Dabetologa (1983) 24:191-195 Dabetologa 9 Sprnger-Verlag 1983 Role of Glucose and nsuln n the Dynamc Regulaton of Glucagon Re by the Perfused Rat Pancreas V. Leclercq-Meyer, J. Marchand andw. J. Malasse Laboratory of xpermental Medcne, Free Unversty of Brussels, Brussels, Belgum Summary. The effect of glucose upon the re of glucagon and nsuln from the perfused rat pancreas n vtro was studed by varyng both the concentraton of glucose (from 3.3 to 4.6, 8.5, or 11.1 mmol/1) and the tme of exposure to an elevated concentraton of the sugar (5, 10 or 23 ran). The results suggest that the amount of nsuln red durng the early perod of stmulaton could contrbute to both the speed and extent of the nhbton n glucagon re. The rate of recovery from nhbton n the A cell, however, appeared to be ndependent of nsuln and was related, n a dose-dependent and tme-dependent manner, only to the glucose stmulus. t s suggested that a drect effect of glucose upon the A cell s nvolved n the physologcal regulaton of glucagon secreton. An ndrect effect of glucose, as medated va nsuln re, may contrbute to the rapdty and magntude of nhbton n A cell secretory actvty. Key words. Glucose, rat, glucagon re, nsuln re, perfused rat pancreas. The secretory responsveness of the pancreatc A cell to glucose [1-5] s apparently altered n dabetes melltus, snce both the suppresson of glucagon re by glucose and ts stmulaton n response to hypoglycaema are mpared [1, 2, 6]. Several explanatons have been proposed to account for such anomales, ncludng the lack of nsuln n the perpheral crculaton or wthn the slets [2, 7, 8], a dsrupton of the normal topographcal relatonshp between dfferent cell types n the slets [7, 8], an ntrnsc A cell defect at the level of ether a hypothetcal glucoreceptor [9] or the metabolsm of glucose [10], or autonomc neuropathy [11]. t should be realzed, however, that a delayed secretory response of the A cell to a decrease n extracellular glucose concentraton may also occur n the absence of dabetes, e.g. when the extracellular glucose level n vtro s suddenly lowered after a pror exposure of normal pancreatc tssue to a hgh concentraton of the sugar [4, 12-16] or n vvo towards the end of an oral or ntravenous glucose tolerance test [17-20]. n the present study we have nvestgated whether the magntude and duraton of changes n the extracellular glucose concentraton affect the rapdty and extent of the A cell secretory response. For ths purpose the dynamcs of glucagon re by the normal rat pancreas were examned n vtro n response to changes of varable ampltude and duraton n the glucose concentraton of the perfusate. Materal and Methods Anmals and Technque The technque used to perfuse the rat pancreas has been descrbed prevously [12]. Brefly, fed female albno rats (Wstar or Sprague- Dawley strans; mean _+ SM body weght: 273 _+ 5 g; n = 32) were anaesthetzed wth sodum barbtal (42 mg/kg, P). The entre pancreas was perfused through the coelac and mesenterc arteres va a cannula nserted nto the aorta, all adjacent organs, ncludng the duodenum, beng excluded. MeNo~ The basal medum contaned (n mmol/1) NAC1:118.5, KC1:4.7, KH2PO4:1.2, MgSO4:1.2, CaC2: 2, NaHCO3 : 25 and glucose: 3.3. t was supplemented wth dextran T70, 40g/1 (Pharmaca, Uppsala, Sweden) and bovne albumn fracton V, 5 g/l, (Sgma Chemcals, St. Lous, USA or Armour, astbourne, UK). t was equlbrated aganst a mxture of 02 and CO2 (95: 5), and entered the pancreas wth a ph of 7.4 and a temperature of 37~ The flow rate averaged 1.98_+ 0.01 ml/mn (n = 32) and the pressure amounted to 27.9 _+ 1.0 mmhg (3.7 + 0.1 kpa; n = 32). The glucose pulses were admnstered through sde-arm syrnges workng at a flow rate of 0.075 ml/mn (Braun nfuson pump, Melsungen, FRG). n a frst seres of experments, glucose was admnstered n hgh concentraton for ether 23 mn, 10 mn or only 5 mn. n the latter case the concentraton of glucose, whch was ntended to be 11.1 mmol/l, only reached that of 8.5 mmol/1 due to the dynamcs of our perfuson system. n a second seres of experments, the concentraton of glucose vared n a narrow range from 3.3 to 4.6 mmol/1, the latter concentraton beng mantaned for 23 ran. Durng the pre- and post-stmulatory perods, the osmolarty of the medum was adjusted to that obtaned n the presence of glucose by the nfuson of an equosmolar amount of NaC.
