Cell culture and treatments
With the approval of the Ethics Committee of Shandong University Qilu Hospital, we obtained fresh human umbilical cords from healthy newborns. All of the participants gave informed consent for the use of umbilical cords in this study. After washing the umbilical cords for thrice in normal saline, we removed blood vessels and cut Wharton’s jelly into small pieces with sterile scissors. These pieces were then spread in cell culture flasks with α-MEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco) and 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. We changed the culture medium every 3 d until the cells reached 80% confluency. The third to the fifth passage of cells were used for identification, administration, and TranswellTM co-culturing.
Human embryo lung fibroblasts (HELFs) were obtained from the China Cell Culture Center (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high-glucose medium (Gibco, USA) supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C and in a 5% CO2 incubator.
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 and antibiotics. After reaching 70–80% confluency, α-cells were stimulated with PA (0.5 mM) for 48 h, followed by treatment with hucMSCs or HELF for 24 h via a TranswellTM system (Catalog No. 3450; Corning, USA). To further explore the mechanism of hucMSCs, α-cells were pre-treated with SIRT1 inhibitor (EX-527; 20 µM, Selleck, Shanghai, China), siR-SIRT1, and siR-FoxO3 before treatment with hucMSCs.
Animal models and hucMSC administration
Six-week-old male C57BL/6J mice were purchased from the Model Animal Research Center of Shandong University (Jinan, China). The mice were housed with a 12-h light/dark cycle at a temperature (22°C–25°C)- and humidity (55 ± 5%)-controlled environment. The T2DM mice model was established by a continuously HFD for 16 weeks followed by a single dose of streptozocin (STZ, 100 mg/kg, Cat. No. S0130; Sigma-Aldrich) intraperitoneally after fasting for 12 h. Four-week-old male db/db mice were purchased from Synergy Pharmaceutical Bioengineering Co., Ltd. (Nanjing, China) and were fed a normal chow diet. T2DM was identified as fasting glucose ≥ 16.7 mmol/L twice in succession. Then, hucMSCs (1×10^6 cells/mouse) were suspended in PBS and injected into both kinds of T2DM mice via the tail vein every 7 d for 6 cycles. To evaluate the mechanism of hucMSCs in vivo, HFD and STZ-induced T2DM mice were also administered intraperitoneally with EX-527 (10 mg/kg) every 3 d for 6 weeks. All of the animal experiments were approved by the Animal Ethics Committee of Shandong University (Ethical No. DWLL-2019-016).
The intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed after hucMSC administration. Each group contained four to six mice selected via randomization procedure. During IPGTT, tail vein blood glucose levels were measured by Accu-Chek® Performa (Roche Life Science, USA) at 0, 15, 30, 60, 90, 120, and 180 min after i.p. injection of 2 g/kg body weight of glucose. During IPITT, tail vein blood glucose levels were measured at 0, 30, 60, 90, 120, and 180 min after i.p. injection of 0.75U/kg body weight of insulin. 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. The sera of mice were stored at −80°C for glucagon enzyme-linked immunosorbent assay (ELISA; Cloud-Clone, Wuhan, China) and insulin ELISA (ALPCO, USA) test.
Immunofluorescence staining of the pancreas
The pancreatic tissues that were fixed in 4% paraformaldehyde were embedded in paraffin, sectioned at 5-µm thickness, and mounted on glass slides. The slides were dewaxed, after which 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. No. 66198-1-Ig, 1:1,000) and anti-rabbit glucagon antibody (Proteintech, Cat. No.15954-1-AP, 1:200) overnight at 4°C. The slides were then incubated with goat anti-rabbit FITC (Zhongshan, Beijing, China, Cat. No. ZF-0311, 1:200) and goat anti-mouse TRITC (Zhongshan, Cat. No. 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 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 the total islet area.
After different kinds of intervention, supernatants were removed and αTC1-6 cells were balanced in Krebs-Ringer bicarbonate HEPES (KRBH) buffer (120 mM NaCl, 0.75 mM CaCl2·2H2O, 4 mM KH2PO4, 10 mM NaHCO3, 1 mM MgSO4·7H2O, 30 mM HEPES, 1% BSA) containing no glucose for 0.5 h in 37°C, followed by incubation in KRBH buffer containing 25 mmol/L glucose for 2 h. Subsequently, supernatants were collected and kept at −80°C for glucagon ELISA (Cloud-Clone, Wuhan, China).
Real-time quantitative PCR analysis
Total RNA from 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 of proglucagon were sense 5’-GCACATTCACCAGCGACTAC-3’ and antisense 5’-CTGGTGGCAAGATTGTCCAG-3’. The primer sequences of β-actin were sense 5’-AAGAGCTATGAGCTGCCTGA-3’ and antisense 5’-TACGGATGTCAACGTCACAC-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 β-actin as control.
