The glucagon-secreting α-cell is vital for the maintenance of glucose homeostasis and prevention of hypoglycaemia. Despite its importance many aspects of α-cell physiology are disputed. Thus, in this thesis, I aimed to elucidate several features of α-cell function - exploring how autonomic signals are integrated by the cell and how diabetes leads to its dysfunction. The autonomic response to hypoglycaemia results in increased acetylcholine and adrenaline in the islet vicinity, which stimulate glucagon secretion. The mechanisms underlying these effects were investigated using live [Ca²⁺]_i imaging and patch-clamp electrophysiology. Adrenaline was found to target the α-cell via a Î2-adrenergic mechanism, inducing TPC2-mediated Ca²⁺ release from the (endo)lysosomal stores, which triggered calcium-induced calcium release from the endoplasmic reticulum (ER). Acetylcholine also induced ER Ca²⁺-release via muscarinic G_q signalling. However, a component of the effect resulted from activation of a nicotinic pathway that evoked P/Q-type Ca²⁺ channel influx. The glucagon response to hypoglycaemia is lost in diabetes. To investigate the effect of hyperglycaemia on α-cell function at low glucose, the Fh1Î2KO type 2 diabetic mouse model was used. In these mice, prolonged hyperglycaemia led to blunted glucagon secretion at low glucose. Using live pH imaging, it was shown that this was caused by hyperglycaemia increasing flux through Na⁺ coupled glucose transporters (SGLTs), disrupting Na⁺-dependent acid extrusion and inducing cytoplasmic acidification. The resulting build-up of protons was speculated to compromise mitochondrial ATP production leading to the observed glucagon secretory defects. The effects of insulin-induced hypoglycaemia on δ-cell [Ca²⁺]i activity were also investigated. Increased SGLT2 transport and low [K⁺]_o, features of insulin-induced hypoglycaemia, were both shown to increase [Ca²⁺]i activity in δ-cells, stimulating somatostatin secretion and consequently suppressing glucagon secretion. Together these data suggest that glucagon secretion at low glucose is lost due to the combined effects of hyperglycaemia-driven intrinsic dysfunction and insulin-induced somatostatin secretion.

BACKGROUND: Elevation of glucagon levels and increase in alpha-cell mass are associated with states of hyperglycemia in diabetes. However, little is known about the mechanisms that control glucagon secretion and alpha-cell mass expansion in normal or diabetogenic conditions. Glucagon is secreted during the fasting state, when glucose levels are low, to stimulate glycogenolysis and gluconeogenesis in the liver to increase the blood glucose level. Amino acids, also, stimulate-glucagon secretion and alpha-cell mass. Amino acids increase glucagon secretion via activation of mTORC1 in alpha-cells. A critical step for mTOR activation is the localization of mTORC1 to the lysosome where it meets Rheb for activation. Amino acids are unique in their ability to localize mTORC1 to the lysosomal membrane for activation through their interaction with a variety of amino acid sensors, such as Sestrin2, which modulates mTORC1 activity via its interaction with GATOR2. Integral to mTORC1’s localization is the Ragulator complex, more specifically, p18, which provides the essential scaffolding necessary for lysosomal docking. Amino acids sensors work upstream of mTORC1 and sense amino acid concentrations with different affinities and are specific to certain amino acids and relay this information to mTORC1. OBJECTIVE: To investigate the role that p18, a component of the Ragulator complex, and GATOR2, a component of an amino acid sensor complex, play in amino-acid dependent mTORC1 lysosomal localization and its effect on alpha-cell function and glucagon secretion. METHODS: Generation of Knockout mice for p18 and GATOR2 in alpha-cells were produced by crossing Glu-Cre mice with P18(flox/flox) and GATOR2(flox/flox). Blood glucose, glucagon, and insulin levels were evaluated during fed, fasting, and insulin-induced hypoglycemic conditions to evaluate glucagon secretion. Isolated islets were also exposed to media containing different glucose or nutrient concentrations to evaluate the effect on glucagon secretion. RESULTS: Our data shows that knockdown experiments in alpha-cells for p18 and GATOR2 have demonstrated the role of these proteins in amino acid dependent localization of mTORC1 to the