Differences In Oxygen Consumption Rates Research Articles

Protein kinase N1 deficiency results in upregulation of cerebral energy metabolism and is highly protective in in vivo and in vitro stroke models

Background and aimWe recently identified protein kinase N1 (PKN1) as a master regulator of brain development. However, its function in the adult brain has not been clearly established. In this study, we assessed the cerebral energetic phenotype of wildtype (WT) and global Pkn1 knockout (Pkn1−/−) animals under physiological and pathophysiological conditions. MethodsCerebral energy metabolism was analyzed by 13C6-glucose tracing in vivo and real time seahorse analysis of extracellular acidification rates as well as mitochondrial oxygen consumption rates (OCR) of brain slice punches in vitro. Isolated WT and Pkn1−/− brain mitochondria were tested for differences in OCR with different substrates. Metabolite levels were determined by mass spectrometric analysis in brain slices under control and energetic stress conditions, induced by oxygen-glucose deprivation and reperfusion, an in vitro model of ischemic stroke. Differences in enzyme activities were assessed by enzymatic assays, western blotting and bulk RNA sequencing. A middle cerebral artery occlusion stroke model was used to analyze lesion volumes and functional recovery in WT and Pkn1−/− mice. ResultsPkn1 deficiency resulted in a remarkable upregulation of cerebral energy metabolism, in vivo and in vitro. This was due to two separate mechanisms involving an enhanced glycolytic flux and higher pyruvate-induced mitochondrial OCR. Mechanistically we show that Pkn1−/− brain tissue exhibits an increased activity of the glycolysis rate-limiting enzyme phosphofructokinase. Additionally, glucose-1,6-bisphosphate levels, a metabolite that increases mitochondrial pyruvate uptake, were elevated upon Pkn1 deficiency. Consequently, Pkn1−/− brain slices had more ATP and a greater accumulation of ATP degradation metabolites during energetic stress. This translated into increased phosphorylation and activity of adenosine monophosphate (AMP)-activated protein kinase (AMPK) during in vitro stroke. Accordingly, Pkn1−/− brain slices showed a post-ischemic transcriptional upregulation of energy metabolism pathways and Pkn1 deficiency was strongly protective in in vitro and in vivo stroke models. While inhibition of mitochondrial pyruvate uptake only moderately affected the protective phenotype, inhibition of AMPK in Pkn1−/− slices increased post-ischemic cell death in vitro. ConclusionThis is the first study to comprehensively demonstrate an essential and unique role of PKN1 in cerebral energy metabolism, regulating glycolysis and mitochondrial pyruvate-induced respiration. We further uncovered a highly protective phenotype of Pkn1 deficiency in both, in vitro and in vivo stroke models, validating inhibition of PKN1 as a promising new therapeutic target for the development of novel stroke therapies.

Metformin impairs trophoblast metabolism and differentiation in a dose-dependent manner.

Metformin is a widely prescribed medication whose mechanism of action is not completely defined and whose role in gestational diabetes management remains controversial. In addition to increasing the risk of fetal growth abnormalities and preeclampsia, gestational diabetes is associated with abnormalities in placental development including impairments in trophoblast differentiation. Given that metformin impacts cellular differentiation events in other systems, we assessed metformin's impact on trophoblast metabolism and differentiation. Using established cell culture models of trophoblast differentiation, oxygen consumption rates and relative metabolite abundance were determined following 200µM (therapeutic range) and 2000µM (supra-therapeutic range) metformin treatment using Seahorse and mass-spectrometry approaches. While no differences in oxygen consumption rates or relative metabolite abundance were detected between vehicle and 200µM metformin-treated cells, 2000µM metformin impaired oxidative metabolism and increased the abundance of lactate and TCA cycle intermediates, α-ketoglutarate, succinate, and malate. Examining differentiation, treatment with 2000μM, but not 200µM metformin, impaired HCG production and expression of multiple trophoblast differentiation markers. Overall, this work suggests that supra-therapeutic concentrations of metformin impair trophoblast metabolism and differentiation whereas metformin concentrations in the therapeutic range do not strongly impact these processes.

