Culture of rat bone marrow mesenchymal stem cells (BM-MSCs)
Rat primary bone marrow MSCs (BM-MSCs) were obtained by isolating the femurs of rats and flushing the marrow. Then, they were cultured in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F12) (Gibco, USA) with 20% fetal bovine serum (FBS; Gibco) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco). The culture medium was changed every 2 days until the cells reached 80–90% confluency. After the first passage, the MSCs were cultured in DMEM/F12 with 10% FBS. Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 in air.
Identification and differentiation of BM-MSCs and preparation of MSC-conditioned medium
Flow cytometry analysis was performed to identify MSCs according to characteristics that they are positive for CD90 and CD44 (BD, USA) and negative for CD34 (BD), and CD45 (Santa Cruz Biotechnology, USA). The BM-MSCs were analyzed for their ability to differentiate into adipocytes and osteoblasts by Oil Red O (Sigma-Aldrich, USA) staining of lipid droplets and Alizarin Red S staining (Sigma-Aldrich) of calcium nodes. Once the MSCs reached 80–90% confluency, the medium was replaced with serum-free medium and harvested for 24 h. Subsequently, the supernatant was collected and concentrated 20 times using 10-kDa MW cutoff ultrafiltration membranes (Ultra-15 10K; Millipore). Finally, the bmMSC-CM was sterilized by filtration through a 0.22-µm filter and stored at − 80 °C.
Animal models and bmMSC-CM administration
Six-week-old male C57BL/6J mice were purchased from the Model Animal Research Center of Shandong University (Jinan, China) (Ethical number: DWLL-2019-016). The mice were housed with a 12-h light/dark cycle at a temperature (22–25 °C)- and humidity (55 ± 5%)-controlled environment. After one-week of adaptive feeding, the mice were randomly divided into the NCD group (which were fed a normal chow diet) and the HFD group (which were fed a 45% high-fat diet, purchased from Botai Hongda Biotechnology Co., Ltd, Beijing, China). After nine months of HFD, we seperated them into two groups: HFD + PBS and HFD + bmMSC-CM. Approximately 200 µL of the bmMSC-CM concentrate, which is equivalent to a total of 100 µg of bmMSC-CM protein per mouse, were injected into the mice via their tail vein every 3 days for 10 cycles.
The intraperitoneal glucose tolerance test (IPGTT) was performed after bmMSC-CM administration. Each group contained four mice selected via randomization procedure. Tail vein blood glucose levels were measured by Accu-Chek® Performa (Roche Life Science, USA) at 0, 15, 30, 60, 90, 120, and 150 min after i.p. injection of 2 g/kg body weight of glucose. The area under the curve (AUC) was calculated using the trapezoidal rule. After the mice were anaesthetized, their pancreas were fixed in 4% paraformaldehyde for immunofluorescence staining for structural assessment of islets. The sera of mice were stored at − 80 °C for glucagon enzyme-linked immunosorbent assay (ELISA; Bluegene, Shanghai, China).
Immunofluorescence staining of the pancreas
The pancreas that were fixed in 4% paraformaldehyde were embedded in paraffin, sectioned at 5-µm thickness, and mounted on glass slides. The slides were dewaxed, and then antigen retrieval was performed using antigen unmasking buffer. After blocking for 30 min at room temperature in a protein-blocking solution (10% normal goat serum), the slides were incubated with anti-mouse insulin antibody (Proteintech, China, Cat: 66198-1-Ig, 1:1,000) and anti-rabbit glucagon antibody (Proteintech, Cat:15954-1-AP, 1:200) overnight at 4 °C. The slides were then incubated with goat anti-rabbit FITC (Zhongshan, Beijing, China, Cat: ZF-0311, 1:200) and goat anti-mouse TRITC( Zhongshan, Cat: ZF-0313, 1:200) for 60 min at room temperature and then stained for DAPI for 5 min. Fluorescence was observed and captured using a fluorescence microscope (Olympus BX53, Japan). The areas of the islets, α cells, and β cells were analyzed by Image Pro Plus software. The α-or β-cell ratio in islets was measured by the glucagon or insulin-positive area divided by both-positive area (n = 3 mice and 45 islets).
