HuVECs were incubated for 21 days in normal (NG) (5mmol/L) or high glucose (HG) (25mmol/L) conditions, and treated with 50¦ÌM GPL-1 for 1h before their harvesting. Differences in antioxidant genes, ER stress/UPR markers, and apoptosis/proliferation genes were assessed by qRT-PCR and immunoblotting.
Chronic hyperglycemia induces the expression of the antioxidant genes HO-1 (NG vs HG P=0.01) and NQO-1 (NG vs HG P=0.07), as well as increases their protein levels. An equivalent activation of both genes was also found after GLP-1 addition in normal glucose concentration (NG vs NG+GLP-1 P=0.05 and P=0.07, HO-1 and NQO-1, respectively). GLP-1 loses this capacity when the HuVECs are exposed to high glucose (HG vs HG+GLP-1 P=1.00 and P=0.70, HO-1 and NQO-1, respectively). Furthermore, we confirm that hyperglycemia also induces gene expression of several ER stress markers in HuVECs, such as BIP, CHOP and PERK (NG vs HG P=0.01, P=0.05 and P=0.03, respectively), as well as eIF2a phosphorylation, and that GLP-1 activates some of them in normal glucose concentration. Consistently, we observe an attenuation or a complete loss of GLP-1 capacity in HuVECs after they are exposed to high glucose. Finally, as expected, hyperglycemia diminishes both gene (NG vs HG P=0.01) and protein expression of BCL-2, while the expression CDKN1a at mRNA level is increased (NG vs HG P=0.01). In both cases, GLP-1 does not exert its effects after chronic exposure to high glucose concentration (NG vs NG+GLP-1 P=0.02 and P=0.06, BCL-2 and CDKN1A, respectively. And HG vs HG+GLP-1 P=0.30 and P=0.74, BCL-2 and CDKN1A, respectively).
Our preliminary findings suggest that endothelial cells, after a long of exposure to high glucose, lose their ability to respond normally to the GLP-1 action, in terms of increasing their antioxidant capacity and regulating ER function and UPR response. This effect is probably due to the development of resistance to GLP-1 during high glucose chronic exposure.