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Endocrinology Vol. 144, No. 9 4154-4163
Copyright © 2003 by The Endocrine Society

Saturated Fatty Acids Synergize with Elevated Glucose to Cause Pancreatic ß-Cell Death

Wissal El-Assaad, Jean Buteau, Marie-Line Peyot, Christopher Nolan, Raphael Roduit, Serge Hardy, Erik Joly, Ghassan Dbaibo, Lawrence Rosenberg and Marc Prentki

Departments of Nutrition and Biochemistry, University of Montréal (W.E.-A., J.B., M.-L.P., C.N., R.R., S.H., E.J., M.P.), and Department of Surgery, McGill University (L.R.), Montréal, Québec, Canada H2L 4MI; and Department of Pediatrics (G.D.) American University of Beirut, 113/6044, B21 Beirut, Lebanon

Address all correspondence and requests for reprints to: Dr. Marc Prentki, CR-CHUM, Pavillon de Sève, Y4603, 1560 Sherbrooke East, Montreal, Quebec, Canada H2L 4M1. E-mail: marc.prentki{at}umontreal.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have proposed the "glucolipotoxicity" hypothesis in which elevated free fatty acids (FFAs) together with hyperglycemia are synergistic in causing islet ß-cell damage because high glucose inhibits fat oxidation and consequently lipid detoxification. The effects of 1–2 d culture of both rat INS 832/13 cells and human islet ß-cells were investigated in medium containing glucose (5, 11, 20 mM) in the presence or absence of various FFAs. A marked synergistic effect of elevated concentrations of glucose and saturated FFA (palmitate and stearate) on inducing ß-cell death by apoptosis was found in both INS 832/13 and human islet ß-cells. In comparison, linoleate (polyunsaturated) synergized only modestly with high glucose, whereas oleate (monounsaturated) was not toxic. Treating cells with the acyl-coenzyme A synthase inhibitor triacsin C, or the AMP kinase activators metformin and 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside that redirect lipid partitioning to oxidation, curtailed glucolipotoxicity. In contrast, the fat oxidation inhibitor etomoxir, like glucose, markedly enhanced palmitate-induced cell death. The data indicate that FFAs must be metabolized to long chain fatty acyl-CoA to exert toxicity, the effect of which can be reduced by activating fatty acid oxidation. The results support the glucolipotoxicity hypothesis of ß-cell failure proposing that elevated FFAs are particularly toxic in the context of hyperglycemia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE 2 DIABETES is characterized by progressive deterioration in insulin secretory function, diminished hepatic glucoregulation, and peripheral insulin resistance (1, 2, 3, 4, 5). Most often it is associated with, and usually preceded by central obesity and dyslipidemia (5, 6, 7). Insulin resistance is usually compensated by islet ß-cells adaptation with both increased ß-cell number and/or volume (ß-cell mass) and augmented ß-cell function (5, 8, 9, 10). It is only when insulin resistance is accompanied by failure of the islet ß-cell to compensate, that type 2 diabetes (5) and gestational diabetes (11) develop. Although the pathogenesis of the ß-cell failure is not well understood, it is becoming increasingly evident from animal models (12, 13) and human autopsy series (14, 15, 16, 17) that, in addition to ß-cell dysfunction, reduction in ß-cell mass is involved. Importantly, in a recently reported autopsy series (14), with adequate subject number and predeath clinical data, islet ß-cell mass was shown to be reduced in type 2 diabetic subjects and associated with evidence of increased apoptosis. Thus, both types 1 and 2 diabetes are increasingly viewed as ß-cell mass defects.

In recent years, both elevated glucose "glucotoxicity" and lipids "lipotoxicity" have been implicated in the failure of ß-cells in type 2 diabetes (4, 18). Before ß-cell failure, glucose levels are usually within the normal range in the fasting state. However, abnormal glucose excursions frequently occur in the postprandial state, consistent with the impaired glucose tolerance (19). Although not all patients show dyslipidemia in the pre-type 2 diabetic stage, circulating free fatty acids (FFAs) and triglycerides (TGs) concentrations are commonly elevated particularly within overweight subjects (2, 4, 5, 19). Thus, postprandial hyperglycemia and dyslipidemia are common features that occur before the development of insulin secretory defect in the natural history of this disease. Hence, both alterations might be implicated in the pathogenesis of type 2 diabetes (3, 4, 20). Further supporting a pathogenic role for dyslipidemia, elevated FFAs have been shown to be predictive of conversion from normal glucose tolerance and impaired glucose tolerance to diabetes (21, 22). With regard to FFAs and insulin secretion, evidence from both in vitro and in vivo studies support the suggestion that chronically elevated plasma FFAs contribute to ß-cell dysfunction in type 2 diabetes (23, 24, 25, 26, 27). Relevant to loss of ß-cell mass, several reports have documented that chronic exposure to high FFA levels can induce apoptosis in pancreatic ß-cells (28, 29, 30, 31), as can elevated glucose (29, 32).

