Physioh~,gy & Beharior, Vol. 22, pp. 85--93. Pergamon Press and Brain Re,arch Publ., 1979. Printed in the U.S.A.

Development of VMH Obesity in Vagotomized Rats R I C H A R D S. W A M P L E R

Psychology Department, Indiana University-Purdue University, Fort Wayne, IN 46805 AND C H A R L E S T. S N O W D O N

Psychology Department, University of Wisconsin, Madison, WI 53706 (Received 29 July 1977) WAMPLER, R. S. AND C. T. SNOWDON. Development of VMH obesity in vagotomized rats. PHYSIOL. BEHAV. 22(!) 85-93, 1979.mln 3 different experiments, vagotomy failed to reverse or prevent the development of hyperphagia and obesity following VMH lesions. In the first experiment, previously vagotomized male rats were given bilateral VMH lesions after testing for completeness of vagotomy. In a second experiment on male rats, vagotomy preceded or followed VMH lesions, and testing for completeness of vagotomy was not carded out until the end of the experiment. In a third experiment obese VMH female rats were vagotomized before or after VMH lesions, and completeness of vagotomy was assessed histologically. In each experiment, vagotomized VMH rats displayed hyperlahagiaand became obese. Discrepancies between these results and those reported by others are discussed in terms of diet offered, surgical procedures, and the possibility of continuing malaise after vagotomy. Vagotomy

Obesity

Ventromedialhypothalamus

RECENTLY, there have been several reports that the obesity which follows damage to the ventromedial nucleus of the hypothalamus (VMH) can be reversed or prevented by subdiphragmatic vagotomy [4, 13, 21, 22, 26, 27]. Since obese Zucker rats (fatty) do not show this weight loss after vagotomy, it appears that their genetic obesity is not the result of changes in vagai function [20]. These studies have attracted interest because they provide a potentially simple explanation of VMH obesity (i.e., increased insulin secretion which results in hyperphagia and consequent obesity) which does not require hypothetical changes in body weight setpoint or modification of some presumed hypothalamic glucostatic or lipostatic mechanism. Additional evidence that VMH obesity depends upon vagally-mediated hyperinsulinemia has been provided by Inoue, Bray, and Mullen [14]. Using inbred Lewis rats, they destroyed the beta cells of the pancreas with streptozotocin and, then, transplanted fetal pancreatic tissue under the capsule of the kidney. The graft became functional but remained free from neural control. In animals with successful grafts, the blood glucose level returned to normal. Obesity did not

VMH

develop after VMH lesions in such animals, and the investigators concluded that the development of VMH obesity depends upon a neural signal, via the vagus or sympathetic system (no distinction could be made between these two routes). Unfortunately, no attempt was made to assess whether the grafted pancreatic tissue was capable of secreting sufficient insulin to handle the large quantities of food normally ingested by VMH rats (Bray, personal communication). Thus, this study does not provide the crucial evidence that neural release of insulin is critical for the development of VMH obesity. Although the hypothesis that vagotomy blocks hyperinsulinemia in VMH rats is attractive in its simplicity, the cause of VMH hyperinsulinemia is not clear cut. Dynamic and static-phase obese VMH rats are hyperinsulinemic, as are rats which are pair-fed with normal rats or force-fed amounts of food eaten by normal rats [6, 7, 9, 10, 11]. On the other hand, hyperinsulinemia does not develop in fasting VMH rats during the first day after VMH lesions [17], a period in which they are commonly hyperphagic. Although hyperinsulinemia has been demonstrated in VMH rats on

'We thank Michael Murry, Jon Dull, Kathleen Schweitzer, and Judith Hollander for assistance in conducting these studies. Sirra

Ranasinghe, M.D., pathologist at St. Joseph's Hospital, Ft. Wayne, sectioned, stained and evaluated the esophagi from animals in Experiment 3. This research was supported by U.S.P.H.S. Grant AM 17,171 and grants from the Nutrition Foundation (428 and 483). Manuscript preparation was carried out while the first author was a Purdue University XL and Summer Research Grant Fellow. Purified vegetable fats used in the experiments were supplied by Dr. Steven A. Ziller, Procter and Gamble. Reprint address: R. S. Wampler, Psychology, IU-PU, Ft. Wayne, IN 46805.

