Single-cell lactate production rate as a measure of glycolysis in endothelial cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Harrison D
PROVIDER: S-EPMC8433287 | biostudies-literature | 2021 Sep
REPOSITORIES: biostudies-literature
Publications
Single-cell lactate production rate as a measure of glycolysis in endothelial cells.
Harrison Devin D Wu David D Huang Jun J Fang Yun Y
STAR protocols 20210908 3
Heterogeneous metabolism supports critical single-cell functions. Here, we describe deep-learning-enabled image analyses of a genetically encoded lactate-sensing probe which can accurately quantify metabolite levels and glycolytic rates at the single-cell level. Multiple strategies and test data have been included to obviate possible obstacles including successful sensor expression and accurate segmentation. This protocol reliably discriminates between metabolically diverse subpopulations which ...[more]
Similar Datasets
Project description:Previous studies have shown that chronic hyperglycemia impairs glucose and fatty acid oxidation in cultured human myotubes. To further study the hyperglycemia-induced suppression of oxidation, lactate oxidation, mitochondrial function and glycolytic rate were evaluated. Further, we examined the intracellular content of reactive oxygen species (ROS), production of lactate and conducted pathway-ANOVA analysis on microarray data. In addition, the roles of the pentose phosphate pathway (PPP) and the hexosamine pathway were evaluated. Lactic acid oxidation was suppressed in hyperglycemic versus normoglycaemic myotubes. No changes in mitochondrial function or ROS concentration were observed. Pathway-ANOVA analysis indicated several upregulated pathways in hyperglycemic cells, including glycolysis and PPP. Functional studies showed that glycolysis and lactate production were higher in hyperglycemic than normoglycaemic cells. However, there were no indications of involvement of PPP or the hexosamine pathway. In conclusion, hyperglycemia reduced substrate oxidation while increasing glycolysis and lactate production in cultured human myotubes.
| S-EPMC6770141 | biostudies-literature
Project description:ObjectivePediatric obesity predisposes children and adolescents to early onset insulin resistance and dysglycemia. In the last 20 years this has led to a rise in the prevalence of prediabetes, diabetes and fatty liver in youngsters, due to the high degree of insulin resistance experienced by these patients and the consequent high availability of glucose. As glucose accesses the liver, it is partly metabolized through glycolysis, whose main product is pyruvate that is then converted into Acetyl CoA and lactate. Therefore, lactate production rate (LPR) represents the best proxy for the assessment of glycolysis. Since to date there are not methods to estimate postprandial LPR, here we proposed a novel oral glucose-lactate model to estimate LPR during an oral glucose tolerance test and tested it in 24 youth with and without obesity.MethodsThe model is based on the oral glucose minimal model and assumes that LPR is a fraction (fr) of glucose disposal rate, proportional to glucose concentration and controlled by insulin action.ResultsThe model well fitted the glucose and lactate data, and provided both precise parameter estimates (e.g., fr = 22.5 [12.6-54.1]%, median [IQR]), CV = 18 [13-25]%) and LPR time course.ConclusionsThe proposed model is a valid tool to assess LPR, and thus glycolysis, during OGTT in nondiabetic subjects.SignificanceThe proposed methodology will allow to assess postprandial LPR in simple and cost-effective way.
| S-EPMC9016183 | biostudies-literature
Project description:Following activation, T cells rapidly divide and acquire effector functions. This energetically demanding process depends upon the ability of T cells to undergo metabolic remodeling from oxidative phosphorylation to aerobic glycolysis, during which glucose is converted into lactate and released extracellularly. In this article, we demonstrate that extracellular lactate can be used to dynamically assess human T cell responses in vitro. Extracellular lactate levels strongly correlated with T cell proliferation, and measuring lactate compared favorably with traditional methods for determining T cell responses (i.e., [3H]thymidine incorporation and the use of cell proliferation dyes). Furthermore, we demonstrate the usefulness of measuring lactate as a read-out in conventional suppression assays and high-throughput peptide-screening assays. Extracellular lactate was stably produced over 7 d, and results were reproducibly performed over several freeze-thaw cycles. We conclude that the use of extracellular lactate measurements can be a sensitive, safe, stable, and easy-to-implement research tool for measuring T cell responses and cellular metabolic changes in vitro.
| S-EPMC5776880 | biostudies-literature
Project description:Metabolic reprogramming plays a crucial role in cancer biology and the mechanisms underlying its regulation represent a promising study area. In this regard, the discovery of non-coding RNAs opened a new regulatory landscape, which is in the early stages of investigation. Using a differential expression model of HOTAIR, we evaluated the expression level of metabolic enzymes, as well as the metabolites produced by glycolysis and glutaminolysis. Our results demonstrated the regulatory effect of HOTAIR on the expression of glycolysis and glutaminolysis enzymes in colorectal cancer cells. Specifically, through the overexpression and inhibition of HOTAIR, we determined its influence on the expression of the enzymes PFKFB4, PGK1, LDHA, SLC1A5, GLUD1, and GOT1, which had a direct impact on lactate and glutamate production. These findings indicate that HOTAIR plays a significant role in producing "oncometabolites" essential to maintaining the bioenergetics and biomass necessary for tumor cell survival by regulating glycolysis and glutaminolysis.
| S-EPMC11898799 | biostudies-literature
Project description:In solid tumors, hypoxia can trigger aberrant expression of transcription factors and genes, resulting in abnormal biological functions such as altered energetic pathways in cancer cells. Glucose metabolism is an important part of this phenomenon, which is associated with changes in the functional expression of transporters and enzymes involved in the glycolysis pathway. The latter phenomenon can finally lead to the lactate accumulation and pH dysregulation in the tumor microenvironment and subsequently further invasion and metastasis of cancer cells. Having capitalized on the computational modeling, in this study, for the first time, we aimed to investigate the effects of hypoxia-induced factor-1 (HIF-1) mediated hypoxia on the magnitude of functional expression of all the enzymes and transporters involved in the glycolysis process. The main objective was to establish a quantitative relationship between the hypoxia intensity and the intracellular lactate levels and determine the key regulators of the glycolysis pathway. This model clearly showed an increase in the lactate concentration during the oxygen depletion. The proposed model also predicted that the phosphofructokinase-1 and phosphoglucomutase enzymes might play the most important roles in the regulation of the lactate production.
| S-EPMC7280308 | biostudies-literature
Project description:The most common positron emission tomography (PET) radio-labeled probe for molecular diagnostics in patient care and research is the glucose analog, 2-deoxy-2-[F-18]fluoro-D-glucose (18F-FDG). We report on an integrated microfluidics-chip/beta particle imaging system for in vitro18F-FDG radioassays of glycolysis with single cell resolution. We investigated the kinetic responses of single glioblastoma cancer cells to targeted inhibitors of receptor tyrosine kinase signaling. Further, we find a weak positive correlation between cell size and rate of glycolysis.
| S-EPMC4728151 | biostudies-literature
Project description:Vascular endothelial cells have important tissue-specific functions in fibrosis and regeneration. In the salivary gland, endothelial cells are required for proper development, but their roles within adult glands are largely unknown. To identify ligand-receptor interactions between endothelial cells and other cell types that may be important during fibrosis and regeneration, we used a reversible ductal ligation injury. To induce injury, a clip was applied to the primary ducts for 14 d, and to induce a regenerative response, the clip was subsequently removed for 5 d. To identify endothelial cell-produced factors, we used single-cell RNA sequencing of stromal-enriched cells from adult female submandibular and sublingual salivary glands. Transcriptional profiles of homeostatic salivary gland endothelial cells were compared to endothelial cells of other organs. Salivary gland endothelial cells expressed many unique genes and displayed the highest overlap in gene expression with other fenestrated endothelial cells from the colon, small intestine, and kidney. Comparison of the 14-d ligated, mock-ligated, and 5-d deligated stromal-enriched transcripts and lineage tracing revealed that endothelial cells retain their identity following ligation and recovery from injury. CellChat and NATMI were used to predict changes in ligand-receptor interactions from endothelial cells to other cells in response to ligation and deligation. CellChat and NATMI predicted that after ligation, interactions with fibroblasts, epithelial cells, and glial cells were increased, and following deligation, interactions with pericyte, glia, fibroblasts, and immune cells were increased. Some of the highest-ranked interactions predicted in ligated compared to mock endothelial cells were between glial cells via Col4a2-Cd93 and Jag2-Notch1, as well as epithelial cells via Pecam1-Cd38, while in deligated compared to ligated endothelial cells, the top interactions were between fibroblasts via Ntf3-Ntrk2, glial cells via Hspg2-Itgb1, and pericytes via Jam2-F11r. Understanding salivary gland endothelial cell signaling will inform future endothelial cell-based regenerative therapies.
| S-EPMC10985389 | biostudies-literature
Project description:Vascular endothelial cells have important functions in fibrosis via direct and indirect methods and in regeneration through secretion of tissue-specific, paracrineacting angiocrine factors. In the salivary gland, endothelial cells are required for proper development, but their roles within adult glands are largely unknown. The goal of this work was to identify ligand-receptor interactions between endothelial cells and other cell types that are important during homeostasis, fibrosis, and regeneration. To model salivary gland fibrosis and regeneration, we utilized a reversible ductal ligation. To induce injury, a clip was applied to the primary ducts for 14 days, and to induce a regenerative response, the clip was subsequently removed for 5 days. To identify endothelial cell-produced factors, we used single-cell RNA-sequencing of stromal-enriched cells from adult submandibular and sublingual salivary glands. Transcriptional profiles of homeostatic salivary gland endothelial cells were compared to endothelial cells of other organs. Salivary gland endothelial cells were found to express unique genes and displayed the highest overlap in gene expression with other fenestrated endothelial cells from the colon, small intestine, and kidney. Comparison of the 14-day ligated, mock ligated, and 5-day deligated stromal-enriched transcripts and lineage tracing were used to identify evidence for a partial endoMT phenotype, which was observed in a small number of endothelial cell subsets with ligation. CellChat was used to predict changes in ligand-receptor interactions in response to ligation and deligation. CellChat predicted that after ligation, endothelial cells are sources of protein tyrosine phosphatase receptor type m, tumor necrosis factor ligand superfamily member 13, and myelin protein zero signaling and targets for tumor necrosis factor signaling. Following deligation, CellChat predicted that endothelial cells are sources of chemokine (C-X-C motif) and EPH signaling to promote regenerative responses. These studies will inform future endothelial cell-based regenerative therapies.