To reconcile and integrate the roles of elevated glucose and FFAs in the pathogenesis of type 2 diabetes, we proposed the glucolipotoxicity hypothesis (3, 4, 33). This hypothesis states that the toxic actions of elevated FFAs on various tissues will become particularly apparent in the context of hyperglycemia. This derives from the fact that high glucose curtails fat oxidation and consequently the cellular detoxification of fatty acids. Thus, glucose induces and activates enzymes and transcription factors involved in fat synthesis and storage and simultaneously switches off fat oxidation via the accumulation of malonyl-coenzyme A (34, 35, 36) and reduction in the expression level of peroxisomal proliferator-activated receptor {alpha} (37). Sustained inhibition of fat oxidation results in an accumulation of long chain fatty acyl-coenzyme A (LC-CoA), which is thought to mediate the effects of chronically elevated FFAs (3). Whether LC-CoA accumulation affects the ß-cell directly or indirectly via TG deposition or act as precursors for other lipid-signaling metabolites such as phosphatidate, diacylglycerol, and ceramide is unknown. For early ß-cell failure to occur, we proposed that high normal postprandial glucose excursions, together with hyperlipidemia and elevated postprandial FFA levels act in synergy to cause the initial ß-cell failure (4). The process is then likely to accelerate once hyperglycemia has developed. This concept is also applicable to type 1 diabetes in which glucolipotoxicity may synergize with the immune attack of the ß-cell or to transplanted patients (4).

To date the actions of glucose and fatty acids on ß-cell growth and death have been studied mostly independently. In this study, we tested the effects of different classes of FFA in the presence or absence of elevated glucose on pancreatic ß-cell death. We show that fatty acids are poorly toxic at low glucose but saturated FFAs synergize with elevated glucose in causing INS 832/13 cells and human islet apoptosis. Furthermore, the various FFAs have different effects on cell death ranging from nontoxicity (oleate) to high toxicity (palmitate and stearate).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Regular and glucose-free RPMI medium and cell culture supplements were purchased from Invitrogen (Burlington, Ontario, Canada). The caspase-3 substrate N-acetyl-Asp-Glu-Val-Asp-amino trifluoromethyl coumarin (Ac-DEVD-AFC), the pan-caspase inhibitor z-VAD-fmk, and the acyl-coenzyme A synthase inhibitor triacsin C were purchased from Biomol (Plymouth Meeting, PA). Fatty acids were purchased from {nu}-Check Prep (Elysian, MN). Fatty acid-free BSA (fraction V), etomoxir, AICAR, metformin, Hoechst 33342 (bis-benzamide), and propidium iodide (PI) were obtained from Sigma (St. Louis, MO).

Cell culture
INS 832/13 cells (kindly provided by Dr. C. B. Newgard, Duke University) (38) between passages 36 and 70 were grown as previously described (39) in monolayer at 37 C with 5% CO2 in regular RPMI-1640 medium at 11 mM glucose supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10 mM HEPES (pH 7.4), 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µM ß-mercaptoethanol (complete medium). To measure glucose- and FFA-induced cell death, cells were seeded at a density of 4 x 104 cells/cm2 and grown for 2 d in complete RPMI. After the culture establishment period, medium was removed, and cells were then incubated in RPMI medium supplemented as above with 1% FBS (described hereafter as RPMI incubation medium), at 5, 11, or 20 mM glucose in the presence of 0.5% BSA alone (control) or with various FFAs (0.25, 0.3, or 0.4 mM) bound to 0.5% BSA as indicated. In some experiments, following culture establishment, cells were pretreated with the inhibitors, zVAD-fmk (50 µM), etomoxir (0.2 mM), or triacscin C (5 µM) for 1–2 h as indicated in complete RPMI containing 10% FBS, followed by FFA treatment at 5 or 20 mM glucose in RPMI incubation medium for 24 h, and in the presence of the inhibitor. The antidiabetic agent metformin (0.5 mM) and the AMP-activated protein kinase (AMPK) activator 5-amino-imidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) (1 mM) were added together with the FFAs for 24 h in RPMI incubation medium.

Preparation of BSA-bound fatty acids
Stock solutions of fatty acids bound to BSA were prepared as described by Roche et al. (40). Briefly, a saturating quantity of the sodium salt fatty acid was dissolved at 37 C for 16 h under a nitrogen atmosphere in Krebs-Ringer bicarbonate buffer containing 10 mM HEPES (pH 7.4) buffer and 5% (wt/vol) fatty acid-free BSA. Solutions were then filtered through a 0.2 µm filter. BSA-bound FFA was quantitated using the NEFA C kit (Wako Chemicals GmbH, Neuss, Germany) and stock solutions were finally adjusted to 4 mM FFA using 5% fatty acid-free BSA in Krebs-Ringer bicarbonate buffer containing 10 mM HEPES (pH 7.4) buffer and stored at -20 C.

