Title: Designing Cell Biosensors to Detect Incremental Glucagon Expression
Abstract: Hypoglycemia is a highly prevalent complication for patients with Type 1 Diabetes. Research into alpha cell function and glucagon release has been hampered because current methods used to measure glucagon, such as ELISA, are limited in sensitivity and specificity. In order to uncover the targets and pathways involved in alpha cell dysfunction in type 1 diabetes, we have designed multipurpose glucagon biosensors to detect changes in glucagon release on a cellular level and in real time. To generate the glucagon biosensors, HEK‐293 cells were transfected with a GCGR (RFP‐Tagged) Human Glucagon Receptor vector, along with GCamP6s calcium reporter (GFP). Once transfected, they were maintained and selected for using media consisting of puromycin and G418. This selection allows the cells with the incorporated vectors to be isolated. We previously found that treatment of these cells with recombinant glucagon led to clearly visible increases in GCamP6s (GFP) fluorescence. To better simulate hypoglycemic conditions, immortalized alpha cells were co‐cultured with the glucagon biosensors, and the glucagon response was measured. This system could provide a novel model for evaluating the regulation of glucagon release from alpha cells as well as the dynamics of glucagon release following exposure to hypoglycemic conditions. In future experiments, we will expose the cells to environmental stressors, such as a pro‐inflammatory cytokines, to determine their effects on glucagon release. In summary, the glucagon biosensors can offer enlightening information about sensitivity along with specificity of cellular interactions in real‐time. Support or Funding Information Funding is supported by the Helmsley Charitable Trust
Publication Year: 2020
Publication Date: 2020-04-01
Language: en
Type: article
Indexed In: ['crossref']
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