- Email: [email protected]
Failure of portal glucose and adrenaline infusions or liver denervation to affect food intake in dogs
Physiology & Behavior, Vol. 16, pp. 299--304. Pergamon Press and Brain Research Publ., 1976. Printed in the U.S.A.
Failure of Portal Glucose and Adrenaline Infusions or Liver Denervation to Affect Food Intake in Dogs L. L. BELLINGER 2'3, G. J. TRIETLEY AND L. L. BERNARDIS
Departments o f Surgery and Pathology, State University o f New York at Buffalo Buffalo, N Y 14215 (Received 18 July 1975) BELLINGER, L. L., G. J. TRIETLEY AND L. L. BERNARDIS. Failureof portal glucose and adrenaline infusions or Hver denervation to affect food intake in dogs. PHYSIOL. BEHAV. 16(3) 299-304, 1976. - Total liver denervations were attempted on 5 mongrel dogs. At the same time, the hepatic portal vein was cannulated with a polyethylene cannula which was exteriorized. Five sham operated, cannulated dogs served as controls. Both denervated and sham operated dogs returned to preoperative food intake within 8 days post surgery. After recovery, intraportal infusions of 6 to 25 g of glucose prior to food presentation in 23 hr fasted sham or denervated dogs resulted in no anorexia. The portal vein and jugular vein were cannulated in an additional 4 dogs. Jugular blood samples were taken prior to and after portal infusion of 20 g of glucose in 23 hr fasted dogs. Both jugular blood glucose and insulin concentration increased significantly 1 to 2 min after portal infusion. The dogs were presented with food 3 to 5 min after the cessation of infusion, yet they showed no anorexia. These same 4 dogs were given portal infusions of either 0.5 #g/kg, 1.0/~g/kg or 1.5 #g/kg of adrenaline after a 23 hr fast. When food was presented 10 min after the infusions were stopped, the dogs ate immediately showing no signs of anorexia. These results question the role or the existence of hepatic glucoreceptors in the control of food intake in the dog. Food intake
lntraportal glucose
Adrenaline
Liver-denervation
WHILE the existence of central nervous, i.e. hypothalamic glucoreceptors [ 16] had been known and used as a working hypothesis for some time, the existence of hepatic glucoreceptors was postulated by Russek in 1963 [24]. These receptors were thought to participate in the control of food intake. (For review see Russek, 1971 [29] ). The Russek hypothesis was based on his earlier work [26] where, following an intrapertoneal injection of adrenaline, anorexia was produced, conceivably by raising the hepatic intracellular glucose concentration. This change would be perceived by intraceUular receptors, i.e. nerve endings [29] which would discharge upon a decrease of hepatic glucose concentration. This discharge would be conveyed via relays to appropriate central nervous system loci and initiate feeding behavior. A lack of discharge of the receptors, in turn, would produce satiation [24,28]. Rodriquez-Zendejas et aL [23] reported later using cats that intraportal adrenaline infusion ( 0 . 0 0 1 - 0 . 5 t~g/kg) decreases the liver output of glucose with the maximum decrease occurring with the 0.5 ug/kg dose. Higher doses of adrenaline result in an increased output of hepatic glucose. In the same study [23] intraperitoneal injections of 300
Glucoreceptors
mg/kg of glucose also resulted in a decreased hepatic output of glucose which lasted for 1 0 - 3 5 min. The duration of this decreased glucose output by the liver corresponds to the interval of anorexia reported by Russek's group [28] when 300 mg/kg of glucose is given to normal dogs. Russek's group suggests the decreased hepatic glucose output after the above mentioned doses of adrenaline and glucose corresponds to an increased hepatic intracellular uptake of glucose. He further suggests the animals should be anorexia during the increased hepatic intracellular glucose uptake [23, 28, 29] which, according to the hypothesis, should decrease neural activity of the proposed hepatic glucose receptors resulting in anorexia. Anatomical as well as functional support for Russek's hypothesis has come from Niijima's work [19]. Using an isolated perfused guinea pig liver preparation with intact vagus, he recorded neural discharges whose frequency was inversely related to the glucose concentrations in the perfusate. Isotonic saline or other monosacharide solutions were without effect. While all of the above was only indirect evidence for Russek's hypothesis, direct support was presented by
This research was supported in part by National Institute of General Medical Sciences Grant 15768. 2N.I.H. individual postdoctoral fellowship 1 F22 NS 02625-01-NEURA. 3Reprint requests should be sent to Dr. L. L. Bellinger, Department of Surgery, SUNY at Buffalo Clinical Center, 462 Grider Street, Buffalo, New York, 14215. 299
300 himself [25] when he reported that hepatic intraportal infusions of 3 - 1 2 g of glucose (30% glucose solution given at a rate of 10 cc/min) resulted in anorexia that lasted from 4 0 - 7 5 min in 2 dogs that had been fasted for 22 hr previously. The same amount of glucose infused into the jugular vein did not cause anorexia. Additional evidence [20] was forthcoming when it was demonstrated that intraportal infusions of 2-Deoxy-D-glucose (2 DG) which causes intracellular glucoprivation, initiated feeding in rabbits in a shorter time than when the compound was given via the jugular vein. The intrahepatic infusions also caused significant food intake during the hour following the infusion. It has been Russek's contention that the hepatic glucoreceptors are a major controller of feeding behavior and account for the day-to-day control of food intake [29]. This hypothesis is an attractive one and well in keeping with the fact that animals terminate feeding before major changes have occurred in the systemic levels of various metabolites derived from a meal just consumed. This termination of feeding is thought not be caused by the activity of gastric stretch receptors [22]. The hypothesis allows for small amounts of glucose to go to the liver where it is sensed by the proposed glucoreceptors, thus stopping further feeding. Nevertheless, further verification of receptor afferents is necessary before this hypothesis can be finally accepted. On a neural level, transection of the nerves to the liver should result in either aphagia or markedly reduced food intake if the hepatic glucoreceptors are indeed a major controller of food intake. Russek attempted this demonstration in the rabbit [29] in which he sectioned the abdominal vagi, celiac ganglia and all visible nerves to the liver. Some, but not all of the animals were reported to be either quasi-aphagic or to have prolonged anorexia. However, the celiac ganglion and vagus nerves have many neural connections with various visceral areas [21]. It is thus unclear as to whether the observed effects in some of his experimental animals were due to liver denervation or to the denervation of other receptors involved in the control of food intake elsewhere in the gut, i.e. intestinal glucose receptors and/or amino acid receptors [31], or intestinal osmoreceptors [ 13]. Firmer support for the Russek hypothesis could come from experiments in which only the afferent nerves from the liver, and only those, were severed. This would then eliminate any possible effect of transection of afferents from other gut receptors that may affect food intake. Although no measurements of food intake were given in a series of liver transplant studies performed on pigs [4,5], monkeys [61, and in man [7] in which all liver nerve fibers must have been transected, several of the subjects recovered without apparent impairments of food intake. The purpose of the present study was twofold: firstly, to see if liver denervation would affect food intake in the dog and secondly, to examine if, after hepatic denervation, hepatic infusions of glucose or adrenaline would still cause anorexia in the case any of the denervated dogs were eating. The results were most puzzling, dogs with denervated livers showed no signs of anorexia at any time after the operation when compared with appropriately shamoperated controls. Furthermore, and more disturbingly so, was our lack of finding any anorexia in control dogs upon infusion of 6 to 25 g of glucose or 0.5 ~g/kg to 1.5 /~g/kg of adrenaline.
