Relationship between the displacement of phosphatidate phosphohydrolase from the membrane-associated compartment by chlorpromazine and the inhibition of the synthesis of triacylglycerol and phosphatidylcholine in rat hepatocytes

Glycerolipid synthesis was studied in isolated hepatocytes by using 177 microM [14C]oleate and 1 mM [3H]glycerol. Chlorpromazine (25-400 microM) inhibited the synthesis of phosphatidylcholine and triacylglycerol. This was accompanied by an average increase of 12-fold in the accumulation of the labelled precursors in phosphatidate at 200 microM chlorpromazine and a decrease in the conversion of phosphatidate to diacylglycerol of 76%. These results indicate that part of the inhibition of the synthesis of phosphatidylcholine and triacylglycerol occurs at the level of phosphatidate phosphohydrolase. The relative rate of triacylglycerol synthesis at different concentrations of chlorpromazine was approximately proportional to the rate of conversion of phosphatidate to diacylglycerol. Phosphatidylcholine synthesis increased at higher rates of conversion of phosphatidate to diacylglycerol, but it was relatively independent of the latter rate when this was inhibited by more than about 30% with chlorpromazine. The addition of oleate to the hepatocytes caused a translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Chlorpromazine had the opposite effect and displaced the phosphohydrolase from the membranes in the presence or absence of oleate. There was a highly significant correlation between the activity of phosphatidate phosphohydrolase that was associated with the membranes of the hepatocytes and the calculated conversion of [3H]phosphatidate to diacylglycerol. Chlorpromazine also antagonized the association of the phosphohydrolase with microsomal membranes when cell-free preparations were incubated with combinations of oleate and spermine. Furthermore, it inhibited the transfer of the soluble phosphohydrolase to microsomal membranes that were labelled with [14C]phosphatidate and thereby decreased diacylglycerol production. It is concluded that part of the action of chlorpromazine in inhibiting the synthesis of triacylglycerol and phosphatidylcholine occurs because it prevents the interaction of the soluble phosphatidate phosphohydrolase with the membranes on which glycerolipid synthesis occurs. This in turn prevents the conversion of phosphatidate to diacylglycerol.     

The effects of cortisol, corticotropin and thyroxine on the synthesis of glycerolipids and on the phosphatidate phosphohydrolase activity in rat liver

1. Male rats were injected daily for 5 days with 0.15m-NaCl, corticotropin, cortisol or l-thyroxine and the rates of glycerolipid synthesis were measured in the livers after intraportal injection of [(14)C]palmitate and [(3)H]glycerol. 2. Injection of all three hormones decreased the rates of body-weight gain. 3. Cortisol treatment increased the weight of the liver relative to body weight. 4. Thyroxine treatment increased the relative rate of triacylglycerol synthesis from [(3)H]glycerol and decreased the relative accumulation of (3)H and (14)C in diacylglycerol. It did not significantly alter the accumulation of these isotopes in phosphatidate nor the activity of the soluble phosphatidate phosphohydrolase in the total liver. However, this activity increased by 1.5-fold when expressed relative to the soluble protein of the liver. The increased triacylglycerol synthesis appears to be related to a general increase in the turnover of fatty acids in the liver. 5. Treatment with cortisol and corticotropin increased the relative rate of triacylglycerol synthesis from [(3)H]glycerol, decreased the accumulation of (3)H in phosphatidate and increased the flux of both isotopes from phosphatidate to diacylglycerol. This appeared to be caused by the increased activity of the soluble phosphatidate phosphohydrolase that was observed in the livers of the cortisol-treated rats. 6. It is proposed that cortisol could be directly or indirectly involved in increasing the activity of hepatic phosphatidate phosphohydrolase in starvation, diabetes, laparotomy, subtotal hepatectomy, liver damage, ethanol feeding and in obesity. This enzyme adaptation could contribute to the potential of the liver to increase its synthesis and accumulation of triacylglycerols or to secrete very-low-density lipoproteins.     

Enzymatic aspects of the reduction of microsomal glycerolipid biosynthesis after perfusion of the liver with dibutyryl adenosine-3',5'-monophosphate.

