The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity, cholesterol esterification and the expression of low-density lipoprotein receptors in cultured monocyte-derived macrophages.

Human blood monocytes cultured in medium containing 20% whole serum showed the greatest activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and [14C]acetate incorporation into non-saponifiable lipids around the 7th day after seeding, the period of greatest growth. Although there was enough low-density lipoprotein (LDL) in the medium to saturate the LDL receptors that were expressed by normal cells at that time, HMG-CoA reductase activity and acetate incorporation were as high in normal cells as in cells from familial-hypercholesterolaemic (FH) patients. Both the addition of extra LDL, which interacted with the cells by non-saturable processes, and receptor-mediated uptake of acetylated LDL significantly reduced reductase activity and increased incorporation of [14C]oleate into cholesteryl esters in normal cells and cells from FH patients ('FH cells'), and reduced the expression of LDL receptors in normal cells. Pre-incubation for 20h in lipoprotein-deficient medium apparently increased the number of LDL receptors expressed by normal cells but reduced the activity of HMG-CoA reductase in both normal and FH cells. During subsequent incubations the same rate of degradation of acetylated LDL and of non-saturable degradation of LDL by FH cells was associated with the same reduction in HMG-CoA reductase activity, although LDL produced a much smaller stimulation of oleate incorporation into cholesteryl esters. In normal cells pre-incubated without lipoproteins, receptor-mediated uptake of LDL could abolish reductase activity and the expression of LDL receptors. The results suggested that in these cells, receptor-mediated uptake of LDL might have a greater effect on reductase activity and LDL receptors than the equivalent uptake of acetylated LDL. It is proposed that endogenous synthesis is an important source of cholesterol for growth of normal cells, and that the site at which cholesterol is deposited in the cells may determine the nature and extent of the metabolic events that follow.     

INCORPORATION OF LIPIDS INTO SUBCELLULAR FRACTIONS OF BRAIN OF QUAKING MUTANT

The synthesis of lipids and their assembly into subcellular membrane fractions of the myelin deficient Quaking mutant and control brains was studied in 18-, 24- and 41-day-old animals using a double label methodology with¹⁴C and ³H acetate as precursors. As a general procedure, Quaking mutants were injected intracranially with 50 μCi [¹⁴C]acetate and their littermate controls with 300 μCi [³H]acetate. The animals were killed 3 h post-injection, their brains were pooled and subcellular fractions prepared from the common homogenate. An 80-90% decrease in the incorporation of acetate into eleven lipids of myelin in the Quaking mutant was found. This occurred in the face of apparent normal incorporation (relative to microsomes) into lipids of the other main subcellular fractions (nuclear. mitochondrial and synaptosomal) with the exception of decreased incorporation into the myelin-like fraction at 18 and 24 days. Cholesterol and cerebroside were less readily incorporated into Quaking myelin than the other lipids. Although the microsomal synthesis of cholesterol and cerebroside was depressed by about 30% in the Quaking mutant, the incorporation of cholesterol into nuclear, synaptosomal and mitochondrial fractions was unaffected in the mutant. This indicates that sufficient cholesterol is synthesized for the normal assembly of these organelles. In contrast the incorporation of acetate into cholesterol and cerebroside of Quaking myelin was decreased much more than microsomal synthesis. This latter result is consistent with a defect in the process of myclin membrane assembly     

The regulation of ubiquinone synthesis in fibroblasts: The effect of modulators of β-hydroxy-β-methylglutaryl-coenzyme A reductase activity

The effect of inhibitors of β-hydroxy-β-methylglutaryl-coenzyme A (HMG-CoA) reductase such as low-density lipoprotein (LDL) and compactin were tested for their effects on the biosynthesis of ubiquinone in fibroblasts using [2-¹⁴C]acetic acid as a labeled precursor. LDL added to fibroblasts incubated in lipoprotein-deficient serum inhibited acetate incorporation into ubiquinone by 35%. Compactin, 2.5 μm, inhibited acetate incorporation by 60%. Further increases in compactin concentration up to 20 μm gradually increased the extent of inhibition but leveled off between 70 and 80%. The incorporation of ³H]mevalonic acid and 4-[U-¹⁴C]hydroxybenzoic acid into ubiquinone were determined with a range of compactin concentrations. Whereas the incorporation of [³H]mevalonate showed an apparent increase in response to compactin, the incorporation of 4-[U-¹⁴C]hydroxybenzoate into ubiquinone decreased. Both curves leveled off at concentrations of 5 μm did not significantly change with further increases in compactin concentration approaching 20 μm. Thus, the inhibition of acetate and 4-hydroxybenzoate incorporation into ubiquinone by compactin showed similar patterns. Cells incubated in lipoprotein-deficient serum compared to whole human serum showed inhibition of acetate incorporation similar to that observed previously for 4-hydroxybenzoate (9), thereby suggesting the presence of a stimulatory factor for ubiquinone biosynthesis in whole human serum. These data confirm and extend our earlier conclusions that inhibition of HMG-CoA reductase greatly affects ubiquinone synthesis in fibroblasts.     

