Energy metabolism in respiration-deficient and wild type Chinese hamster fibroblasts in culture.

This paper presents a comparison of energy metabolism in wild type and respiration-deficient Chinese hamster cells. From previous work (DeFrancesco et. al., '75) it was concluded that the mutant satisfies essentially all of its energy requirements from glycolysis and in this study we measure precisely the amount of glucose consumed and lactate produced per milligram increment of protein in exponentially growing cultures. From these measurements we calculate the amount of ATP derived from glycolysis (and hence the total energy requirement for normal proliferation) to be 105 +/- 15 mumoles ATP/delta mg protein in the mutant. It is 63 +/- 10 mumoles ATP/delta mg protein derived from glycolysis in wild type cells. We present evidence that the total energy requirement of wild type cells is similar to that of the mutant suggesting that approximately 40% of the energy requirement is derived from respiration. The oxidation of glutamine appears to be more significant than the complete oxidation of glucose to CO2 in these Chinese hamster fibroblasts. The amount of ATP required by the mutant cells per milligram increment of protein is relatively independent of pH.     

Glucose oxidation by adherent and mechanically detached cultured cells.

This study examined the effect of mechanical detachment from the growth surface on energy metabolism of cultured cells. Oxidation of [1(-14)C]glucose measured by production of 14CO2 by adherent neuroblastoma (123 +/- 5 nmol/mg protein per minute), glioma (128 +/- 10 nmol/mg protein per minute), and fibroblast (137 +/- 5 nmol/mg protein per minute) cultures was similar. Removing cells from the culture flask by scraping reduced glucose oxidation by 62, 30, and 82% in neuroblastoma, glioma, and fibroblast cultures, respectively. Transferring cells from a culture flask to a test tube, to control for diffusional surface area, did not further reduce glucose oxidation. Detaching cells from the growth surface destroyed the extensive process formation and disrupted the normal spatial organization on the culture plate. These results indicate that it is essential to maintain these aspects of cellular architecture when evaluating metabolic properties of cultured cells.     

Fatty acids are not an important fuel for coronary microvascular endothelial cells

Summary The metabolism by coronary microvascular endothelial cells (CMEC) of the heart typical substrates palmitate and lactate was compared to that of glucose and glutamine. Confluent cultures of CMEC were used. Palmitate oxidation was saturable and independent of the exogenous albumin concentration. Palmitate, 300 M, lactate, 1 mM, and glutamine, 0.5 mM, were oxidized to 35, 46, and 56 nmol CO₂/h × mg protein. These oxidation rates were decreased by 80, 66, and 48% in presence of 5 mM glucose. The largest energy yield was obtained by glycolytic breakdown of glucose. Glucose, 5 mM, was degraded to lactate by 99%, and oxidized in the Krebs cycle by only 0.04%. 1% was catabolized via the hexose monophosphate pathway. The rate of glucose oxidation in the Krebs cycle could be 30-fold increased by the uncoupler 2,4-dinitrophenol, 30 µM. At concentrations lower than 1 mM the amount of glucose oxidized in the Krebs cycle also grew, indicating existence of the Crabtree effect. The energy demand of CMEC seems to be of the same order as that of the arrested heart.     

More monensin-sensitive, ammonia-producing bacteria from the rumen

Two monensin-sensitive bacteria which utilized carbohydrates poorly and grew rapidly on amino acids were isolated from the bovine rumen. The short rods (strain SR) fermented arginine, serine, lysine, glutamine, and threonine rapidly (greater than 158 nmol/mg of protein per h) and grew faster on casein digest containing short peptides than on free amino acids ().34 versus 0.29 h(-1)). Gelatin hydrolysate, an amino acid source containing an abundance of long peptides, was unable to support growth or ammonia production, but there was a large increase in ammonia production if strain SR was cocultured with peptidase-producing ruminal bacteria (Bacteroides ruminicola or Streptococcus bovis). Cocultures showed no synergism with short peptides. Strain SR washed out of continuous culture ().1 h(-1)) at pH 5.9. The irregularly shaped organisms (strain F) deaminated glutamine, histidine, glutamate, and serine rapidly (greater than 137 nmol/mg of protein per min) and grew faster on free amino acids than on short peptides ().43 versus 0.21 h(-1)). When strain F was provided with casein or gelatin hydrolysate and cocultured with peptidase-producing bacteria, there was a more than additive increase in ammonia production. Strain F grew in continuous culture (0.1 h(-1)) when the pH was as low as 5.3. The irregularly shaped cells and short rods were present at less than 10(9)/ml in vivo, but they ahd very high specific activities of ammonia production (greater than 310 nmol of ammonia/mg of protein per min) and could play an important role in ruminal amino acid fermentation.     

