Chronic exposure of pancreatic β-cells to saturated non-esterified fatty acids can lead to inhibition of insulin secretion and apoptosis. Several previous studies have demonstrated that saturated fatty acids such as PA (palmitic acid) are detrimental to β-cell function compared with unsaturated fatty acids. In the present study, we describe the effect of the polyunsaturated AA (arachidonic acid) on the function of the clonal pancreatic β-cell line BRIN-BD11 and demonstrate AA-dependent attenuation of PA effects. When added to β-cell incubations at 100 μM, AA can stimulate cell proliferation and chronic (24 h) basal insulin secretion. Microarray analysis and/or real-time PCR indicated significant AA-dependent up-regulation of genes involved in proliferation and fatty acid metabolism [e.g. Angptl (angiopoietin-like protein 4), Ech1 (peroxisomal Δ3,52,4-dienoyl-CoA isomerase), Cox-1 (cyclo-oxygenase-1) and Cox-2, P<0.05]. Experiments using specific COX and LOX (lipoxygenase) inhibitors demonstrated the importance of COX-1 activity for acute (20 min) stimulation of insulin secretion, suggesting that AA metabolites may be responsible for the insulinotropic effects. Moreover, concomitant incubation of AA with PA dose-dependently attenuated the detrimental effects of the saturated fatty acid, so reducing apoptosis and decreasing parameters of oxidative stress [ROS (reactive oxygen species) and NO levels] while improving the GSH/GSSG ratio. AA decreased the protein expression of iNOS (inducible NO synthase), the p65 subunit of NF-κB (nuclear factor κB) and the p47 subunit of NADPH oxidase in PA-treated cells. These findings indicate that AA has an important regulatory and protective β-cell action, which may be beneficial to function and survival in the ‘lipotoxic’ environment commonly associated with Type 2 diabetes mellitus.

INTRODUCTION

T2DM (Type 2 diabetes mellitus) is a metabolic disorder characterized by elevated blood glucose concentrations. Most cases of T2DM are also characterized by elevated circulating fatty acid concentrations due to high dietary lipid intake, which is associated with decreased GSIS (glucose-stimulated insulin secretion) [1,2]. The impairment of insulin secretion has been attributed to insults and desensitization associated with chronic exposure of pancreatic β-cells to elevated glucose and NEFA (non-esterified fatty acid) levels, which can lead to reduction in β-cell mass (glucolipotoxicity) [3,4]. It is possible that NEFAs inhibit insulin secretion by slowing glucose uptake, metabolism and oxidation via Randle cycle effects, where an increase in availability of NEFA favours their oxidation leading to impaired glucose metabolism via glycolytic inhibition [5,6]. The exact mechanism of the cytotoxic effect of NEFA remains unclear, but it has been suggested that formation of free radicals [7,8], generation of ceramides [9], activation of endoplasmic reticulum stress and of stress-related response genes, such as that encoding NF-κB (nuclear factor κB) [10], could be involved in triggering apoptosis. In vitro studies have demonstrated that saturated fatty acids such as PA (palmitic acid) and stearic acid are more toxic than unsaturated fatty acid such as oleic and AA (arachidonic acid), although unsaturated fatty acids are not entirely free of cytotoxic effects at elevated concentrations [1114]. NEFAs, however, in low concentrations are essential for GSIS by potentiation of GSIS and can be used as an energy substrate for β-cells during periods of fasting and starvation.

PA is one of the most abundant saturated fatty acids in the human diet and is the major fatty acid synthesized de novo in the liver; in addition, its levels are elevated in the plasma in T2DM [15,16]. Several studies have demonstrated the detrimental effect of chronic exposure (usually 24 h) of different pancreatic β-cell lines and rodent islets, to PA [17]. By contrast, AA is suggested to be an important modulator of pancreatic β-cell function, enhancing insulin secretion and cell proliferation in vitro [18]. The metabolism of AA by various isoforms of COX (cyclo-oxygenase) generates lipid products that can increase insulin secretion [16]. A recent study showed that concomitant incubation of BRIN-BD11 β-cells with inhibitors of AA mobilization altered glucose-induced insulin secretion when compared with cells incubated in the presence of AA [19]. BRIN-BD11 β-cells represent a useful model for such studies, since they are stable in culture and have well-characterized metabolic, signalling, insulin secretory and cell viability responses to glucose, amino acids and numerous other modulators of β-cell function (see [20,21] for details). Additionally, recently published work has reported that palmitic acid and cytokines induce effects on insulin secretion and p47phox expression to a similar extent in both BRIN-BD11 cells and mouse islets [22].

We have now extended these studies to investigate the roles of AA in the regulation of β-cell functional integrity, insulin secretion, gene expression, ROS (reactive oxygen species) production and protection from the detrimental effects of PA.

MATERIALS AND METHODS

Reagents

RPMI 1640 medium, penicillin/streptomycin, FBS (fetal bovine serum) and glutamine were obtained from Gibco. The WST-1 (water-soluble tetrazolium salt 1) cell viability assay was obtained from Roche Diagnostics. The rat insulin ELISA kit was obtained from Mercodia. The Griess Reagent System for nitrite detection was obtained from Promega. All other reagents were obtained from Sigma–Aldrich unless otherwise stated.

Cell culture

BRIN-BD11 cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, 0.1% antibiotics (100 units/ml penicillin and 0.1 mg/ml streptomycin) and 2 mM glutamine and were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air using a Forma Scientific incubator. Cells were kept between 1×105 and 1×106 cells/ml. For the experiments, cells (1.5×105) were seeded in a 24-well plate or containing 2 ml of medium or 1.5×106 in six-well plates containing 5 ml of medium and allowed to adhere overnight before treatment in the presence or absence of fatty acids. A stock solution of each fatty acid (100 mM) was prepared using ethanol as solvent. The final concentration of ethanol added to the cell culture medium was always less than 0.5%, a concentration that was not toxic to the cells (results not shown). In some experiments, PA and AA were prepared by mixing with 90% ethanol at room temperature (20 °C) to produce stock solutions of 90 mM. The fatty acid preparations were then bound to 10% fatty-acid-free BSA (MP Biomedicals) by incubation for 1 h at 37 °C. The mixture was added to RPMI 1640 medium (containing 11 mM glucose) deprived of FBS. The final concentrations present in the cell environment were 1% for BSA and 0.5% for ethanol. The cells were seeded into six-well plates at densities of 105 cells/well and incubated for 24 h in complete RPMI 1640 medium. The medium was then removed and replaced with relevant fatty acid–BSA complexes (in RPMI 1640 devoid of FBS) for a further 24 h. Controls received BSA and vehicle only.

Cell viability and relative cell number

Cell viability (or relative cell number) was assessed using an assay based on the reduction of WST-1, a tetrazolium salt, to formazan by cell-dependent mitochondrial dehydrogenase activity. An increase in the number of viable cells results in an increase in the overall activity of mitochondrial dehydrogenases in the sample. Briefly, BRIN-BD11 cells (2×104) were plated in a 96-well plate and allowed adhere overnight. Cells were treated for 24 h, and at the end of incubation, the cell proliferation assaying reagent WST-1 (10 μl) was added to each well. The cells were incubated for 1 h in the incubator, and the formazan dye produced was quantified by measuring its absorbance at 450 nm using a kinetic plate reader (Spectramax Plus; Molecular Devices). For cell viability studies using flow cytometry, fatty acids were bound to 10% fatty-acid-free BSA by incubation at 37 °C for 1 h, and the final concentration of BSA was maintained at 1%. For individual experiments, the culture medium was removed and replaced by serum-free RPMI 1640 containing appropriate fatty acid–BSA complexes. Controls received BSA and vehicle only.

