Effects of dexamethasone and insulin on the synthesis of triacylglycerols and phosphatidylcholine and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by monolayer cultures of rat hepatocytes.

Rat hepatocytes in monolayer culture were preincubated for 19 h with 1 microM-dexamethasone, and the incubation was continued for a further 23 h with [14C]oleate, [3H]glycerol and 1 microM-dexamethasone. Dexamethasone increased the secretion of triacylglycerol into the medium in particles that had the properties of very-low-density lipoproteins. The increased secretion was matched by a decrease in the triacylglycerol and phosphatidylcholine that remained in the hepatocytes. Preincubating the hepatocytes for the total 42 h period with 36 nM-insulin decreased the amount of triacylglycerol in the medium and in the cells after the final incubation for 23 h with radioactive substrates. However, insulin had no significant effect on the triacylglycerol content of the cell and medium when it was present only in the final 23 h incubation. Insulin antagonized the effects of dexamethasone in stimulating the secretion of triacylglycerol from the hepatocytes, especially when it was present throughout the total 42 h period. The labelling of lysophosphatidylcholine in the medium when hepatocytes were incubated with [14C]oleate and [3H]glycerol was greater than that of phosphatidylcholine. The appearance of this lipid in the medium, unlike that of triacylglycerol and phosphatidylcholine, was not stimulated by dexamethasone, or inhibited by colchicine. However, the presence of lysophosphatidylcholine in the medium was decreased when the hepatocytes were incubated with both dexamethasone and insulin. These findings are discussed in relation to the control of the synthesis of glycerolipids and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by the liver, particularly in relation to the interactions of glucocorticoids and insulin.     

Precursor supply and hepatic enzyme activities as regulators of triacylglycerol synthesis in isolated hepatocytes and perfused liver

The relative significance of alterations in precursor supply and enzyme activities for the rate of triacylglycerol synthesis was studied in isolated hepatocytes and perfused livers. Precursor availability was varied in vitro by changing the fatty acid concentration in the incubation medium or adding ethanol to the perfusion medium in order to increase the cellular glycerol 3-phosphate concentration. The rate of glycerolipid synthesis in hepatocytes, measured in terms of the label incorporated into the various lipid classes from tritiated glycerol, was strongly dependent on the fatty acid concentration up to 2 mm of oleate (fatty acid/albumin molar ratio $\text{7}\text{1}$). Ethanol in vitro increased the incorporation of labeled oleate into phosphatidic acid and diacylglycerol in the isolated perfused liver, but its effect on the incorporation into triacylglycerol was small. Ethanol in vitro increased the label incorporation into both diacylglycerol and triacylglycerol in the livers from cortisol-treated rats. Although cortisol treatment increased the soluble phosphatidate phosphohydrolase activity 4.4-fold in the hepatocytes, it had no effect on the rate of triacylglycerol synthesis, whereas fasting increased this rate about 3-fold, although only a moderate concomitant increase in soluble phosphatidate phosphohydrolase activity was observed. Neither cortisol treatment nor fasting affected the microsomal glycerol-3-phoshate acyltransferase activity. The results demonstrate that substrate availability can override enzyme modulations in the regulation of triacylglycerol synthesis and that phosphatidate phosphohydrolase is not the main regulator of triacylglycerol synthesis.     

Effect of ethanol on lipid metabolism in cultured hepatocytes.

Isolated rat hepatocytes were cultured in a modified HI-WO/BA medium for 16 h. In the following 24 h oleate or oleate plus ethanol was added to the medium. After this period the medium was changed again and the cultures were further incubated with [1-14C]oleate alone or with [1-14C]oleate plus ethanol for 6 h. This allowed a comparison of effects of short-term (6 h) and long-term (24 + 6 h) exposure to ethanol on fatty acid metabolism. The increased intracellular accumulation of triacylglycerol in the presence of ethanol was quantitatively accounted for by increased fatty acid uptake, by decreased fatty acid oxidation in the tricarboxylic acid cycle and by decreased VLDL (very-low-density lipoprotein)-triacylglycerol secretion. Ketone-body production was not affected. After short-term exposure the rate of accumulation of triacylglycerol was increased by 50%. This increase was accounted for by increased fatty acid uptake (44%), decreased tricarboxylic acid-cycle activity (49%) and decreased VLDL-triacylglycerol secretion (7%). After long-term exposure, the rate of accumulation of triacylglycerol was increased by 74%. This increase was accounted for by increased fatty acid uptake (34%), decreased tricarboxylic acid-cycle activity (34%) and decreased VLDL-triacylglycerol secretion (32%). The larger increase in accumulation of triacylglycerol after long-term exposure to ethanol was entirely accounted for by increased inhibition of secretion of VLDL-triacylglycerol. The biochemical mechanisms underlying the observations are discussed.     

