Capacity Of Respiratory Chain Research Articles

Maintained respiratory chain capacity in mice with severely reduced levels of mitochondrial respirasomes

Maintained respiratory chain capacity in mice with severely reduced levels of mitochondrial respirasomes

Transcriptomic analysis reveals mitochondrial pathways associated with distinct adolescent behavioral phenotypes and stress response

Adolescent individuals exhibit great variability in cortical dynamics and behavioral outcomes. The developing adolescent brain is highly sensitive to social experiences and environmental insults, influencing how personality traits emerge. A distinct pattern of mitochondrial gene expression in the prefrontal cortex (PFC) during adolescence underscores the essential role of mitochondria in brain maturation and the development of mental illnesses. Mitochondrial features in certain brain regions account for behavioral differences in adulthood. However, it remains unclear whether distinct adolescent behavioral phenotypes and the behavioral consequences of early adolescent stress exposure in rats are accompanied by changes in PFC mitochondria-related genes and mitochondria respiratory chain capacity. We performed a behavioral characterization during late adolescence (postnatal day, PND 47–50), including naïve animals and a group exposed to stress from PND 31–40 (10 days of footshock and 3 restraint sessions) by z-normalized data from three behavioral domains: anxiety (light–dark box tests), sociability (social interaction test) and cognition (novel-object recognition test). Employing principal component analysis, we identified three clusters: naïve with higher-behavioral z-score (HBZ), naïve with lower-behavioral z-score (LBZ), and stressed animals. Genome-wide transcriptional profiling unveiled differences in the expression of mitochondria-related genes in both naïve LBZ and stressed animals compared to naïve HBZ. Genes encoding subunits of oxidative phosphorylation complexes were significantly down-regulated in both naïve LBZ and stressed animals and positively correlated with behavioral z-score of phenotypes. Our network topology analysis of mitochondria-associated genes found Ndufa10 and Cox6a1 genes as central identifiers for naïve LBZ and stressed animals, respectively. Through high-resolution respirometry analysis, we found that both naïve LBZ and stressed animals exhibited a reduced prefrontal phosphorylation capacity and redox dysregulation. Our findings identify an association between mitochondrial features and distinct adolescent behavioral phenotypes while also underscoring the detrimental functional consequences of adolescent stress on the PFC.

Preserved respiratory chain capacity and physiology in mice with profoundly reduced levels of mitochondrial respirasomes

The mammalian respiratory chain complexes I, III2, and IV (CI, CIII2, and CIV) are critical for cellular bioenergetics and form a stable assembly, the respirasome (CI-CIII2-CIV), that is biochemically and structurally well documented. The role of the respirasome in bioenergetics and the regulation of metabolism is subject to intense debate and is difficult to study because the individual respiratory chain complexes coexist together with high levels of respirasomes. To critically investigate the invivo role of the respirasome, we generated homozygous knockin mice that have normal levels of respiratory chain complexes but profoundly decreased levels of respirasomes. Surprisingly, the mutant mice are healthy, with preserved respiratory chain capacity and normal exercise performance. Our findings show that high levels of respirasomes are dispensable for maintaining bioenergetics and physiology in mice but raise questions about their alternate functions, such as those relating to the regulation of protein stability and prevention of age-associated protein aggregation.

Brain mitochondrial diversity and network organization predict anxiety-like behavior in male mice

The brain and behavior are under energetic constraints, limited by mitochondrial energy transformation capacity. However, the mitochondria-behavior relationship has not been systematically studied at a brain-wide scale. Here we examined the association between multiple features of mitochondrial respiratory chain capacity and stress-related behaviors in male mice with diverse behavioral phenotypes. Miniaturized assays of mitochondrial respiratory chain enzyme activities and mitochondrial DNA (mtDNA) content were deployed on 571 samples across 17 brain areas, defining specific patterns of mito-behavior associations. By applying multi-slice network analysis to our brain-wide mitochondrial dataset, we identified three large-scale networks of brain areas with shared mitochondrial signatures. A major network composed of cortico-striatal areas exhibited the strongest mitochondria-behavior correlations, accounting for up to 50% of animal-to-animal behavioral differences, suggesting that this mito-based network is functionally significant. The mito-based brain networks also overlapped with regional gene expression and structural connectivity, and exhibited distinct molecular mitochondrial phenotype signatures. This work provides convergent multimodal evidence anchored in enzyme activities, gene expression, and animal behavior that distinct, behaviorally-relevant mitochondrial phenotypes exist across the male mouse brain.

