Effect of gastrointestinal hormones on choleresis from the isolated perfused rat liver

Regulatory Peptides, 10 (1985) 237-242

237

Elsevier RPT00346

Effect of gastrointestinal hormones on choleresis from the isolated perfused rat liver Keiichi Y a m a t a n i a, N o r i h i r o Sato a, Kenji T a k a h a s h i ~, M a s a o H a r a b a n d H i d e o Sasaki" aThird Department of Internal Medicine, Yamagata University School of Medicine, Yamagata 990-23, and bDepartment of Internal Medicine, Nanyo General Hospital, Nanyo 992-04, Japan

(Received 5 September 1984; revisedmanuscriptreceived 3 December 1984; accepted for publication 5 December 1984)

Summary Using isolated perfused rat liver, the direct effect of secretin, glucagon, caerulein, insulin and somatostatin on choleresis was investigated. When the liver was perfused in the absence of sodium taurocholate, the bile volumes were: control, 0.33 4- 0.01 (mean + S.E.M.) ml/10 g liver per 50 rain; secretin 0.05 U/ml, 0.39 4. 0.01 (P < 0.01); glucagon 10 - l ° M, 0.44 4. 0.02 (P < 0.01); caerulein 10 -s M, 0.34 + 0.03 (n.s.); insulin 1 mU/ml, 0.35 -4- 0.02 (n.s.); glucagon plus somatostatin 10 -7 M, 0.46 4- 0.03 (n.s. vs. glucagon alone), respectively. When 10 -5 M sodium taurocholate was present in the perfusate, the bile volumes were: control, 0.61 4- 0.03; secretin, 0.63 -4- 0.01 (n.s.); glucagon, 0.70 4. 0.01 (P < 0.05); caerulein, 0.55 4- 0.01 (n.s.); insulin, 0.62 -4- 0.04 (n.s.); somatostatin, 0.59 4- 0.01 (n.s.); respectively. Glucagon increased glucose output and cyclic A M P in the effluent from the liver neither of which were suppresscd by somatostatin. Secretin increased cyclic A M P but not glucose output. These results indicate that glucagon has the most potent action on bile acid-independent canalicular bile, that caerulein and insulin do not act on canalicular bile production directly and that somatostatin docs not directly suppress canalicular bile production nor hepatic glucose output produced by glucagon in rats. glucagon; secretin; caerulein; insulin; somatostatin; canalicular bile

Address for correspondence: Keiichi Yamatani, M.D., The Third Department of Internal Medicine,

Yamagata UniversitySchool of Medicine,Yamagata 990-23, Japan. 0167-0115/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)

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Introduction

Several gastrointestinal hormones have been known to participate in the regulation of bile secretion [1]. From studies on the component of bile in vivo, secretin is thought to increase mainly ductular bile [2,3] and, in part, bile acid-independent canalicular bile [4]. Glucagon is thought to increase more bile acid-independent canalicular bile than ductular bile [2,5], and caerulein is reported to increase both bile acid-dependent canalicular bile and ductular bile [6]. However, the exact effect(s) of each hormone on bile secretion is (are) still not fully understood, because gastrointestinal hormones have many biological effects including neuronal effects and interaction with each other. Part of the confusion is due to the different experimental models and experimental conditions reported in a variety of species. The choleretic effect of insulin [7-11] and the cholestatic effect of somatostatin [12-18] are particularly controversial. Therefore, isolated liver perfusion was performed to investigate the direct effect of gastrointestinal hormones such as secretin, glucagon, caerulein, insulin and somatostatin on bile secretion in rats.

