Differential effects of saturated and unsaturated fatty acids on autophagy in pancreatic β-cells

in Journal of Molecular Endocrinology
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  • 1 Institute of Biomedical & Clinical Science, University of Exeter Medical School, Exeter, UK
  • 2 The Medical School, Newcastle University, Newcastle Upon Tyne, UK
  • 3 Living Systems Institute, University of Exeter, Exeter, UK

Correspondence should be addressed to S Dhayal or N G Morgan: s.dhayal2@exeter.ac.uk or n.g.morgan@exeter.ac.uk

Long-chain saturated fatty acids are lipotoxic to pancreatic β-cells, whereas most unsaturates are better tolerated and some may even be cytoprotective. Fatty acids alter autophagy in β-cells and there is increasing evidence that such alterations can impact directly on the regulation of viability. Accordingly, we have compared the effects of palmitate (C16:0) and palmitoleate (C16:1) on autophagy in cultured β-cells and human islets. Treatment of BRIN-BD11 β-cells with palmitate led to enhanced autophagic activity, as judged by cleavage of microtubule-associated protein 1 light chain 3-I (LC3-I) and this correlated with a marked loss of cell viability in the cells. In addition, transfection of these cells with an mCherry-YFP-LC3 reporter construct revealed the accumulation of autophagosomes in palmitate-treated cells, indicating an impairment of autophagosome-lysosome fusion. This was also seen upon addition of the vacuolar ATPase inhibitor, bafilomycin A1. Exposure of BRIN-BD11 cells to palmitoleate (C16:1) did not lead directly to changes in autophagic activity or flux, but it antagonised the actions of palmitate. In parallel, palmitoleate also improved the viability of palmitate-treated BRIN-BD11 cells. Equivalent responses were observed in INS-1E cells and in isolated human islets. Taken together, these data suggest that palmitate may cause an impairment of autophagosome-lysosome fusion. These effects were not reproduced by palmitoleate which, instead, antagonised the responses mediated by palmitate suggesting that attenuation of β-cell stress may contribute to the improvement in cell viability caused by the mono-unsaturated fatty acid.

Abstract

Long-chain saturated fatty acids are lipotoxic to pancreatic β-cells, whereas most unsaturates are better tolerated and some may even be cytoprotective. Fatty acids alter autophagy in β-cells and there is increasing evidence that such alterations can impact directly on the regulation of viability. Accordingly, we have compared the effects of palmitate (C16:0) and palmitoleate (C16:1) on autophagy in cultured β-cells and human islets. Treatment of BRIN-BD11 β-cells with palmitate led to enhanced autophagic activity, as judged by cleavage of microtubule-associated protein 1 light chain 3-I (LC3-I) and this correlated with a marked loss of cell viability in the cells. In addition, transfection of these cells with an mCherry-YFP-LC3 reporter construct revealed the accumulation of autophagosomes in palmitate-treated cells, indicating an impairment of autophagosome-lysosome fusion. This was also seen upon addition of the vacuolar ATPase inhibitor, bafilomycin A1. Exposure of BRIN-BD11 cells to palmitoleate (C16:1) did not lead directly to changes in autophagic activity or flux, but it antagonised the actions of palmitate. In parallel, palmitoleate also improved the viability of palmitate-treated BRIN-BD11 cells. Equivalent responses were observed in INS-1E cells and in isolated human islets. Taken together, these data suggest that palmitate may cause an impairment of autophagosome-lysosome fusion. These effects were not reproduced by palmitoleate which, instead, antagonised the responses mediated by palmitate suggesting that attenuation of β-cell stress may contribute to the improvement in cell viability caused by the mono-unsaturated fatty acid.

Introduction

Chronic exposure of pancreatic β-cells to long-chain saturated fatty acids in vitro is associated with a phenomenon often referred to as ‘lipotoxicity’ in which the cells display secretory dysfunction and, ultimately, an increased rate of apoptosis (Abdulkarim et al. 2017, Ciregia et al. 2017, Mehmeti et al. 2017, Ruan et al. 2018, Wang et al. 2019). By contrast, long-chain mono- or poly-unsaturated fatty acids are much less toxic to β-cells and they can be overtly cytoprotective; even in the face of an otherwise lipotoxic stimulus (Sargsyan et al. 2016, Sramek et al. 2017, Bellini et al. 2018, Nemecz et al. 2018, Liu et al. 2019b). For example, incubation of β-cells in vitro with elevated concentrations of palmitate (C16:0) leads to loss of viability over a period of 18–36 h, whereas treatment of these cells for the same period of time with a still higher total concentration of free fatty acid (FFA), yet comprising a mixture of palmitate plus palmitoleate (C16:1), is associated with the maintenance of cell viability (Welters et al. 2004, Diakogiannaki et al. 2007, Dhayal et al. 2008). The molecular basis of the cytoprotective response seen under these conditions has not been defined fully, but it has been reported that unsaturated FFAs attenuate some of the chronic stress responses associated with incubation of cells under more lipotoxic conditions. This has been studied principally in relation to the regulation of endoplasmic reticulum (ER) stress, where it has been shown that palmitoleate causes a reduction in the expression of pro-apoptotic transcription factors, such as ATF4 and CHOP, in cells treated with palmitate (Diakogiannaki et al. 2008, Green & Olson 2011, Nemcova-Furstova et al. 2011).

ER stress is not the only mechanism involved in the regulation of cellular homeostasis in the face of environmental stressors and another important process which may become activated under these conditions is macroautophagy (hereafter termed ‘autophagy’) (Hu et al. 2016, Trudeau et al. 2016, Chen et al. 2017, Varshney et al. 2017, Assali et al. 2019). Indeed, increasing evidence implies that ER stress and autophagy exist in a state of mutual counter-balance to promote the maintenance of cell viability (Demirtas et al. 2016, Bugliani et al. 2019). Autophagy is a process by which the degradation and recycling of macronutrients is facilitated within cells and it involves the formation of double membrane bound vesicles (’autophagosomes’) which ultimately enclose relevant substrates and fuse with lysosomes to mediate degradation (Eskelinen 2019).

Several groups have reported that autophagy is affected when pancreatic β-cells are incubated with long-chain saturated fatty acids but several conundrums remain unresolved. For example, some investigators have concluded that autophagy increases upon exposure of β-cells to palmitate, whereas others report an impairment of autophagic flux under these conditions (Sharma & Alonso 2014, Hu et al. 2016, Varshney et al. 2017, Assali et al. 2019). Less attention has been paid to the effects of long-chain unsaturated fatty acids on autophagy but, again, a firm consensus is lacking. The response of cells to combinations of these molecules has been studied even less extensively. By way of illustration, Choi et al. have provided evidence that exposure of β-cells to palmitate leads to alterations in autophagy and have suggested that this may occur as an early adaptive response designed to minimise the impact of lipotoxic stress (Choi et al. 2009). In support of this they, and others, noted that palmitate-mediated cytotoxicity was attenuated by rapamycin, an agent well known as an inducer of autophagy (Bachar et al. 2009, Las et al. 2011, Bugliani et al. 2019). A similar response was also reported recently in bone marrow mesenchymal cells (Liu et al. 2018). On this basis, it seems possible that regulation of autophagy may provide a mechanism by which the viability of β-cells can be influenced by environmental stressors and that saturated and unsaturated fatty acids may regulate autophagy differentially. In this context it is reported that additional cytoprotective agents such as spermidine and glucagon-like peptide-1 (GLP-1) can also regulate autophagy (Sharma et al. 2011, Tirupathi Pichiah et al. 2011, Zummo et al. 2017, Liu et al. 2019a). Therefore, in the present work we have studied the effects of saturated and an unsaturated fatty acids on autophagy in two clonal rat β-cell lines, BRIN-BD11 and INS-1E cells, in which FFAs are known to influence viability differentially according to their degree of saturation.

Materials and methods

Materials

RPMI-1640 medium, penicillin/streptomycin, glutamine, NuPAGE® Novex® Bis-Tris Gels and NuPAGE® MES running SDS buffer were from Invitrogen, foetal calf serum from PAA Laboratories (Yeovil, England); fatty acid-free BSA and palmitoleate from MP Biomedicals (Thame, England). Docosahexanoic acid (DHA) and arachidonic acid (AA) were from Enzo Life Sciences (Exeter, England). Palmitate, myristate, laurate, the methyl-esters of palmitate, palmitoleate, DHA, and AA; rapamycin, tunicamycin, bafilomycin A1 and antiserum against LC3 (L7543) were purchased from Sigma. PVDF membrane and Mini-Protean TGX gels were from Bio-Rad; stripping buffer (Re-Blot Plus-Strong) from Millipore. Antiserum against LC3 (ab58610) was purchased from Abcam and other antibodies (phospho-mTOR (2971S); total mTOR (2972S), phospho S6K (9234S) and total S6K (9202S)), pEIF2α (3398P), total pEIF2α (5324P) were purchased from Cell Signalling. Antiserum against GAPDH (5G4) was purchased from Hytest. pBABE-puro mCherry-EGFP-LC3B was a gift from Jayanta Debnath (25) (Addgene plasmid # 22418).

Cell culture, fatty acid treatment and viability analysis

BRIN-BD11 and INS-1E cell lines are derived from rat insulinoma by methods reported by McClenaghan et al. (1996) and Merglen et al. (2004). Both cell lines were cultured in RPMI-1640 medium containing 11 mM glucose supplemented with 10% (v/v) foetal bovine serum, 2 mM l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. INS-1E culture medium was also supplemented with 50 µM β-mercaptoethanol. For cell viability experiments, cells were seeded at a density of 0.5 × 105 cells/mL in complete medium for 24 h. After this period, the medium was removed and replaced by serum-free medium containing appropriate fatty acid-BSA complexes. Fatty acid stocks were prepared in ethanol and then bound to fatty acid-free BSA by incubation at 37°C for 1 h. The final concentration of BSA used for cell incubations was maintained at 1% (w/v) and ethanol was 0.5% (v/v). Relevant vehicle (BSA plus ethanol) controls were included in each experiment. Cell death was quantified by flow cytometry after propidium iodide staining (Dhayal & Morgan 2011) or by propidium iodide staining via fluorescence microscopy (Zummo et al. 2017).

Human islets

Human islets were isolated from four non-diabetic donors at the Clinical Islet Lab, University of Alberta, Canada, or the Islet Isolation Unit, Kings College London, UK, with appropriate ethical approval. Upon arrival, islets were maintained in CMRL media supplemented with 0.5% human albumin serum and 50 U/mL penicillin and 50 μg/mL streptomycin for 24 h prior to treatment with palmitate or palmitoleate as for the cell lines detailed above.

Western blotting

BRIN-BD11 cells were seeded into 25 cm3 flasks at a density of 2 × 106 cells/mL in complete medium for 24 h and INS-1E cells were seeded into 12-well plates and cultured in complete medium for 24 h. The medium was then removed and replaced by serum free medium containing appropriate fatty acid-BSA complexes. After the required incubation period, whole-cell protein was extracted and Western blotting performed as described previously (Dhayal & Morgan 2011) using antisera directed against LC3, phospho-mTOR, phospho-S6K, phospho-eIF2α, as described in individual experiments. For loading controls, membranes were stripped and probed for the presence of beta-actin, GAPDH, total mTOR, total S6K or total eIF2α.

Imaging using tandem fluorescent-tagged LC3 constructs (mCherry-EGFP-LC3/mCherry-YFP-LC3)

BRIN-BD11 cells were seeded into fluorophore dishes at a density of 4 × 105 cells/mL in complete medium for 24 h. Thereafter, the cells were transiently transfected with an mCherry-YFP-LC3 construct. Culture medium was removed after 24 h and replaced by serum-free medium containing appropriate fatty acid-BSA complexes for a duration of 4 h. Bafilomycin A1 was added 1 h prior to the introduction of fatty acids. Images were captured with a Leica DMI8 confocal microscope using Leica Application Suite X software. Images were semi-automatically analysed using custom-built scripts in MATLAB in order to determine the number of LC3 puncta and level of autophagic flux per treatment.

For INS-1E, a stable cell line expressing mCherry-EGFP-LC3 was generated as previously described (Zummo et al. 2017). INS-1E (mCherry-EGFP-LC3) cells were plated on chambered coverslips and nuclei stained with 4.5 µg/mL Hoechst for 5 min. Live cell imaging was performed in a heated environmental chamber (37°C, 5% CO2) using a Nikon TE2000 microscope (×100). Images were processed using NIS Element Imaging software and puncta quantified using Volocity software (Quorum Technologies).

Statistical analysis

Experiments were performed on either two or three separate occasions using either duplicates or triplicates for each experimental condition. Results are presented either as representative Western blots or, for viability and autophagy analysis studies (n = 3–6), as mean values ± s.e.m. For BRIN-BD11 cells, pairwise statistical comparisons are performed using a two-sample t-test, whereas for INS-1E cells, values are compared by one or two-way ANOVA followed by a Bonferroni’s test.

Results

Rapamycin improves cell viability and increases LC3-II accumulation in cultured β-cells

Initially, we sought to determine whether the anti-apoptotic effects of rapamycin reported previously in INS-1 cells and in foetal β-cells incubated under lipotoxic conditions (Choi et al. 2009, Las et al. 2011) are also observed in BRIN-BD11 cells. As expected, incubation of BRIN-BD11 cells with palmitate for 18 h caused a dose-dependent loss of viability (Fig. 1), while treatment with rapamycin alone did not lead to cell death over this time period. More specifically, the presence of rapamycin (up to 50 ng/mL) was associated with a net improvement in cell viability. This was seen both under relatively benign conditions when BRIN-BD11 cells were incubated with vehicle alone (BSA plus ethanol; Fig. 1) and also when cells were exposed to palmitate. To establish whether the cytoprotective action of rapamycin correlated with the induction of structural changes in proteins associated with autophagy in BRIN-BD11 cells, the cleavage of LC3-I was measured. Increased formation of LC3-II (from LC3-I) was observed in BRIN-BD11 cells within 15 min of exposure to rapamycin (Fig. 2), and this response was sustained for at least 8 h.

Figure 1
Figure 1

BRIN-BD11 cells were exposed to either 20 ng/mL (Rap20) or 50 ng/mL rapamycin (Rap50) in the absence or presence of 25 µM or 100 µM palmitate, as shown. Cell viability was assessed at the end of 18-h incubation period by flow cytometry after staining with propidium iodide. *P < 0.01 relative to control (0); #P < 0.001 relative to control (0); **P < 0.001 relative to 25 µM palmitate alone; ***P < 0.001 relative to 100 µM palmitate alone.

Citation: Journal of Molecular Endocrinology 63, 4; 10.1530/JME-19-0096

Figure 2
Figure 2

BRIN-BD11 cells were incubated with 20 ng/mL rapamycin for increasing periods of time (up to 8 h). Proteins were extracted from the cells and analysed by Western blotting using an antiserum directed against LC3. Beta actin was probed as a loading control and the bands were quantified by densitometry. *P < 0.005 relative to condition 0 h.

Citation: Journal of Molecular Endocrinology 63, 4; 10.1530/JME-19-0096

Palmitoleate improves cell viability and decreases LC3-II accumulation caused by palmitate

Given that rapamycin caused an improvement in cell viability during exposure of cells to palmitate, we next confirmed that palmitoleate protected BRIN-BD11 cells against the lipotoxic effects of palmitate (Fig. 3A). Similar results were also observed in INS-1E cells (Fig. 3B). However, unlike the situation with rapamycin, no increase in LC3-II generation was detected during 8 h of exposure of BRIN-BD11 cells to palmitoleate (Fig. 4A). By contrast, LC3-II was generated in cells exposed to palmitate alone (Fig. 4A, B and C).

Figure 3
Figure 3

(A) BRIN-BD11 cells were exposed to increasing concentrations of palmitoleate in the presence of 250 µM palmitate for 18 h. Cell viability was then assessed by flow cytometry after staining with propidium iodide. #P < 0.001 relative to BSA control; *P < 0.05 or **P < 0.005 or ***P < 0.001 relative to palmitate alone. (B) INS-1E cells were exposed to increasing concentrations of palmitoleate in the presence of 500 μM palmitate for 18 h. Cell viability was then assessed by microscopic analysis after staining with propidium iodide. #P < 0.05, ##P < 0.01 relative to BSA control; *P < 0.05 relative to palmitate alone.

Citation: Journal of Molecular Endocrinology 63, 4; 10.1530/JME-19-0096

Figure 4
Figure 4

BRIN-BD11 cells were incubated with (A) Either 250 µM palmitate (P) for 4 h or 250 µM palmitoleate for increasing time periods (up to 8 h). #P < 0.05 relative to BSA control; *P < 0.05 relative to palmitate alone; or (B) Vehicle (C) or 250 µM of palmitate (P) or palmitoleate (PO) either alone or in combination (P + PO) for 6 h. #P < 0.005 relative to BSA control; *P < 0.005 relative to palmitate alone. Or (C) to increasing concentration of palmitoleate in the presence of 250 µM palmitate for 6 h. *P < 0.01 and **P < 0.001 relative to palmitate alone (0 h). (D) 250 µM palmitate for increasing time periods (up to 8 h). *P < 0.001 relative to condition at 0 h and #P < 0.005 relative to condition at 6 h. Protein extracts were probed with an antiserum to LC3. Beta actin was used as a loading control and the bands were quantified by densitometry.

Citation: Journal of Molecular Endocrinology 63, 4; 10.1530/JME-19-0096

To investigate these phenomena further, the extent of LC3-II generation was examined in cells exposed to either palmitate or palmitoleate alone, or to the two FFAs in combination (Fig. 4B and C). The data confirmed that palmitate caused a time-dependent increase in LC3-II formation (Fig. 4A, B, C and D), whereas palmitoleate did not. More strikingly, incubation of cells with 250 μM palmitate and increasing concentrations of palmitoleate (Fig. 4C) was associated with a reduction in palmitate-induced LC3-I cleavage. An equivalent reduction in LC3-II generation was not seen when cells were treated with the combination of palmitate and rapamycin (not shown), despite the fact that these incubation conditions were associated with an improvement in viability.

Likewise, in INS-1E cells, increased LC3-II generation was also observed in response to palmitate, whereas palmitoleate did not induce LC3-I cleavage and was able to inhibit the LC3-I cleavage induced by palmitate in these cells (Fig. 5A). Most importantly, the results were replicated in human islets (Fig. 5B). Culture of isolated human islets with 250 μM palmitate in the presence of 20 mM glucose increased LC3-I cleavage, while co-incubation with 250 μM palmitoleate attenuated this response (Fig. 5B).

