Diabetologia. Review Articles. Secretion of Somatostatin from the Normal and Diabetic Pancreas. Diabetologia 19, (1980)

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1 Dabetologa 19, (1980) Dabetologa 9 by Sprnger-Verlag 1980 Revew Artcles Secreton of Somatostatn from the Normal and Dabetc Pancreas Studes In Vtro K. Hermansen Second Unversty Clnc of Internal Medcne, Kommunehosptalet, Aarhus, Denmark Key words: Pancreas, dog pancreas, dabetes, somatostatn, solated perfused pancreas. Rapd progress n the study of pancreatc somatostatn secreton has taken place, snce ts dscovery n 1976 [65, 66, 79]. It has been shown that pancreatc D cell functon s subject to a multtude of control mechansms. Ths revew, whch deals mostly wth n vtro results from mammals, wll concentrate on three aspects: a) the mechansm and dynamcs of pancreatc somatostatn secreton; b) the factors that nfluence ts release; and c) secretory abnormaltes of the D cell n expermental dabetes. The perfused dog pancreas preparaton of Iversen and Mles [45] has turned out to be a useful and vald preparaton.for ths purpose. Methods for Studyng Pancreatc Somatostatn Secreton In Vtro Methods The applcaton of n vtro technques provdes a controlled envronment free of the hepatc, humoral and nervous regulatory mechansms present n vvo and makes t possble to dssect out ndvdual factors whch nfluence pancreatc somatostatn secreton. The solated perfused pancreas preparaton offers several advantages Over the other n vtro technques employed: a) t allows assessment of moment-tomoment changes n secreton; b) t makes possble the collecton of control and expermental data from the same pancreas; c) t has consderable functonal ntegrty wth ntact crculaton to the slets; d) t s exposed to mnmal ~nzymatc leakage from acnar tssue whch could result n mpared slet cell responses. Methods usng solated slets and monolayer cultures are defcent n these respects. A further problem arses n ncubaton studes where slet hormones accumulatng n the medum may nterfere wth the normal hormone secreton. In many perfuson methods currently used a porton of duodenum remans attached [17, 19, 20, 30, 36, 38, 64, 95]. The queston has often been rased whether the endocrne cells of the duodenum may contrbute to or modfy the secreton of pancreatc somatostatn measured n the pancreatco-duodenal efflux thereby makng nterpretaton of results more dffcult [40, 72, 76, 77]. It has, however, been found that n the dog dentcal release patterns of somatostatn as well as of nsuln and glucagon were obtaned whether or not the duodenum was excluded durng ether basal or stmulated condtons [25] (Fg. 1). For the evaluaton of somatostatn secreton perfused pancreas preparatons from dog [30, 38, 76], pg [73], rat [17, 19, 20, 36, 46], and chcken [36] have been used. The perfused chcken pancreas s perhaps napproprate f ones nterest s prmarly n mammalan systems snce slet morphology and pancreatc hormone responses of the avan class dffer dstnctly from those of the mammalan e. g. nsuln stmulates the pancreatc somatostatn secreton n the chcken [37] whle leavng t unaffected n mammals [34, 46, 63, 67, 106]. Comparson of the secretory pattern of somatostatn from perfused dog and pg pancreas wth that from perfused rat pancreas ndcates that the latter s a relatvely nsenstve model wth rather ndstnct somatostatn responses to the varous challenges and wth basal somatostatn levels often at or below the detecton lmts of the assay [19, 36, 46] X/80/0019/0492/$02.60

2 K. Hermansen: Pancreatc Somatostatn Secreton g ~o :D E Z CO 5 Z.=_. E z CO 9 O CO f 6 Ft'] 1 3'0 6'Omn. Fg. 1. Lack of effect of duodenal excluson on tolbutamde (T) (50 9g/ml) - stmulated somatostatn and nsuln release from the solated, perfused dog pancreas. The clampng off of the duodenum s ndcated by arrows It s obvously necessary to be sure that the somatostatn mmunoreactvty released from the pancreas s "true" somatostatn. Valdaton has come from the followng fndngs: the somatostatn lke mmunoreaetvty n pancreatc effluent samples shows parallelsm n bndng percentage wth the synthetc standard on seral dlutons [8, 19, 26], and chromatographs n a sngle peak correspondng to that of synthetc cyclc somatostatn [8, 19, 26]. Further support has been obtaned by purfcaton and chemcal structural analyss of the extracted mmunoreactve materal [57, 98]. In Vvo Methods It s obvous that there must be reservatons when tryng to extrapolate n vtro fndngs to the n vvo state. Unfortunately the nterpretaton of the results of perpheral plasma somatostatn determnatons n physologcal and pathophysologcal stuatons s hampered by varous dsadvantages. The wdespread locaton of somatostatn secretng cells [53] mples that changes n the levels of somatostatn n the perpheral crculaton may derve from varatons n the release from almost anywhere. Moreover, n vew of the portal-perpheral gradent [5, 86] and of the short half-lfe of somatostatn [71, 87, 97] t s apparent that changes n crculatng somatostatn levels r-~ may bear only a slender relaton to changes takng place n the pancreas. These problems can n part be crcumvented by cathersaton of the pancreatcoduodenal ven n large anmals [84, 86, 88, 91]. Even such results cannot, however, be regarded as wholly quanttatve snce blood flow measurements were not performed smultaneously wth blood samplng. Furthermore, the dffculty n obtanng relable and senstve somatostatn assays for measurements n plasma has slowed the progress of studes n ntact man and anmal. Mechansm of Somatostatn Secreton The term 'stmulus-secreton couplng' [13] has been used to cover the events occurrng n a secretory cell exposed to ts mmedate stmulus. Accordng to ths concept calcum s regarded as a substance whch couples the secretory sgnal to the dscharge of the secretory product from endocrne cells [13]. Another potental messenger s cyclc AMP (camp). The queston arses as to whether the effects of dfferent modulators of pancreatc somatostatn secreton are also medated va one or both of these second messengers. In ths context the effects of catons and cyclc AMP upon somatostatn secreton are relevant to our understandng of the mechansms of D cell secreton. Cyclc AMP and Somatostatn Secreton There s persuasve evdence that the release of somatostatn by the pancreatc D cell may be nfluenced by cyclc AMP dependent mechansms. Barden et al. [3] frst reported that hgh concentratons of 8-Br-cAMP stmulated somatostatn secreton from solated slets n the presence of a normal glucose level. Agents that are supposed to ncrease the cellular level of the nucleotde, such as glucagon [11, 46, 63, 65, 67, 78, 79, 105, 106],/3-adrenergc effectors [30, 33, 76, 77] and phosphodesterase nhbtors (theophyllne) [3, 20, 63, 78] ncrease the somatostatn secreton. Moreover dbutyryl cyclc AMP enhances somatostatn release [63]. Some evdence suggests, however, that elevated D cell cyclc AMP concentraton alone s not a suffcent stmulus to somatostatn release. Thus the very hgh concentraton of glucagon at 5 ~tg/ml [63, 78] and 1 mmol/1 theophyllne [20] were unable to cause a sustaned stmulaton of somatostatn release at a non-stmulatory glucose level. These fndngs conflct wth other studes where glucagon at a low glucose [106] and theophyllne n the absence of glucose [78] both elcted a modest somatostatn secreton.

