Developing a high-throughput assay to study glucagon secretion.

Abstract

Glucose homeostasis is necessary to sustain cellular function in a healthy individual. This is obtained by the coordinated action of two hormones, insulin and glucagon, that are produced and secreted by cells in the pancreatic islet. Diabetes is the condition that develops when insulin action is insufficient to achieve glucose homeostasis. The resulting disease is addressed by administering drugs that target insulin secretion and insulin receptor signaling at large, or by injection of insulin itself. Recently there is interest in understanding how we can control glucagon secretion to provide an alternative avenue to manage diabetes and maintain euglycemia. Modulators of glucagon secretion would be highly desirable for this purpose. A barrier that prevents the application of drug discovery methods to search for glucagon inhibitors is the fact that the glucagon producing alpha-cells constitute only 10% of islet cells. In addition, glucagon concentration in the plasma is measured in picograms/ml. Lastly, tissue culture cell lines poorly mimic the behaviors of alpha-cells, which limits many experiments to isolated primary culture alpha cells and greatly complicates large-scale approaches. Here we present the results of our efforts to build faster and more sensitive methods to monitor the glucagon output of islet alpha-cells. We explore the use of genetically encoded reporters for glucagon secretion, validate this reporter system using viral transfection of primary alpha cells from transgenic mice, and translate the approach to a novel reporter cell line. To facilitate this work, we redesigned and miniaturized a luciferase glucagon secretion assay to make it scalable and take advantage of automation where possible. The long-term goal is to perform the first large scale drug-screening study looking for compounds that modulate glucagon secretion.