The authors thank Marcia Boulina from the DRI imaging core facility for help with the microscopes. This work was supported by NIH grants 1K01DK111757-01 (JA), F31668418 (MM), R01 DK111538, R33 ES025673 and R56 DK084321 (AC).

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
<ol>
	<li>Islet culture: prepare the islet culture media: Connaught Medical Research Laboratories (CMRL) 1066, 10% (v/v) FBS and 2 mM L-Glutamine.
	<ol>
		<li>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...

<ol>
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J., Martinez-Sanchez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+beta-cell+identity,+glucose+sensing+and+the+control+of+insulin+secretion.">Pancreatic beta-cell identity, glucose sensing and the control of insulin secretion.</a> Biochem J. 466 (2), 203-218 (2015).</li><li>Rorsman, P., Renstrom, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+granule+dynamics+in+pancreatic+beta+cells.">Insulin granule dynamics in pancreatic beta cells.</a> Diabetologia. 46 (8), 1029-1045 (2003).</li><li>Takahashi, N., Kishimoto, T., Nemoto, T., Kadowaki, T., Kasai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+pore+dynamics+and+insulin+granule+exocytosis+in+the+pancreatic+islet.">Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet.</a> Science. 297 (5585), 1349-1352 (2002).</li><li>Ramirez, D. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+insulin+vesicle+dynamics+during+pulsatile+secretion.">Human insulin vesicle dynamics during pulsatile secretion.</a> Diabetes. 56 (5), 1277-1288 (2007).</li><li>Ohara-Imaizumi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+exocytosis+and+endocytosis+of+insulin+secretory+granules+in+the+pancreatic+beta-cell+line+MIN6+using+pH-sensitive+green+fluorescent+protein+(pHluorin)+and+confocal+laser+microscopy.">Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy.</a> Biochem J. 363 (Pt 1), 73-80 (2002).</li><li>Whim, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+beta+cells+synthesize+neuropeptide+Y+and+can+rapidly+release+peptide+co-transmitters.">Pancreatic beta cells synthesize neuropeptide Y and can rapidly release peptide co-transmitters.</a> PLoS One. 6 (4), e19478 (2011).</li><li>Tsuboi, T., Rutter, G. 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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...

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 using electrophysiological approaches, such as membrane capacitance measurements, and microscopic approaches in combination with fluorescent molecules. Membrane capacitance measurements have good temporal resolution and allow single cell recordings. However, changes in the capacitance reflect the net surface change of the cell and do not capture individual fusion events or distinguish insulin granule fusion from other non-insulin secretory vesicles3. Microscopic approaches, such as two-photon or total internal reflection fluorescence (TIRF) microscopy in combination with fluorescent probes and vesicle cargo proteins, provide additional detail. These techniques capture single exocytotic events and also the pre- and post-exocytotic stages and can be used for studying exocytotic patterns in populations of cells3.
Fluorescent reporters can be of three types: 1) extracellular, 2) cytoplasmic, or 3) vesicular. 1) Extracellular reporters are polar tracers (e.g., dextrans, sulforhodamine B (SRB), lucifer yellow, pyranine) that can be introduced through the extracellular milieu4. The use of polar tracers allows for the investigation of the fusion pore in a population of cells and captures various intercellular structures such as blood vessels. However, they do not report on vesicle cargo behavior. 2) Cytoplasmic reporters are fluorescent probes coupled to membrane-associated proteins that face the cytoplasm and are involved in docking and exocytosis. Examples include members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family that have been successfully used in neuroscience for studying neurotransmitter release5. Such proteins have multiple binding partners and are not insulin-granule specific. 3) Vesicular reporters are fluorescent probes fused to vesicular cargo proteins that allow for the investigation of cargo-specific vesicle behavior. Insulin-granule specific cargo proteins include insulin, c-peptide, islet amyloid polypeptide, and NPY among others6,7. NPY is only present in insulin containing granules, and is co-released with insulin, making it an excellent partner for a fluorescent reporter8.
The fusion of different fluorescent proteins to NPY has been previously employed to study various aspects of exocytosis in neuroendocrine cells, such as the requirement of specific synaptotagmin isoforms9,10 and how the time-course of release depends on the actin cytoskeleton and on myosin II11,12. In this study, we chose pHluorin as the fluorescent reporter, which is a modified GFP that is non-fluorescent at the acidic pH inside dense core granules but becomes brightly fluorescent upon exposure to the neutral extracellular pH13. Mature insulin granules have an acidic pH below 5.5. Once the granule fuses with the plasma membrane and opens, its cargo is exposed to the neutral extracellular pH of 7.4, allowing the use of the pH-sensitive proteins pHluorin as a reporter7,14.
In view of the pH sensitive nature of pHluorin and the selective expression of NPY in insulin granules, the NPY-pHluorin fusion construct can be used to study various properties of insulin granule exocytosis. The viral delivery of the fusion construct ensures high transfection efficiency and works on primary beta cells or cell lines as well as on isolated islets. This method can also be used as a guideline for studying exocytosis in any other cell type with NPY-containing vesicles. It can also be combined with any transgenic mouse model to study effects of certain conditions (knockdowns, overexpression, etc.) on exocytosis. This technique has been previously used to characterize spatial and temporal patterns of insulin granule secretion in beta cell populations in human islets15.

