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^2,3,4.

Although insulin secretion has been extensively studied, its regulatory mechanisms remain poorly understood. A critical area of investigation has been identification of novel modulators of insulin secretion by beta cells^5,6,7,8. These studies require a better understanding of the coupling relationship between glucose stimulation and insulin secretion. Therefore, the ability to accurately monitor and quantify the levels of glucose-stimulated insulin secretion (GSIS) has been essential. To date, however, only a limited number of methods were available to allow quantification of GSIS using insulin-secreting cell lines and/or pancreatic islets. One is radioimmunoassay (RIA), which utilizes radioisotope-tagged insulin and antibodies. The main limitations of this approach include safety issues due to the handling and disposal of radioactive materials. Additionally, this method is labor-intensive, involving multiple long washing and incubation steps. Enzyme-linked immunosorbent assay (ELISA) is another costly and labor-intensive approach that utilizes antibodies for insulin detection. Variation in antibody affinities and in the efficiency of recognizing insulin are limiting factors of this method and can affect the reproducibility of the results. Neither ELISA nor RIA was designed for high-throughput experiments. AlphaScreen is a homogeneous assay used for detecting and measuring levels of insulin secretion. AlphaScreen technology is based on the conversion of ambient oxygen into an excited oxygen singlet state that can react with chemiluminescent species, resulting in the generation of chemiluminescence. Because the assay is homogenous, many of the washing steps associated with RIA and ELISA are eliminated. However, the instability of the signal due to the nature of the reaction is a limiting factor that may affect the readout of the assay. (TR-PINCER, developed by Heyduk and colleagues⁹, is another homogenous approach to insulin measurement based on the binding of two separate antibodies to different epitopes on the insulin molecule. The antibodies are each chemically linked to double stranded DNA with short complementary single stranded DNA overhangs. Binding of the antibodies to insulin brings them together and leads to a double stranded DNA duplex. Each antibody is also associated with a respective donor or acceptor fluorophore, and the association of the DNA duplex brings together these fluorophores to generate Förster resonance energy transfer (FRET). One potential limitation of TR-PINCER, however, rests with the FRET itself. The inability to rapidly dissipate background fluorescence during the FRET reaction may lead to relatively high levels of background fluorescence and a low signal to noise ratio within the assay. Therefore, a need still exists for a reliable, robust, and cost-effective assay for quantifying GSIS in a high-throughput manner.

Recent advances in biophysics have culminated in the development of a homogeneous time-resolved fluorescence energy resonance transfer (HTRF) based assay. Specifically, while the energy transfer within the assay may be described as FRET-based, more accurately, HTRF relies on luminescence energy resonance transfer (LRET)¹⁰ which is the non-radiative transfer of energy between the donor and acceptor species^11,12,13. This distinction is important, since the timing of a fluorescence or quenching based FRET interaction is much different than it is for LRET, though the same types of gating can be used for FRET and LRET. Moreover, the use of rare earth lanthanide cryptate compounds such as europium or terbium cryptate in HTRF produces long fluorescence half-lives^12,14. This offers the unique advantage of the introduction of a time delay (µsec) between donor excitation and the measurement of emission from the acceptor (i.e., time-resolved assay). This time delay allows for sufficient time for the background fluorescence to dissipate prior to measurement of acceptor emission fluorescence. Consequently, the readout is free of non-specific fluorescence and thus, a high signal-to-noise ratio is achieved (Figure 1). Furthermore, the homogenous nature of HTRF eliminates the need for washing steps to wash off the unbound species, making the assay much more rapid than ELISA or RIA-based methods.

Figure 1: Schematic of the mechanism for HTRF insulin detection. Two independently generated monoclonal antibodies specifically recognize and bind to insulin at separate sites. These antibodies are conjugated to either the europium cryptate donor or the XL665 acceptor. Excitation of the donor at 337 nm results in emission at 620 nm. The resulting energy transfer causes XL665 to emit at a longer wavelength, 665 nm. Please click here to view a larger version of this figure.

Here, we provide a detailed protocol for using an HTRF-based approach to determine the levels of GSIS from INS-1E cells, a well-established insulin-secreting beta cell-derived rat insulinoma cell line¹⁵. In addition, this assay may be used for identifying the pharmacological profile of molecular regulators of insulin secretion. We apply this HTRF-based insulin assay to examine dopamine D₂-like receptor regulation of GSIS. Increasing studies have revealed that the neurotransmitter dopamine is an important regulator of GSIS^{8,16,17,18,19,20,21,22}. Dopamine affects GSIS in a negative autocrine/paracrine manner via actions on the dopamine D₂-like receptors (D₂, D₃, D₄ receptors) expressed at the surface of beta pancreatic cells^8,16,19. Using this assay, we confirm dopamine's role as a negative regulator of GSIS and demonstrate that the dopamine D₂-like receptor agonists bromocriptine and quinpirole also reduce GSIS.

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 mL PBS to trypsinize the cells and incubate for 3 - 4 min at 37 ˚C. Deactivate trypsin by adding 9 mL complete media and transfer cells to a 15 mL centrifuge tube by pipetting.
3. Pellet cells by centrifugation and re-suspend the cell pellet in approximately 5 mL fresh media.
4. Take 10 µL of the re-suspended cells and mix with 10 µL Trypan Blue vital dye to check for cell viability. Count live and dead cells in 10 µL of this mixture using a hemocytometer. Viability level should be above 90%. Dilute the re-suspended cells in fresh media to 1 million cells per mL.
5. Seed 0.5 mL INS-1E cells per well in a poly-L-Lysine pre-coated 24-well plate, at a density of 500,000 cells/well.
6. Day 2: Remove media 18 - 24 h after plating and add 500 µL/well of fresh RPMI 1640 media. Incubate the cells for another 24 h to allow cells to fully recover from the prior passaging step. This additional time permits the cells to spread on the tissue culture plate.

