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Modification of mitochondrial content demands the synthesis of hundreds of proteins encoded by nuclear and mitochondrial genomes. The responsibility for coordination of this process falls to nuclear-encoded master regulators of transcription. DNA-binding proteins and coactivators integrate information from energy-sensing pathways and hormones to alter mitochondrial gene expression. In mammals, the signaling cascade for mitochondrial biogenesis can be described as follows: hormonal signals and energetic information are sensed by protein-modifying enzymes that in turn regulate the post-translational modification of transcription factors. Once activated, transcription-factor complexes form on promoter elements of many of the nuclear-encoded mitochondrial genes, recruiting proteins that remodel chromatin and initiate transcription. One master regulator in mammals, PGC-1α, is well studied because of its role in determining the metabolic phenotype of muscles, but also due to its importance in mitochondria-related metabolic diseases. However, relatively little is known about the role of this pathway in other vertebrates. These uncertainties raise broader questions about the evolutionary origins of the pathway and its role in generating the diversity in muscle metabolic phenotypes seen in nature. Key words: mitochondria, energetics, gene expression, hypoxia, cytochrome c oxidase, metabolism. La modification du contenu mitochondrial necessite la synthese de centaines de proteines encodees par les genomes nucleaire et mitochondrial, la coordination de ce processus etant assuree par des maitres regulateurs de la transcription codes par le genome nucleaire. Des proteines se liant a l'ADN et des coactivateurs integrent l'information provenant de voies de detection de l'energie et d'hormones pour ensuite modifier l'expression des genes mitochondriaux. Chez les mammiferes, la cascade de signalisation pour la biogenese mitochondriale peut etre decrite comme suit : des enzymes de modification de proteines detectent des signaux hormonaux et de l'information energetique et regulent la modification post-traduction des facteurs de transcription. Une fois actives, des complexes de facteurs de transcription se forment sur des elements promoteurs de nombreux genes mitochondriaux a encodage nucleaire, recrutant des proteines qui remodelent la chromatine et amorcent la transcription. Un des maitres regulateurs chez les mammiferes, le PGC-1α, fait l'objet de nombreuses etudes en raison de son role dans la determination du phenotype metabolique des muscles, mais aussi en raison de son importance dans les maladies metaboliques associees aux mitochondries. Les connaissances sur le role de cette voie chez d'autres vertebres sont toutefois limitees. Ces incertitudes soulevent des questions plus larges concernant les origines evolutives de cette voie et son role dans la production de la diversite de phenotypes metaboliques musculaires observee dans la nature. [Traduit par la Redaction] Mots-cles : mitochondrie, energetique, expression genique, hypoxie, cytochrome c oxydase, metabolisme., Introduction The ability of a cell to survive stress often hinges on its capacity to modulate energy production to match demands. Regulation of energy production can occur at numerous points, [...]

We used expression and reporter gene analysis to understand how changes in transcription factors impinge on mitochondrial gene expression during myogenesis of cultured murine myoblasts (C2C12 and So18). The mRNA levels for nuclear respiratory factor-1 (NRF-1) and NRF-2[alpha] increased 60% by the third day of myogenesis, whereas NRF-1 and NRF-2 reporter gene activity increased by fivefold over the same period. Although peroxisome proliferator activated receptor (PPAR[alpha]) mRNA levels increased almost 10-fold, the activity of a PPAR reporter was unchanged during myogenesis. The PPAR coactivator PPAR-[gamma] coactivator-1[alpha] (PGC1[alpha]), a master controller of mitochondrial biogenesis, was not expressed at detectable levels. However, the mRNA for both PGC1[alpha]-related coactivator and PGC1[beta] was abundant, with the latter increasing by 50% over 3 days of differentiation. We also conducted promoter analysis of the gene for citrate synthase (CS), a common mitochondrial marker enzyme. The proximal promoter (~2,100 bp) of the human CS lacks binding sites for PPAR, NRF-1, or NRF-2. Deletion mutants, a targeted mutation, and an Sp1 site-containing reporter construct suggest that changes in Sp1 regulation also participate in mitochondrial biogenesis during myogenesis. Because most mitochondrial genes are regulated by PPARs, NRF-1, and/or NRF-2, we conducted inhibitor studies to further support the existence of a distinct pathway for CS gene regulation in myogenesis. Although both LY-294002 (a phosphatidylinositol 3-kinase inhibitor) and SB-203580 (a p38-MAPK inhibitor) blocked myogenesis (as indicated by creatine phosphokinase activity), only SB-203580 prevented the myogenic increase in cytochrome oxidase activity, whereas only LY-294002 blocked the increase in CS (enzyme and reporter gene activities). Collectively, these studies help delineate the roles of some transcriptional regulators involved in mitochondrial biogenesis associated with myogenesis and underscore an import role for posttranscriptional regulation of transcription factor activity. citrate synthase; muscle; transcription factor; coactivator; metabolism

