Adenoviruses carrying PPARγ1(1-182 aa)-SUMO-1, PPARγ1(1-182 aa), and adeno-association viruses carrying Tie2-PPARγ1(1-182 aa)-SUMO-1, Tie2-PPARγ1 (1-182 aa) were constructed and obtained from GeneChem (Shanghai, CHN). Primary antibodies against p-PI3K (Tyr 458), PI3K, p-Akt (Ser 473), Akt, p-eNOS (Ser 1177), eNOS, p-FOXO1 (Ser 256), FOXO1, His-tag, β-actin, and PCNA were purchased from Cell Signalling Technology (MA, USA); antibodies against p-IKKα/β (Ser 176), IKKα/β were from Santa Cruz Biotechnology (CA, USA). Dulbecco’s modified Earle’s medium (DMEM), radio-immunoprecipitation assay (RIPA) buffer and phenylmethanesulfonyl fluoride (PMSF) was purchased from Solarbio (Beijing, CHN), and foetal bovine serum (FBS) and penicillin-streptomycin were obtained from Gibco (CA, USA). The kits for nitric oxide (NO) assays and ELISA kits for endothelin-1 (ET-1) detection were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, CHN). A kit used for chromatin immunoprecipitation was purchased from Cell Signalling Technology (MA, USA). Recombinant human insulin, acetylcholine (Ach) and sodium nitroprusside (SNP) were purchased from Sigma Aldrich (MO, USA).
Cell culture and establishment of IR model induced by HF/HG in vitro
HUVECs were obtained from the American Type Culture Collection (ATCC, Catalogue No. CRL-1730, USA), and primary rat thoracic aorta endothelial cells (RTAECs) were obtained according to a method described in previous studies [16, 17]. Cells were cultured in 100 mm dishes with 5.5 mM low-glucose Dulbecco’s modified Earle’s medium(L-DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U/mL). In additional, heparin (100 μg/mL) as well as endothelial cell growth factor (20 μg/mL) were needed for RTAECs. Cells were cultured in an incubator filled with 5% CO2 at a humidified atmosphere and a constant temperature at 37 °C.
The IR model was established with HF/HG in vitro as described in our previous report with a few improvements . Briefly, the cells were first cultured in DMEM containing 22 mM of glucose and 0.25 mM palmitic acid (PA) for 48 h. Next, they were washed twice with PBS (pH 7.40) and cultured in FBS-free DMEM for 4 h. Subsequently, the cells were treated with 5 mIU/L insulin for 10 min. Finally, the cell supernatant and cells were harvested for measurements of the levels of nitrite (equivalent to NO) and ET-1, p-PI3K/PI3K, p-Akt/Akt, and p-eNOS/eNOS.
Adenovirus infection in cells and experimental design
The cells infected with adenoviruses carrying a truncation of PPARγ1 (GenBank Accession NM_138711) with intact AF1 domain and a DNA binding domain (DBD) fused with SUMO-1 (GenBank Accession NM_003352), namely Ad-PPARγ1(1-182 aa)-SUMO-1, were used as the PPARγ1-K77 SUMOylation mimic group (Mimic), while the cells infected with PPARγ1(1-182 aa) unfused with SUMO-1, namely Ad-PPARγ1(1-182 aa), were used as the vehicle control group (Veh). The extent of sequence amplification and purification, titer levels, and cell infection were measured according to the instructions provided by GeneChem Technologies, Inc. In the PPARγ1-K77 SUMOylation-imitating IR experiment, HUVECs cultured in either 5.5 mM low-glucose DMEM or 22 mM glucose combined with 0.25 mM PA (22/0.25 mM, HG/HF) for 48 h were respectively considered as the control (Ctrl) or IR group; cells infected with either Ad-PPARγ1(1-182 aa)-SUMO-1 or Ad-PPARγ1(1-182 aa) (MOI=100) and cultured in normal medium for 48 h were used as the SUMOylation mimic (Mimic) or the vehicle (Veh) group, respectively. In the PPARγ1-K77 SUMOylation-worsening IR experiment, treatments in the Ctrl and IR groups were the same as those in the SUMOylation-imitating IR experiment. However, the cells infected with either Ad-PPARγ1(1-182 aa)-SUMO-1 or Ad-PPARγ1(1-182 aa) followed by exposure to HF/HG for 48 h were respectively used as the IR+Mimic or IR+Veh groups. Before evaluated, all these cells were finally stimulated with insulin (5 mIU/L) for 10 min in serum-free medium.
Establishment of IR model in vivo
Male SD rats (6-7 weeks old; 180-200 g) were purchased from Nanchang University School of Medicine. All animal procedures were approved by the Institutional Animal Care and Use Committee of Nanchang University School of Medicine and conducted in accordance with the guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No.85-23, revised 1996). All animals were acclimated to the SPF environment in a 12 h light-dark alternate cycle and approximately 25 °C and 50% humidity and provided with sterilized water. IR was induced by high-fat and high-sucrose (HFS) diets in vivo, as described in our previous study with minor modifications . Briefly, after adaptation for 1 week, the rats were fed the HFS diet for 24 weeks. Serum NO and ET-1 levels were tested; protein levels of the PI3K-Akt-eNOS pathway in aortic tissues were measured.
Adeno-associated virus construction and infection and experimental design
AAV9 and the endothelial cell-specific promoter Tie2 were used to construct AAV-Tie2-His-PPARγ1(1-182 aa)-SUMO-1 and AAV-Tie2-His-PPARγ1(1-182 aa). Recombinant virus (1.25×1013 v.g.) was diluted in 0.2 mL saline solution and injected into the tail vein of rats. In the PPARγ1-K77 SUMOylation simulating IR experiment, 40 male rats were randomly assigned to the normal group (30 rats) that were fed with regular chow, and the IR group (10 rats), which was fed with HFS diets for 24 weeks. After 24 weeks of feeding, the rats on the regular diet were randomly allocated into control (Ctrl), negative control (Veh), and PPARγ1-K77 SUMOylation mimic (Mimic) groups. The rats in the Mimic group were intravenously infected with AAV-Tie2-His-PPARγ1 [1-182 aa]-SUMO-1, whereas the rats in the Veh group were intravenously infected with AAV-Tie2-His-PPARγ1 [1-182 aa]. After infection, all animals were fed a normal diet for an additional 4 weeks. In the PPARγ1-K77-SUMOylation- worsening IR experiment, in addition to the Ctrl and IR groups, an additional 20 IR-rats were first injected with AAV-Tie2-His-PPARγ1(1-182 aa) (IR+Veh group) or AAV-Tie2-His- PPARγ1(1-182 aa)-SUMO-1 (IR+Mimic group), respectively, and then fed with HFS diets for an additional 24 weeks.
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of ET-1 in rats serum (the total blood column samples with visible hemolysis were excluded) or cell supernatants were detected using a specific ELISA kit. Measurements were performed according to the manufacturer’s instructions.
Western blot assay
Cells or aortic tissues isolated from rats were washed three times by pre-cold PBS and lysis buffer was prepared as radio-immunoprecipitation assay (RIPA) buffer supplemented with phenylmethanesulfonyl fluoride (PMSF) (PMSF: RIPA=1:100). Another phosphatase inhibitor cocktail tablet (APPLYGEN, Beijing, CHN) was added for phosphorylation protein detection (1:100). Then, the samples were lysed on ice for 30 min, centrifuged at 14,000 rpm for 15 min at 4 °C, and the supernatants containing total protein were collected immediately for quantification using the BCA protein assay kit (TIANGEN, Beijing, CHN). Equal amounts of protein were loaded and separated on an 8 %–12% SDS-PAGE gel by electrophoresis. The protein samples were transferred onto a polyvinylidene fluoride membrane (Millipore, MA, USA), which was then blocked with 7% skimmed milk solution for 1.5 h, and subsequently incubated at 4 °C overnight with specific primary antibodies at a 1:1000 dilution, under gentle shaking. HRP-conjugated secondary antibody (diluted 1:3000) was incubated for 1 h at room temperature, immunolabeled with an ECL detection kit (FUDE, Hangzhou, CHN) and visualized in the dark. The intensity of the bands was determined using a gel imaging system (Bio-Rad Laboratories, CA, USA), and lanes were quantified using Image J software.
Protein samples were prepared for western blot assay, and a suitable quantity of samples was transferred to a new tube as input, and the remaining samples were incubated with the primary antibody under gentle shaking on a rotating shaker at 4 °C overnight. The next day, protein A/G-beads (Millipore) were washed twice with PBS and then added to samples containing antigen-antibody, which was then incubated for 3 h at 4 °C. The beads were washed three times with immunoprecipitation buffer, and the samples were assessed by western blotting.
