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Hyperpolarized carbon‐13 (13C) magnetic resonance imaging (MRI) has shown promise for non‐invasive assessment of the cerebral metabolism of [1‐13C]pyruvate in both healthy volunteers and patients. The exchange of pyruvate to lactate catalysed by lactate dehydrogenase (LDH) and that of pyruvate flux to bicarbonate through pyruvate dehydrogenase (PDH) are the most widely studied reactions in vivo. Here we show the potential of the technique to probe additional enzymatic activity within the brain. Approximately 50 s after intravenous injection of hyperpolarized pyruvate, high‐flip‐angle pulses were used to detect cerebral 13C‐labelled carbon dioxide (13CO2), in addition to the 13C‐bicarbonate (H13CO3−) subsequently formed by carbonic anhydrase (CA). Brain pH measurements, which were weighted towards the extracellular compartment, were calculated from the ratio of H13CO3− to 13CO2 in seven volunteers using the Henderson–Hasselbalch equation, demonstrating an average pH ± SD of 7.40 ± 0.02, with inter‐observer reproducibility of 0.04. In addition, hyperpolarized [1‐13C]aspartate was also detected, demonstrating irreversible pyruvate carboxylation to oxaloacetate by pyruvate carboxylase (PC) and subsequent transamination by aspartate aminotransferase (AST), with the average flux being on average 11% ± 3% of that through PDH. A hyperpolarized [1‐13C]alanine signal was also detected, but this was localized to extracranial muscle tissue in keeping with skeletal alanine aminotransferase (ALT) activity. The results demonstrate the potential of hyperpolarized 13C‐MRI to assess cerebral and extracerebral [1‐13C]pyruvate metabolism in addition to LDH and PDH activity. Non‐invasive measurements of brain pH could be particularly important in assessing cerebral pathology given the wide range of disease processes that alter acid–base balance. [ABSTRACT FROM AUTHOR]

Fumarate is an unsaturated dicarboxylic acid useful as a raw material for unsaturated polyester resins, polybutylene succinate (PBS), poly(propylene fumarate) (PPF), plasticisers, and other products. Biodegradable plastics derived from fumarate are an attractive solution to the serious environmental pollution caused by plastic disposal. A new fumarate production from CO2 and biobased pyruvate using enzymes in aqueous media under ambient temperature and pressure is an environmental approach to overcome plastic pollution and achieve CO2 capture, utilization and storage (CCUS). In this work, fumarate production from pyruvate and low-concentration CO2 below 15% captured from the gas phase using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH buffer solution with a multi-enzyme system consisting of pyruvate carboxylase from a bovine liver (PC; EC 6.4.1.1), recombinant malate dehydrogenase from bacteria (MDH; EC 1.1.1.37) and fumarase from a porcine heart (FUM; EC 1.1.1.37) in the presence of adenosine 5′-triphosphate (ATP) and NADH was investigated. It was found that pyruvate can be converted into L -malate in high yields (more than 80%) directly using 15% CO2 equivalent to exhaust gas as a carboxylating agent using a dual-enzyme system consisting of PC and MDH in the presence of ATP and NADH after 5 h incubation. Moreover, fumarate production from 15% CO2 and pyruvate as raw materials was accomplished using a dual-enzyme system consisting of PC and MDH. [ABSTRACT FROM AUTHOR]

Defective glucose-stimulated insulin secretion (GSIS) and β-cell senescence are hallmarks in diabetes. The mitochondrial enzyme pyruvate carboxylase (PC) has been shown to promote GSIS and β-cell proliferation in the clonal β-cell lines, yet its physiological relevance remains unknown. Here, we provide animal and human data showing a role of PC in protecting β-cells against senescence and maintaining GSIS under different physiological and pathological conditions. β-cell-specific deletion of PC impaired GSIS and induced β-cell senescence in the mouse models under either a standard chow diet or prolonged high-fat diet feeding. Transcriptomic analysis indicated that p53-related senescence and cell cycle arrest are activated in PC-deficient islets. Overexpression of PC inhibited hyperglycemia-and aging-induced p53-related senescence in human and mouse islets as well as INS-1E β-cells, whereas knockdown of PC provoked senescence. Mechanistically, PC interacted with MDM2 to prevent its degradation via the MDM2 binding motif, which in turn restricts the p53-dependent senescent program in β-cells. On the contrary, the regulatory effects of PC on GSIS and the tricarboxylic acid (TCA) anaplerotic flux are p53-independent. We illuminate a function of PC in controlling β-cell senescence through the MDM2-p53 axis. [ABSTRACT FROM AUTHOR]

Introduction: Metabolic adaptability, including glucose metabolism, enables cells to survive multiple stressful environments. Glycogen may serve as a critical storage depot to provide a source of glucose during times of metabolic demand during the metastatic cascade; therefore, understanding glycogen metabolism is critical. Our goal was to determine mechanisms driving glycogen accumulation and its role in metastatic (MCF10CA1a) compared to nonmetastatic (MCF10A-ras) human breast cancer cells. Methodology:13C6-glucose flux analysis in combination with inhibitors of the gluconeogenic pathway via phosphoenolpyruvate carboxykinase (PCK), the anaplerotic enzyme pyruvate carboxylase (PC), and the rate-limiting enzyme of the pentose phosphate pathway (PPP) glucose 6-phosphate dehydrogenase (G6PD). To determine the requirement of glycogenolysis for migration or survival in extracellular matrix (ECM) detached conditions, siRNA inhibition of glycogenolysis (liver glycogen phosphorylase, PYGL) or glycophagy (lysosomal enzyme α-acid glucosidase, GAA) enzymes was utilized. Results: Metastatic MCF10CA1a cells had 20-fold greater glycogen levels compared to non-metastatic MCF10A-ras cells. Most glucose incorporated into glycogen of the MCF10CA1a cells was in the five13C-containing glucose (M+5) instead of the expected M+6 glycogen-derived glucose moiety, which occurs through direct glucose conversion to glycogen. Furthermore,13C6-glucose in glycogen was quickly reduced (~50%) following removal of13C-glucose. Incorporation of 13C6-glucose into the M+5 glucose in the glycogen stores was reduced by inhibition of PCK, with additional contributions from flux through the PPP. Further, inhibition of PC reduced total glycogen content. However, PCK inhibition increased total unlabeled glucose accumulation into glycogen, suggesting an alternative pathway to glycogen accumulation. Inhibition of the rate-limiting steps in glycogenolysis (PYGL) or glycophagy (GAA) demonstrated that both enzymes are necessary to support MCF10CA1a, but not MCF10A-ras, cell migration. GAA inhibition, but not PYGL, reduced viability of MCF10CA1a cells, but not MCF10A-ras, in ECM detached conditions. Conclusion: Our results indicate that increased glycogen accumulation is primarily mediated through the gluconeogenesis pathway and that glycogen utilization is required for both migration and ECM detached survival of metastatic MCF10CA1a cells. These results suggest that glycogen metabolism may play an important role in the progression of breast cancer metastasis. [ABSTRACT FROM AUTHOR]

Deficiencies in the electron transport chain (ETC) lead to mitochondrial diseases. While mutations are distributed across the organism, cell and tissue sensitivity to ETC disruption varies, and the molecular mechanisms underlying this variability remain poorly understood. Here we show that, upon ETC inhibition, a non-canonical tricarboxylic acid (TCA) cycle upregulates to maintain malate levels and concomitant production of NADPH. Our findings indicate that the adverse effects observed upon CI inhibition primarily stem from reduced NADPH levels, rather than ATP depletion. Furthermore, we find that Pyruvate carboxylase (PC) and ME1, the key mediators orchestrating this metabolic reprogramming, are selectively expressed in astrocytes compared to neurons and underlie their differential sensitivity to ETC inhibition. Augmenting ME1 levels in the brain alleviates neuroinflammation and corrects motor function and coordination in a preclinical mouse model of CI deficiency. These studies may explain why different brain cells vary in their sensitivity to ETC inhibition, which could impact mitochondrial disease management. Mitochondrial diseases caused by ETC deficiencies affect cells differently. Here, the authors show that ETC inhibition activates a non-canonical TCA cycle to maintain NADPH levels, which explains the differential sensitivity observed between astrocytes and neurons. [ABSTRACT FROM AUTHOR]

