Differentiating between apparent and actual rates of H2O2 metabolism by isolated rat muscle mitochondria to test a simple model of mitochondria as regulators of H2O2 concentration

AbstractMitochondria are often regarded as a major source of reactive oxygen species (ROS) in animal cells, with H2O2 being the predominant ROS released from mitochondria; however, it has been recently demonstrated that energized brain mitochondria may act as stabilizers of H2O2 concentration (Starkov et al. [1]) based on the balance between production and the consumption of H2O2, the later of which is a function of [H2O2] and follows first order kinetics. Here we test the hypothesis that isolated skeletal muscle mitochondria, from the rat, are able to modulate [H2O2] based upon the interaction between the production of ROS, as superoxide/H2O2, and the H2O2 decomposition capacity. The compartmentalization of detection systems for H2O2 and the intramitochondrial metabolism of H2O2 leads to spacial separation between these two components of the assay system. This results in an underestimation of rates when relying solely on extramitochondrial H2O2 detection. We find that differentiating between these apparent rates found when using extramitochondrial H2O2 detection and the actual rates of metabolism is important to determining the rate constant for H2O2 consumption by mitochondria in kinetic experiments. Using the high rate of ROS production by mitochondria respiring on succinate, we demonstrate that net H2O2 metabolism by mitochondria can approach a stable steady-state of extramitochondrial [H2O2]. Importantly, the rate constant determined by extrapolation of kinetic experiments is similar to the rate constant determined as the [H2O2] approaches a steady state

Estimates of metabolic rate and major constituents of metabolic demand in fishes under field conditions: Methods, proxies, and new perspectives

Metabolic costs are central to individual energy budgets, making estimates of metabolic rate vital to understanding how an organism interacts with its environment as well as the role of species in their ecosystem. Despite the ecological and commercial importance of fishes, there are currently no widely adopted means of measuring field metabolic rate in fishes. The lack of recognized methods is in part due to the logistical difficulties of measuring metabolic rates in free swimming fishes. However, further development and refinement of techniques applicable for field-based studies on free swimming animals would greatly enhance the capacity to study fish under environmentally relevant conditions. In an effort to foster discussion in this area, from field ecologists to biochemists alike, we review aspects of energy metabolism and give details on approaches that have been used to estimate energetic parameters in fishes. In some cases, the techniques have been applied to field conditions; while in others, the methods have been primarily used on laboratory held fishes but should be applicable, with validation, to fishes in their natural environment. Limitations, experimental considerations and caveats of these measurements and the study of metabolism in wild fishes in general are also discussed. Potential novel approaches to FMR estimates are also presented for consideration. The innovation of methods for measuring field metabolic rate in free-ranging wild fish would revolutionize the study of physiological ecology

The Mitochondrial Contribution to Animal Performance, Adaptation, and Life-History Variation

We thank the National Science Foundation (grant IOS1738378 to W.R.H. and K.S.), SICB’s division of Comparative Physiology and Biochemistry and Comparative Endocrinology, the Company of Biologists, the Society of Experimental Biology, and the Canadian Society of Zoology for funding the symposium.  Peer reviewedPostprin

Effects of repeated daily acute heat challenge on the growth and metabolism of a cold-water stenothermal fish.

Temperature is an important environmental factor influencing fish physiology that varies both spatially and temporally in ecosystems. In small north-temperate lakes, cold water piscivores rely on nearshore prey; however, this region exceeds the optimal temperature of the foraging species during summer. To cope, piscivores make short excursions into the nearshore to feed and return to cold water to digest, but the physiological impacts of these repeated acute exposures to warm water are not well understood. We exposed juvenile lake trout ( ) to treatments where they were held at ≈10°C and exposed to either 17 or 22°C for 5 - 10 min daily for 53 days mimicking warm-water forays. Control fish, held at an average temperature of ≈10°C but not exposed to thermal variation, consumed more food and grew slightly faster than heat challenged fish, with no clear differences in body condition, hepatosomatic index, ventricle mass, or muscle concentrations of lactate dehydrogenase and cytochrome c oxidase. Aerobic metabolic rates measured at 10°C indicated that standard metabolic rates (SMR) were similar among treatments; however, fish that were repeatedly exposed to 17°C had higher maximum metabolic rates (MMR) and aerobic scopes (AS) than control fish and those repeatedly exposed to 22°C. There were no differences in MMR or AS between fish exposed to 22°C and control fish. These results suggest that although SMR of fish are robust to repeated forays into warmer environments, MMR displays plasticity, allowing fish to be less constrained aerobically in cold water after briefly occupying warmer waters

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

The accumulation, synthetic capacity and intertissue distribution of trimethylamine oxide in deep-sea fish and the cold adapted smelt (Osmerus mordax)

Trimethylamine oxide (TMAO) is common to most marine fishes; however, the role TMAO plays in the physiology of marine fish is not well understood. I have used two distinct TMAO accumulating fish 'types', deep-sea fish and smelt (Osmerus mordax), to compare differences in the levels, intertissue distribution and capacity for synthesis of TMAO in fish with high and low levels of TMAO. Several consistencies were found. The intertissue distribution of TMAO showed a trend of locomotory muscle > heart > liverliver ≅ kidney ≅ brain. Levels of trimethylamine oxidase, the enzyme required for TMAO synthesis, did not correlate with higher tissue TMAO content indicating that enhanced endogenous synthetic capacity is not responsible for elevated TMAO content. Finally, evidence for the active uptake of TMAO into striated muscle and the regulation of TMAO concentration in white muscle is presented, possibly due to some role TMAO plays in muscle function

The accumulation and metabolism of methylamine organic osmolytes in elasmobranch fishes

It is widely accepted that marine elasmobranch fishes accumulate the methylamine compounds trimethylamine oxide (TMAO), glycine-betaine (betaine) and sarcosine as osmolytes. The metabolism and accumulation of these compounds has received relatively little study in elasmobranchs, and as such the purpose of this thesis was to investigate aspects of methylamine metabolism, retention and accumulation. Experimental approaches range from multispecies comparisons to directed study of a single species (the winter skate, Leucoraja ocellata ) at different levels of organization from the whole animal down to subcellular components. -- Marine elasmobranchs and a euryhaline species in freshwater accumulate methylamines as the predominant intracellular non-urea organic osmolytes in muscle, whereas, freshwater species preferentially accumulated the β-amino acid taurine. All elasmobranch species examined in this thesis had measurable enzymatic capacity for betaine synthesis in the liver and strong correlation between hepatic betaine synthesizing enzymes and muscle betaine content was found. Only one species of the seven examined had measurable TMAO synthetic capacity and a phylogenetic explanation is proposed for the distribution of TMAO synthesis in elasmobranch fishes. -- In a detailed study of TMAO metabolism in the winter skate, it was concluded that the presence of flavin-containing monoxygenase activity does not indicate the capacity for the synthesis of TMAO. Winter skates lack measurable endogenous TMAO synthesis, apparently obtaining this compound in the diet, and maintain levels without feeding as a result of very low whole animal TMAO losses (<1% day -1 ). Betaine synthesis was also examined in detail in the winter skate, where the liver and kidney are the likely sites of synthesis. The enzymes of betaine synthesis from choline, choline dehydrogenase and betaine aldehyde dehydrogenase, are localized in the mitochondria, as is the case in other animals. Winter skates do not activate betaine synthetic capacity in response to food deprivation or accumulate betaine in the muscle when exposed to a hyperosmotic challenge; however, muscle betaine content increases when they are fed a high betaine diet, suggesting exogenous methylamine supply may be a key determinant in muscle methylamine accumulation in elasmobranchs