Low‐dose caffeine administration increases fatty acid utilization and mitochondrial turnover in C2C12 skeletal myotubes

Caffeine has been shown to directly increase fatty acid oxidation, in part, by promoting mitochondrial biogenesis. Mitochondrial biogenesis is often coupled with mitophagy, the autophagy‐lysosomal degradation of mitochondria. Increased mitochondrial biogenesis and mitophagy promote mitochondrial turnover, which can enhance aerobic metabolism. In addition, recent studies have revealed that cellular lipid droplets can be directly utilized in an autophagy‐dependent manner, a process known as lipophagy. Although caffeine has been shown to promote autophagy and mitochondrial biogenesis in skeletal muscles, it remains unclear whether caffeine can increase lipophagy and mitochondrial turnover in skeletal muscle as well. The purpose of this study was to determine the possible contribution of lipophagy to caffeine‐dependent lipid utilization. Furthermore, we sought to determine whether caffeine could increase mitochondrial turnover, which may also contribute to elevated fatty acid oxidation. Treating fully differentiated C2C12 skeletal myotubes with 0.5 mM oleic acid (OA) for 24 hr promoted an approximate 2.5‐fold increase in cellular lipid storage. Treating skeletal myotubes with 0.5 mM OA plus 0.5 mM caffeine for an additional 24 hr effectively returned cellular lipid stores to control levels, and this was associated with an increase in markers of autophagosomes and autophagic flux, as well as elevated autophagosome density in TEM images. The addition of autophagy inhibitors 3‐methyladenine (10 mM) or bafilomycin A1 (10 μM) reduced caffeine‐dependent lipid utilization by approximately 30%. However, fluorescence and transmission electron microscopy analysis revealed no direct evidence of lipophagy in skeletal myotubes, and there was also no lipophagy‐dependent increase in fatty acid oxidation. Finally, caffeine treatment promoted an 80% increase in mitochondrial turnover, which coincided with a 35% increase in mitochondrial fragmentation. Our results suggest that caffeine administration causes an autophagy‐dependent decrease in lipid content by increasing mitochondrial turnover in mammalian skeletal myotubes. Low‐dose caffeine promoted autophagy in skeletal myotubes. Inhibiting autophagy reduced caffeine‐dependent lipid droplet breakdown by 30%. Caffeine did not directly increase lipophagy in skeletal myotubes.