Dexamethasone increases the activity but not the amount of choline-phosphate cytidylyltransferase in fetal rat lung

The activity of choline-phosphate cytidylyltransferase is increased by glucocorticoids in late gestation fetal lung in association with increased phosphatidylcholine biosynthesis. Previous indirect data had suggested that the stimulatory effect of the hormone was due to activation of existing enzyme rather than synthesis of new cytidylyltransferase protein. Using a rabbit antibody raised against purified rat liver choline-phosphate cytidylyltransferase, we have now quantitated the amount of the enzyme in fetal rat lung explants cultured with and without dexamethasone. Our results show that the hormone increased the activity of the enzyme but not the amount of cytidylyltransferase protein. Thus the stimulatory effect of dexamethasone on cytidylyltransferase is due to activation of existing enzyme rather than induction of enzyme synthesis.     

Glucocorticoid stimulation of choline-phosphate cytidylyltransferase activity in fetal rat lung: receptor-response relationships

A number of previous studies using in vivo and cultured fetal lung models have shown that the activity of choline-phosphate cytidylyltransferase, the enzyme which catalyzes a rate-limiting reaction in de novo phosphatidylcholine synthesis, is increased by glucocorticoids and other hormones which accelerate fetal lung maturation. To examine the mechanism of this glucocorticoid action further, we examined the effect of dexamethasone on cytidylyltransferase activity in cultured fetal rat lung explants and related it to specific dexamethasone binding. Dexamethasone stimulated cytidylyltransferase activity in the homogenate, microsomal and 105,000 X g supernatant fractions. The hormone did not alter the subcellular distribution of the enzyme, however; the bulk of the activity was in the supernatant fraction in both the control and dexamethasone-treated cultures. The dose-response curves for stimulation of cytidylyltransferase activity in the supernatant fraction and specific nuclear binding of dexamethasone were similar and both plateaued at approx. 20 nM. The EC50 for cytidylyltransferase stimulation was 6.6 nM and the Kd for dexamethasone binding was 6.8 nM. The relative potencies of various steroids for stimulating choline-phosphate cytidylyltransferase and for specific nuclear glucocorticoid binding were the same: dexamethasone greater than cortisol = corticosterone = dihydrocorticosterone greater than progesterone. The stimulation by dexamethasone of cytidylyltransferase activity and of choline incorporation into phosphatidylcholine were both abolished by actinomycin D. These data show that the stimulatory effect of dexamethasone on fetal rat lung choline-phosphate cytidylyltransferase activity is largely on the enzyme in the supernatant fraction and does not involve enzyme translocation to the microsomes as has been reported for cytidylyltransferase activation in some other systems. This effect of dexamethasone is a receptor-mediated process dependent on RNA and protein synthesis.     

Effects of glucocorticoid and thyroid hormones on regulatory enzymes of fatty acid synthesis and glycogen metabolism in developing fetal rat lung

Although glucocorticoid and thyroid hormones are known to act synergistically to stimulate surfactant production, they have opposite effects on other parameters of fetal lung maturation. We recently reported that the developmental increases in de novo fatty acid synthesis and glycogen accumulation in fetal rat lung were accelerated by dexamethasone but prevented by triiodothyronine and that the dexamethasone-induced increases were diminished when the two hormones were administered together. We have now examined the effects of maternal administration of these hormones on activities of enzymes of lung fatty acid synthesis and glycogen metabolism in the rat. There was a developmental increase in fatty-acid synthase activity between 19 and 21 days gestation. This activity was increased by dexamethasone but decreased by triiodothyronine. When the two hormones were administered together the stimulatory effect of dexamethasone was decreased from 56% to 29%. The stimulatory effect on fatty-acid synthase was also observed in fetal lung explants cultured in the presence of dexamethasone. This shows that the effect of the hormone was directly on the fetal lung. Dexamethasone had no effect on liver fatty-acid synthase. There was a developmental decrease in acetyl-CoA carboxylase activity but it was not affected by the hormones. These data show that the developmental and hormone-induced changes in fetal lung de novo fatty acid synthesis are mediated by fatty-acid synthase. Although there were developmental changes in fetal lung 6-phosphofructokinase, glycogen synthase and glycogen phosphorylase activities, these enzymes were not affected by the hormones.     

