Accumulation Of Α-ketoglutarate Research Articles

Inhibition of fatty acid oxidation enables heart regeneration in adult mice.

Postnatal maturation of cardiomyocytes is characterized by a metabolic switch from glycolysis to fatty acid oxidation, chromatin reconfiguration and exit from the cell cycle, instating a barrier for adult heart regeneration1,2. Here, to explore whether metabolic reprogrammingcan overcome this barrier and enable heart regeneration, we abrogate fatty acid oxidation in cardiomyocytes by inactivation of Cpt1b. We find that disablement of fatty acid oxidation in cardiomyocytes improves resistance to hypoxia and stimulates cardiomyocyte proliferation, allowing heart regeneration after ischaemia-reperfusion injury. Metabolic studies reveal profound changes in energy metabolism and accumulation of α-ketoglutarate in Cpt1b-mutant cardiomyocytes, leading to activation of the α-ketoglutarate-dependent lysine demethylase KDM5 (ref. 3). Activated KDM5 demethylates broad H3K4me3 domains in genes that drive cardiomyocyte maturation, lowering theirtranscription levels and shifting cardiomyocytes into a less mature state, thereby promoting proliferation. We conclude that metabolic maturation shapes the epigenetic landscape of cardiomyocytes, creating a roadblock for further cell divisions. Reversal of this process allows repair of damaged hearts.

Novel SLC13A3 Variants and Cases of Acute Reversible Leukoencephalopathy and α-Ketoglutarate Accumulation and Literature Review.

Acute reversible leukoencephalopathy with increased urinary alpha-ketoglutarate (ARLIAK) is a recently described autosomal recessive leukoencephalopathy caused by pathogenic variants in the SLC13A3 gene. ARLIAK is characterized by acute neurologic involvement, often precipitated by febrile illness, with largely reversible clinical symptoms and imaging findings. Three patients have been reported in the literature to date. Our objective is to report newly identified patients and their genetic variants and phenotypes and review published literature on ARLIAK. This report contributes 4 additional patients to the literature; describes novel variants in SLC13A3; and reviews genetic, biochemical, clinical, and radiologic features of all published patients with ARLIAK. We provide additional genetic, imaging, and laboratory insights into ARLIAK, an atypical leukodystrophy with clinical and radiologic findings that can normalize. Our case series highlights the importance of reanalysis of next-generation sequencing in the diagnostic workup.

ALKBH5 modulates hematopoietic stem and progenitor cell energy metabolism through m6A modification-mediated RNA stability control

N6-methyladenosine (m6A) RNA modification controls numerous cellular processes. To what extent these post-transcriptional regulatory mechanisms play a role in hematopoiesis has not been fully elucidated. We here show that the m6A demethylase alkB homolog 5 (ALKBH5) controls mitochondrial ATP production and modulates hematopoietic stem and progenitor cell (HSPC) fitness in an m6A-dependent manner. Loss of ALKBH5 results in increased RNA methylation and instability of oxoglutarate-dehydrogenase (Ogdh) messenger RNA and reduction of OGDH protein levels. Limited OGDH availability slows the tricarboxylic acid (TCA) cycle with accumulation of α-ketoglutarate (α-KG) and conversion of α-KG into L-2-hydroxyglutarate (L-2-HG). L-2-HG inhibits energy production in both murine and human hematopoietic cells invitro. Impaired mitochondrial energy production confers competitive disadvantage to HSPCs and limits clonogenicity of Mll-AF9-induced leukemia. Our study uncovers a mechanism whereby the RNA m6A demethylase ALKBH5 regulates the stability of metabolic enzyme transcripts, thereby controlling energy metabolism in hematopoiesis and leukemia.

Case Report: Compound Heterozygous Variants of SLC13A3 Identified in a Chinese Patient With Acute Reversible Leukoencephalopathy and α-Ketoglutarate Accumulation

Background: SLC13A3 gene encodes the Na+/dicarboxylate cotransporter 3 (NaDC3), which locates on the plasma membrane and is mainly expressed in kidney, astrocytes and the choroid plexus. It imports four to six carbon dicarboxylates together with three Na+ ions into the cytosol. Nowadays, pathogenic variants of SLC13A3 gene were found to cause acute reversible leukoencephalopathy and α-ketoglutarate accumulation (ARLIAK) in patients. Here, we report two novel SLC13A3 variants c.185C>T (p.T62M) and c.331C>T (p.R111*) identified in a Chinese patient with ARLIAK.Case Presentation: The patient was a Chinese girl aged 13 years and 7 months old, who had acute, recurrent neurological deterioration during two febrile episodes. She presented with reversible leukoencephalopathy and increased urinary excretion of α-ketoglutarate. Genetic studies revealed compound heterozygous variants (c.185C>T, p.T62M, and c.331C>T, p.R111*) in SLC13A3, which had not been reported previously.Conclusions: These findings expand the variant spectrum of SLC13A3, providing the basis for the further study of this rare disease.