Insight into the characterization of commercial oenological tannins

The characterization in terms of phenolics, antioxidant capacity and oxygen consumption rate (OCR) was carried out for 15 commercial oenological tannins of different origin. A NMR approach was used to evaluate their average molecular weight, and their glycosidic and aromatic moieties. The investigated oenological tannins showed wide differences in their chemical properties. Total phenol index (TPI) ranged from 461±28 and 1018±57 mg gallic acid/g for cherry tannin and nut gall tannin, respectively. The antioxidant capacity, positively correlated with TPI, was higher for hydrolysable tannins ranging 3.05±0.06 and 12.06±0.71 mM Trolox/g for ABTS assay, and from 3.70±0.23 and 10.94±1.28 mM Trolox/g for DPPH assay. Relevant differences in OCRs were found and chestnut tannin showed the highest OCR. Wide range of molecular weights were found with nut gall 1 tannin showing the highest one, ranging from 790 to 1900 Da. This study improves and expands the actual knowledge of tannins supporting the suitability of NMR for the characterization of oenological tannins.

Investigating the Role of Salicylic Acid in Determining the Lifespan of Short-Lived and Long-Lived Flax Species and Lines

Introduction: Reactive oxygen species (ROS) accumulation has been related to aging in both plant and human physiology. Leaked electrons from the electron transport system (ETS) react with molecular oxygen producing ROS, such as hydrogen peroxide (H2O2) and superoxide anions. There are numerous antioxidative mechanisms present in plants that aid in redox balance, one of which is the mild uncoupling of mitochondria. Uncoupling agents, such as salicylic acid (SA), can abolish the connection between oxidation and phosphorylation allowing electron transport to take place without the need for parallel ATP synthesis. “Uncoupling to Survive” Hypothesis states that uncoupling activity should be greater in long-lived species since uncoupled mitochondria result in reduced ROS production thereby increasing lifespan. Since ROS accumulation has been linked to lifespan in the Oxidative Stress Theory of Aging, the idea that long-lived species should exhibit greater antioxidative capacity than short-lived species is the foundation upon which this hypothesis is established. We tested this hypothesis in various flax species and lines differing in lifespan. Methods: Spectrophotometry was used to determine SA levels in plant tissue while respiration rates were measured using a dissolving oxygen electrode. Results: The “Uncoupling to Survive” Hypothesis does not seem to hold for flax since long-lived and short-lived species did not exhibit significant differences in oxygen consumption rates that would indicate differences in the uncoupling activity of their mitochondria. Discussion: However, early flowering lines exhibited lower levels of SA suggesting that flax plants do not require elevated SA expression to display the early flowering phenotype as seen in Arabidopsis species. In addition, SA significantly increased the oxygen consumption rates in wild-type and early flowering plants which further provides evidence for its role as an uncoupling agent. Conclusion: Our findings add to our understanding of the many roles that SA plays in plant physiology. Advantages of SA are well established in the cosmetic and medical communities and investigating its effects on plants can be beneficial. We also looked at the "Uncoupling to Survive Hypothesis" in plants, which helped us get a better grasp of the antioxidant mechanisms that may eventually reduce oxidative stress and lead to senescence.

Assessment of Mitochondrial Respiratory Function in Isolated Nephron Segments of Dahl SS Rats in Salt Induced Hypertension

RationaleKidney nephrons actively reabsorb 99% of the filtrate to maintain body fluid and solute homeostasis. A large amount of ATP synthesis and O2 consumption must work coordinal to support this demand. We hypothesized that mitochondrial function of proximal tubules (PT) and medullary thick ascending limbs (mTAL) is altered during the development of salt induced hypertension in the Dahl salt‐sensitive (SS) rat.MethodThe mitochondrial oxygen consumption rates (OCR) were determined in freshly bulk isolated PT and mTAL segments from SS rats fed 0.4% NaCl diet (low salt; LS) or 4% NaCl diet (high salt; HS) for 7 days. A protocol was developed to determine basal levels of OCR and the effects of various key circulating substrates (glucose, lactate, pyruvate, glutamine, and palmitate) upon mitochondrial respiration (OCR) using the Agilent Seahorse XF96 Extracellular Flux Analyzer combined with the Agilent Seahorse XF Cell Mito Stress Test protocol.ResultsAnalysis of the renal tubular mitochondrial OCR data for five different circulating substrates confirmed that there were clear differences in the substrate preference between the PT and mTAL which were altered by HS feeding. It was found that PT preferred lactate and glutamine while mTAL preferred glucose and pyruvate and that both segments could effectively utilize palmitate as a mitochondrial substrate. PT isolated from SS rats fed HS displayed higher OCR levels compared to PT from LS fed rats, but HS diet did not result in significant differences in individual substrate effects. In contrast, isolated mTAL from HS fed rats exhibited increased OCR in response to glutamine and palmitate compared to mTAL from LS fed rats although no basal differences in OCR were observed.ConclusionThese observations demonstrated that renal tubular mitochondrial respiratory function is altered in SS rats during the development of salt‐induced hypertension and that both PT and mTAL exhibit an increased respiratory capacity in response to HS feeding. These changes in metabolism appear necessary to meet the increased tubular workloads and need for greater ATP production and O2 utilization in both PT and mTAL. We conclude, however, that different metabolic strategies are utilized by each of these tubular segments to meet these increased metabolic requirements.