192 no7 A n=4 B n:: V. Leclercq-Meyer C et al.: Glucose, nsuln and Glucagon Re [ --::: 52 T t311 C " t- O 1.5,5 = c- O 1.0 O,5 ~ 0.. 1 Tme (ran) _ J L... / L l L Fg. 1. ffects of glucose, admnstered at a hgh (up to 11.1 mmol/1; A and C) or a low concentraton (up to 4.6 mmol/1; B) and for dfferent lengths of tme (23 ran n A and B, 10 mn n C), upon the re of glucagon and nsuln from the perfused rat pancreas. Results are shown as mean _+ SM, together wth the number of ndvdual experments (n) t} o "-. l J, ~ 5 0 16,-2... 12 - =' 5 ~ a 4 o 0.4 u 0.2 l '-", t [ 35 4043 50 60 Tme (ran) Fg.2. ffects of a short (5 mn) pulse of glucose upon the re of glucagon and nsuln from the perfused rat pancreas (= =, n = 9). Also shown are the results of control experments performed n the presence of 3.3 mmol/1 glucose throughout (o o, n = 7). Same presentaton as n Fg. 1 Samples of the pancreatc effluent were collected, wthout recyclng, at 1 mn ntervals n chlled glass tubes contanng 2 000 KU aprotnn (Trasylol, Bayer, Brussels, Belgum) and frozen at -25 ~ untl the tme of assay. Glucagon and nsuln levels were estmated usng ndvdual assays for these hormones [21]. n the glucagon assay, the 125-glucagon tracers were obtaned from several sources (Centre Natonal de Transfuson Sangune, Pars, France; New ngland Nuclear, Boston, USA; Novo, Bagsvaerd, Denmark). Porcne glucagon (Novo) was used as the standard and a N- or C-termnal antbody from our laboratory was employed (AGS and 2601, respectvely)9 nsuln was estmated usng 125-nsuln (lnsttut Natonal des Radoelements, Fleurus, Belgum or Radochemcal Centre, Amersham, UK), a gunea-pg ant-nsuln serum (a gft from Dr. P. H. Wrght, ndanapols, USA) and rat nsuln standard R170 (a gft from Dr. J. Sehlchtkrull, Novo). n the case of glucagon, t mght be noted that the basal rates of glucagon re (. e. those observed at 3.3 mmol/1 glucose) were about fve tmes greater n our earler experments (performed n 1979; Fgs 1 and 3) than n the more recent perfusons (performed n 1980M 981 ; Fg. 2). The reasons for such a quanttatve dfference were not found. Glucose was assayed n the pancreatc effluent wth a glucose-oxdase method (GOD-PAP, Boehrnger, Mannhem, FGR) usng an AA- Techncon analyzer. Statstcal Analyss The results are presented as mean_+ SM together wth the number of ndvdual determnatons (n). ntegrated glucagon and nsuln re durng each secretory phase and the amount of glucose whch had perfused the pancreas were computed from the areas under the curves. The separaton between the early and late phases of nsuln re was judged from the lowest secretory rate recorded alter the ntal peak. n each ndvdual experment, the magntude of the nhbtory effect of glucose upon glucagon output was calculated by comparng the last measurement durng the control perod (ran 43) and the mean steady-state value reached durng exposure to the hgher concentraton of glucose. The latter value was computed between mnute 46 and ether mnute 50 (5 ran stmulaton), mnute 54 (10 mn stmulaton) or mnute 67 (23 ran stmulaton) nclusve when the glucose concentraton was rased from 3.3 to 8.5 mmol/1 or more, and between mnutes 50 and 67 when the glucose concentraton was rased for 23 mn from 3.3 to only 4.6 mmol/1. The tme requred to reach a steady-state glucagon output was estmated by graphcal examnaton of each ndvdual experment. Ths method was valdated by the fact that the concentraton of glucagon n the frst sample consdered as representatve of the new equlbrum stage was much lower than that found n the mmedately precedng sample. ndeed, the pared dfference n glucagon output between these two samples averaged 32.9 + 7.5% (geometrc mean; n = 24; p < 0.001) of the correspondng mean stcady-state value recorded durng exposure to the hgh concentraton of glucose. Statstcal analyses were conducted usng the two-taled non-pared t-test and the correlaton coeffcent r [22].