Flow cytometry

Following incubation of cells with relevant fatty acids, both attached and floating cells were harvested and centrifuged at 300 g for 5 min. The pellet was resuspended in 200 μl of medium. PI (propidium iodide) staining solution was prepared by mixing 20 μg/ml PI with FACS buffer (PBS, 2% FBS and 10 mM sodium azide). A portion (200 μl) of this PI solution was then added to the samples and incubated on ice for 10 min. The samples were then analysed on a Beckman Coulter Epics XL.MCL flow cytometer and analysed using EXPO32 ADC software (Applied Cytometry Systems).

Insulin secretion

After cell incubation, in the absence or presence of NEFA for 24 h, the medium was removed from each well and, in some cases, stored for later insulin measurement. The cells were then washed with PBS, and acute insulin secretion was determined after cell incubation for 40 min in KRB (Krebs Ringer buffer), pH 7.4, containing 1.1 mM D-glucose, followed by subsequent stimulation for 20 min in KRB pH 7.4 containing 16.7 mM glucose plus 10 mM alanine (a robust and reproducible insulinotropic stimulus) unless otherwise stated. The medium was then collected, and insulin secretion was determined using a Mercodia Ultra-sensitive Rat Insulin ELISA kit.

RT–PCR (reverse transcription–PCR)

Total RNA was isolated from cells using TRI REAGENT (Molecular Research Center) according to standard protocols provided by the manufacturer. cDNA synthesis was carried out on the total RNA using the Superscript Preamplification System II (Invitrogen Life Technologies). The primers used were Srebp-1c (sterol-regulatory-element-binding protein-1c) (forward primer, 5′-TCAGTTCCAGCATGGCTACC-3′; reverse primer, 5′-GCAGTTGATGTAGAGGCTAA-3′), Ppara (peroxisome-proliferator-activated receptor-α) (forward primer, 5′-GGTCCGATTCTTCCACTGC-3′; reverse primer, 5′-TCCCCTCCTGCAACTTCTC-3′), Ppard (peroxisome-proliferator-activated receptor-δ) (forward primer, 5′-GCAGGCTCTAGAATTCCATC-3′; reverse primer, 5′-GCCGTGTCTGGAGTGTTGTG-3′), Ampk (AMP-activated protein kinase) (forward primer, 5′-TGCTTTGCTGTGTGGAACTC-3′; reverse primer, 5′-TGTTGTACAGGCAGCTGAGG-3′), Gck (glucokinase) (forward primer, 5′-CAGTGGAGCGTGAAGACAAA-3′; reverse primer, 5′-ACCATCCGGTCATACTCCAG-3′), and Ucp-2 (uncoupling protein-2) (forward primer, 5′-ATTGCACGAGAGGAAGGGAT-3′; reverse primer, 5′-ATTACTACGTTCAGGATCC-3′). Expression of genes of interest were normalized to 18S rRNA amplified from the same samples. 18S rRNA expression levels were not altered by any treatment reported in the present study.

Microarray

Total RNA from the cells was isolated using RNeasy kit (Qiagen). RNA samples were prepared for microarray analysis using the GeneChip System (Affymetrix). Briefly, cDNA synthesis was performed using the First-Strand cDNA Synthesis Kit. The double-stranded cDNA was then cleaned using the GeneChip Sample Cleanup module. Synthesis of biotin-labelled cRNA was then prepared using the GeneChip IVT Labelling kit. Subsequently, the biotin-labelled cRNA was cleaned using the GeneChip Sample Cleanup module and following quantification was fragmented at 94 °C prior to hybridization with Rat Genome 230 2.0 Array. After hybridization, the arrays were washed and stained on a GeneChip Fluidics Station (Affymetrix). The preliminary preprocessing and the comparative gene expression analysis of this data was carried out using freeware from the Bioconductor website (www.bioconductor.org). Transcripts representing genes with a value of >1.7-fold change between control and AA-treated samples and a P value <0.05 were deemed to be significantly differentially expressed.

Real-time PCR

Total RNA was isolated from cells using TRI REAGENT (Molecular Research Center) according to the manufacturer's protocol. cDNA synthesis was carried out on the total RNA using the Superscript Preamplification System II (Invitrogen Life Technologies). Real-time PCR TaqMan assay was used to quantify the relative expression levels of genes identified as differentially expressed by microarray data. To eliminate the possibility of genomic DNA contamination, RNA samples (1 μg) were digested with DNase I before reverse transcription. DNase-treated RNA samples were then used as templates for first-strand cDNA synthesis, using SuperScript II reverse transcriptase and random primers; 2 μl of cDNA was used for real-time PCR analysis. The probes for the target genes were labelled with the fluorescent dye, FAM on the 5′-end and a non-fluorescent quencher on the 3′-end. 18S rRNA was used as an endogenous control for normalization of the target genes. PCR reactions were set up with Taqman Universal PCR Master Mix from Applied Biosystems. cDNA was amplified on the 7900HT Sequence Detection System (Applied Biosystems). Results were analysed using the relative standard curve method of analysis/ΔCt (threshold cycle) method of analysis.

ROS detection

After incubation, cells were washed with PBS and lysed with lysis buffer (containing 1% Triton-X100 and 40 mM EDTA). Lysates were then transferred to a 96-well opaque plate and incubated for 30 min with 10 μM H2DCFDA (2′,7′,di-hydrochlorofluorescein-diacetate) (Molecular Probes). Its oxidation by ROS releases DCF (2′,7′-dichlorofluorescein), which is fluorescent (excitation/emission at 495/527 nm). Fluorescence was measured in a Spectramax Plus Fluorescence Plate Reader (Molecular Devices).

Superoxide production

Cells were seeded in an opaque 96-well plate, and after treatment, 10 μM DHE (dihydroethidine) was added and incubated for 30 min in the dark and under agitation. This probe is oxidized in the presence of superoxide, and ethidium is released. Ethidium is a fluorescent compound, which binds to the DNA. Its fluorescence was measured using a Spectramax Plus Fluorescence Plate Reader (Molecular Devices).

Nitrite production

NO production was measured indirectly by nitrite quantification, which is a stable NO oxidation product. Nitrite was measured using the Griess Reagent System (Promega) by following the manufacturer's protocol.

Intracellular GSH (reduced glutathione) and GSSG (oxidized glutathione) measurement

Intracellular GSH and GSSG content was determined using a protocol described by Rahman et al. [23]. The assay is based on the reaction of GSH with DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)] (also known as Ellman's reagent), which produces the TNB (2-nitro-5-thiobenzoic acid) chromophore, which has a maximal absorbance at 412 nm, and GSSG–TNB adduct). The rate of formation of TNB, measured at 412 nm, is proportional to the concentration of GSH in the sample. The disulfide product (GSSG–TNB) is then reduced by glutathione reductase in the presence of NADPH, recycling GSH back into the reaction.

Briefly, cells were lysed in an extraction buffer (0.1% Triton X-100 and 0.6% sulfosalicylic acid in 0.1 M potassium phosphate buffer with 5 mM EDTA disodium salt, pH 7.5) for 15 min under agitation and centrifuged at 12000 g for 10 min. The supernatant was collected and stored at −80 °C. An aliquot was kept for protein measurement.

For GSH measurement, samples and GSH standards were added to a 96-well plate with a reaction mixture containing DTNB, β-NADPH and glutathione reductase, and the absorbance was read at 412 nm using a Spectramax Plus Plate Reader (Molecular Devices). Results are expressed as μg/mg of protein.

For GSSG measurement, samples and GSSG standards were incubated with 2-vinylpyridine and triethanolamine for 1 h at room temperature and then transferred to a 96-well plate and read at 412 nm. Results are expressed as μg/mg of protein.

Western blot analysis

After the incubation period, cells were trypsinized and centrifuged at 1200 g for 10 min and homogenized in extraction buffer (100 mM Tris/HCl, pH 7.0, 10 mM EDTA, 100 mM NaF, 10 mM sodium pyrophosphate and 10 mM sodium orthovanadate). The samples were sonicated and SDS (10%) was added, followed by 30 min of incubation on ice. After that, samples were centrifuged at 12000 g, for 40 min, at 4 °C. Aliquots of supernatants were used for measurement of total protein content as previously described [18]. Equal amounts of protein from each sample were separated using SDS/PAGE. Western blotting was carried out using standard protocols.

