Pyruvate Carboxylation Research Articles

Hydrolysis of the acetyl-CoA allosteric activator by Staphylococcus aureus pyruvate carboxylase.

Pyruvate carboxylase (PC) catalyzes the carboxylation of pyruvate to oxaloacetate which serves as an important anaplerotic reaction to replenish citric acid cycle intermediates. In most organisms, the PC-catalyzed reaction is allosterically activated by acetyl-coenzyme A. It has previously been reported that vertebrate PC can catalyze the hydrolysis of acetyl-CoA, offering a potential means for the enzyme to attenuate its allosteric activation. However, in the years since this initial report, there has been no further investigation of this phenomenon. The allosteric binding site for acetyl-CoA is now well characterized, enabling more detailed studies on acetyl-CoA hydrolysis at the allosteric site. Here, we confirm that slow acetyl-CoA hydrolysis is catalyzed by a bacterial PC from Staphylococcus aureus, indicating that this phenomenon is a broad feature of PC enzymes spanning the domains of life. Surprisingly, the enzyme can hydrolyze acetyl-CoA even when the binding site for the acetyl moiety is eliminated through truncation of the biotin carboxylase domain. This suggests that an alternative site for acetyl-CoA binding and hydrolysis may be present in the carboxyltransferase domain of S. aureus PC. We conclude that PC has evolved to minimize the rate of acetyl-CoA hydrolysis at the allosteric site and update the description of PC-catalyzed acetyl-CoA hydrolysis to suggest that this reaction is unlikely to play a significant physiological, metabolic or catalytic role.

Structure based exploration of mitochondrial alpha carbonic anhydrase inhibitors as potential leads for anti-obesity drug development.

Obesity has emerged as a major health challenge globally in the last two decades. Dysregulated fatty acid metabolism and de novo lipogenesis are prime causes for obesity development which ultimately trigger other co-morbid pathological conditions thereby risking life longevity. Fatty acid metabolism and de novo lipogenesis involve several biochemical steps both in cytosol and mitochondria. Reportedly, the high catalytically active mitochondrial carbonic anhydrases (CAVA/CAVB) regulate the intercellular depot of bicarbonate ions and catalyze the rapid carboxylation of pyruvate and acetyl-co-A to acetyl-co-A and malonate respectively, which are the precursors of fatty acid synthesis and lipogenesis. Several in vitro and in vivo investigations indicate inhibition of mitochondrial carbonic anhydrase isoforms interfere in the functioning of pyruvate, fatty acid and succinate pathways. Targeting of mitochondrial carbonic anhydrase isoforms (CAVA/CAVB) could thereby modulate gluconeogenetic as well as lipogenetic pathways and pave way for designing of novel leads in the development pipeline of anti-obesity medications. The present review unveils a diverse chemical space including synthetic sulphonamides, sulphamates, sulfamides and many natural bioactive molecules which selectively inhibit the mitochondrial isoform CAVA/CAVB with an emphasis on major state-of-art drug design strategies. More than 60% similarity in the structural framework of the carbonic anhydrase isoforms has converged the drug design methods towards the development of isoform selective chemotypes. While the benzene sulphonamide derivatives selectively inhibit CAVA/CAVB in low nanomolar ranges depending on the substitutions on the phenyl ring, the sulpamates and sulpamides potently inhibit CAVB. The virtual screening and drug repurposing methods have also explored many non-sulphonamide chemical scaffolds which can potently inhibit CAVA. The review could pave way for the development of novel and effective anti-obesity drugs which can modulate the energy metabolism.

Oxaloacetate anaplerosis differently contributes to pathogenicity in plant pathogenic fungi Fusarium graminearum and F. oxysporum.

