PARTX. MICROTUBULES

AND

HUMORAL SECRETION

ROLE OF MICROTUBULES IN THE PHASIC PATTERN OF INSULIN RELEASE * W. J. Malaise, F. Malaise-Lagae, E. Van Obberghen, G. Somers, G. Devis, M. Ravazzola, and L. Orci Laboratory of Experimental Medicine Brussels University Brussels, Belgium and Institutes of Histology and Embryology, and of Biochimie Clinique Geneva, Switzerland

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 45calciumnet 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. OF THE B-CELL THE ULTRASTRUCTURAL ORGANIZATION MICROTUBULAR-MICROFILAMENTOUS SYSTEM

In the B-cells of adult animals, microtubules have been recognized by several investigator~.~~-l~ As in other tissues, the microtubules (21-25 nm in diameter) display a hollow appearance, with a dense wall limiting a center of lower density, and are surrounded by a clear halo. The microtubules are scattered in the cytoplasm. They are often prominent in the ectoplasmic, paranuclear and Golgi areas (FIGURE1). The spatial organization of the B-cell microtubulcr apparatus is still poorly documented in several respects. First, it is unknown whether the microtubules are disposed in the B-cell to form an aster-like configuration, with microtubules radiating from the cytocenter to the cortex of

* The experimental work under review was supported in part by Grants 20001 from the FRSM (Brussels), 1180 from the FGWO (Brussels), and 3.8080.72, 3.0310.73 and 3.0280.73 from the FNRS (Bern). 630

FIGURE 1. Isolated rat islets. (a) Central region of a B-cell. Microtubules (arrows) are evident in the juxtanuclear and Golgi regions. N=nucleus; G=Golgi complex; sg=secretory granules. ( x I8,OOO) ( b ) The microfilamentous cell web is delineated by the dotted line. Microtubules are seen both in longitudinal (dotted arrows) and cross sections (solid arrows) ( x 84,000).

632

Annals New York Academy of Sciences

the cell, as described in some other cell types.lS-l7 Second, little is known concerning the close association between microtubules and secretory granules. Microtubules are occasionally seen in close proximity to secretory granules (FIGURE 2); but the possible existence of cross-bridges between these two organelles, comparable to those described between microtubules and synaptic vesicles lo remains to be fully assessed. Last, the detailed relationship between the microtubular apparatus and the microfilamentous cell web (see below)

FIGURE 2. Monolayer culture of neonatal rat pancreas. Several microtubules (arrows) are seen intermingled with and ending within the cell web (cw) ( x 81,000). In the insert, the clear halo surrounding a microtubule appears interrupted by short microfilamentous structures that seem to connect the denser part of the microtubule with a granule membrane ( x 73,000).

deserves further evaluation. These questions are of obvious interest in considering the possible role of microtubules as guiding elements for the translocation of secretory granules. The cell web occupies cytoplasmic areas of variable thickness (50-300 nm) 1). The characteristic component just beneath the plasma membrane (FIGURE of these areas, which extend into the core of microvillous processes, consists of a network of microfilaments, 4-7 nm in diameter, generally disposed to form irregularly shaped polygons (FIGURE 3). The cell web is virtually devoid of

Malaisse et al.: Phasic Pattern of Insulin Release

633

large cytoplasmic organelles, but may contain free ribosomes, profiles of endoplasmic reticulum, pinocytotic microvesicles and microtubules, as well as some secretory granules. .Individual microfilaments in the network may have an apparent insertion on the plasma membrane (FIGURE 3), microtubules, microvesicles and the surface of secretory granules, as if the cell web were to interconnect these different structures.20-22 In monolayer cultures of rat endocrine pancreas, an impressive increase in magnitude of the cell web and in the number of cytoplasmic microtubules (FIGURES 2-4) is enc~untered.'~In this model, &granules may be seen arranged in highly ordered rows and frequently interdigitating with microtubules and filaments coursing towards and ending in the cell web (FIGURE 4). It is tempting, in view of such an arrangement, to ascribe to the microtubularmicrofilamentous system a role in the translocation and release of secretory granules. Thus, the microtubules may serve as a guiding cytoskeleton for the oriented migration of secretory granules towards the cell surface, whereas the cell web may control the access of the secretory granules to sites of exocytosis at the plasma membrane.', 24-26

