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. 2014 Sep;155(9):3352-64.
doi: 10.1210/en.2013-2134. Epub 2014 Jul 8.

Evidence of contribution of iPLA2β-mediated events during islet β-cell apoptosis due to proinflammatory cytokines suggests a role for iPLA2β in T1D development

Xiaoyong Lei  1 , Robert N Bone, Tomader Ali, Sheng Zhang, Alan Bohrer, Hubert M Tse, Keshore R Bidasee, Sasanka Ramanadham
Affiliations

Evidence of contribution of iPLA2β-mediated events during islet β-cell apoptosis due to proinflammatory cytokines suggests a role for iPLA2β in T1D development

Xiaoyong Lei et al. Endocrinology. 2014 Sep.

Abstract

Type 1 diabetes (T1D) results from autoimmune destruction of islet β-cells, but the underlying mechanisms that contribute to this process are incompletely understood, especially the role of lipid signals generated by β-cells. Proinflammatory cytokines induce ER stress in β-cells and we previously found that the Ca(2+)-independent phospholipase A2β (iPLA2β) participates in ER stress-induced β-cell apoptosis. In view of reports of elevated iPLA2β in T1D, we examined if iPLA2β participates in cytokine-mediated islet β-cell apoptosis. We find that the proinflammatory cytokine combination IL-1β+IFNγ, induces: a) ER stress, mSREBP-1, and iPLA2β, b) lysophosphatidylcholine (LPC) generation, c) neutral sphingomyelinase-2 (NSMase2), d) ceramide accumulation, e) mitochondrial membrane decompensation, f) caspase-3 activation, and g) β-cell apoptosis. The presence of a sterol regulatory element in the iPLA2β gene raises the possibility that activation of SREBP-1 after proinflammatory cytokine exposure contributes to iPLA2β induction. The IL-1β+IFNγ-induced outcomes (b-g) are all inhibited by iPLA2β inactivation, suggesting that iPLA2β-derived lipid signals contribute to consequential islet β-cell death. Consistent with this possibility, ER stress and β-cell apoptosis induced by proinflammatory cytokines are exacerbated in islets from RIP-iPLA2β-Tg mice and blunted in islets from iPLA2β-KO mice. These observations suggest that iPLA2β-mediated events participate in amplifying β-cell apoptosis due to proinflammatory cytokines and also that iPLA2β activation may have a reciprocal impact on ER stress development. They raise the possibility that iPLA2β inhibition, leading to ameliorations in ER stress, apoptosis, and immune responses resulting from LPC-stimulated immune cell chemotaxis, may be beneficial in preserving β-cell mass and delaying/preventing T1D evolution.

