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B and Endoplasmic Reticulum Stress
Laboratory of Experimental Medicine (I.K., L.L., A.K.C., Z.D., M.C., D.L.E.) and Division of Endocrinology (M.C.), Erasmus Hospital, Université Libre de Bruxelles, B-1070 Brussels, Belgium
Address all correspondence and requests for reprints to: Décio L. Eizirik, Laboratory of Experimental Medicine, Université Libre de Bruxelles, Route de Lennik, 808 CP 618, B-1070 Brussels, Belgium. E-mail: deizirik{at}ulb.ac.be.
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B (NF-B) activation. Previous studies suggested that in T2DM high glucose and free fatty acids (FFAs) are ß-cell toxic also via NF-B activation. The aims of this study were to clarify whether common mechanisms are involved in FFA- and cytokine-induced ß-cell apoptosis and determine whether TNF
, an adipocyte-derived cytokine, potentiates FFA toxicity through enhanced NF-B activation. Apoptosis was induced in insulinoma (INS)-1E cells, rat islets, and fluorescence-activated cell sorting-purified ß-cells by oleate, palmitate, and/or cytokines (IL-1ß, interferon-
, TNF). Palmitate and IL-1ß induced a similar percentage of apoptosis in INS-1E cells, whereas oleate was less toxic. TNF did not potentiate FFA toxicity in primary ß-cells. The NF-B-dependent genes inducible nitric oxide synthase and monocyte chemoattractant protein-1 were induced by IL-1ß but not by FFAs. Cytokines activated NF-B in INS-1E and ß-cells, but FFAs did not. Moreover, FFAs did not enhance NF-B activation by TNF. Palmitate and oleate induced C/EBP homologous protein, activating transcription factor-4, and immunoglobulin heavy chain binding protein mRNAs, X-box binding protein-1 alternative splicing, and activation of the activating transcription factor-6 promoter in INS-1E cells, suggesting that FFAs trigger an endoplasmic reticulum (ER) stress response. We conclude that apoptosis is the main mode of FFA- and cytokine-induced ß-cell death but the mechanisms involved are different. Whereas cytokines induce NF-B activation and ER stress (secondary to nitric oxide formation), FFAs activate an ER stress response via an NF-B- and nitric oxide-independent mechanism. Our results argue against a unifying hypothesis for the mechanisms of ß-cell death in T1DM and T2DM. |
Introduction |
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, IL-6, leptin, resistin, and adiponectin. TNF has direct cytotoxic effects on pancreatic ß-cells, especially in combination with other cytokines (16, 17).
In type 1 diabetes mellitus (T1DM), the autoimmune assault on pancreatic ß-cells leads to progressive ß-cell loss. Macrophages and T cells, attracted to the islets, secrete soluble mediators such as oxygen free radicals, nitric oxide (NO), and the cytokines IL-1ß, interferon (IFN)- and TNF. Increasing evidence suggests that these mediators induce apoptosis, the main mode of ß-cell death in the development of T1DM (18).
ß-Cell apoptosis may thus be a common feature of T1DM and T2DM, but the mechanisms leading to initiation of the cell death program remain to be clarified. The transcription factor nuclear factor-B (NF-B) plays a crucial role in cytokine- and double-stranded RNA + cytokine-induced apoptosis (19, 20, 21). Cytokine-induced activation of NF-B leads to: 1) loss of differentiated ß-cell functions by down-regulation of Isl-1 and pancreatic duodenal homeobox-1 (Pdx-1) (22, 23), 2) up-regulation of inducible nitric oxide synthase (iNOS) (24) and excessive NO production, 3) up-regulation of chemokines such as monocyte chemoattractant protein-1 (MCP-1) (25, 26), and 4) down-regulation of the Ca2+ pump sarcoendoplasmic reticulum Ca2+ ATPase type 2b (SERCA-2b) (22, 23). Decreased SERCA-2b expression leads to endoplasmic reticulum (ER) calcium depletion and ER stress (23, 27, 28) (Cardozo, A. K., F. Ortis, Y.-M. Feng, J. Rasschaert, F. Van Eylen, J. Storling, T. Mandrup-Poulsen, A. Herchuelz, and D. L. Eizirik, submitted for publication). The cellular response to ER stress is mediated by several ER transmembrane proteins including the kinase, inositol requiring and ER-to-nucleus signaling kinase 1, PKR-like ER kinase (PERK), activating transcription factor (ATF)-6, and X-box binding protein-1 (XBP-1) (29). The activation of the transcription factor XBP-1 through its alternative splicing by IRE1 is an important indicator of ER stress (30), and XBP-1 alternative splicing is detected in both thapsigargin- and cytokine-treated ß-cells (Cardozo, A. K., F. Ortis, Y.-M. Feng, J. Rasschaert, F. Van Eylen, J. Storling, T. Mandrup-Poulsen, A. Herchuelz, and D. L. Eizirik, submitted for publication). Thapsigargin is a well-known inducer of ER stress, previously shown to cause apoptosis of insulin-producing cells (31, 32). ß-Cell exposure to IL-1ß + IFN or thapsigargin also triggers expression of the mRNA for growth arrest and DNA damage (GADD) 153, also known as C/EBP homologous protein (CHOP) (GADD153/CHOP) (22, 23) (Cardozo, A. K., F. Ortis, Y.-M. Feng, J. Rasschaert, F. Van Eylen, J. Storling, T. Mandrup-Poulsen, A. Herchuelz, and D. L. Eizirik, submitted for publication), a transcription factor involved in ER stress-induced apoptosis (33).
