Diabetologia 2001) 44: 575±584 Ó Springer-Verlag 2001 Association between islets of Langerhans and pancreatic ductal system in adult rat. Where endocrine and exocrine meet together? E.Bertelli1, M.Regoli1, D.Orazioli1, M.Bendayan2 1 Department of Biomedical Sciences, University of Siena, Siena, Italy 2 Department of Pathology and Cell Biology, University of Montreal, Montreal, Canada Abstract to the ducts. For each series, 93.48 5.43% of the is- lets contacting the duct tree were associated with Aims/hypothesis. Studies on the functional and mor- small-sized ducts or centroacinar cells. Electron mi- phological relations between exocrine and endocrine croscopy revealed that some insulin and somatostatin pancreas have been conducted mainly to disclose the cells do face the duct lumen. Insulin was detected influence of islets of Langerhans on acinar parenchy- within the duct lumen and in the endosomal compart- ma. Less attention has been paid to the relations oc- ment of the duct cells. curring between islets and pancreatic ducts. Conclusions/interpretation. The finding that most is- Methods. A series of consecutive sections of normal lets are connected with the duct system in the adult adult rat pancreas were double stained with islet hor- pancreas is discussed in terms of hormone secretion mones) and duct cytokeratin 20) markers. Electron into the ducts, islet histogenesis and the relation microscopy was conducted to investigate the ultra- among the three tissue components of the pancreas, structural features of duct-islet relations and anti-in- the endocrine, the exocrine and the duct system. sulin immunogold labelling was carried out to reveal [Diabetologia 2001) 44: 575±584] the presence of insulin in the pancreatic duct system. Results. Consecutive double-stained sections demon- Keywords Pancreas, islet cells, duct epithelial cells, strated that 73.60 2.97% of the islets were attached cytokeratin, glucagon, somatostatin, insulin, rat. It is common to describe the endocrine and the exo- dreds of thousands variable-sized clusters of endo- crine pancreas as if they were two distinct and inde- crine cells islets of Langerhans), has evolved to facil- pendent entities. However the pancreas is an inte- itate interactions between the two glandular compo- grated organ involved in the digestion and absorption nents [1]. In order to determine reciprocal influences, of nutrients on the one hand, and in the regulation of many aspects of the relations occurring between exo- blood glucose homeostasis, on the other. Most proba- crine and endocrine pancreas have been investigated bly, the unique anatomical architecture of this organ, in the past three decades [2, 3]. Because of the obser- characterized by the dispersion of the endocrine com- vation that, in contrast to ªtele-insularº acini, ªperi- ponent throughout the acinar parenchyma as hun- insularº acini are formed by larger cells, containing more zymogen granules [2, 4±6], numerous studies have addressed the roles) played by the islet hor- Received: 12 October 2000 and in revised form: 18 January mones in tuning acinar cell secretion and the anatom- 2001 ical pathways, the so called ªislet-acinar axisª, through which this influence is exerted [2, 3, 7]. A Corresponding author: Dr. Eugenio Bertelli, Dept. of Biomed- large body of evidence indicates that a continuous ical Sciences, University of Siena, Via Aldo Moro, I-53100 Sie- na, Italy insulo-acinar venous portal system conveys hormone Abbreviations: AP, Alkaline phosphatase; CK20, cytokeratin enriched blood from the islets to the acinar parenchy- 20; MAb, monoclonal antibody; PAb, polyclonal antibody ma [2, 3, 8, 9]. The amount of acinar tissue that is ac- 576 E.Bertelli et al.: Association between islets and pancreatic ducts tually involved in this portal system and its physiolog- AP)-conjugated anti-rabbit IgG was from Roche Diagnostics ic relevance are, however, still issues of debate [10, Monza, Italy) and AP-conjugated rat serum absorbed anti- 11]. mouse IgG from Sigma-Aldrich Milan, Italy). In contrast, much less attention has been paid to Animals and tissues. A total of 10 male adult Sprague-Dawley the relations between the endocrine pancreas and rats 3 to 9 months of age), fed with a standard diet, were the ductal system. In normal conditions, only ªextra- used for this study. Principles of laboratory animal care were insularº endocrine tissue, represented by single cells followed as well as the Italian law on animal experiments. Af- or small buds of endocrine cells, is usually thought to ter anaesthesia, four animals were killed by decapitation and be associated with the duct lining [12±16]. Islets of the pancreatic tissue was sampled. The pancreas of each ani- Langerhans, by contrast, have been reported to be mal was divided into four pieces, fixed overnight in 1% glu- taraldehyde, dehydrated and embedded in methylmethacry- connected with the ductal system only in the fetal late. The pancreas of three other animals were