Anaplerosis refers to enzymatic reactions or pathways replenishing metabolic intermediates in the tricarboxylic acid (TCA) cycle. Pyruvate carboxylase (PYC) plays an important anaplerotic role by catalyzing pyruvate carboxylation, forming oxaloacetate. Although PYC orthologs are well conserved in prokaryotes and eukaryotes, their pathobiological functions in filamentous pathogenic fungi have yet to be fully understood. Here, we delve into the molecular functions of the ortholog gene PYC1 in Fusarium graminearum and F. oxysporum, prominent fungal plant pathogens with distinct pathosystems, demonstrating variations in carbon metabolism for pathogenesis. Surprisingly, the PYC1 deletion mutant of F. oxysporum exhibited pleiotropic defects in hyphal growth, conidiation, and virulence, unlike F. graminearum, where PYC1 deletion did not significantly impact virulence. To further explore the species-specific effects of PYC1 deletion on pathogenicity, we conducted comprehensive metabolic profiling. Despite shared metabolic changes, distinct reprogramming in central carbon and nitrogen metabolism was identified. Specifically, alpha-ketoglutarate, a key link between the TCA cycle and amino acid metabolism, showed significant down-regulation exclusively in the PYC1 deletion mutant of F. oxysporum. The metabolic response associated with pathogenicity was notably characterized by S-methyl-5-thioadenosine and S-adenosyl-L-methionine. This research sheds light on how PYC1-mediated anaplerosis affects fungal metabolism and reveals species-specific variations, exemplified in F. graminearum and F. oxysporum.

Metabolic cycles: A unifying concept for energy transfer in the heart

It is still debated whether changes in metabolic flux are cause or consequence of contractile dysfunction in non-ischemic heart disease. We have previously proposed a model of cardiac metabolism grounded in a series of six moiety-conserved, interconnected cycles. In view of a recent interest to augment oxygen availability in heart failure through iron supplementation, we integrated this intervention in terms of moiety conservation. Examining published work from both human and murine models, we argue this strategy restores a mitochondrial cycle of energy transfer by enhancing mitochondrial pyruvate carrier (MPC) expression and providing pyruvate as a substrate for carboxylation and anaplerosis. Metabolomic data from failing heart muscle reveal elevated pyruvate levels with a concomitant decrease in the levels of Krebs cycle intermediates. Additionally, MPC is downregulated in the same failing hearts, as well as under hypoxic conditions. MPC expression increases upon mechanical unloading in the failing human heart, as does contractile function. We note that MPC deficiency also alters expression of enzymes involved in pyruvate carboxylation and decarboxylation, increases intermediates of biosynthetic pathways, and eventually leads to cardiac hypertrophy and dilated cardiomyopathy. Collectively, we propose that an unbroken chain of moiety-conserved cycles facilitates energy transfer in the heart. We refer to the transport and subsequent carboxylation of pyruvate in the mitochondrial matrix as an example and a proposed target for metabolic support to reverse impaired contractile function.

Production of succinate with two CO2 fixation reactions from fatty acids in Cupriavidus necator H16

BackgroundBiotransformation of CO2 into high-value-added carbon-based products is a promising process for reducing greenhouse gas emissions. To realize the green transformation of CO2, we use fatty acids as carbon source to drive CO2 fixation to produce succinate through a portion of the 3-hydroxypropionate (3HP) cycle in Cupriavidus necator H16.ResultsThis work can achieve the production of a single succinate molecule from one acetyl-CoA molecule and two CO2 molecules. It was verified using an isotope labeling experiment utilizing NaH13CO3. This implies that 50% of the carbon atoms present in succinate are derived from CO2, resulting in a twofold increase in efficiency compared to prior methods of succinate biosynthesis that relied on the carboxylation of phosphoenolpyruvate or pyruvate. Meanwhile, using fatty acid as a carbon source has a higher theoretical yield than other feedstocks and also avoids carbon loss during acetyl-CoA and succinate production. To further optimize succinate production, different approaches including the optimization of ATP and NADPH supply, optimization of metabolic burden, and optimization of carbon sources were used. The resulting strain was capable of producing succinate to a level of 3.6 g/L, an increase of 159% from the starting strain.ConclusionsThis investigation established a new method for the production of succinate by the implementation of two CO2 fixation reactions and demonstrated the feasibility of ATP, NADPH, and metabolic burden regulation strategies in biological carbon fixation.

Metals and other ligands balance carbon fixation and photorespiration in chloroplasts.

