Glucocorticoid induction of fatty-acid synthase mediates the stimulatory effect of the hormone on choline-phosphate cytidylyltransferase activity in fetal rat lung

Fetal lung fatty-acid synthase and choline-phosphate cytidylyltransferase activities are increased by glucocorticoids. There is evidence that the hormone increases synthesis of fatty-acid synthase but only increases the catalytic activity of the cytidylyltransferase. Free fatty acids and a number of phospholipids have been reported to stimulate cytidylyltransferase activity in several organs, including the lung. We have addressed the question of whether glucocorticoid induction of fatty-acid synthase mediates the stimulatory effect of the hormone on choline-phosphate cytidylyltransferase activity. Explants of 18-day fetal rat lung were cultured for 48 h with dexamethasone and inhibitors of de novo fatty acid biosynthesis (agaric acid and hydroxycitric acid) being included in the medium for the final 20 h. Dexamethasone increased the activities of fatty acid synthase and choline-phosphate cytidylyltransferase by 84% and 60%, respectively. Agaric acid and hydroxycitric acid completely abolished the stimulatory effect of the hormone on cytidylyltransferase but not on fatty-acid synthase. The inhibitors had no effect on cytidylyltransferase activity in control cultures. Fetal lung choline-phosphate cytidylyltransferase can be maximally stimulated by inclusion of phosphatidylglycerol in the assay mixture and under this condition, cytidylyltransferase activity in control and dexamethasone-treated cultures in the presence and absence of the inhibitors were all increased to the same level. Therefore, the inhibitors did not diminish the capacity of cytidylyltransferase to be fully activated. We suggest that the glucocorticoid induction of fatty-acid synthase in fetal lung results in increased synthesis of fatty acids which in turn, either as free acids or after incorporation into phospholipids, activate choline-phosphate cytidylyltransferase.     

De novo fatty acid synthesis in developing rat lung

The rate of de novo fatty acid synthesis in developing rat lung was measured by the rate of incorporation of 3H from 3H2O into fatty acids in lung slices and by the activity of acetyl-CoA carboxylase in fetal, neonatal and adult lung. Both tritium incorporation and acetyl-CoA carboxylase activity increased sharply during late gestation, peaked on the last fetal day, and declined by 50% 1 day after birth. In the adult, values were only one-half the peak fetal rates. In vitro regulation of acetyl-CoA carboxylase activity in fetal lung was similar to that described in adult non-pulmonary tissues: activation by citrate and inhibition by palmitoyl-CoA. Similarly, incubation conditions that favored enzyme phosphorylation inhibited acetyl-CoA carboxylase activity in lung while dephosphorylating conditions stimulated activity. Incorporation of [U-14 C]glucose into lung lipids during development was influenced heavily by incorporation into fatty acids, which generally paralleled the rate of tritium incorporation into fatty acids. The relative utilization of acetyl units from exogenous glucose for overall fatty acid synthesis was greater in adult lung than in fetal or neonatal lung, suggesting that other substrates may be important for fatty acid synthesis in developing lung. In fetal lung explants, de novo fatty acid synthesis was inhibited by exogenous palmitate. Taken together, these data suggest that de novo synthesis may be an important source of saturated fatty acids in fetal lung but of lesser importance in the neonatal period. Furthermore, the regulation of acetyl-CoA carboxylase activity and fatty acid synthesis in lung may be similar to non-pulmonary tissues.     

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.     

Hormonal regulation of lipogenic enzymes in chick embryo hepatocytes in culture. Expression of the fatty acid synthase gene is regulated at both translational and pretranslational steps