The mechanisms involved in FFA-induced ß-cell apoptosis in T2DM remain to be clarified. We have previously shown that FFA cytotoxicity is inversely related with cytoplasmic triglyceride accumulation (15). This suggests that cytoplasmic accumulation of fatty acyl-CoA is directly ß-cell toxic, whereas their esterification probably functions as a protective mechanism (15). Increased ß-cell FFA levels might lead to de novo ceramide formation (34, 35) and mitochondrial cytochrome C release (14, 36). Other proposed mechanisms for FFA-induced ß-cell death are the activation of protein kinase C (37), inhibition of protein kinase B activity (38), and activation of calpain-10 (39). Moreover, microarray studies of global gene expression in FFA-treated ß-cells (40, 41, 42) indicate similarities between several of the genes induced by FFAs and those observed downstream of cytokine-induced NF-B, including ornithine decarboxylase, b-2 microglobulin, DNA-binding protein A, and MCP-1 (22, 26). In line with these observations, a recent study reported that palmitate induces a pronounced (>10-fold) NF-B activation in insulinoma (INS)-1 cells, comparable with the activation observed with TNF (43). Based on these and additional observations obtained in islets cultured at high glucose concentrations, it has been proposed that prolonged exposure to excessive concentrations of nutrients results in a proinflammatory ß-cell response, contributing to ß-cell damage and death in T2DM (35, 44, 45, 46). A unifying hypothesis has also been suggested for the mechanisms of nutrient- and cytokine-induced ß-cell death in T1DM and T2DM, in which activation of NF-B is a common and crucial step for both proapoptotic stimuli (45, 46).
Against this background, the aim of this study was to clarify whether common mechanisms are indeed involved in FFA- and cytokine-induced ß-cell apoptosis. The questions we asked were: 1) is FFA-induced apoptosis associated with the activation of the transcription factor NF-B and the induction of its downstream genes iNOS and MCP-1, as is the case for cytokines; 2) does TNF potentiate the toxic effects of FFAs via a potentiation of NF-B activation; and 3) do these FFA-mediated effects result in the induction of an ER stress response in ß-cells? To address these questions, we exposed the insulinoma cell line INS-1E, rat islets, and fluorescence-activated cell sorting (FACS)-purified ß-cells to oleate or palmitate, alone or in combination with TNF. For comparative purposes, these cells were also exposed to IL-1ß or, in some experiments, IL-1ß + IFN or thapsigargin.
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Materials and Methods |
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(100 U/ml; R&D Systems, Oxon, UK); rat TNF (1000 U/ml; Innogenetics, Gent, Belgium); or the FFAs oleate and palmitate. Oleate and palmitate (sodium salt, Sigma) were solubilized in 90% ethanol, heated to 60 C, and used in a 1:100 dilution in culture medium as previously described (15, 51). The control condition contained a similar dilution of ethanol, which by itself does not affect any of the variables evaluated in the present study (data not shown). The FFAs and cytokine concentrations were selected based on our previous time course and dose-response studies and the calculation of unbound FFA concentrations derived from the total FFA and albumin concentrations (15, 18, 51, 52).
Determination of cell viability
The percentage of viable, apoptotic, and necrotic cells was determined after 24–72 h exposure to FFAs or cytokines. For this purpose, islets, FACS-purified ß-cells, and INS-1E cells were incubated for 15 min with the DNA binding dyes propidium iodide (PI, 10 µg/ml, Sigma) and Hoechst 33342 (HO 342, 20 µg/ml, Sigma) (15, 52, 53). The cells were examined by inverted microscopy with UV excitation at 340–380 nm. Viable cells were identified by their intact nuclei with blue fluorescence (HO 342), necrotic cells by their intact nuclei with yellow-red fluorescence (HO 342 + PI), and apoptotic cells by their fragmented nuclei, exhibiting either a blue (HO 342; early apoptosis) or yellow-red fluorescence (HO 342 + PI; late apoptosis) (53). This fluorescence assay is quantitative for single ß-cells and has been validated by systematic comparison with electron microscopy (53). This method has previously been used to evaluate apoptosis/necrosis in rat (15, 21, 53, 54) (and present data), mouse (55), and human (56) ß-cells and INS-1 cells (52).
