CTRP6 promotes the macrophage inflammatory response, and its deficiency attenuates LPS-induced inflammation.

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors. We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesity-linked inflammation. However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown. Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs). Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression. We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6. Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion. Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs. Importantly, in accordance with our in vitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion. Finally, loss of CTRP6 in mice also dampened LPS-induced inflammation and hypothermia. Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts.

Long non-coding RNA MIR17HG impedes FOSL2-mediated transcription activation of HIC1 to maintain a pro-inflammatory phenotype of microglia during intracerebral haemorrhage.

Activation and polarization of microglia play decisive roles in the progression of intracerebral haemorrhage (ICH), and lactate exposure correlates with microglia polarization. This study explores molecules influencing lactate production and microglia phenotype alteration following ICH. A murine model of ICH was induced by intracerebral injection of collagenase. The mice experienced autonomous neurological function recovery, haematoma resolution and rapid lactate production, along with a gradual increase in angiogenesis activity, neuronal recovery and an M1-to-M2 phenotype change of microglia. Galloflavin, a lactate dehydrogenase antagonist, suppressed this phenotype change and the functional recovery in mice. FOS like 2 (FOSL2) was significantly upregulated in the brain tissues from day 7 post-ICH. Overexpression of FOSL2 induced an M1-to-M2 phenotype shift in microglia and accelerated lactate production in vivo and in haemoglobin-treated microglia in vitro. Long non-coding RNA MIR17HG impeded FOSL2-mediated transcription activation of hypermethylated in cancer 1 (HIC1). MIR17HG overexpression induced pro-inflammatory activation of microglia in mice, which was blocked by further HIC1 overexpression. Overall, this study demonstrates that MIR17HG maintains a pro-inflammatory phenotype of microglia during ICH progression by negating FOSL2-mediated transcription activation of HIC1. Specific inhibition of MIR17HG or upregulation of FOSL2 or HIC1 may favour inflammation inhibition and haematoma resolution in ICH.

Circular RNA circ_0000592 elevates ANXA4 expression via sponging miR-1179 to facilitate tumor progression in gastric cancer.

Increasing evidence indicated that dysregulated circular RNAs were implicated in the progression of multiple malignancies. However, the function of circ_0000592 in gastric cancer (GC) progression and its associated mechanism remain poorly understood. Quantitative real-time PCR and Western blot assay were performed to detect RNA and protein expression. Cell proliferation, migration and invasion were analyzed by 5-Ethynyl-2'-deoxyuridine staining assay, Transwell migration assay and Transwell invasion assay, respectively. The glucose/lactate assay kit was used to assess the rates of glucose consumption and lactate production. The interaction between microRNA-1179 (miR-1179) and circ_0000592 or Annexin A4 (ANXA4) was confirmed by dual-luciferase reporter assay and RNA pull-down assay. Xenograft tumor model was established to investigate the effect of circ_0000592 on tumor growth in vivo. Circ_0000592 expression was elevated in GC tissues and cells. Circ_0000592 knockdown hampered cell proliferation, migration, invasion and glycolysis of GC cells. MiR-1179 was a direct target of circ_0000592, and circ_0000592 silencing-mediated effects in GC cells were partly reversed by the knockdown of miR-1179. MiR-1179 interacted with the 3' untranslated region (3'UTR) of ANXA4. Circ_0000592 silencing reduced ANXA4 expression partly by upregulating miR-1179 in GC cells. ANXA4 overexpression partly overturned circ_0000592 knockdown-induced effects in GC cells. Circ_0000592 depletion markedly suppressed xenograft tumor growth in vivo. Circ_0000592 contributed to GC progression through regulating miR-1179/ANXA4 axis, which provided novel potential biomarkers and therapeutic targets for GC treatment.

Tubulin alpha 1c promotes aerobic glycolysis and cell growth through upregulation of yes association protein expression in breast cancer.

