Protective role of the ELOVL2/docosahexaenoic acid axis in glucolipotoxicity-induced apoptosis in rodent beta cells and human islets
Dietary n-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), are known to influence glucose homeostasis. We recently showed that Elovl2 expression in beta cells, which regulates synthesis of endogenous DHA, was associated with glucose tolerance and played a key role in insulin secretion. The present study aimed to examine the role of the very long chain fatty acid elongase 2 (ELOVL2)/DHA axis on the adverse effects of palmitate with high glucose, a condition defined as glucolipotoxicity, on beta cells.
We detected ELOVL2 in INS-1 beta cells and mouse and human islets using quantitative PCR and western blotting. Downregulation and adenoviral overexpression of Elovl2 was carried out in beta cells. Ceramide and diacylglycerol levels were determined by radio-enzymatic assay and lipidomics. Apoptosis was quantified using caspase-3 assays and poly (ADP-ribose) polymerase cleavage. Palmitate oxidation and esterification were determined by [U-14C]palmitate labelling.
We found that glucolipotoxicity decreased ELOVL2 content in rodent and human beta cells. Downregulation of ELOVL2 drastically potentiated beta cell apoptosis induced by glucolipotoxicity, whereas adenoviral Elovl2 overexpression and supplementation with DHA partially inhibited glucolipotoxicity-induced cell death in rodent and human beta cells. Inhibition of beta cell apoptosis by the ELOVL2/DHA axis was associated with a decrease in ceramide accumulation. However, the ELOVL2/DHA axis was unable to directly alter ceramide synthesis or metabolism. By contrast, DHA increased palmitate oxidation but did not affect its esterification. Pharmacological inhibition of AMP-activated protein kinase and etomoxir, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), the rate-limiting enzyme in fatty acid β-oxidation, attenuated the protective effect of the ELOVL2/DHA axis during glucolipotoxicity. Downregulation of CPT1 also counteracted the anti-apoptotic action of the ELOVL2/DHA axis. By contrast, a mutated active form of Cpt1 inhibited glucolipotoxicity-induced beta cell apoptosis when ELOVL2 was downregulated.
Our results identify ELOVL2 as a critical pro-survival enzyme for preventing beta cell death and dysfunction induced by glucolipotoxicity, notably by favouring palmitate oxidation in mitochondria through a CPT1-dependent mechanism.
KeywordsAMPK Apoptosis Ceramide DHA ELOVL2 Glucolipotoxicity Mitochondrial β-oxidation Pancreatic beta cells Type 2 diabetes
AMP-activated protein kinase
Carnitine palmitoyltransferase 1
Very long chain fatty acid elongase 2
G-protein coupled receptor 120
Monounsaturated fatty acid
Poly (ADP-ribose) polymerase
Polyunsaturated fatty acid
Small interfering RNA
Pancreatic beta cell dysfunction plays a central role in the pathogenesis of type 2 diabetes. Islets of Langerhans can increase insulin secretion and their mass to limit the effect of insulin resistance associated with obesity  and therefore maintain glycaemia. Type 2 diabetes onset occurs at the time of beta cell failure due to insufficient production and secretion of insulin  and beta cell apoptosis [3, 4, 5]. Studies have shown that saturated NEFA are responsible for the defective adaptation of beta cell turnover [6, 7, 8]. Importantly, the chronic adverse effects of saturated NEFA on beta cell function and viability are potentiated by the presence of hyperglycaemia, a phenomenon that has been termed ‘glucolipotoxicity’ . The molecular mechanisms involved in the pathogenesis of glucolipotoxicity in beta cells include an important role of ceramide synthesis [10, 11].
By contrast, monounsaturated fatty acids (MUFAs) exhibited opposite effects and counteracted the toxic effects of glucolipotoxicity induced by saturated NEFA [7, 12]. Another class of fatty acids, namely n-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA; C22:6 n-3), has also been shown to modulate lipotoxicity, possibly by enhancing insulin signalling in peripheral tissues  and preventing the development of liver steatosis [14, 15, 16, 17]. Currently, the role of endogenously synthesised n-3 PUFAs compared with those taken up from the diet on (gluco)lipotoxicity is relatively unknown, especially in beta cells.
