Physiology & Behavior, Vol. 15, pp. 137--143. Pergamon Press and Brain Research Publ., 1975. Printed in the U.S.A.
Elevated Blood Glucose Levels and Satiety in the Rat W. H. WILSON AND H. C. HELLER
Department o f Biological Sciences, Stanford University, Stanford, CA 94305
(Received 23 September 1974) WILSON, W. H. AND H. C. HELLER. Elevated blood glucose levels and satiety in the rat. PHYSIOL. BEHAV. 15(2) 137-143, 1975. - Thirteen rats were placed on a feeding schedule of two 3 hr periods of food availability daily. Blood glucose levels of the experimental animals were altered by intraperitoneal injection of 8 ml of 2, 5, 8, 12, 16, 20 and 25 percent glucose solutions and by intragastric loading of 8 ml of 25, 50 and 65 percent glucose solutions in 10 different feeding experiments. In 4 additional experiments, experimental animals received intraperitoneal injections of 8 ml of 12, 16, 20 and 25 percent solutions of mannitol, a non-metabolizable sugar-alcohol. Controls always received identical quantities of mammalian Ringer's solution administered via the same route as in the experimentals. Another set of 20 rats was used to determine glucose tolerance curves for each concentration of glucose and mannitol administered. No food intake depression occurred following intraperitoneal injections of 2, 5, 8 and 12 percent glucose, 12 percent mannitol and following intragastric loading of 25, 50 and 65 percent glucose. However, intraperitoneal injections of 16, 20 and 25 percent glucose and mannitol caused depression of food intake. No depression of food intake occurred following intragastric loading of 50 and 65 percent glucose solutions which raised blood glucose levels a minimum of 43 mg percent and 55 mg percent above basal, respectively, for the duration of the 3 hr feeding period. These minimum blood glucose levels greatly exceed normal values following a meal. It was concluded that blood glucose level per se is not an important feedback parameter in the long-term control of food intake, and the depression in food intake following intraperitoneal injections of 16, 20 and 25 percent glucose and mannitol solutions was due to an abnormal physiological condition. Glucose Anorexia
Arterial blood glucose
Intraperitoneal injection
Food intake
Intragastrie loading
gations suggested that the action of nutrients in the duodenum was more important in the control of short-term feeding behavior than the nutrient's action postabsorptively. In contrast, Booth et al. [2] found that intraperitoneal (IP) glucose injections depressed feeding significantly more than comparable intragastric (IG) loads. They suggested that the effect of IP-administered glucose was due to its interaction initially or mainly with the liver. Panksepp [10] observed that 8 ml of 25 percent glucose-administered IP depressed feeding more than IG loads and that feeding was depressed 2 to 3 days postinjection. He also noted that animals with experimentally created lesions in the VMH were less sensitive to the anorexigenic effects of IP injections of glucose. He concluded that this indicated the involvement of the VMH glucoreceptors and blood glucose level in control of long-term satiety. The series of experiments described in this paper was prompted by the conflicting data published on the role of blood glucose as a feedback parameter in the control of feeding behavior. Our intent was to delineate the relationship between blood glucose levels and food intake and to assess the involvement of glucose in control of long-term feeding behavior. We began by repeating Panksepp's experiments. If, as Panksepp hypothesized, blood glucose level is
THE glucostatic hypothesis of regulation of food intake postulates the presence of glucoreceptors in the ventromedial nuclei of the hypothalamus (VMH) which respond to decreased glucose availability bY stimulating an increase in food intake [6,17]. The presence of these glucose-sensitive cells has been supported by the selective destruction of cells in the VMH by microinjection of gold thioglucose. The resulting hyperphagia seen in rats was assumed to reflect the inactivation of these glucoreceptors [15,16]. Later work has suggested the existence of peripheral glucoreceptors located in the liver [12,13]. This has been supported by the induction of anorexia in fasted dogs by intraportal injection of glucose and by the induction of hyperphagia by infusion of the glucose analog, 2-deoxy-D-glucose, into the hepatic-portal vein [8,14]. Numerous investigators have observed anorexia after administering glucose via various routes [2, 10, 13, 14, 20], but intrajugular injections of similar concentrations of glucose have been without effect [15]. Vanderweele et al. [21] found that hepatic-portal injections of up to 30 percent glucose solutions did not alter food consumption in free-feeding rabbits. However, similar concentrations administered intra-duodenally reduced food intake during the first hour after perfusion. These investi137
138
WILSON AND HELLER
related to long-term regulation of food intake, lower concentrations of glucose injected intraperitoneally would be expected to produce proportionately less depression of long-term food intake than higher glucose concentrations. To test this hypothesis, we injected rats IP with different concentrations of glucose. Concurrent with this, blood samples were collected for analysis of blood glucose levels. Additional experimentation with mannitol was done to determine if it could induce similar concentration-dependent effects on food intake. Finally, we wished to compare the effects of IP and IG administration of glucose on blood glucose levels and on food intake. If blood glucose level per se is a primary feedback parameter in the regulation of food intake, it should make no difference whether the route of glucose administration is IP or IG, as long as similar blood levels are achieved. METHOD A n i m a l s a n d Procedure