α-Cell culture and processing
Palmitate (PA; Sigma-Aldrich, USA) was prepared by dissolving and heating equimolar amounts of NaOH and palmitate in distilled water to a concentration of 500 mmol/liter palmitate. This was further diluted with 5% bovine serum albumin (BSA) (fatty acid free; Sigma) to make a stock solution of 50 mmol/liter palmitate. The stock solution was filter-sterilized and stored at -20 °C, and palmitate solution was freshly prepared before each experiment.
αTC1-6 cells were obtained from the American Type Culture Collection (ATCC Number: CRL-2934™) and cultured in DMEM/high-glucose medium (Gibco, USA) supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C, in a 5% CO2 incubator. InR1G9 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, USA) with 10% FBS and antibiotics. Both α cells were cultured to 70–80% confluency before treatment. In detail, α cells were randomly divided into the following three groups: normal control group, PA treatment group (PA,0.5 mM), and rescue group with addition of bmMSC-CM (PA + bmMSC-CM).
Isolation and purification of islets from mice
Islets were isolated from normal C57BL/6J mice by collagenase V (1.5 mg/mL, Cat. no. C8170; Solarbio) and DNase Ⅰ (62.5 U/mL, Cat. no. EN0521; Thermo Fisher) digestion followed by hand picking under a stereoscopic microscope (Olympus SZX7). Briefly, we sacrificed the mice and separated the pancreas followed by washing with ice-cold Hanks solution. Then, the pancreas were incubated in 1 mL Hanks solution that included collagenase V (1.5 mg/mL) and DNase Ⅰ (62.5 U/mL) under 37℃ with strong oscillation for about 12 min, until it was disintegrated into a fine sand-like suspension. Subsequently, we added 5 mL of ice-cold Hanks with 10% FBS to stop the digestion, mixed them, and then kept them on ice. After three minutes of natural settlement, we discarded the supernatant and washed the sediment with ice-cold Hanks solution thrice. To purify the isolated islets, we picked the islets into DMEM under a stereoscopic microscope. After incubation overnight, the islets were exposed to 1 mM PA and bmMSC-CM for 48 h and used to glucagon secretion assay.
After stimulation with PA and bmMSC-CM, both of α-cells and isolated islets were incubated for 2 h in Krebs-Ringer buffer (KRB) containing 2.5 mmol/L glucose. Supernatants were collected and kept at − 80 °C for glucagon ELISA.
Preparation of cell lysate and western blot analysis
After washing with ice-cold PBS, the cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime, Shanghai, China) for approximately 30 min. Then, the samples were centrifuged at 12,000 rpm at 4 °C for 15 min. Protein concentration was determined using the bicinchoninic acid (BCA) method (Beyotime, China). Subsequently, proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (EpiZyme, China) and transferred onto polyvinylidene difluoride (PVDF) membranes (IPVH00010 0.45 µm, Millipore, USA). Then, the membranes were blocked with 5% skim milk in Tris-buffered saline solution containing Tween-20 (Sigma-Aldrich, USA) for 1 h at room temperature and incubated in specific primary antibodies at 4 °C overnight. After detection by horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, the proteins were visualized using enhanced chemiluminescence.
The primary antibodies were as follows: PTEN (Abcam, USA, Cat. no. ab32199, 1:10000), Phosphor-AKT (CST, USA, Cat. no. 4060S, 1:2000), AKT (Abcam, Cat. no. ab179463, 1:10000), GADPH (Boster, China, Cat. no. BA2913, 1:500).
