Ghislain Devis

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.

REGULATION OF CALCIUM FLUXES AND THEIR REGULATORY ROLES IN PANCREATIC ISLETS

It is today considered as crystal clear that calcium plays an essential role in the regulation of insulin release by the pancreatic B-cell. Some of the major issues concerning such a role are as follows: ( i ) what is the detailed mechanism by which secretagogues are susceptible to influence the handling of calcium in the B-cell; (ii) what is the nature and location of the critical pool of calcium that controls insulin release; (iii) what is the relative and respective contribution of calcium influx, efflux, and subcellar distribution in the regulation of such a pool; and (iv) how does calcium influence the process by which secretory granules migrate to the cell boundary and are extruded via exocytosis in the interstitial fluid.’ In the present report, for the sake of clarity, we will restrict the discussion of these questions to the process of glucose-induced insulin release, with the main emphasis on the possible significance of passive ionophoretic movements. The process by which glucose provokes insulin release can be viewed as a sequence of three major events, namely: ( i ) the recognition or identification of glucose by the B-cell; (ii) the subsequent remodelling of cationic fluxes; and (iii) the activation by calcium of an effector system controlling the migration and exocytosis of secretory granules.2 The role of calcium in insulin release is here considered within the framework of such a sequential view.

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.

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.

Stimulation of insulin release by calcium

Abstract In the absence of secretagogue, Ca 2+ (2 to 10 mM) provokes a short-lived release of insulin in the perfused rat pancreas first exposed to EGTA. The secretory response is abolished by verapamil and enhanced by theophylline. These findings afford the first demonstration that Ca 2+ itself can trigger insulin release.

The stimulus-secretion coupling of glucose-induced insulin release. Metabolic and functional effects of NH4+ in rat islets.

NH4+ caused a dose-related, rapid, and reversible inhibition of glucose-stimulated insulin release by isolated rat islets. It also inhibited glyceraldehyde-, Ba2+-, and sulfonylurea-stimulated insulun secretion. NH4+ failed to affect glucose utilization and oxidation, glucose-stimulated proinsulin biosynthesis, the concentration of ATP, AD, and AMP, and the intracellular pH. NH4+ also failed to affect the ability of theophylline and cytochalasin B to augment glucose-induced insulin release. However, in the presence and absence of glucose, accumulation of NH4+ in islet cells was associated with a fall in the concentration of NADH and HADPH and a concomitant alteration of 86Rb+ and 45Ca2+ (or 133Ba2+) handling. These findings suggest that reduced pyridine nucleotides, generated by the metabolism of endogenous of exogenous nutrients, may modulate ionophoretic processes in the islet cells and by doing so, affect the net uptake of Ca2+ and subsequent release of insulin.

The stimulus secretion coupling of glucose-induced insulin release.

In isolated rat pancreatic islets, exogenous l-lactate causes a dose-related enhancement of glucose-induced insulin release and shifts the sigmoidal curve relating insulin output to ambient glucose concentrations to the left. l-Lactate also enhances α-ketoisocaproate-induced insulin release and glucose-induced proinsulin biosynthesis. l-Lactate rapidly accumulates in the islet cells, is converted to pyruvate and CO2, and raises the intracellular concentration of both ATP and NAD(P)H. On a molar basis, the insulinotropic capacity of nutrients ranges as follows d-glucose ⪢ l-lactate > pyruvate = d/l-lactate > d-lactate and does not correlate with their respective oxidation rates. However, when allowance is made for the intracellular interconversion of these exogenous nutrients, for their reciprocal influence upon oxidation rates, and for their sparing action on the utilization of endogenous fuels, a close correlation is found between the aptitude of glucose, l-lactate, and pyruvate to generate reducing equivalents and to stimulate insulin release. It is proposed that the concentration of NAD(P)H in islet cells affects the ionophoretic fluxes of cations (K+, Ca2+) across membrane systems and, hence, regulates the net uptake of Ca2+ and subsequent release of insulin. The effect of l-lactate upon Ca2+ handling is sufficiently rapid to account for the immediate secretory response to this nutrient.

The stimulus -secretion coupling of glucose-induced insulin release. XIX. The insulinotropic effect of glyceraldehyde

Glyceraldehyde is known to stimulate insulin release. Its influence on various parameters of islet function was investigated in order to assess the possible significance of glycolsis in the insulinotropic action of glucose. In the absence of glucose, glyceraldehyde (5-20 mM), but neither dihydroxyacetone nor glycerol stimulated insulin release in rat isolated islets. The glucose-like effect glyceraldehyde (10 mM) was characterized by a shift to the left of the curve relating insulin release to glucose concentration, without any significant increase in the maximal velocity of the secretory process. In the isolated perfused rat pancreas, glyceraldehyde provoked a biphasic secretory response. Glyceraldehyde-induced insulin release was inhibited in the absence of calcium or in the presence of epinephrine, unaffected by mannoheptulose or 3,3-tetramethyleneglutaric acid, and enhanced by theophylline and cytochalism B. Glyceraldehyde also stimulated to pro-insulin biosynthesis and 45Ca net uptake by isolated islets, the latter effect being apparently due, in part at least, to inhibition of calcium outward transport across the cell membrane. At concentrations of nearly equivalent insulinotropic potency, glucose and glyceraldehyde were metabolized at rates yielding comparable output of both lactate and 14CO2. The data indicate that glyceraldehyde mimics many effects of glucose on islet function, suggesting that the insulinotropic action of glucose may be related to its metabolism through the glycolytic pathway.

Calcium-antagonists and islet function. IV. Effect of D600.

SummaryD600 (2 to 20 μM;α-isopropyl-α [(N-methyl-N-homoveratril)-γ-aminopropyl]-3,4,5 -trimethoxyphenyl-acetonitril) caused a dose-related, rapid and reversible inhibition of glucose-induced insulin release. It also suppressed the insulinotropic action of a sulphonylurea but failed to affect the enhancing action of theophylline upon glucose-induced release. The inhibitory effect of D600 was enhanced at low extracellular Ca2+ concentration. D600 reduced both basal and glucose-stimulated45calcium net uptake, whilst failing to affect the efflux of45calcium from perifused islets. The recognition of glucose by the B-cell was also unaffected by D600 as judged by the effect of the sugar upon both 45 calcium efflux and net uptake in the isolated islets. These findings are compatible with the hypothesis that the primary mode of action of D600 is to inhibit Ca2+entry in the B-cell.

The stimulus-secretion coupling of glucose-induced insulin release. XLV. The anion-osmotic hypothesis for exocytosis.

Replacement of extracellular chloride by the impermeant anion isethionate, increase in extracellular osmotic strength by addition of sucrose, or exposure to the anion transport blocking agent probenecid inhibited insulin release evoked by glucose or α-ketoisocaproate in rat isolated islets. The inhibition of insulin release due to Cl− substitution was associated with a modest decrease in glucose oxidation, but no significant change in glucose-stimulated45Ca net uptake by the islets. In the isolated perfused rat pancreas, the isethionate- or sucrose-induced inhibition of insulin release was a rapid and rapidly reversible phenomenon. Chloride substitution by isethionate inhibited more severely the second than the first phase of the secretory response to glucose, and failed to affect the insulin response to gliclazide. A chemosmotic mechanism for exocytosis, as proposed for epinephrine and parathyroid hormone release, may also be involved in insulin release.