The cellular mechanism of glucocorticoid acceleration of fetal lung maturation. Fibroblast-pneumonocyte factor stimulates choline-phosphate cytidylyltransferase activity

The cellular mechanism by which glucocorticoids stimulate phosphatidylcholine biosynthesis has been studied in the fetal rat lung in vivo and in cultured fetal rat lung cells of varying levels of complexity. Administration of dexamethasone to pregnant rats at 18 days gestation resulted in a significant increase in saturated phosphatidylcholine content in fetal lung 24 h after injection. Dexamethasone administration increased the activity of fetal lung choline-phosphate cytidylyltransferase by 34%. It had no effect on the activities of fetal lung choline kinase and choline phosphotransferase. Exposure of fetal lung type II cells in organotypic cultures (which contain both type II cells and fibroblasts) to cortisol resulted in a 1.6-fold increase in the incorporation of [Me-3H]choline into saturated phosphatidylcholine. The activities of the enzymes in the choline pathway for the de novo biosynthesis of phosphatidylcholine were not significantly altered except for a 105% increase in choline-phosphate cytidylyltransferase activity. Treatment of monolayer cultures of fetal type II cells with cortisol-conditioned medium from fetal lung fibroblasts resulted in a 1.5-fold increase in saturated phosphatidylcholine production. This effect correlated with a doubling of choline-phosphate cytidylyltransferase activity. Additional evidence that this stimulatory action is mediated by fibroblast-pneumonocyte factor, produced by fetal lung fibroblasts in response to cortisol, was obtained. The factor was partially purified from cortisol-conditioned medium of fetal lung fibroblasts by gel filtration and affinity chromatography. Based on biological activity, a 3000-fold purification was obtained. Stimulation of saturated phosphatidylcholine synthesis in type II cells by fibroblast-pneumonocyte factor was maximal within 60 min of incubation. Pulse-chase experiments indicated that the stimulatory effect was correlated with an increased conversion of choline phosphate into CDP choline. Moreover, the enhanced phosphatidylcholine formation by fetal type II cells in response to fibroblast-pneumonocyte factor was accompanied by decreased levels of cellular choline phosphate. These findings further support the concept that glucocorticoid action on surfactant-associated phosphatidylcholine synthesis occurs ultimately at the level of the alveolar type II cell and involves fibroblast-pneumonocyte factor which stimulates the activity of choline-phosphate cytidylyltransferase.     

Pre-translational regulation by glucocorticoid of fatty acid and phosphatidylcholine synthesis in type II cells from fetal rat lung.

Exposure to fibroblast-conditioned cortisol-containing medium increased fatty acid synthase activity and fatty acid synthase, acetyl-CoA carboxylase and ATP citrate lyase mRNA abundance in fetal type II alveolar epithelial cells. Both fibroblast conditioning and cortisol in the medium were required for maximal effect on the mRNA levels, indicating involvement of mesenchymal-epithelial interaction in the cortisol effects. The observed effects provide evidence for an earlier hypothesis that increased activity of CTP:phosphocholine cytidylyltransferase in lung tissue caused by glucocorticoid is due to increased fatty acid synthesis. However, evidence suggesting pre-translational regulation of this enzyme by glucocorticoid was also found.     

Stimulation of cholinephosphate cytidylyltransferase activity by estrogen in fetal rabbit lung is mediated by phospholipids

We have investigated the mechanism by which estrogen stimulates phosphatidylcholine synthesis in fetal rabbit lung. The hormone increased the activity of cholinephosphate cytidylyltransferase in the 105 000 X g supernatant fraction but had no effect on the activities of this enzyme in the homogenate or other subcellular fractions. Although microsomal cytidylyltransferase has been reported to regulate phosphatidylcholine synthesis in other systems, and translocation of the enzyme from cytosol to microsomes has been reported in association with increased phosphatidylcholine synthesis, we found no evidence of this in the case of estrogen-stimulated phosphatidylcholine synthesis in the fetal lung. Cytosolic cytidylyltransferase activity was dependent on phospholipids. Extraction with acetone/butanol drastically reduced its activity as well as the stimulatory effect of estrogen. The activity and the effect of estrogen were restored on re-addition of lipids extracted with chloroform/methanol from additional supernatants. Fractionation of the total lipids revealed that the stimulatory effect was entirely associated with the phospholipids; neutral lipids and glycolipids did not stimulate. Treatment of the phospholipid fraction with phospholipase C abolished the stimulatory effect. The stimulatory effect of estrogen, however, could not be attributed to any individual phospholipid species but appeared to require the entire phospholipid mixture. We conclude that estrogen stimulates fetal lung phosphatidylcholine synthesis by increasing the activity of cytosolic cytidylyltransferase and this activation in turn is mediated by cytosolic phospholipids.     

