Seahorse XFe24 Extracellular Flux Analyzer Research Articles

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.

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.

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 < 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

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.

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.

Modulation of energy metabolism to overcome drug resistance in chronic myeloid leukemia cells through induction of autophagy

Tyrosine kinase inhibitors (TKIs) such as imatinib (IM) are key drugs for treatment of chronic myeloid leukemia (CML). Development of drug resistance to TKIs due to BCR-ABL mutation, especially T315I mutation, poses a major challenge in the clinical treatment of CML. The purpose of this study was to test metabolic modulation as a potential strategy to overcome imatinib resistance based on the possible crosstalk between BCR-ABL signaling and metabolic changes in CML. 2-deoxy-d-glucose (2-DG) was used to modulate the glucose metabolism in CML cells sensitive to IM (KBM5 cell line) and resistant to imatinib with BCR-ABL T315I mutation (KBM5-T315I cell line). Seahorse XFe24 extracellular flux analyzer to quantify oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was used to measure cellular energy metabolism. Cell proliferation was analyzed by CCK-8 assay and MTS assay. Annexin V/PI staining was used to evaluate cell apoptosis. Autophagy-related proteins and enzyme/proteins were detected by Western blotting. Cellular ATP concentration was detected using an ATP-based Cell Titer Kit. The combined action of 2-DG and IM was evaluated by calculating the drug combination index. Our results found that inhibition of glucose metabolism by 2-DG significantly impaired the viability of CML cells and co-treatment with 2-DG and imatinib induced a synergistic inhibition of KBM5 and KBM5-T315I cells. 2-DG induced cell death by autophagy, not by apoptosis, as evidenced by increased expression of Beclin1 and LC3AII and lack of annexin V/PI-positive cells. At the biochemical level, 2-DG inhibited glycolysis and mitochondrial oxygen consumption manifested by a significant decrease in ECAR and OCR, and a depletion of ATP. The severe metabolic stress induced by 2-DG in CML cells led to autophagic cell death. Our results suggested a metabolic vulnerability of CML cells that could be targeted by a combination of 2-DG and imatinib as an alternative treatment for imatinib-resistant CML.

Real-Time Analysis of Mitochondrial Electron Transport Chain Function in Toxoplasma gondii Parasites Using a Seahorse XFe96 Extracellular Flux Analyzer.

The mitochondrial electron transport chain (ETC) performs several critical biological functions, including maintaining mitochondrial membrane potential, serving as an electron sink for important metabolic pathways, and contributing to the generation of ATP via oxidative phosphorylation. The ETC is important for the survival of many eukaryotic organisms, including intracellular parasites such as the apicomplexan Toxoplasma gondii. The ETC of T. gondii and related parasites differs in several ways from the ETC of the mammalian host cells they infect, and can be targeted by anti-parasitic drugs, including the clinically used compound atovaquone. To characterize the function of novel ETC proteins found in the parasite and to identify new ETC inhibitors, a scalable assay that assesses both ETC function and non-mitochondrial parasite metabolism (e.g., glycolysis) is desirable. Here, we describe methods to measure the oxygen consumption rate (OCR) of intact T. gondii parasites and thereby assess ETC function, while simultaneously measuring the extracellular acidification rate (ECAR) as a measure of general parasite metabolism, using a Seahorse XFe96 extracellular flux analyzer. We also describe a method to pinpoint the location of ETC defects and/or the targets of inhibitors, using permeabilized T. gondii parasites. We have successfully used these methods to investigate the function of T. gondii proteins, including the apicomplexan parasite-specific protein subunit TgQCR11 of the coenzyme Q:cytochrome c oxidoreductase complex (ETC Complex III). We note that these methods are also amenable to screening compound libraries to identify candidate ETC inhibitors.

Monitoring Mitochondrial Respiration in Mouse Cerebellar Granule Neurons.

