Physiology & Behavior, Vol. 20, pp. 643-651. Pergamon Press and Brain Research Publ., 1978. Printed in the U.S.A
Hyperphagia and Obesity following Ventromedial Hypothalamic Lesions in Rats with Subdiaphragmatic Vagotomy BRUCE M. KING, RICHARD G. C A R P E N T E R , BARBARA A. STAMOUTSOS, LAWRENCE A. FROHMAN* AND SEBASTIAN P. GROSSMAN 1
Department o f Behavioral Sciences, Committee on Biopsychology, University o f Chicago, Chicago, IL 60637 *Division o f Endocrinology and Metabolism and Department o f Medicine University o f Chicago, Chicago, IL 60616 (Received 23 January 1978)
KING, B. M., R. G. CARPENTER, B. A. STAMOUTSOS, L. A. FROHMAN AND S. P. GROSSMAN. Hyperphagia and obesity following ventromedial hypothalamic lesions in rats with subdiaphragmatic vagotomy. PHYSIOL. BEHAV. 20(5) 643-651, 1978. - Bilateral subdiaphragmatic vagotomy chronically reduced body weight to 85-90% of sham vagotomy weight levels in female rats maintained on a standard pellet diet (observed for 114 days). Ventromedial hypothalamic lesions 70 days after vagotomy resulted in marked hyperphagia and obesity, although the increases were not as great as those following lesions in nonvagotomized animals. When the order of surgery was reversed, vagotomy reduced the body weight of obese VMH-lesioned rats to vagotomized control levels, with no evidence of recovery after 90 days. These results suggest that while enhanced vagal activity and/or vagally mediated hyperinsulinemia contribute to VMH lesion-induced overeating and weight gains, they are not necessary for the manifestation of either the hyperphagia or obesity. The importance of adaptation to the effects of vagal transections for the appearance of hypothalamic hyperphagia and obesity is discussed. Ventromedial hypothalamus
Vagusnerve
Hyperphagia
Obesity
Noting that vagotomy eliminated the hyperinsulinemia and gastric hyperacidity that is characteristic of rats with VMH lesions, Powley and Opsaht [32] suggested that VMH obesity was the result of an alteration in vagally mediated control of metabolism, particularly increased pancreatic insulin release. In support of this hypothesis it was noted that several experiments have reported that VMH-lesioned rats are hyperinsulinemic even during food restriction [17,27], and that lesioned rats pair-fed to control levels frequently display obesity [14, 15, 16]. However, while VMH obesity can be prevented by prior destruction of pancreatic beta cells [7, 43, 44], substantial weight gains have been observed in diabetic VMH-lesioned rats (compared to nonlesioned diabetic animals) during controlled insulin administration [7, 41, 44], demonstrating that VMH obesity is not entirely the result of hyperinsulinemia. Evidence that the metabolic alterations characteristic of VMH obesity can develop in the absence of hyperinsulinemia has also been presented [ 10]. In addition to impaired gastric motility, which no doubt
IT HAS recently been found that the subdiaphragmatic vagotomy completely eliminates ventromedial hypothalamic obesity in male and female rats [18, 32, 34], confirming an earlier report by Brooks, Lockwood and Wiggins [2]. Inoue and Bray [18] observed that pair-feeding nonvagotomized VMH-lesioned rats with vagotomized animals similarly abolished obesity, and suggested that the loss in body weight following vagotomy was primarily due to a decrease in food intake. Vagotomized animals show evidence of severe gastrointestinal dysfunction, displaying distended stomachs even after 2 4 h r food deprivation [18]. The decreased food consumption in VMH rats, however, does not appear to be due to impaired gastric motility imposing an upper limit on food intake, for vagotomy does not reverse the excess body weight of genetically obese [30] or ovariectomized animals [34]. Moreover, VMH-lesioned rats with vagotomy have been reported to compensate for a cellulose-diluted diet with an increase in food intake [33] and display overeating when offered a high-fat diet [32,42].
This research was supported by USPHS grants MH 26934 to Dr. Grossman and AM 17046 and AM 20595 to Dr. Frohman. Requests for reprints should be addressed to S. P. Grossman, Department of Behavioral Sciences, University of Chicago, 5848 South University Avenue, Chicago, IL 60637. 643
KING ET AL.
644 imposes some level of discomfort, vagotomized animals have difficulty swallowing [4] and show a history of recovery similar to that following lesions of the lateral hypothalamus [36]. The difficulty in swallowing is more severe the higher the transection [4] and impairs water as well as food intake [4,23]. Apparently overlooked in the discussion of altered vagal control of metabolism as the cause of weight losses [32] is the fact that VMH-lesioned animals are hyperreactive to a variety of dietary manipulations. When offered a quinine-adulterated or unpalatable diet, rats with VMH lesions are anorexic and defend their body weight only at the same subnormal body weight levels as control animals [5,6]. Similarly, the otherwise hyperphagic lesioned animals display marked decreases in food intake when required to work for food [22, 28, 40]. It is possible, therefore, that vagotomy eliminates VMH overeating (and thus obesity) at least in part because of an overreactivity to the subsequent difficulty and discomfort experienced in eating. As both finickiness and unwillingness to work for food are substantially alleviated by preoperative adaptation to the unpalatable diet or work requirement [21, 22, 38], the present experiment examined the possibility that the effects of vagotomy in VMH-lesioned rats would be similarly attenuated by transecting the vagus prior to VMH lesions.
GE N E R A L METHOD
Animals Sixty female Long-Evans hooded rats (Simonsen Laboratories, Gilroy, CA) were used. The animals were 1 0 0 - 1 2 0 days old and weighed 2 7 0 - 3 2 5 g at the beginning of the experiment. All animals were individually caged in a temperature controlled colony ( 2 1 - 2 4 ° C ) with a 1 2 h r light/dark cycle throughout the course of the experiment.
VMH Lesion Bilateral ventromedial hypothalamic lesions were produced under sodium pentobarbital (Nembutal) anesthesia (5 0 m g / k g ) by passing a 1.5 mA anodal current between the 0.5 mm uninsulated tip of a teflon insulated stainless steel electrode (No. 0 insect pin) and a rectal cathode for 20 sec. With the upper incisor bar positioned 5 mm above the interaural line, the electrodes were stereotaxically positioned 0.8 mm posterior to bregma, 0.7 mm lateral to the midsagittal suture, and 1 0 . 0 m m below the surface of the skull. As newly lesioned animals often attempt to eat before fully recovering from the anesthesia [2], sometimes resulting in death by choking, all rats were deprived of food for the first 12 hr following surgery. Upon completion of the experiment, animals with VMH lesions were anesthetized and intracardially perfused with isotonic saline followed by a 10% formol saline solution. Histological analysis was performed by light microscopic examination of cresyl violet stained 80 u coronal sections, cut on a freezing microtome. The atlas of Pellegrino and Cushman [31] was used in estimating the extent of the lesions.
Vago to my Bilateral subdiaphragmatic vagotomies were performed
by making a 3 cm midline incision directly inferior to the sternum. The stomach was pulled gently toward the posterior and the lobes of the liver held aside in order to expose the esophagus. With the aid of a dissecting microscope, the two subdiaphragmatic branches of the vagus were separated from the esophagus and severed. The esophagus was then carefully examined with the dissecting microscope and any smaller trunks of the vagus were additionally severed. Great care was taken not to damage the esophagus. In animals with sham vagotomy the vagus was separated from the esophagus but not transected.
Tests for Completeness o f Vagotomy Our experience with the gastric acid secretion test used by others [18,32] has shown it to be a highly unreliable test for completeness of vagotomy. In a pilot study, we performed deliberately incomplete vagotomies in which one small branch of the anterior vagus (innervating the stomach) was spared. Such rats did not show a measurable increase in gastric HC1 secretion during electrical stimulation of the cervical vagus. It should be noted that VMH-lesioned rats classified as incompletely vagotomized by Inoue and Bray [18] similarly did not display a significant increase in HC1 secretion over basal levels in response to stimulation of the cervical vagus. Powley and Opsahl [32] did not report any data for their incompletely vagotomized group. Our rats that were given incomplete vagotomies (and classified as complete by the HC1 test) did show a small area of remaining vagal innervation on a neutral red test. Therefore, this test, as described by Legros and Griffith [25], was used in all our animals as a necessary criterion for the success of our operations. Animals were deprived of food for 24 hr prior to the neutral red test. They were then anesthetized and their stomachs exposed, opened, and washed with saline warmed to 37°C. White filter paper was then held in contact with the mucosal surface of the corpus and neutral red (1 ml of a 1% solution) injected into the jugular vein. After 15 