Clenbuterol induces muscle-specific attenuation of atrophy through effects on the ubiquitin-proteasome pathway.

The ubiquitin-proteasome pathway is primarily responsible for myofibrillar protein degradation during hindlimb unweighting (HU). Beta-adrenergic agonists such as clenbuterol (CB) induce muscle hypertrophy and attenuate muscle atrophy due to disuse or inactivity. However, the molecular mechanism by which CB exerts these effects remains poorly understood. The aims of this study were to investigate whether CB attenuates HU-induced muscle atrophy through an inhibition of the ubiquitin-proteasome pathway and whether insulin-like growth factor I (IGF-I) mediates this inhibition. Rats were randomized to the following groups: weight-bearing control, 14-day CB-treated, 14-day HU, and CB + HU. HU-induced atrophy was associated with increased proteolysis and upregulation of components of the ubiquitin-proteasome pathway (ubiquitin conjugates, ubiquitin conjugating enzyme E2-14 kDa, and 20S proteasome activity). Upregulation of the ubiquitin proteasome occurred in all muscles tested but was more pronounced in muscles composed primarily of slow-twitch fibers (soleus) than in fast-twitch muscles (plantaris and tibialis anterior). Although CB induced hypertrophy in all muscles, CB attenuated the HU-induced atrophy and reduced ubiquitin conjugates only in the fast plantaris and tibialis anterior and not in the slow soleus muscle. CB did not elevate IGF-I protein content in either of the muscles examined. These results suggest that CB induces hypertrophy and alleviates HU-induced atrophy, particularly in the fast muscles, at least in part through a muscle-specific inhibition of the ubiquitin-proteasome pathway and that these effects are not mediated by the local production of IGF-I in skeletal muscle.     

Response of the ubiquitin-proteasome pathway to changes in muscle activity

The ubiquitin-proteasome pathway plays a critical role in the adaptation of skeletal muscle to persistent decreases or increases in muscle activity. This article outlines the basics of pathway function and reviews what we know about pathway responses to altered muscle use. The ubiquitin-proteasome pathway regulates proteolysis in mammalian cells by attaching ubiquitin polymers to damaged proteins; this targets the protein for degradation via the 26S proteasome. The pathway is constitutively active in muscle and continually regulates protein turnover. Conditions of decreased muscle use, e.g., unloading, denervation, or immobilization, stimulate general pathway activity. This activity increase is caused by upregulation of regulatory components in the pathway and leads to accelerated proteolysis, resulting in net loss of muscle protein. Pathway activity is also increased in response to exercise, a two-phase response. An immediate increase in selective ubiquitin conjugation by constitutive pathway components contributes to exercise-stimulated signal transduction. Over hours-to-days, exercise also stimulates a delayed increase in general ubiquitin conjugating activity by inducing expression of key components in the pathway. This increase mediates a late-phase rise in protein degradation that is required for muscle adaptation to exercise. Thus the ubiquitin-proteasome pathway functions as an essential mediator of muscle remodeling, both in atrophic states and exercise training.     

Chronic contractile activity upregulates the proteasome system in rabbit skeletal muscle.

Remodeling of skeletal muscle in response to altered patterns of contractile activity is achieved, in part, by the regulated degradation of cellular proteins. The ubiquitin-proteasome system is a dominant pathway for protein degradation in eukaryotic cells. To test the role of this pathway in contraction-induced remodeling of skeletal muscle, we used a well-established model of continuous motor nerve stimulation to activate tibialis anterior (TA) muscles of New Zealand White rabbits for periods up to 28 days. Western blot analysis revealed marked and coordinated increases in protein levels of the 20S proteasome and two of its regulatory proteins, PA700 and PA28. mRNA of a representative proteasome subunit also increased coordinately in contracting muscles. Chronic contractile activity of TA also increased total proteasome activity in extracts, as measured by the hydrolysis of a proteasome-specific peptide substrate, and the total capacity of the ubiquitin-proteasome pathway, as measured by the ATP-dependent hydrolysis of an exogenous protein substrate. These results support the potential role of the ubiquitin-proteasome pathway of protein degradation in the contraction-induced remodeling of skeletal muscle.     