BELLINGER, TRIETLEY AND BERNARDIS EXPERIMENT 1 METHOD
Animals Ten adult male mongrel dogs were obtained 6 weeks prior to the experiment proper and wormed and vaccinated against hepatitis. During the 6 weeks prior to the start of the experiment, they were fed a mixture of canned and dry commercial dog food (Big Bet Pet Foods, Teklad Mills) ad lib, beginning at 1400 hr each day. When their body weight had stabilized, the study proper was started.
Procedure At the beginning of the experiment, the animals (body weight range 1 6 - 2 2 kg) were presented with food for only 1 hr per day beginning at 1400 hr. After food presentation the time of onset of eating was recorded. Then at 1500 hr the food was removed and weighed. This period of measurement lasted 7 to 8 days.
Surgery At the end of the measurement period, the dogs were anesthetized with sodium pentobarbital (1 gin/2 kg body weight) and a laparotomy was performed. Subsequently, a complete denervation of the liver was attempted on 5 of the dogs. In the dog, nerve fibers reach and leave the liver by various routes. The vagus has an anterior and a posterior trunk [ 17]. The former supplies the ventral surface of the stomach where it sends branches along the lesser curvature of the stomach to within 2 cm of the pylorus. This trunk also gives off hepatic branches - one, sometimes two, which pass high up in the lesser o m e n t u m to the liver [17]. These fibers originate from the region of the esophagus or cardiac portion of the stomach [ 17]. The posterior trunk of the dog vagus goes to the stomach where it sends branches to within a short distance of the pylorus [17]. At the cardiac level of the stomach a branch from the posterior trunk enters the lesser omentum, passing obliquely-ventrally to the liver [9,18]. A second division of the posterior vagus gives off branches that reach the celiac ganglion and the pancreas. Fibers originating from the posterior vagal trunk reach the liver from the celiac ganglion by way of the hepatic artery [21] or by crossing over to the portal vein [ 1,18]. The celiac ganglion also sends fibers from the spanchnic nerves to the liver [ 18,21]. These fibers travel with the ones originating from the vagus along the hepatic artery to the liver. They also cross over to the hepatic portal vein [ 18,21 ]. The phrenic nerve also travels to the liver region from the esophagus or the cardiac portion of the stomach where it courses through the omentum to the hepatic portal vein and then on the liver region. A second possible branch may cross to the liver directly from the esophagus [21 ]. The denervation was attempted in the following manner: the hepatic artery was isolated and all nerves on it or in the surrounding connective tissue were transected and 1 to 2 cm removed. Next, the hepatic portal vein was isolated and stripped of all nerves and, again, 1 to 2 cm were removed. The stripping was done within 1 to 2 cm of the liver. The nerves to the gallbladder were left intact. Then any branches from the ventral or dorsal vagus or phrenic nerve to the liver between the diaphragm and stomach were cut
D EN E R VATI ON AND I N T R A P O R T A L GLUCOSE ON FEEDING and again 1 to 2 cm of nerve removed. Vagal branches from the stomach to the liver were then isolated and 1 to 2 cm were cut and removed. Usually, there was one large branch at the cardiac portion and one at the pyloric end of the stomach. To insure that any smaller vagal branches to the liver from the stomach were not missed, the o m en t u m between the stomach and the liver was carefully cut. A modification of the Bard-Parker Company, Model No. 3411, (O.D. 3.58 mm) polyethylene cannula was then inserted into either the portal vein or a branch of the portal system, i.e. the splenic vein. The cannulas were modifed by placing a small tight fitting polyethylene band around the outside cannula wall so that it would act to anchor the cannula into the portal or splenic vein. The cannulas were tied in using a purse-string suture so that the vessel was not occuluded. Additional anchor bands were placed where we brought the cannula through the abdominal wall. The cannulas were then passed under the skin to the neck region where they were anchored again before they were exteriorized. Finally the cannulas were stoppered and filled with saline. The dogs' necks were then wrapped to protect the ends of the cannulas. To insure against possible nerve regeneration, which has been reported to take several months [ 15], the dogs were used in experiments only for 1 month after the denervation. While it was impossible to ascertain whether all nerves to the liver had been severed we believe that, if we did not in fact cut all the nerves, the majority of them were certainly severed. The 5 remaining dogs were sham operated, i.e. the various visceral organs were moved and handled in the same manner as was done in the dogs in which the nerves had been severed, and were then cannulated. On the first postoperative day, the animals were fed during their usual feeding time, i.e. 1400 hr. After the offering of food the onset of continuous eating was recorded and the food intake was recorded at 1500 hr. Continuous eating was arbitrarily defined by us to be a meal at least 3 min in length. After the animals had recovered to their preoperative food intake levels both the denervated and sham operated dogs were infused with one of the following glucose concentrations: 6 g, 12 g, 18 g (given in a 30% glucose solution) or 20 g or 25 g (given in a 50% glucose solution). See Table 1 for number of trials at each concentration. Saline solutions were made up at each of the glucose
301
osmotic concentrations. Each animal that received a glucose infusion also received the same volume and osmotic load of saline. Only one infusion trial of glucose or saline was performed on any one day. Both glucose and saline were injected using a syringe at the same rate that Russek [25] used which was 10 ml per minute. Infusions were made just prior to the time the animals were normally fed, after a 23 hr fast at 1400 hr in a room adjacent to where the dogs were kept. After the infusions were stopped, the animals were returned to their home cages. Three to 10 min were allowed to elapse from the end of the infusion until the dogs were presented with food. They were closely observed to determine the time of onset of continuous eating and their food intake was recorded at 1 500 hr. RESULTS All sham and denervated dogs returned to within 1 SD of their preoperative food intake levels by 8 days postsurgery. The difference between groups was not statistically significant (sham 3.6 -+ 1.0 days; denervated 5.4 _+ 1.0 days). The pertinent point is that the denervated dogs shortly returned to normal preoperative food intake levels. The sham operated dogs were infused with 12 to 25 g of glucose in 22 separate trials (Table 1) and in every case the dogs showed no anorexia and ate within 30 sec after being fed as did the denervated dogs after receiving 6 - 2 5 g of glucose in the 20 trials performed on them. In all cases, the dogs which are generally meal eaters continued to eat for at least 3 min after being fed. When the hourly intakes of the sham operated and liver denervated dogs after hepatic infusion of 12 g and 20 g of glucose were compared to their control hourly intakes, the sham dogs given glucose ate 94.1 _+ 13.6% and 100 _+ 11.2% respectively of their control intake while the denervated dogs ate 97.2 +_ 13.8% and 82.4 -+ 4.9% of their control days.
EXPERIMENT 2 METHOD
Animals and Surgery An additional 4 dogs (body weight range 1 6 - 2 0 kg) were treated prior to surgery as described in Experiment 1. At the time of surgery 1 polyethylene cannula was inserted
TABLE 1 FOOD INTAKE IN 23 HR FASTED LIVER DENERVATEDOR SHAM OPERATED DOGS 3 TO I0 MINS AFTERINTRAPORTALINFUSIONSOF 6 TO 25 g OF GLUCOSE Amount of glucose infused into the portal vein 6g 12 g 18 g 20 g 25 g Sham-operated
(0) * --
Denervation
(1) 1/1 = 100%
(5)
(1)
13/13 =
1/1 =
6/6=
(3)
2/2=
(2)
100%? (4) 12/12 = 100%
100% (2) 2/2= 100%
100% (1) 4/4= 100%
100% (1) 1/1 = 100%
*Number of animals used shown in parentheses. ?Percent of trials in which animals ate within 30 sec. of food presentation.
