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Evidence Against the Presence of Tight Junctions in Normal Endocrine Pancreas P. A. in't VELD, D. G. PIPELEERS, AND W. GEPTS SUMMARY Tight junctional fibrils were absent in freeze-fracture replicas of rat and human islets in situ, but were easily discerned in collagenase-isolated islets. Disruption of the pancreatic gland and its exposure to trypsin were each found to induce tight junction formation in rat is- lets. The amount of tight junctions between islet cells declined progressively during culture, but tight junc- tional structures remained detectable after 1 day of culture. It is suggested that rather than being involved in normal islet cell function tight junctions provide an adaptive mechanism intended to seal and hence to protect islet microdomains against sudden perturba- tions in local interstitial fluid. DIABETES 33:101-104, January 1984. T ight junctions are membrane specializations that represent foci of plasma membrane fusion between adjacent cells and are recognized as fibrils on freeze-fracture replicas of membrane P-faces. 1 They have been mainly identified between cells that separate body fluid compartments of different chemical composition, where they probably help maintain compartmentalization in sealing off the intercellular spaces. Such a role has been extensively supported by in vitro experiments; wherein tight junctions were acting as diffusion barriers in the extracellular space, as well as in the plane of the membrane. 23 Tight junctions have also been described in isolated islets of Langerhans where they occur between homologous and heterologous cells. 4 6 The amount of these structures was altered by exposing islets to enzymes or to glucose, but it remains unclear whether tight junctions participate in islet cell function. 79 In a more recent report Kawai et al. sug- From the Department of Pathology (P.A.V., W.G.) and the Department of Me- tabolism and Endocrinology (D.G.P.), Vrije Universiteit Brussel, Brussels, Bel- gium. Address reprint requests to Dr. P. A. in't Veld, Department of Pathology, A.Z. - V.U.B., Laarbeeklaan 101, B-1090 Brussels, Belgium. Received for publication 4 October 1983. gested that tight junctions might create compartments within the in vivo pancreatic islet and could thus delineate islet microdomains that determine the degree of paracrine com- munication. 10 Such a hypothesis assumes the existence of tight junctions in the in vivo endocrine pancreas, which is, so far, merely an assumption. We therefore investigated the occurrence of these membrane structures in freeze-fracture replicas of islets in vivo; as we recently observed that the islet isolation procedure itself decreased the amount of gap junctional structures between islet cells, 11 we also compared the values obtained in situ with those after islet isolation and culture. MATERIALS AND METHODS Studies on islets in situ. All rat experiments were carried out on fed adult Sprague-Dawley rats (males, 250-300 g). The rat pancreas was perfusion fixed through the aorta using 2% glutaraldehyde in 0.1 M cacodylate, pH 7.35, for 5 min, followed by a 5-min perfusion with the same fixative containing 0.12 mM dithizone (Merck, West Germany). A dithizone stock-solution was prepared by dissolving 10 mg dithizone in 3 ml ethanol (99%) and adding three drops 25% ammonia in water; 12 dithizone treatment results in a bright red staining of the islets. In control experiments, perfusions were performed without dithizone and islets were examined after random fracturing of glutaraldehyde-fixed pancreata. Human islets were examined under similar conditions. Pancreatic tissue was obtained from a brain-dead kidney donor, and tissue blocks were microperfused with glutaral- dehyde and dithizone; islets were microdissected and freeze fractured. Rat islets in situ were also examined after exposure to agents known to affect pancreatic exocrine or endocrine function. The agents were administered via a left ventricle perfusion of nembutal-anesthetized rats; the medium con- sisted of Earle's salts and 2.8 mM glucose, was buffered with 10 mM Hepes and 14.3 mM bicarbonate (pH 7.35 at 37°C), and supplemented with 0.2% albumin (BSA, fraction V; Sigma, St. Louis, Missouri), 1 mg/ml procaine, and 1 U/ ml heparin. Experimental conditions included addition of 25 DIABETES, VOL. 33, JANUARY 1984 101

