XFe24 Extracellular Flux Analyzer Research Articles

Cellular oxygen consumption in patients with diabetic ketoacidosis

BackgroundDiabetic ketoacidosis (DKA) is a potentially life-threatening disorder associated with severe alterations in metabolism and acid–base status. Mitochondrial dysfunction is associated with diabetes and its complications. Thiamine and coenzyme Q10 (CoQ10) are important factors in aerobic metabolism. In this study, we measured cellular oxygen consumption rates (OCRs) and the effects of in vitro administration of thiamine and CoQ10 on OCRs in patients with DKA versus healthy controls.MethodsBlood samples were collected from a prospective cohort of patients with DKA and from controls. Cellular OCRs were measured in peripheral blood mononuclear cells (PBMC) without treatment and after treatment with thiamine, CoQ10, or both. The mitochondrial profile was measured using an XFe96 Extracellular Flux Analyzer and XF Cell Mito Stress Test Kit (Seahorse Bioscience). A linear quantile mixed model was used to compare OCRs and estimate treatment effects.ResultsA total of 62 patients with DKA and 48 controls were included in the study. The median basal and maximal OCRs were lower in the DKA group than in the control group (basal: 4.7 [IQR: 3.3, 7.9] vs. 7.9 [5.0, 9.5], p = 0.036; maximal: 16.4 [9.5, 28.1] vs. 31.5 [20.6, 46.0] pmol/min/µg protein, p < 0.001). In DKA samples, basal and maximal OCRs were significantly increased when treated with thiamine, CoQ10, or both. In controls, basal and maximal OCR were significantly increased only with thiamine treatment.ConclusionMitochondrial metabolic profiles of patients with DKA demonstrated lower cellular oxygen consumption when compared to healthy controls. Oxygen consumption increased significantly in cells of patients with DKA treated with thiamine or CoQ10. These results suggest that thiamine and CoQ10 could potentially have therapeutic benefits in DKA via their metabolic effects on mitochondrial cellular respiration.

CircRNA6448-14/miR-455-3p/OTUB2 axis stimulates glycolysis and stemness of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a gastrointestinal malignancy with high incidence. This study aimed to reveal the complete circRNA-miRNA-mRNA regulatory network in ESCC and validate its function mechanism. Expression of OTU Domain-Containing Ubiquitin Aldehyde-Binding Protein 2 (OTUB2) in ESCC was analyzed by bioinformatics to find the binding sites between circRNA6448-14 and miR-455-3p, as well as miR-455-3p and OTUB2. The binding relationships were verified by RNA Immunoprecipitation (RIP) and dual-luciferase assay. The expressions of circRNA6448-14, miR-455-3p, and OTUB2 were detected by quantitative real-time polymerase chain reaction (qRT-PCR). MTT assay measured cell viability, and the spheroid formation assay assessed the ability of stem cell sphere formation. Western blot (WB) determined the expression of marker proteins of stem cell surface and rate-limiting enzyme of glycolysis. The Seahorse XFe96 extracellular flux analyzer measured the rate of extracellular acidification rate and cellular oxygen consumption. Corresponding assay kits assessed cellular glucose consumption, lactate production, and adenosine triphosphate (ATP) generation. In ESCC, circRNA6448-14 and OTUB2 were highly expressed in contrast to miR-455-3p. Knocking down circRNA6448-14 could prevent the glycolysis and stemness of ESCC cells. Additionally, circRNA6448-14 enhanced the expression of OTUB2 by sponging miR-455-3p. Overexpression of OTUB2 or silencing miR-455-3p reversed the inhibitory effect of knockdown of circRNA6448-14 on ESCC glycolysis and stemness. This research demonstrated that the circRNA6448-14/miR-455-3p/OTUB2 axis induced the glycolysis and stemness of ESCC cells. Our study revealed a novel function of circRNA6448-14, which may serve as a potential therapeutic target for ESCC.

Prdx1 regulates macrophage polarization by maintaining mitochondrial homeostasis

In order to investigate the role of Prdx1 in macrophage polarization, mouse leukemia cells of monocyte macrophage (RAW264.7) were treated with lipopolysaccharides (LPS)+ interferon gamma (IFNγ) or IL-4 to induce type 1 macrophage (M1) and type 1 macrophage (M2) macrophages, respectively. The Prdx1 gene knockout cells (Prdx1-/-) were used for the study. Flow cytometry was conducted to detect M1/M2 macrophage markers, and ELISA kits were used to measure M1/M2 cytokine levels. Inducible nitric-oxide synthase (iNOS) activity, arginase-1 (Arg-1) activity, and oxidative damage were also assessed. The Seahorse XFe24 Extracellular Flux Analyzer was employed to measure extracellular acidification rate and oxygen consumption rate. The mitochondrial membrane potential was analyzed using the mitochondrial membrane potential dye (JC-1) fluorescent probe, and mitochondrial superoxide was detected through fluorescence staining. Additionally, the impact of adding a mitochondrial reactive oxygen species (ROS) scavenger on RAW264.7 macrophage polarization was examined. The results demonstrated an increase in ROS, hydrogen peroxide, and 8-hydroxy-2 deoxyguanosine (8-OHDG). Cytotoxicity and mitochondrial toxic effects, including mitochondrial superoxide accumulation, decreased adenosine-triphosphate (ATP) production, reduced mitochondrial membrane potential, and decreased mitochondrial DNA copy number, were observed. Furthermore, down-regulation of translocase of inner mitochondrial membrane 23 (TIM23) mitochondrial protein and mitochondrial stress protein heat shock protein 60 (HSP60) was noted. The extra cellular acidification rate (ECAR) in M1 macrophage polarization in RAW264.7 cells was increased, while oxygen consumption rate (OCR) in M2 macrophages was reduced. These findings indicate that Prdx1 knockout in RAW264.7 cells can inhibit M2 macrophage polarization but promote M1 macrophage polarization by impairing mitochondrial function and reducing oxidative phosphorylation.