Statistical analysis

The results are presented as means±S.E.M. and were analysed by one-way or two-way ANOVA with Tukey or Bonferroni post-hoc tests respectively. The level of significance was set at P<0.05.

RESULTS

Effect of AA on relative cell number

Incubation of BRIN-BD11 cells with AA (up to 150 μM) for 24 h did not impair cell viability when compared with control, as determined by WST-1 analysis or flow cytometry. WST-1 is widely used to determine mitochondrial dehydrogenase activity, making the assumption that viable cells contain fully functional mitochondria, and therefore WST-1 absorbance is proportional to cell number. The addition of 100 μM of AA, however, stimulated cell proliferation, increasing the cell population by 19% when compared with the control. Incubation of the BRIN-BD11 cells with a relatively high level of 150 μM AA resulted in a decrease in cell viability and therefore a reduction in relative cell number when compared with cells incubated with 100 μM, but not when compared with the vehicle control (Figure 1).

Effect of AA on relative cell number

Figure 1
Effect of AA on relative cell number

BRIN-BD11 β-cells were incubated in the absence or presence of various concentrations of AA for 24 h (50–150 μM AA). Relative cell number was assessed using an assay based on the reduction of WST-1, a tetrazolium salt, to formazan by cellular mitochondrial dehydrogenase activity. An increase in the number of viable cells results in an increase in the overall mitochondrial dehydrogenase activity. Results are normalized to 100% and are expressed as means±S.E.M., for six independent experiments. *P<0.05 compared with WST-1 absorbance from control cells.

Figure 1
Effect of AA on relative cell number

BRIN-BD11 β-cells were incubated in the absence or presence of various concentrations of AA for 24 h (50–150 μM AA). Relative cell number was assessed using an assay based on the reduction of WST-1, a tetrazolium salt, to formazan by cellular mitochondrial dehydrogenase activity. An increase in the number of viable cells results in an increase in the overall mitochondrial dehydrogenase activity. Results are normalized to 100% and are expressed as means±S.E.M., for six independent experiments. *P<0.05 compared with WST-1 absorbance from control cells.

Effect of AA on acute (20 min) insulin secretion

A significant increase in basal- (1.1 mM glucose), but not nutrient-, (16.7 mM glucose and 10 mM alanine) stimulated (20 min) insulin secretion (P<0.05) was observed following 24 h exposure to 100 μM AA, when compared with the vehicle control (Figure 2).

Effect of AA on acute (20 min) insulin secretion

Figure 2
Effect of AA on acute (20 min) insulin secretion

BRIN-BD11 β-cells were incubated in the absence (Ctrl) or presence of 100 μM AA for 24 h. Subsequently, an acute (20 min) determination of basal- (1.1 mM glucose; black bars) and nutrient- (16.7 mM glucose and 10 mM alanine; white bars) stimulated insulin secretion was made. Results are expressed as means±S.E.M., for six independent experiments. *P<0.05 compared with basal insulin secretion from control cells.

Figure 2
Effect of AA on acute (20 min) insulin secretion

BRIN-BD11 β-cells were incubated in the absence (Ctrl) or presence of 100 μM AA for 24 h. Subsequently, an acute (20 min) determination of basal- (1.1 mM glucose; black bars) and nutrient- (16.7 mM glucose and 10 mM alanine; white bars) stimulated insulin secretion was made. Results are expressed as means±S.E.M., for six independent experiments. *P<0.05 compared with basal insulin secretion from control cells.

Effect of AA on gene expression

RT–PCR analysis comparing Srebp-1c, Ppard, Ppara, Ins (proinsulin), Ucp-2, Ampk and Gck mRNA expression in BRIN-BD11 cells treated in the absence or presence of increasing concentrations of AA for 24 h was performed. A significant (P<0.05) increase in the relative expression of Ampk mRNA was observed in BRIN-BD11 cells stimulated for 24 h with 100 μM AA, compared with untreated control cells.

There was a significant (P<0.01) decrease in relative expression of Srebp-1c following 24-h exposure to 50 or 100 μM AA compared with cells cultured in control medium, whereas the relative expression levels of Ppara and Ppard mRNA in cells treated with 50 or 100 μM AA was also significantly (P<0.05) reduced compared with control cells. However, there was no significant change in proinsulin, Ucp-2 or Gck mRNA expression following 24-h stimulation with AA.

Microarray analysis and subsequent data analysis (using freeware from the Bioconductor website (www.bioconductor.org) revealed that four genes were significantly altered in response to chronic (24 h) treatment of BRIN-BD11 cells with 100 μM AA (Table 1). The down-regulation of Scd-2 (stearoyl-CoA desaturase) and Abcg1 (ATP-binding cassette subfamily G member 1) observed using microarray analysis was confirmed using real-time PCR (Figures 3A and 3B). The up-regulation of Ech1 (peroxisomal Δ3,52,4-dienoyl-CoA isomerase) and Angptl4 (angiopoietin-like protein 4), both detected by microarray analysis, was also confirmed by using real-time PCR (Figures 3C and 3D).

Table 1
Microarray gene expression analysis following 24 h of AA treatment

List of BRIN-BD11 β-cell genes differentially regulated >1.7-fold following 24-h incubation with 100 μM AA compared with vehicle control.

FunctionGenBank® accession no.Gene nameFold changeP value
Lipid metabolism NM_031841 Stearoyl-CoA desaturase-2 (Scd-2−3.87 0.0018 
Cholesterol transport/lipid trafficking NM_053502 ATP-binding cassette transporter subfamily G member 1 (Abcg1−1.7 0.0284 
Angiogenesis/differentiation AA818262 Angiopoietin-like protein 4 (Angptl4+2.72 0.0134 
Fatty acid metabolism mitochondrial and peroxisomal NM_022594 Peroxisomal Δ3,52,4-dienoyl CoA isomerase (Ech1+1.73 0.0284 
FunctionGenBank® accession no.Gene nameFold changeP value
Lipid metabolism NM_031841 Stearoyl-CoA desaturase-2 (Scd-2−3.87 0.0018 
Cholesterol transport/lipid trafficking NM_053502 ATP-binding cassette transporter subfamily G member 1 (Abcg1−1.7 0.0284 
Angiogenesis/differentiation AA818262 Angiopoietin-like protein 4 (Angptl4+2.72 0.0134 
Fatty acid metabolism mitochondrial and peroxisomal NM_022594 Peroxisomal Δ3,52,4-dienoyl CoA isomerase (Ech1+1.73 0.0284 

Effect of AA on gene expression as determined by real-time PCR.

Figure 3
Effect of AA on gene expression as determined by real-time PCR.

BRIN-BD11 β-cells were cultured in the absence (Ctrl) or presence of 100 μM AA for 24 h. Expression of (A) Scd-2, (B) Abcg-1, (C) Ech1 and (D) Angptl4 was determined by real-time PCR and normalized to 18S rRNA amplified from the same samples. Results are means±S.E.M. for three individual experiments where, for each condition, they were performed in duplicate. The relative ratio for the control cells was arbitrarily set to 1. *P<0.05 and **P<0.01 compared with the control cells.

Figure 3
Effect of AA on gene expression as determined by real-time PCR.

BRIN-BD11 β-cells were cultured in the absence (Ctrl) or presence of 100 μM AA for 24 h. Expression of (A) Scd-2, (B) Abcg-1, (C) Ech1 and (D) Angptl4 was determined by real-time PCR and normalized to 18S rRNA amplified from the same samples. Results are means±S.E.M. for three individual experiments where, for each condition, they were performed in duplicate. The relative ratio for the control cells was arbitrarily set to 1. *P<0.05 and **P<0.01 compared with the control cells.

