1. Volume 136, Issue 2, Pages 663-672.e4 (February 2009)
Tissue Inhibitor of Metalloproteinase 3 Deficiency Causes Hepatic Steatosis and Adipose Tissue Inflammation in Mice 
Rossella Menghini, Stefano Menini, Roberta Amoruso, Loredana Fiorentino, Viviana Casagrande, Valeria Marzano, Federica Tornei, Pierfrancesco Bertucci, Carla Iacobini, Matteo Serino, Ottavia Porzio, Marta L. Hribal, Franco Folli, Rama Khokha, Andrea Urbani, Renato Lauro, Giuseppe Pugliese, Massimo Federici 
Gastroenterology 
Volume 136, Issue 2, Pages e4 (February 2009)
DOI: /j.gastro
Copyright © 2009 AGA Institute Terms and Conditions

2. Figure 1
Effects of short-term regular diet on WT, Timp3−/−, Insr+/−, Insr+/−Timp3−/−, and Insr+/−Tace+/−mice. (A) Glucose, insulin, adipokine, and transaminases levels. (B) Intraperitoneal glucose and insulin tolerance tests; n = 5 per group, data are mean ± SD.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

3. Figure 2
Timp3 deficiency exacerbates metabolic defects caused by interaction of HFD with predisposition to genetic insulin resistance. (A) Fasting and fed glucose and insulin (*P < .05, ***P < .001 for Insr+/−Timp3−/− compared with other groups; #P < .05 for Insr+/−and Timp3−/− compared with WT and Insr+/−Tace+/−; 1-way ANOVA, n = 9 per group). (B) intraperitoneal glucose and insulin tolerance test (***P < .001 for Insr+/−Timp3−/− compared with other groups; 2-way ANOVA, n = 9 per group). (C) HOMA IR (*P < .05 for Insr+/−Timp3−/− compared with other groups, 1-way ANOVA, n = 9 per group). (D) Adipokines and transaminases (*P < .05, 1-way ANOVA, for Insr+/−Timp3−/− compared with other groups, n = 9 per group). Data are mean ± SD.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

4. Figure 3
Interaction between predisposition to genetic insulin resistance and HFD with Timp3 deficiency promotes inflammation in WAT: morphologic characteristics. (A) WAT tissue morphology from WT (left column), Insr+/−, Timp3−/−Insr+/−Timp3−/−, and Insr+/−Tace+/−: H&E staining (100×); Insr+/−Timp3−/− showed increased inflammatory infiltrate and pericellular fibrosis as suggested by a particular of the dotted area (higher magnifications); trichrome (100×), Insr+/−Timp3−/− showed increased collagen staining and dilated interstitial spaces as suggested by a particular from the dotted area (250×); MCP-1 immunostaining (250×) is present in Insr+/−Timp3−/− but not in WT, Insr+/−, Timp3−/−, and Insr+/−Tace+/−; F4/80 immunostaining (400×), Insr+/−Timp3−/− showed increased macrophage accumulation as suggested by a particular (1000×). (B) Mean adipocyte area (**P < .01, 1-way ANOVA, for Insr+/−Timp3−/− compared with other groups). Data are mean ± SD.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

5. Figure 4
Interaction between predisposition to genetic insulin resistance and HFD with Timp3 deficiency promotes inflammation in white adipose tissue: molecular aspects. (A) Insulin-stimulated phosphorylation of AKT is reduced in Insr+/−Timp3−/− compared with WT, Insr+/−, Timp3−/−, and Insr+/−Tace+/− fed an HFD (**P < .01, 1-way ANOVA, for Insr+/−Timp3−/− compared with other groups); JNK and IKK phosphorylation are increased in Insr+/−Timp3−/− compared with other groups (**P < .01, 1-way ANOVA, for Insr+/−Timp3−/− compared with other groups). (B) mRNA expression of MCP-1, CCR2, CD68, interleukin-6, CXCL16, RbP4, ADRP, SCD-1, and SOCS-3 is increased in Insr+/−Timp3−/− compared with WT, Insr+/−, Timp3−/−, and Insr+/−Tace+/− (*P < .05, **P < .01, 1-way ANOVA, for Insr+/−Timp3−/− compared with other groups); n = 5 per group. Data are mean ± SD.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

