Effects of rabbit anti-α-melanocyte-stimulating hormone (α-MSH) immunoglobulins on α-MSH signaling related to food intake control

Neuropeptides 48 (2014) 21–27

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Neuropeptides journal homepage: www.elsevier.com/locate/npep

Effects of rabbit anti-a-melanocyte-stimulating hormone (a-MSH) immunoglobulins on a-MSH signaling related to food intake control Nicolas Lucas a,b, Romain Legrand a,b, Wassila Ouelaa a,b, Jonathan Breton a,b, Naouel Tennoune a,b, Christine Bole-Feysot a,b, Pierre Déchelotte a,b,c, Sergueï O. Fetissov a,b,⇑ a b c

Inserm UMR1073, Nutrition, Gut and Brain Laboratory, Rouen 76183, France Institute for Research and Innovation in Biomedicine (IRIB), Rouen University, Normandy University, Rouen 76183, France Rouen University Hospital, CHU Charles Nicolle, 76183 Rouen, France

a r t i c l e

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Article history: Received 14 August 2013 Accepted 24 October 2013 Available online 1 November 2013 Keywords: Melanocortin system Energy homeostasis Hypothalamus Animal models Autoantibodies Affinity kinetics

a b s t r a c t Anti-a-melanocyte-stimulating hormone (a-MSH) polyclonal antibodies have been used for a-MSH neutralization in functional studies, but the results are sometime inconsistent with the antibody expected blocking properties. The present study aimed to determine if rabbit (Rb) anti-a-MSH immunoglobulins (Ig) may inhibit or enhance a-MSH signaling on melanocortin receptor type 4 (MC4R) and a-MSH-induced anorexigenic effect if presented as immune complexes with a-MSH. Polyclonal Rb anti-a-MSH IgG were commercially available and their ability to bind a-MSH has been confirmed by the immunohistochemical detection of a-MSH neurons in the rat hypothalamus. In vitro assay of the cyclic-adenosine mono-phosphate (cAMP) secreted by cells transfected with MC4R was performed to analyze effect of Rb IgG on a-MSH-induced cAMP production. We found that adding Rb IgG to a-MSH resulted in stimulation of cAMP detected at lower peptide concentrations as compared to a-MSH alone. To determine effects of Rb IgG on food intake, rats were injected into the arcuate hypothalamic nucleus with either a-MSH, Rb IgG alone or Rb IgG preincubated with a-MSH. During 2 days after injections, food intake was increased in both groups of rats receiving Rb IgG. However, during following 4 days when food was restricted to 1 h/day, only the Rb IgG group displayed higher food intake. Furthermore, after the refeeding, 24 h food intake was lower in rats receiving Rb IgG - a-MSH immune complexes. This group of rats was also characterized by higher number of immunopositive neurons in the arcuate nucleus expressing a-MSH and agouti-related protein but not tyrosine hydroxylase. Taken together, these results show that Rb anti-a-MSH antisera, although efficient for immunohistochemical detection of a-MSH, does not always display a-MSH blocking properties but, in contrast, may enhance a-MSH binding to MC4R and increase a-MSH anorexigenic effects when presented as immune complexes with the peptide. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Functional studies of neuropeptide signaling employ sometime neutralization of endogenous neuropeptides by polyclonal immunoglobulins (Ig) issued from immunized animals, most commonly rabbits (Rb). Obtained results sometime, however, may contradict to the expected decrease of corresponding neuropeptide effects, instead, neuropeptide agonist-like effects are observed such as reported for a-melanocyte-stimulating hormone (a-MSH) which is a 13 amino acid acylated neuropeptide (Harris and Lerner, 1957). a-MSH is involved in multiple physiological functions (Catania et al., 2000; Bertolini et al., 2009) and its peripheral and central ⇑ Corresponding author. Address: Inserm UMR1073, Faculté de MédecinePharmacie, 22 Bld. Gambetta, Rouen 76183, France. Tel.: +33 (0)2 35 14 82 55; fax: +33 (0)2 35 14 82 26. E-mail address: [email protected] (S.O. Fetissov). 0143-4179/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.npep.2013.10.017

