MMP-9 activity is increased by adiponectin in primary human hepatocytes but even negatively correlates with serum adiponectin in a rodent model of non-alcoholic steatohepatitis

https://doi.org/10.1016/j.yexmp.2011.07.001Get rights and content

Abstract

Adiponectin protects from inflammation and fibrosis in metabolic liver disease. In the present study we analyzed whether this adipokine may directly affect the activity of matrix metalloproteinases (MMPs), central regulators of fibrinolysis, in hepatocytes. Global gene expression analysis indicated upregulation of MMP-9 and tissue inhibitor of metalloproteinases-1 (TIMP-1) expression in primary human hepatocytes (PHH) in response to stimulation with adiponectin, and these results were confirmed by real-time RT-PCR. Furthermore, gelatin zymography revealed that MMP-9 activity was significantly induced in supernatants of adiponectin stimulated PHHs. In a murine model of hepatic steatosis and in human steatotic liver samples hepatic MMP-9 activity was not significantly altered. However, in two different murine models of non-alcoholic steatohepatitis (NASH) MMP-9 activity was significantly elevated compared to chow fed control mice. Of note, MMP-9 activity did not or even negatively, respectively, correlate with adiponectin serum levels in these models.

The current data indicate that in NASH hepatic inflammation and fibrosis but not hepatic steatosis induce liver MMP-9 activity, and this induction seems to be related to the anti-inflammatory activity of adiponectin rather than its effect on hepatocellular MMP-9 expression.

Highlights

Adiponectin protects from inflammation and fibrosis in metabolic liver disease, and here it is shown that this adipokine induces matrix metalloproteinase-9 (MMP-9) mRNA and activity in primary human hepatocytes (PHH). ► MMP-9 activity is increased in two models of non-alcoholic stetaohepatitis but is not altered in simple fatty liver. ► MMP-9 activity even negatively correlates with adiponectin serum levels in a murine model of non-alcoholic stetaohepatitis.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome with a higher prevalence in obesity and type 2 diabetes (Schaffler et al., 2005, Tilg, 2010). Adiponectin is an adipose tissue released protein which ameliorates insulin resistance, and thereby, may protect from NAFLD. However, systemic levels are reduced in these patients (Hui et al., 2004, Targher et al., 2004). Systemic adiponectin levels are inversely associated with the degree of hepatic steatosis, necroinflammation and fibrosis in NAFLD, and this correlation is even independent of insulin resistance and body mass index (BMI) (Hui et al., 2004, Targher et al., 2004). In animal models of non-alcoholic steatohepatitis (NASH) adiponectin lowers inflammation, reactive oxygen species production and liver fibrosis (Schaffler et al., 2005, Xu et al., 2003), and thereby, may protect from progressive liver disease.

Fibrotic liver is characterized by increased deposition of extracellular matrix (ECM). Matrix-metalloproteinases (MMPs) degrade ECM but upregulation of tissue inhibitors of metalloproteinases (TIMPs) in liver fibrosis may impair their activity (Hemmann et al., 2007, Tomita et al., 2006). Just a few of the so far described MMPs are expressed in the human liver. These include MMP-1 (collagenase 1), MMP-2 (gelatinase A) and MMP-9 (gelatinase B) (Hemmann et al., 2007), as well as MMP-10 and MMP-11 (Garciade Leon Mdel et al., 2006, Lichtinghagen et al., 2003). Expression of MMPs may vary during progression of fibrotic liver disease. Thus, in chronic hepatitis C MMP-2 increases with the stage of fibrosis while MMP-9 is only transiently elevated in early fibrosis (Lichtinghagen et al., 2003).

Studies analyzing MMPs in human NAFLD are rare. MMP-9 mRNA is significantly higher in NASH while MMP-2, -10 and -11 are similar to control tissues (Ljumovic et al., 2004). MMP-9 in plasma is comparably increased in NASH and hepatitis C infected patients (D'Amico et al., 2010) while hepatic MMP-9 mRNA is significantly higher in NASH compared to a cohort of patients suffering from hepatitis B or C virus infection (Ljumovic et al., 2004).

So far mainly mRNA expression of these enzymes has been studied. However, higher mRNA levels of MMPs and TIMPs (Lo et al., 2011) and the fact that MMPs are secreted as zymogens, which have to be activated by proteolysis, do not allow to conclude whether MMP activity is altered in NAFLD.