Figure 5
Figure 5

(A) INS-1E were incubated with vehicle (C) or 250 µM palmitoleate (PO) or 500 µM of palmitate (P) either alone or in combination (P + PO) for 6 h. (B) Human islets were incubated with vehicle (C) or palmitoleate (PO) or 25 mM glucose containing 500 µM of palmitate (GLT - glucolipotoxicity) either alone or with 250 µM palmitoleate (GLT + PO) for 48 h. Protein extracts were probed with an antiserum to LC3. GAPDH was used as a loading control and the bands were quantified by densitometry. Results normalised to control and are expressed as fold change. #P < 0.05, ##P < 0.01 relative to control; *P < 0.05, **P < 0.01 relative to palmitate alone.

Citation: Journal of Molecular Endocrinology 63, 4; 10.1530/JME-19-0096

Generation of LC3-II reflects an increase in autophagic activity but, counter-intuitively, this can reflect either an increase or a decrease in net autophagic flux. Therefore, we examined the changes in autophagic flux more directly using dual reporter fluorescence constructs (mCherry-YFP-LC3 and mCherry-EGFP-LC3). Following expression of these constructs, an increase in the total number of red puncta is seen when autophagic activity is increased, whereas, an increase in the proportion of yellow puncta is observed under conditions when fusion of autophagosomes with lysosomes is inhibited. Both of these can be useful measures of autophagic flux, but the latter may be taken either as an indication of enhanced flux prior to lysosomal fusion or as evidence of blockage in the fusion mechanism. We have previously validated the quantification of red and yellow puncta as a measure of flux in INS-1E cells (Zummo et al. 2017). In the present study each of the constructs was expressed and the cells were then imaged following exposure to vehicle or fatty acids. Palmitate treatment increased the number of co-localised (yellow) puncta per cell (Fig. 6B), suggesting a potential impairment of autophagic flux, while concomitantly promoting overall autophagic activity (as judged by the total number of puncta; Fig. 6B). By contrast, palmitoleate failed to elicit these effects. Indeed, palmitoleate decreased the number of yellow puncta found in cells exposed to palmitate, and it also lowered the palmitate-induced increase in autophagic activity (total red puncta; Fig. 6B). Palmitoleate similarly antagonised the effects of palmitate on autophagic flux (i.e. it reduced the number of yellow puncta) in INS-1 cells although treatment of these cells with palmitate did not increase the number of red puncta (Fig. 7A and B).

Figure 6
Figure 6

(A) BRIN-BD11 cells expressing mCherry-YFP-LC3 construct were incubated with Vehicle (C), 250 µM of palmitate (P), 250 µM of palmitoleate (PO) or a combination of palmitate and palmitoleate (P + PO) for 4 h. Cells were imaged using fluorescence microscopy and LC3-positive puncta counted using custom-built MATLAB scripts. Scale bar = 10 µm. (b) Quantification of overall autophagic activity, measured by the total number of puncta per cell (red channel) and the number of colocalised puncta per cell (yellow channel). Represented are mean values ± s.e.m. for the number of experiments (n = 3). *P < 0.05, **P < 0.005 or ***P < 0.001 for comparing FA treatments to their respective control treatments (C); ΔP < 0.05, ΔΔP < 0.005 or ΔΔΔP < 0.001 for comparing palmitate + palmitoleate (P + PO) treatments relative to their respective palmitate alone (P) treatments. All remaining pairwise tests show no significant differences.

Citation: Journal of Molecular Endocrinology 63, 4; 10.1530/JME-19-0096

Figure 7
Figure 7

(A) INS-1E cells expressing mCherry-GFP-LC3 construct were incubated with BSA control or 25 mM glucose with 500 μM palmitate (glucolipotoxicity - GLT) either alone or in combination with 25 μM or 250 μM palmitoleate for 4 h. Cells were imaged using fluorescence microscopy and autophagic flux quantified by counting of red only and yellow puncta using Volocity software. White arrows indicate colocalising green and red (yellow) puncta. Scale bar = 10 μm. (B) Quantification of autophagic flux, measured by the number of red only puncta per cell (red only) and the number of colocalised puncta per cell (yellow). (C) Cells were imaged using fluorescence microscopy and autophagic flux quantified by counting of total red and yellow puncta using Volocity software. Results are normalised to control. Represented are mean values ± s.e.m. for the number of experiments (n = 5). *P < 0.05 for comparing FA treatments to their respective control treatments (C); ΔP < 0.05 for comparing GLT + PO treatments relative to their respective GLT alone treatments. All remaining pairwise tests show no significant differences.

Citation: Journal of Molecular Endocrinology 63, 4; 10.1530/JME-19-0096

Rapamycin and palmitate induce LC3-I cleavage by different mechanisms

Examination of the time-course of palmitate-mediated LC3-II generation revealed that the response occurred much less rapidly than when cells were exposed to rapamycin (compare Figs 2B and 4D) since a minimum of 4-h exposure to palmitate was required before significant LC3 cleavage was observed (Fig. 4D), whereas this occurred within a few minutes of addition of rapamycin (Fig. 2B). This implies that the mechanisms involved in the actions of rapamycin and palmitate may be different; a proposition that was studied by examination of the extent of phosphorylation of mammalian target of rapamycin (mTOR) and ribosomal S6-kinase in response to each agent (Fig. 8A). As its name implies, the protein kinase, mTOR, is a principal target for rapamycin and, consistent with this, its phosphorylation was reduced by the drug in BRIN-BD11 cells (Fig. 8A). By contrast, palmitate failed to influence mTOR phosphorylation at any time point studied (Fig. 8A). mTOR phosphorylation was similarly unaffected by the presence of palmitoleate (Fig. 8B). Additionally, the extent of phosphorylation of the downstream mTOR substrate, S6K, was abolished during exposure of BRIN-BD11 cells to rapamycin (Fig. 8A), but palmitate did not alter S6K phosphorylation.

Figure 8
Figure 8

BRIN-BD11 cells were exposed to (A) either 20 ng/mL rapamycin (R) for 8 h or 250 µM palmitate for increasing time lengths (up to 8 h). Protein extracts were probed with antisera to phospho mTOR (P-mTOR) and phospho S6K (P-S6K). *P < 0.005 relative to condition 0 h. Or (B) 250 µM palmitoleate for increasing time lengths (up to 8 h). Protein extracts were probed with antisera to phospho mTOR (P-mTOR). Total mTOR (T-mTOR) and total S6K (T-S6K) were used to demonstrate that changes in phosphorylated forms of the proteins were not due to alterations in the respective total protein levels within the cells.

Citation: Journal of Molecular Endocrinology 63, 4; 10.1530/JME-19-0096

Alterations in LC3-I cleavage by fatty acids mirrors their effects on cell viability

To establish whether the ability of long-chain fatty acids to regulate β-cell viability correlates with their effects on autophagy, we next studied a range of structurally modified fatty acid derivatives having differential effects on viability. Exposure of BRIN-BD11 cells to the methyl-ester of palmitate failed to promote LC3-I cleavage (Fig. 9A). In addition, two shorter chain saturated FFAs which are better tolerated than palmitate by BRIN-BD11 cells, myristate (C14:0) and laurate (C12:0) were also ineffective in this assay. It was also observed that the methyl-ester of palmitoleate failed to induce LC3-II generation but, intriguingly, in common with its unesterified parent fatty acid, the methyl-ester of palmitoleate prevented the cleavage of LC3-I seen in cells exposed to palmitate (Fig. 9B). The actions of two poly-unsaturated FFAs, arachidonic acid (C20:4) and docosahexaenoic acid (DHA; C22:6) as well as their respective methyl-esters, mirrored those of palmitoleate, in that, when added alone, each failed to induce LC3-I cleavage, but they markedly reduced the response in palmitate-treated cells (Fig. 9C).

Figure 9
Figure 9

BRIN-BD11cells were incubated under control conditions (C) or with (A) 250 µM of each of palmitate (C16:0) (P), myristate (C14:0) (M), laurate (C12:0) (L) or the methyl-ester of palmitate (Me-P) for 6 h. #P < 0.005 relative to BSA control; NSnon-significant relative to control. (B) 250 µM palmitate (P) in the absence or presence of 250 µM palmitoleate (P + PO) or methyl pamitoleate (P + Me-PO) for 6 h. *P < 0.005 relative to palmitate. (C) 250 µM of each of arachidonate (A), arachidonate methyl ester (Me-A), DHA (D) or methyl-DHA (Me-D) either alone or in the presence of palmitate (P) for 6 h. #P < 0.005 relative to BSA control; *P < 0.005 relative to palmitate. Protein extracts were probed with an antiserum to LC3. Beta actin was used as a loading control and the bands were quantified by densitometry.

Citation: Journal of Molecular Endocrinology 63, 4; 10.1530/JME-19-0096

Palmitoleate prevents the palmitate-induced increase in LC3-1 cleavage by resolution of ER stress

We have previously demonstrated that palmitoleate can alleviate ER stress induced by palmitate in β-cells (Diakogiannaki et al. 2008, Dhayal & Morgan 2011). Therefore, we also explored the effects of ER stressors on LC3 cleavage. As expected, tunicamycin, a known inducer of ER stress increased the phosphorylation of eIF2α in INS-1E and a similar response was seen with palmitate. Palmitoleate was able to alleviate this stress response in cells exposed to palmitate (Fig. 10A) and palmitoleate also inhibited LC3-I cleavage induced by another ER stressor, tunicamycin (Fig. 10B).

