Partial liver denervations dissociate the inhibitory effects of pancreatic glucagon and epinephrine on feeding

Physiology & Behavior, Vol. 35, pp. 233-237. Copyright©Pergamon Press Ltd., 1985. Printed in the U.S.A.

0031-9384/85 $3.00 + .00

Partial Liver Denervations Dissociate t]he Inhibitory Effects of Pancreatic Glucagon and Epinephrine on Feeding LAURA MAcISAAC AND NORI GEARY 1

Department o f Psychology, Box 28, Schermerhorn Hall, Columbia University, N e w York, N Y 10027 R e c e i v e d 12 O c t o b e r 1984 MAclSAAC, L. AND N. GEARY. Partial liver denervations dissociate the inhibitor), effects of pancreatic glucagon and epinephrine on feeding. PHYSIOL BEHAV 35(2) 233-237, 1985.---We compared the roles of different components of the liver's innervation in the inhibitory effects of pancreatic glucagon and epinephrine on feeding by testing the effects on meal size of intraperitoneal injections of these hormones in rats with selective abdominal vagotomies of only the hepatic branch, with partial liver denervations that spared only the hepatic branch of the vagus, and with sham operations. Pancreatic glucagon (100-400/xg/kg) inhibited size of evaporated milk test meals equally in rats with partial liver denervations sparing the hepatic vagus and in sham-operated rats, but had no effect on feeding in rats with selective hepatic vagotomies. In contrast, epinephrine (25-100/xg/kg) inhibited meal size equally in all rats. These data suggest that the hepatic vagus is the necessary and sufficient contribution of the liver's innervation to pancreatic glucagon's satiety effect and that hepatic innervation does not contribute to epinephrine's inhibitory effect on meal size. Thus, different peripheral neural mechanisms appear to mediate the effects of these hormones on feeding. Further, the data fail to support the hypothesis that abdominal vagotomies and coeliac ganglionectomies attenuate epinephrine's effect on feeding by disconnecting hepatic afferents. Epinephrine

Feeding

Pancreatic glucagon

Vagotomy

P E R I P H E R A L injection of either pancreatic glucagon [8,11] or epinephrine [17, 18, 24, 25] elicits a rapid inhibition of feeding in rats and other experimental animals. Although the mechanisms of these effects are unknown, in each case attention has focused on the liver as a site where a metabolic signal inhibiting food intake may arise. The rationale for this is that both hormones potently stimulate hepatic glycogenolysis and glucose production [5, 23, 26, 29] and that, in at least some situations, glycogenolysis and increased blood glucose are associated with their inhibitory effects on feeding [6, 15, 18, 19]. Further, because there are hepatic neural afferents sensitive to such metabolic events [13, 14, 20], neural signals mediating the inhibition of feeding after glucagon or epinephrine injections may originate in the liver. Lesion studies provide some support for this hypothesis. Pancreatic glucagon's inhibitory effect on feeding is blocked by total abdominal vagotomy [12] and by selective disconnection of only the hepatic branch of the abdominal vagus [9], although, paradoxically, it appears to survive total liver denervation [4]. Epinephrine's inhibitory effect is attenuated, but not blocked, by total abdominal vagotomy or by coeliac ganglionectomy, but is apparently not affected by hepatic vagotomy [24,25]. To further localize the peripheral neural mechanisms of pancreatic glucagon's and epinephrine's inhibitory effects on feeding and to test whether both are mediated by a single neural mechanism, we compared the ability of partial neural disconnections of the liver to block these effects. Our results

Liver denervation

Satiety

confirm and extend the finding that pancreatic glucagon's satiety effect depends on the hepatic branch of the abdominal vagus, but they fail to support either the hypothesis that epinephrine's inhibitory effect on feeding depends on hepatic innervation or the hypothesis that pancreatic glucagon's and epinephrine's feeding effects are mediated by a single mechanism. METHOD

Subjects Fifty-one adult male Sprague Dawley rats (Charles River Breeding, Wilmington, MA) were individually housed in hanging wire cages in a colony room that was brightly illuminated from 0700--1900 hr daily. Rats were fed pelleted diet (Purina, St. Louis, MO) and water ad lib for at least 2 weeks prior to surgery. At 1000 hr food pellets were removed and evaporated milk (Carnation, Los Angeles, CA) was presented in graduated drinking tubes (Wahman Manufacturing, Timonium, MD). Saline (1 ml/kg) was intraperitoneally injected before milk presentation using tuberculin syringes with 25 ga needles (Becton Dickenson, Rutherford, NJ). Milk intakes were measured each 30 min for 2-3 hr. Milk was then removed and food pellets returned.

