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Am J Physiol Gastrointest Liver Physiol 294: G27-G38, 2008. First published October 18, 2007; doi:10.1152/ajpgi.00296.2007
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LIVER AND BILIARY TRACT

A new model for nonalcoholic steatohepatitis in the rat utilizing total enteral nutrition to overfeed a high-polyunsaturated fat diet

January N. Baumgardner,1,4 Kartik Shankar,1,4 Leah Hennings,3 Thomas M. Badger,1,2,4 and Martin J. J. Ronis1,4

1Departments of Pharmacology and Toxicology, 2Physiology and Biophysics and 3Pathology, University of Arkansas for Medical Sciences; and 4Arkansas Children's Nutrition Center, Little Rock, Arkansas

Submitted 28 June 2007 ; accepted in final form 15 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have used total enteral nutrition (TEN) to moderately overfeed rats high-polyunsaturated fat diets to develop a model for nonalcoholic steatohepatitis (NASH). Male Sprague-Dawley rats were fed by TEN a 187 kcal·kg–3/4·day–1 diet containing 5% (total calories) corn oil or a 220 kcal·kg–3/4·day–1 diet in which corn oil constituted 5, 10, 25, 35, 40, or 70% of total calories for 21 or 65 days. Rats fed the 5% corn oil, 220 kcal·kg–3/4·day–1 diet had greater body weight gain (P ≤ 0.05), fat mass (P ≤ 0.05), and serum leptin and glucose levels (P ≤ 0.05), but no liver pathology. A dose-dependent increase in hepatic triglyceride deposition occurred with increase in percent corn oil in the 220 kcal·kg–3/4·day–1 groups (P ≤ 0.05). Steatosis, macrophage infiltration, apoptosis, and focal necrosis were present in the 70% corn oil group, accompanied by elevated serum alanine aminotransferase (ALT) levels (P ≤ 0.05). An increase in oxidative stress (thiobarbituric acid-reactive substances) and TNF-{alpha} expression (P ≤ 0.05) was observed in the 70% corn oil group, as well as an increase in hepatic CYP2E1 and CYP4A1 expression (P ≤ 0.05). Significant positive correlations were observed between the level of dietary corn oil and the degree of pathology, ALTs, oxidative stress, and inflammation. Liver pathology was progressive with increased necrosis, accompanied by fibrosis, observed after 65 days of TEN. Increased expression of CD36 and L-FABP mRNA suggested development of steatosis was associated with increased fatty acid transport. These data suggest that intragastric infusion of a high-polyunsaturated fat diet at a caloric level of 17% excess total calories results in pathology similar to clinical NASH.

obesity; oxidative stress; tumor necrosis factor-alpha, CD36; liver fatty acid binding protein

NONALCOHOLIC FATTY LIVER DISEASE (NAFLD) is one of the most common liver pathologies and is closely associated with obesity and metabolic syndrome. NAFLD has been estimated to occur in 35% of lean and up to 70% of obese patients (83). As the incidence of obesity increased throughout the United States, there was a concomitant increase of NAFLD. There is a wide spectrum of NAFLD pathologies, ranging from simple reversible steatosis to nonalcoholic steatohepatitis (NASH) in which steatosis is accompanied by inflammation, necrosis, apoptosis, and fibrosis. Research into the molecular mechanisms underlying development of NASH pathology has been hampered by the lack of a suitable experimental animal model that mimics the natural course and etiological background of this disease in patients (19, 45). Three types of animal models have been pursued: 1) Genetic models that result in obesity have been studied, such as models of hyperphagia [the JCR:LA-corpulent rat (24)]; models in which leptin or the leptin receptor are absent [ob/ob and db/db mice and fa/fa Zucker rats (7, 65)]; and models in which hepatic lipid metabolism is disrupted [peroxisome proliferator activator receptor {alpha} (PPAR{alpha}) knockout mice (12)] or fatty acid synthesis is enhanced [sterol regulatory element binding protein-1c (SREBP-1c) transgenic mice (76)]. 2) Pharmacological models using drugs to target hepatic triglyceride export by inhibiting apolipoprotein synthesis and assembly of very-low-density lipoprotein (43). 3) Dietary deficiency models such as choline + methionine deficiency (18, 81). A common problem with all of these models is that they represent manipulations that do not reflect the natural etiological setting in which NAFLD/NASH develops clinically. More natural nutritional models in which animals have been fed diets high in sucrose, fructose, or fat ad libitum generally only result in obesity and hepatic steatosis without inflammation or necrosis (62, 77).

