1. Volume 125, Issue 6, Pages 1796-1807 (December 2003)
Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin 
Yoshihiro Kamada, Shinji Tamura, Shinichi Kiso, Hitoshi Matsumoto, Yukiko Saji, Yuichi Yoshida, Koji Fukui, Norikazu Maeda, Hitoshi Nishizawa, Hiroyuki Nagaretani, Yoshihisa Okamoto, Shinji Kihara, Jun-ichiro Miyagawa, Yasuhisa Shinomura, Tohru Funahashi, Yuji Matsuzawa 
Gastroenterology 
Volume 125, Issue 6, Pages (December 2003)
DOI: /j.gastro

2. Figure 1
Experimental protocol. (A ) Twelve male wild-type (WT) mice and 12 male adiponectin-knockout (KO) mice (25–30 g bw; 6–8 weeks old) were used. Mice were divided into 4 groups (6 mice per group), and each was injected with a dose of CCl4 (300 or 1000 μL/kg bw) intraperitoneally twice a week for 12 weeks to induce liver fibrosis. Mice were killed 3 days after the final CCl4 injection. (B) Male WT mice were used in this study (24–29 g bw; 6–8 weeks old). Mice were injected with CCl4 (1000 μL/kg bw) twice a week for 12 weeks. Controls were mice that had never been treated with CCl4 (n = 6); group 1 included mice treated with CCl4 for 12 weeks (n = 6); group 2 included mice treated with CCl4 for 12 weeks after AdADN infusion (n = 6); group 3 included mice treated with CCl4 for 12 weeks after AdLacZ infusion (n = 6); group 4 included mice treated with CCl4 for 12 weeks with AdADN infusion at 6 weeks (n = 6); group 5 included mice treated with CCl4 for 12 weeks with AdLacZ infusion at 6 weeks (n = 6); group 6W included mice treated with CCl4 for 6 weeks (n = 6); and group AdADN included mice 12 weeks after AdADN infusion without CCl4 injection (n = 3). Mice were killed 3 days after the final CCl4 injection.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
Adiponectin-knockout (KO) mice developed more extensive fibrosis compared with wild-type (WT) mice; 6- to 8-week-old male WT and adiponectin-KO mice were each injected with a dose of CCl4 (300 or 1000 μL/kg bw) twice a week for 12 weeks to induce liver fibrosis. Controls were WT mice never treated with CCl4 (n = 6); WT300 were WT mice treated with 300 μL/kg bw CCl4 (n = 6); KO300 were KO mice treated with 300 μL/kg bw (n = 6); WT1000 were WT mice treated with 1000 μL/kg bw CCl4 (n = 6); and KO1000 were KO mice treated with 1000 μL/kg bw CCl4 (n = 6). (A ) Picrosirius red staining of mouse livers. (B) Histographic representation of quantified data. KO mice injected with 300 μL/kg bw of CCl4 developed a more extensive fibrosis compared with WT mice. The fibrosis areas were measured as described in the Materials and Methods section. The degree of fibrosis was expressed as the percentage of the total area measured. The fibrosis areas were significantly increased in KO mice compared with WT mice injected with 300 μL/kg bw of CCl4 (P < 0.05). However, there were no statistical differences in liver fibrosis between KO mice and WT mice injected with 1000 μL/kg bw of CCl4. For all panels, bars show mean ± SD. ∗P < 0.05; ∗∗∗P < 0.001; analysis of variance with the Scheffé test. (Original magnification, 40×.)
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