rain the filter paper was examined for evidence of neutral red, which is secreted from the area of the corpus with remaining vagal innervation. In addition to the neutral red test, we attempted to demonstrate the absence of vagally mediated insulin secretion in some animals by stimulating the cervical vagi under pentobarbital anesthesia (40 mg/kg) and collecting heparinized blood samples for measurement of plasma insulin [8]. Rats were adrenalectomized immediately before the test in order to eliminate the suppression of insulin secretion by adrenal catecholamines. The change in plasma insulin levels five rain after the onset of vagal stimulation was significantly greater in intact animals (N = 5, • plasma insulin, uU/ml, mean _+ SEM = 7.4 + 4.3) than in those classified as having been completely vagotomized using other criteria (N = 6, 8.2 ± 3.9) (p
Hyperphagia and Obesity following Ventromedial Hypothalamic Lesions in Rats with Subdiaphragmatic Vagotomy BRUCE M. KING, RICHARD G. C A R P E N T E R , BARBARA A. STAMOUTSOS, LAWRENCE A. FROHMAN* AND SEBASTIAN P. GROSSMAN 1
Department o f Behavioral Sciences, Committee on Biopsychology, University o f Chicago, Chicago, IL 60637 *Division o f Endocrinology and Metabolism and Department o f Medicine University o f Chicago, Chicago, IL 60616 (Received 23 January 1978)
KING, B. M., R. G. CARPENTER, B. A. STAMOUTSOS, L. A. FROHMAN AND S. P. GROSSMAN. Hyperphagia and obesity following ventromedial hypothalamic lesions in rats with subdiaphragmatic vagotomy. PHYSIOL. BEHAV. 20(5) 643-651, 1978. - Bilateral subdiaphragmatic vagotomy chronically reduced body weight to 85-90% of sham vagotomy weight levels in female rats maintained on a standard pellet diet (observed for 114 days). Ventromedial hypothalamic lesions 70 days after vagotomy resulted in marked hyperphagia and obesity, although the increases were not as great as those following lesions in nonvagotomized animals. When the order of surgery was reversed, vagotomy reduced the body weight of obese VMH-lesioned rats to vagotomized control levels, with no evidence of recovery after 90 days. These results suggest that while enhanced vagal activity and/or vagally mediated hyperinsulinemia contribute to VMH lesion-induced overeating and weight gains, they are not necessary for the manifestation of either the hyperphagia or obesity. The importance of adaptation to the effects of vagal transections for the appearance of hypothalamic hyperphagia and obesity is discussed. Ventromedial hypothalamus
Vagusnerve
Hyperphagia
Obesity
Noting that vagotomy eliminated the hyperinsulinemia and gastric hyperacidity that is characteristic of rats with VMH lesions, Powley and Opsaht [32] suggested that VMH obesity was the result of an alteration in vagally mediated control of metabolism, particularly increased pancreatic insulin release. In support of this hypothesis it was noted that several experiments have reported that VMH-lesioned rats are hyperinsulinemic even during food restriction [17,27], and that lesioned rats pair-fed to control levels frequently display obesity [14, 15, 16]. However, while VMH obesity can be prevented by prior destruction of pancreatic beta cells [7, 43, 44], substantial weight gains have been observed in diabetic VMH-lesioned rats (compared to nonlesioned diabetic animals) during controlled insulin administration [7, 41, 44], demonstrating that VMH obesity is not entirely the result of hyperinsulinemia. Evidence that the metabolic alterations characteristic of VMH obesity can develop in the absence of hyperinsulinemia has also been presented [ 10]. In addition to impaired gastric motility, which no doubt
IT HAS recently been found that the subdiaphragmatic vagotomy completely eliminates ventromedial hypothalamic obesity in male and female rats [18, 32, 34], confirming an earlier report by Brooks, Lockwood and Wiggins [2]. Inoue and Bray [18] observed that pair-feeding nonvagotomized VMH-lesioned rats with vagotomized animals similarly abolished obesity, and suggested that the loss in body weight following vagotomy was primarily due to a decrease in food intake. Vagotomized animals show evidence of severe gastrointestinal dysfunction, displaying distended stomachs even after 2 4 h r food deprivation [18]. The decreased food consumption in VMH rats, however, does not appear to be due to impaired gastric motility imposing an upper limit on food intake, for vagotomy does not reverse the excess body weight of genetically obese [30] or ovariectomized animals [34]. Moreover, VMH-lesioned rats with vagotomy have been reported to compensate for a cellulose-diluted diet with an increase in food intake [33] and display overeating when offered a high-fat diet [32,42].
This research was supported by USPHS grants MH 26934 to Dr. Grossman and AM 17046 and AM 20595 to Dr. Frohman. Requests for reprints should be addressed to S. P. Grossman, Department of Behavioral Sciences, University of Chicago, 5848 South University Avenue, Chicago, IL 60637. 643
KING ET AL.
