Xanthine oxidase inhibition attenuates skeletal muscle signaling following acute exercise but does not impair mitochondrial adaptations to endurance training.

The aim of this research was to examine the impact of the xanthine oxidase (XO) inhibitor allopurinol on the skeletal muscle activation of cell signaling kinases' and adaptations to mitochondrial proteins and antioxidant enzymes following acute endurance exercise and endurance training. Male Sprague-Dawley rats performed either acute exercise (60 min of treadmill running, 27 m/min, 5% incline) or 6 wk of endurance training (5 days/wk) while receiving allopurinol or vehicle. Allopurinol treatment reduced XO activity to 5% of the basal levels (P < 0.05), with skeletal muscle uric acid levels being almost undetectable. Following acute exercise, skeletal muscle oxidized glutathione (GSSG) significantly increased in allopurinol- and vehicle-treated groups despite XO activity and uric acid levels being unaltered by acute exercise (P < 0.05). This suggests that the source of ROS was not from XO. Surprisingly, muscle GSSG levels were significantly increased following allopurinol treatment. Following acute exercise, allopurinol treatment prevented the increase in p38 MAPK and ERK phosphorylation and attenuated the increase in mitochondrial transcription factor A (mtTFA) mRNA (P < 0.05) but had no effect on the increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor-2, GLUT4, or superoxide dismutase mRNA. Allopurinol also had no impact on the endurance training-induced increases in PGC-1α, mtTFA, and mitochondrial proteins including cytochrome c, citrate synthase, and β-hydroxyacyl-CoA dehydrogenase. In conclusion, although allopurinol inhibits cell signaling pathways in response to acute exercise, the inhibitory effects of allopurinol appear unrelated to exercise-induced ROS production by XO. Allopurinol also has little effect on increases in mitochondrial proteins following endurance training.

Wireless instantaneous neurotransmitter concentration system-based amperometric detection of dopamine, adenosine, and glutamate for intraoperative neurochemical monitoring - laboratory investigation

Object  In a companion study, the authors describe the development of a new instrument named the Wireless Instantaneous Neurotransmitter Concentration System (WINCS), which couples digital telemetry with fast-scan cyclic voltammetry (FSCV) to measure extracellular concentrations of dopamine. In the present study, the authors describe the extended capability of the WINCS to use fixed potential amperometry (FPA) to measure extracellular concentrations of dopamine, as well as glutamate and adenosine. Compared with other electrochemical techniques such as FSCV or high-speed chronoamperometry, FPA offers superior temporal resolution and, in combination with enzyme-linked biosensors, the potential to monitor nonelectroactive analytes in real time.

Methods  The WINCS design incorporated a transimpedance amplifier with associated analog circuitry for FPA; a microprocessor; a Bluetooth transceiver; and a single, battery-powered, multilayer, printed circuit board. The WINCS was tested with 3 distinct recording electrodes: 1) a carbon-fiber microelectrode (CFM) to measure dopamine; 2) a glutamate oxidase enzyme–linked electrode to measure glutamate; and 3) a multiple enzyme–linked electrode (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to measure adenosine. Proof-of-principle analyses included noise assessments and in vitro and in vivo measurements that were compared with similar analyses by using a commercial hardwired electrochemical system (EA161 Picostat, eDAQ; Pty Ltd). In urethane-anesthetized rats, dopamine release was monitored in the striatum following deep brain stimulation (DBS) of ascending dopaminergic fibers in the medial forebrain bundle (MFB). In separate rat experiments, DBS-evoked adenosine release was monitored in the ventrolateral thalamus. To test the WINCS in an operating room setting resembling human neurosurgery, cortical glutamate release in response to motor cortex stimulation (MCS) was monitored using a large-mammal animal model, the pig.

Results   The WINCS, which is designed in compliance with FDA-recognized consensus standards for medical electrical device safety, successfully measured dopamine, glutamate, and adenosine, both in vitro and in vivo. The WINCS detected striatal dopamine release at the implanted CFM during DBS of the MFB. The DBS-evoked adenosine release in the rat thalamus and MCS-evoked glutamate release in the pig cortex were also successfully measured. Overall, in vitro and in vivo testing demonstrated signals comparable to a commercial hardwired electrochemical system for FPA.

