Human Islet Amyloid Polypeptide Overexpression in INS-1E Cells Influences Amylin Oligomerization under ER Stress and Oxidative Stress.

Human amylin or islet amyloid polypeptide (hIAPP) is synthesized in the pancreatic ß-cells and has been shown to contribute to the pathogenesis of type 2 diabetes (T2D) in vitro and in vivo. This study compared amylin oligomerization/expression and signal transduction under endoplasmic reticulum (ER) stress and oxidative stress. pCMV-hIAPP-overexpressing INS-1E cells presented different patterns of amylin oligomerization/expression under ER stress and oxidative stress. Amylin oligomerization/expression under ER stress showed three amylin oligomers of less than 15 kDa size in pCMV-hIAPP-overexpressing cells, while one band was detected under oxidative stress. Under ER stress conditions, HIF1α, p-ERK, CHOP, Cu/Zn-SOD, and Bax were significantly increased in pCMV-hIAPP-overexpressing cells compared to the pCMV-Entry-expressing cells (control), whereas p-Akt, p-mTOR, Mn-SOD, catalase, and Bcl-2 were significantly decreased. Under oxidative stress conditions, HIF1α, p-ERK, CHOP, Mn-SOD, catalase, and Bcl-2 were significantly reduced in pCMV-hIAPP-overexpressing cells compared to the control, whereas p-mTOR, Cu/Zn-SOD, and Bax were significantly increased. In mitochondrial oxidative phosphorylation (OXPHOS), the mitochondrial complex I and complex IV were significantly decreased under ER stress conditions and significantly increased under oxidative stress conditions in pCMV-hIAPP-overexpressing cells compared to the control. The present study results demonstrate that amylin undergoes oligomerization under ER stress in pCMV-hIAPP-overexpressing cells. In addition, human amylin overexpression under ER stress in the pancreatic ß cells may enhance amylin protein aggregation, resulting in ß-cell dysfunction.

Physiological concentrations of cyanide stimulate mitochondrial Complex IV and enhance cellular bioenergetics.

In mammalian cells, cyanide is viewed as a cytotoxic agent, which exerts its effects through inhibition of mitochondrial Complex IV (Cytochrome C oxidase [CCOx]). However, the current report demonstrates that cyanide's effect on CCOx is biphasic; low (nanomolar to low-micromolar) concentrations stimulate CCOx activity, while higher (high-micromolar) concentrations produce the "classic" inhibitory effect. Low concentrations of cyanide stimulated mitochondrial electron transport and elevated intracellular adenosine triphosphate (ATP), resulting in the stimulation of cell proliferation. The stimulatory effect of cyanide on CCOx was associated with the removal of the constitutive, inhibitory glutathionylation on its catalytic 30- and 57-kDa subunits. Transfer of diluted Pseudomonas aeruginosa (a cyanide-producing bacterium) supernatants to mammalian cells stimulated cellular bioenergetics, while concentrated supernatants were inhibitory. These effects were absent with supernatants from mutant Pseudomonas lacking its cyanide-producing enzyme. These results raise the possibility that cyanide at low, endogenous levels serves regulatory purposes in mammals. Indeed, the expression of six putative mammalian cyanide-producing and/or -metabolizing enzymes was confirmed in HepG2 cells; one of them (myeloperoxidase) showed a biphasic regulation after cyanide exposure. Cyanide shares features with "classical" mammalian gasotransmitters NO, CO, and H2S and may be considered the fourth mammalian gasotransmitter.

miR-204/COX5A axis contributes to invasion and chemotherapy resistance in estrogen receptor-positive breast cancers.

Breast cancer is the most common cancer among women worldwide, with 70% being estrogen receptor-positive (ER+). Although ER-targeted treatment is effective in treating ER + breast cancer, chemoresistance and metastasis still prevail. Outcome-predictable biomarkers can help improve patient prognosis. Through the analysis of the Array Express database, The Cancer Genome Atlas-Breast Cancer datasets, and breast tumor tissue array results, we found that cytochrome c oxidase subunit 5a (COX5A) was related to poor prognosis of ER + breast cancer. Further studies revealed that COX5A was positively associated with metastasis and chemoresistance in ER + breast cancer. In vitro experiments showed that knockdown of COX5A was accompanied by a decrease in ERα expression, cell cycle arrest, and epithelial-mesenchymal transition blockade, resulting in an inhibition of proliferation and invasion. Knockdown of COX5A enhanced the chemosensitivity of breast cancer cells by decreasing adenosine triphosphate and increasing reactive oxygen species levels. We report that miR-204 can target and inhibit the expression of COX5A, thus, reversing the functions of COX5A in ER + breast cancer cells. We found that COX5A may serve as a prognostic biomarker in ER + breast cancer.

