Antibodies and Reagents
Antibodies against p-IRTyr1189/1190 (#3024), IR (#3025), p-AKTThe308 (#13038), AKT (#4685), p-AS160Thr462 (#8881), AS160 (#2670) were purchased from Cell Signaling Technology (Massachusettes, USA). Antibodies against SIRT1 (#13161-1-AP) and WARS (#16081-1-AP) were purchased from Proteintech (Rosemont, USA). Antibodies against Flag (#M20008) and HA (#M20003) were purchased from Abmart (Berkeley Heights, USA). Antibody against Actin (A00702-100) was purchased from GenScript (Piscataway, USA).
Insulin (#I9278), methyl-tryptophan (#447439), anti-Flag M2 affinity Gel (#A2200) were purchased from Sigma-Aldrich (Darmstadt, Germany). Protein-G-Agarose (#16–266) and Protein-A-Agarose (#16–125) were purchased from Millipore (Danvers, USA). Kynurenine (#R134932) and 5-HT (#H303833) were purchased from Aladdin (Shanghai, CHN).
The anti-IRW−K1209 antibody was generated by ABclonal Technology Co., Ltd. Briefly, synthetic peptides (PVRWMAPESLKTrpDGVFTTSSDM) were conjugated to keyhole limpet hemocyanin (KLH) as antigen. Rabbits were immunized by four doses of subcutaneous injection at two weeks interval before the rabbits were sacrificed for anti-sera. Antibody was immunoaffinity purified by antigen and tested for specificity by dot blot assay.
Male C57BL/6J mice were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Mice were housed in a specific pathogen-free facility under a 12 h light/dark cycle with ad libitum access to food and water. Mice were randomly divided into control and experimental groups. The control mice were fed with standard chow (SC) and the experimental group were fed with 1% tryptophan chow (Trp), which was manufactured from Research Diets (New Brunswick, USA), for 12 weeks.
Mice were fasted for 6 hours, and then were euthanized 15 minutes after intraperitoneal injection of insulin (0.5 units/kg). The inguinal fats were collected according to standard procedure for western blot analysis.
Gtt And Itt
Assays were performed using male C57BL/6J mice after 16 weeks of treatments. For GTT analysis, mice were intraperitoneally injected with glucose (1 g/kg) after 16 h of fasting, and the blood was sampled 0, 15, 30, 60, 90, and 120 min after glucose injection. For ITT analysis, mice were intraperitoneally injected with 0.5 units/kg of insulin after 6 h of fasting, and the blood was sampled 0, 15, 30, 60, 90, and 120 min after insulin injection.
HEK293T cells, HeLa cells, HepG2 cells and Hep3B cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, USA), 100 units/ml penicillin (Invitrogen, Carlsbad, USA) and 100 µg/ml streptomycin (Invitrogen, Carlsbad, USA). CHO cells were cultured in Ham’s F12-K medium (Invitrogen) supplemented with 10% FBS, 100 units/ml penicillin and 100 µg/ml streptomycin.
HPA-v cells were cultured in preadipocyte medium (PAM, #7211, ScienCell Research Laboratories) supplemented with 5% FBS, 100 units/ml penicillin and 100 µg/ml streptomycin. Two days after confluence, cells were switched into differentiation medium containing 5% FBS, 0.5 mM IBMX (Sigma-Aldrich, #I5879), 5 µg/ml insulin, 1 µM dexamethasone (Sigma-Aldrich, #D4902), for 2 days, and then replaced with DMEM containing 5% fetal bovine serum, 5 µg/ml Insulin for 2 days. Cells were switched to DMEM containing 5% FBS and cultured for another 4 days, then be used for experiments.
Methyl-tryptophan treatment to cells was achieved by incubating cells in DMEM medium without serum for 2 hours, followed by incubating in amino acids-free medium (US biological life sciences) for another 1 hour, and then treated with methyl-tryptophan as indicated by experiments respectively, 100 nM insulin was used to stimulate insulin signaling for 10 minutes before harvest of cells.
Kynurenine (KYN) and 5-HT treatments was performed by incubating cells in DMEM medium without serum for 2 hrs, followed by incubating in amino acids-free medium for another hour, and then treated with KYN or 5-HT for 1 hour, 100 nM insulin was used to stimulate insulin signaling for 10 minutes before harvest of the cells.
IDO and TDO inhibition was carried out by pre-incubating cells with 100 nM INCB024360 or 2.5 µM LM for 48 hours, then incubating cells in DMEM medium without serum for 2 hours, followed by incubating in amino acids-free medium for 1 hour. Cells were treated with Me-Trp for another 1 hour, and cells were harvested after 10 minutes stimulation with 100 nM insulin.