Preparation of cell lysate and western blot analysis
After being washed by ice-cold PBS, αTC1-6 cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime, Shanghai, China) for approximately 30 min. Then, the samples were centrifuged at 13,500 rpm at 4°C for 20 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: Glucagon(Abcam, Cat. No. ab92517, 1:1,000), SIRT1(Proteintech, China, Cat. No.13161-1-AP, 1:1,000), FoxO3a(CST, USA, Cat. No. 2497S, 1:1,000), Glucose transporter type 1(GLUT1, Bioss, China, Cat. No.bs0472R, 1:1,000), Glucokinase(GCK, Abcam, Cat. No. ab88056, 1:1,000), NDUFB8(Proteintech, Cat. No.14794-1-AP, 1:1,000), SDHB(Proteintech, Cat. No.10620-1-AP, 1:5,000), UQCRC2(Proteintech, Cat. No.14742-1-AP, 1:1,000), MTCO2(Proteintech, Cat. No.55070-1-AP, 1:1,000), ATP5A1(Proteintech, Cat. No.14676-1-AP, 1:2,000), HSP90(Santa Cruz, USA, Cat. No.sc13119, 1:500), β-actin (CST, Cat. No. 3700S, 1:1,000).
Transmission electron microscopy (TEM)
αTC1-6 cells were exposed to PA, followed by treatment with hucMSCs or HELFs, and then collected by trypsinization. Afterward, cells were fixed with 2.5% glutaraldehyde and post-fixed in 1% phosphate-buffered osmium tetroxide. After being embedded, sectioned, and double-stained with uranyl acetate and lead citrate, electron photomicrographs of αTC1-6 cell ultrastructure were taken via TEM (JEM-1200EX II, JEOL; Tokyo, Japan).
Glucose uptake assay
Glucose uptake ability of αTC1-6 cells was evaluated using fluorescent glucose 2-NBDG (Maokang Bio, Shanghai, China, Cat. No. MX4511) following the manufacturer’s instructions. After intervention, αTC1-6 cells were gently washed with KRBH and incubated with 50 µM 2-NBDG at 37°C for 30 min. Fluorescent intensity of the cells was detected on a microplate reader (Excitation wavelength: 465 nm; Emission wavelength: 540 nm).
Determination of intracellular ATP
Intracellular ATP content was determined using an ATP content kit (Beyotime, China) following the manufacture’s instruction. ATP levels were further normalized by protein content.
The Mito Stress Test Kit (Agilent, Cat. No. 103015-100) was used to detect the oxygen consumption rate (OCR) according to the procedure provided by the manufacturer. αTC1-6 cells were seeded onto each well of an XF96 cell culture microplate at 2*10^4 cells/well. The OCR was assessed in Seahorse XF DMEM Medium (pH 7.4) containing 10-mM glucose, 2-mM pyruvate glutamine, and 1-mM pyruvate according to the manufacturer’s instructions. The concentrations of oligomycin, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and antimycin A/rotenone were 1.5, 1, and 0.5 µM, respectively. OCR was determined and analyzed on the Agilent Seahorse Bioscience XF96 Extracellular Flux Analyzer (Agilent Technologies).
Small interfering RNA transfections
Small interfering RNA (siRNA) oligonucleotides were synthesized by Shanghai GenePharma Co., Ltd. The sequences of negative control (NC) siRNA were as follows: sense 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense 5'-ACGUGACACGUUCGGAGAATT-3'. The sequences for the SIRT1 siRNA were sense 5'-GGGAUCAAGAGGUUGUUAATT-3' and antisense 5'-UUAACAACCUCUUGAUCCCTT-3'. The sequences for the FoxO3 siRNA were as follows: sense 5'-GGAGCUUGGAAUGUGACAUTT-3' and antisense 5'-AUGUCACAUUCCAAGCUCCTT-3'.
αTC1-6 cells were transfected with siRNA for SIRT1 and FoxO3 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 siRNA for SIRT1 and FoxO3 (125 nM) for 6 h. Subsequently, the culture medium was replaced by DMEM medium with 10% FBS, and the cells were treated with hucMSCs. After 24 h, the cells and supernatant were collected for the next experiments.
All of the data were presented as the mean ± SEM. Differences between two groups were evaluated using unpaired Student’s t-test; for three groups or more, a one-way ANOVA was performed followed by Bonferroni’s test for multiple comparisons. P < 0.05 was considered to be statistically significant. GraphPad Prism 8 was used to conduct all of the statistical analyses and to create the graphs.