Recently, three new nutritional animal models for NASH have been described. In 2004, Lieber et al. (45) published a study in which male Sprague-Dawley rats were fed a high-fat liquid diet (71% energy from corn oil-olive oil-safflower oil) for 21 days; however, resulting hepatic pathology remained mild and no increase in serum alanine aminotransferase (ALT) activity occurred (45). This appears to be due to satiety limiting consumption of this high-fat diet ad libitum. In a second study, Deng et al. (19) utilized intragastric overfeeding of a 37% corn oil diet at 185% normal caloric intake over 9 wk to induce NASH pathology in male C57BL/6 mice. Development of obesity and insulin resistance was accompanied by liver hyperplasia, steatosis, inflammation, necrosis, and fibrosis. However, this model represents excessive overfeeding and some of the biochemical changes observed in the liver did not mimic those seen in NASH patients. For example, cytochrome P-450 2E1 (CYP2E1) expression was downregulated rather than upregulated and so was PPAR{alpha} expression (19). Most recently, Wang et al. (80) compared feeding solid purified casein-based diets enriched with starch, sucrose, corn oil, or lard oil to male Wistar Crl(WI)BR rats for up to 24 wk. Sucrose, corn oil, and lard diets all resulted in steatosis; however, increased serum ALTs and apoptosis were observed in sucrose- and lard-fed rats, associated with evidence of endoplasmic reticulum stress, but did not occur in the corn oil-fed animals. Unlike the normal course of NASH in patients, severity of steatosis did not increase with time, liver damage developed rapidly prior to increases in body weight, and adiposity or TNF-{alpha} in these rats and was independent of changes in insulin signaling or mitochondrial function. Significant differences in molecular responses were observed between these dietary NASH models, and it is unclear whether these differences were related to species, rat strain, diet composition, or differences in experimental design or how reflective these models are of the human condition.

In the present study, we have examined the effects of moderate (17%) caloric excess and high dietary corn oil content on the development of NASH in male rats using a model in which overfeeding is accomplished by total enteral nutrition (TEN). This model is unique in that the TEN system represents a low-stress model in which the rats are essentially sedentary and NASH pathology very similar to that described clinically develops in response to feeding polyunsaturated fats (PUFA), despite homeostatic changes in fatty acid metabolism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Potassium chloride, potassium phosphate, and potassium ferricyanide were purchased from Fisher Scientific (Hampton, NH). ECL for chemiluminescent detection in Western blotting was purchased from Amersham Biosciences (Piscataway, NJ). TRI Reagent used for RNA extraction was obtained from Molecular Research Center (Cincinnati, OH). Reagents for assessment of RNA quality using the Agilent Bioanalyzer were acquired from Agilent Technologies (Foster City, CA).

Experimental animals and diets. Male Sprague-Dawley rats (175 g) were purchased from Harlan Sprague Dawley (Indianapolis, IN). Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care approved animal facility. Animal maintenance and experimental treatments were conducted in accordance with ethical guidelines for animal research and were approved by the Institutional Animal Care and Use Committee at University of Arkansas for Medical Sciences. Rats had an intragastric cannula surgically inserted and were allowed 7 days to recover before diet infusion as described previously (2–5, 69). Animals had ad libitum access to water throughout the experiment.

Animals were randomly assigned to groups and infused diets containing 5% corn oil for 21 days at 187 kcal·kg–3/4·day–1, previously shown to maintain body weight gains equivalent to ad libitum chow-fed rats (2, 68) or were overfed isocaloric 220 kcal·kg–3/4·day–1 diets (17% excess calories) containing 5, 10, 25, 35, 40, or 70% corn oil substituted for carbohydrate calories. Diet composition is given in Table 1. Protein, vitamin, and mineral content was the same in all diets (2). All diets met caloric and nutritional recommendations established by the National Research Council (NRC). One important aspect of this model is that the rats were essentially sedentary. Rats in metabolism cages have little opportunity for physical activity, except to eat and drink. When fed via TEN, the rats are even more sedentary because they do not need to work for food or water. We have documented this. Activity levels of rats were studied in metabolic cages equipped with a series of invisible light beams traversing each cage at difference distances from the cage floor. When a rat moved, one or more beams were broken and registered on a computer. The number of beam breaks per 24 h was used to calculate an activity that could be used to compare different treatment groups. Adult male Sprague-Dawley rats fed by TEN (n = 6) had 3,611.5 ± 186.3 units/24 h and rats having ad libitum access to standard commercially rodent foods and water (n = 5) had 6,285.4 ± 476.6 units/24 h (P ≤ 0.05). In addition, the TEN system was designed to be very low stress. The animals were preconditioned to regular handling and housing in metabolic cages prior to cannulation and were also acclimated to the tether and to further handling after surgery and before the studies began. The unique headpiece design disperses the weight of the lightweight spring directly over the animal and permits the rat to assume all body positions for sleep, drinking, and 360° movement without any impairment of movement or pulling of the skin such as occurs with tether attached on the back or the neck. We have successfully utilized this system previously to study effects of chronic ethanol infusion on temporal sexually dimorphic patterns of growth hormone secretion, a parameter notoriously sensitive to stress (4).


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  		Table 1. Diet composition

 
Body weight gain was measured twice a week and at the beginning and end of the study. Total body fat composition was assessed by magnetic resonance imaging (Echo, Houston, TX). Rats were euthanized after 21 or 65 days of infusion, and serum and livers were collected and stored at –20 and –70°C, respectively.

