Tatsuro Egawa

Caffeine activates preferentially α1-isoform of 5'AMP-activated protein kinase in rat skeletal muscle.

Aim:  Caffeine activates 5′AMP‐activated protein kinase (AMPK), a signalling intermediary implicated in the regulation of glucose, lipid and energy metabolism in skeletal muscle. Skeletal muscle expresses two catalytic α subunits of AMPK, α1 and α2, but the isoform specificity of caffeine‐induced AMPK activation is unclear. The aim of this study was to determine which α isoform is preferentially activated by caffeine in vitro and in vivo using rat skeletal muscle.

Evidence for differential regulation of lactate metabolic properties in aged and unloaded rat skeletal muscle

Skeletal muscles of elderly individuals show fatigue resistance and reduced lactate accumulation compared with those of young subjects during activities that recruit a small amount of muscle mass. To explore the mechanism underlying the functional changes in aged muscle, we focused on lactate metabolic properties, including monocarboxylate transporter (MCT) 1 and MCT4, in muscles from old and young control rats and hindlimb-suspended young rats. MCT1 expression was lower in soleus (SOL) of old rats than in SOL of young control rats, but was similar in young control and hindlimb-suspended rats. MCT4 expression was lower in extensor digitorum longus (EDL) of old rats than in that of young control rats, but did not differ between young control and hindlimb-suspended rats. The ratio of lactate dehydrogenase to citrate synthase activities was higher in SOL of hindlimb-suspended and old rats than in SOL of young control rats, and was lower in EDL of old rats than in those of young control and hindlimb-suspended rats. Our data suggest that aging causes metabolic changes that can reduce lactate accumulation during exercise and increase fatigue resistance in skeletal muscle, and that these changes result from aging rather than from inactivity.

Involvement of AMPK in regulating slow-twitch muscle atrophy during hindlimb unloading in mice

AMPK is considered to have a role in regulating skeletal muscle mass. However, there are no studies investigating the function of AMPK in modulating skeletal muscle mass during atrophic conditions. In the present study, we investigated the difference in unloading-associated muscle atrophy and molecular functions in response to 2-wk hindlimb suspension between transgenic mice overexpressing the dominant-negative mutant of AMPK (AMPK-DN) and their wild-type (WT) littermates. Male WT (n = 24) and AMPK-DN (n = 24) mice were randomly divided into two groups: an untreated preexperimental control group (n = 12 in each group) and an unloading (n = 12 in each group) group. The relative soleus muscle weight and fiber cross-sectional area to body weight were decreased by ∼30% in WT mice by hindlimb unloading and by ∼20% in AMPK-DN mice. There were no changes in puromycin-labeled protein or Akt/70-kDa ribosomal S6 kinase signaling, the indicators of protein synthesis. The expressions of ubiquitinated proteins and muscle RING finger 1 mRNA and protein, markers of the ubiquitin-proteasome system, were increased by hindlimb unloading in WT mice but not in AMPK-DN mice. The expressions of molecules related to the protein degradation system, phosphorylated forkhead box class O3a, inhibitor of κBα, microRNA (miR)-1, and miR-23a, were decreased only in WT mice in response to hindlimb unloading, and 72-kDa heat shock protein expression was higher in AMPK-DN mice than in WT mice. These results imply that AMPK partially regulates unloading-induced atrophy of slow-twitch muscle possibly through modulation of the protein degradation system, especially the ubiquitin-proteasome system.

Up-Regulation of Adiponectin Expression in Antigravitational Soleus Muscle in Response to Unloading Followed by Reloading, and Functional Overloading in Mice

The purpose of this study was to investigate the expression level of adiponectin and its related molecules in hypertrophied and atrophied skeletal muscle in mice. The expression was also evaluated in C2C12 myoblasts and myotubes. Both mRNA and protein expression of adiponectin, mRNA expression of adiponectin receptor (AdipoR) 1 and AdipoR2, and protein expression of adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif 1 (APPL1) were observed in C2C12 myoblasts. The expression levels of these molecules in myotubes were higher than those in myoblasts. The expression of adiponectin-related molecules in soleus muscle was observed at mRNA (adiponectin, AdipoR1, AdipoR2) and protein (adiponectin, APPL1) levels. The protein expression levels of adiponectin and APPL1 were up-regulated by 3 weeks of functional overloading. Down-regulation of AdipoR1 mRNA, but not AdipoR2 mRNA, was observed in atrophied soleus muscle. The expression of adiponectin protein, AdipoR1 mRNA, and APPL1 protein was up-regulated during regrowth of unloading-associated atrophied soleus muscle. Mechanical loading, which could increase skeletal muscle mass, might be a useful stimulus for the up-regulations of adiponectin and its related molecules in skeletal muscle.

