Báo cáo khoa học: Peroxisome proliferator-activated receptor a–retinoid X receptor agonists induce beta-cell protection against palmitate toxicity doc

Peroxisome proliferator-activated receptor a–retinoid X receptor agonists induce beta-cell protection against palmitate toxicity Karine Hellemans1, Karen Kerckhofs1, Jean-Claude Hannaert1, Geert Martens1, Paul Van Veldhoven2 and Daniel Pipeleers1 Diabetes Research Center (DRC), Brussels Free University-VUB, Belgium Afdeling Farmakologie, Departement Moleculaire Celbiologie, K U Leuven, Belgium Keywords cytotoxicity; fibrate; free fatty acid; pancreatic beta-cells; peroxisome proliferator-activated receptor a Correspondence K Hellemans, Diabetes Research Center, Brussels Free University-VUB, Laarbeeklaan 103, 1090 Brussels, Belgium Fax: +32 4774545 Tel: +32 4774541 E-mail: karine.hellemans@vub.ac.be (Received 11 July 2007, revised October 2007, accepted October 2007) doi:10.1111/j.1742-4658.2007.06131.x Fatty acids can stimulate the secretory activity of insulin-producing betacells At elevated concentrations, they can also be toxic to isolated betacells This toxicity varies inversely with the cellular ability to accumulate neutral lipids in the cytoplasm To further examine whether cytoprotection can be achieved by decreasing cytoplasmic levels of free acyl moieties, we investigated whether palmitate toxicity is also lowered by stimulating its b-oxidation Lower rates of palmitate-induced beta-cell death were measured in the presence of l-carnitine as well as after addition of peroxisome proliferator-activated receptor a (PPARa) agonists, conditions leading to increased palmitate oxidation In contrast, inhibition of mitochondrial b-oxidation by etomoxir increased palmitate toxicity A combination of PPARa and retinoid X receptor (RXR) agonists acted synergistically and led to complete protection; this was associated with enhanced expression levels of genes involved in mitochondrial and peroxisomal b-oxidation, lipid metabolism, and peroxisome proliferation PPARa–RXR protection was abolished by the carnitine palmitoyl transferase inhibitor etomoxir These observations indicate that PPARa and RXR regulate beta-cell susceptibility to long-chain fatty acid toxicity by increasing the rates of b-oxidation and by involving peroxisomes in fatty acid metabolism Under normal circumstances, long-chain fatty acids serve as regulators of beta-cell function [1,2] At sustained, elevated concentrations, they can exert cytotoxic actions on beta-cells, and this has led to the view that they could be, at least in part, responsible for the loss of beta-cells in diabetes [3,4] Several in vitro and in vivo studies have supported this lipotoxicity concept and extended into experiments aimed at pharmacologic prevention of this process [3,5] We previously reported that pancreatic beta-cells possess defense mechanisms against oxidative damage [6] and could be induced to provide cytoprotection [7] It is still uncertain whether they also exhibit such properties when exposed to cytotoxic fatty acid concentrations, and if so, whether they can be activated through similar or other mechanisms Indirect evidence for the presence of such a protective mechanism comes from the observation that fatty acid-induced toxicity was limited to a subpopulation of beta-cells, and apparently related to the cellular ability to accumulate neutral lipids in the cytoplasm [7] We thus proposed that the formation of cytoplasmatic lipids could reduce fatty acid-induced toxicity by Abbreviations CPT1, carnitine palmitoyl transferase 1, liver; GPAT, glycerol-3-phosphate acyltransferase, mitochondrial; Pex2, peroxisomal biogenesis factor 2; Pex3, peroxisomal biogenesis factor 3; Pex11a, peroxisomal biogenesis factor 11a; Pex14, peroxisomal biogenesis factor 14; Pex16, peroxisomal biogenesis factor 16; PPARa, peroxisome proliferator-activated receptor a; PMP70, peroxisomal membrane protein 70; RA, retinoic acid; RXRa, retinoid X receptor; SCD1, stearoyl-CoA desaturase 1; SCD2, stearoyl-CoA desaturase 6094 FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS K Hellemans et al Results Specificity of palmitate toxicity When rat beta-cells were cultured with palmitate, timeand concentration-dependent cytotoxicity was measured At 50 and 100 lm, no toxic effect was detected after days, and only 10–20% cells were damaged after days (data not shown) At 250 and 500 lm, cytotoxicity was noticed after days and resulted, after days, in, respectively, 38 ± 2% and 75 ± 4% dead cells (Fig 1) It was noticed that with exposure longer than days at 500 lm, the slope of the toxicity curve declined The percentage of surviving cells tended to stabilize around 25% after days, despite administration of a new bolus of fatty acid every 48 h With 250 lm, the fraction of cell survival stabilized around 60% The cytotoxic effect of palmitate did not vary with the glucose concentration in the medium (comparison of 5, 10 and 20 mm glucose, data not shown) Subsequent studies were conducted at 10 mm glucose for days with 500 lm palmitate (acute toxicity) and for days at 250 lm (chronic toxicity) Islet endocrine nonbeta-cells exhibited lower susceptibility to palmitate toxicity: cytotoxicity was ± 0.5% after days at 500 lm, and 16 ± 3% after 100 Cytotoxicity (%) preventing a rise in toxic free acyl moieties [7] and ⁄ or fatty acid metabolites such as ceramides [8,9] Along this line, one can further hypothesize that an increased rate of fatty acid oxidation also lowers the formation of these cytotoxic mediators and could thus also act as a cytoprotective mechanism To test this hypothesis, we examined whether palmitate toxicity can be reduced by increasing its oxidation rates We first assessed whether protection could be conferred by l-carnitine, a rate-limiting component for long-chain fatty acid transport into the mitochondria, or suppressed by etomoxir, an irreversible carnitine palmitoyl transferase (CPT1) inhibitor [10] In a second set of experiments, we assessed the effects of agonists for peroxisome proliferator-activated receptor a (PPARa) and retinoid X receptor (RXR) PPARa–RXR dimers can be activated by both PPARa and RXR agonists PPARa–RXR dimers typically regulate the expression of multiple genes involved in mitochondrial and peroxisomal b-oxidation as well as lipoprotein metabolism [11] PPARa is expressed in primary rat beta-cells [12], and has been shown to activate fatty acid oxidation [13,14] PPARa agonists have been reported to prevent fatty acid-induced beta-cell