3-Mercaptopyruvate sulfurtransferase disruption in dermal fibroblasts facilitates adipogenic trans-differentiation

Transitioning from a differentiated state to a higher-order of plasticity, by partial rather than full reactivation of pluripotency genes, might be a better approach in regenerative medicine. Hydrogen sulfide plays a crucial role in the maintenance and differentiation of mesenchymal stem cells (MSC) that have the potential to differentiate to a diverse group of mesenchymally derived cells. It was shown that these cells show a heavy reliance on cystathionine-β-synthase (CBS)-derived hydrogen sulfide (H2S) during differentiation. We have found that expression and activity of 3-mercaptopyruvate sulfurtransferase (MPST), one of three enzymes that hat regulates H2S biosynthesis, is significantly lower in MSC as compared with lineage-restricted dermal fibroblasts. Here, we tested the hypothesis that suppression of MPST in dermal fibroblasts might induce plasticity-related changes and broaden the transdifferentiation potency. Inactivation of MPST with phenylpyruvate (PP) or by siRNA silencing led to diminished H2S production associated with increased production of reactive oxygen species (ROS) and lactic acid. Accumulation of α-ketoglutarate (α-KG), a key metabolite required for the expression of ten-eleven translocation hydroxylase (TET), was associated with stimulated transcription of pluripotency related genes including OCT4, KLF4, SOX2, and NANOG. Suppression of TET1 gene and inhibition of glycolysis with glucose analog, 2-desoxy-d-glucose, or hexokinase II inhibitor significantly reduced expression of pluripotency genes following MPST inactivation or knockdown. MPST disruption promoted the conversion of fibroblasts into adipocytes as evidenced by a significant increase in expression of adipocyte-specific genes, PPARγ, and UCP1, and intracellular accumulation of oil Red-O positive fat droplets. Inhibition of glycolysis inhibited these changes. Under induced differentiation conditions, fibroblasts with disrupted MPST show the potency to differentiate to white adipogenic lineage. Thus, MPST inactivation or silencing enhances the plasticity of dermal fibroblasts in a TET1 and glycolysis dependent manner and promotes adipogenic transdifferentiation.

Suppressed fate

Morris IV et al. adapted a mouse model of human pancreatic ductal adenocarcinoma and found that p53-dependent metabolic changes restrain tumour progression through α-ketoglutarate accumulation and promotion of a premalignant cell fate.

Distinctive Krebs cycle remodeling in iPSC-derived neural and mesenchymal stem cells

Mitochondria play a vital role in proliferation and differentiation and their remodeling in the course of differentiation is related to the variable energy and metabolic needs of the cell. In this work, we show a distinctive mitochondrial remodeling in human induced pluripotent stem cells differentiated into neural or mesenchymal progenitors. While leading to upregulation of the citrate synthase-α-ketoglutarate dehydrogenase segment of the Krebs cycle and increased respiratory chain activities and respiration in the mesenchymal stem cells, the remodeling in the neural stem cells resulted in downregulation of α-ketoglutarate dehydrogenase, upregulation of isocitrate dehydrogenase 2 and the accumulation of α-ketoglutarate. The distinct, lineage-specific changes indicate an involvement of these Krebs cycle enzymes in cell differentiation.

AβPP-Transgenic 2576 Mice Mimic Cell Type-Specific Aspects of Acetyl-CoA-Linked Metabolic Deficits in Alzheimer's Disease.

