Pyruvate Production Research Articles

Circular RNA PLCE1 promotes epithelial mesenchymal transformation, glycolysis in colorectal cancer and M2 polarization of tumor-associated macrophages

ABSTRACT Plentiful studies have clarified that circular RNAs (circRNAs) are crucial in colorectal cancer (CRC)’s occurrence and development, but its function has not been fully elucidated. The purpose of this study was to investigate the biological functions of circPLCE1 on epithelial mesenchymal transformation (EMT) and glycolysis in CRC, and tumor-associated macrophage (TAM) polarization. The results affirmed augment of circPLCE1 and γ-Actin Gene (ACTG1) but decline of miR-485-5p in CRC. Knockdown circPCLE1 refrained CRC proliferation, glucose consumption, lactic acid and pyruvate production, M2 macrophage markers (IL-10, MRC1), N-cadherin, Snail, reduced the proportion of CD206+ and CD168+ macrophages, but expedited M1 macrophage markers (TNF-α, IL-6) and E-cadherin, while descending miR-485-5p expedited EMT, glycolysis in CRC and TAM M2 polarization . Additionally, it was affirmed that the repression or motivation of depressive or elevated circPCLE1 on EMT, glycolysis in CRC and TAM M2 polarization were reversed via facilitated ACTG1 and miR-485-5p, separately. Mechanism studies have clarified that circPCLE1 as a competitive endogenous RNA adsorbed miR-485-5p to mediate ACTG1. It was assured that refrained circPCLE1 constrained CRC tumor growth, EMT and TAM M2 polarization. In brief, circPCLE1 expedites EMT, glycolysis in CRC and TAM M2 polarization via modulating the miR-485-5p/ACTG1 axis, and is supposed to be a latent molecular target for CRC therapy later.

Automated online monitoring of lactate and pyruvate in tamoxifen resistant MCF-7 cells using sequential-injection capillary electrophoresis with contactless conductivity detection (SI-CE-C4D) and correlation with MCT1 and MCT4 genes expression

Breast cancer is among the most common cancer types worldwide. The first and second line treatment protocols for various stages of breast cancer in females rely on tamoxifen. Until now, the development of tamoxifen resistance is not entirely understood. In this study, an automated sequential injection capillary electrophoresis with capacitively coupled contactless conductivity detector was developed for online levels monitoring of both lactate and pyruvate from the supernatant media of MCF-7 cells developing tamoxifen resistance. Changes in concentration of the two metabolites were simultaneously monitored from three model cell cultures and two control untreated cells. The electrophoretic separation was performed under reversed electroosmotic flow conditions. The system delivers high sample throughput at low sample consumption of 20 µL per analysis. The method is robust and achieved high sensitivity and resolution. Limits of detection were 8.0 and 17.0 nM and linear ranges were 0.15–5 and 0.01–1 mM with a correlation coefficient of 0.9951 and 0.9963 for lactate and pyruvate, respectively. Inter-run and intra-run accuracy were in the range of 95.37–107.02% with precision (RSD, %) of ≤9.84%. The method was completely validated and the data obtained were in agreement with results achieved using the lactate and pyruvate assay kits. The highly informative generated data revealed a significant increase in lactate and pyruvate production in the three tamoxifen resistant MCF-7 cells models compared to the two control cells starting from 5.8 hours and 6.8 hours culturing times, respectively. The increase in concentrations of both lactate and pyruvate were correlated with an increase in MCT1 and MCT4 genes expression.

Metabolomic Signatures of In Vitro Biofilm Maturation of Streptococcus mutans.

