| The digestive capacity of starch is limited in the intestine of dairy cow.Only 40-62% of small intestine starch can be digested.If a large amount of rumen protected starch used in diet of dairy cow,it will be caused diarrhea and stimulated the secretion of insulin which is not good for lactation.The reason is that the lack of amylase secretion in pancreas of dairy cows.Studies have found that leucine and phenylalanine can regulate the pancreatic exocrine function in dairy goats and dairy cows,and improve the digestive function of the small intestine as well as increase the energy efficiency,especially the utilization efficiency of starchy feeds,thereby improving the performance and maintaining a healthy body condition in special physiological stages.However,the specific regulation mechanisms of leucine and phenylalanine in the pancreas of dairy cows have not been elucidated.It is necessary to start with specific molecular signals to study relevant signaling networks.In this paper,in vitro cell experiment and tissue segments incubation experiment were used to study the effects of different concentrations of leucine and phenylalanine in the medium on the process of the transcription,translation and secretion of digestive enzymes.The methods of spectrophotometry,transmission electron microscopy,real-time fluorescence quantitative PCR,Western Blot,Elisa,proteomic analysis and other methods were used in the present study.So that the mechanism of amino acids regulates pancreatic enzyme synthesis and secretion were explored in order to provide a theoretical basis for improving intestinal digestive capacity of dairy cows.Exp.1 The molecular mechanism of phenylalanine regulates enzyme synthesis and secretion in pancreatic acinar cells of dairy cows.This experiment was conducted by adding different concentrations of phenylalanine in culture media,which were 0,0.15,0.45 mM,respectively.The incubation time was 120 min.Present study focused on the digestive enzymes synthesis and secretion,mRNA and mTOR signaling pathway factors expression.The results showed that as the concentration of phenylalanine increased in the media,the secretion and gene expression of amylase and the phosphorylation levels of S6K1 and 4EBP1 were increased significantly(P < 0.05).After inhibiting the activation of S6K1 protein,the amylase activity in the phenylalanine-treated group was significantly decreased(P < 0.05).CCK can directly stimulate acinar cells to secrete amylase,while the mechanism of action was different from that of phenylalanine.The results of this experiment indicated that the high concentration of phenylalanine(0.45 mM)promotes the formation of protein translation initiation complexes by increasing the mRNA expression of amylase,phosphorylation of S6K1 and 4EBP1 proteins,and ultimately increasing the exocrine ability of pancreatic acinar cells.Exp.2 The molecular mechanism of phenylalanine regulates the exocrine function in pancreatic tissue of dairy cows.The experiment was carried out by in vitro incubation of pancreatic tissue to study the effects of phenylalanine on digestive enzyme activity,mRNA expression and mTOR signaling pathway in pancreatic tissue of dairy cows.The healthy Holstein milk calves of 2 months old was selected,and the pancreas was collected after bloodletting and immediately subjected to tissue incubation experiment.The concentration of phenylalanine treatment was determined according to the Exp.1: control group(0 mM)and phenylalanine group(0.35 mM),with 6 repetitions per treatment,incubation time of 180 min,and samples were taken every 60 min.The results showed that phenylalanine significantly regulated the synthesis of digestive enzymes in pancreatic tissue of dairy cows and its gene expression,including ?-amylase,lipase and protease,and phosphorylation levels of S6 K and 4EBP1.The mechanism may be that the phenylalanine promotes the formation of a transcriptional initiation complex and then increases the synthesis of protein in the pancreatic tissue of dairy cow.Exp.3 The molecular mechanism of leucine regulates enzyme synthesis and secretion in pancreatic acinar cells of dairy cows.This experiment was conducted by adding different concentrations of leucine to the culture media,which were 0,0.23,0.45,0.90 mM,respectively,and incubation time was 60 min.Present study focused on the synthesis of digestive enzymes,effects of PI3K/Akt,mTOR,GCN2 and other protein expression in primary acinar cells of pancreas.The results showed that with increasing leucine concentration from 0 to 0.45 mM,intracellular and extracellular amylase activity,media leucine consumption,number and volume of zymogen particles,and phosphorylation of PI3 K,Akt,mTOR,and S6K1 were significantly increased(P < 