Abstract
Glucagon is a counterregulatory hormone, produced by the pancreatic alpha cells, that is essential for protection against hypoglycemia. In patients with Type 1 Diabetes, failure of appropriate glucagon responses can lead to severe hypoglycemia, coma, and death. Studies into the regulation of glucagon secretion have been hindered by a lack of assays that exhibit adequate specificity and sensitivity. To overcome this limitation, we constructed glucagon biosensors expressing the glucagon receptor and the calcium sensor, GCamP6s. First, HEK‐293 cells were transfected with a GCGR (RFP‐Tagged) Human Glucagon Receptor vector, followed by transfection with the GCamP6s calcium reporter (GFP). After transfection, the cells were then passaged and maintained with media containing puromycin and G418 to isolate the cells that took up both vectors. Expression of the vectors could then be detected and monitored for changes stimulated by glucagon under different glucose conditions. After applying varied concentrations of glucagon, the GFP signal was detected using microscopy. The images produced clearly showed the receptor was visible in the membrane, and when glucagon was present the GCamP6s sensor was activated. In future studies, these glucagon‐sensing cells can be utilized to detect and monitor glucagon release from isolated islets, offering an alternative to conventional detection methods such as ELISA.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Published Version
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