Mechanisms involved in the multihormonal regulation of fatty acid synthase have been investigated by comparing levels of its mRNA with rates of enzyme synthesis in chick embryo hepatocytes in culture. Triiodothyronine or insulin caused about a 2.5-fold increase in the relative rate of synthesis of fatty acid synthase. Together, these hormones were synergistic, stimulating enzyme synthesis by nearly 40-fold (Fischer, P.W.F., and Goodridge, A.G. (1978) Arch. Biochem. Biophys. 190, 332-344). Addition of triiodothyronine stimulated increases in mRNA levels comparable to increases in enzyme synthesis whether insulin was present or not. Thus, triiodothyronine regulates fatty acid synthase primarily by controlling the amount of its mRNA. Addition of insulin, in the presence of triiodothyronine, stimulated enzyme synthesis by 14-fold and mRNA levels by only 2-fold. In the absence of triiodothyronine, insulin had no effect on mRNA levels. Thus, insulin has a major effect on the translation of fatty acid synthase mRNA. After the addition of triiodothyronine, fatty acid synthase mRNA accumulated with sigmoidal kinetics, approaching a new steady state about 48 h after the addition of hormone. Puromycin, an inhibitor of protein synthesis, blocked the effect of triiodothyronine. We suggest that the abundances of both fatty acid synthase and malic enzyme mRNAs are regulated by a common triiodothyronine-induced peptide intermediate which has a relatively long half-life. Glucagon caused an 80% decrease in the synthesis of fatty acid synthase (Fischer, P.W.F., and Goodridge, A.G. (1978) Arch. Biochem. Biophys. 190, 332-344) and a 60% decrease in the level of fatty acid synthase mRNA. Thus, glucagon regulates fatty acid synthase by controlling the concentration of its mRNA. The synthesis of malic enzyme also was inhibited by glucagon at a pretranslational step, but the inhibition was almost complete. Thus, despite coordinated regulation of the concentrations of these enzymes during starvation and refeeding, individual hormones sometimes regulate synthesis of the two enzymes at the same step and to about the same degree and sometimes at different steps or to very different degrees.     

Insulin regulation of glycogen synthase phosphatase in primary cultures of hepatocytes

Activation of glycogen synthase in the perfused rat liver is defective in severely diabetic rats. In the present study, activation of glycogen synthase by glucose and increased incorporation of [14C]glucose into glycogen by insulin are defective in hepatocytes isolated from alloxan diabetic rats. Acute activation of glycogen synthase in hepatocytes isolated from diabetic rats was restored by treatment of the rats with insulin in vivo. Restoration of synthase activation was not achieved by incubation of hepatocytes in the presence of insulin in vitro for up to 12 h. When isolated hepatocytes from diabetic rats were placed in primary culture in a serum-free defined medium over a 3-day period, glycogen synthesis was partially restored by cortisol and triiodothyronine and dramatically increased by insulin. Concomitant with restoration of [14C]glycogen synthesis was an insulin-mediated increase in glycogen synthase I and synthase phosphatase activity. Restoration of regulation of glycogen synthesis in primary cultures of hepatocytes from diabetic rats by insulin required the presence of cortisol and triiodothyronine. Primary cultures of hepatocytes from normal rats did not require triiodothyronine for insulin to effect glycogenesis over a 3-day period. These data demonstrate that insulin acts in a chronic manner in concert with other hormones to control synthase phosphatase activity, an effect which may be influencing acute control of hepatic glycogen synthesis.     

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.     

Fatty acid synthesis in the fetal lung: relationship to surfactant lipids

The aims of this study were to investigate the control of fatty acid synthesis and its relationship to surfactant production in the fetal lung during alteration of hormonal and substrate conditions. Lung explants from 18 day fetuses (term = 22 days) which were cultured 2 days in the presence of 10 mM lactate showed parallel acceleration of de novo fatty acid synthesis (3H2O incorporation) and [14C]choline incorporation into disaturated phosphatidylcholine (DSPC) compared to culture of explants in glucose. Both the cultured and fresh explants were resistant to the classical short term (4 h) cAMP inhibition of fatty acid synthesis with 3 mM dibutyryl cAMP or 0.5 mM aminophylline. In the cultured explants short term cAMP elevation increased DSPC production, and long term (2 day) cAMP elevation caused a further increase in DSPC synthesis and also stimulated fatty acid synthesis. In cultured explants from 17 day fetuses, dexamethasone (0.1 microM) caused a synergistic increase with aminophylline in both fatty acid synthesis and DSPC production whereas, in explants from 18 day fetuses, dexamethasone inhibited both processes and reduced the level of stimulation of DSPC and fatty acid synthesis seen with aminophylline alone. Dexamethasone also reduced the stimulation of both DSPC and fatty acid synthesis produced in the culture of 18 day explants with bacitracin (0.5 mg/ml), whereas the combination of bacitracin and aminophylline resulted in a synergistic increase in DSPC production. Culture with glucagon (0.1 microM) also stimulated DSPC synthesis but at physiological levels insulin had no effect on either DSPC or fatty acid synthesis. These data show that lung fatty acid synthesis exhibits unique features of fatty acid synthesis regulation compared to other lipogenic tissues and also suggest a link between de novo fatty acid synthesis and surfactant production during the critical period of accelerated lung maturation.     