Effect of COX/LOX (lipoxygenase) inhibitors in combination with AA on insulin secretion

In order to determine whether the effects of AA on β-cell function were mediated by the products of AA metabolism or by AA itself, specific COX enzyme inhibitors and a non-selective LOX enzyme inhibitor were added to the cell culture medium for 24 h prior to the determination of acute nutrient stimulated insulin secretion (Figure 4). The selectivity of various COX inhibitors has recently been reviewed [24].

Effect of COX/LOX inhibitors in combination with AA on insulin secretion

Figure 4
Effect of COX/LOX inhibitors in combination with AA on insulin secretion

BRIN-BD11 β-cells were incubated in the absence (Ctrl) or presence of 100 μM AA or 100 μM AA in combination with 100 μM acetaminophen (Acet) or 100 μM indomethacin (Indo) or 100 μM NS-398 or 10 μM SC-560 for 24 h. Subsequently, an acute (20 min) determination of basal- and nutrient- (D-glucose and L-alanine) stimulated insulin secretion was obtained. Results are expressed as means±S.E.M. for six independent experiments. *P<0.05 and **P<0.01 compared with cells incubated with control medium; #P<0.05 and ##P<0.01 compared with cells incubated with 100 μM AA and stimulated with 16.7 mM D-glucose and 10 mM L-alanine.

Figure 4
Effect of COX/LOX inhibitors in combination with AA on insulin secretion

BRIN-BD11 β-cells were incubated in the absence (Ctrl) or presence of 100 μM AA or 100 μM AA in combination with 100 μM acetaminophen (Acet) or 100 μM indomethacin (Indo) or 100 μM NS-398 or 10 μM SC-560 for 24 h. Subsequently, an acute (20 min) determination of basal- and nutrient- (D-glucose and L-alanine) stimulated insulin secretion was obtained. Results are expressed as means±S.E.M. for six independent experiments. *P<0.05 and **P<0.01 compared with cells incubated with control medium; #P<0.05 and ##P<0.01 compared with cells incubated with 100 μM AA and stimulated with 16.7 mM D-glucose and 10 mM L-alanine.

Selective inhibition of COX-1 by 100 μM acetaminophen decreased significantly (P<0.05) the extent of nutrient-stimulated insulin secretion in the presence of 100 μM AA. This finding suggests that the inhibition of the COX-1 pathway results in a loss of AA-derived metabolites, which contribute to D-glucose- and L-alanine-stimulated insulin secretion. No differences were found in basal insulin secretion. Inhibition of COX-1 with 10 μM SC-560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole] in combination with 100 μM AA, however, significantly (P<0.05) enhanced insulin secretion measured under non-stimulating conditions compared with control. There was no significant effect on nutrient-stimulated insulin secretion from cells incubated with SC-560 and AA.

Combinations of 100 μM indomethacin (a non-specific COX and LOX inhibitor) with 100 μM AA significantly reduced basal- (P<0.01) and nutrient- (P<0.01) stimulated insulin secretion when compared with control.

Selective inhibition of COX-2 via 100 μM NS-398 [N-(2-cyclohexyloxyl-4 nitrophenyl)methanesulfonamide] in the presence of 100 μM AA significantly enhanced basal- (P<0.05) and nutrient- (P<0.01) stimulated insulin secretion when compared with control. The increase in nutrient-stimulated insulin secretion in the presence of NS-398 and exogenous AA was also significantly elevated (P<0.01) when compared with BRIN-BD11 cells incubated with 100 μM AA alone.

Effect of PA or AA on expression levels of COX-1 and COX-2

The incubation of BRIN-BD11 cells with PA for 24 h did not change the expression levels of Cox-1 or Cox-2 mRNA (Figure 5). Incubation with AA (100 μM) alone or in combination with PA, significantly increased the expression of both enzymes when compared with the no-AA control. Additionally, concomitant incubation of PA and AA (100 μM) for 24 h significantly reduced the expression levels of Cox-1 and Cox-2 mRNA, compared with AA alone, in a PA-concentration-dependent manner.

Effect of PA or AA on expression levels of Cox-1 and Cox-2

Figure 5
Effect of PA or AA on expression levels of Cox-1 and Cox-2

BRIN-BD11 cells were incubated and in the absence or presence of 100 μM AA and in the absence or presence of increasing concentrations of PA as indicated for 24 h. After this time, the cells were harvested, and the expression levels of Cox-1 (A) or Cox-2 (B) were determined by real-time PCR. Expression levels were subsequently normalized to Gapdh (glyceraldehyde-3-phosphate dehydrogenase) expression. Results are means±S.E.M. for three individual experiments. A significant improvement in cell viability (*P<0.05 and **P<0.001) was observed at all concentrations of AA tested.

Figure 5
Effect of PA or AA on expression levels of Cox-1 and Cox-2

BRIN-BD11 cells were incubated and in the absence or presence of 100 μM AA and in the absence or presence of increasing concentrations of PA as indicated for 24 h. After this time, the cells were harvested, and the expression levels of Cox-1 (A) or Cox-2 (B) were determined by real-time PCR. Expression levels were subsequently normalized to Gapdh (glyceraldehyde-3-phosphate dehydrogenase) expression. Results are means±S.E.M. for three individual experiments. A significant improvement in cell viability (*P<0.05 and **P<0.001) was observed at all concentrations of AA tested.

Rescue of PA-incubated cells by AA

Incubation of BRIN-BD11 cells with PA for 24 h decreased relative cell number in a dose-dependent manner as determined by WST-1 analysis (P<0.001; Table 2). However, concomitant incubation with AA protected BRIN-BD11 cells from the toxic effect of PA in a dose-dependent manner as determined either by WST-1 analysis (Table 2) or by flow cytometry (Figure 6A). The two experiments described in Table 2 and Figure 6(A) were performed using different methods of fatty acid delivery. The fatty acids were added to the incubation medium in ethanol only in the experiment described in Table 2, while the fatty acids were delivered complexed to BSA in the experiment described in Figure 6(A). The latter revealed that the cytoprotective response was extremely potent with an EC50 ~2 μM (total AA concentration) under the experimental conditions used and that AA caused complete protection against the cytotoxicity induced by 250 μM PA.

Rescue of PA-incubated cells by AA determined by flow cytometry (A), and effect of PA and AA on chronic (24 h) insulin secretion (B)

Figure 6
Rescue of PA-incubated cells by AA determined by flow cytometry (A), and effect of PA and AA on chronic (24 h) insulin secretion (B)

(A) BRIN-BD11 cells were incubated in the presence of 250 μM PA complexed to BSA in the absence or increasing concentrations of AA as indicated for 24 h. After this time, the cells were harvested and their viability assessed by flow cytometry after staining with PI. A significant improvement in cell viability (P<0.01) was observed at all concentrations of AA tested. (B) BRIN-BD11 β-cells were incubated in the presence of various concentrations of PA (50–150 μM non-complexed) in the absence or presence of 100 μM AA for 24 h. After the 24-h incubation period, an aliquot of the medium was removed, and the concentration of insulin was determined. The results are expressed as means±S.E.M., for three separate determinations each measured in duplicate, relative to cells incubated in the absence of fatty acid (#P<0.05) or compared with the equivalent PA-only-treated cells (*P<0.05).

Figure 6
Rescue of PA-incubated cells by AA determined by flow cytometry (A), and effect of PA and AA on chronic (24 h) insulin secretion (B)

(A) BRIN-BD11 cells were incubated in the presence of 250 μM PA complexed to BSA in the absence or increasing concentrations of AA as indicated for 24 h. After this time, the cells were harvested and their viability assessed by flow cytometry after staining with PI. A significant improvement in cell viability (P<0.01) was observed at all concentrations of AA tested. (B) BRIN-BD11 β-cells were incubated in the presence of various concentrations of PA (50–150 μM non-complexed) in the absence or presence of 100 μM AA for 24 h. After the 24-h incubation period, an aliquot of the medium was removed, and the concentration of insulin was determined. The results are expressed as means±S.E.M., for three separate determinations each measured in duplicate, relative to cells incubated in the absence of fatty acid (#P<0.05) or compared with the equivalent PA-only-treated cells (*P<0.05).

