Brain Research 986 (2003) 139–147www.elsevier.com/ locate/brainres

Research report

A nabolic androgenic steroid nandrolone decanoate reduceshypothalamic proopiomelanocortin mRNA levels

a c c b¨Jonas Lindblom , Anna M.S. Kindlundh , Fred Nyberg , Lena Bergstrom , Jarlb ,*E.S. Wikberg

aDepartment of Neuroscience, Division of Pharmacology, Uppsala University, Box 591 BMC, S-751 24 Uppsala, SwedenbDepartment of Pharmaceutical Biosciences, Division of Pharmacology, Uppsala University, Box 593 BMC, S-751 24 Uppsala, Sweden

cDepartment of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Uppsala University, Box 591 BMC, S-751 24Uppsala, Sweden

Accepted 26 June 2003

Abstract

Supratherapeutical doses of anabolic androgenic steroids (AASs) have dramatic effects on metabolism in humans, and also inhibitfeeding and reduce the rate of body weight gain in rats. In order to test the hypothesis that the AAS metabolic syndrome is accompaniedby alterations in the central melanocortin system, we evaluated body weight, food intake and hypothalamic agouti-related protein (AgRP)and proopiomelanocortin (POMC) mRNA levels following administration of different doses of the anabolic androgenic steroidnandrolone decanoate. In order to distinguish changes induced by the steroid treatment per se from those resulting from the reduced foodintake and growth rate, we also compared the effect of nandrolone decanoate on AgRP and POMC mRNA expression with both normallyfed, and food restricted control groups. We here report that administration of nandrolone specifically reduces arcuate nucleus POMCmRNA levels while not affecting the expression level of AgRP. The effect on POMC expression was not observed in the food restrictedcontrols, excluding the possibility that the observed effect was a mere response to the reduced food intake and body weight. These resultsraise the possibility that some of the metabolic and behavioural consequences of AAS abuse may be the result of alterations in themelanocortin system. 2003 Elsevier B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters, and receptors

Topic: Peptides: biosynthesis, metabolism, and biochemical characterization

Keywords: Anabolic androgenic steroid; Nandrolone decanoate; Agouti-related protein; Pro-opiomelanocortin;a-Melanocyte stimulating hormone

1 . Introduction the central control of body weight homeostasis.a-MSH-synthesizing neurons in the arcuate nucleus of the hypo-

Abuse of anabolic androgenic steroids (AASs) is some- thalamus receive inputs concerning the current metabolictimes associated with a form of ‘reverse anorexia’, which state from the periphery. One important such signal is theis characterized by a drive to increase lean body mass circulating protein leptin, which is secreted from adiposewhile reducing body fat[21]. AASs have been demon- tissue in proportion to its fat mass. When the energystrated to induce a number of metabolic changes in non- balance is positive POMC neurons are believed to conveyexercised male rats, including lack of appetite and, conse-a-MSH mediated anorexigenic signals to other nucleiquently, a reduced rate of body weight gain[34]. involved in the control of feeding.a-MSH is an endogen-

The neuropeptides derived from proopiomelanocortin ous agonist to the MC receptors. Other neurons in the(POMC), especially the melanocortin (MC)a-melanocyte arcuate nucleus synthesize an endogenous antagonist to thestimulating hormone (a-MSH), are known to participate in MC receptors called agouti related protein (AgRP). These

neurons are inactivated by leptin, and mediate orexigenicsignals[7].*Corresponding author. Tel.:146-18-471-4238; fax:146-18-559-718.

E-mail address: jarl.wikberg@farmbio.uu.se(J.E.S. Wikberg). We hypothesized that at least part of the effect of AAS

0006-8993/03/$ – see front matter 2003 Elsevier B.V. All rights reserved.doi:10.1016/S0006-8993(03)03223-2

140 J. Lindblom et al. / Brain Research 986 (2003) 139–147

on food intake and body weight is mediated by dysregula- vehicle (n58) for 14 days. We used a commerciallytion of the central melanocortin system. The objective of available nandrolone decanoate solution of 50 mg/ml thatthe present study was to investigate whether the metabolic was diluted with peanut oil to appropriate concentrationssyndrome induced by nandrolone decanoate is accom- for the lower doses to keep the volume of injectionpanied by changes in the melanocortin system. First, we constant. However, the animals receiving the highest doseinvestigated the effect of different doses of nandrolone on of nandrolone decanoate were administered a volume ofbody weight, AgRP and POMC mRNA levels. In sub- 0.36 ml, distributed at four different injection sites. There-sequent studies we used a dose that proved to effectively fore we added a control (n54) receiving 0.36 ml vehicle.inhibit body weight gain and caused a reduction in POMC Body weight was monitored daily. After treatment, the ratsmRNA levels. However, changes in POMC mRNA levels were killed by decapitation, the brains rapidly removed[15] and MC receptor binding[10] have also been reported and frozen in chilled (220 to 230 8C) 2-methyl butane.as a result of food restriction. In order to distinguishchanges induced by the steroid treatment per se from those2 .2.1. In situ hybridizationresulting from the reduced food intake and growth rate, we The brains were cut at219 8C and 10-mm sections wereincluded a control group subjected to food restriction, collected on polylysine slides (Menzel, Germany) atmatched to the food intake of the nandrolone treated bregma23.8 mm. The sections were air-dried, thenanimals. In this experiment we also investigated whether immediately stored at280 8C until used. The oligonucleo-or not steroid treatment causes changes in plasma leptin tides, synthesized by TIB Molbiol (Berlin, Germany), werelevels compared to the food restricted, body weight complementary to the nucleotides 72–122 of POMCmatched group. In addition, as nandrolone previously has mRNA[6] and 73–112 of AgRP mRNA (AF206017)[16].been reported to affect kidney mass[34], we dissected a The oligonucleotides (40 ng) were labelled with 40mCi

