All animal experiments were performed according to procedures approved by the Ethical Committees of the State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University (approved in 2017, approval number WCHSIRB-D-2017-183). 8-week-old male C57BL/6 mice were purchased from Chengdu DaShuo Biotechnology Co., Ltd. 10-week-old male wild type (WT) C57BL/6 mice and 10-week-old male obese C57BL/6 (ob/ob) mice were purchased from Model Animal Research Center of Nanjing University. Animals were housed in a plexiglass cage (5 per cage) at a temperature (22 ± 3ºC) and humidity (55 ± 15%). Animals were provided with food and sterile water and kept on a 12-hour light-dark cycle acclimated for 1 week before the study. β3-adrenergic agonist CL-316,243 (Sigma, U.S.A.) was intraperitoneally injected into mice every day at 1mg/kg bodyweight for 5 days. For acute cold exposure, mice were individually caged with food withdrawn and water provided, placed in a 4℃ cold room, and core body temperature was measured with a thermometer (Taishi, TES-1310, China). The experiment was conducted in a random manner. All the mice of the same sex and weight in a certain range were randomly divided into three groups. Four animals of the same sex in the same nest and with similar body weight were used as the compatibility group. After the allocation, the number of animals in each group was equal. The weight of each group was similar, so as to reduce the experimental error. According to the allocation of different stages of the experiment, there are corresponding records on the label outside the cage. Correspondingly, the result evaluation and data analysis are analyzed according to the random cage unit.
Preparation of plasma
Plasma was collected from the tail vein (mice or rat) with syringe pre-treated with heparin sulfate. Blood was span down at 2000g, 20min at room temperature. 500µl of freshly collected plasma was incubated with 500µl of 2x sample buffer at 95℃ for 10 min. Before the analysis, 1µl of each sample was added to 49 µl of 1x sample buffer and further incubated at at 95℃ for 10 min and then were used to perform SDS-PAGE.
In vivo adenovirus associated virus injection
Adenovirus associated virus (AAV) expressing N-terminally EGFP-tagged NPM3 genes (AAV-NPM3-EGFP) was designed and synthesized by Hanbio Co. Ltd. AAV containing only EGFP (AAV-CTR) was used as a negative control. For in vivo injection, 8-week-old male wild-type C57BL/6 mice were used. The interscapular brown adipose tissue (BAT) was injected with AAV at 5 different sites in each side with 1.0×10^10 transducing units per site to cover the whole tissue. Two weeks after injection, the mice were sacrificed and the BAT tissues were obtained. BAT transplantation was carried out as previously described1. In brief, AAV-NPM3-EGFP or AAV-CTR infected BAT was isolated and cut into several pieces and transplanted into 8-week-old male C57BL/6 mice. For each recipient mouse, a total of 1.0 g of the resulting slices of fat were transplanted into the inguinal area. One week later, the mice were sacrificed and the iWAT and adjacent muscle
3T3-L1 preadipocytes Differentiation and Treatment
3T3-L1 preadipocytes were obtained from Kunming Cell Bank, Chinese Academy of Sciences and maintained in DMEM with 10%FBS. For adipogenic induction, 3T3-L1 preadipocytes were incubated with 10% FBS-DMEM medium supplemented with 0.5mM isobutylmethylxanthine (Sigma, USA), 1mM dexamethasone (Sigma, USA) and 5mg/ml insulin (Merck, USA) for five days. Then the cells were treated with induction medium supplemented with rosiglitazone (2µM) for browning induction for another five days.
3T3-L1 preadipocytes (5×104 per well) were seeded in 24-well plates and cultured overnight. The cells transfected with 50 nM non-targeting siRNA or three different NPM3-targeting siRNAs (Ruibo, China) using the Lipofectamine 3000 transfection reagent following the manufacturer's instructions (Life Technologies, USA). The negative control group was treated only with transfection reagent. After transfection, cells were used for RNA extraction for qPCR. The knockdown efficiency of NPM3 targeting siRNAs was evaluated by qPCR 72h post-transfection. siRNA #2 and siRNA #3 showed >90% inhibition of NPM3 mRNA expression compared to the control siRNA. Therefore, NPM3 siRNA #2 and #3 was used in all of the experiments. In vivo study, 15 nmol Cholesterol-modified siNPM3 (Ribobio) dissolved in diluted water were injected directly into the BAT of 8 week-old male C57BL/6 mice (n=3) every day by local injection at multiple points. 5 days later, BAT was collected for sEVs isolation.
