Choline deficiency causes translocation of CTP:phosphocholine cytidylyltransferase from cytosol to endoplasmic reticulum in rat liver

The choline-deficient rat liver has been chosen as a physiologically relevant model system in which to study the regulation of phosphatidylcholine biosynthesis. When 50-g rats were placed on a choline-deficient diet for 3 days, the activity of CTP:phosphocholine cytidylyltransferase (CT) was increased 2-fold in the microsomes and decreased proportionately in the cytosol. A low titer antibody to CT was obtained from chickens and used to identify the amount of CT protein in cytosol from rat liver. The amount of CT recovered from the choline-deficient cytosol was significantly less than in cytosol from choline-supplemented rats. When hepatocytes were prepared from choline-deficient livers, supplementation of the medium of the cells with choline caused CT to move from the membranes to cytosol within 1-2 h. The activity of another translocatable enzyme of glycerolipid metabolism, phosphatidate phosphohydrolase, was unchanged in cytosol from choline-deficient rat livers, and the microsomal activity of this enzyme was only minimally increased. When the livers were fractionated into endoplasmic reticulum and Golgi, there was a 2-fold increase in the activity on the endoplasmic reticulum from choline-deficient livers but no change in activity associated with Golgi. Thus, the increased association of CT with endoplasmic reticulum in choline-deficient livers appears to be specific to that subcellular fraction, and the subcellular location of other enzymes may not be affected.     

Evidence that the rate of phosphatidylcholine catabolism is regulated in cultured rat hepatocytes

The regulation of phosphatidylcholine (PC) catabolism has been studied in choline-deficient rat hepatocytes. Supplementation of choline-deficient hepatocytes, prelabeled with [3H]choline, with 100 microM choline increased the rate of PC catabolism by approx. 2-fold. The major product of PC degradation was glycerophosphocholine in both choline-deficient and choline-supplemented cells. Choline supplementation decreased the radioactivity recovered in lysoPC by 50%. This effect was accompanied by a 2-fold increase of labeled glycerophosphocholine. Comparable results were obtained when PC of the cells was prelabeled with [3H]methionine or [3H]glycerol. The activity of phospholipase A in cytosol, mitochondria and microsomes isolated from choline-deficient rat liver was similar to the activity in control liver, when determined with [3H]PC vesicles as the substrate. Measurement of the activity of phospholipase A with endogenously [3H]choline-labeled PC showed that the formation of lysoPC in mitochondria isolated form choline-supplemented cells was 40% lower than in choline-deficient cells. Alternatively, the formation of [3H]glycerophosphocholine and [3H]choline in microsomes from choline-supplemented cells was significantly higher (1.4-fold) than in microsomes from choline-deficient cells. These results suggest that the rate of PC catabolism is regulated in rat hepatocytes and that the concentration of PC might be an important regulatory factor.     

Effect of sodium chloride concentration on fluid-phase assembly and stability of the C3 convertase of the classical pathway of the complement system.

The specificity of the phospholipid head-group for feedback regulation of CTP: phosphocholine cytidylyltransferase was examined in rat hepatocytes. In choline-deficient cells there is a 2-fold increase in binding of cytidylyltransferase to cellular membranes, compared with choline-supplemented cells. Supplementation of choline-deficient cells with choline, dimethylethanolamine, monomethylethanolamine or ethanolamine resulted in an increase in the concentration of the corresponding phospholipid. Release of cytidylyltransferase into cytosol was only observed in hepatocytes supplemented with choline or dimethylethanolamine. The apparent EC50 values (concn. giving half of maximal effect) for cytidylyltransferase translocation were similar for choline and dimethylethanolamine (25 and 27 microM respectively). The maximum amount of cytidylyltransferase released into cytosol with choline supplementation (1.13 m-units/mg membrane protein) was twice that (0.62) observed with dimethylethanolamine. Supplementation of choline-deficient hepatocytes with NN'-diethylethanolamine, N-ethylethanolamine or 3-aminopropanol also did not cause release of cytidylyltransferase from cellular membranes. The translocation of cytidylyltransferase appeared to be mediated by the concentration of phosphatidylcholine in the membranes and not the ratio of phosphatidylcholine to phosphatidylethanolamine. The results provide further evidence for feedback regulation of phosphatidylcholine biosynthesis by phosphatidylcholine.     

