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Leptin's effect on hyperactivity: Potential downstream effector mechanisms
Physiology & Behavior 94 (2008) 689–695
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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Leptin's effect on hyperactivity: Potential downstream effector mechanisms J.J.G. Hillebrand a,b,⁎, M.J.H. Kas a, A.A. van Elburg b,c, H.W. Hoek d,e,f, R.A.H. Adan a a
Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Centre, Utrecht, The Netherlands Rintveld Centre for Eating Disorders, Altrecht Mental Health Institute, Zeist, The Netherlands c Rudolf Magnus Institute of Neuroscience, Department of Child and Adolescent Psychiatry, University Medical Centre, Utrecht, The Netherlands d Parnassia Psychiatric Institute, The Hague, The Netherlands e Department of Psychiatry, University Medical Centre Groningen, University of Groningen, The Netherlands f Department of Epidemiology, Columbia University, New York, NY, USA b
A R T I C L E
I N F O
Article history: Received 2 April 2008 Accepted 2 April 2008 Keywords: Activity-based anorexia Anorexia Nervosa Hyperactivity Leptin Translational research Hypothalamus Reward Dopamine NPY Naltrexone
A B S T R A C T Up to 80% of patients with Anorexia Nervosa (AN) demonstrate hyperactivity. Hyperactivity counteracts weight gain during treatment and is associated with poor outcome of the disease. We hypothesized that hyperactivity in AN patients has a neurobiological basis and used an animal model-based translational approach to gain insight in mechanisms underlying this hyperactivity. Previously we and others showed that leptin treatment attenuates hyperactivity in the rat activity-based anorexia (ABA) model. The mechanisms involved in this process are, however, unknown. Here we describe potential downstream effector mechanisms involved in the attenuation of hyperactivity by leptin treatment in ABA rats. © 2008 Elsevier Inc. All rights reserved.
1. Hyperactivity in Anorexia Nervosa patients Excessive physical activity is frequently observed in Anorexia Nervosa (AN) patients and might even be called a hallmark of the disorder, even though not part of the DSM IV criteria for AN [1]. The prevalence of hyperactivity varies widely between 31–80%, due to lack of a clear definition of hyperactivity [2]. Hyperactivity might take several forms and durations, ranging from excessive walking, fidgeting, and subjective or motor restlessness to compulsive exercising. Accordingly, different terms of hyperactivity are being used (i.e. over activity, excessive exercising, compulsive exercise, motor/subjective restlessness) and also different strategies are undertaken to examine this behavior (i.e. semi-structured interviews, self ratings, visual analogue scales (VAS) and accelerometers). At least one study suggests that before becoming ill, AN patients display higher physical activity than healthy controls, suggesting that increased physical activity may underlie AN in a subset of patients [3]. Historically, hyperactivity in AN patients has been regarded as a conscious attempt to lose body weight (BW). Hyperactivity may, ⁎ Corresponding author. ETH Zurich, Institute of Animal Science, Physiology and Behaviour, Schorenstrasse 16 8603 Schwerzenbach Switzerland. Tel.: +41 44 655 73 90; fax: +41 44 655 72 06. E-mail address: [email protected] (J.J.G. Hillebrand). 0031-9384/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.04.011
however, also be triggered by an unconscious biological drive, and may involve parts of several behaviors and an altered neurobiological profile [4]. From an evolutionary point of view, hyperactivity may be an expression of foraging behavior, which in itself is rewarding [5]. During the acute phase of AN, hyperactivity has been found to be negatively correlated with food intake (FI) and to complicate BW gain [6–8]. On the long-term, hyperactivity often stays present despite recovery of BW and may be associated with poor outcome [3,9–12]. Therefore it is important to understand the biological underpinnings of this seemingly paradoxical behavior. 2. Hyperactivity in a rat model of anorexia: the activity-based anorexia model The activity-based anorexia (ABA) model mimics various characteristics of hyperactive AN patients and has been forwarded as a potential animal model of AN. ABA is also called semi starvation-induced hyperactivity, activity stress or activity anorexia and although some differences between the paradigms exist (duration of food access or running wheel access), all studies are referred to as ABA in this paper. In ABA, voluntary access to a running wheel along with restricted feeding (RF) (60–120 min/d) leads to an increase in running wheel activity (RWA) in almost direct proportion to a decrease in FI, resulting in substantial BW loss (N20%). This behavior was already described in
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the 1950s, but was termed ABA and claimed as an animal model of human AN in the 1980s [13]. Not only total RWA is increased during ABA, but also the distribution of activity throughout the day is disturbed. High levels of RWA are observed just prior to food access (often during the light phase), a phenomenon that is called foodanticipatory activity (FAA) [14]. In contrast to ABA rats, control ad libitum fed rats with continuous access to running wheels show stable levels of RWA (without FAA) and increase FI to compensate for increased energy expenditure (EE) due to wheel running. Importantly, control rats on the same RF schedule (60–120 min/d) but without running wheel access eat more than ABA rats in 1 h and show only marginal BW loss [15,16]. In addition to increased RWA, reduced FI and BW loss, ABA rats also show hypothermia, loss of estrous cycle, and stomach ulceration (when BW loss N30%) and will eventually die of emaciation [17,18]. When food is again ad libitum accessible, rats will quickly reduce RWA and
increase FI and BW. Thus the anorexic phenotype in rats can be easily rescued, in contrast to humans [18]. 3. The role of leptin on hyperactivity 3.1. General The identification of leptin, the gene mutated in the ob/ob mouse, opened a major new chapter in feeding research [19]. Leptin (16 kDa), mainly produced by adipocytes, regulates neural circuits controlling feeding behavior, EE (incl. thermogenesis), BW, and neuroendocrine function. Central or peripheral administration of leptin reduces meal size and total FI, decreases BW loss and fat mass loss, and increases EE in rodents [20–22]. The absence of leptin or its receptor, lepr, leads to hyperphagia, obesity, hypoactivity and neuroendocrine and metabolic malfunction [20,21,23,24]. During starvation leptin levels fall rapidly
Fig. 1. Chronic leptin treatment reduces running wheel activity in activity-based anorexia rats, but not in ad libitum fed rats. Running wheel activity (RWA) in rats after vehicle or leptin (4 μg/d) treatment. (A, B) Distribution of RWA during the day in (A) activity-based anorexia (ABA) rats and (B) ad libitum-fed rats with vehicle (black, n = 8/n = 8) or leptin treatment (gray, n = 7/n = 8). Leptin treatment started at d 0. (C, D) Total dark phase RWA per d in (C) ABA rats and (D) ad libitum-fed rats with vehicle (black, n = 8/n = 8) or leptin treatment (white, n = 7/n = 8). (E, F) Total light phase RWA per d in (E) ABA rats and (F) ad libitum-fed rats with vehicle (black, n = 8/n = 8) or leptin treatment (white, n = 7/n = 8). ⁎Different from vehicle. Repeated measurements followed by t tests, p b 0.05 (with permission from [16]).
