Home Liver Diseases AdipoR1/AdipoR2 dual agonist recovers nonalcoholic steatohepatitis and related fibrosis via endoplasmic reticulum-mitochondria axis

AdipoR1/AdipoR2 dual agonist recovers nonalcoholic steatohepatitis and related fibrosis via endoplasmic reticulum-mitochondria axis

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AdipoR1/AdipoR2 dual agonist recovers nonalcoholic steatohepatitis and related fibrosis via endoplasmic reticulum-mitochondria axis

Peptides synthesis

According to the standard method of solid-phase peptide synthesis (SPPS), polypeptide gAd155 – 161 and a series of analog (JT001 – JT006) sequences with free N-terminals were synthesized using MBHA amide resin (GL Biochem, Shanghai, China). Purification and atomic accumulation process were reverse phase high performance liquid chromatography mass spectrometric (RP-HPLC-MS, Agilent Technologies, USA). The analysis results are shown in Supplementary Figs. 11 and 12.

In vitro cytotoxicity assay

The in vitro cytotoxicity of candidate peptides was tested using CCK8 cell proliferation assay kit (Beyotime Biotechnology, Shanghai, China). Cells were seeded in flat bottom 96-well plates (5000 cells per well for LX2) with 100 μL medium per well. Candidate peptides were added to each well to get desired final concentration (16, 32, 64, 128, 256, 512, 1,024 μM). After incubation for 48 h at 37 °C in a humidified atmosphere of 5% CO2, 10 μL CCK8 reagent was added to each well and the plate was incubated at 37 °C for another 4 h. At the end of the incubation, the absorbance was read at 490 nm on Flex Station 3 plate reader (Molecular Devices, USA).

Hemolytic activity assay

The hemolytic activity of candidate peptides was assessed with mouse red blood cells49. Candidate peptides were incubated with mouse red blood cells (4% hematocrit in PBS) in indicated concentrations (2, 4, 8, 16, 32, 64, 128, 256, 512, 1,024, 2,048, 4,096 μM) at 37 °C for 1 h. Centrifugation for 10 min at 1000 xg to isolate the supernatants, the absorbance was determined at 490 nm. PBS was used as the positive control and 0.2% Triton X-100 which could cause 100% hemolysis was used as the negative control.

Serum stability assay

The stability of candidate peptides was investigated following procedure50. 5 mM peptides were incubated with mouse serum for 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4,12 or 24 h at 37 °C. Ice-cold acetonitrile was added to terminate the reaction. Vortex and stand for 1 min, deionized water with 1% TFA was added to the mixture to further terminate the reaction. Mixtures were then centrifuged (13,000g, 10 min) to obtain the supernatant. RP-HPLC-MS was applied to analysis the content of candidate peptides. The serum stability of candidate peptides was represented as the retention rate of each peptide.

Cell confocal imaging

For in vitro receptor binding assay, 4E4 HepG2 cells or 2E4 LX2 cells were seeded on 35 mm glass bottom dish with 150 μL complete medium. Then cells were incubated with FITC labeled peptide for 4 h. The nuclei were stained with Hoechst 33342 (ThermoFisher, Scientific, USA). After washing with PBS, cells were visualized in a laser confocal microscope (Olympus FV3000, Japan). Cells with suppressed AdipoR1 and AdipoR2 expression with each specific siRNA were used as negative control.

RNA interference

AdipoR1 and AdipoR2 siRNA as well as mock siRNA were chemically synthesized (GenePharma, China) and transfected into 60–70% confluent HepG2 and LX2 cells using Lipofectamine 3000 (ThermoFisher Scientific, USA). Dilution of siRNA and Lipofectamine 3000 reagents as well as the transfection of cells were performed following the manufacturer’s protocol. For HepG2 cell model, 5E5 cells were plated in 6-well plates and transfected with 5 μL of 20 μM stock siRNA. At 48 h following transfection, the cells were processed for 0.25 mM PA and/or 200 μM JT003 treated for another 24 h. BSA was used as the negative control. For LX2 cell model, 2E5 cells were plated in 6-well plates and transfected with 5 μL of 20 μM stock siRNA. At 48 h following transfection, the cells were processed for JT003 or PBS treated for another 48 h. Next, cells were harvested for either western blot or qPCR assay.

