Measurement Of Oxygen Consumption Rate Research Articles

Evaluation of mitochondrial respiratory function in highly glycolytic glioma cells reveals low ADP phosphorylation in relation to oxidative capacity.

High-grade gliomas are aggressive and intensely glycolytic tumors. In the present study, we evaluated the mitochondrial respiratory function of glioma cells (T98G and U-87MG) and fresh human glioblastoma (GBM) tissue. To this end, measurements of oxygen consumption rate (OCR) were performed under various experimental conditions. The OCR of T98G and U-87MG cells was well coupled to ADP phosphorylation based on the ratio of ATP produced per oxygen consumed of ~2.5. In agreement, the basal OCR of GBM tissue was also partially associated with ADP phosphorylation. The basal respiration of intact T98G and U-87MG cells was not limited by the supply of endogenous substrates, as indicated by the increased OCR in response to a protonophore. These cells also displayed a high affinity for oxygen, as evidenced by the values of the partial pressure of oxygen when respiration is half maximal (p 50). In permeabilized glioma cells, ADP-stimulated OCR was only approximately 50% of that obtained in the presence of protonophore, revealing a significant limitation in oxidative phosphorylation (OXPHOS) relative to the activity of the electron transport system (ETS). This characteristic was maintained when the cells were grown under low glucose conditions. Flux control coefficient analyses demonstrated that the impaired OXPHOS was associated with the function of both mitochondrial ATP synthase and the adenine nucleotide translocator, but not the phosphate carrier. Altogether, these data indicate that the availability and metabolism of respiratory substrates and mitochondrial ETS are preserved in T98G and U-87MG glioma cells even though these cells possess a relatively restrained OXPHOS capability.

Attenuation of Staphylococcal enterotoxin-B (SEB)-induced lung injury (ALI) by resveratrol results from miR-193a down-regulation and alterations in metabolic profile

Abstract Resveratrol, (RES), a phytoalexin, is well-known for its anti-inflammatory and anti-oxidant properties. SEB, a superantigen, is known to trigger ALI and cause mortality. In the current study, we tested the effect of RES in a dual-dose model of SEB exposure that triggers ALI and causes 100% mortality in C3H/HeJ-mice. The data revealed RES attenuated SEB-induced ALI and prevented mortality. Forty eight hours post-SEB exposure, lung-infiltrating mononuclear cells were tested for microRNA expression profile to determine the epigenetic regulation by resveratrol. SEB-activated splenocytes were pre-treated with 50 μM of RES or vehicle for metabolic profile analysis by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). We also noted significant decline in miR-193a in the lungs of RES-treated SEB group, which targeted and caused an increase in TGFβ2 and TGFβR3, potent inhibitors of T-cell proliferation, by using RT-PCR and validation by transfection studies. RES-induced downregulation of miR-193a also influenced the activity of mechanistic target of rapamycin (mTOR) as well as pyruvate kinase muscle isozyme2 (PKM2) genes, and caused RES-treated SEB-activated T cells to be quiescent metabolically in comparison to the energetic vehicle-treated SEB-activated T cells. Together, RES caused inhibition in the proliferation of SEB-activated T-cells by alterations in miR expression and metabolic profiles. (Supported by NIH grants P01AT003961, R01AT006888, R01ES019313, R01MH094755, P20GM103641 and R01AI129788).

Differential nutrient uptake by lung resident and spleen effector memory T cells following influenza virus infection

Abstract Pulmonary virus infection generates a distinct population of CD8 T cells that reside within the lungs following antigen clearance. Resident memory T cells (TRM) are critical mediators of host defense, however, their ability to provide protection wanes over time. The protective immunity provided directly corresponds to the number of antigen-specific CD8+ TRM within the lungs at the time of secondary infection. Mechanisms underlying the differential loss of CD8 lung TRM remain poorly understood. Recent work has highlighted the importance of cellular metabolism in relation to cellular function and survival, but little is known about the metabolic phenotype of lung TRM following influenza infection. We investigated whether metabolic differences could explain the differential loss of lung TRM compared to circulating TEM. Using fluorescently labeled glucose and palmitate, we compared the nutrient uptake capacity of nucleoprotein (NP)-specific CD8 T cells in bronchealveolar lavage (BAL), mediastinal lymph node (mLN), spleen and lung TRM in vivo following influenza infection. We find lung TRM have differential capacity for nutrient uptake compared to NP-specific cells in other tissues. In addition, we utilized the Seahorse XF analyzer to measure oxygen consumption rate (OCR) and determine the spare respiratory capacity (SRC) of lung TRM compared to circulating TCM and TEM. We find that lung TRM tend to have lower OCR at baseline, as well as decreased SRC. Taken together, these data show that lung TRM have a different metabolic profile than circulating memory T cells. This work highlights the need to understand the differential metabolic requirements of TRM, which could facilitate future vaccine design strategies aimed at these pathways.

