Mass spectrometric characterization of arachidonate-containing plasmalogens in human pancreatic islets and in rat islet .beta.-cells and subcellular membranes

Abstract

Pancreatic islets, when stimulated with D-glucose, secrete insulin by processes requiring glycolytic metabolism and generation of ATP. Hydrolysis of membrane phospholipids also occurs in glucose-stimulated islets, resulting in accumulation of nonesterified arachidonate, which facilitates Ca2+ entry and the rise in beta-cell [Ca2+] that triggers insulin secretion. Glucose-induced hydrolysis of arachidonate from islet phospholipids is mediated in part by an ATP-stimulated, Ca(2+)-independent (ASCI) phospholipase A2 (PLA2) which prefers plasmenyl over diacyl phospholipid substrates. Here we characterize the endogenous plasmalogen content of islet cells and subcellular membranes. Fast atom bombardment mass spectrometric analyses demonstrated that three of the most abundant molecular species of ethanolamine phospholipids in rat pancreatic islets were plasmalogens with sn-2 arachidonate residues and palmitic, oleic, or stearic aldehyde residues, respectively, in the sn-1 position. Purified populations of beta-cells prepared by fluorescence-activated cell sorting were also found to contain these plasmenylethanolamine molecular species in abundance similar to that in intact islets and greater than that in islet alpha-cells. Both islet plasma membranes (PM) and endoplasmic reticulum (ER) also contained these plasmenylethanolamine species, which accounted for 42% (PM) to 64% (ER) of the ethanolamine phospholipid arachidonate content of these membranes, as measured by stable isotope dilution mass spectrometry. Plasmenylethanolamine species were also abundant constituents of human pancreatic islets (accounting for 58% of their ethanolamine phospholipid arachidonate content) and were hydrolyzed more rapidly than diacyl ethanolamine phospholipid by human islet cytosolic ASCI-PLA2. Both secretagogue-induced eicosanoid release and insulin secretion from human islets were attenuated by an ASCI-PLA2 suicide substrate which sterically resembles plasmalogens. These observations are consistent with the hypotheses that islet beta-cell ASCI-PLA2-catalyzed hydrolysis of arachidonate from endogenous plasmenylethanolamine substrates may occur in membrane compartments which participate in regulation of the beta-cell cytosolic [Ca2+] and that this may be an intermediary biochemical event in the induction of insulin secretion.

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

“…Upon activation, iPLA 2 ␤ hydrolyzes the sn-2 substituent from phospholipids substrates, such as phosphatidylcholine, to yield a free fatty acid and a lysophospholipid, such as LPC (44,54,55). Because arachidonate is the most abundant sn-2 substituent in ␤-cell membrane phospholipids (23,25,26), activation of iPLA 2 ␤ upon stimulation of islets with glucose results in liberation of arachidonate as the preponderant free fatty acid (7). Stably transfected INS-1 cells that overexpress iPLA 2 ␤ (INS1-iPLA 2 ␤) exhibit amplified insulin secretory responses to glucose and other secretatogues (21,56).…”

“…The phospholipid content of esterified AA is higher in pancreatic islets than in other tissues (23,25,26,29), and arachidonate is hydrolyzed from phospholipids upon stimulation of islets with secretagogues (13,14,27,31,36,44) and accumulates to micromolar concentrations (7,27,28) that are sufficient to modulate delayed rectifier Kv channel FIGURE 5. Effects of the muscarinic agonist carbachol on INS-1 cell-delayed rectifier currents.…”

“…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).…”

Electrospray ionization/tandem quadrupole mass spectrometric studies on phosphatidylcholines: The fragmentation processes

“…P hospholipids are essential components of cellular membranes, and distinct molecular species are differentially distributed among tissue and within subcellular compartment [1][2][3][4]. Among the phospholipids found in mammalian cell membranes, phosphatidylcholine species are the most abundant constituents.…”