Livers from fed male rats were perfused in a nonrecycling system for 60 min with a medium containing 100 mg/dl glucose, 3 g/dl bovine serum albumin, and ~0.5 mm oleic acid, with or without 20 μm dibutyryl cyclic adenosine-3′,5′-monophosphate (Bt₂cAMP). At the termination of the experiment, microsomes were isolated from these livers. In agreement with data reported previously, Bt₂cAMP decreased output of triacylglycerol, but stimulated ketogenesis and output of glucose; uptake of free fatty acid was unaffected by the nucleotide. Perfusion with Bt₂AMP decreased the biosynthesis of triacylglycerol, diacylglycerol, and phosphatidate from sn-[U-¹⁴C]glycerol-3-phosphate by microsomes isolated from these livers. Perfusion with Bt₂cAMP also decreased incorporation of sn-glycerol-3-phosphate into phosphatidate by microsomes isolated from the livers, when the microsomes were incubated with NaF to inhibit phosphatidate phosphohydrolase, and when fatty acid, coenzyme A and ATP were replaced by the acyl coenzyme A derivative; the formation of phosphatidate under these conditions was used as an estimate of the activity of sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15). However, the activities of microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) and diacylglycerol acyltransferase (EC 2.3.1.20), measured with microsomal bound substrate, were increased by Bt₂cAMP. These data have been interpreted to mean that Bt₂cAMP inhibits hepatic microsomal synthesis of triacylglycerol at a step prior to the formation of phosphatidate, presumably at the glycerophosphate acyltransferase (EC 2.3.1.15) step(s).     

Translocation of Mg2+-dependent phosphatidate phosphohydrolase between cytosol and endoplasmic reticulum in a permanent cell line from human lung

Incubation of A549 cells with digitonin for 4 min resulted in the release of over 90% of the lactate dehydrogenase activity into the medium. Approximately 80% of the Mg2+-dependent but only 7% of the Mg2+-independent phosphatidate phosphohydrolase activity was released in the presence of digitonin. Pretreatment of the cells with oleate reduced the efflux of the Mg2+-dependent phosphatidate phosphohydrolase activity to approximately 5% of total. Oleate did not affect the release of lactate dehydrogenase or the release of the Mg2+-independent phosphohydrolase activity. Incubation of A549 cells with [3H]oleate for 60 min led to incorporation of the label into phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, diacylglycerol, monoacylglycerol, and triacylglycerol, in ascending order. When the level of exogenous oleate was increased to over 2.0 mM, there was a marked increase in the incorporation into monoacylglycerol and diacylglycerol. Only small amounts of radioactivity were associated with phosphatidic acid. Time course studies revealed that the amount of radioactive phosphatidate remained low throughout the incubation period. These investigations were interpreted to indicate that free fatty acids can promote the translocation of the Mg2+-dependent phosphatidate phosphohydrolase activity from cytosol to membrane fractions. This translocation could, at least theoretically, function to facilitate the metabolism of increased amounts of phosphatidate.     

Possible relationship of the hepatic microsomal ATP-dependent calcium pump to sex differences in triacylglycerol synthesis

Microsomes were isolated from livers of fed male and female rats and the rates of incorporation of sn-[¹⁴C]-glycerol-3-phosphate into phosphatidate, diacylglycerol and triacylglycerol by the microsomes were measured. Simultaneously, microsomal ATP-dependent uptake of calcium was evaluated and correlated with synthesis of phosphatidate from sn-glycerol-3-phosphate. The rate of glycerolipid synthesis by hepatic microsomes from female rats was greater than that of microsomes from male rats. By contrast, the active accumulation of calcium and subsequent inhibition of synthesis of phosphatidate from glycerol-3-phosphate was lower in microsomes from livers of female rats than from male animals. This reciprocal relationship between uptake of calcium and incorporation of sn-glycerol-3-phosphate into phosphatidate as reported earlier (Biochem. Biophys. Res. Commun. $\text{78}$, 1053–1059 (1977)) may, in part, be responsible for the differences in the rates of hepatic triacylglycerol synthesis between livers from male and female rats.     

Effects of vasopressin and corticosterone on fatty acid metabolism and on the activities of glycerol phosphate acyltransferase and phosphatidate phosphohydrolase in rat hepatocytes.