The ubiquitin-proteasome proteolytic pathway in heart vs skeletal muscle: effects of acute diabetes

The ubiquitin-proteasome system is thought to play a major role in normal muscle protein turnover and to contribute to diabetes-induced protein wasting in skeletal muscle. However, its importance in cardiac muscle is not clear. We measured heart muscle mRNA for ubiquitin and for the C2 and C8 proteasomal subunits, the amount of free ubiquitin and the proteasome chymotrypsin-like proteolytic activity in control and diabetic rats. Results were compared to those in skeletal muscle (rectus). Heart ubiquitin, C2 and C8 subunit mRNA and proteolytic activity were significantly greater than in skeletal muscle (P 相似文献   

Downregulation of ubiquitin-dependent proteolysis by eicosapentaenoic acid in acute starvation.

A number of acute wasting conditions are associated with an upregulation of the ubiquitin-proteasome system in skeletal muscle. Eicosapentaenoic acid (EPA) is effective in attenuating the increased protein catabolism in muscle in cancer cachexia, possibly due to inhibition of 15-hydroxyeicosatetraenoic acid (15-HETE) formation. To determine if a similar pathway is involved in other catabolic conditions, the effect of EPA on muscle protein degradation and activation of the ubiquitin-proteasome pathway has been determined during acute fasting in mice. When compared with a vehicle control group (olive oil) there was a significant decrease in proteolysis of the soleus muscles of mice treated with EPA after starvation for 24 h, together with an attenuation of the proteasome "chymotryptic-like" enzyme activity and the induction of the expression of the 20S proteasome alpha-subunits, the 19S regulator and p42, an ATPase subunit of the 19S regulator in gastrocnemius muscle, and the ubiquitin-conjugating enzyme E2(14k). The effect was not shown with the related (n-3) fatty acid docosahexaenoic acid (DHA) or with linoleic acid. However, 2,3,5-trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504), an inhibitor of 5-, 12- and 15-lipoxygenases also attenuated muscle protein catabolism, proteasome "chymotryptic-like" enzyme activity and expression of proteasome 20S alpha-subunits in soleus muscles from acute fasted mice. These results suggest that protein catabolism in starvation and cancer cachexia is mediated through a common pathway, which is inhibited by EPA and is likely to involve a lipoxygenase metabolite as a signal transducer.     

Glycogen overload by postexercise insulin administration abolished the exercise-induced increase in GLUT4 protein

Summary To elucidate the role of muscle glycogen storage on regulation of GLUT4 protein expression and whole-body glucose tolerance, muscle glycogen level was manipulated by exercise and insulin administration. Sixty Sprague-Dawley rats were evenly separated into three groups: control (CON), immediately after exercise (EX0), and 16 h after exercise (EX16). Rats from each group were further divided into two groups: saline- and insulin-injected. The 2-day exercise protocol consisted of 2 bouts of 3-h swimming with 45-min rest for each day, which effectively depleted glycogen in both red gastrocnemius (RG) and plantaris muscles. EX0 rats were sacrificed immediately after the last bout of exercise on second day. CON and EX16 rats were intubated with 1 g/kg glucose solution following exercise and recovery for 16 h before muscle tissue collection. Insulin (0.5 μU/kg) or saline was injected daily at the time when glucose was intubated. Insulin injection elevated muscle glycogen levels substantially in both muscles above saline-injected group at CON and EX16. With previous day insulin injection, EX0 preserved greater amount of postexercise glycogen above their saline-injected control. In the saline-injected rats, EX16 significantly increased GLUT4 protein level above CON, concurrent with muscle glycogen supercompensation. Insulin injection for EX16 rats significantly enhanced muscle glycogen level above their saline-injected control, but the increases in muscle GLUT4 protein and whole-body glucose tolerance were attenuated. In conclusion, the new finding of the study was that glycogen overload by postexercise insulin administration significantly abolished the exercise-induced increases in GLUT4 protein and glucose tolerance.     

