Steatohepatitis develops rapidly in transgenic mice overexpressing Abcb11 and fed a methionine-choline-deficient diet

Shikha S. Sundaram,1 Peter F. Whitington,1 and Richard M. Green2

1Division of Gastroenterology and Hepatology and Nutrition, Departments of Pediatrics, and 2Division of Hepatology, Department of Internal Medicine, Northwestern University, Feinberg School of Medicine, Chicago, Illinois

Submitted 8 October 2004 ; accepted in final form 11 January 2005


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Nonalcoholic fatty liver disease is the most common reason for abnormal liver chemistries in the United States. The factors that lead from benign steatosis to nonalcoholic steatohepatitis are poorly understood. Transthyretin-Abcb11 (TTR-Abcb11) transgenic mice overexpress the bile salt transporter Abcb11 and hypersecrete biliary lipids. Thus the aim of this study is to employ feeding of the methionine-choline-deficient (MCD) diet to TTR-Abcb11 transgenic mice to further determine the mechanisms responsible for the development of steatohepatitis. FVB/NJ and TTR-Abcb11 mice were fed control or MCD diets for up to 30 days. Serum aminotransferase levels, serum and hepatic triglyceride content, cytokines, markers of oxidative stress, and expression of selective genes were examined. MCD diet-fed TTR-Abcb11, but not wild-type, mice have elevated serum aminotransferase levels when compared after 7 days. They also have significantly lower hepatic triglyceride levels at all time points studied. After 14 days on the MCD diet, TTR-Abcb11 mice have 3-fold increases in TNF-{alpha} mRNA and 3.9-fold increases in IL-6 mRNA compared with FVB/NJ mice. TTR-Abcb11 mice also had a greater increase in cytochrome P-450 2E1 expression. A greater decrease in sterol regulatory element binding protein-1c and fatty acid synthase mRNA expression was also seen in TTR-Abcb11 compared with wild-type mice fed an MCD diet. They also have enhanced TNF-{alpha}, IL-6, and cytochrome P-450 2E1 expression. We conclude that TTR-Abcb11 mice develop a more rapid hepatitis with less steatosis.

nonalcoholic steatohepatitis; oxidative stress; cytokines


NONALCOHOLIC FATTY LIVER DISEASE (NAFLD) is the most common cause of abnormal liver chemistries in the United States and other developed countries and is a significant cause of cryptogenic cirrhosis (3). NAFLD represents a wide spectrum of liver injury, including simple steatosis, steatohepatitis, fibrosis, and cirrhosis. Hepatic steatosis occurs in up to 25% of individuals and can progress to cirrhosis in a significant minority of these patients (26). Hepatic steatosis is commonly associated with obesity, type 2 diabetes, and hypertriglyceridemia (25). The pathophysiological mechanisms leading to the progression from simple steatosis to nonalcoholic steatohepatitis (NASH), however, remain poorly elucidated.

A current hypothesis to help explain the progression from steatosis to NASH is the "two-hit hypothesis." The first hit is chronic accumulation of excessive hepatic fat secondary to a metabolic disturbance, such as insulin resistance (14). The second hit has been suggested to be toxic lipid peroxidation and oxidative stress due to reactive oxygen species accumulation (7). Inflammatory cytokine-mediated liver injury has also been implicated as a second hit (7, 33, 49). NASH, however, is most likely a disease of multifactorial etiology, in which disease pathogenesis involves "multiple hits" or pathogenic factors.

Mice fed a diet deficient in methionine and choline (MCD) have been shown to develop steatosis, necroinflammation, and eventually progressive fibrosis. This MCD dietary model of NASH has been extensively used in previous studies since this nutritional model creates progressive, fibrosing steatohepatitis in rodents (19, 21, 23, 32, 33). Both methionine and choline are essential precursors of hepatic phosphatidylcholine (PC) synthesis, and the hepatic steatohepatitis induced by this diet may be due, in part, to impairments in PC synthesis. Figge et al. (13) recently developed the transthyretin-Abcb11 (TTR-Abcb11) transgenic mouse model, which overexpresses the hepatic canalicular ATP-dependent bile salt transporter Abcb11. Hepatobiliary secretion of bile salt is the primary driving force for bile formation, and enhanced canalicular expression of Abcb11 [often termed bile salt export pump (BSEP)] results in increased bile flow and increased biliary lipid secretion. In particular, TTR-Abcb11 mice exhibit an increase in biliary secretion of bile salts, cholesterol, and PC. In addition, when fed a lithogenic diet (high in fat, cholesterol, and cholic acid), these mice demonstrate significantly less hepatic steatosis than wild-type FVB/NJ controls (13).