Thirteen male Sprague-Dawley rats (Group A) with a mean body weight of 279 + 11.2 g were used to determine the effect of IP or IG administration of glucose on food intake. The effect of the IP administration of mannitol on food intake was also studied in this group of rats. Each animal was housed in an aluminum cage 25 by 29 by 15.5 cm with a wire mesh top. Animals were maintained on a 12 hr light/dark cycle with lights on at 8:00 a.m. and at an environmental temperature of 21 +_ 2°C. During the experiments, water was available ad lib and between experiments both water and Purina Laboratory Chow were available ad lib. The length of the animal's feeding schedule was chosen to assure sufficient time for the animals to consume a normal quantity of food and to approximate the animal's normal feeding behavior. Shorter feeding schedules resulted in either body weight loss or slowed body weight gain in comparison to the ad lib regime. Prior to any experimentation, the animals were adjusted to the schedule for 16 days. During this adjustment period, body weight increased at a normal rate as compared to animals on ad lib feeding, and food consumption stabilized at approximately 7 + 2 g from 8:30 to 11:30 a.m. and at approximately 9 + 2 g from 5:30 to 8:30 p.m. During all subsequent experiments, the animals displayed normal body weight and food consumption as compared to this initial adjustment period. For each experiment, Group A was randomly divided into 7 experimental and six control animals. Consequently, an animal chosen as an experimental during one experiment had a random probability of becoming an experimental animal during the subsequent experiment, with the exception that no animal was an experimental in more than two consecutive experiments. The animals were then placed on a feeding schedule of two 3 hr periods of food availability per day. Food was weighed in tared dishes and placed in the cages from 8: 30 to I 1:30 a.m. and from 5:30 to 8: 30 p.m. At the end of each feeding period, the uneaten food was carefully collected and weighed and the intake recorded. It was assumed that any significant reduction in food intake during any portion of the 3 hr feeding period would be reflected in the total food intake at the end of the feeding period. This feeding schedule was begun at least 4 feeding periods prior to each experiment to re-adapt the animals, and it was continued for 3 to 6 feeding periods postadministration. IP or IG administration of test solutions began at
5:00 p.m. and was completed by 5:30 p.m. At the end of each experiment the animals were returned to ad lib feeding for at least 4 days to allow normalization of food intake before the next experiment was begun. Twenty different male Sprague-Dawley rats (Group B) with a mean body weight of 268 -+ 12.4 g were used to determine changes in blood glucose levels induced by IP or IG administration of glucose and the IP injection of mannitol. Group B animals were used for assay of blood glucose levels only, and Group A animals for intake experiments only. Both procedures were not performed simultaneously. Group B animals were kept in groups of 5 in four aluminum cages measuring 30 by 50 by 15.5 cm with lighting and temperature conditions as previously noted for Group A. These animals had water and Purina Laboratory Chow ad lib. Each of the Group B animals used for the determination of blood glucose levels was placed in a rat restrainer constructed out of a 6.4 cm dia. plastic tube with a wire mesh covering over one end. The tip of the restrained animal's tail was quickly cut with a razor blade and the tail gently stroked. Approximately 50 ~1 of blood were collected in a capillary tube for each sample and immediately centrifuged to obtain cell-free plasma. Ten/al of the blood plasma were used in a coupled glucose-oxidase-peroxidase reaction as modified by W. Werner et al. [21] (Biochemica Test Kit, Boehringer Mannlieim Corporation, San Francisco, California, U.S.A.). The development of a green color in the reaction was proportional to the concentration of glucose in the plasma. The optical density of the sample was read at 650 n m on a model 240 Gilford Spectrophotometer. To Group A experimentals, 8 ml of 2, 5, 8, 12, 16, 20 or 25 percent d-glucose solutions (w/v) were administered intraperitoneally in 7 different experiments. The three highest concentration experiments were followed by the lower concentration experiments. Sufficient time elapsed between experiments to prevent conditioning. Each animal received only one injection of a particular glucose concentration. The solutions were freshly prepared, millipore filtered and sterilized prior to each trial. The controls received an injection of 8 ml of mammalian Ringer's solution at the same time that the experimentals received glucose. All solutions were maintained at a pH between 7 and 8. Blood glucose assays were performed on 3 animals from Group B for each of the 7 glucose concentrations. For each analysis, blood samples were taken 5 min prior to injection and at 5, 20, 40, 60, 90, 120, 150 and 180 min postinjection. In three additional experiments, experimental animals from Group A received IP injections of 8 ml of 16, 20 and 25 percent mannitol (Invenex), and controls received 8 ml IP of mammalian Ringer's solution. Blood glucose tolerance curves were performed on 3 animals from Group B for each concentration of mannitol. Finally, we investigated the effect of IG loads of dglucose solutions on food intake. IG loads of 8 ml of 25, 50 and 65 percent glucose were given to Group A experimentals in 4 different experiments. Controls received an IG load of 8 ml of mammalian Ringer's solution. Blood glucose analyses were done on 3 animals from Group B for each glucose concentration. During every experiment, the food intake of the experimentals after administration of solutions was compared to the food intake of the controls and to the preadministration intake of the experimentals. The choice of internal or external controls did not affect the results.
