gold thioglucose-induced hypothalamic damage, hyperphagia, and obesity: dependence on the adrenal...

0013-7227/82/1106-2024$02.00/0EndocrinologyCopyright © 1982 by The Endocrine Society

Vol. 110, No. 6Printed in U.S.A.

Gold Thioglucose-Induced Hypothalamic Damage,Hyperphagia, and Obesity: Dependence on theAdrenal Gland*A. F. DEBONS, E. SICLARI, K. C. DAS, AND B. FUHR

Nuclear Medicine Service, Veterans Administration Medical Center, and The State University of NewYork, Downstate Medical Center, Brooklyn, New York 11209

ABSTRACT. Previous studies from our laboratory suggestedthat adrenal hormones may participate, directly or indirectly, inthe hypothalamic mechanism involved in the regulation of foodintake. In the present studies, the effect of adrenalectomy on thedevelopment of gold thioglucose (GTG)-induced hyperphagiaand obesity in mice was investigated. As expected, damage tothe ventromedial hypothalamus by GTG was followed by hy-perphagia and obesity. Ablation of the adrenal glands after theadministration of GTG prevented the onset and development ofhyperphagia and obesity. The administration of cortisone com-

pletely restored the hyperphagia and weight gain of GTG-treatedadrenalectomized mice. The administration of desoxycorticos-terone not only failed to restore the hyperphagia and obesity inthese mice but, rather, led to a suppression of food intake, weightloss, and death. It is concluded that 1) GTG-induced hypotha-lamic hyperphagia and obesity are dependent on adrenal gluco-corticoids, and 2) the ability of adrenal glucocorticoids to restorehyperphagia and obesity in hypothalamic lesioned adrenalecto-mized mice indicates that adrenal glucocorticoids can act oncomponents outside of the ventromedial hypothalamus involvedin the control of food intake. {Endocrinology 110: 2024, 1982)

THE ADMINISTRATION of gold thioglucose(GTG) to mice induces bilateral necrosis of a cir-

cumscribed region of the ventromedial hypothalamus(VMH) (1). This lesion, like that induced by electrocau-tery in this area in rats (2, 3), leads to hyperphagia andobesity (1-5). We have established that the sensitivity ofthe VMH to GTG-induced necrosis is dependent oninsulin (6), and the administration of insulin, either iv ordirectly, into the VMH of alloxan-diabetic mice canrestore the sensitivity of the VMH to GTG (7, 8). Wehave also found that adrenalectomy, which is known todiminish the effects of insulin deficiency on carbohydratemetabolism, can restore the sensitivity of the VMH ofalloxan-diabetic mice to GTG and reduce the elevatedfood intake of these mice to normal levels (9). When theadrenal status of alloxan-diabetic adrenalectomized micewas restored by the administration of adrenal glucocor-ticoids, resistance of the VMH to GTG necrosis (9) andhyperphagia were found to return. More recently, wehave found that under special conditions in which GTG-induced damage to the VMH is restricted to neuralelements in the area (in the absence of general necrosis),

Received August 10, 1981.Address requests for reprints to: Dr. A. F. Debons, Veterans Admin-

istration Medical Center, Nuclear Medicine Service, 800 Poly Place,Brooklyn, New York 11209.

* This work was supported by V.A. Medical Research Funds MRIS1788 and USPHS Grant AM-12479.

the administration of adrenal glucocorticoids blockedthese changes (10). These findings strongly suggest thatadrenal hormones may be acting directly at the level ofthe VMH in altering the sensitivity of the VMH to GTGas well as feeding behavior. The possibility remains,nevertheless, that adrenal hormones may also be actingon areas outside of the VMH, either in the centralnervous system or at peripheral sites, in bringing aboutchanges in feeding behavior.

In the present studies, we sought to determine whetherthe hyperphagia resulting from GTG-induced damage tothe VMH, like the hyperphagia accompanying insulindeficiency, was dependent on the adrenal gland. Thesestudies, it was thought, might enable us to discernwhether the hyperphagia of diabetes, which is dependenton the adrenal gland in the presence of an intact VMH,was different from that resulting from damage to theVMH which might be expected to be independent of theadrenal gland.

Materials and Methods

Animals

Female CF1 mice, approximately 3 months of age, obtainedfrom Charles River Breeding Laboratories, Inc. (Wilmington,MA), were used in these studies. They were maintained onPurina mouse chow and given tap water freely. They were

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ADRENAL DEPENDENCY OF GTG OBESITY 2025

housed in a constant temperature room maintained at 22.5 Cwith a 12-h light and 12-h dark cycle.

Drugs and chemicals

Sodium pentobarbital (100 mg/kg) used for anesthesia, wasgiven in a single ip injection as a 10 mg/ml aqueous solution.

