Metabolism of palmitate in perfused rat liver. Isolation of subcellular fractions containing triacylglycerol.

1. The metabolism of [1-14C]palmitate in rat liver was studied in a single-pass perfusion system at concentrations of 0.2 or 1 mM. 2. After the perfusion the liver was homogenized and the floating fat was isolated. The incorporation of [1-14C]palmitate into triacylglycerol in this pool increased 9-fold when the palmitate concentration in the medium was increased from 0.2 to 1 mM. In time studies with 1 mM-[1-14C]palmitate 75% of the total accumulation of triacylglycerol occurred in this pool. Our results support the concept that the floating-fat fraction contains the storage pool of triacylglycerol, i.e. the cytoplasmic lipid droplets. 3. In a particulate preparation consisting mainly of mitochondria and microsomal fraction the incorporation of [1-14C]palmitate into triacylglycerol was proportional to the fatty acid concentration. Triacylglycerol in the perfusate medium and in the particulate fraction was in isotopic equilibrium, which indicates that the particulate fraction contained the precursor pool for secreted triacylglycerol, i.e. the pool in endoplasmic reticulum and Golgi apparatus. 4. The oxidation to labelled water-soluble products and to CO2 was increased 14-fold by the 5-fold increase in palmitate concentration.     

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.     

Mechanism of the hypertriglyceridemia induced by tumor necrosis factor administration to rats

4 h after intravenous injection of recombinant HuTNF-alpha to fed rats, an increase in heart, diaphragm, and plasma lipoprotein lipase activity was observed. At the same time, a 40-60% decrease in enzymic activity in epididymal fat pad and kidney and 40% decrease in hepatic lipase activity in liver had occurred. Similar results were obtained 20 h after injection of recombinant HuTNF-alpha into fasted rats. Pretreatment with Indomethacin did not affect the changes in tissue lipoprotein lipase activity observed following recombinant HuTNF-alpha administration. Serum triacylglycerol concentration increased by 2- and 6-fold; 4 and 20 h after recombinant HuTNF-alpha administration. Disappearance of 14C-labeled triacylglycerol from the circulation after injection of small chylomicrons, biosynthetically labeled in their triacylglycerol and cholesterol moieties, was lower in TNF-treated than in control rats. However, the clearance rate of triacylglycerol was the same or even higher in recombinant HuTNF-alpha treated rats (assuming that 14C-labeled chylomicron triacylglycerol represents the serum triacylglycerol pool). The livers of recombinant HuTNF-alpha-treated rats and controls contained similar amounts of 14C-labeled lipids, but less [3H]cholesterol, suggesting that in recombinant HuTNF-alpha-treated rats, the liver took up chylomicron remnant particles enriched with triacylglycerol. Separation of the d less than 1.04 g/ml fraction of serum obtained from control and recombinant HuTNF-alpha treated rats by zonal ultracentrifugation revealed that in recombinant HuTNF-alpha-treated rats the lipoprotein particles were less lipolyzed than in controls. The secretion rate of [3H]triacylglycerol into the serum was determined 90 min after injection of [3H]palmitate albumin complex and Triton WR 1339. In recombinant HuTNF-alpha-treated rats, the secretion of [3H]triacylglycerol into plasma was 48% higher than in controls. It is suggested that the increase in lipoprotein lipase activity of heart and diaphragm resulted from an indirect effect of TNF. It is concluded that the increase in serum triacylglycerol in the recombinant HuTNF-alpha-treated rats is due mainly to an increased secretion of triacylglycerol by the liver. Impaired lipolysis, probably due to a fall in hepatic lipase could also contribute to the rise in plasma triacylglycerol.     

Brain cholesterol XVIII: EFFECt of methylphenidate (Ritalin) on [U-14C] glucose and [2-3H] acetate incorporation.

The effect of a single injection of methylphenidate (Ritalin, 4 mg/kg) on precursor ([2-3H]acetate and [U-14C]glucose) incorporation into brain cholesterol was studied. The drug caused a steady decrease in the concentration of brain cholesterol during the 24-hr period examined. Incorporation studies during this time with [U-14C]glucose indicated higher than normal incorporation for all time periods studied. The most significant incorporation increases took place 2 and 4 hr after drug injection. Experiments using [2-3H]acetate as the sterol precursor gave incorporation values which tended (not significantly) to be lower than control values at 2 and 4 h. The values after 12 hr were less than normal, while the 24-hr group indicated an increase to or slightly higher than normal values. These data suggest that the pharmacological effect of methylphenidate may be due to lowering of brain cholesterol levels directly or on some more basic metabolic process leading to a decreased level of membrane sterols.     

