enwronmental toxicology ana pharmacotogy

ELSEVIER European Journal of Pharmacology Environmental Toxicology and Pharmacology Section 293 ( 1995 ) 309- 317

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced anorexia and wasting syndrome in rats: aggravation after ventromedial hypothalamic lesion

Jouni T. Tuomisto *, Raimo Pohjanvirta, Mikko Unkila, Jouko Tuomisto National Public Health Institute, Department of Toxicology. P.O. Box 95, FIN-70701 guopio, Finland

Received 13 February 1995; revised 19 May 1995; accepted 23 May 1995

Abstract

Long-term regulation of body weight and food intake were studied after rats were subjected to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which causes hypophagia and body weight loss, and to ventromedial hypothalamic lesion, which causes hyperphagia, metabolic changes and obesity. These two factors appeared to have an interaction, as ventromedial hypothalamic lesion initially aggravated the effects of TCDD on body weight and food intake. This was seen in both TCDD-resistant and TCDD-susceptible rat strains. In contrast, if TCDD was given several weeks before the lesion and body weight had stabilized to a low level, no aggravation was seen, but TCDD completely blocked the effects of ventromedial hypothalamic lesion. Thus, TCDD seems to affect the same regulation chain that is involved in the lesioning of the ventromedial hypothalamus. TCDD might serve as a tool in studying different mechanisms of long-term food intake and body weight regulation.

Keywords: 2,3,7,8-Tetrachlorodibenzo-p-dioxin; Vena'omedial hypothalamic nucleus; Body weight; Appetite; Eating; (Rat)

1. Introduction

The common environmental contaminant, 2,3,7,8-tetra- chlorodibenzo-p-dioxin (TCDD) is a polychlorinated aro- matic hydrocarbon and the most potent congener of its group. Thus it is used as a model compound for dioxin toxicity. The most striking facet of the acute toxicity of TCDD is a wasting syndrome with hypophagia, or even aphagia, and weight loss. TCDD is actually the most potent anorectic compound known. After a single sublethal dose to rats, food intake returns to almost normal in about 3 weeks, but after a lethal dose, hypophagia and weight loss continue until the animals die ca. 2-3 weeks post exposure. The animals exposed to TCDD seem to change their body weight set-point, as there is no defect in the ability to make appropriate adjustments to food intake or to body weight when challenged by fasting or overeating. The mechanism of this wasting syndrome is unknown

* Preliminary results of some of these experiments were presented in Eurotox Congress held in Uppsala, Sweden, June 30-July 3, 1993.

• Corresponding author. Tel.: +358-71-201305; Fax: +358-71- 201265; e-mail: jouni.momisto@ktl.fi.

0926-6917/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 9 2 6 - 6 9 1 7 ( 9 5 ) 0 0 0 3 3 - X

(Seefeld et al., 1984; for review, see Peterson et al., 1984; Pohjanvirta and Tuomisto, 1994). Nausea and changes in general behavior have been studied as possible mecha- nisms, but the results were negative (Pohjanvirta et al., 1994; Sirkka et al., 1992).

There are great differences in susceptibility to the wast- ing syndrome of TCDD among species and strains. The most susceptible and most resistant rat strains known are Long-Evans (Turku AB: inbred; L-E) and Han/Wistar (Kuopio: outbred; H / W ) with LD5o values ca. l0 p ,g /kg for females and more than 9600 /~g/kg for both genders, respectively (Pohjanvirta and Tuomisto, 1990a; Pohjan- virta et al., 1993; Unkila et al., 1994). Except for lethality, H / W rats display most other biochemical and morphologi- cal effects of TCDD at doses that are effective in L-E rats (Pohjanvirta et al., 1989).

Hypothalamus has been known as a major centre of appetite regulation since the nineteenth century observa- tions in patients with hypophyseal or hypothalamic tu- mours. Bilateral lesions of the rat ventromedial hypothala- mus cause hyperphagia and weight gain (Hetherington and Ranson, 1940). The obesity is thought to ensue as a result of increased food intake, reduced sympathetic and in- creased vagal activity, and hyperinsulinemia (Bray and

310 J T. Tuo tms lo ~.t al .. E , r J. P h a r m a c o L Ent t ton To,tool . Pharmuco l . Sectt t ,n 2 9 3 1 1 9 9 5 ~ 3t)9-_?1 ~

York, 1979; Bray et al., 1989). Also numerous other hypothalamic and extrahypothalamic sites have been found to take part in appetite regulation, and they seem to form a most complicated interacting regulator3,, network (Blundell, 1991). Monoamines. histamine and several peptides, such as neuropeptide Y, corticotropin-releasing factor, and en- dorphins, are important neurotransmitters in the hypothala- mus in modulating short-term feeding (Leibowitz and Shor-Posner, 1986: Levine and Billington, 1989: Morley, 1989; Angel, 1990; Ookuma et al., 1993).

