Agmatine stimulates hepatic fatty acid oxidation: a possible mechanism for up-regulation of ureagenesis

We demonstrated previously in a liver perfusion system that agmatine increases oxygen consumption as well as the synthesis of N-acetylglutamate and urea by an undefined mechanism. In this study our aim was to identify the mechanism(s) by which agmatine up-regulates ureagenesis. We hypothesized that increased oxygen consumption and N-acetylglutamate and urea synthesis are coupled to agmatine-induced stimulation of mitochondrial fatty acid oxidation. We used 13C-labeled fatty acid as a tracer in either a liver perfusion system or isolated mitochondria to monitor fatty acid oxidation and the incorporation of 13C-labeled acetyl-CoA into ketone bodies, tricarboxylic acid cycle intermediates, amino acids, and N-acetylglutamate. With [U-13C16] palmitate in the perfusate, agmatine significantly increased the output of 13C-labeled beta-hydroxybutyrate, acetoacetate, and CO2, indicating stimulated fatty acid oxidation. The stimulation of [U-13C16]palmitate oxidation was accompanied by greater production of urea and a higher 13C enrichment in glutamate, N-acetylglutamate, and aspartate. These observations suggest that agmatine leads to increased incorporation and flux of 13C-labeled acetyl-CoA in the tricarboxylic acid cycle and to increased utilization of 13C-labeled acetyl-CoA for synthesis of N-acetylglutamate. Experiments with isolated mitochondria and 13C-labeled octanoic acid also demonstrated that agmatine increased synthesis of 13C-labeled beta-hydroxybutyrate, acetoacetate, and N-acetylglutamate. The current data document that agmatine stimulates mitochondrial beta-oxidation and suggest a coupling between the stimulation of hepatic beta-oxidation and up-regulation of ureagenesis. This action of agmatine may be mediated via a second messenger such as cAMP, and the effects on ureagenesis and fatty acid oxidation may occur simultaneously and/or independently.     

Accumulation of carnitine esters of beta-oxidation intermediates during palmitate oxidation by rat-liver mitochondria

Rat-liver mitochondria were incubated with [14C]palmitate in the presence of L-malate, fluorocitrate, and L-carnitine. The specific activities of acetyl groups incorporated into citrate, ketone bodies and acetyl-L-carnitine were measured. During state-4 oxidation of [1--14C]palmitate the specific activity of the acetyl-CoA pool was 1.3-times higher than that of the average acetyl group of palmitate, indicating an incomplete breakdown of the palmitate molecule. Accumulation of carnitine esters was observed in this condition. The acyl moieties of carnitine esters formed during the state-4 oxidation of [U-14C]palmitate or [16(-14)C]palmitate were analysed by radioactive gas-chromatography. Substantial amounts of beta-oxidation intermediates were found. The accumulation of carnitine esters of C6-C14 intermediates can quantitatively explain the high specific activity of the acetyl-CoA pool during the state-4 oxidation of [1(-14)C] palmitate. The localization and control of beta-oxidation are discussed.     

Peroxisomal fatty acid oxidation is a substantial source of the acetyl moiety of malonyl-CoA in rat heart

Little is known about the sources of acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of mitochondrial fatty acid oxidation in the heart. In perfused rat hearts, we previously showed that malonyl-CoA is labeled from both carbohydrates and fatty acids. This study was aimed at assessing the mechanisms of incorporation of fatty acid carbons into malonyl-CoA. Rat hearts were perfused with glucose, lactate, pyruvate, and a fatty acid (palmitate, oleate or docosanoate). In each experiment, substrates were (13)C-labeled to yield singly or/and doubly labeled acetyl-CoA. The mass isotopomer distribution of malonyl-CoA was compared with that of the acetyl moiety of citrate, which reflects mitochondrial acetyl-CoA. In the presence of labeled glucose or lactate/pyruvate, the (13)C labeling of malonyl-CoA was up to 2-fold lower than that of mitochondrial acetyl-CoA. However, in the presence of a fatty acid labeled in its first acetyl moiety, the (13)C labeling of malonyl-CoA was up to 10-fold higher than that of mitochondrial acetyl-CoA. The labeling of malonyl-CoA and of the acetyl moiety of citrate is compatible with peroxisomal beta-oxidation forming C(12) and C(14) acyl-CoAs and contributing >50% of the fatty acid-derived acetyl groups that end up in malonyl-CoA. This fraction increases with the fatty acid chain length. By supplying acetyl-CoA for malonyl-CoA synthesis, peroxisomal beta-oxidation may participate in the control of mitochondrial fatty acid oxidation in the heart. In addition, this pathway may supply some acyl groups used in protein acylation, which is increasingly recognized as an important regulatory mechanism for many biochemical processes.     