35number of organs in order to investigate whether this effect [a- S]dATP (1250 Ci /mmol) using terminal deoxynu-was an effect of the reductions in body weight gain. In a cleotidyl transferase (60 U) at 378C for 90 min andseparate experiment, we monitored individual food and purified with Nensorb 20 columns (NEN Life Sciencewater intake after 10 days of treatment, when changes in Products, London, UK). The brain sections were air-dried

6body weight were becoming apparent. and hybridized with 10 cpm labelled probe/ml in ahybridization solution containing 50% deionized form-amide for 20 h at 428C. Control sections were coincubated

2 . Methods with 0.5mg/ml non-labelled oligonucleotide. After hybrid-ization, the sections were washed 4315 min in 0.15 M

2 .1. Animal treatment NaCl, 15 mM sodium citrate, pH 7.0, at 568C anddehydrated. Labelled sections and plastic standards (Au-

14Male Sprague–Dawley rats (250–350 g) were purchased toradiographic [ C]Microscales (30–880 nCi /g, Amer-from Alab, Sollentuna, Sweden. The animals were housed sham, Stockholm) were placed in X-ray cassettes andin groups of two to four per cage in air-conditioned rooms exposed to autoradiographic film (Hyperfilm-bmax, Amer-(12-h dark/ light cycle) at 22–238C and a humidity of sham, Stockholm). After 6 weeks the films were developed55%. The animals had free access to water and R36 food manually (Kodak D19, Unifix). The autoradiograms werepellets (Labfor, Lactamin, Vadstena, Sweden), with the digitized and the optical density calibrated to pmol /mg wetexception of one group subjected to food restriction. The weight based on the coexposed standards using NIH-Imageanimals received daily intramuscular injections of nan- software (NIH Image 1.62, NIMH, Bethesda, MD, USA),drolone decanoate (Deca-Durabol, Organon, Oss, The and the binding was quantified in the arcuate nucleus ofNetherlands) or vehicle, i.e. peanut oil alone (Apoteket, the hypothalamus.

˚Umea, Sweden) for 14 days, 2 h after lights on. Thevolume of injection was 0.1 ml unless otherwise stated. 2 .3. Comparison of steroid effects with food restrictionThe rats were killed by decapitation. The animal experi-ments were approved by the local ethical committee in In order to distinguish changes induced by steroidUppsala. treatment per se from those resulting from the reduced

food intake and body weight gain, 43 rats were randomized2 .2. Dose response study into three groups. Animals in one group (n514) were

given daily intramuscular injections with 15 mg/kg nan-A dose–response study was undertaken to probe the drolone decanoate in peanut oil. This dose was chosen

effect of different doses of nandrolone decanoate on body because it effectively caused a reduction in body weightweight and mRNA levels of AgRP and POMC. Rats were gain and POMC mRNA levels. Control rats (n514)randomized into five groups and kept in groups of four. received peanut oil. The third group (n515) also receivedThey received daily i.m. injections of 1 (n58), 5 (n58), peanut oil, but their food intake was restricted to the15 (n58) and 45 (n54) mg/kg nandrolone decanoate or amounts ingested by the nandrolone treated rats. The

J. Lindblom et al. / Brain Research 986 (2003) 139–147 141

animals were kept in groups of two or three animals per to the samples and incubation for 1 h at 48C. The samplescage. were centrifuged for 10 min at 12 0003g in a Beckman