Quercetin, a natural product supplement, impairs mitochondrial bioenergetics and locomotor behavior in larval zebrafish (Danio rerio)

Quercetin is a natural product that is sold as a supplement in health food stores. While there are reported benefits for this flavonoid as a dietary supplement due to antioxidant properties, the full scope of its biological interactions has not been fully addressed. To learn more about the mechanisms of action related to quercetin, we exposed zebrafish (Danio rerio) embryos to 1 and 10μg/L quercetin for 96h starting at 3h post fertilization. Quercetin up to 10μg/L did not induce significant mortality in developing fish, but did increase prevalence of an upward-curved dorsal plane in hatched larvae. To determine whether this developmental defect was potentially related to mitochondrial bioenergetics during development, we measured oxygen consumption rate in whole embryos following a 24-hour exposure to quercetin. Basal mitochondrial and ATP-linked respiration were decreased at 1 and 10μg/L quercetin, and maximal respiration was decreased at 10μg/L quercetin, suggesting that quercetin impairs mitochondrial bioenergetics. This is proposed to be related to the deformities observed during development. Due to the fact that ATP production was affected by quercetin, larval behaviors related to locomotion were investigated, as well as transcriptional responses of six myogenesis transcripts. Quercetin at 10μg/L significantly reduced the swimming velocity of zebrafish larvae. The expression levels of both myostatin A (mstna) and myogenic differentiation (myoD) were also altered by quercetin. Mstna, an inhibitory factor for myogenesis, was significantly increased at 1μg/L quercetin exposure, while myoD, a stimulatory factor for myogenesis, was significantly increased at 10μg/L quercetin exposure. There were no changes in transcripts related to apoptosis (bcl2, bax, casp3, casp7), but we did observe a decrease in mRNA levels for catalase (cat) in fish exposed to each dose, supporting an oxidative stress response. Our data support the hypothesis that quercetin may affect locomotion and induce deformities in zebrafish larvae by diminishing ATP production and by altering the expression of transcripts related to muscle formation and activity.

Four-week cold acclimation in adult humans shifts uncoupling thermogenesis from skeletal muscles to brown adipose tissue.

Muscle-derived thermogenesis during acute cold exposure in humans consists of a combination of cold-induced increases in skeletal muscle proton leak and shivering. Daily cold exposure results in an increase in brown adipose tissue oxidative capacity coupled with a decrease in the cold-induced skeletal muscle proton leak and shivering intensity. Improved coupling between electromyography-determined muscle activity and whole-body heat production following cold acclimation suggests a maintenance of ATPase-dependent thermogenesis and decrease in skeletal muscle ATPase independent thermogenesis. Although daily cold exposure did not change the fibre composition of the vastus lateralis, the fibre composition was a strong predictor of the shivering pattern evoked during acute cold exposure. We previously showed that 4weeks of daily cold exposure in humans can increase brown adipose tissue (BAT) volume by 45% and oxidative metabolism by 182%. Surprisingly, we did not find a reciprocal reduction in shivering intensity when exposed to a mild cold (18°C). The present study aimed to determine whether changes in skeletal muscle oxidative metabolism or shivering activity could account for these unexpected findings. Nine men participated in a 4week cold acclimation intervention (10°C water circulating in liquid-conditioned suit, 2hday-1 , 5daysweek-1 ). Shivering intensity and pattern were measured continuously during controlled cold exposure (150min at 4°C) before and after the acclimation. Muscle biopsies from the m. vastus lateralis were obtained to measure oxygen consumption rate and proton leak of permeabilized muscle fibres. Cold acclimation elicited a modest 21% (P<0.05) decrease in whole-body and m. vastus lateralis shivering intensity. Furthermore, cold acclimation abolished the acute cold-induced increase in proton leak. Although daily cold exposure did not change the fibre composition of the m. vastus lateralis, fibre composition was a strong predictor of the shivering pattern evoked during acute cold. We conclude that muscle-derived thermogenesis during acute cold exposure in humans is not only limited to shivering, but also includes cold-induced increases in proton leak. The efficiency of muscle oxidative phosphorylation improves with cold acclimation, suggesting that reduced muscle thermogenesis occurs through decreased proton leak, in addition to decreased shivering intensity as BAT capacity and activity increase. These changes occur with no net difference in whole-body thermogenesis.