Conclusions  By incorporating FPA, the chemical repertoire of WINCS-measurable neurotransmitters is expanded to include glutamate and other nonelectroactive species for which the evolving field of enzyme-linked biosensors exists. Because many neurotransmitters are not electrochemically active, FPA in combination with enzyme-linked microelectrodes represents a powerful intraoperative tool for rapid and selective neurochemical sampling in important anatomical targets during functional neurosurgery.

cGMP phosphodiesterase inhibition improves the vascular and metabolic actions of insulin in skeletal muscle

There is considerable support for the concept that insulin-mediated increases in microvascular blood flow to muscle impact significantly on muscle glucose uptake. Since the microvascular blood flow increases with insulin have been shown to be nitric oxide-dependent inhibition of cGMP-degrading phosphodiesterases (cGMP PDEs) is predicted to enhance insulin-mediated increases in microvascular perfusion and muscle glucose uptake. Therefore, we studied the effects of the pan-cGMP PDE inhibitor zaprinast on the metabolic and vascular actions of insulin in muscle. Hyperinsulinemic euglycemic clamps (3 mU·min−1·kg−1) were performed in anesthetized rats and changes in microvascular blood flow assessed from rates of 1-methylxanthine metabolism across the muscle bed by capillary xanthine oxidase in response to insulin and zaprinast. We also characterized cGMP PDE isoform expression in muscle by real-time PCR and immunostaining of frozen muscle sections. Zaprinast enhanced insulin-mediated microvascular perfusion by 29% and muscle glucose uptake by 89%, while whole body glucose infusion rate during insulin infusion was increased by 33% at 2 h. PDE2, -9, and -10 were the major isoforms expressed at the mRNA level in muscle, while PDE1B, -9A, -10A, and -11A proteins were expressed in blood vessels. Acute administration of the cGMP PDE inhibitor zaprinast enhances muscle microvascular blood flow and glucose uptake response to insulin. The expression of a number of cGMP PDE isoforms in skeletal muscle suggests that targeting specific cGMP PDE isoforms may provide a promising avenue for development of a novel class of therapeutics for enhancing muscle insulin sensitivity.

Skeletal muscle reactive oxygen species: a target of good cop/bad cop for exercise and disease

Metabolic stresses associated with disease, ageing, and exercise increase the levels of reactive oxygen species (ROS) in skeletal muscle. These ROS have been linked mechanistically to adaptations in skeletal muscle that can be favourable (i.e. in response to exercise) or detrimental (i.e. in response to disease). The magnitude, duration (acute versus chronic), and cellular origin of the ROS are important underlying factors in determining the metabolic perturbations associated with the ROS produced in skeletal muscle. In particular, insulin resistance has been linked to excess ROS production in skeletal muscle mitochondria. A chronic excess of mitochondrial ROS can impair normal insulin signalling pathways and glucose disposal in skeletal muscle. In contrast, ROS produced in skeletal muscle in response to exercise has been linked to beneficial metabolic adaptations including mitochondrial biogenesis and muscle hypertrophy. Moreover, unlike insulin resistance, exercise-induced ROS appears to be primarily of non-mitochondrial origin. The present review summarizes the diverse ROS-targeted metabolic outcomes associated with insulin resistance versus exercise in skeletal muscle, thus, presenting two contrasting perspectives of pathologically harmful versus physiologically beneficial ROS. Here, we discuss the key sites of ROS production during exercise and the effect of ROS in skeletal muscle of people with type 2 diabetes.

Modulating exercise-induced hormesis: does less equal more?

Hormesis encompasses the notion that low levels of stress stimulate or upregulate existing cellular and molecular pathways that improve the capacity of cells and organisms to withstand greater stress. This notion underlies much of what we know about how exercise conditions the body and induces long-term adaptations. During exercise, the body is exposed to various forms of stress, including thermal, metabolic, hypoxic, oxidative, and mechanical stress. These stressors activate biochemical messengers, which in turn activate various signaling pathways that regulate gene expression and adaptive responses. Historically, antioxidant supplements, nonsteroidal anti-inflammatory drugs, and cryotherapy have been favored to attenuate or counteract exercise-induced oxidative stress and inflammation. However, reactive oxygen species and inflammatory mediators are key signaling molecules in muscle, and such strategies may mitigate adaptations to exercise. Conversely, withholding dietary carbohydrate and restricting muscle blood flow during exercise may augment adaptations to exercise. In this review article, we combine, integrate, and apply knowledge about the fundamental mechanisms of exercise adaptation. We also critically evaluate the rationale for using interventions that target these mechanisms under the overarching concept of hormesis. There is currently insufficient evidence to establish whether these treatments exert dose-dependent effects on muscle adaptation. However, there appears to be some dissociation between the biochemical/molecular effects and functional/performance outcomes of some of these treatments. Although several of these treatments influence common kinases, transcription factors, and proteins, it remains to be determined if these interventions complement or negate each other, and whether such effects are strong enough to influence adaptations to exercise.

Short-term intensified cycle training alters acute and chronic responses of PGC1α and cytochrome C oxidase IV to exercise in human skeletal muscle

Reduced activation of exercise responsive signalling pathways have been reported in response to acute exercise after training; however little is known about the adaptive responses of the mitochondria. Accordingly, we investigated changes in mitochondrial gene expression and protein abundance in response to the same acute exercise before and after 10-d of intensive cycle training.