Adrenoleukodystrophy. The chain shortening of erucic acid (22:1(n-9)) and adrenic acid (22:4(n-6)) is deficient in neonatal adrenoleukodystrophy and normal in X-linked adrenoleukodistrophy skin fibroblasts

The metabolism of long chain unsaturated fatty acids was studied in cultured fibroblasts from patients with X-linked adrenoleukodystrophy (ALD) and with neonatal ALD. By using [14-14C] erucic acid (22:1(n-9)) as substrate it was shown that the peroxisomal beta-oxidation, measured as chain shortening, was impaired in cells from patients with neonatal ALD. The beta-oxidation of adrenic acid (22:4(n-6)), measured as acid-soluble products, was also reduced in the neonatal ALD cells. The peroxisomal beta-oxidation of [14-14C]erucic acid (22:1(n-9)) and [2-14C]adrenic acid (22:4(n-6)) was normal in cells from X-ALD patients. The beta-oxidation, esterification and chain elongation of [1-14C]arachidonic acid (20:4(n-6)) and [1-14C]eicosapentaenoic acid (20:5(n-3)) was normal in both X-linked ALD and in neonatal ALD. Previous studies suggest that the activation of very long chain fatty acids by a lignoceryl (24:0)-CoA ligase is deficient in X-linked ALD, while the peroxisomal beta-oxidation enzymes are deficient in neonatal ALD. The present results suggest that the peroxisomal very long-chain acyl-CoA ligase is not required for activation of unsaturated C20 and C22 fatty acids and that these fatty acids can be efficiently activated by the long chain acyl-(palmityl)-CoA ligase.     

Peroxisomal beta-oxidation of branched chain fatty acids in human skin fibroblasts.

Human skin fibroblasts in suspension are able to degrade [1-14C]-labeled alpha- and gamma-methyl branched chain fatty acids such as pristanic and homophytanic acid. Pristanic acid was converted to propionyl-CoA, whereas homophytanic acid was beta-oxidized to acetyl-CoA. Incubation of skin fibroblasts with [1-14C]-labeled fatty acids for longer periods produced radiolabeled carbon dioxide, presumably by further degradation of acetyl-CoA or propionyl-CoA generated by beta-oxidation. Under the same conditions similar products were produced from very long chain fatty acids, such as lignoceric acid. Inclusion of digitonin (> 10 micrograms/ml) in the incubations strongly inhibited carbon dioxide production but stimulated acetyl-CoA or propionyl-CoA production from fatty acids. ATP, Mg2+, coenzyme A, NAD+ and L-carnitine stimulated acetyl-CoA or propionyl-CoA production from [1-14C]-labeled fatty acids in skin fibroblast suspensions. Branched chain fatty acid beta-oxidation was reduced in peroxisome-deficient cells (Zellweger syndrome and infantile Refsum's disease) but they were beta-oxidized normally in cells from patients with X-linked adrenoleukodystrophy (ALD). Under the same conditions, lignoceric acid beta-oxidation was impaired in the above three peroxisomal disease states. These results provide evidence that branched chain fatty acid, as well as very long chain fatty acid, beta-oxidation occurs only in peroxisomes. As the defect in X-linked ALD is in a peroxisomal fatty acyl-CoA synthetase, which is believed to be specific for very long chain fatty acids, we postulate that different synthetases are involved in the activation of branched chain and very long chain fatty acids in peroxisomes.     

Peroxisomal and mitochondrial oxidation of fatty acids in the heart, assessed from the 13C labeling of malonyl-CoA and the acetyl moiety of citrate

We previously showed that a fraction of the acetyls used to synthesize malonyl-CoA in rat heart derives from partial peroxisomal oxidation of very long and long-chain fatty acids. The 13C labeling ratio (malonyl-CoA)/(acetyl moiety of citrate) was >1.0 with 13C-fatty acids, which yields [13C]acetyl-CoA in both mitochondria and peroxisomes and < 1.0 with substrates, which yields [13C]acetyl-CoA only in mitochondria. In this study, we tested the influence of 13C-fatty acid concentration and chain length on the labeling of acetyl-CoA formed in mitochondria and/or peroxisomes. Hearts were perfused with increasing concentrations of labeled docosanoate, oleate, octanoate, hexanoate, butyrate, acetate, or dodecanedioate. In contrast to the liver, peroxisomal oxidation of 1-13C-fatty acids in heart does not form [1-13C]acetate. With [1-13C]docosanoate and [1,12-13C2]dodecanedioate, malonyl-CoA enrichment plateaued at 11 and 9%, respectively, with no detectable labeling of the acetyl moiety of citrate. Thus, in the intact rat heart, docosanoate and dodecanedioate appear to be oxidized only in peroxisomes. With [1-13C]oleate or [1-13C]octanoate, the labeling ratio >1 indicates the partial peroxisomal oxidation of oleate and octanoate. In contrast, with [3-13C]octanoate, [1-13C]hexanoate, [1-13C]butyrate, or [1,2-13C2]acetate, the labeling ratio was <0.7 at all concentrations. Therefore, in rat heart, (i) n-fatty acids shorter than 8 carbons do not undergo peroxisomal oxidation, (ii) octanoate undergoes only one cycle of peroxisomal beta-oxidation, (iii) there is no detectable transfer to the mitochondria of acetyl-CoA from the cytosol or the peroxisomes, and (iv) the capacity of C2-C18 fatty acids to generate mitochondrial acetyl-CoA decreases with chain length.     

The effects of alkylthioacetic acids (3-thia fatty acids) on fatty acid metabolism in isolated hepatocytes

Long-chain alkylthioacetic acids (3-thia fatty acids) inhibit fatty acid synthesis from [1-14C]acetate in isolated hepatocytes, while fatty acid oxidation is nearly unaffected or even stimulated. Desaturation of [1-14C]stearate (delta 9-desaturase) is also unaffected. [1-14C]Dodecylthioacetic acid (a 3-thia fatty acid) is incorporated in triacylglycerol and in phospholipids more efficiently than [1-14C]palmitate in isolated hepatocytes. The metabolism of [1-14C]dodecylthioacetic acid to acid-soluble products (by omega-oxidation) is slow compared to the oxidation of [1-14C]palmitate. In hepatocytes from adapted rats (rats fed tetradecylthioacetic acid for 4 days) the rate of [1-14C]palmitate oxidation is increased and its rate of esterification is decreased. Stearate desaturation is also decreased. The rate of cyanide-insensitive peroxisomal fatty acid beta-oxidation is several-fold increased. The metabolic effects of long-chain 3-thia fatty acids are discussed and it is concluded that they behave essentially like normal fatty acids except for their slow breakdown due to the sulfur atom in the 3 position, which blocks normal beta-oxidation.     

