Pattern of substrate utilization in vascular smooth muscle using 13C isotopomer analysis of glutamate

1998 ◽  
Vol 275 (6) ◽  
pp. H2227-H2235 ◽  
Author(s):  
Tara M. Allen ◽  
Christopher D. Hardin
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Acetyl Coa ◽  
Contractile State ◽  
The Tca Cycle ◽  
Direct Measurements ◽  
Isotopomer Analysis ◽  
13C Isotopomer Analysis

Although vascular smooth muscle (VSM) derives the majority of its energy from oxidative phosphorylation, controversy exists concerning which substrates are utilized by the tricarboxylic acid (TCA) cycle. We used 13C isotopomer analysis of glutamate to directly measure the entry of exogenous [13C]glucose and acetate and unlabeled endogenous sources into the TCA cycle via acetyl-CoA. Hog carotid artery segments denuded of endothelium were superfused with 5 mM [1-13C]glucose and 0–5 mM [1,2-13C]acetate at 37°C for 3–12 h. We found that both resting and contracting VSM preferentially utilize [1,2-13C]acetate compared with [1-13C]glucose and unlabeled substrates. The entry of glucose into the TCA cycle (30–60% of total entry via acetyl-CoA) exhibited little change despite alterations in contractile state or acetate concentrations ranging from 0 to 5 mM. We conclude that glucose and nonglucose substrates are important oxidative substrates for resting and contracting VSM. These are the first direct measurements of relative substrate entry into the TCA cycle of VSM during activation and may provide a useful method to measure alterations in VSM metabolism under physiological and pathophysiological conditions.

Fatty acid, tricarboxylic acid cycle metabolites, and energy metabolism in vascular smooth muscle

1994 ◽  
Vol 267 (2) ◽  
pp. H764-H769 ◽  
Author(s):  
J. T. Barron ◽  
S. J. Kopp ◽  
J. Tow ◽  
J. E. Parrillo
Keyword(s):  
Smooth Muscle ◽  
Fatty Acid ◽  
Energy Metabolism ◽  
Carotid Artery ◽  
Vascular Smooth Muscle ◽  
Lactate Production ◽  
Tca Cycle ◽  
High Energy ◽  
Tricarboxylic Acid ◽  
O2 Consumption

The influence of octanoate on O2 consumption, tricarboxylic acid (TCA) cycle intermediates, and high-energy phosphates was examined in intact resting porcine carotid artery to investigate the role of fatty acid in energy metabolism and its integration with glucose metabolism in vascular smooth muscle. Incubation of resting arteries with octanoate (0.5 mM), which was previously shown to inhibit aerobic glycolysis (6), inhibited lactate production by 64% and increased O2 consumption by 30%. The increase in O2 consumption with octanoate was approximately equal to that calculated to account for the ATP production lost by inhibition of aerobic lactate production by octanoate. In glucose-free medium, the level of high-energy phosphate was reduced but was restored when octanoate was included in the incubation medium. This was associated with an increase in O2 consumption. These results suggest that the energy requirements of resting carotid artery can be largely met by the oxidative metabolism of fatty acid. Octanoate induced anaplerosis of the TCA cycle, as indicated by a 70% increase in the level of citrate. Extracellular glucose was necessary for octanoate-induced anaplerosis, probably by providing the extra carbon via pyruvate carboxylation, whereas a coupled transamination involving aspartate was a less important anaplerotic mechanism.

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.

IGF-I promotes a shift in metabolic flux in vascular smooth muscle cells

2002 ◽  
Vol 283 (3) ◽  
pp. E465-E471 ◽  
Author(s):  
Jennifer L. Hall ◽  
Gary H. Gibbons ◽  
John C. Chatham
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Metabolic Flux ◽  
Tca Cycle ◽  
Muscle Cells ◽  
Glucose Flux ◽  
The Tca Cycle ◽  
Igf I

13C-nuclear magnetic resonance (NMR) spectroscopy was used to test our hypothesis that insulin-like growth factor I (IGF-I) stimulates glucose flux into both nonoxidative and oxidative pathways in vascular smooth muscle cells (VSMC). Rat VSMC were exposed to uniformly labeled [13C]glucose ([U-13C]glucose; 5.5 mM) and [3-13C]pyruvate (1 mM) in the presence and absence of IGF-I (100 ng/ml). IGF-I increased glucose flux through glycolysis and the tricarboxylic acid (TCA) cycle as well as total anaplerotic flux into the TCA cycle. Previous work in our laboratory identified an increase in GLUT1 content and glucose metabolism in neointimal VSMC that was sufficient to promote proliferation and inhibit apoptosis. To test whether IGF-I could potentiate the GLUT1-induced increased flux in the neointima, we utilized VSMC harboring constitutive overexpression of GLUT1. Indeed, IGF-I markedly potentiated the GLUT1-induced increase in glucose flux through glycolysis and the TCA cycle. Taken together, these findings demonstrate that upregulation of glucose transport through either IGF-I or increased GLUT1 content stimulates glucose flux through both nonoxidative and oxidative pathways in VSMC.

Production of lactic acid and energy metabolism in vascular smooth muscle: effect of dichloroacetate

1995 ◽  
Vol 268 (2) ◽  
pp. H713-H719 ◽  
Author(s):  
J. T. Barron ◽  
J. E. Parrillo
Keyword(s):  
Smooth Muscle ◽  
Lactic Acid ◽  
Vascular Smooth Muscle ◽  
Energy Demand ◽  
Glucose Oxidation ◽  
Lactate Production ◽  
Muscle Metabolism ◽  
Tca Cycle ◽  
The Tca Cycle ◽  
Optimal Coordination

Vascular smooth muscle metabolism is characterized by substantial production of lactic acid even under fully oxygenated conditions. The role the aerobic production of lactate plays in the energetics of smooth muscle is obscure and was investigated in this study. Helical strips of porcine carotid arteries were incubated in medium containing 1 mM dichloroacetate (DCA), an agent that stimulates pyruvate dehydrogenase and promotes the oxidation of glucose. Lactate production in resting muscle was decreased in the presence of DCA (0.033 +/- 0.006 vs. 0.111 +/- 0.014 mumol.g-1.min-1, P < 0.02), indicating diversion of glucose metabolism from lactate production to enhanced glucose oxidation. This was associated with reduction in the level of ATP+phosphocreatine (PCr) (0.99 +/- 0.01 vs. 1.40 +/- 0.09 mumol/g, P < 0.05) and cataplerosis of the tricarboxylic acid (TCA) cycle. Contraction by KCl was also associated with reduced lactate production in the presence of DCA (0.086 +/- 0.017 vs. 0.20 +/- 0.002 mumol.g-1.min-1, P < 0.01), but ATP+PCr normalized, and there was anaplerosis of the TCA cycle. Glycogen in control arteries declined by approximately 1.3 mumol/g over 30 min K+ contraction but was unchanged in the presence of DCA. By calculation, the glycogen spared could be accounted for by the quantity of glucose diverted from lactate production to glucose oxidation during contraction. It is concluded that the aerobic production of lactate is a mechanism affording optimal coordination and modulation of glucose supply and oxidative energy production with energy demand.