Real-time quantitative PCR analysis
Total RNA from BM-MSCs and αTC1-6 cells was extracted using an E.Z.N.A. MicroElute Total RNA Kit (Cat. no. R6831-01; Omega BioTek, USA) following the manufacturer’s instructions. Next, 1 µg RNA was reverse-transcribed into cDNA using a Prime Script RT Reagent Kit (Cat. no. RR047A; Takara, Japan). Primers were chemically synthesized by GenePharma (Shanghai, China) Co., Ltd. The primer sequences were as follows: rno-mir-181a-5p, sense 5'-ATTCTCTCAAACATTCAACGCTG-3' and antisense 5'-TATGGTTGTTCTGCTCTCTGTGTC-3'; U6, sense 5'-CAGCACATATACTAAAATTGGAACG-3' and antisense 5'-ACGAATTTGCGTGTCATCC-3'. Real-time PCR was conducted with the SYBR Green PCR kit (Cat. no. RR420A; Takara), gene expression changes were determined with the comparative CT (2-ΔΔCt) method, and quantification was achieved by normalization using U6 as control.
Luciferase reporter assay
Luciferase reporter assay was conducted as previously described . Wild-type PTEN 3' UTR firefly luciferase reporter plasmids or PTEN 3' UTR firefly luciferase reporter plasmids with the potential miR-181a-5p binding site mutated were co-transfected with miR-181a-5p mimics or miR-NC mimics into αTC1-6 cells using Lipofectamine 2000 (Invitrogen), respectively. After 48 h of transfection, the luciferase assay was performed using a Dual-Glo Luciferase Assay Kit (Promega, Cat: E2920) according to the manufacturer's protocol.
To study the effect of miR-181a-5p secreted by BM-MSCs and its target PTEN on glucagon secretion of α cells, we altered their expression by transfecting miR-181a-5p mimics and a negative control (NC), inhibitors and an miRNA inhibitor negative control (inhibitor NC), small interfering RNA (siRNA) for PTEN and its NC into αTC1-6 cells, respectively. All oligonucleotides were synthesized by Shanghai GenePharma Co., Ltd. Their sequences were as follows: rno-miR-181a-5p mimics, sense 5'-AACAUUCAACGCUGUCGGUGAGU-3' and antisense 5'-UCACCGACAGCGUUGAAUGUUUU-3'; NC, sense 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense 5'-ACGUGACACGUUCGGAGAATT-3'; rno-miR-181a-5p inhibitors 5'-ACUCACCGACAGCGUUGAAUGUU-3'; inhibitor NC 5'-CAGUACUUUUGUGUAGUACAA-3'; siRNA-PTEN, sense 5'-CCCACCACAGCUAGAACUUTT-3' and antisense 5'-AAGUUCUAGCUGUGGUGGGTT-3'; and siRNA-NC, sense 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense 5'-ACGUGACACGUUCGGAGAATT-3'.
αTC1-6 cells were transfected with miR-181a-5p oligomer and siRNA for PTEN using Lipofectamine 2000 transfection reagent (Invitrogen, USA) according to the manufacturer’s instructions. Briefly, the αTC1-6 cells were plated in 6-well plates until they reached 80–90% confluency. The medium was then removed and replaced by Opti-MEM I Reduced Serum Medium (Gibco) mixed with miR-181a-5p mimics (12.5 nM), inhibitor (150 nM), or siRNA for PTEN (125 nM) for 6 h. Subsequently, the culture medium was replaced by RPMI-1640 medium with 10% FBS, and the cells were treated with PA or bmMSC-CM. After 48 h, the cells and supernatant were collected for the next experiments. RT-qPCR analysis was performed to assess transfection efficiency, whereas ELISA and western blot analysis were used for the detection of supernatant glucagon and PTEN/AKT signaling pathway, respectively.
Differences between two groups were evaluated using the t test; for three groups or more, a one-way ANOVA was performed. Differences between the values were considered to be significant at P < 0.05. All of the statistical analyses were carried out with GraphPad Prism 5, and all of the graphs were also created with this software.