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Evidence Against the Presence ofTight Junctions inNormal Endocrine PancreasP. A. in't VELD, D. G. PIPELEERS, AND W. GEPTS

SUMMARYTight junctional fibrils were absent in freeze-fracturereplicas of rat and human islets in situ, but were easilydiscerned in collagenase-isolated islets. Disruption ofthe pancreatic gland and its exposure to trypsin wereeach found to induce tight junction formation in rat is-lets. The amount of tight junctions between islet cellsdeclined progressively during culture, but tight junc-tional structures remained detectable after 1 day ofculture. It is suggested that rather than being involvedin normal islet cell function tight junctions provide anadaptive mechanism intended to seal and hence toprotect islet microdomains against sudden perturba-tions in local interstitial fluid. DIABETES 33:101-104,January 1984.

Tight junctions are membrane specializations thatrepresent foci of plasma membrane fusion betweenadjacent cells and are recognized as fibrils onfreeze-fracture replicas of membrane P-faces.1

They have been mainly identified between cells that separatebody fluid compartments of different chemical composition,where they probably help maintain compartmentalization insealing off the intercellular spaces. Such a role has beenextensively supported by in vitro experiments; wherein tightjunctions were acting as diffusion barriers in the extracellularspace, as well as in the plane of the membrane.23

Tight junctions have also been described in isolated isletsof Langerhans where they occur between homologous andheterologous cells.4 6 The amount of these structures wasaltered by exposing islets to enzymes or to glucose, but itremains unclear whether tight junctions participate in isletcell function.79 In a more recent report Kawai et al. sug-

From the Department of Pathology (P.A.V., W.G.) and the Department of Me-tabolism and Endocrinology (D.G.P.), Vrije Universiteit Brussel, Brussels, Bel-gium.Address reprint requests to Dr. P. A. in't Veld, Department of Pathology,A.Z. - V.U.B., Laarbeeklaan 101, B-1090 Brussels, Belgium.Received for publication 4 October 1983.

gested that tight junctions might create compartments withinthe in vivo pancreatic islet and could thus delineate isletmicrodomains that determine the degree of paracrine com-munication.10 Such a hypothesis assumes the existence oftight junctions in the in vivo endocrine pancreas, which is,so far, merely an assumption. We therefore investigated theoccurrence of these membrane structures in freeze-fracturereplicas of islets in vivo; as we recently observed that theislet isolation procedure itself decreased the amount of gapjunctional structures between islet cells,11 we also comparedthe values obtained in situ with those after islet isolation andculture.

MATERIALS AND METHODSStudies on islets in situ. All rat experiments were carriedout on fed adult Sprague-Dawley rats (males, 250-300 g).

The rat pancreas was perfusion fixed through the aortausing 2% glutaraldehyde in 0.1 M cacodylate, pH 7.35, for5 min, followed by a 5-min perfusion with the same fixativecontaining 0.12 mM dithizone (Merck, West Germany). Adithizone stock-solution was prepared by dissolving 10 mgdithizone in 3 ml ethanol (99%) and adding three drops 25%ammonia in water;12 dithizone treatment results in a brightred staining of the islets. In control experiments, perfusionswere performed without dithizone and islets were examinedafter random fracturing of glutaraldehyde-fixed pancreata.

Human islets were examined under similar conditions.Pancreatic tissue was obtained from a brain-dead kidneydonor, and tissue blocks were microperfused with glutaral-dehyde and dithizone; islets were microdissected and freezefractured.

Rat islets in situ were also examined after exposure toagents known to affect pancreatic exocrine or endocrinefunction. The agents were administered via a left ventricleperfusion of nembutal-anesthetized rats; the medium con-sisted of Earle's salts and 2.8 mM glucose, was bufferedwith 10 mM Hepes and 14.3 mM bicarbonate (pH 7.35 at37°C), and supplemented with 0.2% albumin (BSA, fractionV; Sigma, St. Louis, Missouri), 1 mg/ml procaine, and 1 U/ml heparin. Experimental conditions included addition of 25

DIABETES, VOL. 33, JANUARY 1984 101

EVIDENCE AGAINST PRESENCE OF TIGHT JUNCTIONS ENDOCRINE PANCREAS

FIGURE 1. Tight junction strands (TJ) associated with gap junctions (GJ) on a membrane P-face in the central region of an islet cultured for 20 hat 5.6 mM glucose (a; bar, 0.1 jim) or after exposure to trypsin in situ (b; bar, 1 jxm).

