Biotin concentration affects anaplerotic reactions functioning in glutamic acid production in Corynebacterium glutamicum.

The study investigates the effect of biotin concentration on the role of anaplerotic reactions catalysed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) in glutamic acid production by Corynebacterium glutamicum. C. glutamicum requires biotin for its growth, and its glutamic acid production can be induced by the addition of Tween 40 or penicillin or by biotin limitation. The biotin enzyme PC and the non-biotin enzyme PEPC catalyse two anaplerotic reactions to supply oxaloacetic acid to the TCA cycle in C. glutamicum. Therefore, they are crucial for glutamic acid production in this bacterium. In this study, we investigated the contribution of each anaplerotic reaction to Tween 40- and penicillin-induced glutamic acid production using disruptants of PEPC and PC. In the presence of 20 µg l-1 biotin, which is sufficient for growth, the PEPC-catalysed anaplerotic reaction mainly contributed to Tween 40- and penicillin-induced glutamic acid production. However, when increasing biotin concentration 10-fold (i.e. 200 µg l-1), both PC- and PEPC-catalysed reactions could function in glutamic acid production. Western blotting revealed that the amount of biotin-bound PC was reduced by the addition of Tween 40 and penicillin in the presence of 20 µg l-1. However, these induction treatments did not change the amount of biotin-bound PC in the presence of 200 µg l-1 biotin. These results indicate that both anaplerotic reactions are functional during glutamic acid production in C. glutamicum and that biotin concentration mainly affects which anaplerotic reactions function during glutamic acid production.

Compensatory activity of the PC-ME1 metabolic axis underlies differential sensitivity to mitochondrial complex I inhibition.

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.

The mitochondrial enzyme pyruvate carboxylase restricts pancreatic ß-cell senescence by blocking p53 activation.

Defective glucose-stimulated insulin secretion (GSIS) and ß-cell senescence are hallmarks in diabetes. The mitochondrial enzyme pyruvate carboxylase (PC) has been shown to promote GSIS and ß-cell proliferation in the clonal ß-cell lines, yet its physiological relevance remains unknown. Here, we provide animal and human data showing a role of PC in protecting ß-cells against senescence and maintaining GSIS under different physiological and pathological conditions. ß-cell-specific deletion of PC impaired GSIS and induced ß-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 ß-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 ß-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 ß-cell senescence through the MDM2-p53 axis.

Circ-CAMTA1 regulated by Ca2+ influx inhibited pyruvate carboxylase activity and modulate T cell function in patients with systemic lupus erythematosus.

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 Ca²âº influx modulated T cell function in patients with SLE.

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.

The role of anaplerotic metabolism of glucose and glutamine in insulin secretion: A model approach.

We propose a detailed computational beta cell model that emphasizes the role of anaplerotic metabolism under glucose and glucose-glutamine stimulation. This model goes beyond the traditional focus on mitochondrial oxidative phosphorylation and ATP-sensitive K+ channels, highlighting the predominant generation of ATP from phosphoenolpyruvate in the vicinity of KATP channels. It also underlines the modulatory role of H2O2 as a signaling molecule in the first phase of glucose-stimulated insulin secretion. In the second phase, the model emphasizes the critical role of anaplerotic pathways, activated by glucose stimulation via pyruvate carboxylase and by glutamine via glutamate dehydrogenase. It particularly focuses on the production of NADPH and glutamate as key enhancers of insulin secretion. The predictions of the model are consistent with empirical data, highlighting the complex interplay of metabolic pathways and emphasizing the primary role of glucose and the facilitating role of glutamine in insulin secretion. By delineating these crucial metabolic pathways, the model provides valuable insights into potential therapeutic targets for diabetes.

Ex-Vivo 13C NMR Spectroscopy of Rodent Brain: TNF Restricts Neuronal Utilization of Astrocyte-Derived Metabolites.