PNU-91325 increases fatty acid synthesis from glucose and mitochondrial long chain fatty acid degradation: a comparative tracer-based metabolomics study with rosiglitazone and pioglitazone in HepG2 cells

The mitochondrial membrane protein termed “mitoNEET,” is a putative secondary target for insulin-sensitizing thiazolidinedione (TZD) compounds but its role in regulating metabolic flux is not known. PNU-91325 is a thiazolidinedione derivative which exhibits high binding affinity to mitoNEET and lowers cholesterol, fatty acid and blood glucose levels in animal models. In this study we report the stable isotope-based dynamic metabolic profiles (SIDMAP) of rosiglitazone, pioglitazone and PNU-91325 in a dose-matching, dose-escalating study. One and 10 μM concentrations 1 and 10 μM drug concentrations were introduced into HepG2 cells in the presence of either [1,2−¹³C₂]-D-glucose or [U−¹³C₁₈]stearate, GC/MS used to determine positional tracer incorporation (mass isotopomer analysis) into multiple metabolites produced by the Krebs and pentose cycles, de novo fatty acid synthesis, long chain fatty acid oxidation, chain shortening and elongation. Rosiglitazone and pioglitazone (10 μM) increased pentose synthesis from [U−¹³C₁₈]stearate by 127% and 185%, respectively, while PNU-91325 rather increased glutamate synthesis in the Krebs cycle by 113% as compared to control vehicle treated cells. PNU-91325 also increased stearate chain shortening into palmitate by 59%. Glucose tracer-derived de novo palmitate and stearate synthesis were increased by 1 and 10 μM rosiglitazone by 41% and 83%, respectively, and by 63% and 75% by PNU-91325. Stearate uptake was also increased by 10 μM PNU-91325 by 15.8%. We conclude that the entry of acetyl Co-A derived from long-chain fatty acid β-oxidation into the mitochondria is facilitated by the mitoNEET ligand PNU-91325, which increases glucose-derived long chain fatty acid synthesis and breakdown via β-oxidation and anaplerosis in the mitochondria.     

Intermediates of peroxisomal beta-oxidation of [U-14C]hexadecanedionoate. A study of the acyl-CoA esters which accumulate during peroxisomal beta-oxidation of [U-14C]hexadecanedionate and [U-14C]hexadecanedionoyl-mono-CoA.

1. The oxidation of [U-14C]hexadecanedionoyl-mono-CoA was stimulated by CoA, by carnitine in the absence of CoA and by the presence of an NAD(+)-regenerating system. 2. Substrate inhibition was observed with respect to [U-14C]hexadecanedionoyl-mono-CoA at concentrations greater than 35 microM. 3. Acetyl-CoA and the dicarboxyl-CoA esters of chain length C6-16 were detected by HPLC under standard incubation conditions. 4. In the absence of the NAD(+)-regenerating system, 2-enoyl-CoA and 3-hydroxacyl-CoA esters were detected. 5. In general, the peroxisomal beta-oxidation of dicarboxylates is very similar to that of monocarboxylates [Bartlett, K., Hovik, R., Eaton, S., Watmough, N. J. & Osmundsen, H. (1990) Biochem. J. 270, 175-180] except that chain shortening does not proceed beyond C6. 6. We conclude that the peroxisomal beta-oxidation of dicarboxylates is regulated by the redox state of the peroxisomal matrix and CoA availability.     

Metabolism of saturated and polyunsaturated very-long-chain fatty acids in fibroblasts from patients with defects in peroxisomal beta-oxidation.

