Hepatocyte-specific clusterin overexpression attenuates diet-induced nonalcoholic steatohepatitis

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Abstracts

Clusterin is a multifunctional glycoprotein that plays important roles and is up-regulated in liver diseases such as hepatitis and hepatocellular carcinoma. However, little is known about the significance of clusterin in the pathogenesis of non-alcoholic steatohepatitis (NASH). The aim of this study is to examine the role of clusterin in progression of steatohepatitis in mice fed a methionine and choline deficient (MCD) diet. We generated hepatocyte-specific clusterin overexpression (hCLU-tg) mice, and hCLU-tg mice showed lower levels of hepatic triglycerides, less infiltration of macrophages and reduction of TNF-α, activation of Nrf-2 than wild-type littermates fed the MCD diet. Also, sustained clusterin expression in liver ameliorated hepatic fibrogenesis by reducing the activation of hepatic stellate cells by MCD diet. Sustained expression of clusterin in liver functioned as a preconditioning stimulus and prevented MCD diet-induced severe steatohepatitis injury via Nrf2 activation. These results demonstrate a novel function of clusterin as an immune preconditioning regulator in various inflammatory diseases including steatohepatitis.

Introduction

Clusterin is a disulfide-linked heterodimeric glycoprotein of 75–80 kDa originally isolated from ram rete testis fluid [1], [2]. Since then, it has been reported to be ubiquitously expressed in a variety of tissues and found in almost all body fluids at low levels under normal physiological conditions [2], [3]. In addition, clusterin has been shown to be significantly up-regulated in diverse tissues and/or fluids undergoing patho-physiological disturbances such as ischemia, inflammation [4], [5]. In particular, clusterin is highly induced during the acute phase of pancreatitis, and protects cells from inflammation of the exocrine pancreas by reducing the expression of pro-inflammatory cytokines such as TNF-α and also suppressing NF-κB activation [6]. However, it was later demonstrated that clusterin conferred resistance to TNF-α mediated cell death in breast cancer cells by activating the NF-κB pathway [7]. In addition, other studies demonstrated that clusterin prevented cell death induced by oxidative stress [8]. In sum, while these findings draw attention to a potentially protective association of clusterin in modulating oxidative and inflammatory diseases, the effect of clusterin on regulation of the NF-κB pathway is unclear and needs to be further refined.

Non-alcoholic fatty liver disease (NAFLD), is a type of chronic liver disease associated with metabolic diseases such as obesity, diabetes. NAFLD can be classified into simple steatosis and NASH. The pathological feature of simple steatosis is that the total fat content accounts for over 5% of the liver due to excessive fat deposition. However, the pathological feature of NASH is oxidative stress and inflammation. During the progression to NASH, oxidative stress resulting from elevated intracellular reactive oxygen species (ROS) is a major contributing factor that can further aggravate inflammation characterized by the activation of macrophages [9]. In addition, it has been shown that the toll-like receptor 4 (TLR4) signaling pathway, is critical in the pathogenesis of NASH, and deficiency of TLR4 signaling reduces hepatic damage and inflammation in MCD diet-induced NASH [10], [11].

In a previous study, we demonstrated that TLR4 signaling is required for clusterin-induced TNF-α in macrophages [12]. We have also reported that clusterin functions as a cytokine with chemotactic activity, and induces migration of macrophages and NF-κB activity [13], [14]. In contrast to the anti-oxidative and anti-inflammatory effect of clusterin studied in vivo [4], [5], [6], [15], our in vitro studies suggested that clusterin induced both oxidative stress and pro-inflammatory cytokines in macrophages through the activation of TLR4 signaling pathway [12], [13], [14], [16]. In this study, we generated transgenic mice to constitutively express clusterin in liver (hCLU-tg), and examined its controversial role in an experimentally-induced oxidative and inflammatory liver disease in mice.

The present study shows that clusterin is up-regulated during pathogenesis resulting in NASH induced by the MCD diet, and that sustained expression of clusterin in liver prevents MCD diet-induced severe steatohepatitis injury through the reduction of pro-inflammatory cytokines via TRL4.

Section snippets

Animals and dietary experiments

All animal experiments were performed under the guidelines of the Institutional Animal Care and Use Committee of Korea University (KUIACUC-2015-257). 6 week-old male mice were fed a MCD diet (#518810 Dyets, Inc) or control diet for 3 weeks.

RT-PCR and western blot analysis

RT-PCR and Western blot were performed as described previously [14], [16].

Blood analysis

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglyceride were measured using their assay kits (#K752-100-1, K753-100-1, K622-100-1 Biovision, USA)

Up-regulation of clusterin in livers of mice on MCD diet

We found that clusterin mRNA and protein level increased 2-fold in the liver of mice with steatohepatitis induced by MCD diet (Fig. 1A and B). Based on these results, we performed immunohistochemistry to examine which cells express clusterin. As shown in Fig. 1C, weak immunoreactivity against clusterin was detected only in hepatocytes in the liver of mice fed with control diet. However, clusterin immunoreactivity was greatly increased in hepatocytes of mice fed with MCD diet. In addition,

Discussion

In this study, we demonstrated the protective role of clusterin on the pathogenesis of NASH induced by MCD diet. Clusterin was found to be highly upregulated in parallel with the development of steatohepatitis. Moreover, its expression level was inversely related to the degree of liver damage exhibited by individual mice, suggesting that it might play an important role in the progression of steatohepatitis. To assess the induction of clusterin as a possible physiologic early response to

Author contributions

J,-S. Park, as the first authors, contributed to the interpretation of the data, analysis of the data, and drafting the manuscript. B,-H. Min, as corresponding author, contributed to the design and conceptualization of the study. J,-S. Park and B,-H. Min wrote the paper. Y,-J. Shim and B,-H. Kang, participated in the animal study. W,-K. Lee, contributed to the design and generation of transgenic mice. All authors prepared and approved the paper for submission.

Conflicts of interest

The authors declare no competing financial interests.

Acknowledgements

This research was supported by the National Research Foundation of Korea (NRF-2015R1D1A1A01058467).

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