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, [H.sub.2].[O.sub.2] production, or state 4 proton leak respiration. These data suggest most mitochondrial qualitative properties are conserved across fiber types. However, there remained modest differences (~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. skeletal muscle; oxidative phosphorylation; reactive oxygen species; membrane fluidity; proton leak; energy metabolism

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 hypertension; mitochondria; mitochondrial deoxyribonucleic acid; angiotensin-converting enzyme inhibitor; spontaneously hypertensive rats; cytochrome oxidase; oxidative stress

The mechanisms by which cold exposure alters the expression of cytochrome-c oxidase (CCO) in the cold stenothermal Antarctic eelpout Pachycara brachycephalum and the eurythermal eelpout Zoarces viviparus were investigated. Results suggested that seasonal cold acclimation of Z. viviparus leads to an alteration in the relationship between transcription and translation or posttranslational processes. In the cold-adapted P. brachycephalum, the CCO expression was found to have similar characteristics with that of warm-acclimated confamilial species.

Cultured L6 myotubes were used to analyze the molecular mechanisms of glucagon-like peptide-1 (GLP-1) action in muscle. GLP-1 enhanced glycogen synthesis by 140% while stimulating CO2 production and lactate formation by 150%. In L6 myotubes transfected with pancreatic GLP-1 receptor, GLP-1 increased cAMP levels and inhibited glycogen synthesis. These findings suggested that the insulinomimetic effects of GLP-1 in L6 cells may be mediated by a receptor different from the GLP-1 receptor in the pancreas.

The differentiation of immortalized rodent cells such as C2C12 and 3T3 was analyzed to determine the relationship between mitochondrial ultrastructure, enzyme activity and gene expression. Cell differentiation of murine skeletal muscle-derived C2C12 and rat fibroblast-derived 3T3 cells induced distinct alterations in mitochondrial enzyme activity based on the location of the genes that encode the proteins. Furthermore, mitochondrial enzyme activity exhibited linear increases in C2C12 cells and exponential increases in 3T3 cells.

The influence of the calcium (Ca2+)-binding site of troponin C (cTnC) in striated muscle myofibrils on muscle contraction was investigated. Cloning and sequencing of cTnC isolated from cold-water salmonid, Onchorhynchus mykiss, revealed that the Ca2+-binding site is involved in the regulation of contraction by influencing the interaction of cTnC with other troponin proteins in vertebrate muscle.

Analyses of the influence of temperature and pH on rainbow trout myofibrillar Ca2+ sensitivity of myofibrillar ATPase show that temperature has little influence on Ca2+ sensitivity. Higher inherent Ca2+ sensitivity of poikilotherm myofibrils surmount desensitization due to temperature. Trout exhibits a greater inherent Ca2+ sensitivity than rat.