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(even faster than body fat mass) and adaptive responses like a reduction of EE, suppression of the gonadal and thyroid axis, and activation of the adrenal axis are observed. These effects of starvation are blunted by leptin treatment in both mice and men [24,25]. Leptin conveys information on adiposity, or rather starvation, to the brain where it enters at the level of the hypothalamus. The hypothalamus has long been known as a central regulator of feeding behavior. In the hypothalamic arcuate nucleus (ARC) at least two populations of neurons exist, that seem to play antagonistic roles in control of feeding behavior and EE. Both types of neurons express the long form of lepr, leprb, which is the isoform crucial for leptin action using JAKSTAT and PI3 kinase signaling pathways [26]. In the ARC, binding of leptin to leprb, inhibits neurons expressing agouti-related protein (AgRP) and neuropeptide Y (NPY), both orexigenic neuropeptides, and stimulates neurons expressing proopiomelanocortin (POMC), that encodes the anorexigenic α-melanocyte-stimulating hormone (α-MSH), and cocaine- and amphetamine-regulated transcript (CART) [27]. Leprb is also expressed in other hypothalamic nuclei, like the dorsomedial hypothalamic nucleus (DMH) and the lateral hypothalamus (LH) as well as in the cortex, hippocampus, midbrain, and caudal brainstem [22,28]. 3.2. Leptin and activity-based anorexia During ABA, RWA significantly increases while plasma leptin levels drop [15,29,30]. This observation led us and others to the hypothesis that by increasing circulating leptin levels in ABA rats the typical increase in RWA could be prevented. Indeed, leptin treatment in the ABA model suppresses hyperactivity [16,30]. We showed that female Wistar rats (220 g) chronically treated with 4 μg leptin/d (intracerebroventricular in the lateral ventricle (ilvt) using subcutaneously (sc) implanted osmotic minipumps) during RF (1 h food access/d) and voluntary wheel running do not develop hyperactivity. In fact, both RWA in the dark phase and in the light phase (i.e. FAA) is attenuated in leptin-treated ABA rats (Fig. 1). Furthermore, leptin-treated ABA rats reduced 1 h FI vs. vehicle-treated rats, tended to lose more BW and showed a normal body temperature (instead of hypothermia) during the first four d of the experiment. Unfortunately, reduced FI and increased EE (by thermogenesis) resulted in a rapidly worsening condition. RWA or locomotor activity (LMA) levels in ad libitum fed rats or RF controls were not affected by leptin treatment (Fig. 1) [16]. Using a slightly different design, it was shown before that leptin treatment (31 μg leptin/d sc via osmotic minipumps) also prevents hyperactivity in male ABA rats (230 g), both during the dark and light phase (using a milder food restriction, 60% of baseline FI/d) [30]. Leptin-treated ABA rats in this experiment showed comparable FI intake to vehicle-treated controls because food access was not limited by time. Leptin-treated ABA rats lost a similar amount of BW as vehicle-treated controls despite reduced RWA, suggesting an attenuated reduction of metabolic rate or increased thermogenesis. It was also nicely shown that leptin treatment (by doubling fasting leptin levels, starting at d 5) rescued RWA when ABA had already developed. Interestingly other rodent models of impaired leptin signaling also show deficits in physical activity. For instance, ob/ob mice lacking leptin are obese and show low activity, which can be reversed by leptin treatment [21]. Whereas we and others showed that leptin treatment (4 μg leptin ilvt by chronic infusions) in ad libitum fed rats does not influence RWA or LMA [16,31], it was recently shown that daily injections of 10 μg leptin (ilvt) increase LMA, mainly in the dark phase [32]. In addition, re-expressing lepr specifically in the ARC of lepr lacking mice normalizes their reduced LMA, suggesting that the ARC is involved in leptin-regulated effects on LMA [33]. Attenuation of hyperactivity by leptin treatment in ABA rats, however, suggests that during ABA, leptin treatment affects LMA/RWA oppositely or by different downstream targets. One explanation is that leptin's effects on (hyper)activity depend on the state of energy balance.
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3.3. Leptin and hyperactivity in Anorexia Nervosa patients Plasma and cerebrospinal fluid (csf) leptin levels are low in AN patients, corresponding to reduced BW and (sc) fat mass [34–36]. During BW gain, circulating leptin levels increase and leptin secretion becomes variable, depending on the rate and amount of BW gain. When the patient reaches target BW, leptin levels may be disproportional high (upon adjustment for BMI and fat mass) compared to healthy controls. Hyperleptinemia is, however, not observed universally, which is probably due to between-study differences in treatment units‘ BW gain criteria (amount and rate of BW gain) and differences in leptin measurements (leptin assay used, time of blood sampling) [37]. Hyperleptinemia may imply a risk for renewed BW loss (by reduced caloric intake and increased EE) and poor outcome [38]. A relation between hyperactivity and leptin levels has been observed in ABA rats as well as in AN patients. Subjective measurements of motor restlessness in AN patients are highest during admission, when leptin levels are lowest [30]. Moreover, it has been shown that physical activity of AN patients (as determined by the structured inventory for anorexia and bulimia, SIAB) are negatively correlated with (lg10) leptin levels, with leptin (not BMI) explaining 37% of the variation in physical activity [39]. Leptin levels of (adolescent) AN patients at admission have also been found to be negatively correlated with their physical activity measurements using accelerometers, and with their subjective selfreports of inner restlessness (by VAS) or motor restlessness (by a 5-point Likert scale), and leptin levels have been found to predict all three hyperactivity measurements [40]. We found that expert ratings of hyperactivity (using a VAS examining motor restlessness and excessive exercise), that were validated with accelerometer scores, are more legitimate than adolescent AN patients' self-reports of hyperactivity [41]. At admission, leptin levels of adolescent AN patients are negatively correlated to expert ratings of hyperactivity, while during treatment this relation develops into a positive one in recovering AN patients [42]. 4. Downstream effector mechanisms of leptin 4.1. General As described above, peripheral leptin signals to (amongst other) the ARC where it influences the activity of AgRP/NPY and POMC/CART expressing neurons. In a first attempt to gain more insight in attenuation of hyperactivity by leptin treatment in ABA rats, we analyzed expression levels of these neuropeptides in the ARC. In our standard design we start with 1 d of food deprivation (d 0) followed by a varying number of d of 1 h food access/d (mainly dependent on the rate of BW loss). During RF, food is always presented at dark onset and sacrificing of the rats always takes place 1–2 h before dark onset (and food access). AgRP and NPY mRNA levels are increased and CART mRNA levels are decreased in ABA rats compared to ad libitum fed or RF controls (at ABA d 3), suggesting increased orexigenic signaling [29]. Yet ABA rats eat less than ad libitum fed and RF controls. An interesting transient upregulation of POMC expression has been observed during the first d of ABA (i.e. ABA d 0–2; at 1 d food deprivation, RF d 1–2), followed by a significant down-regulation (at d 3–6). This finding suggests increased activity of the melanocortin (MC) system during the first three d (at d 0– 2) of exposure to the ABA model [15,43]. Chronic ilvt leptin treatment reduces the up-regulation of NPY and AgRP and increases POMC mRNA levels vs. vehicle-treated ABA rats (at d 4) and thus leads to a net result of reduced orexigenic signaling [16]. We further explored the role of systems downstream of leptin on the attenuation of hyperactivity by leptin treatment, starting with the MC system. 4.2. Melanocortins MC binding sites in the ventromedial hypothalamus (VMH) of ABA rats are increased compared to controls (at d 6) [15]. This suggests
