Lipid peroxidation, stellate cell activation and hepatic fibrogenesis in a rat model of chronic steatohepatitis

Journal of Hepatology 39 (2003) 756–764 www.elsevier.com/locate/jhep

Lipid peroxidation, stellate cell activation and hepatic fibrogenesis in a rat model of chronic steatohepatitis Jacob George*, Natasha Pera, Nghi Phung, Isabelle Leclercq, Jing Yun Hou, Geoffrey Farrell Storr Liver Unit, Westmead Millennium Institute, University of Sydney, Westmead Hospital, Sydney, New South Wales, Australia

Background/Aims: We explored the involvement of cell types, cytokines and lipid peroxidation in a rat dietary model of fibrosing steatohepatitis. Methods: Male rats were fed a high fat diet deficient in methionine and choline (MCD) for up to 17 weeks. Whole liver, hepatocytes and non-parenchymal cells were analysed for reduced glutathione (GSH) levels, products of lipid peroxidation (thiobarbituric acid reactive substances, TBARS), liver injury, and fibrosis. Results: MCD diet-fed rats developed hepatic steatosis at week 2 and focal necroinflammatory change by week 5, while pericellular fibrosis evolved and progressed between weeks 12 and 17. Collagen a1(1) gene expression was upregulated by week 5 and increased fivefold by week 17. Stellate cells were the unique source of collagen gene expression. TIMP-1 and -2 were increased at week 12. Livers of MCD diet-fed rats exhibited lowered levels of GSH and elevated TBARS. Hepatocytes were the source of lipid peroxidation, and mRNA levels for TGFb1 were increased only in this cell type. Conclusions: The MCD model of ‘fibrosing steatohepatitis’ replicates the histologic features of human steatohepatitis, and the sequence of steatosis, inflammatory cell injury and fibrogenesis. The temporal sequence is consistent with a concept for involvement of oxidative injury in inflammatory recruitment and pathogenesis of hepatic fibrogenesis. q 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Steatohepatitis; Hepatic fibrosis; Lipid peroxidation; Collagen; Stellate cells; Hepatocytes; Transforming growth factor-beta

1. Introduction Liver injury resulting from metabolic disorders is common and is exemplified by non-alcoholic steatohepatitis (NASH) in individuals with truncal obesity, type 2 diabetes, hypertriglyceridemia [1 –3], and other features of the insulin resistance syndrome. In all forms of Received 17 December 2002; received in revised form 11 June 2003; accepted 8 July 2003 * Corresponding author. Department of Medicine, Westmead Hospital, Sydney, NSW 2145, Australia. Tel.: þ61-2-9845-7705; fax: þ 61-2-96357582. E-mail address: [email protected] (J. George). Abbreviations: a-SMA, alpha smooth muscle actin; ALT, alanine aminotransferase; cDNA, complementary DNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSH, reduced glutathione; MCD, methionine and choline deficient; mRNA, messenger RNA; NASH, non-alcoholic steatohepatitis; PCR, polymerase chain reaction; TBARS, thiobarbituric acid reactive substances; TGFb, transforming growth factor beta; TIMP, tissue inhibitor of metalloproteinases.

steatohepatitis, as well as in alcoholism and jejuno – ileal bypass, hepatic lipid accumulation is associated with oxidative stress and lipid peroxidation, processes that cause hepatocellular injury and mediate fibrogenesis. However, while there has recently been an explosion of interest about why hepatic steatosis develops in humans, little is known about the mechanistic basis for the clinically most important outcome, progressive hepatic fibrosis [4,5]. There are few animal models that replicate the pathology and chronicity of fibrosing human liver diseases. We report here the characterisation of a chronic (17 weeks) rodent model of hepatic fibrosis, which follows ingestion of a high-fat diet deficient in methionine and choline (MCD). The model is associated pathologically with steatohepatitis and a pattern of perivenous and pericellular hepatic fibrosis around lipid-laden hepatocytes; the latter features are found in all causes of

0168-8278/$30.00 q 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0168-8278(03)00376-3

J. George et al. / Journal of Hepatology 39 (2003) 756–764

steatohepatitis in humans. In this model, we have now characterised hepatocytes as the site of lipid peroxidation that develops during the evolution of hepatic fibrosis. This precedes fibrogenesis, and is associated with up-regulation of TGFb1 at the mRNA level, and activation of hepatic stellate cells that were confirmed to be the unique cell type elaborating matrix components.