Regulation of Mammalian 13-Subunit Cytochrome c Oxidase and Binding of other Proteins: Role of NDUFA4.

Cytochrome c oxidase (CcO) is the final oxygen accepting enzyme complex (complex IV) of the mitochondrial respiratory chain. In contrast to the other complexes (I, II, and III), CcO is highly regulated via isoforms for six of its ten nuclear-coded subunits, which are differentially expressed in species, tissues, developmental stages, and cellular oxygen concentrations. Recent publications have claimed that NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 (NDUFA4), originally identified as subunit of complex I, represents a 14th subunit of CcO. Results on CcO composition in tissues from adult animals and the review of data from recent literature strongly suggest that NDUFA4 is not a 14th subunit of CcO but may represent an assembly factor for CcO or supercomplexes (respirasomes) in mitochondria of growing cells and cancer tissues.

Low Cytochrome Oxidase 1 Links Mitochondrial Dysfunction to Atherosclerosis in Mice and Pigs.

BACKGROUND: Cytochrome oxidase IV complex regulates energy production in mitochondria. Therefore, we determined the relation of COX genes with atherosclerosis in mice and pigs. METHODS AND RESULTS: First, we compared atherosclerosis in the aortic arch of age-matched (24 weeks) C57BL/6J control (n = 10), LDL-receptor deficient (n = 8), leptin-deficient ob/ob (n = 10), and double knock-out (lacking LDL-receptor and leptin) mice (n = 12). Low aortic mitochondria-encoded cytochrome oxidase 1 in obese diabetic double knock-out mice was associated with a larger plaque area and higher propensity of M1 macrophages and oxidized LDL. Caloric restriction increased mitochondria-encoded cytochrome oxidase 1 and reduced plaque area and oxidized LDL. This was associated with a reduction of titer of anti-oxidized LDL antibodies, a proxy of systemic oxidative stress. Low of mitochondria-encoded cytochrome oxidase 1 was related to low expression of peroxisome proliferative activated receptors α, δ, and γ and of peroxisome proliferative activated receptor, gamma, co-activator 1 alpha reflecting mitochondrial dysfunction. Caloric restriction increased them. To investigate if there was a diabetic/obesity requirement for mitochondria-encoded cytochrome oxidase 1 to be down-regulated, we then studied atherosclerosis in LAD of hypercholesterolemic pigs (n = 37). Pigs at the end of the study were divided in three groups based on increasing LAD plaque complexity according to Stary (Stary I: n = 12; Stary II: n = 13; Stary III: n = 12). Low mitochondria-encoded cytochrome oxidase 1 in isolated plaque macrophages was associated with more complex coronary plaques and oxidized LDL. Nucleus-encoded cytochrome oxidase 4I1 and cytochrome oxidase 10 did not correlate with plaque complexity and oxidative stress. In mice and pigs, MT-COI was inversely related to insulin resistance. CONCLUSIONS: Low MT-COI is related to mitochondrial dysfunction, oxidative stress and atherosclerosis and plaque complexity.

