acetyl-CoA Flux Research Articles

Effect of ethanol and hydrogen peroxide on poly(3-hydroxybutyrate) biosynthetic pathway in Cupriavidus necator H16

Exposition of Cupriavidus necator to ethanol or hydrogen peroxide at the beginning of the stationary phase increases poly(3-hydroxybutyrate) (PHB) yields about 30%. Hydrogen peroxide enhances activity of pentose phosphate pathway that probably consequently increases intracellular ratio NADPH/NADP(+). This effect leads to stimulation of the flux of acetyl-CoA into PHB biosynthetic pathway and to an increase of enzymatic activities of β-ketothiolase and acetoacetyl-CoA reductase while activity of PHB synthase remains uninfluenced. During ethanol metabolisation, in which alcohol dehydrogenase is involved, acetyl-CoA and reduced coenzymes NAD(P)H are formed. These metabolites could again slightly inhibit TCA cycle while flux of acetyl-CoA into PHB biosynthetic pathway is likely to be supported. As a consequence of TCA cycle inhibition also less free CoA is formed. Similarly with hydrogen peroxide, activities of β-ketothiolase and acetoacetyl-CoA reductase are increased which results in over-production of PHB. Molecular weight of PHB produced under stress conditions was significantly higher as compared to control cultivation. Particular molecular weight values were dependent on stress factor concentrations. This could indicate some interconnection among activities of β-ketothiolase, acetoacetyl-CoA reductase and PHB molecular weight control in vivo.

ATP limitation in a pyruvate formate lyase mutant of Escherichia coli MG1655 increases glycolytic flux to d-lactate

A derivative strain of Escherichia coli MG1655 for D-lactate production was constructed by deleting the pflB, adhE and frdA genes; this strain was designated "CL3." Results show that the CL3 strain grew 44% slower than its parental strain under nonaerated (fermentative) conditions due to the inactivation of the main acetyl-CoA production pathway. In contrast to E. coli B and W3110 pflB derivatives, we found that the MG1655 pflB derivative is able to grow in mineral media with glucose as the sole carbon source under fermentative conditions. The glycolytic flux was 2.8-fold higher in CL3 when compared to the wild-type strain, and lactate yield on glucose was 95%. Although a low cell mass formed under fermentative conditions with this strain (1.2 g/L), the volumetric productivity of CL3 was 1.31 g/L h. In comparison with the parental strain, CL3 has a 22% lower ATP/ADP ratio. In contrast to wild-type E. coli, the ATP yield from glucose to lactate is 2 ATP/glucose, so CL3 has to improve its glycolytic flux in order to fulfill its ATP needs in order to grow. The aceF deletion in strains MG1655 and CL3 indicates that the pyruvate dehydrogenase (PDH) complex is functional under glucose-fermentative conditions. These results suggest that the pyruvate to acetyl-CoA flux in CL3 is dependent on PDH activity and that the decrease in the ATP/ADP ratio causes an increase in the flux of glucose to lactate.

Cardioselective dominant-negative thyroid hormone receptor (Δ337T) modulates myocardial metabolism and contractile efficiency

Dominant-negative thyroid hormone receptors (TRs) show elevated expression relative to ligand-binding TRs during cardiac hypertrophy. We tested the hypothesis that overexpression of a dominant-negative TR alters cardiac metabolism and contractile efficiency (CE). We used mice expressing the cardioselective dominant-negative TRbeta(1) mutation Delta337T. Isolated working Delta337T hearts and nontransgenic control (Con) hearts were perfused with (13)C-labeled free fatty acids (FFA), acetoacetate (ACAC), lactate, and glucose at physiological concentrations for 30 min. (13)C NMR spectroscopy and isotopomer analyses were used to determine substrate flux and fractional contributions (Fc) of acetyl-CoA to the citric acid cycle (CAC). Delta337T hearts exhibited rate depression but higher developed pressure and CE, defined as work per oxygen consumption (MVo(2)). Unlabeled substrate Fc from endogenous sources was higher in Delta337T, but ACAC Fc was lower. Fluxes through CAC, lactate, ACAC, and FFA were reduced in Delta337T. CE and Fc differences were reversed by pacing Delta337T to Con rates, accompanied by an increase in FFA Fc. Delta337T hearts lacked the ability to increase MVo(2). Decreases in protein expression for glucose transporter-4 and hexokinase-2 and increases in pyruvate dehydrogenase kinase-2 and -4 suggest that these hearts are unable to increase carbohydrate oxidation in response to stress. These data show that Delta337T alters the metabolic phenotype in murine heart by reducing substrate flux for multiple pathways. Some of these changes are heart rate dependent, indicating that the substrate shift may represent an accommodation to altered contractile protein kinetics, which can be disrupted by pacing stress.

