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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2011 Dec;92(6):413–421. doi: 10.1111/j.1365-2613.2011.00793.x

Pathology of the liver in obese and diabetic ob/ob and db/db mice fed a standard or high-calorie diet

Viviane Trak-Smayra *, Valérie Paradis †, Julie Massart ‡, Selim Nasser §, Victor Jebara , Bernard Fromenty ‡
PMCID: PMC3248077  PMID: 22118645

Abstract

Non-alcoholic fatty liver disease (NAFLD) is one of the commonest liver diseases in Western countries. Although leptin deficient ob/ob and db/db mice are frequently used as murine models of NAFLD, an exhaustive characterization of their hepatic lesions has not been reported to date, particularly under calorie overconsumption. Thus, liver lesions were characterized in 78 ob/ob and db/db mice fed either a standard or high-calorie (HC) diet, for one or three months. Steatosis, necroinflammation, apoptosis and fibrosis were assessed and the NAFLD activity score (NAS) was calculated. Steatosis was milder in db/db mice compared to ob/ob mice and was more frequently microvesicular. Although necroinflammation was usually mild in both genotypes, it was aggravated in db/db mice after one month of calorie overconsumption. Apoptosis was observed in db/db mice whereas it was only detected in ob/ob mice after HC feeding. Increased apoptosis was frequently associated with microvesicular steatosis. In db/db mice fed the HC diet for three months, fibrosis was aggravated while steatosis, necroinflammation and apoptosis tended to alleviate. This was associated with increased plasma β-hydroxybutyrate suggesting an adaptive stimulation of hepatic mitochondrial fatty acid oxidation (FAO). Nevertheless, one-third of these db/db mice had steatohepatitis (NAS ≥ 5), whereas none of the ob/ob mice developed non-alcoholic steatohepatitis under the same conditions. Steatosis, necroinflammation, apoptosis and fibrosis are modulated by calorie overconsumption in the context of leptin deficiency. Association between apoptosis and microvesicular steatosis in obese mice suggests common mitochondrial abnormalities. Enhanced hepatic FAO in db/db mice is associated with fibrosis aggravation.

Keywords: animal models, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, obese mice

Non-alcoholic fatty liver disease is currently one of the most common liver diseases in western countries, paralleling the continuous increase in the prevalence of obesity (Farrell & Larter 2006; Fabbrini et al. 2010). Importantly, NAFLD is associated with insulin resistance and the metabolic syndrome. In most cases, NAFLD follows a benign course with the presence of isolated fatty change, but in a few patients non-alcoholic steatohepatitis (NASH) develops and can progress to cirrhosis and hepatocellular carcinoma (Farrell & Larter 2006). Despite numerous experimental and clinical investigations, the physiopathology of obesity-related fatty liver and NASH is still poorly understood although insulin resistance, mitochondrial dysfunction and oxidative stress seem to play a prominent role (Robertson et al. 2001; Begriche et al. 2006; Fabbrini et al. 2010).

Many of the investigations pertaining to obesity and related metabolic disorders are carried out in genetic leptin-deficient ob/ob mice and leptin-resistant db/db mice (Anstee & Goldin 2006; Lindström 2007). Ob/ob mice carry a mutation resulting in the synthesis of a truncated, non-functional leptin protein. On the other hand, db/db mice harbour a mutation which induces the lack of the long isoform of the leptin receptor (Ob-Rb). As leptin plays a major role in food intake and energy expenditure, total leptin deficiency or leptin resistance is leading to massive obesity, type 2 diabetes, dyslipidaemia and fatty liver (Anstee & Goldin 2006; Lindström 2007; Fromenty et al. 2009). However, despite the extensive utilization of ob/ob and db/db mice for experimental purposes, a comprehensive characterization of the different hepatic lesions has not been reported thus far for these murine models of NAFLD. Hence, the first aim of the current study was to fully characterize the different liver lesions in ob/ob and db/db mice including macrovacuolar and microvesicular steatosis, necroinflammation, apoptosis as well as portal and perisinusoidal fibrosis. Moreover, the obese animals were also fed a high-calorie diet for 3 months to determine whether ob/ob and db/db mice respond differently to calorie overconsumption.

