F. Malaisse-Lagae

ROLE OF MICROTUBULES IN THE PHASIC PATTERN OF INSULIN RELEASE

The release of insulin evoked by glucose and other insulinotropic agents in the pancreatic B-cell represents the outcome of a sequence of cellular events including the recognition of the secretagogue, the subsequent modification of cationic fluxes, and the eventual extrusion of secretory granules into the extracellular space.l Investigations on 45calcium net uptake, subcellular distribution, and efflux in isolated islets have led to the concept that, whatever the stimulatory agent used, the secretory response is invariably mediated through an accumulation of calcium in some critical site, possibly the cytosol of the B-cell.2-s This raises the question as to the link between the accumulation of calcium and the resulting exocytotic release of insulin. It has been proposed that such a link might be a collapse of the electrostatic potential energy barrier to granule/ membrane interactions0 Alternatively, it was suggested that calcium might trigger insulin secretion by activating a microtubular-microfilamentous system involved in the translocation and exocytosis of secretory granules.1o* l1 It is the aim of the present report to review the experimental data in support of the latter hypothesis, and to present a model for the participation of microtubules and microfilamentous structures in the phasic pattern of insulin release.

Dynamics of Insulin Release and Microtubular-Microfilamentous System. I. EFFECT OF CYTOCHALASIN B

In order to assess the participation of the microfilamentous cell web in the multiphasic response of the pancreatic beta cell, the effect of cytochalasin B upon both glucose- and sulfonylurea-induced insulin release was investigated in the perfused isolated pancreas. Cytochalasin B failed to affect the basal rate of insulin release, but enhanced the initial and later phases of insulin secretion in response to either glucose or gliclazide. In addition, cytochalasin B lowered the threshold concentration for the stimulant action of glucose upon insulin release. Ultrastructural studies supported the concept of a specific interaction of cytochalasin B with the microfilamentous cell web of the beta cell. It is concluded that the integrity of such a structure is equally important for both the initial and later secretory responses of the beta cell to various insulinotropic agents.

Exocrine pancreas: evidence for topographic partition of secretory function

The pattern of amylase, lipase, and chymotrpsinogen content found in pancreatic exocrine tissue surrounding the islets of Langerhans (periinsular halos) differs from that of the rest, or teleinsular part, of the pancreas. Such a topographic partition of secretory function may play a role in the regulation of pancreatic juice composition.

Dynamics of Insulin Release and Microtubular-Microfilamentous System

SummaryIn order to document the participation of microtubules in the dynamics of insulin release, the secretory response of the isolated perfused rat pancreas was measured after various times of exposure to vincristine (2.10−5M). After a short exposure time (25 min), both phases of glucose-induced insulin release were increased. After longer pretreatment (60 min), this facilitating effect disappeared and a slight, insignificant reduction of both phases of the secretory response to glucose was observed. A still longer exposure time (120 min) provoked a more marked and significant inhibition of the early and late phases of insulin release. The same enhancing effect after short pretreatment with vincristine was noticed when gliclazide was used as the insulinotropic agent. The ultrastructural studies indicated a progressive disappearance of microtubules concomitantly with an increase in number and size of vincristine-induced paracrystalline deposits. These findings suggest that microtubules indeed participate in the dynamics of insulin release, a reduction of both phases of insulin secretion being caused by an extended disruption of the microtubular apparatus, whereas a more limited disturbance of the microtubular system appears to be associated with facilitated insulin release in response to either glucose or sulfonylurea.

Dynamics of Insulin Release and Microtubular-microfilamentous System: VII. Do Microfilaments Provide the Motive Force for the Translocation and Extrusion of Beta Granules?

The active role played by beta-cell microfilamentous structures in the dynamics of insulin secretion was investigated by examining the influence of cytochalasin B upon various parameters of hormonal release by the isolated perfused rat pancreas. The view that the cytochalasin-induced changes in insulin release are due to a primary biophysical effect on microfilaments, rather than to an unrelated biochemical alteration of the beta-cell glucose-sensor device, was strengthened by the following observations: (1) the onset and disappearance of the cytochalasin B-induced facilitating action upon insulin release followed a time-course parallel to that characterizing the ultrastructural changes provoked by the drug in the distribution of beta-cell microfilamentous material; and (2) cytochalasin B facilitated leucine-induced insulin release in the presence of a very low glucose concentration. The mold metabolite was also found to transform transient secretory responses into biphasic ones and to prevent the reduction that normally affects the early response to insulinotropic agents when the pancreas is stimulated a few minutes after a prior and short exposure to glucose. The release of insulin evoked by either glucose or gliclazide was abolished in the absence of extracellular calcium, whether in the presence or absence of cytochalasin B. Theophylline and cytochalasin B exerted a synergistic effect upon glucose-induced insulin release. These data support the concept that calcium-dependent contractile events involving cytochalasin B-sensitive microfilamentous structures provide the motive force for both the intracellular translocation and exocytotic release of beta granules.

