- Open Access
Long term highly saturated fat diet does not induce NASH in Wistar rats
© Romestaing et al; licensee BioMed Central Ltd. 2007
Received: 06 September 2006
Accepted: 21 February 2007
Published: 21 February 2007
Understanding of nonalcoholic steatohepatitis (NASH) is hampered by the lack of a suitable model. Our aim was to investigate whether long term high saturated-fat feeding would induce NASH in rats.
21 day-old rats fed high fat diets for 14 weeks, with either coconut oil or butter, and were compared with rats feeding a standard diet or a methionine choline-deficient (MCD) diet, a non physiological model of NASH.
MCDD fed rats rapidly lost weight and showed NASH features. Rats fed coconut (86% of saturated fatty acid) or butter (51% of saturated fatty acid) had an increased caloric intake (+143% and +30%). At the end of the study period, total lipid ingestion in term of percentage of energy intake was higher in both coconut (45%) and butter (42%) groups than in the standard (7%) diet group. No change in body mass was observed as compared with standard rats at the end of the experiment. However, high fat fed rats were fattier with enlarged white and brown adipose tissue (BAT) depots, but they showed no liver steatosis and no difference in triglyceride content in hepatocytes, as compared with standard rats. Absence of hepatic lipid accumulation with high fat diets was not related to a higher lipid oxidation by isolated hepatocytes (unchanged ketogenesis and oxygen consumption) or hepatic mitochondrial respiration but was rather associated with a rise in BAT uncoupling protein UCP1 (+25–28% vs standard).
Long term high saturated fat feeding led to increased "peripheral" fat storage and BAT thermogenesis but did not induce hepatic steatosis and NASH.
Non-alcoholic fatty liver diseases (NAFLD) are characterized by triglyceride accumulation in hepatocytes (i.e., liver steatosis). In some cases, steatosis becomes complicated by inflammation and can evolve to apoptosis, necrosis and fibrosis. This association of steatosis to other lesions is called non-alcoholic steatohepatitis or NASH , and may evolve into cirrhosis and hepatocellular carcinoma.
NASH is a disease of emerging importance and is now considered as the most common cause of chronic liver disease in the USA [2, 3]. While the pathogenesis of NASH is poorly understood, the hypothesis of two "hits" is recognized . Fat accumulation in the liver represents the "first hit". The factor responsible for the second "hit" is hepatic oxidative stress due to ROS emission and/or increased cytokine release, enhancing lipid peroxidation, mitochondrial DNA and respiratory chain damages [5, 6]. Currently, no defined therapy is known to alter the course of NASH [5, 7–10].
The study of the pathogenic factor involved in NASH is difficult because of the lack of a suitable experimental animal model . Currently, available animal models are rodents either with a genetic defect (ob/ob mice or Fa/Fa rat)  or fed a methionine and choline deficient diet (MCD diet) . The latter model is commonly used but induces a nutritional deficiency that is not observed in patients with NASH. The major disadvantage of these models is that they fail to reflect the multi-factorial features of NASH observed in patients. High caloric intake and obesity are factors frequently associated with NASH in humans. In rodents, however, the situation is less clear as rats fed high fat diets were shown to develop hepatic steatosis in some studies [14–16] but not in others [17, 18]. Comparisons of the protocols used showed that the composition and the palatability of the diets may play an important role in the development of the obesity and NASH. To overcome these difficulties, some authors gave diet ad libitum while others strictly controlled the caloric intake through intragastric diet infusion or force-feeding. Lieber et al. used a liquid high fat diet given ad libitum to rats  whereas Zou et al. controlled daily fat intake by force-feeding rats . In these two cases, high fat diet induced mild steatosis (two fold increase in hepatic triacylglycerol compared to control) and huge hepatic inflammation. The main fat component of these two diets was corn oil, (consisted of 13% (w/w) saturated fatty acid (SFA), 24% monounsaturated fatty acid (MUFA) and 59% polyunsaturated fatty acid (PUFA)). These PUFA were almost entirely composed by pro-inflammatory n-6 polyunsaturated fatty acids which are known to be involved in liver oxidative stress . These models do not really mimic human NASH diet features since a study reported that patients with NASH usually have a diet with higher levels of SFA (13.7% instead of 10.0% total kcal) and cholesterol, and low levels of PUFA (3.5% (w/w) .
