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Cadenas 2006 Biochim Biophys Acta + (At low oxygen levels, mitochondrial respir … At low oxygen levels, mitochondrial respiration is controlled by the nitric oxide (NO)-cytochrome c oxidase (COX) signaling pathway, since NO is a membrane-permeant second messenger and competitive inhibitor of COX (1). It is now well established that oxygraphs, with Teflon-coated stirrer bars and other plastic materials of high oxygen solubility, yield high rates of oxygen back-diffusion into the chamber when oxygen levels decline, causing artefacts of respiratory measurements. High-resolution respirometry with the Oroboros O2k reduces such back-diffusion by at least an order of magnitude, and incorporates automatic instrumental background corrections, treating the ‘closed’ chamber essentially as an open system with oxygen transport between the aqueous phase and the system boundary (2). For measurement of NO in experimental chambers, however, the same instrumental problem of gas exchange between hydrophobic plastic materials and the aqueous medium has not been addressed, despite the high partition coefficient of NO between aqueous and organic phases (3). To address these problems, we incorporated an NO sensor (ISO-NOP, WPI) into a Hansatech oxygraph chamber and a high-resolution respirometer (O2k), for simultaneous recording of respiration and NO. The NO sensor was calibrated by addition of known concentrations of KNO2 under reducing conditions (KI/H2SO4) at 37 ºC and the response of the NO sensor in terms of accuracy, stability and reproducibility of the signal was compared between the two chambers. Measurements were taken in 1 ml (Hansatech) or 2 ml (O2k) closed chambers at 37 ºC, using their standard Teflon- or PEEK-coated stirrer bars, respectively. The titanium stopper of the O2k chamber was replaced by a polyvinylidenfluorid (PVDF) stopper, including a second inlet (2 mm diameter) for the NO sensor in addition to the capillary used for extrusion of gas bubbles and titration of chemicals. The PVDF stopper showed identical characteristics to titanium in terms of minimum back-diffusion of oxygen in aerobic-anaerobic transitions, can be cleaned with 70 % and pure ethanol, and offers increased flexibility for accommodation of various additional electrodes for multi-sensor applications. We compared the response of the NO sensor in the determination of the release of NO from a chemical source (DETA-NO) and the endogenous release from controlled intracellular NO production. We determined the inhibition of respiration caused by NO under physiological oxygen concentrations using conventional and high-resolution respirometry (2).ntrations using conventional and high-resolution respirometry (2).)
Reijne 2016 PLOS ONE + (At old age, humans generally have declinin … At old age, humans generally have declining muscle mass and increased fat deposition, which can increase the risk of developing cardiometabolic diseases. While regular physical activity postpones these age-related derangements, this is not always possible in the elderly because of disabilities or risk of injury. Whole-body vibration (WBV) training may be considered as an alternative to physical activity particularly in the frail population. To explore this possibility, we characterized whole-body and organ-specific metabolic processes in 6-month and 25-month old mice, over a period of 14 weeks of WBV versus sham training. WBV training tended to increase blood glucose turnover rates and stimulated hepatic glycogen utilization during fasting irrespective of age. WBV was effective in reducing white fat mass and hepatic triglyceride content only in old but not in young mice and these reductions were related to upregulation of hepatic mitochondrial uncoupling of metabolism (assessed by high-resolution respirometry) and increased expression of uncoupling protein 2. Because these changes occurred independent of changes in food intake and whole-body metabolic rate (assessed by indirect calorimetry), the liver-specific effects of WBV may be a primary mechanism to improve metabolic health during aging, rather than that it is a consequence of alterations in energy balance.sequence of alterations in energy balance.)
Miller 2017 PLOS ONE + (At the annual Iditarod Race, Alaskan Huski … At the annual Iditarod Race, Alaskan Huskies repeatedly run for up to 8 hours at 16 km/h to complete 1600 km. We previously demonstrated high rates of mitochondrial protein synthesis in Alaskan Huskies, which we suspected allowed rapid remodeling of mitochondrial proteins in response to energetic stress. The purpose of this study was to examine mitochondrial respiration in permeabilized skeletal muscle fibers of Alaskan Huskies in the offseason (Non-raced) and following the 1600 km Iditarod Sled Dog Race (Raced). We hypothesized that compared to Non-raced Huskies, raced Huskies that completed a 1600 km race would have greater mitochondrial respiratory capacities, and improvements in capacities of oxidative phosphorylation (OXPHOS) based on NADH-generating substrates as compared to fatty acids. Using high-resolution respirometry (HRR) we investigated the respiration of permeabilized muscle fibers from Alaskan Huskies. Maximum capacities were 254±26 pmol.s-1.mg-1 for OXPHOS (coupled, P) and 254±37 pmol.s-1.mg-1 for the electron transfer-pathway (ET-pathway; non-coupled, E). After racing respiratory capacities from NADH-linked substrates, but not fat-derived substrates increased. Finally, the OXPHOS to ET capacity ratio (P/E) increased after racing from 0.90±0.03 to 0.97±0.02. From our previous studies and the current study, we conclude that Alaskan Huskies maintain high mitochondrial protein turnover to facilitate rapid adaptation to environmental extremes and energetic challenges.urnover to facilitate rapid adaptation to environmental extremes and energetic challenges.)
Koendjbiharie 2021 FEMS Microbiol Rev + (At the junction between the glycolysis and … At the junction between the glycolysis and the tricarboxylic acid cycle-as well as various other metabolic pathways-lies the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node (PPO-node). These three metabolites form the core of a network involving at least eleven different types of enzymes, each with numerous subtypes. Obviously, no single organism maintains each of these eleven enzymes; instead, different organisms possess different subsets in their PPO-node, which results in a remarkable degree of variation, despite connecting such deeply conserved metabolic pathways as the glycolysis and the tricarboxylic acid cycle. The PPO-node enzymes play a crucial role in cellular energetics, with most of them involved in (de)phosphorylation of nucleotide phosphates, while those responsible for malate conversion are important redox enzymes. Variations in PPO-node therefore reflect the different energetic niches that organisms can occupy. In this review, we give an overview of the biochemistry of these eleven PPO-node enzymes. We attempt to highlight the variation that exists, both in PPO-node compositions, as well as in the roles that the enzymes can have within those different settings, through various recent discoveries in both bacteria and archaea that reveal deviations from canonical functions.eveal deviations from canonical functions.)
National Academies of Sciences, Engineering, and Medicine 2023 Navigating infodemics + (At the request of the Centers for Disease … At the request of the Centers for Disease Control and Prevention (CDC), the National Academies of Sciences, Engineering, and Medicine hosted a two-day public workshop on April 10-11, 2023 to examine the history of public health infodemics, the impact of infodemics on trust in the public health enterprise, and tools and practices used to address infodemics.At the outset of the workshop, Howard Koh, Harvard T.H. Chan School of Public Health, described the term “infodemic” as the rapid spread of large amounts of sometimes conflicting or inaccurate information that can impede the ability of individuals, communities, and authorities to protect health and effectively respond in a crisis. Even a deluge of accurate information can overwhelm the public. In times of emergency, there may also be situations in which people do not have access to the information they need. In his closing remarks, Griffis called for increased understanding of how to resource institutions at all levels to reduce the harmful effects of mis- and disinformation. To this end, CDC conducts regular meetings with other federal agencies to coordinate responses to emerging misinformation. Furthermore, CDC is working to: (1) build improved misinformation monitoring and alert systems; (2) develop a more systematic approach to misinformation through the agency; (3) create, in collaboration with academic institutions, a system to increase sentinel data collection and social listening; (4) establish rapid response infrastructure to provide the public with accurate information from trusted community sources; and (5) develop tools for public health to predict the virality of vaccine misinformation. CDC continually strives to disseminate accurate information as it is needed through appropriate channels in order to empower the public to make decisions that support their health and wellbeing.s that support their health and wellbeing.)
