FUNCTIONAL ASPECTS OF THE EXOCRINE PANCREAS IN RELATION...

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FUNCTIONAL ASPECTS OF THE EXOCRINE PANCREAS IN RELATION TO THE ISLETS OF LANGERHANS Effects of Islet Hormones on the Synthesis, Storage and Secretion of Amylase AKADEMISK AVHANDLING som med vederbörligt tillstånd av medicinska fakulteten vid Umeå Universitet för ernående av medicine doktorsgrad offentligt försvaras i anatomi-histologi-institutionens föreläsningssal lördagen den 18 maj 1974 kl. 9.00 av ÂKE DANIELSSON med. kand.

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FUNCTIONAL ASPECTS OF THE EXOCRINE PANCREAS IN RELATION TO THE ISLETS

OF LANGERHANSEffects o f Islet Hormones on the Synthesis, Storage and

Secretion o f Amylase

AKADEMISK AVH AN D LIN G

som med vederbörlig t tills tånd av

medicinska fakulteten vid Umeå Universitet

fö r ernående av medicine doktorsgrad

o ffe n tlig t försvaras

i anatom i-histolog i-institu tionens föreläsningssal

lördagen den 18 maj 1974 kl. 9.00

av

ÂKE DANIELSSON

med. kand.

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UMEÅ UNIVERSITY MEDICAL DISSERTATIONS

No 10 1974

From the Department o f H istology, University o f Umeå, Umeå, Sweden

FUNCTIONAL ASPECTS OF THE EXOCRINE PANCREAS IN RELATION TO THE ISLETS

OF LANGERHANSEffects o f Islet Hormones on the Synthesis, Storage and

Secretion o f Amylase

by

ÂKE DANIELSSON

Umeå 1974

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The present thesis is based on the fo llow ing publications, reference to whichw ill be made by c itation o f the appropria te Roman numerals.

I Danielsson, A: Techniques fo r Measuring Amylase Secretion from Pieces o f Mouse Pancreas. Anal. Biochem. in press.

II Danielsson, Å: Effects o f Glucose, Insulin and G lucagon on Amylase Secretion from Incubated Mouse Pancreas. Pflügers Arch, in press.

III Danielsson, A: Effects o f N u tritiona l State and o f Adm in istra tion of Glucose, G libenclam ide o r D iazoxide on the Storage o f Amylase in Mouse Pancreas. Digestion in press.

IV Carlsöö, B., Danielsson, A. and Helander, H. F.: Effects o f Starvation on Amylase Storage in Mouse Pancreas and Parotid G land. A Biochemical and M orphom etrie Study. Acta Hepato-Gastroenterol. 21, 48, 1974.

V Danielsson, Å. and Sehlin J.: Transport and O xida tion o f Am ino Acids and Glucose in the Isolated Exocrine Mouse Pancreas: Effects o f Insulin and Pancreozymin. Acta Physiol.Scand. in press.

VI Danielsson, A., Marklund, S. and Stigbrand, T.: Effects o f Starvation and Islet Hormones on the Synthesis o f Amylase in Isolated Exocrine Panc­reas o f the Mouse. Acta Hepato-Gastroenterol. in press.

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BACKGROUND

In the pancreas o f the rabb it small accumulations o f cells, d iffe ren t from the acinar cell, were described by Paul Langerhans in 1869. Later these accumulations were termed the islets o f Langerhans and were found to constitute an endocrine gland which produces hormones active in the blood sugar homeostasis. In a ll species o f birds and mammals the islets, which produce insulin, glucagon and probab ly gastrin, are dispersed throughout the exocrine parenchyma. This arrangem ent provides a very large area o f contact between the two types o f tissue, and it is natural to de liberate on whether the intim ate re lation between the endocrine and exocrine parts o f the g land has any physio log ica l im plications. In the adrenal, another example o f closely associated tissues w ith distinct secretory functions, the corticosteroids are considered im portant fo r the synthesis o f adrenaline in the medulla (W urtman, 1966).

