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.

• MeSH
• beta-buňky cytologie   účinky léků   metabolismus   sekrece   MeSH
• biosenzitivní techniky MeSH
• buněčné linie MeSH
• fluorescenční mikroskopie MeSH
• glukosa farmakologie   MeSH
• hypoglykemika farmakologie   MeSH
• inzulin metabolismus   sekrece   MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• myši MeSH
• rekombinantní fúzní proteiny metabolismus   sekrece   MeSH
• reportérové geny MeSH
• tolbutamid farmakologie   MeSH
• vápník metabolismus   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH