FUNCTIONAL ASPECTS OF THE EXOCRINE PANCREAS IN RELATION TO THE ISLETS
OF LANGERHANS
Effects o f Islet Horm ones on the Synthesis, Storage and Secretion o f Am ylase
AKADEM ISK A V H A N D L IN G som med v e d e rb ö rlig t tills tå n d av m edicinska fa ku lte te n v id Umeå U niversitet
fö r ernående av m edicine d o kto rs g ra d o ffe n tlig t försvaras
i a n a to m i-h isto lo g i-in stitu tio n e n s föreläsn ing ssal lö rda gen den 18 maj 1974 kl. 9.00
av
ÂKE DANIELSSON med. kand.
UMEÅ UNIVERSITY M EDICAL DISSERTATIONS N o 10 1974
From the D e pa rtm e nt o f H isto log y, U niversity o f Umeå, Umeå, Sweden
FUNCTIONAL ASPECTS OF THE EXOCRINE PANCREAS IN RELATION TO THE ISLETS
OF LANGERHANS
Effects o f Islet Horm ones on the Synthesis, Storage and Secretion o f A m ylase
by ÂKE DANIELSSON
Umeå 1974
The present thesis is based on the fo llo w in g pu b lica tio n s, reference to w hich w ill be m ade by c ita tio n o f the a p p ro p ria te Roman numerals.
I Danielsson, A: Techniques fo r M easuring Am ylase Secretion fro m Pieces o f Mouse Pancreas. Anal. Biochem. in press.
II Danielsson, Å: Effects o f G lucose, Insulin and G luca go n on Am ylase Secretion fro m Incubated Mouse Pancreas. Pflügers Arch, in press.
III Danielsson, A: Effects o f N u tritio n a l State and o f A d m in is tra tio n o f Glucose, G lib e n c la m id e o r D ia zo xid e on the Storage o f Am ylase in Mouse Pancreas. Digestion in press.
IV Carlsöö, B., Danielsson, A. and Helander, H. F.: Effects o f S tarva tion on Am ylase Storage in M ouse Pancreas and Parotid G la n d . A Biochem ical and M o rp h o m e trie Study. Acta Hepato-Gastroenterol. 21, 48, 1974.
V Danielsson, Å. and Sehlin J.: Transport and O x id a tio n o f A m in o Acids and G lucose in the Isolated Exocrine Mouse Pancreas: Effects o f Insulin and Pancreozym in. Acta Physiol.Scand. in press.
VI Danielsson, A., M arklund, S. and Stigbrand, T.: Effects o f S tarvation and Islet Horm ones on the Synthesis o f A m ylase in Isolated Exocrine Panc
reas o f the Mouse. Acta Hepato-Gastroenterol. in press.
BACKGROUND
In the pancreas o f the ra b b it sm all accum ulations o f cells, d iffe re n t fro m the a c in a r cell, w ere described by Paul Langerhans in 1869. Later these accum ulations w ere term ed the islets o f Langerhans and w ere fou nd to constitute an en docrine gla n d w hich produces hormones active in the b lo o d sugar homeostasis. In a ll species o f birds and m ammals the islets, which produce insulin, gluca gon and p ro b a b ly gastrin, are dispersed th ro u g h o u t the exocrine parenchym a. This a rra ng em en t provides a very la rge area o f con tact between the tw o types o f tissue, and it is na tural to d e lib e ra te on w h ethe r the in tim a te re la tio n between the en do crine and exocrine parts o f the g la n d has any p h ysio lo g ica l im p lica tion s. In the a d re n a l, a n othe r exam ple o f closely associated tissues w ith d istinct secretory functions, the cortico ste roids are considered im p o rta n t fo r the synthesis o f a d re n a lin e in the m edulla (W urtm an, 1966).
