Scalable workflows for high-throughput respirometry.

Paracetamol is a potent analgesic and antipyretic with limited side effects compared to the nonsteroidal anti-inflammatory drugs (NSAIDs) and opiates. Worldwide paracetamol is commonly used to treat pain and fever in both children and adults. Although, this drug has been in clinical use for more than a century, the mechanisms of action are not fully understood. Historically some of the actions of paracetamol were attributed to the inhibition of central cyclooxygenase (COX-1 and COX-2) enzymes however given the weak inhibitory effects on COX-1 and COX-2 enzymes, alternative targets have been suggested including a possible novel COX-3. The inhibition of COX-2 is accepted as the mechanism by which paracetamol reduces core temperature (Tc) in febrile animals. However, in non-febrile animals where COX-2 is not induced, paracetamol has also been shown to cause hypothermia by a mechanism that is not fully understood. Both the reduction of pyresis and induction of hypothermia can only occur when peripheral metabolic rate decreases and/or heat loss increases. In terms of antipyresis and hypothermia, the inhibition of lipolysis, fatty acid oxidation and mitochondria function are obvious alternative targets. Studies were undertaken to identify and characterise the putative COX-3 at protein and mRNA level using western blot analysis and reverse transcription polymerase chain reaction (RT-PCR) in mouse brain endothelial cells (b.End3) and whole brain tissues isolated from male C57BL/6 mice. Additional studies were also undertaken to assess if the hypothermic properties of paracetamol could be attributed to direct inhibition of thermogenic pathways in both 3T3-L1 adipocytes and primary brown adipocytes isolated from male Wistar rats. Adipocytes and isolated mitochondria were exposed to paracetamol and lipolysis, fatty acid oxidation (FAO), mitochondrial electron transport chain (ETC), assessed by measuring oxygen consumption rate (OCR). In these studies no expression of the COX-3 protein could be detected in brain endothelial cells and homogenates and no evidence of a COX-3 was detected at mRNA level. However, paracetamol caused a significant decrease (upto 70%; P<0.01, from control) in both basal and stimulated lipolysis at 1, 3 and 24 hours without affecting cell viability. Paracetamol (10 mM) and its metabolite N-acetyl-p-benzoquinone imine (NAPQI) at 50 µM also significantly (P<0.01, from control), reduced endogenous and exogenous FAO by 50% and 70% respectively. NAPQI (50 µM) had limited effect on mitochondrial uncoupling. Finally, paracetamol and other antipyretic compounds also significantly reduced ETC activity (upto 90%; P<0.01, from control). Both the maintenance of normal body temperature (Tb) and the induction of pyresis require increased mitochondrial ETC activity normally initiated centrally and driven peripherally by reduction of substrates such as fatty acids and glucose. The failure to identify the COX-3 protein and the direct inhibition of lipolysis, FAO and ETC activity indicate that antipyretic actions of paracetamol could partly be attributed to it actions on peripheral energy generation systems and provide new drug targets for reducing fever and chemically inducing hypothermia.

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.

