Male C57BL/6J WT mice were obtained from Shanghai Laboratory Animal Co., Ltd. (Shanghai, China). Leptin receptor-mutated (db/db) mice were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). ERAP1 hybrid knockout (Erap1+/−) mice were generated using CRISPR/Cas9 technology, and the guide RNAs (gRNAs) were targeted at the intron 2 and intron 3 of Erap1 (Shanghai Model Organisms Center, Shanghai, China).
Erap1+/− mice were back-crossed to WT mice for at least two generations to generate Erap1 global KO mice. For all experiments, littermates of the same sex (male) were randomly assigned to experimental groups. For the HFD feeding experiment, 4-week-old WT mice were fed either a control diet or 60% HFD (Research Diets, NJ, USA) for 4 months. For ERAP1 inhibitor, thimerosal52 experiments, mice were intraperitoneally (i.p.) injected with thimerosal (Sigma, MO, USA) at a dose of 6 mg/kg53 or PBS for 6 days. For neutralizing antibody injection, mice were singly i.p. injected with anti-ERAP1 neutralizing antibodies or control IgG (R&D system, USA) at a dose of 1 mg/kg 30 min before experiments. Mice were maintained with a 12-h light/dark cycle at 23 °C. All anim al experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences (CAS).
Cell culture and treatments
HEK 293 (ATCC, CRL-1573), Hep G2 (ATCC, HB-8065), C3H/10T1/2 (ATCC, CCL-226), and C2C12 (ATCC, CRL-1772) cell lines were purchased from Cell Bank of Shanghai Institute of Cell Biology, CAS. Mouse primary hepatocytes were prepared by collagenase perfusion as described previously54. Cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate. The differentiation of C2C12 myoblasts55 and C3H/10T1/2 preadipocytes56 was induced, as previously described. C2C12 myotubes, C3H/10T1/2 adipocytes, or primary hepatocytes were incubated with CM-ERAP157 or rmERAP1 (R&D system, USA)58 at the indicated concentration for the indicated time. To detect insulin signaling, C2C12 myotubes, primary hepatocytes, and C3H/10T1/2 adipocytes were incubated with 100 nM insulin for 20 min27,55,59.
The DNA fragments encoding ERAP1 and ADRB2 were amplified from mouse liver cDNA. The recombinant adenovirus expressing mouse ERAP1 (Ad-ERAP1) or ADRB2 (Ad-ADRB2) was generated using the AdEasy™ Adenoviral Vector System (Qbiogene, Irvine, CA, USA) and Ad-NC or Ad-shERAP1 was generated using the BLOCK-iT™ Adenoviral RNAi Expression System (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The shRNA sequence for mouse ERAP1 was 5′- CCAGCACCATTATTATGCATAGTCA-3′. Purified high-titer stocks of amplified recombinant adenoviruses were diluted in PBS and injected via the tail vein at a dose of 1 ´ 109 pfu /mice for a single injection54.
High-ERAP1 conditioned medium
HepG2 cells were infected with Ad-GFP or Ad-ERAP1 at a dose of 107 pfu/well in 12-well plates and changed with fresh medium 24 h later54; CM-ERAP1 was collected at 48 and 72 h, as described previously60.
Insulin resistance associated parameters
Blood glucose was measured with a Glucometer Elite monitor (Optium Xceed, IL, USA). Serum insulin was determined based on an ELISA using the Mercodia Ultrasensitive Rat Insulin ELISA kit (ALPCO Diagnostic, NH, USA) in accordance with the manufacturer’s instructions. After 14 h of overnight fasting, GTTs were conducted via the i.p. injection of 2 g/kg glucose. For ITTs, mice were injected with 0.75 or 1 U/kg insulin after 4 h of fasting. The HOMA-IR index was calculated as follows: [fasting glucose levels (mmol/l)] ´ [fasting serum insulin (μU/ml)] / 22.554.
In vivo insulin signaling assay
Mice were fasted for 6 h prior to insulin injection, as previously described27. Small sections of the soleus muscle, WAT, and liver were excised from anesthetized live mice and kept as untreated controls. Insulin was injected at a dose of 2 U/kg into WT mice or at 5 U/kg in db/db mice via the portal vein; a small piece of the liver section was excised for western blot analysis after 3 min. Another side WAT and soleus muscle were excised after 4 and 5 min.
Western blot analysis
Western blot analysis was performed as previously described27. Primary antibodies obtained are as follows: anti-p-IR (tyr1150/1151), anti-IR, anti-p-AKT (ser473), anti-AKT, anti-p-GSK3β (ser 9), anti-GSK3β, and anti-p-PKA substrates (Cell Signaling Technology, Beverly, MA, USA); anti-ERAP1 (Abcam, Cambridge, England); anti-ADRB2 and anti-β-actin (Protein Tech, Chicago, USA); anti-α-tubulin and anti-ADRB1 (Sigma-Aldrich, St. Louis, USA); anti-ADRB3 (Signalway Antibody, Maryland, USA). All of these assays were performed according to the manufacturer’s instructions.
Relative RT-PCR and Illumina deep sequencing
Total RNA was extracted from mouse tissue samples using TRIzol reagent (Invitrogen, Waltham, MA, USA) as previously described54. mRNA levels were examined by RT-PCR with thebprimers described in Table S1. The samples were also sequenced using the Illumina HiSeq™ 4000 system at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China). The data were analyzed with the free online platform Majorbio Cloud Platform (www.majorbio.com)
Quantification and statistical analysis
Statistical analysis was performed using GraphPad Prism, version 8.0 (GraphPad Software, San Diego, CA). All data are expressed as the mean ± SEM. Significant differences were assessed either by an unpaired two-tailed student t-test or one-way ANOVA followed by the Student-Newman-Keuls (SNK) test, as indicated. For GTTs and ITTs, a t-test or one-way ANOVA was used to compare the difference between groups at each time points examined. P < 0.05 was considered statistically significant.