Exendin-4, a GLP-1 agonist, was purchased from Sigma Aldrich (St. Louis, MO, United States). The non-obese NASH model mice received MCD, while the control group mice received a methionine-choline-sufficient diet (MCS). Both the diets were purchased in powdered form (Oriental Yeast Co., LTD., Tokyo, Japan). Exendin-4 was diluted in saline to achieve a concentration of 2 μg/mL (as used in a previous study[14]).

Six-week-old male db/db mice were purchased from Japan SLC Inc. (Hamamatsu, Japan). After a 1-wk acclimatization period on a basal diet (Oriental Yeast), 44 mice were divided into 3 groups and received 1 of the following diets: (1) MCS; (2) MCD with saline; or (3) MCD with exendin-4 (20 μg/kg per day, ip) for 4 or 8 wk. The exendin-4 dose was determined according to a previous study[14]. All mice were given free access to water and the experimental diets. Body weight and food consumption of the mice in each group were recorded weekly. Protocols describing the use of mice were approved by the Institutional Animal Care and Use Committee of Aichi Medical University, and were in accordance with the National Institutes of Health’s “Guide for the Care and Use of Laboratory Animals.” After 4 or 8 wk of the experimental diet, the mice were euthanized and their livers were rapidly excised and fixed either in buffered formalin (10%) or frozen in liquid nitrogen, and stored at -80 °C. Blood samples were collected from the left ventricle and centrifuged, and the serum samples were stored at -80 °C.

Serum alanine aminotransferase (ALT) and fasting blood glucose levels were determined using commercially available kits (Wako, Osaka, Japan). Serum immunoreactive insulin (IRI) levels were measured using a Mouse Insulin ELISA kit (Funakoshi, Tokyo, Japan), and then the homeostasis model assessment-insulin resistance (HOMA-IR) was calculated.

The stored liver samples (100 mg) were lysed and homogenized in 2 mL of a solution containing 150 mmol/L NaCl, 0.1% TritonX-100, and 10 mmol/L Tris using a polytron homogenizer (NS-310E; MicroTech Nichion, Tokyo, Japan) for 1 min. Hepatic triglyceride (TG) and free fatty acid (FFA) content was measured using the Triglyceride Detection Kit and Free Fatty Acids Detection Kit (Wako), respectively. The levels of hepatic malondialdehyde (MDA), a marker of reactive oxygen species (ROS), were measured using an MDA Assay Kit (Northwest Life Science, Vancouver, WA, United States).

Five-micrometer-thick sections from the liver tissues fixed in formalin and embedded in paraffin were examined in all the experimental groups. Hematoxylin and eosin staining was performed to assess hepatic inflammation. Oil Red O staining was performed using a standard technique to assess hepatic fat deposition. Hepatic inflammation was scored from 0 to 3, based on the histological scoring system for NAFLD edited by Kleiner et al[21], and averaged over 5 fields per slide at 50× magnification. The Oil Red O positive area was quantified in 5 randomly selected fields per section. The percentage of Oil Red O-positive areas was measured using a computerized image analysis system with Image-Pro Plus, version 4.5 (Media Cybernetics, Silver Spring, MD, United States).

For immunohistochemical analysis, endogenous peroxidase activity was blocked with hydrogen peroxide, and nonspecific binding was blocked with 10% normal goat serum in phosphate-buffered saline. After blocking, the liver sections were incubated overnight with anti-F4/80 (rat monoclonal, 1:50 dilution; Abcam, Cambridge, MA, United States), a macrophage marker. Antigen-antibody complexes were detected using the avidin-biotin peroxidase method (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, United States). The number of F4/80 positive cells was counted independently by 2 pathologists (Nakade Y and Fukuzawa Y), who were blinded to the source of the specimens, and averaged over 5 fields per slide at 50 × magnification.