Betamethasone activation of CTP: Cholinephosphate cytidylyltransferase is mediated by fatty acids

The purpose of the present study was to determine the mechanisms by which glucocorticoids increase the activity of CTP: cholinephosphate cytidylyltransferase, a key enzyme required for the synthesis of surfactant phosphatidylcholine. Lung cytidylyltransferase exists as an inactive, light form low in lipids (L-form) and an active, heavy form high in lipid content (H-form). In vivo, fatty acids stimulate and aggregate the inactive L-form to the active H-form. In vivo, betamethasone increases the amount of H-form while decreasing the amount of L-form in fetal lung. There is also a coordinate increase in total free fatty acids in the H-form. In the present study, we used gas chromatography–mass spectrometry to measure the fatty acid species associated with the H-forms in fetal rat lung after the mothers were treated with betamethasone (1 mg/kg). In vivo, betamethasone increased the total amount of free fatty acids associated with the H-form by 62%. Further, the hormone selectively increased the mass of myristic and oleic acids in H-form by 52 and 82%, respectively. However, betamethasone produced the greatest increase in the amount of H-form linoleic acid, which increased fourfold relative to control. In vitro, each of the fatty acids increased L-form activity in a dose-dependent manner; however, linoleic acid was the most potent. Linoleic and oleic acids also effectively increased L-form aggregations. These observations suggest that in vivo glucocorticoids elevate the level of specific fatty acids which convert cytidylyltransferase to the active form. © 1995 Wiley-Liss, Inc.     

Influence of dexamethasone on the lipid distribution of newly synthesized fatty acids in fetal rat lung

There is a developmental increase in fatty acid biosynthesis and surfactant production in late-gestation fetal lung and both are accelerated by glucocorticoids. We have examined the distribution of the newly synthesized fatty acids to determine whether they are preferentially incorporated into surfactant. Explants of 18 day fetal rat lung were cultured with and without dexamethasone for 48 h and then with [3H]acetate for 4 h after which labeled fatty acids were measured. Incorporation of radioactivity from acetate was considered a measure of newly synthesized fatty acids. Phospholipids contained 86% of the newly synthesized fatty acids of which approx. 80% were in phosphatidylcholine. Phosphatidylcholine and disaturated phosphatidylcholine contained a much greater percentage of the labeled fatty acids than of the phospholipid mass determined by phosphorus assay while phosphatidylethanolamine, phosphatidylserine and sphingomyelin contained less. Dexamethasone increased the rate of acetate incorporation into total lipid fatty acids but it had little effect on fatty acid distribution, except that it increased the percentages in phosphatidylglycerol and disaturated phosphatidylcholine. The hormone also increased the mass of these two phospholipids to a greater extent than that of the total. These data suggested that the newly synthesized fatty acids are preferentially incorporated into surfactant phospholipids and that this process is accelerated by dexamethasone. However, since phosphatidylcholine and phosphatidylglycerol are not exclusive to surfactant, we compared isolated lamellar bodies with a residual fraction not enriched in surfactant. The rate of acetate incorporation into fatty acids in lamellar body phosphatidylcholine as well as its specific activity (radioactivity per unit phosphorus) were both increased by dexamethasone. Specific activity, however, was no greater in the lamellar bodies than in the residual fraction in both control and dexamethasone-treated cultures. Therefore, there is no preferential incorporation of newly synthesized fatty acids into phospholipids in surfactant as opposed to those in other components of the lung.     

CTP:phosphorylcholine cytidylyltransferase in rat lung. The effect of free fatty acids on the translocation of activity between microsomes and cytosol

Phosphatidylglycerol and oleic acid had differential effects on cytidylyltransferase activity in cytosol and microsomes. The low-molecular-weight cytidylyltransferase in cytosol was stimulated more by phosphatidylglycerol than by oleic acid, whereas microsomal activity was stimulated more by oleic acid than by phosphatidylglycerol. Microsomal activity was stimulated by several unsaturated fatty acids but was not stimulated by saturated fatty acids. Bovine serum albumin decreased cytidylyltransferase activity in microsomes in the presence or absence of oleic acid but did not alter the activity measured in the presence of phosphatidylglycerol. The addition of oleic acid to albumin/microsome mixtures in amounts exceeding the binding capacity of albumin lead to complete recovery of the oleic acid stimulation. The addition of oleic acid to postmitochondrial supernatants resulted in a translocation of cytidylyltransferase activity from cytosol to microsome. The magnitude of the shift was severalfold greater with fetal preparations than adult. The free fatty acid content of microsomes increased coincident with the translocation. Bovine serum albumin, added to postmitochondrial supernatants, caused a release of cytidylyltransferase from microsomes to cytosol and a corresponding decrease in microsomal free fatty acid content. The amount of cytidylyltransferase activity in microsomes increased shortly after birth. The increase was accompanied by an increase in free fatty acid content of the microsomes. The increase in cytidylyltransferase activity and free fatty acids which occurred in vivo following birth was nearly identical to that obtained by adding oleic acid to postmitochondrial supernatants from fetal lung. We conclude that free fatty acids may affect the intracellular activity of cytidylyltransferase by promoting the translocation of inactive cytosolic forms to microsomes as well as by stimulating microsomal bound activity.     

