We would like to thank Renee L. Pasker and Harpreet K. Brar for expert technical assistance on the protocols described in this work. Furthermore, we would like to acknowledge the mentoring of Christopher B. Newgard at Duke University and Alan D. Attie at the University of Wisconsin-Madison, along with the support of their laboratory members, which allowed us the time and support necessary to optimize the described protocols. In particular, we thank Hans Hohmeier, Danhong Lu, and Helena Winfield in the Newgard Laboratory and Mary Rabaglia in the Attie Laboratory for productive discussions and advice. This work was supported by NIH grant DK080845 and Juvenile Diabetes 594 
Research Foundation grant 17-2011-608 (to M.E.K.)

All animal experiments were executed in compliance with all relevant guidelines, regulations and regulatory agencies. The protocol being demonstrated was performed under the guidance and approval of the Institutional Animal Care and Use Committee (IACUC) of the University of Wisconsin-Madison.
1. Preparation of Solutions
<ol>
	<li>The method of euthanasia in this protocol is exsanguination under Avertin anesthesia (for a review of alternatives, see Discussion). To make Avertin, add 0.625 g (...

<ol>
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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. 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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. 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Authors have nothing to disclose.

With the prevalence of diabetes projected to affect 7.7% of the world&#8217;s population, the requirement of novel research techniques is imperative to both understand and treat diabetes18. The present islet isolation is a well-established protocol used for in vitro experimentation and has been presented previously with slight modifications11,14,16. Although insulin secretion is a common downstream application for isolated islets, focusing on upstream constituents, such as cAMP, may help de...

The strict maintenance of euglycemia is imperative to prevent morbidities such as neuropathy, nephropathy, and retinopathy, which are all hallmarks of the pathology of uncontrolled type 1 and 2 diabetes1. Reduced &#946;-cell function and mass in both type 1 and 2 diabetes perturb blood glucose concentrations2. Whereas immune-mediated type 1 diabetes results from a devastating loss of insulin-producing &#946;-cells, impaired &#946;-cell insulin secretion and peripheral insulin signaling in type 2 diabetes together promote hyperglycemia, dyslipidemia, and increased hepatic glucose production, which eventually results in both loss of &#946;-cell mass and insulin secretory capacity from individual &#946;-cells3. Understanding the underlying &#946;-cell mechanisms in the progression of type 1 and 2 diabetes will hopefully give rise to novel therapies to prevent and treat these diseases.
In vitro tissue culture models, such as the INS-1 and MIN6 immortalized &#946;-cell lines, can be useful tools for understanding specific &#946;-cell functions. However, the interactions among the different cell types within the islet may themselves regulate &#946;-cell function. For example, the paracrine influence of glucagon (released from &#945;-cells) and somatostatin (released from &#948;-cells) in increasing and decreasing insulin secretion, respectively, demonstrates the importance of cell cell proximity in the endocrine response4. Moreover, gap junctions between &#946;-cells potentiate the release of insulin5. Furthermore, although strides have been made in generating insulinoma lines that better replicated the physiological response of isolated islets to glucose (e.g., the INS-1- derived 832/13 and 832/3 cell lines), their glucose responsiveness still differs from normal rat islets6,7. Moreover, the response of these clonal insulinoma cell lines to glucagon-like peptide-1 (GLP-1) agonists can differ dramatically from one another, as well as from normal islets6. Therefore, immortalized cell lines may not represent the best model for assaying agents that impact on cAMP production.
In contrast to the insulinoma-derived cell lines, studying &#946;-cell function solely in whole animal models offers its own set of complications. One of the biggest challenges in working with endocrine tissue is measuring the precise concentration of hormone released. Specifically, the liver plays a major role metabolizing insulin, and the pancreas blood flow goes directly to the liver. Thus, a plasma insulin measurement may not accurately portray the amounts of insulin being secreted from the pancreas itself or the impact of different treatments on the rate of insulin secretion8. Furthermore, renal metabolism of glucagon may limit the reliability of glucagon output from islet &#945;-cells9. Therefore, isolating primary mouse islets for in vitro experimentation provides a more precise understanding of how the islet is responding to specific stimuli to complement measurements made in vivo.
The present protocol for the isolation of mouse islets is a well-established protocol used by a number of groups (with slight modifications that may help to increase success)10,11. In addition, the determination of cAMP production allows for a direct read-out of the incretin responsiveness of the &#946;-cells. In conjunction with cAMP measurement, protein content and insulin secretion can also be quantified from the same cAMP sample prep, helping to determine whether a defect in &#946;-cell function lies proximal or distal to cAMP10. The final cAMP content and insulin secretion application in this protocol can be a very powerful tool for understanding the influence of pharmaceutical and dietary constituents, among others, on cAMP and insulin secretion. In addition to stimulation from glucose alone, other compounds can be used to measure changes in cAMP and insulin secretion10,11.
Finally, although insulin is the primary hormone we assay from isolated islets, other hormones, such as glucagon and somatostatin, as well as cytokines, eicosanoids, and cyclic adenosine monophosphate, can also be measured, either by a transient stimulation assay or by quantification of their levels in culture medium12. Finally, although outside of the scope of this manuscript, islet isolation with the described collagenase isolation method allows for islet preservation so that many other downstream applications may be pursued, such as islet transplantation, RNA isolation for quantitative real time PCR or microarray analyses, protein isolation for Western blotting, islet embedding and immunofluorescent imaging, and [3H]-thymidine incorporation as a measure of islet cell replication, some of which have been described in previous JoVE articles13-16. Overall, following the islet isolation procedure described in the protocol may provide a researcher with important and useful information for developing therapies and promoting drug discovery aimed at enhancing &#946;-cell function.