The aim of this study was to find out ho~ these two body weight changing factors, i.e. ventromedial hypothala- mic lesion and TCDD, affect weight and food intake together. The effects could be additive suggesting indepen- dent mechanisms or an interaction with either blockade or enhancement of changes: this would suggest that these factors participate in the same chain of mechanism(s). It is possible to administer ver), high doses of TCDD to H / W rats without lethality, to ensure maximal effect. On the other hand. a possible alleviation of wasting syndrome would best be seen in L-E rats with a small dose that alread) causes the wasting syndrome and some lethality, and aggravation could also be observable in this setting. Differences in the outcome in the two strains might give valuable hints for explaining the wide interstrain difference in the potency of TCDD.

2. Materials and methods

2. I. Chemicals

TCDD was > 99% pure as determined by gas chro- matography-mass spectrometry. It was dissolved in corn oil as described previously (Pohjanvirta et al., 1987). a-Methyl-p-tyrosine methyl esther hydrochloride (a - methyltyrosine) was purchased from Sigma Chemicals (St. Louis, MO) and was dissolved in isotonic saline. All chemicals in high pressure liquid chromatograph (HPLC) analyses were of at least analytical grade.

2.2. Animal husbandt3'

Female H / W and L-E rats (male H / W rats in experi- ment 3) were obtained from the breeding colony of the National Public Health Institute, Division of Environmen- tal Health, Kuopio, Finland. The rats were 11-12 weeks of age in the beginning of the experiments. They were kept in metabolic cages (Tecniplast, Buguggiate, Italy) or in modi- fied single-rat wire mesh cages enabling accurate measure- ment of food intake and food spillage. When food intake was not recorded, the rats were housed in regular single-rat wire mesh cages. The food was powdered while food intake was monitored and otherwise pelleted (R3, Ewos, SiSdert~tlje, Sweden). The lights were on from 7.00 a.m. to

7.00 p.m. The ambient room temperature was 21.5 + I~C and the humidity 55 + 10%.

2.3. Experimental design

Four sets of experiments were performed. In experiment 1, TCDD-resistant female H / W rats were ventromedial hypothalamic lesioned or sham operated. 2 weeks later, when the lesioned rats had become obese, the rats were administered TCDD I000 / . tg/kg i.p., or vehicle (corn oil 5 ml /kg) only. A similar procedure was used in experi- ment 2, where TCDD-susceptible female L-E rats were exploited. The dose of TCDD was 8.5 /,tg/kg, which is slightly below their LDs0 value. In experiment 3, ventro- medial hypothalamic lesions were performed to male H / W rats, and TCDD (1000 p,g/kg) was administered 4 days post operationally, when no significant differences in body weights had emerged. In experiment 4, TCDD (1000 /xg/kg) was given to female H / W rats 7 weeks before the operation, In each experiment body weights were mea- sured ever), other day and food intake every day.

2.4. SurgeD'

The ventromedial hypothalamic lesions were performed under ketamine (60 m g / k g i.p., Ketalar, Parke Davis, Barcelona, Spain) and medetomidine (0.5 m g / k g s.c.. Domitor, Orion-Farmos, Turku, Finland) anaesthesia. In experiment 3, pentobarbitai (50 m g / k g i.p.) anaesthesia was used, but the stability of this anaesthesia was not satisfactory and it was therefore not used in other experi- ments. The rats were placed on a stereotaxic device with the incisor bar 3.5 mm lower than interaurai line; thus, the lambda and the bregma were at the same horizontal level. The coordinates changed slightly from one experiment to another, as lesion verification enabled the adjustment of lesions more precisely in later experiments. The coordi- nates for ventromedial hypothalamus were 2.4__+ 0.1 mm (2.8 mm in experiment 1) posterior, 0.6 mm lateral, and 9.6 + 0.1 mm ventral to bregma. The lesion was made by lowering an insulated stainless steel electrode (diameter 0.4 ram) into the brain and passing through 1.0-1.3 mA anodal current for 20 s. The cathode was attached against the right hind leg with a cloth soaked with saline. The sham rats were operated similarly except that the electrode was lowered I mm less and no current was passed through. After the operation, the ketamine-medetomidine-anaesthe- tized rats were awakened with atipamezole (0.25 mg/ra t i,m., Antisedan, Orion-Farmos, Turku, Finland),

