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Functional recovery after liver resection
Veteläinen, R.L.
Publication date
2006
Link to publication
Citation for published version (APA):
Veteläinen, R. L. (2006). Functional recovery after liver resection.
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Reetaa Vetelainen
Arlènee van Vliet
Thomass M. van Gulik
ABSTRACT T
Background:: Choline deficient (CD) and methione-choline deficient (MCD) diets are rodentt models for steatosis, with potentially dissimilar biochemical backgrounds. The aim off this study was to assess the metabolic and pathological derangements in rats fed CD andd MCD diets.
Methods:: Male Wistar rats received CD or MCD diet up to 7 weeks. Nutritional status, liverr histopathology, Kupffer cell-mediated inflammation and injury, oxidative stress via thiobarbituricc reactive species (TBARS), hepatic and plasma glutathione (GSH) and insulin homeostasiss were assessed.
Results:: In CD-fed rats, mainly microvesicular steatosis developed with occasional inflammatoryy cells. In MCD-fed rats, macrovesicular steatosis progressed to steatohepa-titiss (collagen deposition, activated stellate cells). Hepatic TBARS was increased and GSH decreasedd in the MCD-fed rats compared to no changes in the CD-fed rats. The CD-fed ratss developed obesity, dyslipidemia and insulin resistance, in contrast to undetectable plasmaa lipids, unaffected insulin homeostasis and loss of body weight in the MCD-fed rats. .
Conclusions:: The CD diet induced uncomplicated steatosis as compared to progressive inflammationn and fibrinogenesis in the MCD diet. CD and MCD diets represent two patho-genicallyy different models of steatosis. Although equivalence for the outcome of both dietss can be found in clinical steatosis, the results of models using these diets should be comparedd with caution.
Introduction n
Nonalcoholicc fatty liver disease (NAFLD) is the most common chronic liver disorder in the world,, with a prevalence of approximately 20% in the general population and up to 95% amongg those with obesity.1-3 NAFLD represents a broad spectrum of liver disease ranging fromm mild steatosis to steatohepatitis featuring severe steatosis (>60% hepatocytes affected),, hepatocellular injury, progressive chronic inflammation and fibrosis.3,4 Until recently,, NAFLD was identified as a mild and reversible disorder without impact on liver surgery.55 However, several clinical studies have shown the significance of steatosis as patientss having any degree of steatosis have an increased risk of postoperative mortality andd morbidity after liver resection or liver transplantation.6,7
Animaii models have greatly contributed to the understanding of mechanisms behind thee increased susceptibility of steatotic livers to injury. However, the large variety of experimentall models of steatosis is confusing and potentially obscures the comparison and interpretationn of results. The most commonly used models are based on nutritional changes orr on leptin deficiency (Zucker rats, ob/ob mice).8 However, in leptin-deficient rodents, the leptinn deficiency and not steatosis per se leads to impaired hepatic regeneration after injuryy or resection.9'10 Therefore, the use of leptin-deficient rodents in surgical models relatingg to liver regeneration is questionable.
Nutritionall models based on choline deficiency (CD) or methione and choline deficiency (MCD)) have been widely applied in surgical models of organ preservation,11 ischemia-reperfusionn injury12"14 and liver regeneration.15,16 Existing data show that the impaired formationn of very-low-density-lipoproteins (VLDL) contributes to the development of steatosiss in MCD diet.17 However, choline can be biosynthesized via methione and choline deficiencyy atone does not seem to impair the VLDL excretion.18 Therefore, the clinically relevantt pathological features such as increased oxidative stress and the Kupffer cell-mediatedd inflammatory response contributing to the development of steatohepatitis, ass described with the MCD diet, are not necessarily similarly induced by the CD diet.19 Furthermore,, overall metabolic derangements induced by both diets are not well described inn the light of new insights into NAFLD pathogenesis such as altered insulin homeostasis, aa potential pathogenic feature of NAFLD.20-22
Ourr hypothesis is that the steatosis induced with the CD diet and that with the MCD diett are distinct in respect to clinically relevant pathological and metabolic derangements suchh as hepatic steatosis, oxidative stress, Kupffer cell-mediated hepatocellular injury and inflammatoryy response, insulin homeostasis, antioxidant capacity and the development off steatohepatitis. The aim of this study, therefore, was to assess these metabolic and pathologicall derangements in rats fed with these different diets.
