[Methods for preparation and functional analysis of islet beta-cells].
Regulation of blood glucose is a fundamental homeostasis in the body. Insulin is released from pancreatic beta-cells in response to changes in blood glucose, the defect of which leads to impaired insulin secretion and diabetes mellitus. Pancreatic beta-cells that release insulin occupy approximately 70% of the islet cells, while alpha, delta, PP-cells are also present in islets. Therefore, analysis of beta-cells is a direct approach for studying mechanisms of insulin secretion. Insulin secretion is regulated by cytosolic Ca(2+) in beta-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, beta-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 beta-cell lines including HIT, MIN6, INS1, RIN, and beta TC are used; however, they do not necessarily retain normal responsiveness to glucose. Therefore, analysis of physiologic functions of beta-cells requires the use of acutely isolated or primary cultured beta-cells from normal animals. The methods for preparation of islets and single beta-cells using collagenase can be applied to a variety of animals of small to large sizes, which can produce islets and beta-cells with physiological responsiveness to glucose. These primary cultured beta-cells can be used for elucidating signal transduction mechanisms, and evaluating effects of drugs, providing excellent tools for physiological, pharmacological, and disease-oriented studies.
Kakei
Masafumi
M
Department of Internal Medicine, Division of Endocrinology, Diabetes Geriatric Medicine, Akita University School of Medicine.
Dezaki
Katsuya
K
Yada
Toshihiko
T
D000818:Animals; D002118:Calcium / Q000032:analysis*; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D005947:Glucose / Q000032:analysis*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D015398:Signal Transduction / Q000502:physiology
D007328:Insulin; D005947:Glucose; D002118:Calcium