Consequently, to analyse NASH pathogenesis, the aims of the present study were (i) to study the development of steatosis following a SFA-rich diet, ii) to study the possible evolution from steatosis to NASH and iii) to determine the possible liver adaptations to this new condition. We tested two high saturated fat diets with either coconut oil, which contains roughly 86% SFA, or butter, which contains 51% SFA. These diets were compared with the MCD diet, the most common diet used to mimic NASH in rodents.
Animals and diets
Components (g/kg) of the standard, coconut, butter and methionine-choline deficient (MCD) diets and their fatty acid (FA) composition.
Histological analyses were achieved in the gastroenterology service of Caen hospital (France). Liver specimens were fixed in 4% buffered formalin for 24 to 48 h. They were then embedded in paraffin, cut at 5 μm, and routinely stained with hematoxylin-eosin (H&E) and reticulin. Severity of steatosis was evaluatedusing the percentage of macrovesicular fat within hepatocytes: mild (5 to 30 %); moderate (30 to 60 %); severe (more than 60%) .
Mitochondrial preparation and utilisation
The liver was rapidly removed and finely minced and washed with ice-cold isolation medium containing 250 mM sucrose, 2 mM KH2PO4, 1 mM EGTA and 20 mM Tris-HCl (pH 7.2). Liver mitochondria were prepared by standard differential centrifugation procedures . Mitochondrial protein content was determined by the Biuret method  with serum albumin as standard. For the determination of oxygen consumption, mitochondria were incubated at a concentration of 2 mg/mL in an oxygraph vessel with a Clark electrode, thermostatically controlled at 37°C, in a medium containing 125 mM KCl, 1 mM EGTA, 2 mM KH2PO4, 20 mM Tris-HCl with 0.1% fatty acid-free Bovin Serum Albumin (pH 7.2). The control state of respiration was initiated by the addition of 5 mM succinate/0.5 mM malate, in the presence of rotenone (1.25 μM) while the addition of 1 mM ADP initiated the active state of respiration (state 3). Oligomycin (1.25 μg/mg protein) was then added to the mitochondrial suspension to determine the non-phosphorylating respiratory rate (state 4).
Hepatocyte isolation and closed vials incubation
Hepatocytes were isolated from 20–24 h starved rats as previously described by Berry and Friend (Berry, 1969) and modified by Groen et al. , by a two-step in situ collagenase perfusion technique. Hepatocytes (10 mg/mL dry weight) were incubated at 37°C in closed vials containing a Krebs-bicarbonate buffer (120 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 24 mM NaHCO3, pH 7.4) saturated with 95% O2/5% CO2 containing BSA (2%, w/v) and 2.4 mM CaCl2. Experiments were performed with 20 mM dihydroxyacetone, with or without fatty acids, 4 mM octanoate or 2 mM oleate. After 20 min, oxygen uptake (J O2) was measured polarographically at 37°C with a Clark electrode before and after the addition of 6 μg/ml oligomycin. At t = 0 and 30 min, samples of hepatocyte suspension were taken, quenched in HClO4 (4% v/v final concentration) and neutralized with 2 M KOH/0.3 M MOPS for later enzymatic measure of 3-hydroxybutyrate, acetoacetate and glucose as described by Bergmeyer .
Adipocytes were prepared from retroperitoneal white fat pads. 1.5 g of fat tissue was digested for 30 min at 37°C with 2 mg/mL of type II collagenase. The digestion medium was a Krebs-Ringer medium (139 mM NaCl, 5.4 mM KCl, 1 mM NaH2PO4, 1 mM MgSO4, 2.2 mM CaCl2, pH 7.4) buffered with 20 mM Hepes, containing 2% (w/v) BSA with 7 mM glucose. Isolated cells were obtained by filtration through a coarse nylon mesh (250 μm) before being washed twice with a 2% BSA buffer. Adipocytes were then observed under a microscope and the determination of the diameter was measured on pictures of roughly 200–250 cells using a calibrated scale.
