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Studies of phospholipid metabolism, proliferation, and secretion of stably transfected insulinoma cells that overexpress group VIA phospholipase A2

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Lipids

Abstract

A cytosolic 84 kDa Group VIA phospholipase A2 (iPLA2β) that does not require Ca2+ for catalysis was cloned from Chinese hamster ovary (CHO) cells, murine P388D1 cells, pancreatic islet β-cells, and other sources. Proposed iPLA2β functions include participation in phosphatidylcholine (PC) homeostasis by degrading excess PC generated in CHO cells that overexpress CTP:phosphocholine cytidylyltransferase (CT), which catalyzes the rate-limiting step in PC biosynthesis; participation in biosynthesis of arachidonate-containing PC species in P388D1 cells by generating lysophosphatidylcholine (IPC) acceptors for arachidonate incorporation; and participation in signaling events in insulin secretion from islet β-cells. To further examine iPLA2β functions in β-cells, we prepared stably transfected INS-1 insulinoma cell lines that overexpress iPLA2β activity eightfold compared to parental INS-1 cells or to INS-1 cells transfected with an empty retroviral vector that did not contain iPIA2β cDNA. The iPLA2β-overexpressing cells exhibit a twofold increase in CT activity compared to parental cells but little change in rates of [3H] choline incorporation into or disappearance from PC. Electrospray ionization (ESI) tandem mass spectrometric measurements indicate that iPLA2β-overexpressing cells have 1.5-fold higher LPC levels than parental INS-1 cells but do not exhibit increased rates of [3H]arachidonate incorporation into phospholipids, and incorporation is unaffected by a bromoenol lactone (BEL) suicide substrate inhibitor of iPLA2β. The rate of appearance of arachidonate-containing phosphatidylethanolamine species visualized by ESI mass spectrometry is also similar in iPLA2β-overexpressing and parental INS-1 cells incubated with supplemental arachidonic acid, and this process is unaffected by BEL. Compared to parental INS-1 cells, iPLA2β-overexpressing cells proliferate more rapidly and exhibit amplified insulin secretory responses to a protein kinase C-activating phorbol ester, glucose, and a cAMP analog. These findings suggest that iPLA2β plays a signaling role in β-cells that differs from housekeeping functions in PC biosynthesis and degradation in P388D1 and CHO cells.

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Abbreviations

BEL:

bromoenol lactone suicide substrate

BSA:

bovine serum albumin

CAD:

collisionally activated dissociation

CHO:

Chinese hamster ovary

cPLA:

Group IV phospholipase A2

CT:

CTP:phosphocholine cytidylyltransferase

dBcAMP:

dibutyryl cyclic AMP

dpm:

disintegration per minute

ESI:

electrospray ionization

FBS:

fetal bovine serum

HPLC:

high-performance liquid chromatography

iPLA2β:

Group VIA phospholipase A2

KRB:

Krebs-Ringer bicarbonate buffer

LPA:

lysophosphatidic acid

LPC:

lysophosphatidylcholine

MS:

mass spectrometry

MS/MS:

tandem mass spectrometry

NP-HPLC:

normal-phase high-performance liquid chromatography

PBS:

phosphate-buffered saline

PC:

phosphatidylcholine

PE:

phosphatidylethanolamine

PLA2 :

phospholipase A2

PMA:

phorbol myristate acetate

sPLA2 :

secretory phospholipase A2

TLC:

thin-layer chromatography

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Authors and Affiliations

  1. Mass Spectrometry Resource, Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Washington University School of Medicine, 660 S. Euclid Ave., St., Box 8127, 63110, Louis, MO

    Zhongmin Ma, Alan Bohrer, Mary Wohltmann, Sasanka Ramanadham, Fong-Fu Hsu & John Turk

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  1. Zhongmin Ma
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  2. Alan Bohrer
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  3. Mary Wohltmann
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  4. Sasanka Ramanadham
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  5. Fong-Fu Hsu
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  6. John Turk
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Correspondence to John Turk.

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Ma, Z., Bohrer, A., Wohltmann, M. et al. Studies of phospholipid metabolism, proliferation, and secretion of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 . Lipids 36, 689–700 (2001). https://doi.org/10.1007/s11745-001-0774-9

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  • DOI: https://doi.org/10.1007/s11745-001-0774-9

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