Integrative analysis of transcriptional profile reveals LINC00052 as a suppressor of breast cancer cell migration.

Long-non-coding RNAs, a class of transcripts with lengths > 200nt, play key roles in tumour progression. Previous reports revealed that LINC00052 (long intergenic non-coding RNA 00052) was strongly downregulated during breast cancer multicellular spheroids formation and suggested a role in cell migration and oxidative metabolism. To examine the function of LINC00052 in MCF-7 breast cancer cells. Loss-of-function studies were performed to evaluate LINC00052 role on MCF-7 breast cancer cells. Microarray expression assays were performed to determine genes and cellular functions modified after LINC00052 knockdown. Next, the impact of LINC00052 depletion on MCF-7 cell respiration and migration was evaluated. 1,081 genes were differentially expressed upon LINC00052 inhibition. Gene set enrichment analysis, Gene Ontology and Key Pathway Advisor analysis showed that signalling networks related to cell migration and oxidative phosphorylation were enriched. However, whereas LINC00052 knockdown in MCF-7 cells revealed marginal difference in oxygen consumption rates when compared with control cells, LINC00052 inhibition enhanced cell migration in vitro and in vivo, as observed using a Zebrafish embryo xenotransplant model. Our data show that LINC00052 modulates MCF-7 cell migration. Genome-wide microarray experiments suggest that cancer cell migration is affected by LINC00052 through cytoskeleton modulation and Notch/β-catenin/NF-κB signalling pathways.

Unveiling the contribution of the reproductive system of individual Caenorhabditis elegans on oxygen consumption by single-point scanning electrochemical microscopy measurements

Metabolic analysis in animals is usually either evaluated as whole-body measurements or in isolated tissue samples. To reveal tissue specificities in vivo, this study uses scanning electrochemical microscopy (SECM) to provide localized oxygen consumption rates (OCRs) in different regions of single adult Caenorhabditis elegans individuals. This is achieved by measuring the oxygen reduction current at the SECM tip electrode and using a finite element method model of the experiment that defines oxygen concentration and flux at the surface of the organism. SECM mapping measurements uncover a marked heterogeneity of OCR along the worm, with high respiration rates at the reproductive system region. To enable sensitive and quantitative measurements, a self-referencing approach is adopted, whereby the oxygen reduction current at the SECM tip is measured at a selected point on the worm and in bulk solution (calibration). Using genetic and pharmacological approaches, our SECM measurements indicate that viable eggs in the reproductive system are the main contributors in the total oxygen consumption of adult Caenorhabditis elegans. The finding that large regional differences in OCR exist within the animal provides a new understanding of oxygen consumption and metabolic measurements, paving the way for tissue-specific metabolic analyses and toxicity evaluation within single organisms.

Reduced adenosine diphosphate sensitivity in skeletal muscle mitochondria increases reactive oxygen species production in mouse models of aging and oxidative stress but not denervation.