Figure 10
Figure 10

INS-1E were incubated with Vehicle (C), 500 µM of palmitate (P) or 5 μg/mL tunicamycin (TU) with or without 250 μM palmitoleate (PO) for 18 h. Protein extracts were probed with an antiserum to p-eIF2α and total eIF2α (A) or LC3 (B). GAPDH was used as a loading control and the bands were quantified by densitometry. Results normalised to control and are expressed as fold change. #P < 0.05, ##P < 0.01 relative to control; **P < 0.01 relative to palmitate alone.

Citation: Journal of Molecular Endocrinology 63, 4; 10.1530/JME-19-0096

Discussion

Activation of autophagy is increasingly implicated in the regulation of cell viability, including in pancreatic β-cells, and a number of studies have suggested that alterations in autophagic activity occur in response to the addition of long-chain fatty acids to these cells (Sharma & Alonso 2014, Janikiewicz et al. 2015, Zummo et al. 2017, Assali et al. 2019, Bugliani et al. 2019). However, there is little consensus about the impact of fatty acids on autophagy in β-cells. Some reports suggest that palmitate-induced toxicity is due to an increase in autophagic flux (Martino et al. 2012, Chen et al. 2013, Wu et al. 2017), whereas others suggest that palmitate impairs autophagic flux to induce cell death (Hong et al. 2018, Liu et al. 2018, RostamiRad et al. 2018, Xu et al. 2018). In the present work, we confirm that autophagy is increased when cultured rodent β-cells are exposed to palmitate. We have employed two different approaches to reach these conclusions and our results imply that the loss of viability caused by palmitate in β-cells is due to an increase in cell stress responses associated with the changes in autophagic activity. This is evidenced by an increase in LC3-I cleavage (yielding LC3-II) monitored by Western blotting following exposure to palmitate and by quantification of the yellow puncta seen in cells transfected with dual reporter constructs (either mCherry-YFP-LC3 or mCherry-GFP-LC3) whose number was increased by palmitate. An increase in yellow puncta occurs when the rate of fusion between autophagosomes and lysosomes is attenuated, since successful fusion leads to quenching of the yellow fluorescence due to the acidic intraluminal environment of the resultant autolysophagosome. Thus, a rise in the number of yellow puncta implies a net reduction in flux through the pathway.

By contrast with palmitate, the long-chain unsaturated fatty acid, palmitoleate, was inert in both assays of autophagy (LC3-I cleavage and changes in reporter fluorescence) suggesting that it does not directly alter the rate of autophagy in β-cells. Despite this, the presence of palmitoleate markedly affected the autophagic response to palmitate since, when the two fatty acids were present in combination, palmitate was much less effective at causing LC3-I cleavage and enhanced yellow puncta formation. Thus, although palmitoleate does not, itself, appear to influence the rate of autophagy directly in β-cells in the absence of palmitate, it clearly has the capacity to antagonise palmitate’s ability to regulate autophagy.

Based on these observations, we were able to then consider, in more detail, the mechanisms by which palmitate and palmitoleate induce their differential effects on cell viability in β-cells and the extent to which changes in autophagy may contribute to these effects. To address these issues, a range of additional reagents were employed. Initially, we compared the responses of β-cells to rapamycin and the two fatty acids since rapamycin is a well-known inducer of autophagy, but it has also been reported to protect beta cells against cytotoxic stimuli. In this respect, its actions appear to mirror those of palmitoleate. However, as noted above, unlike rapamycin, palmitoleate failed to stimulate β-cell autophagy. Indeed, in the presence of palmitate, rapamycin protected the cells while increasing LC3-I cleavage, whereas palmitoleate exerted its protective actions while simultaneously preventing LC3-I cleavage. Thus, there was a clear dissociation between the effects of rapamycin and palmitoleate on LC3-I cleavage in β-cells. There was an equally strong dissociation between rapamycin and palmitate with respect to the induction of autophagy since 18-h exposure to rapamycin was associated with cytoprotection, whereas, in the case of palmitate, cell death ensued. The simplest interpretation of these data is that a rise in autophagic activity is not directly cytotoxic to β-cells. This is consistent with emerging data in many cell types that autophagy is a process designed to maintain viability in the face of otherwise threatening environmental conditions.

At first sight, this conclusion does not, however, seem readily compatible with the present (and other) evidence indicating that an induction in autophagic activity occurs in β-cells exposed to palmitate, under conditions when cell viability is compromised. However, as noted above, use of dual reporter fluorescence constructs provided important information about the rates of autophagosome fusion with lysosomes in the distal phase of the autophagic pathway, in palmitate-treated cells. This revealed that, while palmitate causes a rise in autophagy in β-cells, the process does not go to completion but is interrupted by an impairment in the rate of autophagosome–lysosome fusion. By this means, palmitate causes an increase in overall β-cell stress, as further evidenced by the enhancement of eIF2α phosphorylation seen under these conditions. Thus, it seems likely that the loss of viability deriving from exposure to palmitate occurs as a result of a cumulative increase in total cellular stress associated with impaired autophagic flux and dysfunctional ER responses. Electron microscopic analysis of palmitate-treated BRIN-BD11 cells supports this by revealing a dramatic dysregulation of ER architecture (Diakogiannaki et al. 2008).

Importantly, these cellular stress responses were minimised when cells were exposed simultaneously to both palmitate and palmitoleate. Thus, the cleavage of LC3-I was blocked under these conditions and the formation of yellow puncta was also attenuated in cells transfected with dual reporter constructs. Similarly, the palmitate-mediated increase in phosphorylation of eIF2α was also prevented by palmitoleate (as was the distension of ER seen under the electron microscope) (Diakogiannaki et al. 2008, Dhayal & Morgan 2011). Indeed, we show here that the effects of another well-established ER stressor, tunicamycin, on eIF2α phosphorylation were also attenuated by palmitoleate. On this basis, we propose that the capacity of palmitoleate to sustain β-cell viability (despite the presence of an excess of palmitate or other stressors) relates to its ability to minimise the development of ER and autophagic stress responses. It may even be the case that palmitoleate can reverse stress responses after they have been initiated since it attenuates the toxic effects of palmitate even when introduced into the culture medium several hours after initial exposure of cells to the saturated fatty acid (Dhayal et al. 2008).

Consideration of these issues then raises a further important matter; namely, the mechanism by which palmitoleate reduces β-cell stress and autophagy in palmitate-treated cells. The present work has not disclosed this mechanism in full but we have shown that a similar response is exerted both by other long-chain unsaturated fatty acids and by their equivalent methyl-ester derivatives. This is important since the methyl-esters of long-chain fatty acids are not activated by esterification to co-enzyme A in β-cells and, as a consequence, they cannot be readily metabolised. This implies that their actions are likely to be mediated by more direct interaction with specific cellular targets in a manner equivalent to receptor activation (Morgan & Dhayal 2009, 2010). The identity of these putative molecular targets is still unknown, but our results suggest that the quest to discover β-cell molecules that can mediate anti-apoptotic effects upon ligation of relevant unsaturated fatty acids is a worthwhile endeavour.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

The authors are grateful to Diabetes UK for financial support via project grants 14/0005093 and 15/0005156 (to N G M) and a PhD studentship (14/0005093) to Patricia Thomas. They also thank Dr Jon Lane (University of Bristol) for the kind gift of a dual-fluorescence LC3 reporter construct.

References

  • Abdulkarim B, Hernangomez M, Igoillo-Esteve M, Cunha DA, Marselli L, Marchetti P, Ladriere L & Cnop M 2017 Guanabenz sensitizes pancreatic beta cells to lipotoxic endoplasmic reticulum stress and apoptosis. Endocrinology 16591670. (https://doi.org/10.1210/en.2016-1773)

    • Search Google Scholar
    • Export Citation
  • Assali EA, Shlomo D, Zeng J, Taddeo EP, Trudeau KM, Erion KA, Colby AH, Grinstaff MW, Liesa M, Las G, et al. 2019 Nanoparticle-mediated lysosomal reacidification restores mitochondrial turnover and function in beta cells under lipotoxicity. FASEB Journal 41544165. (https://doi.org/10.1096/fj.201801292R)

    • Search Google Scholar
    • Export Citation
  • Bachar E, Ariav Y, Ketzinel-Gilad M, Cerasi E, Kaiser N & Leibowitz G 2009 Glucose amplifies fatty acid-induced endoplasmic reticulum stress in pancreatic beta-cells via activation of mTORC1. PLoS ONE e4954. (https://doi.org/10.1371/journal.pone.0004954)

    • Search Google Scholar
    • Export Citation
  • Bellini L, Campana M, Rouch C, Chacinska M, Bugliani M, Meneyrol K, Hainault I, Lenoir V, Denom J, Veret J, et al. 2018 Protective role of the ELOVL2/docosahexaenoic acid axis in glucolipotoxicity-induced apoptosis in rodent beta cells and human islets. Diabetologia 17801793. (https://doi.org/10.1007/s00125-018-4629-8)

    • Search Google Scholar
    • Export Citation
  • Bugliani M, Mossuto S, Grano F, Suleiman M, Marselli L, Boggi U, De Simone P,, Eizirik DL, Cnop M, Marchetti P, et al. 2019 Modulation of autophagy influences the function and survival of human pancreatic beta cells under endoplasmic reticulum stress conditions and in type 2 diabetes. Frontiers in Endocrinology 52. (https://doi.org/10.3389/fendo.2019.00052)