The pyruvate-derived acetyl-CoA is a principal direct precursor substrate for bulk energy synthesis in the brain. Deficits of pyruvate dehydrogenase in the neocortex are common features of Alzheimer's disease and other age-related encephalopathies in humans. Therefore, amyloid-β overload in brains of diverse transgenic Alzheimer's disease model animals was investigated as one of neurotoxic compounds responsible for pyruvate dehydrogenase inhibition yielding deficits of cholinergic neurotransmission and cognitive functions. Brains of aged, 14-16-month-old Tg2576 mice contained 0.6 μmol/kg levels of amyloid-β1 - 42. Activities of pyruvate dehydrogenase complex, choline acetyltransferase, and several enzymes of acetyl-CoA and energy metabolism were found to be unchanged in both forebrain mitochondria and synaptosomes of Tg2576 mice, indicating preservation of structural integrity at least in cholinergic neuronal cells. However, in transgenic brain synaptosomes, pyruvate utilization, mitochondrial levels, and cytoplasmic acetyl-CoA levels, as well as acetylcholine content and its quantal release, were all found to be decreased by 25-40% . On the contrary, activation of pyruvate utilization was detected and no alterations in acetyl-CoA content and citrate or α-ketoglutarate accumulation were observed in transgenic whole brain mitochondria. These data indicate that amyloid-β evoked deficits in acetyl-CoA are confined to mitochondrial and cytoplasmic compartments of Tg2576 nerve terminals, becoming early primary signals paving the path for further stages of neurodegeneration. On the other hand, acetyl-CoA synthesis in mitochondrial compartments of glial cells seems to be activated despite amyloid-β accumulated in transgenic brains.

Acetyl-CoA deficit in brain mitochondria in experimental thiamine deficiency encephalopathy

Several pathologic conditions are known to cause thiamine deficiency, which induce energy shortages in all tissues, due to impairment of pyruvate decarboxylation. Brain is particularly susceptible to these conditions due to its high rate of glucose to pyruvate-driven energy metabolism. However, cellular compartmentalization of a key energy metabolite, acetyl-CoA, in this pathology remains unknown. Pyrithiamine-evoked thiamine deficiency caused no significant alteration in pyruvate dehydrogenase and 30% inhibition of α-ketoglutarate dehydrogenase activities in rat whole forebrain mitochondria. It also caused 50% reduction of the metabolic flux of pyruvate through pyruvate dehydrogenase, 78% inhibition of its flux through α-ketoglutarate dehydrogenase steps, and nearly 60% decrease of intramitochondrial acetyl-CoA content, irrespective of the metabolic state. State 3 caused a decrease in citrate and an increase in α-ketoglutarate accumulation. These alterations were more evident in thiamine-deficient mitochondria. Simultaneously thiamine deficiency caused no alteration of relative, state 3-induced increases in metabolic fluxes through pyruvate and α-ketoglutarate dehydrogenase steps. These data indicate that a shortage of acetyl-CoA in the mitochondrial compartment may be a primary signal inducing impairment of neuronal and glial cell functions and viability in the thiamine-deficient brain.

A systematic investigation of Escherichia coli central carbon metabolism in response to superoxide stress

BackgroundThe cellular responses of bacteria to superoxide stress can be used to model adaptation to severe environmental changes. Superoxide stress promotes the excessive production of reactive oxygen species (ROS) that have detrimental effects on cell metabolic and other physiological activities. To antagonize such effects, the cell needs to regulate a range of metabolic reactions in a coordinated way, so that coherent metabolic responses are generated by the cellular metabolic reaction network as a whole. In the present study, we have used a quantitative metabolic flux analysis approach, together with measurement of gene expression and activity of key enzymes, to investigate changes in central carbon metabolism that occur in Escherichia coli in response to paraquat-induced superoxide stress. The cellular regulatory mechanisms involved in the observed global flux changes are discussed.ResultsFlux analysis based on nuclear magnetic resonance (NMR) and mass spectroscopy (MS) measurements and computation provided quantitative results on the metabolic fluxes redistribution of the E. coli central carbon network under paraquat-induced oxidative stress. The metabolic fluxes of the glycolytic pathway were redirected to the pentose phosphate pathway (PP pathway). The production of acetate increased significantly, the fluxes associated with the TCA cycle decreased, and the fluxes in the glyoxylate shunt increased in response to oxidative stress. These global flux changes resulted in an increased ratio of NADPH:NADH and in the accumulation of α-ketoglutarate.ConclusionsMetabolic flux analysis provided a quantitative and global picture of responses of the E. coli central carbon metabolic network to oxidative stress. Systematic adjustments of cellular physiological state clearly occurred in response to changes in metabolic fluxes induced by oxidative stress. Quantitative flux analysis therefore could reveal the physiological state of the cell at the systems level and is a useful complement to molecular systems approaches, such as proteomics and transcription analyses.