Copyright ~ 1979 Brain Research Publications Inc.--0031-9384/79/010085-09502.00/0

86

W A M P L E R AND SNOWDON TABLE 1 SEQUENCE OF PROCEDURES IN EACH EXPERIMENT Experiment

Group

1

1

Vagotomy - (2 weeks) fistulation - (1-2 weeks) testing (4-5 weeks) VMH lesions - (74 days) termination

2

Vagotomy - (2 months) fistulation - (1-2 weeks) testing (6-7 weeks) VMH lesions - (135 days) termination

control 2

3

Sequence (time between procedures in parentheses)

entry - (136 days) termination

VMH-vag

VMH lesions - (6 weeks) deprivation - (6 weeks) vagotomy (6 weeks) deprivation - (2 weeks) fistulation - (1-2 weeks) testing (3 weeks) termination

vag-VMH

Vagotomy - (3.5 weeks) VMH lesions - (6 weeks) deprivation (2 weeks) fistulation - ( 1 - 2 weeks) testing - (3 weeks) termination

VMH-vag

VMH lesions - (1 month) vagotomy - (2 months) restricted intake (2 months) refeeding - (2 months) termination

vag-VMH

Vagotomy - (1 month) VMH lesions - (1 month) restricted intake (5 months) refeeding - (2 months) termination

restricted rations [12,18], it can be reversed when food intake is more severely restricted and the VMH rat is losing weight [13]. Clearly, the availability o f insulin is a limiting factor in the development o f V M H obesity since diabetic V M H rats do not become obese [5, 8, 34, 35]; however, diabetic rats on insulin replacement treatment become obese when compared to appropriate control rats [5, 33, 34]. Thus, the hyperinsulinemia found in freely-feeding VMH rats may reflect increased insulin secretion in response to increased food intake and/or increased secretion as body fat stores increase and insulin resistance develops in addition to neurally.mediated insulin release. W e have been unable to confirm the general finding that vagotomy reverses or blocks the development o f obesity in rats with V M H lesions. The studies reported here have been conducted over a period ot'6 years at diffe~nt laboratories and with different experimenters. In each study, vagotomy did not block the development o f VMH obesity nor did it reverse an already existing obese state. Some, but not all, of our results have been confirmed in a recent study [ 15] which was conceived and carried out independently of the present research. EXPERIMENT 1

Method AnimaLs'. Experimental animals in the first study were 32 male rats (CD strain, Charles River) approximately 4 months of age at the time o f vagotomy. Male albino rats (N =29) of the same strain served as intact controls. These control rats were approximately 6 months o f age when introduced into the experiment, an age which fell between the ages o f the two experimental groups at the time o f their VMH lesions. Vagotomy. The sequence of procedures for all experiments is outlined in Table 1. All surgical procedures were carried out under sodium pentobarbital anesthesia (Nembutal, 45 mg/kg). Vagotomies were performed on all experimental animals within a 4-day period using S n o w d o n ' s procedure 128-301. Vagotomy was performed by making a 4 cm incision directly inferior to the sternum. The stomach was

lifted gently through this opening and covered with gauze pads soaked in normal saline. The esopham..,,us was lifted and held outside the peritoneum using a pair o f large curved forceps slipped beneath the esophagus. Using fine dissecting forceps, the gastric artery and vein were stripped from the side of the esophagus, and the anterior and posterior branches of the vagus were removed for a length o f at least 1.5 cm anteriorly from the esophageal-gastric junction. All visible branches of the vagi were stripped also, but a dissecting microscope was not employed. The entire procedure required less than 20 rain for completion. Further procedural details are given in [29]. The vagotorny procedure eliminated both the gastric and coeliac portions o f the vagat ~ t d b u t i o n since vagal removal began at the e s o p h a p a i , g a s t r i c j u n c t i o n and continued anteriorly as close as possible to the diaphragm. No attempt was made to visualize the hepatic branch o f the vagus and the lobes o f the liver were not reflected out of the peritoneal cavity althoullh the esopheg0s was stretched gently with the large forceps to increase the length o f the esophagus which could be visualized. Completeness of vagotomy was determined using the stomach acid secretion test of Snowdon and Epstein [30]. At present. there is no reliable, independent test of completeness o f removal of the cocliac or hepatic branches o f the vagus [15]. While it is possible to selectively denervate the stomach [16. 26. 271, the surgical procedure e m p l o y e d would not selectively spare the coeliac branch and was similar to vagotomy procedures reported by others [4, 13, 15, 20-22, 26-30]. Fistulation. in Group 1, stainless steel gastric fistulas (modified from [29]) were implanted 2 weeks after surgery, and tests for the completeness o f vagotomy began 1-2 weeks later. In Group 2, fistulas were not installed until 2 months following vagotomy. The difference in time of installation of the fistulas provided a control for the possibility o f vagal regrowth "after tests in Group I. To test for completeness o f vagotomy, the animals were deprived o f food for 24 hr. At 9 a.m. the fistula was opened. and 25-50 mi o f warmed distilled water was flushed through the opening o f the fistula. An additional 25-50 ml was flushed through the stomach via an oral-gastric tube (French No. 8

