High-fat emulsion-induced rat model of nonalcoholic steatohepatitis

Life Sciences 79 (2006) 1100 – 1107 www.elsevier.com/locate/lifescie

High-fat emulsion-induced rat model of nonalcoholic steatohepatitis Yuhong Zou, Jun Li ⁎, Chao Lu, Jianqing Wang, Jinfang Ge, Yan Huang, Lei Zhang, Yuanyuan Wang Institute of Clinical Pharmacology, School of Pharmacy, Anhui Medical University, Heifei, 230032, Anhui Province, China Received 21 December 2005; accepted 9 March 2006

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 highfat 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. © 2006 Elsevier Inc. All rights reserved. Keywords: Animal model; Nonalcoholic steatohepatitis; Tumor necrosis factor α; Insulin resistance; Oxidative stress

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

⁎ Corresponding author. Tel.: +86 551 5161001; fax: +86 551 5161001. E-mail address: [email protected] (J. Li). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.03.021

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.

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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. Materials and methods 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 Table 1 The composition and caloric content of the high-fat emulsion diet ingested via gavage in NASH model rats Component

High-fat emulsion

Corn oil (g) Saccharose (g) Total milk powder (g) Cholesterol (g) Sodium deoxycholate (g) Tween 80 (g) Propylene glycol (g) Vitamin mixture (g) Cooking salt (g) Mineral mixture (g) Distilled water (ml) Total energy (kcal/l)

400 150 80 100 10 36.4 31.1 2.5 10 1.5 300 4342

1101

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 ± 20 g) were supplied by the Experimental Animal Center, Anhui Medical University. They were housed in plastic cages at a room temperature of 22 ± 1 °C under a 12-h light–dark cycle. Our studies were carried out in accordance with the guidelines for humane treatment of animals set by the Association of Laboratory Animal Sciences and the Center for Laboratory Animal Sciences, Anhui Medical University. Rats were divided into normal control group (NC group) and high-fat emulsion model group (HF group). All rats were provided with standard rodent chow and water. In addition, they were allowed free access to a saccharose solution (18%). The model rats were orally treated with the high-fat emulsion (10 ml/kg) once per day. The NC rats were given equal volumes of saline via gavage daily. Rats were sacrificed post-feeding either by exsanguination (under light pentobarbital anesthesia) or by decapitation after 6 weeks. Blood samples were collected from the aorta ventralis. The livers were immediately removed and weighed. Analytical procedures The concentration of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), triglyceride (TG) and glucose in the serum were measured by an automatic analyzer. The levels of serum-free fatty acids (FFA), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), liver homogenate malondialdehyde (MDA), liver superoxide dismutase (SOD), and liver FFA were detected with commercial analysis kits obtained from the Jiancheng Institute of Biotechnology (Nanjing, China) and Furui Institute of Biotechnology (Beijing, China). Serum insulin and tumor necrosis factor α (TNF-α) levels were measured by radioimmunoassay kit obtained from the Northern Bioengineering Institute (Beijing, China). Hepatic concentrations of TC and TG were also measured after chloroform–methanol extraction (Folch et al., 1957). Hyperinsulinemic euglycemic clamp Whole-body insulin sensitivity was determined using the hyperinsulinemic euglycemic clamp technique as described previously (D'Angelo et al., 2005; Wan et al., 2005). All rats were fasted overnight for 8 h before experiments were conducted. Clamps were performed with insulin continuously infused at 8 mU/kg/min for 2 h. The blood glucose concentration was clamped at 2.5 ± 0.2 mmol/l by estimating the blood glucose level at 5-min intervals and adjusting the

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Table 2 Body weight, weight gain and liver index (liver weight/body weight) in rats of NC and HF groups after high-fat emulsion treatment for 6 weeks Parameter

NC group

HF group

Body weight (g) Starting Final Body weight gain (g) Liver index (%)

237.0 ± 18.9 375.63 ± 38.8 139.0 ± 33.2 2.59 ± 0.18

243.9 ± 10.0 433.6 ± 54.1⁎⁎ 189.3 ± 57.0⁎ 3.46 ± 0.27⁎⁎

NC: normal control diet group; HF: high-fat emulsion group; values are given as means ± S.D. of 12 rats. ⁎P < 0.05, ⁎⁎P < 0.01 compared with NC group.