Recently, Jacobsson and colleagues demonstrated that the major in vivo NEFA produced by very long chain fatty acid elongase 2 (ELOVL2) are DHA and docosapentaenoic acid . Interestingly, we recently identified Elovl2 as a key islet gene associated with glucose intolerance and insulin secretion . In the present study, we determined the effect of glucolipotoxicity on ELOVL2 levels in rodent and human beta cells. We then explored whether the ELOVL2/DHA axis could regulate beta cell apoptosis induced by glucolipotoxicity.
Cell culture conditions
Rat insulinoma INS-1 cells (mycoplasma-free) kindly provided by Merck–Serono (Merck Biopharma, Lyon, France) were cultured as already described . Human islets were isolated from ten non-diabetic organ donors (age 61 ± 31 years; BMI 25 ± 12 kg/m2) in Pisa, Italy, with the approval of the local ethics committee. Human dispersed islet cells were obtained through trypsin digestion. INS-1 cells were transiently transfected with 30 nmol of sequence-specific small interfering RNA (siRNA) against rat Elovl2 and control siRNA using Lipofectamine RNAiMAX (Invitrogen, Paris, France). Human dispersed islet cells were transiently transfected with 30 nmol of sequence-specific small interfering RNA (siRNA) against human ELOVL2 and control siRNA (Dharmacon, Lafayette, France) using Lipofectamine RNAiMAX (Invitrogen, Paris, France). INS-1 cells and human dispersed islet cells were transfected with human ELOVL2 (SignaGen Laboratories, Le Perray-en-Yvelines, France). Tests were performed 48 h after infection. Fatty acids (palmitate, DHA and eicosapentaenoic acid) were prepared as already described . The molar ratio of palmitate to BSA was 5:1. The fatty acid stock solutions were diluted in RPMI-1640 Medium (Invitrogen, Paris, France) supplemented with 1% (vol./vol.) FBS to obtain the required final concentration. In some experiments, gfp transfected cells were used as control and further stimulated with DHA. Other materials are described in ESM Materials.
Cells lysates were obtained and analysed by immunoblotting, as described . The following antibodies were used: anti-poly (ADP-ribose) polymerase (PARP), anti-ELOVL2, anti-V5, anti-AMP-activated protein kinase (AMPK), anti-phospho-AMPK, anti-acetyl-CoA carboxylase (ACC), anti-phospho-ACC, anti-carnitine palmitoyltransferase 1 (CPT1) A and anti-β-actin. More details are given in ESM Materials.
Measurement of caspase-3/7 activity
Caspase-3/7 activity assays were performed using the Promega (Promega, Charbonnières-les-Bains, France) Apo-ONE Homogeneous Caspase-3/7 Assay kit as described previously . Caspase-3/7 specific activity was expressed as the slope of the kinetic in arbitrary units. More details are given in ESM Materials.
RNA isolation, cDNA synthesis and mRNA quantification of the seven Elovl elongases were carried out in INS-1 cells, whereas ELOVL2 was quantified in human islets as described . A list of the primer sequences used is shown in ESM Table 1. mRNA transcript level of Rpl19 housekeeping gene was assayed and used for normalisation of other transcripts. More details are given in ESM Materials.
Lipid extraction and ceramide measurement
Cellular lipids were extracted by a modified Bligh and Dyer procedure . Analysis of ceramide species was performed by LC-MS/MS as described previously . Total ceramide levels were measured by the diacylglycerol kinase enzymatic method and total phospholipids were quantified as previously described . More details are given in ESM Materials.
Palmitate oxidation and esterification rates were determined during the last 2 h of culture in the presence of 250 μmol/l [U-14C]palmitate (2 Ci/mol) bound to 0.5% (wt/vol.) fatty acid-free BSA and 1 mmol/l carnitine. The medium was collected to determine [14C]CO2 and [14C]acid-soluble products (ASP) . Cells extracted with chloroform/methanol (2:1, vol./vol.) were used to determine [14C]triacylglycerol, [14C]phospholipid and [14C]diacylglycerol separated by thin-layer chromatography on silica gel plates .
Data are expressed as means ± SEM. Results were compared using one-way or two-way ANOVA on GraphPad Prism version 5.0 (GraphPad Sofware, La Jolla, CA USA). Bonferroni post-hoc analysis was used to determine p values; a p value of <0.05 was considered to be statistically significant.