The effects of vasopressin on the short-term control of fatty acid metabolism were studied in isolated rat hepatocytes. Vasopressin increased the oxidation of oleate to CO2 and decreased the formation of ketones in hepatocytes from Wistar rats, but not from Brattleboro rats. Incubation with vasopressin for 30 min increased the conversion of oleate into triacylglycerol by 17% and 32% in hepatocytes from Wistar and Brattleboro rats respectively. The corresponding increases for the phospholipid fraction were 19% and 42%. When Wistar-rat hepatocytes were incubated with corticosterone for 6 h there was a 19% increase in triacylglycerol synthesis, and a 52% increase if vasopressin was added 30 min before the end of the incubation. Glycerol phosphate acyltransferase activity was not significantly increased by vasopressin. Incubation for 5-60 min with vasopressin increased the Vmax. of phosphatidate phosphohydrolase by 48% and 32% respectively in hepatocytes from Wistar and Brattleboro rats. These increases were antagonized if EGTA was added to the medium used for incubating the hepatocytes. The replacement of vasopressin by 5 microM-ionophore A23187 produced a significant increase of 13% in the phosphohydrolase activity. It is therefore likely that the effects of vasopressin on the phosphohydrolase are mediated by Ca2+. These results are discussed in relation to the possible function of phosphatidate phosphohydrolase in controlling the turnover of phosphoinositides, the synthesis of phosphatidylethanolamine, phosphatidylcholine and triacylglycerol, and the secretion of very-low-density lipoproteins.     

Effects of ethynyloestradiol on the metabolism of [1-14C]-oleate by perfused livers and hepatocytes from female rats

Normal female rats were given 15mug of ethynyloestradiol/kg body wt. for 14 days and were killed on day 15 after starvation for 12-14h. The livers were isolated and were perfused with a medium containing washed bovine erythrocytes, bovine serum albumin, glucose and [1-(14)C]oleic acid; 414mumol of oleate were infused/h during a 3h experimental period. The output of bile and the flow of perfusate/g of liver were decreased in livers from animals pretreated with ethynyloestradiol, whereas the liver weight was increased slightly. The rates of uptake and of utilization of [1-(14)C]oleate were measured when the concentration of unesterified fatty acid in the perfusate plasma was constant. The uptake of unesterified fatty acid was unaffected by pretreatment of the animal with oestrogen; however, the rate of incorporation of [1-(14)C]oleate into hepatic and perfusate triacylglycerol was stimulated, whereas the rate of conversion into ketone bodies was impaired by treatment of the rat with ethynyloestradiol. Pretreatment of the rat with ethynyloestradiol increased the output of very-low-density lipoprotein triacylglycerol, cholesterol, phospholipid and protein. The production of (14)CO(2) and the incorporation of radioactivity into phospholipid, cholesteryl ester and diacylglycerol was unaffected by treatment with the steroid. The net output of glucose by livers from oestrogen-treated rats was impaired despite the apparent increased quantities of glycogen in the liver. The overall effect of pretreatment with oestrogen on hepatic metabolism of fatty acids is the channeling of [1-(14)C]oleate into synthesis and increased output of triacylglycerol as a moiety of the very-low-density lipoprotein, whereas ketogenesis is decreased. The effect of ethynyloestradiol on the liver is apparently independent of the nutritional state of the animal from which the liver was obtained. It is pertinent that hepatocytes prepared from livers of fed rats that had been treated with ethynyloestradiol produced fewer ketone bodies and secreted more triacylglycerol than did hepatocytes prepared from control animals. In these respects, the effects of the steroid were similar in livers from fed or starved (12-14h) rats. Oestrogens may possibly inhibit hepatic oxidation of fatty acid, making more fatty acid available for the synthesis of triacylglycerol, or may stimulate the biosynthesis of triacylglycerol, or may be active on both metabolic pathways.     

The effect of dietary carbohydrate and fat on the activities of some enzymes responsible for glycerolipid synthesis in rat liver.

1. Male rats were fed for 14 days on diets containing (by wt.) 53% of starch, or on diets in which 20% of the starch was replaced by sucrose, corn oil or lard. 2. The hepatic activities of the microsomal glycerol phosphate acyltransferase, dihydroxyacetone phosphate acyltransferase, phosphatidate cytidylyltransferase, diacylglycerol acyltransferase and choline phosphotransferase, and of the soluble phosphatidate phosphohydrolase, were measured. 3. The soluble phosphatidate phosphohydrolase activity was higher in those rats fed on lard than in those fed on the starch diet. Choline phosphotransferase activity was higher in the rats fed on corn oil than in those fed on the starch diet. 4. The rate of hepatic glycerolipid synthesis was measured in vivo 1 min after injection of [1,3-3H]glycerol and [1-14C]palmitate into the portal veins. 5. The relative rate of phosphatidylcholine synthesis in vivo was increased after feeding with corn oil and the higher specific activity of choline phosphotransferase may contribute to this result. The equivalent rate of triacylglycerol synthesis was increased by feeding with lard rather than corn oil, and the increased activity of phosphatidate phosphohydrolase may partly explain this. The latter changes probably contribute to the increased concentration of triacylglycerol which other authors have observed in the livers and sera of animals fed on saturated and monounsaturated fats.     

