Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle

Alves, Tiago C.; Pongratz, Rebecca L.; Zhao, Xiaojie; Yarborough, OrLando H.; Sereda, Sam; Shirihai, Orian; Cline, Gary W.; Mason, Graeme F.; Kibbey, Richard G.

doi:10.1016/j.cmet.2015.08.021

Cited by 117 publications

(169 citation statements)

References 28 publications

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“…However, the amount of ATP-released in the medium by PYR was smaller than that induced by PEP. It has been concluded after mass-isotopomeric flux analysis of glycolytic flux in insulinoma INS-1 cells that anaplerotic carboxylation of pyruvate represents only a minority of the overall citric acid cycle and that the carbon flow from pyruvate to oxaloacetate/malate and back to PEP/pyruvate does not mix with glycolytic pyruvate [20]. It might explain the low secretory capacity of exogenously added pyruvate as due to its poor metabolism in the citric acid cycle because of a limitation of the cycle anaplerotic replenishment under the prevalent experimental conditions.…”

Section: Discussionmentioning

confidence: 99%

“…However, the extracellular ATP accumulation differed among the citric acid cycle intermediates but it was nevertheless increased by their presence (Table 2). This seems to be at variance with the lack of response to glucose of oligomycin-dependent oxygen consumption in INS-1 cells, attributed to an increased mitochondrial proton leak [20]. If this were to apply also to primary β-cells, an increased glycolytic production of ATP might, alternatively, be more specifically linked to the stimulation of insulin secretion by glucose.…”

Section: Discussionmentioning

confidence: 99%

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Direct Stimulation of Islet Insulin Secretion by Glycolytic and Mitochondrial Metabolites in KCl-Depolarized Islets

et al. 2016

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We have previously demonstrated that islet depolarization with 70 mM KCl opens Cx36 hemichannels and allows diffusion of small metabolites and cofactors through the β-cell plasma membrane. We have investigated in this islet “permeabilized” model whether glycolytic and citric acid cycle intermediates stimulate insulin secretion and how it correlates with ATP production (islet content plus extracellular nucleotide accumulation). Glycolytic intermediates (10 mM) stimulated insulin secretion and ATP production similarly. However, they showed differential sensitivities to respiratory chain or enzyme inhibitors. Pyruvate showed a lower secretory capacity and less ATP production than phosphoenolpyruvate, implicating an important role for glycolytic generation of ATP. ATP production by glucose-6-phosphate was not sensitive to a pyruvate kinase inhibitor that effectively suppressed the phosphoenolpyruvate-induced secretory response and islet ATP rise. Strong suppression of both insulin secretion and ATP production induced by glucose-6-phosphate was caused by 10 μM antimycin A, implicating an important role for the glycerophosphate shuttle in transferring reducing equivalents to the mitochondria. Five citric acid cycle intermediates were investigated for their secretory and ATP production capacity (succinate, fumarate, malate, isocitrate and α-ketoglutarate at 5 mM, together with ADP and/or NADP+ to feed the NADPH re-oxidation cycles). The magnitude of the secretory response was very similar among the different mitochondrial metabolites but α-ketoglutarate showed a more sustained second phase of secretion. Gabaculine (1 mM, a GABA-transaminase inhibitor) suppressed the second phase of secretion and the ATP-production stimulated by α-ketoglutarate, supporting a role for the GABA shuttle in the control of glucose-induced insulin secretion. None of the other citric acid intermediates essayed showed any suppression of both insulin secretion or ATP-production by the presence of gabaculine. We propose that endogenous GABA metabolism in the “GABA-shunt” facilitates ATP production in the citric acid cycle for an optimal insulin secretion.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Direct Stimulation of Islet Insulin Secretion by Glycolytic and Mitochondrial Metabolites in KCl-Depolarized Islets

et al. 2016

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“…Pyruvate enters the mitochondria through the pyruvate carrier [44] and is oxidised and decarboxylated into acetyl CoA, producing CO 2 and NADH [45]. Citrate, formed by joining the acetyl group of acetyl CoA to oxaloacetate, then enters the tricarboxylic acid (TCA) cycle [46]. While this process may classically be viewed as a catabolic prelude to ATP generation by aerobic respiration, the large array of intermediates involved serve as important biosynthetic precursors [34], including for the synthesis of non-essential amino acids and fatty acids [47].…”

