NESS038C6, a novel selective CB1 antagonist agent with anti-obesity activity and improved molecular profile

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Behavioural Brain Research 234 (2012) 192– 204

Contents lists available at SciVerse ScienceDirect

Behavioural Brain Research

j ourna l ho me pa ge: www.elsev ier .com/ locate /bbr

esearch report

ESS038C6, a novel selective CB1 antagonist agent with anti-obesity activity andmproved molecular profile

ndrea Mastinua,b,∗, Marilena Piraa,c, Luca Pania,1, Gérard Aimè Pinnac, Paolo Lazzari c,d,∗∗

CNR, Istituto di Farmacologia Traslazionale, UOS Cagliari, Edificio 5, Loc. Piscinamanna, 09010 Pula, ItalyDipartimento Scienze Biomediche, Università degli Studi di Cagliari, s.p. 8 Monserrato-Sestu Km. 0, 700 – 09042 – Monserrato Cagliari, ItalyDipartimento di Chimica e Farmacia, Università degli Studi di Sassari, Via F.Muroni 23/A, 07100 Sassari, ItalyNeuroscienze Pharmaness S.c.ar.l., Edificio 5, Loc. Piscinamanna, 09010 Pula, Italy

i g h l i g h t s

NESS038C6 produced a significant weight loss in DIO mice fed with a fat diet.Chronic treatment with NESS038C6 improved cardiovascular risk factors.NESS038C6 regulated the molecular pathways between hypothalamus and fat tissue.NESS038C6 upregulated metabolic enzymes and PPAR-� mRNA in the liver.Our compound upregulated monoaminergic transporters and neurotrophic factors mRNA.

r t i c l e i n f o

rticle history:eceived 14 June 2012eceived in revised form 27 June 2012ccepted 28 June 2012vailable online 4 July 2012

eywords:ESS038C6io micelood parameterseptin pathwayonoaminergic transporterseurotrophic factors

a b s t r a c t

The present work aims to study the effects induced by a chronic treatment with a novel CB1 antago-nist (NESS038C6) in C57BL/6N diet-induced obesity (DIO) mice. Mice treated with NESS038C6 and fedwith a fat diet (NESS038C6 FD) were compared with the following three reference experimental groups:DIO mice fed with the same fat diet used for NESS038C6 and treated with vehicle or the reference CB1antagonist/inverse agonist rimonabant, “VH FD” and “SR141716 FD”, respectively; DIO mice treated withvehicle and switched to a normal diet (VH ND).

NESS038C6 chronic treatment (30 mg/kg/day for 31 days) determined a significant reduction in DIOmice weight relative to that of VH FD. The entity of the effect was comparable to that detected in bothSR141716 FD and VH ND groups.

Moreover, if compared to VH FD, NESS038C6 FD evidenced: (i) improvement of cardiovascular riskfactors; (ii) significant decrease in adipose tissue leptin expression; (iii) increase in mRNA expressionof hypothalamic orexigenic peptides and a decrease of anorexigenic peptides; (iv) expression increaseof metabolic enzymes and peroxisome proliferator-activated receptor-� in the liver; (v) normalization

of monoaminergic transporters and neurotrophic expression in mesolimbic area. However, in contrastto the case of rimonabant, the novel CB1 antagonist improved the disrupted expression profile of geneslinked to the hunger-satiety circuit, without altering monoaminergic transmission.

In conclusion, the novel CB1 antagonist compound NESS038C6 may represent a useful candidate agentfor the treatment of obesity and its metabolic complications, without or with reduced side effects relativeto those instead observed with rimonabant.

∗ Corresponding author at: CNR, Istituto di Farmacologia Traslazionale, UOSagliari, Edificio 5, Loc. Piscinamanna, 09010 Pula, Italy. Tel.: +39 0709242026;

ax: +39 0709242206.∗∗ Corresponding author at: Neuroscienze PharmaNess S.c.a.r.l., Edificio 5, Loc.iscinamanna, 09100 Pula (CA), Italy. Tel: +39 0709242025; fax: +39 0709242206.

E-mail addresses: [email protected] (A. Mastinu),[email protected] (P. Lazzari).1 Current address: Agenzia Italiana del Farmaco (AIFA), Via del Tritone 181, 00187oma, Italy.

166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bbr.2012.06.033

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Obesity has become a major public health concern in indus-trialized countries. Worldwide there are 1.1 billion overweightpeople with a BMI between 25 kg/m2 and 30 kg/m2 and 312 mil-lion with a BMI >30 kg/m2 [20]. A projection for the year 2030

estimates that 366 million people will suffer from obesity [54]. Inaddition, abdominal obesity is significantly associated with vari-ous metabolic abnormalities, including insulin resistance, impairedglucose tolerance/type-2 diabetes, and atherogenic dyslipidaemia

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rain Research 234 (2012) 192– 204 193

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Table 1Oligonucleotides TaqMan® MGB probe genes with assay ID used for semi-quantitative real-time PCR amplifications in the the brain and peripheral tissues.