BackgroundMitochondrial bioenergetics are sensitive to adenosine diphosphate (ADP) concentration. Reactive oxygen species (ROS) production and respiration [oxygen consumption rate (OCR)] are altered at physiological ADP concentrations (i.e. ADP insensitivity) in aged human muscle. Here, we investigate ADP sensitivity in mouse muscle mitochondria.MethodsWe measured OCR and ROS production in permeabilized gastrocnemius fibres using an ADP titration protocol and the Oroboros O2k respirometer and fluorometer. We measured changes in ADP sensitivity in muscle from mice at different ages, after sciatic nerve transection (denervation), and in response to increased oxidative stress (Sod1−/− mice). Further, we asked whether the mitochondrial-targeted peptide SS-31 can modulate ADP insensitivity and contractile function in the Sod1−/− mouse model.ResultsReduced ADP sensitivity is associated with increases in mitochondrial ROS production in aged (62%) and Sod1−/− (33%) mice. The maximal capacity to produce ROS does not increase with age, and there is no effect of age on ADP sensitivity for OCR in mouse gastrocnemii. Denervation does not induce ADP insensitivity for either ROS generation or OCR. Treatment of Sod1−/− mice with SS-31 increases ADP sensitivity for both OCR and ROS, decreases maximal ROS production (~40%), and improves resistance to muscle fatigue.ConclusionsAdenosine diphosphate sensitivity for ROS production decreases in aged mouse gastrocnemius muscle fibres, although aged mice do not exhibit a difference in OCR. Denervation does not induce ADP insensitivity; however, insensitivity to ADP is induced in a model of oxidative stress. ADP insensitivity could contribute to muscle fatigue, and SS-31 may be the first drug capable of targeting this aging phenotype.

Passaging capability of human corneal endothelial cells derived from old donors with and without accelerating cell attachment

In a recent report, we showed that it is possible to establish the culture of Human Corneal Endothelial Cells (HCEnCs) from older donor corneas (usually over 65 year olds) when left to attach in the presence of a viscoelastic solution, potentially increasing the donor pool for culturing HCEnCs. Therefore, we set out to evaluate the outcome of using a viscoelastic solution (Viscoat) to accelerate the attachment of passaged cultured human corneal endothelial cells (HCEnCs). The cells from 28 donor tissues were isolated using peel-and-digest method and evenly seeded into two wells of an 8-well chamber slide. The cells were left to attach after topical application of Viscoat. At confluence, one well was subjected to end-stage characterization, whereas the other well was passaged into another two wells. The cells at P1 were attached with and without the use of Viscoat. The growth rate was monitored; and at confluence, morphometric analysis, corneal endothelial specific (CD166-Tag1A3 & PRDX6-Tag2A12), mitochondrial and respiration assessment (Tom-20 and Seahorse); function-associated (Na+/K+ATPase & ZO-1); proliferative (Ki-67) marker analysis, and viability (Hoechst, Ethidium Homodimer and Calcein AM-HEC) studies were performed. Cells at P0 (with Viscoat) showed 100% confluence at day 9. Cells at P1 with and without Viscoat showed significant difference of confluence 67.0% v 18.8% respectively (p < 0.05). Confluence rate, cell density, hexagonality, Ki-67 positivity and mitochondrial intensity was significantly higher (p < 0.05), whereas cell-area and polymorphism was significantly lower (p < 0.05) in the cells attached with Viscoat compared with the cells attached without Viscoat. There was no significant difference in oxygen consumption rate between the groups. In conclusion, we observed that acceleration in the attachment of passaged HCEnCs with the assistance of Viscoat, could be beneficial for the propagation of HCEnCs isolated from older donors, to increase their propensity to proliferate, without loss of the expression of vital proteins and heterogeneity in cellular morphology.

An inducible knockout of Dicer in adult mice does not affect endurance exercise-induced muscle adaptation

The contractile and metabolic properties of adult skeletal muscle change in response to endurance exercise. The mechanisms of transcriptional regulation in exercise-induced skeletal muscle adaptation, including fiber-type switching and mitochondrial biogenesis, have been investigated intensively, whereas the role of microRNA (miRNA)-mediated posttranscriptional gene regulation is less well understood. We used tamoxifen-inducible Dicer1 knockout (iDicer KO) mice to reduce the global expression of miRNAs in adult skeletal muscle and subjected these mice to 2 wk of voluntary wheel running. Dicer mRNA expression was completely depleted in fast-twitch plantaris muscle after tamoxifen injection. However, several muscle-enriched miRNAs, including miR-1 and miR-133a, were reduced by only 30-50% in both the slow and fast muscles. The endurance exercise-induced changes that occurred for many parameters (i.e., fast-to-slow fiber-type switch and increases in succinate dehydrogenase, respiratory chain complex II, and citrate synthase activity) in wild type (WT) also occurred in the iDicer KO mice. Protein expression of myosin heavy chain IIa, peroxisome proliferator-activated receptor-γ coactivator-1α, and cytochrome c complex IV was also increased in the iDicer KO mice by the voluntary running. Furthermore, there was no significant difference in oxygen consumption rate in the isolated mitochondria between the WT and iDicer KO mice. These data indicate that muscle-enriched miRNAs were detectable even after 4 wk of tamoxifen treatment and there was no apparent specific endurance-exercise-induced muscle phenotype in the iDicer KO mice.