Liver triacylglycerol concentration was estimated from glycerol release after ethanolic KOH hydrolysis , using a commercial colorimetric kit (Biomerieux, France).
Western blot analysis
Mitochondria from brown adipose tissue (BAT) were prepared as described previously. 40 μg of BAT mitochondrial proteins were separated by SDS-PAGE (12.8% acrylamide) and transferred to polyvinylidene fluoride membranes (Immobilon-P, Millipore). Immunological detection was performed using a rabbit antiserum against UCP1 (α-diagnostics UCP11-S (1:15000), USA). The detection was realized with a horseradish peroxidase-coupled anti-rabbit (Bio-Rad 170–6516 (1:5000)) secondary antibody and an enhanced chemiluminescence (ECL) detection kit (Amersham, UK). Quantification of autoradiographs was performed by scanning densitometry.
BAT samples (40–50 mg) were immediately homogenized at +4°C in 0.3 M phosphate buffer containing 0.05% bovine serum albumin (pH 7.7) using a glass Potter-Elvehjem homogenizer. Then, they were frozen at -80°C and thawed three times to disrupt the mitochondrial membrane. 3-hydroxyacyl-CoA dehydrogenase (HAD, EC 220.127.116.11) was spectrophotometrically determined as previously described by Lowry & Passonneau . Enzyme activity was determined at 25°C and expressed as micromoles of substrate per minute per milligram of protein.
Differences between groups were determined using non-parametric Mann & Whitney tests. Data were expressed as mean values ± SEM and differences between means were considered significant when P < 0.05.
Caloric intake, body and tissue mass
Percentage of the main fatty acid (FA) measured in the coconut oil and butter.
Lipids, protein and carbohydrate repartition from the standard, coconut and butter diet
% from control diet
% from high fat diet
% from control diet
% from high fat diet
% from control diet
% from high fat diet
An opposite picture was observed with MCD diet. Rats fed a MCD diet showed a smaller food intake (Fig. 1C) and a drop in body mass (Fig. 1D) as classically observed with this diet [30–32]. The mass of retroperitoneal white adipose tissue was dramatically reduced (-44%) in MCD rats (Fig. 2) which clearly indicates no fat accumulation in white adipose tissue. Conversely, the liver mass of MCD rat was higher than that of controls (4.5 ± 0.2 vs 3.4 ± 0.1 g/100g; P < 0.05).
Liver histology and lipid content
Ability of the liver to oxidize fatty acids
Changes in dietary lipid can modify cell membrane composition that can, in turns, alter cell integrity resulting in a metabolism decrease. Hence, we studied gluconeogenesis which is controlled by, and therefore reflects, hepatocyte energy state. Using dihydroxyacetone as substrate, glucose production from isolated hepatocytes was not different in high fat diet groups as compared with standard group (Fig. 5C).
To verify that whole cell respiration measurement was not biased by the oxygen consumption of other organelles such as peroxisomes, the respiration capacities of isolated liver mitochondria were measured. Bovine serum albumin was added to eliminate any artefactual uncoupling effect of free fatty acids. No difference was found between mitochondrial oxygen consumption from high fat and standard diet fed rats (Fig. 5D). Therefore, resistance to steatosis was not explained by an enhanced ability of the liver to oxidize fatty acids.