Incorporation of labeled small molecules into rubratoxin

A sterile glucose-mineral salts broth was inoculated with conidia of Penicillium rubrum P-13 and P-3290. Radiolabeled compounds were added to some cultures, these being incubated quiescently at 28° C for 14 days. Other stationary cultures were grown for 21 days, received labeled compounds, and were then grown for 5 more days. The remaining cultures were inoculated with 72-h-old mycelial pellets, received labeled materials and were incubated with shaking for 60 h. Rubratoxin was resolved by thin-layer chromatography. Labeled [1¹⁴C]acetate, [1,5¹⁴C]citrate, [2¹⁴C]malonate, [1¹⁴C]glucose, [U¹⁴C]glucose or [1¹⁴C]hexanoate were incorporated into rubratoxins A and B by P. rubrum 3290 and into rubratoxin B by P. rubrum 13. Incorporation of [1¹⁴C]acetate and [2¹⁴C]malonate increased when exogenous unlabeled acetate, malonate, pyruvate, or phosphoenol-pyruvate was added. Acetate incorporation was influenced by cultural conditions, attaining maximum amounts in quiescent cultures which received labeled acetate after 21 days of incubation. Acetate incorporation in shake cultures was enhanced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) and by unlabeled exogenous citrate.Abbreviations GMS glucose-mineral salts - RCM replacement culture medium - TCA tricarboxylic acid - PEP phosphoenolpyruvate - RIC relative isotopic content - PI percent incorporation     

Lipid synthesis by oligodendrocytes from adult pig brain maintained in long-term culture

Oligodendrocytes were isolated from adult pig brain and cultivated for 18–24 days. [¹⁴C]acetate, [³H]galactose or [³⁵S]sulfate were added to the medium for an additional 24 h. Lipids were extracted and separated by high-performance thin-layer chromatography. The labeled lipids were studied by fluorography and scintillation counting. [¹⁴C]acetate was incorporated in decreasing order into neutral lipids, phosphatidylcholine, ethanolamine phosphatides, galactocerebrosides, phosphatidylinositol, phosphatidylserine, sulfatides and sphingomyelin. From the [¹⁴C]acetate incorporated into ethanolamine and choline phosphatides, 71.6 and 14.8%, respectively, were found in plasmalogens. Among neutral lipids, [¹⁴C]acetate labeled not only cholesterol but also large amounts of triglycerides. No cholesterol esters were synthesized. [³H]galactose primarily labeled galactocerebrosides, sulfatides, and monogalactosyl diglyceride. [³⁵S]sulfate incorporation was restricted to sulfatides. Together with our previous results concerning proteins, these data show that: (1) oligodendrocytes remain highly differentiated in long-term cultures; (2) they are able to synthesize the major components of myelin; (3) they synthesize surprisingly high amounts of triglycerides and of monogalactosyl diglyceride, a marker for myelination.     

PROVENANCE OF THE ACETYL GROUP OF ACETYLCHOLINE AND COMPARTMENTATION OF ACETYL-CoA AND KREBS CYCLE INTERMEDIATES IN THE BRAIN IN VIVO