Swimming Energy Economy in Bottlenose Dolphins Under Variable Drag Loading

Instrumenting animals with tags contributes additional resistive forces (weight, buoyancy, lift and drag) that may result in increased energetic costs; however, additional metabolic expense can be moderated by adjusting behaviour to maintain power output. We sought to increase hydrodynamic drag for near-surface swimming bottlenose dolphins, to investigate the metabolic effect of instrumentation. In this experiment, we investigate whether (1) metabolic rate increases systematically with hydrodynamic drag loading from tags of different sizes or (2) whether tagged individuals modulate speed, swimming distance and/or fluking motions under increased drag loading. We detected no significant difference in oxygen consumption rates when four male dolphins performed a repeated swimming task, but measured swimming speeds that were 34% (>1 m s-1) slower in the highest drag condition. To further investigate this observed response, we incrementally decreased and then increased drag in six loading conditions. When drag was reduced, dolphins increased swimming speed (+1.4 m s-1; +45%) and fluking frequency (+0.28 Hz; +16%). As drag was increased, swimming speed (-0.96 m s-1; -23%) and fluking frequency (-14 Hz; 7%) decreased again. Results from computational fluid dynamics simulations indicate that the experimentally observed changes in swimming speed would have maintained the level of external drag forces experienced by the animal. Together, these results indicate that dolphins may adjust swimming speed to modulate the drag force opposing their motion during swimming, adapting their behaviour to maintain a level of energy economy during locomotion.

Comparison of Oxygen Consumption Rates of Nondegenerate and Degenerate Human Intervertebral Disc Cells.

In vitro measurements of the oxygen consumption rates (OCR) of human intervertebral disc (IVD) cells. The aim of this study was to determine the differences in the OCR of nondegenerate and degenerate human annulus fibrosus (AF), nucleus pulposus (NP), and cartilage endplate (CEP) cells at different glucose concentrations. The avascular nature of the IVD creates a delicate balance between rate of nutrient transport through the matrix and rate of disc cell consumption necessary to maintain tissue health. Previous studies have shown a dependence of OCR for animal (e.g., bovine and porcine) IVD cells on oxygen level and glucose concentration. However, the OCR of nondegenerate human IVD cells compared to degenerate human IVD cells at different glucose concentrations has not been investigated. IVD cells were isolated from the AF, NP, and CEP regions of human cadaver spines and surgical samples. The changes in oxygen concentration were recorded when cells were sealed in a metabolic chamber. The OCR of cells was determined by curve fitting using the Michaelis-Menton equation. Under identical cell culture conditions, the OCR of degenerate human IVD cells was three to five times greater than that of nondegenerate human IVD cells. The degenerate IVD cells cultured in low-glucose medium (1 mmol/L) exhibited the highest OCR compared to degenerate cells cultured at higher glucose levels (i.e., 5 mmol/L, 25 mmol/L), whereas no significant differences in OCR were found among the nondegenerate IVD cells for all glucose levels. Considering the significantly higher OCR and unique response to glucose of degenerate human IVD cells, the degeneration of the IVD is associated with a cell phenotypic change related to OCR. The OCR of IVD cells reported in this study will be valuable for understanding human IVD cellular behavior and tissue nutrition in response to disc degeneration. N/A.

Elevated nitrate alters the metabolic activity of embryonic zebrafish.

