Nos gustaría agradecer a Renee L. Pasker y Harpreet K. Brar para la asistencia técnica de expertos sobre los protocolos descritos en este trabajo. Además, nos gustaría agradecer la tutoría de Christopher B. Newgard en la Universidad de Duke y Alan D. Attie en la Universidad de Wisconsin-Madison, junto con el apoyo de los miembros de su laboratorio, que nos permitió el tiempo y el apoyo necesarios para optimizar la protocolos descritos. En particular, agradecemos a Hans Hohmeier, Danhong Lu, y Helena Winfield en el Laboratorio Newgard y María Rabaglia en el Laboratorio Attie para los debates y asesoramiento de producción. Este trabajo fue apoyado por el NIH subvención DK080845 y Diabetes Juvenil 594 Fundación de Investigación de subvención 17-2011-608 (a MEK)

<ol>
	<li>Cryer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoglycaemia:+the+limiting+factor+in+the+glycaemic+management+of+Type+I+and+Type+II+diabetes.">Hypoglycaemia: the limiting factor in the glycaemic management of Type I and Type II diabetes.</a> Diabetologia. 45, 937-948 (2002).</li><li>Alberti, K. G., Zimmet, P. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition,+diagnosis+and+classification+of+diabetes+mellitus+and+its+complications.+Part+1:+diagnosis+and+classification+of+diabetes+mellitus+provisional+report+of+a+WHO+consultation.">Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation.</a> Diabet Med. 15, 539-553 (1998).</li><li>Muoio, D. 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=Mechanisms+of+disease:+molecular+and+metabolic+mechanisms+of+insulin+resistance+and+beta-cell+failure+in+type+2+diabetes.">Mechanisms of disease: molecular and metabolic mechanisms of insulin resistance and beta-cell failure in type 2 diabetes.</a> Nat Rev Mol Cell Biol. 9, 193-205 (2008).</li><li>Kelly, C., McClenaghan, N. H., Flatt, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+islet+structure+and+cellular+interactions+in+the+control+of+insulin+secretion.">Role of islet structure and cellular interactions in the control of insulin secretion.</a> Islets. 3, 41-47 (2011).</li><li>Meda, P., Halban, P., Perrelet, A., Renold, A. E., Orci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junction+development+is+correlated+with+insulin+content+in+the+pancreatic+B+cell.">Gap junction development is correlated with insulin content in the pancreatic B cell.</a> Science. 209, 1026-1028 (1980).</li><li>Hohmeier, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+INS-1-derived+cell+lines+with+robust+ATP-sensitive+K++channel-dependent+and+-independent+glucose-stimulated+insulin+secretion.">Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion.</a> Diabetes. 49, 424-430 (2000).</li><li>Hohmeier, H. E., 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=Cell+lines+derived+from+pancreatic+islets.">Cell lines derived from pancreatic islets.</a> Mol Cell Endocrinol. 228, 121-128 (2004).</li><li>Field, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+insulin+by+liver.">Extraction of insulin by liver.</a> Annu Rev Med. 24, 309-314 (1973).</li><li>Emmanouel, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucagon+metabolism+in+the+rat.">Glucagon metabolism in the rat.</a> J Clin Invest. 62, 6-13 (1978).</li><li>Kimple, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+GalphaZ+protein+protects+against+diet-induced+glucose+intolerance+via+expansion+of+beta-cell+mass.">Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.</a> J Biol Chem. 287, 20344-20355 (2012).</li><li>Rabaglia, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-Ketoisocaproate-induced+hypersecretion+of+insulin+by+islets+from+diabetes-susceptible+mice.">Alpha-Ketoisocaproate-induced hypersecretion of insulin by islets from diabetes-susceptible mice.</a> Am J Physiol Endocrinol Metab. 289, 218-224 (2005).