Free CoA-mediated regulation of intermediary and central metabolism: An hypothesis which accounts for the excretion of α-ketoglutarate during aerobic growth of Escherichia coli on acetate

During growth of Escherichia coli on acetate, phosphotransacetylase and α-ketoglutarate dehydrogenase are in direct competition for their common co-factor, HS-CoA. Such competition is resolved in favour of phosphotransacetylase, thus rendering α-ketoglutarate dehydrogenase rate-limiting (controlling) and, in turn, creating a bottleneck at the level of α-ketoglutarate in the Krebs cycle. Accumulation of α-ketoglutarate is then balanced by its excretion. Addition of pyruvate, glucose or any glycolytic intermediate to acetate-grown culture relieves such a bottleneck by reversing carbon flow through phosphotransacetylase to give acetyl phosphate and much-needed HS-CoA. The urgent need for HS-CoA by the primordial organism might therefore have provided the selective pressure that led to the co-evolution of phosphotransacetylase and the two-malate synthase isoenzymes.

Feedback Interactions in the Control of Citric Acid Cycle Activity in Rat Heart Mitochondria

Factors regulating the citric acid cycle have been investigated in rat heart mitochondria oxidizing pyruvate plus malate or acetylcarnitine plus malate as substrates. Effects caused by changing the NAD oxidation-reduction state, the intramitochondrial ATP:ADP ratio, and the respiratory rate were studied in five different metabolic states produced by additions of ADP, oligomycin, or uncouplers. The accumulations of cycle intermediates and the mitochondrial content of CoA derivatives and pyridine nucleotides were measured in extracts prepared from the whole incubation medium or after rapid separation of the mitochondria from the medium. These data, together with measurements of substrate utilization, were used to calculate flux through the individual steps of the citric acid cycle. Pyruvate dehydrogenase was found not to be rate limiting for citric acid cycle activity. Flux through citrate synthase in State 3 was approximately the same (100 nmoles per min per mg) whether pyruvate or acetylcarnitine were used to generate acetyl-CoA, whereas acetyl-CoA levels were higher with pyruvate as substrate. Flux in State 4 with either substrate was diminished by 75 to 85% relative to State 3 and was associated during early incubation times with elevated levels of both NADH and acetyl-CoA, consonant with regulation of pyruvate dehydrogenase by product inhibition. A comparison of flux through citrate synthase and changes in the levels of intramitochondrial malate, citrate, ATP, and the NAD oxidation-reduction state showed that increased flux associated with a State 4 to 3 transition could be accounted for largely by an increased availability of oxalacetate to citrate synthase rather than by ATP inhibition. On the other hand, comparisons between states in which phosphate acceptor was not rate limiting for electron transport (State 3, uncoupled or uncoupled plus oligomycin) showed that oxalacetate availability remained high because of the highly oxidized state of the pyridine nucleotides, and indicated a regulation of citrate synthase under conditions of low acetyl-CoA availability by an energy-linked process dependent on substrate level phosphorylation. Thus, a 55% inhibition of flux through citrate synthase was obtained after addition of oligomycin to the uncoupled state with acetylcarnitine plus malate as substrate, but only a 25% inhibition was achieved with pyruvate plus malate as substrate. The intramitochondrial ATP:ADP ratio increased from 0.3 to 4:6 with either substrate under these conditions. A kinetic evaluation of the data indicated that the energy-dependent inhibition was not caused by a direct effect of ATP on citrate synthase, but was due to changes of the succinyl-CoA content relative to that of acetyl-CoA. This conclusion is based (a) on the finding of an accumulation of a short chain acyl-CoA compound identifiable as succinyl-CoA by direct enzyme assay under conditions of diminished citrate synthase flux, (b) a correlation between decreased flux and lowered acetyl-CoA levels in the different experiments, and (c) the observation that succinyl-CoA is an inhibitor of citrate synthase competitive with acetyl-CoA. A comparison of succinyl-CoA levels in mitochondria incubated in the different metabolic states indicated that its content was regulated by the phosphorylation state of the adenine nucleotides, presumably via the effect of altered GTP:GDP ratios on succinate thiokinase. Product inhibition of α-ketoglutarate dehydrogenase by succinyl-CoA was evidenced from an inverse relationship between α-ketoglutarate accumulation and succinyl-CoA levels. The conclusion is reached that feedback from the electron transport chain to the citric acid cycle is mediated by a combination of factors which include the phosphorylation state of the adenine and guanine nucleotides and the oxidation-reduction state of the pyridine nucleotides. Changes of these parameters secondarily affect the intramitochondrial concentrations of oxalacetate, acetyl-CoA, and succinyl-CoA, which are the direct regulators of citrate synthase activity.

The effect of cobalt ions on energy metabolism in the rat.