Studies on the cat have shown that the exocrine pancreatic cells situated close to the islets d isplay a pronounced granu la tion ; the granule population is furtherm ore resistant to discharge by vagal nerve stim ulation or choliner­gic drugs (Sergeyeva, 1938). In the mouse these juxta insular cells are larger, as are the ir nuclei and nucleoli and they also exhib it more nucleoli than those acinar cells which are located fa rther away from the islets (Heilman, W a llg ren and Petersson, 1962). The m orpholog ica l characteristics suggest tha t acinar cells in the periinsular region — the "ha lo zone” — have a higher functional activ ity than other acinar cells. This has been attribu ted to a high local concentration o f insulin in the ” halo zone” (Ferner, 1958). In support o f this hypothesis the adm in istra tion o f a lloxan — a ß-cell cytotoxin — w ill abolish the ” halo zone” phenomenon in rats (Kramer and Tan, 1968). M oreover, the duck pancreas contains two types o f islets, only one o f which produces insulin and is surrounded by a ” halo zone” (Ferner, 1958; W a llg ren and Heilman, 1962).

A high local concentration o f insulin in the surrounding exocrine tissue could be due either to d iffusion o f the hormone from the islets or to a specia­lized arrangem ent o f the vascular system. Reports in the relevant literature are rather contrad ictory where this matter is concerned. In a number o f species, vascular connections have been demonstrated between the islets and the exocrine pancreas (Beck and Berg, 1931; W harton, 1932; Ferner, 1958; Fujita, 1973; Fujita and M urakam i, 1973). It has even been claimed that the b lood supply to the exocrine pancreas is exclusively derived from the islet c ircu lation (W harton, 1932; Fujita, 1973). However, other authors have fa iled to demonstrate an intim ate vascular connection between the two portions o f the pancreas (Brunfeldt, Hunhammer and Skouby, 1958; Bunnag, Bunnag and W arner, 1963).

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Diabetes mellitus is often accompanied by m orpho log ica l changes in the exocrine pancreas (W arren, LeCompte and Legg, 1966). Furthermore, the to ta l we ight o f the g land is reduced (Vartia inen, 1944) and the exocrine secretion is often im paired (Chey, Shay and Shuman, 1963; Vacca, Henke and Knight, 1964). A lloxan-d iabetic rats display a decreased content o f pancreatic amylase (Christophe et al., 1971). Ben A b d e ljlil, Palla and Desnuelle (1965) and Palla, Ben A b d e ljlil and Desnuelle (1968) reported that a lloxan reduces the amylase content but increases the level o f chymotrypsi- nogen. Daily injections o f insulin restored the amylase content, but did not reverse the effect on the content o f chymotrypsinogen. Adm in istra tion o f insulin to a lloxan-d iabetic rats has been reported to enhance the incorpora­tion o f labelled am ino acids into rat pancreatic amylase (Söling and Unger, 1972) and to ta l prote in (Palla, Ben A bd e ljlil and Desnuelle, 1968).

The main physio log ica l stimuli o f secretion from the exocrine pancreas are the gastro intestinal hormones. It is o f interest that both cholecystokinin- pancreozymin (CCK-PZ) and secretin have also been found to stimulate in ­sulin secretion (Dupré et al., 1969). Hinz et al. (1971) and Goberna et al. (1971) found that this effect on insulin release is dependent on an adequate function o f the exocrine parenchyma.

