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The effect of oxyntomodulin (Glucagon-37) and glucagon on exocrine pancreatic secretion in the conscious rat
Peptides, Vol. 8, pp. 967-972. ©PergamonJournals Ltd., 1987. Printedin the U.S.A.
0196-9781/87$3.00 + ,00
The Effect of Oxyntomodulin (Glucagon-37) and Glucagon on Exocrine Pancreatic Secretion in the Conscious Rat T. M. BIEDZINSKI, D. BATAILLE,* M. A. D E V A U X A N D H. S A R L E S * Unit6 1 . N . S . E . R . M . U-31 de Pathologie Digestive, 46, B d de la Gaye, 13009 Marseille, France • Centre C N R S - I N S E R M de Pharmacologie-Endocrinologie, Rue de la Cardonille 34094 Montpellier Cedex, France R e c e i v e d 26 M a r c h 1987 BIEDZINSKI, T. M., D. BATAILLE, M. A. DEVAUX AND H. SARLES. The effect t~foxyntomodulin (glucagon-37) and glucagon on exocrinepancreatic secretion h~the conscious rat. PEPTIDES 8(6) 967-972, 1987.--The inhibitory effect of glucagon on exocrine pancreas has been the subject of controversial reports. On the other hand, oxyntomodulin (bioactive enteroglucagon or glucagon-37), a 37 amino acid peptide isolated from porcine lower intestine, has been shown to be 10-20 times more potent than glucagon in inhibiting gastric acid secretion in the rat. In view of this, the effect of glucagon and oxyntomodulin on basal and caerulein-stimulated pancreatic secretion has been studied, during re-introduction of pancreatic juice into duodenum, in the conscious rat provided with pancreatic and duodenal fistulas. A depression of pancreatic function was observed with both peptides on the three parameters studied: (volume of juice secreted, bicarbonate and protein output), either under basal conditions or during stimulation by caerulein. In all the experimental conditions used, oxyntomodulinwas ca. ten times more potent than glucagon in its inhibitory effect. The fact that oxyntomodulin,as what is observed in the stomach, is one order of magnitude more potent than glucagon in inhibiting pancreatic secretion suggests that the biological mechanisms by which the peptides of the glucagon-family act on exocrine pancreas are similar, or related to that present at the gastric level. Oxyntomodulin
Glucagon-37
Enteroglucagon
Glucagon
Inhibition
Exocrine pancreatic secretion
suits were obtained by others [1, 25, 28]. It is the purpose of the present paper to investigate the effect of glucagon and oxyntomodulin on the basal and cerulein-stimulated exocrine pancreatic secretion in the conscious rat, animal in which oxyntomodulin has been shown to be one order of magnitude more potent than glucagon on gastric acid secretion [11].
OXYNTOMODULIN (glucagon-37), a 37 amino acid glucagon-containing peptide isolated from porcine lower intestine [2], represents a part of the "Enteroglucagon" concept (for reviews see [4,19]). It has been shown to display a tissue-specific pattern of activity which is different from that of glucagon. Whereas the former peptide is about one order of magnitude less potent than the latter in liver and other tissues [3-5], it is 10-20 times more potent than glucagon on stomach, both in vitro in modulating the gastric glands cyclic AMP [3,4] and in vivo in inhibiting the pentagastrinstimulated acid secretion in the anesthetized [ll] and the conscious rat [20]. This different tissue specificity between the 37- and the 29-amino acid peptides is related to the existence of two different receptor sites. In liver and other tissues, the well known glucagon-receptor [27] is present with affinities of 1:10 for oxyntomodulin and glucagon, respectively [5], whereas a receptor with respective affinities of 10:1 was described in acid-secreting gastric glands [4,9]. As far as the effect of glucagon on exocrine pancreatic secretion is concerned, it is noticeable that most authors [12, 15, 22, 29] have reported an inhibitory effect of this peptide on flow rate, bicarbonate and protein output, whereas negative re-
METHOD Male Sprague-Dawley rats weighing 300-350 g were fed a standard diet. After an 18-hr fast, the rats were anesthetized with ether and the abdominal cavity was opened. The common bile duct was ligated under the liver on a polyethylene catheter (No. 3, Biotrol, France) the other end of which being inserted into the duodenum to insure a normal flow of bile into the duodenum. The bilio-pancreatic duct was ligated and cannulated proximal to the duodenum papilla to collect pancreatic juice (Venocath 18, 4718, Abbot, USA). Another catheter (Silastic medical-grade tubing 602175, Dow Coming, USA) was introduced into the duodenum at the level of the duodenal papilla. The latter two were exteriorized
~Requests for reprints should he addressed to H. Sarles, I.N.S.E.R.M. U 31, 46 Bd de la Gaye, 13009 Marseille, France.
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FIG. 1. Effect of oxyntomodulin (OX), left panels, or glucagon (G), right panels, on basal pancreatic secretion during reintroduction of pancreatic juice. Data on volume (A), HCO:~ (B) and protein output (C). Peptides were injected as a bolus at the following doses: Oxyntomodulin: 0.1 (©), 0.21 (&), 0.43 (5)and 0.86 (11)nmol.kg-~;Glucagon: 2.14 (©), 4.30 (&), 8.60 ([3)and 17.1 (11) nmol.kg-'; Control injection of NaCI (e). Results are expressed as means_+S.D, of A values. The statistical difference (filled stars) was calculated from plateau values (*p<0.05, **p<0.025), n=5.
