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ABSTRACT: We conducted a study coupling metabolomics and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. Rat
livers were perfused with lactate or pyruvate ± aminooxyacetate or mercaptopicolinate in the presence of 40% enriched NaH13CO3. Other livers were perfused with dimethyl [1,4-13C2]succinate ± mercaptopicolinate. In this first of two companion articles, we show that a substantial fraction of gluconeogenic
carbon leaves the liver as citric acid cycle intermediates, mostly α-ketoglutarate. The efflux of gluconeogenic carbon ranges
from 10 to 200% of the rate of liver gluconeogenesis. This cataplerotic efflux of gluconeogenic carbon may contribute to renal
gluconeogenesis in vivo. Multiple crossover analyses of concentrations of gluconeogenic intermediates and redox measurements expand previous reports
on the regulation of gluconeogenesis and the effects of inhibitors. We also demonstrate the formation of adducts from the
condensation, in the liver, of (i) aminooxyacetate with pyruvate, α-ketoglutarate, and oxaloacetate and (ii) mercaptopicolinate
and pyruvate. These adducts may exert metabolic effects unrelated to their effect on gluconeogenesis.
Journal of Biological Chemistry 08/2008; 283(32):21978-21987. · 4.77 Impact Factor
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ABSTRACT: In this second of two companion articles, we compare the mass isotopomer distribution of metabolites of liver gluconeogenesis and citric acid cycle labeled from NaH(13)CO(3) or dimethyl [1,4-(13)C(2)]succinate. The mass isotopomer distribution of intermediates reveals the reversibility of the isocitrate dehydrogenase + aconitase reactions, even in the absence of a source of alpha-ketoglutarate. In addition, in many cases, a number of labeling incompatibilities were found as follows: (i) glucose versus triose phosphates and phosphoenolpyruvate; (ii) differences in the labeling ratios C-4/C-3 of glucose versus (glyceraldehyde 3-phosphate)/(dihydroxyacetone phosphate); and (iii) labeling of citric acid cycle intermediates in tissue versus effluent perfusate. Overall, our data show that gluconeogenic and citric acid cycle intermediates cannot be considered as sets of homogeneously labeled pools. This probably results from the zonation of hepatic metabolism and, in some cases, from differences in the labeling pattern of mitochondrial versus extramitochondrial metabolites. Our data have implications for the use of labeling patterns for the calculation of metabolic rates or fractional syntheses in liver, as well as for modeling liver intermediary metabolism.
Journal of Biological Chemistry 07/2008; 283(32):21988-96. · 4.77 Impact Factor
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ABSTRACT: We conducted a study coupling metabolomics and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. Rat livers were perfused with lactate or pyruvate +/- aminooxyacetate or mercaptopicolinate in the presence of 40% enriched NaH(13)CO(3). Other livers were perfused with dimethyl [1,4-(13)C(2)]succinate +/- mercaptopicolinate. In this first of two companion articles, we show that a substantial fraction of gluconeogenic carbon leaves the liver as citric acid cycle intermediates, mostly alpha-ketoglutarate. The efflux of gluconeogenic carbon ranges from 10 to 200% of the rate of liver gluconeogenesis. This cataplerotic efflux of gluconeogenic carbon may contribute to renal gluconeogenesis in vivo. Multiple crossover analyses of concentrations of gluconeogenic intermediates and redox measurements expand previous reports on the regulation of gluconeogenesis and the effects of inhibitors. We also demonstrate the formation of adducts from the condensation, in the liver, of (i) aminooxyacetate with pyruvate, alpha-ketoglutarate, and oxaloacetate and (ii) mercaptopicolinate and pyruvate. These adducts may exert metabolic effects unrelated to their effect on gluconeogenesis.
Journal of Biological Chemistry 07/2008; 283(32):21978-87. · 4.77 Impact Factor
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ABSTRACT: Anaplerosis from propionate was investigated in rat hearts perfused with 0-2mM [(13)C(3)]propionate and physiological concentrations of glucose, lactate, and pyruvate. The data show that when the concentration of [(13)C(3)]propionate was raised from 0 to 2mM, total anaplerosis increased from 5% to 16% of the turnover of citric acid cycle intermediates. Then, [(13)C(3)]propionate abolished anaplerosis from endogenous substrates, glucose, lactate, and pyruvate. Also, while the contents of propionyl-CoA and methylmalonyl-CoA increased with [(13)C(3)]propionate concentration, the content of succinyl-CoA decreased, presumably via activation of succinyl-CoA hydrolysis by a decrease in free CoA. Under our conditions, [(13)C(3)]propionate was a purely anaplerotic substrate since there was no labeling of mitochondrial acetyl-CoA, reflected by the labeling of the acetyl moiety of citrate.