Genes Tissues

Cannabinoid receptor 1,Mm00432321 s1, CB1

Hypothalamus, Liver, Visceral fat

Neuropeptide Y, Mm00445771 m1, NPY HypothalamusAgouti-related protein,

Mm00475829 g1, AGRPHypothalamus

Cocaine and amphetamine regulatedtranscript, Mm00489086 m1, CART

Hypothalamus

Proopiomelanocortin, Mm00435874 m1,POMC

Hypothalamus

Serotonin transporter,Mm00439391 m1, SERT

Nucleus accumbens, Amygdala,Prefrontal cortex

Norepinephrine transporter,Mm00436661 m1, NET

Nucleus accumbens, Amygdala,Prefrontal cortex

Dopamine transporter,Mm00438388 m1, DAT

Nucleus accumbens, Amygdala,Prefrontal cortex

Brain derived neurotrophic factor,Mm04230607 s1, BDNF

Hippocampus

Nerve growth factor, Mm00443039 m1,NGF

Hippocampus

Insulin receptor, Mm00439694 m1,INS-R

Hypothalamus

Leptin receptor, Mm 00440181 m1,LEP-R

Hypothalamus

Adiponectin, Mm01343606 m1, ADIPOQ Visceral fatLeptin, Mm 00434759 m1, LEP Visceral fatPeroxisome proliferator-activated

receptor-�, Mm00440939 m1, PPAR-�Liver

Peroxisome proliferator-activatedreceptor-�, Mm01184322 m1, PPAR-�

Visceral fat

Fatty acid synthase, Mm00662319 m1,FAS

Liver, Visceral fat

A. Mastinu et al. / Behavioural B

ith low high-density lipoprotein (HDL) cholesterol, high triglyc-rides, and increased small dense low-density lipoprotein (LDL)holesterol [45,54,59]. These metabolic disorders may be attributedn part to increased endocannabinoid activity [55]. The selectiveannabinoid 1 (CB1) receptor antagonist/inverse agonist rimona-ant has been shown to reduce body weight, waist circumference,

nsulin resistance, triglycerides, dense LDL, and blood pressure,nd to increase HDL and adiponectin concentrations in bothon-diabetic and diabetic overweight/obese patients [62]. Despiteimonabant withdrawn from European market in 2008, principallyue to its adverse effects on CNS, including depression and anxi-ty, the development of anti-obesity drugs targeting CB1R has beenecently relaunched. The new strategies are principally based onhe discovery of novel CB1 antagonist compounds selectively actingt peripheral level in order to eliminate CNS side effects, and main-aining therapeutic benefits in metabolic syndrome and associatediseases [65].

In this work, we tested the effects of a new CB1 antagonist,ESS038C6, on the metabolic pathways in a mouse model ofiet-induced obesity (DIO mice). The novel CB1 derivative was com-ared to rimonabant. We first measured the impact of NESS038C6n body weight, caloric intake and body mass index. Moreover, wexamined the effect of CB1 blockade on both biochemical bloodarameters and mRNA expression of metabolic markers in the livernd in the visceral fat associated to the hypothalamic pathway ofood intake. In addition, we determined in the several mesolimbicreas the gene expression of monoamine transporters and neu-otrophic factors involved in the metabolic syndrome.

. Materials and methods

.1. Chemicals

NESS038C6 (N-piperidinyl-7-bromo-1-(2′ ,4′-dichlorophenyl)-4,5-dihydro-1H-hieno[2,3-g] indazol-3-carboxamide) was synthesized according to the previouslyeported procedure described in US Patent 7,485,730 [33]. The compoundshowed CB1 and CB2 affinity expressed as Ki of 4,47 and 36,75 nM, respec-ively. CB1 antagonism behavior was previously highlighted for NESS038C6 byoth isolated organ assays and in vivo test based on rat intestinal motility (dataot shown). CB1 antagonist/inverse agonist N-piperidinyl-5-(4-chlorophenyl)--(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (rimonabant) wasurchased by KEMPROTECH Limited, Middlesbrough, UK.

For in vivo assays, daily prepared formulations were employed by solubilizationf the compounds in 0.9% Saline containing Tween 80 (Sigma Aldrich, Milan, Italy),atio of 8:1 (v/v).

.2. Animals

The experiments were performed according to the UE (CEE N◦86/609) guidelinesor the care and use of experimental animals.

A total of 48 male C57BL/6N DIO mice (30–40 g) were purchased from Charlesiver (Calco, Lecco, Italy) at 10 weeks of age. The mice were housed one animaler cage in environmentally controlled conditions (22◦ ± 1 ◦C, and maintained on aeverse 12:12 h light/dark cycle) and fed ad libitum with a high fat diet (D12492,0% fat, 20% carbohydrate, 20% protein, total 5.24 kcal/g; Research Diets Inc., Newrunswick) for 1 week. DIO mice were then weighed and divided into 4 groups of2 animals each and exposed to a 31 days chronic treatment:

“VH FD”: DIO mice fed with diet D12492 and treated with vehicle (Tween 80 inaline solution, ratio of 8:1 v/v);

“NESS038C6 FD”: DIO mice fed with diet D12492 and treated with NESS038C630 mg/kg);

“SR141716 FD”: DIO mice fed with diet D12492 and treated with rimonabant10 mg/kg);

“VH ND”: DIO mice switched to a normal chow (D12450B, 10% fat, 70% carbohy-rate, 20% protein, total 3.85 kcal/g; Research Diets Inc., New Brunswick) and treatedith vehicle.

.3. Chronic treatment

Rimonabant, NESS038C6, and vehicle were administered by oral gavage once peray. Rimonabant and NESS038C6 were administered at a dose of 10–30 mg/kg/day,espectively. The dosage of rimonabant was chosen according to previously reportedata [34], while that of NESS038C6 was selected on the basis of the value of the CB1i compare to that of the reference compound. Individual body weight, caloric intake

Glucokinase, Mm01183091 m1, GLUK LiverPyruvate kinase, Mm00443090 m1, PYK Liver

and cage food consumption were recorded daily. Body length was measured at thestart and at the end treatment for the calculation of body mass index (BMI) accord-ing to the formula reported elsewhere [14]. At the end of the experimental period,blood samples were collected for immediate assessment of serum biochemicalparameters. The brain regions (hypothalamus, prefrontal cortex, amygdala, nucleusaccumbens, hippocampus), the liver and white total adipose tissues (epididymal,lumbar, and perirenal) were removed, weighed and immediately stored at −80 ◦Cuntil RNA analysis.

