Decreased Lactate Production Research Articles

Intracellular glucose starvation inhibits osteogenic differentiation in human periodontal ligament cells

Periodontal ligament cells (PDLCs), as mesenchymal cells in the oral cavity, are closely linked to periodontal tissue regeneration. However, the effect of local glucose deficiency on periodontal tissue regeneration, such as immediately post-surgery, remains unknown. In the present study, we investigated the effect of a low-glucose environment on the proliferation and osteogenic differentiation of PDLCs. We used media with five glucose concentrations (100, 75, 50, 25, and 0mg/dL) and focused on the effects of a low-glucose environment on the proliferation, osteogenic differentiation, and autophagy of PDLCs. Additionally, we focused on changes in lactate production in a low-glucose environment and investigated the involvement of lactate with AZD3965, a monocarboxylate transporter-1 (MCT-1) inhibitor. The low-glucose environment inhibited PDLCs proliferation, migration, and osteogenic differentiation, and induced the expression of the autophagy-related factors LC3 and p62. Lactate and ATP production were decreased under low-glucose conditions. The addition of AZD3965 (MCT-1 inhibitor) in normal glucose conditions caused a similar trend as in low-glucose conditions on PDLCs. Our results suggest lactate production through glucose metabolism in the osteogenic differentiation of PDLCs. A low-glucose environment decreased lactate production, inhibiting cell proliferation, migration, and osteogenic differentiation and inducing autophagy in PDLCs.

Air–liquid interface cultures trigger a metabolic shift in intestinal epithelial cells (IPEC-1)

An improved oxygen availability in air–liquid interface (ALI) cultures of enterocytes of the small intestine seems to be primarily responsible for morphological, metabolic, and functional changes. Intestinal porcine epithelial cells 1 (IPEC-1) are less investigated and are rarely used as model for intestinal barrier but showed a profound change of cell shape during ALI cultivation. We aim to answer the following question: Are the observed morphological effects accompanied by changes in metabolic function? A microarray analysis of submerged culture (SMC) and ALI cultures identified 830 significantly regulated genes. Subsequent functional clustering revealed alterations in 31 pathways, with the highest number of regulated genes in metabolic pathways, carbon metabolism, glycolysis, and hypoxia-inducible factor (HIF) signaling. Furthermore, HIF-1α as a mediator of a metabolic switch between glycolysis and oxidative phosphorylation showed a trend of increased mRNA levels in ALI in contrast to a reduced nuclear HIF-1α content in the nucleus. Candidate genes of oxidative phosphorylation such as a mitochondrial marker exhibited enhanced mRNA levels, which was confirmed by western blot analysis. Cytochrome C oxidase (COX) subunit 5B protein was decreased in ALI, although mRNA level was increased. The oxidation of ferrocytochrome C to ferricytochrome C was used for detection of cytochrome C oxidase activity of isolated mitochondria and resulted in a trend of higher activity in ALI. Furthermore, quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. To evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenized cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In contrast, blocking with 2-desoxy-d-glucose (2DG) significantly reduced ATP content in ALI and SMC. These results indicate a metabolic shift in IPEC-1 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis.

Dysregulated glycolysis underpins high-fat-associated endometrial decidualization impairment during early pregnancy in mice

Pregnancy complications are more likely to occur in obese women because of defective decidualization. However, the specific mechanism of glycolysis in decidual modulation associated with obesity remains unknown. Therefore, we explored the role of glycolysis in the endometrium of obese pregnant mice during decidualization. C57BL/6J mice were fed a high-fat diet (HFD) to induce obesity. All obesity related parameters were significantly higher in the HFD mice than control. Furthermore, the HFD mice had fewer implantation sites, a smaller decidual area growth, and decreased decidualization marker protein expression than control. The HFD mice also had significantly decreased lactate production and glycolytic enzyme expression. To confirm the functional role of glycolysis during the decidual period in obese pregnant mice, we extracted endometrial stromal cells (ESCs) and treated them with oleic acid (OA) and palmitic acid (PA) to mimic a high-fat environment. Decidualization and glycolysis were significantly restricted in the OA-and PA-treated groups. Moreover, we administered a glycolytic inhibitor, 2-DG, and an agonist, pioglitazone. 2-DG treatment considerably decreased the cells' glycolysis and decidualization. However, pioglitazone treatment improved glycolysis and alleviated defective decidualization. In conclusion, obesity-induced endometrial glycolysis modifications and key glycolytic enzyme downregulation during early pregnancy might cause abnormal decidualization, leading to an unsustainable pregnancy.