GTG (800 mg/kg) was given in a single ip injection as an 80mg/ml aqueous solution. This material was supplied to us bySchering Corp. (Bloomfield, NJ). Cortisone acetate (Cortone,Merck, Sharp, and Dohme, West Point, PA; 20 mg/kg) wasgiven ip as a 2 mg/ml suspension in isotonic saline.

Desoxycorticosterone pivalate (Percorten, Ciba Pharmaceu-tical Co., Summit, NJ; 0.3 mg/kg) was given ip as a 0.03 mg/mlsuspension in isotonic saline.

Presence of GTG-induced lesions in the VMH

At the completion of the studies, GTG-treated and controlmice were sacrificed by decapitation. The brain of each mousewas carefully removed from the skull, fixed overnight in Bouin'sfluid, processed histologically, and subsequently examined forthe presence of lesions in the VMH. The remaining carcassesfrom some of the animals in each of the experimental groupswere assayed for total lipid content.

Measurement of lipid content of decapitated carcasses

After mice were decapitated, the remaining carcasses of someof the mice in each of the experimental groups were stripped ofthe gastrointestinal tract, minced, and homogenized in a chlo-roform-methanol solution (2:1) in a Waring blender (model CB-

6). The homogenates were then treated, in accord with theprocedures described by Folch et al. (11), for measurement oftotal lipids.

Adrenalectomy procedure

Control and GTG-treated mice were anesthetized with so-dium pentobarbital. The mice were then adrenalectomized bythe lumbar approach. The excised adrenal glands were fixed in100 /ul/ml buffered formalin for subsequent gross examinationof the adrenal under a dissection microscope. Sham-adrenal-ectomized experimental and control mice were exposed to thesame operative procedure, except for removal of the glands.GTG-treated mice were adrenalectomized 10 days after treat-ment with GTG. Adrenalectomy was conducted at this time toallow the mice to recover from the transient inanition and lossof body weight which occurs during the first week after theinjection of GTG. All adrenalectomized mice were suppliedwith a 9 mg/ml NaCl solution in place of tap water for drinking.Adrenalectomized mice, when administered cortisone or desox-ycorticosterone, were maintained on tap water for drinking.

Food intake

Mice in all of the experimental groups were housed in indi-vidual cages, and food intake was measured daily.

Results

Effect of adrenalectomy on GTG-induced hypothalamichyperphagia and obesity

Thirty-four mice were injected with GTG. Ten dayslater, at a time when the mice began to exhibit a pro-nounced hyperphagia and weight gain, half of the micewere adrenalectomized. Control groups, consisting of 12untreated and 12 adrenalectomized mice, were also pre-pared. Two additional groups, consisting of 12 sham-adrenalectomized and 12 GTG-treated sham-adrenalec-tomized mice, were followed to rule out any effects of theoperative procedure. Food intake and body weights wererecorded daily.

The results of this experiment are shown in Fig. 1.Mice treated with GTG exhibited a significant (P < 0.01)transient reduction in food intake and loss in body weightduring the first week after treatment. However, by the10th day, the GTG-treated mice had regained the weightthey had lost and began to exhibit hyperphagia. Asexpected, all of the mice treated with GTG developedhyperphagia and obesity. Adrenalectomy prevented theonset of hyperphagia and the consequent obesity in 14(-85%) of the 17 GTG-treated mice. It is particularlynoteworthy that 4 (-29%) of the GTG-treated adrenal-ectomized nonobese mice developed anorexia, lostweight, and died. The death of these animals did notappear to be due to surgical complications followingadrenalectomy, since we did not experience any mortalityin the adrenalectomized control group. The food intakeand body weights of the nonobese, GTG-treated adre-nalectomized mice were not significantly different fromthose of the adrenalectomized control mice. Three of theGTG-adrenalectomized mice which became obese ex-hibited variable weight gains of 8, 12, and 18 g, respec-tively. The sham-adrenalectomized control mice and theGTG-treated sham-adrenalectomized mice responded inthe same manner as their corresponding unoperatedgroups, with the exception of a slight transient decreasein food intake and body weight on the day after theoperation (results not shown).

Gross examination of the excised adrenal glands underthe dissection microscope confirmed that in the case ofthe GTG-treated adrenalectomized nonobese mice, theadrenal glands were removed intact. In the case of theobese mice, however, the adrenal glands were found tobe fragmented, and pieces of adrenal tissue may haveremained in the abdominal cavity.

The presence of bilateral lesions in the VMH wasconfirmed upon histological examination of all brains ofmice treated with GTG.

As expected, more than 80% of the increased bodyweight of the GTG-treated obese mice could be ac-counted for by an increase in carcass fat content (Table


2026 DEBONS ET AL. Endo • 1982Vol 110 • No 6

40 r

BODY WEIGHT

3 0 -

FIG. 1. Responses in body weight andfood intake of untreated, adrenalecto-mized, GTG-treated, and GTG-treatedadrenalectomized mice. GTG (800 mg/kg, ip) was given on day 0; adrenalecto-mies were performed on the 10th day.The values shown represent the meanresponse of 10-17 mice in each group.