V. Leclercq-Meyer et al.: Glucose, nsuln and Glucagon Re 193 Table 1. ffect of glucose upon glucagon and nsuln re by the perfused rat pancreas. Glucose Glucagon re nsuln re Maxmal Length of Total amount Degree of Rapdty of concentraton admnstraton perfused nhbton nhbton (mmol/1) (mn) (mmol) (%) (ran) arly phase (ng) Late phase (ng) Total (early + late phase) (rg) 11.8+-0.3(7) 23 0.60 70.2 3.6 4.6+-0.1 ~ 23 0.26 66.1 8.5 11.2 10 0.26 89.6 3.3 8.5+_0.1(9) 5 0.06 69.7 :3.8 54.3 + 7.4 2.9 + 0.2 78.4+13.3 31.6 + 8.1 327 +_62 2.9+_ 0.7 71.9+ 4.5 6.1+ 0.7 382 + 13 5.8 + 0.2 150 +9 38 +_3 Results expressed as mean +- SM; number of ndvdual experments shown n parentheses 2.OF >20 ' ' l, l l,, r r r 0 1.5- ~6._ ~ v U 1.0- =4 0.5-2[ Q...,, ~.Or J 0 -- 0 ~..... 58 42, 9 : 0 47 9... Tme (mn) 9176149,..o,."" 38 43 47 o Q; 5 m 0 U Fg,3. Dynamcs of the varatons n the concentraton of glucose (o... o), glucagon (0 0) and nsuln (o o) n the pancreatc effluent durng the early mnutes of the admnstraton of glucose. Mean values refer to 11 (left panel) and 4 (rght panel) ndvdual experments respectvely Results Tme- and Dose-Dependent nhbton of Glucagon Re The rse n the concentraton of glucose resulted n an nhbton of glucagon re n all experments (Fgs. 1 and 2). Such an nhbton was least marked when the output of nsuln durng the early mnutes of the nfuson of glucose was lowest and most marked when the output of nsuln durng the early perod was hghest (Table 1). There was a postve correlaton between the percentage nhbton of glucagon re and ether the peak nsuln concentraton or the amount of nsuln red durng the early perod of stmulaton (Table 2). No correlaton was found wth other varables such as the amount of nsuln red durng the late phase, the total amount of glucose admnstered and the maxmal levels of glucose reached durng the experments. A sgnfcant fall n glucagon output was always observed at mnute 44 ether at the tme when the concentraton of glucose and nsuln n the effluent were frst ncreased or 1 ran later (Fg. 3). The nhbton of glucagon re never preceded the stmulaton of nsuln re. t clearly took less tme to acheve a new steady-state level of glucagon secreton when the concentraton of glucose was ncreased to > 8.3 mmol/1 than n response to a more modest ncrease n glucose concentraton (Table 1, p < 0.001). There were sgnfcant correlatons between the rapdty of the nhbton of glucagon secreton and both the amount of nsuln red durng the early perod of stmulaton and the maxmal levels of glucose reached durng the perfusons (Table 2). Tme- and Dose-Dependent Reversal of the Glucose-nduced nhbton of Glucagon Re The reversal from the glucose-nduced nhbton of glucagon re appeared to be more a tme-dependent than a dose-related phenomenon. Thus, when glucose was admnstered for 23 mn, whether at a hgh or a low concentraton, the glucagon secretory rates recorded 15 mn after restoraton of a low glucose concentraton (mnute 80 at the end of the perfuson) amounted to only 40.6 + 8.8% and 47.6 + 6.0% respectvely of the secretory rates whch had been recorded mmedately before the onset of nhbton of glucagon secreton (mnute 43). On the other hand, when glucose was admnstered for shorter tmes (such as 10 or 5 ran) these late secretory rates amounted to 86.5 + 7.5% and 74.0 + 6.9%, respectvely. These late secretory rates of glucagon re were comparable to those seen towards the end of the perfuson n control experments performed throughout n the presence of a fxed 3.3 mmol/1 glucose concentraton (Fg. 2). There was a sgnfcant nverse correlaton between the rate of reversal from the glucose-nduced nhbton of glucagon re and the tme of exposure