Transgenic mice overexpressing GLUT-1 protein in muscle exhibit increased muscle glycogenesis after exercise

The purpose of the present study was to determine the rates of muscle glycogenolysis and glycogenesis during and after exercise in GLUT-1 transgenic mice and their age-matched littermates. Male transgenic mice (TG) expressing a high level of human GLUT-1 and their nontransgenic (NT) littermates underwent 3 h of swimming. Glycogen concentration was determined in gastrocnemius and extensor digitorum longus (EDL) muscles before exercise and at 0, 5, and 24 h postexercise, during which food (chow) and 10% glucose solution (as drinking water) were provided. Exercise resulted in approximately 90% reduction in muscle glycogen in both NT (from 11.2 +/- 1.4 to 2. 1 +/- 1.3 micromol/g) and TG (from 99.3 +/- 4.7 to 11.8 +/- 4.3 micromol/g) in gastrocnemius muscle. During recovery from exercise, the glycogen concentration increased to 38.2 +/- 7.3 (5 h postexercise) and 40.5 +/- 2.8 micromol/g (24 h postexercise) in NT mice. In TG mice, however, the increase in muscle glycogen concentration during recovery was greater (to 57.5 +/- 7.4 and 152.1 +/- 15.7 micromol/g at 5 and 24 h postexercise, respectively). Similar results were obtained from EDL muscle. The rate of 2-deoxyglucose uptake measured in isolated EDL muscles was 7- to 10-fold higher in TG mice at rest and at 0 and 5 h postexercise. There was no difference in muscle glycogen synthase activation measured in gastrocnemius muscles between NT and TG mice immediately after exercise. These results demonstrate that the rate of muscle glycogen accumulation postexercise exhibits two phases in TG: 1) an early phase (0-5 h), with rapid glycogen accumulation similar to that of NT mice, and 2) a progressive increase in muscle glycogen concentration, which differs from that of NT mice, during the second phase (5-24 h). Our data suggest that the high level of steady-state muscle glycogen in TG mice is due to the increase in muscle glucose transport activity.     

Skeletal muscle RNA synthesis following endurance and sprint exercise

Two groups of male Wistar endurance- and sprint-acclimatized rats were used to study the time course of uridine uptake into skeletal muscle RNA following acute exercise. Endurance and sprint animals were killed at 0, 2, 18, 24, and 48 hr following 1 hr of either endurance (30 m X min-1) or sprint running (90 m X min-1). Red vastus (RV) and white vastus (WV) muscle samples were incubated for 30 min in a medium containing 1 microCi 5-[14C]uridine. Uridine uptake was determined in the myofibrillar-nuclear, mitochondrial, microsomal, and soluble fractions of skeletal muscle via liquid scintillation counting. A significant decrease in whole muscle uridine uptake into RNA was observed in RV muscles following endurance exercise as well as in WV of sprint-exercised rats. Sprint-exercised RV had significantly greater uridine uptake into RNA in the homogenate and myofibrillar-nuclear fraction 2-18 hr post exercise. Increased mitochondrial uridine incorporation into RNA was observed in endurance- and sprint-exercised muscles between 18 and 48 hr post exercise. A very large increment in microsomal uridine uptake was observed in sprint-exercised WV at 24 hr. These data suggest that while whole muscle RNA synthesis may decline immediately following acute exercise overload, increases are observed in specific muscle fractions. These changes appear to coincide with protein-specific adaptations to sprint and endurance exercise.     

