Palmitate (C16:0) induces apoptosis of insulin-secreting -cells by processes that involve generation of reactive oxygen species, and chronically elevated blood long chain free fatty acid levels are thought to contribute to -cell lipotoxicity and the development of diabetes mellitus. (C18:0/hydroxyeicosatetraenoic acid)-glycerophosphoethanolamine, and these effects were blunted in INS-1 cells that overexpress iPLA2, consistent with iPLA2-mediated removal of oxidized phospholipids. C16:0 also induced iPLA2 association with INS-1 cell mitochondria, consistent with a role in mitochondrial repair. These findings indicate that iPLA2 confers significant protection of -cells against C16:0-induced injury. oxidase complex IV (COX IV) (1:1000 dilution of antibody 4844 from Cell Signaling Technology, Beverly, MA), anti-FLAG (1:1000; Sigma, F1804), and anti-polyhistidine (1:1000; Sigma, H1029). Secondary antibody concentration was 1:10,000. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL). Caspase-3 Activation Caspase-3 activation was measured as described (19, 20, 22) in INS-1 cells after incubation with palmitate or vehicle by homogenizing the cells and analyzing extracted protein electrophoretically on a 4C20% Tris-glycine gel (Invitrogen, EC6028PK5). The activated 17-kDa isoform (p17) was detected with antibody against caspase-3 (H-277) (Santa Cruz Biotechnology). A luminescence-based assay was performed (31) with a commercial kit (G8090, Promega, Madison, WI) for isolated islets according to the manufacturer’s instructions. Quantitative Real Time PCR As described (26, 27, 30), total RNA was extracted from INS-1 cells using a Qiagen RNeasy Mini kit (catalog number 74104), and aliquots from samples for each condition were prepared that contained equal amounts of RNA. SuperScript III (Invitrogen, catalog number 18080-044) enzyme was used to generate cDNA from the RNA template. PCR amplification mixtures (25 l) contained SYBR Green PCR Master Mix (12.5 l, 2, Applied Biosystems, catalog number 4309155), a 2,3-Dimethoxybenzaldehyde mixture (1.5 l) of reverse and forward primers (30 nm), water (9 l), and cDNA template (2 l). Real time quantitative PCR was performed using the GeneAmp 5700 Sequence Detection System (PerkinElmer Life Sciences) with the following cycling parameters: polymerase activation (10 min at 95 C) and amplification (40 cycles of 15 s at 95 C and then 1 min at 60 C). Relative expression levels were normalized to the endogenous control 18 S rRNA. Primer sets used were: 1) C/EBP homologous protein (CHOP) (forward, 5-CTC ATC CCC AGG AAA CGA AG-3; reverse, 5-GAA CTC TGA CTG GAA TCT GGA G-3); 2) activating transcription factor 4 (ATF4) (forward, 5-CCA AGC ACT TCA AAC CTC ATG-3; reverse, 5-GTC 2,3-Dimethoxybenzaldehyde CAT TTT CTC CAA CAT CCA ATC-3), and 3) inducible nitric-oxide synthase (iNOS) (forward, 5-CGTGTG CCT GCT GCC TIC CTG CTG T-3; reverse, 5-GTA ATC CTCAAC CTG CTC CTC ACT C-3). Lipid Extraction As referred to (24, Fertirelin Acetate 26), islets or INS-1 cells had been placed in a remedy (2 ml) of chloroform/methanol (1:1, v/v), homogenized, and sonicated on snow (20% power, 5-s bursts for 60 s; Vibra Cell probe sonicator; Materials and Sonics, Danbury, CT). After centrifugation (2,800 432) inner regular (500 ng) as referred to (32). After Vortex-mixing and centrifugation (800 335 to 182 and 338 to 182 had been 2,3-Dimethoxybenzaldehyde supervised for 4-HNE-DNPH and (20 l; 200 m) and H2O2 (20 l; 250 m) had been then added, as well as the blend was incubated (37 C, under atmosphere, 1 h). Through the incubation, H2O2 was added at 15-min intervals (last focus, 100 m). Lipid removal above was performed as, and concentrated extracts were reconstituted (chloroform/methanol, 1:1, v/v; 200 l) and analyzed by ESI/MS on a ThermoElectron TSQ 2,3-Dimethoxybenzaldehyde Vantage triple quadrupole mass spectrometer in negative ion mode. Cardiolipin Hydrolysis by iPLA2 Purified recombinant iPLA2 (2 g) was added to hydrolysis buffer (50 l;.