through the skin. Between experiments, the two catheters were connected so that the pancreatic juice could enter the duodenum. The femoral vein was cannulated to allow intravenous infusion. All rats were kept in Bollman restraining cages and were allowed free access to water. Experiments were not performed on the rats until 24 hr after surgery. The pancreatic juice was collected in graduated pipettes for 30-min periods, an equal volume of the juice having been simultaneously introduced into the duodenum via the duodenal catheter. The first two samples were discarded and the mean of the next two 30-min periods were taken as the basal secretion. After obtaining a steady state of pancreatic secretion, graded doses from 2.14 to 17.1 nmol.kg -~ of glucagon and from 0.1 to 0.86 nmol.kg -1 of oxyntomodulin were administered as bolus IV injections through a polyethylene catheter. Peptides were dissolved in saline immediately before injection. In pancreatic stimulation experiments, caerulein (Farmitalia) was given at doses of 0.15/xg.kg-l.hr -1 as a continuous IV infusion. In all experiments, pancreatic secretion was studied during a 180-rain period, after a stable flow rate had
been obtained (60-90 min). Only one experiment was performed on each rat per day. The volume of each sample was recorded to the nearest 10/zl directly from the pipette. The bicarbonate concentration was determined by heating an aliquot of each sample after adding one volume of 0.01 N HCI and backtitration to pH 7.0 with 0.02 N NaOH using an automatic titrator (Titrimat TAT 5 Tacussel, Villeurbanne, France), the bicarbonate concentrations being obtained by regression analysis on a standard curve obtained with NaHCO~. The protein concentration was estimated using an absorption spectrophotometer at 280 nm (E 1%/1 cm=20). Highly purified natural porcine oxyntomodulin, displaying a single peak in high performance liquid chromatography, was obtained as previously described [2]. Precise quantification of the peptides was assessed by two means: (1) integration by a computing integrator (SP 4100, Spectra-Physics) of the UV-absorbing HPLC peaks [2]. (2)radioimmunoassay of glucagon in which oxyntomodulin and glucagon have the same molar reaction [21]. Both methods gave the same results. For a perfect comparison of the potencies between oxyntomodulin and
O X Y N T O M O D U L I N A N D EXOCRINE PANCREAS
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glucagon, the glucagon batch used in this study (from the Novo Research Institute, Copenhagen, Denmark) was assayed using the same methods as that used for oxyntomodulin. The same peptide batches were used for previous studies on stomach [10,20]. Statistical analysis was performed using the Student's t-test for paired and non-paired values, p <0.05 being taken as the level of significance. RESULTS
Effect of Oxyntomodulin and Glucagon on Basal Pancreatic Secretion During Reintroduction of Pancreatic Juice (Fig. 1) Volume. Small graded doses of oxyntomodulin (0,1, 0.21, 0.43, 0.86 nmol.kg -~) or glucagon (2.14, 4.3, 8.6 or 17.1 nmol.kg -1) were administered. Both peptides depressed the pancreatic flow rate within the first 30 minutes after injec-
tion. Although the statistical analysis indicated that a significant effect was observed only for the two highest doses of oxyntomodulin and the three highest doses of glucagon, it was possible to draw a dose-effect relationship for both peptides (Fig. 2). This representation shows that, in the early period, oxyntomodulin, with a IDs0 of 0.27 nmol.kg -1 was 9 times more potent than glucagon (ID~0=2,4 nmoi-kg-a). Furthermore, the maximal level of inhibition observed was similar for both peptides (21% for oxyntomodulin and 19% for glucagon). Thus, on this parameter, oxyntomodulin and glucagon display potencies of ca. 10 to 1, respectively, and with the same efficacy. The longest duration of the inhibitory effects were noticed at 0.43 nmol.kg -I of oxyntomodulin and at a 10-fold higher dose of glucagon (4.3 nmol.kg-1). Bicarbonate output. The effect on bicarbonate output of graded doses of oxyntomodulin was similar to that observed
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Volume. Bolus injections of oxyntomodulin or glucagon were superimposed to a caerulein perfusion (0.15 /zg.kg ~hr-'). The injection of doses, of 0.43 nmol.kg-; of oxyntomodulin or of 4.3 nmol.kg-' of glucagon totally suppressed the stimulatory effect of caerulein: the resulting flow rates observed were at the level of or below the basal values. At the higher dose of oxyntomodulin (0.86 nmol.kg -;) or of glucagon (8.6 nmol-kg-'), much less pronounced inhibitory effects were noticed, which did not prove to be statistically significant. As for the effect on basal secretion (see Fig. 1), oxyntomodulin was effective at doses of peptide that were one order of magnitude lower than the effective doses of glucagon. Bicarbonate output. The results with both peptides were similar to that obtained for the flow rate: the two lowest doses (0.43 nmoi.kg ' of oxyntomodulin and 4.3 nmol.kg - ' of glucagon) completely inhibited the effect of caerulein on bicarbonate output and the two highest doses of peptides induced a lower inhibition. Protein output. Both oxyntomodulin and glucagon induced, whatever the dose used, a profound inhibition of protein output which dropped to levels that were below the basal secretion. In all cases, a significant inhibition lasted for at least 120 minutes.
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carbonate output was observed with glucagon only at 4.3 nmol.kg -~ (p <0.05). Protein output. The lowest doses of oxyntomodulin, 0.1 and 0.21 nmol.kg -1, did not significantly affect the protein output. When a dose of 0.43 nmol.kg ~ was administered, a significant inhibition of protein output occurred (p<0.05), but this maximal inhibitory effect (18.5%) was restricted to the first 60 min following peptide injection. After administration of 0.86 nmol-kg -~, the observed inhibition was followed by a significant increase in protein output which occurred at 120 min. (p<0.025). Glucagon was an inhibitor of protein output at the two highest doses (4.3 and 8.6 nmol.kg-J).
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FIG. 3. Effect of oxyntomodulin (OX), left panels and glucagon (G), right panels, on cerulein-stimulated (150 ng.kg-~.hr -~) pancreatic secretion in the rat during re-introduction of pancreatic juice. Data on volume (A), HCO~- (B) and protein output (C). Peptides were injected as a bolus at the following doses: Oxyntomodulin: 0.43 nmol.kg-~ ([]) and 0.86 nmol.kg-~ ( i ) ; Glucagon: 4.30 nmol.kg-~ ([]) and 8.60 nmol.kg-~ (11); Control injection of NaC1 (e). Results are expressed as means_+S.D, of A values. The statistical difference (filled stars) was calculated from plateau values (*p<0.05, **p <0.025), n=5.
for water secretion. The two lowest doses (0.1 and 0.21 nmol.kg -1) did not significantly depress bicarbonate output. The maximal inhibition (approximately 50%) was similar at doses of 0.43 nmol.kg -1 (p<0.01) and 0.86 nmol.kg -~ (p<0.01). The apparent IDs0 for oxyntomodulin on this parameter was 0.16 nmol.kg-'. A significant inhibition of bi-
The present study clearly demonstrates that pancreatic glucagon is an inhibitor of both basal and stimulated exocrine pancreatic secretion. It can be seen from the contradictory data reported so far, that inhibition of external pancreatic function by glucagon is not universally accepted: Shaw et al. [28] found in the rat that this peptide, given IV, significantly increased pancreatic water secretion, as well as bicarbonate output, during feeding but depressed the pancreatic response in the fasting state. However, Youngs [31] and Lankisch et al. [23] reported that the resting pancreas was stimulated by glucagon administration, while the actively secreting pancreas was inhibited by this peptide. According to Glasser et al. [18], immediately after a single IV injection of glucagon, the secretory rate of the pancreas during caerulein stimulation was transiently accelerated and then depressed for a longer period of time. Similar results were obtained by Necheles [26]. It was also reported by Fitzgerald et al. [14] that in a totally isolated canine pancreas, glucagon, given as a bolus injection on a background of secretin or CCK stimulation, did not alter the secretory response.