Hepatobiliary secretion of PC is high, and it has been estimated that the entire hepatic PC content is secreted into bile each day (42, 43). Thus mice with enhanced biliary secretion of PC, such as TTR-Abcb11 mice, may be more susceptible to rapidly develop hepatic injury induced by the MCD diet. Steatosis, however, is likely an essential precursor for the development of steatohepatitis. Mice that are more resistant to the development of hepatic steatosis may be more resistant to oxidative stress, hepatocyte injury, and steatohepatitis. Therefore, the primary aim of this study was to employ TTR-Abcb11 transgenic mice to further determine the mechanisms responsible for the development of steatohepatitis.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Animal protocol. Male FVB/NJ mice were purchased from Jackson Laboratories (Bar Harbor, ME). Male TTR-Abcb11 transgenic mice, in an FVB/NJ background, were bred from founder lines at Northwestern University or the Lakeside Veterans Affairs Medical Center (Chicago, IL). All animal protocols were approved by the Northwestern University Animal Care and Use Committee (ACUC) and the Lakeside Veterans Affairs Medical Center ACUC and conformed to standard procedures set out by the ACUC. Mice were euthanized by CO2 inhalation in accordance with recommendations of the American Veterinary Medical Association. All animals were 6–8 wk old when experiments were initiated, and mice were fed a control diet or MCD diet (ICN Biomedicals, Aurora, OH) for up to 30 days. Mice were given free access to food and water. Body weights were recorded at the start and end of each experimental protocol. Blood was collected by cardiac puncture and centrifuged at 7,000 rpm for 6 min to collect serum. Livers were rapidly excised, rinsed in ice-cold saline, and weighed, and aliquots were snap-frozen in liquid nitrogen and kept at –80°C until analyzed. A portion of each liver was fixed in 10% formalin for histology.

Histological evaluation. Formalin-fixed liver was embedded in paraffin, and 5-µm sections were stained with hematoxylin and eosin. An investigator (R. M. Green) blinded to experimental conditions examined sections for steatosis and inflammation as follows: 1) steatosis: grade 0, none present; grade 1, steatosis of <25% of parenchyma; grade 2, steatosis of 26–50% of parenchyma; grade 3, steatosis of 51–75% of parenchyma; grade 4, steatosis of >76% of parenchyma; 2) inflammation: grade 0, no inflammatory foci; grade 1, 1 inflammatory foci/high-powered field (hpf); grade 2, 2–3 inflammatory foci/hpf; grade 3, >4 inflammatory foci/hpf.

Measurement of serum and liver triglyceride levels and chemistries. Liver samples were homogenized in 50 mM Tris·HCl buffer, pH 7.4, containing 150 mM NaCl, 1 mM EDTA, and 1 mM 1,1,1-trichloro-2,2-bis[p-chlorophenyl]ethane. Liver triglyceride and cholesterol content was measured using a spectrophotometric assay from Sigma Diagnostics (St. Louis, MO) as per instructions from the manufacturer and was expressed as milligrams of lipid per milligrams of liver protein. Liver thiobarbituric acid reactive substance (TBARS) content was measured by spectrophotometric assay using a kit from Zeptometrix (Buffalo, NY) and expressed as nanomoles of TBARS per milligrams of protein. Reduced glutathione levels were measured in liver homogenate prepared in 5% metaphosphoric acid using a spectrophotometric assay purchased from Calbiochem (San Diego, CA) per instructions from the manufacturer and expressed as micromoles of glutathione per milligrams of liver protein. Serum alanine aminotransferase (ALT) was determined using spectrophotometric assay kits purchased from Sigma Diagnostics as per instructions from the manufacturer. Protein concentration of liver homogenate was measured, employing a Coomassie assay reagent (Pierce, IL).

Western blot analysis. Liver samples were homogenized in a lysis buffer (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 25 mM EDTA, 5 mM EGTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, and 1 mM DTT) containing a protease inhibitor cocktail (Calbiochem). Homogenates were centrifuged at 12,000 rpm for 5 min at 4°C. Samples were mixed with 5x reducing electrophoresis buffer (50 mM Tris·HCl, pH 6.8, containing 10% glycerol, 2% SDS, 1% {beta}-mercaptoethanol, and 0.02% bromophenol blue) and heated at 95°C for 5 min. Samples containing 10–50 µg of protein were separated by 10% SDS polyacrylamide gel electrophoresis. Proteins were then transferred onto a nitrocellulose membrane overnight by electrophoresis. Protein detection was performed using a polyclonal rabbit anti-human cytochrome P-450 2E1 (CYP2E1) antibody (1:2,000 dilution, Chemicon, Temecula, CA) or rabbit polyclonal {gamma}-glutamyl cysteine synthetase ({gamma}-GCS) antibody (1:200 dilution, Neomarkers, Fremont, CA). Bound primary antibody was detected using a horseradish peroxidase conjugated secondary antibody (1:1,000 dilution, Amersham, Arlington Heights, IL) and enhanced chemiluminescence method. Blots were also stripped and reprobed for tubulin using anti-tubulin antibody (1:200 dilution, Sigma-Aldrich, St. Louis, MO). Quantification of protein levels was performed by densitometric analysis using an Eagle Eye II system (Stratagene, La Jolla, CA) and normalized for tubulin expression.

Quantification of {gamma}-GCS enzymatic activity. {gamma}-GCS enzymatic activity was determined in a reaction mixture that contained sodium L-glutamate (10 mM), L-{alpha}-aminobutyrate (10 mM), MgCl2 (20 mM), Na2ATP (5 mM), Na2EDTA (2 mM), Tris·HCl buffer (100 mM; pH 8.2), and bovine serum albumin (10 µg) in a final volume of 0.5 ml. The reaction was initiated by adding enzyme, incubated for 10 min at 37°C, and terminated by adding 0.5 ml of trichloroacetic acid (35). The inorganic phosphate generated was then determined by spectrophotometric absorbance at 720 nm absorbance, as described by Taussky and Shorr (40). {gamma}-GCS enzymatic activity was expressed per milligrams of liver protein.

Real-time quantitative PCR analysis. The mRNA expression of sterol regulatory element binding protein (SREBP)-1c, TNF-{alpha}, IL-6, fatty acid synthase (FAS), and GAPDH was assessed using real-time PCR. Total RNA from liver samples was isolated using TRIzol reagent (GIBCO-BRL, Grand Island, NY). For reverse transcription PCR, 2 µg of total RNA were reverse transcribed using 2 µl of SuperScript II RNaseH reverse transcriptase and 2 µl of random hexamers (Invitrogen, Carlsbad, CA). Real-time PCR was performed using 4 µl of total cDNA in a 50-µl reaction mixture containing Quantitect SYBR Green PCR Mastermix (Qiagen, Valencia, CA), along with primers specific for mouse TNF-{alpha}, IL-6, and FAS, and the housekeeping gene GAPDH (Integrated Technologies, Coralville, IA). The primer sequence used for SREBP-1c forward was 5'-ATC GGC GCG GAA GCT GTC GGG GTA GCG TC-3' and reverse 5'-ACT GTC TTG GTT GTT GAT GAG CTG GAG CAT-3'. The primer sequence used for FAS forward was 5'-TGC TCC CAG CTG CAG GC-3' and reverse 5'-GCC CGG TAG CTC TGG GTG TA-3'. The primer sequence used for TNF-{alpha} forward was 5'-CTG GGA CAG TGA CCT GGA CT-3' and reverse 5'-GCA CCTCAGGGAAGA GTC TG-3'. The primer sequence used for IL-6 forward was 5'-AGT TGC CTT CTT GGG ACT GA-3' and reverse 5'-TCC ACG ATT TCC CAG AGA AC-3'. The primer sequence used for GAPDH forward was 5'-GTC GTG GAT CTG ACG TGC C-3' and reverse 5'-TGC CTG CTT CAC CAC CTT C-3'. Each PCR amplification was performed on five to seven mice in the experimental group and a minimum of three separate mice in the control group. Reported data reflect a minimum of two distinct runs on each mouse liver. Amplification was performed in duplicate for each sample in an ABI Prism 5700 sequence detector (PE Applied Biosystems). All data were normalized for GAPDH expression. Relative gene expression levels from real-time PCR data were analyzed as recently reported using the comparative threshold cycle method, as described in the Applied Biosystems Sequence Detection Systems instruction guide (2, 20, 32, 33).

Statistical analysis. Data are presented as means ± SE. Comparison between groups was performed using Student's t-test or ANOVA analysis.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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The effect of the MCD diet on serum ALT levels. After 7 days on the MCD diet, TTR-Abcb11 mice had significantly elevated serum ALT levels (82.6 ± 11.2 IU), which were higher than those observed in FVB/NJ mice (38.9 ± 7.3 IU), P = 0.005. After 14 days on the MCD diet, serum ALT levels were significantly elevated to a comparable degree in both the TTR-Abcb11 mice (144.2 ± 13.9 IU) and FVB/NJ (161.2 ± 13.9 IU) controls. Histologically, TTR-Abcb11 mice also showed more inflammation after 14 days on the MCD diet (1.3 ± 0.5 IU), compared with FVB/NJ mice (0.8 ± 0.5 IU), P = 0.05. Elevated ALT levels persisted through 30 days in both TTR-Abcb11 (111.9 ± 13.9 IU) and FVB/NJ (102.3 ± 9.0 IU) mice. When fed a control diet for up to 30 days, both TTR-Abcb11 and FVB/NJ wild-type controls maintained normal serum ALT levels (39.6 ± 8.1 and 37.3 ± 7.2 IU, respectively) (see Fig. 1).



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Fig. 1. Serum aminotransferase (ALT) levels in mice fed the methionine-choline-deficient (MCD) diet. Transthyretin (TTR)-Abcb11 and wild-type FVB/NJ mice were fed an MCD diet for 2 wk, and serum ALT levels were measured. TTR-Abcb11 mice developed markedly elevated serum ALT levels after 7 days on the MCD diet. Values are means ± SE. *P = 0.005 compared with FVB/NJ mice.

 
The effect of the MCD diet on hepatic triglyceride and cholesterol levels. TTR-Abcb11 mice fed an MCD diet had lower hepatic triglyceride levels than FVB/NJ mice after 7 (0.16 ± 0.03 vs. 0.33 ± 0.06 mg/mg protein), 14 (0.2 ± 0.03 vs. 0.37 ± 0.06 mg/mg protein), and 30 days (0.34 ± 0.08 vs. 0.58 ± 0.2 mg/mg protein), P = 0.001. Consistent with this finding, histologically FVB/NJ mice also demonstrated more steatosis than TTR-Abcb11 mice at 7 (2.3 ± 0.5 vs. 1.7 ± 0.5 mg/mg protein) and 14 (3.0 ± 0.6 vs. 1.5 ± 0.5 mg/mg protein) days, P < 0.05 (see Fig. 2). As illustrated in Fig. 3, both mouse strains demonstrate progressive hepatic triglyceride deposition over time, results that we confirmed histologically (P < 0.05). Hepatic triglyceride content in mice fed a control chow diet for up to 30 days was minimal and similar in both TTR-Abcb11 (0.04 ± 0.006 mg/mg protein) and FVB/NJ (0.06 ± 0.02 mg/mg protein) mice. Serum triglyceride levels were unchanged.



