prevention of autoimmune ß-cell destruction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell isolation and culture
Islets of Langerhans were isolated from adult male Wistar rats and dissociated with trypsin (20 µg/ml), and ß- and non-ß-cells were sorted by flow cytometry (FACStar, Becton Dickinson and Co., Sunnyvale, CA), as previously described (19). Purified ß-cells (>95% ß-cells) were either reaggregated for 1.5 h in a rotatory shaking incubator (19) and further suspension-cultured under static incubation in Lux dishes (Nunc, Naperville, IL) at a density of 1–2 x 105 cells/dish containing 3 ml medium, or cultured as single cells in polylysine-coated Falcon 96-well microtiter plates (Becton Dickinson and Co., Rutherford, NJ) at a density of 3–5 x 103 cells in 0.2 ml medium. ß-Cells were cultured (20) in serum-free Ham’s F-10 medium (Life Technologies, Inc., Paisley, Scotland) supplemented with 2 mM L-glutamine, 50 µM 3-isobutyl-1-methylxanthine (Jansen Chimica, Beerse, Belgium), 75 µg/ml penicillin, 100 µg/ml streptomycin, 10 mM glucose, and 1% charcoal-extracted BSA (fraction V, RIA grade, Sigma, St. Louis, MO); the cells were precultured 24 h before exposure to cytokines.

MIN6 cells (passages 20–25) were cultured in DMEM containing 25 mM glucose and supplemented with 15% heat-inactivated FBS (Life Technologies, Inc.), 50 µM 2-mercaptoethanol, 75 µg/ml penicillin, and 100 µg/ml streptomycin. MIN6 cells (106) were seeded in 25-cm2 tissue culture flasks (Falcon, Becton Dickinson and Co.) containing 5 ml medium. They were cultured for 3 d before treatment with cytokines. RINm5F cells were cultured in RPMI 1640 medium containing 11 mM glucose supplemented with 2 mM L-glutamine, 10 mM HEPES, penicillin (75 µg/ml), streptomycin (100 µg/ml), and 10% heat-inactivated FBS. In propagating and harvesting cell lines, cells were detached by 2-min incubation at 37 C in prewarmed PBS containing 1 mM EDTA and 1% BSA, and single cells were obtained by pipetting.

Cytokine treatment
Primary ß-cells and MIN6 cells were treated with recombinant mouse IFN{gamma} (1000 U/ml, 10 U/ng) and recombinant human IL-1ß (30 U/ml, 38 U/ng), gifts from Dr. D. L. Eizirik (Free University of Brussels, Brussels, Belgium). Culture medium from singly cultured ß-cells was collected after 72 h for spectrophotometric determination of nitrite (as a measure for the total amount NO formed), using the Griess reaction (21). In all other cases, the culture medium was renewed, and fresh cytokines were added every 48 h. The NO dependency of ß-cell viability and ß-cell protein expression was evaluated through the addition of 0.5 mM N-methyl-L-arginine (NMA; Sigma), which is known to specifically inhibit iNOS activity and prevent nitrite production. Suspension- cultured ß-cells were collected after 24 and 72 h of culture in the presence or absence of cytokines for RNA or protein extraction.

Determination of apoptosis
The number of live, apoptotic, and necrotic ß-cells was directly counted in populations of single cells, cultured for 3 or 7 d under normal conditions or in the presence of cytokines (with or without NMA). This was done by means of staining with the DNA dyes Hoechst 3342 and propidium iodide (PI; 10 µg/ml each) and microscopic examination of fluorescence, as previously described (10, 13). From the obtained values, apoptotic and necrotic indexes were calculated (10, 13).

Induction of apoptosis in cultures of MIN6 cells was assessed through measurement of the fraction of subG1 (apoptotic) nuclei, using the PI-lysis/FACS method (22). Total dissociated MIN6 cells (from both attached and floating cell populations) were collected in a volume of 7 ml in 15-ml conical Falcon tubes. To 100-µl samples (±5 x 104 cells) of these cell suspensions, 1 ml cold propidium iodide lysis buffer [50 µg/ml PI, 0.1% (wt/vol) trisodium citrate, and 0.1% (vol/vol) Triton X-100] was added. For cell lysis and nuclear staining, the aliquots were briefly vortexed and stored overnight at 4 C. The percentage of subG1 nuclei was then quantified by FACS on the basis of the PI fluorescence intensity (FL-2H histograms).

