Glucagon-like peptide-1 affects human umbilical vein endothelial cells in high glucose by the PI3K/Akt/eNOS signaling pathway

Reagents

GLP-1, exendin, wortmannin and L-NAME were purchased from Sigma (USA). P-Akt antibody [phospho-Akt (Ser473) antibody: #9271] and p-eNOS antibody [phospho-eNOS (Ser1177) antibody: #9571] were purchased from Cell Signaling Technology, Inc. (USA). Annexin-V/PI double staining kits and NO assay kit were obtained from Beyotime (China). Exendin, wortmannine and L-NAME were purchased from ApexBio (USA).

Phospho-Akt (Ser473) antibody, a mouse polyclonal antibody, can detect endogenous levels of Akt1 only when phosphorylated at Ser473 of Akt from human by Western blot. Phospho-eNOS (Ser1177) antibody, a rabbit polyclonal antibody, can detect endogenous levels of eNOS only when phosphorylated at Ser1177 of Akt from human by Western blot.

Cell culture

Primary human umbilical vein endothelial cells (HUVECs, Catalog Number: C-12200) from normal human placenta umbilical cord tissues were obtained from Promo Cell GmbH (Heidelberg, Germany) as cryopreserved primary cultures. After purchased, HUVECs were recovered and sub-cultured in endothelial cell medium containing 5% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., USA) and 1% endothelial cell growth supplement in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. After added different experimental factors, HUVECs in logarithmic growth phase were divided into six groups. It is as follows: physiological concentration of glucose (control: 5 mmol/L of glucose), high glucose (HG: 33 mmol/L of glucose), high glucose and GLP-1 (HG+G: 33 mmol/L of glucose and 10 nmol/L of GLP-1), physiological concentration of glucose and GLP-1 (control+G: 5 mmol/L of glucose and 10 nmol/L of GLP-1), high glucose, GLP-1and exendin (HG+G+E: 33 mmol/L of glucose, 10 nmol/L of GLP-1 and 5 nmol/L of exendin), high glucose, GLP-1 and wortmannine (HG+G+W: 33 mmol/L of glucose, 10 nmol/L of GLP-1 and 100 nmol/L of wortmannine), and high glucose, GLP-1 and L-NAME (HG+G+L: 33 mmol/L of glucose, 10 nmol/L of GLP-1 and 100 μmol/L of L-NAME). Exendin, wortmannine and L-NAME were added in 3 min ago.

The detection of cell apoptosis rate

The flow cytometry was used to detect the cell apoptosis rate. The HUVECs in logarithmic growth phase were inoculated into six-well plates, and a suspension of HUVECs at 4×10⁵ cells/mL in a 2-mL volume of culture medium was incubated at 37°C. The experimental groups were based on the above method, and every group was in triplicate. After HUVECs incubated for 48 h, the culture solution was discarded. Next, after washed with D-Hank’s and digested with pancreatic enzymes successively, the cells were gathered. After the cell solution was centrifuged (10,000×g, 10 min, 4°C), the supernatant was removed. After washed with phosphate buffer saline (PBS) precooling at 4°C, the cell solution was centrifuged (10,000×g, 10 min, 4°C) again, and then the supernatant PBS was removed. The washing was repeated twice. According to the operating instructions of Annexin-V/PI double staining kits, the HUVECs were stained and quantitatively analyzed by the flow cytometry (FACS Calibur 95, BD Biosciences). The data were presented as dot plots showing fluorescence intensity of Annexin-V and PI.