Antifibrolytic effects of adiponectin have already been described. In cultured human chondrocytes adiponectin increases the release of MMP-3 by activating NFκB (Tong et al., 2011). Induction of MMP-3 by adiponectin has been confirmed in a second study, and here, elevated MMP-1 and MMP-13 levels are also detected (Kang et al., 2010). Upregulation of these MMPs is blocked by inhibitors of AMP-activated protein kinase and c-Jun N-terminal kinase (Kang et al., 2010, Tong et al., 2011). TIMP-1 levels are not affected in chondrocytes upon adiponectin treatment (Kang et al., 2010). These findings suggest that adiponectin-mediated upregulation of MMPs contributes to degradation of ECM thereby enhancing cartilage destruction during arthritis. ECM degradation in atherosclerosis, however, is suggested to be impaired by adiponectin. Here adiponectin specifically increases TIMP-1 mRNA expression while MMP-9 is not affected (Kumada et al., 2004). Adiponectin also stimulates invasion of trophoblasts by elevating MMP-2 and MMP-9 activities while simultaneously suppressing TIMP-2 mRNA (Benaitreau et al., 2010).

Until now, no information regarding direct effects of adiponectin on MMPs and TIMPs in hepatocytes is available. Therefore, global gene expression analysis was performed to identify MMPs and TIMPS regulated at the mRNA level by this adipokine.

Section snippets

Culture media and reagents

Dulbecco's modified eagle medium (DMEM) was from PAA (Karlsruhe, Germany), RNeasy Mini Kit was from Qiagen (Hilden, Germany) and oligonucleotides were synthesized by Metabion (Planegg-Martinsried, Germany). LightCycler FastStart DNA Master SYBR Green I was purchased from Roche (Mannheim, Germany). Palmitate acid and oleate were ordered from Sigma (Deisenhofen, Germany). GAPDH antibody was from New England Biolabs GmbH (Frankfurt, Germany). Recombinant full-length human adiponectin, MMP-1

Adiponectin upregulates MMP-9 and TIMP-1 mRNA and MMP-9 activity in primary human hepatocytes

Primary human hepatocytes (PHH) of three different donors were treated with 10 μg/ml adiponectin or PBS as solvent control for 24 h. Hybridization of the Affymetrix Gene Chip HgU133 Plus was performed with RNA isolated from these cells. Values with a “present call” indicating a reliable hybridization signal were used to calculate differential gene expression in adiponectin treated hepatocytes relative to controls, and MMP-1, MMP-9 and TIMP-1 mRNA levels were found increased in the adiponectin

Discussion

Adiponectin has been shown to enhance MMP activity in trophoblasts and chondrocytes (Benaitreau et al., 2010, Kang et al., 2010, Tong et al., 2011), and here it is demonstrated that adiponectin increases MMP-9 activity in primary human hepatocytes. This effect seems to be partly related to increased MMP-9 mRNA expression, which is about 8-fold higher in the adiponectin-incubated cells. TIMP-1 mRNA is about 2-fold upregulated by adiponectin but induction seems not to be efficient to block MMP-9

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

The expert help of Yvonne Hader is greatly appreciated. The study was supported by the Regensburger Forschungsförderung in der Medizin (ReForM C) and the Deutsche Forschungsgemeinschaft (BU 1141/3-3).

References (30)

  • D. Benaitreau

    Effects of adiponectin on human trophoblast invasion

    J. Endocrinol.

    (2010)
  • C. Dorn

    Expression of fatty acid synthase in nonalcoholic fatty liver disease

    Int. J. Clin. Exp. Pathol.

    (2010)
  • C. Garciade Leon Mdel

    Hepatocyte production of modulators of extracellular liver matrix in normal and cirrhotic rat liver

    Exp. Mol. Pathol.

    (2006)
  • J.M. Hui

    Beyond insulin resistance in NASH: TNF-alpha or adiponectin?

    Hepatology

    (2004)
  • M. Illemann

    MMP-9 is differentially expressed in primary human colorectal adenocarcinomas and their metastases

    Mol. Cancer Res.

    (2006)
  • Cited by (0)

    1

    These authors contributed equally.

    View full text