Defects in mitochondrial oxidative phosphorylation have been observed in numerous neurodegenerative disorders and are linked to bioenergetic crises leading to neuronal death. The distinct metabolic profile of neurons is predominantly oxidative, which is characterized by the oxidation of glucose or its metabolites in the mitochondria to produce ATP. This process involves the tricarboxylic acid cycle, electron transfer in the respiratory chain, and oxygen consumption. Therefore, measurement of oxygen consumption rates (OCR) can be accurately applied to assess the rate of mitochondrial respiration. In this chapter, we describe our optimized protocol for the assessment of OCR specifically in primary mouse cerebellar granule neurons (CGN). The protocol includes isolation and manipulation of mouse CGNs followed by real-time assessment of mitochondrial OCR using a Seahorse XFe96 extracellular flux analyzer.

Curcumin pre-treatment may protect against mitochondrial damage in LRRK2-mutant Parkinson's disease and healthy control fibroblasts

Mitochondrial dysfunction has been proposed as one of the pathobiological underpinnings in Parkinson's disease. Environmental stressors, such as paraquat, induce mitochondrial dysfunction and promote reactive oxygen species production. Targeting oxidative stress pathways could prevent mitochondrial dysfunction and thereby halt the neurodegeneration in Parkinson's disease. Since curcumin is touted as an antioxidant and neuroprotective agent, the aim of this study was to investigate if curcumin is a suitable therapy to target mitochondrial dysfunction in Parkinson's disease using a paraquat-toxicity induced model in fibroblasts from LRRK2-mutation positive Parkinson's disease individuals and healthy controls. The fibroblasts were exposed to five treatment groups, (i) untreated, (ii) curcumin only, (iii) paraquat only, (iv) pre-curcumin group: with curcumin for 2hr followed by paraquat for 24hr and (v) post-curcumin group: with paraquat for 24hr followed by curcumin for 2hr. Mitochondrial function was determined by measuring three parameters of mitochondrial respiration (maximal respiration, ATP-associated respiration, and spare respiratory capacity) using the Seahorse XFe96 Extracellular Flux Analyzer. As expected, paraquat effectively disrupted mitochondrial function for all parameters. Pre-curcumin treatment improved maximal and ATP-associated respiration whereas, post-curcumin treatment had no effect. These findings indicate that curcumin may be most beneficial as a pre-treatment before toxin exposure, which has implications for its therapeutic use. These promising findings warrant future studies testing different curcumin dosages, exposure times and curcumin formulations in larger sample sizes of Parkinson's disease and control participants.

Lactate oxidative phosphorylation by annulus fibrosus cells: evidence for lactate-dependent metabolic symbiosis in intervertebral discs

BackgroundIntervertebral disc degeneration contributes to low back pain. The avascular intervertebral disc consists of a central hypoxic nucleus pulpous (NP) surrounded by the more oxygenated annulus fibrosus (AF). Lactic acid, an abundant end-product of NP glycolysis, has long been viewed as a harmful waste that acidifies disc tissue and decreases cell viability and function. As lactic acid is readily converted into lactate in disc tissue, the objective of this study was to determine whether lactate could be used by AF cells as a carbon source rather than being removed from disc tissue as a waste byproduct.MethodsImport and conversion of lactate to tricarboxylic acid (TCA) cycle intermediates and amino acids in rabbit AF cells were measured by heavy-isotope (13C-lactate) tracing experiments using mass spectrometry. Levels of protein expression of lactate converting enzymes, lactate importer and exporter in NP and AF tissues were quantified by Western blots. Effects of lactate on proteoglycan (35S-sulfate) and collagen (3H-proline) matrix protein synthesis and oxidative phosphorylation (Seahorse XFe96 Extracellular Flux Analyzer) in AF cells were assessed.ResultsHeavy-isotope tracing experiments revealed that AF cells imported and converted lactate into TCA cycle intermediates and amino acids using in vitro cell culture and in vivo models. Addition of exogenous lactate (4 mM) in culture media induced expression of the lactate importer MCT1 and increased oxygen consumption rate by 50%, mitochondrial ATP-linked respiration by 30%, and collagen synthesis by 50% in AF cell cultures grown under physiologic oxygen (2-5% O2) and glucose concentration (1-5 mM). AF tissue highly expresses MCT1, LDH-H, an enzyme that preferentially converts lactate to pyruvate, and PDH, an enzyme that converts pyruvate to acetyl-coA. In contrast, NP tissue highly expresses MCT4, a lactate exporter, and LDH-M, an enzyme that preferentially converts pyruvate to lactate.ConclusionsThese findings support disc lactate-dependent metabolic symbiosis in which lactate produced by the hypoxic, glycolytic NP cells is utilized by the more oxygenated AF cells via oxidative phosphorylation for energy and matrix production, thus shifting the current research paradigm of viewing disc lactate as a waste product to considering it as an important biofuel. These scientifically impactful results suggest novel therapeutic targets in disc metabolism and degeneration.