Role of the ubiquitin-proteasome pathway in sepsis-induced muscle catabolism

Several lines of evidence suggest that the ubiquitin-proteasome pathway is involved in sepsis-induced muscle catabolism. The gene expression of ubiquitin and several of the proteasome subunits was increased in muscle from both septic rats and patients. In other studies, the activity of isolated 20S proteasomes was stimulated in septic muscles. Sepsis-induced increase in muscle total and myofibrillar protein breakdown was inhibited with specific proteasome blockers. Although the ubiquitin-proteasome pathway is upregulated in septic muscle, it is still unclear how the myofibrillar proteins actin and myosin are ubiquitinated and become substrates for the 26S proteasome. Recent studies suggest that a calcium-dependent, calpain-mediated process releases myofilaments from the Z-disks during sepsis. It is possible that this process exposes destabilizing N-terminal residues on actin and myosin, making them suitable substrates for the N-end rule pathway involving the 14 kD ubiquitin-conjugating enzyme E214k and the ubiquitin-protein ligase E3.     

Estrogen effect on post-exercise skeletal muscle neutrophil infiltration and calpain activity

We hypothesized that estrogen administration would attenuate skeletal muscle neutrophil infiltration, indices of muscle membrane disruption, and muscle calpain activity shortly after the termination of exercise. Ovariectomized female rats were implanted with either an estogen pellet (25 mg beta-estradiol) or a placebo pellet. Two weeks postimplant, animals were killed either at rest or 1 h after running exercise (60 min at 21 m x min(-1), 12% grade). The 4 experimental groups (n = 12) used were: unexercised placebo (UP), unexercised estrogen (UE), exercised placebo (EP), and exercised estrogen (EE). Blood samples were analyzed for creatine kinase (CK) activity and estradiol content. Plantaris and gastrocnemius muscles were removed and histochemical determination of neutrophil content or biochemical determination of myeloperoxidase (MPO), glucose-6-phosphate dehydrogenase (G6PD), and calpain-like activity determined. Estrogen supplemented animals had 10-20-fold higher circulating estradiol levels than placebo animals. EP animals had significantly higher (P < 0.05) circulating CK activities than EE or unexercised animals. Muscle neutrophil concentrations were significantly (P < 0.01) elevated in EP and EE groups compared with unexercised controls, with EP muscle neutrophil levels also being over 60% greater (P < 0.05) than in EE animals. EP animals also had higher (P < 0.05) muscle MPO activities than unexercised or EE animals. Muscle G6PD activities were not significantly different between any groups. Muscle caplain-like activities were 80% higher (P < 0.01) in EP animals than EE animals with calpain-like activities in EE animals similar to unexercised groups. These results indicate that estrogen supplementation in ovariectomized rats attenuated 1-h post-exercise serum CK activities, muscle neutrophil infiltration, MPO activities, and calpain-like activities when compared with exercised, unsupplemented animals. This supports the possibility of a relationship between estrogen, calpain dependent production of neutrophil chemo-attractant peptides, and 1-h post-exercise skeletal muscle neutrophil infiltration.     

Eccentric exercise-induced injury to rat skeletal muscle

These experiments were designed to study skeletal muscle pathology resulting from eccentric-biased exercise in rats. The effects on the muscles of running on a treadmill on a 0 degrees incline (similar amounts of concentric and eccentric contractions), down a 16 degrees incline (primarily eccentric contractions), and up a 16 degrees incline (primarily concentric contractions) at 16 m . min-1 for 90 min were assessed by following postexercise changes in 1) plasma creatine kinase and lactate dehydrogenase activities, 2) glucose-6-phosphate dehydrogenase (G-6-PDase) activity (bio- and histochemically) in the physiological extensor muscles, and 3) histological appearance of the muscles. The data indicate the following. 1) Whereas all exercise protocols resulted in elevations of plasma enzymes immediately after running, only eccentric exercise caused late phase elevations 1.5-2 days postexercise. 2) Significant increases in muscle G-6-PDase activity, which were always associated with accumulations of mononuclear cells, always occurred within some muscles of each extensor group 1-3 days following downhill and uphill running and did not occur following level running; the increases in activity were usually of lower magnitude in the muscles of uphill runners than in those of downhill runners; the deeply located, predominantly slow-twitch muscles were most affected by both down- and uphill running. 3) Muscle histology demonstrated localized disruption of normal banding patterns of some fibers immediately after exercise and accumulations of macrophages in the interstitium and in some (less than 5%) muscle fibers by 24 h postexercise in the deep slow muscles of the antigravity groups. Although the data generally indicated that eccentric exercise causes greater injury to the muscles, questions remain.     