Nine untrained, healthy participants (mean±SD; VO2peak 44.1±17.6 ml/kg/min) performed a 60 min bout of cycling exercise at 164±18 W (72% of pre-training VO2peak). Muscle biopsies were obtained from the vastus lateralis muscle at rest, immediately and 3 h after exercise. The participants then underwent 10-d of cycle training which included four high-intensity interval training sessions (6×5 min; 90–100% VO2peak) and six prolonged moderate-intensity sessions (45–90 min; 75% VO2peak). Participants repeated the pre-training exercise trial at the same absolute work load (64% of pre-training VO2peak). Muscle PGC1-α mRNA expression was attenuated as it increased by 11- and 4- fold (P<0.001) after exercise pre- and post-training, respectively. PGC1-α protein expression increased 1.5 fold (P<0.05) in response to exercise pre-training with no further increases after the post-training exercise bout. RIP140 protein abundance was responsive to acute exercise only (P<0.01). COXIV mRNA (1.6 fold; P<0.01) and COXIV protein expression (1.5 fold; P<0.05) were increased by training but COXIV protein expression was decreased (20%; P<0.01) by acute exercise pre- and post-training.

These findings demonstrate that short-term intensified training promotes increased mitochondrial gene expression and protein abundance. Furthermore, acute indicators of exercise-induced mitochondrial adaptation appear to be blunted in response to exercise at the same absolute intensity following short-term training.

Copper in disorders with neurological symptoms: Alzheimer`s, Menkes, and Wilson Diseases

Copper is an essential element for the activity of a number of physiologically important enzymes. Enzyme-related malfunctions may contribute to severe neurological symptoms and neurological diseases: copper is a component of cytochrome c oxidase, which catalyzes the reduction of oxygen to water, the essential step in cellular respiration. Copper is a cofactor of Cu/Zn-superoxide-dismutase which plays a key role in the cellular response to oxidative stress by scavenging reactive oxygen species. Furthermore, copper is a constituent of dopamine-β-hydroxylase, a critical enzyme in the catecholamine biosynthetic pathway. A detailed exploration of the biological importance and functional properties of proteins associated with neurological symptoms will have an important impact on understanding disease mechanisms and may accelerate development and testing of new therapeutic approaches. Copper binding proteins play important roles in the establishment and maintenance of metal-ion homeostasis, in deficiency disorders with neurological symptoms (Menkes disease, Wilson disease) and in neurodegenerative diseases (Alzheimer’s disease). The Menkes and Wilson proteins have been characterized as copper transporters and the amyloid precursor protein (APP) of Alzheimer’s disease has been proposed to work as a Cu(II) and/or Zn(II) transporter. Experimental, clinical and epidemiological observations in neurodegenerative disorders like Alzheimer’s disease and in the genetically inherited copper-dependent disorders Menkes and Wilson disease are summarized. This could provide a rationale for a link between severely dysregulated metal-ion homeostasis and the selective neuronal pathology.

Utility of mitochondrial DNA sequences from four gene regions for systematic studies of Australian freshwater crayfish of the Genus Cherax (Decapoda: Parastacidae)

One of the most important requirements for systematic and phylogenetic studies is the identification of gene regions with the appropriate level of variation for the question of interest. Molecular phylogenetic and systematic studies of freshwater crayfish have made use of DNA sequences mainly from ribosomal genes, especially the 16S rRNA gene region. Thus, little information is available on other potentially useful mitochondrial gene regions for systematic studies in these animals. In this study, we look at nucleotide variation and phylogenetic relations within and between four species of freshwater crayfish of the genus Cherax from the southwest of Western Australia using four fragments amplified from the 16S rRNA, 12S rRNA, Cytochrome Oxidase I (COI), and Cytochrome b (Cyt b) gene regions. Samples of Engaeus strictifrons, Euastacus bispinosus, and Geocharax falcata were also sequenced for comparative purposes. The size of the fragments varied from 358 bp to 600 bp. Across all samples, the four fragments showed significant phylogenetic signal and showed similar proportions of variable sites (28.81–37.33%). Average divergence within species for the mitochondrial gene regions varied from 1.18% to 4.91%, with the 16S rRNA being the least variable and Cyt b the most variable. Average divergence between species ranged 7.63–15.53%, with 16S rRNA being the least variable and COI the most variable. At the generic level, average divergence ranged 17.21–23.82%. Phylogenetic analyses of the 16S rRNA, 12S rRNA, and COI regions generated four clades consistent with the presence of four species previously identified on the basis of allozyme and morphological studies. The relationships among samples were largely congruent across the data set, although some relationships remained unresolved. Not all samples could be amplified using the Cyt b primers, and some of those that were showed quite anomalous relationships, suggesting that one or more Cyt b pseudogenes were being amplified.