GLP-1 and Muscarinic Receptor Mediated Activation of ERK1/2 in Pancreatic β-cells

Glucagon like peptide-1 (GLP-1) and acetylcholine (ACh), acting thourough their GPCRs on β-cells, potentiate glucose stimulated insulin secretion and mediate ERK1/2 activation. Both agonists have also been demonstrated to stimulate proliferation, neogenesis and increased transcription potentially thourough ERK1/2 dependent pathways. ERK1/2 has been shown to play an important role in regulating pancreatic β-cell function and mass and mediating gene transcription. Therefore, this thesis aims to elucidate the mechanism by which GLP-1, via the GLP-1R, and carbachol, via Gq-coupled mAChR activation, leads to the phosphorylation of ERK1/2 in the pancreatic β-cell line MIN6. I demonstrate that both GLP-1R and mAChR stimulation mediate Ca2+-dependent ERK1/2 activation requiring the activation of the L-type VGCC. GLP-1 causes a sustained activation of ERK1/2 that requires continual activation of the L-type VGCC and the sustained elevation of local Ca2+ around the mouth of the channel. Importantly, ERK1/2 activation stimulated by L-type VGCCs mediated Ca2+ influx is required for GLP-1 stimulated insulin transcription. The mechanism by which the L-type VGCC signals to ERK1/2 was also investigated. I demonstrate that L-type VGCC-dependent ERK1/2 activation mediated by local Ca2+ is Ras-independent. However, a global rise in Ca2+ mediated by a depolarising stimulus is capable of mediating ERK1/2 activation by a Ras-dependent mechanism. I have also demonstrated that the down-regulation of DAG-sensitive proteins significantly inhibits GLP-1 stimulated ERK1/2 activation, but this is not thourough the down regulation or inactivation of classical or novel PKC isoforms. mAChR activation mediates the rapid and transient phosphorylation of ERK1/2 which is dependent upon PLC and a rise in [Ca2+]i, but independent of PKC activation. Importantly, the rise in [Ca2+]i is mediated from multiple sources including: the efflux of Ca2+ from the ER by IP3R activation, the influx of extracellular Ca2+ thourough store operated channels (SOC) and L-type VGCC activation. I provide evidence that the activation of the L-type VGCC is partially mediated by the inhibition of KATP channels via PIP2 depletion, as increasing PIP2 levels partially inhibits carbachol-stimulated increases in [Ca2+]i and ERK1/2 activation. However, carbachol stimulated ERK1/2 activation appears to have, like a depolarising stimulus, a Ras-dependent and a Rasindependent pathway mediating ERK1/2 activation, potentially due to the L-type VGCC activation initiated by carbachol. Overall this thesis demonstrates that the L-type VGCC is a key mediator in ERK1/2 activation in β-cells. Both GLP-1 and mAChR stimulation requires the activity of the Ltype VGCC to mediate Ca2+-dependent ERK1/2 activation, and I have provided evidence that a Ras-independent Ca2+-dependent pathway leading to ERK1/2 activation is initiated within the microdomain of the L-type VGCC and can stimulate transcription.