2.4. Blood analysis

Blood samples (300 �L, approximately) were collected from orbital sinus andcentrifuged at 1000 g for 10 min. Triglycerides (TG), glucose (GLU), total cholesterol(CHO), and transaminase ALT and AST, were determined in the serum samples bya KEYLAB LiquiVet® Analyzer using kits from BPC BioSed srl (Castelnuovo di Porto,Rome, Italy).

2.5. RNA extraction and cDNA synthesis

Frozen dissected central and peripheral tissues were rapidly thawed on ice.Total RNA was extracted by Trizol (Life technologies, Monza, Italy) following man-ufacturer instructions. The extraction procedure was an improvement of the RNAisolation method by Chomczynski and Sacchi [7]. RNA concentration and purity wereestimated by absorbance at 260–280 nm. After digestion with DNAse, RNA (0.5 �g)was retrotranscribed into a cDNA molecule by SuperScript® VILOTM cDNA SynthesisKit (Life technologies, Monza, Italy). An aliquote (3.5 �l) of cDNA was then amplifiedby Real time PCR.

2.6. Real time PCR

Real time PCR was used to evaluate mRNA expression levels of genes in the brainand peripheral tissues showed in Table 1. Beta-actin was used as housekeeping gene.TaqMan® MGB probe genes were amplified in single, parallel reactions to normalizeany variation of RNA or cDNA quality or quantity. PCR was performed in duplicatein 96 wells optical plates (Life technologies, Monza, Italy) by the ABI Prism 7000

Sequence Detection instrument (Life technologies, Monza, Italy). PCR conditionswere: 95 ◦C, 10 min; 50 cycles 95 ◦C, 15 s and 60 ◦C, 1 min. Relative quantificationwas performed using the comparative C(T) method also referred to as the 2 (−��CT)

method.

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Fig. 1. Evolution of body weight and caloric intake in DIO mice after rimonabant, NESS38C6 treatment, and switched to a normal diet. Body weight (a), caloric intake (b)i ESS0c .M. n =w *p < 0.

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n DIO mice during a 31 days chronic treatment with rimonabant (10 mg/kg) and Nompared to those of groups VH FD and VH ND. Values are expressed as mean ± S.Eas used to determine the significance of the pharmacological effect, ***p < 0.001, *

.7. Statistical analysis

Data are expressed as mean ± S.E.M. (n = 12). Two-way Repeated MeasuresNOVA with Bonferroni post test was used to determine the significance of theharmacological effect on the body weight and caloric intake during the chronicreatment. One-way ANOVA with Newman–Keuls post test was instead adopted toetermine: body weight gain, fat mass, BMI and total caloric intake detected at thend of the treatment; the statistical difference between biochemical blood param-ters; the relationship between mRNA expression levels of genes in the brain anderipheric tissues. All statistical analyses were performed using GraphPad Prismersion 5.00 for Windows, (GraphPad Software, San Diego CA).

. Results

.1. Body weight, visceral fat and body mass index

DIO mice were weighed and divided into 4 groups of 12 animalsach, the baseline body weight of the four groups of mice were com-arable (p > 0.05, VHFD = 33.21 g ± 1.85; VHND = 33.19 g ± 1.85;R141716 FD = 32.99 g ± 2.91; NESS038C6 FD = 33.11 g ± 1.23). DIOice were exposed to a 31 days chronic treatment with rimona-

ant (10 mg/kg/day), or with NESS038C6 (30 mg/kg/day), or withehicle, by oral gavage once per day.

Fig. 1 reports body weight (Fig. 1a) and caloric intake (Fig. 1b)ecorded day by day during the chronic treatment. According torevious reported results [34], the switching of DIO mice from a fato a normal diet determined a fast weight reduction in the first fiveays of the treatment [Finteraction (90, 682) = 3.9, p < 0.0001; Ftreatment

3, 682) = 610.1, p < 0.0001; Fdays (30, 682) = 2.8, p < 0.0001] whichs related to a significant decrease of caloric intake in the first three

ays [Finteraction (90, 992) = 1.3, p = 0.03; Ftreatment (3, 992) = 43.4,

< 0.0001; Fdays (30, 992) = 6.4, p < 0.0001] (Fig. 1b). During thereatment, caloric consumption for VH ND reached the same val-es of VH FD, even if the weight of the mice was maintained at

38C6 (30 mg/kg) by oral gavage (o.g.). The data obtained with CB1 antagonists are 12 for each group. Two-way Repeated Measures ANOVA with Bonferroni post test01, *p < 0.05 vs VHFD.

significant lower level. Both the CB1 antagonist compounds assuredweight control of DIO mice fed with fat diet. From the 7th up tothe tenth day of treatment also weight reduction of SR141716 FDand NESS038C6 FD were statistically significant relative to VH FD(Fig. 1a). As in the case of VH ND, significant reduction of caloricintake was highlighted in the first three days of chronic treatmentwith rimonabant and NESS038C6 (Fig. 1b). In the aftermath, caloricintake was substantially equivalent for all the experimental groups.

At the end of chronic treatment, body weight gain, total fat mass,and BMI were comparable for SR141716 FD, NESS038C6 FD, and VHND (Fig. 2). In all the three cases, all the parameters were significantlower than those detected for VH FD [final body weight (Ftreatment

(3, 22) = 32.6, p < 0.0001), visceral fat mass (Ftreatment (3, 22) = 19.6,p < 0.0001), and BMI (Ftreatment (3, 22) = 15.1, p < 0.0001)] (Fig. 2a–c).In contrast, no significant difference in the total caloric consump-tion between experimental groups has been highlighted at the endof DIO mice treatment (Ftreatment (3, 22) = 17.3, p = 0.21)] (Fig. 2d).