2.5. Lesion t'erification

Ventromedial hypothalamic lesions were verified mi- croscopically with the help of a brain atlas (Paxinos and Watson, 1982). At termination the brain was removed,

J.T. Tuomisto et a l . / Eur. J. Pharmacol. Enuiron. Toxicol. Pharmacol. Section 293 (1995) 309-317 311

frozen in -70°C isopentane and liquid nitrogen, cut into 25 p,m thick slices with a cryostat and stained with cresyl violet. The location of the lesion was estimated without knowledge of the group of the rat. Rats were accepted for or rejected from the experiments before exposure on the basis of weight gain and food intake. However, a few rats were rejected afterwards when the lesions appeared to be located outside the ventromedial hypothalamus (see Re- sults).

2.6• Biochemical assays

Plasma insulin was measured with rat insulin radioim- munoassay kit supplied by Novo Nordisk, Bagsveerd, Den- mark. The blood samples were taken within a few minutes from the tail vein under ether anaesthesia on day 66 post exposure. Blood glucose was determined in samples bled from the tip of the tail with an Accutrend (Boehringer Mannheim, Germany) portable refractometer. Also sympa- thetic activity was estimated in experiment 3. tx-Methyl- tyrosine blocks catecholamine synthesis and thus decreases noradrenaline reservoirs in organs at a rate related to sympathetic activity (Saito et al., 1989; Bray et al., 1989). Each group (n = 6-7) was divided into a-methyltyrosine (300 mg/kg i.p.) rats and saline (5 ml/kg i.p.) rats. 4 h after injection the rats were decapitated and the sub- mandibular salivary gland, heart, spleen, and brown adi- pose tissue were removed. Noradrenaline was measured by an HPLC method (Mefford, 1982) after an activated alu- mina procedure (Elo and MacDonald, 1989). The change in noradrenaline was calculated by comparing average noradrenaline concentrations of ot-methyltyrosine-injected rats and saline-injected rats within each group.

2.7. Statis~cs

The groups were statistically compared with the analy- sis of variance (with repeated measures where appropriate) at specific time points and periods along the time course. Student's t test was used when only two groups were compared. Body weights were used as such, but food intake was averaged mostly over consecutive 10-day blocks.

3. Results

3.1. Response to TCDD in obese uentromedial hypothala- mic-lesioned rats

The experiment was accomplished with both TCDD- susceptible L-E rats and TCDD-resistant H / W rats. The rats were allowed to gain weight for two weeks after ventromedial hypothalamic lesioning. Food intake and body weight gain increased in both rat strains during this period (Fig. 1, Fig. 2 and Table 1).

35 Food intake, g/day

30 '~

25

20

15

10

5

0 I- i I

~ .... *" '" Sham/'I'CDD

=~ ~ ' ;' • • Lealon/I"CDO

I ' I ' I

300 Body weight, g

250

200 _ -, , ,4

VMH lesion 100 ; " : " " : - : " t I I t

-20 -10 10 20 30 40 S0 60 Days alter TCDD

Fig. I. Food intake and body weights (mean -t- SD) of obese venlromedial hypothalamic-lesioned female H / W rats (experiment 1). The rats were

operated on day - 14 (arrow) and exposed to TCDD (1000 / z g / k g i.p.)

on day 0. The death of each non-survivor rat is marked with a single symbol indicating the day, body weight and group. Only a few standard deviat ions are shown for clarity, n = 5-6 .

In the sham/TCDD group the outcome after TCDD (1000 p,g/kg i.p.) was typical of the wasting syndrome in H / W rats: after the initial drop, food intake returned almost to the original level in two weeks, but body weight remained subnormal (Fig. 1 ). In contrast, the lesion/TCDD rats stopped eating completely by day 3-11 post exposure. Body weight declined until it was significantly lower than that of the sham/TCDD group. Three lesion/TCDD rats resumed eating 5-19 days later and recovered their origi- nal weights, but the other three died after a total fast of 19-25 days. One sham/TCDD rat died on day 42 post exposure. The food consumption of the lesion/control group returned slowly down to the pre-operation level over 4 weeks, but the body weight remained high.