</li><li>Collins, S. C., Salehi, A., Eliasson, L., Olofsson, C. S., Rorsman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+exposure+of+mouse+pancreatic+islets+to+oleate+or+palmitate+results+in+reduced+glucose+induced+somatostatin+and+oversecretion+of+glucagon.">Long-term exposure of mouse pancreatic islets to oleate or palmitate results in reduced glucose induced somatostatin and oversecretion of glucagon.</a> Diabetologia. 51, 1689-1693 (2008).</li><li>Fueger, P. T., Hernandez, A. M., Chen, Y. C., Colvin, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+Replication+and+Beta+Cell+Function+in+Adenovirally-transduced+Isolated+Rodent+Islets.">Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets.</a> J Vis Exp. , (2012).</li><li>Szot, G. L., Koudria, P., Bluestone, J. A. <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. , (2007).</li><li>Szot, G. L., Koudria, P., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+pancreatic+islets+into+the+kidney+capsule+of+diabetic+mice.">Transplantation of pancreatic islets into the kidney capsule of diabetic mice.</a> J Vis Exp. , (2007).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> J Vis Exp. , (2011).</li><li>Kimple, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+G(z)+in+pancreatic+islet+beta-cell+biology.">A role for G(z) in pancreatic islet beta-cell biology.</a> J Biol Chem. 280, 31708-31713 (2005).</li><li>Shaw, J. E., Sicree, R. A., Zimmet, P. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+the+prevalence+of+diabetes+for+2010+and+2030.">Global estimates of the prevalence of diabetes for 2010 and 2030.</a> Diabetes Res Clin Pract. 87, 4-14 (2010).</li><li>Kirstetter, P., Lagneau, F., Lucas, O., Krupa, Y., Marty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelium+in+the+modulation+of+isoflurane-induced+vasodilatation+in+rat+thoracic+aorta.">Role of endothelium in the modulation of isoflurane-induced vasodilatation in rat thoracic aorta.</a> Br J Anaesth. 79, 84-87 (1997).</li><li>Brown, E. T., Umino, Y., Loi, T., Solessio, E., Barlow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+can+cause+sustained+hyperglycemia+in+C57/BL6J+mice.">Anesthesia can cause sustained hyperglycemia in C57/BL6J mice.</a> Vis Neurosci. 22, 615-618 (2005).</li><li>Vaupel, D. B., McCoun, D., Cone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phencyclidine+analogs+and+precursors:+rotarod+and+lethal+dose+studies+in+the+mouse.">Phencyclidine analogs and precursors: rotarod and lethal dose studies in the mouse.</a> J Pharmacol Exp Ther. 230, 20-27 (1984).</li><li>Davis, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxM1+is+up-regulated+by+obesity+and+stimulates+beta-cell+proliferation.">FoxM1 is up-regulated by obesity and stimulates beta-cell proliferation.</a> Mol Endocrinol. 24, 1822-1834 (2010).</li><li>Dionne, K. E., Colton, C. K., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hypoxia+on+insulin+secretion+by+isolated+rat+and+canine+islets+of+Langerhans.">Effect of hypoxia on insulin secretion by isolated rat and canine islets of Langerhans.</a> Diabetes. 42, 12-21 (1993).</li><li>Linetsky, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+human+islet+isolation+using+a+new+enzyme+blend,+liberase.">Improved human islet isolation using a new enzyme blend, liberase.</a> Diabetes. 46, 1120-1123 (1997).</li><li>Vaithilingam, V., Sundaram, G., Tuch, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+cell+transplantation.">Islet cell transplantation.</a> Curr Opin Organ Transplant. 13, 633-638 (2008).</li><li>Brandhorst, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+comparison+of+Liberase+HI+and+collagenase+NB1+utilized+for+human+islet+isolation.">Large-scale comparison of Liberase HI and collagenase NB1 utilized for human islet isolation.</a> Cell Transplant. 19, 3-8 (2010).</li></ol>

Los autores tienen nada que revelar.

Con el predominio de la diabetes proyectados para afectar el 7,7% de la población mundial, la obligación de aplicar técnicas de investigación novedosas es imprescindible tanto para comprender y tratar la diabetes 18. El actual aislamiento de islotes es un protocolo bien establecido utilizado para la experimentación in vitro y se ha presentado con anterioridad, con ligeras modificaciones 11,14,16. Aunque la secreción de insulina es una aplicación común aguas abajo de islotes aislado...

El mantenimiento estricto de euglucemia es imperativo para prevenir la morbilidad como la neuropatía, nefropatía y retinopatía, que son todas las características de la patología de la diabetes 1 no controlada de tipo 1 y 2. Disminución de la función de las células β y la masa, tanto en la diabetes tipo 1 y 2 perturban las concentraciones de glucosa en la sangre 2. Considerando que el tipo inmunomediada 1 diabetes resulta de una devastadora pérdida de β-células productoras de insulina, la secreción de insulina de las células β alterada y la señalización de la insulina periférica en la diabetes tipo 2 en conjunto promueven la hiperglucemia, la dislipidemia y aumento de la producción hepática de glucosa, lo que finalmente se traduce en tanto la pérdida de la capacidad de la masa de células β y secretora de insulina a partir de β-células individuales 3. La comprensión de los mecanismos de las células β subyacentes en la progresión de la diabetes tipo 1 y 2 con suerte, dará lugar a nuevas terapias para prevenir y tratar estas enfermedades. En ti vitromodelos de cultivo ncidencia, como el INS-1 y MIN6 inmortalizados líneas de células β, pueden ser herramientas útiles para la comprensión de las funciones específicas de las células β. Sin embargo, las interacciones entre los diferentes tipos de células dentro de la isleta pueden ellos mismos regular la función de las células β. Por ejemplo, la influencia paracrina de glucagón (liberado de α-células) y la somatostatina (liberado de las células δ) en el aumento y la disminución de la secreción de insulina, respectivamente, demuestra la importancia de la proximidad de células de células en la respuesta endocrina 4. Por otra parte, los cruces brecha entre β-células potencian la liberación de insulina 5. Además, si bien se ha avanzado en la generación de líneas de insulinoma que mejor replican la respuesta fisiológica de los islotes aislados en glucosa (por ejemplo, el INS-1 derivado de 832/13 y 832/3 líneas celulares), su capacidad de respuesta de la glucosa aún difiere de rata normal islotes 6,7. Por otra parte, la respuesta de estas líneas celulares de insulinoma clonalesa péptido-1 (GLP-1) agonistas de tipo glucagón puede diferir drásticamente el uno del otro, así como de los islotes normales 6. Por lo tanto, las líneas de células inmortalizadas pueden no representar el mejor modelo para ensayar agentes que influyen en la producción de cAMP. En contraste con las líneas celulares de insulinoma-derivado, el estudio de la función de células β únicamente en modelos animales enteros ofrece su propio conjunto de complicaciones. Uno de los mayores retos en el trabajo con el tejido endocrino es la medición de la concentración exacta de la hormona liberada. En concreto, el hígado juega un papel importante el metabolismo de la insulina, y el flujo de sangre páncreas va directamente al hígado. Por lo tanto, una medición de la insulina en plasma no puede representar con precisión las cantidades de insulina secretada por el páncreas en sí o el impacto de diferentes tratamientos sobre la tasa de secreción de insulina 8. Además, el metabolismo renal de glucagón puede limitar la fiabilidad de la producción de glucagón de las células de los islotes α-9. Por lo tanto, el aislamiento de islotes primarios de ratón para la experimentación in vitro proporciona una comprensión más precisa de cómo el islote está respondiendo a los estímulos específicos para complementar las mediciones realizadas in vivo. El presente protocolo para el aislamiento de islotes de ratón es un protocolo bien establecido utilizado por varios grupos (con ligeras modificaciones que pueden ayudar a aumentar el éxito) 10,11. Además, la determinación de la producción de cAMP permite una lectura directa de la capacidad de respuesta de la incretina de las células β. En conjunción con la medición de AMPc, contenido de proteína y la secreción de insulina también se puede cuantificar a partir de la misma preparación de muestras de cAMP, lo que ayuda a determinar si un defecto en la función de las células β se encuentra proximal o distal a AMPc 10. El contenido de cAMP final y aplicación secreción de insulina en este protocolo puede ser una herramienta muy poderosa para la comprensión de la influencia de los componentes farmacéuticos y alimenticios, entre otross, el cAMP y la secreción de insulina. Además de la estimulación de glucosa sola, otros compuestos pueden ser utilizados para medir los cambios en el AMPc y la secreción de insulina 10,11. Por último, aunque la insulina es la principal hormona que del ensayo de islotes aislados, otras hormonas, tales como glucagón y somatostatina, así como citoquinas, eicosanoides, y monofosfato de adenosina cíclico, puede también ser medido, ya sea mediante un ensayo de estimulación transitoria o por cuantificación de sus niveles en el medio de cultivo 12. Por último, aunque fuera del alcance de este manuscrito, aislamiento de islotes con el método de aislamiento de colagenasa descrito permite la preservación de los islotes de manera que muchas otras aplicaciones posteriores pueden llevarse a cabo, tales como el trasplante de islotes, el aislamiento de ARN para cuantitativa en tiempo real PCR o análisis de microarrays, proteínas aislamiento para el Western Blot, la incrustación de los islotes y de formación de imágenes de inmunofluorescencia, y la incorporación de [3H]-timidina como una medida de la replicación de células de islotescatiónico, algunos de los cuales se han descrito en artículos anteriores JoVE 13-16. En general, siguiendo el procedimiento de aislamiento de los islotes se describe en el protocolo puede proporcionar un investigador con información útil e importante para el desarrollo de terapias y promover el descubrimiento de medicamentos destinada a mejorar la función de las células β.

