Springer-Verlag 1981

Transcription

1 Diabetlgia (1981) 21: Springer-Verlag 1981 The Priming Effect f Glucse n nsulin Secretin frm slated slets f Langerhans J. P. Ashby and D. Shirling Metablic Unit, University Department f Medicine, Western General Hspital, Edinburgh, Sctland Summary. Biphasic insulin secretin frm perifused rat islets f Langerhans was enhanced if islets had previusly been stimulated with glucse 16.6 mml/1. The priming effect f glucse was reduced if mannheptulse (16.6 mml/1), deuterium xide (D; 98% v/v) r adrenaline (1 ~tml/l) was included in the medium during the initial stimulatin perid, r if Calcium was mitted. Glyceraldehyde (16.6 mml/1) but nt thephylline (5 mml/1) culd substitute fr glucse during the initial stimulatin and make islets mre respnsive t subsequent stimulatin. The results suggest that the priming effect f glucse n insulin secretin may be related t 1) glucse metablism and 2) Ca fluxes in the B cell and the cnsequent activatin f the micrtubular system. Neither the generatin f intracellular cyclic AMP nr the release f insulin per se appears t be invlved in the priming prcess. Key wrds: Biphasic insulin secretin, perifused islets, glucse priming, mannheptulse, glyceraldehyde, calcium, deuterium xide, cyclic AMP, adrenaline. Acute expsure f the rat pancreatic B cell t glucse leads t biphasic secretin which is characterised by a shrt-lived burst f insulin release (phase 1) fllwed by a slwer prgressive rise in the secretry rate knwn as phase 2 [1-4]. Studies in viv in man [5] and in vitr with the perfused rat pancreas [6] and perifused islets f Langerhans [7] have shwn that a shrt perid f glucse stimulatin may als ptentiate the secretry respnse f the B cell t a secnd pulse f glucse given shrtly afterwards. Althugh it has been suggested that this effect ccurs nly if the cncentratin f glucse present during the initial stimulatin perid is in excess f apprximately 14 mml/1 in man [5] r 11 mml/1 in perifused rat islets [7], the bichemical mechanisms mediating this phenmenn are prly understd. n the present study the pssible rles f 1) glucse metablism 2) calcium and the micrtubular system, and 3) cyclic AMP have been examined. The experiments were carried ut using perifused rat islets f Langerhans. Materials and Methds slatin and Perifusin f slets f Langerhans Male Wister albin rats weighing apprximately g were fed ad libitum n a standard labratry diet cntaining 48% carbhydrate, 21.3% prtein and 3.4% fat. slets were islated by cllagenase digestin [8]. The medium used fr extractin and subsequent experiments was a bicarbnate-buffered salt slutin with the fllwing inic cmpsitin: Na + 141; K + 5.9; Ca ; Mg ; PO~- 1.2; C- 11 and HCO mml/1; ph 7.4. The medium was supplemented with the sdium salts f glutamic, lactic and fumaric acids at a cncentratin f 5 rnml/l [9]. The gas phase was O2:CO2 (95:5). The calcium cncentratin f 'calcium-free' media was.3mml/1 as determined by atmic absrptin spectrphtmetry. When islets were expsed t deuterium xide (D) the inic cmpsitin f the medium was unchanged but D replaced H t give a final cncentratin f 98% (v/v). The ph was adjusted t that f the cntrl medium. The basal cncentratin f glucse present during islet islatin and perifusin was 2.7 mml/1. Thirty islets were laded int each channel f a multi-channel perifusin system [7] and equilibrated by perifusin with medium cntaining basal glucse fr 4 rain befre the administratin f the first stimulus. The flw rate in each channel was apprximately 1 ml/min and was mnitred during the curse f each experiment. Minr variatins in flw between channels were allwed fr when secretry rates were calculated. Samples f perifusate were cllected ver 2 min perids, except when the cncentratin f glucse was increased abve basal when they were btained at 1 rain intervals fr 1 min after the change. The average time interval between the remval f the pancreas and the start f the experiment was 2 h. Experimental Design Every experiment was carried ut n fur separate ccasins, using pls f islets btained frm tw cllagenase digests each cntaining pancreatic tissue frm three rats. On each ccasin X/81/21/23/$1.

2 J. P. Ashby and D. Shirling: Glucse Priming and Biphasic nsulin Secretin 231 islets were assigned t ne f three treatment grups. The cntrl grup was subjected t tw stimulatins with glucse 16.6 mml/1 applied between 4-6 and 8-1 min f perifusin. The medium bathing the tw experimental grups was mdified as indicated in the text between 38 and 64 min. All experimental islets were stimulated with glucse 16.6 mml/1 disslved in nrmal medium between 8-1 min. On each ccasin tw t fur channels were allcated t each treatment grup and the mean rate f insulin secretin calculated fr each grup. The ttal insulin secreted by cntrl islets during the initial stimulatin perid with glucse 16.6 mml/1 varied frm day t day, with mean values ranging frm.91 t 4. ~g insulin/ min per islet. T accunt fr this variatin results were nrmalised [1]: the mean secretry rates f cntrl and experimental islets measured n any particular ccasin were expressed as percentages f the mean ttal secretin bserved frm cntrl islets during the initial stimulatin with glucse 16.6 mml/1 n that same ccasin. Nrmalised results btained n different ccasins were cmbined and expressed as means + SEM. The statistical significance f results was assessed by tw-tailed t-test fr paired data, cmparing the mean respnse f experimental islets prepared n any particular ccasin with that f cntrls n the same ccasin. Analytical Methds nsulin was assayed by radiimmunassay [9] using purified rat insulin as standard (Nv Research nstitute, Cpenhagen, Denmark). The cncentratin f glucse in all media was measured using a glucse xidase methd. Results Shaded areas in Figures 1-4 illustrate that when cntrl islets were expsed t tw cnsecutive stimulatins with glucse 16.6 mml/1 applied rain apart, insulin secretin in respnse t the secnd stimulus was always greater than that bserved in respnse t the first. During the secnd perid f stimulatin the cntributin f phase 1 t the ttal secretin als increased. (phase 1 as percentage f ttal: stimulus 1, 9.28 _+ 1.42; stimulus 2, _+ 1.41; p <.1; data frm Fig. 1 A). Rle f Calcium and the Micrtubular System The missin f calcium frm the perifusin medium during the initial stimulatin perid inhibited bth the acute secretry respnse t glucse and its priming effect n the islet respnse t subsequent stimulatin (Fig. 2A). O (98% v/v) inhibited insulin secretin during the initial stimulatin perid with glucse (Fig. 2B), and a rebund f secretin was bserved when D was remved frm the medium. The priming effect f glucse was als reduced by this treatment, since the maximum phase 1 secretin rate bserved frm D-treated islets was significantly lwer than cntrl values during the secnd perid f stimulatin (maximum nrmalised secretry rates: phase 1 cntrl, ; DzOtreated, ; p <.1). Hwever, since the maximum phase 1 secretry rate was still significantly (p <.5) greater than that bserved frm islets nt previusly stimulated with glucse but expsed t D alne ( , nrmalised data), the priming effect f glucse was nt cmpletely ablished by D. Effect f Thephylline The presence f thephylline (5 mml/1) during the initial stimulatin perid greatly enhanced the acute secretry respnse t glucse, but did nt influence its priming effect n the islet respnse t subsequent stimulatin (Fig. 3). The inclusin f thephylline in medium cntaining basal glucse between min f perifusin did nt increase insulin secretin acutely r appear t have any effect n insulin release induced by glucse between 8-1 min. During the latter perid the cntributin f the phase 1 respnse t the ttal secretin f thephylline-treated islets ( %) was nt significantly different frm that bserved in cntrls during the initial perid f stimulatin with glucse alne (9.13 _+ 3.