administration in rats, among other effects, will activate midbrain and hypothalamic dopaminergic neurons (Lichtensteiger and Lienhart, 1977; Lindblom et al., 2001) and will decrease food intake (Fan et al., 1997; Forbes et al., 2001; Poggioli et al., 1986; Murphy et al., 1998; Trivedi et al., 2003). Paradoxically, peripheral administration of Rb anti-a-MSH antisera in rats was shown to activate the same groups of dopaminergic neurons in a similar way as administration of the exogenous a-MSH (Monnet and Lichtensteiger, 1981). Moreover, peripheral injections of Rb anti-a-MSH IgG in rats with methotrexate-induced anorexia resulted in prolongation of the anorectic state (Coquerel et al., 2012). Furthermore, intracerebroventricular (ICV) injections in rats of Rb anti-a-MSH IgG was virtually inactive to modulate stress-associated memory (van Wimersma Greidanus et al., 1978) and also in non-stressed rats, ICV a-MSH antiserum did not change anxiety-like behavior (Kokare et al., 2006). Additionally, conflicting results have been reported regarding effects of Rb anti- a-MSH IgG on prolactin

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secretion in rats showing its stimulation (Khorram et al., 1984, 1985) or inhibition (Watanobe et al., 2003). Hypothalamic arcuate nucleus (ARC) neurons synthesize a-MSH (Jacobowitz and O’Donohue, 1978) which plays a critical role in signaling satiety by binding to the melanocortin receptor 4 (MC4R) (Cone, 2006) but, surprisely, no studies reported possible central effects of anti-a-MSH IgG on food intake, which may help to better clarify the role of endogenous a-MSH in food intake regulation. Molecular mechanisms responsible for the opposite to expected effects of Rb anti-a-MSH IgG injections have not been formally clarified. However, one reason might be directly related to the properties of anti-neuropeptide Ig such as their affinity and ability to form immune complexes. Indeed, the clinical use of some antipeptide monoclonal antibodies relies on their high affinities responsible for antagonizing peptide binding to their receptors (Carter, 2006). However, the affinity of polyclonal Ig for neuropeptides used for experimental studies is an information which cannot be found in the product data sheet. Yet, affinity kinetics may be important to assure neuropeptide neutralizing effects. In fact, animals naturally display a variety of anti-neuropeptide IgG or autoantibodies (autoAbs) including those that react with a-MSH (Fetissov et al., 2008a) and, hence, a mixture of IgG issued from immunized animals eventually contains IgG with different affinities resulting in an uncertain avidity of the final antiserum. Furthermore, plasma levels of a-MSH-reactive IgG in rats were shown to correlate positively with a-MSH peptide concentrations and with a-MSH-mediated satiety and anxiety suggesting that such low affinity natural IgG may increase a-MSH actions for example by forming a-MSH IgG immune complexes (IC) which could transport a-MSH eventually protecting it from degradation by peptidases (Coquerel et al., 2012; Fetissov et al., 2008b; Hamze Sinno et al., 2009) in a similar way as was shown for ghrelin (Takagi et al., 2013). Thus, in the present work to better understand the mechanism of action of Rb anti-a-MSH as relevant to food intake, we measured affinity kinetics of commercially available Rb anti-a-MSH IgG and studied in vitro effects of a-MSH IC to activate MC4R. Furthermore, we studied effects of administration in rats of Rb anti-a-MSH IgG as free antibody or as a-MSH IC in the ARC on food intake and ARC neurons histochemistry. 2. Experimental procedures 2.1. Antiserum In all experiments of this study we used polyclonal anti-a-MSH Rb IgG purchased from Peninsula Laboratories (San Carlos, CA). IgG were purified by the manufacturer from whole serum by protein A chromatography. 2.2. Affinity kinetics of Rb anti-a-MSH IgG Affinity kinetics of Rb anti-a-MSH IgG were analyzed by a biospecific interaction analysis (BIA) based on the surface plasmon resonance (SPR) phenomenon on a BIAcore 1000 instrument (GE Healthcare, Piscataway, NJ). a-MSH (Bachem AG, Bubendorf, Switzerland) was diluted 0.5 mg/ml in 10 mM sodium acetate buffer, pH 5.0 (GE Healthcare) and was covalently coupled on the CM5 sensor chip (GE Healthcare), using the amine coupling kit (GE Healthcare). For the affinity kinetic analysis, a multi-cycle method was run with seven serial dilutions of Rb IgG: 3360, 1680, 840, 420, 210, 105 and 53 (nmol) and a blank (buffer only) Dilution of 3360 nmol corresponds to 0.5 mg/ml of IgG. Each cycle included 2 min of analyte injection and 5 min of dissociation with flow speed 30 ll/min at 25 °C. Between injections of each sample, the

