Oxygen Consumption Rate Values Research Articles

HDAC/H3K27ac-mediated transcription of NDUFA3 exerts protective effects on high glucose-treated human nucleus pulposus cells through improving mitochondrial function

Diabetes mellitus (DM) is a well-documented risk factor of intervertebral disc degeneration (IVDD). The current study was aimed to clarify the effects and mechanisms of NADH: ubiquinone oxidoreductase subunit A3 (NDUFA3) in human nucleus pulposus cells (HNPCs) exposed to high glucose. NDUFA3 was overexpressed in HNPCs via lenti-virus transduction, which were co-treated with high glucose and rotenone (a mitochondrial complex I inhibitor) for 48 h. Cell activities were assessed for cell viability, cell apoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) ratio, oxygen consumption rate (OCR) and mitochondrial complexes I activities. High glucose decreased cell viability, increased apoptotic cells, increased ROS production, decreased MMP levels and OCR values in HNPCs in a dose-dependent manner. Rotenone co-treatment augmented the high glucose-induced injuries on cell viability, apoptosis, ROS production and mitochondrial function. NDUFA3 overexpression counteracted the high glucose-induced injuries in HNPCs. HDAC/H3K27ac mechanism was involved in regulating NDUFA3 transcription. NDUFA3 knockdown decreased cell viability and increased apoptotic cells, which were reversed by ROS scavenger N-acetylcysteine. HDAC/H3K27ac-mediated transcription of NDUFA3 protects HNPCs against high glucose-induced injuries through suppressing cell apoptosis, eliminating ROS, improving mitochondrial function and oxidative phosphorylation. This study sheds light on candidate therapeutic targets and deepens the understanding of molecular mechanisms behind DM-induced IVDD.

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

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

Efficient single-cell oxygen consumption rate characterization based on frequency domain fluorescence lifetime imaging microscopy measurement and microfluidic platform.

Cell metabolism is critical in regulating normal cell functions to maintain energy homeostasis. In order to monitor cell metabolism, the oxygen consumption rate (OCR) of cells has been characterized as an important factor. In conventional cell analysis, the cells are characterized in bulk due to technical limitations. However, the heterogeneity between the cells cannot be identified. Therefore, single-cell analysis has been proposed to reveal cellular functions and their heterogeneity. In this research, an approach integrating a microfluidic device and widefield frequency domain fluorescence imaging lifetime microscopy (FD-FLIM) for single-cell OCR characterization in an efficient manner is developed. The microfluidic device provides an efficient platform to trap and isolate single cells in microwells with the buffer saline containing an oxygen-sensitive phosphorescent dye. The oxygen tension variation within the microwells can be efficiently estimated by measuring the fluorescence lifetime change using the FD-FLIM, and the OCR values of the single cells can then be calculated. In the experiments, breast cancer (MCF-7) cells are exploited for the OCR measurement. The results demonstrate the functionality of the developed approach and show the heterogeneity among the cells. The developed approach possesses great potential to advance cellular metabolism studies with single-cell resolution.

A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy.

To develop and optimize a method to monitor real-time mitochondrial function by measuring the oxygen consumption rate (OCR) in murine corneal biopsy punches with a Seahorse extracellular flux analyzer. Murine corneal biopsies were obtained using a biopsy punch immediately after euthanasia. The corneal metabolic profile was assessed using a Seahorse XFe96 pro analyzer, and mitochondrial respiration was analyzed with specific settings. Real-time adenosine triphosphate rate assay showed that mitochondrial oxidative phosphorylation is a major source of adenosine triphosphate production in ex vivo live murine corneal biopsies. Euthanasia methods (carbon dioxide asphyxiation vs. overdosing on anesthetic drugs) did not affect corneal OCR values. Mouse corneal biopsy punches in 1.5-mm diameter generated higher and more reproducible OCR values than those in 1.0-mm diameter. The biopsy punches from the central and off-central cornea did not show significant differences in OCR values. There was no difference in OCR reading by the tissue orientations (the epithelium side up vs. the endothelium side up). No significant differences were found in corneal OCR levels between sexes, strains (C57BL/6J vs. BALB/cJ), or ages (4, 8, and 32 weeks). Using this method, we showed that the wound healing process in the mouse cornea affected mitochondrial activity. The present study validated a new strategy to measure real-time mitochondrial function in fresh mouse corneal tissues. This procedure should be helpful for studies of the ex vivo live corneal metabolism in response to genetic manipulations, disease conditions, or pharmacological treatments in mouse models.