Gliding through the warm south-western Indian Ocean, green turtles live a peaceful life feasting on seagrass meadows before returning to their deep coral reef homes. But their idyllic lifestyle is under threat from human activity; so understanding how these enigmatic creatures stay in tune with their environment is becoming increasingly important. ‘If they don't balance their energy budget there is going to be a problem for the individual and later on at a population level,’ explains Manfred Enstipp from the DEPE/CNRS Laboratory in Strasbourg, France. However, conventional methods for measuring energy expenditure may be unreliable in turtles, so Enstipp and Jean-Yves Georges decided to try another approach: measuring the animals' movements with accelerometers to estimate their energy use. First, the team had to discover whether there was a measurable relationship between the turtle's acceleration and their oxygen consumption in order to extrapolate the animals' energy use from their activity (p. 4010).However, when Enstipp and his colleagues embarked on the study they weren't even sure that they could measure the turtle's oxygen consumption rate. ‘They had to find this hole that is 40×40 cm to breathe so that we could measure the oxygen consumption rate,’ explains Enstipp, but many of his colleagues thought that the freely swimming animals could not be trained to surface repeatedly at a single point in long channel.Travelling to Stephane Ciccione's Kelonia Turtle Aquarium on Reunion Island, Enstipp isolated a 13 m-long swim channel in the turtles' enclosure and then, over a period of months, he and Benoit Gineste slowly covered the channel's surface with sections of metal fence until the turtles were content to surface and breathe at the respirometry dome, which covered the channel's only remaining open surface. Despite his colleagues' misgivings, Enstipp found that the turtles successfully learned to surface and breathe at the respirometry dome. Then, Enstipp velcroed a two-axis accelerometer onto a turtle's shell before recording her oxygen consumption and movements as she swam to and fro.Returning to Reunion Island with Gineste, Myriam Milbergue and Virginie Plot in the summer and winter, Enstipp successfully measured the oxygen consumption rates of six adult turtles at temperatures ranging from 24.8 to 30.1°C while also measuring their acceleration patterns. But could he use the acceleration traces to accurately estimate the turtles' oxygen consumption?Developing an equation to calculate the turtles' oxygen consumption based on their acceleration and the temperature of the surrounding water – which determines turtle metabolic rate – Enstipp calculated the turtles' oxygen consumption rates and compared them with the animals' measured oxygen consumption rates. They agreed well: the team could use the turtle's acceleration pattern to calculate their oxygen consumption, which they could use as a proxy for the animal's energy consumption.But how does the captive turtle's activity level compare with that of free-ranging green turtles grazing their seagrass meadows? ‘We are trying to apply this to Katia Ballorain's data from turtles foraging around Mayotte Island,’ Enstipp reports. However, early evidence suggests that the captive turtles were no couch potatoes and were every bit as active as their free-ranging cousins, offering Enstipp and his colleagues hope that accelerometery could provide much needed measurements of green turtles' energy consumption in the wild.

Mitochondrial metabolism is of central importance to diverse aspects of cell and developmental biology. Defects in mitochondria are associated with many diseases, including cancer, neuropathology, and infertility. Our understanding of mitochondrial metabolism in situ and dysfunction in diseases are limited by the lack of techniques to measure mitochondrial metabolic fluxes with sufficient spatiotemporal resolution. Herein, we developed a new method to infer mitochondrial metabolic fluxes in living cells with subcellular resolution from fluorescence lifetime imaging of NADH. This result is based on the use of a generic coarse-grained NADH redox model. We tested the model in mouse oocytes and human tissue culture cells subject to a wide variety of perturbations by comparing predicted fluxes through the electron transport chain (ETC) to direct measurements of oxygen consumption rate. Interpreting the fluorescence lifetime imaging microscopy measurements of NADH using this model, we discovered a homeostasis of ETC flux in mouse oocytes: perturbations of nutrient supply and energy demand of the cell do not change ETC flux despite significantly impacting NADH metabolic state. Furthermore, we observed a subcellular spatial gradient of ETC flux in mouse oocytes and found that this gradient is primarily a result of a spatially heterogeneous mitochondrial proton leak. We concluded from these observations that ETC flux in mouse oocytes is not controlled by energy demand or supply, but by the intrinsic rates of mitochondrial respiration.

Mitochondrial metabolism is of central importance to diverse aspects of cell and developmental biology. Defects in mitochondria are associated with many diseases, including cancer, neuropathology, and infertility. Our understanding of mitochondrial metabolism in situ and dysfunction in diseases are limited by the lack of techniques to measure mitochondrial metabolic fluxes with sufficient spatiotemporal resolution. Herein, we developed a new method to infer mitochondrial metabolic fluxes in living cells with subcellular resolution from fluorescence lifetime imaging of NADH. This result is based on the use of a generic coarse-grained NADH redox model. We tested the model in mouse oocytes and human tissue culture cells subject to a wide variety of perturbations by comparing predicted fluxes through the electron transport chain (ETC) to direct measurements of oxygen consumption rate. Interpreting the fluorescence lifetime imaging microscopy measurements of NADH using this model, we discovered a homeostasis of ETC flux in mouse oocytes: perturbations of nutrient supply and energy demand of the cell do not change ETC flux despite significantly impacting NADH metabolic state. Furthermore, we observed a subcellular spatial gradient of ETC flux in mouse oocytes and found that this gradient is primarily a result of a spatially heterogeneous mitochondrial proton leak. We concluded from these observations that ETC flux in mouse oocytes is not controlled by energy demand or supply, but by the intrinsic rates of mitochondrial respiration.

