Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

Summary

This protocol describes how to perform rapid low-cost luciferase assays at medium-throughput using an insulin-linked Gaussia luciferase as a proxy for insulin secretion from beta cells. The assay can be performed with most luminescence plate readers and multichannel pipettes.

Abstract

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, depending on the specific assay. Secreted luciferase assays expedite results and lower the assay cost per sample substantially. Here we present a relatively underused approach to gauge insulin secretory activity from pancreatic β cells by using Gaussia luciferase genetically inserted within the C-peptide. During proteolytic processing of proinsulin, the C-peptide is excised releasing the luciferase within the insulin secretory vesicle where it is co-secreted with insulin. Results can be obtained within minutes after sample collection because of the speed of luciferase assays. A limitation of the assay is that it is a relative measurement of insulin secretion and not an absolute quantitation. However, this protocol is economical, scalable, and can be performed using most standard luminescence plate readers. Analog and digital multichannel pipettes facilitate multiple steps of the assay. Many different experimental variations can be tested simultaneously. Once a focused set of conditions are decided upon, insulin concentrations should be measured directly using antibody-based assays with standard curves to confirm the luciferase assay results.

Introduction

The method presented here allows insulin secretion from a genetically-modified beta cell line to be assayed rapidly and affordably in 96-well-plate format. The key to this protocol is a modified version of insulin with the naturally-secreted Gaussia luciferase (GLuc, ~18 kDa) inserted (see Figure 1) into the C-peptide to generate insulin-Gaussia (InsGLuc)^1,2. Other larger proteins, such as GFP (~25 kDa), have been successfully inserted into the C-peptide of insulin and exhibited the expected post-translational processing from proinsulin-GFP to insulin and GFP-C-peptide^3,4. For the assay in this protocol, GLuc has been codon-optimized for mammalian expression and two mutations have been introduced to enhance glow-like kinetics^5,6. Multiple combinations and replicates of treatment conditions can be easily tested in 96-well-plate format and the secretion results can be obtained immediately following the experiment.

A major advantage, as previously noted², is the low cost of this luciferase-based secretion measurement (< $0.01/well) which differentiates it from the relatively higher costs and technical aspects of enzyme-linked immunosorbent assays (ELISAs) (> $2/well) and homogenous time-resolved fluorescence (HTRF) or other Förster resonance energy transfer (FRET)-based antibody (> $1/well) assays. In comparison to these antibody-based assays, which measure the concentration of insulin by referencing a standard curve, the InsGLuc assay measures secretory activity as a relative comparison to control wells on the plate. For that reason, every experiment requires the inclusion of proper controls. This distinction is a trade-off to allow rapid and inexpensive measurements. However, InsGLuc secretion has been demonstrated to be highly correlated with insulin secretion as measured by ELISA^1,2. This technology has been scaled up for high-throughput screening^1,2,7 and has led to the identification of novel modulators of insulin secretion including a voltage-gated potassium channel inhibitor⁷ as well as a natural product inhibitor of β cell function, chromomycin A₂⁸. The use of InsGLuc is most appropriate for researchers who plan to continually test many different treatment conditions for their impact on insulin secretion. In follow-up experiments it is necessary to repeat key findings in a parental β cell line, and optimally in murine or human islets, and measure insulin secretion using an antibody-based assay.

Protocol

1. Preparation of reagents, media and buffers (Table 1)

1. Prepare MIN6 complete media in 500 mL of high-glucose (4.5 g/L) Dulbecco's modified Eagle medium (DMEM) with the following additives: 15% fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin, 292 μg/mL L-glutamine, and 50 μM β-mercaptoethanol.
   NOTE: The stable cell line in this case is maintained in 250 µg/mL of G418 antibiotic.
2. Prepare Krebs-Ringer bicarbonate buffer (KRBH) by making a solution containing 5 mM KCl, 120 mM NaCl, 15 mM HEPES (pH 7.4), 24 mM NaHCO₃, 1 mM MgCl₂, 2 mM CaCl₂, and 1 mg/mL radioimmunoassay-grade bovine serum albumin (BSA). Glucose is to be added where specified from a 2 M stock.
   NOTE: KRBH is used to incubate the cells with and without stimulation in order to assess insulin and/or Gaussia luciferase secretion.
3. Prepare coelenterazine (CTZ) stock solution as follows. Prepare acidified methanol by adding 106 µL of concentrated HCl to 10 mL of methanol. Next, dissolve lyophilized CTZ in acidified methanol at 1 mg/mL and store at -80 °C in screw-cap tubes.
   NOTE: These stocks retain sufficient activity in routine luciferase assays, even after 1 year of proper storage.
4. Prepare Gaussia luciferase (GLuc) assay buffer based upon the literature⁹ as well as patent information¹⁰ to aid in half-life of the Gaussia luciferase assay in 96 well plate format. Use the formula: 25 mM Tris pH 8, 1 mM EDTA, 5% glycerol, 1 mg/mL Na₂PO₄, 300 mM sodium ascorbate, 200 mM Na₂SO₃ in water. Freeze stocks at -20 °C. After thawing, store the buffer at 4 °C.
5. To prepare Gaussia luciferase working solution, add 4.2 µL/mL of the 1 mg/mL (2.36 mM) CTZ stock solution to GLuc assay buffer. This results in a 2x working solution of 10 µM CTZ which will have a 5 µM final concentration in the assay.