Can Single Cell Respiration be Measured by Scanning Electrochemical Microscopy (SECM)?

Ultramicroelectrode (UME), or, equivalently, microelectrode, probes are increasingly used for single-cell measurements of cellular properties and processes, including physiological activity, such as metabolic fluxes and respiration rates. Major challenges for the sensitivity of such measurements include: (i) the relative magnitude of cellular and UME fluxes (manifested in the current); and (ii) issues around the stability of the UME response over time. To explore the extent to which these factors impact the precision of electrochemical cellular measurements, we undertake a systematic analysis of measurement conditions and experimental parameters for determining single cell respiration rates via the oxygen consumption rate (OCR) in single HeLa cells. Using scanning electrochemical microscopy (SECM), with a platinum UME as the probe, we employ a self-referencing measurement protocol, rarely employed in SECM, whereby the UME is repeatedly approached from bulk solution to a cell, and a short pulse to oxygen reduction reaction (ORR) potential is performed near the cell and in bulk solution. This approach enables the periodic tracking of the bulk UME response to which the near-cell response is repeatedly compared (referenced) and also ensures that the ORR near the cell is performed only briefly, minimizing the effect of the electrochemical process on the cell. SECM experiments are combined with a finite element method (FEM) modeling framework to simulate oxygen diffusion and the UME response. Taking a realistic range of single cell OCR to be 1 × 10-18 to 1 × 10-16 mol s-1, results from the combination of FEM simulations and self-referencing SECM measurements show that these OCR values are at, or below, the present detection sensitivity of the technique. We provide a set of model-based suggestions for improving these measurements in the future but highlight that extraordinary improvements in the stability and precision of SECM measurements will be required if single cell OCR measurements are to be realized.

P.122: Development of an Oxygen Durability Model to Overcome Hypoxia in Encapsulated Islets Within a Functionalized Oxygenated Biomaterial

Introduction: Post-transplantation islet survival is inherently limited by the lack of suitable support matrix and insufficient oxygen supply (hypoxia), which is aggravated in macroencapsulation devices.Graft failure can be overcome by cell encapsulation in highly oxygenated functionalized biomaterials able to provide oxygen and extracellular matrix (ECM) support. An estimation of the oxygen durability in these encapsulation matrices is critical to define the cell density and implant size for optimal graft survival, especially until the macroencapsulation device is sufficiently re-vascularized. The aim of this study was to develop a predictive mathematical model that could estimate the oxygen lifetime of an oxygenated biomaterial (Oxygel) encapsulating different diabetes-relevant cell lines. Methods: Oxygel was formulated by the shear mixing of a physically crosslinked native Hyaluronic acid hydrogel and a Perfluocarbon/lipoid nanoemulsion. Oxygel was oxygenated by flushing oxygen using a customized static Y-mixer while the gel was infused with a syringe pump or using a designed scaled-up oxygenator. Post-oxygenation O2 levels of Oxygel and controls were monitored by microinvasive fibre optic needle-type oxygen microsensors (n=3). Oxygen diffusion coefficient (Dv) was estimated by the experimental fitting of the oxygen release profiles to a diffusion model governed by Fick’s second law. Oxygen durability predictions were performed using a non-diffusion-controlled model considering: (i) recorded initial oxygen tension, (ii) gel concentration and Ostwald oxygen solubility constants of different gel components, (iii) cell density and (iv) oxygen consumption rate (OCR) of the studied cell line. Different diabetes-relevant cell lines (INS-1E, iPS derived B-cells, rat islets, human islets) were modelled using experimental (Agilent Seahorse analyzer) and/or bibliographic OCR values. Summary of results: A self-stable and shear-thinning gel was successfully formulated and oxygenated. A high initial oxygen tension was achieved (650–700 torr, 87–94 % O2 saturation), that was diminished after the cell encapsulation/dilution step (520–550 torr, 70–74 % O2 saturation, Fig 1). Acellular oxygel released oxygen for 100 h until stabilization at atmospheric levels, presenting a 14-times smaller Dv in Oxygel than in PBS. The impact of the model parameters in cell lines with different OCR values was investigated, showing shorter oxygen lifetime in islets (or IEQ) compared to single cell analogues.Conclusion: A highly oxygenated encapsulation matrix for enhanced islet survival was developed and their properties as oxygen carrier evaluated, showing a high oxygen payload and prolonged sustained release properties. Model predictions shows the importance of islet O2 metabolism in the design of macroencapsulation devices and supporting ECM. G.P.D. and D.A.D.-L. acknowledge the DELIVER project that received funding from the European Union’s Horizon 2020 Marie Skłodowska-Curie Actions programme under grant agreement number 812865. G.P.D. acknowledges funding from Science Foundation Ireland under grant SFI/12/RC/2278, Advanced Materials and Bioengineering Research (AMBER) Centre and financial support from the College of Medicine, Nursing and Health Sciences, National University of Ireland Galway.

