Mechanisms responsible for sex difference in insulin release

The etiology of type 2 diabetes mellitus (T2DM) involves the deregulation and un-synchronization of insulin secretion from pancreatic ß-cells and insulin sensitivity in the peripheral tissues. The incidence of T2DM in humans has a clear sexual dimorphism with diabetes and impaired fasting glycaemia being more common in men than in women 30-69 years of age but reversed in the elderly (>70 years of age). Similarly, sexual dimorphism in diabetes has been described in rodent models with females having a higher insulin sensitivity compared to males. Few mouse models indicated a direct effect of sex hormones on pancreatic ß-cell function nevertheless a thorough study investigating the mechanisms responsible for the higher insulin release in females has not been published. Insulin release from pancreatic ß-cells depends on Ca2+ influx through high voltage-gated Ca2+ channels (HVCC). HVCC Ca2+ influx also regulate insulin synthesis, insulin granule priming at the cell membrane and contribute to ß-cell electrical activity. Recently we demonstrated that genetic deletion of a2d-1 HVCC Ca2+ channel subunit in mice leads to diabetes in a sex-specific manner (Mastrolia et al. 2017, Diabetes). Although the insulin sensitivity was similar in males and females, the higher insulin release in females partially alleviated the diabetes phenotype in a2d-1-/- mice. Here we propose to identify the molecular mechanisms responsible for the higher insulin release in wild-type and a2d-1-/- females compared to males. We will use a battery of state of the art methods like electrophysiology, confocal microscopy, fluorescence recovery after photo bleaching, and single cell electrical capacitance measurements to characterize the sex-differences in pancreatic ß-cell electrical activity in response to glucose, ß-cell Ca2+ amplification, pancreatic islet Ca2+ dynamics, connectivity of ß-cells in the islet of Langerhans, insulin granule priming and release. The successful completion of this project will have immediate scientific relevance as it will unveil molecular mechanisms capable of increasing insulin release from pancreatic ß-cells, and help better understand the role of a2d-1 subunit in particular and HVCC in general on ß-cell insulin release. Several patients carrying a2d-1 loss-of-function mutations have been identified and according to our recently published data we predict that these patients, especially men, are at high risk of developing diabetes. The current proposal will identify the risk level for the female patients and the possible molecular pathways that could be pharmacologically enhanced to increase insulin secretion in both males and females.

Type 2 Diabetes mellitus (T2DM) is a metabolic disorder characterized by hyperglycemia. While peripheral insulin resistance might be the initiator, impaired or total lack of insulin release from pancreatic ß-cells is the ultimate cause. In humans, the incidence of T2DM is lower in premenopausal women compared to men, a phenotype recapitulated by many rodent models. Yet, the molecular mechanism responsible for T2DM protection in females is unknown. Previously we have shown that female pancreatic islets of WT mice secrete significantly more insulin compared to males (Mastrolia et al, 2017). Insulin release is a finely -tuned process coordinated by the activity of a multitude of ion channels with the high voltage -gated Ca2+ channels (HVCC) occupying the central role in vesicles exocytosis. The HVCC complex is formed by the pore-forming a1 subunit and the auxiliary subunits ß and a2d that modulate the channel gating properties as well as trafficking to the membrane. Previously we showed that genetic deletion of a2d-1, the main a2d isoform expressed in pancreatic islets, equally affected the Ca2+ current properties in both male and female ß-cells. However, insulin secretion from a2d-1-/- pancreatic islets was significantly higher in females compared to males. In this project we found that WT female ß-cells display a higher glucose-induced membrane depolarization in all stimulatory glucose concentrations compared to males despite similar Ca2+, KATP, Ca2+ dependent and K2P K+ currents. However, a significantly larger Kv2.1 current density in male ß-cells causes a more hyperpolarized membrane potential that leads to a range of functional alterations; the lower male ß-cell resting membrane potential causes shorter AP-bursts durations but increases the HVCC availability and intracellular Ca2+ concentration while the higher membrane potential in female ß-cells alters the incretin pathway. Following a2d-1 ablation ß-cells from both male and female mice showed similar HVCC Ca2+, Ca2+-dependent K+, KATP, Kv and Nav current densities that consequently led to similar glucose -induced membrane depolarization. Yet, female a2d-1-/- ß-cells still showed higher electrical activity caused by a significantly increased Kv channel availability at physiologically relevant membrane potentials. Cumulative our results demonstrate for the first time that modulation of Kv channel density or availability increases the glucose-induced electrical activity in female ß- cells leading to a higher insulin release both in healthy and a Diabetes Mellitus mouse model. The intrinsic islet cell properties are strongly regulated by other hormones among which catecholamines play a key role. Adrenaline inhibits insulin secretion and promotes a-cell glucagon release that increases blood glucose. Here we show that a2d-1 genetic deletion also reduces HVCC Ca2+ influx in mouse chromaffin cells (MCCs) but leads to a paradoxical increase in induced MCCs electrical activity. Combined with an otherwise unaltered vesicle exocytosis this could potentially exacerbate DM phenotype observed in a2d-1-/- mice.