dysfunction and apoptosis in human islets [15], and improve beta-cell function in insulin-resistant rodent models [16] Beta-cell protection against palmitate toxicity 75 250 M C16:0 500 M C16:0 50 25 Days of exposure 10 Fig Time course analysis for palmitate cytotoxicity Primary rat beta-cells were exposed to 250 or 500 lM palmitate Cytotoxicity was measured on a daily basis (n ¼ 4, vertical bars represent SEM) days at 250 lm After days at 500 lm, the cytotoxicity increased to 46 ± 8% (results not shown) In the latter condition, more than 95% of purified beta-cells died, which means that approximately 20% of the dead cells in the nonbeta-cell fraction correspond to beta-cells, as the nonbeta-cell fraction is contaminated to this extent by beta-cells; consequently, the palmitate toxicity for the islet nonbeta-cells is calculated to be about 25% after days at 500 lm Palmitate toxicity in beta-cells was compared with that of equimolar concentrations of other fatty acids in the presence of 1% BSA (Table 1) Oleate, an unsaturated long-chain fatty acid, was less toxic, and the shorter-chain molecules butyrate (C4), hexanoate (C6) and octanoate (C8) were only marginally toxic The 2-methyl and 3-methyl derivatives of palmitate were virtually nontoxic: < 5% after days and < 10% after days, both at 250 and at 500 lm, whereas Table Cytotoxicity of fatty acids for rat beta-cells Beta-cells were exposed to the following fatty acids at 500 lM for days or at 250 lM for days: palmitate (C16:0), oleate (C18:1), butyrate (C4:0), hexanoate (C6:0), octanoate (C8:0), 2-methylhexadecanoic acid (2-Me-C16:0), 3-methylhexadecanoic acid (3-Me-C16:0), 2-bromopalmitate (Br-C16:0) Data represent means ± SD (n ¼ 3–6) *P < 0.001 as compared to C16:0 (student’s t-test) Percentage cytotoxicity Fatty acid 500 lM for days 250 lM for days C16:0 C18:1 C4:0 C6:0 C8:0 2-Me-C16:0 3-Me-C16:0 Br-C16:0 24 11 58 38 28 11 92 FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS ± ± ± ± ± ± ± ± 2* 2* 1* 1* 1* 4* 6* ± ± ± ± ± ± ± ± 3* 1* 1* 1* 4* 4* 3* 6095 Beta-cell protection against palmitate toxicity K Hellemans et al Table Metabolism of [14C]palmitate and [14C]glucose in beta-cells and islet nonbeta-cells at 3.3 and 20 mM glucose Palmitate and glucose metabolism was measured in freshly isolated beta-cells and islet nonbeta-cells incubated for h with 50 lM [14C]palmitate or lCi of 14 14 D-[U- C]glucose C incorporation was measured in CO2 and small metabolic intermediates, as well as in the lipid and protein fractions and expressed as (pmol per h per 103 cells) Data are indicated as mean ± SD (n ¼ 5) Italic: P < 0.05 for 20 mM glucose as compared to 3.3 mM glucose *P < 0.05 for nonbeta-cells as compared to beta-cells, **P < 0.001 for nonbeta-cells as compared to beta-cells (Student’s t-test) 14 C recovery as: CO2 From [14C]palmitate Beta-cells Glucose 3.3 mM Glucose 20 mM Nonbeta-cells Glucose 3.3 mM Glucose 20 mM From [14C]glucose Beta-cells Glucose 3.3 mM Glucose 20 mM Nonbeta-cells Glucose 3.3 mM Glucose 20 mM Intermediates Protein 0.52 ± 0.04 0.10 ± 0.01 0.62 ± 0.05 0.06 ± 0.01 1.71 ± 0.11 1.73 ± 0.04 0.21 ± 0.06 0.13 ± 0.08 0.63 ± 0.04* 0.46 ± 0.04* 0.18 ± 0.003* 0.10 ± 0.01 1.18 ± 0.04* 1.06 ± 0.07* 0.13 ± 0.08 0.07 ± 0.03 0.29 ± 0.08 2.16 ± 0.72 0.40 ± 0.02 3.16 ± 0.99 0.13 ± 0.03* 1.13 ± 0.55 0.19 ± 0.04* 0.45 ± 0.07** 5.49 ± 0.31 31.15 ± 1.86 4.41 ± 0.17 19.90 ± 1.96 0.96 ± 0.22** 3.82 ± 0.95** 1.20 ± 0.11** 3.78 ± 0.48** 2-bromopalmitate was markedly more toxic than palmitate There was no difference in the percentage of living cells following vehicle treatment when compared to the standard control Palmitate and glucose metabolism by beta-cells and islet nonbeta-cells Palmitate and glucose metabolism was measured in freshly isolated beta-cells and in islet nonbeta-cells that were incubated for h with 50 lm [14C]palmitate or lCi of d-[U-14C]glucose 14C incorporation was measured in CO2 and small metabolic intermediates, as well as in the lipid and protein fractions (Table 2) After incubation with [14C]palmitate at 3.3 mm glucose, the largest amount was recovered in the lipid-soluble fraction (55%) and the lowest in the protein fraction, whereas comparable amounts were converted to CO2 (17%) and to small metabolic intermediates (20%); at 20 mm glucose, the total amounts in the lipid fraction (representing 82%) and protein fraction were not significantly different from those at mm glucose, but the production of 14CO2 and of 14C intermediates was, respectively, five- and 10-fold lower (Table 2) Analysis of the fate of [14C]glucose indicated that, at 3.3 mm glucose, the tracer was converted to CO2 and to small intermediates, and that high glucose increased this rate five-fold, and also increased seven-fold the 14C incorporation into the lipid and protein fraction Islet endocrine nonbeta-cells exhibit much lower rates of glucose oxidation and utilization than islet 6096 Lipid beta-cells; the values shown in Table are an overestimation, in view of the contamination of this fraction by 20–25% beta-cells; the CO2 production from glucose that is calculated for islet nonbeta-cells is thus less than 10% of that in beta-cells On the other hand, their level of CO2 production from palmitate is higher, in particular at 20 mm glucose, where four-fold higher rates were measured than in beta-cells (Table 2) In contrast to the situation in beta-cells, 30% of the labeled palmitate was converted to CO2 independently of the glucose concentration Effect of regulators of palmitate metabolism on palmitate toxicity in beta-cells When beta-cells were precultured for 24 h with the CPT1 activator l-carnitine before measurement of their palmitate oxidation during h of incubation in the further presence of l-carnitine, 14CO2 formation was six-fold higher (to 0.83 ± 0.14 pmol per h per 103 cells, n ¼ 4, P < 0.05) than in control cells cultured and incubated with the solvent (0.12 ± 0.03) This stimulatory effect was preserved when 250 lm palmitate was added to the preculture medium (0.78 ± 0.19 pmol per h per 103 cells, n ¼ 4, P < 0.01) (Fig 2A) It was associated with an eight-fold elevation of 14C incorporation into metabolic intermediates (from 0.15 ± 0.05 to 0.95 ± 0.07 pmol per h per 103 cells, P < 0.05) (results not shown) Preculture with mm l-carnitine protected beta-cells from palmitate toxicity during a FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS K Hellemans et al B 50 vehicle L-carnitine C16:0 + L-carnitine ** 1.00 ** 0.50 30 *** 20 10 *** 0.00 500 C % Cytotoxicity 60 Effect