Methods for preparation and functional analysis of islet β-cells

Abstract

Regulation of blood glucose is a fundamental homeostasis in the body. Insulin is released from pancreatic β-cells in response to changes in blood glucose, the defect of which leads to impaired insulin secretion and diabetes mellitus. Pancreatic β-cells that release insulin occupy approximately 70% of the islet cells, while α, δ, PP-cells are also present in islets. Therefore, analysis of β-cells is a direct approach for studying mechanisms of insulin secretion. Insulin secretion is regulated by cytosolic Ca2+ in β-cells, and its concentration and localization can be measured in real time by fluorescence imaging using indicators. Glucose metabolism is assessed by measurements of NAD (P) H by its autofluorescence. Furthermore, β-cells are equipped with ion channels, which transduce glucose-evoked metabolic signals to electric signals. Electrophysiological analysis by patch clamp techniques detects the activity of various ion channels and membrane potential. Several β-cell lines including HIT, MIN6, INS1, RIN, and βTC are used; however, they do not necessarily retain normal responsiveness to glucose. Therefore, analysis of physiologic functions of β-cells requires the use of acutely isolated or primary cultured β-cells from normal animals. The methods for preparation of islets and single β-cells using collagenase can be applied to a variety of animals of small to large sizes, which can produce islets and β-cells with physiological responsiveness to glucose. These primary cultured β-cells can be used for elucidating signal transduction mechanisms, and evaluating effects of drugs, providing excellent tools for physiological, pharmacological, and disease-oriented studies.