Early decreased respiratory chain capacity in resuscitated experimental sepsis is a major contributor to lactate production.

Increased plasma lactate levels in patients with sepsis may be due to insufficient oxygen delivery, but mitochondrial dysfunction or accelerated glycolysis may also contribute. We studied the effect of the latter on muscle metabolism by using microdialysis in a sepsis model with sustained oxygen delivery and decreased energy consumption or mitochondrial blockade. Pigs were subjected to continuous E. coli infusion (sepsis group, n = 12) or saline infusion (sham group, n = 4) for 3 hours (h). Protocolized interventions were applied to normalize the oxygen delivery and blood pressure. Microdialysis catheters were used to monitor muscle metabolism (naïve). The same catheters were used to block the electron transport chain with cyanide or the Na+/K+-ATPase inhibitor, ouabain locally. All pigs in the sepsis group had positive blood cultures and a Sequential Organ Failure Assessment (SOFA) score increase by at least 2, fulfilling the sepsis criteria. Plasma lactate was higher in the sepsis group than in the sham group (p < 0.001), whereas muscle glucose was lower in the sepsis group (p < 0.01). There were no changes in muscle lactate levels over time but lactate to pyruvate ratio (LPR) was elevated in the Sepsis vs. the Sham group (p < 0.05). Muscle lactate, LPR, and glutamate levels were higher in the sepsis group than in the sham group in the cyanide catheters (p < 0.001, all comparisons) and did not normalize in the former group. In this experimental study on resuscitated sepsis, we observed increased aerobic metabolism and preserved mitochondrial function. Sepsis and electron transport chain inhibition led to increased LPR, suggesting a decreased mitochondrial reserve capacity in early sepsis.

STF-083010 an inhibitor of IRE1α endonuclease activity affects mitochondrial respiration and generation of mitochondrial membrane potential

STF-083010 is an inhibitor of endonuclease activity of inositol requiring-enzyme 1α (IRE1α) that is involved in activation of IRE1α-XBP1 axis of the unfolded protein response after ER stress. STF-083010 was tested as a possible antitumor agent in some previous studies exhibiting the ability either to induce death of tumour cells or to increase sensitivity of tumours cells to other neoplastic agents. STF-083010 exhibits also hepatoprotective effects in different models of liver injury and hepatic steatohepatitis. We have shown that STF-083010 has significant impact on mitochondrial functions that is not dependent on the way of STF-083010 application. We have observed that STF-083010 decrease of both maximal respiration (representing maximal electron transfer capacity of mitochondrial respiratory chain) and spare respiratory capacity after either incubation of the SH-SY5Y cells with STF-083010 or direct addition of STF-083010 to the respiration medium. In addition, we have documented impact of STF-083010 on generation of mitochondrial membrane potential (ΔΨm) that could be a result of decreased mitochondrial substrate level phosphorylation. Finally, increased sensitivity of ΔΨm to uncoupler in the presence of STF-083010 was documented. Our results indicate that STF-083010 has important impact on mitochondrial functions independently of its ability to inhibit endonuclease activity of IRE1α that is involved in activation of IRE1α-XBP1 axis of the unfolded protein response after ER stress. The impact of STF-083010 on mitochondrial functions could be associated with its possible off-target effect.