Archives of Biochemistry and Biophysics 08/2007; 463(1):110-7. · 2.93 Impact Factor
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ABSTRACT: We developed gas chromatography-mass spectrometry assays for the relative concentration and for the mass isotopomer distribution of gluconeogenic and citric acid cycle intermediates in tissues. The assay involves (i) spiking the sample with one or more internal standards, (ii) chloroform–methanol extraction at −25°C, (iii) Folch wash of the extract, (iv) treatment of the water-methanol phase with methoxylamine, (v) evaporation and trimethylsilyl derivatization, and (vi) ammonia positive chemical ionization gas chromatography-mass spectrometry. For metabolomic computations, indices of concentrations for all compounds assayed are calculated as (Area of analyte)/(Area of reference compound). The assay was applied to a study of the effect of mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, on the profile of gluconeogenic intermediates in rat livers perfused with pyruvate. Crossover analysis of concentrations indices, compared to a control group, yielded very similar profiles as previous enzymatic assays, and correctly identified the site of action of mercaptopicolinate. Principal component analysis distinguished between control and drug treated samples. A loadings plot was used to identify the site of action of the drug in the metabolic pathway. Since metabolite concentrations do not address the flux through a pathway, perfusions with [1,4-13C2] succinate dimethylester were conducted to assess fluxes around PEPCK. This allowed a dynamic metabolomics analysis which indicated that considerable flux through the pathway remained in the presence of mercaptopicolinate. This study illustrates the power of dynamic metabolomics to complement concentration based metabolomic studies.
Metabolomics 05/2006; 2(2):85-94. · 4.51 Impact Factor
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ABSTRACT: We reported previously that a substantial fraction of the acetyl groups used to synthesize malonyl-CoA in rat heart is derived from peroxisomal beta-oxidation of long-chain and very-long-chain fatty acids. This conclusion was based on the interpretation of the 13C-labelling ratio (malonyl-CoA)/(acetyl moiety of citrate) measured in the presence of substrates that label acetyl-CoA in mitochondria only (ratio < 1.0) or in both mitochondria and peroxisomes (ratio > 1.0). The goals of the present study were to test, in rat livers perfused with [1-(13C)]octanoate or [3-(13C)]octanoate, (i) whether peroxisomal beta-oxidation contributes acetyl groups for malonyl-CoA synthesis, and (ii) the degree of labelling homogeneity of acetyl-CoA proxies (acetyl moiety of citrate, acetate, beta-hydroxybutyrate, malonyl-CoA and acetylcarnitine). Our data show that (i) octanoate undergoes two cycles of peroxisomal beta-oxidation in liver, (ii) acetyl groups formed in peroxisomes contribute to malonyl-CoA synthesis, (iii) the labelling of acetyl-CoA proxies is markedly heterogeneous, and (iv) the labelling of C1+2 of beta-hydroxybutyrate does not reflect the labelling of acetyl-CoA used in the citric acid cycle.
Biochemical Journal 08/2005; 389(Pt 2):397-401. · 4.90 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 04/2005; 280(10):9265-71. · 4.77 Impact Factor
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ABSTRACT: Measurement of fractional lipogenesis by condensation polymerization methods assumes constant enrichment of lipogenic acetyl-CoA in all hepatocytes. mass isotopomer distribution analysis (MIDA) and isotopomer spectral analysis (ISA) represent such methods and are based on the combinatorial analyses of mass isotopomer distributions (MIDs) of fatty acids and sterols. We previously showed that the concentration and enrichment of [13C]acetate decrease markedly across the dog liver because of the simultaneous uptake and production of acetate. To test for zonation of the enrichment of lipogenic acetyl-CoA, conscious dogs, prefitted with transhepatic catheters, were infused with glucose and [1,2-13C2]acetate in a branch of the portal vein. Analyses of MIDs of fatty acids and sterols isolated from liver, bile, and plasma very low density lipoprotein by a variant of ISA designed to detect gradients in precursor enrichment revealed marked zonation of enrichment of lipogenic acetyl-CoA. As control experiments where no zonation of acetyl-CoA enrichment would be expected, isolated rat livers were perfused with 10 mm [1,2-13C2]acetate. The ISA analyses of MIDs of fatty acids and sterols from liver and bile still revealed a zonation of acetyl-CoA enrichment. We conclude that zonation of hepatic acetyl-CoA enrichment occurs under a variety of animal models and physiological conditions. Failure to consider gradients of precursor enrichment can lead to underestimations of fractional lipogenesis calculated from the mass isotopomer distributions. The degree of such underestimation was modeled in vitro, and the data are reported in the companion paper (Bederman, I. R., Kasumov, T., Reszko, A. E., David, F., Brunengraber, H., and Kelleher, J. K. (2004) J. Biol. Chem. 279, 43217-43226).