of PPARa–RXR agonists on palmitate cytotoxicity C16:0 C16:0 + L-carnitine 40 % Cytotoxicity Palmitate oxidation (pmol/Kc/2h) A 1.50 Beta-cell protection against palmitate toxicity M Fatty acid Fatty acid + etomoxir - 2d 250 M - 8d ** *** 40 20 500 M - 2d 250 C4:0 M - 8d 500 M 250 - 2d C16:0 M - 8d Fig Effect of regulators of fatty acid metabolism on fatty acid toxicity (A) Effect of L-carnitine on palmitate oxidation Beta-cells were precultured for 24 h with the CPT1 activator L-carnitine (1 mM) in the absence or presence of 250 lM palmitate before measurement of [14C]palmitate oxidation during a h incubation in the further presence of L-carnitine (B) Effect of L-carnitine on palmitate cytotoxicity Cells were exposed for or days to 500 or 250 lM palmitate in the presence of L-carnitine following 24 h of preculture with mM L-carnitine (C) Effect of a CPT1 inhibitor on fatty acid toxicity Cells were exposed for or days to 500 or 250 lM butyrate or palmitate alone, or in combination with 200 lM etomoxir Data are presented as mean ± SEM (n > 4); **P < 0.01, ***P < 0.001 (Student’s t-test) subsequent exposure to 500 lm palmitate for days or 250 lm for days by, respectively, 70% and 40% (Fig 2B) On the other hand, when beta-cells were exposed to palmitate in the presence of the CPT1 inhibitor etomoxir (200 lm) (Fig 2C), their survival was further decreased Lower concentrations of etomoxir (1, and 50 lm), which are known to stimulate PPARa, failed to show an effect on palmitate toxicity Etomoxir (200 lm) did not affect beta-cell survival in the presence of butyrate (C4), which is known to enter mitochondria independently of CPT1 Addition of l-cycloserine (100 lm), an inhibitor of serine palmitoyl transferase, was also found to reduce palmitate toxicity, both after days at 500 lm (from 24 ± 2% to 10 ± 1%), and after days at 250 lm (from 38 ± 2% to 20 ± 4%), but not that of oleate (results not shown) The effects of PPARa–RXR agonists clofibrate, ciprofibrate and 9-cis-retinoic acid (9-cis-RA) were examined by adding these compounds to the and day culture media Clofibrate alone (tested at 100, 250 and 500 lm, two-way ANOVA, P < 0.001) reduced palmitate toxicity at both time points, with a maximal effect at 250 lm (Fig 3A) Protection from palmitate toxicity was also observed after treatment with 9-cis-RA alone at all tested concentrations (0.5, or lm, two-way ANOVA, P < 0.001); at lm, the effect was comparable to that of 250 lm clofibrate, namely 60% protection after days at 500 lm and 70% after days at 250 lm (Fig 3A) Combinations of different concentrations of both agents further reduced palmitate toxicity; the maximal level of protection, reducing palmitate-induced cell death to only 5%, was reached using a combination of lm 9-cis-RA and 100 lm clofibrate Higher clofibrate concentrations in the presence of 9-cis-RA did not lower palmitate toxicity any further (Fig 3A) For all further experiments, a combination of 250 lm clofibrate and lm 9-cis-RA was used, resulting in a reduction of palmitate toxicity by 90% (Fig 3B) The efficacy of this combination did not significantly differ from that of 250 lm clofibrate plus lm 9-cis-RA The same level of protection as found in the presence of 10 mm glucose was found at low (5 mm) and high (20 mm) glucose concentrations (results not shown) Under the same conditions, clofibrate and 9-cis-RA were also found to protect against oleate toxicity (from 25 ± 3% to 13 ± 2% for 500 lm after days, and from 32 ± 3% to 14 ± 4% for 250 lm after days, P < 0.01, results not shown) Ciprofibrate (10, 50 and 100 lm) mimicked the effect of clofibrate, with a maximal effect at 100 lm, and similar additive protection by 9-cis-RA (Fig 3B) Addition of fibrate and ⁄ or 9-cis-RA to palmitate-free control medium did not influence cell survival during culture (data not shown) When endocrine nonbeta-cells were exposed to 500 lm palmitate for days with or without 250 lm clofibrate plus lm 9-cis-RA, no differences in toxicity were noticed, indicating the absence of a protective effect of the supplement (results not shown) Effect of PPARa–RXR agonists on palmitate metabolism Preculture (24 h) of beta-cells with 250 lm clofibrate plus lm 9-cis-RA increased palmitate oxidation during a subsequent h incubation with 50 lm FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS 6097 Beta-cell protection against palmitate toxicity 500 M–2 days 9-cis RA ( M) 0.5 20 B 40 10 % cytotoxicity % cytotoxicity % cytotoxicity A K Hellemans et al 250 M–8 days 40 30 30 C16:0 + Clofibrate + Ciprofibrate + 9-cis RA + Clofibrate / 9-cis RA + Ciprofib / 9-cis RA # # 20 # # # # 10 20 #$ #$ #$ #$ 10 500 250 100 clofibrate ( M - days 250 M - days 500 M) Fig Effect of PPARa and RXR agonists on palmitate toxicity (A) Effect of clofibrate and 9-cis-RA on palmitate toxicity Primary rat betacells were exposed to 500 lM palmitate for days, or to 250 lM palmitate for days, in the presence or absence of clofibrate (100, 250, 500 lM) and ⁄ or 9-cis-RA (0.5, 2, lM) (n ¼ 4–8) Vertical bars represent SEM (B) Comparison between the protective effect of clofibrate (250 lM) and of ciprofibrate (100 lM) against palmitate toxicity, alone or in combination with lM 9-cis-RA Data are indicated as mean ± SEM (n > 5) #P < 0.001 as compared to palmitate; $P < 0.001 as compared to single agonist treatment (clofibrate, ciprofibrate or 9-cis-RA) (two-way ANOVA) Table Effect of palmitate and PPARa–RXR agonists on [14C]palmitate metabolism Beta-cells were precultured for 24 h in the presence or absence of 250 lM palmitate and ⁄ or 250 lM clofibrate (Clof) plus lM 9-cis-RA (RA) Incorporation of the 14C label into CO2, lipid intermediates, lipids and proteins was measured during a h incubation with 50 lM [14C]palmitate, and expressed as a percentage of the control condition (vehicle only, 10 mM glucose) *P < 0.001 as compared to control, **P < 0.001 as compared to cytochrome P250, Student’s t-test (n ¼ 5) 14 C-recovery as Treatment CO2 Intermediates Lipid Protein C16:0 C16:0 + Clof ⁄ RA Clof ⁄ RA 59 ± 13* 94 ± 12** 181 ± 27* 80 ± 18 78 ± 10 94 ± 27 101 ± 94 ± 108 ± 93 ± 12 77 ± 27 83 ± 24 [14C]palmitate by 80% (P < 0.001) (Table 3) This effect was also seen when palmitate (250 lm) was present during preculture (Table 3) The lower 14CO2 values measured in this condition when compared with the control reflect isotopic dilution in palmitate-pretreated cells No differences were found for the incorporation of label in lipids, intermediates or proteins Effect of PPARa–RXR agonists on gene