Total No. of Trials 22/22 = 100%
20/20 = 100%
302
BELLINGER, TRIETLEY AND BERNARDIS
into the portal vein and a second cannula into the jugular vein. Procedure After the dogs had recovered from the operation and were eating normally, the following studies were performed. (1). After a 23 hr fast a jugular blood sample was taken on 3 of the dogs after 1 jugular cannula had become unoperational. All 4 dogs were then infused into the portal vein over 4 min with 20 g of glucose (50% glucose solution). One to 2 min after the end of the infusion, a second jugular sample was taken. The dogs were then presented with food 3 to 5 min after the glucose infusion was stopped. They were then closely observed to determine the onset of continuous feeding. A total of 3 trials (on separate days) were performed on each dog. The blood was centrifuged and plasma glucose was determined on a Technican Auto Analyzer and Insulin was measured using an insulin kit purchased from Schwartz Mann. (2). After a 23 hr fast the 4 dogs were infused into the portal vein with adrenaline (1 ug/cc) starting with a concentration of 0.5 ug/kg, then 1.0 ug/kg and finally 1.5 ug/kg (given on separate days). Two trials were run at each adrenaline concentration before the next higher dose was used. The adrenaline was given over a period of 3 min. Ten min after the end of the infusion, the dogs were presented with food and onset of continuous eating was noted. Because 3 of the four cannulas had become unusable, blood glucose and insulin jugular could not be determined. At the end of both Experiments 1 and 2, the dogs were sacrificed and placement and patency of the portal cannulas were verified. They were all found to be functional. Attempts were made to determine if any nerves to the liver had remained functional, but because of the many adhesions it was impossible to verify total denervation. All data was analyzed using the Student's t method. RESULTS After portal infusion of 20 g of glucose the 4 dogs ate within 30 sec of food presentation in t2/12 trials. Jugular blood was collected on 3 dogs pre- and posthepatic infusion after 1 dog's cannula had become unoperational. Plasma glucose increased from 97.6 -+ 3.0 mg% preportal glucose infusion to 513.9 +_ 16.0 mg% (p<0.001) 1 to 2 min after infusion ended. Plasma insulin concentration increased from 22.2 -+ 3.1 uU/ml preglucose infusion to 125.0 _+28.4 uU/ml (p<0.001) 1 to 2 min after infusion was terminated. Ten minutes after the 23 hr fasted dogs received either 0.5 ug/kg, 1.0 ug/kg or 1.5 ug/kg of adrenaline, the animals ate within 30 sec of food presentation in all 24 trials performed. DISCUSSION It was surprising to us that liver denervation had no effect on food intake in view of Russek's claim that hepatic glucoreceptors are a major controller of food intake [25]. While it was impossible for us to ascertain that all nerves to and/or from the liver were cut, we firmly believe that we must have severed the majority of fibers. It would seem likely that removal of the greater part of liver innervation would have had some affect on the animals' food intake since it is well known that even partial damage to sensory
fibers of peripheral nerves produces hyperesthesia [34]. It is also recognized [12] that larger lesions of the ventromedial hypothalamus (VMH) which destroy more of the central glucoreceptors of Mayer [ 16 ] cause a greater degree of hyperphagia and obesity than smaller lesions which destroy less tissue and hence less receptors. Lesion size of the VMH has also been positively correlated with graded changes in certain endocrine, i.e. insulin and growth hormone [2] and metabolic parameters [3]. In the liver denervation in this study, we did not observe any anorexia or any graded reduction of food consumption which we would have expected after removing the majority of the hypothesized receptors innervation. We are left with several possibilities to explain the lack of any observable effect: (1) We have left enough glucose receptors innervated so that they could take up the function of the receptors that were denervated. However, as we have intimated above, we believe that removal of the majority of receptor innervation should have resulted in some sort of graded reduction in food intake. (2) The receptors are real and we did, in fact, functionally remove their influence on food intake but other mechanisms of controlling food intake compensated for their loss. These might have included gastric stretch receptors [22 ], central glucoreceptors [ 16 ] or influence of gastrointestinal hormones such as cholecystokin [10]. If this were the case, then hepatic glucose receptors would not be the major controller of food intake but only a component of a many factorial control system. (3) There are no hepatic glucose receptors that affect food intake in the dog. The most disturbing part of this study was the failure of intraportal injections of glucose to result in anorexia. According to Russek [25] 3 - 1 2 g of intraportally infused glucose should result in anorexia for approximately 4 0 - 7 5 min. We did not find any anorexia in dogs similarly treated in this study. Three to 10 min after receiving 6 to 25 g of glucose intraportally, all animals began eating within 30 sec of being presented with food. We are at a loss to explain the discrepancy between our data and the data of Russek [25]. Our dogs were given a mixture of chow and canned dog food, whereas Russek were fed only chow. Also, our dogs were fed in their home cages while Russek ran his trials on the dogs outside their home cages. On this latter difference one would suspect that a truer test of the existence of the proposed glucose receptors could be obtained by feeding the dogs after glucose infusions in their home cages. This would allow the dogs to be in a familiar environment and in one where they have been accustomed to feeding. Russek has reported [28] higher doses of intraperitoneal injected adrenaline (which supposedly increased intracellular hepatic glucose levels resulting in decreased glucose receptor activity and thus satiety) were needed to produce anorexia [28] in dogs given a more palatable diet than chow. Thus, the possibility exists that, because we fed our dogs a mixture of canned dog food and chow, higher hepatic intracellular glucose levels would be needed to produce anorexia. However, the 25 g glucose dose was 8 1/3 times the dose that Russek [25] reported would produce anorexia for at least 40 min, yet we found no anorexia. This 25 g dose of glucose representing approximately 100 kcal was given over 4 min and far exceeds any absorption of calories that would occur normally in the dog. It is of interest that in Experiment 2, the dogs ate within 30 sec of food presentation even though jugular
DENERVATION AND INTRAPORTAL GLUCOSE ON FEEDING plasma glucose was 5 times the normal concentration. However, this is not surprising in view of the early work of Janowitz [ 14] (for review of this subject see [ 11 ] ). What is noteworthy here is that of the 20 g of glucose given our dogs only approximately 4 g remained in the blood (if one assumes that the dogs weighed 20 kg and had a blood volume that approximated 8% of their body weight). The large majority of the remaining approximate 16 g of infused glucose would most likely be taken up by the liver [8]. Thus these dogs ate within 30 sec of food presentation even though in the liver the intracellular concentration of glucose must have been very high. It also should be noted that Stephens and Baldwin [32] have reported that intraportal infusions of 500 ml of 15% glucose failed to produce any anorexia in pigs, while VanderWheele e t al. [33] have reported that intraportal glucose infusions in the free feeding rabbit have also failed to cause anorexia. Finally Russel and Mogenson [30] have recently reported that food intake changes after infusion of 2 DG was unrelated to either portal or jugular sites of infusion. They suggest that while the hepatic contribution to food intake control is poorly understood their data were inconsistent with reports of hepatic participation in feeding. Russek has reported that intraperitoneal injections of adrenaline at doses of 0.1 to 0.3 mg]kg cause dogs to become anorexic for 45 min to 2 hr by increasing hepatic glucose concentration [28]. Russek's group also reported in the anesthetized animal that intraportal infusions of glucose at 10 mg/kg [27] or adrenaline [23] at a dose of 0.5 ug/kg resulted in a drop in arterial glucose while higher doses of either increased arterial glucose. He suggests this drop in arterial glucose occurs as a result of increased hepatic utilization of glucose which he interprets as being suggestive of increased glucoreceptor uptake of glucose. The drop of arterial glucose (from increased receptor utilization) also
303
corresponds to the anorexic period he reports for IP injections of glucose [28]. However, in the present study we have intraportally infused several unanesthetized 23 hr fasted dogs with 0.5 ug/kg of adrenaline and the dogs exhibited no anorexia on feeding as should have occurred according to Russek's group [23,29]. When the dose was increased to 1.0 and 1.5 ug/kg, the dogs still exhibited no anorexia even though hepatic output of glucose according to Russek [29] should have increased and therefore complicating the issue by possibly involving central glucoreceptors [ 16]. Thus it is unclear whether minute amounts of adrenaline infused into the liver can produce anorexia in the unanesthetized dog as suggested by Russek and his group [23,29]. We have observed (unpublished observations) in dogs that were given IP adrenaline at a dose of 0.2 mg/kg that about one half of the dogs were anorexic when presented with food after a 23 hr fast. However, the dogs that were anorexic panted excessively, clawed at their cages, howled and some of them vomited. It is difficult to determine whether adrenaline caused anorexia by causing hepatic hyperglycemia, i.e. affected the proposed hepatic glucoreceptors or whether it caused a general stress response in which case one would not expect the animals to eat. The above data are clearly in conflict with Russek's data and question the existence and/or the role which the hypothesized glucoreceptors play in the control of food intake in the dog. It is our hope that further work will be done in this area to clarify the discrepancy between the present data and those of Russek's. ACKNOWLEDGEMENT We wish to thank William O'Donnell and Steve Brooks for excellent technical assistance.