Phosphatidylethanolamine levels and regulation of phosphatidylethanolamine N-methyltransferase

The activity of phosphatidylethanolamine (PE) N-methyltransferase in liver microsomes, measured using endogenous microsomal PE as a substrate, was elevated 2-fold in the choline-deficient state. However, methyltransferase activity assayed in the presence of a saturating concentration of phosphatidyl-N-mono-methylethanolamine or microsomal PE was unchanged by choline deficiency. Accompanying the increase in methyltransferase activity in liver homogenates and microsomes were increased PE concentrations and an increased PE to phosphatidylcholine ratio. The concentration of other phospholipids was unchanged. Immunoblot analysis of choline-deficient and choline-supplemented rat liver microsomes using a rabbit polyclonal anti-PE N-methyltransferase antibody revealed that the amount of enzyme protein was unaltered. The regulation of methyltransferase by PE levels was also investigated in cultured hepatocytes obtained from choline-deficient rat livers. Supplementation of deficient hepatocytes with 200 microM methionine resulted in a 50% reduction in cellular PE levels over a 12-h period. PE N-methyltransferase activity assayed with endogenous PE was also reduced by 50%, but phosphatidyl-N-monomethylethanolamine-dependent activity was unchanged. A 4-h supplementation with choline did not affect PE levels or methyltransferase activity. Either methionine or choline supplementation resulted in net synthesis of cellular phosphatidylcholine. Immunoblotting of membranes from methionine-supplemented hepatocytes revealed no change in enzyme protein, a further indication that enzyme mass was constitutive, and activity was regulated by the concentration of PE.     

Evidence that remodeling of the fatty acids of phosphatidylcholine is regulated in isolated rat hepatocytes and involves both the sn-1 and sn-2 positions

The remodeling of the fatty acyl moieties of phosphatidylcholine (PC) has been studied in choline-deficient and choline-supplemented hepatocytes prepared from a choline-deficient rat. Choline-deficient hepatocytes were prelabeled with [Me-3H]choline for 30 min and subsequently incubated for up to 12 h in the presence or absence of choline. Analysis of the molecular species of PC from choline-deficient cells showed that, at the end of the pulse, approx. 75% of the label was incorporated into palmitate-containing species and only approx. 16% of the labeled species contained stearate. During the chase period there was a redistribution of label and after 12 h approx. 56% of the total radioactivity was associated with palmitate containing species and 37% was recovered in stearate-containing species. A similar distribution of radioactivity was observed in choline-supplemented cells. Measurement of the specific radioactivity of the major molecular species of PC was consistent with a precursor-product relationship between palmitate-containing species and stearate-containing species with arachidonate or linoleate on the sn-2 position. A model is presented which takes into account remodeling of both the sn-1 and sn-2 positions of PC.     

LECITHIN SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION IN RAT LIVER : IV. A Radioautographic and Biochemical Study of Choline-Deficient Rats Injected with Choline-3H

Injection of choline-³H into choline-deficient rats resulted in an enhanced incorporation of the label into liver lecithin, as compared to the incorporation of label into liver lecithin of normal rats. The results obtained with the use of different lecithin precursors indicate that in the intact liver cell, both in vivo and in vitro, exchange of choline with phosphatidyl-choline is not significant. The synthesis and secretion of lecithins by the choline-deficient liver compare favorably with the liver of choline-supplemented rats, when both are presented with labeled choline or lysolecithin as lecithin precursors. Radioautography of the choline-deficient liver shows that 5 min after injection of choline-³H the newly synthesized lecithin is found in the endoplasmic reticulum (62%), mitochondria (13%), and at the "cell boundary" (20%). The ratio of the specific activity of microsomal and mitochondrial lecithin, labeled with choline, glycerol, or linoleate, was 1.53 at 5 min after injection, but the ratio of the specific activity of phosphatidyl ethanolamine (PE), labeled with ethanolamine, was 5.3. These results indicate that lecithin and PE are synthesized mainly in the endoplasmic reticulum, and are transferred into mitochondria at different rates. The site of a precursor pool of bile lecithin was studied in the intact rat and in the perfused liver. Following labeling with choline-³H, microsomal lecithin isolated from perfused liver had a specific activity lower than that of bile lecithin, but the specific activity of microsomal linoleyl lecithin was comparable to that of bile lecithin between 30 and 90 min of perfusion. It is proposed that the site of the bile lecithin pool is located in the endoplasmic reticulum and that the pool consists mostly of linoleyl lecithin.     