The frozen liver specimens were crushed in TRIzol reagent (Life Technologies, Tokyo, Japan). RNA extraction was performed using an RNeasy Mini Kit (Qiagen, Tokyo, Japan). RNA was resuspended in 40 μL of RNase-free water and quantified by spectrophotometry [optical density (OD) 260 and low-mass gel electrophoresis] (Invitrogen, Tokyo, Japan). Total RNA was reverse transcribed to cDNA using a High Capacity cDNA Reverse Transcriptional Kit (Applied Biosystems, Foster City, CA, United States) according to the manufacturer’s instructions. Real time quantitative polymerase chain reaction (PCR) was carried out with the ABI Step One Sequence Detection System (Applied Biosystems) using the TaqMan Gene Expression Assays [acyl-coenzyme A oxidase 1 (ACOX1), Mm01246834_m1; microsomal triglyceride transfer protein (MTTP), Mm00435015_m1; sterol regulatory element-binding protein-1c (SREBP-1c), Mm01495763_g1; fatty acid transport protein (FATP) 2, Mm0128768_m1; FATP4, Mm01327413_g1; tumor necrosis factor-α (TNF-α), Mm00443258_m1; monocyte chemotactic protein-1 (MCP-1), Mm00441242_m1; cc-chemokine receptor 2 (CCR-2), Mm99999051_Gh] and the TaqMan Universal PCR Master Mix (Applied Biosystems), according to the manufacturer’s instructions. The detailed protocol for TaqMan PCR was determined based on a previous study[22].

The liver tissue samples (100 mg) were lysed in a sodium dodecylsulphate (SDS) sample buffer, separated on a 10% SDS-acrylamide gel, and electrotransferred to the nitrocellulose membranes. After blocking with 5% nonfat dry milk in TBST buffer [10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% Tween-20], the membranes were probed with anti-rabbit polyclonal FATP4 antibody (1:400; Abcam), and incubated using HRP-conjugated anti-rabbit or anti-mouse immunoglobulin G secondary antibodies (1:2000; DAKO Japan, Tokyo, Japan). Antibody binding was then visualized using an enhanced chemiluminescence reagent (GE Healthcare, Tokyo, Japan), and the band images detected using the LAS1000 system (Fuji Film, Tokyo, Japan) were densitometrically analyzed using Image Gauge (Fuji Film).

All results are expressed as mean ± SE. Values in 2 groups were compared using the Student’s t-test. Multiple group comparison was performed using analysis of variance (ANOVA) followed by Fisher’s protected least significant difference post-hoc test. A P value < 0.05 was considered statistically significant.

The daily intake of MCS was more than that of MCD in the 4- and 8-wk experiments (Table 1). The MCD-fed mice had lost weight whereas the MCS-fed mice had gained weight over the 8-wk experimental period (Figure 1). MCD resulted in an Oil Red O-positive area at 4 wk, and this area had reduced in size at 8 wk (Figure 2). The hepatic TG and FFA content was significantly higher in the MCD-fed mice than in the MCS-fed mice at both 4 and 8 wk (Table 1).

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Figure 1 Time course changes in body weight of mice fed methionine-choline-sufficient diet (n = 6), methionine-choline-deficient diet with saline (n = 8), and methionine-choline-deficient diet with exendin-4 (n = 8). Data are expressed as mean SE. (^bP < 0.01, vs MCS, ^cP < 0.05, ^dP < 0.01, vs MCD with saline). MCD: Methionine-choline-deficient diet; MCS: Mice fed methionine-choline-sufficient diet .

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Figure 2 Evaluation of hepatic lipid contents of mice fed mice fed methionine-choline-sufficient diet, methionine-choline-deficient diet with exendin-4 or saline for 4 and 8 wk (A) and quantitative analysis of changes in Oil Red O positive area in respective groups (B). Frozen liver sections were stained with Oil Red O. Data are expressed as mean ± SE. (^bP < 0.01 vs respective groups). Original magnification, 100 ×. MCD: Methionine-choline-deficient diet; MCS: Mice fed methionine-choline-sufficient diet.