Pimpinellin ameliorates macrophage inflammation by promoting RNF146-mediated PARP1 ubiquitination.

Macrophage inflammation plays a central role during the development and progression of sepsis, while the regulation of macrophages by parthanatos has been recently identified as a novel strategy for anti-inflammatory therapies. This study was designed to investigate the therapeutic potential and mechanism of pimpinellin against LPS-induced sepsis. PARP1 and PAR activation were detected by western blot or immunohistochemistry. Cell death was assessed by flow cytometry and western blot. Cell metabolism was measured with a Seahorse XFe24 extracellular flux analyzer. C57, PARP1 knockout, and PARP1 conditional knock-in mice were used in a model of sepsis caused by LPS to assess the effect of pimpinellin. Here, we found that pimpinellin can specifically inhibit LPS-induced macrophage PARP1 and PAR activation. In vitro studies showed that pimpinellin could inhibit the expression of inflammatory cytokines and signal pathway activation in macrophages by inhibiting overexpression of PARP1. In addition, pimpinellin increased the survival rate of LPS-treated mice, thereby preventing LPS-induced sepsis. Further research confirmed that LPS-induced sepsis in PARP1 overexpressing mice was attenuated by pimpinellin, and PARP1 knockdown abolished the protective effect of pimpinellin against LPS-induced sepsis. Further study found that pimpinellin can promote ubiquitin-mediated degradation of PARP1 through RNF146. This is the first study to demonstrate that pimpinellin inhibits excessive inflammatory responses by promoting the ubiquitin-mediated degradation of PARP1.

Enhancement of de novo lipogenesis by the IDH1 and IDH2-dependent reverse TCA cycle maintains the growth and angiogenic capacity of bone marrow-derived endothelial progenitor cells under hypoxia

BackgroundBone marrow-derived endothelial progenitor cells (EPCs) play a dynamic role in maintaining the structure and function of blood vessels. But how these cells maintain their growth and angiogenic capacity under bone marrow hypoxic niche is still unclear. This study aims to explore the mechanisms from a perspective of cellular metabolism. MethodsXFe96 Extracellular Flux Analyzer was used to analyze the metabolic status of EPCs. Gas Chromatography-Mass Spectrometry (GC-MS) was used to trace the carbon movement of 13C-labeled glucose and glutamine under 1 % O2 (hypoxia) and ∼20 % O2 (normoxia). Moreover, RNA interference, targeting isocitrate dehydrogenase-1 (IDH1) and IDH2, was used to inhibit the reverse tricarboxylic acid (TCA) cycle and analyze metabolic changes via isotope tracing as well as changes in cell growth and angiogenic potential under hypoxia. The therapeutic potential of EPCs under hypoxia was investigated in the ischemic hindlimb model. ResultsCompared with normoxic cells, hypoxic cells showed increased glycolysis and decreased mitochondrial respiration. Isotope metabolic tracing revealed that under hypoxia, the forward TCA cycle was decreased and the reverse TCA cycle was enhanced, mediating the conversion of α-ketoglutarate (α-KG) into isocitrate/citrate, and de novo lipid synthesis was promoted. Downregulation of IDH1 or IDH2 under hypoxia suppressed the reverse TCA cycle, attenuated de novo lipid synthesis (DNL), elevated α-KG levels, and decreased the expression of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor A (VEGFA), eventually inhibiting the growth and angiogenic capacity of EPCs. Importantly, the transplantation of hypoxia-cultured EPCs in a mouse model of limb ischemia promoted new blood vessel regeneration and blood supply recovery in the ischemic area better than the transplantation of normoxia-cultured EPCs. ConclusionsUnder hypoxia, the IDH1- and IDH2-mediated reverse TCA cycle promotes glutamine-derived de novo lipogenesis and stabilizes the expression of α-KG and HIF-1α, thereby enhancing the growth and angiogenic capacity of EPCs.

Real-Time Assessment of Mitochondrial Function in Cytotrophoblast and Syncytialized Trophoblast Cells Using the Seahorse XFe24 Extracellular Flux Analyzer.