<table border="0" cellpadding="0" cellspacing="0" width="502">
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	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Collagenase: Collagenase from Clostridium histolyticum suitable for isolating active islets</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C7657</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Ficoll 400</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>F9378</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hanks Balanced Salt Solution 10X</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td>14065-056</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hepes</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>H3375</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">RPMI 1640 (powder)</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">31800-022</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Albumin from Bovine Serum (BSA)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>A7888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3/0 Silk Suture Thread</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>18020-30</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dumont #5 Forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>11251-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">0.8 mm Forceps&#160;&#160;</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">11050-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Curved Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14061-10</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Vannas-T&#252;bingen Spring Scissors - Straight/Sharp/8.5 cm/5 mm Cutting Edge</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">15003-08</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dissecting Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14002-14</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">5ml BD Luer-Lok Syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309646</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">1ml BD syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309628</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">30 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305106</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">27 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305109</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sharpening Stone</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">29008-01</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-2-2 tribromoethanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">T48402-25G</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-methyl-2-butanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>240486-100mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sodium Chloride (NaCl)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>S9888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Potassium Chloride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>P3911</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:235px;">Monopotassium Phosphate (KH2PO4)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">P0662</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">Sodium Bicarbonate (NaCHO3)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">S6014</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">CaCl2 *2H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C3881</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">MgSO4 *7H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">M9397</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Penicillin-Streptomycin</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">15140-122</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:235px;">Heat Inactivated Fetal Bovine Serum (H.I. FBS)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:136px;">SH30088.03HI</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">5879-100MG</td>
		</tr>
	</tbody>
</table>

a method for mouse pancreatic islet isolation and intracellular camp determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

To ensure a high islet yield during isolation, surgical techniques outlined in the protocol should be followed closely. Although the techniques presented here will be tailored to each laboratory, there are a few critical steps that will lead to a successful isolation. In order to make the common bile duct easily accessible, it is recommended that the organs be displaced to the right side of the mouse (Figure 1). Moreover, this will allow the pancreas to inflate with a smaller amount of resistance since t...

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

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

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

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JoVE Journal

Biology

64.9K 조회수.  University of Wisconsin-Madison.  랑게르한스의 고립 된 마우스 독도를 이용하여 체외 β-세포 기능을 시금 당뇨병의 병태 생리 및 치료의 연구에 중요한 구성 요소입니다. 많은 다운 스트림 응용 프로그램을 사용할 수 있지만,이 프로토콜은 특히 β-세포의 기능을 결정하는 중요한 매개 변수로 세포 내 순환 아데노신 monophosphate (캠프)의 측정에 대해 설명합니다. 