The overall objective of this proposal is to elucidate ionic mechanisms regulating secretagogue induced changes in (-cell electrical activity and their effect on insulin secretion. Glucagon-like peptide 1 (GLP-1) stimulates islet cAMP production and augments postprandial insulin secretion. However, a clear role for GLP-1 in modulating islet electrical activity and calcium fluctuations is lacking. I have determined that GLP-1 stimulation of islets causes a significant reduction in glucose and/or tolbutamide (to block KATP) induced (-cell action potential (AP) firing frequency while increasing AP duration, which correlates precisely with fast intracellular calcium concentration changes. In contrast, tolbutamide induced islet AP frequency is significantly increased in response to glucose, which also causes a transient hyperpolarization of (-cells. Both glucose and GLP-1 increase islet cAMP, a molecule that regulates voltage-gated calcium channels (CaVs) in muscle through phosphorylation. Therefore, the modulation of islet calcium fluctuations by secretagogues could occur through phosphorylation of CaV channels. Glucose also activates an anesthetic sensitive leak-potassium conductance from islet (-cells, causing a transient hyperpolarization, which closely resembles Task-1 and Task-3 potassium channels. Based on these preliminary studies this proposal will investigate two mechanisms including A. Modulation of islet electrical activity by L-type calcium channel phosphorylation and B. Glucose dependent activation of Task-1 and Task-3 channels and both of their roles in regulating (-cell insulin secretion. This will be investigated using: 1. (-cell-attached whole islet current clamp recordings in combination with high speed calcium imaging to measure changes in APs and calcium fluctuations caused by glucose, GLP-1, and phosphatase regulation. 2. Phospho-specific antibodies directed to phosphorylation sequences of CaV1.2 and CaV1.3 will be employed together with voltage clamp experiments on rodent and human (-cells in combination with glucose and GLP-1 stimulation to address changes in L-type calcium channel amplitude and recovery from inactivation regulated by phosphorylation. Specific kinase inhibitors, phosphatase inhibitors, and siRNA will also be utilized to clarify the regulatory pathways shown to regulate CaV1.2 and CaV1.3. 3. An shRNA targeted approach to determine if Task-1 and Task-3 combine to form a glucose activated potassium channel in (-cells and their role together and/or individually during glucose stimulated insulin secretion (GSIS). Specific knockout and diabetic mouse models will also be employed in all three aims.
KATP is an essential ion channel for normal GSIS, however, rodents lacking KATP and diabetic patients treated with blockers to KATP still exhibit glucose regulated insulin secretion. Therefore this project seeks to define the ion channels, which are independent of KATP, which can also help regulate glucose induced calcium influx and insulin secretion. Understanding how secretagogues regulate islet electrical activity distinctly from KATP may help to develop new therapies that target specific elements of the excitation-secretion pathway.
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|Dadi, Prasanna K; Vierra, Nicholas C; Days, Emily et al. (2017) Selective Small Molecule Activators of TREK-2 Channels Stimulate Dorsal Root Ganglion c-Fiber Nociceptor Two-Pore-Domain Potassium Channel Currents and Limit Calcium Influx. ACS Chem Neurosci 8:558-568|
|Vierra, Nicholas C; Dadi, Prasanna K; Milian, Sarah C et al. (2017) TALK-1 channels control ? cell endoplasmic reticulum Ca2+ homeostasis. Sci Signal 10:|
|Dickerson, Matthew T; Vierra, Nicholas C; Milian, Sarah C et al. (2017) Osteopontin activates the diabetes-associated potassium channel TALK-1 in pancreatic ?-cells. PLoS One 12:e0175069|
|Boortz, Kayla A; Syring, Kristen E; Dai, Chunhua et al. (2016) G6PC2 Modulates Fasting Blood Glucose In Male Mice in Response to Stress. Endocrinology 157:3002-8|
|Vierra, Nicholas C; Dadi, Prasanna K; Jeong, Imju et al. (2015) Type 2 Diabetes-Associated K+ Channel TALK-1 Modulates ?-Cell Electrical Excitability, Second-Phase Insulin Secretion, and Glucose Homeostasis. Diabetes 64:3818-28|
|Dadi, Prasanna K; Luo, Brooke; Vierra, Nicholas C et al. (2015) TASK-1 Potassium Channels Limit Pancreatic ?-Cell Calcium Influx and Glucagon Secretion. Mol Endocrinol 29:777-87|
|Dadi, Prasanna K; Vierra, Nicholas C; Ustione, Alessandro et al. (2014) Inhibition of pancreatic ?-cell Ca2+/calmodulin-dependent protein kinase II reduces glucose-stimulated calcium influx and insulin secretion, impairing glucose tolerance. J Biol Chem 289:12435-45|
|Dadi, Prasanna K; Vierra, Nicholas C; Jacobson, David A (2014) Pancreatic ?-cell-specific ablation of TASK-1 channels augments glucose-stimulated calcium entry and insulin secretion, improving glucose tolerance. Endocrinology 155:3757-68|
|Egnatchik, Robert A; Leamy, Alexandra K; Jacobson, David A et al. (2014) ER calcium release promotes mitochondrial dysfunction and hepatic cell lipotoxicity in response to palmitate overload. Mol Metab 3:544-53|
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