Materials and methods

Animals, diets and collection of blood and liver samples

Thirty eight male C57BL/KsJ-db/db mice (henceforth referred to as db/db mice) aged 7 weeks and forty male C57BL/6J-ob/ob mice (henceforth referred to as ob/ob mice) of the same age were purchased from Janvier laboratories (Le Genest St Isle, France). After a week of acclimatization and feeding with a standard diet, animals were divided into four groups according to their strain and type of diet. Whereas 19 db/db mice and 20 ob/ob mice were fed a standard calorie diet (henceforth referred herein to as standard chow), 19 other db/db mice and 20 other ob/ob mice received a high-calorie (HC) diet. All mice were allowed free access to tap water and food pellets purchased from SAFE laboratory (Augy, France). Whereas the standard chow (A04) contains 16% proteins, 3% lipids, 60% carbohydrates (mainly starch) and provides 2900 kcal/kg, the HC diet (also referred to as Western diet) contains 16% proteins, 16% lipids, 46% carbohydrates (including 11% saccharose) and provides 3920 kcal/kg. Composition of the standard and high-calorie diets is detailed in Table 1. Western diets enriched in fat and simple carbohydrates (such as saccharose) promote fatty liver not only by increasing the delivery of lipids to this tissue but also by upregulating hepatic de novo lipogenesis, which is favoured by insulin resistance (Begriche et al. 2006; Harmancey et al. 2010).

Table 1.

Detailed composition of the standard and high-calorie diets

Standard A04 diet High-calorie diet
Moisture (%) 11 10
Crude protein (%) 16 16
Crude oil (%) 3.1 16
Nitrogen free extracts (%) 60 47
 Of which starch (%) 45.8 29
 Of which total sugars (%) 2 12
Crude fibres (%) 3.9 2.4
Total minerals (%) 5.1 7.8
Calcium (mg/kg) 8400 1500
Phosphorus (mg/kg) 5700 2700
Sodium (mg/kg) 2500 8000
Potassium (mg/kg) 6400 3000
Manganese (mg/kg) 70 3.6
Copper (mg/kg) 17 2.7
Vitamin A (IU/kg) 6600 4700
Vitamin D3 (IU/kg) 900 500
Vitamin E (mg/kg) 30 20
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The mean body weight of mice ± standard error of the mean (SEM), at the beginning of the experiment, was 39.5 ± 1.2 g for db/db mice fed a SC diet, 39.1 ± 0.8 g for db/db under HC diet, 36.6 ± 1.9 g for ob/ob mice fed a SC diet and 39.1 ± 1 g for ob/ob mice under HC diet. All mice were weighed twice a week during the experimental period which lasted one or 3 months. Accordingly, half of the mice were killed after 1 month while the remaining animals were euthanized after 3 months. Killing of the animals in the fed state was performed by cervical dislocation, under anaesthesia. Immediately before killing, blood samples were collected from the retroorbital sinus and centrifuged to obtain plasma that was stored at −20 °C until analysis. Following killing, the liver was dissected and small fragments of liver tissue were either frozen at −80 °C or immediately fixed in 10% neutral formalin, embedded in paraffin and routinely processed for histopathological analysis.

Blood parameters

Plasma triglycerides, glucose, alanine aminotransferase (ALT), lactate dehydrogenase (LDH), total cholesterol, β-hydroxybutyrate, non-esterified fatty acids (NEFAs) and total antioxidant status (expressed as Trolox equivalents) were measured on an automatic analyzer (Olympus AU400: Olympus Diagnostics, Rungis, France.) as previously described (Begriche et al. 2008a,b;). Triglycerides, glucose, ALT, LDH and total cholesterol were measured with commercial kits (OSR6133, OSR6121, OSR6107, OSR6126, OSR6116, respectively) from Olympus Diagnostic (Rungis, France), whereas β-hydroxybutyrate, NEFAs and total antioxidant status were measured with commercial kits (RB1007, FA115 and NX2332, respectively) from RANDOX Diagnostic (Montpellier, France). Plasma leptin in db/db mice was measured using double antibody RIA kit (ML-82K) purchased from Linco Research (St Charles, MO, USA).