binding surface was regenerated with 10 mM NaOH resulting in a return of the sensorgram to the same baseline level. The affinity kinetic data were analyzed using BiaEvaluation 4.1.1 program (GE Healthcare). For fitting kinetic data, the Langmuir’s 1:1 model was used and the sample values were corrected by subtracting the blank values resulting from the injection of HBS-EP buffer. 2.3. In vitro cAMP assay Stable cell line of human embryonic kidney (HEK) 293 cells expressing human MC4R was generated using a lentiviral transduction technology and purchased from Amsbio (Oxon, UK). High expression of MC4R mRNA in transfected cells was validated by RT-PCR in Amsbio and in our laboratory. The presence of the transgene in cells before each experiment was verified by the visualization at a fluorescence microscope of green-fluorescent protein (GFP) which gene was inserted in the same with MC4R lentivector but under a different promoter. The day before experiment, Rb IgG was diluted to 0.5 and to 1.0 mg/ml in PBS. The a-MSH peptide (Bachem, in doses of 0.6, 3, 4.5, 6, 15, 30, 45, 60 and 120 pmol) was diluted in the induction buffer: PBS, 500 lM IBMX, 100 lM RO 20-1724 (Sigma–Aldrich, Gillingham, UK), 20 mM MgCl2 and one blank sample was also included. Each dilution of a-MSH was incubated with 3 lL of Rb IgG (0.5 or 1.0 mg/ml) or with PBS at 4 °C overnight. After unfreezing, the cells were cultured in 250 ml tissue culture flasks (BD-Falcon, Beckton-Dickison, Bedford, MA) in Dubecco’s Modified Eagle Medium 4,5 g/l glucose (Eurobio, Courtaboeuf, France) supplemented with (2 mM L-glutamine; 10% FCS; 0,1 mM non-essential amino-acids; 1% penicillin-streptavidin) in humidified cell culture incubator at 37 °C, 5% CO2 for 8–10 days. At the day of experiment, cultured MC4R HEK293 cells were treated with 0,25% trypsin–EDTA (Sigma–Aldrich) and cell pellets were resuspended in PBS to obtain about 5000 cells par well (10 lL) in a non-treated bioluminescence white 96-microwell plate (Nunc, Roskilde, Denmark). The cyclic adenosine monophosphate (cAMP) production by MC4R expressing HEK 293 cells was measured using the bioluminescent assay cAMP-Glo™ Max Assay kit (Promega, Madison, WI) according to the manufacturer’s instructions. Briefly, the cells were incubated with different dilutions of a-MSH peptide alone or a-MSH with Rb IgG for 15 min at room temperature. Serial dilutions of cAMP standard (provided by the kit) were assayed on the same microplate. cAMP detection solution was added to each well, then the cells were homogenized by agitation and centrifuged 2 min at 1000 r.p.m. and then incubated for 20 min at 23 °C. Kinase-Glo reagent substrate was added in each well and after 10 min of incubation at 23 °C, the luminescence was read with a bioluminescence detection instrument (Safas Spectrometer, Monaco). Three tests for each dilution were performed in separate wells and were repeated at three separate days resulting in n = 9 for each point of the cAMP activation curve. The negative controls included incubation of a-MSH peptide with anti-a-MSH Rb IgG on HEK 293 cells which were not transfected with MC4R (Amsbio). 2.4. Central administration of Rb IgG in rats and food intake Rat care and experimentation were in accordance with guidelines established by the National Institutes of Health, USA and complied with both French and European Community regulations (Official Journal of European Community L 358, 18/12/1986). 200–250 g male Sprague–Dawley rats (Janvier Labs, L’Arbresle, France) were maintained at 24 °C with a 12:12-h light–dark cycle (light period 7–19 h) in a specialized air-conditioned animal facility. When housed in standard holding cages (3 rats per cage) animals were fed with standard pelleted rodent chow (RM1 diet, SDS, UK), when kept individually in metabolism cages (Techniplast,

