Physiology & Behavior, Vol. 19,
pp. 461466.
Pergamon Press and Brain Research
Publ.,
1977.
Printed
in the U.S.A.
Decreased Feeding in Rats Following Hepatic-Portal Infusion of Glucagonl JAMES R. MARTIN2
AND DONALD NOVIN
Department of Psychology and Brain Research Institute, University of California, Los Angeles, CA 90024 (Received 23 February
1977)
MARTIN, J. R. AND D. NOVIN. Decreased feeding in rats following hepatic-portal infusion of glucagon. PHYSIOL. BEHAV. 19(4) 461-466, 1977. -Male rats received a single hepatic-portal injection of glucagon following mild food deprivation. Cumulative food intake measured after 0.5-20 hr was decreased by the hormone. The absence of a concomitant decrease in water intake suggested a specific effect of glucagon on feeding. This specificity was further demonstrated by the use of an hepatic-portal infusion of glucagon as the unconditioned stimulus for the formation of a conditioned taste aversion which failed to produce avoidance of a novel taste. In contrast, pairing the taste with an intraperitoneal injection of lithium chloride did produce a learned taste aversion. Thus, the decreased feeding following infusion of a low concentration of pancreatic glucagon through a chronic hepatic-portal cannula cannot be attributed to visceral malaise. The relatively specific effect of this hormone on short-term feeding probably results from the activation of hepatic glycogenolysis, with the long-term effect on feeding possibly due to gluconeogenesis. Satiety
Hepatic-portal infusion
Glucagon
Short-term food intake
glycerol from adipose tissue and increases uptake and oxidation of fatty acids in liver and muscle [ 2 1 ] . Results from a number of studies have suggested that pancreatic glucagon plays some role in controlling feeding, Sudsaneh and Mayer [27] intravenously infused glucagon in fasted rats and noted inhibition of gastric contractions. Salter [ 181 demonstrated that chronic subcutaneous administration of glucagon in rats produced a slight reduction in feeding concomitant with drastically decreased weight gain, decreased protein and fat synthesis, and increased hepatic glycogen storage. In another investigation, intraperitoneal injection of a large dose of glucagon delayed the onset of feeding in contrast to a more rapid onset following insulin administration [ 21. In addition, Balagura and Hoebel [ 11 demonstrated that glucagon administration decreased the self-stimulation rate for lateral hypothalamic electrodes that had previously been demonstrated to elicit feeding. A number of clinical investigations have also implicated glucagon in the control of feeding. Glucagon reduced hunger sensations, abolished gastric contractions, raised blood glucose, and increased peripheral arteriovenous glucose differences in humans [ 25,261. In other clinical studies, chronic glucagon administration resulted in reduced caloric intake and decreased body weight [ 13,201. Interpretation of previous studies concerning the role of glucagon in modulating feeding are limited by the high concentrations used, often injected repeatedly over long time periods, and by administration of the hormone via unphysiological routes [ 1, 2, 7, 181. The present work was
NATURAL ingestion of food, as well as infusion of nutrients directly into the gastrointestinal system, stimulates the release of numerous hormonal substances [ 14,15 I. The administration of several of these hormones produces modulation of meal pattern and magnitude. The injection of an intestinal extract identified as enterogasterone resulted in a short-term decrease in food intake in rabbits [9]. Similarly, administration of a preparation of porcine enterogasterone was shown to suppress feeding in severely fasted mice [ 191. The intestinal hormone cholecystokinin has been extensively studied and has been found to reduce feeding in food-deprived rats [4] and in rats with an open gastric fistula that permitted the continuous drainage of a liquid diet as it was ingested [S] . In addition, reduction of feeding following injection of cholecystokinin has been demonstrated in rhesus monkeys [3] but was not obtained in man [6]. Administration of caerulein, a decapeptide chemically and physiologically similar to cholecystokinin, has also been shown to decrease food intake in rats [ 241. The behavioral effects of pancreatic glucagon have received relatively little attention in comparison to the extensive literature concerned with its physiological actions [8]. Under natural conditions, pancreatic glucagon is released in response to hypoglycemia and stimulates increased glucose output from the liver by means of increased glycogenolysis, increased gluconeogenesis, and decreased glycogen synthesis; the relative contribution of each of these metabolic processes to overall glucose production varies with physiological circumstances. In addition, glucagon stimulates the mobilization of free-fatty acids and
designed
to
further
investigate
the
role
of glucagon
in
’ Supported by Grant NS 7687 from the National Institute of Neurological Diseases and Stroke to D.N. ‘Supported by NIMH Postdoctoral Fellowship MH 5101. Present address and address for reprints: Department of Psychology, University of California, Los Angeles, CA 90024. 461
controlling food intake. A dose-response relation was generated, with glucagon infused through a chronic hepatic-portal cannula, to closely approximate its physiological route. The specificity of the satiety induced by glucagon was assessed by measuring both food and water intake. In addition, a toxiphobia paradigm similar to that described by Rozin [ 171 was used to determine whether visceral discomfort could account for the suppression of feeding observed subsequent to hepatic-portal infusions of glucagon. This latter control was considered particularly important because injection of a high concentration of glucagon into severely food-deprived rats can act as an unconditioned stimulus for the formation of a learned taste aversion [ 161, indicating that under some experimental circumstances glucagon administration can result in aversive consequences. EXPERIMENT
1
The first experiment was conducted to demonstrate that an injection of glucagon through the hepatic-portal vein of male rats suppresses feeding. Several concentrations of glucagon were used to establish the dose-response relation between the reduction in food intake and the concentration of glucagon. To assess the specificity of this effect, water intake was also monitored. METHOD
The Long-Evans hooded male rats used in this experiment were individually housed in a room with a 12: 12 hr light-dark cycle. Prior to surgery, the animals were maintained on ad lib Purina powdered lab chow and tap water. Of an initial group of 54 animals, 44 survived the surgical intervention; of these survivors, 21 completed testing with a cannula verified as intravenous (IV). Although the surgical procedure was relatively stressful and accounted for some of the above losses, the majority of the discarded animals completed all tests but their cannulae were found to empty into the peritoneal cavity. The animals that completed testing with an IV cannula had a mean body weight of 364 g prior to surgery, 360 g at the start of testing, and 380 g’when sacrificed. Following sodium pentobarbital anesthesia (50 mg/kg), all animals were surgically implanted with a Silastic cannula (Dow Corning: 0.30 mm I.D. x 0.65 mm O.D.) inserted into a collecting vessel of the hepatic-portal vein. The collecting vessel was permanently ligated above the point of cannula entry. Fascia was dissected from the vein and it was elevated by the placement of blunt forceps underneath. The vessel was cut midway between two loosely tied sutures that were weakly pulled in opposite directions to minimize bleeding once a hole was made. The cannula was then threaded approximately 40 mm through the collecting vessel and into the hepatic-portal vein. The placement of the cannula was verified by withdrawal of blood. The remainder of the cannula extended through the sutured abdominal opening and then proceeded subcutaneously to the skull, where it was attached to a blunted 25ga needle. Dental acrylic cement was applied to bind the blunted needle to four stainless-steel screws fastened into the skull. The tip of a Luer-Lok syringe, with the center sealed, provided an airtight cap. A similar procedure developed for use in rabbits has previously been described [ 111. Following surgery, the cannulae were injected with 0.5 ml of 10% Sodium Heparin in 0.9% NaCl once each day for 2-4 days. Thereafter, the animals received a daily
injection of physiological saline, except on days when a ie>: solution of glucagon was infused. The animals were allowctl to recover for l-2 wk after cannula implantation to per-n11t a return to preoperative body weight. During the Iattei phase of recovery, the rats were food deprived each day for a period of four hr, beginning two hr after the start of the light portion of the light-dark cycle. At the end of thr deprivation period, the animals received saline injections to maintain cannula patency. Powdered food was then introduced into each cage in a metal cup attached to a Petri dish of 150 mm diameter. The testing procedure was similar to that of the latter phase of recovery. However, on every third day a test solution (saline, 25, 35, 40, 45, 55, or 65 &g/kg glucagon, 0.2% b.w.) was infused according to a counterbalanced design. The pancreatic glucagon (Calbiochem, bovine origin) was dissolved in 0.9% NaCl. The animals were also food deprived on all non-test days and received a saline injection prior to presentation of food. On the test days, food cups and water bottles were weighed to the nearest 0.1 g. Food consumption was recorded 0.5, 1, 2,3, and 20 hr after each of the seven test infusions. Water intake was measured 1. 3. and 20 hr subsequent to infusion. Following the completion of the full sequence of test injections, all animals were sacrificed with sodium pentobarbital. Each cannula was injected with dye during autopsy to confirm that it was functional and that the tip was located within the hepatic-portal vein.
RESULTS
Food intake was significantly reduced by hepatic-portal infusion of glucagon without any concomitant effect on water intake. Because of the relatively large number of individual comparisons made, overall significance was first tested with analysis of variance (animals x treatments) computed separately for each measurement period. Significant differences were found for food intake measured after 0.5, 1, 2, and 3 hr. Failure to meet the assumption of homogeneity of variance resulted in analysis of 20-hr food intake and 3-hr water intake with the Friedman two-way analysis of variance; only the former analysis yielded a significant result. Thus, significant overall differences were revealed for food intake after 0.5, 1, 2, 3, and 20 hr (p
pp. 461466.
Pergamon Press and Brain Research
Publ.,
1977.
Printed
in the U.S.A.
Decreased Feeding in Rats Following Hepatic-Portal Infusion of Glucagonl JAMES R. MARTIN2
AND DONALD NOVIN
Department of Psychology and Brain Research Institute, University of California, Los Angeles, CA 90024 (Received 23 February
1977)
MARTIN, J. R. AND D. NOVIN. Decreased feeding in rats following hepatic-portal infusion of glucagon. PHYSIOL. BEHAV. 19(4) 461-466, 1977. -Male rats received a single hepatic-portal injection of glucagon following mild food deprivation. Cumulative food intake measured after 0.5-20 hr was decreased by the hormone. The absence of a concomitant decrease in water intake suggested a specific effect of glucagon on feeding. This specificity was further demonstrated by the use of an hepatic-portal infusion of glucagon as the unconditioned stimulus for the formation of a conditioned taste aversion which failed to produce avoidance of a novel taste. In contrast, pairing the taste with an intraperitoneal injection of lithium chloride did produce a learned taste aversion. Thus, the decreased feeding following infusion of a low concentration of pancreatic glucagon through a chronic hepatic-portal cannula cannot be attributed to visceral malaise. The relatively specific effect of this hormone on short-term feeding probably results from the activation of hepatic glycogenolysis, with the long-term effect on feeding possibly due to gluconeogenesis. Satiety
Hepatic-portal infusion
Glucagon
Short-term food intake
glycerol from adipose tissue and increases uptake and oxidation of fatty acids in liver and muscle [ 2 1 ] . Results from a number of studies have suggested that pancreatic glucagon plays some role in controlling feeding, Sudsaneh and Mayer [27] intravenously infused glucagon in fasted rats and noted inhibition of gastric contractions. Salter [ 181 demonstrated that chronic subcutaneous administration of glucagon in rats produced a slight reduction in feeding concomitant with drastically decreased weight gain, decreased protein and fat synthesis, and increased hepatic glycogen storage. In another investigation, intraperitoneal injection of a large dose of glucagon delayed the onset of feeding in contrast to a more rapid onset following insulin administration [ 21. In addition, Balagura and Hoebel [ 11 demonstrated that glucagon administration decreased the self-stimulation rate for lateral hypothalamic electrodes that had previously been demonstrated to elicit feeding. A number of clinical investigations have also implicated glucagon in the control of feeding. Glucagon reduced hunger sensations, abolished gastric contractions, raised blood glucose, and increased peripheral arteriovenous glucose differences in humans [ 25,261. In other clinical studies, chronic glucagon administration resulted in reduced caloric intake and decreased body weight [ 13,201. Interpretation of previous studies concerning the role of glucagon in modulating feeding are limited by the high concentrations used, often injected repeatedly over long time periods, and by administration of the hormone via unphysiological routes [ 1, 2, 7, 181. The present work was
NATURAL ingestion of food, as well as infusion of nutrients directly into the gastrointestinal system, stimulates the release of numerous hormonal substances [ 14,15 I. The administration of several of these hormones produces modulation of meal pattern and magnitude. The injection of an intestinal extract identified as enterogasterone resulted in a short-term decrease in food intake in rabbits [9]. Similarly, administration of a preparation of porcine enterogasterone was shown to suppress feeding in severely fasted mice [ 191. The intestinal hormone cholecystokinin has been extensively studied and has been found to reduce feeding in food-deprived rats [4] and in rats with an open gastric fistula that permitted the continuous drainage of a liquid diet as it was ingested [S] . In addition, reduction of feeding following injection of cholecystokinin has been demonstrated in rhesus monkeys [3] but was not obtained in man [6]. Administration of caerulein, a decapeptide chemically and physiologically similar to cholecystokinin, has also been shown to decrease food intake in rats [ 241. The behavioral effects of pancreatic glucagon have received relatively little attention in comparison to the extensive literature concerned with its physiological actions [8]. Under natural conditions, pancreatic glucagon is released in response to hypoglycemia and stimulates increased glucose output from the liver by means of increased glycogenolysis, increased gluconeogenesis, and decreased glycogen synthesis; the relative contribution of each of these metabolic processes to overall glucose production varies with physiological circumstances. In addition, glucagon stimulates the mobilization of free-fatty acids and
designed
to
further
investigate
the
role
of glucagon
in
’ Supported by Grant NS 7687 from the National Institute of Neurological Diseases and Stroke to D.N. ‘Supported by NIMH Postdoctoral Fellowship MH 5101. Present address and address for reprints: Department of Psychology, University of California, Los Angeles, CA 90024. 461
