1. Volume 122, Issue 5, Pages 1399-1410 (May 2002)
Leptin receptor–mediated signaling regulates hepatic fibrogenesis and remodeling of extracellular matrix in the rat 
Kenichi Ikejima, Yoshiyuki Takei, Hajime Honda, Miyoko Hirose, Mutsuko Yoshikawa, Yan-Jun Zhang, Tie Lang, Toru Fukuda, Shunhei Yamashina, Tsuneo Kitamura, Nobuhiro Sato 
Gastroenterology 
Volume 122, Issue 5, Pages (May 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Induction of leptin in thioacetamide-induced fibrosis in the liver. (A) RT-PCR Male Wistar rats were given TAA (200 mg/kg, intraperitoneally) 3 times per week for 4 weeks. Leptin mRNA in the liver was detected by RT-PCR as described in Materials and Methods. α1(I) procollagen (COL1A1) and β-actin mRNA were detected in the same cDNA as a marker of hepatic fibrosis and a housekeeping gene, respectively. PCR products were electrophoresed in 1.5% agarose gels and stained with ethidium bromide. Lane 1, size marker (ΦX174/HaeIII); lane 2, control liver; lane 3, TAA treatment for 4 weeks; lane 4, negative control (no RNA). (B–D) Immunohistochemistry. Leptin and αSMA were detected simultaneously in the liver 4 weeks after TAA treatment by immunohistochemistry using anti-leptin and anti-αSMA antibodies. (B) Expression of leptin in the liver; an FITC-conjugated secondary antibody was used. (C) Expression of αSMA in the same section; a rhodamine-conjugated secondary antibody was used. (D) Merged image of leptin and αSMA. Representative images captured under a confocal microscope are shown (original magnification 600×).
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
TAA-induced liver fibrosis in leptin receptor–deficient Zucker rats. (A–D) TAA-induced fibrosis in the liver. Experimental design as in Figure 1 except male Zucker (fa/fa) rats and their lean littermates (+/?) were used, and TAA was given up to 8 weeks. Photographs of (A and B) gross appearance of the liver and (C and D) histology of the liver by Azan staining after 8-week TAA treatment are shown. (A and C) Lean littermates (+/?) given TAA for 8 weeks. (B and D) Zucker rats given TAA for 8 weeks. (E) RPA for α1(I) procollagen. Twenty micrograms of total liver RNA was analyzed by dual probe RPA for α1(I) procollagen (COL1A1) and rat cyclophilin as a housekeeping gene. Yeast transfer RNA was used as a negative control. Arrows indicate protected bands for COL1A1 (374 nt) and cyclophilin (103 nt). (F) RPA for TGF-β1. TGF-β1 mRNA in the liver was measured similarly by RPA. Representative photographs of protected bands for TGF-β1 and cyclophilin are shown.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Expression of αSMA in the liver after TAA treatment and in vitro transactivation of HSCs isolated from Zucker rats. (A–D) Immunohistochemistry for αSMA in the liver. Experimental design as in Figure 2. Expression of αSMA in the liver was detected by immunohistochemistry using an anti-αSMA antibody as described in Materials and Methods. (A) Lean controls (+/?) given physiological saline solution (PSS) for 4 weeks. (B) Zucker (fa/fa) rats given PSS for 4 weeks. (C) Lean controls (+/?) given TAA for 4 weeks. (D) Zucker (fa/fa) rats given TAA for 4 weeks. Representative photographs from 4 individual experiments were shown (original magnification 100×). (E and F) Immunocytochemistry for αSMA in cultured HSCs. Expression of αSMA in 7-day cultured HSCs isolated from lean controls (E) and Zucker rats (F) was detected by immunocytochemistry using an anti-αSMA antibody as described in detail in Materials and Methods (original magnification 1000×). (G) Western blotting for αSMA. Expression of αSMA in 7-day cultured HSCs from lean controls (+/?) and Zucker rats (fa/fa) was analyzed by Western blotting. Specific bands at 42 kilodaltons were shown. (H and I) Immunocytochemistry for type I collagen in cultured HSCs. Expression of type I collagen in 7-day cultured HSCs from (H) lean (+/?) controls and (I) Zucker (fa/fa) rats was detected by indirect immunofluorescence analysis using an anti–type I collagen antibody (original magnification 1000×). Photographs represent typical experiments from 4 different preparations.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