3.2. Blood parameters

The effect of CB1 blockade treatments on GLU, TG, CHO, ALT,and AST serum levels of DIO mice were assessed at the termina-tion of the 31th day regimen. The results are reported in Fig. 3. Thefat diet induced GLU, TG, CHO, ALT, and AST values of 162.3 mg/dL,211.5 mg/dL, 230.3 mg/dL, 125.5 U/L and 87.5 U/L, respectively, inDIO mice. As in the case of diet switching to normal chow, treat-ment with rimonabant and NESS038C6 resulted in a significantreduction of GLU (Ftreatment (3, 46) = 5.51, p = 0.003), TG (Ftreatment

(3, 44) = 25.90, p < 0.0001), and CHO (Ftreatment (3, 46) = 23.86,p < 0.0001) levels compared to VH FD (Fig. 3a–c). The effect ofNESS038C6 in TG reduction appeared significantly higher thanthat of rimonabant (Fig. 3b). Moreover, SR141716 FD, NESS038C6

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Fig. 2. Effect of rimonabant and NESS038C6 on body weight gain, fat mass, BMI and kcal. Total body weight (a), final fat mass (b), body mass index (c), and total kcal (d)detected in DIO mice at the end of 31 days of chronic treatment by rimonabant (10 mg/kg) and NESS038C6 (30 mg/kg). The data obtained with CB1 antagonists are comparedt ean ±t

Fia(dCew

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o those of groups VH FD and VH ND. n = 12 for each group. Values are expressed as mhe statistical difference ***p < 0.001, **p < 0.01, *p < 0.05 vs VHFD.

D, and VH ND groups showed statistically significant decreas-ng of the transaminase ALT (Ftreatment (3, 38) = 23.14, p < 0.0001)nd AST (Ftreatment (3, 47) = 9.6, p < 0.0001) values relative to VH FDFig. 3d–e). NESS038C6 showed lower efficacy in both ALT and ASTecreasing if compared to VH ND (Fig. 3d–e). The effect of the novelB1 antagonist compound resulted comparable to that of the ref-rence CB1 antagonist compound in ALT reduction (Fig. 3d), but itas of lower entity in the case of AST (Fig. 3e).

.3. Gene expression in visceral fat

In order to investigate the peripheral effect of NESS038C6nd rimonabant in visceral fat we studied the mRNA expressionf major regulators of adiposity and lipid metabolism, i.e. CB1eceptor, leptin, adiponectin, and fatty acid synthase (FAS) andPAR-�. Fig. 4 shows that both chronic treatment with rimon-bant or NESS038C6, and the diet switch from fat to regularhow were able to affect all the assayed parameters comparedo VH FD. In particular, with reference to VH FD, the treatmentsith CB1 antagonists and the feeding with normal diet inducedRNA expression reduction of CB1 receptor (Ftreatment (3, 41) = 9.15,

< 0.0001) (Fig. 4a), PPAR-� (Ftreatment (3, 43) = 12.99, p < 0.0001)Fig. 4b), FAS (Ftreatment (3, 43) = 17.57, p < 0.0001) (Fig. 4c), and lep-in (Ftreatment (3, 43) = 14.30, p < 0.0001) (Fig. 4d). The change of theiet exerted a higher effect than the CB1 blockade in the decreas-

ng of PPAR-� (Fig. 4b). It determined also lower value of mRNA

xpression of FAS compared to SR141716 FD, but not to NESS038C6D (Fig. 4c). Moreover, one of the most interesting consequences ofESS038C6 and rimonabant treatment was the significant increasef adiponectin mRNA relative to the case of VH FD (Ftreatment

S.E.M, One-way ANOVA with Newman–Keuls post test was performed to determine

(3, 43) = 13.15, p < 0.0001) (Fig. 4e). The effect induced by CB1 antag-onist treatments was comparable to that due to diet switching.

3.4. Gene expression in liver

If compared to VH FD, NESS038C6 FD displayed decreasing ofmRNA expression of CB1 receptor (Fig. 5a) and up-regulation ofmRNA expression of glycolytic enzymes, glucokinase (Ftreatment

(3, 44) = 7.85, p = 0.0003) (Fig. 5b) and pyruvate kinase (Ftreatment

(3, 41) = 11.87, p < 0.0001) (Fig. 5c), lipogenic enzyme fatty acidsynthase (Ftreatment (3, 40) = 4.56, p = 0.008) (Fig. 5d), and PPAR-�(Ftreatment (3, 44) = 18.10, p < 0.0001) (Fig. 5e) in the liver. The samebehavior was highlighted for SR141716 FD, even if the referenceCB1 antagonist compound appeared less active then NESS08C6 inthe up-regulation of pyruvate kinase (Fig. 5c). Analogue effectswere detected also for VH ND, even if with the exclusion of themRNA expression of both glucokinase and PPAR-�. These parame-ters were in fact not significantly affected by switching from fat tonormal diet (Fig. 5b and e). Moreover, with reference to NESS038C6FD, VH ND showed lower efficacy in pyruvate kinase up-regulation(Fig. 5c).