Tubulin alpha 1c (TUBA1C) as a member of α-tubulin was identified to take part in the occurrence and development of hepatocellular carcinoma and pancreatic cancer. Using the bioinformatics, we noticed that TUBA1C level was also increased in breast cancer was also demonstrated. Here, we explored TUBA1 role in modulation of breast cancer cell aerobic glycolysis, growth and migration and explored whether yes association protein (YAP) was involved. Fifty-five matched breast cancer tissues and the para-carcinoma normal tissues were included in this study and used to verify TUBA1C expression using quantitative reverse transcription-PCR and western blotting. ATP level, lactate secretion and glucose consumption were used to assess aerobic glycolysis. Cell growth, invasion, migration and tumorigenesis were detected using cell count kit-8, transwell, wound healing and animal assays. TUBA1 was upregulated in breast cancer, which associated with advanced primary tumor, lymph node, metastasis stage and tumor size. Silencing of TUBA1C with sh-TUBA1C infection led to significant inhibitions in ATP level, lactate secretion, glucose consumption, cell growth, migration, invasion and tumorigenesis, as well as declined YAP expression, while TUBA1C overexpression induced a opposite result. And, the above tendencies induced by TUBA1C downregulation were reversed by YAP overexpression. This study revealed that TUBA1C was overexpressed in breast cancer and promoted aerobic glycolysis and cell growth through upregulation of YAP expression.

NOX4-derived ROS-induced overexpression of FOXM1 regulates aerobic glycolysis in glioblastoma.

BACKGROUND: Increased expression of the transcription factor Forkhead box M1 (FOXM1) has been reported to play an important role in the progression and development of multiple tumors, but the molecular mechanisms that regulate FOXM1 expression remain unknown, and the role of FOXM1 in aerobic glycolysis is still not clear. METHODS: The expression of FOXM1 and NADPH oxidase 4 (NOX4) in normal brain tissues and glioma was detected in data from the TCGA database and in our specimens. The effect of NOX4 on the expression of FOXM1 was determined by Western blot, qPCR, reactive oxygen species (ROS) production assays, and luciferase assays. The functions of NOX4 and FOXM1 in aerobic glycolysis in glioblastoma cells were determined by a series of experiments, such as Western blot, extracellular acidification rate (ECAR), lactate production, and intracellular ATP level assays. A xenograft mouse model was established to test our findings in vivo. RESULTS: The expression of FOXM1 and NOX4 was increased in glioma specimens compared with normal brain tissues and correlated with poor clinical outcomes. Aberrant mitochondrial reactive oxygen species (ROS) generation of NOX4 induced FOXM1 expression. Mechanistic studies demonstrated that NOX4-derived MitoROS exert their regulatory role on FOXM1 by mediating hypoxia-inducible factor 1α (HIF-1α) stabilization. Further research showed that NOX4-derived MitoROS-induced HIF-1α directly activates the transcription of FOXM1 and results in increased FOXM1 expression. Overexpression of NOX4 or FOXM1 promoted aerobic glycolysis, whereas knockdown of NOX4 or FOXM1 significantly suppressed aerobic glycolysis, in glioblastoma cells. NOX4-induced aerobic glycolysis was dependent on elevated FOXM1 expression, as FOXM1 knockdown abolished NOX4-induced aerobic glycolysis in glioblastoma cells both in vitro and in vivo. CONCLUSION: Increased expression of FOXM1 induced by NOX4-derived MitoROS plays a pivotal role in aerobic glycolysis, and our findings suggest that inhibition of NOX4-FOXM1 signaling may present a potential therapeutic target for glioblastoma treatment.

Tumor Lymphatic Interactions Induce CXCR2-CXCL5 Axis and Alter Cellular Metabolism and Lymphangiogenic Pathways to Promote Cholangiocarcinoma.

Cholangiocarcinoma (CCA), or cancer of bile duct epithelial cells, is a very aggressive malignancy characterized by early lymphangiogenesis in the tumor microenvironment (TME) and lymph node (LN) metastasis which correlate with adverse patient outcome. However, the specific roles of lymphatic endothelial cells (LECs) that promote LN metastasis remains unexplored. Here we aimed to identify the dynamic molecular crosstalk between LECs and CCA cells that activate tumor-promoting pathways and enhances lymphangiogenic mechanisms. Our studies show that inflamed LECs produced high levels of chemokine CXCL5 that signals through its receptor CXCR2 on CCA cells. The CXCR2-CXCL5 signaling axis in turn activates EMT (epithelial-mesenchymal transition) inducing MMP (matrix metalloproteinase) genes such as GLI, PTCHD, and MMP2 in CCA cells that promote CCA migration and invasion. Further, rate of mitochondrial respiration and glycolysis of CCA cells was significantly upregulated by inflamed LECs and CXCL5 activation, indicating metabolic reprogramming. CXCL5 also induced lactate production, glucose uptake, and mitoROS. CXCL5 also induced LEC tube formation and increased metabolic gene expression in LECs. In vivo studies using CCA orthotopic models confirmed several of these mechanisms. Our data points to a key finding that LECs upregulate critical tumor-promoting pathways in CCA via CXCR2-CXCL5 axis, which further augments CCA metastasis.