Histopathological analysis and in situ detection of apoptosis

Paraffin sections of liver tissue were stained with haematoxylin-eosin (H&E) and Sirius red. Steatosis, necroinflammation as well as portal and perisinusoidal fibrosis were then assessed. Regarding steatosis, three different parameters were determined namely intensity, distribution and type of lipid accumulation in the liver lobules. The intensity of lipid accretion was evaluated as the percentage of hepatocytes containing one or several lipid vacuoles in 10 randomly chosen different medium power fields (× 200). Whereas the distribution of steatosis was categorized as centrilobular, mediolobular or panlobular, its type was classified as microvesicular, macrovacuolar or mediovesicular. While microvesicular steatosis is defined by the presence of minute cytoplasmic lipid droplets around a centrally positioned nucleus, macrovacuolar steatosis refers to as a single large cytoplasmic lipid vacuole displacing the nucleus to the periphery of the hepatocyte (Figure 1a,c). In the current study, steatosis was considered as mediovesicular when several medium-sized lipid vacuoles were present in the cytoplasm of the hepatocytes (Figure 1b).

Figure 1.

Figure 1

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Representation of macrovacuolar (a) mediovesicular (b) and microvesicular (c) steatosis (H&E, × 400).

Necroinflammation was scored 0 (absent), 1 (mild) or 2 (moderate) depending on the number of inflammatory infiltrates and apoptotic bodies. Mild necroinflammation referred to few lobular aggregates of inflammatory cells with or without apoptotic bodies. Necroinflammation was considered moderate when at least one lobular area contained two or more of such aggregates (Figure 2a,b).

Figure 2.

Figure 2

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Representation of mild (a) and moderate (b) necroinflammation (H&E, × 200), mild (c) and moderate (d) portal fibrosis (Sirius Red × 100) and mild (e) and moderate (f) perisinusoidal fibrosis (Sirius Red × 200).

Non-alcoholic fatty liver disease activity score (NAS) was calculated according to the definition of the Pathology Committee of the NASH Clinical Research Network (Kleiner et al. 2005), by adding the scores of steatosis, hepatocellular ballooning and inflammation. According to this semi-quantitative histological scoring system, NAS equal or superior to five is diagnostic of NASH. However, NAS in the current study was evaluated only by the sum of steatosis and necroinflammation scores because ballooning degeneration was not observed in mouse liver, whatever the experimental group. Although NAS has originally been established and validated in human adult and paediatric patients (Kleiner et al. 2005), recent animal studies have used this score to assess NASH (Piguet et al. 2009; Martín-Castillo et al. 2010; Kuwashiro et al. 2011).

Sections stained with Sirius red were used to evaluate fibrosis. Portal and perisinusoidal fibrosis were scored as absent (0), mild (1) or moderate (2) depending on the extent of extracellular matrix deposition (Figure 2c–f).

Finally, in situ detection of apoptosis was performed with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay by using the ApopTag Plus Peroxydase In Situ detection kit (Millipore, France), according to the manufacturer's instructions. Four micrometer sections of paraffin-embedded liver tissue were first deparaffinized with xylene and alcohol and then washed with PBS. Proteinase K was applied to the slides and endogenous peroxidase was subsequently quenched with 1% hydrogen peroxide solution. Cells were then permeabilized using an equilibration buffer supplied by the manufacturer and incubated with TdT enzyme for 1 h at 37 °C. After washing with the supplied stop/wash buffer and PBS, the slides were incubated with an anti-digoxygenin conjugate, washed and incubated again with a peroxidase substrate. Light green was used for counterstaining. The percentage of stained nuclei was subsequently assessed for each liver sample, using light microscope (Figure 3).

Figure 3.

Figure 3

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Apoptosis as assessed by terminal deoxynucleotidyl transferase nick-end labelling (TUNEL): weak (a) and intense (b) staining (× 400). Positive nuclei are stained in brown colour.

