If you're having problems viewing this message, please
click here
for additional help.
December, 2007
Glucose - Stimulated Insulin Secretion Assays
At Asterand, we have developed an assay to study the effects of drugs and drug candidates on glucose-stimulated insulin secretion (GSIS) in human isolated pancreatic islets. This forms a key part of our Metabolic Disorders Package which has been designed to target our clients’ needs in the disease areas of diabetes, obesity and metabolic syndrome.
Type 2 diabetes accounts for more than 90% of all diabetes and affects over 200 million people worldwide. At present, therapy for type 2 diabetes relies mainly on approaches intended to reduce hyperglycemia by increasing glucose absorption in target tissues. Development of compounds able to enhance GSIS is one alternative approach to providing effective management of type 2 diabetes. The ability to assess effects of these compounds at their native targets in human isolated pancreatic islets can support pre-clinical drug discovery and development and build confidence in the selection of compounds that will have the desired effects in man.
From within Asterand’s extensive human tissue network we are able to regularly access high quality human pancreatic islets, consented for use in research. This has enabled us to develop our GSIS assay such that we can generate data quickly and efficiently. Human pancreatic islets are isolated in specialized laboratories according to established protocols in clinical use. Their purity, viability and integrity are assessed immediately post-isolation. Islet viability is measured by plasma membrane integrity and assessed using a number of inclusion and exclusion dyes e.g. trypan blue, calcein or propidium iodide. Islets are then left to “rest” in culture conditions (CO
2
incubators at 30
0
C) for a period of 24 hours. This period of rest allows the islets to recover to their pre-isolation state and reduces the basal level of secreted insulin.
Upon receipt in our laboratories islets are assessed again for their microscopic integrity and level of purity. Only islets without excess exocrine tissue are used for further work. Islet viability is checked using staining by dithizone - a dye that chelates zinc atoms present in the intracellular insulin. Islets are then counted again using the concept of “islet equivalents”, a virtual measure of the number of islets of a diameter of 150 microns. We do not pick islets by hand but instead we work using suspensions of islets. The number of islet equivalents in the suspension is calculated and an appropriate aliquot added to each well. The assumption is that each such aliquot will have a mixture of small and large islets but that the overall mass (and therefore overall number of functional ß-cells) will be the same in each well.
Figure 1.
Human isolated islets stained with dithizone
Islets are then used for GSIS assays within 48 hours from receipt. We have developed a robust and reliable assay format. All assays are run in 96-well microplates which allows the simultaneous generation of a high number of data points. Typically, up to 64 compounds at one concentration can be tested in one batch of islets. Each treatment is performed in quadruplicate.
Islet functionality is routinely tested in a range of glucose concentrations. Typically, islets are incubated in at least “low glucose” (1.67mM), “medium glucose” (6.7mM) and “high glucose” (16.7mM). Intermediate glucose concentrations are incorporated on a study-by-study basis and to meet clients’ requirements.
Figure 2.
Effect of glucose on insulin secretion from human isolated pancreatic islets. “*” indicates statistically significant (p<0.05) when compared with 1.67mM glucose
A number of control compounds can be included in each assay to confirm good islet functionality. GLP-1 and its Gila monster analog, Exendin-4, are known to lower postprandial hyperglycemia by a number of mechanisms one of which is the increase of insulin secretion. Potentiation of insulin secretion by one of these compounds in the GSIS assay not only confirms islet function but also provides a good positive control for testing compounds with activities similar to GLP-1.
Theophylline can be also included in the assay. It is a potent phosphodiesterase inhibitor and as such confirms that the studied islets have retained their sensitivity to increases in cAMP.
Figure 3.
Effects of glucose, GLP-1 and theophylline on insulin secretion from human isolated pancreatic islets. “*” indicates statistically significant (p<0.05) when compared with 1.67mM glucose. “**” indicates statistically significant when compared with ‘glucose only’
Efficiency of insulin release in response to elevated blood glucose levels is variable among humans and depends on an individual’s physiology, state of health and medical history. It is not surprising therefore to observe a high variability among batches of islets isolated from different donors. The differences are first of all observed in the concentration of glucose giving the maximal insulin secretion. In some batches, the peak of insulin release is observed in the presence of high glucose. In islets from other donors, the insulin secretion cannot be driven as high and reaches its peak already at lower glucose levels.
Figure 4.
Effect of glucose on insulin secretion from human pancreatic islets isolated from two different donors. “*” indicates statistically significant (p<0.05) when compared with 1.67mM glucose
There is also variability in the level of potentiation by secretagogues. Additional increase of insulin secretion upon stimulation with GLP-1 can be observed in the presence of medium glucose concentrations in some cases and at high glucose concentrations in islets from other donors.
Figure 5.
Effect of glucose on insulin secretion from human pancreatic islets isolated from two different donors. “*” indicates statistically significant (p<0.05) when compared with 1.67mM glucose. “**” indicates statistically significant when compared with ‘glucose only’
Further development:
A number of novel assays are currently under development to complement our routine GSIS assays. Longer-term cultures are being developed where islets are kept functional for more than 6 days. A continuous treatment with GLP-1 will be also incorporated in these longer-term cultures as GLP-1 potentially protects ß-cells from apoptosis.
More reference compounds will be routinely included in our assays such as sulfonylureas, e.g. chlorpropamide, or compounds that actually inhibit the glucose-stimulated insulin release. Including these types of control treatments will, we believe, help our clients to investigate the functionality pattern of their drug candidates.
Compounds which are tested in the GSIS assay are potential drug candidates for treatment of type 2 diabetes and a comparison of their behaviour in islets from healthy donors and from individuals who are already diabetic would be of great value. However, it has always been a challenge to obtain pancreatic islets from diabetic donors since the primary source of islets for research are pancreata which have been isolated for clinical use but have proven to be unsuitable for transplantation. Obviously no diabetic pancreas could be used for transplantation. With Asterand’s extended human tissue network we can now access diabetic islets although with much lower frequency than islets from healthy individuals. We hope this will be of great interest to our clients.
For further information, please e-mail:
advantage@asterand.com
.
ASTERAND SUPPORTS HUMAN DRUG DISCOVERY AND TRANSLATIONAL MEDICINE THROUGH THE PROVISION OF HIGH QUALITY WELL CHARACTERIZED HUMAN BIOMATERIALS AND PRECLINICAL RESEARCH SERVICES
Visit us at www.Asterand.com