Effects Of Sheep Musclin Gene On Glucose Metabolism And Insulin Function In Myoblasts

Musclin is a skeletal muscle-derived secretory factor considered to be involved in cellular glucose metabolism,fat metabolism and insulin resistance.At present,researches on musclin are mainly focused on model organisms such as mice and rats.There is no report on the glucose metabolism effects of musclin in sheep.Here,the eukaryotic expression vector of musclin gene(pc DNA3.1-Musclin),which was constructed by our group in previous study,was used to investigate the effects of sheep musclin gene on glucose metabolism and insulin function in sheep myoblasts.Experiment 1: The structure and physicochemical properties of sheep musclin protein were predicted by using various bioinformatics-related softwares.The results showed that: musclin gene was a conserved gene with high homology among mammals.Musclin protein contained a 27 amino acid signal peptide and had a restriction site at the 28 amino acid site.Subcellular localization showed that it was in the extracellular space,and belonged to the secretory protein.Its secondary structure mainly included alpha helixes and random coils.Experiment 2: Sheep myoblasts were divided into four groups: pc DNA3.1,pc DNA3.1-Musclin,pc DNA3.1 + Insulin(80 n M),and pc DNA3.1-Musclin + Insulin(80 n M).According to grouping protocol,sheep myoblasts were treated separately with insulin or PBS in differentiation medium(2%horse serum)after transfection for 6 h.The culture medium and cells were collected for subsequent detection,respectively after 48 h or 72 h.The expressions of related genes were detected by q RT-PCR.The results showed that: Overexpression of sheep musclin gene significantly reduced glucose uptake and glycogen synthesis or accelerated glycogenolysis in normal or insulin-treated sheep myoblasts.The sheep musclin gene inhibited the mRNA expressions of IRS1,AKT1,AKT2,GLUT1,GLUT4 mRNA,and promoted the expression of GSK3β mRNA in normal or insulin-treated sheep myoblasts induced to differentiate for 72 h,while only the expression of the GLUT4 gene was affected after cells were induced to differentiate for 48 h.In conclusion,the overexpression of the sheep musclin gene affected the glucose metabolism and attenuated the effects of insulin in sheep myoblasts,and they coordinated with each other to maintain the homeostasis of glucose metabolism.And the functional mechanism of sheep musclin involved the expressional changes of the above related genes and were related to the differentiation state of sheep myoblasts.