Nitrate accumulation in aquatic reservoirs from agricultural pollution has often been overlooked as a water quality hazard, yet a growing body of literature suggests negative effects on human and wildlife health following nitrate exposure. This research seeks to understand differences in oxygen consumption rates between different routes of laboratory nitrate exposure, whether via immersion or injection, in zebrafish (Danio rerio) embryos. Embryos were exposed within 1 h post fertilization (hpf) to 0, 10, and 100 mg/L NO3-N with sodium nitrate, or to counter ion control (CIC) treatments using sodium chloride. Embryos in the immersion treatments received an injection of 4 nL of appropriate treatment solution into the perivitelline space. At 24 hpf, Oxygen Consumption Rates (OCR) were measured and recorded invivo using the Agilent Technologies XFe96 Extracellular Flux Analyzer and Spheroid Microplate. Immersion exposures did not induce significant changes in OCR, yet nitrate induced significant changes when injected through the embryo chorion. Injection of 10 and 100 mg/L NO3-N down-regulated OCR compared to the control treatment group. Injection of the 100 mg/L CIC also significantly down-regulated OCR compared to the control treatment group. Interestingly, the 100 mg/L NO3-N treatment further down-regulated OCR compared to the 100 mg/L CIC treatment, suggesting the potential for additive effects between the counter ion and the ion of interest. These data support that elevated nitrate exposure can alter normal metabolic activity by changing OCR in 24 hpf embryos. These results highlight the need for regularly examining the counter ion of laboratory nitrate compounds while conducting research with developing zebrafish, and justify examining different routes of laboratory nitrate exposure, as the chorion may act as an effective barrier to nitrate penetration in zebrafish, which may lead to conservative estimates of significant effects in other species for which nitrate more readily penetrates the chorion.

Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

Pyruvate kinase M2 (PKM2) regulates glycolysis and oxidative phosphorylation; however, the role of PKM2 in ovarian cancer remains largely unknown. We investigated whether ovarian cancer metabolism could provide insight into the development of therapeutic strategies. We performed immunohistochemical staining for PKM2 on a tissue microarray for multivariate analysis. It revealed that patients exhibiting higher PKM2 expression were significantly associated with malignancy groups (p < 0.001) and pathogenesis models (p < 0.001), had poor progression-free survival rates (p = 0.01) as compared with patients exhibiting lower PKM2 levels, and yielded a hazard ratio of death of 2.02 (95% confidence interval: 0.70–5.85). In cell lines, PKM2 inhibitor significantly inhibited the glycolytic rate according to cellular glucose consumption (p < 0.001). We also utilized Seahorse assays to assess metabolism-related cell-specific factors and the impact of PKM2 inhibitors. Energy shifts as per Seahorse analysis showed attenuation of the extracellular acidification rate (p < 0.05) and no significant difference in oxygen-consumption rate in SKOV3 cells. Treatment with PKM2 inhibitor suppressed ovarian cancer growth and cell migration in vitro and inhibited tumor growth without significant toxicity in a xenograft study. PKM2 inhibition disturbed Warburg effects and inhibited ovarian cancer cell growth. Targeting PKM2 may constitute a promising therapy for patients with ovarian cancer, and clinical trials involving shikonin are warranted.

Metabolic and reproductive plasticity of core and marginal populations of the eurythermic saline water bug Sigara selecta (Hemiptera: Corixidae) in a climate change context

Ongoing climate change is driving dramatic range shifts in diverse taxa worldwide, and species responses to global change are likely to be determined largely by population responses at geographical range margins. Here we investigate the metabolic and reproductive plasticity in response to water temperature and salinity variation of two populations of the eurythermic saline water bug Sigara selecta: one population located close to the northern edge of its distribution, in a relatively cold, thermally stable region (SE England – ‘marginal’), and one close to the range centre, in a warmer and more thermally variable Mediterranean climate (SE Spain – ‘core’). We compared metabolic and oviposition rates and egg size, following exposure to one of four different combinations of temperature (15 and 25°C) and salinity (10 and 35gL−1). Oviposition rate was significantly higher in the marginal population, although eggs laid were smaller overall. No significant differences in oxygen consumption rates were found between core and marginal populations, although the marginal population showed higher levels of plasticity in both metabolic and reproductive traits. Our results suggest that population-specific responses to environmental change are complex and may be mediated by differences in phenotypic plasticity. In S. selecta, the higher plasticity of the marginal population may facilitate both its persistence in current habitats and northward expansion with future climatic warming. The less plastic core population may be able to buffer current environmental variability with minor changes in metabolism and fecundity, but could be prone to extinction if temperature and salinity changes exceed physiological tolerance limits in the future.