Journal of Biological Chemistry 11/2004; 279(41):43207-16. · 4.77 Impact Factor
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ABSTRACT: In the companion report (Bederman, I. R., Reszko, A. E., Kasumov, T., David, F., Wasserman, D. H., Kelleher, J. K., and Brunengraber, H. (2004) J. Biol. Chem. 279, 43207-43216), we demonstrated that, when the hepatic pool of lipogenic acetyl-CoA is labeled from [13C]acetate, the enrichment of this pool decreases across the liver lobule. In addition, estimates of fractional synthesis calculated by isotopomer spectral analysis (ISA), a nonlinear regression method, did not agree with a simpler algebraic two-isotopomer method. To evaluate differences between these methods, we simulated in vitro the synthesis of fatty acids under known gradients of precursor enrichment, and known values of fractional synthesis. First, we synthesized pentadecanoate from [U-13C3]propionyl-CoA and four gradients of [U-13C3]malonyl-CoA enrichment. Second, we pooled the fractions of each gradient. Third, we diluted each pool with pentadecanoate prepared from unlabeled malonyl-CoA to simulate the dilution of the newly synthesized compound by pre-existing fatty acids. This yielded a series of samples of pentadecanoate with known values of (i) lower and upper limits for the precursor enrichment, (ii) the shape of the gradient, and (iii) the fractional synthesis. At each step, the mass isotopomer distributions of the samples were analyzed by ISA and the two-isotopomer method to determine whether each method could correctly (i) detect gradients of precursor enrichment, (ii) estimate the gradient limits, and (iii) estimate the fractional synthesis. The two-isotopomer method did not identify gradients of precursor enrichment and underestimated fractional synthesis by up to 2-fold in the presence of gradients. ISA uses all mass isotopomers, correctly identified imposed gradients of precursor enrichment, and estimated the expected values of fractional synthesis within the constraints of the data.
Journal of Biological Chemistry 11/2004; 279(41):43217-26. · 4.77 Impact Factor
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Aneta E Reszko,
Takhar Kasumov, France David,
Katherine R Thomas,
Kathryn A Jobbins,
Jie-Fei Cheng,
Gary D Lopaschuk,
Jason R B Dyck,
Mireya Diaz,
Christine Des Rosiers,
William C Stanley,
Henri Brunengraber
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ABSTRACT: The goal of this study was to test the relationship between malonyl-CoA concentration and its turnover measured in isolated rat hearts perfused with NaH(13)CO(3). This turnover is a direct measurement of the flux of acetyl-CoA carboxylation in the intact heart. It also reflects the rate of malonyl-CoA decarboxylation, i.e. the only known fate of malonyl-CoA in the heart. Conditions were selected to result in stable malonyl-CoA concentrations ranging from 1.5 to 5 nmol.g wet weight-(1). The malonyl-CoA concentration was directly correlated with the turnover of malonyl-CoA, ranging from 0.7 to 4.2 nmol.min(-) (1).g wet weight(-1) (slope = 0.98, r(2) = 0.94). The V(max) activities of acetyl-CoA carboxylase and of malonyl-CoA decarboxylase exceeded the rate of malonyl-CoA turnover by 2 orders of magnitude and did not correlate with either concentration or turnover of malonyl-CoA. However, conditions of perfusion that increased acetyl-CoA supply resulted in higher turnover and concentration, demonstrating that malonyl-CoA turnover is regulated by the supply of acetyl-CoA. The only condition where the activity of malonyl-CoA decarboxylase regulated malonyl-CoA kinetics was when the enzyme was pharmacologically inhibited, resulting in increased malonyl-CoA concentration and decreased turnover. Our data show that, in the absence of enzyme inhibitors, the rate of acetyl-CoA carboxylation is the main determinant of the malonyl-CoA concentration in the heart.