Enhanced PHB production and scale up studies using cheese whey in fed batch culture of Methylobacterium sp. ZP24

Methylobacterium sp. ZP24 produced polyhydroxybutyrate (PHB) from disaccharides like lactose and sucrose. As Methylobacterium sp. ZP24 showed growth associated PHB production, an intermittent feeding strategy having lactose and ammonium sulfate at varying concentration was used towards reaching higher yield of the polymer. About 1.5-fold increase in PHB production was obtained by this intermittent feeding strategy. Further increase in PHB production by 0.8-fold could be achieved by limiting the dissolved oxygen (DO) levels in the fermenter. The decreased DO is thought to increase flux of acetyl CO-A towards PHB accumulation over TCA cycle. Cheese whey, a dairy waste product and being a rich source of utilizable sugar and other nutrients, when used in the bioreactor as a main substrate replacing the lactose, led to further increase in the PHB production by 2.5-fold. A total of 4.58-fold increase in the PHB production was obtained using limiting DO conditions with processed cheese whey supplemented with ammonium sulfate in fed batch culture of Methylobacterium sp. ZP24. The present investigation therefore reflects on the possibility of developing a cheap biological route for production of green thermoplastics.

Redirecting metabolic fluxes through cofactor engineering: Role of CoA-esters pool during l(−)-carnitine production by Escherichia coli

Cofactor engineering, defined as the purposeful modification of the pool of intracellular cofactors, has been demonstrated to be a very suitable strategy for the improvement of l(−)-carnitine production in Escherichia coli strains. The overexpression of CaiB (CoA-transferase) and CaiC (CoA-ligase), both enzymes involved in coenzyme A transfer and substrate activation during the bioprocess, led to an increase in l(−)-carnitine production. Under optimal concentrations of inducer and fumarate (used as electron acceptors) yields reached 10- and 50-fold, respectively, that obtained for the wild type strain. However, low levels of coenzyme A limited the activity of these two enzymes since the addition of pantothenate increased production. Growth on substrates whose assimilation yields acetyl-CoA (such as acetate or pyruvate) further inhibited l(−)-carnitine production. Interestingly, control steps in the metabolism of acetyl-CoA of E. coli were detected. The glyoxylate shunt and anaplerotic pathways limit the bioprocess since strains carrying deletions of isocitrate lyase and isocitrate dehydrogenase phosphatase/kinase yielded 20–25% more l(−)-carnitine than the control. On the other hand, the deletion of phosphotransacetylase strongly inhibited the bioprocess, suggesting that an adequate flux of acetyl-CoA and the connection of the phosphoenolpyruvate–glyoxylate cycle together with the acetate metabolism are crucial for the biotransformation.

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.

Poly-beta-hydroxybutyrate biosynthesis in the facultative methylotroph methylobacterium extorquens AM1: identification and mutation of gap11, gap20, and phaR.

Methylobacterium extorquens AM1, a serine cycle facultative methylotroph, accumulates poly-beta-hydroxybutyrate (PHB) as a carbon and energy reserve material during growth on both multicarbon- and single-carbon substrates. Recently, the identification and mutation of the genes involved in the biosynthesis and degradation of PHB have been described for this bacterium, demonstrating that two of the genes of the PHB cycle (phaA and phaB) are also involved in C(1) and C(2) metabolism, as part of a novel pathway for glyoxylate regeneration in the serine cycle (N. Korotkova and M. E. Lidstrom, J. Bacteriol. 183:1038-1046, 2001; N. Korotkova, L. Chistoserdova, V. Kuksa, and M. E. Lidstrom, J. Bacteriol. 184:1750-1758, 2002). In this work, three new genes involved in PHB biosynthesis in this bacterium have been investigated via mutation and phenotypic analysis: gap11, gap20, and phaR. We demonstrate that gap11 and gap20 encode two major granule-associated proteins (phasins) and that mutants with mutations in these genes are defective in PHB production and also in growth on C(2) compounds, while they show wild-type growth characteristics on C(1) or multicarbon compounds. The phaR mutant shows defects in both PHB accumulation and growth characteristics when grown on C(1) compounds and has defects in PHB accumulation but grows normally on C(3) and C(4) compounds, while both PHB accumulation and growth rate are at wild-type levels during growth on C(2) compounds. Our results suggest that this phenotype is due to altered fluxes of acetyl coenzyme A (CoA), a major intermediate in C(1), C(2), and heterotrophic metabolism in M. extorquens AM1, as well as the entry metabolite for the PHB cycle. Therefore, it seems likely that PhaR acts to control acetyl-CoA flux to PHB in this methylotrophic bacterium.

