Substrate metabolism of isolated jejunal epithelium: conservation of three-carbon units

1986 ◽  
Vol 250 (2) ◽  
pp. C191-C198 ◽  
Author(s):  
R. T. Mallet ◽  
J. K. Kelleher ◽  
M. J. Jackson
Keyword(s):  
Ketone Bodies ◽  
Tca Cycle ◽  
Substrate Metabolism ◽  
Metabolic Substrates ◽  
The Tca Cycle ◽  
Carbon Compounds ◽  
Consumption Rates ◽  
14Co2 Production ◽  
Tca Cycle Intermediates ◽  
Labeling Pattern

This study characterizes the substrate metabolism of isolated jejunal epithelial cells. Utilization of substrates was assessed by spectrophotometric assay. Significant quantities of glucose, glutamine, and ketone bodies were consumed in a 1-h period; lactate and ammonia were produced. [U-14C]glucose was metabolized in this medium to approximately three moles of lactate per mole of CO2. The pattern of tricarboxylic acid (TCA) cycle metabolism was analyzed utilizing media containing different concentrations of potential metabolic substrates and trace quantities of [14C]- succinate. O2 consumption rates indicated that glutamine can serve as an energy source in the absence of other substrates. Relative 14CO2 production from [1,4-14C]succinate versus [2,3-14C]succinate, which estimates flux of TCA cycle intermediates to products other than CO2, was increased more than twofold when glutamine was the only major substrate available. Alanine was produced from TCA cycle intermediates. Analysis of the citrate labeling pattern in the presence of [2,3-14C] succinate suggested that carbon from the TCA cycle does not form a significant fraction of acetyl-CoA used for citrate synthesis and that glutamine carbon was not completely oxidized to CO2. These findings suggest that glucose and glutamine are converted to three-carbon compounds by the jejunal epithelium.

scholarly journals Evidence for reverse flux through pyruvate kinase in skeletal muscle

2009 ◽  
Vol 296 (4) ◽  
pp. E748-E757 ◽  
Author(s):  
Eunsook S. Jin ◽  
A. Dean Sherry ◽  
Craig R. Malloy
Keyword(s):  
Skeletal Muscle ◽  
Glucose Production ◽  
Tca Cycle ◽  
Hepatic Glycogen ◽  
Direct Transfer ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates ◽  
Minced Muscle ◽  
Labeling Pattern

Conversion of lactate to glucose was examined in myotubes, minced muscle tissue, and rats exposed to 2H2O or 13C-enriched substrates. Myotubes or minced skeletal muscle incubated with [U-13C3]lactate released small amounts of [1,2,3-13C3]- or [4,5,6-13C3]glucose. This labeling pattern is consistent with direct transfer from lactate to glucose without randomization in the tricarboxylic acid (TCA) cycle. After exposure of incubated muscle to 2H2O, [U-13C3]lactate, glucose, and glutamine, there was minimal release of synthesized glucose to the medium based on a low level of 2H enrichment in medium glucose but 50- to 100-fold greater 2H enrichment in glucosyl units from glycogen. The 13C enrichment pattern in glycogen from incubated skeletal muscle was consistent only with direct transfer of lactate to glucose without exchange in TCA cycle intermediates. 13C nuclear magnetic resonance (NMR) spectra of glutamate from the same tissue showed flux from lactate through pyruvate dehydrogenase but not flux through pyruvate carboxylase into the TCA cycle. Carbon from an alternative substrate for glucose production that requires metabolism through the TCA cycle, propionate, did not enter glycogen, suggesting that TCA cycle intermediates do not exchange with phospho enolpyruvate. In vivo, the 13C labeling patterns in hepatic glycogen and plasma glucose after administration of [U-13C3]lactate did not differ significantly. However, skeletal muscle glycogen was substantially enriched in [1,2,3-13C3]- and [4,5,6-13C3]glucose units that could only occur through skeletal muscle glyconeogenesis rather than glycogenesis. Lactate serves as a substrate for glyconeogenesis in vivo without exchange into symmetric intermediates of the TCA cycle.

