original article nicotine induces negative energy balance through hypothalamic...

Nicotine Induces Negative Energy Balance ThroughHypothalamic AMP-Activated Protein KinasePablo B. Martínez de Morentin,

1,2Andrew J. Whittle,

3Johan Fernø,

4,5Rubén Nogueiras,

1,2

Carlos Diéguez,1,2

Antonio Vidal-Puig,3and Miguel López

1,2

Smokers around the world commonly report increased bodyweight after smoking cessation as a major factor that interfereswith their attempts to quit. Numerous controlled studies in bothhumans and rodents have reported that nicotine exerts a markedanorectic action. The effects of nicotine on energy homeostasishave been mostly pinpointed in the central nervous system, butthe molecular mechanisms controlling its action are still notfully understood. The aim of this study was to investigate theeffect of nicotine on hypothalamic AMP-activated protein kinase(AMPK) and its effect on energy balance. Here we demonstratethat nicotine-induced weight loss is associated with inactivationof hypothalamic AMPK, decreased orexigenic signaling in thehypothalamus, increased energy expenditure as a result of in-creased locomotor activity, increased thermogenesis in brownadipose tissue (BAT), and alterations in fuel substrate utilization.Conversely, nicotine withdrawal or genetic activation of hypotha-lamic AMPK in the ventromedial nucleus of the hypothalamus re-versed nicotine-induced negative energy balance. Overall thesedata demonstrate that the effects of nicotine on energy balanceinvolve specific modulation of the hypothalamic AMPK-BAT axis.These targets may be relevant for the development of new thera-pies for human obesity. Diabetes 61:807–817, 2012

Nicotine, the main addictive component of to-bacco, shows anorexigenic properties and pro-motes body weight reduction in humans androdents (1–4). In keeping with these points,

smoking cessation is associated with hyperphagia andweight gain in over 70% of people who attempt to do so. Infact, in many cases, the prospect of weight gain itself hasacted as an incentive for persevering in the smoking habit(1,2,4). Therefore, it is clear that smoking leads to a stateof negative energy balance and one that might be exploitedfor therapeutic purposes if its molecular mechanisms werebetter understood.

Maintaining energy balance depends on the efficiency oftightly regulated processes involved in 1) energy intake(feeding); 2) energy expenditure (EE) comprising basalmetabolism, adaptive thermogenesis, and physical (loco-motor) activity (LA); and 3) nutrient partitioning (5,6). Thehypothalamus is a region within the central nervous sys-tem that plays a major role in the modulation of energybalance. The hypothalamic arcuate nucleus (ARC) expressesthe orexigenic (feeding-promoting) neuropeptides agouti-related protein (AgRP) and neuropeptide Y (NPY) and theanorexigenic precursors proopiomelanocortin (POMC) andcocaine and amphetamine-regulated transcript (CART)(5,6). In addition to controlling food intake, the hypothala-mus modulates EE in brown adipose tissue (BAT) (7,8),as well as glucose (9,10) and lipid metabolism in peripheraltissues (11,12) through the autonomic nervous system(7,8,12,13).

An important mediator of all of these effects is hypo-thalamic AMP-activated protein kinase (AMPK), a cellulargauge that controls whole-body energy balance and is ac-tivated in conditions of low energy (14,15). When activated,AMPK optimizes energy utilization by increasing energy-producing and reducing energy-wasting processes at thewhole-body level. In the hypothalamus, AMPK activationdrives feeding by modulating mitochondrial fatty acid oxi-dation and intracellular levels of reactive oxygen species.This untimely regulates neuropeptide expression in theARC (16–18) and also suppresses EE and thyroid-inducedthermogenesis in the BAT (7,8). Activated AMPK is also animportant regulator of fatty acid biosynthesis, suppressingde novo lipogenesis by phosphorylation and inactivation ofacetyl-CoA carboxylase (ACC) (7,8,14,15,17,18).

Whether the effects of nicotine on energy balance aremediated by modulation of hypothalamic AMPK is cur-rently unknown. However, there are some clues that mightindicate the existence of such an action. Nicotine cor-egulates most of the processes controlled by AMPK. Forinstance, chronic nicotine exposure in rats reduces theexpression of NPY (19) and consistently blunts NPY-induced hyperphagia (20). Nicotine withdrawal also increa-ses hypothalamic NPY and AgRP expression, mechanismsthat contribute to increased feeding after nicotine cessation(20,21). Current evidence has demonstrated that nicotinealso upregulates the expression of POMC (22) and decreasesfeeding through activation of POMC neurons (3). Besidesits actions on hypothalamic feeding circuits nicotine alsomodulates peripheral metabolism in rats, increasing BATthermogenesis through activation of the sympathetic ner-vous system (SNS) and increased expression of uncouplingprotein 1 (UCP1) (23,24).

Based on this evidence, we hypothesized that the effectof nicotine on energy homeostasis could be mediated atleast in part by hypothalamic AMPK. The aim of this studywas to investigate the effect of nicotine on hypothalamic

From the 1Department of Physiology, School of Medicine-CIMUS, Universityof Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago deCompostela, A Coruña, Spain; the 2CIBER Fisiopatología de la Obesidad yNutrición (CIBERobn), Santiago de Compostela, Spain; the 3Institute ofMetabolic Science, Metabolic Research Laboratories, NIHR Cambridge Bio-medical Research Centre Addenbrooke’s Hospital, University of Cambridge,Cambridge, U.K.; the 4Dr. Einar Martens’ Research Group for BiologicalPsychiatry, Department of Clinical Medicine, University of Bergen, Bergen,Norway; and the 5Center for Medical Genetics and Molecular Medicine,Haukeland University Hospital, Bergen, Norway.

