The Mechanism Of Microsomal Triglyceride Transfer Protein(MTTP)in Cadmium Induced High Triglycerides Levels In Hepatocyte

Environmental pollution and occupational exposure to cadmium（Cd）can cause abnormal lipid metabolism and increase the incidence of cardiovascular diseases,such as hypertension and atherosclerosis,as well as fatty liver,pancreatitis and hyperuricemia.The previous studies of lipids metabolism disorder under Cd exposure have demonstrated the molecular mechanism of elevated cholesterol level induced by Cd.However,the underlying mechanisms of Cd exposure affects triglyceride（TG）levels remain unclear.Our previous results confirmed that Cd exposure increased plasma TG levels and induced microrosomal triglyceride transfer protein（MTTP）expression in mouse liver tissues.The present study aims to reveal the role of MTTP in Cd exposures-increased in TG levels and its regulatory mechanism.In this study,we firstly determined the TG levels in both intracellular and conditioned medium of Cd-exposed human liver cells（Hep G2）.The results showed that the TG levels increased in both intracellular and conditioned medium under Cd exposure,and it suggests that Cd induces TG and exportation in Hep G2 cells.Real-time quantitative RT-PCR and Western blotting showed that Cd exposure did not affect the m RNA level of MTTP in Hep G2 cells,but increased the protein level of MTTP.Furthermore,si RNA interference experiments confirmed that increase of the TG levels was medated by MTTP protein,which is induced by Cd.Protein degradation is a major posttranslational regulation pathway.The proteasome pathway and the autophagy-lysosome pathway are two major prtein degradation pathways.The studies showed that proteasomal inhibitor MG132 and lysosomal inhibitor Chloroquine（CQ）had synergistic effects on the protein level of MTTP with Cd treatment,which indicated that Cd exposure could promote the protein stability of MTTP by inhibiting proteasomal and lysosomal protein degradation pathways.In addition,both autophagy inducer Rapamycin and autophagy inhibitor 3-MA could inhibit the accumulation of MTTP protein induced by Cd.This suggests that Cd can affect the accumulation of MTTP protein and TG levels by interfering with autophagy-lysosomal pathway.To further clarify the mechanism of lysosomal pathway in Cd-induced MTTP accumulation and elevated TG levels,we examined the effect of Cd on calcium ion(Ca²⁺)levels in Hep G2 cells and on lysosomal acidity.The lysosomal acidic environment,which is necessary for lysosomal degradation pathway,was disrupted by Cd exposure but restored by pre-treatment of intracellular Ca²⁺chelator BAPTA/AM.Furthermore,Cd-disrupted lysosomal acidity was restored by pre-treatment of the inositol 1,4,5-trisphosphate receptor（IP3R）inhibitor 2-APB to prevent from endoplasmic reticulum（ER）Ca²⁺release.Thapsigargin（Tg）,the inhibitor of ER Ca²⁺reuptake pump sarco（endo）plasmic reticulum Ca²⁺-ATPase（SERCA）,also disrupted acid environment of lysosomes.Moreover,Tg increased MTTP protein and TG levels as proton pump V-ATPase inhibitor Bafilomycin A1（Baf A1）,while Cd-induced MTTP protein and TG levels were significantly reduced by pretreatments of 2-APB and SERCA agonist CDN1163.Taken together,these results suggest that Cd-induced ER Ca²⁺release impaired the lysosomal acidity,which caused MTTP protein accumulation and contributed to increased TG levels.