expression of enzymes involved in fatty acid metabolism and peroxisomal membrane proteins RT-PCR analysis was performed to investigate whether the agonist combination (250 lm clofibrate 6098 plus lm 9-cis-RA) increased expression of genes coding for enzymes involved in peroxisomal or mitochondrial lipid metabolism or of peroxisomal membrane proteins (Table 4) Palmitate 250 lm alone induced the mRNA expression of CPT1 (two-fold increase over control, P < 0.05), and decreased that of PPARa, stearoyl-CoA desaturase (SCD1), stearoyl-CoA desaturase (SCD2), and the peroxisomal membrane proteins peroxisomal biogenesis factor (Pex2) and peroxisomal biogenesis factor 14 (Pex14) (P < 0.05) Addition of clofibrate plus 9-cis-RA further increased mRNA levels of CPT1 (1.7-fold, P < 0.001) and induced the expression of the mitochondrial enzymes acyl-CoA dehydrogenase (medium chain), acyl-CoA dehydrogenase (long chain), and mitochondrial acetylCoA acetyltransferase (P < 0.01), as well as of the peroxisomal enzymes peroxisomal acetyl-CoA acetyltransferase 1, palmitoyl-CoA oxidase 1, and pristanoyl-CoA oxidase (P < 0.01), and the prolipogenic endoplasmatic reticulum enzymes glycerol-3-phosphate acyltransferase, mitochondrial (GPAT), SCD1 and SCD2 (P < 0.01) This combination was also found to increase the mRNA levels of peroxisomal biogenesis factor (Pex3), peroxisomal biogenesis factor 16 (Pex16), and peroxisomal biogenesis factor 11a (Pex11a), as well as of Pex2, Pex14 and peroxisomal membrane protein 70 (PMP70) Comparable changes in expression were also found after stimulation with clofibrate and 9-cis-RA in the absence of palmitate FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS K Hellemans et al Beta-cell protection against palmitate toxicity Table Effect of palmitate and PPARa–RXR agonists on mRNA expression levels Beta-cells were exposed for days to 250 lM palmitate and ⁄ or 250 lM clofibrate plus lM 9-cis-RA or vehicle (control) qPCR values were normalized to actin and calculated as DDCt values relative to the indicated control conditions Unpaired student t-test, two-tailed, mean ± SD, n ¼ 4–6, *P < 0.05, **P < 0.01, ***P < 0.001 Protein Gene transcription Peroxisome proliferator-activated receptor a Mitochondrial b-oxidation Carnitine palmitoyl transferase Acyl-CoA dehydrogenase, long chain Acyl-CoA dehydrogenase, medium chain Acyl-CoA dehydrogenase, short chain Mitochondrial acetyl-CoA acetyltransferase Peroxisomal fatty acid oxidation Peroxisomal acetyl-CoA acetyltransferase Palmitoyl-CoA oxidase Pristanoyl-CoA oxidase a-Methylacyl-CoA racemase Lipid synthesis Stearoyl-CoA desaturase Stearoyl-CoA desaturase Glycerol-3-phosphate acyltransferase Peroxisomal membrane proteins Peroxisomal biogenesis factor Peroxisomal biogenesis factor 16 Peroxisomal biogenesis factor 11a Peroxisomal membrane protein 70 Peroxisomal biogenesis factor 14 Peroxisomal biogenesis factor C16:0, compared to control Clofibrate +9-cis-RA, compared to control C16:0 + clofibrate + 9-cis-RA, compared to C16:0 0.7 ± 0.2 1.6 ± 0.2*** 1.9 ± 0.4 *** 1.9 1.4 0.8 1.0 1.3 ± ± ± ± ± 0.6** 0.7 0.2 0.1 0.4 2.6 1.3 1.3 1.8 2.5 ± ± ± ± ± 0.4*** 0.2 0.3 0.6* 1.0** 1.9 1.5 1.6 1.1 1.6 ± ± ± ± ± 0.2*** 0.2** 0.3** 0.2 0.1*** 0.8 1.0 1.4 1.1 ± ± ± ± 0.2 0.3 0.9 0.2 0.9 1.6 2.0 0.9 ± ± ± ± 0.5 0.2*** 0.8* 0.1 1.6 1.6 1.5 1.2 ± ± ± ± 0.1*** 0.4** 0.2*** 0.2 0.5 ± 0.2*** 0.7 ± 0.1*** 1.1 ± 0.3 4.5 ± 2.5** 1.8 ± 0.3*** 1.5 ± 0.2** 5.8 ± 2.7*** 2.6 ± 0.6*** 1.5 ± 0.2** 1.2 0.9 1.0 0.9 0.7 0.7 1.2 1.7 1.3 1.4 2.6 1.4 1.5 1.4 1.6 2.0 1.4 1.4 ± ± ± ± ± ± 0.5 0.1 0.2 0.3 0.1** 0.2* Effect of etomoxir on palmitate toxicity in the presence of PPARa–RXR agonists Discussion This study confirms that sustained exposure to palmitate causes time- and dose-dependent toxicity on rat beta-cells Over an day culture period, palmitate, at 250 or 500 lm, progressively reduced the number of surviving cells, involving both necrotic and apoptotic pathways [7], but a significant fraction remained resistant These survival curves show that primary betacells can be less susceptible or even resistant to fatty 0.6 0.5** 0.2* 0.3** 1.0* 0.5 ± ± ± ± ± ± 0.3* 0.2** 0.3** 0.6** 0.2** 0.2* C16:0 C16:0 + Clofibrate / 9-cis RA C16:0 + Clof / 9RA + etomoxir 30 % Cytotoxicity In the presence of etomoxir, the protective effect of clofibrate and 9-cis-RA was abolished, and the cytotoxicity of palmitate 500 lm for days increased four-fold, i.e from ± 3% to 17 ± 5% (Fig 4) This toxicity was also increased when the cells were precultured for 24 h with 200 lm etomoxir prior to their incubation with the palmitate ⁄ clofibrate ⁄ RA mixture for days (19 ± 3%, P < 0.05 versus ± 2%) The effect of preculture with etomoxir was lost after prolonged subsequent culture in its absence (250 lm palmitate for days) 40 ± ± ± ± ± ± $ 20 $ *** 10 *** *** 500 M - 2d 250 M - 8d Fig Effect of etomoxir on PPARa–RXR protection against palmitate toxicity Primary beta-cells were cultured with 500 lM palmitate for days, or 250 lM palmitate for days, in the absence or presence of 250 lM clofibrate plus lM 9-cis-RA, or in combination with 200 lM etomoxir Data are presented as mean ± SEM (n ¼ 4) ***P < 0.001 as compared to palmitate; $P < 0.01 for etomoxir as compared to clofibrate plus 9-cis-RA (Student’s t-test) acid toxicity, and that this property is heterogeneously expressed, like other cell functions [17,18] We previously reported that addition of oleate increases the FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS 6099 Beta-cell protection against palmitate toxicity K Hellemans et al resistance of rat beta-cells to palmitate-induced cell death [7] This oleate effect was also observed in human beta-cells [19] and in other cell types [20–23] It appeared to be correlated with the formation of triglycerides, supporting the view that fatty acid incorporation into neutral lipids prevents the accumulation of toxic free palmitoyl acyl moieties [22], and ⁄ or ceramide derivatives [24,25] A role of ceramides in palmitate toxicity is supported by the observed protection by l-cycloserine, an inhibitor of serine palmitoyl transferase and thus of ceramide synthesis At variance with other cytotoxic conditions [26], no protective effect could be attributed to glucose, as similar palmitate toxicities were measured following culture at or 10 mm glucose The percentage of dead cells was not increased when palmitate exposure was assessed at excessive glucose levels (20 mm), which contrasts with observations in beta-cell lines [27–30] The latter discrepancy might