Glucolipotoxicity reduced ELOVL2 expression in INS-1 beta cells and mouse islets
ELOVL2 expression regulated glucolipotoxicity-induced INS-1 beta cell apoptosis
The ELOVL2/DHA axis inhibited ceramide accumulation induced by glucolipotoxicity 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
The ELOVL2/DHA axis required CPT1 activity to protect INS-1 beta cells against glucolipotoxicity
ELOVL2/DHA axis regulated glucolipotoxicity-induced apoptosis in human dispersed islet cells
Saturated NEFA are known to mediate beta cell dysfunction and apoptosis, which contribute to the development of type 2 diabetes [24, 32]. There is growing evidence pointing to the beneficial effect of NEFA taken up from the diet, such as n-3 PUFAs, on glucose homeostasis . Recently, we identified ELOVL2, which produces n-3 PUFAs, as a novel regulator of beta cell function . In the present study, we found that glucolipotoxicity downregulated ELOVL2 in both rodent and human beta cells. Interestingly, downregulation of ELOVL2 significantly increased beta cell apoptosis induced by glucolipotoxicity. By contrast, ELOVL2 overproduction in beta cells partially protected them from glucolipotoxicity-induced apoptosis. Previous studies have shown that n-3 PUFAs could counteract the deleterious effect of palmitate on beta cell dysfunction . We found that DHA, an n-3 PUFA, completely inhibited beta cell apoptosis induced by glucolipotoxicity. Together, these results suggest that the ELOVL2/DHA axis is a new regulator of rodent and human beta cell fate under conditions of glucolipotoxicity.
We found, counter to what has been proposed recently, that the protective mechanism of the ELOVL2/DHA axis was not mediated by G-protein coupled receptor 120 (GPR120) . GPR120 agonists were unable to inhibit glucolipotoxicity-induced caspase-3/7 activation, and addition of a GPR120 antagonist did not counteract the inhibitory effect of DHA on caspase-3/7 activation by glucolipotoxicity (ESM Fig. 5a, b). By contrast, we found that the ELOVL2/DHA axis inhibited glucolipotoxicity-increased ceramide levels, which have been shown to mediate apoptosis [24, 32]. Apoptosis induced by ELOVL2 downregulation potentiated ceramide accumulation during glucolipotoxicity and was completely abolished by inhibition of de novo ceramide synthesis. Glucolipotoxicity has been shown to induce the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content [20, 35]. We found that DHA blocked the accumulation of pro-apoptotic ceramide species induced by glucolipotoxicity. However, the ELOVL2/DHA axis was unable to inhibit conversion of sphingosine into ceramide and beta cell apoptosis induced by sphingosine. Sphingosine-induced apoptosis is associated with its conversion into ceramide by ceramide synthases , suggesting that the ELOVL2/DHA axis was not blocking ceramide synthase activities in beta cells. Additionally, the protective effect of the ELOVL2/DHA axis was not linked to ceramide metabolism, as the conversion of ceramide into non-toxic sphingolipids [22, 25] did not affect glucolipotoxicity-induced apoptosis when ELOVL2 was upregulated. Together, these results suggest that the ELOVL2/DHA axis does not act directly on ceramide synthesis.
Esterification of saturated fatty acids into neutral lipid such as triacylglycerol has been shown to prevent glucolipotoxicity-induced beta cell apoptosis [27, 28]. We found that palmitate and high glucose increased palmitate esterification into triacylglycerol and its precursor diacylglycerol in beta cells. DHA slightly decreased esterification of palmitate into diacylglycerol, while it had no effect on triacylglycerol synthesis. Moreover, DHA reduced accumulation of diacylglycerol species incorporating palmitate molecules, suggesting that the ELOVL2/DHA axis probably does not prevent beta cell apoptosis by stimulating neutral lipid synthesis from palmitate. It is known that inhibition of mitochondrial NEFA β-oxidation potentiates glucolipotoxicity-induced apoptosis . Indeed, high glucose levels have been shown to decrease NEFA β-oxidation, through synthesis of malonyl-CoA, an allosteric inhibitor of CPT1 . Nevertheless, etomoxir, an inhibitor of CPT1, could still potentiate glucolipotoxicity-induced beta cell apoptosis, suggesting a partial inactivation of CPT1 by high glucose. We found that DHA significantly increased palmitate oxidation, even in the presence of high levels of glucose and palmitate, suggesting a role for this pathway in the anti-apoptotic effect of the ELOVL2/DHA axis. Indeed, etomoxir prevented the protective effect of the ELOVL2/DHA axis in rodent and human beta cells and was associated with higher ceramide accumulation during glucolipotoxicity. Overexpression of a mutated form of Cpt1, which is insensitive to malonyl-CoA inhibition , counterbalanced Elovl2 downregulation-induced apoptosis and ceramide accumulation in beta cells during glucolipotoxicity. Moreover, specific downregulation of CPT1 totally prevented the protective effect of the ELOVL2/DHA axis. Together, our data suggest that the entry of palmitate into mitochondria through CPT1 contributes to the protective effect of the ELOVL2/DHA axis against glucolipotoxicity in beta cells.
