Clusterin overexpression protects against western diet-induced obesity and NAFLD

Figures

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  Figure 1. Clusterin is upregulated in metabolic diseases induced by WD. (A) Serum clusterin expression level in wild type mice with metabolic diseases induced by WD. (B) Western blot analysis of clusterin expression in various metabolic tissues, including Liver, eWAT, BAT of wild type mice fed CHOW or WD. (C) Immunostaining for clusterin in various tissues of wild type mice fed CHOW or WD. Clusterin expression level was approximately 2.5–3 times higher in the liver, 2 times higher in the eWAT, and 3–4 times higher in the BAT of WD-fed wild type mice than in Chow-fed wild type mice. Data are expressed as standard error (± SEM), and eight mice were used per group. Three independent experiments were performed. **P < 0.01; ***P < 0.001 vs CHOW -fed WT mice. Magnification, 400x.

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  Figure 2. Clusterin overexpression in various tissues of wCLU-tg mice. (A) Serum clusterin expression level in wild type mice and wCLU-tg mice fed CHOW. (B) western blot analysis and (C) Immunostaining for clusterin in various tissues of wild type mice and wCLU-tg mice fed CHOW. Clusterin expression level was approximately 3–4 times higher in the liver and 2.5–3 times higher in the both eWAT and BAT of CHOW-fed wCLU-tg mice than in Chow-fed wild type mice. The liver tissue, eWAT, and BAT of CLU-KO mice were used as negative controls. **P < 0.01; ***P < 0.001; ###P < 0.001 vs CHOW -fed WT mice. Magnification, 200x, 400x.

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  Figure 3. Role of clusterin in weight gain and related parameters induced by WD. (A,B) Weight gain and calorie intake in the two groups of mice fed CHOW or WD for 15 weeks. Body weight of WD-fed wCLU-tg mice was less than that of WD-fed wild type mice despite no difference in calorie intake. (C,D) Analysis of fat mass and lean mass using a fat/lean analyzer. Fat mass of WD-fed wCLU-tg mice was lower than that of WD-fed wild type mice while there was no significant difference in lean mass. (E,F) Oxygen consumption (VO2), CO2 emission (VCO2), and RQ (respiratory quotient) values were measured using an indirect calorimeter for 3 h at intervals of 30 min every 24 h. (G,H) H&E (Hematoxylin & Eosin) staining for eWAT and BAT of the two groups mice fed CHOW and WD. (I) F4/80 staining in eWAT. *P < 0.05; **P < 0.01; ***P < 0.001 vs WD-fed WT mice. ns: not significant. Magnification, × 200, × 400.

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  Figure 4. Protective effect of clusterin in the pathology of WD-induced NAFLD. (A) H&E staining in the liver of wild type, wCLU-tg, and CLU-KO mice fed CHOW or WD. (B–E) Immunostaining for F4/80, Nrf2, p-Nrf2, and α-SMA in the liver of wild type, wCLU-tg, and CLU-KO mice fed CHOW or WD. (F) Picro-Sirius red staining in the liver of wild type, wCLU-tg, and CLU-KO mice fed CHOW or WD. *P < 0.05; **P < 0.01; ***P < 0.001. Magnification, × 200, × 400.

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  Figure 5. Correlation between clusterin expression and AMPK activation. (A,B) Western blotting analysis of AMPK phosphorylation in the liver and eWAT of wild type and wCLU-tg mice fed CHOW or WD for 15 weeks. GAPDH was used as an internal control and was used to determine the relative ratio of AMPK phosphorylation. (C,D) Huh7 cells were incubated in 6-well plates. After 24 h, cells were incubated in serum-free DMEM. AMPK phosphorylation increased in a time- (0, 0.5, 1, 4, 12, 24 h) and dose- (0, 0.1, 0.5, 1, 2, 4 µg/ml) dependent manner in Huh7 cells. (E) Huh7 cells were pretreated with ML385 at 1, 2, 5, and 10 µM concentrations for 1 h and then treated with clusterin (2 µg/ml) for 48 h. (F) Huh7 cells were pretreated with Compound C at concentrations of 1, 2, 5, and 10 µM for 1 h and then treated with clusterin (2 µg/ml) for 48 h. (G) Huh7 cells were pretreated with TLR4 inhibitors, CLI-095 (1, 5 µM), and OxPAPC (15, 30 µg/ml), for 30 min and then treated with clusterin (2 µg/ml) for 24 h. *P < 0.05; **P < 0.01; ***P < 0.001.

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  Figure 6. Schematic of the protective mechanism of clusterin via preconditioning. Clusterin induces a mild inflammatory response and oxidative stress, which enhances AMPK phosphorylation and Nrf2 expression, producing a preconditioning status.