Nickel 2018 IOC130 + (At the request of the author, this abstract is not made available online.)
Can 2018 IOC130 + (At the request of the author, this abstract is not made available online.)
Janowska 2018 IOC134 + (At the request of the author, this abstract is not made available online.)
Ganetzky 2018 IOC134 + (At the request of the author, this abstract is not made available online.)
Revenco 2019 Abstract IOC141 + (At the request of the author, this abstract is not made available online.)
Mueller 2023 Abstract IOC160 + (At the request of the author, this abstract is not made available online.)
Edman 2023 Abstract IOC160 + (At the request of the author, this abstract is not made available online.)
Rice 2023 Abstract IOC160 + (At the request of the author, this abstract is not made available online.)
Alves 2023 Abstract IOC162 + (At the request of the author, this abstract is not made available online.)
Panajatovic 2017 IOC124 + (At the request of the authors, this abstract is not made available online.)
Simon 2022 Function (Oxf) + (At-risk alcohol use is associated with mul … At-risk alcohol use is associated with multisystemic effects and end-organ injury, and significantly contributes to global health burden. Several alcohol-mediated mechanisms have been identified, with bioenergetic maladaptation gaining credence as an underlying pathophysiological mechanism contributing to cellular injury. This evidence-based review focuses on the current knowledge of alcohol-induced bioenergetic adaptations in metabolically active tissues: liver, cardiac and skeletal muscle, pancreas, and brain. Alcohol metabolism itself significantly interferes with bioenergetic pathways in tissues, particularly the liver. Alcohol decreases states of respiration in the electron transport chain, and activity and expression of respiratory complexes, with a net effect to decrease ATP content. In addition, alcohol dysregulates major metabolic pathways, including glycolysis, the tricarboxylic acid cycle, and fatty acid oxidation. These bioenergetic alterations are influenced by alcohol-mediated changes in mitochondrial morphology, biogenesis, and dynamics. The review highlights similarities and differences in bioenergetic adaptations according to tissue type, pattern of (acute vs. chronic) alcohol use, and energy substrate availability. The compromised bioenergetics synergizes with other critical pathophysiological mechanisms, including increased oxidative stress and accelerates cellular dysfunction, promoting senescence, programmed cell death, and end-organ injury.ogrammed cell death, and end-organ injury.)
Masci 2008 Biochim Biophys Acta + (Ataxia Telangiectasia (AT) patients are pa … Ataxia Telangiectasia (AT) patients are particularly sensitive to oxidative–nitrosative stress. Nitric oxide (NO) controls mitochondrial respiration via the reversible inhibition of complex IV. The mitochondrial response to NO of AT lymphoblastoid cells was investigated. Cells isolated from three patients and three intrafamilial healthy controls were selected showing within each group a normal diploid karyotype and homogeneous telomere length. Different complex IV NO-inhibition patterns were induced by varying the electron flux through the respiratory chain, using exogenous cell membrane permeable electron donors. Under conditions of high electron flux the mitochondrial NO inhibition of respiration was greater in AT than in control cells (P ≤ 0.05). This property appears peculiar to AT, and correlates well to the higher concentration of cytochrome c detected in the AT cells. This finding is discussed on the basis of the proposed mechanism of reaction of NO with complex IV. It is suggested that the peculiar response of AT mitochondria to NO stress may be relevant to the mitochondrial metabolism of AT patients.e mitochondrial metabolism of AT patients.)
Scheede-Bergdahl 2017 Can J Physiol Pharmacol + (Atherosclerosis is one of the leading caus … Atherosclerosis is one of the leading causes of morbidity and mortality in the Western world. Although the clinical manifestations of this disease are well documented, the etiology and progression remain to be fully understood. Recently, the mitochondria have been implicated in important cellular processes involved in development of atherosclerosis. Despite the link between mitochondria and atherosclerosis, early-phase mechanisms of the disease have yet to be elucidated. The aim of this project was to explore the role of mitochondria in vascular smooth muscle (VSMC) dedifferentiation. A murine ''in vitro'' model, involving organ culture of aortic tissue in serum-free media, was used. Mitochondrial function was measured by high-resolution respirometry. Proteins associated with the VSMC phenotype switch, as well as mitochondrial density, were assessed by immunoblotting. The findings show that intrinsic mitochondrial Complex I activity is significantly upregulated during VSMC dedifferentiation. Diminished coupling between phosphorylation and oxidation was also found, indicating a greater ADP:ATP ratio. This data suggests increased leak in the electron transport chain and altered mitochondrial function specifically at Complex I. This project provides important information regarding the role of mitochondria in the early atherosclerotic process and that detectable changes in mitochondrial function and expression are related to VSMC dedifferentiation.ion are related to VSMC dedifferentiation.)
Chowdhury 2010 Am J Physiol Endocrinol Metab + (Atherosclerotic cardiovascular disease is … Atherosclerotic cardiovascular disease is the leading cause of mortality in the Western world. Dysfunction of the mitochondrial respiratory chain and overproduction of reactive oxygen species (ROS) are associated with atherosclerosis and cardiovascular disease. Oxidation increases the atherogenecity of LDL. Oxidized LDL may be apoptotic or nonapoptotic for vascular endothelial cells (EC), depending on the intensity of oxidation. A previous study demonstrated that nonapoptotic oxidized LDL increased activity of mitochondrial complex I in human umbilical vein EC. The present study examined the impact of extensively oxidized LDL (eoLDL) on oxygen consumption and the activities of key enzymes in the mitochondrial respiratory chain of cultured porcine aortic EC. Oxygraphy detected that eoLDL significantly reduced oxygen consumption in various mitochondrial complexes. Treatment with eoLDL significantly decreased NADH-ubiquinone dehydrogenase (complex I), succinate cytochrome c reductase (complex II/III), ubiquinone cytochrome c reductase (complex III), and cytochrome c oxidase (complex IV) activities and the NAD+-to-NADH ratio in EC compared with mildly oxidized LDL, LDL, or vehicle. Butylated hydroxytoluene, a potent antioxidant, normalized eoLDL-induced reductions in complex I and III enzyme activity in EC. Mitochondria-associated intracellular ROS and release of ROS from EC were significantly increased after eoLDL treatment. These findings suggest that eoLDL impairs enzyme activity in mitochondrial respiratory chain complexes and increases ROS generation from mitochondria of arterial EC. Collectively, these effects could contribute to vascular injury and atherogenesis under conditions of hypercholesterolemia and oxidative stress.nditions of hypercholesterolemia and oxidative stress.)
Leo 2021 Conserv Physiol + (Atlantic herring (''Clupea harengus'') is … Atlantic herring (''Clupea harengus'') is a benthic spawner, therefore its eggs are prone to encounter different water conditions during embryonic development, with bottom waters often depleted of oxygen and enriched in CO2. Some Atlantic herring spawning grounds are predicted to be highly affected by ongoing Ocean Acidification and Warming with water temperature increasing by up to +3°C and CO2 levels reaching ca. 1000 μatm (RCP 8.5). Although many studies investigated the effects of high levels of CO2 on the embryonic development of Atlantic herring, little is known about the combination of temperature and ecologically relevant levels of CO2. In this study, we investigated the effects of Ocean Acidification and Warming on embryonic metabolic and developmental performance such as mitochondrial function, respiration, hatching success (HS) and growth in Atlantic herring from the Oslo Fjord, one of the spawning grounds predicted to be greatly affected by climate change. Fertilized eggs were incubated under combinations of two PCO2 conditions (400 μatm and 1100 μatm) and three temperatures (6, 10 and 14°C), which correspond to current and end-of-the-century conditions. We analysed HS, oxygen consumption (MO2) and mitochondrial function of embryos as well as larval length at hatch. The capacity of the electron transport system (ETS) increased with temperature, reaching a plateau at 14°C, where the contribution of Complex I to the ETS declined in favour of Complex II. This relative shift was coupled with a dramatic increase in MO2 at 14°C. HS was high under ambient spawning conditions (6-10°C), but decreased at 14°C and hatched larvae at this temperature were smaller. Elevated PCO2 increased larval malformations, indicating sub-lethal effects. These results indicate that energetic limitations due to thermally affected mitochondria and higher energy demand for maintenance occur at the expense of embryonic development and growth.mally affected mitochondria and higher energy demand for maintenance occur at the expense of embryonic development and growth.)