PDK1- and PDK2-mediated metabolic reprogramming contributes to the TGFβ1-promoted stem-like properties in head and neck cancer

BackgroundResistance to chemotherapeutic drugs is a key factor for cancer recurrence and metastases in head and neck cancer (HNC). Cancer stem cells (CSCs) in tumors have self-renewal, differentiation, and higher drug resistance capabilities, resulting in a poor prognosis for patients. In glucose metabolism, pyruvate dehydrogenase kinase (PDK) inhibits pyruvate dehydrogenase and impedes pyruvate from being metabolized into acetyl-CoA and entering the tricarboxylic acid cycle to generate energy. Studies have reported that PDK1 and PDK2 inhibition suppresses the growth, motility, and drug resistance of cancer cells. Furthermore, while TGFβ1 levels are persistently elevated in HNC patients with poor prognosis, the role of PDK isoforms in the TGFβ1-promoted progression and stem-like properties of HNC is unclear.MethodsLevels of PDK1 and PDK2 were evaluated in HNC tissue microarrays by immunohistochemistry to explore potential clinical relevance. PDK1 and PDK2 were knocked down by the lentivirus shRNA system to investigate their role in TGFβ1-promoted tumor progression in vitro.ResultsWe found that PDK2 levels were increased in the later stage of HNC tissues compared to constant PDK1 expression. After PDK1 and PDK2 knockdown, we discovered increased ATP production and decreased lactate production in TGFβ1-treated and untreated HNC cells. However, only PDK2 silencing significantly inhibited the clonogenic ability of HNC cells. We subsequently found that TGFβ1-promoted migration and invasion capabilities were decreased in PDK1 and PDK2 knockdown cells. The tumor spheroid-forming capability, motility, CSC genes, and multidrug-resistant genes were downregulated in PDK1 and PDK2 silencing CSCs. PDK1 and PDK2 inhibition reversed cisplatin and gemcitabine resistance of CSCs, but not paclitaxel resistance.ConclusionThe results demonstrated that the PDK1- and PDK2-mediated Warburg effect contributes to the TGFβ1-enhanced stemness properties of HNC. Therefore, PDK1 and PDK2 may serve as molecular targets for the combination therapy of HNC.

Targeting the bicarbonate transporter SLC4A4 overcomes immunosuppression and immunotherapy resistance in pancreatic cancer

Solid tumors are generally characterized by an acidic tumor microenvironment (TME) that favors cancer progression, therapy resistance and immune evasion. By single-cell RNA-sequencing analysis in individuals with pancreatic ductal adenocarcinoma (PDAC), we reveal solute carrier family 4 member 4 (SLC4A4) as the most abundant bicarbonate transporter, predominantly expressed by epithelial ductal cells. Functionally, SLC4A4 inhibition in PDAC cancer cells mitigates the acidosis of the TME due to bicarbonate accumulation in the extracellular space and a decrease in lactate production by cancer cells as the result of reduced glycolysis. In PDAC-bearing mice, genetic or pharmacological SLC4A4 targeting improves T cell-mediated immune response and breaches macrophage-mediated immunosuppression, thus inhibiting tumor growth and metastases. In addition, Slc4a4 targeting in combination with immune checkpoint blockade is able to overcome immunotherapy resistance and prolong survival. Overall, our data propose SLC4A4 as a therapeutic target to unleash an antitumor immune response in PDAC.

The DEAD-box RNA helicase Dhx15 controls glycolysis and arbovirus replication in Aedes aegypti mosquito cells.