Statistical analysis

Data pertaining to body weight, plasma parameters, steatosis and apoptosis are presented as means ± standard error of the mean (SEM). For body weight and plasma parameters, a two-way analysis of variance (anova) with the factors of time (1 or 3 months) and diet (standard chow or HC) was performed to assess statistical significances. When the anova indicated a significant interaction between factors, individual means were compared with least significant difference (LSD) post hoc test. A P value inferior to 0.05 was considered as significant.

Results

Body weight and biological parameters

As expected, body weight of db/db and ob/ob mice significantly increased with time and under-feeding with a HC diet (Table 2). In general, body weight of ob/ob mice tended to be higher compared to db/db mice except after 1 month of HC diet (Table 2). Regarding plasma parameters, β-hydroxybutyrate was dramatically increased in db/db mice after 3 months of the HC diet (Table 2), suggesting a stimulation of mitochondrial fatty acid oxidation (FAO) in liver (Begriche et al. 2008a,b;). In addition, HC feeding significantly enhanced total cholesterol in db/db mice whereas the other parameters were unchanged (Table 2). However, it is noteworthy that the large variation observed for ALT and LDH values may have precluded statistical differences between the standard chow and HC groups. In ob/ob mice, β-hydroxybutyrate was not increased after 3 months of the HC diet (Table 2). In contrast, plasma NEFAs were significantly increased after 1 month of HC diet (Table 2), suggesting enhanced lipolysis (i.e. hydrolysis of adipose triglycerides and the subsequent release of NEFAs into the blood) (Fromenty et al. 2009). However, plasma NEFAs were normalized after 3 months of HC diet. Finally, whereas HC feeding enhanced total cholesterol in plasma in ob/ob mice there was a time-dependent increase in the antioxidant status whatever the diet (Table 2).

Table 2.

Body weight and plasma parameters in db/db and ob/ob mice after 1 and 3 months of standard chow or high-calorie (HC) diet

Standard chow HC diet
1 month 3 months 1 month 3 months
db/db mice n = 9 n = 10 n = 10 n = 9
Body weight (g) 44.5 ± 2.3 52.3 ± 1.0* 53.1 ± 0.9† 58.6 ± 1.9*†
Leptin (ng/ml) 144 ± 18 215 ± 15 208 ± 27 179 ± 35
β-Hydroxybutyrate (mM) 0.22 ± 0.05 0.20 ± 0.03 0.14 ± 0.03 1.90 ± 0.97*†
NEFAs (mM) 1.58 ± 0.43 1.62 ± 0.66 1.29 ± 0.07 1.49 ± 0.21
Triglycerides (mM) 2.17 ± 0.16 2.32 ± 0.40 1.89 ± 0.12 2.00 ± 0.26
Total cholesterol (mM) 3.49 ± 0.28 4.33 ± 0.24 7.80 ± 0.42† 8.31 ± 1.17†
Glucose (mM) 58.2 ± 4.7 64.5 ± 8.2 60.0 ± 4.9 57.0 ± 4.3
Antioxidant status (mM) 1.16 ± 0.08 1.36 ± 0.09 1.33 ± 0.08 1.34 ± 0.13
ALT (UI/l) 660 ± 229 506 ± 158 979 ± 335 794 ± 369
LDH (UI/l) 8234 ± 2468 4400 ± 672 7440 ± 2526 5643 ± 2075
ob/ob mice n = 10 n = 10 n = 10 n = 10
Body weight (g) 47.5 ± 1.3 56.2 ± 1.0* 51.1 ± 1.4† 59.2 ± 1.0*†
β-Hydroxybutyrate (mM) 0.08 ± 0.02 0.07 ± 0.01 0.07 ± 0.02 0.07 ± 0.01
NEFAs (mM) 0.92 ± 0.09 0.97 ± 0.09 1.37 ± 0.13† 0.99 ± 0.09*
Triglycerides (mM) 1.10 ± 0.10 1.22 ± 0.09 1.42 ± 0.20 1.21 ± 0.14
Total cholesterol (mM) 5.02 ± 0.14 5.24 ± 0.53 6.26 ± 0.39† 5.91 ± 0.34†
Glucose (mM) 23.0 ± 2.9 20.3 ± 1.8 23.3 ± 4.0 21.3 ± 2.0
Antioxidant status (mM) 1.16 ± 0.05 1.20 ± 0.05 1.04 ± 0.07 1.30 ± 0.09*
ALT (UI/l) 681 ± 225 592 ± 118 218 ± 39 400 ± 212
LDH (UI/l) 6982 ± 3070 5217 ± 772 5386 ± 812 3827 ± 1094
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Blood was drawn in the fed state. Results are means ± SEM for the number of mice specified in the table, excepted for leptin which was assessed in 4–8 db/db mice per group.