Regeneration of injured skeletal muscle in heat shock transcription factor 1-null mice

The purpose of this study was to investigate a role of heat shock transcription factor 1 (HSF1)‐mediated stress response during regeneration of injured soleus muscle by using HSF1‐null mice. Cardiotoxin (CTX) was injected into the left muscle of male HSF1‐null and wild‐type mice under anesthesia with intraperitoneal injection of pentobarbital sodium. Injection of physiological saline was also performed into the right muscle. Soleus muscles were dissected bilaterally 2 and 4 weeks after the injection. The relative weight and fiber cross‐sectional area in CTX‐injected muscles of HSF1‐null, not of wild‐type, mice were less than controls with injection of physiological saline 4 weeks after the injury, indicating a slower regeneration. Injury‐related increase of Pax7‐positive muscle satellite cells in HSF1‐null mice was inhibited versus wild‐type mice. HSF1‐deficiency generally caused decreases in the basal expression levels of heat shock proteins (HSPs). But the mRNA expression levels of HSP25 and HSP90α in HSF1‐null mice were enhanced in response to CTX‐injection, compared with wild‐type mice. Significant up‐regulations of proinflammatory cytokines, such as interleukin (IL) ‐6, IL‐1β, and tumor necrosis factor mRNAs, with greater magnitude than in wild‐type mice were observed in HSF1‐deficient mouse muscle. HSF1 and/or HSF1‐mediated stress response may play a key role in the regenerating process of injured skeletal muscle. HSF1 deficiency may depress the regenerating process of injured skeletal muscle via the partial depression of increase in Pax7‐positive satellite cells. HSF1‐deficiency‐associated partial depression of skeletal muscle regeneration might also be attributed to up‐regulation of proinflammatory cytokines.

Heat Shock Transcription Factor 1-Deficiency Attenuates Overloading-Associated Hypertrophy of Mouse Soleus Muscle

Hypertrophic stimuli, such as mechanical stress and overloading, induce stress response, which is mediated by heat shock transcription factor 1 (HSF1), and up-regulate heat shock proteins (HSPs) in mammalian skeletal muscles. Therefore, HSF1-associated stress response may play a key role in loading-associated skeletal muscle hypertrophy. The purpose of this study was to investigate the effects of HSF1-deficiency on skeletal muscle hypertrophy caused by overloading. Functional overloading on the left soleus was performed by cutting the distal tendons of gastrocnemius and plantaris muscles for 4 weeks. The right muscle served as the control. Soleus muscles from both hindlimbs were dissected 2 and 4 weeks after the operation. Hypertrophy of soleus muscle in HSF1-null mice was partially inhibited, compared with that in wild-type (C57BL/6J) mice. Absence of HSF1 partially attenuated the increase of muscle wet weight and fiber cross-sectional area of overloaded soleus muscle. Population of Pax7-positive muscle satellite cells in HSF1-null mice was significantly less than that in wild-type mice following 2 weeks of overloading (p<0.05). Significant up-regulations of interleukin (IL)-1β and tumor necrosis factor mRNAs were observed in HSF1-null, but not in wild-type, mice following 2 weeks of overloading. Overloading-related increases of IL-6 and AFT3 mRNA expressions seen after 2 weeks of overloading tended to decrease after 4 weeks in both types of mice. In HSF1-null mice, however, the significant overloading-related increase in the expression of IL-6, not ATF3, mRNA was noted even at 4th week. Inhibition of muscle hypertrophy might be attributed to the greater and prolonged enhancement of IL-6 expression. HSF1 and/or HSF1-mediated stress response may, in part, play a key role in loading-induced skeletal muscle hypertrophy.

Caffeine and contraction synergistically stimulate 5′‐AMP‐activated protein kinase and insulin‐independent glucose transport in rat skeletal muscle