Against this background o f evidence suggestive o f functional in te rre la ­tionships between the endocrine and exocrine pancreas, it was decided to try and establish whether the islet hormones affect the synthesis and release o f pancreatic enzymes in the mouse pancreas. Amylase is a m ajor prote in component o f the mouse pancreas (Danielsson, M arklund and Stig- brand, 1974) and most authors have found that the d iffe ren t pancreatic enzymes are released concom itantly (W ormsley and G oldberg, 1972). For these reasons amylase was used as an index o f the exocrine pancreatic function. During the course o f the work, the scope o f the study had to be w idened to include various method developments as well as certain com ple­mentary m orphologica l and biochemical studies on the pancreas. The main purposes o f the investigation may be summarized as fo llow s:

1. The development o f methods fo r a) the m icrodeterm ination o f amylase activ ity, b) the pu rifica tion o f pancreatic amylase, and c) the in vitro incubation o f pancreas pieces.

2. The study o f the effects o f starvation as well as m odifiers o f insulin release on the synthesis and storage o f amylase.

3. The study o f the in vitro effects o f insulin, glucagon and glucose on the synthesis and release o f amylase.

4. The study o f the in vitro effects o f insulin, glucose and CCK-PZ on the oxidation o f glucose and on the uptake and ox ida tion o f a lanine and leucine.

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METHODS

ANIM ALS A N D EXPERIMENTAL CO NDITIONS

A ll experiments were perform ed on pancreatic glands o f mice obtained from a colony bred in our labo ra to ry (Heilman, 1965). This stock carries a gene, ob, which in the homozygous state gives rise to a syndrome o f obesity and hyperglycemia. However, in the present investigations only fem ale mice o f normal phenotype were used.

For the in vivo investigations (III, IV, VI) mice were given in traperitoneal injections o f test substances, and the contro l animals received the same amounts o f solvent. A fte r starvation or treatm ent the anim al was brie fly etherized and decapitated. Blood was collected fo r serum analyses o f glucose and insulin (III) and the pancreas was quickly removed. Tissue spe­cimens fo r ligh t and electron m icroscopy (IV) as well as fo r amylase determ inations were collected. For the in vitro studies the glands were prepared in d iffe rent ways according to the type o f experimental design employed.

ASSAY PROCEDURES

Serum glucose was determ ined according to Lowry et al. (1964) by recording the fluorescence o f NADPH 2 in a hexokinase/glucose-6-phosphate dehydro­genase system. Insulin was assayed rad io im m unolog ica lly using separation o f free and antibody-bound insulin w ith ethanol precip ita tion (Heding, 1966). A m odifica tion o f the Kissane and Robins (1958) procedure was used to measure DNA fluo rom etrica lly . Q uantita tion o f 14C- and 3H-labelled compounds was perform ed in a Packard Tri-Carb Liquid Scin tilla tion Spectro­meter.

Amylase was determ ined using a m icrom odification o f the d in itrosa licy la te (DNS) method (I). Samples (10 [A each) were incubated at 25° C fo r 10 min w ith 10 /d 2 % starch solution. The reaction was interrupted by adding 20 /d DNS-reagent. A fte r bo iling fo r 10 min, the samples were diluted w ith 200 /d d istilled w ater and were measured spectrophotom etrically The results were calculated from a standard curve o f maltose. One unit o f amylase was defined as the enzyme activ ity libera ting reducing groups corresponding to 1 ^m o l o f maltose per min.

LIGHT A N D ELECTRON MICROSCOPIC TECHNIQUES (IV).

The tissue samples were fixed in buffered 4 % glutara ldehyde, postfixed in O s 0 4 and embedded in Epon. Pancreatic acini and cells were selected

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from areas distant from the islets to avoid an influence on the measurements from the ” halo zones” . For quantita tive ligh t m icroscopy roughly 1 ^ thick sections were ligh tly stained w ith to lu id ine blue and examined by phase contrast microscopy. In m icrographs o f random ly selected regions, the surface area and the number o f granules w ith in this defined area were estimated and expressed as granules per [a2. For quantita tive electron microscopy about 800 Å thick sections were contrasted and examined in the electron microscope. Exocrine cells which displayed a portion o f the apical and basal cell surfaces as w ell as the nucleus were photographed. The volume densities o f the secretory granules and o f the nuclei were calculated using the po int-counting method (W eibel and Elias, 1967). In add ition , the secretory granule profiles were measured in a partic le size analyser and the results used to calculate the mean diam eter o f the granules according to the method o f Bach (1967).