By the use of FACS-purified ß-cells (95% purity) in some of our present experiments, we avoided the problem of apoptosis/necrosis detection in non-ß-cells, inherent to studies performed in whole islets. In each experimental condition a minimum of 500 cells was counted by at least two observers, one of them blinded for the sample identity. Of note, exact quantification of cell death in whole islets is difficult due to the superposition between cells. The data obtained in whole islets thus provide an approximate percentage of dead cells (54, 55). There was a more than 90% agreement between observers in the different assessments of viability, confirming the reproducibility of the method. Findings obtained using this apoptosis assay were corroborated by a second method, namely the cell death detection ELISAPLUS (Roche Diagnostics, Mannheim, Germany). This method detects enrichment of mono- and oligonucleosomes in the cytoplasm of apoptotic cells, indicating apoptosis-associated DNA degradation. The method was validated for detection of apoptosis in our experimental conditions by systematic comparison with the HO 342 + PI method (described above) in purified ß-cells exposed either to a proapoptotic stimulus (the ER stress inducer thapsigargin; 1 µM) or a pronecrotic stimulus (the mitochondrial poison antimycin A, 0.5 µg/ml) (data not shown). We found that the cell death detection ELISAPLUS provides reliable and reproducible data in single cells and cell monolayers (ß-cells or INS-1 cells) but not in whole islets, probably due to the islet dispersion procedure.
Analysis of mRNA expression by real-time PCR
Poly(A)+ RNA was isolated from islets and INS-1E cells, using oligo(dT)25-coated polystyrene Dynabeads (Dynal, Oslo, Norway) and reverse transcribed as previously described (25). The real-time PCR amplification reaction was done in 20 µl containing 3 mM MgCl2, 0.5 µM forward and reverse primers, 2 µl FastStart SYBR Green mix (Roche Diagnostics), and 2 µl cDNA. Standards for each gene were prepared using appropriate primers in a conventional PCR and purified for subsequent analyses. PCR products were quantified fluorometrically using SYBR Green, and their concentration was calculated as copies per microliter by comparison with a standard curve (57). The expression level of the gene of interest was corrected for that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (57). The mRNAs analyzed were: iNOS, MCP-1, Pdx-1, GADD153/CHOP, BiP, ATF-4, and uncoupling protein-2 (UCP-2). The specific primers used and their respective PCR fragment lengths were as follows: GAPDH, forward, 5'-AGTTCAACGGCACAGTCAAG-3', reverse, 5'-TACTCAGCACCAGCATCACC-3' (118 bp); iNOS, forward, 5'-GGGAGCCAGAGCAGTACAAG-3', reverse, 5'-GGCTGGACTTCTCACTCTGC-3' (138 bp); MCP-1, forward, 5'-CTTCTGGGCCTGTTGTTCA-3', reverse, 5'-CCAGCCGACTCATTGGGATCA-3' (127 bp); Pdx-1, forward, 5'-GGTATAGCCAGCGAGATGCT-3', reverse, 5'-TCAGTTGGGAGCCTGATTCT-3' (153 bp); GADD153, forward, 5'-CCAGCAGAGGTCACAAGCAC-3', reverse, 5'-CGCACTGACCACTCTGTTTC-3' (125 bp); UCP-2, forward, 5'-CGAAATGCCATTGTCAACTG-3', reverse, 5'-CAAGGGAGGTCGTCTGTCAT-3' (100 bp); ATF-4, forward, 5'-GTTGGTCAGTGCCTCAGACA-3', reverse, 5'-CATTCGAAACAGAGCATCGA-3' (109 bp); and BiP, forward, 5'-CCACCAGGATGCAGACATTG-3', reverse, 5'-AGGGCCTCCACTTCCATAGA-3' (100 bp).
Analysis of XBP-1 activation and ATF-6 promoter activity
Alternative splicing of XBP-1 mRNA during ER stress leads to the loss of a Pst1 restriction site (58, 59), and the activation of this transcription factor can be evaluated by restriction analysis after PCR amplification. The XBP-1 cDNA fragment (601 bp) encompassing the region of restriction site was amplified by conventional PCR using previously described primers (58). The purified PCR product was incubated with the Pst1 restriction enzyme for 5 h at 37 C, followed by separation of the restriction digests on a 2% agarose gel with ethidium bromide. The gels were photographed under UV transillumination using a Digital Science DC 290 camera (Kodak, Rochester, NY) and quantified by densitometry. The amount of 601-bp material (indicative of XBP-1 activation and thus of ER stress) was expressed as a percent of the total amount of amplified material, considering the sum of the two bands in each lane as 100%.
For the determination of ATF-6 promoter activation, another indicator of ER stress, a reporter plasmid containing the luciferase gene under the control of five ATF-6 binding sites (60) was kindly provided by Prof. Prywes (Columbia University, New York, NY). Of note, it has been reported that this construct might also be activated by XBP-1 under some experimental conditions (61). INS-1E cells were cotransfected with luciferase test plasmids and pRL-CMV (Promega, Madison, WI) by lipofection with Lipofectamine 2000 (Invitrogen, Baesley, Scotland) (24), and luciferase activities were assayed with the dual-luciferase reporter assay system (Promega) as previously described (24, 26). Test values were corrected for the luciferase value of the internal control plasmid pRL-CMV.
EMSA and immunofluorescence of NF-B activation
Nuclear extracts were obtained from INS-1E cells as described (62, 63, 64). Extracted nuclear protein (4 µg) was preincubated for 10 min at 4 C with 1 µg of poly(dIdC) in 20 µl medium containing 10 mM HEPES (pH 7.9), 50 mM KCl, 5 mM MgCl2, 0.05 mM EDTA, 0.5 mM dithiothreitol, and 10% glycerol before addition of the radiolabeled probe (40,000 cpm) for the NF-B consensus sequence 5'-AGCTTCAGAGGGGACTTTCCGAGA (64). Specificity of protein-oligo binding was tested by addition of 50 molar excess of cold oligonucleotide. After 20 min incubation at 4 C, the samples were electrophoresed on 5% polyacrylamide gels in 25 mM Tris, 25 mM boric acid, and 0.5 mM EDTA.
NF-B nuclear translocation was also determined, as previously described (20, 65) by immunofluorescent staining using an anti-p65 antibody (sc-372x; Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:5000 dilution. The percentage of cells with nuclear staining (indicating NF-B activation) was determined in at least 200 cells by two observers, one of them blinded for sample identity.
Statistical analysis
Data are presented as means ± SE. Comparisons vs. the control conditions were performed by the Student’s paired t test or ANOVA followed by paired t test with the Bonferroni correction for multiple comparisons. P 
0.05 was considered statistically significant.
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). In the presence of 0.25 mM palmitate, apoptosis was detected from 24 h on. Palmitate induced more apoptosis than a similar concentration (0.5 mM) of oleate, and it was as cytotoxic to INS-1E cells as IL-1ß (30 U/ml). The percentage of necrotic cells did not change significantly after FFA treatment (data not shown). Exposure of rat islets to palmitate decreased islet cell viability only after 72 h (Fig. 2), whereas increased cell death in the presence of IL-1ß was already detected at 24 h (Fig. 2).
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potentiates the deleterious effect of FFAs on INS-1E and FACS-purified ß-cell viability. Confirming the findings shown in Figure 1
, treatment of INS-1E cells for 24–48 h with oleate or palmitate induced apoptosis. TNF alone induced a minor increase in apoptosis rates, which did not reach statistical significance (Fig. 3A). The combination of palmitate with TNF induced a mild increase in cell death after 24 h, compared with palmitate alone, whereas there was a TNF-induced increase in oleate-triggered apoptosis after 48 h only (Fig. 3A). Oleate and palmitate induced apoptosis in FACS-purified ß-cells, but this effect was not augmented by TNF (Fig. 3B). The observation of FFA-induced apoptosis in pure ß-cell preparations (Fig. 3B) (15) and clonal insulin-producing cells (Fig. 3A) strongly suggests that the deleterious effects of FFAs in whole islets (Fig. 2) occur at least in part at the ß-cell level. FFA-induced ß-cell apoptosis was confirmed by the cell death detection ELISAPLUS. After treatment of purified ß-cells with oleate or palmitate (same experimental conditions as in Fig. 3B), there was a respective 2.4 ± 0.4- and 4.1 ± 0.6-fold increase in apoptosis, compared with control (n = 4; P < 0.05), and similar results were obtained in INS-1E cells. In primary ß-cells, there was again no potentiating effect by TNF (2.7 ± 0.7- and 3.1 ± 0.8-fold increase in apoptosis for oleate + TNF and palmitate + TNF, NS vs. FFAs alone), whereas in INS-1E cells, TNF tended to increase oleate-mediated cell death by 50% at 48 h (data not shown).
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on FFA-induced primary ß-cell death, and the presence of only a minor additive effect on INS-1E cells at some time points, contrasts with previous observations showing that oleate increases by more than 2-fold IL-1ß toxicity in INS-1E cells (66). Both IL-1ß and TNF induce NF-B expression and NF-B-dependent genes in ß-cells, but the effects of TNF are less pronounced than the effects of IL-1ß (63, 67). Moreover, and as shown in the present study (Figs. 1 and 3), IL-1ß alone, but not TNF, induces INS-1E cell death. This might explain these somewhat divergent observations.
NF-B activation plays a crucial role for both IL-1ß + IFN- or IFN + double-stranded RNA-induced ß-cell death (18, 19, 20, 21). This, and the fact that microarray analysis of cytokine- or FFA-treated ß-cells indicated some similarities in the pattern of gene expression (22, 23, 26, 40, 41, 42), prompted us to assess the role of NF-B for FFA-induced ß-cell gene expression and apoptosis. In a first series of experiments, we studied two genes previously shown to be regulated by NF-B in ß-cells, namely iNOS and MCP-1 (24, 26). Because the deleterious effects of FFAs were detected earlier in INS-1E cells, compared with whole islets (see Figs. 1 and 2), we selected 6 h as the first time point for mRNA determination in INS-1E cells, whereas 24 h was selected for rat islets. iNOS gene expression was not induced after exposure of INS-1E cells to oleate or palmitate, whereas a clear induction was observed with the positive control IL-1ß (Fig. 4A). Similarly, rat islets exposed for 24 h to 0.5 mM oleate or palmitate did not increase iNOS expression, whereas IL-1ß induced iNOS mRNA (Fig. 4B). In line with these results, there was no increased accumulation of nitrite in the culture medium of FFA-treated cells, whereas IL-1ß induced a marked increase in nitrite production (data not shown).
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Oleate and palmitate also failed to modify MCP-1 mRNA expression. Thus, there was no induction of MCP-1 mRNA in INS-1E cells (Fig. 5A) or in rat islets (Fig. 5B) after 6–48 h exposure to FFAs. IL-1ß treatment, on the other hand, led to a clear induction of MCP-1 expression in both cell types, in good agreement with previous observations (25, 26, 52).
