Effects And Mechanisms Of The Dipeptide Pro-Gly On Hepatic IGF-1 Expression And Secretion

Insulin-like growth factor 1(IGF-1)is an important endocrine factor that promotes the growth and development of animals and is mainly synthesized and secreted by the liver.The level of IGF-1 in the blood is mainly regulated by growth hormone(GH)secreted by the pituitary.New research shows that nutrients in the blood,such as proteins and their products,amino acids and small peptides,also have important effects on the secretion of IGF-1 in the liver.Pro-Gly(PG)is a naturally-occurring dipeptide in human serum.It has biological functions such as promoting protein synthesis of fibroblasts.So far,whether Pro-Gly can affect the secretion of IGF-1 in the liver has not been see the report.To study the effect of dipeptide Pro-Gly on the expression and secretion of hepatic IGF-1,C57BL/6J female mice were first selected as the test.The following two experiments were designed.(1)Short-term effects of intraperitoneal injection of Pro-Gly on expression and secretion of IGF-1 in mouse liver: 18 6-week-old female mice were randomly divided into 3 groups(control Group,Pro-Gly group,and Pro+Gly group),were injected intraperitoneally with normal saline,100 mg/kg Pro-Gly,58 mg/kg Pro+43 mg/kg Gly(a composite group of two amino acids).One hour after injection,the liver tissue and serum of the mice were collected.The mRNA expression of IGF-1 was detected by RT-qPCR,and the protein expression of IGF-1 was detected by Western blot.Radioimmunoassay(RIA)was used to detect the serum IGF-1 in mice.The results showed that after acute intraperitoneal injection of Pro-Gly for 1 h,the mRNA and protein levels of IGF-1 in the liver and serum IGF-1 levels were significantly increased,and the mRNA expression of insulin-like growth factor binding protein 1(IGF-BP1)and insulin-like growth factor binding protein 3(IGF-BP3)was significantly decreased(P<0.05);(2)Long-term effects of repeated intraperitoneal injection of Pro-Gly on IGF-1 expression and secretion in mice liver: Thirty 4-week-old female mice were randomly divided into 3 groups(control group,Pro-Gly group,and Pro+Gly group),were injected intraperitoneally with normal saline,150 mg/kg Pro-Gly,87 mg/kg Pro+57 mg/kg Gly.Injection once every other day,sampling after 35 days.The results showed that long-term intraperitoneal injection of dipeptide Pro-Gly significantly increased body weight and body weight gain in mice compared with Pro+Gly group(P<0.05).Compared with saline group,injection of Pro-Gly significantly reduced the content of fat in mice(P<0.05)and had no significant effect on muscle content(P>0.05).And injection of Pro-Gly significantly increased the index of brown adipose tissue in mice,and reduced periocular lipid index(P<0.05),and a rising trend of tibia index(P=0.06).Pro-Gly significantly promoted mouse liver IGF-1 gene copy number,protein expression and serum IGF-1 content(P<0.05).In order to verify the effect of Pro-Gly on hepatic IGF-1 secretion at the cellular level,the human hepatocellular carcinoma cell line HepG2 cells were treated with Pro-Gly dipeptide at different concentrations(0,0.2,0.5,and 1 mM)for 24 h in culture medium(n=6).IGF-1 related genes mRNA expression were detected by RT-qPCR,Western blot was used to detect the protein expression of IGF-1,and RIA was used to detect the content of IGF-1 in cell supernatant.The results showed that the effect of 0.5 mM Pro-Gly was most significant and stable,and it could significantly promote the mRNA and protein expression of IGF-1,and the content of IGF-1 in the cell supernatant(P<0.05),and decreased the mRNA expression of IGF-BP3(P<0.05).Pro+Gly,a combination of two amino acids,did not promote the expression and secretion of IGF-1(P>0.05).The above results indicate that dipeptide Pro-Gly could promote the growth performance of C57BL/6J female mice,increase the expression and secretion of IGF-1 in mouse liver and HepG2 cells,but the composite treatment of monomeric amino acids(Pro or Gly)did not have these effects.To investigate the mechanism of Pro-Gly's promotion of IGF-1 expression and secretion in the liver,co-immunoprecipitation(co-IP),immunocytochemistry(ICC),and Western blot were used to examine the effects of signaling pathways related to IGF-1 secretion.The study found that after 6 h of Pro-Gly treatment of HepG2 cells,the binding between JAK2 and STAT5 protein was significantly enhanced,and the effect of p-STAT5 translocation into the nucleus was significantly enhanced;mice were injected intraperitoneally with 100 mg/kg Pro-Gly and long-term intraperitoneal injection of 150 mg/kg Pro-Gly could significantly increase the phosphorylation of JAK2/STAT5 signaling pathway in the liver(P<0.05).JAK2 blocking assays were further performed at in virto and in vivo tests using the JAK2 blocker AZD1480 respectively.(1)In vitro test: After 6 or 24 hours of treatment with 1 ?M AZD1480 and 0.5 mM Pro-Gly in HepG2 cells,phosphorylation of JAK2 and STAT5 was significantly inhibited(P<0.05),p-STAT5 translocated into the nucleus and the expression and secretion of IGF-1 was reversed(P>0.05).(2)In vivo test: After mice were injected intraperitoneally with Pro-Gly(100 mg/kg,1 h)and AZD1480(30 mg/kg,3 h),the phosphorylation of JAK2,STAT5 protein in the liver of mice was significantly reduced(P<0.05),Pro-Gly no longer promoted the expression and secretion of IGF-1(P>0.05).The above results indicate that the JAK2/STAT5 signaling pathway mediates Pro-Gly-promoted IGF-1 expression and secretion.In the above experiments,it was also found that Pro-Gly can significantly upregulate the mRNA expression level of oligopeptide transporter PepT1 in mouse liver and HepG2 cells(P<0.05).In order to verify whether Pro-Gly functions through PepT1,siRNA was used to test PepT1 interference in HepG2 cells.The results showed that phosphorylation of JAK2,STAT5 and IGF-1 protein expression were significantly inhibited after treatment with PepT1 siRNA and 0.5 mM Pro-Gly for 24 h(P<0.05),and Pro-Gly no longer promoted the protein expression of IGF-1(P>0.05).These results indicate that the effect of Pro-Gly on the expression of IGF-1 in HepG2 cells through JAK2/STAT5 pathway is dependent on PepT1.Collectively,this study found that dipeptide Pro-Gly can promote the expression and secretion of IGF-1 in the liver through the activation of PepT1 and increase the activity of JAK2/STAT5 signaling pathway,ultimately promoting the expression and secretion of IGF-1.This study provides experimental basis for further understanding the molecular nutrition mechanism of pro-Gly dipeptide regulation of hepatic IGF-1 expression and secretion,and provides new ideas for the further research and development of nutritional regulation technology to promote animal growth and development.