2. Materials and methods 2.1. Animals and diet This study was approved by the Animal Ethics Committee and all procedures complied with international standards of humane care in animal experimentation. Male Sprague– Dawley rats weighing at least 400 g (ARC, Canning Vale, Australia) were maintained on a 12-h light/dark cycle, under conditions of constant temperature (22 8C) and humidity. They were fed ad libitum a high-fat, methionine and choline deficient (MCD) diet (ICN Biomedicals, Sydney, Australia; catalogue number: 960439) for up to 17 weeks. Controls were pair-fed the same diet supplemented with choline chloride (2 g/kg) and DL -methionine (3 g/kg). Animals were weighed daily; coat appearance, activity and other aspects of well being were noted.

2.2. Tissue preparation

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2.2.4. Histology Five-micron sections were deparaffinised, stained with haematoxylin and eosin (H&E), and examined to determine the degree of steatosis and inflammation. Fibrosis was assessed following incubation for 30 min in a solution of saturated picric acid containing 0.1% direct red 80 (Sirius Red F3B) and 0.1% Fast Green FCF (Sigma Aldrich, Sydney, Australia) [8]. Expression of a-smooth muscle actin (a-SMA) by activated stellate cells was determined by incubating sections with a mouse monoclonal anti-aSMA primary antibody (1:400 dilution, clone 1A4, Sigma), followed by detection using a mouse ABC staining kit (Santa Cruz Biotechnology, Santa Cruz, USA).

2.2.5. Collagen and a-smooth muscle actin quantitation in liver sections Collagen deposition in Sirius red-stained sections was quantitated by morphometry as a percentage (%) of the total cross-sectional surface area occupied by collagen using Optimas 6.5 software (Media Cybernetics L.P., Silver Spring, USA). Ten random fields centred on the central vein were examined at 100 £ magnification for each animal. a-SMA in immunostained sections was similarly assessed by examining ten random fields at 400 £ magnification.

2.2.6. Biochemical analyses Serum alanine aminotransferase (ALT) levels were measured by the Department of Clinical Chemistry, Westmead Hospital, using automated techniques. Thiobarbituric acid reactive substances (TBARS) were assessed using the method of Ohkawa et al. [9]. Reduced glutathione (GSH) levels were determined according to Hissin and Hilf [10]. TBARS levels from cell isolates were standardised against protein concentrations (DC Protein assay, Bio-Rad Laboratories, Hercules, USA).

2.2.1. Blood and whole liver Rats were anaesthetised with ketamine (10 mg/100 g body wt.) and xylazine (0.4 mg/100 g body wt.). Blood was collected from the inferior vena cava, serum was prepared and aliquots stored at 270 8C until analysis. Livers were perfused with L-15 salts solution (137 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 0.44 mM KH2PO4, pH 7.4), excised and cut into small pieces. Some tissue was fixed in 4% buffered formaldehyde, embedded in paraffin and used for histological analysis. The remainder was snap frozen and stored at 270 8C.

2.2.2. Isolation of rat hepatocytes Hepatocytes were isolated by collagenase perfusion [6]. After perfusion, the liver was excised, minced and passed through a 100-mm nylon mesh. Hepatocytes were washed three times in L-15 salts and separated from non-parenchymal cells by centrifugation at 50 £ g for 2 min. In all preparations, the viability and purity of cells, as determined by microscopy and trypan blue exclusion, was .95%. Cell aliquots were snap frozen and stored at 270 8C until analysis.