Connecting links between the urea cycle and the TCA cycle: a tutorial exercise

Some interesting points arose in a discussion on the links between the urea cycle and the TCA cycle. That aspartate is regenerated from fumarate is well known. One of the prime precursors of urea, the bicarbonate ion, is also formed from the CO 2, which is generated by the TCA cycle. The flux of acetyl CoA through the TCA cycle can indirectly affect the urea cycle by altering the levels of N-acetyl glutamate.

Photosynthetic accumulation of poly-(hydroxybutyrate) by cyanobacteria—the metabolism and potential for CO 2 recycling

Regulatory mechanism in PHB [poly-(hydroxybutyrate)] accumulation by cyanobacteria, especially by a thermophilic isolate, Synechococcus MA19 was reviewed in comparison with a genetically engineered strain. The strain, MA19 accumulates PHB under nitrogen starved and photoautotrophic conditions (MA19-N). Little PHB synthase activity was detected in crude extracts from the cells grown in nitrogen sufficient conditions (MA19+N). The activity was detected exclusively in membrane fractions from MA19+N. The change of the enzyme activity was insensitive to chloramphenicol, which suggests post-translational activation. In vitro, acetyl phosphate activated PHB synthase in membrane fractions from MA19+N, and the extent of activation depended on the concentration of acetyl phosphate. Phosphotransacetylase which catalyzes the conversion of acetyl-CoA to acetyl phosphate was detected in crude extracts from MA19-N but not in those from MA19+N. These results suggested that intracellular acetyl phosphate concentration could be controlled, depending on C–N balance and intracellular acetyl-CoA concentration. On the contrary, in genetically-engineered cyanobacterium (transformant with PHB synthesizing genes from Ralstonia eutropha), it did not seem to be PHB synthase but acetyl-CoA flux that limits PHB synthesis. The closer association of PHB granules with thylakoid membranes in MA19 is suggested than that in the genetically-engineered cyanobacterium, which may reflect the difference of distribution of PHB synthase. Transposon-mutagenesis was used to acquire mutants of its altered PHB regulatory mechanism. PHA production by cyanobacteria was considered from the aspects of photobioreactors.

Reduced effects of L-carnitine on glucose and fatty acid metabolism in myocytes isolated from diabetic rats.

Depressed glucose utilization and over-reliance of muscle tissues on fat represents a major metabolic disturbance in diabetes. This study was designed to investigate the relationship between fatty acid oxidation and glucose utilization in diabetic hearts and to examine the role of L-Carnitine on the utilization of these substrates in diabetes. 14CO2 release from [1-14C]pyruvate (an index of PDH activity), [2-14C]pyruvate and [6-14C]glucose (an index of acetyl-CoA flux through the Krebs cycle), [U-14C]glucose (an index of both PDH and acetyl-CoA flux through the Krebs cycle), and [1-14C]palmitate oxidation were studied in cardiac myocystes isolated from normal and streptozotocin-injected rats. Palmitate oxidation was increased twofold in diabetic myocytes compared to normal cells (5.4 +/- 1.45 vs 2.35 +/- 0.055 nmol/mg protein/30 min, p > 0.05). L-Carnitine (5 mM) significantly increased palmitate oxidation (60-70%) in normal cells but had no effect on diabetic cells. The activity of PDH and acetyl-CoA flux through the Krebs cycle was severely depressed in diabetes (58.14 +/- 20.27 and 8.63 +/- 0.62 in diabetes vs 128.75 +/- 11.47 and 24.84 +/- 7.81 nmol/mg protein/30 min in controls, p > 0.05, respectively). The efflux of acetylcarnitine, a by-product of PDH activity was also much lower in diabetic cells than in normal cells but had no effect in diabetes. L-Carnitine also had no effect on 14CO2 release from [U-14C]glucose but significantly decreased that from [6-14C]glucose, which reflects oxidative metabolism suggesting that L-Carnitine decreases oxidative glucose utilization. Thus, these data suggest that the overreliance on fat in diabetes may be in part secondary to a reduction of carbohydrate-generated acetyl-CoA through the Krebs cycle.