Methylation-associated silencing of miR-9-1 promotes nasopharyngeal carcinoma progression and glycolysis via HK2.

Characteristically, cancer cells metabolize glucose through aerobic glycolysis, known as the Warburg effect. Accumulating evidence suggest that during cancer formation, microRNAs (miRNAs) could regulate such metabolic reprogramming. In the present study, miR-9-1 was identified as significantly hypermethylated in nasopharyngeal carcinoma (NPC) cell lines and clinical tissues. Ectopic expression of miR-9-1 inhibited NPC cell growth and glycolytic metabolism, including reduced glycolysis, by reducing lactate production, glucose uptake, cellular glucose-6-phosphate levels, and ATP generation in vitro and tumor proliferation in vivo. HK2 (encoding hexokinase 2) was identified as a direct target of miR-9-1 using luciferase reporter assays and Western blotting. In NPC cells, hypermethylation regulates miR-9-1 expression and inhibits HK2 translation by directly targeting its 3' untranslated region. MiR-9-1 overexpression markedly reduced HK2 protein levels. Restoration of HK2 expression attenuated the inhibitory effect of miR-9-1 on NPC cell proliferation and glycolysis. Fluorescence in situ hybridization results indicated that miR-9-1 expression was an independent prognostic factor in NPC. Our findings revealed the role of the miR-9-1/HK2 axis in the metabolic reprogramming of NPC, providing a potential therapeutic strategy for NPC.

Sugar or Fat? Renal Tubular Metabolism Reviewed in Health and Disease.

The kidney is a highly metabolically active organ that relies on specialized epithelial cells comprising the renal tubules to reabsorb most of the filtered water and solutes. Most of this reabsorption is mediated by the proximal tubules, and high amounts of energy are needed to facilitate solute movement. Thus, proximal tubules use fatty acid oxidation, which generates more adenosine triphosphate (ATP) than glucose metabolism, as its preferred metabolic pathway. After kidney injury, metabolism is altered, leading to decreased fatty acid oxidation and increased lactic acid generation. This review discusses how metabolism differs between the proximal and more distal tubular segments of the healthy nephron. In addition, metabolic changes in acute kidney injury and chronic kidney disease are discussed, as well as how these changes in metabolism may impact tubule repair and chronic kidney disease progression.

The Warburg effect as a therapeutic target for bladder cancers and intratumoral heterogeneity in associated molecular targets.

Bladder cancer is the 10th most common cancer worldwide. For muscle-invasive bladder cancer (MIBC), treatment includes radical cystectomy, radiotherapy, and chemotherapy; however, the outcome is generally poor. For non-muscle-invasive bladder cancer (NMIBC), tumor recurrence is common. There is an urgent need for more effective and less harmful therapeutic approaches. Here, bladder cancer cell metabolic reprogramming to rely on aerobic glycolysis (the Warburg effect) and expression of associated molecular therapeutic targets by bladder cancer cells of different stages and grades, and in freshly resected clinical tissue, is investigated. Importantly, analyses indicate that the Warburg effect is a feature of both NMIBCs and MIBCs. In two in vitro inducible epithelial-mesenchymal transition (EMT) bladder cancer models, EMT stimulation correlated with increased lactate production, the end product of aerobic glycolysis. Protein levels of lactate dehydrogenase A (LDH-A), which promotes pyruvate enzymatic reduction to lactate, were higher in most bladder cancer cell lines (compared with LDH-B, which catalyzes the reverse reaction), but the levels did not closely correlate with aerobic glycolysis rates. Although LDH-A is expressed in normal urothelial cells, LDH-A knockdown by RNAi selectively induced urothelial cancer cell apoptotic death, whereas normal cells were unaffected-identifying LDH-A as a cancer-selective therapeutic target for bladder cancers. LDH-A and other potential therapeutic targets (MCT4 and GLUT1) were expressed in patient clinical specimens; however, positive staining varied in different areas of sections and with distance from a blood vessel. This intratumoral heterogeneity has important therapeutic implications and indicates the possibility of tumor cell metabolic coupling.

Excess exogenous pyruvate inhibits lactate dehydrogenase activity in live cells in an MCT1-dependent manner.