TARGETING CD4+ T CELL METABOLISM PROVIDES AN EFFECTIVE AND AGE-SPECIFIC IMMUNOSUPPRESSION

Targeting T-cell metabolism may provide an effective immunosuppression. We delineated the correlation of aging and T cell metabolism. Moreover, we defined the consequences of metabolic changes in T-cell compartments on alloimmunity and delineated specific and novel immunosuppressive targets on CD-4+ T-cells. We collected naïve CD4+ T cells (splenocytes) of young and old (3 and 18 months) C67BL/6 mice. Following activation with anti-CD3 and anti-CD28 (24 hrs), we evaluated oxidative phosphorylation (OXPHOS) and aerobic glycolysis by oxygen consumption (OCR) and extracellular acidification (ECAR) using a Seahorse XFe96 extracellular flux analyzer. Graft survival, T-cell specificity and a detailed immune profiling were recorded serially. Upon activation in-vitro, a compromised utilization of OXPHOS and glycolysis pathways was observed in old CD4+ T cells (p<0.0001), indicating failure of old CD4+ T cells to exhibit adequate metabolic reprogramming. Further analysis of metabolic pathways revealed a critical role of glutaminolysis in T cell activation. 6-diazo-5-oxo-l-norleucine (DON), an analog of glutamine inhibiting critical enzymes of glutaminolysis resulted into an age-specific reduced activation and proliferative capacities of CD4+T cells. Notably, lower doses of DON inhibited IL-2 production and CFSE-determined proliferation only in old but not young CD4+ T cells (p<0.001). Utilizing a pre-clinical fully mismatched transplantation model, we observed inversed CD4:CD8 ratio in old recipients following DON (1.6mg/kg every other day). Graft survival was prolonged in young recipients, from 7 to 18 days (p< 0.01). Notably, was a drastic and age-specific effect of DON on graft skin graft survival in old recipients to 42 days (n=7, p<0.001). Targeting metabolic pathways was specific to CD4+ T-cells. Adoptively transferring young CD4+ T cells reversed the prolonged survival observed in old recipients. Depletion of CD8+ T cells did not alter transplant outcomes of DON in either young or old recipients. Taken together, we introduce a detailed and age-specific analysis of metabolic pathways in T cells and introduce a novel concept for an optimized immunosuppression in old recipients.

Nicotinamide Riboside Inhibits Alcohol-Induced Inflammation and Oxidative Stress in Macrophages