Abstract Objectives To investigate the roles of Ca2+ influx-regulated circular RNAs (circRNAs) in T cells from patients with systemic lupus erythematosus (SLE). Methods The expression profile of circRNAs in Jurkat cells, co-cultured with and without ionomycin, was analyzed by next-generation sequencing and validated using real-time polymerase chain reaction. The identified Ca2+ influx-regulated circRNAs were further examined in T cells from 42 patients with SLE and 23 healthy controls. The biological function of specific circRNA was investigated using transfection and RNA pull-down assay. Results After validation, we confirmed that the expression levels of circ-ERCC4, circ-NFATC2, circ-MYH10, circ-CAMTA1, circ-ASH1L, circ-SOCS7, and circ-ASAP1 were consistently increased in Jurkat cells following Ca2+ influx. The expression levels of circ-CAMTA1, circ-ASH1L, and circ-ASAP1 were significantly lower in T cells from patients with SLE, with even lower levels observed in those with higher disease activity. Interferon (IFN)-α was found to suppress the expression of circ-CAMTA1. Circ-CAMTA1 bound to pyruvate carboxylase and inhibited its biological activity. Overexpression of circ-CAMTA1, but not its linear form, significantly decreased extracellular glucose levels. Furthermore, increased expression of circ-CAMTA1, but not its linear form, decreased miR-181c-5p expression, resulting increased IL-2 secretion. Conclusion Three Ca2+ influx-regulated circ-RNAs—circ-CAMTA1, circ-ASH1L, and circ-ASAP1 —were significantly reduced in T cells from patients with SLE and associated with disease activity. IFN-α suppressed the expression of circ-CAMTA1, which interacted with pyruvate carboxylase, inhibited its activity, affected glucose metabolism, and increased IL-2 secretion. These findings suggest that circ-CAMTA1 regulated by Ca²⁺ influx modulated T cell function in patients with SLE.

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. Author summary: Pyruvate carboxylase plays a crucial role in replenishing oxaloacetate in the tricarboxylic acid cycle, a fundamental carbon metabolic pathway. While PYC is present in both prokaryotes and eukaryotes, its specific contribution to the development and pathogenesis of fungal pathogens remains poorly understood. Our study focuses on elucidating the function of PYC, particularly investigating the effects of deleting the ortholog gene PYC1 in two important fungal plant pathogens, Fusarium graminearum and F. oxysporum, which have distinct pathosystems. By conducting comprehensive genetic and metabolic profiling, we observed different impacts on fungal developmental processes and pathogenicity between the two species. In particular, the deletion of the PYC1 gene in F. oxysporum led to a unique metabolic profile and the subsequent loss of pathogenicity. This research provides valuable insights into the distinct effects of PYC1-mediated anaplerosis on fungal metabolism in pathogenic fungi, contributing to a more profound understanding of the molecular mechanisms governing fungal pathogenesis. [ABSTRACT FROM AUTHOR]

Anaplerotic reactions catalyzed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) have important roles in the production of l -lysine to replenish oxaloacetic acid (OAA) in Corynebacterium glutamicum. However, the relative contributions of these enzymes to l -lysine production in C. glutamicum are not fully understood. In this study, using a parent strain (P) carrying a feedback inhibition-resistant aspartokinase with the T311I mutation, we constructed a PC gene-deleted mutant strain (PΔPC) and a PEPC gene-deleted mutant strain (PΔPEPC). Although the growth of both mutant strains was comparable to the growth of strain P, the maximum l -lysine production in strains PΔPC and PΔPEPC decreased by 14% and 49%, respectively, indicating that PEPC strongly contributed to OAA supply. l -Lysine production in strain PΔPC slightly decreased during the logarithmic phase, while production during the early stationary phase was comparable to production in strain P. By contrast, strain PΔPEPC produced l -lysine in an amount comparable to the production of strain P during the logarithmic phase; l -lysine production after the early stationary phase was completely stopped in strain PΔPEPC. These results indicate that OAA is supplied by both PC and PEPC during the logarithmic phase, while only PEPC can continuously supply OAA after the logarithmic phase. [Display omitted] [ABSTRACT FROM AUTHOR]

The ischemia–reperfusion process of a donor heart during heart transplantation leads to severe mitochondrial dysfunction, which may be the main cause of donor heart dysfunction after heart transplantation. Pyruvate carboxylase (PC), an enzyme found in mitochondria, is said to play a role in the control of oxidative stress and the function of mitochondria. This research examined the function of PC and discovered the signaling pathways controlled by PC in myocardial IRI. We induced IRI using a murine heterotopic heart transplantation model in vivo and a hypoxia–reoxygenation cell model in vitro and evaluated inflammatory responses, oxidative stress levels, mitochondrial function, and cardiomyocyte apoptosis. In both in vivo and in vitro settings, we observed a significant decrease in PC expression during myocardial IRI. PC knockdown aggravated IRI by increasing MDA content, LDH activity, TUNEL-positive cells, serum cTnI level, Bax protein expression, and the level of inflammatory cytokines and decreasing SOD activity, GPX activity, and Bcl-2 protein expression. PC overexpression yielded the opposite findings. Additional research indicated that reducing PC levels could block the Wnt/β-catenin pathway and glutamine metabolism by hindering the movement of β-catenin to the nucleus and reducing the activity of complex I and complex II, as well as ATP levels, while elevating the ratios of NADP+/NADPH and GSSG/GSH. Overall, the findings indicated that PC therapy can shield the heart from IRI during heart transplantation by regulating glutamine metabolism through the Wnt/β-catenin pathway. [ABSTRACT FROM AUTHOR]

A growing number of studies indicate that mitochondrial dysfunction serves as a pathological mechanism for periodontitis. Therefore, this two-sample Mendelian randomization (MR) study was carried out to explore the causal associations between mitochondrial biological function and periodontitis, because the specific nature of this causal relationship remains inconclusive in existing MR studies. Inverse variance weighting, Mendelian randomization-Egger, weighted mode, simple mode, and weighted median analyses were performed to assess the causal relationships between the exposure factors and periodontitis. The results of the present study revealed a causal association between periodontitis and medium-chain specific acyl-CoA dehydrogenase (MCAD), malonyl-CoA decarboxylase (MLYCD), glutaredoxin 2 (Grx2), oligoribonuclease (ORN), and pyruvate carboxylase (PC). Notably, MCAD and MLYCD are causally linked to periodontitis, and serve as protective factors. However, Grx2, ORN, and PC function as risk factors for periodontitis. Our study established a causal relationship between mitochondrial biological function and periodontitis, and such insights may provide a promising approach for treating periodontitis via mitochondrial regulation. [ABSTRACT FROM AUTHOR]

Skeletal muscle metabolism has implications for swine feed efficiency (FE); however, it remains unclear if the metabolic profile of skeletal muscle changes during postnatal growth. To assess the metabolic changes, samples were collected from the longissimus dorsi (LD, glycolytic muscle), latissimus dorsi (LAT, mixed muscle), and masseter (MS, oxidative muscle) at 20, 53, 87, 120, and 180 days of age from barrows. Muscles were assessed to determine the abundance of several metabolic enzymes. Lactate dehydrogenase (LDHα) decreased in all muscles from 20 to 87 d (p < 0.01), which may be attributed to the muscles being more glycolytic at weaning from a milk-based diet. Pyruvate carboxylase (PC) increased in all muscles at 53 d compared to the other time points (p < 0.01), while pyruvate dehydrogenase α 1 (PDHα1) increased at 87 and 180 d in MS compared to LD (p < 0.05), indicating that potential changes occur in pyruvate entry into the tricarboxylic acid (TCA) cycle during growth. Isolated mitochondria from each muscle were incubated with 13C-labeled metabolites to assess isotopomer enrichment patterns of TCA intermediates. Citrate M + 2 and M + 4 derived from [13C3]-pyruvate increased at 87 d in LAT and MS mitochondria compared to LD mitochondria (p < 0.05). Regardless of the muscle, citrate M+3 increased at 87 d compared to 20, 53, and 120 d, while 180 d showed intermediate values (p < 0.01). These data support the notion that pyruvate metabolism is dynamic during growth. Our findings establish a metabolic fingerprint associated with postnatal muscle hypertrophy. [ABSTRACT FROM AUTHOR]