—The origin of the acetyl group in acetyl-CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate +cis-aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were [1-¹⁴C]acetate, [2-¹⁴C]acetate, [4-¹⁴C]acetoacetate, [1-¹⁴C]butyrate, [1, 5-¹⁴C]citrate, [2-¹⁴C]glucose, [5-¹⁴C]glutamate, 3-hydroxy[3-¹⁴C]butyrate, [2-¹⁴C]lactate, [U-¹⁴C]leucine, [2-¹⁴C]pyruvate and [³H]acetylaspartate. The highest specific radioactivity of the acetyl group of ACh was observed 4 min after the injection of [2-¹⁴C]pyruvate. The contribution of pyruvate, lactate and glucose to the biosynthesis of ACh is considerably higher than the contribution of acetoacetate, 3-hydroxybutyrate and acetate; that of citrate and leucine is very low. No incorporation of label from [5-¹⁴C]glutamate into ACh was observed. Pyruvate appears to be the most important precursor of the acetyl group of ACh. The incorporation of label from [1, 5-¹⁴C]citrate into ACh was very low although citrate did enter the cells, was metabolized rapidly, did not interfere with the metabolism of ACh and the distribution of radioactivity from it in subcellular fractions of the brain was exactly the same as from [2-¹⁴C]pyruvate. It appears unlikely that citrate, glutamate or acetate act as transporters of intramitochondrially generated acetyl groups for the biosynthesis of ACh. Carnitine increased the incorporation of label from [1-¹⁴C]acetate into brain lipids and lowered its incorporation into ACh. Differences in the degree of labelling which various radioactive precursors produce in brain glutamine as compared to glutamate, previously described after intravenous, intra-arterial, or intraperitoneal administration, were confirmed using direct administration into the cerebrospinal fluid. Specific radioactivities of brain glutamine were higher than those of glutamate after injections of [1-¹⁴C]acetate, [2-¹⁴C]acetate, [1-¹⁴C]butyrate, [1,5-¹⁴C]citrate, [³H]acetylaspartate, [U-¹⁴C]leucine, and also after [2-¹⁴C]pyruvate and [4-¹⁴C]acetoacetate. The intracisternal route possibly favours the entry of substrates into the glutamine-synthesizing (‘small’) compartment. Increasing the amount of injected [2-¹⁴C]pyruvate lowered the glutamine/glutamate specific radioactivity ratio. The incorporation of ¹⁴C from [1-¹⁴C]acetate into brain lipids was several times higher than that from other compounds. By the extent of incorporation into brain lipids the substrates formed four groups: acetate > butyrate, acetoacetate, 3-hydroxybutyrate, citrate > pyruvate, lactate, acetylaspartate > glucose, glutamate. The ratios of specific radioactivity of ‘citrate’ over that of ACh and of glutamine over that of ACh were significantly higher after the administration of [1-¹⁴C]acetate than after [2-¹⁴C]pyruvate. The results indicate that the [1-¹⁴C]acetyl-CoA arising from [1-¹⁴C]acetate does not enter the same pool as the [1-¹⁴C]acetyl-CoA arising from [2-¹⁴C]pyruvate, and that the cholinergic nerve endings do not form a part of the acetate-utilizing and glutamine-synthesizing (‘small’) metabolic compartment in the brain. The distribution of radioactivity in subcellular fractions of the brain after the injection of [1-¹⁴C]acetate was different from that after [1, 5-¹⁴C]citrate. This suggests that [1-¹⁴C]acetate and [1, 5-¹⁴C]citrate are utilized in different subdivisions of the ‘;small’ compartment.     

Anaerobic microbial activities including hydrogen mediated acetogenesis within natural gas transmission lines

Microbially influenced corrosion (MIC) is being increasingly recognised as a serious problem. To investigate the role of MIC, radiotracer activity and lipid biomass measurements were performed on samples from offshore and on‐shore natural gas transmission systems. These measurements evaluated the biomass and metabolism of microbial communities residing inside transmission pipelines. Aqueous and nonaqueous hydrocarbon samples from liquid separators, sludge catchers and nodules attached to pipe walls were aseptically recovered and inoculated into anaerobic tubes for radiotracer time course experiments or preserved with chloroform‐methanol for total lipid analyses. MPN enrichments and phospholipid biomass determinations estimated microbial populations of 10⁴—10⁷ cells per gram in several samples. General microbial metabolism was demonstrated by [l‐¹⁴C]acetate incorporation into lipids and by [¹⁴C]CO₂ production from [U‐¹⁴C]glucose. [¹⁴C]Acetate was slowly mineralised to ¹⁴CO₂ without significant methane production. [¹⁴C]Acetate was produced by fermentation of [¹⁴C]glucose, [¹⁴C]palmitate and by hydrogen mediated acetogenesis in the presence of [^I4C]CO₂. In one location acetogenesis from hydrogen and carbon dioxide accounted for 0–7 mmol.l^‐1 of acetate production per week. These results demonstrated that microorganisms could utilise natural gas impurities to produce organic acids. This activity could adversely affect the structural integrity (MIC) of high pressure natural gas pipelines.     