Symbols indicate statistical significance between groups

*

Different from 1 month (P<0.05).

†

Different from standard chow (P<0.05).

ALT, alanine aminotransferase; LDH, lactate dehydrogenase; NEFA, non-esterified fatty acids.

Steatosis

Next, we performed a thorough pathological examination of the liver with respect to the mouse genotype and the diet. We first noticed that all mice showed marked steatosis except for a single db/db mouse which had virtually no lipid accumulation after 1 month of standard chow (this mouse was nevertheless considered for the calculation of the mean percentage of steatosis in this group). However, fatty change in ob/ob mice was more severe than in db/db mice whatever the diet but this difference was more obvious after 1 month of standard chow (Table 3). Interestingly, while the HC diet enhanced the severity of fatty change in db/db mice after 1 month, steatosis was subsequently reduced after 3 months of HC feeding (Table 3). It was also noteworthy that the type of steatosis was somewhat different between ob/ob and db/db mice. In particular, microvesicular steatosis was more frequently observed in db/db mice compared to ob/ob mice whatever the diet. However, the number of hepatocytes with small lipid droplets tended to decrease with time in both db/db and ob/ob mice fed the standard chow or the HC diet (Table 3). In contrast, mediovesicular steatosis was more prevalent in ob/ob mice than in db/db mice and this trend was observed whatever the diet (Table 3). Interestingly, macrovacuolar steatosis was about similar between ob/ob and db/db mice with a number of hepatocytes harbouring a large lipid vacuole increasing with time in both genotypes (Table 3). Steatosis was mainly localized in the centrilobular and mediolobular areas in ob/ob and db/db except for 2 db/db mice that showed only mediolobular steatosis.

Table 3.

Pathological features of liver lesions in db/db and ob/ob after 1 and 3 months of standard chow or high-calorie (HC) diet

Standard chow HC diet
ob/ob db/db ob/ob db/db
1 month 3 months 1 month 3 months 1 month 3 months 1 month 3 months
Steatosis
 Percentage 82 ± 1 80 ± 3 58 ± 8† 69 ± 3† 92 ± 2§ 89 ± 2§ 86 ± 2†§ 76 ± 2†§*
 Type
  Micro ++ + +++ ++ ++ + +++ ++
  Medio ++ ++ + + ++ ++ + +
  Macro + ++ + ++ + ++ + ++
Necroinflammation
 0 1 1 4 0 0 0 0 2
 1 8 7 5 8 9 10 8 4
 2 1 2 0 2 1 0 2 3
Apoptosis Percentage 3 ± 1 0 ± 0* 13 ± 2† 15 ± 1† 10 ± 1§ 7 ± 1§ 15 ± 2 11 ± 1
Portal fibrosis
 0 6 0 5 1 1 1 1 0
 1 4 10 4 9 9 9 9 7
 2 0 0 0 0 0 0 0 2
Perisinusoidal fibrosis
 0 10 5 9 6 8 4 10 2
 1 0 4 0 4 2 5 0 6
 2 0 1 0 0 0 1 0 1
NAS
 0–2 0 0 5 0 0 0 0 0
 3–4 9 8 4 8 9 10 8 6
 5–8 1 2 0 2 1 0 2 3
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Results for steatosis and apoptosis are means ± SEM for 9–10 mice. Results for necroinflammation and fibrosis are given as the number of mice presenting a score of 0, 1 or 2 respectively. Results for NAS (non-alcoholic fatty liver disease activity score) are given as the number of mice presenting scores of 0–2, 3–4 or 5–8 respectively.

