Regulation of glycogen synthase and phosphorylase during recovery from high-intensity exercise in the rat

The aim of this study was to determine the role of the phosphorylation state of glycogen synthase and glycogen phosphorylase in the regulation of muscle glycogen repletion in fasted animals recovering from high-intensity exercise. Groups of rats were swum to exhaustion and allowed to recover for up to 120 min without access to food. Swimming to exhaustion caused substantial glycogen breakdown and lactate accumulation in the red, white and mixed gastrocnemius muscles, whereas the glycogen content in the soleus muscle remained stable. During the first 40 min of recovery, significant repletion of glycogen occurred in all muscles examined except the soleus muscle. At the onset of recovery, the activity ratios and fractional velocities of glycogen synthase in the red, white and mixed gastrocnemius muscles were higher than basal, but returned to pre-exercise levels within 20 min after exercise. In contrast, after exercise the activity ratios of glycogen phosphorylase in the same muscles were lower than basal, and increased to pre-exercise levels within 20 min. This pattern of changes in glycogen synthase and phosphorylase activities, never reported before, suggests that the integrated regulation of the phosphorylation state of both glycogen synthase and phosphorylase might be involved in the control of glycogen deposition after high-intensity exercise.

Slow component of VO2 kinetics: the effect of training status, fibre type, UCP3 mRNA and citrate synthase activity

Examines the relationship between the magnitude of the relative slow component (SC) of pulmonary oxygen uptake VO[sub 2], citrate synthase activity, UCP2 and UCP3 mRNA levels and muscle fiber composition in both endurance-trained and recreationally active subjects. Magnitude of the relative SC of the Tr group; Indicators of aerobic fitness; High negative correlations between the magnitude of the relative SC and citrate synthase activity and VO[sub 2] peak.

Identification of a by-product of nitric oxide synthase activity in human acute brain injury with in vivo proton magnetic resonance spectroscopy

BACKGROUND AND PURPOSE: Laboratory studies have been used to identify nitric oxide as a notable mediator in neuronal death after acute brain injury. To our knowledge, this has not previously been confirmed with in vivo study in humans. Our purpose was to seek in vivo evidence for the induction of nitric oxide synthase (NOS) in human acute brain injury by using proton MR spectroscopy.

METHODS: In vitro proton MR spectra were obtained in neural extracts from 30 human cadavers, and in vivo spectra were obtained in 20 patients with acute brain injury and in a similar number of control subjects.

RESULTS: We identified a unique peak at 3.15 ppm by using in vivo proton MR spectroscopy in eight of 20 patients with acute brain injury but not in 20 healthy volunteers (P < .002). On the basis of in vitro data, we have tentatively assigned this peak to citrulline, a NOS by-product.

CONCLUSION: To our knowledge, our findings suggest, for the first time, that excitotoxicity may occur in human acute brain injury. Confirmation with the acquisition of spectra in very early acute cerebral injury would provide a rationale for the use of neuroprotective agents in these conditions, as well as a new noninvasive method for quantification.

Effect of AICAR treatment on gycogen metabolism in skeletal muscle

AMP-activated protein kinase (AMPK) is proposed to stimulate fat and carbohydrate catabolism to maintain cellular energy status. Recent studies demonstrate that pharmacologic activation of AMPK and mutations in the enzyme are associated with elevated muscle glycogen content in vivo. Our purpose was to determine the mechanism for increased muscle glycogen associated with AMPK activity in vivo. AMPK activity and glycogen metabolism were studied in red and white gastrocnemius muscles from rats treated with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) in vivo, and also in muscles incubated with AICAR in vitro. In vivo AICAR treatment reduced blood glucose and increased blood lactate compared with basal values. AICAR increased muscle α2 AMPK activity, glycogen, and glucose-6-phosphate concentrations. Glycogen synthase activity was increased in the red gastrocnemius but was decreased in the white gastrocnemius. Glycogen phosphorylase activity increased in both muscles, with an inhibition initially observed in the red gastrocnemius. In vitro incubation with AICAR activated α2 AMPK but had no effect on either glycogen synthase or glycogen phosphorylase. These results suggest that AICAR treatment does not promote glycogen accumulation in skeletal muscle in vivo by altering glycogen synthase and glycogen phosphorylase. Rather, the increased glycogen is due to the well-known effects of AICAR to increase glucose uptake.

