The physiological maintenance of normal blood glucose levels (euglycaemia) and prevention of hypoglycaemia is controlled by the intricate process of hormone release from the pancreatic islets of Langerhans, including the secretion of glucose-raising hormone (glucagon) from the islet α-cells. The defective control of glucagon release is commonly associated with both hyperglycaemia and recurrent hypoglycaemia in individuals with diabetes mellitus (DM). There is increasing evidence that α-cells, alongside intra-islet and autonomic control, are directly glucose-sensing to control glucagon secretion, This is includes evidence to suggest the activation of a critical energy sensor, AMP-activated protein kinase (AMPK), is at least partly involved in the stimulation of glucagon release. The mechanisms behind how α-cells intrinsically regulate glucagon secretion, and how these defective regulatory mechanisms could contribute toward the pathophysiology of DM are relatively under-explored. Providing greater understanding of α-cell physiology can therefore aid the development of small molecule compounds to pharmacologically boost α-cell glucose sensing and restore the α-cell functionality observed in DM.

Here, the overarching aim of this thesis was to characterise the bioenergetic and secretory effects of pharmacological AMPK activation in pancreatic α-cells by using novel small molecules, including R481, O-304 and BI-9774. By doing so, this will widen the current knowledge on α-cell functional dynamics in both physiological and pathophysiological contexts, including recurrent hypoglycaemia. In addition to this, a literature review was conducted to highlight the putative role of AMPK and potential application of small molecule activators as a therapeutic avenue in cystic fibrosis-related diabetes (CFRD).

Evidence suggests that, for the first time, AMPK could be a potent modulator of α-cell metabolic function and substrate utilisation in nutrient-depleted conditions. Here, AMPK activator exposure differentially controlled the glucagon secretory response and total glucagon content in α-cells, suggesting a role of AMPK in regulating glucagon granule dynamics and mechanism-of-action of such small molecules. Similarly, novel findings indicated that α-cells exposed to recurrent hypoglycaemic-like conditions (RLG) had intrinsic glycolytic and mitochondrial adaptations during low glucose and upon recovery, possibly to maintain energy status. The dysregulated intra-islet responses to glycaemia are similarly observed in other DM sub-types, such as CFRD, and contribute to worsened glucose tolerance. The author therefore discussed how the defective glucose-sensing in several tissues could contribute towards worsened glycaemia in CF/CFRD and postulated if the therapeutic application for small molecule AMPK activators could alleviate glucose-related complications, including inflammation,
associated with CFRD.

Altogether, the themes in this thesis explored the complex and wide-ranging metabolic targets of small molecule AMPK activators in the pancreatic α-cell. Furthermore, evidence suggested α-cells metabolically respond and adapt, respectively, to acute and recurrent nutrient depletion, and thereby pose an important therapeutic avenue in restoring glucose homeostasis in DM.

AimWe evaluated the efficacy of a novel brain permeable “metformin-like” AMP-activated protein kinase activator, R481, in regulating glucose homeostasis.Materials and MethodsWe used glucose sensing hypothalamic GT1-7 neuronal cells and pancreatic αTC1.9 α-cells to examine the effect of R481 on AMPK pathway activation and cellular metabolism. Glucose tolerance tests and hyperinsulinemic-euglycemic and hypoglycemic clamps were used in Sprague-Dawley rats to assess insulin sensitivity and hypoglycemia counterregulation, respectively.ResultsIn vitro, we demonstrate that R481 increased AMPK phosphorylation in GT1-7 and αTC1.9 cells. In Sprague-Dawley rats, R481 increased peak glucose levels during a glucose tolerance test, without altering insulin levels or glucose clearance. The effect of R481 to raise peak glucose levels was attenuated by allosteric brain permeable AMPK inhibitor SBI-0206965. This effect was also completely abolished by blockade of the autonomic nervous system using hexamethonium. During hypoglycemic clamp studies, R481 treated animals had a significantly lower glucose infusion rate compared to vehicle treated controls. Peak plasma glucagon levels were significantly higher in R481 treated rats with no change to plasma adrenaline levels. In vitro, R481 did not alter glucagon release from αTC1.9 cells, but increased glycolysis. Non brain permeable AMPK activator R419 enhanced AMPK activity in vitro in neuronal cells but did not alter glucose excursion in vivo.ConclusionsThese data demonstrate that peripheral administration of the brain permeable “metformin-like” AMPK activator R481 increases blood glucose by activation of the autonomic nervous system and amplifies the glucagon response to hypoglycemia in rats. Taken together, our data suggest that R481 amplifies the counterregulatory response to hypoglycemia by a central rather than a direct effect on the pancreatic α-cell. These data provide proof-of-concept that central AMPK could be a target for future drug development for prevention of hypoglycemia in diabetes.