A choline-deficient diet in mice inhibits neither the CDP-choline pathway for phosphatidylcholine synthesis in hepatocytes nor apolipoprotein B secretion

Phosphatidylcholine is a major component of very low density lipoproteins (VLDLs) secreted by the liver. Hepatic phosphatidylcholine is synthesized from choline via the CDP-choline pathway and from the phosphatidylethanolamine N-methyltransferase pathway. Elimination of the methyltransferase in male mice reduces hepatic VLDL secretion. Our objective was to determine whether inhibition of the CDP-choline pathway for phosphatidylcholine synthesis (by restricting the supply of choline) also impaired VLDL secretion. In mice fed a choline-deficient (CD), compared with a choline-supplemented, diet for 21 days, the amounts of plasma apolipoproteins (apo) B100 and B48 were reduced and the liver triacylglycerol content was increased. Hepatocytes were isolated from male mice that had been fed the CD diet for 3 or 21 days, and the cells were incubated with or without choline. The secretion of apoB100 and B48 from CD hepatocytes was not reduced, and triacylglycerol secretion was only modestly decreased, compared with that from cells supplemented with choline. Remarkably, in light of widely held assumptions, the rate of phosphatidylcholine synthesis from the CDP-choline pathway was not decreased in CD hepatocytes. Rather, there was a trend toward increased phosphatidylcholine synthesis that might be explained by enhanced CTP:phosphocholine cytidylyltransferase activity. Although the concentration of phosphocholine in CD hepatocytes was reduced, the size of the phosphocholine pool remained well above the K for the cytidylyltransferase. Moreover, the amount and m activity of the cytidylyltransferase and methyltransferase were increased. The reduction in plasma apoB in mice deprived of dietary choline cannot, therefore, be attributed to decreased apoB secretion.     

Effects of chronic (dietary) choline availability on the transport of choline across the blood-brain barrier

The effects of dietary choline availability on the transport of choline across the blood-brain barrier (BBB) were investigated using the intracarotid injection technique. Maintenance of rats on choline-deficient, basal choline, or choline-supplemented diets for 28-32 days led to respective increases in blood levels of choline and correlative increases in the velocity of transport of choline measured using a buffer injectate. When serum from these rats was included in the injectate and transport determined in control animals, there was a marked inhibition of choline transport that was related to the concentration of choline in the diets. Results suggest that the activity of the choline carrier at the BBB is antagonized by an inhibitory substance in serum whose concentration or activity may be modified by chronic alterations in circulating levels of choline and whose presence may normally regulate the velocity of choline transport.     

Subcellular distribution and regulation of hepatic bilirubin UDP-glucuronyltransferase

We have investigated the subcellular location and regulation of hepatic bilirubin UDP-glucuronyltransferase, which has been presumed to be located largely in the smooth endoplasmic reticulum. Purity of subcellular membrane fractions isolated from rat liver was assessed by electron microscopy and marker enzymes. Bilirubin UDP-glucuronyltransferase activity was measured by radiochemical assay using a physiologic concentration of [14C]bilirubin, and formation rates of bilirubin diglucuronide and monoglucuronides (C-8 and C-12 isomers) were determined. Activity of the enzyme was widely distributed in subcellular membranes, the majority being found in smooth and rough endoplasmic reticulum, with small amounts in nuclear envelope and Golgi membranes. No measurable activity was found in plasma membranes or in cytosol. Synthesis of bilirubin diglucuronide as a percentage of total conjugates and the ratio of C-8/C-12 bilirubin monoglucuronide isomers formed were comparable in all membranes, suggesting that the same enzyme is present in all locations. However, the regulation of bilirubin UDP-glucuronyltransferase activity differed among intracellular membranes; enzyme activity measured in the presence of the allosteric effector uridine 5'-diphospho-N-acetylglucosamine exhibited latency in smooth endoplasmic reticulum and Golgi membranes, but not in rough endoplasmic reticulum and nuclear envelope. Since rough membranes comprise 60% of hepatocyte endoplasmic reticulum and bilirubin UDP-glucuronyltransferase activity in vitro is maximal in this membrane fraction under presumed physiologic conditions, it is likely that the rough endoplasmic reticulum represents the major site of bilirubin glucuronidation in hepatocytes.     