The scombroid fish (tunas, mackerels and billfish) display many examples of specializations in muscle energetics, including high activities of mitochondrial enzymes. We are investigating the origins of variation in the mitochondrial matrix enzyme citrate synthase (CS), a marker for mitochondrial content, between clades within this group and also between fiber-types within a species. Our approach includes i) comparative sequence analyses to determine if differences in activity can be attributed to structural differences in the enzyme ii) assessment of the relationship between CS activity and gene dosage and iii) an evaluation of the variation in transcriptional regulation of the CS gene. Preliminary analyses of the coding region of CS indicate that molecular evolution of the enzyme is not responsible for higher activity in the tunas. Within a species, differences in CS activity per gram tissue between fiber types are almost entirely accounted for by gene dosage. For example, swordfish white muscle and heater organ have a 30-fold difference in CS per gram tissue but only a 3-fold difference in CS per CS gene. Conversely, inter-species differences in CS activity (approximately 2-3 fold between homologous tissues) remain when gene dosage is accounted for. This indicates that inter-tissue differences in CS activity are mainly due to constitutive expression while clade-specific differences are the result of evolutionary variation in regulatory control. Between species, CS mRNA levels do not correlate with CS enzyme activity, suggesting little transcriptional regulation. The post-transcriptional mechanisms by which variations in CS content are mediated are currently under investigation. Funded by NSERC and NMFS.

Sexually mature male bluegill sunfish (Lepomis macrochirus) display two distinct life histories. Some individuals delay reproduction until ca. 7 years of age, build nests and actively court females. Other males mature at a younger age and smaller size, and adopt a cuckolder tactic. We hypothesized that these smaller 'sneaker' males would compensate for their size disadvantage at the gametic level. We examined the motility and energy stores of sperm from males of both life-history strategies. During the first 15 seconds following activation, sperm from sneaker males tended to swim faster than sperm from parental males. This greater swimming speed disappeared by ca. 45 seconds, at which point the sperm of parental males swam faster. Higher initial swimming speeds of sperm from sneaker males likely resulted from the sperm's higher starting ATP levels. The rate at which ATP was utilized by sperm did not, however, differ between the two reproductive tactics, suggesting the existence of a trade-off between sperm swimming speed and swimming duration. Our data suggest that sneaker males may be compensating for their size disadvantage by producing physiologically superior spermatozoa.

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We investigated if seasonal changes in rainbow trout muscle energetics arise in response to seasonal changes in erythrocyte properties. We assessed if skeletal muscle mitochondrial enzymes changed (1) acutely in response to changes in erythrocyte abundance, or (2) seasonally when we altered the age profile of erythrocytes. Rainbow trout were treated with pheynylhydrazine, causing a 75% reduction in hematocrit within 4 days. After erythropoiesis had returned hematocrit to normal, treated and control fish were subjected to a seasonal cold acclimation regime to assess the impact of erythrocyte age on skeletal muscle remodeling. Anemia (i.e. phenylhydrazine treatment) did not alter the specific activities (U g-1 tissue) of mitochondrial enzymes in white or red muscle. Anemic pretreatment did not alter the normal pattern of cold-induced mitochondrial proliferation in skeletal muscle, suggesting erythrocyte age was not an important influence on seasonal remodeling of muscle. Anemia and cold acclimation both induced a 25-30% increase in relative ventricular mass. The increase in relative ventricular mass with phenylhydrazine treatment was accompanied by a 35% increase in DNA content (mg DNA per ventricle), suggesting an increase in number of cells. In contrast, the increase in ventricular mass with cold temperature acclimation occurred without a change in DNA content (mg DNA per ventricle), suggesting an increase in cell size. Despite the major increases in relative ventricular mass, neither anemia nor seasonal acclimation had a major influence on the specific activities of a suite of mitochondrial enzymes in heart. Collectively, these studies argue against a role for erythrocyte dynamics in seasonal adaptive remodeling of skeletal muscle energetics. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Examines the induction of thermotolerance by anoxia in the locust flight system. Effects of heat shock and anoxia on cellular responses; Use of electrophysiology test; Respiration of isolated mitochondria; Rates od substrate oxidation.