Studies on the cat have shown th a t the exocrine pa n cre a tic cells situated close to the islets d is p la y a pronounced g ra n u la tio n ; the gra n u le p o p u la tio n is fu rth e rm o re resistant to discharge by v a g a l nerve stim ulatio n o r c h o lin e r
gic drugs (Sergeyeva, 1938). In the mouse these ju xta in su la r cells are la rg e r, as are th e ir nuclei and nucleoli and they also e x h ib it m ore nucleoli than those a c in a r cells w hich are lo cated fa rth e r aw ay from the islets (Heilm an, W a llg re n and Petersson, 1962). The m o rp h o lo g ic a l cha racteristics suggest th a t a cin a r cells in the p e riin su la r region — the "h a lo zo n e ” — have a high er fu n ctio n a l a c tiv ity than o th e r a cin a r cells. This has been a ttrib u te d to a high local con cen tra tion o f insulin in the ” ha lo zon e” (Ferner, 1958).
In s up po rt o f this hypothesis the a d m in is tra tio n o f a llo x a n — a ß-cell cytotoxin — w ill abolish the ” ha lo zo n e ” phenom enon in rats (Kram er and Tan, 1968). M ore ove r, the duck pancreas contains tw o types o f islets, o n ly one o f which produces insulin and is surrounded by a ” ha lo zo n e ” (Ferner, 1958; W a llg re n and H eilm an, 1962).
A high local con cen tra tion o f insulin in the surrounding exocrine tissue could be due eith er to d iffu sio n o f the horm one fro m the islets o r to a specia
lized arra ng em en t o f the vascular system. Reports in the releva nt lite ra tu re are ra th e r c o n tra d ic to ry w here this m atter is concerned. In a num ber o f species, vascular connections have been dem onstrated between the islets and the exocrine pancreas (Beck and Berg, 1931; W h a rto n , 1932; Ferner, 1958; Fujita, 1973; Fujita and M u ra k a m i, 1973). It has even been claim ed th a t the b lo o d supply to the exocrine pancreas is exclusively derived fro m the islet c irc u la tio n (W h a rto n , 1932; Fujita, 1973). How ever, o th er authors have fa ile d to dem onstrate an in tim a te vascu la r connection between the tw o po rtion s o f the pancreas (B runfeldt, Hunham m er and Skouby, 1958;
Bunnag, Bunnag and W a rn e r, 1963).
Diabetes m ellitus is often acco m pa nie d by m o rp h o lo g ic a l changes in the exocrine pancreas (W arren , LeCompte and Legg, 1966). Furtherm ore, the to ta l w e ig h t o f the g la n d is reduced (V artia in en, 1944) and the exocrine secretion is often im p a ire d (Chey, Shay and Shuman, 1963; Vacca, Henke and Knight, 1964). A llo x a n -d ia b e tic rats disp la y a decreased content o f pa ncre atic am ylase (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) re p o rte d th a t a llo x a n reduces the am ylase content but increases the level o f chym otrypsi- nogen. D a ily injections o f insulin restored the am ylase content, but did not reverse the e ffe ct on the content o f chym otrypsinogen. A d m in is tra tio n o f insulin to a llo x a n -d ia b e tic rats has been rep orted to enhance the in c o rp o ra tio n o f la b e lle d am ino acids in to ra t p a ncre atic am ylase (Söling and Unger, 1972) and to ta l pro te in (Palla, Ben A b d e ljlil and Desnuelle, 1968).
The main ph ysio lo g ica l stim uli o f secretion fro m the exocrine pancreas are the g a stro in te stin a l horm ones. It is o f interest th a t both cholecystokinin- pancreozym in (CCK-PZ) and secretin have also been fo u n d to stim ulate in sulin secretion (Dupré et al., 1969). H inz et al. (1971) and G ob erna et al.
(1971) fou nd th a t this e ffe c t on insulin release is dependent on an adequate fu n ctio n o f the exocrine parenchym a.
A g a in s t this ba ckgro und o f evidence suggestive o f fu n ctio n a l in te rre la tionships between the en do crine and exocrine pancreas, it was decided to try and establish w h ethe r the islet hormones a ffe c t the synthesis and release o f pa n cre a tic enzymes in the mouse pancreas. Am ylase is a m a jo r pro te in com ponent o f the mouse pancreas (Danielsson, M a rklu n d and Stig- bra nd , 1974) and most authors have fou nd th a t the d iffe re n t pa ncre atic enzymes are released co n co m ita n tly (W orm sley and G o ld b e rg , 1972). For these reasons am ylase was used as an index o f the exocrine pa ncre atic fun ction. D uring the course o f the w o rk, the scope o f the study had to be w iden ed to include variou s m ethod developm ents as w e ll as certain com p le
m entary m o rp h o lo g ic a l and biochem ical studies on the pancreas. The main purposes o f the in ve stig a tio n m ay be sum m arized as fo llo w s :
1. The de velopm ent o f m ethods fo r a) the m ic ro d e te rm in a tio n o f am ylase a ctiv ity, b) the p u rific a tio n o f pa n cre a tic am ylase, and c) the in vitro incub ation o f pancreas pieces.
2. The study o f the effects o f s tarvatio n as w e ll as m od ifie rs o f insulin release on the synthesis and storage o f am ylase.
3. The study o f the in vitro effects o f insulin, gluca gon and glucose on the synthesis and release o f am ylase.
4. The study o f the in vitro effects o f insulin, glucose and CCK-PZ on the o x id a tio n o f glucose and on the uptake and o x id a tio n o f a la n in e and leucine.
METHODS
A N IM A LS A N D EXPERIMENTAL C O N D IT IO N S
A ll experim ents w ere pe rfo rm ed on pa ncre atic glands o f mice o b ta in e d fro m a colon y bred in o u r la b o ra to ry (Heilm an, 1965). This stock carries a gene, ob, w hich in the hom ozygous state gives rise to a syndrom e o f obesity and hyp erg lycem ia. H ow ever, in the present investigations o n ly fem ale mice o f norm al phenotype w ere used.
For the in vivo investigations (III, IV, VI) mice w ere given in tra p e rito n e a l injections o f test substances, and the con tro l anim als received the same amounts o f solvent. A fte r starvatio n o r treatm e nt the a n im a l was b rie fly etherized and d e cap itated . Blood was collecte d fo r serum analyses o f glucose and insulin (III) and the pancreas was q u ic kly rem oved. Tissue spe
cimens fo r lig h t and electron m icroscopy (IV) as w e ll as fo r am ylase d e term in ation s w ere collected. For the in vitro studies the glands w ere p re p a re d in d iffe re n t ways acco rd in g to the type o f exp erim e ntal design em ployed.
ASSAY PROCEDURES
Serum glucose was determ ined a cco rd in g to Lowry et al. (1964) by reco rdin g the fluorescence o f N A D P H2 in a hexokinase/glucose-6-phosphate d e h y d ro genase system. Insulin was assayed ra d io im m u n o lo g ic a lly using sep ara tion o f free and a n tib o d y-b o u n d insulin w ith ethanol p re c ip ita tio n (Heding, 1966). A m o d ific a tio n o f the Kissane and Robins (1958) procedure was used to measure D N A flu o ro m e tric a lly . Q u a n tita tio n o f 14C- and 3H -la b e lle d com pounds was pe rfo rm ed in a Packard T ri-C a rb Liquid S cin tilla tio n S pectro
meter.
Am ylase was determ ined using a m ic ro m o d ific a tio n o f the d in itro s a lic y la te (DNS) m ethod (I). Samples (10 [A each) w ere incubated a t 25° C fo r 10 min w ith 10 /d 2 % starch solution. The reaction was in te rru p te d by a d d in g 20 /d D N S-reagent. A fte r b o ilin g fo r 10 min, the samples w ere d ilu te d w ith 200 /d d is tille d w a te r and w ere measured s p e ctro p h o to m e trica lly The results w ere calcula ted fro m a stan dard curve o f m altose. O ne unit o f am ylase was d e fine d as the enzyme a c tiv ity lib e ra tin g reducing groups corre spo nding to 1 ^ m o l o f m altose per min.