Table 2
Rescue of PA-incubated cells by AA as determined by WST-1 absorbance analysis

BRIN BD11 β-cells were incubated in the absence or presence of various concentrations of PA and/or AA at 100 μM for 24 h (50–150 μM PA). Cell viability and proliferation was assessed using an assay based on the reduction of WST-1, a tetrazolium salt, to formazan by cellular mitochondrial dehydrogenase activity and was normalized to 100%. An increase in the number of viable cells results in an increase in the overall activity of mitochondrial dehydrogenases in the sample. Results are expressed as normalized means±S.E.M. for six independent experiments. *P<0.05, **P<0.01 and ***P<0.0001 compared with cell viability from control cells (no fatty acid)

Relative mitochondrial dehydrogenase activity (%)
TreatmentControlPA (μM)…50100150
−100 μM AA 100.00±1.1 93.96±0.5 70.84±1.86*** 58.55±1.03*** 
+100 μM AA 119.0±2.75* 98.33±0.66 83.46±1.54* 67.56±5.36** 
Relative mitochondrial dehydrogenase activity (%)
TreatmentControlPA (μM)…50100150
−100 μM AA 100.00±1.1 93.96±0.5 70.84±1.86*** 58.55±1.03*** 
+100 μM AA 119.0±2.75* 98.33±0.66 83.46±1.54* 67.56±5.36** 

Effect of PA and AA on insulin secretion measured over 24 h

PA decreased cumulative insulin secretion over a 24-h incubation period in a dose-dependent manner (P<0.05; Figure 6B). Concomitant incubation of PA and AA (100 μM) significantly increased insulin secretion at all concentrations of PA used compared with incubation with PA only (P<0.0001; Figure 6B).

Influence of PA and AA on oxidative stress

An increase in the production of total ROS and superoxide was observed in BRIN-BD11 cells following incubation with 100 or 150 μM PA for 24 h. Incubation with 100 μM AA alone did not alter either the production of ROS or superoxide when compared with control. AA, however, when added in combination with 100 or 150 μM PA, significantly decreased the level of superoxide and, at 150 μM PA, significantly decreased the level of ROS (Figures 7A and 7B).

Influence of PA and AA on oxidative stress and nitrite production

Figure 7
Influence of PA and AA on oxidative stress and nitrite production

BRIN-BD11 cells were incubated for 24 h in the presence of different concentrations of PA and 100 μM AA. (A) ROS production was quantified using the H2DFCH probe. Results are expressed as means±S.E.M., for three independent experiments. (B) Superoxide production was measured using the DHE probe. Results are expressed as means±S.E.M., for five independent experiments.#P<0.0001 compared with the no-fatty-acid control. (C) Nitrite production was measured using the Griess Reagent kit according to the manufacturer's instructions. Results are expressed as means±S.E.M., for n=5 independent experiments. #P<0.0001 compared with the no-fatty-acid control. *P<0.0001 compared with the respective group without 100 μM AA.

Figure 7
Influence of PA and AA on oxidative stress and nitrite production

BRIN-BD11 cells were incubated for 24 h in the presence of different concentrations of PA and 100 μM AA. (A) ROS production was quantified using the H2DFCH probe. Results are expressed as means±S.E.M., for three independent experiments. (B) Superoxide production was measured using the DHE probe. Results are expressed as means±S.E.M., for five independent experiments.#P<0.0001 compared with the no-fatty-acid control. (C) Nitrite production was measured using the Griess Reagent kit according to the manufacturer's instructions. Results are expressed as means±S.E.M., for n=5 independent experiments. #P<0.0001 compared with the no-fatty-acid control. *P<0.0001 compared with the respective group without 100 μM AA.

Effect of PA and AA on nitrite production

NO was determined by quantifying nitrite, an oxidative product of NO. Cells increased their production of nitrite when incubated with 100 or 150 μM of PA (P<0.0001). However, concomitant incubation of AA and PA decreased the production of nitrite when compared with PA-only-treated cells (P<0.001) (Figure 7C).

Effect of PA and AA on protein expression

PA increased the protein expression of subunit p65 of NF-κB and subunit p47 of NADPH oxidase in a dose-dependent manner. AA, when added concomitantly with PA, decreased the expression of these proteins compared with PA-only-treated cells.

The protein level of the enzyme responsible for NO generation, iNOS (inducible NO synthase), was increased following incubation in the presence of PA. A combination of AA and PA significantly decreased iNOS expression compared with PA-only-treated cells (Figure 8).

Effect of PA and AA on protein expression

Figure 8
Effect of PA and AA on protein expression

BRIN-BD11 cells were incubated for 24 h in the presence of different concentrations of PA and 100 μM AA. Expression of the p65 subunit of NF-κB, p47 of NADPH oxidase and iNOS was analysed by Western blotting after gel-based protein separation (A). The absorbances of the bands were measured and normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) protein expression (B–D). Results are expressed as means±S.E.M., for three independent experiments. P<0.05 compared with cells incubated in the absence of fatty acid; #P<0.05 compared with the equivalent PA-only-treated cells.

Figure 8
Effect of PA and AA on protein expression

BRIN-BD11 cells were incubated for 24 h in the presence of different concentrations of PA and 100 μM AA. Expression of the p65 subunit of NF-κB, p47 of NADPH oxidase and iNOS was analysed by Western blotting after gel-based protein separation (A). The absorbances of the bands were measured and normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) protein expression (B–D). Results are expressed as means±S.E.M., for three independent experiments. P<0.05 compared with cells incubated in the absence of fatty acid; #P<0.05 compared with the equivalent PA-only-treated cells.

Effect of PA and AA on glutathione metabolism

Incubation of cells in the presence of PA resulted in a decrease in intracellular GSH in a dose-dependent manner, while the content of GSSG was increased. AA addition to cells, which were also treated with PA, increased the concentration of GSH and decreased the concentration of GSSG, contributing to an increase in the ratio of GSH/GSSG when compared with the group incubated with PA only (Figure 9).

Effect of PA and AA on glutathione metabolism

Figure 9
Effect of PA and AA on glutathione metabolism

BRIN-BD11 cells were incubated for 24 h in the presence of different concentrations of PA and 100 μM AA. (A) Intracellular content of GSH was measured. (B) Intracellular content of GSGG was measured. (C) GSH/GSSG ratio. Results are expressed as means±S.E.M., for three independent experiments. *P<0.0001 compared with the no-fatty-acid control. In (A and B). #P<0.0001 compared with the respective group without 100 μM AA.

Figure 9
Effect of PA and AA on glutathione metabolism

BRIN-BD11 cells were incubated for 24 h in the presence of different concentrations of PA and 100 μM AA. (A) Intracellular content of GSH was measured. (B) Intracellular content of GSGG was measured. (C) GSH/GSSG ratio. Results are expressed as means±S.E.M., for three independent experiments. *P<0.0001 compared with the no-fatty-acid control. In (A and B). #P<0.0001 compared with the respective group without 100 μM AA.

DISCUSSION

The effects of NEFAs on β-cell function are complex. Pancreatic islets exposed to high concentrations of NEFAs for periods of 24–48 h [3] display enhanced insulin secretion in the presence of a non-stimulatory glucose concentration, decreased insulin synthesis, depletion of stored insulin and an impaired response of the β-cell to stimulation by high glucose concentrations, all of which are characteristic of the altered β-cell function associated with T2DM. Rat and human islets exposed to elevated levels of NEFAs for 48 h also demonstrated increased basal insulin release (glucose concentration was 3 mM), but decreased release at an elevated (stimulatory) glucose concentration (27 mM) [25].