The influence of insulin on glucose and fatty acid metabolism in the isolated perfused rat hind quarter.

Glucose and fatty acid metabolism of resting skeletal muscle were studied by perfusion of the isolated rat hind leg with a hemoglobin-free medium. Tissue integrity was demonstrated by normal ATP, ADP and creatine phosphate levels, by a sufficient oxygen supply, and by a normal appearance of perfused muscle specimens under the electron microscope. The rates of glucose and fatty acid uptake, and of lactate, alanine, glycerol and fatty acid release were constant over a perfusion period of 60 min. Insulin (1 unit/l) caused a more than threefold increase in glucose uptake, a stimulation of lactate production, and a 20% increase in the muscular glycogen levels. Fatty acids and alanine release were significantly diminished by insulin, but glycerol release did not change. The uptake of oleate by the rat hind leg was dependent on the medium concentration in a range of 0.7-1.9mM oleate, and was stimulated by insulin. Glucose uptake was not influenced by oleate, whether sodium was present or not. When the leg was perfused with [1-14C]oleate, 75% of the incorporated fatty acids were found in muscle lipids, 10% were oxidized to CO2, and 5% were recovered in bone lipids. The absolute amount of oleate oxidation was not altered by insulin. In all experiments with and without glucose in the medium, 70-80% of the 14C label incorporated into muscle lipids was found in the triglyceride fraction. In the presence of glucose, insulin significantly increased the incorporation of [1-14C]oleate into muscle triglycerides, whereas no insulin effect, either on fatty acid uptake or on triglyceride formation, could be observed when glucose was omitted from the perfusate. The present results indicate that a "glucose-fatty acid cycle" as found in rat heart muscle does not operate in resting peripheral skeletal muscle tissue. They also demonstrate that the stimulating effect of insulin on muscular fatty acid uptake and triglyceride synthesis is dependent on glucose supply. This finding can be intrepreted as a stimulation of fatty acid esterification by sn-glycerol 3-phosphate derived from an increased glucose turnover, which is in turn due to insulin.     

Factors regulating the secretion of lysophosphatidylcholine by rat hepatocytes compared with the synthesis and secretion of phosphatidylcholine and triacylglycerol. Effects of albumin, cycloheximide, verapamil, EGTA and chlorpromazine.

1. The synthesis and secretion of glycerolipid by monolayer cultures of rat hepatocytes was measured by determining the incorporations of [3H]glycerol, [3H]oleate and [14C]choline and by the absolute concentration of triacylglycerol. 2. The presence of albumin in the medium stimulated the accumulation of lysophosphatidylcholine in the medium by 11-13-fold. 3. Cycloheximide did not significantly alter the accumulation of lysophosphatidylcholine. 4. This process was particularly sensitive to inhibition by chlorpromazine and verapamil, compared with the secretion of triacylglycerol and phosphatidylcholine. By contrast, it was relatively less sensitive to EGTA. 5. It is suggested that intracellular Ca2+ may be important in the production of lysophosphatidylcholine, which then accumulates in the medium by binding to albumin. In vivo this lysophosphatidycholine may be a means of delivering choline and polyunsaturated fatty acids to other organs.     

Alpha-adrenergic suppression of very-low-density-lipoprotein triacylglycerol secretion by isolated rat hepatocytes.

The effect of adrenaline on triacylglycerol synthesis and secretion was examined in isolated rat hepatocytes. Cells were incubated with 0.5 mM-[1-14C]oleate, and the accumulation of triacylglycerol and [14C]triacylglycerol was measured in the incubation medium. Triacylglycerol appearing in the medium was present in a form with properties similar to very-low-density lipoproteins. Triacylglycerol, [14C]triacylglycerol and [14C]phospholipid contents of hepatocytes were also determined. Addition of 10 microM-(-)adrenaline decreased accumulation of glycerolipid in the incubation medium and also decreased cellular [14C]phospholipid content. Prazosin abolished these effects, whereas propranolol did not. The hormone did not affect cellular triacylglycerol content or rates of incorporation of [1-14C]oleate into cell triacylglycerol. The effect of adrenaline on the removal of newly secreted triacylglycerol and the secretion of synthesized glycerolipid was also examined. The catecholamine did not affect rates of removal of newly secreted triacylglycerol. Adrenaline did inhibit the secretion of pre-synthesized lipid by the cells, as assessed by the appearance of radiolabelled triacylglycerol from hepatocytes that had been preincubated with [1,2,3-3H]-glycerol. Adrenaline did not affect rates of fatty acid uptake by hepatocytes, but did stimulate oxidation of [1-14C]oleate, principally to 14CO2.     