Aedes aegypti mosquitoes are responsible for the transmission of arthropod-borne (arbo)viruses including dengue and chikungunya virus (CHIKV) but in contrast to human hosts, arbovirus-infected mosquitoes are able to efficiently control virus replication to sub-pathological levels. Yet, our knowledge of the molecular interactions of arboviruses with their mosquito hosts is incomplete. Here, we aimed to identify and characterize novel host genes that control arbovirus replication in Aedes mosquitoes. RNA binding proteins (RBPs) are well-known to regulate immune signaling pathways in all kingdoms of life. We therefore performed a knockdown screen targeting 461 genes encoding predicted RBPs in Aedes aegypti Aag2 cells and identified 15 genes with antiviral activity against Sindbis virus. Amongst these, the three DEAD-box RNA helicases AAEL004419/Dhx15, AAEL008728, and AAEL004859 also acted as antiviral factors in dengue and CHIKV infections. Here, we explored the mechanism of Dhx15 in regulating an antiviral transcriptional response in mosquitoes by silencing Dhx15 in Aag2 cells followed by deep-sequencing of poly-A enriched RNAs. Dhx15 knockdown in uninfected and CHIKV-infected cells resulted in differential expression of 856 and 372 genes, respectively. Interestingly, amongst the consistently downregulated genes, glycolytic process was the most enriched gene ontology (GO) term as the expression of all core enzymes of the glycolytic pathway was reduced, suggesting that Dhx15 regulates glycolytic function. A decrease in lactate production indicated that Dhx15 silencing indeed functionally impaired glycolysis. Modified rates of glycolytic metabolism have been implicated in controlling the replication of several classes of viruses and strikingly, infection of Aag2 cells with CHIKV by itself also resulted in the decrease of several glycolytic genes. Our data suggests that Dhx15 regulates replication of CHIKV, and possibly other arboviruses, by controlling glycolysis in mosquito cells.

Melatonin Treatment Triggers Metabolic and Intracellular pH Imbalance in Glioblastoma.

Metabolic rewiring in glioblastoma (GBM) is linked to intra- and extracellular pH regulation. In this study, we sought to characterize the role of melatonin on intracellular pH modulation and metabolic consequences to identify the mechanisms of action underlying melatonin oncostatic effects on GBM tumor initiating cells. GBM tumor initiating cells were treated at different times with melatonin (1.5 and 3.0 mM). We analyzed melatonin's functional effects on GBM proliferation, cell cycle, viability, stemness, and chemo-radiosensitivity. We then assessed the effects of melatonin on GBM metabolism by analyzing the mitochondrial and glycolytic parameters. We also measured the intracellular and extracellular pH. Finally, we tested the effects of melatonin on a mouse subcutaneous xenograft model. We found that melatonin downregulated LDHA and MCT4, decreasing lactate production and inducing a decrease in intracellular pH that was associated with an increase in ROS and ATP depletion. These changes blocked cell cycle progression and induced cellular death and we observed similar results in vivo. Melatonin's cytotoxic effects on GBM were due, at least in part, to intracellular pH modulation, which has emerged as a newly identified mechanism, providing new insights into the oncostatic effect of melatonin on GBM.

NRG1/ErbB2 axis regulated mitochondrial function and antioxidant enzymes of neural stem cells in the cochlear nucleus partially through PGC-1α

Neuregulin-1 (NRG1)/erythroblastic leukaemia viral oncogene homologues 2 (ErbB2) pathway had been implicated in promoting differentiation and suppressing apoptosis of neuronal stem cells (NSCs) isolated from cochlear nucleus. In the current study, we aimed at determining the effects of NRG1/ErbB2 on mitochondrial (mt) function of NSCs. As expected, NRG1 increased the expression of mitofusin (Mfn) 1 and Mfn2 and decreased the expression of mitochondrial fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1). However, after ErbB2 knockout, Mfn1 and Mfn2 expression decreased while Fis1 and Drp1 increased. Moreover, the increased mtDNA copy number and intracellular ATP level, elevated ATPase activities as well as decreased lactate production induced by NRG1 were partially reversed by ErbB2 knockout. Additionally, NRG1 treatment increased the activities of catalase, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and upregulated the protein expression of catalase, manganese superoxide dismutase (MnSOD), peroxisome proliferator-activated receptor-γ coactlvator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1) and transcription factor A, mitochondrial (TFAM), which were also reversed by ErbB2 knockout. Furthermore, PGC-1α overexpression partially reversed the above effects of ErbB2 knockout. In conclusion, these findings suggest that the promotion of mitochondrial function of NRG1/ErbB2 axis is at least in part mediated by PGC-1α in NSCs from cochlear nucleus.