Analysis of mRNA and protein expression
mRNA expression was analyzed in vitro after a 24- or 72-h culture of 105 reaggregated rat ß-cells in the absence or presence of IL-1ß and IFN{gamma}. Polyadenylated RNA was purified with oligo(deoxythymidine)-coated magnetic microbeads (Dynabeads, DynAl, Oslo, Norway). RNA was reverse transcribed with the GeneAmp RNA PCR kit using random hexamer primers and Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer Corp., Norwalk, CT), followed by PCR in standard conditions for 30–34 cycles using the PCR primers listed in Table 1Go for specific amplification of the Bcl-2, Bax, and Bcl-xL genes and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Amplified fragments were cloned in pGEM-T vector (Promega Corp., Madison, WI), and their DNA sequences were determined. The lengths of PCR-generated fragments are indicated in Table 1Go. Their sequences showed more than 98% identity to deposited sequences in GenBank (Table 1Go). For determining expression levels of Bcl-2, Bax-{alpha}, and Bcl-xL mRNA, each target was amplified within the linear amplification range, and the amounts of PCR product were analyzed on ethidium bromide-stained agarose gels that were photographed under UV transillumination using a Kodak Digital Science DC40 camera (Eastman Kodak Co., Rochester, NY). The primers employed to measure Bax-{alpha} mRNA (Table 1Go) are complementary to sequences spanning intron 5 of the Bax gene. However, under the experimental conditions used in the present study to measure Bax-{alpha} mRNA (PCR including up to 34 cycles), a signal corresponding to the alternatively spliced Bax-{omega} mRNA could not be detected (not shown). The results from RT-PCR determinations for Bcl-2, Bax-{alpha}, and Bcl-xL mRNA were expressed relative to the signal obtained for the mRNA encoding GAPDH.


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  		Table 1. Primers and PCR products

 
To examine whether the protein levels of Bax-{alpha}, Bax-{omega}, Bcl-2, and stress protein GRP-75 were modulated by IL-1ß and IFN{gamma}, cell-free extracts were prepared from 2 x 105 reaggregated ß-cells that had been exposed for 24 or 72 h and probed by immunoblotting using rabbit polyclonal anti-Bax antibodies P-19 and N-20, anti-Bcl-2 antibody N-19, as well as goat polyclonal antibodies for GRP-75 and actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Anti-Bax antibodies N-20 and P-19 are directed against epitopes mapping at the amino-terminus of Bax, comprising amino acids 1–11 and 43–61, respectively. Specific binding of these antibodies was verified using the corresponding blocking peptides (Santa Cruz Biotechnology, Inc.). Expression of Bax proteins was analyzed in homogenates of freshly isolated rat tissues and in FACS-enriched islet non-ß- and ß-cells and compared with the expression pattern in mouse (MIN6) and rat (RIN) insulinoma cell lines (23, 24). Immunoprecipitation of Bax from 200 µg total MIN6 cell proteins was carried out by rotation overnight at 4 C in the presence of 5 µl anti-Bax N-20 antibody, followed by a 1-h incubation with 25 µl protein A-Sepharose beads. Preparation of protein extracts, Western blotting, and immunoprecipitation were carried out as previously described (25).

Enrichment of apoptotic MIN6 cells
Subconfluent MIN6 cells were exposed to IL-1ß plus IFN{gamma} for 24 h. They were stained with 10 µg/ml Hoechst-3342 (HO-3342, Calbiochem, La Jolla, CA) and incubated at 37 C for 1 h. The detached and adherent cells were harvested separately. They were then stained with 10 µg/ml PI (Sigma) for 5 min at 37 C, and both preparations were FACS-sorted for cells that were PI negative and HO-3342 positive. Aliquots of the sorted cells (concentrated by centrifugation) were placed on glass slides, and the nuclear morphology of these cells was examined under the inverted fluorescence microscope by HO-3342 fluorescence under UV light (filter 340–380 nm); the remaining sorted MIN6 cells were collected in 3-ml Falcon tubes, centrifuged, and rinsed with PBS, and cell pellets were stored at -20 C until used in Western blotting.