The ventromedial hypothalamic lesion was shown to aggravate the wasting syndrome in L-E rats in a manner similar to that in H / W rats (Fig. 2 and Table 1). Food consumption in lesioned TCDD-treated L-E rats (8.5 /zg/kg i.p.) declined to a level significantly lower than that in sham-operated rats showing interaction between ventromedial hypothalamic lesion and TCDD exposure throughout the observation period (P < 0.05). However, the average body weight did not fall much below that of sham groups, as the rats died when they reached a critical weight level of about 140 g. All lesion/TCDD rats died (except an outlier, see below) before the end of the experi-

312 J.T. l-uomisto et al Eur J. Pharmacol. Ent tron. To.ttcol. Pharmacol Scctton 2v3 t 1995 J .?tt9 3 / 7

- - - - - Les,on/Control 50 Food =ntake. g/day

4 5 , j~ . . . . ~ " " " S h a m / C o n t r o l

40 i-~ ~t , . . . -A . . . Snam/rCDD Ih 35 ~ '~l Ill. / I

30 F , t" I ' ¢ • LesiorV'rCDD

25 ~ , ~ 1 " ',' ' ' ~ " ' , , l " a l . - , ~ " ~ ,

20 , - I v

10 t 5 t

0 I ' l , , I ' ' ' I •

450 Body weight, g

400

350

300

2 5 0

20O

1 5 0

V M H lesion 100 m i

-20 -10 0

~ ~ . C _ . , . . . ~ . ÷ . . > C ' C ' e ' ' ; ' ~

" A ' J ~ t . l ~ , .A- . A ' A ' ~

I I I O

10 20 30 40

Days after TCDD

Fig. 2. Food intake and body weught of obese ventromedial hypothalamic-lesioned female L-E rats (experiment 2). The rats were operated on day - 14 (arrow) and exposed to TCDD (8.5 p.g/kg i.p.) on day 0. One rat in the lesion/TCDD group paradoxically increased food intake and body weight after TCDD; these data are shown separately (dashed line). The death of each non-sur~,ivor rat is marked with a single s)mbol indicating the day, body weight and group. Only a few standard deviations are shown for clarity, n = 5-7.

ment (day 42 post exposure), but two out of five s h a m / T C D D rats survived.

There was one L-E rat in the l e s i o n / T C D D group that behaved in an opposite way to that of the others and is shown separately in Fig. 2. After TCDD, it increased its food intake and weight gain rate. By the end of the

experiment , it was fatter than any of the rats m the l e s ion /con t ro l group. As an aberrant outlier, the rat was excluded from statistical analyses. However, the lesion was proved to be in the correct place in ventromedial h.~pothalamus. In addit ion, the exposure was conf i rmed b~

taking an abdominal fat sample and by measuring the T C D D concentra t ion with gas chromatograph-mass spec-

trometer. For compar ison, samples were also taken from one l e s ion / con t ro l rat, two s h a m / T C D D rats (combined

sample) and one rat outside the experiment . The concentra- tions were 1.20, 0.1 1, 12.6, and 0.01 n g / g fat, respec-

tivel2~. Dilut ion in substant ial ly expanded adipose tissue in

the lesioned rat explains the difference in T C D D concen- trations (1.20 vs. 12.6 n g / g ) in exposed animals.

An interest ing clinical difference was seen between L-E and H / W rats after ventromedial hypothalamic lesion and TCDD. The resistant H / W rats typically fasted totally for weeks and dropped their weights far below the level of sham-operated rats and became seriously debili tated and had dirty fur for days. Suddenly, however, some of them started to eat, groom and gain weight again. In contrast, the TCDD-suscept ib le L-E rats typically reduced food intake down to a few grams (not zero) and lost weight in a rather good shape until they became comatous and died within hours.

3.2 . R e s p o n s e to T C D D in n o n - o b e s e c e n t r o m e d i a l hy -

p o t h a l a m i c - l e s i o n e d ra t s

The aggravat ion of the wast ing syndrome by ventrome- dial hypothalamic lesion could be caused either by altered body weight regulat ion or by the changed body weight itself. Also sex could be a modi fy ing factor. During the first days after the lesion the hyperphagic response can already be seen but no statistically signif icant body weight changes have yet occurred. In this exper iment male H / W rats underwent ventromedial hypothalamic lesion or sham operation 4 days before TCDD.