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B activation and NO production (22), and we next evaluated the effects of FFAs or IL-1ß on Pdx-1 mRNA expression. In line with our previous findings (22), there was a decrease in Pdx-1 expression in both INS-1 cells and pancreatic islets after 24 h treatment with IL-1ß (Fig. 6). On the other hand, neither oleate nor palmitate decreased Pdx-1 expression (Fig. 6).
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The data presented above indicate that FFAs do not induce the expression of NF-B-dependent genes in INS-1E cells or pancreatic islets. The fact that palmitate and oleate, under similar experimental conditions, induced both UCP-2 mRNA expression and apoptosis in these cells confirms that the FFAs presently used are biologically active. Moreover, the observation that IL-1ß affects expression of iNOS, MCP-1, and Pdx-1, as previously described (22, 23, 24, 25, 26), confirms that the primers and RT-PCR conditions used for the detection of these mRNAs were adequate. Thus, lack of FFA-induced iNOS or MCP-1 expression is a true biological phenomenon and not an artifact induced by inadequate experimental conditions.
We next performed EMSA and immunofluorescence to determine the nuclear translocation of activated NF-B and thus directly assess a putative involvement of NF-B on the effects of FFA. Neither oleate nor palmitate induced NF-B activation in INS-1E cells examined after 30 min, 4 h, or 12 h of FFA exposure (Fig. 7, lanes 2 and 3). In contrast, specific DNA binding by nuclear NF-B was observed at all time points studied in INS-1E cells exposed to TNF (used as a positive control for NF-B activation) (Fig. 7, lane 4). The specificity of DNA binding was confirmed by competition between labeled and cold target sequence in 50-fold molar excess in cells treated with TNF for 30 min (Fig. 7, lane 7) and by supershift after incubation with antibodies directed against either the NF-B p50 or p65 subunits (data not shown). The time course observed for TNF-induced NF-B activation, namely early (30 min) activation followed by progressive decrease after 4–12 h, is consistent with our previous observations (62, 63). The addition of FFAs to TNF did not affect the magnitude or time course of NF-B activation (Fig. 7, lanes 5 and 6), arguing against a synergistic effect between FFAs and TNF on NF-B activation. These observations were confirmed by immunostaining for subcellular localization of p65 (20), an NF-B subunit, in primary ß-cells (Fig. 8) and INS-1E cells (data not shown). A 30-min exposure to IL-1ß or TNF induced NF-B nuclear migration in nearly 90% of the ß-cells, whereas no activation of NF-B was observed with oleate or palmitate at 30 min or at 12 h (Fig. 8). Moreover, and in line with the gel shift results (Fig. 7), the FFAs did not modify TNF-induced NF-B activation after 30 min or 12 h. Similar results were obtained in INS-1E cells after 30 min, 4 h, or 12 h of exposure to FFA and/or TNF (data not shown). These observations, using two complementary methods to determine NF-B activation and taken together with the mRNA expression data (see above), make it unlikely that NF-B activation plays a role in FFA-induced ß-cell dysfunction and death.
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), whereas palmitate effects were most marked after 48 h (Fig. 9). GADD153/CHOP induction by IL-1ß was already present after 6 h (Fig. 9). This effect of IL-1ß was potentiated by the addition of IFN, leading to a 4- to 5-fold increase in GADD153/CHOP, compared with control cells (data not shown).
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). In view of the induction of the downstream ER stress effectors GADD153/CHOP and ATF-4, we examined whether other specific ER stress mediators such as XBP-1, ATF-6, and BiP were also activated in ß-cells by FFAs.
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) by its PCR amplification followed by incubation with Pst1. In the control condition, most of the XBP-1 PCR product was cut by the Pst1 restriction enzyme into two fragments of 300 bp, corresponding to native form of XBP-1, and only 23 ± 2% and 27 ± 1% of the total were detected as the 601-bp form at 24 and 48 h, respectively (n = 3). In INS-1E cells exposed to FFAs, a larger proportion of the XBP-1 PCR product kept its full 601-bp length (for 0.5 mM oleate 50 ± 5% at 24 h and 42 ± 4% at 48 h, n = 3, P < 0.05 vs. control; for 0.5 mM palmitate 56 ± 3% at 24 h and 51 ± 6% at 48 h, n = 3, P < 0.05 vs. control), indicating partial XBP-1 activation (Fig. 11) and presence of ER stress. The combination of cytokines (IL-1ß + IFN) as well as thapsigargin, which blocks SERCA-2b (31), was used as positive controls for an ER stress response. These compounds also induced a clear XBP-1 activation (for cytokines 50 ± 2% at 24 h and 50 ± 3% at 48 h, n = 2; for thapsigargin 84 ± 2% at 24 h and 82 ± 1% at 48 h, n = 2), more pronounced in the case of thapsigargin than the activation induced by FFA (Fig. 11).
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). The ER chaperone BiP is a known target of ATF-6 (74), and, in agreement with the ATF-6 promoter data, INS-1E cells exposed for 24 or 48 h to FFA had a 2-fold increase in BiP mRNA expression (Fig. 13).