Body weight and collective food intake (i.e. food intake/ GS-15R centrifuge, the supernatant discarded and thecage) was monitored daily during the 14 day treatment. At radioactivity in the remaining pellet counted in a Wallacday 15, the rats were killed by decapitation and the brains 1470 Wizardg-counter.were rapidly removed. Brains destined for melanocortinreceptor autoradiography and in situ hybridization (eight 2 .4. Food and water intakenandrolone decanoate, eight controls and nine food re-stricted) were frozen in chilled (220 to230 8C) 2-methyl A total of 16 animals destined for the food intake studybutane. Sections were collected and were used for in situ were randomized into two groups. Rats were treated withhybridization as described above. The brains destined for peanut oil (n58) or 15 mg/kg nandrolone decanoate (n5peptide analysis (all groupsn56) were removed and the 8). This dose was chosen from the dose response study,following structures dissected using a brain matrix (Activa- where it effectively inhibited body weight gain. At day 10,tional System, Mortella Drive Warren, MI, USA) and when changes in body weight are becoming apparent, therapidly frozen on dry ice: the ventral hypothalamus animals were, just prior to lights off, placed individually in(VHT), nucleus accumbens (Acb), caudate putamen (CPu), metabolic and diuresis cages (Tecniplast, Buguggia, Italy).dorsal hypothalamus (DHT), amygdala (Am), ventral Intake of food and water and excretion of urine and faecestegmental area (VTA), periaqueductal grey area (PAG) and were monitored after 12, 24, 36 and 48 h. Food and watersuperior colliculus (SC). The tissues were stored at280 8C intake were corrected for spillage. Dark and light phaseuntil used. At decapitation, blood samples were collected, data were pooled, and all data were normalized to themixed with 1/10 volume 1% EDTA in 0.9% NaCl, stored weight of the animal.on ice, then centrifuged at 7353g for 2310 min. Theplasma was collected and stored at280 8C until used for 2 .5. Statisticsleptin determination as described below.

Animals destined for the peptide analysis (all groups Body weight data from the dose–response study and then56) were dissected for examination of peripheral organ comparison with food restriction were analyzed by analysismass. Tissues chosen were kidney, adrenal gland, heart, of variance (ANOVA) for repeated measures followed byliver, thymus, spleen, testicles, bi-testicles and abdominal Fisher’s protected least significant difference (PLSD) test,fat pads. where appropriate. In situ hybridization, radioimmunoas-

say and organ weight data were analyzed by factorial2 .3.1. Peptide analysis ANOVA. A correlationZ-test was used to correlate relative

Plasma samples were used to analyze the levels of leptin body weight gain and POMC expression levels in the doseimmunoreactivity (i.r.) using a rat leptin RIA kit (Linco response study. An unpaired Student’st-test was used toResearch, Missouri, USA) according to the instructions of evaluate data from the food and water intake study. APthe manufacturer. value,0.05 was used as the criterion of statistical

The dissected brain tissues were processed by boiling in significance. StatView 4.51 software for Macintosh was1 M acetic acid for 5 min, cooled on ice and homogenised used for the statistical analysis.by sonication using a Branson sonifier, and then reheatedfor 5 min. The homogenates were kept on ice andcentrifuged for 10 min at 12 0003g in a Beckman GS-15R 3 . Resultscentrifuge. The supernatants were collected and dried in avacuum centrifuge. The samples and peptide standards3 .1. Dose response effects of nandrolone decanoatewere dissolved in methanol–0.1 M HCl (1:1, v /v). 25ml treatment on body weight and AgRP/POMC expressionsample or peptide standard were incubated with 100ml levelsrabbit anti-a-MSH (EuroDiagnostica, France) and 100ml

125[ I]a-MSH (̄ 4500 cpm; EuroDiagnostica, France) at There was no difference in body weight gain between4 8C over night. The antia-MSH had a 100% cross- the groups receiving 0.1 or 0.36 ml vehicle (F 50.003,1,10

reactivity with a-MSH and des-acetyl-a-MSH, but less P50.96). Therefore these two groups were merged in thethan 0.004% cross reactivity with ACTH(1–24, 1–39), statistics. The dose–response experiment indicated that theb-MSH andg-MSH. The antiserum and labelled peptide highest 3 doses of nandrolone decanoate used (5, 15 andwere diluted in a 0.05 M sodium phosphate buffer con- 45 mg/kg daily) inhibited body weight gain when ad-taining 0.15 M NaCl, 0.02% sodium azide, 0.1% gelatine, ministered for 14 days (F 57.4, P,0.001). The 5- and4.34

0.1% Triton X-100 and 0.1% bovine serum albumin. 15-mg/kg values were equally potent, but with 15 mg/kgSeparation of free and antibody-bound peptides was per- the effect appeared 1 day earlier than for the lower dose,formed by adding 100ml sheep anti-rabbit antiserum effects seen at day 9 and 10, respectively. The highest(Pharmacia Decanting Suspension, Pharmacia Diagnostics) dose, 45 mg/kg, caused a fast response, reducing the rate

142 J. Lindblom et al. / Brain Research 986 (2003) 139–147

of body weight gain already at day 4. After day 6, theanimals treated with the highest dose started to lose bodyweight. Body weight curves from the dose response studyare presented inFig. 1a.Analysis of the autoradiograms ofthe in situ experiment indicated that there was no signifi-cant effect in Arc AgRP mRNA levels at any dose ofnandrolone treatment (F 50.84, P50.51). Arc POMC4,23

mRNA levels, however, were reduced at the two highestdoses of nandrolone (F 55.14, P,0.01; Fig. 2). More-4,35

over, there was a significant positive correlation betweenrelative body weight gain at day 14 and POMC expressionlevels (Z52.634,P,0.01;Fig. 3). Interestingly, when datafrom the food restricted animals were added to thisanalysis (see below), there was no correlation whatsoever(Z50.649,P50.52).