Mapping cold-water coral biomass: an approach to derive ecosystem functions

This study presents a novel approach resulting in the first cold-water coral reef biomass maps, used to assess associated ecosystem functions, such as carbon (C) stock and turnover. We focussed on two dominant ecosystem engineers at the Mingulay Reef Complex, the coral Lophelia pertusa (rubble, live and dead framework) and the sponge Spongosorites coralliophaga. Firstly, from combining biological (high-definition video, collected specimens), environmental (extracted from multibeam bathymetry) and ecosystem function (oxygen consumption rate values) data, we calculated biomass, C stock and turnover which can feed into assessments of C budgets. Secondly, using those values, we employed random forest modelling to predictively map whole-reef live coral and sponge biomass. The whole-reef mean biomass of S. coralliophaga was estimated to be 304 T (range 168–440 T biomass), containing 10 T C (range 5–18 T C) stock. The mean skeletal mass of the coral colonies (live and dead framework) was estimated to be 3874 T (range 507–9352 T skeletal mass), containing a mean of 209 T of biomass (range 26–515 T biomass) and a mean of 465 T C (range 60–1122 T C) stock. These estimates were used to calculate the C turnover rates, using respiration data available in the literature. These calculations revealed that the epi- and microbial fauna associated with coral rubble were the largest contributor towards C turnover in the area with a mean of 163 T C year−1 (range 149–176 T C year−1). The live and dead framework of L. pertusa were estimated to overturn a mean of 32 T C year−1 (range 4–93 T C year−1) and 44 T C year−1 (range 6–139 T C year−1), respectively. Our calculations showed that the Mingulay Reef overturned three to seven (with a mean of four) times more C than a soft-sediment area at a similar depth. As proof of concept, the supply of C needed from surface water primary productivity to the reef was inferred. Since 65–124 T C year−1 is supplied by natural deposition and our study suggested that a mean of 241 T C year−1 (range 160–400 T C year−1), was turned over by the reef, a mean of 117–176 T C year−1 (range 36–335 T C year−1) of the reef would therefore be supplied by tidal downwelling and/or deep-water advection. Our results indicate that monitoring and/or managing surface primary productivity would be a key consideration for any efforts towards the conservation of cold-water coral reef ecosystems.

Feeding mode influence on treatment performance of unsaturated and partially saturated vertical flow constructed wetland

This study aimed to evaluate the influence of two different feeding modes on wastewater treatment performance and oxygen consumption rate (OCR) of the unsaturated (UVF wetland) and partially saturated (SVF wetland) vertical flow constructed wetlands operated in parallel under subtropical climate for four years. Each wetland had a superficial area of 7.5 m2 and was planted with Typha domingensis. Both units have a filter media depth of 0.75 m, composed by sand (effective diameter of 0.29 mm and uniformity of 4). UVF wetland operated typically unsaturated, while SVF wetland had the bottom part saturated (57% of total depth). Two feeding modes were evaluated for both wetlands. The feeding mode was operated within the limits recommended by the German standard, with a hydraulic loading rate (HLR) of 75 mm d−1 and specific pulse volume (SPV) of 19 L m−2 for both wetlands and a specific hydraulic loading rate (SHLR) of 8 and 9 L m−2 min−1 for UVF and SVF wetlands, respectively. Meanwhile, the second feeding mode was applied for both wetlands, being an HLR of 103 mm d−1, 26 L m−2 of SPV, and 4 L m−2 min−1 of SHLR. The load removal efficiency of SVF wetland was higher than the UVF wetland for all parameters and feeding modes. No statistical difference was identified for OCR values between wetlands and feeding mode. The results showed that operating UVF and SVF wetlands with a SHLR around 4 L m−2 min−1 and SPV equal of 26 L m−2 is preferable. This fact could represent a significant reduction in inlet pumping power requirements and also less superficial area requirements.