METABOLIC EFFECTSOF MICROTUBULAR-MICROFILAMENTOUS POISONS Most of our knowledge concerning the participation of the B-cell microtubular-microfilamentous system in the process of insulin release is based on the use of various drugs-cytochalasin B, vincristine, colchicine, D20-supposed to interfere with the structure and/or function of these organelles. It was important, therefore, to establish whether these drugs also affect early events in the secretory sequence, such as the recognition of insulinotropic agents and the subsequent changes in calcium handling by the B-cell. Neither D,O, vincristine, nor cytochalasin B exert any obvious effect upon the glucose-induced net uptake of *5calcium by isolated 27 The stimulant action of glucose upon calcium net uptake is thought to be due mainly to an inhibition of outward calcium transport across the B-cell membrane.O As illustrated in FIGURE 5, in the presence of either D20 or cytochalasin B, the immediate inhibitory effect of glucose on outward calcium transport displays its normal zR Likewise, the biphasic change in 45calcium efflux and the increase in 45calcium net uptake normally provoked by sulfonylurea are also observed in the presence of D20.5ys Lastly, in the presence of glucose, the addition of cytochalasin B to the perifusate does not exert any obvious influence upon 45calcium efflux from the perifused islets.2R These observations suggest that the alterations of insulin release observed in the presence of cytochalasin B, vincristine, and D 2 0 cannot be ascribed to any major abnormality in the handling of calcium by the B-cell. In other terms, the present data support the concept that microtubular-microfilamentous poisons modify insulin secretion by influencing the response to calcium rather than its provision to the secretory 30

The latter statement does not imply that all metabolic events in the B-cell are unaffected by these microtubular-microfilamentous poisons. As a matter of fact, untoward metabolic effects have indeed been reported. For instance, both D,O and vincristine were found to inhibit [*H]leucine incorporation in islet peptides, although the preferential stimulatory action of glucose on proinsulin biosynthesis was unaffected." In the absence of glucose, heavy water

FIGURE 4. Monolayer culture of neonatal rat pancreas. (a) Low power magnification of a B-cell. Secretory granules appear arranged in roughly parallel rows ( x 11,000). The area framed by the rectangle is shown at a higher magnification in b. (b) In the portion of cytoplasm delineated by two rows of secretory granules, a microtubule is indicated by the arrows ( x 41,000). FIGURE 3. Monolayer culture of neonatal rat pancreas. (a) The microfilamentous cell web is prominent ( x 25,000). The insert shows the network of filaments at high magnification. Microfilaments also fill the core of a microvillous process. The encircled area emphasizes the relationship of microfilaments with the inner leaflet of the plasma membrane (cm) ( x 103,000). (b) The arrows point to several profiles of microtubules in longitudinal section; sg=secretory granules ( x 28,000).

636

Annals New York Academy of Sciences

TIME (min)

FIGURE5. Effect of glucose upon "calcium efflux from perifused rat islets. Mean

values ( a SEM) for '%alcium efilux are expressed in percent of the mean value found within the same experiment between the 40th and 44th minute and refer to 4 individual experiments. The glucose concentration of the perifusate was raised at the The experiments were 45th minute (dotted line) from zero (Go)to 3.0 mg/ml (G3). performed at normal calcium concentration (NCa; 2 mEq/l) or in calcium-depleted media enriched with a calcium-chelating agent (aCa). Control data (left panels) are compared to those obtained either in media in which all &O was replaced by deuterium oxide (D20) or in the presence of cytochalasin B (Cy; 10 pg/ml).