scholarly journals Analysis of tricarboxylic acid-cycle metabolism of hepatoma cells by comparison of 14CO2 ratios

1987 ◽  
Vol 246 (3) ◽  
pp. 633-639 ◽  
Author(s):  
J K Kelleher ◽  
B M Bryan ◽  
R T Mallet ◽  
A L Holleran ◽  
A N Murphy ◽  
...  
Keyword(s):  
Steady State ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Hepatoma Cells ◽  
Acid Cycle ◽  
The Tca Cycle ◽  
14Co2 Production ◽  
Metabolic Properties ◽  
Tca Cycle Intermediates

The CO2-ratios method is applied to the analysis of abnormalities of TCA (tricarboxylic acid)-cycle metabolism in AS-30D rat ascites-hepatoma cells. This method utilizes steady-state 14CO2-production rates from pairs of tracers of the same compound to evaluate TCA-cycle flux patterns. Equations are presented that quantitatively convert CO2 ratios into estimates of probability of flux through TCA-cycle-related pathways. Results of this study indicated that the ratio of 14CO2 produced from [1,4-14C]succinate to 14CO2 produced from [2,3-14C]succinate was increased by the addition of glutamine (5 mM) to the medium. An increase in the succinate CO2 ratio is quantitatively related to an increased flux of unlabelled carbon into the TCA-cycle-intermediate pools. Analysis of 14C distribution in [14C]citrate derived from [2,3-14C]succinate indicated that flux from the TCA cycle to the acetyl-CoA-derived carbons of citrate was insignificant. Thus the increased succinate CO2 ratio observed in the presence of glutamine could only result from an increased flux of carbon into the span of the TCA cycle from citrate to oxaloacetate. This result is consistent with increased flux of glutamine to alpha-oxoglutarate in the incubation medium containing exogenous glutamine. Comparison of the pyruvate CO2 ratio, steady-state 14CO2 production from [2-14C]pyruvate versus [3-14C]pyruvate, with the succinate 14CO2 ratio detected flux of pyruvate to C4 TCA-cycle intermediates in the medium containing glutamine. This result was consistent with the observation that [14C]aspartate derived from [2-14C]pyruvate was labelled in C-2 and C-3. 14C analysis also produced evidence for flux of TCA-cycle carbon to alanine. This study demonstrates that the CO2-ratios method is applicable in the analysis of the metabolic properties of AS-30D cells. This methodology has verified that the atypical TCA-cycle metabolism previously described for AS-30D-cell mitochondria occurs in intact AS-30D rat hepatoma cells.

scholarly journals Differential and shared effects of eicosapentaenoic acid and docosahexaenoic acid on serum metabolome in subjects with chronic inflammation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wan-Chi Chang ◽  
Jisun So ◽  
Stefania Lamon-Fava
Keyword(s):  
Metabolic Syndrome ◽  
Docosahexaenoic Acid ◽  
Eicosapentaenoic Acid ◽  
Chronic Inflammation ◽  
Cell Function ◽  
Tca Cycle ◽  
Differential Effects ◽  
Epa And Dha ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates

AbstractThe omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affect cell function and metabolism, but the differential effects of EPA and DHA are not known. In a randomized, controlled, double-blind, crossover study, we assessed the effects of 10-week supplementation with EPA-only and DHA-only (3 g/d), relative to a 4-week lead-in phase of high oleic acid sunflower oil (3 g/day, defined as baseline), on fasting serum metabolites in 21 subjects (9 men and 12 post-menopausal women) with chronic inflammation and some characteristics of metabolic syndrome. Relative to baseline, EPA significantly lowered the tricarboxylic acid (TCA) cycle intermediates fumarate and α-ketoglutarate and increased glucuronate, UDP-glucuronate, and non-esterified DHA. DHA significantly lowered the TCA cycle intermediates pyruvate, citrate, isocitrate, fumarate, α-ketoglutarate, and malate, and increased succinate and glucuronate. Pathway analysis showed that both EPA and DHA significantly affected the TCA cycle, the interconversion of pentose and glucuronate, and alanine, and aspartate and glutamate pathways (FDR < 0.05) and that DHA had a significantly greater effect on the TCA cycle than EPA. Our results indicate that EPA and DHA exhibit both common and differential effects on cell metabolism in subjects with chronic inflammation and some key aspects of metabolic syndrome.