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.     

Fibroblast growth factor upregulates PGG/H synthase in rabbit microvascular endothelial cells by a glucocorticoid independent mechanism.

The present study was undertaken to determine the effects of acidic fibroblast growth factor (aFGF) on eicosanoid synthesis in microvessel endothelial cells derived from rabbit left ventricular muscle (RCME cells). We observed that aFGF increased AA conversion to PGE2 in a time- and dose-dependent manner, and the stimulatory effect was abolished by actinomycin D and cycloheximide. Acidic FGF increased the recovery of PGG/H synthase activity following aspirin treatment, suggesting an action on de novo PGG/H synthase synthesis. Acidic FGF increased the incorporation of [35S] methionine into a 70 kD immunoreactive PGG/H synthase band. PGG/H synthase synthesis following aspirin treatment was also increased by transforming growth factor beta, while epidermal growth factor basic FGF and platelet derived growth factor were without effect. In addition, the actions of aFGF on de novo PGG/H synthase were compared in several endothelial preparations. Acidic FGF treatment of aspirin treated endothelial cells from rabbit lung microvessels and small pulmonary artery and from human lung microvessels all showed an increase in PGG/H synthase recovery. In contrast, similar treatment of human umbilical vein endothelial cells was without effect. Pretreatment of RCME cells with dexamethasone (1 microM) did not alter the aFGF induction of PGG/H synthase activity. We conclude that aFGF stimulates PGE2 production by a mechanism that includes the de novo synthesis of PGG/H synthase. This mechanism appears to be distinct from previously described glucocorticoid sensitive translational controls of PG synthase synthesis by epidermal growth factor in smooth muscle and mesangial cells.     

Control of glycogen metabolism by gluconeogenic and ketogenic substrates in isolated hepatocytes from fed rats.

1. This study was conducted to examine the effects of gluconeogenic and ketogenic substrates on the activities of the glycogen-metabolizing enzymes and on glycogenolysis in isolated hepatocytes from fed rats. 2. Gluconeogenic substrates like fructose, dihydroxyacetone or lactate turned out to stimulate the glucose-induced activation of glycogen synthase and this effect may be linked, to some extent, to the increase of the cellular glucose 6-phosphate concentration. 3. The effect of fructose was accompanied by the onset of glycogen synthesis. 4. Energetic substrates like fatty acids were also potent activators of glycogen synthase, especially in the presence of glucose. 5. When fatty acids were added alone or together with a physiological concentration of glucose, they induced or potentiated the inhibition of glycogen phosphorylase-a. 6. This inhibitory effect was mediated by a decrease of lactate release. 7. The stimulatory effect of amino acids on glycogen synthase seemed to be direct, non mediated by an inhibition of the phosphorylase-a activity although hepatic glycogenolysis markedly decreased. 8. Moreover, the amino acid action could be linked to their capacities to induce cell swelling and/or to limit proteolysis.     

Insulin action on protein phosphatase-1 activation is enhanced by the antidiabetic agent pioglitazone in cultured diabetic hepatocytes

Effect of the antidiabetic agent pioglitazone on the insulin-mediated activation of protein phosphatase-1 was examined in diabetic hepatocytes. Streptozotocin-induced diabetes in Sprague Dawley rats caused a significant decrease in the activation of glycogen synthase in hepatocytes isolated from these animals. There was an inverse correlation between the in vivo hyperglycemic condition and the in vitro activation of glycogen synthase in liver cells (r = 0.93, p > 0.001). Long term incubation of diabetic hepatocytes with insulin and dexamethasone caused significant (p > 0.001) improvement in the activation of glycogen synthase activation. When incubated along with hormones, pioglitazone enhanced their action (p > 0.05-0.01). Diabetic hepatocytes were also characterized by 50% decrease in the activity of protein phosphatase-1, the enzyme which dephosphorylates and activates glycogen synthase. Pioglitazone potentiated the acute stimulatory effect of insulin on protein phosphatase-1 in normal hepatocytes but not in diabetic hepatocytes. Long term incubation of diabetic hepatocytes with insulin ameliorated the decrease in the protein phosphatase -1 activity in these cells. This stimulatory long-term effect of insulin was significantly (p > 0.05) enhanced by the antidiabetic agent pioglitazone.     

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.     