Leo 2018 Conserv Physiol + (Atlantic herring (Clupea harengus) is a be … Atlantic herring (Clupea harengus) is a benthic spawner, therefore its eggs are prone to encounter different water conditions during embryonic development, with bottom waters often depleted of oxygen and enriched in CO2. Some Atlantic herring spawning grounds are predicted to be highly affected by ongoing Ocean Acidification and Warming with water temperature increasing by up to +3°C and CO2 levels reaching ca. 1000 μatm (RCP 8.5). Although many studies investigated the effects of high levels of CO2 on the embryonic development of Atlantic herring, little is known about the combination of temperature and ecologically relevant levels of CO2. In this study, we investigated the effects of Ocean Acidification and Warming on embryonic metabolic and developmental performance such as mitochondrial function, respiration, hatching success (HS) and growth in Atlantic herring from the Oslo Fjord, one of the spawning grounds predicted to be greatly affected by climate change. Fertilized eggs were incubated under combinations of two PCO2 conditions (400 μatm and 1100 μatm) and three temperatures (6, 10 and 14°C), which correspond to current and end-of-the-century conditions. We analysed HS, oxygen consumption (MO2) and mitochondrial function of embryos as well as larval length at hatch. The capacity of the electron transport system (ETS) increased with temperature, reaching a plateau at 14°C, where the contribution of Complex I to the ETS declined in favour of Complex II. This relative shift was coupled with a dramatic increase in MO2 at 14°C. HS was high under ambient spawning conditions (6-10°C), but decreased at 14°C and hatched larvae at this temperature were smaller. Elevated PCO2 increased larval malformations, indicating sub-lethal effects. These results indicate that energetic limitations due to thermally affected mitochondria and higher energy demand for maintenance occur at the expense of embryonic development and growth.pense of embryonic development and growth.)
Koopman 2022 Abstract Bioblast + (Attachment of cargo molecules to lipophili … Attachment of cargo molecules to lipophilic triphenylphosphonium (TPP+) cations is a widely applied key technology for mitochondrial targeting. We previously demonstrated that the vitamin E-derived antioxidant (Trolox; 500 nM; 96 h) increases the levels of active mitochondrial Complex I (CI), the first complex of the electron transfer system (ETS), in primary human skin fibroblasts (PHSFs) of Leigh Syndrome (LS) patients with isolated CI deficiency. Primed by this finding, we here studied the cellular effects of mitochondria-targeted Trolox (MitoE10), mitochondria-targeted ubiquinone (MitoQ10) and their mitochondria-targeting moiety decylTPP (C10-TPP+). Relative to vehicle (DMSO), chronic treatment (100 nM, 96 h) with these molecules of PHSFs from a healthy subject and an LS patient with isolated CI deficiency (''NDUFS7-V122M'' mutation) did not greatly affect cell viability. Unexpectedly, this treatment significantly reduced CI levels/activity, lowered the amount of ETS supercomplexes, inhibited mitochondrial oxygen consumption, increased extracellular acidification, altered mitochondrial morphology and stimulated the levels of hydroethidine-oxidizing ROS.We conclude that the mitochondria-targeting decylTPP moiety is responsible for the observed effects and advocate that every study employing alkylTPP-mediated mitochondrial targeting should routinely include control experiments with the corresponding alkylTPP moiety. routinely include control experiments with the corresponding alkylTPP moiety.)
Poole 2020 J Physiol + (August Krogh twice won the prestigious int … August Krogh twice won the prestigious international Steegen Prize, for nitrogen metabolism (1906) and overturning the concept of active transport of gases across the pulmonary epithelium (1910). Despite this, at the beginning of 1920, the consummate experimentalist was relatively unknown worldwide and even among his own University of Copenhagen faculty. But, in early 1919, he had submitted three papers to Dr Langley, then editor of The Journal of Physiology in England. These papers coalesced anatomical observations of skeletal muscle capillary numbers with O2 diffusion theory to propose a novel active role for capillaries that explained the prodigious increase in blood-muscle O2 flux from rest to exercise. Despite his own appraisal of the first two papers as "rather dull" to his friend, the eminent Cambridge respiratory physiologist, Joseph Barcroft, Krogh believed that the third one, dealing with O2 supply and capillary regulation, was "interesting". These papers, which won Krogh an unopposed Nobel Prize for Physiology or Medicine in 1920, form the foundation for this review. They single-handedly transformed the role of capillaries from passive conduit and exchange vessels, functioning at the mercy of their upstream arterioles, into independent contractile units that were predominantly closed at rest and opened actively during muscle contractions in a process he termed 'capillary recruitment'. Herein we examine Krogh's findings and some of the experimental difficulties he faced. In particular, the boundary conditions selected for his model (e.g. heavily anaesthetized animals, negligible intramyocyte O2 partial pressure, binary open-closed capillary function) have not withstood the test of time. Subsequently, we update the reader with intervening discoveries that underpin our current understanding of muscle microcirculatory control and place a retrospectroscope on Krogh's discoveries. The perspective is presented that the imprimatur of the Nobel Prize, in this instance, may have led scientists to discount compelling evidence. Much as he and Marie Krogh demonstrated that active transport of gases across the blood-gas barrier was unnecessary in the lung, capillaries in skeletal muscle do not open and close spontaneously or actively, nor is this necessary to account for the increase in blood-muscle O2 flux during exercise. Thus, a contemporary model of capillary function features most muscle capillaries supporting blood flow at rest, and, rather than capillaries actively vasodilating from rest to exercise, increased blood-myocyte O2 flux occurs predominantly via elevating red blood cell and plasma flux in already flowing capillaries. Krogh is lauded for his brilliance as an experimentalist and for raising scientific questions that led to fertile avenues of investigation, including the study of microvascular function.uding the study of microvascular function.)
Radenkovic 2017 Biochem Pharmacol + (Auranofin is a thiol-reactive gold (I)-con … Auranofin is a thiol-reactive gold (I)-containing compound with potential asa chemotherapeutic. Auranofin has the capacity to selectively inhibit endogenous antioxidant enzymes thioredoxin reductase (TrxR) and glutathione peroxidase (GPx), resulting in oxidative stress and the initiation of a pro-apoptotic cascade. The effect of Auranofin exposure on TrxR and GPx, and the potential for cellular protection through selenium supplementation was examined in the non-cancerous human cell line Swan-71. Auranofin exposure resulted in a concentration dependent differential inhibition of selenoprotein antioxidants. Significant inhibition of TrxR was observed at 20nM Auranofin with inhibition of GPx from 10µM. Significant increases in reactive oxygen species (ROS) were associated with antioxidant inhibition at Auranofin concentrations of 100nM (TrxR inhibition) and 10µM (TrxR and GPx inhibition), respectively. Evaluation of mitochondrial respiration demonstrated significant reductions in routine and maximal respiration at both 100nM and 10μM Auranofin. Auranofin treatment at concentrations of 10μM and higher concentrations resulted in a ∼68% decrease in cellular viability and was associated with elevations in pro-apoptotic markers cytochrome c flux control factor (FCFc) at concentration of 100nM and mitochondrial Bax at 10μM. The supplementation of selenium (100nM) prior to treatment had a generalized protective affect through the restoration of antioxidant activity with a significant increase in TrxR and GPx activity, a significant reduction in ROS and associated improvement in mitochondrial respiration and cellular viability (10µM ∼48% increase). Selenium supplementation reduced the FCFc at low doses of Auranofin (<10μM) however no effect was noted on either FCFc or Bax at concentrations above 10μM. The inhibition of antioxidant systems in non-cancerous cells by Auranofin is strongly dose dependent, and this inhibition can be altered by selenium exposure. Therefore, Auranofin dose and the selenium status of patients are important considerations in the therapeutic use of Auranofin as an agent of chemosensitization. Auranofin as an agent of chemosensitization.)