Pathological evaluation. Liver pathology was assessed by hematoxylin-eosin and Oil Red O staining of liver sections and scored via blinded samples by a board-certified pathologist (L. Hennings). The pathology calculation was on the basis of ballooning degeneration (0–2), presence or absence of serum markers of necrosis (ALT score: cutoff was 55, based on data from Charles River Laboratories and our baseline values, <55 = 0, >55 = 1), the lipidosis score (based on MCID imaging analysis of Oil Red O-stained slides), and lobular inflammation/necrosis (0 = 0 foci, 1 = <2 foci, 2 = 2–4 foci, 3 = >4 foci). Apoptosis was assessed by in situ end labeling of free 3'-hydroxyl ends generated during apoptosis [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)] using a commercial kit (Frag-EL DNA Fragmentation Detection Kit, Fisher Scientific, Hampton, NH). The sections were counterstained with Gill's hematoxylin. Apoptotic bodies and cells appeared brown. At least 2,000 cells were counted from each liver section (5). Apoptosis was also assessed by immunohistochemical assessment of caspase 3 activation. Sections were pretreated with Dako EDTA antigen retrieval (pH 9.0) in a Dako tissue decloaker (Dako, Carpenteria, CA) according to manufacturer's directions for 20 min. Sections were then incubated in Dako Peroxidase block (Dako) for 10 min at room temperature, and rinsed three times in Tris-buffered salmet 0.1% Tween 20 (TBST). A 10% normal goat serum protein block (Vector Laboratories, Burlingame, CA) in TBST was applied for 30 min at room temperature. Sections were incubated in primary polyclonal antibody to cleaved caspase-3 (Asp175, Cell Signaling, Danvers, MA) at 1:00 dilution for 1 h at room temperature, rinsed three times with TBST, and incubated in secondary antibody [biotinylated goat anti-rabbit (Vector Laboratories)] at 1:400 dilution for 30 min at room temperature. Sections were rinsed three times with TBST, and the ABC reagent (Vector Laboratories) was applied for 30 min at room temperature. Sections were rinsed three times in TBST, and incubated in diaminobenzidine (Dako) for 3 min at room temperature, then rinsed in TBST, counterstained with Mayer's hematoxylin, and coverslipped. Slides were examined under a light microscope, and positive cells were counted in 10 randomly chosen x200 fields. In rats infused with TEN diets for 65 days, portal and lobular fibrosis was detected by picrosirius red staining of collagen (72). Collagen staining was quantified in five randomly chosen x40 fields using MCID Imaging Software version 7.0 linked to an Olympus Bx50 microscope.

Biochemical analysis. Nonesterified fatty acids (NEFA) were measured using the NEFA C kit from Waco Chemicals (Richmond, VA). Serum triglycerides were measured with Triglyceride Reagent (IR141; Synermed, Westfield, IN). Triglyceride was extracted from whole liver homogenates with chloroform-methanol (2:1, vol/vol) and analyzed using Triglyceride Reagent (IR141; Synermed). Serum glucose levels were measured with Glucose Reagent (IR071-072; Synermed, Westfield, IN). Serum insulin and leptin levels were measured using ELISA kits from Linco Research (St. Charles, MO) according to manufacturer's protocols. Serum adiponectin levels were measured with an ELISA kit from B-Bridge International (Sunnyvale, CA) according to manufacturer's protocols. Serum ALT levels were measured at death by using the Infinity ALT liquid stable reagent (Thermo Electron, Waltham, MA) according to manufacturer's protocols. Liver microsomes were prepared by differential centrifugation and stored at –70°C until analysis. Protein concentrations of the microsomes were determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Liver lipid peroxidation was assessed as a measure of oxidative stress as described by Ohkawa et al. (59). Western immunoblot analysis of apoprotein expression for CYP2E1 and CYP4A1 was conducted as previously described (67) except that cross-reactive proteins were detected by enhanced chemiluminescence using horseradish peroxidase-linked goat antibody to rabbit IgG or rabbit antibody to sheep IgG in the case of CYP4A1. CYP2E1 was a gift from the laboratory of Dr. Magnus Ingelman-Sundberg (Karolinska Institute, Stockholm, Sweden) (37). CYP4Al was detected by using a polyclonal sheep antibody to rat CYP4A1 (78), which was a gift from Dr. Gordon Gibson (University of Surrey, Guildford, UK). TNF-{alpha} protein was stained in liver tissue sections with anti-TNF-{alpha} antibody (Abcam, Cambridge, MA).

Real-time reverse transcription-polymerase chain reaction. Total RNA was extracted from livers by using TRI Reagent and cleaned with RNeasy mini columns (Qiagen, Valencia, CA). RNA quality was ascertained spectrophotometrically (ratio of A260 to A280) and also by checking the ratio of 28S to 18S ribosomal RNA using the RNA Nano Chip on a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Total RNA (1 µg) was reverse transcribed by using the iScript Reverse Transcription kit (Bio-Rad Laboratories) according to manufacturer's instructions. The reverse transcribed cDNA (10 ng) was utilized for real-time PCR using the 2x SYBR green master mix and monitored on a ABI Prism 7000 sequence-detection system (Applied Biosystems, Foster City, CA). Gene-specific probes were designed by use of Primer Express Software (Applied Biosystems; Table 2), and the relative amounts of gene expression were quantitated by using a standard curve according to manufacturer's instructions.


