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).

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 p....

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

List of materials used in this article

Name	Company	Catalog Number	Comments
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-Mercapt–thanol	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