Background: Metabolic plasticity mediates breast cancer survival, growth, and immune evasion during metastasis. However, how tumor cell metabolism is influenced by and feeds back to regulate breast cancer progression are not fully understood. We identify hypoxia-mediated suppression of pyruvate carboxylase (PC), and subsequent induction of lactate production, as a metabolic regulator of immunosuppression. Methods: We used qPCR, immunoblot, and reporter assays to characterize repression of PC in hypoxic primary tumors. Steady state metabolomics were used to identify changes in metabolite pools upon PC depletion. In vivo tumor growth and metastasis assays were used to evaluate the impact of PC manipulation and pharmacologic inhibition of lactate transporters. Immunohistochemistry, flow cytometry, and global gene expression analyzes of tumor tissue were employed to characterize the impact of PC depletion on tumor immunity. Results: PC is essential for metastatic colonization of the lungs. In contrast, depletion of PC in tumor cells promotes primary tumor growth. This effect was only observed in immune competent animals, supporting the hypothesis that repression of PC can suppress anti-tumor immunity. Exploring key differences between the pulmonary and mammary environments, we demonstrate that hypoxia potently downregulated PC. In the absence of PC, tumor cells produce more lactate and undergo less oxidative phosphorylation. Inhibition of lactate metabolism was sufficient to restore T cell populations to PC-depleted mammary tumors. Conclusions: We present a dimorphic role for PC in primary mammary tumors vs. pulmonary metastases. These findings highlight a key contextual role for PC-directed lactate production as a metabolic nexus connecting hypoxia and antitumor immunity. [ABSTRACT FROM AUTHOR]

Using the previously engineered adipate-secreting Escherichia MG1655 lacIQ, ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, PL-SDφ10-atoB, Ptrc-ideal-4-SDφ10-fadB, ∆fadE, PL-SDφ10-tesB, ∆yciA, Ptrc-ideal-4-SDφ10-fabI, PL-SDφ10-paaJ, ∆aceBAK, ∆glcB as the core strain, the derivatives capable of enhanced synthesis of the target compound from glucose via the reversed fatty acid β-oxidation pathway were obtained. The respective effect was achieved due to the intensification of the tricarboxylic acid cycle in the cells. Prevention of multiple cycle turnovers, resulting from the inactivation of succinate dehydrogenase, had no pronounced effect on the formation of adipic acid by the recombinant. Upon intensification of the cycle due to enhancing anaplerotic oxaloacetic acetic acid formation from phosphoenolpyruvate, resulting from the increased expression of the native ppc gene, the synthesis of adipic acid increased by 1.2-fold up to ~390 μM. Enabling the formation of oxaloacetate from pyruvic acid, by introducing heterologous Bacillus subtilis pyruvate carboxylase in the cells , resulted in a 1.5-fold intensification of the cycle, concomitantly with the proportional increase in adipic acid secretion to ~496 μM. Subsequent inactivation of sdhAB genes in the strain increased the secretion of the target compound only slightly, and the adipic acid titer reached ~520 μM. The obtained data indicated a direct dependence of the efficiency of adipic acid synthesis by the engineered strains on the degree of intensification of the tricarboxylic acid cycle. [ABSTRACT FROM AUTHOR]

Objectives: Colitis is a global disease usually accompanied by intestinal epithelial damage and intestinal inflammation, and an increasing number of studies have found natural products to be highly effective in treating colitis. Anemoside B4 (AB4), an abundant saponin isolated from Pulsatilla chinensis (Bunge), which was found to have strong anti-inflammatory activity. However, the exact molecular mechanisms and direct targets of AB4 in the treatment of colitis remain to be discovered. Methods: The anti-inflammatory activities of AB4 were verified in LPS-induced cell models and 2, 4, 6-trinitrobenzene sulfonic (TNBS) or dextran sulfate sodium (DSS)-induced colitis mice and rat models. The molecular target of AB4 was identified by affinity chromatography analysis using chemical probes derived from AB4. Experiments including proteomics, molecular docking, biotin pull-down, surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA) were used to confirm the binding of AB4 to its molecular target. Overexpression of pyruvate carboxylase (PC) and PC agonist were used to study the effects of PC on the anti-inflammatory and metabolic regulation of AB4 in vitro and in vivo. Results: AB4 not only significantly inhibited LPS-induced NF-κB activation and increased ROS levels in THP-1 cells, but also suppressed TNBS/DSS-induced colonic inflammation in mice and rats. The molecular target of AB4 was identified as PC, a key enzyme related to fatty acid, amino acid and tricarboxylic acid (TCA) cycle. We next demonstrated that AB4 specifically bound to the His879 site of PC and altered the protein's spatial conformation, thereby affecting the enzymatic activity of PC. LPS activated NF-κB pathway and increased PC activity, which caused metabolic reprogramming, while AB4 reversed this phenomenon by inhibiting the PC activity. In vivo studies showed that diisopropylamine dichloroacetate (DADA), a PC agonist, eliminated the therapeutic effects of AB4 by changing the metabolic rearrangement of intestinal tissues in colitis mice. Conclusion: We identified PC as a direct cellular target of AB4 in the modulation of inflammation, especially colitis. Moreover, PC/pyruvate metabolism/NF-κB is crucial for LPS-driven inflammation and oxidative stress. These findings shed more light on the possibilities of PC as a potential new target for treating colitis. [ABSTRACT FROM AUTHOR]

Although a high titre of malic acid is achieved by filamentous fungi, by‐product succinic acid accumulation leads to a low yield of malic acid and is unfavourable for downstream processing. Herein, we conducted a series of metabolic rewiring strategies in a previously constructed Myceliophthora thermophila to successfully improve malate production and abolish succinic acid accumulation. First, a pyruvate carboxylase CgPYC variant with increased activity was obtained using a high‐throughput system and introduced to improve malic acid synthesis. Subsequently, shifting metabolic flux to malate synthesis from mitochondrial metabolism by deleing mitochondrial carriers of pyruvate and malate, led to a 53.7% reduction in succinic acid accumulation. The acceleration of importing cytosolic succinic acid into the mitochondria for consumption further decreased succinic acid formation by 53.3%, to 2.12 g/L. Finally, the importer of succinic acid was discovered and used to eliminate by‐product accumulation. In total, malic acid production was increased by 26.5%, relative to the start strain JG424, to 85.23 g/L and 89.02 g/L on glucose and Avicel, respectively, in the flasks. In a 5‐L fermenter, the titre of malic acid reached 182.7 g/L using glucose and 115.8 g/L using raw corncob, without any by‐product accumulation. This study would accelerate the industrial production of biobased malic acid from renewable plant biomass. [ABSTRACT FROM AUTHOR]

Background and Aims: Disorders in liver lipid metabolism have been implicated in a range of metabolic conditions, including fatty liver and liver cancer. Altered lipid distribution within the liver, shifting from the pericentral to the periportal zone under pathological circumstances, has been observed; however, the underlying mechanism remains elusive. Iron, an essential metal, exhibits a zonal distribution in the liver similar to that of lipids. Nevertheless, the precise relationship between iron and lipid distribution, especially in the pericentral and periportal zones, remains poorly understood. Methods: We conducted comprehensive in vitro and in vivo experiments, combining with in situ analysis and RNA sequencing, aiming for a detailed exploration of the causal relationship between iron accumulation and lipid metabolism. Results: Our research suggests that iron overload can disrupt the normal distribution of lipids within the liver, particularly in the periportal zone. Through meticulous gene expression profiling in both the pericentral and periportal zones, we identified pyruvate carboxylase (PC) as a pivotal regulator in iron overload-induced lipid accumulation. Additionally, we revealed that the activation of cyclic adenosine monophosphate response element binding protein (CREB) was indispensable for Pc gene expression when in response to iron overload. Conclusions: In summary, our investigation unveils the crucial involvement of iron overload in fostering hepatic lipid accumulation in the periportal zone, at least partly mediated by the modulation of Pc expression. These insights offer new perspectives for understanding the pathogenesis of fatty liver diseases and their progression. [ABSTRACT FROM AUTHOR]