be related to differences in experimental protocols, such as free fatty acid concentrations, free fatty acid ⁄ BSA ratios [7], or the use of serum, but could also result from differences between primary beta-cells and cell lines Glucose cytotoxicity has also been seen in cell lines [27,31], whereas increased glucose exerts cytoprotective effects in primary beta-cells, at least in conditions that cause an oxidative shift in their metabolic state [32–35] Our data suggest that glucose-induced changes in the cellular metabolic redox state not alter cellular susceptibility to palmitate toxicity At a nontoxic palmitate concentration (50 lm), glucose suppresses its oxidation, probably due to dynamic regulation of the malonyl-CoA ⁄ CPT1 axis [3], but this seems not to be accompanied by a toxic effect; in fact, this may lead to the formation of fatty acid derivatives with physiologic action in the presence of glucose [36– 38], or in protective accumulation in the form of neutral lipids [7,39] Furthermore, palmitate induced the expression of CPT1 two-fold, suggesting that beta-cells have the inherent capacity to adapt their oxidation rate to elevated fatty acid levels, independently of the suppressive effect of glucose Our data indicate that palmitate toxicity can be reduced by increasing its oxidation through mitochondrial and ⁄ or peroxisomal pathways Mitochondrial oxidation of long-chain fatty acids is known to be rate limited at the level of CPT1 [10] Viral overexpression of CPT1 has been shown to enhance palmitate oxidation in INS-1 cells and islets [40,41] In our study, culture with l-carnitine, an essential component of CPT1, stimulated palmitate oxidation and reduced its toxicity, while etomoxir, an inhibitor of CPT1, increased the toxicity of palmitate (C16:0) but not of butyrate (C4:0), which enters mitochondria independently of 6100 CPT1 In fact, no toxicity was measured for any of the tested shorter-chain fatty acids, raising the possibility that shortening the palmitate chain represents another and perhaps more important mechanism for inducing cytoprotection It is so far unclear to what extent peroxisomes in beta-cells contribute to fatty acid metabolism That they could be involved is suggested by the absence of cytotoxicity for the 2-methyl and 3-methyl derivatives of palmitate Like other branched fatty acids, these compounds are known to be preferentially transported to the peroxisomes, where 2-methyl-C16 undergoes b-oxidation and 3-methyl-C16 will be a-oxidized before being transported to the mitochondria [42] Fibrates are known to regulate genes involved in mitochondrial, as well as peroxisomal, fatty acid oxidation and to induce peroxisome proliferation and maturation in multiple cell types [11] Both clofibrate and ciprofibrate were found to increase palmitate breakdown and to reduce its toxicity; their combination with 9-cis-RA resulted in complete protection This protective action of 9-cis-RA against palmitate toxicity contrasts with the proapoptotic effect observed in MIN6 cells at a 10-fold higher concentration [43] Clofibrate plus 9-cis-RA was found to provide the same level of protection at all examined glucose concentrations This effect correlated with induced expression of CPT1 and mitochondrial and peroxisomal b-oxidation enzymes, and resulted in normalization of palmitate oxidation In further support of this, inhibition of CPT1 by etomoxir was found to abolish the protective action of clofibrate plus 9-cis-RA PPARa:RXR agonists also induced expression of GPAT, SCD1 and SCD2 mRNA, which might mediate incorporation of the fatty acid into (phospho)lipids [44] Increased SCD1 expression has been previously noticed in palmitate-resistant MIN6 cells but has not yet been directly correlated with a cytoprotective diversion of palmitate into lipid formation [39]; esterification of palmitate was shown to result in accumulation of insoluble tripalmitin and to correlate with endoplasmic reticulum stress and apoptosis [45,46] The PPARa–RXR agonists were also found to act at a third level of potential relevance, namely the expression of proteins involved in peroxisome biogenesis (Pex3 and Pex16), proliferation (Pex11a) and maturation (PMP70, Pex2 and Pex14) [47] By inducing the peroxisomal compartment, they might indeed increase the channeling of palmitate through the first cycles of chain shortening before further breakdown in mitochondria This view is consistent with the virtual absence of toxicities for short-chain fatty acids Peroxisomes might thus represent a key site for reducing the FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS K Hellemans et al toxicity of palmitate in primary beta-cells Their activity might be low in normal circumstances, as judged by the low catalase activity in islet beta-cells [48,49] Addition of clofibrate was shown to increase catalase activity in INS-1 cells together with palmitate oxidation [50] Clofibrate plus 9-cis-RA did not provide protection in the nonbeta-cells where, independently of glucose, a substantially higher proportion of palmitate was converted to CO2 Our data supplement previous in vivo findings or observations in cell lines, and more specifically indicate to what extent they reflect effects on the survival of primary beta-cells and, if so, through which mechanism these can then be explained Although fatty acids, and particularly palmitate, are classically seen as mediators of lipotoxicity at the level of the beta-cells, it is often not clear whether the reported derangements are the result of beta-cell death and ⁄ or dysfunction Consequently, the protective action of PPARa agonists with or without RXR agonists was not always well specified in these terms [51] Adenoviral coexpression of PPARa and RXRa synergistically – and in a dose- and liganddependent manner ) potentiated glucose-stimulated insulin secretion from INS-1E cells while increasing their expression of genes involved in free fatty acid uptake and b-oxidation [52,53] An increase of PPARadriven b-oxidation in response to topiramate was also found to protect INS-1E cells from oleate toxicity [54] When administered in vivo, PPAR–RXR ligands induced expression of b-oxidation enzymes and stimulated palmitate oxidation in isolated islets [13] Fibrate treatment restored the coupling between insulin secretion and action in glucose-intolerant rats on a high-fat diet [55] and prevented diabetes in obese OLETF rats [56,57] Combination therapy with PPARa and a-agonists, or dual agonists, ameliorated insulin secretion and