Section: Biochemical Foundations Of Nad(p)h Fluorescence Measurementsmentioning

confidence: 99%

Investigating mitochondrial redox state using NADH and NADPH autofluorescence

Blacker

Duchen

2016

Free Radical Biology and Medicine

316

297

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The redox states of the NAD and NADP pyridine nucleotide pools play critical roles in defining the activity of energy producing pathways, in driving oxidative stress and in maintaining antioxidant defences. Broadly speaking, NAD is primarily engaged in regulating energy-producing catabolic processes, whilst NADP may be involved in both antioxidant defence and free radical generation. Defects in the balance of these pathways are associated with numerous diseases, from diabetes and neurodegenerative disease to heart disease and cancer. As such, a method to assess the abundance and redox state of these separate pools in living tissues would provide invaluable insight into the underlying pathophysiology. Experimentally, the intrinsic fluorescence of the reduced forms of both redox cofactors, NADH and NADPH, has been used for this purpose since the mid-twentieth century. In this review, we outline the modern implementation of these techniques for studying mitochondrial redox state in complex tissue preparations. As the fluorescence spectra of NADH and NADPH are indistinguishable, interpreting the signals resulting from their combined fluorescence, often labelled NAD(P)H, can be complex. We therefore discuss recent studies using fluorescence lifetime imaging microscopy (FLIM) which offer the potential to discriminate between the two separate pools. This technique provides increased metabolic information from cellular autofluorescence in biomedical investigations, offering biochemical insights into the changes in time-resolved NAD(P)H fluorescence signals observed in diseased tissues.

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“…The solvents and optimized gradient conditions for LC were as follows: solvent A, 50% methanol with 0.05% acetic acid; solvent B, 90% acetonitrile with 10 mM ammonium acetate, pH 7.0; elution gradient, 99.5% B (0 min), 50% B (12 to 14 min), 0% B (15 to 19 min); post-run time for equilibration, 5 min in 99.5% B. The MS-MS experiment was performed in positive/negative switching electrospray mode, as described previously (16). The optimized operating electrospray ionization (ESI) conditions were as follows: gas temperature, 230°C (nitrogen); gas flow, 15 liters/min; nebulizer pressure, 40 lb/in 2 ; sheath gas temperature, 350°C; and sheath gas flow, 12 liters/min.…”

Section: Generation Of Mpc1 Conditional Knock-in and Deletion Allelesmentioning

confidence: 99%

Requirement for the Mitochondrial Pyruvate Carrier in Mammalian Development Revealed by a Hypomorphic Allelic Series

Bowman

Zhao

Hartung

et al. 2016

Molecular and Cellular Biology

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bGlucose and oxygen are two of the most important molecules transferred from mother to fetus during eutherian pregnancy, and the metabolic fates of these nutrients converge at the transport and metabolism of pyruvate in mitochondria. Pyruvate enters the mitochondrial matrix through the mitochondrial pyruvate carrier (MPC), a complex in the inner mitochondrial membrane that consists of two essential components, MPC1 and MPC2. Here, we define the requirement for mitochondrial pyruvate metabolism during development with a progressive allelic series of Mpc1 deficiency in mouse. Mpc1 deletion was homozygous lethal in midgestation, but Mpc1 hypomorphs and tissue-specific deletion of Mpc1 presented as early perinatal lethality. The allelic series demonstrated that graded suppression of MPC resulted in dose-dependent metabolic and transcriptional changes. Steady-state metabolomics analysis of brain and liver from Mpc1 hypomorphic embryos identified compensatory changes in amino acid and lipid metabolism. Flux assays in Mpc1-deficient embryonic fibroblasts also reflected these changes, including a dramatic increase in mitochondrial alanine utilization. The mitochondrial alanine transaminase GPT2 was found to be necessary and sufficient for increased alanine flux upon MPC inhibition. These data show that impaired mitochondrial pyruvate transport results in biosynthetic deficiencies that can be mitigated in part by alternative anaplerotic substrates in utero. E mbryonic development in eutherian mammals is defined by ongoing metabolic interactions between mother and fetus. Placentas have evolved a sophisticated suite of adaptations to ensure adequate nutrient, gas, and waste exchange between fetus and mother, as well as hormonal and immunological communication (1, 2). To meet the fetal requirements for nutrient and oxygen consumption during pregnancy, maternal cardiac output increases such that uteroplacental blood flow accounts for ϳ25% of total cardiac output (3). Glucose and oxygen are arguably the two most important molecules transferred from mother to fetus, in terms of both concentration and the multitude of physiological adaptations in place to ensure their adequate transport; however, the requirement for mitochondrial oxidative metabolism during embryonic development remains poorly defined.While oxygen tension in utero is low relative to atmospheric levels, measurement of oxygen consumption and lactate uptake in fetal lambs provided evidence that the fetus is a net consumer rather than a producer of lactate (4). Consistent with this, fetal myocardium consumes a large amount of lactate, in addition to glucose, indicating that oxidative metabolism of carbohydrates is an important energy source in the developing heart (5, 6). Additionally, lactate utilization is high in the neonatal brain, and the capacity for lactate import is higher in the neonatal brain than in the adult brain (7). Together, these observations provide evidence for the importance of mitochondrial oxidative metabolism early in mammalian development. S...

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Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle

Cited by 117 publications

References 28 publications

Direct Stimulation of Islet Insulin Secretion by Glycolytic and Mitochondrial Metabolites in KCl-Depolarized Islets

Direct Stimulation of Islet Insulin Secretion by Glycolytic and Mitochondrial Metabolites in KCl-Depolarized Islets

Investigating mitochondrial redox state using NADH and NADPH autofluorescence

Requirement for the Mitochondrial Pyruvate Carrier in Mammalian Development Revealed by a Hypomorphic Allelic Series

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