Elsevier

Life Sciences

Volume 79, Issue 11, 8 August 2006, Pages 1100-1107
Life Sciences

High-fat emulsion-induced rat model of nonalcoholic steatohepatitis

https://doi.org/10.1016/j.lfs.2006.03.021Get rights and content

Abstract

Non-alcoholic fatty liver disease (NAFLD) is emerging as a common medical problem. Nonalcoholic steatohepatitis (NASH) is the critical turning point at which NAFLD progresses to more advanced stages such as hepatic fibrosis, cirrhosis and even hepatocellular carcinoma. However, the study of the pathogenic or therapeutic factors involved in NASH has been hampered by the absence of a suitable experimental model. The aim of the present work was to establish a high-fat emulsion-induced rat model of NASH. Male Sprague–Dawley rats were fed a high-fat emulsion via gavage for 6 weeks. Animals were examined for weight gain, serum and hepatic biochemistry, insulin sensitivity, hepatic malondialdehyde (MDA), superoxide dismutase (SOD) and tissue morphology, as well as cytochrome P-450 2E1 (CYP2E1) and peroxisome proliferator-activated receptor α (PPARα) expression in the liver. The results showed that rats treated with high-fat emulsion became obese, demonstrated abnormal aminotransferase activity, hyperlipoidemia, hyperinsulinemia, hyperglycemia and insulin resistance. The model rats exhibited an increased concentration of serum TNF-α, total cholesterol (TC), triglyceride (TG), MDA and reduced SOD levels in the liver. Immunoblot analysis showed that the expression of CYP2E1 was increased, whereas PPARα was reduced in the NASH model rat liver. Moreover, morphological evaluation revealed that hepatic steatosis, inflammation and mitochondrial lesions were also reproduced in this model. In conclusion, a practical and repeatable new rat model of steatohepatitis was established by feeding with high-fat emulsion via gavage. This model provides a valuable research tool and reproduces many of the clinical indices of human NASH.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is now recognized as the most common type of liver disease and might lead to important public health problems (Clark et al., 2002). This disease, with an unclear natural history, can be severe and is characterized by a wide spectrum of pathological lesions. These lesions closely resemble those induced by alcohol, but are also observed in patients without excessive alcohol consumption. Steatosis alone does not appear to be progressive, while nonalcoholic steatohepatitis (NASH) does, since the latter often progresses to more severe stages of liver injury such as fibrosis, cirrhosis and even hepatocellular carcinoma. Thus far, no therapy for NASH has clearly been proven effective (Fong et al., 2000, Angulo, 2002). Animal models of steatohepatitis, obesity, insulin resistance and dyslipidemia are valuable for studying the pathogenesis and treatment of NASH as well as its relationship to metabolic syndrome. However, for a long time, studies have failed to clearly define the molecular and physiological changes that mediate the presumed transition from hepatic steatosis to steatohepatitis due to the lack of animal models carrying key features of human NASH. The three models that have been most extensively studied in order to define mechanisms for steatosis and steatohepatitis were reviewed by Koteish and Diehl (2002). In all these three models steatosis develops spontaneously, but the progression to steatohepatitis and cirrhosis is very different. In addition, a major disadvantage of these animal models is that they, to some degree, fail to reflect the natural multi-factorial etiological setting of the most common form of NAFLD in patients.

Obesity is frequently associated with NAFLD and other metabolic syndromes in humans. In addition to the genetic component of obesity, high caloric intake is another important factor. Many studies have demonstrated that normal rats become obese and develop hepatic steatosis when fed with a high-fat diet ad libitum (Hill et al., 1983, Corbett et al., 1986, Watarai et al., 1988, Harris, 1994). In contrast, several other studies demonstrated that it is relatively difficult to induce obesity in normal rats and mice via high-fat diet ad libitum (Maegawa et al., 1986, Pedersen et al., 1991, Harrold et al., 2000). The composition, palatability and consumption of specific diets in animal models might provide crucial insights into the role of diet in the pathogenesis of obesity-related hepatic steatosis. The difference in body weight gain between control and high-fat fed rats is likely attributed to differences in daily diet intake. Due to the self-limiting nature of ad libitum feeding, rats fed with the high-fat diet eat less and thus take in less energy compared to rats on the control diet (Corbett et al., 1986, Maegawa et al., 1986, Watarai et al., 1988, Lieber et al., 2004). To control daily diet intake, an intragastric diet infusion technique has been applied to avoid limited consumption of the high-fat diet offered ad libitum (Akiyama et al., 1996, Deng et al., 2005). However, this technique requires technical training (surgery) and special equipment (infusion pumps).

The aim of the present study is to produce a practical and repeatable experimental rat model for steatohepatitis by designing a high-fat emulsion that contains high fat, sucrose and protein. Moreover, this high-fat emulsion is administered via gavage (not by feeding ad libitum) in order to overcome natural aversion to the high-fat diet due to its taste. This model is more likely to reproduce the key features of NASH in humans.

Section snippets

Preparation of fat emulsion

High-fat emulsion diet was prepared, which derives 77% of its energy from fat, 14% from total milk powder and 9% from carbohydrates. The composition of macronutrients in this emulsion is shown in Table 1. In this emulsion, proteins were provided by total milk powder, carbohydrates by saccharose and fat by corn oil. Each diet was supplemented with a vitamin and mineral mixture. This emulsion was stored at 4 °C, heated in a 42 °C water bath and fully mixed before use.

Animals and treatment

Male Sprague–Dawley rats (230 ±

Changes of body weight and liver weight in rats fed with high-fat emulsion

The NASH model was induced by feeding rats with high-fat emulsion for 6 weeks, and the general condition of the rats remained satisfactory. All rats showed a steady increase in body weight. The body weight gain and liver index in the HF group was significantly greater than that in the NC group (Table 2).

Serum biochemistry and TNF-α change after high-fat emulsion treatment

Following continuous feeding with high-fat emulsion for 6 weeks, serum levels of AST, ALT, TC, TG, FFA, LDL-C, blood glucose, insulin and TNF-α in the HF group were significantly higher than

Discussion

Animal models have greatly contributed to the understanding of many human diseases. While several models of steatosis exist, very few models of steatohepatitis are available (Nanji, 2004a). In the present study, a new rat model of NASH was induced by feeding rats with a high-fat emulsion. This model successfully reproduced several typical aspects of NASH, such as obesity, abnormal aminotransferase, hyperlipidemia, hyperinsulinemia, hyperglycemia and insulin resistance. Moreover, the typical

Acknowledgements

We thank Mr. Christopher M. Spring, MSc from Department of Transfusion Medicine, Saint Michael's Hospital, University of Toronto for careful presentation of the manuscript. We also thank Dr. Weiling He at School of Pharmacy, University of Wyoming for her constructive suggestions.

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