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increased sensitivity to anorexigenic α-MSH during ABA, which can be reduced by binding the endogenous inverse agonist AgRP [44]. In two separate experiments we have shown that AgRP(83–132) (5.6 μg/d ilvt) increases 1 h FI of ABA rats, reduces hypothermia, and seems to reduce LMA, thereby attenuating ABA [15,43]. Chronic infusion of αMSH (24 μg/d ilvt) enhances ABA by reducing FI, resulting in increased BW loss. In addition, α-MSH-treated rats show slightly increased FAA [45]. SHU9119 treatment (0.5 μg/d ilvt) does, however, not influence the development of ABA, suggesting that inverse agonism of MC receptors might be important in the development/rescue from ABA, or alternatively that AgRP has an effect on its own, independent of antagonism of MC receptors [43]. Although some of the results above suggest that a hyperactive MC system triggers ABA, these results are not compatible with the effects of leptin treatment on ABA. However, the association of variations in the AgRP gene with AN supports the notion that the MC system affects the susceptibility to develop AN [46]. 4.3. Neuropeptide Y RWA in ad libitum fed rats increases mRNA levels of NPY in the ARC, DMH and medial preoptic area (MPO), similar to weight-matched RF rats [47]. Negative energy balance either achieved by increased EE or reduced FI thus seems to activate the NPY system in the hypothalamus. Centrally injected NPY not only increases FI, but also increases food seeking behavior in rodents and recently it was shown that blocking the Y1 receptor by Y1 antagonist 1229U91 reverses the stimulatory effect of food deprivation on wheel running for food in hamsters [48–50]. The above findings suggest that the NPY system might be involved in (leptin's effect on) hyperactivity in ABA rats. Indeed, daily injections of NPY (10 μg ilvt prior to dark onset) facilitate ABA in 2 h fed female rats [51]. It was shown that NPYtreated ABA rats (do not increase but) reduce FI, lose more BW and are more hyperactive than csf-treated ABA controls. Since NPY-treated ad libitum fed rats increase FI without changing RWA, behavioral effects of NPY treatment seem to depend on the rat's energy state. We could not confirm the above mentioned results of facilitation of ABA by ilvt NPY treatment. In our hands, female ABA rats chronically treated with NPY (10 μg/d ilvt) show no differences in 1 h FI, RWA during dark or light phase, or BW loss (Table 1). Discrepancies between both studies might be explained by differences in the RF paradigm and/ or differences in administration of NPY. Daily injections of NPY prior to food access might be more effective in increasing RWA than chronic infusions as we did. Major differences have been reported between acute and chronic interference with the NPY system [52]. Since the Table 1 Chronic neuropeptide Y (NPY) treatment does not influence activity-based anorexia in rats NPY (μg/d) 7 d survival
0 10 NPY (μg/d)
0 10
50 % (n = 4) 50 % (n = 4)
BW loss
FI (g)
RWA (rev)
(d 0–4)
(Sum d 1–4)
(Sum d 0–4)
22.8% ± 1.8 23.1% ± 1.5
17.5 ± 1.3 17.4 ± 1.2
28045 ± 5400 22820 ± 4786
BW loss
FI (g)
RWA (rev)
(During d 3–4)
(Sum d 3–4)
(Sum d 3–4)
8.4% ± 1.1 8.1% ± 1.0
11.6 ± 0.7 11.7 ± 0.6
18346 ± 3062 14166 ± 2950
After 10 d of ad libitum feeding and wheel running, ilvt cannula were implanted (connected to sc osmotic minipumps delivering neuropeptide Y (NPY)) in female rats (230 g). After surgery food was removed and presented again the next d for 1 h at dark onset (procedures as described in [45]). The exposure to the ABA model lasted for 1 wk. However, because some ABA animals had to be sacrificed before end of the study due to excessive BW loss (N 25%), only data (mean and SE) of the first 5 d of ABA is shown. Although pumps worked immediately after surgery, NPY (10 µg/d) only reached the brain during the second d of RF. Therefore results (mean and SE) are separately presented for RF d 3 and 4. BW= body weight, FI = food intake, RWA = running wheel activity, rev = revolutions.
(presumably) facilitation of ABA by NPY treatment is compatible with the effects of leptin treatment on ABA, the role of the NPY system in ABA deserves further studying. 4.4. Corticotrophin-releasing hormone Another neuropeptide of interest in relation to attenuation of hyperactivity by leptin treatment in ABA rats is corticotrophinreleasing hormone (CRH). Centrally injected CRH reduces FI [53]. Furthermore central leptin injections as well as treadmill running increase CRH mRNA in the paraventricular nucleus of the hypothalamus (PVN) [54,55]. Stressful situations, like ABA, might increase CRH expression and release, leading (in)directly to activation of dopamine (DA) neurons in the ventral tegmental area (VTA) and noradrenergic (NA) neurons in the locus coeruleus (LC), brain areas implicated with reward mechanisms and selective attention [56]. CRH antagonist, α-hCRH, attenuates leptin-induced anorexia and also blocks exercise-induced anorexia [54,57]. We and others found, however, no evidence for increased CRH expression in ABA rats (at d 3 and at 25% BW loss) [29,58,59]. Increased activity of the adrenal axis has nevertheless been described in ABA rats (at d 6 and at 25% BW loss), i.e. increased corticosterone and ACTH levels with increased adrenal gland weight [45,58]. It should be mentioned that these effects have generally been observed when animals were close to emaciation, questioning the supposed involvement of the adrenal axis in development of ABA. The adrenal axis in AN patients is clearly activated, with increased CRH and cortisol levels, which may be related to patients' hyperactivity levels [60,61]. 4.5. Melanin concentrating hormone and orexins Melanin concentrating hormone (MCH) and orexin neurons in the LH regulate feeding, energy metabolism and arousal state [62,63]. Deficiency of either (prepro)MCH or the MCH receptor 1 results in a lean and hyperactive phenotype displaying FAA under RF conditions [64–66]. This suggests that MCH plays an inhibitory role on RWA and is not involved in FAA. In contrast, ablation of orexin neurons results in a narcoleptic, hypophagic and obese phenotype, with diminished FAA under RF conditions, implying that orexin neurons contribute to FAA [67,68]. Expression levels of MCH are slightly increased in ABA rats (at d 3), however, it seems unlikely that MCH plays a major role in attenuation of hyperactivity by leptin treatment [29]. No studies have yet been published on direct pharmacological interference with the MCH system in ABA rats. Preproorexin mRNA does not seem to be changed during ABA (at d 3), but the orexins (receptor) peptide levels might be regulated [29]. Since others reported altered FAA following manipulation with the orexin system [67,68], further studies on orexins' possible role in attenuation of hyperactivity by leptin treatment should be encouraged. 4.6. Opioids and dopamine Voluntary wheel running seems to be rewarding for ABA rats via activation of the opioid system and similarly exercising might be rewarding for AN patients [69,70]. Hypothalamic levels of endogenous opioid β-endorphin are increased in ABA rats when lost 25% BW. βEndorphin, like α-MSH also a product of POMC, seems to be mainly involved in incentive motivation to acquire food reinforcers, rather than in homeostatic control of feeding behavior (like α-MSH) [71]. Antagonizing the opioid system with naloxone blocks RWA in ad libitum fed rats [72], and μ-opioid receptor deficient mice (with reduced β-endorphin signaling) display attenuated FAA during RF [5]. We attempted to attenuate the development of hyperactivity in ABA rats by chronic treatment with opioid antagonist naltrexone (NTX). Chronic peripheral NTX treatment (0, 0.3 or 1.0 mg/kg/d) did, however, not influence RWA, FI or BW loss in female ABA rats on a 1 h feeding schedule (Table 2).