Methods s
Diett a n d animals
Malee Wistar rats (250-300 g) (Harlan CPB, Zeist, the Netherlands) were acclimatized to laboratoryy conditions for a minimum of 7 days, maintained at constant C with 12 h
light-darkk cycle and fed a standard rodent chow (Hope Farms, Woerden, the Netherlands) with waterr ad libitum. After acclimatization, the rats were randomized into three experimental groupss (n = 4 - 6 per time point/group) and fed with either choline deficient (CD) diet orr methione and choline deficient (MCD) diet (Harlan Teklad, Madison, Wl, USA) up to 77 weeks ad libitum. A control group received isocaloric chow, containing adequate levels off choline and methione, ad libitum (Harlan Teklad).
Thee daily food consumption and body weight were measured and after the designated periodd rats were killed. Blood was collected by vena cava puncture, centrifuged (10 min, 30000 r.p.m., 4'JC) and plasma was stored at . Livers were removed, weighed and thinn slices of all liver lobes were immersed in 10% formalin for light microscopy (HE and Siriuss red staining) or frozen immediately in liquid nitrogen for immunohistochemistry or Oill Red O staining.
Duringg all procedures the animals were treated according to the guidelines of the Dutchh legislation and international standards for animal care and handling. The protocol wass approved by the Animal Ethics Committee of the University of Amsterdam, the Netherlands. .
Biochemicall analysis of plasma
Plasmaa samples were analyzed in the Department of Clinical Chemistry using standard laboratoryy methods for alanine aminotransferase (ALT), total bilirubin (T-Bil), albumin, triglyceride,, cholesterol and glucose.
Assessmentt of hepatic lipid concentration
Liverr samples were homogenized in phosphate-buffered saline (pH 7,2) and centrifuged (40000 g, 10 min, . Hepatic lipids were extracted bythechloroform:methanol extraction methodd according to Folch eta!23 and measured enzymatically using commercial kits accordingg to manufacturer's instructions (Trig/GB, Roche, Switzerland; Cholesterol Biomerïeux,, Boxtel, Netherlands). The protein concentration in the homogenates was measuredd with BCA Protein Assay kit (Pierce, Rockford, IL, USA) and expressed in mmol/ mgg protein.
Assessmentt of oxidative stress and antioxidant response
Hepaticc and systemic oxidative stress was assessed by measuring lipid peroxidation as determinedd by malondialdehyde and measured as total thiobarbituric acid reactive substancess {TBARS) according to Asakawa and Matsushita.24 Assays were performed in thee presence of the antioxidant butylated hydrotoluene limiting the generation of new TBARSS during the assay. Antioxidant response was assessed by plasma and hepatic total glutathionee (GSH) concentrations. Liver samples were homogenized in meta-phosphoric bufferr (pH 6.0), centrifuged (4000 g. 10 min, ) and analyzed for GSH as previously describedd and expressed in nmol/mg protein.2^
Assessmentt of insulin homeostasis
Fastingg plasma insulin levels were measured with enzyme-linked immunosorbent assay (Ratt Insulin ELISA, Lmco, St Charles, MO, USA). All samples were measured in duplicate in aa 96-well mtcrotiter plate and the concentrations were calculated from a standard curve. Forr assessment of insulin secretion and sensitivity, homeostasis model assessment (HOMA) andd quantitative insulin sensitivity check index (QUICKI) were used, which correlate very welll with the gold standard of the hyperglycemic clamp, the intravenous glucose tolerance testt and the oral glucose tolerance test.2 6 2 7
Thee calculations were made as follows: HOMA-R = fasting insulin lU/mL) * fasting glucose (mmol/L)/22.55 and QUICKI = 1/[log (fasting insulin) + log (fasting glucose)].
Proinflammatoryy cytokine response
Kupfferr cell activation was assessed by plasma and hepatic tumor necrosis factor (TNF)-« contentt using an ELISA (Duoset Rat TNF-«, RnD Systems, Abingdon, UK) according to the manufacturer'ss instructions. Liver samples were homogenized in buffer (NaPi 5 mmol/L, pHH 6.0), centrifuged (10 000 g, , 10 min), measured in duplicate in a 96-well microtiter plate.. The concentrations were calculated from a standard curve and expressed in pg/mg protein. .
Immunohistochemistry y
Briefly,, after formaldehyde fixation and paraffin-embedding, 4 « m sections were deparaffinized.. For the detection of activated stellate cells, sections incubated with «-smoothh muscle actin (a-SMA) antibody (dilution 1:200, 60 min, DAKO Cytomation, Copenhagen,, Denmark) or ED-1 for both hepatic resident macrophages (Kupffer cells) andd circulatory macrophages (1:100, 60 min; Southern Biotechnologies, Birmingham, AL, USA).. After incubation with secondary antibody (GAM-lgG2A, 1:100, 60 min; Southern Biotechnologies),, Fast DAB (3,3-diaminobezidine; Sigma, Munich, Germany) was used togetherr with hematoxylin counterstaining. The number of positive cells was calculated in 300 high power fields (HPF) at magnification of *100 and was expressed as positive cells/ HPF. .
Histopathology y
Liverr sections were stained with hematoxylin-eosin for routine morphology and with Siriuss red for collagen staining (0.1% Fast red in picric acid; Immunotech, Mijdrecht, the Netherlands).. Liver histopathology was examined by light microscopy in 30 HPF per sample withh a magnification of *40.
Twoo blinded investigators evaluated the slides on a semiquantitative basis as follows. For steatosis:: grade 0, none; grade 1, <25%, grade 2, 26-50%; grade 3, 51-75%; grade 4, >75%.. For inflammation: grade 0, none; grade 1, scattered inflammatory cells; grade 2, <55 foci of inflammatory cells per HPF; grade 3, >5 inflammatory foci per HPF. For fibrosis: gradee 0, no fibrosis; grade 1, moderately thickened centrilobular vein (CLV); grade 2, markedlyy thickened CLV (annular appearance of the vein wall with numerous fibrous extensionss between hepatocytes); grade 3, cirrhosis.28
Statisticall analysis
Dataa analysis was performed with GraphPad Prism 3.02 for Windows (GraphPad Software, Sann Diego, CA, USA). The results are presented as means SEM. Differences between groupss were tested using the unpaired Student's f-test or A NOVA (with appropriate post hoc analysis)) for multiple comparisons. P-values less than 0.05 were considered significant.
Results s
Generall features
Inn all groups, physical activity was similar and appearance of animals remained healthy. Thee mean body weight of the MCD-fed rats was significantly less than that of CD-fed rats orr controls. The liver-to-body weight ratio was increased in CD-fed rats at 1 and 3 weeks comparedd to the MCD-fed rats and controls (P<0.05) but after 7 weeks, showed no differencess in all groups (P = 0.054). During the MCD diet, the rats sustained significant weightt loss in contrast to a significant weight gain in the CD-fed rats compared to controls (MCDD 221 11 g, CD 442 13 g, controls 367 6 g; P< 0.05). No changes in plasma albuminn levels were observed in both groups, indicating unaffected protein synthesis (dataa not shown).
Inductionn of steatosis and steatohepatitis
Inn controls, no pathological changes were seen and the inflammation score was significantly lowerr at all time points compared to the CD- and MCD-fed rats (Table 1).
Tablee 1 Steatosis and inflammation in rats fed either a choline deficient (CD) or methione-choline deficient
11 week 33 weeks 55 weeks 77 weeks Control l 0.033 0.03 0 . 3 + 0 . 1 1 0.55 1 0.66 + 0.04 0.011 +0.01 1.9 9 0.66 2 1.00 0.1 1.44 1 4 4 1.11 5 1.55 9 1.88 7 2.11 0.04 0.066 1 1.66 0.1 2.33 1 2.88 5 2.99 5
Inn the CD-fed rats, an increase of microvesicular steatosis with few inflammatory cells was observedd at 1 and 3 weeks (P< 0.05; Fig. 1a). After 5 weeks, the CD-fed rats showed aa microvesicular steatosis with few macrovesicular hepatocytes and inflammatory foci
(P<(P< 0.05). At 7 weeks in the CD-fed rats, only a slight increase in macrovesicular steatosis
wass detected with a slight increase in inflammatory cells (Fig. 1c).