Data analysis
The OD of the signals on films or on agarose gels was obtained with minimal exposure times and measured using NIH Image 1.60 software (NIH, Bethesda, MD). Data are the mean ± SE for the number of experiments stated. Statistical significance of differences between experimental groups was assessed using the paired two-tailed t test. Statistical analysis in multiple comparisons was performed by one-way ANOVA with Bonferonni’s method.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NO dependence of cytokine-induced apoptosis
In vitro exposure of single rat ß-cells to IL-1ß (30 U/ml) and IFN{gamma} (1000 U/ml) for 7 d resulted in 28 ± 2% apoptotic cells (Fig. 1Go), which is significantly higher than after 3 d (<10%; P < 0.001, by ANOVA; Fig. 1Go). Only a small percentage of necrotic cells were noticed (11 ± 2% after 7 d). Both forms of cell death were nearly completely blocked by the addition of 0.5 mM NMA, which is known to inhibit NO production by iNOS. The NMA suppression of NO formation is complete during the first 3 d of culture, which precedes the appearance of apoptotic cells (Fig. 1Go). We next examined the effects of cytokines on the expression of Bcl-2-related proteins during the first 3d of culture.



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  		Figure 1. Effect of cytokines on ß-cell death and NO formation. Single rat ß-cells were exposed to IL-1ß (30 U/ml) and IFN{gamma} (1000 U/ml) in the absence or presence of 0.5 mM NMA. Indexes for apoptosis and necrosis were determined after 3 and 7 d of culture using nuclear staining with Hoechst 3342 and PI. Medium nitrite accumulation was determined after 3 d of culture. Data are represented as the mean ± SE (n = 6). *, P < 0.001, by ANOVA.

 
Expression of Bcl-2-related proteins in ß-cells
Immunoblots of extracts from rat ß-cells indicated the presence of several proteins of the Bcl-2 family (Fig. 2Go). In addition to the more classical members, such as Bcl-2, Bax-{alpha}, and Bcl-xL (detected at mRNA level, see Fig. 3Go), we noticed the Bax-{alpha}-related protein Bax-{omega}. Use of two heterologous anti-Bax antibodies, namely N-20 and P-19, identified a 30-kDa protein besides the expected 21-kDa Bax-{alpha} species (Fig. 2AGo). Both the 30-kDa (p30) and the 21-kDa (p21) proteins were also expressed in MIN6 cells, a mouse ß-cell-derived cell line. Their immunoprecipitation by antibody N-20 was blocked by the N-20 blocking peptide (Fig. 2AGo). Furthermore, the N-20-precipitated p21 and p30 proteins were immunodetected with the P-19 antibody (Fig. 2AGo). The 30-kDa protein thus corresponds to Bax-{omega}, a recently described Bax-{alpha} splice variant that was shown to be specifically recognized by antibody P-19 as a 30-kDa band (26). When different rat tissues were examined with the N-20 antibody for the expression of Bax proteins, Bax-{alpha} (p21) appeared ubiquitously expressed, whereas high levels of Bax-{omega} (p30) were only observed in MIN6 cells and in enriched or purified rat ß-cell preparations. The ß-cell-specific expression of Bax-{omega} also became apparent by its virtual absence in islet endocrine non-ß-cells and in pancreatic exocrine cells, both acinar and ductal, as well as in the poorly differentiated rat insulinoma cell line RINm5F (24). As a number of other ß-cell proteins (27), Bax-{omega} was also detected in rat brain, although it had a weak signal (Fig. 2BGo).



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  		Figure 2. Bax protein expression in the rat pancreas. A, Immunoprecipitation of Bax-{alpha} and Bax-{omega} from MIN6 cell extracts by antibody N-20 and subsequent immunoblot detection by antibody P-19. Immunoprecipitation of both Bax proteins was blocked by the N-20 antigenic peptide (left panel). Immunoblot detection of Bax-{alpha} and Bax-{omega} proteins in total extracts of rat ß-cells, using anti-Bax antibodies P-19 and N-20 is also shown (right). B, Immunoblot detection of Bax-{alpha} and Bax-{omega} in various rat tissues using antibody N-20. Application of 25 µg total protein from extracts of rat brain, liver, pancreas (Pancr.), pancreatic duct cell (Duct), and acinar cell (Acinar) preparations; islet cells (Islet); purified islet non-ß-cells (nonß); purified islet ß-cells (ß); rat insulinoma cells RINm5F (RIN); and the mouse cell line MIN6 (MIN).

