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Abstract

Introduction

Protocol

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Materials

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Biology

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Published: June 25th, 2014

DOI:

10.3791/50374

1Department of Nutrional Sciences, University of Wisconsin-Madison, 2Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin-Madison, 3School of Pharmacy, University of Waterloo

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

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 β-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 β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-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.

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 β-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 β-cells, impaired β-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 β-cell mas....

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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

  1. 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 (.......

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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.......

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With the prevalence of diabetes projected to affect 7.7% of the world’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.......

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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 5....

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

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