LIGHT A N D ELECTRON M ICRO SCO PIC TECHNIQUES (IV).
The tissue samples w ere fixe d in b u ffe re d 4 % g lu ta ra ld e h y d e , postfixed in O s 0 4 and em bedded in Epon. Pancreatic acini and cells w ere selected
fro m areas d ista nt fro m the islets to a vo id an influence on the measurements fro m the ” ha lo zones” . For q u a n tita tiv e lig h t m icroscopy ro u g h ly 1 ^ thick sections w ere lig h tly stained w ith to lu id in e blue and exam ined by phase contrast m icroscopy. In m icro grap hs o f ra n d o m ly selected regions, the surface area and the num ber o f granules w ith in this d e fine d area w ere estim ated and expressed as granules per [a2. For q u a n tita tiv e electron m icroscopy a b o u t 800 Å thick sections w ere contrasted and exam ined in the electron m icroscope. Exocrine cells w hich displa yed a p o rtio n o f the a p ica l and basal cell surfaces as w e ll as the nucleus w ere ph o to g ra p h e d . The volum e densities o f the secretory granules and o f the nuclei w ere calcula ted using the p o in t-c o u n tin g m ethod (W eib el and Elias, 1967). In a d d itio n , the secretory gra nu le p ro file s w ere measured in a p a rtic le size an alyser and the results used to calcula te the mean diam e te r o f the granules a cco rding to the m ethod o f Bach (1967).
BIOSYNTHESIS OF AMYLASE A N D TOTAL PROTEIN (VI).
Pieces o f m icrodissected exocrine pancreas w ere incubated in a supple
m ented Krebs-Ringer b ic a rb o n a te b u ffe r also c o n ta in in g 20 am ino acids acco rd in g to C am pagne and G ru b e r (1962), as w e ll as L-(U-14C)leucine (7.5 m C i/m m ol). A fte r incub ation the tissue was hom ogenized by son ica tion in 50 mM phosphate b u ffe r (pH 6.9) and am ylase was isolated fro m o th er pa ncre atic proteins and fro m free L-(U-14C)leucine in one step by isoelectric focusing in a thin la yer o f A m p h o lin e R-co n ta in in g p o ly a c ry la m id e gel. The am ylase co n ta in in g gel strip was cut out and dissolved in H2O2. Total pa ncre atic proteins w ere o b ta in e d by p re c ip ita tio n and repeated w ashing w ith TCA. In co rp o ra tio n o f L-(U-14C)leucine in to am ylase and to ta l pro te in was expressed in re la tio n to the tissue content o f D N A.
AMYLASE SECRETION
A Krebs-Ringer b ic a rb o n a te b u ffe r (pH 7.4) supplem ented w ith pyruvate, g lutam a te and fu m a ra te as w e ll as glucose and album in was used fo r batch- -incubations and perifusions. In the b a tch -in cu b a tio n experim ents the pancreas pieces were incubated in vessels spe cia lly designed fo r continuous e q u ilib ra tio n o f the medium w ith O2-C O2 (95:5) (I). Am ylase activities w ere determ ined in in cub ation m edia and supernatants o f pancreas hom ogenates, and related to the w et w e ig h t o f incubated tissue. The dynam ics o f the release process w ere in vestig ated in a spe cia lly designed pe rifusion system (I).
M ETABOLISM OF GLUCOSE A N D A M IN O ACIDS (V).