The Streptococcus mutans is commonly find in oral environment in both symbiont and dysbiotic conditions, where for the last one it causes the break in homeostatic balance and, in association with other microorganisms' community, results in dental caries process. Additionally, it is important to determine the low molecular weight metabolites profile from Streptococcus mutans to distinguish the endogenous and exogenous compounds from patient subjected to salivary metabolomic studies. Thus, the objective of the present study was to characterize the in vitro metabolomic profile of the maturation of a single-species Streptococcus mutans biofilm using metabolomic approach by 1H-nuclear magnetic resonance (NMR) spectroscopy. A distinct metabolomic profile was observed after 2days of biofilm maturation, independently of the presence of enamel substrate. Sucrose, lactate, and fructose were the main metabolites responsible for the distinction. The sucrose was consumed by S. mutans in higher levels in the initial experimental periods than at 6days of biofilm growth. Lactate and fructose were the main compounds secreted, regardless of the type of growth, but it was also observed production of propionate, iso-butyrate, and pyruvate. Pyruvate metabolism and glycolysis/gluconeogenesis were the main pathways related to biofilm growth. The results contribute to the determination of compounds that are resulted from oral microbial activity and help to guide further metabolomics studies.

MiR-93-5P Represses the Gluconeogenesis of Hepatocellular Carcinoma while Boosting Its Glycolysis and Malignant Progression by Suppressing PCK1.

Herein, we explored effects of miR-93-5p and gluconeogenic rate-limiting enzyme PCK1 on HCC cells. Bioinformatics analysis and cell experiments confirmed that, compared with expression in normal tissue and cells, miR-93-5p in HCC was abnormally upregulated while PCK1 expression was remarkably downregulated. PCK1 overexpression repressed proliferation, migration, and invasion of HCC cells, and blocked cell cycle in G0/G1 phase. During this process, glucose production was boosted while the production of pyruvate, lactic acid, citric acid, and malic acid was reduced, suggesting that the effect was related to inhibition of glycolysis and induction of gluconeogenic pathways. Elevated miR-93-5p level promoted proliferation, migration, and invasion of HCC cells, accelerated development of cell cycle, activated glycolysis, and suppressed gluconeogenesis. In addition, when miR-93-5p and PCK1 were concurrently upregulated, the abovementioned promoting effects were canceled out. These investigations demonstrated that promoting effect of miR-93-5p on HCC cell growth may be carried out by inhibiting the PCK1 expression, suggesting that miR-93-5p and PCK1 could be applied as new biomarkers or novel therapeutic targets for HCC diagnosis.

Tissue-specific responses that constrain glucose oxidation and increase lactate production with the severity of hypoxemia in fetal sheep.

Fetal hypoxemia decreases insulin and increases cortisol and norepinephrine concentrations and may restrict growth by decreasing glucose utilization and altering substrate oxidation. Specifically, we hypothesized that hypoxemia would decrease fetal glucose oxidation and increase lactate and pyruvate production. We tested this by measuring whole body glucose oxidation and lactate production, and molecular pathways in liver, muscle, adipose, and pancreas tissues of fetuses exposed to maternal hypoxemia for 9 days (HOX) compared with control fetal sheep (CON) in late gestation. Fetuses with more severe hypoxemia had lower whole body glucose oxidation rates, and HOX fetuses had increased lactate production from glucose. In muscle and adipose tissue, expression of the glucose transporter GLUT4 was decreased. In muscle, pyruvate kinase (PKM) and lactate dehydrogenase B (LDHB) expression was decreased. In adipose tissue, LDHA and lactate transporter (MCT1) expression was increased. In liver, there was decreased gene expression of PKLR and MPC2 and phosphorylation of PDH, and increased LDHA gene and LDH protein abundance. LDH activity, however, was decreased only in HOX skeletal muscle. There were no differences in basal insulin signaling across tissues, nor differences in pancreatic tissue insulin content, β-cell area, or genes regulating β-cell function. Collectively, these results demonstrate coordinated metabolic responses across tissues in the hypoxemic fetus that limit glucose oxidation and increase lactate and pyruvate production. These responses may be mediated by hypoxemia-induced endocrine responses including increased norepinephrine and cortisol, which inhibit pancreatic insulin secretion resulting in lower insulin concentrations and decreased stimulation of glucose utilization.NEW & NOTEWORTHY Hypoxemia lowered fetal glucose oxidation rates, based on severity of hypoxemia, and increased lactate production. This was supported by tissue-specific metabolic responses that may result from increased norepinephrine and cortisol concentrations, which decrease pancreatic insulin secretion and insulin concentrations and decrease glucose utilization. This highlights the vulnerability of metabolic pathways in the fetus and demonstrates that constrained glucose oxidation may represent an early event in response to sustained hypoxemia and fetal growth restriction.

Metabolic flux analysis of the non-transitory starch tradeoff for lipid production in mature tobacco leaves

The metabolic plasticity of tobacco leaves has been demonstrated via the generation of transgenic plants that can accumulate over 30% dry weight as triacylglycerols. In investigating the changes in carbon partitioning in these high lipid-producing (HLP) leaves, foliar lipids accumulated stepwise over development. Interestingly, non-transient starch was observed to accumulate with plant age in WT but not HLP leaves, with a drop in foliar starch concurrent with an increase in lipid content. The metabolic carbon tradeoff between starch and lipid was studied using 13CO2-labeling experiments and isotopically nonstationary metabolic flux analysis, not previously applied to the mature leaves of a crop. Fatty acid synthesis was investigated through assessment of acyl-acyl carrier proteins using a recently derived quantification method that was extended to accommodate isotopic labeling. Analysis of labeling patterns and flux modeling indicated the continued production of unlabeled starch, sucrose cycling, and a significant contribution of NADP-malic enzyme to plastidic pyruvate production for the production of lipids in HLP leaves, with the latter verified by enzyme activity assays. The results suggest an inherent capacity for a developmentally regulated carbon sink in tobacco leaves and may in part explain the uniquely successful leaf lipid engineering efforts in this crop.

CRISPRi enables fast growth followed by stable aerobic pyruvate formation in Escherichia coli without auxotrophy.

CRISPR interference (CRISPRi) was applied to enable the aerobic production of pyruvate in Escherichia coli MG1655 under glucose excess conditions by targeting the promoter regions of aceE or pdhR. Knockdown strains were cultivated in aerobic shaking flasks and the influence of inducer concentration and different sgRNA binding sites on the production of pyruvate was measured. Targeting the promoter regions of aceE or pdhR triggered pyruvate production during the exponential phase and reduced expression of aceE. In lab‐scale bioreactor fermentations, an aceE silenced strain successfully produced pyruvate under fully aerobic conditions during the exponential phase, but loss of productivity occurred during a subsequent nitrogen‐limited phase. Targeting the promoter region of pdhR enabled pyruvate production during the growth phase of cultivations, and a continued low‐level accumulation during the nitrogen‐limited production phase. Combinatorial targeting of the promoter regions of both aceE and pdhR in E. coli MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 resulted in the stable aerobic production of pyruvate with non‐growing cells at YP/S = 0.36 ± 0.029 gPyruvate/gGlucose in lab‐scale bioreactors throughout an extended nitrogen‐limited production phase.

Actin-related protein 4: An unconventional negative regulator of mitochondrial calcium in protozoan parasite Leishmania

Regulation of mitochondrial calcium import is less understood in evolutionarily distinct protozoan parasites, such as Leishmania, as some of the mitochondrial calcium uniporter complex proteins are either missing or functionally diverged. Here, we show that Actin-related protein4 (ARP4), localizes exclusively into the Leishmania mitochondrion and depletion of this protein causes cells to accumulate calcium in the mitochondrion. The ARP4 depleted cells show increased activation of pyruvate dehydrogenase and production of ATP. Overall, our results indicate that ARP4 negatively regulates calcium uptake in the Leishmania mitochondrion.

An oxidative metabolic pathway of 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEHU) from alginate in an alginate-assimilating bacterium

Alginate-assimilating bacteria degrade alginate into an unsaturated monosaccharide, which is converted into 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEHU). DEHU is reduced to 2-keto-3-deoxy-D-gluconate by a DEHU-specific reductase using NAD(P)H. This is followed by pyruvate production via the Entner-Doudoroff pathway. Previously, we identified FlRed as a DEHU reductase in an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. Here, we showed that FlRed can also catalyze the oxidation of DEHU with NAD+, producing 2-keto-3-deoxy-D-glucarate (KDGR). FlRed showed a predilection for NADH and NAD+ over NADPH and NADP+, respectively, and the Km value for NADH was approximately 2.6-fold less than that for NAD+. Furthermore, we identified two key enzymes, FlDet and FlDeg, for KDGR catabolism. FlDet was identified as an enzyme of the ribonuclease activity regulator A family, which converts KDGR to α-ketoglutaric semialdehyde (α-KGSA). FlDeg, a type II α-KGSA dehydrogenase, generated α-ketoglutaric acid by oxidizing the aldehyde group of α-KGSA using NAD(P)+. Consequently, unlike the conventional DEHU reduction pathway, DEHU can be directly converted to α-ketoglutaric acid without consuming NAD(P)H. Alginate upregulated the expression of not only FlRed and two enzymes of the DEHU-reduction pathway, but also FlDet and FlDeg. These results revealed dual pathways of DEHU metabolism involving reduction or oxidation by FlRed.

Lactate and pyruvate levels correlation with lactate dehydrogenase gene expression and glucose consumption in Tamoxifen-resistant MCF-7 cells using capillary electrophoresis with contactless conductivity detection (CE-C4 D).

Breast cancer is the second leading cause of cancer death in women after lung cancer. The first-line treatment of metastatic breast cancer in premenopausal women relies on tamoxifen. The development of tamoxifen resistance is not fully understood. In this study, capillary electrophoresis with capacitively coupled contactless conductivity detector was developed to monitor the changes in lactate and pyruvate levels in supernatant media of three models of developed MCF-7 tamoxifen-resistant cells and correlate these metabolites changes with lactate dehydrogenase genes expression and glucose consumption. The electrophoretic separation was achieved under reversed electroosmotic flow conditions. The linear ranges were 0.15-5 and 0.01-1mM with a correlation coefficient of 0.9966 and 0.9971 and the limits of detection were 0.01 and 0.02μM for lactate and pyruvate, respectively. Inter- and intrarun accuracy were in the range of 96.88-105.94% with precision (CV, %) of ≤7.35%. The method was completely validated and the results were in agreement with those obtained using the lactate and glucose assay kits. The results revealed a significant increase in both lactate and pyruvate production in the three tamoxifen-resistant MCF-7 cells models compared to control cells. This increase was correlated with the increase of lactate dehydrogenase genes expression and the increase of glucose consumption.

Development of novel media formulation using rice straw and corn steep liquor for pyruvate production

Development of novel media formulation using rice straw and corn steep liquor for pyruvate production

The pyruvate kinase activity in peripheral and uterine blood in women with atypical endometrial hyperplasia and endometrial cancer

IntroductionPyruvate kinase in an enzyme that catalyzes the production of pyruvate and ATP as the final step of the glycolysis. Potential role of Pyruvate kinase in tumorigenesis was previously suggested, due to its altered activity in several tumors.Material and methodsThis study looks at M2 isozyme of pyruvate kinase activity (M2-PK) measured in peripheral and uterine blood plasma in various stages of endometrial cancer (EC) as well as in precancerous state of atypical endometrial hyperplasia (AEH). Measurements were performed using spectrophotometric method in citrate plasma samples from peripheral and uterine blood. Study group included 84 patients with endometrial cancer, 28 patients with atypical endometrial hyperplasia and 23 non-cancerous controls.ResultsPyruvate kinase activity in EC group was 3-fold higher than in control group both in peripheral and uterine blood samples. Pyruvate kinase activity was also 3-fold higher in uterine blood when compared to peripheral blood samples. We also found statistically significant correlation between FIGO staging and detected activity with the M2-PK activity being 2-fold higher for FIGO1 than for FIGO3. We also describe a paradox in which the M2-PK activity in patients with atypical endometrial hyperplasia is lower than M2-PK activity in control group in peripheral blood samples, but higher in uterine blood samples.ConclusionsThe measurement of citrate plasma pyruvate kinase metabolic activity varies greatly between samples collected from different sites and samples collected from patients with varied tumor staging. Further studies are needed in order to elucidate molecular pathways that are responsible for observed differences.