Overexpression of rat long chain acyl-coa synthetase 1 alters fatty acid metabolism in rat primary hepatocytes

Long chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for both anabolic and catabolic pathways. We have hypothesized that each of the five ACSL isoforms partitions FA toward specific downstream pathways. Acsl1 mRNA is increased in cells under both lipogenic and oxidative conditions. To elucidate the role of ACSL1 in hepatic lipid metabolism, we overexpressed an Acsl1 adenovirus construct (Ad-Acsl1) in rat primary hepatocytes. Ad-ACSL1, located on the endoplasmic reticulum but not on mitochondria or plasma membrane, increased ACS specific activity 3.7-fold. With 100 or 750 mum [1-(14)C]oleate, Ad-Acsl1 increased oleate incorporation into diacylglycerol and phospholipids, particularly phosphatidylethanolamine and phosphatidylinositol, and decreased incorporation into cholesterol esters and secreted triacylglycerol. Ad-Acsl1 did not alter oleate incorporation into triacylglycerol, beta-oxidation products, or total amount of FA metabolized. In pulse-chase experiments to examine the effects of Ad-Acsl1 on lipid turnover, more labeled triacylglycerol and phospholipid, but less labeled diacylglycerol, remained in Ad-Acsl1 cells, suggesting that ACSL1 increased reacylation of hydrolyzed oleate derived from triacylglycerol and diacylglycerol. In addition, less hydrolyzed oleate was used for cholesterol ester synthesis and beta-oxidation. The increase in [1,2,3-(3)H]glycerol incorporation into diacylglycerol and phospholipid was similar to the increase with [(14)C]oleate labeling suggesting that ACSL1 increased de novo synthesis. Labeling Ad-Acsl1 cells with [(14)C]acetate increased triacylglycerol synthesis but did not channel endogenous FA away from cholesterol ester synthesis. Thus, consistent with the hypothesis that individual ACSLs partition FA, Ad-Acsl1 increased FA reacylation and channeled FA toward diacylglycerol and phospholipid synthesis and away from cholesterol ester synthesis.     

Effects of ethanol feeding on hepatic lipid synthesis

Rats were fed a high-fat, liquid diet containing either 36% of total calories as ethanol or an isocaloric amount of sucrose, for a period up to 35 days. At different time intervals we measured the effects of ethanol administration on the activities of a number of key enzymes involved in hepatic lipid synthesis. At the start of the experimental period the activities of acetyl-CoA carboxylase and fatty acid synthase, measured in liver homogenates, increased in the control as well as in the ethanol-fed group. After 35 days these enzyme activities were still elevated but there were no significant differences between the two groups. In hepatocytes isolated from controls as well as from ethanol-fed rats, short-term incubations with ethanol induced an increase in the rate of fatty acid synthesis and in the activities of acetyl-CoA carboxylase and fatty acid synthase. However, no alterations in the regulation of these enzymes by short-term modulators of lipogenesis were apparent in hepatocytes isolated from alcohol-treated animals. The results do not indicate a major role for the enzymes of de novo fatty acid synthesis in the development of the alcoholic fatty liver. The amount of liver triacylglycerols increased in ethanol-fed rats during the entire treatment period, whereas the hepatic levels of phosphatidylcholine and phosphatidylethanolamine were not affected by ethanol ingestion. Ethanol administration for less than 2 weeks increased the activities of phosphatidate phosphohydrolase, diacylglycerol acyltransferase, and microsomal phosphocholine cytidylyltransferase, whereas the cytosolic activity of phosphocholine cytidylyltransferase was slightly decreased. Upon prolonged ethanol administration the activities of these enzymes were slowly restored to control values after 35 days, suggesting development of some kind of adaptation. It is interesting that, although the activities of phosphatidate phosphohydrolase and diacylglycerol acyltransferase were restored to the levels found in the control rats, this effect was not accompanied by a stabilization or decrease of the concentration of hepatic triacylglycerols.     