AussieMit 2018 Melbourne AU + (AussieMit 2018, Melbourne, Australia, 2018)

AussieMit 2020 Sydney AU + (AussieMit 2020, Sydney, Australia, 2020)

AussieMit 2022 Sydney AU + (AussieMit 2022, Sydney, Australia, 2022)
Gama Perez 2023 MiP2023 + (Authors: [[Gama Perez Pau]] … Authors: [[Gama Perez Pau]] Chronic overfeeding has a profound metabolic impact on multiple tissues. Consequently, unraveling the differential adaptations in each of them is fundamental to understand the progression of obesity-related comorbidities. In our laboratory we have tackled this issue in a model of obesity and weight loss induced by a combined nutritional and exercise intervention. This model has enabled us to identify visceral adipose tissue as the most vulnerable organ to such stress, not only by the magnitude of changes observed in the obese state but most importantly, because of the permanent alterations we observe even after the restoration of adequate weight and metabolic health. Whether this fingerprint is a distinctive trait of the visceral fat or it is affecting other depots is still unsolved, although the recognized developmental, morphological as well as functional differences among fat depots might drive a differential response.To this end, we aim to explore the subcutaneous adipose tissue behavior in our model, characterizing those significant indicators of vulnerability already identified in the visceral depot. These include linear regression models to correlate tissue mass and body weight, histological and immunohistochemical analysis to characterize the morphological remodeling of the tissue, the assessment of transcriptional changes in both tissues, as well as the impact on mitochondria through the evaluation of OXPHOS capacities and the quantification of mitochondrial DNA.This comparative analysis suggests that unlike visceral fat, the detrimental impact of chronic overfeeding is blunted in subcutaneous adipose tissue, with no apparent consequences on its metabolic plasticity. Among the important points to consider, these findings could represent a relevant concern for the study of obesity-related pathophysiology in humans since, thus far, most longitudinal studies exploring adipose tissue responses to weight fluctuations have been addressed in subcutaneous biopsies due to ethical constrains.ed in subcutaneous biopsies due to ethical constrains.)
Yardeni 2021 Proc Natl Acad Sci U S A + (Autism spectrum disorders (ASDs) are chara … Autism spectrum disorders (ASDs) are characterized by a deficit in social communication, pathologic repetitive behaviors, restricted interests, and electroencephalogram (EEG) aberrations. While exhaustive analysis of nuclear DNA (nDNA) variation has revealed hundreds of copy number variants (CNVs) and loss-of-function (LOF) mutations, no unifying hypothesis as to the pathophysiology of ASD has yet emerged. Based on biochemical and physiological analyses, it has been hypothesized that ASD may be the result of a systemic mitochondrial deficiency with brain-specific manifestations. This proposal has been supported by recent mitochondrial DNA (mtDNA) analyses identifying both germline and somatic mtDNA variants in ASD. If mitochondrial defects do predispose to ASD, then mice with certain mtDNA mutations should present with autism endophenotypes. To test this prediction, we examined a mouse strain harboring an mtDNA ND6 gene missense mutation (P25L). This mouse manifests impaired social interactions, increased repetitive behaviors and anxiety, EEG alterations, and a decreased seizure threshold, in the absence of reduced hippocampal interneuron numbers. EEG aberrations were most pronounced in the cortex followed by the hippocampus. Aberrations in mitochondrial respiratory function and reactive oxygen species (ROS) levels were also most pronounced in the cortex followed by the hippocampus, but absent in the olfactory bulb. These data demonstrate that mild systemic mitochondrial defects can result in ASD without apparent neuroanatomical defects and that systemic mitochondrial mutations can cause tissue-specific brain defects accompanied by regional neurophysiological alterations.y regional neurophysiological alterations.)
Kim 2018 Pflugers Arch + (Autophagy and mitophagy are important for … Autophagy and mitophagy are important for training-inducible muscle adaptations, yet it remains unclear how these systems are regulated throughout the adaptation process. Here, we studied autophagic and mitophagic flux in the skeletal muscles of Sprague-Dawley rats (300-500 g) exposed to chronic contractile activity (CCA; 3 h/day, 9 V, 10 Hz continuous, 0.1 ms pulse duration) for 1, 2, 5, and 7 days (N = 6-8/group). In order to determine the flux rates, colchicine (COL; 0.4 mg/ml/kg) was injected 48 h before tissue collection, and we evaluated differences of autophagosomal protein abundances (LC3-II and p62) between colchicine- and saline-injected animals. We confirmed that CCA resulted in mitochondrial adaptations, including improved state 3 respiration as early as day 1 in permeabilized muscle fibers, as well significant increases in mitochondrial respiratory capacity and marker proteins in IMF mitochondria by day 7. Mitophagic and autophagic flux (LC3-II and p62) were significantly decreased in skeletal muscle following 7 days of CCA. Notably, the mitophagic system seemed to be downregulated prior (day 3-5) to changes in autophagic flux (day 7), suggesting enhanced sensitivity of mitophagy compared to autophagy with chronic muscle contraction. Although we detected no significant change in the nuclear translocation of TFEB, a regulator of lysosomal biogenesis, CCA increased total TFEB protein, as well as LAMP1, in skeletal muscle. Thus, chronic muscle activity reduces mitophagy in parallel with improved mitochondrial function, and this is supported by enhanced lysosomal degradation capacity.y enhanced lysosomal degradation capacity.)
Kim 2019 Pflugers Arch + (Autophagy and mitophagy are important for … Autophagy and mitophagy are important for training-inducible muscle adaptations, yet it remains unclear how these systems are regulated throughout the adaptation process. Here, we studied autophagic and mitophagic flux in the skeletal muscles of Sprague-Dawley rats (300-500 g) exposed to chronic contractile activity (CCA; 3 h/day, 9 V, 10 Hz continuous, 0.1 ms pulse duration) for 1, 2, 5, and 7 days (N = 6-8/group). In order to determine the flux rates, colchicine (COL; 0.4 mg/ml/kg) was injected 48 h before tissue collection, and we evaluated differences of autophagosomal protein abundances (LC3-II and p62) between colchicine- and saline-injected animals. We confirmed that CCA resulted in mitochondrial adaptations, including improved state 3 respiration as early as day 1 in permeabilized muscle fibers, as well significant increases in mitochondrial respiratory capacity and marker proteins in IMF mitochondria by day 7. Mitophagic and autophagic flux (LC3-II and p62) were significantly decreased in skeletal muscle following 7 days of CCA. Notably, the mitophagic system seemed to be downregulated prior (day 3-5) to changes in autophagic flux (day 7), suggesting enhanced sensitivity of mitophagy compared to autophagy with chronic muscle contraction. Although we detected no significant change in the nuclear translocation of TFEB, a regulator of lysosomal biogenesis, CCA increased total TFEB protein, as well as LAMP1, in skeletal muscle. Thus, chronic muscle activity reduces mitophagy in parallel with improved mitochondrial function, and this is supported by enhanced lysosomal degradation capacity.y enhanced lysosomal degradation capacity.)
