Rate Of Lactate Production Research Articles

Lactylation of nuclear receptor coactivator 4 promotes ferritinophagy and glycolysis of neuronal cells after cerebral ischemic injury.

Ischemic stroke remains a major cause of disability and mortality. Nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy is involved in cerebral ischemic injury. Additionally, lactylation regulates the progression of ischemia injury. This study aimed to investigate the impact of NCOA4 on ferritinophagy and glycolysis of hippocampal neuron cells and its lactylation modification. Middle cerebral artery occlusion (MCAO) mouse and oxygen-glucose deprivation (OGD)-treated HT22 cell models were generated. Ferritinophagy was evaluated via detecting ferrous iron (Fe 2+ ), glutathione, malondialdehyde, and protein levels. Glycolysis was assessed by examining the glucose consumption, lactate production, and extracellular acidification rate. The lactylation was evaluated using immunoprecipitation and immunoblotting. Brain injury in vivo was analyzed by measuring brain infarct and neurological function. The results showed that NCOA4 expression was increased in the blood of patients with acute ischemia stroke, the peri-infarct region of the brain in MCAO mice (increased percentage: 142.11%) and OGD-treated cells (increased percentage: 114.70%). Knockdown of NCOA4 inhibited ferritinophagy and glycolysis of HT22 cells induced by OGD. Moreover, OGD promoted the lactylation of NCOA4 at lysine (K)450 sites, which enhanced NCOA4 protein stability. Additionally, interfering with NCOA4 attenuated brain infarction and neurological dysfunction in MCAO mice. Lactylation of NCOA4 at K450 sites promotes ferritinophagy and glycolysis of hippocampal neuron cells, thereby accelerating cerebral ischemic injury. These findings suggest a novel pathogenesis of ischemic stroke.

LMP1 enhances aerobic glycolysis in natural killer/T cell lymphoma

Natural killer/T cell lymphoma (NKTCL) exhibits highly aggressive clinical behavior, and the outcomes for relapsed/refractory patients are still poor. Recently, the mechanism underlying the effect of Epstein-Barr virus (EBV) infection, which has not been fully defined in NKTCL, has attracted great attention. We explored how LMP1 promoted aerobic glycolysis via metabolic sequencing combined with mRNA sequencing and immunoprecipitation coupled to mass spectrometry. Experimental assays were used to determine the effects of LMP1 and its downstream pathway on the function and glucose metabolism of NKTCL cells. The correlations between LMP1 expression in patients and their clinical features, treatment response, and prognosis were analyzed. Results show that LMP1 enhances NKTCL cell proliferation in vitro and in vivo, inhibits apoptosis, and decreases gemcitabine sensitivity. In addition, LMP1 also enhances aerobic glycolysis in NKTCL cells, as indicated by increases in glucose uptake, lactate production, and extracellular acidification rate. Clinically, LMP1 expression is correlated with risk stratification, treatment response, and prognosis, and higher LMP1 expression indicates greater SUVmax for NKTCL patients. Mechanistically, LMP1 competitively binds to TRAF3 to promote cell proliferation and aerobic glycolysis by regulating the noncanonical NF-κB pathway. The application of an NF-κB pathway inhibitor or reactivation of the NF-κB pathway affects aerobic glycolysis and the biological function of NKTCL cells. In summary, this study is the first to describe and define in detail how LMP1 affects glucose metabolism in NKTCL and might provide a novel perspective for further treatment.

Buyang Huanwu Decoction suppresses ischemic stroke by suppressing glycolysis and cell apoptosis in rat brain microvascular endothelial cells

