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Effects of Secretin on Insulin Secretion and Glucose Tolerance J. D U P R ~ D., J . CHISHOLM, T. J. MCDONALD, A N D A. RABINOVITCH McQ;ifl I/tzivcr,rify Clinic, Royak Victoria Hospitak, Montreal, Qeiebec FdA 1AI Received August 22, 1975
DUPR~ J .,, CHISHOLM, D. J., MCDONALL), T. Q., and RAB(NOVITCH, A. 1975. Effects of secretin on insulin secretion and glucose tolerance. Can. J. Physiol. Pharmacol. 53. 1115-1 121. Effects of intravenous (IV) infusion of secretin during IV infusion of glucose were examined in normal men. Secretin was administered according to three schedules: with each schedule a comparable priming dose was delivered in the first minute, but this was followed by a maintained (120 min) infusion d secretin at a relatively high rate, or by maintained infusion at one-third that rate, or by brief (15 mill) infusion at the lower rate. The lower infusion rate produced increments in secretin in the blood within the range attainable during endogenous secretion. By comparison with effects of glucose alone each secretin infusion enhanced the increments of immunoreactive insulin in the blood. Enhancement of the early release (0-5 min) of insulin was similar with each type of secretin infusion, brat the integrated changes in insulin levels through the total infusion period were related tcs the total doses of secretin. With each dose of secretin glucose tolerance was improved but the three mean glucose curves observed during infusions of secretin were not distinguishable from one another in spite of widely different integrated insulin responses. Secretin did not modify suppression of immunoreactive glucagon or free fatty acids in the blood during hyperglycemia. The results suggest that the effect of continuous administration of secretin on glucose tolerance is not si~nplyrelated to its integrated insulinotropic action. It is suggested that the effect may be highly dependent on enhancement of insulin secretion early in the response to glycemia, or that it may be due to effects of secretin on glucose production or disposal which are not mediated by insulin. D U P R J~. ,? CHISEIOI~M, D. Q.,MCDONAI-D, T. J. et RABINOVI-~~1.1, A. 1975. Effects of secretin on insulin secretion and glucose tolerance. Can. J . Physiol. Pharmacol. 53, 11 15-1 121. Les effets d'infusion de la secretine par vcaie intraveineuse, durant une infusion intraveineuse dal gliacose, ant ete etudiis chez des individus normalex. Ida secrktine a ete administree suivant trois rnodalites, cavec pour chaque modalite une dose initiale semblable. Aprks la dose initiale, dans le premier cas l'infusion (120 rnin) comprenait une quantite de secretine relativement tleveet dans le deuxikme cas, cette quantite de sicrktine aetC reduite 2 un tiers de la quantite precedente; dans le troisieme cas, I'adrninistration de la sicretine a ete effectuee dans les rnZmes conditions que dans le cas N o 2, mais la duree de I'infusion a ete limitee h 15 min. Ees infusions h faible dose ont produit dans le sang une augmentation du taux de la secretine se situant dans les limites physicalogiques probables. Cornparativernent aux effets du glucose seul, chaque infusion de secrktine prodiaisait dans le sang une augmentation significative du taux de 19insuline immunoreactive. A la phase du debut, I'augmentation de la liberation d'insuline a ete similaire dans les trois CBS,mais le changement total des taux de I'insmline durant la periode intkgrale de I'infusion a ete proportionnee aux doses infusees de secritine. Chaque dose de secretine ameliorait Ba tolerance am glucose, rnais les moyennes de trois courbes de tolkrances n'etaient pas bien distinctes une de I'autre, malyre une grande difference obtenue pour les valeurs totales de I'insuIine. Dr~rantl'hyperglycemie, la secretine n'a pas modifie dans le sang la suppression du glucagon immunoreactif ou des acides gras libres. Ces resultats suggerent que 19effetsur la tolerance ael glucose d'une administration continue de secretine n'est pas simplement relie $ son action insulinotropique integrale, mais peut dependre en grande partie de I'augrnentation de la secretion de I'insuli~nedans la phase du debut, csu en partie d'un effet de skcretine nonattribuable a I'insuiine.
Introduction Dupr6 et wl. 1966, 19691, but its eflccts on X have not been clearly dclined. Secretin is insulinotropic in fasting man and ~ ~ U C O tolerance In earlier ~tudies highly purified secretin had can the secretion of insulin in reno effcct on the blood glucose during relatively sponse to 11~1glucose ( D ~ and ~ ~Beck c 1966; brief (25-40 min) IV infusions of gl~rcose " 'g69; Chisholm e' ". 196''). In 'Abbreviations: FFA, free fatly acids; IRI, serum ( D u ~ r B insulin; IRG, a.adioimmunoassay for glucagon: IV, preliminary reports of the present experirlaents intravenous. (Duprk and Chisholin 19'70) and in another
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CAN. J . PHYSIOL,. PHARMACOL. VOL. 53, 1975
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Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by McMaster University on 01/13/15 For personal use only.
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CAN. J. PHYSHOL. PHARMACOL. VOL. 53, 1975
study (Enk and Deckert l 9 7 3 ) , more prolonged (120 min) IV infusions of secretin resulted in improvement in glucose tolerance. We report here the effects of various doses of secreti-n delivered during IV infusions of glucose of 2 h duration in normal man and show that even the lowest dose, which enhanced insulin secretion only transiently, resulted in substantial and sustained improvement in glucose tolerance.