Enhanced hepatic respiratory capacity and altered lipid metabolism support metabolic homeostasis during short-term hypoxic stress

BackgroundTissue hypoxia is a key feature of several endemic hepatic diseases, including alcoholic and non-alcoholic fatty liver disease, and organ failure. Hypoxia imposes a severe metabolic challenge on the liver, potentially disrupting its capacity to carry out essential functions including fuel storage and the integration of lipid metabolism at the whole-body level. Mitochondrial respiratory function is understood to be critical in mediating the hepatic hypoxic response, yet the time-dependent nature of this response and the role of the respiratory chain in this remain unclear.ResultsHere, we report that hepatic respiratory capacity is enhanced following short-term exposure to hypoxia (2 days, 10% O2) and is associated with increased abundance of the respiratory chain supercomplex III2+IV and increased cardiolipin levels. Suppression of this enhanced respiratory capacity, achieved via mild inhibition of mitochondrial complex III, disrupted metabolic homeostasis. Hypoxic exposure for 2 days led to accumulation of plasma and hepatic long chain acyl-carnitines. This was observed alongside depletion of hepatic triacylglycerol species with total chain lengths of 39-53 carbons, containing palmitic, palmitoleic, stearic, and oleic acids, which are associated with de novo lipogenesis. The changes to hepatic respiratory capacity and lipid metabolism following 2 days hypoxic exposure were transient, becoming resolved after 14 days in line with systemic acclimation to hypoxia and elevated circulating haemoglobin concentrations.ConclusionsThe liver maintains metabolic homeostasis in response to shorter term hypoxic exposure through transient enhancement of respiratory chain capacity and alterations to lipid metabolism. These findings may have implications in understanding and treating hepatic pathologies associated with hypoxia.

The Relationship between Mitochondrial Reactive Oxygen Species Production and Mitochondrial Energetics in Rat Tissues with Different Contents of Reduced Coenzyme Q.

We investigated the relationship between mitochondrial production of reactive oxygen species (ROS) and mitochondrial energetics in various rat tissues with different contents of the reduced coenzyme Q (Q) pool (Q9 + Q10). Our results indicate that similar to the tissue level, mitochondrial H2O2 release under nonphosphorylating conditions was strongly dependent on the amount of the reduced Q pool. Namely, in brain and lung mitochondria, less H2O2 release corresponded to a less reduced Q pool, while in liver and heart mitochondria, higher H2O2 release corresponded to a more reduced Q pool. We can conclude that the differences observed in rat tissues in the size of the reduced Q pool reflect different levels of ROS production and hence may reflect different demands for reduced Q as an antioxidant. Moreover, differences in mitochondrial H2O2 release were observed in different types of rat mitochondria during the oxidation of succinate (complex II substrate), malate plus glutamate (complex I substrate), and their mixture under phosphorylating and nonphosphorylating conditions. Our results indicate the existence of a tissue-specific maximum respiratory chain capacity in ROS production, possibly related to the membrane potential-mediated control of oxidative phosphorylation. We propose the use of a new parameter for the study of isolated mitochondria, RCRROS, the ratio between the formation of mitochondrial ROS under nonphosphorylating and phosphorylating conditions, which represents the maximum factorial increase in mitochondrial ROS formation that can be achieved after all ADP is phosphorylated.

MCL-1Matrix maintains neuronal survival by enhancing mitochondrial integrity and bioenergetic capacity under stress conditions

Mitochondria play a crucial role in neuronal survival through efficient energy metabolism. In pathological conditions, mitochondrial stress leads to neuronal death, which is regulated by the anti-apoptotic BCL-2 family of proteins. MCL-1 is an anti-apoptotic BCL-2 protein localized to mitochondria either in the outer membrane (OM) or inner membrane (Matrix), which have distinct roles in inhibiting apoptosis and promoting bioenergetics, respectively. While the anti-apoptotic role for Mcl1 is well characterized, the protective function of MCL-1 Matrix remains poorly understood. Here, we show MCL-1OM and MCL-1Matrix prevent neuronal death through distinct mechanisms. We report that MCL-1Matrix functions to preserve mitochondrial energy transduction and improves respiratory chain capacity by modulating mitochondrial oxygen consumption in response to mitochondrial stress. We show that MCL-1Matrix protects neurons from stress by enhancing respiratory function, and by inhibiting mitochondrial permeability transition pore opening. Taken together, our results provide novel insight into how MCL-1Matrix may confer neuroprotection under stress conditions involving loss of mitochondrial function.