Maternal administration of dexamethasone stimulates choline-phosphate cytidylyltransferase in fetal type II cells.

Administration of dexamethasone to pregnant rats at 19 days gestation increased phosphatidylcholine synthesis (45%) from radioactive choline in type II cells. This enhanced synthesis of phosphatidylcholine was accompanied by an increased conversion of choline phosphate into CDP-choline. Similar results were obtained by incubating organotypic cultures of 19-day-fetal rat lung with cortisol. The increased conversion of choline phosphate into CDP-choline correlated with an enhanced choline-phosphate cytidylyltransferase activity (31% after dexamethasone treatment; 47% after cortisol exposure) in the cell homogenates. A similar increase (26% after dexamethasone treatment; 39% after cortisol exposure) was found in the microsomal-associated enzyme. No differences in cytosolic enzyme activity were observed. The specific activity of the microsomal enzyme was 3-4 times that of the cytosolic enzyme. Most of the enzyme activity was located in the microsomal fraction (58-65%). The treatments had no effect on the total amount of enzyme recovered from the cell homogenates. These results, taken collectively, are interpreted to indicate that the active form of cytidylyltransferase in type II cells is the membrane-bound enzyme and that cytidylyltransferase activation in type II cells from fetal rat lung after maternal glucocorticoid administration occurs by binding of inactive cytosolic enzyme to endoplasmic reticulum.     

Kinetic and biochemical properties of CTP:choline-phosphate cytidylyltransferase from the rat brain

In order to investigate the mechanisms involved in some brain disorders at the membrane level, we studied the kinetics and biochemical properties of brain CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15), the rate-limiting enzyme of the two-step biosynthesis of phosphatidylcholine. This enzyme catalyzes the biosynthesis of CDPcholine from choline phosphate and CTP. We found that its subcellular localization (mainly in microsomal and cytosolic fractions) was different from that of phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17), the enzyme of the alternative pathway for phosphatidylcholine synthesis. CTP:choline-phosphate cytidylyltransferase showed a Km of 10 mM for CTP and 0.3 mM for choline phosphate and exhibited a random mechanism. CDPcholine, the reaction product, was a competitive inhibitor of choline phosphate and CTP utilization and had a Ki of 0.090 mM. Both particulate and soluble enzymes required Mg2+ and exhibited an optimal pH at about 7. Cytosolic activity was enhanced by addition of unsaturated fatty acids or phospholipids extracted from brain membranes. Such an enhancement was increased with the centrifugation time used for preparing the soluble enzyme.     

The transfer of fatty acids across the sheep placenta

Maternal and fetal plasma concentrations of free fatty acids, triacylglycerols and phospholipids and the profile of their fatty acids were measured in three catheterized and unanaesthetized sheep. Fetal concentrations of all three lipid fractions were low and did not correlate with maternal concentrations. There were no measurable umbilical venous-arterial differences. Linoleic acid concentrations were low in both mother and fetus. The fatty acid composition of fetal adipose tissue, liver, lung and cerebellum of five animals was analysed. Again linoleic acid levels were very low, but phospholipids contained 2-8% arachidonic acid. [14C] linoleic acid and [3H] palmitic acid were infused intravenously into three ewes. Only trace amounts of labelled fatty acids were found in fetal plasma and these were confined to the free fatty acids. 14C-label was incorporated into fetal tissue lipids, but most of this probably was due to fetal lipid synthesis from [14C] acetate or other water-soluble products of maternal [14C] linoleic acid catabolism. It is concluded that only trace amounts of fatty acids cross the sheep placenta. They are derived mainly from the maternal plasma free fatty acids and might just be sufficient to be the source of the small amounts of essential fatty acids found in the lamb fetus, but are insignificant in terms of energy supply or lipid storage.     