OXYNTOMODULIN AND EXOCRINE PANCREAS Most studies, however, reported an inhibitory action of glucagon on exocrine pancreatic secretion. Sweeting [30] has shown that this peptide inhibits the pancreatic response to duodenal acidification. Dyck et al. [13] has found that small doses of glucagon produced a dose-related inhibition of pancreatic flow rate and enzyme concentration occurring in response to secretin alone or secretin + CCK. The study by Singer et al. [29], contrary to that by Shaw and Heath [28], confirmed the inhibitory effect of glucagon on food stimulated canine pancreatic secretion and demonstrated that this inhibition is dose-dependent. A similar dose-dependent response in inhibition of pancreatic secretion by glucagon was obtained by Konturek et al. [22]. In our study, the inhibitory effect of glucagon occurred in the two different protocols: under basal conditions and following caerulein stimulation during reintroduction of juice. In the basal condition, the maximal inhibitory effect of glucagon was usually observed during the 30-min period after its injection. This was then followed by an increase in pancreatic secretion. This biphasic glucagon action probably depends on a rebound effect. Adler [1] also reported a biphasic response of the rat pancreas to prolonged glucagon infusion: an initial transient inhibition and then subsequent stimulation of enzyme release. He concluded that high circulating levels of glucagon cannot be considered as a persistent factor with respect to the pancreatic function. The modulation of pancreatic exocrine functions by gut factors such as "enteroglucagon" is poorly understood because of the difficulties encountered in isolating such factors. The isolation from distal porcine small bowel of a 37-amino acid glucagon-related peptide referred to as glucagon-37 or oxyntomodulin [2-5] and its availability for biological studies opened a new field of research on the biological role of "enteroglucagon." Its hormonal status is strongly supported by the unambiguous demonstration of its presence in the lower bowel mucosa of human [24] and rat [21] and in the plasma of human [4] and rat [21]. Oxyntomodulin contains the entire sequence of pancreatic glucagon plus a C-terminal octapeptide and seems to have its own physiological role, different from that of pancreatic glucagon [3, 4, 11, 20]. One of these roles appears to be the control of gastric acid secretion, as this peptide powerfully inhibits this secretion, being 15-20 times more potent than pancreatic glucagon [11] in doing so. In this respect, the effect of glucagon on gastric function [6, 8, 16, 17] appears more pharmacological than physiological. If we compare the two peptides in our experimental conditions, it appears that oxyntomodulin and glucagon are clear-cut inhibitors of pancreatic secretion. As far as the first type of protocol is concerned (basal secretion), comparison of the results from two groups of rats which received either glucagon or oxyntomodulin, with reintroduction of pancreatic juice, revealed that, whatever the parameter studied (volume, bicarbonate output or protein output), the IDs0 for oxyntomodulin was about 1/10 that for glucagon, and that the
971 maximal inhibitory activity was similar. When the pancreatic secretion was stimulaed with caerulein, it is noteworthy that oxyntomodulin at 0.43 nmol.kg -1 or glucagon at 4.3 nmol.kg -~ totally suppressed with stimulatory effect of caerulein on water or bicarbonate secretion, the resulting secretion being similar to basal levels. A paradoxical effect was observed at the higher dose of either peptide, 0.86 nmol.kg -~ oxyntomodulin or 8.6 nmol.kg -l glucagon being less efficient on either parameter than the lower dose. In contrast, the two doses of each peptide were equally active on protein output. On this parameter, both peptides were able not only to suppress the stimulatory effect of caerulein, but also to decrease the secretion below the basal level. Although it is not possible to draw, from our data on stimulated secretion, a dose-response curve, it is clear that the same effect was noticed, in all cases, with a dose of oxyntomodulin and the 10-fold higher dose of glucagon. Thus, as for the basal experiments, oxyntomodulin appears as being 10-fold more potent than glucagon. The mechanism by which oxyntomodulin and glucagon inhibit exocrine pancreatic secretion is unknown. On the other hand, it has been clearly established that oxyntomodulin is able to inhibit stimulated gastric acid secretion and that it is 10-20 times more potent than pancreatic glucagon in doing so. Since secretin and CCK increase the pancreatic response to duodenal acidification, the decrease in acid secretion induced by oxyntomodulin could indirectly depress pancreatic secretion. The study by Chiba [7] invoked another possible mechanism. He demonstrated a significant correlation between the increase in gastric somatostatin release and decrease in gastrin secretion induced by glucagon, secretin and VIP. This may also be true for oxyntomodulin, which raises the possibility that somatostatin mediates or facilitates the observed inhibition of pancreatic secretion. Concerning the latter possibility, it is noticeable that a clear-cut potentiation by somatostatin of the inhibitory effect of oxyntomodulin on gastric acid secretion has been observed in the rat [10]. A similar mechanism may exist at the pancreatic level. On the other hand, the possibility of a direct effect of oxyntomodulin and glucagon on exocrine pancreatic tissue should be checked on in vitro models, as already shown for stomach [4,9]. Whatever the precise mechanisms involved in the inhibition of pancreatic secretion by oxyntomodulin, the details of which require further investigations, our data suggest that this peptide, besides its probable importance in controlling gastric secretion, plays a role in regulating the intestinal phase of exocrine pancreatic secretion. ACKNOWLEDGEMENTS Supported by I.N.S.E.R.M. (CRL 82 7 009). The authors would like to thank Dr. Robin Kennedy for providing helpful manuscript review and Dr. Nathalie Debbas for mathematical aid.
REFERENCES 1. Adler, G. Effect of glucagon on the secretory process in the rat exocrine pancreas. Cell Tissue Res 182: 193-204, 1977. 2. Bataille, D., A. M. Coudray, M. Carlqvist, G. Rosselin and V. Mutt. Isolation of Glucagon-37 (bioactive Enteroglucagon/ Oxyntomodulin) from porcine jejuno-ileum. Isolation of the peptide. FEBS Lett 146: 73-78, 1982.
