The Effect Of Transmissible Gastroenteritis Virus Infection On Glucose And Arginine Uptake In Porcine Intestinal Columnar Epithelial Cells

Transmissible gastroenteritis(TGE)is a highly contagious infectious disease of pigs that is characterized by vomiting,diarrhea,and dehydration.Notably,the mortality rate of this disease in seronegative suckling piglets can reach up to 100%.TGE is caused by the TGE virus(TGEV),which infects the gastrointestinal epithelial cells.The gastrointestinal epithelial cells also play an important role in nutrients absorption,including glucose and arginine.little is known about the effect of TGEV infection on the intestinal epithelial cell nutrient uptake in vitro.Porcine small intestinal epithelial cell line(IPEC-J2)is an intestinal columnar epithelial cell line that was isolated from the mid-jejunum of a neonatal piglet.It offers researchers with a unique model for swine-based virus and nutritional studies in vitro.In this study,we aimed to explore the effects of TGEV infection on glucose and arginine uptake and the regulation mechanism in IPEC-J2 cells.We conducted the following studies:1)The effect on TGEV infection on glucose uptake in IPEC-J2 cellsTGE is caused by the TGE virus(TGEV),whichinfects the gastrointestinal tract,causing villus atrophy and crypt hyperplasia,and disrupting intestinal nutrition absorption.Glucose is among the nutrients absorbed in the intestinal epithelium,and glucose uptake in epithelial cells depends on two types of glucose transporters,the apically expressed Na-dependent glucose transporter 1(SGLT1)and basolaterally expressed facilitative glucose transporter 2(GLUT2).Compared with control group,TGEV infection increased glucose uptake of IPEC-J2 cells after 48h(p<0.05).Further,while there were no marked changes in the mRNA expression of the SGLT1 and GLUT2 genes after TGEV infection,there were significant increases in the protein expression of both SGLT1 and GLUT2 at 48 and 60 hpi(p<0.05).We also exclude the possibility that cell viability and proliferation may affect glucose uptake.In summary,TGEV infection promoted the uptake of glucose by increasing the expression of glucose transporter SGLT-1 and GLUT2 protein.2)EGFR regulated glucose uptake in IPEC-J2 cellsIt has been reported that EGFR is an important signaling protein involved in the regulation of glucose transporter SGLT1 and GLUT2 expression.We formed EGFR lentiviral expression vector amdexplored the expression of EGFR.SGLT1 and GLUT2 in mocked infected and TGEV infected IPEC-J2 cells respectively.The results showed that the protein expression of SGLT1 was decreased by inhibiting EGFR and the protein level of SGLT1 was increased by overexpression of EGFR in the mock infected IPEC-J2 cells(p<0.05).EGFR did not affect the protein expression of GLUT2.In TGEV infected cells,interference and overexpression of EGFR showed the positive correlation between EGFR protein level and SGLT1,GLUT2 protein expression.On the other hand,it was found that high concentration of glucose could promote TGEV replication and release.3)The effect of TGEV infection on arginine uptake of IPEC-J2 cellsMost published studies focus on the effect of TGEV on intestinal immune.However,little is known about the effect of TGEV infection on the intestinal epithelial cell nutrient uptake in vitro,especially for amino acids.The present study investigated the mechanism of TGEV affecting arginine absorption by IPEC-J2 as models in vitro.High performance liquid chromatography(HPLC)detection found that TGEV-infected cells presented significant decreased arginine uptake compared to mock-infected cells after 48 h.p.i(P<0.05).Quantitative reverse transcription-PCR and Western analysis further confirmed that TGEV-induced changes in arginine transport were accompanied by changes in the expression of cationic amino acid transporter(CAT)-1 protein instead of CAT-2.Phorbol 12myristate 13-acetate(PMA)further reduced CAT-1 protein expression and arginine uptake though its activation effects on the protein kinase C(PKC)in TGEV-infected cells(p<0.05).CAT-1 protein expression and arginine uptake were increased by inhibition of phosphorylated epidermal growth factor receptor(p-EGFR)and Caveolin-1 the protein levels between Caveolin-1 and CAT-1 were bidirectional and were negatively correlated to each other.They might formed p-EGFR-PKC-Caveolin-1 signaling pathway involved in regulation of TGEV infection-decreased arginine uptake.