Tumor necrosis factor (TNF) has well-established roles in neuroinflammatory disorders, but the effect of TNF on the biochemistry of brain cells remains poorly understood. Here, we microinjected TNF into the brain to study its impact on glial and neuronal metabolism (glycolysis, pentose phosphate pathway, citric acid cycle, pyruvate dehydrogenase, and pyruvate carboxylase pathways) using 13C NMR spectroscopy on brain extracts following intravenous [1,2-13C]-glucose (to probe glia and neuron metabolism), [2-13C]-acetate (probing astrocyte-specific metabolites), or [3-13C]-lactate. An increase in [4,5-13C]-glutamine and [2,3-13C]-lactate coupled with a decrease in [4,5-13C]-glutamate was observed in the [1,2-13C]-glucose-infused animals treated with TNF. As glutamine is produced from glutamate by astrocyte-specific glutamine synthetase the increase in [4,5-13C]-glutamine reflects increased production of glutamine by astrocytes. This was confirmed by infusion with astrocyte substrate [2-13C]-acetate. As lactate is metabolized in the brain to produce glutamate, the simultaneous increase in [2,3-13C]-lactate and decrease in [4,5-13C]-glutamate suggests decreased lactate utilization, which was confirmed using [3-13C]-lactate as a metabolic precursor. These results suggest that TNF rearranges the metabolic network, disrupting the energy supply chain perturbing the glutamine-glutamate shuttle between astrocytes and the neurons. These insights pave the way for developing astrocyte-targeted therapeutic strategies aimed at modulating effects of TNF to restore metabolic homeostasis in neuroinflammatory disorders.

Hypoxia-mediated repression of pyruvate carboxylase drives immunosuppression.

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.

Contributions of the anaplerotic reaction enzymes pyruvate carboxylase and phosphoenolpyruvate carboxylase to l-lysine production in Corynebacterium glutamicum.

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.

Metabolic profiling of glioblastoma stem cells reveals pyruvate carboxylase as a critical survival factor and potential therapeutic target.

BACKGROUND: Glioblastoma (GBM) is a highly aggressive tumor with unmet therapeutic needs, which can be explained by extensive intra-tumoral heterogeneity and plasticity. In this study, we aimed to investigate the specific metabolic features of Glioblastoma stem cells (GSC), a rare tumor subpopulation involved in tumor growth and therapy resistance. METHODS: We conducted comprehensive analyses of primary patient-derived GBM cultures and GSC-enriched cultures of human GBM cell lines using state-of-the-art molecular, metabolic, and phenotypic studies. RESULTS: We showed that GSC-enriched cultures display distinct glycolytic profiles compared with differentiated tumor cells. Further analysis revealed that GSC relies on pyruvate carboxylase (PC) activity for survival and self-renewal capacity. Interestingly, inhibition of PC led to GSC death, particularly when the glutamine pool was low, and increased differentiation. Finally, while GSC displayed resistance to the chemotherapy drug etoposide, genetic or pharmacological inhibition of PC restored etoposide sensitivity in GSC, both in vitro and in orthotopic murine models. CONCLUSIONS: Our findings demonstrate the critical role of PC in GSC metabolism, survival, and escape to etoposide. They also highlight PC as a therapeutic target to overcome therapy resistance in GBM.

Discovery of a New Anti-Inflammatory Agent from Anemoside B4 Derivatives and Its Therapeutic Effect on Colitis by Targeting Pyruvate Carboxylase.

Anemoside B4 (AB4), a triterpenoidal saponin from Pulsatilla chinensis, shows significant anti-inflammatory activity, and may be used for treating inflammatory bowel disease (IBD). Nevertheless, its application is limited due to its high molecular weight and pronounced water solubility. To discover new effective agents for treating IBD, we synthesized 28 AB4 derivatives and evaluated their cytotoxic and anti-inflammatory activities in vitro. Among them, A3-6 exhibited significantly superior anti-inflammatory activity compared to AB4. It showed a significant improvement in the symptoms of DSS-induced colitis in mice, with a notably lower oral effective dose compared to AB4. Furthermore, we discovered that A3-6 bound with pyruvate carboxylase (PC), then inhibited PC activity, reprogramming macrophage function, and alleviated colitis. These findings indicate that A3-6 is a promising therapeutic candidate for colitis, and PC may be a potential new target for treating colitis.

