Decreased NPC1L1 expression in the liver from Chinese female gallstone patients

Abstract

Background: Cholesterol gallstone disease is a very common disease in both industrialized and developing countries. Many studies have found that cholesterol gallstones are more common in women than men. The molecular mechanisms underlying the relationship between female gallstone disease and hepatic sterol transporters are still undergoing definition and have not been evaluated in humans.

Aims: The aim of this study is to probe for underlying hepatic molecular defects associated with development of gallstones in female.

Methods/results: Fifty-seven nonobese, normolipidemic Chinese female gallstone patients (GS) were investigated with 12 age- and body mass index-matched female gallstone-free controls (GSF). The bile from the female GS had higher cholesterol saturation than that from the female GSF. The hepatic NPC1L1 mRNA levels were lower in female GS, correlated with SREBP2 mRNA. NPC1L1 downregulation was confirmed at protein levels. Consistently, immunohistochemistry showed decreased NPC1L1 expression in female GS.

Conclusions: The decreased hepatic NPC1L1 levels in female GS might indicate a downregulated reabsorption of biliary cholesterol in the liver, which, in turn, leads to the cholesterol supersaturation of bile. Our data are consistent with the possibility that hepatic NPC1L1 may be mediated by SREBP2.

Figure 1

Quantitative mRNA expression levels in genes from the liver in female gallstone patients (GS) and female gallstone-free patients (GSF). A: Relative gene expression between female GS (n = 57) and female GSF (n = 12). The dotted line at value 1 represents the mean gene expression level in female GSF, which was arbitrarily set to 1 (A.U.); the black bars represent the gene expression levels in female GS (means ± SD). B: Correlation between hepatic NPC1L1 and SREBP2 mRNA levels (n = 69). C: Correlation between hepatic ABCG5 and ABCG8 mRNA levels (n = 69). D: Correlation between hepatic LXRα and ABCG5 mRNA levels (n = 69). E: Correlation between hepatic LXRα and ABCG8 mRNA levels (n = 69).

Figure 2

Hepatic expression of NPC1L1 protein in female gallstone patients (GS) and female gallstone-free patients (GSF). A: Correlation between NPC1L1 mRNA and protein levels in 18 female subjects (P < 0.01). AU, arbitrary units. B: NPC1L1 protein expression in female patients with gallstone disease (n = 11), and without gallstone disease (n = 7). GAPDH is shown as a control. R represents a human liver membrane sample that was used as a reference for each gel. C: NPC1L1 protein level was lower in female GS than in female GSF. Data show means ± SD of the value obtained from the quantitation of the blots shown in 2B. A.U., arbitrary units.

Figure 3

Quantitative immunohistochemistry to detect NPC1L1 from a specimen from a female gallstone-free control using ImageJ. After stripping the original digital image of non-white background (A), colour deconvolution is used to separate the haematoxylin component (B) from the 3',3'-diaminobenzidine (DAB) component (C). The minimum and maximum mean optical density values of eight positively stained cells selected from the DAB analysis are used to set the threshold manually (D) for sample optical density measurement. original magnification 40×.

Figure 4

Presence of hepatic NPC1L1 in female gallstone patients (GS) and female gallstone-free patients (GSF). Tissues were immunohistochemically stained for NPC1L1 (A, B). Hepatic specimens shown are from female GS (A) and female GSF (B). Panel C shows results in the absence of primary antibody for NPC1L1. Panels A, B are superimposed with optical density results (red colour) derived from ImageJ. All samples were counterstained with haematoxylin; original magnification 10× (A, B) and 40× (C).