Malaisse et al.: Phasic Pattern of Insulin Release

637

also interferes with the intracellular translocation of calcium normally evoked by t h e ~ p h y l l i n e . ~Lastly, ~ inhibition of glucose uptake and oxidation was recently observed during prolonged incubation (120 to 240 minutes) of isolated islets in the presence of cytochalasin B.32 A dose-dependent deleterious effect of cytochalasin B on islet glucose metabolism would explain why the facilitating action of the mold metabolite upon glucose-induced insulin release fades out as its concentration is raised u p to 50 pglml,?' and why inhibition of glucoseinduced secretion takes place when cytochalasin B is used at a 200 pglml con~entration.~3Anyhow, it should be stressed, as outlined in greater detail that none of these metabolic as distinct from biophysical abnormalities can account, on its own, for the changes in insulin release now to be considered. EFFECTOF CYTOCHALASIN B ON INSULIN RELEASE Although the participation in insulin secretion of the microfilamentous component of the B-cell translocator-releasing system 34 may appear somewhat beyond the scope of the present conference, it will nevertheless be briefly dealt with here, since, in our view, the microtubular-microfilamentous apparatus should be looked upon as an integrated functional unit. Cytochalasin B, which is said to interfere with the contractile function of microfilaments, was used in order to assess the possible role of B-cell microfilamentous structures in insulin secretion. Ultrastructurally, cytochalasin B, at a 10 pglml concentration provokes striking alterations of the cell web, which, instead of being disposed as a discrete band just beneath the plasma membrane, now appears in several places as large masses of closely packed short microfilaments having lost their prevailing characteristic polygonal arrangement. These masses extend far in the cytoplasmic space and may contain roundish clumps of densely packed material. Perhaps as a result of this reorganization of microfilaments, the peripheral region of B-cells exposed to cytochalasin B shows noticeable alterations; although the length and number of true microvillous processes decrease, there are frequent outward and inward irregular extensions of the plasma membrane, and also margination of secretory granules (FIGURE6) .20-?? In the perfused rat pancreas, some of these ultrastructural changes are already observed within 3 minutes of exposure to cytochalasin B.35 Most of the cytochalasin-induced microfilamentous masses apparently disappear within 5 minutes after removal of the mold metabolite from the perfusion 3G From the functional standpoint, cytochalasin B (10 pglml) was found to enhance insulin release in response to a variety of insulinotropic agents including glucose, leucine, sulfonylurea and the combination of glucose and theophylline.2112?,3?,37. 3S The magnitude of this facilitating effect, although somewhat variable, generally corresponds to a doubling of the control rate of secretion (FIGURE7 ) . The mold metabolite may also lower the threshold concentration for the stimulant action of glucose ? ? or leucine 32 upon insulin release. The facilitating effect of cytochalasin B is detected in the 1.0 to 20.0 pglml range and fades out at both lower and higher concentrations. Cytochalasin B enhances both the initial and late phase of insulin release in response to either glucose or sulfonylurea.22*3iIt tends to mask the decrease in the secretory response usually observed when the pancreas is exposed repeatedly to sul-

638

Annals New York Academy of Sciences

Malaisse et al.: Phasic Pattern of Insulin Release FIRST INCUBATION CONTROL

639

SECOND INCUBATION

h

CYTO B

... .. . i p ..................

COL VIN I I I

VIN + D20

I I I I I I

D20

I

0

50

100

150

200

INSULIN

50

100

150

200

OUTPUT (% of control)

FIGURE 7. Mean values (and SEM) for insulin output induced by glucose (16.7 mM) over two successive periods of incubation of 90 minutes each in islet tissue exposed to cytochalasin B (CYTO B), colchicine (COL), vincristine (VIN), and deuterium oxide (DzO) are shown as percent of the control rate of secretion. The data were selected to illustrate the sustained facilitating effect of cytochalasin B, the slowly induced and not immediately reversible inhibitory effect of colchicine and vincristine, the protective effect of D20 against vincristine, and the rebound phenomenon observed after the DaO-induced inhibition of release. More detailed data, including dose-action relationships and other combinations of the microtubular-microfilamentous poisons are given elsewhere (see references 21 and 27).

f ~ n y l u r e a .The ~ ~ facilitating effect of cytochalasin B is rapidly reversible.?17 37 It also takes place within less than 1-2 minutes. Thus, in the isolated perfused rat pancreas, when cytochalasin B is introduced in the perfusate during the second phase of the secretory response to glucose, it produces both a short-lived secretory peak superimposed on the progressively increasing pattern of insulin secretion otherwise observed at that time, and a marked enhancement of the subsequent and steadily increasing rates of secreti0n.3~9d o These ultrastructural and functional findings have been interpreted to suggest that microfilamentous structures, especially the cell web, control the access of secretory granules to the cell membrane. More precisely, it was suggested that the cell web may act as a sphincter, being both a barrier to and an effector of exocytotic insulin release.?O*91 3 j u