Surgery In the first experiment, rats received either selective

hepatic vagotomies (n= 19) or sham operations (n= 12). Body

1Requests for reprints should be addressed to N. Geary.

233

234 weights were 290-370 g on the day of surgery. Rats were anesthetized with chloral hydrate and pentobarbital (Chloropent, 2.5 ml/kg, Fort Dodge Laboratories, Fort Dodge, IA) and laporotomized. To expose the abdominal vagus, the stomach was retracted caudally with a stay suture, mesenteric connections between the ventral surface of the stomach and the right and central lobes of the liver were cut, and those liver lobes were reflected rostrally. The esophageal trunks of the vagus were identified just below the diaphragmatic hiatus with the aid of an operating microscope (Jenoptik, Jena, GDR). Two 4-0 silk sutures were placed 2-5 mm apart around the hepatic branch of the vagus distal to its division from the right (anterior) vagal trunk, and the nerve between the sutures was lesioned. Further details of the procedure have been described previously [9]. For sham operations, the hepatic branch was similarly exposed, but not sutured or cut. In the second experiment, rats received either partial liver denervations (n=10) or sham operations (n=10). The procedure was modified from Bellinger and Williams' [3,4] technique for total liver denervations so as to achieve a disconnection of all neural connections to the liver except for the hepatic branch of the vagus. Body weights were 440590 g on the day of surgery. After laparotomy and retraction of the stomach, all mesenteric connections between the liver and the stomach or proximal duodenum were cut. The hepatic artery was cut between two 4-0 silk ligatures placed 1-3 mm apart distal to the point of the bifurcation of the gastroduodenal and hepatic arteries. Finally, approximately 5 mm of the bile duct and the portal vein in the gap created by the section of the hepatic artery were stripped of all adhering tissue with the aid of the operating microscope. The stripped area was swabbed with a 9% phenol, 47% ethanol solution for 2 min and then rinsed with saline. For sham operations, the surgical field was similarly exposed, but not further manipulated. In addition, the right renal vein was treated with phenolethanol as above.

Procedure Saline injections and milk presentation were resumed a few days after surgery. Testing began when during 4 consecutive sessions: (1) the group mean 30 min intake did not vary significantly and (2) SDs of individual rats' 30 min milk intakes were less than 30% of their mean intake. Epinephrine chloride (Parke Davis, Morris Plains, NJ) doses of 12.5, 25, 50, and 100 /~g/kg and pancreatic glucagon (Eli Lilly, Indianapolis, IN) doses of 50, 100, 200, and 400/~g/kg were injected just prior to milk presentation. These dosages have produced rapid inhibitions of feeding in a variety of test situations [6, 7, 11, 17, 24, 25]. Drugs were dissolved in a vehicle of 3% dimethyl sulfoxide (DMSO, Burdick and Jackson Laboratories, Muskegon, MI) and 0.9% saline. All injection volumes were 1 ml/kg. One epinephrine and one pancreatic glucagon dose were compared to a vehicle injection day each week. Doses were tested in descending order of magnitude. To measure the latency to meal initiation, rats' behaviors were observed once each 90 sec for at least 6 min after injections (behavioral observations were continued for 60 min in the hepatic vagotomy experiment). Tests were begun with hepatic vagotomized rats two weeks after surgery. Body weight of the vagotomized rats was 336-+6 g (mean-+SE), or 100.4-+ 1.9% of the sham operated rats' mean. Ambient temperature in the colony room ranged from 21-25°C during this experiment. Tests of partial