Fiber-specific responses of muscle glycogen repletion in fasted rats physically active during recovery from high-intensity physical exertion

Mild physical activity performed immediately after a bout of intense exercise in fasting humans results in net glycogen breakdown in their slow oxidative (SO) muscle fibers and glycogen repletion in their fast twitch (FT) fibers. Because several animal species carry a low proportion of SO fibers, it is unclear whether they can also replenish glycogen in their FT fibers under these conditions. Given that most skeletal muscles in rats are poor in SO fibers (<5%), this issue was examined using groups of 24-h fasted Wistar rats (n = 10) that swam for 3 min at high intensity with a 10% weight followed by either a 60-min rest (passive recovery, PR) or a 30-min swim with a 0.5% weight (active recovery, AR) preceding a 30-min rest. The 3-min sprint caused 61–79% glycogen fall across the muscles examined, but not in the soleus (SOL). Glycogen repletion during AR without food was similar to PR in the white gastrocnemius (WG), where glycogen increased by 71%, and less than PR in both the red and mixed gastrocnemius (RG, MG). Glycogen fell by 26% during AR in the SOL. Following AR, glycogen increased by 36%, 87%, and 37% in the SOL, RG, and MG, respectively, and this was accompanied by the sustained activation of glycogen synthase and inhibition of glycogen phosphorylase in the RG and MG. These results suggest that mammals with a low proportion of SO fibers can also replenish the glycogen stores of their FT fibers under extreme conditions combining physical activity and fasting.

Post-exercise muscle glycogen repletion in the extreme : effect of food absence and active recovery

Glycogen plays a major role in supporting the energy demands of skeletal muscles during high intensity exercise. Despite its importance, the amount of glycogen stored in skeletal muscles is so small that a large fraction of it can be depleted in response to a single bout of high intensity exercise. For this reason, it is generally recommended to ingest food after exercise to replenish rapidly muscle glycogen stores, otherwise one's ability to engage in high intensity activity might be compromised. But what if food is not available? It is now well established that, even in the absence of food intake, skeletal muscles have the capacity to replenish some of their glycogen at the expense of endogenous carbon sources such as lactate. This is facilitated, in part, by the transient dephosphorylation-mediated activation of glycogen synthase and inhibition of glycogen phosphorylase. There is also evidence that muscle glycogen synthesis occurs even under conditions conducive to an increased oxidation of lactate post-exercise, such as during active recovery from high intensity exercise. Indeed, although during active recovery glycogen resynthesis is impaired in skeletal muscle as a whole because of increased lactate oxidation, muscle glycogen stores are replenished in Type IIa and IIb fibers while being broken down in Type I fibers of active muscles. This unique ability of Type II fibers to replenish their glycogen stores during exercise should not come as a surprise given the advantages in maintaining adequate muscle glycogen stores in those fibers that play a major role in fight or flight responses.

Postexercise muscle glycogen resynthesis in obese insulin-resistant Zucker rats

We determined the effect of an acute bout of swimming (8 × 30 min) followed by either carbohydrate administration (0.5 mg/g glucose ip and ad libitum access to chow; CHO) or fasting (Fast) on postexercise glycogen resynthesis in soleus muscle and liver from female lean (ZL) and obese insulin-resistant (ZO) Zucker rats. Resting soleus muscle glycogen concentration ([glycogen]) was similar between genotypes and was reduced by 73 (ZL) and 63% (ZO) after exercise (P < 0.05). Liver [glycogen] at rest was greater in ZO than ZL (334 ± 31 vs. 247 ± 16 μmol/g wet wt; P < 0.01) and fell by 44 and 94% after exercise (P < 0.05). The fractional activity of glycogen synthase (active/total) increased immediately after exercise (from 0.22 ± 0.05 and 0.32 ± 0.04 to 0.63 ± 0.08 vs. 0.57 ± 0.05; P < 0.01 for ZL and ZO rats, respectively) and remained elevated above resting values after 30 min of recovery. During this time, muscle [glycogen] in ZO increased 68% with CHO (P < 0.05) but did not change in Fast. Muscle [glycogen] was unchanged in ZL from postexercise values after both treatments. After 6 h recovery, GLUT-4 protein concentration was increased above resting levels by a similar extent for both genotypes in both fasted (∼45%) and CHO-supplemented (∼115%) rats. Accordingly, during this time CHO refeeding resulted in supercompensation in both genotypes (68% vs. 44% for ZL and ZO). With CHO, liver [glycogen] was restored to resting levels in ZL but remained at postexercise values for ZO after both treatments. We conclude that the increased glucose availability with carbohydrate refeeding after glycogen-depleting exercise resulted in glycogen supercompensation, even in the face of muscle insulin-resistance.