Administration of exendin-4 did not change the daily intake of MCD until 4 and 8 wk (Table 1). Additionally, it did not change the body weight of the MCD-fed mice until the end of the 4-wk experiment; however, it significantly reduced the body weight of these mice from 5 to 8 wk (Figure 1). Exendin-4 administration also significantly attenuated the MCD-induced Oil Red O-positive area in the MCD + exendin-4 group compared to in the saline group at 4 and 8 wk (Figure 2). Moreover, it significantly reduced the hepatic TG content in MCD-fed mice at both 4 and 8 wk (Table 1). Furthermore, extendin-4 administration showed a tendency to decrease hepatic FFA content in the MCD-fed mice at both 4 and 8 wk (Table 1).

SREBP-1c mRNA (Figure 3A) and ACOX1 mRNA levels (Figure 3B) tended to decrease in the MCD-fed mice at 4 and 8 wk. MTTP mRNA levels were unchanged in the MCD-fed mice at 4 and 8 wk (Figure 3C). FATP2 mRNA levels tended to increase, and FATP4 mRNA levels significantly increased in the MCD-fed mice at 4 wk (Figure 3D and E). FATP2 and FATP4 mRNA levels tended to increase in the MCD-fed mice at 8 wk (Figure 3D and E). FATP4 protein levels were significantly increased in the MCD-fed mice at 4 wk (Figure 3F and G).

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Figure 3 Evaluation of hepatic lipid metabolism-related genes of mice fed methionine-choline-sufficient diet, methionine-choline-deficient diet with exendin-4, or saline for 4 wk. The relative mRNA expression of SREBP-1c (A), ACOX1 (B), MTTP (C), FATP2 (D) and FATP4 (E) were evaluated. Immunoblot of FATP4 protein expressions in the liver (F); To control for loading, the blot was stripped and reprobed for β-actin. FATP4 levels in each group, after a 4-wk treatment, were expressed relative to β-actin (G). Data are expressed as mean ± SE. (^aP < 0.05, ^bP < 0.01 vs respective groups). MCD: Methionine-choline-deficient diet; MCS: Mice fed methionine-choline-sufficient diet; SREBP: Sterol regulatory element-binding protein; ACOX1: Acyl-coenzyme A oxidase 1; MTTP: Microsomal triglyceride transfer protein; FATP: Fatty acid transport protein.

Administration of exendin-4 decreased SREBP-1c mRNA levels in MCD-fed mice at both 4 and 8 wk (Figure 3A). Additionally, it tended to reverse the MCD-induced decrease of ACOX1 mRNA levels at both 4 and 8 wk (Figure 3B); however, it did not alter MTTP mRNA levels in MCD-fed mice at 4 and 8 wk (Figure 3C). Exendin-4 administration slightly decreased the FATP2 mRNA levels augmented by 4- and 8-wk MCD, and significantly depressed the FATP4 mRNA levels augmented by 4-wk MCD (Figure 3D and E). The 8-wk exendin-4 administration tended to decrease the FATP2 mRNA levels altered by MCD (Figure 3D and E). Moreover, extendin-4 significantly inhibited the augmentation of FATP4 protein levels altered by the 4-wk MCD (Figure 3F and G).

Serum ALT levels were significantly higher in the MCD-fed mice than in the MCS-fed mice at 4 wk (Table 1). Hepatic inflammatory foci appeared in the 4-wk MCD-fed mice (Figure 4A). The hepatic inflammation score was significantly augmented in the 4-wk MCD-fed mice, and was further exacerbated in the 8-wk MCD-fed mice (Figure 4B). F4/80 positive cells in the liver appeared in the 4-wk MCD-fed mice (Figure 4C and D). The hepatic MDA and TNF-α mRNA levels were significantly increased by MCD at both 4 and 8 wk (Figure 4E and F). Hepatic MCP-1 and CCR-2 mRNA levels were significantly increased in MCD-fed mice at both 4 and 8 wk (Figure 4G and H). In contrast, exendin-4 did not significantly alter the serum ALT levels that were increased by MCD at 4 wk; however, it significantly improved these hepatic indices at 8 wk (Table 1). It prevented the MCD-induced augmentation of the hepatic inflammation score at both 4 and 8 wk. Furthermore, it significantly decreased the increased number of F4/80 positive cells due to MCD at 4 wk (Figure 4B and D). Additionally, exendin-4 administration significantly attenuated the hepatic MDA and TNF-α mRNA levels enhanced by MCD at both 4 and 8 wk (Figure 4E and F). Furthermore, it significantly attenuated the MCD-induced increase in hepatic MCP-1 and CCR-2 mRNA levels at both 4 and 8 wk (Figure 4G and H).