Materials and Methods

Male Wistar rats weighing 200-250 g, fed ad libitum, were killed and the liver was isolated according to the method of Sugano et al. [19]. The perfusion medium was a hemoglobin-free mixture of 115 mM NaC1, 5.9 mM KC1, 1.2 mM MgCI2, 1.2 mM NaH2PO4, 1.2 mM Na2SO4, 2.5 mM CaCI2 and 25 mM NaHCO3 gassed with a mixture of 95% 02/5% CO2. The liver was perfused non cyclically at a rate of 30 ml/min through the portal vein. The effluent was collected from the inferior vena cava every minute. After the liver was preperfused for 30 min, porcine glucagon (Novo AS, Copenhagen), synthetic porcine seeretin (Eisai, Tokyo), caerulein (Kyowa Hakko, Tokyo), porcine insulin (Novo AS, Copenhagen), sodium taurocholate (Nakarai Chemicals, Kyoto) and somatostatin (Protein Research Foundation, Osaka) were administered alone or together through a side arm via the portal vein to the liver for 50 min. The bile volume was measured gravimetrically from a cannula inserted into the bile duct. Glucose and cyclic AMP in the effluent were measured respectively by the glucose oxidase method and a radioimmunoassay kit (Yamasa Shoyu, Choshi). Each value in the results is shown as mean + S.E.M. Student's ttest was used for statistical analysis.

Results and Discussion

The basal bile production from the isolated perfused rat liver was 0.33 4- 0.01 ml/g liver per 50 min (Table I). Addition of 10- s M sodium taurocholat¢ to the perfusate increased the bile volume to 0.61 -t- 0.03 ml/g liver per 50 min (Table II). The increment in the bile volume produced by 10 - t ° M glucagon was the same in either the presence or absence of sodium taurocholate. This result coincides with the reports

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TABLE I Effect o f glucagon, secretin, caerulein and insulin on the bile volume from the isolated perfused rat liver when sodium tauroeholate is not included in the perfusate No.

Bile volume ml/10 g liver per 50 rain (mean 4- S.E.)

Control Glucagon 10 - t ° M Secretin 0.05 U / m l Caerulein 10 - s M Insulin I m U / m l

5 5 5 3 3

0.33 0.44 0.39 0.34 0.35

44444-

0.01 0.02"** 0.01" 0.03 0.03

* P < 0.01 vs. control, ** P < 0.01 vs. secretin

T A B L E II Effect of glucagon, secretin, caerulein, insulin and somatostatin on the bile volume from the isolated perfused rat liver when sodium tauroeholate is included in the perfusate No.

Bile volume ml/10 g liver per 50 rain (mean + S.E.)

Sodium Sodium + Sodium + Sodium + Sodium + Sodium +

taurocholate taurocholate glucagon taurocholate secretin taurocholate caerulein taurocholate insulin taurocholate somatostatin

10 -5 M 10 - s M 10 - 1 ° M 10 -5 M 0.05 U / m l 10 - s M 10 - s M 10 -5 M 1 mU/ml 10 - s M 10 - 7 M

4 4

0.61 4- 0.03 0.70 4- 0.01"

4

0.63 4- 0.01

4

0.55 4- 0.01

3

0.62 + 0.04

3

0.59 4- 0.01

* P < 0.05 vs. 10 -5 M sodium taurocholate.

that glucagon increases mainly the bile acid-independent canalicular bile [2,5]. The increment in the bile volume produced by 0.05 U/ml secretin (approximately 10 - 9 M) was less than that brought about by glucagon (Table I). This result is conceivable if secretin acts mainly on ductular bile production [2-4]. Since the blood circulation to the bile ducts or ductules is supplied from the propria hepatic artery, seeretin administered through the portal vein would perfuse the ductule less than the hepatocyte. Therefore, it is conceivable that the results with sodium taurocholate-free perfusate indicate the bile salt-independent canalicular bile production and the results with sodium taurocholate-contained perfusate indicate the bile salt-dependent canalicular bile production. It is likely that a small increment in bile produced by seeretin