controlling food intake. A dose-response relation was generated, with glucagon infused through a chronic hepatic-portal cannula, to closely approximate its physiological route. The specificity of the satiety induced by glucagon was assessed by measuring both food and water intake. In addition, a toxiphobia paradigm similar to that described by Rozin [ 171 was used to determine whether visceral discomfort could account for the suppression of feeding observed subsequent to hepatic-portal infusions of glucagon. This latter control was considered particularly important because injection of a high concentration of glucagon into severely food-deprived rats can act as an unconditioned stimulus for the formation of a learned taste aversion [ 161, indicating that under some experimental circumstances glucagon administration can result in aversive consequences. EXPERIMENT
1
The first experiment was conducted to demonstrate that an injection of glucagon through the hepatic-portal vein of male rats suppresses feeding. Several concentrations of glucagon were used to establish the dose-response relation between the reduction in food intake and the concentration of glucagon. To assess the specificity of this effect, water intake was also monitored. METHOD
The Long-Evans hooded male rats used in this experiment were individually housed in a room with a 12: 12 hr light-dark cycle. Prior to surgery, the animals were maintained on ad lib Purina powdered lab chow and tap water. Of an initial group of 54 animals, 44 survived the surgical intervention; of these survivors, 21 completed testing with a cannula verified as intravenous (IV). Although the surgical procedure was relatively stressful and accounted for some of the above losses, the majority of the discarded animals completed all tests but their cannulae were found to empty into the peritoneal cavity. The animals that completed testing with an IV cannula had a mean body weight of 364 g prior to surgery, 360 g at the start of testing, and 380 g’when sacrificed. Following sodium pentobarbital anesthesia (50 mg/kg), all animals were surgically implanted with a Silastic cannula (Dow Corning: 0.30 mm I.D. x 0.65 mm O.D.) inserted into a collecting vessel of the hepatic-portal vein. The collecting vessel was permanently ligated above the point of cannula entry. Fascia was dissected from the vein and it was elevated by the placement of blunt forceps underneath. The vessel was cut midway between two loosely tied sutures that were weakly pulled in opposite directions to minimize bleeding once a hole was made. The cannula was then threaded approximately 40 mm through the collecting vessel and into the hepatic-portal vein. The placement of the cannula was verified by withdrawal of blood. The remainder of the cannula extended through the sutured abdominal opening and then proceeded subcutaneously to the skull, where it was attached to a blunted 25ga needle. Dental acrylic cement was applied to bind the blunted needle to four stainless-steel screws fastened into the skull. The tip of a Luer-Lok syringe, with the center sealed, provided an airtight cap. A similar procedure developed for use in rabbits has previously been described [ 111. Following surgery, the cannulae were injected with 0.5 ml of 10% Sodium Heparin in 0.9% NaCl once each day for 2-4 days. Thereafter, the animals received a daily
injection of physiological saline, except on days when a ie>: solution of glucagon was infused. The animals were allowctl to recover for l-2 wk after cannula implantation to per-n11t a return to preoperative body weight. During the Iattei phase of recovery, the rats were food deprived each day for a period of four hr, beginning two hr after the start of the light portion of the light-dark cycle. At the end of thr deprivation period, the animals received saline injections to maintain cannula patency. Powdered food was then introduced into each cage in a metal cup attached to a Petri dish of 150 mm diameter. The testing procedure was similar to that of the latter phase of recovery. However, on every third day a test solution (saline, 25, 35, 40, 45, 55, or 65 &g/kg glucagon, 0.2% b.w.) was infused according to a counterbalanced design. The pancreatic glucagon (Calbiochem, bovine origin) was dissolved in 0.9% NaCl. The animals were also food deprived on all non-test days and received a saline injection prior to presentation of food. On the test days, food cups and water bottles were weighed to the nearest 0.1 g. Food consumption was recorded 0.5, 1, 2,3, and 20 hr after each of the seven test infusions. Water intake was measured 1. 3. and 20 hr subsequent to infusion. Following the completion of the full sequence of test injections, all animals were sacrificed with sodium pentobarbital. Each cannula was injected with dye during autopsy to confirm that it was functional and that the tip was located within the hepatic-portal vein.
RESULTS
Food intake was significantly reduced by hepatic-portal infusion of glucagon without any concomitant effect on water intake. Because of the relatively large number of individual comparisons made, overall significance was first tested with analysis of variance (animals x treatments) computed separately for each measurement period. Significant differences were found for food intake measured after 0.5, 1, 2, and 3 hr. Failure to meet the assumption of homogeneity of variance resulted in analysis of 20-hr food intake and 3-hr water intake with the Friedman two-way analysis of variance; only the former analysis yielded a significant result. Thus, significant overall differences were revealed for food intake after 0.5, 1, 2, 3, and 20 hr (p