Physiological processes utilize variable amounts of energy required for optimal growth, development, and survival. This energy is supplied by total intracellular adenosine triphosphate (ATP) and is mainly generated by mitochondrial oxidative phosphorylation and to a lesser extent via glycolysis. Here, we provide a detailed protocol for obtaining measurements of energy metabolism using the Seahorse XFe24 Extracellular Flux Analyzer. Specifically, this assay measures mitochondrial oxidative phosphorylation based on oxygen consumption rate (OCR) and glycolysis by analyzing the extracellular acidification rate (ECAR) via real-time live cell analysis. Using trophoblast cell lines, this protocol focuses on analyzing mitochondrial respiration for both cytotrophoblasts and syncytiotrophoblasts.

A potential adverse role for leptin and cardiac leptin receptor in the right ventricle in pulmonary arterial hypertension: effect of metformin is BMPR2 mutation-specific.

Pulmonary arterial hypertension is a fatal cardiopulmonary disease. Leptin, a neuroendocrine hormone released by adipose tissue, has a complex relationship with cardiovascular diseases, including PAH. Leptin is thought to be an important factor linking metabolic syndrome and cardiovascular disorders. Given the published association between metabolic syndrome and RV dysfunction in PAH, we sought to determine the association between leptin and RV dysfunction. We hypothesized that in PAH-RV, leptin influences metabolic changes via leptin receptors, which can be manipulated by metformin. Plasma leptin was measured in PAH patients and healthy controls from a published trial of metformin in PAH. Leptin receptor localization was detected in RV from PAH patients, healthy controls, animal models of PH with RV dysfunction before and after metformin treatment, and cultured cardiomyocytes with two different BMPR2 mutants by performing immunohistochemical and cell fractionation studies. Functional studies were conducted in cultured cardiomyocytes to examine the role of leptin and metformin in lipid-driven mitochondrial respiration. In human studies, we found that plasma leptin levels were higher in PAH patients and moderately correlated with higher BMI, but not in healthy controls. Circulating leptin levels were reduced by metformin treatment, and these findings were confirmed in an animal model of RV dysfunction. Leptin receptor expression was increased in PAH-RV cardiomyocytes. In animal models of RV dysfunction and cultured cardiomyocytes with BMPR2 mutation, we found increased expression and membrane localization of the leptin receptor. In cultured cardiomyocytes with BMPR2 mutation, leptin moderately influences palmitate uptake, possibly via CD36, in a mutation-specific manner. Furthermore, in cultured cardiomyocytes, the Seahorse XFe96 Extracellular Flux Analyzer and gene expression data indicate that leptin may not directly influence lipid-driven mitochondrial respiration in BMPR2 mutant cardiomyocytes. However, metformin alone or when supplemented with leptin can improve lipid-driven mitochondrial respiration in BMPR2 mutant cardiomyocytes. The effect of metformin on lipid-driven mitochondrial respiration in cardiomyocytes is BMPR2 mutation-specific. In PAH, increased circulating leptin can influence metabolic signaling in RV cardiomyocytes via the leptin receptor; in particular, it may alter lipid-dependent RV metabolism in combination with metformin in a mutation-specific manner and warrants further investigation.

Characterization of the effect of pomegranate crude extract, and its post-harvesting preservation procedures, on redox tone, cellular growth and metabolic profile of MDA-MB-231 cell line

BackgroundPomegranate is known for its beneficial properties due to its high content in antioxidants and might constitute a natural option for preventing and treatment of different pathologies including cancer. Since mitochondria are involved in tumorigenesis through ROS production and modulation of oxidative metabolism, we investigated the biological effects of pomegranate on cellular redox state, proliferation and metabolism in the breast cancer cell line MDA-MB-231 (MDA).MethodsMDA were treated for 24 h with graded concentration of filtered Pomegranate juice (PJ) and tested for metabolic Flux Analysis with XFe96 Extracellular Flux Analyzer, for proliferation using the xCELLigence System Real-Time Cell Analyzer and for intracellular ROS content by Confocal Microscopy Imaging.ResultsCells-treatment with freshly prepared pomegranate juice (PJ) resulted in a significant reduction of the intracellular ROS content already at the lower concentration of PJ tested. Additionally, it enhanced mitochondria respiration, and decreased glycolysis at high concentrations, inhibiting at the same time cell proliferation. As pomegranate is a seasonal fruit, assessment of optimum storage conditions preserving its bio-active properties was investigated. Our results indicated that storage conditions under controlled atmosphere for 30 days was able to enhance mitochondrial respiration at the same extent than freshly extracted PJ. Conversely, freezing procedure, though retaining the antioxidant and cell-growth inhibitory property, elicited an opposite effect on the metabolic profile as compared with fresh extract.ConclusionOverall, the results of our study, on the one hand, confirms the preventive/therapeutic potential of PJ, as well as of its post-harvested processing, for cancer management. On the other hand, it highlights the intrinsic difficulties in attaining mechanistic insights when a multiplicity of effects is elicited by a crude mixture of bio-active compounds.

Nutritional Composition and Pharmacological Activity of Musa balbisiana Colla Seed: An Insight into Phytochemical and Cellular Bioenergetic Profiling.

Musa balbisiana Colla belongs to the family Musaceae which is well-known for its nutritional and pharmacological properties. Here, we have analysed the phytochemical content and evaluated the nutritional, antioxidant, anti-glycation, α-amylase, and α-glucosidase inhibition potential. Moreover, for the first time, we have studied the bioenergetic profiles of the bioactive fractions of M. balbisiana seeds extract against oxidative stress-related mitochondrial and cellular dysfunction using XFe24 extracellular flux analyzer. M. balbisiana seeds have high nutritional values with significant levels of carbohydrates, starch, protein, and minerals (Ca, Na, Mg, Cu, Fe, and Zn). Bioactivity-guided fractionation of the methanolic extract of M. balbisiana seeds revealed that the ethyl acetate fraction (EAF) showed the highest antioxidant, anti-glycation, and phytochemical content as compared to other fractions. Moreover, the EAF showed a lower α-amylase inhibition and a higher α-glucosidase inhibitory activity. Most importantly, our GC-MS analyses of EAF revealed the presence of unique and previously unreported 14 phytochemical compounds. A strong correlation between the biological activities and total phenolic/tannin content was observed. In addition, the bioactive fraction of M. balbisiana seeds (EAF) improved the bioenergetic profiles of free fatty acid-induced oxidative stress with a concomitant increase in ATP production, and respiratory and glycolytic capacity. Altogether, our findings suggest that M. balbisiana seeds can be used as a natural supplement to boost antioxidant levels and combat oxidative stress and non-enzymatic glycation.

O-197 Vertical transmission of maternal mitochondrial DNA via extracellular vesicles (EVs) modulates embryo bioenergetics during the periconceptional period

Abstract Study question Does maternal endometrial mitochondrial (mt) DNA cargo of EVs modulate embryo bioenergetics during embryo implantation? Summary answer We demonstrate the vertical transmission of maternal mtDNA within endometrial-derived EVs and their uptake by the trophoblast which reduces mitochondrial respiration and ATP production. What is known already The release and uptake of membrane-enclosed compartments with specific cargos, commonly known as EVs, represents a novel cell-to-cell communication mechanism in physiological and pathogenic conditions. EVs are generally classified into three populations based on their biogenetic pathways, composition, and physical characteristics: apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs). Among other contents, EVs contain single- and double-stranded DNA, with their relative abundance varying depending on the cell and vesicle type. The vertical transmission of EV-associated DNA has been proposed as a novel genetic material transfer mechanism that may impact genome evolution and tumorigenesis. Study design, size, duration Prospective observational multicenter analysis in which EVs were obtained from endometrial fluid from healthy donors aged 18–35 years (n = 10) during the receptive phase of their natural cycle and under hormonal replacement therapy in pre-receptive (P + 2), receptive (P + 5), post-receptive (P + 8) stages (n = 13). Participants/materials, setting, methods Endometrial EVs isolated using ultracentrifugation and classified according to `parameters obtained from electron microscopy, Western blotting, and size distribution analysis. DNA copy number identified using high throughput sequencing. EV-associated DNA tagged with 5-ethynyl-2’-deoxyuridine was followed by confocal imaging after co-incubation of EVs with murine embryos (n = 200). ATP levels assessed using the FLASC luciferase reporter system, and the Seahorse XFE96 extracellular flux analyzer used to measure embryo oxygen consumption rate (OCR) (n = 400). Main results and the role of chance The human endometrium secretes all three EV types - ABs, MVs, and EXOs - into the human endometrial fluid. Deep sequencing revealed that EVs encapsulated nuclear and mtDNA. When analyzing endometrial biopsies, we observed the reduced mtDNA content of endometrial cells and the activation of mitochondrial clearance mechanisms, which coincided with the time of embryo implantation together with specific enrichment in endometrial MVs secreted during the periconceptional period. EVs were internalized and DNA was transferred to the cytoplasm and nuclei of trophectoderm of murine embryos. We analyzed ATP concentrations in murine embryos and found a significant reduction in ATP levels following the coculture of embryos with a combination of all EVs types compared to control embryos cultured without endometrial EVs (p &amp;lt; 0.001). Finally, we demonstrated a reduction in the OCR in embryos treated with endometrial EVs obtained during the receptive phase compared with the pre-receptive phase. In conclusion, maternal EVs modulate the bioenergetics of the preimplantation embryo by increasing the embryo's metabolic rate and oxygen consumption during the periconceptional period. Limitations, reasons for caution These results were obtained using a combination of a human endometrial model and a murine embryo model. Wider implications of the findings Our results suggest that the vertical transmission of maternal mtDNA encapsulated within EVs to the trophectoderm might energetically assist the preimplantation embryo through the implantation process. Trial registration number N/A

The Role of PGK1 in Promoting Ischemia/Reperfusion Injury-Induced Microglial M1 Polarization and Inflammation by Regulating Glycolysis