Materials sand Methods Studies were carried out in normal nonobese male volunteers. Each received the infusions in varied order (after overnight fasting) within a period of 3 weeks. Highly purified porcine secretin (Gastrointestinal Laboratories, Karolinska Institute, Stockholm) was dissolved in infusates immediately before use. Glucose was infused IV' at a constant rate (66 g in 120 min). Blood samples were drawn from a contralateral vein before, during, and after infusions. Whole blood glucose was estimated by the ferricyanide method on a Technicon Auto Analyzer. Serum FFA were estimated by the Dole titration (Dole and MeinerQz 1960). IRI was estimated by radioimmunoassay (Herbert et a / . 1965). Tn assays for immunoreactive glucagon (IRG) an antiserum believed to be specific for glucagon (Aguilar-Parada et ul. 1949) was employed (antibody 30K supplied by Dr. K. H. Unger, Dallas, Texas): in preparation of specimens for immunoassay Trasylol. 500 KIU/ml was added to blood immediately upon sampling. Specimens from all studies on each subject were assayed together. IRI in serum was fractionated by chromatography on Sephadex G 50 (Gorden and Koth 8969). Secretin was administered according Qs three schedules. With 6pprlonged high-dose infusions', 3.5 ciinical units per minute of secretin (0.9 pg/min) were delivered through 124) min. With 'Bow-dose infusions9, constant-rate infusions of 1.2 U/min (0.3 ,ug/min) were administered, but these infusions were initiated with a priming dose of five clinical units (1.2 ,ug) in the first minute. With 'prolonged low-dose infusions9, delivery of secretin at the lower rate was continued through 120 min, and with 'brief low-dose infusions', infusion of secretin at the same lower rate was continued for 15 min only. A group of the subjects also received the prolonged high-dose secretin infusion in saline vehicle. With an immunoassay system for secretin employing an apparently specific antiserum (donated by Dr. L. Lazarus and Dr. D. Byrnes, Garvan Institute, Sydney, Australia) ir was established that infusion of secretin at the lower dose rate raised the level of secretin in the blood to a plateau of 100-200 pg/rnl, matching endogenous responses to acidification of the duodenurn quantitated in the same assay (unpublished observations of the authors). Plasma levels sf secretin fell1 after completion of infusions with a half-life of less than 5 min.
TIME (rntn)
TIME ( m ~ n l
FIG. 1. Mean blood glucose and serum l R I in subjects given glucose alone (O), glucose plus prolonged high-dose secretin ( A ), glucose pl~msprolonged Icw-dose secretin (m), or glucose plus brief low-dose secretin ( g). Bars show SEM of mean values with glucose alone. A total of 11 subjects took part in the study. All received the standard glucose infusion. Eleven received the prolonged high-dose secretin infusion in glucose. Seven received infusions of secretin according to each of the three secretin dose schedules. Five received the prolonged high-dose secretin infusion in saline vehicle.
Results Mean values for blood glucose and serum IRI1 during infusions of glucose alone or with secretin are illustratecl in Fig. 1. In the present study and in previous 'paired' studies in this laboratory (Dupr6 eb ak. 1969) the basal ( 0 time) values for glucose and IRI showecl little variation, and changes during IV infusions of glucose exhibited least variation when treated as differences from basal values. The statistical significance of differences bctwcen effects of glucose alone and of glucose with different doses of secretin was evaluated by analysis of paired changes at each time. The means and standard errors of these paired differences are shown in Table 1, together with the results of analysis of the paired incremental differences. The overall changes in blood glucose and serum IRI were also treated as the integrated changes through the 120 min infusion period and these results are shown in Table 2. 'To validate these comparisons six of the subjects received a second infusion of glucose alone; the same analyses revealed no significant differences between the efIects of the two infusions. Administration of secretin in saline according to the 'prolonged high-doseq scheclule raised serum IRP transiently but had no effect on the
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blood glucose (Table 1 ) . When secretin was infused at the high-dose rate together with glucose the integrated rise (0-120 min) was greatly enhanced by comparison with that in response to infusion of glucose alone (+85%, p < 0.001). At individual sample times this effect was significant at 5 rnin and through 75 min. This was associated with reduction in the integrated change (0-120 min) in blood glucose concentration (-22 % , p < 8.05), and with significant lowering of the incremental glucose at sample times 45 min through 90 min. When the 'prolonged low-dose' infusion of secretin was administered together with glucose the early rise in IWI in serum ( 5 min) was again significantly greater than that evoked by glucose alone ( p < 0.05). The mean increment in IRI was greater than that with infusion of glucose alone throughout the infusion period, but this difference was significant only at 5 rnin and through the interval 38-60 min. The mean integrated change (0-120 min) in IRI was significantly increased (4-35 %, p < 0.01 ). The 'prolonged low-dose' infusion of secretin also resulted in significant lowering of the mean increment in blood glucose in the interval 30-1 05 rnin and the integrated change in blood glucose was significantly reduced ( -3 8 % , p < 0.001). When secretin was delivered according to the 'brief low-dose' schedule, at 5 rnin the early rise in IRI in serum was again significantly greatcr than that observed when glucose was given alone ( p < 0.05) but enhancement of the mean rise in IRI was maintained only through the short period of administration of secretin, and the integrated change in IRI through the 120 rnin period of infusion of glucose was not significantly greater than that evoked by glucose alone. Nevertheless, this dose of secretin also caused a significant reduction of the rise in blood glucose at all times in the interval 30-105 min and the integrated rise in blood glucose was rcduced by 42% ( p < 0.001 '
>
Eflects of Secretin on Serum lmmuntsreactive Glucagon Serum IRG was estimated in samples drawn at time 0 and at 5, 10, 30, 45, and 75 rnin in subjects recciving glucose, or glucose with 'prolonged high-dose' secretin, or glucose with
FIG.2 . Mean changes in serum glucagon-like immunoreactivity in subjects given glucose alone ( 0) , glucose plus brief low-dose secretin ( 0), glucose plus prolonged high-dose secsetin ( A ) , or prolonged highdose secsetin alone ( ). . Bars show SEM of mean values with glucose alone and with prolonged highdose secretin alone.
'brief low-dose' secretin. The infusions of secretin did not modify the suppression of serum IRG during hyperglycemia (Fig. 2 ) .
Chromatographic Properties of Secretinstimulated IRI Serum drawn from each subject at 15 and 30 rnin (pooled) and at 45 and 60 rnin (pooled) was chromatographed on Sephadex G50. Mean overall recovery of IRI was 103 2 6.1 % (SB) . In every instance more than 90% of the IRI was recovered in fractions corresponding to monomeric insulin in eluates from subjects receiving 'prolonged high-dose' infusions or 'brief low-dose' infusions of the hormone. Eflects of Glucose and Secretin on Serum F F A Serum total F F A were titrated in specimens from subjects who received glucose alone and glucose with 'prolonged high-dose' or 'brief low-dose' infusions of secretin. Secretin did not modify the suppression of FFA (Fig. 3 ) . Discussion Although it has been previously demonstrated that secretin is potentially insulinotropic and can enhance the insulin response to glycemia in man, irnprovcment of glucose tolerance as a result of administration of purified secretin during IV1 infusions of glucose has not hitherto been clearly established. The present studies show that secretin can produce substantial improvement in tolerance to IV glucose during prolonged IV infusions of the sugar. The mean blood glucose curves during infusion of the threc different doses of secretin were
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CAN. J. PHYSIOL. PHARMAGOL. VCBI,. 53, 1975
Fnc;. 3. Mean total serum FFA in subjects receiving glucose alone (Q), glucose plus brief Bow-dose secretin ( 0 ) , or glucose plus prolonged high-dose secretin ( A ) . Bars show SEM of mean values with gBucsse alone.
lower than the curve during administration of glucose alone, but were not significantly different froin one another. This effect of secretin was not apparent before 30-40 min of infusion of glucose. Thus the failure of secretin to affect the concentration of glucose in the blood during infusions in earlier studies may be related to the shorter periods of administration of glucose employed by Bupr6 et a/. (1969) (40 min) and by Chisholm e& ak. (1969) (25 min) . The three schedules for infusion of secretin delivered similar doses during the first 2 mila and produced similar enhancement of the insulin response at the 5 min time point. Administration sf sccretin in saline vehicle also resulted in transient elevation of immunoreactive insulin in the blood. Although the increments in TRT at 5 min after starting the infusions of glucose with secretin were greater in the mean than the sum of responses to glucose alone and secretin alone, at this time the response to tlae combined stimuli was not significantly greater than the sum of their individual effects. However, stimulation of insulin release by administration of secretin in saline was transient, and the enhancement of insulin responses to glucose with the prolonged administration of secretin at high or low dose rates clearly represented potentiation of the response to glucose. This effect of secretin was related to the dose administered. Thus the integrated change in HRI in response to 'pro-
longed high-dose' infusions of secretin was significantly greater than that in response to 'prolonged low-dose' infusion of secretin ( p < 0.02)' and the latter was significantly greater than that in response to 'brief low-dose' infusion of secretin ( p < 0.05). The chromatographic properties of IWH detected in the blood during administration of secsetin at high and low rates were sinsilas, suggesting that immunsreactive iilsulins with different biological potencies were not released. Suppression of IWG and FFA during infusiomss of glucose with or without secretin in high or low dosage was similar, suggesting that net disposal sf glucose was not affected by metabolic actions of glucagon or of FFA. Thus these studies identified no reason for the lack of a simple relationship between thc total quantity of secretln administered and the associated glucose tolerance, in spite of dose-related effects of secretin on integrated changes in IRI. It is possible that the enhancement of the early release of insulin that was common to the three types sf secretin infusions may have been the major determinant of the glucose tolerance curve. Although such an effect of transient enhancement of endogenous secretion of insulin during continuous IV infusion of glucose has not been reported previously, it has been suggested that early rapid release of insulin may be important in determining the physiologic effects of the hormone (Lerner and Porte 197B ) . The inferred importance of rapid early delivery of insulin is supported by the results of recent studies of effects of HV infusion of insulin on oral glucose tolerance in diabetes mellitus (Albisser et al. 1974). However, while the effect of secretin on glucose tolerance might be attributed to enhancement of the secretion of iilsulin at an early stage of the response, it is important to consider the possibility that secretin may exert metabolic effects other than those directly dependent on its insulinotropic action. Thus, there is evidence of an interaction of secretin with insulin in muscle leading to increased glycolysis (Chisholm el al. 1972, 1975). It has long been recognized that secretin affects the external secretions of the liver, and the identification of specific receptor sites for secretin on liver cell membranes has recently been reported (Bataille et al. 1974). It is therefore possible that sccretin affects the
