eNOS-wild-type (WT)—The cDNA sequence of human eNOS (kindly provided by Dr. Yong Xia) was subcloned into pCDN3.1 (His-tagged). eNOS-S1177A, eNOS-T866A, eNOS-S867A and eNOS-S738A are point mutant of eNOS-WT were constructed by site-directed mutagenesis (Ser1177, Thr866, Ser867, Ser738 mutated to Ala). All mutations were performed by TransGen (TransGen Biotech, BeiJing, China) and were confirmed by direct sequencing.
Cell Culture, Transfection, and Hypoglycemia Induction
Bovine aortic endothelial cells (BAECs, passages 6–10) and human embryonic kidney (HEK293) cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) FBS (Invitrogen) and 1 % (v/v) penicillin/streptomycin (PS) (Beyotime Biotechnology, JiangSu, China) in a humidified incubator at 37 °C with 5% CO2. For cell transfection, BAECs grown in 100-mm dishes were transfected with plasmids encoding eNOS-WT and eNOS mutants using Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions and then analyzed 48 h after transfection. HEK293 cells in 6-well cell culture plates were transfected with plasmids encoding eNOS-WT or the eNOS mutants using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions and then analyzed 24 h after transfection. To induce hypoglycemia (LG), BAECs and HEK293 cells were cultured with medium containing 1 mmol/L glucose.
AMPKα1 Gene Silencing in BAECs
The sense (5′-GAUCCAUCAUAUAGCUCAAdTdT-3′) and antisense (3′-UUGAGCUAUAUGAUGGAUCdTdT-5′) Small interfering RNA (siRNA) strands of AMPKα1 were purchased from Biomic (Nanjing, China). siRNA oligonucleotides (75 nM) were delivered into cells using Opti-MEM (Invitrogen) and Lipofectamine 2000 according to the manufacturer’s protocol. After transfection for 48 h, cells were subjected to further experiments.
Preparation of Protein Samples from Cells
After culturing, cells were harvested by scraping. Harvested cells were washed three times with ice-cold PBS, resuspended in 100 μl lysis buffer (Beyotime) containing protease inhibitor cocktail (Beyotime) and PUGNAc (Sigma Aldrich, St. Louis, MO, USA) in a clean Eppendorf tube, and kept on ice for 1 h. The homogenate was then centrifuged at 13800 × g and 4 °C for 15 min and the supernatant were recovered. The concentration of supernatant was determined by the Bradford protein assay.
Preparation of Protein Samples from Rat Aortae
Male and Female Sprague-Dawley rats approximately 6 to 8 weeks of age and 180 to 220 g weight were purchased from Experimental Animal Center of Chongqing Medical University. All animal procedures were carried out according to the guidelines of the China Animal Protection Law and were approved of by the Institutional Ethics Committee of Chongqing Medical University [Permit No. SCXK (Chongqing) 2007–0001] and the State Science and Technology Commission of China. Animals were housed individually and maintained on a 12 h light/12 h dark schedule at 22–23 °C with ad libitum access to food and water.
After overnight fasted, rats (200–250 g) were randomly divided into 4 groups (5 rats in each group): control group, hypoglycemia for 3, 6 and 9 h group. In hypoglycemia groups, rats were treated by glargine insulin (Lantus-Solostar, Paris, France) with subcutaneous injection (200 g /U). In control group, rats were treated by PBS with subcutaneous injection (200 g /1 ml). Blood glucose was monitored every 30 min from 0 to 9 hours after insulin injection via tail prick, hypoglycemia was considered when glycemic < 3 mmol/l (14, 15). Rats were anesthetized by 2 % sodium pentobarbital solution with subcutaneous injection (0.002 ml /g). The thoracic aorta was quickly removed, and washed by ice cold PBS. After washing with ice-cold PBS, rat thoracic aortae were cut into pieces. Tissues were resuspended in lysis buffer containing protease inhibitor cocktail and PUGNAc in a clean Eppendorf tube. Tubes were placed on a cracker and the tissues were homogenized for 70 min at 4 °C. Lysate supernatants were collected by centrifugation at 13800 × g for 15 min at 4 °C. The concentration of supernatant was determined by the Bradford protein assay.
For immunoprecipitation, aliquots of cell homogenates were incubated with polyclonal antibodies targeting OGT (4 μg/ml) (Abcam, Cambridge, UK) overnight at 4 °C. Protein G-Sepharose beads (GE Healthcare, Chicago, IL, USA) were added to the supernatant and incubated for 3 h. The beads were washed thoroughly with lysis buffer and then eluted by boiling for 3 min in SDS-PAGE sample buffer (Beyotime).
eNOS Pull-down Assay
In BAEC and rat thoracic aorta, eNOS was extracted from the lysate by affinity precipitation using 2′,5′-ADP Sepharose beads (GE Healthcare). Cell/tissue lysates were and mixed with prepared 2′,5′-ADP Sepharose resins (50% slurry) at 4 °C for 2 h with gentle shaking. The mixture was then centrifuged at 13800 × g for 1 min. The supernatant was discarded, and the resins were washed with washing buffer (PBS supplemented with 500 mM NaCl) three times. Bound proteins were eluted by boiling the resins in 50 μl SDS-PAGE sample buffer for 10 min. For transfected cells, eNOS was extracted by affinity precipitation after transfection using the His-tag protein purification Kit (Beyotime) per the manufacturer’s protocol.