194 V. Leclercq-Meyer et al.: Glucose, nsuln and Glucagon Re Table2. The correlaton between selected varables n ndvdual experments Part Varables r t p 1 nhbton of glucagon re (%) Versus early phase of nsuln 0.560 3.167 < 0.01 re Versus early peak n nsuln re- 0.602 3.538 < 0.01 Versus late phase of nsuln re- 0.007 0.031 NS Versus early + late phases of 0.094 0.444 NS nsuln re Versus total amount of glu- 0.101 0.466 NS cose admnstered Versus maxmal glucose level 0.353 1.730 NS 2 Rapdty of the nhbton of glucagon re Versus early phase of nsuln - 0.425 2.200 < 0.05 re Versus early peak n nsuln re- - 0.411 2.116 < 0.05 Versus maxmal glucose level -0.687 4.326 < 0.001 3 Percent reversal of the nhbton of glucagon re Versus early + late phases of 0.177 0.806 NS nsuln re Versus total amount of glu- -- 0.472 2.450 < 0.05 cose admnstered Versus maxmal glucose level - 0.182 0.345 NS Versus tme of exposure to - 0.615 3.658 < 0.01 hgh glucose Twenty-four experments were performed n all cases to the glucose stmulus (Table 2). A somewhat lower correlaton was found between the rate of reversal and the total amount of glucose whch had perfused the pancreas (Table 2). The lesser sgnfcance of the latter relatonshp was corroborated by the observaton that an extensve reversal ether occurred (Fg. 1 C) or faled to do so (Fg. 1 B) although the total amount of glucose nfused through the pancreas n ether 10 or 23 mn was the same (0.26 mmol) n these two sets of experments. Dscusson The results of the present experments confrm that the nhbton of glucagon re by glucose may fal to be rapdly reversed n a normal, non-dabetc, expermental preparaton. Thus, a delayed recovery from nhbton n the A cell s not restrcted to pathologcal condtons. The role of nsuln n medatng the response of the pancreatc A cell to changes n the envronmental glucose concentraton has remaned a matter of debate over many years. On the one hand, t has been postulated that the A cells were devod of ntrnsc glucosesensng capacty, and that the effects of glucose were medated ndrectly, through the glucose-nduced changes n nsuln secreton [2, 7, 8]. On the other, there s evdence that glucagon secreton mght not depend solely on nsuln [1, 6]. The results of the present experments are compatble wth the vew that both glucose and nsuln could be nvolved n the regulaton of glucagon re. The relatve mportance of these two regulatory factors may dffer, however, n the suppresson of glucagon re by glucose and stmulaton of glucagon re n response to hypoglycaema, respectvely. The relatonshps found, between both the extent and the rapdty of the nhbton of glucagon re and the amount of nsuln red durng the early perod of exposure to glucose, are compatble wth the vew, but do not demonstrate, that nsuln s nvolved n the nhbton of A cell secretory actvty n response to an ncrease n glucose concentraton. A causal relatonshp between changes n nsuln and glucagon re respectvely s also compatble wth the observaton that the nhbton of glucagon output dd not precede the stmulaton of nsuln re. Ths s not always the case, glucagon re beng reported to precede nsuln re n response to argnne [23]. Alternatvely, t could be postulated that glucose exerts a drect effect upon the secretory actvty of the A cell. Ths vew s supported by the fndng that nhbton of glucagon re occurred even when nsuln was red at a very low rate. When the concentraton of glucose was ncreased to 4.6 retool/1 for 23 mn, the ntegrated output of nsuln dd not exceed 5.8 ng (or 3.2 mu/1). That glucose can nhbt glucagon re n the vrtual absence of nsuln has been documented prevously by other nvestgators (3, 24-28]. The concept of a dual control of glucagon re by both glucose and nsuln cannot be ruled out. n several studes performed n normal pancreatc tssue, no drect effect of exogenous nsuln upon glucagon re could be observed [24, 28-31]. However, n the presence of glucose, exogenous nsuln was found to decrease glucagon output n pancreatc tssue removed from streptozotocn-treated gunea pgs and rats [31-33]. Recent studes suggest that the transport of glucose [34] and 3-O-methyl-glucose [35] n non-b slet cells occur more slowly than n B cells. Hence, the transport of glucose could represent a rate-lmtng factor for ts metabolsm