Pieces o f exocrine pancreas, p ra c tic a lly devoid o f islets, were incubated in a Krebs-Ringer b ica rb o n a te bu ffe r. O x id a tio n studies w ere pe rfo rm ed w ith d iffe re n t concentrations o f 14C-la,belled D-glucose, L-alanine o r L-leucine, as w e ll as un la b e lle d test substances. A fte r being tra p p e d in hyam ine the
produced 14C02 was measured. The uptake and tra n sp o rt o f L-àlanine and L-leucine w ere studied w ith 3H -la b e lle d am ino acids using a d o u b le -la b e l technique w ith 14C -la b e lle d L-glucose as an e x tra c e llu la r space m arker. This m ade it possible to correct fo r e x tra c e llu la r and c o n ta m in a tin g 3H -am ino acids w ith o u t w ashing the tissue. The data w ere expressed as mm ol substrate o xid iz e d o r taken up per kg dry w e ig h t o f pancreas.
STATISTICS
In the in vitro experim ents (I, II, III, V, VI) statistica l significances w ere d e te r
m ined by com p utin g t-values fro m the mean ± S.E.M. o f the differences between p a ire d test and con tro l incubations in a series o f repeated but separate experim ents. In the in vivo studies (III, IV, VI) differences between gro u p means w ere tested by the o rd in a ry Student’s t-test.
RESULTS A ND DISCUSSION
CHARACTERISTICS OF THE IN VITRO INCUBATED EXOCRINE PANCREAS Because the exocrine cells synthesize and secrete proteins at a high rate, the im p ortan ce o f oxygen and exogenous substrates fo r the adequate fu n ctio n o f pancreas pieces in vitro was e xp lo red (I). D in itro p h e n o l (DNP), an uncoupler o f o x id a tiv e p h o sp h o ry la tio n , o r an oxia reduced the basal secretion and abolishe d carb a m ylch o lin e -stim u la te d release o f am ylase.
Continuous e q u ilib ra tio n o f the m edium w ith O2-C O2 (95:5) du rin g the in cub atio n was necessary to achieve optim um secretory responses to ca rb a m ylch o lin e . The secretory responses w ere la rg e r and m ore re p ro d u cib le in m edia supplem ented w ith pyruvate, glutam a te , fu m a ra te and glucose than in m edia c o n ta in in g glucose alone. These results are consistent w ith the fin d in g s th a t a e ro b ic glycolysis accounts fo r less than 1 0 % o f the ATP p ro d u ctio n in ra t exocrine cells (Bauduin, C olin and Dum ont, 1969), and th a t the o x id a tio n 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 supplem ented b u ffe r, both ba tch-in cuba ted and perifused pieces o f mouse pancreas responded to known ch o lin e rg ic and g a stro in te stin a l secretogogues. H ow ever, continuous s tim ulatio n o f secretion fro m perifused mouse pancreas w ith CCK-PZ o r c a rb a m ylc h o lin e produced an in itia l rise o f am ylase release, fo llo w e d by a d e clin e in the release curve. S im ila r results w ere o b ta in e d by M atthew s, Petersen and W illia m s (1973) a fte r stim ulatio n o f the perifused ra t pancreas w ith acetylcho lin e. Repeated pulse-stim ulations seemed to be m ore effective in p ro vo k in g am ylase secretion (I). The reasons fo r the transien t nature o f the secretory response to p h y sio lo g ic a l stim uli are not clear.
S tim ulation o f secretion by CCK-PZ was accom panied by an increased rate o f o x id a tio n o f D-glucose, L-alanine and L-leucine (V). An enhanced o x id a tio n o f p a lm ita te has pre vio usly been observed in CCK-PZ stim ulated pigeon pancreas (W ebster, Gunn and Tyor, 1966). The increased rate o f o x id a tio n o f am ino acids was not caused by an enhanced uptake, since this process was un affected by CCK-PZ in the mouse pancreas (V). The o x id a tio n o f D-glucose in the exocrine cells was lo w com pared to th a t o f L-alanine and L-leucine (V).