Dutta 2013 Autophagy + (Autophagy is a cellular self-digestion pro … Autophagy is a cellular self-digestion process that mediates protein quality control and serves to protect against neurodegenerative disorders, infections, inflammatory diseases and cancer. Current evidence suggests that autophagy can selectively remove damaged organelles such as the mitochondria. Mitochondria-induced oxidative stress has been shown to play a major role in a wide range of pathologies in several organs, including the heart. Few studies have investigated whether enhanced autophagy can offer protection against mitochondrially-generated oxidative stress. We induced mitochondrial stress in cardiomyocytes using antimycin A (Ama), which increased mitochondrial superoxide generation, decreased mitochondrial membrane potential and depressed cellular respiration. In addition, Ama augmented nuclear DNA oxidation and cell death in cardiomyocytes. Interestingly, although oxidative stress has been proposed to induce autophagy, treatment with Ama did not result in stimulation of autophagy or mitophagy in cardiomyocytes. Our results showed that the MTOR inhibitor rapamycin induced autophagy, promoted mitochondrial clearance and protected cardiomyocytes from the cytotoxic effects of Ama, as assessed by apoptotic marker activation and viability assays in both mouse atrial HL-1 cardiomyocytes and human ventricular AC16 cells. Importantly, rapamycin improved mitochondrial function, as determined by cellular respiration, mitochondrial membrane potential and morphology analysis. Furthermore, autophagy induction by rapamycin suppressed the accumulation of ubiquitinylated proteins induced by Ama. Inhibition of rapamycin-induced autophagy by pharmacological or genetic interventions attenuated the cytoprotective effects of rapamycin against Ama. We propose that rapamycin offers cytoprotection against oxidative stress by a combined approach of removing dysfunctional mitochondria as well as by degrading damaged, ubiquitinated proteins. We conclude that autophagy induction by rapamycin could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes. stress-mediated damage in cardiomyocytes.)
Leduc-Gaudet 2023 Nat Commun + (Autophagy is a critical process in the reg … Autophagy is a critical process in the regulation of muscle mass, function and integrity. The molecular mechanisms regulating autophagy are complex and still partly understood. Here, we identify and characterize a novel FoxO-dependent gene, d230025d16rik which we named Mytho (Macroautophagy and YouTH Optimizer), as a regulator of autophagy and skeletal muscle integrity ''in vivo''. Mytho is significantly up-regulated in various mouse models of skeletal muscle atrophy. Short term depletion of MYTHO in mice attenuates muscle atrophy caused by fasting, denervation, cancer cachexia and sepsis. While MYTHO overexpression is sufficient to trigger muscle atrophy, MYTHO knockdown results in a progressive increase in muscle mass associated with a sustained activation of the mTORC1 signaling pathway. Prolonged MYTHO knockdown is associated with severe myopathic features, including impaired autophagy, muscle weakness, myofiber degeneration, and extensive ultrastructural defects, such as accumulation of autophagic vacuoles and tubular aggregates. Inhibition of the mTORC1 signaling pathway in mice using rapamycin treatment attenuates the myopathic phenotype triggered by MYTHO knockdown. Skeletal muscles from human patients diagnosed with myotonic dystrophy type 1 (DM1) display reduced Mytho expression, activation of the mTORC1 signaling pathway and impaired autophagy, raising the possibility that low Mytho expression might contribute to the progression of the disease. We conclude that MYTHO is a key regulator of muscle autophagy and integrity.gulator of muscle autophagy and integrity.)
De Castro IP 2013 Cell Death Dis + (Autophagy is a critical regulator of organ … Autophagy is a critical regulator of organellar homeostasis, particularly of mitochondria. Upon the loss of membrane potential, dysfunctional mitochondria are selectively removed by autophagy through recruitment of the E3 ligase Parkin by the PTEN-induced kinase 1 (PINK1) and subsequent ubiquitination of mitochondrial membrane proteins. Mammalian sequestrome-1 (p62/SQSTM1) is an autophagy adaptor, which has been proposed to shuttle ubiquitinated cargo for autophagic degradation downstream of Parkin. Here, we show that loss of ''ref(2)P'', the ''Drosophila'' orthologue of mammalian ''P62'', results in abnormalities, including mitochondrial defects and an accumulation of mitochondrial DNA with heteroplasmic mutations, correlated with locomotor defects. Furthermore, we show that expression of Ref(2)P is able to ameliorate the defects caused by loss of Pink1 and that this depends on the presence of functional Parkin. Finally, we show that both the PB1 and UBA domains of Ref(2)P are crucial for mitochondrial clustering. We conclude that Ref(2)P is a crucial downstream effector of a pathway involving Pink1 and Parkin and is responsible for the maintenance of a viable pool of cellular mitochondria by promoting their aggregation and autophagic clearance.heir aggregation and autophagic clearance.)
Setz 2018 Hear Res + (Autophagy is a highly evolutionary conserv … Autophagy is a highly evolutionary conserved quality control defense mechanism within cells, which has also been implicated in cell death processes. In the mammalian inner ear, autophagy has been shown to play a role during early morphogenesis as well as in adult cochlear hair cells exposed to ototoxic insults. Mitophagy, a selective autophagic cell process targeting mitochondria, hasn't been studied in the inner ear so far. On this work, we searched for molecular indicators of mitophagy within House Ear Institute-Organ of Corti-1 (HEI-OC1) cells as well as in the organ of Corti (OC). We first tested for the expression of ''Pink1''/''Park2'' mRNA in 5-day-old C57BL/6 mice's cochleae using RT-PCR. We focused on the induction of mitophagy in HEI-OC1 cells as well as in the OC and investigated a possible mitophagic potential of the aminoglycoside agent gentamicin. The induction of mitophagy in HEI-OC1 cells was detected by objectivizing the translocation of fluorescence-tagged LC3 to mitochondria using confocal microscopy after a 6-h incubation with a well-described mitochondrial uncoupler and mitophagy-inducing agent: carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Incubation with gentamicin generated no mitochondrial translocation of LC3. Protein levels of COXIV, Atg5/12 and LC3 were evaluated by an immunoblot analysis after a 24-h CCCP treatment as well as gentamicin. We demonstrated mitophagy after CCCP exposure in HEI-OC1 cells by showing a downregulation of COXIV. A downregulation of COXIV could also be visualized in the OC after CCCP. A significant oxygen consumption rate (OCR) changed in cells treated with CCCP as well as significant morphological changes of mitochondria by electron microscopy (EM) strengthen this assumption. Gentamicin exposure generated no impact on OCR or mitochondrial morphological changes by EM. Finally, we demonstrated changes in the expression of Atg12 and LC3 proteins in both the OC and HEI-OC1 cells after CCCP exposure but not after gentamicin. Our data indicate that gentamicin had no impact in the activation of mitophagy-neither in the HEI-OC1 cell line nor in the OC. Therefore, we speculate that mitophagic-independent mechanisms may underly aminoglycoside ototoxicity.ms may underly aminoglycoside ototoxicity.)
Kataura 2022 Dev Cell + (Autophagy is an essential catabolic proces … Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction. lysosomal, and mitochondrial dysfunction.)
Wilson 2023 Trends Cell Biol + (Autophagy is an intracellular degradation … Autophagy is an intracellular degradation pathway that recycles subcellular components to maintain metabolic homeostasis. NAD is an essential metabolite that participates in energy metabolism and serves as a substrate for a series of NAD+-consuming enzymes (NADases), including PARPs and SIRTs. Declining levels of autophagic activity and NAD represent features of cellular ageing, and consequently enhancing either significantly extends health/lifespan in animals and normalises metabolic activity in cells. Mechanistically, it has been shown that NADases can directly regulate autophagy and mitochondrial quality control. Conversely, autophagy has been shown to preserve NAD levels by modulating cellular stress. In this review we highlight the mechanisms underlying this bidirectional relationship between NAD and autophagy, and the potential therapeutic targets it provides for combatting age-related disease and promoting longevity.e-related disease and promoting longevity.)