13C isotopomer model for estimation of anaplerotic substrate oxidation via acetyl-CoA

1996 ◽  
Vol 271 (4) ◽  
pp. E788-E799 ◽  
Author(s):  
F. M. Jeffrey ◽  
C. J. Storey ◽  
A. D. Sherry ◽  
C. R. Malloy
Keyword(s):  
Biochemical Analysis ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Acetyl Coa ◽  
Acid Cycle ◽  
Propionate Metabolism ◽  
13C Nuclear Magnetic Resonance ◽  
Isotopomer Analysis ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Anaplerotic Pathway

A previous model using 13C nuclear magnetic resonance isotopomer analysis provided for direct measurement of the oxidation of 13C-enriched substrates in the tricarboxylic acid cycle and/or their entry via anaplerotic pathways. This model did not allow for recycling of labeled metabolites from tricarboxylic acid cycle intermediates into the acetyl-CoA pool. An extension of this model is now presented that incorporates carbon flow from oxaloacetate or malate to acetyl-CoA. This model was examined using propionate metabolism in the heart, in which previous observations indicated that all of the propionate consumed was oxidized to CO2 and water. Application of the new isotopomer model shows that 2 mM [3-13C]propionate entered the tricarboxylic acid cycle as succinyl-CoA (an anaplerotic pathway) at a rate equal to 52% of tricarboxylic acid cycle turnover and that all of this carbon entered the acetyl-CoA pool and was oxidized. This was verified using standard biochemical analysis; from the rate (mumol.min-1.g dry wt-1) of propionate uptake (4.0 +/- 0.7), the estimated oxygen consumption (24.8 +/- 5) matched that experimentally determined (24.4 +/- 3).

scholarly journals Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

2021 ◽  
Author(s):  
Joy Omini ◽  
Izabela Wojciechowska ◽  
Aleksandra Skirycz ◽  
Hideaki Moriyama ◽  
Toshihiro Obata
Keyword(s):  
Malate Dehydrogenase ◽  
Metabolic Flux ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Feedback Regulation ◽  
Dynamic Structure ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Enzyme Complex ◽  
Acetyl Coa

Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle 'metabolon' which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was presented together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

scholarly journals Staphylococcus epidermidis Polysaccharide Intercellular Adhesin Production Significantly Increases during Tricarboxylic Acid Cycle Stress

2005 ◽  
Vol 187 (9) ◽  
pp. 2967-2973 ◽  
Author(s):  
Cuong Vuong ◽  
Joshua B. Kidder ◽  
Erik R. Jacobson ◽  
Michael Otto ◽  
Richard A. Proctor ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Redox Status ◽  
Tricarboxylic Acid ◽  
Polysaccharide Intercellular Adhesin ◽  
Low Oxygen ◽  
Acid Cycle ◽  
The Tca Cycle ◽  
High Concentrations

ABSTRACT Staphylococcal polysaccharide intercellular adhesin (PIA) is important for the development of a mature biofilm. PIA production is increased during growth in a nutrient-replete or iron-limited medium and under conditions of low oxygen availability. Additionally, stress-inducing stimuli such as heat, ethanol, and high concentrations of salt increase the production of PIA. These same environmental conditions are known to repress tricarboxylic acid (TCA) cycle activity, leading us to hypothesize that altering TCA cycle activity would affect PIA production. Culturing Staphylococcus epidermidis with a low concentration of the TCA cycle inhibitor fluorocitrate dramatically increased PIA production without impairing glucose catabolism, the growth rate, or the growth yields. These data lead us to speculate that one mechanism by which staphylococci perceive external environmental change is through alterations in TCA cycle activity leading to changes in the intracellular levels of biosynthetic intermediates, ATP, or the redox status of the cell. These changes in the metabolic status of the bacteria result in the attenuation or augmentation of PIA production.

scholarly journals CBMT-49. OXALOACETATE ALTERS GLUCOSE METABOLISM IN GLIOBLASTOMA: 13C ISOTOPOMER STUDY

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi43-vi44
Author(s):  
Omkar Ijare ◽  
David Conway ◽  
Alan Cash ◽  
David Baskin ◽  
Kumar Pichumani
Keyword(s):  
Glucose Metabolism ◽  
Cancer Cells ◽  
Neuronal Cell ◽  
Tca Cycle ◽  
Positive Effects ◽  
Isotopomer Analysis ◽  
Carboxylation Pathway ◽  
13C Isotopomer Analysis ◽  
Positive Effect