More monensin-sensitive, ammonia-producing bacteria from the rumen.

Two monensin-sensitive bacteria which utilized carbohydrates poorly and grew rapidly on amino acids were isolated from the bovine rumen. The short rods (strain SR) fermented arginine, serine, lysine, glutamine, and threonine rapidly (greater than 158 nmol/mg of protein per h) and grew faster on casein digest containing short peptides than on free amino acids ().34 versus 0.29 h(-1)). Gelatin hydrolysate, an amino acid source containing an abundance of long peptides, was unable to support growth or ammonia production, but there was a large increase in ammonia production if strain SR was cocultured with peptidase-producing ruminal bacteria (Bacteroides ruminicola or Streptococcus bovis). Cocultures showed no synergism with short peptides. Strain SR washed out of continuous culture ().1 h(-1)) at pH 5.9. The irregularly shaped organisms (strain F) deaminated glutamine, histidine, glutamate, and serine rapidly (greater than 137 nmol/mg of protein per min) and grew faster on free amino acids than on short peptides ().43 versus 0.21 h(-1)). When strain F was provided with casein or gelatin hydrolysate and cocultured with peptidase-producing bacteria, there was a more than additive increase in ammonia production. Strain F grew in continuous culture (0.1 h(-1)) when the pH was as low as 5.3. The irregularly shaped cells and short rods were present at less than 10(9)/ml in vivo, but they ahd very high specific activities of ammonia production (greater than 310 nmol of ammonia/mg of protein per min) and could play an important role in ruminal amino acid fermentation.     

Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts

Human diploid fibroblasts utilize both glucose and glutamine as energy sources. The utilization of glutamine by fibroblasts is regulated by glucose, and vice versa. This conclusion is supported by the following observations: (1) essentially identical growth rates were observed in Eagle's minimum essential medium (MEM)3 in which the glucose concentration was either 5.5 mM or was maintained between 25 and 40 micrometer, (2) the total glutamine utilization by fibroblasts increase at least 30% in medium with 25 micrometer to 70 micrometer glucose compared to medium with 5.5 mM glucose, while the rate of glutamine-1 or 5-14C oxidation to CO2 increased 5-fold as the glucose concentration was decreased to zero, (3) 2 mM glutamine inhibited glucose-6-14C oxidation by 88% and stimulated glucose-1-14C by 77% in log phase cells and (4) glutamine oxidation in normal medium contributed approximately 30% of the energy requirement of human diploid fibroblasts.     

Fermentation of peptides and amino acids by a monensin-sensitive ruminal Peptostreptococcus

A monensin-sensitive ruminal peptostreptococcus was able to grow rapidly (growth rate of 0.5/h) on an enzymatic hydrolysate of casein, but less than 23% of the amino acid nitrogen was ever utilized. When an acid hydrolysate was substituted for the enzymatic digest, more than 31% of the nitrogen was converted to ammonia and cell protein. Coculture experiments and synergisms with peptide-degrading strains of Bacteroides ruminicola and Streptococcus bovis indicated that the peptostreptococcus was unable to transport certain peptides or hydrolyze them extracellularly. Leucine, serine, phenylalanine, threonine, and glutamine were deaminated at rates of 349, 258, 102, 95, and 91 nmol/mg of protein per min, respectively. Deamination rates for some other amino acids were increased when the amino acids were provided as pairs of oxidized and reduced amino acids (Stickland reactions), but these rates were still less than 80 nmol/mg of protein per min. In continuous culture (dilution rate of 0.1/h), bacterial dry matter and ammonia production decreased dramatically at a pH of less than 6.0. When dilution rates were increased from 0.08 to 0.32/h (pH 7.0), ammonia production increased while production of bacterial dry matter and protein decreased. These rather peculiar kinetics resulted in a slightly negative estimate of maintenance energy and could not be explained by a change in fermentation products. Approximately 80% of the cell dry matter was protein. When corrections were made for cell composition, the yield of ATP was higher than the theoretical maximum value. It is possible that mechanisms other than substrate-level phosphorylation contributed to the energetics of growth.     

Fermentation of peptides and amino acids by a monensin-sensitive ruminal Peptostreptococcus.