For western blotting, proteins were fractionated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocking for 1.5 h in blocking buffer (20 mM Tris-HCl (pH 7.5),150 mM NaCl, 5% skim milk powder, 0.1% (v/v) Tween-20) at room temperature, the membranes were reacted with the appropriate primary antibodies (primary antibodies 1:2000; diluted in blocking buffer) at 4 °C overnight. Thereafter, membranes were thoroughly washed three times with TBST (20 mM Tris-Cl (pH 7.5), 150 mM NaCl, 0.1% (v/v) Tween-20) for 10 min per wash. Membranes were then incubated in 1:5000 diluted secondary antibody for 1.5 h, thoroughly washed three times (10 min per wash) with TBST, and finally detected by chemiluminescence (Advansta Inc., San Jose, CA, USA).
Primary antibodies targeting phospho-eNOS Ser1177, phospho-eNOS Ser633, phospho-eNOS Thr495, and eNOS were purchased from Cell Signaling Technology (Danvers, MA, USA); O-GlcNAc antibody (RL2) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies targeting O-GlcNAcase (OGA), Threonine, phospho-AMPK Thr172, and anti-AMPKα1 were purchased from Abcam (Cambridge, UK); β-actin antibody was purchased from Proteintech (Rosemont, IL, USA). Goat anti-mouse IgG and Goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and Proteintech, respectively.
Preparation of eNOS for HPLC-MS
eNOS was extracted by affinity precipitation from eNOS-WT-transfected HEK293 cells using the His-tag protein purification Kit. Affinity-precipitated eNOS was fractionated by SDS-PAGE, and the gel was stained with Coomassie brilliant blue. Stained eNOS was excised from the gel and stored in a clean Eppendorf tube after the excised gel was proven to contain eNOS by immunoblotting using the Micro Protein PAGE Recovery Kit (Sangon Biotechnology, Shanghai, China). Gels in the Eppendorf tubes were decolorized and swelled. After de-coloration and swelling, absolute acetonitrile was added to shrink and solidify the gel. At last, the acetonitrile was then absorbed and the gel was heat-dried. Dithiothreitol (DTT) solution were added to the tubes and mixed thoroughly, then incubated at 56 °C for 1 h; after incubation, the solution was discarded and the sample was heat-dried. Indole-3-acetic acid (IAA) solution was added, mixed well, and the mixture was incubated at room temperature for 30 min; thereafter, the solution was discarded and DTT was added. The mixture was incubated at room temperature for 15 min for neutralize any remaining IAA; the solution was then discarded and the sample was heat-dried. Chymotrypsin was added to the sample at an enzyme/protein ratio of 1:20 in a reaction system containing 200 μl 50 mmol/L ammonium bicarbonate solution, and incubated at 37 °C for 14 h. After the enzymatic digestion was completed, the enzymatic digestion solution was successively added with 1% TFA solution, 60% acetonitrile solution, 0.1% TFA solution, and absolute acetonitrile solution. The reaction was conducted at 37 °C for 1 h respectively, and the reaction solution was combined.
HPLC-MS for Enrichment of eNOS O-Glycosylation
Digested samples were dissolved in A solution (deionized H2O containing 0.1% formic acid) and centrifuged at 1000 × g for 5 min; the supernatants were then tested using the Thermo Orbitrap Lumos HPLC-MS system with a data collection time of 120 min, spray voltage of 2.20 kV, capillary temperature of 320 °C, collision energy of 50%, first-level mass range of collection of 300–1800 m/z, and second-level scanning range of 100–1400 m/z. Data from HPLC-MS for the enrichment of eNOS O-glycosylation were provided by the Beijing Proteome Research Center Tandem Mass Spectrometry (MS/MS) laboratory (China).
Measurement of NO
The concentration of NO released from BAECs in culture medium was measured in terms of the concentration of nitrate and nitrite using a modified Griess reaction method with the Total Nitric Oxide Assay Kit (Beyotime). Aorta blood samples from normal and hypoglycemic rats were collected using vacuum hemostix. Plasma was stored at -80 °C until assay. NO concentrations were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) per the manufacturer’s protocol.
eNOS Activity Assay
eNOS activity was measured from the conversion rate of L-[14C] Arginine to L-[14C] Citrulline in cells. The conversion was monitored at a total volume of 300 ul buffer containing 50 mM Tris-HCl, pH 7.4, 5 μM L-[14C] arginine (Moravek, Brea, CA, USA), 45 μM L-arginine, 0.5 mM NADPH, 10 μM BH4, 10 μg/ml calmodulin, and 10 nM eNOS. The reactions were initiated by adding L-[14C] arginine and terminated by stop buffer (20 mM HEPES, 2 mM EDTA) after 1 h incubation at 37 °C. L-[14C] Citrulline was separated by passing the reaction mixture through a Dowex AG 50W-X8 (Na+ form) (Sigma) cation exchange columns and quantitated by liquid scintillation counting. N(gamma)-nitro-L-arginine methyl ester (L-NAME; 5 mM) inhibitory activity was analyzed to determine the concentration of L-[14C] citrulline converted by eNOS.
All data are presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using SPSS by two-way analysis of variance (ANOVA), one-way ANOVA, or unpaired Student’s t-test. A value of P < 0.05 was considered statistically significant. Data analysis was performed using SPSS version 25 (SPSS Inc/IBM, Chicago, Ill, USA).