BackgroundBuyang Huanwu Decoction (BHD) is widely used in Chinese clinical practice for the treatment and prevention of ischemic cerebral vascular diseases. This study was designed to investigate the effects of BHD on ischemic stroke (IS) and its underlying mechanism. MethodsThe middle cerebral artery occlusion (MCAO) rat model and oxygen-glucose deprivation and reoxygenation (OGD/R) rat brain microvascular endothelial cell (RBMVEC) models were established. Brain infarction size and neurological score were calculated following MCAO surgery. Evans blue was used to measure blood brain barrier (BBB) permeability. Cell counting kit-8 (CCK-8) and TUNEL assays were performed to evaluate the cell viability and apoptosis of RBMVECs. Dual-luciferase reporter assay was used to analyze the transcriptional activities of apoptosis-related genes. ResultsResults showed that higher infarction volume, neurological scores, and BBB permeability in the MCAO group rats were reduced after BHD treatment. Drug serum (DS) treatment had no impact on the normal RBMVECs’ cell viability and cell apoptosis. Besides, DS treatment decreased the lactate production, glucose uptake, and extracellular acidification rate in normal and OGD/R-induced RBMVECs. DS treatment downregulated the protein levels of pan-lysine lactylation (kla), histone H3 lysine 18 lactylation (H3K18la), and the transcriptional of apoptotic protease activating factor-1 (Apaf-1) in OGD/R-treated RBMVECs. In addition, Apaf-1 overexpression decreased cell viability and increased apoptosis and glycolysis activity of OGD/R-treated RBMVECs. ConclusionIn summary, BHD inhibited glycolysis and apoptosis via suppressing the pan-kla and H3K18la protein levels and the Apaf-1 transcriptional activity, thus restraining the progression of IS.

Modeling of cell cultivation for monoclonal antibody production processes considering lactate metabolic shifts.

Demand for monoclonal antibodies (mAbs) is rapidly increasing. To achieve higher productivity, there have been improvements to cell lines, operating modes, media, and cultivation conditions. Representative mathematical models are needed to narrow down the growing number of process alternatives. Previous studies have proposed mechanistic models to depict cell metabolic shifts (e.g., lactate production to consumption). However, the impacts of variations of some operating conditions have not yet been fully incorporated in such models. This paper offers a new mechanistic model considering variations in dissolved oxygen and glutamine depletion on cell metabolism applied to a novel Chinese hamster ovary (CHO) cell line. Expressions for the specific rates of lactate production, glutamine consumption, and mAb production were formulated for stirred and shaken-tank reactors. A deeper understanding of lactate metabolic shifts was obtained under different combinations of experimental conditions. Lactate consumption was more pronounced in conditions with higher DO and low glutamine concentrations. The model offers mechanistic insights that are useful for designing advanced operation strategies. It can be used in design space generation and process optimization for better productivity and product quality.

Astragaloside IV inhibits cell viability and glycolysis of hepatocellular carcinoma by regulating KAT2A-mediated succinylation of PGAM1

BackgroundAstragaloside IV (AS-IV) is one of the basic components of Astragali radix, that has been shown to have preventive effects against various diseases, including cancers. This study aimed to explore the role of AS-IV in hepatocellular carcinoma (HCC) and its underlying mechanism.MethodsThe cell viability, glucose consumption, lactate production, and extracellular acidification rate (ECAR) in SNU-182 and Huh7 cell lines were detected by specific commercial kits. Western blot was performed to analyze the succinylation level in SNU-182 and Huh7 cell lines. The interaction between lysine acetyltransferase (KAT) 2 A and phosphoglycerate mutase 1 (PGAM1) was evaluated by co-immunoprecipitation and immunofluorescence assays. The role of KAT2A in vivo was explored using a xenografted tumor model.ResultsThe results indicated that AS-IV treatment downregulated the protein levels of succinylation and KAT2A in SNU-182 and Huh7 cell lines. The cell viability, glucose consumption, lactate production, ECAR, and succinylation levels were decreased in AS-IV-treated SNU-182 and Huh7 cell lines, and the results were reversed after KAT2A overexpression. KAT2A interacted with PGAM1 to promote the succinylation of PGAM1 at K161 site. KAT2A overexpression promoted the viability and glycolysis of SNU-182 and Huh7 cell lines, which were partly blocked following PGAM1 inhibition. In tumor-bearing mice, AS-IV suppressed tumor growth though inhibiting KAT2A-mediated succinylation of PGAM1.ConclusionAS-IV inhibited cell viability and glycolysis in HCC by regulating KAT2A-mediated succinylation of PGAM1, suggesting that AS-IV might be a potential and suitable therapeutic agent for treating HCC.

Mitochondrial dysfunction significantly contributes to the sensitivity of tumor cells to anoikis and their metastatic potential

It is well-known that the survival of metastatic cells during their dissemination plays an important role in metastasis. However, does this mean that the final result of the metastatic cascade (the volume of metastatic damage to distant organs and tissues) depends on, or at least correlates with, the degree of resistance to anoikis (distinctive hallmarks of metastatic cells)? This question remains open.The aim of the work was to study in vitro the changes in the survival rates, proliferative activity, oxidative stress, and glycolysis intensity during three days of anchorage-dependent and anchorage-independent growth of two Lewis lung carcinoma cell lines (LLC and LLC/R9) and compare these changes with the status of mitochondria and metastatic potential of the cells in vivo. MethodsThe number and volume of lung metastases were estimated for each cell line after intramuscular inoculation of the cells in C57Bl/6 mice. For the in vitro study, the cells were seeded on Petri dishes pretreated with poly-HEMA or untreated dishes and then allowed to grow for 3 days. Cell viability, cell cycle progression, the level of reactive oxygen species (ROS), glucose consumption and lactate production rates were investigated daily in both growth conditions. An electron microscopy study of intracellular structures was carried out. ResultsThe study showed (as far as we know for the first time) a correlation between the metastatic potential of cells (determined in vivo) and their sensitivity to anoikis (assessed in vitro). The transition of LLC/R9 cells with an inherently defective mitochondrial system to the conditions of anchorage-independent growth was characterized by a decrease in survival, a slowdown in growth rates, an increase in both glucose consumption rate and intracellular ROS levels and manyfold lower metastatic potential, compared to highly metastatic LLC cells with the normal mitochondrial system.

Nucleolin myocardial-specific knockout exacerbates glucose metabolism disorder in endotoxemia-induced myocardial injury.

Sepsis-induced myocardial injury, as one of the important complications of sepsis, can significantly increase the mortality of septic patients. Our previous study found that nucleolin affected mitochondrial function in energy synthesis and had a protective effect on septic cardiomyopathy in mice. During sepsis, glucose metabolism disorders aggravated myocardial injury and had a negative effect on septic patients. We investigated whether nucleolin could regulate glucose metabolism during endotoxemia-induced myocardial injury. The study tested whether the nucleolin cardiac-specific knockout in the mice could affect glucose metabolism through untargeted metabolomics, and the results of metabolomics were verified experimentally in H9C2 cells. The ATP content, lactate production, and oxygen consumption rate (OCR) were evaluated. The metabolomics results suggested that glycolytic products were increased in endotoxemia-induced myocardial injury, and that nucleolin myocardial-specific knockout altered oxidative phosphorylation-related pathways. The experiment data showed that TNF-α combined with LPS stimulation could increase the lactate content and the OCR values by about 25%, and decrease the ATP content by about 25%. However, interference with nucleolin expression could further decrease ATP content and OCR values by about 10-20% and partially increase the lactate level in the presence of TNF-α and LPS. However, nucleolin overexpression had the opposite protective effect, which partially reversed the decrease in ATP content and the increase in lactate level. Down-regulation of nucleolin can exacerbate glucose metabolism disorders in endotoxemia-induced myocardial injury. Improving glucose metabolism by regulating nucleolin was expected to provide new therapeutic ideas for patients with septic cardiomyopathy.

Fibroblast growth factor pathway promotes glycolysis by activating LDHA and suppressing LDHB in a STAT1-dependent manner in prostate cancer