Journal of Biological Chemistry 09/2004; 279(33):34298-301. · 4.77 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 05/2004; 279(19):19574-9. · 4.77 Impact Factor
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Takhar Kasumov,
Laura L Brunengraber,
Blandine Comte,
Michelle A Puchowicz,
Kathryn Jobbins,
Katherine Thomas, France David,
Renee Kinman,
Suzanne Wehrli,
William Dahms,
Douglas Kerr,
Itzhak Nissim,
Henri Brunengraber
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ABSTRACT: Phenylbutyrate is used to treat inborn errors of ureagenesis, malignancies, cystic fibrosis, and thalassemia. High-dose phenylbutyrate therapy results in toxicity, the mechanism of which is unexplained. The known metabolites of phenylbutyrate are phenylacetate, phenylacetylglutamine, and phenylbutyrylglutamine. These are excreted in urine, accounting for a variable fraction of the dose. We identified new metabolites of phenylbutyrate in urine of normal humans and in perfused rat livers. These metabolites result from interference between the metabolism of phenylbutyrate and that of carbohydrates and lipids. The new metabolites fall into two categories, glucuronides and phenylbutyrate beta-oxidation side products. Two questions are raised by these data. First, is the nitrogen-excreting potential of phenylbutyrate diminished by ingestion of carbohydrates or lipids? Second, does competition between the metabolism of phenylbutyrate, carbohydrates, and lipids alter the profile of phenylbutyrate metabolites? Finally, we synthesized glycerol esters of phenylbutyrate. These are partially bioavailable in rats and could be used to administer large doses of phenylbutyrate in a sodium-free, noncaustic form.
Drug Metabolism and Disposition 02/2004; 32(1):10-9. · 3.73 Impact Factor
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ABSTRACT: While a number of studies underline the importance of anaplerotic pathways for hepatic biosynthetic functions and cardiac contractile activity, much remains to be learned about the sites and regulation of anaplerosis in these tissues. As part of a study on the regulation of anaplerosis from propionyl-CoA precursors in rat livers and hearts, we investigated the degree of reversibility of the reactions of the propionyl-CoA pathway. Label was introduced into the pathway via NaH13CO3, [U-13C3]propionate, or [U-13C3]lactate + [U-13C3]pyruvate, under various concentrations of propionate. The mass isotopomer distributions of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA revealed that, in intact livers and hearts, (i) the propionyl-CoA carboxylase reaction is slightly reversible only at low propionyl-CoA flux, (ii) the methylmalonyl-CoA racemase reaction keeps the methylmalonyl-CoA enantiomers in isotopic equilibrium under all conditions tested, and (iii) the methylmalonyl-CoA mutase reaction is reversible, but its reversibility decreases as the flow of propionyl-CoA increases. The thermodynamic dis-equilibrium of the combined reactions of the propionyl-CoA pathway explains the effectiveness of anaplerosis from propionyl-CoA precursors such as heptanoate.
Journal of Biological Chemistry 10/2003; 278(37):34959-65. · 4.77 Impact Factor
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ABSTRACT: The current dietary treatment of long-chain fatty acid oxidation defects (high carbohydrate with medium-even-chain triglycerides and reduced amounts of long-chain fats) fails, in many cases, to prevent cardiomyopathy, rhabdomyolysis, and muscle weakness. We hypothesized that the apparent defect in energy production results from a depletion of the catalytic intermediates of the citric acid cycle via leakage through cell membranes (cataplerosis). We further hypothesized that replacing dietary medium-even-chain fatty acids (precursors of acetyl-CoA) by medium-odd-chain fatty acids (precursors of acetyl-CoA and anaplerotic propionyl-CoA) would restore energy production and improve cardiac and skeletal muscle function. We fed subjects with long-chain defects a controlled diet in which the fat component was switched from medium-even-chain triglycerides to triheptanoin. In three patients with very-long-chain acyl-CoA dehydrogenase deficiency, this treatment led rapidly to clinical improvement that included the permanent disappearance of chronic cardiomyopathy, rhabdomyolysis, and muscle weakness (for more than 2 years in one child), and of rhabdomyolysis and weakness in the others. There was no evidence of propionyl overload in these patients. The treatment has been well tolerated for up to 26 months and opens new avenues for the management of patients with mitochondrial fat oxidation disorders.