Microbial activities in deep subsurface environments

Activities of microorganisms residing in terrestrial deep subsurface sediments were examined in 46 sediment samples from three boreholes. Radiolabeled time course experiments assessing in situ microbial activities were initiated within 30 min of core recovery. [1‐C⁴] Acetate incorporation into lipids, [ methyl‐³H] thymidine incorporation into DNA, [2‐¹⁴C]acetate, and [U‐¹⁴C]glucose mineralization in addition to microbial enrichment and enumeration studies were examined in surface and subsurface sediments. Surface soils contained the greatest biomass and activities, followed by the shallow aquifer zones. Water‐saturated subsurface sands exhibited three to four orders of magnitude greater activity and culturable microorganisms than the dense clay zones, which had low permeability. Regardless of depth, sediments that contained more than 20% clays exhibited the lowest activities and culturable microorganisms.     

Lipid biosynthesis in warm- and cold-acclimatized sea anemones, Metridium senile (L.)

Sea anemones, Metridium senile (L.), naturally acclimatized to warm (18°C) and cold (0°C) conditions, were exposed to either [2-¹⁴C]acetate, [16-¹⁴C]palmitate, or [4-¹⁴C]cholesterol for periods up to 24 h. Isotope incorporation into triglyceride (TG), wax esters (WE), and polar lipid (PL) was recorded. Compared to warm-acclimatized groups, incorporation of [2-¹⁴C]acetate into WE of cold anemones was dramatically reduced, while TG incorporation remained at about the same levels. Highest values were recorded for PL in both groups. Using radiolabeled palmitate, incorporation values for WE were very low in both acclimatization groups though TG uptake remained comparatively high. Also noteworthy was a significant decrease in PL activity in cold anemones. Fatty acid analysis of total lipid, wax ester, triglyceride and phospholipid fractions showed a general shift towards increased chain length and unsaturation in cold-acclimatized anemones.     

In vitro age-dependent incorporation of [1-14C]acetate into lipid subclasses in rat ventral prostate

The [1-14C]acetate incorporation into different lipid subclasses by the rat prostate gland was lineal between 20 and 80 mg of wet tissue. The in vitro [1-14C]acetate incorporation into lipid subclasses was a development-dependent process. The highest values of [1-14C]acetate incorporation into triacylglycerols, free cholesterol and esterified cholesterol were observed at puberty, but radioactivity incorporation into phospholipids was similar in both prepuberty and puberty, then decreasing in maturity. The relationship between triacylglycerols, free cholesterol and esterified cholesterol with respect to total lipids was about 12, 10 and 3.5%, respectively, values being maintained during the animal development. The in vitro [1-14C]acetate incorporation into lipid subclasses in castrated rats decreased considerably as compared with normal rats.     

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells

The time course of incorporation of [¹⁴C]arachidonic acid and [³H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [¹⁴C]arachidonic acid and [³H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

Effects of inhibitors of lipid synthesis on the replication of rous sarcoma virus. A specific effect of cerulenin on the processing of major non-glycosylated viral structural proteins

The effects of two inhibitors of lipid biosynthesis on the replication of Rous sarcoma virus Prague C strain in chick embryo fibroblasts have been examined in media containing delipidated serum. 25-Hydroxycholesterol, which markedly inhibits the incorporation of [1-¹⁴C]acetate into sterols, had no effect on the formation of infectious virions or on the synthesis and processing of intracellular virion proteins. Cerulenin strongly inhibited [1-¹⁴C]acetate incorporation into fatty acids and partially inhibited its incorporation into sterols in chick embryo cells. Rous sarcoma virus production as measured by focus formation and by the production of [³⁵S]methionine-labeled virions was strongly inhibited within 5 h after cerulenin addition to infected cultures. Examination of extracts of these cells revealed the accumulation of the 76 000 dalton precursor (Pr76) of the major non-glycosylated virion structural proteins, p27, p19, p15 and p12. The failure to process the 76 000 dalton precursor was coincident in time with the decrease in viron production. Neither whole serum nor mixtures of fatty acids plus cholesterol were able to reverse the effects of cerulenin.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

Sphingolipid-induced enhancement of receptor-mediated uptake of low density lipoproteins in normal and receptor-deficient human skin fibroblasts