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Fig. 2. Hepatic histology in mice fed control and MCD diet. A: normal hepatic architecture in FVB/NJ mice fed a control diet. B: normal hepatic architecture in TTR-Abcb11 mice fed a control diet. C: severe steatosis and mild inflammation in FVB/NJ mice fed an MCD diet for 14 days. D: mild steatosis and more severe inflammation in TTR-Abcb11 mice fed an MCD diet for 14 days.

 


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Fig. 3. Hepatic triglyceride content of mice fed the MCD diet. TTR-Abcb11 and FVB/NJ mice were fed an MCD diet for 2 wk, and hepatic triglyceride levels were measured. TTR-Abcb11 mice had lower hepatic triglyceride levels than wild-type mice fed the MCD diet. Values are means ± SE. *P = 0.001 compared with FVB/NJ mice.

 
FVB/NJ mice showed progressive increases in hepatic cholesterol content from baseline levels (0.03 ± 0.01 mg/mg protein) after both 7 days (0.05 ± 0.01 mg/mg protein) and 14 days (0.06 ± 0.01 mg/mg protein) on an MCD diet, P < 0.05. Hepatic cholesterol levels in TTR-Abcb11 mice, however, remained similar to baseline levels (0.04 ± 0.02 mg/mg protein) after either 7 days (0.05 ± 0.01 mg/mg protein) or 14 days (0.05 ± 0.01 mg/mg protein) on the MCD diet. Serum cholesterol levels were similar in chow-fed FVB/NJ (152.9 ± 33.1 mg/dl) and TTR-Abcb11 mice (175.3 ± 11.6 mg/dl). Serum cholesterol levels decreased after 7 days (127.1 ± 20.7 mg/dl) and 14 days (79.4 ± 12.7 mg/dl) on the MCD diet in FVB/NJ mice, P < 0.05. Similarly, serum cholesterol levels also decreased after 7 days (93.3 ± 13.3 mg/dl) and 14 days (95.7 ± 26.0 mg/dl) on the MCD diet in TTR-Abcb11 mice, P < 0.05.

Cytokine analysis using real-time PCR. Expression of TNF-{alpha} mRNA was assayed using real-time PCR. After 14 days on the MCD diet, TTR-Abcb11 mice demonstrated a threefold increase in TNF-{alpha} mRNA expression compared with FVB/NJ mice (P = 0.03). A 3.9-fold increase in IL-6 mRNA expression was also seen in TTR-Abcb11 compared with FVB/NJ mice after 14 days on the MCD diet. There was no significant difference in gene expression of both TNF-{alpha} and IL-6 mRNA at baseline in control diet-fed mice and in mice fed an MCD diet for only 7 days (see Table 1).


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Table 1. Hepatic gene expression of TTR-Abcb11 and FVB/NJ mice fed the MCD diet

 
The effect of the MCD diet on oxidative stress. Hepatic TBARS, glutathione levels, and expression of CYP2E1 were analyzed as markers of oxidative stress. Minimally elevated TBARS occurred at 7 days on the MCD diet in both FVB/NJ (0.76 ± 0.24 nM/mg protein) and TTR-Abcb11 (1.34 ± 0.68 nM/mg protein) mice (P = not significant). By 14 days on the MCD diet, however, FVB/NJ mice had significantly greater TBARS compared with both control-fed mice and TTR-Abcb11 mice fed an MCD diet. TBAR levels were 4.05 ± 0.32 nM/mg protein in FVB/NJ mice vs. 1.79 ± 0.4 nM/mg protein in TTR-Abcb11 mice (see Fig. 4). After 30 days on the MCD diet, both FVB/NJ and TTR-Abcb11 mice had significantly elevated TBARS with no strain-specific differences. TBARS in control diet-fed mice were similar and minimal in both FVB/NJ and TTR-Abcb11 mice fed a chow diet.



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Fig. 4. Hepatic thiobarbituric acid reactive substance (TBARS) in mice administered the MCD diet. TTR-Abcb11 and FVB/NJ mice were fed an MCD diet, and TBARS were measured. TTR-Abcb11 mice had lower hepatic TBARS than wild-type mice after 14 days on the MCD diet. Values are means ± SE. *P < 0.005 compared with FVB/NJ mice.

 
Both mouse strains showed dramatic increases in CYP2E1 protein expression over time (P < 0.0005). After 14 days on the MCD diet, FVB/NJ mice showed a twofold increase in CYP2E1 protein expression, whereas this was increased fourfold in TTR-Abcb11 mice. This pattern persisted at 30 days on the MCD diet. On the chow diet, hepatic CYP2E1 protein expression showed no strain-specific differences (see Fig. 5).



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Fig. 5. Cytochrome P-450 2E1 (CYP2E1) expression in TTR-Abcb11 and FVB/NJ mice fed an MCD diet. Protein densitometry was performed on Western blots examining CYP2E1 expression and was normalized for tubulin expression. Both mouse strains showed dramatic increases in CYP2E1 protein expression over time, although it was greater in TTR-Abcb11 mice. P < 0.001 by ANOVA.

 
Baseline hepatic glutathione levels in TTR-Abcb11 mice were higher (6.87 mM/mg protein) than in FVB/NJ mice (5.01 mM/mg protein), P < 0.05. Both mouse strains demonstrated minimal increases, rather than decreases, in hepatic glutathione over 30 days (see Fig. 6). The TTR-Abcb11 mice maintained higher levels of hepatic glutathione at all time points up to 30 days (P < 0.0001). Although increased glutathione production could account for these effects, we observed no corresponding increases of {gamma}-GCS enzymatic activity or protein expression.



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Fig. 6. Hepatic glutathione (GTH) levels in TTR-Abcb11 and FVB/NJ mice fed the MCD diet. TTR-Abcb11 and FVB/NJ mice were fed an MCD diet, and hepatic GTH levels were measured. FVB/NJ mice had lower GTH levels than TTR-Abcb11 mice at all time points. Values are means ± SE. P < 0.001 by ANOVA.

 
The effect of the MCD diet on SREBP-1c and FAS gene expression. SREBP-1c regulates many hepatic metabolic factors, including the rate of hepatic FAS (10, 16, 17, 34). After 7 days on the MCD diet, TTR-Abcb11 mice had a fourfold decrease in SREBP-1c mRNA expression (P < 0.001), and this effect persisted at 14 days (P = 0.002). Similar decreases of SREBP-1c mRNA expression were not observed in the FVB/NJ mice fed the MCD diet for up to 14 days. TTR-Abcb11 mice fed an MCD diet for 7 days had significantly lower SREBP-1c mRNA expression than FVB/NJ mice (P < 0.001). This difference between strains was not noted after 14 days of feeding of the MCD diet. Baseline expression of hepatic SREBP-1c mRNA was similar in FVB/NJ and TTR-Abcb11 mice fed a chow diet (see Table 1).

We also examined the expression of the SREBP-1c downstream target gene FAS. Consistent with SREBP-1c expression, after 7 days on the MCD diet, TTR-Abcb11 mice had a strong trend toward a decrease in FAS gene expression, with gene expression being reduced almost twofold (P = 0.07). By 14 days of MCD diet feeding, TTR-Abcb11 mice had a highly significant 2.6-fold decrease in FAS expression (P < 0.01). Baseline expression of hepatic FAS mRNA was similar in FVB/NJ and TTR-Abcb11 mice fed a chow diet (see Table 1).


    DISCUSSION
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The MCD diet induces the rapid development of hepatitis in TTR-Abcb11 transgenic mice, despite relative steatosis resistance. Both FVB/NJ strain controls and TTR-Abcb11 mice eventually manifest progressive hepatic triglyceride deposition, marked elevation of serum ALT, and histologically evident inflammation. A more pronounced increase in mRNA expression of both TNF-{alpha} and IL-6 was also observed in the TTR-Abcb11 mice at 1 and 2 wk compared with the FVB/NJ mice. In contrast, FVB/NJ mice exhibited elevated markers of oxidative stress sooner than the TTR-Abcb11 mice. A disassociation exists between the degree of oxidative stress and degree of early hepatitis. When followed over a longer period of time, however, both mouse strains show evidence of ongoing lipid peroxidation, as demonstrated by TBAR elevation and increased CYP2E1 protein expression. Alterations in FAS likely contribute to this phenomenon, as evidenced by decreased mRNA expression of SREBP-1c and FAS in the TTR-Abcb11 mice on the MCD diet.

TTR-Abcb11 mice fed an MCD diet demonstrate a relative resistance to steatosis. This is the second steatogenic dietary model in which the TTR-Abcb11 mouse shows resistance to steatosis (13, 31). Steatosis induced by the MCD diet provides the "first hit" in NASH pathogenesis. Choline is an essential substrate for PC synthesis, the primary phospholipid in liver and bile. The MCD diet likely induces hepatic injury by restricting PC synthesis via the Kennedy pathway and preventing rescue by the phosphatidylethanolamine N-methyltransferase pathway. In fact, phosphatidylethanolamine N-methyltransferase knockout mice fed a choline-deficient diet develop lethal hepatic injury and failure (1). TTR-Abcb11 mice hypersecrete PC and thus have an enhanced need for PC synthesis. They may be particularly susceptible to early injury and hepatocyte damage when placed on an MCD diet. Our observations further emphasize the critical role of phospholipid metabolism in hepatocyte viability.

Abcb11 encodes for the liver BSEP, the major canalicular bile salt transporter (28, 41). TTR-Abcb11 mice not only overexpress BSEP, but have increased hydrophobicity of the bile salt pool and enhanced secretion of bile salts into bile that is coupled with increases in biliary phospholipids and cholesterol secretion (5, 13, 44). The increased PC excretion in this transgenic strain would be expected to increase dependence on PC synthesis to maintain hepatocyte stores. Unfortunately, we were technically unable to measure biliary PC secretion in TTR-Abcb11 mice on the MCD diet. Our observation that TTR-Abcb11 mice develop increased ALT levels more rapidly than control mice on the MCD diet serves to highlight the important role of PC metabolism in the pathogenesis of experimental steatohepatitis.

Hepatic steatosis is likely affected by SREBPs, transcription factors that regulate enzymes needed for fatty acid, triglyceride, and cholesterol synthesis (16). SREBP-1c controls the rate of hepatic FAS (10, 16, 17, 34). Overexpression of SREBP-1c enhances FAS and induces lipogenic enzymes like FAS that may control triglyceride accumulation and result in fatty liver disease (36, 3739, 48). FAS expression is downregulated by higher levels of triglyceride accumulation. The hepatic steatosis and elevated hepatic triglyceride levels evident in the TTR-Abcb11 mice in our study correlate well with downregulation of the regulatory genes SREBP-1c and FAS mRNA. This suggests the physiological importance of a negative feedback system. This negative feedback likely works in conjunction with insulin to affect SREBP-1c expression (4).