The metabolism of [1-14C]lignoceric acid (C24:0) and [1-14C]tetracosatetraenoic acid (C24:4, n-6) was studied in normal skin fibroblast cultures and in cultures from patients with defects in peroxisomal beta-oxidation (but normal peroxisomal numbers). Cells from X-linked adrenoleukodystrophy (ALD) patients with a presumed defect in a peroxisomal acyl-CoA synthetase, specific for fatty acids of carbon chain lengths greater than 22 (very-long-chain fatty acids; VLCFA), showed a relatively normal production of radiolabelled CO2 and water-soluble metabolites from [1-14C]C24:0. However, the products of synthesis from acetate de novo (released by beta-oxidation), i.e. C16 and C18 fatty acids, were decreased, and carbon chain elongation of the fatty acid was increased. In contrast, cell lines from two patients with an unidentified lesion in peroxisomal beta-oxidation (peroxisomal disease, PD) showed a marked deficiency in CO2 and water-soluble metabolite production, a decreased synthesis of C16 and C18 fatty acids and an increase in carbon chain elongation. The relatively normal beta-oxidation activity of ALD cells appears to be related to low uptake of substrate, as a defect in beta-oxidation is apparent when measurements are performed on cell suspensions under high uptake conditions. Oxidation of [1-14C]C24:4 was relatively normal in ALD cells and in the cells from one PD patient but abnormal in those from the other. Our data suggest that, despite the deficiency in VLCFA CoA synthetase, ALD cells retain a near normal ability to oxidize both saturated and polyunsaturated VLCFA under some culture conditions. However, acetate released by beta-oxidation of the saturated VLCFA and, to a much lesser degree, the polyunsaturated VLCFA, appears to be used preferentially for the production of CO2 and water-soluble products, and acetate availability for fatty acid synthesis in other subcellular compartments is markedly decreased. It is likely that the increased carbon chain elongation of the saturated VLCFA which is also observed reflects the increased availability of substrate (C24:0) and/or an increase in microsomal elongation activity in ALD cells.     

The incorporation of acetate,stearate and d(?)-β-hydroxybutyrate into milk fat by the isolated perfused mammary gland of the goat

1. Mammary glands of lactating goats were perfused for 12.5-15hr. with heparinized whole blood and infused with a substrate mixture of glucose, acetate and amino acids (and sometimes chylomicra) containing either [1-(14)C]acetate, d(-)-beta-hydroxy[1-(14)C]butyrate or [U-(14)C]stearate. 2. There was a substantial net uptake of acetate by the glands and transfer of radioactivity into milk fat. Acetate was extensively utilized for the synthesis of milk fatty acids of chain length up to C(14) and to a smaller extent for the synthesis of palmitate. 3. There was a small and variable net uptake of stearate and beta-hydroxybutyrate and negligible oxidation of these substrates. However, tissue uptake was demonstrated by a substantial fall in specific radioactivity across the glands and an extensive transfer of radioactivity into milk fatty acids. 4. With beta-hydroxybutyrate the labelling of milk fat was very similar to that with acetate, but the distribution of radioactivity suggested a cleavage into C(2) fragments of about 40%. 5. Labelled stearate gave rise to highly labelled stearate and oleate in the milk fat. Small amounts of radioactivity were detected in stearate of plasma triglycerides and oleate of plasma free fatty acids. 6. In experiments where there was a decline in milk-fat secretion late in perfusion, the milk fatty acids showed a marked decline in the proportion of stearate and oleate and a rise in the proportion of myristate and palmitate. This did not occur in experiments where milk-fat secretion was maintained at a higher level. 7. The present results confirm that there is a large pool of long-chain fatty acids in mammary tissue that can act as an endogenous source of these substrates.     

Properties of a collagenase inhibitor partially purified from cultures of smooth muscle cells

1. The metabolism of [14-14C]erucate and [U-14C]palmitate has been investigated in perfused heart from rats fed 0.3% clofibrate for 10 days and from control rats. 2. The total uptake of fatty acids in the heart increased in the clofibrate fed group. Clofibrate increased the oxidation of [14-14C]erucic acid by 100% and the oxidation of [U-14C]palmitic acid by 30% compared to controls. 3. The chain-shortening of erucate to C20:1 and C18:1 fatty acids in the perfused heart was stimulated at least two-fold by clofibrate feeding. 4. The activity of the peroxisomal marker enzyme catalase increased 60%, the activity of cytochrome oxidase increased approx. 16% and the content of total coenzyme A increased 30% in heart homogenates from rats fed clofibrate compared to controls. 5. The isolated mitochondrial fraction from clofibrate fed rats showed an increased capacity for oxidation of palmitoylcarnitine and decanoylcarnitine, while the oxidation of erucoylcarnitine showed little change. 6. It is suggested that clofibrate increases the oxidation of [14-14C]erucic acid in the perfused heart by increasing the capacity for chain-shortening of [14-14C]erucate in the peroxisomal beta-oxidation system.     