<table><tbody><tr><td>Collagenase from &lt;em&gt;Clostridium histolyticum&lt;/em&gt; suitable for isolating active islets</td><td>Sigma-Aldrich</td><td>C7657</td><td></td></tr><tr><td>Ficoll 400</td><td>Sigma-Aldrich</td><td>F9378</td><td></td></tr><tr><td>Hanks Balanced Salt Solution 10X</td><td>Invitrogen (Gibco)</td><td>14065-056</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>RPMI 1640 (powder)</td><td>Invitrogen (Gibco)</td><td>31800-022</td><td></td></tr><tr><td>Albumin from Bovine Serum (BSA)</td><td>Sigma-Aldrich</td><td>A7888</td><td></td></tr><tr><td>3/0 Silk Suture Thread</td><td>Fine Science Tools</td><td>18020-30</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>0.8 mm Forceps&amp;nbsp;&amp;nbsp;</td><td>Fine Science Tools</td><td>11050-10</td><td></td></tr><tr><td>Curved Scissors</td><td>Fine Science Tools</td><td>14061-10</td><td></td></tr><tr><td>Vannas-T&amp;uuml;bingen Spring Scissors - Straight/Sharp/8.5 cm/5 mm Cutting Edge</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>Dissecting Scissors</td><td>Fine Science Tools</td><td>14002-14</td><td></td></tr><tr><td>5 ml BD Luer-Lok Syringe</td><td>BD</td><td>309646</td><td></td></tr><tr><td>1 ml BD syringe</td><td>BD</td><td>309628</td><td></td></tr><tr><td>30 G BD Needle 1/2&quot; Length</td><td>BD</td><td>305106</td><td></td></tr><tr><td>27 G BD Needle 1/2&quot; Length</td><td>BD</td><td>305109</td><td></td></tr><tr><td>Sharpening Stone</td><td>Fine Science Tools</td><td>29008-01</td><td></td></tr><tr><td>2-2-2-tribromoethanol</td><td>Sigma-Aldrich</td><td>T48402-25G</td><td></td></tr><tr><td>2-methyl-2-butanol</td><td>Sigma-Aldrich</td><td>240486-100mL</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>Monopotassium Phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P0662</td><td></td></tr><tr><td>Sodium Bicarbonate (NaCHO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C3881</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#183;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M9397</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen (Gibco)</td><td>15140-122</td><td></td></tr><tr><td>Heat Inactivated Fetal Bovine Serum (H.I. FBS)</td><td>Fisher Scientific</td><td>SH30088.03HI</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine (IBMX)</td><td>Sigma-Aldrich</td><td>5879-100MG</td><td></td></tr></tbody></table>

a method for mouse pancreatic islet isolation and intracellular camp determination

Glucemia no controlada es una característica de la diabetes mellitus y promueve morbilidades como la neuropatía, nefropatía, y retinopatía. Con la creciente prevalencia de la diabetes, tanto inmune tipo 1 y relacionada con la obesidad de tipo 2, los estudios dirigidos a la delimitación de la fisiopatología de la diabetes y los mecanismos terapéuticos son de importancia crítica. Los β-células de los islotes pancreáticos de Langerhans son responsables de la secreción de insulina apropiadamente en respuesta a concentraciones elevadas de glucosa en sangre. Además de la glucosa y otros nutrientes, las células β también son estimulados por hormonas específicas, denominado incretinas, que se secretan en el intestino en respuesta a una comida y actúan sobre los receptores de células β que aumentan la producción de monofosfato de adenosina cíclico intracelular ( AMPc). Disminución de la función de las células β, la masa y la capacidad de respuesta de la incretina son bien entendidos para contribuir a la fisiopatología de la diabetes tipo 2, y también se están cada vez más vinculados widiabetes tipo 1 ª. El presente ratón aislamiento de islotes y el protocolo de determinación de cAMP puede ser una herramienta para ayudar a delinear los mecanismos que promueven la progresión de la enfermedad y las intervenciones terapéuticas, en particular los que están mediadas por los receptores de la incretina o receptores que actúan a través de la modulación de la producción de cAMP intracelular relacionado. Si bien sólo se describirán las medidas de cAMP, el protocolo de aislamiento de islotes descrito crea una preparación limpia que permite también para muchas otras aplicaciones posteriores, incluyendo la glucosa estimula la secreción de insulina, [3 H]-timidina incorporación, la abundancia de proteínas, y la expresión del ARNm.