Afterr 1 week of diet intake, the MCD-fed rats initially developed mainly microvesicular steatosis,, with a macrovesicular component, predominantly in the acinar zone 3 (Fig. 1b). Att 3 weeks, macrovesicular steatosis was the main feature of steatosis showing scattered focii of inflammatory cells. After 5 weeks of diet intake, the MCD-fed rats showed a diffusee and extensive macrovesicular steatosis with increased numbers of mononuclear inflammatoryy cell foci and with occasional spotty necrosis. At 7 weeks, extensive
Figuree 1 Representative histology of liver tissue (original magnification ><20). HE-stained liver tissue of choline deficientt (CD) diet-fed rat at (a) 1 week and (c) 7 weeks shows mainly microvesicular steatosis without prominentt inflammation. HE-stained liver tissue of methione-choline deficient (MCD) diet-fed rat at (b) 1 weekk and (d) 7 weeks shows many inflammatory foci. Sinus red stained liver tissue of (e) CD-fed and (f) MCD-fedd rats at 7 weeks shows increased collagen deposition in the MCD-fed rats (arrows).
macrovesicularr steatosis throughout the section was observed. Steatosis was most evident inn centrilobular areas together with accumulation of cellular debris and numerous clusters off inflammatory cells (P < 0.03; Fig. 1d)
Furthermore,, in the MCD-fed rats, Sirius red staining showed increased perivenular fibrosiss (Fig. 1e) with occasional centrilobular fibrosis (Fig. 1f). Activated stellate cells were detectedd by a-SMA immunohistochemistry in the portal areas and occasionally also in the parenchymaa (data not shown).
Biochemicall parameters
Plasmaa ALT was slightly increased in the CD-fed rats at 3 and 5 weeks but no difference wass seen at 7 weeks compared to controls. However, in the MCD-fed rats ALT increased 10-foldd after 5 weeks (P<0.05; Fig. 2). Plasma bilirubin increased in the MCD-fed rats afterr only 1 week being fourfold higher after 7 weeks, in contrast to the CD-fed rats in whichh a twofold increase was seen only after 7 weeks (P < 0.05, data not shown).
500-, , 400<~<~ 300
-II I
## 200 i 1 week ii i 3 weeks ii i 5 weeks ii i 7 weeksM M
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Figuree 2 Effect of choline deficient (CD) and methione-cholinee deficient (MCD) diets on hepatocytes.. Hepatocellular damage assessed by plasmaa ALT was more prominent in the MCD-fed rats.. Values are expressed as mean + SEM. "P< 0.05 comparedd to controls (CO); **P< 0.05 compared to thee other diet (ANOVA).
EE E E E
1 1
co o 111 week ]] 3 weeks 155 weeks -- : CDD D M C D D D EE -= CDD D MCDD D uu O cc » uu "5 Q.. E i i CO O CDD D M C D D D D D .== o nn E P-- E CO O: :
: :
CDD D MCDD D Figuree 3 Effect of choline deficient (CD) and methione-choline deficient (MCD) diets on (a) plasma triglycerides,, (b) cholesterol, (c) hepatic tissue triglycerides and (d) cholesterol. In the CD-fed rats, a significant increasee in hepatic triglycerides was observed in contrast to undetectable plasma triglyceride and cholesterol levelss at 7 weeks in the MCD-fed rats. Values are expressed as mean SEM. 'P < 0.05 compared to controls (CO);; '*P< 0.05 compared to the other diet (ANOVA).Inn the CD-fed rats plasma triglyceride and cholesterol levels increased, whereas in MCD-fedd rats the levels decreased below the detection limit (0.1 mg/dL)
(Fig.. 3a,b) Hepatic triglyceride content was increased in the MCD-fed rats at all time points comparedd to the controls and to the CD-fed rats, corresponding with the histopathological changess (P < 0.01; Fig. 3c). Hepatic cholesterol content was increased in the CD and MCD-fedd rats at all time points compared to controls and was higher in the MCD-fed rats at 1 andd 3 weeks compared to the CD-fed rats (P < 0.01; Fig. 3d).
Oxidativee stress and antioxidant response
Hepaticc TBARS was at all time points increased in the MCD-fed and the CD-fed rats as comparedd to controls and was significantly higher in the MCD-fed rats (P < 0.01, Fig. 4a). Thee plasma and hepatic glutathione levels were reduced in the MCD-fed rats (P< 0.05; Fig.. 4b,c) whereas no significant changes were observed in rats having the CD diet, althoughh there was a tendency towards increased levels at 3 and 5 weeks.