Deprivation and repletion of androgen in vivo modifies triacylglycerol synthesis by rat hepatocytes

Given the same quantity of fatty acid, livers from male rats esterify less fatty acid and secrete less triacylglycerol in very-low-density lipoprotein than do livers from female animals. To elucidate the role of testosterone in maintenance of this male pattern, conversion of [1-14C]oleic acid into triacylglycerol was assessed in vitro by rat hepatocytes (male) following gonadectomy and replacement with testosterone. Following castration, incorporation of fatty acid into triacylglycerol was increased. In contrast, esterification of exogenous fatty acid into phospholipid, cholesteryl esters, and diacylglycerol was unchanged. Treatment with testosterone (75 micrograms/day) reduced incorporation of exogenous fatty acid into triacylglycerol. Higher doses of testosterone (200 or 100 micrograms/day) modified the effect, such that inhibition was observed only at low oleate (0.5 mM) concentrations. At higher substrate concentrations (1.0-2.0 mM) the inhibitory effect was no longer observed. Further, a similar dose-dependent effect of testosterone was observed following in vivo treatment of castrate females with testosterone. These data support the concept of a regulatory role of testosterone in hepatic triacylglycerol synthesis. These findings also demonstrate a biphasic effect of testosterone, an effect that is dependent not only upon the dose of testosterone administered, but also on the concentration of fatty acid to which the hepatocyte is exposed in vitro.     

Relationships between fatty acid synthesis and lipid secretion in the isolated perfused rat liver: effects of hyperthyroidism, glucose and oleate

Various studies on the effects of thyroid status on hepatic fatty acid synthesis have produced conflicting results. Several variables (e.g., plasma free fatty acid and glucose concentrations) are altered simultaneously by thyroid status and can affect fatty acid synthesis. To evaluate the effects of these variables, hepatic fatty acid synthesis (lipogenesis) was studied in isolated perfused livers from normal and triiodothyronine-treated rats. Livers were perfused with media containing either 5.5 or 25 mM glucose without fatty acid, or 5.5 mM glucose and 0.7 mM oleate. Rates of lipogenesis were determined by measurement of incorporation of 3H2O into fatty acids. Lipogenesis in livers from hyperthyroid animals exceeded that of controls, when perfused with 5.5 mM glucose with or without oleate. Perfusion with 25 mM glucose increased lipogenesis in both euthyroid and hyperthyroid groups to the same level, abolishing this difference between them. Perfusion with oleate reduced rates of lipogenesis by livers from euthyroid and hyperthyroid rats to a similar extent, but stimulated secretion of radioactive fatty acid in phospholipid and free fatty acid fractions. Oleate increased ketogenesis by livers from normal and triiodothyronine-treated rats, with higher rates of ketogenesis in the triiodothyronine-treated group. When oleate was omitted, ketogenesis in the presence of 5.5 mM glucose by the hyperthyroid group was similar to that of euthyroid controls, while ketogenesis was decreased in the hyperthyroid group relative to controls when perfused with 25 mM glucose. About 30% of the radioactivity incorporated into the total fatty acid of both groups was recovered in palmitate, with the remainder in longer chain saturated and unsaturated fatty acids. In both euthyroid and hyperthyroid groups, the ratio of triacylglycerol:phospholipid fatty acid radioactivity was not only less than predicted (based on synthetic rates of PL and TG) but also was decreased in perfusions with exogenous oleate compared to perfusions without oleate. In perfusions with oleate, both groups incorporated twice as much radioactivity into phospholipid as into triacylglycerol. The data suggest the following concepts: while hepatic fatty acid synthesis and oxidation are increased simultaneously in the hyperthyroid state, de novo synthesized fatty acids seem to be poorer substrates for oxidation than are exogenous fatty acids, and are preferentially incorporated into phospholipid, while exogenous fatty acids are better substrates for oxidation and esterification to triacylglycerol. The preferential utilization of de novo synthesized fatty acid for phospholipid synthesis may be an important physiologic adaptation insuring a constant source of fatty acid for membrane synthesis.     

Interactions of insulin, glucagon and dexamethasone in controlling the activity of glycerol phosphate acyltransferase and the activity and subcellular distribution of phosphatidate phosphohydrolase in cultured rat hepatocytes.

