Comparative biochemistry of cytochrome c oxidase in animals.

Cytochrome c oxidase (COX), the terminal enzyme of the electron transport system, is central to aerobic metabolism of animals. Many aspects of its structure and function are highly conserved, yet, paradoxically, it is also an important model for studying the evolution of the metabolic phenotype. In this review, part of a special issue honouring Peter Hochachka, we consider the biology of COX from the perspective of comparative and evolutionary biochemistry. The approach is to consider what is known about the enzyme in the context of conventional biochemistry, but focus on how evolutionary researchers have used this background to explore the role of the enzyme in biochemical adaptation of animals. In synthesizing the conventional and evolutionary biochemistry, we hope to identify synergies and future research opportunities. COX represents a rare opportunity for researchers to design studies that span the breadth of biology: molecular genetics, protein biochemistry, enzymology, metabolic physiology, organismal performance, evolutionary biology, and phylogeography.

Fiber-type differences in muscle mitochondrial profiles.

Although striated muscles differ in mitochondrial content, the extent of fiber-type specific mitochondrial specializations is not well known. To address this issue, we compared mitochondrial structural and functional properties in red muscle (RM), white muscle (WM), and cardiac muscle of rainbow trout. Overall preservation of the basic relationships between oxidative phosphorylation complexes among fiber types was confirmed by kinetic analyses, immunoblotting of native holoproteins, and spectroscopic measurements of cytochrome content. Fiber-type differences in mitochondrial properties were apparent when parameters were expressed per milligram mitochondrial protein. However, the differences diminished when expressed relative to cytochrome oxidase (COX), possibly a more meaningful denominator than mitochondrial protein. Expressed relative to COX, there were no differences in oxidative phosphorylation enzyme activities, pyruvate-based respiratory rates, H2O2 production, or state 4 proton leak respiration. These data suggest most mitochondrial qualitative properties are conserved across fiber types. However, there remained modest differences ( approximately 50%) in stoichiometries of selected enzymes of the Krebs cycle, beta-oxidation, and antioxidant enzymes. There were clear differences in membrane fluidity (RM > cardiac, WM) and proton conductance (H+/min/mV/U COX: WM > RM > cardiac). The pronounced differences in mitochondrial content between fiber types could be attributed to a combination of differences in myonuclear domain and modest effects on the expression of nuclear- and mitochondrially encoded respiratory genes. Collectively, these studies suggest constitutive pathways that transcend fiber types are primarily responsible for determining most quantitative and qualitative properties of mitochondria.

Origins and consequences of mitochondrial decline in nucleated erythrocytes.

Cellular aging in nucleated erythrocytes from lower vertebrates is accompanied by losses in mitochondria but it remains unclear (i) how these losses accrue (ii) if these changes alter energetics and (iii) whether such changes increase the propensity for apoptosis. We addressed these questions using trout erythrocytes that were separated into age classes using inherent differences in buoyant density. The oldest cells showed a profound decline in mtDNA transcripts, due to reductions in both transcription (90% decline in total RNA) and mtDNA copy number (35%). No alterations in the ratio of 16S rRNA to COX I mRNA were detected, nor was there an accumulation of unprocessed mtDNA transcripts. While older cells had reduced basal respiration, there were no changes in mitochondrial enzymes stoichiometries, tissue ATP levels or dinitrophenol-induced (maximal) respiration rates. Apoptosis could not be induced in either whole blood, young or old erythrocytes by pro-oxidants, mitochondrial inhibitors or staurosporine. In contrast, cyclosporin A (CsA) caused caspase 3 activation, DNA laddering and LDH leakage, but only in young cells. Both CsA and a combination of azide, oligomycin and dinitrophenol cause mitochondrial depolarization and caspase 9 activation, but only CsA induced caspase 3 and apoptosis. Caspase inhibitor studies support the conclusion that mitochondrial changes may accompany CsA-induced cell death, but are not essential in its progression. While pifithrin failed to induce cell death, it enhanced the effects of CsA, implicating a role for p53. Collectively, these studies suggest that the mitochondrial changes with aging do not compromise cellular function, although trout erythrocytes can initiate apoptosis by non-mitochondrial pathways.

Bioenergetic remodeling of heart during treatment of spontaneously hypertensive rats with enalapril.

We used spontaneously hypertensive rats to study remodeling of cardiac bioenergetics associated with changes in blood pressure. Blood pressure was manipulated with aggressive antihypertensive treatment combining low dietary salt and the angiotensin-converting enzyme inhibitor enalapril. Successive cycles of 2 wk on, 2 wk off treatment led to rapid, reversible changes in left ventricular (LV) mass (30% change in <10 days). Despite changes in LV mass, specific activities of bioenergetic (cytochrome-c oxidase, citrate synthase, lactate dehydrogenase) and reactive oxygen species (ROS) (total cellular superoxide dismutase) enzymes were actively maintained within relatively narrow ranges regardless of treatment duration, organismal age, or transmural region. Although enalapril led to parallel declines in mitochondrial enzyme content and ventricular mass, total ventricular mtDNA content was unaffected. Altered enzymatic content occurred without significant changes in relevant mRNA and protein levels. Transcript levels of gene products involved in mtDNA maintenance (Tfam), mitochondrial protein degradation (LON protease), fusion (fuzzy onion homolog), and fission (dynamin-like protein, synaptojanin-2alpha) were also unchanged. In contrast, enalapril-mediated ventricular and mitochondrial remodeling was accompanied by a twofold increase in specific activity of catalase, an indicator of oxidative stress, suggesting that rapid cardiac adaptation is accompanied by tight regulation of mitochondrial enzyme activities and increased ROS production.

Efflux of T4 from the in situ perfused liver of rainbow trout: effect of T4, dithiothreitol and cysteine in the perfusate.

A Cortland saline-perfused rainbow trout (Oncorhynchus mykiss) liver model was used to study aspects of T4 efflux from the intact organ system. There was a consistent efflux of T4 in the absence of T4 in the perfusate, and the T4 efflux was increased in the presence of T4 in the perfusate, but the efflux was not T4-dose dependent. The addition of the thiol-containing compound dithiothreitol (DTT, 2 mM) to the perfusate had no significant effect on the flux of T4 from the liver, whereas the addition of cysteine (2 mM), a thiol-containing amino acid suppressed T4 efflux. The results are consistent with the known mechanisms of thyroid hormone trafficking across cell membranes, and suggest that organ systems, such as the liver, may act as a major reserve of hormone, thus participating in plasma thyroid hormone homeostasis.

Allometric scaling of RNA, DNA, and enzyme levels: an intraspecific study.

The activities of oxidative and glycolytic enzymes show body size-dependent relationships across a wide variety of taxa; however, the mechanistic basis remains unknown. We sampled white epaxial muscle from rainbow trout (Oncorhynchus mykiss) spanning a 100-fold range in body mass. We measured activities of enzymes from aerobic and anaerobic metabolic pathways, RNA [total RNA and mRNA, pyruvate kinase (PK), citrate synthase (CS), and MyoD mRNA], and total DNA. Total RNA and DNA showed a biphasic relationship with body size, with a break point occurring after fish reached 1 yr of age. In contrast, total RNA/total DNA was constant across the entire size range. Neither CS activity nor CS mRNA levels scaled with body mass. PK activity and PK mRNA levels increased in parallel in yearling fish only (r(2) = 0.91, P < 0.01). This suggests that although PK expression is transcriptionally regulated in yearlings, the molecular mechanisms regulating expression change with growth and age. This was supported by a positive correlation between MyoD and PK mRNA levels (r(2) = 0.17, P < 0.05).

Evaluation of thyroid hormone economy in elasmobranch fishes, with measurements of hepatic 5'-monodeiodinase activity in wild dogfish.

This paper reviews the current understanding of thyroid hormone economy and homeostasis in elasmobranch fishes and considers those measures of the activity of the hypothalamic-pituitary gland-thyroid gland-peripheral tissue axis that are necessary for adequate assessment of thyroid hormone physiology. In particular, we focus on the value of measuring hepatic 5'-monodeiodinase (5'-MDA) activity as an indicator of the animal's cellular production rate of the active thyroid hormone, triiodo-L-thyronine (T(3)). We also examine the characteristics of hepatic 5'-MDA activity, in vitro, in adult female dogfish (Squalus acanthias) collected from Passamaquoddy Bay, New Brunswick, Canada, and in the embryos that they were carrying. T(3) production from T(4) by hepatic homogenates in vitro was time- and temperature-dependent, and was enhanced by the presence of a thiol donor. Michaelis constant (K(m)) and maximum reaction velocity (V(max)) values were 3.8 x 10(-7) M and 0.29 nM T(3)/mg protein/hr, respectively. The inclusion of trimethylamine-N-oxide (TMAO) or a mixture of urea, TMAO, betaine and sarcosine significantly enhanced T(3) production. Hepatic 5'-MDA activity was depressed in fish fasted for 7 days. J. Exp. Zool. 284:492-499, 1999.

