The Regulatory Mechanism of Endothelin-1 on Cholesterol Homeostasis in Hepatocyte

• Main Authors: Chang-Ming Yang, 楊程茗
• Other Authors: Chi-Chang Juan
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/94322415897878340339

碩士 === 國立陽明大學 === 生理學研究所 === 104 === Endothelin-1 (ET-1), a powerful vasoconstricting polypeptide, primarily produced and secreted by vascular endothelial cells. Our studies have demonstrated that ET-1 impaired insulin signal pathway and subsequent glucose uptake in vitro and in vivo. Besides, we also demonstrated that ET-1 stimulated lipolysis in adipocytes. These findings suggested that ET-1 could regulate lipid-associated metabolism. However, the regulation of ET-1 on hepatic lipid metabolism is still not clear. Reverse cholesterol transport (RCT) is a mechanism regulating the removal of excess body cholesterol through hepatocytes. RCT is a complex mechanism including the efflux of cellular cholesterol to high density lipoprotein (HDL) from peripheral cells, the transport of lipoprotein-cholesterol in blood to the liver, and the delivery of cholesterol esters to hepatocytes from HDL for its metabolism and biliary excretion. Hepatic scavenger receptor type B class I (SR-BI), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), low density lipoprotein receptor related protein 1 (LRP1) and low density lipoprotein receptor (LDLR) play important roles in the RCT. The purpose of this study is to explore the regulatory mechanism of ET-1 on RCT in HepG2 cells. We first investigated the effect of ET-1 on expression of SR-BI, LDLR, LRP1, ABCA1 by western blot. Effects of ET-1 on cholesterol efflux and cholesterol uptake were also determined by using TOPFLOUR-cholesterol and DiI-HDL. To further clarify the mechanism mediating ET-1 actions on RCT, ET-1 receptor antagonists or specific signal inhibitors were administrated and ET-1-regulated expression of SR-BI, LDLR, LRP1, ABCA1, and cholesterol efflux and cholesterol uptake were measured. Our results showed that ET-1 significantly decreased ABCA1 protein expression and cholesterol effux in HepG2 cells. However, ET-1 has no effect on SR-B1, LDLR, LRP1 protein expression. Pretreatment of ET type B receptor (ETBR) blocker BQ788, but not ETAR blocker BQ610, successfully prevented ET-1-suppressed ABCA1 expression and cholesterol efflux. In addition, pretreatment of PI3K inhibitor (LY294002), but not ERK inhibitor (PD98059), JNK inhibitor (SP600125) or PKC inhibitor (Ro318220), or p38MAPK inhibitor (SB203580), prevented ET-1-suppressed ABCA1 expression. Moreover, pretreatment of LY294002 also blocked ET-1-reduced cholesterol efflux. Besides, cells were pretreated with protein synthesis inhibitors cycloheximide and effect of ET-1 and ABCA1 was evaluated. The result showed that ET-1 significantly decreased ABCA1 protein levels and suggested that ET-1 could decrease ABCA1 protein stability. In conclusion, ET-1, acting via the ETBR, decreased ABCA1 protein expression and cholesterol efflux through PI3K-depended pathway.