3. Bataille, D., C. Gespach, A. M. Coudray and G. Rosselin. "Enteroglucagon": A specific effect on gastric glands isolated from the rat fundus. Evidence for an "Oxyntomodulin" action. Biosci Rep 1: 151-155, 1981. 4. Bataille, D., C. Jarrousse, A. Kervran, C. Depigny and M. Dubrasquet. The biological significance of "'Enteroglucagon." Present status. Peptides 7: Suppl 1, 37-42, 1986.
972 5. Bataille, D., K. Tatemoto, C. Gespach, H. Jornvall, G. Rosselin and V. Mutt. Isolation of Glucagon-37 (bioactive Enteroglucagon/Oxyntomodulin) from porcine jejuno-ileum. Characterisation of the peptide. FEBS Lett 146: 79-86, 1982. 6. Becker, H. D., D. D. Reeder and J. C. Thompson. Effect of glucagon on circulating gastrin. Gastroenterology 65: 28--35, 1973. 7. Chiba, T., T. Taminato and S. Kadawaki et al. Effect of glucagon, secretin and vasoactive intestinal polypeptide on gastric somatostatin and gastrin release from isolated perfused rat stomach. Gastroenterology 79: 67-71, 1980. 8. Clarke, S. D., D. W. Neil and R. B. Welburn. Effect ofglucagon on gastric secretion in the dog. Gut 1: 146-148, 1960. 9. Depigny, C., B. Lupo, A. Kervran and D. Bataille. Evidence for a specific binding site for glucagon-37 (oxyntomodulin/bioactive enteroglucagon) in rat oxyntic glands. C R Seances Aead Sei [111] 299: 677-680, 1984. 10. Dubrasquet, J., M. P. Audousset-Puech, J. Martinez and D. Bataille. Somatostatin enhances the inhibitory effect of Oxyntomodulin and its C-terminal octapeptide on acid secretion. Peptides 7: Suppl 1,257-259, 1986. 11. Dubrasquet, M., D. Bataille and C. Gespach. Oxyntomodulin (Glucagon-37 or bioactive Enteroglucagon): A potent inhibitor of pentagastrin-stimulated acid secretion in rat. Biosci Rep 2: 391-395, 1982. 12. Dyck, W. P., J. Rudick, B. Hoexter and H. D. Janowitz. Influence of glucagon on pancreatic exocrine secretion. Gastroenterology 56: 531-537, 1969. 13. Dyck, W. P., E. C. Texter, J. M. Lasater and N. C. Hightower. Influence of glucagon on pancreatic exocrine secretion in man. Gastroenterology 58: 532-539, 1970. 14. Fitzgerald, O., K. F. McGeeney and J. J. Murphy. An endocrine exocrine interaction in the isolated canine pancreas. J Physiol (Lond) 239: 59, 1974. 15. Fontana, G., P. L. Costa, R. Tessari and G. Labo. Effect of glucagon on pure human exocrine pancreatic secretion. Am J Gastroenterol 63: 490-494, 1975. 16. Gerner, T. and J. F. W. Haffner. The significance of distension for the effect of glucagon on the fundic and antral motility in isolated guinea pig stomach. Scand J Gastroenterol [Suppl] 26: 5]-53, 1974. 17. Ginsberg, B., R. A. Levine and D. E. Wilson. The effect of glucagon on histamine and pentagastrin-stimulated canine gastric secretion and mucosal blood flow. Gastroenterology 60: 775, 1971.
BIEDZINSKI ET AL. 18. Glesser, A. G., L. Dorigotti and G. Vaghi. Inhibitory activity of glucagon on caerulein exocrine stimulation, independent of hyperglycemia. Adv Exp Med Biol 21: 347-355, 1972. 19. Holst, J. J. Gut glucagon, enteroglucagon, gut glucagon-like immunoreactivity, glicentin-current status. Gastroenterology 84: 1602-1613, 1983. 20. Jarrousse, C., M-P. Audousset-Puech, M. Dubrasquet, H. Niel, J. Martinez and D. Bataille. Oxyntomodulin (Glucagon-37) and its C-terminal octapeptide inhibit gastric secretion. FEBS Lett 188: 81-84, 1985. 21. Kervran, A., C. Jarrousse and D. Bataille. Oxyntomodulin (G-37) and Glucagon (G-29): distribution in the gastrointestinal tract and the plasma of the rat. Diabetologia 27: 295-296, 1984. 22. Konturek, S. J., J. Tasler and W. Obtulowicz. Characteristics of inhibition of pancreatic secretion by glucagon. Digestion 10: 138-149, 1974. 23. Lankisch, P. G., K. Winckler and H. Schmidt. Glucagon Behandlung der Akuten Pankreatitis. Dtseh Med Wsehr 100: 845846, 1975. 24. Munck, A., A. Kervran, J. C. Marie, D. Bataille and G. Rosselin. Glucagon-37 (oxyntomodulin) and glucagon-29 (pancreatic glucagon) in human bowel: Analysis by HPLC and radioreceptorassay. Peptides 5: 553-561, 1984. 25. Murphy, J. J. and K. F. McGeeney. The effects ofglucagon on the exocrine secretion of the perfused canine pancreas, Isr J Med Sci 143: 37-41, 1974. 26. Necheles, H., L. Walker and W. Mabin. Effects of glucagon on external secretion of the pancreas. Am J Physiol 191: 585-597, 1957. 27. Rodbell, M., H. M. S. Krans, S. L. Pohl and L. Birnbaumer. The glucagon sensitive adenyl cyclase system in plasma membrane of rat liver. III. Binding of glucagon: Method of assay and specificity. J Biol Chem 246: 1861-1871, 1971. 28. Shaw, H. M. and T. J. Heath. The effect of glucagon on the formation of pancreatic juice and bile in the rat. Can J Physio[ Pharmacol 51: I-5, 1973. 29. Singer, M. V., O. M. Tiscornia, J. P. Mendes de Olivera, P. Demol, D. Levesque and H. Sarles. Effect of glucagon on canine exocrine pancreatic secretion stimulated by a test meal. Can J Physiol 56: 1-6, 1978. 30. Sweeting, J. G. The effect of glucagon on bile and pancreatic juice. Gastroenterology 54: 1276, 1968. 31. Youngs, G. Hormonal control of pancreatic endocrine and exocrine secretion. Gut 13: 154-161, 1972.
0196-9781/87$3.00 + ,00
The Effect of Oxyntomodulin (Glucagon-37) and Glucagon on Exocrine Pancreatic Secretion in the Conscious Rat T. M. BIEDZINSKI, D. BATAILLE,* M. A. D E V A U X A N D H. S A R L E S * Unit6 1 . N . S . E . R . M . U-31 de Pathologie Digestive, 46, B d de la Gaye, 13009 Marseille, France • Centre C N R S - I N S E R M de Pharmacologie-Endocrinologie, Rue de la Cardonille 34094 Montpellier Cedex, France R e c e i v e d 26 M a r c h 1987 BIEDZINSKI, T. M., D. BATAILLE, M. A. DEVAUX AND H. SARLES. The effect t~foxyntomodulin (glucagon-37) and glucagon on exocrinepancreatic secretion h~the conscious rat. PEPTIDES 8(6) 967-972, 1987.--The inhibitory effect of glucagon on exocrine pancreas has been the subject of controversial reports. On the other hand, oxyntomodulin (bioactive enteroglucagon or glucagon-37), a 37 amino acid peptide isolated from porcine lower intestine, has been shown to be 10-20 times more potent than glucagon in inhibiting gastric acid secretion in the rat. In view of this, the effect of glucagon and oxyntomodulin on basal and caerulein-stimulated pancreatic secretion has been studied, during re-introduction of pancreatic juice into duodenum, in the conscious rat provided with pancreatic and duodenal fistulas. A depression of pancreatic function was observed with both peptides on the three parameters studied: (volume of juice secreted, bicarbonate and protein output), either under basal conditions or during stimulation by caerulein. In all the experimental conditions used, oxyntomodulinwas ca. ten times more potent than glucagon in its inhibitory effect. The fact that oxyntomodulin,as what is observed in the stomach, is one order of magnitude more potent than glucagon in inhibiting pancreatic secretion suggests that the biological mechanisms by which the peptides of the glucagon-family act on exocrine pancreas are similar, or related to that present at the gastric level. Oxyntomodulin
Glucagon-37
Enteroglucagon
Glucagon
Inhibition
Exocrine pancreatic secretion
suits were obtained by others [1, 25, 28]. It is the purpose of the present paper to investigate the effect of glucagon and oxyntomodulin on the basal and cerulein-stimulated exocrine pancreatic secretion in the conscious rat, animal in which oxyntomodulin has been shown to be one order of magnitude more potent than glucagon on gastric acid secretion [11].
OXYNTOMODULIN (glucagon-37), a 37 amino acid glucagon-containing peptide isolated from porcine lower intestine [2], represents a part of the "Enteroglucagon" concept (for reviews see [4,19]). It has been shown to display a tissue-specific pattern of activity which is different from that of glucagon. Whereas the former peptide is about one order of magnitude less potent than the latter in liver and other tissues [3-5], it is 10-20 times more potent than glucagon on stomach, both in vitro in modulating the gastric glands cyclic AMP [3,4] and in vivo in inhibiting the pentagastrinstimulated acid secretion in the anesthetized [ll] and the conscious rat [20]. This different tissue specificity between the 37- and the 29-amino acid peptides is related to the existence of two different receptor sites. In liver and other tissues, the well known glucagon-receptor [27] is present with affinities of 1:10 for oxyntomodulin and glucagon, respectively [5], whereas a receptor with respective affinities of 10:1 was described in acid-secreting gastric glands [4,9]. As far as the effect of glucagon on exocrine pancreatic secretion is concerned, it is noticeable that most authors [12, 15, 22, 29] have reported an inhibitory effect of this peptide on flow rate, bicarbonate and protein output, whereas negative re-
METHOD Male Sprague-Dawley rats weighing 300-350 g were fed a standard diet. After an 18-hr fast, the rats were anesthetized with ether and the abdominal cavity was opened. The common bile duct was ligated under the liver on a polyethylene catheter (No. 3, Biotrol, France) the other end of which being inserted into the duodenum to insure a normal flow of bile into the duodenum. The bilio-pancreatic duct was ligated and cannulated proximal to the duodenum papilla to collect pancreatic juice (Venocath 18, 4718, Abbot, USA). Another catheter (Silastic medical-grade tubing 602175, Dow Coming, USA) was introduced into the duodenum at the level of the duodenal papilla. The latter two were exteriorized
~Requests for reprints should he addressed to H. Sarles, I.N.S.E.R.M. U 31, 46 Bd de la Gaye, 13009 Marseille, France.
967
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FIG. 1. Effect of oxyntomodulin (OX), left panels, or glucagon (G), right panels, on basal pancreatic secretion during reintroduction of pancreatic juice. Data on volume (A), HCO:~ (B) and protein output (C). Peptides were injected as a bolus at the following doses: Oxyntomodulin: 0.1 (©), 0.21 (&), 0.43 (5)and 0.86 (11)nmol.kg-~;Glucagon: 2.14 (©), 4.30 (&), 8.60 ([3)and 17.1 (11) nmol.kg-'; Control injection of NaCI (e). Results are expressed as means_+S.D, of A values. The statistical difference (filled stars) was calculated from plateau values (*p<0.05, **p<0.025), n=5.