Cellular pyruvate is an essential metabolite at the crossroads of glycolysis and oxidative phosphorylation, capable of supporting fermentative glycolysis by reduction to lactate mediated by lactate dehydrogenase (LDH) among other functions. Several inherited diseases of mitochondrial metabolism impact extracellular (plasma) pyruvate concentrations, and [1-13C]pyruvate infusion is used in isotope-labeled metabolic tracing studies, including hyperpolarized magnetic resonance spectroscopic imaging. However, how these extracellular pyruvate sources impact intracellular metabolism is not clear. Herein, we examined the effects of excess exogenous pyruvate on intracellular LDH activity, extracellular acidification rates (ECARs) as a measure of lactate production, and hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates across a panel of tumor and normal cells. Combined LDH activity and LDHB/LDHA expression analysis intimated various heterotetrameric isoforms comprising LDHA and LDHB in tumor cells, not only canonical LDHA. Millimolar concentrations of exogenous pyruvate induced substrate inhibition of LDH activity in both enzymatic assays ex vivo and in live cells, abrogated glycolytic ECAR, and inhibited hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates in cellulo. Of importance, the extent of exogenous pyruvate-induced inhibition of LDH and glycolytic ECAR in live cells was highly dependent on pyruvate influx, functionally mediated by monocarboxylate transporter-1 localized to the plasma membrane. These data provided evidence that highly concentrated bolus injections of pyruvate in vivo may transiently inhibit LDH activity in a tissue type- and monocarboxylate transporter-1-dependent manner. Maintaining plasma pyruvate at submillimolar concentrations could potentially minimize transient metabolic perturbations, improve pyruvate therapy, and enhance quantification of metabolic studies, including hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging and stable isotope tracer experiments.

Identification of novel inhibitors of GLUT1 by virtual screening and cell-based assays.

In order to fuel the uncontrolled cell proliferation and division, tumor cells reprogram the energy metabolism to Warburg effect, where glucose is preferably converted by glycolysis even in the presence of oxygen. However, the high energetic demand of tumor cells require upregulating the expression of glucose transporters, notably GLUT1, which substantially increases glucose uptake into cytoplasm. GLUT1 is overexpressed in a variety of tumor cells and is likely to be a potential drug target in the treatment of pan-cancers. Although many small molecules were reported to inhibit the glucose uptake function by various measurements, several shortcomings such as weak binding affinity, low specificity of the known inhibitors demand the identification of alternative inhibitors with novel scaffolds. In this study, we performed a virtual screening campaign by docking each compound from Chemdiv database to the glucose binding pocket based on the crystal structure of GLUT1 (PDB ID 4PYP) and four small molecules with novel scaffolds were identified to inhibit the glucose uptake of cancer cells at the sub-micromole level. The identified compounds may serve as starting points for the development of anti-cancer drugs via the manipulation of the energy metabolism.

Fat Induces Glucose Metabolism in Nontransformed Liver Cells and Promotes Liver Tumorigenesis.

Hepatic fat accumulation is associated with diabetes and hepatocellular carcinoma (HCC). Here, we characterize the metabolic response that high-fat availability elicits in livers before disease development. After a short term on a high-fat diet (HFD), otherwise healthy mice showed elevated hepatic glucose uptake and increased glucose contribution to serine and pyruvate carboxylase activity compared with control diet (CD) mice. This glucose phenotype occurred independently from transcriptional or proteomic programming, which identifies increased peroxisomal and lipid metabolism pathways. HFD-fed mice exhibited increased lactate production when challenged with glucose. Consistently, administration of an oral glucose bolus to healthy individuals revealed a correlation between waist circumference and lactate secretion in a human cohort. In vitro, palmitate exposure stimulated production of reactive oxygen species and subsequent glucose uptake and lactate secretion in hepatocytes and liver cancer cells. Furthermore, HFD enhanced the formation of HCC compared with CD in mice exposed to a hepatic carcinogen. Regardless of the dietary background, all murine tumors showed similar alterations in glucose metabolism to those identified in fat exposed nontransformed mouse livers, however, particular lipid species were elevated in HFD tumor and nontumor-bearing HFD liver tissue. These findings suggest that fat can induce glucose-mediated metabolic changes in nontransformed liver cells similar to those found in HCC. SIGNIFICANCE: With obesity-induced hepatocellular carcinoma on a rising trend, this study shows in normal, nontransformed livers that fat induces glucose metabolism similar to an oncogenic transformation.