Islet isolation and culture conditions
Human islets were isolated as described previously (41) from organ donors at the Department of Surgery, Montréal General Hospital, McGill University Health Center (three batches from three separate donors received). Human ethics approval was obtained through the McGill University Health Center ethics committee. Donors were between 50 and 65 yr old and none had a history of diabetes or metabolic disorder. Islets were separated from the surrounding exocrine tissue by enzymatic digestion with 2.5 mg/ml Liberase CI (Roche Molecular Biochemicals, Indianapolis, IN) in Hank’s balanced salt solution supplemented with 0.1 mg/ml DNase I (Roche Molecular Biochemicals) at 37 C using a semiautomated technique. Purification was achieved by density gradient separation in a three-step discontinuous EuroFicoll gradient using a CORE 2991 cell processor (COBE BCT, Denver, CO). Contamination by exocrine cells was less than 10% as observed by dithizone staining (Sigma). After isolation, islets were rested overnight in complete RPMI-1640 medium at 11 mM glucose containing 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37 C and 5% CO2. Glucose-induced insulin secretion at 3 and 20 mM glucose over a 45-min time period, and secretion in response to 35 mM KCl at 3 mM glucose were determined routinely on rested human islets to ensure a healthy state of the islets following isolation. The secretion protocol was as previously described (42).

Quantification of cell death
INS 832/13 cell viability was determined by the ability of cells to exclude trypan blue. All cells (adherent and detached) were collected and combined before staining and counting by a blinded observer. To discriminate between necrosis and apoptosis, cells were double stained with the fluorescent DNA-staining dyes Hoechst 33342 and PI. INS 832/13 cells (8 x 104 cells) were seeded on 12-mm glass coverslips placed in 24-well plates in complete RPMI medium for 2 d. Medium was changed to RPMI incubation medium with 0.5% BSA, at 5, 11, or 20 mM glucose with or without the various FFAs (0.25, 0.3, 0.4 mM) as indicated for an additional 24 h. At the end of the treatment period, cells were stained with Hoechst 33342 (10 µg/ml) and PI (1 µg/ml) in media simultaneously for 30 min at 37 C. Coverslips were then placed on slides, and the stained nuclei were immediately visualized by fluorescence microscopy with a Axioskop microscope (Carl Zeiss, Gottingen, Germany) using Hoechst and PI filter sets. Cells were defined as apoptotic when they exhibited a condensed nuclear chromatin or a fragmented nuclear membrane when visualized with Hoechst 33342. Necrotic cells were characterized by nuclear PI staining but without condensed chromatin or fragmented nuclear membranes. Cells without apoptotic or necrotic features were considered viable.

Quantitative analysis of each sample was performed by a blinded observer randomly choosing five fields and by counting at least 200 cells per assay condition. In the case of human islets, apoptosis indices were assessed by counting Hoechst 33342 (chromatin condensation) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) (DNA fragmentation) positive cells using a fluorescence in situ death detection kit (Roche Molecular Biochemicals) according to the manufacturer’s protocol with few modifications. After a 20-h resting period, 80 human islets per condition were dispersed into single cells and small clusters by trypsinization (43). They were then seeded on poly-ornithine-treated glass coverslips and cultured overnight in complete RPMI at 11 mM glucose containing 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37 C in the presence of 5% CO2. The next day, the coverslips were gently washed with PBS, and islet cells were incubated for a further 24 h in RPMI incubation medium containing 0.5% BSA at 5 or 25 mM glucose in the absence or presence of 0.4 mM palmitate. The islet cells were then fixed for 1 h using 4% paraformaldehyde, washed with PBS, and incubated in a permeabilization solution (0.1% Triton X-100 in PBS) for 2 min at room temperature. The cells were then blocked with 1% BSA in PBS for 10 min and incubated for 1 h at room temperature with TUNEL reaction mixture containing a mouse antiinsulin primary antibody (10 µg/ml) (Sigma) to identify ß- from non-ß-cells. After four washes (5 min each) in PBS, cells were stained with a rhodamine-conjugated donkey antimouse secondary antibody (Jackson Immunoresearch, West Grove, CA). Hoechst staining was performed by exposing the coverslips to 0.5 mg/ml Hoechst 33342 for 10 min at room temperature. The slides were then rinsed several times with PBS and mounted with antifade reagent. The fluorescence of Hoechst, TUNEL, and bound antiinsulin antibodies were visualized under a microscope at x400 magnification using their respective filter set. At least 200 cells were analyzed for each assay condition per experiment by a blinded observer.

In vitro caspase-3 activity assay
Caspase-3 activity was measured using the substrate Ac-DEVD-AFC and was assayed according to the manufacturer’s protocol. Briefly, INS 832/13 cells established in culture were incubated for 24 h in RPMI incubation medium with 0.5% BSA at 5 or 20 mM glucose with or without various fatty acids (0.25, 0.3, 0.4 mM) as indicated in the figure legends. Both adherent and unattached cells were then harvested and combined. After sedimentation at 500 g for 10 min, the cells were washed twice with ice-cold PBS, lysed for 10 min on ice with a cell lysis buffer (Invitrogen), and centrifuged (10 min, 15,000 x g, 4 C) to remove debris. Fifty micrograms of proteins determined by the BCA protein quantification kit (Pierce) were incubated for 30 min with 50 µM Ac-DEVD-AFC at 30 C. Fluorescence was analyzed using a FluoStar-Optima microplate reader (BMG Lab Technologies, Offenburg, Germany) in fluorescence mode using an excitation filter of 380 nm (10 nm bandpass) and an emission filter of 505 nm (bandpass 10 nm). The reaction was allowed to proceed for 30 min with a reading every minute. Caspase-3 activities were obtained by calculating the slope of the reaction over 30 min and reporting the slope for each condition.