3 .2. Effects of nandrolone decanoate and foodrestriction on body weight and effects of nandrolonedecanoate on continuous food intake

Treatment of rats with daily injections of 15 mg/kgnandrolone decanoate for 14 days caused a reduction in therate of body weight gain (F 55.2, P,0.05). Post hoc2,28

analysis indicated that the effect was significant alreadyfrom day 6. The food restricted animals’ growth rate wassomewhat lower than the nandrolone treated animals’; forthe latter group the effect was evident already at day 5.Body weight data from this experiment are summarized inFig. 1b.

Average daily food intake was computed by dividing themeasured food consumption/cage with the number ofanimals in each cage. The food restricted group wasexcluded from the analysis as the food intake in this groupwas paired to that of the nandrolone treated animals.Analysis of variance for repeated measurements indicatedthat there was a significant difference in food consumptionbetween the vehicle and the nandrolone treated groups(F 55.76, P,0.001; Fig. 1c). Post hoc analysis indi-1,13

cated that—with two exceptions—the food intake wassignificantly reduced from day 9 in the nandrolone treatedgroup. However, there were large distributions in meanfood intake, probably stemming from intergroup bodyweight variations and intragroup variations in food spil-lage.

Fig. 1. Changes in body weight during the 14 days of treatment. (a)Different doses of nandrolone decanoate treatment compared to vehicle.(b) Nandrolone decanoate treatment (15 mg/kg) compared with vehicle3 .3. Effects of nandrolone decanoate and foodand food restriction. All values in (a) and (b) are normalized to initial

restriction on AgRP and POMC mRNA levels body weight. (c) Daily food intake after nandrolone decanoate treatment(15 mg/kg) compared to vehicle. The data in (c) are presented as g/cagedivided by the number of rats. * Significantly different from vehicleAnalysis of the autoradiograms of the in situ hybridiza-

[treated animals,P,0.05. Significantly different from vehicle treatedtion experiment confirmed our previous results that nan-animals,P,0.01.drolone treatment (15 mg/kg) does not cause any change

in Arc AgRP mRNA levels. The same result was obtainedfor the food restricted rats (F 50.89, P50.43). Arc 4.6,P,0.05). Representative autoradiograms from the in2,20

POMC mRNA levels, however, were decreased in nan- situ hybridization experiment are shown inFig. 4, and thedrolone treated rats, but not in food restricted rats (F 5 results are summarized inFig. 5.2,20

J. Lindblom et al. / Brain Research 986 (2003) 139–147 143

35Fig. 2. Levels of AgRP (a) and POMC (b) [ S]oligonucleotide binding in the arcuate nucleus after treatment with 1, 5, 15 and 45 mg/kg nandrolone[decanoate or vehicle for 14 days. The data are normalized to the mean of vehicle levels. Columns and error bars represent mean6S.E.M. Significantly

different from vehicle treated animals,P,0.01.

3 .4. Effects of nandrolone decanoate on plasma leptin group. In the Acb (F 50.74,P50.49), DHT (F 51.3,2,15 2,15

and brain a-MSH levels P50.30), VHT (F 50.05,P50.95), Am (F 51.8, P52,15 2,13

0.20), VTA (F 52.19, P50.15), PAG (F 50.06, P52,15 2,14

Results from the plasma leptin analysis showed that 0.94) and SC (F 50.44, P50.65) there was no signifi-2,13

there was a significant difference between the groups cant change. For control animals, meana-MSH i.r. levels(F 55.4, P,0.05). Post-hoc analysis indicated that both were in the Acb 1.02, DHT 60.45, VHT 23.23, Am 2.20,2,15

nandrolone treated (P,0.05) and food restricted rats (P, CPu 0.36, VTA 0.91, PAG 7.12 and SC 0.28 fmol /mg.0.01) had reduced leptin levels compared to vehicle treateda-MSH i.r. data are summarized inFig. 6b.animals. There was no significant difference betweennandrolone treated and food restricted animals (P50.31). 3 .5. Effects of nandrolone decanoate on peripheralThe results from the leptin assay are presented inFig. 6a. organ sizea-MSH immunoreactivity (i.r.) levels were significantly

affected in the CPu (F 56.6, P,0.05), where post-hoc The effect of the nandrolone treatment and food restric-2,13

analysis indicated an increase in the nandrolone treated tion on peripheral organ mass are shown inTable 1.The

Fig. 3. Correlation of Arc POMC mRNA levels (normalized to the mean of controls) and the relative body weight gain after treatment with 1, 5, 15 and 45mg/kg nandrolone treatment or vehicle for 14 days (Z52.634,P,0.01). When data from the food restriction study ([) was added to the statistical analysis,the correlation was lost (Z50.649,P50.52).

144 J. Lindblom et al. / Brain Research 986 (2003) 139–147

3 .6. Effects of nandrolone decanoate treatment for 10days on food and water intake

The feeding study indicated that the nandrolone treatedanimals had a lower food intake during the dark phasecompared to the controls. During the light phase however,there was no difference in food intake. The oppositephenomenon was observed for water intake, which wasincreased during the light phase but not during the darkphase. These differences were evident also when statisticswere performed on absolute numbers (instead of usingnormalized numbers as presented here), proving that theeffect is not an artifact due to differences in body weightbetween the two groups. Faeces and urine excretionparallelled food and water intake, respectively. Faecesexcretion was reduced during both the dark and lightphase, and urine excretion increased during the light phase.The data from the feeding study is presented inTable 2.

Fig. 4. Representative autoradiograms from agouti-related protein(AgRP) and pro-opiomelanocortin (POMC) in situ hybridization in thearcuate nucleus (Arc) of control (vehicle), food restricted and nandrolone

4 . Discussiondecanoate treated animals. Scale bar: 0.5 mm.

We have here investigated the effect of the anabolicandrogenic steroid nandrolone decanoate on feeding be-

nandrolone treatment had a dramatic effect on kidney and haviour, body weight and different components of thethymus mass. Kidney mass thus increased (F 526.3, melanocortin system in the male rat brain. In an initial2,15

P,0.001), while thymus mass decreased (F 563.2,P, dose–response study we concluded that 14 days of treat-2,15

0.001). There was also a slight but significant decrease in ment with 5, 15 and 45 mg/kg nandrolone decanoatethymus mass in the food restricted group. Both nandrolone significantly reduces body weight gain. Moreover, thetreatment and food restriction caused a decrease in ab- highest two doses also caused a reduction in Arc POMCdominal fat (F 512.5, P,0.001) and liver (F 512.8, mRNA levels, whereas no dose affected AgRP transcrip-2,15 2,15

P,0.001) mass. Neither of the treatments had any effect tion. In subsequent studies, we chose the dose 15 mg/kgon heart (F 50.56,P50.58), adrenal gland (F 51.82, nandrolone decanoate, based on the results from the dose–2,15 2,15

P50.20), testicles (F 50.77, P50.48), bitesticles response study. The 15 mg/kg dose of nandrolone de-2,15

(F 51.0, P50.39) or spleen (F 50.26, P50.77) canoate corresponds well to the heavy abuse of AAS[20].2,15 2,15

mass. Treatment with nandrolone for 10 days reduced foodintake during the dark (active) phase, supporting the notionthat anabolic steroids reduce body weight at least partly by

inhibiting food intake. This was also supported by the factthat nandrolone treatment caused a decrease in abdominalfat mass that was identical to that of the food restrictedrats. The nandrolone treatment also affected the thymusand kidney mass. Here we have shown that these phenom-ena are not results of the lower food intake and bodyweight in steroid treated animals.

The present results indicate that a high dose of nan-drolone decanoate causes a reduction of POMC mRNAlevels in the caudal (B23.8 mm) Arc. Food restriction,weight loss and leptin are factors known to affect POMCbiosynthesis[7]. The effect of nandrolone treatment on Arc

Fig. 5. Levels of agouti-related protein (AgRP) and pro-opiomelanocortin POMC mRNA levels was not observed in the food-re-35(POMC) [ S]oligonucleotide binding in the arcuate nucleus of control stricted group. Moreover, the correlation between relative

(filled bars), food restricted (squared bars) and nandrolone treated (slashedweight gain at day 14 and relative POMC mRNA levelsbars) animals. The data are normalized to the mean of control levels.

was lost when data from the food-restricted rats wasColumns and error bars represent mean6S.E.M. * Significantly differentc pooled with the dose–response data. The nandrolonefrom vehicle treated animals,P,0.05. Significantly different from food

restricted animals,P,0.01. treatment significantly reduced plasma leptin levels. How-

J. Lindblom et al. / Brain Research 986 (2003) 139–147 145

Fig. 6. (a) Levels of plasma leptin i.r. for vehicle treated, food restricted and nandrolone treated (15 mg/kg) rats after 14 days of treatment. (b) Levels ofa-MSH i.r. in eight different brain regions of control, food restricted and nandrolone decanoate treated animals. The data are normalized to the mean of

f [control levels. Columns and error bars represent mean6S.E.M. Significantly different from vehicle treated animals,P,0.01. Significantly different fromfood restricted animals,P,0.05. Abbreviations: Acb, nucleus accumbens; CPu, caudate putamen; DHT, dorsal hypothalamus; HT, ventral hypothalamus;Am, amygdala; VTA, ventral tegmental area; PAG, periaqueductal grey area; SC, superior colliculus.

T able 1 ever, so did food restriction, and the reduction in leptinEffects of nandrolone treatment and food restriction on peripheral organ levels was not different from that of the nandrolone treatedweight rats. Thus, the effects of nandrolone treatment on the

Vehicle Food Nandrolone POMC system could not be attributed to alterations in foodrestriction decanoate intake, body weight or leptin secretion. The lack of any

Heart 1.4360.05 1.3560.03 1.3760.08 effect on POMC or AgRP mRNA levels in the foodfcKidney 2.6160.06 2.5160.08 3.2960.10 restricted group is not surprising, as the present protocol of

Adrenal gland 0.05260.004 0.05260.006 0.04160.005 food restriction was relatively mild, following the foodTesticles 2.9760.07 2.9160.07 2.8560.06

consumption of the nandrolone treated rats (Fig. 1).Bitesticles 0.2760.02 0.2960.01 0.3060.02Hypothalamic POMC levels are believed to fluctuate inSpleen 0.7960.04 0.7860.04 0.7460.05

fcThymus 0.6660.04 0.5660.03* 0.2260.02 a circadian pattern dependent on sex hormones[28,31].f fLiver 13.9160.42 11.0360.43 11.2060.50 Castration of male rats causes a rapid reduction in POMCf fAbdominal fat 6.4060.38 4.8060.21 4.2060.35 mRNA levels, followed by an increase after 1–4 weeks

Absolute weight of peripheral organs after nandrolone decanoate treat- [29]. The drop in POMC mRNA levels 4 days afterment or food restriction. Data are shown as g and are presented ascastration was prevented by administration of testosteronemean6S.E.M.

or estrogen[4], as was the increase in POMC biosynthesis* Significantly different from vehicle treated animals,P,0.05.f 3–4 weeks after castration[2,18]. AASs—including theSignificantly different from vehicle treated animals,P,0.01.c Significantly different from food restricted animals,P,0.01. nonaromatizable 5a-dihydrotestosterone—may exert es-

T able 2Effects of nandrolone treatment on food and water intake and faeces and urine excretion

Food intake (g/kg) Water intake (g/kg) Faeces excretion (g/kg) Urine excretion (g/kg)

Dark Light Dark Light Dark Light Dark Light

Nandrolone 42.863.0 16.561.1 67.465.0 30.062.1 16.961.3 17.261.3 44.164.3 54.663.7Vehicle 52.561.0 16.961.0 68.363.6 21.562.4 21.260.8 21.760.8 37.963.7 42.264.0

P-value 0.008 0.75 0.89 0.02 0.014 0.011 0.30 0.039

Effect on the intake of food and water and the excretion of faeces and urine during dark and light phase after treatment with nandrolone decanoate for10–11 days. Data are shown as g/kg body weight and are presented as mean6S.E.M.

146 J. Lindblom et al. / Brain Research 986 (2003) 139–147

trogen-like effects[13] and estrogen is known to reduce panied by a release of dopamine in mesolimbic terminalPOMC mRNA levels[30]. Moreover, as nandrolone is regions, including the nucleus accumbens[26], as arereadily aromatized, although the enzymatic process is administration of alcohol, amphetamine, cocaine and mor-slower than for testosterone[3], a nandrolone metabolite phine[27]. In fact, patients with eating disorders ofteninteraction with estrogen receptors is a distinct possibility. exhibit addiction to food[23], and dopamine has beenInterestingly, in previous studies showing that high doses suggested to be involved in this phenomenon[11].of testosterone reduce food intake and weight gain this The strong correlation between POMC mRNA levelseffect was attributed to the aromatization of testosterone and body weight gain in nandrolone treated rats was ainto estrogen[9]. However, only a small fraction of POMC quite interesting finding, as a reduced synthesis of theneurons express androgen[8] or estrogen receptors[8,25], anorexigenic POMC producta-MSH would be expected toindicating that sex hormones act indirectly on POMC rather result in an increase in food intake and increasedneurons. In one study, the negative effect of estrogen on gain in body weight. It could be argued that POMC is alsoPOMC biosynthesis was blocked bya -adrenoceptor the precursor of the orexigenic peptideb-endorphin[19]1

blockade, suggesting that the effect is mediated by norad- and that a change in the activity of POMC neurons couldrenergic mechanisms[24]. It should be noted, though, that be interpreted as a shift in the balance ofa-MSH andat the high doses used in the present study, nandrolone andb-endorphin output as suggested elsewhere[22]. Neverthe-its metabolites are likely to interact also with other steroid less, entire deficiency of POMC transcription is correlatedreceptors, such as the progesterone and corticosteroid to obesity in mice[33], indicating that the main effect ofreceptors[13]. Glucocorticoids may inhibit POMC tran- reduced levels of POMC would be increased levels ofscription in the anterior pituitary[5], and AAS may body fat. The present finding reinforces the complexdisplace glucocorticoids from the glucocorticoid receptor patterns of biochemical and neurobiological alterations[32]. However, testosterone and 17a-methyltestosterone caused by nandrolone, the sum of all effects that mayare antiglucocorticoids[13]. Provided that this is true also contribute to the altered behaviours it induces.for nandrolone, we would expect an increase in POMC In summary we have provided evidence that administra-biosynthesis if an interaction with the glucocorticoid tion of supratherapeutical doses of the AAS nandrolonereceptor was relevant in this treatment regimen. decanoate reduces hypothalamic POMC biosynthesis and