In Vitro Evaluation of Probiotic Properties of Lactobacillus plantarum UBLP40 Isolated from Traditional Indigenous Fermented Food.

In this study, traditional indigenous fermented food isolate Lactobacillus plantarum UBLP40 was screened for in vitro probiotic properties, antibiotic susceptibility, hemolytic activity, production of lactic acid, hydrogen peroxide, bile salt hydrolase and phytase, and antioxidative activity. Results showed that Lact. plantarum UBLP40 can survive simulated gastrointestinal conditions, adhere to mucin, possess a hydrophobic cell surface, ability to auto-aggregation, and possessed antimicrobial activity against Micrococcus luteus MTCC 106, methicillin-resistant Staphylococcus aureus subsp. aureus ATCC® BAA-1720, Pseudomonas aeruginosa MTCC 1688, and Escherichia coli MTCC 1687. Lact. plantarum UBLP40 produced 48.59 U/mg phytase and 1.78 ± 0.01 gm % lactic acid and showed the ability to produce hydrogen peroxide and bile salt hydrolase. Moreover, the usual antibiotic susceptible profile and non-hemolytic activity indicated the safety of the strain. The intracellular extract of UBLP40 showed 13.8 ± 1.4% (equivalent to ~8 µM butylated hydroxytoluene) α,α-diphenyl-ß-picrylhydrazyl (DPPH) radical scavenging activity, reducing activity equivalent to 1 µg L-cysteine, Fe2+ chelation equivalent to 5 µM ethylenediaminetetraacetic acid, and exhibited 17.73 ± 4.40 µM glutathione per gram of protein. In conclusion, this study demonstrates that Lact. plantarum UBLP40 is a potential probiotic candidate.

Glutaminolysis is a metabolic route essential for survival and growth of prostate cancer cells and a target of 5α-dihydrotestosterone regulation.

PURPOSE: Resistance to androgen-deprivation therapies and progression to so-called castrate-resistant prostate cancer (CRPC) remain challenges in prostate cancer (PCa) management and treatment. Among other alterations, CRPC has been associated with metabolic reprogramming driven by androgens. Here, we investigated the role of androgens in regulating glutaminolysis in PCa cells and determined the relevance of this metabolic route in controlling the survival and growth of androgen-sensitive (LNCaP) and CRPC (DU145 and PC3) cells. METHODS: PCa cells (LNCaP, DU145 and PC3) and 3-month old rats were treated with 5α-dihydrotestosterone (DHT). Alternatively, LNCaP cells were exposed to the glutaminase inhibitor BPTES, alone or in combination with the anti-androgen bicalutamide. Biochemical, Western blot and extracellular flux assays were used to evaluate the viability, proliferation, migration and metabolism of PCa cells in response to DHT treatment or glutaminase inhibition. RESULTS: We found that DHT up-regulated the expression of the glutamine transporter ASCT2 and glutaminase, both in vitro in LNCaP cells and in vivo in rat prostate cells. BPTES diminished the viability and migration of PCa cells, while increasing caspase-3 activity. CRPC cells were found to be more dependent on glutamine and more sensitive to glutaminase inhibition. BPTES and bicalutamide co-treatment had an additive effect on suppressing LNCaP cell viability. Finally, we found that inhibition of glutaminolysis differentially affected glycolysis and lipid metabolism in both androgen-sensitive and CRPC cells. CONCLUSION: Our data reveal glutaminolysis as a central metabolic route controlling PCa cell fate and highlight the relevance of targeting glutaminase for CRPC treatment.

ATGL activity regulates GLUT1-mediated glucose uptake and lactate production via TXNIP stability in adipocytes.

