O‐GlcNAcylation Mediates Glucose‐Induced Alterations in Endothelial Cell Phenotype in Human Diabetes Mellitus

Background Posttranslational protein modification with O‐linked N‐acetylglucosamine (O‐GlcNAc) is linked to high glucose levels in type 2 diabetes mellitus (T2DM) and may alter cellular function. We sought to elucidate the involvement of O‐GlcNAc modification in endothelial dysfunction in patients with T2DM. Methods and Results Freshly isolated endothelial cells obtained by J‐wire biopsy from a forearm vein of patients with T2DM (n=18) was compared with controls (n=10). Endothelial O‐GlcNAc levels were 1.8‐ford higher in T2DM patients than in nondiabetic controls (P=0.003). Higher endothelial O‐GlcNAc levels correlated with serum fasting blood glucose level (r=0.433, P=0.024) and hemoglobin A1c (r=0.418, P=0.042). In endothelial cells from patients with T2DM, normal glucose conditions (24 hours at 5 mmol/L) lowered O‐GlcNAc levels and restored insulin‐mediated activation of endothelial nitric oxide synthase, whereas high glucose conditions (30 mmol/L) maintained both O‐GlcNAc levels and impaired insulin action. Treatment of endothelial cells with Thiamet G, an O‐GlcNAcase inhibitor, increased O‐GlcNAc levels and blunted the improvement of insulin‐mediated endothelial nitric oxide synthase phosphorylation by glucose normalization. Conclusions Taken together, our findings indicate a role for O‐GlcNAc modification in the dynamic, glucose‐induced impairment of endothelial nitric oxide synthase activation in endothelial cells from patients with T2DM. O‐GlcNAc protein modification may be a treatment target for vascular dysfunction in T2DM.


Introduction
We evaluated eNOS activation by determining eNOS phosphorylation at Ser1177. The supplemental material is sought to determine NO levels by 5,6diaminofluorescein diacetate (DAF-2 DA) fluorescence in the endothelial cells from non-diabetic patients treated by Thiamet G, a high-specific O-GlcNAcase (OGA) inhibitor. This would support a functional correlation between O-GlcNAc and eNOS activity.

Subjects
Patients who underwent elective cardiac catheterization from November 2019 to January 2020 at the National Defense Medical College Tokorozawa, Japan were enrolled in the supplemental study. Exclusion criteria included ongoing treatment for diabetes mellitus, malignant tumor and hemodialysis.
The study protocol was approved by the National Defense Medical College Review Board, and all participants provided written informed consent. The details of diagnostic criteria, laboratory examinations were shown in our previous study. 27

Endothelial cells
Fresh arterial endothelial cells were harvested from patients without diabetes mellitus as described previously. 27 Briefly, an inner dilator of a radial catheter sheath used for coronary angiography was extracted under a sterile technique.
In the laboratory, endothelial cells adherent to the disposable device were removed by washing with an erythrocyte lysing kit (R&D Systems) and the pellet was obtained by centrifugation. The pellet was washed again with endothelial basal medium (EBM-2; Lonza, Basel, Switzerland) and plated on poly-L-lysinecoated microscope slides (Sigma, St. Louis, MO).

Nitric oxide detection
Endothelial nitric oxide levels were visualized and quantified using DAF-2 DA staining, a membrane-permeable fluorescent indicator for nitric oxide by a Slide images of a fluorescence microscope at × 40 magnification were captured (KEYENCE, Osaka, Japan). Exposure time was constant, and image intensity was corrected for background fluorescence. Fluorescent intensity was quantified by a software (KEYENCE, Osaka, Japan) in 20 cells from each slide and averaged. Wilcoxon Signed Rank test was used for comparing paired samples of freshly isolated endothelial cells from a same patient to assess treatment effects. The data of experimental studies were expressed as mean ± standard error. A 2-sided P value < 0.05 was considered statistically significant.

Immunofluorescence
Freshly isolated endothelial cells were treated with or without Thiamet G in chamber slides for 8 hours and preserved in a refrigerator at -80°C after fixing with 4% paraformaldehyde. In another day, the slides were incubated overnight at 4°C with primary antibodies against O-GlcNAc (RL2) (1:50 dilution; Santa-Cruz, Dallas, TX). The slides were double-stained with anti-von Willebrand Factor (vWF) antibody (1:300 dilution; Thermo Fisher Scientific) for identification of endothelial cells. After incubation, the slides were washed and incubated for 3 h at 37 °C with corresponding Alexa Fluor-488 and Alexa Fluor-594 antibodies (1:200 dilution; Invitrogen, Carlsbad, CA). The detail of immunofluorescence microscope was described previously. 24,27

Results
The freshly isolated arterial endothelial cells taken from patients were incubated with or without Thiamet G 1 μM for 8 hours. The characteristics of the patients were shown in Table S1. The increase of insulin-mediated DAF-2 intensities was lower in the cells treated by Thiamet G than the controls (Figure S1). We also confirmed that intracellular O-GlcNAc levels were increased (1.4-fold) by Thiamet G (Figure S2). Table S1. Clinical Characteristics.