BackgroundThe initiation of fibroblast growth factor 1 (FGF1) expression coincident with the decrease of FGF2 expression is a well-documented event in prostate cancer (PCa) progression. Lactate dehydrogenase A (LDHA) and LDHB are essential metabolic products that promote tumor growth. However, the relationship between FGF1/FGF2 and LDHA/B-mediated glycolysis in PCa progression is not reported. Thus, we aimed to explore whether FGF1/2 could regulate LDHA and LDHB to promote glycolysis and explored the involved signaling pathway in PCa progression.MethodsIn vitro studies used RT‒qPCR, Western blot, CCK-8 assays, and flow cytometry to analyze gene and protein expression, cell viability, apoptosis, and cell cycle in PCa cell lines. Glycolysis was assessed by measuring glucose consumption, lactate production, and extracellular acidification rate (ECAR). For in vivo studies, a xenograft mouse model of PCa was established and treated with an FGF pathway inhibitor, and tumor growth was monitored.ResultsFGF1, FGF2, and LDHA were expressed at high levels in PCa cells, while LDHB expression was low. FGF1/2 positively modulated LDHA and negatively modulated LDHB in PCa cells. The depletion of FGF1, FGF2, or LDHA reduced cell proliferation, induced cell cycle arrest, and inhibited glycolysis. LDHB overexpression showed similar inhibitory effect on PCa cells. Mechanistically, we found that FGF1/2 positively regulated STAT1 and STAT1 transcriptionally activated LDHA expression while suppressed LDHB expression. Furthermore, the treatment of an FGF pathway inhibitor suppressed PCa tumor growth in mice.ConclusionThe FGF pathway facilitates glycolysis by activating LDHA and suppressing LDHB in a STAT1-dependent manner in PCa.

Modelling and assessment of glucose-lactate kinetics in youth withoverweight, obesityand metabolic dysfunction-associated steatotic liver disease: A pilot study.

In this study, we investigated glucose and lactate kinetics during a 75 g oral glucose tolerance test (OGTT) in 23 overweight and obese adolescents and assessed putative differences among participants with and without metabolic dysfunction-associated steatotic liver disease (MASLD). We enrolled 23 young people (six girls) with obesity [body mass index 33 (29-37)]. Glucose-lactate kinetics parameters (disposal glucose insulin sensitivity, SID; fraction of glucose converted into lactate, fr; fractional lactate clearance rate, kL) and lactate production rate (LPR) were estimated using the oral glucose-lactate minimal model. MASLD presence was assessed using the proton density fat fraction. We analysed glucose, lactate and LPR time to peak, peak values and area under the curve and evaluated differences using the Wilcoxon test. MASLD and no-MASLD participants were compared using the Mann-Whitney test. Correlations between parameters were assessed using the Spearman correlation coefficient (ρ). We also tested the performance of two (4 or 3 h OGTT) protocols in estimating oral glucose-lactate minimal model and LPR parameters. Glucose peaks 30 min earlier than lactate (p = .0019). This pattern was present in the no-MASLD group (p < .001). LPR peaks 30 min later in the MASLD group (p = .02). LPR and kL were higher in MASLD, suggesting higher glycolysis and lactate utilization. SID and fr correlate significantly (ρ = -0.55, p = .008). SID and fr were also correlated with the body mass index, (ρ = -0.45, p = .04; and ρ = 0.45; p = .03). The protocol duration did not influence the estimates of the parameters. Youth with MASLD showed a delayed glucose metabolism, possibly because of greater utilization of the underlying substrates. A 3-h OGTT may be used to assess lactate metabolism effectively.

Culture Shock! Investigating the Importance of Physiological Tumour-Like Conditions on Cultured Cancer Cells

