Genetic modulation of islet β-cell iPLA₂β expression provides evidence for its impact on β-cell apoptosis and autophagy

Abstract

β-cell apoptosis is a significant contributor to β-cell dysfunction in diabetes and ER stress is among the factors that contributes to β-cell death. We previously identified that the Ca2+-independent phospholipase A2β (iPLA2β), which in islets is localized in β-cells, participates in ER stress-induced β-cell apoptosis. Here, direct assessment of iPLA2β role was made using β-cell-specific iPLA2β overexpressing (RIP-iPLA2β-Tg) and globally iPLA2β-deficient (iPLA2β-KO) mice. Islets from Tg, but not KO, express higher islet iPLA2β and neutral sphingomyelinase, decrease in sphingomyelins, and increase in ceramides, relative to WT group. ER stress induces iPLA2β, ER stress factors, loss of mitochondrial membrane potential (∆Ψ), caspase-3 activation, and β-cell apoptosis in the WT and these are all amplified in the Tg group. Surprisingly, β-cells apoptosis while reduced in the KO is higher than in the WT group. This, however, was not accompanied by greater caspase-3 activation but with larger loss of ∆Ψ, suggesting that iPLA2β deficiency impacts mitochondrial membrane integrity and causes apoptosis by a caspase-independent manner. Further, autophagy, as reflected by LC3-II accumulation, is increased in Tg and decreased in KO, relative to WT. Our findings suggest that (1) iPLA2β impacts upstream (UPR) and downstream (ceramide generation and mitochondrial) pathways in β-cells and (2) both over- or under-expression of iPLA2β is deleterious to the β-cells. Further, we present for the first time evidence for potential regulation of autophagy by iPLA2β in islet β-cells. These findings support the hypothesis that iPLA2β induction under stress, as in diabetes, is a key component to amplifying β-cell death processes.

Calcium-independent phospholipases A2 and their roles in biological processes and diseases

“…Ceramides are part of the sphingolipid family and act as lipid messengers that can suppress cell growth and induce apoptosis (247)(248)(249), and as expected, inhibition or knockdown of NSMase-2-mitigated ceramide accumulation ( 103,104,137 ). Unexpectedly, inhibition, knockdown, or iPLA 2 ␤ defi ciency attenuated, and iPLA 2 ␤ overexpression exacerbated, NSMase-2 induction and ceramide accumulation ( 103,104,136,138,246 ). These fi ndings indicated that NSMase-2-catalyzed ceramide accumulations during ␤ -cell Fig.…”

“…During the same period, Polonsky's group reported that ER stress-induced death of insulinoma cells occurs independently of Ca 2+ and is mediated by a metabolite of AA ( 245 ). Intriguingly, while short-term (minutes) stimulation of iPLA 2 ␤ in ␤ -cells has a benefi cial effect of enhancing glucose-stimulated insulin secretion ( 4, 105,177,201,203,204 ), long-term (hours) exposures to pro-infl ammatory cytokines, hyperglycemia, and ER stress induce iPLA 2 ␤ at the message, protein, and activity levels in ␤ -cells ( 98, [136][137][138]246 ) and lead to deleterious consequences that ultimately cause ␤ -cell death ( 98, 103, 104, 136-138, 246 ). ␤ -Cell apoptosis is a critical contributor to the onset and progression of diabetes and it is in this context that our group has focused on iPLA 2 ␤ biology to understand the underlying molecular mechanisms by which iPLA 2 ␤ -derived lipids promote ␤ -cell death during T1D development, and our current understanding is illustrated in Fig.…”

“…1 . iPLA 2 ␤ and sphingolipids. Stress stimuli induce the intrinsic (mitochondrial) apoptotic pathway, which is mitigated by suppressing iPLA 2 ␤ expression/activity ( 103,104,136,138,246 ). Intriguingly, the primary lipid signals promoting mitochondrial decompensation were ceramides, derived through hydrolysis of sphingomyelins by neutral sphingomyelinase-2 (NSMase-2) ( 103,104 ).…”

Polarization of Macrophages toward M2 Phenotype Is Favored by Reduction in iPLA2β (Group VIA Phospholipase A2)

“…The NOD mice were all generated as described (16,29). Before experimentation, the mice were genotyped as described (30,78). Animal experiments were conducted according to approved Institution Animal Care and Use Committee (IACUC) guidelines at the University of Alabama at Birmingham.…”

“…NOD Islet iPLA 2 ␤ RT-qPCR Analyses-Islets were isolated from female spontaneous diabetes-prone NOD, immune-deficient and diabetes-resistant NOD.Rag, and NOX-derived superoxide-deficient NOD (NOD.Ncf1 m1j ) mice, as described (30). Total RNA was isolated and cDNA was prepared for iPLA 2 ␤ RT-qPCR analyses as described (29).…”