The effects of high concentrations of NEFA on isolated islets and clonal pancreatic insulin-secreting cells are dependent on the structure (i.e. whether saturated or unsaturated) of the fatty acid and the period of exposure during cell culture. Acute exposure (1–3 h) of pancreatic islets to saturated NEFA enhances insulin secretion [26] and plays a critical role in modulating the stimulatory effect of glucose on insulin release [27]. Acute removal of exogenous NEFA may, however, result in excessively high rates of glucose-stimulated insulin secretion [28]. The latter results may argue in favour of a buffering role for NEFA, such that islet cells utilize some glucose (in the form of a downstream metabolite, glycerol 3-phosphate) for formation of TAG (triacylglycerol), so as to ensure that excessively high rates of insulin secretion do not occur. Expression of UCP-2 in β-cells is increased by NEFAs. UCPs are located in the mitochondrial inner membrane and act as proton channels. They uncouple the electrochemical gradient produced by the respiratory chain from ATP synthesis [29]. Up-regulation of UCP-2 expression or overactivity of UCP-2 leads to inhibition of ATP synthesis and alteration in levels of ROS generation. NEFAs increase UCP-2 mRNA and protein levels in several cells including β-cells. As a result, ATP synthesis is reduced, and GSIS is blunted. Ucp-2-knockout mice have lower fasting blood glucose and elevated insulin levels when fed with a high-fat diet compared with WT (wild-type) mice [30]. Exposure to PA reduced GSIS in WT islets, whereas Ucp-2−/− islets had enhanced GSIS. While saturated NEFAs may induce detrimental effects on β-cell function in part via increased UCP-2 expression, we clearly demonstrated in the present study (microarray and PCR data) that the polyunsaturated NEFA AA did not increase Ucp-2 expression, therefore avoiding any UCP-2-dependent deleterious effects associated with saturated NEFAs. Saturated NEFAs can lead to excessive ROS generation from mitochondrial and extramitochondrial sources such as NADPH oxidase in β-cells, accompanied by the loss of antioxidant defence via glutathione depletion [3133]. Additionally, NO generation in pancreatic β-cells may be stimulated through ligation and signalling via the NEFA receptor GPR40 (G-protein-related protein 40) [34], a possible regulator of iNOS expression [35]. NO competes with molecular oxygen at Complex IV of the electron-transport chain, leading to impaired oxidative phosphorylation and ATP generation, which can induce dysregulation of cellular Ca2+ homoeostasis resulting in endoplasmic reticulum stress [3638].

In contrast with the deleterious effects associated with elevated saturated fatty acids, reports suggest that non-esterified AA is critical for normal pancreatic β-cell function. For example, studies in which the release of endogenous AA was attenuated by inhibiting PLA2 (phospholipase A2) activity, revealed a significant reduction in GSIS from human islets [39]. The release of endogenous AA from the plasma membrane via PLA2-dependent hydrolysis of phospholipids has been identified as an important signalling event [4042]. Exogenous addition of AA has been reported to augment insulin secretion from pancreatic β-cells [43], which is in agreement with results obtained for chronic (24 h) insulin secretion in the present study (Figure 6B). We have now provided further mechanistic data on the action of AA. The positive effects of AA described here could result from the direct second messenger functions of this molecule, or they could derive from metabolism of AA to biologically active products. Metabolism of non-esterified AA by COX enzymes yields PGs (prostaglandins), while LOX enzymes generate LTs (leukotrienes) and HETEs (hydroxyeicosatetraenoic acids). Previous experiments evaluating the functional effects of PGs (most notably PGE2) on the β-cell have implicated PGs in the inhibition of glucose-stimulated insulin secretion from a hamster-derived cell line [44]. However, HETEs and LTs produced from LOX enzyme activity augment GSIS [45]. In the present study, we have demonstrated that inhibition of the COX-1 pathway of AA metabolism resulted in an inhibition of D-glucose- and L-alanine-stimulated insulin secretion from BRIN-BD11 cells, in the presence of exogenously added AA. This is consistent with the possibility that COX-2 metabolites (which will be preferentially generated in these conditions) are inhibitory to nutrient-stimulated insulin secretion. In contrast, selective inhibition of COX-2 using NS-398 (100 μM) in combination with 100 μM exogenously added AA significantly (P<0.05) enhanced both basal- and nutrient-stimulated insulin secretion compared with BRIN-BD11 cells incubated with control media (Table 1). This may indicate that COX-1 metabolites (which will be preferentially generated under these conditions) are stimulatory to basal- and nutrient-stimulated insulin secretion. We also demonstrated that BRIN-BD11 cell COX-1 and COX-2 expression was not altered when incubated with PA. In contrast, AA significantly increased the expression of both enzymes. However, PA was able to reduce enhanced AA expression in a dose-dependent manner. The latter findings correlate well with the insulinotropic effects of addition of both AA and PA over 24 h of incubation (Figure 6B)

The ability of AA to cause down-regulation of Scd-2 and Abcg1 and the up-regulation of Ech1 and Angptl4 gene expression (Figure 3) would infer a decrease in synthesis of unsaturated fatty acids (Scd-2) and a decrease in cholesterol transport to HDL (high-density lipoprotein) (Abcg1). An increase in isomerization activity (Ech1) would favour the entry of unsaturated fatty acids into the pathway of β-oxidation, whereas an increase in Angptl4 signals a potential increase in cholesterol synthesis. The expression changes identified by microarray analysis and confirmed with real-time PCR would therefore predict greater levels of unsaturated fatty acid oxidation but a potential increase in cholesterol synthesis and transport in the presence of AA.

The AA-mediated rescue of BRIN-BD11 cells from PA-mediated dysfunction, as reported in the present work, is remarkable and supports earlier data obtained in MIN6 cells [46] This response may be mediated by a reduction in oxidative and inflammatory stress as indicated by reductions in NF-κB, iNOS and NADPH oxidase subunit levels and a reduction in NO and ROS levels respectively, resulting in a corresponding increase in the GSH/GSSG ratio. The positive impact of AA with respect to its effect on cellular oxidative stress is a novel finding and must be considered as a key mechanism of cell protection with respect to fatty-acid-induced β-cell dysfunction. The molecular mechanisms could involve AA- or PG-mediated changes in signal transduction, subsequent gene expression and their resulting impact on redox and inflammatory signalling pathways.

It is clear that further work on the mechanisms of cytoprotection by AA (and/or downstream metabolites) and its ability to modulate insulin secretion is required. This may reveal novel therapeutic options for the treatment of T2DM.

FUNDING

This work was supported by the Health Research Board of Ireland [grant number RP/2002/184], European Foundation for Study of Diabetes, Diabetes Research and Wellness Foundation (U.K.) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.

We thank Dr Mauricio da Silva Krause, Rodolfo Sumayao and Niamh Mullooly for their scientific assistance and discussions.

Abbreviations

     
  • AA

    arachidonic acid

  •  
  • Abcg1

    ATP-binding cassette subfamily G member 1

  •  
  • Ampk

    AMP-activated protein kinase

  •  
  • COX

    cyclo-oxygenase

  •  
  • DHE

    dihydroethidine

  •  
  • Ech1

    peroxisomal Δ3,52,4-dienoyl-CoA isomerase

  •  
  • FBS

    fetal bovine serum

  •  
  • Gck

    glucokinase

  •  
  • GSIS

    glucose-stimulated insulin secretion

  •  
  • HETE

    hydroxyeicosatetraenoic acid

  •  
  • iNOS

    inducible NO synthase

  •  
  • LT

    leukotriene

  •  
  • NEFA

    non-esterified fatty acid

  •  
  • NF-κB

    nuclear factor κB

  •  
  • NS-398

    N-(2-cyclohexyloxyl-4 nitrophenyl)methanesulfonamide

  •  
  • PA

    palmitic acid

  •  
  • PI

    propidium iodide

  •  
  • PG

    prostaglandin

  •  
  • PLA2

    phospholipase A2

  •  
  • Ppara

    peroxisome-proliferator-activated receptor α

  •  
  • Ppard

    peroxisome-proliferator-activated receptor δ

  •  
  • ROS

    reactive oxygen species

  •  
  • RT–PCR

    (reverse transcription–PCR

  •  
  • SC-560

    5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole

  •  
  • Scd-2

    stearoyl-CoA desaturase-2

  •  
  • Srebp-1c

    sterol-regulatory-element-binding protein-1c

  •  
  • T2DM

    Type 2 diabetes mellitus

  •  
  • TNB

    2-nitro-5-thiobenzoic acid

  •  
  • UCP

    uncoupling protein

  •  
  • WST-1

    water-soluble tetrazolium salt 1

  •  
  • WT

    wild-type

AUTHOR CONTRIBUTION

Philip Newsholme was the principal investigator responsible for this work and takes full responsibility for the manuscript. Deirdre Keane and Hilton Kenji Takahashi completed the majority of experiments described. Shalinee Dhayal completed the experiment described in Figure 6(A). Noel Morgan, Rui Curi and Philip Newsholme co-wrote and revised the manuscript.