increased insulin stores in genetically obese diabetic db ⁄ db mice [58] The present work has shown that PPARa–RXR agonists can protect primary beta-cells against the cytodestructive effects of palmitate It has provided evidence that this protection is achieved by stimulating mitochondrial and peroxisomal pathways for palmitate breakdown Further work is needed to assess the functional properties of these protected beta-cells and to evaluate the influence of the agonists at nontoxic palmitate concentrations Experimental procedures Materials Palmitate, oleate, butyrate, hexanoate, octanoate (sodium salts), 2-bromohexadecanoic acid, clofibrate, ciprofibrate, Beta-cell protection against palmitate toxicity 9-cis-RA, l-carnitine and l-cycloserine were purchased from Sigma-Aldrich (Bornem, Belgium) Branched 2-methylhexadecanoic acid and 3-methylhexadecanoic acid were prepared as described previously [59,60] Stock solutions of fatty acids (25 mm, 50 mm) were made in 90% ethanol by heating to 60 °C, except for 2-bromopalmitate, which was dissolved at room temperature Stock solutions of clofibrate (200 mm), ciprofibrate (20 mm) and 9-cis-RA (10 mm) were dissolved in absolute ethanol Etomoxir was a gift from V Grill (Trondheim University, Norway) and dissolved in saline d-[U-14C]glucose (287–311 mCiỈmmol)1; mCi per mL) was purchased from Amersham Biosciences (Roosendaal, Belgium), and [U-14C]palmitic acid (850 mCiỈ mmol)1; 0.1 mCiỈmL)1) from Perkin Elmer Life Sciences (Zaventem, Belgium) Preparation and culture of rat beta-cells Adult male Wistar rats were bred according to Belgian regulations on animal welfare Experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC) Pancreatic islets were isolated, dissociated and purified into single beta-cells (purity 88 ± 4% insulin-positive cells) and endocrine nonbeta-cells (70 ± 11% alpha-cells, 23 ± 3% beta-cells) by autofluorescence-activated cell sorting [61] For studies on cytoxicity, isolated cells were cultured in polylysine-coated microtiter plates (2500–3000 cells per well) with Ham’s F10 medium containing 10 mm glucose (unless stated otherwise), 1% charcoal-extracted BSA (fraction V, radioimmunoassay grade; Sigma-Aldrich), mm l-glutamine, 50 mm 3-isobutyl-1-methylxanthine, 0.075 mgỈmL)1 penicillin and 0.1 mgỈmL)1 streptomycin [7] Test reagents were added to the culture medium, with control conditions receiving similar dilutions of solvent After and days of culture, the medium was changed and fresh reagents were added Percentages of living and dead cells were determined by vital staining using neutral red [7] For metabolic and gene expression studies, freshly isolated cells were reaggregated and cultured in suspension as previously described [62] Measurement of glucose and palmitate metabolism Duplicate samples of · 104 rat beta-cells were incubated for h at 37 °C using Ham’s F10 medium containing 0.5% BSA, mm l-glutamine and 10 mm Hepes for measuring glucose metabolism (5 lCi of d-[U-14C]glucose with different concentrations of unlabeled d-glucose) [63] Palmitate metabolism was measured using KRBH medium, containing 0.2% BSA (fraction V), mm calcium chloride, and 10 mm Hepes (0.5 lCi of [U-14C]palmitic acid, with unlabeled palmitate up to 50 lm in order to achieve the same ratio of free fatty acid over BSA as in the cytotoxicity experiments with 250 lm palmitate) The rate of FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS 6101 Beta-cell protection against palmitate toxicity K Hellemans et al d-[U-14C]glucose or [U-14C]palmitic acid oxidation was assessed through the formation of 14CO2 [63] Cells were incubated in a siliconized tube trapped in an airtight glass vial After h, 20 lL of m HCl was injected, and 250 lL of Hyamine (Packard Bioscience, Groningen, the Netherlands) added to capture 14CO2 for h at room temperature The 14C incorporation into lipids, proteins and metabolic intermediates was measured as previously described [64] Gene expression analysis Total beta-cell RNA was extracted with TRIzol Reagent (Gibco BRL, Carlsbad, CA, USA) and its quality was assessed on a 2100 Bioanalyzer (Agilent, Waldbronn, Germany), taking a minimal cutoff RNA integrity number of RNA clean-up was performed with the Turbo DNA Free Kit (Ambion, Austin, TX, USA) and cDNA prepared with the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) Real-time PCR was performed using an ABI Prism Sequence Detector (Applied Biosystems) Primers were obtained from Applied Biosystems (Table 4) For each RT-PCR reaction, the cycle threshold (Ct) was determined with sds 1.9.1 software DDCt values were calculated versus b-actin Fold changes were calculated starting from DDCt values of a minimum of four independent experiments performed in duplicate Data analysis Data are presented as mean ± SEM, or as mean ± SD of n independent experiments Statistical analysis was performed using Student’s t-test, unless stated otherwise Differences were considered significant for P < 0.05 Acknowledgements This work was supported by the Research Foundation Flanders (Fonds Voor Wetenschappelijk OnderzoekVlaanderen, Grant FWO-G.0357.03, Grant FWO-1.5.195.05 and PhD grant FWOTM277 to K Kerckhofs) and by the Inter-University Poles of Attraction Program (IUAP P5 ⁄ 17) from the Belgian Science Policy The Diabetes Research Center is a partner of the Juvenile Diabetes Research Center for Beta Cell Therapy in Diabetes References Haber EP, Procopio J, Carvalho CR, Carpinelli AR, Newsholme P & Curi R (2006) New insights into fatty acid modulation of pancreatic beta-cell function Int Rev Cytol 248, 1–41 Prentki M & Nolan CJ (2006) Islet beta cell failure in type diabetes J Clin Invest 116, 1802–1812 6102 Prentki M, Joly E, El Assaad W & Roduit R (2002) Malonyl-CoA signaling, lipid partitioning, and glucolipotoxicity: role in beta-cell adaptation and failure in the etiology of diabetes Diabetes 51 (Suppl 3), S405– S413 Unger RH & Zhou YT (2001) Lipotoxicity of beta-cells in obesity and in other causes of fatty acid spillover Diabetes 50 (Suppl 1), S118–S121 Robertson RP, Harmon J, Tran PO & Poitout V (2004) Beta-cell glucose toxicity, lipotoxicity, and chronic oxidative stress in type diabetes Diabetes 53 (Suppl 1), S119–S124 Martens G, Cai Y, Hinke S, Stange G, Van De CM & Pipeleers D (2005) Nutrient sensing in pancreatic beta cells suppresses mitochondrial superoxide generation and its contribution to apoptosis Biochem Soc Trans 33, 300–301 Cnop M, Hannaert JC, Hoorens A, Eizirik DL & Pipeleers DG (2001) Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation Diabetes 50, 1771– 1777 Kharroubi I, Ladriere L, Cardozo AK, Dogusan Z, Cnop M & Eizirik DL (2004) Free fatty acids and cytokines induce pancreatic beta-cell apoptosis by different mechanisms: role of nuclear factor-kappaB and endoplasmic reticulum stress Endocrinology 145, 5087–5096 Kelpe CL, Moore PC, Parazzoli SD, Wicksteed B, Rhodes CJ & Poitout V (2003) Palmitate inhibition of insulin gene expression is mediated at the transcriptional level via ceramide synthesis J Biol Chem 278, 30015– 30021 10 Kerner J & Hoppel C (2000) Fatty acid import into mitochondria Biochim Biophys Acta 1486, 1–17 11 Desvergne B & Wahli W (1999) Peroxisome proliferator-activated receptors: nuclear control of metabolism Endocr Rev 20, 649–688 12 Schuit F, Flamez D, De Vos A & Pipeleers D (2002) Glucose-regulated gene expression maintaining the glucose-responsive state of beta-cells Diabetes 51 (Suppl 3), S326–S332 13 Zhou YT, Shimabukuro M, Wang MY, Lee Y, Higa M, Milburn JL, Newgard CB & Unger RH (1998) Role of peroxisome proliferator-activated receptor alpha in disease of pancreatic beta cells Proc Natl Acad Sci USA 95, 8898–8903 14 Gremlich S, Nolan C, Roduit R, Burcelin R, Peyot ML, Delghingaro-Augusto V, Desvergne B, Michalik L, Prentki M & Wahli W (2005) Pancreatic islet adaptation to fasting is dependent on peroxisome proliferator-activated receptor alpha transcriptional up-regulation of fatty acid oxidation Endocrinology 146, 375–382 15 Lalloyer F, Vandewalle B, Percevault F, Torpier G, Kerr-Conte J, Oosterveer M, Paumelle R, Fruchart JC, Kuipers F, Pattou F et al (2006) Peroxisome prolifera- FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS K Hellemans et al 16 17 18 19 20 21 22 23 24 25 26 tor-activated receptor {alpha} improves pancreatic adaptation to insulin resistance in obese mice and reduces lipotoxicity in human islets Diabetes 55, 1605– 1613 Koh EH, Kim MS, Park JY, Kim HS, Youn JY, Park HS, Youn JH & Lee KU (2003) Peroxisome proliferator-activated receptor (PPAR)-alpha activation prevents diabetes in OLETF rats: comparison with PPARgamma activation Diabetes 52, 2331–2337 Pipeleers D, Kiekens R, Ling Z, Wilikens A & Schuit F (1994) Physiologic relevance of heterogeneity in the pancreatic beta-cell population Diabetologia 37 (Suppl 2), S57–S64 Ling Z, Wang Q, Stange G, In’t Veld P & Pipeleers D (2006) Glibenclamide treatment recruits beta-cell subpopulation into elevated and sustained basal insulin synthetic activity Diabetes 55, 78–85 Maedler K, Oberholzer J, Bucher P, Spinas G & Donath MY (2003) Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human beta-cell turnover and function Diabetes 52, 726–733 Welters HJ, Tadayyon M, Scarpello JH, Smith SA & Morgan NG (2004) Mono-unsaturated fatty acids protect against beta-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure FEBS Lett 560, 103–108 Eitel K, Staiger H, Brendel MD, Brandhorst D, Bretzel RG, Haring HU & Kellerer M (2002) Different role of saturated and unsaturated fatty acids in beta-cell apoptosis Biochem Biophys Res Commun 299, 853– 856 Listenberger LL, Han X, Lewis SE, Cases S, Farese RV Jr, Ory DS & Schaffer JE (2003) Triglyceride accumulation protects against fatty acid-induced lipotoxicity Proc Natl Acad Sci USA 100, 3077–3082 Beeharry N, Chambers JA & Green IC (2004) Fatty acid protection from palmitic acid-induced apoptosis is lost following PI3-kinase inhibition Apoptosis 9, 599– 607 Shimabukuro M, Higa M, Zhou YT, Wang MY, Newgard CB & Unger RH (1998) Lipoapoptosis in beta-cells of obese prediabetic fa ⁄ fa rats Role of serine palmitoyltransferase overexpression J Biol Chem 273, 32487– 32490 Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M, Santangelo C, Patane G, Boggi U, Piro S, Anello M et al (2002) Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated Diabetes 51, 1437–1442 Pipeleers D & Van De Winkel M (1986) Pancreatic B cells possess defense mechanisms against cell-specific toxicity Proc Natl Acad Sci USA 83, 5267–5271 Beta-cell protection against palmitate toxicity 27 El Assaad W, Buteau J, Peyot ML, Nolan C, Roduit R, Hardy S, Joly E, Dbaibo G, Rosenberg L & Prentki M (2003) Saturated fatty acids synergize with elevated glucose to cause pancreatic beta-cell death Endocrinology 144, 4154–4163 28 Maestre I, Jordan J, Calvo S, Reig JA, Cena V, Soria B, Prentki M & Roche E (2003) Mitochondrial dysfunction is involved in apoptosis induced by serum withdrawal and fatty acids in the beta-cell line INS-1 Endocrinology 144, 335–345 29 Briaud I, Harmon JS, Kelpe CL, Segu VB & Poitout V (2001) Lipotoxicity of the pancreatic beta-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids Diabetes 50, 315–321 30 Zhou YP & Grill VE (1994) Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle J Clin Invest 93, 870–876 31 Buteau J, El Assaad W, Rhodes CJ, Rosenberg L, Joly E & Prentki M (2004) Glucagon-like peptide-1 prevents beta cell glucolipotoxicity Diabetologia 47, 806–815 32 Ling Z, Kiekens R, Mahler T, Schuit FC, PipeleersMarichal M, Sener A, Kloppel G, Malaisse WJ & Pipeleers DG (1996) Effects of chronically elevated glucose levels on the functional properties of rat pancreatic beta-cells Diabetes 45, 1774–1782 33 Hoorens A, Van De Casteele M, Kloppel G & Pipeleers D (1996) Glucose promotes survival of rat pancreatic beta cells by activating synthesis of proteins which suppress a constitutive apoptotic program J Clin Invest 98, 1568–1574 34 Khaldi MZ, Guiot Y, Gilon P, Henquin JC & Jonas JC (2004) Increased glucose sensitivity of both triggering and amplifying pathways of insulin secretion in rat islets cultured for wk in high glucose Am J Physiol Endocrinol Metab 287, E207–E217 35 Martens GA, Cai Y, Hinke S, Stange G, Van De Casteele M & Pipeleers D (2005) Glucose suppresses superoxide generation in metabolically responsive pancreatic beta cells J Biol Chem 280, 20389–20396 36 Nolan CJ, Madiraju MS, Delghingaro-Augusto V, Peyot ML & Prentki M (2006) Fatty acid signaling in the {beta}-cell and insulin secretion Diabetes 55 (Suppl 2), S16–S23 37 Yaney GC & Corkey BE (2003) Fatty acid metabolism and insulin secretion in pancreatic beta cells Diabetologia 46, 1297–1312 38 Warnotte C, Nenquin M & Henquin JC (1999) Unbound rather than total concentration and saturation rather than unsaturation determine the potency of fatty acids on insulin secretion Mol Cell Endocrinol 153, 147–153 39 Busch AK, Gurisik E, Cordery DV, Sudlow M, Denyer GS, Laybutt DR, Hughes WE & Biden TJ (2005) Increased fatty acid desaturation and enhanced