Dynamics of insulin release and microtubular-microfilamentous system: IV. Effect of colchicine upon sulphonylurea-induced insulin secretion

Abstract. The effect of colchicine upon glucose‐induced insulin release by the isolated perfused rat pancreas was examined in order to characterize the participation of the β‐cell microtubular apparatus in the biphasic insulin secretory response to glucose. After a short pretreatment (25 min.) with a low colchicine concentration (2.10−5 M), both the early and late phases of glucose‐induced insulin secretion were markedly enhanced. As either the concentration of colchicine or the length of the pretreatment period with this mitotic spindle‐inhibitor were increased, the facilitating effect faded out and partial inhibition of insulin release was observed. However, colchicine, even at high concentration, markedly enhanced the residual early release of insulin evoked by glucose in the presence of diazoxide (0.05 mg/ml). These findings suggest that (i) a fraction of the early phase of insulin secretion completely escapes from the inhibitory effect of colchicine and may correspond, therefore, to a modality of hormonal release which does not involve the oriented intracellular translocation of secretory granules; and (ii) extensive disruption of the microtubular apparatus is associated with inhibition of insulin release, especially during the late phase of the secretory response to glucose.

Effect of a local anesthetic upon calcium uptake and insulin secretion by isolated islets of langerhans

Abstract Tetracaine, a local anesthetic, inhibits the stimulant action of glucose upon both calcium uptake and insulin secretion by isolated islets of Langerhans. This finding is consistent with the concept that glucose-induced insulin release is triggered by an influx of calcium in the beta-cell.

Can desensitization of the B-cell to D-glucose be simulated in cultured pancreatic islets?

SummaryIn order to investigate the phenomenon of B-cell desensitization to D-glucose, rat pancreatic islets were cultured for 20–44h in the presence of increasing concentrations of D-glucose in the 5.6 to 27.8 mM range, and then incubated for 30 to 120 min for measurement of secretory, metabolic and ionic variables. After culture in the presence of 5.6 mM D-glucose, the release of insulin evoked by D-glucose (16.7 mM) was less marked than that seen in islets cultured in the presence of 11.1 mM D-glucose. In the latter islets, the secretory response to D-glucose (8.3 mM or more) was still modest, especially over short periods of incubation, but was markedly enhanced by either theophylline or forskolin. The release of insulin evoked by D-glucose in the presence of theophylline was little affected by either Ca2+ concentration of the culture medium or length of culture period (20hvs 44h). The culture-induced alteration in the responsiveness to D-glucose coincided with a smaller relative increase of D-[53H]glucose utilization, D-[U-14C]glucose oxidation or net45Ca uptake at increasing concentrations of the hexose. It contrasted with a well-preserved secretory response to nonnutrient secretagogues. Although these findings could be interpreted as evidence of B-cell desensitization to D-glucose, the fact that the secretory behavior of the islets was not vastly different whether they were first cultured at physiological (8.3 mM) or higher (11.1 to 27.8 mM) concentrations of D-glucose suggests that this experimental design may not be an optimal model for the functional alteration of the B-cell in hyperglycemic non-insulin-dependent diabetic subjects.

Reduced glutamate decarboxylase activity in rat islet β cells which survived streptozotocin-induced cytotoxicity

Rat pancreatic β cells exhibit a 16‐fold higher glutamate decarboxylase (GAD) activity than islet non‐β cells, but a similar glutamate dehydrogenase (GDH) activity, β Cells which survive exposure to 2 mM streptozotocin only contain 10 percent of the GAD activity of control cells, but their GDH activity remains unaltered. Culture of streptozotocin‐treated β cell preparations with 2 mM nicotinamide reduces the number of dead cells and prevents in part the decline in GAD activity of surviving β cells. These data indicate that loss in activity of the β cell specific enzyme GAD can serve as marker for β cells which survived a destructive process. It is furthermore demonstrated that nicotinamide increases the percent surviving cells and decreases their loss in GAD activity.