BIOSYNTHESIS OF AMYLASE AN D TOTAL PROTEIN (VI).

Pieces o f microdissected exocrine pancreas were incubated in a supple­mented Krebs-Ringer b icarbonate buffe r also contain ing 20 am ino acids according to Campagne and G ruber (1962), as well as L-(U-14C)leucine (7.5 mCi/mmol). A fte r incubation the tissue was homogenized by sonication in 50 mM phosphate buffe r (pH 6.9) and amylase was isolated from other pancreatic proteins and from free L-(U-14C)leucine in one step by isoelectric focusing in a thin layer o f A m pholineR-conta in ing polyacrylam ide gel. The amylase contain ing gel strip was cut out and dissolved in H2O 2. Total pancreatic proteins were obtained by precip ita tion and repeated washing w ith TCA. Incorporation o f L-(U-14C)leucine in to amylase and to ta l protein was expressed in re lation to the tissue content o f DNA.

AMYLASE SECRETION

A Krebs-Ringer b icarbonate buffe r (pH 7.4) supplemented w ith pyruvate, g lutam ate and fum arate as well as glucose and albumin was used fo r batch- -incubations and perifusions. In the batch-incubation experiments the pancreas pieces were incubated in vessels specially designed fo r continuous equ ilib ra tion o f the medium w ith O 2-CO 2 (95:5) (I). Amylase activities were determ ined in incubation media and supernatants o f pancreas homogenates, and related to the wet we ight o f incubated tissue. The dynamics o f the release process were investigated in a specia lly designed perifusion system (I).

METABOLISM OF GLUCOSE A N D A M IN O ACIDS (V).

Pieces o f exocrine pancreas, p ractica lly devoid o f islets, were incubated in a Krebs-Ringer b icarbonate buffer. O x ida tion studies were perform ed w ith d iffe ren t concentrations o f 14C-la,belled D-glucose, L-alanine or L-leucine, as well as unlabelled test substances. A fte r being trapped in hyamine the

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produced 14C02 was measured. The uptake and transport o f L-àlanine and L-leucine were studied w ith 3H-labelled am ino acids using a double-label technique w ith 14C-labelled L-glucose as an ex trace llu la r space marker. This made it possible to correct fo r extrace llu la r and contam inating 3H-amino acids w ithou t washing the tissue. The data were expressed as mmol substrate oxidized o r taken up per kg dry we ight o f pancreas.

STATISTICSIn the in vitro experiments (I, II, III, V, VI) statistical significances were deter­mined by computing t-values from the mean ± S.E.M. o f the differences between paired test and control incubations in a series o f repeated but separate experiments. In the in vivo studies (III, IV, VI) differences between group means were tested by the ord inary Student’s t-test.

RESULTS AND DISCUSSION

CHARACTERISTICS OF THE IN VITRO INCUBATED EXOCRINE PANCREAS

Because the exocrine cells synthesize and secrete proteins at a high rate, the im portance o f oxygen and exogenous substrates fo r the adequate function o f pancreas pieces in vitro was explored (I). D in itrophenol (DNP), an uncoupler o f oxidative phosphorylation, or anoxia reduced the basal secretion and abolished carbam ylcholine-stim ulated release o f amylase. Continuous equ ilib ra tion o f the medium w ith O 2-CO 2 (95:5) during the in­cubation was necessary to achieve optimum secretory responses to carbam ylcholine. The secretory responses were la rger and more reproducib le in media supplemented w ith pyruvate, glutam ate, fum arate and glucose than in media contain ing glucose alone. These results are consistent w ith the find ings that aerobic glycolysis accounts fo r less than 10 % o f the ATP production in rat exocrine cells (Bauduin, Colin and Dumont, 1969), and that the ox idation o f D-glucose is fa ir ly slow in the isolated exocrine pancreas o f the mouse (V).