(xg/ml trypsin (70 U/mg; Boehringer, West Germany), 20 mMglucose plus 1 mM theophylline, or 0.4 mg/ml pilocarpine.After a 10-min perfusion at 37°C, the Earle's medium wasreplaced with glutaraldehyde and fixation was carried outas outlined earlier.Studies on isolated and cultured islets. Pancreatic isletswere isolated using the collagenase-digestion technique ofLacy and Kostianovsky.13 Crude collagenase was purchasedfrom Boehringer (West Germany) and used at 3.5 mg/ml.Two to three hundred islets were obtained per pancreaswithin 2 h after decapitation. The medium used throughoutthe isolation procedure was composed of Earle's salts andsupplemented with 10 mM Hepes, 0.2% BSA, and 2.8 mMglucose (pH 7.35 at room temperature). The isolated isletswere either fixed with glutaraldehyde for freeze fracture andelectron microscopy, or cultured for periods up to 20 h at37°C and 5% CO2 in air. Groups of 200 islets were distributedover 35-mm petri dishes containing 3 ml CMRL-1066 (Gibco,Scotland) supplemented with 0.1 mg/ml glutamine, 0.1 mg/ml streptomycin, 0.1 mg/ml penicillin, and 10% (vol/vol)heat-inactivated fetal calf serum.

In one series of experiments, islets were dissociated intosingle cells14 in order to compare islet cell diameters aftervarious culture periods.Electron microscopy and freeze fracture. Glutaraldehyde-fixed islets were prepared for electron microscopy by post-fixation in 1 % (wt/vol) osmium tetroxide, en-bloc staining with

0.5% (wt/vol) uranylacetate in acetate buffer (pH 5.0), andembedding in araldite.

For freeze fracture, the glutaraldehyde-fixed material waswashed in 0.1 M cacodylate, impregnated with 30% caco-dylate-buffered glycerol (pH 7.35), frozen in a liquid-solidnitrogen interface, and fractured in a Balzers BAF 301 freeze-fracture apparatus (-Balzers High Vacuum Corp., Liechten-stein). Carbon-platinum replicas were examined in a ZeissEM 9 S electron microscope. Areas of membrane P-facewere quantified on micrographs at a final magnification of5000 x with the aid of an IBAS I (Kontron, Munchen, WestGermany) semiautomatic image analyzer; magnificationswere calibrated with a line grid (54,800 lines/inch, BalzersCorp.). Peripheral and central areas of rat islets were sep-arately analyzed as these regions are known to differ in isletcell composition.815 The length of tight junctional fibrils andlinear particle aggregates was measured at a final magni-fication of 63,250 x . Data were expressed as |xm tight junc-tion (TJ) per unit of membrane area, and as (xm particle arrayper unit membrane area. Results are expressed as mean± SEM, from at least three separate experiments. The signifi-cance of differences was determined by unpaired t testing.

RESULTSTight junctions in rat islets: comparison of in situ and invitro conditions. No tight junctional fibrils were observed infreeze-fracture replicas of islets that were examined after

102 DIABETES, VOL. 33, JANUARY 1984

P. A. in t VELD, D. G. PIPELEERS, AND W. GEPTS

TABLE 1Amount of tight junctions in islets

Islet preparation

In situNo perfusionPerfusion

Earle's mediumEarle's medium + trypsin

Isolated isletsFreshly isolatedCulture

2h20 h

0

039.0

9.0

6.40.7

n tight junction/100 jim2 membrane

B-cell area

± 5.4

± 1.4

± 0.7*± 0 . 2 *

P-face

(7)