Glutamine metabolism in isolated incubated adipocytes of the rat.

1. Phosphate-dependent glutaminase activity in the epididymal fat-pad was 15.1 nmol/min per mg of protein. Glutaminase activity demonstrated differences with respect to adipose-tissue sites. Considerable variation was found in different sites of adipose tissue from lean control and Zucker obese animals. 2. Adipocytes incubated in the presence of 2 mM-glutamine utilized glutamine at a rate of 1.8 mumol/h per g dry wt., and glutamate, ammonia, lactate and alanine were produced. Addition of glucose plus insulin increased the rates of glutamine utilization and glutamate, ammonia, lactate and alanine production. Isoprenaline alone or plus glucose further stimulated the rate of glutamine utilization and formation of end products. 3. The rate of incorporation of 14C from glutamine into CO2 was similar to that of glucose, but the rate of incorporation into triacylglycerol was much less. Addition of unlabelled glucose or glucose plus insulin stimulated the rate of incorporation of [14C]glutamine into triacylglycerol, but had no effect on that of 14CO2 formation. Isoprenaline plus glucose increased the rate of incorporation of [14C]glutamine into CO2, but decreased the rate of incorporation into triacylglycerol. 4. In the absence of insulin, the rate of [14C]glutamine incorporation into triacylglycerol was related to the glucose concentration (0-10 mM). However, in the presence of insulin, the rate of incorporation of [14C]glutamine was maximal at 1 mM-glucose.     

Lipid and lipoprotein metabolism in Hep G2 cells

Lipid composition, lipid synthesis and lipoprotein secretion by the Hep G2 cell line have been studied with substrate and insulin supplied under different conditions. The lipid composition of Hep G2 cells was close to that of normal human liver, except for a higher content in sphingomyelin (P less than 0.005) and a lower phosphatidylcholine/sphingomyelin ratio. Most of the [14C]triacylglycerols secreted into the medium were recovered by ultracentrifugation at densities of 1.006 to 1.020 g/ml. The main apolipoproteins secreted were apo B-100 and apo A-I. Hep G2 mRNA synthesized in vitro the pro-apolipoproteins A-I and E. Triacylglycerol secretion was 7.38 +/- 1.04 micrograms/mg cell protein per 20 h with 5.5 mM glucose in the medium and increased linearly with glucose concentration. Oleic acid (1 mM) increased the incorporation of [3H]glycerol into the medium and cell triacylglycerols by 251 and 899%, with a concomitant increment in cell triacylglycerols and cholesterol ester. Insulin (1 mU or 7 pmol/ml) inhibited triacylglycerol secretion and [35S]methionine incorporation into secreted protein by 47 and 28%, respectively, with a corresponding increase in the cells. Preincubation of cells with 2.5-10 mM mevalonolactone decreased the incorporation of [14C]acetate into cholesterol 6.2-fold, indicating an inhibitory effect on HMG-CoA reductase. It is concluded that in spite of some differences between Hep G2 and normal human hepatocytes, this line offers an alternative and reliable model for studies on liver lipid metabolism.     

Occurrence of squalene and sterols in Cellulomonas dehydrogenans (Arnaudi 1942) comb. nov. Hester 1971.

The neutral lipid fraction of the photochromogenic, coryneform bacterium Cellulomonas dehydrogenans (Arnaudi 1942) comb. nov. contains the sterol precursor squalene and at least two sterols, cholesterol and beta-sitosterol. The compounds were characterized by mass spectrometry and combination gas-liquid chromatography--mass spectrometry. De novo sterol biosynthetic ability was shown from incorporation of 14C from D-[U-14C]glucose into squalene and the sterol fraction. The squalene concentration approximated 0.002 to 0.005% of the total dry cell weight, and the sterols approximated 0.03 to 0.05%.     