Multiple studies have underscored the importance of inflammatory cytokine signaling and stimulation in the pathogenesis of NASH through mediation of hepatic inflammation, apoptosis, and necrosis (6, 9, 12, 15, 27, 29, 50). Patients with NASH demonstrate increased hepatic and serum TNF-{alpha} expression, a proinflammatory cytokine (8, 22). Experimental treatments against TNF-{alpha} or receptor deletions are known to attenuate the NASH disease process (18, 24, 50). Additionally, rodents on the MCD diet have higher TNF-{alpha} levels and are more sensitive to endotoxin-mediated injury (6). In our experimental model, TTR-Abcb11 mice fed the MCD diet demonstrated threefold increases in TNF-{alpha} RNA expression compared with wild-type controls. TNF-{alpha} may also trigger the production of other cytokines, such as IL-6 (15). The higher levels of cytokine expression resulting from 7 days compared with 14 days of MCD diet feeding may occur because, in this nutritional model, cytokines may be primarily elevated during the initial phases of inflammation. The upregulation of hepatic TNF-{alpha} and IL-6 mRNA seen in the TTR-Abcb11 mouse likely reflects the importance of this cytokine-mediated injury.

The substantial rise in TBARS seen after 14 days in the FVB/NJ mice and both strains after 30 days on the MCD diet emphasizes the role of lipid peroxidation in hepatic injury. It is not surprising that the TTR-Abcb11 mice initially manifest lower TBARS than their wild-type strain controls because they have less substrate (triglyceride) available for lipid peroxidation. In addition, there may be disassociation between the degree of steatosis and the severity of hepatitis (19). The TTR-Abcb11 mice, however, manifest significant hepatitis without concurrent elevations in TBARS after 7 days on the MCD diet. These data demonstrate a novel disassociation between the severity of hepatitis and the degree of oxidative stress.

NASH shares many pathophysiological characteristics with alcoholic steatohepatitis. In alcoholic steatohepatitis, evidence exists that upregulation of CYP2E1 results in lipid peroxidation and resultant accumulation of reactive oxygen species (7, 11, 30). Several studies have underscored the role of CYP2E1 in the pathogenesis of steatohepatitis (7, 23, 45, 46). Leclercq et al. (23) have further emphasized the importance of microsomal enzymes in the development of NASH by demonstrating their induction in a dietary model of NASH. The significant increase in CYP2E1 observed in TTR-Abcb11 mice fed an MCD diet is consistent with the role of CYP2E1 in the pathogenesis of steatohepatitis.

As reactive oxygen species are generated, antioxidant defense by glutathione may occur through scavenging of free radicals, and removing lipid peroxides (47). The imbalance of prooxidants, like microsomal CYPE2E1, ROS release from mitochondria, and antioxidants like glutathione, results in hepatic damage (7). A reduction in glutathione may contribute to hepatic damage caused by oxidative stress, abrogated by secondary insults from cytokines such as TNF-{alpha} and IL-6. In our MCD dietary model of NASH, the expected decrease in glutathione activity or functional protein expression did not occur. The known antioxidant properties of glutathione could be responsible for the attenuated inflammation and injury seen in this model.

The TTR-Abcb11 transgenic mouse hypersecretes biliary lipids and is resistant to steatosis. It provides an animal model with which to investigate the pathogenic mechanisms of NASH. Our study is consistent with the observation in humans that the absolute amount of fat is not primarily responsible for the transition from bland hepatic steatosis to steatohepatitis. Rather, beyond a certain threshold of steatosis required for injury, the second hit becomes central to NASH development and progression. Therapy directed toward these latter pathological mechanisms could enhance the care of patients with NASH.


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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-59580 (to R. M. Green).