Table I Body weights in grams (mean ± SD) of TCDD groups in each experiment at several time points" weight as percentage of the initial weight is shown m parentheses; the numbers of rats on day 0 and day 40 are also shown

Experiment Operation Pre-lesion Day 0 Day 24 Day 40

Weight (g) Weight (g) n Wenght (gl Weight (g) n

Obese H/W (Exp. I) Lesion 195+ 14 252:1:25(129%) ac 6 140-1- 18(74%) .~ac 184+ 10(98%) ~ 3 Sham 200+ II 212+9(106%) 5 184+ 14(.92%) 191 _t 17 (95%) 5

Obese L-E(Exp. 2) Lesion 190::t: 14 240+ 17(126%) BC 7 157-t- 15(81%) AC 140+ I1 (75%) ~ 2 Sham 188+6 204::1:6(108%) c 5 159± 10(86%) ~c 177± 1(96%) '~ 2

Non-obese H/W (Exp. 3) Lesion 316+18 335+25(106%) 6 256+29(81%) at'c 264±24(84%) ~t,c 6 Sham 331 _1:21 322 -I- 26(97%'1 6 306-.t: 26(93%) a 310±31 (94%) A 6

POSt-TCDD H/W (Exp. 4) '~ Lesion 208± 14 192±22(93%) '~' 9 199+25(97%) a 193::1: 14(95%)A 3 Sham 208+ 18 198 ::t: 19(96%) A 10 212 Zt: 24(102%) ~ 216±20(106%) a 7

Statistical significance vs. corresponding control (non-TCDD) group; lowercase: P < 0.05; uppercase: P < 0.01; b statistical sngnificance vs. correspond- ing sham (non-lesion) group; ~ significance vs. pre-lesion values; d in these groups, the columns denote for days 0, 41, 65, and 82, respectively, i.e. pre-TCDD and pre-lesion weights, and weights 24 and 41 days after the lesion.

J.T. Tuomisto et al. / Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. Section 293 (1995) 309-317 313

Food intake, g/day

25 ~ Lesion/Control

2O

0 ~ ' ' I I ' I ' ; " ; " : ' ! '

500 Body weight, g

450 .~

40O

35O " & . = ~

300

25O

20O

150 I

100 i -10 0 10 20 30 40 50 60 70

Days after TCDD

Fig. 3. Food intake and body weight of non-obese ventromedial hypotha- lamic-lesioned male H /W rats (experiment 3). The rats were operated on day - 4 (arrow) and exposed to TCDD (1000/.Lg/kg i.p.) on day 0. Only a few standard deviations axe shown for clarity, n = 6.

35

30

25

20

15

10.

5

0

350

300

250

200

150

100

Food intake, g/day

Control/Lesion

.... a--. Control/Sham

... . A.-- TCDD/Sham

• TCDD/Leaaon~

Body weight, g

VMH lesion

0 10 20 30 40 50 60 70 80 90

Days after TCDD

Fig. 4. Food intake and body weight of ventromedial hypothalamic-le- sioned H/W rats (experiment 4) that were exposed to TCDD (1000 /,tg/kg i.p. on day 0) before ventromedial hypothalamic operation (day 41, arrow). The death of each non-survivor rat is marked with a single symbol indicating the day, body weight and group. Only a few standard deviations are shown for clarity, n = 9-10.

Vent romedia l hypothalamic lesion increased daily food

intake, but after T C D D (1000 / z g / k g i.p.) the amount fell

f rom about 26 g to 10 g with the lowest value o f 5.3 g on

day 10 post exposure . Dal ly food intake dec l ined also in

s h a m / T C D D group but only from about 19 g to 15 g (Fig.

3). The T C D D - e x p o s e d groups lost weight; les ioned rats at

a h igher rate than sham rats. The body weights o f the

fo rmer group dec l ined to a s ignif icant ly lower level than

those o f the latter (Fig. 3). The l e s i o n / c o n t r o l group

gained weight more than the s h a m / c o n t r o l group, al-

though the dif ference was not statistically significant. This

reveals that all lesions were not opt imal . Yet some individ-

uals gained weight up to 187% from pre- lesion values. The

interaction be tween vent romedia l hypotha lamic lesion and

T C D D exposure was s ignif icant in both food intake and

body weight ( P = 0.009 and P = 0.023, respectively) .