644 imposes some level of discomfort, vagotomized animals have difficulty swallowing [4] and show a history of recovery similar to that following lesions of the lateral hypothalamus [36]. The difficulty in swallowing is more severe the higher the transection [4] and impairs water as well as food intake [4,23]. Apparently overlooked in the discussion of altered vagal control of metabolism as the cause of weight losses [32] is the fact that VMH-lesioned animals are hyperreactive to a variety of dietary manipulations. When offered a quinine-adulterated or unpalatable diet, rats with VMH lesions are anorexic and defend their body weight only at the same subnormal body weight levels as control animals [5,6]. Similarly, the otherwise hyperphagic lesioned animals display marked decreases in food intake when required to work for food [22, 28, 40]. It is possible, therefore, that vagotomy eliminates VMH overeating (and thus obesity) at least in part because of an overreactivity to the subsequent difficulty and discomfort experienced in eating. As both finickiness and unwillingness to work for food are substantially alleviated by preoperative adaptation to the unpalatable diet or work requirement [21, 22, 38], the present experiment examined the possibility that the effects of vagotomy in VMH-lesioned rats would be similarly attenuated by transecting the vagus prior to VMH lesions.
GE N E R A L METHOD
Animals Sixty female Long-Evans hooded rats (Simonsen Laboratories, Gilroy, CA) were used. The animals were 1 0 0 - 1 2 0 days old and weighed 2 7 0 - 3 2 5 g at the beginning of the experiment. All animals were individually caged in a temperature controlled colony ( 2 1 - 2 4 ° C ) with a 1 2 h r light/dark cycle throughout the course of the experiment.
VMH Lesion Bilateral ventromedial hypothalamic lesions were produced under sodium pentobarbital (Nembutal) anesthesia (5 0 m g / k g ) by passing a 1.5 mA anodal current between the 0.5 mm uninsulated tip of a teflon insulated stainless steel electrode (No. 0 insect pin) and a rectal cathode for 20 sec. With the upper incisor bar positioned 5 mm above the interaural line, the electrodes were stereotaxically positioned 0.8 mm posterior to bregma, 0.7 mm lateral to the midsagittal suture, and 1 0 . 0 m m below the surface of the skull. As newly lesioned animals often attempt to eat before fully recovering from the anesthesia [2], sometimes resulting in death by choking, all rats were deprived of food for the first 12 hr following surgery. Upon completion of the experiment, animals with VMH lesions were anesthetized and intracardially perfused with isotonic saline followed by a 10% formol saline solution. Histological analysis was performed by light microscopic examination of cresyl violet stained 80 u coronal sections, cut on a freezing microtome. The atlas of Pellegrino and Cushman [31] was used in estimating the extent of the lesions.
Vago to my Bilateral subdiaphragmatic vagotomies were performed
by making a 3 cm midline incision directly inferior to the sternum. The stomach was pulled gently toward the posterior and the lobes of the liver held aside in order to expose the esophagus. With the aid of a dissecting microscope, the two subdiaphragmatic branches of the vagus were separated from the esophagus and severed. The esophagus was then carefully examined with the dissecting microscope and any smaller trunks of the vagus were additionally severed. Great care was taken not to damage the esophagus. In animals with sham vagotomy the vagus was separated from the esophagus but not transected.
Tests for Completeness o f Vagotomy Our experience with the gastric acid secretion test used by others [18,32] has shown it to be a highly unreliable test for completeness of vagotomy. In a pilot study, we performed deliberately incomplete vagotomies in which one small branch of the anterior vagus (innervating the stomach) was spared. Such rats did not show a measurable increase in gastric HC1 secretion during electrical stimulation of the cervical vagus. It should be noted that VMH-lesioned rats classified as incompletely vagotomized by Inoue and Bray [18] similarly did not display a significant increase in HC1 secretion over basal levels in response to stimulation of the cervical vagus. Powley and Opsahl [32] did not report any data for their incompletely vagotomized group. Our rats that were given incomplete vagotomies (and classified as complete by the HC1 test) did show a small area of remaining vagal innervation on a neutral red test. Therefore, this test, as described by Legros and Griffith [25], was used in all our animals as a necessary criterion for the success of our operations. Animals were deprived of food for 24 hr prior to the neutral red test. They were then anesthetized and their stomachs exposed, opened, and washed with saline warmed to 37°C. White filter paper was then held in contact with the mucosal surface of the corpus and neutral red (1 ml of a 1% solution) injected into the jugular vein. After 15 rain the filter paper was examined for evidence of neutral red, which is secreted from the area of the corpus with remaining vagal innervation. In addition to the neutral red test, we attempted to demonstrate the absence of vagally mediated insulin secretion in some animals by stimulating the cervical vagi under pentobarbital anesthesia (40 mg/kg) and collecting heparinized blood samples for measurement of plasma insulin [8]. Rats were adrenalectomized immediately before the test in order to eliminate the suppression of insulin secretion by adrenal catecholamines. The change in plasma insulin levels five rain after the onset of vagal stimulation was significantly greater in intact animals (N = 5, • plasma insulin, uU/ml, mean _+ SEM = 7.4 + 4.3) than in those classified as having been completely vagotomized using other criteria (N = 6, 8.2 ± 3.9) (p
