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 placing it into a tissue culture incubator for at least 30 min.</li>
 <li>Immediately following rat islet isolation 18,19, place 100-200 islets into individual wells of the 6-well non-tissue culture coated plate. Sixty islets are required for the insulin secretion and thymidine incorporation assays. The remaining islets can be used for RNA isolation for gene expression studies or protein isolation for immunoblotting.
 </li>
 </ol>
 [Note: From this point forward, please follow institutional protocols for the handling, use, and disposal of biohazardous materials.]
 <ol start="4">
 <li>Gently swirl the plate to bring the islets to the center of the well.</li>
 <li>Pipette the adenovirus directly onto the islets in the center of the dish. Use 100-500 multiplicities of infection (MOI, the ratio of target cells to viral plaque-forming units).</li>
 <li>Let the islets rest for 5 min.</li>
 <li>Place the plate in the tissue culture incubator (37 &deg;C, 5% CO2).</li>
 <li>After 24 h, gently swirl the plate to bring the islets to the centers of the wells and transfer the islets using a P200 micropipette to a new well containing fresh media. If the islets become attached to the plate, they can be gently dislodged with the pipette tip.
 </li>
 </ol>
 [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.]
 <ol start="9"> 
 <li>Culture the islets for an additional 24-72 h, depending on the desired timing of the experiment from optimization pilot studies. For example, induction of a proliferative response may require times ranging from 24-72 h or knockdown of the gene of interest may require 48 or 72 hours. Transfer the islets to fresh media each day.</li>
 <li>For the final 24 h of the experiment, culture the islets in media containing 1 &mu;Ci [methyl-3H]-thymidine/ml media (generally 1 &mu;l thymidine/ml media).
 </li>
</ol>
[Note: From this point forward, please follow institutional protocols for the handling, use, and disposal of radioactive materials.]
2. Insulin Secretion Assay
<ol>
 <li>Prepare the secretion assay buffer (SAB) 10X stock solution (1.14 M NaCl, 47 mM KCl, 12 mM KH2PO4, 11.6 mM MgSO4) and CaCl2 100X stock solution (0.25 M CaCl2). These stock solutions may be prepared ahead of time and stored at room temperature.</li>
 <li>Freshly prepare 50 ml of the working SAB (5 mL of 10X SAB, 1 ml of 1 M HEPES, 0.5 ml of 100X CaCl2, 0.28 ml of 35% BSA, 0.11 g NaHCO3, and sterile water to 50 ml) in a 50-ml conical tube and warm to 37 &deg;C by placing in a 37 &deg;C waterbath.</li>
 <li>Pipette 10 ml of the working SAB into a 15-ml conical tube and add 66.8 &mu;l of 2.5 M D-glucose to prepare the high glucose (16.7 mM) SAB.</li>
 <li>Add 44.8 &mu;l of 2.5 M D-glucose to the remaining 40 ml of the working SAB to prepare the low glucose (2.8 mM) SAB.</li>
 <li>Label three 1.7-ml microcentrifuge tubes for each well of the 6-well plate and add 1 ml of phosphate-buffered saline (PBS).
 </li>
 </ol>
 [Note: As the islets are radioactive, please follow institutional protocols for the handling, use, and disposal of radioactive materials.]
 <ol start="6">
 <li>Place 20 islets into each microcentrifuge tube. Make every attempt to add comparably sized islets to each microcentrifuge tube. For example, each tube may contain 5 small-, 10 medium-, and 5 large-sized islets (see Figure 1).
 </li>
 </ol>
 [Note: Islets can be visualized using either a dissecting stereoscope or a standard microscope.]
 <ol start="7">
 <li>After the islets have settled on the bottom of the tube by gravity (~2 min), aspirate the PBS with a micropipette and discard.
 </li>
 </ol>
 [Note: As an alternative to settling by gravity, the tubes may be centrifuged at 300 x g for 1 min.]
 <ol start="8">
 <li>For pre-incubation, add 400 &mu;l of the low glucose SAB, place the tubes (with their caps open) into the tissue culture incubator (37 &deg;C, 5% CO2), and pre-incubate for 60 min. Aspirate the pre-incubation low glucose SAB and discard.</li>
 <li>For basal insulin secretion, add 400 &mu;l of the low glucose SAB, place the tubes (with their caps open) into the tissue culture incubator (37 &deg;C, 5% CO2), and incubate for 60 min. Collect the low glucose SAB and save for the insulin radioimmunoassay.</li>
 <li>For stimulated insulin secretion, add 400 &mu;l of the high glucose SAB, place the tubes (with their caps open) into the tissue culture incubator (37 &deg;C, 5% CO2), and incubate for 60 min. Collect the high glucose SAB and save for the insulin radioimmunoassay.</li>
</ol>
3. Thymidine Incorporation Assay
<ol>
 <li>Add 1 ml PBS; after the islets have settled on the bottom of the tube by gravity, aspirate the PBS with a micropipette, discard, and repeat this step once.</li>
 <li>Add 500 &mu;l ice cold trichloroacetic acid (TCA, 10% w/v) and incubate on ice for 30 min.</li>
 <li>Centrifuge the tubes at 16 000 x g for 3 min at 4 &deg;C.</li>
 <li>Aspirate the TCA, add 80 &mu;l of 0.3 N NaOH, and incubate for 30 min at room temperature. During this time, vigorously vortex the samples for 5-10 s every 10 min.</li>
 <li>Add 4 ml of Econo-safe counting cocktail to 7 ml liquid scintillation counting tubes.</li>
 <li>Add 50 &mu;l of the sample to the scintillation counting tube, cap the tube, shake briefly, and count in a liquid scintillation counter.</li>
 <li>Measure the protein concentration using the bicinchoninic acid (BCA) assay and 10 &mu;l of sample according to the manufacturer's protocol.</li>
</ol>
4. Data Analysis
<ol>
 <li>Perform the insulin radioimmunoassay following the manufacturer's protocol.</li>
 <li>Normalize the insulin secretion and thymidine incorporation data with the protein concentration.</li>
</ol>
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.
<img src="/files/ftp_upload/4080/4080fig1.jpg" alt="Figure 1"/> 
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.
<img src="/files/ftp_upload/4080/4080fig2.jpg" alt="Figure 2"/> 
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.

<ol>
	<li>Ferrannini, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Cell+function+in+subjects+spanning+the+range+from+normal+glucose+tolerance+to+overt+diabetes:+a+new+analysis.">beta-Cell function in subjects spanning the range from normal glucose tolerance to overt diabetes: a new analysis.</a> J. Clin. Endocrinol. Metab. 90, 493-500 (2005).</li><li>Weyer, C., Bogardus, C., Mott, D. M., Pratley, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+history+of+insulin+secretory+dysfunction+and+insulin+resistance+in+the+pathogenesis+of+type+2+diabetes+mellitus.">The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus.</a> J. Clin. Invest. 104, 787-794 (1999).</li><li>Keenan, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+insulin+production+and+pancreatic+ss-cell+turnover+after+50+years+of+diabetes:+Joslin+Medalist+Study.">Residual insulin production and pancreatic ss-cell turnover after 50 years of diabetes: Joslin Medalist Study.</a> Diabetes. 59, 2846-2853 (2010).</li><li>Bain, J. R., Schisler, J. C., Takeuchi, K., Newgard, C. B., Becker, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adenovirus+vector+for+efficient+RNA+interference-mediated+suppression+of+target+genes+in+insulinoma+cells+and+pancreatic+islets+of+langerhans.">An adenovirus vector for efficient RNA interference-mediated suppression of target genes in insulinoma cells and pancreatic islets of langerhans.</a> Diabetes. 53, 2190-2194 (2004).</li><li>Fueger, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trefoil+factor+3+stimulates+human+and+rodent+pancreatic+islet+beta-cell+replication+with+retention+of+function.">Trefoil factor 3 stimulates human and rodent pancreatic islet beta-cell replication with retention of function.</a> Mol. Endocrinol. 22, 1251-1259 (2008).</li><li>Schisler, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+human+and+rat+islet+beta-cell+proliferation+with+retention+of+function+by+the+homeodomain+transcription+factor+Nkx6.1.">Stimulation of human and rat islet beta-cell proliferation with retention of function by the homeodomain transcription factor Nkx6.1.</a> Mol. Cell Biol. 28, 3465-3476 (2008).</li><li>Chan, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+uncoupling+protein+2+inhibits+glucose-stimulated+insulin+secretion+from+rat+islets.">Overexpression of uncoupling protein 2 inhibits glucose-stimulated insulin secretion from rat islets.</a> Diabetes. 48, 1482-1486 (1999).</li><li>Cozar-Castellano, I., Takane, K. K., Bottino, R., Balamurugan, A. N., Stewart, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+beta-cell+proliferation+and+retinoblastoma+protein+phosphorylation+in+rat+and+human+islets+using+adenovirus-mediated+transfer+of+cyclin-dependent+kinase-4+and+cyclin+D1.">Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1.</a> Diabetes. 53, 149-159 (2004).</li><li>Icyuz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+infection+activates+akt1+and+induces+cell+proliferation+in+pancreatic+islets1.">Adenovirus infection activates akt1 and induces cell proliferation in pancreatic islets1.</a> Transplantation. 87, 821-824 (2009).</li><li>Kaneto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+hexosamine+pathway+leads+to+deterioration+of+pancreatic+beta-cell+function+through+the+induction+of+oxidative+stress.">Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress.</a> J. Biol. Chem. 276, 31099-31104 (2001).</li><li>Antinozzi, P. A., Berman, H. K., O'Doherty, R. M., Newgard, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+engineering+with+recombinant+adenoviruses.">Metabolic engineering with recombinant adenoviruses.