Stroke is a leading cause of death, with a continuously increasing incidence. As a metabolic process that catabolizes glucose pyruvate and provides adenosine triphosphate (ATP), glycolysis plays a crucial role in different diseases. Phosphoglycerate kinase 1 (PGK1) facilitates energy production with biosynthesis in many diseases, including stroke. However, the exact role of PGK1/glycolysis in stroke remains to be elucidated. A rat model of middle cerebral artery occlusion (MCAO) was used to mimic ischemia/reperfusion injuries. Oxygen glucose deprivation/re-oxygenation (OGD/R) was used to induce injury to highly aggressively proliferating immortalized (HAPI) rat microglial cells. The extracellular acidification rate (ECAR) was determined using an XFe24 Extracellular Flux Analyzer. ATP, lactate dehydrogenase, tumor necrosis factor-alpha, and interleukin-6 levels were measured using commercial kits. Chromatin immunoprecipitation assay was performed to examine the interaction between H3K27ac or p300 and the PGK1 promoter region. PGK1 was either knocked down or overexpressed by lentivirus. Thus, to examine its role in stroke, real-time polymerase chain reaction and immunoblotting were used to measure gene expression. The expression of PGK1 was increased and associated with M1 polarization and glycolysis in MCAO rat models. OGD/R promoted M1 polarization and HAPI microglial cell inflammation by regulating glycolysis. Silencing PGK1 reduced OGD/R-increased M1 polarization, inflammation, and glycolysis. Conversely, the overexpression of PGK1 promoted HAPI microglial cell inflammation by regulating glycolysis. The mechanism showed that histone acetyltransferase p300 promoted PGK1 expression through H3K27 acetylation. Finally, data indicated that silencing PGK1 inhibited microglia M1 polarization, inflammation, and glycolysis in MCAO rat models. PGK1 could promote ischemia/reperfusion injury-induced microglial M1 polarization and inflammation by regulating glycolysis, which might provide a novel direction in developing new therapeutic medications for preventing or treating stroke.

1,5-Anhydroglucitol promotes pre-B acute lymphocytic leukemia progression by driving glycolysis and reactive oxygen species formation

BackgroundPrecursor B-cell acute lymphoblastic leukemia (pre-B ALL) is the most common hematological malignancy in children. Cellular metabolic reorganization is closely related to the progression and treatment of leukemia. We found that the level of 1,5-anhydroglucitol (1,5-AG), which is structurally similar to glucose, was elevated in children with pre-B ALL. However, the effect of 1,5-AG on pre-B ALL was unclear. Here, we aimed to reveal the roles and mechanisms of 1,5-AG in pre-B ALL progression.MethodsThe peripheral blood plasma level of children with initial diagnosis of pre-B ALL and that of healthy children was measured using untargeted metabolomic analysis. Cell Counting Kit-8 assay, RNA sequencing, siRNA transfection, real-time quantitative PCR, and western blot were performed using pre-B ALL cell lines Reh and HAL-01. Cell cycle, cell apoptosis, ROS levels, and the positivity rate of CD19 were assessed using flow cytometry. Oxygen consumption rates and extracellular acidification rate were measured using XFe24 Extracellular Flux Analyzer. The lactate and nicotinamide adenine dinucleotide phosphate levels were measured using kits. The effect of 1,5-AG on pre-B ALL progression was verified using the In Vivo Imaging System in a xenotransplantation leukemia model.ResultsWe confirmed that 1,5-AG promoted the proliferation, viability, and intracellular glycolysis of pre-B ALL cells. Mechanistically, 1,5-AG promotes glycolysis while inhibiting mitochondrial respiration by upregulating pyruvate dehydrogenase kinase 4 (PDK4). Furthermore, high levels of intracellular glycolysis promote pre-B ALL progression by activating the reactive oxygen species (ROS)-dependent mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Conversely, N-acetylcysteine or vitamin C, an antioxidant, effectively inhibited 1,5-AG-mediated progression of leukemia cells.ConclusionsOur study reveals a previously undiscovered role of 1,5-AG in pre-B ALL, which contributes to an in-depth understanding of anaerobic glycolysis in the progression of pre-B ALL and provides new targets for the clinical treatment of pre-B ALL.

BUTYRATE-INDUCED MITOCHONDRIAL FUNCTION IMPROVES BARRIER FUNCTION IN INFLAMMATORY BOWEL DISEASE (IBD)