(1) The receptor mediated endocytosis of homologous LDL by human skin fibroblasts can be significantly enhanced by prior incubation of the cells with sphingolipids. Gangliosides G_M1 or G_D1a, their desialylated derivatives and sphingosine stimulate binding and uptake to LDL by up to 40% of normal values. The effect is observed in normal fibroblasts, LDL receptor deficient fibroblasts or in tunicamycin-treated cells with a reduced number of functional receptors but is dependent on the time of preincubation of the cells and the concentration of the sphingolipid in the medium. (2) Detailed studies on the ganglioside effect revealed, that cell bound gangliosides intensify the LDL-induced supression of [¹⁴C]acetate incorporation into cholesterol. (3) The receptor dependence and relative receptor specificity of the sphingolipid effect is evident from the fact that (a) after complete suppression of receptor synthesis gangliosides fail to stimulate uptake of LDL, that (b) fatty acids or lipids not containing sphingosine are without effect and that (c) the receptor specific internalisation of α₂-macroglobulin or epidermal growth factor is not influenced by exogenous sphingolipids.     

Further investigations of the incorporation of [1-14C]acetate into the lipids of the silkworm Bombyx mori L

1. After the injection of sodium [1-¹⁴C]acetate, the highest incorporation of ¹⁴C into the lipids of the silkworm was observed after 24hr. 2. The specific radioactivity of the palmitic acid fraction was greater and increased more rapidly than that of the stearic acid fraction, which was consistent with the precursor–product relationship to be expected on the basis of current concepts of fatty acid synthesis in vivo. 3. The results indicate the probability of synthesis of lipid components in tissues other than the fat body. 4. Fractionation studies indicate considerable differences in the rate of incorporation of [1-¹⁴C]acetate into neutral lipids and phospholipids between larvae and pupae as well as among tissues of larvae. 5. The rate of incorporation of [1-¹⁴C]acetate remains constant throughout pupal development.     

The effects of retinoic acid on [14C]acetate incorporation into lipids of normal and transformed hamster fibroblasts

This study examined the effects of retinoic acid (RA) on [14C]acetate incorporation and fatty acid composition of hamster embryo fibroblasts (HEF) and two cell lines derived from the same inbred strain but transformed by herpes simplex-2 virus (HSV) or polyoma virus (HFT). Cells were exposed to all trans RA, or dimethylsulfoxide (DMSO), the vehicle for RA, and the lipids labeled with [14C]acetate. Lipids were extracted from the cells, separated by paper chromatography, located by autoradiography, and acetate incorporation determined by liquid scintillation spectrometry. The distribution of fatty acids in total cell lipids was examined by gas chromatography. HEF cells incorporated more acetate into cholesterol than either transformed cell type. The HFT line incorporated more acetate into triglycerides and less into total phospholipids than either the HSV line or the HEF line. RA caused a significant decrease in incorporation of acetate into cholesterol and sphingomyelin in all three cell lines. HEF and HSV cells had decreased incorporation into phosphatidyl inositol-phosphatidyl serine and increased incorporation into triglycerides, changes not evident in the HFT cell. The control fatty acid profiles of the HEF and HSV cells were similar, while the HFT cells had a larger proportion of C16:0 and 18:1 fatty acids. Following treatment with RA all three cell types showed an increase in palmitic and a decrease in oleic acids. The three related cell types showed different [14C]acetate labeling patterns which did not respond uniformly to RA. On the other hand, exposure elicited some like responses in all cell types.     

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.     

Effects of the calcium ionophore A 23187 on low-density lipoprotein processing and lipid metabolism in cultured human fibroblasts

Pretreatment of cultured human fibroblasts with the calcium ionophore A 23187 resulted in a decrease in low-density lipoprotein internalization. This effect was dose-dependent and did not occur in a medium devoid of calcium. About 2-fold reduction was observed with 10(-5)M A 23187. In contrast, the low-density lipoprotein binding was only slightly affected. The incorporation of [14C]acetate and [14C]oleate into all classes of lipids (sterol, triacylglycerols and phospholipids) was strikingly reduced by ionophore pretreatment.     

THE METABOLISM OF LEUCINE BY DEVELOPING RAT BRAIN: EFFECT OF LEUCINE AND 2-OXO-4-METHYLVALERATE ON LIPID SYNTHESIS FROM GLUCOSE AND KETONE BODIES

