Introduction:
We recently reported the impact of glucagon-like peptide-1 (GLP-1) on myocardial remodeling observed in type 2 diabetic mice (T2DM) via cyclic-AMP-dependent activation of autophagy in myocardium; however, it remains unclear whether GLP-1 may modulates capillary formation in heart.
Hypothesis/AIM:
We thus evaluated the impact of GLP-1 on angiogenesis and its link to endothelial autophagy.
Methods:
T2DM was treated with Ex4 (24 nmole/kg/day for 4 weeks). Cardiac capillary density was measured by immunohistology. Cultured HUVECs were used for in vitro experiments. Changes in activities of autophagy (LC3-turnover assay and protein levels of p62 and Beclin1), and angiogenesis (tube formation assay and Akt/AMPK/eNOS activity), were evaluated.[[Unable to Display Character: 
]] Role of PKA was assessed by CREB phosphorylation and RNA interference (siRNA for catalytic subunit of PKA). Effect of autophagy was assessed by use of pharmacological inhibitor 3MA and siRNA of autophagy-related gene (ATG) 5 , ATG7, and p62.
Results:
Immunohistochemical analyses revealed that T2DM exhibited reduced cardiac capillary density, which was reversed by Ex-4 with concomitant amelioration of systemic diabetic condition. Ex-4 treated heart exhibited increase in myocardial cyclic AMP concentration. We thus observed direct impact of Ex-4 and cyclic AMP elevation on HUVECs, in which GLP-1 receptor expression was confirmed by immunoblot and QPCR. In vitro angiogenesis assay revealed that Ex-4 and PKA enhancers (10 μM forskolin and 1 mM 8-bromo-cyclic AMP) facilitated angiogenesis and autophagy in HUVECs. The PKA/AMPK/eNOS phosphorylation levels of Ex-4-treated HUVECs were elevated. Of note, each Akt activity remains unchanged. PKA inhibitors (H89, RP-cAMP, siRNA) abrogated the increase in phosphorylation of AMPK/eNOS axis induced by Ex-4 in HUVECs. Tube formation assay revealed that Ex-4 and PKA enhancers augmented in vitro angiogenesis, which were abrogated by inhibition of autophagy and AMPK using pharmacological inhibitors (3MA and compound C) and siRNA for autophagy-related gene (ATG) 5 , ATG7, p62, and catalytic subunit of AMPK. [[Unable to Display Character: 
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Conclusions:
GLP-1 directly promoted angiogenesis via the PKA/AMPK-dependent autophagic activation.
A cyclic AMP related gene network in microglia is inversely regulated by morphine tolerance and withdrawal