%). Rle f Glucse Metablism The presence f mannheptulse (16 mml/1) during the initial stimulatin perid inhibited bth the acute secretry respnse t glucse and its priming effect n the islet respnse t subsequent stimulatin (Fig. 1A). Figure 1B demnstrates that glyceraldehyde (16.6 mml/1) between 4-6 min f perifusin acutely stimulated insulin secretin, and enhanced the secretry respnse t a subsequent glucse lad applied between 8-1 min f perifusin. The priming effect f glyceraldehyde was cmparable t that bserved in islets initially primed with glucse 16.6 mml/1 alne. Effect f Adrenaline nsulin secretin was inhibited when adrenaline (1 ~tml/1) was included in the perifusin medium during the initial stimulatin perid with glucse (Fig. 4). The priming effect f glucse was als reduced by this treatment since maximal rates f phase 1 secretin by adrenaline-treated islets during the secnd stimulatin perid were significantly lwer than cntrl values (maximum nrmalised secretry rates: phase 1 cntrl, _+.9; adrenaline-treated 4.85 _+ 1.11; p <.5). Hwever, the maximum phase 1 secretry rate was still significantly higher (p < '.2) than that bserved frm

3 232 J.P. Ashby and D. Shirling: Glucse Priming and Biphasic nsulin Secretin _ 8 "5 4 E Z -. s UJ b9 z 16 _J z m ~2 A. 35 +M GLUCOSE(16.6mmL/L GLUCOSE16.6mm/) 1 -M l ' :~iii~i~h:i!i!i!: n: ).::F... ":~ii: ~i ~!i,iii... ~'.#, :~! ~ :i!..:i~i~:-- T/a'4)~,~... #h t :ii! i: :ii~.:~iiii~: [... /... ', ii: ii B.!GLYCERALOEHYOE OR GLUCOSE rnmt/t) i OLUCOSE(16.6mml/[ )] :~iiiiii!i!iii~ ::iiiiiiiii!i!ii!iiiiiiii~i~ J TME (rain) Fig. 1. Upperpaneh nhibitry effect f mannheptulse 16.6 mml/1 (M) n the priming actin f glucse n insulin secretin frm islated islets f Langerhans. Between 4-6 min f perifusin cntrl islets were stimulated with high glucse alne (shaded area) while experimental islets were expsed t either high glucse + mannheptulse (e) r mannheptulse disslved in medium cntaining basal glucse (). Mannheptulse was present in experimental media during the perid indicated by arrws. All islets were expsed t high glucse alne between 8-1 min. Lwerpaneh Enhancement f glucse-induced insulin secretin frm islated islets f Langerhans fllwing stimulatin with glyceraldehyde 16.6 mml/1. Between 4-6 min f perifusin cntrl islets were stimulated with high glucse alne (shaded area) while experimental islets were expsed t glyceraldehyde in the presence f basal glucse (e) r basal glucse alne (). All islets were expsed t high glucse alne between 8-1 min. All results represent means _+ SEM fr nrmalised data / -5 8 E 4 Z U.J LL LO.J z_ ~2 A. GLUCOSE(16.6mm/) GLUCOSE(16.6mm/l)],.;:!i!i!i!~!!i~jl!!: ::ii!i!iiii~i~i!ili!il!i~ -?= +i~..., ili:...,....,......,.,,.....,......,,., :::::::::::::::::::::..:.:+x+:.:.: ~zii;ili;iiiiiii;iiiili~ii~i~i: :~iii!iii!iiiiiiii~:: ]r/~ :,~. :~ill,~iliiiiiiii7, if! ~:: ::iils.;iiii:" ~! iii :i: :i!iiiiii~i~ iil; % if!i::.:f i: ij :iiiii~! T~(n=4... ~" :! iii :!i ~ ::iil ~... % :i;.i%;::...::~iilf, :i~::. i i!i ~]~ - B.,GLUCOSE(16.Omrnl/[)] _,., GLUCOSE(16.6mmt/ +D,,-D2u.:l: :if: ( n = 4 )..., :~iiii::: ~i....:.. :"!!!i!. : ::i:" ] S i :t _./-~ A. ~ s ::!$h...:: %: TME (rain) Fig. 2. Upperpaneh nhibitry effect f calcium deprivatin n the priming actin f glucse n insulin secretin frm islated islets f Langerhans. Between 4-6 rain f perifusin cntrl islets were stimulated with high glucse in nrmal medium (shaded area) while experimental islets were expsed t either high glucse disslved in calcium-free medium () r calcium-free medium alne cntaining basal glucse (). Calcium was excluded frm experimental media during the perid indicated by arrws. All islets were expsed t high glucse alne between 8-1 rain. Lwer panel: Effect f deuterium xide (D) (98%; v/v) n the priming actin f glucse n insulin secretin frm islated islets f Langerhans. Between 4-6 min f perifusin cntrl islets were stimulated with high glucse alne (shaded area) while experimental islets were expsed t either high glucse disslved in medium cntaining D () r t medium cntaining D () and basal glucse. D:O was present in experimental islets during the perid indicated by the arrws. All islets were expsed t high glucse alne between 8-1 min. All results represent means _+ SEM fr nrmalised data