The behavior of many plant enzymes depends on the metals and other ligands to which they are bound. A previous study demonstrated that tobacco Rubisco binds almost equally to magnesium and manganese and rapidly exchanges one metal for the other. The present study characterizes the kinetics of Rubisco and the plastidial malic enzyme when bound to either metal. When Rubisco purified from five C3 species was bound to magnesium rather than manganese, the specificity for CO2 over O2, (Sc/o) increased by 25% and the ratio of the maximum velocities of carboxylation / oxygenation (Vcmax/Vomax) increased by 39%. For the recombinant plastidial malic enzyme, the forward reaction (malate decarboxylation) was 30% slower and the reverse reaction (pyruvate carboxylation) was three times faster when bound to manganese rather than magnesium. Adding 6-phosphoglycerate and NADP+ inhibited carboxylation and oxygenation when Rubisco was bound to magnesium and stimulated oxygenation when it was bound to manganese. Conditions that favored RuBP oxygenation stimulated Rubisco to convert as much as 15% of the total RuBP consumed into pyruvate. These results are consistent with a stromal biochemical pathway in which (1) Rubisco when associated with manganese converts a substantial amount of RuBP into pyruvate, (2) malic enzyme when associated with manganese carboxylates a substantial portion of this pyruvate into malate, and (3) chloroplasts export additional malate into the cytoplasm where it generates NADH for assimilating nitrate into amino acids. Thus, plants may regulate the activities of magnesium and manganese in leaves to balance organic carbon and organic nitrogen as atmospheric CO2 fluctuates.

Metabolic adaptation in epithelial ovarian cancer metastasis

Epithelial ovarian cancer (EOC) is highly lethal due to its unique metastatic characteristics. EOC spheroids enter a non-proliferative state, with hypoxic cores and reduced oncogenic signaling, all of which contribute to tumour dormancy during metastasis. We investigated the metabolomic states of EOC cells progressing through the three steps to metastasis. Metabolomes of adherent, spheroid, and re-adherent cells were validated by isotopic metabolic flux analysis and mitochondrial functional assays to identify metabolic pathways that were previously unknown to promote EOC metastasis. Although spheroids were thought to exist in a dormant state, metabolomic analysis revealed an unexpected upregulation of energy production pathways in spheroids, accompanied by increased abundance of tricarboxylic acid (TCA) cycle and electron transport chain proteins. Tracing of 13C-labelled glucose and glutamine showed increased pyruvate carboxylation and decreased glutamine anaplerosis in spheroids. Increased reductive carboxylation suggests spheroids adjust redox homeostasis by shuttling cytosolic NADPH into mitochondria via isocitrate dehydrogenase. Indeed, we observed spheroids have increased respiratory capacity and mitochondrial ATP production. Relative to adherent cells, spheroids reduced serine consumption and metabolism, processes which were reversed upon spheroid re-adherence. The data reveal a distinct metabolism in EOC spheroids that enhances energy production by the mitochondria while maintaining a dormant state with respect to growth and proliferation. The findings advance our understanding of EOC metastasis and identify the TCA cycle and mitochondrional activity as novel targets to disrupt EOC metastasis, providing new approaches to treat advanced disease.

Hyperpolarized [2-13C, 3-2H3]Pyruvate Detects Hepatic Gluconeogenesis In Vivo.

The feasibility of hyperpolarized [2-13C, 3-2H3]pyruvate for probing gluconeogenesis in vivo was investigated in this study. Whereas hyperpolarized [1-13C]pyruvate has clear access to metabolic pathways that convert pyruvate to lactate, alanine, and bicarbonate, its utility for assessing pyruvate carboxylation and gluconeogenesis has been limited by technical challenges, including spectral overlap and an obscure enzymatic step that decarboxylates the labeled carbon. To achieve unambiguous detection of gluconeogenic products, the carbonyl carbon in pyruvate was labeled with 13C. To prolong the T1 relaxation time, [2-13C, 3-2H3]pyruvate was synthesized and dissolved with D2O after dynamic nuclear polarization. The T1 of [2-13C, 3-2H3]pyruvate in D2O could be improved by 76.9% (79.6 s at 1 T and 74.5 s at 3 T) as compared to [2-13C]pyruvate in water. Hyperpolarized [2-13C, 3-2H3]pyruvate with D2O dissolution was applied to rat livers in vivo under normal feeding and fasting conditions. A gluconeogenic product, [2-13C]phosphoenolpyruvate, was observed at 149.9 ppm from fasted rats only, highlighting the utility of [2-13C, 3-2H3]pyruvate in detecting key gluconeogenic enzyme activities such as pyruvate carboxylase and phosphoenolpyruvate carboxykinase in vivo.