Triacylglycerol metabolism in isolated rat kidney cortex tubules

Triacylglycerol metabolism has been studied in kidney cortex tubules from starved rats, prepared by collagenase treatment. Triacylglycerol was determined by a newly developed fully enzymic method. Incubation of tubules in the absence of fatty acids led to a decrease of endogenous triacylglycerol by about 50% in 1h. Addition of albuminbound oleate or palmitate resulted in a steady increase of tissue triacylglycerol over 2h. The rate of triacylglycerol synthesis was linearly dependent on oleate concentration up to 0.8mm, reaching a saturation at higher concentrations. Triacylglycerol formation from palmitate was less than that from oleate. This difference was qualitatively the same when net synthesis was compared with incorporation of labelled fatty acids. Quantitatively, however, the difference was less with the incorporation technique. Gluconeogenic substrates, which by themselves had no effect on triacylglycerol concentrations, stimulated neutral lipid formation from fatty acids. Glucose and lysine did not have such a stimulatory effect. Inhibition of gluconeogenesis from lactate by mercaptopicolinic acid likewise inhibited triacylglycerol formation. This inhibitory effect was seen with oleate as well as with oleate plus lactate. When [2-¹⁴C]lactate was used the incorporation of label into triacylglycerol was found in the glycerol moiety exclusively. Addition of dl-β-hydroxybutyrate (5mm) to the incubation medium in the presence of oleate or oleate plus lactate led to a significant increase in triacylglycerol formation. In contrast with the gluconeogenic substrates, dl-β-hydroxybutyrate had no stimulatory effect on fatty acid uptake. The results suggest that renal triacylglycerol formation is a quantitatively important metabolic process. The finding that gluconeogenic substrates, but not glucose, increase lipid formation, indicates that the glycerol moiety is formed by glyceroneogenesis in the proximal tubules. The effect of ketone bodies seems to be caused by the sparing action of these substrates on fatty acid oxidation. The decrease of triacylglycerol in the absence of exogenous substrates confirms previous conclusions that endogenous lipids provide fatty acids for renal energy metabolism.     

Storage, mobilization and secretion of cytosolic triacylglycerol in hepatocyte cultures. The role of insulin.

Cytosolic triacylglycerol labelled from [3H]oleate accounted for almost 50% (57 +/- 22 nmol/mg of protein) of the total cellular triacylglycerol which was newly synthesized by cultured hepatocytes during a 24 h incubation. Insulin decreased the export of triacylglycerol as very-low-density lipoprotein (VLDL) during this period. This resulted in a sequestration of newly synthesized triacylglycerol in the cytosol, rather than in the particulate fraction of the cell. Longer periods of incubation with [3H]oleate resulted in increased concentrations of newly synthesized triacylglycerol within the cell, most of which (78 +/- 3% after 48 h; 80 +/- 3% after 72 h) was located within the cytosolic fraction. The quantity of newly synthesized triacylglycerol in the cell cytosol was further increased by insulin. During these periods there were decreases in the amounts of triacylglycerol associated with the particulate fraction of the cell, irrespective of the presence or absence of insulin. In no case was a decrease in VLDL triacylglycerol secretion in response to insulin accompanied by an increased triacylglycerol content in the particulate fraction of the cell. In some experiments, the fate of the cytosolic triacylglycerol was studied by pulse labelling with [3H]oleate. In these cases, when insulin was removed from the medium of cells to which they had previously been exposed, more newly synthesized triacylglycerol was secreted compared with cells which had not been exposed to insulin. This extra triacylglycerol was mobilized from the cytosolic rather than from the particulate fraction of the cell. Subsequent addition of insulin to the medium prevented the mobilization of cytosolic triacylglycerol. These results suggest that insulin enhances the storage of hepatocellular triacylglycerol in a cytosolic pool. Deficiency of insulin in the medium stimulates the mobilization of this pool which is channelled into the secretory pathway, entering the extracellular medium as VLDL.     