The role of mitochondrial dysfunction in the pathophysiology of ICU-acquired weakness

Methods Muscle biopsies were obtained from critically ill patients with severe ICUAW (defined as MRC score < 24 on two separate tests) and from otherwise healthy controls undergoing elective hip replacement. 50-100mg of the native sample was homogenized by a technique which perserves mitochondria in their cytosolic context [3]. Oxygen consumption was measured by high-resolution respirometry (Oxygraph-2Km Oroboros) under two occasions: 1. homogenate enriched with substrates was sequentially treated with an ATPase inhibitor (oligomycine), an uncoupler (FCCP) and KCN. This allowed us to measure oxidative phosphorylation, respiratory chain capacity, proton leak through inner mitochondrial membrane and non-mitochondrial O2 consumption. 2. skeletal muscle homogenate enriched with abundant ADP was treated with sequential addition of substrates and inhibitors of complexes I, II, III, and IV, respectively. Integrity of outer membrane was assessed by measuring the response to addition of cytochrome c. Oxygen consumption rate was normalized to citrate-synthase activity and total protein content.

Pro-oxidant effect of ALA is implicated in mitochondrial dysfunction of HepG2 cells.

Heme biosynthesis begins in the mitochondrion with the formation of delta-aminolevulinic acid (ALA). In acute intermittent porphyria, hereditary tyrosinemia type I and lead poisoning patients, ALA is accumulated in plasma and in organs, especially the liver. These diseases are also associated with neuromuscular dysfunction and increased incidence of hepatocellular carcinoma. Many studies suggest that this damage may originate from ALA-induced oxidative stress following its accumulation. Using the MnSOD as an oxidative stress marker, we showed here that ALA treatment of cultured cells induced ROS production, increasing with ALA concentration. The mitochondrial energetic function of ALA-treated HepG2 cells was further explored. Mitochondrial respiration and ATP content were reduced compared to control cells. For the 300 μM treatment, ALA induced a mitochondrial mass decrease and a mitochondrial network imbalance although neither necrosis nor apoptosis were observed. The up regulation of PGC-1, Tfam and ND5 genes was also found; these genes encode mitochondrial proteins involved in mitochondrial biogenesis activation and OXPHOS function. We propose that ALA may constitute an internal bioenergetic signal, which initiates a coordinated upregulation of respiratory genes, which ultimately drives mitochondrial metabolic adaptation within cells. The addition of an antioxidant, Manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), resulted in improvement of maximal respiratory chain capacity with 300 μM ALA. Our results suggest that mitochondria, an ALA-production site, are more sensitive to pro-oxidant effect of ALA, and may be directly involved in pathophysiology of patients with inherited or acquired porphyria.

Normalizing Glutamine Concentration Causes Mitochondrial Uncoupling in an In Vitro Model of Human Skeletal Muscle