Kupfferr cell activation
Afterr 1 week, a twofold increase in hepatic TNF-a was seen in both the CD- and MCD-fed ratss compared to controls (P < 0.05; Fig. 5). After 7 weeks, TNF-« levels had increased
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Figuree 4 Effect of choline deficient (CD) and methione-cholinee deficient (MCD) diets on (a) activationn of hepatic lipid peroxidation as measured byy free TBARS in liver, and antioxidant response in (b)) plasma and (c) liver as measured by total GSH. Hepaticc TBARS was increased and both plasma and hepaticc GSH decreased in the MCD-fed rats. Values aree expressed as mean SEM. *P < 0.05 compared to controlss (CO); **P < 0.05 compared to the other diet
(ANOVA). . CO O CDD D MCDD D oii o yy E
I I
CO Op p
z z
CDD D J , , MCDD D 22 Eit t
i s s
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CDD D Ü r m m MCDD D45 5
Figuree 5 Ef
fect of choline deficient (CD) and methione-cholinee deficient (MCD) diets on hepatic Kupfferr cell activation measured by tissue TNF-o levels.. A significant and progressive increase was observedd in the MCD-fed rats as compared to only a slightt increase in the CD-fed rats. Values are expressed ass mean SEM. *P < 0.05 compared to controls (CO),
**P**P < 0.05 compared to the other diet (ANOVA).
COO CDD MCDD
foldd in the MCD-fed rats but stayed at the same level in the CD-fed rats throughout the wholee diet period. In the MCD-fed rats, the levels were significantly higher after 5 and 77 weeks compared to the CD-fed rats (P < 0.05).
Thee increased hepatic TNF-a in the MCD-fed rats corresponded with an increased number off ED-1 positive macrophages in liver parenchyma compared to no differences between thee CD-fed rats and controls (data not shown). Plasma TNF-a was undetectable in controls (detectionn level 5 pg/mL) but was increased 10-fold in both the CD and the MCD-fed rats afterr 1 week compared to controls and was highest after 5 and 7 weeks in the MCD-fed ratss (P < 0.05) (data not shown).
Assessmentt of insulin homeostasis
HOMA-IRR and QUICKI did not significantly differ from the controls in the MCD-fed rats att all time points, whereas in the CD-fed rats, significant increases in both indexes were observedd after only 1 week (P < 0.05; Fig. 6).
COO CDD MCDD C 0 CDD MCDD
Figuree 6 Effect of choline deficient (CD) and methione-choline deficient (MCD) diets on insulin homeostasis ass assessed by (a) HOMA-IR and (b) QUICKI. In the CD-fed rats, QUICKI and HOMA showed significantly disruptedd insulin homeostasis. No changes were detected in MCD-fed rats Values are expressed as meann SEM. *P < 0.05 compared to controls (CO); * * P < 0.05 compared to the other diet (ANOVA).
Discussion n
Thee present study is the first to report essential differences in steatosis induced by CD andd MCD diets in a rat model. The induction of hepatic steatosis, oxidative stress, insulin resistance,, cytokine mediated hepatocellular damage and progression to steatohepatitis differedd significantly between animals fed with one of the two diets. The MCD-fed rats
developedd the highest degree of steatosis corresponding with increased oxidative stress, decreasedd antioxidant response and progression to steatohepatitis. Consistent with previouss studies, a significant weight loss was seen in the MCD-fed rats.16,17'29 In contrast, thee CD-fed rats developed steatotic features similar to the human metabolic syndrome characterizedd by insulin resistance, dyslipidemia and obesity.
Thee proposed biochemical basis of fatty accumulation in choline and/or methione deficiencyy is impaired phosphatidyl choline (PC) synthesis, which is essential for hepatic VLDLL secretion.30 PC synthesis occurs via two pathways: through direct incorporation off preformed choline into phosphatidyl compounds (CDP pathway) or through stepwise
methylationn of adenosyl methione.31 Interestingly, a recent study reported unimpaired PCC synthesis via the CDP pathway in choline-deficient mice.32 The authors suggest that thiss is due to compensatory activation of the methylation pathway to maintain the PC synthesiss and VLDL excretion, corresponding with the observed increased plasma lipids in thee CD-fed rats. The exact mechanism of hepatic lipid accumulation in the CD-fed remains
unclear. .