Abstract BACKGROUND Short-chain fatty acids (SCFAs), end-product of gut microbial metabolism, play an important role in colonic homeostasis. Butyrate is a prime SCFA that fuels intestinal epithelial cells (IECs). However, this energy source is deficient in inflammatory bowel disease (IBD) due to dysbiotic microbiota. In this study, we posit that butyrate supplementation drives mitochondrial respiration induced barrier function in IBD. METHODS Normal human Colon mucosal (NCM460) epithelial cells were treated overnight with different concentration of sodium butyrate (SB; 0.5-5.0mM) with or without TNF (1ng/ml). To model mitochondrial deficiency, TFAM (mitochondrial transcriptional factor A), was silenced (shTFAM) in NCM460 cells. For mitochondrial respiration assessment, NCM cells were maintained and treated with SB (-/+TNF) under glucose-free medium and Oxygen consumption rate (OCR) was measured using high-throughput Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies). Gene expression analysis was determined by RT-qPCR from total RNA isolated from untreated and treated NCM cells as well as in mitochondria-deficient shTFAM NCMs compared to shControl cells. All assays were performed in triplicate and fold changes were calculated using the ΔΔCT method. RESULT Seahorse analysis showed significant increase in Maximal respiratory capacity, spare respiratory capacity and ATP production at sub-optimal (2mM) and optimal (5mM) SB concentrations, suggesting a SB induced mitochondrial respiration and capacity to overcome oxidative stress. At molecular level, SB significantly increased the expression of genes associated with mitochondrial biogenesis (TFAM), complex I (Ndufa1, Ndufa4, Ndufa6), complex IV (Cox6A1) and complex V (ATP5E, ATP8), at optimal 5mM concentration. The effect of SB was more pronounced under inflammatory condition when NCM cells were treated in the presence of low dose TNF. Further transcriptomic analysis of TFAM-deficient NCMs demonstrated significant decrease in mRNA of major butyrate transporters (MCT1, MCT4, ABCG2) compared to TFAM-proficient shControl cells, irrespective of any treatment. Similar decreased expression pattern was observed for tight junction genes (ZO1 and Occludin) in shTFAM compared to shControl cells. However, butyrate transporters and ZO1 mRNA increased significantly in both shTFAM and shControl cells after SB treatment at sub-optimal concentration (2mM) in the presence of TNF. Interestingly, only shTFAM cells showed increase in transcript level of Occludin after SB+TNF treatment, with no effect in shControl. CONCLUSION These data suggest that butyrate metabolism is a critical player in promoting oxidative health of mitochondria, which further drives transporter-mediated butyrate utilization and promotes barrier integrity under inflammatory conditions as in IBD.

Paeonol represses A549 cell glycolytic reprogramming and proliferation by decreasing m6A modification of Acyl-CoA dehydrogenase.

Aberrant glycolytic reprogramming is involved in lung cancer progression by promoting the proliferation of non-small cell lung cancer cells. Paeonol, as a traditional Chinese medicine, plays a critical role in multiple cancer cell proliferation and inflammation. Acyl-CoA dehydrogenase (ACADM) is involved in the development of metabolic diseases. N6-methyladenosine (m6A) modification is important for the regulation of messenger RNA stability, splicing, and translation. Here, we investigated whether paeonol regulates the proliferation and glycolytic reprogramming via ACADM with m6A modification in A549 cells (human non-small cell lung cancer cells). Cell counting kit 8, 5-Bromo-2-deoxyuridine, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, flow cytometry analysis, western blotting and seahorse XFe24 extracellular flux analyzer assays showed that paeonol had a significant inhibitory effect against A549 cell proliferation and glycolysis. Mechanistically, ACADM was a functional target of paeonol. We also showed that the m6A reader YTH domain containing 1 plays an important role in m6A-modified ACADM expression, which is negatively regulated by paeonol, and is involved in A549 cell proliferation and glycolytic reprogramming. These results indicated the central function of paeonol in regulating A549 cell glycolytic reprogramming and proliferation via m6A modification of ACADM.

Taurine protects R28 cells from hypoxia/re-oxygenation-induced damage via regulation of mitochondrial energy metabolism

Oxidative-induced damage and hypoxia/re-oxygenation (H/R) injury are common causes of irreversible visual impairment. The goals of this study were to explore the effects of taurine on R28 cells under the two damage models and the underlying mechanisms. Low doses of taurine supplementation promoted cell viability, mitochondrial membrane potential (MMP), SOD levels, ATP contents and attenuated cytotoxicity and intracellular ROS generation of the R28 cells under the two kinds of damage. The expression level of GTPBP3, a mitochondrial-tRNA (mt-tRNA) modification enzyme that catalyzes the taurine involved modification, was decreased under the two damage and taurine could reverse the reduction. After knocking down GTPBP3, the R28 cells become vulnerable to damage. The viability, cytotoxicity, MMP and intracellular ROS level of knockdown cells changed more obviously under the H/R injury than those of control cell. We also found that knockdown of GTPBP3 significantly decreased mitochondrial energy metabolism by measuring the oxidative respiration rate by the Seahorse XFe24 extracellular flux analyzer. The protection of low doses of taurine disappeared on knockdown R28 cells, indicating that GTPBP3 is crucial in the protection mechanisms of taurine. However, the impacts of the reduction of GTPBP3 level can be reversed by relatively high doses of taurine, implying the protection effects of taurine were dose-dependent, and there were more complicated mechanisms remain to be explored. This study explored a new mechanism of the neuroprotective effects of taurine, which depend on the GTPBP3-mediated taurine modification of mt-tRNAs and the promotion of mitochondrial energy metabolism.