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It has been previously shown that NO production, followed by SERCA2b inhibition, is the main mechanism by which cytokines cause ER stress in ß-cells (22, 27, 28) (Cardozo, A. K., F. Ortis, Y.-M. Feng, J. Rasschaert, F. Van Eylen, J. Storling, T. Mandrup-Poulsen, A. Herchuelz, and D. L. Eizirik, submitted for publication). As discussed above, FFAs induce neither iNOS expression nor NO production in ß-cells, suggesting that FFAs induce ER stress by a different mechanism. Oleate and palmitate have been shown to affect Ca2+ homeostasis in the ER in other cell types (76, 77, 78), which might impair the processing and folding of proteins (30, 58) and contribute to ER stress. Additional studies are required to clarify the mechanisms involved in FFA-induced ER stress.
Previous studies have reported similarities between nutrient- and cytokine-induced ß-cell apoptosis (35, 44, 45, 46), and the hypothesis of a common cell death mechanism involving activation of NF-B has been proposed (43, 45, 46). Against this background, we presently compared the mechanisms involved in FFA-induced ß-cell death with the mechanisms of ß-cell death induced by IL-1ß and, in some experiments, IL-1ß + IFN. The main conclusions of this study are the following: 1) cytokines, but not FFAs, induce expression of the NF-B-dependent genes iNOS and MCP-1; 2) cytokines, but not FFAs, induce NF-B activation; 3) FFAs do not potentiate TNF-induced NF-B activation; and 4) both FFAs and cytokines induce ER stress in INS-1E cells, but ER stress seems to be triggered by different mechanisms. These observations indicate that FFAs and cytokines lead to ß-cell death by fundamentally distinct mechanisms, namely an NF-B-dependent mechanism that culminates in caspase-3 activation in the case of cytokines (18) and an NF-B-independent mechanism in the case of FFAs. FFA-induced apoptosis may be mediated by activation of caspases via other pathways (34), but recent evidence indicates that FFA-induced ß-cell death may be related to calpain-10 activation and does not necessarily require caspase-3 (39). As a whole, the present findings argue against a unifying hypothesis for the mechanisms of ß-cell death in T1DM and T2DM. Moreover, and if the present in vitro observations are indeed representative of the clinical situation, the findings suggest that different approaches should be pursued when attempting to prevent ß-cell death in T1DM and T2DM.
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Acknowledgments |
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Footnotes |
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Abbreviations: ATF, Activating transcription factor; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; FACS, fluorescence-activated cell sorting; FFA, free fatty acid; GADD, growth arrest and DNA damage; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO 342, Hoechst 33342; IFN, interferon; iNOS, inducible nitric oxide synthase; INS, insulinoma; MCP, monocyte chemoattractant protein; NF-B, nuclear factor-B; NO, nitric oxide; Pdx-1, pancreatic duodenal homeobox-1; PI, propidium iodide; SERCA-2b, sarcoendoplasmic reticulum Ca2+ ATPase type 2b; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus; UCP, uncoupling protein-2; XBP-1, X-box binding protein-1.
Received April 14, 2004.
Accepted for publication July 30, 2004.
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References |
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S.-E Choi, S.-M. Lee, Y.-J. Lee, L.-J. Li, S.-J. Lee, J.-H. Lee, Y. Kim, H.-S. Jun, K.-W. Lee, and Y. Kang Protective Role of Autophagy in Palmitate-Induced INS-1 {beta}-Cell Death Endocrinology, January 1, 2009; 150(1): 126 - 134. [Abstract] [Full Text] [PDF]
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T. Hartley, J. Brumell, and A. Volchuk Emerging roles for the ubiquitin-proteasome system and autophagy in pancreatic {beta}-cells Am J Physiol Endocrinol Metab, January 1, 2009; 296(1): E1 - E10. [Abstract] [Full Text] [PDF]
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 M. C. Akerfeldt, J. Howes, J. Y. Chan, V. A. Stevens, N. Boubenna, H. M. McGuire, C. King, T. J. Biden, and D. R. Laybutt Cytokine-Induced {beta}-Cell Death Is Independent of Endoplasmic Reticulum Stress Signaling Diabetes, November 1, 2008; 57(11): 3034 - 3044. [Abstract] [Full Text] [PDF]
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C. S. Nunemaker, M. Chen, H. Pei, S. D. Kimble, S. R. Keller, J. D. Carter, Z. Yang, K. M. Smith, R. Wu, M. H. Bevard, et al. 12-Lipoxygenase-knockout mice are resistant to inflammatory effects of obesity induced by western diet Am J Physiol Endocrinol Metab, November 1, 2008; 295(5): E1065 - E1075. [Abstract] [Full Text] [PDF]
