Effect Of High-fat Environment Combined With PDTC On The Expression Of Amino Acid Transporter In Rat Hepatocytes

Background and purpose: Studies show that the fasting plasma branched-chain amino acid(BCAA)level increases in patients with type 2 diabetes and insulin resistance,but the mechanism is unclear.The liver is the key organ for BCAA metabolism and plasma amino acids go into and out of hepatocytes relying on the coordination of amino acid transport proteins,therefore amino acid transport proteins may change.In addition,obesity causes a chronic inflammatory response and activates nuclear factor-κB(NF-κB).Our previous studies showed that plasma BCAA levels increased in obese insulin-resistant rats with high-fat diet,and pyrrolidine dithiocarbamate(PDTC),an inhibitor of the NF-κB signaling pathway,reduced the increase in BCAA levels.To elucidate the cause of the elevated fasting plasma branched-chain amino acid(BCAA)level,it is necessary to investigate the changes of the amino acid transporter protein and amino acid metabolism in hepatocytes under high-fat environment.This study intends to investigate whether high-fat environment combined with PDTC influences the expression of amino acid transporter protein and amino acid metabolism in hepatocyte in vitro.Materials and methods: BRL-3A hepatocyte were grown and maintained MEM medium.Differentiated cells were starved for 4h and then placed in MEM medium.The specific grouping was as follows: 1.Regular MEM(containing 5mmol/L glucose,0.1mM palmitic acid),further divided into two groups: normal control group,without adding any other drugs in the culture medium(group N);PDTC 0.1μM was added to the culture solution(group NP);2.High-fat MEM(containing 5mmol/L glucose,0.5mM palmitic acid),also further divided into two groups: high-fat control group,without adding any other drugs in the culture medium(group H);PDTC 0.1μM was added to the culture medium(group HP).Each group was set duplicates and incubated for 24 hours.After collecting the samples,the gene expression of P65,RelB,P-P65,P-RelB,SLC38A2,SLC38A3,SLC38A4,SLC38A7,SLC7A7,SLC3A2,SLC43A1,BCAT1,BCAT2,BCKDH-E2 were detected by fluorescent quantitative RT-PCR and the protein content of P65,RelB,P-P65,P-RelB,SLC38A2,SLC38A3,SLC38A4,SLC7A7,SLC3A2,SLC43A1 were detected by western blot.Research result:1.P65,P-P65 and P-RelB proteins were significantly increased(P<0.05)in the H group compared to the group N.P65 protein was significantly increased(P<0.05)and P-P65 protein was significantly decreased(P<0.05)in the group HP compared to the group H.No significant effect was shown on RelB and P-RelB(P>0.05).2.The expression of BCAT1 and BCAT2 mRNA were significantly decreased(P<0.01),BCKDHE2 mRNA was significantly increased(P<0.05)in group H compared to group N.BCAT1 mRNA expression was significantly increased(P<0.05)and BCKDH-E2 mRNA expression was significantly decreased(P<0.05)in group HP compared to group H.BCAT2 mRNA expression was significantly decreased(P<0.05)in group HP compared to group NP.3.The protein content of SLC38A3,SLC38A4,SLC7A7,SLC3A2 were significantly lower in group H compared with group N(p<0.05);the protein content of SLC38A2 was significantly lower in group HP compared with group H(p<0.05).The protein content of SLC38A3,SLC38A4,SLC7A7,SLC43A1,SLC3A2 did not show significant difference(p>0.05).Conclusion: 1.High-fat environment activated the NF-κB classical pathway and non-classical pathway activity of hepatocyte.PDTC inhibited the activity of classical pathway and did not affect the non-classical pathway.2.High-fat environment reduced SLC38A3,SLC38A4,SLC7A7,SLC3A2 protein content in hepatocyte,and the inflammatory inhibitor PDTC reduced the protein content of SLC38A2.