Role of inositol pyrophosphates in pancreatic beta cell function

Abstract: Inositol pyrophosphates are high energy diphosphate containing molecules that are ubiquitous in eukaryotic cells. They have been implicated in diverse cellular processes ranging from DNA repair, telomere length regulation, ribosome synthesis, cell cycle regulation and apoptosis to osmoregulation, phosphate homeostasis, insulin sensitivity, vesicle trafficking, cytoskeletal rearrangement and exocytosis. The inositol pyrophosphate diphosphoinositol pentakisphosphate (IP7) is present in high levels in pancreatic β-cells. These cells secrete insulin to regulate blood glucose homeostasis. Previous work has shown that IP7 is important for maintaining the immediate exocytotic capacity of β-cells and thus the potential to secrete insulin. However, the physiological regulation and role of IP7 in these cells, especially in response to glucose, remained unexplored. The aims of this Ph.D. work were to investigate the dependence of IP7 on cellular bioenergetic status, the consequent action of IP7 in glucose-induced insulin secretion and IP7’s broader role in cellular regulation. We initially discovered the dependence of IP7 on the cellular ATP/ADP levels in insulin secreting HIT-T15 cells. Off-target reduction in ATP/ADP, upon use of a selection of signal transduction inhibitors, decreased IP7 levels. The compounds tested included inhibitors of phosphatidylinositol 3-kinase, PI3K, (wortmannin, LY294002), phosphatidylinositol 4-kinase, PI4K, (Phenylarsine Oxide, PAO), phospholipase C, PLC, (U73122) and the insulin receptor (HNMPA). We demonstrated for the first time a direct positive correlation between intracellular changes in endogenous ATP/ADP and IP7, pinpointing the regulation of IP7 by the cellular bioenergetic status. This is in agreement with the enzymatic properties of the inositol hexakisphosphate kinases (IP6Ks) that synthesize IP7. Their high Km for ATP makes IP6Ks sensitive to ATP changes. We have also revealed that some inhibitors (PAO, U73122 and LY294002) directly inhibit IP6Ks. We then investigated how physiological changes in ATP/ADP regulate IP7 production in β-cells. Glucose stimulation induced a transient increase in IP7 levels in insulin secreting cell lines and primary islets. Other secretagogues known to increase ATP/ADP, e.g. leucine, also increased IP7 levels. Silencing IP6K1, but not IP6K2, decreased glucose-mediated IP7 production and first phase insulin secretion. Therefore, IP6K1 acts as a key metabolic sensor. In diabetic ob/ob mouse islets the deranged ATP/ADP levels were mirrored by perturbed IP7 production and insulin secretion. Altogether these studies show that metabolic changes in the β-cells are reflected in IP7 levels, which consequently affect exocytosis under both physiological and pathophysiological conditions. IP7 inhibition of Akt/PKB had been described in insulin-sensitive tissues, such as liver, muscle and white fat. β-cells are also regulated by insulin. Therefore, we examined the role of IP7 in modulating the activity of Akt/PKB. To our surprise, the increase in IP7 generated by IP6K1 in glucose-stimulated β-cells was associated with higher Akt/PKB phosphorylation on the T308 and S473 sites. This indicates that IP7 activates Akt/PKB. The results cannot be explained by a direct effect of IP7 on Akt/PKB, because of the inhibitory nature of this interaction. Instead, we propose that Akt/PKB is indirectly activated by IP7 through the IP7-induced increase of insulin secretion and the consequent potentiation of the insulin feedback signaling on β-cell insulin receptors. In conclusion, the dependence of IP7 on cellular bioenergetics status suggests that IP6K1, i.e. the kinase that produces IP7 under glucose stimulation, is a new metabolic sensor in β-cells and is a likely hostage to the disrupted metabolism of type-2 diabetes. The work on Akt/PKB also exposes the complexity of inositol pyrophosphate signaling in different biological settings. Collectively our new findings have considerably broadened the understanding of IP7 regulation and function in β-cells and islets under both physiological and diabetic conditions.