Oxygen Consumption Rate as a Measurement of Freeze-Induced Damage to Chicken Spermatozoa

The study of human ovarian tissue transplantation and cryopreservation has advanced significantly. Autotransplantation of human pre-antral follicles isolated from cryopreserved cortical tissue is a promising option for the preservation of fertility in young cancer patients. The purpose of the present study was to reveal the effect of vitrification after low-temperature transportation of human pre-antral follicles by using the oxygen consumption rate (OCR). Cortical tissues from 9 ovaries of female-to-male transsexuals were vitrified after transportation (6 or 18 h). The follicles were enzymatically isolated from nonvitrified tissue (group I, 18 h of transportation), vitrified-warmed tissue (group II, 6 and 18 h of transportation) and vitrified-warmed tissue that had been incubated for 24 h (group III, 6 and 18 h of transportation). OCR measurement and the LIVE/DEAD viability assay were performed. Despite the ischemic condition, the isolated pre-antral follicles in groupI consumed oxygen, and the mean OCRs increased with developmental stage. Neither the transportation time nor patient age seemed to affect the OCR in this group. Meanwhile, the mean OCR was significantly lower (P < 0.05) in group II but was comparable to that of group I after 24 h of incubation. The integrity of vitrified-warmed primordial and primary follicles was clearly corroborated by the LIVE/DEAD viability assay. These results demonstrate that the OCR can be used to directly estimate the effect of vitrification on the viability of primordial and primary follicles and to select the viable primordial and primary follicles from vitrified-warmed follicles.

Oxygen consumption is a fundamental characteristic of staphylococcal physiology reflecting the energy and metabolic state of the bacterial cell. During aerobic growth, oxygen consumption rates (OCR) depend on nutrient availability and vary at different growth stages. The measurement of oxygen consumption rates provides a versatile tool to characterize the impact of various mutations, environmental cues, and antibiotics on bacterial growth and fitness. In this chapter, we describe a MitoXpress® Xtra-based oxygen consumption assay for fast and reliable determination of respiration rates in Staphylococcus aureus. This highly reproducible and simple method requires a minimal set of reagents and allows rapid screening of multiple samples through real-time determination of the OCR with an oxygen-sensing probe and fluorescence plate reader.