Stimulation of sphingomyelin biosynthesis by brefeldin A and sphingomyelin breakdown by okadaic acid treatment of rat hepatocytes.

Studies on sphingomyelin metabolism in rat hepatocytes were facilitated by the use of choline-deficient cells which allowed for the rapid labeling of phosphatidylcholine and as a result sphingomyelin. Pulse and pulse-chase studies with [methyl-3H]choline and [methyl-3H]methionine demonstrated that both compounds were effectively used for sphingomyelin biosynthesis and that newly made and pre-existing phosphatidylcholine could be used for sphingomyelin biosynthesis. When hepatocytes were incubated with brefeldin A, there was a 2.4-fold stimulation of the conversion of phosphatidylcholine into sphingomyelin. Since brefeldin A causes collapse of the cis/medial Golgi into the endoplasmic reticulum the stimulation of sphingomyelin biosynthesis could be due to more rapid access of the labeled phosphatidylcholine in the endoplasmic reticulum to sphingomyelin synthase in the collapsed Golgi. Forskolin inhibited the brefeldin A-induced stimulation of sphingomyelin biosynthesis. To investigate whether or not phosphorylation reactions regulate sphingomyelin metabolism, hepatocytes were incubated with okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A. Rather than stimulating sphingomyelin biosynthesis, okadaic acid enhanced the catabolism of sphingomyelin. In contrast, a cyclic AMP analogue and forskolin had no effect on sphingomyelin biosynthesis or catabolism. Surprisingly, other pulse-chase studies demonstrated that okadaic acid stimulated the catabolism of only newly made sphingomyelin. The brefeldin A and okadaic acid effects were independent of lysosomal involvement. Subcellular fractionation studies revealed that brefeldin A and okadaic acid effects were generalized in all sphingomyelin containing membranes. The brefeldin A studies suggest that the rate of transfer of phosphatidylcholine from the endoplasmic reticulum to the Golgi might be limiting for sphingomyelin biosynthesis. The okadaic acid studies indicate that the catabolism of sphingomyelin by a sphingomyelinase is regulated by an unidentified protein kinase and by either protein phosphatase 1 and/or 2A activity in hepatocytes.     

Head group specificity in the regulation of phosphatidylcholine catabolism in rat hepatocytes

Previous studies have shown that the catabolism of PC is regulated in choline-deficient hepatocytes and the concentration of phosphatidylcholine (PC) might be an important regulatory factor (Tijburg, L.B.M., Nishimaki-Mogami, T. and Vance, D.E. (1991) Biochim. Biophys. Acta, 1085, 167-177). In the present study we investigated the head group specificity of the regulation of PC catabolism. Supplementation of choline-deficient rat hepatocytes, prelabeled with [3H]choline, with dimethylethanolamine increased the catabolism of PC by 1.6-fold after 6 h. This effect was accompanied by a 2.5-fold increase in the production of [3H]glycerophosphocholine (GPC). Radioactivity associated with lysoPC was decreased by 50% in dimethylethanolamine-treated cells. Supplementation of the cells with monomethylethanolamine had little effect on the degradation of PC. In other experiments choline-deficient cells were prelabeled with [3H]methionine. Treatment of the cells with dimethylethanolamine increased the formation of [3H]GPC by 5-fold, while the production of lysoPC was inhibited by 60%. Supplementation of the medium with monomethylethanolamine resulted in a 2-fold increase in labeled GPC, with a concomitant decrease of [3H]lysoPC by approx. 25%. We conclude that the formation of phosphatidyldimethylethanolamine from its corresponding base mimics the effect of the synthesis of PC from choline in increasing PC catabolism, whereas the effect of monomethylethanolamine is much less pronounced.     