Dynamic metabolic engineering of Escherichia coli improves fermentation for the production of pyruvate and its derivatives

Pyruvate is a key intermediate that is involved in various synthetic metabolic pathways for microbial chemical and fuel production. It is widely used in the food, chemical, and pharmaceutical industries. However, the microbial production of pyruvate and its derivatives compete with microbial cell growth, as pyruvate is an important metabolic intermediate that serves as a hub for various endogenous metabolic pathways, including gluconeogenesis, amino acid synthesis, TCA cycle, and fatty acid biosynthesis. To achieve a more efficient bioprocess for the production of pyruvate and its derivatives, it is necessary to reduce the metabolic imbalance between cell growth and target chemical production. For this purpose, we devised a dynamic metabolic engineering strategy within an Escherichia coli model, in which a metabolic toggle switch (MTS) was employed to redirect metabolic flux from the endogenous pathway toward the target synthetic pathway. Through a combination of TCA cycle interruption through MTS and reduction of pyruvate consumption in endogenous pathways, we achieved a drastic improvement (163mM, 26-fold) in pyruvate production. In addition, we demonstrated the redirection of metabolic flux from excess pyruvate toward isobutanol production. The final isobutanol production titer of the strain harboring MTS was 26% improved compared with that of the control strain.

Zhoushi Qi Ling decoction represses docetaxel resistance and glycolysis of castration-resistant prostate cancer via regulation of SNHG10/miR-1271-5p/TRIM66 axis

Docetaxel resistance developed in half of castration-resistant prostate cancer (CRPC) patients hinders its long-term clinical application. The current study was designed to investigate the effects of Chinese medicine Zhoushi Qi Ling decoction on the docetaxel resistance of prostate cancer as well as elucidate the underlying molecular mechanism. In our study, Qi Ling significantly decreased viability and colony formation as well as increased apoptosis of docetaxel-resistant (DR) CRPC cells. Qi Ling-treated DR cells exhibited decreased glucose consumption, lactate release and pyruvate production. Moreover, lncRNA SNHG10 was upregulated in DR tissues of CRPC patients and was negatively correlated with the progression-free survival. Bioinformatics analysis indicated miR-1271-5p as the associated miRNA possibly binding with SNHG10. miR-1271-5p up-regulation dramatically decreased the luciferase activity of SNHG10 in DR cells. SNHG10 knockdown sharply increased the expression of miR1271-5p in DR cells. Targetscan predicted TRIM66 as one of the downstream targets of miR-1271-5p. miR-1271-5p up-regulation drastically reduced luciferase activity as well as TRIM66 expression in DR cells. Also, the knockdown of SNHG10 remarkably suppressed the expression of TRIM66 in DR cells. Additionally, Qi Ling treatment reduced SNHG10 and TRIM66, while increased miR1271-5p, in DR cells. In summary, Qi Ling inhibited docetaxel resistance and glycolysis of CRPC possibly via SNHG10/miR-1271-5p/TRIM66 pathway.

Using systems biology approaches to identify signalling pathways activated during chronic wound initiation.

Chronic wounds are a significant health problem worldwide. However, nothing is known about how chronic wounds initiate and develop. Here we use a chronic wound model in diabetic mice and a Systems Biology Approach using nanoString nCounter technology and weighted gene correlation network analysis (WGCNA), with tissues collected at 6, 12, 24 and 48 h post-wounding, to identify metabolic signalling pathways involved in initiation of chronicity. Normalized counts obtained from the nanoString nCounter Mouse Metabolic Panel were used for the WGCNA, which groups genes into co-expression modules to visualize the correlation network. Genes with significant module membership and gene trait significance (p < 0.05) were used to identify signalling pathways that are important for the development of chronicity. The pathway analysis using the Reactome database showed stabilization of PTEN, which down-regulates PI3K/AKT1, which in turn down-regulates Nrf2, as shown by ELISA, thus disabling antioxidant production, resulting in high oxidative stress levels. We find that pathways involved in inflammation, including those that generate pro-inflammatory lipids derived from arachidonic acid metabolism, IFNγ and catecholamines, occur. Moreover, HIF3α is over-expressed, potentially blocking Hif1α and preventing activation of growth factors and cytokines that promote granulation tissue formation. We also find that FGF1 is under-expressed, while thrombospondin-1 is over-expressed, resulting in decreased angiogenesis, a process that is critical for healing. Finally, enzymes involved in glycolysis are down-regulated, resulting in decreased production of pyruvate, a molecule critical for ATP production, leading to extensive cell death and wound paralysis. These findings offer new avenues of study that may lead to the development of novel treatments of CW to be administered right after debridement.

Circ\u03b2-catenin promotes tumor growth and Warburg effect of gallbladder cancer by regulating STMN1 expression

Gallbladder cancer (GBC) is the most malignant cancer of the biliary tract cancer and presents poor prognosis. CircRNAs have been identified as critical regulators of multiple stages in tumor progression. In the study, we first demonstrated that circular RNA circβ-catenin expression was upregulated in GBC tissues when compared to adjacent normal tissues and associated with advanced clinical stage and poor prognosis in GBC patients. Silencing of circβ-catenin obviously suppressed GBC cell proliferation and cell cycle progression in vitro, but circβ-catenin overexpression had the opposite effects. In vivo, silencing of circβ-catenin inhibited tumor growth. Furthermore, we also found that circβ-catenin promoted GBC cell lactate production, pyruvate production, ATP quantity, and extracellular acidification rate (ECAR), which suggested that circβ-catenin regulated Warburg effect in GBC. Mechanistic analysis further highlighted that circβ-catenin promoted Stathmin 1 (STMN1) expression through sponging miR-223 in GBC progression. In addition, knockdown of STMN1 inhibited cell growth and Warburg effect in GBC. In summary, our findings indicated that circβ-catenin/miR-223/STMN1 axis could regulate cell growth and Warburg effect in GBC. Targeting circβ-catenin might be a potential therapeutic strategy for GBC.

CAR T-Cells Depend on the Coupling of NADH Oxidation with ATP Production.

The metabolic milieu of solid tumors provides a barrier to chimeric antigen receptor (CAR) T-cell therapies. Excessive lactate or hypoxia suppresses T-cell growth, through mechanisms including NADH buildup and the depletion of oxidized metabolites. NADH is converted into NAD+ by the enzyme Lactobacillus brevis NADH Oxidase (LbNOX), which mimics the oxidative function of the electron transport chain without generating ATP. Here we determine if LbNOX promotes human CAR T-cell metabolic activity and antitumor efficacy. CAR T-cells expressing LbNOX have enhanced oxygen as well as lactate consumption and increased pyruvate production. LbNOX renders CAR T-cells resilient to lactate dehydrogenase inhibition. But in vivo in a model of mesothelioma, CAR T-cell’s expressing LbNOX showed no increased antitumor efficacy over control CAR T-cells. We hypothesize that T cells in hostile environments face dual metabolic stressors of excessive NADH and insufficient ATP production. Accordingly, futile T-cell NADH oxidation by LbNOX is insufficient to promote tumor clearance.

Semi-rational design of L-amino acid deaminase for production of pyruvate and D-alanine by Escherichia coli whole-cell biocatalyst.