Glucose consumption measurements
The cell culture medium of 3T3-L1 preadipocytes after browning induction was collected at different time points (0, 12, 24, 36, 48h). The concentration of glucose in the medium was determined using EnzyChromTM Glucose Assay Kit (BioAssay, USA) following the manufacturer's instructions. The absorbance was measured at 570nm with a spectrophotometer (MultiskanGO, Thermo Scientific).
RNA immunoprecipitation (RIP) assay
3T3-L1 preadipocytes (5×104 per well) were seeded in 24-well plates and cultured overnight. FLAG-NPM3 fusion protein expression plasmids and empty plasmids with the same backbone (GeneCopoeia, USA) were used. The cells were transfected with 1µg plasmids using the Lipofectamine 3000 transfection reagent (Life Technologies, USA) following the manufacturer's instructions. After 48h, the cells were used to perform a RIP experiment using an anti-FLAG antibody (CST, USA) or isotype-matched control antibody (normal rabbit IgG; Sigma). Following the recovery of antibodies using protein A/G beads, qRT-PCR was performed on the precipitates to detect the gene expression. RNA immunoprecipitation (RIP) assay was performed using a Magna RIP Kit (EMD Millipore, Billerica, MA, USA) according to the manufacturer’s instructions.
mRNA stability analysis
NPM3 overexpressed 3T3-L1 preadipocytes cells (5×104 per well) were induced for browning in a 24-well plate for 10 days. 3T3-L1 preadipocytes cells (5×104 per well) were also treated with siNPM3 for 2 days and subsequently induced for browning in a 24-well plate for 10 days. Then cells were treated with actinomycin D (5mg/ml), total intracellular RNA was harvested at different times (0, 1, 2, 3, 4 and 5h). qRT-PCR analysis was performed to calculate relative mRNA expression using the 2−ΔΔCT method. mRNA levels were calibrated to the 0h time point.
sEVs used in this study were isolated using the Total Exosome Isolation reagent with minor modifications. Briefly, 5g of adipose tissue were collected from 8-week-old male C57BL/6 mice, washed extensively with sterile phosphate-buffered saline (PBS) to remove the debris and red blood cells. The tissue was cut into small pieces (1–2mm3) under aseptic condition and then treated with 10ml 0.075% collagenase (type I) for 30 min at 37°C. The digested adipose tissue was centrifuged at 300g for 10min and the supernatant (SN-AT) was collected, filtered (0.22µm filter) to remove the debris of cells. Then the supernatant was concentrated with Amicon® Ultra-15 Centrifugal Filter Units (10,000Mw cut off the membrane, Millipore, USA) at the speed of 5,000g for 30min (4℃, Beckman Avanti J-26S XP centrifuge, JS5.30). The concentrated medium was mixed with 0.5 volume of Total Exosome IsolationTM reagent (Life Technologies, USA), incubated overnight at 4°C and spun down for 1h at 10,000g at 4°C. The pellet was re-suspended in 100µL and used for cell treatment or injection in vivo.
EV analysis with sequential centrifugation
The supernatant from digested adipose tissue (SN-AT) was analyzed by sequentially centrifuged. SN-AT was firstly centrifuged at 2,000g for 10 min to collect large EVs (lEVs), then the supernatant (SNI) was collected and further centrifuged at 20,000g for 30 min (4℃, Beckman Avanti J-26S XP centrifuge, JA25.50, polyamide tube (Cat. 357003)). The pellet was collected as mixed EVs (mEVs), and the supernatant (SNII) was further ultracentrifuged at 120,000g for 2h (4℃, Himac CP 70MX centrifuge, P40ST, Kadj:328.96, polyamide tube (Cat. 332901A)) to collect sEVs. The supernatant (SNIII) was also collected for western blot analysis. The pellets in every centrifugation step were collected and re-suspended in 100µL PBS for further analysis.
