Figure 4.

Determination of the signaling mechanisms involved in AdipoRon-mediated regulation of cardiomyocyte autophagosome formation in high-fat-induced diabetic mice. AdipoRon caused significant PRKAA2 phosphorylation (A). Representative western blots (B) and densitometry analysis (C) showing AdipoRon caused significant phosphorylation of ACACA, PIK3C3 (Ser164), BECN1 (Ser93) and BECN1 (Thr119), an effect blocked by compound C (intravenously administered 10 min before AdipoRon), an PRKAA inhibitor. AdipoRon caused significant activation of PtdIns3K, an effect blocked by compound C (D). An in vitro kinase assay demonstrated that activation of PRKAA2 by AMP caused significant BECN1 phosphorylation at Ser93 as well as Thr119 (E: Western blot). AdipoRon promoted BECN1-BCL2L1 dissociation in NC and DB mice (F). DB mice were subjected to sham MI-R or MI-R. Ten min before reperfusion, animals were randomized to receive either vehicle or AdipoRon (intravenous bolus injection). (G) Determination of PRKAA2-induced BECN1 phosphorylation at different sites upon BECN1-BCL2L1 interaction. (A-D) N = 7–8 mice/group; (E-G) representative results from 3–4 repeated experiments. Data were analyzed by one-way ANOVA followed by the Tukey post hoc test for pairwise comparisons. *P < 0.05, **P < 0.01 between vehicle and AdipoRon-treated MI-R animals; ^##P < 0.01 between AdipoRon-treated animals with or without compound C pretreatment (C,D).