The tumour microenvironment (TME) is complex and dynamic, characterized by poor vascularization, limited nutrient availability, hypoxia, and an acidic pH. This environment plays a critical role in cancer progression, driving hallmark changes in cancer cell metabolism, morphology, proliferation, and motility. Although mammalian cell culture is a foundational tool for investigating cancer cell biology in vitro, standard cell culture practices fall short of replicating the in vivo conditions of tumours. Recently, ‘physiologic’ cell culture media that closely resemble human plasma (e.g., Plasmax, HPLM) have been developed, along with more frequent adoption of physiological oxygen conditions (2-8% O2) rather than standard atmosphere (~18% O2). Although an improvement, these conditions are not representative of all in vivo cellular environments, particularly that of the TME. Considering the tight regulatory capacity of cancer cells to sense and respond to changes in metabolic availability, we hypothesized that alterations in nutrient and oxygen availability, even if within the physiological range, may subtly rewire the metabolism in cultured cancer cells. This may necessitate further refinement of cell-specific physiologic culture conditions to better maintain the in vivo biology of cells. In this study, we directly address this hypothesis by describing the development of a tumour microenvironment-like medium (TMEM) based on metabolomic profiling of murine pancreatic ductal adenocarcinoma tumour interstitial fluid from relevant literature. We found that murine pancreatic ductal adenocarcinoma (KPCY) cells cultured under TME-like conditions (TMEM, pH 7.0, 1.5% O2) exhibited increased glucose uptake and lactate production rates compared to those cultured under healthy plasma-like conditions (Mouse Plasma-like Medium, pH 7.4, 5% O2), despite lower initial glucose concentrations in TMEM (2.3 mM vs. 4.4 mM in Mouse Plasma-like Medium). Moreover, cell proliferation rates were lower in TMEM, suggesting an overall reduction in ATP consumption fueling cell division. Seahorse Extracellular Flux Analysis is being performed to determine effects on cellular bioenergetics. Metabolomic amino acid uptake using LCMS is also being performed to further assess the effect of TMEM on nutrient utilization. Using RNA-sequencing, we showed that culture in TMEM increased expression of genes associated with angiogenesis, amino acids biosynthesis, cell migration, and endothelial-to-mesenchymal transition. Migration assays confirmed increased motility in KPCY cells cultured in TMEM. Taken together, these results demonstrate that growth in a medium designed to simulate the TME alters KPCY cell biology in ways that are relevant to their pathology, which highlights the significance of maintaining TME-like conditions in culture. This research was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant from JAS and a NSERC Canadian Graduate Student (Masters) Grant awarded to GLG. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The mechanism of PFK-1 in the occurrence and development of bladder cancer by regulating ZEB1 lactylation

BackgroundBladder cancer (BC) is one of the most common malignancies of the genitourinary system. Phosphofructokinase 1 (PFK-1) is one of member of PFK, which plays an important role in reprogramming cancer metabolism, such as lactylation modification. Zinc finger E-box-binding homeobox 1 (ZEB1) has been demonstrated to be a oncogene in many cancers. Therefore, this study was performed to explore the effects of PFK-1 on the lactylation of ZEB1 in BC development.MethodsCell viability was measured using the CCK-8 kit. The glucose assay kit and lactate assay kit were used to detect glucose utilization and lactate production. The DNA was purified and quantified by qRT-PCR.ResultsIn the present study, we found that ZEB1 expression levels were significantly elevated in bladder cancer cells. Impaired PFK-1 expression inhibits proliferation, migration, and invasion of BC cells and suppresses tumour growth in vivo. We subsequently found that knockdown of PFK-1 decreases glycolysis, including reduced glucose consumption, lactate production and total extracellular acidification rate (ECAR). Mechanistically, PFK-1 inhibits histone lactylation of bladder cancer cells, and thus inhibits the transcription activity of ZEB1.ConclusionOur results suggest that PFK-1 can inhibit the malignant phenotype of bladder cancer cells by mediating the lactylation of ZEB1. These findings suggested PFK-1 to be a new potential target for bladder cancer therapy.

Biochemical features of glycolysis in cancer cells with different metastatic potential

It is known that, compared with normal cells, cancer cells are characterized by an intensification of glycolysis and an increase in glucose uptake to ensure high proliferative activity. Meanwhile, the question of the relationship between the intensity of glycolysis and the metastatic potential of tumor cells remains open. The aim of the study was to determine the rate of glucose uptake (GCR) and lactate production (LPR) by Lewis lung carcinoma cells with high (LLC) and low metastatic potential (LLC/R9) during 1-day growth in vitro under standard incubation conditions. The results showed that glycolysis intensity was significantly higher in low-metastatic Lewis lung carcinoma cells compared to the high-metastatic cells.

Metabolic Responses of Lung Adenocarcinoma Cells to Survive under Stressful Conditions Associated with Tumor Microenvironment.