Simple Summary: In intensive farming, due to economic and management reasons, piglets have been progressively weaned earlier at 3 to 4 weeks of age. Early weaning usually leads to low feed intake and weight loss, which causes undernutrition and energy deficits. However, the effects of early weaning on hepatic glucose metabolism in piglets remain unclear. The objective of this study was to evaluate the dynamic changes in the hepatic glucose metabolism of 60 piglets at 14 days postweaning. We found that early weaning remodeled the content of metabolites related to glycometabolism and enhanced gluconeogenesis to produce more hepatic glucose. Further study showed that early weaning reduced the content of oxaloacetate and succinate, which suppressed ATP (adenosine triphosphate) production. Our findings enrich weaning stress theory and might provide a reference for dietary intervention. This study aimed to investigate the dynamic changes in hepatic glucose metabolism in response to early weaning. A total of 60 piglets were randomly selected and weaned at 21 days old. Six piglets were slaughtered on the weaning day (d0) and at 1 (d1), 4 (d4), 7 (d7), and 14 (d14) days postweaning. The results illustrated that body weight significantly increased from d4 to d14 (p < 0.001). Serum glucose fell sharply after weaning and then remained at a low level from d1 to d14 (p < 0.001). Serum insulin decreased from d4 (p < 0.001), which caused hepatic glycogen to be broken down (p = 0.007). The glucose-6-phosphatase activity increased from d0 to d4 and then decreased from d4 to d14 (p = 0.039). The pyruvate carboxylase activity presented a significant sustained increase from d0 to d14 (p < 0.001). The succinate (p = 0.006) and oxaloacetate (p = 0.003) content on d4 was lower than that on d0. The succinate dehydrogenase activity (p = 0.008) and ATP (p = 0.016) production decreased significantly on d4 compared to that on d0. Taken together, these findings reveal the dynamic changes of metabolites and enzymes related to hepatic glycometabolism and the TCA (tricarboxylic acid) cycle in piglets after weaning. Our findings enrich weaning stress theory and might provide a reference for dietary intervention. [ABSTRACT FROM AUTHOR]

The ischemia–reperfusion process of a donor heart during heart transplantation leads to severe mitochondrial dysfunction, which may be the main cause of donor heart dysfunction after heart transplantation. Pyruvate carboxylase (PC), an enzyme found in mitochondria, is said to play a role in the control of oxidative stress and the function of mitochondria. This research examined the function of PC and discovered the signaling pathways controlled by PC in myocardial IRI. We induced IRI using a murine heterotopic heart transplantation model in vivo and a hypoxia–reoxygenation cell model in vitro and evaluated inflammatory responses, oxidative stress levels, mitochondrial function, and cardiomyocyte apoptosis. In both in vivo and in vitro settings, we observed a significant decrease in PC expression during myocardial IRI. PC knockdown aggravated IRI by increasing MDA content, LDH activity, TUNEL-positive cells, serum cTnI level, Bax protein expression, and the level of inflammatory cytokines and decreasing SOD activity, GPX activity, and Bcl-2 protein expression. PC overexpression yielded the opposite findings. Additional research indicated that reducing PC levels could block the Wnt/β-catenin pathway and glutamine metabolism by hindering the movement of β-catenin to the nucleus and reducing the activity of complex I and complex II, as well as ATP levels, while elevating the ratios of NADP+/NADPH and GSSG/GSH. Overall, the findings indicated that PC therapy can shield the heart from IRI during heart transplantation by regulating glutamine metabolism through the Wnt/β-catenin pathway.

The biosynthesis of succinic acid from glucose by the previously engineered E. coli strain SUC1.0 (pMW119-kgd) (MG1655 ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆ptsG, PLglk, PtacgalP, ∆aceBAK, ∆glcB, ∆sdhAB, pMW119-kgd) was optimized. The yield of the target substance was increased, upon the activation in the strain of the tricarboxylic acid cycle variant mediated by the action of heterologous 2-ketoglutarate decarboxylase, due to the intensification of the anaplerotic formation of oxaloacetic acid. Inactivation of the nonspecific thioesterase YciA in the strain did not considerably change the biosynthetic characteristics of the producer. The enhancement of the expression of native phosphoenolpyruvate carboxylase led to an increase in the yield of the target compound by the recombinant synthesizing succinic acid via the reactions of the native tricarboxylic acid cycle from 25 to 42%, and from 67 to 75% upon the induced expression of Mycobacterium tuberculosis 2-ketoglutarate decarboxylase. The expression of the Bacillus subtilis pyruvate carboxylase gene in the strain resulted in an increase in the yield of succinic acid up to 84%. While functioning in whole-cell biocatalyst mode, the engineered strain SUC1.0 PL-pycA (pMW119-kgd) demonstrated a substrate-to-target product conversion ratio reaching 93%, approaching the corresponding theoretical maximum. [ABSTRACT FROM AUTHOR]

Jinhua lean ham (LH), a dry-cured ham made from the defatted hind legs of pigs, has become increasingly popular among consumers with health concerns. However, the influence of fat removal on the quality of Jinhua ham is still not fully understood. Therefore, a label-free proteomics strategy was used to explore the protein differential profile between Jinhua fatty ham (FH) and lean ham (LH). Results showed that 179 differential proteins (DPs) were detected, including 82 up-regulated and 97 down-regulated DPs in LH vs. FH, among which actin, myosin, tropomyosin, aspartate aminotransferase, pyruvate carboxylase, and glucose-6-phosphate isomerase were considered the key DPs. GO analysis suggested that DPs were mainly involved in binding, catalytic activity, cellular process, and metabolic process, among which catalytic activity was significantly up-regulated in LH. Moreover, the main KEGG-enriched pathways of FH focused on glycogen metabolism, mainly including the TCA cycle, pyruvate metabolism, and glycolysis/gluconeogenesis. However, amino acid metabolism and oxidative phosphorylation were the main metabolic pathways in LH. From the protein differentiation perspective, fat removal significantly promoted protein degradation, amino acid metabolism, and the oxidative phosphorylation process. These findings could help us to understand the effects of fat removal on the nutritional metabolism of Jinhua hams and provide theoretical supports for developing healthier low-fat meat products. [ABSTRACT FROM AUTHOR]

Carbon-concentrating mechanisms enhance the carboxylase efficiency of Rubisco by providing supra-atmospheric concentrations of CO2 in its surroundings. Beside the C4 photosynthesis pathway, carbon concentration can also be achieved by the photorespiratory glycine shuttle which requires fewer and less complex modifications. Plants displaying CO2 compensation points between 10 ppm and 40 ppm are often considered to utilize such a photorespiratory shuttle and are termed 'C3–C4 intermediates'. In the present study, we perform a physiological, biochemical, and anatomical survey of a large number of Brassicaceae species to better understand the C3–C4 intermediate phenotype, including its basic components and its plasticity. Our phylogenetic analysis suggested that C3–C4 metabolism evolved up to five times independently in the Brassicaceae. The efficiency of the pathway showed considerable variation. Centripetal accumulation of organelles in the bundle sheath was consistently observed in all C3–C4-classified taxa, indicating a crucial role for anatomical features in CO2-concentrating pathways. Leaf metabolite patterns were strongly influenced by the individual species, but accumulation of photorespiratory shuttle metabolites glycine and serine was generally observed. Analysis of phospho enol pyruvate carboxylase activities suggested that C4-like shuttles have not evolved in the investigated Brassicaceae. Convergent evolution of the photorespiratory shuttle indicates that it represents a distinct photosynthesis type that is beneficial in some environments. [ABSTRACT FROM AUTHOR]