Metabolites and amino acids affecting cellular cofactor concentrations and Poly-β-hydroxybutyrate biosynthesis in Alcaligenes eutrophus

Additon of pyruvate or leucine was found to be efficient for increasing the intracellular ratios of NADH/NAD and NADPH/NADP while reducing the coenzyme A concentration during the cultivation of Alcaligenes eutrophus. Poly-β-hydroxybutyrate (PHB) accumulation was enhanced more than 2-fold since metabolic flux of acetyl-CoA into PHB synthetic pathway could be facilitated by the changes of the cofactor concentrations.

Regulation of poly-β-hydroxybutyrate biosynthesis by nicotinamide nucleotide in Alcaligenes eutrophus

Poly-β-hydroxybutyrate biosynthesis was studied in Alcaligenes eutrophus under various nutrient-limiting conditions. When the cells were cultivated in nitrogen-limited media, both the levels of NAD(P)H and the ratios of NAD(P)H/NAD(P) were higher than those under nitrogen-sufficient conditions. The specific poly-β-hydroxybutyrate production rate was found to increase with the values of both NADH/NAD and NADPH/NADP, indicating that poly-β-hydroxybutyrate synthesis is directly regulated by the ratios of nicotinamide nucleotides. The effects of nicotinamide nucleotides on poly-β-hydroxybutyrate biosynthesis was investigated with regard to enzyme kinetics. Citrate synthase activity was significantly inhibited by NADH and NADPH, indicating that poly-β-hydroxybutyrate accumulation could be enhanced by facilitating the metabolic flux of acetyl-CoA to poly-β-hydroxybutyrate synthetic pathway. It was also found that cellular NADPH was a limiting substrate for NADPH-linked reductase, controlling the overall biosynthetic activity of poly-/3-hydroxybutyrate in this strain.

Regulation of poly-β-hydroxybutyrate biosynthesis by nicotinamide nucleotide in Alcaligenes eutrophus

Poly-β-hydroxybutyrate biosynthesis was studied in Alcaligenes eutrophus under various nutrient-limiting conditions. When the cells were cultivated in nitrogen-limited media, both the levels of NAD(P)H and the ratios of NAD(P)H/NAD(P) were higher than those under nitrogen-sufficient conditions. The specific poly-β-hydroxybutyrate production rate was found to increase with the values of both NADH/NAD and NADPH/NADP, indicating that poly-β-hydroxybutyrate synthesis is directly regulated by the ratios of nicotinamide nucleotides. The effects of nicotinamide nucleotides on poly-β-hydroxybutyrate biosynthesis was investigated with regard to enzyme kinetics. Citrate synthase activity was significantly inhibited by NADH and NADPH, indicating that poly-β-hydroxybutyrate accumulation could be enhanced by facilitating the metabolic flux of acetyl-CoA to poly-β-hydroxybutyrate synthetic pathway. It was also found that cellular NADPH was a limiting substrate for NADPH-linked reductase, controlling the overall biosynthetic activity of poly-/3-hydroxybutyrate in this strain.

Effect of acetic acid on poly-(3-hydroxybutyrate-CO-3-hydroxyvalerate) synthesis in recombinantEscherichia coli

The synthesis of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer by recombinantEscherichia coli was studied in the medium containing glucose and valeric acid as carbon sources. A recombinantE. coli strain (fadR atoC) harboring a stable high-copy number plasmid containing theAlcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes was constructed for the production of the copolymer P(3HB-co-3HV). Accumulation of acetic acid not only had a detrimental effect on cell growth but also decreased the flux of acetyl-CoA into the P(3HB-co-3HV) biosynthetic pathway. Reducing specific growth rate by increasing the initial acetic acid concentration resulted in enhanced copolymer synthesis due to less accumulation of acetic acid. Initial acetic acid concentration of 50 mM was found to be optimal at 20 g/l glucose and 20 mM valeric acid concentration. The fraction of 3-hydroxyvalerate (3HV) increased with decreasing growth temperature. The ratios of 3HV to 3HB in the copolymer could be controlled by altering the concentrations of valeric acid and glucose in the medium. Catabolite repression was in part responsible for the inefficient copolymer synthesis. Various nutritional components were examined for their ability to relieve catabolite repression. An addition of oleic acid resulted in threefold increase of the 3HV fraction in the copolymer. An addition of a small amount of tryptone and peptone considerably promoted P(3HB-co-3HV) synthesis.