Table 2 Blood glucose, plasma insulin and sympathetic activity in non-obese ventromedial hypothalamic-lesioned rats in experiment 3 (mean + SD)

Parameter Time/organ Lesion/ Lesion/ Sham/ Sham/ TCDD Control TCDD Control

Blood glucose (raM) Before lesion 6.9 5:0.8 6.8 ± 0.5 7.0 + 0.5 6.9 + 0.2 Before TCDD 6.7 ± 0.7 6.5 5:0.3 7.0 ± 0.5 6.5 5- 0.4 Day 10 5.2 + 0.8 6.2 5:0.7 5.7 + 0.6 6.0 5:0.3 Day 63 5.6 + 0.4 5.9 + 0.3 5.9 ± 0.3 6.2 ± 0.4

Plasma insulin (ng/ml) Day64 9.9±2.6 ^ 17.5±4.6 a 7.1:1:1.7 a 10.9 ± 2.3

Noradrenaline 4 h after a-methyltyrosine vs. vehicle (%; day 66) Submandibular gland 94 + 47 67:1:9 84 + 15 75 5:6 Heart 105 + 17 84 -t- 14 100 + 25 100 5" 13 Spleen 106 -I- 40 83 + 33 94 ± 16 73 5:18 Brown adipose tissue 59:1:26 63 ± 36 60 ± 30 55 5- 19

Blood glucose was taken at several time points during the experiment. Sympathetic activity was estimated by comparing tissue noradrenaline concentrations 4 h after a-methyltyrosine or vehicle, n = 6 except for sympathetic activity n = 3. = statistical significance vs. corresponding control (non-TCDD) group; lowercase: P < 0.05; uppercase: P < 0.01; b statistical significance vs. corresponding sham (non-lesion) group.

314 J. 1-. Tuomt ~to el al. . Eur. J. P h a r m a t ol. Ent tron. Touc o l . P h a r m a c o l Sectt t ,n 293 t 19951 309-3l

3.3. Effect.~ o ft,entromedial hypothalamic lesion performed after TCDD treatment

pose tissue ~ere 3.91, 1.26. 1.88. and 1.39 /.tg/g tissue. respectively.

Thus ventromedial hypothalamic lesion aggravated wasting syndrome in two rat strains and even if there was no initial weight difference between lesioned and sham operated rats. Does TCDD have a permanent effect on feeding regulation that could be aggravated by a late ventromedial h2ypothalamic lesion, and does the aggrava- tion after the lesion also occur when body weight has already stabilized to a lower le~,el after TCDD? To answer these questions, TCDD was administered to resistant H / W rats (1000 /Ltg/kg i.p.) and the lesions were performed 41 days later. This is about two half-lives (provided that the elimination of TCDD at this huge dose is similar to that after low doses), i.e. about one-fourth of the dose remain- ing in the body (Pohjanvirta et al., 1990b).

Mortality from operation within 24 h was high in the TCDD/lesion group (four rats) while only one rat in the control/lesion group and one in the TCDD/sham group died (Fig. 4). The preoperational size of each group was nine to ten rats. One rat from the TCDD/sham group and two rats from the TCDD/lesion groups died during the follow-up time (43 days).

TCDD reduced body weight permanently (Fig. 4). Ven- tromedial hypothalamic lesion increased both food intake and body weight in control animals. In contrast, there was no difference in food intake between TCDD groups, and body weight decreased only slightly more in lesioned TCDD rats (significant only by repeated measures analysis of variance, P = 0.046). However, also this experiment revealed an interaction between ventromedial hypothala- mic lesion and TCDD exposure (P < 0.05) in both food intake and body weight.

3.4. Effects on blood glucose, plasma insulin and periph- eral noradrenaline turnocer

Glucose was measured four times in blood samples from rats in experiment 3, and plasma insulin and sympa- thetic activity were measured at the end of this experiment (Table 2). Neither TCDD nor ventromedial hypothalamic lesion affected blood glucose levels. In contrast, ventrome- dial hypothalamic lesion increased plasma insulin and TCDD decreased it 64 days post exposure. These effects were interactive ( P = 0.015), as TCDD blocked the rise of insulin caused by ventromedial hypothalamic lesion. One rat from sham/TCDD group was excluded because of excess delay in sampling. Also sympathetic activity was estimated by measuring noradrenaline decrease in several organs after the injection of a-methyltyrosine, a cate- cholamine synthesis blocker. The decreases were small in every organ measured, the greatest being in the brown adipose tissue, and no differences occurred between the groups. The control levels of noradrenaline in the sub- mandibular salivary gland, heart, spleen and brown adi-

3.5. Lesions

After ventromedial hypothalamic operation, rats with poor performance in weight gain and food intake were rejected before the rats were randomized in TCDD and control groups. In experiment 1, six lesioned rats and one sham-operated rat were rejected. In experiment 2. three lesioned and two sham-operated L-E rats died after the operation, and six lesioned and two sham-operated L-E rats were rejected. In experiment 3, one lesioned rat died within 24 h from the operation, and six lesioned rats were rejected. In experiment 4, no exclusion of unsuccessfully lesioned rats was possible due to the setting of the experi- ment. One TCDD rat died before the operation.

Afterwards the lesions were verified microscopically. The lesions were mostly symmetrical and successfully positioned in the ventromedial hypothalamus. However, in experiment 3, two rats from both TCDD and control groups had asymmetrical ventromedial hypothalamic le- sions, but the? were accepted in the experiment. Four rats (one from experiment 2, one from experiment 3, and two from experiment 4) were excluded, because the lesions were outside the ventromedial hypothalamus.

4. Discussion

The present study revealed two intriguing features of body weight regulation after TCDD treatment. Firstly, an otherwise nonlethal dose of TCDD to ventromedial hy- pothalamic-lesioned H / W rats paradoxically caused a complete aphagia and greater weight loss than the same dose to sham-operated rats, and this led to exceptionally high mortality. If TCDD was given soon after operation to non-obese rats, aphagia was not complete, and there was no mc "tality, but the reductions in food intake and body weight were still potentiated in lesioned rats. Hence obe- sity was not necessary for this phenomenon, even though it fortified the effect. The lesioned rats did not resume eating until their weights had declined significantly below the level of sham-operated rats. All this implies that ventrome- dial hypothalamic lesion aggravated the effects of TCDD. The phenomenon was also seen in both male and female H / W rats, as well as in female L-E rats.

Secondly, the targeted long-term body weight and food intake levels after TCDD seemed to be the same for both sham-operated and surviving ventromediai hypothalamic- lesioned rats (although in L-E rats high mortality interfered with at late time points). This suggests that TCDD totally reversed the increasing effect of ventromedial hypothala- mic lesion on the putative body weight set-point. Con- versely, the impact of TCDD on this set-point was thus

J.T. Tuomisto et a l . / Fur. J. Pharmacol. Environ. Toxicol. Pharmacol. Section 293 (19951309-317 315

unaffected by ventromedial hypothalamic lesion. It is note- worthy that if TCDD was given several weeks before the lesion, there was no enhancement of wasting syndrome. Thus, ventromedial hypothalamic lesion seems to aggra- vate the TCDD wasting syndrome during the dynamic period when body weight decreases, but it seems to have less impact on the static phase when the low weight level has already been achieved.

Even though there is a wealth of experimental evidence in favour of the set-point hypothesis in body weight regu- lation (Keesey and Powley, 1986), the monitored factors are unknown. The proposed variables include body fat (Kennedy, 1953) or its correlate. A recent report described positional cloning of mouse ob gene (Zhang et al., 1994), which causes severe obesity if both alleles are mutated (Friedman et al., 1991). The ob protein was found to be produced solely in white adipose tissue and its structure is suited for secretion (Zhang et al., 1994). Thus it could be (one of) the factor(s) that transmit information about body weight to regulating systems such as the hypothalamus.

Ventromedial hypothalamic lesion causes changes in brain functions and deletes certain neuronal pathways that affect endocrine system and autonomic nervous system. Thus it alters physiological functions, and the metabolic derangement eventually leads to obesity (Bray et al., 1989). Along the regulatory chain there seems to be a point at which TCDD exerts its effect, which may explain the interaction between TCDD and ventromedial hypothalamic lesion described above. This point clearly cannot be the ventromedial hypothalamus, as the effect of TCDD was not attenuated and the strain susceptibility difference did not disappear after the ventromedial hypothalamus was destroyed. It could be elsewhere either in the central nervous system or in the periphery.

In the central nervous system an inhibitory path from the paraventricular hypothalamic nucleus to feeding regu- latory centres in the brain stem is damaged by ventrome- dial hypothalamic lesions (Wellman et al., 1993), and this is thought to be a partial cause of ventromedial hypothala- mic obesity. The interaction of TCDD with ventromedial hypothalamic lesion could be understood, if TCDD had a direct inhibitory (or stimulatory to an inhibitory tract) effect on the brain stem or another site downstream the regulatory path from the ventromedial hypothalamus.

