We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.
Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.
The role of beta-cell dysfunction in disease has been widely recognized 3,4. Cell lines such as the MIN6 and INS-1 are useful tools to understand the biology of beta cell behavior. However, the physiological control of insulin secretion takes place within the islets of Langerhans. These islets contain thousands of tightly packed beta cells, as well as blood vessels and other endocrine cell types. This environment within the islet influences insulin secretion and is likely to be important in diabetes. Therefore, to understand the physiological control of insulin secretion, and the pathophysiology of disease, it is essential to study intact islets.
Islet isolation
Live human islets and, in particular, human islets from type 2 diabetes patients are difficult to obtain. In addition, human islets have limited possibilities for experimental molecular manipulation. Researchers have therefore employed islets from animals and animal models of type 2 diabetes. One such disease model is the db/db mouse. This is a spontaneous mutation that models type 2 diabetes with a phenotype progression that closely parallels human disease 5,6. The protocol presented here for islet isolation from diabetic db/db mice has many steps in common with other groups with some refinements for better yield, purification and enhanced islet survival.
2 photon imaging
The live-cell 2-photon assay described here enables researchers to quantify the number and evaluate the characteristics of single insulin-containing granules from many cells of diabetic7 and wild type islets 8,9.
NOTE: All present experiments were performed according to local animal ethics procedures of the University of Queensland (approved by the University of Queensland, Anatomical Biosciences Ethics Committee).
1. Islet Isolation
2. Live Cell 2-Photon Imaging
Islet yield and purification
For a normal wild type mouse, about 200 islets are expected. Healthy islets look bright, round shaped and have a smooth border. An over-digested isolation batch usually has small and fuzzy islets while an under-digested batch has fewer islets and acinar cell attached islets (Figure 3). For diabetic db/db mice, the islet yield and appearance depends on the disease progression with better glycemic mice have bigger, brighter and more islets (up to 300 islets) compared to worse glycemic mice which have smaller islets with a translucent appearance (under 100 islets)
2-photon assay
2-photon imaging is an indirect assay to measure insulin secretion at the level of single insulin granule fusion. In response to stimuli such as glucose or high potassium, insulin granules fuse with the plasma membrane and the extracellular dye SRB enters the granules which results in the appearance of a sudden fluorescent spot ~400 nm in diameter (Figure 5). Various experiments have been carried out to validate this assay as a measurement of fusion of insulin-containing granules like the glucose dose dependent of increasing number of granule fusion events with the expected amount of insulin secretion, the consistent size of the responded cells in the 2 photon with that of a beta cell, the similar granule diameter measured by the 2 photon and the electron microscope, the colocalization of the dye uptake and insulin staining 8.
Within the time of recording, all the exocytic events in response to the stimulus are identified and the location of the events, the time of the appearance, the duration and type of the fusion events like kiss-and-run or full fusion are characterized (Figure 5, 6). These characteristics of the fusion process are valuable for assessing any defect in the granule fusion in models of type 2 diabetes. Our previous report using this method shows that the defect in diabetic db/db islets is the loss of full granule fusion and not a change in the characteristics of the kinetics of individual granule fusion 7. Furthermore, we show that the biggest factor in the decreased in insulin secretion in disease is a reduction in the number of responsive cells 7.
Figure 1: A diagram of the sites of clamping and injection. Inject the enzymes to the common bile duct near the junction of the common hepatic duct and cystic duct. Clamp the ampulla of Valter by a vascular clamp to facilitate the injected enzyme to the pancreatic duct.
Figure 2: The injection process in the mice. Top figure illustrates the needle inside the common bile duct with a vascular clamp applied at the ampulla of Valter. Bottom figure shows the liberase-perfused pancreas after injection.
Figure 3: Typical images of Wild type and db/db isolated islets. (A) Wild type isolated islets. (B) db/db isolated islets. (C) Under-digested islets with attached acinar cells (arrows). (D) Over-digested islets with small and fuzzy islets (arrows). Please click here to view a larger version of this figure.
Figure 4: The main components of the custom-made 2 photon microscope. Please click here to view a larger version of this figure.
Figure 5: Example of a single insulin granule fusion event as recorded with 2 photon microscopy. Left figure represents the cell before (A) and after (B) the appearance of an exocytic event (arrow) in response to 15 mM glucose stimulation. (C) illustrates the SRB labeling with the entering of the extracellular dye SRB into the granule when it fuses with the plasma membrane. (D) shows the fluorescence intensity over a region of interest of an exocytic event as well as the sequential images of this exocytic event (scale bar 1 μm). Image modified from Do et al 2014 7. Please click here to view a larger version of this figure.