Cobalt treatment for 8 days significantly depressed respiration of isolated rat heart mitochondria when citrate or α-ketoglutarate but not succinate was utilized as added substrate. The depressed respiration of the cardiac mitochondria in citrate media was accompanied by an increased accumulation of α-ketoglutarate in the incubation media. Cobalt treatment also reduced the ferricyanide-linked ketoglutarate dehydrogenase activity of isolated rat heart mitochondria. For the first 3 days, isolated diaphragm segments from cobalt-treated rats utilized glucose and pyruvate at rates higher than the controls but respired at reduced rates if ketoglutarate or octanoate was used as the substrate. After 4 days of cobalt-treatment, oxygen uptake was consistently lower in diaphragm segments for all four substrates and remained so for the next 3 days of treatment. By contrast, heart slices from cobalt-treated rats had a consistently lower oxygen uptake for glucose, pyruvate, α-ketoglutarate, and octanoate, commencing with the first day of cobalt injection and continuing for the next 7 days. Significant differences were found in resting glycogen (decreased), glucose uptake (increased), and glycogen deposition (decreased) in isolated diaphragm taken from cobalt-treated rats. Cobalt treatment had no effect on blood pyruvate and lactate levels.

Accumulation of α-ketoglutaric acid in yeast mutants requiring lysine

Accumulation of a-ketoglutaric acid in two nonallelic yeast mutants (Ly 7 and Ly 4) requiring lysine has been demonstrated. In addition to α-ketoglutarate, Ly 7 accumulates homocitrate, and Ly 4 accumulates both homocitrate and cis-homoaconitate. The cell-free preparations of Ly 4 lack homoacunitase activity, but Ly 7 has homocitrate synthetase and homoaconitase activities. The mutants that fail to accumulate homoisocitrate have so far been found to accumulate α-ketoglutarate, which is not accumulated in wild-type culture. The accumulation of α-ketoglutarate has been confirmed in radioactive and nonradioactive experiments, and the mutant product has been identified by various Chromatographic and chemical procedures. The accumulation data for these mutants as well as for Ly 12 suggest that there are two different enzymic steps to convert homocitrate to homisocitrate. The accumulation of α-ketoglutarate has been found in mutants which are blocked at steps preceding the synthesis of homoisocitrate.

The pathway of glutamate oxidation by mitochondria isolated from different tissues

1. The pathway of glutamate oxidation by mitochondria isolated from rat liver, Ehrlich ascites cells, rat heart and pigeon-breast muscle has been investigated. 2. The average inhibition of glutamate oxidation by malonate was 70% with rat-liver mitochondria, 82% with ascites-cell mitochondria, 97% with rat-heart sarcosomes and more than 97% with pigeon-breast-muscle sarcosomes. Almost complete inhibition was also found by fluoride with pigeon-breast-muscle sarcosomes and by arsenite with rat-heart sarcosomes. 3. Accumulation of α-ketoglutarate during the oxidation of glutamate by rat-liver, rat-heart and ascites-cell mitochondria was negligible, the ΔO/Δα-ketoglutarate being larger than 100 in nearly all experiments. 4. Glutamic dehydrogenase activity, measured spectrophotometrically, was very high in rat-liver mitochondria and moderate in ascites-cell mitochondria, while no activity could be demonstrated in rat-heart or pigeon-breast-muscle sarcosomes. The rate of glutamate formation by the Krebs-Cohen dismutation ran roughly parallel to the glutamic dehydrogenase activity. 5. During glutamate oxidation in the absence of inhibitors aspartate was formed. The average Δ glutamate/Δ aspartate value, determined with bacterial decar☐ylases, was 1.0 for ascites-cell mitochondria and 1.1 for rat-liver, rat-heart and pigeon-breast-muscle mitochondria. 6. It is concluded that under the experimental conditions used, the predominant pathway for glutamate oxidation in the mitochondria studied is conversion to aspartate by means of glutamate-oxaloacetate transaminase followed by that part of the Krebs-cycle lying between α-ketoglutarate and oxaloacetate. In rat-heart and pigeon-breast-muscle sarcosomes the transamination pathway is the only one available for rapid glutamate oxidation.

Hormonal effects on pyridine nucleotide metabolism in human placenta.

Abstract The addition of estradiol to homogenates of human term placenta leads to increases in incorporation of inorganic P 32 into ATP, utilization of citrate, accumulation of α-ketoglutarate, and oxygen consumption. Placental mitochondria have a P:O ratio of 1.3 when DPNH is oxidized and 0.2 when TPNH is oxidized. The placenta contains DPN largely in its oxidized form and TPN largely in its reduced form. The relation of these observations to estrogen action in vivo is discussed.