Lrp6 Genotype affects Individual Susceptibility to Nonalcoholic Fatty Liver Disease and Silibinin Therapeutic Response via Wnt/β-catenin-Cyp2e1 Signaling

Abstract

Background: Nonalcoholic fatty liver disease (NAFLD) is a serious threat to human health worldwide, with a high genetic susceptibility. Rs2302685, a functional germline variant of LRP6, has been recently found to associate with NAFLD risk. This study was aimed to clarify the underlying mechanism associated with rs2302685 risk and its impact on pharmacotherapy in treatment of NAFLD. Methods: Venous blood samples were collected from NAFLD and non-NAFLD patients for SNP genotyping by using mass spectrometry. The Lrp6-floxdel mouse (Lrp6^(+/-)) was generated to model the partial function associated with human rs2302685. The liver injury and therapeutic effects of silibinin were compared between Lrp6^(+/-) and Lrp6^(+/+) mice received a methionine-choline deficient (MCD) diet or normal diet. The effect of Lrp6 functional alteration on Wnt/β-catenin-Cyp2e1 signaling activities was evaluated by a series of cellular and molecular assays. Results: The T allele of LRP6 rs2302685 was confirmed to associate with a higher risk of NAFLD in human subjects. The carriers of rs2302685 had reduced level of AST and ALT as compared with the noncarriers. The Lrp6^(+/-) mice exhibited a less severe liver injury induced by MCD but a reduced response to the treatment of silibinin in comparison to the Lrp6^(+/+) mice, suggesting Lrp6 as a target of silibinin. Wnt/β-catenin-Cyp2e1 signaling together with ROS generation could be exacerbated by the overexpression of Lrp6, while decreased in response to Lrp6 siRNA or silibinin treatment under NAFLD modeling. Conclusions: The Lrp6 function affects individual susceptibility to NAFLD and the therapeutic effect of silibinin through the Wnt/β-catenin-Cyp2e1 signaling pathway. The present work has provided an underlying mechanism for human individual susceptibility to NAFLD associated with Lrp6 polymorphisms as well as a rationale for the effective use of silibinin in NAFLD patients.

Keywords: cytochrome P450 2e1; genotype; low-density lipoprotein receptor-related protein 6; nonalcoholic fatty liver disease; reactive oxygen species.

Figure 1

LRP6 rs2302685 was associated with a reduced liver injury in NAFLD patients. (A) Distribution of 22 SNPs likely contributing to the individual risk of NAFLD based on the 'two-hit hypothesis'. (B) Comparison of the serum levels of ALT and AST between the LRP6 rs2302685 T allele carriers (n=47) and C allele carriers (n=239) in NAFLD subjects.

Figure 2

Lrp6 haploinsufficiency (Lrp6^(+/-)) ameliorated liver injury and reduced the efficacy of silibinin in NAFLD mice. (A) Comparison of liver function, as determined from the levels of enzymatic markers, and liver/body weight ratio between the Lrp6^(+/+) and the Lrp6^(+/-) mice received MCD diet. The MCD diet was used to induce liver injury and NAFLD in mice. (B) Effect of Lrp6 genotypes on liver histological injury in mice received different diets and treatments. The histology of liver tissues was examined after H&E staining. The macrovesicular steatosis was indicated with arrows, while necroinflammatory foci were indicated with circular broken lines (Scale bar, 1μM for the above row, and 5μM for the below row in each group). (C) Effects of diets MCS and MCD on liver injury in mice. The hepatic steatosis, hepatocyte ballooning, lobular inflammation and NAS score were examined under microscopy and compared between the Lrp6^(+/+) and the Lrp6^(+/-) mice with and without silibinin treatment. (D) Comparison of the change rate for injury indicators between the Lrp6^(+/+) and the Lrp6^(+/-) mice groups. * P < 0.05; ** P < 0.01; *** P < 0.001; NS, No significance.

Figure 3

Lrp6 haploinsufficiency (Lrp6^(+/-)) affected the therapeutic effect of silibinin in NAFLD mice. (A) Comparison of the liver function, as determined from the levels of enzymatic markers, and liver/body weight ratio between the Lrp6^(+/+) and the Lrp6^(+/-) mice received MCD diet with and without the treatment of silibinin. (B) Change of the liver function and liver/body weight ratio by silibinin treatment between the Lrp6^(+/+) and Lrp6^(+/-) mice. * P < 0.05; NS, No significance.

Figure 4

Lrp6 was a target for silibinin to inhibit the Wnt/β-catenin-Cyp2e1 signaling pathway and the generation of ROS. (A) Comparison of the Wnt/β-catenin signaling activity between the silibinin-treated and untreated HL7702 cells. The Wnt/β-catenin signaling activity was determined from the fluorescence intensity induced by LiCl treatment or Wnt3a-conditioned medium in reporter gene assays. XAV939, a characterized Wnt/β-catenin pathway inhibitor, served as a positive control. (B) Comparison of the Wnt/β-catenin signaling activity between the silibinin-treated and untreated TOPFlash HEK293 cells overexpressing Lrp6 W (wide-type) or Lrp6 M (mutant). The Wnt/β-catenin signaling activity was determined by reporter gene assays for the TOPFlash cells, as described in Methods. (C) Comparison of the oleat uptake between the silibinin-treated and untreated HL7702 cells overexpressing Lrp6 W (wide-type) or Lrp6 M (mutant) (Scale bar, 5μM). (D) Comparison of ROS generation induced by oleat treatment between the silibinin-treated and untreated HL7702 cells transfected with Lrp6 siRNA or Lrp6 W vector (Scale bar, 1μM). * P < 0.05; ** P < 0.01; *** P < 0.001; NS, No significance.

Figure 5

Effects of Lrp6 expression and silibinin treatment on the expression and activity of Cyp2e1. (A) Protein expression of Lrp6, β-catenin and Cyp2e1 among different treatment groups in mice. (B) Expression change of Lrp6, β-catenin and Cyp2e1 proteins by silibinin treatment between the Lrp6^(+/+) and the Lrp6^(+/-) mice. (C) Effects of Lrp6 genotypes and silibinin treatment on the activity of Cyp2e1 in mice received MCD diet. (D) The activity change of Cyp2e1 by silibinin treatment between the Lrp6^(+/+) and Lrp6^(+/-) mice. (E) Effects of Lrp6 expression on the expression changes of Lrp6, β-catenin and Cyp2e1 in HL7702 cells with and without oleat treatment. The cells were transfected with a control vector, the vectors containing Lrp6 siRNA or Lrp6 W. (F) The scheme showing that Lrp6 functional alteration confers different susceptibility to NAFLD and affects the therapeutic effect of silibinin by modulating Wnt/β-catenin-CYP2E1 signaling. * P < 0.05; ** P < 0.01; *** P < 0.001; NS, No significance.

Figure 5

Effects of Lrp6 expression and silibinin treatment on the expression and activity of Cyp2e1. (A) Protein expression of Lrp6, β-catenin and Cyp2e1 among different treatment groups in mice. (B) Expression change of Lrp6, β-catenin and Cyp2e1 proteins by silibinin treatment between the Lrp6^(+/+) and the Lrp6^(+/-) mice. (C) Effects of Lrp6 genotypes and silibinin treatment on the activity of Cyp2e1 in mice received MCD diet. (D) The activity change of Cyp2e1 by silibinin treatment between the Lrp6^(+/+) and Lrp6^(+/-) mice. (E) Effects of Lrp6 expression on the expression changes of Lrp6, β-catenin and Cyp2e1 in HL7702 cells with and without oleat treatment. The cells were transfected with a control vector, the vectors containing Lrp6 siRNA or Lrp6 W. (F) The scheme showing that Lrp6 functional alteration confers different susceptibility to NAFLD and affects the therapeutic effect of silibinin by modulating Wnt/β-catenin-CYP2E1 signaling. * P < 0.05; ** P < 0.01; *** P < 0.001; NS, No significance.

Figure 5

Effects of Lrp6 expression and silibinin treatment on the expression and activity of Cyp2e1. (A) Protein expression of Lrp6, β-catenin and Cyp2e1 among different treatment groups in mice. (B) Expression change of Lrp6, β-catenin and Cyp2e1 proteins by silibinin treatment between the Lrp6^(+/+) and the Lrp6^(+/-) mice. (C) Effects of Lrp6 genotypes and silibinin treatment on the activity of Cyp2e1 in mice received MCD diet. (D) The activity change of Cyp2e1 by silibinin treatment between the Lrp6^(+/+) and Lrp6^(+/-) mice. (E) Effects of Lrp6 expression on the expression changes of Lrp6, β-catenin and Cyp2e1 in HL7702 cells with and without oleat treatment. The cells were transfected with a control vector, the vectors containing Lrp6 siRNA or Lrp6 W. (F) The scheme showing that Lrp6 functional alteration confers different susceptibility to NAFLD and affects the therapeutic effect of silibinin by modulating Wnt/β-catenin-CYP2E1 signaling. * P < 0.05; ** P < 0.01; *** P < 0.001; NS, No significance.