Studies proposed here concern a novel phospholipase C-epsilon (PLC?) that we propose mediates beneficial blood glucose-lowering actions of the glucagon-like peptide-1 receptor (GLP-1R) agonist Byetta in patients with type 2 diabetes mellitus (T2DM). The central hypothesis we present is that there exists coupling of the pancreatic beta-cell GLP-1R to cAMP production with consequent activation of PLC? in order to potentiate glucose-stimulated insulin secretion (GSIS) from the islets of Langerhans. By understanding the nature of this unconventional cAMP signaling mechanism, we hope to further drug discovery efforts that seek to identify GLP-1R agonists that are pure insulin secretagogues and that do not induce dangerous side effects such as pancreatitis and cancer.
Aim 1 : Byetta might restore insulin secretion in T2DM by facilitating a "late step" of exocytosis that is under te control of PLC?. Using human islets or islets of PLC? KO mice, this hypothesis will be tested in perfusion or static incubation assays of GSIS. A first goal is to determine if PLC? mediates the action of Byetta to potentiate 1st and/or 2nd phase GSIS, or to potentiate "triggering" and "amplification" mechanisms of GSIS. Next, single cell patch clamp assays in combination with 2-photon confocal microscopy of secretory granule dynamics will be performed to test if PLC? activation explains diacylglycerol (DAG) and protein kinase C (PKC) mediated actions of Byetta to facilitate exocytosis. To evaluate the in vivo action of Byetta, glucoregulation will be studied using Pdx-1-hGLP1R:Glpr-/- mice in which there is a beta-cell specific KO of PLC?. Since Pdx-1-hGLP1R:Glpr-/- mice express the GLP-1R only in the pancreas, specific activation of the beta-cell GLP-1R by administered Byetta will be possible. We predict that a beta-cell specific KO of PLC? will disrupt the action of Byetta to potentiate GSIS in vivo.
Aim 2 : Byetta might also restore insulin secretion in patients with T2DM by sensitizing beta cells to the stimulatory effect of glucose metabolism. More specifically, we propose that Byetta acts via Epac2, Rap1, and PLC? to restore glucose metabolism-dependent closure of K-ATP channels in beta cells of T2DM patients. Our hypothesis embraces a new model of stimulus-secretion coupling in which the sulfonylurea receptor-1 (SUR1) subunit of K-ATP channels acts as a molecular scaffold to allow the formation of a signal transduction complex comprised of Epac2, Rap1, and PLC?. Importantly, we demonstrate that cAMP sensor Epac2 binds to SUR1, and that this interaction is facilitated by H-Ras GTPase acting at a Ras-association (RA) domain of Epac2. Thus, we hypothesize that Byetta acts in concert with growth factors or possibly secreted insulin to activate PLC?, to stimulate PIP2 hydrolysis, and to modulate the ATP and Mg-ADP sensitivity of K-ATP channels in order to close the channels. This hypothesis concerning a novel mechanism of ion channel modulation will be tested in assays of K-ATP channel activity using human islets or islets of Epac2 and PLC? knockout (KO) mice. Summary: The long-term goal of this project concerns our interest in determining the molecular basis for beneficial blood glucose-lowering properties of GLP-1R agonists in patients with T2DM.
A new strategy for the treatment of type 2 diabetes mellitus (T2DM) involves the administration of a metabolically stable GLP-1 mimetic (Byetta) or a GLP-1 analog (Victoza), each of which stimulates insulin secretion by activating a pancreatic beta-cell GLP-1 receptor positively linked to the production of cAMP. The main goal of the studies outlined here is to identify the beta-cell cAMP signal transduction pathway that mediates the rapid action of GLP-1 receptor agonists to restore the missing first phase kinetic component of glucose-stimulated insulin secretion in patients diagnosed with T2DM. The significance of such studies is that they have the potential to dramatically enhance our understanding of how GLP-1-based pharmaceuticals exert beneficial antidiabetogenic actions to stimulate insulin secretion, and to lower levels of blood glucose, in patients diagnosed with T2DM.
|Chepurny, Oleg G; Leech, Colin A; Tomanik, Martin et al. (2016) Synthetic small molecule GLP-1 secretagogues prepared by means of a three-component indole annulation strategy. Sci Rep 6:28934|
|Kolic, Jelena; Manning Fox, Jocelyn E; Chepurny, Oleg G et al. (2016) PI3 kinases p110Î± and PI3K-C2Î² negatively regulate cAMP via PDE3/8 to control insulin secretion in mouse and human islets. Mol Metab 5:459-71|
|Chepurny, Oleg G; Holz, George G; Roe, Michael W et al. (2016) GPR119 Agonist AS1269574 Activates TRPA1 Cation Channels to Stimulate GLP-1 Secretion. Mol Endocrinol 30:614-29|
|Henry, Kelly E; Kerwood, Deborah J; Allis, Damian G et al. (2016) Solution Structure and Constrained Molecular Dynamics Study of Vitamin B12 Conjugates of the Anorectic Peptide PYY(3-36). ChemMedChem 11:1015-21|
|Takeda, Yukari; Shimayoshi, Takao; Holz, George G et al. (2016) Modeling analysis of inositol 1,4,5-trisphosphate receptor-mediated Ca2+ mobilization under the control of glucagon-like peptide-1 in mouse pancreatic Î²-cells. Am J Physiol Cell Physiol 310:C337-47|
|Henry, Kelly E; Elfers, Clinton T; Burke, Rachael M et al. (2015) Vitamin B12 conjugation of peptide-YY(3-36) decreases food intake compared to native peptide-YY(3-36) upon subcutaneous administration in male rats. Endocrinology 156:1739-49|
|Bonaccorso, Ron L; Chepurny, Oleg G; Becker-Pauly, Christoph et al. (2015) Enhanced Peptide Stability Against Protease Digestion Induced by Intrinsic Factor Binding of a Vitamin B12 Conjugate of Exendin-4. Mol Pharm 12:3502-6|
|Schwede, Frank; Chepurny, Oleg G; Kaufholz, Melanie et al. (2015) Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion. Mol Endocrinol 29:988-1005|
|Holz, George G; Leech, Colin A; Chepurny, Oleg G (2014) New insights concerning the molecular basis for defective glucoregulation in soluble adenylyl cyclase knockout mice. Biochim Biophys Acta 1842:2593-600|
|Nadkarni, Prashant; Chepurny, Oleg G; Holz, George G (2014) Regulation of glucose homeostasis by GLP-1. Prog Mol Biol Transl Sci 121:23-65|
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