Abstract
In McA-RH7777 cells, the oleate-stimulated assembly and secretion of very low density lipoproteins (VLDL) was associated with enhanced deacylation of phospholipids, which was markedly decreased by inactivation of the cellular phospholipase A(2). Treatment of the cells with antagonists or antisense oligonucleotide of the Ca(2+)-independent phospholipase A(2) (iPLA(2)) significantly inhibited secretion of apoB100-VLDL and triglyceride. Similar inhibitory effect of the iPLA(2) antagonists was observed on apoB48-VLDL secretion, but secretion of high density lipoprotein particles (such as apoAI- and apoB48-high density lipoprotein) or proteins in general was unaffected. The iPLA(2) antagonist did not affect the synthesis of apoB100 or triglyceride, nor did it affect the activities of phospholipase D, phosphatidate phosphohydrolase, or microsomal triglyceride transfer protein. Inactivation of iPLA(2) resulted in impaired apoB100-VLDL assembly as shown by decreased apoB100-VLDL and triglyceride within the microsomal lumen, with concomitant increase in apoB100 association with the microsomal membranes. The inhibitory effect of iPLA(2) antagonists on apoB100-VLDL assembly/secretion could be abated by pretreatment of cells with oleate. Analysis of molecular species of microsomal phosphatidylcholine and phosphatidylethanolamine by electron spray tandem mass spectrometry revealed that the enrichment of oleoyl moieties was altered by the treatment of iPLA(2) antagonist. These results suggest that the oleate-induced VLDL assembly/secretion may depend upon the establishment of membrane glycerolipids enriched in oleoyl chain, a process mediated by the iPLA(2) activity.
Highlights
The post-translational model for very low density lipoproteins (VLDL) assembly is established on the basis of kinetic analysis that observes the time taken by apoB and TG to be assembled
Analysis of molecular species of microsomal phosphatidylcholine and phosphatidylethanolamine by electron spray tandem mass spectrometry revealed that the enrichment of oleoyl moieties was altered by the treatment of independent phospholipase A2 (iPLA2) antagonist. These results suggest that the oleate-induced VLDL assembly/secretion may depend upon the establishment of membrane glycerolipids enriched in oleoyl chain, a process mediated by the iPLA2 activity
Deacylation—We used cumulative flotation ultracentrifugation technique [40] to resolve B100-VLDL1 (Sf Ͼ100) and B100VLDL2 (Sf 20 –100) from intermediate density lipoproteins (IDL)/LDL and high density lipoproteins (HDL) that were synthesized by human apoB100 (hB100)-transfected McA-RH7777 cells
Summary
Materials—[35S]Methionine/cysteine (1000 Ci/mmol), 2-[3H]palmitoyl-phosphatidylcholine (PC) (89 Ci/mmol), [3H]oleic acid (9 Ci/mmol), [3H]glycerol (1.1 Ci/mmol), [3H]myristic acid (53 Ci/mmol), [14C]triolein (57 mCi/mmol), [3H]choline chloride (40 mCi/mmol), and [14C]phosphatidic acid (100 –200 mCi/mmol) were purchased from Amersham Pharmacia Biotech and NEN Life Science Products. Cells were treated with 0 –100 M PLA2 antagonists (i.e. BEL or MAFP) for 30 min and labeled with 100 Ci/ml [35S]methionine/cysteine for up to 2 h in a methionine- and cysteine-free DMEM containing 20% fetal bovine serum and 0.4 mM oleate. Enzyme Assays—For PLA2, phosphatidate phosphohydrolase (PAPH), and MTP activity assays, cells were treated with BEL (100 M) for 30 min, followed by incubation with oleate (0.4 mM) plus BEL (100 M) for 1 h. Thin Layer Chromatography (TLC)—Lipids in total cells, subcellular fractions, or enzyme assay mixtures (e.g. PLA2 and PLD) were extracted with chloroform/methanol/acetic acid/saturated NaCl/H2O (4:2: 0.01:1:2; by volume), and resolved by TLC using Silica Gel 60 plates. Both were synthesized by Life Technologies, Inc. Cells (20% confluence) were incubated with 10 M oligonucleotides (freshly prepared prior to use) in serum-free DMEM for 4 h. The fatty acid composition of each molecular species was determined by daughter ion analysis in the negative ion mode
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