Ljubojevic-Holzer 2021 Cardiovasc Res + (Autophagy protects against the development … Autophagy protects against the development of cardiac hypertrophy and failure. While aberrant Ca2+ handling promotes myocardial remodelling and contributes to contractile dysfunction, the role of autophagy in maintaining Ca2+ homeostasis remains elusive. Here, we examined whether Atg5 deficiency-mediated autophagy promotes early changes in subcellular Ca2+ handling in ventricular cardiomyocytes, and whether those alterations associate with compromised cardiac reserve capacity, which commonly precedes the onset of heart failure.RT-qPCR and immunoblotting demonstrated reduced Atg5 gene and protein expression and decreased abundancy of autophagy markers in hypertrophied and failing human hearts. The function of ATG5 was examined using cardiomyocyte-specific Atg5-knockout mice (Atg5-/-). Before manifesting cardiac dysfunction, Atg5-/- mice showed compromised cardiac reserve in response to β-adrenergic stimulation. Consequently, effort intolerance and maximal oxygen consumption were reduced during treadmill-based exercise tolerance testing. Mechanistically, cellular imaging revealed that Atg5 deprivation did not alter spatial and functional organization of intracellular Ca2+ stores or affect Ca2+ cycling in response to slow pacing or upon acute isoprenaline administration. However, high frequency stimulation exposed stunted amplitude of Ca2+ transients, augmented nucleoplasmic Ca2+ load and increased CaMKII activity, especially in the nuclear region of hypertrophied Atg5-/- cardiomyocytes. These changes in Ca2+ cycling were recapitulated in hypertrophied human cardiomyocytes. Finally, ultrastructural analysis revealed accumulation of mitochondria with reduced volume and size distribution, meanwhile functional measurements showed impaired redox balance in Atg5-/- cardiomyocytes, implying energetic unsustainability due to overcompensation of single mitochondria, particularly under increased workload.Loss of cardiac Atg5-dependent autophagy reduces mitochondrial abundance and causes subtle alterations in subcellular Ca2+ cycling upon increased workload in mice. Autophagy-related impairment of Ca2+ handling is progressively worsened by β-adrenergic signalling in ventricular cardiomyocytes, thereby leading to energetic exhaustion and compromised cardiac reserve.ed impairment of Ca2+ handling is progressively worsened by β-adrenergic signalling in ventricular cardiomyocytes, thereby leading to energetic exhaustion and compromised cardiac reserve.)
Morselli 2011 J Cell Biol + (Autophagy protects organelles, cells, and … Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy independent of SIRT1 in human and yeast cells as well as in nematodes. Although resveratrol and spermidine ignite autophagy through distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant modifications of the acetylproteome. Both agents favor convergent deacetylation and acetylation reactions in the cytosol and in the nucleus, respectively. Both resveratrol and spermidine were able to induce autophagy in cytoplasts (enucleated cells). Moreover, a cytoplasm-restricted mutant of SIRT1 could stimulate autophagy, suggesting that cytoplasmic deacetylation reactions dictate the autophagic cascade. At doses at which neither resveratrol nor spermidine stimulated autophagy alone, these agents synergistically induced autophagy. Altogether, these data underscore the importance of an autophagy regulatory network of antagonistic deacetylases and acetylases that can be pharmacologically manipulated.that can be pharmacologically manipulated.)
Van Bergen 2011 PLoS One + (Autosomal Dominant Optic Atrophy (ADOA) is … Autosomal Dominant Optic Atrophy (ADOA) is the most common inherited optic atrophy where vision impairment results from specific loss of retinal ganglion cells of the optic nerve. Around 60% of ADOA cases are linked to mutations in the OPA1 gene. OPA1 is a fission-fusion protein involved in mitochondrial inner membrane remodelling. ADOA presents with marked variation in clinical phenotype and varying degrees of vision loss, even among siblings carrying identical mutations in OPA1. To determine whether the degree of vision loss is associated with the level of mitochondrial impairment, we examined mitochondrial function in lymphoblast cell lines obtained from six large Australian OPA1-linked ADOA pedigrees. Comparing patients with severe vision loss (visual acuity [VA]<6/36) and patients with relatively preserved vision (VA>6/9) a clear defect in mitochondrial ATP synthesis and reduced respiration rates were observed in patients with poor vision. In addition, oxidative phosphorylation (OXPHOS) enzymology in ADOA patients with normal vision revealed increased complex II+III activity and levels of complex IV protein. These data suggest that OPA1 deficiency impairs OXPHOS efficiency, but compensation through increases in the distal complexes of the respiratory chain may preserve mitochondrial ATP production in patients who maintain normal vision. Identification of genetic variants that enable this response may provide novel therapeutic insights into OXPHOS compensation for preventing vision loss in optic neuropathies.or preventing vision loss in optic neuropathies.)
Lesage 2016 Am J Hum Genet + (Autosomal-recessive early-onset parkinsoni … Autosomal-recessive early-onset parkinsonism is clinically and genetically heterogeneous. The genetic causes of approximately 50% of autosomal-recessive early-onset forms of Parkinson disease (PD) remain to be elucidated. Homozygozity mapping and exome sequencing in 62 isolated individuals with early-onset parkinsonism and confirmed consanguinity followed by data mining in the exomes of 1,348 PD-affected individuals identified, in three isolated subjects, homozygous or compound heterozygous truncating mutations in vacuolar protein sorting 13C (VPS13C). VPS13C mutations are associated with a distinct form of early-onset parkinsonism characterized by rapid and severe disease progression and early cognitive decline; the pathological features were striking and reminiscent of diffuse Lewy body disease. In cell models, VPS13C partly localized to the outer membrane of mitochondria. Silencing of VPS13C was associated with lower mitochondrial membrane potential, mitochondrial fragmentation, increased respiration rates, exacerbated PINK1/Parkin-dependent mitophagy, and transcriptional upregulation of PARK2 in response to mitochondrial damage. This work suggests that loss of function of VPS13C is a cause of autosomal-recessive early-onset parkinsonism with a distinctive phenotype of rapid and severe progression.Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.shed by Elsevier Inc. All rights reserved.)
Angebault 2015 Am J Hum Genet + (Autosomal-recessive optic neuropathies are … Autosomal-recessive optic neuropathies are rare blinding conditions related to retinal ganglion cell (RGC) and optic-nerve degeneration, for which only mutations in ''TMEM126A'' and ''ACO2'' are known. In four families with early-onset recessive optic neuropathy, we identified mutations in ''RTN4IP1'', which encodes a mitochondrial ubiquinol oxydo-reductase. ''RTN4IP1'' is a partner of ''RTN4'' (also known as NOGO), and its ortholog Rad8 in ''C. elegans'' is involved in UV light response. Analysis of fibroblasts from affected individuals with a ''RTN4IP1'' mutation showed loss of the altered protein, a deficit of mitochondrial respiratory complex I and IV activities, and increased susceptibility to UV light. Silencing of RTN4IP1 altered the number and morphogenesis of mouse RGC dendrites ''in vitro'' and the eye size, neuro-retinal development, and swimming behavior in zebrafish ''in vivo''. Altogether, these data point to a pathophysiological mechanism responsible for RGC early degeneration and optic neuropathy and linking ''RTN4IP1'' functions to mitochondrial physiology, response to UV light, and dendrite growth during eye maturation.and dendrite growth during eye maturation.)