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Figures

Figure 1.
Figure 1.
IL-1β+IFNγ (CTK) induces ER stress factors, iPLA2β, and mSREBP-1 in human pancreatic islets. Islets (500/condition) were treated with either vehicle (DMSO) or IL-1β+IFNγ (CTK), harvested at 24 and 48 hours, and processed for immunoblotting analyses for GRP78, pPERK, iNOS, iPLA2β, and mSREBP-1. Tubulin was used as loading control. A and C, Analyses were repeated 3 times and representative blots are presented. B and D, Densitometry quantitation. (*Significantly different vs corresponding DMSO groups, P < .05; §vs other groups, P < .05; # and † vs corresponding DMSO groups, P < .01 and P < .05, respectively, n = 3.)
Figure 2.
Figure 2.
IL-1β+IFNγ (CTK) induces iPLA2β and mSREBP-1 in islets. Islets (500/condition, n = 3–5) were treated as in Figure 1 for 24 hours. A, Human islet iPLA2β mRNA was determined by RT-qPCR. (*Significantly different from DMSO, P < .05). B, iPLA2β specific activity (SA) was determined in cytosol of islets after treatment with vehicle or CTK ± S-BEL and presented as mean pmol/min/mg protein ± SEM: DMSO, 11 ± 0.5; S-BEL, 3 ± 3; CTK and CTK, 25 ± 6.5; CTK + S-BEL, 1.5 ± 1.5. (*†Significantly different from other groups, P < .05). C, iPLA2β and mSREBP-1 protein. Cytosol of human and mouse islets (n = 3 each) after treatment with vehicle or CTK ± S-BEL was processed for immunoblotting analyses and representative blots from mouse islets are presented (actin was used as loading control). D, Densitometry quantitation. (* and †Significantly different from corresponding DMSO and S-BEL groups, P < .005 and P < .05, respectively, n = 6.)
Figure 3.
Figure 3.
IL-1β+IFNγ (CTK) promotes ceramide generation in human islets by an iPLA2β-mediated induction of NSMase2. Islets (500/condition) were treated as in Figure 1 for 24 hours and processed for ceramide analyses by ESI/MS/MS. Representative spectra from (3–5 replicates) are presented, where individual ceramide molecular species ions (m/z) and their (ratios), relative to internal standard (IS, 14:0/14:0-CM), are identified in each spectrum. A, DMSO vehicle. B, CTK. C, CTK + S-BEL (iPLA2β inhibitor). D, CTK + GW4869 (NSMase2 inhibitor). Quantitation of these analyses is presented in Figure 4.
Figure 4.
Figure 4.
IL-1β+IFNγ (CTK) induces neutral sphingomyelinase-2 (NSMase2), and ceramide and LPC generation by an iPLA2β-dependent manner in human islets. Islets (500/condition) were processed after 24 hours exposure to CTK for NSMase2 mRNA by RT-qPCR (A), and for ceramide (B), sphingomyelin (C), and LPC (D) molecular species ± S-BEL by ESI/MS/MS analyses. To determine the content of each species, standard curves were generated from a series of samples containing a fixed amount of internal standard (IS) and varied amounts of individual molecular species standards. The relative abundance of individual species, relative to IS, was measured by ESI-MS/MS scanning. Standard curves were then plotted as a ratio of individual species relative to the IS and a linear regression equation generated. From a given experimental condition, the ratio of a molecular species to the added IS was calculated and applied to the equation. The same amount of IS was added to each of the samples and the highest intensity ion (IS or a sample molecular species) was taken as 100%. The ratio of individual molecular species to IS was contained within the linear range of the standard curves. This value was then divided by the molecular species mass and normalized to lipid phosphorus content in the given sample to obtain amount of molecular species/nmol PO4 for each molecular species identified, as described (14–16, 19). These were then summed to obtain a pooled value for each lipid class. Basal ceramide, sphingomyelin, and LPC pools ranged from 4 to 24 nmol/nmol PO4, 123 to 409 pmol/nmol PO4, and 0.03 to 0.25 pmol/nmol PO4 respectively. Because of subject-to-subject variation in basal ceramide, sphingomyelin, and LPC molecular species in human islets, the values for each subject were normalized to corresponding DMSO control and the data are presented as % change, relative to control. (*Significantly different from corresponding minus S-BEL groups, P < .05; †significantly different from DMSO minus S-BEL groups, P < .05; and §significantly different from other groups, P < .05, n = 3–5).
Figure 5.
Figure 5.