Metabolic changes induced during adaptation of Saccharomyces cerevisiae to a water stress

When exponentially growing Saccharomyces cerevisiae was transferred from a normal high water activity growth medium (a_w 0.997) to a medium containing 8% NaCl low water activity growth medium (a_w 0.955), glycerol accumulation during the first eight hours of the adaptation was both retarded and greatly diminished in magnitude. Investigation of the underlying reasons for the slow onset of glycerol accumulation revealed that not only was overall glycerol production reduced by salt transfer, but also the rates of ethanol production and glucose consumption were reduced. Measurement of glycolytic intermediates revealed an accumulation of glucose-6-phosphate, fructose-6-phosphate, fructose 1,6 bisphosphate and phosphoenolpyruvate in S. cerevisiae 3 to 4 h after transfer to salt, suggesting that one or more glycolytic enzymes were inhibited. Potassium ions accumulated in S. cerevisiae after salt transfer and reached a maximum about 6 h after transfer, whereas the sodium ion content increased progressively during the adaptation period. The trehalose content also increased in adapting cells. It is suggested that inhibition of glycerol production during the initial period of adaptation could be due to either the inhibition of glycerol-3-phosphate dehydrogenase by increased cation content or the inhibitin of glycolysis, glycerol being produced glycolytically in S. cerevisiae. The increased accumulation of glycerol towards the end of the 8-h period suggests that the osmoregulatory response of S. cerevisiae involves complex sets of adjustments in which inhibition of glycerol-3-phosphate dehydrogenase must be relieved before glycerol functions as a major osmoregulator.     

Insulin and oxytocin effects on phosphoinositide metabolism in adipocytes

The effects of hormones on phosphoinositide metabolism were examined in rat adipocytes prelabeled with 32Pi or [3H]inositol. Oxytocin and vasopressin produced large decreases in labeled polyphosphoinositides and increases in phosphatidic acid and inositol phosphates, whereas insulin was without effect, although it stimulated lipogenesis from glucose. Likewise, insulin did not elevate 1,2-diacylglycerol measured chemically by high pressure liquid or thin-layer chromatography in fat cells or pads. It also did not increase the radioactivity in 1,2-diacylglycerol in ghosts prepared from fat cells previously labeled with [3H]arachidonic acid, although oxytocin and vasopressin increased this. It is therefore concluded that insulin does not stimulate the breakdown of polyphosphoinositides to yield 1,2-diacylglycerol and inositol phosphates in adipocytes and that the insulin-like actions of oxytocin must be due to other changes. Insulin induced small, but significant and equal increases (40% at 30 min) in the incorporation of [3H] inositol into phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in adipocytes. The effects were not dependent upon glucose and were not evident before 15 min. Oxytocin also produced large increases in the labeling of the three phosphoinositides. Insulin stimulated the incorporation of [3H]glycerol into the three phosphoinositides and also phosphatidic acid, phosphatidylserine, and phosphatidylethanolamine by 50-100% in cells incubated without glucose. No changes in the labeling of glycerol 3-phosphate, lysophosphatidic acid, phosphatidylcholine, and triacylglycerol were detected, and there was a small increase (30%) in 1,2-diacylglycerol labeling. It is concluded that insulin increases the synthesis of phosphatidylinositol, phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, phosphatidylethanolamine, and phosphatidylserine in fat cells partly by stimulating a reaction(s) located between glycerol 3-phosphate and phosphatidic acid in the biosynthetic pathway.     

Mechanisms of intestinal triacylglycerol synthesis

Triacylglycerols are a major source of stored energy that are obtained either from the diet or can be synthesized to some extent by most tissues. Alterations in pathways of triacylglycerol metabolism can result in their excessive accumulation leading to obesity, insulin resistance, cardiovascular disease and nonalcoholic fatty liver disease. Most tissues in mammals synthesize triacylglycerols via the glycerol 3-phosphate pathway. However, in the small intestine the monoacylglycerol acyltransferase pathway is the predominant pathway for triacylglycerol biosynthesis where it participates in the absorption of dietary triacylglycerol. In this review, the enzymes that are part of both the glycerol 3-phosphate and monoacylglycerol acyltransferase pathways and their contributions to intestinal triacylglycerol metabolism are reviewed. The potential of some of the enzymes involved in triacylglycerol synthesis in the small intestine as possible therapeutic targets for treating metabolic disorders associated with elevated triacylglycerol is briefly discussed.     

