Postmenopausal women are prone to develop obesity and insulin resistance, which might be related to skeletal muscle mitochondrial dysfunction. In a rat model of ovariectomy (OVX), skeletal muscle mitochondrial function was examined at short- and long-term periods after castration. Mitochondrial parameters in the soleus and white gastrocnemius muscle fibers were analyzed. Three weeks after surgery, there were no differences in coupled mitochondrial respiration (ATP synthesis) with pyruvate, malate, and succinate; proton leak respiration; or mitochondrial reactive oxygen species production. However, after 3 wk of OVX, the soleus and white gastrocnemius muscles of the OVX animals showed a lower use of palmitoyl-carnitine and glycerol-phosphate substrates, respectively, and decreased peroxisome proliferator-activated receptor-γ coactivator-1α expression. Estrogen replacement reverted all of these phenotypes. Eight weeks after OVX, ATP synthesis was lower in the soleus and white gastrocnemius muscles of the OVX animals than in the sham-operated and estrogen-treated animals; however, when normalized by citrate synthase activity, these differences disappeared, indicating a lower muscle mitochondria content. No differences were observed in the proton leak parameter. Mitochondrial alterations did not impair the treadmill exercise capacity of the OVX animals. However, blood lactate levels in the OVX animals were higher after the physical test, indicating a compensatory extramitochondrial ATP synthesis system, but this phenotype was reverted by estrogen replacement. These results suggest early mitochondrial dysfunction related to lipid substrate use, which could be associated with the development of the overweight phenotype of ovariectomized animals.

Both acute exercise and excessive training can cause oxidative stress. The resulting increase in free radicals and the inadequate response from antioxidant systems can lead to a framework of cellular damage. An association between affected tissue and the biomarkers of oxidative stress that appear in plasma has not been clearly established. The aim of this study was to evaluate the source of oxidative stress biomarkers found in the plasma of untrained rats after a single bout of swimming exercise at 2 different intensities: low intensity (SBLIE) or high intensity (SBHIE). Immediately after the exercise, aspartate transaminase (AST), alanine transaminase (ALT), γ-glutamyltransferase (GGT), and lactate dehydrogenase (LDH) were measured in plasma to characterize cell damage. Oxidative stress was assessed using protein carbonylation (PC), total antioxidant capacity (TAC), and thiobarbituric acid reactive substances (TBARS) quantified by malondialdehyde concentration. SBHIE raised levels of plasma AST (93%) and ALT (17%), and both exercise regimens produced an increase in GGT (7%) and LDH (∼55%). Plasma levels of PC and TBARS were greater in the SBHIE group; there were no changes in TAC. SBLIE caused only a modest increase in TBARS. In muscle, there were no changes in TAC, PC, or TBARS, regardless of exercise intensity, In the liver, TAC and TBARS increased significantly in both the SBLIE and SBHIE groups. This indicates that the oxidative stress biomarkers measured in the plasma immediately after a single bout of swimming exercise were generated primarily in the liver, not in muscle.

New Findings What is the central question of this study?What are the temporal responses of mitochondrial respiration and mitochondrial responsivity to insulin in soleus muscle fibres from mice during the development of obesity and insulin resistance? What is the main finding and its importance?Short‐ and long‐term feeding with a high‐fat diet markedly reduced soleus mitochondrial respiration and mitochondrial responsivity to insulin before any change in glycogen synthesis. Muscle glycogen synthesis and whole‐body insulin resistance were present after 14 and 28 days, respectively. Our findings highlight the plasticity of mitochondria during the development of obesity and insulin resistance. Abstract Recently, significant attention has been given to the role of muscle mitochondrial function in the development of insulin resistance associated with obesity. Our aim was to investigate temporal alterations in mitochondrial respiration, H2O2 emission and mitochondrial responsivity to insulin in permeabilized skeletal muscle fibres during the development of obesity in mice. Male Swiss mice (5–6 weeks old) were fed with a high‐fat diet (60% calories from fat) or standard diet for 7, 14 or 28 days to induce obesity and insulin resistance. Diet‐induced obese (DIO) mice presented with reduced glucose tolerance and hyperinsulinaemia after 7 days of high‐fat diet. After 14 days, the expected increase in muscle glycogen content after systemic injection of glucose and insulin was not observed in DIO mice. At 28 days, blood glucose decay after insulin injection was significantly impaired. Complex I (pyruvate + malate) and II (succinate)‐linked respiration and oxidative phosphorylation (ADP) were decreased after 7 days of high‐fat diet and remained low in DIO mice after 14 and 28 days of treatment. Moreover, mitochondria from DIO mice were incapable of increasing respiratory coupling and ADP responsivity after insulin stimulation in all observed periods. Markers of mitochondrial content were reduced only after 28 days of treatment. The mitochondrial H2O2 emission profile varied during the time course of DIO, with a reduction of H2O2 emission in the early stages of DIO and an increased emission after 28 days of treatment. Our data demonstrate that DIO promotes transitory alterations in mitochondrial physiology during the early and late stages of insulin resistance related to obesity.