(6)(4)

(10)

(5)(12)

Non-I

0

11.9 ±

4.3 ±0.9 ±

B-cell area

1.4

2.4t0.6*

(5)

(3)

(3)(6)

Results are expressed as mean ± SE based on (N) islets. Signifi-cance of differences with freshly isolated islets: *NS; tP < 0.05;tP < 0.001.

random fracturing of perfusion-fixed rat pancreas. Identicalfindings were obtained for isleis that were microdissectedfrom perfusion-fixed and dithizone-stained pancreas. Allsubsequent experiments on islets in situ were therefore car-ried out on dithizone-stained material.

In contrast to islets in situ, freshly isolated islets containedan abundant amount of tight junctional fibrils in both centraland peripheral regions. These structures were mostly focallydistributed and closely associated to gap junctions (Figure1a). Culture of isolated islets at 5.6 mM glucose resulted ina progressive decline in the amount of tight junctions perunit of membrane area (Table 1), but did not affect islet celldiameters.

The linear particle arrays that were discerned between isletcells, both in situ and in vitro, do not fulfill the criteria ofclassic tight junctions,1 but appear to be related to gap junc-tional structures:9 they are composed of 9-nm particles (justas gap junctions) and apparently evolve into gap junctionsor are continuous with them (Figure 2). Quantitatively, thesestructures were only present in small amounts both in thecentral (1.8 ± 0.5 (xm/100 |xm2 P-face) and peripheral (0.9± 0.3 |xm/100 JJUTI2 P-face) islet regions in situ.Induction of tight junctions in the endocrine pancreas insitu. In situ perfusion of a rat pancreas with trypsin (25 jxg/ml) induced within 10 min large amounts of tight junctionsbetween islet cells (Table 1, Figure 1b); medium alone waswithout effect. Tight junctional fibrils did not develop after a10-min perfusion with B-cell secretagogues (20 mM glucoseplus 1 mM theophylline) nor with a stimulator of the exocrinepancreas (pilocarpine).

Tight junctions could also be induced, especially in theislet periphery, by injecting 15 ml physiologic Earle's mediuminto the pancreatic duct, after its ligation near the duodenum.This injection produced a severe disruption of the gland withdamage to exocrine cells.Tight junctions in human endocrine pancreas. The com-bination of immersion and microperfusion fixation of pan-creatic fragments resulted in a well-preserved ultrastructureof the endocrine cells. Freeze-fracture replicas of central isletregions were devoid of tight junctional fibrils; as in rat isletssmall amounts of linear particle arrays were observed (0.5ixm/lOO |j,m2 P-face, investigated area: 1974 JJUTI2). Identicalobservations were made in the peripheral regions of eight

of nine islets; however, in one islet a few tight junctional fibrilswere detected on the surface of one cell.

DISCUSSIONTight junctions have been previously identified in isolatedislets of Langerhans, where they occur between homologousand heterologous cell types.4"6 In both rodent and humanislets, the tight junctional fibrils are grouped in several small,irregular, and apparently unconnected patches;46 this focaldistribution contrasts with the continuous belt-like structures

FIGURE 2. Varying organization of linear particle arrays, possibly indi-cating intermediate stages in gap junction formation. Suggested se-quence of events from a to c (bar, 0.1 itm).

DIABETES, VOL. 33, JANUARY 1984 103

EVIDENCE AGAINST PRESENCE OF TIGHT JUNCTIONS IN ENDOCRINE PANCREAS

that are formed by the interconnecting junctions in intestinal,respiratory, and urogenital epithelia. In the present study, notight junctional fibrils were detected in rat or human isletsexamined in situ; this observation cannot be attributed todithizone staining, as this step was introduced after glutar-aldehyde fixation, and as analogous conclusions werereached in unstained perfusion-fixed pancreas. Instead, thetight junctional structures that have been described so farin isolated islets, appear to result from the isolation proce-dure rather than to express an in vivo intercellular organi-zation.

