Protection of pancreatic beta-cells by group VIA phospholipase A(2)-mediated repair of mitochondrial membrane peroxidation

Abstract

Mitochondrial production of reactive oxygen species and oxidation of cardiolipin are key events in initiating apoptosis. We reported that group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) localizes in and protects beta-cell mitochondria from oxidative damage during staurosporine-induced apoptosis. Here, we used iPLA(2)beta-null (iPLA(2)beta(-/-)) mice to investigate the role of iPLA(2)beta in the repair of mitochondrial membranes. We show that islets isolated from iPLA(2)beta(-/-) mice are more sensitive to staurosporine-induced apoptosis than those from wild-type littermates and that 2 wk of daily ip administration of staurosporine to iPLA(2)beta(-/-) mice impairs both the animals' glucose tolerance and glucose-stimulated insulin secretion by their pancreatic islets. Moreover, the iPLA(2)beta inhibitor bromoenol lactone caused mitochondrial membrane peroxidation and cytochrome c release, and these effects were reversed by N-acetyl cysteine. The mitochondrial antioxidant N-t-butyl hydroxylamine blocked staurosporine-induced cytochrome c release and caspase-3 activation in iPLA(2)beta(-/-) islets. Furthermore, the collapse of mitochondrial membrane potential in INS-1 insulinoma cells caused by high glucose and fatty acid levels was attenuated by overexpressing iPLA(2)beta. Interestingly, iPLA(2)beta was expressed only at low levels in islet beta-cells from obesity- and diabetes-prone db/db mice. These findings support the hypothesis that iPLA(2)beta is important in repairing oxidized mitochondrial membrane components (e.g. cardiolipin) and that this prevents cytochrome c release in response to stimuli that otherwise induce apoptosis. The low iPLA(2)beta expression level in db/db mouse beta-cells may render them vulnerable to injury by reactive oxygen species.

Figure 1

Effects of staurosporine on iPLA₂β^−/− islets. Islets isolated from 6-month-old iPLA₂β^−/− mice, and their normal littermates were treated with 1 μm staurosporine for up to 12 h. After extraction of mitochondria (Mito.) and cytosol (Cyt.) fractions, the released cytochrome c (A) and caspase-3 activities (B) were measured. Data are presented as the mean ± sem of three independent experiments. *, P < 0.05. INS-1 cells and iPLA₂β-overexpression INS-1 cells (iPLA₂-INS) were treated with 1 μm staurosporine over time. Then the cell lysates were prepared for analysis of cytochrome (Cyt. c) by the Western blot analysis (C), and the cells were stained with annexin-V and immediately analyzed by flow cytometry (D). Data are presented as the mean ± sem of six independent experiments. *, P < 0.05.

Figure 2

Effects of staurosporine (STS) on the metabolism of iPLA₂β^−/− mice. Six-month-old iPLA₂β^−/− (C57BL6 background) mice and their normal littermates were randomly divided into control (n = 10) and staurosporine (n = 10) groups. Control mice were injected ip with 0.5% sodium carboxymethyl cellulose at a volume of 1 ml/kg, and staurosporine groups were injected ip with staurosporine (0.4 mg/kg) daily for 2 wk. A, IPGTT (2 g/kg body weight). Data are presented as mean ± sem (n = 10); *, P < 0.05, iPLA₂β^−/− + STS vs. all other groups; #, P < 0.05, iPLA₂β^−/− control or WT + STS vs. the WT control. B, IAUCs of the GTTs. Data are presented as mean ± sem (n = 10); *, P < 0.001, STS-treated groups vs. their controls; #, P < 0.001, iPLA₂β^−/− groups vs. corresponding WT groups. C, ITT. Data are presented as mean ± sem; P > 0.05. D, Glucose-stimulated insulin secretion by isolated islets ex vivo. The islets were isolated from the mice described above for insulin secretion assays. Insulin secretion of each group in 16.7 mm glucose is significantly higher than that in 1.7 mm glucose (P < 0.01). *, P < 0.05, compared with WT 16.7 mm glucose; #, P < 0.05, iPLA₂β^−/− + STS vs. all of other groups.