REFERENCES
1. Alexander, W. The innervation of the billary system. J. comp. Neurol. 72: 357-370, 1940. 2. Bernardis, L. and L. Frohman. Effect of lesion size in the ventromedial hypothalamus on growth hormone and insulin levels in weanling rats. Neuroendocrinology 6:319-328, 1970. 3. Bemardis, L. and J. Goldman. Effect of hypothalamic lesion localization and size on metabolic alterations in weanling rat adipose tissue. J. Neuro-Viscer. Relat. 32: 253-269, 1972. 4. Calne, R., H. White, D. Yoffa, R. Maginn, R. Binns, J. Samuel and V. Molina. Observations of orthotopic liver transplantation in the pig. Br. med. J. 2: 478-480, 1967. 5. Calne, R., R. Sells, J. Pena, D. Davis, P. Millard, B. Herbertson, R. Binns and D. Davies. Induction of immunological tolerance by procine liver allografts. Nature 223: 472-476, 1969. 6. Calne, R., D. Davis, J. Pena, H. Balner, M. DeVries, B. Herbertson, V. Joysey, P. Millard, M. Seaman, J. Samuel, J. Stibbe and D. Westbroek. Hepatic allografts and xenografts in primates. Lancet 1: 103-106, 1970. 7. Calne, R., R. Williams,J. Dawson, I. Ansell, D. Evans, P. Flute, B. Herbertson, V. Joysey, G. Keates, R. Knill-Jones, S. Mason, P. Millard, J. Pena, B. Pentlow, J. Salaman, R. Sells and P. Cullum. Liver transplantation in Man-II, A report of two orthotopic liver transplants in adult recipients. Br. reed. J. 4: 541-546, 1968. 8. Camu, F. Hepatic balances of glucose and insulin in response to physiological increments of endogenous insulin during glucose infusions in dogs. Eur. J. clin. lnvest. 5: 101-108, 1975. 9. Chiu, S. The superficial hepatic branches of the vagi and their distribution to the extrahepatic billary tract in certain mammals. Anat. Rec. 86: 149-155, 1943.
10. G~bs, J., R. Young and G. Smith. Cholecystokinin decreases food intake in rats. J. comp. physiol. Psychol. 84: 488-495, 1973. 11. Grossman, M. Integration of current views on the regulation of hunger and appetite. Ann. N. Y. Acad. Sci. 63: 76-91, 1955. 12. Hetherington, A. and S. Ranson. Hypothalamic lesions and adiposity in the rat. Anat. Rec. 78: 149-172, 1940. 13. Hsiao, S. and D. Langenes. Liquid intake, initiation and amount of eating as determined by osmolality of drinking liquids. Physiol. Behav. 7: 233-237, 1971. 14. Janowitz, H., M. Hanson, M. Grossman. Effect of intravenously administered glucose on food intake in the dog. Am. Z Physiol. 156: 87-91, 1949. 15. Lubinska, L. Axoplasmic streaming in regenerating and in normal nerve fibers. In: Mechanisms o f neural regeneration. Prog. in Brain Res. Vol. 13, edited by M. Singer and J. Schade. New York: Elsevier, 1964, pp. 1-66. 16. Mayer, J. and D. Thomas. Regulation of food intake and obesity. Science 156: 328-337, 1967. 17. McCrea, E. The abdominal distribution of the vagus. J. Anat. 59: 18-40, 1924. 18. Mitchell, G. Anatomy o f the autonomic nervous system, Edinburgh and London: E. and S. Livingstone Ltd., 1953, pp. 120-283. 19. Niijima, A. Afferent impulse discharges from glucoreceptors in the liver of the guinea pig. Ann. N. Y. Acad. Sci. 157: 690-700, 1969. 20. Novin, D., D. VanderWeele and M. Rezek. Infusion of 2-deoxy-D-glucoseinto the hepatic-portal system causes eating: Evidence for peripheral glucoreceptors. Science 181 : 858-860, 1973.
304 21. Pick, J. The Autonomic Nervous System. Philadelphia: J. B. Lippincott, 1970, pp. 313-331. 22. Quigley, J. The role of the digestive tract in regulating the ingestion of food. Ann. N. Y. Acad. Sci. 63: 6 - 1 4 , 1955. 23. Rodriquez-Zendejas, A., A. Vega, L. Soto-Mora, S. Pina and M. Russek. Some effects of intrapertoneal glucose and of intraportal glucose and adrenaline. Physiol. Behav. 3: 2 5 9 - 2 6 4 , 1968. 24. Russek, M. and S. Pina. Conditioning of adrenaline-induced anorexia. Nature 193: 1296-1297, 1962. 25. Russek, M. Demonstration of the influence of a hepatic glucosensitive mechanism on food intake. Physiol. Behav. 5: 1207-1209, 1970. 26. Russek, M. and S. Pina. Conditioning of adrenaline-induced anorexia. Nature 193: 1296-1297, 1962. 27. Russek, M. and L. Soto-Mora. Evidence de la existencia de glucoreceptores hepaticos. Acta. physiol, latinoam. 16: 8 5 - 8 6 , 1965. 28. Russek. M., A. Rodrigquez-Zendejas and S. Pina. Hypothetical liver receptors and the anorexia caused by adrenaline and glucose. Physiol. Behav. 3: 249-257, 1968.
BELLINGER, TRIETLEY AND BERNARDIS 29. Russek. M. Hepatic receptors and the neurophysiological mechanisms controlling feeding behavior. In: Neurosciences Res. Vol. 4, edited by S. Ehrenpreis. New York: Acad. Press, 1971, pp. 213-282. 30. Russell, P. and G. Mogenson. Drinking and feeding induced by jugular and portal infusions of 2-deoxy-d-glucose. Am. J. Physiol. 229: 1014-1018, 1975. 31. Sharma, K. Receptor mechanisms in the alimentary tract: Their excitation and functions. In: Control o f Food and Water Intake. Handbook of Physiology Sec. 6, Alimentary Canal, edited by C. Code, Washington, D.C.: Am. Physiol. Society, 1967, 225-237. 32. Stephens, D. and B. Baldwin. The lack of effect of intrajugular or intraportal injections of glucose or amino acids on food intake in pigs. Physiol. Behav. 12: 9 2 3 - 9 2 9 , 1974. 33. Vanderweele, D., D. Novin, M. Rezck and J. Sanderson. Duodenal or Hepatic-Portal Glucose Perfusion: Evidence for Duodenally-based Satiety. Physiol. Behav. 12: 4 6 7 - 4 7 3 , 1974. 34. Willis, W. and R. Grossman. Medical Neurobiology. Saint Louis: C. V. Mosby., 1973, pp. 355.