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Figure 4 Evaluation of hepatic inflammation-related parameters of mice fed methionine-choline-sufficient diet, methionine-choline-deficient with exendin-4, or saline for 4 and 8 wk. A: Representative images showing the inflammatory foci for MCD-induced liver injury at 4 wk. Original magnification, 200 ×; B: The number of inflammatory foci per 20 × field was enumerated on each section; C: Representative images of F4/80 immunoreactivity; positive cells are indicated by arrows. Original magnification, 200 ×; D: Quantitative analysis of changes in F4/80 positive cells in the respective groups; E: Evaluation of hepatic MDA levels and hepatic inflammation-related genes of mice fed MCS, MCD with exendin-4, or saline for 4 and 8 wk. Hepatic MDA levels, a marker of oxidative stress, were examined. Relative mRNA expression of TNF-α (F), MCP-1 (G), and CCR-2 (H) was evaluated in the liver. Data are expressed as mean ± SE. (^aP < 0.05, ^bP < 0.01, vs respective groups). MCD: Methionine-choline-deficient diet; MCS: Mice fed methionine-choline-sufficient diet ; MDA: Malondialdehyde; TNF-α: Tumor necrosis factor-α; MCP-1: Monocyte chemotactic protein-1; CCR-2: cc-chemokine receptor 2.

The fasting blood glucose, IRI, and HOMA-IR levels were significantly higher in the MCS-fed mice than in the MCD-fed mice at both 4 and 8 wk (Table 2). The administration of exendin-4 tended to further decrease the fasting blood glucose level, and it significantly decreased the IRI level at both 4 and 8 wk (Table 2). The HOMA-IR level in the MCD + exendin-4 group was significantly decreased compared to that in the MCD + saline group (Table 2).

Although there is no promising therapy for nonalcoholic steatohepatitis (NASH), it has been reported that glucagon-like peptide-1 (GLP-1) analogues may decrease hepatic steatosis in high fat diet-induced NASH models. Several studies on the effects of GLP-1 analogues in NASH models have been conducted; however, it remains unclear whether GLP-1 analogues improve hepatic steatosis in non-obese NASH models.

Previous studies have shown that GLP-1 analogues reduce hepatic steatosis in high fat diet-induced NASH models. Additionally, GLP-1 analogues stimulate hepatic lipid oxidation induced by a high fat diet in NASH models.

To our knowledge, this is the first study evaluating the effects of a GLP-1 analogue in a non-obese NASH model. The authors showed that the GLP-1 analogue prevented not only hepatic steatosis but also hepatic inflammation in a methionine choline deficient diet (MCD)-induced non-obese NASH model.

Regarding hepatic inflammation, a previous study has shown that the number of F4/80 positive cells was increased in NASH models. Those data showed that F4/80 positive cells were significantly increased by MCD. The presence of F4/80 positive cells suggests that hepatic steatosis and inflammation induced the recruitment of macrophages in the liver.

A MCD has been reported to induce steatohepatitis, which is morphologically similar to NASH except for body weight gain. MCD-induced adipose tissue lipolysis resulted in an increase in serum free fatty acid levels and hepatic free fatty acid content in rodents.

The authors demonstrated that a GLP-1 analogue, exendin-4, prevented hepatic steatosis and inflammation, and decreased hepatic free fatty acid influx in a non-obese NASH model. The title reflects the topic and contents of the study. The authors clearly state the purpose of the manuscript. The manuscript is well structured and concise. The methods used are appropriate.