Body mass shapes mitochondrial NADH and NADPH sources in mammalian skeletal muscle.

Mitochondrial threshold for H2O2 release in skeletal muscle of mammals

Histochemical, biochemical, and contractile properties of red, white, and intermediate fibers.

Iron deficiency anemia: mitochondrial alpha-glycerophosphate dehydrogenase in guinea pig skeletal muscle.

Aralar is a mitochondrial calcium-regulated aspartate-glutamate carrier mainly distributed in brain and skeletal muscle, involved in the transport of aspartate from mitochondria to cytosol, and in the transfer of cytosolic reducing equivalents into mitochondria as a member of the malate-aspartate NADH shuttle. In the present study, we describe the characteristics of aralar-deficient (Aralar-/-) mice, generated by a gene-trap method, showing no aralar mRNA and protein, and no detectable malate-aspartate shuttle activity in skeletal muscle and brain mitochondria. Aralar-/- mice were growth-retarded, exhibited generalized tremoring, and had pronounced motor coordination defects along with an impaired myelination in the central nervous system. Analysis of lipid components showed a marked decrease in the myelin lipid galactosyl cerebroside. The content of the myelin lipid precursor, N-acetylaspartate, and that of aspartate are drastically decreased in the brain of Aralar-/- mice. The defect in N-acetylaspartate production was also observed in cell extracts from primary neuronal cultures derived from Aralar-/- mouse embryos. These results show that aralar plays an important role in myelin formation by providing aspartate for the synthesis of N-acetylaspartate in neuronal cells.

Reduced nicotinamide adenine dinucleotide (NADPH) is a functionally important metabolite required to support numerous cellular processes. However, despite the identification of numerous NADPH-producing enzymes, the mechanisms underlying how the organellar pools of NADPH are maintained remain elusive. Here, we have identified glucose-6-phosphate dehydrogenase (G6PDH) as an important source of NADPH in mitochondria. Activity analysis, submitochondrial fractionation, fluorescence microscopy, and protease sensitivity assays revealed that G6PDH is localized to the mitochondrial matrix. 6-ANAM, a specific G6PDH inhibitor, depleted mitochondrial NADPH pools and increased oxidative stress revealing the importance of G6PDH in NADPH maintenance. We also show that glucose availability and differences in metabolic state modulate the enzymatic sources of NADPH in mitochondria. Indeed, cells cultured in high glucose (HG) not only adopted a glycolytic phenotype but also relied heavily on matrix-associated G6PDH as a source of NADPH. In contrast, cells exposed to low-glucose (LG) concentrations, which displayed increased oxygen consumption, mitochondrial metabolic efficiency, and decreased glycolysis, relied predominantly on isocitrate dehydrogenase (ICDH) as the principal NADPH-producing enzyme in the mitochondria. Culturing glycolytic cells in LG for 48 h decreased G6PDH and increased ICDH protein levels in the mitochondria, further pointing to the regulatory role of glucose. 2-Deoxyglucose treatment also prevented the increase of mitochondrial G6PDH in response to HG. The role of glucose in regulating enzymatic sources of mitochondrial NADPH pool maintenance was confirmed using human myotubes from obese adults with a history of type 2 diabetes mellitus (post-T2DM). Myotubes from post-T2DM participants failed to increase mitochondrial G6PDH in response to HG in contrast to mitochondria in myotubes from control participants (non-T2DM). Hence, we not only identified a matrix-associated G6PDH but also provide evidence that metabolic state/glucose availability modulate enzymatic sources of NADPH.

The cytoplasmic serum of Hevea brasiliensis latex contains two glyceraldehyde‐3‐phosphate dehydrogenases. These enzymes have been isolated and purified. One of them has an absolute specificity for NAD (EC 1.2.1.12); the other utilizes mainly NADP and reacts only slowly with NAD; it is similar to the non‐phosphorylating enzyme discovered by Arnon (EC 1.2.1.9). The NAD‐dependent enzyme has a molecular weight which is analogous to that of the other species of glyceraldehyde‐3‐phosphate dehydrogenases. Its optimum is around 8 and its Km‐values for the substrate, NAD and Pi respectively are: 0.1 mM, 0.12 mM and 2.6 mM.The NADP‐dependent enzyme is probably composed of 4 subunits and its molecular weight is estimated to be about 200000. Its optimum pH is around 8.6 and it catalyses a reaction that is irreversible. Its Km‐values for the substrate, NADP and NAD respectively are: 1.2 mM, 10 μM and 12 mM.Influence of some effectors like NADH, NADPH and adenosine phosphates on the enzymatic reaction has been studied. Only NADH has an influence. It inhibits competitively the reaction and the Ki value of the order 13 μM. It appears on the other hand, taking into account the influence of cysteine, that the NADP‐dependent enzyme is more resistant to xodative agents than the NAD‐dependent enzyme. NADP‐dependent enzyme is also inhibited by PO42‐, Mg2+, Ca2+, 3‐phosphoglycerate, glyceraldehyde iodoacetamide and citrate.