 


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  		Figure 3. Effects of cytokines on the expression of Bax-{alpha}, Bcl-2, and Bcl-xL mRNA in rat ß-cells. Rat ß-cells were cultured for 24 and 72 h with or without IL-1ß (30 U/ml) and IFN{gamma} (1000 U/ml), as indicated. Bcl-2, Bax-{alpha}, and Bcl-xL mRNA levels were determined by RT-PCR and expressed relative to internal signals obtained for GAPDH. Data are the mean ± SE from six independent experiments. *, P < 0.01 vs. control.

 
Effect of IL-1ß and IFN{gamma} on expression of Bcl-2- related proteins
Exposure to IL-1ß and IFN{gamma} for 24 h reduced mRNA expression of the Bcl-2 antiapoptotic protein; this effect was more pronounced after 72 h (Fig. 3Go). Parallel analysis of mRNA for Bax-{alpha} showed no effect after 72 h (Fig. 3Go). No alterations were seen in the expression of the Bcl-xL antiapoptotic gene.

At the protein level, 72-h culture with IL-1ß and IFN{gamma} reduced the expression of Bcl-2 and Bax-{omega} by, respectively, more than 60% and 80%, whereas that of Bax-{alpha} or the apoptosis-unrelated mitochondrial protein GRP-75 was unaffected (Fig. 4Go, A and B). This decrease in abundance of the two antiapoptotic proteins was not yet present after the first 24 h of exposure to the cytokines (data not shown). Suppression of Bax-{omega} protein appears to require signals from both cytokines, as neither IL-1ß nor IFN{gamma} alone influenced Bax-{omega} expression (Fig. 4CGo). One of these signals seemed to be NO dependent, as suggested by the protective effect of NMA on the cytokine-induced inhibition of Bcl-2 and Bax-{omega} expression (Fig. 4AGo).



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  		Figure 4. Effect of cytokines on the expression of Bcl-2 and Bax-{omega}. Rat ß-cells were cultured for 72 h without cytokines (-) or in the presence of IL-1ß (30 U/ml) and IFN{gamma} (1000 U/ml), either without (CK) or with 0.5 mM NMA (CK+NMA). A, Representative Western blotting experiment for evaluating the expression of Bcl-2, Bax-{omega}, Bax-{alpha}, and GRP-75 protein, with actin as the internal control. B, Relative expression levels as determined by densitometry and expressed relative to the signal obtained for actin (n = 6). *, P < 0.01 vs. control. Data are expressed as the mean ± SE. JK, Jurkat cells, Bcl-2-positive control. C, Level of Bax-{omega} expression in rat ß-cells exposed for 72 h to either IL-1ß (30 U/ml) or IFN{gamma} (1000 U/ml) alone (IL and IFN, respectively) or to both (IL+IFN) in four independent experiments. ¶, P < 0.01 vs. control.

 
Association of cytokine-induced apoptosis with a preceding decrease in cellular expression of Bax-{omega} and Bcl-2 proteins
Exposure of subconfluent cultures of MIN6 cells to IL-1ß (30 U/ml) and IFN{gamma} (1000 U/ml) resulted within 24 h in apoptosis of a fraction of the cells, as shown by the proportion of hypodiploid (fragmented) nuclei (Fig. 5AGo). The apoptotic and nonapoptotic cells were separated by FACS sorting on the basis of the cellular fluorescence for the Hoechst and PI fluorescent compounds (Fig. 5BGo). The two subpopulations were examined by Western blotting for their respective abundance in Bax-{omega} and Bcl-2 proteins. Both proteins were markedly suppressed in the subpopulation that was sorted as apoptotic cells (Fig. 5CGo).