Solid tumors frequently present a heterogeneous tumor microenvironment. Because tumors have the potential to proliferate quickly, the consequence is a reduction in the nutrients, a reduction in the pH (<6.8), and a hypoxic environment. Although it is often assumed that tumor clones show a similar growth rate with little variations in nutrient consumption, the present study shows how growth-specific rate (µ), the specific rates of glucose, lactate, and glutamine consumption (qS), and the specific rates of lactate and glutamate production (qP) of 2D-cultured lung tumor cells are affected by changes in their environment. We determined in lung tumor cells (A427, A549, Calu-1, and SKMES-1) the above mentioned kinetic parameters during the exponential phase under different culture conditions, varying the predominant carbon source, pH, and oxygen tension. MCF-7 cells, a breast tumor cell line that can consume lactate, and non-transformed fibroblast cells (MRC-5) were included as controls. We also analyzed how cell-cycle progression and the amino acid transporter CD98 expression were affected. Our results show that: (1) In glucose presence, μ increased, but qS Glucose and qP Lactate decreased when tumor cells were cultured under acidosis as opposed to neutral conditions; (2) most lung cancer cell lines consumed lactate under normoxia or hypoxia; (3) although qS Glutamine diminished under hypoxia or acidosis, it slightly increased in lactate presence, a finding that was associated with CD98 upregulation; and (4) under acidosis, G0/G1 arrest was induced in A427 cancer cells, although this phenomenon was significantly increased when glucose was changed by lactate as the predominant carbon-source. Hence, our results provide an understanding of metabolic responses that tumor cells develop to survive under stressful conditions, providing clues for developing promising opportunities to improve traditional cancer therapies.

Metabolic switching, growth kinetics and cell yields in the scalable manufacture of stem cell-derived insulin-producing cells

BackgroundDiabetes is a disease affecting over 500 million people globally due to insulin insufficiency or insensitivity. For individuals with type 1 diabetes, pancreatic islet transplantation can help regulate their blood glucose levels. However, the scarcity of cadaveric donor islets limits the number of people that could receive this therapy. To address this issue, human pluripotent stem cells offer a potentially unlimited source for generating insulin-producing cells through directed differentiation. Several protocols have been developed to make stem cell-derived insulin-producing cells. However, there is a lack of knowledge regarding the bioprocess parameters associated with these differentiation protocols and how they can be utilized to increase the cell yield.MethodsWe investigated various bioprocess parameters and quality target product profiles that may influence the differentiation pipeline using a seven-stage protocol in a scalable manner with CellSTACKs and vertical wheel bioreactors (PBS-Minis).ResultsCells maintained > 80% viability through all stages of differentiation and appropriately expressed stage-specific markers. During the initial four stages leading up to the development of pancreatic progenitors, there was an increase in cell numbers. Following pancreatic progenitor stage, there was a gradual decrease in the percentage of proliferative cells, as determined by Ki67 positivity, and a significant loss of cells during the period of endocrine differentiation. By minimizing the occurrence of aggregate fusion, we were able to enhance cell yield during the later stages of differentiation. We suggest that glucose utilization and lactate production are cell quality attributes that should be considered during the characterization of insulin-producing cells derived from stem cells. Our findings also revealed a gradual metabolic shift from glycolysis, during the initial four stages of pancreatic progenitor formation, to oxidative phosphorylation later on during endocrine differentiation. Furthermore, the resulting insulin-producing cells exhibited a response to several secretagogues, including high glucose.ConclusionThis study demonstrates process parameters such as glucose consumption and lactate production rates that may be used to facilitate the scalable manufacture of stem cell-derived insulin-producing cells.

High sensitivity and automatic chemiluminescence detection of glucose and lactate using a spin-disc paper-based device.

This paper reports a spin-disc paper-based device with 10 individual detection units containing electromagnetic modules controlling the sample incubation time before chemiluminescence (CL) signal detection. After the sample was added to the top paper chip and incubated with the enzyme, the electromagnet was turned off to allow contact between the top and bottom paper. The H2O2 generated by the sample flowed vertically to the bottom paper and initiated the oxidase of the luminol to generate the CL signal. After one detection the disc was automatically rotated to the next position to repeat the above detection. The advantage of using the device over the lateral flow and the in situ detection was firstly proved using the detection of H2O2 and the glucose/lactate sample with 5 minute incubation. The CL intensity was increased 300 times/1000 times as the glucose/lactate was incubated for 5 minutes compared to the non-incubated samples. Afterward, the device was employed to separately detect glucose and lactate diluted in PBS, artificial sweat, artificial saliva, and fresh cell culture media. Finally, the device was employed to detect the glucose and lactate in the media collected over the 24 hour culture of PC3 cells. The uptake and production rates of glucose and lactate were correspondingly determined as 0.328 ± 0.015 pmol h-1 per cell and 1.254 ± 0.053 pmol h-1 per cell, respectively. The reported device has wide application potential due to its capabilities in automatic detection of multiple samples with very high sensitivity and small sample volume (down to 0.5 μL).