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. M. Green, Associate Professor of Medicine, Northwestern Univ., The Feinberg School of Medicine, 303 East Chicago Ave., Tarry Bldg. 14-711, Chicago, IL 60611 (E-mail: r-green2{at}northwestern.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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  1. Agellon LB, Walkey CJ, Vance DE, Kuipers F, and Verkade HJ. The unique acyl chain specificity of biliary phosphatidylcholines in mice is independent of their biosynthetic origin in the liver. Hepatology 30: 725–729, 1999.[CrossRef][ISI][Medline]
  2. Applied Biosystems. Comparative CT method for relative quantification. In: Sequence Detection Systems Chemistry Guide. Foster City, CA: Applied Biosystems, sect. 3.2, p. 3–35 and 33–39.
  3. Angulo P. Nonalcoholic fatty liver disease. N Engl J Med 346: 1221–1231, 2002.[Free Full Text]
  4. Bobard A, Hainault I, Ferre P, Foufelle F, and Bossard P. Differential regulation of sterol regulatory element-binding protein 1c transcriptional activity by insulin and liver X receptor during liver development. J Biochem 280: 199–206, 2005.
  5. Carey MC. The Liver: Biology and Pathobiology. New York: Raven, 1994.
  6. Chawla RK, Watson WH, Eastin CE, Lee EY, Schmidt J, and McClain CJ. S-adenosylmethionine deficiency and TNF-{alpha} in lipopolysaccharide-induced hepatic injury. Am J Physiol Gastrointest Liver Physiol 275: G125–G129, 1998.[Abstract/Free Full Text]
  7. Chitturi S and Farrell GC. Etiopathogenesis of nonalcoholic steatohepatitis. Semin Liver Dis 21: 27–41, 2001.[CrossRef][ISI][Medline]
  8. Crespo J, Cayon A, Fernandez-Gil P, Hernandez-Guerra M, Mayorga M, Dominguez-Diez A, Fernandez-Escalante JC, and Pons-Romero F. Gene expression of tumor necrosis factor alpha and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients. Hepatology 34: 1158–1163, 2001.[CrossRef][ISI][Medline]
  9. Eastin CE, McClain CJ, Lee EY, Bagby GJ, and Chawla RK. Choline deficiency augments and antibody to tumor necrosis factor-alpha attenuates endotoxin-induced hepatic injury. Alcohol Clin Exp Res 21: 1037–1041, 1997.[ISI][Medline]
  10. Edwards PA, Tabor D, Kast HR, and Venkateswaran A. Regulation of gene expression by SREBP and SCAP. Biochim Biophys Acta 1529: 103–113, 2000.[ISI][Medline]
  11. Ekstrom G and Ingelman-Sundberg M. Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1). Biochem Pharmacol 38: 1313–1319, 1989.[CrossRef][ISI][Medline]
  12. Fernandez-Checa JC, Kaplowitz N, Garcia-Ruiz C, Colell A, Miranda M, Mari M, Ardite E, and Morales A. GSH transport in mitochondria: defense against TNF-induced oxidative stress and alcohol-induced defect. Am J Physiol Gastrointest Liver Physiol 273: G7–G17, 1997.[Abstract/Free Full Text]
  13. Figge A, Lammert F, Paigen B, Henkel A, Matern S, Korstanje R, Shneider BL, Chen F, Stoltenberg E, Spatz K, Hoda F, Cohen DE, and Green RM. Hepatic overexpression of murine Abcb11 increases hepatobiliary lipid secretion and reduces hepatic steatosis. J Biol Chem 279: 2790–2799, 2004.[Abstract/Free Full Text]
  14. Green RM. NASH–hepatic metabolism and not simply the metabolic syndrome. Hepatology 38: 14–17, 2003.[CrossRef][ISI][Medline]
  15. Hill MR and McCallum RE. Identification of tumor necrosis factor as a transcriptional regulator of the phosphoenolpyruvate carboxykinase gene following endotoxin treatment of mice. Infect Immun 60: 4040–4050, 1992.[Abstract]
  16. Horton JD, Goldstein JL, and Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 109: 1125–1131, 2002.[Free Full Text]
  17. Horton JD and Shimomura I. Sterol regulatory element-binding proteins: activators of cholesterol and fatty acid biosynthesis. Curr Opin Lipidol 10: 143–150, 1999.[CrossRef][ISI][Medline]
  18. Iimuro Y, Gallucci RM, Luster MI, Kono H, and Thurman RG. Antibodies to tumor necrosis factor alpha attenuate hepatic necrosis and inflammation caused by chronic exposure to ethanol in the rat. Hepatology 26: 1530–1537, 1997.[ISI][Medline]
  19. Ip E, Farrell GC, Robertson G, Hall P, Kirsch R, and Leclercq I. Central role of PPAR alpha-dependent hepatic lipid turnover in dietary steatohepatitis in mice. Hepatology 38: 123–132, 2003.[CrossRef][ISI][Medline]
  20. Koppe SW, Sahai A, Malladi P, Whitington PF, and Green RM. Pentoxifylline attenuates steatohepatitis induced by the methionine choline deficient diet. J Hepatol 41: 592–598, 2004.[CrossRef][ISI][Medline]
  21. Koteish A and Diehl AM. Animal models of steatosis. Semin Liver Dis 21: 89–104, 2001.[CrossRef][ISI][Medline]
  22. Kugelmas M, Hill DB, Vivian B, Marsano L, and McClain CJ. Cytokines and NASH: a pilot study of the effects of lifestyle modification and vitamin E. Hepatology 38: 413–419, 2003.[CrossRef][ISI][Medline]
  23. Leclercq IA, Farrell GC, Field J, Bell DR, Gonzalez FJ, and Robertson GR. CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis. J Clin Invest 105: 1067–1075, 2000.[Abstract/Free Full Text]
  24. Li Z, Yang S, Lin H, Huang J, Watkins PA, Moser AB, Desimone C, Song XY, and Diehl AM. Probiotics and antibodies to TNF inhibit inflammatory activity and improve nonalcoholic fatty liver disease. Hepatology 37: 343–350, 2003.[CrossRef][ISI][Medline]
  25. Marchesini G, Bugianesi E, Forlani G, Cerrelli F, Lenzi M, Manini R, Natale S, Vanni E, Villanova N, Melchionda N, and Rizzetto M. Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology 37: 917–923, 2003.[CrossRef][ISI][Medline]