BLOOD GLUCOSE AND SATIETY
139
The Olivetti Underwood Programma 101 was used for all statistical analysis. A T-test for the significance o f the difference between two sample means was used (program No. 5.20): p < 0 . 0 5 was taken as significant. RESULTS Effect on Food Intake o f IP and IG Administration o f Glucose and IP Injections o f Mannitol. The food intakes of animals in Group A were measured at 4 feeding periods before IP or IG administrations and for 3 feeding periods postadministration. The animals were closely observed after the IP or IG administrations. They displayed no apparent abnormalities and commenced feeding immediately upon presentation o f food. Intraperitoneal glucose injections of 8 ml o f 2, 5, 8, and 12 percent did not significantly depress food intake when compared to the intake of controls or when compared to the preinjection intake of the experimentals (Fig. 1). A significant depression o f food intake was observed, however, after IP injections of 8 ml of 16, 20 and 25 percent glucose solutions (Fig. 1). F o o d intake was depressed for the first feeding period after injection o f 16 percent glucose solution (p< 0.01). Depression was observed for two periods foTlowing injection of 20 percent glucose solution (p
Elevated Blood Glucose Levels and Satiety in the Rat W. H. WILSON AND H. C. HELLER
Department o f Biological Sciences, Stanford University, Stanford, CA 94305
(Received 23 September 1974) WILSON, W. H. AND H. C. HELLER. Elevated blood glucose levels and satiety in the rat. PHYSIOL. BEHAV. 15(2) 137-143, 1975. - Thirteen rats were placed on a feeding schedule of two 3 hr periods of food availability daily. Blood glucose levels of the experimental animals were altered by intraperitoneal injection of 8 ml of 2, 5, 8, 12, 16, 20 and 25 percent glucose solutions and by intragastric loading of 8 ml of 25, 50 and 65 percent glucose solutions in 10 different feeding experiments. In 4 additional experiments, experimental animals received intraperitoneal injections of 8 ml of 12, 16, 20 and 25 percent solutions of mannitol, a non-metabolizable sugar-alcohol. Controls always received identical quantities of mammalian Ringer's solution administered via the same route as in the experimentals. Another set of 20 rats was used to determine glucose tolerance curves for each concentration of glucose and mannitol administered. No food intake depression occurred following intraperitoneal injections of 2, 5, 8 and 12 percent glucose, 12 percent mannitol and following intragastric loading of 25, 50 and 65 percent glucose. However, intraperitoneal injections of 16, 20 and 25 percent glucose and mannitol caused depression of food intake. No depression of food intake occurred following intragastric loading of 50 and 65 percent glucose solutions which raised blood glucose levels a minimum of 43 mg percent and 55 mg percent above basal, respectively, for the duration of the 3 hr feeding period. These minimum blood glucose levels greatly exceed normal values following a meal. It was concluded that blood glucose level per se is not an important feedback parameter in the long-term control of food intake, and the depression in food intake following intraperitoneal injections of 16, 20 and 25 percent glucose and mannitol solutions was due to an abnormal physiological condition. Glucose Anorexia
Arterial blood glucose
Intraperitoneal injection
Food intake
Intragastrie loading
gations suggested that the action of nutrients in the duodenum was more important in the control of short-term feeding behavior than the nutrient's action postabsorptively. In contrast, Booth et al. [2] found that intraperitoneal (IP) glucose injections depressed feeding significantly more than comparable intragastric (IG) loads. They suggested that the effect of IP-administered glucose was due to its interaction initially or mainly with the liver. Panksepp [10] observed that 8 ml of 25 percent glucose-administered IP depressed feeding more than IG loads and that feeding was depressed 2 to 3 days postinjection. He also noted that animals with experimentally created lesions in the VMH were less sensitive to the anorexigenic effects of IP injections of glucose. He concluded that this indicated the involvement of the VMH glucoreceptors and blood glucose level in control of long-term satiety. The series of experiments described in this paper was prompted by the conflicting data published on the role of blood glucose as a feedback parameter in the control of feeding behavior. Our intent was to delineate the relationship between blood glucose levels and food intake and to assess the involvement of glucose in control of long-term feeding behavior. We began by repeating Panksepp's experiments. If, as Panksepp hypothesized, blood glucose level is
THE glucostatic hypothesis of regulation of food intake postulates the presence of glucoreceptors in the ventromedial nuclei of the hypothalamus (VMH) which respond to decreased glucose availability bY stimulating an increase in food intake [6,17]. The presence of these glucose-sensitive cells has been supported by the selective destruction of cells in the VMH by microinjection of gold thioglucose. The resulting hyperphagia seen in rats was assumed to reflect the inactivation of these glucoreceptors [15,16]. Later work has suggested the existence of peripheral glucoreceptors located in the liver [12,13]. This has been supported by the induction of anorexia in fasted dogs by intraportal injection of glucose and by the induction of hyperphagia by infusion of the glucose analog, 2-deoxy-D-glucose, into the hepatic-portal vein [8,14]. Numerous investigators have observed anorexia after administering glucose via various routes [2, 10, 13, 14, 20], but intrajugular injections of similar concentrations of glucose have been without effect [15]. Vanderweele et al. [21] found that hepatic-portal injections of up to 30 percent glucose solutions did not alter food consumption in free-feeding rabbits. However, similar concentrations administered intra-duodenally reduced food intake during the first hour after perfusion. These investi137
138
WILSON AND HELLER
related to long-term regulation of food intake, lower concentrations of glucose injected intraperitoneally would be expected to produce proportionately less depression of long-term food intake than higher glucose concentrations. To test this hypothesis, we injected rats IP with different concentrations of glucose. Concurrent with this, blood samples were collected for analysis of blood glucose levels. Additional experimentation with mannitol was done to determine if it could induce similar concentration-dependent effects on food intake. Finally, we wished to compare the effects of IP and IG administration of glucose on blood glucose levels and on food intake. If blood glucose level per se is a primary feedback parameter in the regulation of food intake, it should make no difference whether the route of glucose administration is IP or IG, as long as similar blood levels are achieved. METHOD A n i m a l s a n d Procedure
Thirteen male Sprague-Dawley rats (Group A) with a mean body weight of 279 + 11.2 g were used to determine the effect of IP or IG administration of glucose on food intake. The effect of the IP administration of mannitol on food intake was also studied in this group of rats. Each animal was housed in an aluminum cage 25 by 29 by 15.5 cm with a wire mesh top. Animals were maintained on a 12 hr light/dark cycle with lights on at 8:00 a.m. and at an environmental temperature of 21 +_ 2°C. During the experiments, water was available ad lib and between experiments both water and Purina Laboratory Chow were available ad lib. The length of the animal's feeding schedule was chosen to assure sufficient time for the animals to consume a normal quantity of food and to approximate the animal's normal feeding behavior. Shorter feeding schedules resulted in either body weight loss or slowed body weight gain in comparison to the ad lib regime. Prior to any experimentation, the animals were adjusted to the schedule for 16 days. During this adjustment period, body weight increased at a normal rate as compared to animals on ad lib feeding, and food consumption stabilized at approximately 7 + 2 g from 8:30 to 11:30 a.m. and at approximately 9 + 2 g from 5:30 to 8:30 p.m. During all subsequent experiments, the animals displayed normal body weight and food consumption as compared to this initial adjustment period. For each experiment, Group A was randomly divided into 7 experimental and six control animals. Consequently, an animal chosen as an experimental during one experiment had a random probability of becoming an experimental animal during the subsequent experiment, with the exception that no animal was an experimental in more than two consecutive experiments. The animals were then placed on a feeding schedule of two 3 hr periods of food availability per day. Food was weighed in tared dishes and placed in the cages from 8: 30 to I 1:30 a.m. and from 5:30 to 8: 30 p.m. At the end of each feeding period, the uneaten food was carefully collected and weighed and the intake recorded. It was assumed that any significant reduction in food intake during any portion of the 3 hr feeding period would be reflected in the total food intake at the end of the feeding period. This feeding schedule was begun at least 4 feeding periods prior to each experiment to re-adapt the animals, and it was continued for 3 to 6 feeding periods postadministration. IP or IG administration of test solutions began at