Single Cell Examination of Membrane Fluidity and Cellular Respiration

Bulk measurements of oxygen consumption rates by bacteria have previously been shown to be related to the cells' average membrane fluidity (as measured by the diffusion of fluorescently-labeled molecules). Within a bacterial population, there is also significant cell-to-cell variation in both the diffusion coefficient of membrane probes and oxygen consumption rates of individual cells. One possibility is the diffusion of ubiquinone, an electron carrier in the electron transport chain (ETC), is being limited by the fluidity of the membrane which is causing the heterogeneity.We describe Fluorescence Recovery After Photobleaching (FRAP) measurements to monitor cell-to-cell variation in membrane diffusion coefficient. These results are compared to two approaches to look at what influence it might have on respiration at the single cell level. The first uses a fluorescent indicator of the activity of the ETC. The second directly measures the consumption of oxygen by individual cells using a phosphorescent Pt-porphyrin dye.

Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness.

Mitochondrial respiration is the most important generator of cellular energy under most circumstances. It is a process of energy conversion of substrates into ATP. The Seahorse equipment allows measuring oxygen consumption rate (OCR) in living cells and estimates key parameters of mitochondrial respiration in real-time mode. Through use of mitochondrial inhibitors, four key mitochondrial respiration parameters can be measured: basal, ATP production-linked, maximal, and proton leak-linked OCR. This approach requires application of mitochondrial inhibitors-oligomycin to block ATP synthase, FCCP-to make the inner mitochondrial membrane permeable for protons and allow maximum electron flux through the electron transport chain, and rotenone and antimycin A-to inhibit complexes I and III, respectively. This chapter describes the protocol of OCR assessment in the culture of primary myotubes obtained upon satellite cell fusion.

AG311, a small molecule inhibitor of complex I and hypoxia-induced HIF-1α stabilization

Cancer cells have a unique metabolic profile and mitochondria have been shown to play an important role in chemoresistance, tumor progression and metastases. This unique profile can be exploited by mitochondrial-targeted anticancer therapies. A small anticancer molecule, AG311, was previously shown to possess anticancer and antimetastatic activity in two cancer mouse models and to induce mitochondrial depolarization. This study defines the molecular effects of AG311 on the mitochondria to elucidate its observed efficacy. AG311 was found to competitively inhibit complex I activity at the ubiquinone-binding site. Complex I as a target for AG311 was further established by measuring oxygen consumption rate in tumor tissue isolated from AG311-treated mice. Cotreatment of cells and animals with AG311 and dichloroacetate, a pyruvate dehydrogenase kinase inhibitor that increases oxidative metabolism, resulted in synergistic cell kill and reduced tumor growth. The inhibition of mitochondrial oxygen consumption by AG311 was found to reduce HIF-1α stabilization by increasing oxygen tension in hypoxic conditions. Taken together, these results suggest that AG311 at least partially mediates its antitumor effect through inhibition of complex I, which could be exploited in its use as an anticancer agent.

BaroFuse, a novel pressure-driven, adjustable-throughput perfusion system for tissue maintenance and assessment.