infusion rate of a 10% glucose solution. Clamp was achieved by 60 min and maintained for 30 min. The final seven samples obtained over this 30-min period were averaged and reported as the glucose infusion rate (ml/kg/ min) required to maintain euglycemic conditions in the case of hyperinsulinemia. Preparation of microsomes Individual livers were homogenized (20% w/v) in ice-cold Tris–acetate buffer (pH 7.4) containing 1.15% KCl. The homogenate was centrifuged at 10,000 × g for 30 min at 4 °C. The supernatant was then centrifuged at 100,000 × g for 60 min at 4 °C. Microsomal pellets were re-suspended by homogenization in a glass homogenizer and again centrifuged at 100,000g. Microsomal pellets were again recovered, snap frozen, and stored at − 70 °C for later use. Hepatic protein extract Hepatic protein extract was performed as described by Bordji et al. (2000) with slight modifications. Liver samples were homogenized in extraction buffer (25 mM HEPES, 400 mM KCl, 1 mM EDTA, 1.5 mM MgCl2) supplemented with protease and phosphatase inhibitors (1 mmol/l phenylmethyl sulfonyl fluoride, 0.1 mmol/l N-tosyl-L-phenylalanine chloromethyl ketone, 1 mg/ml aprotinin, 1 mg/ml pepstatin, 0.5 mg/ml leupeptin, 1 mmol/l NaF, 1 mmol/l Na4P2O4, 2 mmol/ l Na3VO4). The extract was centrifuged at 10,000 × g for 10 min at 4 °C, and protein concentration was determined in the supernatant. Immunoblot analysis Proteins of homogenates or microsomes were separated by electrophoresis on a 10 × 20 cm, 10% sodium dodecyl sulfate polyacrylamide gel for 1.5 h at 120 V. The proteins were transferred to a polyvinylidene difluoride membrane at 150 V in a transfer buffer consisting of 25 mmol/l Tris–HCl, 192 mmol/ l glycine, and 20% methanol for 3 h at 4 °C. The membrane was blocked overnight at 4 °C by immersion in buffer (TBST-20) containing 10 mmol/l Tris–HCl, 150 mmol/l NaCl, 0.08% Tween 20, and 10% nonfat dry milk. The membrane was then incubated for 2 h with a 1:1000 dilution of a rabbit polyclonal anti-rat cytochrome P-450 2E1 (CYP2E1) antibody (Chemicon

ICN, Temecula, CA, USA) or a 1:1000 dilution of a rabbit polyclonal anti-mouse peroxisome proliferator-activated receptor α (PPARα) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membrane was rinsed several times with TBST-20 buffer and then incubated with a 1:4000 dilution of horseradish peroxidase-coupled goat anti-rabbit secondary antibody (Santa Cruz Biotechnology). The blots were visualized using enhanced chemiluminescence kit by Pierce (Rockford, IL). Morphological evaluation Liver specimens were taken immediately after the rats were sacrificed. Paraffin-embedded sections of liver were fixed in 10% formalin and stained with hematoxylin–eosin to detect hepatic steatosis, inflammation and necrosis. Formalinfixed (not embedded in paraffin) and frozen fresh samples were cut in cryostat for intracellular lipid detection by staining with Oil Red O. Fatty change was graded according to the percentage of hepatocytes containing macrovesicular fat (grade 1: 0–25%; grade 2: 26–50%; grade 3: 51–75%; grade 4, 76–100%) (Kirsch et al., 2003). The degree of inflammation and necrosis was expressed as the mean of 10 different fields within each slide that had been classified on a scale of 0–3 (0: normal; 1: mild; 2: moderate; 3: severe) (Avni et al., 2004). Pathology was scored in a blinded manner by two independent pathologists with expertise in rodent liver. For ultrastructural analysis, 1-mm3 samples were fixed in glutaraldehyde (2.5%), postfixed in osmium tetraoxide (2%) and embedded in Spurr's resin. Sections were stained with uranyl acetate (2%, w/v) and lead citrate prior to analysis with electron microscopy (Zeiss EM 109; Oberkochen, Germany). Morphometric measurement of the mitochondrial surface area was performed on the five largest mitochondria per photograph using an image analysis and measuring system (Image Pro Plus V 5.2) (Kirsch et al., 2003). Statistical analysis Quantitative data are expressed as mean ± S.D. and compared using Student's t-test and Mann–Whitney rankTable 3 Serum biochemical indices change after high-fat emulsion treatment for 6 weeks Parameter

NC group

HF group

AST (U/l) ALT (U/l) TC (mmol/l) TG (mmol/l) FFA (mmol/l) HDL-C (mg/dl) LDL-C (mg/dl) Blood glucose (mmol/l) Insulin (μIU/ml)

28.64 ± 9.61 23.2 ± 8.13 1.88 ± 0.35 1.57 ± 0.54 0.36 ± 0.08 42.83 ± 7.01 33.76 ± 9.05 5.36 ± 1.02 22.19 ± 4.33

87.32 ± 21.63⁎⁎ 70.52 ± 24.85⁎⁎ 3.56 ± 0.97⁎⁎ 2.28 ± 0.74⁎ 1.13 ± 0.26⁎⁎ 28.12 ± 7.50⁎⁎ 75.90 ± 25.85⁎⁎ 8.36 ± 1.46⁎⁎ 34.16 ± 7.49⁎⁎

Measurements obtained in the fasting state. NC: normal control diet group; HF: high-fat emulsion group; values are given as means ± S.D. of 12 rats. ⁎P < 0.05, ⁎⁎P < 0.01 compared with NC group.