FIGURE 6. Effect of cytochalasin B upon the B-cell. (a) Perfused rat pancreas exposed to cytochalasin B (10 pg/ml) for 60 minutes. A microfilamentous mass (delineated by the dotted line) extends far into the cytoplasm ( x 33,000). ( b ) Monolayer culture of neonatal rat pancreas exposed to cytochalasin B. The cell surface displays outward irregular cytoplasmic expansions containing secretory granules (x

25,000).

Annals New York Academy of Sciences

640

EFFECTOF COLCHICINE AND VINCRISTINE ON INSULINRELEASE

Colchicine or vincristine provoke a reduction in the number of cytoplasmic microtubules in the B-cell.l39 4 2 In the case of colchicine, the remaining microtubules often seem altered: they appear more dense, having lost their normal hollow appearance, and are often deprived of their clear h a l 0 . ~ ~ 9 " ~ Vincristine, in addition to causing the disappearance of microtubules, characteristically provokes a massive precipitation of crystalline-like material presumably derived from the microtubular protein (FIGURE8). l5,4 z 4~3 As illustrated in TABLE 1, these effects of vincristine (2.104 M ) d o not immediately reach their full magnitude, but take place progressively during exposure to the mitotic spindle-inhibitor.l5I 4 2 In the B-cell, there does not appear to exist any preferential topographical location of the vincristine-induced deposits. No other obvious deleterious effect could be disclosed, even after prolonged exposure (120 minutes) of islet tissue to high concentrations (10-4-l@3M) of colchicine.l5,39, 4 1 , 4 2 TABLE 1

EFFECTOF VlNCRISTlNE

UPON

MICROTUBULES IN B-CELLS *

Exposure Time to Vincristine

Microtubules Frequency

Nil 25 to 30 min 135 min

47.3k3.1

-

16.520.9

7.9f1.4 18.7f1.5

4.220.6

Paracrystalline Deposits Volume Density (

* Isolated perfused rat pancreases were exposed to vincristine (2.10-6 M). Mean values (k SEM) for 10 individual measurements refer to the frequency of microtubular profiles (each individual value represents the number of microtubular profiles, excluding transverse sections, in 5 randomly taken micrographs from the same islet representing a total cytoplasmic area of approximately 80 p') and to the volume density of paracrystalline deposits in the cytoplasm (as determined by point counting in the same micrographs). The major functional effect of mitotic spindle-inhibitors is to inhibit insulin release in response to glucose or the combination of glucose and theophylline (FIGURE 7) lz?27 The inhibition of glucose-induced insulin release has been documented in various in vitro systems including the incubation of pieces of p a n c r e a ~ ,27 ~ ,the incubation 12,2 7 or perfusion 4 4 of isolated islets, the perifusion of the isolated pancreas 42, 45 and the incubation of monolayer cultures of pancreatic endocrine cells.4s The degree of inhibition increases as a function of the time of exposure to colchicine or vincristine prior to stimulation with glucose.27.41s 4 2 For a given time of exposure, the degree of inhibition is to related to the concentration of colchicine in the M range (FIGURE 9) 41 These findings are thought to reflect the dose- and time-related disorganization of the microtubular apparatus and, as such, support the view that an extended disruption of the latter system is incompatible with the maintenance of a normal secretory response to glucose. Incidentally, the inhibitory effect of mitotic spindle-inhibitors does not appear to be rapidly reversible, since after .49

413

.27p

Malaisse et al.: Phasic Pattern of Insulin Release

641

FIGURE8. Isolated rat islet exposed to vincristine (2.10.' M ) for 40 minutes. Peripheral portions of two adjacent B-cells. Two vincristine-induced paracrystalline deposits are seen surrounded by secretory granules and cisternae of the rough endoplasmic reticulum ( x 46,000).