917-98 Regulation of Glucose Utilization by L-Carnitine in Cardiac Myocytes

L-Carnitine has been shown to protect the myocardium from ischemic injury, and also improves cardiac function in cardiomyopathies associated with carnitine deficiency and diabetes. However, the exact mechanism whereby carnitine exerts its effect is not fully understood. The present study was designed to investigate the effect of carnitine on the regulation of glucose utilization in the heart. These studies were performed by determining the effect of L-carnitine (5 mM) on the 14 CO 2 release from [1- 14 C]pyruvate (an index of pyruvate dehydrogenase complex, PDH), [2- 14 C]pyruvate (an index of acetyl-CoA flux through Kreb's cycle), and [6- 14 C]glucose (an index of the oxidative utilization of glucose). in rat myocytes. Substrate Rate of Oxidation (nmol/mg protein130 min) Control L-Carnitine [1- 14 C]pyruvate 141.80 ± 8.50 212 ± 28.80 * [2- 14 C]6436pyruvate 39.40 ± 7.90 21.1 ± 4.40 * [6- 14 C]glucose 1.56 ± 0.05 0.95 ± 0.05 * Values are presented as the mean ± SD of at least three different experiments. Asterisks represent a p < 0.05 of the effect of carnitine vs control. These data show that L-carnitine stimulates PDH activity by 50%. However, the flux of acetyl-CoA and glucose through Kreb's cycle was significantly decreased by this compound. These results suggest that L-carnitine enhances the removal of acetyl-CoA produced from glucose metabolism out of the mitochondrial matrix, thereby, activating the PDH complex.

Isotopomer studies of gluconeogenesis and the Krebs cycle with 13C-labeled lactate.

Fasted rats were intragastrically infused with either [2,3-13C]lactate or [1,2,3-13C]lactate. The infusate also contained 14C-labeled lactate and [3-3H]glucose. Glucose, alanine, glutamate, and glutamine were isolated from liver and blood. There was near complete equilibration of lactate and alanine, and the relative specific activities and relative enrichments were the same in blood and liver. Glucose was cleaved enzymatically to lactate. The compounds were examined by gas chromatography-mass spectroscopy. From the mass isotopomer spectra of the lactate, glutamate, and glutamine and their cleavage fragments the positional isotopomer composition of these compounds was obtained. The enrichment and isotopomer pattern in the lactate from cleaved glucose represents that in phosphoenolpyruvate (PEP). When [1,2,3-13C]lactate was infused the mass isotopomer spectrum of glutamates consisted only of compounds containing either one, two, or three 13C carbons per molecule (m1, m2, and m3). There was little 13C in C-4 and C-5 of glutamate. The rate of pyruvate decarboxylation is low, and 3-4% of the acetyl-CoA flux in the Krebs cycle is contributed by lactate carbon. The major isotopomers in lactate, alanine, and PEP were m3 and m2 with 13C in C-2 and C-3. The predominant isotopomer in PEP from [2,3-13C]lactate was m2 with 13C in C-2 and C-3. There was much more of m1 isotopomer with 13C in C-3 and C-2 than the m1 isotopomer with 13C in C-1. There was very little m3, the isotopomer with 13C in all three carbons. Most of the 13C in C-3 and C-4 of glucose and C-1 of glutamate was introduced via 13CO2 fixation. From the isotopomer distribution and the rate of glucose turnover we deduced, applying the analysis described in the "Appendix," the absolute rates of gluconeogenic pathways, recycling of PEP and the Cori cycle, and flux in the Krebs cycle. The flux from oxaloacetate (OAA)-->PEP was seven times that of OAA-->citrate, and about half of PEP was recycled to pyruvate via pyruvate kinase. The mass isotopomer patterns in glutamate and glutamine were similar but differed from those of lactate and glucose. It appears that the glutamates are derived from alpha-ketoglutarate from a different Krebs cycle pool than PEP. The flux from OAA to PEP in this pool was two to three times that of OAA to citrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolism of Tryptophan to Niacin in Saccharomyces uvarum.