In our previous study, one-step pyruvate and D-alanine production from D,L-alanine by a whole-cell biocatalyst Escherichia coli expressing L-amino acid deaminase (Pm1) derived from Proteus mirabilis was investigated. However, due to the low catalytic efficiency of Pm1, the pyruvate titer was relatively low. Here, semi-rational design based on site-directed saturation mutagenesis was carried out to improve the catalytic efficiency of Pm1. A novel high-throughput screening (HTS) method for pyruvate based on 2,4-dinitrophenylhydrazine indicator was then established. The catalytic efficiency (kcat/Km) of the mutant V437I screened out by this method was 1.88 times higher than wild type. Next, to improve the growth of the engineered strain BLK07, the genes encoding for Xpk and Fbp were integrated into its genome to construct non-oxidative glycolysis (NOG) pathway. Finally, the CRISPR/Cas9 system was used to integrate the N6-pm1-V437I gene into the genome of BLK07. Pyruvic acid titer of the plasmid-free strain reached 42.20g/L with an L-alanine conversion rate of 77.62% and a D-alanine resolution of 82.4%. This work would accelerate the industrial production of pyruvate and D-alanine by biocatalysis, and the HTS method established here could be used to screen other Pm1 mutants with high pyruvate titers.

High Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Correlating the Metabolic Changes of Lactate, Pyruvate and L-Glutamine with Induced Tamoxifen Resistant MCF-7 Cell Line Potential Molecular Changes

Breast cancer is one of the most prevalent cancers worldwide usually treated with Tamoxifen. Tamoxifen resistance development is the most challenging issue in an initially responsive breast tumor, and mechanisms of resistance are still under investigation. The objective of this study is to develop and validate a selective, sensitive, and simultaneous high performance liquid chromatography–tandem mass spectrometry method to explore the changes in substrates and metabolites in supernatant media of developed Tamoxifen resistance MCF-7 cells. We focus on the determination of lactate, pyruvate, and L-glutamine which enables the tracking of changes in metabolic pathways as a result of the resistance process. Chromatographic separation was achieved within 3.5 min. using a HILIC column (4.6 × 100 mm, 3.5 µm particle size) and mobile phase of 0.05 M acetic acid–ammonium acetate buffer solution pH 3.0: Acetonitrile (40:60 v/v). The linear range was 0.11–2.25, 0.012–0.227, and 0.02–0.20 mM for lactate, pyruvate, and L-glutamine, respectively. Within- and between-run accuracy was in the range 98.94-105.50% with precision (CV, %) of ≤0.86%. The results revealed a significant increase in both lactate and pyruvate production after acquiring the resistant. An increase in L-glutamine levels was also observed and could be attributed to its over production or decline in its consumption. Therefore, further tracking of genes responsible of lactate, pyruvate, and glutamine metabolic pathways should be performed in parallel to provide in-depth explanation of resistance mechanism.

Glutamate-Oxaloacetate Transaminase 1 Impairs Glycolysis by Interacting with Pyruvate Carboxylase and Further Inhibits the Malignant Phenotypes of Glioblastoma Cells

Glycolysis is an important metabolic manner in glioblastoma multiforme (GBM)'s rapid growth. It has been reported that glutamate-oxaloacetate transaminase 1 (GOT1) is low-expressed in GBM and patients with high-expressed GOT1 have better prognosis. However, the effect and mechanism of GOT1 on glycolysis and malignant phenotypes of GBM cells are still unclear. The expression differences of GOT1 between GBM parenchyma and adjacent tissues were detected. The prognosis and clinical data with different levels of GOT1 were also analyzed. The glucose consumption, production of lactate and pyruvate were measured after GOT1 was knocked down or overexpressed. The effects of GOT1 on GBM cell's malignant phenotypes were analyzed by Western blot, CCK-8 assay, and flow cytometry. The relationship between GOT1 and pyruvate carboxylase (PC) was examined by immunoprecipitation and immunofluorescence. GOT1 was expressed little in GBM, and patients with highly expressed GOT1 had longer survival periods. Overexpressed GOT1 inhibited the glycolysis and malignant phenotypes of GBM cells. 2-DG treatment could partially reverse the enhancement of malignant phenotypes caused by knockdown of GOT1. The expression of GOT1 was positively correlated with PC. The inhibitory effect of GOT1 on glycolysis could be partially reversed by PC's knockdown. GOT1 could impair glycolysis by interacting with PC and further inhibit the malignant phenotypes of GBM cells.