</a> Annu. Rev. Nutr. 19, 511-544 (1999).</li><li>Newgard, C. B., Becker, T. C., Berman, H. K., O'Doherty, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+overexpressed+hexokinases+in+liver+and+islet+cells.">Regulation of overexpressed hexokinases in liver and islet cells.</a> Biochem. Soc. Trans. 25, 118-122 (1997).</li><li>Becker, T. C., BeltrandelRio, H., Noel, R. J., Johnson, J. H., Newgard, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+hexokinase+I+in+isolated+islets+of+Langerhans+via+recombinant+adenovirus.+Enhancement+of+glucose+metabolism+and+insulin+secretion+at+basal+but+not+stimulatory+glucose+levels.">Overexpression of hexokinase I in isolated islets of Langerhans via recombinant adenovirus. Enhancement of glucose metabolism and insulin secretion at basal but not stimulatory glucose levels.</a> J. Biol. Chem. 269, 21234-21238 (1994).</li><li>Csete, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+gene+transfer+to+pancreatic+islets+does+not+alter+islet+function.">Adenoviral-mediated gene transfer to pancreatic islets does not alter islet function.</a> Transplant Proc. 26, 756-757 (1994).</li><li>Csete, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+to+pancreatic+islets+mediated+by+adenoviral+vectors.">Efficient gene transfer to pancreatic islets mediated by adenoviral vectors.</a> Transplantation. 59, 263-268 (1995).</li><li>Meng, Z. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+liver+X+receptors+inhibits+pancreatic+islet+beta+cell+proliferation+through+cell+cycle+arrest.">Activation of liver X receptors inhibits pancreatic islet beta cell proliferation through cell cycle arrest.</a> Diabetologia. 52, 125-135 (2009).</li><li>Ronnebaum, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyruvate+cycling+pathway+involving+cytosolic+NADP-dependent+isocitrate+dehydrogenase+regulates+glucose-stimulated+insulin+secretion.">A pyruvate cycling pathway involving cytosolic NADP-dependent isocitrate dehydrogenase regulates glucose-stimulated insulin secretion.</a> J. Biol. Chem. , (2006).</li><li>Milburn, J. L. <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+in+obesity.+Evidence+for+induction+of+functional,+morphologic,+and+metabolic+abnormalities+by+increased+long+chain+fatty+acids.">Pancreatic beta-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids.</a> J. Biol. Chem. 270, 1295-1299 (1995).</li><li>Szot, G., Koudria, P., Bluestone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+Pancreatic+Islet+Isolation.">Murine Pancreatic Islet Isolation.</a> J. Vis. Exp. (7), e255 (2007).</li></ol>

No conflicts of interest declared.

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 Aufrufe.  Indiana University School of Medicine. Dieses Protokoll erlaubt es, Faktoren, die funktionelle Betazellmasse modulieren, um potentielle therapeutische Ziele für die Behandlung von Diabetes zu finden identifizieren. Das Protokoll besteht aus einem optimierten Verfahren zur Insel Replikation und Beta-Zellfunktion in isolierten Ratteninseln nach Manipulation der Genexpression mit Adenoviren zu beurteilen.

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

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

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.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

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, ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer&#039;s Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...