Simple Summary: This study discusses the impacts of postruminally infusing palm oil or flaxseed oil in conjunction with propionate or acetate on blood metabolites and hepatic pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PCK1) expression. The flaxseed infusion changed plasma C18:3n-3 cis but did not affect PC or PCK1 expression. The palm oil infusion did not affect plasma metabolites or PC or PCK1 expression. Palmitic (C16:0), α-linolenic acid (C18:3n-3 cis), and propionate regulate bovine pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PCK1) expression in vitro. The objective of this experiment was to determine the impact of C16:0, C18:3n-3 cis, propionate, and acetate postruminal infusions on hepatic PC and PCK1 expression. We hypothesized that circulating fatty acids alter hepatic PC and PCK1 in lactating dairy cows. Acetate, propionate, palm oil, and flaxseed oil were supplied postruminally to lactating cows (n = 4) using two 4 × 4 Latin square studies. For Experiment 1, cows were infused on an hourly basis with either a bolus of propionate, acetate, or the combination of propionate and palm oil, or acetate and palm oil, and Experiment 2 was similar, but flaxseed oil replaced palm oil. Flaxseed infusions increased plasma concentration and the molar percent of C18:3n-3 cis and decreased C16:0 but did not affect PC or PCK1 expression. Palm infusions did not affect blood metabolites or the hepatic expression of PC or PCK1. The lack of responses to short-chain fatty acid infusions and changes in circulating long-chain fatty acids in mature cattle are not suitable models to study the effects of α-linolenic acid and propionate on bovine PC and PCK1 expression previously observed in vitro. [ABSTRACT FROM AUTHOR]

Renal metabolism is essential for kidney functions and energy homeostasis in the body. The TCA cycle is the hub of metabolism, but the metabolic activities of the cycle in the kidney have rarely been investigated. This study is to assess metabolic processes at the level of the TCA cycle in the kidney based on isotopomer distributions in multiple metabolites. Isolated rat kidneys were perfused with media containing common substrates including lactate and alanine for an hour. One group of kidneys received [U‐13C3]lactate instead of natural abundance lactate while the other group received [U‐13C3]alanine instead of natural abundance alanine. Perfused kidneys and effluent were prepared for analysis using NMR spectroscopy. 13C‐labeling patterns in glutamate, fumarate, aspartate and succinate from the kidney extracts showed that pyruvate carboxylase and oxidative metabolism through the TCA cycle were comparably very active, but pyruvate cycling and pyruvate dehydrogenase were relatively less active. Isotopomer analyses with fumarate and malate from effluent, however, indicated that pyruvate carboxylase was much more active than the TCA cycle and other metabolic processes. The reverse equilibrium of oxaloacetate with four‐carbon intermediates of the cycle was nearly complete (92%), based on the ratio of [2,3,4‐13C3]/[1,2,3‐13C3] in aspartate or malate. 13C enrichment in glucose with 13C‐lactate supply was higher than that with 13C‐alanine. Isotopomer analyses with multiple metabolites (i.e., glutamate, fumarate, aspartate, succinate and malate) allowed us to assess relative metabolic processes in the TCA cycle in the kidney supplied with [U‐13C3]lactate. Data from the analytes were generally consistent, indicating highly active pyruvate carboxylase and oxidative metabolism through the TCA cycle. Different 13C‐labeling patterns in analytes from the kidney extracts versus effluent suggested metabolic compartmentalization. [ABSTRACT FROM AUTHOR]

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.

Background and aims The introduction of crassulacean acid metabolism (CAM) into C3 crops has been considered as a means of improving water-use efficiency. In this study, we investigated photosynthetic and leaf structural traits in F 1 hybrids between Cymbidium ensifolium (female C3 parent) and C. bicolor subsp. pubescens (male CAM parent) of the Orchidaceae. Methods Seven F 1 hybrids produced through artificial pollination and in vitro culture were grown in a greenhouse with the parent plants. Structural, biochemical and physiological traits involved in CAM in their leaves were investigated. Key results Cymbidium ensifolium accumulated very low levels of malate without diel fluctuation, whereas C. bicolor subsp. pubescens showed nocturnal accumulation and diurnal consumption of malate. The F 1s also accumulated malate at night, but much less than C. bicolor subsp. pubescens. This feature was consistent with low nocturnal fixation of atmospheric CO2 in the F 1s. The δ13C values of the F 1s were intermediate between those of the parents. Leaf thickness was thicker in C. bicolor subsp. pubescens than in C. ensifolium , and those of the F 1s were more similar to that of C. ensifolium. This was due to the difference in mesophyll cell size. The chloroplast coverage of mesophyll cell perimeter adjacent to intercellular air spaces of C. bicolor subsp. pubescens was lower than that of C. ensifolium , and that of the F 1s was intermediate between them. Interestingly, one F 1 had structural and physiological traits more similar to those of C. bicolor subsp. pubescens than the other F 1s. Nevertheless, all F 1s contained intermediate levels of phosphoenolpyruvate carboxylase but as much pyruvate, Pi dikinase as C. bicolor subsp. pubescens. Conclusions CAM traits were intricately inherited in the F 1 hybrids, the level of CAM expression varied widely among F 1 plants, and the CAM traits examined were not necessarily co-ordinately transmitted to the F 1s. [ABSTRACT FROM AUTHOR]

Deregulated oxidative metabolism is a hallmark of leukaemia. While tyrosine kinase inhibitors (TKIs) such as imatinib have increased survival of chronic myeloid leukaemia (CML) patients, they fail to eradicate disease-initiating leukemic stem cells (LSCs). Whether TKI-treated CML LSCs remain metabolically deregulated is unknown. Using clinically and physiologically relevant assays, we generate multi-omics datasets that offer unique insight into metabolic adaptation and nutrient fate in patient-derived CML LSCs. We demonstrate that LSCs have increased pyruvate anaplerosis, mediated by increased mitochondrial pyruvate carrier 1/2 (MPC1/2) levels and pyruvate carboxylase (PC) activity, in comparison to normal counterparts. While imatinib reverses BCR::ABL1-mediated LSC metabolic reprogramming, stable isotope-assisted metabolomics reveals that deregulated pyruvate anaplerosis is not affected by imatinib. Encouragingly, genetic ablation of pyruvate anaplerosis sensitises CML cells to imatinib. Finally, we demonstrate that MSDC-0160, a clinical orally-available MPC1/2 inhibitor, inhibits pyruvate anaplerosis and targets imatinib-resistant CML LSCs in robust pre-clinical CML models. Collectively these results highlight pyruvate anaplerosis as a persistent and therapeutically targetable vulnerability in imatinib-treated CML patient-derived samples. The persistence of leukemic stem cells (LSCs) is known to limit the success of imatinib in patients with chronic myeloid leukaemia (CML). Here, the authors identify a reliance of these persisting LSCs on pyruvate carboxylase mediated pyruvate anaplerosis for survival after imatinib and demonstrate the therapeutic efficacy of targeting this using an inhibitor of mitochondrial pyruvate carrier. [ABSTRACT FROM AUTHOR]

Aim: The purpose of this study was to investigate the function and mechanism of a novel peptide derived from adipose‐derived stem cell‐conditioned medium (ADSC‐CM). Methods: Mass spectrometry was applied to identify expressed peptides in ADSC‐CM obtained at different time points. The cell counting kit‐8 assay and quantitative reverse transcription polymerase chain reactions were performed to screen the functional peptides contained within ADSC‐CM. RNA‐seq, western blot, a back skin excisional model of BALB/c mice, the peptide pull‐down assay, rescue experiments, untargeted metabolomics, and mixOmics analysis were performed to thoroughly understand the functional mechanism of selected peptide. Results: A total of 93, 827, 1108, and 631 peptides were identified in ADSC‐CM at 0, 24, 48, and 72 h of conditioning, respectively. A peptide named ADSCP2 (DENREKVNDQAKL) derived from ADSC‐CM inhibited collagen and ACTA2 mRNAs in hypertrophic scar fibroblasts. Moreover, ADSCP2 facilitated wound healing and attenuated collagen deposition in a mouse model. ADSCP2 bound with the pyruvate carboxylase (PC) protein and inhibited PC protein expression. Overexpressing PC rescued the reduction in collagen and ACTA2 mRNAs caused by ADSCP2. Untargeted metabolomics identified 258 and 447 differential metabolites in the negative and positive mode, respectively, in the ADSCP2‐treated group. The mixOmics analysis, which integrated RNA‐seq and untargeted metabolomics data, provided a more holistic view of the functions of ADSCP2. Conclusion: Overall, a novel peptide derived from ADSC‐CM, named ADSCP2, attenuated hypertrophic scar fibrosis in vitro and in vivo, and the novel peptide ADSCP2 might be a promising drug candidate for clinical scar therapy. [ABSTRACT FROM AUTHOR]

