Animals and experimental design
Four-week-old male C57BL/6 mice were purchased from Shanghai Jihui Laboratory Animal Care. After 1 week adaptation to the environment, rats were randomized to fed with a standard chow-fat diet (10 % of calories from fat) or a high-fat diet (HFD: 60 % of calories from fat). Mice fed with chow-fat diet served as normal controls. 10 weeks later, the HFD mice were then randomized into 3 groups and received three times tail vein injection at d0, d4, d9 with either 1) phosphate buffered saline (PBS)(n=5), 2) 5×105 ADSCs from normal control mice (N-ADSCs) (n=5), 3) 5×105 ADSCs from obese mice (O-ADSCs) (n=6). Body weight and blood glucose levels were continuous monitored and % change was calculated. At 2 months after cell infusion, intraperitoneal glucose tolerance (IPGTT) and insulin releasing test (IRT) were performed after 12-hours fast. At the end, the mice were sacrificed and tissues of adipose, liver, and skeletal muscle were rapidly removed and stored at -80℃.
ADSCs isolation and culture
Fresh inguinal fats were isolated from normal chow or obese mice and were washed with Dulbecco's phosphate buffered saline (D-PBS, Gibico). The adipose tissues were cut into small fragments and then digested with 2mg/ml type Ⅰ collagenase (Sigma-Aldrich) for 30 minutes at 37ºC on a shaker. Cells were resuspended after mixtures centrifuged at 1500 rpm for 5 min. Next the cell suspension was filtered through a 40-mm nylon filter mesh (BD Falcon) to remove tissue residues. After twice washes, cells were resuspended with alpha-MEM medium (Gibico) plus 10% fetal bovine serum (Gibico) and seeded in 75-cm2 tissue-culture flasks (BD Falcon). Cells were cultured in a humidified 5% CO2 incubator at 37ºC. Floating cells were removed at 24 hours and mediums were changed per 3 days. At 90% confluence, cells were detached with 0.25% trypsin-EDTA for passage.
Intraperitoneal glucose tolerance and insulin releasing test
For IPGTT, all mice received intraperitoneal injection with 2 g/kg of glucose after an overnight fast. At 0, 15, 30, 60, 90, and 120 minutes after glucose load, blood glucose levels were measured with a glucometer (Roche). At 0, 15 and 30 minutes, 0.1mL blood samples were collected from the orbital venous plexus. The samples were centrifuged immediately and plasma were stored at -80°C. Subsequent insulin assay was performed by ELISA (Alpco) according to the manufacturer’s protocol. HOMA-IR index was calculated by the equation: HOMA-IR index = (FBG [in mmol/L] × FINS [in units/L])/22.5.
RNA extraction and quantitative RT-PCR
Total RNA was extracted from liver, muscle and adipose, then converted into first-strand cDNA with the first-strand cDNA synthesis kit (Takara). Quantitative RT-PCR was performed using SYBR Master Mix (Takara) and a LightCycler 480 System (Roche). The quantity of mRNA was normalized to β-actin in liver and muscle and to 36B4 in adipose.
Statistical analyses
Statistical calculations were performed using SPSS version 25 and Graph Pad Prism 8. Data were expressed as mean±SD. Student’s t test or ANOVA was performed to analyze the differences. The area under the curve (AUC) of the IPGTT and IRT were calculated. P<0.05 was considered statistically significant.