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* GIR(ml/kg/min)

TNF-α(ng/ml)

0.9

1103

0.6

0.3

10 8

*

6 4 2 0

0

NC

NC

HF

HF

Fig. 1. Serum content of TNF-α changes after high-fat emulsion treatment for 6 weeks. TNF-α concentration was significantly higher in HF group than those in NC group. NC: normal control diet group; HF: high-fat emulsion group; Values are given as means ± S.D. of 12 rats. ⁎P < 0.01 compared with NC group.

Fig. 2. Hyperinsulinemic–euglycemic clamp study revealed a decreased insulinstimulated glucose disposal of the whole-body level. NC: normal control diet group; HF: high-fat emulsion group; GIR: glucose injection rate. Values are given as means ± S.D. of 6 rats. ⁎P < 0.01 compared with NC group.

sum test where applicable. P < 0.05 was considered statistically significant.

Insulin resistance sensitivity assay by hyperinsulinemic– euglycemic clamp in rats

Results

A hyperinsulinemic–euglycemic clamp technique was used to assay the effect of high-fat emulsion on insulin resistance in rats. The results showed that the glucose infusion rate needed to maintain homeostasis of blood sugar in rats of the HF group decreased compared with that of the NC group (Fig. 2). This result indicated that regular consumption of high-fat emulsion leads to insulin resistance in the HF rats.

Changes of body weight and liver weight in rats fed with highfat 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 those in the NC group, whereas HDL-C was lower (Table 3, Fig. 1).

Expression of CYP2E1 and PPAR-α protein in the liver Previous studies showed that alterations of CYP2E1 and peroxisomal proliferation contributed to NAFLD (Weltman et al., 1996; Yeon et al., 2004). Therefore, we analyzed the protein expression of CYP2E1 and PPAR-α in the liver. During the 6 weeks of diet treatment, the protein expression of CYP2E1 was significantly increased in the HF group, whereas the PPAR-α protein expression was gradually reduced compared to that in the NC group (Fig. 3). Morphological analysis

Hepatic biochemistry change after high-fat emulsion treatment After 6 weeks of treatment, the hepatic content of TC, TG, FFA and MDA was markedly increased in the HF group compared to those in the NC group, while hepatic SOD activity was significantly lower in the HF group than that in NC group (Table 4). Table 4 Hepatic biochemical indices, concentration of MDA and SOD change after high-fat emulsion treatment for 6 weeks Parameter

NC group

HF group

TC (mmol/kg tissue) TG(mmol/kg tissue) FFA(mmol/kg tissue) MDA (nmol/mg protein) SOD (NU/mg protein)

2.20 ± 0.51 14.72 ± 4.32 5.03 ± 0.98 4.83 ± 1.02 23.35 ± 4.90

9.57 ± 1.52⁎ 22.41 ± 3.42⁎ 14.54 ± 2.11⁎ 12.08 ± 1.82⁎ 8.13 ± 2.93⁎

NC: normal control diet group; HF: high-fat emulsion group; values are given as means ± S.D. of 12 rats. ⁎P < 0.01 compared with NC group.

Rats fed with high-fat emulsion for 6 weeks developed a higher degree of steatosis. Livers of the HF group (Fig. 4B) were grossly larger compared with those in the NC group and were beige in color (Fig. 4A). This higher steatosis was confirmed by hematoxylin–eosin staining of the liver sections, and severe cytoplasmic vacuoles in the hepatocytes were NC

HF

CYP2E1

α PPARα

Fig. 3. Immunoblot analysis reveals the protein expression of CYP2E1 was significantly increased in the HF group compared with the NC group. In contrast, the expression of PPARα was decreased in HF group. NC: normal control diet group; HF: high-fat emulsion group.

