The adipokine orosomucoid alleviates adipose tissue fibrosis via the AMPK pathway

Abstract

The excess deposition of underlying extracellular matrix (ECM) in adipose tissue is defined as adipose tissue fibrosis that is a major contributor to metabolic disorder such as obesity and type 2 diabetes. Anti-fibrosis therapy has received much attention in the treatment of metabolic disorders. Orosomucoid (ORM) is an acute-phase protein mainly produced by liver, which is also an adipokine. In this study, we investigated the effects of ORM on adipose tissue fibrosis and the potential mechanisms. We showed that ORM1-deficient mice exhibited an obese phenotype, manifested by excessive collagen deposition in adipose tissues and elevated expression of ECM regulators such as metalloproteinases (MMP-2, MMP-13, MMP-14) and tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3). Administration of exogenous ORM (50 mg· kg^-1· d^-1, ip) for 7 consecutive days in high-fat diet (HFD)-fed mice and leptin receptor (LepR)-deficient db/db mice attenuated these abnormal expressions. Meanwhile, ORM administration stimulated AMP-activated protein kinase (AMPK) phosphorylation and decreased transforming growth factor-β1 (TGF-β1) level in adipose tissues of the mice. In TGF-β1-treated 3T3-L1 fibroblasts, ORM (10 μg/mL) improved the impaired expression profiles of fibrosis-related genes, whereas a selective AMPK inhibitor dorsomorphin (1 μmol/mL) abolished these effects. Together, our results suggest that ORM exerts a direct anti-fibrosis effect in adipose tissue via AMPK activation. ORM is expected to become a novel target for the treatment of adipose tissue fibrosis.

Keywords: 3T3-L1 fibroblasts; AMPK; ORM; TGF-β1; adipose tissue; dorsomorphin; fibrosis; metabolic disorders; obesity.

Fig. 1. ORM1-deficient mice exhibit an obese phenotype with adiposity and excessive collagen deposition.

a Representative coronal views (from back to abdomen) of 21-week-old male Orm1^+/+ and Orm1^−/− mice were acquired by fat-selective whole-body ¹H NMR pseudocolor images. Adipose tissues: red and yellow. Lean tissues and organs: blue. b Fat and lean body mass were quantified according to the MRI body composition analysis of the mice in a (n = 3 per group). c Representative H&E staining and Masson’s trichrome staining of iWAT from 21-week-old male Orm1^+/+ and Orm1^−/− mice. d The average adipocyte size in c (n = 3 per group). e The average Masson’s trichrome stain-positive area (% iWAT area) in c (n = 3 per group). f The hydroxyproline levels in iWAT from 21-week-old male Orm1^+/+ and Orm1^−/− mice (n = 7–8 per group). g Quantitative PCR analysis of fibrogenic genes encoding Col1a1, Col3a1, Col6a3, fibronectin, and Lox in iWAT. h Western blot analysis of iWAT extracted from 21-week-old male Orm1^+/+ and Orm1^−/− mice (n = 3 per group). i–m Densitometric quantification of the data in h. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.

Fig. 2. ORM1 deficiency increases the expression of ECM regulators involved in adipose tissue fibrosis.

a, b The mRNA levels of the ECM regulators Mmp-2, Mmp-13, Mmp-14, Timp-1, Timp-2, and Timp-3 in iWAT extracted from 21-week-old male Orm1^+/+ and Orm1^−/− mice (n = 3 per group). c Western blot analysis of iWAT extracted in a (n = 3 per group). d–i Densitometric quantification of the data in c. *P < 0.05, **P < 0.01, ***P < 0.001 by the Student’s t test.

Fig. 3. ORM administration ameliorates the abnormal synthesis and degradation of ECM components in the iWAT of HFD mice.

a The mRNA levels of Col1a1, Col3a1, and Col6a3 in iWAT extracted from C57BL/6J mice fed a chow diet or 60% HFD (12 weeks) and treated with intraperitoneal injection of vehicle or 50 mg/kg ORM for 7 consecutive days (n = 4 per group). b, c The mRNA levels of the ECM regulators Mmp-2, Mmp-13, Mmp-14, Timp-1, Timp-2, and Timp-3 in iWAT extracted from the mice in a (n = 4 per group). d Western blot analysis of iWAT extracted from the mice in a (n = 3 per group). e–m Densitometric quantification of the data in d. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with the Tukey’s test.

Fig. 4. ORM administration ameliorates the abnormal synthesis and degradation of ECM components in the iWAT of db/db mice.

a The mRNA levels of Col1a1, Col3a1, and Col6a3 in iWAT extracted from C57BL/6J mice and db/db mice treated with intraperitoneal injection of vehicle or 50 mg/kg ORM for 7 consecutive days (n = 3–4 per group). b, c The mRNA levels of the ECM regulators Mmp-2, Mmp-13, Mmp-14, Timp-1, Timp-2, and Timp-3 in iWAT extracted from the mice in a (n = 3–4 per group). d Western blot analysis of iWAT extracted from the mice in a (n = 3 per group). e–g Densitometric quantification of the data in d. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with the Tukey’s test.

Fig. 5. ORM stimulates AMPK activity and inhibits TGF-β1 signaling in the iWAT of obese mice.

a Western blot analysis of iWAT extracted from C57BL/6J mice fed a chow diet or 60% HFD (12 weeks) and treated with intraperitoneal injection of vehicle or 50 mg/kg ORM for 7 consecutive days (n = 3). b Densitometric quantification of the data in a. c The mRNA levels of Tgf-β1 of iWAT extracted in a (n = 4). d Western blot analysis of iWAT extracted from C57BL/6J mice and db/db mice treated with intraperitoneal injection of vehicle or 50 mg/kg ORM for 7 consecutive days (n = 3). e Densitometric quantification of the data in d. f The mRNA levels of Tgf-β1 of iWAT extracted in d (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with the Tukey’s test.

Fig. 6. AMPK mediates the antifibrotic effect of ORM in vitro.

Relative mRNA levels of a–c collagen (Col1a1, Col3a1, and Col6a3), d, e ECM regulators (Mmp-13 and Timp-1) and f the collagen fiber cross-linking marker Lox in 3T3-L1 cells treated with 5 ng/mL TGF-β1 for 24 h in the absence or presence of 10 μg/mL ORM and/or dorsomorphin (1 μmol/mL), as indicated (n = 4 per group). g Representative immunofluorescence images of the cells treated in f. h Quantification of LOX fluorescence shown in g (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with the Tukey’s test.

Fig. 7. Proposed mechanism by which ORM is involved in adipose tissue fibrosis.

ORM1-deficient mice showed significant increases in fat mass and excessive collagen deposition. Exogenous administration of ORM activates adipose tissue AMPK, inhibits TGF-β1 levels, and reduces the expression of collagen and ECM mediators, thus alleviating adipose tissue fibrosis.