fixed for 2 h in pancreas as a short-lived result of endocrine histogen- Bouin's fluid, dehydrated and embedded in paraffin. esis [17±19]. In adults, the eventual retention and the Tissues from three additional rats were used for electron extent of such associations is controversial [20, 21] microscopy. Perfusion was carried out for 20 min with 1% glu- because no specific study has addressed this subject, taraldehyde in 0.1 mol/l sodium cacodylate buffer pH 7.35) apart from one investigator's pioneer work in guinea through the thoracic aorta. The pancreas was sampled, trim- pigs [22]. A study of this issue appears particularly med, further fixed for 2 h with the same fixative at 4C, post- fixed with 1% OsO4 in cacodylate buffer for 2 h at 4C, dehy- important considering that islets of Langerhans dif- drated and embedded in Epon 812. ferentiate and develop from precursor cells hosted in the duct lining. These precursors or stem cells are be- Conventional histology, immunocytochemistry and transmis- lieved to persist in the pancreas of adult individuals sion electron microscopy. Blocks of methylmethacrylate-em- within the wall of the duct system [23±25]. Moreover, bedded pancreatic tissue were cut leaving a step between experimental studies have indicated that islet hor- each section for a total length of about 100 mm. Thirty random sections 3.5 mm thick) were routinely stained with 0.1% tolui- mones are possible modulators of ductal secretion dine blue. [26, 27]. Indeed, whereas insulin appears to increase A total of 5 series of 25 consecutive sections 4 mm thick) secretin-induced pancreatic juice secretion [26±28], were cut from five different blocks of paraffin-embedded pan- glucagon, pancreatic polypeptide as well as soma- creas and used for immunocytochemistry. The first section of tostatin seem to have an inhibitory affect on duct each series and the last one of the fifth series were considered cell functions [26, 27]. Many other ªnon-classicalº is- as ªrandom sectionsº. Double immunocytochemical staining was carried out with the anti-CK20 MAb and with a cocktail let hormones such as pancreastatin, neuropeptide ty- of anti-somatostatin and anti-glucagon antisera. Sections were rosine NPY) and peptide tyrosine tyrosine YY) deparaffinazed, rehydrated, treated with 0.1% trypsin to re- could also be involved in duct cell secretion [27], trieve CK20 antigenicity as previously reported [31], washed with peptide YY, in particular, inhibiting spontaneous in PBS and incubated overnight at 4C with the anti-CK20 and secretin-stimulated bicarbonate secretion [29]. MAb dilution 1/100). After rinsing with PBS, sections were In recent studies, cytokeratin 20 CK20) has been incubated with AP-conjugated anti-mouse IgG and the AP re- action was developed with NBT/BCIP stock solution as chro- found to be expressed by all rat pancreatic duct cells mogen diluted in 0.1 mol/l TRIS buffer, pH 9.5, 0.05 mol/l including centroacinar cells) regardless of the size MgCl2, 0.1 mol/l NaCl, 2 mmol/l levamisole. Slides were then of the ducts [30, 31]. Taking advantage of cytokeratin rinsed with distilled water to stop the reaction, equilibrated 20's reliability as a ductal marker, we studied the ex- with PBS, and incubated with 0.25 mol/l EDTA for 30 min to tent of the topographical associations occurring be- inactivate AP as previously reported [32]. After washing with tween islets and ducts in the adult rat pancreas. Using PBS, slides were incubated overnight at 4C with the cocktail double immunocytochemical labellings, which reveal of anti-somatostatin and anti-glucagon antibodies both at a di- lution of 1/50. Sections were subsequently rinsed in PBS, incu- both CK20 and islet hormone immunoreactive cells, bated with AP-conjugated anti-rabbit IgG and the reaction we were able to demonstrate that most islets of was developed with Fast-Red tablets Sigma-Aldrich, Milan, Langerhans of the adult rat pancreas are directly con- Italy). nected with the pancreatic ductal system. This finding For transmission electron microscopic analysis, consecutive adds a new and important piece of information to the ultrathin sections were routinely stained and observed with a mosaic of the multiple relations occurring among the Philips 201 or 410 electron microscope. For the immunocyto- chemical detection of insulin at the electron microscope level, different components of the pancreas. we followed previously published techniques [7]. Briefly, ultra- thin sections of 1% glutaraldehyde-1% OsO4 fixed, epon-em- bedded adult rat pancreatic tissue were treated with a saturat- Materials and methods ed aqueous solution of sodium metaperiodate followed, after rinsing with PBS, by a 2 h incubation with a guinea-pig anti-bo- Antibodies. Rabbit polyclonal antibodies PAb) to somatosta- vine insulin antibody dilution 1/200). After rinsing with PBS, tin and glucagon were purchased from Novocastra Lab. New- the sections were incubated with the protein A-gold complex castle upon Tyne, UK).