Statistical analysis
Data were analyzed by one-way and two-way ANOVA as indicated with Bonferroni post hoc adjustment for multiple testing. P < 0.05 was considered significant. Data are expressed as mean ± SE of the mean.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Various fatty acids have differential effects on ß-cell death, which for some are highly glucose dependent
INS 832/13 cells were treated for 24 h with different concentrations of palmitate at 5 and 20 mM glucose. At 5 mM glucose, palmitate, even at high physiological concentrations (0.4 mM), showed minimal toxicity (Fig. 1AGo). At 20 mM glucose, however, palmitate caused cell death at concentrations greater than 0.2 mM (Fig. 1AGo). Thus, 0.3 or 0.4 mM palmitate was used to run subsequent experiments with all FFAs used except for stearate, which was used at 0.25 mM. INS 832/13 cells were then exposed for 24 h to 5, 11, or 20 mM glucose in the absence or presence of the three most abundant FFAs found in the blood, i.e. saturated palmitate (C 16:0), monounsaturated oleate (C 18:1), and polyunsaturated linoleate (C 18:2) (Fig. 1BGo). In the absence of exogenous FFAs, cells exposed to 5, 11, and 20 mM glucose showed minimal cell death (less than 7%) as measured by trypan blue exclusion. At 5 mM glucose, FFA toxicity did not reach statistical significance. Higher glucose concentrations markedly increased lipotoxicity of palmitate but had no effect on toxicity of oleate. There was a trend for the toxicity of linoleate to increase at high glucose, which did not reach statistical significance. Thus, a dramatic synergistic action of palmitate and glucose on cell death was observed at both 11 and 20 mM glucose (Fig. 1BGo). In marked contrast, oleate showed low cytotoxicity at all glucose concentrations examined. Various fatty acids, therefore, are not equivalent with respect to their action on cell death and the cytotoxicity of some fatty acids is markedly glucose dependent.



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  		FIG. 1. Effect of various FFAs at different glucose concentrations on ß-cell death. INS 832/13 cells were cultured for 24 h in RPMI incubation medium as described in Materials and Methods containing 0.5% BSA at A. Five (G5) and 20 (G20) mM glucose in the absence (control) or presence of 0.05, 0.1, 0.2, 0.3, and 0.4 mM palmitate, B, 5 (G5), 11 (G11), or 20 (G20) mM glucose in the absence (control) or presence of 0.4 mM palmitate (Pal), oleate (Ol), or linoleate (Lin). After treatment, both adherent and floating cells were collected and combined, and the cells were stained with trypan blue. Positive (dead) and negative (live) cells were counted. Results are expressed as percent of total cell number. Data are mean ± SE of three different experiments. A, Two-way ANOVA; glucose effect, P < 0.0001; palmitate effect, P < 0.0001; and glucose-palmitate interaction, P < 0.0001. Bonferroni post hoc test; #, P < 0.05 and ##, P < 0.001, compared with palmitate at 5 mM glucose. B, One-way ANOVA with Bonferroni post hoc test; *, P < 0.001, compared with control at same glucose concentration. **, P < 0.001, compared with corresponding palmitate at 5 and 11 mM glucose.

 
Saturated fatty acids are particularly efficient in synergizing with glucose to induce ß-cell apoptosis
To determine the mode of cell death induced by various FFAs at elevated glucose, INS 832/13 cells were stained with the dyes Hoechst 33342 and PI to distinguish apoptosis from necrosis (Fig. 2Go, A and B). Under all tested experimental conditions (various fatty acids at different glucose concentrations), the percentage of necrotic cells was modest (less than 7%), except for palmitate at 20 mM glucose (18%) (Fig. 2BGo). There was a nonsignificant trend for elevated glucose to increase apoptosis in the absence of fatty acids at 24 h (Fig. 2AGo). Palmitate, stearate, and, in this experiment, linoleate induced cell death primarily by apoptosis, and, as for cell death examined by trypan blue (Fig. 1BGo), FFA-induced apoptosis was strongly glucose concentration dependent (Fig. 2Go). The saturated FFAs, palmitate and stearate, were more potent, compared with linoleate at high glucose, and unlike linoleate, were able to induce apoptosis at intermediate (11 mM), compared with low (5 mM) glucose. Linoleate caused significant apoptosis (30%) only at elevated glucose (20 mM). Oleate was not toxic both in terms of apoptosis and necrosis.