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 K. Larade, Z. Jiang, Y. Zhang, W. Wang, S. Bonner-Weir, H. Zhu, and H. F. Bunn Loss of Ncb5or Results in Impaired Fatty Acid Desaturation, Lipoatrophy, and Diabetes J. Biol. Chem., October 24, 2008; 283(43): 29285 - 29291. [Abstract] [Full Text] [PDF]
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E. Sargsyan, H. Ortsater, K. Thorn, and P. Bergsten Diazoxide-induced {beta}-cell rest reduces endoplasmic reticulum stress in lipotoxic {beta}-cells J. Endocrinol., October 1, 2008; 199(1): 41 - 50. [Abstract] [Full Text] [PDF]
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 D. A. Cunha, P. Hekerman, L. Ladriere, A. Bazarra-Castro, F. Ortis, M. C. Wakeham, F. Moore, J. Rasschaert, A. K. Cardozo, E. Bellomo, et al. Initiation and execution of lipotoxic ER stress in pancreatic {beta}-cells J. Cell Sci., July 15, 2008; 121(14): 2308 - 2318. [Abstract] [Full Text] [PDF]
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 K. D. Jeffrey, E. U. Alejandro, D. S. Luciani, T. B. Kalynyak, X. Hu, H. Li, Y. Lin, R. R. Townsend, K. S. Polonsky, and J. D. Johnson Carboxypeptidase E mediates palmitate-induced {beta}-cell ER stress and apoptosis PNAS, June 17, 2008; 105(24): 8452 - 8457. [Abstract] [Full Text] [PDF]
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E. Diakogiannaki, H. J Welters, and N. G Morgan Differential regulation of the endoplasmic reticulum stress response in pancreatic {beta}-cells exposed to long-chain saturated and monounsaturated fatty acids J. Endocrinol., June 1, 2008; 197(3): 553 - 563. [Abstract] [Full Text] [PDF]
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 V. Poitout and R. P. Robertson Glucolipotoxicity: Fuel Excess and {beta}-Cell Dysfunction Endocr. Rev., May 1, 2008; 29(3): 351 - 366. [Abstract] [Full Text] [PDF]
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D. Scheuner and R. J. Kaufman The Unfolded Protein Response: A Pathway That Links Insulin Demand with {beta}-Cell Failure and Diabetes Endocr. Rev., May 1, 2008; 29(3): 317 - 333. [Abstract] [Full Text] [PDF]
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S. C. Martinez, K. Tanabe, C. Cras-Meneur, N. A. Abumrad, E. Bernal-Mizrachi, and M. A. Permutt Inhibition of Foxo1 Protects Pancreatic Islet {beta}-Cells Against Fatty Acid and Endoplasmic Reticulum Stress-Induced Apoptosis Diabetes, April 1, 2008; 57(4): 846 - 859. [Abstract] [Full Text] [PDF]
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E. Lai, G. Bikopoulos, M. B. Wheeler, M. Rozakis-Adcock, and A. Volchuk Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic {beta}-cells Am J Physiol Endocrinol Metab, March 1, 2008; 294(3): E540 - E550. [Abstract] [Full Text] [PDF]
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D. L. Eizirik, A. K. Cardozo, and M. Cnop The Role for Endoplasmic Reticulum Stress in Diabetes Mellitus Endocr. Rev., February 1, 2008; 29(1): 42 - 61. [Abstract] [Full Text] [PDF]
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 Y. Kamijo, K. Hora, K. Kono, K. Takahashi, M. Higuchi, T. Ehara, K. Kiyosawa, H. Shigematsu, F. J. Gonzalez, and T. Aoyama PPAR{alpha} Protects Proximal Tubular Cells from Acute Fatty Acid Toxicity J. Am. Soc. Nephrol., December 1, 2007; 18(12): 3089 - 3100. [Full Text] [PDF]
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 M. E. Tushuizen, M. C. Bunck, P. J. Pouwels, S. Bontemps, J. H. T. van Waesberghe, R. K. Schindhelm, A. Mari, R. J. Heine, and M. Diamant Pancreatic Fat Content and {beta}-Cell Function in Men With and Without Type 2 Diabetes Diabetes Care, November 1, 2007; 30(11): 2916 - 2921. [Abstract] [Full Text] [PDF]
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 M. F. Gregor and G. S. Hotamisligil Thematic review series: Adipocyte Biology. Adipocyte stress: the endoplasmic reticulum and metabolic disease J. Lipid Res., September 1, 2007; 48(9): 1905 - 1914. [Abstract] [Full Text] [PDF]
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M. J. Pagliassotti, Y. Wei, and D. Wang Insulin Protects Liver Cells from Saturated Fatty Acid-Induced Apoptosis via Inhibition of c-Jun NH2 Terminal Kinase Activity Endocrinology, July 1, 2007; 148(7): 3338 - 3345. [Abstract] [Full Text] [PDF]
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S. S. Choe, A H. Choi, J.-W. Lee, K. H. Kim, J.-J. Chung, J. Park, K.-M. Lee, K.-G. Park, I.-K. Lee, and J. B. Kim Chronic Activation of Liver X Receptor Induces {beta}-Cell Apoptosis Through Hyperactivation of Lipogenesis: Liver X Receptor-Mediated Lipotoxicity in Pancreatic {beta}-Cells Diabetes, June 1, 2007; 56(6): 1534 - 1543. [Abstract] [Full Text] [PDF]
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 S. Horke, I. Witte, P. Wilgenbus, M. Kruger, D. Strand, and U. Forstermann Paraoxonase-2 Reduces Oxidative Stress in Vascular Cells and Decreases Endoplasmic Reticulum Stress-Induced Caspase Activation Circulation, April 17, 2007; 115(15): 2055 - 2064. [Abstract] [Full Text] [PDF]