0.05);isoleucine depletion and GCN2 expression were significnatly decreased(P < 0.05);the phosphorylation level of 4EBP1 and eIF2? was not affected(P > 0.05).The results of rapamycin inhiitors showed that mTOR phosphorylation and intracellular amylase synthesis were significantly reduced(P < 0.05).The consumption of leucine and isoleucine and the phosphorylation of PI3K/Akt were significantly decreased in the 0.90 mM leucine group compared with the 0.45 group(P < 0.05).Label-free proteomic analysis found that there were 2009 differentially expressed proteins be identified in the four treatments.Cellular component,molecular function,biological process and KEGG pathway analysis revealed that leucine significantly affected oxidative phosphorylation in mitochondria of acinar cells(P < 0.01).Cluster analysis showed that the intracellular mitochondrial tricarboxylic and cycle and oxidative phosphorylation process gradually increased with the increase of leucine concentration(P < 0.01).The activities of citrate synthase,isocitrate dehydrogenase,and cytochrome C oxidase increased significantly with increasing leucine concentration(P < 0.05).ATP content was significantly higher in 0.45 and 0.90 mM leucine treatments than that of the 0 and 0.23 mM leucine treatments(P < 0.05).Leucine significantly increased the activity of the protein transport signaling pathway-SEC signaling pathway(P < 0.05).The results indicated that leucine enhancd the amylase synthesis by promoting the protein synthesis signal network,and increased the amylase secretion by promoting the TCA cycle-mediated ATP production,as well as simultaneously activating SEC signaling pathway,thereby promoting amylase secretion.Exp.4 The molecular mechanism of leucine regulates the synthesis of digestive enzymes in pancreatic tissue of dairy cows.In this experiment,pancreatic tissue culture was used to study the effects of leucine on digestive enzyme activity,mRNA expression and mTOR signaling pathway in pancreatic tissue of dairy cows.Pancreatic tissue incubation method was the same as test 2,leucine treatment concentration was set according to Exp.3: control group(0 mM)and leucine addition group(0.45 mM),6 replicates per treatment,incubation time was 180 min,and samples were taken every 60 min.The results showed that leucine could enhance the synthesis of ?-amylase(P < 0.05)and trypsin(P < 0.05)in the pancreas of dairy cows,and stimulate the synthesis of ?-amylase and trypsin through the mTOR pathway as a nutrient signal.Exp.5 The regulation mechanism of leucine and phenylalanine mixed regulates the exocrine function in pancreatic tissue of dairy cow.The methods of pancreatic tissue segments incubation was used to study the effects of leucine and phenylalanine on digestive enzymes synthesis and secreation as well as its mRNA expression,and the activation of mTOR signaling pathway.Pancreas was obtiened from two-month-old healthy Holstein dairy bull calves.Three treatments were set up for the experiment: a control group(no amino acid),a mixed group(0.45 mM leucine + 0.35 mM phenylalanine),and a function amino acids group(0.45 mM leucine + 0.35 mM phenylalanine + 0.23 mM valine+ 0.22 mM isoleucine).The incubation time was 180 min.Samples were collected every 60 min and biological replicates were set to 6.The results showed that mixed addition of leucine and phenylalanine significantly increased the activity of trypsin(P < 0.05)and chymotrypsin(P < 0.05)at 2 h,3 h,and decreased amylase at 1 h and 2 h(P < 0.05);significantly increased 1 h,2 h trypsin,1 h chymotrypsin and lipase mRNA expression;meanwhile,increased mTOR pathway target molecule phosphorylation,and reduced amylase activity;functional amino acid treatment had no significant effect on enzyme activity.It was indicated that the combination of leucine and phenylalanine promotes the secretion of proteases,but inhibits the secretion of amylase,and is not suitable for simulataneous improvement in the utilization of starch in small intestines of dairy cow.This study can get two conclusions:(1)Phenylalanine regulates the synthesis and secretion of digestive enzymes by affecting pancreatic enzyme mRNA expression and phosphorylation of S6K1 and 4EBP1 proteins.(2)Leucine could increase the synthesis of digestive enzymes by promoting the PI3K/Akt-mTOR signaling pathway in the pancreatic acinar cells of dairy cows;on the other hand,leucine also regulates the secretion of enzymes by increased the ATP content which produced by tricarboxylic cycle and oxidative phosphorylation process thereby simultaneously activating the SEC signaling pathway which can be enhanced the enzymes processing and export from intracellular to extracellular. |
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