    • Search Google Scholar
    • Export Citation
  • Chen YY, Sun LQ, Wang BA, Zou XM, Mu YM & Lu JM 2013 Palmitate induces autophagy in pancreatic beta-cells via endoplasmic reticulum stress and its downstream JNK pathway. International Journal of Molecular Medicine 14011406. (https://doi.org/10.3892/ijmm.2013.1530)

    • Search Google Scholar
    • Export Citation
  • Chen E, Tsai TH, Li L, Saha P, Chan L & Chang BH 2017 PLIN2 is a key regulator of the unfolded protein response and endoplasmic reticulum stress resolution in pancreatic beta cells. Scientific Reports 40855. (https://doi.org/10.1038/srep40855)

    • Search Google Scholar
    • Export Citation
  • Choi SE, Lee SM, Lee YJ, Li LJ, Lee SJ, Lee JH, Kim Y, Jun HS, Lee KW & Kang Y 2009 Protective role of autophagy in palmitate-induced INS-1 beta-cell death. Endocrinology 126134. (https://doi.org/10.1210/en.2008-0483)

    • Search Google Scholar
    • Export Citation
  • Ciregia F, Bugliani M, Ronci M, Giusti L, Boldrini C, Mazzoni MR, Mossuto S, Grano F, Cnop M, Marselli L, et al. 2017 Palmitate-induced lipotoxicity alters 410 acetylation of multiple proteins in clonal beta cells and human pancreatic islets. Scientific Reports 13445. (https://doi.org/10.1038/s41598-017-13908-w)

    • Search Google Scholar
    • Export Citation
  • Demirtas L, Guclu A, Erdur FM, Akbas EM, Ozcicek A, Onk D & Turkmen K 2016 Apoptosis, autophagy and endoplasmic reticulum stress in diabetes mellitus. Indian Journal of Medical Research 515524. (https://doi.org/10.4103/0971-5916.200887)

    • Search Google Scholar
    • Export Citation
  • Dhayal S & Morgan NG 2011 Structure-activity relationships influencing lipid-induced changes in eIF2alpha phosphorylation and cell viability in BRIN-BD11 cells. FEBS Letters 22432248. (https://doi.org/10.1016/j.febslet.2011.05.045)

    • Search Google Scholar
    • Export Citation
  • Dhayal S, Welters HJ & Morgan NG 2008 Structural requirements for the cytoprotective actions of mono-unsaturated fatty acids in the pancreatic beta-cell line, BRIN-BD11. British Journal of Pharmacology 17181727. (https://doi.org/10.1038/bjp.2008.43)

    • Search Google Scholar
    • Export Citation
  • Diakogiannaki E, Dhayal S, Childs CE, Calder PC, Welters HJ & Morgan NG 2007 Mechanisms involved in the cytotoxic and cytoprotective actions of saturated versus monounsaturated long-chain fatty acids in pancreatic {beta}-cells. Journal of Endocrinology 283291. (https://doi.org/10.1677/JOE-07-0082)

    • Search Google Scholar
    • Export Citation
  • Diakogiannaki E, Welters HJ & Morgan NG 2008 Differential regulation of the endoplasmic reticulum stress response in pancreatic beta-cells exposed to long-chain saturated and monounsaturated fatty acids. Journal of Endocrinology 553563. (https://doi.org/10.1677/JOE-08-0041)

    • Search Google Scholar
    • Export Citation
  • Eskelinen EL 2019 Autophagy: supporting cellular and organismal homeostasis by self-eating. International Journal of Biochemistry and Cell Biology 110. (https://doi.org/10.1016/j.biocel.2019.03.010)

    • Search Google Scholar
    • Export Citation
  • Green CD & Olson LK 2011 Modulation of palmitate-induced endoplasmic reticulum stress and apoptosis in pancreatic beta-cells by stearoyl-CoA desaturase and Elovl6. American Journal of Physiology: Endocrinology and Metabolism E640E649. (https://doi.org/10.1152/ajpendo.00544.2010)

    • Search Google Scholar
    • Export Citation
  • Hong YJ, Ahn HJ, Shin J, Lee JH, Kim JH, Park HW & Lee SK 2018 Unsaturated fatty acids protect trophoblast cells from saturated fatty acid-induced autophagy defects. Journal of Reproductive Immunology 5663. (https://doi.org/10.1016/j.jri.2017.12.001)

    • Search Google Scholar
    • Export Citation
  • Hu M, Yang S, Yang L, Cheng Y & Zhang H 2016 Interleukin-22 alleviated palmitate-induced endoplasmic reticulum stress in INS-1 cells through activation of autophagy. PLoS ONE e0146818. (https://doi.org/10.1371/journal.pone.0146818)

    • Search Google Scholar
    • Export Citation
  • Janikiewicz J, Hanzelka K, Kozinski K, Kolczynska K & Dobrzyn A 2015 Islet beta-cell failure in type 2 diabetes – within the network of toxic lipids. Biochemical and Biophysical Research Communications 491496. (https://doi.org/10.1016/j.bbrc.2015.03.153)

    • Search Google Scholar
    • Export Citation
  • Las G, Serada SB, Wikstrom JD, Twig G & Shirihai OS 2011 Fatty acids suppress autophagic turnover in beta-cells. Journal of Biological Chemistry 4253442544. (https://doi.org/10.1074/jbc.M111.242412)

    • Search Google Scholar
    • Export Citation
  • Liu Y, Wang N, Zhang S & Liang Q 2018 Autophagy protects bone marrow mesenchymal stem cells from palmitateinduced apoptosis through the ROSJNK/p38 MAPK signaling pathways. Molecular Medicine Reports 14851494. (https://doi.org/10.3892/mmr.2018.9100)

    • Search Google Scholar
    • Export Citation
  • Liu P, De La Vega MR, Dodson M, Yue F, Shi B, Fang D, Chapman E, Liu L & Zhang DD 2019a Spermidine confers liver protection by enhancing NRF2 signaling through a MAP1S-mediated non-canonical mechanism. Hepatology 372388. (https://doi.org/10.1002/hep.30616)

    • Search Google Scholar
    • Export Citation
  • Liu X, Zeng X, Chen X, Luo R, Li L, Wang C, Liu J, Cheng J, Lu Y & Chen Y 2019b Oleic acid protects insulin-secreting INS-1E cells against palmitic acid-induced lipotoxicity along with an amelioration of ER stress. Endocrine 512524. (https://doi.org/10.1007/s12020-019-01867-3)

    • Search Google Scholar
    • Export Citation
  • Martino L, Masini M, Novelli M, Beffy P, Bugliani M, Marselli L, Masiello P, Marchetti P & De Tata V 2012 Palmitate activates autophagy in INS-1E beta-cells and in isolated rat and human pancreatic islets. PLoS ONE e36188. (https://doi.org/10.1371/journal.pone.0036188)

    • Search Google Scholar
    • Export Citation
  • McClenaghan NH, Barnett CR, Ah-Sing E, Abdel-Wahab YH, O’Harte FP, Yoon TW, Swanston-Flatt SK & Flatt PR 1996 Characterization of a Novel Glucose-Responsive Insulin-Secreting Cell Line, BRIN-BD11, Produced by Electrofusio. Diabetes 45 1132. (https://doi.org/10.2337/diab.45.8.1132)

    • Search Google Scholar
    • Export Citation
  • Mehmeti I, Lortz S, Avezov E, Jorns A & Lenzen S 2017 ER-resident antioxidative GPx7 and GPx8 enzyme isoforms protect insulin-secreting INS-1E beta-cells against lipotoxicity by improving the ER antioxidative capacity. Free Radical Biology and Medicine 121130. (https://doi.org/10.1016/j.freeradbiomed.2017.07.021)

    • Search Google Scholar
    • Export Citation
  • Merglen A, Theander S, Rubi B, Chaffard G, Wollheim CB & Maechler P 2004 Glucose Sensitivity and Metabolism-Secretion Coupling Studied during Two-Year Continuous Culture in INS-1E Insulinoma Cells. Endocrinology 145 667. (https://doi.org/10.1210/en.2003-1099)

    • Search Google Scholar
    • Export Citation
  • Morgan NG & Dhayal S 2009 G-protein coupled receptors mediating long chain fatty acid signalling in the pancreatic beta-cell. Biochemical Pharmacology 14191427. (https://doi.org/10.1016/j.bcp.2009.07.020)

    • Search Google Scholar
    • Export Citation
  • Morgan NG & Dhayal S 2010 Unsaturated fatty acids as cytoprotective agents in the pancreatic beta-cell. Prostaglandins, Leukotrienes, and Essential Fatty Acids 231236. (https://doi.org/10.1016/j.plefa.2010.02.018)

    • Search Google Scholar
    • Export Citation
  • Nemcova-Furstova V, James RF & Kovar J 2011 Inhibitory effect of unsaturated fatty acids on saturated fatty acid-induced apoptosis in human pancreatic beta-cells: activation of caspases and ER stress induction. Cellular Physiology and Biochemistry 525538. (https://doi.org/10.1159/000329954)

    • Search Google Scholar
    • Export Citation
  • Nemecz M, Constantin A, Dumitrescu M, Alexandru N, Filippi A, Tanko G & Georgescu A 2018 The distinct effects of palmitic and oleic acid on pancreatic beta cell function: the elucidation of associated mechanisms and effector molecules. Frontiers in Pharmacology 1554. (https://doi.org/10.3389/fphar.2018.01554)

    • Search Google Scholar
    • Export Citation
  • Rostamirad A, Ebrahimi SSS, Sadeghi A, Taghikhani M & Meshkani R 2018 Palmitate-induced impairment of autophagy turnover leads to increased apoptosis and inflammation in peripheral blood mononuclear cells. Immunobiology 269278. (https://doi.org/10.1016/j.imbio.2017.10.041)