Effect of leptin on in vitro transactivation in hepatic stellate cells. (A–D) Immunocytochemistry for αSMA. Effect of leptin on induction of αSMA in cultured HSCs was evaluated by immunocytochemistry. HSCs isolated from Wistar rats were incubated with recombinant rat leptin (10–100 nmol/L) for 72 hours (from day 4 to day 7 after inoculation), and expression of αSMA was detected by immunocytochemistry. Representative photographs of (A) controls, (B) 10 nmol/L, (C) 50 nmol/L, and (D) 100 nmol/L of leptin for 72 hours were shown (original magnification 1000×). (E) Western blotting for αSMA. Experimental design as described above. Expression of αSMA in the culture was detected by Western blotting. Specific bands for αSMA (42 kilodaltons) were shown. (F and G) Western blotting for phospho-STAT3. Primary cultured SECs (day 3 of culture) and 3-day or 7-day cultured HSCs were incubated with leptin (100 nmol/L) for 1 hour, and phospho-STAT3 (Tyr 705) was detected by Western blotting using an anti–phospho-(Tyr 705) STAT3 antibody as described in Materials and Methods. Representative photographs of (F) specific bands and (G) the ratio of phospho-(Tyr) STAT3 vs. total STAT3 were shown (*P ≤ 0.05 vs. control levels indicated as a dotted line, n = 4). (H) RPA for COL1A1. Experimental design as described above. Total RNA (10 μg) from day 7 HSCs incubated with leptin for 72 hours was analyzed by RPA as similar to Figure 2E. A representative photograph from 4 separate experiments was shown. Arrows indicate protected bands for COL1A1 (374 nt) and cyclophilin (103 nt).
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Expression of long-form leptin receptor (Ob-Rb) in sinusoidal endothelial cells. (A) RT-PCR expression of Ob-Ra and Ob-Rb mRNA in isolated HSCs (7-day cultured), SECs (3-day cultured), and Kupffer cells (1-day cultured) from Wistar rats were detected by RT-PCR. The same cDNA was used for amplification of α1(I) procollagen (COL1A1), Kupffer cell receptor (KCR), and β-actin as a marker of activated HSCs, Kupffer cells, and a housekeeping gene, respectively. PCR products were electrophoresed in 1.5% agarose gels and stained with ethidium bromide. (B and C) Electrophoretic mobility shift assay for STAT3. (B) STAT3 DNA binding activity was detected in the nuclear extracts from 3-day cultured rat SECs treated with recombinant rat leptin (100 nmol/L) for 1 hour. (C) STAT3 DNA binding activity was detected in LSE cells treated with recombinant human leptin (10–100 nmol/L) and recombinant human IL-6 (10 ng/mL) as a positive control. Hundred-fold amount of cold oligonucleotides was used for competition assay (comp.).
Gastroenterology  , DOI: ( /gast )
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7. Fig. 6
Leptin induces uPA and TGF-β1 mRNA in sinusoidal endothelial cells through activation of transcription factors AP-1 and Ets-1. (A and B) Electrophoretic mobility shift assay. DNA binding activities of (A) AP-1 and (B) Ets-1 were detected in nuclear extracts from LSE cells treated with recombinant human leptin (10–100 nmol/L) and basic fibroblast growth factor (bFGF) (10 ng/mL, a positive control) for 1 hour. Excess amount of cold oligonucleotides (250-fold) were used for competition assay (comp.). (C) RPA for uPA. Steady-state mRNA levels of uPA were detected by RPA in LSE cells treated with recombinant human leptin (100 nmol/L) for 3 hours. Cyclophilin as a housekeeping gene. Yeast transfer RNA was used as a negative control. (D and E) Northern blotting for TGF-β1. TGF-β1 mRNA was detected in LSE cells treated with leptin (10–100 nmol/L) for 3–6 hours. Glyceraldehyde-3-phosphate dehydrogenase as a housekeeping gene. Representative photographs from experiments repeated 4 times were shown.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Leptin increases TGF-β1 mRNA in isolated Kupffer cells. Steady-state mRNA levels of TGF-β1 were detected by RPA in primary cultured rat Kupffer cells incubated with recombinant rat leptin (100 nmol/L) for 3–6 hours. Cyclophilin as a housekeeping gene. Yeast transfer RNA was used as a negative control. Representative photographs from experiments repeated 4 times were shown.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions