Study of the effect of metformin on the level and expression of endocan in diabetic conditions

Abstract

Background: Diabetes mellitus is a chronic metabolic disease with complicated vascular injuries. Endocan is a novel endothelium-derived proteoglycan and may play a role in endothelial cells activity under diabetic conditions. Objective: Here, we evaluated the effect of high glucose concentration on endocan level in presence or absence of metformin. Methods: Human Umbilical Vein Endothelial Cells (HUVECs) viability was assessed by MTT. Cell migration was studied by scratch test. Endocan expression and protein levels were evaluated by RT-PCR, ELISA and flow cytometry. Griess reaction was used to measure nitric oxide (NO) levels. Functional activity of cells was monitored using Dil-Ac-LDL uptake. BALB/c mice (35-40 g) were divided into 4 groups (n=6): Control, diabetic group was injected Streptozocin, and groups were given 50 and 100 mg/kg metformin orally, once daily for 2 weeks. Endocan was detected in tissues by Immunofluorescence (IF) analysis. Phosphorylation of AMPK was assessed using western blotting. Histological examination was performed to follow the von Willebrand factor (vWF) expression. Results: High glucose concentration (30mM) reduced endothelial cells’ viability and migration, whereas these features were improved with metformin. Metformin increased endocan transcription and protein levels, NO production, and LDL uptake capacity in diabetic condition (p<0.05). Western blotting confirmed the increase of p-AMPK/AMPK ratio in metformin-treated cells. ELISA assay showed elevated levels of endocan by metformin in tissues. IF and Histological examination of kidneys showed that the increase of endocan protein coincided with the promotion of vWF factor in metformin-treated mice (p<0.05). Western blotting confirmed the phosphorylation of AMPK by metformin in tissues. Conclusion: Metformin could change the endocan levels possibly by the modulation of p-AMPK/AMPK axis and through its effect on vWF promotes angiogenic potential of endothelial cells.