induced activation of AMPK negatively regulates myotube hypertrophy through the HSP72-mediated pathway in C2C12 skeletal muscle cells. Am J Physiol Endocrinol Metab 306: E344 -E354, 2014. First published December 17, 2013; doi:10.1152/ajpendo.00495.2013.-5=-AMP-activated protein kinase (AMPK) plays an important role as a negative regulator of skeletal muscle mass. However, the precise mechanism of AMPK-mediated regulation of muscle mass is not fully clarified. Heat shock proteins (HSPs), stress-induced molecular chaperones, are related with skeletal muscle adaptation, but the association between AMPK and HSPs in skeletal muscle hypertrophy is unknown. Thus, we investigated whether AMPK regulates hypertrophy by mediating HSPs in C2C12 cells. The treatment with AICAR, a potent stimulator of AMPK, decreased 72-kDa HSP (HSP72) expression, whereas there were no changes in the expressions of 25-kDa HSP, 70-kDa heat shock cognate, and heat shock transcription factor 1 in myotubes. Protein content and diameter were less in the AICARtreated myotubes in those without treatment. AICAR-induced suppression of myotube hypertrophy and HSP72 expression was attenuated in the siRNA-mediated AMPK␣ knockdown myotubes. AICAR increased microRNA (miR)-1, a modulator of HSP72, and the increase of miR-1 was not induced in AMPK␣ knockdown condition. Furthermore, siRNA-mediated HSP72 knockdown blocked AICARinduced inhibition of myotube hypertrophy. AICAR upregulated the gene expression of muscle Ring-finger 1, and this alteration was suppressed in either AMPK␣ or HSP72 knockdown myotubes. The phosphorylation of p70 S6 kinase Thr 389 was downregulated by AICAR, whereas this was attenuated in AMPK␣, but not in HSP72, knockdown myotubes. These results suggest that AMPK inhibits hypertrophy through, in part, an HSP72-associated mechanism via miR-1 and protein degradation pathways in skeletal muscle cells. microRNA; heat shock transcription factor 1; muscle rRing-finger 1; atrogin-1; acetyl-CoA carboxylase SKELETAL MUSCLE HAS A GREATER CAPACITY to adapt to various stimuli. Increased loading, such as resistance training and mechanical stretching, stimulates protein synthesis and reduces protein degradation, thereby inducing muscle hypertrophy (10). On the other hand, decrease of use, such as immobilization, denervation, aging, and/or various pathological conditions, attenuates protein synthesis and increases protein degradation, resulting in atrophy (12,32). Although the process of skeletal muscle adaptation to hypertrophic and atrophic stimuli has been studied, the molecular mechanism involved in this process is not fully understood yet.5=-AMP-activated protein kinase (AMPK) is well known as a sensor for cellular energy status and metabolic stress, such as muscle contraction, fasting, hypoxia, ischemia, and/or oxidative and osmotic stresses and as a signaling intermediary that controls the use of glucose and fatty acids in skeletal muscle (8,14,15). In addition, several studies in the past decade have suggested that AMPK plays an important rol...

-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 ϭ 24) and AMPK-DN (n ϭ 24) mice were randomly divided into two groups: an untreated preexperimental control group (n ϭ 12 in each group) and an unloading (n ϭ 12 in each group) group. The relative soleus muscle weight and fiber cross-sectional area to body weight were decreased by ϳ30% in WT mice by hindlimb unloading and by ϳ20% 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 B␣, 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. AMP-activated protein kinase; protein degradation; autophagy; ubiquitin-proteasome; microRNA; heat shock protein SKELETAL MUSCLE IS THE LARGEST TISSUE IN THE BODY, accounting for ϳ40% of the total body mass, and has a crucial role in metabolism as well as locomotion. Skeletal muscle has a high ability to adapt to multiple stimuli. Increased loading, such as resistance training and mechanical stretching, leads to skeletal muscle hypertrophy (18,75). In contrast, aging, poor nutrition, several diseases such as diabetes, cancer, sepsis, and chronic renal failure, and decreased loading, such as inactivity, lead to skeletal muscle atrophy (23,34,39). Skeletal muscle atrophy occurs as a result of changes in protein turnover: decreased protein synthesis, increased protein degradation, or a combination of both (17). The coordination of protein turnover in the atrophic process is regulated by complicated molecular responses, and the molecular mechanism involved in this process in skeletal muscle is not yet completely understood and remains to be elucidated.5=-AMP-activated protein kinase (AMPK) is well established as a metabolic sensor that helps maintain cellular energy homeostasis by modulating glucose, lipid, and protein metabolism (16,26,28). AMPK is a heterotrimeric kinase comprising a catalytic ␣-subunit and two regulatory subunits, the ␤-and ␥-subunits. Two ...

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 Thr 389 , 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.