  26. Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, and McCullough AJ. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 116: 1413–1419, 1999.[ISI][Medline]
  27. McClain CJ, Hill DB, Song Z, Chawla R, Watson WH, Chen T, and Barve S. S-adenosylmethionine, cytokines, and alcoholic liver disease. Alcohol 27: 185–192, 2002.[CrossRef][ISI][Medline]
  28. Meier PJ and Stieger B. Bile salt transporters. Annu Rev Physiol 64: 635–661, 2002.[CrossRef][ISI][Medline]
  29. Mizuhara H, O'Neill E, Seki N, Ogawa T, Kusunoki C, Otsuka K, Satoh S, Niwa M, Senoh H, and Fujiwara H. T cell activation-associated hepatic injury: mediation by tumor necrosis factors and protection by interleukin 6. J Exp Med 179: 1529–1537, 1994.[Abstract/Free Full Text]
  30. Nanji AA, Zhao S, Sadrzadeh SM, Dannenberg AJ, Tahan SR, and Waxman DJ. Markedly enhanced cytochrome P450 2E1 induction and lipid peroxidation is associated with severe liver injury in fish oil-ethanol-fed rats. Alcohol Clin Exp Res 18: 1280–1285, 1994.[ISI][Medline]
  31. Rinella MGR. TTR-Abcb11 transgenic mice fed a high fat diet are obese, insulin resistant and develop hepatic steatosis (Abstract). Hepatology 38: 703, 2003.[ISI][Medline]
  32. Sahai A, Malladi P, Melin-Aldana H, Green RM, and Whitington PF. Upregulation of osteopontin expression is involved in the development of nonalcoholic steatohepatitis in a dietary murine model. Am J Physiol Gastrointest Liver Physiol 287: G264–G273, 2004.[Abstract/Free Full Text]
  33. Sahai A, Malladi P, Pan X, Paul R, Melin-Aldana H, Green RM, and Whitington PF. Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin. Am J Physiol Gastrointest Liver Physiol 287: G1035–G1043, 2004.[Abstract/Free Full Text]
  34. Sakakura Y, Shimano H, Sone H, Takahashi A, Inoue N, Toyoshima H, Suzuki S, Yamada N, and Inoue K. Sterol regulatory element-binding proteins induce an entire pathway of cholesterol synthesis. Biochem Biophys Res Commun 286: 176–183, 2001.[CrossRef][ISI][Medline]
  35. Sekura R and Meister A. {gamma}-Glutamylcysteine synthetase. Further purification, "half of the sites" reactivity, subunits, and specificity. J Biol Chem 252: 2599–2605, 1977.[Abstract]
  36. Shimano H, Horton JD, Hammer RE, Shimomura I, Brown MS, and Goldstein JL. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a. J Clin Invest 98: 1575–1584, 1996.[Abstract/Free Full Text]
  37. Shimano H, Horton JD, Shimomura I, Hammer RE, Brown MS, and Goldstein JL. Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells. J Clin Invest 99: 846–854, 1997.[Abstract/Free Full Text]
  38. Shimomura I, Bashmakov Y, and Horton JD. Increased levels of nuclear SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus. J Biol Chem 274: 30028–30032, 1999.[Abstract/Free Full Text]
  39. Shimomura I, Shimano H, Korn BS, Bashmakov Y, and Horton JD. Nuclear sterol regulatory element-binding proteins activate genes responsible for the entire program of unsaturated fatty acid biosynthesis in transgenic mouse liver. J Biol Chem 273: 35299–35306, 1998.[Abstract/Free Full Text]
  40. Taussky HH and Shorr E. A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem 202: 675–685, 1953.[Free Full Text]
  41. Trauner M and Boyer JL. Bile salt transporters: molecular characterization, function, and regulation. Physiol Rev 83: 633–671, 2003.[Abstract/Free Full Text]
  42. Vance DE and Walkey CJ. Roles for the methylation of phosphatidylethanolamine. Curr Opin Lipidol 9: 125–130, 1998.[CrossRef][ISI][Medline]
  43. Walkey CJ, Yu L, Agellon LB, and Vance DE. Biochemical and evolutionary significance of phospholipid methylation. J Biol Chem 273: 27043–27046, 1998.[Abstract/Free Full Text]
  44. Wang DQ, Lammert F, Paigen B, and Carey MC. Phenotypic characterization of lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice. Pathophysiology of biliary lipid secretion. J Lipid Res 40: 2066–2079, 1999.[Abstract/Free Full Text]
  45. Weltman MD, Farrell GC, Hall P, Ingelman-Sundberg M, and Liddle C. Hepatic cytochrome P450 2E1 is increased in patients with nonalcoholic steatohepatitis. Hepatology 27: 128–133, 1998.[CrossRef][ISI][Medline]
  46. Weltman MD, Farrell GC, and Liddle C. Increased hepatocyte CYP2E1 expression in a rat nutritional model of hepatic steatosis with inflammation. Gastroenterology 111: 1645–1653, 1996.[ISI][Medline]
  47. Wu G, Fang YZ, Yang S, Lupton JR, and Turner ND. Glutathione metabolism and its implications for health. J Nutr 134: 489–492, 2004.[Abstract/Free Full Text]
  48. Yahagi N, Shimano H, Hasty AH, Matsuzaka T, Ide T, Yoshikawa T, Amemiya-Kudo M, Tomita S, Okazaki H, Tamura Y, Iizuka Y, Ohashi K, Osuga J, Harada K, Gotoda T, Nagai R, Ishibashi S, and Yamada N. Absence of sterol regulatory element-binding protein-1 (SREBP-1) ameliorates fatty livers but not obesity or insulin resistance in Lep(ob)/Lep(ob) mice. J Biol Chem 277: 19353–19357, 2002.[Abstract/Free Full Text]
  49. Yang SQ, Lin HZ, Lane MD, Clemens M, and Diehl AM. Obesity increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis. Proc Natl Acad Sci USA 94: 2557–2562, 1997.[Abstract/Free Full Text]
  50. Yin M, Wheeler MD, Kono H, Bradford BU, Gallucci RM, Luster MI, and Thurman RG. Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice. Gastroenterology 117: 942–952, 1999.[ISI][Medline]




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