D'Souza 2018 J Lipid Res + (Autotaxin (ATX) is an adipokine that gener … Autotaxin (ATX) is an adipokine that generates the bioactive lipid, lysophosphatidic acid (LPA). ATX-LPA signaling has been implicated in diet-induced obesity and systemic insulin resistance. However, it remains unclear whether the ATX-LPA pathway influences insulin function and energy metabolism in target tissues, particularly skeletal muscle, the major site of insulin-stimulated glucose disposal. The objective of this study was to test whether the ATX-LPA pathway impacts tissue insulin signaling and mitochondrial metabolism in skeletal muscle during obesity. Male mice with heterozygous ATX deficiency (ATX+/-) were protected from obesity, systemic insulin resistance, and cardiomyocyte dysfunction following high-fat high-sucrose (HFHS) feeding. HFHS-fed ATX+/- mice also had improved insulin-stimulated AKT phosphorylation in white adipose tissue, liver, heart, and skeletal muscle. Preserved insulin-stimulated glucose transport in muscle from HFHS-fed ATX+/- mice was associated with improved mitochondrial pyruvate oxidation in the absence of changes in fat oxidation and ectopic lipid accumulation. Similarly, incubation with LPA decreased insulin-stimulated AKT phosphorylation and mitochondrial energy metabolism in C2C12 myotubes at baseline and following palmitate-induced insulin resistance. Taken together, our results suggest that the ATX-LPA pathway contributes to obesity-induced insulin resistance in metabolically relevant tissues. Our data also suggest that LPA directly impairs skeletal muscle insulin signaling and mitochondrial function.directly impairs skeletal muscle insulin signaling and mitochondrial function.)
Albertini 2012 Aging (Albany NY) + (Availability of methionine is known to mod … Availability of methionine is known to modulate the rate of aging in model organisms, best illustrated by the observation that dietary methionine restriction extends the lifespan of rodents. However, the underlying mechanisms are incompletely understood. In eukaryotic cells, methionine can be converted to cysteine through the reverse transsulfuration pathway thereby modulating intracellular methionine availability. Whereas previous results obtained in yeast and fruit flies suggest that alterations in the reverse transsulfuration pathway modulate the rate of aging, it is not known whether this function is conserved in evolution. Here we show that depletion of cystathionine beta synthase (CBS), a rate limiting enzyme in the reverse transsulfuration pathway, induces premature senescence in human endothelial cells. We found that CBS depletion induces mild mitochondrial dysfunction and increases the sensitivity of endothelial cells to homocysteine, a known inducer of endothelial cell senescence and an established risk factor for vascular disease. Our finding that CBS deficiency induces endothelial cell senescence ''in vitro'', involving both mitochondrial dysfunction and increased susceptibility of the cells to homocysteine, suggests a new mechanism linking CBS deficiency to vascular aging and disease. deficiency to vascular aging and disease.)
Ravera 2018 Biol Cell + (BACKGROUND INFORMATION: Energy demand in h … BACKGROUND INFORMATION: Energy demand in human platelets is very high, to carry out their functions. As for most human cells, the aerobic metabolism represents the primary energy source in platelets, even though mitochondria are negligibly represented. Following the hypothesis that other structures could be involved in chemical energy production, in this work, we have investigated the functional expression of an extramitochondrial aerobic metabolism in platelets.RESULTS: Oximetric and luminometric analyses showed that platelets consume large amounts of oxygen and produce ATP in the presence of common respiring substrates, such as pyruvate + malate or succinate, although morphological electron microscopy analysis showed that these contain few mitochondria. However, evaluation of the anaerobic glycolytic metabolism showed that only 13% of consumed glucose was converted to lactate. Interestingly, the highest OXPHOS activity was observed in the presence of NADH, not a readily permeant respiring substrate for mitochondria. Also, oxygen consumption and ATP synthesis fuelled by NADH were not affected by atractyloside, an inhibitor of the adenine nucleotide translocase, suggesting that these processes may not be ascribed to mitochondria. Functional data were confirmed by immunofluorescence microscopy and Western blot analyses, showing a consistent expression of the β subunit of F1 Fo -ATP synthase and COXII, a subunit of Complex IV, but a low signal of translocase of the inner mitochondrial membrane (a protein not involved in OXPHOS metabolism). Interestingly, the NADH-stimulated oxygen consumption and ATP synthesis increased in the presence of the physiological platelets agonists, thrombin or collagen.CONCLUSIONS: Data suggest that in platelets, aerobic energy production is mainly driven by an extramitochondrial OXPHOS machinery, originated inside the megakaryocyte, and that this metabolism plays a pivotal role in platelet activation.SIGNIFICANCE: This work represents a further example of the existence of an extramitochondrial aerobic metabolism, which can contribute to the cellular energy balance.contribute to the cellular energy balance.)
Regueira 2009 Liver Int + (BACKGROUND/AIMS: Genes encoding for some o … BACKGROUND/AIMS:Genes encoding for some of the mitochondrial proteins are under the control of the transcriptional factor hypoxia inducible factor-1 alpha (HIF-1 alpha), which can accumulate under normoxic conditions in inflammatory states. The aim of this study was to evaluate the effects of cobalt chloride (CoCl2, a hypoxia mimicking agent), tumour necrosis factor-alpha (TNF-alpha) and toll-like receptor (TLR) -2, -3 and -4 agonists on HIF-1 alpha accumulation, and further on HIF-1 alpha-mediated modulation of mitochondrial respiration in cultured human hepatocytes.METHODS:The human hepatoma cell line HepG2 was used in this study. Cells were treated with CoCl2, TNF-alpha and TLR-2, -3 and -4 agonists. HIF-1 alpha was determined by Western blotting and mitochondrial respiration in stimulated cells by high-resolution respirometry.RESULTS:CoCl2, TNF-alpha and TLR agonists induced the expression of HIF-1 alpha in a time-dependent fashion. TNF-alpha and CoCl2, but not TLR agonists, induced a reduction in complex I-, II- and IV-dependent mitochondrial oxygen consumption. TNF-alpha-associated reduction of cellular oxygen consumption was abolished through inhibition of HIF-1 alpha activity by chetomin (CTM). Pretreatment with cyclosporine A prevented CoCl2-induced reduction of complex I- and II-dependent mitochondrial oxygen consumption and TNF-alpha-induced reduction of complex-I-dependent respiration, implicating the involvement of the mitochondrial permeability transition pore openings. TNF-alpha and TLR-2, -3 and -4 agonists induced the expression of vascular endothelial growth factor, which was partially abolished by the blockage of HIF-1 alpha with CTM.CONCLUSIONS:The data suggest that HIF-1 alpha modulates mitochondrial respiration during CoCl2 and TNF-alpha stimulation, whereas it has no effect when induced with TLR-2, -3 and -4 agonists.>2 and TNF-alpha stimulation, whereas it has no effect when induced with TLR-2, -3 and -4 agonists.)
Escribano-Lopez 2019 Cell Physiol Biochem + (BACKGROUND/AIMS: Mitochondria-targeted ant … BACKGROUND/AIMS:Mitochondria-targeted antioxidants such as mitoquinone (MitoQ) have demonstrated protective effects against oxidative damage in several diseases. The increase in reactive oxygen species (ROS) production during glucose metabolism in β cells can be exacerbated under hyperglycaemic conditions such as type 2 diabetes (T2D), thus contributing to β cell function impairment. In the present work, we aimed to evaluate the effect of MitoQ on insulin secretion, oxidative stress, endoplasmic reticulum (ER) stress and nuclear factor kappa B (NFκB) signalling in a pancreatic β cell line under normoglycaemic (NG, 11.1 mM glucose), hyperglycaemic (HG, 25 mM glucose) and lipidic (palmitic acid (PA), 0.5mM) conditions.METHODS:We incubated the pancreatic β cell line INS-1E with or without MitoQ (0.5µM) under NG, HG and PA conditions. We then assessed the following parameters: glucose-induced insulin secretion, O₂ consumption (with a Clark-type electrode); mitochondrial function, oxidative stress parameters and calcium levels (by fluorescence microscopy); ER stress markers and NFκB-p65 protein levels (by western blotting).RESULTS:MitoQ increased insulin secretion and prevented the enhancement of ROS production and O₂ consumption and decrease in GSH levels that are characteristic under HG conditions. MitoQ also reduced protein levels of ER stress markers (GRP78 and P-eIF2α) and the proinflammatory nuclear transcription factor NFκB-p65, both of which increased under HG. MitoQ did not significantly alter ER stress markers under lipidic conditions.CONCLUSION:Our findings suggest that treatment with MitoQ modulates mitochondrial function, which in turn ameliorates endoplasmic reticulum stress and NFκB activation, thereby representing potential benefits for pancreatic β cell function.l benefits for pancreatic β cell function.)