The co-importance of physical mixing and biogeochemical consumption in controlling water cap oxygen levels in Base Mine Lake

Bitumen extraction in the Athabasca oil sands region of Alberta Canada has resulted in 1 billion m3 of tailings. Base Mine Lake (BML), the first commercial-scale pit lake (PL) in the Alberta oil sands, is currently being assessed as a potential fluid fine tailings (FFT) reclamation strategy. Here, field-based, BML water cap depth-dependent oxygen consumption rate (OCR) determination, geochemical characterization and modelling of physical mixing were combined to identify the key processes driving the oxygen mass balance and distribution. The highest OCR values (66.9 ± 5.9 O2/m2/d) occurred at the FFT water interface and were driven by dissolved methane and ammonia concentrations. Given the extensive mass of these reductants within the FFT, their biogeochemical redox cycling is anticipated to impact BML oxygen concentrations for decades. The integration of OCR and modelling results revealed the importance of physical mixing as a mechanism to maintain an oxic hypolimnion and FFT water interface. The oxygen mass balance indicates that without the physical movement of oxygen into the hypolimnion, the OCR values currently observed would result in anoxia in the lower 3.5 m of the BML water cap, migrating upwards of the oxic-anoxic boundary and enabling the generation of new oxygen-consuming constituents directly within the water cap through anaerobic redox transformations. Results here guide the development and application of water-capped tailings as a reclamation strategy for not only the oil sands industry, but also for other anthropogenically impacted or engineered systems with high oxygen demand.

Overcoming Radiation Resistance by FePt Nanoparticles via Mitochondrial Targeting

The glutathione (GSH) overexpression small-cell-lung-cancer cell lines, SR3A-13 and SR3A-14, transfected with recombinant DNA encoding g-glutamylcysteine synthetase (γ-GCSh), are more resistant to ionizing radiation (IR) as compared with their parental cell, SR3A. We investigated the mechanism of how FePt NPs sensitize these cells to irradiation. Small cell lung cancer cell, SR3, and its γ-GCSh-transfected stable cells, SR3A-13 and SR3A-14, were studied. Uptakes of FePt NPs in these cells were studied by plasma optical emission spectrometry (ICP-OES) and transmission electron microscopy (TEM). Effects of the combination of FePt NPs and IR were investigated by colony formation assay. Intracellular reactive oxygen species (ROS) were assessed by using fluorescent probes and imaged by a FACS caliber flow cytometer. Mitochondrial membrane potential was monitored by using fluorescent probes and imaged by confocal laser scanning microscopy. Oxygen consumption rate (OCR) in mitochondria after FePt NP and IR treatment was investigated by a Seahoarse XF24 cell energy metabolism analyzer. Uptakes of FePt NPs by SR3A-13 and -14 cells were 3 to 3.5 folds higher than that of SR3A cells, measured with inductively coupled ICP-OES and TEM. After FePt NP and IR, SR3A-13 and -14 cells produced numerous irregularly-shaped mitochondria, as visualized by TEM imaging. Increased ROS production by FePt NPs and IR were found in SR3A-13 and -14 cells, accompanied with low signals of tetramethylrhodamine methyl ester (TMRM), indicating malfunction of the mitochondria. Furthermore, OCR values in SR3A-13 and -14 cell after FePt NPs and IR were significantly lower than that of SR3A, and the OCR of the 72h group in SR3A-13 cell had a rapid and pronounced reduction in basal and maximal mitochondrial oxygen consumption. FePt NPs produce overproduction of ROS and overcome radiation resistance in glutathione-overexpressing cells via mitochondria targeting.