Cellular cholesterol metabolism in mitogen-stimulated lymphocytes--requirement for de novo synthesis

The relationship between cholesterol synthesis and uptake in proliferating lymphocytes has been examined. [14C]Acetate incorporation into lymphocytes cultured under lipoprotein-deficient conditions increased initially in response to mitogen, decreased after 24 h, and increased rapidly between 72 and 96 h. Addition of LDL (10 micrograms/ml) to the culture during the 'trough' period caused [14C]acetate incorporation to return rapidly to baseline, while at peak periods LDL suppression of cholesterol synthesis was minimal. Lymphocytes cultured in the presence of the HMG-CoA reductase inhibitor, mevinolin, exhibited a time-dependent increase in their capacity to incorporate [14C]acetate into cholesterol, evident when mevinolin was removed by washing prior to assay. PHA enhanced 125I-labelled LDL receptor-mediated binding by lymphocytes cultured in lipoprotein-deficient medium over a 4 day period and mevinolin augmented the effect. [3H]Thymidine incorporation into mitogen-stimulated lipoprotein-deficient cultures was inhibited up to 75% by mevinolin (1 mumol/l). LDL (2.5-10 micrograms/ml) substantially reversed this inhibition in 72 h cultures, but only partially overcame inhibition in cells cultured for 96 h. Results suggest that endogenous cholesterol synthesis may be obligatory for lymphocyte proliferation after the initial round of cell division.     

Effects of 1,25-dihydroxyvitamin D-3 on phospholipid metabolism in chick myoblasts

1,25-Dihydroxyvitamin D-3 has been shown to increase phosphatidylcholine and decrease phosphatidylethanolamine levels of myoblasts. Recent studies have suggested that the metabolite stimulates the methylation of phosphatidylethanolamine into phosphatidylcholine. In addition, the sterol increases the arachidonate content of phosphatidylcholine. Experiments were carried out to identify the steps of muscle cell lipid metabolism affected by 1,25-dihydroxyvitamin D-3. Primary cultures of chick embryo myoblasts pretreated with physiological concentrations of 1,25-dihydroxyvitamin D-3 were labelled with [14C]ethanolamine. The sterol increased the incorporation of precursor into dimethylphosphatidylethanolamine and phosphatidylcholine, whereas it decreases the labelling of phosphatidylethanolamine. Prior treatment with cycloheximide and actinomycin D blocked these changes. 1,25-Dihydroxyvitamin D-3 also stimulated the incorporation of [14C]ethanolamine into CDP-ethanolamine. In addition, the sterol increased the incorporation of [3H]arachidonic acid into the phosphatidylcholine fraction but did not affect the incorporation of [14C]palmitic acid. The incorporation of labelled fatty acids into diacylglycerol was not changed by the sterol, whereas it stimulated incorporation of both precursors into triacylglycerol. The data indicate that 1,25-dihydroxyvitamin D-3 enhances the synthesis of phosphatidylcholine through a stimulation of de novo synthesis and methylation of phosphatidylethanolamine via a nuclear mechanism. The sterol may also increase the polyunsaturated fatty acid content of phosphatidylcholine by means of an activation of its deacylation-reacylation cycle.     

Effect of CO 2 concentration on phospholipid metabolism in the isolated perfused rat lung

Studies have been carried out on the incorporation of [U-(14)C]glucose, [2-(14)C]pyruvate, [2-(14)C]acetate, and [1-(14)C]-palmitate into the phospholipids of the isolated perfused rat lung in the presence of either 6 or 45 mm total CO(2) concentration in the perfusion medium. Incorporation of [U-(14)C]glucose into total phospholipid and into the phosphatidylcholine fraction was increased 19-53% over the 2-hr perfusion period in lungs perfused with medium containing 45 as compared with 6 mm CO(2). The incorporation of [2-(14)C]acetate, [2-(14)C]-pyruvate, and [1-(14)C]palmitate was not affected by the change in medium CO(2) concentration. Increased incorporation of [U-(14)C]glucose combined with a shift toward greater incorporation into the fatty acids of the phosphatidylcholine fraction produced a maximum increase of 90% in [U-(14)C]glucose incorporation into the fatty acids of phosphatidylcholine after 2 hr of perfusion in the presence of medium containing 45 mm CO(2) as compared with 6 mm CO(2). The increase in medium CO(2) concentration produced as much as a 150% increase in [U-(14)C]glucose incorporation into palmitate derived from the phosphatidylcholine fraction. The results provide evidence that glucose functions as an important precursor of palmitate in the phosphatidylcholine fraction of lung phospholipids and that the CO(2) concentration of the perfusion medium affects the incorporation of glucose into palmitate.     