3 494 K. Hermansen: Pancreatc Somatostatn Secreton Catons and Somatostatn Release The Role of Calcum. The key role of calcum for somatostatn secreton has been establshed n studes wth the perfused dog pancreas preparaton [24, 26, 30-33]. Extracellular calcum s an absolute requrement for the stmulaton of somatostatn release nduced by glucose [33], argnne [33], soproterenol [33], ouaban [24, 32], potassum [24, 31], and veratrdne [24, 26]. The somatostatn responses ncrease progressvely when perfusate calcum s ncreased from 0 through 1.25 and 2.5 to 5 mmol/1 [24, 30]. It s suggested that the ncrease n extracellular calcum leads to an ncrease n ntracellular calcum manly va an ncreased calcum nflux. The dea of a drect regulatory role of calcum s supported by the fact that a rse of the extracellular concentraton of calcum augments somatostatn release n the absence of the glucose [30]. When evaluatng the role of calcum n somatostatn secreton ts relaton to other catons especally the monovalent Na + and K + must be consdered. The Role of Na Partal replacement of extracellular Na + by cholne (100mmol/1 cholne, 40mmol/l Na +) provokes enhanced somatostatn secreton n the presence of extracellular calcum [24, 26]. It s possble that ths somatostatn secreton s due to a cholnergc effect of the hgh concentraton of cholne, snce acetylcholne has been shown to stmulate D cell secreto n [30]. The fact that the ncrease n somatostatn secreton whch results from a decrease n extracellular Na + concentraton s unaffected by the presence of 1 mmol/1 atropne seems to rule out ths possblty. When extracellular calcum s omtted from the medum, the somatostatn response to a drop n extracellular Na concentraton, however, dsappears [24, 26]. Ths ponts to the exstence n the D cell membrane of a Na+-Ca ++ counter transport mechansm analogous to that descrbed for other exctable tssues [6]. The somatostatn responses nduced by Na + deprvaton may thus be caused by an ncreased nflux of calcum nto the D cell coupled to an ncreased egress of Na +. These fndngs emphasze the mportance of extracellular Na + n somatostatn secreton. The rote of ntracellular sodum has been nvestgated by blockade of the Na+/K + pump. Ths process, whch ncreases ntracellular Na + content n a wde varety of cells ncludng pancreatc slet cells [93, 94], stmulates somatostatn secreton [24, 32]. Thus both the addton of ouaban and the omsson of extracellular K + from the medum elct an mmedate somatostatn response [24, 32]. In addton, the alkalod, veratrdne, whch ncreases Na + and calcum uptake through Na D- Z O3 9 9 CO o E E 0 ' 6'0 ' ran 1~0 Fg. 2. Effect of 10 ran ncreases of perfusate potassum from 4.4 to 6.9, 11.3 and to 18.2 mmol/1 on pancreatc somatostatn release from the solated dog pancreas. Perfusate calcum and glucose concentratons were 1.3 mmol/l and 5.5mmol/l, respectvely. Mean + SEM (n = 3) E O.. z t-- O _ E 0 E 6 3'0 6'0 mn Fg. 3. Lack of effect of 1 ~tmol/1 phentolamne (P) on potassum (18.2 mmol/1)-nduced somatostatn release from the solated dog pancreas. Glucose concentraton was 5.5 mmol/1 and calcum concentraton was 1.3 mmol/l. Mean + SEM (n = 3) channels n exctable tssue nduces a bphasc somatostatn response [24, 26]. The response to veratrdne s counteracted by tetrodotoxn (TTX) [24, 26], whch selectvely blocks Na+-channels and thereby the nward Na + current n exctable membranes.

4 K. Hermansen: Pancreatc Somatostatn Secreton 495 A clearcut dstncton exsts between effects obtaned n the presence and n the absence of extracellular calcum. Thus no ncrease n somatostatn secreton occurs n the absence of calcum when veratrdne [24, 26] or ouaban [24, 32] are added to the perfusate or K + s omtted [24, 32]. An ncrease n ntracellular Na + s supposed to lead to somatostatn secreton manly by causng a depolarzaton and an openng of voltage senstve calcum channels and an ncreased calcum uptake nto the D cell. Our fndngs make t unlkely, however, that an ncrease n ntracellular Na + evokes somatostatn secreton per se or va moblzaton of ntracellularly bound calcum. The Role of K +. An elevaton of the potassum concentraton resulted n a dose-dependent ncrease n the secreton of somatostatn [24, 31]. Even a small ncrease n K + from 4.4 to 6.9 mmol/1 results n ncreased somatostatn secreton (Fg. 2). It has prevously been demonstrated that a hgh external K + concentraton nduces depolarzaton [10] and rased calcum uptake n pancreatc slets [56]. The mmedate somatostatn response to the sudden rse of external K + may therefore be the consequence of an ncreased calcum nflux n the D cell. Ths s further supported by the fndng that the absence of extracellular calcum abolshes somatostatn secreton n response to hgh K + [24, 31]. Hgh potassum concentratons may also cause the release of neurotransmtters from nerve termnals. The queston arses therefore whether the somatostatn response to hgh extracellular K + s also due to neurotransmtter lberaton from nerve endngs n the slets. It s unlkely that acetylcholne release or catecholamne release from the parasympathetc and sympathetc nerve termnals s responsble snce hgh concentratons of atropne [24, 31], propranolol [24, 31], and phentolamne (Fg. 3) leave the potassum-nduced somatostatn secreton unaffected. There s also no mmunohstochemcal evdence for the presence of somatostatn contanng nerves n the pancreas [14]. The slets are, however, rchly nnervated wth other peptdergc nerve fbres whch contan somatostatn stmulatng peptdes such as CCK, VIP, and substance P [50, 52]. It cannot therefore be ruled out that such peptdergc nerves may to some degree be nvolved n the observed K + evoked somatostatn release. Conclusons The role of cyclc AMP n the control of somatostatn release may be prmarly that of a modulator wthout beng an essental factor for ntaton of somatostatn release. Much work s, however, stll requred to elucdate the exact nature of the role of cyclc AMP n the secretory mechansm of the D cell. All of the present evdence, however, ponts to a key regulatory role for calcum n the cascade of events that proceeds to the somatostatn secreton [24, 26, 30-33]. The data ndcate that the changes n somatostatn secreton provoked by alteratons n the extra- and ntracellular levels of Na + and K + are secondary to changes n the ntracellular level of calcum n the D cell. Cauton should, however, be exercsed n deducng from the results from caton fluxes n whole slets because of the mxed cell populaton studed. They cannot be assumed to represent the responses of the D cell alone because these cells make up only 5-15% of the total cell mass n the slet. Dynamcs of Somatostatn Secreton The secretory characterstcs of somatostatn release have much n common wth those of nsuln release. Thus n the solated perfused dog pancreas a square wave pulse of hgh glucose nduces a bphasc ncrease n the secreton of somatostatn [27, 29, 36, 105]. It conssts of a peak release startng 1-2 mn after the glucose challenge (Frst phase); after about 5 ran ths s followed by a lesser more prolonged secreton (Second phase). Although one group has reported to the contrary [81, 82], the omsson of glucose results n an mmedate declne n somatostatn release [17, 29, 36, 38, 105]. The dynamcs of the somatostatn release are concentraton-dependent. Low concentratons of glucose thus merely elct a transent burst of secretory actvty, and the bphasc nature of the secretory response manfests tself only as the glucose level s ncreased [29]. Bphasc profles of somatostatn secreton are also obtaned wth hgh concentratons of agents such as argnne [19, 20, 30, 106], leucne [38], glucagon [105], CCK-8-S [27, 40], substance P [27], soproterenol [30], and tolbutamde [38]. Usng the dog pancreas preparaton t has been found that not all responses are typcally bphasc. Thus the responses to potassum [31], dhydroxyacetone [29], and D-glyceraldehyde [29] dffer from the usual response pattern. A monophasc response s prompted by potassum ([31] Fgs. 2 and 3), whle a gradually ncreasng release s seen n response to dhydroxyacetone [29]. The underlyng mechansm responsble for the bphasc somatostatn response s obvously of nterest. It may be due to the exstence of two pools of somatostatn analogous to the proposals for the pancreatc B cell [9, 69]. Another suggeston s that

