Group VIB Phospholipase A₂ Promotes Proliferation of INS-1 Insulinoma Cells and Attenuates Lipid Peroxidation and Apoptosis Induced by Inflammatory Cytokines and Oxidant Agents

Figure 6

HPLC-ESI-MS/MS analysis of oxidized lipid molecular species that accumulate in INS-1 cells incubated with streptozotocin. Control INS-1 cells (light bars) or iPLA₂γ-knockdown INS-1 cells (dark bars) were incubated with vehicle only or with STZ (5 mM) for 16 hr as in Figure 5, and lipids were then extracted and analyzed by HPLC-LC-MS/MS as described in Experimental Procedures. Panel (a) illustrates the MS/MS transition monitored, which is the production of the hydroxyeicosatetraenoate (HETE) [M-H]⁻ ion (m/z 319.3) from the parent (C18:0/C20:4)-GPE [M-H]⁻ ion (m/z 782.76) upon collisionally activated dissociation. Prior survey scans monitoring parents of oxidized linoleate (C18:2) and docosahexaenoate (C22:6) species had revealed that HETE (HO-C20:4) was the dominant oxidized fatty acid residue esterified in INS-1 cell phospholipids. Panel (b) is an MS/MS scan over the m/z range 400–2000 in which parent ions that generate the HETE anion (m/z 319.3) are monitored, and m/z 782.76 is the vastly predominant parent, which was found to represent the (C18:0/C20:4)-GPE [M-H]⁻ ion upon analysis of the complete MS/MS spectrum/ Panel (c) is an expansion of that mass spectrum over the m/z range 782.5 to 785.0 to illustrate the [¹³C] isotopomer distribution of the [M-H]⁻ ion. In panels (d) and (e) represent HPLC/ESI/MS/MS scans in which the transition m/z 782.76 to m/z 319.3 is monitored as a function of LC retention time to quantitate the (C18:0/C20:4)-GPE content of control INS-1 cells (panels (d) and (e)) or iPLA₂γ-knockdown (KD) INS-1 cells (panels (f) and (g)) incubated without (panels (d) and (f)) or with STZ (panels (e) and (g)). Panel (h) represents a summary of four such experiments, and mean values are displayed and SEM indicated. An asterisk (*) denotes a significant (𝑃<0.05) difference between control and iPLA₂γ-KD INS-1 cell lines, and an X denotes a significant difference between cells incubated with or without STZ.

(a) MRM of the transition 782.76 → 319.3 for oxy-pe

(b) Parents of m/z 319.3 MS/MS scan

(c) {[M-H]⁻ [¹³C]} isotopomers parents of m/z 319.3 scan

(d) LC/MS/MS MRM m/z 782.76 → m/z 319.3 control INS-1 cells

(e) LC/MS/MS m/z 782.76 → m/z 319.3 control INS-1 cells incubated with STZ

(f) LC/MS/MS MRM m/z 782.76 → m/z 319.3 iPLA₂γ-knockdown INS-1 cells

(g) LC/MS/MS m/z 782.76 → m/z 319.3 INS-1-KD cells treated with STZ

(h) Streptozotocin effect on INS-1 cell oxidized pe