2.1 Human pancreas tissue sections and human islets
Human pancreas tissue sections (3 µm, paraffin embedded) with T2DM or non-diabetes were obtained from the Human Islet Resource Center (HIRC, China), Tianjin First Central Hospital, People of the Republic of China, with informed research consent. Human islets were prepared in HIRC, China, from the pancreases from organ donors with informed research consent. Human islets were isolated by Collagenase NB1 (SERVA, Heidelberg, Germany) and Neutral Protease NB (SERVA, Heidelberg, Germany) digestion and continuous density purification, according to our earlier documents[10, 11]. High purity islets (>80%) were collected and cultured in CMRL-1066 medium (Corning, Manassas, VA, USA), supplemented with 10% Human Serum Albumin (Baxter, Vienna, Austria), 100 U/mL penicillin, and 100 mg/mL streptomycin, at 37 ℃ in 5% CO2. All study protocols were approved by the Medical Ethical Committee of Tianjin First Central Hospital.
2.2 Mice islet isolation and treatment
Male ICR, 8 to 10 weeks, were purchased from Beijing Huafukang Biosciences (Beijing, China). The islets used in the in vitro experiments were isolated from male ICR mice because of their high yield of islets. All mice were fed standard chow and maintained on a 12-h light-dark cycle (lights on at 7:00 AM). Islets were isolated from ICR mice, and the pancreatic tissue was perfused with collagenase P (0.5 mg/ml, Roche, Basel, Switzerland) containing either 0.1% DMSO (control; Solarbio, Beijing, China) or 100 mM CORM-2 (Sigma-Aldrich, St. Louis, MO, USA) and incubated on ice for 30 min, followed by digestion at 37℃for 11 min and purification by density gradient (Histopaque 1077, Sigma-Aldrich, St Louis, MO, USA). Isolated islets were either used immediately or cultured in RPMI 1640, supplemented with 1% penicillin/and streptomycin, 10% fetal bovine serum (FBS), 37℃ and 5% CO2.
2.3 Cell culture
Mouse pancreatic β cell lines βTC-6 (ATCC CRL-11506TM) and NIT-1 (ATCC CRL-2055TM) were used in this study. βTC-6 was cultured in DMEM medium, supplemented with 10% FBS, and NIT-1 cells were cultured in DMEM/F-12 medium, supplemented with 10% FBS at 37℃ in 5% CO2. Both media were supplied with 100 U/mL penicillin and 100 μg/mL streptomycin.
2.4 Drug treatment
SAFit2 (Aobious, AOB6548) was dissolved in dimethyl sulfoxide (DMSO, MilliporeSigma, D2650) to a final concentration of 10 mM. Cells were treated with DMSO, 1 μM SAFit2 for 8 h, 16 h, and 24 h. SAFit2 was added to fresh culture medium When the cell confluency reached 80% and then added to the 12-well plates as part of the medium change.
2.5 siRNA knockdown of FKBP5/FOXO1
Human islets, mouse islets or NIT cells were cultured as described above. Small interfering RNA (siRNA) against FKBP5 and FOXO1 for human and Fkbp5/Foxo1 for mouse were purchased from GenePharma (Shanghai, China). Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used to transfect siRNA, according the manufacturer instructions. mRNA and protein expression examination were performed at 24 h or 48 h after transfection.
2.6 RNA isolation and qRT-PCR
Total RNA was extracted from tissue samples or cultured cells using Trizol Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Total RNA was reverse transcribed to cDNA using reverse transcriptase (RT) reaction kit (Takara, Kohoku-Cho, Kusatsu, Japan). qRT-PCR was performed using FastStart Essential DNA Green Master on a LightCycler96 machine (Roche, Basel, Switzerland). The relative expression of mRNA to internal control (β-actin RNA) was calculated using the 2-ΔΔCt method. The primers used in this study, human genes included: 1) 36B4-F: 5'-AGGCGTCCTCGTGGAAGTGA-3'; 36B4-R: 5'-GCGGATCTGCTGCATCTGCT-3′ ;2) FKBP5-F: 5'-CTCCCTAAAATTCCCTCGAATGC-3'; FKBP5-R: 5'-CCCTCTCCTTTCCGTTTGGTT-3', 3) NKX6.1-F:5'-GGGCTCGTTTGGCCTATTCGTT-3'; NKX6.1-R: 5'-CCACTTGGTCCGGCGGTTCT-3'; 4) MAFA-F: 5'-GCTCTGGAGTTGGCACTTCT-3'; MAFA-F: 5'-CTTCAGCAAGGAGGAGGTCA-3'; 5) INS-F: 5'-GCAGCCTTTGTGAACCAACAC-3'; INS-R: 5'-CCCCGCACACTAGGTAGAGA-3'. Mouse primer used included: 1) β-actin-F: 5'-GTGACGTTGACATCCGTAAAGA-3', β-actin-R: 5’- GCCGGACTCATCGTACTCC-3'; 2) Fkbp5-F: 5'-TTTGAAGATTCAGGCGTTATCCG-3'; Fkbp5'-R: 5'-GGTGGACTTTTACCGTTGCTC-3'; 3) Nkx6.1-F: 5'-CTGCACAGTATGGCCGAGATG-3′; Nkx6.1-R: 5'-CCGGGTTATGTGAGCCCAA-3′; 4) Mafa-F: 5'-AGGAGGAGGTCATCCGACTG-3′; Mafa-R: 5'-CTTCTCGCTCTCCAGAATGTG-3′;5) Ins-F: 5'-CAATCATAGACCATCAGCAAGC-3′; Ins-R: 5'-AGAAACCACGTTCCCCAC-3′.