expres- FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS 6103 Beta-cell protection against palmitate toxicity 40 41 42 43 44 45 46 47 48 49 50 51 K Hellemans et al sion of stearoyl coenzyme A desaturase protects pancreatic beta-cells from lipoapoptosis Diabetes 54, 2917– 2924 Herrero L, Rubi B, Sebastian D, Serra D, Asins G, Maechler P, Prentki M & Hegardt FG (2005) Alteration of the malonyl-CoA ⁄ carnitine palmitoyltransferase I interaction in the beta-cell impairs glucose-induced insulin secretion Diabetes 54, 462–471 Rubi B, Antinozzi PA, Herrero L, Ishihara H, Asins G, Serra D, Wollheim CB, Maechler P & Hegardt FG (2002) Adenovirus-mediated overexpression of liver carnitine palmitoyltransferase I in INS1E cells: effects on cell metabolism and insulin secretion Biochem J 364, 219–226 Van Veldhoven PP, Casteels M, Mannaerts GP & Baes M (2001) Further insights into peroxisomal lipid breakdown via alpha- and beta-oxidation Biochem Soc Trans 29, 292–298 Wente W, Brenner MB, Zitzer H, Gromada J & Efanov AM (2006) Activation of liver X receptors and retinoid X receptors induces growth arrest and apoptosis in insulin secreting cells Endocrinology 148, 1843–1849 Man WC, Miyazaki M, Chu K & Ntambi J (2006) Colocalization of SCD1 and DGAT2: implying preference for endogenous monounsaturated fatty acids in triglyceride synthesis J Lipid Res 47, 1928–1939 Moffitt JH, Fielding BA, Evershed R, Berstan R, Currie JM & Clark A (2005) Adverse physicochemical properties of tripalmitin in beta cells lead to morphological changes and lipotoxicity in vitro Diabetologia 48, 1819– 1829 Karaskov E, Scott C, Zhang L, Teodoro T, Ravazzola M & Volchuk A (2006) Chronic palmitate but not oleate exposure induces endoplasmic reticulum stress, which may contribute to INS-1 pancreatic beta-cell apoptosis Endocrinology 147, 3398–3407 Wanders RJ (2004) Peroxisomes, lipid metabolism, and peroxisomal disorders Mol Genet Metab 83, 16–27 Parker SM, Moore PC, Johnson LM & Poitout V (2003) Palmitate potentiation of glucose-induced insulin release: a study using 2-bromopalmitate Metabolism 52, 1367–1371 Xu B, Moritz JT & Epstein PN (1999) Overexpression of catalase provides partial protection to transgenic mouse beta cells Free Radic Biol Med 27, 830–837 Tordjman K, Standley KN, Bernal-Mizrachi C, Leone TC, Coleman T, Kelly DP & Semenkovich CF (2002) PPARalpha suppresses insulin secretion and induces UCP2 in insulinoma cells J Lipid Res 43, 936–943 Roduit R, Nolan C, Alarcon C, Moore P, Barbeau A, Delghingaro-Augusto V, Przybykowski E, Morin J, Masse F, Massie B et al (2004) A role for the malonylCoA ⁄ long-chain acyl-CoA pathway of lipid signaling in 6104 52 53 54 55 56 57 58 59 60 61 the regulation of insulin secretion in response to both fuel and nonfuel stimuli Diabetes 53, 1007–1019 Ravnskjaer K, Boergesen M, Dalgaard LT & Mandrup S (2006) Glucose-induced repression of PPARalpha gene expression in pancreatic beta-cells involves PP2A activation and AMPK inactivation J Mol Endocrinol 36, 289–299 Ravnskjaer K, Boergesen M, Rubi B, Larsen JK, Nielsen T, Fridriksson J, Maechler P & Mandrup S (2005) Peroxisome proliferator-activated receptor alpha (PPARalpha) potentiates, whereas PPARgamma attenuates, glucose-stimulated insulin secretion in pancreatic beta-cells Endocrinology 146, 3266–3276 Frigerio F, Chaffard G, Berwaer M & Maechler P (2006) The antiepileptic drug topiramate preserves metabolism–secretion coupling in insulin secreting cells chronically exposed to the fatty acid oleate Biochem Pharmacol 72, 965–973 Holness MJ, Smith ND, Greenwood GK & Sugden MC (2005) Interactive influences of peroxisome proliferator-activated receptor alpha activation and glucocorticoids on pancreatic beta cell compensation in insulin resistance induced by dietary saturated fat in the rat Diabetologia 48, 2062–2068 Jia D & Otsuki M (2003) Bezafibrate, a peroxisome proliferator-activated receptor (PPAR)-alpha activator, prevents pancreatic degeneration in obese and diabetic rats Pancreas 26, 286–291 Lee HJ, Choi SS, Park MK, An YJ, Seo SY, Kim MC, Hong SH, Hwang TH, Kang DY, Garber AJ et al (2002) Fenofibrate lowers abdominal and skeletal adiposity and improves insulin sensitivity in OLETF rats Biochem Biophys Res Commun 296, 293–299 Yajima K, Hirose H, Fujita H, Seto Y, Fujita H, Ukeda K, Miyashita K, Kawai T, Yamamoto Y, Ogawa T et al (2003) Combination therapy with PPARgamma and PPARalpha agonists increases glucose-stimulated insulin secretion in db ⁄ db mice Am J Physiol Endocrinol Metab 284, E966–E971 Vanhove G, Van Veldhoven PP, Vanhoutte F, Parmentier G, Eyssen HJ & Mannaerts GP (1991) Mitochondrial and peroxisomal beta oxidation of the branched chain fatty acid 2-methylpalmitate in rat liver J Biol Chem 266, 24670–24675 Croes K, Casteels M, De Hoffmann E, Mannaerts GP & Van Veldhoven PP (1996) alpha-Oxidation of 3-methyl-substituted fatty acids in rat liver Production of formic acid instead of CO2, cofactor requirements, subcellular localization and formation of a 2-hydroxy-3methylacyl-CoA intermediate Eur J Biochem 240, 674– 683 Pipeleers DG, In’t Veld PA, van de Winkel M, Maes E, Schuit FC & Gepts W (1985) A new in vitro model for the study of pancreatic A and B cells Endocrinology 117, 806–816 FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS K Hellemans et al 62 Ling Z & Pipeleers DG (1994) Preservation of glucoseresponsive islet beta-cells during serum-free culture Endocrinology 134, 2614–2621 63 Schuit F, De Vos A, Farfari S, Moens K, Pipeleers D, Brun T & Prentki M (1997) Metabolic fate of glucose in purified islet cells Glucose-regulated anaplerosis in beta cells J Biol Chem 272, 18572–18579 Beta-cell protection against palmitate toxicity 64 Flamez D, Berger V, Kruhoffer M, Orntoft T, Pipeleers D & Schuit FC (2002) Critical role for cataplerosis via citrate in glucose-regulated insulin release Diabetes 51, 2018–2024 FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS 6105 ... Cytotoxicity 60 Effect of PPARa–RXR agonists on palmitate cytotoxicity C16:0 C16:0 + L-carnitine 40 % Cytotoxicity Palmitate oxidation (pmol/Kc/2h) A 1.50 Beta-cell protection against palmitate toxicity. .. days with 500 lm palmitate (acute toxicity) and for days at 250 lm (chronic toxicity) Islet endocrine nonbeta-cells exhibited lower susceptibility to palmitate toxicity: cytotoxicity was ± 0.5%... Hellemans et al Beta-cell protection against palmitate toxicity Table Effect of palmitate and PPARa–RXR agonists on mRNA expression levels Beta-cells were exposed for days to 250 lM palmitate and