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France) they were fed with the same RM1 but ground chow (SDS). Drinking water was always available ad libitum. Rats were randomly divided into four groups depending on their treatment (n = 6 in each group) and were kept 1 week for acclimatization in holding cages with food available ad libitum. Then, all rats were placed in metabolism cages and body weight, food intake and water intake were monitored daily. At day 0, rats were anaesthetized by an intraperitoneal injection of ketamine (75 mg/kg)/xylasine (5 mg/kg) solution (3:1 vol – 0,1 mL/100 g body weight) and bilateral stereotaxic injections were performed using 5 lL Hamilton syringes (Hamilton, Reno, NV). In order to target the hypothalamic ARC, stereotaxic coordinates for injections were: ±0.5 mm lateral to the midline, 2.85 mm posterior to Bregma and 9.5 mm below the surface of the skull (Swanson, 1998). The ‘a-MSH0 group received 2  1 lL of a-MSH peptide (Bachem) at a dose of 1 lg/ll, the ‘IgG’ group received 2  1 lL of Rb anti-a-MSH IgG in PBS (1:1, 0,5 lg/lL) and the ‘Immunocomplexes’ group (a-MSH IC) received 2  1 lL of the same Rb anti-a-MSH IgG pre-incubated with the a-MSH peptide overnight at 4 °C. Control group (Ctrl) received 2  1 lL of PBS. After awakening from the operation, rats were returned in their metabolism cages. Food and water were available ad libitum from day 0 to day 3. Then, food was given only for 1 h/day (between 10 and 11 h AM) for 4 days. Finally, at day 7, food was given again ad libitum for refeeding during 24 h. During all experiments, food and water intake were measured daily. 2.5. Tissue preparation In order to immunohistochemically visualize cell bodies of ARC neurons producing AgRP and a-MSH, we blocked centrifugal axonal transport by colchicine. Next day after the refeeding (day 8), rats were anaesthetized by an intraperitoneal injection of ketamine (75 mg/kg) and xylasine (5 mg/kg) (3:1 vol – 0.1 mL/100 g body weight). Colchicine (Sigma) was injected in the brain lateral ventricle (120 lg in 20 lL of 0.9% NaCl). The stereotaxic coordinates were +1.6 mm lateral to the midline, 1.0 mm posterior to Bregma and 4.5 mm below the surface of the skull. The next day, all rats were anaesthetized by an intraperitoneal injection of the same ketamine/xylasine solution (3:1, vol – 0.1 mL/100 g body weight) and perfused via the ascending aorta with Tyrode’s solution (0.68% NaCl, 0.04% KCl, 0.015% MgCl2.6H20, 0.01% MgSO4.7H20, 0.019% NaH2PO4) at 37 °C, followed by fixation with a mixture of 4% paraformaldehyde and 0.4% picric acid in a 0.16 M phosphate buffer (pH 6.9 at 37 °C) and then the same ice-cold fixative. Brains were rapidly dissected out and immersed in the same fixative for 90 min, then placed overnight in 0.1 M PBS containing 10% sucrose (pH 7.4). Brains were frozen during 1 min in methyl-butane (Sigma–Aldrich) pre-cooled to 45 °C in liquid nitrogen. Successive coronal brain sections (14 lm) including the ARC were cut on a cryostat (Leica Microsystems, Nanterre, France) and mounted on chrome alum-gelatine-coated glass slides, then air-dried for 2 h. 2.6. Immunohistochemistry Sections were rehydrated in PBS and then treated with 0.03% H2O2, followed by 5% bovine serum albumin (Sigma) diluted in PBS. The tyramide signal amplification (TSA) immunohistochemical technique (Adams, 1992) was employed. Briefly, sections were incubated with primary antisera overnight at 4 °C, followed by anti-rabbit horseradish peroxidase-conjugated swine anti-rabbit antibodies (1:200; Dako Cytomation, Glostrup, Denmark), and then processed using the TSA-plus Fluorescein System (NEN, Boston, MA). Primary polyclonal antisera (raised in rabbits) were against a-MSH (1:8000; Bachem), against AgRP (1:4000; Phoenix Pharmaceuticals, Belmont, CA) and against tyrosine hydroxylase (TH, Chemicon International Inc., Billerica, MA).