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Fig. 4. Steatohepatitis was induced by high-fat emulsion diet. Macroscopic observation of the liver of HF group (B) showed a markedly enlarged size and paler color compared with the NC group (A).

observed in the HF group (Fig. 5B), whereas the histology appeared normal in the NC group (Table 5, Fig. 5A). Feeding with high-fat emulsion also resulted in more prominent inflammation and necrosis in the HF group, which was corroborated by morphological analysis of the abundance of inflammatory cells and pericentral necrosis (Table 5, Fig. 5C). Livers stained with Oil red O from the HF group showed widespread deposition of lipid droplets of different sizes inside the parenchymal cells (Fig. 6B, C). In contrast, livers from the NC group showed scattered, negligible droplets of fat which were not visibly stained with Oil red O (Fig. 6A). Electron microscopic examination showed that HF rats had aberrant mitochondria, characterized by profound mitochondrial swelling, rarefied matrix and loss of cristae (Table 5, Fig. 7B). These gross changes in the mitochondria were not observed in the NC group (Fig. 7A). 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 hepatic lesions of NASH, steatosis and inflammation, were also reproduced with this model. Although the pathogenesis of NAFLD remains poorly understood, metabolic syndrome is one of the most reproducible factors in the development of NAFLD. The five components that compose the metabolic syndrome are central (truncal) obesity, hyperglycemia, hypertension, hypertriglyceridemia, and low levels of HDL-cholesterol. Subjects with specified values for at least three of these components are considered to have metabolic syndrome (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 2001). In this model, high-fat emulsion diet caused an accentuated increase in body weight when compared to a standard diet. After overnight fasting, serum levels of TG, blood glucose and insulin in the HF group remained significantly elevated, while HDL-C markedly decreased compared to the NC group. These results indicated that the rat model successfully recapitulated several key features of human metabolic syndrome. The results of the hyperinsulinemic–euglycemic clamp experiments showed that the glucose infusion rate was indeed decreased in rats fed a high-fat emulsion, indicating that insulin resistance is achieved in the HF group. The current popular theory on NASH pathogenesis is the “two-hit” hypothesis (Day and James, 1998). The first hit leads to hepatic fat accumulation. One major factor promoting this first hit is insulin resistance, which is present in most patients with NASH (Chitturi et al.,

Fig. 5. Steatohepatitis was induced by high-fat emulsion diet. The liver samples were stained with hematoxylin–eosin, and the magnification is 200. The liver of rats fed the standard rat chow is normal and shows few inflammatory cells (A). The livers of the rats fed high-fat emulsion showed pronounced hepatic steatosis (B) and abundant inflammatory cells (C).

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Table 5 Average histological grades of steatosis, inflammation, necrosis and mitochondrial surface area in rats of NC and HF groups after high-fat emulsion treatment for 6 weeks Parameter

NC group

HF group

Steatosis Inflammation Necrosis Mitochondrial area (μm2)

1.00 ± 0.00 0 0 0.32 ± 0.07

3.67 ± 0.49⁎ 1.83 ± 0.39⁎ 1.67 ± 0.49⁎ 0.78 ± 0.13⁎

NC: normal control diet group; HF: high-fat emulsion group; values are given as means ± S.D. of 12 rats. ⁎P < 0.01 compared with NC group.

2002). Marchesini et al. found that insulin resistance was a strong predictor of NAFLD, even in the absence of glucose intolerance (Marchesini et al., 2001). In obese and diabetic patients, increased plasma FFA levels were observed, due to abnormal release by insulin-resistant adipocytes (Shepherd and Kahn, 1999). With insulin resistance, the combination of elevated plasma concentrations of glucose and fatty acids promotes hepatic fatty acid synthesis and impairs β-oxidation, which further leads to hepatic steatosis (Samuel et al., 2004). In our current experiment, rat hepatic concentration of MDA increased, while SOD activity decreased after treatment with a high-fat emulsion diet for 6 weeks. These results indicate that the imbalance between pro-oxidant and antioxidant chemical species leads to oxidative damage. According to the “two-hit” hypothesis, the second hit leads to steatohepatitis in which oxidative stress and subsequent lipid peroxidation play a key role in the pathogenesis of NASH. There is evidence of increased lipid peroxidation in human patients with NASH (Robertson et al., 2001; Sanyal et al., 2001). Lipid peroxidation results in the formation of aldehyde by-products such as MDA and 4-hydroxynonenol (Esterbauer et al., 1991). The proinflammatory effects of the aldehyde end products can account for all of the typical histological features observed in NASH (James and Day, 1999). The protein expression of CYP2E1 was significantly increased in the HF group compared to the NC group in this model. CYP2E1 is a major microsomal source of oxidative stress. In vitro inhibition studies demonstrated that CYP2E1 is the major catalyst for the formation of lipid peroxides in mice (Leclercq et al., 2000). CYP2E1 has been shown to play a key