642

Annals

New York

Academy of Sciences

pretreatment for 90 minutes with vincristine, the rate of glucose-induced insulin release over the ensuing 90 minutes is equally depressed whether measured in the presence or absence of vincristine.zi A more detailed insight into the effects of mitotic spindle-inhibitors upon insulin release was recently gained from studies performed with the isolated perfused rat p a n c r e a ~41, . ~4~2 - ~4iThese investigations have revealed that, at least in this system, a short exposure (25 minutes) to a low concentration (2.10-5 M) of either colchicine or vincristine produced a facilitation of both the early and late phases of glucose-induced insulin 4 2 Although no satisfactory explanation can yet be given for such an unexpected facilitating effect, the dual action of mitotic spindle-inhibitors was used to investigate further the participation of the microtubular apparatus in the multiphasic pattern of insulin release in response to glucose stimulation. Data were obtained that

n

&? 100

W

c

z W z

B5

0 0 W

75

50

c a

T

d

a

25

3

G

u)

w

a

FIGURE9. Effect of vincristine (shaded columns) and colchicine (open columns) upon glucoseinduced insulin release by the isolated perfused rat pancreas. The residual mean values for insulin release are shown as percent of their respective mean control values. The data refer to the late component of insulin release and were derived from the experimental values after correction for the facilitating action of mitotic spindleinhibitors (see reference 47). Also shown are the concentration of and time of exposure to the inhibitors, prior to stimulation with glucose.

0 CONCENTRATION (M)

indicate that (1) a n underlying facilitating effect is invariably present whatever the time of exposure to and concentration of either colchicine or vincristine used prior to stimulation, and (2) the concomitant inhibition of insulin release, which under suitable experimental conditions is sufficiently pronounced to become the dominant feature, only affects that component of the secretory response that is responsible for the late and progressive build-up in secretion rate normally seen during prolonged stimulation by glucose.4i In contrast, the early component of the secretory response, characteristic of the immediate peakshaped release of insulin, is not susceptible to inhibition by the mitotic spindleinhibitors. This behavior explains why the short-lived secretory response evoked by sulfonylureas in the absence of glucose or at low glucose concentration is not inhibited but actually enhanced by either colchicine or v i n c r i ~ t i n e .4~2 ,~l,i It also implies that a net reduction in the integrated amount of insulin released

Malaisse et al.: Phasic Pattern of Insulin Release

643

over a prolonged period of incubation is only observed under those conditions normally associated with a sustained and high rate of secretion (such as that induced by glucose at high concentration) and an extended disruption of the microtubular apparatus (such as that observed after a long pretreatment with mitotic spindle-inhibitors) . These considerations were taken into account in designing our model for the phasic pattern of insulin release, to be outlined later in this report (see AN INTEGRATED MODELFOR THE PARTICIPATION OF THE B-CELL MICROTUBULAR-MICROFILAMENTOUS SYSTEM IN INSULIN RELEASE).

EFFECT OF DEUTERIUM OXIDE

ON

INSULIN RELEASE

Eflect of D,O Alone

Exposure of islet tissue to D 2 0 apparently increases the number of microtubules identified in the B-cell. With the exception of a moderate dilatation of the cisternae of the endoplasmic reticulum and the presence of autophagosomes enclosing secretory granules, no other obvious deleterious effect of D20 is obser~ed.3,15, 2 5 , 36 36, ** leucine Heavy water inhibits insulin release in response to and sulfonylurea.5p 4 L In all cases, the degree of inhibition (expressed in percent) is approximately the same as the relative concentration of D,O (also expressed in percent, v/v), complete or almost complete inhibition being obtained when all H,O is replaced by D20.?' Deuterium oxide abolishes both the early and late phases of insulin release in response to either glucose or sulfonylurea.30p4 1 The inhibitory effect of D?O is an immediate phenomenon; 4 4 it is also immediately reversible,"* - ( I a rebound in secretory rate being generally observed when the deuteriated medium is removed (FIGURE 7) 36 Other microtubule-stabilizers, such as 2-methyl-2, 4-pentanediol and ethanol mimic the inhibitory effect of D,O.?' It would appear from these ultrastructural and functional data that D,O abolishes insulin release by overstabilizing the microtubular 2i The rebound phenomenon suggests that cellular events normally leading to insulin release (e.g. glucose metabolism and calcium accumulation in the B-cell) still occur during D 2 0 treatment, so that the B-cell is primed to release large amounts of insulin on relief from the D,O-induced blockade of the microtubular That the inhibitory effect of D?O is somewhat more complex will be shown later, when considering the combined influence of D20 and cytochalasin B upon insulin release (see Cornbined Effecf of D,O and Cytochalasin B ) . 3B9