It is well known that tight junctions can be rapidly inducedor (dis)organized by a variety of conditions.16~22 A massiveproliferative assembly of new tight junction strands wasnoted, for example, during a 5-min in vitro incubation ofprostatic tissue.23 Similarly, tight junctional fibrils developedbetween islet cells during collagenase digestion of the pan-creas. Among many possibilities, disruption of the pan-creatic gland and its exposure to trypsin were each foundto represent likely explanations for such alteration. Trypsinis a known constituent of the collagenase preparations used;it might also be released and activated during pancreaticdamage or digestion. Orci et al.7 have demonstrated pre-viously that proteolytic enzymes increase the number of tightjunctions in isolated islets, but did not correlate this findingwith the presence of tight junctions in islets after their col-lagenase isolation. Similarly, we assume that the tight junc-tional fibrils, which were observed in the peripheral area ofone human islet, did develop as a result of the excision,which preceded fixation in the human material.

The rapidity of tight junction formation in islets suggeststhe availability of an easily accessible pool of subunits fromwhich junctional structures can be formed. Assuming a lipidnature of these subunits,24 it is conceivable that the cleavageof membrane proteins—as might occur with trypsin—leadsto a phase-transition of membrane lipids, which then changetheir bilayer configuration into particulate or tube-like struc-tures.25-27

Although one can only speculate on the pathophysiologicsignificance of these phenomena, it seems attractive to con-sider tight junction formation in islets as an adaptive mech-anism, intended to seal and hence to protect islet micro-domains against sudden alterations in local interstitial fluid.

In summary, there is so far no experimental evidence in-dicating a participation of tight junctions in the normal proc-ess of pancreatic hormone release and its regulation bycirculating or local factors; tight junctional fibrils were notdetected in normal rat and human islets examined in situ.The present data indicate that islet tight junctions can berapidly formed after severe perturbations in the local inter-stitial fluid. It is suggested that the function of these newlyformed tight junctions consists primarily in sealing off theintercellular islet space when the tissue is exposed to a com-partment of different chemical composition.

ACKNOWLEDGMENTSWe thank D. Van Ossel for excellent technical assistance.This study was supported by grants from the Belgian Fondsvoor Wetenschappelijk Geneeskundig Onderzoek (20.02 9

and 3.0021.80) and from the Belgian Ministerie voor Weten-schapsbeleid (80-85/9).

REFERENCES1 Staehelin, L. A.: Further observations on the structure of freeze

cleaved tight junctions. J. Cell Sci. 1973; 13:763-86.2 Claude, P., and Goodenough, D. A.: Fracture faces of zonulae oc-

cludentes from "tight" and "leaky" epithelia. J. Cell Biol. 1973; 58:390-400.3 Dragsten, P. R., Blumenthal, R., and Handler, J. S.: Membrane asym-

metry in epithelia: is the tight junction a barrier to diffusion in the plasmamembrane. Nature 1981; 294:718-22.

4 Orci, L, Unger, R. H., and Renold, A. E.: Structural coupling betweenpancreatic islet cells. Experientia 1973; 29:1015-18.

5 Orci, L, Malaisse-Lagae, F., Ravazzola, M., Rouiller, D., Renold,A. E., Perrelet, A., and Unger, R.: A morphological basis for intercellularcommunication between alpha and p-cells in the endocrine pancreas. J. Clin.Invest. 1975; 56:1066-70

6 Orci, L, Malaisse-Lagae, F., Amherdt, M., Ravazzola, M., Weis-swange, A., Dobbs, R., Perrelet, A., and Unger, R.: Cell contacts in humanislets of Langerhans. J. Clin. Endocrinol. Metab. 1975; 41:841-44.

7 Orci, L, Amherdt, M., Henquin, J. C, Lambert, A. E., Unger, R. H., andRenold, A. E.: Pronase effect on pancreatic beta cell secretion and mor-phology. Science 1973; 180:647-49.

8 Orci, L: Morphofunctional aspects of the islets of Langerhans, themicroanatomy of the islets of Langerhans. Metabolism 1976; 25 (Suppl.1):1303-13.

9 Orci, L., and Perrelet, A.: Morphology of membrane systems in pan-creatic islets. In The Diabetic Pancreas. Volk, B. W., and Wellmann, K. F, Eds.New York, Plenum Press, 1977:171-210.

10 Kawai, K., Ipp, E., Orci, L., Perrelet, A., and Unger, R. H.: Circulatingsomatostatin acts on the islets of Langerhans by way of a somatostatin-poorcompartment. Science 1982; 218:477-78.

11 In't Veld, P. A., Pipeleers, D. G., and Gepts, W.: Cyclic-AMP-involve-ment in gap junction formation between pancreatic B-cells. Diabetologia 1983;25:166

12 Bonnevie-Nielsen, V, Skovgaard, L. T, and Lernmark, A.: (J-cell func-tion relative to islet volume and hormone content in the isolated perfusedmouse pancreas. Endocrinology 1983; 112:1049-56.

13 Lacy, P. E., and Kostianovsky, M.: Method for the isolation of intactislets of Langerhans from the rat pancreas. Diabetes 1967; 16:35-39

14 Pipeleers, D. G., and Pipeleers-Marichal, M. A.: A method for thepurification of single A, B and D cells and for the isolation of coupled cellsfrom isolated rat islets. Diabetologia 1981; 20:654-63.

15 Ferner, H.: Das Inselsystem des Pankreas. Stuttgard, Thieme Ver-slag, 1952.

16 Montesano, R., Gabbani, G., Perrelet, A., and Orci, L: In vivo in-duction of tight junction proliferation in rat liver. J. Cell Biol. 1976; 68:793-98.

17 Elias, P. M., and Friend, D. S.: Vitamin-A-induced mucous metaplasia.J. Cell Biol. 1976; 68:173-88.

18Shimono, M., and Clementi, R: Intercellular junctions of oral epithe-lium. II. Ultrastructural changes in rat buccal epithelium induced by trypsindigestion. J. Ultrastruct. Res. 1977; 59:101-12.

19 Porvaznik, M., Johnson, R. G., and Sheridan, J. D.: Tight junctionsdevelopment between cultured hepgtoma cells: possible stages in assemblyand enhancement with dexamethasone. J. Supramol. Struct. 1979; 10:13—30.

20 Posalaky, Z., Meyer, R., and McGinley, D.: The effects of follicle-stimulating hormone (FSH) on Sertoli cell junctions in vitro: a freeze fracturestudy. J. Ultrastruct. Res. 1981; 74:241-54.

21 Rassat, J., Robenek, H., and Themann, H.: Alterations of tight andgap junctions in mouse hepatocytes following administration of colchicine.Cell Tissue Res. 1982; 223:187-200.

22Kitajima, Y, Eguchi, K., Ohno, T, Mori, S., and Yaoita, H.: Tightjunctions of human keratinocytes in primary culture: a freeze-fracture study.J. Ultrastruct. Res. 1983; 82:309-13.

23 Kachar, B., and Pinto Da Silva, P.: Rapid massive assembly of tightjunction strands. Science 1981; 213:541-43.

24 Kachar, B., and Reese, T. S.: Evidence for the lipidic nature of tightjunction strands. Nature 1982; 296:464-66.

25 Verkleij, A. J., Mombers, C, Leunissen-Bijvelt, J., and Vervegaert,P. H. J. Th.: Lipidic intramen branous particles. Nature 1979; 279:162-63.

26 De Grip, W. J., Drenthe, E. H. S., Van Echtels, C. J. A., De Kruyff,B., and Verkley, A. J.: A possible role of rhodopsin in maintaining bilayerstructure in the photoreceptor membrane. Biochim. Biophys. Acta 1979;558:330-37.

27 Venetie, R. van, and Verkley, A. J.: Analysis of the hexagonal II phaseand its relations to lipidic particles and the lamellar phase. Biochim. Biophys.Acta 1981; 645:262-69.

104 DIABETES, VOL. 33, JANUARY 1984