Journal of Clinical Investigation 08/2002; 110(2):259-69. · 15.39 Impact Factor
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ABSTRACT: We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA in tissues. The assays involves perchloric acid extraction of the tissue, spiking the extract with [(2)H(5)]propionyl-CoA and [(2)H(4)]succinyl-CoA internal standards, and isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge. Propionyl-CoA is reacted with sarcosine and the formed N-propionylsarcosine is assayed as its pentafluorobenzyl derivative. Methylmalonyl-CoA and succinyl-CoA are hydrolyzed and the corresponding acids assayed as tert-butyl dimethylsilyl derivatives. The assay was applied to a study of [U-(13)C(3)]propionate metabolism in perfused rat livers. While propionyl-CoA is only M3 labeled, succinyl-CoA is M3, M2, and M1 labeled because of isotopic exchanges in the citric acid cycle. Methylmalonyl-CoA is M3 and M2 labeled, reflecting reversal of S-methylmalonyl-CoA mutase. Thus, our assays allow measuring the turnover of the coenzyme A derivatives involved in anaplerosis of the citric acid cycle via precursors of propionyl-CoA, i.e., propionate, odd-chain fatty acids, isoleucine, threonine, and valine.
Analytical Biochemistry 07/2002; 305(1):90-6. · 3.00 Impact Factor
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ABSTRACT: A high-fat, almost carbohydrate-free diet is used in children with intractable epilepsy to help control seizures by inducing a permanent state of ketosis. Esters of ketone bodies have been previously studied for their potential as parenteral and enteral nutrients. We tested in conscious dogs whether ketosis could be induced by repeated ingestion of R,S-1,3-butanediol diacetoacetate with or without carbohydrates. This ester is a water-soluble precursor of ketone bodies. Two constraints were imposed on this preclinical study: The rate of ester administration was limited to one half of the daily caloric requirement and to one half of the capacity of the liver to oxidize butanediol derived from ester hydrolysis. Under these conditions, the level of ketosis achieved in this dog model (0.8 mM) was lower than the level measured in children whose seizures were controlled by the ketogenic diet (1–3 mM). However, because humans may have a lower capacity for ketone body utilization than dogs, the doses of R,S-butanediol diacetoacetate used in the present study might induce higher average ketone body concentrations in humans than in dogs.
The Journal of Nutritional Biochemistry 06/2000; · 3.89 Impact Factor
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ABSTRACT: We previously reported (Previs, S. F., Fernandez, C. A., Yang, D., Soloviev, M. V., David, F., and Brunengraber, H. (1995)
J. Biol. Chem. 270, 19806–19815) that glucose made in isolated livers from starved rats perfused with physiological concentrations of lactate,
pyruvate, and either [2-13C]- or [U-13C3]glycerol had a mass isotopomer distribution incompatible with glucose being made from a homogeneously labeled pool of triose
phosphates. Similar data were obtained in live rats infused with [U-13C3]glycerol. We ascribed the labeling heterogeneity to major decreases in glycerol concentration and enrichment across the liver.
We concluded that [13C]glycerol is unsuitable for tracing the contribution of gluconeogenesis to total glucose production. We now report isotopic
heterogeneity of gluconeogenesis in hepatocytes, even when all cells are in contact with identical concentrations and enrichments
of gluconeogenic substrates. Total rat hepatocytes were incubated with concentrations of glycerol, lactate, and pyruvate that
were kept constant by substrate infusions. To modulate competition between substrates, the (glycerol)/(lactate + pyruvate)
infusion ratio ranged from 0.23 to 3.60. Metabolic and isotopic steady states were achieved in all cases. The apparent contribution
of gluconeogenesis to glucose production (f) was calculated from the mass isotopomer distribution of glucose. When all substrates were13C-labeled, f was 97%, as expected in glycogen-deprived hepatocytes. As the infusion ratio ([13C]glycerol)/(lactate + pyruvate) increased,f increased from 73% to 94%. In contrast, as the infusion ratio (glycerol)/([13C]lactate + [13C]pyruvate) increased, f decreased from 93% to 76%. In all cases, f increased with the rate of supply of the substrate that was labeled. Variations in fshow that the 13C labeling of triose phosphates was not equal in all hepatocytes, even when exposed to the same substrate concentrations and
enrichments. We also showed that zonation of glycerol kinase activity is minor in rat liver. We conclude that zonation of
other processes than glycerol phosphorylation contributes to the heterogeneity of triose phosphate labeling from glycerol
in rat liver.