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  		FIG. 2. Quantification of the effect of various FFAs at different glucose concentrations on ß-cell apoptosis and necrosis. INS 832/13 cells were cultured for 24 h as described in Fig. 1Go at 5 (G5), 11 (G11), or 20 mM (G20) glucose in the absence (control) or presence of 0.4 mM palmitate (Pal), oleate (Ol), linoleate (Lin), or 0.25 mM stearate (St). Cells were then stained with the dyes Hoechst 33342 and PI, analyzed by fluorescence microscopy, and scored according to the morphology and type of the fluorescence of the nucleus, as described in Materials and Methods. Results are expressed as percent of total cell number for A, Apoptotic cells. B, Necrotic cells. Data represent means ± SE of four different experiments. **, P < 0.001, compared with control at same glucose concentration. *, P < 0.05 and ***, P < 0.001, compared with control at 5 mM glucose. #, P < 0.05, compared with stearate at 5 mM glucose. ##, P < 0.001, compared with corresponding FFA at 5 mM glucose (one-way ANOVA with Bonferroni post hoc test).

 
Elevated glucose enhances palmitate cytotoxicity in human islets
Experiments were carried out to determine whether the ß-cell glucolipotoxicity concept is applicable to normal human islet tissue. Dispersed human islet cells were used after a 20-h recovery period from trypsin treatment. Following incubation for 24 h at 5 and 25 mM glucose in the absence or presence of 0.4 mM palmitate, dispersed cells on coverslips were double stained with Hoechst 33342 (Fig. 3Go, A and B), TUNEL (Fig. 3CGo), and insulin to assess apoptotic death in ß-cells. At 5 mM glucose, there was 14 ± 3% Hoechst-positive and 22 ± 4% TUNEL-positive ß-cells. This relatively high basal apoptosis is comparable with that reported previously at 5 mM glucose in isolated intact humans islets (~9%) (44) or islets after dispersion (~22%) (45). Elevated glucose alone or palmitate at 5 mM glucose did not significantly alter the percentage of apoptotic cells. In agreement with the INS 832/13 cell data, however, elevated glucose and palmitate synergized in promoting human ß-cell apoptosis that reached a value of about 47 ± 9% (Hoechst) and 62 ± 9% (TUNEL) of total ß-cell number. Similar observations were made when apoptosis was assessed as the total (ß-plus non-ß) islet cell population (data not shown).



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  		FIG. 3. Palmitate synergizes with elevated glucose to cause human ß-cell apoptosis. Dispersed human islet cells were attached onto coverslips and incubated for 24 h in RPMI incubation medium containing 0.5% BSA at 5 (G5) or 25 mM (G25) glucose in the absence or presence of 0.4 mM palmitate (Pal). Apoptosis was assessed by Hoechst staining (A, B) and TUNEL staining (C), and the ß-cell population was identified by insulin immunostaining (as described in Materials and Methods). Representative photographs for Hoechst and insulin immunostaining are shown for each condition in A. Arrows indicate apoptotic nuclei corresponding to insulin-positive cells in each field. Mean values ± SE of three separate experiments. *, P < 0.05, compared with G25; **, P < 0.001, compared with G5 (one-way ANOVA with Bonferroni post hoc test).

 
Palmitate at elevated glucose induces caspase-3 activation
To confirm that these morphological changes were part of an apoptotic process, we evaluated the effect of the various FFAs on the activation of caspase-3, a hallmark of apoptosis (Fig. 4Go). None of the tested FFAs significantly increased caspase-3 activity at low glucose. In INS 832/13 cells, palmitate and stearate synergized with high glucose to increase caspase-3 activity by 8- and 11-fold, respectively. Oleate did not cause activation of caspase-3, and linoleate, together with high glucose, increased caspase 3 activity by about 4-fold (not significant). Saturated fatty acids at elevated glucose also caused poly (ADP-ribose) polymerase cleavage with the formation of the 85-Kda fragment, characteristic of apoptosis (data not shown).



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  		FIG. 4. The action of various FFAs on caspase-3 activation is glucose dependent. INS 832/13 cells were cultured for 24 h as described in Fig. 1Go at 5 or 20 mM glucose in the absence (control) or presence of 0.4 mM palmitate (Pal), oleate (Ol), linoleate (Lin), or 0.25 mM stearate (St). Cell lysates were incubated with the caspase-3 substrate Ac-DEVD-AFC. Fluorescence of released AFC was measured as described in Materials and Methods. Results are presented as fold increase over control and are means ± SE of four separate experiments. *, P < 0.05, compared with control at 5 mM glucose. **, P < 0.05, compared with corresponding FFA at 5 mM glucose. #, P < 0.001, compared with control at 20 mM glucose (one-way ANOVA with Bonferroni post hoc test).

 
To determine whether inhibiting caspases could provide a means by which ß-cell death induced by elevated glucose and fatty acids, singly or combined, could be reduced, apoptotic and necrotic events were quantitated in the absence and presence of the pancaspase inhibitor zVAD-FMK. As expected, z-VAD-FMK was highly effective at reducing apoptosis induced by elevated glucose together with palmitate (Fig. 5AGo). This, however, did not translate into inhibition of cell death (Fig. 5BGo) because of a dramatic shift from apoptosis to necrosis (Fig. 5AGo). The same observation was made with stearate and linoleate at high glucose (data not shown). In accordance with these observations, experiments using trypan blue also showed that zVAD-FMK did not prevent cell death induced by the combined presence of elevated glucose plus palmitate (data not shown). Altogether, this series of experiments establishes that an apoptotic pathway is induced in INS 832/13 cells by high glucose together with some fatty acids and that, in particular, saturated fatty acids are markedly synergistic with elevated glucose in this effect.



