Pancreatic islets and insulinoma cells express a novel isoform of group VIA phospholipase A2 (iPLA2 beta) that participates in glucose-stimulated insulin secretion and is not produced by alternate splicing of the iPLA2 beta transcript.
Many cells express a group VIA 84 kDa phospholipase A(2) (iPLA(2)beta) that is sensitive to inhibition by a bromoenol lactone (BEL) suicide substrate. Inhibition of iPLA(2)beta in pancreatic islets and insulinoma cells suppresses, and overexpression of iPLA(2)beta in INS-1 insulinoma cells amplifies, glucose-stimulated insulin secretion, suggesting that iPLA(2)beta participates in secretion. Western blotting analyses reveal that glucose-responsive 832/13 INS-1 cells express essentially no 84 kDa iPLA(2)beta-immunoreactive protein but predominantly express a previously unrecognized immunoreactive iPLA(2)beta protein in the 70 kDa region that is not generated by a mechanism of alternate splicing of the iPLA(2)beta transcript. To determine if the 70 kDa-immunoreactive protein is a short isoform of iPLA(2)beta, 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)beta tryptic peptides. Peptide sequences identified in the 70 kDa tryptic digest include iPLA(2)beta residues 7-53, suggesting that the N-terminus is preserved. We also report here that the 832/13 INS-1 cells express iPLA(2)beta 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)beta activity expressed in 832/13 INS-1 cells is attributable to a short isoform of iPLA(2)beta 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)beta-immunoreactive protein suggests that a signal transduction role of iPLA(2)beta in the native beta-cell might be attributable to a 70 kDa isoform of iPLA(2)beta.
Ramanadham
Sasanka
S
Mass Spectrometry Resource, Division of Endocrinology, Diabetes, and Metabolism, Washington University School of Medicine, St. Louis, Missouri 63110, USA. sramanad@im.wustl.edu
Song
Haowei
H
Hsu
Fong-Fu
FF
Zhang
Sheng
S
Crankshaw
Mark
M
Grant
Gregory A
GA
Newgard
Christopher B
CB
Bao
Shunzhong
S
Ma
Zhongmin
Z
Turk
John
J
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000737:chemistry; D001483:Base Sequence; D002118:Calcium / Q000737:chemistry / Q000502:physiology; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D005947:Glucose / Q000737:chemistry*; D054513:Group IV Phospholipases A2; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma / Q000201:enzymology* / Q000378:metabolism; D007515:Islets of Langerhans / Q000201:enzymology* / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009281:Naphthalenes / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics*; D054467:Phospholipases A2; D011753:Pyrones / Q000737:chemistry; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007328:Insulin; D007527:Isoenzymes; D009281:Naphthalenes; D010713:Phosphatidylcholines; D011753:Pyrones; D012333:RNA, Messenger; D016718:Arachidonic Acid; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; D054467:Phospholipases A2; D005947:Glucose; D002118:Calcium