Partial characterization of steryl ester biosynthesis in spinach leaves

Acetone powders of a 20,000g pellet fraction from spinach leaves (Spinacia oleracea L.) synthesized [4-(14)C]cholesteryl esters when incubated with [4-(14)C]cholesterol. The reaction was inhibited by digitonin. There was a reciprocal relationship between the decline of label in cholesterol and its incorporation into cholesteryl ester, indicating that free cholesterol was the direct precursor for cholesteryl ester biosynthesis. The hydrolysis of cholesteryl [1-(14)C]palmitate into free cholesterol and [1-(14)C]palmitate was not detected in these acetone powder preparations. Exogenous cholesteryl palmitate had no effect on the esterification of [4-(14)C]cholesterol. The data indicate that an esterase-type mechanism was not involved in the biosynthesis of these steryl esters. Label from [1-(14)C]palmitoyl-CoA was incorporated into steryl esters when incubated with spinach leaf acetone powder preparations. The optimal buffer for steryl ester biosynthesis was 2-(N-morpholino)ethanesulfonate and the optimal pH was 6. Iodoacetamide, N-ethylmaleimide, and dithiothreitol had no effect on the esterification reaction. Ethylenediaminetetraacetate, MgCl(2), CaCl(2), MnCl(2), and ZnSO(4) inhibited at concentrations of 10 to 30 mm.     

Incorporation of mevalonate into dolichol and other isoprenoids during estrogen-induced chick oviduct differentiation

Incorporation of [14C]mevalonate into dolichol and other isoprenoid compounds by chick oviduct explants has been studied. A reliable assay of dolichol biosynthesis employing several chromatographic procedures, including two-dimentional TLC, was developed. Incorporation of [14C]mevalonate into dolichol by oviduct explants was linear for at least 6 h. The effect of estrogen-induced differentiation was studied by incubation of explants obtained from chicks treated for various periods of time with diethylstilbestrol. Mevalonate incorporation into dolichol, when expressed as cpm per g of tissue, was not affected by estrogen treatment, but since the oviduct increased about 100-fold in mass during differentiation, each oviduct synthesizes about 100-fold more dolichol. In most tissues, the major product of mevalonate incorporation is cholesterol. However, although approx. 90% of the non-saponifiable 14C-labeled compounds were in the so-called 'cholesterol fraction', oviduct explants from estrogenized chicks synthesized little, if any, cholesterol. A number of cholesterol biosynthetic intermediates were observed, with compounds comigrating with squalene and lanosterol accounting for about 50% of the total. Since the estrogenized chick has serum cholesterol levels in the range of 800-900 mg/dl, these results suggest that oviduct has secondary control points which allow it to inhibit cholesterol synthesis when mevalonate is used as the precursor. In support of this hypothesis is the observation that explants from untreated chicks can incorporate mevalonate into cholesterol.     

Effect of 25-hydroxycholesterol and bile acids on the regulation of cholesterol metabolism in Hep G2 cells.

The effect of 25-hydroxycholesterol (25-OH-cholesterol) and chenodeoxycholic (CDC) acid on apoprotein secretion, low-density lipoprotein receptor activity, and [3H]triacylglycerol secretion in Hep G2 cells was studied. Both 25-OH-cholesterol and CDC acid increased the secretion of apolipoprotein (apo) E by Hep G2 cells. The secretion of apo A-I was slightly lowered (less than 10% disease). The maximal increase in apo E secretion was observed in culture medium containing 2 micrograms of 25-OH-cholesterol/ml or 10 micrograms of CDC acid/ml plus 10% fetal calf serum. Cholesterol, 7-OH-cholesterol and other bile acids were ineffective in inducing increases in apo E secretion. Another cholesterol synthesis inhibitor, mevinolin, was also ineffective in generating an increase in apoprotein secretion. The data indicated a specific interaction between 25-OH-cholesterol or CDC acid and apo E secretion in Hep G2 cells. Cholesterol synthesis, as measured by the incorporation of [14C]acetic acid into sterols, was repressed in Hep G2 cells in the presence of 25-OH-cholesterol (17% of control value). CDC acid, on the other hand, increased [14C]acetic acid incorporation (156% of control value). The number of LDL receptors in Hep G2 cells was decreased after incubation with 25-OH-cholesterol (62% of control value), but increased significantly after incubation with CDC acid (149% of control value). The secretion of [3H]triacylglycerol by Hep G2 cells incubated with 25-OH-cholesterol was greatly increased (248% of control value). On the contrary, CDC acid did not cause any increase in [3H]triacylglycerol secretion. The above results suggest that 25-OH-cholesterol and CDC acid have different effects on lipid metabolism in Hep G2 cells. The mRNA levels of apo E increased in cells preincubated with 25-OH-cholesterol and CDC acid, which suggested that the increase in apo E secretion is at least partly due to an increase in synthesis.     