<table width="845">
	<tbody>
		<tr height="63">
			<td height="63" style="width:280px;height:63px;">Upright laser-scanning confocal microscope</td>
			<td style="width:232px;">Leica Microsystems, Wetzlar, Germany</td>
			<td style="width:167px;">TCS-SP5</td>
			<td style="width:167px;">includes LAS AF, the image acquisition software</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Imaging chamber</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">RC-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Imaging chamber platform</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">PH-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">22x40 glass coverslips</td>
			<td style="width:232px;">Daiggerbrand</td>
			<td style="width:167px;">G15972H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Vacuum silicone grease</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">Z273554-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Multichannel perfusion system</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">VC-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Single inline solution heater</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">SH-27B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Temperature controller</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">TC-324C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Peristaltic Suction pump</td>
			<td style="width:232px;">Pharmacia</td>
			<td style="width:167px;">P-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:280px;height:42px;">35 mm Petri dish, non-tissue culture treated</td>
			<td style="width:232px;">VWR</td>
			<td style="width:167px;">10861-586</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">CMRL Medium, no glutamine</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">11530037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">FBS, heat inactivated</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">16140071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">L-Glutamine 200mM</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">5M NaCl solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">S5150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">3M KCl solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">60135</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:280px;height:25px;">1M CaCl2 solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">21115</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:280px;height:25px;">1M MgCl2 solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">M1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Bovine Serum Albumin</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">1M HEPES solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Vacuum filter</td>
			<td style="width:232px;">VWR</td>
			<td style="width:167px;">431098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">D-Glucose</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Poly-D-lysine hydrobromide</td>
			<td style="width:232px;">Sigma-aldrich</td>
			<td style="width:167px;">P6407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Di-8-ANNEP</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">D3167</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">3-isobutyl-1-methylxanthine (IBMX)</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">I5879</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Forskolin</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">F3917</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

confocal imaging of neuropeptide y-phluorin: a technique to visualize insulin granule exocytosis in intact murine and human islets

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 &#181;m 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).

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...

Watch this Scientific Journal Video about Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets at JoVE.com

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

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.