A Case Report of a Patient with Pyruvate Carboxylase Deficiency

Background: Pyruvate carboxylase catalyzes the carboxylation of pyruvate to oxaloacetate, a crucial intermediate of the tricarboxylic acid (TCA) cycle and the initial step in converting pyruvate to glucose (gluconeogenesis). Pyruvate carboxylase deficiency is a rare metabolic disorder characterized by lactic acidosis, failure to thrive, development delay, and recurrent seizures at an early age in severely affected patients. The onset and severity of pyruvate carboxylase deficiency have been classified as severe neonatal (type B), usually fatal, less severe infantile (type A), compatible with survival but with impaired neurologic development, and milder, later onset (type C) with some residual impairment. Clinical manifestations include hypotonia, mixed hypertonia, ataxia, choreoathetosis, microcephaly, and other signs of impaired white matter development. Case Report: A 7-day-old baby with a birth weight of 3kg, born to related parents, presented with clinical symptoms such as lethargy, poor feeding, and grunting since birth. Additionally, he experienced a drop in O2 saturation and cyanosis during his hospitalization. Test results revealed lactic acidosis and hyperammonemia. Furthermore, serum amino acids chromatography- HPLC indicated an increase in lysine and citrulline. The patient succumbed after 16 days due to multi-organ damage. Genetic analysis identified pyruvate carboxylase enzyme deficiency. Conclusion: Pyruvate carboxylase deficiency is a rare inborn error of metabolism that can lead to developmental delay and failure to thrive, typically beginning in the neonatal or early infantile period. The possibility of pyruvate carboxylase deficiency should be considered in any child presenting with lactic acidosis and neurological abnormalities, particularly if associated with hypoglycemia, hyperammonemia, or ketosis.

Metabolic Fluxes in the Renal Cortex Are Dysregulated InVivo in Response to High-Fat Diet.

Diabetes and obesity are risk factors for kidney disease. While renal glucose production increases in diabetes, recent data suggest that gluconeogenic and oxidative capacity decline in kidney disease. Thus, metabolic dysregulation caused by diet-induced insulin resistance may sensitize the kidney for a loss in function. Here we examined how diet-induced insulin resistance disrupts mitochondrial metabolic fluxes in the renal cortex in vivo. C57Bl/6J mice were rendered insulin resistant through high-fat (HF) feeding; anaplerotic, cataplerotic, and oxidative metabolic fluxes in the cortex were quantified through 13C-isotope tracing during a hyperinsulinemic-euglycemic clamp. As expected, HF-fed mice exhibited increased body weight, gluconeogenesis, and systemic insulin resistance compared to chow-fed mice. Relative to the citric acid cycle, HF-feeding increased metabolic flux through pyruvate carboxylation (anaplerosis) and phosphoenolpyruvate carboxykinase (cataplerosis) while decreasing flux through the pyruvate dehydrogenase complex in the cortex. Furthermore, the relative flux from non-pyruvate sources of acetyl-CoA profoundly increased in the cortex of HF-fed mice, correlating with a marker of oxidative stress. The data demonstrate that HF-feeding spares pyruvate from dehydrogenation at the expense of increasing cataplerosis, which may underpin renal gluconeogenesis during insulin resistance; the results also support the hypothesis that dysregulated oxidative metabolism in the kidney contributes to metabolic disease.

Hepatic malonyl-CoA synthesis restrains gluconeogenesis by suppressing fat oxidation, pyruvate carboxylation, and amino acid availability

Acetyl-CoA carboxylase (ACC) promotes prandial liver metabolism by producing malonyl-CoA, a substrate for de novo lipogenesis and an inhibitor of CPT-1-mediated fat oxidation. We report that inhibition of ACC also produces unexpected secondary effects on metabolism. Liver-specific double ACC1/2 knockout (LDKO) or pharmacologic inhibition of ACC increased anaplerosis, tricarboxylic acid (TCA) cycle intermediates, and gluconeogenesis by activating hepatic CPT-1 and pyruvate carboxylase flux in the fed state. Fasting should have marginalized the role of ACC, but LDKO mice maintained elevated TCA cycle intermediates and preserved glycemia during fasting. These effects were accompanied by a compensatory induction of proteolysis and increased amino acid supply for gluconeogenesis, which was offset by increased protein synthesis during feeding. Such adaptations may be related to Nrf2 activity, which was induced by ACC inhibition and correlated with fasting amino acids. The findings reveal unexpected roles for malonyl-CoA synthesis in liver and provide insight into the broader effects of pharmacologic ACC inhibition.