Sepsis-induced muscle proteolysis is prevented by a proteasome inhibitor in vivo

Sepsis-induced muscle proteolysis mainly reflects ubiquitin-proteasome-dependent protein degradation. The effect of in vivo administration of a proteasome inhibitor on muscle protein breakdown during sepsis is not known. We treated rats with the proteasome inhibitor N-benzyloxycarbonyl-Ile-Glu-(O-t-butyl)-Ala-leucinal (PSI) or corresponding volume of vehicle i.p. 2 h before sham-operation or induction of sepsis by cecal ligation and puncture. The sepsis-induced increase in total and myofibrillar muscle protein breakdown was inhibited in rats treated in vivo with PSI and a maximal effect was seen following 15 mg/kg of the proteasome inhibitor. Results from in vitro experiments in which incubated muscles were treated with 100 microM PSI suggest that the drug has a direct effect on muscle and that the effect is specific for the proteasome. The results are important because they suggest that it may be possible to prevent or improve the cachectic response in skeletal muscle during sepsis by treatment with a proteasome inhibitor.     

Treatment of rats with calpain inhibitors prevents sepsis-induced muscle proteolysis independent of atrogin-1/MAFbx and MuRF1 expression

Muscle wasting in sepsis is a significant clinical problem because it results in muscle weakness and fatigue that may delay ambulation and increase the risk for thromboembolic and pulmonary complications. Treatments aimed at preventing or reducing muscle wasting in sepsis, therefore, may have important clinical implications. Recent studies suggest that sepsis-induced muscle proteolysis may be initiated by calpain-dependent release of myofilaments from the sarcomere, followed by ubiquitination and degradation of the myofilaments by the 26S proteasome. In the present experiments, treatment of rats with one of the calpain inhibitors calpeptin or BN82270 inhibited protein breakdown in muscles from rats made septic by cecal ligation and puncture. The inhibition of protein breakdown was not accompanied by reduced expression of the ubiquitin ligases atrogin-1/MAFbx and MuRF1, suggesting that the ubiquitin-proteasome system is regulated independent of the calpain system in septic muscle. When incubated muscles were treated in vitro with calpain inhibitor, protein breakdown rates and calpain activity were reduced, consistent with a direct effect in skeletal muscle. Additional experiments suggested that the effects of BN82270 on muscle protein breakdown may, in part, reflect inhibited cathepsin L activity, in addition to inhibited calpain activity. When cultured myoblasts were transfected with a plasmid expressing the endogenous calpain inhibitor calpastatin, the increased protein breakdown rates in dexamethasone-treated myoblasts were reduced, supporting a role of calpain activity in atrophying muscle. The present results suggest that treatment with calpain inhibitors may prevent sepsis-induced muscle wasting.     