Effects of Lactate Transport Inhibition by AZD3965 in Muscle-Invasive Urothelial Bladder Cancer.

The Warburg Effect is characterized by high rates of glucose uptake and lactate production. Monocarboxylate transporters (MCTs) are crucial to avoid cellular acidosis by internal lactate accumulation, being largely overexpressed by cancer cells and associated with cancer aggressiveness. The MCT1-specific inhibitor AZD3965 has shown encouraging results in different cancer models. However, it has not been tested in urothelial bladder cancer (UBC), a neoplasm where rates of recurrence, progression and platinum-based resistance are generally elevated. We used two muscle-invasive UBC cell lines to study AZD3965 activity regarding lactate production, UBC cells' viability and proliferation, cell cycle profile, and migration and invasion properties. An "in vivo" assay with the chick chorioallantoic membrane model was additionally performed, as well as the combination of the compound with cisplatin. AZD3965 demonstrated anticancer activity upon low levels of MCT4, while a general lack of sensitivity was observed under MCT4 high expression. Cell viability, proliferation and migration were reduced, cell cycle was arrested, and tumor growth "in vivo" was inhibited. The compound sensitized these MCT4-low-expressing cells to cisplatin. Thus, AZD3965 seems to display anticancer properties in UBC under a low MCT4-expression setting, but additional studies are necessary to confirm AZD3965 activity in this cancer model.

Modulation of Designed Gut Bacterial Communities by Prebiotics and the Impact of Their Metabolites on Intestinal Cells.

The impact of prebiotics on human health is associated with their capacity to modulate microbiota, improving beneficial microbiota-host interactions. Herein, the prebiotic potential of microbial-fructo-oligosaccharides (microbial-FOSs) produced by a co-culture of Aspergillus ibericus plus Saccharomyces cerevisiae was evaluated on seven- and nine-strain bacterial consortia (7SC and 9SC, respectively), designed to represent the human gut microbiota. The 7SC was composed of Bacteroides dorei, Bacteroides vulgatus, Bifidobacterium adolescentis, Bifidobacterium longum, Escherichia coli, Lactobacillus acidophilus, and Lactobacillus rhamnosus. The 9SC also comprised the aforementioned bacteria, with the addition of Bacteroides thetaiotaomicron and Roseburia faecis. The effect of microbial-FOSs on the metabolic activity of intestinal Caco-2/HT29-MTX-E12 co-culture was also assessed. The results showed that microbial-FOS selectively promoted the growth of probiotic bacteria and completely suppressed the growth of E. coli. The microbial-FOSs promoted the highest production rates of lactate and total short-chain fatty acids (SCFA) as compared to the commercial prebiotic Frutalose® OFP. Butyrate was only produced in the 9SC consortium, which included the R. faecis-a butyrate-producing bacteria. The inclusion of this bacteria plus another Bacteroides in the 9SC promoted a greater metabolic activity in the Caco-2/HT29-MTX-E12 co-culture. The microbial-FOSs showed potential as promising prebiotics as they selectively promote the growth of probiotic bacteria, producing high concentrations of SCFA, and stimulating the metabolic activity of gut cells.