2. Culture of InsGLuc MIN6 cells and seeding for secretion assays

1. To culture MIN6 cells, trypsinize and seed the cells once per week using standard cell culture techniques. Change media on the cells every two to three days. Include appropriate selection antibiotic, such as 250 µg/mL of G418 in the media.
   1. To provide a sufficient number of cells weekly for experiments, maintain the cells in T75 flasks. Seeding 6 x 10⁶ cells per T75 in 10 mL of media will typically yield 30 x 10⁶ to 40 x 10⁶ cells total per T75 after 7 days of culture.
2. To prepare cells for plating into 96-well plates, wash a confluent T75 of InsGLuc MIN6 cells twice with PBS and add 2 mL of trypsin. Incubate at 37 °C for ~5 min or until the cells dissociate from the flask. Determine the cell concentration per milliliter.
   1. Dilute the cells in complete media to 1 x 10⁶ cells/mL to result in 1 x 10⁵ cells in 100 µL per well in a 96-well plate. The cells should be sufficiently confluent for the assay after 3–4 days.
      NOTE: To extend the culture period, half the cell concentration can be plated. Media changes are not required prior to the day of the assay unless the cells are to be subjected to experimental treatments.

3. Glucose-stimulated Gaussia luciferase secretion assay

1. On the day of the assay prepare enough KRBH for the experiment (step 1.2). A minimum of 50 mL of KRBH per 96-well plate is required, therefore prepare at least 75 mL to ensure sufficient buffer for the experiment. Prepare extra buffer if different combinations of drug treatment conditions will require KRBH for dilution.
2. Prepare a reservoir with glucose-free KRBH. Decant the medium from 96-well plate(s) by quickly inverting the plate over a laboratory sink and then blot firmly on a stack of paper towels to remove excess medium.
3. Using either an electronic or manual 8-channel pipette, pipette 100 µL /well KRBH from the reservoir across the 96-well plate(s). Repeat for a total of two washes.
4. (Optional step of acute compound treatments) If not performing drug treatments, proceed with steps 3.5 and 3.6 without modification. To test the effects of small molecules on insulin secretion, compounds can be added to the cells during the preincubation period, stimulation period, or both.
   1. One technique is to add compounds in batch to the KRBH in 1.5 mL tubes and use an adjustable 8-channel digital pipette to transfer the drug-KRBH from the tubes to cells in 96-well plates.
      NOTE: If an adjustable pipette is not available, a replica 96-well plate of drug-KRBH can be made and a standard 8-channel pipette can be used to transfer buffer. Further modifications to the treatment paradigm can be made to treat cells for 24 h in media prior to the assay, as previously described^1,8.
5. Add 100 µL of KRBH containing the desired concentration of glucose or compounds and place the dish in the 37 °C incubator for 1 h.
   NOTE: Depending on the experimental layout, it is extremely helpful to have an electronic multichannel pipette that allows transitioning the channel distances from a column of eight 1.5-mL tubes to the 8 rows of a 96-well plate.
6. After the 1 h of preincubation, decant the buffer into the sink and blot firmly on paper towel. Add 100 µL of glucose-free KRBH per well to wash away accumulated background of Gaussia luciferase. Decant the plate again and add control and stimulatory conditions to the plate at 100 µL per well. Place the dish in the 37°C incubator for 1 h.
7. Carefully collect 50 µL of supernatant using a multichannel pipette, changing tips between treatment conditions as necessary, and transfer the supernatant to a clean opaque white 96-well assay plate.
   NOTE: White-walled clear-bottom plates can be used if necessary, although a significant amount of luciferase signal will be lost.
8. After collection of 50 µL of KRBH supernatant, the sample can be assayed immediately. If necessary, seal and store samples at 4 °C for a few days (GLuc activity half-life ~6 days) or -20 °C for up to one month^11,12.