Rat hepatocytes were incubated in monolayer culture for 8 h. Glucagon (10nM) increased the total phosphatidate phosphohydrolase activity by 1.7-fold. This effect was abolished by adding cycloheximide, actinomycin D or 500 pM-insulin to the incubations. The glucagon-induced increase was synergistic with that produced by an optimum concentration of 100 nM-dexamethasone. Theophylline (1mM) potentiated the effect of glucagon, but it did not affect the dexamethasone-induced increase in the phosphohydrolase activity. The relative proportion of the phosphohydrolase activity associated with membranes was decreased by glucagon when 0.15 mM-oleate was added 15 min before the end of the incubations to translocate the phosphohydrolase from the cytosol. This glucagon effect was not seen at 0.5 mM-oleate. Since glucagon also increased the total phosphohydrolase activity, the membrane-associated activity was maintained at 0.15 mM-oleate and was increased at 0.5 mM-oleate. This activity at both oleate concentrations was also increased in incubations that contained dexamethasone, particularly in the presence of glucagon. Insulin increased the relative proportion of phosphatidate phosphohydrolase that was associated with membranes at 0.15 mM-oleate, but not at 0.5 mM-oleate. It also decreased the absolute phosphohydrolase activity on the membranes at both oleate concentrations in incubations that also contained glucagon and dexamethasone. None of the hormonal combinations significantly altered the total glycerol phosphate acyltransferase activity. However, glucagon significantly increased the microsomal activities, and insulin had the opposite effect. Glucagon also decreased the mitochondrial acyltransferase activity. There was a highly significant correlation between the total phosphatidate phosphohydrolase activity and the synthesis of neutral lipids from glycerol phosphate and 0.5 mM-oleate in homogenates of cells from all of the hormonal combinations. Phosphatidate phosphohydrolase activity is increased in the long term by glucocorticoids and also by glucagon through cyclic AMP. In the short term, glucagon increases the concentration of fatty acid required to translocate the cytosolic reservoir of activity to the membranes on which phosphatidate is synthesized. Insulin opposes the combined actions of glucagon and glucocorticoids. The long-term events explain the large increases in the phosphohydrolase activity that occur in vivo in a variety of stress conditions. The expression of this activity depends on increases in the net availability of fatty acids and their CoA esters in the liver.     

The effect of fatty acid exposure on the biosynthesis of glycerolipids by cultured hepatocytes

Incubation of hepatocyte monolayers with oleate or palmitate (1.0 mM) for 2-48 h, increased (20 to 80%) the incorporation of [1,3-14C]glycerol and palmitate into triacyglycerol but not phosphatidylcholine. The effect of fatty acids on liver cell triacylglycerol formation correlated well (r = 0.990) with a simultaneous rise (2-4-fold) in phosphatidate phosphatase (EC 3.1.3.4) activity. Phosphatidate phosphatase activity and triacylglycerol biosynthesis are also increased (2-fold) after hepatocyte monolayers are incubated for 24 h with cyclic GMP in the absence of fatty acids. Fatty acid-dependent increases in liver cell triacylglycerol formation and phosphatidate phosphatase activity are not blocked by cycloheximide. Phosphatidylcholine biosynthesis was also elevated in homogenates of liver cells exposed (24-48 h) to 1.0 mM oleate when exogenous CDPcholine was added to the incubation mixture. Apparently, the phosphatidate phosphatase-dependent rise in diacylglycerols that occurs after fatty acid exposure is primarily shunted into triacylglycerols because liver cell CDPcholine content is not correspondingly increased, and high levels of diacylglycerol acyltransferase (EC 2.3.1.20) and fatty acyl-CoA derivatives are present.     

Regulation of the translocation of phosphatidate phosphohydrolase between the cytosol and the endoplasmic reticulum of rat liver. Effects of unsaturated fatty acids, spermine, nucleotides, albumin and chlorpromazine.