3.5. Gene expression in hypothalamus

Hypothalamus was proposed as hunger/satiety center in CNS[3,72]. In order to elucidate the molecular mechanism of action

of NESS038C6, we evaluated its effect on modulation of mRNAexpression of markers in the hypothalamus, i.e. CB1 receptor, lep-tin receptor, insulin receptor, orexigenic markers Neuropeptide Y(NPY) and Agouti-related protein (AGRP), anorexigenic peptides

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196 A. Mastinu et al. / Behavioural Brain Research 234 (2012) 192– 204

Fig. 3. Effects of weight management treatments on plasma markers. Plasma levels of glucose (a), cholesterol (b), triglycerides (c), transaminase AST (d), transaminase ALT( /kg). TV VA wi*

PuDrabiaNHb

e), after treatment of DIO mice with rimonabant (10 mg/kg) and NESS038C6 (30 mgH ND. n = 12 for each group. Values are expressed as mean ± S.E.M, One-way ANO**p < 0.001, **p < 0.01, *p < 0.05 vs VHFD.

roopiomelancortin (POMC) and Cocaine and Amphetamine reg-lated transcript (CARTPT). Chronic treatment with NESS038C6 inIO mice fed with fat diet compared to VH FD determined: down-

egulation expression of mRNA of CB1 (Fig. 6a), leptin (Fig. 6b),nd insulin (Fig. 6c) receptor; up-regulation of mRNA expression ofoth orexigenic markers NPY (Fig. 6d) and AGRP (Fig. 6e); decreas-

ng of mRNA expression of anorexigenic peptides POMC (Fig. 6f)

nd CARTPT (Fig. 6g). The marker profiles of SR141716 FD and VHD were similar to that detected in the case of NESS038C6 FD.owever, in contrast to the novel CB1 antagonist, both rimona-ant treatment and diet switching did not significantly alter the

he data obtained with CB1 antagonists are compared to those of groups VH FD andth Newman–Keuls post test was performed to determine the statistical difference

mRNA expression of Neuropeptide Y compared to VH FD (Fig. 6d).A deeper comparison between NESS038C6 FD, SR141716 FD, andVH ND, highlighted: higher down-regulation of CB1 receptor mRNAexpression by SR141716 FD (Fig. 6a), significant increase of AGRPmRNA expression due to the administration of NESS038C6 (Fig. 6e).

3.6. Gene expression of monoamminergic transporters and

neurotrophins in CNS

Dysregulation of the endocannabinoid system is known tointerfere with emotional processing of stressful events in the

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A. Mastinu et al. / Behavioural Brain Research 234 (2012) 192– 204 197

Fig. 4. Gene expression in visceral fat. mRNA expression by PCR Real time of CB1 receptor (a), PPAR-� (b), fatty acid synthase (FAS) (c), leptin (d), and adiponectin (e) in thevisceral fat of DIO mice switched to regular diet (VH ND) or fed with a fat diet and treated for 31 days with rimonabant (10 mg/kg), NESS038C6 (30 mg/kg) or vehicle (VH FD).V est wav

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alues are expressed as mean ± S.E.M, One-way ANOVA with Newman–Keuls post ts VHFD.

esolimbic system [32]. To determine potential interactions of thendocannabinoid system with monoaminergic and neurotrophinystems we measured mRNA expression of the monoaminergicransporters and neurotrophic factors after CB1 antagonist treat-

ent. In particular, we studied mRNA expression of dopamineransporter (DAT), norepineprhine transporter (NET) and sero-onine transporter (SERT) in nucleus accumbens (ACC) (Fig. 7),mygdala (AMY) (Fig. 8), and prefrontal cortex (PCX) (Fig. 9).oreover we detected brain derived neurotrophic factor (BDNF)

nd nerve growth factor (NGF) expression in hippocampus (HIPP)Fig. 10).

Rimonabant treatment generated a significant increase ofonoamine transporters (MATs) in mesolimbic pathway relative

o VH FD:ACC (Fig. 7): DAT (Ftreatment (3, 43) = 14.58, p < 0.0001), NET

Ftreatment (3, 45) = 41.28, p < 0.0001), SERT (Ftreatment (3, 43) = 10.43,

< 0.0001);

PCX (Fig. 8): DAT (Ftreatment (3, 43) = 16.17, p < 0.0001), NETFtreatment (3, 45) = 36.27, p < 0.0001), SERT (Ftreatment (3, 43) = 19.53,

< 0.0001);

s performed to determine the statistical difference ***p < 0.001, **p < 0.01, *p < 0.05

AMY (Fig. 9): DAT (Ftreatment (3, 43) = 8.52, p < 0.0001), NET(Ftreatment (3, 39) = 53.49, p < 0.0001), SERT (Ftreatment (3, 42) = 29.30,p < 0.0001).

Moreover SR141716 FD mice showed significantly lower mRNAexpression of BDNF (Ftreatment (3, 40) = 16.35, p < 0.0001) and NGF(Ftreatment (3, 43) = 23.25, p < 0.0001) in hippocampus DIO mice com-pared to both VH FD and VH ND (Fig. 10).

It is important to note, that in contrast to rimonabant treatment,the administration of the novel CB1 antagonist NESS038C6, as inthe case of VH ND, induced no effect on DAT, NAT, and SERT in ACC,PCX, and AMY; at the same time it improved mRNA expressionof neurotrophic factors BDNF and NGF relative to both VH FD andVH ND.

4. Discussion

According to the main standard procedures for the screen-ing and the evaluation of potential applications of cannabinoidCB1 receptor antagonists, [35,71] this paper investigates the

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F cokinad g/kg),O atistic

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ig. 5. Gene expression in the liver. Liver mRNA expression of CB1 receptor (a), gluiet (VH ND) or fed with a fat diet and treated for 31 days with rimonabant (10 mne-way ANOVA with Newman–Keuls post test was performed to determine the st

harmacological properties of a novel CB1 antagonist (NESS038C6)rom molecular to entire animal level.