beta-Oxidation of polyunsaturated fatty acids by rat liver peroxisomes. A role for 2,4-dienoyl-coenzyme A reductase in peroxisomal beta-oxidation

beta-Oxidation of unsaturated fatty acids was studied with isolated solubilized or nonsolubilized peroxisomes or with perfused liver isolated from rats treated with clofibrate. gamma-Linolenic acid gave the higher rate of beta-oxidation, while arachidonic acid gave the slower rate of beta-oxidation. Other polyunsaturated fatty acids (including docosahexaenoic acid) were oxidized at rates which were similar to, or higher than, that observed with oleic acid. Experiments with 1-14C-labeled polyunsaturated fatty acids demonstrated that these are chain-shortened when incubated with nonsolubilized peroxisomes. Spectrophotometric investigation of solubilized peroxisomal incubations showed that 2,4-dienoyl-CoA esters accumulated during peroxisomal beta-oxidation of fatty acids possessing double bond(s) at even-numbered carbon atoms. beta-Oxidation of [1-14C]docosahexaenoic acid by isolated peroxisomes was markedly stimulated by added NADPH or isocitrate. This fatty acid also failed to cause acyl-CoA-dependent NADH generation with conditions of assay which facilitate this using other acyl-CoA esters. These findings suggest that 2,4-dienoyl-CoA reductase participation is essential during peroxisomal beta-oxidation if chain shortening is to proceed beyond a delta 4 double bond. Evidence obtained using arachidionoyl-CoA, [1-14C]arachidonic acid, and [5,6,8,9,11,12,14,15-3H]arachidonic acid suggests that peroxisomal beta-oxidation also can proceed beyond a double bond positioned at an odd-numbered carbon atom. Experiments with isolated perfused livers showed that polyunsaturated fatty acids also in the intact liver are substrates for peroxisomal beta-oxidation, as judged by increased levels of the catalase-H2O2 complex on infusion of polyunsaturated fatty acids.     

The regulation of peroxisomal enzyme systems of Tetrahymena pyriformis by fatty acid composition, glucose and oxygen in the medium

The effect of the chain length of fatty acids on peroxisomal enzyme activities of Tetrahymena pyriformis was investigated. The growth of cells and the activities of peroxisomal enzymes were inhibited markedly by the addition of medium-chain fatty acids (C6-C12) to the culture medium, whereas the addition of longer-chain fatty acids (C14-C18) resulted in a slight increase of growth and in the marked stimulation of enzyme activities concerned with fatty acid beta-oxidation and the glyoxylate cycle in peroxisomes. Peroxisomal beta-oxidation (fatty acyl-CoA oxidase) was more potent towards longer-chain fatty acids than the mitochondrial activity (fatty acyl-CoA dehydrogenase). The induction of the peroxisomal beta-oxidation system by palmitate was repressed both by the addition of glucose and the aeration of the culture medium, whereas that of the peroxisomal glyoxylate cycle was repressed only by the addition of glucose to the medium. These results indicate that peroxisomal enzyme systems related to the beta-oxidation of fatty acids and the glyoxylate cycle are regulated by the compositions of fatty acids, glucose, and oxygen in the medium.     