ATP generation in a host cell in early-phase infection is increased by upregulation of cytochrome c oxidase activity via the p2 peptide from human immunodeficiency virus type 1 Gag.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) must take advantage of its own proteins with two or more functions to successfully replicate. Although many attempts have been made to determine the function of viral proteins encoded in the HIV-1 genome, the role of the p2 peptide, a spacer between the capsid and the nucleocapsid in HIV-1 Gag in early-phase HIV infection still remains unclarified. RESULTS: In this study, we show that the p2 peptide enhances HIV-1 acute infection by increasing intracellular ATP production via the activation of mitochondrial cytochrome c oxidase (MT-CO) involved in the respiratory chain. We found that cell-permeable p2-peptide-treated cells were more effectively infected by HIV-1 than control cells. To characterize the effect of the p2 peptide on HIV-1 replication in MAGIC-5 cells, various HIV-1 cDNA products were measured by quantitative real-time PCR. The levels of the late (R/gag), 2-LTR circular (2-LTR), and integrated (Alu) forms of viral cDNAs increased in the presence of the p2 peptide. Interestingly, yeast two-hybrid analysis revealed a novel interaction between the p2 peptide and the mitochondrial intermembrane space domain (N(214)-F(235)) of MT-CO subunit I (MT-CO1). Mutational analysis indicated that Gln(6) in the p2 peptide is important for the interaction with MT-CO1. The p2 peptide activated MT-CO1 in vitro in a concentration-dependent manner, and fluorescence-microscopy analysis demonstrated that the p2 peptide had a significant effect on mitochondrial targeting. Furthermore, the analysis of HIV-1 lacking a functional p2 peptide demonstrated the inhibition of intracellular ATP production in MT-4 cells and monocyte-derived macrophages (MDMs) and a decrease in reverse transcription efficiency following infection of MT-4 cells and MDMs. CONCLUSIONS: These findings provide evidence that the p2 peptide is a viral positive allosteric modulator of MT-CO and the increased intracellular ATP production after HIV infection in a p2-peptide-dependent manner is essential for efficient reverse transcription in early-phase HIV-1 infection.

LACK, a RACK1 ortholog, facilitates cytochrome c oxidase subunit expression to promote Leishmania major fitness.

Leishmania are kinetoplastid parasites that cause the sandfly-transmitted disease leishmaniasis. To maintain fitness throughout their infectious life cycle, Leishmania must undergo rapid metabolic adaptations to the dramatically distinct environments encountered during transition between sandfly and vertebrate hosts. We performed proteomic and immunoblot analyses of attenuated L. major strains deficient for LACK, the Leishmania ortholog of the mammalian receptor for activated c kinase (RACK1), that is important for parasite thermotolerance and virulence. This approach identified cytochrome c oxidase (LmCOX) subunit IV as a LACK-dependent fitness protein. Consistent with decreased levels of LmCOX subunit IV at mammalian temperature, and in amastigotes, LmCOX activity and mitochondrial function were also impaired in LACK-deficient L. major under these conditions. Importantly, overexpression of LmCOX subunit IV in LACK-deficient L. major restored thermotolerance and macrophage infectivity. Interestingly, overexpression of LmCOX subunit IV enhanced LmCOX subunit VI expression at mammalian temperature. Collectively, our data suggest LACK promotes Leishmania adaptation to the mammalian host environment by sustaining LmCOX subunit IV expression and hence energy metabolism in response to stress stimuli such as heat. These findings extend the repertoire of RACK1 protein utility to include a role in mitochondrial function.

A mutation of COX6A1 causes a recessive axonal or mixed form of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy characterized by clinical and genetic heterogeneity. Although more than 30 loci harboring CMT-causing mutations have been identified, many other genes still remain to be discovered for many affected individuals. For two consanguineous families with CMT (axonal and mixed phenotypes), a parametric linkage analysis using genome-wide SNP chip identified a 4.3 Mb region on 12q24 showing a maximum multipoint LOD score of 4.23. Subsequent whole-genome sequencing study in one of the probands, followed by mutation screening in the two families, revealed a disease-specific 5 bp deletion (c.247-10_247-6delCACTC) in a splicing element (pyrimidine tract) of intron 2 adjacent to the third exon of cytochrome c oxidase subunit VIa polypeptide 1 (COX6A1), which is a component of mitochondrial respiratory complex IV (cytochrome c oxidase [COX]), within the autozygous linkage region. Functional analysis showed that expression of COX6A1 in peripheral white blood cells from the affected individuals and COX activity in their EB-virus-transformed lymphoblastoid cell lines were significantly reduced. In addition, Cox6a1-null mice showed significantly reduced COX activity and neurogenic muscular atrophy leading to a difficulty in walking. Those data indicated that COX6A1 mutation causes the autosomal-recessive axonal or mixed CMT.

Cytochrome c oxidase dysfunction in oxidative stress.

