Animal housing and procedures were approved by the Animal Experiments Ethics Committee of the Sungkyunkwan University, Kangbuk Samsung Hospital. Otsuka Long-Evans Tokushima Fatty (OLETF) and age-matched Long-Evans Tokushima Otsuka (LETO) rats were purchased from Otsuka Pharmaceutical Company (Tokushima, Japan). Rats at 4 weeks of age were maintained in a temperature and humidity-controlled specific pathogen-free facility on a 12-h light/12-h dark cycle and had unrestricted access to water and a standard irradiated rodent chow diet (PicoLab Rodent Diet 20 5053, 5% wt/wt fat, Purina Mills, Richmond, IN, USA). To investigate the role of CB1 receptor blockade via rimonabant in hepatic lipid accumulation and inflammation, 20 rats at 32 weeks of age were treated with either PBS as vehicle or rimonabant (10 mg·kg^-1·day^-1, Sanofi-Aventis, Paris, France) by daily oral gavage for an additional 6 weeks. Pair-fed controls were given a daily amount of food equal to that consumed by rimonabant-treated counterparts over the previous 24 h period. Body weight and food intake were monitored daily. At the end of 6-week animal experiment, 38-week-old LETO and OLETF rats were fasted overnight and anesthetized with intraperitoneal Zoletil/Rompun. Blood was collected from abdominal aorta and liver tissues were dissected, immediately frozen in liquid nitrogen, and stored at −80°C until further analysis.

An oral glucose tolerance test (OGTT) was performed after 16 h fasting. Rats received an oral administration of glucose solution (2 g/kg body weight) by stomach gavage. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after the glucose challenge, using a Glucocard X-Meter (Arkray, Kyoto, Japan). The area under the curve (AUC) was calculated and the difference (ΔAUC) was reported.

Serum glucose, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were analyzed by enzymatic enzyme methods (Sigma-Aldrich, St. Louis, MO, USA). For hepatic triglyceride (TG) quantification, lipid was extracted as described in a previous study [28]. Briefly, liver tissue was homogenized in 1 mL solution containing 5% Nonidet P-40 (NP-40) substitute (Amresco, Solon, OH, USA). The homogenates were slowly heated to 80–100°C in a water bath for 2–5 min until the NP-40 became cloudy, then cooled down to room temperature. Samples were then centrifuged for 2 min to remove any insoluble materials. Hepatic TG levels were measured by enzymatic assay (Sigma-Aldrich) and normalized to their respective protein concentrations. Lipid peroxidation (MDA) levels in the liver tissue were measured by a OxiSelect^TM TBARS assay kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s protocol, normalized to their respective protein concentrations, and expressed as fold change compared to LETO control group.

Dissected liver tissues were fixed with 10% neutral formalin buffer overnight, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and Masson's trichrome. Digital images were acquired with a microscope (Olympus BX51 light microscope, Tokyo, Japan). To investigate the severity of NAFLD or fibrosis, a pathologist blinded to the experiment measured the NAFLD activity score (NAS) or fibrosis score. NAS was calculated by measuring three features of NAFLD and adding up their scores: steatosis (0–3), ballooning (0–2), and inflammation (0–3) [29]. To illustrate the fibrosis stage, fibrosis score was calculated by summing up scores: no fibrosis (0), perisinusoidal or periportal fibrosis (1), perisinusoidal and portal/periportal fibrosis (2), bridging fibrosis (3), and cirrhosis (4) [30].

The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed using an In Situ Cell Death Detection Kit, TMR red (Roche, Indianapolis, IN, USA), according to the manufacturer’s instruction. The TUNEL assay is based on the detection of DNA fragmentation by labeling 3'-hydroxyl ends in double-stranded DNA breaks in the early stages of apoptosis. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 10 min, and the slides were observed and visualized on a LSM700 confocal microscope (Carl Zeiss, Jena, Germany) at 800 X magnification. Results were expressed as the number of TUNEL-positive cells per microscopic field.