Plasmids Construction And Transfection
Whole length human WARS, IR and SIRT1 were amplified HEK293T cDNA and cloned into Xho I and EcoR I sites of the pcDNA3.1-Flag/HA vector using CloneExpress MultiS One Step Cloning Kit (Vazyme, #C113-02, CHN). The mutants were generated by site-directed mutagenesis using the Mut Express MultiS Fast Mutagenesis Kit (Vazyme, #C215-01, CHN) according to the manufacturer’s instructions. The primers used as follows:
Reverse, 5’-TAGTCCAGTGTGGTGGAATTCCTGAAAGTCGAAGGACAGCTTC C-3’
All other primers used in this study were list in Supplementary Table 1.
Small Rna Interference
Synthetic oligos were used for siRNA-mediated silencing of WARS and SIRT1, scramble siRNA was used as a control. Cells were transfected with siRNA using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s manual. siRNA sequences were as follows: siWARS: 5’-CCAGGAUCCUUACUUUAGAdTdT-3’;siSIRT1: 5’-GAAGUUGACCUCCUCAUUGUdTdT-3’.
Crispr/cas9 Generation Of Knockout Cells
To generate WARS and IR knock cells, the following guide sequence targeting the human WARS forward: 5′-CACCGAACTGCCCAGCGTGACCAG-3′, reverse: 5′-AAACCTGGTCACGCTGGGCAGTTC-3′, and the human IR forward: 5′-CACCGG CGGTGGCCGCGCTGCTACT-3′; reverse: 5′-AAACAGTAGCAGCGCGGCCACC GCC-3′ were used, following standard CRISPR/Cas9 gene editing protocols.
Crispr/cas9 Genomic Knock-in Cell Lines
IRK1209R knock-in cell line was generated by using CRISPR/Cas9 mediated-mutagenesis method. Briefly, IRK1209R-sgRNA-pX458 and ssODNs were co-transfected into cells using Lipofectamine 3000 (Invitrogen). 48 hours after transfection, FACS selection was performed to sort GFP-positive single cells into 96-well plates and then allowed them to expand for 2–3 weeks. PCR analysis was carried out to identify cells with success knock-in, and verified by both Sanger sequencing of genomic DNA and western blotting. The guide sequence targeting IRK1209 were: Forward, 5’-CACCG CACCGGAGTCCCTGAAGGAT-3’; Reverse, 5’-AAACATCCTTCAGGG ACTCCG GTGC-3’.
The non-radioactive 2-dexoyglucose uptake was detected using a Glucose Uptake-Glo™ Assay Kit (J1341, Promega, Madison, USA) according to manufactured instruction. Briefly, HPA-v cells were seeded on 96-well plates and differentiated to mature adipocytes, and then serum starved for 2 hours followed by amino acids starvation for 1 hour before the assay. Cells were treated with methyl-tryptophan for 40 minutes, and stimulated with 100 nM insulin and incubated with 1 mM 2-DG in PBS for additional 15 minutes at 37°C. Cells were then lysed in stop buffer and neutralized with neutralization buffer. Lysates were then centrifuged at 15000 g for 15 minutes at 4°C, and the supernatant were incubated with 2DG6P detection reagent for 1 hour at 25°C, then recorded luminescence with 0.3-1 second integration on a luminometer (Glomax96, Promega, Madison, USA).
Cultured cells or cells extracted from mice inguinal fats were homogenized with 0.5% NP-40 buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Nonidet P-40, and a mixture of protease inhibitors (Sigma-Aldrich). After centrifugation at 12,000 rpm at 4°C for 15 minutes, the supernatant was collected for western blotting according to the standard procedures. Membranes were developed using ECL-Plus (Thermo Fisher Scientific) and visualized using Typhoon FLA 9500 (GE Healthcare, UK).
Cells were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Nonidet P-40, and a mixture of protease inhibitors (Sigma-Aldrich). After centrifugation at 12,000 rpm at 4°C for 15 minutes, the supernatant was incubated with anti-Flag beads at 4°C for 4 hours with rotation. The binding complexes were washed with 0.5% NP-40 buffer three times and mixed with loading buffer for SDS-PAGE.
In vitro tryptophanylation assay
The in vitro tryptophanylation reactions were carried out in a 30 µl reaction mix that contained 50 mM HEPES (pH7.5), 25 mM KCL, 2 mM MgCl2, 5 mM tryptophan, 4 mM ATP, 10 nM WARS, and 0.05 µg/µl synthetic substrate peptide. The mixture was incubated at 37°C for 2 hours, and the peptide was then desalted by passing through a C18 ZipTip (Millipore) before subjecting to analysis by a MALDI-TOF/TOF mass spectrometer (SCIEeX-5800).
Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, Inc. San Diego, USA). A two-tailed Student’s t-test was used to evaluate statistical significance between two groups. Data are shown as means ± S.E.M. P < 0.05 was considered statistically significant, and the significance was indicated as: *, P < 0.05; **, P < 0.01; ***, P < 0.001.