Glucagon-like peptide-1 (GLP-1) protects vascular endothelial cells against advanced glycation end products (AGEs)-induced apoptosis

Abstract

Background: The peptide glucagon-like peptide-1 (GLP-1) is a hormone secreted by intestinal L cells in response to food intake. GLP-1 has been proposed as the basis of emerging therapy for patients with type 2 diabetes. However, the effects of GLP-1 on vascular injury in diabetes have not been identified. Advanced glycation end products (AGEs) induce endothelial cell apoptosis and have been implicated in the process of vascular complications from diabetes.

Material/methods: The aim of this work was to investigate whether and how GLP-1 protects endothelial cells from apoptosis induced by AGEs. Human umbilical vein endothelial cells (HUVECs) were treated with AGEs (200 µg/mL) for 48 h in the presence or absence of GLP-1. Cell morphology, viability, apoptosis, ratio of Bcl-2 protein to Bax protein, cytochrome c release, and activity of caspase-9 and -3 were determined.

Results: Treatment of cells with AGEs led to cell morphology changes and decreased cell viability, resulting in apoptosis. GLP-1 alone increased cell viability in a concentration-dependent manner. GLP-1 partially inhibited AGEs-induced apoptosis in HUVECs. GLP-1 increased Bcl-2/Bax ratio, reduced cytochrome c levels in the cytoplasm, and reduced the activity of caspase-9 and -3 in AGEs-treated HUVECs.

Conclusions: AGEs induces apoptosis via the mitochondrion-cytochrome c-caspase protease pathway, and GLP-1 protects endothelial cells by interfering with this mechanism. GLP-1 may represent an anti-apoptotic agent in the treatment of vascular complications arising from diabetes.

Figure 1

Peptide glucagon-like peptide-1 (GLP-1) increases cell viability and reduces advanced glycation end products (AGEs)-induced toxicity in human umbilical vein endothelial cells (HUVECs). (A) GLP-1 increases cell viability. Cells were incubated with GLP-1 (10, 50, 100, 150 nmol/L) for 48 h. Total cell viability was measured with the MTT assay. (B) GLP-1 (50, 100, 150 nmol/L) reduces AGEs-induced cell toxicity. n=3, * p<0.05, and ** p<0.01 vs. control; # p<0.05 and ^## p<0.01 vs. AGEs-treated cells.

Figure 2

Effects of GLP-1 on AGEs-induced apoptosis in HUVECs. Cells were treated with 200 μg/mL AGEs or non-glycated BSA in the presence or absence of GLP-1. (A) Morphological changes in HUVECs. Cells were detected by Hoechst 33258 staining. Apoptotic cells were observed as blue intact round nuclei and fragmented/condensed nuclei. Original magnification, ×200. Quantification of apoptotic cells with Hoechst 33258 staining (B) and Annexin-FITC/PI assay (C). n=3, * p<0.05, and ** p<0.01 vs. control; ^## p<0.01 vs. AGEs-treated cells.

Figure 3

Inhibitory effects of GLP-1 on AGEs-induced apoptotic protein expression and mitochondrial permeability in HUVECs. (A) Western blot analysis of apoptosis-provoking proteins in response to AGEs (200 μg/mL) and GLP-1 (100 nmol/L) for 48 h. (B) Relative levels of cytochrome c (in the cytosolic fraction) normalized to levels of β-actin. (C) Ratio of Bcl-2 protein to Bax protein. n=3, ** p<0.01 vs. control; ^## p<0.01 vs. AGEs-treated cells.

Figure 4

Inhibitory effects of GLP-1 on AGEs-induced caspase-9 and −3 activation. n=3, ** p<0.01 vs. control; ^## p<0.01 vs. AGEs-treated cells.