Glucolipotoxicity reduced ELOVL2 expression in INS-1 beta cells and mouse islets
In INS-1 beta cells, we found that 5 mmol/l glucose with or without 0.4 mmol/l palmitate had no effect on Elovl2 mRNA levels after 24 h of treatment (Fig. 1a). By contrast, 30 mmol/l glucose transiently decreased Elovl2 mRNA levels at 6 h, with a progressive return to control levels at 24 h. Interestingly, we found that 0.4 mmol/l palmitate with 30 mmol/l glucose (conditions of glucolipotoxicity) induced a time-dependent decrease in Elovl2 mRNA levels (Fig. 1a). Glucolipotoxicity decreased Elovl2 mRNA levels by 41% after 6 h, and this effect persisted until 24 h, where a 60% reduction was observed. In agreement, western blot analysis showed that glucolipotoxicity reduced ELOVL2 protein levels in INS-1 cells (Fig. 1b). We also found that glucolipotoxicity decreased Elovl2 mRNA and ELOVL2 protein levels in mouse islets of Langerhans after 48 h of treatment (Fig. 1c, d). Gene expression of other elongases (Elovl1 to Elovl7) was not significantly altered by glucolipotoxicity in INS-1 cells (ESM Fig. 1). Together, these results suggest that glucolipotoxicity downregulates specifically Elovl2 expression in beta cells.
ELOVL2 expression regulated glucolipotoxicity-induced INS-1 beta cell apoptosis
Palmitate is known to stimulate beta cell apoptosis in the presence of high glucose concentrations [6, 20, 24]. Indeed, caspase-3/7 activity and PARP cleavage were increased with palmitate and high glucose (Fig. 1e, f). A specific Elovl2 siRNA significantly reduced Elovl2 mRNA and ELOVL2 protein levels in INS-1 cells (Fig. 2a, b). Interestingly, Elovl2 siRNA potentiated glucolipotoxicity-induced caspase activation (4.7-fold increase compared with control siRNA) (Fig. 2c) and cleavage of PARP in INS-1 cells (Fig. 2d). Adenoviral overexpression of human ELOVL2 significantly increased human ELOVL2 mRNA and ELOVL2 protein levels in INS-1 cells (Fig. 2e, f). In INS-1-overexpressing Elovl2 cells, induction of caspase-3/7 activation by glucolipotoxicity was significantly inhibited by 33% compared with Ad-gfp-transfected cells (Fig. 2g), while glucolipotoxicity was unable to induce PARP cleavage (Fig. 2h). Since ELOVL2 has been shown to be responsible for the synthesis of DHA , we explored whether addition of DHA to the culture medium could also inhibit glucolipotoxicity-induced apoptosis. DHA 10 μmol/l significantly inhibited caspase-3/7 activation (Fig. 2i) and PARP cleavage (Fig. 2j) induced by glucolipotoxicity. Together, these results suggest that ELOVL2, and the consequent synthesis of endogenous n-3 PUFAs, such as DHA, counteracts glucolipotoxicity-induced apoptosis of beta cells.
The ELOVL2/DHA axis inhibited ceramide accumulation induced by glucolipotoxicity in INS-1 beta cells
Glucolipotoxicity has been shown to induce beta cell apoptosis through ceramide accumulation via de novo ceramide synthesis [7, 24]. In agreement, we observed that glucolipotoxicity increased ceramide levels in INS-1 cells (Fig. 3a; ESM Fig. 2d). Elovl2 overexpression in INS-1 cells decreased ceramide accumulation induced by glucolipotoxicity (Fig. 3a). Addition of DHA, at 100 μmol/l and as low as 10 μmol/l, significantly inhibited ceramide accumulation during glucolipotoxicity (Fig. 3b; ESM Fig. 2d). During glucolipotoxicity, de novo ceramide biosynthesis led to the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content . DHA treatment generally had no effect on ceramide species levels by itself, with the exception of reducing C16:0 and increasing C24:0 ceramide species (ESM Fig. 2a–c). However, during glucolipotoxicity, DHA decreased accumulation of specific ceramide species, namely C18:0, C22:0 and C24:0 ceramides, which have been previously linked to the glucolipotoxic pro-apoptotic effects (Fig. 3c) . Interestingly, downregulation of Elovl2 expression via Elovl2 siRNA significantly increased ceramide levels under glucolipotoxic stimulation (Fig. 3d). Inhibition of ceramide synthesis with both l-cycloserine and fumonisin B1, which block serine palmitoyltransferase and ceramide synthase activity, respectively (Fig. 4a), partially protected INS-1 cells from glucolipotoxicity-induced apoptosis (Fig. 3e, f). In conditions where levels of ELOVL2 were downregulated, l-cycloserine and fumonisin B1 still decreased glucolipotoxicity-induced apoptosis by 40% and 70%, respectively (Fig. 3e, f). In agreement, both inhibitors efficiently diminished glucolipotoxicity-induced ceramide accumulation when ELOVL2 was downregulated (Fig. 3g, h). DHA treatment did not modify gene expression of serine palmitoyltransferase subunit and of ceramide synthases (data not shown). Together, our results suggest that the ELOVL2/DHA axis counteracts glucolipotoxicity-induced death by controlling accumulation of ceramides in INS-1 beta cells.
The ELOVL2/DHA axis did not regulate ceramide and neutral lipid metabolism in INS-1 beta cells under conditions of glucolipotoxicity
We previously showed that palmitate stimulates ceramide accumulation in beta cells either by increasing de novo synthesis  or by modulating ceramide metabolism . We found that dl-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide synthases (Fig. 4a), potentiated glucolipotoxic caspase-3/7 activation (Fig. 4b) but was unable to inhibit the effect of Ad-Elovl2 and DHA on beta cell survival under conditions of glucolipotoxicity (Fig. 4b). Inhibition of sphingosine kinase 1 by PF-543  exacerbated glucolipotoxicity-induced apoptosis of INS-1 beta cells (ESM Fig. 3a) but did not counteract the anti-apoptotic effect of Ad-Elovl2 or DHA during glucolipotoxicity (ESM Fig. 3a). As the protective effects of ELOVL2/DHA appear independent of ceramide metabolism, an alternative hypothesis is that the ELOVL2/DHA axis was blocking the accumulation of pro-apoptotic ceramides at an early step in the de novo synthesis pathway. We found that sphingosine treatment induced accumulation of endogenous ceramides and caspase-3/7 activation when INS-1 cells were under conditions of glucolipotoxicity (Fig. 4c, d). DHA and Ad-Elovl2 were unable to block either ceramide accumulation or caspase-3/7 activation induced by sphingosine with glucolipotoxicity (Fig. 4c, d). These data suggest that the ELOVL2/DHA axis is unable to inhibit ceramide synthase, which converts sphingosine into ceramides (Fig. 4a ), to block caspase activity induced by glucolipotoxicity. Finally, we found that the ELOVL2/DHA axis was also unable to counteract caspase-3/7 activation (ESM Fig. 3b) induced by the C2-ceramide analogue. C2 ceramides were transformed into endogenous ceramides (Fig. 4a ; ESM Fig. 2e, 3c), which were not affected by the presence of Ad-Elovl2 and DHA (ESM Fig. 2e, 3b). In beta cells, palmitate could be esterified into triacylglycerols, a neutral form of fatty acid storage which has been shown to be non-toxic to the cells [27, 28]. In conditions of glucolipotoxicity, [U-14C]palmitate esterification into phospholipid was decreased, whereas esterification into diacylglycerol and triacylglycerol was increased in INS-1 beta cells (Fig. 4e). In these conditions, DHA led to a reduction in [U-14C]palmitate esterification into [14C]diacylglycerol but not into [14C]triacylglycerol (Fig. 4e). Using lipidomic analysis, we found that DHA inhibited the accumulation of diacylglycerol, the precursor of triacylglycerol synthesis, induced by glucolipotoxicity in INS-1 cells (Fig. 4f). ELOVL2/DHA preferentially inhibited diacylglycerol species incorporating palmitate (ESM Fig. 4a). Together, these data suggest that the ELOVL2/DHA axis inhibited ceramide accumulation without affecting the enzymatic machinery responsible for ceramide synthesis or metabolism in INS-1 cells. Moreover, it appears that the ELOVL2/DHA axis did not favour palmitate esterification into neutral lipids in order to mediate its protective effect against glucolipotoxicity in INS-1 beta cells.
The ELOVL2/DHA axis stimulated fatty acid oxidation to protect INS-1 beta cells against glucolipotoxicity
CoA esterification of palmitate, the first step of NEFA metabolism, is required for the toxic action of NEFA at elevated glucose concentrations . Palmitoyl-CoA is the precursor for ceramide synthesis but could also enter the mitochondrial NEFA β-oxidation pathway that is protective against beta cell apoptosis induced by glucolipotoxicity . In INS-1 beta cells, we found that palmitate metabolism was mainly directed towards its esterification (93%), leaving only 7% being oxidised in basal conditions (ESM Fig. 4c). Looking at [U-14C]palmitate oxidation into [14C]CO2, [14C]ASP and their sum, we found, as expected, that high glucose with or without palmitate drastically decreased this pathway (Fig. 5a). Interestingly, DHA by itself significantly increased palmitate oxidation in basal conditions, and this stimulation remained even in the presence of palmitate and/or high glucose (Fig. 5a), suggesting a potent protective mechanism in INS-1 cells. Etomoxir, an inhibitor of CPT1, the rate-limiting-step enzyme of NEFA β-oxidation, allowed palmitate at 5 mmol/l glucose to induce caspase activation at a similar level to that caused by the combined presence of high glucose and palmitate (Fig. 5b). Etomoxir was also able to potentiate (fivefold) caspase-3/7 activity induced by glucolipotoxicity (Fig. 5b). Interestingly, etomoxir partially inhibited the protective effect of Ad-Elovl2 and DHA on caspase-3/7 activation (Fig. 5b, d; −45% and −44%) and PARP cleavage (Fig. 5c, e) induced by glucolipotoxicity. Moreover, in the presence of etomoxir, ceramide accumulation during glucolipotoxicity is significantly increased, and Ad-Elovl2 or DHA treatment was unable to inhibit this accumulation (Fig. 5f, g). Together, these data strongly suggest that palmitate oxidation could play a central role in the protective effect of the ELOVL2/DHA axis against glucolipotoxicity in INS-1 beta cells.
The ELOVL2/DHA axis required CPT1 activity to protect INS-1 beta cells against glucolipotoxicity
5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMPK that increases NEFA β-oxidation , drastically inhibited caspase-3/7 activation (Fig. 6a) induced by glucolipotoxicity. Moreover, AICAR partially inhibited glucolipotoxicity-induced caspase activation when Elovl2 was downregulated (Fig. 6a). This inhibitory effect of AICAR was associated with a decrease in glucolipotoxicity-induced ceramide accumulation (Fig. 6b). As expected, in INS-1 cells, AICAR induced AMPK phosphorylation which inhibited by phosphorylation its downstream target, ACC (Fig. 6c). Interestingly, DHA and Ad-Elovl2 overexpression also increased AMPK and ACC phosphorylation in INS-1 cells even in conditions of glucolipotoxicity (Fig. 6d). Pharmacological inhibition of AMPK, which blocked AMPK phosphorylation in response to DHA and Ad-Elovl2 (Fig. 6e), totally prevented the protective effect on PARP cleavage induced by glucolipotoxicity in INS-1 cells (Fig. 6). ACC activity is known to play a central role in malonyl-CoA synthesis, a physiological inhibitor of CPT1 that regulates mitochondrial NEFA oxidation . We directly determined whether CPT1 activity plays a role in glucolipotoxicity-induced apoptosis when Elovl2 is downregulated, by using a mutated form of Cpt1, Cpt1-mutated (Cpt1-m) that is active but insensitive to malonyl-CoA inhibition . Overexpression of Cpt1-m partially inhibited caspase-3/7 activation (−33%) (Fig. 6f) and PARP cleavage induced by glucolipotoxicity (Fig. 6g). Interestingly, these effects correlated with a diminution in ceramide accumulation (Fig. 6h). When Elovl2 was downregulated in INS-1 cells, Cpt1-m overexpression also induced a large decrease in glucolipotoxicity-induced caspase-3/7 activation (−58%) (Fig. 6f) and PARP cleavage (Fig. 6g). This inhibitory effect of Cpt1-m on glucolipotoxicity-induced apoptosis was also associated with reduced ceramide accumulation during glucolipotoxicity (Fig. 6h). When Cpt1a is downregulated by a specific Cpt1 siRNA (ESM Fig. 4b), both DHA and Ad-Elovl2 were unable to prevent glucolipotoxicity-induced PARP cleavage (Fig. 6i vs Fig. 2h, j). Together, these data strongly suggest that regulation of CPT1 activity by AMPK plays a central role in the protective effect of the ELOVL2/DHA axis against glucolipotoxicity in INS-1 beta cells.
ELOVL2/DHA axis regulated glucolipotoxicity-induced apoptosis in human dispersed islet cells
We observed that human dispersed islet cells treated for 24 h with high glucose in the presence of palmitate also displayed a significant decrease in ELOVL2 mRNA levels (Fig. 7a). With reference to caspase-3/7 activity, palmitate at low glucose concentrations was not toxic, while in association with high glucose concentrations it significantly stimulated caspase-3/7 activity in human dispersed islets (Fig. 7b). Upon ELOVL2 downregulation (Fig. 7c), caspase-3/7 activation in human dispersed islets was significantly increased by glucolipotoxicity (Fig. 7e). Conversely, ELOVL2 overproduction (Fig. 7d) or DHA addition inhibited glucolipotoxicity-induced caspase-3/7 activation (−55% and −99%, respectively) in human dispersed islets (Fig. 7f, g). Etomoxir treatment of human dispersed islets increased glucolipotoxicity-induced caspase-3/7 activity twofold (Fig. 7h). Importantly, Ad-ELOVL2 or DHA addition were less effective at inhibiting glucolipotoxicity-induced caspase-3/7 activation (−25% and −34%, respectively) when human dispersed islets were treated with etomoxir (Fig. 7h, i). Together, these results support a protective role of the ELOVL2/DHA axis, mediated through CPT1 activity, in human islets against glucolipotoxicity-induced apoptosis.