Depletion of B cell-activating factor attenuates hepatic fat accumulation in a murine model of nonalcoholic fatty liver disease

Figures

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  Figure 1. Lipid distribution in adipose tissue and liver is altered in BAFF−/− mice fed an HFD. (A) Changes in body weight of BAFF−/− and WT mice fed an ND or HFD (n = 25–55/group). (B) Organ weight [(organ weight/body weight) × 100%] in BAFF−/− and WT mice fed an ND (n = 6/group) or HFD (n = 7/group) for 24 weeks. For all bar plots shown, data are expressed as the mean ± SD, and significance was determined by two-tailed Student’s t test (A) and Mann–Whitney U test (B). ♯♯♯P < 0.001 compared to WT-ND mice. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to WT-HFD mice. BAFF, B cell-activating factor; WT, wild-type; ND, normal diet; HFD, high-fat diet; SAT, subcutaneous adipose tissue; EAT, epididymal adipose tissue; RpAT, retroperitoneal adipose tissue; VAT, visceral adipose tissue; BAT, brown adipose tissue.

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  Figure 2. BAFF−/− mice are protected against glucose intolerance and insulin resistance. (A) Fasting blood glucose, plasma insulin, and HOMA-IR values in HFD-fed BAFF−/− and WT mice over the course of 12 weeks (n = 6/group). (B) Glucose- and (C) insulin-tolerance tests (left) and corresponding area under the curve (AUC) values during the tests (right) (n = 7–14/group). For all bar plots, data are expressed as the mean ± SD, and significance was determined by the Mann–Whitney U test. *P < 0.05, **P < 0.01, and ***P < 0.001. BAFF, B cell-activating factor; WT, wild-type; ND, normal diet; HFD, high-fat diet; HOMA-IR, calculated homeostasis model assessment of insulin resistance.

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  Figure 3. Adipose tissue inflammation is alleviated in BAFF−/− mice. (A) Representative H&E staining of EAT (left). The arrows point to the CLSs formed by macrophage aggregation. Quantitative measurement of CLSs (right) (scale bar, 200 μm; images from six different fields; n = 5/group). (B,D) Relative mRNA levels of the indicated genes in the EAT (n = 12/group). (C) Representative flow cytometry plots showing frequencies of CD11c+ F4/80+ macrophages among stromal cells isolated from EATs from HFD-fed BAFF−/− and WT mice (left). Quantification of Cd11c+ F4/80+ cells isolated from EAT (right) (n = 9–12/group). (E) Serum resistin concentrations (n = 14, 15/group). For all bar plots, data are expressed as the means ± SEM. *P < 0.05 and ***P < 0.001 (Mann–Whitney U test). BAFF, B cell-activating factor; WT, wild-type; H&E, haematoxylin and eosin; CLS, crown-like structure; HFD, high-fat diet; EAT, epididymal adipose tissue.

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  Figure 4. Interstitial fibrosis in white adipose tissue is reduced in BAFF−/− mice. (A) The median adipocyte size (left) and the distribution of adipocyte size (right) in EAT from HFD-fed BAFF−/− and WT mice. Representative (B) Sirius red staining and (C) quantification of Sirius red-positive areas of EAT (scale bar, 100 μm; images from 10 different fields; n = 6/group). (D) Total collagen contents in EAT (n = 9–13/group). (E) Expression of genes related to fibrosis in EAT (n = 14–15/group). For all bar plots, data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (Mann-Whitney U test). BAFF, B cell-activating factor; HFD, high-fat diet; WT, wild-type; EAT, epididymal adipose tissue; TGF, transforming growth factor; SMA, smooth muscle actin; Col, Collagen.

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  Figure 5. Hepatic steatosis is ameliorated in HFD-fed BAFF−/− mice. (A) Representative H&E staining of livers from mice after 24 weeks of ND or HFD consumption (scale bar, 500 μm). (B) Representative OsO4 staining (left) and quantification of the OsO4-positive areas (right) of livers (scale bar, 500 μm; images from eight different fields; n = 8/group). (C) Hepatic TG and (D) total cholesterol levels (n = 5/group). For all bar plots, data are expressed as the mean ± SEM, and significance was determined by the Mann–Whitney U test. *P < 0.05 and ***P < 0.001. BAFF, B cell-activating factor; WT, wild-type; ND, normal diet; HFD, high-fat diet; TG, triglyceride.

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  Figure 6. The expression of genes related to steatosis is downregulated in the livers of BAFF−/− mice. (A,C,D) The expression of genes related to lipogenesis (A), inflammation (C), and fibrosis (D) in mouse livers after 24 weeks of HFD consumption (n = 9–14/group). (B) The expression of proteins related to lipogenesis (n = 4/group). Original gel documents of western blot were shown in Supplementary Fig. S4. Protein levels were normalized to those of β-actin, and those of WT mice were set at 1. For all bar plots, data are expressed as the mean ± SEM, and significance was determined by Mann–Whitney U test. *P < 0.05 and ***P < 0.001. BAFF, B cell-activating factor; WT, wild-type; HFD, high-fat diet; FAS, fatty acid synthase; SREBP1, sterol regulatory element-binding protein; ACC, acetyl-CoA carboxylase; SCD, stearoyl-CoA desaturase; ACS, acetyl-CoA synthetase; MTP, microsomal triglyceride transfer protein; TNF, tumour necrosis factor.

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  Figure 7. Hepatocyte fat deposition is reduced in BAFF−/− mice. (A) The expression of genes related to lipogenesis in primary hepatocytes from HFD-fed BAFF−/− and WT mice. (B) Hepatocyte fat deposition. Representative Sudan III staining of primary hepatocytes from BAFF−/− and WT mice exposed to BSA or PA (left). Quantification of TG in primary hepatocytes exposed to PA (right) (n = 8/group). For all bar plots, data are expressed as the mean ± SEM. ***P < 0.001 (Mann–Whitney U test). BAFF, B cell-activating factor; WT, wild-type; FAS, Fatty acid synthase; SREBP1, sterol regulatory element-binding protein; ACC, acetyl-CoA carboxylase; SCD, stearoyl-CoA desaturase; MTP, microsomal triglyceride transfer protein; BSA, bovine serum albumin; PA, palmitate, TG; triglyceride.