through the skin. Between experiments, the two catheters were connected so that the pancreatic juice could enter the duodenum. The femoral vein was cannulated to allow intravenous infusion. All rats were kept in Bollman restraining cages and were allowed free access to water. Experiments were not performed on the rats until 24 hr after surgery. The pancreatic juice was collected in graduated pipettes for 30-min periods, an equal volume of the juice having been simultaneously introduced into the duodenum via the duodenal catheter. The first two samples were discarded and the mean of the next two 30-min periods were taken as the basal secretion. After obtaining a steady state of pancreatic secretion, graded doses from 2.14 to 17.1 nmol.kg -~ of glucagon and from 0.1 to 0.86 nmol.kg -1 of oxyntomodulin were administered as bolus IV injections through a polyethylene catheter. Peptides were dissolved in saline immediately before injection. In pancreatic stimulation experiments, caerulein (Farmitalia) was given at doses of 0.15/xg.kg-l.hr -1 as a continuous IV infusion. In all experiments, pancreatic secretion was studied during a 180-rain period, after a stable flow rate had
been obtained (60-90 min). Only one experiment was performed on each rat per day. The volume of each sample was recorded to the nearest 10/zl directly from the pipette. The bicarbonate concentration was determined by heating an aliquot of each sample after adding one volume of 0.01 N HCI and backtitration to pH 7.0 with 0.02 N NaOH using an automatic titrator (Titrimat TAT 5 Tacussel, Villeurbanne, France), the bicarbonate concentrations being obtained by regression analysis on a standard curve obtained with NaHCO~. The protein concentration was estimated using an absorption spectrophotometer at 280 nm (E 1%/1 cm=20). Highly purified natural porcine oxyntomodulin, displaying a single peak in high performance liquid chromatography, was obtained as previously described [2]. Precise quantification of the peptides was assessed by two means: (1) integration by a computing integrator (SP 4100, Spectra-Physics) of the UV-absorbing HPLC peaks [2]. (2)radioimmunoassay of glucagon in which oxyntomodulin and glucagon have the same molar reaction [21]. Both methods gave the same results. For a perfect comparison of the potencies between oxyntomodulin and
O X Y N T O M O D U L I N A N D EXOCRINE PANCREAS
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glucagon, the glucagon batch used in this study (from the Novo Research Institute, Copenhagen, Denmark) was assayed using the same methods as that used for oxyntomodulin. The same peptide batches were used for previous studies on stomach [10,20]. Statistical analysis was performed using the Student's t-test for paired and non-paired values, p <0.05 being taken as the level of significance. RESULTS
Effect of Oxyntomodulin and Glucagon on Basal Pancreatic Secretion During Reintroduction of Pancreatic Juice (Fig. 1) Volume. Small graded doses of oxyntomodulin (0,1, 0.21, 0.43, 0.86 nmol.kg -~) or glucagon (2.14, 4.3, 8.6 or 17.1 nmol.kg -1) were administered. Both peptides depressed the pancreatic flow rate within the first 30 minutes after injec-
tion. Although the statistical analysis indicated that a significant effect was observed only for the two highest doses of oxyntomodulin and the three highest doses of glucagon, it was possible to draw a dose-effect relationship for both peptides (Fig. 2). This representation shows that, in the early period, oxyntomodulin, with a IDs0 of 0.27 nmol.kg -1 was 9 times more potent than glucagon (ID~0=2,4 nmoi-kg-a). Furthermore, the maximal level of inhibition observed was similar for both peptides (21% for oxyntomodulin and 19% for glucagon). Thus, on this parameter, oxyntomodulin and glucagon display potencies of ca. 10 to 1, respectively, and with the same efficacy. The longest duration of the inhibitory effects were noticed at 0.43 nmol.kg -I of oxyntomodulin and at a 10-fold higher dose of glucagon (4.3 nmol.kg-1). Bicarbonate output. The effect on bicarbonate output of graded doses of oxyntomodulin was similar to that observed
970
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Volume. Bolus injections of oxyntomodulin or glucagon were superimposed to a caerulein perfusion (0.15 /zg.kg ~hr-'). The injection of doses, of 0.43 nmol.kg-; of oxyntomodulin or of 4.3 nmol.kg-' of glucagon totally suppressed the stimulatory effect of caerulein: the resulting flow rates observed were at the level of or below the basal values. At the higher dose of oxyntomodulin (0.86 nmol.kg -;) or of glucagon (8.6 nmol-kg-'), much less pronounced inhibitory effects were noticed, which did not prove to be statistically significant. As for the effect on basal secretion (see Fig. 1), oxyntomodulin was effective at doses of peptide that were one order of magnitude lower than the effective doses of glucagon. Bicarbonate output. The results with both peptides were similar to that obtained for the flow rate: the two lowest doses (0.43 nmoi.kg ' of oxyntomodulin and 4.3 nmol.kg - ' of glucagon) completely inhibited the effect of caerulein on bicarbonate output and the two highest doses of peptides induced a lower inhibition. Protein output. Both oxyntomodulin and glucagon induced, whatever the dose used, a profound inhibition of protein output which dropped to levels that were below the basal secretion. In all cases, a significant inhibition lasted for at least 120 minutes.
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carbonate output was observed with glucagon only at 4.3 nmol.kg -~ (p <0.05). Protein output. The lowest doses of oxyntomodulin, 0.1 and 0.21 nmol.kg -1, did not significantly affect the protein output. When a dose of 0.43 nmol.kg ~ was administered, a significant inhibition of protein output occurred (p<0.05), but this maximal inhibitory effect (18.5%) was restricted to the first 60 min following peptide injection. After administration of 0.86 nmol-kg -~, the observed inhibition was followed by a significant increase in protein output which occurred at 120 min. (p<0.025). Glucagon was an inhibitor of protein output at the two highest doses (4.3 and 8.6 nmol.kg-J).
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FIG. 3. Effect of oxyntomodulin (OX), left panels and glucagon (G), right panels, on cerulein-stimulated (150 ng.kg-~.hr -~) pancreatic secretion in the rat during re-introduction of pancreatic juice. Data on volume (A), HCO~- (B) and protein output (C). Peptides were injected as a bolus at the following doses: Oxyntomodulin: 0.43 nmol.kg-~ ([]) and 0.86 nmol.kg-~ ( i ) ; Glucagon: 4.30 nmol.kg-~ ([]) and 8.60 nmol.kg-~ (11); Control injection of NaC1 (e). Results are expressed as means_+S.D, of A values. The statistical difference (filled stars) was calculated from plateau values (*p<0.05, **p <0.025), n=5.
for water secretion. The two lowest doses (0.1 and 0.21 nmol.kg -1) did not significantly depress bicarbonate output. The maximal inhibition (approximately 50%) was similar at doses of 0.43 nmol.kg -1 (p<0.01) and 0.86 nmol.kg -~ (p<0.01). The apparent IDs0 for oxyntomodulin on this parameter was 0.16 nmol.kg-'. A significant inhibition of bi-
The present study clearly demonstrates that pancreatic glucagon is an inhibitor of both basal and stimulated exocrine pancreatic secretion. It can be seen from the contradictory data reported so far, that inhibition of external pancreatic function by glucagon is not universally accepted: Shaw et al. [28] found in the rat that this peptide, given IV, significantly increased pancreatic water secretion, as well as bicarbonate output, during feeding but depressed the pancreatic response in the fasting state. However, Youngs [31] and Lankisch et al. [23] reported that the resting pancreas was stimulated by glucagon administration, while the actively secreting pancreas was inhibited by this peptide. According to Glasser et al. [18], immediately after a single IV injection of glucagon, the secretory rate of the pancreas during caerulein stimulation was transiently accelerated and then depressed for a longer period of time. Similar results were obtained by Necheles [26]. It was also reported by Fitzgerald et al. [14] that in a totally isolated canine pancreas, glucagon, given as a bolus injection on a background of secretin or CCK stimulation, did not alter the secretory response.
