Perifusion of Isolated Rat Islets in Vitro: Participation of the Microtubular System in the Biphasic Release of Insulin

Abstract

A simple perifusion system for in vitro studies on the rate of insulin secretion of isolated rat islets is described. A biphasic pattern of insulin secretion was produced by glucose whereas tolbutamide stimulated only the first phase of insulin secretion. The perifusion system was used to determine the effect of anti-mitotic agents on the biphasic pattern of insulin secretion. Yinblastine and colchicine destroy microtubules whereas deuterium oxide (D Q 0) produces stabilization and interference with the function of microtubules. Vinblastine (10' 4 M) and D 2 O (100 per cent) inhibited completely the first and second phase of glucose-induced insulin secretion. The inhibitory effect of D 2 O was reversible and a biphasic pattern of secretion occurred following the replacement of D 2 O with water. Colchicine (10" 3 M) produced significant inhibition of only the second phase of glucose-induced insulin secretion. The first phase of tolbutamide-induced insulin secretion was inhibited by vinblastine and D 2 O and the inhibitory effect of D. 2 O was reversible. Colchicine did not inhibit the first phase of tolbutamide-induced insulin release. These findings indicate that the microtubular system is involved in both phases of insulin secretion. The first phase of secretion may be due to the release of beta granules already associated with the microtubular system and the second phase could be the result of stored and newlysynthesized granules becoming associated with the system. DIABETES 21:987-98, October, 1972.In previous studies, we proposed that the microtubular-microfilament system was involved in the intracellular transport of beta granules following glucose stimulation of insulin secretion. 1 This hypothesis was based upon the ultrastructural demonstration of a microtubular-microfilament system in beta cells and the finding that mitotic spindle-inhibitors would inhibit the secretion of insulin by isolated rat islets maintained in vitro. A subsequent series of studies by Malaisse et ai B>3

Ciliary dysfunction impairs beta-cell insulin secretion and promotes development of type 2 diabetes in rodents

“…In line with these observations, nocodazole dissolved the MT skeleton including the ciliary axoneme of the MIN6m9 b-cell line, whereas colchicine did not disassemble the ciliary axoneme ( We cannot exclude an effect of colchicine on ciliogenesis, although a parsimonious explanation might be the improved visibility of ciliary axonemes because of the absence of other acetylated MT structures that otherwise mask the axonemes. These findings not only corroborate our previous results and strongly implicate a specific role for the cilium in first phase insulin release; they also offer an explanation for an almost 40-year-old observation, the differential effect of colchicine and nocodazole on insulin secretion [26][27][28] . show several phenotypes characteristic of T2DM susceptibility.…”

“…Early investigations into the mechanism of insulin release had revealed that the disruption of the MT network resulted in loss of insulin release, indicating that MT transport is essential for insulin secretion 25 . In contrast to MT effectors like nocodazole, treatment with colchicine abolished second phase insulin release but rendered the first phase intact [26][27][28][29] . We sought to confirm these findings and found a loss of second but not first phase insulin release for colchicine-treated MIN6m9 cells and isolated murine islets, whereas there was no insulin released in the case of treatment with nocodazole (data not shown).…”

Microtubules and beta cell function: effect of colchicine on microtubules and insulin secretion in vitro by mouse beta cells.

“…(2) hypothesized that Mts play an important role in secretory activity of the beta cell . These studies have been confirmed (see reference 3 for review) and extended (4)(5)(6) to show that colchicine retards the movement of newly formed insulin from the site of synthesis in the rough endoplasmic reticulum to the plasma membrane (7,8) . There is a second putative pool of insulin (9), presumably located in granules in the periphery of the beta cells, which does not require Mts for secretion.…”

Effect of diltiazem on insulin secretion

“…In perifused islets of Langerhans from rats, Lacy et al suggested that the first phase of insulin secretion following stimulation with glucose may be due to the release of beta granules already associated with the microtubular system and the second phase of secretion could be the result of stored or newly synthesized granules becoming associated with the system (19). In any case, activation of the microtubular-microfilament system in the B-cell is assumed to be due to calcium ion which flows into the cell following stimulation with glucose (20).…”