Isolation of RNA from liver tissues was performed using an RNeasy Mini Kit (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer’s protocol. cDNA was synthesized from isolated total RNA using a high-capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA). The polymerase chain reaction (PCR) was performed using a LightCycler 480 Probes Master Mix and a Lightcycler 480 system (Roche). Primers used are shown in Table 1. The PCR parameters were as follows: pre-denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s and annealing/extension at 60°C for 20 s. Data were analyzed using the ΔΔCt method for relative quantification [31]. Expression of each target gene was normalized to the housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal RNA (18S) and expressed as fold change relative to LETO control group.

Total protein was isolated from liver tissues by homogenization in an ice-cold lysis buffer containing 20 mM HEPES-KOH (pH 7.9), 125 mM NaCl, 10% glycerol, 0.3% Triton X-100, 1 mM EDTA, 0.5% NP-40, 10 mM β-phosphoglycerate, 1 mM Na₃VO₄, 5 mM NaF, 1 mM aprotinin, 1 mM phenylmethanesulfonylfluoride, and 1 mM leupeptin. After centrifugation, supernatants were collected and subjected to Western blot analysis. Equal amounts of protein were separated by electrophoresis on 12% or 14% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membrane (Millipore, Marlborough, MA, USA). Membranes were blocked in 5% nonfat dry milk in Tris-buffered saline/Tween-20 (50 mM Tris pH 7.5, 500 mM sodium chloride, and 0.05% Tween-20) for 1 h at room temperature. Membranes were incubated overnight at 4°C with the following primary antibodies: anti-β actin (AbClon, Seoul, South Korea), anti-HO-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), NQO1 (Santa Cruz Biotechnology), anti-phospho AMPK (Cell Signaling Technology, Danvers, MA, USA), and anti-AMPK (Cell Signaling Technology). Membranes were then exposed to an anti-rabbit secondary antibody conjugated to horseradish peroxidase (Cell Signaling Technology) for 1 h at room temperature. Signals were detected by enhanced chemiluminescence lighting solution (Young In Frontier, Seoul, South Korea). Densitometry analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Data are expressed as mean ± standard error of mean (SEM). Statistical differences among groups were determined by Student’s t-test for the comparison of two groups or by one-way analysis of variance (ANOVA) following Tukey multiple comparison post hoc test. Statistical significance was defined as P < 0.05 using PASW Statistics 18 (SPSS, Chicago, IL, USA).

At 32 weeks of age, initial body weights of OLETF groups (OLETF con, OLETF Rimonabant, and OLETF Pair-feeding) were not statistically different (Fig 1A). Six-week administration of rimonabant did not change final body weight, even though rimonabant-treated rats significantly decreased their food intake compared to OLETF control rats (Fig 1A and 1B). Regardless of rimonabant administration, a significant difference was observed between initial (597.7 ± 8.38 g) and final body weights (544.2 ± 8.61 g) in OLETF control animals fed a chow diet (P < 0.05). Based on decreased final body weight, 38-week-old OLETF rats can be considered to be a rat model of severely uncontrolled diabetes. In addition, CB1 receptor blockade did not improve glycemic control, as indicated by OGTT, AUC, and fasting serum glucose concentration (Fig 2) in severely uncontrolled diabetic OLETF rats.

Next, we investigated whether CB1 receptor blockade can improve hepatic steatosis in conditions of unexpected weight loss and uncontrolled hyperglycemia, which are features of severe uncontrolled diabetes. At 38 weeks of age, OLETF control rats showed larger lipid droplets and ballooning in hepatocytes than age-matched LETO controls; these features were attenuated in rimonabant-treated OLETF rats (Fig 3A). Similar to these features, there was a significant reduction of NAFLD activity score (NAS) in rimonabant-treated OLETF rats (Fig 3B). In addition, Masson’s trichrome staining showed little or mild fibrosis in 38 week- old OLETF rats. Without statistical difference, an increasing trend of fibrosis score in the liver tissues from OLETF rats was reversed by 6-week rimonabant administration (P = 0.06, Fig 3C and 3D). Moreover, hepatic TG concentrations were significantly decreased in rimonabant-treated OLETF rats (Fig 3E). In addition, pair-feeding did not lead to a significant reduction in hepatic TG levels (Fig 3E), indicating that decreased hepatic fat accumulation in rimonabant-treated rats might be independent of rimonabant-induced food reduction in severely uncontrolled diabetic OLETF rats. Furthermore, OLETF control rats showed significantly higher serum liver enzyme markers such as ALT (39.0 ± 3.2 IU/L) and AST (427.5 ± 30.6 IU/L) compared to LETO control animals (ALT, 6.4 ± 0.2; AST, 353.7 ± 74.0 IU/L), which were markedly reduced by rimonabant (Fig 3F). Therefore, 38-week-old severely uncontrolled diabetic OLETF rats might be a rodent model of early stage of NASH representing hepatic steatosis as demonstrated by fat accumulation and increased serum ALT and AST concentrations, and little fibrosis. Six-week rimonabant administration alleviated hepatic fat deposition and fibrosis in OLETF rats in the conditions of severely uncontrolled diabetes and early stage of NASH.

To investigate the involvement of CB1 receptor in hepatic oxidative stress and damage, we next measured the amount of hepatocellular lipid peroxidation and cell death present in OLETF rats having weight depletion and uncontrolled hyperglycemia. A marker of oxidative stress as detected by MDA levels was significantly increased in 38-week-old OLETF rats fed with a chow diet, which was reversed after 6-week rimonabant administration (P < 0.05) (Fig 4A). In addition, increased TUNEL-positive cells was observed in severely uncontrolled diabetic OLETF control rats (Fig 4B). The number of TUNEL-positive cells was significantly higher in OLETF control group compared to rimonabant-treated OLETF group (Fig 4C). CB1 receptor blockade significantly decreased the number of TUNEL-positive cells (Fig 4B and 4C), suggesting a beneficial effect of rimonabant on liver death in OLETF rats with severely uncontrolled diabetes.

We evaluated whether rimonabant significantly reduced hepatic TG levels by regulating genes involved in fatty acid oxidation. mRNA levels of CPT1α and peroxisome proliferator-activated receptor α (PPARα), which are involved in β-oxidation, were significantly upregulated by 6 weeks of rimonabant administration in severely uncontrolled diabetic OLETF rats (Fig 5A). Next, to investigate the inhibitory effects of rimonabant on liver fibrosis and cellular death, gene expression involved in inflammation and fibrosis was measured. CB1 receptor blockade significantly downregulated proinflammatory CD11 antigen-like family member b (CD11b), EGF-like module-containing mucin-like hormone receptor-like 1 (F4/80), CCR2 monocyte chemoattractant protein 1 (MCP1), and tumor necrosis factor α (TNFα) gene expression (Fig 5B). Consistent with significant reductions in mRNA abundance related to inflammation, gene levels of negative inflammatory mediators [32, 33] such as suppressor of cytokine signaling 1 (SOCS1) and SOCS3, were significantly downregulated by rimonabant (Fig 5C). In addition, rimonabant administration significantly suppressed hepatic profibrogenic transforming growth factor β (TGFβ), collagen type 1, and tissue inhibitor of metalloproteinase 1 (TIMP1) gene levels (Fig 5D).

In relation to hepatic inflammation, mRNA levels of hepatic Nrf2 and its downstream genes such as NQO1, HO-1, GSTA, and TXNRD1, regulators of a cellular defense system against oxidative stress, were determined in the present study. Rimonabant-treated OLETF rats showed significantly increased hepatic mRNA levels of Nrf2 and NQO1, compared to OLETF control animals (Fig 6A). In accordance with increased gene expression, hepatic protein levels of NQO1 and HO-1 also significantly upregulated by 6-week rimonabant administration in OLETF rats (Fig 6B and 6C). Next, we examined whether CB1 receptor antagonist affects AMPK activation in addition to gene expression of Nrf2 and its downstream effectors. As shown in Fig 6D and 6E, there was a further stimulatory effect of CB1 receptor blockade on liver AMPK activation, as shown as AMPK phosphorylation.