Abstract
We have demonstrated that hepatic very low density lipoprotein (VLDL) secretion requires active phosphatidylcholine (PC) synthesis via either the CDP-choline pathway or phosphatidylethanolamine (PE) methylation pathway (Yao, Z., and Vance, D.E. (1988) J. Biol. Chem. 263, 2998-3004). In the present work, the head group specificity of phospholipid synthesis required for lipoprotein secretion was investigated in cultured hepatocytes isolated from choline-deficient rats. When N-monomethylethanolamine (0.1 mM) or N,N-dimethylethanolamine (0.1 mM) was added to the culture medium, the cells synthesized correspondingly phosphatidylmonomethylethanolamine (PMME) or phosphatidyldimethylethanolamine (PDME). However, the synthesis of PDME could correct the impaired VLDL secretion only to a limited extent, whereas the synthesis of PMME inhibited VLDL secretion. Although dimethylethanolamine did not promote VLDL secretion as well as choline, dimethylethanolamine altered the increased triacylglycerol synthesis in the choline-deficient cells as effectively as choline. Supplementation of the culture medium with ethanolamine (0.1 mM) had little effect on cellular PE or PC levels, nor was normal VLDL secretion resumed. However, the amounts of cellular PC and PE were both decreased when the medium was supplemented with N-monomethylethanolamine or N,N-dimethylethanolamine. These results suggest that the choline head group moiety of PC is specifically required for normal VLDL secretion and cannot be replaced with ethanolamine, monomethylethanolamine, or dimethylethanolamine. In addition, the impaired VLDL secretion from the choline-deficient hepatocytes could also be corrected by supplementation of betaine (0.2 mM) and homocysteine (0.2 mM), indicating the utilization of a methyl group from betaine for PC formation via methylation of PE.
Highlights
We have demonstratedthat hepatic very low density major site for the formation and export of PC [2]
In the absence metabolic present in of choline nolamine or N,N-dimethylethanolamine.These results and methionine, rat hepatocytes synthesize significant suggest that the choline head group moiety of PC is amounts of phospholipids containing N-monomethylethanolspecifically required for normal VLDL secretion and amine or N,N-dimethylethanolaminefrom the corresponding cannot be replaced with ethanolamine, monomethyl- free bases [7], presumably via the cytidine diphosphate pathethanolamine, or dimethylethanolamine
Way used for PC and PEsynthesis [8].It has been speculated the impaired VLDL secretion from the choline-deficient hepatocytes could be corrected by supplementation of betaine (0.2 mM) and homocysteine (0.2 mM), indicating the utilizationof a methyl group from that the formation of CDP esters of the N-monomethylated andN,N-dimethylated bases is catalyzed by different enzymes, probably phosphoethanolamine cytidylyltransferase betaine for PC formation via methylatioofnPE
Summary
Preparation and Treatment of Hepatocytes-Male Sprague-Dawley Phospholipids in Hepatocytes-Hepatocytes obtained from rats (initially weighing 45-50 g) were fed a choline-deficient diet for 3 days. Hepatocytes were isolated, by collagenase perfusion, from a single rat and cultured in a choline- and methionine-free medium as described previously [5]. The viability the choline-deficient rats were incubated with various phospholipid bases, and the concentrations of four major cellular phospholipids (PC, PE, PMME, and PDME) during a 20-h incubation were determined (Fig. 1).Whenthe cells were of the cells incubated under different conditions was identical Preparation of [methyL3H]Betaine-[ methyL3H]Betainewas prepared enzymatically from [methyl-3H]cholineby treatment with choline oxidase and catalase [20]. Choline oxidase (1mg of protein) and catalase (50 pg of protein) were incubated with 200 pCi of [methyland 60 nmol/mg cell protein, respectively. The betaine was visualized with iodine, scraped, and eluted 15 times with 2 ml of methanol. The methanol was evaporated, and the sample was redissolved in 3 ml of Hz0 and stored at -20 “C
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