Studies on cholesterol and bile acid metabolism in Chinese cholesterol gallstone patients

Sammanfattning: Liver and intestine are important organs for the metabolism of cholesterol and bile acids in the body. Dysfunction in cholesterol and bile acid metabolism may lead to diseases such as cholesterol gallstone (GS) disease and atherosclerosis. The projects presented in this thesis aim to gain further insights into hepatic and intestinal cholesterol and bile acid metabolism and their role in the regulation of lipoprotein metabolism. We found that: Paper I. Chinese patients with GS disease had higher mRNA expression of hepatic ABCG5, ABCG8 and LXRalpha, which correlated with biliary cholesterol molar percentage and cholesterol saturation index (CSI). The mRNA and protein levels of the hepatic scavenger receptor B type I (SRBI) were increased and a significant correlation was found between the protein levels and the CSI as well. The results suggest that an upregulation of ABCG5/ABCG8 in Chinese GS patients, possibly mediated by increased LXRalpha, may contribute to the cholesterol supersaturation of bile. The data also indicate the possibility that increased amount of biliary cholesterol may originate from HDL by an enhanced transfer via SRBI. Paper II. The intestinal mRNA expression of NPC1L1 and ACAT2 were significantly higher in Chinese GS patients than in gallstone-free (GSF) controls. Our data suggest that the increased NPC1L1 and ACAT2 mRNA levels in GS patients might indicate an upregulation of cholesterol absorption and esterification in the small intestine. Paper III. Analysis of liver biopsies from Chinese GS and GSF patients revealed strong positive correlations between NPC1L1 and SREBP2 and HNF4alpha mRNA. Further studies were carried out to reveal the transcriptional regulation of NPC1L1 by these transcription factors. We showed a dosedependent regulation by SREBP2 on the NPC1L1 promoter activity and mRNA expression in HuH7 cells. Chromatin immuno-precipitation (CHIP) assay confirmed the binding of SREBP2 to the promoter in vivo. Moreover, HNF1alpha increased both NPC1L1 promoter activity and the gene expression, and an important HNF1alpha binding site (-158/-144 nt) was identified within the human NPC1L1 promoter. CHIP assays also confirmed that HNF1alpha binds to the NPC1L1 promoter in vivo. Paper IV. In human, hepatic microsomal ACAT2 activity was significantly lower in females than in males. Moreover, the activity of ACAT2 correlated negatively with plasma HDL cholesterol and Apo A1. This is the first description of a gender-related difference of hepatic ACAT2 activity suggesting, a possible role for ACAT2 activity in the regulation of the cholesterol metabolism in humans. The negative correlation between ACAT2 activity and HDL cholesterol or Apo A1 may reflect such regulation. Since ACAT2 activity has been found to be pro-atherogenic, the observed gender-related difference may contribute to the protection of coronary heart disease in females. Paper V. In this study, we found that female mice had significantly higher hepatic ACAT2 activity and mRNA expression and hepatic cholesteryl esters than male mice, independently of the presence/absence of estrogen receptor alpha (ERA) or beta (ERB). The presence of ERA was associated to a higher mRNA of Acat2 in mice. Furthermore, the interaction analysis showed that in female mice, presence of ERA was associated with a higher Acat2 mRNA level. Hepatic ABCA1 protein was also lower in female mice, independently of ERA or ERB. ABCA1 protein correlated positively with the free cholesterol content in the plasma membrane and negatively with hepatic ACAT2 activity. Our data suggest that ERA seems to regulate Acat2 at the mRNA level but not the activity. The observed relation between ABCA1 protein, free cholesterol in the plasma membrane and hepatic ACAT2 activity suggest a possible regulatory pathway in mice.