J.J.G. Hillebrand et al. / Physiology & Behavior 94 (2008) 689–695 Table 2 Chronic naltrexone (NTX) treatment does not influence activity-based anorexia in rats NTX (mg/kg/d)
0 0.3 1.0
6 d survival
100% (n = 8) 75% (n = 6) 62.5% (n = 5)
BW loss
FI (g)
RWA (rev)
(d 0–3)
(Sum d 1–3)
(Sum d 0–3)
18.0% ± 1.3 20.3% ± 1.3 19.1% ± 1.7
13.9 ± 1.4 12.7 ± 1.0 13.0 ± 0.4
20776 ± 4761 26103 ± 5616 23687 ± 5529
After 10 d of ad libitum feeding and wheel running, osmotic minipumps delivering naltrexone (NTX) at a dose of 0, 0.3 or 1.0 mg/kg/d sc were implanted in female rats (230 g). After surgery food was removed and presented again the next d, for 1 h at dark onset (procedures as described in [89]). The exposure to the ABA model lasted for 6 d. However, because 5 NTX-treated rats had to be sacrificed before end of the study due to excessive BW loss (N25%), only data (mean and SE) of the first 4 d of ABA is shown. BW = body weight, FI=food intake, RWA = running wheel activity, rev=revolutions.
Not only wheel running, but also RF influences reward. For example, leptin itself reverses RF-induced sensitization of brain stimulation reward in rats, and attenuates drug-seeking behavior after RF [73,74]. These findings suggest the existence of a pathway between peripheral leptin that signals adiposity and the midbrain DA system, involved in motivational and rewarding aspects of drugs of abuse and exploration [75]. The attenuation of RWA in leptin-treated ABA rats might be explained by a direct effect of leptin on the midbrain DA system. Lepr are present on DA VTA neurons projecting to the nucleus accumbens (NAcc) [76,77]. Leptin infusion into the rat VTA reduces firing of DA neurons, leading to decreased DA levels in the NAcc and consequently reduced FI without affecting LMA [77,78]. Long-term genetic knockdown of lepr in the VTA using an adenoassociated virus containing short-hairpin lepr RNA (AAV-shlepr) increases FI without affecting BW. It has been reported that these knockdown rats are more active, especially in the dark phase, than rats injected with the control virus and are also more sensitive to highly palatable food [77]. This suggests that decreased lepr signaling increases LMA followed by increased FI (or vice versa) and, likewise that increased leptin signaling in the VTA might underlie attenuation of hyperactivity by central or peripheral leptin treatment in ABA rats. Future studies should therefore be aimed at direct interference with leptin signaling in the VTA in ABA rats. Dopamine turnover in the medial basal hypothalamus is reduced during RF, but seems increased or normalized in ABA rats (at 30% BW loss) [79]. The attenuation of hyperactivity by leptin treatment compares with the effects of pimozide treatment in ABA rats, a drug with strong dopamine receptor 2 (D2) affinity [80]. Ill AN patients have reduced csf levels of DA metabolite homovanillic acid, which persist after recovery [81]. In addition, recovered AN patients have been found to display increased D2/D3 receptor binding in the anteroventral striatum (including NAcc), which may imply altered reward processing and may affect physical activity and eating behavior in AN [82]. 4.7. Serotonin Physical activity and RF are both related to increased serotonergic (5-hydroxytryptamine, 5HT) turnover [83,84]. ABA rats (at 30% BW loss) show increased 5HT activity, with stable or increased 5HT levels and increased levels of 5HT metabolite 5-hydroxy-indole-acetic acid (5HIAA) in the medial basal hypothalamus [79]. Reducing 5HT activity by treating ABA rats with 8-OH-DPAT, an agonist of the 5HT1A autoreceptor, does not influence FI but reduces RWA (both in the dark and light phase) and thereby attenuates BW loss [85]. Oppositely, D-fenfluramine, a 5HT releaser and uptake inhibitor does not affect RWA, but reduces FI and thereby increases BW loss in ABA rats [86,87]. We, however, could not replicate these effects in our ABA design [88]. The data mentioned above suggests that a reduction of 5HT activity attenuates ABA. Interestingly, both ABA rats and AN patients reduce
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hyperactivity when treated with the atypical antipsychotic olanzapine, antagonizing both DA and 5HT systems [89]. The 5HT system has been extensively studied in AN patients. Csf levels of 5HIAA are reduced in ill AN patients, but elevated after recovery in AN patients, suggesting that disturbed 5HT signaling might play a role in the pathogenesis and/or maintenance of the disorder. Functional brain imaging has furthermore confirmed pre-existing or persistent changes in 5HT receptor function that suggest impaired signaling in brain reward centers in recovered AN patients [90]. 5. Discussion Hyperactivity is frequently observed in AN patients and influences treatment outcome by hindering BW gain and increasing the probability of relapse. We took a translational approach to gain knowledge of this particular aspect of the pathogenesis of AN by mimicking hyperactivity in the ABA model. The ABA model is considered an animal model of AN with considerable face validity, nevertheless, it is used to explain only certain aspects of the disorder, present in a subset of AN patients. Leptin treatment attenuates hyperactivity in ABA rats and has for this reason been suggested as a putative therapeutic agent to treat severe hyperactivity in AN patients [30]. Since the first aim of AN treatment is to increase BW and obtain normal eating behavior, leptin treatment (if applicable) should certainly not be used during the initial phases of treatment, but (if so) only once BW and FI have been normalized. In the above we gave an overview of possible downstream effectors of leptin's effect on hyperactivity. We, however, want to emphasize that this overview includes most obvious downstream effectors but is not complete (lacking e.g. ghrelin, brain derived neurotrophic factor, histamine, and noradrenaline). Evidence has been reported that during ad libitum feeding, the ARC may be involved in leptin's effect on LMA [33]. MC peptides, derived from the ARC, do play a role in ABA, but mainly affect FI not RWA. ARC NPY may, however, be one of leptin's downstream candidates involved in adapted FI and RWA during ABA. The strong effect of leptin treatment on RWA in ABA rats could be mediated in the VTA, where leptin can bind lepr on DA neurons, consequently silencing these neurons and reducing motivation to run. Future studies should investigate the precise role of these neurons during ABA, by focusing on direct effects of leptin as well as hypothalamic neuropeptides (e.g. orexins, CART) on these neurons in relation to hyperactivity. Molecular determinants of attenuation of hyperactivity by leptin treatment might also be further unraveled using specific mice strains of interest, now that the mouse model of ABA has been developed [91]. Furthermore, since hyperactivity in AN patients is also related to e.g. an anxious phenotype, neurobiological systems underlying hyperactivity and anxiety might be similar or at least connected [6]. Further unraveling of the behavioral phenotype of AN patients offers additional challenging translational opportunities to study mechanisms underlying the hyperactivity in AN. References [1] American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 4th ed. Washington (DC): APA; 1994. [2] Hebebrand J, Exner C, Hebebrand K, Holtkamp C, Casper RC, Remschmidt H, et al. Hyperactivity in patients with anorexia nervosa and in semistarved rats: evidence for a pivotal role of hypoleptinemia. Physiol Behav 2003;79:25–37. [3] Kron L, Katz JL, Gorzynski G, Weiner H. Hyperactivity in anorexia nervosa: a fundamental clinical feature. Compr Psychiatry 1978;19:433–40. [4] Davis C. Eating disorders and hyperactivity: a psychobiological perspective. Can J Psychiatry 1997;42:168–75. [5] Kas MJ, van den Bos R, Baars AM, Lubbers M, Lesscher HM, Hillebrand JJ, et al. Muopioid receptor knockout mice show diminished food-anticipatory activity. Eur J Neurosci 2004;20:1624–32. [6] Holtkamp K, Hebebrand J, Herpertz-Dahlmann B. The contribution of anxiety and food restriction on physical activity levels in acute anorexia nervosa. Int J Eat Disord 2004;36:163–71.
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Contents lists available at ScienceDirect
Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Leptin's effect on hyperactivity: Potential downstream effector mechanisms J.J.G. Hillebrand a,b,⁎, M.J.H. Kas a, A.A. van Elburg b,c, H.W. Hoek d,e,f, R.A.H. Adan a a
Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Centre, Utrecht, The Netherlands Rintveld Centre for Eating Disorders, Altrecht Mental Health Institute, Zeist, The Netherlands c Rudolf Magnus Institute of Neuroscience, Department of Child and Adolescent Psychiatry, University Medical Centre, Utrecht, The Netherlands d Parnassia Psychiatric Institute, The Hague, The Netherlands e Department of Psychiatry, University Medical Centre Groningen, University of Groningen, The Netherlands f Department of Epidemiology, Columbia University, New York, NY, USA b
A R T I C L E
I N F O
Article history: Received 2 April 2008 Accepted 2 April 2008 Keywords: Activity-based anorexia Anorexia Nervosa Hyperactivity Leptin Translational research Hypothalamus Reward Dopamine NPY Naltrexone
A B S T R A C T Up to 80% of patients with Anorexia Nervosa (AN) demonstrate hyperactivity. Hyperactivity counteracts weight gain during treatment and is associated with poor outcome of the disease. We hypothesized that hyperactivity in AN patients has a neurobiological basis and used an animal model-based translational approach to gain insight in mechanisms underlying this hyperactivity. Previously we and others showed that leptin treatment attenuates hyperactivity in the rat activity-based anorexia (ABA) model. The mechanisms involved in this process are, however, unknown. Here we describe potential downstream effector mechanisms involved in the attenuation of hyperactivity by leptin treatment in ABA rats. © 2008 Elsevier Inc. All rights reserved.
1. Hyperactivity in Anorexia Nervosa patients Excessive physical activity is frequently observed in Anorexia Nervosa (AN) patients and might even be called a hallmark of the disorder, even though not part of the DSM IV criteria for AN [1]. The prevalence of hyperactivity varies widely between 31–80%, due to lack of a clear definition of hyperactivity [2]. Hyperactivity might take several forms and durations, ranging from excessive walking, fidgeting, and subjective or motor restlessness to compulsive exercising. Accordingly, different terms of hyperactivity are being used (i.e. over activity, excessive exercising, compulsive exercise, motor/subjective restlessness) and also different strategies are undertaken to examine this behavior (i.e. semi-structured interviews, self ratings, visual analogue scales (VAS) and accelerometers). At least one study suggests that before becoming ill, AN patients display higher physical activity than healthy controls, suggesting that increased physical activity may underlie AN in a subset of patients [3]. Historically, hyperactivity in AN patients has been regarded as a conscious attempt to lose body weight (BW). Hyperactivity may, ⁎ Corresponding author. ETH Zurich, Institute of Animal Science, Physiology and Behaviour, Schorenstrasse 16 8603 Schwerzenbach Switzerland. Tel.: +41 44 655 73 90; fax: +41 44 655 72 06. E-mail address: [email protected] (J.J.G. Hillebrand). 0031-9384/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.04.011
however, also be triggered by an unconscious biological drive, and may involve parts of several behaviors and an altered neurobiological profile [4]. From an evolutionary point of view, hyperactivity may be an expression of foraging behavior, which in itself is rewarding [5]. During the acute phase of AN, hyperactivity has been found to be negatively correlated with food intake (FI) and to complicate BW gain [6–8]. On the long-term, hyperactivity often stays present despite recovery of BW and may be associated with poor outcome [3,9–12]. Therefore it is important to understand the biological underpinnings of this seemingly paradoxical behavior. 2. Hyperactivity in a rat model of anorexia: the activity-based anorexia model The activity-based anorexia (ABA) model mimics various characteristics of hyperactive AN patients and has been forwarded as a potential animal model of AN. ABA is also called semi starvation-induced hyperactivity, activity stress or activity anorexia and although some differences between the paradigms exist (duration of food access or running wheel access), all studies are referred to as ABA in this paper. In ABA, voluntary access to a running wheel along with restricted feeding (RF) (60–120 min/d) leads to an increase in running wheel activity (RWA) in almost direct proportion to a decrease in FI, resulting in substantial BW loss (N20%). This behavior was already described in
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the 1950s, but was termed ABA and claimed as an animal model of human AN in the 1980s [13]. Not only total RWA is increased during ABA, but also the distribution of activity throughout the day is disturbed. High levels of RWA are observed just prior to food access (often during the light phase), a phenomenon that is called foodanticipatory activity (FAA) [14]. In contrast to ABA rats, control ad libitum fed rats with continuous access to running wheels show stable levels of RWA (without FAA) and increase FI to compensate for increased energy expenditure (EE) due to wheel running. Importantly, control rats on the same RF schedule (60–120 min/d) but without running wheel access eat more than ABA rats in 1 h and show only marginal BW loss [15,16]. In addition to increased RWA, reduced FI and BW loss, ABA rats also show hypothermia, loss of estrous cycle, and stomach ulceration (when BW loss N30%) and will eventually die of emaciation [17,18]. When food is again ad libitum accessible, rats will quickly reduce RWA and
increase FI and BW. Thus the anorexic phenotype in rats can be easily rescued, in contrast to humans [18]. 3. The role of leptin on hyperactivity 3.1. General The identification of leptin, the gene mutated in the ob/ob mouse, opened a major new chapter in feeding research [19]. Leptin (16 kDa), mainly produced by adipocytes, regulates neural circuits controlling feeding behavior, EE (incl. thermogenesis), BW, and neuroendocrine function. Central or peripheral administration of leptin reduces meal size and total FI, decreases BW loss and fat mass loss, and increases EE in rodents [20–22]. The absence of leptin or its receptor, lepr, leads to hyperphagia, obesity, hypoactivity and neuroendocrine and metabolic malfunction [20,21,23,24]. During starvation leptin levels fall rapidly
Fig. 1. Chronic leptin treatment reduces running wheel activity in activity-based anorexia rats, but not in ad libitum fed rats. Running wheel activity (RWA) in rats after vehicle or leptin (4 μg/d) treatment. (A, B) Distribution of RWA during the day in (A) activity-based anorexia (ABA) rats and (B) ad libitum-fed rats with vehicle (black, n = 8/n = 8) or leptin treatment (gray, n = 7/n = 8). Leptin treatment started at d 0. (C, D) Total dark phase RWA per d in (C) ABA rats and (D) ad libitum-fed rats with vehicle (black, n = 8/n = 8) or leptin treatment (white, n = 7/n = 8). (E, F) Total light phase RWA per d in (E) ABA rats and (F) ad libitum-fed rats with vehicle (black, n = 8/n = 8) or leptin treatment (white, n = 7/n = 8). ⁎Different from vehicle. Repeated measurements followed by t tests, p b 0.05 (with permission from [16]).