Body mass scaling of passive oxygen diffusion in endotherms and ectotherms

The area and thickness of respiratory surfaces, and the constraints they impose on passive oxygen diffusion, have been linked to differences in oxygen consumption rates and/or aerobic activity levels in vertebrates. However, it remains unclear how respiratory surfaces and associated diffusion rates vary with body mass across vertebrates, particularly in relation to the body mass scaling of oxygen consumption rates. Here we address these issues by first quantifying the body mass dependence of respiratory surface area and respiratory barrier thickness for a diversity of endotherms (birds and mammals) and ectotherms (fishes, amphibians, and reptiles). Based on these findings, we then use Fick's law to predict the body mass scaling of oxygen diffusion for each group. Finally, we compare the predicted body mass dependence of oxygen diffusion to that of oxygen consumption in endotherms and ectotherms. We find that the slopes and intercepts of the relationships describing the body mass dependence of passive oxygen diffusion in these two groups are statistically indistinguishable from those describing the body mass dependence of oxygen consumption. Thus, the area and thickness of respiratory surfaces combine to match oxygen diffusion capacity to oxygen consumption rates in both air- and water-breathing vertebrates. In particular, the substantially lower oxygen consumption rates of ectotherms of a given body mass relative to those of endotherms correspond to differences in oxygen diffusion capacity. These results provide insights into the long-standing effort to understand the structural attributes of organisms that underlie the body mass scaling of oxygen consumption.

Cell bioenergetics in Leghorn male hepatoma cells and immortalized chicken liver cells in response to 4-hydroxy 2-nonenal-induced oxidative stress

The major objectives of this study were to compare cell bioenergetics in 2 avian liver cell lines under control conditions and in response to oxidative stress imposed by 4-hydroxy 2-nonenal (4-HNE). Cells in this study were from a chemically immortalized Leghorn male hepatoma (LMH) cell line and a spontaneously immortalized chicken liver (CELi) cell line. Oxygen consumption rate (OCR) was monitored in specialized microtiter plates using an XF24 Flux Analyzer (Seahorse Bioscience, Billerica, MA). Cell bioenergetics was assessed by sequential additions of oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and antimycin-A that enables the determination of a) OCR linked to adenosine triphosphate (ATP) synthase activity, b) mitochondrial oxygen reserve capacity, c) proton leak, and d) nonmitochondrial cytochrome c oxidase activity. Under control (unchallenged) conditions, LMH cells exhibited higher basal OCR and higher OCR attributed to each of the bioenergetic components listed above compared with CELi cells. When expressed as a percentage of maximal OCR (following uncoupling with FCCP), LMH cells exhibited higher OCR due to ATP synthase and proton leak activity, but lower mitochondrial oxygen reserve capacity compared with CELi cells; there were no differences in OCR associated with nonmitochondrial cytochrome c oxidase activity. Whereas the LMH cells exhibited robust ATP synthase activity up to 50 μM 4-HNE, CELi cells exhibited a progressive decline in ATP synthase activity with 10, 20, and 30 μM 4-HNE. The CELi cells exhibited higher mitochondrial oxygen reserve capacity compared with LMH cells with 0 and 20 μM 4-HNE but not with 30 μM 4-HNE. Both cell lines exhibited inducible proton leak in response to increasing levels of 4-HNE that was evident with 30 μM 4-HNE for CELi cells and with 40 and 50 μM 4-HNE in LMH cells. The results of these studies demonstrate fundamental differences in cell bioenergetics in 2 avian liver-derived cell lines under control conditions and in response to oxidative challenge due to 4-HNE.

Effects of Fiber Type and Size on the Heterogeneity of Oxygen Distribution in Exercising Skeletal Muscle