PS1528 INCREASED T CELLS GLYCOLYSIS IN PATIENTS WITH ACUTE GRAFT VERSUS HOST DISEASE AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL

Background:Acute graft‐versus‐host disease (aGVHD) is a major complication limiting successful allogeneic hematopoietic stem cell transplantation (allo‐HSCT), the pathogenesis of aGVHD is commonly considered to be T cell‐mediated. Cell metabolism determines T cell fate and function, the metabolic profile of T cells varies in different immunological disorders such as lupus, rheumatoid arthritis. Previous murine transplantation study found that alloreactive donor T cells increased both oxidative phosphorylation (OXPHOS) and aerobic glycolysis and appeared bioenergetically similar to the lymphocytes that mediate lupus. Recently, another murine study suggested that alloreactive donor T cells use glycolysis as the predominant metabolic process. However, the metabolic profile of T cells in aGVHD patients still remains unclear.Aims:To determine the metabolic adaptations that occur in activated T cells in patients with aGVHD and approach for modulating their metabolism.Methods:In this prospective case‐control study, a total of 20 patients with aGVHD after allo‐HSCT were enrolled, for each case, 1 matched transplant recipients without aGVHD (non‐aGVHD; N = 20) were selected at random. CD3+ T cells were isolated by microbeads, the metabolic profile of sorted T cells was determined using a Seahorse XF96 Analyze. RNA sequencing (RNA‐Seq) was performed to analyze the gene expression profiles of the T cells. Glucose consumption and lactate production rates were detected by glucose assay kit and lactate assay kit. The levels of intracellular cytokines, such as IFN‐r, IL‐4 and IL‐17, were detected by flow cytometry. Protein expressions for CTP1a and PFKFB3 were measured by flow cytometry. 3‐PO, as a glycolysis inhibitor at PFKFB3, was administrated to T cells from aGVHD patients.Results:Firstly, we detected the intracellular levels of proinflammatory cytokines (IFN‐γ, and IL‐17) in the peripheral T cells. There were significantly elevated proportions of Tc1(CD8+IFN‐γ+), Th1 (CD4+IFN‐γ+) and Th17(CD4+IL17+) in patients with aGVHD. Switching from OXPHOS to glycolysis is the hallmark of in vitro–activated T cell metabolism. Hence, we determined the rates of glycolysis and OXPHOS in T cells by measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). T cells isolated from aGVHD patients exhibited comparable OCR values and higher ECAR values. In addition, glucose consumption rate and lactate production rate were both higher in aGVHD group, implying higher glycolytic activity of T cells in aGVHD patients. Moreover, RNA‐seq were performed to further understand the metabolic profile of T cells in aGVHD patients. Consistent with the elevated glycolytic activity observed in vitro, the levels of the mRNAs encoding metabolic enzymes involved in the glycolytic pathway were substantially elevated in aGVHD group. Protein expression of PFKFB3, which is a glycolytic activator, was significantly higher in T cells, especially in naïve T cells of aGVHD patients. Furthermore, 3‐PO significantly diminished PFKFB3 expression, glycolytic activity of T cells, and reduced the expression of proinflammatory cytokines in T cells from aGVHD patients.Summary/Conclusion:In the current study, we found that T cells in patients with aGVHD preferentially depend on glycolysis to meet bioenergetic demands. Furthermore, inhibition of glycolysis through targeting PFKFB3 ameliorates the activity of T cells from aGVHD patients. Therefore, the current work indicated that glycolysis promises to be a novel therapeutic target for aGVHD patients after allo‐HSCT.

The Effects of Nitric Oxide Synthase Inhibitors on Mitochondrial Respiration in Isolated Mouse Brain Mitochondria

ObjectiveNitric oxide synthase (NOS) plays an important role in intracellular and intercellular signaling pathways. Experimental studies in mitochondria from various tissues, including the brain, have shown the possibility of the presence of a novel mitochondrial variant of NOS (mtNOS) located within the mitochondrial matrix. We hypothesized that administration of NOS inhibitors significantly affects the mitochondrial respiration in isolated brain mitochondria.MethodsMitochondria were isolated from the mice aged 8–12 weeks old and plated in 24‐well microplates for oxygen consumption rate (OCR) readings in the Seahorse XFe24 Analyzer. Mitochondria were incubated with inhibitors of nNOS (ARL‐17477 and N‐ω‐propyl‐L‐arginine), eNOS (N5‐(1‐iminoethyl)‐L‐ornithine), or a non‐specific NOS inhibitor (L‐NG‐Nitroarginine methyl ester) for 30 minutes prior to the mitochondrial stress test in the XFe24 Analyzer and compared to an untreated control group.ResultsPharmacological inhibition of nNOS in isolated brain mitochondria reduced basal respiration and ATP production. Treatment with nNOS‐specific and non‐specific NOS inhibitors showed reduced basal OCR compared to control mitochondria. Control OCR values of 90.76±1.89 pmol/min were significantly higher than treatment with L‐NAME (74.17±2.12 pmol/min), NPA (77.92±2.83 pmol/min) and ARL (76.60±2.85 pmol/min). eNOS‐specific inhibitor treatment showed no significant change in mitochondrial respiration. ATP production as a bioenergetics parameter represents the portion of basal oxygen consumption that was used to drive ATP synthesis. Isolated mitochondria treated with non‐specific inhibitor L‐NAME and nNOS‐specific ARL‐17477 showed a significant decrease in ATP production. Isolated mitochondria treated with NPA show a slight trend towards decreased ATP production, but the statistical analysis did not show significance. Treatment with NIO demonstrated no significant change in ATP production.ConclusionsPharmacological inhibition of NOS isoforms affects brain mitochondrial respiration. Given the significant effects demonstrated by both nNOS‐specific inhibitors and L‐NAME, it may be postulated that mtNOS may be a new isoform of NOS sharing specific properties of the traditional three isoforms.Support or Funding InformationAmerican Heart Association (PVG: 14SDG20490359 and VNS: 16PRE31450006); National Institute of Health: National Institute of Neurological Disorders and Stroke and National Institute of General Medical Sciences (PVK: R01NS094834); and National Institute of Diabetes and Digestive and Kidney Diseases (RS: DK107694).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Culture Cells for Assessment of the Energy Metabolism.

Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Cancer cells are known to reprogram their metabolism using different strategies to meet energetic and anabolic needs ( Koppenol et al., 2011 ; Zheng, 2012). Additionally, each cancer tissue has its own individual metabolic features. Mitochondria not only play a key role in energy metabolism but also in cell cycle regulation of cells. Therefore, mitochondria have emerged as a potential target for anticancer therapy since they are structurally and functionally different from their non-cancerous counterparts (D'Souza et al., 2011). We detail a protocol for measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements in living cells, utilizing the Seahorse XF24 Extracellular Flux Analyzer (Figure 1). The Seahorse XF24 Extracellular Flux Analyzer continuously measures oxygen concentration and proton flux in the cell supernatant over time ( Wu et al., 2007 ). These measurements are converted in OCR and ECAR values and enable a direct quantification of mitochondrial respiration and glycolysis. With this protocol, we sought to assess basal mitochondrial function and mitochondrial stress of three different cancer cell lines in response to the cytotoxic test lead compound mensacarcin in order to investigate its mechanism of action. Cells were plated in XF24 cell culture plates and maintained for 24 h. Prior to analysis, the culture media was replaced with unbuffered DMEM pH 7.4 and cells were then allowed to equilibrate in a non-CO2 incubator immediately before metabolic flux analysis using the Seahorse XF to allow for precise measurements of Milli-pH unit changes. OCR and ECAR were measured under basal conditions and after injection of compounds through drug injection ports. With the described protocol we assess the basic energy metabolism profiles of the three cell lines as well as key parameters of mitochondrial function in response to our test compound and by sequential addition of mitochondria perturbing agents oligomycin, FCCP and rotenone/antimycin A. Figure 1.Overview of seahorse experiment.

The Role of Oxygen in Avascular Tumor Growth.

The oxygen status of a tumor has significant clinical implications for treatment prognosis, with well-oxygenated subvolumes responding markedly better to radiotherapy than poorly supplied regions. Oxygen is essential for tumor growth, yet estimation of local oxygen distribution can be difficult to ascertain in situ, due to chaotic patterns of vasculature. It is possible to avoid this confounding influence by using avascular tumor models, such as tumor spheroids, a much better approximation of realistic tumor dynamics than monolayers, where oxygen supply can be described by diffusion alone. Similar to in situ tumours, spheroids exhibit an approximately sigmoidal growth curve, often approximated and fitted by logistic and Gompertzian sigmoid functions. These describe the basic rate of growth well, but do not offer an explicitly mechanistic explanation. This work examines the oxygen dynamics of spheroids and demonstrates that this growth can be derived mechanistically with cellular doubling time and oxygen consumption rate (OCR) being key parameters. The model is fitted to growth curves for a range of cell lines and derived values of OCR are validated using clinical measurement. Finally, we illustrate how changes in OCR due to gemcitabine treatment can be directly inferred using this model.