Cytochrome c oxidase (CcO) is the terminal oxidase of the mitochondrial electron transport chain. This bigenomic enzyme in mammals contains 13 subunits of which the 3 catalytic subunits are encoded by the mitochondrial genes. The remaining 10 subunits with suspected roles in the regulation, and/or assembly, are coded by the nuclear genome. The enzyme contains two heme groups (heme a and a3) and two Cu(2+) centers (Cu(2+) A and Cu(2+) B) as catalytic centers and handles more than 90% of molecular O(2) respired by the mammalian cells and tissues. CcO is a highly regulated enzyme which is believed to be the pacesetter for mitochondrial oxidative metabolism and ATP synthesis. The structure and function of the enzyme are affected in a wide variety of diseases including cancer, neurodegenerative diseases, myocardial ischemia/reperfusion, bone and skeletal diseases, and diabetes. Despite handling a high O(2) load the role of CcO in the production of reactive oxygen species still remains a subject of debate. However, a volume of evidence suggests that CcO dysfunction is invariably associated with increased mitochondrial reactive oxygen species production and cellular toxicity. In this paper we review the literature on mechanisms of multimodal regulation of CcO activity by a wide spectrum of physiological and pathological factors. We also review an array of literature on the direct or indirect roles of CcO in reactive oxygen species production.

Inhaled hydrogen sulfide improves graft function in an experimental model of lung transplantation.

OBJECTIVES: Ischemia/reperfusion injury (IRI) is a common complication of lung transplantation (LTx). Hydrogen sulfide (H(2)S) is a novel agent previously shown to slow metabolism and scavenge reactive oxygen species, potentially mitigating IRI. We hypothesized that pretreatment with inhaled H(2)S would improve graft function in an ex vivo model of LTx. METHODS: Rabbits (n = 10) were ventilated for 2 h prior to heart-lung bloc procurement. The treatment group (n = 5) inhaled room air (21% O(2)) supplemented with 150 ppm H(2)S while the control group (n = 5) inhaled room air alone. Both groups were gradually cooled to 34°C. All heart-lung blocs were then recovered and cold-stored in low-potassium dextran solution for 18 h. Following storage, the blocs were reperfused with donor rabbit blood in an ex vivo apparatus. Serial clinical parameters were assessed and serial tissue biochemistry was examined. RESULTS: Prior to heart-lung bloc procurement, rabbits pretreated with H(2)S exhibited similar oxygenation (P = 0.1), ventilation (P = 0.7), and heart rate (P = 0.5); however, treated rabbits exhibited consistently higher mean arterial blood pressures (P = 0.01). During reperfusion, lungs pretreated with H(2)S had better oxygenation (P < 0.01) and ventilation (P = 0.02), as well as lower pulmonary artery pressures (P < 0.01). Reactive oxygen species levels were lower in treated lungs during reperfusion (P = 0.01). Additionally, prior to reperfusion, treated lungs demonstrated more preserved mitochondrial cytochrome c oxidase activity (P = 0.01). CONCLUSIONS: To our knowledge, this study represents the first reported therapeutic use of inhaled H(2)S in an experimental model of LTx. After prolonged ischemia, lungs pretreated with inhaled H(2)S exhibited improved graft function during reperfusion. Donor pretreatment with inhaled H(2)S represents a potentially novel adjunct to conventional preservation techniques and merits further exploration.

Supramolecular organisation of the mitochondrial respiratory chain: a new challenge for the mechanism and control of oxidative phosphorylation.

Recent experimental evidence has replaced the random diffusion model of electron transfer with a model of supramolecular organisation based on specific interactions between individual respiratory complexes. These supercomplexes are detected by blue-native electrophoresis and are found to be functionally relevant by flux control analysis; moreover, they have been isolated and characterised by single-particle electron microscopy. The supramolecular association of individual complexes strongly depends on membrane lipid amount and composition and is affected by lipid peroxidation; it also seems to be modulated by membrane potential and protein phosphorylation. Supercomplex association confers several new properties with respect to the non-associated respiratory complexes to the respiratory chain: the most obvious is substrate channelling, specifically addressing Coenzyme Q and cytochrome c to interact directly with the partner enzymes without the need of a less efficient random diffusion step; in addition, supramolecular association may provide a further rate advantage by conferring long-range conformational changes to the individual complexes. Additional properties are stabilisation of Complex I, as evidenced by the destabilising effect on Complex I of mutations in either Complex III or Complex IV, and prevention of excessive generation of reactive oxygen species. On the basis of the properties described above, we hypothesise that an oxidative stress acts primarily by disassembling supercomplex associations thereby establishing a vicious circle of oxidative stress and energy failure, ultimately leading to cell damage and disease. We provide evidence that in physiological ageing and in some disease states, characterised by oxidative stress and mitochondrial damage, such as heart failure, neurodegenerative disorders and cancer, a loss of supercomplex association occurs, in line with our working hypothesis.