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  		Table 2. Primer sequences for real-time RT-PCR

 
Hepatic gene expression analysis. Hepatic gene expression profiles were assessed using Affymetrix RGU34A GeneChip microarrays (Affymetrix, Santa Clara, CA) containing 8,800 genes, with ~7,000 full-length transcripts and 1,800 EST sequences. Livers were excised from rats fed 5 or 70% corn oil diets (n = 3–4) containing 187 or 220 kcal·kg–3/4·day–1, respectively. Total RNA was extracted and prepared as described above. The intensity values of different probe sets (genes) generated by Affymetrix GeneChip Operating Software were imported into GeneSpring version 7.2 software (Silicon Genetics, Redwood City, CA) for data analysis. The data files (CEL files) containing the probe level intensities were processed by the robust multiarray analysis algorithm (GeneSpring) for background adjustment, normalization, and log2-transformation of perfect-match values (36). Subsequently, the data were subjected to per-chip and per-gene normalization by GeneSpring normalization algorithms. For comparison analysis, genes were filtered on the basis of minimum 1.5-fold ratio change and P value ≤0.05 using Student's t-test. Data analyses were performed using Microsoft Excel and GeneSpring GX v7.2. Correlation-based hierarchical clustering between treatment groups and visualization of data were done by using GeneSpring GX v7.2. Known biological functions of genes were queried and acquired from Affymetrix online data analysis resource NetAffx and gene ontology (GO) analyses performed using Affymetrix GO Browser (89). The original CEL files have been submitted to NCBI-GEO database and are accessible as series record GSE8253 [NCBI GEO] . Confirmation of microarray gene expression data was done by real-time RT-PCR.

EMSA. The nuclear extracts were isolated from livers frozen at –70°C using a nuclear extraction kit from Sigma. Protein concentration of the nuclear extracts was determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad). Electrophoretic mobility shift assays (EMSAs) using double-stranded oligonucleotides coding for the acyl CoA oxidase (ACO)-peroxisome proliferator response element (PPRE), gatcCTCCCGAACGTGACCTTTGTC CTGGTCCAgatc, were performed as described previously (5, 87).

Statistical analysis. Data are expressed as means ± SE. Densitometric quantitation of Western blot autoradiograms was performed with Quantity One software (Bio-Rad). SigmaStat software package version 3.0 (SPSS, Chicago, IL) was used to perform all statistical tests. The data were tested with Levene's test for equality of variance. Pearson product moment correlation was performed with SigmaStat software. Group differences were evaluated via one- or two-way analysis of variance followed by Student-Newman-Keuls post hoc comparisons unless otherwise stated. P values ≤0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of overfeeding and high-fat intake on body weight and adiposity. As in previous studies with the TEN model (2–5, 66, 68), infusion of diets at 187 kcal·kg–3/4·day–1 resulted in weight gains similar to those in ad libitum chow-fed rats. Feeding a diet containing 220 kcal·kg–3/4·day–1 represents a caloric intake of ~17% in excess of NRC recommended levels. Twenty-one days of overfeeding resulted in increases in final body weight from 281 ± 10 to 342 ± 3 g (P ≤ 0.05). However, isocaloric increases in fat content from 5 to 70% at 220 kcal3/4/day had no further effect on body weight (344 ± 11 g). The 2-fold increase in body weight gain in the 220 kcal3/4/day groups (P ≤ 0.05) (Fig. 1), was accompanied by increased fat mass as % of body weight compared with those fed the 187 kcal·kg–3/4·day–1 calorie diet (P ≤ 0.05) (Fig. 1).


Figure 1
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  		Fig. 1. Effects of overfeeding and high-fat intake on body weight and adiposity. A: body weight gain (g/day). Data represent means ± SE (n = 8–10). *P ≤ 0.05 vs. 5% corn oil, 187 kcal·kg–3/4·day–1. B: change in fat mass as % body weight (pre- and poststudy). Data represent means ± SE (n = 8–10). *P ≤ 0.05 prestudy vs. poststudy, !P ≤ 0.05 vs. 5% corn oil, 187 kcal·kg–3/4·day–1.

 
Effects of high-fat intake in the context of modest overfeeding on serum parameters. Increased caloric intake from 187 to 220 kcal·kg–3/4·day–1 at 5% corn oil increased serum leptin (3.9 ± 0.4 to 7.2 ± 0.6 ng/ml) (P ≤ 0.05) and serum glucose (92 ± 3.9 to 102 ± 5.8 mg/dl) (P ≤ 0.05). Increasing dietary fat content isocalorically from 5 to 70% in the 220 kcal3/4/day group resulted in no significant additional increases in serum leptin or glucose (leptin 8.9 ± 1.6 ng/ml, glucose 109 ± 3.8 mg/dl), but did significantly increase serum NEFAs and triglycerides (P ≤ 0.05) (Table 4). In addition, serum insulin and adiponectin levels showed evidence of hyperinsulinemia and hypoadiponectinemia in the 70% corn oil, 220 kcal·kg–3/4·day–1 group compared with the 5% corn oil, 220 kcal·kg–3/4·day–1 group (P ≤ 0.05) (Table 4).


