Lelio Orci

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.

Role of microtubules in the synthesis, conversion, and release of (pro)insulin. A biochemical and radioautographic study in rat islets.

In the pancreatic B cell, microtubules are thought to be involved in the process of insulin release. Their possible participation in the sequence of events leading from the biosynthesis and conversion of proinsulin to the release of newly synthesized insulin was investigated in rat isolated islets exposed to colchicine (0.1 mM). When the islets were preincubated for 30 min with colchicine and [3H]-leucine and, thereafter, incubated for two successive periods of 90 min each, still in the presence of colchicine, the release of preformed insulin was progressively inhibited and that of newly synthesized hormone delayed. When the islets were preincubated for 120 min with colchicine, subsequently pulse-labeled with [3H]leucine, and eventually examined by ultrastructural autoradiography, the export of newly synthesized proinsulin out of the rough endoplasmic reticulum, its transit through the Golgi complex, and its eventual packaging in secretory granules were all retarded. This situation was associated with a delayed conversion of proinsulin to insulin. Under the same experimental conditions, colchicine failed to affect the oxidation of glucose and adenylate charge in the islets. The effect of colchicine upon the release of preformed and newly synthesized insulin was not reproduced by lumicolchicine. It is concluded that colchicine interferes with the system controlling the intracellular transfer of secretory material from site of synthesis to site of release. This interference is likely to be linked to the effect of colchicine on microtubules.

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.