    • Search Google Scholar
    • Export Citation
  • Ruan JS, Lin JK, Kuo YY, Chen YW & Chen PC 2018 Chronic palmitic acid-induced lipotoxicity correlates with defective trafficking of ATP sensitive potassium channels in pancreatic beta cells. Journal of Nutritional Biochemistry 3748. (https://doi.org/10.1016/j.jnutbio.2018.05.005)

    • Search Google Scholar
    • Export Citation
  • Sargsyan E, Artemenko K, Manukyan L, Bergquist J & Bergsten P 2016 Oleate protects beta-cells from the toxic effect of palmitate by activating pro-survival pathways of the ER stress response. Biochimica and Biophysica Acta 11511160. (https://doi.org/10.1016/j.bbalip.2016.06.012)

    • Search Google Scholar
    • Export Citation
  • Sharma RB & Alonso LC 2014 Lipotoxicity in the pancreatic beta cell: not just survival and function, but proliferation as well? Current Diabetes Reports 492. (https://doi.org/10.1007/s11892-014-0492-2)

    • Search Google Scholar
    • Export Citation
  • Sharma S, Mells JE, Fu PP, Saxena NK & Anania FA 2011 GLP-1 analogs reduce hepatocyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy. PLoS ONE e25269. (https://doi.org/10.1371/journal.pone.0025269)

    • Search Google Scholar
    • Export Citation
  • Sramek J, Nemcova-Furstova V, Pavlikova N & Kovar J 2017 Effect of saturated stearic acid on MAP kinase and ER stress signaling pathways during apoptosis induction in human pancreatic beta-cells is inhibited by unsaturated oleic acid. International Journal of Molecular Sciences E2313. (https://doi.org/10.3390/ijms18112313)

    • Search Google Scholar
    • Export Citation
  • Tirupathi Pichiah PB, Suriyakalaa U, Kamalakkannan S, Kokilavani P, Kalaiselvi S, Sankarganesh D, Gowri J, Archunan G, Cha YS & Achiraman S 2011 Spermidine may decrease ER stress in pancreatic beta cells and may reduce apoptosis via activating AMPK dependent autophagy pathway. Medical Hypotheses 677679. (https://doi.org/10.1016/j.mehy.2011.07.014)

    • Search Google Scholar
    • Export Citation
  • Trudeau KM, Colby AH, Zeng J, Las G, Feng JH, Grinstaff MW & Shirihai OS 2016 Lysosome acidification by photoactivated nanoparticles restores autophagy under lipotoxicity. Journal of Cell Biology 2534. (https://doi.org/10.1083/jcb.201511042)

    • Search Google Scholar
    • Export Citation
  • Varshney R, Gupta S & Roy P 2017 Cytoprotective effect of kaempferol against palmitic acid-induced pancreatic beta-cell death through modulation of autophagy via AMPK/mTOR signaling pathway. Molecular and Cellular Endocrinology 120. (https://doi.org/10.1016/j.mce.2017.02.033)

    • Search Google Scholar
    • Export Citation
  • Wang Y, Xie T, Zhang D & Leung PS 2019 GPR120 protects lipotoxicity-induced pancreatic beta-cell dysfunction through regulation of PDX1 expression and inhibition of islet inflammation. Clinical Science 101116. (https://doi.org/10.1042/CS20180836)

    • Search Google Scholar
    • Export Citation
  • Welters HJ, Tadayyon M, Scarpello JH, Smith SA & Morgan NG 2004 Mono-unsaturated fatty acids protect against beta-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure. FEBS Letters 103108. (https://doi.org/10.1016/S0014-5793(04)00079-1)

    • Search Google Scholar
    • Export Citation
  • Wu J, Wu Q, Li JJ, Chen C, Sun S, Wang CH & Sun SR 2017 Autophagy mediates free fatty acid effects on MDA-MB-231 cell proliferation, migration and invasion. Oncology Letters 47154721. (https://doi.org/10.3892/ol.2017.6807)

    • Search Google Scholar
    • Export Citation
  • Xu K, Liu XF, Ke ZQ, Yao Q, Guo S & Liu C 2018 Resveratrol modulates apoptosis and autophagy induced by high glucose and palmitate in cardiac cells. Cellular Physiology and Biochemistry 20312040. (https://doi.org/10.1159/000489442)

    • Search Google Scholar
    • Export Citation
  • Zummo FP, Cullen KS, Honkanen-Scott M, Shaw JAM, Lovat PE & Arden C 2017 Glucagon-like peptide 1 protects pancreatic beta-cells from death by increasing autophagic flux and restoring lysosomal function. Diabetes 12721285. (https://doi.org/10.2337/db16-1009)

    • Search Google Scholar
    • Export Citation

 

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    BRIN-BD11 cells were exposed to either 20 ng/mL (Rap20) or 50 ng/mL rapamycin (Rap50) in the absence or presence of 25 µM or 100 µM palmitate, as shown. Cell viability was assessed at the end of 18-h incubation period by flow cytometry after staining with propidium iodide. *P < 0.01 relative to control (0); #P < 0.001 relative to control (0); **P < 0.001 relative to 25 µM palmitate alone; ***P < 0.001 relative to 100 µM palmitate alone.

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    BRIN-BD11 cells were incubated with 20 ng/mL rapamycin for increasing periods of time (up to 8 h). Proteins were extracted from the cells and analysed by Western blotting using an antiserum directed against LC3. Beta actin was probed as a loading control and the bands were quantified by densitometry. *P < 0.005 relative to condition 0 h.

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    (A) BRIN-BD11 cells were exposed to increasing concentrations of palmitoleate in the presence of 250 µM palmitate for 18 h. Cell viability was then assessed by flow cytometry after staining with propidium iodide. #P < 0.001 relative to BSA control; *P < 0.05 or **P < 0.005 or ***P < 0.001 relative to palmitate alone. (B) INS-1E cells were exposed to increasing concentrations of palmitoleate in the presence of 500 μM palmitate for 18 h. Cell viability was then assessed by microscopic analysis after staining with propidium iodide. #P < 0.05, ##P < 0.01 relative to BSA control; *P < 0.05 relative to palmitate alone.

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    BRIN-BD11 cells were incubated with (A) Either 250 µM palmitate (P) for 4 h or 250 µM palmitoleate for increasing time periods (up to 8 h). #P < 0.05 relative to BSA control; *P < 0.05 relative to palmitate alone; or (B) Vehicle (C) or 250 µM of palmitate (P) or palmitoleate (PO) either alone or in combination (P + PO) for 6 h. #P < 0.005 relative to BSA control; *P < 0.005 relative to palmitate alone. Or (C) to increasing concentration of palmitoleate in the presence of 250 µM palmitate for 6 h. *P < 0.01 and **P < 0.001 relative to palmitate alone (0 h). (D) 250 µM palmitate for increasing time periods (up to 8 h). *P < 0.001 relative to condition at 0 h and #P < 0.005 relative to condition at 6 h. Protein extracts were probed with an antiserum to LC3. Beta actin was used as a loading control and the bands were quantified by densitometry.

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    (A) INS-1E were incubated with vehicle (C) or 250 µM palmitoleate (PO) or 500 µM of palmitate (P) either alone or in combination (P + PO) for 6 h. (B) Human islets were incubated with vehicle (C) or palmitoleate (PO) or 25 mM glucose containing 500 µM of palmitate (GLT - glucolipotoxicity) either alone or with 250 µM palmitoleate (GLT + PO) for 48 h. Protein extracts were probed with an antiserum to LC3. GAPDH was used as a loading control and the bands were quantified by densitometry. Results normalised to control and are expressed as fold change. #P < 0.05, ##P < 0.01 relative to control; *P < 0.05, **P < 0.01 relative to palmitate alone.

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    (A) BRIN-BD11 cells expressing mCherry-YFP-LC3 construct were incubated with Vehicle (C), 250 µM of palmitate (P), 250 µM of palmitoleate (PO) or a combination of palmitate and palmitoleate (P + PO) for 4 h. Cells were imaged using fluorescence microscopy and LC3-positive puncta counted using custom-built MATLAB scripts. Scale bar = 10 µm. (b) Quantification of overall autophagic activity, measured by the total number of puncta per cell (red channel) and the number of colocalised puncta per cell (yellow channel). Represented are mean values ± s.e.m. for the number of experiments (n = 3). *P < 0.05, **P < 0.005 or ***P < 0.001 for comparing FA treatments to their respective control treatments (C); ΔP < 0.05, ΔΔP < 0.005 or ΔΔΔP < 0.001 for comparing palmitate + palmitoleate (P + PO) treatments relative to their respective palmitate alone (P) treatments. All remaining pairwise tests show no significant differences.

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    (A) INS-1E cells expressing mCherry-GFP-LC3 construct were incubated with BSA control or 25 mM glucose with 500 μM palmitate (glucolipotoxicity - GLT) either alone or in combination with 25 μM or 250 μM palmitoleate for 4 h. Cells were imaged using fluorescence microscopy and autophagic flux quantified by counting of red only and yellow puncta using Volocity software. White arrows indicate colocalising green and red (yellow) puncta. Scale bar = 10 μm. (B) Quantification of autophagic flux, measured by the number of red only puncta per cell (red only) and the number of colocalised puncta per cell (yellow). (C) Cells were imaged using fluorescence microscopy and autophagic flux quantified by counting of total red and yellow puncta using Volocity software. Results are normalised to control. Represented are mean values ± s.e.m. for the number of experiments (n = 5). *P < 0.05 for comparing FA treatments to their respective control treatments (C); ΔP < 0.05 for comparing GLT + PO treatments relative to their respective GLT alone treatments. All remaining pairwise tests show no significant differences.