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  		Table 4. Biochemical effects of modest overfeeding of high-unsaturated fat diets

 
Liver histopathology. Pathological examination demonstrated no histological evidence of fat accumulation or inflammation in the 187 kcal·kg–3/4·day–1 (data not shown) or 220 kcal·kg–3/4·day–1, 5% corn oil groups (Fig. 2, Table 3), but steatosis, macrophage infiltration, and focal necrosis was present in the 220 kcal·kg–3/4·day–1, 70% corn oil group (Fig. 2, Table 3), accompanied by elevated serum ALT levels (P ≤ 0.05) (Table 4) indicating necrotic injury. Increased levels of hepatocyte apoptosis were also present as determined by TUNEL analysis (P ≤ 0.05) and a nonsignificant increase in activated caspase 3 expression (Table 4). Oil Red O staining and biochemical analysis of triglycerides showed a dose-dependent increase in triglyceride deposition with increase in percent of dietary corn oil (5 < 10 < 35 < 70%, only 5 and 70% shown) in 220 kcal·kg–3/4·day–1 diets (P ≤ 0.05) (Fig. 2, Table 3 and Table 4).


Figure 2
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  		Fig. 2. Representative hematoxylin-eosin (H&E) and Oil Red O-stained liver sections. Representative H&E-stained liver sections of 5% corn oil, 220 kcal·kg–3/4·day–1 (A) and 70% corn oil, 220 kcal·kg–3/4·day–1 (B). Representative Oil Red O-stained liver sections of 5% corn oil, 220 kcal·kg–3/4·day–1 (C) and 70% corn oil, 220 kcal·kg–3/4·day–1 (D).

 

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  		Table 3. Hepatic pathology scores after 21-day TEN feeding

 
Effects of 65 days overfeeding of TEN diets high in PUFA. A more chronic study of 65 days, overfeeding of 220 kcal·kg–3/4·day–1 high-corn oil TEN diets, demonstrated that development of NASH pathology and necrosis was not only dose responsive, but progressive with length of infusion. Feeding of diets containing 40% corn oil, 220 kcal·kg–3/4·day–1 resulted in similar pathology to that observed with 70% corn oil, 220 kcal·kg–3/4·day–1 for 3 wk (data not shown) and was accompanied by increased serum ALTs (P ≤ 0.05) (Fig. 3). Histological examination of livers from the 65-day 70% corn oil-fed group revealed portal/periportal and lobular fibrosis as indicated by increased picrosirius red staining of collagen (P ≤ 0.05) in addition to steatohepatitis, similar to that observed in grade 3 clinical NASH, accompanied by greater serum ALT values (P ≤ 0.05) (Fig. 3).


Figure 3
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  		Fig. 3. Effects of 65-days total enteral nutrition (TEN) infusion. A: alanine aminotransferase activity levels over 65 days TEN infusion. Data represent means ± SE (n = 8–10). *P ≤ 0.05 vs. 5% corn oil, 220 kcal·kg–3/4·day–1. B: representative liver sections of H&E-stained control liver (I); H&E stained liver after 65 days TEN infusion of 70% corn oil and 220 kcal·kg–3/4·day–1 diet (II); and picrosirius red staining of portal and lobular collagen in liver after 65-days TEN infusion of 70% corn oil, 220 kcal·kg–3/4·day–1 diet (III). C: quantitation of picrosirius red collagen staining by image analysis in control liver and after 65-day TEN infusion of 70% corn oil, 220 kcal·kg–3/4·day–1 diet. A.D.U., arbitrary densitometric units.

 
Effects of overfeeding high-fat diets on hepatic oxidative stress and inflammation. In the 70% corn oil, 220 kcal·kg–3/4·day–1 group, an increase in oxidative stress [thiobarbituric acid-reactive substances (TBARSs); P ≤ 0.05] and inflammation, as evidenced by TNF-{alpha} mRNA and protein induction (P ≤ 0.05) was observed (Table 4). An increase in CYP2E1 and CYP4A1 expression (P ≤ 0.05) (Fig. 4), sources of reactive oxygen species (ROS), was also observed.


Figure 4
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  		Fig. 4. Effects on hepatic CYP2E1 and CYP4A1 expression. A: densitometric quantitation of CYP2E1 protein levels from rats fed 5 or 70% corn oil, 220 kcal·kg–3/4·day–1 and representative Western blot. Data represent means ± SE (n = 5). Each lane represents the liver microsomal protein from individual rats. *P ≤ 0.05 vs. 5% corn oil, 220 kcal·kg–3/4·day–1. B: densitometric quantitation of CYP4A1 protein levels from rats fed 5 or 70% corn oil, 220 kcal·kg–3/4·day–1 and representative Western blot. Data represent means ± SE (n = 5). Each lane represents the liver microsomal protein from individual rats. *P ≤ 0.05 vs. 5% corn oil, 220 kcal·kg–3/4·day–1.