ObjectivesMicrofluidic perfusion systems are used for assessing cell and tissue function while assuring cellular viability. Low perfusate flow rates, desired both for conserving reagents and for extending the number of channels and duration of experiments, conventionally depend on peristaltic pumps to maintain flow yet such pumps are unwieldy and scale poorly for high-throughput applications requiring 16 or more channels. The goal of the study was to develop a scalable multichannel microfluidics system capable of maintaining and assessing kinetic responses of small amounts of tissue to drugs or changes in test conditions.MethodsHere we describe the BaroFuse, a novel, multichannel microfluidics device fabricated using 3D-printing technology that uses gas pressure to drive large numbers of parallel perfusion experiments. The system is versatile with respect to endpoints due to the translucence of the walls of the perifusion chambers, enabling optical methods for interrogating the tissue status. The system was validated by the incorporation of an oxygen detection system that enabled continuous measurement of oxygen consumption rate (OCR).ResultsStable and low flow rates (1–20 μL/min/channel) were finely controlled by a single pressure regulator (0.5–2 psi). Control of flow in 0.2 μL/min increments was achieved. Low flow rates allowed for changes in OCR in response to glucose to be well resolved with very small numbers of islets (1–10 islets/channel). Effects of acetaminophen on OCR by precision-cut liver slices of were dose dependent and similar to previously published values that used more tissue and peristaltic-pump driven flow.ConclusionsThe very low flow rates and simplicity of design and operation of the BaroFuse device allow for the efficient generation of large number of kinetic profiles in OCR and other endpoints lasting from hours to days. The use of flow enhances the ability to make measurements on primary tissue where some elements of native three-dimensional structure are preserved. We offer the BaroFuse as a powerful tool for physiological studies and for pharmaceutical assessment of drug effects as well as personalized medicine.

Mitochondrial Spare Respiratory Capacity Is Negatively Correlated with Nuclear Reprogramming Efficiency

Nuclear reprogramming efficiency has been shown to be highly variable among different types of somatic cells and different individuals, yet the underlying mechanism remains largely unknown. Several studies have shown that reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) requires remodeling of mitochondria and a metabolic shift from an oxidative state to a more glycolytic state. In this study, we evaluated the nuclear reprogramming efficiency in relation to mitochondrial bioenergetic parameters of fibroblasts from seven different human individuals. Using the Seahorse extracellular energy flux analyzer, we measured oxygen consumption rate (OCR) profiles of the cells, along with their nuclear reprogramming efficiency into iPSCs. Our results showed that fibroblasts with the lowest mitochondrial spare respiratory capacity (SRC) had the highest nuclear reprogramming efficiency, opposed to fibroblasts with the highest mitochondrial SRC, which showed lowest reprogramming efficiency. Furthermore, we found that targeted fluorescent tagging of endogenous genes (MYH6 and COL2A1) by CRISPR/Cas9-mediated homologous recombination was accompanied by an increase in the SRC level of the modified fibroblasts and impaired reprogramming efficiency. Our findings indicate a negative correlation between high mitochondrial SRC in somatic cells and low reprogramming efficiencies. This type of analysis potentially allows screening and predicting reprogramming efficiency before reprogramming, and further suggests that nuclear reprogramming might be improved by approaches that modulate the SRC.

Development of a new clinically applicable device for embryo evaluation which measures embryo oxygen consumption

Does a new system-the chip-sensing embryo respiration monitoring system (CERMs)-enable evaluation of embryo viability for potential application in a clinical IVF setting? The system enabled the oxygen consumption rate of spheroids, bovine embryos and frozen-thawed human embryos to be measured, and this rate corresponded to the developmental potential of embryos. To date, no reliable and clinically suitable objective evaluation methods for embryos are available, which circumvent the differences in inter-observer subjective view. Existing systems such as the scanning electrochemical microscopy (SECM) technique, which enables the measurement of oxygen consumption rate in embryos, need improvement in usability before they can be applied to a clinical setting. This is a prospective original research study. The feasibility of measuring the oxygen consumption rate was assessed using CERMs for 9 spheroids, 9 bovine embryos and 30 redundant frozen-thawed human embryos. The endpoints for the study were whether CERMs could detect a dissolved oxygen gradient with high sensitivity, had comparable accuracy to the SECM measuring system with improved usability, and could predict the development of an embryo to a blastocyst by measuring the oxygen consumption rate. The relationship between the oxygen consumption rate and standard morphological evaluation was also examined. We developed a new CERMs, which enables the oxygen consumption rate to be measured automatically using an electrochemical method. The device was initially used for measuring a dissolved oxygen concentration gradient in order to calculate oxygen consumption rate using nine spheroids. Next, we evaluated data correlation between the CERMs and the SECM measuring systems using nine bovine embryos. Finally, the oxygen consumption rates of 30 human embryos, which were frozen-thawed on 2nd day after fertilization, were measured by CERMs at 6, 24, 48, 72 and 96 h after thawing with standard morphological evaluation. Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed. The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P =0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P =0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 hafter thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2)=0.6537 (P =0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently. Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown. Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality embryos by CERMs may enable the omission of long-term in vitro embryo culture to the blastocyst stage. CERMs is scalable technology that can be integrated into incubators and/or other embryo evaluation systems, such as the time-lapse systems, due to its chip-based architecture. Thus, CERMS would enable automatic measurement of oxygen consumption, under 5% CO2, in the near future, in order to reduce oxidative stress from exposure to atmospheric air. This study was supported by grants from the Health and Labor Sciences Research Grant (H24-Hisaichiiki-Shitei-016). The authors have no conflicts of interest. Not applicable.

