Caffeine Affects Myotube Size As Well As Regulates Protein Degradation and Protein Synthesis Pathways in C2C12 Skeletal Muscle Cells

Abstract

[Background]: Caffeine has been implicated in energy metabolism regulation in skeletal muscle. However, it is unclear whether caffeine affects the regulation of skeletal muscle size. In the present study, we evaluated the effect of caffeine on muscle size as well as accompanied changes of ubiquitin–proteasome system and Akt/mammalian target of rapamycin (mTOR)/p70 s6 kinase (p70S6K) signaling. [Methods]: Differentiated C2C12 myotubes were incubated with caffeine (0, 0.1, 1.0, 3.0 mM) for 24 hours. We then estimated the protein content, myotube diameter, and the expression levels of muscle RING finger 1 (MuRF1) messenger RNA (mRNA), atrogin-1/muscle atrophy F-box (MAFbx) mRNA, K48-linked polyubiquitin, phosphorylated 5′-AMP-activated protein kinase (AMPK) α Thr172, 72-kDa heat shock protein (HSP72), HSP72 mRNA, inhibitor κBα (IκBα), phosphorylated forkhead box class O3a (FoxO3a) Ser253, myogenin mRNA, microRNA (miR)-23a, phosphorylated Akt Ser473, and phosphorylated p70S6K Thr389. [Results]: Protein content and myotube diameter were lower in myotubes treated with caffeine (≥1 mM) compared with untreated cells. The expression levels of MuRF1 and atrogin-1/MAFbx mRNA and K48-linked polyubiquitin were increased by caffeine treatment. However, phosphorylated AMPKα Thr172, HSP72 protein and mRNA, IκBα, phosphorylated FoxO3a Ser253, and miR-23a expression were not affected by caffeine treatment. Myogenin mRNA expression was upregulated in response to caffeine treatment. The expressions of phosphorylated Akt Ser473 and p70S6K Thr389 were suppressed by caffeine. [Conclusions]: Caffeine might affect muscle size by stimulating ubiquitin–proteasome system and inhibiting Akt/mTOR/p70S6K signaling, partly through an AMPK-independent mechanism.