A complete separation of dimethylaminoazobenzenesulphonyl-amino acids. Amino acid analysis with low nanogram amounts of polypeptide with dimethylaminoazobenzenesulphonyl chloride.

Triacylglycerol synthesis by glycogen-depleted hepatocytes from fed rats that have low glycerol 3-phosphate contents was stimulated by the addition of glycerol 3-phosphate precursors. Glucagon decreased triacylglycerol synthesis only when it also lowered glycerol 3-phosphate content. The hyperbolic-like relationship between glycerol 3-phosphate content and rates of triacylglycerol synthesis was identical in the absence or presence of glucagon, indicating that the glucagon effect on triacylglycerol synthesis was not mediated through changes in enzyme activities of the esterification pathway but through changes in cellular glycerol 3-phosphate content.     

Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea lanceolata.

Embryos of Cuphea lanceolata have more than 80 mol% of decanoic acid ('capric acid') in their triacylglycerols, while this fatty acid is virtually absent in phosphatidylcholine (PtdCho). Seed development was complete 25-27 days after pollination, with rapid triacylglycerol deposition occurring between 9 and 24 days. PtdCho amounts increased until day 15 after pollination. Analysis of embryo lipids showed that the diacylglycerol (DAG) pool consisted of mainly long-chain molecular species, with a very small amount of mixed medium-chain/long-chain glycerols. Almost 100% of the fatty acid at position sn-2 in triacylglycerols (TAG) was decanoic acid. When equimolar mixtures of [14C]decanoic and [14C]oleic acid were fed to whole detached embryos, over half of the radioactivity in the DAG resided in [14C]oleate, whereas [14C]decanoic acid accounted for 93% of the label in the TAG. Microsomal preparations from developing embryos at the mid-stage of TAG accumulation catalysed the acylation of [14C]glycerol 3-phosphate with either decanoyl-CoA or oleoyl-CoA, resulting in the formation of phosphatidic acid (PtdOH), DAG and TAG. Very little [14C]glycerol entered PtdCho. In combined incubations, with an equimolar supply of [14C]oleoyl-CoA and [14C]decanoyl-CoA in the presence of glycerol 3-phosphate, the synthesized PtdCho species consisted to 95% of didecanoic and dioleic species. The didecanoyl-glycerols were very selectively utilized over the dioleoylglycerols in the production of TAG. Substantial amounts of [14C]oleate, but not [14C]decanoate, entered PtdCho. The microsomal preparations of developing embryos were used to assess the acyl specificities of the acyl-CoA:sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) and the acyl-CoA:sn-1-acyl-glycerol-3-phosphate acyltransferase (LPAAT, EC 2.3.1.51) in Cuphea lanceolata embryos. The efficiency of acyl-CoA utilization by the GPAT was in the order decanoyl = dodecanoyl greater than linoleoyl greater than myristoyl = oleoyl greater than palmitoyl. Decanoyl-CoA was the only acyl donor to be utilized to any extent by the LPAAT when sn-decanoylglycerol 3-phosphate was the acyl acceptor. sn-1-Acylglycerol 3-phosphates with acyl groups shorter than 16 carbon atoms did not serve as acyl acceptors for long-chain (greater than or equal to 16 carbon atoms) acyl-CoA species. On the basis of the results obtained, we propose a schematic model for triacylglycerol assembly and PtdCho synthesis in a tissue specialized in the synthesis of high amounts of medium-chain fatty acids.     