Inn the MCD-fed rats, increased oxidative stress and a decreased antioxidant response were observedd in the presence of steatohepatitis (activation of hepatic stellate cells and collagen deposition)) but these features were significantly less induced in the CD-fed rats. Oxidative stresss and lipid peroxidation are generally considered as the most pathogenic mechanisms off nonalcoholic steatohepatitis (NASH).33 For prevention of oxidative stress, a balance betweenn reactive oxygen species (ROS) production and antioxidant response is essential. Thee important sources for oxidative stress are intracellular fatty acids, via direct toxicity or viaa induction of cytochrome p-450 microsomal lipoxygenase 2E1 (CYP2E1) and perixomal beta-- oxidation.34,35 CYP2E1 and beta-oxidation generate ROS promoting cell membrane damagee via malondialdehyde, the end-product of lipid peroxidation. Malondialdehyde causess further structural changes in the mitochondrial matrix leading to mitochondrial dysfunction.36'377 These further activate the proinflammatory cytokine release of Kupffer cellss and stellate cells leading to necroinflammatory changes in hepatic parenchyma3738 Indeed,, in the MCD-fed rats the observed increased lipid peroxidation corresponded withh marked hepatocellular damage and increased hepatic TNF-a. In the CD-fed rats, the antioxidantt scavenging of ROS was apparently sufficient as less lipid peroxidation and hepatocellularr damage was observed.
Interestingly,, another important pathogenic feature proposed to play a role in progression off NASH, insulin resistance, was observed in the CD-fed rats. This insulin resistance is probablyy due to a combination of factors determining hepatocyte sensitivity to insulin suchh as increased TNF-a and hypertriglyceridemia. In the CD-fed rats, no significant inflammationn was found but another source for TNF-a in the absence of inflammation, namelyy adipose tissue, was present. TNF-a reduces the expression of Glut4, an insulin-dependentt glucose transporter and decreases the phosphorylation of insulin-receptor substrate-11 (IRS-1).39 Also hypertriglyceridemia potentially contributed to the insulin resistancee in the CD-fed rats as triglycerides directly inhibit peripheral glucose uptake via down-regulationn of IRS-1.22
Itt has been hypothesized that, in the absence of mitochondrial dysfunction, peripheral insulinn resistance will not lead to the development of NASH but only to uncomplicated
steatosis.. Consistent with this hypothesis, less lipid peroxidation, also indirectly reflecting mitochondriall damage, was detected in the CD-fed rats w i t h insulin resistance. Opposite too this hypothesis, no insulin resistance was present in the MCD-fed rats, as has also beenn previously reported, even though extensive lipid peroxidation and fibrinogenesis was observed.4 00 It seems that insulin resistance in this model does not play a crucial role in the developmentt of NASH. However, additional studies are needed to clarify this aspect. Thee profound differences in metabolic derangements must have significant impact when thesee diets are applied in surgical models of steatosis as used in ischemia-reperfusion and partiall hepatectomy studies. Impaired glucose metabolism alters liver metabolism and affectss the immune system and is also considered to be an independent risk factor for acutee liver failure after extended liver resection.4 1'4 2 Furthermore, oxidative stress and lipid peroxidationn (important pathogenic features of human NAFLD) are already present in MCD-fedd rats several weeks before necroinflammatory changes are seen at histopathological examination.. In contrast, CD-fed rats do not develop significant inflammation or fibrosis norr show mitochondrial changes. This might lead to underestimation of the actual implicationss of steatosis particularly in situations w h e n unaltered mitochondrial function is cruciall such as for hepatocyte proliferation.
InIn conclusion, this study shows that steatosis induced by the CD or MCD diets differ profoundlyy in features clinically relevant to human NAFLD and NASH. The CD diet induced uncomplicatedd steatosis w i t h features of the metabolic syndrome in contrast to the MCD diett w i t h induction of progressive Kupffer cell-mediated inflammation and fibrinogenesis. A l t h o u g hh CD and MCD diets represent t w o pathogenically different models of steatosis, equivalencee w i t h clinical steatosis can be found for the outcome of both diets. However, thee results of studies applying these diets in rats should be compared w i t h caution.
A c k n o w l e d g m e n t t
Thee authors want to thank Jaap Rip (Department of Vascular Medicine) for his assistance inn the analytical work.
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