13C tracer analysis reveals the landscape of metabolic checkpoints in human CD8+ T cell differentiation and exhaustion

IntroductionNaïve T cells remain in an actively maintained state of quiescence until activation by antigenic signals, upon which they start to proliferate and generate effector cells to initiate a functional immune response. Metabolic reprogramming is essential to meet the biosynthetic demands of the differentiation process, and failure to do so can promote the development of hypofunctional exhausted T cells.MethodsHere we used 13C metabolomics and transcriptomics to study the metabolism of CD8+ T cells in their complete course of differentiation from naïve over stem-like memory to effector cells and in exhaustion-inducing conditions. ResultsThe quiescence of naïve T cells was evident in a profound suppression of glucose oxidation and a decreased expression of ENO1, downstream of which no glycolytic flux was detectable. Moreover, TCA cycle activity was low in naïve T cells and associated with a downregulation of SDH subunits. Upon stimulation and exit from quiescence, the initiation of cell growth and proliferation was accompanied by differential expression of metabolic enzymes and metabolic reprogramming towards aerobic glycolysis with high rates of nutrient uptake, respiration and lactate production. High flux in anabolic pathways imposed a strain on NADH homeostasis, which coincided with engagement of the proline cycle for mitochondrial redox shuttling. With acquisition of effector functions, cells increasingly relied on glycolysis as opposed to oxidative phosphorylation, which was, however, not linked to changes in mitochondrial abundance. In exhaustion, decreased effector function concurred with a reduction in mitochondrial metabolism, glycolysis and amino acid import, and an upregulation of quiescence-associated genes, TXNIP and KLF2, and the T cell suppressive metabolites succinate and itaconate. DiscussionOverall, these results identify multiple metabolic features that regulate quiescence, proliferation and effector function, but also exhaustion of CD8+ T cells during differentiation. Thus, targeting these metabolic checkpoints may be a promising therapeutic strategy for both prevention of exhaustion and promotion of stemness of anti-tumor T cells.

Continuous culture of anaerobic fungi enables growth and metabolic flux tuning without use of genetic tools

Anaerobic gut fungi (AGF) have potential to valorize lignocellulosic biomass owing to their diverse repertoire of carbohydrate-active enzymes (CAZymes). However, AGF metabolism is poorly understood, and no stable genetic tools are available to manipulate growth and metabolic flux to enhance production of specific targets, e.g., cells, CAZymes, or metabolites. Herein, a cost-effective, Arduino-based, continuous-flow anaerobic bioreactor with online optical density control is presented to probe metabolism and predictably tune fluxes in Caecomyces churrovis. Varying the C. churrovis turbidostat setpoint titer reliably controlled growth rate (from 0.04 to 0.20 h−1), metabolic flux, and production rates of acetate, formate, lactate, and ethanol. Bioreactor setpoints to maximize production of each product were identified, and all continuous production rates significantly exceed batch rates. Formate spike-ins increased lactate flux and decreased acetate, ethanol, and formate fluxes. The bioreactor and turbidostat culture schemes demonstrated here offer tools to tailor AGF fermentations to application-specific hydrolysate product profiles.

Hsa_circ_0052611 and mir-767-5p guide the warburg effect, migration, and invasion of BRCA cells through modulating SCAI.

Noncoding RNAs are key regulators in the Warburg Effect, an emerging hallmark of cancer. We intended to investigate the role and mechanism of circular RNA hsa_circ_0052611 (circ_0052611) and microRNA (miR)-767-5p in breast cancer (BRCA) hallmarks, especially the Warburg Effect. Expression of circ_0052611 and SCAI was downregulated, and miR-767-5p was upregulated in human BRCA tissues and cells; moreover, circ_0052611 acted as a miR-767-5p sponge to modulate the expression of miR-767-5p-targeted SCAI. Functionally, re-expressing circ_0052611 suppressed migration, invasion, glucose uptake, lactate production, and extracellular acidification rate (ECAR) in BRCA cells, and promoted apoptotic rate. These effects were accompanied by decreased Vimentin, N-cadherin, Bcl-2, and LDHA, and increased E-cadherin and Bax. Consistently, exhausting miR-767-5p exerted similar effects in BRCA cells. High miR-767-5p could counteract the role of circ_0052611 overexpression, and low SCAI likewise blocked the role of miR-767-5p deletion. In vivo, upregulating circ_0052611 delayed tumor growth of BRCA cells by altering miR-767-5p and SCAI expression. circ_0052611/miR-767-5p/SCAI axis might boycott the malignancy of BRCA cells.