Abstract Anhydrous enol-oxaloacetate (AEO) has demonstrated the ability to enhance neuronal cell bioenergetics and activate brain mitochondrial biogenesis. Since oxaloacetate has demonstrated positive effects on brain bioenergetics in neurodegenerative diseases we have begun to investigate whether AEO may also have a positive effect on the altered cellular metabolism found in cancer cells, particularly Glioblastoma multiforme. The “Warburg effect” describes an abnormal metabolic state in cancer, distinct from normal tissue, in which energy is generated through enhanced conversion of pyruvate to lactate even in the presence of oxygen during glycolysis. Oxaloacetate (OAA) is a key anaplerotic substrate that is required to maintain TCA cycle flux. The role of oxaloacetate supplementation on the energy metabolism is not known in cancer cells. Goal of this study is to investigate the changes in metabolic fluxes in glucose metabolism with and without the presence of OAA in patient-derived GBM cells. We use GC-MS based 13C isotopomer analysis for this study. GBM cells are grown in 15mM glucose containing DMEM medium supplemented with 2mM oxaloacetate for 10 days. 6 hours prior to harvesting, [U-13C]glucose is introduced to the medium. 13C isotopomer analysis of GC-MS data showed that OAA supplementation for 10 days drastically decreased Warburg glycolysis by reducing 13C labeling (M+3) by 19.7% and 48.8% in pyruvate and lactate pools respectively in comparison with cells not treated with OAA. M+3 13C labeled pyruvate entered TCA cycle via acetyl-CoA, where we also observed reduced levels of M+2 13C labeled citrate (20.5%) and glutamate (23.9%) isotopomers. Pyruvate can also enter TCA cycle via pyruvate carboxylation pathway and this activity was also found to be slightly decreased in the OAA treated cells. All the differences were statistically significant. These results indicate that OAA can be used to alter bioenergetics of GBM cells, specifically glucose oxidation.

scholarly journals Metabolic engineering of Bacillus amyloliquefaciens for enhanced production of S-adenosylmethionine by coupling of an engineered S-adenosylmethionine pathway and the tricarboxylic acid cycle

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Liying Ruan ◽  
Lu Li ◽  
Dian Zou ◽  
Cong Jiang ◽  
Zhiyou Wen ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Bacillus Amyloliquefaciens ◽  
Tricarboxylic Acid Cycle ◽  
Fold Increase ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Scale Production ◽  
Free Medium ◽  
Enhanced Production ◽  
The Tca Cycle

Abstract Background S-Adenosylmethionine (SAM) is a critical cofactor involved in many biochemical reactions. However, the low fermentation titer of SAM in methionine-free medium hampers commercial-scale production. The SAM synthesis pathway is specially related to the tricarboxylic acid (TCA) cycle in Bacillus amyloliquefaciens. Therefore, the SAM synthesis pathway was engineered and coupled with the TCA cycle in B. amyloliquefaciens to improve SAM production in methionine-free medium. Results Four genes were found to significantly affect SAM production, including SAM2 from Saccharomyces cerevisiae, metA and metB from Escherichia coli, and native mccA. These four genes were combined to engineer the SAM pathway, resulting in a 1.42-fold increase in SAM titer using recombinant strain HSAM1. The engineered SAM pathway was subsequently coupled with the TCA cycle through deletion of succinyl-CoA synthetase gene sucC, and the resulted HSAM2 mutant produced a maximum SAM titer of 107.47 mg/L, representing a 0.59-fold increase over HSAM1. Expression of SAM2 in this strain via a recombinant plasmid resulted in strain HSAM3 that produced 648.99 mg/L SAM following semi-continuous flask batch fermentation, a much higher yield than previously reported for methionine-free medium. Conclusions This study reports an efficient strategy for improving SAM production that can also be applied for generation of SAM cofactors supporting group transfer reactions, which could benefit metabolic engineering, chemical biology and synthetic biology.

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