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  		FIG. 5. Inhibition of palmitate-induced caspase activation is coupled with a switch to necrosis. A, INS 832/13 cells were grown in complete RPMI medium containing 10% FBS at 11 mM glucose on coverslips for 2 d. They were then pretreated with the caspase inhibitor z-VAD (50 µM) for 2 h in complete RPMI medium. Medium was changed to RPMI incubation medium containing 0.5% BSA at 5 (G5) or 20 mM (G20) glucose in the absence (control) or presence of 0.4 mM palmitate (Pal) and z-VAD-fmk for 24 h. Cells were then stained with Hoechst 33342 and PI, and viable, apoptotic (A), and necrotic (N) cells were counted using fluorescent microscopy from five fields chosen randomly. Results are expressed as percent of total cell number. B, Results are expressed as total cell death (sum of apoptotic and necrotic cells). Data represent four different duplicate experiments and are expressed as mean ± SE. *, P < 0.01, compared with palmitate at 5 mM glucose. **, P < 0.01, compared with control at 20 mM glucose. #, P < 0.001, compared with same condition without z-VAD-fmk (one-way ANOVA with Bonferroni post hoc test).

 
Metabolism of palmitate but not its mitochondrial ß-oxidation is required for glucolipotoxicity
Treating INS 832/13 cells with triacsin-C, an inhibitor of acyl-CoA synthase (46), blocked cell death induced by palmitate at 20 mM glucose (Fig. 6Go). For reasons that are unclear, triacsin C exhibited cytotoxicity in the absence of exogenous FFAs but not in their presence. Perhaps in the absence of exogenous FFAs, triacsin C competes with the small amount of endogenously released FFAs for reactions that are limiting for various essential cell function; these processes would not be limited by the drug when exogenous FFAs are provided. Triacsin C cytotoxicity in the absence of exogenous fatty acids has been described earlier in other cell systems (47). In any event, the data indicate that coenzyme A esterification of fatty acids, the first step of FFA metabolism, is required for the toxic action of fatty acids at elevated glucose. In addition, the results show that glucolipotoxicity is not the result of an unspecific toxic effect of FFAs themselves. Treating INS 832/13 cells with etomoxir, an inhibitor of carnitine palmitoyltransferase I (CPT-I) that catalyzes the rate-limiting step of the ß-oxidation of FFAs, markedly amplified cell death (Fig. 7AGo), and caspase-3 activation (Fig. 7BGo) induced by palmitate (0.3 mM) at elevated glucose. Interestingly, etomoxir alone at low glucose was not toxic in the absence of exogenous palmitate but allowed lipotoxicity of palmitate at low glucose that was similar in its extent to the glucolipotoxicity caused by the combined presence of high glucose and palmitate (Fig. 7AGo). This suggests that the ß-oxidation of fatty acids is not involved in the mechanism by which palmitate induces apoptosis at elevated glucose but rather that glucose inhibition of fat oxidation (3, 4) is involved in this process.



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  		FIG. 6. Palmitate must be metabolized to synergize with elevated glucose to induce ß-cell death. INS 832/13 cells were preincubated (as described in Fig. 4Go) for 1 h with or without the acyl-coenzyme A synthase inhibitor triacsin C (5 µM) and subsequently cultured for 24 h in RPMI incubation medium with or without triacsin C at 5 (G5) or 20 mM (G20) glucose in the absence or presence of 0.3 mM palmitate (Pal). Cell death was evaluated with trypan blue staining. Mean ± SE of four determinations (two separate duplicate experiments). *, P < 0.001, compared with Pal(-) and Triacsin(-) at 5 mM and 20 mM glucose. #, P < 0.01, compared with Pal(-), Triacsin (+) at 20 mM glucose. **, P < 0.001, compared with Pal(+), Triacsin(-) at 5 mM glucose. ##, P < 0.001, compared with Pal(+), Triacsin(-) at 20 mM glucose (one-way ANOVA with Bonferroni post hoc test).

 


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  		FIG. 7. The fatty acid ß-oxidation inhibitor etomoxir induces palmitate toxicity at low glucose and enhances palmitate toxicity at high glucose. A, INS 832/13 cells were preincubated as described in Fig. 4Go for 1 h with or without etomoxir (0.2 mM) and subsequently cultured for 24 h in RPMI incubation medium at 5 (G5) or 20 mM (G20) glucose in the absence or presence of 0.3 mM palmitate (Pal) with etomoxir. Dead (trypan blue positive) cells were counted and reported as percentage of total cells counted. Mean ± SE of three separate duplicate experiments. B, Cells were treated as in A, and caspase-3 activity was measured as described in Fig. 3Go. Mean ± SE of three separate experiments. *, P < 0.05, compared with Pal(-), Etomoxir(-) at 5 mM glucose control; {dagger}, P < 0.01, compared with Pal(+), etomoxir(-) at 5 mM glucose; #, P < 0.001, compared with Pal(+), Etomoxir(-) at 20 mM glucose.