6. Figure 5
Timp3 deficiency induces fatty liver disease in mice combining defects from HFD and predisposition to genetic insulin resistance: histologic aspects. Liver tissue H&E staining (250×) from WT, Insr+/−, Timp3−/−, Insr+/−Timp3−/−, and Insr+/−Tace+/−; Insr+/−Timp3−/− showed mixed microvesicular and macrovesicular steatosis; arrow indicates ballooning degeneration in Insr+/−Timp3−/− mice. Oil Red O staining (20×), trichrome (400×), MCP-1 (250×), and F4/80 (250×) staining; Insr+/−Timp3−/− mice showed perisinusoidal fibrosis (dotted line) and increased MCP-1 and F4/80 expression. cv, central vein; p, portal region. Representative images are shown, 5 mice per group were analyzed.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

7. Figure 6
Insulin-signaling in livers from WT, Timp3−/−, Insr+/−, Insr+/−Timp3−/−, and Insr+/−Tace+/− fed an HFD. Insulin-stimulated phosphorylation of AKT Ser 473 is reduced in Insr+/−Timp3−/− compared with WT, Insr+/−, Timp3−/−, and Insr+/−Tace+/− fed an HFD (**P < .01, 1-way ANOVA, for Insr+/−Timp3−/− compared with other groups). Insulin-stimulated phosphorylation of FoxO1Ser256 is reduced in Insr+/−Timp3−/− compared with WT, Insr+/−, Timp3−/−, and Insr+/−Tace+/− fed an HFD (*P < .05, 1-way ANOVA, for Insr+/−Timp3−/− compared with other groups), n = 5 per group. Data are mean ± SD.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

8. Figure 7
Timp3 deficiency exacerbates activation of inflammatory pathways linked to steatosis in mice combining defects from HFD and predisposition to genetic insulin resistance. Insr+/−Timp3−/− compared with WT, Insr+/−, Timp3−/−, and Insr+/−Tace+/− fed an HFD showed decreased phosphorylation of AMPK, increased phosphorylation of JNK, IKKα/β, EGFR, and SOCS-3 expression and increased nuclear translocation p65 subunit of nuclear factor κ B (*P < .05, ***P < .001, 1-way ANOVA, for Insr+/−Timp3−/− compared with other groups; n = 3 per group).
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

9. Figure 8
Expression of genes linked to steatosis in mice combining defects from HFD and predisposition to genetic insulin resistance. (A) mRNA expression of metabolic genes PPARγ, CD36, PGC1α, Nur77, Nurr1, NOR1, Fbp1, and SCD-1 are increased whereas Akt2 is decreased in Insr+/−Timp3−/− compared with WT, Insr+/−, Timp3−/−, and Insr+/−Tace+/−. (B) mRNA expression of inflammatory genes SOCS-3, CD68, CX3CR1, CXCL16, TNFα, and inducible NO synthase are increased in Insr+/−Timp3−/− compared with WT, Insr+/−, Timp3−/−, and Insr+/−Tace+/− (*P < .05, **P < .01, 1-way ANOVA for Insr+/−Timp3−/− compared with other groups); n = 5 per group. Data are mean ± SD.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

10. Supplementary Figure 1
Effect of insulin stimulation on FoxA2 expression and translocation in cytoplasmic and nuclear liver fractions from WT, Insr+/−, Timp3−/−, Insr+/−Timp3−/−, and Insr+/−Tace+/−; n = 2 per group. Data are mean ± SD.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

11. Supplementary Figure 2
Interaction between genetic and HFD-induced insulin resistance with Timp3 deficiency promotes fatty streak formation. (A) Aortic root histology from WT, Insr+/−, Timp3−/−, Insr+/−Timp3−/−, and Insr+/−Tace+/−. Oil Red O staining (50×): arrows indicate fatty streaks that were observed only in Insr+/−Timp3-/; Weigert–Van Gieson staining (50×): arrows indicate fatty streaks that were observed only in Insr+/−Timp3-/; F4/80 staining of aortic roots (magnification, 250×): arrows indicate sites of macrophage accumulation that were observed only in the Insr+/−Timp3−/−group; MCP-1 staining of aortic roots: staining is more intense in Insr+/−Timp3−/− compared with other groups (magnification, 400×); note in Insr+/−Timp3−/−intense MCP-1 staining at both the endothelial and subendothelial layers, whereas WT show scarce staining only at the endothelial level, no staining was observed in Insr+/−Tace+/−mice. RAGE staining of aortic roots (magnification, 250×): RAGE was expressed abundantly at the endothelium level in Insr+/−Timp3−/− mice. (B) Fatty streak area is increased in Insr+/−Timp3−/− compared with WT, Insr+/−, Timp3−/−, and Insr+/−Tace+/− (P < .05, 1-way ANOVA). Representative images are shown; 5 mice per group were analyzed.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions