Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Summary

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

Abstract

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved Förster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved Förster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D₂-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Introduction

The regulation of energy metabolism is fine-tuned by a major anabolic hormone, insulin. Insulin is synthesized and released by pancreatic beta cells in response to increased extracellular glucose levels. The released insulin triggers the uptake of glucose by insulin-sensitive tissues^1,2. Physiologically, this is linked to the elevation of glucose concentration after a meal, followed by secretion of insulin to regulate glucose uptake. Disturbances in glucose homeostasis lead to metabolic impairments culminating in insulin resistance and ultimately in the onset of type 2 diabetes^2,

Protocol

1. INS-1E Cells: Maintenance and Plating

1. Maintain INS-1E cells in a humidified 37 ˚C/5% CO₂ incubator and cultured with RPMI 1640 medium supplemented with 5% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/mL Penicillin/Streptomycin solution, 50 µM β-mercaptoethanol. Culture cells in 10 mL complete RPMI 1640 medium (per plate), until they reach 80 - 90% confluence, when they can be trypsinized and passaged or used for the insulin secretion assay.
2. Day 1: Aspirate media and wash cells once with 5 mL of pre-warmed PBS. Add 0.5 mL of trypsin (0.025%) diluted 1:1 in 0.5 ....

Results

We validated our insulin HTRF assay by generating an insulin standard curve using purified human insulin standards of predefined concentrations (Figure 2). Generation of the standard curve permitted us to extrapolate the ratiometric fluorescence readings and thus to determine the secreted insulin levels in response to the drug treatments (Figure 2). Intraplate variation for curve fitting was minimal (R² = 0.99.......

Discussion

The HTRF insulin assay described here offers a rapid, efficient system to measure insulin secretion from a cultured cell-based system. Among its most important advantages, this assay offers a low background signal due to the high signal-to-noise ratio. Additionally, we have confirmed that the HTRF signal is stable for extended periods of time (>24 h)⁷. Nevertheless, since the insulin-binding monoclonal antibodies quickly reach binding saturation after addition to the assay, kinetics of antibod.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well white half area plate	Greiner Bio-One	82050-042
384-well white low-volume, round-bottom plate	Corning	15100157
Insulin High Range Kit	Cisbio Bioassays	62IN1PEH	10,000 test HTRF-based insulin assay kit
PHERAstar FSX plate reader	BMG Labtech	FSX	Plate reader adapted for HTRF-based assay readings
Plate sealers	Fisher Scientific	DY992	To seal plate while antibodies incubate
Hemocytometer	Hausser Scientific	3100
RPMI Medium 1640 (1x)	Gibco	11875-093	[+] L-glutamine
Trypsin 0.05%	Corning	25-052-CI	0.53 mM EDTA, (-) sodium bicarbonate
DPBS (Dulbecco's phosphate buffered saline)	Corning	21-031-CV	Without calcium and magnesium
Fetal Bovine Serum	Corning	35-010-CV
HEPES	Gibco	156-30-080
Sodium pyruvate	Gibco	11360070
Penicillin/Streptomycin solution 100x	Corning	30-002 CT
2-mercaptoethanol	Sigma	M1348
Trypan Blue Stain (0.4%)	Gibco	15250-061
Dopamine hydrochloride	Sigma	8502
Bromocriptine mesylate	Tocris	427
(-)-Quinpirole hydrochloride	Tocris	1061
Bovine Serum Albumin	Calbiochem	12659
Poly-L-Lysine	Sigma	P4832
Glucose	Sigma	G7021
DMSO (Dimethyl sulfoxide)	Sigma	276855