A monensin-sensitive ruminal peptostreptococcus was able to grow rapidly (growth rate of 0.5/h) on an enzymatic hydrolysate of casein, but less than 23% of the amino acid nitrogen was ever utilized. When an acid hydrolysate was substituted for the enzymatic digest, more than 31% of the nitrogen was converted to ammonia and cell protein. Coculture experiments and synergisms with peptide-degrading strains of Bacteroides ruminicola and Streptococcus bovis indicated that the peptostreptococcus was unable to transport certain peptides or hydrolyze them extracellularly. Leucine, serine, phenylalanine, threonine, and glutamine were deaminated at rates of 349, 258, 102, 95, and 91 nmol/mg of protein per min, respectively. Deamination rates for some other amino acids were increased when the amino acids were provided as pairs of oxidized and reduced amino acids (Stickland reactions), but these rates were still less than 80 nmol/mg of protein per min. In continuous culture (dilution rate of 0.1/h), bacterial dry matter and ammonia production decreased dramatically at a pH of less than 6.0. When dilution rates were increased from 0.08 to 0.32/h (pH 7.0), ammonia production increased while production of bacterial dry matter and protein decreased. These rather peculiar kinetics resulted in a slightly negative estimate of maintenance energy and could not be explained by a change in fermentation products. Approximately 80% of the cell dry matter was protein. When corrections were made for cell composition, the yield of ATP was higher than the theoretical maximum value. It is possible that mechanisms other than substrate-level phosphorylation contributed to the energetics of growth.     

Rates of utilization and fates of glucose, glutamine, pyruvate, fatty acids and ketone bodies by mouse macrophages.

The concentrations of ATP and the ATP/AMP concentration ratios were maintained in thioglycollate-elicited mouse peritoneal macrophages incubated in vitro for 90 min in the presence or absence of added substrate: rates of glycolysis, lactate formation and glutamine utilization were approximately linear with time for at least 60 min of incubation. The rate of oxygen consumption by macrophages was only increased above the basal rate (i.e. that in the absence of added substrate) by addition of succinate or pyruvate, or by addition of the uncoupling agent carboxyl cyanide m-chlorophenylhydrazone ('CCCP'); it was decreased by 75% by the addition of KCN. These findings suggest that metabolism of endogenous substrate can provide most, if not all, of the energy requirement of these cells, at least for a short period. The rates of glucose and glutamine utilization by incubated macrophages were approx. 300 and 100 nmol/min per mg of protein respectively. A large proportion of the glutamine that is utilized is converted into glutamate and aspartate, and very little (perhaps less than 10%) is oxidized. Similarly almost all of the glucose that is utilized is converted into lactate and very little is oxidized. This characteristic is similar to that of resting lymphocytes and rapidly dividing cells; in non-proliferating macrophages it may be a mechanism to provide precision in control of the rate of biosynthetic processes that utilize intermediates of these pathways, e.g. purines and pyrimidines for mRNA for the synthesis of secretory proteins and glycerol 3-phosphate for phospholipid synthesis for membrane recycling. No utilization of acetoacetate or 3-hydroxybutyrate by macrophages was detected. In contrast, both butyrate and oleate were oxidized. The rate of [14C]oleate conversion into 14CO2 (1.3 nmol/h per mg of protein) could account for most of the oxygen consumption by incubated macrophages, suggesting that long-chain fatty acids might provide an important fuel in situ. This may be one explanation for the secretion of lipoprotein lipase by these cells, to provide fatty acids for oxidation from the degradation of local triacylglycerol.     

Neuronal Glutamine Utilization: Pathways of Nitrogen Transfer tudied with [15N]Glutamine

Gas chromatography-mass spectrometry was used to evaluate the metabolism of [15N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate greater than 9 nmol/min/mg of protein. The metabolism of [15N]glutamine generated 15N-labelled glutamate, aspartate, and gamma-aminobutyric acid (GABA). An efflux of both [15N]glutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of 15N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2-oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70-80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.     

Glutamine Oxidation by Dissociated Cells and Homogenates of Rat Brain: Kinetics and Inhibitor Studies

The rates of [U-14C]glutamine oxidation to 14CO2 were determined under a variety of experimental conditions using whole homogenates and dissociated cells from rat brain. The pattern of glutamine oxidation by homogenates differed from that by dissociated brain cells in several respects. The rates of glutamine oxidation by dissociated brain cells showed saturation kinetics with an apparent Km of 0.30 mM. Lineweaver-Burk plots of glutamine oxidation by homogenates revealed two linear segments with two apparent Km values (0.58 mM and 3.0 mM). In the presence of aminooxyacetate, however, the Lineweaver-Burk plots for homogenates were linear with a single Km of 0.47 mM. The oxidation of glutamine by homogenates was inhibited by both rotenone and antimycin A (80-85%), as were glutamate and glucose oxidation, suggesting that a significant amount of glutamine is oxidized via the tricarboxylic acid cycle. In the presence of aminooxyacetate, glutamine oxidation was inhibited less than 40%, whereas the oxidation of glutamate was inhibited 75%; in contrast, glucose oxidation was enhanced 50%. The rates of glutamine oxidation by homogenates were highest in the presence of high levels of potassium (50 mM) and low levels of sodium (2.5 mM). Varying ionic composition, however, had little or no effect on the rates of glutamine oxidation by dissociated brain cells. Measurements of glutamine oxidation by homogenates prepared from 2-, 10-, 15-, 25-, and 90-day-old rats revealed little or no age-dependent difference. In contrast, the oxidation by dissociated brain cells from 2-day-old animals was significantly less than that obtained for animals 10 days or older (7.76 vs. 15.6 nmol/h/mg).(ABSTRACT TRUNCATED AT 250 WORDS)     

The kinetics of glutamine transport in bovine lymphocytes.

The transport of glutamine was examined in bovine peripheral lymphocytes which had been cultured in the presence or absence of Concanavalin A (Con A). Glutamine transport was mediated by a triphasic transport system in both cell populations. The calculated kinetic parameters were: Km 1.0, 4.7 and 12.7 mM and Vmax 4.5, 6.0 and 9.0 nmol/min per mg protein respectively. Con A augmented the capacity rather than the affinity of the glutamine transport systems (Vmax rates being 8.0, 12.2 and 38.0 nmol/min per mg protein respectively). Transporter I displayed Michaelis-Menton kinetics, while transporters II and III were co-operative carriers possessing Hill coefficients of 2.3 and 9.5 respectively. Preliminary studies using amino acid and ion inhibition studies suggested that transporter I was a system ASC-type carrier, transporter III a system L carrier, while the nature of transporter II was unclear.     

Utilization of medium-chain triglycerides and octanoic acid by cultured human fibroblasts and lymphoblasts

Cultured human fibroblasts and lymphoblasts were incubated with emulsions containing 14C-trioctanoin or 14C-tripalmitin. Both cell types were able to hydrolyse the medium-chain triglyceride but not the long-chain triglyceride to the corresponding fatty acids. At the end of a 3 days incubation period, 25-30% of the initial amount of 10 nmol/ml trioctanoin were present as triglyceride. The observed hydrolysis seems to be mediated by an esterase secreted into the culture medium, as was shown by the use of cell-conditioned medium. CO2 production from octanoic acid was below 2 nmol per mg protein and day, demonstrating that these cells have a low capacity to use this substrate for their energy metabolism.     

Rates of utilization of glucose, glutamine and oleate and formation of end-products by mouse peritoneal macrophages in culture.

1. The metabolism of mouse thioglycollate-elicited peritoneal macrophages was studied in culture for up to 96 h. 2. The rates of glycolysis, lactate formation and glutamine utilization were approximately linear with time for at least 80 h of culture. 3. The rates of glucose and glutamine utilization by cultured macrophages were approx. 500 and 90 nmol/h per mg of protein respectively. This rate of glucose utilization is at least 50% greater than that previously reported for macrophages during 60 min incubation in a shaking flask; and it is now increased by addition of glutamine to the culture medium. The rate of glutamine utilization in culture is similar to that previously reported for macrophages during 60 min incubation. The major end-product of glucose metabolism is lactate, and those of glutamine metabolism are CO2, glutamate, ammonia and alanine. 4. Oleate was utilized by these cells: 14C from [14C]oleate was incorporated into CO2 and cellular lipid. The highest rate of oleate utilization was observed when both glucose and glutamine were present in the culture medium. The presence of oleate in the culture medium did not affect the rates of utilization of either glucose or glutamine. Of the [14C]oleate incorporated into lipid, approx. 80% was incorporated into triacylglycerol and only 18% into phospholipid. 5. The turnover rate for the total ATP content of the macrophage in culture is about 10 times per minute: the value for the perfused isolated maximally working rat heart is 22. This indicates a high metabolic rate for macrophages, and consequently emphasizes the importance of the provision of fuels for their function in an immune response.     