Symbols indicate statistical significance between groups

*

Different from 1 month (P<0.05).

†

Different from ob/ob mice of the same age (P<0.05).

§

Different from standard chow (P<0.05).

Necroinflammation

On the whole, hepatic necroinflammation was not prominent in ob/ob and db/db although some differences were observed between groups. In particular, necroinflammation tended to be more frequently detected in ob/ob mice compared to db/db mice after 1 month of standard chow but this trend was no longer observed after 3 months (Table 3). When db/db mice were fed the HC diet, we noticed that necroinflammation was aggravated after 1 month when compared to the standard feeding but this was not observed in ob/ob mice. However, necroinflammation tended to alleviate with time in db/db fed the HC diet.

NAFLD activity score (NAS)

Non-alcoholic fatty liver disease activity score was calculated as indicated in the Materials and Methods to evaluate the presence of NASH. With this histological scoring system, steatohepatitis (NAS ≥ 5) was observed in two of 10 ob/ob and db/db mice, after 3 months of a standard diet (Table 3). However, when mice where fed the HC diet for 3 months, NASH was not observed in ob/ob mice whereas it was present in three of 9 db/db mice (Table 3).

Apoptosis

Apoptosis was also assessed in this study using the TUNEL assay. When mice were fed the standard chow, apoptosis was rare or absent in ob/ob mice whereas the percentage of cells undergoing apoptosis in db/db mice was about similar with time (Table 3). It was also noteworthy that the HC diet induced some apoptosis in ob/ob mice while the number of apoptotic hepatocytes was similar in db/db mice after 1 month of HC diet compared to standard chow (Table 3). However, apoptosis tended to be less prominent in db/db and ob/ob mice after 3 months of feeding with the HC diet, although the difference did not reach statistical significance (Table 3). We also noticed that high number of apoptotic hepatocytes could be detected in areas with prominent microvesicular or mediovesicular steatosis (Figure 3).

Portal and perisinusoidal fibrosis

Finally, portal and perisinusoidal fibrosis were evaluated in the different groups of animals. Importantly, both types of fibrosis could be detected in a significant number of ob/ob and db/db mice although this liver lesion was usually mild (Table 3). It is also worth mentioning that fibrosis was overall about similar in ob/ob and db/db mice fed the standard chow. In particular, we noticed that the number of mice without portal or perisinusoidal fibrosis decreased with time in both genotypes (Table 3). When mice were fed the HC diet, the most significant change was observed for portal fibrosis in particular after 1 month. Indeed, at this time only one animal in each genotype was without portal fibrosis. In addition, two db/db mice fed the HC diet developed moderate portal fibrosis after 3 months (Table 3). In contrast, perisinusoidal fibrosis was less affected by calorie overconsumption except for db/db mice after 3 months of HC feeding. Indeed, in this group there were only two mice without perisinusoidal fibrosis whereas 6 db/db mice did not present this liver lesion after 3 months of standard chow.

Discussion

To the best of our knowledge, the current study is the first one to provide a thorough characterization of the different liver lesions present in obese and diabetic ob/ob and db/db mice fed a standard chow or a high-calorie diet. Overall, our data pertaining to liver pathology in these animals (Table 3) can be summarized as follows: (i) Under normal diet, fatty change in db/db mice was milder compared to ob/ob mice but this difference between genotypes weakened when the obese animals were fed a HC diet; (ii) Microvesicular steatosis was more frequently observed in db/db mice whatever the diet and the duration of feeding; (iii) Necroinflammation was usually mild in both genotypes but calorie overconsumption for 1 month aggravated this lesion in db/db mice; (iv) Apoptosis was rare in ob/ob mice fed a normal chow but was more frequently detected after HC feeding; (v) Increased number of apoptotic hepatocytes was frequently associated with microvesicular steatosis; (vi) Although usually mild, portal fibrosis was more frequently detected in both genotypes when compared to perisinusoidal fibrosis; and (vii) When db/db mice were fed a HC diet, fibrosis aggravated over time, whereas steatosis, necroinflammation and apoptosis tended to improve. Nevertheless, one-third of db/db mice presented steatohepatitis (NAS ≥ 5) after 3 months of HC diet, whereas none of the ob/ob mice developed NASH in the same conditions. Some of these observations will be further discussed below.