The hydroxyproline content and the gene expressions of TGF-β1 and CTGF were increased in KO mice compared with WT mice. Mice were treated as illustrated in Figure 1A. (A ) The hydroxyproline content in the liver. Two samples from each mouse were analyzed for each group. In KO mice, hydroxyproline content was significantly increased compared with WT mice injected with the same amount of CCl4 (300 μL/kg bw). However, the difference between KO mice and WT mice with 1000 μL/kg bw of CCl4 injection was not statistically significant. Quantification of TGF-β1 (B) and CTGF (C ) mRNA in mouse liver by using semiquantitative RT-PCR is shown. Both TGF-β1 and CTGF mRNA levels in mouse livers treated with 300 μL/kg bw of CCl4 were significantly higher in KO mice than in WT mice but were not different between KO and WT mice that had been treated with 1000 μL/kg bw of CCl4. For all panels, bars show mean ± SD. ∗P < 0.05; ∗∗∗P < 0.001; analysis of variance with the Scheffé test.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Immunohistochemical analysis of α-SMA. Mice were treated as illustrated in Figure 1A. In KO mice injected with CCl4 (300 μL/kg bw), α-SMA-positive cells were significantly increased compared with WT mice injected with the same amount of CCl4. However, there were no significant differences between KO mice and WT mice that had received 1000 μL/kg bw injections of CCl4 (Original magnification, 100×).
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Plasma adiponectin concentrations in mice decrease during CCl4 treatment. Plasma adiponectin levels after 12 weeks of CCl4 treatment in WT mice were detected by immunoblotting. Plasma adiponectin concentrations were not changed in the WT mice injected with 300 μL/kg bw of CCl4 but were significantly decreased in the WT mice treated with 1000 μL/kg bw of CCl4, compared with controls. For all panels, bars show mean ± SD. ∗∗∗P < 0.001; analysis of variance with the Scheffé test.
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
Expression of adiponectin protein in WT mice after AdADN infusion. (A ) Immunoblotting of plasma adiponectin. Immunoblot analysis shows the enhanced expression of adiponectin at approximately 30 kilodaltons (arrow) up to 12 weeks after the infusion of AdADN (7 × 107 plaque-forming units) from a tail vein. Expression of adiponectin in the mouse liver was detected by immunohistochemical staining by using rabbit polyclonal antibody against mouse adiponectin as the primary antibody. (B) Before infusion of AdADN, there was no expression of adiponectin in mouse liver. (C ) Two weeks after the infusion of AdADN, adiponectin protein was detected in mouse liver. (D) Representative immunoblotting of liver protein for adiponectin.
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 7
Adiponectin has preventive and therapeutic effects on CCl4-induced liver fibrosis in WT mice. Mice were treated as illustrated in Figure 1B. (A ) Picrosirius red staining of mouse livers. (Original magnification, 40×.) (B) Histographic representation of quantified data. The fibrosis areas were measured as described in the Materials and Methods section. The fibrosis areas were significantly decreased in mice injected with AdADN before CCl4 injection (group 2) and mice injected with AdADN at 6 weeks (group 4) compared with mice injected with AdLacZ before CCl4 injection (group 3) and mice injected with AdLacZ at 6 weeks (group 5). The fibrosis areas in group 4 had a tendency to be smaller than those at 6 weeks of CCl4 treatment (group 6W). For all panels, bars show mean ± SD. ∗P < 0.05; ∗∗P < 0.01; analysis of variance with the Scheffé test.
Gastroenterology  , DOI: ( /j.gastro )