Clonostachys rosea is an important mycoparasitism biocontrol agent that exhibits excellent control efficacy against numerous fungal plant pathogens. Transcriptomic sequencing may be used to preliminarily screen mycoparasitism-related genes of C. rosea against fungal pathogens. The present study sequenced and analyzed the transcriptome of C. rosea mycoparasitizing a Basidiomycota (phylum) fungal pathogen, Rhizoctonia solani, under three touch stages: the pre-touch stage, touch stage and after-touch stage. The results showed that a number of genes were differentially expressed during C. rosea mycoparasitization of R. solani. At the pre-touch stage, 154 and 315 genes were up- and down-regulated, respectively. At the touch stage, the numbers of up- and down-regulated differentially expressed genes (DEGs) were 163 and 188, respectively. The after-touch stage obtained the highest number of DEGs, with 412 and 326 DEGs being up- and down-regulated, respectively. Among these DEGs, ABC transporter-, glucanase- and chitinase-encoding genes were selected as potential mycoparasitic genes according to a phylogenetic analysis. A comparative transcriptomic analysis between C. rosea mycoparasitizing R. solani and Sclerotinia sclerotiorum showed that several DEGs, including the tartrate transporter, SDR family oxidoreductase, metallophosphoesterase, gluconate 5-dehydrogenase and pyruvate carboxylase, were uniquely expressed in C. rosea mycoparasitizing R. solani. These results significantly expand our knowledge of mycoparasitism-related genes in C. rosea and elucidate the mycoparasitism mechanism of C. rosea. [ABSTRACT FROM AUTHOR]

Clostridium ljungdahlii is a representative autotrophic acetogen capable of producing multiple chemicals from one-carbon gases (CO2/CO). The metabolic and regulatory networks of this carbon-fixing bacterium are interesting, but still remain minimally explored. Here, based on bioinformatics analysis followed by functional screening, we identified a RpiR family transcription factor (TF) that can regulate the autotrophic growth and carbon fixation of C. ljungdahlii. After deletion of the corresponding gene, the resulting mutant strain exhibited significantly impaired growth in gas fermentation, thus reducing the production of acetic acid and ethanol. In contrast, the overexpression of this TF gene could promote cell growth, indicating a positive regulatory effect of this TF in C. ljungdahlii. Thus, we named the TF as GssR (growth and solvent synthesis regulator). Through the following comparative transcriptomic analysis and biochemical verification, we discovered three important genes (encoding pyruvate carboxylase, carbon hunger protein CstA, and a BlaI family transcription factor) that were directly regulated by GssR. Furthermore, an upstream regulator, BirA, that could directly bind to gssR was found; thus, these two regulators may form a cascade regulation and jointly affect the physiology and metabolism of C. ljungdahlii. These findings substantively expand our understanding on the metabolic regulation of carbon fixation in gas-fermenting Clostridium species. [ABSTRACT FROM AUTHOR]

Background: Pyruvate carboxylase (PC) is a key enzyme for gluconeogenesis. PC deficiency (PCD) is an extremely rare autosomal recessive metabolic disease and is divided into three types. Type B PCD is clinically featured by lactic acidosis, hyperammonemia, hypercitrullinemia, hypotonia, abnormal movement, and seizures. Case presentation: Here, we report the first case of type B PCD in China, presenting with intractable lactic acidosis shortly after birth. A compound heterozygous mutation in the PC gene was identified by whole-exome sequencing, NM_001040716.2: c.1154_1155del and c.152G>A, which were inherited from her asymptomatic parents, respectively. Furthermore, prenatal neuroradiological presentations including widened posterior horns of lateral ventricles, huge subependymal cysts, and increased biparietal diameter and head circumference were concerned. Symptomatic treatment was taken and the infant died at 26 days. Conclusion: To our knowledge, this is the minimum gestational age (22w5d) that's when the prenatal onset of the neuroradiologic phenotype of PCD was observed. PCD has a poor prognosis and lacks an effective treatment, so this paper is shared to highlight the importance of PCD prenatal diagnosis in the absence of family history. [ABSTRACT FROM AUTHOR]

Aspergillus nidulans has been more extensively characterized than other Aspergillus species considering its morphology, physiology, metabolic pathways, and genetic regulation. As it has a rapid growth rate accompanied by simple nutritional requirements and a high tolerance to extreme cultural conditions, A. nidulans is a promising microbial cell factory to biosynthesize various products in industry. However, it remains unclear for whether it is also a suitable host for synthesizing abundant L-malic acid. In this study, we developed a convenient and efficient double-gene-editing system in A. nidulans strain TN02A7 based on the CRISPR–Cas9 and Cre-loxP systems. Using this gene-editing system, we made a L-malic acid-producing strain, ZQ07, derived from TN02A7, by deleting or overexpressing five genes (encoding Pyc, pyruvate carboxylase; OahA, oxaloacetate acetylhydrolase; MdhC, malate dehydrogenase; DctA, C4-dicarboxylic acid transporter; and CexA, citric acid transporter). The L-malic acid yield in ZQ07 increased to approximately 9.6 times higher (up to 30.7 g/L titer) than that of the original unedited strain TN02A7, in which the production of L-malic acid was originally very low. The findings in this study not only demonstrate that A. nidulans could be used as a potential host for biosynthesizing organic acids, but also provide a highly efficient gene-editing strategy in filamentous fungi. [ABSTRACT FROM AUTHOR]

Pyruvate carboxylase (PC) is an enzyme catalyzing the carboxylation of pyruvate to oxaloacetate. The enzymatic generation of oxaloacetate, an intermediate of the Krebs cycle, could provide the cancer cells with the additional anaplerotic capacity and promote their anabolic metabolism. Recent studies revealed that several types of cancer cells express PC. The gained anaplerotic capability of cells mediated by PC correlates with their expedited growth, higher aggressiveness, and increased metastatic potential. By immunohistochemical staining and immunoblotting analysis, we investigated PC expression among samples of different types of human brain tumors. Our results show that PC is expressed by the cells in glioblastoma, astrocytoma, oligodendroglioma, and meningioma tumors. The presence of PC in these tumors suppose that PC could support the anabolic metabolism of their cellular constituents by its anaplerotic capability. [ABSTRACT FROM AUTHOR]

Pyruvate carboxylase (PC) is a mitochondrial, biotin-containing enzyme catalyzing the ATP-dependent synthesis of oxaloacetate from pyruvate and bicarbonate, with a critical anaplerotic role in sustaining the brain metabolism. Based on the studies performed on animal models, PC expression was assigned to be glia-specific. To study PC distribution among human neural cells, we probed the cultured human astrocytes and brain sections with antibodies against PC. Additionally, we tested the importance of PC for the viability of cultured human astrocytes by applying the PC inhibitor 3-chloropropane-1,2-diol (CPD). Our results establish the expression of PC in mitochondria of human astrocytes in culture and brain tissue and also into a subpopulation of the neurons in situ. CPD negatively affected the viability of astrocytes in culture, which could be partially reversed by supplementing media with malate, 2-oxoglutarate, citrate, or pyruvate. The provided data estimates PC expression in human astrocytes and neurons in human brain parenchyma. Furthermore, the enzymatic activity of PC is vital for sustaining the viability of cultured astrocytes. [ABSTRACT FROM AUTHOR]