IL-1β+IFNγ (CTK) induces mitochondrial membrane potential (ΔΨ) loss in human islet cells in an iPLA2β-dependent manner. Islets (500/condition) were treated as in Figure 1 for 24 hours, dispersed into individual cells, and ΔΨ monitored by MitoFlow (stable analog of TMRE, tetramethylrhodamine, ethyl ester) fluorescence or DiOC6(3) staining. A, Flow cytometry analyses. Representative spectra (top panels) were obtained from analyses of 10,000 cells and the percentage of cells with compromised ΔΨ is indicated by M1. Quantitation of cells with MMP loss ± S-BEL (bottom panel). The percentage of cells with ΔΨ loss, relative to total number of cells, is presented as mean ± SEM. (*Significantly different from other groups, P < .05, n = 3–5). B, DiOC6(3) staining. Cells were loaded with green mitochondrial [DiOC6(3)] stains and examined by confocal microscopy. Representative images are shown (top panels). DiOC6(3) fluorescence intensity was quantitated using ImageJ Software and is presented as mean ± SEM (bottom panel). (*Significantly different from other groups, P < .05, n = 6).
Figure 6.
Figure 6.
IL-1β+IFNγ (CTK) induces activated caspase-3 (aC3) and TUNEL-positivity in human islet β-cells in an iPLA2β-dependent manner. Islets (500/condition) were treated with CTK ± S-BEL for 48 hours and then processed for aC3 and apoptosis analyses. A, Activated casp-3 (aC3) was assessed by immunofluorescence (DAPI, blue; insulin, green; and aC3, red). Representative images of individual and merged fluorescence islet images are presented. B, Apoptosis incidence was assessed by TUNEL staining and the percentage of TUNEL-positive cells, relative to total number of cells (DAPI-stained), was determined in a minimum of 6 fields on each slide from separate experiments and presented as mean ± SEM. (* Significantly different from groups, P < .05, n = 4–5).
Figure 7.
Figure 7.
iPLA2β expression modulates ER stress and islet cell apoptosis induced by IL-1β+IFNγ (CTK). Islets (500/condition) isolated from RIP-iPLA2β-Tg and iPLA2β-KO mice were treated with vehicle (DMSO) alone or CTK for up to 48 hours and then processed for ER stress markers (pPERK, IRE1α, and ATF6α) and apoptosis indices [activated caspase-3 (aC3) and TUNEL positivity] analyses. A, Representative immunoblots of pPERK, IRE1α, ATF6α, and loading control actin. B, Densitometry quantitation, abundances of the ER stress markers, relative to corresponding actin bands ± CTK in each group, are plotted as mean ± SEM (n = 3). (pPERK, *, #, ¶, and † significantly different from corresponding DMSO group, P < .001; P < .0001; P < .01; and P < .05, respectively, a and b significantly different from corresponding WT group, P < .001 and P < .05, respectively; IRE1α, †, *, and # significantly different from corresponding DMSO group, P < .05; P < .001; and P < .0001, respectively; c significantly different from corresponding WT group, P < .0005; and ATF6α, † and § significantly different from corresponding DMSO group, P < .05 and P < .005, respectively; d and b significantly different from corresponding WT group, P < .05 and P < .01, respectively.) C, aC3 activity. Islet lysates were prepared and activity in 30 μg aliquot of protein was assayed using a colorimetric-based protocol. (§ and † significantly different from corresponding DMSO groups, P < .01 and P < .005, respectively; *significantly different from corresponding WT and KO groups, P < .05; and # significantly different from Tg-DMSO and Tg-48h groups, P < .05, n = 3–6.) D, TUNEL analyses. The percentage of TUNEL-positive cells relative to total number of cells (DAPI-stained) was determined in a minimum of 6 fields on each slide from 3 separate experiment and are presented as mean ± SEM. (#* Significantly different from corresponding DMSO group, P < .01 and P < .05, respectively; † and § significantly different from corresponding WT and KO groups, P < .005 and P < .01, respectively.)
Figure 8.
Figure 8.
Proposed model of iPLA2β involvement in the autoimmune destruction of β-cells in T1D. Based on our present findings, we propose that proinflammatory cytokines cause ER stress leading to SREBP-1 activation and induction of iPLA2β. Alternatively, iPLA2β induction may also be promoted by downstream products of proinflammatory cytokines activation. It is suggested that the iPLA2β-derived lipid signals promote β-cell death by a) triggering the intrinsic apoptotic pathway via ceramide generation and by serving as chemoattractants to enhance islet infiltration of immune cells and b) exacerbating ER stress and consequential downstream events. It is speculated that the iPLA2β-derived lipid signals amplify proinflammatory cytokine-induced β-cell apoptosis and that inhibition of iPLA2β may be beneficial in reducing the impact of proinflammatory cytokines on β-cells and mitigating autoimmune destruction of β-cells, thus preventing or delaying the onset and/or progression of T1D. (Dashed arrows reflect undefined pathways.)
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