Effects of silkworm pupa protein on apoptosis and energy metabolism in human colon cancer DLD-1 cells

ObjectiveTo investigate the effects of silkworm pupa (Bombyx mori) protein (SPP) on cell proliferation, apoptosis and energy metabolism in human colon cancer cells DLD-1. MethodsCCK-8 was used to detect cell proliferation rate after 72 h of cell culture for the control group (normal cultured DLD-1 cells) and SPP dose groups; Annexin-V/PI was applied to observe cell apoptosis; XFe24 Extracellular Flux Analyzer was used to detect cell mitochondrial respiratory function and glycolytic function. ResultsComparing with the control, SPP significantly inhibited the proliferation of DLD-1 cells with all the dosage tested (P < 0.01); flow cytometry showed that SPP significantly promoted apoptosis (P < 0.05). Additionally, SPP could significantly inhibited mitochondrial metabolism and glycolysis of DLD-1 cells and decreased cell energy metabolism in all groups treated with different doses. ConclusionSPP can cause oxidative damage, promote apoptosis, and reduce mitochondrial respiratory and glycolysis rate in colon cancer DLD-1 cells, which reveals that SPP has the potential to serve as the anti-cancer drugs in the future, but further experimental evidence is needed.

The Effect of Nicotinamide Riboside, an NAD + Precursor, on Glycolysis and NAD + Salvage Pathway in Alcohol-Stimulated Macrophages

ObjectivesWe demonstrated that ethanol decreased cellular NAD+ levels not only by consuming it but by repressing the expression of genes involved in the NAD+ salvage pathway, consequently altering cellular bioenergetics in macrophages. Therefore, we explored whether nicotinamide riboside (NR), an NAD+ precursor, could restore cellular NAD + and reverse changes in glycolytic capacities triggered by ethanol alone or ethanol with FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway. MethodsRAW 264.7 macrophages were treated with 80 mM ethanol with or without 1 mM of NR and 50 nM FK866 for 72 h. Expression of genes involved in the NAD+ salvage pathway and glycolysis and cellular NAD+ levels were measured. Parameters related to glycolysis were determined using a Seahorse XFe24 Extracellular Flux analyzer. Sirtuin 1 (SIRT1) was inhibited by its inhibitor sirtinol or siRNA-mediated knockdown or activated by resveratrol to evaluate its contribution to the effects of ethanol on the NAD+ salvage pathway. ResultsNR significantly increased, but NAMPT inhibition decreased cellular NAD+ content in control and ethanol-treated cells. However, ethanol decreased, and NAMPT inhibition further lowered NAD+ content. Decreases in cellular NAD+ and mRNA levels of genes in the NAD+ salvage pathway, such as Nmrk1, Nampt, Nmnat1, and Nmnat3, by ethanol and FK866 were attenuated by NR. Interestingly, both NR and FK866 diminished increases in glycolytic capacity, glycolytic reserve, and non-glycolytic acidification caused by ethanol. Ethanol increased the expression and activity of hypoxia-inducible factor 1α (HIF1α) and glycolytic genes, and pyruvate dehydrogenase (PDH) phosphorylation, all of which were attenuated by NR and FK866, with the latter being more potent. Ethanol and FK866, to a much greater extent, repressed Sirt1 expression. SIRT1 inhibition reduced Nampt expression, but SIRT1 activation and NR led to the opposite effects in ethanol-exposed macrophages. ConclusionsNR attenuates decreases in cellular NAD + content and glycolytic capacities by ethanol alone or ethanol with NAMPT inhibition at least partly through the activation of the SIRT1 pathway in macrophages. Funding SourcesUniversity of Connecticut College of Agriculture, Health and Natural Resources & USDA multistate Hatch W4002.

MiR-99a-5p inhibits glycolysis and induces cell apoptosis in cervical cancer by targeting RRAGD.

Cervical cancer (CC) is one of the most common gynecological malignancies that endangers women's health. A negative effect of glycolysis is that it contributes to abnormal tumor growth. MicroRNA (miR)-99a expression has been found to be decreased in CC. The present study aimed to investigate the role of miR-99a-5p in glycolysis in CC. For this purpose, the association between miR-99a and the prognosis of patients with CC from The Cancer Genome Atlas database was analyzed using Kaplan-Meier analysis. miR-99a-5p expression and Ras-related GTP binding D (RRAGD) expression were assessed using reverse transcription-quantitative PCR and western blot analysis. Cell proliferation and apoptosis were examined using an MTT assay and flow cytometry, respectively. Glucose uptake, lactate concentration and extracellular acidification rate were measured using a glucose uptake colorimetric assay, a lactate colorimetric assay and a Seahorse XFe96 extracellular flux analyzer, respectively. The association between miR-99a-5p and RRAGD was predicted using TargetScan 7.1, and was confirmed using dual luciferase reporter assay. The results revealed that miR-99a-5p expression was decreased and that of RRAGD was increased in CC tissues and cell lines. RRAGD was negatively regulated by miR-99a-5p. The overexpression of miR-99a-5p induced apoptosis and inhibited glycolysis in CC cells by targeting RRAGD. On the whole, the present study revealed a novel mechanism through which miR-99a-5p regulates cell apoptosis and glycolysis in CC, thus providing a potential therapeutic target for CC.

Atovaquone: An Inhibitor of Oxidative Phosphorylation as Studied in Gynecologic Cancers.