Using a system w ith continuous gassing o f a supplemented buffer, both batch-incubated and perifused pieces o f mouse pancreas responded to known cholinerg ic and gastro intestinal secretogogues. However, continuous stim ulation o f secretion from perifused mouse pancreas w ith CCK-PZ or carbam ylcholine produced an in itia l rise o f amylase release, fo llow ed by a decline in the release curve. S im ilar results were obtained by Matthews, Petersen and W illiam s (1973) a fte r stim ulation o f the perifused rat pancreas w ith acetylcholine. Repeated pulse-stimulations seemed to be more effective in provoking amylase secretion (I). The reasons fo r the transient nature of the secretory response to physio logical stimuli are not clear.

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Stimulation o f secretion by CCK-PZ was accompanied by an increased rate o f oxida tion o f D-glucose, L-alanine and L-leucine (V). An enhanced oxida tion o f pa lm ita te has previously been observed in CCK-PZ stimulated pigeon pancreas (Webster, Gunn and Tyor, 1966). The increased rate o f oxidation o f am ino acids was not caused by an enhanced uptake, since this process was unaffected by CCK-PZ in the mouse pancreas (V). The oxidation o f D-glucose in the exocrine cells was low compared to that o f L-alanine and L-leucine (V).

EFFECTS OF STARVATION O N THE EXOCRINE PANCREAS

There has been a certain amount o f controversy concerning the effects o f nu tritional state on prote in synthesis in the pancreas. On the one hand, starvation was found to reduce the amylase content (W ebster et al., 1972) and to inh ib it the prote in synthesis in the ra t (Morisset and W ebster, 1972) and the pigeon pancreas (W ebster and Tyor, 1966); in the pigeon pancreas, starvation also induced a reduction o f the polysome content and a reduced capacity o f the polysomes to synthesize protein (Black Jr and W ebster, 1973). On the other hand, Poort and Kramer (1969) claimed that the rate o f protein synthesis in the mammalian pancreas is fa ir ly unaffected by varia tion o f the nutritional state including starvation. In the present study, starvation fo r 24 hrs c learly reduced the rate o f incorporation o f L-(U-14C)leucine into pancreatic amylase and to ta l prote in (VI). This inh ib ition o f the synthesis may explain why starvation also induced a considerable fa ll in the to ta l amylase content (III) and decreased the number o f granules per unit cell area (IV). Refeeding restored the amylase content w ith in 12 hrs (III).

EFFECTS OF GLUCOSE A N D ISLET HORMONES O N THE SYNTHESIS AN D

STORAGE OF AMYLASE

Starvation decreased the rate o f amylase and to ta l prote in synthesis (VI) as well as the content o f amylase (III) and zymogen granules (IV) in the pancreas. These effects may be due to the lowering o f blood sugar, since the adm in istra tion o f glucose to starved mice increased the amylase content (III) and enhanced the synthesis o f both amylase and to ta l protein (VI). The capacity o f injected glucose to increase the amylase synthesis and content was abolished by concom itant adm in istra tion o f d iazoxide, a potent inh ib ito r o f insulin secretion. It is therefore conceivable that the effects o f glucose on amylase synthesis and content were mediated by stim ulation o f insulin release. In vivo adm in istra tion o f insulin has previously been shown to rest­ore the reduced amylase content and the decreased rates o f amylase and