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FIG. 1. The effects of pancreatic glucagon (upper panel) and epinephrine (lower panel) on meal size in rats with selective hepatic vagotomies (n= 16) and neurologically intact rats (n=9). Values are mean-+ SE. *Meal size after hormone injection significantly less than control value, Tukey's test after significant ANOVA, p<0.05; **p <0.01. reflect of hormone significantly different in operated and control rats, post-hoc t-test, p<0.05.

liver denervation rats were begun 21/2 weeks after surgery, when their body weight was 542-+35 g, or 97.4-+6.2% of the control mean. Ambient temperature varied from 18--20°C on test days. At the end of behavioral testing, vagotomized rats weighed 100.8-+2.9% of their controls and partial liver denervation rats weighed 96.8-+9.0 of theirs.

PARTIAL L I V E R D E N E R V A T I O N AND SATIETY

235 found between the liver and the esophagus in a vagotomized rat or between the liver and the stomach or duodenum or along the portal vein or bile duct in a partially denervated rat, we assumed the disconnection was incomplete. This verification procedure should detect all but very small neural connections.

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FIG. 2. The effects of pancreatic glucagon (upper panel) and epinephrine (lower panel) on meal size in rats with liver denervations that spare only the hepatic branch of the vagus (n=5) and neurologically intact rats (n=9). Values are mean---SE. *Meal size after hormone injection significantly less than control value, Tukey's test after significant ANOVA, p<0.05; **p<0.01.

There were three criteria for inclusion of test data in the statistical analysis. The anatomical criterion was that the neural disconnection was verified to be complete. The behavioral criteria were that the rat began feeding within 3 min of injection and that intake during the initial bout of feeding after control injections was at least 3 ml. The behavioral criteria were included to preclude false negative findings because pancreatic glucagon's potency to inhibit feeding is reduced or absent when injections precede meal onset by 5-8 min [8] and because milk intake measurement error was 1-2 ml. Results are reported for 16 hepatic vagotomized rats (2 vagotomies were incomplete and 1 rat did not meet the behavioral criteria) and for all 12 sham vagotomized rats. Results are given for 5 rats with partial liver denervations (3 rats failed to recover from surgery and 2 denervations were incomplete) and for 9 sham denervated rats (1 did not meet the behavioral criteria). The effects of pancreatic glucagon and epinephrine were separately analysed with repeated measures A N O V A [30]. Test intakes were expressed as percent of control for analysis because intakes after vehicle injections tended to increase during the course of testing. For example, in the first experiment, meal size after control injections increased during the four weeks of testing from 11.4--- 1.4 to 15.4-+ 1.4 ml, m e a n - S E , F(3,24)=7.03, p<0.01, in sham operated rats, and from 9.7-+--0.7 to 13.6-+1.0 ml, F(3,45)=7.11, p<0.01, in hepatic vagotomized rats. There was a similar, but less consistent, pattern in the second experiment, in which overall mean control meal sizes were 16.4___2.2 ml in sham operated rats and 12.2-+3.2 ml in rats with partial liver denervations, t(12)= 1.08, p>0.05. When significant (p<0.05) A N O V A effects were detected, effects of individual pancreatic glucagon and epinephrine doses were compared to control values with Tukey's tests. Significant differences are reported when 2 a<0.05. The hypothesis that hepatic vagotomy or partial liver denervations attenuated the inhibitory effects of individual drug doses was tested with planned t-tests. Because this is a directional hypothesis, differences are reported significant when a<0.05. The observed effects were clearest in the 30 min food intake data. Whenever behavioral observations were continued, 30 min food intakes were identical to meal size defined by the biological criterion suggested by Smith and Gibbs [21], that is, the amount eaten by the rat before resting. Therefore, 30 min food intakes are referred to below as meals. RESULTS

Hepatic Vagotomy

Verification Neural disconnections were anatomically verified during post mortem examinations. Rats were anesthetized, beheaded, and bled. The operated areas were searched under 6-30x magnification. When any ambiguous connection was

