Compounds

All chemical compounds were synthesized by the laboratory of Prof. Jiang from East China Normal University (Shanghai, China) and were dissolved in dimethyl sulfoxide (DMSO) prior to use.

Mouse models

Four-week-old, body weight 18 ± 1 g, male C57BL/6 J mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and housed in the specific pathogen free (SPF) facility under controlled environmental conditions (temperature 24–26 °C; relative humidity 60–70%). After importing into the facility, all mice were acclimatized to their environment for two weeks. For generating diet-induced obese (DIO) mice, six-week-old mice were given a high-fat diet (HFD) from Research Diets, Inc. (New Brunswick, NJ, USA) for a total period of 16 weeks before experiments. The DIO mice were randomly divided into two groups (n = 5/group) and were intraperitoneal injected (i.p.) with TOIDC (10 mg/kg body weight) or vehicle (DMSO) once daily for weeks. All animal studies were conducted in accordance with the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation, and the study procedures were approved by the Animal Experimentation Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital.

Mouse serum assay and liver function analyses

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured with the tail vein blood to assess liver injury using the commercial assay kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Hepatic lipid extraction and analysis

For quantification of intrahepatic lipid, tissues were homogenized with phosphate-buffered saline (PBS) and centrifuged, then the liquid phases were transferred into clean tubes. The lipid levels were determined using TG assay kit from Solarbio (Beijing, China), respectively, and normalized to the tissue weight.

Histological analyses

Liver tissues were embedded in 4% paraformaldehyde fix solution (PFA) or OCT compound, then stained with H&E or Nile Red to visualize the lipid accumulation in liver. The histological features were observed under a light microscope and imaged.

Glucose and insulin tolerance tests

Before glucose tolerance test (GTT), mice had free access to drinking water but were fasted 16 h. The fasting blood glucose of each mouse was measured followed by an i.p. injection of D-glucose (20% in saline, 1.5 g/kg body weight). Insulin tolerance tests (ITT) were conducted in mice fasted 4 h by i.p. injection of insulin (0.75 IU/kg body weight). The vein blood was tested at 0, 15, 30, 60, 90 and 120 min after injection.

Cell culture

HepG2 cells were purchased from Shanghai institute of biochemistry and cell biology (Shanghai, China) and maintained in high glucose DMEM containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. Primary hepatocytes were isolated from 6-week-old male C57BL/6 J mice. The mice were anesthetized and perfused with perfusion buffer and collagenase-I through the portal vein at 37 °C. The lysates filtered through a 100 mm cell strainer (Thermo Fisher Scientific, Waltham, MA, USA) and spun at 400 g/min for 3 min at 4 °C. The cells were then resuspended in mixture containing 43% Percoll and 4.8% 10 × PBS and spun at 700 g/min for 10 min at 4 °C. Finally, cells were resuspended in low glucose medium (Yuanpei, Shanghai, China) and plated at the specific density. A mixture of 0.2 mM oleic acid (OA) and 0.1 Mm palmitic acid (PA) (Sigma-Aldrich, St. Louis, MO, USA) in 0.5% BSA was added into low glucose media containing only 2% FBS to establish an in vitro model of lipid accumulation in hepatocytes overnight.

In vitro compounds treatment

To investigate the effects of the compounds, hepatocytes were treated with vehicle or the compounds for 12–36 h. For Nile Red staining, hepatocytes were incubated with a medium containing OA and PA (total 0.3 mM); after 12–18 h, cells were then incubated with vehicle or TOIDC for another 36 h. For quantification of mRNA and protein level, hepatocytes were treated with vehicle or TOIDC for 12–36 h. For the activator’s experiment, hepatocytes were treated with a mixture of TOIDC and 15 or 30 µM T0901317 (MedChemExpress, Monmouth Junction, NJ, USA) for 12–36 h.

Viability assay

Cell viability was accessed by the CCK8 assay (Beyotime, Shanghai, China). Hepatocytes were treated with compounds at about 5 × 103 cells/well in 96-well plates. After 32 h, cells were incubated with 10% CCK8 reagent for another 4 h. The cell viability was detected using micro-plate spectrophotometer at OD 450 nm.

MitoTracker staining

The cultured medium of hepatocytes was removed and added fresh medium containing 200 nM MitoTracker Red probe (Invitrogen, Carlsbad, CA, USA) for 30 min at 37 °C, then wash the dish with PBS for 3 times and incubated with fresh culture medium. Images was captured by the fluorescence microscope.

Immunoblotting

Liver tissues or hepatocytes lysates were extracted with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl, 50 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate 8.0) including 0.5% protease inhibitors and 1% phosphorylation inhibitors. The protein samples were loaded into a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane. After 5% bovine serum albumin (BSA) blocked for 1 h, the membrane was incubated with primary antibodies at 4 °C overnight, and then secondary antibodies for 1 h at room temperature. Immunoreactivities were detected with enhanced chemiluminescent autoradiography (Merck Millipore, Billerica, MA, USA). Chemiluminescence was determined using the AI600 System (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The antibodies used for immunoblotting specific to SREBP1 (1:1000, 14,088–1-AP, Proteintech), FASN (1:1000, SC-48357, Santa Cruz), carnitine palmitoyl transferase 1 alpha (CPT1α, 1:1000, 15,184–1-AP, Proteintech), SCD1 (1:1000, SC-515875, Santa Cruz), GAPDH (1:1000, 60,004–1-Ig, Proteintech), α-Actinin (1:1000, Cat No. 11313–2-AP, Proteintech).

qRT-PCR

Total RNA was extracted from hepatocytes or mouse liver tissues through the Trizol reagent (Invitrogen, Carlsbad, CA, USA). Then, to obtain cDNA, reverse transcription was performed with RNA using cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China). The relative abundance of specific gene was normalized to 18S and analyzed by quantitative real-time PCR system (Roche, Switzerland) using SYBR green PCR mix (Vazyme Biotech, Nanjing, China). qRT-PCR primers are available in Additional file 5: Table S2.

High content screening (HCS)

For HCS, the primary hepatocytes were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature and then incubated with Nile Red buffer purchased from Yeasen (Shanghai, China) for 15 min at 37 °C, afterward the cells were washed three times with PBS and mounted with DAPI. Images were photographed in a continuous field of view autonomously and analyzed using the High content screen system (Image Xpress Micro 4, Molecular Devices, San Francisco, CA, USA).

Molecular docking

Crystal structures of Farnesoid X receptor (FXR) were obtained from PDB database (ID: 6HL1). Then the water molecules, redundant chains, and the original ligand molecules were deleted. The three-dimensional structure of TOIDC was constructed by Chimera software (UCSF, San Francisco, CA, USA). The energy of the receptor structure was minimized using Dunbrack 2010 rotamers. Molecular docking was then performed by AutoDock Vina software to identify potential binding sites. The binding pose of TOIDC with the lowest score was chosen, and hydrogen bonds were analyzed between TOIDC and surrounding amino acid residues.

Statistical analysis

All data are presented as means ± SE of the means (SEM). Differences between the two groups were determined by Student’s two-tailed t-test or two-way ANOVA. When more than two groups were examined, one-way ANOVA followed by Tukey's test was used. P < 0.05 was considered statistically significant. Statistical analysis was carried out using SPSS version 22.0 (IBM, Armonk, NY, USA).