Key role for ceramides in mediating insulin resistance in human muscle cells

Elevated non-esterified fatty acids, triglyceride, diacylglycerol, and ceramide have all been associated with insulin resistance in muscle. We set out to investigate the role of intramyocellular lipid metabolites in the induction of insulin resistance in human primary myoblast cultures. Muscle cells were subjected to adenovirus-mediated expression of perilipin or incubated with fatty acids for 18 h, prior to insulin stimulation and measurement of lipid metabolites and rates of glycogen synthesis. Adenovirus-driven perilipin expression lead to significant accumulation of triacylglycerol in myoblasts, without any detectable effect on insulin sensitivity, as judged by the ability of insulin to stimulate glycogen synthesis. Similarly, incubation of cells with the monounsaturated fatty acid oleate resulted in triacylglycerol accumulation without inhibiting insulin action. By contrast, the saturated fatty acid palmitate induced insulin resistance. Palmitate treatment caused less accumulation of triacylglycerol than did oleate but also induced significant accumulation of both diacylglycerol and ceramide. Insulin resistance was also caused by cell-permeable analogues of ceramide, and palmitate-induced resistance was blocked in the presence of inhibitors of de novo ceramide synthesis. Oleate co-incubation completely prevented the insulin resistance induced by palmitate. Our data are consistent with ceramide being the agent responsible for insulin resistance caused by palmitate exposure. Furthermore, the triacylglycerol derived from oleate was able to exert a protective role in sequestering palmitate, thus preventing its conversion to ceramide.     

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of insulin, adrenaline and some metabolites in vitro

1. Adipose tissues from rats fed a balanced diet were incubated in the presence of glucose (20mm) with the following additions: insulin, anti-insulin serum, insulin+acetate, insulin+pyruvate, insulin+lactate, insulin+phenazine methosulphate, insulin+oleate+albumin, insulin+adrenaline+albumin, insulin+6-N-2'-O-dibutyryl 3':5'-cyclic AMP+albumin. 2. Measurements were made of the whole tissue concentrations of adenine nucleotides, hexose phosphates, triose phosphates, glycerol 1-phosphate, 3 phosphoglycerate, 6-phosphogluconate, long-chain fatty acyl-CoA, acid-soluble CoA, citrate, isocitrate, malate and 2-oxoglutarate, and of the release into the incubation medium of lactate, pyruvate and glycerol after 1h of incubation. 3. Fluxes of [(14)C]glucose carbon through the major pathways of glucose metabolism were calculated from the yields of (14)C in various products after 2h of incubation. Fluxes of [(14)C]acetate, [(14)C]pyruvate or [(14)C]lactate carbon in the presence of glucose were also determined. 4. Measurements were also made of the whole-tissue concentrations of metabolites in tissues taken directly from Nembutal-anaesthetized rats. 5. Whole tissue mass-action ratios for phosphofructokinase, phosphoglucose isomerase and the combined (aldolasextriose phosphate isomerase) reaction were similar in vivo and in vitro. The reactants of phosphofructokinase appeared to be far from mass-action equilibrium. In vitro, the reactants of hexokinase also appeared to be far from mass-action equilibrium. 6. Correlation of observed changes in glycolytic flux with changes in fructose 6-phosphate concentration suggested that phosphofructokinase may show regulatory behaviour. The enzyme appeared to be activated in the presence of oleate or adrenaline and to be inhibited in the presence of lactate or pyruvate. 7. Evidence is presented that the reactants of lactate dehydrogenase and glycerol 1-phosphate dehydrogenase may be near to mass-action equilibrium in the cytoplasm. 8. No satisfactory correlations could be drawn between the whole-tissue concentrations of long-chain fatty acyl-CoA, citrate and glycerol 1-phosphate and the observed rates of triglyceride and fatty acid synthesis. Under the conditions employed, the concentration of glycerol 1-phosphate appeared to depend mainly on the cytoplasmic [NAD(+)]/[NADH] ratios. 9. Calculated hexose monophosphate pathway flux rates roughly correlated with fatty acid synthesis rates and with whole tissue [6-phosphogluconate]/[glucose 6-phosphate] ratios. The relative rates of production of NADPH for fatty acid synthesis by the hexose monophosphate pathway and by the ;malic enzyme' are discussed. It is suggested that all NADH produced in the cytoplasm may be used in that compartment for reductive synthesis of fatty acids, lactate or glycerol 1-phosphate.     

Effect of hydrazine exposure on hepatic triacylglycerol biosynthesis.

Acute hydrazine exposure elevated rat liver triacylglycerol content and produced a rapid rise in triacylglycerol production from sn-[1,3-14C]glycerol 3-phosphate by liver homogenate and microsomal fractions. Hydrazine treatment also increased the incorporation of [1,3-14C]glycerol into hepatic triacylglycerol by the intact animal. Homogenates of hepatocyte monolayers exposed to hydrazine in vitro also exhibited an increased capacity to form triacylglycerol from sn-[1,3-14C]glycerol 3-phosphate. Hydrazine-dependent increases in hepatic triacylglycerol production measured in vitro correlated well with an increase in microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) activity. Therefore, the fatty liver associated with hydrazine exposure may be explained in part by a rise in the enzymatic capacity of hepatic triacylglycerol biosynthesis.     