Although both pancreatic glucagon and epinephrine inhibited the size of the first postinjection meal in sham operated rats, only epinephrine inhibited meal size in hepatic vagotomized rats. Selective hepatic vagotomy completely blocked pancreatic glucagon's satiety effect (Fig. 1, upper panel). A N O V A revealed a main effect of surgical group on the inhibition of initial meal size after glucagon,

236

MAcISAAC A N D GEARY

F(1,23)=8.24, p<0.01. In sham operated rats, glucagon doses of 100 p.g/kg or more significantly inhibited meal size, but in vagotomized rats no glucagon dose affected meal size. Giucagon also inhibited meal duration in sham operated rats, F(1,23)=5.11, p<0.05, but not in vagotomized rats. In contrast to the results after pancreatic glucagon injection, epinephrine inhibited meal size with equal potency in hepatic vagotomized and sham operated rats; main effect of surgical group, F(1,23)=0.17, p>0.25 (Fig. 1, lower panel). Epinephrine doses of 25/~g/kg or more inhibited meal size and meal duration; main effects of treatment, F(4,92)=7.82, p <0.01, and F(4,92) =6.05, p <0.01, respectively. Rats often took a second milk meal during the test period. No significant effects of either hormone, however, were detected on intermeal interval or second meal size. Further, the initial inhibitory effects of both hormones on cumulative milk intake were no longer significant 3 hr after injections (despite that cumulative food intake after epinephrine injection was still as much as 20% reduced). Partial L i v e r D e n e r v a t i o n

Selective liver denervations that spared only the hepatic branch of the vagus had no detectable influence on the behavioral effects of either pancreatic glucagon or epinephrine (Fig. 2). The main effect of glucagon on 30 min food intake was significant, F(4,48)=8.15, p<0.01, but neither the main effect of surgical group, F(1,12)=0.37, p>0.25, nor the interaction effect of glucagon x surgical group, F(4,48)=0.90, p>0.25, was significant. The same pattern of results was observed after epinephrine injections: epinephrine injection effect, F(4,48)=12.88, p<0.01; surgical group effect, F(1,12)=1.21, p>0.25; and interaction, F(4,48)=0.53, p>0.25. These initial inhibitory effects of glucagon and epinephrine appeared to erode after the initial 30 min measurement in this experiment as previously, but because some of the data were lost, statistical analyses were not done. DISCUSSION

it has been argued that hepatic innervation mediates the inhibition of feeding both after pancreatic glucagon and after epinephrine injection. The present data confirm and extend the former conclusion, but fail to support the latter. Geary and Smith [9] reported that selective vagotomy of only the hepatic branch of the abdominal vagus blocked the satiety effect of exogenous pancreatic glucagon, whereas vagotomy of the gastric and coeliac branches that spared the hepatic branch did not affect glucagon's satiety effect. This suggests that the hepatic branch is the necessary and sufficient contribution of the abdominal vagus to the peripheral nervous mechanism that mediates glucagon's satiety effect. The present experiment replicates the finding that hepatic vagotomy blocks glucagon's satiety effect. It also further localizes the neural mechanism involved. Partial liver denervations that spared only the hepatic branch of the abdominal vagus did not affect pancreatic glucagon's potency to inhibit food intake. This denervation should have disconnected the sympathetic innervation of the liver, which communicates with the liver via the coeliac plexus and nerve bundles along the hepatic artery and bile duct, as well as disconnecting any parasympathetic innervation outside the hepatic branch of the vagus [2, 3, 4]. The failure of this lesion to block pancreatic glucagon's satiety effect suggests that the hepatic branch of the vagus is sufficient for this effect in the absence of any contribution from other hepatic innervation.