The metabolism of cholesterol in the presence of liver mitochondria from normal and thyroxine-treated rats

1. [26-(14)C]- and [4-(14)C]-Cholesterol were incubated with liver mitochondria from normal and thyroxine-treated rats, and the radioactivity was measured in the carbon dioxide evolved during the incubation, in a butanol extract of the incubation mixture and in a volatile fraction containing substances of low molecular weight derived from the side chain of cholesterol. The butanol extract was separated by paper chromatography into three radioactive fractions, one of which contained the steroids more polar than cholesterol. 2. The butanol extract from incubations with [4-(14)C]cholesterol contained a radioactive substance moving with the same R(F) as cholic acid on thin-layer chromatography. 3. After incubation with [26-(14)C]-cholesterol, 60-80% of the radioactivity extracted by steam-distillation of the incubation mixture at acid pH was recovered as [(14)C]propionic acid. 4. In the presence of [26-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced more radioactivity in carbon dioxide and in the volatile fraction, and less radioactivity in the fraction containing the polar steroids, than did mitochondria from normal rats. In the presence of [4-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced the same amount of radioactivity in the polar steroids as did normal mitochondria. 5. Thyroxine treatment had no effect on the capacity of the mitochondria to oxidize propionate to carbon dioxide. 6. These results are best explained by supposing that thyroxine stimulates a rate-limiting reaction leading to the cleavage of the side chain of cholesterol, but has little or no influence on the hydroxylations of the ring system or on the oxidation of the C(3) fragment removed from the side chain.     

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.     

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

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

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems.     

Effects of lysosomotropic agents on lipogenesis

Chloroquine, quinine, and NH4Cl are lysosomotropic agents which inhibit lysosomal function, apparently by raising the intralysosomal pH. We found that preincubation of cultured human skin fibroblasts with these lysosomotropic agents under serum-free conditions induced about a 10-fold stimulation of lipogenesis. A similar stimulatory effect on the incorporation of 3H2O, [14C]acetate, [14C]pyruvate, [14C]palmitate, and [14C]choline into cellular lipids was observed. The effect was both time and dose dependent, and was reversible. The concentrations of chloroquine, quinine, and NH4Cl resulting in half-maximal stimulation were about 3 microM, 30 microM, and 9 mM, respectively. At these concentrations, stimulation of lipogenesis correlated with impairment of lysosomal function. At a concentration of 10 microM chloroquine, the half-time for maximal stimulation was about 4 h. Most of the [14C]acetate was incorporated into phosphatidylcholine and other cellular lipids; less than 10% was found in cholesterol and cholesterol ester. Nevertheless, incorporation of [14C]acetate into cholesterol showed a chloroquine-induced stimulation parallel to that observed for phospholipids, suggesting that stimulation of both lipogenesis and cholesterogenesis occurred. The stimulatory effect of lysosomotropic agents on lipogenesis appeared to depend on active synthesis of cellular proteins. In the presence of cycloheximide, an inhibitor of protein synthesis; the stimulation was completely abolished.     

In vitro and in vivo synthesis of long-chain fatty acids from (1-14C) acetate in the renal papillae of rats.

1. The relationship between the rate of [1-14C] acetate incorporation into the fatty acids of renal papillary lipids and the acetate concentration in the medium has been measured. 2. [1-14C] acetate was incorporated mainly into fatty acids of phospholipids and triacylglycerols. Only a few per cent of the radioactivity was found in the free fatty acid fraction. 3. The major part of the [1-14C] acetate was found to be incorporated by a chain elongation of prevalent fatty acids. The major component of the poly-unsaturated fatty acids in triacylglycerols and the major product of fatty acid synthesis from [1-14C] acetate in vitro was demonstrated by mass spectrometry to be docosa-7,10,13,16-tetraenoic acid. 4. The radioactivity of docosa-7,10,13,16-tetraenoic acid accounted for 40% of total radioactivity in triacylglycerol fatty acids (lipid droplet fraction) and 20% of total radioactivity in membrane phospholipid fatty acids.