Intracellular processing of cytidylyltransferase in Krebs II cells during stimulation of phosphatidylcholine synthesis. Evidence that a plasma membrane modification promotes enzyme translocation specifically to the endoplasmic reticulum

After a 3-h incubation of Krebs II ascitic cells in the presence of phospholipase C from Clostridium welchii under nonlytic conditions, the incorporation of [3H] choline into phosphatidylcholine was increased 1.7-fold as compared to untreated cells. The total amounts of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were unchanged up to 3 h of incubation. The limiting step in phosphatidylcholine biosynthesis was the formation of CDP-choline catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) as monitored by the decrease in phosphocholine labeling following phospholipase C treatment of cells prelabeled with [3H]choline. The specific activity of homogenate cytidylyltransferase was increased about 1.6-fold in phospholipase C-treated cells. Specific activity of the membrane fraction was increased 2-fold, whereas cytosolic specific activity decreased in phospholipase C-treated cells. The activation of cytidylyltransferase was concomitant with translocation of the enzyme from the cytosol to the membrane fraction. The latter was further fractionated using a Percoll gradient that allowed an efficient separation between endoplasmic reticulum and other subcellular membranes. In control cells, particulate cytidylyltransferase activity co-migrated with the endoplasmic reticulum and ribosome markers and not with the plasma membrane. Also, in treated cells, the stimulation of cytidylyltransferase activity occurred at the endoplasmic reticulum level and did not involve either the external cell membrane or other cellular organelles including the Golgi apparatus, lysosomes, or mitochondria. Thus, our results demonstrate that a stimulus acting on the plasma membrane promotes the translocation of the soluble form of cytidylyltransferase specifically to the endoplasmic reticulum.     

Environmental temperature and choline requirement in rats. I. Choline deficiency in rats at various temperatures

Rats were maintained at 2 degrees, 21 degrees, and 33 degrees for 3 weeks on a choline-supplemented or a choline-deficient diet. In contrast to the findings of some other workers, choline deficiency produced fatty livers at all temperatures. The ratio of the total liver lipid to the total food intake was the same in all choline-supplemented rats. In choline-deficient rats this ratio was always higher and varied directly with temperature.     

Head group specificity in the requirement of phosphatidylcholine biosynthesis for very low density lipoprotein secretion from cultured hepatocytes

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.     

Influence of Dietary Choline Availability and Neuronal Demand on Acetylcholine Synthesis by Rat Brain

The main objective of this study was to test the hypothesis that the chronic administration of choline supplements a bound pool of choline from which free choline can be mobilized and used to support acetylcholine synthesis when the demand for precursor is increased. For these experiments, brain slices from rats fed diets containing different amounts of choline were incubated in a choline-free buffer and acetylcholine synthesis was measured under resting conditions and in the presence of K+-induced increases in acetylcholine synthesis and release. Rats fed the choline-supplemented diet had circulating choline levels that were 52% greater than the controls, and striatal and cerebral cortical slices from this group produced significantly more free choline during the incubation than slices from the controls. However, the synthesis and release of acetylcholine by these tissues did not differ from those by controls, during either resting or K+-evoked conditions. In contrast, acetylcholine synthesis and release by striatal and hippocampal slices from choline-deficient rats, animals that had circulating choline levels that were 80% of control values, decreased significantly; the production of free choline by these tissues was also depressed. Results indicate that, despite an increased production of free choline by brain slices from choline-supplemented rats, the synthesis of acetylcholine was unaltered, even in the presence of an increased neuronal demand. In contrast, the choline-deficient diet led to a decreased release of free choline from bound stores and an impaired ability of brain to synthesize acetylcholine.     

Studies on vinblastine-induced autophagocytosis in mouse liver. IV. Origin of membranes

The origin of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied by cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). The marker enzymes used were adenosine triphosphatase for the plasma membrane, glucose-6-phosphatase for the endoplasmic reticulum and thiamine pyrophosphatase for the Golgi apparatus and the endoplasmic reticulum. All the three enzymes showed a characteristic localization in both control and vinblastine-treated hepatocytes. The space between the limiting membranes of a few apparently newly formed AV's showed weak glucose-6-phosphatase activity. Neither adenosine triphosphatase nor thiamine pyrophosphatase activities were observed on or between the AV membranes. It was suggested that endoplasmic reticulum membranes may be used as a source of AV membranes in hepatocytes. The lack of glucose-6-phosphatase activity in the limiting membranes even of most of the newly formed AV's suggests a transformation process of the membranes destined to form AV, during which the enzyme activity characteristic for endoplasmic reticulum may disappear from them.     

