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Medicine

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

Published: June 15th, 2013

DOI:

10.3791/50365

1Pediatric Diabetes Research Center, University of California, San Diego, 2Janssen Research & Development, 3Department of Medicine, University of California, San Diego

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.

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.

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).

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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.......

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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.......

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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.......

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This work was supported by National Institutes of Health grant R01DK080971 and Juvenile Diabetes Research Foundation grant 37-2009-44. We also appreciate support received from the UCSD Pediatric Diabetes Research Center (PDRC).

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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  

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