References

1
Poitout
 
V.
Robertson
 
R. P.
 
Glucolipotoxicity: fuel excess and β-cell dysfunction
Endocr. Rev.
2008
, vol. 
29
 (pg. 
351
-
366
)
2
McGarry
 
J. D.
Dobbins
 
R. L.
 
Fatty acids, lipotoxicity and insulin secretion
Diabetologia
1999
, vol. 
42
 (pg. 
128
-
138
)
3
Haber
 
E. P.
Ximenes
 
H. M.
Procopio
 
J.
Carvalho
 
C. R.
Curi
 
R.
Carpinelli
 
A. R.
 
Pleiotropic effects of fatty acids on pancreatic β-cells
J. Cell. Physiol.
2003
, vol. 
194
 (pg. 
1
-
12
)
4
Yaney
 
G. C.
Corkey
 
B. E.
 
Fatty acid metabolism and insulin secretion in pancreatic β-cells
Diabetologia
2003
, vol. 
46
 (pg. 
1297
-
1312
)
5
Randle
 
P. J.
Garland
 
P. B.
Hales
 
C. N.
Newsholme
 
E. A.
 
The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus
Lancet
1963
, vol. 
i
 (pg. 
785
-
789
)
6
Randle
 
P. J.
Kerbey
 
A. L.
Espinal
 
J.
 
Mechanisms decreasing glucose oxidation in diabetes and starvation: role of lipid fuels and hormones
Diabetes Metab. Rev.
1988
, vol. 
4
 (pg. 
623
-
638
)
7
Robertson
 
R. P.
Harmon
 
J.
Tran
 
P. O.
Tanaka
 
Y.
Takahashi
 
H.
 
Glucose toxicity in β-cells: type 2 diabetes, good radicals gone bad, and the glutathione connection
Diabetes
2003
, vol. 
52
 (pg. 
581
-
587
)
8
Newsholme
 
P.
Haber
 
E. P.
Hirabara
 
S. M.
Rebelato
 
E. L.
Procopio
 
J.
Morgan
 
D.
Oliveira-Emilio
 
H. C.
Carpinelli
 
A. R.
Curi
 
R.
 
Diabetes associated cell stress and dysfunction: role of mitochondrial and non-mitochondrial ROS production and activity
J. Physiol.
2007
, vol. 
583
 (pg. 
9
-
24
)
9
Lupi
 
R.
Dotta
 
F.
Marselli
 
L.
Del Guerra
 
S.
Masini
 
M.
Santangelo
 
C.
Patane
 
G.
Boggi
 
U.
Piro
 
S.
Anello
 
M.
, et al 
Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that β-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated
Diabetes
2002
, vol. 
51
 (pg. 
1437
-
1442
)
10
Kharroubi
 
I.
Ladriere
 
L.
Cardozo
 
A. K.
Dogusan
 
Z.
Cnop
 
M.
Eizirik
 
D. L.
 
Free fatty acids and cytokines induce pancreatic β-cell apoptosis by different mechanisms: role of nuclear factor-κB and endoplasmic reticulum stress
Endocrinology
2004
, vol. 
145
 (pg. 
5087
-
5096
)
11
Welters
 
H. J.
Tadayyon
 
M.
Scarpello
 
J. H.
Smith
 
S. A.
Morgan
 
N. G.
 
Mono-unsaturated fatty acids protect against β-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure
FEBS Lett.
2004
, vol. 
560
 (pg. 
103
-
108
)
12
Maedler
 
K.
Spinas
 
G. A.
Dyntar
 
D.
Moritz
 
W.
Kaiser
 
N.
Donath
 
M. Y.
 
Distinct effects of saturated and monounsaturated fatty acids on β-cell turnover and function
Diabetes
2001
, vol. 
50
 (pg. 
69
-
76
)
13
Opara
 
E. C.
Garfinkel
 
M.
Hubbard
 
V. S.
Burch
 
W. M.
Akwari
 
O. E.
 
Effect of fatty acids on insulin release: role of chain length and degree of unsaturation
Am. J. Physiol.
1994
, vol. 
266
 (pg. 
E635
-
E639
)
14
Cnop
 
M.
Hannaert
 
J. C.
Hoorens
 
A.
Eizirik
 
D. L.
Pipeleers
 
D. G.
 
Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation
Diabetes
2001
, vol. 
50
 (pg. 
1771
-
1777
)
15
Stein
 
D. T.
Stevenson
 
B. E.
Chester
 
M. W.
Basit
 
M.
Daniels
 
M. B.
Turley
 
S. D.
McGarry
 
J. D.
 
The insulinotropic potency of fatty acids is influenced profoundly by their chain length and degree of saturation
J. Clin. Invest.
1997
, vol. 
100
 (pg. 
398
-
403
)
16
Rasmussen
 
O.
Lauszus
 
F. F.
Christiansen
 
C.
Thomsen
 
C.
Hermansen
 
K.
 
Differential effects of saturated and monounsaturated fat on blood glucose and insulin responses in subjects with non-insulin-dependent diabetes mellitus
Am. J. Clin. Nutr.
1996
, vol. 
63
 (pg. 
249
-
253
)
17
Haber
 
E. P.
Hirabara
 
S. M.
Gomes
 
A. D.
Curi
 
R.
Carpinelli
 
A. R.
Carvalho
 
C. R.
 
Palmitate modulates the early steps of insulin signalling pathway in pancreatic islets
FEBS Lett.
2003
, vol. 
544
 (pg. 
185
-
188
)
18
Dixon
 
G.
Nolan
 
J.
McClenaghan
 
N. H.
Flatt
 
P. R.
Newsholme
 
P.
 
Arachidonic acid, palmitic acid and glucose are important for the modulation of clonal pancreatic β-cell insulin secretion, growth and functional integrity
Clin. Sci.
2004
, vol. 
106
 (pg. 
191
-
199
)
19
Keane
 
D.
Newsholme
 
P.
 
Saturated and unsaturated (including arachidonic acid) non-esterified fatty acid modulation of insulin secretion from pancreatic β-cells
Biochem. Soc. Trans.
2008
, vol. 
36
 (pg. 
955
-
958
)
20
McClenaghan
 
N. H.
 
Physiological regulation of the pancreatic β cell: functional insights for understanding and therapy of diabetes
Exp. Physiol.
2007
, vol. 
92
 (pg. 
481
-
496
)
21
Newsholme
 
P.
Gaudel
 
C.
McClenaghan
 
N. H.
 
Nutrient regulation of insulin secretion and β cell functional integrity
Adv. Exp. Med. Biol.
2010
, vol. 
654
 (pg. 
91
-
114
)
22
Michalska
 
M.
Wolf
 
G.
Walther
 
R.
Newsholme
 
P.
 
The effects of pharmacologic inhibition of NADPH oxidase and iNOS on pro-inflammatory cytokine, palmitic acid or H2O2-induced mouse islet or clonal pancreatic β cell dysfunction
Biosci. Rep.
2010
, vol. 
30
 (pg. 
445
-
453
)
23
Rahman
 
I.
Kode
 
A.
Biswas
 
S. K.
 
Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method
Nat. Protoc.
2006
, vol. 
1
 (pg. 
3159
-
65
)
24
Sobolewski
 
C.
Cerella
 
C.
Dicato
 
M.
Ghibelli
 
L.
Diederich
 
M.
 