5 496 K. Hermansen: Pancreatc Somatostatn Secreton somatostatn released durng the frst phase nhbts subsequent secreton. Thus Ipp et al. [43] have demonstrated an nhbtory effect of somatostatn on somatostatn secreton by the dog pancreas. Recently, Stagner et al. [99] made the nterestng observaton n the perfused dog pancreas of regular, sustaned oscllatons of somatostatn wth a 10 mnute perod. The monophasc cycle curves n vtro were not produced by adrenergc or cholnergc mechansms and were not a result of glucose changes [99]. The drve may come from spontaneous frng of peptdergc nerves n pancreas [50, 52]. By consderng the pancreatc D cell as a neuroendocrne cell [68] spontaneous actvty n the D cell tself becomes another possblty. The nature of the pacemaker that serves to drve slet cycles s, however, not known. Factors Modfyng Normal Pancreatc Somatostatn Secreton Carbohydrates Glucose stmulates somatostatn secreton n vtro [17, 19, 27, 29, 33, 38, 63, 79, 105] although one group of nvestgators were not able to demonstrate any ncrease n somatostatn release from ncubated rat slets [3]. Increased perfusate glucose also markedly enhances the somatostatn response to agents such as calcum [24, 30], and substance P [27]. A sgmodal curve characterzes the relatonshp between the glucose concentraton of the extracellular medum and the rate at whch somatostatn s released [29]. The D cell threshold for glucose s close to 5 mmol/1 n the solated dog pancreas [28, 29, 38] and to 10 mmol/1 n ncubated rat pancreatc slets [19, 78]. In the dog half maxmal release occurs between 5 and 10 mmol/1 and maxmal release s reached at about 20 mmol/1 [28, 29]. The steep porton of the dose-response curve for somatostatn release from the dog pancreas [29] corresponds to the range of glucose found n the blood postprandally, whch suggests a possble physologcal role for the glucose-nduced somatostatn release. How the pancreatc D cell recognzes and responds to glucose s unknown. In analogy wth the suggested models for the B cell glucoreceptor [10, 49, 70] at least three explanatons can be offered. Somatostatn release may be trggered by the ntact glucose molecule tself or t may be that the metabolsm of glucose wthn the D cell provdes ntracellular sgnals whch ntate somatostatn secreton; fnally the two models may functon smultaneously. Several lnes of evdence seem to ndcate that the "E z ~- 100 ~9 0 9 co 0 j, I t f I f I! 0 30 E! 60ran. Fg. 4. Effect of 10 ran nfuson of 3 and 10 mmol/1 DL-3-hydroxybutyrc acd (BOH) on the somatostatn secreton from the solated perfused dog pancreas. Perfusate glucose was 5.5 mmol/l somatostatn stmulatory acton of glucose s dependent on ts metabolsm. Frst, the troses, dhydroxyacetone and D-glyceraldehyde, whch enter the glyocolytc pathway at the trose phosphate level, both stmulate somatostatn secreton n the absence of glucose [28, 29]. D- Glyceraldehyde seems, however, to exert a dual acton on pancreatc somatostatn secreton snce hgh concentratons suppress D cell secreton [28, 29, 54, 78]. The nhbtory effect of large concentratons of D-glyceraldehyde could be explaned by a decrease n slet cell levels of ATP [2]. Secondly, mannoheptulose, whch markedly nhbts slet glucose metabolsm but not that of D- glyceraldehyde, dramatcally nhbts glucose stmulated somatostatn secreton [28, 29, 79, 83] whle t has no effect on the D cell response to D-glyceraldehyde [28, 29]. Thrdly, the D cell secreton s unaffected by nonor poorly metabolzed sugars such as rbose [29], fructose [29], and galactose [29, 63]. Non-carbohydrate Nutrents Argnne [17, 19, 20, 30, 33, 63, 66], leucne [38, 78], a 10 amno acd mxture [38] and the amno-acd metabolte a-ketosocaproc acd [78] are all powerful stmulators of somatostatn secreton. The mplcatons of the fndng that a-ketosocaproc acdstmulated D cell secreton s nhbted by epnephrne [83] but not by mannoheptulose [83] s uncertan. The ketone body 3-hydroxybutyrate also augments somatostatn secreton by the solated dog pancreas (Fg. 4). In ths context t s nterestng to note that heparn and ntralpd n vvo cause a clearcut ncrease n crculatng somatostatn levels [104]. The ncrease may be attrbuted to ether free fatty acds or to ketone bodes.

6 K. Hermansen: Pancreatc Somatostatn Secreton 497 Effects of Pancreatc Islet and Gut Hormones It s apparent from n vvo studes [86, 90] that the responses of pancreatc somatostatn to the admnstraton of nutrents are medated, by other factors as well. These addtonal medators could be endocrne or nervous. Thus a somatostatn stmulatng peptde present n nerves and/or endocrne cells of the slets may be an mportant component of a physologcal somatostatn response wthout showng alteratons n the perpheral plasma levels. Islet Hormones. Insuln [34, 46, 63, 65, 67, 106] and PP [39] have no demonstrable effects on somatostatn secreton by mammalan pancreas. In contrast, glucagon has consstently been reported to stmulate pancreatc somatostatn secreton [11, 46, 63, 65, 67, 79, 105], the effect beng dose dependent [46, 105]. The queston whether somatostatn exerts a drect nhbtory acton on ts own secreton s of obvous mportance snce such a feed-back mechansm may contrbute to the bphasc nature of the somatostatn response to varous agents. Two somatostatn analogues ((D-TrpS-D-Cys14)-somatostatn and AsnS-(D - TyrS)-somatostatn) whch are non-mmunoreactve to certan somatostatn antbodes nhbt D as well as A and B cell secreton n response to glucose, argnne, and CCK-PZ [43]. Accordng to the paracrne theory of Orc and Unger [61] such a decrease n somatostatn secreton could, however, theoretcally be promoted ndrectly va a suppresson of endogenous glucagon secreton. The fndng that a hgh analogue concentraton s also capable of blockng D cell secreton despte the coperfuson of 10 rg/ ml glucagon, however, ponts towards a self-nhbtng acton on the natve hormone [43]. The fnal proof must awat the development of an analogue wth a selectve effect on the D cell. Gastrontestnal Polypeptdes. The somatostatn releasng potency of a large number of gastrontestnal polypeptdes has been examned n vtro. Among these all of the establshed hormones, CCK-PZ [38], gastrn [40, 73], gastrc nhbtory polypeptde (GIP) [40], and secretn [40] augment the secreton of somatostatn from the solated perfused pancreas. In addton the synthetc octapeptde (CCK-8), exerts an enhancng effect on somatostatn release [27, 40]. Smaller molecular forms of CCK also stmulate D cell hormone secreton. Thus CCK-3, CCK-4, and CCK-5 at doses rangng between 10-7 to 10-3 mmol/1 cause a dose-related somatostatn secreton from the solated porcne pancreas [73]. CCK- 4 s far the most potent of these peptdes [73]. Somatostatn secreton s also stmulated by vasoactve ntestnal polypeptde (VIP) [27, 41] and sub- stance P [27], both of whch are found n neurones n the pancreas [50, 52]. It s unknown whether the stmulant acton on the D cell s aded by a vasodlator effect on slet capllares. Our results are consstent wth the vew that substance P even at concentratons whch can be delvered to the pancreatc slet cells by the crculaton stmulates the secreton of somatostatn n a dose-related manner [27]. In contrast bombesn, a newly recognzed tetradecapeptde contaned n endocrne cels n duodenum, has no effect on somatostatn secreton [27]. The reported effects of neurotensn are, however, puzzlng. Dolas-Ktabg [11, 12] found that 100nmol/1 neurotensn stmulated somatostatn release at low glucose (3 mmol/1), that t dd not alter somatostatn secreton n response to hgh glucose (12 mmol/1) and glucagon (2 ~tg/ml) and ndeed nhbted D cell secreton durng stmulaton wth very hgh glucose (23 mmol/1) or argnne (20 mmol/1) concentratons. Snce the D cells of ther preparaton of solated slets are unresponsve to varaton n glucose from 3 to 23 mmol/1 and to addton of 2 ~tg/ml glucagon [11, 12] the results must be vewed cautously. The demonstraton of mmunoreactve enkephaln n pancreas [50] ponts to a possble regulatory role of ths opod on slet cell functon. Ipp et al. [42] have reported that another endogenous opod, /3- endorphn, and the exogenous opate, morphne sulphate, exert nhbtory effects on somatostatn secreton, whch could be abolshed by the antagonst naloxone hydrochlorde. Whether locally secreted enkephaln nfluences such hypothetcally opate receptors n the slets s, however, unknown. The physologcal meanng of the observatons of peptde-nduced alteraton n D cell secreton s stll obscure snce no data are avaable on the concentraton of the peptdes surroundng the slet cells n dfferent physologcal condtons. Nervous Control of Pancreatc Somatostatn Secreton In most mammals three man types of nerves have been demonstrated n the pancreas: adrenergc, cholnergc and peptdergc [50, 52]. Usng the solated dog pancreas, Samols et al. have shown that a-adrenergc stmulaton nhbted somatostatn secreton [76, 77], a concluson that s supported by the results of Ipp et al. [39]. Furthermore, Samols et al. [76, 77] showed that the /3- adrenergc effects of epnephrne and soproterenol were to enhance somatostatn secreton. Our studes confrm ths latter fndng [30, 33]. The admnstraton of epnephrne alone, at doses of 2 ~tg/ml and

7 498 K. Hermansen: Pancreatc Somatostatn Secreton 10ng/ml, respectvely, elcts an nhbton n somatostatn release from rat slets [79, 83] and dog pancreas [39]. Usng the smaller epnephrne dose of 2 ng/ml Samols et al. [76, 77], however, could fnd no sgnfcant change n somatostatn secreton. Dopamne, whch s present n pancreatc slets n hgh concentratons [107] has also been shown to alter somatostatn secreton. Dopamne (10-4mmol/1) nhbts somatostatn release, an acton whch can be prevented by the dopamnergc antagonst (+) butaclamol ( mmol/1) [4]. It s not known f dopamne s a neurotransmtter n the pancreas, and the sgnfcance of these fndngs reman therefore to be elucdated, but a role for other adrenergc agents seems possble. Acetylcholne, the transmtter of the parasympathetc nervous system, exerts only a mnor and varable effect on hormone secreton from D cells as compared to the acton on the A, B, and PP cell secreton [35]. Samols et al. [77] reported a mld suppressve effect of 10 ~tmol/1 acetylcholne on somatostatn secreton. In contrast we found a modest stmulaton of somatostatn release by 1 ~tmol/1 acetylcholne [30, 33]. By usng another source of acetylcholne [35], we, however, were unable to demonstrate any effect on the somatostatn release. A sgnfcant pont n ths context may be that there s large nterndvdual dfferences between pancreases n the senstvty of the D cells to acetylcholne. Thus current experments wth the more stable carbamylcholne chlorde demonstrate that whle a defnte dose-related stmulaton of somatostatn release s obtaned n some pancreata, others are refractory (K. Hermansen, unpublshed observatons). The reason for ths puzzlng phenomenon s unknown, but t seems that cholnergc mechansms play no major regulatory role n pancreatc D cell secreton. Peptdergc nerves are also found n the pancreas. They can release substance P, VIP, enkephaln, and gastrn/cck [50, 52]. The effects of these peptdes on the pancreatc somatostatn secreton have already been descrbed. No somatostatn contanng nerves are, however, present wthn the pancreas [14]. e. the somatostatn measured n the efflux from slets or the entre pancreas derves exclusvely from endocrne cells. The fact that certan of the effects on pancreatc hormone secreton nduced by pancreatc nerve stmulaton cannot be prevented by adrenergc or cholnergc blockade, strongly suggests an mportant regulatory role of the peptdergc nerves. In concluson, the rates at whch somatostatn s released from the pancreas s now known to be modfed by the autonomc nnervaton. However, the evdence s as yet fragmentary and the extent to whch neural mechansms may be ntegrated n the producton of the somatostatn responses to physologcal stmul certanly needs further exploraton. Somatostatn Release n Response to Pharmacologcal Agents Ipp et al. [38] have demonstrated that tolbutamde n the presence of 5.5mmol/1 glucose stmulates somatostatn secreton. Smlarly Samols et al. [74] found a stmulatory effect of tolbutamde (130 ~tmol/1) on D cell secreton, greater, however, at low (0.8 mmol/1) than at normal glucose levels (4.9 mmol/1). The somatostatn response to the sulfonurea, glbenclamde, was also found to be lowest at hgh glucose concentratons [15]. Furthermore, glbenclamde (1 ~tg/ml) sgnfcantly enhanced the effect of argnne on somatostatn release [16]. In contrast, the hyperglycemc sulfonamde, dazoxde (130~tmol/1) nhbts somatostatn secreton [77]. Wth respect to the effects of sulphonylureas, there s thus agan a strkng smlarty between D and B cell responses. Functonal Role of Pancreatc Somatostatn Local Role? The presence n pancreatc slets of D cells n juxtaposton to the B and n partcular the A cells had led to the postulate of a local regulatory role for somatostatn on the pancreatc release of glucagon and nsuln, that s a paracrne functon of the D cell [61]. The facts that the pancreatc D cells secrete somatostatn and that slet cells can be nfluenced by the secretory products of other slet cells make ths an attractve possblty. The asserton of Larsson et al. [51] that the pancreatc D cells have nonlumnal cytoplasmc processes servng as pathways for paracrne secreton suggest that the D cells may regulate secreton of neghbourng slet cells through somatostatn release from both the cell body and cytoplasmc processes. The presence of tght junctons [60] and gap junctons [62] may also be of functonal mportance. The demonstraton by Kohen et al. [48] of a glucose-enhanced transfer of fluorescen from an slet cell drectly nto neghbourng slet cells supports the vew that cell-to-cell communcaton may be mportant for the secretory functon of all slet cells. Whether somatostatn tself or ons or metaboltes serve as sgnals between D cells s unknown. One approach to answer the queston about drect wthn-slet actons has been the attempt to

8 K. Hermansen: Pancreatc Somatostatn Secreton 499 neutralze pancreatc hormones wth antbodes. In vvo, admnstraton of ant-somatostatn serum left both basal and stmulated glucagon and nsuln levels unchanged, although plasma growth hormone levels rose [1, 89, 102]. Smlarly, actve mmunzaton resulted n an ncrease of serum growth hormone levels, but dd not affect basal and stmulated secreton of glucagon [100]. Ths has been nterpreted as ndcatng that any nfluence of endogenous somatostatn on hormone secreton from the A and B cells takes place va a pathway naccessble to the blood borne antsomatostatn serum [1, 89]. The dssocaton of the effects of antsomatostatn serum on slet hormones and growth hormone levels may, however, reflect a dfferental senstvty to somatostatn. Thus nsuffcent somatostatn antbody may have been admnstered to neutralze enough somatostatn to alter nsuln and glucagon secreton. Hgher concentratons of slet hormone antbodes can, however, be obtaned n the vcnty of the slet cells when antbodes are appled to solated slets. Usng ths technque Tanguch et al. [101] found that nsuln secreton was ncreased at low (3.3 mmol/1) and moderate (8.3 mmol/1) glucose levels, whle t was unchanged at hgh glucose (16.7mmol/1). In contrast, Barden et al. [4] found no elevaton of nsuln secreton at a normal glucose level (5.5 mmol/1) whereas a marked ncrease n glucagon release appeared followng neutralzaton of endogenous somatostatn. Antglucagon serum has also been shown to produce a small decrease n basal secreton of somatostatn from rat slets whereas attempts to neutralze nsuln leave somatostatn release unaffected [63]. It has been suggested that f neutralsaton of an slet hormone alters the release of one of the other slet hormones ths favours the exstence of a paracrne system of secretory control wthn the pancreatc slets [1, 63]. Ths s, however, not necessarly correct snce t remans probable that the detected changes n release of slet hormones are due solely to neutralsaton of the hormone stored between and not wthn slets. Other anatomcal features could also be relevant to a local role for somatostatn. The local portal vessel system n the slets [18] may allow secreted somatostatn to act on slet cells wthn the same slet. Although local such a mechansm should be consdered as endocrne. The qualtatve heterogenety of the responses of A, B, and D cells to dfferent agents seems, however, dffcult to nterpret n terms of ntra-slet feed-back mechansms. The development of specfc D cell agonsts or antagonsts may help to gan further nsght to ths problem. Endocrne Role? In addton to potental actons wthn the slets several lnes of evdence pont to an extra-sletendocrne role for pancreatc somatostatn. Frst of all, pancreatc somatostatn secreton s stmulated by nutrents and gastrontestnal hormones both n vvo [74, 86, 88] and n vtro [4, 17, 19, 27, 30, 33, 38, 40, 63, 65, 66, 79, 105, 106]. Also, n the perpheral crculaton varatons n the somatostatn level can be seen n response to physologcal stmul. Secondly, bologcal actons can be produced by physologcal ncrements n the somatostatn level. The ntraportal nfuson of synthetc somatostatn at a rate whch nduces a physologcal rse n perpheral somatostatn s assocated wth a sgnfcant decrease n the rate of entry of nutrents from the gut nto the crculaton [85]. Moreover, nhbton of nsuln and glucagon secreton from the solated dog pancreas s elcted by somatostatn concentratons whch can easly be acheved n the perphery n vvo n response to varous physologcal stmul [35]. Although somatostatn has a short half-lfe [71, 87] and despte the dffcultes n adressng the multple actons of somatostatn on endocrne and exocrne functons (for revews see [22, 53]) substantal evdence ndcates that the acton of ths peptde extends beyond those of a potental paracrne nature. In addton to regulatng nutrent nflux from the gastrontestnal tract, somatostatn may have other actons whch contrbute to nutrent homeostass. Thus t may affect glucose metabolsm by drectly or ndrectly alterng hepatc glucose output (for revew see [55]), glucose dsposal to tssues [44] or central nervous system actons on glucoregulaton [7]. At present, a cybernetc system wth pancreatc somatostatn actng as a regulatory messenger n the gastroenteronsular axs seems possble. In response to ngeston of nutrents, the gastrontestnal hormones, the gastrontestnal nervous system and the absorbed substrates may determne the secretory rate of pancreatc somatostatn whch n turn acts as modulator of the secreton of gastrontestnal hormones (for revew see [22, 53]), of neuronal functon (for revew see [53]), and of substrate absorpton [85, 92, 103]. Fastng and D Cell Functon Fastng results n a decrease n portal somatostatn level n rats [47, 96] ndcatng a decreased secretory actvty of pancreatc D cells durng fastng. These two studes are not consstent wth the n vtro results of Schauder et al. [80] whch showed that slets from fasted rats release sgnfcantly more somatostatn

9 500 K. Hermansen: Pancreatc Somatostatn Secreton than controls n the presence of 3.3 mmol/1 glucose, whereas the release at 10 or 25 mmol/l glucose was n the normal range. However, basal somatostatn secreton from the perfused rat pancreas s attenuated [95]. Of nterest s the fndng that n the fasted state somatostatn release s apparently ndependent of the prevalng glucose concentraton (3.3, 10 or 25 mmol/1) [80] suggestng that the fasted slet D cells have at least temporarly lost ther ablty to recognze glucose, whereas they stll respond to argnne [95]. 03 Q. z 9 co Dabetes and D Cell Functon The observatons of both an ncrease n the slet and pancreatc contents of somatostatn [58, 64], of an augmentaton of D cell number [59] and of ncreased plasma somatostatn levels n nsuln-defcent dabetes [47, 84] has rased the possblty that abnormaltes n pancreatc somatostatn secreton may be present n dabetes. Ths suggeston s supported n a recent report of Schusdzarra et al. [90] demonstratng that the varatons n perpheral plasma somatostatn levels n alloxan dabetc dogs dffered from those of normal dogs. In the expermental dabetc dogs the rse n plasma somatostatn levels to a proten rch meal s delayed, but surpasses that seen n normals [90]. The postprandal as well as the fastng plasma somatostatn levels were unchanged n normals [90] whle they were dmnshed although not to normal, n the dabetc dogs durng nsuln admnstraton [90]. In contrast to what s seen n normal dogs no sgnfcant change n plasma comatostatn level appeared n response to orally or parenterally admnstered glucose [90]. We have also found that nether somatostatn nor glucagon secreton from the solated pancreas of streptozotocn dabetc dogs altered n response to varatons n perfusate glucose, manoeuvres whch n pancreata from normal anmals are nvarably accompaned by swft but opposte changes n hormone release from the D and A cells [34]. In contrast other substances such as argnne, the fl-adrenergc agonst soproterenol, calcum, and glucagon (Fg. 5), all stmulated the release of somatostatn from the dabetc pancreas [34] as n the normal pancreas. Hara et al. [22] found that both basal and argnne stmulated somatostatn release from perfused pancreas of alloxan dabetc rats were approxmately twofold ncreased as compared to normals, whereas the glucagon levels were decreased. It s not clear whether the D and A cell abnormaltes descrbed [34] are secondary to nsuln defcency. However, the fndng that nsuln treatment n I BLUCA60N 3rm I I '5 ran. Fg. 5. Somatostatn secreton from the solated pancreas of streptozotocn dabetc dogs (n = 5) durng 10 mn nfuson of 3 nmol/1 glucagon. The glucose concentraton was 8.3 mmol/1. The dogs were operated on 3 to 5 days after the admnstraton of streptozotocn (50 mg/kg) vtro dd not cause even a hnt of normalzaton of D and A cell secretory responses to glucose favours the vew that the D and A cell dysfunctons represent abnormaltes ndependent of nsuln secreton [34]. The dversty between n vvo [90] and n vtro results [34] concernng the effect of nsuln on the somatostatn level n dabetes remans unexplaned. However, smlar secretory characterstcs exst for glucagon. It could be speculated that the ncreased somatostatn levels n acute expermental dabetes [47, 84, 90] were at least partly nduced by ether ncreased concentratons of ketone bodes or hgh glucagon levels, the latter beng able to augment somatostatn secreton from the solated pancreas of streptozocn dabetc dogs (Fg. 5). A possble explanaton s that expermental dabetes mght be an overall slet cell dsease due to a common, rather selectve dysfuncton of the glucoreceptor, whch n ths context ncludes both models: the regulator ste and the substrate ste models [70]. The metabolc abnormaltes characterzng the dabetc state may be caused by a dysfuncton of D as well as of A and B cells n the pancreas (or possbly of all cells?) [34]. To explore the molecular bass of the D cell mparement n expermental dabetes we have nvestgated the nfluence of varous carbohydrates on somatostatn secreton n vtro and compared them wth the normal responses [28]. The glucose metaboltes D-glyceraldehyde (1.25 and 2.5 mmol/1) and dhydroxyacetone (11 mmol/l) whch normally stmulate somatostatn secreton do not augment

10 K. Hermansen: Pancreatc Somatostatn Secreton 501 somatostatn secreton from the perfused pancreas of streptozotocn dabetc dogs [28]. Although D-glyceraldehyde at hgh doses causes an nhbton of somatostatn secreton n normal [28, 29, 54, 78] as well as n dabetc pancreas [28] ths may well be due to nonspecfc effects through ATP depleton [2]. In addton mannoheptulose (5 mmol/1) has no effect on somatostatn secreton from the dabetc dog pancreas [28]. Thus the dabetc D cell has lost ts ablty to respond to glucose and ts metaboltes. Whether the D cell abnormaltes are caused by a drect acton of streptozotocn, by longstandng nsuln defcency n the vcnty of the D cell or are secondary to alteratons n ntermedary metabolsm remans to be solved. If one physologcal functon of pancreatc somatostatn s to restran nutrent entry from gut as suggested by Unger et al. [92, 95, 103] t could be expected that abnormaltes n pancreatc somatostatn secreton would also contrbute to the metabolc derangement n expermental dabetes. If the normal ncrease n plasma somatostatn to glucose s extnct, one would predct, that the negatve feed-back control over the rate of glucose entry should dsappear. Ths would result n a more rapd entry of glucose and an augmentaton of the arteral concentraton of glucose. The absent ncrease n somatostatn secreton to glucose may further ndrectly contrbute to the metabolc abnormaltes n dabetes due to the potental loss of glucagon suppresson thereby allowng enhanced heptatc glucose producton and ketogeness. On the other hand the hypersomatostatnaema n nsulnopenc dabetes may reflect a futle compensatory effort to correct the hyperglycaema. Although stll at an early stage studes on D cell functon n normal and dabetc anmal models seem to be a fertle feld. The results ndcate that much can be learned from such studes and wth respect to dabetes t may prove frutful to use other expermental models. References 1. Armura A, Coy DH, Chhara M, Fernandez-Durango R, Samols E, Chhara K, Meyers CA, Schally AV (I978) Somatostatn. In: Bloom SR (ed) Gut hormones. Churchll Lvngstone, Ednburgh London New York, p Ashcroft SJA, Chatra L, Weerasnghe C, Randle PJ (1973) Interrelatonshp of slet metabolsm, adenosne trphosphate content and nsuln release. Bochem J 132: Barden N, Alvarado-Urbna G, Cote J-P, Dupont A (1976) Cyclc AMP-dependent stmulaton of somatostatn secreton by solated rat slets of Langerhans. Bochem Bophys Res Commun 71: Barden N, Cote J-P, Lavoe M, Dupont A (1978) Secreton of somatostatn by rat slets of Langerhans and gastrc mucosa and a role for pancreatc somatostatn n the regulaton of glucagon release. Metabolsm 27 [Suppl 1]: Berelowtz M, Kronhem S, Pmstone B, Shapro B (1978) Somatostatn-lke mmunoreactvty n rat blood. 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12 K. Hermansen: Pancreatc Somatostatn Secreton 503 the ntercellular communcaton between a- and fl-cells n the endocrne pancreas. J Cln Invest 56: Orc L, Unger RH (1975) Functonal subdvson of slets of Langerhans and possble role of D-cells. Lancet II: Orc L, Unger RH, Renold AE (1973) Structural couplng between pancreatc slet cells. Experenta 29: Patel YC, Amherdt M, Orc L (1979) Somatostatn secreton from monolayer cultures of neonatal rat pancreas. Endocrnology 104: Patel YC, Wer GC (1976) Increased somatostatn content of slets from streptozotocn-dabetc rats. Cln Endocrnol (Oxf) 5: Patton GS, Dobbs RE, Orc L, Vale W, Unger RH (1976) Stmulaton of pancreatc mmunoreactve somatostatn (IRS) release by glucagon. Metabolsm 25 [Suppl 1]: Patton GS, Ipp E, Dobbs RE, Orc L, Vale W, Unger RH (1976) Response of pancreatc mmunoreactve somatostatn to argnne. Lfe Sc 19: Patton GS, Ipp E, Dobbs RE, Orc L, Vale W, Unger RH (1977) Pancreatc mmunoreactve somatostatn release. 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13 504 K. Hermansen: Pancreatc Somatostatn Secreton Isolaton and characterzaton of somatostatn from pgeon pancreas. Proc Natl Acad Sc USA 76: Stagner JI, Samols E, Wer GC (1980) Sustaned oscllatons of nsuln, glucagon, and somatostatn from the solated canne pancreas durng exposure to a constant glucose concentraton. J Cln Invest 65: Stewart JK, Stener RA, Barber J, Koerker DJ, Goodner CJ, Game GC (1977) Effects of actve mmunsaton aganst somatostatn on secreton of growth hormone and nsuln n adolescent male baboons. Program of 59th Annual Meetng of the Endocrne Socety, 348A 101. Tanguch H, Utsnm M, Hasegawa M, Kobayash T, Watanabe Y, Murakam K, Sek M, Tsutou A, Makmura H, Sakoda M, Baba S (1977) Physologc role of somatostatn. Insuln release from rat slets treated by somatostatn antserum. Dabetes 26: Tannenbaum GS, Epelbaum J, Colle E, Brazeau P, Martn JB (1978) Dssocaton of effects of somatostatn antserum on growth hormone and nsuln secreton. Metabolsm 27 [Suppl 1]: Unger RH, Ipp E, Schusdzarra V, Orc L (1977) Hypothess: physologc role of pancreatc somatostatn and the contrbuton of D-cell dsorders to dabetes melltus. Lfe Sc 20: Wasada T, Petr A, Raskn P, Dobbs R, Unger RH (1980) Effect of free fatty acds (FFA) on the gastrc and pancreatc secreton of somatostatn-lke mmunoreactvty. Dabetes 29 [Suppl 2]: 9A 105. Wer GC, Samols E, Day JA, Patel YC (1978) Glucose and glucagon stmulate the secreton of somatostatn from the perfused canne pancreas. Metabolsm 27 [Suppl 1]: Wer GC, Samols E, Loo S, Patel YC, Gabbey KH (1979) Somatostatn and pancreatc polypeptde secreton: effects of glucagon, nsuln, and argnne. Dabetes 28: Zern RT, Foster LB, Blalock JA, Feldman JM (1979) Characterstcs of the dopamnergc and noradrenergc systems of the pancreatc slets. Dabetes 28: Receved: September 9, 1980 K. Hermansen 2nd Unversty Clnc of Internal Medcne Kommunehosptalet DK-8000 Aarhus C Denmark