Glutamine has been considered essential for rapidly dividing cells, but its effect on mitochondrial function is unknown. Human myoblasts were isolated from skeletal muscle biopsy samples (n = 9) and exposed for 20 days to 6 different glutamine concentrations (0, 100, 200, 300, 500, and 5000 µM). Cells were trypsinized and manually counted every 5 days. Seven days before the end of exposure, half of these cells were allowed to differentiate to myotubes. Afterward, energy metabolism in both myotubes and myoblasts was assessed by extracellular flux analysis (Seahorse Biosciences, Billerica, MA). The protocol for myoblasts was optimized in preliminary experiments. To account for different mitochondrial density or cell count, data were normalized to citrate synthase activity. Fastest myoblast proliferation was observed at 300 µM glutamine, with a significant reduction at 0 and 100 µM. Glutamine did not influence basal oxygen consumption, anaerobic glycolysis or respiratory chain capacity. Glutamine significantly (P = .015) influenced the leak through the inner mitochondrial membrane. Efficiency of respiratory chain was highest at 200-300 µM glutamine (~90% of oxygen used for adenosine triphosphate synthesis). Increased glutamine concentration to 500 or 5000 µM caused mitochondrial uncoupling in myoblasts and myotubes, decreasing the efficiency of the respiratory chain to ~70%. Glutamine concentrations, consistent with moderate clinical hypoglutaminemia (300 µM), bring about an optimal condition of myoblast proliferation and for efficiency of aerobic phosphorylation in an in vitro model of human skeletal muscle. These data support the hypothesis of hypoglutaminemia as an adaptive phenomenon in conditions leading to bioenergetic failure (eg, critical illness).

Influence of sprint interval training on skeletal muscle mitochondria as determined by high‐resolution respirometry

Short‐term sprint interval training (SIT) improves skeletal muscle mitochondrial function, as reflected by increased maximal activity of several oxidative enzymes. To date, no studies have determined directly the influence of SIT on mitochondrial respiration. 6 sedentary, but healthy adults (3 males; 25±2 years, peak oxygen uptake: 39±2 ml/kg/min (mean±SE)) completed 9 sessions of 4–8, 30‐second sprints, over 21 days. Vastus lateralis samples were obtained prior to and 48‐hours following SIT. Mitochondrial respiratory function was determined in saponin permeabilized fibers using high‐resolution respirometry, combined with a variety of substrate, uncoupler and inhibitor combinations. Measures of maximal complex‐I+II (all substrates) supported mitochondrial respiration revealed 9–13% increases in respiratory efficiency following SIT (P&lt;0.05). Total respiratory chain capacity per mg of muscle was unaltered, as was oxidative phosphorylation (OXPHOS) capacity and efficiency with palmitoylcarnitine + malate (fatty acid) as substrate. Conversely, OXPHOS capacity and efficiency were increased by 16–25% when pyruvate + malate (carbohydrate) was used as substrate, either in the presence or absence of fatty acid (P&lt;0.05). These preliminary data indicate that 3 weeks of SIT selectively improves the efficiency of muscle carbohydrate oxidation, with no appreciable effect on fatty acid oxidation or total muscle respiratory capacity.

Mitochondrial fission induced by platelet-derived growth factor regulates vascular smooth muscle cell bioenergetics and cell proliferation

Vascular smooth muscle cells (VSMCs) develop a highly proliferative and synthetic phenotype in arterial diseases. Because such phenotypic changes are likely integrated with the energetic state of the cell, we hypothesized that changes in cellular metabolism regulate VSMC plasticity. VSMCs were exposed to platelet-derived growth factor-BB (PDGF) and changes in mitochondrial morphology, proliferation, contractile protein expression, and mitochondrial metabolism were examined. Exposure of VSMCs to PDGF resulted in mitochondrial fragmentation and a 50% decrease in the abundance of mitofusin 2. Synthetic VSMCs demonstrated a 20% decrease in glucose oxidation, which was accompanied by an increase in fatty acid oxidation. Results of mitochondrial function assays in permeabilized cells showed few changes due to PDGF treatment in mitochondrial respiratory chain capacity and coupling. Treatment of VSMCs with Mdivi-1—an inhibitor of mitochondrial fission—inhibited PDGF-induced mitochondrial fragmentation by 50% and abolished increases in cell proliferation; however, it failed to prevent PDGF-mediated activation of autophagy and removal of contractile proteins. In addition, treatment with Mdivi-1 reversed changes in fatty acid and glucose oxidation associated with the synthetic phenotype. These results suggest that changes in mitochondrial morphology and bioenergetics underlie the hyperproliferative features of the synthetic VSMC phenotype, but do not affect the degradation of contractile proteins. Mitochondrial fragmentation occurring during the transition to the synthetic phenotype could be a therapeutic target for hyperproliferative vascular disorders.