It is not known which neurons or neurotransmitters are involved in the long-term regulation of feeding. Most pharmacological research deals with short-term regulation where the monoamines noradrenaline and serotonin, the diamine histamine, and several peptides such as neuropep- tide Y and endorphins have a crucial role (see introduc- tion). By pharmacological manipulation of these systems it is possible to achieve a reduction in appetite, but it is quite temporary and has proven to be of limited clinical value (Morley and Levine, 1985; Keesey and Powley, 1986; Blundell, 1991). In contrast, TCDD-treated animals exhibit a hypersensitive satiety response and a blunted hunger

response to feeding challenges several months after a single dose (Pohjanvina and Tuomisto, 1990b,c; Pohjan- virta et al., 1991). Hence the effect is quite different from that produced by, e.g., short-acting amphetamine-like or D-fenfluramine-like drugs (Samanin and Garattini, 1993).

Previously, several neurotransmitters were measured in hypothalamic nuclei and larger brain areas up to 10 days after TCDD administration, but the only consistent change, detectable at lethal doses, was increased serotonin turnover (Tuomisto et ai., 1990,1991; Unkila et al., 1993,1994). Depletion of brain serotonin did not, however, affect the wasting syndrome (Stahl et al., 1991). Thus, it seems unlikely that the key mediator in the long-term regulation would be serotonin, histamine, or a catecholamine.

In the periphery, ventromedial hypothalamic lesion causes metabolic disturbances with reduced sympathetic and increased parasympathetic activity, and a rise in blood insulin levels (Bray et al., 1989). TCDD could interact with these changes. TCDD rats have been reported to have low plasma insulin concentration, but increased insulin sensitivity (Gorski and Rozman, 1987; Pohjanvirta et ai., 1990a), and blood glucose is decreased partly because of energy depletion (Gorski et al., 1990). Ventromedial hy- pothalamic lesion tended to raise the insulin level also in TCDD-exposed rats, although this difference was not sta- tistically significant (note that the measurement took place 68 days after the lesion). It is not clear whether the subtle upward trend was involved in the exacerbated wasting syndrome in these rats. To confirm this hypothesis insulin levels should be measured at early time points when recovery of feeding and body weight has not yet started.

Ventromedial hypothalamic-lesioned rats have been shown to be unable to reduce basal metabolism normally when challenged with energy restriction (Vilberg and Keesey, 1990). This could account for the increased weight loss rate in lesioned TCDD-exposed animals, but cannot explain the aggravation in food intake reduction.

Estimates for sympathetic activity showed no statisti- cally significant changes. Although ventromedial hypotha- iamic-lesioned rats have decreased sympathetic activity, they may have unaltered or even increased noradrenaline turnover possibly due to changed noradrenaline metabolism (Bray et al., 1989). This is in accordance with the results with unchanged noradrenaline turnover rate in ventrome- dial hypothalamic-lesioned rats obtained here. The role of the autonomic nervous system in the interaction of TCDD exposure with ventromedial hypothalamic lesion remains uncertain.

The cause of the numerous deaths of TCDD-pretreated animals after ventromedial hypothalamic lesioning is not clear. It might be due to chance, but also to increased sensitivity to insulin (Gorski and Rozman, 1987; Pohjan- virta et al., 1990a). Ketamine-medetomidine anaesthesia increased blood glucose in TCDD rats and controls (our own observations). Insulin might abruptly rise (after the a 2 stimulation is terminated by atipamezole) because of re-

316 J T. Tuorntsto el al . . Eur J. Pharmaco l . Ent Iron. T o u c o l P harn ,aco l S~ t tton 293 t 1095 J 30tJ 31 -

flection of hyperglycemia or acute insulin secretion caused b} ventromedial hypothalamic lesioning.

In summary, there is a distinct difference in the pattern of bod3, weight changes between ventromedial h)pothala- mic-lesioned and sham-operated rats after TCDD. The effects of ventromedial hypothalamic lesion and TCDD on body weight are interactive. This implies that TCDD af- fects the regulation path distally from the ~entromedial hypothalamus, i.e. serially. The obesit2~ caused by ventro- medial h~pothalamic lesion can be dramatically cancelled off by TCDD, but the lesion leaves the ultimate target level of body weight untouched in TCDD-treated rats. Interactions of TCDD with various experimental models of modified food intake could provide a useful tool for a better understanding of the regulation of appetite and body weight and also for studying mechanisms resulting in the wasting s.v ndrome.

Acknowledgements

We wish to thank Dr. Risto Huupponen for analysing the insulin samples and Ms. Minna Holopainen and Ms. Arja Tamminen for their excellent technical assistance. Supported by research grant from the Academy of Finland, Research Council for Environmental Sciences (Grant no. 5410/4011/89).

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