The relatively milder fatty change in C57BL/KsJ-db/db mice compared to C57BL/6J-ob/ob mice is puzzling as these mice present full-blown diabetes and hyperglycaemia (Table 2), which is expected to stimulate hepatic de novo lipogenesis through the activation of carbohydrate response element-binding protein (ChREBP) (Iizuka & Horikawa 2008; Begriche et al. 2009). However, a recent study suggests that hepatic lipid synthesis could be impaired in C57BL/KsJ-db/db mice (Davis et al. 2010), although hepatic ChREBP activity has not been assessed in this study. Interestingly, an apparent stimulation of hepatic mitochondrial FAO occurred when db/db mice were fed the HC diet as plasma β-hydroxybutyrate was dramatically increased after 3 months of calorie overconsumption (Table 2). This suggests that hepatic FAO in db/db mice can be greatly stimulated in some metabolic contexts such as lipid overload. Furthermore, this may explain (at least in part) why steatosis in db/db mice was alleviated after 3 months of calorie overconsumption (Table 3). Indeed, enhanced FAO is one adaptive mechanism that can be set up in liver to curb lipid accretion (Begriche et al. 2008a, 2009). In the current study, plasma β-hydroxybutyrate was measured in the fed state which is normally associated with lower rate of mitochondrial FAO and ketogenesis. Hence, further investigations will be needed to determine whether milder fatty liver in db/db mice could also be explained by higher hepatic FAO during fasting periods.

Interestingly, the apparent improvement of steatosis in db/db mice after 3 months of HC feeding was associated with some aggravation of fibrosis whereas necroinflammation and apoptosis tended to be alleviated (Table 3). Although we do not have definite explanation for this observation, it is noteworthy that lipid intermediates such as free fatty acids can induce cell death and inflammation (Joshi-Barve et al. 2007; Cazanave et al. 2009) and that mitochondrial FAO can generate high amount of reactive oxygen species (ROS) (Begriche et al. 2009; Seifert et al. 2010). Moreover, mitochondrial ROS could be involved in hepatic fibrogenesis (Wang & Weinman 2006; Mitchell et al. 2009). Thus, the combination of reduced levels of deleterious lipid intermediates and higher generation of ROS caused by higher mitochondrial FAO could explain the opposite evolution of necroinflammation and apoptosis on one hand and fibrosis on the other hand.

In the current study, intensity of lipid accretion was evaluated in H&E-stained sections as the percentage of hepatocytes containing one or several lipid vacuoles. However, it would be interesting to quantify hepatic triglycerides by biochemical quantification (Begriche et al. 2008a,b;), or to assess lipid content in hepatocytes with digital image analysis quantification of oil-red O-stained sections (Levene et al. 2010). Indeed, utilization of these more accurate methods could be valuable to better characterize the evolution of steatosis in ob/ob and db/db mice under standard chow and HC diet.

Microvesicular steatosis is deemed to be the consequence of severe impairment of mitochondrial function, in particular FAO and respiratory chain activity (Fromenty & Pessayre 1995; Rinaldo et al. 1999; Begriche et al. 2011). Thus, mitochondrial dysfunction could affect a larger number of hepatocytes in db/db mice than in ob/ob mice although further investigations will be required to address this issue. In addition, it will be interesting to determine why microvesicular steatosis alleviates with time in both genotypes whatever the diet (Table 3). Different investigations have shown that several mitochondrial and metabolic adaptations are taking place in the liver of obese rodents (including ob/ob mice) and patients, in particular regarding FAO and oxidative stress (Robin et al. 2005; Garcia-Ruiz et al. 2007; Begriche et al. 2009). These adaptive changes may be set up to curb lipid accumulation, as previously discussed but also to limit oxidative stress in the liver (Robin et al. 2005; Begriche et al. 2009). Thus, the current study may suggest that mitochondrial function taken as a whole could improve with age in both ob/ob and db/db mice.

