Aquaporin-8 ameliorates hepatic steatosis through farnesoid X receptor in obese mice

Abstract

Aquaporin-8(AQP8), is a transmembrane channel protein that abounds in liver, which mainly promotes water transport, modulating bile acid formation. However, its role in hepatic lipid metabolism remains unclear. In this study, we found the expression of AQP8 was reduced in liver specimens of patients with NAFLD, high-fat diet (HFD)-induced mice and genetically obese db/db mice. Knockdown of AQP8 in hepatocytes exacerbated the intracellular lipid accumulation induced by free fatty acid (FFA) mixtures. In contrast, hepatic AQP8 overexpression activated farnesoid X receptor (FXR), inhibiting gene expression associated with lipogenesis, which further reduced intrahepatic triglyceride overload in obese mice. FXR knockout abrogated the ameliorating effect of AQP8 overexpression on NAFLD in mice. These findings indicate that AQP8 overexpression protects against fatty liver through activating the FXR pathway.

Keywords: Cell biology; Endocrinology; Molecular biology.

Graphical abstract

Figure 1

Downregulation of AQP8 in the livers of NAFLD patients and obese mice and effects of hepatic AQP8 overexpression in vitro Hepatic mRNA and protein levels of AQP8 decreased in patients and obese mouse model: (A) NAFLD patients (n = 8 per group), (B) db/db mice (n = 6 per group), (C) high fat diet (HFD) mice (n = 7 per group), (D) short-term HFD mice (n = 5 per group) (See also Figure S2A), (E) HepG2 cells without or with FFA(OA:PA = 2:1) treatment for the indicated time(See also Figure S2B). Primary mouse hepatocytes (PMHs) and HepG2 cells were transfected with either ad-GFP or ad-AQP8 with or without FFA treatment. (F) AQP8 mRNA expression of PMHs. (G) Analysis of AQP8 and lipogenic genes expression (See also Figure S2C). (H) Oil red O staining (200 X) of HepG2 cells. (I) TG measurement of PMHs. The data represent the mean ± SEM values. ∗p < 0.05 ∗∗p < 0.01 ∗∗∗p < 0.001.

Figure 2

Effects of AQP8 on body weight and lipid metabolism in db/db mice db/db mice were i.v. injected with either ad-GFP or ad-AQP8 and mice were sacrificed at day 10. (A) Relative AQP8 mRNA levels in liver tissue samples. (B) Body weight of the four groups were monitored for 10 days. (C) Food intake of the four groups were monitored for 10 days. (D–I) Serum levels of FBG, TG, TC, LDL-c, TBA and β-OHB. The data represent the mean ± SEM values, n = 8–11. ∗p<0.05 ∗∗p<0.01 ∗∗∗p<0.001.

Figure 3

Histological and molecular changes in the livers of db/db mice with AQP8 overexpression (A) Histological analysis of hematoxylin-eosin-stained liver sections, magnification: 200×, scale bar, 100 μm. (B) The analysis of liver weight in four groups. The count is liver weight per kilogram of body weight. (C) Quantification of hepatic TG contents. (D–G) Analysis of mRNA levels on hepatic lipogenesis, fatty acid oxidation, bile acid metabolism and inflammation-related genes; β-actin was used as an internal control. (H and I) Western blot assays analysis of the accumulation of AQP8 and lipogenic genes in the liver tissue sample of wildtype littermates and db/db mice. The data represent the mean ± SEM values, n = 8–11. ∗p<0.05 ∗∗p<0.01 ∗∗∗p<0.001.

Figure 4

Effects of AQP8 on body weight and lipid metabolism in HFD mice HFD mice were i.v. injected with either ad-GFP or ad-AQP8 and mice were sacrificed at day 10. (A) Relative AQP8 mRNA levels in liver tissue samples. (B) Body weight of the four groups were monitored for 10 days. (C) Food intake of the four groups were monitored for 10 days. (D–H) Serum levels of TG, TC, LDL-c, TBA and β-OHB. The data represent the mean ± SEM values, n = 8–10. ∗p<0.05 ∗∗p<0.01 ∗∗∗p<0.001.

Figure 5

Histological and molecular changes in the livers of HFD mice with AQP8 overexpression (A) Histological analysis of hematoxylin-eosin-stained liver sections, magnification: 200×, scale bar, 100 μm. (B) The analysis of liver weight in four groups. The count is liver weight per kilogram of body weight. (C) Quantification of hepatic TG contents. (D–G), Analysis of mRNA levels on hepatic lipogenesis, fatty acid oxidation, bile acid metabolism and inflammation-related genes; β-actin was used as an internal control. (H and I) Western blot assays analysis of the levels of AQP8 and lipogenic genes in the liver tissue sample of NCD and HFD mice. The data represent the mean ± SEM values, n = 8–10. ∗p<0.05 ∗∗p<0.01 ∗∗∗p<0.001.

Figure 6

FXR regulates AQP8 expression and FXR knockout inhibits the improvement of AQP8 in NAFLD (A) PMHs were treated with DMSO, PPARα agonist Fenofibrate, PPARβ/δ agonist GW0742, PPARγ agonist Rosiglitazone, FXR agonist GW4064, LXR agonist T0901317 and RT–PCR analysis of AQP8 gene expression. (B) Dual-luciferase reporter assay was conducted to determine the effect of FXR agonist GW4064 on AQP8 activity. (C) Dual-luciferase reporter assay was conducted to determine the effect of FXR overexpression on AQP8 activity. (D) The interaction of FXR with AQP8 promoter containing putative FXRE was confirmed by chromatin immunoprecipitation (ChIP) in HepG2 cells (n = 3 individual experiments) using an FXR antibody, IgG was used as a negative control. (E) Relative AQP8 mRNA levels in the livers of FXR wild-type (WT) and knockout (KO) mice. (F) FXR-WT and FXR-KO were fed an HFD for two weeks and then i.v injected with either ad-GFP or ad-AQP8 and relative AQP8 mRNA levels in the livers of three groups. (G–J) Serum levels of TBA, TG, TC, LDL-c. (K) The analysis of liver weight in three groups. The count is liver weight per kilogram of body weight. (L) Quantification of hepatic TG contents. (M) Histological analysis of hematoxylin-eosin-stained liver sections, magnification: 200×, scale bar, 100 μm. The data represent the mean ± SEM values. ∗p<0.05 ∗∗p<0.01 ∗∗∗p<0.001.

Figure 7

Molecular changes in the liver of FXR knockout mice with AQP8 overexpression (A–D) Analysis of mRNA on hepatic lipogenesis, fatty acid oxidation, bile acid metabolism and inflammation-related genes; β-actin was used as an internal control. (E) Western blot assays analysis of the accumulation of AQP8 and lipogenic genes in the liver tissue samples of the three groups. (p)SREBP-1c, precursors SREBP-1c; (n)SREBP-1c, nuclear SREBP-1c. The data represent the mean ± SEM values, n = 9–11.∗p<0.05 ∗∗p<0.01 ∗∗∗p<0.001.