Akihiro Ikuta

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.

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.

Activation of adiponectin receptors has negative impact on muscle mass in C2C12 myotubes and fast-type mouse skeletal muscle

This study investigated the effects of AdipoRon, which is an agonist for adiponectin receptor 1 (AdipoR1) and AdipoR2, on the protein content, myotube diameter, and number of nuclei per myotube of C2C12 cells and skeletal muscle mass in C57BL/6J mice. AdipoRon suppressed the protein content, myotube diameter, and number of nuclei per myotube of C2C12 cells of C2C12 myotubes in a dose-dependent manner. Adiponectin-associated decline of protein content, diameter, and number of nuclei per myotube in C2C12 myotubes was partially rescued by knockdown of AdipoR1 and/or AdipoR2. Phosphorylation level of AMPK showed a trend to be increased by AdipoRon. A significant increase in phosphorylation level of AMPK was observed at 20 μM AdipoRon. Knockdown of AdipoR1 and/or AdipoR2 rescued AdipoRon-associated decrease in protein content of C2C12 myotubes. AdipoRon-associated increase in phosphorylation level of AMPK in C2C12 myotubes was suppressed by knockdown of AdipoR1 and/or AdipoR2. Successive intravenous injections of AdipoRon into mice caused a decrease in the wet weight of plantaris muscle (PLA), but not in soleus muscle (SOL). Mean fiber cross-sectional area of PLA, but not of SOL, was significantly decreased by AdipoRon administration. On the one hand, the expression level of phosphorylated AMPK and ubiquitinated protein in SOL and PLA muscles was upregulated by AdipoRon administration. On the other hand, AdipoRon administration induced no changes in the expression level of puromycin-labeled proteins in both SOL and PLA muscles. Expression level of adiponectin in extensor digitorum longus (EDL) muscle was increased by aging, but not in SOL muscle. Aging had no effect on the expression level of AdipoR1 and AdipoR2 in both muscles. Phosphorylation level of AMPK in EDL was increased by aging, but not SOL muscle. Results from this study suggest that high level of circulating adiponectin may induce skeletal muscle atrophy, especially fast-type muscle.