1Department of Pediatrics, Indiana University School of Medicine, 2Department of Cellular & Integrative Physiology, Indiana University School of Medicine
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Fueger, P. T., Hernandez, A. M., Chen, Y. C., Colvin, E. S. Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets. J. Vis. Exp. (64), e4080, doi:10.3791/4080 (2012).
Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes 1-3. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.
Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.
By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass 4-6. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets 4,7-15. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function 5,6,8,9,16,17.
1. Adenoviral Transduction and Culturing of Rat Islets
[Note: From this point forward, please follow institutional protocols for the handling, use, and disposal of biohazardous materials.]
[Note: To verify adequate transduction efficiency, the use of a control virus expressing GFP is beneficial, as islets can then be imaged via confocal microscopy to verify penetration of the adenovirus into the islet core.]
[Note: From this point forward, please follow institutional protocols for the handling, use, and disposal of radioactive materials.]
2. Insulin Secretion Assay
[Note: As the islets are radioactive, please follow institutional protocols for the handling, use, and disposal of radioactive materials.]
[Note: Islets can be visualized using either a dissecting stereoscope or a standard microscope.]
[Note: As an alternative to settling by gravity, the tubes may be centrifuged at 300 x g for 1 min.]
3. Thymidine Incorporation Assay
4. Data Analysis
5. Representative Results
An example of the experiment to assess islet replication and beta cell function in rat islets is shown in Figure 2. This example shows that adenoviral overexpression of hypothetical "Gene #6" robustly stimulates islet replication without altering beta cell function. In the top panel, the results from the thymidine incorporation assay demonstrate that increasing the expression of "Gene #6" increases DNA synthesis, as measured by the incorporation of thymidine. Because most of the cells in the rat islet are beta cells, it is likely that this increase in thymidine incorporation indicates an increase in beta cell replication. However, confirmatory experiments must be performed to firmly establish this. In the bottom panel, the results from the insulin secretion assay demonstrate that overexpression of "Gene #6" did not alter one of the primary beta cell functions, i.e., insulin secretion at low and high glucose. The quality of the islet isolation and health of the islets following treatment with adenoviruses is indicated by the fold increase in insulin secretion at low and high glucose concentrations. If increasing the expression of "Gene #6" impaired beta cell function, this would likely be reflected as a decrease in the insulin secreted at high, stimulatory glucose concentrations (16.7 mM). A dose-response curve for varying glucose concentrations could also be performed.
Figure 1. Overview of protocol to assess islet replication and beta cell function in isolated rat islets following adenoviral-mediated alterations in gene expression. Freshly isolated rat islets are exposed to adenoviruses for 24 h and then cultured up to 96 h. Thymidine incorporation is assessed in the final 24 h, followed by the measurement of insulin secretion at low and high glucose.
Figure 2. Results from an experiment using a control adenovirus and an adenovirus overexpressing a hypothetical gene labeled as "Gene #6". The top panel shows the thymidine incorporation and the bottom panel the insulin secretion.
Establishing pathways that can be modulated to stimulate the replication and enhance the function of beta cells are relevant to both major forms of diabetes. Because functional beta cell mass is dependent on the existence and function of insulin-secreting cells, assessing these determinants simultaneously has its advantages. This protocol describes a streamlined protocol for identifying whether overexpression or suppression of a protein leads to changes in functional beta cell mass in vitro, which can then be tested for efficacy in vivo.
One limitation of this protocol is that the islet is a micro-organ consisting of many cell types, including but not limited to alpha, beta, delta, epsilon, and PP cells. A change in islet replication may not completely translate to a change in beta cell replication because 80-90% of the cells in the rat islet are beta cells. The possibility that an observed change in islet replication is due to non-beta cell replication exists. Thus, a logical and confirmatory next step to this protocol might be examining beta cell replication with the use of thymidine analogs coupled to immunofluorescence or FACS analysis 5,6. This confirmatory analysis can also alleviate the potential concern of non-specific thymidine incorporation into islets independent of proliferation.
Another potential drawback lies in the transduction efficiency of adenoviruses. A transduction efficiency of 60-70% is reasonable to achieve, but a key determinant of the effectiveness is the timing of adenoviral transduction. It is essential to culture the isolated islets in the adenoviruses as soon as possible to maximize transduction efficiency. Within a few hours after the islet isolation the islet begins to contract, thereby limiting the ability of the adenovirus to penetrate deep into the core of the islet. The use of a reporter construct, such as an adenovirus expressing GFP, coupled with confocal microscopy may be beneficial for evaluating transduction efficiency.
The primary advantages of this protocol are: 1) the efficiency of testing multiple determinants of functional beta cell mass on the same pool of islets and 2) the small numbers of islets required to perform the protocol (a typical rat islet isolation yields 400 islets). These advantages allow this protocol to be used as a screening tool for multiple genes at a moderate pace.
No conflicts of interest declared.
This work was supported by grant DK078732 from the NIH (to P.T.F).
|RPMI 1640 media||Gibco||11879|
|6-well plate||BD-Falcon||35-1146||Non-TC treated|
|[methyl-3H]-thymidine||Perkin Elmer||NET027Z001MC||1 mCi/ml|
|Micro-centrifuge tubes||Denville||C2170||1.7 ml|
|HEPES||Sigma||H0887||1 M solution|
|TCA||Fisher Scientific||SA9410-1||10% w/v|
|Scintillation counting tube||Sarstedt||58.536||7 ml, PP|
|Scintillation counting tube cap||Sarstedt||65.816|
|Econo-Safe counting cocktail||RPI||111175|
|BCA Assay Kit||Thermo Scientific||23250|
|Scintillation counting tube rack||Sarstedt||93.1431.001|
|Liquid scintillation counter||Perkin Elmer||Tri-Carb 2910TR|