lysosomal membrane. More specifically, our data demonstrate that animals with a knockdown for p18 or GATOR2 demonstrated decreased glucagon secretion during hypoglycemic conditions. Mice with a knockdown for p18 also demonstrate decreased glucagon secretion in the presence of glucagon secretion stimulators such as arginine and also demonstrated decreased insulin secretion. CONCLUSIONS: Loss of essential components of the amino acid signaling and lysosomal localization in the mTORC1 pathway results in impaired function of alpha-cells and glucagon secretion. Loss of p18 in alpha-cells potentially results in an inability of mTORC1 to dock and bind to the lysosomal membrane, whereas loss of GATOR2 potentially results in chronic inhibition of mTORC1 via GATOR1. Loss of each of these components results in the lost or impaired ability for mTORC1 to migrate and bind to the lysosomal membrane. / 2022-06-02T00:00:00Z

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Le diabète de type 2 (DT2) résulte de l’incapacité des cellules β sécrétrices d’insuline à compenser la résistance à l’insuline qui s’installe chez les patients obèses. Un traitement potentiel viserait donc à augmenter la sécrétion d’insuline. Dans ce sens, les récepteurs aux acides gras GPR120 et GPR40 potentialisent la sécrétion d’insuline. Cependant, la signalisation GPR120 dans les îlots est méconnue. L’activation de GPR120 diminue la sécrétion de somatostatine (SST), un inhibiteur de la sécrétion d’insuline, par les cellules δ. Ces deux récepteurs régulent l’homéostasie du glucose et sont donc possiblement complémentaires. Nos objectifs étaient d’étudier la signalisation GPR120 dans les îlots pancréatiques, ainsi que la complémentarité des récepteurs GPR120 et GPR40 dans le contrôle de l’homéostasie glucidique. À l’aide d’îlots isolés de souris n’exprimant pas GPR120, constitutivement ou uniquement dans les cellules δ, nous avons étudié le rôle de GPR120 dans les sécrétions d’insuline, glucagon et de SST. Nous avons ensuite étudié des souris n’exprimant pas GPR40, GPR120 ou les deux, sous une diète riche en gras pendant 12 semaines pour étudier la complémentarité des deux récepteurs. L’activation de GPR120 diminue la sécrétion de SST et stimule les sécrétions d’insuline et de glucagon dans les îlots isolés. Cet effet est aboli par la délétion de GPR120 dans les cellules δ in vitro, et la double délétion de GPR120 et GPR40 ne révèle pas d’action complémentaire dans l’homéostasie glucidique. Ces résultats suggèrent que la signalisation GPR120 dans les cellules δ est responsable de l’amélioration de la fonction des îlots. Une meilleure compréhension du rôle joué par GPR120 dans la fonction des îlots et l’homéostasie du glucose est cruciale et pourrait permettre le développement de nouvelles options thérapeutiques dans le traitement du diabète. / In obese patients, type 2 diabetes stems from the failure of the insulin-secreting beta cells to compensate for insulin resistance. Increasing insulin secretion is therefore a viable treatment strategy. In this regard, G protein-coupled receptors (GPCR) are proven therapeutic targets. Activation of the GPCR for long-chain saturated and unsaturated fatty acid GPR40 and GPR120 increase insulin secretion in response to glucose. However, exactly how GPR120 potentiates insulin secretion is unknown. GPR120 and GPR40 both regulate glucose homeostasis and therefore could act in a complementary manner. We aimed to decipher GPR120 signalling in the pancreatic islets and study the complementary roles of GPR120 and GPR40 in maintaining glucose homeostasis. To this aim, we first measured insulin, glucagon and somatostatin secretion following GPR120 activation in isolated islets from mice with a global or somatostatin-cell-specific knock-out of GPR120. Then we studied glucose metabolism in mice with global deletion of GPR120, GPR40 or both, under a high fat diet for 12 weeks. We observed increased insulin and glucagon secretions mirrored by a decreased in somatostatin release following GPR120 activation in isolated islets, an effect abolished by a global or δ-specific deletion of GPR120. A double deletion of GPR120 and GPR40 did not have more impact on glucose metabolism or beta-cell function compared to a simple deletion of either receptor. A better understanding of the GPR120 role in islet function is crucial and could lead to the discovery of new therapeutic options.