| S-EPMC10327062 | biostudies-literature
Project description:IntroductionMicrobiome plays roles in lung adenocarcinoma (LUAD) development and anti-tumor treatment efficacy. Aberrant glycolysis in tumor might promote lactate production that alter tumor microenvironment, affecting microbiome, cancer cells and immune cells. We aimed to construct intratumor microbiome score to predict prognosis of LUAD patients and thoroughly investigate glycolysis and lactate signature's association with LUAD immune cell infiltration.MethodsThe Cancer Genome Atlas-LUAD (TCGA-LUAD) microbiome data was downloaded from cBioPortal and analyzed to examine its association with overall survival to create a prognostic scoring model. Gene Set Enrichment Analysis (GSEA) was used to find each group's major mechanisms involved. Our study then investigated the glycolysis and lactate pattern in LUAD patients based on 19 genes, which were correlated with the tumor microenvironment (TME) phenotypes and immunotherapy outcomes. We developed a glycolysis-lactate risk score and signature to accurately predict TME phenotypes, prognosis, and response to immunotherapy.ResultsUsing the univariate Cox regression analysis, the abundance of 38 genera were identified with prognostic values and a lung-resident microbial score (LMS) was then developed from the TCGA-LUAD-microbiome dataset. Glycolysis hallmark pathway was significantly enriched in high-LMS group and three distinct glycolysis-lactate patterns were generated. Patients in Cluster1 exhibited unfavorable outcomes and might be insensitive to immunotherapy. Glycolysis-lactate score was constructed for predicting prognosis with high accuracy and validated in external cohorts. Gene signature was developed and this signature was elevated in epithelial cells especially in tumor mass on single-cell level. Finally, we found that the glycolysis-lactate signature levels were consistent with the malignancy of histological subtypes.DiscussionOur study demonstrated that an 18-microbe prognostic score and a 19-gene glycolysis-lactate signature for predicting prognosis of LUAD patients. Our LMS, glycolysis-lactate score and glycolysis-lactate signature have potential roles in precision therapy of LUAD patients.
| S-EPMC10469687 | biostudies-literature
Project description:BackgroundMost normal cells in the presence of oxygen utilize glucose for mitochondrial oxidative phosphorylation. In contrast, many cancer cells rapidly convert glucose to lactate in the cytosol, a process termed aerobic glycolysis. This glycolytic phenotype is enabled by lactate dehydrogenase (LDH), which catalyzes the inter-conversion of pyruvate and lactate. The purpose of this study was to identify and characterize potent and selective inhibitors of LDHA.MethodsHigh throughput screening and lead optimization were used to generate inhibitors of LDHA enzymatic activity. Effects of these inhibitors on metabolism were evaluated using cell-based lactate production, oxygen consumption, and 13C NMR spectroscopy assays. Changes in comprehensive metabolic profile, cell proliferation, and apoptosis were assessed upon compound treatment.Results3-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl) amino) benzoic acid was identified as an NADH-competitive LDHA inhibitor. Lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 nM and 10 to 80-fold selectivity over LDHB. Molecules in this family rapidly and profoundly inhibited lactate production rates in multiple cancer cell lines including hepatocellular and breast carcinomas. Consistent with selective inhibition of LDHA, the most sensitive breast cancer cell lines to lactate inhibition in hypoxic conditions were cells with low expression of LDHB. Our inhibitors increased rates of oxygen consumption in hepatocellular carcinoma cells at doses up to 3 microM, while higher concentrations directly inhibited mitochondrial function. Analysis of more than 500 metabolites upon LDHA inhibition in Snu398 cells revealed that intracellular concentrations of glycolysis and citric acid cycle intermediates were increased, consistent with enhanced Krebs cycle activity and blockage of cytosolic glycolysis. Treatment with these compounds also potentiated PKM2 activity and promoted apoptosis in Snu398 cells.ConclusionsRapid chemical inhibition of LDHA by these quinoline 3-sulfonamids led to profound metabolic alterations and impaired cell survival in carcinoma cells making it a compelling strategy for treating solid tumors that rely on aerobic glycolysis for survival.
| S-EPMC4178217 | biostudies-literature