Failure of Portal Glucose and Adrenaline Infusions or Liver Denervation to Affect Food Intake in Dogs L. L. BELLINGER 2'3, G. J. TRIETLEY AND L. L. BERNARDIS
Departments o f Surgery and Pathology, State University o f New York at Buffalo Buffalo, N Y 14215 (Received 18 July 1975) BELLINGER, L. L., G. J. TRIETLEY AND L. L. BERNARDIS. Failureof portal glucose and adrenaline infusions or Hver denervation to affect food intake in dogs. PHYSIOL. BEHAV. 16(3) 299-304, 1976. - Total liver denervations were attempted on 5 mongrel dogs. At the same time, the hepatic portal vein was cannulated with a polyethylene cannula which was exteriorized. Five sham operated, cannulated dogs served as controls. Both denervated and sham operated dogs returned to preoperative food intake within 8 days post surgery. After recovery, intraportal infusions of 6 to 25 g of glucose prior to food presentation in 23 hr fasted sham or denervated dogs resulted in no anorexia. The portal vein and jugular vein were cannulated in an additional 4 dogs. Jugular blood samples were taken prior to and after portal infusion of 20 g of glucose in 23 hr fasted dogs. Both jugular blood glucose and insulin concentration increased significantly 1 to 2 min after portal infusion. The dogs were presented with food 3 to 5 min after the cessation of infusion, yet they showed no anorexia. These same 4 dogs were given portal infusions of either 0.5 #g/kg, 1.0/~g/kg or 1.5 #g/kg of adrenaline after a 23 hr fast. When food was presented 10 min after the infusions were stopped, the dogs ate immediately showing no signs of anorexia. These results question the role or the existence of hepatic glucoreceptors in the control of food intake in the dog. Food intake
lntraportal glucose
Adrenaline
Liver-denervation
WHILE the existence of central nervous, i.e. hypothalamic glucoreceptors [ 16] had been known and used as a working hypothesis for some time, the existence of hepatic glucoreceptors was postulated by Russek in 1963 [24]. These receptors were thought to participate in the control of food intake. (For review see Russek, 1971 [29] ). The Russek hypothesis was based on his earlier work [26] where, following an intrapertoneal injection of adrenaline, anorexia was produced, conceivably by raising the hepatic intracellular glucose concentration. This change would be perceived by intraceUular receptors, i.e. nerve endings [29] which would discharge upon a decrease of hepatic glucose concentration. This discharge would be conveyed via relays to appropriate central nervous system loci and initiate feeding behavior. A lack of discharge of the receptors, in turn, would produce satiation [24,28]. Rodriquez-Zendejas et aL [23] reported later using cats that intraportal adrenaline infusion ( 0 . 0 0 1 - 0 . 5 t~g/kg) decreases the liver output of glucose with the maximum decrease occurring with the 0.5 ug/kg dose. Higher doses of adrenaline result in an increased output of hepatic glucose. In the same study [23] intraperitoneal injections of 300
Glucoreceptors
mg/kg of glucose also resulted in a decreased hepatic output of glucose which lasted for 1 0 - 3 5 min. The duration of this decreased glucose output by the liver corresponds to the interval of anorexia reported by Russek's group [28] when 300 mg/kg of glucose is given to normal dogs. Russek's group suggests the decreased hepatic glucose output after the above mentioned doses of adrenaline and glucose corresponds to an increased hepatic intracellular uptake of glucose. He further suggests the animals should be anorexia during the increased hepatic intracellular glucose uptake [23, 28, 29] which, according to the hypothesis, should decrease neural activity of the proposed hepatic glucose receptors resulting in anorexia. Anatomical as well as functional support for Russek's hypothesis has come from Niijima's work [19]. Using an isolated perfused guinea pig liver preparation with intact vagus, he recorded neural discharges whose frequency was inversely related to the glucose concentrations in the perfusate. Isotonic saline or other monosacharide solutions were without effect. While all of the above was only indirect evidence for Russek's hypothesis, direct support was presented by
This research was supported in part by National Institute of General Medical Sciences Grant 15768. 2N.I.H. individual postdoctoral fellowship 1 F22 NS 02625-01-NEURA. 3Reprint requests should be sent to Dr. L. L. Bellinger, Department of Surgery, SUNY at Buffalo Clinical Center, 462 Grider Street, Buffalo, New York, 14215. 299
300 himself [25] when he reported that hepatic intraportal infusions of 3 - 1 2 g of glucose (30% glucose solution given at a rate of 10 cc/min) resulted in anorexia that lasted from 4 0 - 7 5 min in 2 dogs that had been fasted for 22 hr previously. The same amount of glucose infused into the jugular vein did not cause anorexia. Additional evidence [20] was forthcoming when it was demonstrated that intraportal infusions of 2-Deoxy-D-glucose (2 DG) which causes intracellular glucoprivation, initiated feeding in rabbits in a shorter time than when the compound was given via the jugular vein. The intrahepatic infusions also caused significant food intake during the hour following the infusion. It has been Russek's contention that the hepatic glucoreceptors are a major controller of feeding behavior and account for the day-to-day control of food intake [29]. This hypothesis is an attractive one and well in keeping with the fact that animals terminate feeding before major changes have occurred in the systemic levels of various metabolites derived from a meal just consumed. This termination of feeding is thought not be caused by the activity of gastric stretch receptors [22]. The hypothesis allows for small amounts of glucose to go to the liver where it is sensed by the proposed glucoreceptors, thus stopping further feeding. Nevertheless, further verification of receptor afferents is necessary before this hypothesis can be finally accepted. On a neural level, transection of the nerves to the liver should result in either aphagia or markedly reduced food intake if the hepatic glucoreceptors are indeed a major controller of food intake. Russek attempted this demonstration in the rabbit [29] in which he sectioned the abdominal vagi, celiac ganglia and all visible nerves to the liver. Some, but not all of the animals were reported to be either quasi-aphagic or to have prolonged anorexia. However, the celiac ganglion and vagus nerves have many neural connections with various visceral areas [21]. It is thus unclear as to whether the observed effects in some of his experimental animals were due to liver denervation or to the denervation of other receptors involved in the control of food intake elsewhere in the gut, i.e. intestinal glucose receptors and/or amino acid receptors [31], or intestinal osmoreceptors [ 13]. Firmer support for the Russek hypothesis could come from experiments in which only the afferent nerves from the liver, and only those, were severed. This would then eliminate any possible effect of transection of afferents from other gut receptors that may affect food intake. Although no measurements of food intake were given in a series of liver transplant studies performed on pigs [4,5], monkeys [61, and in man [7] in which all liver nerve fibers must have been transected, several of the subjects recovered without apparent impairments of food intake. The purpose of the present study was twofold: firstly, to see if liver denervation would affect food intake in the dog and secondly, to examine if, after hepatic denervation, hepatic infusions of glucose or adrenaline would still cause anorexia in the case any of the denervated dogs were eating. The results were most puzzling, dogs with denervated livers showed no signs of anorexia at any time after the operation when compared with appropriately shamoperated controls. Furthermore, and more disturbingly so, was our lack of finding any anorexia in control dogs upon infusion of 6 to 25 g of glucose or 0.5 ~g/kg to 1.5 /~g/kg of adrenaline.
BELLINGER, TRIETLEY AND BERNARDIS EXPERIMENT 1 METHOD
Animals Ten adult male mongrel dogs were obtained 6 weeks prior to the experiment proper and wormed and vaccinated against hepatitis. During the 6 weeks prior to the start of the experiment, they were fed a mixture of canned and dry commercial dog food (Big Bet Pet Foods, Teklad Mills) ad lib, beginning at 1400 hr each day. When their body weight had stabilized, the study proper was started.