2. Insulin Secretion Assay (Day 3)

1. Prepare KRB buffer: 132.2 mM NaCl, 3.6 mM KCl, 5 mM NaHCO₃, 0.5 mM NaH₂PO₄, 0.5 mM MgCl₂, 1.5 mM CaCl₂, and 0.001 g/mL bovine serum albumin (BSA), pH 7.4.
2. Aspirate media from cells and wash twice with pre-warmed PBS.
3. For the glucose starvation step, add 450 µL/well KRB (containing BSA) without glucose for 1 h at 37 ˚C/5% CO₂.
4. During the glucose starvation step, prepare serial dilutions of the drug(s) in KRB containing 200 mM Glucose (10x concentration).
   1. Prepare drug at 10x the final concentration in KRB supplemented with 200 mM glucose (also 10x the final assay glucose concentration). If the drug stock (plus the added 10x glucose) is in DMSO, make sure DMSO percentage is kept consistent throughout the assay (ideally final percentage of less than 0.1% DMSO).
   2. For dopamine and quinpirole treatments, use a final assay drug concentration range of 100 µM to 100 pM (from highest to lowest concentration), with the last point of the dose response containing no drug. For bromocriptine, use a final assay concentration range of 10 µM to 10 pM, with the last point of the dose response being the drug-free control.
5. After glucose starvation, add drug serial dilutions to the assay to produce a dose response.
6. Add 50 µL/well of each serial dilution to the corresponding wells (total assay volume 500 µL).
7. For the glucose stimulation step, incubate cells with the respective drug serial dilutions (in the presence of 20 mM glucose) for 90 min at 37 ˚C/5% CO_2. Include a set of control wells: (1) stimulation with 20 mM glucose alone in the absence of any additional drug and (2) cells that are neither stimulated with drug nor glucose (which provides a basal rate of secretion).
8. After the stimulation step, carefully remove the supernatants (use directly or store at 4 ˚C).
   NOTE: An additional 5 min gentle centrifugation step (600 x g, 1 min) may be introduced at this point to remove any remaining cells in the assay supernatants.

3. HTRF to Measure Insulin Secretion

1. Dilute assay supernatants 1:10 in KRB (without BSA), preferably in clear 96-well plates.
2. Prepare the insulin standard curve for the HTRF insulin assay (Table 1).

Standard stock solution 500 ng/ml	Serial dilutions	Working [insulin] ng/ml
STD 7	30 µl Stock + 140 µl KRB	150
STD 6	30 µl STD 7 + 45 µl KRB	60
STD 5	30 µl STD 6 + 45 µl KRB	24
STD 4	30 µl STD 5 + 45 µl KRB	9.6
STD 3	30 µl STD 4 + 45 µl KRB	3.84
STD 2	30 µl STD 3 + 45 µl KRB	1.54
STD 1	30 µl STD 2 + 45 µl KRB	0.61
STD 0	45 µl KRB	0
Note: STD Stock is 500 ng/ml

Table 1. Serial dilutions to make the insulin standard curve.

3. Add the standard curve samples and the diluted assay supernatants to the HTRF plate. The measurement of secreted insulin by HTRF can be carried out in either a 96-well or a 384-well plate format, keeping in mind that the assay volume has to be re-adjusted. Use 10 µL/well sample in 96-well white half-area plates or 5 µL/well in a 384-well white low-volume, round-bottom plate (see Table of Materials).
4. Prepare antibody mix in detection buffer (see Table of Materials) in a 1:2 donor (cryptate)/acceptor (XL-665) ratio.
   NOTE: Further specific details about the HTRF assay are available from the manufacturer.
5. Add antibody mix to the assay at 30 µL/well (for a 96 well-plate assay format) or 15 µL/well (for a 384-well plate assay format).
6. Seal the plate and incubate at room temperature.
7. Read plate after 2 h, 4 h, and/or overnight incubation with antibodies using the plate reader and the appropriate HTRF optic module (337 665 620 nm) (see Table of Materials and manufacturer's instructions). Set the integration start at 60 µs and the integration time at 400 µs. Use 200 flashes per well.
   NOTE: These parameters were based on the use of our particular reader. The readout at 620 nm and 665 nm may vary between different instruments. This is one of the reasons we suggest using the 665/620 ratio. In calculating this ratio, any potential differences from reader to reader will be normalized and provide consistent values irrespective of the instrument used to measure HTRF.

4. Data Analysis and Normalization

1. Calculate the insulin concentrations of the assay wells via extrapolation of ratiometric fluorescence readings (665 nm/620 nm) to a second order quadratic polynomial curve (Figure 2).

Figure 2: Insulin standard curve. Human insulin stock of known concentrations was used to generate the insulin standard curve. The resulting HTRF ratios (665 nm/ 620 nm) were plotted against the insulin concentrations. The data was best fit to a second order quadratic polynomial curve (R² = 0.99996). This is a representative standard curve. Error bars = SEM. Please click here to view a larger version of this figure.

2. With the extrapolated data as ng/mL of insulin secreted, normalize (insulin secreted in response to increasing ligand concentrations) to the average value of the % maximum insulin secretion assay wells (20 mM glucose alone condition).
3. Use the curve fit (R²) from a single experiment to calculate the intraplate variation. Within the experiment, derived the individual R² values from the intra-experimental duplicates, allowing calculation the standard error of the mean for the curve fit.
4. For determining the interplate variation, use the data from at least three individual experiments to calculate the standard error of the mean for the R² value of the collective curve.

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

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