Presents a study which examined the effects of cell age on metabolism in the nucleated red blood cells of rainbow trout. Methodology; Results of the study; Discussion.

Abstract: Billfishes (Scombroidei) and tunas (Scombridae), both considered part of the suborder Scombroidei, have long been studied by biologists largely because of their remarkable physiological and anatomical muscular adaptations associated with regional endothermy and continuous swimming. These attributes, combined with analyses of other morphological and molecular data, have led to a general perception that tunas and billfishes are close relatives, though this hypothesis has been vigorously debated. Using Bayesian phylogenetic analysis of nine mitochondrial and three nuclear loci (>7000bp), we show that billfishes are only distantly related to tunas, but rather share strong evolutionary affinities with flatfishes (Pleuronectiformes) and jacks (Carangidae). This phylogenetic relationship is striking because of the marked variation in phenotype and niche across these trans-ordinal groups of fishes. Billfishes and flatfishes have each evolved radically divergent morphological and physiological features: elongated bills and extraocular heater organs in billfishes, and cranial asymmetry with complete eye migration during ontogenetic development in flatfishes. Despite this divergence, we identify synapomorphies consistent with the hypothesis of a common billfish/flatfish/jack ancestor. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Peter Hochachka was an early pioneer in the field of comparative biochemistry. He passed away in 2002 after 4 decades of research in the discipline. To celebrate his contributions and to coincide with what would have been his 80th birthday, a group of his former students organized a symposium that ran as a satellite to the 2017 Canadian Society of Zoologists annual meeting in Winnipeg, Manitoba (Canada). This Special Issue of CBP brings together manuscripts from symposium attendees and other authors who recognize the role Peter played in the evolution of the discipline. In this article, the symposium organizers and guest editors look back on his career, celebrating his many contributions to research, acknowledging his role in training of generations of graduate students and post-doctoral fellows in comparative biochemistry and physiology. [ABSTRACT FROM AUTHOR]

Remodeling the muscle metabolic machinery in mammals in response to energetic challenges depends on the energy sensor AMP-activated protein kinase (AMPK) and its ability to phosphorylate PPAR γ coactivator 1 α (PGC1α), which in turn coactivates metabolic genes through direct and indirect association with DNA-binding proteins such as the nuclear respiratory factor 1 (NRF1) (Wu et al., 1999). The integrity of this axis in fish is uncertain because PGC1α i) lacks the critical Thr 177 targeted by AMPK and ii) has mutations that may preclude binding NRF1. In this study we found no evidence that AMPK regulates mitochondrial gene expression through PGC1α in zebrafish and goldfish. AICAR treatment of zebrafish blastula cells increased phosphorylation of AMPK and led to changes in transcript levels of the AMPK targets mTOR and hexokinase 2. However, we saw no increases in mRNA levels for genes associated with mitochondrial biogenesis, including PGC1α, NRF1, and COX7C, a cytochrome c oxidase subunit. Further, AMPK phosphorylated mammalian peptides of PGC1α but not the corresponding region of zebrafish or goldfish in vitro . In vivo cold acclimation of goldfish caused an increase in mitochondrial enzymes, AMP and ADP levels, however AMPK phosphorylation decreased. In fish, the NRF1-PGC1α axis may be disrupted due to insertions in fish PGC1α orthologs within the region that serves as NRF1 binding domain in mammals. Immunocopurification showed that recombinant NRF1 protein binds mammalian but not fish PGC1α. Collectively, our studies suggest that fish have a disruption in the AMPK-PGC1α-NRF1 pathway due to structural differences between fish and mammalian PGC1α. [ABSTRACT FROM AUTHOR]

