In type II diabetes, the insulin secreting beta cells of pancreatic islets fail to secrete insulin in sufficient quantities to maintain normal blood glucose levels. The resulting hyperglycemia can lead to many serious complications. Therefore, understanding the mechanisms that mediate insulin secretion could lead to new therapies to prevent the onset and complications of Type II diabetes. Two sub-classes of L-type calcium channels, Cav1.2 and Cav1.3 are expressed in pancreatic beta cells. We have developed a "knock in" method to introduce Cav1.2 and Cav1.3 mutant channels that are insensitive to the dihydropyridine (DHP) class of L-type channel blockers into the insulinoma cell line INS-1. In this system, the endogenous L-type channels can be "shut off" with DHP drugs, thus pharmacologically isolating either Cav1.2 of Cav1.3 channels. Using this system, we have shown that Cav1.3 but not Cav1.2 channels can mediate glucose-stimulated insulin secretion. Insulin secretion is potentiated by the hormone GLP-1, by its binding to the GLP-1 receptor. We have identified a short peptide, derived from the GLP-1 receptor primary amino acid sequence, that can mimic some, but not all of the actions of GLP-1. We hypothesize that in the context of the receptor this peptide comprises an autoactivation domain that is unmasked upon ligand binding.
In Aim 1 of this project, we will characterize both the activity of this peptide as a small molecule agonist, and its contribution to GLP-1 receptor activation in the context of the GLP-1 receptor structure.
In Aim 2, we will further examine the mechanisms that couple L-type calcium channels to insulin secretion and beta cell proliferation, and how they are modulated by GLP-1 receptor activation. Although most of this work will be done in the INS-1 cell model, viral vectors have been developed to introduce mutant channels and channel fragments into rat primary beta cells. This proposal will utilize techniques such as patch clamp whole-cell electrophysiology, Fluorescence Lifetime Imaging, Total Internal Reflection Fluorescence Microscopy, insulin secretion assays, immunoprecipitation assays, and western blot assays. This proposal is consistent with the PI's long term goal of understanding L-type calcium channel modulation and cellular function.
- This project will explore a set of novel molecules that stimulate beta cells of the pancreas to secret insulin in the presence of glucose, and may also promote the proliferation of beta cells. It will also examine the mechanisms by which the influx of Ca2+ into beta cells through specific types of Ca2+ channels regulates insulin secretion. The results of these studies may provide information for the development of new drugs to treat type 2 diabetes.
|Wang, Yuchen; Jarrard, Rachel E; Pratt, Evan P S et al. (2014) Uncoupling of Cav1.2 from Ca(2+)-induced Ca(2+) release and SK channel regulation in pancreatic ?-cells. Mol Endocrinol 28:458-76|
|Jarrard, Rachel E; Wang, Yuchen; Salyer, Amy E et al. (2013) Potentiation of sulfonylurea action by an EPAC-selective cAMP analog in INS-1 cells: comparison of tolbutamide and gliclazide and a potential role for EPAC activation of a 2-APB-sensitive Ca2+ influx. Mol Pharmacol 83:191-205|
|Lin, Min; Aladejebi, Oluyemi; Hockerman, Gregory H (2011) Distinct properties of amlodipine and nicardipine block of the voltage-dependent Ca2+ channels Ca v 1.2 and Ca v 2.1 and the mutant channels Ca v 1.2/Dihydropyridine insensitive and Ca v 2.1/Dihydropyridine sensitive. Eur J Pharmacol 670:105-13|
|Jacobo, Sarah Melissa P; Guerra, Marcy L; Jarrard, Rachel E et al. (2009) The intracellular II-III loops of Cav1.2 and Cav1.3 uncouple L-type voltage-gated Ca2+ channels from glucagon-like peptide-1 potentiation of insulin secretion in INS-1 cells via displacement from lipid rafts. J Pharmacol Exp Ther 330:283-93|
|Jacobo, Sarah Melissa P; Guerra, Marcy L; Hockerman, Gregory H (2009) Cav1.2 and Cav1.3 are differentially coupled to glucagon-like peptide-1 potentiation of glucose-stimulated insulin secretion in the pancreatic beta-cell line INS-1. J Pharmacol Exp Ther 331:724-32|
|Liu, Guohong; Jacobo, Sarah Melissa P; Hilliard, Nathan et al. (2006) Differential modulation of Cav1.2 and Cav1.3-mediated glucose-stimulated insulin secretion by cAMP in INS-1 cells: distinct roles for exchange protein directly activated by cAMP 2 (Epac2) and protein kinase A. J Pharmacol Exp Ther 318:152-60|