Individual biochemical behaviour versus biological robustness: spotlight on the regulation of cytochrome c oxidase.

During evolution from prokaryotes to eukaryotes, the main function of cytochrome c oxidase (COX), i.e., the coupling of oxygen reduction to proton translocation without the production of ROS (reactive oxygen species) remained unchanged demonstrating its robustness. A new regulation of respiration by the ATP/ADP ratio was introduced in eukaryotes based on nucleotide interaction with the added COX subunit IV. This allosteric ATP-inhibition was proposed to keep the mitochondrial membrane potential (ΔΨ(m)) at low healthy values and thus prevents the formation of ROS at complexes I and III. ROS have been implicated in various degenerative diseases. The allosteric ATP-inhibition of COX is reversibly switched on and off by phosphorylation of COX at a serine or threonine. In more than 100 individual preparations of rat heart and liver mitochondria, prepared under identical conditions, the extent of allosteric ATP-inhibition varied. This variability correlates with the variable inhibition of uncoupled respiration in intact isolated mitochondria by ATP. It is concluded that in higher organisms the allosteric ATP-inhibition is continually switched on and off by neuronal signalling in order to change oxidative phosphorylation from optimal efficiency with lower rate of ATP synthesis under resting conditions (low ΔΨ(m) and ROS production) to maximal rate of ATP synthesis under active (working, stress) conditions (elevated ΔΨ(m) and ROS production).

Phosphorylation of mammalian cytochrome c and cytochrome c oxidase in the regulation of cell destiny: respiration, apoptosis, and human disease.

The mitochondrial oxidative phosphorylation (OxPhos) system not only generates the vast majority of cellular energy, but is also involved in the generation of reactive oxygen species (ROS), and apoptosis. Cytochrome c (Cytc) and cytochrome c oxidase (COX) represent the terminal step of the electron transport chain (ETC), the proposed rate-limiting reaction in mammals. Cytc and COX show unique regulatory features including allosteric regulation, isoform expression, and regulation through cell signaling pathways. This chapter focuses on the latter and discusses all mapped phosphorylation sites based on the crystal structures of COX and Cytc. Several signaling pathways have been identified that target COX including protein kinase A and C, receptor tyrosine kinase, and inflammatory signaling. In addition, four phosphorylation sites have been mapped on Cytc with potentially large implications due to its multiple functions including apoptosis, a pathway that is overactive in stressed cells but inactive in cancer. The role of COX and Cytc phosphorylation is reviewed in a human disease context, including cancer, inflammation, sepsis, asthma, and ischemia/reperfusion injury as seen in myocardial infarction and ischemic stroke.

Cytochrome c oxidase and its role in neurodegeneration and neuroprotection.

A hallmark of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, and stroke is a malfunction of mitochondria including cytochrome c oxidase (COX), the terminal enzyme complex of the respiratory chain. COX is ascribed a key role based on mainly two regulatory mechanisms. These are the expression of isoforms and the binding of specific allosteric factors to nucleus--encoded subunits. These characteristics represent a unique feature of COX compared with the other respiratory chain complexes. Additional regulatory mechanisms, such as posttranslational modification, substrate availability, and allosteric feedback inhibition by products of the COX reaction, control the enzyme activity in a complex way. In many tissues and cell types, COX represents the rate-limiting enzyme of the respiratory chain which further emphasizes the impact of the regulation of COX as a central site for regulating energy metabolism and oxidative stress. Two of the best-analyzed regulatory mechanisms of COX to date are the allosteric feedback inhibition of the enzyme by its indirect product ATP and the expression of COX subunit IV isoforms. This ATP feedback inhibition of COX requires the expression of COX isoform IV-1. At high ATP/ADP ratios, ADP is exchanged for ATP at the matrix side of COX IV-1 leading to an inhibition of COX activity, thus enabling COX to sense the energy level and to adjust ATP synthesis to energy demand. However, under hypoxic, toxic, and degenerative conditions, COX isoform IV-2 expression is up-regulated and exchanged for COX IV-1 in the enzyme complex. This COX IV isoform switch causes an abolition of the allosteric ATP feedback inhibition of COX and consequently the loss of sensing the energy level. Thus, COX activity is increased leading to higher levels of ATP in neural cells independently of the cellular energy level. Concomitantly, ROS production is increased. Thus, under pathological conditions, neural cells are provided with ATP to meet the energy demand, but at the expense of elevated oxidative stress. This mechanism explains the functional relevance of COX subunit IV isoform expression for cellular energy sensing, ATP production, and oxidative stress levels. This, in turn, affects neural cell function, signaling, and -survival. Thus, COX is a crucial factor in etiology, progression, and prevalence of numerous human neurodegenerative diseases and represents an important target for developing diagnostic and therapeutic tools against those diseases.