Interactions between bioenergetics and mitochondrial biogenesis.

We studied the interaction between energy metabolism and mitochondrial biogenesis during myogenesis in C2C12 myoblasts. Metabolic rate was nearly constant throughout differentiation, although there was a shift in the relative importance of glycolytic and oxidative metabolism, accompanied by increases in pyruvate dehydrogenase activation state and total activity. These changes in mitochondrial bioenergetic parameters observed during differentiation occurred in the absence of a hypermetabolic stress. A chronic (3 day) energetic stress was imposed on differentiated myotubes using sodium azide to inhibit oxidative metabolism. When used at low concentrations, azide inhibited more than 70% of cytochrome oxidase (COX) activity without changes in bioenergetics (either lactate production or creatine phosphorylation) or mRNA for mitochondrial enzymes. Higher azide concentrations resulted in changes in bioenergetic parameters and increases in steady state COX II mRNA levels. Azide did not affect mtDNA copy number or mRNA levels for other mitochondrial transcripts, suggesting azide affects stability, rather than synthesis, of COX II mRNA. These results indicate that changes in bioenergetics can alter mitochondrial genetic regulation, but that mitochondrial biogenesis accompanying differentiation occurs in the absence of hypermetabolic challenge.

Regulation of muscle mitochondrial design.

Mitochondria are responsible for the generation of ATP to fuel muscle contraction. Hypermetabolic stresses imposed upon muscles can lead to mitochondrial proliferation, but the resulting mitochondria greatly resemble their progenitors. During the mitochondrial biogenesis that accompanies phenotypic adaptation, the stoichiometric relationships between functional elements are preserved through shared sensitivities of respiratory genes to specific transcription factors. Although the properties of muscle mitochondria are generally thought to be highly conserved across species, there are many examples of mitochondrial differences between muscle types, species and developmental states and even within single cells. In this review, we discuss (1) the nature and regulation of gene families that allow coordinated expression of genes for mitochondrial products and (2) the regulatory mechanisms by which mitochondrial differences can arise over physiological and evolutionary time.

The application of an in vitro perfused liver preparation to examine the effects of epinephrine and bovine thyroid-stimulating hormone on triiodo-L-thyronine release from the liver of rainbow trout (Oncorhynchus mykiss).

An isolated, perfused rainbow trout liver preparation was developed to investigate the action of nonthyroidal hormones on hepatic thyroid hormone metabolism. Several assessments were made of the stability and viability of the preparations under a range of conditions, including measures of lactate dehydrogenase flux and tissue ATP and glycogen content, all of which indicated that the perfused liver was stable for the 60-min perfusion period. Moreover, the liver preparations were responsive to an epinephrine challenge and, throughout the series of experiments, sustained hepatic glucose release. Triiodo-L-thyronine (T3) flux from the liver preparation was significantly increased by the provision of thyroxine (T4) substrate. Epinephrine and bovine thyroid stimulating hormone (TSH) were perfused alone and in combination with T4 to evaluate the effect of these hormones on T3 flux from the liver. Both epinephrine and TSH significantly enhanced hepatic T3 flux in the absence of T3 substrate, but neither had an additional effect on T3 flux when perfused in combination with T4. The results of the study suggest that a relationship exists between the circulating levels of nonthyroid hormones and peripheral thyroid hormone metabolism that may be receptor-mediated.

Direct effects of 3,5,3'-triiodothyronine and 3,5-diiodothyronine on mitochondrial metabolism in the goldfish Carassius auratus.

Direct effects of 3,5,3'-triiodothyronine (T3) and 3,5-diiodothyronine (T2) on the metabolism of the goldfish (Carassius auratus) were assessed using mitochondria isolated from liver and red muscle. Following a 5-min incubation with either T3 or T2, the oxidation rates of substrates involved in amino acid and carbohydrate metabolism and lipid catabolism were measured. State 3 oxidation of pyruvate was significantly higher for liver mitochondria treated with T2 and for red muscle mitochondria incubated with T3 when compared to control mitochondria. Rapid elevation of state 3 rates of substrate oxidation by thyroid hormones may be important in mediating diurnal changes in mitochondrial metabolism. Significant increases in liver and red muscle mitochondrial state 4 rates were also observed for pyruvate in T2- and T3-treated mitochondria and for glutamate in T3-treated mitochondria.