OXYNTOMODULIN AND EXOCRINE PANCREAS Most studies, however, reported an inhibitory action of glucagon on exocrine pancreatic secretion. Sweeting [30] has shown that this peptide inhibits the pancreatic response to duodenal acidification. Dyck et al. [13] has found that small doses of glucagon produced a dose-related inhibition of pancreatic flow rate and enzyme concentration occurring in response to secretin alone or secretin + CCK. The study by Singer et al. [29], contrary to that by Shaw and Heath [28], confirmed the inhibitory effect of glucagon on food stimulated canine pancreatic secretion and demonstrated that this inhibition is dose-dependent. A similar dose-dependent response in inhibition of pancreatic secretion by glucagon was obtained by Konturek et al. [22]. In our study, the inhibitory effect of glucagon occurred in the two different protocols: under basal conditions and following caerulein stimulation during reintroduction of juice. In the basal condition, the maximal inhibitory effect of glucagon was usually observed during the 30-min period after its injection. This was then followed by an increase in pancreatic secretion. This biphasic glucagon action probably depends on a rebound effect. Adler [1] also reported a biphasic response of the rat pancreas to prolonged glucagon infusion: an initial transient inhibition and then subsequent stimulation of enzyme release. He concluded that high circulating levels of glucagon cannot be considered as a persistent factor with respect to the pancreatic function. The modulation of pancreatic exocrine functions by gut factors such as "enteroglucagon" is poorly understood because of the difficulties encountered in isolating such factors. The isolation from distal porcine small bowel of a 37-amino acid glucagon-related peptide referred to as glucagon-37 or oxyntomodulin [2-5] and its availability for biological studies opened a new field of research on the biological role of "enteroglucagon." Its hormonal status is strongly supported by the unambiguous demonstration of its presence in the lower bowel mucosa of human [24] and rat [21] and in the plasma of human [4] and rat [21]. Oxyntomodulin contains the entire sequence of pancreatic glucagon plus a C-terminal octapeptide and seems to have its own physiological role, different from that of pancreatic glucagon [3, 4, 11, 20]. One of these roles appears to be the control of gastric acid secretion, as this peptide powerfully inhibits this secretion, being 15-20 times more potent than pancreatic glucagon [11] in doing so. In this respect, the effect of glucagon on gastric function [6, 8, 16, 17] appears more pharmacological than physiological. If we compare the two peptides in our experimental conditions, it appears that oxyntomodulin and glucagon are clear-cut inhibitors of pancreatic secretion. As far as the first type of protocol is concerned (basal secretion), comparison of the results from two groups of rats which received either glucagon or oxyntomodulin, with reintroduction of pancreatic juice, revealed that, whatever the parameter studied (volume, bicarbonate output or protein output), the IDs0 for oxyntomodulin was about 1/10 that for glucagon, and that the
971 maximal inhibitory activity was similar. When the pancreatic secretion was stimulaed with caerulein, it is noteworthy that oxyntomodulin at 0.43 nmol.kg -1 or glucagon at 4.3 nmol.kg -~ totally suppressed with stimulatory effect of caerulein on water or bicarbonate secretion, the resulting secretion being similar to basal levels. A paradoxical effect was observed at the higher dose of either peptide, 0.86 nmol.kg -~ oxyntomodulin or 8.6 nmol.kg -l glucagon being less efficient on either parameter than the lower dose. In contrast, the two doses of each peptide were equally active on protein output. On this parameter, both peptides were able not only to suppress the stimulatory effect of caerulein, but also to decrease the secretion below the basal level. Although it is not possible to draw, from our data on stimulated secretion, a dose-response curve, it is clear that the same effect was noticed, in all cases, with a dose of oxyntomodulin and the 10-fold higher dose of glucagon. Thus, as for the basal experiments, oxyntomodulin appears as being 10-fold more potent than glucagon. The mechanism by which oxyntomodulin and glucagon inhibit exocrine pancreatic secretion is unknown. On the other hand, it has been clearly established that oxyntomodulin is able to inhibit stimulated gastric acid secretion and that it is 10-20 times more potent than pancreatic glucagon in doing so. Since secretin and CCK increase the pancreatic response to duodenal acidification, the decrease in acid secretion induced by oxyntomodulin could indirectly depress pancreatic secretion. The study by Chiba [7] invoked another possible mechanism. He demonstrated a significant correlation between the increase in gastric somatostatin release and decrease in gastrin secretion induced by glucagon, secretin and VIP. This may also be true for oxyntomodulin, which raises the possibility that somatostatin mediates or facilitates the observed inhibition of pancreatic secretion. Concerning the latter possibility, it is noticeable that a clear-cut potentiation by somatostatin of the inhibitory effect of oxyntomodulin on gastric acid secretion has been observed in the rat [10]. A similar mechanism may exist at the pancreatic level. On the other hand, the possibility of a direct effect of oxyntomodulin and glucagon on exocrine pancreatic tissue should be checked on in vitro models, as already shown for stomach [4,9]. Whatever the precise mechanisms involved in the inhibition of pancreatic secretion by oxyntomodulin, the details of which require further investigations, our data suggest that this peptide, besides its probable importance in controlling gastric secretion, plays a role in regulating the intestinal phase of exocrine pancreatic secretion. ACKNOWLEDGEMENTS Supported by I.N.S.E.R.M. (CRL 82 7 009). The authors would like to thank Dr. Robin Kennedy for providing helpful manuscript review and Dr. Nathalie Debbas for mathematical aid.
REFERENCES 1. Adler, G. Effect of glucagon on the secretory process in the rat exocrine pancreas. Cell Tissue Res 182: 193-204, 1977. 2. Bataille, D., A. M. Coudray, M. Carlqvist, G. Rosselin and V. Mutt. Isolation of Glucagon-37 (bioactive Enteroglucagon/ Oxyntomodulin) from porcine jejuno-ileum. Isolation of the peptide. FEBS Lett 146: 73-78, 1982.
3. Bataille, D., C. Gespach, A. M. Coudray and G. Rosselin. "Enteroglucagon": A specific effect on gastric glands isolated from the rat fundus. Evidence for an "Oxyntomodulin" action. Biosci Rep 1: 151-155, 1981. 4. Bataille, D., C. Jarrousse, A. Kervran, C. Depigny and M. Dubrasquet. The biological significance of "'Enteroglucagon." Present status. Peptides 7: Suppl 1, 37-42, 1986.
972 5. Bataille, D., K. Tatemoto, C. Gespach, H. Jornvall, G. Rosselin and V. Mutt. Isolation of Glucagon-37 (bioactive Enteroglucagon/Oxyntomodulin) from porcine jejuno-ileum. Characterisation of the peptide. FEBS Lett 146: 79-86, 1982. 6. Becker, H. D., D. D. Reeder and J. C. Thompson. Effect of glucagon on circulating gastrin. Gastroenterology 65: 28--35, 1973. 7. Chiba, T., T. Taminato and S. Kadawaki et al. Effect of glucagon, secretin and vasoactive intestinal polypeptide on gastric somatostatin and gastrin release from isolated perfused rat stomach. Gastroenterology 79: 67-71, 1980. 8. Clarke, S. D., D. W. Neil and R. B. Welburn. Effect ofglucagon on gastric secretion in the dog. Gut 1: 146-148, 1960. 9. Depigny, C., B. Lupo, A. Kervran and D. Bataille. Evidence for a specific binding site for glucagon-37 (oxyntomodulin/bioactive enteroglucagon) in rat oxyntic glands. C R Seances Aead Sei [111] 299: 677-680, 1984. 10. Dubrasquet, J., M. P. Audousset-Puech, J. Martinez and D. Bataille. Somatostatin enhances the inhibitory effect of Oxyntomodulin and its C-terminal octapeptide on acid secretion. Peptides 7: Suppl 1,257-259, 1986. 11. Dubrasquet, M., D. Bataille and C. Gespach. Oxyntomodulin (Glucagon-37 or bioactive Enteroglucagon): A potent inhibitor of pentagastrin-stimulated acid secretion in rat. Biosci Rep 2: 391-395, 1982. 12. Dyck, W. P., J. Rudick, B. Hoexter and H. D. Janowitz. Influence of glucagon on pancreatic exocrine secretion. Gastroenterology 56: 531-537, 1969. 13. Dyck, W. P., E. C. Texter, J. M. Lasater and N. C. Hightower. Influence of glucagon on pancreatic exocrine secretion in man. Gastroenterology 58: 532-539, 1970. 14. Fitzgerald, O., K. F. McGeeney and J. J. Murphy. An endocrine exocrine interaction in the isolated canine pancreas. J Physiol (Lond) 239: 59, 1974. 15. Fontana, G., P. L. Costa, R. Tessari and G. Labo. Effect of glucagon on pure human exocrine pancreatic secretion. Am J Gastroenterol 63: 490-494, 1975. 16. Gerner, T. and J. F. W. Haffner. The significance of distension for the effect of glucagon on the fundic and antral motility in isolated guinea pig stomach. Scand J Gastroenterol [Suppl] 26: 5]-53, 1974. 17. Ginsberg, B., R. A. Levine and D. E. Wilson. The effect of glucagon on histamine and pentagastrin-stimulated canine gastric secretion and mucosal blood flow. Gastroenterology 60: 775, 1971.
BIEDZINSKI ET AL. 18. Glesser, A. G., L. Dorigotti and G. Vaghi. Inhibitory activity of glucagon on caerulein exocrine stimulation, independent of hyperglycemia. Adv Exp Med Biol 21: 347-355, 1972. 19. Holst, J. J. Gut glucagon, enteroglucagon, gut glucagon-like immunoreactivity, glicentin-current status. Gastroenterology 84: 1602-1613, 1983. 20. Jarrousse, C., M-P. Audousset-Puech, M. Dubrasquet, H. Niel, J. Martinez and D. Bataille. Oxyntomodulin (Glucagon-37) and its C-terminal octapeptide inhibit gastric secretion. FEBS Lett 188: 81-84, 1985. 21. Kervran, A., C. Jarrousse and D. Bataille. Oxyntomodulin (G-37) and Glucagon (G-29): distribution in the gastrointestinal tract and the plasma of the rat. Diabetologia 27: 295-296, 1984. 22. Konturek, S. J., J. Tasler and W. Obtulowicz. Characteristics of inhibition of pancreatic secretion by glucagon. Digestion 10: 138-149, 1974. 23. Lankisch, P. G., K. Winckler and H. Schmidt. Glucagon Behandlung der Akuten Pankreatitis. Dtseh Med Wsehr 100: 845846, 1975. 24. Munck, A., A. Kervran, J. C. Marie, D. Bataille and G. Rosselin. Glucagon-37 (oxyntomodulin) and glucagon-29 (pancreatic glucagon) in human bowel: Analysis by HPLC and radioreceptorassay. Peptides 5: 553-561, 1984. 25. Murphy, J. J. and K. F. McGeeney. The effects ofglucagon on the exocrine secretion of the perfused canine pancreas, Isr J Med Sci 143: 37-41, 1974. 26. Necheles, H., L. Walker and W. Mabin. Effects of glucagon on external secretion of the pancreas. Am J Physiol 191: 585-597, 1957. 27. Rodbell, M., H. M. S. Krans, S. L. Pohl and L. Birnbaumer. The glucagon sensitive adenyl cyclase system in plasma membrane of rat liver. III. Binding of glucagon: Method of assay and specificity. J Biol Chem 246: 1861-1871, 1971. 28. Shaw, H. M. and T. J. Heath. The effect of glucagon on the formation of pancreatic juice and bile in the rat. Can J Physio[ Pharmacol 51: I-5, 1973. 29. Singer, M. V., O. M. Tiscornia, J. P. Mendes de Olivera, P. Demol, D. Levesque and H. Sarles. Effect of glucagon on canine exocrine pancreatic secretion stimulated by a test meal. Can J Physiol 56: 1-6, 1978. 30. Sweeting, J. G. The effect of glucagon on bile and pancreatic juice. Gastroenterology 54: 1276, 1968. 31. Youngs, G. Hormonal control of pancreatic endocrine and exocrine secretion. Gut 13: 154-161, 1972.