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protein synthesis in a lloxan-d iabetic rats (Palla, Ben A bd e ljlil and Desnuelle, 1968; Söling and Unger, 1972). In non-d iabetic rats, in jection o f insulin enhances both the secretion and the synthesis o f rat pancreatic proteins. Couture, Dunnigan and M orisset (1972) proposed that these effects are not due to insulin acting d irectly on the pancreas but to hypoglycem ia causing stim ulation o f the vagal nerve and the release o f gastro intestinal factors. This hypothesis was not supported by the present studies, since stim ulation o f amylase synthesis and content was observed in hyperglycemic animals w ith increased serum levels o f endogenous insulin (III, VI). On the other hand, studies on the in vitro effects o f glucose, insulin and glucagon did not reveal any changes in amylase and to ta l protein synthesis (VI), suggestig tha t the in vivo effects o f glucose may not be mediated by a d irect action o f the sugar itself or o f insulin on the pancreas. Reservations must, however, be made fo r the possibilities that the responsiveness o f the pancreas may be a ltered by the incubation procedure and that the incubation time may be too short in re lation to the conceivable latency o f direct insulin effects.

EFFECTS OF GLUCOSE AN D ISLET HORMONES O N THE SECRETION OF

AMYLASE

In perifusions as well as in batch-incubation experiments, glucose rap id ly inh ib ited both the basal and CCK-PZ-stimulated amylase secretion from the mouse pancreas (II).Insulin too, at very high concentrations, exerted a sim ilar action. However, the glucose-induced reduction o f secretion did not seem to be mediated by the release o f insulin, as d iazoxide did not counteract the effect o f glucose. Further support fo r the independence o f the glucose and insulin effects was obtained by studying secretion from microdissected pieces o f pancreas (VI). The inh ib ition o f secretion seems to be confined to the secretory process, since, as described above, glucose and insulin in vitro had no effect on amylase and to ta l prote in synthesis. Cyclic AMP has been shown to be an in trace llu la r e ffector o f pancreatic amylase secre­tion (Kulka and Sternlicht, 1968; Bauduin et al., 1971) and insulin decreases the content o f cAMP o f the liver (Exton et al., 1966). It is not known, however, whether insulin affects cAMP in the exocrine pancreas.

G lucagon did not a ffect the basal or CCK-PZ-stimulated amylase secretion from incubated pancreas (II). This is in contrast to results obtained in vivo. The adm in istra tion o f small doses o f glucagon to man and dog induced a marked reduction o f protein output from the pancreas during continuosly stimulated secretion (Dyck et al., 1969, 1970). The la tte r observation may perhaps be expla ined by the glycogenolytic action o f glucagon, leading to hyperglycemia. The increased serum glucose level m ight reduce the dis­charge o f pancreatic enzymes, either alone or in conjunction w ith the accompanying stim ulation o f insulin secretion.

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EFFECTS OF GLUCOSE A N D INSULIN O N THE METABOLISM OF THE

EXOCRINE PANCREAS

Glucose inh ib ited the oxida tion o f L-alanine. This effect may have been due to the inh ib ition o f uptake, which was also noted in the experiments (V). The rate o f ox ida tion o f glucose itself was re la tive ly low compared to that o f L-alanine and L-leucine (V). Insulin stimulated the ox idation o f D-glucose but did not a ffec t the ox ida tion o f L-alanine or L-leucine. Insulin also fa iled to enhance the uptake o f L-alanine and L-leucine in vitro, which is o f interest in relation to the suggestion that insulin stimulates the synthesis o f pancreas proteins in vivo by enhancing am ino acid uptake (Soling and Unger, 1972).

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SUMMARY AND CONCLUSIONS

1. a) A m icrom odification o f the d in itrosalicy la te method fo r measuring reducing groups made it possible to assay an amylase activ ity libera ting less than 10 /ug o f maltose equivalents.

b) By means o f isoelectric focusing in A m pholineR-contain ing po lyacrylam ide gels, amylase was high ly purified from extracts o f tissue samples w ith less than 1 mg dry weight.

c) Routines were worked out fo r the incubation o f pancreas pieces in closed vials and in a perifusion system fo r the study o f secretory dyna­mics. Continuous equ ilib ra tion w ith O 2-CO 2 (95:5) and the supplementa­tion o f incubation buffers w ith glucose, pyruvate, glutam ate and fum a- rate were benefic iary fo r the m agnitude and rep roduc ib ility o f amylase secretory responses to established secretogogues.

