Molecular Mechanism Of Threonine Regulating Lipid Deposition In Liver Of Meat Ducks

Threonine（Thr）is the third-limiting amino acid of poultry,can balance amino acids,improve feed intake,promote growth,maintain intestinal health,regulate fat metabolism,and improve disease resistance.It was found that dietary threonine deficiency increased liver lipid deposition in Peking ducks.However,the molecular mechanism of threonine deficiency regulating liver lipid deposition in meat ducks remains unclear.This study was conducted in vivo and in vitro to investigate the mechanism of threonine deficiency in regulating lipid deposition in liver of meat ducks from the aspects of growth performance,plasma biochemistry,liver triglyceride,gene and protein expression and protein phosphorylation levels through two experiments,in order to provide a theoretical basis for the rational addition of threonine in meat duck diet.in vivo experiments wasdesigned to investigate the effect of dietary threonine on liver lipid deposition,protein expression,phosphorylated protein expression and gene expression in meat ducks,and to preliminarily reveal the molrcular mechanism of threonine deficiency increasing lipid deposition in liver of ducks at the in vivo level.This experiment adopted a single-factor completely randomized experimental design with three dietary threonine supplementation levels（0,0.25%and 0.50%）.240 1-day-old healthy male Pekin ducks were randomly assigned to the above 3 treatments with 10 replicates in each treatment and 8 ducks in each replicate.The feeding cycle was 1-21 days old.The results showed that dietary threonine deficiency reduced the average body weight,average feed intake and average daily gain of meat ducks（P<0.0001）,and increased the ratio of feed to weight gain,liver triglyceride and plasma cholesterol,low density lipoprotein-cholesterol and high density lipoproteincholesterol concentrations（P<0.05）;Dietary threonine excess（added 0.05%threonine）had no effect on average body weight,average feed intake and average daily gain,feed-to-gain ratio,liver triglyceride and plasma low density lipoprotein-cholesterol,cholesterol and high density lipoprotein-cholesterol concentrations（P>0.05）.Dietary without threonine up-regulated the mRNA expressions of STAT1,STAT5B,ADIPOR,PDGFRB,TYK2,COX17,MPC1,PDK2,ACLY,PPARα,ELOVL2,ELOVL7,FABP1,ACACA,DGAT2 and FASN in liver（P<0.05）;down-regulated the expressions of STAT3 and OXSM（P<0.05）,but had no effect on the expressions of JAK1,JAK2,FABP3,AACS,SREBF1,SCD,FASD1,FADS2,ACAD11 and ACADSB（P>0.05）.Dietary threonine excess（added 0.50%threonine）up-regulated the expressions of PDGFRB and FASN and down-regulated the expressions of STAT3,TYK2,ADIPOR and OXSM（P<0.05）.and had no effect on the expressions of STAT1,STAT5B,PDK2,ACLY,ELOVL2,ELOVL7,FABP1,ACACA and DGAT2.（P>0.05）.176 differential proteins and 198 differential phosphorylated proteins were identified by liver proteomic and phosphorymic proteomic sequencing,of which 102 and 116 were up-regulated protein and phosphorylated protein,and 74 and 82 were down-regulated protein and phosphorylated protein.These differentially expressed phosphorylated proteins and proteins were enriched in glycolysis/gluconeogenesis,fatty acid biosynthesis,fatty acid metabolism,energy metabolism pathways and JAK-STAT pathways.Threonine deficiency up-regulated ADIPOR,TYK2,ELOVL2,FABP1,COX17 and MPC1 protein expressions,as well as protein phosphorylation levels of HK,GAPDH,FBP,FASN,ACAC and ACSL;down-regulated STAT5B,PDGFRB,SDSL,AACS,ACLY and PDK2 protein expressions,as well as protein phosphorylation levels STAT3,STAT1 and IRS2.It can be seen that threonine may regulate liver lipid deposition in meat ducks through the JAK-STAT pathway.in vitro experiments were designed to investigate the effect of threonine on cell activity,triglyceride deposition,lipid-related gene expression and phosphorylated protein levels in HepG2 cells（Human hepatocellular carcinoma cell lines）,as well as the effect of STAT3 on triglyceride in HepG2 cells after Stattic inhibition,so as to reveal that threonine may regulate liver lipid deposition of HepG2 cells through STAT3 phosphorylation.The results showed that with the increase of threonine concentration,the cell activity gradually increased;after culturing HepG2 cells with 0,10,25,400,800μM threonine medium for 36 h,it was found that with the increase of threonine concentration,the triglyceride content decreased gradually.Low threonine group（0,10μM and 25μM）up-regulated the mRNA expressions of STAT1,STAT3,STAT5B,ADIPOR,TYK2,JAK1,JAK2,COX17,MPC1,PPARα,OXSM,AACS,ACACA,FAS,DGAT2 and SDSL in HepG2 cells（P<0.05）,down-regulated the expressions of PGDFRB,ACLY,SCD,FASN and FABP3（P<0.05）.High threonine group（400μM and 800μM）upregulated the expressions of PGDFRB,ACLY,and FABP3（P<0.05）,but had no effect on the expressions of PDK2,SREBF1 and ACADSB（P>0.05）.Threonine deficiency reduced STAT3 total protein,STAT3 Ser727 and STAT3 Tyr705 phosphorylation levels（P<0.05）STAT3 protein and STAT3 gene expression trends were opposite,which may be regulated at translation level and post-transcriptional level.The results of Stattic inhibitor experiment showed that inhibiting STAT3 activity could increase triglyceride deposition in HepG2 cells,and after STAT3 activity was inhibited,the increase of threonine concentration could not change the triglyceride deposition in HepG2 cells,indicating that the inhibition of STAT3 activity is the key to regulating the lipid energy of HepG2 cells.In conclusion,threonine can regulate triglyceride deposition in HepG2 cells through STAT3 phosphorylation.