Antimanic-like activity of candesartan in mice: possible involvement of antioxidant, anti-inflammatory and neurotrophic mechanisms

Activation of the brain angiotensin II type 1 receptor (AT1R) triggers pro-oxidant and pro-inflammatory mechanisms which are involved in the neurobiology of bipolar disorder (BD). Candesartan (CDS) is an AT1 receptor antagonist with potential neuroprotective properties. Herein we investigated CDS effects against oxidative, neurotrophic inflammatory and cognitive effects of amphetamine (AMPH)-induced mania. In the reversal protocol adult mice were given AMPH 2mg/kg i.p. or saline and between days 8 and 14 received CDS 0.1, 0.3 or 1mg/kg orally, lithium (Li) 47.5mg/kg i.p., or saline. In the prevention treatment, mice were pretreated with CDS, Li or saline prior to AMPH. Locomotor activity and working memory performance were assessed. Glutathione (GSH), thiobarbituric acid-reactive substance (TBARS) and TNF-α levels were evaluated in the hippocampus (HC) and cerebellar vermis (CV). Brain-derived neurotrophic factor (BDNF) and glycogen synthase kinase 3-beta (GSK-3beta) levels were measured in the HC. CDS and Li prevented and reversed the AMPH-induced increases in locomotor activity. Only CDS prevented and reversed AMPH-induced working memory deficits. CDS prevented AMPH-induced alterations in GSH (HC and CV), TBARS (HC and CV), TNF-α (HC and CV) and BDNF (HC) levels. Li prevented alterations in BDNF and phospho-Ser9-GSK3beta. CDS reversed AMPH-induced alterations in GSH (HC and CV), TBARS (HC), TNF-α (CV) and BDNF levels. Li reversed AMPH-induced alterations in TNF-α (HC and CV) and BDNF (HC) levels. CDS is effective in reversing and preventing AMPH-induced behavioral and biochemical alterations, providing a rationale for the design of clinical trials investigating CDS׳s possible therapeutic effects.

Effect of short-term training on GLUT-4 mRNA and protein expression in human skeletal muscle

Six untrained, male subjects (23 ± 1 years old, 84 ± 5 kg, V_O2peak= 3.7 ± 0.8 l min^–1) exercised for 60 min at 75 ± 1%V_O2peak on 7 consecutive days.  Muscle samples were obtained before the start of cycle exercise training and 24 h after the first and seventh exercise sessions and analysed for citrate synthase activity, glycogen and glucose transporter 4 (GLUT-4) mRNA and protein expression. Exercise training increased (P < 0.05) citrate synthase by ~20% and muscle glycogen concentration by ~40%. GLUT-4 mRNA levels 24 h after the first and seventh exercise sessions were similar to those  measured before the start of exercise training. In contrast, GLUT-4 protein expression was increased after 7 days of exercise training (12.4 ± 1.5 versus 3.4 ± 1.0 arbitray units (a.u.), P < 0.05) and although it tended to be higher 24 h after the first exercise session (6.0 ± 3.0 versus 3.4 ± 1.0 a.u.), this was not significantly different (P= 0.09). These results support the suggestion that the adaptive increase in skeletal muscle GLUT-4 protein expression with short-term exercise training arises from the repeated, transient increases in GLUT-gene transcription following each exercise bout leading to a gradual accumulation of GLUT-4 protein, despite GLUT-4 mRNA returning to basal levels between exercise stimuli.

Creatine supplementation increases glycogen storage but not GLUT-4 expression in human skeletal muscle

It has been speculated that creatine supplementation affects muscle glucose metabolism in humans by increasing muscle glycogen storage and up-regulating GLUT-4 protein expression. In the present study, we assessed the effects of creatine loading and prolonged supplementation on muscle glycogen storage and GLUT-4 mRNA and protein content in humans. A total of 20 subjects participated in a 6-week supplementation period during which creatine or a placebo was ingested. Muscle biopsies were taken before and after 5 days of creatine loading (20 g.day(-1)) and after 6 weeks of continued supplementation (2 g.day(-1)). Fasting plasma insulin concentrations, muscle creatine, glycogen and GLUT-4 protein content as well as GLUT-4, glycogen synthase-1 (GS-1) and glycogenin-1 (Gln-1) mRNA expression were determined. Creatine loading significantly increased total creatine, free creatine and creatine phosphate content with a concomitant 18 +/- 5% increase in muscle glycogen content (P<0.05). The subsequent use of a 2 g.day(-1) maintenance dose for 37 days did not maintain total creatine, creatine phosphate and glycogen content at the elevated levels. The initial increase in muscle glycogen accumulation could not be explained by an increase in fasting plasma insulin concentration, muscle GLUT-4 mRNA and/or protein content. In addition, neither muscle GS-1 nor Gln-1 mRNA expression was affected. We conclude that creatine ingestion itself stimulates muscle glycogen storage, but does not affect muscle GLUT-4 expression.