Spirulina platensis organic extract increases spare respiratory capacity in RAW 264.7 macrophages to exert an anti‐inflammatory effect

Recent evidence supports that crosstalk between energy metabolism and inflammatory processes is a critical determinant for macrophage phenotype. Macrophage energy metabolism greatly influences their activation state. For example, M1, pro‐inflammatory macrophages largely depend on glycolysis while M2, anti‐inflammatory macrophages prefer mitochondrial oxidative phosphorylation (OXPHOS) for their energy generation. We previously demonstrated that organic extract of Spirulina platensis (SPE), a blue‐green algae, exerts a potent anti‐inflammatory effect in vitro and ex vivo. Our recent study also suggests that the anti‐inflammatory effect of SPE is at least partly attributable to the induction of an endotoxin tolerance‐like state in macrophages. Interestingly, although SPE repressed LPS‐induced pro‐inflammatory gene expression as potently as endotoxin tolerant (ET) macrophages, there were some differences between SPE‐treated and ET macrophages. The objective of this study was to determine if SPE alters macrophage energy metabolism to exert its anti‐inflammatory effect in addition to its role in endotoxin tolerance. To determine the energy phenotype, we measured oxygen consumption rate (OCR) and extra cellular acidification rate (ECAR) of naïve, SPE‐treated, and ET macrophages by XF Mito Stress tests using a Seahorse XFe24 Analyzer. Treatment of RAW 264.7 macrophages with 25 mg/mL of SPE or endotoxin tolerization with 10 ng/mL of lipopolysaccharide (LPS) for 12 h increased basal ECAR compared to naïve cells with concomitant increases in the expression of interleukin‐1β and tumor necrosis factor‐α, suggesting that both SPE and LPS activated macrophages to a certain degree. The basal OCR values of naïve cells were the highest, followed by SPE‐treated and then ET cells, which indicates that SPE and LPS decreased cell's dependence on OXPHOS. Interestingly, RAW 264.7 macrophages treated with 25 mg/mL of SPE had the highest spare respiratory capacity (SRC), which is a measure of how much ATP may be produced through OXPHOS in response to a sudden increase in energy demand. When naïve, SPE‐pretreated and ET RAW 264.7 macrophages were stimulated with 100 ng/mL of LPS for 12 h, the SRC was significantly reduced in all treatment groups. During LPS‐stimulation, SPE‐pretreated cells may be better off to meet the energy demands of LPS stimulation as SPE pretreatment significantly increased the SRC. In conclusion, we found that SPE‐pretreated macrophage, but not ET macrophages, had increased the SRC, which may contribute to the potent anti‐inflammatory effects of SPE in macrophages.Support or Funding InformationUSDA AFRI (2014‐01870)

Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells.

The maximal capacity of the mitochondrial electron transport system (ETS) in intact cells is frequently estimated by promoting protonophore-induced maximal oxygen consumption preceded by inhibition of oxidative phosphorylation by oligomycin. In the present study, human glioma (T98G and U-87MG) and prostate cancer (PC-3) cells were titrated with different concentrations of the protonophore CCCP to induce maximal oxygen consumption rate (OCR) within respirometers in a conventional growth medium. The results demonstrate that the presence of oligomycin or its A-isomer leads to underestimation of maximal ETS capacity. In the presence of oligomycin, the spare respiratory capacity (SRC), i.e., the difference between the maximal and basal cellular OCR, was underestimated by 25 to 45%. The inhibitory effect of oligomycin on SRC was more pronounced in T98G cells and was observed in both suspended and attached cells. Underestimation of SRC also occurred when oxidative phosphorylation was fully inhibited by the ATP synthase inhibitor citreoviridin. Further experiments indicated that oligomycin cannot be replaced by the adenine nucleotide translocase inhibitors bongkrekic acid or carboxyatractyloside because, although these compounds have effects in permeabilized cells, they do not inhibit oxidative phosphorylation in intact cells. We replaced CCCP by FCCP, another potent protonophore and similar results were observed. Lower maximal OCR and SRC values were obtained with the weaker protonophore 2,4-dinitrophenol, and these parameters were not affected by the presence of oligomycin. In permeabilized cells or isolated brain mitochondria incubated with respiratory substrates, only a minor inhibitory effect of oligomycin on CCCP-induced maximal OCR was observed. We conclude that unless a previously validated protocol is employed, maximal ETS capacity in intact cells should be estimated without oligomycin. The inhibitory effect of an ATP synthase blocker on potent protonophore-induced maximal OCR may be associated with impaired metabolism of mitochondrial respiratory substrates.