Measurement of flow of carbon atoms from glucose and glycogen glucose to glyceride glycerol and glycerol in rat heart and epididymal adipose tissue: Effects of insulin, adrenaline and alloxan-diabetes

1. Flow of carbon atoms from glucose and glycogen glucose to glyceride glycerol, glyceride fatty acids and glycerol was calculated in the perfused rat heart and incubated epididymal adipose tissue from the incorporation of (14)C from [U-(14)C]-glucose (into glyceride glycerol, glyceride fatty acids and glycerol in the medium), and from measurements of the specific activity of l-glycerol 3-phosphate, and the effects of insulin, adrenaline and alloxan-diabetes were studied. Measurements were also made of the uptake of glucose and the outputs of lactate, pyruvate and glycerol. 2. New methods are described for the measurement of radioactivity in small amounts of metabolites (glycerol, glucose 6-phosphate and fructose 6-phosphate and l-glycerol 3-phosphate) in which use has been made of alterations in charge induced by enzymic conversions to effect resolution by ion-exchange chromatography. 3. In hearts the specific activity of l-glycerol 3-phosphate was less than that of glucose in the medium but similar to that of lactate released during perfusion. Because repeated measurements of the specific activity of l-glycerol 3-phosphate was impracticable, the specific activity of lactate has been used as an indirect measurement of glycerol phosphate specific activity. 4. In fat pads, specific activity of lactate was the same as that of glucose in the medium and thus the specific activity of l-glycerol 3-phosphate was taken to be the same as that of medium glucose. 5. In hearts from alloxan-diabetic rats, despite decreased glucose uptake and l-glycerol 3-phosphate concentration, flow of carbon atoms through l-glycerol 3-phosphate to glyceride glycerol was increased about threefold. 6. In fat pads, flow of carbon atoms through l-glycerol 3-phosphate to glyceride glycerol was increased by insulin (twofold), by adrenaline in the presence of insulin (fivefold) and by diabetes in pads incubated with insulin (1.5-fold). These increases could not be correlated either with increases in glucose uptake, which was unchanged by adrenaline and decreased in diabetes, or with the concentration of l-glycerol 3-phosphate, which was decreased by adrenaline and unchanged in diabetes. 7. These results are discussed in relation to the control of glyceride synthesis in heart and adipose tissue and to the regulation of glyceride fatty acid oxidation in the perfused rat heart.     

Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes.

We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30 min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC/PE labelling by [3H]arachidonic acid, [3H]myristic acid, and [14C]choline. Phenylephrine, ionophore A23187 and phorbol esters did not increase [2-3H]glycerol incorporation into DAG or other glycerolipids in 2-h-prelabelling experiments; thus activation of the phospholipase C which hydrolyses phosphatidylinositol, its mono- and bis-phosphate, Ca2+ mobilization, and protein kinase C activation, appear to be ruled out as mechanisms to explain the insulin effect on synthesis de novo of PA, DAG and PC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gene expression profiles and intracellular contents of stress protectants in Saccharomyces cerevisiae under ethanol and sorbitol stresses

In response to osmotic stress, proline is accumulated in many bacterial and plant cells. During various stresses, the yeast Saccharomyces cerevisiae induces glycerol or trehalose synthesis, but the fluctuations in gene expression and intracellular levels of proline in yeast are not yet well understood. We previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. In this study, we examined the relationships between the gene expression profiles and intracellular contents of glycerol, trehalose, and proline under stress conditions. When yeast cells were exposed to 1 M sorbitol stress, the expression of GPD1 encoding glycerol-3-phosphate dehydrogenase is induced, leading to glycerol accumulation. In contrast, in the presence of 9% ethanol, the rapid induction of TPS2 encoding trehalose-6-phosphate phosphatase resulted in trehalose accumulation. We found that intracellular proline levels did not increase immediately after addition of sorbitol or ethanol. However, the expressions of genes involved in proline synthesis and degradation did not change during exposure to these stresses. It appears that the elevated proline levels are due primarily to an increase in proline uptake from a nutrient medium caused by the induction of PUT4. These results suggest that S. cerevisiae cells do not accumulate proline in response to sorbitol or ethanol stress different from other organisms.     

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.