EFFECTS OF STARVATION O N THE EXOCRINE PANCREAS
There has been a certain am ount o f controversy concerning the effects o f n u tritio n a l state on p ro te in synthesis in the pancreas. O n the one hand, starvatio n was fou nd to reduce the am ylase content (W ebster et al., 1972) and to in h ib it the p ro te in synthesis in the ra t (M orisset and W ebste r, 1972) and the pigeon pancreas (W ebster and Tyor, 1966); in the pigeon pancreas, starvatio n also induced a reduction o f the polysom e content and a reduced ca p a city o f the polysom es to synthesize p ro te in (Black J r and W ebster, 1973).
O n the o th e r hand, Poort and K ram er (1969) claim ed th a t the rate o f pro te in synthesis in the m am m alian pancreas is fa ir ly un affected by v a ria tio n o f the n u tritio n a l state in clu d in g s tarvatio n. In the present study, starvatio n fo r 24 hrs c le a rly reduced the rate o f in c o rp o ra tio n o f L-(U-14C)leucine in to pa ncre atic am ylase and to ta l p ro te in (VI). This in h ib itio n o f the synthesis m ay exp la in w h y starvatio n also induced a con sid erab le fa ll in the to ta l am ylase content (III) and decreased the num ber o f granules per unit cell area (IV). Refeeding restored the am ylase content w ith in 12 hrs (III).
EFFECTS OF GLUCOSE A N D ISLET H O RM O NES O N THE SYNTHESIS A N D STORAGE OF AMYLASE
S tarvation decreased the rate o f am ylase and to ta l p ro te in synthesis (VI) as w e ll as the content o f am ylase (III) and zym ogen granules (IV) in the pancreas. These effects m ay be due to the lo w e rin g o f b lo o d sugar, since the a d m in is tra tio n o f glucose to starved mice increased the am ylase con tent (III) and enhanced the synthesis o f both am ylase and to ta l p ro te in (VI). The ca p a city o f injected glucose to increase the am ylase synthesis and content was ab olishe d by c o n com ita nt a d m in is tra tio n o f d ia zo x id e , a potent in h ib ito r o f insulin secretion. It is th e re fo re conceivable th a t the effects o f glucose on am ylase synthesis and content w ere m ediated by stim ulatio n o f insulin release. In vivo a d m in is tra tio n o f insulin has previously been shown to rest
ore the reduced am ylase content and the decreased rates o f am ylase and
pro te in synthesis in a llo x a n -d ia b e tic rats (Palla, Ben A b d e ljlil and Desnuelle, 1968; Söling and Unger, 1972). In n o n -d ia b e tic rats, in jectio n o f insulin enhances both the secretion and the synthesis o f ra t pa n cre a tic proteins.
Couture, D unnigan and M orisset (1972) proposed th a t these effects are not due to insulin actin g d ire c tly on the pancreas but to hyp og lycem ia causing stim ulatio n o f the v a g a l nerve and the release o f ga stro in te stina l factors.
This hypothesis was not supported by the present studies, since stim ulatio n o f am ylase synthesis and content was observed in hyp erg lycem ic anim als w ith increased serum levels o f endogenous insulin (III, VI). O n the o th e r hand, studies on the in vitro effects o f glucose, insulin and gluca gon d id not reveal any changes in am ylase and to ta l p ro te in synthesis (VI), suggestig th a t the in vivo effects o f glucose may not be m ediated by a d ire ct actio n o f the sugar its e lf o r o f insulin on the pancreas. Reservations must, how ever, be m ade fo r the po ssibilities th a t the responsiveness o f the pancreas m ay be a lte re d by the in cub ation procedure and th a t the in cub ation tim e may be to o short in re la tio n to the con ceivable latency o f d ire ct insulin effects.
EFFECTS OF GLUCOSE A N D ISLET HO RM O NES O N THE SECRETION OF AMYLASE
In perifusions as w e ll as in ba tch -in cu b a tio n experim ents, glucose ra p id ly in h ib ite d both the basal and CC K-PZ-stim ulated am ylase secretion fro m the mouse pancreas (II).Insulin too, a t very high concentrations, exerted a s im ila r action. H ow ever, the glucose-induced reduction o f secretion did not seem to be m ediated by the release o f insulin, as d ia z o x id e did not counteract the effe c t o f glucose. Further sup po rt fo r the independence o f the glucose and insulin effects was o b ta in e d by studying secretion fro m m icrodissected pieces o f pancreas (VI). The in h ib itio n o f secretion seems to be con fine d to the secretory process, since, as described above, glucose and insulin in vitro had no effe ct on am ylase and to ta l pro te in synthesis. Cyclic AMP has been shown to be an in tra c e llu la r e ffe c to r o f p a ncre atic am ylase secre
tion (Kulka and S ternlicht, 1968; Bauduin et al., 1971) and insulin decreases the content o f cAMP o f the live r (Exton et al., 1966). It is not know n, how ever, w hethe r insulin affects cAMP in the exocrine pancreas.
G luca go n did not a ffe c t the basal o r CC K-PZ-stim ulated am ylase secretion from incubated pancreas (II). This is in contrast to results o b ta in e d in vivo.
The a d m in is tra tio n o f sm all doses o f gluca gon to man and dog induced a m arked reduction o f p ro te in ou tp u t fro m the pancreas d u rin g continuosly stim ulated secretion (Dyck et al., 1969, 1970). The la tte r observa tion m ay perhaps be exp la ine d by the g ly c o g e n o ly tic actio n o f gluca gon , le ad ing to hyp erglycem ia. The increased serum glucose level m ight reduce the dis
charge o f p a ncre atic enzymes, eith er alone o r in conjunction w ith the accom panying s tim ulatio n o f insulin secretion.
EFFECTS OF GLUCOSE A N D INSULIN O N THE METABOLISM OF THE EXOCRINE PANCREAS
G lucose in h ib ite d the o x id a tio n o f L-alanine. This e ffe ct m ay have been due to the in h ib itio n o f uptake, w hich was also noted in the experim ents (V).
The rate o f o x id a tio n o f glucose itself was re la tiv e ly lo w com pared to th a t o f L-alanine and L-leucine (V). Insulin stim ulated the o x id a tio n o f D-glucose but did not a ffe c t the o x id a tio n o f L-alanine o r L-leucine. Insulin also fa ile d to enhance the uptake o f L-alanine and L-leucine in vitro, w hich is o f interest in re la tio n to the suggestion th a t insulin stim ulates the synthesis o f pancreas proteins in vivo by enhancing am ino acid uptake (Soling and Unger, 1972).
SUMMARY A N D CONCLUSIONS
1. a) A m ic ro m o d ific a tio n o f the d in itro s a lic y la te m ethod fo r m easuring reducing groups m ade it possible to assay an am ylase a c tiv ity lib e ra tin g less than 10 /ug o f m altose equivalents.
b) By means o f isoelectric focusing in A m p h o lin e R-co n ta in in g p o ly a c ry la m id e gels, am ylase was h ig h ly p u rifie d fro m extracts o f tissue samples w ith less than 1 mg d ry w e igh t.
c) Routines w ere w o rke d out fo r the in cub ation o f pancreas pieces in closed vials and in a pe rifusion system fo r the study o f secretory d y n a mics. Continuous e q u ilib ra tio n w ith O2-C O2 (95:5) and the supplem enta
tion o f in cub ation buffers w ith glucose, pyruvate, glu ta m a te and fum a- rate w ere b e n e fic ia ry fo r the m agnitude and re p ro d u c ib ility o f am ylase secretory responses to established secretogogues.
2. S tarva tion decreased the synthesis and the pancreas content o f am ylase and dim inished the p o p u la tio n o f zym ogen granules. The decreases in am ylase synthesis and content w ere counteracted by in jections o f glucose to starved mice. This e ffe c t o f glucose was in turn ab olished by in jectin g d ia zo x id e , an in h ib ito r o f glucose-induced insulin release, in d ic a tin g th a t the glucose e ffe ct was m ediated via the discharge o f insulin.