SIRT1 regulated hexokinase-2 promoting glycolysis is involved in hydroquinone-enhanced malignant progression in human lymphoblastoid TK6 cells

Reprogramming of cellular metabolism is a vital event during tumorigenesis. The role of glycolysis in malignant progression promoted by hydroquinone (HQ), one of the metabolic products of benzene, remains to be understood. Recently, we reported the overexpression of sirtuin 1 (SIRT1) in HQ-enhanced malignant progression of TK6 cells and hypothesized that SIRT1 might contribute to glycolysis and favor tumorigenesis. Our data showed that acute exposure of TK6 cells to HQ for 48 h inhibited glycolysis, as indicated by reduction in glucose consumption, lactate production, hexokinase activity, and the expression of SIRT1 and glycolytic enzymes, including HIF-1α, hexokinase-2 (HK-2), ENO-1, glucose transporter 1 (Glut-1), and lactic dehydrogenase A (LDHA). Knockdown of SIRT1 or inhibition of glycolysis using the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) downregulated the levels of SIRT1 and glycolytic enzymes and significantly enhanced HQ-induced cell apoptosis, although knockdown of SIRT1 or 2-DG alone had little effect on apoptosis. Furthermore, immunofluorescence and Co-IP assays demonstrated that SIRT1 regulated the expression of HK-2, and HQ treatment caused a decrease in SIRT1 and HK-2 binding to mitochondria. Importantly, we found that glycolysis was promoted with increasing HQ treatment weeks. Long-term HQ exposure increased the expression of SIRT1 and several glycolytic enzymes and promoted malignant cell progression. Moreover, compared with the PBS group, glucose consumption and lactate production increased after 10 weeks of HQ exposure, and the protein levels of SIRT1 and HK-2 were increased after 15 weeks of HQ exposure, while those of Glut-1, ENO-1, and LDHA were elevated. In addition, SIRT1 knockdown HQ 19 cells exhibited decreased lactate production, glucose consumption, glycolytic enzymes expression, cell growth, and tumor formation in nude mice. Our findings identify the high expression of SIRT1 as a strong oncogenic driver that positively regulates HK-2 and promotes glycolysis in HQ-accelerated malignant progression of TK6 cells.

Arthrospira Cell Residues for Lactic Acid Fermentation as Bioproducts From Waste Utilization

Arthrospira (Spirulina) platensis is a freshwater cyanobacterium that is commercially produced as a food source on a global scale and considered safe for human consumption. After C-phycocyanin (C-PC) extraction, the waste cell residue (WCR) is composed of nutrients with 30% protein content. Here, the potential of WCR as a nitrogen source for Bacillus coagulans cultivation and bioproducts was evaluated. Nitrogen substitute from WCR under different conditions of 20—100 g L−1 was performed. B. coagulans cultivation was achieved with maximal viable cells at 7.6–9.5 log CFU mL−1. Increasing WCR gave decreasing lactate production. The highest lactate production at 27 g L−1 was achieved from WCR 20 g L−1, highlighting the potential use of A. platensis waste biomass residue as a nitrogen source for the growth of lactic acid bacteria and zero-waste biotechnology methodology. The utilization of renewable resources is a crucial step toward developing a sustainable industrial society.

Resveratrol reduces lactate production and modifies the ovarian cancer immune microenvironment.

Tumor cells show deregulated metabolism leading to an enrichment of lactate in the tumor microenvironment (TME). This lactate-rich environment has been reported to impair effector T cells. However, T-regulatory cells (Tregs) show metabolic advantages in lactate-rich TME that maintain a strong suppression of effector T cells, which leads to tumor immune evasion. Therefore, the glycolytic process of tumors could represent a therapeutic target, and agents that modify the energy metabolism of tumor cells have therapeutic potential. Resveratrol is a naturally occurring polyphenol that has been confirmed to suppress tumor cells' glycolytic metabolism. In this study, we show that resveratrol induces metabolic reprogramming in ovarian cancer cells. Resveratrol increases oxidative and decreases glycolysis, in association with decreased lactate production both in vitro and in vivo. Lactate reduction in TME weakens the suppressive function of Tregs, and subsequently restores anti-tumor immunity. Significantly, combined resveratrol and PD-1 blockade promote anti-tumor efficacy. These data suggest that resveratrol's anti-tumor actions in ovarian cancer could be explained, in part, through modification of the anti-tumor immunity, and indicate a novel treatment strategy for improving immune checkpoint blockade therapy using resveratrol.