Studies on vinblastine-induced autophagocytosis in mouse liver

Summary The origin of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied by cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). The marker enzymes used were adenosine triphosphatase for the plasma membrane, glucose-6-phosphatase for the endoplasmic reticulum and thiamine pyrophosphatase for the Golgi apparatus and the endoplasmic reticulum. All the three enzymes showed a characteristic localization in both control and vinblastine-treated hepatocytes. The space between the limiting membranes of a few apparently newly formed AV's showed weak glucose-6-phosphatase activity. Neither adenosine triphosphatase nor thiamine pyrophosphatase activities were observed on or between the AV membranes. It was suggested that endoplasmic reticulum membranes may be used as a source of AV membranes in hepatocytes. The lack of glucose-6-phosphatase activity in the limiting membranes even of most of the newly formed AV's suggests a transformation process of the membranes destined to form AV, during which the enzyme activity characteristic for endoplasmic reticulum may disappear from them.     

Phosphatidylcholine and choline homeostasis

Phosphatidylcholine (PC) is made in mammalian cells from choline via the CDP-choline pathway. Animals obtain choline primarily from the diet or from the conversion of phosphatidylethanolamine (PE) to PC followed by catabolism to choline. The main fate of choline is the synthesis of PC. In addition, choline is oxidized to betaine in kidney and liver and converted to acetylcholine in the nervous system. Mice that lack choline kinase (CK) alpha die during embryogenesis, whereas mice that lack CKbeta unexpectedly develop muscular dystrophy. Mice that lack CTP:phosphocholine cytidylyltransferase (CT) alpha also die during early embryogenesis, whereas mice that lack CTbeta exhibit gonadal dysfunction. The cytidylyltransferase beta isoform also plays a role in the branching of axons of neurons. An alternative PC biosynthetic pathway in the liver uses phosphatidylethanolamine N-methyltransferase to catalyze the formation of PC from PE. Mice that lack the methyltransferase survive but die from steatohepatitis and liver failure when placed on a choline-deficient diet. Hence, choline is an essential nutrient. PC biosynthesis is required for normal very low density lipoprotein secretion from hepatocytes. Recent studies indicate that choline is recycled in the liver and redistributed from kidney, lung, and intestine to liver and brain when choline supply is attenuated.     

On the mechanism of the okadaic acid-induced inhibition of phosphatidylcholine biosynthesis in isolated rat hepatocytes.

The mechanism of inhibition of phosphatidylcholine biosynthesis by okadaic acid was investigated in suspension cultures of isolated rat hepatocytes. Cells were pulsed with [methyl-3H]choline and chased in the absence or presence of 1 microM okadaic acid for up to 120 min. Phosphatidylcholine biosynthesis was inhibited after 15 min of chase. To see if okadaic acid altered the degree of phosphorylation of cytidylyltransferase (CT), hepatocytes were incubated with 32P(i) and chased in the absence or presence of okadaic acid. Okadaic acid caused a rapid (within 15 min) increase in the phosphorylation state of the cytosolic enzyme. Two-dimensional peptide map analysis revealed an increase in the phosphorylation of several peptides in okadaic acid-treated hepatocytes compared with controls. After 15 min of incubation of hepatocytes with okadaic acid, membrane CT activity was decreased and a corresponding increase in cytosolic CT activity was observed. In hepatocytes incubated with okadaic acid and oleate a correlation between membrane CT activity, diacylglycerol level, and phosphatidylcholine biosynthesis was observed. These data suggest that the concentration of diacylglycerol is responsible for the increase in membrane CT activity and subsequently phosphatidylcholine biosynthesis in oleate-treated cells. We postulate that the okadaic acid-induced decrease in phosphatidylcholine biosynthesis is due to an increase in the phosphorylation state of CT which promotes a translocation of CT activity from the membranes to the cytosol.