Journal of Biological Chemistry 07/1998; 273(27):16853-16859. · 4.77 Impact Factor
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ABSTRACT: Mass isotopomer distribution analysis allows studying the synthesis of polymeric biomolecules from N, C-, or 2H-labeled monomeric units in the presence of unlabeled polymer. The mass isotopomer distribution of the polymer allows calculation
of (i) the enrichment of the monomer and (ii) the dilution of the newly synthesized polymer by unlabeled polymer. We tested
the conditions of validity of mass isotopomer distribution analysis of glucose labeled from [U-C3]lactate, [U-C3]glycerol, and [2-C]glycerol to calculate the fraction of glucose production derived from gluconeogenesis. Experiments were conducted in perfused
rat livers, live rats, and live monkeys. In all cases, [C]glycerol yielded labeling patterns of glucose that are incompatible with glucose being formed from a single pool of triose
phosphates of constant enrichment. We show evidence that variations in the enrichment of triose phosphates result from (i)
the large fractional decrease in physiological glycerol concentration in a single pass through the liver and (ii) the release
of unlabeled glycerol by the liver, presumably via lipase activity. This zonation of glycerol metabolism in liver results
in the calculation of artifactually low contributions of gluconeogenesis to glucose production when the latter is labeled
from [C]glycerol. In contrast, [U-C3]lactate appears to be a suitable tracer for mass isotopomer distribution analysis of gluconeogenesis in vivo, but not in the perfused liver.
In other perfusion experiments with [2H5]glycerol, we showed that the rat liver releases glycerol molecules containing one to four 2H atoms. This indicates the operation of a substrate cycle between extracellular glycerol and liver triose phosphates, where
2H is lost in the reversible reactions catalyzed by α-glycerophosphate dehydrogenase, triose-phosphate isomerase, and glycolytic
enzymes. This substrate cycle presumably involves α-glycerophosphate hydrolysis.
Journal of Biological Chemistry 08/1995; 270(34):19806-19815. · 4.77 Impact Factor
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ABSTRACT: We conducted an extensive mass isotopomer analysis of citric acid cycle and gluconeogenic metabolites isolated from livers
of overnight fasted rats perfused with 4 mM glucose, 0.2 mM octanoate, 1 mM [U-C3]lactate, and 0.2 mM [U-C3]pyruvate, in the anterograde or retrograde mode. In both perfusion modes, two distinct isotopomer patterns were observed:
(i) those of phosphoenolpyruvate, glucose, malate, and aspartate and (ii) those of citrate, α-ketoglutarate, glutamate, and
glutamine. Key citric acid cycle parameters and, hence, rates of gluconeogenesis, calculated (Lee, W.-N. P. (1989) J. Biol. Chem. 264, 13002-13004 and Lee, W.-N. P. (1993) J. Biol. Chem. 268, 25522-25526) from our mass isotopomer data did not only vary, but lead to conclusions inconsistent with Lee's citric
acid cycle model. Compared to lactate and pyruvate uptake, which sets an upper limit to glucose production, rates of gluconeogenesis
calculated (i) with the phosphoenolpyruvate and citrate data were similar, but those calculated (ii) with the glutamate data
amounted to only 60%, which is unlikely. All these conclusions are independent of the perfusion modes. We provide evidence
that the following processes contribute to the observed labeling discrepancy: (i) the reversibility of the isocitrate dehydrogenase
reaction and (ii) an active citrate cleavage pathway for the transfer of the oxaloacetate carbon skeleton from mitochondria
to the cytosol. Also, a good fit of our labeling data was obtained with a model of citric acid cycle and gluconeogenesis which
we developed to incorporate the above reactions (Fernandez, C. A., and Des Rosiers, C. (1995) J. Biol. Chem. 270, 10037-10042). The following conclusions can be drawn from the calculated reaction rates: (i) about half of the lactate
conversion to glucose occurs via the citrate cleavage pathway, (ii) the flux through the reversal of the isocitrate dehydrogenase
reaction is almost as fast as that through the citrate synthase reaction, and (iii) the flux through citrate synthase and
α-ketoglutarate dehydrogenase is 1.6- and 3.2-fold that through pyruvate carboxylase, respectively.
Journal of Biological Chemistry 04/1995; 270(17):10027-10036. · 4.77 Impact Factor
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ABSTRACT: The concentration of acetone dissolved in liver perfusion medium was determined by injection of the sample into a gas chromatograph equipped with a Carbopack/Carbowax-packed glass column. Interference from labile acetoacetate which readily decomposes to acetone was eliminated by treating the samples with NaBH4 prior to the analysis. Acetone was detected and quantified as 2-propanol. Separation of labeled 2-propanol in the sample by high-performance liquid chromatography allowed the determination of its specific activity. These methods make possible the convenient and rapid determination of acetone concentration and specific activity in biological samples.
Analytical Biochemistry 152(2):256-261. · 3.00 Impact Factor