An analysis of the metabolism and cell wall composition of Candida albicans during germ-tube formation

The uptake of nutrients (glucose, glutamine, and N-acetylglucosamine), the intracellular concentrations of metabolites (glucose-6-phosphate, cyclic AMP, amino acids, trehalose, and glycogen) and cell wall composition were studied in Candida albicans. These analyses were carried out with exponential-phase, stationary-phase, and starved yeast cells, and during germ-tube formation. Germ tubes formed during a 3-h incubation of starved yeast cells (0.8 X 10(8) cells/mL) at 37 degrees C during which time the nutrients glucose plus glutamine or N-acetylglucosamine (2.5 mM of each) were completely utilized. Control incubations with these nutrients at 28 degrees C did not form germ tubes. Uptake of N-acetylglucosamine and glutamine was inhibited by cycloheximide which suggests that de novo protein synthesis was required for the induction of these uptake systems. The glucose-6-phosphate content varied from 0.4 nmol/mg dry weight for starved cells to 2-3 nmol/mg dry weight for growing yeast cells and germ tube forming cells. Trehalose content varied from 85 nmol/mg dry weight (growing yeast cells and germ tube forming cells) to 165 nmol/mg weight (stationary-phase cells). The glycogen content decreased during germ-tube formation (from 800 to 600 nmol glucose equivalent/mg dry weight) but increased (to 1000 nmol glucose equivalent/mg dry weight) in the control incubation of yeast cells. Cyclic AMP remained constant throughout germ-tube formation at 4-6 pmol/mg dry weight. The total amino acid pool was similar in exponential, starved, and germ tube forming cells but there were changes in the amounts of individual amino acids. The overall cell wall composition of yeast cells and germ tube forming cells were similar: lipid (2%, w/w); protein (3-6%), and carbohydrate (77-85%). The total carbohydrates were accounted for as the following fractions: alkali-soluble glucan (3-8%), mannan (20-23%), acid-soluble glucan (24-27%), and acid-insoluble glucan (18-26%). The relative amounts of the alkali-soluble and insoluble glucan changed during starvation of yeast cells, reinitiation of yeast-phase growth, and germ-tube formation. Analysis of the insoluble glucan fraction from cells labelled with [14C]glucose during germ-tube formation showed that the chitin content of the cell wall increased from 0.6% to 2.7% (w/w).     

Stimulation of Synaptosomal γ-Aminobutyric Acid Synthesis by Glutamate and Glutamine

gamma-Aminobutyric acid (GABA) synthesis was studied in rat brain synaptosomes by measuring the increase of GABA level in the presence of the GABA-transaminase inhibitor gabaculine. The basal rate of synaptosomal GABA synthesis in glucose-containing medium (25.9 nmol/h/mg of protein) was only 3% of the maximal activity of glutamate decarboxylase (GAD; 804 +/- 83 nmol/h/mg of protein), a result indicating that synaptosomal GAD operates at only a small fraction of its catalytic capacity. Synaptosomal GABA synthesis was stimulated more than threefold by adding 500 microM glutamine. Glutamate also stimulated GABA synthesis, but the effect was smaller (1.5-fold). These results indicate that synaptosomal GAD is not saturated by endogenous levels of its substrate, glutamate, and account for part of the unused catalytic capacity. The greater stimulation of GABA synthesis by glutamine indicates that the GAD-containing compartment is more accessible to extrasynaptosomal glutamine than glutamate. The strong stimulation by glutamine also shows that the rates of uptake of glutamine and its conversion to glutamate can be sufficiently rapid to support GABA synthesis in nerve terminals. Synaptosomes carried out a slow net synthesis of aspartate in glucose-containing medium (7.7 nmol/h/mg of protein). Aspartate synthesis was strongly stimulated by glutamate and glutamine, but in this case the stimulation by glutamate was greater. Thus, the larger part of synaptosomal aspartate synthesis occurs in a different compartment than does GABA synthesis.     