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DUPRE ET AI,.: EFFECTS ON INSULIN SECRETION 4 N D GLUCOSE TOLERANCE
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BQDEN, C . , and CHEY, W. Y. 1973. Preparation and specificity of antiserum to synthetic secretion and its use in a radioimmunoassay . Endocrinology, 92, 1617-1624. BROWN,J. C., DRYBURGH, J . , ROSS, S. A., and DUPRB,J. 1975. Identification and actions of gastric inhibitory polypeptide. Recent Prog. Hormone Res. 31.487-532. CATALAND, S., CROCKETT,S. E . , BROWN,J. C., and MAZZAFERRI.1974. Gastric inhibitory polypeptide stimulation by oral glucose in man. J . Clin. Endocrinol. Metab. 39,223-228. CIIISHOL~M, D. J., KI,ASSEN,G. A., DUPRE, I . , and PCPZEFSKY, T. 1972. Modification of the effect of insulin on muscle by secretin. Clin. Res. 20,945 (Abstr.). 1975. Interaction of secretin and insulin on human forearm n~etabolism.Eur. J. Clin. Invest. In press. CHISHOLM, D. J., YOUNG,J . D., LAZARUS, L . 1969. The gastrointestinal stimulus to insulin release I Secretin. J. Clin. Invest. 48, 1453-1460. DOLE,V . P., and MEINERTZ, H. 1960. Microdetermination of long-chain fatty acids in plasma and tissues. J . Biol. Chem. 235,2595-2599. IIuPRE, J . , and BECK,J. C . 1966. Stimulation of reiease of insulin by an extract of intestinal mucosa. Diabetes, 15,555-559. U U P R ~J .,, and CHISHOIM,D. J. 1970. Effects of secretin on glucose tolerance in man. Diabetes, 20, 322 (Abstr. j. DUPRI?,J . , CURTIS,J . D., UNGER,R. H., WADDELL,R. W., and BECK,J . C. 1969. Effects of secretin, pancreozymin, or gastrin on the response of the endocrine pancreas to administration of glucose or arginine in man. J . Clin. Invest. 48,745-757. DUPRB,J., KOJAS,I.,., WHITE,J . J., UNGER,R. H . , and BECK,J. C. 1966. Effects of secretin on insulin and glucagon in portal and peripheral blood in man. Lancet, 2, 26-27. DUPRE,J., ROSS, S. A., WA.Y.SON, D., and BROWN,J . C. 1973. Stimulation of insulin secretion by gastric inhibitory polypeptide in man. J . Clin. Endocrinol. Metab. 37,826828. ENK, B., and DECKEW?','r. 1973. Modification of glucose-induced insulin secretion after secretin. Acta Endocrinol. Mbh. Suppl. 177,323 (Abstr.). CORDEN,P., and ROTH, J. 1969. Plasma insulin: fluctuations in the "'bigq9insulin comnlponent after glucose and other stimuli. J. Clin. Invest. 43,2225-2234. AGUILAR-PARADA, E., EBSENTRAUT, A. M., and UNGER, HERBERT,V . , LAU, M. S., GOTTLIEB,C. W.. and BI,EICIER, S. J. 1965. Coated charcoal immunoassay R. H. 1969. Pancreatic glucagon secretion in normal of insulin. J. Clin. Endocrinol. Metab. 25,1375-1384. and diabetic subjects. Am. J. Med. Sci. 257,415-419. EERNER,R., and P O R ~ ~D. E ,1971. Relationships between AL.BISSER,A. M., LIEBEL, B. S . , EWAKI, T. G.. intravenous glucose loads, insulin response and gluDAVIDORAC, Z., B o T ~ C. , K., and ZINGG,W. 1974. cose disappearance rate. I. Clin. Endocrinoi. Metab. An artificial endocrine pancreas. Diabetes, 23, 38933,409417. 396. IACKLEY,H. L . A., CHISHOLM,D. J., RAHINQVITCH, A., BATAII.L.E, D. P., FREYCHET, P., and R o s s ~ r , r G. ~ , 1974. WEXLXR,M., and D U P R ~J., 1975. Effects of porInteractions of glucagon, gut glucagon, vasoactive intacaval anastomosis on glucose tolerance in the dog: testinal polypeptide and secretin with liver and fat cell evidence of an interaction between the gut and the membranes: binding t o specific sites and stimulation liver in oral glucose disposal. Metabolism, 24, s f adenylate cyclase . Endocrinology, 9 5 , 7 13-72 1 . 1157-1 168. BLOOM,S. 1974. Hormones of the gastrointestinal tract. Br. Med. Bull. 30.62-67.
metabolic functions sf the liver, and evidence sf an effect of unidentified huinoral secretions of the intestine on hepatic functions affecting glucose tolerance has recently been reported (Lickley et al. 1975). It is clear that the mechanism of the improvement of glucose tolerance resulting from administration of secretin requires further elucidation. Release of secretin in man after ingestion of glucose alone was suggested in earlier studies with a radioimmunoassay for secretin (Chisholm et al. 1969) but has not been confirmed (Bloom 1974; Boden et al. 1972; Buchanan, K. B. : personal communica~ion; unpublished observations of the authors). Recent evidence suggests that gastric inhibitory polypeptide may be the intestinal hormone that potentiates insuBin secretion after ingestion of glucose alone (Bupr.6 et al. 1973; Cataland et ale 1974; Brown et QE. 1975). However, after ingestion of a normal meal the effects of secretin presumably contribute to the physiological response. These effects may be related to the maintenance of relatively low blood glucose levels during absorption of the glucose component of a meal, by comparison with glucose levels observed after ingestion of glucose alonc. The results of the present study with secretin point out the possible importance of transient endocrine secretory responses of the intestine in relation to effects on glucose disposal under physiological coiaditions. It is clear that a better understanding of the actions of this hormone must await examination of its possible effects on the production of glucose and on net and regional disappearance of glucose during absorption of the sugar from the gut.
Effects of Secretin on Insulin Secretion and Glucose Tolerance J. D U P R ~ D., J . CHISHOLM, T. J. MCDONALD, A N D A. RABINOVITCH McQ;ifl I/tzivcr,rify Clinic, Royak Victoria Hospitak, Montreal, Qeiebec FdA 1AI Received August 22, 1975
DUPR~ J .,, CHISHOLM, D. J., MCDONALL), T. Q., and RAB(NOVITCH, A. 1975. Effects of secretin on insulin secretion and glucose tolerance. Can. J. Physiol. Pharmacol. 53. 1115-1 121. Effects of intravenous (IV) infusion of secretin during IV infusion of glucose were examined in normal men. Secretin was administered according to three schedules: with each schedule a comparable priming dose was delivered in the first minute, but this was followed by a maintained (120 min) infusion d secretin at a relatively high rate, or by maintained infusion at one-third that rate, or by brief (15 mill) infusion at the lower rate. The lower infusion rate produced increments in secretin in the blood within the range attainable during endogenous secretion. By comparison with effects of glucose alone each secretin infusion enhanced the increments of immunoreactive insulin in the blood. Enhancement of the early release (0-5 min) of insulin was similar with each type of secretin infusion, brat the integrated changes in insulin levels through the total infusion period were related tcs the total doses of secretin. With each dose of secretin glucose tolerance was improved but the three mean glucose curves observed during infusions of secretin were not distinguishable from one another in spite of widely different integrated insulin responses. Secretin did not modify suppression of immunoreactive glucagon or free fatty acids in the blood during hyperglycemia. The results suggest that the effect of continuous administration of secretin on glucose tolerance is not si~nplyrelated to its integrated insulinotropic action. It is suggested that the effect may be highly dependent on enhancement of insulin secretion early in the response to glycemia, or that it may be due to effects of secretin on glucose production or disposal which are not mediated by insulin. D U P R J~. ,? CHISEIOI~M, D. Q.,MCDONAI-D, T. J. et RABINOVI-~~1.1, A. 1975. Effects of secretin on insulin secretion and glucose tolerance. Can. J . Physiol. Pharmacol. 53, 11 15-1 121. Les effets d'infusion de la secretine par vcaie intraveineuse, durant une infusion intraveineuse dal gliacose, ant ete etudiis chez des individus normalex. Ida secrktine a ete administree suivant trois rnodalites, cavec pour chaque modalite une dose initiale semblable. Aprks la dose initiale, dans le premier cas l'infusion (120 rnin) comprenait une quantite de secretine relativement tleveet dans le deuxikme cas, cette quantite de sicrktine aetC reduite 2 un tiers de la quantite precedente; dans le troisieme cas, I'adrninistration de la sicretine a ete effectuee dans les rnZmes conditions que dans le cas N o 2, mais la duree de I'infusion a ete limitee h 15 min. Ees infusions h faible dose ont produit dans le sang une augmentation du taux de la secretine se situant dans les limites physicalogiques probables. Cornparativernent aux effets du glucose seul, chaque infusion de secrktine prodiaisait dans le sang une augmentation significative du taux de 19insuline immunoreactive. A la phase du debut, I'augmentation de la liberation d'insuline a ete similaire dans les trois CBS,mais le changement total des taux de I'insmline durant la periode intkgrale de I'infusion a ete proportionnee aux doses infusees de secritine. Chaque dose de secretine ameliorait Ba tolerance am glucose, rnais les moyennes de trois courbes de tolkrances n'etaient pas bien distinctes une de I'autre, malyre une grande difference obtenue pour les valeurs totales de I'insuIine. Dr~rantl'hyperglycemie, la secretine n'a pas modifie dans le sang la suppression du glucagon immunoreactif ou des acides gras libres. Ces resultats suggerent que 19effetsur la tolerance ael glucose d'une administration continue de secretine n'est pas simplement relie $ son action insulinotropique integrale, mais peut dependre en grande partie de I'augrnentation de la secretion de I'insuli~nedans la phase du debut, csu en partie d'un effet de skcretine nonattribuable a I'insuiine.
Introduction Dupr6 et wl. 1966, 19691, but its eflccts on X have not been clearly dclined. Secretin is insulinotropic in fasting man and ~ ~ U C O tolerance In earlier ~tudies highly purified secretin had can the secretion of insulin in reno effcct on the blood glucose during relatively sponse to 11~1glucose ( D ~ and ~ ~Beck c 1966; brief (25-40 min) IV infusions of gl~rcose " 'g69; Chisholm e' ". 196''). In 'Abbreviations: FFA, free fatly acids; IRI, serum ( D u ~ r B insulin; IRG, a.adioimmunoassay for glucagon: IV, preliminary reports of the present experirlaents intravenous. (Duprk and Chisholin 19'70) and in another
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by McMaster University on 01/13/15 For personal use only.
CAN. J . PHYSIOL,. PHARMACOL. VOL. 53, 1975
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by McMaster University on 01/13/15 For personal use only.
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Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by McMaster University on 01/13/15 For personal use only.
1118
CAN. J. PHYSHOL. PHARMACOL. VOL. 53, 1975
study (Enk and Deckert l 9 7 3 ) , more prolonged (120 min) IV infusions of secretin resulted in improvement in glucose tolerance. We report here the effects of various doses of secreti-n delivered during IV infusions of glucose of 2 h duration in normal man and show that even the lowest dose, which enhanced insulin secretion only transiently, resulted in substantial and sustained improvement in glucose tolerance.
Materials sand Methods Studies were carried out in normal nonobese male volunteers. Each received the infusions in varied order (after overnight fasting) within a period of 3 weeks. Highly purified porcine secretin (Gastrointestinal Laboratories, Karolinska Institute, Stockholm) was dissolved in infusates immediately before use. Glucose was infused IV' at a constant rate (66 g in 120 min). Blood samples were drawn from a contralateral vein before, during, and after infusions. Whole blood glucose was estimated by the ferricyanide method on a Technicon Auto Analyzer. Serum FFA were estimated by the Dole titration (Dole and MeinerQz 1960). IRI was estimated by radioimmunoassay (Herbert et a / . 1965). Tn assays for immunoreactive glucagon (IRG) an antiserum believed to be specific for glucagon (Aguilar-Parada et ul. 1949) was employed (antibody 30K supplied by Dr. K. H. Unger, Dallas, Texas): in preparation of specimens for immunoassay Trasylol. 500 KIU/ml was added to blood immediately upon sampling. Specimens from all studies on each subject were assayed together. IRI in serum was fractionated by chromatography on Sephadex G 50 (Gorden and Koth 8969). Secretin was administered according Qs three schedules. With 6pprlonged high-dose infusions', 3.5 ciinical units per minute of secretin (0.9 pg/min) were delivered through 124) min. With 'Bow-dose infusions9, constant-rate infusions of 1.2 U/min (0.3 ,ug/min) were administered, but these infusions were initiated with a priming dose of five clinical units (1.2 ,ug) in the first minute. With 'prolonged low-dose infusions9, delivery of secretin at the lower rate was continued through 120 min, and with 'brief low-dose infusions', infusion of secretin at the same lower rate was continued for 15 min only. A group of the subjects also received the prolonged high-dose secretin infusion in saline vehicle. With an immunoassay system for secretin employing an apparently specific antiserum (donated by Dr. L. Lazarus and Dr. D. Byrnes, Garvan Institute, Sydney, Australia) ir was established that infusion of secretin at the lower dose rate raised the level of secretin in the blood to a plateau of 100-200 pg/rnl, matching endogenous responses to acidification of the duodenurn quantitated in the same assay (unpublished observations of the authors). Plasma levels sf secretin fell1 after completion of infusions with a half-life of less than 5 min.
TIME (rntn)
TIME ( m ~ n l
FIG. 1. Mean blood glucose and serum l R I in subjects given glucose alone (O), glucose plus prolonged high-dose secretin ( A ), glucose pl~msprolonged Icw-dose secretin (m), or glucose plus brief low-dose secretin ( g). Bars show SEM of mean values with glucose alone. A total of 11 subjects took part in the study. All received the standard glucose infusion. Eleven received the prolonged high-dose secretin infusion in glucose. Seven received infusions of secretin according to each of the three secretin dose schedules. Five received the prolonged high-dose secretin infusion in saline vehicle.
Results Mean values for blood glucose and serum IRI1 during infusions of glucose alone or with secretin are illustratecl in Fig. 1. In the present study and in previous 'paired' studies in this laboratory (Dupr6 eb ak. 1969) the basal ( 0 time) values for glucose and IRI showecl little variation, and changes during IV infusions of glucose exhibited least variation when treated as differences from basal values. The statistical significance of differences bctwcen effects of glucose alone and of glucose with different doses of secretin was evaluated by analysis of paired changes at each time. The means and standard errors of these paired differences are shown in Table 1, together with the results of analysis of the paired incremental differences. The overall changes in blood glucose and serum IRI were also treated as the integrated changes through the 120 min infusion period and these results are shown in Table 2. 'To validate these comparisons six of the subjects received a second infusion of glucose alone; the same analyses revealed no significant differences between the efIects of the two infusions. Administration of secretin in saline according to the 'prolonged high-doseq scheclule raised serum IRP transiently but had no effect on the
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blood glucose (Table 1 ) . When secretin was infused at the high-dose rate together with glucose the integrated rise (0-120 min) was greatly enhanced by comparison with that in response to infusion of glucose alone (+85%, p < 0.001). At individual sample times this effect was significant at 5 rnin and through 75 min. This was associated with reduction in the integrated change (0-120 min) in blood glucose concentration (-22 % , p < 8.05), and with significant lowering of the incremental glucose at sample times 45 min through 90 min. When the 'prolonged low-dose' infusion of secretin was administered together with glucose the early rise in IWI in serum ( 5 min) was again significantly greater than that evoked by glucose alone ( p < 0.05). The mean increment in IRI was greater than that with infusion of glucose alone throughout the infusion period, but this difference was significant only at 5 rnin and through the interval 38-60 min. The mean integrated change (0-120 min) in IRI was significantly increased (4-35 %, p < 0.01 ). The 'prolonged low-dose' infusion of secretin also resulted in significant lowering of the mean increment in blood glucose in the interval 30-1 05 rnin and the integrated change in blood glucose was significantly reduced ( -3 8 % , p < 0.001). When secretin was delivered according to the 'brief low-dose' schedule, at 5 rnin the early rise in IRI in serum was again significantly greatcr than that observed when glucose was given alone ( p < 0.05) but enhancement of the mean rise in IRI was maintained only through the short period of administration of secretin, and the integrated change in IRI through the 120 rnin period of infusion of glucose was not significantly greater than that evoked by glucose alone. Nevertheless, this dose of secretin also caused a significant reduction of the rise in blood glucose at all times in the interval 30-105 min and the integrated rise in blood glucose was rcduced by 42% ( p < 0.001 '
>
Eflects of Secretin on Serum lmmuntsreactive Glucagon Serum IRG was estimated in samples drawn at time 0 and at 5, 10, 30, 45, and 75 rnin in subjects recciving glucose, or glucose with 'prolonged high-dose' secretin, or glucose with
FIG.2 . Mean changes in serum glucagon-like immunoreactivity in subjects given glucose alone ( 0) , glucose plus brief low-dose secretin ( 0), glucose plus prolonged high-dose secsetin ( A ) , or prolonged highdose secsetin alone ( ). . Bars show SEM of mean values with glucose alone and with prolonged highdose secretin alone.
'brief low-dose' secretin. The infusions of secretin did not modify the suppression of serum IRG during hyperglycemia (Fig. 2 ) .
Chromatographic Properties of Secretinstimulated IRI Serum drawn from each subject at 15 and 30 rnin (pooled) and at 45 and 60 rnin (pooled) was chromatographed on Sephadex G50. Mean overall recovery of IRI was 103 2 6.1 % (SB) . In every instance more than 90% of the IRI was recovered in fractions corresponding to monomeric insulin in eluates from subjects receiving 'prolonged high-dose' infusions or 'brief low-dose' infusions of the hormone. Eflects of Glucose and Secretin on Serum F F A Serum total F F A were titrated in specimens from subjects who received glucose alone and glucose with 'prolonged high-dose' or 'brief low-dose' infusions of secretin. Secretin did not modify the suppression of FFA (Fig. 3 ) . Discussion Although it has been previously demonstrated that secretin is potentially insulinotropic and can enhance the insulin response to glycemia in man, irnprovcment of glucose tolerance as a result of administration of purified secretin during IV1 infusions of glucose has not hitherto been clearly established. The present studies show that secretin can produce substantial improvement in tolerance to IV glucose during prolonged IV infusions of the sugar. The mean blood glucose curves during infusion of the threc different doses of secretin were
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CAN. J. PHYSIOL. PHARMAGOL. VCBI,. 53, 1975
Fnc;. 3. Mean total serum FFA in subjects receiving glucose alone (Q), glucose plus brief Bow-dose secretin ( 0 ) , or glucose plus prolonged high-dose secretin ( A ) . Bars show SEM of mean values with gBucsse alone.
lower than the curve during administration of glucose alone, but were not significantly different froin one another. This effect of secretin was not apparent before 30-40 min of infusion of glucose. Thus the failure of secretin to affect the concentration of glucose in the blood during infusions in earlier studies may be related to the shorter periods of administration of glucose employed by Bupr6 et a/. (1969) (40 min) and by Chisholm e& ak. (1969) (25 min) . The three schedules for infusion of secretin delivered similar doses during the first 2 mila and produced similar enhancement of the insulin response at the 5 min time point. Administration sf sccretin in saline vehicle also resulted in transient elevation of immunoreactive insulin in the blood. Although the increments in TRT at 5 min after starting the infusions of glucose with secretin were greater in the mean than the sum of responses to glucose alone and secretin alone, at this time the response to tlae combined stimuli was not significantly greater than the sum of their individual effects. However, stimulation of insulin release by administration of secretin in saline was transient, and the enhancement of insulin responses to glucose with the prolonged administration of secretin at high or low dose rates clearly represented potentiation of the response to glucose. This effect of secretin was related to the dose administered. Thus the integrated change in HRI in response to 'pro-
longed high-dose' infusions of secretin was significantly greater than that in response to 'prolonged low-dose' infusion of secretin ( p < 0.02)' and the latter was significantly greater than that in response to 'brief low-dose' infusion of secretin ( p < 0.05). The chromatographic properties of IWH detected in the blood during administration of secsetin at high and low rates were sinsilas, suggesting that immunsreactive iilsulins with different biological potencies were not released. Suppression of IWG and FFA during infusiomss of glucose with or without secretin in high or low dosage was similar, suggesting that net disposal sf glucose was not affected by metabolic actions of glucagon or of FFA. Thus these studies identified no reason for the lack of a simple relationship between thc total quantity of secretln administered and the associated glucose tolerance, in spite of dose-related effects of secretin on integrated changes in IRI. It is possible that the enhancement of the early release of insulin that was common to the three types sf secretin infusions may have been the major determinant of the glucose tolerance curve. Although such an effect of transient enhancement of endogenous secretion of insulin during continuous IV infusion of glucose has not been reported previously, it has been suggested that early rapid release of insulin may be important in determining the physiologic effects of the hormone (Lerner and Porte 197B ) . The inferred importance of rapid early delivery of insulin is supported by the results of recent studies of effects of HV infusion of insulin on oral glucose tolerance in diabetes mellitus (Albisser et al. 1974). However, while the effect of secretin on glucose tolerance might be attributed to enhancement of the secretion of iilsulin at an early stage of the response, it is important to consider the possibility that secretin may exert metabolic effects other than those directly dependent on its insulinotropic action. Thus, there is evidence of an interaction of secretin with insulin in muscle leading to increased glycolysis (Chisholm el al. 1972, 1975). It has long been recognized that secretin affects the external secretions of the liver, and the identification of specific receptor sites for secretin on liver cell membranes has recently been reported (Bataille et al. 1974). It is therefore possible that sccretin affects the