GLP-1 stimulates glucose-derived de novo fatty acid synthesis and chain elongation during cell differentiation and insulin release

Glucagon-like peptide-1 (GLP-1, 7-36) is capable of restoring normal glucose tolerance in aging, glucose-intolerant Wistar rats and is a potent causal factor in differentiation of human islet duodenal homeobox-1-expressing cells into insulin-releasing beta cells. Here we report stable isotope-based dynamic metabolic profiles of rat pancreatic epithelial (ARIP) and human ductal tumor (PANC-1) cells responding to 10 nM GLP-1 treatment in 48 h cultures. Macromolecule synthesis patterns and substrate flow measurements using gas chromatography/mass spectrometry (MS) and the stable [1,2-13C2]glucose isotope as the tracer showed that GLP-1 induced a significant 20% and 60% increase in de novo fatty acid palmitate synthesis in ARIP and PANC-1 cells, respectively, and it also induced a significant increase in palmitate chain elongation into stearate utilizing glucose as the primary substrate. Distribution of 13C in other metabolites indicated no changes in the rates of nucleic acid ribose synthesis, glutamate oxidation, or lactate production. Tandem high-performance liquid chromatography-ion trap MS analysis of the culture media demonstrated mass insulin secretion by GLP-1-treated tumor cells. Metabolic profile changes in response to GLP-1-induced cell differentiation include selective increases in de novo fatty acid synthesis from glucose and consequent chain elongation, allowing increased membrane formation and greater insulin availability and release.     

Glucose effects on lung surfactant kinetics in conscious pigs

The primary goal of this study was to investigate the effects of glucose infusion on surfactant phosphatidylcholine (PC) metabolic kinetics in the lungs. A new stable isotope tracer model was used in which [1,2-(13)C(2)]acetate and uniformly labeled [U-(13)C(16)]palmitate were infused in 12 normal overnight-fasted pigs to quantify lung surfactant kinetics with or without glucose infusion (24 mg. kg(-1). min(-1)). With glucose infusion, the rate of surfactant PC incorporation from de novo synthesized palmitate increased from the control value of 2.1 +/- 0.2 to 15.5 +/- 1.9 nmol PC-bound palmitate. h(-1). g wet lung(-1) (P < 0.05), whereas the incorporation rate from plasma preformed palmitate decreased from the control value of 20.9 +/- 1.9 to 11.6 +/- 1.1 nmol palmitate. h(-1). g wet lung(-1) (P < 0.05). The palmitate composition in lamellar body surfactant PC increased from the control value of 61.7 +/- 2.1% to 75.9 +/- 0.6% (P < 0.05). The surfactant PC secretion rate decreased from the control value of 239.0 +/- 26.1 to 81.9 +/- 5.3 nmol PC-bound palmitate. h(-1). g wet lung(-1) (P < 0.05). We conclude that, whereas surfactant secretion was inhibited by glucose infusion, neither total surfactant PC synthesis nor the surfactant PC pool size was significantly affected due to an increased reliance on de novo synthesized fatty acids.     

Lipid biosynthesis in developing and germinating soybean cotyledons: The formation of palmitate and stearate by chopped tissue and supernatant preparations

Chopped tissue from developing soybean cotyledons incorporated [1-¹⁴C]acetate into palmitate, stearate, oleate, and linoleate, but with germinating cotyledons much less [1-¹⁴C]acetate was incorporated and the principal labeled products were palmitate, stearate, and oleate. When supernatant fractions from developing cotyledons were incubated with [1-¹⁴C]acetate or [2-¹⁴C]malonate the principal labeled products were palmitate and stearate. Supernatant fractions from germinating seed incorporated [2-¹⁴C]malonate into palmitate and also into short chain fatty acids including decanoate, laurate, and myristate. Supernatants from developing cotyledons required acyl carrier protein (ACP), ATP, CoA, and reduced pyridine nucleotides for maximal rates of incorporation of either [1-¹⁴C]acetate or [2-¹⁴C]malonate into palmitate and stearate. The de novo fatty acid synthetase which converts acetyl- and malonyl-ACP's to palmityl ACP was active in supernatant fractions from both young and old developing cotyledons. The elongation system, converting palmityl ACP to stearyl ACP, was more active in supernatants from younger than from older developing cotyledons. In experiments with chopped tissue the elongation system appeared equally active throughout the development process. These results are consistent with the view that the de novo and elongation systems are separate entities and that the elongation system in older cotyledons is less stable to the methods used to prepare supernatant fractions.     