5:00 p.m. and was completed by 5:30 p.m. At the end of each experiment the animals were returned to ad lib feeding for at least 4 days to allow normalization of food intake before the next experiment was begun. Twenty different male Sprague-Dawley rats (Group B) with a mean body weight of 268 -+ 12.4 g were used to determine changes in blood glucose levels induced by IP or IG administration of glucose and the IP injection of mannitol. Group B animals were used for assay of blood glucose levels only, and Group A animals for intake experiments only. Both procedures were not performed simultaneously. Group B animals were kept in groups of 5 in four aluminum cages measuring 30 by 50 by 15.5 cm with lighting and temperature conditions as previously noted for Group A. These animals had water and Purina Laboratory Chow ad lib. Each of the Group B animals used for the determination of blood glucose levels was placed in a rat restrainer constructed out of a 6.4 cm dia. plastic tube with a wire mesh covering over one end. The tip of the restrained animal's tail was quickly cut with a razor blade and the tail gently stroked. Approximately 50 ~1 of blood were collected in a capillary tube for each sample and immediately centrifuged to obtain cell-free plasma. Ten/al of the blood plasma were used in a coupled glucose-oxidase-peroxidase reaction as modified by W. Werner et al. [21] (Biochemica Test Kit, Boehringer Mannlieim Corporation, San Francisco, California, U.S.A.). The development of a green color in the reaction was proportional to the concentration of glucose in the plasma. The optical density of the sample was read at 650 n m on a model 240 Gilford Spectrophotometer. To Group A experimentals, 8 ml of 2, 5, 8, 12, 16, 20 or 25 percent d-glucose solutions (w/v) were administered intraperitoneally in 7 different experiments. The three highest concentration experiments were followed by the lower concentration experiments. Sufficient time elapsed between experiments to prevent conditioning. Each animal received only one injection of a particular glucose concentration. The solutions were freshly prepared, millipore filtered and sterilized prior to each trial. The controls received an injection of 8 ml of mammalian Ringer's solution at the same time that the experimentals received glucose. All solutions were maintained at a pH between 7 and 8. Blood glucose assays were performed on 3 animals from Group B for each of the 7 glucose concentrations. For each analysis, blood samples were taken 5 min prior to injection and at 5, 20, 40, 60, 90, 120, 150 and 180 min postinjection. In three additional experiments, experimental animals from Group A received IP injections of 8 ml of 16, 20 and 25 percent mannitol (Invenex), and controls received 8 ml IP of mammalian Ringer's solution. Blood glucose tolerance curves were performed on 3 animals from Group B for each concentration of mannitol. Finally, we investigated the effect of IG loads of dglucose solutions on food intake. IG loads of 8 ml of 25, 50 and 65 percent glucose were given to Group A experimentals in 4 different experiments. Controls received an IG load of 8 ml of mammalian Ringer's solution. Blood glucose analyses were done on 3 animals from Group B for each glucose concentration. During every experiment, the food intake of the experimentals after administration of solutions was compared to the food intake of the controls and to the preadministration intake of the experimentals. The choice of internal or external controls did not affect the results.
BLOOD GLUCOSE AND SATIETY
139
The Olivetti Underwood Programma 101 was used for all statistical analysis. A T-test for the significance o f the difference between two sample means was used (program No. 5.20): p < 0 . 0 5 was taken as significant. RESULTS Effect on Food Intake o f IP and IG Administration o f Glucose and IP Injections o f Mannitol. The food intakes of animals in Group A were measured at 4 feeding periods before IP or IG administrations and for 3 feeding periods postadministration. The animals were closely observed after the IP or IG administrations. They displayed no apparent abnormalities and commenced feeding immediately upon presentation o f food. Intraperitoneal glucose injections of 8 ml o f 2, 5, 8, and 12 percent did not significantly depress food intake when compared to the intake of controls or when compared to the preinjection intake of the experimentals (Fig. 1). A significant depression o f food intake was observed, however, after IP injections of 8 ml of 16, 20 and 25 percent glucose solutions (Fig. 1). F o o d intake was depressed for the first feeding period after injection o f 16 percent glucose solution (p< 0.01). Depression was observed for two periods foTlowing injection of 20 percent glucose solution (p