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P. Pirot, F. Ortis, M. Cnop, Y. Ma, L. M. Hendershot, D. L. Eizirik, and A. K. Cardozo Transcriptional Regulation of the Endoplasmic Reticulum Stress Gene Chop in Pancreatic Insulin-Producing Cells Diabetes, April 1, 2007; 56(4): 1069 - 1077. [Abstract] [Full Text] [PDF]
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M. Cnop, L. Ladriere, P. Hekerman, F. Ortis, A. K. Cardozo, Z. Dogusan, D. Flamez, M. Boyce, J. Yuan, and D. L. Eizirik Selective Inhibition of Eukaryotic Translation Initiation Factor 2{alpha} Dephosphorylation Potentiates Fatty Acid-induced Endoplasmic Reticulum Stress and Causes Pancreatic beta-Cell Dysfunction and Apoptosis J. Biol. Chem., February 9, 2007; 282(6): 3989 - 3997. [Abstract] [Full Text] [PDF]
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N. M. Borradaile, X. Han, J. D. Harp, S. E. Gale, D. S. Ory, and J. E. Schaffer Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death J. Lipid Res., December 1, 2006; 47(12): 2726 - 2737. [Abstract] [Full Text] [PDF]
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S. M. Turpin, G. I. Lancaster, I. Darby, M. A. Febbraio, and M. J. Watt Apoptosis in skeletal muscle myotubes is induced by ceramides and is positively related to insulin resistance Am J Physiol Endocrinol Metab, December 1, 2006; 291(6): E1341 - E1350. [Abstract] [Full Text] [PDF]
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 F. Ortis, A. K. Cardozo, D. Crispim, J. Storling, T. Mandrup-Poulsen, and D. L. Eizirik Cytokine-Induced Proapoptotic Gene Expression in Insulin-Producing Cells Is Related to Rapid, Sustained, and Nonoscillatory Nuclear Factor-{kappa}B Activation Mol. Endocrinol., August 1, 2006; 20(8): 1867 - 1879. [Abstract] [Full Text] [PDF]
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E. Karaskov, C. Scott, L. Zhang, T. Teodoro, M. Ravazzola, and A. Volchuk Chronic Palmitate But Not Oleate Exposure Induces Endoplasmic Reticulum Stress, Which May Contribute to INS-1 Pancreatic {beta}-Cell Apoptosis Endocrinology, July 1, 2006; 147(7): 3398 - 3407. [Abstract] [Full Text] [PDF]
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L. I. Rachek, N. P. Thornley, V. I. Grishko, S. P. LeDoux, and G. L. Wilson Protection of INS-1 Cells From Free Fatty Acid-Induced Apoptosis by Targeting hOGG1 to Mitochondria. Diabetes, April 1, 2006; 55(4): 1022 - 1028. [Abstract] [Full Text] [PDF]
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 J R Porter and T G Barrett Monogenic syndromes of abnormal glucose homeostasis: clinical review and relevance to the understanding of the pathology of insulin resistance and {beta} cell failure J. Med. Genet., December 1, 2005; 42(12): 893 - 902. [Abstract] [Full Text] [PDF]
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M. Cnop, N. Welsh, J.-C. Jonas, A. Jorns, S. Lenzen, and D. L. Eizirik Mechanisms of Pancreatic {beta}-Cell Death in Type 1 and Type 2 Diabetes: Many Differences, Few Similarities Diabetes, December 1, 2005; 54(suppl_2): S97 - S107. [Abstract] [Full Text] [PDF]
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E. H. Leiter and C.-H. Lee Mouse Models and the Genetics of Diabetes: Is There Evidence for Genetic Overlap Between Type 1 and Type 2 Diabetes? Diabetes, December 1, 2005; 54(suppl_2): S151 - S158. [Abstract] [Full Text] [PDF]
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J. G. Granneman, P. Li, Z. Zhu, and Y. Lu Metabolic and cellular plasticity in white adipose tissue I: effects of {beta}3-adrenergic receptor activation Am J Physiol Endocrinol Metab, October 1, 2005; 289(4): E608 - E616. [Abstract] [Full Text] [PDF]
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D. K. Hagman, L. B. Hays, S. D. Parazzoli, and V. Poitout{paragraph} Palmitate Inhibits Insulin Gene Expression by Altering PDX-1 Nuclear Localization and Reducing MafA Expression in Isolated Rat Islets of Langerhans J. Biol. Chem., September 16, 2005; 280(37): 32413 - 32418. [Abstract] [Full Text] [PDF]
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H. Wang, G. Kouri, and C. B. Wollheim ER stress and SREBP-1 activation are implicated in {beta}-cell glucolipotoxicity J. Cell Sci., September 1, 2005; 118(17): 3905 - 3915. [Abstract] [Full Text] [PDF]
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S.-J. Kim, K. Winter, C. Nian, M. Tsuneoka, Y. Koda, and C. H. S. McIntosh Glucose-dependent Insulinotropic Polypeptide (GIP) Stimulation of Pancreatic {beta}-Cell Survival Is Dependent upon Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase B (PKB) Signaling, Inactivation of the Forkhead Transcription Factor Foxo1, and Down-regulation of bax Expression J. Biol. Chem., June 10, 2005; 280(23): 22297 - 22307. [Abstract] [Full Text] [PDF]
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