Recipient cells were seeded into 6-well tissue culture plates (Corning, NY, USA) at a density of 2.5×104 cells per well and allowed to attach overnight. Costar 24 mm Transwell® Permeable Support Inserts with 0.4 µm Polyester Membranes (Corning) were placed on top of each well. Donor cells were seeded into the inserts at a density of 2×105 cells per insert and made up to 1.5 ml final volume of culture medium. For GW4869 (20µM, Selleck, USA) or calpeptine (50nM, MCE, USA) treatment, the compounds were diluted appropriately in DMSO and added to the culture media in the inserts. The plates were incubated for 48h at 37°C, 5% (v/v) CO2.
2.5×104 3T3-L1 cells were seeded into Confocal Dish (Martinsried, Germany) and allowed to attach overnight. Cells were fixed with 4% (w/v) paraformaldehyde and permeabilized with 0.3% (v/v) Triton X-100 for 5min, blocked with 5% (w/v) bovine serum albumin in PBS for 1h at room temperature and incubated with primary antibodies overnight at 4℃ followed by secondary antibodies for 2h. The primary antibodies and dilutions are NPM3 (1:200). The secondary antibodies and dilutions are DAPI (1:1,000); Alexa-555 conjugated anti-rabbit (1:300). Cells were imaged on an Olympus FV1000 confocal microscope. Images were stacked to ensure equal adjustments to all images.
Proteinase K Digestion Assay
sEVs (TEI reagent precipitation) were collected and exposed in suspension to either 1µg/µl Proteinase K (Sigma), or 0.3% v/v Triton-X100, or both, or neither for 15 minutes on ice. After exposure, all samples were incubated with 1mM PMSF proteinase inhibitor (KeyGEN, China) for 15 minutes, before undergoing normal Western blot.
Induction of 3T3-L1 preadipocytes using sEVs
3T3-L1 preadipocytes were plated in 24-well plates at a density of 105 cells/ well, cultured for 24h, then rinsed with PBS and incubated with 2 ml of one of three different culture medium for up to 10 days. The medium were: (1) basal medium (DMEM supplemented with 10% fetal bovine serum (FBS)), as a negative control; (2) basal medium supplemented with sEVs-BAT (50µg/ml); (3) basal medium supplemented with sEVs-BAT-siNPM3 (50µg/ml). The medium was changed every 3 days. The cells were collected on day 10 for qRT-PCR analysis.
sEVs injection in vivo
8-week-old male C57BL/6 mice (purchased from Chengdu DaShuo Biotechnology Co., Ltd) were divided into three groups (n=4). They were injected via the tail vein with sEVs-BAT or sEVs-BAT-siNPM3 (2µg sEVs/g body weight, resuspended in 200µl PBS) isolated from the digested BAT every 2 days and lasted for 2 weeks. The control group (CTR) was injected with 200µl PBS. 2 weeks later, O2 consumption was detected and the iWAT and eWAT were collected for Immunochemical (IHC) staining and qRT-PCR analysis.
To evaluate the effects of sEVs on obesity, a normal diet containing 10% kcal fat (RDI, D12450J) and a high-fat diet containing 60% kcal (RDI, D12492) were purchased from Research Diets, Inc. 8 week-old male C57BL/6 were fed with a high-fat diet for 6 weeks firstly, at 7th week, the weight was approximately 20% more than the normal diet-fed mice, they were injected via the tail vein with sEVs-BAT or sEVs-BAT-siNPM3 (2µg sEVs/g body weight, resuspended in 200µl PBS, n=6) isolated from the digested BAT every 2 days and lasted for 9 weeks. Bodyweight, food intake was monitored weekly. After 9 weeks of injection, glucose tolerance, insulin sensitivity was determined and iWAT and eWAT were collected for immunochemical (IHC) staining and qRT-PCR analysis.