2. Starvation decreased the synthesis and the pancreas content o f amylase and dim inished the population o f zymogen granules. The decreases in amylase synthesis and content were counteracted by injections o f glucose to starved mice. This e ffect o f glucose was in turn abolished by in jecting diazoxide, an inh ib ito r o f glucose-induced insulin release, indica ting that the glucose effect was mediated via the discharge o f insulin.

3. Insulin, glucagon and glucose, when added to the incubation medium, fa iled to a ffect the synthesis o f amylase. However, glucose reduced both the basal and the CCK-PZ-induced amylase secretion in a dose- dependent fashion. Insulin too, at a very high concentration, inhib ited the secretion o f amylase. The effects o f glucose and insulin seemed to be m utually independent.

4. Insulin, at this high concentration, stimulated glucose ox ida tion w ithout a ffecting the ox ida tion o r uptake o f alanine or leucine. CCK-PZ stimu­lated the oxida tion o f glucose, a lanine and leucine in the pancreas pieces. Glucose inhib ited the uptake and ox ida tion o f alanine.

W ith regard to the overrid ing question that in itia ted the present study, it is concluded that the in vivo experiments, notably those demonstrating the effects o f glucose and diazoxide on amylase synthesis and content, support the idea that insulin influences the specific functions o f the acinar cells.

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The in vitro experiments revealed that insulin and glucose reduced amylase secretion. If the la tte r find ing holds true fo r the in vivo situation, glucose a n d /o r insulin may well contribute to the maintenance o f a high content o f amylase in the exocrine pancreas. It is conceivable that the periinsular acinar cells are exposed to high concentrations o f insulin. The observed effects o f insulin on the biosynthesis and secretion o f amylase m ight explain the appearance o f ” halo zones” around the islets. The fa ilu re o f insulin and glucose to a ffect the in vitro biosynthesis o f amylase may indicate tha t the in vivo effect o f glucose is not a d irect one or mediated by release o f in­sulin.

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ACKNOWLEDGEMENTS

I wish to express my sincere thanks to :

Professor Bo Heilman fo r o ffe ring me the priv ilege o f w ork ing at the Department o f H isto logy in Umeå, fo r in itia ting this study and fo r placing splendid labora tory fac ilities at my disposal.

Docent Inge-Bert Täljedal, to whom I am indebted fo r fru itfu l discussions, valuable advice and constructive criticism.

Professor Gunnar D. Bloom fo r kind interest and stim ulating advice during the course o f the work, and Professor Sture Falkmer fo r helpfu l criticism.

My co-authors, Dr. Bengt Carlsöö, Professor Herbert F. Helander, Docent Stefan M arklund, Docent Janove Sehlin and Docent Torgny Stigbrand, fo r inspiring co llabora tion during the various phases o f the investigation.

Dr. Erik G ylfe, Docent Lars-Ake Idahl, Docent Åke Lernmark, Dr. Sam N orlin and Dr. Torkel W ahlin , my other colleagues at the department, who have contributed to the stim ulating atmosphere in which I have had the priv ilege to work.

Miss M arianne Borg, Mrs. Kerstin H jortsberg, Miss Karin Janze, Miss Kristina Karlsson, Mrs. Ann-C harlo tt Lundberg and Miss Ulla N ordm ark fo r skilfu l technical assistance.

Miss Kristina Linder fo r typing the manuscripts.

Mr. Per-O lof Fredriksson and Mr. Erik ö h lu n d fo r valuable help w ith technical problems.

Miss Janice Robbins, Dip. Ed., and Miss Barbara Steele, F. L., fo r linguistic revision o f the manuscripts.

The investigations were supported by grants from the Swedish M edical Research Council (12x-562), the Swedish Diabetes Association and the M e­dical Faculty, University o f Umeå.

13

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