The adult brain is a high-glucose and oxygen-dependent organ, with an extremely organized network of cells and large energyconsuming synapses. To reach this level of organization, early stages in development must include an efficient control of cellular events and regulation of intracellular signaling molecules and ions such as hydrogen peroxide (H 2 O 2 ) and calcium (Ca 2+ ), but in cerebral tissue, these mechanisms of regulation are still poorly understood. Hexokinase (HK) is the first enzyme in the metabolism of glucose and, when bound to mitochondria (mtHK), it has been proposed to have a role in modulation of mitochondrial H 2 O 2 generation and Ca 2+ handling. Here, we have investigated how mtHK modulates these signals in the mitochondrial context during postnatal development of the mouse brain. Using high-resolution respirometry, western blot analysis, spectrometry and resorufin, and Calcium Green fluorescence assays with brain mitochondria purified postnatally from day 1 to day 60, we demonstrate that brain HK increases its coupling to mitochondria and to oxidative phosphorylation to induce a cycle of ADP entry/ATP exit of the mitochondrial matrix that leads to efficient control over H 2 O 2 generation and Ca 2+ uptake during development until reaching plateau at day 21. This contrasts sharply with the antioxidant enzymes, which do not increase as mitochondrial H 2 O 2 generation escalates. These results suggest that, as its use of glucose increases, the brain couples HK to mitochondria to improve glucose metabolism, redox balance and Ca 2+ signaling during development, positioning mitochondria-bound hexokinase as a hub for intracellular signaling control. Abbreviations used: b-HB, b-hydroxybutyrate; b-HBM, b-hydroxybutyrate plus malate; 2-DG, 2-Deoxyglucose; Ap5A, P1,P5-Di(adenosine-5 0 ) pentaphosphate; DTNB, 5,5 0 -Dithiobis(2-nitrobenzoic acid); FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; G6PDH, glucose-6phosphate dehydrogenase; mΔw, mitochondrial membrane potential; mETS, mitochondrial electron transport system; mPTP, mitochondrial permeability transition pore; mtHK, mitochondrial bound hexokinase; ODC, oxoglutarate dehydrogenase complex; Omy, oligomicyn; PDC, pyruvate dehydrogenase complex; ROS, reactive oxygen species; RRID, research resource identifier. 624

The innate immune response plays an important role in the pathophysiology of acute respiratory distress syndrome (ARDS). Glutamine (Gln) decreases lung inflammation in experimental ARDS, but its impact on the formation of extracellular traps (ETs) in the lung is unknown. In a mouse model of endotoxin-induced pulmonary ARDS, the effects of Gln treatment on leukocyte counts and ET content in bronchoalveolar lavage fluid (BALF), inflammatory profile in lung tissue, and lung morphofunction were evaluated in vivo. Furthermore, ET formation, reactive oxygen species (ROS) production, glutathione peroxidase (GPx), and glutathione reductase (GR) activities were tested in vitro. Our in vivo results demonstrated that Gln treatment reduced ET release (as indicated by cell-free-DNA content and myeloperoxidase activity), decreased lung inflammation (reductions in interferon-γ and increases in interleukin-10 levels), and improved lung morpho-function (decreased static lung elastance and alveolar collapse) in comparison with ARDS animals treated with saline. Moreover, Gln reduced ET and ROS formation in BALF cells stimulated with lipopolysaccharide in vitro, but it did not alter GPx or GR activity. In this model of endotoxin-induced pulmonary ARDS, treatment with Gln reduced pulmonary functional and morphological impairment, inflammation, and ET release in the lung.