Background: Despite recent efforts to elucidate the dependence of acute myeloid leukemia (AML) on the bone marrow microenvironment, the contribution of aged bone marrow mesenchymal stem cells (BMMSCs) to AML patient survival and treatment response remains understudied. Deregulated nutrient sensing is an established hallmark of aging and previous works have identified age-dependent metabolic dysregulations in cancer. The dependence of AML on oxidative phosphorylation (OXPHOS) has been well established, as AML adapts to fluctuations of nutrient and oxygen availability in the bone marrow microenvironment. We hypothesized that metabolic pathways, such as oxidative phosphorylation, are critical in aged AML-BMMSCs as they drive pro-tumorigenic properties and drug resistance. Methods: Our group expanded primary Normal Donor (ND)-BMMSCs and AML-BMMSCs (n=9 CBF-AML, n=11 CK-AML, n=7 ND-BMMSCs) under conditions that mimic the bone marrow microenvironment (91% nitrogen, 4% oxygen, and 5% CO2). We isolated DNA and RNA from early passaged cells to perform methylation analyses using 850k Illumina DNA methylation arrays and gene expression analyses using mRNA-seq. We utilized the Horvath Skin and Blood clock to calculate epigenetic age based on DNA methylation. qPCR assessing mRNA expression of OXPHOS-related genes in ND- and AML-BMMSCs was performed to validate sequencing results. Western blot analyses of Total OXPHOS and its complexes were performed to assess expression levels of OXPHOS-related proteins in ND- and AML-BMMSCs. Seahorse assays were performed to obtain live and repeated metabolic measurements of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in ND- and AML-BMMSCs. Lastly, 2D co-culture experiments were performed to assess cell viability of AML cell lines with BMMSCs treated ± OXPHOS inhibitors (IACS-010759 and Tamoxifen) utilizing AO/PI staining. Results: Our integrative omics approach showed that with increased epigenetic age, as measured by the Horvath Skin and Blood clock, AML-BMMSCs reveal upregulations in hallmark gene sets for MYC and OXPHOS when compared to ND-BMMSCs. Western blot analyses confirmed upregulation of MYC and OXPHOS complexes in AML-BMMSCs when compared to controls. Results from seahorse assay revealed an increase in OCR and ATP production in AML-BMMSCs when compared to controls. Lastly, our 2D co-culture experiments in vitro revealed a decrease in cell viability on AML cells that were co-cultured with AML-BMMSCs treated with IACS-010759 and/or Tamoxifen. Conclusions: Our results elucidate age-dependent metabolic dysregulations within the bone marrow microenvironment in AML and provide novel targetable approaches. Future portions of testing aim to identify the mechanism of action of AML-BMMSCs and OXPHOS. Citation Format: Mary E. Fuentes, Julia K. Christian, Amy Kovacs, James R. Lerma, Liang Niu, Alice S. Mims, Christopher C. Oaks, John C. Byrd, Erin K. Hertlein, Amina Abdul-Aziz. Metabolic dysregulations of aged bone marrow mesenchymal cells in Acute Myeloid Leukemia reveal novel therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1542.

Beta-adrenergic agonists (β-AAs) are widely used supplements in beef and pork production to improve feed efficiency and increase lean muscle mass, yet little is known about the molecular mechanism by which β-AAs achieve this outcome. Our objective was to identify the influence of ractopamine HCl and zilpaterol HCl on mitochondrial respiratory activity in muscle satellite cells isolated from crossbred beef steers (N = 5), crossbred barrows (N = 2), Yorkshire-cross gilts (N = 3), and commercial weather lambs (N = 5). Real-time measurements of oxygen consumption rates (OCRs) were recorded using extracellular flux analyses with a Seahorse XFe24 analyzer. After basal OCR measurements were recorded, zilpaterol HCl, ractopamine HCl, or no β-AA was injected into the assay plate in three technical replicates for each cell isolate. Then, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, and rotenone were injected into the assay plate sequentially, each inducing a different cellular state. This allowed for the measurement of OCR at these states and for the calculation of the following measures of mitochondrial function: basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, adenosine triphosphate (ATP)-linked respiration, and spare respiratory capacity. Incubation of bovine cells with either zilpaterol HCl or ractopamine HCl increased maximal respiration (P = 0.046) and spare respiratory capacity (P = 0.035) compared with non-supplemented counterparts. No difference (P > 0.05) was observed between zilpaterol HCl and ractopamine HCl for maximal respiration and spare respiratory capacity in bovine cell isolates. No measures of mitochondrial function (basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, ATP-linked respiration, and spare respiratory capacity) were altered by β-AA treatment in ovine or porcine cells. These findings indicate that β-AAs in cattle may improve the efficiency of oxidative metabolism in muscle satellite cells by modifying mitochondrial respiratory activity. The lack of response by ovine and porcine cells to β-AA incubation also demonstrates differing physiological responses to β-AA across species, which helps to explain the variation in its effectiveness as a growth supplement.