2.7 Western blot
NIT-1 cells and βTC-6 cells were harvested from culture dishes and lysed in RIPA buffer (Thermo Fisher Scientific, 89900, Waltham, MA, USA) supplemented with protease inhibitors and phosphatase inhibitors (Invitrogen, Carlsbad, CA, USA). Protein concentrations were determined using a BCA Protein Assay Kit (Solarbio, Beijing, China), and 10 µg protein was separated by SDS-PAGE (15%/4-20%) for PVDF membrane blotting. The blotted membranes were blocked with 5% skim milk for 60 min at room temperature and incubated with primary antibodies FKBP51 (1:1000, ABclonal, A3863, ABclonal, Woburn, MA, USA), p-FOXO1 (1:1000, NB100-81927, Novus Biological, Cambridge, UK), Foxo1 (1:1000, CST-2880, Cell signaling technology, Danvers, MA, USA), p-AKT(1:1000, CST-4060S, Cell signaling technology, Danvers, MA, USA), AKT(1:1000, CST-9272S, Cell signaling technology, Danvers, MA, USA), PDX1(1:1000, CST-9679S, Cell signaling technology, Danvers, MA, USA), Sqstm1/p62 (1:1000, ab56416, Abcam, Cambridge, MA, USA), LC3I/II (1:1000, CST-12741S, Cell signaling technology, Danvers, MA, USA), BAX(1:1000, ab32503, Abcam, Cambridge, MA, USA), BCL2(1:1000, ab59348, Cambridge, MA, USA), β-actin (1:1000, CST-3700, Cell signaling technology, Danvers, MA, USA) at 4℃ overnight. The immunoblots were visualized by enhanced chemiluminescence using horseradish peroxidase-conjugated IgG secondary antibodies and then quantified using ImageJ. The relative expression level of a specific protein was normalized to β-actin. The expression level in the control group was arbitrarily set at unit 1. The protein expression fold changes in the treatment groups were normalized relative to the control groups. Data from at least three independent experiments were presented following the corresponding blotting images in the figures.
2.8 Insulin secretion assay
Ten islets (human and mouse) were pretreated in 1 mL of 1.67 mM low-glucose Krebs-Ringer bicarbonate buffer (KRB; supplemented with 0.5% BSA, pH 7.4) for 1 h in a 12-well plate, followed by sequential treatment with 1 mL low-glucose KRB solution (1.67 mM) for one h and high-glucose KRB solution (16.7 mM) for 1 h. The media with low- and high-glucose levels were collected separately. Insulin concentration was measured using an ELISA kit (Mercodia, Uppsala, Sweden). Insulin secretion of islets was expressed as the glucose-stimulated index (GSI; insulin secretion at high glucose/insulin secretion at low glucose).
2.9 Immunohistochemistry
Human islets were fixed in 4% paraformaldehyde, embedded with paraffin, and sectioned (3mm). After deparaffinization, sections were treated with EDTA antigen retrieval solution (Solarbio, Beijing, China) in a microwave oven, washed, permeabilized, and blocked. Immunohistochemical staining was performed using FKBP5 (ABclonal, OH, USA), and then incubation with TRITC AffiniPure Goat Anti-Rabbit IgG H&L (1:200, Jackson Immunoresearch Laboratories and Molecular Probes, West Grove, PA, USA) secondary antibodies. Counterstaining was performed with DAPI (Vector, Burlingame, CA, USA). We used Pannoramic MIDI and Pannoramic Viewer (3DHistech, Budapest, Hungary) to scan stained slides and capture images. The Image Pro-Plus software (Media Cybernetics, Silver Spring, Maryland) was used to quantified in a blinded fashion.
For immunostaining in cultured β cells, cells were washed three times in PBS, and the fixed in 4% PFA for 20 min, incubated 15 min in 0.05% Triton X-100, then washed three times in PBS and incubated with rabbit anti-Fkbp5(1:300, ABclonal, OH, USA) and rabbit anti-p-Foxo1 (1:300, NB100-81927, Novus Biological, Cambridge, UK) at 4 ℃ overnight. After washing, cells were incubated with Goat Anti-Rabbit IgG secondary antibodies (1:200, Jackson Immunoresearch Laboratories and Molecular Probes, West Grove, PA, USA) secondary antibodies and then counterstained with DAPI. Images were captured under fluorescent microscope.
2.10 Apoptosis and autophagy analysis
The Annexin V-PE/7-AAD apoptosis detection kit (BD Biosciences, Franklin lakes, NJ, USA) was used to assess the cell apoptosis level. Following the manufacturer’s, cells stained with PE conjugated Annexin V and 7-Amino-Actinomycin D (7-AAD) in dark environment with room temperature for 15 min, and then underwent flow cytometry analysis (FACScan, BD Biosciences, Franklin lakes, NJ, USA). The early staged apoptotic cells (Annexin V-PE+7-AAD-) and late staged apoptotic cells (Annexin V-PE+7-AAD+) were together assumed as apoptotic cells.
Cell autophagy was detected using Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, NY, USA),and propidium iodide (PI) staining was also used to exclude the apoptotic cells. Chloroquine (CQ) was used to block the autophagosome degradation, and the concentration of CQ was selected according to the previous study [11, 12]. Autophagy level was evaluated by flow cytometry, as described previously [13, 14].
2.11 Statistics
Figure drawing and data processing were performed using GraphPad Prism v9.0 (GraphPad Software, 218 La Jolla, CA, USA). Student t test was used for analyzing the group differences. Data are shown as mean ± SEM. p < 0.05 was considered statistically significant.