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    BRIN-BD11 cells were exposed to (A) either 20 ng/mL rapamycin (R) for 8 h or 250 µM palmitate for increasing time lengths (up to 8 h). Protein extracts were probed with antisera to phospho mTOR (P-mTOR) and phospho S6K (P-S6K). *P < 0.005 relative to condition 0 h. Or (B) 250 µM palmitoleate for increasing time lengths (up to 8 h). Protein extracts were probed with antisera to phospho mTOR (P-mTOR). Total mTOR (T-mTOR) and total S6K (T-S6K) were used to demonstrate that changes in phosphorylated forms of the proteins were not due to alterations in the respective total protein levels within the cells.

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    BRIN-BD11cells were incubated under control conditions (C) or with (A) 250 µM of each of palmitate (C16:0) (P), myristate (C14:0) (M), laurate (C12:0) (L) or the methyl-ester of palmitate (Me-P) for 6 h. #P < 0.005 relative to BSA control; NSnon-significant relative to control. (B) 250 µM palmitate (P) in the absence or presence of 250 µM palmitoleate (P + PO) or methyl pamitoleate (P + Me-PO) for 6 h. *P < 0.005 relative to palmitate. (C) 250 µM of each of arachidonate (A), arachidonate methyl ester (Me-A), DHA (D) or methyl-DHA (Me-D) either alone or in the presence of palmitate (P) for 6 h. #P < 0.005 relative to BSA control; *P < 0.005 relative to palmitate. Protein extracts were probed with an antiserum to LC3. Beta actin was used as a loading control and the bands were quantified by densitometry.

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    INS-1E were incubated with Vehicle (C), 500 µM of palmitate (P) or 5 μg/mL tunicamycin (TU) with or without 250 μM palmitoleate (PO) for 18 h. Protein extracts were probed with an antiserum to p-eIF2α and total eIF2α (A) or LC3 (B). GAPDH was used as a loading control and the bands were quantified by densitometry. Results normalised to control and are expressed as fold change. #P < 0.05, ##P < 0.01 relative to control; **P < 0.01 relative to palmitate alone.

  • Abdulkarim B, Hernangomez M, Igoillo-Esteve M, Cunha DA, Marselli L, Marchetti P, Ladriere L & Cnop M 2017 Guanabenz sensitizes pancreatic beta cells to lipotoxic endoplasmic reticulum stress and apoptosis. Endocrinology 16591670. (https://doi.org/10.1210/en.2016-1773)

    • Search Google Scholar
    • Export Citation
  • Assali EA, Shlomo D, Zeng J, Taddeo EP, Trudeau KM, Erion KA, Colby AH, Grinstaff MW, Liesa M, Las G, et al. 2019 Nanoparticle-mediated lysosomal reacidification restores mitochondrial turnover and function in beta cells under lipotoxicity. FASEB Journal 41544165. (https://doi.org/10.1096/fj.201801292R)

    • Search Google Scholar
    • Export Citation
  • Bachar E, Ariav Y, Ketzinel-Gilad M, Cerasi E, Kaiser N & Leibowitz G 2009 Glucose amplifies fatty acid-induced endoplasmic reticulum stress in pancreatic beta-cells via activation of mTORC1. PLoS ONE e4954. (https://doi.org/10.1371/journal.pone.0004954)

    • Search Google Scholar
    • Export Citation
  • Bellini L, Campana M, Rouch C, Chacinska M, Bugliani M, Meneyrol K, Hainault I, Lenoir V, Denom J, Veret J, et al. 2018 Protective role of the ELOVL2/docosahexaenoic acid axis in glucolipotoxicity-induced apoptosis in rodent beta cells and human islets. Diabetologia 17801793. (https://doi.org/10.1007/s00125-018-4629-8)

    • Search Google Scholar
    • Export Citation
  • Bugliani M, Mossuto S, Grano F, Suleiman M, Marselli L, Boggi U, De Simone P,, Eizirik DL, Cnop M, Marchetti P, et al. 2019 Modulation of autophagy influences the function and survival of human pancreatic beta cells under endoplasmic reticulum stress conditions and in type 2 diabetes. Frontiers in Endocrinology 52. (https://doi.org/10.3389/fendo.2019.00052)

    • Search Google Scholar
    • Export Citation
  • Chen YY, Sun LQ, Wang BA, Zou XM, Mu YM & Lu JM 2013 Palmitate induces autophagy in pancreatic beta-cells via endoplasmic reticulum stress and its downstream JNK pathway. International Journal of Molecular Medicine 14011406. (https://doi.org/10.3892/ijmm.2013.1530)

    • Search Google Scholar
    • Export Citation
  • Chen E, Tsai TH, Li L, Saha P, Chan L & Chang BH 2017 PLIN2 is a key regulator of the unfolded protein response and endoplasmic reticulum stress resolution in pancreatic beta cells. Scientific Reports 40855. (https://doi.org/10.1038/srep40855)

    • Search Google Scholar
    • Export Citation
  • Choi SE, Lee SM, Lee YJ, Li LJ, Lee SJ, Lee JH, Kim Y, Jun HS, Lee KW & Kang Y 2009 Protective role of autophagy in palmitate-induced INS-1 beta-cell death. Endocrinology 126134. (https://doi.org/10.1210/en.2008-0483)

    • Search Google Scholar
    • Export Citation
  • Ciregia F, Bugliani M, Ronci M, Giusti L, Boldrini C, Mazzoni MR, Mossuto S, Grano F, Cnop M, Marselli L, et al. 2017 Palmitate-induced lipotoxicity alters 410 acetylation of multiple proteins in clonal beta cells and human pancreatic islets. Scientific Reports 13445. (https://doi.org/10.1038/s41598-017-13908-w)

    • Search Google Scholar
    • Export Citation
  • Demirtas L, Guclu A, Erdur FM, Akbas EM, Ozcicek A, Onk D & Turkmen K 2016 Apoptosis, autophagy and endoplasmic reticulum stress in diabetes mellitus. Indian Journal of Medical Research 515524. (https://doi.org/10.4103/0971-5916.200887)

    • Search Google Scholar
    • Export Citation
  • Dhayal S & Morgan NG 2011 Structure-activity relationships influencing lipid-induced changes in eIF2alpha phosphorylation and cell viability in BRIN-BD11 cells. FEBS Letters 22432248. (https://doi.org/10.1016/j.febslet.2011.05.045)

    • Search Google Scholar
    • Export Citation
  • Dhayal S, Welters HJ & Morgan NG 2008 Structural requirements for the cytoprotective actions of mono-unsaturated fatty acids in the pancreatic beta-cell line, BRIN-BD11. British Journal of Pharmacology 17181727. (https://doi.org/10.1038/bjp.2008.43)

    • Search Google Scholar
    • Export Citation
  • Diakogiannaki E, Dhayal S, Childs CE, Calder PC, Welters HJ & Morgan NG 2007 Mechanisms involved in the cytotoxic and cytoprotective actions of saturated versus monounsaturated long-chain fatty acids in pancreatic {beta}-cells. Journal of Endocrinology 283291. (https://doi.org/10.1677/JOE-07-0082)

    • Search Google Scholar
    • Export Citation
  • Diakogiannaki E, Welters HJ & Morgan NG 2008 Differential regulation of the endoplasmic reticulum stress response in pancreatic beta-cells exposed to long-chain saturated and monounsaturated fatty acids. Journal of Endocrinology 553563. (https://doi.org/10.1677/JOE-08-0041)

    • Search Google Scholar
    • Export Citation
  • Eskelinen EL 2019 Autophagy: supporting cellular and organismal homeostasis by self-eating. International Journal of Biochemistry and Cell Biology 110. (https://doi.org/10.1016/j.biocel.2019.03.010)

    • Search Google Scholar
    • Export Citation
  • Green CD & Olson LK 2011 Modulation of palmitate-induced endoplasmic reticulum stress and apoptosis in pancreatic beta-cells by stearoyl-CoA desaturase and Elovl6. American Journal of Physiology: Endocrinology and Metabolism E640E649. (https://doi.org/10.1152/ajpendo.00544.2010)

    • Search Google Scholar
    • Export Citation
  • Hong YJ, Ahn HJ, Shin J, Lee JH, Kim JH, Park HW & Lee SK 2018 Unsaturated fatty acids protect trophoblast cells from saturated fatty acid-induced autophagy defects. Journal of Reproductive Immunology 5663. (https://doi.org/10.1016/j.jri.2017.12.001)

    • Search Google Scholar
    • Export Citation
  • Hu M, Yang S, Yang L, Cheng Y & Zhang H 2016 Interleukin-22 alleviated palmitate-induced endoplasmic reticulum stress in INS-1 cells through activation of autophagy. PLoS ONE e0146818. (https://doi.org/10.1371/journal.pone.0146818)

    • Search Google Scholar
    • Export Citation
  • Janikiewicz J, Hanzelka K, Kozinski K, Kolczynska K & Dobrzyn A 2015 Islet beta-cell failure in type 2 diabetes – within the network of toxic lipids. Biochemical and Biophysical Research Communications 491496. (https://doi.org/10.1016/j.bbrc.2015.03.153)