Oxygen consumption rates in deep-sea hydrothermal vent scale worms: Effect of life-style, oxygen concentration, and temperature sensitivity

Rationale: Alveolar-capillary crosstalk from the alveolar epithelium to the capillary endothelium leads to hyperpermeability and thus pulmonary edema in pneumonia-induced Acute Lung Injury (ALI). We have shown that mitochondrial depolarization—the loss of negative mitochondrial membrane potential—in the capillary endothelium causes hyperpermeability in multiple animal models of ALI. Herein, we sought to determine the molecular mechanisms connecting mitochondrial depolarization to hyperpermeability. Methods: To determine changes in endothelial mitochondrial metabolism in response to ALI, we used a combination of omics and in vitro approaches. For our omics studies, we induced ALI by instillation of P seudomonas aeruginosa by the intranasal route. Control mice were treated with sterile saline. 24h later, we enriched endothelial cells using magnetic beads to 95% purity. We immediately extracted RNA and performed bulk RNA-sequencing. For targeted metabolomics, we lysed endothelial cells and quantified tricarboxylic acid (TCA) cycle metabolites by liquid chromatography-mass spectrometry. For our in vitro analysis, we depolarized mitochondria in cultured endothelial cells with a 30-min exposure to H2O2. Immediately following, we measured oxygen consumption rate (OCR) under stress conditions with a Seahorse XFp Extracellular Flux Analyzer (Agilent Technologies). Control cells received saline and were analyzed in parallel. Results: In the murine lung endothelium, we identified 2918 up-regulated genes and 2918 down-regulated genes in response to P. aeruginosa pneumonia. Glycolytic pathways were major responders in pathways analysis (-log(p-value)=2-3.5). The following glycolysis-related genes were identified as increased (-logFDR&amp;gt;2): hk1, slc2a1, and gapdh. Targeted metabolomics identified an increase in lactate and citrate in endothelium of mice with P. aeruginosa pneumonia (p&amp;lt;0.05), suggesting a shift from oxidative phosphorylation to glycolysis. Cultured endothelial cells in which mitochondrial were depolarized with H2O2 had a decrease in OCR in response to FCCP (p&amp;lt;0.05), suggesting decreased maximal respiration and a shift to glycolysis. Conclusions: Mitochondrial depolarization is a key event leading to hyperpermeability in ALI. Our work suggests a pathway in which alveolar-capillary crosstalk depolarizes endothelial mitochondria. This mitochondrial depolarization shifts endothelial metabolism to glycolysis, depleting ATP and causing hyperpermeability and pulmonary edema.

This study explores the metabolic response and carbon budget of two cyclopoid copepod species, Diacyclops belgicus Kiefer, 1936 (a stygobitic, groundwater-adapted species) and Diacyclops crassicaudis crassicaudis (Sars G.O., 1863) (a stygophilic, predominantly surface-associated species). We measured oxygen consumption rates (OCRs), carbon requirements (CRs), ingestion (I) rates, and egestion (E) rates at 14 °C and 17 °C, representing current and predicted future conditions in the collection habitats of the two species. Diacyclops belgicus displayed OCRs (28.15 and 18.32 µL O2/mg DW × h at 14 and 17 °C, respectively) and carbon budget (CR: 0.14 and 0.10 µg C/mg × d at 14 and 17 °C) lower than those of D. crassicaudis crassicaudis (OCR: 55.67 and 47.93 µL O2/mg DW × h at 14 and 17 °C; CR: 0.3 and 0.27 µg C/mg × d at 14 and 17 °C). However, D. belgicus exhibited metabolic rates and carbon requirements comparable to those of other epigean species, challenging the assumption that low metabolic rates are universal among stygobitic species. Temperature variations did not significantly affect the metabolic responses and carbon requirements of the two species, suggesting that they may cope with moderate temperature increases.

Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system's capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates.

Field metabolic rate (FMR) is key to understanding individual and population-level responses to environmental changes, but is challenging to measure in field conditions, particularly in aquatic environments. Here we show that FMR can be estimated directly from the isotopic composition of carbon in fish otoliths (δ13Coto). We describe the relationship between δ13Coto values and oxygen consumption rate, and report results from laboratory experiments relating individual-level measurements of oxygen consumption rates to δ13Coto values in Atlantic cod (Gadus morhua). We apply our new δ13Coto metabolic proxy to existing δ13Coto data from wild cod and four deepwater fish species to test the validity of inferred FMR estimates. The δ13Coto metabolic proxy offers a new approach to study physiological ecology in free-ranging wild fishes. Otolith-based proxies for FMR are particularly promising as they allow retrospective assessment of time-integrated, individual-level FMR throughout an individual fish’s life history.