The number of apoptotic hepatocytes was low or nil in ob/ob mice fed a standard chow, thus confirming previous data (Robin et al. 2005; Dey & Cederbaum 2007; Begriche et al. 2008b). Moreover, ethanol intoxication (Robin et al. 2005) and drug-induced induction of cytochrome P450 2E1 (Dey & Cederbaum 2007) are able to activate hepatic apoptosis in ob/ob mice. Thus, it appears that apoptosis can be triggered in ob/ob liver by different toxic or nutritional factors. In contrast to ob/ob mice, apoptosis was more frequently observed in C57BL/KsJ-db/db mice fed with the standard diet (Table 3). In addition, apoptosis was not increased by calorie overconsumption in these mice and the number of apoptotic hepatocytes was even reduced after 3 months of HC diet (Table 3). Thus, further investigations are warranted to determine whether this apparent resistance to apoptosis can occur with other factors such as toxic compounds.

One major mechanism leading to apoptosis is mitochondrial dysfunction (Begriche et al. 2006, 2011; Malhi et al. 2010). Thus, it was worth mentioning that apoptosis was more frequently observed when microvesicular steatosis was more prevalent. Moreover, whereas microvesicular steatosis was less frequently seen with age the same trend was observed for apoptosis, in particular in mice fed the HC diet. Thus, it is conceivable that the same mitochondrial abnormalities could be common cause responsible for both microvesicular steatosis and apoptosis in obese mice.

Previous studies have shown that fibrosis in ob/ob mice is mild and that it is hardly aggravated by different factors including carbon tetrachloride or a diet deficient in methionine and choline (MCD) (Potter et al. 2003; Sahai et al. 2004). This resistance to fibrosis is attributable to the lack of leptin which activates stellate cells and promotes fibrogenesis (Potter et al. 2003; Bethanis & Theocharis 2006). In contrast, the MCD diet is able to aggravate hepatic fibrosis in db/db mice (Sahai et al. 2004) and in the current study, fibrosis tended to be more prevalent in db/db mice fed for 3 months with a HC diet (Table 3). Even though db/db mice harbour a deficiency in the long isoform of the leptin receptor (Ob-Rb), leptin is able to trigger some biological effects in these mice thanks to several short isoforms of this receptor (Harris et al. 2001; Madiehe et al. 2002; Osborn et al. 2010). Moreover, fa/fa rats (which are also deficient in Ob-Rb) developed periportal fibrosis when fed a high-fat diet (Carmiel-Haggai et al. 2005). Thus, further investigations will be required to determine whether aggravation of fibrosis in db/db mice is mediated by the short isoforms of the leptin receptor.

In ob/ob and db/db mice fed a standard chow, necroinflammation and fibrosis are mild to moderate despite extensive lipid deposition in the liver. Steatohepatitis, as defined by NAS ≥ 5, was nevertheless present in some ob/ob and db/db mice, although ballooning degeneration could not be detected despite a thorough histological analysis. Hence, because of the infrequent development of steatohepatitis in standard nutritional conditions, these animal models are sometimes not considered as relevant models of NASH. It is also noteworthy that ob/ob and db/db mice exhibit the metabolic abnormalities that underlie human NAFLD, such as obesity, insulin resistance and dyslipidaemia. This is in contrast to wild-type rodents fed the methionine choline deficient (MCD) diet, which develop NASH with a concomitant loss of body weight and without whole-body insulin resistance (Rinella & Green 2004; Rizki et al. 2006; Ota et al. 2007). Hence, although ob/ob and db/db mice do not display the whole spectrum of human NASH, these obese animals could represent a suitable model to study NAFLD as the majority of obese and diabetic individuals develop simple steatosis without major necroinflammation or extensive fibrosis (Bellentani et al. 2010; Schattenberg & Galle 2010).

Acknowledgments

This research was supported by the ‘Conseil de Recherche de l'Université Saint-Joseph’ (FM 123).

Conflict of interest

The authors declare that they have no conflict of interest.

Institutional approval

The experimental protocol was approved by the research counsel of Saint Joseph University.

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