L-Asparaginase Strongly Affects Bioenergetics in Leukemic Cells

Abstract Abstract 779 L-asparaginase (L-asp) is an important component of childhood acute lymphoblastic leukemia (ALL) therapy. Its cytotoxic effect is based on the depletion of extracellular asparagine and glutamine. Leukemic cells are sensitive to this depletion due to the lower activity of asparagine synthetase compared to healthy cells. However, the mechanism of resistance development remains unclear. The aim of this study was to obtain further insights into the mechanisms underlying the cytotoxic effect of L-asp. We used two models: resistant preB ALL leukemic cells derived from the REH (TEL/AML1[+]; very sensitive) and NALM6 (TEL/PDGFRB[+]; medium sensitive) cell lines by long-term incubation with L-asp. As a second model we used REH and NALM6 incubated with L-asp for 24 hrs, mimicking acute phase of treatment. We performed GEP of REH, res-REH, NALM6 and res-NALM6. There was an overlap of 30 genes changed in both cases. Applying the pathway analysis we found that L-asp influences the regulation of lipid metabolism and apoptosis regulated by mitochondrial proteins. Next we merged our data with GEP data published by Holleman et al. (NEJM, 2004) of ALL patients′ samples sensitive/resistant to L-Asp. Two pathways, one regulating protein translation, the other metabolism, scored very highly. Next to glucose, glutamine is the other major cellular energy source, it is also important for activation of PI3K/Akt/mTOR pathway - the key regulator of translation. Since L-asp also depletes glutamine, we focused on the effect of L-asp treatment on bioenergetics and translation in leukemic cells. To confirm the effect of L-asp on PI3K/Akt/mTOR pathway we measured the amount of Akt protein and P70S6K/p-P70S6K (downstream target of mTOR) in REH and NALM6 treated for 24 hours with L-asp. Akt and p-P70S6K expression fell in both cell lines. Next we were interested if PI3K/mTOR inhibition affects the sensitivity of resistant cell lines. We measured the cytostatic effect of co-treatment: L-asp with mTORC1 inhibitor (rapamycin); with dual inhibitor of mTORC1 and PI3K (LY294002); and with specific inhibitor of PI3K (PX866) on REH, res-REH, NALM6 and res-NALM6. The MTS assay proved the L-asp/rapamycin combination the most effective. It strongly diminished viability of all cell lines, even in those resistant to L-asp. c-Myc as an activator of glutamine catabolism showed a significant decrease in REH and NALM6 after incubation with L-asp. In addition, c-Myc was also decreased in res-REH compared with REH. Importantly, c-Myc activates glycolysis which is a main energy generator in cancer cells rather than oxidative phosphorylation (OXPHOS). We showed diminishement of Glucose transporter type 1 protein expression and 2.5-times reduced level of lactate, the product of cancer cell glycolysis, in media after L-asp exposure. Next we measured cell respiration that mirrors oxidative fosforylation (OXPHOS). REH and NALM6 showed increased capacity of respiratory chain after L-asp exposure, which was observed as an increase of endogenous respiration in uncoupled state. REH boosted the capacity of the respiratory chain from 163 to 236 pmol O2/s/mg (p&lt;0.01). NALM6 have increased the capacity of the respiratory chain from 78 to 155 pmol O2/s/mg (p&lt;0.01). Interestingly, REH has a significantly higher capacity of respiratory chain compared with NALM6 and a lower ability to regulate it. Moreover, res-REH had elevated the level of the endogenous oxygen consumption whereas res-NALM6 levels did not change. Our results indicate that leukemic cells, which normally depend on glycolysis for energy generation rather than OXPHOS, are able to swap these two mechanisms of energy generation under amino acid deprivation stress. The data show that L-asp inhibits mTORC1 and strongly affects bioenergetics pathways of leukemic cells. It decreased c-Myc-regulated glycolysis and increased OXPHOS. We believe that these metabolic changes are mainly caused by the effort of the cells to mobilize another mitochondrial pathway as Krebs cycle and β-oxidation, with the aim to supply depleted amino acids. We conclude from these data that resistance of the cells is caused by better biochemical adaptability to the nutrient deprived environment. Support: GAUK 92710, IGA NT1249, UNCE204012 Disclosures: No relevant conflicts of interest to declare.

