Development of a Cell-Based Fluorescence Polarization Biosensor Using Preproinsulin to Identify Compounds That Alter...

Area of Science:

• Biochemistry
• Cell Biology
• Endocrinology

Background:

• Diabetes affects 9.3% of the U.S. population, costing $245 billion annually.
• Current diabetes therapies have limitations in controlling blood glucose and enhancing insulin sensitivity.
• Innovative therapies for diabetes are urgently needed.

Purpose of the Study:

• To develop a fluorescent insulin reporter system (preproinsulin-mCherry, PPI-mCherry) for tracking live-cell insulin dynamics and secretion.
• To establish a high-throughput screening (HTS) method for sensing insulin granule packaging using a fluorescence polarization (FP)/Förster resonance energy transfer (FRET) biosensor.
• To assess the utility of this biosensor for drug discovery in diabetes.

Main Methods:

• Development of a preproinsulin-mCherry (PPI-mCherry) fluorescent reporter system.
• Utilizing a hybrid cell-based fluorescence polarization (FP)/internal FRET biosensor for insulin granule packaging.
• Testing the biosensor's response to bafilomycin, an inhibitor of insulin granule formation.
• Performing pilot high-throughput screening (HTS) of 1782 FDA-approved small molecules and natural products.

Main Results:

• The PPI-mCherry system successfully tracked live-cell insulin dynamics and secretion in pancreatic beta cells.
• Bafilomycin treatment significantly increased mCherry FP, correlated with decreased insulin granularity.
• Increased FP was attributed to inhibited self-Förster resonant energy transfer (homo-FRET) due to increased intermolecular distance.
• The biosensor demonstrated suitability for HTS with a Z'-factor of 0.52 ± 0.03.

Conclusions:

• The novel PPI-mCherry biosensor enables live-cell assessment of insulin dynamics and granule packaging.
• This system is compatible with conventional FP plate readers and suitable for HTS.
• The developed biosensor facilitates the discovery of novel therapeutic compounds for diabetes treatment.