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DUPRE ET AI,.: EFFECTS ON INSULIN SECRETION 4 N D GLUCOSE TOLERANCE
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BQDEN, C . , and CHEY, W. Y. 1973. Preparation and specificity of antiserum to synthetic secretion and its use in a radioimmunoassay . Endocrinology, 92, 1617-1624. BROWN,J. C., DRYBURGH, J . , ROSS, S. A., and DUPRB,J. 1975. Identification and actions of gastric inhibitory polypeptide. Recent Prog. Hormone Res. 31.487-532. CATALAND, S., CROCKETT,S. E . , BROWN,J. C., and MAZZAFERRI.1974. Gastric inhibitory polypeptide stimulation by oral glucose in man. J . Clin. Endocrinol. Metab. 39,223-228. CIIISHOL~M, D. J., KI,ASSEN,G. A., DUPRE, I . , and PCPZEFSKY, T. 1972. Modification of the effect of insulin on muscle by secretin. Clin. Res. 20,945 (Abstr.). 1975. Interaction of secretin and insulin on human forearm n~etabolism.Eur. J. Clin. Invest. In press. CHISHOLM, D. J., YOUNG,J . D., LAZARUS, L . 1969. The gastrointestinal stimulus to insulin release I Secretin. J. Clin. Invest. 48, 1453-1460. DOLE,V . P., and MEINERTZ, H. 1960. Microdetermination of long-chain fatty acids in plasma and tissues. J . Biol. Chem. 235,2595-2599. IIuPRE, J . , and BECK,J. C . 1966. Stimulation of reiease of insulin by an extract of intestinal mucosa. Diabetes, 15,555-559. U U P R ~J .,, and CHISHOIM,D. J. 1970. Effects of secretin on glucose tolerance in man. Diabetes, 20, 322 (Abstr. j. DUPRI?,J . , CURTIS,J . D., UNGER,R. H., WADDELL,R. W., and BECK,J . C. 1969. Effects of secretin, pancreozymin, or gastrin on the response of the endocrine pancreas to administration of glucose or arginine in man. J . Clin. Invest. 48,745-757. DUPRB,J., KOJAS,I.,., WHITE,J . J., UNGER,R. H . , and BECK,J. C. 1966. Effects of secretin on insulin and glucagon in portal and peripheral blood in man. Lancet, 2, 26-27. DUPRE,J., ROSS, S. A., WA.Y.SON, D., and BROWN,J . C. 1973. Stimulation of insulin secretion by gastric inhibitory polypeptide in man. J . Clin. Endocrinol. Metab. 37,826828. ENK, B., and DECKEW?','r. 1973. Modification of glucose-induced insulin secretion after secretin. Acta Endocrinol. Mbh. Suppl. 177,323 (Abstr.). CORDEN,P., and ROTH, J. 1969. Plasma insulin: fluctuations in the "'bigq9insulin comnlponent after glucose and other stimuli. J. Clin. Invest. 43,2225-2234. AGUILAR-PARADA, E., EBSENTRAUT, A. M., and UNGER, HERBERT,V . , LAU, M. S., GOTTLIEB,C. W.. and BI,EICIER, S. J. 1965. Coated charcoal immunoassay R. H. 1969. Pancreatic glucagon secretion in normal of insulin. J. Clin. Endocrinol. Metab. 25,1375-1384. and diabetic subjects. Am. J. Med. Sci. 257,415-419. EERNER,R., and P O R ~ ~D. E ,1971. Relationships between AL.BISSER,A. M., LIEBEL, B. S . , EWAKI, T. G.. intravenous glucose loads, insulin response and gluDAVIDORAC, Z., B o T ~ C. , K., and ZINGG,W. 1974. cose disappearance rate. I. Clin. Endocrinoi. Metab. An artificial endocrine pancreas. Diabetes, 23, 38933,409417. 396. IACKLEY,H. L . A., CHISHOLM,D. J., RAHINQVITCH, A., BATAII.L.E, D. P., FREYCHET, P., and R o s s ~ r , r G. ~ , 1974. WEXLXR,M., and D U P R ~J., 1975. Effects of porInteractions of glucagon, gut glucagon, vasoactive intacaval anastomosis on glucose tolerance in the dog: testinal polypeptide and secretin with liver and fat cell evidence of an interaction between the gut and the membranes: binding t o specific sites and stimulation liver in oral glucose disposal. Metabolism, 24, s f adenylate cyclase . Endocrinology, 9 5 , 7 13-72 1 . 1157-1 168. BLOOM,S. 1974. Hormones of the gastrointestinal tract. Br. Med. Bull. 30.62-67.
metabolic functions sf the liver, and evidence sf an effect of unidentified huinoral secretions of the intestine on hepatic functions affecting glucose tolerance has recently been reported (Lickley et al. 1975). It is clear that the mechanism of the improvement of glucose tolerance resulting from administration of secretin requires further elucidation. Release of secretin in man after ingestion of glucose alone was suggested in earlier studies with a radioimmunoassay for secretin (Chisholm et al. 1969) but has not been confirmed (Bloom 1974; Boden et al. 1972; Buchanan, K. B. : personal communica~ion; unpublished observations of the authors). Recent evidence suggests that gastric inhibitory polypeptide may be the intestinal hormone that potentiates insuBin secretion after ingestion of glucose alone (Bupr.6 et al. 1973; Cataland et ale 1974; Brown et QE. 1975). However, after ingestion of a normal meal the effects of secretin presumably contribute to the physiological response. These effects may be related to the maintenance of relatively low blood glucose levels during absorption of the glucose component of a meal, by comparison with glucose levels observed after ingestion of glucose alonc. The results of the present study with secretin point out the possible importance of transient endocrine secretory responses of the intestine in relation to effects on glucose disposal under physiological coiaditions. It is clear that a better understanding of the actions of this hormone must await examination of its possible effects on the production of glucose and on net and regional disappearance of glucose during absorption of the sugar from the gut.