The translocation of phosphatidate phosphohydrolase between the cytosol and the microsomal membranes was investigated by using a cell-free system from rat liver. Linoleate, alpha-linolenate, arachidonate and eicosapentenoate promoted the translocation to membranes with a similar potency to that of oleate. The phosphohydrolase that associated with the membranes in the presence of [14C]oleate or 1mM-spermine coincided on Percoll gradients with the peak of rotenone-insensitive NADH-cytochrome c reductase, and in the former case with a peak of 14C. Microsomal membranes were enriched with the phosphohydrolase activity by incubation with [14C]oleate or spermine and then incubated with albumin. The phosphohydrolase activity was displaced from the membranes by albumin, and this paralleled the removal of [14C]oleate from the membranes when this acid was present. Chlorpromazine also displaced phosphatidate phosphohydrolase from the membranes, but it did not displace [14C]oleate. The effects of spermine in promoting the association of the phosphohydrolase with the membranes was inhibited by ATP, GTP, CTP, AMP and phosphate. ATP at the same concentration did not antagonize the translocating effect of oleate. From these results and previous work, it was concluded that the binding of long-chain fatty acids and their CoA esters to the endoplasmic reticulum acts as a signal for more phosphatidate phosphohydrolase to associate with these membranes and thereby to enhance the synthesis of glycerolipids, especially triacylglycerol. The translocation of the phosphohydrolase probably depends on the increased negative charge on the membranes, which could also be donated by the accumulation of phosphatidate. Chlorpromazine could oppose the translocation by donating a positive charge to the membranes.     

Long-chain fatty acids and their acyl-CoA esters cause the translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction of rat liver

A translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction was promoted in cell-free extracts of rat liver by oleate and palmitate and their CoA esters. Oleate was more potent in this respect than palmitate and the CoA esters were more effective than the unesterified acids. Octanoate, octanoyl-CoA and CoA did not cause the translocation. It is proposed that the interaction of phosphatidate phosphohydrolase with the membranes that synthesize glycerolipids causes it to become metabolically active. This enables the liver to increase its capacity for triacylglycerol synthesis in response to an increased supply of fatty acids.     

The acylation of sn-glycerol 3-phosphate and the metabolism of phosphatidate in microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius L.) seed.

Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalysed the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. The resulting phosphatidate was further utilized in the synthesis of diacyl- and tri-acylglycerol by the reactions of the so-called 'Kennedy pathway' [Kennedy (1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940]. Diacylglycerol equilibrated with the phosphatidylcholine pool when glycerol backbone, with the associated acyl groups, flowed from phosphatidate to triacylglycerol. The formation of diacylglycerol from phosphatidate through the action of a phosphatidate phosphohydrolase (phosphatidase) was substantially inhibited by EDTA and, under these conditions, phosphatidate accumulated in the microsomal membranes. The inhibition of the phosphatidase by EDTA was alleviated by Mg2+. The presence of Mg2+ in all incubation mixtures stimulated quite considerably the synthesis of triacylglycerol in vitro. Microsomal preparations incubated with acyl-CoA, sn-glycerol 3-phosphate and EDTA synthesized sufficient phosphatidate for the reliable analysis of its intramolecular fatty acid distribution. In the presence of mixed acyl-CoA substrates the sn-glycerol 3-phosphate was acylated exclusively in position 1 with the saturated fatty acids, palmitate and stearate. The polyunsaturated fatty acid linoleate was, however, utilized largely in the acylation of position 2 of sn-glycerol 3-phosphate. The affinity of the enzymes involved in the acylation of positions 1 and 2 of sn-glycerol 3-phosphate for specific species of acyl-CoA therefore governs the non-random distribution of the different acyl groups in the seed triacylglycerols. The acylation of sn-glycerol 3-phosphate in position 1 with saturated acyl components also accounts for the presence of these groups in position 1 of sn-phosphatidylcholine through the equilibration of diacylglycerol with the phosphatidylcholine pool, which occurs when phosphatidate is utilized in the synthesis of triacylglycerol. These results add further credence to our previous proposals for the regulation of the acyl quality of the triacylglycerols that accumulate in developing oil seeds [Stymne & Stobart (1984) Biochem. J. 220, 481-488; Stobart & Stymne (1985) Planta 163, 119-125].     

Accumulation of diacylglycerol induced in platelets by exogenous fatty acids

Free fatty acids added in ethanol to human platelets prelabelled with [${}^{14}\text{C}$]arachidonate induce an accumulation of radioactive diacylglycerol. Unsaturated fatty acids are ten times more potent than palmitate. Ethanol alone does not alter the distribution of radioactivity. Increasing the concentration of arachidonate leads to increased diacylglycerol formation. The fatty acid effect is independent of thrombin, which itself causes a relatively small change in diacylglycerol levels. Neither the labelled triacylglycerol nor the labelled free fatty acid appears to be the source of the diacylglycerol formed which may arise from the activation of phosphatidylinositol phosphodiesterase.     