In Saccharomyces uvarum, the effect of metabolic intermediates of the tryptophan-NAD pathway on the niacin-production was investigated. Exogenously added kynurenine and 3-hydroxyanthranilic acid raised the content of total niacin of the cells 2-fold as compared to the control cells, although anthranilic acid and tryptophan were less effective. Tryptophan was taken up into the cells faster than kynurenine, and the intracellular pool of tryptophan was larger than that of kynurenine. Of kynurenine (0.05 mM) added to the medium, 55% went through the transaminase flux (2-H liberation), 20% through the kynureninase flux, but none through the acetyl-CoA flux. As for tryptophan, only 2% went through the kynureninase flux. The products through the transaminase flux were identified as kynurenic acid (85%) and xanthurenic acid. 3-Hydroxykynurenine, 3-hydroxyanthranilic acid, quinolinic acid and niacin were also detected. The metabolism of tryptophan via the kynureninase flux reached a plateau above 0.05 mM. The production of kynurenine and kynurenic acid gradually increased above 0.05 mM. Tryptophol was formed in parallel with the amount of tryptophan consumed, while the rate of niacin production increased after glucose and tryptophan were exhausted. Based on the data obtained, a possible regulatory mechanism of the tryptophan-NAD pathway was discussed.

Measurement of the rates of acetyl-CoA hydrolysis and synthesis from acetate in rat hepatocytes and the role of these fluxes in substrate cycling

1. Acetyl-CoA hydrolysis, acetyl-CoA synthesis from acetate and several related fluxes were measured in rat hepatocytes. 2. In contrast with acetyl-CoA hydrolysis, most of the acetyl-CoA synthesis from acetate occurred in the mitochondria. 3. Acetyl-CoA hydrolysis was not significantly affected by 24 h starvation or (-)-hydroxycitrate. 4. In the cytoplasm there was a net flux of acetyl-CoA to acetate, and substrate cycling between acetate and acetyl-CoA in this compartment was very low, accounting for less than 0.1% of the total heat production by the animal. 5. A larger cycle, involving mitochondrial and cytoplasmic acetate and acetyl-CoA, may operate in fed animals, but would account for only approx 1% of total heat production. 6. It is proposed that the opposing fluxes of mitochondrial acetate utilization and cytoplasmic net acetate production may provide sensitivity, feedback and buffering, even when these fluxes are not linked to form a conventional substrate cycle.

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.

TCA Cycle Models: Implications for Tracer Estimates of Gluconeogenesis

A fundamental lueasurement in the assessment of the metabolic status of man and experimental animal is the rate of gluconeogenesis. Yet, reliable methods for measaring this important indicator are not available. A common experimental procedure is to expose gluconeogenic tissue to a labeled carbon molecule that is metabolized to glucose (acetate or pyruvate, for example) and measure the rate of appearance of labeled carbon in glucose. This method requires a correction factor for isotope dilution as the labeled carbon intersects with the tricarboxylic acid (TCA) cycle en route to glucose. Correction for isotope dilution in this instance requires a modeling approach to evaluate quantitatively the intersection of the two pathways. An important feature of TCA cycle models in current use for estimating isotope dilution is the assumption that no carbon enters the TCA cycle except the gluconeogenic flux from pyruvate to oxaloacetate and the flux of acetyl CoA to citrate that resupplies two carbon units to the cycle. We have evaluated the impact of additional fluxes exchanging carbon with TCA cycle pools in the pathway between citrate and oxaloacetate (example, exchange of glutamate with 2-oxyglutarate). Our major findings are: (1) Estimates of the flux of pyruvate to oxaloacetate (y) derived from the glucose labeling ratio are affected by this assumption. Thus, values of y obtained from the classic model are incorrect if additional exchange of carbon occurs. (2). Estimates of gluconeogenesis from tracer pyruvate are strongly dependent on this assumption that carbon enters the TCA cycle only from pyruvate and acetyl CoA. (3). Estimates of glucoeogenesis from carbon tracers yielding labeled acetyl CoA are not affected by this assumption and no error is introduced due to additional fluxes into and out of the TCA cycle. This work illustrates the value of algebraic models of the TCA cycle and related pathways as an aid in developing improved practical tracer methods for estimating gluconeogenesis.