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Sections were mounted in a mixture (3:1, vol) of glycerol/PBS (0.1 M, pH 7.4) containing 2.5% 1,4-diazabicyclo [2.2.2.] octane, DABCO (Sigma) as an anti-fading agent. After processing, sections were examined in a Zeiss Imager Z1 fluorescence microscope equipped with an AxioCam digital camera (Carl Zeiss Jena GmbH, Germany). Digital images were optimized for brightness and contrast and were merged between adjacent sections after assigning artificial colour using Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA). Mean number of a-MSH and AgRP-immunopositive cells were counted from five sections during microscopy using objective 10 in the middle part of the whole unilateral ARC; number of TH-immunopositive cells were counted only in the dorsomedial part of the ARC that contains dopaminergic neurons. 2.7. Statistical analysis Data were analyzed and the graphs were plotted using the GraphPad Prism 5.02 (GraphPad Software Inc., San Diego, CA). Normality was evaluated by the Kolmogorov–Smirnov test. Group differences were analyzed by the analysis of variance (ANOVA) or the non-parametric Kruskal–Wallis (K–W) test with the Tukey’s or Dunn’s post-tests, according to the normality results. Body weight changes were analyzed with two-way repeated measurements (RM) ANOVA and the Bonferroni post-tests. Where appropriate, individual groups were compared using the Student’s t-test or the Mann–Whitney (M–W) test according to the normality results. Results of affinity kinetics were analyzed by Chi2 test from BiaEvaluation program. The cAMP production was analyzed using a nonlinear regression fit (log(a-MSH) vs. normalized cAMP response) with following equation: Y = 100/(1 + 10^((LogEC50-X)⁄HillSlope)). Inflection point for each curve was calculated by determining the intersection between cAMP curve second derivative and a-MSH concentrations. For all tests, p < 0.05 was considered statistically significant. 3. Results 3.1. Immunohistochemical detection of a-MSH by Rb IgG The same Rb IgG that were used in this work for functional studies have been tested for immunohistochemical detection of a-MSH in the rat brain sections. We found that these Rb IgG produced selective staining of the ARC neurons typical for a-MSH expression and enhanced by colchicine pre-treatment (Fig. 1). 3.2. Affinity kinetics of Rb anti-a-MSH IgG The affinity kinetics parameters were determined between Rb IgG and a-MSH peptide, using surface plasmon resonance showing the dissociation equilibrium constant (KD) 8.25  108 M, the association rate (small Ka) 959 M1s1 and the dissociation rate (small Kd) 7.91  105 s1, Chi2 51.4. 3.3. In vitro cAMP assay

a-MSH dose dependently stimulated cAMP production by HEK293 cells overexpressing MC4R (Fig. 2A). Adding a-MSH together with Rb IgG (0.5 and 1.0 mg/ml) to MC4R overexpressing cells also resulted in an a-MSH dose-dependent increase in cAMP production, but the stimulation curves where shifted to the left from the a-MSH stimulation curve (Fig. 2A). The left shift was observed with the IgG from both concentrations of IgG but it was more pronounced at the 1.0 mg/ml of IgG. Accordingly, with 1.0 mg/ml of Rb IgG, significant increase in cAMP was observed