Hypoxic in vitro culture reduces histone lactylation and impairs pre-implantation embryonic development in mice

BackgroundDynamic changes of histone posttranslational modifications are important contexts of epigenetic reprograming after fertilization in pre-implantation embryos. Recently, lactylation has been reported as a novel epigenetic modification that regulates various cellular processes, but its role during early embryogenesis has not been elucidated.ResultsWe examined nuclear accumulation of H3K23la, H3K18la and pan histone lactylation in mouse oocytes and pre-implantation embryos by immunofluorescence with specific antibodies. All of the three modifications were abundant in GV stage oocytes, and both H3K23la and pan histone lactylation could be detected on the condensed chromosomes of the MII oocytes, while H3K18la were not detected. After fertilization, the nuclear staining of H3K23la, H3K18la and pan histone lactylation was faint in zygotes but homogeneously stained both of the parental pronuclei. The signal remained weak in the early cleavage stage embryos and increased remarkably in the blastocyst stage embryos. Comparison of the embryos cultured in four different conditions with varying concentrations of oxygen found that H3K23la, H3K18la and pan histone lactylation showed similar and comparable staining pattern in embryos cultured in atmospheric oxygen concentration (20% O2), gradient oxygen concentration (5% O2 to 2% O2) and embryos obtained from in vivo, but the modifications were greatly reduced in embryos cultured in hypoxic condition (2% O2). In contrast, nuclear accumulation of H3K18ac or H3K23ac was not significantly affected under hypoxic condition. Moreover, the developmental rate of in vitro cultured embryo was significantly reduced by low oxygen concentration and small molecule inhibition of LDHA activity led to decreased lactate production, as well as reduced histone lactylation and compromised developmental rate.ConclusionsWe provided for the first time the dynamic landscape of H3K23la, H3K18la and pan histone lactylation in oocytes and pre-implantation embryos in mice. Our data suggested that histone lactylation is subjected to oxygen concentration in the culture environment and hypoxic in vitro culture reduces histone lactylation, which in turn compromises developmental potential of pre-implantation embryos in mice.

Enzymatic activation of pyruvate kinase increases cytosolic oxaloacetate to inhibit the Warburg effect.

Pharmacological activation of the glycolytic enzyme PKM2 or expression of the constitutively active PKM1 isoform in cancer cells results in decreased lactate production, a phenomenon known as the PKM2 paradox in the Warburg Effect. Here we show that oxaloacetate (OAA) is a competitive inhibitor of human lactate dehydrogenase A (LDHA) and that elevated PKM2 activity increases de novo synthesis of OAA through glutaminolysis, thereby inhibiting LDHA in cancer cells. We also show that replacement of human LDHA with rabbit LDHA, which is relatively resistant to OAA inhibition, eliminated the paradoxical correlation between the elevated PKM2 activity and the decreased lactate concentration in cancer cells treated with a PKM2 activator. Furthermore, rabbit LDHA-expressing tumors displayed resistance to the PKM2 activator compared to human LDHA-expressing tumors in mice. These findings describe a mechanistic explanation for the PKM2 paradox by showing that OAA accumulates to inhibit LDHA following PKM2 activation.

MYC-mediated early glycolysis negatively regulates proinflammatory responses by controlling IRF4 in inflammatory macrophages.

SUMMARYMYC activates different metabolic programs in a cell-type- and cell-status-dependent manner. However, the role of MYC in inflammatory macrophages has not yet been determined. Metabolic and molecular analyses reveal that MYC, but not hypoxia inducible factor 1 (HIF1), is involved in enhancing early glycolytic flux during inflammatory macrophage polarization. Ablation of MYC decreases lactate production by regulating lactate dehydrogenase (LDH) activity and causes increased inflammatory cytokines by regulating interferon regulatory factor 4 (IRF4) in response to lipopolysaccharide. Moreover, myeloid-specific deletion of MYC and pharmacological inhibition of the MYC/LDH axis enhance inflammation and the bacterial clearance in vivo. These results elucidate the potential role of the MYC/LDH/IRF4 axis in inflammatory macrophages by connecting early glycolysis with inflammatory responses and suggest that modulating early glycolytic flux mediated by the MYC/LDH axis can be used to open avenues for the therapeutic modulation of macrophage polarization to fight against bacterial infection.

The PFKFB3 Inhibitor AZ67 Inhibits Angiogenesis Independently of Glycolysis Inhibition.