Brown adipose tissue, fatty acid oxidation and uncoupling protein 1
In rodents, we succeeded in increasing lipid and caloric intake by a very large amount with an ad libitum access to diet. Nevertheless, this nutritional manipulation did not reproduce the typical hepatic lesion of NASH, i.e. steatosis, inflammation and fibrosis. No accumulation of triacylglycerols was observed in the liver of rats fed coconut oil containing 90% of SFA or butter with 51% of SFA. Such ability to overcome excessive energy intake may be related to rat ability to dissipate excess energy as heat. It is particularly true for young rats that resist becoming obese when fed a cafeteria-diet by increasing energy expenditure  through thermogenic processes occurring in liver  and BAT . Our results show that, in rats fed a high fat diet, the ability of the liver to oxidize fatty acid, as assessed by i) ketone body formation, and ii) hepatocyte and mitochondrial respiration, is not enhanced. In our model, the liver of Wistar rats appears very mildly affected by an overload in lipid intake and we can assume that fatty acid exportation from the liver is sufficient to favour peripheral storage. High fat feeding probably induced an increased capacity to export triacylglycerol in the form of VLDL. Indeed it has been shown that feeding a diet with 20% hydrogenated coconut oil was shown to increase VLDL and LDL levels by 15–17% in rats and by 44% in mice. Furthermore plasma ApoE and ApoB were increased while hepatic ApoE mRNA and ApoB mRNA were unchanged . Moreover, in our study, the high fat fed rats had increased white adipose tissue mass, which is in accordance with a higher triacylglycerol export from the liver to the adipose tissue. Similarly, feeding rats with various high fat diets (coconut oil, olive oil, menhaden oil, etc.) was shown to increase their epididymal fat mass [37–39]. However, we noted that fat accumulation in white adipose tissue was different depending on the type of fatty acid in the diets, and more precisely the length of the carbon chain. Indeed, coconut oil rich in medium chain saturated fat (mainly: lauric acid; C12:0; 44.6%) led to a lower peripheral accumulation than butter diet rich in longer chain saturated fat (mainly palmitic acid; C16:0; 21.7%). Many studies demonstrated that medium chain triglycerides (MCTs), such as in coconut oil, cause significant reduction in body weight or fat pad size in animals and humans [40–42]. This reduction of fat pad could be explained by the fact that MCT are transported directly to the liver via the portal vein and thus do not pass the adipose tissue before hepatic disposal. These characteristics could be responsible for the different rates of MCT oxidation versus LCT , and then could partly account for the difference in fat accumulation observed in white adipose tissue.
Another way to explain the lack of hepatic steatosis during lipid overload is peripheral utilisation. Mammals possess specialised thermogenic BAT that is characterized by a high amount of mitochondria containing high levels of UCP1, an uncoupling protein, located in the mitochondrial inner membrane . UCP activation (by coldness or diet) results in the uncoupling of substrate oxidation from ADP phosphorylation , with a resultant increase in heat production . In that thermogenic process, fatty acids act not solely as substrates for β-oxidation but are also involved in the uncoupling process by activating UCP1 transcription and activity . UCP1 expression is regulated by a fatty acid activated transcriptional factor: peroxisome proliferator-activated receptor (PPAR) . In our study interscapular BAT is larger, or tends to be larger, in the high fat fed groups, concomitantly with and increased content in UCP1, suggesting the implication of this tissue in fatty acid oxidation. BAT thermogenesis and UCP1 expression are known to increase during high-fat feeding, possibly to dissipate energy and to regulate body weight [35, 47–49]. We can therefore postulate that rats can adapt to excessive lipid ingestion: firstly, by increasing the storage of fatty acids in peripheral white adipose tissues, and secondly by over-expressing the UCP1-related thermogenesis in BAT.
At this time, the reference model for the study of NASH is the MCD diet [31, 32]. We confirm here that such diet induces a striking steatosis, demonstrated by a massive increase in hepatic triacylglycerol content. In the MCD diet-fed rats, steatohepatitis is the consequence of both the high-fat content and the methionine and choline deficiency. The lack of methionine reduces glutathione synthesis and impairs antioxidant defences against radical attacks. In addition, the choline deficiency impairs lipid exportation by decreasing the phosphatidylcholine synthesis, leading to a reduction in the fatty acid export from the liver . The fact that, after an MCD diet, the high steatosis is associated with the blocking of the lipid export from the liver consorts with our hypothesis that, in our study, high-fat fed rats are resistant to liver injury thanks to a very efficient lipid exportation. Apart from steatohepatitis, the key feature of human NASH, the MCD diet fails to induce the other characteristics of NASH, i.e. abdominal obesity and increased calorie intake. Therefore, the MCDD model is adequate to study the consequence of fat accumulation and inflammation in hepatocytes but is inadequate to study the pathogenesis of steatohepatitis.