The effects of CB1 receptor blockade by NESS038C6 andimonabant were highlighted on mice (C57BL/6) previouslyxposed to a long-term fat diet (DIO mice). During all the1 days of chronic treatment with both the CB1 antagonistompounds, DIO mice fed with fat diet (NESS038C6 FD andR14176A FD) evidenced weight loss profiles equivalent to thatetermined in the DIO mice experimental group switched toegular chow and treated with vehicle (VH ND). In contrasthe administration of a fat diet without cannabinoid antago-ist treatment determined a significant increase of the miceeight during the period of the study (Fig. 1a). The action

xerted on food intake by both acute and sub-chronic rimon-bant treatment was previously reported for both rats [8,9,58]nd DIO mice models [44,47,48,70]. The behavior highlightedith NESS038C6 was comparable to those previously reported

or other CB1 antagonist compounds, particularly for rimona-ant [4,6,8,12,17,22,47,64,66]. According to previous reported datay our and other groups concerning rimonabant chronic treat-ent [34,49], NESS038C6 displayed a marked hypophagic action

n the first days of the treatment, with evidence of short timeolerance effect (Fig. 1b). After the fifth day, the caloric intakeetermined with the novel CB1 antagonist compound or rimon-bant, was in fact normalized to the same level of that highlighted

se (b), pyruvate kinase (c), FAS (d), and PPAR-� (e) of DIO mice switched to regular NESS038C6 (30 mg/kg) or vehicle (VH FD). Values are expressed as mean ± S.E.M,al difference ***p < 0.001, **p < 0.01, *p < 0.05 vs VHFD.

with VH FD. The profile of caloric intake determined withNESS038C6 chronic treatment was also comparable to that exertedby diet switching in vehicle treated mice (VH ND). Also in thislast case it was in fact evidenced a fast reduction of caloric intakein the first three days of the study, with a normalization of thelevel in the aftermath. As previously reported, this effect in VHND group appears to be due to the habituation of the mice tothe novel chow. Despite no significant difference was recordedfor caloric intake within all the experimental groups during thewidest part of the chronic treatment, weight control in fat dietmice was assured with NESS038C6 administration at the samelevel of that obtained by diet switching (VH ND). The same profilewas obtained with SR14176A FD. According to previously reportedresults, [9,10,26,47,48,63], by comparison of the behaviors dis-played in Fig. 1a and b, it appears that chronic treatments with bothCB1 antagonist compounds allow the achievement of an equilib-rium state of the metabolism in which caloric intake is counteractedby a higher stimulation of energy expenditure. Evidences of theinstauration of this stabilized situation with NESS038C6 FD as forSR14176A FD are easily derived from Figs. 2 and 3. In agreementwith previous studies concerning rimonabant [19,42], in fact, we

found that chronic administration of the novel CB1 antagonist com-pound reduced the total fat mass and BMI, compared to vehicle FD.Moreover it determined an improvement of cardiovascular risk fac-tors in serum, as previously reported for rimonabant [11,57,61].

Page 8

A. Mastinu et al. / Behavioural Brain Research 234 (2012) 192– 204 199

Fig. 6. Gene expression in the hypothalamus. Hypothalamic mRNA expression of CB1 receptor (a), leptin receptor (b), insulin receptor (c), Neuropeptide Y (d), Agouti-relatedprotein (e), Proopiomelanocortin (f), and Cocaine and amphetamine regulated transcript (e) of DIO mice switched to regular diet (VH ND) or fed with a fat diet and treatedfor 31 days with rimonabant (10 mg/kg), NESS038C6 (30 mg/kg) or vehicle (VH FD). Values are expressed as mean ± S.E.M, One-way ANOVA with Newman–Keuls post testwas performed to determine the statistical difference ***p < 0.001, **p < 0.01, *p < 0.05 vs VHFD.

Page 9

200 A. Mastinu et al. / Behavioural Brain Research 234 (2012) 192– 204

Fig. 7. Gene expression in the nucleus accumbens. mRNA expression of dopamine transporter (a), norepinephrine transporter (b) and serotonin transporter (c), genee D) or

( VA w*

Ics

taetcvimrsaePubitwml

xpression in the nucleus accumbens of of DIO mice switched to regular diet (VH N30 mg/kg) or vehicle (VH FD). Values are expressed as mean ± S.E.M, One-way ANO**p < 0.001, **p < 0.01, *p < 0.05 vs VHFD.

n general the entity of the effects induced by NESS038C6 wasomparable to that exerted by both rimonabant treatment and thewitch from fat to regular diet.

Further evidences have been provided by our new data onhe correlation of CB1 receptor blockade and peripheral metabolicnd molecular changes in liver, adipose tissue and blood param-ters. We particularly showed that NESS038C6 and rimonabantreatments normalize blood values of glucose, triglycerides, totalholesterol, GOT and GPT transaminases (Fig. 3). Despite it was pre-iously reported that the liver metabolism is differently regulatedn response to diet-induced obesity and to CB1 antagonist treat-

ent [5,16,39], a comparable effect was detected on transaminaseegulation in NESS038C6 FD, SR14176A FD, and VH ND. For this rea-on in order to investigate the regulation of energetic metabolismfter treatment with NESS038C6, this work analyzed the genexpression modifications of the glucolytic-lipogenic enzymes andPAR-� in the liver (Fig. 5). Our results clearly indicate that thep-regulation of liver CB1 receptors induced by fat diet is reversedy NESS038C6 treatment, suggesting that the observed metabolic

mprovement could be mediated by the blockade of this recep-

or. The same behavior was also detected in DIO mice treatedith rimonabant, even if with an effect of lower entity. Treat-ent with rimonabant and NESS038C6, especially, normalized the

iver parameters related to carbohydrate and lipid metabolism, i.e.

fed with a fat diet and treated for 31 days with rimonabant (10 mg/kg), NESS038C6ith Newman–Keuls post test was performed to determine the statistical difference

glucokinase, pyruvate kinase, fatty acid synthase and PPAR-�. Inaddition, we observed no stimulation of the mRNA expression ofglucolytic and lipogenic enzymes in the liver of DIO mice fed withfat diet. In line with this behavior, it is important to note that ithas been previously hypothesized that the provision of saturatedfatty acid rich diets led to reduced expression of mRNA of lipogenicgenes [28,60]. Moreover, Jourdan et al. [28] reported that this typeof diet increases the concentration of free fatty acids in the bloodcausing liver steatosis. Additionally, our histological analysis of theliver of VH FD mice (data not showed) evidenced marked steatosiscompared to the corresponding organs of both DIO mice treatedwith CB1 antagonists (SR14176 FD and NESS038C6 FD groups) andVH ND.

Recently, many studies on animal and human [13,36–38,46,69]established that diet induced obesity determines an over-activationof endocannabinoid system in visceral fat. In general agreementwith previous data by Starowicz et al. [56], our results evidencedthat CB1 receptor blockade exerts specific effects on visceralfat metabolism. Indeed we observed a significant CB1 receptorup-regulation in visceral fat of VH FD, and a decreased mRNA

expression of the receptors in the tissues obtained from otherexperimental groups (Fig. 4). Furthermore, CB1 antagonist treat-ments normalized the mRNA expression level of lipogenic genes invisceral fat. As consequence, deficit in adipogenesis anddecreasing

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A. Mastinu et al. / Behavioural Brain Research 234 (2012) 192– 204 201

Fig. 8. Gene expression in the amygdala. mRNA expression of dopamine transporter (a), norepinephrine transporter (b), and serotonin transporter (c) of DIO mice switchedt nabanm termi

oncNoatbltab

tbrtsis[ipratt

o regular diet (VH ND) or fed with a fat diet and treated for 31 days with rimoean ± S.E.M, One-way ANOVA with Newman–Keuls post test was performed to de

f the fat storage have been highlighted. DIO mice switched toormal diet (VH ND) showed the same expression levels of glu-olytic and lipogenic enzymes of mice receiving rimonabant andESS038C6, fed with fat diet. These results are in line with previ-usly reported data [63] and they are supported by visceral fat gains reported in Fig. 2. According to previously reported considera-ions [30], the remarkable decrease of body weight accomplishedy both a reduction of fat mass and an improvement of cardiovascu-

ar parameters in DIO mice treated with CB1 antagonists detected inhis study, could be elucidated through the up-regulation of genescting at different levels of the lipogenic and glicolytic pathways inoth the liver and visceral fat.

Among peripheral targets related to metabolic pathways, lep-in and adiponectin represents important signaling moleculesetween fat tissue and central areas involved in weight and feedingegulation. It has been confirmed that both the hormones appearo be involved in cannabinoids action on food intake and homeo-tasis regulation. Leptin inhibits appetite by acting on receptorsn the hypothalamus, where it counteracts the effects of feedingtimulants secreted by cells in the gut and in the hypothalamus23]. Adiponectin is an important regulator of insulin sensitiv-ty. Adiponectin expression is reduced in obese patients and lowlasma adiponectin levels are related to the development of insulin

esistance [25,68]. In addition, rimonabant treatment in isolateddipocytes increased expression of adiponectin [2], and normalizedhe leptin levels in the blood of DIO mice [57]. In agreement withhe above reported behavior, we have demonstrated that treatment

t (10 mg/kg), NESS038C6 (30 mg/kg) or vehicle (VH FD). Values are expressed asne the statistical difference ***p < 0.001, **p < 0.01, *p < 0.05 vs VHFD.

of DIO mice with NESS038C6 decreased leptin and up-regulatedadiponectin mRNA expression in visceral fat (Fig. 4). Leptin circu-lates in levels proportional to body fat and it is considered the mainendocrine adiposity signal in mammals together with insulin hor-mone [53]. Furthermore high levels of adiponectin were associatedto glucose homeostasis, visceral fat reduction and cardiovascularimprovement [29]. Consequently, the weight loss and the improve-ment of cardiovascular risk factors in blood detected for NESS038C6FD and VH ND could be also explained according to the leptin andadiponectin expression profiles.

Moreover, this work analyzes the expression of the hypotha-lamic genes regulated by leptin and insulin signaling and CB1receptor expression (Fig. 6). Our data showed a down-regulationof leptin, insulin and CB1 receptors in hypothalamus of DIOmice treated with NESS038C6 and rimonabant. As confirmed ina previous study by our group [34], leptin plays an importantrole in the control of feeding behavior and energy expenditurein hypothalamic neurons. In addition, it was reported that cir-culating levels of leptin and insulin are significantly high inDIO mice due to increased body fat mass and insulin resis-tance [52]. According to our data, it appears that reductionof leptin and increase of adiponectin levels in visceral fat ofNESS038C6 FD, SR14176A FD, and VH ND induce the down-

regulation of both leptin and insulin receptors in hypothalamus.Concordant findings from both this study and literature sup-port the hypothesis that CB1 receptor blockade as the switchingto regular chow restore circulating levels of leptin and insulin

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202 A. Mastinu et al. / Behavioural Brain Research 234 (2012) 192– 204

Fig. 9. Gene expression in the prefrontal cortex. mRNA expression of dopamine transporter (a), norepinephrine transporter (b), and serotonin transporter (c) of DIO mices rimona deter

brtYnabanavchwanitw“apCssa

witched to regular diet (VH ND) or fed with a fat diet and treated for 31 days withs mean ± S.E.M, One-way ANOVA with Newman–Keuls post test was performed to

y lowering the expression of their hypothalamic correspondingeceptors. Hypothalamic leptin pathway was also associated tohe expression of both the orexigenic neuropeptides neuropeptide

(NPY) and agouti-related protein (AGRP), and the anorexigeniceuropeptides pro-opiomelanocortin (POMC) and cocaine- andmphetamine-regulated transcript (CART) [27,51]. Leptin regulatesoth NPY/AGRP and POMC/CART systems by inhibiting NPY/AGRPnd stimulating POMC/CART [51]. We observed that weight lossormalizes leptin mRNA in visceral fat of DIO mice after NESS038C6nd rimonabant treatments or diet switch. In line with our pre-ious study concerning rimonabant chronic treatment [34], asonsequence of expression reduction of leptin receptor mRNA inypothalamus of DIO mice elicited by NESS038C6 administration,e showed that NPY and AGRP mRNA expression was increased,

nd those of POMC and CART reduced (Fig. 6). It is important toote that NESS038C6 appeared more effective than rimonabant

n the up-regulation of mRNA of both the orexigenic neuropep-ides. With this study we then support the proposed mechanism inhich leptin-hypothalamic pathway plays an important role from

sensor” to “generator” in the negative regulation of energy intakend body weight [31]. A decreased orexigenic effect via NPY-AGRPathway associated with a possible anorexigenic effect via the

ART-POMC pathway can be related to the increased mRNA expres-ion of leptin in the visceral fat of VH FD. NESS038C6 treatmentignificantly improves this signaling acting both in hypothalamusnd in adipose tissue.

abant (10 mg/kg), NESS038C6 (30 mg/kg) or vehicle (VH FD). Values are expressedmine the statistical difference ***p < 0.001, **p < 0.01, *p < 0.05 vs VHFD.

In addition to the positive effects on appetite regulation of CB1antagonist compounds, it has been reported on the psychiatricside effects induced by rimonabant, i.e. depression, anxiety andirritability [18,24]. These rimonabant adverse effects, which areassociated to the block of CB1 receptor in central nervous system,induced anxiety-like responses and an alteration of monoaminergicneurotransmission in both rat and mice [18]. Moreover, the expres-sion of neurotrophic factors, BDNF and NGF, which are involved inthe depression development, was not improved after CB1 antag-onist treatment [1,21]. In line with these data, to determine thecentral effect on monoaminergic transporters and neurotrophicfactors after CB1 antagonist treatments, we studied mRNA expres-sion profile of DAT, NET, SERT in nucleus accumbens, amygdala,and prefrontal cortex of all the assayed mice groups. Moreoverwe determined mRNA expression of the two neurotrophins BDNFand NGF in hippocampus. We observed increased mRNA expres-sion of monoamine transporters (MATs) in all the investigated areaof mesolimbic system after rimonabant treatment. No significanteffect has been instead elicited by NESS038C6 administration rel-ative to VH ND and VH FD (Figs. 7–9). MATs are the targets ofmany therapeutic drugs related with mood disorders because theyare responsible for the reuptake of their associated amine neuro-

transmitters serotonin, dopamine and norepinephrine. Metabolicsyndrome modifies the monoaminergic transmission by generatingan increment of food intake for its own gratification [15]. Fur-thermore, obese human and animal show amineneurotransmitter

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A. Mastinu et al. / Behavioural Brain R

Fig. 10. Gene expression in the hippocampus. mRNA expression of brain derivedneurotrophic (BDNF) factor (a), and nerve growth factor (NGF) (b) of DIO miceswitched to regular diet (VH ND) or fed with a fat diet and treated for 31 dayswith rimonabant (10 mg/kg), NESS038C6 (30 mg/kg) or vehicle (VH FD). Values areepV

dwmbtDt

hait(wFrmeo

m

[

[

[

[

[

[

[

[

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[[

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xpressed as mean ± S.E.M, One-way ANOVA with Newman–Keuls post test waserformed to determine the statistical difference ***p < 0.001, **p < 0.01, *p < 0.05 vsHFD.

ecrease and altered corresponding reuptake [40,41,50]. In thisork, rimonabant markedly influenced the expression of the threeonoamine transporters in all the investigated area of DIO mice

rain, with possible negative effects on animal behavior. In con-rast no effect has been instead detected on mRNA expression ofAT, NET, SERT in the mesolimbic system by NESS038C6 chronic

reatment.It has been reported that obesity impairs neurogenesis in the

ippocampus [43,67] by lowering neurotrophin expression. Ingreement with Assareh et al. [1] we did not observe a normal-zation of BDNF and NGF mRNA in hippocampus after a chronicreatment with rimonabant or a diet switching to regular chowFig. 10). SR14176A FD showed mRNA expression of neurotrophinshich are even significantly lower than those of VH ND and VH

D. In contrast, chronic treatment with NESS038C6 improved neu-otrophin expression by markedly increasing both BDNF and NGFRNA expression. Positive effect on mood disorders could be then

xpected in the case of the administration of the novel CB1 antag-nist compound.

In conclusion, NESS038C6 determined an improvement ofetabolic syndrome in DIO mice, by restoring the signaling

[

[

esearch 234 (2012) 192– 204 203

between visceral fat and hipothalamus. Moreover, NESS038C6, butnot rimonabant, normalized MATs and improved neurotrophinmRNA expression. According to the highlighted profile, NESS038C6appears to be a hopeful candidate for the treatment of obesity andmetabolic disorder without the adverse effects instead observedwith the reference CB1 antagonist/inverse agonist rimonabant.

Acknowledgments

MIUR is acknolwledged for economic support (Project:“Cannabinoidi e obesità”; DM28141).

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