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  		Figure 5. Effect of cytokines on Bcl-2 and Bax-{omega} expression in MIN6 cells and relation to apoptotic cells. A, Induction of apoptosis. Using the PI/lysis-FACS method, the percentage of subG1 nuclei (gating for subG1 fluorescence represented by a horizontal line) was measured in subconfluent cultures of MIN6 cells after exposure to IL-1ß (30 U/ml) and IFN{gamma} (1000 U/ml) for 24 h or in control cultures (CTRL). Data are expressed as the mean ± SE from eight independent experiments. P < 0.001 vs. CTRL. B, Separation of normal (A and B) and apoptotic (C and D) MIN6 cells from cytokine-treated cultures. Normal or apoptotic subpopulations were obtained by FACS sorting of PI-negative/Hoechst-positive cells that were adherent or nonadherent after cytokine treatment, respectively. After sorting, cells were concentrated by centrifugation for viewing under visible light (A and C) or fluorescent light (B and D). Subpopulations repeatedly contained more than 70% of either normal (compare A and B) or apoptotic (compare C and D) MIN6 cells, based on their cellular and nuclear morphologies. Fragmented or condensed nuclei (D) were indicative of apoptosis, compared with nuclei of normal cells (B). C, Western blotting for Bcl-2, Bax-{omega} and Bax-{alpha} protein in control MIN6 cells (CTRL), in the total population of MIN6 cells (Tot) after 24-h cytokine exposure, or in cell preprations enriched for either apoptotic (Ap) or normal (Nm) MIN6 cells by FACS sorting, as shown in B. Protein (25 µg) from each of these four cell preparations was loaded on the gel; actin was detected to evaluate loading. The results shown are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cytokines can cause ß-cell death in vitro (1, 3, 4, 5, 11). Depending on their composition and the selected experimental conditions, they may induce cellular necrosis or apoptosis (10, 12, 17, 28). It seems likely that the presence of high NO levels at the ß-cell membranes leads to necrosis within 3 d, whereas lower levels may contribute to the development of apoptosis after longer exposure times (10, 12). In the presently used model of isolated rat ß-cells, the combination of IL-1ß and IFN{gamma} results predominantly in apoptosis, starting after 3 d of exposure and slowly affecting more cells. This cytokine-induced apoptosis seems dependent on signals from both cytokines. NO is one of the mediators, as apoptosis was suppressed in the presence of NMA; it is certainly not the only mediator, because no apoptosis was induced by IL-1ß alone. Apoptosis induction by the combination of the three cytokines, IL-1ß, IFN{gamma}, and TNF{alpha}, has been found to occur independently of NO formation, at least in other species (11, 28, 29). It is conceivable that TNF{alpha} elicits signals that dissociate apoptosis from NO formation. In NIT-1 insulinoma cells, for example, TNF{alpha} alone can induce apoptosis (30).

Induction of apoptosis in primary ß-cells seems to require a sustained exposure to the causative conditions. During culture with cytokines as well as at low glucose levels, apoptotic cells only became detectable after 3 d and then increase slowly in number during the subsequent 7 d (11, 13, 31). In lymphoid cells and primary hepatocytes, apoptosis was noticed within 24 h of incubation with Fas or TNF{alpha} (32, 33). Exposure of the ß-cells to cytokines is expected to rapidly generate proapoptotic signals, as indicated by the activation of nuclear factor-{kappa}B and MAPKs and by up-regulation of iNOS within 24 h (31, 34). Thus, whereas potent proapoptotic signals such as NO are generated early in cytokine-exposed ß-cells, their death by apoptosis is delayed for at least 3 d, suggesting that ß-cells are protected by antiapoptotic mechanisms. In a previous study we proposed that this protection is maintained by glucose-induced synthesis of antiapoptotic proteins. In view of the specificity of a number of glucose-stimulated proteins in ß-cells, it is conceivable that some of these antiapoptotic factors are also specific in terms of their occurrence in glucose-regulated ß-cells. As cytokines are known to alter the protein profile (35), as well as the glucose sensitivity and phenotype of ß-cells (36), they could be suspected to affect the expression of antiapoptotic proteins in ß-cells. The present study shows that this is indeed the case, at least in rat ß-cells.

Exposure to IL-1ß and IFN{gamma} causes a down-regulation of the antiapoptotic protein Bcl-2, whereas the expression of Bcl-xL or the proapoptotic Bax-{alpha} was unaffected. Consequently, the Bax-{alpha} over Bcl-2 protein ratio was 2.5-fold increased after 72 h, which in other cell types is considered an apoptosis-inducing condition (14). The analysis of Bax-{alpha}-immunoreactive proteins in ß-cells indicated that one related protein is particularly abundant in this cell type, namely Bax-{omega}. The latter member of the Bcl-2 family was shown to be antiapoptotic after overexpression in stably transfected cell lines (26). The present study suggests that the presence of Bax-{omega} in primary ß-cells might represent one of the factors that protect ß-cells against the rapid onset of apoptosis. In addition to its relative abundance in ß-cells compared with other cell types, Bax-{omega} expression was strongly reduced after 72-h culture with the IL-1ß plus IFN{gamma} combination that is known to cause apoptosis during the subsequent days. This combination also induced a down-regulation of Bcl-2 and Bax-{omega} in MIN6 cells, which was directly associated with the fraction of apoptotic cells. In MIN6 cells, these processes were already prominent after 24 h. The kinetics of apoptosis induction in ß-cells may well be determined by the basal expression levels as well as by the turnover rates of the antiapoptotic proteins, Bcl-2 and Bax-{omega}. The higher resistance of primary ß-cells to cytokine-induced apoptosis may thus be related to the higher ratio of Bax-{omega} over Bax-{alpha} in these cells.

The cytokine-induced suppression of Bax-{omega} and Bcl-2 was not observed when only one of the two cytokines was administered or when NO production was inhibited with NMA. These findings correlate with the respective rates of apoptosis at later times, and thus support the view that reduced expression of these two antiapoptotic members of the Bcl-2 family is responsible for or at least contributes to the development of apoptosis in cytokine-exposed ß-cells. It has been shown previously that Bcl-2-transfected human ß-cells or mouse ß-cell lines are protected against cytokine-induced destruction (15, 16). Our studies suggest that environmental factors can regulate the expression of Bax-{omega} and Bcl-2 in ß-cells and thus determine their susceptibility to apoptosis. Inhibition of NO synthesis decreases apoptosis, the expression of Fas, and the activation of caspases induced by cytokines (30, 37, 38, 39). Thus, additional alternative pathways, either in association with or independent of a Bcl-2-like protein, may contribute to apoptosis. Whether reduced expression of Bax-{omega} and Bcl-2 is sufficient to induce or accelerate apoptosis needs to be investigated by a genetic approach using specific down-regulation or loss of function. It should then also become clear to what extent the specific expression of Bax-{omega} in ß-cells is predominantly responsible for the survival of these cells. The present study provides the first evidence for a role of Bax-{omega} in the physiological regulation of ß-cell survival, but the underlying mechanism remains to be clarified. It is known that Bax-{omega} can bind to both Bcl-2 and Bax-{alpha}. It lacks the COOH-terminal hydrophobic sequence by which Bcl-2 and Bax-{alpha} proteins can dock onto intracellular membranes (26). It should therefore be examined whether Bax-{omega} could function as a cytosolic decoy binding partner for Bax-{alpha}, which would reduce the potency to trigger apoptosis.

Previous work has indicated that pancreatic ß-cells possess defense mechanisms against cell death (36, 40). They are protected against apoptosis by glucose-induced proteins (13). The present findings demonstrate that ß-cells express two antiapoptotic proteins, Bcl-2 and Bax-{omega}, which are down-regulated by cytokines before the onset of apoptosis. It remains to be determined whether the survival of ß-cells is primarily dependent on the more ubiquitous Bcl-2 protein or on the more specific Bax-{omega} protein. This knowledge will be useful to search for conditions that protect ß-cells against cell death.


   Acknowledgments
 
The authors thank Décio Eizirik for helpful discussions on cytokines, Geert Stangé and Natascha Caluwé for excellent support, Ying Mei Feng and Johan Guns for performing cell line cultures, and Meng-Chi Chen for providing GAPDH PCR primers. We are grateful to Dr Ishihara (University of Tokyo, Tokyo, Japan) for sending us MIN6 cells.


   Footnotes
 
This work was supported by grants from the Belgian Fund for Scientific Research Flanders (F.W.O. 0376.97) and the services of the Prime Minister (Interuniversity Attraction Pole P4/21). M.V.D.C. and H.H. are recipients of postdoctoral research fellowships from the Fund for Scientific Research-Flanders.

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; IFN{gamma}, interferon-{gamma}; iNOS, inducible nitric oxide synthase; NMA, N-methyl-L-arginine; NO, nitric oxide; PI, propidium iodide.

Received July 10, 2001.

Accepted for publication September 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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