 
Correlation between cell death, steatosis, oxidative stress, and inflammation. The correlations between ALTs (cell death) vs. Oil Red O staining (steatosis), TBARSs (oxidative stress) vs. steatosis, oxidative stress vs. cell death, and oxidative stress vs. TNF-{alpha} (inflammation) were significant (P ≤ 0.05) (Table 5).


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  		Table 5. Correlation between characteristics of NASH observed in 70% corn oil groups

 
Fatty acid homeostasis. The appearance of steatosis in the TEN model after 21 days infusion was accompanied by significant decreases in mRNA expression for the rate limiting enzymes in de novo fatty acid (FA) synthesis: FA synthase (FAS) and acetyl CoA carboxylase (ACC), in the 220 kcal·kg–3/4·day–1, 70% corn oil group relative to the 220 kcal·kg–3/4·day–1, 5% corn oil group (P ≤ 0.05) (Table 6). In addition, we observed decreases in mRNA encoding SREBP-1c and carbohydrate response element binding protein (ChREBP) (P ≤ 0.05) (Table 6). In contrast, we observed significant increases in mRNAs for alcohol dehydrogenase (ADH) class I and PPAR{alpha}-regulated enzymes involved in {omega}-oxidation of FA: CYP4A1 (endoplasmic reticulum FA {omega}-oxidation), ACO (peroxisomal FA β-oxidation), and mitochondrial trifunctional protein, alpha subunit (mitochondrial FA β-oxidation) and FA mitochondrial transport: carnitine palmitoyl transferase (Table 6). EMSA showed an increase in PPAR{alpha} binding to its ACO-PPRE in the 70% corn oil group (P ≤ 0.05) (Fig. 5). Microarray analysis of livers from the 187 kcal·kg–3/4·day–1 5% corn oil diet compared with 220 kcal·kg–3/4·day–1 70% corn oil diets was conducted using Affymetrix GeneChip microarray system. Seventy-seven genes were altered in the 70% corn oil group, using a 1.5-fold cutoff (Fig. 6). Many of these were genes involving fatty acid metabolism and transport. Steroyl CoA desaturase, known to be involved in regulation of fatty acid synthesis was downregulated 25-fold whereas mitochondrial acyl CoA thioesterase (MTE1), an enzyme involved in fatty acid β oxidation was upregulated 10-fold. This is consistent with homeostatic effects of steatosis on fatty acid synthesis and degradation (Table 7). A major gene to be upregulated fivefold was the scavenger receptor and known fatty acid transporter, CD36. We confirmed the regulation of these mRNAs using real-time PCR (Table 7). In addition, a second gene known to be involved in regulation of fatty acid transport, liver fatty acid binding protein (L-FABP), was upregulated in the 70% corn oil group (P ≤ 0.05) (Table 6).


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  		Table 6. Effects of 21-day TEN feeding on fatty acid synthesis, degradation, and transport

 

Figure 5
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  		Fig. 5. EMSA analysis of PPAR{alpha} binding to PPRE element of acyl CoA oxidase. A: representative EMSA showing the effect of rats fed 5 or 70% corn oil, 220 kcal·kg–3/4·day–1 on hepatic PPAR{alpha} binding to the PPRE in the ACO promoter. Lanes 1–5 represent 5% corn oil, 220 kcal·kg–3/4·day–1 and lanes 6–10 represent 70% corn oil, 220 kcal·kg–3/4·day–1. Each lane is nuclear extract from an individual rat. Specificity of EMSA signal confirmed by competition with unlabeled and labeled oligonucleotides (not shown). Data represent means ± SE. Individual densitometry values of 5.8 ± 0.6 for 5% corn oil, 220 kcal·kg–3/4·day–1 and 14.3 ± 1.4 for 70% corn oil, 220 kcal·kg–3/4·day–1 are significantly different, P ≤ 0.05.

 

Figure 6
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  		Fig. 6. Gene expression profiling of genes modulated by the NASH TEN model. A: heatmap representation of relative expression levels of genes modulated by 5% corn oil, 187 kcal·kg–3/4·day–1 or 70% corn oil, 220 kcal·kg–3/4·day–1 diet. Each horizontal line represents a single gene and each column represents a single sample. The relative expression of each gene is color coded as high (red) or low (blue); 77 genes are significant, P ≤ 0.05 and 1.5-fold. B: heatmap clusters of gene expression of genes with biological, molecular, and cellular functions related to lipid metabolism; 25 genes were altered as classified by gene ontology.

 

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  		Table 7. Microarray analysis of the NASH TEN model

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have attempted to produce NASH pathology in the rat using modest overfeeding of a diet high in corn oil. The rats were housed in metabolic cages and enterally fed under low-stress, sedentary conditions. Overfeeding alone increased body weight, weight gain, and adiposity; however, no hepatic pathology, including little or no steatosis, was observed in either rat group fed 5% corn oil for 21 days despite the development of obesity, hyperleptinemia, and hyperglycemia in the overfed 220 kcal·kg–3/4·day–1 group. In contrast, when dietary fat content was increased in the presence of overfeeding, a dose-dependent increase in macro- and microsteatosis, lobular inflammation, necrosis, and apoptosis was observed. In chronic 65-day studies we observed significant further increases in plasma ALT and the appearance of fibrosis, suggesting that the development of NASH pathology was also progressive with time.