Cornbined Effects of D 2 0 arid Mitotic Spindle-Inhibitors

In certain systems, D 2 0 protects the microtubular system against the deleterious effect of mitotic spindle-inhibitors..4sr When exposed to both D,O and vincristine, the B-cell contains vincristine-induced microcrystalline deposits, but microtubules can still be visualized.13 Under these conditions, namely after prior exposure for 90 minutes to both D,O (50 or loo%, v/v) and a mitotic spindle-inhibitor (colchicine M; vincristine 3 . 1 W to 4.10-" M ) , pieces of pancreatic tissue transferred to normal media containing no D,O secrete in

FIGURE10. Perfused rat pancreas exposed to cytochalasin B for the first 20 minutes and to both cytochalasin B and D1Ofrom the 21st to 60th minute. The glucose concentration of the perfusate was raised from 0.3 to 3.0 mg/ml at the 45th minute

Malaisse et al. : Phasic Pattern of * Insulin Release

645

response to glucose significantly more insulin over the ensuing 90 minutes than do pieces first exposed to colchicine or vincristine alone, the rate of secretion being-under suitable experimental conditions-identical to that found in pieces incubated throughout in normal media (FIGURE 7) .27 Such a protective influence of D,O against the inhibitory action of mitotic spindle-inhibitors is compatible with the concept of a microtubule-stabilizing effect of D20 in the B-cell. Combined Effect of D,O and Cytochalasin B

There is an apparent discrepancy between the results obtained after prolonged pretreatment with colchicinc and those collected during exposure to D,O, respectively. I n the first case, the early component of insulin release is not inhibited despite extensive disruption of the microtubular apparatus. I n the second case, both phases of insulin release are abolished, a suppression so far ascribed to the overstabilization of microtubules. A possible explanation for these findings could be that D,O, in addition to overstabilizing the microtubular apparatus, also impairs the ability of microfilamentous structures to generate the motive force for the release of secretory granules. In order to test such a hypothesis, we have recently examined the combined effect of D,O and cytochalasin B upon insulin release by the isolated perfused rat pancreas.36 When pancreases were fixed during concomitant exposure to D,O (loo%, v/v) and cytochalasin B (10 pg/ml), the ultrastructural changes normally induced by either of these agents, when given alone, were both encountered. Thus, on one hand, there was an apparent increase in the number of microtubules probably due to the exposure to D,O. O n the other hand, the typical cytochalasin-induced alterations of the cell web were also noticed, including the shortening of microvillous processes and the appearance of large filamentous masses (FIGURE 10) As far as insulin release is concerned, when the perfused pancreas was first exposed for 25 minutes to D,O and, thereafter, to both D,O and cytochalasin B, the secretory response to either glucose or glucazide (introduced at the 35th minute) remained abolished, as would be the case if D,O alone had been administered throughout (FIGURE 11) .RG This finding confirms a prior observation performed in isolated islets.21 In contrast, when the pancreas was first exposed for 20 minutes to cytochalasin B alone and, thereafter, to both cytochalasin B and D,O, an escape phenomenon from the D,O-induced inhibition was invariably observed, as illustrated in FIGURE 11. Under these experimental conditions, the secretory response, which was proved not to be due to a leakage phenomenon, occurred with an abnormally long delay after the addition of the insulinotropic agent to the perfusate, and was characterized by rates of insulin release much lower than those normally observed in this system. That cytochalasin B is responsible for the escape phenomenon suggests that D,O itself may interfere with the microfilaments’ function. However, the B-cell was still 11 upper and right panel). and the pancreas fixed at the 60th minute (see FIGURE (a) Several microtubular profiles (arrows) and a dense mass (asterisk) are present in the same B-cell (x 35,000). ( b ) The arrows point at secretory granules at different stages of extrusion in the extracellular space (x 23,000). (c) A microtubular profile in cross section (arrows) is enclosed within a cytochalasin-induced dense mass ( x 76,000).