 
Metformin and AICAR rescue INS 832/13 cells from death induced by glucose and palmitate
Metformin, in addition to having antioxidant properties (48), has recently been shown to be an inducer of AMPK (49), and activators of AMPK are known to increase ß-oxidation of fatty acids (50). We investigated the effect of this agent and AICAR, also an activator of AMPK (51), on INS 832/13 cell death induced by glucolipotoxicity. Both metformin (0.5 mM) and AICAR (1 mM) significantly inhibited cell death induced by palmitate at 5 and 20 mM glucose (Fig. 8Go, A and B), and this effect is by inhibiting apoptosis (Fig. 8CGo). The data suggest that by activating AMPK, most probably by its effect on fatty acid ß-oxidation, glucolipotoxicity can be reduced. Although other mechanisms of action of metformin and AICAR may also be involved, these results, in addition to the results of the triacsin C and etomoxir experiments, strongly suggest that fatty acid partitioning toward oxidation and away from esterification processes can protect ß-cells from glucolipotoxicity.



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  		FIG. 8. Metformin and AICAR, activators of AMPK, protect from glucolipotoxicity. INS 832/13 cells were cultured as described in Fig. 1Go for 24 h at 5 mM (G5) or 20 mM (G20) glucose in the absence or presence of 0.3 mM palmitate (Pal) with or without A [0.5 mM metformin (Met)] or B and C [1 mM AICAR]. Cell death (A and B) was evaluated with trypan blue staining, and apoptosis was evaluated by Hoechst staining (C). Mean ± SE of four determinations (two separate duplicate experiments). *, P <0.001, compared with Pal(-), Met(-) or Pal(-), AICAR(-) at 20 mM glucose; **, P < 0.001, compared with Pal(+), Met(-), or Pal(+), AICAR(-) at 5 mM glucose; {dagger}, P < 0.001, compared with Pal(+), Met(-), or Pal(+), AICAR(-) at 5 mM glucose; #, P < 0.001, compared with Pal(+), Met(-), or Pal(+), AICAR(-) at 20 mM glucose (one-way ANOVA with Bonferroni post hoc test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate for the first time a marked synergistic effect of high glucose and saturated FFAs on inducing cell death by apoptosis in both INS 832/13 and human islet ß-cells. Elevated glucose and FFAs together were much more toxic than either of these factors alone. In addition, we found differential effects of the various fatty acids tested at high glucose, with palmitate and stearate (saturated FFA) dramatically having the most toxicity, linoleate (polyunsaturated FFA) having modest toxicity, and oleate (monounsaturated FFA) having poor or no toxicity. Thus, for saturated fatty acids in particular, the results are consistent with the glucolipotoxicity hypothesis (3, 4, 52). This observation provides strong support for the concept that this process might be implicated in the progressive loss of ß-cell mass by apoptosis, which is involved in the pathogenesis of type 2 diabetes (14). Another component of the glucolipotoxicity hypothesis not addressed in this study is that which causes ß-cell dysfunction. In support of this component, Jacqueminet et al. (35) showed in rat islets that 72 h of culture in the presence of palmitate at low glucose did not affect insulin mRNA and insulin biosynthesis, but at high glucose these parameters were reduced. Similarly, the palmitate effect on decreasing IDX-1 expression, a ß-cell differentiation factor, was shown to be glucose dependent (53).

Hoechst and PI staining, poly (ADP-ribose) polymerase cleavage, and caspase-3 experiments showed that apoptosis is the major pathway leading to ß-cell death caused by elevated glucose and FFAs combined. When caspase-3 activation was inhibited using the pan-caspase inhibitor z-VAD-fmk, apoptosis induced by high glucose and palmitate was blocked; however, cell death was not blocked because z-VAD-fmk caused a switch from apoptosis to necrosis. This has been observed earlier in other systems (54, 55). This suggests that glucolipotoxicity, in comparison with gluco- or lipotoxicity, exerts a very severe insult to the ß-cell.

The mechanism of FFA-induced apoptosis is not well understood. However, according to the glucolipotoxicity hypothesis (4), it is predicted that, at elevated glucose, LC-CoA derived from simultaneously elevated FFAs will accumulate because of the inhibitory effect of the glucose on lipid detoxification via ß-oxidation. This results in increased LC-CoA partitioning toward toxic cellular processes either directly or indirectly via acylation and/or esterification products. In support of this concept, treating cells with the fatty acyl-coenzyme A synthetase inhibitor triacsin C demonstrated, in accordance with previous results in ZDF rat islets (30), that apoptosis induced by saturated fatty acids is not caused by the FFAs themselves, but by LC-CoA and/or metabolites derived from them. Furthermore, etomoxir, an inhibitor of CPT-I, the rate-limiting enzyme in the ß-oxidation of FFA, enhanced cell death and caspase-3 activation at both low and elevated glucose levels, suggesting that the toxicity of fatty acids at elevated glucose is not mediated via their oxidation. Interestingly, etomoxir allowed lipotoxicity of palmitate at low glucose at a level equivalent to the glucolipotoxicity caused by the combination of high glucose and palmitate. Also consistent with the view that glucose-induced inhibition of fat oxidation is important in the mechanism whereby glucose and FFAs synergize to cause ß-cell death, long-term exposure of ß-cells to glucose causes sustained accumulation of the CPT-I inhibitor malonyl-coenzyme A (56), chronically suppresses fat oxidation (34), and stimulates fat esterification processes (34). In the ß-cell, we (4) and others (35) have also reported that elevated glucose and palmitate or oleate synergize in promoting lipid esterification with increases particularly in triglyceride esterification and deposition.