Abstract Objectives Macrophages play an essential role in the development of alcohol-induced inflammation. The objective of this study was to investigate whether nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD+) precursor naturally found in milk, can attenuate alcohol-induced inflammation and oxidative stress in macrophages with the elucidation of mechanisms of action. Methods RAW 264.7 macrophages and mouse bone marrow-derived macrophages (BMDMs) were stimulated with 80 mM ethanol with or without 1 mM of NR for 72 h. Expression of genes associated with inflammation and oxidative stress and cellular reactive oxygen species (ROS) accumulation were measured. Also, to evaluate the contribution of sirtuin 1 (SIRT1) to the NR's effect, cellular NAD + level (a cofactor of SIRT1), SIRT1 activity, and mitochondrial DNA copy number were measured. SIRT1 activity was inhibited or activated by sirtinol and resveratrol, respectively, to confirm SIRT1 functions further. Parameters related to mitochondrial respiration were determined using a Seahorse XFe24 Extracellular Flux analyzer. Results NR significantly decreased ethanol-induced inflammatory gene expression, with a concomitant decrease in nuclear translocation of nuclear factor kB p65 in macrophages. Increased cellular ROS levels by ethanol were also attenuated concomitantly with decreased expression of NADPH oxidase 2, a ROS-producing enzyme, by NR in both macrophage cell types. Ethanol decreased SIRT1 mRNA, protein and activity, cellular NAD + level, and mitochondrial DNA, all of which were markedly attenuated by NR. SIRT1 inhibition by sirtinol augmented the inflammatory effects of ethanol, while SIRT1 activation by resveratrol elicited the opposing results. Ethanol increased mitochondrial respiration, ATP production, and proton leak, but decreased maximal respiration and spare respiratory capacity. The ethanol-induced changes in mitochondrial respiration were abolished by NR. Conclusions NR showed anti-inflammatory and antioxidant properties in ethanol-treated macrophages by counteracting the effect of ethanol on lowering SIRT1 expression and cellular NAD+ levels. Therefore, NR may be a potential therapeutic agent for alcohol-induced inflammation and oxidative stress. Funding Sources This work is supported by the NIH 3R01DK108254-04S1.

Astaxanthin Inhibits Alcohol-Induced Inflammation and Oxidative Stress by Mediating the Sirtuin 1 and Histone Deacetylase 4 Axis in Macrophages

Abstract Objectives We previously demonstrated that astaxanthin (ASTX), a xanthophyll carotenoid, exerts anti-inflammatory and antioxidant properties in macrophages exposed to ethanol. In this study, to gain mechanistic insight, we explored the role of sirtuin 1 (SIRT1) and histone deacetylase 4 (HDAC4) in the prevention of ethanol-induced inflammation and oxidative stress by ASTX in macrophages. Methods RAW 264.7 macrophages and bone marrow-derived macrophages (BMDMs) isolated from wild-type (WT) and mice with macrophage specific-deletion of HDAC4 (Hdac4MKO) were used. Cells were stimulated with 80 mM ethanol in the absence or presence of 25 mM of ASTX for 72 h. The expression of genes associated with inflammation, SIRT1 and HDAC4 was measured. RAW 264.7 macrophages were treated with sirtinol or resveratrol, which are known inhibitor or activator of SIRT1 activity, respectively, to determine the effect of SIRT1 activity on HDAC4 expression. Parameters related to mitochondrial respiration were determined using a Seahorse XFe24 Extracellular Flux analyzer in both macrophage cell types. Results Ethanol decreased mRNA and protein levels of SIRT1 but increased those of HDAC4, which was attenuated by ASTX in RAW 264.7 macrophages. ASTX abolished an increase in acetylated histone H3 by ethanol in RAW 264.7 macrophages. Knockdown of HDAC4 increased SIRT1 expression, concomitantly decreasing ethanol-induced inflammatory gene expression. Furthermore, BMDMs from Hdac4MKO mice showed significant decreases in ethanol-induced inflammatory genes but an increase in SIRT1 expression compared with WT BMDMs. Ethanol increased mitochondrial respiration, ATP production, and proton leak, but decreased maximal respiration and spare respiratory capacity. The changes by ethanol were abolished by ASTX in RAW 264.7 macrophages. Compared with WT BMDMs, the ethanol-induced alterations in mitochondrial respiration were abrogated in Hdac4MKO BMDMs. Conclusions The anti-inflammatory and antioxidant properties of ASTX in ethanol-treated macrophages may be mediated, at least in part, by the crosstalk between SIRT1 and/HDAC4. This study, therefore, provides new mechanisms in preventing the alcohol-induced inflammation and oxidative stress through the crosstalk between SIRT1 and HDAC4. Funding Sources This study was supported by NIH 1R01DK108254–01.

Oxygen consumption rate of Caenorhabditis elegans as a high-throughput endpoint of toxicity testing using the Seahorse XFe96 Extracellular Flux Analyzer

Caenorhabditis elegans presents functioning, biologically relevant phenotypes and is frequently used as a bioindicator of toxicity. However, most C. elegans in vivo effect-assessment methods are laborious and time consuming. Therefore, we developed a novel method to measure the oxygen consumption rate of C. elegans as a sublethal endpoint of toxicity. This protocol was tested by exposing 50 larval stage one C. elegans individuals for 48 h (at 20 °C) to different concentrations of two toxicants i.e. benzylcetyldimethylammonium chloride (BAC-C16) and cadmium (Cd). Following exposures, the oxygen consumption rate of the C. elegans individuals were measured using the high-throughput functionality of the Seahorse XFe96 Extracellular Flux Analyzer. Dose-response curves for BAC-C16 (R2 = 0.93; P = 0.001) and Cd (R2 = 0.98; P = 0.001) were created. Furthermore, a strong, positive correlation was evidenced between C. elegans oxygen consumption rate and a commonly used, ecologically relevant endpoint of toxicity (growth inhibition) for BAC-C16 (R2 = 0.93; P = 0.0001) and Cd (R2 = 0.91; P = 0.0001). The data presented in this study show that C. elegans oxygen consumption rate can be used as a promising functional measurement of toxicity.

Astaxanthin stimulates mitochondrial biogenesis in insulin resistant muscle via activation of AMPK pathway.

BackgroundSkeletal muscle is mainly responsible for insulin‐stimulated glucose disposal. Dysfunction in skeletal muscle metabolism especially during obesity contributes to the insulin resistance. Astaxanthin (AX), a natural antioxidant, has been shown to ameliorate hepatic insulin resistance in obese mice. However, its effects in skeletal muscle are poorly understood. The current study aimed to investigate the molecular target of AX in ameliorating skeletal muscle insulin resistance.MethodsWe fed 6‐week‐old male C57BL/6J mice with normal chow (NC) or NC supplemented with AX (NC+AX) and high‐fat‐diet (HFD) or HFD supplemented with AX for 24 weeks. We determined the effect of AX on various parameters including insulin sensitivity, glucose uptake, inflammation, kinase signaling, gene expression, and mitochondrial function in muscle. We also determined energy metabolism in intact C2C12 cells treated with AX using the Seahorse XFe96 Extracellular Flux Analyzer and assessed the effect of AX on mitochondrial oxidative phosphorylation and mitochondrial biogenesis.ResultsAX‐treated HFD mice showed improved metabolic status with significant reduction in blood glucose, serum total triglycerides, and cholesterol (p< 0.05). AX‐treated HFD mice also showed improved glucose metabolism by enhancing glucose incorporation into peripheral target tissues, such as the skeletal muscle, rather than by suppressing gluconeogenesis in the liver as shown by hyperinsulinemic–euglycemic clamp study. AX activated AMPK in the skeletal muscle of the HFD mice and upregulated the expressions of transcriptional factors and coactivator, thereby inducing mitochondrial remodeling, including increased mitochondrial oxidative phosphorylation component and free fatty acid metabolism. We also assessed the effects of AX on mitochondrial biogenesis in the siRNA‐mediated AMPK‐depleted C2C12 cells and showed that the effect of AX was lost in the genetically AMPK‐depleted C2C12 cells. Collectively, AX treatment (i) significantly ameliorated insulin resistance and glucose intolerance through regulation of AMPK activation in the muscle, (ii) stimulated mitochondrial biogenesis in the muscle, (iii) enhanced exercise tolerance and exercise‐induced fatty acid metabolism, and (iv) exerted antiinflammatory effects via its antioxidant activity in adipose tissue.ConclusionsWe concluded that AX treatment stimulated mitochondrial biogenesis and significantly ameliorated insulin resistance through activation of AMPK pathway in the skeletal muscle.