Previous studies have shown evidence of genomic incompatibility and mitochondrial enzyme dysfunction in hybrids of bluegill ( Lepomis macrochirus Rafinesque) and pumpkinseed ( Lepomis gibbosus Linnaeus) sunfish (Davies et al., 2012 Physiol. Biochem. Zool. 85, 321–331). We assessed if these differences in mitochondria had an impact on metabolic processes that depend on mitochondrial function, specifically hypoxia tolerance and recovery from burst exercise. Bluegill, pumpkinseed, and their hybrids showed no difference in the critical oxygen tension (P crit ) and no differences in tissue metabolites measured after exposure to 10% O 2 for 30 min. In contrast, loss of equilibrium (LOE) measurements showed that hybrids had reduced hypoxia tolerance and lacked the size-dependence in hypoxia tolerance seen in the parental species. However, we found no evidence of systematic differences in metabolite levels in fish after LOE. Furthermore, there were abundant glycogen reserves at the point of loss of equilibrium. The three genotypes did not differ in metabolite status at rest, showed an equal disruption at exhaustion, and similar metabolic profiles throughout recovery. Thus, we found no evidence of a mitochondria dysfunction in hybrids, and mitochondrial differences and oxidative metabolism did not explain the variation in hypoxia tolerance seen in the hybrid and two parental species. [ABSTRACT FROM AUTHOR]

Modification of mitochondrial content demands the synthesis of hundreds of proteins encoded by nuclear and mitochondrial genomes. The responsibility for coordination of this process falls to nuclear-encoded master regulators of transcription. DNA-binding proteins and coactivators integrate information from energy-sensing pathways and hormones to alter mitochondrial gene expression. In mammals, the signaling cascade for mitochondrial biogenesis can be described as follows: hormonal signals and energetic information are sensed by protein-modifying enzymes that in turn regulate the post-translational modification of transcription factors. Once activated, transcription-factor complexes form on promoter elements of many of the nuclear-encoded mitochondrial genes, recruiting proteins that remodel chromatin and initiate transcription. One master regulator in mammals, PGC-1α, is well studied because of its role in determining the metabolic phenotype of muscles, but also due to its importance in mitochondria-related metabolic diseases. However, relatively little is known about the role of this pathway in other vertebrates. These uncertainties raise broader questions about the evolutionary origins of the pathway and its role in generating the diversity in muscle metabolic phenotypes seen in nature. [ABSTRACT FROM AUTHOR]

Abstract: Many cancer cells have an unusual ability to grow in hypoxia, but the origins of this metabolic phenotype remain unclear. We compared the metabolic phenotypes of three common prostate cancer cell models (LNCaP, DU145, PC3), assessing energy metabolism, metabolic gene expression, and the response to various culture contexts (in vitro and xenografts). LNCaP cells had a more oxidative phenotype than PC3 and DU145 cells based upon respiration, lactate production, [ATP], metabolic gene expression, and sensitivity of these parameters to hypoxia. PC3 and DU145 cells possessed similar Complex II and mtDNA levels, but lower Complex III and IV activities, and were unresponsive to dinitrophenol or dichloroacetate, suggesting that their glycolytic phenotype is due to mitochondrial dysfunction rather than regulation. High passage under normoxia converted LNCaP from oxidative to glycolytic cells (based on respiration and lactate production), and altered metabolic gene expression. Though LNCaP-derived cells differed from the parental line in mitochondrial enzyme activities, none differed in mitochondrial content (assessed as cardiolipin levels). When LNCaP-derived cells were grown as xenografts in immunodeficient mice, there were elements of a hypoxic response (e.g., elevated VEGF mRNA) but line-specific changes in expression of select glycolytic, mitochondrial and fatty acid metabolic genes. Low oxygen in vitro did not influence the mRNA levels of SREBP axis, nor did it significantly alter triglyceride production in any of the cell lines suggesting that the pathway of de novo fatty acid synthesis is not directly upregulated by hypoxic conditions. Collectively, these studies demonstrate important differences in the metabolism of these prostate cancer models. Such metabolic differences would have important ramifications for therapeutic strategies involving metabolic targets. [Copyright &y& Elsevier]