Fig. 7. Electron microscopy of hepatic mitochondria. Mitochondria in the rats fed the high-fat emulsion diet (B) showed degenerative changes with rarefied matrix and loss of cristae (arrows). These changes were prominent in most of the mitochondria in the rats fed the high-fat emulsion diet but were rare in the mitochondria in the rats fed the standard diet ad libitum (A). The magnification is 15,000.

role in the pathogenesis of alcoholic liver injury, including alcoholic steatohepatitis (Lieber, 1997). Its pathogenic role in NASH has also been recognized in animal experiments and in human studies (Weltman et al., 1996; Chalasani et al., 2003). Rats fed with a lipid-rich, methionine- and choline-deficient diet (MCD) developed steatohepatitis and increased hepatic expression of CYP2E1 (Weltman et al., 1996). Mitochondrial lesions were also reproduced in this model. Mitochondrial β-oxidation is the dominant oxidative pathway for disposing of fatty acids under normal physiological conditions but can also be a major source of reactive oxygen species (ROS) (Reddy and Mannaerts, 1994). Ultrastructural mitochondrial abnormalities have been documented in patients with NASH (Caldwell et al., 1999). The ultrastructural mitochondrial defects in patients with NAFLD may be indicative of defective oxidative phosphorylation, because these patients also have reduced mitochondrial respiratory chain activity (Perez-Carreras et al., 2003) and impaired ATP synthesis after a fructose challenge (Cortez-Pinto et al., 1999). In this study, serum TNF-α levels were also found to be significantly increased after high-fat emulsion treatment for 6 weeks. TNF-α plays a central role in the development of metabolic syndrome (Hotamisligil, 1999). Increased expression

Fig. 6. Steatohepatitis was induced by high-fat emulsion diet. Intracellular lipids were stained with Oil red O, and the magnification is 200. The liver of rats fed the standard rat chow shows scattered small lipid droplets (A). Formalin fixed (B) and frozen fresh (C) liver samples of the rats fed high-fat emulsion diet show widespread deposition of lipid droplets of different sizes inside parenchymal cells.

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of TNF-α is associated with insulin resistance, obesity, hypertriglyceridemia and glucose intolerance (Hotamisligil, 1999; Winkler et al., 2003). In addition, TNF-α has been noted to be an especially important proinflammatory cytokine contributing to the development of many forms of liver injury, including steatohepatitis (Diehl, 2002; Zoccali et al., 2003). In the steatotic livers of our model, the expression of nuclear receptor lipolytic transcription factor (PPARα) was also suppressed. PPAR is a nuclear receptor that controls a variety of genes in several lipid metabolism pathways, including fatty acid uptake and transport by cells, intracellular binding, storage, and catabolism (Desvergne and Wahli, 1999; Bocher et al., 2002). The reduced expression of PPARα is indicative of impaired β-oxidation of fatty acids which may further influence the imbalance of lipid metabolism toward lipid accumulation in the case of induced lipogenic transcription (Reddy, 2001). In the present study, we developed a new formula for a highfat emulsion diet rich in unsaturated fat using corn oil as a dietary source of fat based on the insulin-resistant animal models established by other laboratories (Ai et al., 2005). Studies in experimental alcoholic liver disease have highlighted the roles of unsaturated fatty acids in promoting ethanolinduced liver injury (Nanji, 2004b). Unsaturated fatty acids (corn oil, fish oil) exacerbate alcoholic liver injury by accentuating oxidative stress, whereas saturated fatty acids are protective (Purohit et al., 2004). The diet rich in unsaturated fatty acids may likely have contributed to the rapid development of the pathologic changes of NASH (Lieber et al., 2004). Other nutritional models use a high-fat diet ad libitum yet do not produce significant liver pathology, presumably because they failed to ensure that sufficient fat is ingested. In this experiment, to avoid the possibility of natural aversion to a high-fat diet due to decreased appetite, our high-fat emulsion was administered via gavage. The model described in this study has three major advantages: (1) fat intake is easily controlled; (2) the model reproduces clinical indices of pathology, including inflammation; and (3) the model is simple, reproducible, and does not require surgery. In conclusion, a new rat model of steatohepatitis was established that involves treating rats with a high-fat emulsion diet via gavage. This model provides new opportunities to study the pathogenesis and treatment of metabolic syndrome associated with steatohepatitis. 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. References Ai, J., Wang, N., Yang, M., Du, Z.M., Zhang, Y.C., Yang, B.F., 2005. Development of Wistar rat model of insulin resistance. World Journal of Gastroenterology 11 (24), 3675–3679.

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