The contribution of the hepatic branch may be afferent because pharmacological blockade of postganglionic muscarinic receptors with atropine methylnitrate failed to block glucagon's satiety effect [9]. However, since vagal efferent function has recently been found to include peptidergic as well as cholinergic mechanisms (e.g., [1]), this suggestion needs to be tested with separate surgical lesions of vagal efferents and afferents [22]. One result does not support the conclusion that the hepatic branch of the vagus is the necessary and sufficient component of the liver's innervation for pancreatic glucagon's satiety effect. Bellinger and Williams [4] recently reported that pancreatic glucagon inhibits feeding in rats with total liver denervations. The reason for the apparent discrepancy between the results after selective versus total liver denervations is not clear. One possibility is that different components of the hepatic innervation interact such that the role of one component is evident only when the second is active. Thus, for example, perhaps input from the hepatic vagus normally contributes to a neural state that is required for pancreatic glucagon to elicit satiety, whereas hepatic innervation outside the hepatic vagus inhibits this state. Then the loss of excitatory input from the hepatic vagus might prevent pancreatic glucagon from eliciting satiety, whereas the loss of both the hepatic vagal and extra-vagal liver innervation might produce no net change in this state and thus not affect glucagon's potency for satiety. In any case, the resolution of the results of partial and total liver denervations on pancreatic glucagon's satiety effect is an important open problem. Similarly, little can now be said either about the interface of physiological and hepatic vagal events required for pancreatic glucagon to elicit satiety or about the relationship between exogenous pancreatic glucagon's satiety effect and the apparent satiety effect of endogenous pancreatic glucagon I10]. In contrast to the results for pancreatic glucagon, these experiments did not suggest any role for hepatic innervation in mediating the inhibitory effect of epinephrine on feeding. No differences in food intake after epinephrine injections were detected between control rats and rats with either selective hepatic vagotomies or partial liver denervations that spared the hepatic branch. Thus, hepatic innervation does not appear to be a necessary component of the nervous mechanism mediating epinephrine's inhibitory effect on feeding. This dissociates the peripheral neural mechanisms for the feeding effects of epinephrine and pancreatic glucagon because, under conditions in which the hepatic branch of the vagus was necessary for pancreatic glucagon's satiety effect, it was not required for epinephrine's inhibitory effect on feeding. This does not support Russek and Racotta's [16,18] hypothesis that the feeding effects of both hormones depend on hepatic neural afferents that are sensitive to a common effect they have on liver metabolism. Finally, the failure of selective liver denervations to affect epinephrine's inhibitory effect on feeding does not support Tordoffet al. 's [24,25] hypothesis that this effect is mediated by hepatic vagal afferents that reach the esophageal vagal trunks via the coeliac ganglion. Tordoff et al. [24,25] base this hypothesis on the finding that total abdominal vagotomy and coeliac ganglionectomy similarly attenuate epinephrine's effects on feeding. If disconnection of hepatic afterents were the critical lesion producing this attenuation, then either selective vagotomy of the hepatic branch of the vagus or selective liver denervation sparing the hepatic branch should reproduce the attenuation. The lack of such an effect

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237

in these e x p e r i m e n t s suggests that coeliac g a n g l i o n e c t o m y and total abdominal v a g o t o m y do not attenuate epinephrine's feeding effects by disconnecting hepatic innervation. Ritter and her colleagues [27,28] have recently reported that intrahepatic injection o f a nondiabetogenic dose o f alloxan abolishes pancreatic g l u c a g o n ' s satiety effect, but does not attenuate the inhibition of feeding elicited by epinephrine. T h e s e data, like the results of the present experiment, are consistent with the possibility that a hepatic m e c h a n i s m is required for the inhibition o f feeding by pancreatic glucagon, but not by epinephrine. In s u m m a r y , in neurologically intact rats and in rats with liver denervations sparing the hepatic branch of the vagus, both pancreatic glucagon and epinephrine inhibited feeding, w h e r e a s in rats with selective v a g o t o m i e s of only the hepatic

branch of the vagus, epinephrine still inhibited feeding but pancreatic glucagon did not. Thus, despite the similarity of pancreatic glucagon's and epinephrine's effects on heaptic glucose metabolism, it appears that the feeding effects of the two h o r m o n e s are separate neurobehavioral problems.

ACKNOWLEDGEMENTS We thank Manuel Esguerra, Veronica Hinton, Mark Licht and James Santoro for help with experiments and Francesca Marraro for preparing the manuscript. A preliminary report of these findings was given at the 13th Annual Meeting of the Society for Neuroscience, Boston, MA, November, 1983 [7]. This work was supported by Research Grant AM 32448 to N.G.

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