A possible link between obesity and NASH is a role for disrupted adipokine signaling (11, 14, 33). Serum leptin levels were significantly increased in the present study in all the overfed groups. Leptin is recognized as a multifunctional protein (32), and it has been shown in rats that, even in the absence of the leptin signaling cascade, steatohepatitis and augmentation of oxidative stress could be observed; however, without leptin signaling, neither fibrosis nor hepatocellular carcinoma developed, suggesting that leptin plays a pivotal role in the progression of fibrosis and carcinogenesis in NASH (40). Serum adiponectin was significantly reduced by overfeeding increasing percent PUFA in this model. Serum adiponectin has also been shown to be reduced in NASH patients correlating with the severity of pathology and independent of the development of insulin resistance (33, 53, 70). Recent studies have reported that insulin resistance is present in almost all patients with NAFLD (15) and that the presence of central obesity, type 2 diabetes, or the severity of insulin resistance are predictors of NASH independent of the severity of steatosis (83). This study showed increasing the percentage of corn oil in the 220 kcal·kg–3/4·day–1 diets produced significant increases in serum insulin, reductions in hepatic SREBP-1c, and increases in hepatic ADH I expression all consistent with development of systemic and hepatic insulin resistance (31).

NASH pathology in patients is characterized by evidence of oxidative stress including the appearance of oxidized proteins, DNA, and lipids (1, 11, 15, 29, 75) and the magnitude of oxidative stress has been shown to correlate with disease severity (1, 75). It has been suggested that oxidative stress represents a "second hit" whereby simple reversible steatosis progresses to the more severe pathologies of NASH and fibrosis (16). We observed a significant increase in oxidative stress (TBARSs). Oxidative stress may result from a number of sources but has been particularly linked to mitochondria. There is increasing evidence of mitochondrial dysfunction in patients with NASH (6, 10, 61, 70) and the appearance of megamitochondria in hepatocytes is common in NASH patients (6, 54). Oxidative damage to mitochondria results in reduced ATP formation and permeabilization of the mitochondrial membrane (42, 48). This membrane permeability transition results in release of death mediators such as cytochrome c, which can stimulate both apoptosis and necrosis depending on ATP concentrations (48). Both apoptosis and necrosis were significantly increased in our model. In addition to being an important cellular target of oxidative stress, uncoupled mitochondrial respiration is a major source of ROS and we have evidence for increased mitochondrial fatty acid oxidation in the present model (42, 48). Peroxisomal β-oxidation of fatty acids can also generate ROS (54). The cytochrome P-450 enzymes CYP4A1 and CYP2E1 both catalyze "leaky" redox cycles, which can also produce ROS during fatty acid metabolism in the endoplasmic reticulum or even in the absence of substrate (22, 41, 64, 74). CYP2E1 induction has been shown to occur in NASH patients (8, 84, 85) and is significantly increased in our NASH model. In this regard our rat data differ from the mouse intragastric overfeeding model of NASH developed by Deng et al. (19) in which CYP2E1 was found to be suppressed and where fatty acid oxidation appears downregulated. This may in part reflect species differences in hepatic responses to insulin and glucose. For example, the hepatotoxicity of xenobiotics in diabetic rats and mice are exactly the opposite as a result of differences in PPAR{alpha}-mediated compensatory repair under conditions of disrupted insulin signaling and hyperglycemia. The response in human liver appears more similar to that seen in the rat (82).

Other pathways for NASH progression have also been suggested with oxidative stress representing only an epiphenomenal consequence of cellular injury (54). Cytokines, in particular TNF-{alpha}, have received much attention in the progression of NAFLD to NASH and also in development of steatosis itself (25). TNF-{alpha} protein and mRNA were both significantly increased in this study (Table 4). TNF-{alpha} expression is increased in both plasma and liver in NASH patients (13, 33) and animal models (17, 19) and is capable of producing insulin resistance, apoptosis/necrosis, and stellate cell activation (11). However, the role of TNF-{alpha} remains controversial. Anti-TNF therapy reduces NAFLD in the mouse ob/ob mouse (44) and steatosis was abolished in TNFR1 (–/–) mice fed high-carbohydrate diets (27); however, TNFR1 –/– mice have recently been shown not to be protected against development of liver pathology either in the intragastric overfed mouse model (19) or in methionine/choline-deficient mouse NASH models (17).