Figure 3

Inhibition of iPLA₂β induced the peroxidation of mitochondrial membranes. A, BEL-induced apoptosis at high concentrations. INS-1 and iPLA₂-INS cells were incubated with increasing concentrations of BEL in the presence of 11 mm glucose for 48 h. The cells were then stained with annexin-V and analyzed by flow cytometry. B, BEL-induced apoptosis was blocked by NAC. INS-1 cells were treated with 25 μm of BEL in the presence of 11 mm of glucose for 48 h. Apoptosis was analyzed by Annexin V-staining and fluorescence-activated cell sorter (*, compared with control). C, TBARS assay of mitochondrial membrane peroxidation in INS-1 cells. INS-1 cells were incubated with 25 μm BEL in the presence of 11 mm glucose for 48 h. Mitochondria (Mito) were isolated, and the Mito and non-Mito fractions were subjected to TBARS assay (*, compared with non-Mito). D, Cytochrome c release assay. P < 0.05 (n = 4); *, compared with control. Data are presented as the mean ± sem of three independent experiments.

Figure 4

Effect on apoptosis of blocking the peroxidation of mitochondrial membranes. A, Isolated islets from iPLA₂β^−/− mice were treated with staurosporine in the presence or absence of NtBHA over time. Cytochrome c release (A) and caspase-3 activity (B) were measured. Data are presented as the mean ± sem of three independent experiments. *, P < 0.05 compared with the control.

Figure 5

iPLA₂β prevented the loss of mitochondrial membrane potential in INS-1 cells in response to glucose or fatty acids. A, Flow cytometry analysis of glucose-induced loss of mitochondrial membrane potential. INS-1 cells and iPLA₂-INS were treated with or without (control) 22 mm glucose for 72 h. The cells were then stained with JC-1 and analyzed by flow cytometry. B, Microscopic analysis of living cells treated with palmitate. INS-1 cells and iPLA₂-INS were treated with or without 0.5 mm palmitate for 48 h. The cells were then stained with JC-1, analyzed by fluorescent microscopy, and quantified by flow cytometry. Living cells are stained both red and green, apoptotic cells are stained with green only. Data are presented as the mean ± sem of nine independent experiments. *, P < 0.05, treated groups vs. the controls; #, P < 0.05, the treated iPLA₂-INS vs. the treated INS-1 cells. Pal, Palmitate; Glc, glucose.

Figure 6

Immunohistochemical analysis of iPLA₂β expression in the pancreases of db/db mice. A, Six- to 10-wk-old normal (a) and db/db (C57BL/KsJ-Leprdb) mice (b–d) were killed, and the pancreases were fixed and sliced for iPLA₂ staining with goat anti-iPLA₂β antibodies. B, iPLA₂β activity analysis. The islets were isolated from normal and db/db mice at the ages of 6–10 wk, and cell lysates were prepared for iPLA₂β analysis (n = 4); *, P < 0.05, compared with control (Contr.) in the absence of BEL. Contr, Control.

Figure 7

Proposed model for the role of iPLA₂β in mitochondrial function. (1) Cardiolipin remodeling. Nascent cardiolipin is remodeled to mature cardiolipin through a cycle of deacylation by iPLA₂β and reacylation by monolysocardiolipin acyltransferase (MLCLAT). (2) Repair of cardiolipin oxidation. Under low oxidative stress, mitochondrial cardiolipin is oxidized, which may lower ROS levels, and is repaired promptly by iPLA₂β and MLCLAT-mediated deacylation and reacylation. Under high oxidative stress, cardiolipin oxidation frees cytochrome c and initiates apoptosis. (3) Facilitation of apoptotic process. The permanent activation of iPLA₂β by caspase-3 may facilitate clearance of apoptotic cells by releasing lysophosphatidylcholine (LPC). sFA, Saturated fatty acids; usFA, unsaturated fatty acids. Gray arrows, The biochemical reactions catalyzed by iPLA₂β; black arrows, stimulatory pathway; dashed arrow, inhibitory pathway.