Inhibition of pyruvate dehydrogenase accelerates anaerobic glycolysis under postmortem simulating conditions.

This research aimed to explore the potential influence of mitochondria on the rate of anaerobic glycolysis. We hypothesized that mitochondria could reduce the rate of anaerobic glycolysis and pH decline by metabolizing a portion of glycolytic pyruvate. We utilized an in vitro model and incorporated CPI-613 and Avidin to inhibit pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), respectively. Four treatments were tested: 400 µM CPI-613, 1.5 U/ml Avidin, 400 µM CPI-613 + 1.5 U/ml Avidin, or control. Glycolytic metabolites and pH of the in vitro model were evaluated throughout a 1440-min incubation period. CPI-613-containing treatments, with or without Avidin, decreased pH levels and increased glycogen degradation and lactate accumulation compared to the control and Avidin treatments (P < 0.05), indicating increased glycolytic flux. In a different experiment, two treatments, 400 µM CPI-613 or control, were employed to track the fates of pyruvate using [13C6]glucose. CPI-613 reduced the contribution of glucose carbon to tricarboxylic acid cycle intermediates compared to control (P < 0.05). To test whether the acceleration of acidification in reactions containing CPI-613 was due to an increase in the activity of key enzymes of glycogenolysis and glycolysis, we evaluated the activities of glycogen phosphorylase, phosphofructokinase, and pyruvate kinase in the presence or absence of 400 µM CPI-613. The CPI-613 treatment did not elicit an alteration in the activity of these three enzymes. These findings indicate that inhibiting PDH increases the rate of anaerobic glycolysis and pH decline, suggesting that mitochondria are potential regulators of postmortem metabolism.

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.

Anemoside B4, a new pyruvate carboxylase inhibitor, alleviates colitis by reprogramming macrophage function.

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.

tRF-1:30-Gly-CCC-3 inhibits thyroid cancer via binding to PC and modulating metabolic reprogramming.

tRFs and tiRNAs (tRNA-derived fragments) are an emerging class of small noncoding RNAs produced by the precise shearing of tRNAs in response to specific stimuli. They have been reported to regulate the pathological processes of numerous human cancers. However, the biofunction of tRFs and tiRNAs in the development and progression of papillary thyroid cancer (PTC) has not been reported yet. In this study, we aimed to explore the biological roles of tRFs and tiRNAs in PTC and discovered that a novel 5'tRNA-derived fragment called tRF-1:30-Gly-CCC-3 (tRF-30) was markedly down-regulated in PTC tissues and cell lines. Functionally, tRF-30 inhibited the proliferation and invasion of PTC cells. Mechanistically, tRF-30 directly bound to the biotin-dependent enzyme pyruvate carboxylase (PC), downregulated its protein level, interfered with the TCA cycle intermediate anaplerosis, and thus affected metabolic reprogramming and PTC progression. These findings revealed a novel regulatory mechanism for tRFs and a potential therapeutic target for PTC.

In silico Analysis of Two Novel Variants in the Pyruvate Carboxylase (PC) Gene Associated with the Severe Form of PC Deficiency.