HTRF (homogeneous time resolved fluorescence)

The expression plasmid of AdipoR1 and AdipoR2 were synthesized and codon optimized by Genescript (Nanjing, China) for their expression in HEK293 cell line. The concentration of FITC-JT003 was 0, 0.1, 0.25, 0.5, 1, 2.5 or 5 μM respectively. Unlabeled peptide JT003 was used as the control to measure the non-specific signal. 10E4 AdipoR1 and AdipoR2 overexpression cells, anti-His-Tb (ThermoFisher Scientific, USA) and JT003-FITC were added into a 384-well plate, with a total volume of 25μl. The mixtures were incubated at room temperature for 30 min in the dark. Fluorescent values at 520 nm and 620 nm were measured for Kd calculation.

Competition binding assay

Different concentration of JT003 were incubated with AdipoR1 or AdipoR2 overexpression HEK293 cell line for 4 h. After that, 50 μg ml−1 gAd155–161 were added for another 4 h incubation. The binding of gAd155–161 to the cells were measured via western blot assay.

Cell model

Hepatoma carcinoma cell line HepG2 and human hepatic activated stellate cell line LX2 used in this study were purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), supplemented with 1% penicillin–streptomycin (ThermoFisher Scientific, USA) at 37 °C in a humidified atmosphere of 5% CO2. In PA (Sigma, USA) induced lipid accumulation cell model, HepG2 cells were seeded into a 6-well plate at 5E5 cells per well. 0.25 mM PA and/or 200 μM candidate peptides were co-incubated with cells for 24 h. Then Oil Red O staining was performed to evaluate the accumulation of lipid droplets. BSA (Sigma, USA) was used in this experiment as the negative control. For LX2 cells, 2E5 cells were seeded, then the adherent cells were exposed to 200 μM candidate peptides for another 48 h. Next, the cell protein was obtained and western blotting was performed. Candidate peptides were diluted in DMEM in a stock concentration of 1 M.

Computational docking

Molecular Operating Environment (MOE) version 2013.8 was used to perform the automated docking studies and ECL-1 was selected as the activity domain. The initial structures of JT003 were pre-calculated a three-dimensional grid in Discovery Studio (DS) version 3.5. Then the structures were energetically minimized with Powell method via default parameters. 20 possible three-dimensional conformations were generated during this phase. Afterward, automated docking calculations were carried out using MOE via default parameters, and the binding free energies of the agonists within the receptors were evaluated. For each possible three-dimensional conformation of JT003, 20 promising docking poses were generated and all docked poses were clustered using a tolerance of 2 Å in positional root-mean-square-deviation (RMSD), and were sorted by binding docking energies. The most favorable free energy of binding was considered as the resultant complex structure.

Animal models and tissue collection

Male C57BL/6J mice of 6–8 weeks of age (20–24 g) were used in these experiments including the NAFLD model and liver fibrosis model. The mice were purchased from Guangdong Medical Laboratory Animal Center and housed in standard isolator cages and maintained on a room temperature of 25 °C and a 12 h:12 h light/dark cycle. At the end of the experiments, mice were fasted 8 h prior to sacrifice. Liver tissue and/or epididymal adipose tissue were flash frozen in liquid nitrogen (with or without RNAlater (TransGen Biotech, China)) and stored at −80 °C for future use, or fixed in 10% paraformaldehyde for histological assays. Blood samples were obtained from the posterior venous plexus of the eyes of anesthetized mice, Blood was separated by centrifugation at 1000g for 15 min Serum was then stored at −80 °C. The ethical approval has been obtained from Animal Care and Use Committee of the Sun Yat-sen University. All animals were maintained in pathogen-free conditions and cared for in accordance with policies and certification of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).