3. Insulin, gluca gon and glucose, when adde d to the in cub ation medium, fa ile d to a ffe c t the synthesis o f amylase. How ever, glucose reduced both the basal and the CCK-PZ-induced am ylase secretion in a dose- dependent fashion. Insulin too , at a very high con cen tra tion , in h ib ite d the secretion o f am ylase. The effects o f glucose and insulin seemed to be m utually independent.
4. Insulin, at this high con cen tra tion , stim ulated glucose o x id a tio n w ith o u t a ffe c tin g the o x id a tio n o r uptake o f a lan ine o r leucine. CCK-PZ stim u
lated the o x id a tio n o f glucose, a la n in e and leucine in the pancreas pieces. G lucose in h ib ite d the uptake and o x id a tio n o f alan ine .
W ith reg a rd to the o v e rrid in g question th a t in itia te d the present study, it is concluded th a t the in vivo experim ents, n o ta b ly those dem on stra ting the effects o f glucose and d ia zo xid e on am ylase synthesis and content, support the idea th a t insulin influences the specific functions o f the a cin a r cells.
The in vitro experim ents revealed th a t insulin and glucose reduced am ylase secretion. If the la tte r fin d in g holds true fo r the in vivo situa tion , glucose a n d /o r insulin m ay w e ll co n trib u te to the m aintenance o f a high content o f am ylase in the exocrine pancreas. It is conceivable th a t the p e riin s u la r a c in a r cells are exposed to high concentrations o f insulin. The observed effects o f insulin on the biosynthesis and secretion o f am ylase m ig ht e xp la in the ap pearance o f ” ha lo zones” aro un d the islets. The fa ilu re o f insulin and glucose to a ffe c t the in vitro biosynthesis o f am ylase m ay in dica te th a t the in vivo effe ct o f glucose is not a d ire c t one o r m ediated by release o f in sulin.
ACKNOWLEDGEMENTS
I wish to express my sincere thanks to :
Professor Bo H eilm an fo r o ffe rin g me the p riv ile g e o f w o rk in g a t the D epartm ent o f H is to lo g y in Umeå, fo r in itia tin g this study and fo r placin g splendid la b o ra to ry fa c ilitie s a t my disposal.
Docent Inge-Bert T ä lje d a l, to whom I am in de bted fo r fru itfu l discussions, va lu a b le advice and constructive criticism .
Professor G un n a r D. Bloom fo r kind interest and stim ulatin g advice du rin g the course o f the w o rk, and Professor Sture Falkm er fo r h e lp fu l criticism .
M y co-authors, Dr. Bengt C arlsöö, Professor H e rbe rt F. H e lan de r, Docent Stefan M a rk lu n d , Docent Janove Sehlin and D ocent T orgny S tigb ran d, fo r in spiring c o lla b o ra tio n du rin g the various phases o f the investig ation .
Dr. Erik G y lfe , Docent Lars-Ake Ida hl, Docent Åke Lernm ark, Dr. Sam N o rlin and Dr. T orkel W a h lin , my o th e r colleagues a t the departm ent, w ho have co n trib u te d to the stim u la tin g atm osphere in w hich I have had the p riv ile g e to w o rk.
Miss M a ria n n e Borg, Mrs. Kerstin H jortsbe rg , Miss Karin Janze, Miss Kristina Karlsson, Mrs. A n n -C h a rlo tt Lundberg and Miss U lla N o rd m a rk fo r skilfu l technical assistance.
Miss K ristina Linder fo r typ in g the m anuscripts.
M r. P e r-O lo f Fredriksson and M r. Erik ö h lu n d fo r va lu a b le help w ith technical problem s.
Miss Janice Robbins, Dip. Ed., and Miss B arb ara Steele, F. L., fo r lin g u istic revision o f the m anuscripts.
The investigations w ere supported by grants fro m the Swedish M ed ica l Research C ouncil (12x-562), the Swedish Diabetes A ssociation and the M e dical Faculty, U niversity o f Umeå.
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