Ghrelin receptor agonist GHRP-2 prevents arthritis-induced increase in E3 ubiquitin-ligating enzymes MuRF1 and MAFbx gene expression in skeletal muscle

Chronic arthritis is a catabolic state associated with an inhibition of the IGF system and a decrease in body weight. Cachexia and muscular wasting is secondary to protein degradation by the ubiquitin-proteasome pathway. The aim of this work was to analyze the effect of adjuvant-induced arthritis on the muscle-specific ubiquitin ligases muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx) as well as on IGF-I and IGF-binding protein-5 (IGFBP-5) gene expression in the skeletal muscle. We also studied whether the synthetic ghrelin receptor agonist, growth hormone releasing peptide-2 (GHRP-2), was able to prevent arthritis-induced changes in the skeletal muscle. Arthritis induced an increase in MuRF1, MAFbx (P < 0.01), and tumor necrosis factor (TNF)-alpha mRNA (P < 0.05) in the skeletal muscle. Arthritis decreased the serum IGF-I and its gene expression in the liver (P < 0.01), whereas it increased IGF-I and IGFBP-5 gene expression in the skeletal muscle (P < 0.01). Administration of GHRP-2 for 8 days prevented the arthritis-induced increase in muscular MuRF1, MAFbx, and TNF-alpha gene expression. GHRP-2 treatment increased the serum concentrations of IGF-I and the IGF-I mRNA in the liver and in the cardiac muscle and decreased muscular IGFBP-5 mRNA both in control and in arthritic rats (P < 0.05). GHRP-2 treatment increased muscular IGF-I mRNA in control rats (P < 0.01), but it did not modify the muscular IGF-I gene expression in arthritic rats. These data indicate that arthritis induces an increase in the activity of the ubiquitin-proteasome proteolytic pathway that is prevented by GHRP-2 administration. The parallel changes in muscular IGFBP-5 and TNF-alpha gene expression with the ubiquitin ligases suggest that they can participate in skeletal muscle alterations during chronic arthritis.     

Selected Contribution: IGF-I antibody prevents increases in protein synthesis in epitrochlearis muscles from refed, diabetic rats.

The purpose of this study was to examine whether immune neutralization of muscle-produced insulin-like growth factor I (IGF-I) would prevent an appropriate anabolic response to refeeding in diabetic rats. Male Sprague-Dawley rats were made diabetic by partial pancreatectomy and were randomly assigned to be either control-fed, fasted, or fasted-refed (n = 7-8 per group). Diabetes decreased rates of protein synthesis and increased rates of protein degradation in incubated epitrochlearis muscles (P < 0.05). In both groups of rats, fasting lowered protein synthesis and increased proteolysis and subsequent refeeding returned both parameters to near basal values (P < 0.05). Neutralization of muscle IGF-I by the addition of IGF-I antibody to the incubation medium reduced protein synthesis an average of 22% for all groups (P < 0.05). However, rates of protein degradation were not affected. In nondiabetic rats, refeeding increased protein synthesis in both control and antibody-treated muscles (P < 0.05). Refeeding also increased protein synthesis in the control muscles from diabetic rats (P < 0.01). In contrast, muscles from diabetic rats that were incubated with anti-IGF-I did not increase protein synthesis in response to refeeding. These data suggest that immune neutralization of muscle IGF-I in hypoinsulinemic rats negated the ability of endogenous IGF-I to promote protein synthesis and thereby prevented an appropriate anabolic response.     

Adding protein to a carbohydrate supplement provided after endurance exercise enhances 4E-BP1 and RPS6 signaling in skeletal muscle.

To examine the role of both endurance exercise and nutrient supplementation on the activation of mRNA translation signaling pathways postexercise, rats were subjected to a 3-h swimming protocol. Immediately following exercise, the rats were provided with a solution containing either 23.7% wt/vol carbohydrates (CHO), 7.9% wt/vol protein (Pro), 31.6% wt/vol (23.7% wt/vol CHO + 7.9% wt/vol Pro) carbohydrates and Pro (CP), or a placebo (EX). The rats were then killed at 0, 30, and 90 min postexercise, and phosphorylation states of mammalian target of rapamycin (mTOR), ribosomal S6 kinase (p70(S6K)), ribosomal protein S6 (rpS6), and 4E-binding protein 1 (4E-BP1), were analyzed by immunoblot analysis in the red and white quadriceps muscle. Results demonstrated that rat groups provided with any of the three nutritional supplements (CHO, Pro, CP) transiently increased the phosphorylation states of mTOR, 4E-BP1, rpS6, and p70(S6K) compared with EX rats. Although CHO, Pro, and CP supplements phosphorylated mTOR and p70(S6K) after exercise, only CP elevated the phosphorylation of rpS6 above all other supplements 30 min postexercise and 4E-BP1 30 and 90 min postexercise. Furthermore, the phosphorylation states of 4E-BP1 (r(2) = 0.7942) and rpS6 (r(2) = 0.760) were highly correlated to insulin concentrations in each group. These results suggest that CP supplementation may be most effective in activating the mTOR-dependent signaling pathway in the postprandial state postexercise, and that there is a strong relationship between the insulin concentration and the activation of enzymes critical for mRNA translation.     