Open-Loop Control of Oxidative Phosphorylation in Skeletal and Cardiac Muscle Mitochondria by Ca2+

Endurance exercise training is known to increase mitochondrial respiration in skeletal muscle. However, the molecular mechanisms behind this are not fully understood. Myoglobin (Mb) is a member of the globin family, which is highly expressed in skeletal and cardiac muscles. We recently found that Mb localizes inside mitochondria in skeletal muscle and interacts with cytochrome c oxidase subunit IV (COXIV), a subunit of mitochondrial complex IV, which regulates respiration by augmenting complex IV activity. In the present study, we investigated the effect of endurance training on Mb-COXIV interaction within mitochondria in rat skeletal muscle. Eight-week-old male Wistar rats were subjected to 6-week treadmill running training. Forty-eight hours after the last training session, the plantaris muscle was removed under anesthesia and used for biochemical analysis. The endurance training increased mitochondrial content in the skeletal muscle. It also augmented complex IV-dependent oxygen consumption and complex IV activity in isolated mitochondria from skeletal muscle. Furthermore, endurance training increased Mb expression at the whole muscle level. Importantly, mitochondrial Mb content and Mb-COXIV binding were increased by endurance training. These findings suggest that an increase in mitochondrial Mb and the concomitant enhancement of Mb interaction with COXIV may contribute to the endurance training-induced upregulation of mitochondrial respiration by augmenting complex IV activity.

The author's goal was to develop a pathophysiological model for neuroleptic malignant syndrome with greater explanatory power than the alternative hypotheses of hypothalamic dopamine antagonism (elevated set point) and direct myotoxicity (malignant hyperthermia variant). Published clinical findings on neuroleptic malignant syndrome were integrated with data from human and animal studies of muscle physiology, thermoregulation, and autonomic nervous system function. The data show that the sympathetic nervous system's latent capacity for autonomous activity is expressed when tonic inhibitory inputs from higher central nervous system centers are disrupted. These tonic inhibitory inputs are relayed to preganglionic sympathetic neurons by way of dopaminergic hypothalamospinal tracts. The sympathetic nervous system mediates hypothalamic coordination of thermoregulatory activity and is a primary regulator of muscle tone and thermogenesis, augmenting both of these when stimulated. In addition, the sympathetic nervous system modulates all of the other end-organs that function abnormally in neuroleptic malignant syndrome. There is substantial evidence to support the hypothesis that dysregulated sympathetic nervous system hyperactivity is responsible for most, if not all, features of neuroleptic malignant syndrome. A predisposition to more extreme sympathetic nervous system activation and/or dysfunction in response to emotional or psychological stress may constitute a trait vulnerability for neuroleptic malignant syndrome, which, when coupled with state variables such as acute psychic distress or dopamine receptor antagonism, produces the clinical syndrome of neuroleptic malignant syndrome. This hypothesis provides a more comprehensive explanation for existing clinical data than do the current alternatives.

In order to study branched chain alpha-keto acid oxidative decarboxylation in skeletal muscle mitochondria, an improved procedure was developed for isolating muscle mitochondria. The procedure uses the protease Nagase in mannitol sucrose media (Procedure A). These mitochondria exhibited high rates of oxygen consumption, good respiratory control ratios, and improved rates of branched chain alpha-keto acid oxidation. At 20 microM [1-14C]alpha-ketoisovalerate (KIV), rates were 1.99 +/- 0.09 nmol/mg of mitochondrial protein/min versus 0.85 +/- 0.02 in mitochondria prepared in electrolyte media without Nagase treatment (Procedure B). The apparent kinetic constants for KIV and alpha-ketoisocaproate (KIC) oxidation were determined. In the presence of ATP, the Vmax and K0.5 for KIV were 17.7 +/- 2.5 nmol/mg of mitochondrial protein/min and 82 microM, respectively. The K0.5 for KIV was at least 2-fold higher than for KIC as were apparent Vmax values. Branched chain alpha-keto acid oxidative decarboxylation in skeletal muscle mitochondria was compared to the activity in mitochondria isolated from liver, heart, and kidney. Rates of KIV and KIC oxidative decarboxylation were highest in heart mitochondria and quite similar in skeletal muscle, liver, and kidney mitochondria. It is the low mitochondrial content of mixed skeletal muscle, not the specific activity of the branched chain alpha-keto acid dehydrogenase, that limits muscle oxidative capacity. The data also indicate that the total activity in muscle has been routinely underestimated. Addition of ATP which increased the matrix pH (increases delta pH) stimulated the rate of oxidative decarboxylation of branched chain alpha-keto acids. On the other hand, addition of uncoupler which decreased the delta pH inhibited the rate of oxidation. Nigericin in low K+ medium inhibited oxidation to about the same degree as uncoupler, while addition of valinomycin in high K+ medium, which will decrease the electrical potential, had little effect on oxidation rates. Transport of branched chain alpha-keto acids should be sensitive to the mitochondrial pH gradient. Hence, the effects of ATP and the mitochondrial inhibitors on rates of branched chain alpha-keto acid oxidation suggest that mitochondrial transport may be partially rate-controlling for oxidation at physiological concentrations of the branched chain alpha-keto acids.

