Effects Of Glucose And Lysine Levels On The Lysine Absorption And Transportation In Intestinal Epithelial Cells Of Piglets

Glucose and amino acids as the main precusor substance of energy and protein,however,they cannot be directly through the intestinal mucosa,and transport into the cell inside by the intestinal mucosal carrier protein.The small intestine acts as a primary site for the digestion,absorption and transport of nutrients,which plays a key role in maintaining the energy and protein balance of humans and animals.Therefore,to evaluate the law of absorption and transport of the glucose and lysine in intestinal epithelial cells of piglets,three experiments were conducted in this study.The intestinal epithelial cellls of piglets(IPEC-J2)was used to be a model,the effects of different levels glucose and lysine on cell proliferation and the expression of transporters,including b0,+AT,y+LAT1(IPEC-J2)were detected in this study,And discusses the law of absorption and transportion of lysine.This may improve the use of nitrogen and provide the data support for the animal production.Experiment Ⅰ:to evaluate the effects of different levels of glucose and lysine on cells proliferation,the IPEC-J2 cells were treated with glucose(25,75 and 225mM/L),lysine(0,0.5,1,2 and 4mM/L)and the combination of glucose and lysine,respectively.After 20,40,80 and 160 min,the cells proliferation was detected by RTCA.Each experiment was performed in triplicate and repeated in three independent experiments.The results showed that the cells proliferation was significanted increased when the IPEC-J2 cells were treated with lysine(4mM/L)and glucose(75mM/L),respectively compared to other groups.However,the proliferation was inhibited when the IPEC-J2 cells were treated with 225 mM/L glucose.For the combination,75mM/L glucose and 4mM/L lysine inhibited the IPEC-J2 cells proliferation.In order to determine the optimal glucose,lysine concentration and time for the growth of IPEC-J2 cells cells,we conducted a second experiment.Experiment Ⅱ: to determine the optimum glucose,lysine concentration and time point,the effects of different glucose and lysine levels on the mRNA relative expression of lysine transporters in pig intestinal cells were detected.Experiment design and the IPEC-J2 cells was treament as experiment I.Total RNA was extrated by using Trizol reagent.Then total RNA was reverse-transcribed to cDNA using the reverse transcription kit and the qPCR was performed.Each experiment was performed in triplicate and repeated in three independent experiments.The results showed that suitable glucose and lysine levels could increased the mRNA expression of b0,+AT,y+LAT1,And the interaction effect between glucose and lysine.At 40 mins after treatment,the mRNA expression of b0,+AT、y+LAT1 were the highest when the cells treated with 75mM/L glucose and 2mM/L lysine.Experiment Ⅲ: to determine the optimum glucose,lysine concentration and time point,the effects of different glucose and lysine levels on the proteinexpression of lysine transporters in pig intestinal cells were detected.The IPEC-J2 cells was treament as experiment I and the treatment was 40 mins.Total protein was extracted from cells using RIPA buffer,and the protein expression was detected by western blot.Based on the western blot,the lysine transport vectors,including b0,+AT,y+LAT1 were significant increased in cells treated with 75mM/L glucose and 2mM/L lysine.Of the above research results show that the suitable glucose and lysine combination can promote the absorption、transportion of lysine in IPEC-J2 cell.And these results have an important significance to further explore the digestion,absorption and utilizing system of energy and protein in piglets.