Role of mitochondrial complex I and protective effect of CoQ10 supplementation in propofol induced cytotoxicity.

Propofol (2,6-diisopropylphenol) is an anaesthetic widely used for human sedation. Due to its intrinsic antioxidant properties, rapid induction of anaesthesia and fast recovery, it is employed in paediatric anaesthesia and in the intensive care of premature infants. Recent studies have pointed out that exposure to anaesthesia in the early stage of life might be responsible of long-lasting cognitive impairment. The apoptotic neurodegeneration induced by general anaesthetics (GA) involves mitochondrial impairment due to the inhibition of the OXPHOS machinery. In the present work, we aim to identify the main mitochondrial respiratory chain target of propofol toxicity and to evaluate the possible protective effect of CoQ10 supplementation. The propofol effect on the mitochondrial functionality was assayed in isolated mitochondria and in two cell lines (HeLa and T67) by measuring oxygen consumption rate. The protective effect of CoQ10 was assessed by measuring cells viability, NADH-oxidase activity and ATP/ADP ratio in cells treated with propofol. Our results show that propofol reduces cellular oxygen consumption rate acting mainly on mitochondrial Complex I. The kinetic analysis of Complex I inhibition indicates that propofol interferes with the Q module acting as a non-competitive inhibitor with higher affinity for the free form of the enzyme. Cells supplemented with CoQ10 are more resistant to propofol toxicity. Propofol exposure induces cellular damages due to mitochondrial impairment. The site of propofol inhibition on Complex I is the Q module. CoQ10 supplementation protects cells against the loss of energy suggesting its possible therapeutic role to minimizing the detrimental effects of general anaesthesia.

A comparative analysis of the metabolism and energetics of darters (percidae).

Ecological niche theory suggests that, because closely related species cannot coexist in the same ecological niche, subtle differences exist among species that appear to cohabitate. Metabolic rate can explain variations that potentially enable species coexistence. This study evaluated the coexistence of highly sympatric darter fishes by examining interspecific variation in metabolism and behavior.Intermittent-flow respirometry measured oxygen consumption rate of greenside (Etheostoma blennioides) and variegate (E. variatum) darters over 48 h, and activity was analyzed using time-lapse videos. E. blennioides had a significantly greater mean metabolic rate (p=0.0006; 154.64 ± SE 52.54 mg O2·kg-1·hr-1; n=14) than E. variatum (92.51 ± SE 32.70 mg O2·kg-1·hr-1; n=15). The greenside darter’s metabolic rate oscillated more over time, whereas the metabolic rate of the variegate darter decreased. The greenside darter had consistently elevated activity compare to the variegate darter, but was not significantly different over time (p=0.5173), while the variegate darter’s activity was significantly different over time (p=0.0004). The results suggest that the greenside darter is a bolder and more active species than the variegate darter, which exhibited more reserved activity. These physiological and behavioral differences discovered between the greenside and variegate darters could potentially facilitate their coexistence. Therefore, it is a hope that these novel findings for two closely related darter species will lead to future research on darter physiological ecology.