Para asegurar un alto rendimiento durante el aislamiento de los islotes, las técnicas quirúrgicas descritas en el protocolo deben ser seguidos de cerca. Aunque las técnicas que aquí se presentan se adaptarán a cada laboratorio, hay algunos pasos críticos que conduzcan a un aislamiento exitoso. Con el fin de hacer que el conducto biliar común de fácil acceso, se recomienda que los órganos ser desplazados hacia el lado derecho del ratón (Figura 1). Además, esto permitirá que el páncreas se in...

Watch this Scientific Journal Video about A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination at JoVE.com

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

El ensayo in vitro función de las células β usando islotes aislados de ratones de Langerhans es un componente importante en el estudio de la fisiopatología de la diabetes y la terapéutica. Mientras que muchas aplicaciones posteriores están disponibles, este protocolo se describe específicamente la medición de intracelular de adenosina monofosfato cíclico (cAMP) como un parámetro esencial para determinar la función de las células β.

Un método para ratón islotes pancreáticos Aislamiento y cAMP intracelular Determinación

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

a-method-for-mouse-pancreatic-islet-isolation-intracellular-camp

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

65.1K vistas.  University of Wisconsin-Madison.  El ensayo in vitro función de las células β usando islotes aislados de ratones de Langerhans es un componente importante en el estudio de la fisiopatología de la diabetes y la terapéutica. Mientras que muchas aplicaciones posteriores están disponibles, este protocolo se describe específicamente la medición de intracelular de adenosina monofosfato cíclico (cAMP) como un parámetro esencial para determinar la función de las células β. 

Video: A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Un método para el aislamiento de los islotes murinos y Trasplante Renal subcapsular

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Investigación

Medicina

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Análisis transcriptómico de unicelular de células endocrinas pancreáticas de ratón

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Biología del Desarrollo

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.

Lepr<sup&gt; Db</sup&gt; Modelo de ratón de la Diabetes Tipo 2: El aislamiento de los islotes pancreáticos y células en Vivo 2-Photon imágenes de islotes intactos

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 Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Una proyección basadas en células de islotes pancreáticos murinos Diabetogenic ambiental productos químicos

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

Inmunología e Infección

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

Aislamiento de ratón Islet Langerhans utilizando una combinación de colagenasa purificada y proteasa neutra

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Biología

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Visualización de complejos endógenos de mitophagy in Situ en células beta pancreáticas humanas que utilizan el ensayo de ligadura de proximidad

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Un protocolo simple de alta eficiencia para el aislamiento de islotes pancreáticos de ratones

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Imagen de la dinámica del calcio en subpoblaciones de células de islotes pancreáticos de ratón

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

<meta charset="utf8"></head><body>Optimización de la extracción de células endoteliales pancreáticas de ratón para estudios de enfermedades

Isolation of Mouse Pancreatic Endothelial Cells

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.

<meta charset="utf8"></head><body>Aislamiento de células endoteliales pancreáticas de ratón

This protocol describes the isolation of mouse endothelial cells from whole pancreas.

Aislamiento de células endoteliales pancreáticas de ratón

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and ...

Un método para ratón islotes pancreáticos Aislamiento y cAMP intracelular Determinación

Resumen

Explorar más videos

Capítulos en este video

Un método para el aislamiento de los islotes murinos y Trasplante Renal subcapsular

Análisis transcriptómico de unicelular de células endocrinas pancreáticas de ratón

Lepr

Una proyección basadas en células de islotes pancreáticos murinos Diabetogenic ambiental productos químicos

Aislamiento de ratón Islet Langerhans utilizando una combinación de colagenasa purificada y proteasa neutra

Visualización de complejos endógenos de mitophagy in Situ en células beta pancreáticas humanas que utilizan el ensayo de ligadura de proximidad

Un protocolo simple de alta eficiencia para el aislamiento de islotes pancreáticos de ratones

Imagen de la dinámica del calcio en subpoblaciones de células de islotes pancreáticos de ratón

Aislamiento de células endoteliales pancreáticas de ratón

Aislamiento de células endoteliales pancreáticas de ratón