Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Summary

Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method—adapted from a coculture model of synaptogenesis—for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.

Abstract

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.

Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.

Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.

Introduction

We describe here a method to facilitate investigations of how the extracellular domains of specific transmembrane proteins affect insulin secretion. The method probes the effects of interactions of the protein of interest with proteins (or possibly other molecules) on the pancreatic beta-cell surface. The method allows investigations of how cell-surface proteins expressed by beta cells or by other neighboring cells (e.g. endothelial cells, neurons, pancreatic alpha cells) affect beta-cell function through transcellular interactions (i.e. through interactions with interaction partners on the surface of adjacent beta cells).

The cellular plasma membrane contains a complex array of structural and functional proteins serving as bridges to the extracellular environment. By formation of transcellular connections or by initiation of plastic signaling events, interactions between cell-surface proteins can help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within the pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis¹. As revealed, for example, by the importance of extracellular EphA-ephrinA and neuroligin-2 interactions in the regulation of glucose-stimulated insulin secretion, it is becoming ever more clear that increased knowledge of the extracellular interactions occurring between proteins on the surfaces of adjacent beta cells will be of great importance for gaining a full understanding of insulin secretion, beta cell functional maturation and the maintenance of glucose homeostasis^1-3. The goal of the method described here is to enable investigations of the effects on beta cell function of transcellular interactions involving specific transmembrane or otherwise-cell-surface-associated proteins. By co-culturing beta cells with HEK293 cells transfected with different expression constructs, the effects on beta cell function of different cell-surface proteins or mutated variants thereof can be efficiently probed. This is accomplished without having to transfect the beta cells themselves.

Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of beta-cell mRNA and protein expression, potentially affecting beta cell health and/or function in ways that could confound analyses of the effects of specific extracellular interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and, further, do not allow effects due to interactions between proteins on or in the same cell to be distinguished from the effects of transcellular interactions. Here, a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is described. This method is applicable to insulin-secreting beta-cell lines, such as INS-1 cells⁴, and to dissociated primary rodent or human beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers could identify proteins that drive synapse formation^5,6. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell function and functional maturation might be driven by similar transcellular interactions^7-9. In order to probe these interactions, we developed the system described herein in which beta cells are cocultured on a layer of HEK293 cells expressing a protein of interest¹⁰. This system allows the beta-cell cytoplasm to remain untouched while extracellular protein-protein interactions are manipulated.

Protocol

1. Transfection of HEK293 Layer

1. Prepare HEK293 cell medium by adding to 500 ml bottles of DMEM (with 4.5 g/ml glucose and phenol red and without glutamine): 50 ml FBS, 5 ml 100X penicillin/streptomycin solution, 5 ml 100x L-glutamine solution and 500 μl amphotericin B.
2. Plate out HEK293 cells in a 24-well plate using 0.5 ml of HEK293 media per well. Ensure that the cells are spread evenly across the bottom of the plate.
3. When HEK293 cells reach 100% confluency, transfect with 0.8 μg of plasmid coding the protein of interest (inserted in a mammalian expression vector) and 2 μl of Lipofectamine 2000 according to the Lipofectamine protocol. We have opted to use the pcDNA 3.3 backbone designed for expression in mammalian cells.
4. Optionally, after 6 hr of incubation, exchange the transfection media described in the Lipofectamine protocol with normal HEK293 media.
5. To evaluate expression of the transfected protein, a subset of the transfected tissue culture wells can be used for immunofluorescent detection of the expressed protein or for western blot analysis of protein expression using standard techniques.

2. Optional Fixation of HEK293 Cells Expressing Transfected Protein

Transfected HEK293 cells can be gently fixed in order to facilitate coculture in media that might be harmful to living HEK293 cells (e.g. step 3.9 below) or to allow the efficient preparation in advance of plates for several experiments all at once (see also Discussion).

1. Conduct pilot studies to determine the time course of the expression of the protein in the HEK293 cells.
2. After transfecting the HEK293 cell layer, aspirate the media from the cells at the post-transfection time point associated with highest level of expression. If this occurs within the first 24 hr, do not aspirate until 24 hr after transfection.
3. Wash HEK293 cells gently with D-PBS then add 500 μl 4% paraformaldehyde (PFA) and incubate at RT for 30 min. (To make 4% PFA solution, start with 16% solution in 1X PBS and dilute 1:4 with 1X PBS.)
4. Using sterile PBS, gently wash the cells 3 times and incubate in PBS O/N at 4 °C.
5. Wash cells 3 times with sterile PBS, then add PBS and leave at 4 °C until needed (maximum storage time may vary depending on protein expressed. We store HEK293 cells transfected to express neuroligin isoforms for up to 2 weeks without significant changes in observed effects on insulin secretion).

3. Co-culturing of INS-1 Beta Cells with HEK293 Cells

1. Prepare INS-1 media by adding to 500 ml RPMI 1640 (with glucose and phenol red and without glutamine): 50 ml FBS, 5 ml 100X penicillin/streptomycin, 5 ml 100X L-glutamine solution, 5 ml sodium pyruvate, 500 μl amphotericin B, 500 μl beta-mercaptoethanol (this is nearly identical to the medium typically used for primary islet culture except for the addition of beta-mercaptoethanol).
2. Using Cell-stripper (or another non-enzymatic cell remover), harvest INS-1 cells at approximately 70-80% confluency off the bottom of a T75 culture flask. Each flask will provide roughly enough cells for 48 wells.
3. After spinning down cells in the centrifuge for 3 min at 1,200 rpm, resuspend in a 50/50 mix of INS-1 and HEK media. For a 70-80% confluent flask of INS-1 cells, resuspend in approximately 25 ml of mixed media.
4. If using HEK293 cells that have been fixed in paraformaldehyde rather than live HEK293 cells, resuspend the INS-1 cells in 100% INS-1 media rather than mixed media.
5. Using a 10 ml pipette, dislodge the cells from the pellet to make a homogenous cell suspension.
6. Aspirate to remove the media on the HEK293 cells.
7. Using a p1000 pipette, gently add 500 μl of INS-1 cell suspension onto the HEK cell layer along the sides of the well. After every 6 wells, use a 10 ml pipette to resuspend the INS-1 cells again to ensure a homogenous cell suspension. The overall scheme of the coculture set-up is depicted in Figure 1.
8. Incubate the cells for 24-48 hr depending on the experimental protocol.
9. If more than 48 hr coculture period is required, use optional steps in section 2 above to fix the HEK293 cells.
10. If using primary dissociated rodent or human islets [e.g. see prior protocols in JoVE^11-15], ensure that an appropriate media such as RPMI with 5 ml pen/strep, 5 ml L-Glut, 0.5 ml amphotericin-B is used instead of INS-1 media. Although one can use dissociated islet cells in a 24 well plate, in practice because of the large number of islets potentially needed, it is recommended that consideration be given to scaling down to a 48 well plate (necessitating fewer islet cells per well). On a 48 well plate, add approximately 100 islets worth of dissociated cells per plate to start. Depending on the efficacy of dissociation and the method used, fewer islets may be required.

4. Glucose Stimulated Insulin Secretion

1. Preincubate cells in 250 μl of 2.5 mM glucose in KRB (Krebs-Ringer bicarbonate buffer) for a minimum of 1 hr. To preincubate, remove coculture medium and immediately replace with KRB. This exchange should be done one well at a time to minimize exposure of INS-1 cells to environmental oxygen. Aspirate with one hand while pipetting the low (2.5 mM) glucose KRB with the other.
2. Exchange preincubation KRB to 250 μl of either 2.5 mM or 20 mM glucose in KRB buffer one well at a time.
3. If appropriate for the specific experiment, additional secretagogues such as 100 μM IBMX may be added to produce a more robust stimulated insulin secretory response. Dissociated primary islet beta cells in particular are poorly responsive to increased glucose concentrations alone, so here especially addition of IBMX or other secretagogues should be considered^16,17 .
4. After 1 hr of incubation, transfer media to microfuge tubes and spin 1,500 x g for 5 min.
5. Remove 200 μl of the supernatant and use this for analysis by insulin RIA or ELISA.
6. To prepare cell lysate, add 100 μl of RIPA cell lysis buffer (150 mM NaCl, 1.0% IGEPALCA-630 or Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) with protease inhibitors to the plate immediately after removing media and incubate at 4 °C for 20 min.
7. Spin down lysate for 15 min at 10,000 x g and remove 50 μl of supernatant for analysis by insulin RIA or ELISA.

Results

Using the method described here, we have tested the effect of different variants of the protein neuroligin on insulin secretion. This complements our published work investigating the effect of neuroligin-2 on beta-cell function¹⁰. Figure 2, for example, depicts results obtained from coculturing INS-1 beta cells with HEK293 cells transfected to express a neuroligin isoform referred to here as NL-X. This experiment was designed to test the hypothesis that NL-X engages in transcellular interactio...

Discussion

The coculture method described here provides an effective way to determine the physiological importance of specific beta-cell-surface, transmembrane proteins, and specifically of their extracellular domains. By culturing beta cells or insulinoma cells (such as the INS-1 cells employed here) in contact with HEK293 cells displaying a protein of interest on the cell surface, experiments can be designed to determine the effects of extracellular protein-protein interactions without directly disturbing the intracellular milieu...

Materials

Name	Company	Catalog Number	Comments
Name of the reagent	Company	Catalogue number	Comments (optional)
pcDNA 3.3 vector/backbone	Invitrogen	K830001
Lipofectamine 2000	Invitrogen	18324012
DMEM	Mediatech	45001-312
Pen/strep solution	Mediatech	45001-652-1
Amphotericin B	Mediatech	45001-808-1/30
RPMI-1640	Mediatech	45001-404
D-PBS	Mediatech	45001-434
Sodium Pyruvate	Mediatech	45001-710-1
2-Mercaptoethanol	Invitrogen	21985023
Cell stripper	Mediatech	45000-668
T75 Flask	BD	1368065
16% Paraformaldehyde	Electron Microscopy Sciences	50980487
10X PBS	Mediatech	45001-130
Fetal bovine serum	Mediatech	MT35010CV
IBMX	Sigma	I5879-100MG
RIPA lysis buffer	Sigma	R0278