Several model systems were investigated to elucidate the mechanisms by which rat liver phosphatidylcholine synthesis is controlled. CTP: phosphocholine cytidylyltransferase was clearly the key regulatory enzyme for phosphatidylcholine formation from choline. This ambiquitous enzyme was detected in both the cytosolic and microsomal fractions of rat liver, although the majority of the cytidylyltransferase occurred in the soluble fraction. The distribution of cytidylyltransferase between these fractions was altered when the rate of phosphatidylcholine synthesis was perturbed. Translocation of cytidylyltransferase was observed in rat liver during early development, with starvation and during a diurnal rhythm. A redistribution of cytidylyltransferase was also detected in isolated hepatocytes which were treated with glucagon, cAMP analogues or fatty acids bound to albumin. The rate of phosphatidylcholine synthesis was found to reflect the amount of microsomal cytidylyltransferase activity. The inhibition of phosphatidylcholine synthesis by glucagon or cAMP analogues was likely due to phosphorylation and inhibition of the cytidylyltransferase. Several lines of evidence indicated that the cytidylyltransferase in fresh rat liver cytosol was probably phosphorylated and activated upon dephosphorylation by endogenous phosphoprotein phosphatases or alkaline phosphatase from hog intestine. Although the phosphorylation of cytidylyltransferase was apparently kinetically "silent", dephosphorylation resulted in an increased affinity of the enzyme for membranes. Fatty acids stimulated de novo phosphatidylcholine synthesis by acceleration of the cytidylyl-transferase-catalyzed reaction. Fatty acids and their CoA derivatives were shown to stimulate the cytosolic cytidylyltransferase activity. However, these compounds failed to activate partially purified cytidylyltransferase appreciably. Apparently, fatty acids, like dephosphorylation, enhanced the tenacity of cytidylyltransferase for membranes. Upon binding to membranes, cytidylyltransferase activity could be elevated up to 45-fold, and the affinity of the enzyme for the substrate, CTP, was increased 20-fold. The influence of glucagon, cAMP analogues and fatty acids on the synthesis of phosphatidylcholine by successive N-methylation was also examined in isolated rat hepatocytes. Glucagon and cAMP analogues inhibited the methylation pathway in these cells, but the activity of microsomal phosphatidylethanolamine methyltransferase was elevated. Fatty acids also reduced the formation of phosphatidylcholine from phosphatidylethanolamine. Fatty acids and their CoA derivatives directly inhibited the phosphatidylethanolamine methyltransferase in rat liver microsomes. The coordinate control of hepatic phosphatidylcholine synthesis by cAMP and fatty acids may be important during starvation when the intracellular levels of these compounds are increased. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Young male rats fed a choline-deficient diet for three days accumulated triacylglycerol (TG) in the liver, and had reduced very low density lipoprotein (VLDL), but not high density lipoprotein (HDL), levels in the plasma. Cultured hepatocytes obtained from these rats were used as a model system to investigate how choline deficiency affected hepatic lipogenesis, apolipoprotein synthesis and lipoprotein secretion. When the cells were cultured in a medium free of choline and methionine, the secretion of TG and phosphatidylcholine (PC) was impaired. Supplementation of choline, methionine or lysophosphatidylcholine (lysoPC) to the culture medium increased the secretion of these lipids to normal levels, and stimulated PC biosynthesis. Fractionation of the secreted lipoproteins by ultracentrifugation revealed that the reduced secretion of TG and PC from choline-deficient cells was mainly due to impaired secretion of VLDL. The secretion of HDL and lipid-free proteins (for example albumin), however, was not affected by choline and methionine deficiency. Supplementation of betaine and homocysteine also stimulated PC biosynthesis and enhanced hepatic VLDL secretion. However, supplementation of ethanolamine, N-monomethylethanol-amine or N, N-dimethylethanolamine did not correct the impaired VLDL secretion from the hepatocytes, although an active synthesis of phosphatidylmonomethyl-ethanolamine (PMME) and phosphatidyldimethylethanolamine was observed. Choline deficiency had no effect on the rate of incorporation of [³H]leucine into cellular apolipoprotein B, E and C or on the rate of disappearance of radioactivity from the labeled apolipoproteins. These results suggest that biosynthesis of PC is specifically required for hepatic VLDL (but not HDL) secretion, and any one of the three synthetic pathways, the CDP-choline pathway, methylation of phospha-tidylethanolamine (PE) or reacylation of lysoPC, is sufficient to provide the required PC. The total activity of cytidylyltransferase in liver was unchanged in choline deficiency. However, choline deficiency caused an abnormal distribution of cytidylyltransferase activity between rat liver cytosol and microsomes (mainly endoplasmic reticulum), a decrease in the cytosolic enzyme activity and an increase in the microsomal enzyme activity. In cultured hepatocytes from the choline-deficient rat, the abnormally distributed cytidylyltransferase activity could be rapidly reversed by the addition of choline, but not lysoPC, to the culture medium. The stimulated microsomal activity of cytidylyltransferase during choline deficiency might be a mechanism whereby the cells could more effectively utilize phosphocholine to maintain a normal CDP-choline level in the choline-deficient liver. Rat liver PE N-methyltransferase catalyzes all three transmethylation reactions in the conversion of PE to PC. The in vitro activity of PE N-methyltransferase was increased in choline-deficient livers using endogenous PE as the methyl group acceptor. However, no significant changes were observed in the enzyme activity when exogenous PMME was used as the methyl group acceptor. Addition of methionine to the cultured hepatocytes obtained from choline-deficient rats resulted in a concomitant reduction in cellular PE levels and the specific activity of PE-dependent methyltransferase. However, the specific activity of PMME-dependent methyltransferase was not significantly altered upon the addition of methionine. No change in PE N-methyltransferase activity was observed in the cultured hepatocytes supplemented with choline. Immunoblotting of PE N-methyltransferase, in crude liver microsomes and in membrane fractions of cultured hepatocytes, revealed that the enzyme mass was not altered by choline and methionine deficiency. Thus, hepatic PE N-methyltransferase is preserved in choline deficiency, and its activity is probably dependent on the availability of metabolic substrates (i.e. methionine and PE). / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate