Effect of Selective and Truncal Vagotomy on Insulin-Stimulated Bile Secretion in Dogs

Vol. 60, No.4

GASTROENTEROLOGY

Printed in U.S.A.

Copyright © 1971 by The Williams & Wilkins Co.

EFFECT OF SELECTIVE AND TRUNCAL VAGOTOMY ON INSULIN-STIMULATED BILE SECRETION IN DOGS RiCHARD E. GEIST, M.D., AND RAYFORD S. JONES, M.D.

Department of Surgery, Veterans Administration Hospital, and University of California at San Francisco, San Francisco, California

Dogs were prepared by cholecystectomy, ligation of the lesser pancreatic duct, and insertion of Thomas cannulas into the stomach and duodenum. Hepatic bile was collected by cannulating the common bile duct through the duodenal fistula. illsulin, 1 U per kg, increased bile flow and chloride concentration. Neither selective hepatic nor selective extrahepatic vagotomy reduced the stimulation of bile flow produced by insulin. Selective hepatic vagotomy reduced control chloride output, but had no significant effect on insulin choleresis. Selective extrahepatic vagotomy decreased bile bicarbonate concentration and output in both control and postinsulin periods. Truncal vagotomy reduced, but did not abolish, the increase in bile flow following insulin. Control bile bicarbonate concentration was reduced after truncal vagotomy, and truncal vagotomy prevented the increase in bicarbonate output after insulin. These results indicate that an extravagal mechanism is important to insulin-induced choleresis. The formation of bile is a complex process involving secretion from at least two sites: canaliculi and ductules. 1 Canalicular secretion depends upon active transport of bile salts from hepatocytes into the canaliculi. 2 ill addition, recent work has revealed a bile salt-independent inorganic electrolyte canalicular fraction. 3 • 4 Ductular secretion is probably controlled by secretin, gastrin, cholecystokinin, glucagon, and vagal stimulation. 1 Tanturi and Ivy5 demonstrated that elecReceived August 10, 1970. Accepted November 13, 1970. Address requests for reprints to: Dr. Rayford S. Jones, Veterans Administration Hospital, 4150 Clement Street, San Francisco, California 94121. This work was supported by a Clinical Investigator appointment from the Veterans Administration (R. S. J.). The authors are grateful for the expert technical assistance of Elizabeth Stevenson, John Dunphy, and Michael Stewart, and are also indebted to Dr. Morton I. Grossman for advice and assistance in the preparation of the manuscript.

trical stimulation of the vagus nerves caused increased bile secretion. Later, Fritz and Brooks 6 showed that insulin hypoglycemia caused a marked choleresis in conscious dogs and that the insulin response was prevented by anticholinergic drugs or vagotomy. The latter findings indicated that insulin choleresis was vagally mediated. Chemical stimulation of the pyloric antrum has been shown to increase bile secretion in dogs. 7 , 8 However, this observation has recently been disputed. 9 Jones and Brooks 10 found that removal of the pyloric antrum reduced the insulin choleresis in dogs. These findings, plus the observation of Zaterka and Grossman 11 that exogenous gastrin stimulated flow in dogs, suggest that antral gastrin may be important in insulin choleresis. Since it has been clearly demonstrated that vagal stimulation releases gastrin,12 it is of interest to inquire whether vagal choleresis is primarily due to direct vagal effect on the liver, vagal release of antral gastrin, 566

VAGOTOMY AND BILE SECRETION

April 1971

or both processes. The present experiment was done to investigate this question.

Methods Six mongrel dogs weighing 15.5 to 24.6 kg were prepared by cholecystectomy, ligation of the lesser pancreatic duct, insertion of a modified Thomas cannula 13 into the duodenum opposite the opening of the common bile duct, and insertion of another Thomas cannula into the stomach. Experiments were begun no earlier than 2 weeks following surgery and were performed no more than three times weekly, and never on successive days. Mter the dogs fasted for 16 hr the gastric and duodenal ~an­ nulas were unplugged and a polyethylene tube (Intramedic PE-190, outside diameter 1. 7 mm) was inserted 4 to 6 cm into the common bile duct through the duodenal cannula. A 14 F rubber tube was inserted into the lumen of the duodenum with its tip between the duodenal cannula and the pylorus. Both the polyethylene tube in the common bile duct and the rubber tube in the duodenum were brought out through holes in the cork used to occlude the duodenal cannula during experiments. Bile, duodenal, and gastric specimens were collected every 15 min. During experiments each dog received an intravenous infusion at a rate of 50 ml per hr delivered by a calibrated peristaltic pump (Harvard Apparatus Co., Dover, Mass.) through a polyethylene tube (PE-50) in a leg vein. The intravenous infusion was 0.9% NaCI for four 15-min periods, then 0.5% sodium taurocholate (Maybridge Chemical Co., Ltd., Tintagel, North Cornwall, Unitfld Kingdom) was added and infused for the remaining 12 15-min periods of the experiment. After sodium taurocholate had been infused for four 15-min periods, control or insulin injections were given. Regular insulin (Eli Lilly, Indianapolis, Ind.) diluted in 10 ml of 0.9% NaCI was injected in a dose of 1 U per kg through a three-way stopcock in the intravenous tubing. We have previously shown in the dog that 1 U per kg is the maximal choleretic dose of insulin, while 0.5 U per kg is the maximal dose for gastric acid output. 14 Control experiments were performed similarly by injecting 10 ml of 0.9 % NaCl. Volumes of bile and gastric juice were recorded. Bile bicarbonate concentrations were measured by adding 0.5 ml of bile to 1.0 ml of 0.1 N HCI, boiling, cooling, and back titrating to pH 7.0 with an automatic titrator and pH meter (Autoburette, Radiometer, Copenhagen). Chloride concentrations were estimated with a chloridometer

567

(Buchler-Cotlove, Fort Lee, N. J.). Gastric acid concentration was measured by titrating with an automatic titrator to pH 7.0 using 0.2 N NaOH. A venous blood sample was drawn 1 hr following insulin injection and the glucose concentration was measured by the O-toluidine method. '5 The pH of duodenal juice was measured with a glass electrode (Radiometer). Mter two insulin and two control tests had been performed, the dogs were randomly divided into two groups of 3 dogs each. One group of dogs underwent selective hepatic vagotomy. The hepatic branches of the ventral vagus nerve were identified and divided. The second group of dogs underwent selective extrahepatic vagotomy. The hepatic branches of the ventral vagal trunk were identified and spared, but the ventral principal gastric nerves and the dorsal vagal trunks were divided. Mter a 2-week recovery period, two insulin and two control experiments were performed as described; then all dogs underwent truncal supradiaphragmatic vagotomy through a left thoracotomy incision. No gastric drainage procedures were done. One animal died following transthoracic vagotomy. After a 2-week recovery period, two insulin and two control experiments were again performed on each dog.

Results General Duodenal pH. In none of the experiments did the pH of the duodenal specimens fall below 6.0. Blood sugar. The mean plasma glucose concentration for all experiments 1 hr following insulin administration was 39.9 ± 1.7 mg per 100 ml. Effect of insulin before vagotomy (tables 1, 2, and 3). Insulin increased the bile flow rate, chloride concentration, chloride output, and bicarbonate output but did not increase the bicarbonate concentration. Effect of Selective Hepatic Vagotomy (table 1) Selective hepatic vagotomy had no significant effect on control or postinsulin bile flow or composition, except for a decrease in chloride output in control tests. Selective hepatic vagotomy had no significant effect on insulin-stimulated peak acid output (fig. 1).

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GEIST AND JONES TABLE

Vol . 60, No.4

1. Electrolyte composition of control and insulin-stimulated bile before and after selective hepatic vagotomy (3 dogs)a Prevagotomy

Bile flow (mIl15 min) ...... HCO, - concentration (mEq/liter) ... . . . . . HC0 3 - output (/LEq/15 min) ....... . . Cl - concentration (mEq/liter) . ... Cl - output (/LEq/15 min)

Postvagotomy

Control

Insulin

Control

2.0 30.8 65.1 91.5 182.3

3.9 31.5 125.8 103;0 403.3

1.4 24.9 35.7 87.7 71.0·

Insulin

4.1 33.1 137.7 '100.7 417.6

F'

31.39 0.92 9.75 4.09 35.89

LSD'

0.7 47.0 10.9 85.6

a Each value is the mean of two experiments on each of 3 dogs. Peak bile flow occurred in the fourth period following insulin. The control and insulin values during the fourth 15-min period after insulin before and after hepatic vagotomy were compared using analysis of variance. • F, variance ratio. c LSD, least significant difference at the 5% level; F a.ao = 3.29, F a.al 5.42 (degrees of freedom, 3, 15) . " Significantly (P < 0.05) different from prevagotomy value.

I.

TABLE

2. Electrolyte composition of control and insulin-stimulated bile before and after

selective extrahepatic vagotomy (3 dogs)a Prevagotomy

Bile flow (mIl15 min) ... ..... . . . HCO, - concentration (mEq/liter) ... . .. . . HCO, - output (/LEq/15 min) . . .. Cl - concentration (mEq/liter) .. .. " Cl - output (/LEq/15 min)

Postvagotomy

Cont.rol

Insulin

Control

Insulin

1.5 40.7 64.9 77.2 120.6

3.5 41.1 145.5 94.3 332.7

1.5 29.9" 45.5 84.2 135.2

3.4 29.4" 98.9" 96.8 326.5

po

LSD'

50.17 8.62 42.30 5.73 44.67

0.47 6.69 20.30 U.5 52.6

a Each value is the mean of two experiments on each of 3 dogs. Peak bile flow occurred in the fifth period following insulin. The control and insulin values during the fifth 15-min period after insulin before and after extrahepatic vagotomy were compared using analysis of variance. • F, variance ratio. c LSD , least significant difference at the 5% level; Fa."" = 3.29, F a.al 5.42 (degree of freedom 3, 15). " Significantly (P < 0.05) different from prevagotomy value.

TABLE

3. Electrolyte composition of control and insulin-stimulated bile before and after truncal vagotomy (5 dogs) a Prevagotomy

Bile flow (mIl15 min) .. . . HCO, - concentration (mEq/liter) . . .. .. . HCO. - output (/LEq/15 min) Cl- concentration (mEq/liter) . . .. . . .. . Cl - output (/LEq/15 min)

Postvagotomy

Control

Insulin

Control

Insulin

1.7 37.7 68.2 83.1 145.4

3.5 37.7 130.0 97.9 345.4

1.3 14.7" 18.1" 80.2 107.6

2.6" 9.1" 22.7" 94.0 246.3"

po

LSD"

40.15 43.88 39.96 5.94 36.97

0.46 6.60 24.8 10.1 51.0

a Each value is the mean of two experiments on each of 5 dogs. Peak bile flow occurred in the fifth period following insulin. The control and insulin values during the fifth I5-min period after insulin before and after truncal vagotomy were compared using analysis of variance. • F, variance ratio. c LSD, least significant difference at the 5% level; F a.ao = 2.96, F a.al 4.60 (degrees of freedom 3, 27). d Significantly (P < 0.05) different from prevagotomy value.

VAGOTOMY AND BILE SECRETION

April 1971

Effect of Selective Extrahepatic Vagotomy (table 2) Selective extrahepatic vagotomy reduced bicarbonate concentration and output both in control tests and in tests with insulin. Control and postinsulin bile flow rate, chloride concentration, and chloride output were not significantly changed by selective extrahepatic vagotomy. Prevagotomy insulin peak acid output, 2.7 mEq per 15 min, occurred in the second 15-min period after insulin injection. After extrahepatic vagotomy, insulin failed to stimulate acid outputs above basal levels (fig. 2). Effect of Truncal Vagotomy (table 3) Truncal vagotomy caused the same changes in bile secretion as selective extrahepatic vagotomy, namely decreased bicarbonate concentration and output in both control and insulin tests. In addition, truncal vagotomy decreased the bile flow rate and chloride output in response to insulin. Truncal vagotomy completely abolished the insulin-stimulated gastric acid output (fig. 3).

Discussion In this experiment vagotomy did not abolish the choleretic response to insulin hypoglycemia. This observation, plus our

569

previous finding that atropine failed to prevent insulin choleresis,17 means that a nonvagal factor is important in insulin choleresis. This may explain why insulin greatly increases bile flow, while sham feeding 18 has almost no effect. Fritz and Brooks 6 previously demonstrated that truncal vagotomy abolished the choleretic response to insulin. We cannot explain the apparent discrepancy. One possible factor is that the present experiments were performed during intravenous bile salt administration, while the studies of Fritz and Brooks 6 were done without bile salt replacement. In any case, the nonvagal mechanism in insulin choleresis 10

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570

makes it impossible to answer the original question of this experiment regarding the relative roles of direct hepatic vagal effect and vagal release of gastrin. Hepatic vagotomy decreased the control chloride output. Extrahepatic vagotomy decreased control and insulin bicarbonate concentration as well as insulin bicarbonate output, while truncal vagotomy decreased insulin bile flow rate, control and insulin bicarbonate concentration and output, and insulin chloride output. Truncal vagotomy caused the greatest change in bile electrolyte secretion followed by extrahepatic and hepatic vagotomy in that order (table 4). We have no good explanation for the observed changes. The slight change in bile secretion following hepatic vagotomy raises the question of completeness of vagotomy. Unfortunately, there is no known test for completeness of hepatic vagotomy. The extrahepatic and truncal vagotomies abolished the gastric secretory response to insulin. However, following extrahepatic vagotomy there was high basal acid output and a late rise in acid output after insulin. These findings probably indicate incomplete gastric vagotomy in the extrahepatic vagotomy group. Studies on bile secretion must be controlled carefully to prevent entry of gastric acid into the duodenum. Duodenal acidification releases secretin, which stimulates increased bile flow. The open gastric fistula in our experiments was effective in diverting acid, since duodenal pH never fell below 6.0. If complete truncal vagotomy failed to TABLE

Vol. 60, No . 4

GEIST AND JONES

4. The effect of hepatic, extrahepatic, and truncal vagotomy on bile secretion Selective hepatic Con· msutrol lin

Bile flow HCO .. concentration . HCO .. output .... Cl' concentration Cl' output , ....

Selective extrahepatic

Inou· lin

Con· tr01

Insu· lin

0

0

0

0

0

1

0 0 0

0 0 0 0

1

1 1

1 1 1 1

1 1

1

0 0

REFERENCES 1. Wheeler HO: Water and electrolytes in bile,

2. 3. 4.

5.

6. 7. 8. 9.

Truncal

Con· trol

0 0 0

prevent insulin choleresis, then insulin must cause increased bile flow either by acting directly on the liver or indirectly by stimulating release of another choleretic. Insulin caused increased bile chloride concentration without affecting bile bicarbonate concentration. The chloride and bicarbonate concentrations during insulin choleresis are similar to those during glucagon infusion. 19 Unger et al. 20 have shown that insulin stimulates release of glucagon. It is possible that insulin stimulates glucagon release, which causes increased bile flow.

0

1

10.

11.

12.

Handbook of Physiology, sect 6: Alimentary Canal, vol 5. Edited by CF Code. Washington, Arnerican Physiological Society, 1968, 2409-2431 Sperber I: Secretion of organic anions in the formation of urine and bile. Pharmacol Rev 11:109134, 1959 Wheeler HO, Ross ED, Bradley SE: Canalicular bile production in dogs. Arner J Physiol 214 :866874, 1968 Erlinger S, Dhumeaux D, Berthelot P, et al: Effect of inhibitors of sodium transport on bile formation in the rabbit. Amer J Physiol 219:416422, 1970 Tanturi CA, Ivy AC : On the existence of secretory nerves in the vagi for and the reflex excitation and inhibition of the bile secretion. Arner J Physiol 121 :270-283, 1938 Fritz ME, Brooks FP: Control of bile flow in the chloecystectomized dog. Amer J Physiol 204: 825-828, 1963 Jones RS, Powell KC , Brooks FP: The role of the gastric antrum in the control of bile flow. Surg Forum 16:386-387, 1965 Nahrwold DL, Cooke AR, Grossman MI: Choleresis induced by stimulation of the gastric an· trum. Gastroenterology 52:18- 22, 1967 Ross H, Sauberrnann A, Silen W: The effect of gastric antral stimulation upon the secretion of hepatic bile. Proc Soc Exp Bioi Med 130:278283, 1969 Jones RS, Brooks FP: Role of pyloric antrum in choleresis after insulin and feeding. Arner J Physiol 213:1406-1408, 1967 Zaterka S, Grossman MI: The effect of gastrin and histamine on secretion of bile. Gastroenterology 50:500-505, 1966 PeThein M, Schofield B: Release of gastrin from the pyloric antrum following vagal stimulation

April 1971

13.

14.

15.

16.

VAGOTOMY AND BILE SECRETION

by sham feeding in dogs. J Physiol (London) 148:291-305, 1959 Thomas JE: An improved cannula for gastric and intestinal fistulas. Proc Soc Exp Bioi Med 46:260-261, 1941 Jones RS, Geist RE, Hall AD : Comparison of dose-response relations of insulin and 2 deoxyo-glucose for biliary and gastric acid secretion in dogs. Gastroenterology 59:665-670, 1970 Feteris WA: Serum glucose method without protein precipitation Amer J Med Techn 31:17-21, 1965 Snedecor GW, Cochran WG: Statistical Methods. Sixth edition. Ames, The Iowa State University Press. 1967

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17. Geist RE, Jones RS, Hall AD: The effect of atropine on vagally stimulated bile and gastric acid secretion. Clin Res 18:129, 1970 18. Powell KC , Miller LC, Brooks FP: Effect of sham feeding on bile flow in cholecystectomized dogs. Proc Soc Exp Bioi Med 118:481-483, 1965 19. Jones RS , Geist RE, Hall AD: The choleretic effects of glucagon and secretin in the dog. Gastroenterology 60:64-68, 1971 20. Unger RH, Eisentraut AM. McCall MS, et al: Measurements of endogenous glucagon in plasma and the influence of blood glucose concentration upon its secretion. J Clin Invest 41:682-689, 1962