4. Secreted Gaussia luciferase assay

1. To prepare the GLuc assay working solution, pipette the required amount of CTZ stock solution (4.2 µL/mL) into GLuc assay buffer. To prevent warming the CTZ, pipette the CTZ quickly at the -80 °C freezer or keep the tube on dry ice.
2. Using an electronic multichannel pipette, quickly add 50 µL of the GLuc assay working solution per well across the 96-well dish containing the collected KRBH supernatants. If there are any droplets on the sides of any wells, briefly spin the plate in a table-top swing-bucket centrifuge.
3. Read the luminescence in a suitable plate reader within a few minutes and read each well with a 0.1 s integration time.

Results

To gauge the performance of the assay under control conditions, a simple glucose dose-response curve or a stimulation using the diazoxide paradigm can be completed. In the case of the former, pre-incubating the cells for 1 h in glucose-free conditions followed by treating for 1 h with increasing glucose concentrations should result in very little secretory activity at and below 5 mM, while increased secretion is observed above 8 mM glucose (Figure 2). Stimula...

Discussion

Herein we present a method to rapidly assess glucose-stimulated insulin secretion responses from MIN6 β cells. For the best responses in the assay it is important to seed the MIN6 cells at the proper density and allow them to become 85-95% confluent. This improves β cell responses to glucose because of improved cell-cell contacts and synchronization, which  occurs both in primary islets^17,18,19,

Materials

Name	Company	Catalog Number	Comments
Cell culture materials
rIns-GLuc stable MIN6 cells			Parental MIN6 cell line stably expressing pcDNA3.1+rInsp-Ins-eGLuc and maintained in 250 ug/ml G418
DMEM	Sigma	D6429	4.5 g/L glucose media
fetal bovine serum, heat-inactivated	Sigma	F4135
Penicillin/Streptomycin	Thermo-Fisher Scientific	SV30010
beta-mercaptoethanol	Thermo-Fisher Scientific	BP 176-100
glutamine	Thermo-Fisher Scientific	BP379-100
Trypsin-EDTA	Sigma	T3924-500
G418	Gold Biotechnology	G418-10	Stock solution 250 mg/mL in water. Freeze aliquots at -20C.
T75 tissue culture flasks	Fisher Scientific	07-202-000
96 well tissue culture plates	Celltreat	229196
Reagent reservoirs (50 mL)	Corning	4870
Name	Company	Catalog Number	Comments
Secretion assay reagents
BSA (RIA grade)	Thermo-Fisher Scientific	50-146-952
D-(+)-Glucose	Sigma	G8270-1KG
KCl	Thermo-Fisher Scientific	P217-500
NaCl	Thermo-Fisher Scientific	S271-3
Hepes, pH 7.4	Thermo-Fisher Scientific	50-213-365
NaHCO3	Thermo-Fisher Scientific	15568414
MgCl2	Thermo-Fisher Scientific	M9272-500G
CaCl2	Sigma	C-7902
Name	Company	Catalog Number	Comments
Optional drugs for stimulation experiments
Diazoxide	Sigma	D9035	Stock solution: 50 mM in 0.1N NaOH. Add equal amount of 0.1N HCl to any buffer where diazoxide is added.
epinephrine (bitartrate salt)	Sigma	E4375	Stock solution: 5 mM in water
PMA (phorbol 12-myristate)	Sigma	P1585	Stock solution: 100 µM in DMSO
Name	Company	Catalog Number	Comments
Guassia assay materials
Disodium phosphate (Na2HPO4)	Thermo-Fisher Scientific	S374-500
Glycerol	Thermo-Fisher Scientific	G334
Sodium Bromide	Thermo-Fisher Scientific	AC44680-1000
EDTA	Thermo-Fisher Scientific	AC44608-5000	Stock solution: 0.5 M pH 8
Tris base	RPI	T60040-1000.0	Stock solution: 1 M pH 8
Ascorbic Acid	Fisher Scientific	AAA1775922	US Patent US7718389 suggested ascorbate can increase coelenterazine stability.
Na2SO3	Sigma	S0505-250G	US Patent US8367357 suggested sulfite may decrease background due to BSA
Coelenterazine (native)	Nanolight / Prolume	3035MG	Stock solution: 1 mg/ml in acidified MeOH (2.36 mM)
OptiPlate-96, White Opaque 96-well Microplate	Perkin Elmer	6005290	Any opaque white 96 well plate should be sufficient. Clear bottom plates will also work, however some signal will be lost.
Name	Company	Catalog Number	Comments
Equipment
Synergy H1 Hybrid plate reader or equivalent	BioTek	8041000	A plate reader with luminescence detection and 96-well plate capabilities is required.
8-channel VOYAGER Pipette (50-1250 µL)	Integra	4724	An automated multichannel pipette is extremely useful for rapid addition of luciferase reagents and plating cells in 96 well format
8-channel 200 µL pipette	Transferpette S 20-200 µL	2703710