    • Search Google Scholar
    • Export Citation
  • Las G, Serada SB, Wikstrom JD, Twig G & Shirihai OS 2011 Fatty acids suppress autophagic turnover in beta-cells. Journal of Biological Chemistry 4253442544. (https://doi.org/10.1074/jbc.M111.242412)

    • Search Google Scholar
    • Export Citation
  • Liu Y, Wang N, Zhang S & Liang Q 2018 Autophagy protects bone marrow mesenchymal stem cells from palmitateinduced apoptosis through the ROSJNK/p38 MAPK signaling pathways. Molecular Medicine Reports 14851494. (https://doi.org/10.3892/mmr.2018.9100)

    • Search Google Scholar
    • Export Citation
  • Liu P, De La Vega MR, Dodson M, Yue F, Shi B, Fang D, Chapman E, Liu L & Zhang DD 2019a Spermidine confers liver protection by enhancing NRF2 signaling through a MAP1S-mediated non-canonical mechanism. Hepatology 372388. (https://doi.org/10.1002/hep.30616)

    • Search Google Scholar
    • Export Citation
  • Liu X, Zeng X, Chen X, Luo R, Li L, Wang C, Liu J, Cheng J, Lu Y & Chen Y 2019b Oleic acid protects insulin-secreting INS-1E cells against palmitic acid-induced lipotoxicity along with an amelioration of ER stress. Endocrine 512524. (https://doi.org/10.1007/s12020-019-01867-3)

    • Search Google Scholar
    • Export Citation
  • Martino L, Masini M, Novelli M, Beffy P, Bugliani M, Marselli L, Masiello P, Marchetti P & De Tata V 2012 Palmitate activates autophagy in INS-1E beta-cells and in isolated rat and human pancreatic islets. PLoS ONE e36188. (https://doi.org/10.1371/journal.pone.0036188)

    • Search Google Scholar
    • Export Citation
  • McClenaghan NH, Barnett CR, Ah-Sing E, Abdel-Wahab YH, O’Harte FP, Yoon TW, Swanston-Flatt SK & Flatt PR 1996 Characterization of a Novel Glucose-Responsive Insulin-Secreting Cell Line, BRIN-BD11, Produced by Electrofusio. Diabetes 45 1132. (https://doi.org/10.2337/diab.45.8.1132)

    • Search Google Scholar
    • Export Citation
  • Mehmeti I, Lortz S, Avezov E, Jorns A & Lenzen S 2017 ER-resident antioxidative GPx7 and GPx8 enzyme isoforms protect insulin-secreting INS-1E beta-cells against lipotoxicity by improving the ER antioxidative capacity. Free Radical Biology and Medicine 121130. (https://doi.org/10.1016/j.freeradbiomed.2017.07.021)

    • Search Google Scholar
    • Export Citation
  • Merglen A, Theander S, Rubi B, Chaffard G, Wollheim CB & Maechler P 2004 Glucose Sensitivity and Metabolism-Secretion Coupling Studied during Two-Year Continuous Culture in INS-1E Insulinoma Cells. Endocrinology 145 667. (https://doi.org/10.1210/en.2003-1099)

    • Search Google Scholar
    • Export Citation
  • Morgan NG & Dhayal S 2009 G-protein coupled receptors mediating long chain fatty acid signalling in the pancreatic beta-cell. Biochemical Pharmacology 14191427. (https://doi.org/10.1016/j.bcp.2009.07.020)

    • Search Google Scholar
    • Export Citation
  • Morgan NG & Dhayal S 2010 Unsaturated fatty acids as cytoprotective agents in the pancreatic beta-cell. Prostaglandins, Leukotrienes, and Essential Fatty Acids 231236. (https://doi.org/10.1016/j.plefa.2010.02.018)

    • Search Google Scholar
    • Export Citation
  • Nemcova-Furstova V, James RF & Kovar J 2011 Inhibitory effect of unsaturated fatty acids on saturated fatty acid-induced apoptosis in human pancreatic beta-cells: activation of caspases and ER stress induction. Cellular Physiology and Biochemistry 525538. (https://doi.org/10.1159/000329954)

    • Search Google Scholar
    • Export Citation
  • Nemecz M, Constantin A, Dumitrescu M, Alexandru N, Filippi A, Tanko G & Georgescu A 2018 The distinct effects of palmitic and oleic acid on pancreatic beta cell function: the elucidation of associated mechanisms and effector molecules. Frontiers in Pharmacology 1554. (https://doi.org/10.3389/fphar.2018.01554)

    • Search Google Scholar
    • Export Citation
  • Rostamirad A, Ebrahimi SSS, Sadeghi A, Taghikhani M & Meshkani R 2018 Palmitate-induced impairment of autophagy turnover leads to increased apoptosis and inflammation in peripheral blood mononuclear cells. Immunobiology 269278. (https://doi.org/10.1016/j.imbio.2017.10.041)

    • Search Google Scholar
    • Export Citation
  • Ruan JS, Lin JK, Kuo YY, Chen YW & Chen PC 2018 Chronic palmitic acid-induced lipotoxicity correlates with defective trafficking of ATP sensitive potassium channels in pancreatic beta cells. Journal of Nutritional Biochemistry 3748. (https://doi.org/10.1016/j.jnutbio.2018.05.005)

    • Search Google Scholar
    • Export Citation
  • Sargsyan E, Artemenko K, Manukyan L, Bergquist J & Bergsten P 2016 Oleate protects beta-cells from the toxic effect of palmitate by activating pro-survival pathways of the ER stress response. Biochimica and Biophysica Acta 11511160. (https://doi.org/10.1016/j.bbalip.2016.06.012)

    • Search Google Scholar
    • Export Citation
  • Sharma RB & Alonso LC 2014 Lipotoxicity in the pancreatic beta cell: not just survival and function, but proliferation as well? Current Diabetes Reports 492. (https://doi.org/10.1007/s11892-014-0492-2)

    • Search Google Scholar
    • Export Citation
  • Sharma S, Mells JE, Fu PP, Saxena NK & Anania FA 2011 GLP-1 analogs reduce hepatocyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy. PLoS ONE e25269. (https://doi.org/10.1371/journal.pone.0025269)

    • Search Google Scholar
    • Export Citation
  • Sramek J, Nemcova-Furstova V, Pavlikova N & Kovar J 2017 Effect of saturated stearic acid on MAP kinase and ER stress signaling pathways during apoptosis induction in human pancreatic beta-cells is inhibited by unsaturated oleic acid. International Journal of Molecular Sciences E2313. (https://doi.org/10.3390/ijms18112313)

    • Search Google Scholar
    • Export Citation
  • Tirupathi Pichiah PB, Suriyakalaa U, Kamalakkannan S, Kokilavani P, Kalaiselvi S, Sankarganesh D, Gowri J, Archunan G, Cha YS & Achiraman S 2011 Spermidine may decrease ER stress in pancreatic beta cells and may reduce apoptosis via activating AMPK dependent autophagy pathway. Medical Hypotheses 677679. (https://doi.org/10.1016/j.mehy.2011.07.014)

    • Search Google Scholar
    • Export Citation
  • Trudeau KM, Colby AH, Zeng J, Las G, Feng JH, Grinstaff MW & Shirihai OS 2016 Lysosome acidification by photoactivated nanoparticles restores autophagy under lipotoxicity. Journal of Cell Biology 2534. (https://doi.org/10.1083/jcb.201511042)

    • Search Google Scholar
    • Export Citation
  • Varshney R, Gupta S & Roy P 2017 Cytoprotective effect of kaempferol against palmitic acid-induced pancreatic beta-cell death through modulation of autophagy via AMPK/mTOR signaling pathway. Molecular and Cellular Endocrinology 120. (https://doi.org/10.1016/j.mce.2017.02.033)

    • Search Google Scholar
    • Export Citation
  • Wang Y, Xie T, Zhang D & Leung PS 2019 GPR120 protects lipotoxicity-induced pancreatic beta-cell dysfunction through regulation of PDX1 expression and inhibition of islet inflammation. Clinical Science 101116. (https://doi.org/10.1042/CS20180836)

    • Search Google Scholar
    • Export Citation
  • Welters HJ, Tadayyon M, Scarpello JH, Smith SA & Morgan NG 2004 Mono-unsaturated fatty acids protect against beta-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure. FEBS Letters 103108. (https://doi.org/10.1016/S0014-5793(04)00079-1)

    • Search Google Scholar
    • Export Citation
  • Wu J, Wu Q, Li JJ, Chen C, Sun S, Wang CH & Sun SR 2017 Autophagy mediates free fatty acid effects on MDA-MB-231 cell proliferation, migration and invasion. Oncology Letters 47154721. (https://doi.org/10.3892/ol.2017.6807)

    • Search Google Scholar
    • Export Citation
  • Xu K, Liu XF, Ke ZQ, Yao Q, Guo S & Liu C 2018 Resveratrol modulates apoptosis and autophagy induced by high glucose and palmitate in cardiac cells. Cellular Physiology and Biochemistry 20312040. (https://doi.org/10.1159/000489442)

    • Search Google Scholar
    • Export Citation
  • Zummo FP, Cullen KS, Honkanen-Scott M, Shaw JAM, Lovat PE & Arden C 2017 Glucagon-like peptide 1 protects pancreatic beta-cells from death by increasing autophagic flux and restoring lysosomal function. Diabetes 12721285. (https://doi.org/10.2337/db16-1009)

    • Search Google Scholar
    • Export Citation