AICAR-induced activation of AMPK negatively regulates myotube hypertrophy through the HSP72-mediated pathway in C₂C₁₂skeletal muscle cells

Abstract

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...

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

“…6E), suggesting that AMPK does not modulate HSF1 expression in unloading-induced skeletal muscle atrophy. This is supported by previous data showing that pharmacological activation of AMPK did not affect HSF1 expression in skeletal muscle cells (13). Thus, we consider that AMPK-mediated regulation of HSP72 during hindlimb unloading is independent of HSF1.…”

“…Furthermore, a molecular mechanism of the resistance to skeletal muscle atrophy by HSP72 seems to be that HSP72 directly prevents FoxO3a activation during unloading (65,66). We have also demonstrated previously that AMPK negatively regulates HSP72 expression in skeletal muscle cells and that HSP72 controls AMPK-mediated activation of the ubiquitin-proteasome system (13). Considering these findings, it is suggested that a high expression of HSP72 due to the suppression of AMPK activity is a possible mechanism that attenuates the unloading-induced activation of the protein degradation system partly through FoxO3a deactivation.…”

“…A recent study using a knockout mouse model demonstrated that overload-induced muscle hypertrophy was accelerated in AMPK␣1-deficient mice compared with the wildtype mice (50). Correspondingly, we (13) showed in vitro that stimulation with a pharmacological AMPK agonist on cultured skeletal muscle cells inhibited myotube hypertrophy, and this response was attenuated in the AMPK␣1/␣2-knockdown condition. In addition, skeletal muscle-specific AMPK␣1/␣2 double-knockout mice exhibit higher muscle mass than the wild-type mice in normal growth conditions (37,38).…”

“…Previous studies have reported that agonist-induced activation of AMPK enhances protein degradation accompanied by increased MuRF1 and atrogin-1/MAFbx mRNA expressions in cultured myotubes (35,52). In addition, we have demonstrated recently that pharmacological activation of AMPK upregulates MuRF1 mRNA expression, and this upregulation is abolished in AMPK-knockdown cells (13). Thus, AMPK appears to be associated with activation of the ubiquitin-proteasome system, and it is possible that AMPK regulates protein degradation through the ubiquitin-proteasome system during unloading.…”

“…We have also shown that AMPK-mediated inhibition of skeletal muscle hypertrophy is accompanied by upregulation of miR-1 (13). Therefore, we considered that miRNAs might be potent mediators of AMPK-associated regulation of muscle mass during unloading.…”