The pathway of glutamate metabolism in rat brain mitochondria

1. The pathway of glutamate metabolism in non-synaptic rat brain mitochondria was investigated by measuring glutamate, aspartate and ammonia concentrations and oxygen uptakes in mitochondria metabolizing glutamate or glutamine under various conditions. 2. Brain mitochondria metabolizing 10mm-glutamate in the absence of malate produce aspartate at 15nmol/min per mg of protein, but no detectable ammonia. If amino-oxyacetate is added, the aspartate production is decreased by 80% and ammonia production is now observed at a rate of 6.3nmol/min per mg of protein. 3. Brain mitochondria metabolizing glutamate at various concentrations (0-10mm) in the presence of 2.5mm-malate produce aspartate at rates that are almost stoicheiometric with glutamate disappearance, with no detectable ammonia production. In the presence of amino-oxyacetate, although the rate of aspartate production is decreased by 75%, ammonia production is only just detectable (0.3nmol/min per mg of protein). 4. Brain mitochondria metabolizing 10mm-glutamine and 2.5mm-malate in States 3 and 4 were studied by using glutamine as a source of intramitochondrial glutamate without the involvement of mitochondrial translocases. The ammonia production due to the oxidative deamination of glutamate produced from the glutamine was estimated as 1nmol/min per mg of protein in State 3 and 3nmol/min per mg of protein in State 4. 5. Brain mitochondria metabolizing 10mm-glutamine in the presence of 1mm-amino-oxyacetate under State-3 conditions in the presence or absence of 2.5mm-malate showed no detectable aspartate production. In both cases, however, over the first 5min, ammonia production from the oxidative deamination of glutamate was 21-27nmol/min per mg of protein, but then decreased to approx. 1-1.5nmol/min per mg. 6. It is concluded that the oxidative deamination of glutamate by glutamate dehydrogenase is not a major route of metabolism of glutamate from either exogenous or endogenous (glutamine) sources in rat brain mitochondria.     

Developmental changes of gamma-aminobutyric acid (GABA) and putative amino acid neurotransmitters in the organotypic culture of newborn mouse cerebellum

The quantitative changes and metabolism of GABA and putative amino acid neurotransmitters during early developmental stages in the organotypic culture of newborn mouse cerebellum were examined by using the high-performance liquid chromatograph (HPLC) technique. D-[U-14C]Glucose was used as a precursor of amino acids. To analyze amino acid neurotransmitters, explants were incubated for 4 weeks under standard conditions. The amount of GABA linearly increased from 8.7 +/- 1.3 nmol/mg protein (2 days in vitro, 2 DIV) to 26.5 +/- 6.1 nmol/mg protein (15 DIV) and was saturated after that (24.0 +/- 3.6 nmol/mg protein at 30 DIV). During the period of GABA increase, the capability for GABA synthesis from [14C]glucose increased rapidly from 3.03 +/- 0.67 nCi/mg protein (2 DIV, 3 h incubation) to 9.32 +/- 1.34 nCi/mg protein (15 DIV, 3 h incubation). In the case of glutamic acid, a putative neurotransmitter of granule cell parallel fibers in the cerebellum, the amount in explants was nearly constant during incubation, in contrast with the fact that the amount in vivo gradually increased. However, the capability for glutamic acid synthesis from [14C]glucose increased from 10.80 +/- 3.01 nCi/mg protein (2 DIV, 1 h incubation) to 27.62 +/- 4.71 nCi/mg protein (22 DIV, 1 h incubation). In the case of taurine, found in abundance in fetal brain and supposed to play a specific role in the development and maturation of the central nervous system, the amount in explants decreased from 139.8 +/- 4.0 nmol/mg protein (2 DIV) to 54.0 +/- 0.8 nmol/mg protein (30 DIV).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transport of fatty acids into human and rat peroxisomes. Differential transport of palmitic and lignoceric acids and its implication to X-adrenoleukodystrophy.