5′‐Adenosine monophosphate‐activated protein kinase (AMPK) has been identified as a key mediator of contraction‐stimulated insulin‐independent glucose transport in skeletal muscle. Caffeine acutely stimulates AMPK in resting skeletal muscle, but it is unknown whether caffeine affects AMPK in contracting muscle. Isolated rat epitrochlearis muscle was preincubated and then incubated in the absence or presence of 3 mmol/L caffeine for 30 or 120 min. Electrical stimulation (ES) was used to evoke tetanic contractions during the last 10 min of the incubation period. The combination of caffeine plus contraction had additive effects on AMPKα Thr172 phosphorylation, α‐isoform‐specific AMPK activity, and 3‐O‐methylglucose (3MG) transport. In contrast, caffeine inhibited basal and contraction‐stimulated Akt Ser473 phosphorylation. Caffeine significantly delayed muscle fatigue during contraction, and the combination of caffeine and contraction additively decreased ATP and phosphocreatine contents. Caffeine did not affect resting tension. Next, rats were given an intraperitoneal injection of caffeine (60 mg/kg body weight) or saline, and the extensor digitorum longus muscle was dissected 15 min later. ES of the sciatic nerve was performed to evoke tetanic contractions for 5 min before dissection. Similar to the findings from isolated muscles incubated in vitro, the combination of caffeine plus contraction in vivo had additive effects on AMPK phosphorylation, AMPK activity, and 3MG transport. Caffeine also inhibited basal and contraction‐stimulated Akt phosphorylation in vivo. These findings suggest that caffeine and contraction synergistically stimulate AMPK activity and insulin‐independent glucose transport, at least in part by decreasing muscle fatigue and thereby promoting energy consumption during contraction.

Heat stress acutely activates insulin‐independent glucose transport and 5′‐AMP‐activated protein kinase prior to an increase in HSP72 protein in rat skeletal muscle

Heat stress (HS) stimulates heat shock protein (HSP) 72 mRNA expression, and the period after an increase in HSP72 protein is characterized by enhanced glucose metabolism in skeletal muscle. We have hypothesized that, prior to an increase in the level of HSP72 protein, HS activates glucose metabolism by acutely stimulating 5′‐AMP‐activated protein kinase (AMPK). Rat epitrochlearis muscle was isolated and incubated either with or without HS (42°C) for 10 and 30 min. HS for 30 min led to an increase in the level of Hspa1a and Hspa1b mRNA but did not change the amount of HSP72 protein. However, HS for both 10 and 30 min led to a significant increase in the rate of 3‐O‐methyl‐d‐glucose (3MG) transport, and the stimulatory effect of 3MG transport was completely blocked by cytochalasin B. HS‐stimulated 3MG transport was also inhibited by dorsomorphin but not by wortmannin. HS led to a decrease in the concentration of ATP, phosphocreatine, and glycogen, to an increase in the level of phosphorylation of AMPKα Thr172, and to an increase in the activity of both AMPKα1 and AMPKα2. HS did not affect the phosphorylation status of insulin receptor signaling or Ca2+/calmodulin‐dependent protein kinase II. These results suggest that HS acts as a rapid stimulator of insulin‐independent glucose transport, at least in part by stimulating AMPK via decreased energy status. Although further research is warranted, heat treatment of skeletal muscle might be a promising method to promote glucose metabolism acutely.

Potential involvement of dietary advanced glycation end products in impairment of skeletal muscle growth and muscle contractile function in mice

Diets enriched with advanced glycation end products (AGE) have recently been related to muscle dysfunction processes. However, it remains unclear whether long-term exposure to an AGE-enriched diet impacts physiological characteristics of skeletal muscles. Therefore, we explored the differences in skeletal muscle mass, contractile function and molecular responses between mice receiving a diet high in AGE (H-AGE) and low in AGE (L-AGE) for 16 weeks. There were no significant differences between L-AGE and H-AGE mice with regard to body weight, food intake or epididymal fat pad weight. However, extensor digitorum longus (EDL) and plantaris (PLA) muscle weights in H-AGE mice were lower compared with L-AGE mice. Higher levels of N ε -(carboxymethyl)-l-lysine, a marker for AGE, in EDL muscles of H-AGE mice were observed compared with L-AGE mice. H-AGE mice showed lower muscle strength and endurance in vivo and lower muscle force production of PLA muscle in vitro. mRNA expression levels of myogenic factors including myogenic factor 5 and myogenic differentiation in EDL muscle were lower in H-AGE mice compared with L-AGE mice. The phosphorylation status of 70-kDa ribosomal protein S6 kinase Thr389, an indicator of protein synthesis signalling, was lower in EDL muscle of H-AGE mice than that of L-AGE mice. These findings suggest that long-term exposure to an AGE-enriched diet impairs skeletal muscle growth and muscle contractile function, and that these muscle dysfunctions may be attributed to the inhibition of myogenic potential and protein synthesis.

Deficiency of heat shock transcription factor 1 suppresses heat stress-associated increase in slow soleus muscle mass of mice.

Effects of heat shock transcription factor 1 (HSF1) deficiency on heat stress‐associated increase in slow soleus muscle mass of mice were investigated.