Modulation of hepatic autophagy for therapeutic correction of type 2 diabetes

Abstract

Glucagon is produced from pancreatic α-cells to control hepatic glucose production and glucose homeostasis. Hyperglycaemia is an early hallmark of type 2 diabetes, and therefore hepatic glucose production can contribute to the diabetic phenotype. Glucagon also activates hepatic autophagy, generating free amino acids, fatty acids and other small molecule metabolites. Activation of autophagy liberates glucogenic amino acids that can be converted to glucose via gluconeogenesis. The mechanisms of how glucagon stimulates autophagy and how this then contributes to gluconeogenesis remain unclear. This study sought to investigate the mechanism and signalling pathways involved in glucagon-dependent autophagy induction in hepatocytes and how this contributes to gluconeogenesis. In HepG2 cells, glucagon treatment stimulated autophagy under low glucose (5.5 mM) and glucose-free conditions and caused a robust conversion of microtubule-associated protein 1 light chain 3-I (LC3B-I) to microtubule-associated protein 1 light chain 3-II (LC3B-II) and an increase in the formation of LC3B-positive autophagosomes. High glucose levels (25 mM), typical of normal growth culture conditions, did not affect glucagon-mediated stimulation of autophagy, as determined by LC3B-II accumulation. Complete absence of amino acids from the media also had no effect on glucagon-stimulated activation of autophagy. Therefore, at least in a cellular model of liver autophagy, the optimal glucagon- dependent autophagy response in hepatocytes is dependent on extracellular glucose concentration when amino acids are present. Autophagy contributed to glucagon-stimulated gluconeogenesis only in the presence of a low glucose concentration (5.5 mM) and amino acids. When cells were incubated with BafA1 to prevent lysosomal degradation of autophagy cargoes, gluconeogenesis was reduced and there was an accompanying decrease in the expression of the gluconeogenic protein, PCK1. In terms of signalling that controls these processes, calcium calmodulin-dependent protein kinase II (CaMKII) is known as an essential kinase in Ca2+ meditated signalling in many tissues. The activity of CaMKII was significantly increased by glucagon after 60 mins. In addition, starvation induced CaMKII activation led to partial phosphorylation of Beclin1 at Ser30 and correlated with autophagy stimulation in HepG2 cells. Moreover, CaMKII inhibition by KN-93 prevented autophagy induction and gluconeogenesis in both basal and glucagon-stimulated HepG2 cells, indicating the important role of CaMKII in the regulation of autophagy initiation and gluconeogenesis activation. Taken together, these data would suggest that in vivo, glucagon regulates autophagy induction, to provide glucogenic amino acid substrates for gluconeogenesis to maintain glucose homeostasis but also may contribute to hyperglycaemia. These findings provide evidence suggesting that a novel therapeutic strategy to prevent the development of T2DM may be to target hepatic autophagy activity to restrict gluconeogenesis, thus producing a therapeutic euglycemic effect.