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

The overall goal of this assay is to visualize insulin granule exocytosis in real time in intact pancreatic islets by confocal microscopy. This method will answer key questions in the diabetes field. For example, what regulates insulin exocytosis?This is critical because granule fusion is the single most important event in the life of a better cell. The main advantage of this technique is that we can use intact islets, we can see fusion events in cells within their native environment. Madina Mahkmutova, a PhD student from our laboratory will be demonstrating the technique.The begin, upon arrival of pancreatic islets, transfer them to 50 milliliter falcon tubes and centrifuge the islets suspension at 1, 000 rotations per minute for five minutes. The islets will collect at the bottom of the tube, transfer the islets in two milliliters of CMRL culture medium to 35 millimeter non-tissue culture treated petri dishes and incubate them at 37 degree celsius and 5%carbon dioxide for 24 hours before viral infection. Following their isolation, re-suspend approximately mouse pancreatic islet equivalents in two milliliters of CMRL culture medium and transfer them to 35 millimeter non-tissue culture petri dishes.Incubate the tissue at 37 degree celsius and 5%carbon dioxide for 24 hours before viral infection. To carry out viral infection, add five to 10 microliters of stock virus to each 35 millimeter petri dish containing human or mouse islets in two milliliters of CMRL culture medium. After culturing the islets for 24 hours, aspirate the medium and replace it with two milliliters of CMRL culture medium without virus.Culture the islets for four to six days, replacing the medium every three days. Prepare extracellular solution by combining the reagents listed here and then filter sterilize the buffer. Make basal glucose medium to a three millimolar final glucose concentration, by adding 75 microliters of two molar glucose stock to 50 milliliters of extracellular solution.Then to prepare hyperglycemic medium to a 16 millimolar final glucose concentration, add 400 microliters of two molar glucose stock to 50 milliliters of extracellular solution. Dilute any additional stimuli and extracellular solution containing three millimolar glucose. Before starting an experiment, pre treat cover slips with 30 microliters of one milligram per milliliter poly-d-lysine solution and incubate for one hour.Then use water to thoroughly rinse the cover slips. At least one hour before the experiment, transfer a group of islets to a 35 millimeter petri dish containing extracellular solution with three millimolar glucose. Keep the remaining islets at 37 degrees celsius and 5%carbon dioxide.30 minutes before starting an experiment, attach the cover slip to the imaging chamber by using silicon grease to seal it. Then fix the imaging chamber to the imaging platform. Next, using a pipette, transfer 20 to 30 islets to the poly-d-lysine treated area of the cover slip and let the islets adhere to the surface for 20 minutes.This step is critical for the success of the experiment and we collect approximately 20 to 30 islets in less than 10 microliters of buffer and wait for 20 minutes. It is important to not let the cover slip dry completely, to avoid islet damage. While the islets are adhering to the cover slip, prepare the perfusion system by using water to thoroughly rinse it.Then add each solution to a different channel, according to this table. Remove all the bubbles was from the system by opening each channel separately and letting the solution flow for a few minutes. Ensure that the flow is consistent and the tubing is not leaking.Next, connect to the single inline solution heater to the perfusion outlet tube and adjust the temperature of the out flowing buffer to 37 degree celsius. Then, prepare the suction pump. Connect the tubing, turn on the pump and make sure the liquid is being aspirated.Place the imaging platform with the islets onto the microscope stage and connect it to the perfusion system and suction pump. Then, gently fill up the imaging chamber with the extracellular solution containing three millimolar glucose. To carry out confocal imaging, locate the islets in the microscope field with low magnification.Once focused on the islets, switch to higher magnification objectives. Open the acquisition software and activate the resident scanning mode. Select xyzt imaging, then to configure the acquisition settings, turn on the argon laser and set it to 30%and then the 488 nanometer laser line, adjusting the laser power to around 50%for pHluorin excitation.Collect emission at 505 to 555 nanometers, using the EGFP emission spectrum as guide. Choose a resolution of 512 by 512 pixels. Start imaging by pressing the live button and adjust the gain levels.Set the beginning end of the z stack by focusing on the top of islet and choosing begin and then moving to the last plane that can be focused and choosing n. Choose a z stack size of five micrometers. Set the time interval for acquisition of each z stack close to 1.5 to 2 seconds and choose the option, acquire until stopped for continuous imaging.Then, press the start button to initialize imaging. Refer to the text protocol for stimulation protocols to induce granule exocytosis. Using the resident scanner, confocal images of the islet are acquired as timelapse recordings in less than two seconds.In addition, this technique can be combined with dyes for functional imaging, like membra potential or cytosolic free calcium dyes. To determine if NP Y-pHluorin is indeed a suitable tool to monitor insulin granule dynamics, infected islets were immunostained with antibodies against GFP for NP Y-pHluorin and the islet hormones insulin, somatostatin or glucagon. Most cells expressing NP Y-pHluorin were beta cells as they also expressed insulin.Only a few glucagon positive alpha cells or somatostatin positive delta cells were GFP labeled. Importantly in infected cells, the NP Y fusion co-localized with insulin. This figure demonstrates that pHluorin is an effective pH sensor.As increasing the intercellular pH with 50 millimolar ammonium chloride resulted in a greater than 500%increase in the intercellular fluorescence. As the pH inside the granule increases upon fusion with the plasma membrane in the opening of the fusion pore, granules containing NP Y-pHluorin become visible under conditions that stimulate insulin exocytosis such as, membrane de-polarization with potassium chloride. Using confocal timelapse imaging of NP Y-pHluorin infected islets, single secretarial events in beta cells within intact islets can be visualized.These occur in response to direct membrane de-polarization with potassium chloride or several other physiological stimuli. At the basal extracellular glucose concentration, little secretarial activity is observed. However, granule fusion with the plasma membrane is triggered in response to stimulation with high glucose.Following this procedure, other methods like immunohistochemistry can be performed to answer additional questions like, why granule fusion occurs in specific regions of the cell. After its development, this technique has been used to explore the temporal pattern of insulin granule secretion in beta cell populations, in human islets.

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9.0K vistas.  University of Miami. The overall goal of this assay is to visualize insulin granule exocytosis in real time in intact pancreatic islets by confocal microscopy. This method will answer key questions in the diabetes field. For example, what regulates insulin exocytosis?This is critical because granule fusion is the single most important event in the life of a better cell. The main advantage of this technique is that we can use intact islets, we can see fusion events in cells within their native environment. ...