Abstract 215: Metabolic and mitochondrial consequences of p53 upregulation and their relation to cell fate outcomes

Abstract Background: p53 regulates a plethora of biological processes including cellular metabolism and mitochondrial function. Using the small molecule MDM2 agonist MI-63, we previously demonstrated differential p53 upregulation and induction of global metabolomics profiles in malignant MCF-7 and non-malignant MCF-10A mammary epithelial cells undergoing different cell fates (apoptosis vs. cytostasis). In particular, p53 upregulation suppressed glycolytic metabolite pools in MCF-10A, while increasing their levels in MCF-7 cells. We postulated that this opposing metabolic response may be linked to their different cell fate outcomes. In this study, we evaluated the association between p53 induced effects on live cell metabolic flux and cell fate using different p53wt cell lines subjected to comparable p53 upregulation. Methods: We employed four p53wt cell lines: MCF-7, ZR-75-1, SJSA-1 and MCF-10A. Cell fate outcomes upon p53 upregulation (24hr, 10μM MI63) were assessed by Western blotting for cytostasis (p21) and apoptosis markers (cleaved PARP, PUMA). Expression changes in enzymes involved in proline metabolism were similarly measured. Real-time measurements of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) under control and MI-63 treatments were assessed by the Seahorse Extracellular Flux Analyzer, and specific acidification contributions from respiration and glycolysis to total ECAR were computed. Results: p53 upregulation induced apoptosis in the malignant MCF-7, ZR-75-1 and SJSA-1 cells, but not in non-malignant MCF-10A cells. MI-63 treatment decreased basal OCR in MCF-7 cells (t-test p = 0.046), but not the other cell lines. No significant differences in maximum OCR were observed between control and MI-63 treatments in any of the cell lines tested. As well, p53 upregulation did not produce any significant change in total ECAR. However, in MCF-7 cells, glycolytic ECAR was significantly increased (MI-63/Control: 4.36, Tukey test p = 0.001), consistent with our earlier metabolomics conclusions. In contrast, no significant changes in glycolytic ECAR were produced in MCF-10A, ZR-75-1 or SJSA-1 cells (MI-63/Control: 1.27, 1.32 and 1.33 respectively). Interestingly, p53 upregulation induced expression of proline dehydrogenase (PRODH) in all three malignant cell lines, but not in MCF-10A cells; and coordinated changes in other proline catabolizing enzymes (P5C dehydrogenase, P5C reductase) were seen in MCF-7 cells. Conclusion: These findings indicate that p53 induced effects on oxygen consumption and glycolysis are cell context dependent but are not likely responsible for the different cell fate outcomes observed following p53 upregulation. Rather, our evidence suggests a potential mechanistic link between p53 induced effects on proline metabolism and cell fate outcomes, indicating that this stress-activated mitochondrial pathway deserves further study. Citation Format: Yau Christina, Katya Frazier, Daniel Rothschild, Shona Mookerjee, Martin Brand, Gary Scott, Christopher C. Benz. Metabolic and mitochondrial consequences of p53 upregulation and their relation to cell fate outcomes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 215.

Concurrent environmental stressors and jellyfish stings impair caged European sea bass (Dicentrarchus labrax) physiological performances.

The increasing frequency of jellyfish outbreaks in coastal areas has led to multiple ecological and socio-economic issues, including mass mortalities of farmed fish. We investigated the sensitivity of the European sea bass (Dicentrarchus labrax), a widely cultured fish in the Mediterranean Sea, to the combined stressors of temperature, hypoxia and stings from the jellyfish Pelagia noctiluca, through measurement of oxygen consumption rates (MO2), critical oxygen levels (PO2crit), and histological analysis of tissue damage. Higher levels of MO2, PO2crit and gill damage in treated fish demonstrated that the synergy of environmental and biotic stressors dramatically impair farmed fish metabolic performances and increase their health vulnerability. As a corollary, in the current scenario of ocean warming, these findings suggest that the combined effects of recurrent hypoxic events and jellyfish blooms in coastal areas might also threaten wild fish populations.