Synergistic inhibitory effect of nicotine plus oral contraceptive on mitochondrial complex-IV is mediated by estrogen receptor-ß in female rats.

Chronic nicotine and oral contraceptive (NOC) exposure caused significant loss of hippocampal membrane-bound estrogen receptor-beta (ER-ß) in female rats compared with exposure to nicotine alone. Mitochondrial ER-ß regulates estrogen-mediated mitochondrial structure and function; therefore, investigating the impact of NOC on mitochondrial ER-ß and its function could help delineate the harmful synergism between nicotine and OC. In this study, we tested the hypothesis that NOC-induced loss of mitochondrial ER-ß alters the oxidative phosphorylation system protein levels and mitochondrial respiratory function. This hypothesis was tested in hippocampal mitochondria isolated from female rats exposed to saline, nicotine, OC or NOC for 16 days. NOC decreased the mitochondrial ER-ß protein levels and reduced oxygen consumption and complex IV (CIV) activity by 34% and 26% compared with saline- or nicotine-administered groups, respectively. We also observed significantly low protein levels of all mitochondrial-encoded CIV subunits after NOC as compared with the nicotine or saline groups. Similarly, the silencing of ER-ß reduced the phosphorylation of cyclic-AMP response element binding protein, and also reduced levels of CIV mitochondrial-encoded subunits after estrogen stimulation. Overall, these results suggest that mitochondrial ER-ß loss is responsible for mitochondrial malfunction after NOC.

Diesel exhaust particulate extracts inhibit transcription of nuclear respiratory factor-1 and cell viability in human umbilical vein endothelial cells.

Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1-regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17ß-estradiol (E(2)), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription, and this suppression was not ablated by concomitant treatment with E(2), 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E(2) increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects.

Acquired cytochrome C oxidase impairment in apheresis platelets during storage: a possible mechanism for depletion of metabolic adenosine triphosphate.

BACKGROUND: Intracellular adenosine triphosphate (ATP) levels decline significantly during storage of platelet (PLT) products, in part due to PLT degranulation. However, metabolic ATP stores also become depleted during storage through an unclear mechanism. Since both anaerobic glycolysis and oxidative phosphorylation are important for PLT ATP production, it is possible that the reduction in metabolic ATP reflects impaired oxidative phosphorylation. To assess this, we evaluated the kinetic activity and protein expression of cytochrome C oxidase (CcOX) in stored apheresis PLTs. STUDY DESIGN AND METHODS: Apheresis PLTs were collected and stored with agitation at 22 ± 2°C for 7 days. In vitro measurements of PLT metabolic state, function, and activation were performed on Days 0, 2, 4, and 7 of storage. Total PLT ATP content, steady-state CcOX kinetic activity, and protein immunoblotting for CcOX Subunits I and IV were also performed using isolated PLT mitochondria from simultaneously collected samples. RESULTS: Intra-PLT ATP and steady-state PLT CcOX activity declined significantly and in a progressive manner throughout storage while steady-state levels of CcOX I and IV protein remained unchanged. Time-dependent decline in CcOX activity correlated with progressive ATP depletion over time. CONCLUSION: During storage of apheresis PLTs for 7 days, the parallel decline in CcOX function and intra-PLT ATP suggests development of an acquired impairment in PLT oxidative phosphorylation associated with perturbed ATP homeostasis in stored PLTs.

(-)-Epicatechin maintains endurance training adaptation in mice after 14 days of detraining.