n the A cell and nsuln could concevably affect glucagon re by facltatng glucose transport. A prolonged nhbton of glucagon re was noted upon the arrest of glucose nfuson n hgh concentraton and for a prolonged perod of tme. Ths confrms prevous work performed wth the perfused rat pancreas [4, 12-14, 16]. The same phenomenon was observed when glucose was admnstered for the same length of tme but n a much lower concentraton. Only when glucose was admnstered for a shorter tme (5-10 mn) dd the glueagon secretory rate return rapdly to levels close to those seen before the glucose nfuson. The more or less rapd reversblty of glucagon re was apparently ndependent of nsuln re and related only to the envronmental glucose concentraton and, even more, to the length of exposure to the sugar. Once agan the relatonshp between the rate of recovery n glucagon output and the magntude and duraton of the prevous glucose admnstraton could concevably depend on the amount of glucose accumulated
V. Leclercq-Meyer et al.: Glucose, nsuln and Glucagon Re n the A cell durng ths pror treatment. f so, a greater than twofold ncrease n the length of exposure to glucose (from 10 to 23 mn) would seem to cause a greater accumulaton of glucose than an equal ncrease n the concentraton of the sugar (from 4.6 to 11.1 mmol/1). Ths s not an unrealstc postulate snce the relatonshp between glucose concentraton and glucose transport affects a hyperbolc pattern (tendng to saturaton at hgh glucose concentratons) n those tssues n whch glucose transport s ndeed rate-lmtng, e.g. n muscle. n concluson, the present fndngs emphasze the vew that glucose per se apparently plays a crtcal role n the control of glucagon re whereas the possble ndrect effect of glucose, as medated va nsuln re, appears to play, at the most, a modest modulatng effect on the magntude and rapdty of the nhbtory acton of the sugar. Acknowledgements. Ths work was supported n part by grants 3.4528.79 and 3.4519.80 from the Belgan Foundaton for Scentfc Medcal Research. References 1. Gerch J, Charles MA, Grodsky GM (1976) Regulaton of pancreatc nsuln and glucagon secreton. Ann Rev Physol 38: 353-388 2. Unger RH, Dobbs R, Orc L (1978) nsuln, glucagon and somatostatn secreton n the regulaton of metabolsm. Ann Rev Physol 40:307-343 3. Gefch J, Charles MA, Grodsky GM (1974) Characterzaton of the effects of argnne and glucose on glucagon and nsuln re from the perfused rat pancreas. J Cln nvest 54:833-841 4. Paglara AS, Stllngs SN, Hover B, Martn D, Matschnsky FM (1974) Glucose modulaton of amno-acd nduced glucagon and nsuln re from the solated perfused rat pancreas. J Cln nvest 54:819-832 5. Wer GC, Knowlton SD, Martn DB (1974) Glucagon secreton from the perfused rat pancreas. Studes wth glucose and catecholamnes. J Cln nvest 54:1403-1412 6. Reach G, Assan R (1979) Glucagon and dabetes melltus. Dab Mrtab 5:63-70 7. 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Ostenson C-G (1979) Regulaton of glucagon re: effects of nsuln on the pancreatc Az-cell of the gunea pg. Dabetologa 17:325-330 32. Ostenson C-G, Andersson A, Broln S, Petersson B, Hellerstrrm C (1977) ffects of nsuln on the glucagon re, glucose utlzaton and ATP content of the pancreatc A cells of the gunea pg. n: Fog PP, Bajaj JS, Fol NL (eds) Glucagon: ts role n physology and clncal medcne, Sprnger, New York, Hedelberg, Berln, pp 243-254 33. Wer CG, Knowlton SD, Atkns RF, Mc Kennan KX, Martn DB (1976) Glucagon secreton from the perfused pancreas of streptozotocn-treated rats. Dabetes 25 : 275-282 34. dahl L-A (1981) Glucose content n the A-cell slets of pgeon pancreas. Dabetologa 21 : 284-285 (Abstract) 35. Malasse WJ, Gorus FK, Ppeleers DG (1982) Alloxan uptake by pancreatc B and non-b slet cells. Bol Cell 45: 282 (Abstract) Receved: 8 June 1982 and n revsed form: 19 October 1982 Dr. V. Leclercq-Meyer Laboratory of xpermental Medcne Unverst6 Lbre de Bruxelles 115, Boulevard de Waterloo B- 1000 Brussels, Belgum 195