Different levels of autophagy induced by transient serum starvation regulate metabolism and differentiation of porcine skeletal muscle satellite cells

Abstract

This study aimed to investigate the effects of different levels of autophagy induced by transient serum starvation on the metabolism, lipid metabolism, and differentiation of porcine skeletal muscle satellite cells (SMSCs) to preliminary elucidate the role and function of autophagy in the regulatory network of skeletal muscle development. Different levels of autophagy were induced by controlling the serum concentration in the culture system for 24 h. Apoptosis, membrane potential, reactive oxygen species (ROS), ATP, and myogenic and lipogenic differentiation markers were monitored to determine if autophagy affected the metabolism and differentiation of SMSCs. Autophagy was induced in SMSCs via serum starvation (5%, 15%), as evidenced by decreased p62 and mTOR phosphorylation levels and increased LC3B lipidation and AMPK phosphorylation levels. Transmission electron microscopy revealed the presence of autophagosomes, and the rates of morphologically abnormal nuclei and mitochondria gradually increased with the decrease in serum concentration, the number of autophagic lysosomes also increased, indicating that 5% serum starvation induced severe autophagy, while 15% serum starvation induced mild autophagy. Compared with the control group and 15% serum-starved SMSCs, SMSCs undergoing 5% serum starvation had the highest intracellular ATP and ROS levels, the highest percentage of apoptotic cells, and the lowest membrane potential. The 15% serum-starved SMSCs had the highest membrane potential, but the percentage of apoptotic cells did not change significantly compared with the control group. The levels of the myogenic markers MyoD1 and MHC were significantly higher in 15% serum-starved SMSCs than in serum-sufficient SMSCs and the lowest in the 5% serum-starved SMSCs. The lipid contents (measured by Oil Red O staining and quantification of triglycerides) and lipogenic markers Peroxisome Proliferators-activated Receptors γ and Lipoprotein Lipase were also significantly higher in SMSCs undergoing 15% serum starvation than in the control group, and the lowest in the 5% serum-starved SMSCs. Different levels of starvation stress induce different levels of autophagy. Mild autophagy induced by moderate serum starvation promotes the metabolism and differentiation of SMSCs, while severe autophagy renders SMSCs more apoptotic, abnormal metabolism and suppresses SMSC differentiation into adipocytes or myocytes, and reduces lipid metabolisms. Our study suggests that autophagy plays a role in skeletal muscle development and may help design strategies for improving meat production traits in domestic pigs.