Development of glycogen and phospholipid metabolism in fetal and newborn rat lung.

Glucose, a major metabolic substrate for the mammalian fetus, probably makes significant contributions to surface active phospholipid synthesis in adult lung. We examined the developmental patterns of glycogen content, glycogen synthase activity, glycogen phosphorylase activity and glucose oxidation in fetal and newborn rat lung. These patterns were correlated with the development of phosphatidylcholine synthesis, content and the activities of enzymes involved in phosphatidylcholine synthesis. Fetal lung glycogen concentration increased until day 20 of gestation (term is 22 days) after which it declined to low levels. Activity of both glycogen synthase I and total glycogen synthase (I + D) in fetal lung increased late in gestation. Increased lung glycogen concentration preceded changes in enzyme activity. Glycogen phosphorylase a and total glycogen phosphorylase (a + b) activity in fetal lung increased during the period of prenatal glycogen depletion. The activity of the pentose phosphate pathway, as measured by the ratio of CO2 derived from oxidation of C1 and C6 of glucose, declined after birth. Fetal lung total phospholipid, phosphatidycholine and disaturated phosphatidylcholine content increased by 60, 90 and 180%, respectively, between day 19 of gestation and the first postnatal day. Incorporation of choline into phosphatidylcholine and disaturated phosphatidylcholine increased 10-fold during this time. No changes in phosphatidylcholine enzyme activities were noted during gestation, but both choline phosphate cytidylyltransferase and phosphatidate phosphatase activity increased after birth. The possible contributions of carbohydrate derived from fetal lung glycogen to phospholipid synthesis are discussed.     

Activity and properties of CTP: cholinephosphate cytidylyltransferase in adult and fetal rat lung.

Cholinephosphate cytidylyltransferase (CTP : cholinephosphate cytidylyltransferase, EC 2.7.7.15) is located in both the microsomal and supernatant fractions of adult lung when the tissue is homogenized in 0.145 M NaCl. The activity is located predominantly in the supernatant fraction in fetal lung. Cholinephosphate cytidylyltransferase in the supernatant from fetal lung is stimulated 4- to 6-fold by the additions of total lung lipid. Serine phosphoglycerides and inositol phosphoglycerides specifically caused stimulation whereas choline phosphoglycerides and ethanolamine phosphoglycerides produced no stimulation. Lysophosphatidylcholine cause some stimulation, but only at high concentrations. A number of detergents were investigated. All produced inhibition except for the ampholytic detergent, miranol H2M which was not inhibitory. None of the detergents produced any stimulation of activity. Cytidylyltransferase activity in fetal lung when assayed in the absence of lipid is about 25% of the adult. The activity when assayed in the presence of lipid is equal or slightly higher than adult levels. The activity, measured without added phospholipid, increases 5- to 6-fold within 12 h after birth, to values higher than in the adult. The activity, measured in the presence of phospholipid, increased almost linearly from -2 day until +1 day. There is an inverse relationship between the concentration of phospholipid in the fetal lung supernatant and the degree of lipid stimulation. Chromatographic experiments with Biogel A 1.5 columns have shown that cytidylyltransferase can exist in two molecular sizes, a small molecular size that requires phospholipid for activity, and a larger molecular weight species which does not require the addition of phospholipid for activity. Fetal lung has a higher proportion of the low molecular weight form than adult lung. The small molecular weight species can be converted to the larger molecular weight form by the addition of phospholipids.     

Fatty acid synthesis in fetal lung

De novo fatty acid synthesis in lung is significant during fetal growth and development. Specific activity and relative rate of synthesis of fatty acid synthetase increase with the days of gestational age and drop significantly after birth. Fetal lungs contain thyroid hormone receptors and binding capacities of this hormone to the fetal lungs also increase with the days of gestational age. Our results suggest that de novo fatty acid synthesis in fetal lungs may make a significant contribution towards surfactant synthesis.     