The effects of stirring, population levels and a potential anti-nematodal product on the respiration of second-stage juveniles of Meloidogyne incognita measured with different technology

The oxygen consumption rate (OCR) ofMeloidogyne incognitasecond-stage juveniles (J2) was measured using polarographic oxygen sensor (POS) and fibre-optic oxygen sensor (FOS) technology. The effect of stirring speed on the OCR of J2 suspended in tap water was determined using POS. Next, the OCR of different J2 numbers was measured using FOS. Also, the OCR of J2 in tap water using POS and FOS was recorded, and also the effect of SoilBioMuti (SBM) on J2 OCR using FOS. Stirring speed had no pronounced effect on the OCR of J2. FOS recorded the OCR of as few as five J2. The OCR of J2 in sterile tap water was significantly lower for POS than FOS. Using FOS, non-filtered (NF) SBM without J2 had the highest OCR, being 42% higher than that of NF SBM with J2. The two filtered SBM (with and without J2) treatments had the lowest OCR values.

Assessing the bioenergetic profile of human pluripotent stem cells.

Assessing the bioenergetics of human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provides considerable insight into their mitochondrial functions and cellular properties. This might allow exposing potential energetic defects caused by mitochondrial diseases. However, certain challenges have to be met due to unique growth conditions in highly specialized and costly culture media. Here, we describe a method that facilitates the assessment of the bioenergetic profiles of hPSCs in a noninvasive fashion, while requiring only small sample sizes and allowing for several replicates. Basal respiratory and glycolytic capacities are assessed using a XF24 Extracellular Flux Analyzer by simultaneous measurements of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively. In addition, bioenergetic parameters are estimated by monitoring OCR and ECAR values upon metabolic perturbations via the consecutive introduction of mitochondria-specific inhibitors.

Oxygen consumption dynamics in steady-state tumour models.

Oxygen levels in cancerous tissue can have a significant effect on treatment response: hypoxic tissue is both more radioresistant and more chemoresistant than well-oxygenated tissue. While recent advances in medical imaging have facilitated real-time observation of macroscopic oxygenation, the underlying physics limits the resolution to the millimetre domain, whereas oxygen tension varies over a micrometre scale. If the distribution of oxygen in the tumour micro-environment can be accurately estimated, then the effect of potential dose escalation to these hypoxic regions could be better modelled, allowing more realistic simulation of biologically adaptive treatments. Reaction–diffusion models are commonly used for modelling oxygen dynamics, with a variety of functional forms assumed for the dependence of oxygen consumption rate (OCR) on cellular status and local oxygen availability. In this work, we examine reaction–diffusion models of oxygen consumption in spherically and cylindrically symmetric geometries. We consider two different descriptions of oxygen consumption: one in which the rate of consumption is constant and one in which it varies with oxygen tension in a hyperbolic manner. In each case, we derive analytic approximations to the steady-state oxygen distribution, which are shown to closely match the numerical solutions of the equations and accurately predict the extent to which oxygen can diffuse. The derived expressions relate the limit to which oxygen can diffuse into a tissue to the OCR of that tissue. We also demonstrate that differences between these functional forms are likely to be negligible within the range of literature estimates of the hyperbolic oxygen constant, suggesting that the constant consumption rate approximation suffices for modelling oxygen dynamics for most values of OCR. These approximations also allow the rapid identification of situations where hyperbolic consumption forms can result in significant differences from constant consumption rate models, and so can reduce the computational workload associated with numerical solutions, by estimating both the oxygen diffusion distances and resultant oxygen profile. Such analysis may be useful for parameter fitting in large imaging datasets and histological sections, and allows easy quantification of projected differences between functional forms of OCR.

Glycolysis–respiration relationships in a neuroblastoma cell line

BackgroundAlthough some reciprocal glycolysis–respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized. MethodsWe concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions. ResultsUnder conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased. ConclusionsThese data document a novel intracellular glycolysis–respiration effect in which restricting glycolysis flux increases mitochondrial respiration. General significanceSince this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis–respiration effect can practically inform the development of new mitochondrial medicine approaches.