Proteasome function is required to maintain muscle cellular architecture

BACKGROUND INFORMATION: Protein degradation via the UPS (ubiquitin-proteasome system) plays critical roles in muscle metabolism and signalling pathways. The present study investigates temporal requirements of the UPS in muscle using conditional expression of mutant proteasome beta subunits to cause targeted inhibition of proteasome function. RESULTS AND CONCLUSIONS: The Drosophila GeneSwitch system was used, with analyses of the well-characterized larval somatic body wall muscles. This method acutely disrupts proteasome function and causes rapid accumulation of polyubiquitinated proteins, specifically within the muscle. Within 12 h of transgenic proteasome inhibition, there was a gross disorganization of muscle architecture and prominent muscle atrophy, progressing to the arrest of all co-ordinated movement by 24 h. Progressive muscle architecture changes include rapid loss of sarcomere organization, loss of nuclei spacing/patterning, vacuole formation and the accumulation of nuclear and cytoplasmic aggregates at the ultrastructural level. At the neuromuscular junction, the highly specialized muscle membrane folds of the subsynaptic reticulum were rapidly lost. Within 24 h after transgenic proteasome inhibition, muscles contained numerous autophagosomes and displayed highly elevated expression of the endoplasmic reticulum chaperone GRP78 (glucose-regulated protein of 78 kDa), indicating that the loss of muscle maintenance correlates with induction of the unfolded protein response. Taken together, these results demonstrate that the UPS is acutely required for maintenance of muscle and neuromuscular junction architecture, and provides a Drosophila genetic model to mechanistically evaluate this requirement.     

Effect of prolonged intermittent hypoxia and exercise training on glucose tolerance and muscle GLUT4 protein expression in rats

We compared the chronic effect of intermittent hypoxia and endurance training on the glucose tolerance and GLUT4 protein expression in rat skeletal muscle. Thirty-two Sprague-Dawley rats were matched for weight and assigned to one of the following four groups: control, endurance training, hypoxia, or hypoxia followed by endurance training. Hypoxic treatment consisted of breathing 14% O₂ for 12 h/day under normobaric conditions, and the training protocol consisted of making animals swim 2 times for 3 h/day. At the end of the 3rd week, an oral glucose tolerance test (OGTT) was performed 16 h after treatments. At the end of the 4th week, GLUT4 protein, mRNA, and glycogen storage in skeletal muscle were determined. Endurance training significantly improved OGTT results. Glycogen content and GLUT4 protein expression in the plantaris and red gastrocnemius, but not in the soleus or white gastrocnemius muscles, were also elevated. Chronic intermittent hypoxia also improved OGTT results, but did not alter GLUT4 protein expression. Additionally, hypoxia followed by exercise training produced significant increases in GLUT4 protein and mRNA in a greater number of muscles compared to endurance training alone. Both exercise training and hypoxia significantly reduced body mass, and an additive effect of both treatments was found. In conclusion, chronic intermittent hypoxia improved glucose tolerance in the absence of increased GLUT4 protein expression. This treatment facilitated the exercise training effect on muscle GLUT4 expression and glycogen storage. These new findings open the possibility of utilizing intermittent hypoxia, with or without exercise training, for the prevention and clinical treatment of type 2 diabetes or insulin resistance.     