The different topology of palmitoyl-CoA ligase (on the cytoplasmic surface) and of lignoceroyl-CoA ligase (on the luminal surface) in peroxisomal membranes suggests that these fatty acids may be transported in different form through the peroxisomal membrane (Lazo, O., Contreras, M., and Singh, I. (1990) Biochemistry 29, 3981-3986), and this differential transport may account for deficient oxidation of lignoceric acid in X-adrenoleukodystrophy (X-ALD) (Singh, I., Moser, A. B., Goldfisher, S., and Moser, H. W. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4203-4207). To define the transport mechanism for these fatty acids through the peroxisomal membrane and its possible implication to lignoceric acid metabolism in X-ALD, we examined cofactors and energy requirements for the transport of palmitic and lignoceric acids in isolated peroxisomes from rat liver and peroxisomes isolated from X-ALD and control fibroblasts. The similar rates of transport of palmitoyl-CoA (87.6 +/- 6.3 nmol/h/mg protein) and palmitic acid in the fatty acid activating conditions (83.4 +/- 5.1 nmol/h/mg protein) and lack of transport of palmitic acid (4% of palmitoyl-CoA transport) when ATP and/or CoASH were removed or substituted by alpha,beta-methyleneadenosine-5'-triphosphate (AMPCPOP) and/or desulfoCoA-agarose from assay medium clearly demonstrate that transport of palmitic acid requires prior synthesis of palmitoyl-CoA by palmitoyl-CoA ligase on the cytoplasmic surface of peroxisomes. The 10-fold higher rate of transport of lignoceric acid (5.3 +/- 0.6 nmol/h/mg protein) as compared with lignoceroyl-CoA (0.41 +/- 0.11 nmol/h/mg protein) and lack of inhibition of transport of lignoceric acid when ATP and/or CoASH were removed or substituted with AMPCPOP or desulfoCoA-agarose suggest that lignoceric acid is transported through the peroxisomal membrane as such. Moreover, the lack of effect of removal of ATP or substitution with AMPOPCP (a nonhydrolyzable substrate) demonstrates that the translocation of palmitoyl-CoA and lignoceric acid across peroxisomal membrane does not require energy. The transport, activation, and oxidation of palmitic acid are normal in peroxisomes from X-ALD. The deficient lignoceroyl-CoA ligase (13% of control) and oxidation of lignoceric acid (10% of control) as compared with normal transport of lignoceric acid into peroxisomes from X-ALD clearly demonstrates that pathogenomonic accumulation of very long chain fatty acids (greater than C22) in X-ALD is due to the deficiency of peroxisomal lignoceroyl-CoA ligase activity.     

Cellular oxidation of lignoceric acid is regulated by the subcellular localization of lignoceroyl-CoA ligases

The acyl-CoA ligases convert free fatty acids to acyl-CoA derivatives, and these enzymes have been shown to be present in mitochondria, peroxisomes, and endoplasmic reticulum. Because their activity is obligatory for fatty acid metabolism, it is important to identify their substrate specificities and subcellular distributions to further understand the cellular regulation of these pathways. To define the role of the enzymes and organelles involved in the metabolism of very long chain (VLC) fatty acids, we studied human genetic cell mutants impaired for the metabolism of these molecules. Fibroblast cell lines were derived from patients with X-linked adrenoleukodystrophy (X-ALD) and Zellweger's cerebro-hepato-renal syndrome (CHRS). While peroxisomes are present and morphologically normal in X-ALD, they are either greatly reduced in number or absent in CHRS. Palmitoyl-CoA ligase is known to be present in mitochondria, peroxisomes, and endoplasmic reticulum (microsomes). We found enzyme-dependent formation of lignoceroyl-CoA in these same organelles (specific activities were 0.32 +/- 0.12, 0.86 +/- 0.12, and 0.78 +/- 0.07 nmol/h per mg protein, respectively). However, lignoceroyl-CoA synthesis was inhibited by an antibody to palmitoyl-CoA ligase in isolated mitochondria while it was not inhibited in peroxisomes or endoplasmic reticulum (ER). This suggests that palmitoyl-CoA ligase and lignoceroyl-CoA are different enzymes and that mitochondria lack lignoceroyl-CoA ligase. This conclusion is further supported by data showing that oxidation of lignoceric acid was found almost exclusively in peroxisomes (0.17 nmol/h per mg protein) but was largely absent from mitochondria and the finding that monolayers of CHRS fibroblasts lacking peroxisomes showed a pronounced deficiency in lignoceric acid oxidation in situ (1.8% of control). In spite of the observation that lignoceroyl-CoA ligase activity is present on the cytoplasmic surface of ER, our data indicate that lignoceroyl-CoA synthesized by ER is not available for oxidation in mitochondria. This organelle plays no physiological role in the beta-oxidation of VLC fatty acids. Furthermore, the normal peroxisomal oxidation of lignoceroyl-CoA but deficient oxidation of lignoceric acid in X-ALD cells indicates that cellular VLC fatty acid oxidation is dependent on peroxisomal lignoceroyl-CoA ligase. These studies allow us to propose a model for the subcellular localization of various acyl-CoA ligases and to describe how these enzymes control cellular fatty acid metabolism.