Direct "lipotoxicity" of excess circulating NEFAs has also been proposed as a progressor of NASH (73). We observed a significant increase in serum NEFA. Free FAs (FFAs) can produce apoptosis in hepatocytes directly (26, 73) and can destabilize lysosomes resulting in nuclear factor-{kappa}B-dependent TNF-{alpha} synthesis (27). However, lipotoxicity has been linked to long-chain saturated FAs such as palmitate (26, 27, 73) whereas the present study demonstrates that NASH pathology can be produced as the result of feeding corn oil, which consists primarily of PUFA. In alcoholic liver disease models, which have very similar hepatic pathology to NASH, several studies have demonstrated increased pathology associated with feeding PUFA or {omega}-3 FAs in fish oil whereas long- and short-chain saturated fat diets are protective (55, 56, 68, 87). Studies are in progress to assess the effects of dietary fat composition on development of NASH pathology in rat in the TEN model. It has been suggested that the link between obesity, insulin resistance, and NASH risk is explained by increased release of FFA from adipose tissue (15). Insulin resistance increases lipolysis of adipose tissue depots, increasing FFA in NASH patients (49, 70). FFA transport into the liver has been proposed to stimulate PPAR{alpha}, resulting in increased FA {omega}- and β-oxidation pathways as is observed in the present study (15).

Some animal studies of NASH have suggested that fat accumulation is the result of increased de novo FA synthesis. Hepatic FA synthesis has been suggested to be under the regulation by two transcription factors: SREBP-1c, which is regulated by fat and insulin, and ChREBP, which is regulated by dietary carbohydrates (20, 21). Both transcription factors stimulate expression of the rate limiting lipogenic genes FAS and ACC (20, 21). ACC activity is also regulated posttranscriptionally (52). In the overfed intragastric mouse model of NASH and in the JCR-LA corpulent rat SREBP-1c, FAS and ACC expression is increased (19, 23). In contrast, we have observed downregulation of mRNAs for SREBP1c, ChREBP, FAS, and ACC. Recent studies have suggested that suppression of SREBP-1c and ChREBP signaling is a specific hepatocyte response to PUFA, which are the major components of the corn oil utilized in the present study (20, 21, 38). The downregulation of SREBP-1c may also be a consequence of reduced expression of SCD-1 in these livers since this enzyme has been implicated in regulation of SREBP-1c and FA synthesis as the result of its ability to regulate cell signaling through conversion of palmitate to oleate (58, 63). Little is known regarding expression of these enzymes, SREBP-1c, or ChREBP in livers from NASH patients.

Two major proteins involved in FA uptake are L-FABP (34, 47, 57, 81) and the scavenger receptor, CD36 (46). CD36 is normally expressed at low levels in liver (86). Knockout of NADPH-cytochrome P-450 reductase or feeding of high-fat diets have recently been reported to significantly induce hepatic CD36 expression in mice coincident with the development of steatosis (35, 86); however, little is known about hepatic uptake of FFAs in animal models of NASH or in NASH patients. We have observed significant increases in expression of both L-FABP and CD36. This suggests that increased FA uptake plays a major role in the increased steatosis observed in this NASH model.

In summary, NASH is the most rapidly increasing hepatic pathology in the U. S. population and yet molecular mechanisms underlying NASH and methods to protect against it remain incompletely understood. This results from the absence of an animal model which replicates the natural course and etiology of the disease in patients. We have developed a new model of NASH in which hepatic pathology including fibrosis develops in sedentary rats under low-stress conditions as the result of moderate overconsumption of a diet similar to that consumed by Americans. Obviously, enteral feeding of liquid diets is not completely analogous to the human NASH situation. However, the TEN system uniquely allows dissection of the pathological effects of excessive caloric intake from the effects of imbalances in the major macronutrients thought to be involved in NASH and other related disorders of overweight and/or obesity. Pathological, endocrine, and biochemical changes were similar to those observed in NASH patients clinically and develop under conditions of obesity known to increase NASH incidence. However, hepatic steatosis and NASH develop rapidly in obese rats fed via TEN when the percentage of unsaturated dietary fat is increased. The mechanism of steatosis in this model does not involve altered FA synthesis or degradation but rather may involve increased FA transport via CD36 and/or L-FABP, genes known to be regulated via the PPAR transcription factors (34, 35, 46, 47, 57, 81, 86). Several biochemical endpoints including CYP2E1 expression, FA homeostasis, and PPAR{alpha}-signaling differed between the present study other rodent NASH models. This may reflect differences in species, strain, diet composition, and experimental design. NASH appears to be a multifactorial condition in which the same pathological end points can be achieved by different routes. However, as in other animal models and in clinical samples from NASH patients, the appearance of liver pathology correlated with disruption of adipokines, evidence of insulin resistance, increases in hepatic oxidative stress, and increases in hepatic TNF-{alpha} expression. The commonalities between different models suggest that these pathways are of fundamental importance underlying the development of liver pathology in NAFLD.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This research was supported, in part, by RO1 AA 12819 (M. J. J. Ronis), ACNC-USDA-ARS 6251-51000-005D and the National Institute of Environmental Health Sciences Graduate Student Training Grant (J. N. Baumgardner).


   ACKNOWLEDGMENTS
 
We thank the following people for their technical assistance: Matt Ferguson, Jamie Badeaux, Tammy Dallari, James M. Robinette, and Michele Perry.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. J. J. Ronis, Arkansas Children's Nutrition Center, Slot 512-20B, 1212 Marshall St., Little Rock, AR 72202 (e-mail: RonisMartinJ{at}uams.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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