Effects of exhaustive exercise on the expression of PHB1 and the function of mitochondria in rats

Point:Counterpoint: Lactic acid accumulation is an advantage/disadvantage during muscle activity

The role of Sidt2 in the process of glucose and lipid metabolism has been recently reported. However, whether Sidt2 is involved in the metabolic regulation in skeletal muscle remains unknown. In this study, for the first time, using skeletal muscle-selective Sidt2 knockout mice, we found that Sidt2 was vital for the quality control of mitochondria in mouse skeletal muscle. These mice showed significantly reduced muscle tolerance and structurally abnormal mitochondria. Deletion of the Sidt2 gene resulted in decreased expression of mitochondrial fusion protein 2 (Mfn2) and Dynamin-related protein 1 (Drp1), as well as peroxisome proliferator-activated receptor γ coactivator-1 (PGC1-α). In addition, the clearance of damaged mitochondria in skeletal muscle was inhibited upon Sidt2 deletion, which was caused by blockade of autophagy flow. Mechanistically, the fusion of autophagosomes and lysosomes was compromised in Sidt2 knockout skeletal muscle cells. In summary, the deletion of the Sidt2 gene not only interfered with the quality control of mitochondria, but also inhibited the clearance of mitochondria and caused the accumulation of a large number of damaged mitochondria, ultimately leading to the abnormal structure and function of skeletal muscle.

Muscle dysfunction is a common complication and an important prognostic factor in chronic obstructive pulmonary disease (COPD). As therapeutic strategies are still needed to treat this complication, gaining more insight into the process that leads to skeletal muscle decline in COPD appears to be an important issue. This review focuses on mitochondrial involvement in limb skeletal muscle alterations (decreased muscle mass, strength, endurance and power and increased fatigue) in COPD. Mitochondria are the main source of energy for the cells; they are involved in production of reactive oxygen species and activate an important pathway that leads to apoptosis. In COPD patients, skeletal muscles are characterized by decreased mitochondrial density and biogenesis, impaired activity and coupling of mitochondrial respiratory chain complexes, increased mitochondrial production of reactive oxygen species and, possibly, increased apoptosis. Of particular interest, a sedentary lifestyle, hypoxia, hypercapnia, tobacco smoking, corticosteroid therapy and, possibly, inflammation participate in this mitochondrial dysfunction, which is accessible to conventional therapies, such as exercise and tobacco cessation, as well as, potentially, to more innovative approaches, such as antioxidant treatment and supplementation with polyunsaturated fatty acids.

1. The possibility that rapid Ca2+-uptake by skeletal muscle mitochondria may cause local reductions in pHi (by H+/Ca2+ exchange) and so promote lysosomal breakdown has been explored using amphibian and mammalian preparations. Recent studies suggested that such a sequence of events is possible in cardiac muscle. 2. However, extensive muscle damage can still be initiated in skeletal muscle when the mitochondria are uncoupled so that Ca2+-uptake is prevented. 3. DNP alone induces extensive myofilament degradation which is similar to that produced by A23187 and caffeine and described previously. 4. It is suggested that (a) the known action of DNP in promoting lysosomal labilization in living cells is produced by mitochondrial uncoupling and the release of stored Ca2+, (b) raised [Ca2+]i promotes lysosomal breakdown in skeletal muscle, so that the hydrolases released effect myofilament dissolution rapidly. 5. DNP also rapidly causes septation and division of the mitochondria in mammalian skeletal muscle.