Regulation of phospholipid synthesis by intracellular phospholipases in fetal rabbit type II pneumocytes

Exposure of fetal type II pneumocytes to phospholipase A2 inhibitors led to significantly reduced choline uptake and decreased synthesis of total and disaturated phosphatidylcholines from both [methyl-14C]choline and [9,10(n)-3H]palmitate precursors. The percentage of the total synthesized phosphatidylcholine recovered as disaturated phosphatidylcholine was increased when compared to that in control cultures, suggesting that unsaturated phosphatidylcholine synthesis was reduced to a greater extent than that of the disaturated species. Synthesis of sphingomyelin and phosphatidylethanolamine from labeled palmitate was also reduced, whereas that of phosphatidylinositol and phosphatidylglycerol was significantly increased. Addition of phospholipase C resulted in increased synthesis of phosphatidylcholine from both labeled precursors; no significant changes were found in synthesis of most of the other 3H-labeled lipids. Added phospholipase A2 did not lead to any changes in either choline or palmitate incorporation. However, when melittin (a phospholipase A2 activator) was added to the cultures, greater incorporation of both palmitate and choline was observed, along with a significant increase in the percentage of total cellular radioactivity in 14C-labeled lipids, indicating also stimulation of phosphatidylcholine synthesis. A marked increase in CTP: phosphorylcholine cytidylyltransferase activity was found after treatment of the cultures with phospholipase C. Exposure to quinacrine also increased the activity of this enzyme. Addition of phospholipase C and melittin to prelabeled pneumocyte cultures accelerated degradation of cell phospholipids and the release of free fatty acids as the main degradation products. These findings suggest that intracellular phospholipases are regulators of synthesis of surfactant phospholipids in fetal type II pneumocytes, and that activation or inhibition of these phospholipases could represent a mechanism through which hormones and pharmacological agents modify surfactant and other phospholipid synthesis.     

Direct effects of corticotropin-releasing hormone and thyrotropin-releasing hormone on fetal lung explants

Fetal lung produces corticotropin-releasing hormone (CRH) without known direct effects. We tested the hypothesis that CRH can directly regulate lung development. In baboon fetal lung explants, CRH strongly induces surfactant phospholipid synthesis and SP-C immunostaining, plus [³H]thymidine incorporation. CRH receptor mRNA was detected in lung from multiple baboons at e125. Testing thyrotropin (TRH) as a specificity control, we did demonstrate different direct effects with only modest stimulation of surfactant phospholipid synthesis and strong induction of cytidylyltransferase gene expression. Therefore, CRH, similar to ACTH and glucocorticoids, is a potent inducer of cell differentiation in fetal lung.     

Regulation of fatty acid oxidation and triglyceride and phospholipid metabolism by hypolipidemic sulfur-substituted fatty acid analogues

The mechanisms behind the hypotriglyceridemic effect of 1,10-bis(carboxymethylthio)decane (3-thiadicarboxylic acid) and tetradecylthioacetic acid and the development of fatty liver caused by 3-tetradecylthiopropionic acid (Aarsland et al. 1989. J. Lipid Res. 30: 1711-1718.) were studied in the rat. Repeated administration of S-substituted non-beta-oxidizable fatty acid analogues to normolipidemic rats resulted in a time-dependent decrease in plasma triglycerides, phospholipids, and free fatty acids. This was accompanied by an acute reduction in the liver content of triglycerides and an increase in the hepatic concentration of phospholipids. Mitochondrial fatty acid oxidation was stimulated, whereas lipogenesis was inhibited. The activity of phosphatidate phosphohydrolase decreased while the activity of CTP:phosphocholine cytidylyltransferase increased. These results suggest that the observed triglyceride-lowering effect was due to increased mitochondrial fatty acid oxidation accompanied by a reduction in the availability of the substrate i.e., free fatty acid, along with an enzymatic inhibition (phosphatidate phosphohydrolase). Administration of 3-tetradecylthiopropionic acid led to a drastic increase in the hepatic triglyceride content. Levels of plasma triglyceride phospholipid and free fatty acid also increased. Phosphatidate phosphohydrolase activity was stimulated whereas CTP:phosphocholine cytidylyltransferase was inhibited. Mitochondrial fatty acid oxidation was decreased. These data indicate that the development of fatty liver as an effect of 3-tetradecylpropionic acid is probably due to accelerated triglyceride biosynthesis, which is mediated by an increase in the availability of fatty acid along with stimulation of phosphatidate phosphohydrolase. The results of the present study speak strongly in favor of the hypothesis that phosphatidate phosphohydrolase is a major rate-limiting enzyme in triglyceride biosynthesis. Furthermore, they point out that the biosynthesis of triglycerides and phospholipids might be coordinately regulated. Such regulation is possibly mediated via phosphatidate phosphohydrolase and CTP:phosphocholine cytidylyltransferase. Whether the increase in hepatic phospholipids via increased CDP-pathway accounts for an increase of lipid components for proliferation of peroxisomes (3-thiadicarboxylic acid and tetradecylacetic acid) should be considered.