Changes in activity and transcript level of liver and gill metabolic enzymes during smoltification in wild and hatchery-reared masu salmon (Oncorhynchus masou)

It is important for the success of the masu salmon, Oncorhynchus masou, stock enhancement program in Hokkaido (northern Japan) to demonstrate physiological problems in hatchery-reared (hatchery) smolt for artificial release. The present study examined changes in liver and gill metabolic parameters in wild and hatchery masu salmon during smoltification and elucidated differences in hepatic and gill metabolism between wild and hatchery fish. As reference to freshwater-adapted wild and hatchery smolt in this study, metabolic parameters of coastal smolt were studied. Yearling wild and hatchery smolting fish were collected from the Ken-ichi River and the Donan Research Branch, which used Ken-ichi river water for fish culture, at the same time every month from March through May 2008. Coastal smolts were caught from Nemuro Bay of Hokkaido in June. Decreased hepatic glycogen content during smoltification, which was observed in wild fish and revealed activation of glycogenolysis, was not found in hatchery fish. Hatchery fish demonstrated a positive change in hepatic ATP content during smoltification, while wild fish showed negative change in the content, which reflected activated consumption of hepatic ATP stores during smoltification. Increases in gill pyruvate kinase activity during smoltification, which were found in wild fish and indicated activation of glycolysis, were not detected in hatchery fish. There was a difference in increased timing of hepatic citrate synthase activity during smoltification between hatchery and wild fish. Increased gill citrate synthase activity during smoltification, which was observed in wild fish and reflected enhancement of the citric acid cycle, was not found in hatchery fish. Hatchery smolt revealed lower liver cytochrome c oxidase activity and transcript levels of some respiratory chain enzymes compared to wild smolt in May, which suggested lower respiratory chain capacity in hatchery fish at mid-smolt stage. On the other hand, there were no remarkable differences in hepatic and gill 3-hydroxyacyl-coenzyme A dehydrogenase related to lipolysis and creatine kinase activities, which operate in resolution of creatine phosphate, during smoltification between hatchery and wild fish. These results suggested hatchery masu salmon had some metabolic problems with carbohydrate metabolism, the citric acid cycle, and the respiratory chain. Our study will give valuable information to improve physiological quality of hatchery smolt for artificial release.

Estrogen‐related receptor α and PGC‐1‐related coactivator constitute a novel complex mediating the biogenesis of functional mitochondria

Mitochondrial biogenesis, which depends on nuclear as well as mitochondrial genes, occurs in response to increased cellular ATP demand. The nuclear transcriptional factors, estrogen-related receptor alpha (ERRalpha) and nuclear respiratory factors 1 and 2, are associated with the coordination of the transcriptional machinery governing mitochondrial biogenesis, whereas coactivators of the peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family serve as mediators between the environment and this machinery. In the context of proliferating cells, PGC-1-related coactivator (PRC) is a member of the PGC-1 family, which is known to act in partnership with nuclear respiratory factors, but no functional interference between PRC and ERRalpha has been described so far. We explored three thyroid cell lines, FTC-133, XTC.UC1 and RO 82 W-1, each characterized by a different mitochondrial content, and studied their behavior towards PRC and ERRalpha in terms of respiratory efficiency. Overexpression of PRC and ERRalpha led to increased respiratory chain capacity and mitochondrial mass. The inhibition of ERRalpha decreased cell growth and respiratory chain capacity in all three cell lines. However, the inhibition of PRC and ERRalpha produced a greater effect in the oxidative cell model, decreasing the mitochondrial mass and the phosphorylating respiration, whereas the nonphosphorylating respiration remained unchanged. We therefore hypothesize that the ERRalpha-PRC complex plays a role in arresting the cell cycle through the regulation of oxidative phosphorylation in oxidative cells, and through some other pathway in glycolytic cells.