Nutritional and hormonal regulation of mRNA levels of lipogenic enzymes in primary cultures of rat hepatocytes.

The effects of nutrients and hormones on the mRNA levels of acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase were examined in primary cultures of rat hepatocytes during the process of induction. The addition of both glucose and insulin to the culture medium markedly enhanced the lipogenic enzyme mRNA induction due to either of them, in 16 h. Fructose or glycerol proved to be an effective substitute for glucose, suggesting that glycolytic metabolites were involved in the mRNA induction. It is remarkable that mRNA induction of acetyl-CoA carboxylase was the most sensitive to glucose and also to insulin among the lipogenic enzymes. Polyunsaturated fatty acids markedly reduced the mRNA induction of lipogenic enzymes. Dexamethasone enhanced all the lipogenic enzyme mRNA induction by insulin. On the other hand, triiodothyronine addition greatly increased the mRNA concentrations of lipogenic enzymes, but dexamethasone decreased rather than increased the mRNA induction by triiodothyronine. The effects of insulin on the induction of the lipogenic enzyme mRNAs were similar, but those of triiodothyronine were not. Triiodothyronine markedly enhanced malic enzyme mRNA induction by insulin with dexamethasone, and tended to enhance the induction of the acetyl-CoA carboxylase and fatty acid synthase mRNAs, but not that of glucose 6-phosphate dehydrogenase mRNA. It appeared that insulin and triiodothyronine synergistically enhanced lipogenic enzyme mRNA induction by glucose, but the mechanisms were different.     

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.     

Apicoplast and endoplasmic reticulum cooperate in fatty acid biosynthesis in apicomplexan parasite Toxoplasma gondii

Apicomplexan parasites are responsible for high impact human diseases such as malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular pathogens are dependent on both de novo lipid biosynthesis as well as the uptake of host lipids for biogenesis of parasite membranes. Genome annotations and biochemical studies indicate that apicomplexan parasites can synthesize fatty acids via a number of different biosynthetic pathways that are differentially compartmentalized. However, the relative contribution of each of these biosynthetic pathways to total fatty acid composition of intracellular parasite stages remains poorly defined. Here, we use a combination of genetic, biochemical, and metabolomic approaches to delineate the contribution of fatty acid biosynthetic pathways in Toxoplasma gondii. Metabolic labeling studies with [(13)C]glucose showed that intracellular tachyzoites synthesized a range of long and very long chain fatty acids (C14:0-26:1). Genetic disruption of the apicoplast-localized type II fatty-acid synthase resulted in greatly reduced synthesis of saturated fatty acids up to 18 carbons long. Ablation of type II fatty-acid synthase activity resulted in reduced intracellular growth that was partially restored by addition of long chain fatty acids. In contrast, synthesis of very long chain fatty acids was primarily dependent on a fatty acid elongation system comprising three elongases, two reductases, and a dehydratase that were localized to the endoplasmic reticulum. The function of these enzymes was confirmed by heterologous expression in yeast. This elongase pathway appears to have a unique role in generating very long unsaturated fatty acids (C26:1) that cannot be salvaged from the host.     

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.     

Stimulation of glycogen synthesis and lipogenesis by glutamine in isolated rat hepatocytes.

Glutamine stimulated glycogen synthesis and lactate production in hepatocytes from overnight-fasted normal and diabetic rats. The effect, which was half-maximal with about 3 mM-glutamine, depended on glucose concentration and was maximal below 10 mM-glucose. beta-2-Aminobicyclo[2.2.1.]heptane-2-carboxylic acid, an analogue of leucine, stimulated glutaminase flux, but inhibited the stimulation of glycogen synthesis by glutamine. Various purine analogues and inhibitors of purine synthesis were found to inhibit glycogen synthesis from glucose, but they did not abolish the stimulatory effect of glutamine on glycogen synthesis. The correlation between the rate of glycogen synthesis and synthase activity suggested that the stimulation of glycogen synthesis by glutamine depended solely on the activation of glycogen synthase. This activation of synthase was not due to a change in total synthase, nor was it caused by a faster inactivation of glycogen phosphorylase, as was the case after glucose. It could, however, result from a stimulation of synthase phosphatase, since, after the addition of 1 nM-glucagon or 10 nM-vasopressin, glutamine did not interfere with the inactivation of synthase, but did promote its subsequent re-activation. Glutamine was also found to inhibit ketone-body production and to stimulate lipogenesis.