Role of immunodeficient animal models in the development of fructose induced NAFLD

doi:10.1016/j.jnutbio.2013.10.010

The Journal of Nutritional Biochemistry

Volume 25, Issue 2, February 2014, Pages 219-226

https://doi.org/10.1016/j.jnutbio.2013.10.010 Get rights and content

Abstract

Cellular and humoral immunity had been implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). This study was designed to assess if T, B and natural killer (NK) cells are involved in the progress of NAFLD in mouse models after chronic fructose treatment. Mouse models that are deficient in either T cells, B cells or NK cells or lacking both T and B cells were fed with 30% fructose solution for 12 weeks. Typical features of NAFLD, including the relative body weight, food and water intake, biochemical analytes, liver histology, NAFLD activity score, and glucose tolerance and insulin tolerance test were characterized. Further, the percentage of CD3, B220 and NK cells in peripheral-blood mononuclear cell, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, immunodetection for hepatic apoptosis (p53) and for inflammation (TNFα) and quantitative real-time polymerase chain reaction for putative and inflammatory genes involved were determined. Our results conclude that mice deficient in T cells or NK cells fail to develop fructose induced NAFLD whereas the immunocompetent mice and mice with B-cell-specific defect developed NAFLD. Taken together, these data support that the onset of fructose-induced NAFLD is associated with involvement of T cells and NK cells in mice.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and Type 2 diabetes. NAFLD represents a wide spectrum of diseases ranging from fatty liver (hepatic steatosis), steatosis with inflammation and necrosis, to cirrhosis. The animal model to study NAFLD/non-alcoholic steatohepatitis (NASH) is particularly useful to decide the unappreciated events involved in the pathology of the disease. The studies from the established animal models have provided some clues for the pathogenesis of steatosis and steatohepatitis; nevertheless, therapeutic options are still limited as the mechanisms involved in the development of NAFLD are not yet fully understood [1]. Therefore, a better understanding of the biochemical and pathological changes that led to early stages of NAFLD is essential to develop therapeutic strategies [2].

Recent studies in humans show that the diet rich in carbohydrates, particularly fructose may be a significant cause of NAFLD [3]. Fructose rich diet has been postulated to be a key reason in the development of NAFLD [4]. In line with these findings, it has also been shown in animal studies that increased consumption of fructose may result in hepatic steatosis accompanied by insulin resistance, elevated plasma triglyceride levels, and oxidative stress in the liver [5], [6].

Understanding the mechanisms that led to progress from simple steatosis to NASH is essential for designing realistic treatment strategies for patients who develop the progressive disease [7]. In addition, oxidative stress, cytokine production and other pro-inflammatory mediators have been implicated for delivering a second hit during the switch from simple steatosis to NASH [8]. Altogether, hepatic immune responses also play key roles in the pathogenesis of NASH and other progressive diseases [9].

Lymphocytes are part of the adaptive immune response and as such, are crucial for normal immune functions. T or B cell deficiencies are known to result in severe immunodeficiency. T cells are key regulators of adipose inflammation, and thus the adaptive immune system is also crucially important [10]. In mouse models, modulation of T cell function ameliorated not only adipose inflammation but also systemic insulin resistance induced by obesity. On the other hand, natural killer (NK) cells are an important part of the innate immune response. Studies on NK cell deficiency have been carried out in mice models for the development of atherosclerosis and obesity [11] but not NAFLD. Recently, increasing attention has been given to determine the role of T, B cells and NK cells in the development of NAFLD. Although there are a few reports on involvement of B cells, T cells and NK on the development of obesity, inflammation and diabetes mellitus, only few addressed their role in the development of NAFLD. The present study is to understand the role of T cells, B cells and NK cells in the development of NAFLD.

Section snippets

Animals

Experimental procedures for this study were duly approved by the Institutional Animal Ethics Committee. Six-week-old mice (n=5) that were T cell-deficient [NU/J (nude)], T and B cell deficient [B6.129S7-Rag1^tm1Mom/J (Rag-1)], NK cell deficient [C57BL/6J-Lystbg/j (beige)], B cells defect [CBA/CaHN-Btk^xid/J (xid)] and wild type (C57BL/6J (B6)) (Jackson Laboratories, Bar Harbour, USA) were used for this study. The animals were housed in a pathogen-free environment under standard light (14 h light,

Results

To detect any physiological changes in mice in response to treatment with fructose, we analyzed body weight, food and water intake and biochemical parameters of all strain of mice used throughout this study. There was an increase in body weight in B6, nude and xid mice (Supplementary Graph1). There was a significant decrease in food intake in fructose-treated groups in all genotypes, but fluid intake was decreased significantly in fructose-treated B6, xid and nude mice compared to their

Discussion

The present study addresses the question whether the absence of functional T, B and NK cells will affect NAFLD development in immunodeficient mice. High-fructose consumption clearly increases visceral fat in healthy adults and in animal models. In rodent models, a high-fructose diet induces a “metabolic syndrome” that includes development of insulin resistance, hypertriglyceridemia, abdominal obesity, hypertension and hepatic steatosis. Based on the published report on fructose induced NAFLD in

Acknowledgment

The authors wish to thank the director, National Institute of Immunology for providing kind support from the funds originated from Department of Biotechnology, India. The authors also wish to thank Drs. Vineeta Bal, Ashok Mukaphadhayay NII, New Delhi and Nanthakumar Thirunarayanan, USA, for their kind support and advice to carry out this study.

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Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of broadly immunodeficient chimeric mice respond to hypercaloric diets. As early as four weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatosis with ballooning degeneration selectively in the human graft, followed by pericellular fibrosis after eight weeks of hypercaloric feeding. Hepatocytes with the PNPLA3-148M variant, either from a homozygous 148M donor or overexpressed in a 148I donor background, developed microvesicular and severe steatosis with frequent ballooning degeneration, resulting in more active steatohepatitis than 148I hepatocytes. We conclude that PNPLA3-148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetic variant contributions to advanced fatty liver diseases.
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^☆: Funding: National Institute of Immunology, Department of Biotechnology, New Delhi, India.

^☆☆: Conflict of Interest Statement: The authors declare no conflict of interest.

¹: Equally contributed.

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Research articleRole of immunodeficient animal models in the development of fructose induced NAFLD☆,☆☆

Abstract

Introduction

Section snippets

Animals

Results

Discussion

Acknowledgment

J Nutr

J Hepatol

J Hepatol

Clin Liver Dis

Trends Mol Med

Metabolism

J Biol Chem

Genetically modified mouse models for the study of nonalcoholic fatty liver disease

World J Gastroenterol

Role of the inducible nitric oxide synthase in the onset of fructose-induced steatosis in mice

Antioxidants & Redox Signaling.

Consuming fructose-sweetened beverages increases body adiposity in mice

Obes Res

Pathogenesis of steatohepatitis: insights from the study of animal models

Acta Gastroenterol Belg

Research article
Role of immunodeficient animal models in the development of fructose induced NAFLD☆,☆☆