The process of oxygen delivery from capillary to muscle fiber is essential for a tissue with variable oxygen demand, such as skeletal muscle. Oxygen distribution in exercising skeletal muscle is regulated by convective oxygen transport in the blood vessels, oxygen diffusion and consumption in the tissue. Spatial heterogeneities in oxygen supply, such as microvascular architecture and hemodynamic variables, had been observed experimentally and their marked effects on oxygen exchange had been confirmed using mathematical models. In this study, we investigate the effects of heterogeneities in oxygen demand on tissue oxygenation distribution using a multiscale oxygen transport model. Muscles are composed of different ratios of the various fiber types. Each fiber type has characteristic values of several parameters, including fiber size, oxygen consumption, myoglobin concentration, and oxygen diffusivity. Using experimentally measured parameters for different fiber types and applying them to the rat extensor digitorum longus muscle, we evaluated the effects of heterogeneous fiber size and fiber type properties on the oxygen distribution profile. Our simulation results suggest a marked increase in spatial heterogeneity of oxygen due to fiber size distribution in a mixed muscle. Our simulations also suggest that the combined effects of fiber type properties, except size, do not contribute significantly to the tissue oxygen spatial heterogeneity. However, the incorporation of the difference in oxygen consumption rates of different fiber types alone causes higher oxygen heterogeneity compared to control cases with uniform fiber properties. In contrast, incorporating variation in other fiber type-specific properties, such as myoglobin concentration, causes little change in spatial tissue oxygenation profiles.

Mitochondrial oxygen consumption in permeabilized fibers and its link to colour changes in bovine M. semimembranosus muscle

Animal and muscle characteristics were recorded for 41 cattle. The oxygen consumption rate (OCR) of M. semimembranosus was measured between 3.0–6.4h post mortem (PM3-6) and after 3weeks in a vacuum pack at 4°C. Colour change measurements were performed following the 3weeks using reflectance spectra (400–1100nm) and the colour coordinates L*, a* and b*, with the samples being packaged in oxygen permeable film and stored at 4°C for 167h.Significant individual animal differences in OCR at PM3-6 were found for mitochondrial complexes I and II. OCR of complex I declined with increased temperature and time PM, while residual oxygen-consuming side-reactions (ROX) did not. OCR of stored muscles was dominated by complex II respiration. A three-way regression between samples, colour variables collected upon air exposure and OCR of 3weeks old fibres revealed a positive relationship between OCR and complex II activity and also between OCR and OCRROX. The presence of complex I and β-oxidation activities increased metmyoglobin formation.

Growth, metabolism and immune responses to evisceration and the regeneration of viscera in sea cucumber, Apostichopus japonicus

Growth, metabolism and immune responses of sea cucumber, Apostichopus japonicus, following evisceration induced artificially, were investigated in this study. The body weight, specific growth rate (SGR), oxygen consumption rate (OCR), activities of immunoenzymes including acidic phosphatase (ACP), alkaline phosphatase (AKP) from hydrolytic system, and catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and malonyl-dialdehyde (MDA) from antioxidant system in muscle, intestine and respiratory tree were measured to evaluate the physiological responses of A. japonicus to evisceration and regeneration of viscera. The results showed that body weight of sea cucumber significantly dropped following evisceration and then increased gradually with digestive function resumed. Accelerated growth rates were observed in the regeneration group from the 10th day to 20th day, but there was no significant difference between the regeneration group and control group at the end of the experiment. OCR reduced rapidly after evisceration and increased gradually afterwards. Compared to the control, no significant difference in OCR was found on the 45th day (P>0.05). The immune responses of A. japonicus were highly tissue-specific during the regeneration of viscera. Both ACP and AKP activities in muscle peaked on the 10th day after evisceration, then decreased gradually to normal on the 45th day (P>0.05), while those in the regenerated tissues exhibited a differential rising trend from the 20th day after evisceration. SOD activity in muscle was not significantly influenced by evisceration; however, in the regenerated tissues it was enhanced and significantly higher than those in the control on the 45th day (P<0.01). CAT activity in muscle of the regeneration group was significantly higher than in the control during the experiment (P<0.05). Comparatively, CAT activity in the respiration tree increased invariably after evisceration, while in the intestine CAT activity was not significantly affected by evisceration (P>0.05). Significant increase was found in both T-AOC and MDA in all three tissues after evisceration (P<0.05); however, they all returned to normal at the end of the experiment. Results of this study indicated that growth, metabolism and immune responses including hydrolytic and antioxidant enzymes in sea cucumber were significantly influenced by evisceration. Although the regeneration group did not catch up with the control in body weight, SGR, OCR, major non-specific immune parameters resumed to normal level within 45days after evisceration, indicating that A. japonicus might have recovered physiologically from evisceration. Results from this study first presented comprehensive and valuable data of physiological responses to evisceration and the regeneration of viscera in A. japonicus, which would have important implications for those farmers engaged in sea cucumber culture.