Simple SummaryThe Warburg effect in cancer cells (high glucose update and lactate release) is an adaptation that is considered a hallmark of most neoplasms. Blocking oxidative phosphorylation is one way to combat carcinogenesis. Atovaquone is a mitochondrial complex III inhibitor. It is currently FDA-approved for the treatment of malaria, and is a well-tolerated, orally available medication. Our laboratory studied the anti-cancer properties of atovaquone in gynecologic cancers. We found that atovaquone slowed ovarian cancer growth in both cell lines and mouse models. Additional anti-cancer effects were seen, such as the reduced proliferation of cancer stem cells and spheroids implanted in mice. Atovaquone inhibited oxygen consumption and ATP production. Metabolic studies showed that atovaquone shifted glycolysis, electronic transport and the citric acid cycle. Our studies provided the mechanistic understanding and preclinical data to support the further investigation of atovaquone’s potential as a cancer therapy for gynecologic cancers.Oxidative phosphorylation is an active metabolic pathway in cancer. Atovaquone is an oral medication that inhibits oxidative phosphorylation and is FDA-approved for the treatment of malaria. We investigated its potential anti-cancer properties by measuring cell proliferation in 2D culture. The clinical formulation of atovaquone, Mepron, was given to mice with ovarian cancers to monitor its effects on tumor and ascites. Patient-derived cancer stem-like cells and spheroids implanted in NSG mice were treated with atovaquone. Atovaquone inhibited the proliferation of cancer cells and ovarian cancer growth in vitro and in vivo. The effect of atovaquone on oxygen radicals was determined using flow and imaging cytometry. The oxygen consumption rate (OCR) in adherent cells was measured using a Seahorse XFe96 Extracellular Flux Analyzer. Oxygen consumption and ATP production were inhibited by atovaquone. Imaging cytometry indicated that the majority of the oxygen radical flux triggered by atovaquone occurred in the mitochondria. Atovaquone decreased the viability of patient-derived cancer stem-like cells and spheroids implanted in NSG mice. NMR metabolomics showed shifts in glycolysis, citric acid cycle, electron transport chain, phosphotransfer, and metabolism following atovaquone treatment. Our studies provide the mechanistic understanding and preclinical data to support the further investigation of atovaquone’s potential as a gynecologic cancer therapeutic.

Ethanol Shifts Myoblast Bioenergetic Phenotype: Implications for Muscle Regeneration

At‐risk alcohol use is nearly twice as prevalent among people living with HIV (PLWH) as the general population. Alcohol and HIV can independently and synergistically increase risk for metabolic diseases, including myopathy. Decreased functional skeletal muscle (SKM) mass is associated with morbidity and all‐cause mortality. A major focus of our research is to investigate the independent and combined effects of alcohol and HIV on SKM regenerative capacity and its contribution to myopathy development. Previously, we showed that in vitro ethanol decreases myoblast extracellular acidification rate (ECAR), indicative of decreased glycolytic function, and that the decrease in ECAR was associated with decreased myoblast differentiation. The objective of this study was to determine effects of prior and recent alcohol exposure and simian immunodeficiency virus (SIV) on bioenergetic function in myoblasts from naïve and SIV‐infected rhesus macaques. Female rhesus macaques (6‐10 years of age) were administered chronic binge alcohol (CBA; peak blood alcohol concentration=50‐60 mM, 5 days per week) or isovolumetric water vehicle (VEH) beginning 3 months prior to infection with SIVmac251. Daily antiretroviral therapy (ART) was initiated 2.5 months after SIV infection, and macaques were humanely euthanized 9 months later. SKM samples were collected for myoblast isolation prior to CBA or VEH (naïve, N=6) and at study end point (VEH/SIV, N=6; CBA/SIV, N=6). Myoblasts from each group were incubated with ethanol (0 and 50 mM) for 3 days prior to Mito Stress Test, Glycolysis Stress Test, and ATP Rate Assay using an XFe96 Extracellular Flux Analyzer. There were no significant differences due to CBA or SIV on mitochondrial or glycolytic function for any measure. During the Mito Stress Test, ethanol exposure significantly (p&lt;0.05) decreased ECAR at baseline and after the addition of oligomycin (ATP synthase inhibitor), indicative of decreased glycolytic function, without significant changes in mitochondrial function. Similarly, ethanol exposure decreased all parameters in the Glycolysis Stress Test (non‐glycolytic acidification, glycolysis, glycolytic reserve, and glycolytic capacity). An ATP rate assay confirmed that ethanol exposure significantly decreased glycolytic ATP production, contributing to a decrease in total ATP production. These results indicate that recent exposure to an intoxicating concentration of ethanol shifts myoblast bioenergetic phenotype, impairs glycolysis, and produces a bioenergetic deficit regardless of prior alcohol exposure or SIV/ART. The shift away from glycolysis occurs under basal and stressed conditions. We propose that altered glycolytic enzyme activity underlies the alcohol‐mediated decrease in glycolytic function, negatively impacting myoblast differentiation and SKM regenerative capacity.