Carnitine-dependent transport of acetyl coenzyme A in Candida albicans is essential for growth on nonfermentable carbon sources and contributes to biofilm formation

In eukaryotes, acetyl coenzyme A (acetyl-CoA) produced during peroxisomal fatty acid beta-oxidation needs to be transported to mitochondria for further metabolism. Two parallel pathways for acetyl-CoA transport have been identified in Saccharomyces cerevisiae; one is dependent on peroxisomal citrate synthase (Cit), while the other requires peroxisomal and mitochondrial carnitine acetyltransferase (Cat) activities. Here we show that the human fungal pathogen Candida albicans lacks peroxisomal Cit, relying exclusively on Cat activity for transport of acetyl units. Deletion of the CAT2 gene encoding the major Cat enzyme in C. albicans resulted in a strain that had lost both peroxisomal and mitochondrion-associated Cat activities, could not grow on fatty acids or C(2) carbon sources (acetate or ethanol), accumulated intracellular acetyl-CoA, and showed greatly reduced fatty acid beta-oxidation activity. The cat2 null mutant was, however, not attenuated in virulence in a mouse model of systemic candidiasis. These observations support our previous results showing that peroxisomal fatty acid beta-oxidation activity is not essential for C. albicans virulence. Biofilm formation by the cat2 mutant on glucose was slightly reduced compared to that by the wild type, although both strains grew at the same rate on this carbon source. Our data show that C. albicans has diverged considerably from S. cerevisiae with respect to the mechanism of intracellular acetyl-CoA transport and imply that carnitine dependence may be an important trait of this human fungal pathogen.     

Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos

A method involving labeling to isotopic steady state and modeling of the tricarboxylic acid cycle has been used to identify the respiratory substrates in lettuce embryos during the early steps of germination. We have compared the specific radioactivities of aspartate and glutamate and of glutamate C-1 and C-5 after labeling with different substrates. Labeling with [U-14C]acetate and 14CO2 was used to verify the validity of the model for this study; the relative labeling of aspartate and glutamate was that expected from the normal operation of the tricarboxylic acid cycle. After labeling with 14CO2, the label distribution in the glutamate molecule (95% of the label at glutamate C-1) was consistent with an input of carbon via the phosphoenolpyruvate carboxylase reaction, and the relative specific radioactivities of aspartate and glutamate permitted the quantification of the apparent rate of the fumarase reaction. CO2 and intermediates related to the tricarboxylic acid cycle were labeled with [U-14C]acetate, [1-14C] hexanoate, or [U-14C]palmitic acid. The ratios of specific radioactivities of asparate to glutamate and of glutamate C-1 to C-5 indicated that the fatty acids were degraded to acetyl units, suggesting the operation of beta-oxidation, and that the acety-CoA was incorporated directly into citrate. Short-term labeling with [1-14C]hexanoate showed that citrate and glutamate were labeled earlier than malate and aspartate, showing that this fatty acid was metabolized through the tricarboxylic acid cycle rather than the glyoxylate cycle. This was in agreement with the flux into gluconeogenesis compared to efflux as respiratory CO2. The fraction of labeled substrate incorporated into carbohydrates was only about 5% of that converted to CO2; the carbon flux into gluconeogenesis was determined after labeling with 14CO2 and [1-14C]hexanoate from the specific radioactivity of aspartate C-1 and the amount of label incorporated into the carbohydrate fraction. It was only 7.4% of the efflux of respiratory CO2. The labeling of alanine indicates a low activity of either a malic enzyme or the sequence phosphoenolpyruvate carboxykinase/pyruvate kinase. After labeling with [U-14C]glucose, the ratios of specific radioactivities indicated that the labeled carbohydrates contributed less than 10% to the flux of acetyl-CoA. The model indicated that the glycolytic flux is partitioned one-third to pyruvate and two-thirds to oxalacetate and is therefore mainly anaplerotic. The possible role of fatty acids as the main source of acetyl-CoA for respiration is discussed.