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(even faster than body fat mass) and adaptive responses like a reduction of EE, suppression of the gonadal and thyroid axis, and activation of the adrenal axis are observed. These effects of starvation are blunted by leptin treatment in both mice and men [24,25]. Leptin conveys information on adiposity, or rather starvation, to the brain where it enters at the level of the hypothalamus. The hypothalamus has long been known as a central regulator of feeding behavior. In the hypothalamic arcuate nucleus (ARC) at least two populations of neurons exist, that seem to play antagonistic roles in control of feeding behavior and EE. Both types of neurons express the long form of lepr, leprb, which is the isoform crucial for leptin action using JAKSTAT and PI3 kinase signaling pathways [26]. In the ARC, binding of leptin to leprb, inhibits neurons expressing agouti-related protein (AgRP) and neuropeptide Y (NPY), both orexigenic neuropeptides, and stimulates neurons expressing proopiomelanocortin (POMC), that encodes the anorexigenic α-melanocyte-stimulating hormone (α-MSH), and cocaine- and amphetamine-regulated transcript (CART) [27]. Leprb is also expressed in other hypothalamic nuclei, like the dorsomedial hypothalamic nucleus (DMH) and the lateral hypothalamus (LH) as well as in the cortex, hippocampus, midbrain, and caudal brainstem [22,28]. 3.2. Leptin and activity-based anorexia During ABA, RWA significantly increases while plasma leptin levels drop [15,29,30]. This observation led us and others to the hypothesis that by increasing circulating leptin levels in ABA rats the typical increase in RWA could be prevented. Indeed, leptin treatment in the ABA model suppresses hyperactivity [16,30]. We showed that female Wistar rats (220 g) chronically treated with 4 μg leptin/d (intracerebroventricular in the lateral ventricle (ilvt) using subcutaneously (sc) implanted osmotic minipumps) during RF (1 h food access/d) and voluntary wheel running do not develop hyperactivity. In fact, both RWA in the dark phase and in the light phase (i.e. FAA) is attenuated in leptin-treated ABA rats (Fig. 1). Furthermore, leptin-treated ABA rats reduced 1 h FI vs. vehicle-treated rats, tended to lose more BW and showed a normal body temperature (instead of hypothermia) during the first four d of the experiment. Unfortunately, reduced FI and increased EE (by thermogenesis) resulted in a rapidly worsening condition. RWA or locomotor activity (LMA) levels in ad libitum fed rats or RF controls were not affected by leptin treatment (Fig. 1) [16]. Using a slightly different design, it was shown before that leptin treatment (31 μg leptin/d sc via osmotic minipumps) also prevents hyperactivity in male ABA rats (230 g), both during the dark and light phase (using a milder food restriction, 60% of baseline FI/d) [30]. Leptin-treated ABA rats in this experiment showed comparable FI intake to vehicle-treated controls because food access was not limited by time. Leptin-treated ABA rats lost a similar amount of BW as vehicle-treated controls despite reduced RWA, suggesting an attenuated reduction of metabolic rate or increased thermogenesis. It was also nicely shown that leptin treatment (by doubling fasting leptin levels, starting at d 5) rescued RWA when ABA had already developed. Interestingly other rodent models of impaired leptin signaling also show deficits in physical activity. For instance, ob/ob mice lacking leptin are obese and show low activity, which can be reversed by leptin treatment [21]. Whereas we and others showed that leptin treatment (4 μg leptin ilvt by chronic infusions) in ad libitum fed rats does not influence RWA or LMA [16,31], it was recently shown that daily injections of 10 μg leptin (ilvt) increase LMA, mainly in the dark phase [32]. In addition, re-expressing lepr specifically in the ARC of lepr lacking mice normalizes their reduced LMA, suggesting that the ARC is involved in leptin-regulated effects on LMA [33]. Attenuation of hyperactivity by leptin treatment in ABA rats, however, suggests that during ABA, leptin treatment affects LMA/RWA oppositely or by different downstream targets. One explanation is that leptin's effects on (hyper)activity depend on the state of energy balance.
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3.3. Leptin and hyperactivity in Anorexia Nervosa patients Plasma and cerebrospinal fluid (csf) leptin levels are low in AN patients, corresponding to reduced BW and (sc) fat mass [34–36]. During BW gain, circulating leptin levels increase and leptin secretion becomes variable, depending on the rate and amount of BW gain. When the patient reaches target BW, leptin levels may be disproportional high (upon adjustment for BMI and fat mass) compared to healthy controls. Hyperleptinemia is, however, not observed universally, which is probably due to between-study differences in treatment units‘ BW gain criteria (amount and rate of BW gain) and differences in leptin measurements (leptin assay used, time of blood sampling) [37]. Hyperleptinemia may imply a risk for renewed BW loss (by reduced caloric intake and increased EE) and poor outcome [38]. A relation between hyperactivity and leptin levels has been observed in ABA rats as well as in AN patients. Subjective measurements of motor restlessness in AN patients are highest during admission, when leptin levels are lowest [30]. Moreover, it has been shown that physical activity of AN patients (as determined by the structured inventory for anorexia and bulimia, SIAB) are negatively correlated with (lg10) leptin levels, with leptin (not BMI) explaining 37% of the variation in physical activity [39]. Leptin levels of (adolescent) AN patients at admission have also been found to be negatively correlated with their physical activity measurements using accelerometers, and with their subjective selfreports of inner restlessness (by VAS) or motor restlessness (by a 5-point Likert scale), and leptin levels have been found to predict all three hyperactivity measurements [40]. We found that expert ratings of hyperactivity (using a VAS examining motor restlessness and excessive exercise), that were validated with accelerometer scores, are more legitimate than adolescent AN patients' self-reports of hyperactivity [41]. At admission, leptin levels of adolescent AN patients are negatively correlated to expert ratings of hyperactivity, while during treatment this relation develops into a positive one in recovering AN patients [42]. 4. Downstream effector mechanisms of leptin 4.1. General As described above, peripheral leptin signals to (amongst other) the ARC where it influences the activity of AgRP/NPY and POMC/CART expressing neurons. In a first attempt to gain more insight in attenuation of hyperactivity by leptin treatment in ABA rats, we analyzed expression levels of these neuropeptides in the ARC. In our standard design we start with 1 d of food deprivation (d 0) followed by a varying number of d of 1 h food access/d (mainly dependent on the rate of BW loss). During RF, food is always presented at dark onset and sacrificing of the rats always takes place 1–2 h before dark onset (and food access). AgRP and NPY mRNA levels are increased and CART mRNA levels are decreased in ABA rats compared to ad libitum fed or RF controls (at ABA d 3), suggesting increased orexigenic signaling [29]. Yet ABA rats eat less than ad libitum fed and RF controls. An interesting transient upregulation of POMC expression has been observed during the first d of ABA (i.e. ABA d 0–2; at 1 d food deprivation, RF d 1–2), followed by a significant down-regulation (at d 3–6). This finding suggests increased activity of the melanocortin (MC) system during the first three d (at d 0– 2) of exposure to the ABA model [15,43]. Chronic ilvt leptin treatment reduces the up-regulation of NPY and AgRP and increases POMC mRNA levels vs. vehicle-treated ABA rats (at d 4) and thus leads to a net result of reduced orexigenic signaling [16]. We further explored the role of systems downstream of leptin on the attenuation of hyperactivity by leptin treatment, starting with the MC system. 4.2. Melanocortins MC binding sites in the ventromedial hypothalamus (VMH) of ABA rats are increased compared to controls (at d 6) [15]. This suggests
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increased sensitivity to anorexigenic α-MSH during ABA, which can be reduced by binding the endogenous inverse agonist AgRP [44]. In two separate experiments we have shown that AgRP(83–132) (5.6 μg/d ilvt) increases 1 h FI of ABA rats, reduces hypothermia, and seems to reduce LMA, thereby attenuating ABA [15,43]. Chronic infusion of αMSH (24 μg/d ilvt) enhances ABA by reducing FI, resulting in increased BW loss. In addition, α-MSH-treated rats show slightly increased FAA [45]. SHU9119 treatment (0.5 μg/d ilvt) does, however, not influence the development of ABA, suggesting that inverse agonism of MC receptors might be important in the development/rescue from ABA, or alternatively that AgRP has an effect on its own, independent of antagonism of MC receptors [43]. Although some of the results above suggest that a hyperactive MC system triggers ABA, these results are not compatible with the effects of leptin treatment on ABA. However, the association of variations in the AgRP gene with AN supports the notion that the MC system affects the susceptibility to develop AN [46]. 4.3. Neuropeptide Y RWA in ad libitum fed rats increases mRNA levels of NPY in the ARC, DMH and medial preoptic area (MPO), similar to weight-matched RF rats [47]. Negative energy balance either achieved by increased EE or reduced FI thus seems to activate the NPY system in the hypothalamus. Centrally injected NPY not only increases FI, but also increases food seeking behavior in rodents and recently it was shown that blocking the Y1 receptor by Y1 antagonist 1229U91 reverses the stimulatory effect of food deprivation on wheel running for food in hamsters [48–50]. The above findings suggest that the NPY system might be involved in (leptin's effect on) hyperactivity in ABA rats. Indeed, daily injections of NPY (10 μg ilvt prior to dark onset) facilitate ABA in 2 h fed female rats [51]. It was shown that NPYtreated ABA rats (do not increase but) reduce FI, lose more BW and are more hyperactive than csf-treated ABA controls. Since NPY-treated ad libitum fed rats increase FI without changing RWA, behavioral effects of NPY treatment seem to depend on the rat's energy state. We could not confirm the above mentioned results of facilitation of ABA by ilvt NPY treatment. In our hands, female ABA rats chronically treated with NPY (10 μg/d ilvt) show no differences in 1 h FI, RWA during dark or light phase, or BW loss (Table 1). Discrepancies between both studies might be explained by differences in the RF paradigm and/ or differences in administration of NPY. Daily injections of NPY prior to food access might be more effective in increasing RWA than chronic infusions as we did. Major differences have been reported between acute and chronic interference with the NPY system [52]. Since the Table 1 Chronic neuropeptide Y (NPY) treatment does not influence activity-based anorexia in rats NPY (μg/d) 7 d survival
0 10 NPY (μg/d)
0 10
50 % (n = 4) 50 % (n = 4)
BW loss
FI (g)
RWA (rev)
(d 0–4)
(Sum d 1–4)
(Sum d 0–4)
22.8% ± 1.8 23.1% ± 1.5
17.5 ± 1.3 17.4 ± 1.2
28045 ± 5400 22820 ± 4786
BW loss
FI (g)
RWA (rev)
(During d 3–4)
(Sum d 3–4)
(Sum d 3–4)
8.4% ± 1.1 8.1% ± 1.0
11.6 ± 0.7 11.7 ± 0.6
18346 ± 3062 14166 ± 2950
After 10 d of ad libitum feeding and wheel running, ilvt cannula were implanted (connected to sc osmotic minipumps delivering neuropeptide Y (NPY)) in female rats (230 g). After surgery food was removed and presented again the next d for 1 h at dark onset (procedures as described in [45]). The exposure to the ABA model lasted for 1 wk. However, because some ABA animals had to be sacrificed before end of the study due to excessive BW loss (N 25%), only data (mean and SE) of the first 5 d of ABA is shown. Although pumps worked immediately after surgery, NPY (10 µg/d) only reached the brain during the second d of RF. Therefore results (mean and SE) are separately presented for RF d 3 and 4. BW= body weight, FI = food intake, RWA = running wheel activity, rev = revolutions.
(presumably) facilitation of ABA by NPY treatment is compatible with the effects of leptin treatment on ABA, the role of the NPY system in ABA deserves further studying. 4.4. Corticotrophin-releasing hormone Another neuropeptide of interest in relation to attenuation of hyperactivity by leptin treatment in ABA rats is corticotrophinreleasing hormone (CRH). Centrally injected CRH reduces FI [53]. Furthermore central leptin injections as well as treadmill running increase CRH mRNA in the paraventricular nucleus of the hypothalamus (PVN) [54,55]. Stressful situations, like ABA, might increase CRH expression and release, leading (in)directly to activation of dopamine (DA) neurons in the ventral tegmental area (VTA) and noradrenergic (NA) neurons in the locus coeruleus (LC), brain areas implicated with reward mechanisms and selective attention [56]. CRH antagonist, α-hCRH, attenuates leptin-induced anorexia and also blocks exercise-induced anorexia [54,57]. We and others found, however, no evidence for increased CRH expression in ABA rats (at d 3 and at 25% BW loss) [29,58,59]. Increased activity of the adrenal axis has nevertheless been described in ABA rats (at d 6 and at 25% BW loss), i.e. increased corticosterone and ACTH levels with increased adrenal gland weight [45,58]. It should be mentioned that these effects have generally been observed when animals were close to emaciation, questioning the supposed involvement of the adrenal axis in development of ABA. The adrenal axis in AN patients is clearly activated, with increased CRH and cortisol levels, which may be related to patients' hyperactivity levels [60,61]. 4.5. Melanin concentrating hormone and orexins Melanin concentrating hormone (MCH) and orexin neurons in the LH regulate feeding, energy metabolism and arousal state [62,63]. Deficiency of either (prepro)MCH or the MCH receptor 1 results in a lean and hyperactive phenotype displaying FAA under RF conditions [64–66]. This suggests that MCH plays an inhibitory role on RWA and is not involved in FAA. In contrast, ablation of orexin neurons results in a narcoleptic, hypophagic and obese phenotype, with diminished FAA under RF conditions, implying that orexin neurons contribute to FAA [67,68]. Expression levels of MCH are slightly increased in ABA rats (at d 3), however, it seems unlikely that MCH plays a major role in attenuation of hyperactivity by leptin treatment [29]. No studies have yet been published on direct pharmacological interference with the MCH system in ABA rats. Preproorexin mRNA does not seem to be changed during ABA (at d 3), but the orexins (receptor) peptide levels might be regulated [29]. Since others reported altered FAA following manipulation with the orexin system [67,68], further studies on orexins' possible role in attenuation of hyperactivity by leptin treatment should be encouraged. 4.6. Opioids and dopamine Voluntary wheel running seems to be rewarding for ABA rats via activation of the opioid system and similarly exercising might be rewarding for AN patients [69,70]. Hypothalamic levels of endogenous opioid β-endorphin are increased in ABA rats when lost 25% BW. βEndorphin, like α-MSH also a product of POMC, seems to be mainly involved in incentive motivation to acquire food reinforcers, rather than in homeostatic control of feeding behavior (like α-MSH) [71]. Antagonizing the opioid system with naloxone blocks RWA in ad libitum fed rats [72], and μ-opioid receptor deficient mice (with reduced β-endorphin signaling) display attenuated FAA during RF [5]. We attempted to attenuate the development of hyperactivity in ABA rats by chronic treatment with opioid antagonist naltrexone (NTX). Chronic peripheral NTX treatment (0, 0.3 or 1.0 mg/kg/d) did, however, not influence RWA, FI or BW loss in female ABA rats on a 1 h feeding schedule (Table 2).
J.J.G. Hillebrand et al. / Physiology & Behavior 94 (2008) 689–695 Table 2 Chronic naltrexone (NTX) treatment does not influence activity-based anorexia in rats NTX (mg/kg/d)
0 0.3 1.0
6 d survival
100% (n = 8) 75% (n = 6) 62.5% (n = 5)
BW loss
FI (g)
RWA (rev)
(d 0–3)
(Sum d 1–3)
(Sum d 0–3)
18.0% ± 1.3 20.3% ± 1.3 19.1% ± 1.7
13.9 ± 1.4 12.7 ± 1.0 13.0 ± 0.4
20776 ± 4761 26103 ± 5616 23687 ± 5529
After 10 d of ad libitum feeding and wheel running, osmotic minipumps delivering naltrexone (NTX) at a dose of 0, 0.3 or 1.0 mg/kg/d sc were implanted in female rats (230 g). After surgery food was removed and presented again the next d, for 1 h at dark onset (procedures as described in [89]). The exposure to the ABA model lasted for 6 d. However, because 5 NTX-treated rats had to be sacrificed before end of the study due to excessive BW loss (N25%), only data (mean and SE) of the first 4 d of ABA is shown. BW = body weight, FI=food intake, RWA = running wheel activity, rev=revolutions.
Not only wheel running, but also RF influences reward. For example, leptin itself reverses RF-induced sensitization of brain stimulation reward in rats, and attenuates drug-seeking behavior after RF [73,74]. These findings suggest the existence of a pathway between peripheral leptin that signals adiposity and the midbrain DA system, involved in motivational and rewarding aspects of drugs of abuse and exploration [75]. The attenuation of RWA in leptin-treated ABA rats might be explained by a direct effect of leptin on the midbrain DA system. Lepr are present on DA VTA neurons projecting to the nucleus accumbens (NAcc) [76,77]. Leptin infusion into the rat VTA reduces firing of DA neurons, leading to decreased DA levels in the NAcc and consequently reduced FI without affecting LMA [77,78]. Long-term genetic knockdown of lepr in the VTA using an adenoassociated virus containing short-hairpin lepr RNA (AAV-shlepr) increases FI without affecting BW. It has been reported that these knockdown rats are more active, especially in the dark phase, than rats injected with the control virus and are also more sensitive to highly palatable food [77]. This suggests that decreased lepr signaling increases LMA followed by increased FI (or vice versa) and, likewise that increased leptin signaling in the VTA might underlie attenuation of hyperactivity by central or peripheral leptin treatment in ABA rats. Future studies should therefore be aimed at direct interference with leptin signaling in the VTA in ABA rats. Dopamine turnover in the medial basal hypothalamus is reduced during RF, but seems increased or normalized in ABA rats (at 30% BW loss) [79]. The attenuation of hyperactivity by leptin treatment compares with the effects of pimozide treatment in ABA rats, a drug with strong dopamine receptor 2 (D2) affinity [80]. Ill AN patients have reduced csf levels of DA metabolite homovanillic acid, which persist after recovery [81]. In addition, recovered AN patients have been found to display increased D2/D3 receptor binding in the anteroventral striatum (including NAcc), which may imply altered reward processing and may affect physical activity and eating behavior in AN [82]. 4.7. Serotonin Physical activity and RF are both related to increased serotonergic (5-hydroxytryptamine, 5HT) turnover [83,84]. ABA rats (at 30% BW loss) show increased 5HT activity, with stable or increased 5HT levels and increased levels of 5HT metabolite 5-hydroxy-indole-acetic acid (5HIAA) in the medial basal hypothalamus [79]. Reducing 5HT activity by treating ABA rats with 8-OH-DPAT, an agonist of the 5HT1A autoreceptor, does not influence FI but reduces RWA (both in the dark and light phase) and thereby attenuates BW loss [85]. Oppositely, D-fenfluramine, a 5HT releaser and uptake inhibitor does not affect RWA, but reduces FI and thereby increases BW loss in ABA rats [86,87]. We, however, could not replicate these effects in our ABA design [88]. The data mentioned above suggests that a reduction of 5HT activity attenuates ABA. Interestingly, both ABA rats and AN patients reduce
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hyperactivity when treated with the atypical antipsychotic olanzapine, antagonizing both DA and 5HT systems [89]. The 5HT system has been extensively studied in AN patients. Csf levels of 5HIAA are reduced in ill AN patients, but elevated after recovery in AN patients, suggesting that disturbed 5HT signaling might play a role in the pathogenesis and/or maintenance of the disorder. Functional brain imaging has furthermore confirmed pre-existing or persistent changes in 5HT receptor function that suggest impaired signaling in brain reward centers in recovered AN patients [90]. 5. Discussion Hyperactivity is frequently observed in AN patients and influences treatment outcome by hindering BW gain and increasing the probability of relapse. We took a translational approach to gain knowledge of this particular aspect of the pathogenesis of AN by mimicking hyperactivity in the ABA model. The ABA model is considered an animal model of AN with considerable face validity, nevertheless, it is used to explain only certain aspects of the disorder, present in a subset of AN patients. Leptin treatment attenuates hyperactivity in ABA rats and has for this reason been suggested as a putative therapeutic agent to treat severe hyperactivity in AN patients [30]. Since the first aim of AN treatment is to increase BW and obtain normal eating behavior, leptin treatment (if applicable) should certainly not be used during the initial phases of treatment, but (if so) only once BW and FI have been normalized. In the above we gave an overview of possible downstream effectors of leptin's effect on hyperactivity. We, however, want to emphasize that this overview includes most obvious downstream effectors but is not complete (lacking e.g. ghrelin, brain derived neurotrophic factor, histamine, and noradrenaline). Evidence has been reported that during ad libitum feeding, the ARC may be involved in leptin's effect on LMA [33]. MC peptides, derived from the ARC, do play a role in ABA, but mainly affect FI not RWA. ARC NPY may, however, be one of leptin's downstream candidates involved in adapted FI and RWA during ABA. The strong effect of leptin treatment on RWA in ABA rats could be mediated in the VTA, where leptin can bind lepr on DA neurons, consequently silencing these neurons and reducing motivation to run. Future studies should investigate the precise role of these neurons during ABA, by focusing on direct effects of leptin as well as hypothalamic neuropeptides (e.g. orexins, CART) on these neurons in relation to hyperactivity. Molecular determinants of attenuation of hyperactivity by leptin treatment might also be further unraveled using specific mice strains of interest, now that the mouse model of ABA has been developed [91]. Furthermore, since hyperactivity in AN patients is also related to e.g. an anxious phenotype, neurobiological systems underlying hyperactivity and anxiety might be similar or at least connected [6]. Further unraveling of the behavioral phenotype of AN patients offers additional challenging translational opportunities to study mechanisms underlying the hyperactivity in AN. References [1] American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 4th ed. Washington (DC): APA; 1994. [2] Hebebrand J, Exner C, Hebebrand K, Holtkamp C, Casper RC, Remschmidt H, et al. Hyperactivity in patients with anorexia nervosa and in semistarved rats: evidence for a pivotal role of hypoleptinemia. Physiol Behav 2003;79:25–37. [3] Kron L, Katz JL, Gorzynski G, Weiner H. Hyperactivity in anorexia nervosa: a fundamental clinical feature. Compr Psychiatry 1978;19:433–40. [4] Davis C. Eating disorders and hyperactivity: a psychobiological perspective. Can J Psychiatry 1997;42:168–75. [5] Kas MJ, van den Bos R, Baars AM, Lubbers M, Lesscher HM, Hillebrand JJ, et al. Muopioid receptor knockout mice show diminished food-anticipatory activity. Eur J Neurosci 2004;20:1624–32. [6] Holtkamp K, Hebebrand J, Herpertz-Dahlmann B. The contribution of anxiety and food restriction on physical activity levels in acute anorexia nervosa. Int J Eat Disord 2004;36:163–71.
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