Peretinoin, an acyclic retinoid, suppresses steatohepatitis and tumorigenesis by activating autophagy in mice fed an atherogenic high-fat diet

The pathogenesis of non-alcoholic steatohepatitis (NASH) is still unclear and the prevention of the development of hepatocellular carcinoma (HCC) has not been established. We established an atherogenic and high-fat diet mouse model that develops hepatic steatosis, inflammation, fibrosis, and liver tumors at a high frequency. Using two NASH-HCC mouse models, we showed that peretinoin, an acyclic retinoid, significantly improved liver histology and reduced the incidence of liver tumors. Interestingly, we found that peretinoin induced autophagy in the liver of mice, which was characterized by the increased co-localized expression of microtubule-associated protein light chain 3B-II and lysosome-associated membrane protein 2, and increased autophagosome formation and autophagy flux in the liver. These findings were confirmed using primary mouse hepatocytes. Among representative autophagy pathways, the autophagy related (Atg) 5-Atg12-Atg16L1 pathway was impaired; especially, Atg16L1 was repressed at both the mRNA and protein level. Decreased Atg16L1 mRNA expression was also found in the liver of patients with NASH according to disease progression. Promoter analysis revealed that peretinoin activated the promoter of Atg16L1 by increasing the expression of CCAAT/enhancer-binding-protein-alpha. Interestingly, Atg16L1 overexpression in HepG2 cells inhibited palmitate-induced NF-kB activation and interleukin-6-induced STAT3 activation. We showed that Atg16L1 induced the de-phosphorylation of Gp130, a receptor subunit of interleukin-6 family cytokines, which subsequently repressed phosphorylated-STAT3 (Tyr705) levels, and this process might be independent of autophagy function. Thus, peretinoin prevents the progression of NASH and the development of HCC through activating the autophagy pathway by increased Atg16L1 expression, which is an essential regulator of autophagy and anti-inflammatory proteins.


Lipid droplet accumulation
To measure lipid drop accumulation in vivo, frozen liver tissue sections were fixed in 10% formalin for 1 h, washed with 60% isopropanol, and stained with oil red-O (Sigma-Aldrich, St. Louis, MO) solution for 10 min. The tissues sections were then washed repeatedly with water, photographed, and de-stained in 100% isopropanol for 15 min. To assess the effect of lipid droplet accumulation in vitro, cultured cells fixed in 4% paraformaldehyde were stained with BODIPY 493/503 Lipid Probes (Invitrogen, Carlsbad, CA). Nuclei were labeled with DAPI (Vector Laboratories, Burlingame, CA). The tissue sections and cultured cells were viewed using an image analysis system (BIOREVO BZ-9000; KEYENCE).

Blood sampling and analysis
At 20, 38, and 68 weeks, blood samples were obtained from the inferior vena cava of sacrificed mice following a 12-h fast. Enzymatic assays for total cholesterol and triglycerides were performed with kits purchased from Wako Pure Chemical Industries (Osaka, Japan).

Quantitative real-time detection PCR
Total RNA was isolated from frozen liver tissue samples and cultured cells using a High Pure RNA Tissue Kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer's protocol. cDNA was synthesized from 100 ng total RNA using a High-capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA) and then mixed with the TaqMan Universal Master Mix (Applied Biosystems) and each TaqMan probe. The following TaqMan probes were used: FASN, SCD1, PPARγ, CPT1, PDGFB, PDGFC, IL1β, IL6, CCL2, CCL5, CEBPα, TNFα, Atg16L1, Atg5, and Atg7 (Applied Biosystems). Relative expression levels were calculated after normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or 18S rRNA.

Gene expression profiling
Gene expression profiling of mouse liver was performed using a GeneChip Mouse Gene 1.0 ST Array (Affymetrix, Santa Clara, CA).(3) Liver tissue was obtained from mice fed the basal, Ath+HF, or Ath+HF diet containing 0.03% peretinoin for 30 or 60 weeks. Pathway analysis was conducted using MetaCore (Thomson Reuters, New York, NY). Functional ontology enrichment analysis was conducted to compare the Gene Ontology process distribution of the differentially expressed genes.

DNA transfection
The expression vector for Atg16L1 and GP130 with the Halo tag was purchased from KAZUSA DNA Research Institute (Chiba, Japan). Putative Stat3 activating domain site-mutations of the GP130 vector were generated using a PrimeSTAR Mutagenesis Basal Kit (Takara Bio, Inc., Shiga, Japan), according to the manufacturer's instructions.
HepG2 cells were seeded in each well of a 6-well plate; 1 μg plasmid DNA with 2 μL Lipofectamine 2000 (Invitrogen) were added to each well. After 48 h, the culture medium was replaced with serum-free or amino acid-free medium containing 50 ng/mL recombinant human IL6 (PeproTech). After a 6-h incubation, the cells were harvested for analysis.

RNA interference
Small interfering RNAs (siRNAs) specific to Atg16L1 and GP130 and Low DC control siRNA were obtained from Invitrogen. Transfection was performed with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. www.impactjournals.com/oncotarget Supplemental Figure 2. Effect of peretinoin on STAM TM mice. (A) Feeding schedule of the mice. After weaning, STAM TM mice were divided randomly into 3 groups: (i) HF diet, (ii) HF diet supplemented with 0.03% peretinoin, and (iii) HF diet supplemented with 0.06% peretinoin. Liver histology and tumorigenesis were analyzed at 10w and 22w. (B) Macroscopic findings (upper) and hematoxylin and eosin staining (lower) of background liver of STAM TM mice fed the HF diet, HF diet supplemented with 0.03% peretinoin, or HF diet supplemented with 0.06% peretinoin at 10w. (C) Macroscopic liver findings of STAM TM mice fed the HF diet or HF diet supplemented with 0.03% peretinoin at 22w. (D) Relative expression of CCL2, CCL5, IL1β, IL6, and TNFα mRNA in the liver of STAM TM mice fed the HF diet or HF diet supplemented with 0.03% peretinoin at 10w and 22w. (E) Western blotting of LC3B-II, Atg16L1, the Atg5-12 complex, STAT3, and p-STAT3 in the liver of STAM TM mice fed the HF diet or HF diet supplemented with 0.03% peretinoin at 10w and 22w. (F) Immunofluorescence staining of Atg16L1 in the liver of STAM TM mice fed the HF diet or HF diet supplemented with 0.03% peretinoin at 22w.