Short-term effects of sodium arsenite (AsIII) and sodium arsenate (AsV) on carbohydrate metabolism in the perfused rat liver

The actions of arsenite and arsenate on carbohydrate metabolism in the once-through perfused rat liver were investigated. The compound inhibited lactate gluconeogenesis with an IC50 of 25 µM. It also increased glycolysis and fructolysis at concentrations between 10 and 100 µM. This effect was paralleled by strong inhibition of pyruvate carboxylation (IC50 = 4.25 µM) and by a relatively moderate diminution in the ATP levels. The inhibitory action of arsenate on pyruvate carboxylation and lactate gluconeogenesis was 103 times less effective than that of arsenite. For realistic doses and concentrations («1 mM), impairment of metabolism by arsenate can be expected to occur solely after its reduction to arsenite. Arsenite, on the other hand, can be regarded as a strong short-term modifier of lactate gluconeogenesis and other pathways. The main cause of the former is inhibition of pyruvate carboxylation, a hitherto unknown effect of arsenic compounds.

Comprehensive quantification of metabolic flux during acute cold stress in mice

Cold-induced thermogenesis (CIT) is widely studied as a potential avenue to treat obesity, but a thorough understanding of the metabolic changes driving CIT is lacking. Here, we present a comprehensive and quantitative analysis of the metabolic response to acute cold exposure, leveraging metabolomic profiling and minimally perturbative isotope tracing studies in unanesthetized mice. During cold exposure, brown adipose tissue (BAT) primarily fueled the tricarboxylic acid (TCA) cycle with fat in fasted mice and glucose in fed mice, underscoring BAT's metabolic flexibility. BAT minimally used branched-chain amino acids or ketones, which were instead avidly consumed by muscle during cold exposure. Surprisingly, isotopic labeling analyses revealed that BAT uses glucose largely for TCA anaplerosis via pyruvate carboxylation. Finally, we find that cold-induced hepatic gluconeogenesis is critical for CIT during fasting, demonstrating a key functional role for glucose metabolism. Together, these findings provide a detailed map of the metabolic rewiring driving acute CIT.

Current affairs in the use of medical ozone. Biological effects. Mechanisms of action

Introduction. Oxygen-ozone therapy stands as a medically endorsed practice confirmed by numerous international clinical studies. Various authors have illustrated the beneficial clinical outcomes of ozone therapy in terms of its capacity to regulate redox balance, cellular inflammatory responses, and adaptation to ischemia/reperfusion processes. Ozone therapy extends to encompass a range of viral infections, inflammatory disorders, and degenerative ailments, used as both monotherapy and as an adjunct to unified conventional therapies. Material and methods. Narrative literature review study. Bibliographic search was conducted using the PubMed, Hinari, and SpringerLink databases, as well as the National Center of Biotechnology Information and Medline. Articles published between 1990 and 2022 were selected using various combinations of keywords, including “ozone”, “ozone therapy”, “mechanisms of ozone action”, “biological effects of ozone”, “antioxidant effect”, “anti-inflammatory effect” and “immunomodulatory effect.” Information regarding ozone’s mechanisms of action was identified and processed. Following the database information processing and search criteria, a total of 475 full-text articles were found. The final bibliography consists of 52 relevant sources that were deemed representative of the materials published on the topic of this synthesis article. Results. The effects of ozone on oxygen metabolism are explained by changes in the rheological properties of blood, including inhibition of erythrocyte aggregation and stimulation of 2,3-diphosphoglycerate in erythrocytes, favoring the transport and delivery of oxygen to tissues while facilitating the substantial elimination of nitric oxide and increasing blood flow. Intracellular triatomic oxygen enhances the oxidative carboxylation of pyruvate, stimulating ATP production, which also contributes to reducing peripheral vascular resistance. Conclusions. Ozone generates a moderate oxidative stress. Yet, it can set off several beneficial biochemical mechanisms that reactivate both the intra- and extracellular antioxidant systems and reverse chronic oxidative stress in various inflammatory and degenerative processes. Ozone induces a mild activation of the immune system by triggering neutrophil activation and stimulating the synthesis of certain cytokines (IL-2, TNF-α, IL-6, and IFN-γ), thereby initiating a complete cascade of immune responses. Ozone therapy yields the following biological reactions: optimization of blood circulation and oxygen delivery to ischemic tissue, regulation of cellular antioxidant enzymes, initiation of a slight immune system activation, and enhancing the release of growth factors.