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was indistinguishable when the control bile volume was increased remarkably by sodium taurocholate (Table II). It was reported that glucagon induces the bile production through cyclic AMP, and that the infusion of dibutyryl cyclic AMP increased the bile production from the in situ perfused rat liver [7]. The role of cyclic AMP in secretin-induced cholesis was also augumented in humans and baboons, but not in dogs or rodents [20]. In the present study, 10-10 M glucagon increased the glucose output and cyclic AMP in the effluent (Table III), 0.05 U/ml secretin increased cyclic AMP in the effluent (924 + 291 pmol/g liver per 50 min) but not the glucose output from the isolated perfused rat liver as described previously [21], and the increment in the bile volume produced by secretin was less than that produced by glucagon (Tables I and II). 10 - 9 M glucagon and 0.5 U/ml secretin caused a remarkable increment in cyclic AMP but the bile volume was the same as in the case of 10- lo M glucagon and 0.05 U/ml secretin, respectively (data not shown). These results indicate that cyclic AMP produced by secretin is not related to the choleretic effect. There might be a difference in the sensitivity to cyclic AMP between the hepatocyte and the ductular epithelium, or as another possibility, there might be a functional compartmentalization of cyclic AMP in the hepatocyte. In any case, these possibilities must await further examination. Caerulein was reported to increase the bile volume, HCO3- and bile salt in the bile from the dog liver in vivo [6] or to increase the bile volume and H C O 3 - but not bile salt in the bile from the rat liver in vivo [22]. 10-s M caerulein with or without sodium taurocholate did not affect bile volume from the isolated perfused rat liver (Tables I and II). This means that caerulein does not directly affect the bile saltindependent or -dependent canalicular bile production, which coincides with the latter report [22]. There are controversial reports about the choleretic effect of insulin [7-11], which was reported to be mediated by other hormone(s) [7] or hypoglycemic vagal stimulation [8,9]. Others support that insulin has a direct choleretic effect [10,11]. In the present study, 1 mU/ml insulin with or without sodium taurocholate did not affect bile volume (Tables I and II). Thus, at least, it can be said that insulin does not directly affect the bile salt-dependent or -independent canalicular bile production. TABLE III Effect of somatostatin on the bile volume, glucose output and cyclic AMP in the effluent from the isolated perfused rat liver increased by glucagon Bile volume (ml/10 g liver per 50 rain) Glucagon 10 - t ° M

(n

0.44 4- 0.01

Increment in glucose output (/zmol/g liver per 50 rain) 95.4 4- 5.4

Increment in cAMP in effluent (pmol/g liver per 50 rain) 188 + 55

= 5)

Glucagon 10- t0 M S o m a t o s t a t i n 10 - 7 M

0.46 + 0.03

103

4- 5.3

191 4- 98

(n = 5)

Taurocholate is not included in the perfusate. There is no significant difference between the two groups.

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The cholestatic effect of somatostatin is also controversial [12-18]. Many investigators reported that this is an indirect effect because somatostatin did not suppress the choleretic effect of extrinsic pentagastrin [12], glucagon [13] or secretin [14,15] in vivo. It is also reported that the cholestatic effect of somatostatin during intraduodenal acid infusion is related to secretin release [16]. However, there are some reports that somatostatin partially suppressed the choleretic effects of extrinsic secretin in vivo [17] and sodium taurocholate in vitro [18]. In this study it was clarified that 10 -7 M somatostatin did not suppress the bile volume increased by 10 -1° M glucagon (Table III) or sodium taurocholate (Table II), and that somatostatin did not suppress the glucose output or cyclic AMP increased by glucagon (Table III). These results indicate that the cholestatic effect of somatostatin is mediated by the reduction of choleretic hormone induced by somatostatin [12-15]. In conclusion, though of course the present results are limited to rats, choleresis in the isolated perfused liver is mainly due to the canalicular bile production and less due to the ductular bile production. Glucagon was more potent than secretin in the choleresis from the isolated perfused rat liver. Neither caerulein nor insulin directly affect the bile acid-dependent or bile acid-independent canalicular bile production. Somatostatin did not suppress the choleresis produced by sodium taurocholate or glucagon directly. Somatostatin did not suppress the glucose output or cyclic AMP in the effluent from the liver produced by glucagon.

References 1 Glass, G.B~J., Gastrointestinal peptide hormones as modulators of bile secretion, Prog. Liver Dis., 7 (1982) 243-260. 2 Wheeler, H.O. and Mancusi-Ungaro, P.L., Role of bile ducts during secretin choleresis in dogs, Am. J. Physiol., 210 (1966) 1153 1159. 3 Jones, R.S., Geist, R.E. and Hall, A.D., The choleretic effect of glucagon and secretin in the dog, Gastroenterology, 60 (1971) 64-68. 4 Barnhart, J.L. and Combes, B., Erythritol and mannitol clearances with taurocholate and secretininduced choleresis, Am. J. Physiol., 234 (1978) E146-156. 5 Khedis, A., Dumont, M., Duval, M. and Erlinger, S., Influence of glucagon on canalicular bile production in the dog, Biomedicine, 21 (1974) 176-181. 6 Dangoumau, J., Balabaud, C., Bussiere-Leboeuf, C., Dallet-Duverge, F. and Noel, M., Influence of pentagastrine, cholecystokinin and caerulein on bile production in the rat, J. Pharmacol. (Paris), 8 (1977) 197-204. 7 Thomsen, O.13. and Larsen, J.A., The effect of glucagon, dibutyrylic cyclic AMP and insulin on bile production in the intact rat and the perfused rat liver, Acta Physiol. Scand., 111 (1981) 23 30. 8 Fritz, M.E. and Brooks, F.P., Control of bile flow in the cholecystectomized dog, Am. J. Physiol., 204 (1963) 825 828. 9 Kaminski, D.L., Rose, R.C. and Nahrwold, D.L., Effect of cholinergic blockade on insulin choleresis in dogs, Ann. Surg., 179 (1974) 505-509. I0 Meyers, W.C., Hanks, J.B. and Jones, R.S., Insulin and glucagon choleresis during normoglycemia, Surg. Forum, 30 (1979) 398~400. 11 Thomsen, O.13, Larsen, J.A. and Orskov, H., Insulin-induced choleresis in relation to insulin concentrations in plasma and bile in the cat, Scand. J. Gastroenterol., 17 (1982) 297-303. 12 Linsheer, W.G. and Raheja, K.L., Effects of somatostatin on gastric acid secretion and on lipid and carbohydrate metabolism in the rat, Br. J. Pharmacol., 64 (1978) 311 314.

242 13 Garberoglio, C.A., Bickerstaff, K.I., Baker, A.L. and Moossa A.R., Is glucagon a choleretic hormone at physiologic blood levels?, Am. J. Surg., 143 (1982) 61 66. 14 Lewis, M.H., Baker, A.L., Ipp, E. and Moossa, A.R., Effect of somatostatin on determinants of bile flow in unanesthetized dogs, Ann. Surg., 195 (1981) 97-103. 15 Ren~, E., Danzinger, R.G., Hofmann, A.F. and Nakagaki, M., Pharmacologic effect of somatostatin on bile formation in the dog: Enhanced ductular reabsorption as the major mechanism of anticholeresis, Gastroenterology, 84 (1983) 12@129. 16 Kaminski, D.L. and Deshepande, Y.G., Effect of somatostatin and bombesin on secretin-stimulated ductular bile blow in dogs, Gastroenterology, 85 (1983) 1239 1247. 17 Hottinger, S. and Preisig, R., Dual inhibitory effect of somatostatin on canalicular and ductular bile salt-independent bile formation, Gastroenterology, 79 (1980) 1109. 18 Ricci, G.L. and Fevery, J., Cholestatic action ofsomatostatin in the rat: Effect on the different fractions of bile secretion, Gastroenterology, 81 (1981) 552-562 19 Sugano, T., Suda, K., Shimada, M. and Oshino, N., Biochemical and ultrastructual evaluation of isolated rat liver systems perfused with a hemoglobin-free medium, J. Biochera., 83 (1978) 995-1007. 20 Levine, R.A. and Hall, R.C., Cyclic AMP in secretin eholeresis: Evidence for a regulatory role in man and baboons but not in dogs, Gastroenterology, 70 (1976) 537-544. 21 Yamatani, K., Sato, N., Takahashi, K., Hara, M. and Sasaki, H., A rise in cyclic AMP with a lack of glycogenolysis by secretin in the isolated perfused rat liver and its inhibition by epinephrine, Biochem. Biophys. Res. Commun., 115 (1983) 743-748. 22 Jones, R.S. and Grossman, M.I., Cholerctic effect of cholecystokinin, Gastrin II, and caerulcin in the dog, Am. J. Physiol., 219 (1970) 1014-1018.