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)

Nutrient‐Induced Metabolism Dictates Intestinal Epithelial Crypt Proliferation

The intestinal epithelium plays an essential role in nutrient absorption, hormone release and barrier function. Maintenance of the epithelium is driven by continuous cell renewal by stem cells located in the intestinal crypts. The amount and type of diet influence this process and result in changes in the size and cellular make‐up of the tissue. We have found that glucose induces an increase in the rate of proliferation in intestinal epithelial crypts in a concentration dependent manner. The mechanism underlying this nutrient‐driven change in crypt proliferation is not known, but may involve a shift in intracellular metabolism that allows for more nutrients to be used to manufacture new cells. We hypothesized that nutrient availability drives changes in cellular energy metabolism of small intestinal epithelial crypts that could contribute to the increase in crypt proliferation. To test this hypothesis, we utilized primary small intestinal epithelial crypts from C57BL/6J mice to study the effect of glucose on crypt metabolism using a Seahorse XFe96 Extracellular Flux Analyzer for real‐time metabolic measurements. Glycolysis was determined by measuring the extracellular acidification rate (ECAR) in response to glucose and the glycolytic pathway inhibitor 2‐Deoxy‐D‐glucose (2‐DG). Glucose oxidation was determined by measuring oxygen consumption rate (OCR) in response to glucose and the glucose oxidation pathway inhibitor UK‐5099. Glucose‐induced mitochondrial respiration was determined by measuring OCR in response to glucose and modulators of mitochondrial respiration (i.e. ATP synthase inhibitor oligomycin, uncoupler FCCP, complex I inhibitor rotenone, complex III inhibitor antimycin A). The maximum respiration capacity (i.e. the capacity to oxidize any substrates present to meet metabolic challenges) was determined by measuring FCCP‐stimulated OCR. We found that glucose increased ECAR in a concentration dependent manner, and the glycolytic pathway inhibitor 2‐DG reduced glucose‐induced ECAR, indicating that glucose stimulates glycolysis of crypts. However, neither glucose nor the glucose oxidation pathway inhibitor UK‐5099 affected OCR, indicating that glucose oxidation is not active in crypts. Furthermore, glucose increased FCCP‐stimulated OCR, indicating that the presence of glucose is necessary for crypts to utilize substrates for oxidation. These data suggest that high nutrient availability drives metabolism towards utilization of glucose for glycolysis in crypts, and glucose might indirectly induce an increase in the oxidation of other nutrients (e.g. fatty acids, amino acids) in crypts. These metabolic changes may contribute to the nutrient‐induced crypt proliferation.Support or Funding InformationUSDA ILLU‐538‐926

HIF‐1α Stabilization Inhibits Basal and Maximal Mitochondrial Respiration in Fetal Cardiac and Skeletal Myocytes

IntroductionHypoxia is the most common stress experienced by fetuses, and chronic hypoxia affects cardiac function as well as growth of fetal striated muscle. Cardiac muscle is active in utero, while skeletal muscle is relatively inactive, thus the metabolic effects of hypoxia may be differently mediated in these cells. Hypoxia signaling is mediated through both HIF‐1α and depletion of high‐energy phosphates, but the relative regulatory roles of these two signals on metabolism in fetal striated muscle is unknown.HypothesisChronic HIF‐1α stabilization and reduced mitochondrial proton gradient suppress oxidative respiration and promote glycolysis in fetal sheep cardiac and skeletal myocytes.MethodsNear‐term fetal sheep cardiomyocytes and biceps femoris myoblasts were cultured (n=4) and myoblasts were differentiated to myotubes. Cells were treated for 48h with cobalt (II) chloride (to stabilize HIF‐1α) or carbonyl cyanide 4‐trifluoromethoxy phenylhydrazone (FCCP; to diminish the mitochondrial proton gradient). Doses were selected to achieve 80% cell survival; compounds and detached cells were washed out prior to metabolic assessment. The Seahorse XFe96 metabolic analyzer measured oxygen consumption rate (respiration) and H+ production rate (glycolysis), which were normalized to protein content. Data were analyzed within cell type by one‐way analysis of variance followed by Tukey's multiple comparisons test, and between cell type by Student's t‐test. Noted differences were significant at p&lt;0.05.ResultsBasal ATP‐producing respiration (excluding proton leak) was 2.5 fold higher in cardiomyocytes than skeletal myotubes, and maximal respiration was 5.4 fold higher, while glycolysis was not different between cardiac and skeletal myocytes. In skeletal myotubes, basal ATP‐producing respiration was reduced 24% by FCCP and 77% by cobalt. In cardiomyocytes, ATP‐producing respiration was reduced 39% by cobalt. No significant changes occurred in glycolytic capacity following chronic cobalt or FCCP treatment.ConclusionMetabolic changes following treatment were similar between cardiac and skeletal muscle, although the metabolic capacity of fetal cardiomyocytes was many‐fold higher than skeletal myotubes. HIF‐1α signaling, as occurs in chronic hypoxia, significantly reduces the mitochondrial oxidative respiratory capacity of fetal striated muscle. This change may affect cardiac and skeletal muscle function and growth during and following birth.Support or Funding InformationThis research was supported by NICHD under awards numbered R01HD071068 and P01HD034430.