The NAFLD model was established in male mice through feeding a HFD continuously for 16 weeks (n = 6; protein: 18.1%; fat: 61.6%; carbohydrates: 20.3%; D12492, Research Diets, USA). Mice administered an SD diet serves as control group (n = 6; protein: 18.3%; fat: 10.2%; carbohydrates: 71.5%; D12450B, Research Diets, USA). The NASH model was conducted with feeding a HFD for 16 weeks combine with a MCD diet (Methionine/Choline Deficient Diet, Medicience, China) in the last 4 weeks. Mice administered a MCS diet (Methionine/Choline Sufficient Diet, Medicience, China) serves as control group. NAFLD or NASH mice treated with JT003 for 4 weeks were used to evaluate the pharmacological effects (n = 6; 500 μg kg−1; daily intraperitoneal injection). Food and water were provided ad libitum. The body weight of mice was examined every 3 days. The diet was renewed every 7 days. JT003 was diluted in PBS. Supplementary Fig. 5 showed the schematic of experiment procedure.

Male mice liver fibrosis was induced by intraperitoneal injection of 20% CCl4 (Sigma, USA) diluted in corn oil (Aladdin, China) every 3 days for 6 weeks (n = 6; 5 ml kg−1 body weight). Mice injected with equivalent volume of corn oil serves as control group (n = 6). Liver fibrosis mice treated with JT003 for 3 weeks were used to evaluated the pharmacological effects (n = 6; 500 μg kg−1; daily intraperitoneal injection). Supplementary Fig 19 showed the schematic of experiment procedure.

OGTT and ITT

Fasting blood glucose was measured using OneTouch UltraVue Blood Glucose Meter and test strips (Johnson&Johnson, USA) from tail nicks. After HFD fed for 14 weeks and JT003 treated for 2 weeks, oral glucose tolerance tests were performed on 8 h fasted mice. 0.5 g ml−1d-glucose solution (Sigma-Aldrich, USA) were oral injected to mice (2 g kg−1 of bodyweight), blood glucose level was recorded at baseline, 15, 30, 60, 120 min

For insulin tolerance test, one week after OGTT assay, 6 h fasted mice were treated with JT003, and the baseline blood glucose level was measured. An hour later, 0.25 U kg−1 insulin (Novolin R, Denmark) were intraperitoneal injection to mice, blood glucose level was measured at 15, 30, 45, 60 min

Serum biochemical assays

The levels of ALT, AST, TC and TG in serum were determined using certain commercial assay kits (Nanjing Jiancheng Bioengineering Institute, China). Fasting adiponectin and insulin were quantified in plasma samples using corresponding commercially available Elisa kits (Cloud-Clone Crop, USA).

Histological examination

The levels of TG and HYP in liver tissue were detected with certain commercial assay kit (Nanjing Jiancheng Bioengineering Institute, China). Formalin-fixed liver tissues were embedded in paraffin, sectioned into 5 μm slices. After deparaffinization and rehydration of the section slices, they were stained with Haematoxylin and Eosin or Periodic Acid-Schiff. For the detection of liver collagen content, Sirius Red staining for collagen was performed. OCT-embedded frozen liver tissues were sectioned into 12 μm slices, and Oil Red O staining was performed. The cryostat-cut slices were stained with freshly prepared Oil Red O working solution for 7 min Two independent experimenters who were blinded to the treatment evaluated the staining slides and analyzed the score for steatosis, glucogen accumulation and collagen proliferation area.

Immunohistochemistry

Paraffin-embedded liver slices were deparaffinized and rehydrated, antigen retrieval was performed under the high pressure and temperature in 0.01 M sodium citrate buffer (pH = 6.0). To quench the endogenous peroxidase activity, the slices were then incubated with 1% hydrogen peroxide in methanol for 10 min in room temperature. Then slices were incubated with corresponding primary antibodies. After washing, HRP-conjugated secondary antibodies specific to the species of the primary antibodies were used. After the section were developed with DAB, hematoxylin staining was performed for cell counting.