Johnson 2016 Transfusion + (BACKGROUND: Alternatives to room temperat … BACKGROUND:Alternatives to room temperature storage of platelets (PLTs) may be beneficial to extend the limited shelf life and support transfusion logistics in rural and military areas. The aim of this study was to assess the morphologic, metabolic, and functional aspects of PLTs stored at room temperature or in refrigerated conditions or cryopreserved.STUDY DESIGN AND METHODS:A three-arm pool-and-split study was carried out using buffy coat-derived PLTs stored in 30% plasma/70% SSP+. The three matched treatment arms were room temperature stored (20-24°C), cold-stored (2-6°C), and cryopreserved (-80°C with dimethyl sulfoxide). Liquid-stored PLTs were tested over a 21-day period, while cryopreserved PLTs were examined immediately after thawing and after 6 and 24 hours of storage at room temperature.RESULTS:Cold-stored and cryopreserved PLTs underwent a significant shape change, although the cryopreserved PLTs appeared to recover from this during subsequent storage. Glycolytic metabolism was reduced in cold-stored PLTs, but accelerated in cryopreserved PLTs, while oxidative phosphorylation was negatively affected by both storage conditions. PLT aggregation was potentiated by cold storage and diminished by cryopreservation in comparison to room temperature-stored PLTs. Cold storage and cryopreservation resulted in faster clot formation (R-time; thromboelastography), which was associated with an increase in microparticles.CONCLUSION:Cold storage and cryopreservation of PLTs led to morphologic and metabolic changes. However, storage under these conditions appears to maintain or even enhance certain aspects of in vitro PLT function. certain aspects of in vitro PLT function.)
Johnson 2014 Transfusion + (BACKGROUND: Cryopreservation of platelets … BACKGROUND:Cryopreservation of platelets (PLTs) at -80°C with dimethyl sulfoxide (DMSO) can extend the shelf life from 5 days to 2 years. Cryopreserved PLTs are reported to have a greater in vivo hemostatic effect than liquid-stored PLTs. As such, the aim of this study was to understand the mechanisms responsible for the hemostatic potential of cryopreserved PLTs and the contribution of the reconstitution solution to this activity.STUDY DESIGN AND METHODS:DMSO (5% final concentration) was added to buffy coat-derived PLTs, followed by prefreeze removal of DMSO and storage at -80°C. Cryopreserved PLTs (n=8 per group) were thawed at 37°C, reconstituted with either 1 unit of thawed frozen plasma or PLT additive solution (PAS-G). In vitro assays were performed before freezing and after thawing to assess the hemostatic activity of PLTs.RESULTS:Cryopreserved PLTs expressed high levels of phosphatidylserine and contained significantly more phosphatidylserine-positive PLT microparticles than liquid-stored PLTs. This was accompanied by a significant decrease in the time to clot formation and clot strength, as measured by thromboelastography. The supernatant from cryopreserved PLTs was sufficient to reduce the phosphatidylserine-dependent clotting time and increase the thrombin generation potential. Overall, plasma-reconstituted cryopreserved PLTs were more procoagulant than those reconstituted in PAS-G.CONCLUSION:PLT cryopreservation results in the generation of phosphatidylserine-expressing PLT microparticles which contribute to the hemostatic activity. Understanding the hemostatic activity of these components may assist in extending the use of these specialized components beyond military applications.d components beyond military applications.)
Angiulli 2015 Biochim Biophys Acta + (BACKGROUND: ''Leishmania infantum'' is a p … BACKGROUND:''Leishmania infantum'' is a protozoan of the trypanosomatid family causing ''visceral leishmaniasis''. ''Leishmania'' parasites are transmitted by the bite of phlebotomine sand flies to the human host and are phagocyted by macrophages. The parasites synthesize N1-N8-bis(glutationyl)-spermidine (trypanothione, TS2), which furnishes electrons to the tryparedoxin-tryparedoxin peroxidase couple to reduce the reactive oxygen species produced by macrophages. Trypanothione is kept reduced by trypanothione reductase (TR), a FAD-containing enzyme essential for parasite survival.METHODS:The enzymatic activity has been studied by stopped-flow, absorption spectroscopy, and amperometric measurements.RESULTS:The study reported here demonstrates that the steady-state parameters change as a function of the order of substrates addition to the TR-containing solution. In particular, when the reaction is carried out by adding NADPH to a solution containing the enzyme and trypanothione, the KM for NADPH decreases six times compared to the value obtained by adding TS2 as last reagent to start the reaction (1.9 vs. 12 μM). More importantly, we demonstrate that TR is able to catalyze the oxidation of NADPH also in the absence of trypanothione. Thus, TR catalyzes the reduction of O2 to water through the sequential formation of C(4a)-(hydro)peroxyflavin and sulfenic acid intermediates. This NADPH:O2 oxidoreductase activity is shared by ''Saccharomyces cerevisiae'' glutathione reductase (GR).CONCLUSIONS:TR and GR, in the absence of their physiological substrates, may catalyze the electron transfer reaction from NADPH to molecular oxygen to yield water.GENERAL SIGNIFICANCE:TR and GR are promiscuous enzymes.d water. GENERAL SIGNIFICANCE: TR and GR are promiscuous enzymes.)
Montaigne 2014 Circulation + (BACKGROUND: -Obesity and diabetes mellitus … BACKGROUND:-Obesity and diabetes mellitus (DM) are independently associated with the development of heart failure. In this study, we determined the respective effects of obesity, insulin resistance and DM on intrinsic contraction and mitochondrial function of the human myocardium before the onset of cardiomyopathy.METHODS AND RESULTS:-Right atrial myocardium was obtained from 141 consecutive patients, presenting no sign of cardiomyopathy. We investigated (i) ex vivo isometric contraction (ii) mitochondrial respiration and calcium retention capacity (iii) respiratory chain complex activities and oxidative stress status. DM was associated with a pronounced impairment of intrinsic contraction, mitochondrial dysfunction and increased myocardial oxidative stress, irrespective of weight status. By contrast, obesity was associated with less pronounced contractile dysfunction without any significant perturbation of mitochondrial function or oxidative stress status. Tested as continuous variables, glycated haemoglobin (HbA1C), but neither body mass index nor the insulin resistance index HOMA-IR, was independently associated with cardiac mitochondrial function. Furthermore, DM was associated with cardiac mitochondrial network fragmentation and significant decreased expression of the mitochondrial fusion related protein MFN1. Myocardial MFN1 content was inversely proportional to HbA1C.CONCLUSIONS:-Worsening of intrinsic myocardial contraction in the transition from obesity to DM is likely related to worsening of cardiac mitochondrial function, since impaired mitochondrial function and dynamics, as well as contractile dysfunction are observed in diabetic patients but not in "metabolically healthy" obese patients at early stage in insulin resistance.ents at early stage in insulin resistance.)