2.2.3. Isolation of hepatic non-parenchymal cells Non-parenchymal cells were isolated by in situ perfusion with pronase and collagenase (Roche, Sydney, Australia), as previously described [7]. The cell pellet was resuspended in Joklik-modified minimum essential medium (Gibco BRL, Melbourne, Australia), loaded onto a discontinuous gradient of arabinogalactan (Sigma Aldrich, Sydney, Australia), and centrifuged for 35 min at 20,000 rpm (Beckman SW-28 rotor). Stellate cells were collected from the top four layer interfaces to obtain a representative population of stellate cells with different fat content. Kupffer and sinusoidal endothelial cells from the bottom two-layer interfaces were separated by centrifugal elutriation using a J-6M/E centrifuge with a JE 5.0 rotor (Beckman Instruments Inc., Palo Alto, USA) [6]. To characterise the identity of each cell type, an aliquot was cultured for 48 h. Stellate cells were identified by their autofluorescence (purity .98%), Kupffer cells by latex bead phagocytosis (purity .95%) and endothelial cells by their cobblestone morphology (purity .90%) and presence of fenestrae on electron microscopy. The remainder of each cell pellet was resuspended in TRIzol reagent (Gibco BRL, Melbourne, Australia) or snap frozen and stored at 270 8C until analysis.

2.2.7. Determination of collagen a1(1), TIMP-1 and TIMP-2 mRNA levels Total RNA was extracted from whole liver tissue or cell pellets using TRIzol reagent. The integrity of the RNA was confirmed by visualising the 18S and 28S ribosomal fragments on an agarose/MOPS gel. mRNA species were quantified by RNase protection. Antisense riboprobes against TIMP1, TIMP-2 (kindly provided by Professor John Iredale, University of Southampton, UK) and collagen a1(1) were prepared [11] and labelled using [a-32P] cytidine-50 -triphosphate (CTP, .800 Ci/mmol, AmershamPharmacia Biotech, Sydney, Australia). The TIMP-1 [12] probe protected a 0.65-kb fragment, TIMP-2 [13] a 0.97-kb fragment and the collagen a1(1) [14] probe a 0.35-kb fragment. To control for mRNA loading, samples were assayed for S14 mRNA [15]. Probes were hybridised for 16–18 h with either 30 mg (TIMP-1 and TIMP-2), 20 mg (collagen-a1(1)) or 5 mg (S14) of total RNA. Negative controls consisting of equivalent amounts of yeast tRNA (Gibco BRL, Melbourne, Australia), were included with each assay. The hybridisation temperatures were 42 8C for TIMP-1 and TIMP-2, 70 and 55 8C for collagen a1(1) and S14, respectively. After hybridisation and RNase digestion, protected RNA–cRNA hybrid fragments were separated on denaturing polyacrylamide gels, dried and autoradiographed. Protected fragments for each mRNA were quantified using scanning densitometry and ImageQuante software (Molecular Dynamics, Sunny Vale, USA), and normalised against S14.

2.2.8. Determination of levels of TGFb1 mRNA by reverse transcription- and real time-PCR Five micrograms of total RNA from whole liver, hepatocytes or nonparenchymal cells was reverse transcribed using random hexamers (Promega Corporation, Madison, USA), and SuperScript II RNase HReverse Transcriptase (Gibco BRL, Melbourne, Australia). Reverse transcription-PCR was used to visualise differences in TGFb1 mRNA expression in whole liver at different time points. TGFb1 (285 bp; TGFb1 forward, 50 -ATGCTAAAGAGGTCACCC-30 ; TGFb1 reverse: 5 0 CAAAAGACAGCCACTCAG-30 ) was amplified together with a fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 191 bp; GAPDH forward, 50 -AACGACCCCTTCATTGAC-30 ; GAPDH reverse, 5 0 TCCACGACATACTCAGCAC-30 ) over 32 cycles using an annealing temperature of 56 8C. The PCR reaction mixture contained cDNA

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corresponding to 100 ng of total RNA, 1 mM of each specific primer, 0.75 mM MgCl2 and 2 units of Red-Hot DNA polymerase (Advanced Biotechnologies, Epsom, UK). PCR products were separated on a 1.5% agarose gel containing ethidium bromide. Levels of TGFb1 mRNA expression in whole liver and cells isolated at 12 weeks were quantified by real time-PCR using the PE Applied Biosystems (Foster City, USA) Prism 7700 real-time PCR platform. TGFb1 mRNA was amplified from cDNA using SYBRw Green PCR Master Mix (PE Applied Biosystems) and the TGFb1 forward primer, 50 GCCCGAAGCGGACTACTATG-30 and TGFb1 reverse primer, 50 AGATGGCGTTGTTGCGGT-30 (Genbank accession number X52498). Results were normalised against signals for GAPDH which were detected using TaqManw Universal PCR Master Mix (PE Applied Biosystems), the GAPDH forward primer, 50 -GTCGTGGATCTGACGTGCC-30 ; the GAPDH reverse primer, 50 -TGCCTGCTTCACCACCTTCT-30 ; and the fluorogenic TaqManw probe 50 -VIC-CCTGGAGAAACCTGCCAAGTATGATGACA-TAMRA-30 . Cycle parameters were 50 8C for 2 min, then 95 8C for 10 min, followed by 50 cycles of 95 8C for 15 s and 60 8C for 1 min.

(Fig. 2B), macrovesicular steatosis affected zones 2 and 3 with relative sparing of zone 1 (periportal) hepatocytes and foci of mixed inflammatory cell infiltration and hepatocyte necrosis appeared throughout the lobule. Conspicuous inflammation persisted during the subsequent period of MCD feeding (Fig. 2B, inset). 3.2. Steatohepatitis associated with feeding rats a MCD diet causes progressive, pericellular and pericentral fibrosis

3. Results

By week 12, steatohepatitis in MCD diet-fed rat liver was associated with pronounced hepatic fibrosis (n ¼ 9). It was most conspicuous in zone 3, conforming to a pericellular, ‘chicken-wire’ pattern. In Fig. 2D it is evident that fibrotic tendrils surround lipid-laden zone 3 hepatocytes. By weeks 12 and 17, portal–portal and central–portal bridging fibrosis were established (Fig. 2E); macronodular cirrhosis was present in one of seven animals examined at week 17 (Fig. 2F). Quantitation of collagen in Sirius red-stained sections confirmed that the observed fibrosis at week 12 corresponded to a significant increase in collagen protein (Fig. 2G).

3.1. Effects of feeding the MCD diet on body weight, liver enzymes, and hepatic pathology

3.3. Stellate cell activation, and hepatic expression of pro-fibrogenic genes

As previously reported [16], MCD diet-fed rats lost weight compared to isocaloric pair-fed controls (respectively, 32 ^ 1% vs. 3 ^ 2% of initial body weight by week 17), but appeared well and remained active. By week 5, serum ALT levels were elevated threefold in MCD diet-fed rats (Fig. 1); this increase persisted during the 17-week study protocol. Liver morphology remained normal in isocalorically fed control rats (Fig. 2A). In contrast, livers from rats fed the MCD diet showed steatosis by week 2. By week 5

Livers of MCD-fed rats exhibited stellate cell activation at 12 and 17 weeks. Thus a-SMA staining was conspicuously greater than that in control rats at these times (Fig. 3). Furthermore, levels of collagen a1(1) mRNA in whole liver increased . 3-fold (P , 0:01) compared with pair-fed controls at week 5, and rose further by weeks 12 and 17 (Fig. 4). Expression of TIMP-1 and TIMP-2 mRNA species increased later. At week 12, TIMP-1 and TIMP-2 mRNA levels were at least twofold increased in livers from MCDfed rats compared to controls (P , 0:05, Figs. 5 and 6).

2.2.9. Statistical analysis Data are represented as means ^ S.E.M. Differences between treatment and control groups were analysed using the unpaired Student’s t-test, or ANOVA (with appropriate post hoc analysis) for multiple comparisons. Values of P , 0:05 were considered significant.

3.4. Cellular source of pro-fibrogenic gene expression in fibrosing steatohepatitis To determine the cellular source of increased hepatic levels of collagen a1(1) mRNA, we isolated hepatic non-parenchymal cells at weeks 12 and 17. As shown in Fig. 7, collagen a1(1) mRNA was increased 12- and 15-fold in stellate cells at weeks 12 and 17, respectively. There was no significant upregulation of collagen a1(1) in Kupffer and endothelial cells (Fig. 7). TIMP-1 was likewise increased in stellate cells isolated from MCD diet-fed rats (P , 0:05, Fig. 8). 3.5. Expression of TGFb mRNA during MCD dietary feeding Fig. 1. Serum ALT in rats fed the MCD diet (B) and their pair-fed controls (A). Values are expressed as mean 6 S.E.M. for between three and six separate experiments. *P < 0.01. By post hoc analysis, ALT levels in rats fed the MCD diet for 2 weeks were lower (P < 0.05) than at other time points.

The temporal profile of whole liver TGFb1 mRNA expression during intake of the MCD diet shows apparent upregulation by week 5 (Fig. 9); values were significantly

J. George et al. / Journal of Hepatology 39 (2003) 756–764

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Fig. 2. Representative liver sections and collagen quantitation in rats fed the control or MCD diet. (A) Rat fed the control diet for 5 weeks. H&E (original magnification, 60 3 ). (B) Rat fed the MCD diet for 5 weeks. H&E (60 3 ). Steatosis is present predominantly in zones 2 and 3 and there is a mixed inflammatory cell infiltrate in the hepatic lobule (see inset, 400 3 , arrows).(C) Rat fed the control diet for 17 weeks. Sirius red staining (60 3 ). There is no fibrosis. (D) Rat fed the MCD-diet for 12 weeks. Sirius red staining (400 3 ). Peri-cellular ‘chicken-wire’ fibrosis is evident around zone 3 hepatocytes (arrow). Central vein is indicated by CV. (E) Rat fed the MCD-diet for 12 weeks. Sirius red staining (60 3 ). Portal-portal and central portal bridging fibrosis is evident (arrows). (F) Rat fed the MCD-diet for 17 weeks, Sirius red staining (60 3 ). In this liver, cirrhosis is present. (G) Quantitation of collagen deposition in Sirius red-stained sections of rats fed the MCD diet (B) and their pair-fed controls (A). Values are expressed as mean 6 S.E.M. for between three and four separate experiments. *P < 0.05. †The difference between MCD and control rats at week 17 was significant (P 5 0.03) after exclusion of the cirrhotic rat liver sample.

Fig. 3. a-Smooth muscle actin (a-SMA) immunostaining in liver sections. (A) Rat fed the control diet for 17 weeks (original magnification, 200 3 ). a-SMA expression, indicated by the brown staining, is confined to the portal tracts (PT) and central vein (CV). (B) Rat fed the MCD-diet for 12 weeks (200 3 ). a-SMA is also expressed in the hepatic lobule (arrows), distributed around steatotic hepatocytes, in the same distribution as collagen fibrils (see Fig. 2D). (C) Rat fed the MCD-diet for 17 weeks (200 3 ). In this liver, cirrhosis is present and smooth muscle actin staining is more extensive in the lobule. (D) Rat fed the MCD-diet for 17 weeks (400 3 ). Higher power view of the cirrhotic liver lobule. (E) Quantitation of a-smooth muscle actin in liver lobules of rats fed the MCD diet (B) and their pair-fed controls (A). Values are expressed as mean 6 S.E.M. for three separate experiments. *P < 0.05.

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Fig. 4. Expression of collagen a1(I) in whole liver of rats fed the MCD diet (B) and their pair-fed controls (A). (A) A representative RNase protection assay showing samples at 12 and 17 weeks, using 20 mg of total RNA for the collagen a1(I) probe and 5 mg for S14. Yeast tRNA is a negative control. (B) Collagen a1 (I) mRNA expression normalised against S14 mRNA expression. Values are expressed as mean 6 S.E.M. for between three and five separate experiments. *P < 0.05; †the difference between MCD and control rats at week 17 was significant (P 5 0.04) after exclusion of the cirrhotic rat liver sample. By post-hoc analysis, collagen a1(I) mRNA expression in MCD diet-fed rats was different (P < 0.05) from each other at the various time points.

increased at 12 weeks (P , 0:05, Fig. 10). Hepatocytes isolated at the same time showed a corresponding increase in TGFb1 mRNA (P , 0:05), but there was no significant change in TGFb1 mRNA levels in non-parenchymal cell populations. 3.6. Hepatocyte-induced lipid peroxidation precedes the up-regulation of pro-fibrotic genes in experimental steatohepatitis We have previously shown that the MCD diet is associated with CYP2E1-mediated lipid peroxidation, suggestive of an imbalance between proxidants and antioxidant defences in these livers [17,18]. This finding may have mechanistic significance for liver cell injury, hepatic inflammation and fibrogenesis in steatohepatitis [2,4,19]. To determine the temporal relationships between lipid

Fig. 5. Expression of TIMP-1 in whole liver of rats fed the MCD diet (B) for 12 and 17 weeks and their pair-fed controls (A). (A) A representative RNase protection assay using 30 mg of total RNA for the TIMP-1 probe and 5 mg for S14. Yeast tRNA is a negative control. (B) TIMP-1 mRNA expression normalised against S14 mRNA expression. Values are expressed as mean 6 S.E.M. for four separate experiments. *P < 0.05. By post hoc analysis TIMP-1 mRNA expression was not different between week 12 and 17 in MCD diet-fed rats.

peroxidation and hepatic fibrogenesis in MCD-associated steatohepatitis, we determined levels of TBARS, as well as GSH in whole liver homogenates. At all experimental time points, hepatic GSH levels were lower in MCD diet-fed rats compared to controls (Table 1). This reduction was maximal at week 2, when levels were only 49% of controls; at week 17, values were 73% of controls. Associated with this marked early decline in GSH, hepatic TBARS were 16-fold increased at week 2 in MCD-fed rats compared to controls; at week 17, TBARS were increased 47-fold (Fig. 11). To determine the cellular source of lipid peroxidation during MCD diet feeding, we determined TBARS levels in hepatocytes and non-parenchymal cells isolated at 12 weeks. As shown in Fig. 12, TBARS were elevated only in hepatocytes; in none of the non-parenchymal cell populations were TBARS values significantly altered compared to respective controls.

4. Discussion In earlier work [16], we showed that rats fed the MCD diet for 4 weeks develop zone 3 steatosis, scattered foci of

J. George et al. / Journal of Hepatology 39 (2003) 756–764

Fig. 6. Expression of TIMP-2 in whole liver of rats fed the MCD diet (B) for 5, 12 and 17 weeks and their pair-fed controls (A). (A) A representative RNase protection assay using 30 mg of total RNA for the TIMP-2 probe and 5 mg for S14. Yeast tRNA is a negative control. (B) TIMP-2 mRNA expression normalised against S14 mRNA expression. Values are expressed as mean 6 S.E.M. for six separate experiments. *P < 0.05. By post hoc analysis, TIMP-2 mRNA expression in MCD diet-fed rats was greater at weeks 12 and 17 compared to week 5 (P < 0.05), but not different from each other.

inflammation and hepatocyte necrosis; these findings have been replicated by others [20]. In the present study, we demonstrate that the sequence of pathogenic events during prolonged feeding of the MCD diet involves early lipid accumulation and lipid peroxidation in hepatocytes, followed by liver cell injury and inflammation (increased ALT), stellate cell activation, upregulation of profibrotic genes (collagen a1(1) and TIMP-1 and -2) in stellate cells and eventually conspicuous hepatic fibrosis. In this model of ‘fibrosing steatohepatitis’, fibrosis begins in a centrizonal, pericellular location, with fibrotic strands enveloping lipid-laden hepatocytes. These changes are identical to those seen in disorders of lipid-associated hepatic fibrosis in humans, such as in jejuno –ileal bypass, alcoholic liver disease and NASH. On the other hand, the pathology differs from that in simple choline deficiency in which steatosis is associated with progressive portal fibrosis rather than steatohepatitis [21]. In the MCD diet model, steatosis, chronic hepatocyte injury and hepatic inflammation precede activation of stellate cells and