We showed that both metformin and AICAR, agents that activate AMPK and favor fatty acid ß-oxidation (49, 51, 57), dramatically prevented glucolipotoxicity-induced apoptosis. These findings are in agreement with the opposite effects shown with etomoxir, an agent that inhibits ß-oxidation, and are consistent with the glucolipotoxicity model in that a central feature of this model is the effect of glucose on inhibiting fatty acid oxidation. Metformin may also exert an additional effect by reducing oxidative stress-induced apoptosis (58). Consistent with our findings with metformin, this agent has been shown to inhibit lipotoxicity induced insulin secretory dysfunction in isolated human islets (59). Clearly, the role of metformin and new pharmacotherapeutic agents designed to activate AMPK and/or ß-oxidation in preventing glucolipotoxicity warrants further attention.

The minimal toxicity of oleate, an abundant FFA in serum, is particularly interesting. Our findings are consistent with the study of Maedler et al. (29) in human islets in which oleate was not toxic. They showed, in addition, that oleate prevented toxicity of 33.3 mM glucose (29). Unsaturated fatty acids such as oleate and palmitoleate have been shown in other cellular systems to have either no effect on apoptosis (28, 60) or even to protect from palmitate-induced apoptosis (28, 60, 61). The difference between palmitate and oleate on ß-cell cytotoxicity could be attributed to differences in metabolism of the fatty acids, in particular the makeup of the phospholipid pool. Thus, the enrichment of phospholipids by saturated FFAs lowers membrane fluidity, impairing various membrane functions (62, 63). Reduced cardiolipin synthesis is another possibility because palmitate has been shown to decrease the synthesis of cardiolipin in cardiomyocytes (64) and the breast cancer cells MDA-MB231 [Hardy and Prentki (64A )]. The phospholipid cardiolipin binds cytochrome C in the mitochondrion, which prevents the release of cytochrome C into the cytoplasm, this release being an important event in the apoptotic process (65). We previously showed that oleate activates phosphatidylinositol 3-kinase in MDA-MB231 cells and promotes cell proliferation, whereas palmitate had opposite effects (61). Thus, it would be of interest to determine whether palmitate also reduces phosphatidylinositol 3-kinase activity in the ß-cell, considering that this enzyme plays a critical role in the control of cell growth and apoptosis (66). Finally, it may relate to the recently described capacity of oleate to drive palmitate to TG deposition that might be protective (67, 68). In any event, the differential effects of saturated vs. unsaturated FFAs on ß-cell apoptosis is of interest from a nutritional standpoint and provides a plausible explanation for various epidemiological and dietary intervention studies that have consistently led to the recommendation of a low saturated/high monounsaturated fat diet for type 2 diabetes prevention (69).

Our findings with oleate are at variance with a recent study by Wrede et al. (70), which showed a proapoptotic action of oleate at 5 mM glucose. This study, however, was carried out in the absence of serum. We have previously shown that in the absence of serum, INS cells are susceptible to a proapoptotic action of both oleate and palmitate, likely because of the fact that the cells are already stressed under this condition (31). A potentially very important aspect of this work by Wrede et al. (70), however, is the finding that activation of protein kinase B (PKB)/Akt by high glucose, IGF-1, or adenovirus-mediated PKB expression prevented oleate-induced apoptosis in pancreatic ß-cells. Activators of PKB/Akt could potentially protect against ß-cell glucolipotoxicity in which saturated fatty acids are involved. This hypothesis is currently being tested in our laboratory.


   Acknowledgments
 
We thank Dr. Richard Bertrand for helpful discussions.


   Footnotes
 
This work was supported by grants from the Juvenile Diabetes Research Foundation and the Canadian Institute of Health Research (CIHR) (to M.P. and L.R.). W.E.-A. was supported by a studentship from the Association du Diabète du Québec, J.B. by a graduate studentship from the CIHR, R.R. by a postdoctoral fellowship from the Swiss National Science Foundation, and S.H. by a graduate studentship from the Fond de Recherche Scientifique du Québec (FRSQ). M.P. is a CIHR scientist, and L.R. is a National scientist of the FRSQ.

Abbreviations: Ac-DEVD-AFC, N-Acetyl-Asp-Glu-Val-Asp-amino trifluoromethyl coumarin; AICAR, 5-amino-imidazole-4-carboxamide-1-ß-D-ribofuranoside; AMPK, AMP-activated protein kinase; CPT-I, carnitine palmitoyltransferase I; FBS, fetal bovine serum; FFA, free fatty acid; LC-CoA, long-chain fatty acyl-coenzyme A; PI, propidium iodide; PKB, protein kinase B; TG, triglyceride; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling.

Received April 1, 2003.

Accepted for publication June 6, 2003.


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