9. Figure 8
The hydroxyproline content and the gene expressions of TGF-β1 and CTGF were increased in KO mice compared with WT mice. Mice were treated as illustrated in Figure 1B. (A ) The hydroxyproline content in the liver. Two samples from each mouse were analyzed for each group. In group 2, the content was significantly decreased compared with that in group 1 and group 3. In group 4, hydroxyproline content was significantly lower than that in group 5. The hydroxyproline content in the liver was increased by CCl4 administration for the initial 6 weeks (group 6W). The additional 6 weeks of CCl4 treatment led to an increase in the content by approximately 48% (group 1). However, in group 4, the hydroxyproline content was significantly decreased even though CCl4 was given for an additional 6 weeks (total of 12 weeks). (B and C ) Quantification of TGF-β1 (B) and CTGF (C ) mRNA of mouse liver with semiquantitative RT-PCR. Both TGF-β1 and CTGF mRNA expression in group 2 mouse liver had a tendency to be lower compared with those in group 3, and those in group 4 were significantly lower than those in group 5. For all panels, bars show mean ± SD. ∗P < 0.05; ∗∗∗P < 0.001; analysis of variance with the Scheffé test.
Gastroenterology  , DOI: ( /j.gastro )

10. Figure 9
Immunohistochemical analysis of α-SMA. Mice were treated as illustrated in Figure 1B. In group 2, α-SMA-positive cells were decreased compared with group 1 and group 3. In group 4, α-SMA-positive cells were decreased compared with group 6W and group 5. (Original magnification 100×.)
Gastroenterology  , DOI: ( /j.gastro )

11. Figure 10
Effect of adiponectin on PDGF-induced proliferation and migration in activated mouse HSCs. (A ) DNA synthesis was evaluated as the incorporation of [3H] thymidine into DNA as described in the Materials and Methods section. HSCs were treated with the indicated concentrations of adiponectin either in the presence or absence of PDGF-BB for 18 hours and then pulsed for an additional 5 hours with 37 kBq per well of [3H] thymidine in 96-well dishes. Four wells were analyzed for each condition in 3 different experiments. Data are mean ± SD. Adiponectin inhibited PDGF-BB-induced DNA synthesis in a dose-dependent manner. (B) Cell counts were performed on triplicate wells on days 0, 2, 4, and 6 after treatment with the indicated concentrations of adiponectin either in the presence or absence of PDGF-BB by using a hemacytometer. •, adiponectin 0 μg/mL and PDGF-BB 10 ng/mL; ■, adiponectin 10 μg/mL and PDGF-BB 10 ng/mL; ▴, adiponectin 30 μg/mL and PDGF-BB 10 ng/mL; ♦, adiponectin 0 μg/mL and PDGF-BB 0 ng/mL. The medium was changed every 24 hours. Cell number was quantified as the mean number of HSCs observed in 4 fields under a microscope (magnification, 200×). Adiponectin inhibited HSC PDGF-BB-induced proliferation in a dose-dependent manner. Data are the mean ± SD of 3 experiments performed in triplicate. (C ) Effects of adiponectin on PDGF-BB-induced migration of HSCs. HSCs were trypsinized and incubated in the 8-μm-pore-size cell culture inserts for 5 hours at 37°C with or without adiponectin (30 μg/mL) either in the presence or absence of PDGF-BB (10 ng/mL). Values are expressed as mean ± SD of 4 separate fields of 3 different experiments. PDGF-BB-induced HSC migration was completely abolished by treatment with 30 μg/mL of adiponectin. ∗∗P < 0.01; analysis of variance with the Scheffé test.
Gastroenterology  , DOI: ( /j.gastro )

12. Figure 11
The effect of adiponectin on TGF-β1-induced fibrogenic gene expression and the nuclear translocation of Smad2 in activated HSCs. (A and B) The effect of adiponectin on (A ) TGF-β1 and (B) CTGF gene expression in HSCs stimulated with TGF-β1 by using quantitative RT-PCR. HSCs were stimulated with adiponectin (30 μg/mL) either in the presence or absence of TGF-β1 (1 ng/mL). For study of the gene expression, incubation time was 2 hours (TGF-β1) and 8 hours (CTGF). Adiponectin significantly suppressed the fibrogenic gene expression in activated HSCs. Data are the mean ± SD of 3 different experiments. (C ) Effects of adiponectin (30 μg/mL) on the nuclear translocation of Smad2 in HSCs stimulated with TGF-β1. HSCs were stimulated with adiponectin (30 μg/mL) either in the presence or absence of TGF-β1 (1 ng/mL). Nuclear extracts from HSCs were prepared as described in Materials and Methods, and 30 μg of protein from the samples was used for immunoblotting. The 58-kilodalton bands represent nuclear Smad2. Quantification of the protein bands was performed with a densitometer. Adiponectin significantly inhibited TGF-β1-induced nuclear translocation of Smad2. Data are the mean ± SD of 3 different experiments. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; analysis of variance with the Scheffé test.
Gastroenterology  , DOI: ( /j.gastro )