The detection of p-Akt and p-eNOS expression

The Western blot was used to detect the expression of p-Akt and p-eNOS. The HUVECs in logarithmic growth phase were inoculated into 25 cm³ of culture bottle, and a suspension of HUVECs at 1×10⁶ cells/mL in a 5-mL volume of culture medium was incubated at 37°C. The experimental groups were based on the above method, and every group was in triplicate. After HUVECs incubated for 48 h, the HUVECs was washed twice with PBS precooling at 4°C and collected by cell scraper. Next, according to the operating instructions of protein extraction kit, the total protein of each group was extracted and measured by Bradford method. After 40 μg of the total protein in each group was denatured at 100°C for 5 min, the protein was separated by 12% SDS-PAGE and transferred to PVDE membrane, and then sealed by 5% skimmed milk for 2 h. After the membrane was washed, p-Akt antibody (1:1000) and p-eNOS antibody (1:1000) were added and incubated overnight. After that, horseradish peroxidase labeled second antibody was added and incubated at room temperature for 1 h. After washed, the membrane dried, and the target protein was detected by chemiluminescence method. β-Aatin was internal reference and used to calculate the relative content of the target protein. Signal intensity of the bands was analyzed to use ImagePro Plus Software of version 6.0 (Media Cybernetics, Inc., MD, USA).

The detection of NO concentration

The HUVECs in logarithmic growth phase were inoculated into six-well plates, and a suspension of HUVECs at 4×10⁵ cells/mL in a 2-mL volume of culture medium was incubated at 37°C. The experimental groups were based on the above method, and every group was in triplicate. After incubated for 48 h, 0.1 mL of supernatant was taken out. According to the operating instructions of NO assay kit, the absorbance of the supernatant was determined by ELISA at 550 nm to calculate the concentration of NO.

Statistical analysis

The statistical analyses in this work were carried out by SPSS version 17.0. All data were presented as the mean±SD. Comparisons between two groups were performed using student’s t-test. Values of p<0.05 were considered to indicate a statistically significant difference.

The effect of GLP-1 and high glucose for the apoptosis rate of HUVECs

The percentage of apoptotic cells was examined by Annexin-V/PI double staining. The apoptosis rate of HUVECs in HG was 23.8% and significantly higher than 4.8% of control group (p<0.05). The apoptosis rate of HUVECs in HG+G was 4.5% and significantly reduced in comparison with HG. The apoptosis rate of HUVECs in HG+G+E (23.1%), HG+G+W (24.7%) and HG+G+L (21.8%) was markedly higher than HG+G. The all above results were showed in Figure 1.

Figure 1:

The apoptosis rate of HUVECs in the different conditions was labeled with Annexin V/PI and detected by the flow cytometry.

^ap<0.05 compared to control; ^bp<0.05 compared to HG; ^cp<0.05 compared to HG+G. (A) control, (B) HG, (C) control+G, (D) HG+G; (E) HG+G+E; (F) HG+G+W, (G) HG+G+L and (H) a histogram of the apoptosis rate from A to G.

The effect of GLP-1 and high glucose for the p-Akt and p-eNOS expression of HUVECs

The expression of p-Akt and p-eNOS in HG reduced (p<0.05) in comparison with control group, but the expressions in HG+G significantly increased (p<0.05), and the expression in HG+G+E with exendin and HG+G+W with wortmannine also decreased in comparison with HG+G, as showed in Figure 2. The expression of p-Akt in HG+G+L with L-NAME were not significantly different from HG+G (p>0.05), as shown in Figure 2A and B. However, there was a significant difference between HG+G+L and HG+G in the expression of p-eNOS (p<0.05), as shown in Figure 2C and D.

Figure 2:

The effect of GLP-1 and high glucose for the p-Akt and p-eNOS expression of HUVECs.

The histogram (A) showed the relative amount of p-Akt in HUVECs. The images (B) showed the analyzed result of p-Akt in HUVECs by Western blot. The histogram (C) showed the relative amount of p-eNOS in HUVECs. The images (D) showed the analysis result of p-eNOS in HUVECs by Western blot. ^ap<0.05 compared to control; ^bp<0.05 compared to HG; ^cp<0.05 compared to HG+G; ^dp>0.05 compared to HG+G.

The effect of GLP-1 and high glucose for the NO production of HUVECs

The NO production of HG was obviously less than the control group (p<0.05), but the HG+G produced more NO than HG (p<0.05). The NO production of HG+G+E with exendin, HG+G+W with wortmannine or HG+G+L with L-NAME obviously reduced (p<0.05). The all above results were showed in Figure 3.

Figure 3:

The histogram showed the effect of GLP-1 and high glucose for the NO concentration of HUVECs in the different conditions.

^ap<0.05 compared to control; ^bp<0.05 compared to HG; ^cp<0.05 compared to HG+G.