Apoptosis assay

TUNEL assay was performed using 5 μm liver sections and the apoptotic cells were identified using a Cell Death Detection kit (Beyotime Biotechnology, Shanghai, China). And then the slices were counterstained with DAPI (Life science, ThermoFisher Scientific, USA). Six random fields from 6 slides per mice were examined, and the TUNEL-positive red area within the slides were counted by two individuals who were blinded to the treatment.

RNA isolation and gene expression assay

Liver total RNA was extracted by using Trizol reagent following the manufacture’s instruction (TransGen Biotech, China). The concentration of RNA was measured by Nanodrop 2000 (ThermoFisher Scientific, USA). cDNA was transcribed from 10ug total RNA with First Strand cDNA synthesis Super Mix (TransGen Biotech, China). The cDNA was diluted 1/50 and 1.0 uL were used as a template for qPCR. qPCR was performed with SYBR Green qPCR Super Mix (TransGen Biotech, China) in the Light Cycler 480 Real-Time PCR System (Roche, Swiss). Reference gene β-actin was used for data normalization. mRNA expression was analyzed with specific primers listed in Supplementary Table 2.

Protein prepared and western blot analysis

Total protein was isolated from cells or liver samples using RIPA lysis buffer (50.0 mM Tris-HCl (pH = 8.0), 150.0 mM NaCl, 0.02% NaN3, 0.1% SDS, 1% NP-40, 0.5% deoxysodium cholate, 1 mM EDTA, 1 μM RIPA). The protein concentration was quantified with BCA kit (Pierce, ThermoFisher Scientific, USA). Thirty micrograms protein were separated by 10 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis depending on the band of target protein, then transferred onto equilibrated polyvinylidene difluoride membrane (PVDF, Immobilon®-PSQ transfer membranes, Merck Millipore, Germany). Membranes were incubated with primary antibodies overnight at 4 °C. Specific antibodies were listed in Supplementary Table 3. After incubation with the secondary antibody, proteins were detected by enhanced chemiluminescence (GlarityTM Western ECL Substrate, Bio-Rad, USA) and the signals were recorded and quantified with Tanon-Image Software (Shanghai, China). Reference antibody GAPDH were used for data normalization.

Mitochondrial content assay

60–70% confluent LX2 cells and PA induced HepG2 cells were washed with PBS twice and incubated with 100 nM MitoTracker Green FM (YeSan, China) at 37 °C for 30 min For fluorescence intensity assay, cells were harvested by using trypsin/EDTA (ThermoFisher Scientific, USA) and resuspended in PBS. The excitation and emission wavelengths were 490 and 516 nm, respectively, and the intensity values corrected for total protein level (mg ml−1). For mitochondrial visualized assay, after incubated with MitoTracker Green FM, the adherent cells were washed with PBS and a single in-focus optical section was acquired with confocal microscopy (Olympus, Japan). The nucleus was stained with Hochest33342 (ThermoFisher Scientific, USA). For every section n = 50 cells, images were analyzed and quantified by ImageJ.

Pharmacokinetic assay

Male SpragueDawley (SD) rats were randomly divided into two groups (n = 3 per group) and received a single intravenous dose of JT003 (0.5 mg/kg) or ADP355 (1.0 mg kg−1) respectively. Blood samples were collected at 5, 15, 30, 45 min and 1, 2, 3 h following the injection, and then were immediately centrifuged (4000g, 10 min, 4 °C) to obtain plasma samples. Plasma concentrations of the two peptides were measured by LC-MS/MS (ThermoFisher Scientific, USA). Pharmacokinetic parameters of JT003 and ADP355 were calculated using non-compartmental analysis in Phoenix WinNonlin (version 8.0, Pharsight, USA). The concentration values less than the LLOQ were set to zero for the analysis.

Statistical analysis

The positive area of all stained liver slices was quantified by ImageJ (https://imagej.nih.gov/ij/). All data in this study were shown as the mean ± SEM (Standard Error). Student’s two-tailed t test was used to compare the mean of two groups of samples. All statistical analyses were performed with GraphPad Prism software (https://www.graphpad.com/). Significance was considered when p < 0.05. In RNAseq gene expression analysis, false discovery rate <0.01, q value <0.001. The confidence level was 95%.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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