Gluconeogenesis from labeled carbon: estimating isotope dilution

1986 ◽  
Vol 250 (3) ◽  
pp. E296-E305 ◽  
Author(s):  
J. K. Kelleher
Keyword(s):  
Steady State ◽  
Isotope Dilution ◽  
Experimental Tests ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Complex Model ◽  
Acetyl Coa ◽  
The Tca Cycle ◽  
Carbon Compounds ◽  
Mathematical Techniques

To estimate the rate of gluconeogenesis from steady-state incorporation of labeled 3-carbon precursors into glucose, isotope dilution must be considered so that the rate of labeling of glucose can be quantitatively converted to the rate of gluconeogenesis. An expression for the value of this isotope dilution can be derived using mathematical techniques and a model of the tricarboxylic acid (TCA) cycle. The present investigation employs a more complex model than that used in previous studies. This model includes the following pathways that may affect the correction for isotope dilution: 1) flux of 3-carbon precursor to the oxaloacetate pool via acetyl-CoA and the TCA cycle; 2) flux of 4- or 5-carbon compounds into the TCA cycle; 3) reversible flux between oxaloacetate (OAA) and pyruvate and between OAA and fumarate; 4) incomplete equilibrium between OAA pools; and 5) isotope dilution of 3-carbon tracers between the experimentally measured pool and the precursor for the TCA-cycle OAA pool. Experimental tests are outlined which investigators can use to determine whether these pathways are significant in a specific steady-state system. The study indicated that flux through these five pathways can significantly affect the correction for isotope dilution. To correct for the effects of these pathways an alternative method for calculating isotope dilution is proposed using citrate to relate the specific activities of acetyl-CoA and OAA.

Glutamate availability is important in intramuscular amino acid metabolism and TCA cycle intermediates but does not affect peak oxidative metabolism

2008 ◽  
Vol 105 (2) ◽  
pp. 547-554 ◽  
Author(s):  
M. Mourtzakis ◽  
T. E. Graham ◽  
J. González-Alonso ◽  
B. Saltin
Keyword(s):  
Oxidative Metabolism ◽  
Acid Metabolism ◽  
Glutamate Uptake ◽  
Knee Extensor ◽  
Tca Cycle ◽  
Peak Oxygen Consumption ◽  
Peak Exercise ◽  
Early Exercise ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates

Muscle glutamate is central to reactions producing 2-oxoglutarate, a tricarboxylic acid (TCA) cycle intermediate that essentially expands the TCA cycle intermediate pool during exercise. Paradoxically, muscle glutamate drops ∼40–80% with the onset of exercise and 2-oxoglutarate declines in early exercise. To investigate the physiological relationship between glutamate, oxidative metabolism, and TCA cycle intermediates (i.e., fumarate, malate, 2-oxoglutarate), healthy subjects trained (T) the quadriceps of one thigh on the single-legged knee extensor ergometer (1 h/day at 70% maximum workload for 5 days/wk), while their contralateral quadriceps remained untrained (UT). After 5 wk of training, peak oxygen consumption (V̇o2peak) in the T thigh was greater than that in the UT thigh ( P < 0.05); V̇o2peak was not different between the T and UT thighs with glutamate infusion. Peak exercise under control conditions revealed a greater glutamate uptake in the T thigh compared with rest (7.3 ± 3.7 vs. 1.0 ± 0.1 μmol·min−1·kg wet wt−1, P < 0.05) without increase in TCA cycle intermediates. In the UT thigh, peak exercise (vs. rest) induced an increase in fumarate (0.33 ± 0.07 vs. 0.02 ± 0.01 mmol/kg dry wt (dw), P < 0.05) and malate (2.2 ± 0.4 vs. 0.5 ± 0.03 mmol/kg dw, P < 0.05) and a decrease in 2-oxoglutarate (12.2 ± 1.6 vs. 32.4 ± 6.8 μmol/kg dw, P < 0.05). Overall, glutamate infusion increased arterial glutamate ( P < 0.05) and maintained this increase. Glutamate infusion coincided with elevated fumarate and malate ( P < 0.05) and decreased 2-oxoglutarate ( P < 0.05) at peak exercise relative to rest in the T thigh; there were no further changes in the UT thigh. Although glutamate may have a role in the expansion of the TCA cycle, glutamate and TCA cycle intermediates do not directly affect V̇o2peak in either trained or untrained muscle.