Figure 6: Responses of the wild type and diabetic db/db islets under the 2-photon microscope with SRB as the extracellular dye. In these experiments insulin granule fusion events in response to 15mM glucose stimulation are recorded. Yellow circles are sites of the exocytic events during 20 min of recording. Image modified from Do et al 2014 7.
The most critical factor in the islet isolation is the initial perfusion of the pancreas; an under perfused pancreas results in a considerably lower islet yield. Other factors also affect the isolation quality such as the digestion time and the level of shaking which could partly compensate for the level of perfusion. For example, a fully perfused pancreas will need to be incubated at 37 °C for ~18 min 30 sec while shaking gently, in contrast an under digested pancreas may require ~20 min digestion with harder shaking. The mixture of two digestion enzymes in our hands provides better islet isolation than either one alone. For the collagenase only method, the digestion time critically affects the islet yield and time differences of only 30 sec can dramatically affect quality. For liberase only, the yield is acceptable but under-digestion sometimes happens, we believe that the addition of collagenase helps to digest the connective tissue better.
We have found the db/db mice have diverse diabetic phenotypes, even among siblings. As a result, glucose tolerance tests on each animal before sacrificing are routinely performed. This enables classification the animals in terms of resting glucose levels and the area under the curve in response to injected glucose.
There are a number of possible experiments that can be performed after the islets have been isolated. Isolated islets can be fixed in paraformaldehyde or methanol immediately after isolation and used in immunofluorescence staining to determine the location of proteins of interest 10. Isolated islets can be dispersed in to single cells. This can be done on the same day of islet isolation or after a few days of islet culture. The single cells are then plated out and it is this preparation that has been the mainstay for techniques such as patch clamp and total internal reflection microscopy. In experiments that measure insulin secretion, as well as for live-cell 2-photon imaging, we have found that islets require 2 days in culture in order to give reproducible data. What is happening in culture is not well understood but is thought to be a period of recovery from the isolation and digestion process.
The 2-photon technique presented here enables researchers to image the insulin secretory process at the level of individual granule fusion from many cells within intact islets. We have found that the number of granule fusion events and their timing is consistent with the amounts and temporal profile of insulin secretion 8. This suggests the method is detecting the majority of insulin granule fusion events. Given that dye entry in to the fusing granules is not a specific method for detecting insulin granule fusion events we have gone to great lengths to conduct control experiments to demonstrate that insulin granule fusion in beta cells are being studied 7,8. However, a component of this evidence is dependent on the structure of the rodent islet where beta cells are enriched in the core of the islet. The different structure of human islets, where different endocrine cells are scattered throughout the islet volume, would require additional methods to identify beta cells.
There are a number of technical problems with the 2-photon microscope that are generic to all imaging approaches. For example, we carefully balance the opposing factors of trying to get enough light in to the islets so that we can reduce the gain of the detectors, and therefore reduce noise, with putting so much light in to the islets that we cause cell damage. In our system, a Pockels cell is used to attenuate the pulsed laser (Figure 3); all commercial systems have mechanisms to alter the intensity of light that falls on to the specimen.
In summary, the combination of careful isolation of db/db islets and whole-islet 2 photon microscopy present new opportunities to study the processes of insulin secretion and determine the key defective elements in type 2 diabetes.
The authors have nothing to disclose.
This work was supported by an Australian Research Council Grant DP110100642 (to PT) and National Health and Medical Research Council Grants APP1002520 and APP1059426 (to PT and HYG).
Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
Liberase TL 5mg | Roche | -5401020001 | |
Collagenase type IV-1g | Gibco-Life Technologies | 17104-019 | |
Histopaque 1077 500ml | Sigma Aldrich | 10771 | |
RPMI 1640 Medium (10X) 1L | Sigma Aldrich | R1383 | to prepare isolation media |
RPMI 1640 (1X) 500ml | Gibco-Life Technologies | 21870-076 | to prepare cultured media |
Penicillin-Streptomycin 100ml | Gibco-Life Technologies | 15140-122 | to prepare cultured media |
Fetal bovine serum 500ml | Gibco-Life Technologies | 10099-141 | to prepare cultured media |
DMEM (Dulbecco's Modified Eagle Medium) | Gibco-Life Technologies | 11966-025 | to dilute the liberase |
Metamorph program | Molecular Devices, USA | to analyze the 2-photon images |