Oxymax metabolic analysis
sEVs treated mice were acclimated for 12h in the metabolic cages, and their metabolic rates were measured for 24h in an indirect open-circuit calorimeter (Oxymax Comprehensive Lab Animal Monitoring System; Columbus Instruments). O2 consumption was measured at room temperature (RT) and normalized to body weight to account for the disparity in body weight between the groups.
Glucose tolerance test (GTT) and Insulin tolerance test (ITT)
For GTT, mice were fasted for 8 h. After basal glucose measurement, glucose (2g/kg, i.p.) was injected and blood glucose was measured from the tail tip at 15, 30, 60, 90 and 120min by using a glucometer (Accu-Chek, Roche Diagnostics). For ITT, mice were fasted for 5 h. Following basal glucose measurement at 0 min time point, Insulin (0.75 U/kg, i.p.) was injected in both groups and blood glucose from the tail tip was measured at 15, 30, 60, 90, and 120 min.
Immunochemical (IHC) staining
iWAT, eWAT, BAT, liver, and muscle tissues were fixed in 10% neutral-buffered formalin for 24 hours. Tissues were embedded in paraffin and sectioned at 4µm. For IHC, tissues were incubated for 2h at 60℃, deparaffinized, and rehydrated. Antigen retrieval was performed using citrate buffer (pH6) at 97◦C for 20 min. Endogenous peroxidase activity was blocked by incubating the sections with 3% hydrogen peroxide for 10 min at room temperature. Non-specific binding of the antibody was blocked by incubating the slides with 5% normal goat serum in PBS containing 0.1% Tween 20 (PBST) for 1 h at room temperature. The slides were then incubated with primary antibodies against NPM3 (1:200, Zen Bioscience, China), UCP1 (1:200, Abcam, U.K.) overnight at 4℃. After washing, each slide was incubated with the appropriate HRP-labeled secondary antibody, and signals were developed with DAB solution before counterstaining with hematoxylin.
Western blot analysis
Total proteins were extracted by the Total Protein Extraction Kit (KeyGEN, China). 30µg proteins were dissolved in RIPA Lysis Buffer (KeyGEN, China), resolved on a 10% polyacrylamide gel and blotted onto PVDF membrane. The membranes were blocked for 1h and then incubated with primary antibodies (listed in Table S1) at 4°C overnight, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 1h at room temperature. Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) was used for the detection following the manufacturer’s instructions. Signals were visualized by ImageQuant LAS4000 mini (GE Healthcare, USA). Band intensities were determined using Image J software and normalized to internal control ACTB.
Total cellular RNA was extracted using RNAiso Plus (TaKaRa Biotechnology) according to the manufacturer’s instruction. The quantity of RNA was calculated based on the absorbance at 260 nm detected by a NanoDrop 2000 spectrophotometer. 260/280 nm absorbance ratio between 1.8 and 2.0 was considered as good purity RNA and used for further experiment. cDNA was reverse transcribed from 2µg of RNA with First Strand cDNA Synthesis Kit (Thermo Scientific, USA) according to the manufacturer’s instructions with a final volume of 50µl. 1µl out of 50µl reverse transcribed cDNA was used as a template for qPCR with iTaq™ Universal SYBR® Green (BioRad, USA) utilizing Eco Real-time PCR System (Illumina, USA). Reaction conditions were: 95°C for 2min; followed by 40 cycles of 95°C for 5 s, 60°C for 30s. The results were analyzed using the 2 − ΔΔCT relative quantitative method with ACTB as an internal control. Primer sequences are listed in Table S2.
Results are presented as mean ± SEM. All statistical tests were performed using GraphPad Prism 5. Significance between the two groups was assessed by Student’s t-test. The comparisons between multiple groups were carried out using one-way ANOVA followed by Tukey’s test. Linear regression analysis was used to analyze plasma NPM3 levels of mice across different body weight groups. Sample sizes and other statistical parameters are indicated in the figures and texts. *p< 0.05, **p< 0.01, ***p< 0.001. Significance was concluded at p< 0.05.