Spermidine

To gain further insight into the links between altered autophagy and eNOS signaling in diabetes, we used HAECs exposed to physiological (5 mM) and elevated (30 mM) glucose concentrations for 24 hours. HAECs were purchased from Lonza, Inc and maintained with the EGM-2 bullet kit containing 5 mM glucose. Expression of autophagy proteins and insulin-mediated activation of eNOS was assessed by immunofluorescence, as described above. To block autophagy and to probe the level of autophagic flux, HAECs were incubated for 60 min with 10 nM bafilomycin A₁.^{22 (link), 23 (link)} To determine whether activating autophagy would reverse glucose-induced endothelial dysfunction, HAECs were incubated with 30 mM glucose in EGM-2 media for 24 hours and then treated with 3 mM spermidine (Sigma Aldrich) for six hours followed by 18 additional hours of 30 mM glucose exposure before fixing cells for protein expression or examining insulin-induced eNOS activation as described above. In addition, insulin-induced eNOS activation was assessed following treatment with autophagy inhibitors bafilomycin A1 (10 nM) or 3-methyladenine (3-MA; 10 mM; Sigma Aldrich) during insulin stimulation for one hour following spermidine treatment. spermidine activates autophagy via a general increase in the expression of autophagy-related genes, which was confirmed by RT-qPCR, as described below.