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3.4. Central administration of Rb IgG in rats and food intake

Fig. 1. Immunostaining of a-MSH neurons of the arcuate nucleus (ARC) of rat hypothalamus with polyclonal rabbit anti-a-MSH IgG used in this work for in vitro and in vivo studies. Before perfusion, rat was pre-treated with colchicine in ICV. 3v, third ventricle; ME, median eminence; VMN, ventromedial nucleus.

at the smallest dose of 15 pmol of a-MSH which did not result in any stimulation of cAMP with the same dose of a-MSH when peptide was added alone or with 0.5 mg/ml of Rb IgG (Fig. 2B). Furthermore, the inflection point was significantly lower in both groups containing Rb IgG and particularly at higher IgG concentration (Fig. 2C). Adding a-MSH together with Rb IgG (0.5 mg/ml) to HEK293 cells which were not transfected with MC4R did not stimulate cAMP production (Fig. 2A), showing that MC4-R was necessary for a-MSH and Rb IgG activation cAMP secretion.

Next day after the intra-ARC injections, body weight returned to the preoperational level in all animals and it was not statistically significant among the groups throughout the study (Fig. 3A). Because of the period of 4 days of food restriction, all rats lost weight as compared to the preoperational level, however, the rats which received Rb IgG lost less weight (Day 8 vs. Day 0, Controls, 28.7 ± 10,5 g, a-MSH, 29.8 ± 6,9 g, Rb IgG,-13.2 ± 2,7 g and a-MSH IC, 28.3 ± 10,9 g), although these difference did not reach significance (Student’s t-test Rb IgG vs. a-MSH p = 0.05, and p = 0.15 vs. other groups). Mean daily food intake with ad libitum access to food during 2 days after the ARC injections was increased in rats that received Rb IgG as immune complexes (a-MSH IC) with a-MSH (Fig. 3B). Rats receiving the Rb IgG alone also showed a slight increase in food intake, but it was significant only as compared to the group injected with a-MSH (Fig. 3B). However, during the restricted feeding schedule, which lasted 4 days, the rats that received Rb IgG increased their 1 h food intake significantly more than a-MSH and a-MSH IC groups (Fig. 3C). Furthermore after 24 h of refeeding, the a-MSH IC group showed lower food intake as compared to all other groups (Fig. 3D).

3.5. Arcuate nucleus histochemistry After 24 h of refeeding, rats were injected ICV with colchicine and perfused 24 h later for the immunohistochemical study of the ARC neurons. As expected, use of colchicine allowed the visualization of a-MSH and AgRP peptides in the cell bodies of the ARC neurons (Fig. 4A–D) in all four groups of rats. Mean number of both a-MSH and AgRP neurons was higher in rats that received a-MSH IC while number of TH neurons was unchanged (Fig. 4E).

Fig. 2. cAMP assay in HEK 293 cultured cells overexpressing MC4-R after stimulation by a-MSH peptide alone or together with polyclonal Rb anti-a-MSH IgG at 0.5 or 1 mg/ mL preincubated with a-MSH. Non MC4-R transfected HEK 293 cells were used as negative controls. (A) Dose–response curves of cAMP production were normalized from the basal level (each point represents mean ± S.E.M). (B) cAMP production at 15 pmol of a-MSH was statistically analyzed between three groups (ANOVA p < 0.0001, Tukey’s posttest ⁄⁄⁄, p < 0.001, a-MSH + Rb IgG 1.0 mg/ml vs. both groups). (C) Inflection point of a-MSH concentration for each curve (ANOVA p < 0.0001, Tukey’s post-tests ⁄⁄ p < 0.01⁄⁄⁄p < 0.001).

