Pancreatic Islets and Insulinoma Cells Express a Novel Isoform of Group VIA Phospholipase A₂ (iPLA₂β) that Participates in Glucose-Stimulated Insulin Secretion and Is Not Produced by Alternate Splicing of the iPLA₂β Transcript

Abstract

Many cells express a group VIA 84 kDa phospholipase A 2 (iPLA 2 β) that is sensitive to inhibition by a bromoenol lactone (BEL) suicide substrate. Inhibition of iPLA 2 β in pancreatic islets and insulinoma cells suppresses, and overexpression of iPLA 2 β in INS-1 insulinoma cells amplifies, glucose-stimulated insulin secretion, suggesting that iPLA 2 β participates in secretion. Western blotting analyses reveal that glucose-responsive 832/13 INS-1 cells express essentially no 84 kDa iPLA 2 β-immunoreactive protein but predominantly express a previously unrecognized immunoreactive iPLA 2 β protein in the 70 kDa region that is not generated by a mechanism of alternate splicing of the iPLA 2 β transcript. To determine if the 70 kDa-immunoreactive protein is a short isoform of iPLA 2 β, protein from the 70 kDa region was digested with trypsin and analyzed by mass spectrometry. Such analyses reveal several peptides with masses and amino acid sequences that exactly match iPLA 2 β tryptic peptides. Peptide sequences identified in the 70 kDa tryptic digest include iPLA 2 β residues 7-53, suggesting that the N-terminus is preserved. We also report here that the 832/13 INS-1 cells express iPLA 2 β catalytic activity and that BEL inhibits secretagogue-stimulated insulin secretion from these cells but not the incorporation of arachidonic acid into membrane PC pools of these cells. These observations suggest that the catalytic iPLA 2 β † This research was supported in part by grants from National Institutes of Health (R37-DK34388, P41-RR00954, P01-HL57278, P60-DK20579, P30-DK56341) and by an Award (S.R.) from the American Diabetes Association. 1 Abbreviations: AA, arachidonic acid; BEL, bromoenol lactone suicide inhibitor of iPLA 2 β; BME, β-mercaptoethanol; BSA, bovine serum albumin; bp, base pairs; cPLA 2 , Group IV cytosolic phospholipase A 2 ; dpm, disintegrations per minute; ECL, enhanced chemiluminescence; FBS, fetal bovine serum; GPC, glycerophosphocholine; IF, immunofluorescence; iPLA 2 β, β-isoform of Group VIA calcium-independent phospholipase A 2 ; kDa, kilodaltons; LC/ESI, liquid chromatography/electrospray ionization; MALDI/TOF, matrix-assisted laser desorption ionization/time-of-flight; MS, mass spectrometry; OE, iPLA 2 β overexpressing cells; O/N, overnight; PAGE, polyacrylamide gel electrophoresis; Q-TOF, quadrupole-time-of-flight; PBS, phosphate-buffered saline; PC, phosphatidylcholine; piPLA 2 , polyclonal iPLA 2 ; PIC, protease inhibitor cocktail; PLA 2 , phospholipase A 2 ; SDS, sodium dodecyl sulfate; TBS-T, Tris-buffered saline-tween; TLC, thin-layer chromatography; RT, room temperature; RT-PCR, reverse transcriptionpolymerase chain reactions; V, empty vector transfected cells. activity expressed in 832/13 INS-1 cells is attributable to a short isoform of iPLA 2 β and that this isoform participates in insulin secretory but not in membrane phospholipid remodeling pathways. Further, the finding that pancreatic islets also express predominantly a 70 kDa iPLA 2 β-immunoreactive protein suggests that...

Modulation of the Pancreatic Islet β-Cell-delayed Rectifier Potassium Channel Kv2.1 by the Polyunsaturated Fatty Acid Arachidonate

“…Islet phospholipase A 2 (PLA 2 ) enzyme(s) activated by stimulation with glucose and/or by G-protein signaling mechanisms catalyze hydrolysis of arachidonic acid from ␤-cell membrane phospholipids and cause levels of free arachidonic acid to rise by up to 3-fold to the mid-micromolar range (7,13,14,27,28). Such concentrations of arachidonic acid amplify both the rise in ␤-cell [Ca 2ϩ ] and insulin secretion induced by glucose (7, 15-17, 23, 25, 27, 28).Pharmacologic inhibition or molecular biologic suppression of expression of the pancreatic islet ␤-cell Group VIA phospholipase A 2 (iPLA 2 ␤) enzyme (24,29,30) results in attenuation of insulin secretory responses induced by glucose and other secretagogues (18 -22, 24). The products of iPLA 2 ␤ action on its *…”

“…Pharmacologic inhibition or molecular biologic suppression of expression of the pancreatic islet ␤-cell Group VIA phospholipase A 2 (iPLA 2 ␤) enzyme (24,29,30) results in attenuation of insulin secretory responses induced by glucose and other secretagogues (18 -22, 24). The products of iPLA 2 ␤ action on its *…”

“…Pharmacologic, biochemical, and genetic evidence suggests that glucose-stimulated hydrolysis of esterified arachidonic acid from ␤-cell membrane phospholipids is required for physiological insulin secretion (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Pancreatic islet ␤-cells contain high levels of arachidonic acid compared with other tissues (7, 9 -12 24, 25), and about two-thirds of islet ␤-cell glycerophospholipids contain arachidonic acid as the sn-2 substituent (23)(24)(25).…”

“…Pancreatic islet ␤-cells contain high levels of arachidonic acid compared with other tissues (7, 9 -12 24, 25), and about two-thirds of islet ␤-cell glycerophospholipids contain arachidonic acid as the sn-2 substituent (23)(24)(25). Islet phospholipase A 2 (PLA 2 ) enzyme(s) activated by stimulation with glucose and/or by G-protein signaling mechanisms catalyze hydrolysis of arachidonic acid from ␤-cell membrane phospholipids and cause levels of free arachidonic acid to rise by up to 3-fold to the mid-micromolar range (7,13,14,27,28).…”

“…Pharmacologic inhibition or molecular biologic suppression of expression of the pancreatic islet ␤-cell Group VIA phospholipase A 2 (iPLA 2 ␤) enzyme (24,29,30) results in attenuation of insulin secretory responses induced by glucose and other secretagogues (18 -22, 24). The products of iPLA 2 ␤ action on its phospholipid substrates include a nonesterified fatty acid, such as AA, and a 2-lysphophosplipid, such as lysophosphatidylcholine (LPC), and ␤-cell levels of both products correlate with iPLA 2 ␤ expression level (21,22,31,32).…”