Background: Inborne errors of metabolism are a common cause of neonatal death. This study evaluated the acute early-onset metabolic derangement and death in two unrelated neonates. Methods: Whole-exome sequencing (WES), Sanger sequencing, homology modeling, and in silico bioinformatics analysis were employed to assess the effects of variants on protein structure and function. Results: WES revealed a novel homozygous variant, p.G303Afs*40 and p.R156P, in the pyruvate carboxylase (PC) gene of each neonate, which both were confirmed by Sanger sequencing. Based on the American College of Medical Genetics and Genomics guidelines, the p.G303Afs*40 was likely pathogenic, and the p.R156P was a variant of uncertain significance (VUS). Nevertheless, a known variant at position 156, the p.R156Q, was also a VUS. Protein secondary structure prediction showed changes in p.R156P and p.R156Q variants compared to the wild-type protein. However, p.G303Afs*40 depicted significant changes at C-terminal. Furthermore, comparing the interaction of wild-type and variant proteins with the ATP ligand during simulations, revealed a decreased affinity to the ATP in all the variants. Moreover, analysis of Single nucleotide polymorphism impacts on PC protein using Polyphen-2, SNAP2, FATHMM, and SNPs&GO servers predicted both R156P and R156Q as damaging variants. Likewise, free energy calculations demonstrated the destabilizing effect of both variants on PC. Conclusion: This study confirmed the pathogenicity of both variants and suggested them as a cause of type B Pyruvate carboxylase deficiency. The results of this study would provide the family with prenatal diagnosis and expand the variant spectrum in the PC gene,which is beneficial for geneticists and endocrinologists.

The presence of pyruvate carboxylase in the human brain and its role in the survival of cultured human astrocytes.

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.

Isotopomer analyses with the tricarboxylic acid cycle intermediates and exchanging metabolites from the rat kidney.

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-13 C3 ]lactate instead of natural abundance lactate while the other group received [U-13 C3 ]alanine instead of natural abundance alanine. Perfused kidneys and effluent were prepared for analysis using NMR spectroscopy. 13 C-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 [2,3,4-13 C3 ]/[1,2,3-13 C3 ] in aspartate or malate. 13 C enrichment in glucose with 13 C-lactate supply was higher than that with 13 C-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-13 C3 ]lactate. Data from the analytes were generally consistent, indicating highly active pyruvate carboxylase and oxidative metabolism through the TCA cycle. Different 13 C-labeling patterns in analytes from the kidney extracts versus effluent suggested metabolic compartmentalization.

Inhibition of pyruvate carboxylase reverses metformin resistance by activating AMPK in pancreatic cancer.

AIMS: Pyruvate carboxylase (PC) plays a key role in cancer cell metabolic reprogramming. Whether metabolic reprogramming and PC are related in PDAC is unclear. Here, the effect of PC expression on PDAC tumorigenesis and metabolic reprogramming were evaluated. MATERIALS AND METHODS: PC protein expression in PDAC and precancerous tissues was measured through immunohistochemistry. The maximum standardized uptake (SUVmax) of 18F-fluoro-2-deoxy-2-d-glucose (18F-FDG) in PDAC patient PET/CT scans before surgical resection was retrospectively determined. Stable PC-knockdown and PC-overexpressing cells were established using lentiviruses, and PDAC progression was assessed in vivo and in vitro. Lactate content, 18F-FDG cell uptake rate, mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in cells. RNA sequencing revealed and qPCR verified differentially expressed genes (DEGs) after PC knockdown. The signaling pathways involved were determined by Western blotting. KEY FINDINGS: PC was significantly upregulated in PDAC tissues vs. precancerous tissues. A high SUVmax correlated with PC upregulation. PC knockdown significantly inhibited PDAC progression. Lactate content, SUVmax, and ECAR significantly decreased after PC knockdown. Peroxisome proliferator-activated receptor gamma coactivator-one alpha (PGC-1α) was upregulated after PC knockdown; and PGC1a expression promoted AMPK phosphorylation to activate mitochondrial metabolism. Metformin significantly inhibited mitochondrial respiration after PC knockdown, further activated AMPK and downstream carnitine palmitoyltransferase 1A (CPT1A)-regulated fatty acid oxidation (FAO), and inhibited PDAC cells progression. SIGNIFICANCE: PDAC cell uptake of FDG was positively correlated with PC expression. PC promotes PDAC glycolysis, and reducing PC expression can increase PGC1a expression, activate AMPK, and restore metformin sensitivity.

Immunodetection of Pyruvate Carboxylase Expression in Human Astrocytomas, Glioblastomas, Oligodendrogliomas, and Meningiomas.

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.