The role of cyclooxygenase-2 in cell proliferation and cell death in human malignancies
Int. J. Cell Biol.
2010
, vol. 
2010
 pg. 
215158
 
25
Zhou
 
Y. P.
Grill
 
V. E.
 
Palmitate-induced β-cell insensitivity to glucose is coupled to decreased pyruvate dehydrogenase activity and enhanced kinase activity in rat pancreatic islets
Diabetes
1995
, vol. 
44
 (pg. 
394
-
399
)
26
Crespin
 
S. R.
Greenough
 
W. B.
Steinberg
 
D.
 
Stimulation of insulin secretion by long-chain free fatty acids. A direct pancreatic effect
J. Clin. Invest.
1973
, vol. 
52
 (pg. 
1979
-
1984
)
27
Vara
 
E.
Fernandez-Martin
 
O.
Garcia
 
C.
Tamarit-Rodriguez
 
J.
 
Palmitate dependence of insulin secretion, “de novo” phospholipid synthesis and 45Ca2+-turnover in glucose stimulated rat islets
Diabetologia
1988
, vol. 
31
 (pg. 
687
-
693
)
28
Straub
 
S. G.
Sharp
 
G. W.
 
Massive augmentation of stimulated insulin secretion induced by fatty acid-free BSA in rat pancreatic islets
Diabetes
2004
, vol. 
53
 (pg. 
3152
-
3158
)
29
Joseph
 
J. W.
Koshkin
 
V.
Saleh
 
M. C.
Sivitz
 
W. I.
Zhang
 
C. Y.
Lowell
 
B. B.
Chan
 
C. B.
Wheeler
 
M. B.
 
Free fatty acid-induced β-cell defects are dependent on uncoupling protein 2 expression
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
51049
-
51056
)
30
Joseph
 
J. W.
Koshkin
 
V.
Zhang
 
C. Y.
Wang
 
J.
Lowell
 
B. B.
Chan
 
C. B.
Wheeler
 
M. B.
 
Uncoupling protein 2 knockout mice have enhanced insulin secretory capacity after a high-fat diet
Diabetes
2002
, vol. 
51
 (pg. 
3211
-
3219
)
31
Newsholme
 
P.
Morgan
 
D.
Rebelato
 
E.
Oliveira-Emilio
 
H. C.
Procopio
 
J.
Curi
 
R.
Carpinelli
 
A.
 
Insights into the critical role of NADPH oxidase(s) in the normal and dysregulated pancreatic β cell
Diabetologia
2009
, vol. 
52
 (pg. 
2489
-
2498
)
32
Robertson
 
R. P.
 
Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet β-cells in diabetes
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
42351
-
42354
)
33
Kaneto
 
H.
Katakami
 
N.
Kawamori
 
D.
Miyatsuka
 
T.
Sakamoto
 
K.
Matsuoka
 
T. A.
Matsuhisa
 
M.
Yamasaki
 
Y.
 
Involvement of oxidative stress in the pathogenesis of diabetes
Antioxid. Redox Signaling
2007
, vol. 
9
 (pg. 
355
-
366
)
34
Itoh
 
Y.
Kawamata
 
Y.
Harada
 
M.
Kobayashi
 
M.
Fujii
 
R.
Fukusumi
 
S.
Ogi
 
K.
Hosoya
 
M.
Tanaka
 
Y.
Uejima
 
H.
, et al 
Free fatty acids regulate insulin secretion from pancreatic β-cells through GPR40
Nature
2003
, vol. 
422
 (pg. 
173
-
176
)
35
Qader
 
S. S.
Jimenez-Feltstrom
 
J.
Ekelund
 
M.
Lundquist
 
I.
Salehi
 
A.
 
Expression of islet inducible nitric oxide synthase and inhibition of glucose-stimulated insulin release after long-term lipid infusion in the rat is counteracted by PACAP27
Am. J. Physiol. Endocrinol. Metab.
2007
, vol. 
292
 (pg. 
E1447
-
E1455
)
36
Oyadomari
 
S.
Takeda
 
K.
Takiguchi
 
M.
Gotoh
 
T.
Matsumoto
 
M.
Wada
 
I.
Akira
 
S.
Araki
 
E.
Mori
 
M.
 
Nitric oxide-induced apoptosis in pancreatic β-cells is mediated by the endoplasmic reticulum stress pathway
Proc. Natl. Acad. Sci. U.S.A.
2001
, vol. 
98
 (pg. 
10845
-
10850
)
37
Moncada
 
S.
Erusalimsky
 
J. D.
 
Does nitric oxide modulate mitochondrial energy generation and apoptosis?
Nat. Rev. Mol. Cell Biol.
2002
, vol. 
3
 (pg. 
214
-
220
)
38
Erusalimsky
 
J. D.
Moncada
 
S.
 
Nitric oxide and mitochondrial signaling: from physiology to pathophysiology
Arterioscler. Thromb. Vasc. Biol.
2007
, vol. 
27
 (pg. 
2524
-
2531
)
39
Persaud
 
S. J.
Muller
 
D.
Belin
 
V. D.
Kitsou-Mylona
 
I.
Asare-Anane
 
H.
Papadimitriou
 
A.
Burns
 
C. J.
Huang
 
G. C.
Amiel
 
S. A.
Jones
 
P. M.
 
The role of arachidonic acid and its metabolites in insulin secretion from human islets of Langerhans
Diabetes
2007
, vol. 
56
 (pg. 
197
-
203
)
40
Wolf
 
B. A.
Pasquale
 
S. M.
Turk
 
J.
 
Free fatty acid accumulation in secretagogue-stimulated pancreatic islets and effects of arachidonate on depolarization-induced insulin secretion
Biochemistry
1991
, vol. 
30
 (pg. 
6372
-
6379
)
41
Ramanadham
 
S.
Gross
 
R. W.
Han
 
X.
Turk
 
J.
 
Inhibition of arachidonate release by secretagogue-stimulated pancreatic islets suppresses both insulin secretion and the rise in β-cell cytosolic calcium ion concentration
Biochemistry
1993
, vol. 
32
 (pg. 
337
-
346
)
42
Ramanadham
 
S.
Song
 
H.
Bao
 
S.
Hsu
 
F. F.
Zhang
 
S.
Ma
 
Z.
Jin
 
C.
Turk
 
J.
 
Islet complex lipids: involvement in the actions of group VIA calciumindependent phospholipase A2 in β-cells
Diabetes
2004
, vol. 
53
 (pg. 
S179
-
S185
)
43
Band
 
A. M.
Jones
 
P. M.
Howell
 
S. L.
 
Arachidonic acid-induced insulin secretion from rat islets of Langerhans
J. Mol. Endocrinol.
1992
, vol. 
8
 (pg. 
95
-
101
)
44
Robertson
 
R. P.
Tsai
 
P.
Little
 
S. A.
Zhang
 
H. J.
Walseth
 
T. F.
 
Receptor-mediated adenylate cyclase-coupled mechanism for PGE2 inhibition of insulin secretion in HIT cells
Diabetes
1987
, vol. 
36
 (pg. 
1047
-
1053
)
45
Pek
 
S. B.
Walsh
 
M. F.
 
Leukotrienes stimulate insulin release from the rat pancreas
Proc. Natl. Acad. Sci. U.S.A.
1984
, vol. 
81
 (pg. 
2199
-
2202
)
46
Papadimitriou
 
A.
King
 
A. J.
Jones
 
P. M.
Persaud
 
S. J.
 
Anti-apoptotic effects of arachidonic acid and prostaglandin E2 in pancreatic β-cells
Cell Physiol. Biochem.
2007
, vol. 
20
 (pg. 
607
-
616
)

Author notes

1

These authors contributed equally to this work.

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