Angiogenesis is the process of new blood vessel formation. In this complex orchestrated growth, many factors are included. Lately, focus has shifted to endothelial cell metabolism, particularly to the PFKFB3 protein, a key regulatory enzyme of the glycolytic pathway. A variety of inhibitors of this important target have been studied, and a plethora of biological effects related to the process of angiogenesis have been reported. However, recent studies have disputed their mechanism of action, questioning whether all the effects are indeed due to PFKFB3 inhibition. Remarkably, the most well-studied inhibitor, 3PO, does not bind to PFKFB3, raising questions about this target. In our study, we aimed to elucidate the effects of PFKFB3 inhibition in angiogenesis by using the small molecule AZ67. We used isothermal titration calorimetry and confirmed binding to PFKFB3. In vitro, AZ67 did not decrease lactate production in endothelial cells (ECs), nor ATP levels, but exhibited good inhibitory efficacy in the tube-formation assay. Surprisingly, this was independent of EC migratory and proliferative abilities, as this was not diminished upon treatment. Strikingly however, even the lowest dose of AZ67 demonstrated significant inhibition of angiogenesis in vivo. To our knowledge, this is the first study to demonstrate that the process of angiogenesis can be disrupted by targeting PFKFB3 independently of glycolysis inhibition.

Pharmacokinetic and Biochemical Profiling of Sodium Dichloroacetate in Pregnant Ewes and Fetuses.

Sodium dichloroacetate (DCA) is an investigational drug that shows promise in the treatment of acquired and congenital mitochondrial diseases, including myocardial ischemia and failure. DCA increases glucose utilization and decreases lactate production, so it may also have clinical utility in reducing lactic acidosis during labor. In the current study, we tested the ability of DCA to cross the placenta and be measured in fetal blood after intravenous administration to pregnant ewes during late gestation and labor. Sustained administration of DCA to the mother over 72 hours achieved pharmacologically active levels of DCA in the fetus and decreased fetal plasma lactate concentrations. Multicompartmental pharmacokinetics modeling indicated that drug metabolism in the fetal and maternal compartments is best described by the DCA inhibiting lactate production in both compartments, consistent with our finding that the hepatic expression of the DCA-metabolizing enzyme glutathione transferase zeta1 was decreased in the ewes and their fetuses exposed to the drug. We provide the first evidence that DCA can cross the placental compartment to enter the fetal circulation and inhibit its own hepatic metabolism in the fetus, leading to increased DCA concentrations and decreased fetal plasma lactate concentrations during its parenteral administration to the mother. SIGNIFICANCE STATEMENT: This study was the first to administer sodium dichloroacetate (DCA) to pregnant animals (sheep). It showed that DCA administered to the mother can cross the placental barrier and achieve concentrations in fetus sufficient to decrease fetal lactate concentrations. Consistent with findings reported in other species, DCA-mediated inhibition of glutathione transferase zeta1 was also observed in ewes, resulting in reduced metabolism of DCA after prolonged administration.

IRF3 reduces adipose thermogenesis via ISG15-mediated reprogramming of glycolysis

Adipose thermogenesis is repressed in obesity, reducing the homeostatic capacity to compensate for chronic overnutrition. Inflammation inhibits adipose thermogenesis, but little is known about how this occurs. Here we showed that the innate immune transcription factor IRF3 is a strong repressor of thermogenic gene expression and oxygen consumption in adipocytes. IRF3 achieved this by driving expression of the ubiquitin-like modifier ISG15, which became covalently attached to glycolytic enzymes, thus reducing their function and decreasing lactate production. Lactate repletion was able to restore thermogenic gene expression, even when the IRF3/ISG15 axis was activated. Mice lacking ISG15 phenocopied mice lacking IRF3 in adipocytes, as both had elevated energy expenditure and were resistant to diet-induced obesity. These studies provide a deep mechanistic understanding of how the chronic inflammatory milieu of adipose tissue in obesity prevents thermogenic compensation for overnutrition.

