Development of a Cell-Based Fluorescence Polarization Biosensor Using Preproinsulin to Identify Compounds That Alter Insulin Granule Dynamics
A new tool for studying insulin packaging and dynamics
In this paper, we described a fluorescent insulin reporter system (preproinsulin-mCherry, PPI-mCherry) that tracks live-cell insulin dynamics and secretion in pancreatic beta cells with utility for high- content assessment of real-time insulin dynamics. Additionally, we reported a new modality for sensing insulin granule packaging in conventional high-throughput screening (HTS), using a hybrid cell- based fluorescence polarization (FP)/internal FRET biosensor using the PPI-mCherry reporter system.
PPI-mCherry reporter system: (A) Schematic diagram of PPI-mCherry reporter construct. A part of C-peptide and entire A chain of mouse preproinsulin (PPI) II were replaced with mCherry. (B) Immunofluorescent image of transfected INS-1 cells with PPI-mCherry reporter. (C) Colocalization of PPI-mCherry with insulin in INS-1 cells. The yellow/orange color indicated a high extent of colocalization of PPI-mCherry (red) and insulin vesicles (green) in the INS-1 cells. In the human islets, glucagon (green) was not colocalized with the mCherry reporter. (D) The extent of colocalization of PPI-mCherry reporter and insulin (green) was not significant (E). (nuclei = blue).
A positive result
Validation and utilization of the biosensor using a fluorescent PPI reporter. (A) Glucose and exendin-4 stimulated insulin secretion. Inset shows relative fluorescent intensity of secreted mCherry-insulin. (B) Flow cytometry histogram for primary human b-cell sorting. LG, low glucose (5.6mM); HG, high glucose (25mM); Ex, exendin-4 10nM; NF, nifedipine 10mM; RFU, relative fluorescence units.
High Content assessment of insulin granularity using the INS-1 PPI-mCherry reporter system. When Bafilomycin is added, vesicle formation is inhibited and the insulin does not package into normal vesicles.