4 J. P. Ashby and D. Shirling: Glucse Priming and Biphasic nsulin Secretin A ~ 28._~ ~ 21' r z LL n, _z 8 "~ m E 16 t- z 12 ~ 8 z_ 1' _J +T _filucose(16.6mm/) -T GLUCOSE(16.6mm[/) ', "2 1 ' 1 [ ::#ii:::" ~i!: i.t:.:i~iiii::::! ~:~ " :::ii::! '?i[, :i~i~il ~;~... :i::i... / :? :ii:iiiiii ":ii: ~ :!:? ~!' 1 :" ' *' " ::~]::~k-:: :~i:: "::~i~::::.: _. O. ~ - ~. - ~ ". i 35 1' Z, TME (min) GLUCOSE (16.6 mml/[][ +A ', -A ~:. (n=1') ~r i... :.i 9 ::i~:: ~ii t :?... Ji::::... t i ~i. ' :~iii ~" :~i::~...::~r...,...::.. _z 35 1' 1' (, GLUCOSE(16.6mmi/)]...x:!...:.::i:~:~:~: ff,(~~ il.! TME (mini Fig. 3. Effect f thephylline 5 mml/1 n the priming actin f glucse n insulin secretin frm islated islets f Langerhans. Between 4-6 rain f perifusin cntrl islets were stimulated with high glucse alne (shaded area) while experimental islets were expsed t high glucse + thephylline () r thephylline disslved in medium cntaining basal glucse (). Thephylline was present in experimental media during the perid indicated by arrws. All islets were expsed t high glucse alne between 8-1 min. Results represent means SEM fr nrmalised data Fig. 4. Effect f adrenaline 1 ~ml/1 n the priming actin f glucse n insulin secretin frm islated islets f Langerhans. Between 4-6 min f perifusin cntrl islets were stimulated with high glucse alng (shaded area) while experimental islets were expsed t high glucse + adrenaline (e) r adrenaline disslved in medium cntaining basal glucse (). Adrenaline was present in experimental media during the perid indicated by arrws. All islets were expsed t high glucse alne between 8-1 min. Results represent means SEM fr nrmalised data islets which had nt been previusly stimulated with glucse but expsed t adrenaline alne (1.12 _+.81, nrmalised data). The priming effect f glucse was therefre nt cmpletely ablished by adrenaline. Discussin The results cnfirm that a shrt perid f stimulatin with glucse primes the B cell, making it mre respnsive t subsequent stimulatin with glucse [5-7]. The priming effect f glucse culd be reprduced by the glyclytic intermediate glyceraldehyde, and prevented by mannheptulse which inhibits the key glucse phsphrylating enzyme, high-km hexkinase [12]. This cnfirms the findings f thers [13] and suggests that glucse metablism is invlved~in the priming prcess. n cntrast t the findings f an earlier investigatin [ 13] these bservatins suggest that extracellular calcium is essential fr the priming effect f glucse n the B cell. This discrepancy may relate t differing degrees f calcium depletin in the B cell since the acute secretry respnse t glucse, althugh greatly diminished, was nt cmpletely ablished (Fig. 2 A) when calcium was remved frm the medium perfusing the rat pancreas preparatin used in earlier studies [13]. Since the metablism f glucse is inhibited nly slightly in a calcium-free medium [14], the present results suggest that calcium dependent prcesses, such as the activatin f the micrtubular micrfilamentus system [15], may als be invlved in glucse priming. This pssibility is strengthened by the bservatin that D, an agent which reversibly stabilises the micrtubular system [11], reduced the

5 234 J. P. Ashby and D. Shirling: Glucse Priming and Biphasie nsulin Secretin priming effect f glucse (Fig. 2 B). t is unlikely that this was due t an effect n islet glucse metablism since D des nt appear t influence either islet calcium uptake r insulin bisynthesis, bth f which are reduced fllwing inhibitin f glucse metablism [11]. Hwever, it is imprtant t nte that the effect f D may nt be entirely specific since this agent may interfere with the mbilisatin f calcium frm intracellular rganelles in islets [16] and reduce ttal pancreatic xygen cnsumptin [17]. Experiments with thephylline, which elevates islet cyclic AMP levels by inhibiting cyclic nucletide phsphdiesterase [18], did nt prduce any evidence that the generatin f cyclic AMP can induce a primed state in the B cell. Others [13] have shwn that the enhanced secretin f insulin which ccurs after glucse priming is nt accmpanied by a simultaneus increase in islet cyclic AMP frmatin. n cntrast with studies in man [19] the present results shw that adrenaline may impair the priming effect f glucse in vitr (Fig. 4). Althugh the mechanism is uncertain it is tempting t speculate that this may be related t the inhibitry effect f adrenaline n calcium uptake in islets []. Finally, the results f experiments with