Traditionally, lipolysis has been regarded as an enzymatic activity that liberates fatty acids as metabolic fuel. However, recent work has shown that novel substrates, including a variety of lipid compounds such as fatty acids and their derivatives, release lipolysis products that act as signaling molecules and transcriptional modulators. While these studies have expanded the role of lipolysis, the mechanisms underpinning lipolysis signaling are not fully defined. Here, we uncover a new mechanism regulating glucose uptake, whereby activation of lipolysis, in response to elevated cAMP, leads to the stimulation of thioredoxin-interacting protein (TXNIP) degradation. This, in turn, selectively induces glucose transporter 1 surface localization and glucose uptake in 3T3-L1 adipocytes and increases lactate production. Interestingly, cAMP-induced glucose uptake via degradation of TXNIP is largely dependent upon adipose triglyceride lipase (ATGL) and not hormone-sensitive lipase or monoacylglycerol lipase. Pharmacological inhibition or knockdown of ATGL alone prevents cAMP-dependent TXNIP degradation and thus significantly decreases glucose uptake and lactate secretion. Conversely, overexpression of ATGL amplifies the cAMP response, yielding increased glucose uptake and lactate production. Similarly, knockdown of TXNIP elicits enhanced basal glucose uptake and lactate secretion, and increased cAMP further amplifies this phenotype. Overexpression of TXNIP reduces basal and cAMP-stimulated glucose uptake and lactate secretion. As a proof of concept, we replicated these findings in human primary adipocytes and observed TXNIP degradation and increased glucose uptake and lactate secretion upon elevated cAMP signaling. Taken together, our results suggest a crosstalk between ATGL-mediated lipolysis and glucose uptake.

circPVT1 promotes osteosarcoma glycolysis and metastasis by sponging miR-423-5p to activate Wnt5a/Ror2 signaling.

Osteosarcoma (OS) is the most prevalent form of bone cancer. It has a high metastatic potential and progresses rapidly. The molecular mechanisms of OS remain unclear and this study aims to examine the functional role of circPVT1 and miR-423-5p in OS. Quantitative RT-PCR (qRT-PCR) and western blotting were used to examine levels of miR-423-5p, circPVT1, Wnt5a, Ror2, and glycolysis-related proteins, including HK2, PKM2, GLUT1, and LDHA. Colony formation and transwell assays were used to test the roles of miR-423-5p, circPVT1, and Wnt5a/Ror2 in OS cell proliferation, migration, and invasion. Dual luciferase assay and Ago2-RIP were used to validate the interactions of miR-423-5p/Wnt5a, miR-423-5p/Ror2, and circPVT1/miR-423-5p. Glucose uptake assay and measurement of lactate production were performed to assess the glycolysis process. A nude mouse xenograft model was used to evaluate the effects of sh-circPVT1 and miR-423-5p mimics on tumor growth and metastasis in vivo. miR-423-5p was reduced in both OS tissues and OS cell lines, while Wnt5a/Ror2 and circPVT1 were elevated. miR-423-5p bound to 3'-UTR of Wnt5a and Ror2 mRNA, and inhibited glycolysis and OS cell proliferation, migration, and invasion by targeting Wnt5a and Ror2. circPVT1 interacted with miR-423-5p and activated Wnt5a/Ror2 signaling by sponging miR-423-5p. Knockdown of circPVT1 or overexpression of miR-423-5p suppressed OS tumor growth and metastasis in vivo. miR-423-5p inhibited OS glycolysis, proliferation, migration, and metastasis by targeting and suppressing Wnt5a/Ror2 signaling pathway, while circPVT1 promoted those processes by acting as a sponge of miR-423-5p.

Resource allocation explains lactic acid production in mixed-culture anaerobic fermentations.

Lactate production in anaerobic carbohydrate fermentations with mixed cultures of microorganisms is generally observed only in very specific conditions: the reactor should be run discontinuously and peptides and B vitamins must be present in the culture medium as lactic acid bacteria (LAB) are typically auxotrophic for amino acids. State-of-the-art anaerobic fermentation models assume that microorganisms optimise the adenosine triphosphate (ATP) yield on substrate and therefore they do not predict the less ATP efficient lactate production, which limits their application for designing lactate production in mixed-culture fermentations. In this study, a metabolic model taking into account cellular resource allocation and limitation is proposed to predict and analyse under which conditions lactate production from glucose can be beneficial for microorganisms. The model uses a flux balances analysis approach incorporating additional constraints from the resource allocation theory and simulates glucose fermentation in a continuous reactor. This approach predicts lactate production is predicted at high dilution rates, provided that amino acids are in the culture medium. In minimal medium and lower dilution rates, mostly butyrate and no lactate is predicted. Auxotrophy for amino acids of LAB is identified to provide a competitive advantage in rich media because less resources need to be allocated for anabolic machinery and higher specific growth rates can be achieved. The Matlab™ codes required for performing the simulations presented in this study are available at https://doi.org/10.5281/zenodo.4031144.

Effects of sterol-type elicitors on biochemical characterization of polysaccharides from Antrodia cinnamomea.