The regulation of triacylglycerol biosynthesis in cocoa (Theobroma cacao) L.

Developing cocoa cotyledons accumulate initially an unsaturated oil which is particularly rich in oleate and linoleate. However, as maturation proceeds, the characteristic high stearate levels appear in the storage triacylglycerols. In the early stages of maturation, tissue slices of developing cotyledons (105 days post anthesis, dpa) readily accumulate radioactivity from [¹⁴C]acetate into the diacylglycerols and label predominantly palmitate and oleate. In older tissues (130 dpa), by contrast, the triacylglycerols are extensively labelled and, at the same time, there is an increase in the percentage labelling of stearate. Thus, the synthesis of triacylglycerol and the production of stearate are co-ordinated during development. The relative labelling of the phospholipids (particularly phosphatidylcholine) was rather low at both stages of development which contrasts with oil seeds that accumulate a polyunsaturated oil (e.g. safflower). Microsomal membrane preparations from the developing cotyledons readily utilised an equimolar [¹⁴C]acyl-CoA substrate (consisting of palmitate, stearate and oleate) and glycerol 3-phosphate to form phosphatidate, diacylglycerol and triacylglycerol. Analysis of the [¹⁴C]acyl constituents at the sn-1 and sn-2 positions of phosphatidate and diacylglycerol revealed that the first acylase enzyme (glycerol 3-phosphate acyltransferase) selectively utilised palmitate over stearate and excluded oleate, whereas the second acylase (lysophosphatidate acyltransferase) was highly selective for the unsaturated acyl-CoA. On the other hand, the third acylase (diacylglycerol acyltransferase) exhibited an almost equal selectivity for palmitate and stearate. Thus, stearate is preferentially enriched at position sn-3 of triacylglycerol at 120–130 dpa because of the relatively higher selectivity of the diacylglycerol acyltransferase for this fatty acid compared with those of the other two acylation enzymes.Abbreviation dpa days post anthesis We are grateful to Drs. G. Pettipher (Cadbury-Schweppes, Reading, UK), M. End and P. Hadley (Department of Horticulture, University of Reading) for the supply of cocoa pods and to the Agricultural and Food Research Council for financial support. We also wish to thank Dr. S. Stymne (Swedish University of Agricultural Sciences, Uppsala, Sweden) for a generous gift of acyl-CoA substrates.     

Role of glycerol 3-phosphate and glycerophosphate acyltransferase in the nutritional control of hepatic triacylglycerol synthesis

1. Glycerol 3-phosphate content of isolated hepatocytes from starved rats and of glycogen-depleted hepatocytes from fed rats was low and severely limited triacylglycerol synthesis. 2. Raising the glycerol 3-phosphate content by addition of precursors to the cells resulted in a hyperbolic-like relationship between triacylglycerol synthesis and cellular glycerol 3-phosphate content. Statistical analysis of the curves showed no significant differences between the nutritional states either at saturating or at subsaturating glycerol 3-phosphate content. 3. V_max. of glycerophosphate acyltransferase measured in homogenized hepatocytes was decreased by 30–40% in starvation. There was no change in apparent K_m for glycerol 3-phosphate. Since at saturating glycerol 3-phosphate content esterification rates in hepatocytes of both nutritional states were identical, the enzyme is not limiting esterification under this condition. 4. At subsaturating glycerol 3-phosphate content the flux through glycerophosphate acyltransferase necessarily limits esterification. Therefore one would expect a decrease in esterification in starvation under this condition. This was the case when triacylglycerol synthesis was plotted against intracellular glycerol 3-phosphate concentration, calculated from the cellular glycerol 3-phosphate content and the intracellular water space, which was smaller in hepatocytes from starved rats. 5. The data obtained in hepatocytes were extrapolated to the intact liver by using the number of parenchymal cells per g of liver as determined from marker-enzyme analysis and the liver weight per 100g body weight. The extrapolation suggested that glycerol 3-phosphate is limiting esterification in vivo for contents below 0.3–0.4 and 0.5–0.65μmol/g for livers from fed and starved animals respectively. Also for a given fatty acid load and a glycerol 3-phosphate content below 0.3μmol/g the liver may esterify less in the starved state. However, at the glycerol 3-phosphate contents measured in freeze-clamped livers (0.30 and 0.44μmol/g for the fed and starved state respectively), livers in both nutritional states seemed capable of esterifying similar amounts of fatty acids.