These data support the idea that the ELOVL2/DHA axis regulates lipid partitioning in beta cells by channelling palmitate towards β-oxidation instead of ceramide synthesis. A similar effect is well consolidated for MUFAs . PUFAs are known to prevent hepatic insulin resistance in an AMPKα2-dependent manner through regulation of NEFA β-oxidation . Interestingly, AMPK activation, which increased NEFA β-oxidation , partially inhibited glucolipotoxicity-induced caspase activation when Elovl2 was downregulated. The ELOVL2/DHA axis stimulated AMPK as reflected by its phosphorylation and one of its targets, ACC, in beta cells. Inhibition of AMPK totally prevented the anti-apoptotic effect of the ELOVL2/DHA axis during glucolipotoxicity. Together, these data suggest that AMPK activation will reduce malonyl-CoA synthesis and therefore favour palmitate β-oxidation and beta cell survival in response to the ELOVL2/DHA axis.
We recently provided evidence for a role of ELOVL2 in glucose-induced insulin release . In the present study, we showed an anti-apoptotic role of ELOVL2 against the deleterious effects of glucolipotoxicity in both rodent and human beta cells. Indeed, low levels of ELOVL2 and its product DHA predisposed beta cells to glucolipotoxicity-induced apoptosis. This protective effect is mediated by blocking the accumulation of pro-apoptotic ceramides. In fact, the ELOVL2/DHA axis modified lipid partitioning towards a non-toxic utilisation of palmitate by favouring its transport into mitochondria through a CPT1-dependent mechanism, where it can be β-oxidised. Finally, our work suggests that, independently of PUFA intake, modulation of intracellular PUFAs levels in beta cells could constitute a novel therapeutic strategy to prolong their survival and could limit the development of type 2 diabetes.
The authors thank C. K. Ng, University College Dublin, Belfield, Ireland, for help with preparing the manuscript.
HLS and LB contributed to the study concept and design, and to the analysis and interpretation of the data. LB, MCa, CR, NK, JV, KM and JD contributed to the acquisition of the data on caspase activity and the western blot experiments. LB and NK contributed to the acquisition and interpretation of the data from quantitative PCR experiments. LB and IH contributed to generate Elovl2 adenoviruses and interpretation of data using them. VL and CP-B contributed to the acquisition and interpretation of the data on [U-14C]palmitate metabolism. MB contributed to the preparation of human islets and analysis of the data. MCh and AB-Z performed and analysed the lipidomic data. CM, CC-G, PM, MI and BT contributed to the analysis of the data. HLS, LB and CM wrote/edited the manuscript with contributions from CP-B, CC-G, MI, PM and BT. HLS is responsible for the integrity of the work as a whole. All authors revised the article and approved the final version.
This project was supported by grants from the Centre National de la Recherche Scientifique (CNRS), the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 155005 (IMIDIA), and the Agence Nationale de la Recherche (ANR PRCI-15-CE14-0027-01 BetaDiamark). LB received a doctoral fellowship supported by IMIDIA-ENSO. MCa received a doctoral fellowship from the Cardiovasculaire – Obésité – Rein – Diabète – Domaine d’Intérêt Majeur (CORDDIM), Ile de France. JV was supported by a postdoctoral fellowship from Université Paris Diderot.
Duality of interest
All authors declare that they have no duality of interest associated with this manuscript. The study sponsor was not involved in the design of the study, interpretation of the data or writing of the report.