Sterols play crucial roles in the physiological functions of organisms. In this study, we examined the chemical and biological effects of sterol type elicitors, including squalene, cholesterol and stigmasterol, on polysaccharides (PSs) of Antrodia cinnamomea. Characteristic studies revealed that squalene and stigmasterol effectively increased the glucose contents in PSs. Specifically, squalene not only induced glucose content but also increased fucose and mannose levels in PSs. However, cholesterol did not induce changes in sugar content in PSs. We further identified that high dose squalene significantly promoted 20% yield (w/w) of PSs as well as significantly increased the glucose, galactose and fucose contents. In addition, the major PSs species had a molecular weight of 21 kDa, and squalene significantly increased its area percentage to 43.54. The biological effects of PSs (squalenePS) from squalene treated A. cinnamomea presented anticancer activities by inhibiting lung cancer cell viability and colony formation. Functional studies revealed that squalenePS reduced the glucose uptake and lactate secretion may correlate to inhibition of AKT activity and downregulation of glucose transporter (GLUT1) expression. Our findings suggested squalene may play vital roles in regulating the PSs assembling and bioactivities of A. cinnamomea. Moreover, squalene may be a potential supplement for adding the culture medium of A. cinnamomea.

Low glucose enhanced metformin's inhibitory effect on pancreatic cancer cells by suppressing glycolysis and inducing energy stress via up-regulation of miR-210-5p.

To explore mechanisms underlying the discrepancy in anti-tumor effects of metformin on pancreatic cancer cells PANC-1 under different glucose conditions. We cultured PANC-1 cells in 25 mM and 5 mM glucose media, then treated with or without metformin. It showed that metformin significantly inhibited proliferation and viability, induced apoptosis of PANC-1 cells, which was more pronounced in low-glucose than in high-glucose group. Metformin up-regulated the expression of miR-210-5p in low glucose, but not in high glucose. miR-210-5p mimic inhibited the viability of PANC-1 cells and further enhanced the inhibitory effect of metformin. miR-210-5p down-regulated the expression of PFKFB2, a predicted target gene of miR-210-5p, reduced the activity of PFK1 and LDH. Metformin significantly inhibited the expression of phosphorylation-PFKFB2(p-PFKFB2) in the low-glucose group and inhibited the LDH activity both in the low and high glucose groups, thus inhibiting anaerobic glycolysis and inducing energy stress. Cells in the high glucose group could make a compensatory adaptation to the energy stress induced by metformin through increasing glucose consumption. However, due to the limited glucose supply and high dependence on anaerobic glycolysis of cells in the low glucose group, they couldn't make effective adaptive compensation. Therefore, cells in the low-glucose group were more vulnerable to the toxicity of metformin. In conclusion, the enhanced inhibitory effect of metformin on PANC-1 cells cultured in low glucose may be due to the up-regulation of the expression of miR-210-5p, then inhibiting anaerobic glycolytic flux and inducing energy stress via repressing the expression of p-PFKFB2 and activity of LDH. ABBREVIATIONS: PC: pancreatic cancer; DM: diabetes mellitus; PFKFB2: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase2; PFK1: phosphofructokinases; LDH: lactate dehydrogenase; F-2,6-BP: fructose 2,6-bisphosphate.

Development of dual inhibitors targeting pyruvate dehydrogenase kinases and human lactate dehydrogenase A: High-throughput virtual screening, synthesis and biological validation.

Most cancer cells feature an altered glucose metabolism from oxidative phosphorylation to cytoplasmic glycolysis. Pyruvate dehydrogenase kinases (PDKs) and lactate dehydrogenase A (LDHA) play crucial roles in promotion of glycolysis, thus the inhibition of both enzymes is considered a promising strategy for developing of anticancer therapeutics. Herein, we describe the first discovery of series novel dual inhibitors targeting PDKs and LDHA. We identified 6 hits from a library database containing 485465 compounds through a high-throughput virtual screening assay. Hit-to-lead optimization enabled us to discover two compounds, namely 20e and 20k, which inhibited PDKs with IC50 values of 0.8, and 1.6 µM, respectively, and inhibited LDHA with IC50 values of 0.15 and 0.7 µM, respectively. Meanwhile, the two compounds reduced A549 cell proliferation with EC50 values of 13.2, and 15.7 µM. Furthermore, 20e and 20k decreased the lactate formation, and increased oxygen consumption, suggesting the two compounds modulated the glucose metabolic pathways in cancer cells.