Despite the urgent need for assays to visualize insulin secretion there is to date no reliable method available for measuring insulin release from single cells. To address this need, we developed a genetically encoded reporter termed RINS1 based on proinsulin superfolder GFP (sfGFP) and mCherry fusions for monitoring insulin secretion. RINS1 expression in MIN6 β cells resulted in proper processing yielding single-labeled insulin species. Unexpectedly, glucose or drug stimulation of insulin secretion in β cells led to the preferential release of the insulin-sfGFP construct, while the mCherry-fused C-peptide remained trapped in exocytic granules. This physical separation was used to monitor glucose-stimulated insulin secretion ratiometrically by total internal reflection fluorescence microscopy in single MIN6 and primary mouse β cells. Further, RINS1 enabled parallel monitoring of pulsatile insulin release in tolbutamide-treated β cells, demonstrating the potential of RINS1 for investigations of antidiabetic drug candidates at the single-cell level.

Department of Physiology and Pharmacology Oregon Health and Science University Portland OR 97237 USA

Group of Chemical Biology Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo namesti 2 16610 Prague 6 Czech Republic

Interdisciplinary Chemistry Group Cell Biology and Biophysics Unit European Molecular Biology Laboratory Meyerhofstrasse 1 69117 Heidelberg Germany

Citace poskytuje Crossref.org