The purpose of this study was to determine whether (-)-epicatechin (mainly found in cocoa) could attenuate detraining effects in the hindlimb muscles of mice. Thirty-two male mice were randomized into 4 groups: control, trained, trained with 14 d of detraining and vehicle (DT-14-W), and trained with 14 d of detraining and (-)-epicatechin [DT-14-(-)-Epi]. DT-14-(-)-Epi received (-)-epicatechin (1.0 mg/kg 2 ×/d), whereas water was given to the DT-14-W group. The latter 3 groups performed 5 wk of endurance training 5 ×/wk. Hindlimb muscles were harvested, and Western blots, as well as enzyme analyses, were performed. Training significantly increased capillary-to-fiber ratio (≈ 78.8%), cytochrome-c oxidase (≈ 35%), and activity (≈ 144%) compared to controls. These adaptations returned to control levels for the DT-14-W group, whereas the DT-14-(-)-Epi group was able to maintain capillary-to-fiber ratio (≈ 44%), CcO protein expression (≈ 45%), and activity (≈ 108%) above control levels. In addition, the increase in capillarity was related to decreased protein expression of thrombospondin-1, an antiangiogenic regulator. Furthermore, there were no significant differences in endurance capacity between the trained and DT-14-(-)-Epi groups. Our data suggest that (-)-epicatechin may be a suitable compound to maintain exercise-induced improved capillarity and mitochondrial capacity, even when exercise regimens are discontinued.

Ethanol withdrawal hastens the aging of cytochrome c oxidase.

We investigated whether abrupt ethanol withdrawal (EW) age-specifically inhibits a key mitochondrial enzyme, cytochrome c oxidase (COX), and whether estrogen mitigates this problem. We also tested whether this possible effect of EW involves a substrate (cytochrome c) deficiency that is associated with proapoptotic Bcl2-associated X protein (BAX) and mitochondrial membrane swelling. Ovariectomized young, middle age, and older rats, with or without 17ß-estradiol (E2) implantation, underwent repeated EW. Cerebelli were collected to measure COX activity and the mitochondrial membrane swelling using spectrophotometry and the mitochondrial levels of cytochrome c and BAX using an immunoblot method. The loss of COX activity and the mitochondrial membrane swelling occurred only in older rats under control diet conditions but occurred earlier, starting in the young rats under EW conditions. E2 treatment mitigated these EW effects. EW increased mitochondrial BAX particularly in middle age rats but did not alter cytochrome c. Collectively EW hastens but E2 delays the age-associated loss of COX activity. This EW effect is independent of cytochrome c but may involve the mitochondrial overload of BAX and membrane vulnerability.

Low-volume interval training improves muscle oxidative capacity in sedentary adults.

INTRODUCTION: High-intensity interval training (HIT) increases skeletal muscle oxidative capacity similar to traditional endurance training, despite a low total exercise volume. Much of this work has focused on young active individuals, and it is unclear whether the results are applicable to older less active populations. In addition, many studies have used "all-out" variable-load exercise interventions (e.g., repeated Wingate tests) that may not be practical for all individuals. We therefore examined the effect of a more practical low-volume submaximal constant-load HIT protocol on skeletal muscle oxidative capacity and insulin sensitivity in middle-aged adults, who may be at a higher risk for inactivity-related disorders. METHODS: Seven sedentary but otherwise healthy individuals (three women) with a mean ± SD age, body mass index, and peak oxygen uptake (VO(2peak)) of 45 ± 5 yr, 27 ± 5 kg·m(-2), and 30 ± 3 mL·kg(-1)·min(-1) performed six training sessions during 2 wk. Each session involved 10 × 1-min cycling at ∼60% of peak power achieved during a ramp VO(2peak) test (eliciting ∼80%-95% of HR reserve) with 1 min of recovery between intervals. Needle biopsy samples (vastus lateralis) were obtained before training and ∼72 h after the final training session. RESULTS: Muscle oxidative capacity, as reflected by the protein content of citrate synthase and cytochrome c oxidase subunit IV, increased by ∼35% after training. The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α was increased by ∼56% after training, but the transcriptional corepressor receptor-interacting protein 140 remained unchanged. Glucose transporter protein content increased ∼260%, and insulin sensitivity, on the basis of the insulin sensitivity index homeostasis model assessment, improved by ∼35% after training. CONCLUSIONS: Constant-load low-volume HIT may be a practical time-efficient strategy to induce metabolic adaptations that reduce the risk for inactivity-related disorders in previously sedentary middle-aged adults.