Allosteric Site at the Biotin Carboxylase Dimer Interface Mediates Activation and Inhibition in Staphylococcus aureus Pyruvate Carboxylase.

Allosteric regulation of the essential anaplerotic enzyme, pyruvate carboxylase (PC), is vital for metabolic homeostasis. PC catalyzes the bicarbonate- and ATP-dependent carboxylation of pyruvate to form oxaloacetate. Dysregulation of PC activity can impact glucose and redox metabolism, which contributes to the pathogenicity of many diseases. To maintain homeostasis, PC is allosterically activated by acetyl-CoA and allosterically inhibited by l-aspartate. In this study, we further characterize the molecular basis of allosteric regulation in Staphylococcus aureus PC (SaPC) using slowly/nonhydrolyzable dethia analogues of acetyl-CoA and site-directed mutagenesis of residues at the biotin carboxylase homodimer interface. The dethia analogues fully activate SaPC but demonstrate significantly reduced binding affinities relative to acetyl-CoA. Residues Arg21, Lys46, and Glu418 of SaPC are located at the biotin carboxylase dimer interface and play a critical role in both allosteric activation and inhibition. A structure of R21A SaPC in complex with acetyl-CoA reveals an intact molecule of acetyl-CoA bound at the allosteric site, offering new molecular insights into the acetyl-CoA binding site. This study demonstrates that the biotin carboxylase domain dimer interface is a critical allosteric site in PC, serving as a convergence point for allosteric activation by acetyl-CoA and inhibition by l-aspartate.

Observing exocrine pancreas metabolism using a novel pancreas perfusion technique in combination with hyperpolarized [1-13 C]pyruvate.

In a clinical setting, ex vivo perfusions are routinely used to maintain and assess organ viability prior to transplants. Organ perfusions are also a model system to examine metabolic flux while retaining the local physiological structure, with significant success using hyperpolarized (HP) 13 C NMR in this context. We use a novel exocrine pancreas perfusion technique via the common bile duct to assess acinar cell metabolism with HP [1-13 C]pyruvate. The exocrine component of the pancreas produces digestive enzymes through the ductal system and is often neglected in research on the pancreas. Real-time production of [1-13 C]lactate, [1-13 C]alanine, [1-13 C]malate, [4-13 C]malate, [1-13 C]aspartate, and H13 CO3 - was detected. The appearance of these resonances indicates flux through both pyruvate dehydrogenase and pyruvate carboxylase. We studied excised pancreata from C57BL/6J mice and NOD.Rag1-/- .AI4α/β mice, a commonly used model of Type 1 Diabetes (T1D). Pancreata from the T1D mice displayed increased lactate to alanine ratio without changes in oxygen consumption, signifying increased cytosolic NADH levels. The mass isotopologue analysis of the extracted pancreas tissue using gas chromatography-mass spectrometry revealed confirmatory 13 C enrichment in multiple TCA cycle metabolites that are products of pyruvate carboxylation. The methodology presented here has the potential to provide insight into mechanisms underlying several pancreatic diseases, such as diabetes, pancreatitis, and pancreatic cancer.

Harmful effects of chlorhexidine on hepatic metabolism

Chlorhexidine (CHX) is an over-the-counter antiseptic amply used by the population. There are reports that CHX acts in mitochondria as an uncoupler and inhibitor. The purpose of this study was to investigate the short-term effects of CHX on hepatic metabolic pathways linked to energy metabolism in the perfused rat liver. The compound inhibited both glucose synthesis and the urea cycle. Oxygen consumption was raised at low concentrations (up to 10 μM) and diminished at higher ones. A pronounced diminution in the cellular ATP content was observed. Conversely, CHX stimulated glycolysis and enhanced leakage of cellular enzymes (lactate dehydrogenase and fumarase). In isolated mitochondria, this antiseptic inhibited pyruvate carboxylation, oxidases, and oxygen uptake at very low concentrations (2 μM) and promoted uncoupling. The results described herein raise great concerns about the safety of CHX, as the observed effects can induce hypoglycemia, lactic acidosis, ammonemia as well as cell membrane disruption.