Annals N6w York Academy of Sciences

646 160r

-

126

100.

.

76

c 60.

z

\

3

2

26-

'.l t o 5 0

- 1 '

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26

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30

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' 40

' 46

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0

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20

C

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40

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46

00

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66

00

66

10

3

g

cr-D@-

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,+-OLUCOSE+ I

- 1 . :

0

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26

30

36

40

' 46

FIGURE11. Effect of D20 alone (loo%, v/v) and combinations of D20and cytochalasin B (10 pg/ml) upon insulin release evoked by glucose (16.7 mM) in the isolated perfused rat pancreas. Mean values refer to 3 to 17 individual experiments (see reference 36). able to perform the elaborate sequence of molecular events associated with insulin release despite prolonged exposure to D,O. Incidentally, under these as well as other experimental conditions so far examined for this purpose, there was a satisfactory correlation between the stimulation or arrest of insulin release and the presence or absence of emiocytotic figures in the B-cell.??,3G The present ultrastructural and functional observations suggest that microtubular-microfilamentous structures, which are apparently affected by both cytochalasin B and D,O, actively participate in both phases of insulin release, so that a functional paralyzation of these structures by D20, when given alone, results in the suppression of the secretory process.

AN INTEGRATED MODELFOR THE PARTICIPATION OF THE B-CELL MICROTUBULAR-MICROFILAMENTOUS SYSTEM IN INSULIN RELEASE Using the above-mentioned findings as basis, we wish to propose the following scheme concerning the participation of the B-cell microtubular-microfilamentous system in insulin secretion. The release of insulin during both phases

Malaisse et al.: Phasic Pattern of Insulin Release

647

of the secretory process is dependent and apparently entirely dependent on the functional integrity of the microtubular-microfilamentous system taken as a whole (see EFFECTOF DEUTERIUM OXIDEON INSULINRELEASE).The characteristic early secretory response does not depend on the presence of an intact microtubular apparatus (see EFFECT OF COLCHICINE AND VINCRISTINE ON INSULIN RELEASE) ; it could, therefore, correspond to the mobilization of secretory granules that are already located in close vicinity to the cell web/plasma membrane complex. In contrast, the progressive build-up in secretory rate seen during the second phase of glucose-induced insulin release appears to require an intact microtubular apparatus (see EFFECTOF COLCHICINE AND VINCRISTINE ON INSULINRELEASE) ; it could thus correspond to the mobilization of secretory granules transported to the periphery of the B-cell along oriented microtubular pathways. In both cases, the final access of secretory granules to the cell membrane and their extrusion into the extracellular space appear to be placed under the control of the microfilamentous cell web (see EFFECTOF CYTOCHALASIN B ON INSULINRELEASE). These propositions are graphically illustrated in FIGURE

12.

Although the model depicted in FIGURE12 may well be naive and oversimplified, it is nevertheless able to account not only for the experimental data so far outlined, but also for other aspects of the insulin secretory process not yet touched upon in this review.

FIGURE12. Schematic representation of the relationship between secretory granules, microtubules and the microfilamentous cell web. In the unstimulated B-cell, the granules are kept away from the plasma membrane by the cell web (A). During the first phase of the secretory response (B), the release of insulin corresponds, in part, to the extrusion of granules that were already located in close vicinity to the cell web/plasma membrane complex (early component). During the later phase (C),the release of insulin is largely dependent on the provision of secretory granules transported to the cell web along microtubular pathways (late component). Data for insulin output are expressed in percent of the total amount of insulin released over the entire period of stimulation (see reference 47).

