Maternal Low Protein Diet Affects Skeletal Muscle Growth In Weaning Piglets Through Myostatin-Mediated Pathway

There are many reports of the impact of maternal nutrition on skeletal muscle of offspring. So far, the research has focused on the effect of malnutrition during gestation and/or lactation on growth of muscle fibers, muscle fiber type and protein metabolism in offspring muscle fibers. Recent studies show that maternal low protein diet during pregnancy and lactation can affect the concentration of insulin and amino acids in blood, and also affect the Myostatin (MSTN) pathway. However, the effect of maternal low protein diet on gene expression of myogenesis, muscle cell proliferation and apoptosis is unclear. Therefore, the present study was aimed to investigate the effects of maternal low protein diet during gestation and lactation on the levels of insulin, free amino acid, urea nitrogen, total protein and insulin in the plasma of weaning piglets, the effects of gene expression of myogenesis, muscle cell proliferation and apoptosis in weaning piglets skeletal muscle, and the MSTN transcriptional regulation mechanism in weaning piglets skeletal muscle.1. Effects of maternal low protein diet on gene expression of growth-related genes and on apparent indicators in weaning piglets skeletal muscle.In this study, Sixteen Landrace and Large White crossbred sowst of second pregnancy were assigned randomly into standard (SP) and low (LP) protein groups. The sows in the SP group were fed diets containing 15% and 18% crude protein, while those in the LP group were fed diets containing 7.5% and 9% crude protein during gestation and lactation.After childbirth, litter size was adjusted to 7-8 pigs per litter in the same group. Newborn piglets were allowed free access to their mothers and weaned at 28 days (d) of age. Selected one male piglets per litter to collected samples.Weighed piglets’weight before sampling. Serum free amino acids, insulin, three triiodothyronine, thyroxine, cortisol, blood urea nitrogen and total protein levels in blood were measured. Detection RNA, DNA and protein concentrations and contents in weaning piglets longissimus muscle. The results showed that:none of body weight, muscle weight or muscle protein content was affected by maternal dietary protein, so did free amino acid, insulin, T3, T4 or cortisol in plasma. However, the levels of blood urea nitrogen and total protein were decreased in LP piglets at weaning stage (p<0.05). The concentrations of RNA and protein and the contents of RNA were decreased in LP piglets at weaning stage (p<0.05). The contents of DNA showed a trend of decrease in LP piglets at weaning stage (p<0.1). These results suggest that maternal low protein diet may regulate the growth of skeletal muscle of weaning piglets.In order to study the effects of maternal low protein diet on the growth and development of weaning piglets skeletal muscle and its molecular mechanisms, We use the Real-time PCR to tested the gene expression of IGFs, mTOR, PCNA, CCND2, Bcl, Bax, Atrogin1 and MuRF1 in weaning piglets skeletal muscle. We found that IGF2R mRNA levels showed a trend of increase in LP piglets at weaning stage (p<0.1) and CCND2 mRNA levels showed a trend of decrease in LP piglets at weaning stage (p<0.1). Nevertheless, there were no significant differences between the two groups of the other genes expression. The result shows that The result shows that maternal low-protein diet may not be by directly influence the expression of these genes in offspring of skeletal muscle, which affect the growth of skeletal muscle.2 The molecular mechanism of maternal dietary protein level affecting transcriptional regulation of MSTN geneWe detected the expression of genes (MSTN, MyoD, Myf5, Myf6, MEF2 and Myogenin) related to muscle development using Real-time PCR. The results showed that maternal low protein diet in addition to increase the MSTN gene expression, have no effect on the rest of the muscle growth related gene expression. Thus, we examined the expression of MSTN receptor (ACVR2B and ALK5) and the changes of its downstream signal p38-MAPK, ERK, JNK and their phosphorylation forms. The results showed that the phosphorylation levels of p38-MAPK were significantly increased in LP piglets at weaning stage (p<0.05). Suggested that maternal low protein may through MSTN and its downstream p38-MAPK signaling pathways influence offspring skeletal muscle growth. In addition, in order to investigate the transcriptional mechanism of MSTN, We use Real-time PCR and Western blotting methods to detect the expression of transcription factors (FoxO3, FoxO4, GR and PGCla) of LD muscle. The results showed that FoxO4 mRNA expression was increased (p<0.05), FoxO3 and GR mRNA levels showed a trend of decrease (p<0.1) in LP piglets at weaning stage. Western blotting method for the detection, FoxO3 protein expression was significantly higher (p< 0.05) in LP piglets at weaning stage. In order to clarify whether FoxO3 and GR take part in the transcriptional regulation of MSTN, we performed chromatin immunoprecipitation (ChIP) analysis whether FoxO3 and GR were binding to MSTN in LD muscle of pigs at weaning stage. The results revealed significantly (p< 0.05) increased GR and FoxO3 binding to one binding sites on MSTN promoter and a trend of increased (p< 0.1) GR binding to another binding sites on MSTN promoter in LP piglets at weaning stage. The above results suggest:maternal low protein diet may through the MSTN and its downstream p38-MAPK signaling pathways that affect offspring skeletal muscle growth, and FoxO3 and GR involved in MSTN transcription regulation.