Pyruvate transamination and NAD biosynthesis enable proliferation of succinate dehydrogenase-deficient cells by supporting aerobic glycolysis

Succinate dehydrogenase (SDH) is the mitochondrial enzyme converting succinate to fumarate in the tricarboxylic acid (TCA) cycle. SDH acts as a tumor suppressor with germline loss-of-function mutations in its encoding genes predisposing to aggressive familial neuroendocrine and renal cancer syndromes. Lack of SDH activity disrupts the TCA cycle, imposes Warburg-like bioenergetic features, and commits cells to rely on pyruvate carboxylation for anabolic needs. However, the spectrum of metabolic adaptations enabling SDH-deficient tumors to cope with a dysfunctional TCA cycle remains largely unresolved. By using previously characterized Sdhb-deleted kidney mouse cells, here we found that SDH deficiency commits cells to rely on mitochondrial glutamate-pyruvate transaminase (GPT2) activity for proliferation. We showed that GPT2-dependent alanine biosynthesis is crucial to sustain reductive carboxylation of glutamine, thereby circumventing the TCA cycle truncation determined by SDH loss. By driving the reductive TCA cycle anaplerosis, GPT2 activity fuels a metabolic circuit maintaining a favorable intracellular NAD+ pool to enable glycolysis, thus meeting the energetic demands of SDH-deficient cells. As a metabolic syllogism, SDH deficiency confers sensitivity to NAD+ depletion achieved by pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway. Beyond identifying an epistatic functional relationship between two metabolic genes in the control of SDH-deficient cell fitness, this study disclosed a metabolic strategy to increase the sensitivity of tumors to interventions limiting NAD availability.

Differential Inhibition of Anaplerotic Pyruvate Carboxylation and Glutaminolysis-Fueled Anabolism Underlies Distinct Toxicity of Selenium Agents in Human Lung Cancer.

Past chemopreventive human trials on dietary selenium supplements produced controversial outcomes. They largely employed selenomethionine (SeM)-based diets. SeM was less toxic than selenite or methylseleninic acid (MSeA) to lung cancer cells. We thus investigated the toxic action of these Se agents in two non-small cell lung cancer (NSCLC) cell lines and ex vivo organotypic cultures (OTC) of NSCLC patient lung tissues. Stable isotope-resolved metabolomics (SIRM) using 13C6-glucose and 13C5,15N2-glutamine tracers with gene knockdowns were employed to examine metabolic dysregulations associated with cell type- and treatment-dependent phenotypic changes. Inhibition of key anaplerotic processes, pyruvate carboxylation (PyC) and glutaminolysis were elicited by exposure to MSeA and selenite but not by SeM. They were accompanied by distinct anabolic dysregulation and reflected cell type-dependent changes in proliferation/death/cell cycle arrest. NSCLC OTC showed similar responses of PyC and/or glutaminolysis to the three agents, which correlated with tissue damages. Altogether, we found differential perturbations in anaplerosis-fueled anabolic pathways to underlie the distinct anti-cancer actions of the three Se agents, which could also explain the failure of SeM-based chemoprevention trials.

Photosynthetic resource-use efficiency trade-offs triggered by vapour pressure deficit and nitrogen supply in a C4 species

Trade-offs in resource-use efficiency (including water-, nitrogen-, and light-use efficiency, i.e., WUE, NUE, and LUE) are an important acclimation strategy of plants to environmental stresses. C4 photosynthesis, featured by a CO2 concentrating mechanism, is believed to be more efficient in using resources compared to C3 photosynthesis. However, response of photosynthetic resource-use efficiency trade-offs in C4 plants to vapour pressure deficit (VPD) and N supply has rarely been studied. Here, we studied the photosynthetic acclimation of Cleistogenes squarrosa, a perennial C4 grass, to controlled growth conditions with high or low VPD and N supply. High VPD increased WUE by 12% and decreased NUE by 16%, the ratio of net photosynthetic rate (A) to electron transport rate (J) (A/J) by 7% and the apparent quantum yield by 6%. High N supply tended to reduce NUE and increased maximum phosphoenol pyruvate carboxylation rate by 71% and slightly increased WUE. Stomatal conductance showed acclimation to VPD according to the Ball-Berry model, while a balanced cost of carboxylation and transpiration capacity was found across VPD and N treatments based on the least-cost model. WUE correlated negatively with NUE and LUE indicating that there was a trade-off between them, which is likely associated with acclimations in stomatal conductance and CO2 concentrating mechanisms.