The CPu was the only region where a significant increases levels of caudate putamena-MSH i.r., possiblydifference in a-MSH i.r. was observed, the levels of by an interaction with the estrogen receptor. These effectsa-MSH i.r. being doubled after nandrolone treatment. The are specific for nandrolone treatment, as they could not beCPu has an important role in the regulation of motor attributed to the reductions in food intake, body weightbehaviour[17]. Treatment with nandrolone (15 mg/kg for gain or leptin levels. The present results raise the possi-14 days) has been shown to reduce locomotor behaviour in bility that some of the metabolic and behavioural conse-an open-field study[12]. There are a number of studies quences of AAS abuse may be the result of alterations insuggesting that melanocortins have effects in open-field the POMC system.studies, but there is no consensus as to their precise role inlocomotion [35]. However, in the CPu, no fibers con-taining POMC derived peptides have been detected[14],

A cknowledgementsindicating that thea-MSH detected in this area originateseither from adjacent structures (by diffusion) or from the

This work was supported by the Swedish Researchperiphery. Radiolabelleda-MSH has been shown to ac-

Council (04X-05957 and 04X-09459) and Melacure Thera-cumulate in the striatum after peripheral administration[1].

peutics AB. Dr Jonas Lindblom was supported by theThus, the here observed accumulation ofa-MSH in the

Swedish Brain Foundation. We thank Dr Tina Kunz and DrCPu may reflect changes in circulating levels ofa-MSH or

Ingrid Nylander for their technical guidance. We also thankchanges in the permeability of the blood brain barrier.

Helena Wilking and Jenny Nilsson who were of great helpThere was also a tendency for increaseda-MSH i.r. for

in setting up the radioimmunoassay.both nandrolone treated and food restricted rats in the Acband VTA. In the VTA, the effect was close to significant(P50.15). The VTA and Acb are the areas of origin andtermination of the mesolimbic dopamine system. Strong R eferencesevidence exists that the mesolimbic system has a role inthe rewarding effects of positive reinforcers, and that [1] W .A. Banks, A.J. Kastin, Permeability of the blood–brain barrier to

melanocortins, Peptides 16 (1995) 1157–1161.dopamine has a role as a major mediator for motivation[2] M . Blum, J.L. Roberts, S.L. Wardlaw, Androgen regulation ofand rewarding mechanisms in this area[27]. Addictive

proopiomelanocortin gene expression and peptide content in thedrugs are well known strong positive reinforcers, but it is basal hypothalamus, Endocrinology 124 (1989) 2283–2288.also considered that food may function as a positive [3] R .W. Brueggemeier, Male sex hormones, analogs, and antagonists,reinforcer. Thus, the rewarding effects of food are accom- in: M.E. Wolff (Ed.), Burger’s Medicinal Chemistry and Drug

J. Lindblom et al. / Brain Research 986 (2003) 139–147 147

Discovery, 5th Edition, Therapeutic Agents, vol. 3, Wiley, Laguna Intracerebroventricular beta-endorphin increases food intake of rats,Beach, 1996, pp. 445–510. Life Sci. 29 (1981) 1429–1434.

[4] J .A. Chowen, J. Argente, L. Vician, D.K. Clifton, R.A. Steiner, [20] H .G. Pope Jr., D.L. Katz, Affective and psychotic symptomsPro-opiomelanocortin messenger RNA in hypothalamic neurons is associated with anabolic steroid use, Am. J. Psychiatry 145 (1988)increased by testosterone through aromatization to estradiol, Neuro- 487–490.endocrinology 52 (1990) 581–588. [21] H .G. Pope Jr., D.L. Katz, J.I. Hudson, Anorexia nervosa and

[5] J . Drouin, M. Chamberland, J. Charron, L. Jeannotte, M. Nemer, ‘reverse anorexia’ among 108 male bodybuilders, Compr. PsychiatryStructure of the rat pro-opiomelanocortin (POMC) gene, FEBS Lett. 34 (1993) 406–409.

´193 (1985) 54–58. [22] F . Rene, A. Muller, E. Jover, B. Kieffer, B. Koch, J.P. Loeffler,[6] J . Drouin, H.M. Goodman, Most of the coding region of rat ACTH Melanocortin receptors andd-opioid receptor mediate opposite

b-LPH precursor gene lacks intervening sequences, Nature 288 signalling actions of POMC-derived peptides in CATH.a cells, Eur.(1980) 610–613. J. Neurosci. 10 (1998) 1885–1894.

[7] J .K. Elmquist, C.F. Elias, C.B. Saper, From lesions to leptin: [23] E .A. Riley, Eating disorders as addictive behavior. Integrating 12-hypothalamic control of food intake and body weight, Neuron 22 step programs into treatment planning, Nurs. Clin. North Am. 26(1999) 221–232. (1991) 715–726.