HUVECs were seeded on glass coverslips and treated as previously described. Monolayer cells were first cross-linked with 4% paraformaldehyde for 15 min and then washed three times with PBS. The cells were permeabilized with 0.1% Triton X-100-PBS for 20 min. Next, the samples were blocked with goat serum (Boster, Wuhan, CHN) for 1 h at room temperature, and incubated overnight with rabbit anti-FOXO1 (1:100) at 4 °C under gentle shaking. Following three washes with PBS, the cells were incubated with goat anti-rabbit IgG (H+L) CoraLite 594-conjugated secondary antibody (Proteintech, Wuhan, CHN) for 1 h with gentle shaking. Subsequently, the coverslips were covered with 4, 6-diamidino-2-phenylindole (DAPI) at a final concentration of 1 μg/mL (Solarbio Co., Beijing, CHN) for 5 min for staining. After washing three times with PBS, the slices were covered with slides with a drop of mounting medium. Images were captured at random fields from various areas at 200× magnification using a laser scanning confocal microscope (Olympus, Tokyo, Japan).
Chromatin immunoprecipitation (CHIP) assay
ChIP assay was performed using a commercially available kit, as previously reported [19, 20]. Briefly, 37% formaldehyde was added to the dishes at a final concentration of 1%, followed by incubation for 10 min at 37 °C for protein-DNA cross-linking reaction. The reaction was stopped by supplementation with 125 mM glycine for 5 min at room temperature. After cell sonication, the extracted chromatin was digested and randomly fragmented into 150–900 bp, and the fragments were then incubated overnight at 4 °C with ChIP-grade antibodies against His-tag, FOXO1 (1:50), and IgG, which served as the negative control. Then, ChIP-grade protein G agarose beads were added, and the cross-linked DNA-protein complexes were unlinked to obtain a pure DNA fragment for use as the template for PCR. The following oligonucleotides spanning the putative PI3K-binding site were (F) 5′-TGG TGC ATA CCT GTA GTC CC-3′ and (R) 5′-TTT TGA GAT GGA GTC TCG CTT-3′. The bands were obtained by agarose gel electrophoresis. Fold enrichment was calculated by quantitative real-time PCR (q-PCR) to evaluate the degree of PPARγ1 binding to PPRE in the PI3K promoter.
Double luciferase reporter gene assays
For the transactivation assay with a 3×PPRE promoter, HUVECs were seeded on 24-well plates (6×104 cells/well), pre-treated as described above, and co-transfected with 250 ng of reporter plasmid (PMCS-3×PPRE-TK-Luc) and 25 ng of Renilla plasmid (pRL-TK-Renilla) (10:1), which was regarded as the internal control (GeneChem, Shanghai, CHN), using Lipofectamine 3000 reagent (Invitrogen, CA, USA). Twenty-four hours later, the firefly luciferase and Renilla activities were simultaneously measured using a dual luciferase assay kit (Promega, WI, USA) and quantified using a luminometer. Firefly luciferase activity was normalized to Renilla luciferase activity.
Detection of NO concentrations
The concentrations of NO in the cell supernatants or rats serum (the total blood column samples with visible hemolysis were excluded) were measured using specific kits. The measurements were performed according to the manufacturer’s instructions using a microplate reader at the specified optical density.
Detection of vasodilation in vivo
Tests were performed as described in previously published studies, with a few modifications [21-24]. Pre-treated rats were anesthetized with 3-4% isoflurane and administered an intraperitoneal injection of 1 U/kg insulin. Another batch of rats was successively injected with acetylcholine (Ach, 0.05 mg/kg) and sodium nitroprusside (SNP, 0.5 mg/kg). Subsequently, the rats were placed in a supine position on a platform with the left neck exposed to the transducer. To obtain better contact between the skin and transducer, a site on the left side of the neck was shaved and gently smeared with ultrasound gel. Subsequently, the left carotid artery was visualized by placing the probe beside the trachea, and images were recorded using an ultrahigh-resolution small animal ultrasound imaging instrument (Vevo 2100, Toronto, CAN). The inner diameter of the left carotid artery (mm) was measured and used for analysis.
All data were expressed as the means ± standard deviation (SD) and analyzed using GraphPad Prism 6.0. Homogeneity of variance and one-way analysis of variance (ANOVA) were determined where appropriate. Comparisons between inner groups were performed using unpaired Student's t-tests. Differences were considered significant if the P-value was less than 0.05.