scholarly journals Ammonia inhibits energy metabolism in astrocytes in a rapid and glutamate dehydrogenase 2-dependent manner

2020 ◽  
Vol 13 (10) ◽  
pp. dmm047134
Author(s):  
Leonie Drews ◽  
Marcel Zimmermann ◽  
Philipp Westhoff ◽  
Dominik Brilhaus ◽  
Rebecca E. Poss ◽  
...  
Keyword(s):  
Amino Acids ◽  
Energy Metabolism ◽  
Glutamate Dehydrogenase ◽  
Mitochondrial Respiration ◽  
Tca Cycle ◽  
Dependent Manner ◽  
Independent Manner ◽  
The Tca Cycle ◽  
Branched Chain ◽  
Tca Cycle Intermediates

ABSTRACTAstrocyte dysfunction is a primary factor in hepatic encephalopathy (HE) impairing neuronal activity under hyperammonemia. In particular, the early events causing ammonia-induced toxicity to astrocytes are not well understood. Using established cellular HE models, we show that mitochondria rapidly undergo fragmentation in a reversible manner upon hyperammonemia. Further, in our analyses, within a timescale of minutes, mitochondrial respiration and glycolysis were hampered, which occurred in a pH-independent manner. Using metabolomics, an accumulation of glucose and numerous amino acids, including branched chain amino acids, was observed. Metabolomic tracking of 15N-labeled ammonia showed rapid incorporation of 15N into glutamate and glutamate-derived amino acids. Downregulating human GLUD2 [encoding mitochondrial glutamate dehydrogenase 2 (GDH2)], inhibiting GDH2 activity by SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Metabolomic tracking of 13C-glutamine showed that hyperammonemia can inhibit anaplerosis of tricarboxylic acid (TCA) cycle intermediates. Contrary to its classical anaplerotic role, we show that, under hyperammonemia, GDH2 catalyzes the removal of ammonia by reductive amination of α-ketoglutarate, which efficiently and rapidly inhibits the TCA cycle. Overall, we propose a critical GDH2-dependent mechanism in HE models that helps to remove ammonia, but also impairs energy metabolism in mitochondria rapidly.

scholarly journals Tricarboxylic Acid (TCA) Cycle Intermediates: Regulators of Immune Responses

2020 ◽  
Author(s):  
Inseok Choi ◽  
Hyewon Son ◽  
Jea-Hyun Baek
Keyword(s):  
Immune Responses ◽  
Molecular Mechanisms ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Cell Stress ◽  
Danger Signals ◽  
Cellular Processes ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates

Tricarboxylic acid cycle (TCA) is a series of chemical reactions in aerobic organisms used to generate energy via the oxidation of acetyl-CoA derived from carbohydrates, fatty acids, and proteins. In the eukaryotic system, the TCA cycle completely occurs in mitochondria, while the intermediates of the TCA cycle are retained in mitochondria due to their polarity and hydrophilicity. Under conditions of cell stress, mitochondria become disrupted and release their contents, which act as danger signals in the cytosol. Of note, the TCA cycle intermediates may also leak from dysfunctioning mitochondria and regulate cellular processes. Increasing evidence shows that the metabolites of the TCA cycle are substantially involved in the regulation of immune responses. In this review, we aimed to provide a comprehensive systematic overview of the molecular mechanisms of each TCA cycle intermediate that may play key roles in regulating cellular immunity in cell stress and discuss their implications for immune activation and suppression.

scholarly journals Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline

2021 ◽  
Vol 17 (3) ◽  
pp. e1009204
Author(s):  
Oriana Villafraz ◽  
Marc Biran ◽  
Erika Pineda ◽  
Nicolas Plazolles ◽  
Edern Cahoreau ◽  
...  
Keyword(s):  
Carbon Sources ◽  
Tca Cycle ◽  
Central Carbon Metabolism ◽  
Tsetse Fly ◽  
Growth Defect ◽  
Insect Vector ◽  
Metabolic Intermediates ◽  
The Tca Cycle ◽  
Production And Consumption ◽  
Tca Cycle Intermediates

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1–2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.

scholarly journals Tricarboxylic Acid (TCA) Cycle Intermediates: Regulators of Immune Responses

Life ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 69
Author(s):  
Inseok Choi ◽  
Hyewon Son ◽  
Jea-Hyun Baek
Keyword(s):  
Immune Responses ◽  
Molecular Mechanisms ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Cell Stress ◽  
Danger Signals ◽  
Cellular Processes ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates

The tricarboxylic acid cycle (TCA) is a series of chemical reactions used in aerobic organisms to generate energy via the oxidation of acetylcoenzyme A (CoA) derived from carbohydrates, fatty acids and proteins. In the eukaryotic system, the TCA cycle occurs completely in mitochondria, while the intermediates of the TCA cycle are retained inside mitochondria due to their polarity and hydrophilicity. Under cell stress conditions, mitochondria can become disrupted and release their contents, which act as danger signals in the cytosol. Of note, the TCA cycle intermediates may also leak from dysfunctioning mitochondria and regulate cellular processes. Increasing evidence shows that the metabolites of the TCA cycle are substantially involved in the regulation of immune responses. In this review, we aimed to provide a comprehensive systematic overview of the molecular mechanisms of each TCA cycle intermediate that may play key roles in regulating cellular immunity in cell stress and discuss its implication for immune activation and suppression.

Ins and Outs of the TCA Cycle: The Central Role of Anaplerosis

2021 ◽  
Vol 41 (1) ◽  
Author(s):  
Melissa Inigo ◽  
Stanisław Deja ◽  
Shawn C. Burgess
Keyword(s):  
Redox State ◽  
Tca Cycle ◽  
Oxidative Capacity ◽  
Annual Review ◽  
Publication Date ◽  
Energy Status ◽  
Cellular Energy ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates

The reactions of the tricarboxylic acid (TCA) cycle allow the controlled combustion of fat and carbohydrate. In principle, TCA cycle intermediates are regenerated on every turn and can facilitate the oxidation of an infinite number of nutrient molecules. However, TCA cycle intermediates can be lost to cataplerotic pathways that provide precursors for biosynthesis, and they must be replaced by anaplerotic pathways that regenerate these intermediates. Together, anaplerosis and cataplerosis help regulate rates of biosynthesis by dictating precursor supply, and they play underappreciated roles in catabolism and cellular energy status. They facilitate recycling pathways and nitrogen trafficking necessary for catabolism, and they influence redox state and oxidative capacity by altering TCA cycle intermediate concentrations. These functions vary widely by tissue and play emerging roles in disease. This article reviews the roles of anaplerosis and cataplerosis in various tissues and discusses how they alter carbon transitions, and highlights their contribution to mechanisms of disease. Expected final online publication date for the Annual Review of Nutrition, Volume 41 is September 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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