AMPK regulation of cardiomyocyte glycogen handling and autophagic signalling is dependent on extracellular glucose status

Abstract

Background: Glycogen accumulates in diabetic hearts and could contribute to the development of diabetic cardiomyopathy. We have recently demonstrated that a glycogen specific autophagy pathway, ‘glycophagy’, is modulated by metabolic stress conditions. The AMPK and PI3K/Akt signalling pathways are deregulated in diabetes and converge on mTOR, a key component of metabolic processes including macro autophagy. This current study examines the effect of these signalling pathways on glycophagy and glycogen regulation under simulated diabetic conditions in vitro. Methods: Neonatal rat ventricular myocytes were isolated and cultured in 5mM or 30mM glucose for 24 hours. AMPK agonist (AICAR, 1mM) was administered 30 min prior to end of culture period. Western blotting was used to determine protein expression of key signalling markers. Results: AICAR increased phosphorylated AMPK in both 5mM and 30mM glucose conditions (3.6- and 2.8-fold increase respectively, p<0.05). Macro-autophagy marker, LC3BII:I, was decreased with AICAR treatment under both glucose conditions (29% and 33% decrease, p< 0.05). No differences were seen in protein expression levels of the glycophagy marker, GABARAPL1. The glycophagy tagging protein, STBD1, showed a trend towards an increase under 30mM glucose conditions (p=0.061). High extracellular glucose induced a reduction in phosphorylated Akt (33% decrease, p<0.05) with no effect of AICAR. In contrast, phosphorylation of mTOR (Ser2448), increased with AICAR treatment in 30mM glucose conditions only (2-fold increase, p<0.05). Conclusions: This study provides the first evidence that AMPK regulation of mTOR is dependent on extracellular high glucose conditions. In addition, these data suggest that glycophagic tagging and macro-autophagic signalling are differentially regulated by AMPK in cardiomyocytes under metabolic stress. These findings are of particular importance in considering the cardiac implications of current AMPK-related therapeutic interventions in patients with diabetes.