Glyoxylates are essential intermediates in several metabolic pathways and have a broad range of industrial applications. In this study, we propose a novel method for producing glyoxylate from glucose using engineered Escherichia coli BW25113. To direct the production of glyoxylate from glucose, malate synthase A (aceB), malate synthase G (glcB), glyoxylate carboligase (gcl), and glyoxylate/hydroxypyruvate reductase A (ycdW) genes were disrupted, and the glyoxylate shunt was reinforced in the disruptants by the overexpression of citrate synthase (gltA) and isocitrate lyase (aceA). In flask cultivation using M9 medium supplemented with 1% glucose, the disruptant E. coli BW25113 ΔaceB ΔglcB Δgcl ΔycdW produced 0.93 ± 0.17 g/L of glyoxylate. Further overexpression of gltA and aceA in the disruptant resulted in an improvement in glyoxylate production to 1.15 ± 0.02 g/L. By expressing a heterologous gene, pyc, in the engineered E. coli, the accumulation of intracellular oxaloacetate remarkably improved, leading to glyoxylate production of up to 2.42 ± 0.00 g/L with specific productivity at 4.22 ± 0.09 g/g-cell. To date, this is the highest reported titer and specific productivity of glyoxylate in E. coli. [ABSTRACT FROM AUTHOR]

Background: Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. Results: In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol−1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol−1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol−1 sugars and resulted in a final titer of 36.0 g/L 3-HP. Conclusions: The results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane. [ABSTRACT FROM AUTHOR]

Metformin is the most prescribed drug for DM2, but its site and mechanism of action are still not well established. Here, we investigated the effects of metformin on basolateral intestinal glucose uptake (BIGU), and its consequences on hepatic glucose production (HGP). In diabetic patients and mice, the primary site of metformin action was the gut, increasing BIGU, evaluated through PET-CT. In mice and CaCo2 cells, this increase in BIGU resulted from an increase in GLUT1 and GLUT2, secondary to ATF4 and AMPK. In hyperglycemia, metformin increased the lactate (reducing pH and bicarbonate in portal vein) and acetate production in the gut, modulating liver pyruvate carboxylase, MPC1/2, and FBP1, establishing a gut-liver crosstalk that reduces HGP. In normoglycemia, metformin- induced increases in BIGU is accompanied by hypoglycemia in the portal vein, generating a counter-regulatory mechanism that avoids reductions or even increases HGP. In summary, metformin increases BIGU and through gut-liver crosstalk influences HGP. [ABSTRACT FROM AUTHOR]

Pancreatic-β-cell-specifying transcription factor Nkx6.1, indispensable for embryonic development of the pancreatic epithelium and commitment to β-cell lineage, directly controls the expression of a glucose transporter (Glut2), pyruvate carboxylase (Pcx), and genes for insulin processing (endoplasmic reticulum oxidoreductase-1β, Ero1lb; zinc transporter-8, Slc30a8). The Nkx6.1 decline in aging diabetic Goto-Kakizaki rats contributes to β-cell trans-differentiation into δ-cells. Elucidating further Nkx6.1 roles, we studied Nkx6.1 ablation in rat INS-1E cells, prepared by CRISPR/Cas9 gene editing from single colonies. INS-1ENkx6.1–/– cells exhibited unchanged glucose-stimulated insulin secretion (GSIS), moderately decreased phosphorylating/non-phosphorylating respiration ratios at high glucose; unchanged but delayed ATP-elevation responses to glucose; delayed uptake of fluorescent glucose analog, but slightly improved cytosolic Ca2+-oscillations, induced by glucose; despite approximately halved Glut2, Pcx, Ero1lb, and Slc30a8 expression, and reduced nuclear receptors Nr4a1 and Nr4a3. Thus, ATP synthesis was time-compensated, despite the delayed GLUT2-mediated glucose uptake and crippled pyruvate-malate redox shuttle (owing to the PCX-deficiency) in INS-1ENkx6.1–/– cells. Nkx6.1 thus controls the expression of genes that are not essential for acute insulin secretion, the function of which can be compensated for. Considerations that Nkx6.1 deficiency is an ultimate determinant of β-cell pathology beyond cell trans-(de-)differentiation or β-cell identity are not supported by our results. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase V (CA V), a mitochondrial enzyme, was first isolated from guinea‐pig liver and subsequently identified in mice and humans. Later, studies revealed that the mouse genome contains two mitochondrial CA sequences, named Car5A and Car5B. The CA VA enzyme is most highly expressed in the liver, whereas CA VB shows a broad tissue distribution. Car5A knockout mice demonstrated a predominant role for CA VA in ammonia detoxification, whereas the roles of CA VB in ureagenesis and gluconeogenesis were evident only in the absence of CA VA. Previous studies have suggested that CA VA is mainly involved in the provision of HCO3− for biosynthetic processes. In children, mutations in the CA5A gene led to reduced CA activity, and the enzyme was sensitive to increased temperature. The metabolic profiles of these children showed a reduced supply of HCO3− to the enzymes that take part in intermediary metabolism: carbamoylphosphate synthetase, pyruvate carboxylase, propionyl‐CoA carboxylase and 3‐methylcrotonyl‐CoA carboxylase. Although the role of CA VB is still poorly understood, a recent study reported that it plays an essential role in human Sertoli cells, which sustain spermatogenesis. Metabolic disease associated with CA VA appears to be more common than other inborn errors of metabolism and responds well to treatment with N‐carbamyl‐l‐glutamate. Therefore, early identification of hyperammonaemia will allow specific treatment with N‐carbamyl‐l‐glutamate and prevent neurological sequelae. Carbonic anhydrase VA deficiency should therefore be considered a treatable condition in the differential diagnosis of hyperammonaemia in neonates and young children. [ABSTRACT FROM AUTHOR]

[Display omitted] • Arabidopsis over-expressing Tappc3A , a plant-type phospho enol pyruvate carboxylase from common wheat, exhibited irregular stamen formation. • Tappc3 isogenes were highly co-expressed with wheat SEEDSTICKs. • Promoter sequences of Tappc3 isogenes maintain two CArG boxes. We examined whether plant-type phospho enol pyruvate carboxylase (PEPC) is involved in flower organ formation or not by over-expression in Arabidopsis. A wheat PEPC isogene Tappc3A , belonging to the ppc3 group, was targeted due to its preferential expression pattern in pistils and stamens. Transgenic Arabidopsis over-expressing Tappc3A exhibited irregular stamen formation, i.e., a lesser number of stamens per flower and shorter filaments in T 2 and T 3 generations. Irregular stamens were frequently observed in homozygous T 4 lines, but no morphological change was observed in other floral organs. High-degree gene co-expression of Tappc3 isogenes with wheat SEEDSTICKs but not with other homeotic transcription factor genes for flower formation implicates that Tappc3 is under control by the class D genes of the ABCDE model to flower development. In addition, the conservation of CArG box sequences on the Tappc3 promoters supported the developmentally programmed gene expression of ppc3 in wheat flowering organs. Thus, this study provides the first experimental evidence for the critical regulation of plant-type PEPC for flower formation. [ABSTRACT FROM AUTHOR]

Although the shrimp Penaeus vannamei can tolerate oxygen-limited conditions, the organisms may suffer detrimental effects in several processes, including growth, reproduction, immune responses, and others. During hypoxia, the shrimp makes metabolic adjustments in energy use and glucose homeostasis. Glucose is hydrolyzed during glycolysis and synthesized from non-glycosidic metabolites in gluconeogenesis. Although these pathways share many enzymes, the processes are not exactly the reverse of each other. We studied the first and last irreversible steps of glycolysis and gluconeogenesis catalyzed by hexokinase/glucose-6-phosphatase (HK/G6Pase) and pyruvate kinase/pyruvate carboxylase/phosphoenolpyruvate carboxykinase (PK/PC/PEPCK), respectively, in muscle under hypoxia and hypoxia followed by reoxygenation to gain insights about the coordinated metabolic responses that maintain glucose balance during oxygen stress. For the first group, HK gene expression was induced in hypoxia while G6Pase was reduced in hypoxia and reoxygenation, whereas for enzymes activity, HK was maintained and G6Pase was only detected in reoxygenation. For the second group, gene expression increased in PK in hypoxia, in PC in reoxygenation and in both cases, the enzymatic activities were maintained. Mitochondrial PEPCK expression increased during hypoxia and cytosolic PEPCK decreased, while total PEPCK activity was maintained. Intracellular lactate decreased during hypoxia while glucose levels were maintained. Altogether the results show that in limited oxygen, the expression of glycolytic enzymes-genes are induced, the gluconeogenic counterparts are active, although with more varied responses to oxygen limited conditions, and intracellular glucose levels are maintained. • Glycolysis and gluconeogenesis genes/enzymes are active in oxygen limited conditions. • Hypoxia induces HK and PK expression. • Reoxygenation reduces G6Pase1 expression compared to normoxia. • PC and PEPCK expressions are varied in different oxygen conditions. • Intracellular glucose levels are maintained under oxygen limited conditions. [ABSTRACT FROM AUTHOR]

