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内容記述 |
A spatial organization of islet β-cells, a secretion of insulin, and a relationship between them were studied without cellular dissociation by using a video-enhanced contrast differential interference contrast (VEC-DIC) microscope. In the rat pancreas, endocrine islet cells appeared with less contrast than exocrine acinar cells, and contained secretory granules (c.a. 0.5 - 1.2 μm in diameter) more scattered than those in acinar cells. In individual islet cells, the exocytotic response (degranulation) appeared as abrupt light intensity changes of many secretory granules, when the concentration of glucose in the superfusion solution was raised from 1.5 mM to 15 mM. Such responses were observed in most islet cells simultaneously. By counting the number of responses in a unit of time, the time course was studied for quantitative estimation of the secretory activity. The glucose-induced exocytotic response was augmented in the presence of L-arginine (0.3 mM), and was suppressed completely by removal of Ca2+ from the medium. The glucose-sensitive cells were sensitive also to elevation of K+ concentration in the medium from 5 mM to 65 mM, and to addition of A23187 (a Ca2+ ionophore, 1 μM), tolbutamide (a sulfonylurea, 100 μM) and bethanechol (a muscarinic agonist, 100 μM) to the medium. The cells that responded to glucose were immunopositive when fixed in mid-response and processed with an HRP-conjugated anti-insulin antibody. After a glucose-induced degranulatmg response, release of insulin to the extracellular medium was detected by the reverse hemolytic plaque assay. These results indicate that the exocytotic responses induced by glucose stimulation reflect the secretory activity of insulin-containing β-cells.<br /> A three dimensional configuration of β-cells in Ltngerhans islet was best elucidated when cells were completely degranulated by extensive glucose stimulation. Several β-cells adhered each other, and formed many elliptically shaped clusters (40.3 ± 0.8 μm for long axis and 35.1 ± 0.5 μm for short one, n=56). The cluster of β-cells was clearly visualized also by confocal laser microscopy in tissues stained with lucifer yellow or 8-hydroxy-pyrene-1, 3, 6-trisulfonic acid trisodium salt (an anionic fluorescence chromophore) and by light microscopy in tissues immunostained with anti-insulin antibody. The clustered structure was also recognized in the normally circulated pancreas. The clusters were classified roughly into two classes by their morphological characteristics: a small-sized one consisting of 8.3 ± 0.1 cells (n=43) and a large-sized one consisting of 15.5 ± 0.5 cells (n=13). The former were located in the center of islet, and the later in the periphery.<br /> In order to develop a method for analysing secretion of insulin dynamically, the secretory activity of β-cell cluster was examined quantitatively. Almost all β-cells in a single cluster showed patterns of exocytotic responses similar to each other upon glucose stimulation. Different clusters, sometimes even next neighbors, in an islet showed heterogeneous response patterns. This heterogeneity of β-cell clusters was further demonstrated by immunostaining of tissues fixed in the peak of degranulating response. The heterogeneity became less prominent when the clusters were stimulated with K+ -rich solution or A23187-containing solution. The patterns of glucose-induced exocytotic response at 35 ℃ were classified roughly into two types: one showing a rapid and transient response, and the other showing a delayed and persistent response. An ensemble average of the responses observed in 40 clusters revealed a biphasic pattern involving an early phase and a late phase, consistent with the earlier observation. These suggest that the biphasic secretion of insulin was ascribed to heterogeneous activities of different β-cell clusters. The time course of the degranulation pattem depended significantly on the size of cluster: the early phase was mostly found in the small-sized clusters, and the late phase in the large-sized ones.<br /> In Langerhans islet, β-cells were arranged in many clusters. These clusters were subdivided into two groups by their cell population and by their degranulatmon pattern. We concluded that the small-sized cluster of β-cells are mostly responsible for the secretion of insulin during brief stimulation, whereas the large-sized cluster of β-cells may significantly contribute to the total insulin release only after prolonged stimulation. The cluster of β-cells functions as a physiological unit for insulin secretion, and a diversity of individual clusters of β-cells may be an essential basis for a multifactorial control of glucose homeostasis. |
要旨・審査要旨 / Abstract, Screening Result (284.2 kB)