Id

I

LATE COYWNENT

:,L/

TOTAL OUTPUT

i

s ;

b

T I M E (mln)

il

b

1;

648

Annals New York Academy of Sciences The Functional Segregation o f Secretory Granules in the B-cell

One of the implications of our model is the existence of a functional segregation of secretory granules into two separate pools within the B-cell. Grodsky et al.6°-52 were the first to indicate that the biphasic insulin secretory response of the isolated perfused rat pancreas to glucose stimulation could indeed be accounted for by such a compartmentation of granules into a small pool particularly labile to stimulatory agents and a larger and more stable storage pool. Although the validity of such a two-compartmental storage system has been assessed under a variety of experimental conditions, little was known so far of the ultrastructural or biochemical basis for the existence of two separate pools of insulin secretory granules. The present model may now explain why the monophasic and exponentiallike accumulatjon of cytosolic calcium thought to be induced by glucose in the Btell 5 3 eventually causes a biphasic secretory response. Indeed, in our model, the factors responsible for such a biphasicity take place in the secretory sequence at or beyond the site of action of Incidentally, in view of the postulated dual modality for the mobilization of secretory granules, it is conceivable that inherited or acquired abnormalities at either the microfilamentous or microtubular level are responsible for concomitant alterations in the secretory responsiveness of the B-cell. For instance, it was suggested, as a provocative hypothesis, that the excessive insulin response to various stimuli often seen in patients with myotonic dystrophy 54-5i could be due to an abnormality of the B-cell microfilamentous system comparable to that experimentally induced by cytochalasin B.?' Besides, the delayed build-up in glucose-induced secretory rate observed in spiny mice 5s-oo could well be due, in part at least, to the presence of an abnormally low amount of microtubular protein in the B-cells of these diabetes-prone rodents."' The Motive Forces for the Translocation-Release Process

A second implication of our model is that the microfilamentous cell web provides the motive force for the ultimate access of secretory granules to the plasma membrane and their extrusion in the extracellular space. In other terms, we wish to attribute to the cell web a contractile capacity. Such a view is consistent with the two following series of observations. First, and as outlined in greater detail elsewhere,43there are a number of analogies between the process of stimulus-secretion coupling in the B-cell and excitation-contraction coupling in muscle: for instance, both processes are ATP-dependent,62 associated with cell dep~larization,~~ and apparently triggered by a cytosolic accumulation of calcium.2e Second, the cell web is indeed formed by a network of actin-like microfilaments (see THE ULTRASTRUCTURAL ORGANIZATION OF THE B-CELLMICROTUBULAR-MICROFILAMENTOUS SYSTEM).Moreover, by use of an immunofluorescent technique, actin was recently found in the B-cell, with an apparently higher concentration in the peripheral part of the The motive force responsible for the movements of secretory granules along the microtubular pathway is as yet unknown. At least the following hypotheses should be considered. First, and as suggested in a preliminary granules could undergo back-and-forth saltatory movements along microtubular pathways. According to current theories concerning such movements,Gca minor

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number of microfilaments physically linking secretory granules with microtubules would be sufficient for development of the motive force needed to propel particles in saltatory motion. Second, the translocation of secretory granules may be driven by tubule assembly at an initiating site in the cytocenter and concurrent depolymerization into tubule subunits at the cell periphery.lg Lastly, since cytochalasin B also enhances the second phase of glucose-induced insulin release, the possibility should be kept in mind that contractile events taking place at the level of the microfilamentous cell web may affect the cytoplasmic streaming along and/or the spatial distribution of the microtubular apparatusto which the cell web appears to be normally anchored-and, in doing so, may influence the progression of secretory granules.35

The Link Between Motile Events and Insulin Release in the B-cell If our contention is correct that the intracellular transport and eventual exocytosis of secretory granules is entirely dependent on the generation of appropriate motive forces, there should exist a close link between the nature and intensity of motile events in the B-cell and the concomitant pattern of insulin release. In order to test the latter hypothesis, we have recently initiated a study of monolayer cultures of endocrine pancreatic cells examined by timelapse This work is still in progress. The data so far obtained indicate that both the intracellular movement of particulate structures and the motile activity of the cell membrane are indeed influenced, qualitatively and quantitatively, by factors known to affect the secretory activity of the B-cell, such as glucose and other insulinotropic agents, cationic factors and microtubular-microfilamentous poisons. These findings convincingly support the idea that motile events placed under the control of the microtubular-microfilamentous system are intimately involved in the secretory function of the B-cell. REFERENCES 1. MALAISE,W. J. 1973. Insulin secretion: multifactorial regulation for a shgle

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