VAGOTOMY AND VMH OBESITY

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FIG. I. Body weight (g) of vagot0mized-VMH animals (Group 1, solid squares; Group 2, open squares)

and of control animals (high-fat diet, open circles; pellet diet, solid circles).

feeding tube) until no particles could be washed out. An extension tube was fitted in the threaded fistula, a flexible plastic tube attached to the extension, and the stomach contents were allowed to drain for 1 hr, In insulin tests, 1 U/kg body weight of fast-acting crystalline (regular) insulin was injected subcutaneously. In saline tests, an equal volume of 0.9% saline was injected. Gastric secretions were collected for 4 hr in a small, lightweight plastic bottle. The collected secretions were neutralized with 0.05 M NaOH using a phenol red indicator. At least 3 insulin and 2 saline tests were conducted. Vagotomy was considered complete if (a) the animal secreted less than 250/~Eq of H + in 4 hr after insulin injection, and (b) if there was no substantial difference in rate of I-I÷ secretion between saline and insulin tests. In order for an animal to be considered vagotomized, both criteria had to be met [29,30]. VMH lesions. Bilateral VMH lesions were made 2 months (Group 1) or 4 months (Group 2) after vagotomy when the tests for completeness of vagotomy were ended. Lesion parameters were 2 mA anodal DC current passed for 15 sec with the electrode positioned 2.4 mm behind bregma, 0.6-0.7 mm lateral to the midline sinus, and 1 mm dorsal to the floor of the skull. The electrode was an Epoxylite-insulated, 0.36 mm dia. stainless steel orthodonic wire exposed in a crosssection of the tip. Diet. Prior to VMH lesions, experimental animals were maintained on a pelleted diet (Purina Lab Chow) ad lib. To ensure that sudden hyperphagia following VMH lesions would not result in damage to the fistulated stomach, a restricted amount of pellets was given for 8 days after VMH lesions (10 g twice a day). Following this 8-day period, highfat diet, consisting of 2 parts ground Purina Lab Chow to 1 part solidified vegetable fat [2], was available ad lib for 66 (Group 1) or 127 (Group 2) additional days. There were 7 rats with confirmed vagotomies in Group 1, 5 in Group 2. Control

animals (intact) were kept on restricted amounts of pellets for 8 days, and then given ad lib access to pellets (N= 15) or high-fat diet (N= 14) for an additional 128 days. At the end of the experiment, experimental animals were anesthetized with Nembutal and peffused intracardially with 0.9% saline followed by 10% Formalin. Brains were removed immediately after perfusion and stored in 10% Formalin. After embedding in celloidin, 40 ~t sections were taken through the extent of the lesion and stained with cresyl violet. Results

The effect of VMH lesions on body weight of vagotomized animals is summarized in Fig. 1. Following the 8-day period of restricted intake, weight gain was rapid in all groups; however, rats in the vagotomized-VMH groups gained significantly more weight after this initial period ofad lib feeding. Data from the 2 groups of vagotomized-VMH rats were pooled because the weights were not significantly different beyond 20-30 days after lesions. Compared to the body weight of control rats in the high fat group, the weights of the combined group of vagotomized-VMH rats did not differ on the day of surgery nor at the end of the &.day period of restricted intake. However, VMH rats were significantly heavier 47 (approximately 1 month after ad lib access began), 74 (last day for Group 1), and 131 (last week for Group days after VMH lesions (Mann-Whitney U tests: U(12,14)=19, p