Environmental input to the pancreatic β-cells - the role of mechanosensitive and other ion channels

Sammanfattning: Compound input from genetic predisposition, environmental factors and lifestyle lead to beta-cell dysfunction which initiates the development of type 2 diabetes. Understanding the linkage between the environmental input and gene regulatory pathways controlling beta-cell function is key for developing novel therapies against T2D. The pancreatic beta-cell is controlled by ion channels. Voltage-gated Ca2+ channels (VGCC) regulate Ca2+ signaling and insulin secretion. They are assembled with pore-forming alpha1 subunits and auxiliary subunits (alpha2delta, beta, gamma). Very recently, the mechanosensitive channel Piezo1 was suggested as a stimulator of insulin secretion. Mechanotransduction transduces mechanical forces into intracellular signalings and affects various cellular processes, possibly also insulin secretion. Genetic predisposition controls the susceptibility for T2D. The transcription factor TCF7L2 harbors the strongest diabetes risk gene variant and controls gene networks in insulin processing and secretion. MafA is a beta-cell maturation marker, its expression is tightly associated with the differentiation state of beta-cells. However, the exact mechanism behind Piezo1 regulated insulin secretion and how Tcf7l2 and MafA affect ion channels remain unknown.Results: PIEZO1 is significantly upregulated in islets from T2D donors and also under the conditions of developing diabetes. Hyperglycemia triggers translocation of Piezo1 into the nucleus and normoglycemia can reverse this abnormal distribution. Inhibition of Piezo1 by GsMTx4 reduces swelling/glucose-induced Ca2+ signaling, membrane depolarization and insulin secretion. Silencing of Piezo1 reduces Ca2+ handling and impairs glucose-stimulated insulin secretion (GSIS) while yoda1, the specific activator of Piezo1 induces such responses. Piezo1 regulates abundant genes (most notably Cartpt). Next, we generated a beta-cell specific Piezo1 knockout mouse model and ablation of Piezo1 in beta-cells results in an age-dependent effect on glucose utilization and insulin secretion. Piezo1 deletion strongly reduced glucose-stimulated electrical activity in beta-cells. These results highlight Piezo1 as a key regulator of beta-cell function in vivo and in vitro.Tcf7l2 regulates both mRNA and protein levels of alpha2delta-1. Suppression of alpha2delta-1 reduces Ca2+ currents and glucose/depolarization-induced Ca2+ concentration which mimics the effect of silencing of Tcf7l2. Silencing of Cacna2d1 impairs GSIS and overexpression of alpha2delta-1 improves it by alpha2delta-1 regulated Cav1.2 trafficking. Importantly, re-introducing alpha2delta-1 recovers the Tcf7l2-dependent impairment of Ca2+ signaling, but not the reduced insulin secretion. Taken together, these data demonstrate that alpha2delta-1 is the target of Tcf7l2 in controlling Ca2+-signaling.Cav gamma4 is downregulated in islets from hyperglycemic human donors and T2D rodent models. Silencing of Cacng4 inhibits Ca2+ influx and insulin secretion by suppressing the expression of L-type Ca2+ channels (Cav1.2 and 1.3). MafA regulates gamma4 expression by directly binding to its promoter. Cav gamma4 expression is also associated with beta-cell differentiation state verified by testing the de-differentiation marker Aldh1a3. These findings demonstrate that gamma4 is part of MafA mediated beta-cell differentiation and suggest the potential role of gamma4 for correcting beta-cell dysfunction.Conclusions: This thesis presents evidence for novel regulatory pathways involving mechanosensor Piezo1, Tcf7l2 and MafA controlled Cav alpha2delta-1 and gamma4, respectively, for preserving beta-cell function and normal insulin secretion. These findings update the current consensus model of Ca2+-dependent insulin release. Mediating Piezo1 activity to optimize beta-cell response to environmental input, recovering alpha2delta-1 or gamma4 expression to restore beta-cell function may also serve as new potential therapies to T2D.