Abstract— The oxidation of l -[U-¹⁴C]leucine and l -[l-¹⁴C]leucine at varying concentrations from 0.1 to 5mM to CO₂ and the incorporation into cerebral lipids and proteins by brain slices from 1-week old rats were markedly stimulated by glucose. Although the addition of S mM-dl -3-hydroxybutyrate had no effect on the metabolism of [U-¹⁴C]leucine by brain slices from suckling rats, the stimulatory effects of glucose on the metabolism of l -[U-¹⁴C]leucine were markedly reduced in the presence of dl -3-hydroxybutyrate. The stimulatory effect of glucose on leucine oxidation was, however, not observed in adult rat brain. Furthermore, the incorporation of leucine-carbon into cerebral lipids and proteins was also very low in the adult brain. The incorporation of l -[U-¹⁴C]leucine into cerebral lipids by cortex slices was higher during the first 2 postnatal weeks, which then declined to the adult level. During this time span, the oxidation of l -[U-¹⁴C]leucine to CO₂ remained relatively unchanged. The incorporation in vivo of D-3-hydroxy[3-¹⁴C]butyrate into cerebral lipids was markedly decreased by acute hyperleucinemia induced by injecting leucine into 9-day old rats. In in vitro experiments, 5 mM-leucine had no effect on the oxidation of [U-¹⁴C]glucose to CO₂ or its incorporation into lipids by brain slices from 1-week old rats. However, 5 mM-leucine inhibited the oxidation of d -3-hydroxy-[3-¹⁴C]butyrate, [3-¹⁴C]acetoacetate and [1-¹⁴C]acetate to CO₂ by brain slices, but their incorporation into cerebral lipids was not affected by leucine. In contrast 2-oxo-4-methylvalerate, a deaminated metabolite of leucine, markedly inhibited both the oxidation to CO₂ and the incorporation into lipids of labelled glucose, ketone bodies and acetate by cortex slices from 1-week old rats. These findings suggest that the reduction in the incorporation in vivo of d -3-hydroxy[3-¹⁴C]butyrate into cerebral lipids in rats injected with leucine is most likely caused by 2-oxo-4-methylvalerate formed from leucine. Since the concentrations of leucine and 2-oxo-4-methylvalerate in plasma of untreated patients with maple-syrup urine disease are markedly elevated, our findings are compatible with the possibility that an alteration in the metabolism of glucose and ketone bodies in the brain may contribute to the pathophysiology of this disease.     

Metabolism of tween-Fatty Acid esters by cultured soybean cells : kinetics of incorporation into lipids, subsequent turnover, and associated changes in endogenous Fatty Acid synthesis

Uptake of Tween-fatty acid esters and incorporation of the fatty acids into lipids by soybean (Glycine max [L.] Merr.) suspension cultures was investigated, together with subsequent turnover of the incorporated fatty acids and associated changes in endogenous fatty acid synthesis. Tween uptake was saturable, and fatty acids were rapidly transferred from Tweens to all acylated lipids. Patterns of incorporation into glycerolipids were similar in cells treated with Tweens carrying [1-¹⁴C]-fatty acids and in cells treated with [1-¹⁴C]acetate, indicating that exogenous fatty acids were used for glycerolipid synthesis essentially as if they had been made by the cell. In Tween-treated cells neutral lipids (which include Tweens) initially accounted for the majority of lipid radioactivity. Radioactivity was then rapidly transferred to glycerolipids. A transient pool of free fatty acids accounting for up to 10% of lipid radioactivity was observed. This was consistent with the hypothesis that fatty acids are transferred from Tweens to lipids by deacylation of the Tweens, creating a pool of free fatty acids which are then used for lipid synthesis. Sterols were only slightly labeled in cells treated with Tweens, but accounted for nearly 50% of lipid radioactivity in cells treated with acetate. This suggested very little degradation and reutilization of the radioactive fatty acids in cells treated with Tweens. In cells treated with either [1-¹⁴C]acetate or Tween-[1-¹⁴C]-18:1, 70% of the initial fatty acid radioactivity remained in fatty acids after a 100 hour chase. By contrast, fatty acids not normally present disappeared more rapidly, suggesting differential treatment of such fatty acids compared with those normally present. Cells which had incorporated large amounts of exogenous fatty acids altered fatty acid synthesis in three distinct ways: (a) amounts of [1-¹⁴C]acetate incorporated into fatty acids were reduced; (b) cells incorporating exogenous unsaturated fatty acids increased the proportion of [1-¹⁴C]acetate partitioned into saturated fatty acids, while the converse was true of cells which had incorporated exogenous saturated fatty acids; (c) desaturation of 18:1 to 18:2 and 18:3 was reduced in cells which had incorporated unsaturated fatty acids. These results suggest that Tween-fatty acid esters will be useful for supplying fatty acids to cells for a variety of studies related to fatty acid or membrane metabolism.