[8] M . Fodor, H.A. Delemarre-van de Waal, Are POMC neurons targets [24] R . Rosie, B.E. Sumner, G. Fink, Ana1 adrenergic mechanismfor sex steroids in the arcuate nucleus of the rat?, Neuroreport 12 mediates estradiol stimulation of LHRH mRNA synthesis and(2001) 3989–3991. estradiol inhibition of POMC mRNA synthesis in the hypothalamus

[9] R .T. Gentry, G.N. Wade, Androgenic control of food intake and of the prepubertal female rat, J. Steroid Biochem. Mol. Biol. 49body weight in male rats, J. Comp. Physiol. Psychol. 90 (1976) (1994) 399–406.18–25. [25] S .X. Simonian, D.P. Spratt, A.E. Herbison, Identification and

[10] J .A. Harrold, P.S. Widdowson, G. Williams, Altered energy balance characterization of estrogen receptora-containing neurons project-causes selective changes in melanocortin-4 (MC4-R), but not ing to the vicinity of the gonadotropin-releasing hormone perikaryamelanocortin-3 (MC3-R), receptors in specific hypothalamic re- in the rostral preoptic area of the rat, J. Comp. Neurol. 411 (1999)gions: further evidence that activation of MC4-R is a physiological 346–358.inhibitor of feeding, Diabetes 48 (1999) 267–271. [26] G .P. Smith, Dopamine and food reward, Prog. Psychobiol. Physiol.

[11] L . Hernandez, B.G. Hoebel, Food reward and cocaine increase Psychol. 16 (1995) 83–144.extracellular dopamine in the nucleus accumbens as measured by [27] R . Spanagel, F. Weiss, The dopamine hypothesis of reward: past andmicrodialysis, Life Sci. 42 (1988) 1705–1712. current status, Trends Neurosci. 22 (1999) 521–527.

[12] P . Johansson, A. Lindqvist, F. Nyberg, C. Fahlke, Anabolic an- [28] R .A. Steiner, E. Kabigting, K. Lent, D.K. Clifton, Diurnal rhythm indrogenic steroids affects alcohol intake, defensive behaviors and proopiomelanocortin mRNA in the arcuate nucleus of the male rat,brain opioid peptides in the rat, Pharmacol. Biochem. Behav. 67 J. Neuroendocrinol. 6 (1994) 603–608.(2000) 271–279. [29] S .L. Wardlaw, M. Blum, Biphasic effect of orchiectomy on pro-

¨[13] O .A. Janne, Androgen interaction through multiple steroid receptors, opiomelanocortin gene expression in the hypothalamus, Neuroen-in: G. Lin, L. Erinoff (Eds.), Anabolic Steroid Abuse, National docrinology 52 (1990) 521–526.Institute of Drug Abuse Research Monograph Series, vol. 102, US [30] J .N. Wilcox, J.L. Roberts, Estrogen decreases rat hypothalamicDepartment of Health and Human Services, Rockville, MD, 1990, proopiomelanocortin messenger ribonucleic acid levels, Endocrinol-pp. 178–186. ogy 117 (1985) 2392–2396.

[14] H . Khachaturian, M.E. Lewis, K. Tsou, S.J. Watson,b-Endorphin, [31] P .M. Wise, K. Scarbrough, N.G. Weiland, G.H. Larson, Diurnal¨ ¨a-MSH, ACTH, and related peptides, in: A. Bjorklund, T. Hokfelt pattern of proopiomelanocortin gene expression in the arcuate

(Eds.), Handbook of Chemical Neuroanatomy, GABA and Neuro- nucleus of proestrous, ovariectomized, and steroid-treated rats: apeptides in the CNS, vol. 4, Elsevier, Amsterdam, 1985, pp. 216– possible role in cyclic luteinizing hormone secretion, Mol. Endo-272, Part I. crinol. 4 (1990) 886–892.

[15] E .M. Kim, C.C. Welch, M.K. Grace, C.J. Billington, A.S. Levine, [32] F .C. Wu, Endocrine aspects of anabolic steroids, Clin. Chem. 43Chronic food restriction and acute food deprivation decrease mRNA (1997) 1289–1292.levels of opioid peptides in arcuate nucleus, Am. J. Physiol. 270 [33] L . Yaswen, N. Diehl, M.B. Brennan, U. Hochgeschwender, Obesity(1996) R1019–1024. in the mouse model of pro-opiomelanocortin deficiency responds to

[16] J . Korner, S.L. Wardlaw, S.M. Liu, I.M. Conwell, R.L. Leibel, S.C. peripheral melanocortin, Nature Med. 5 (1999) 1066–1070.Chua Jr., Effects of leptin receptor mutation on Agrp gene expres- [34] J .A. Yu-Yahiro, R.H. Michael, D.V. Nasrallah, B. Schofield, Mor-

fsion in fed and fasted lean and obese (LA/N-fa ) rats, Endocrinolo- phologic and histologic abnormalities in female and male ratsgy 141 (2000) 2465–2471. treated with anabolic steroids, Am. J. Sports Med. 17 (1989)

[17] J .P. Martin, The caudate nuclei and locomotion, Ann. R. Coll. Surg. 686–689.Engl. 38 (1966) 166–175. [35] J .E. Zadina, W.A. Banks, A.J. Kastin, Central nervous system effects

[18] C . Matera, S.L. Wardlaw, Aromatization is not required for androgen of peptides, 1980–1985: a cross-listing of peptides and their centralinduced changes in proopiomelanocortin gene expression in the actions from the first six years of the journal Peptides, Peptides 7hypothalamus, Mol. Brain Res. 27 (1994) 275–280. (1986) 497–537.

[19] L .D. McKay, N.J. Kenney, N.K. Edens, R.H. Williams, S.C. Woods,