Lipid characteristics of high fat diets used to induce steatosis and steatohepatitis
30.1% corn oil
17.6% olive oil
1.7% safflower oil
35.7% corn oil
15% corn oil
3.4% corn oil
10% lard oil
SFA (% fat)
MUFA (% fat)
PUFA (% fat)
24.7% corn starch
16.2% dextrin maltose
39% corn starch
Time of diet (week)
The lipid composition of the different diets which induce steatohepatitis (see Table 4) [19, 20, 51, 52, 54], were lard and corn oil, both oils rich in unsaturated fatty acids. We can observe that fat of all the diets inducing steatosis and inflammation (Table 4) were richer in MUFA and PUFA (>30% and >20% of total fat respectively) as compared to our diet (5% and 2%). The injurious effect of unsaturated fatty acids, and particularly n-6 polyunsaturated fatty acids, was associated with enhanced lipid peroxidation and decreased concentrations of antioxidant enzymes, implicating oxidative stress as a causal factor. Indeed, different studies showed the pro-inflammatory effect of polyunsaturated n-6 fatty acids which exacerbate liver oxidative stress [60, 61] and promote the development of NASH.
During the two last century, in Western diets, there has been a huge increase in n-6 fatty acid consumption and, the ratio of n-6 over n-3 fatty acids has increased from 1:1 to 15–20:1 [61, 62]. Arachidonic acid (n-6) and eicosapentanoic acid (n-3) are precursors for the production of eicosanoids, and have opposite metabolic effects. Cardiovascular diseases, diabetes, obesity, cancer and other pathologies are associated with increased production of thromboxane A2, leukotriene B4, Il-1β, IL-6 and TNF. All these factors increase consequently to a rise in n-6 fatty acid intake and decrease with a higher n-3 fatty acid intake . Different studies showed the pro-inflammatory effect of polyunsaturated n-6 fatty acids which exacerbate liver oxidative stress [63, 64] and promote the development of NASH. It follows that the development of steatohepatitis with high fat diet in rats may be facilitated by the use of MUFA and PUFA, especially n-6 fatty acid, than SFA. However, these results contradict the observations made in humans where the daily intake of PUFA is around 5% (w/w) in the general population and 3.5% in NASH patients . A high fat diet with saturated fatty acids is not sufficient to induce a steatosis and then a steatohepatitis. A number of studies showed, that it may be more suitable to use a high mono- and polyunsaturated fatty acid diet to induce NASH in rats. It appears here that the key factor could be the possible induction of the lipid peroxidation and pro-inflammatory cytokine production by the high level of PUFA leading to steatosis and inflammation. Another possibility is that Wistar rat is not a suitable model to study obesity and pathologic modifications in the liver consecutively to a modification of the diet .
In conclusion, Wistar rats have an incredible capacity to adapt to a large increase of lipids in their alimentation. The mechanism underlying this resistance to high fat feeding is complex and involves both a change in body composition, with an increased storage in white adipose tissue, and an activation of lipid oxidation in BAT. We can conclude that, feeding Wistar rats with a high saturated fat diet does not induce liver failure and cannot be used as model of NASH. However, it is a good model for studying the adaptations of the organism to a high fat diet. Hence, it is still necessary to conceive a diet that can induce NASH, maybe by coupling a high fat diet with a stress, like inducing insulino-resistance or increasing ROS production.
This work was supported by the French Ministère de l'Enseignement, de la Recherche et de la Technologie and by a grant from Centre National de la Recherche Scientifique.
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