Procedure At the beginning of the experiment, the animals (body weight range 1 6 - 2 2 kg) were presented with food for only 1 hr per day beginning at 1400 hr. After food presentation the time of onset of eating was recorded. Then at 1500 hr the food was removed and weighed. This period of measurement lasted 7 to 8 days.
Surgery At the end of the measurement period, the dogs were anesthetized with sodium pentobarbital (1 gin/2 kg body weight) and a laparotomy was performed. Subsequently, a complete denervation of the liver was attempted on 5 of the dogs. In the dog, nerve fibers reach and leave the liver by various routes. The vagus has an anterior and a posterior trunk [ 17]. The former supplies the ventral surface of the stomach where it sends branches along the lesser curvature of the stomach to within 2 cm of the pylorus. This trunk also gives off hepatic branches - one, sometimes two, which pass high up in the lesser o m e n t u m to the liver [17]. These fibers originate from the region of the esophagus or cardiac portion of the stomach [ 17]. The posterior trunk of the dog vagus goes to the stomach where it sends branches to within a short distance of the pylorus [17]. At the cardiac level of the stomach a branch from the posterior trunk enters the lesser omentum, passing obliquely-ventrally to the liver [9,18]. A second division of the posterior vagus gives off branches that reach the celiac ganglion and the pancreas. Fibers originating from the posterior vagal trunk reach the liver from the celiac ganglion by way of the hepatic artery [21] or by crossing over to the portal vein [ 1,18]. The celiac ganglion also sends fibers from the spanchnic nerves to the liver [ 18,21]. These fibers travel with the ones originating from the vagus along the hepatic artery to the liver. They also cross over to the hepatic portal vein [ 18,21 ]. The phrenic nerve also travels to the liver region from the esophagus or the cardiac portion of the stomach where it courses through the omentum to the hepatic portal vein and then on the liver region. A second possible branch may cross to the liver directly from the esophagus [21 ]. The denervation was attempted in the following manner: the hepatic artery was isolated and all nerves on it or in the surrounding connective tissue were transected and 1 to 2 cm removed. Next, the hepatic portal vein was isolated and stripped of all nerves and, again, 1 to 2 cm were removed. The stripping was done within 1 to 2 cm of the liver. The nerves to the gallbladder were left intact. Then any branches from the ventral or dorsal vagus or phrenic nerve to the liver between the diaphragm and stomach were cut
D EN E R VATI ON AND I N T R A P O R T A L GLUCOSE ON FEEDING and again 1 to 2 cm of nerve removed. Vagal branches from the stomach to the liver were then isolated and 1 to 2 cm were cut and removed. Usually, there was one large branch at the cardiac portion and one at the pyloric end of the stomach. To insure that any smaller vagal branches to the liver from the stomach were not missed, the o m en t u m between the stomach and the liver was carefully cut. A modification of the Bard-Parker Company, Model No. 3411, (O.D. 3.58 mm) polyethylene cannula was then inserted into either the portal vein or a branch of the portal system, i.e. the splenic vein. The cannulas were modifed by placing a small tight fitting polyethylene band around the outside cannula wall so that it would act to anchor the cannula into the portal or splenic vein. The cannulas were tied in using a purse-string suture so that the vessel was not occuluded. Additional anchor bands were placed where we brought the cannula through the abdominal wall. The cannulas were then passed under the skin to the neck region where they were anchored again before they were exteriorized. Finally the cannulas were stoppered and filled with saline. The dogs' necks were then wrapped to protect the ends of the cannulas. To insure against possible nerve regeneration, which has been reported to take several months [ 15], the dogs were used in experiments only for 1 month after the denervation. While it was impossible to ascertain whether all nerves to the liver had been severed we believe that, if we did not in fact cut all the nerves, the majority of them were certainly severed. The 5 remaining dogs were sham operated, i.e. the various visceral organs were moved and handled in the same manner as was done in the dogs in which the nerves had been severed, and were then cannulated. On the first postoperative day, the animals were fed during their usual feeding time, i.e. 1400 hr. After the offering of food the onset of continuous eating was recorded and the food intake was recorded at 1500 hr. Continuous eating was arbitrarily defined by us to be a meal at least 3 min in length. After the animals had recovered to their preoperative food intake levels both the denervated and sham operated dogs were infused with one of the following glucose concentrations: 6 g, 12 g, 18 g (given in a 30% glucose solution) or 20 g or 25 g (given in a 50% glucose solution). See Table 1 for number of trials at each concentration. Saline solutions were made up at each of the glucose
301
osmotic concentrations. Each animal that received a glucose infusion also received the same volume and osmotic load of saline. Only one infusion trial of glucose or saline was performed on any one day. Both glucose and saline were injected using a syringe at the same rate that Russek [25] used which was 10 ml per minute. Infusions were made just prior to the time the animals were normally fed, after a 23 hr fast at 1400 hr in a room adjacent to where the dogs were kept. After the infusions were stopped, the animals were returned to their home cages. Three to 10 min were allowed to elapse from the end of the infusion until the dogs were presented with food. They were closely observed to determine the time of onset of continuous eating and their food intake was recorded at 1 500 hr. RESULTS All sham and denervated dogs returned to within 1 SD of their preoperative food intake levels by 8 days postsurgery. The difference between groups was not statistically significant (sham 3.6 -+ 1.0 days; denervated 5.4 _+ 1.0 days). The pertinent point is that the denervated dogs shortly returned to normal preoperative food intake levels. The sham operated dogs were infused with 12 to 25 g of glucose in 22 separate trials (Table 1) and in every case the dogs showed no anorexia and ate within 30 sec after being fed as did the denervated dogs after receiving 6 - 2 5 g of glucose in the 20 trials performed on them. In all cases, the dogs which are generally meal eaters continued to eat for at least 3 min after being fed. When the hourly intakes of the sham operated and liver denervated dogs after hepatic infusion of 12 g and 20 g of glucose were compared to their control hourly intakes, the sham dogs given glucose ate 94.1 _+ 13.6% and 100 _+ 11.2% respectively of their control intake while the denervated dogs ate 97.2 +_ 13.8% and 82.4 -+ 4.9% of their control days.
EXPERIMENT 2 METHOD
Animals and Surgery An additional 4 dogs (body weight range 1 6 - 2 0 kg) were treated prior to surgery as described in Experiment 1. At the time of surgery 1 polyethylene cannula was inserted
TABLE 1 FOOD INTAKE IN 23 HR FASTED LIVER DENERVATEDOR SHAM OPERATED DOGS 3 TO I0 MINS AFTERINTRAPORTALINFUSIONSOF 6 TO 25 g OF GLUCOSE Amount of glucose infused into the portal vein 6g 12 g 18 g 20 g 25 g Sham-operated
(0) * --
Denervation
(1) 1/1 = 100%
(5)
(1)
13/13 =
1/1 =
6/6=
(3)
2/2=
(2)
100%? (4) 12/12 = 100%
100% (2) 2/2= 100%
100% (1) 4/4= 100%
100% (1) 1/1 = 100%
*Number of animals used shown in parentheses. ?Percent of trials in which animals ate within 30 sec. of food presentation.
