This work was supported by grant DK078732 from the NIH (to P.T.F).

1. Adenoviral Transduction and Culturing of Rat Islets
<ol>
 <li>Prepare a 6-well non-tissue culture coated plate by adding 2 ml of media (RPMI 1640 media containing 8 mM glucose, 10% fetal bovine serum, 50 units/ml penicillin, and 50 &mu;g/ml streptomycin) to the required number of wells. For example, a typical experiment may require three wells &#x2013; one each for a no-virus control, a virus control (e.g., GFP-expressing adenovirus), and the experimental group.</li>
 <li>Warm the plate to 37 &deg;C by p...

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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 the...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>RPMI 1640 media</td>
 <td>Gibco</td>
 <td>11879</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin/streptomycin</td>
 <td>Gibco</td>
 <td>15140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>6-well plate</td>
 <td>BD-Falcon</td>
 <td>35-1146</td>
 <td>Non-TC treated</td>
 </tr>
 <tr>
 <td>[methyl-3H]-thymidine</td>
 <td>Perkin Elmer</td>
 <td>NET027Z001MC</td>
 <td>1 mCi/ml</td>
 </tr>
 <tr>
 <td>Micro-centrifuge tubes</td>
 <td>Denville</td>
 <td>C2170</td>
 <td>1.7 ml</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma</td>
 <td>59888</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Acros</td>
 <td>42409</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>Acros</td>
 <td>20592</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MgSO4</td>
 <td>Acros</td>
 <td>41348</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CaCl2</td>
 <td>Acros</td>
 <td>34961</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma</td>
 <td>H0887</td>
 <td>1 M solution</td>
 </tr>
 <tr>
 <td>35% BSA</td>
 <td>Sigma</td>
 <td>A7979</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>Acros</td>
 <td>42427</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>d-glucose</td>
 <td>Sigma</td>
 <td>G8769</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TCA</td>
 <td>Fisher Scientific</td>
 <td>SA9410-1</td>
 <td>10% w/v</td>
 </tr>
 <tr>
 <td>NaOH</td>
 <td>Acros</td>
 <td>12426</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Scintillation counting tube</td>
 <td>Sarstedt</td>
 <td>58.536</td>
 <td>7 ml, PP</td>
 </tr>
 <tr>
 <td>Scintillation counting tube cap</td>
 <td>Sarstedt</td>
 <td>65.816</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Econo-Safe counting cocktail</td>
 <td>RPI</td>
 <td>111175</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Insulin RIA</td>
 <td>Siemens</td>
 <td>TKIN2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BCA Assay Kit</td>
 <td>Thermo Scientific</td>
 <td>23250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Eppendorf</td>
 <td>5415R</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Scintillation counting tube rack</td>
 <td>Sarstedt</td>
 <td>93.1431.001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Liquid scintillation counter</td>
 <td>Perkin Elmer</td>
 <td>Tri-Carb 2910TR</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

assessing replication and beta cell function in adenovirally-transduced isolated rodent islets

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.

Watch this Scientific Journal Video about Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets at JoVE.com

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses.

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

assessing-replication-beta-cell-function-adenovirally-transduced

Research

JoVE Journal

Medicine

13.9K visualizações.  Indiana University School of Medicine. Este protocolo permite uma para identificar factores que modulam a massa de células funcional de beta para encontrar potenciais alvos terapêuticos para o tratamento da diabetes. O protocolo é constituído por um método simplificado para avaliar a replicação de ilhotas e função das células beta em ilhéus isolados de ratos após a manipulação da expressão do gene com adenovírus.