Cardiorespiratory upregulation during seawater acclimation in rainbow trout: effects on gastrointestinal perfusion and postprandial responses.

Increased gastrointestinal blood flow is essential for euryhaline fishes to maintain osmotic homeostasis during the initial phase of a transition from freshwater to seawater. However, the cardiorespiratory responses and hemodynamic changes required for a successful long-term transition to seawater remain largely unknown. In the present study, we simultaneously measured oxygen consumption rate (ṀO2), cardiac output (CO), heart rate (HR), and gastrointestinal blood flow (GBF) in rainbow trout (Oncorhynchus mykiss) acclimated to either freshwater or seawater for at least 6 wk. Seawater-acclimated trout displayed significantly elevated ṀO2 (day: 18%, night: 19%), CO (day: 22%, night: 48%), and GBF (day: 96%, night: 147%), demonstrating that an overall cardiorespiratory upregulation occurs during seawater acclimation. The elevated GBF was achieved via a combination of increased CO, mediated through elevated stroke volume (SV), and a redistribution of blood flow to the gastrointestinal tract. Interestingly, virtually all of the increase in CO of seawater-acclimated trout was directed to the gastrointestinal tract. Although unfed seawater-acclimated trout displayed substantially elevated cardiorespiratory activity, the ingestion of a meal resulted in a similar specific dynamic action (SDA) and postprandial GBF response as in freshwater-acclimated fish. This indicates that the capacity for the transportation of absorbed nutrients, gastrointestinal tissue oxygen delivery, and acid-base regulation is maintained during digestion in seawater. The novel findings presented in this study clearly demonstrate that euryhaline fish upregulate cardiovascular function when in seawater, while retaining sufficient capacity for the metabolic and cardiovascular changes associated with the postprandial response.

Usefulness of &lt;i&gt;in vitro&lt;/i&gt; combination assays of mitochondrial dysfunction and apoptosis for the estimation of potential risk of idiosyncratic drug induced liver injury

Drug-induced liver injury (DILI) is one of the serious and frequent drug-related adverse events. This adverse event is a main reason for regulatory action pertaining to drugs, including restrictions in clinical indications and withdrawal from clinical trials or the marketplace. Idiosyncratic DILI especially has become a major clinical concern because of its unpredictable nature, frequent hospitalization, need for liver transplantation and high mortality. The estimation of the potential for compounds to induce idiosyncratic DILI is very difficult in non-clinical studies because the precise mechanism of idiosyncratic DILI is still unknown. Recently, many in vitro assays which indicate a possibility of the prediction of the idiosyncratic DILI have been reported. Among these, some in vitro assays focus on the effects of compounds on mitochondrial function and the apoptotic effects of compounds on human hepatocytes. In this study, we measured oxygen consumption rate (OCR) and caspase-3/7 activity as an endpoint of mitochondrial dysfunction and apoptosis, respectively, with human hepatocytes after treatment with compounds causing idiosyncratic DILI (troglitazone, leflunomide, ranitidine and diclofenac). Troglitazone and leflunomide decreased the OCR but did not affect caspase-3/7 activity. Ranitidine increased caspase-3/7 activity but did not affect the OCR. Diclofenac decreased the OCR and increased caspase-3/7 activity. Acetaminophen and ethanol, which are also hepatotoxicants but do not induce idiosyncratic DILI, did not affect the OCR or caspase-3/7 activity. These results indicate that a combination assay of mitochondrial dysfunction and apoptosis is useful for the estimation of potential risk of compounds to induce idiosyncratic DILI.