thephylline and adrenaline suggest that the magnitude f the acute insulin respnse t glucse is nt related t the magnitude f the priming effect f glucse n insulin secretin. Fr example, thephylline markedly enhanced acute insulin secretin in respnse t glucse but did nt affect the priming respnse. Cnversely, adrenaline cmpletely inhibited the acute insulin respnse t glucse, but nly partially reduced the priming effect f glucse n insulin secretin. Acknwledgements. We are grateful t Prfessr J. A. Strng and Dr. J.D. Baird fr encuragement and helpful criticism during these studies, t Carl-Anne McKechnie, Nicla Christie and Susan Taylr fr expert technical assistance, t the Animal Unit f the Western General Hspital fr prviding the animals used fr this study, and to Mr. C. M. Ferringtn fr assistance with cmputerised handling f data. References 1. Cerasi E, Luft R (1967) The plasma insulin respnse t glucse infusin in healthy subjects and in diabetes mellitus. Acta Endcrinl (Cpenh) 55: Prte D, Pup AA (1969) nsulin respnses t glucse: Evidence fr a tw pl system in man. J Clin nvest 48: Grdsky GM, Bennett LL, Smith D, Nemeehek K (1967) The effect f tlbutamide and glucse n the timed release f insulin frm the islated perfused pancreas. n: Butterfield WJH, Westering W (eds) Tlbutamide after ten years. Excerpta Medica Fundatin, Amsterdam, p Curry DL, Bennett LL, Grdsky GM (1968) Dynamics f insulin secretin by the perfused rat pancreas. Endcrinlgy 83: Cerasi E (1975) Ptentiatin f insulin release by glucse in man.. Quantitative analysis f enhancement f glucseinduced insulin secretin by pretreatment with glucse in nrmal subjects. Acta Endcrinl (Cpenh) 79: Grdsky GM (1972) A threshld distributin hypthesis fr packet strage f insulin and its mathematical mdelling. J Clin nvest 51: Ashby JP, Shirling D (198) Evidence fr priming and inhibitry effects f glucse n insulin secretin frm islated islets f Langerhans. Diabetlgia 18: Lacy PE, Kstianvsky M (1967) Methd fr islatin f intact islets f Langerhans frm the rat pancreas. Diabetes 16: Ashby JP, Speake RN (1975) nsulin and glucagn secretin frm islated islets f Langerhans: Effects f calcium inphres. Biehem J 15: Smers G, Van Obberghen E, Devis G, Ravazzla M, Malaisse-Lagae F, Malaisse WJ (1974) Dynamics f insulin release and the micrtubular-micrfilamentus system.. Effect f clchicine upn glucse-induced insulin secretin. Eur J Clin nvest 4: Malaisse W J, Malaisse-Lagae F, Walker MO, Lacy PE (1971 ) The stimulus-secretin cupling f glucse-induced insulin release. V. The participatin f a micrtubular-micrfilamentus system. Diabetes : Zawalich WS (1979) ntermediary metablism and insulin secretin frm islated rat islets f Langerhans. Diabetes 28: Grill V, Adamsn U, Cerasi E (1978) mmediate and timedependent effects f glucse n insulin release frm rat pancreatic tissue. Evidence fr different mechanisms f actin. J Clin nvest 61: Hellman B, dahl LA, Lernmark A, Sehlin JO, Taljedal B (1974) The pancreatic B cell recgnitin f insulin secretaggues. Effects f calcium and sdium n glucse metablism and insulin release. Bichem J 138: Malaisse WJ (1973) nsulin secretin: multifactrial regulatin fr a single prcess f release. Diabetlgia 9: Malaisse WJ (1973) Thephylline induced translcatin f calcium in the pancreatic beta cell: nhibitin by deuterium xide. Nature 242: Van Obberghen E, Smers G, Devis G, Ravazzla M, Malaisse-Lagae F, Orci L, Malaisse WJ (1974) Dynamics f insulin release and micrtubular micrfilamentus system. V. Effect f DzO. Endcrinlgy 95: Mntague W. Ck JR (1971) The rle f adensine 3' :5' cyclic mnphsphate in the regulatin f insulin release by islated islets f Langerhans. Bichem J 122: Cerasi E (1975) Ptentiatin f insulin release by glucse in man.. Rle f the insulin respnse and enhancement f stimuli ther than glucse. Acta Endcrinl (Cpenh) 79: Malaisse-Lagae F, Malaisse WJ (1971) Stimulus secretin cupling f glucse induced insulin release f. Uptake f 4SCa by islated islets f Langerhans. Endcrinlgy 88:72-8 Received: August 28, 198, and in revised frm: February 5, 1981 Dr. J. P. Ashby Department f Medicine Western General Hspital Edinburgh EH5 2XU, UK