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Fig. 7. Expression of collagen a1(I) in hepatic non-parenchymal cells from rats fed the MCD diet (B) for 12 and 17 weeks, and their pair-fed controls (A). (A) A representative RNase protection assay showing samples at 12 weeks using 20 mg of total RNA for the collagen a1(I) probe and 5 mg for S14. Yeast tRNA is a negative control. (B) Collagen a1 (I) mRNA expression normalised against S14 mRNA expression. Values are expressed as mean 6 S.E.M. for between four and five separate experiments. *P < 0.05. By post hoc analysis, collagen a1(I) mRNA expression in stellate cells was greater (P < 0.005) than that in Kupffer and endothelial cells at the corresponding time point.

fibrosis by several weeks. This sequence of events is analogous to that which occurs in NASH, despite possible differences between the factors, which cause steatosis after feeding the MCD diet and in NASH [22]. Two interrelated processes that could promote fibrogenesis in the face of liver injury are oxidative stress and the resultant liberation of cytokines. In MCD diet-fed rodents, steatosis is a consequence of the high dietary fat content as well as methionine and choline deficiency. In turn, the latter impairs phosphatidylcholine synthesis with consequent reduction in fatty acid export from the liver [23]. Starvation is not the cause for steatosis because levels of bhydroxybutyrate are unaltered [18] and isocalorically fed rats demonstrate normal liver histology. Choline deficiency leads to hepatic steatosis [23], but in our experience [21], inflammation, a hallmark of steatohepatitis in humans is minimal and fibrosis highly variable. The addition of methionine deficiency in this present model reduces glutathione biosynthesis among other likely important effects; depletion of GSH impairs antioxidant defences in the face of generation of pro-oxidants by lipid oxidases (e.g.

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Fig. 10. Expression of TGFb1 mRNA normalised against GAPDH in whole liver and cell isolates of rats pair-fed the MCD- (B) and control(A) diet for 12 weeks. Amplification of cDNA prepared from total RNA was carried out using real time PCR as outlined in Materials and methods. Values are expressed as mean 6 S.E.M. for between three and six separate experiments; *P < 0.05.

Fig. 8. Expression of TIMP-1 in stellate cells from rats fed the MCD diet (B) for 12 and 17 weeks, and their pair-fed controls (A). (A) A representative RNase protection assay using 15 mg of total RNA for the TIMP-1 probe and 5 mg for S14. Yeast tRNA is a negative control. (B) TIMP-1 mRNA expression normalised against S14 mRNA expression. Values are expressed as mean 6 S.E.M. for between three and seven separate experiments; *P < 0.05.

CYPs 2E1 and 4A), and possibly mitochondria and other sources [2]. It is now established that oxidative stress and/or oxidant damage is present in several animal models of steatohepatitis, including MCD diet-fed rats and mice, acyl-CoA oxidase-deficient mice and methionine adenosyltransferase 1A nullizygous mice [24,25]. Oxidative damage is also prominent in the liver of humans with NASH [3,26 – 27], as well as in the histologically similar disorder of alcoholic hepatitis [4,5,19,28,29]. The present work establishes that oxidative damage to cellular lipids occurs at week 2 in the MCD diet model of steatohepatitis, several weeks before there is hepatic inflammation.

Hepatocytes are the major site of lipid peroxidation in this model. While a contribution from Kupffer cells, monocytes and other inflammatory cell types is not excluded, isolated hepatic non-parenchymal cells did not show an increase in products of lipid peroxidation. These findings are consistent with the proposal that hepatocytes are the major site of lipid peroxidation in experimental steatohepatitis; this is also consistent with a proposed role for the generation of prooxidants, from CYP-P450 lipid oxidases and/or mitochondria, both of which are abundant in this cell type and less conspicuous in non-parenchymal cells. An alternative explanation, that lipid accumulation can occur secondary to hepatic pro-inflammatory events (especially activation of the transcription factor, nuclear factor- kappa B, and release of cytokines) would not be consistent with the present results. Hepatic stellate cells can be activated by exposure to products of lipid peroxidation [30]. Furthermore, exposure

Table 1 Reduced glutathione (GSH) levels in whole liver during pair-feeding of the MCD and control diets Duration of feeding (weeks)

Diet

GSH (mmol/g liver)

% MCD relative to control

2

Control MCD Control MCD Control MCD Control MCD

4.28 ^ 0.49a 2.11 ^ 0.17 5.12 ^ 0.33 3.37 ^ 0.08 3.95 ^ 0.17 3.15 ^ 0.20 4.08 ^ 0.17 2.99 ^ 0.11

49

0.01

66

,0.01

80

0.04

73

,0.01

5 12 Fig. 9. Expression of TGFb1 mRNA relative to GAPDH in whole liver of rats fed the MCD or control diets over 17 weeks. Amplification was carried out using reverse transcription PCR over 32 cycles with 56 8C as the annealing temperature.

17

P

a Values are mean ^ S.E.M. for between three and five separate experiments at each time.

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Fig. 11. TBARS levels in whole liver during pair-feeding of the MCD(B) and control- (A) diet. Values are expressed as mean 6 S.E.M. for at least six separate experiments; *P < 0.05.

to prooxidants or end products of lipid peroxidation activates the transcription of collagen genes [30 –33]. The temporal sequence of steatosis/lipid peroxidation, followed by liver cell injury, inflammation, TGFb upregulation and fibrosis is consistent with the proposal that lipid peroxidation may be mechanistically involved in the development of fibrosis in this model. As expected, stellate cells are the main cellular source of extracellular matrix in MCD-diet associated fibrosing steatohepatitis. However, hepatocytes were the only source of elevated TGFb1 mRNA. TGFb1 is the major profibrogenic cytokine in wound healing responses of the liver [34 –37]; however, others have shown that enhanced expression of TGFb mRNA is confined to cells that are the target of injury [38]. Thus TGFb mRNA is elevated in hepatocytes following partial hepatectomy, and in stellate cells following bile duct ligation [38]. The present data conform to this schema because in steatohepatitis caused by MCD, hepatocytes are clearly the site for lipid accumulation, GSH depletion and lipid peroxidation. However, the results do not exclude a

Fig. 12. TBARS levels in cell isolates from rats pair-fed the MCD- (B) and control- (A) diet for 12 weeks. Values are expressed as mean 6 S.E.M. for at least three separate experiments; *P < 0.05.

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contribution to TGFb release and activation by nonparenchymal cells, particularly as the biological effects of TGFb are regulated extensively at a post-translational level and by release and storage of the cytokine. In conclusion, rats fed a high fat MCD-diet develop chronic steatohepatitis that results in hepatic fibrosis in a pattern resembling that found in NASH. In this model, steatosis is due to dietary factors rather than insulin resistance as found in NASH, but the histologic picture, including hepatocellular injury, inflammatory recruitment and pericellular zone 3 hepatic fibrosis mimics that seen in human fibrotic disorders associated with hepatic lipid accumulation. The time course of events is consistent with lipid peroxidation as a pathogenic mechanism for initiation and perpetuation of liver injury, inflammatory recruitment, and as now shown here for the first time, for stellate cell activation and fibrogenesis.

Acknowledgements This work was supported by a grant from the Australian National Health and Medical Research Council (153899), and the Robert W. Storr Bequest of the Medical Foundation, University of Sydney. The authors wish to thank John Iredale (School of Medicine, University of Southampton, Southampton, UK) for the TIMP-1 and –2 probes; Pauline Hall (Department of Anatomical Pathology, University of Cape Town, Cape Town South Africa) and Graham Robertson for stimulating discussions about the pathology and pathogenesis of steatohepatitis; Tina Borovina for assistance with the animals and Jacqueline Field for assisting with surgery and PCR.

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