Total No. of Trials 22/22 = 100%
20/20 = 100%
302
BELLINGER, TRIETLEY AND BERNARDIS
into the portal vein and a second cannula into the jugular vein. Procedure After the dogs had recovered from the operation and were eating normally, the following studies were performed. (1). After a 23 hr fast a jugular blood sample was taken on 3 of the dogs after 1 jugular cannula had become unoperational. All 4 dogs were then infused into the portal vein over 4 min with 20 g of glucose (50% glucose solution). One to 2 min after the end of the infusion, a second jugular sample was taken. The dogs were then presented with food 3 to 5 min after the glucose infusion was stopped. They were then closely observed to determine the onset of continuous feeding. A total of 3 trials (on separate days) were performed on each dog. The blood was centrifuged and plasma glucose was determined on a Technican Auto Analyzer and Insulin was measured using an insulin kit purchased from Schwartz Mann. (2). After a 23 hr fast the 4 dogs were infused into the portal vein with adrenaline (1 ug/cc) starting with a concentration of 0.5 ug/kg, then 1.0 ug/kg and finally 1.5 ug/kg (given on separate days). Two trials were run at each adrenaline concentration before the next higher dose was used. The adrenaline was given over a period of 3 min. Ten min after the end of the infusion, the dogs were presented with food and onset of continuous eating was noted. Because 3 of the four cannulas had become unusable, blood glucose and insulin jugular could not be determined. At the end of both Experiments 1 and 2, the dogs were sacrificed and placement and patency of the portal cannulas were verified. They were all found to be functional. Attempts were made to determine if any nerves to the liver had remained functional, but because of the many adhesions it was impossible to verify total denervation. All data was analyzed using the Student's t method. RESULTS After portal infusion of 20 g of glucose the 4 dogs ate within 30 sec of food presentation in t2/12 trials. Jugular blood was collected on 3 dogs pre- and posthepatic infusion after 1 dog's cannula had become unoperational. Plasma glucose increased from 97.6 -+ 3.0 mg% preportal glucose infusion to 513.9 +_ 16.0 mg% (p<0.001) 1 to 2 min after infusion ended. Plasma insulin concentration increased from 22.2 -+ 3.1 uU/ml preglucose infusion to 125.0 _+28.4 uU/ml (p<0.001) 1 to 2 min after infusion was terminated. Ten minutes after the 23 hr fasted dogs received either 0.5 ug/kg, 1.0 ug/kg or 1.5 ug/kg of adrenaline, the animals ate within 30 sec of food presentation in all 24 trials performed. DISCUSSION It was surprising to us that liver denervation had no effect on food intake in view of Russek's claim that hepatic glucoreceptors are a major controller of food intake [25]. While it was impossible for us to ascertain that all nerves to and/or from the liver were cut, we firmly believe that we must have severed the majority of fibers. It would seem likely that removal of the greater part of liver innervation would have had some affect on the animals' food intake since it is well known that even partial damage to sensory
fibers of peripheral nerves produces hyperesthesia [34]. It is also recognized [12] that larger lesions of the ventromedial hypothalamus (VMH) which destroy more of the central glucoreceptors of Mayer [ 16 ] cause a greater degree of hyperphagia and obesity than smaller lesions which destroy less tissue and hence less receptors. Lesion size of the VMH has also been positively correlated with graded changes in certain endocrine, i.e. insulin and growth hormone [2] and metabolic parameters [3]. In the liver denervation in this study, we did not observe any anorexia or any graded reduction of food consumption which we would have expected after removing the majority of the hypothesized receptors innervation. We are left with several possibilities to explain the lack of any observable effect: (1) We have left enough glucose receptors innervated so that they could take up the function of the receptors that were denervated. However, as we have intimated above, we believe that removal of the majority of receptor innervation should have resulted in some sort of graded reduction in food intake. (2) The receptors are real and we did, in fact, functionally remove their influence on food intake but other mechanisms of controlling food intake compensated for their loss. These might have included gastric stretch receptors [22 ], central glucoreceptors [ 16 ] or influence of gastrointestinal hormones such as cholecystokin [10]. If this were the case, then hepatic glucose receptors would not be the major controller of food intake but only a component of a many factorial control system. (3) There are no hepatic glucose receptors that affect food intake in the dog. The most disturbing part of this study was the failure of intraportal injections of glucose to result in anorexia. According to Russek [25] 3 - 1 2 g of intraportally infused glucose should result in anorexia for approximately 4 0 - 7 5 min. We did not find any anorexia in dogs similarly treated in this study. Three to 10 min after receiving 6 to 25 g of glucose intraportally, all animals began eating within 30 sec of being presented with food. We are at a loss to explain the discrepancy between our data and the data of Russek [25]. Our dogs were given a mixture of chow and canned dog food, whereas Russek were fed only chow. Also, our dogs were fed in their home cages while Russek ran his trials on the dogs outside their home cages. On this latter difference one would suspect that a truer test of the existence of the proposed glucose receptors could be obtained by feeding the dogs after glucose infusions in their home cages. This would allow the dogs to be in a familiar environment and in one where they have been accustomed to feeding. Russek has reported [28] higher doses of intraperitoneal injected adrenaline (which supposedly increased intracellular hepatic glucose levels resulting in decreased glucose receptor activity and thus satiety) were needed to produce anorexia [28] in dogs given a more palatable diet than chow. Thus, the possibility exists that, because we fed our dogs a mixture of canned dog food and chow, higher hepatic intracellular glucose levels would be needed to produce anorexia. However, the 25 g glucose dose was 8 1/3 times the dose that Russek [25] reported would produce anorexia for at least 40 min, yet we found no anorexia. This 25 g dose of glucose representing approximately 100 kcal was given over 4 min and far exceeds any absorption of calories that would occur normally in the dog. It is of interest that in Experiment 2, the dogs ate within 30 sec of food presentation even though jugular
DENERVATION AND INTRAPORTAL GLUCOSE ON FEEDING plasma glucose was 5 times the normal concentration. However, this is not surprising in view of the early work of Janowitz [ 14] (for review of this subject see [ 11 ] ). What is noteworthy here is that of the 20 g of glucose given our dogs only approximately 4 g remained in the blood (if one assumes that the dogs weighed 20 kg and had a blood volume that approximated 8% of their body weight). The large majority of the remaining approximate 16 g of infused glucose would most likely be taken up by the liver [8]. Thus these dogs ate within 30 sec of food presentation even though in the liver the intracellular concentration of glucose must have been very high. It also should be noted that Stephens and Baldwin [32] have reported that intraportal infusions of 500 ml of 15% glucose failed to produce any anorexia in pigs, while VanderWheele e t al. [33] have reported that intraportal glucose infusions in the free feeding rabbit have also failed to cause anorexia. Finally Russel and Mogenson [30] have recently reported that food intake changes after infusion of 2 DG was unrelated to either portal or jugular sites of infusion. They suggest that while the hepatic contribution to food intake control is poorly understood their data were inconsistent with reports of hepatic participation in feeding. Russek has reported that intraperitoneal injections of adrenaline at doses of 0.1 to 0.3 mg]kg cause dogs to become anorexic for 45 min to 2 hr by increasing hepatic glucose concentration [28]. Russek's group also reported in the anesthetized animal that intraportal infusions of glucose at 10 mg/kg [27] or adrenaline [23] at a dose of 0.5 ug/kg resulted in a drop in arterial glucose while higher doses of either increased arterial glucose. He suggests this drop in arterial glucose occurs as a result of increased hepatic utilization of glucose which he interprets as being suggestive of increased glucoreceptor uptake of glucose. The drop of arterial glucose (from increased receptor utilization) also
303
corresponds to the anorexic period he reports for IP injections of glucose [28]. However, in the present study we have intraportally infused several unanesthetized 23 hr fasted dogs with 0.5 ug/kg of adrenaline and the dogs exhibited no anorexia on feeding as should have occurred according to Russek's group [23,29]. When the dose was increased to 1.0 and 1.5 ug/kg, the dogs still exhibited no anorexia even though hepatic output of glucose according to Russek [29] should have increased and therefore complicating the issue by possibly involving central glucoreceptors [ 16]. Thus it is unclear whether minute amounts of adrenaline infused into the liver can produce anorexia in the unanesthetized dog as suggested by Russek and his group [23,29]. We have observed (unpublished observations) in dogs that were given IP adrenaline at a dose of 0.2 mg/kg that about one half of the dogs were anorexic when presented with food after a 23 hr fast. However, the dogs that were anorexic panted excessively, clawed at their cages, howled and some of them vomited. It is difficult to determine whether adrenaline caused anorexia by causing hepatic hyperglycemia, i.e. affected the proposed hepatic glucoreceptors or whether it caused a general stress response in which case one would not expect the animals to eat. The above data are clearly in conflict with Russek's data and question the existence and/or the role which the hypothesized glucoreceptors play in the control of food intake in the dog. It is our hope that further work will be done in this area to clarify the discrepancy between the present data and those of Russek's. ACKNOWLEDGEMENT We wish to thank William O'Donnell and Steve Brooks for excellent technical assistance.
