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.

Stanniocalcin-1 (STC-1) is a glycoprotein hormone involved in calcium/phosphorus metabolism and direct inhibition of bone and muscle growth. The aim of this study was to investigate the STC-1 gene with respect to the regulatory mechanisms of porcine growth metabolic pathways involving autophagy. Western blotting was used to detect the expression of autophagy and mitochondrial function-related proteins, and flow cytometry was used to detect mitochondrial function-related. Changes in the autophagosome and mitochondrial were observed by electron microscopy. The expression of the autophagy-related proteins was detected by confocal microscopy. The results showed that Pink1, Parkin and LC3B expression was increased; SQSTM1/P62 expression was reduced. Electron microscopy revealed that the cells in the serum starvation group all produced autophagosomes. The fluorescence intensity of GFP-LC3B and GFP-Parkin increased. The Bax/Bcl-2 ratio, Pink1 and Parkin protein levels were profoundly reduced in the STC-KO. In addition, the increase in Mfn2, OPA1, DRP1 and LC3B proteins was attenuated; the increase in the apoptosis rate and amount of active oxygen was attenuated; the decrease in membrane potential; the decrease in ATP was reversed; the fluorescence intensity of GFP-LC3B and GFP-Parkin was increased. These results indicate that autophagy can be caused by serum starvation. Knocking out the porcine STC-1 gene had an obvious antiapoptotic effect on cells, the inhibition of serum starvation-induced autophagy. This is the first study to show that the porcine STC-1 gene confers self-protection in the absence of nutrients. To provide a theoretical basis for studying the effect of STC-1 on pig growth and development.

BACKGROUND:Perilipin 1 (PLIN1) is a lipid droplet scaffolding protein that plays a regulatory role in fat decomposition and mitochondrial function.OBJECTIVE:In this study, the effects of PLIN1 gene knockout (PLIN1-KO) and PLIN1 gene overexpression (PLIN1-EX) on cell metabolism and mitochondrial function in porcine skeletal muscle satellite cells were assessed.METHODS:Porcine skeletal muscle satellite cells were used as the control group (NC). The expression of mitochondrial function-related proteins was detected by western blot. Apoptosis, cell cycle, mitochondrial function-related indices, mitochondrial structure, and morphology were measured by flow cytometry.RESULTS:Our results demonstrated that stable expression of the PLIN1 gene in skeletal muscle satellite cells is critical to maintaining cell metabolism and mitochondrial function. After knockout and overexpression of the PLIN1 gene, the anti-apoptotic ability of cells was enhanced, and the metabolic activity of the cells was accelerated, but at the cost of mitochondrial structural damage, reduction in the number of mitochondria, and decreased mitochondrial function.CONCLUSION:This study explored the effect of the PLIN1 gene on the mitochondria and metabolism of porcine skeletal muscle satellite cells and provided a theoretical basis for the subsequent study of the effects of PLIN1 on muscle tissue development and meat quality.

Deoxynivalenol (DON) caused serious cytotoxicity for animal cells. However, genes involved in regulating DON toxicity and the underlying molecular mechanisms remain largely unknown. This study explored the role of SLC4A11 and MFSD3 in alleviating DON toxicity and analyzed the DNA methylation changes of these two genes. Viability and cell cycle analysis showed that DON exposure decreased the IPEC-J2 viability ( P < 0.01), blocked the cell cycle in the G2/M phase ( P < 0.01), and increased the rate of apoptosis ( P < 0.05). Expression of the SLC4A11 and MFSD3 genes was significantly downregulated upon DON exposure ( P < 0.01). Overexpression of SLC4A11 and MFSD3 can enhance the cell viability ( P < 0.01). DNA methylation assays indicated that promoter methylation of SLC4A11 (mC-1 and mC-23) and MFSD3 (mC-1 and mC-12) were significantly higher compared with those in the controls and correlated negatively with mRNA expression ( P < 0.05). Further analysis showed that mC-1 of SLC4A11 and MFSD3 was located in transcription factor binding sites for NF-1 and Sp1. Our findings revealed the novel biological functions of porcine SLC4A11 and MFSD3 genes in regulating the cytotoxic effects induced by DON, and may contribute to the detection of biomarkers and drug targets for predicting and eliminating the potential toxicity of DON.

Abstract The excessive and inappropriate production of reactive oxygen species (ROS) can cause oxidative stress and is implicated in the pathogenesis of lung cancer. Cyclophilin A (CypA), a member of the immunophilin family, is secreted in response to ROS. To determine the role of CypA in oxidative stress injury, we investigated the role that CypA plays in human lung carcinoma (A549) cells. Here, we showed the protective effect of human recombinant CypA (hCypA) on hydrogen peroxide (H2O2)-induced oxidative damage in A549 cells, which play crucial roles in lung cancer. Our results demonstrated that hCypA substantially promoted cell viability, superoxide dismutase (SOD), glutathione (GSH), and GSH peroxidase (GSH-Px) activities, and attenuated ROS and malondialdehyde (MDA) production in H2O2-induced A549 cells. Compared with H2O2-induced A549 cells, Caspase-3 activity in hCypA-treated cells was significantly reduced. Using Western blotting, we showed that hCypA facilitated Bcl-2 expression and inhibited Bax, Caspase-3, Caspase-7, and PARP-1 expression. Furthermore, hCypA activates the PI3K/Akt/mTOR pathway in A549 cells in response to H2O2 stimulation. Additionally, peptidyl-prolyl isomerase activity was required for PI3K/Akt activation by CypA. The present study showed that CypA protected A549 cells from H2O2-induced oxidative injury and apoptosis by activating the PI3K/Akt/mTOR pathway. Thus, CypA might be a potential target for lung cancer therapy.