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Fig. 3. Effects of bilateral injections into the ARC of Rb anti-a-MSH IgG on food intake in rats. Rats received injections of 1lL of either 0,5 lg/lL Rb anti-a-MSH IgG, 0,5 lg/lL Rb anti-a-MSH IgG pre-incubated with 1 lg of a-MSH (a-MSH IC,) 1 lg of a-MSH alone, or PBS as control. Body weight (A) and food intake were monitored daily. Food intake was ad libitum from day 0 to day 3 (B) and then food access was restricted to 1 h/day from day 3 to day 7 (C). After day 8, food was given ad libitum for 24 h (D). (B-D: Student’s t-test ⁄p < 0.05 ⁄⁄p < 0.01; C: Student’s t-test, ⁄p < 0.05, Rb IgG vs. a-MSH (a) and Rb IgG vs. a-MSH IC (b).

4. Discussion This study revealed that in experimental conditions when Rb anti-a-MSH IgG are complexed with a-MSH peptide, they do not block a-MSH-induced activation of MC4R but, instead, enhance it. Since the MC4R is responsible in the brain for mediating a-MSH anorexigenic effects (Cone, 2005), these enhancing properties of a-MSH IC are further supported in the present study by showing lower food intake in a-MSH IC-injected rats during refeeding after food restriction. The role of the IC in enhancing a-MSH effects appears as an important condition, because intrahypothalamic administration of Rb IgG alone resulted in higher food intake than in a-MSH-injected rats, particularly during the food restriction schedule. Since the enhanced anorexigenic effects of a-MSH IC was revealed only following food restriction it is likely that it was related to the increased MC4R expression levels in the hypothalamus triggered by the negative energy balance. Indeed, increased MC4R in the hypothalamus was previously shown in rat models of activity-based anorexia (Gutiérrez et al., 2009) and cancer cachexia (Suzuki et al., 2012). Mechanisms responsible for an increased food intake observed in a-MSH IC-injected ad libitum fed rats during two days after the injections is likely due to activation of NPY/AgRP neurons that are known to express MC3R (Mounien et al., 2005). In fact, activation of both AGRP and POMC neurons by an optogenetic approach was shown to acutely (but not chronically) activate feeding in mice (Atasoy et al., 2012). Affinity assay of Rb IgG for a-MSH showed that their KD was in the micromolar range. This characteristic differentiates the polyclonal IgG from neutralizing monoclonal antibodies which are usually selected based on their nanomolar affinities. It is, hence, possible that relatively low affinity or avidity of Rb IgG may explain the enhancing properties of a-MSH IC, at least on the MC4R. The affinity of Rb IgG appears, nevertheless, sufficient to provide a-MSH selective immunostaining of the arcuate a-MSH neurons and also to cause increased food intake in rats when these IgG

are injected in the mediobasal hypothalamus without preincubation with a-MSH. One of the purposes of this study was to verify if intra-hypothalamic administration of a-MSH IC might induce neuronal loss in agreement with a known role of antigen–antibody IC activation of the complement pathway and cell lysis (Gasque, 2004). Our data ruled out such possibility and, instead, more a-MSH and AgRP positive neurons were detected in the ARC of rats injected with a-MSH IC, which may signify increased peptide productions in these neurons. Although immunohistochemical detection of these peptides was performed after the colchicine treatment, and hence, is somewhat artificial, it still may reflect the activity of peptide synthesis by ARC neurons. Such an increase can be a result of a prolonged stimulation by a-MSH IC of melanocortin receptors expressed directly by a-MSH and AgRP neurons (Jégou et al., 2000; Mounien et al., 2005) or indirectly from the ventromedial nucleus (Sternson et al., 2005) which express MC4R (Mountjoy et al., 1994; Kishi et al., 2003) and MC3R (Roselli-Rehfuss et al., 1993; Jégou et al., 2000). Reciprocally, the VMN was shown to be a downstream target for anorexigenic effects of a-MSH via stimulation of brain-derived neurotrophic factor (Xu et al., 2003) and, hence, can be involved in the a-MSH IC-induced decrease of food intake found in this experiment. Activation of both orexigenic (AgRP) and anorexigenic (POMC) ARC neurons by a-MSH IC, as revealed by immunohistochemistry, may explain lower food intake during refeeding in these rats by a long-term contribution of anorexigenic signal from the POMC neurons in agreement with the optogenetic data showing that chronic but not acute activation of POMC neurons decrease food intake in mice (Atasoy et al., 2012). Enhanced activation of the MC4R by a-MSH IC suggests that these IC may induce an allosteric effect, but the underlying molecular mechanisms need further clarification. A limitation of this in vitro study was that effects of Rb anti-a-MSH IgG have been studied on human MC4R and, hence, it needs to be further validated with human a-MSH-reactive IgG autoantibodies.