U18666A-mediated apoptosis in cultured murine cortical neurons : role of caspases, calpains and kinases

Studies have suggested that cholesterol imbalance in the brain might be related to the development of neurological disorders such as Alzheimer's disease and Niemann–Pick disease type C. Previously, we have reported that U18666A, a cholesterol transport-inhibiting agent, leads to apoptosis and intracellular cholesterol accumulation in primary cortical neurons. In this study, we examined the effects of U18666A-mediated neuronal apoptosis, and found that chronic exposure to U18666A led to the activation of caspases and calpains and hyperphosphorylation of tau. Tau hyperphosphorylation is regulated by several kinases that phosphorylate specific sites of tau in vitro. Surprisingly, the kinase activity of cyclin-dependent kinase 5 decreased in U18666A-treated cortical neurons whereas its protein level remained unchanged. The amount of glycogen synthase kinase 3 and mitogen-activated protein kinases were found to decrease in their phosphorylated states by Western blot analysis. Gene transcription was further studied using microarray analysis. Genes encoding for kinases and phosphatases were differentially expressed with most up-regulated and some down-regulated in expression upon U18666A treatment. The activation of cysteine proteases and cholesterol accumulation with tauopathies may provide clues to the cellular mechanism of the inhibition of cholesterol transport-mediated cell death in neurodegenerative diseases.

New drug targets in depression : inflammatory, cell-mediated immune, oxidative and nitrosative stress, mitochondrial, antioxidant, and neuroprogressive pathways. And new drug candidates—Nrf2 activators and GSK-3 inhibitors

This paper reviews new drug targets in the treatment of depression and new drug candidates to treat depression. Depression is characterized by aberrations in six intertwined pathways: (1) inflammatory pathways as indicated by increased levels of proinflammatory cytokines, e.g. interleukin-1 (IL-1), IL-6, and tumour necrosis factor α. (2) Activation of cell-mediated immune pathways as indicated by an increased production of interferon γ and neopterin. (3) Increased reactive oxygen and nitrogen species and damage by oxidative and nitrosative stress (O&NS), including lipid peroxidation, damage to DNA, proteins and mitochondria. (4) Lowered levels of key antioxidants, such as coenzyme Q10, zinc, vitamin E, glutathione, and glutathione peroxidase. (5) Damage to mitochondria and mitochondrial DNA and reduced activity of respiratory chain enzymes and adenosine triphosphate production. (6) Neuroprogression, which is the progressive process of neurodegeneration, apoptosis, and reduced neurogenesis and neuronal plasticity, phenomena that are probably caused by inflammation and O&NS. Antidepressants tend to normalize the above six pathways. Targeting these pathways has the potential to yield antidepressant effects, e.g. using cytokine antagonists, minocycline, Cox-2 inhibitors, statins, acetylsalicylic acid, ketamine, ω3 poly-unsaturated fatty acids, antioxidants, and neurotrophic factors. These six pathways offer new, pathophysiologically guided drug targets suggesting that novel therapies could be developed that target these six pathways simultaneously. Both nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activators and glycogen synthase kinase-3 (GSK-3) inhibitors target the six above-mentioned pathways. GSK-3 inhibitors have antidepressant effects in animal models of depression. Nrf2 activators and GSK-3 inhibitors have the potential to be advanced to phase-2 clinical trials to examine whether they augment the efficacy of antidepressants or are useful as monotherapy.

Effect of streptozotocin-induced diabetes on glycogen resynthesis in fasted rats post-high-intensity exercise

It has recently been shown that food intake is not essential for the resynthesis of the stores of muscle glycogen in fasted animals recovering from high-intensity exercise. Because the effect of diabetes on this process has never been examined before, we undertook to explore this issue. To this end, groups of rats were treated with streptozotocin (60 mg/kg body mass ip) to induce mild diabetes. After 11 days, each animal was fasted for 24 h before swimming with a lead weight equivalent to 9% body mass attached to the tail. After exercise, the rate and the extent of glycogen repletion in muscles were not affected by diabetes, irrespective of muscle fiber composition. Consistent with these findings, the effect of exercise on the phosphorylation state of glycogen synthase in muscles was only minimally affected by diabetes. In contrast to its effects on nondiabetic animals, exercise in fasted diabetic rats was accompanied by a marked fall in hepatic glycogen levels, which, surprisingly, increased to preexercise levels during recovery despite the absence of food intake.