Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

Summary

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

Abstract

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 µm of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

Introduction

Insulin is produced by the beta cells of the pancreatic islet and it is a key regulator of glucose metabolism¹. Death or dysfunction of beta cells disturbs glucose homeostasis and leads to diabetes². Insulin is packed in dense-core granules that are released in a Ca²⁺-dependent manner³. Elucidating how insulin granule exocytosis is regulated is essential to fully understand what determines insulin secretion and opens new avenues for the identification of novel therapeutic targets for the treatment of diabetes.

Insulin exocytosis has been extensively studied u....

Protocol

The animal ethics committee of the University of Miami has approved all the experiments.

1. Viral Infection of Intact Isolated Human or Mouse Pancreatic Islets

1. Islet culture: prepare the islet culture media: Connaught Medical Research Laboratories (CMRL) 1066, 10% (v/v) FBS and 2 mM L-Glutamine.
   1. Human pancreatic islets are obtained from the Integrated Islet Distribution Program (NIDDK, NIH). Upon arrival, transfer the islets (~ 500 islet equivalents) to 35-mm non-tissue culture treated Petri dishes with 2 mL CMRL culture media at 37 °C, 5%/95% CO₂/O₂ for 24 h before viral infection.
   ....

Results

The entire workflow of the technique is shown in Figure 1. Briefly, mouse or human islets can be infected with adenovirus encoding NPY-pHluorin and imaged, after few days in culture, under a confocal microscope. As granules fuse with plasma membrane and open, an increase in fluorescence is observed and can be quantified (Figure 1). To determine if NPY-pHluorin is indeed a suitable tool to monitor insulin granule dynamics, infecte.......

Discussion

This manuscript describes a technique that can be used to visualize exocytosis of insulin granules in beta cells within intact pancreatic islets by confocal microscopy. It uses NPY-pHluorin as the fluorescent reporter cloned into adenovirus to ensure a high transfection efficiency.

Although the method was highly efficient in our hands, it might require some modifications that primarily depend on two parameters: 1) the quality of islet preparation, and 2) the titer of viral stock with optimizat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Upright laser-scanning confocal microscope	Leica Microsystems, Wetzlar, Germany	TCS-SP5	includes LAS AF, the image acquisition software
Imaging chamber	Warner instruments	RC-26
Imaging chamber platform	Warner instruments	PH-1
22 x 40 glass coverslips	Daiggerbrand	G15972H
Vacuum silicone grease	Sigma	Z273554-1EA
Multichannel perfusion system	Warner instruments	VC-8
Single inline solution heater	Warner instruments	SH-27B
Temperature controller	Warner instruments	TC-324C
Peristaltic Suction pump	Pharmacia	P-1
35 mm Petri dish, non-tissue culture treated	VWR	10861-586
CMRL Medium, no glutamine	ThermoFisher	11530037
FBS, heat inactivated	ThermoFisher	16140071
L-Glutamine 200 mM	ThermoFisher	25030081
5 M NaCl solution	Sigma	S5150
3 M KCl solution	Sigma	60135
1 M CaCl2 solution	Sigma	21115
1 M MgCl2 solution	Sigma	M1028
Bovine Serum Albumin	Sigma	A2153
1 M HEPES solution	Sigma	H0887
Vacuum filter	VWR	431098
D-Glucose	Sigma	G8270
Poly-D-lysine hydrobromide	Sigma-aldrich	P6407
Di-8-ANNEP	ThermoFisher	D3167
3-isobutyl-1-methylxanthine (IBMX)	Sigma	I5879
Forskolin	Sigma	F3917