20

10

UntreatedA drenalectomizedGold ThioglucoseGold Thioglucose6 Adrenal&ctomy

FOOD INTAKE

10 15 20 25DAYS

TABLE 1. Effect of adrenalectomy on weight gain and lipid content incarcasses of GTG-treated mice

TreatmentNo.of

mice

Wt gain(g)

Carcass lipidcontent (g)

None (control)GTGGTG-treated adrenalectomizedAdrenalectomized

5 1.8 ± 0.3 2.55 ± 0.235 15.1 ± 1.7° 12.10 ± 0.94"5 1.9 ± 0.6 2.44 ± 0.315 1.7 ± 0.2 2.29 ± 0.25

Values are expressed as the mean ± SEM. Mice were sacrificed 6weeks after GTG administration.

° Significantly different from the control group (P < 0.001).

1). Adrenalectomy, which prevented hyperphagia andweight gain after GTG administration, also preventedthe increase in carcass fat. Control mice that had beenadrenalectomized did not differ from untreated controlswith respect to weight gain and carcass fat content.

Effect of cortisone on the return of hyperphagia andobesity in GTG-treated adrenalectomized mice

The above findings suggest that adrenal hormones arenecessary for the development of the hypothalamic hy-perphagia and obesity induced by GTG. To determinewhether adrenal glucocorticoids are essential for thedevelopment of GTG-induced hypothalamic hyper-phagia and obesity, nonobese, GTG-treated adrenalec-tomized mice were injected daily with cortisone, and foodintake and body weights were recorded daily. The effectof cortisone on untreated and adrenalectomized controlmice was also followed.

The results of this study are shown in Fig. 2. The dailyadministration of cortisone was accompanied by a return

of hyperphagia and weight gain in the GTG-treatedadrenalectomized mice. The amount of cortisone admin-istered had no effect on the food intake or body weightof untreated (normal) and adrenalectomized (responsenot shown) control mice.

The effect of the mineralocorticoid desoxycorticosteroneon the food intake and body weight response of GTG-treated adrenalectomized mice

To exclude the possibility that prevention of GTG-induced hyperphagia and obesity by adrenalectomy wasdue in part to the loss of adrenal corticoids with miner-alocorticoid activity, GTG-treated adrenalectomizedmice were injected daily with desoxycorticosterone (0.3mg/kg). Food intake and body weight were recordeddaily. The effects of desoxycorticosterone on the foodintake and body weights of control untreated (normal)and adrenalectomized mice were also followed.

The results of these studies are shown in Fig. 3. Thedaily administration of desoxycorticosterone to GTG-treated adrenalectomized mice suppressed (P < 0.001)their food intake and led to a persistent loss in bodyweight. It is thought that the death of these mice, whichoccurred within the course of a few weeks, resulted fromthe anorexia and inability of the mice to maintain theirbody weight. The administration of desoxycorticosteronein the amounts indicated had no effect on the food intakeor body weights of untreated (normal) and adrenalecto-mized (responses not shown) control mice.

Discussion

Our studies indicate that the onset and developmentof hyperphagia and consequent obesity following damage



25

20

10

—• Normals (untreated)Normals 8 CortisoneGTG 3 AdrenalectomyGTG 3 Adrenalectomy3 Cortisone

FOOD INTAKE

FIG. 2. Effect of the daily administration of cortisone (20 mg/kg, ip)on the body weights and food intake response of normal and GTG-treated adrenalectomized mice. The response of adrenalectomized con-trol mice was not significantly different from that of normal controlsand was omitted from the figure. The values shown represent the meanresponse of 10-15 mice in each group.

35

30

25

20

10

BODY WEIGHT

— • Normals (untreated)Normals 8 DOCGTG 8 AdrenalectomyGTG 3 Adrenalectomy8 DOC

FOOD INTAKE

DAYS

FIG. 3. Effect of the daily administration of desoxycorticosterone(DOC; 0.3 mg/kg, ip) on the body weights and food intake of normalarid GTG-treated adrenalectomized mice. The response of adrenalec-tomized control mice was not significantly different from that of normalcontrols and was omitted from the figure. The values shown representthe mean response of 10 mice in each group.

to the VMH by GTG require the presence of the adrenalgland. This dependency on adrenal activity is furthersupported by the restoration of hyperphagia and obesityobserved after the administration of cortisone to GTG-

treated adrenalectomized mice. Mineralocorticoids ap-parently are not involved in GTG-induced hypothalamichyperphagia, since the administration of desoxycorticos-terone was without effect in restoring hyperphagia andobesity in GTG-treated adrenalectomized mice. Unex-pectedly, however, the administration of desoxycorticos-terone led to a suppression of food intake and a persistentloss of body weight to the point where the mice wereunable to maintain their body weight and died within thecourse of a few weeks. This action of desoxycorticoster-one, which was not observed in normal or adrenalecto-mized control mice, cannot be explained at the presenttime.

The manner in which adrenal glucocorticoids partici-pate in the development of hypothalamic hyperphagia isnot known. Our previous findings (6-9) of resistance ofthe VMH to GTG-induced necrosis with accompanyinghyperphagia in alloxan-diabetic mice, and the reversal ofthese parameters {i.e. return of sensitivity of the VMHto GTG and normal food intake) by adrenalectomystrongly suggested that adrenal hormones may be actingdirectly at the level of the VMH in modulating foodintake. Although the possibility remains that the hyper-phagia accompanying insulin deficiency may be mediatedin part by effects of adrenal glucocorticoids on the VMH,the present findings clearly indicate an action of adrenalglucocorticoids on areas outside of the VMH in thecontrol of food intake. There is evidence that the para-ventricular nucleus in the rostral hypothalamus is in-volved in the central nervous system control of foodintake (12-14), and that its activity in this regard isdependent on adrenal hormones (15, 16). Leibowitz (14)has established the presence of a-adrenergic receptors inthis area, which, upon activation by norepinephrine, elicithyperphagia. The norepinephrine-induced activation ofthese receptors was shown to be dependent on adrenalhormones, since adrenalectomy prevented the norepi-nephrine effects on feeding behavior. The administrationof corticosterone restored the norepinephrine-inducedactivation of feeding behavior in adrenalectomized rats(15, 16).

There are conflicting reports as to the importance ofadrenal hormones in the syndrome of hyperphagia andobesity which follows the induction of lesions in theVMH (17-19). Bernardis and Skelton (17) concludedfrom studies on adrenal-demedullated rats with electro-lytic lesions of the VMH that the adrenal gland was notinvolved in the control of food intake. It was subsequentlyreported by York and Bray (18) that obesity followinghypothalamic damage in rats was not affected by adre-nalectomy. On the other hand, Mook et al. (19) foundthat adrenalectomy depressed the hyperphagia andweight gain of rats with electrolytic lesions of the VMH.In the rat, hypophysectomy, which is known to result in


2028 DEBONS ET AL. Endo • 1982VolllO«No6

adrenal deficiency, has not been found to prevent theobesity which normally follows electrolytic lesions of theVMH (18, 20-22). In mice, however, Redding et al. (23)found that hypophysectomy prevented the increase inbody weight and total body fat which follows GTG-induced damage to the VMH. These findings have beenconfirmed by Liebelt and Perry (24). Among the possibleexplanations for the apparently conflicting reports ofadrenal and pituitary effects on hypothalamic hyper-phagia and obesity are the following: 1) VMH lesions inrats were induced by electrocautery, while in mice lesionswere induced chemically; and 2) the presence of varyingamounts of adrenal accessory bodies are known to occurfairly frequently in rodents (25); adrenalectomy alonemay not be sufficient in some species to induce adequateadrenal deficiency. These points are currently underinvestigation in our laboratory.

There is additional evidence in support of adrenalhormones in the control of feeding behavior. Hausberger(26) found that the prolonged administration of adrenalglucocorticoids in mice and guinea pigs led to an increasein body weight and obesity. Bray and York (27) haveshown that the weight of the adrenal gland is increasedin genetically obese rats and mice; the increase is pro-portional to the elevation in food intake and weight gain.Naeser (28) has shown that increased adrenal activity(i.e. elevated levels of corticosterone and adrenal hyper-trophy) accompanies hyperphagia and obesity in geneti-cally obese (ob/ob) mice. Yukimura et al. (29, 30) havereported that adrenalectomy prevented the hyperphagiaand weight gain of genetically obese (ob/ob) mice andZucker fatty rats. Adrenal glucocorticoids have also beenfound by Mook et al. (31) to be required for the elevationof food intake and body weight which follows ovariec-tomy in rats. These workers reported that adrenalec-tomy, but not adrenal demedullation, blocked or reversedthe elevated food intake and weight gain of ovariecto-mized rats. All of these studies emphasize a need for abetter understanding of the mechanism of action of ad-renal glucocorticoids in the regulation of food intake.

Acknowledgments

We wish to thank Drs. M. Steinberg and H. Herzog of the ScheringCorp. for supplying us with GTG for these studies.

R e f e r e n c e s

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24. Liebelt RA, Perry JH 1967 Action of gold thioglucose on the centralnervous system. In: Code CF, Heidel W (eds) Handbook of Physi-ology, sect 6, vol 1. American Physiological Society, WashingtonDC, p 271

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