The metabolic fluxes throughout the tricarboxylic acid cycle (TCAC) are inhibited in the light by the mitochondrial thioredoxin (TRX) system. However, it is unclear how this system orchestrates the fluxes throughout the TCAC and associated pathways in the dark. Here we carried out a13C–HCO 3 labelling experiment in Arabidopsis leaves from wild type (WT) and mutants lacking TRX o1 (trxo1), TRX h2 (trxh2), or both NADPH-dependent TRX reductase A and B (ntra ntrb) exposed to 0, 30 and 60 min of dark or light conditions. No 13C-enrichment in TCAC metabolites in illuminated WT leaves was observed. However, increased succinate content was found in parallel to reductions in Ala in the light, suggesting the latter operates as an alternative carbon source for succinate synthesis. By contrast to WT, all mutants showed substantial changes in the content and 13C-enrichment in TCAC metabolites under both dark and light conditions. Increased 13C-enrichment in glutamine in illuminated trxo1 leaves was also observed, strengthening the idea that TRX o1 restricts in vivo carbon fluxes from glycolysis and the TCAC to glutamine. We further demonstrated that both photosynthetic and gluconeogenic fluxes toward glucose are increased in trxo1 and that the phospho enol pyruvate carboxylase (PEPc)-mediated 13C-incorporation into malate is higher in trxh2 mutants, as compared to WT. Our results collectively provide evidence that TRX h2 and the mitochondrial NTR/TRX system regulate the metabolic fluxes throughout the TCAC and associated pathways, including glycolysis, gluconeogenesis and the synthesis of glutamine in a light-independent manner. • No 13C-enrichment in TCA cycle metabolites was observed in illuminated WT leaves fed with 13C–HCO 3. • The lack of TRX o1 increases the metabolic fluxes throughout the TCA cycle in a light-independent manner. • TRX o1 restricts in vivo carbon fluxes from glycolysis and the TCA cycle to glutamine. • Both photosynthetic and gluconeogenic fluxes toward glucose are increased in the trxo1 mutant, as compared to WT. • The PEPc-mediated 13C-incorporation into malate is higher in the trxh2 mutant, as compared to WT. [ABSTRACT FROM AUTHOR]

Aloidendron dichotomum appears to be undergoing the early stages of a range shift in response to anthropogenic climate change in south-western Africa. High mortality has been recorded in warmer populations, while population expansions have been recorded in cooler poleward parts of its range. This study aimed to determine the key environmental controls on A. dichotomum photosynthesis in areas of population expansion, to inform the potential attribution of directional population expansion to anthropogenic warming. Nocturnal acid accumulation and CO2 assimilation were measured in individuals growing under a range of temperature and watering treatments in a greenhouse experiment. In addition, nocturnal acid accumulation and phospho enol pyruvate carboxylase activity were quantified in two wild populations at the most southerly and south-easterly range extents. Multiple lines of evidence confirmed that A. dichotomum performs Crassulacean acid metabolism. Total nocturnal acid accumulation was highest at night-time temperatures of ~21.5 °C, regardless of soil water availability, and night-time CO2 assimilation rates increased with leaf temperature, suggesting a causal link to the cool southern range limit. Leaf acidity at the start of the dark period was highly predictive of nocturnal acid accumulation in all individuals, implicating light availability during the day as an important determinant of nocturnal acid accumulation. [ABSTRACT FROM AUTHOR]

High temperature inhibits the growth and development of maize (Zea mays L.) and wheat (Triticum aestivum L.) seedlings. The current study attempts to elucidate the underlying mechanisms of exogenous trehalose for the improvement of maize and wheat responses to heat stress. Results show that, in maize seedlings, exogenously-supplied trehalose was able to effectively elevate the capacity of photosynthetic carbon assimilation and improve the carbohydrate content via up-regulating the transcript levels of key enzymes including phosphoenol pyruvate carboxylase (PEPC), NADP-malate dehydrogenase (NADP-MDH), NADP-malic enzyme (NADP-ME), pyruvate phosphate dikinase (PPDK) and Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit. Besides, it was also able to improve C4 pathway enzyme activities, including PEPC, NADP-MDH, NADP-ME and Ribulose-1,5-bisphosphate carboxylase (RUBPCase). In wheat seedlings, exogenously-supplied trehalose promoted photosynthetic carbon assimilation by elevating the activity of RUBPCase. These results show that exogenous trehalose improved photosynthetic carbon assimilation capacities in maize and wheat seedlings under heat stress through different mechanisms. [ABSTRACT FROM AUTHOR]

Pyruvate has two major fates upon entry into mitochondria, the oxidative decarboxylation to acetyl-CoA via the pyruvate decarboxylase complex or the biotin-dependent carboxylation to oxaloacetate via pyruvate carboxylase (Pcx). Here, we have generated mice with a liver-specific KO of pyruvate carboxylase (PcxL-/-) to understand the role of Pcx in hepatic mitochondrial metabolism under disparate physiological states. PCXL-/- mice exhibited a deficit in hepatic gluconeogenesis and enhanced ketogenesis as expected but were able to maintain systemic euglycemia following a 24 h fast. Feeding a high-fat diet to PcxL-/- mice resulted in animals that were resistant to glucose intolerance without affecting body weight. However, we found that PcxL-/- mice fed a ketogenic diet for 1 week became severely hypoglycemic, demonstrating a requirement for hepatic Pcx for long-term glycemia under carbohydrate-limited diets. Additionally, we determined that loss of Pcx was associated with an induction in the abundance of lysine-acetylated proteins in PCXL-/- mice regardless of physiologic state. Furthermore, liver acetyl-proteomics revealed a biased induction in mitochondrial lysine-acetylated proteins. These data show that Pcx is important for maintaining the proper balance of pyruvate metabolism between oxidative and anaplerotic pathways. [ABSTRACT FROM AUTHOR]

The HIF-1 and HIF-2 (HIF1/2) hypoxia responses are frequently upregulated in cancers, and HIF1/2 inhibitors are being developed as anticancer drugs. How could cancers resist anti-HIF1/2 therapy? We studied metabolic and molecular adaptations of HIF-1β-deficient Hepa-1c4, a hepatoma model lacking HIF1/2 signalling, which mimics a cancer treated by a totally effective anti-HIF1/2 agent. [1,2-13C2]-D-glucose metabolism was measured by SiDMAP metabolic profiling, gene expression by TaqMan, and metabolite concentrations by 1H MRS. HIF-1β-deficient Hepa-1c4 responded to hypoxia by increasing glucose uptake and lactate production. They showed higher glutamate, pyruvate dehydrogenase, citrate shuttle, and malonyl-CoA fluxes than normal Hepa-1 cells, whereas pyruvate carboxylase, TCA, and anaplerotic fluxes decreased. Hypoxic HIF-1β-deficient Hepa-1c4 cells increased expression of PGC-1α, phospho-p38 MAPK, and PPARα, suggesting AMPK pathway activation to survive hypoxia. They had higher intracellular acetate, and secreted more H2O2, suggesting increased peroxisomal fatty acid β-oxidation. Simultaneously increased fatty acid synthesis and degradation would have "wasted" ATP in Hepa-1c4 cells, thus raising the [AMP]:[ATP] ratio, and further contributing to the upregulation of the AMPK pathway. Since these tumour cells can proliferate without the HIF-1/2 pathways, combinations of HIF1/2 inhibitors with PGC-1α or AMPK inhibitors should be explored. [ABSTRACT FROM AUTHOR]