Effect of flywheel-based resistance exercise on processes contributing to muscle atrophy during unloading in adult rats.

Flywheel-based resistance exercise (RE) attenuates muscle atrophy during hindlimb suspension. We have previously shown that protein synthesis is elevated in response to RE, but the effect on protein degradation, cell proliferation, or apoptosis was not investigated. We hypothesized that, in addition to affecting protein synthesis, RE inhibits processes that actively contribute to muscle atrophy during hindlimb suspension. Male rats were housed in regular cages (control), tail suspended for 2 wk (HS), or HS with RE every other day for 2 wk (HSRE). Although RE attenuated soleus muscle atrophy during HS, the observed fivefold elevation in apoptosis and the 53% decrease in cell proliferation observed with HS were unaffected by RE. Expression of genes encoding components of the ubiquitin-proteasome pathway of protein degradation were elevated with HS, including ubiquitin, MAFbx, Murf-1, Nedd4, and XIAP, and proteasome subunits C2 and C9. Total ubiquitinated protein was increased with HS, but proteasome activity was not different from control. RE selectively altered the expression of different components of this pathway: MAFbx, Murf-1, and ubiquitin mRNA abundance were downregulated, whereas C2 and C9 subunits remained elevated. Similarly, Nedd4 and XIAP continued to be upregulated, potentially accounting for the observed augmentation in total ubiquitinated protein with RE. Thus a different constellation of proteins is likely ubiquitinated with RE due to altered ubiquitin ligase composition. In summary, the flywheel-based resistance exercise paradigm used in this study is associated with the inhibition of some mechanisms associated with muscle atrophy, such as the increase in MAFbx and Murf-1, but not with others, such as proteasome subunit remodeling, apoptosis, and decreased proliferation, potentially accounting for the inability to completely restore muscle mass. Identifying specific exercise parameters that affect these latter processes may be useful in designing effective exercise strategies in the elderly or during spaceflight.     

Impact of protein coingestion on muscle protein synthesis during continuous endurance type exercise

This study investigates the impact of protein coingestion with carbohydrate on muscle protein synthesis during endurance type exercise. Twelve healthy male cyclists were studied during 2 h of fasted rest followed by 2 h of continuous cycling at 55% W(max). During exercise, subjects received either 1.0 g·kg(-1)·h(-1) carbohydrate (CHO) or 0.8 g·kg(-1)·h(-1) carbohydrate with 0.2 g·kg(-1)·h(-1) protein hydrolysate (CHO+PRO). Continuous intravenous infusions with l-[ring-(13)C(6)]phenylalanine and l-[ring-(2)H(2)]tyrosine were applied, and blood and muscle biopsies were collected to assess whole body protein turnover and muscle protein synthesis rates at rest and during exercise conditions. Protein coingestion stimulated whole body protein synthesis and oxidation rates during exercise by 22 ± 3 and 70 ± 17%, respectively (P < 0.01). Whole body protein breakdown rates did not differ between experiments. As a consequence, whole body net protein balance was slightly negative in CHO and positive in the CHO+PRO treatment (-4.9 ± 0.3 vs. 8.0 ± 0.3 μmol Phe·kg(-1)·h(-1), respectively, P < 0.01). Mixed muscle protein fractional synthetic rates (FSR) were higher during exercise compared with resting conditions (0.058 ± 0.006 vs. 0.035 ± 0.006%/h in CHO and 0.070 ± 0.011 vs. 0.038 ± 0.005%/h in the CHO+PRO treatment, respectively, P < 0.05). FSR during exercise did not differ between experiments (P = 0.46). We conclude that muscle protein synthesis is stimulated during continuous endurance type exercise activities when carbohydrate with or without protein is ingested. Protein coingestion does not further increase muscle protein synthesis rates during continuous endurance type exercise.