REFERENCES
1. Alexander, W. The innervation of the billary system. J. comp. Neurol. 72: 357-370, 1940. 2. Bernardis, L. and L. Frohman. Effect of lesion size in the ventromedial hypothalamus on growth hormone and insulin levels in weanling rats. Neuroendocrinology 6:319-328, 1970. 3. Bemardis, L. and J. Goldman. Effect of hypothalamic lesion localization and size on metabolic alterations in weanling rat adipose tissue. J. Neuro-Viscer. Relat. 32: 253-269, 1972. 4. Calne, R., H. White, D. Yoffa, R. Maginn, R. Binns, J. Samuel and V. Molina. Observations of orthotopic liver transplantation in the pig. Br. med. J. 2: 478-480, 1967. 5. Calne, R., R. Sells, J. Pena, D. Davis, P. Millard, B. Herbertson, R. Binns and D. Davies. Induction of immunological tolerance by procine liver allografts. Nature 223: 472-476, 1969. 6. Calne, R., D. Davis, J. Pena, H. Balner, M. DeVries, B. Herbertson, V. Joysey, P. Millard, M. Seaman, J. Samuel, J. Stibbe and D. Westbroek. Hepatic allografts and xenografts in primates. Lancet 1: 103-106, 1970. 7. Calne, R., R. Williams,J. Dawson, I. Ansell, D. Evans, P. Flute, B. Herbertson, V. Joysey, G. Keates, R. Knill-Jones, S. Mason, P. Millard, J. Pena, B. Pentlow, J. Salaman, R. Sells and P. Cullum. Liver transplantation in Man-II, A report of two orthotopic liver transplants in adult recipients. Br. reed. J. 4: 541-546, 1968. 8. Camu, F. Hepatic balances of glucose and insulin in response to physiological increments of endogenous insulin during glucose infusions in dogs. Eur. J. clin. lnvest. 5: 101-108, 1975. 9. Chiu, S. The superficial hepatic branches of the vagi and their distribution to the extrahepatic billary tract in certain mammals. Anat. Rec. 86: 149-155, 1943.
10. G~bs, J., R. Young and G. Smith. Cholecystokinin decreases food intake in rats. J. comp. physiol. Psychol. 84: 488-495, 1973. 11. Grossman, M. Integration of current views on the regulation of hunger and appetite. Ann. N. Y. Acad. Sci. 63: 76-91, 1955. 12. Hetherington, A. and S. Ranson. Hypothalamic lesions and adiposity in the rat. Anat. Rec. 78: 149-172, 1940. 13. Hsiao, S. and D. Langenes. Liquid intake, initiation and amount of eating as determined by osmolality of drinking liquids. Physiol. Behav. 7: 233-237, 1971. 14. Janowitz, H., M. Hanson, M. Grossman. Effect of intravenously administered glucose on food intake in the dog. Am. Z Physiol. 156: 87-91, 1949. 15. Lubinska, L. Axoplasmic streaming in regenerating and in normal nerve fibers. In: Mechanisms o f neural regeneration. Prog. in Brain Res. Vol. 13, edited by M. Singer and J. Schade. New York: Elsevier, 1964, pp. 1-66. 16. Mayer, J. and D. Thomas. Regulation of food intake and obesity. Science 156: 328-337, 1967. 17. McCrea, E. The abdominal distribution of the vagus. J. Anat. 59: 18-40, 1924. 18. Mitchell, G. Anatomy o f the autonomic nervous system, Edinburgh and London: E. and S. Livingstone Ltd., 1953, pp. 120-283. 19. Niijima, A. Afferent impulse discharges from glucoreceptors in the liver of the guinea pig. Ann. N. Y. Acad. Sci. 157: 690-700, 1969. 20. Novin, D., D. VanderWeele and M. Rezek. Infusion of 2-deoxy-D-glucoseinto the hepatic-portal system causes eating: Evidence for peripheral glucoreceptors. Science 181 : 858-860, 1973.
304 21. Pick, J. The Autonomic Nervous System. Philadelphia: J. B. Lippincott, 1970, pp. 313-331. 22. Quigley, J. The role of the digestive tract in regulating the ingestion of food. Ann. N. Y. Acad. Sci. 63: 6 - 1 4 , 1955. 23. Rodriquez-Zendejas, A., A. Vega, L. Soto-Mora, S. Pina and M. Russek. Some effects of intrapertoneal glucose and of intraportal glucose and adrenaline. Physiol. Behav. 3: 2 5 9 - 2 6 4 , 1968. 24. Russek, M. and S. Pina. Conditioning of adrenaline-induced anorexia. Nature 193: 1296-1297, 1962. 25. Russek, M. Demonstration of the influence of a hepatic glucosensitive mechanism on food intake. Physiol. Behav. 5: 1207-1209, 1970. 26. Russek, M. and S. Pina. Conditioning of adrenaline-induced anorexia. Nature 193: 1296-1297, 1962. 27. Russek, M. and L. Soto-Mora. Evidence de la existencia de glucoreceptores hepaticos. Acta. physiol, latinoam. 16: 8 5 - 8 6 , 1965. 28. Russek. M., A. Rodrigquez-Zendejas and S. Pina. Hypothetical liver receptors and the anorexia caused by adrenaline and glucose. Physiol. Behav. 3: 249-257, 1968.
BELLINGER, TRIETLEY AND BERNARDIS 29. Russek. M. Hepatic receptors and the neurophysiological mechanisms controlling feeding behavior. In: Neurosciences Res. Vol. 4, edited by S. Ehrenpreis. New York: Acad. Press, 1971, pp. 213-282. 30. Russell, P. and G. Mogenson. Drinking and feeding induced by jugular and portal infusions of 2-deoxy-d-glucose. Am. J. Physiol. 229: 1014-1018, 1975. 31. Sharma, K. Receptor mechanisms in the alimentary tract: Their excitation and functions. In: Control o f Food and Water Intake. Handbook of Physiology Sec. 6, Alimentary Canal, edited by C. Code, Washington, D.C.: Am. Physiol. Society, 1967, 225-237. 32. Stephens, D. and B. Baldwin. The lack of effect of intrajugular or intraportal injections of glucose or amino acids on food intake in pigs. Physiol. Behav. 12: 9 2 3 - 9 2 9 , 1974. 33. Vanderweele, D., D. Novin, M. Rezck and J. Sanderson. Duodenal or Hepatic-Portal Glucose Perfusion: Evidence for Duodenally-based Satiety. Physiol. Behav. 12: 4 6 7 - 4 7 3 , 1974. 34. Willis, W. and R. Grossman. Medical Neurobiology. Saint Louis: C. V. Mosby., 1973, pp. 355.