All animals maintained on a 12 h light/dark cycle. To obtain high fat diet induced obesity model, C57/BL6J mice (6 weeks old) were randomly assigned to two groups. Then, the mice were fed either normal chow diet (Chow) with 10 KJ% fat (Keao Xieli, Beijing) as control group, or high fat diet (HFD) with 60 KJ% fat (Keao Xieli, Beijing) as obesity group ad libitum until 16 weeks. Before adipose tissue was collected, the mice were anesthetized with isoflurane and sacrificed after cervical dislocation.
All animal experiments were carried out in accordance with Chinese Council on Animal Care guidelines, and the experimental protocol was approved by the Animal Research Committee of Xinxiang Medical University. Additionally, all methods were performed in accordance with the relevant guidelines and regulations and the study was carried out in compliance with the ARRIVE guidelines.
Body weight and composition analysis
Body weights were measured every week with a digital scale. Body composition of lean body mass (LBM) and fat mass were measured using time-domain nuclear magnetic resonance (TD-NMR) method (the minispec Live Mice Analyzer LF90, Bruker).
Analysis of glucose metabolism in mice
Blood samples were collected from submandibular vein and centrifuged at 2000 g for 10 min. Plasma insulin and leptin were measured by commercial enzyme-linked immunosorbent assay (ELISA) kit (Crystal Chem Inc. #90080, #90030). Plasma triglyceride, free fat acid (FFA), glycerol, and cholesterol levels were measured using commercially available kits (Cayman, 10010303, 700310, 10010755, 10007640).
For glucose tolerance test (GTT), mice were injected intraperitoneally (i. p.) with D-glucose (2 mg/g body weight) after overnight fasting, blood glucose levels were measured using Glucometer (Contour, Bayer) before and following the injection from the facial vein of non-anesthetized animals. For insulin tolerance test (ITT), 6 h fasted mice were injected by i. p. with human recombinant insulin (0.75 U/kg body weight, PAN Biotech), and tail-tip blood glucose was measured before and after the injection.
Metabolic Phenotyping analysis
Sixteen weeks old Chow and HFD mice were acclimated into home cage of Promethion Core System (Sable Systems International) for 24 h before the start of the experimental record. Then, O2 and CO2 exchange were continuously recorded every 9 min for 1 min for 4 days, and locomotor activity was continuously recorded by interruptions of infrared light beams. O2 consumed (VO2), CO2 produced (VCO2), and energy expenditure (EE) were calculated following the manufacture’s manual, and the respiratory exchange ratio (RER) was computed by the ratio of VCO2/VO2. Finally, EE was analyzed either after the normalization by LBM or locomotor activity using covariance (ANCOVA).
Total protein of brown adipose tissue (BAT), subcutaneous white adipose tissues (sWAT), and epididymal white adipose tissues (eWAT) were lysed by RIPA buffer, and quantitated by PierceTM BCA Protein Assay kit (23225, ThermoFisher Scientific). Equal amounts of protein (20 μg) were separated by SDS-PAGE electrophoresis, transferred to PVDF membrane, and blocked in 5% skim milk in TBST. Then, the membrane was incubated overnight with primary antibody solution at 4 ºC. After rinsing the blot, the membrane was incubated in the horseradish peroxidase (HRP)-conjugated secondary antibody solution for 2 h at room temperature. Finally, chemiluminescent signals on the blot were applied with SuperSignal™ West Femto Maximum Sensitivity Substrate (34095, ThermoFisher Scientific), and captured using Amersham Imager 600 imagers (GE Healthcare Life Sciences). The band intensity of protein was read by image analysis software Fiji. The following primary and secondary antibodies were used in this study: Ago2 (RN003M, MBL International, 1:1000), UCP1 (#14670, Cell Signaling Technology, 1:1000), AMPKα (#2532, Cell Signaling Technology, 1:1000), p-AMPKα (#2535, Cell Signaling Technology, 1:1000), GAPDH (ab8245, Abcam, 1:1000), Goat Anti-Rabbit IgG H&L (HRP) (ab6721, Abcam, 1:10000), Goat Anti-Mouse IgG H&L (HRP) (ab6789, Abcam, 1:10000).
Quantitative real-time polymerase chain reaction (qPCR)
Total RNA was isolated from BAT, sWAT, eWAT and inguinal white adipose tissue (ingWAT) using RNeasy plus Mini kit (QIAGEN) and quantitated by Spark® (TECAN). First strand cDNA from total RNA was efficiently synthesized using RevertAid First Strand cDNA Synthesis Kit (#K1621, ThermoFisher Scientific). Quantitation of miR-148a-3p expression level was performed with TaqMan™ MicroRNA Assay (4440887, Applied Biosystems) and normalized to U6 snRNA following the primers from Applied Biosystems: mmu-miR-148a-3p (mmu477814_mir) and U6 snRNA (001973). qPCR of mRNAs was detected using FastStart SYBR Green Master (04673484001, Roche) and normalized to β-actin in StepOne™ Real-Time PCR Systems (Applied Biosystems). The sets of primer sequence were ordered from ThermoFisher Scientific in Supplementary Table S1.
The HEK293T cell line was obtained from CCTCC (1101HUM-PUMC000091, Beijing). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; 10569010, Gibco) supplemented with 10% fetal bovine serum (FBS; abs972, Absin) and 1% penicillin/streptomycin (15140122, Gibco) in a 5% CO2 atmosphere at 37 ◦C.
Luciferase assay and transfection
The 3′-UTR of murine AMPKα was PCR amplified using the following primers 5′-TGG TAG CAT AGC ATA ATG GG-3′ and 5′ -CAA CAG TTT ATA GAG ATA TTC CTC AG-3′ and cloned into the pGL3 Luciferase Reporter Vectors (Promega). HEK293T cells were seeded for co-transfection of AMPKα 3′-UTR plasmid and miR-148a mimics (MC10263, ThermoFisher Scientific) or mirVana™ miRNA Mimic, Negative Control #1 (4464058, ThermoFisher Scientific). After 48 h of transfections, luciferase assays were detected using the Dual-Luciferase® Reporter Assay System (Promega) following the manufacturer's manual.
The adipose tissue pads and liver were dissected from 16-week-old mice, fixed in 4% paraformaldehyde (PFA), and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (H&E) and imaged using a Nikon ECLIPSE 80i microscope.
All experimental data were expressed as mean ± standard error (SEM), statistical analysis was performed using student's t test, two-way ANOVA in GraphPad Prism 8, and ANCOVA in SPSS. P < 0.05 was considered statistically significant (see Supplementary Table S2 online.).