Paradoxical Effect of Mitochondrial Respiratory Chain Impairment on Insulin Signaling and Glucose Transport in Adipose Cells

Adipocyte function is crucial for the control of whole body energy homeostasis. Pathway analysis of differentiating 3T3-L1 adipocytes reveals that major metabolic pathways induced during differentiation involve mitochondrial function. However, it is not clear why differentiated white adipocytes require enhanced respiratory chain activity relative to pre-adipocytes. To address this question, we used small interference RNA to interfere with the induction of the transcription factor Tfam, which is highly induced between days 2 and 4 of differentiation and is crucial for replication of mitochondrial DNA. Interference with Tfam resulted in cells with decreased respiratory chain capacity, reflected by decreased basal oxygen consumption, and decreased mitochondrial ATP synthesis, but no difference in many other adipocyte functions or expression levels of adipose-specific genes. However, insulin-stimulated GLUT4 translocation to the cell surface and subsequent glucose transport are impaired in Tfam knockdown cells. Paradoxically, insulin-stimulated Akt phosphorylation is significantly enhanced in these cells. These studies reveal independent links between mitochondrial function, insulin signaling, and glucose transport, in which impaired respiratory chain activity enhances insulin signaling to Akt phosphorylation, but impairs GLUT4 translocation. These results indicate that mitochondrial respiratory chain dysfunction in adipocytes can cause impaired insulin responsiveness of GLUT4 translocation by a mechanism downstream of the Akt protein kinase.

Mitochondrial transcription factor A regulates mtDNA copy number in mammals.

Mitochondrial DNA (mtDNA) copy number regulation is altered in several human mtDNA-mutation diseases and it is also important in a variety of normal physiological processes. Mitochondrial transcription factor A (TFAM) is essential for human mtDNA transcription and we demonstrate here that it is also a key regulator of mtDNA copy number. We initially performed in vitro transcription studies and determined that the human TFAM protein is a poor activator of mouse mtDNA transcription, despite its high capacity for unspecific DNA binding. Next, we generated P1 artificial chromosome (PAC) transgenic mice ubiquitously expressing human TFAM. The introduced human TFAM gene was regulated in a similar fashion as the endogenous mouse Tfam gene and expression of the human TFAM protein in the mouse did not result in down-regulation of the endogenous expression. The PAC-TFAM mice thus had a net overexpression of TFAM protein and this resulted in a general increase of mtDNA copy number. We used a combination of mice with TFAM overexpression and TFAM knockout and demonstrated that mtDNA copy number is directly proportional to the total TFAM protein levels also in mouse embryos. Interestingly, the expression of human TFAM in the mouse results in up-regulation of mtDNA copy number without increasing respiratory chain capacity or mitochondrial mass. It is thus possible to experimentally dissociate mtDNA copy number regulation from mtDNA expression and mitochondrial biogenesis in mammals in vivo. In conclusion, our results provide genetic evidence for a novel role for TFAM in direct regulation of mtDNA copy number in mammals.

Mitochondrial function and dysfunction in the cell: its relevance to aging and aging-related disease

Mitochondria plays a complex multi-factorial role in the cell. In addition to their primary role in ATP generation, the organelles sequester calcium and both generate and detoxify reactive oxygen species. All these functions are intimately inter-linked through the central bioenergetic parameter of the proton electrochemical gradient across the inner mitochondrial membrane. Subtle changes in respiratory chain capacity, substrate supply, glutathione levels, cytoplasmic calcium and membrane potential occur in aging and in conditions predisposing towards neurodegenerative disease. These interactions are incompletely understood and in this review I present an overview of some of the current research in this area, and its possible relevance to aging and aging-related disease.