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Fig. 4. Merged images from representative immunostaining of a-MSH (red), AgRP (green) and tyrosine hydroxylase (TH, blue) in the arcuate nucleus (ARC) of rats after refeeding and colchicine treatment. Rats were injected with either (A) PBS as control (Ctrl), (B) Rb anti-a-MSH IgG, (C) a-MSH peptide or (D) Rb anti-a-MSH IgG pre-incubated with a-MSH (a-MSH IC). (E) Mean number of a-MSH, AgRP and TH immunopositive neurons in the ARC (ANOVA p = 0.01 for both a-MSH and AgRP staining, Tukey’s post-test ⁄ p < 0.05). 3v, third ventricle; ME, median eminence; VMN, ventromedial nucleus.

These results provide an explanation of previous data describing a-MSH-like effects of Rb anti-a-MSH IgG (Coquerel et al., 2012; Monnet and Lichtensteiger, 1981) suggesting that peripherally administered Rb IgG could form IC with circulating a-MSH peptide and then stimulate MC receptors. The potential IC forming capacities of Rb IgG are probably more effective in the systemic circulation, because their intrahypothalamic injection caused an expected increase in food intake. In fact, in the present study, Rb IgG were particularly effective in increasing food intake in food restricted rats, indicating that during the negative energy balance conditions, rats become very sensitive to endogenous a-MSH signaling satiety. In agreement with this conclusion, it was shown that a-MSH binding is increased in the ventromedial hypothalamus of rats with activity-based anorexia (Kas et al., 2003) and ICV infusion of a-MSH further decreases food intake (Hillebrand et al., 2005) while antagonising MC4R by ICV AgRP infusion improved food intake (Kas et al., 2003) in this rat model of anorexia. These data also indicate that during chronic food restriction such as in anorexia nervosa, increase in a-MSH alone or in complex with a-MSH-reactive autoAbs (Fetissov et al., 2002) may contribute to the maintenance of the anorexigenic state.

Although this study was focused on the Rb IgG against a-MSH, it is possible that relatively low affinity polyclonal IgG raised against other neuropeptides may display similar activating properties when forming IC with neuropeptides. E.g., an enhanced immunostaining is sometime observed after absorption of IgG with the neuropeptide (Fetissov et al., 2002). Furthermore, transporting properties of natural IgG was suggested for several peptide hormones such as NPY (Garcia et al., 2012), oxytocin and vasopressin (Garcia et al., 2011), insulin (Radermecker et al., 2009) and prolactin (Hattori et al., 2008). Existence of such enhancing effects should be considered in functional studies when using polyclonal immunoglobulins against neuropeptides. In conclusion, polyclonal Rb anti-a-MSH IgG administered into the mediobasal hypothalamus of rats increased food intake during food restriction indicating an increased sensitivity of hypothalamic neurons to endogenous a-MSH during starvation. However, when presented as IC with a-MSH peptide, Rb anti- a-MSH IgG did not increase food intake during starvation and even decreased it during refeeding suggesting their agonistic effects on MC4R. Existence of such agonistic effects of Rb anti-a-MSH IgG preincubated with a-MSH has been confirmed in an in vitro assay of cAMP production

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