FSMP-02. CHANGES IN GLUTAMINE METABOLISM INDUCED BY OXALOACETATE IN GLIOBLASTOMA

Anhydrous Enol-Oxaloacetate (AEO) has been shown to significantly increase survival and decrease tumor growth rates in animal models of glioblastoma and hepatocellular carcinoma. In the body, AEO is metabolized to “oxaloacetate” (OAA). Earlier, we demonstrated that AEO drastically reduced Warburg glycolysis in glioblastoma cells which was determined by the increase in pyruvate to lactate ratio and a 48.8% decrease in lactate production in 13C-labeled glucose metabolism studies. We have expanded this previous work to examine 13C-labeled glutamine metabolism. Cultured solid tumor cancer cells strongly rely on both glucose and glutamine to synthesize carbon intermediates for anaplerotic reactions. With treatment of OAA, we hypothesize that glutamine-derived OAA may be reduced which can be tracked through the use GC-MS based 13C-labeled glutamine isotopomer experiments. Patient-derived glioblastoma cells were grown in 15 mM glucose and 2 mM glutamine containing DMEM medium supplemented with 2 mM OAA for 10 days. 24 hours prior to harvesting the cells, 4 mM of [U-13C]glutamine was introduced to the medium. OAA treated cells showed significant decrease in the protein levels of lactate dehydrogenase A and C which indicates the switching off of glycolysis to support the utilization of elevated OAA levels during glutamine metabolism in the TCA cycle. 13C mass distribution analysis showed significant decrease in malate, aspartate and citrate pools (malate: 8.8%, p = 0.0098; aspartate: 9.2%, p = 0.0064; citrate: 9.5%, p = 0.0036) in their M+4 isotopomer labeling in OAA treated group compared to the control group. Decrease in lactate generation may reduce cancer proliferation, migration and invasion. Together, these data provide alternative way to modulate energy metabolism in glioblastoma using AEO treatment. Similarly, AEO treatment in other solid tumors (e.g. pancreatic ductal adenocarcinoma) may produce altered glutamine metabolism which may be of therapeutic value.

NAMPT promotes hepatitis B virus replication and liver cancer cell proliferation through the regulation of aerobic glycolysis.

Nicotinamide phosphoribosyltransferase (NAMPT) is a critical rate-limiting enzyme involved in NAD synthesis that has been shown to contribute to the progression of liver cancer. However, the potential role and mechanism of NAMPT in hepatitis B virus (HBV)-associated liver cancer remain unclear. The present study assessed the expression of NAMPT in HBV-positive and -negative liver cancer cells, and investigated whether HBV-induced NAMPT expression is dependent on HBV X protein (HBx). In addition, the role of NAMPT in HBV replication and transcription, and in HBV-mediated liver cancer cell growth was explored. The effects of NAMPT on the glycolytic pathway were also evaluated. Reverse transcription-quantitative PCR and western blotting results revealed that NAMPT expression levels were significantly higher in HBV-positive liver cancer cells than in HBV-negative liver cancer cells, and this effect was HBx-dependent. Moreover, the activation of NAMPT was demonstrated to be required for HBV replication and transcription. The NAMPT inhibitor FK866 repressed cell survival and promoted cell death in HBV-expressing liver cancer cells, and these effects were attenuated by nicotinamide mononucleotide. Furthermore, the inhibition of NAMPT was associated with decreased glucose uptake, decreased lactate production and decreased ATP levels in HBV-expressing liver cancer cells, indicating that NAMPT may promote the aerobic glycolysis. Collectively, these findings reveal a positive feedback loop in which HBV enhances NAMPT expression and the activation of NAMPT promotes HBV replication and HBV-mediated malignant cell growth in liver cancer. The present study highlights the important role of NAMPT in the regulation of aerobic glycolysis in HBV-mediated liver cancer, and suggests that NAMPT may be a promising treatment target for patients with HBV-associated liver cancer.

LAMC1 is related to the poor prognosis of patients with gastric cancer and facilitates cancer cell malignancies.

Gastric cancer is a common malignancy in the alimentary system. The laminin subunit gamma 1 (LAMC1) is an important oncogene in human cancers. However, how and whether LAMC1 takes part in gastric cancer progression is largely uncertain. This study analyzed the association between clinical factors of patients and LAMC1 expression and explored the influence of LAMC1 silencing on cell proliferation, migration, invasion, the Warburg effect, protein kinase B (AKT) pathway, and mitogen-activated protein kinase (MEK)/extracellular regulated protein kinase (ERK) pathway in gastric cancer cells. Our results showed LAMC1 abundance was enhanced in gastric cancer samples and cells. LAMC1 was related to the clinical stage, tumor depth, lymph node metastasis, and distant metastasis of patients. LAMC1 silencing inhibited cell proliferation, migration, and invasion. Moreover, LAMC1 knockdown suppressed the Warburg effect via decreasing lactate production, lactate dehydrogenase (LDH) activity, and glucose uptake. LAMC1 interference blocked the activation of the AKT and MEK/ERK signaling. Collectively, LAMC1 knockdown constrained cell proliferation, migration, invasion, and the Warburg effect in gastric cancer cells via inactivating the AKT and MEK/ERK pathway.