Corresponding author: Miguel López, [email protected] 2 August 2011 and accepted 8 December 2011.DOI: 10.2337/db11-1079This article contains Supplementary Data online at http://diabetes

.diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1079/-/DC1.� 2012 by the American Diabetes Association. Readers may use this article as

long as the work is properly cited, the use is educational and not for profit,and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

See accompanying commentary, p. 776.

diabetes.diabetesjournals.org DIABETES, VOL. 61, APRIL 2012 807

ORIGINAL ARTICLE

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AMPK and its impact on feeding and physiological param-eters modulating energy balance such as EE, LA, BATfunction, and nutrient partitioning. Our data confirm theexistence of a previously unknown link between nicotine-induced negative energy balance and hypothalamic AMPK.

RESEARCH DESIGN AND METHODS

Animals.We used adult male Sprague-Dawley rats (9–11 weeks old; Animalario-General USC). Rats were housed in a temperature-controlled (22°C) room, witha 12-h light/dark cycle (7:00 to 19:00). The experiments were performed inagreement with the International Law on Animal Experimentation and ap-proved by the USC Bioethics Committee and the Ministry of Science andInnovation of Spain (PS09/01880). For all of the experimental groups andanalytic methods, we used 9–16 rats per group.Nicotine treatment. Nicotine (nicotine hydrogen tartrate salt; Sigma-Aldrich,St. Louis, MO) or vehicle (saline) was given subcutaneously (2 mg/kg per 12 h)in a total volume of 100 mL (25). Rats received a nicotine or vehicle injectionevery 12 h in both subchronic (48 h) and chronic (17 days) experiments. Tovalidate the EE data, all rats were matched for body weight before treatmentbegan. To control nicotine-induced respiratory depression (RD), rats weretreated with nicotine and symptoms of RD were visually controlled from 2 minbefore to 12 h after treatment. For the antagonism of a3b4 nicotinic acetyl-choline receptors, rats were treated previously (30 min) intraperitoneally withmecamylamine (mecamylamine hydrochloride, 1 mg/kg; Sigma-Aldrich) asdescribed recently (3) and subcutaneously with nicotine during 48 h.Intracerebroventricular treatment. Intracerebroventricular cannulae werestereotaxically implanted under ketamine/xylazine anesthesia in the lateralventricle (coordinates 1.2 mm lateral to sagittal suture and 1.0 mm posterior tobregma), as described previously (8,18,26), and demonstrated to be correctlylocated by methylene blue staining. Rats received either a single administra-tion of the AMPK activator AICAR (Sigma-Aldrich; 5 mg, dissolved in 5 mL ofsaline) or vehicle (5 mL saline) (8) at 19:00.Stereotaxic microinjection of adenoviral expression vectors. Rats wereplaced in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) underketamine/xylazine anesthesia. The ventromedial nucleus of the hypothalamus(VMH) was targeted bilaterally using a 25-gauge needle (Hamilton, Reno, NV)connected to a 1-mL syringe. The injection was directed to stereotaxic coor-dinates 2.3/3.3 mm posterior to the bregma (two injections were performed ineach VMH), 60.6 mm lateral to midline and 10.2 mm below the surface of theskull (8,18). Adenoviral vectors (green fluorescent protein [GFP] or AMPKa-CA; Viraquest, North Liberty, IA) were delivered at a rate of 200 nL/min for5 min (1 mL/injection site) (8,18).Conditioned taste aversion. Five days before the test, rats were allowed 2-hdaytime access to water. On the day of the experiment, rats were given accessto 0.15% sodium saccharin rather than water for 30 min. Next, one experimentalgroup was injected intraperitoneally with 0.15 mol/L lithium chloride (LiCl) insaline and subcutaneously with saline (vehicle). The second experimentalgroup was injected intraperitoneally with saline and subcutaneously withnicotine. A third experimental group was injected intraperitoneally and sub-cutaneously with saline. Twenty-four hours later, rats were given a 2-h access toa two-bottle choice test of 0.15% saccharin versus water. Conditioned tasteaversion (CTA) was expressed as percent saccharin preference ratio [100 3saccharin intake/(saccharin intake + water intake)]. A saccharin preferenceratio ,50% was considered as a signal of CTA (26).Temperature measurements. Body temperature was recorded with a rectalprobe connected to digital thermometer (BAT-12 Microprobe-Thermometer;Physitemp, Clifton, NJ). Skin temperature surrounding BAT was recorded withan infrared camera (E60bx: Compact-Infrared-Thermal-Imaging-Camera; FLIR,West Malling, Kent, U.K.) and analyzed with a specific software package (FLIR-Tools-Software; FLIR). We used eight animals per group, and for each animal,three or four pictures were analyzed. The skin temperature surrounding BAT forone particular animal was calculated as the average temperature recorded byanalyzing those pictures.In situ hybridization. Coronal brain sections (16 mm) were probed as pub-lished (8,18,26). We used specific oligonucleotides for AgRP, CART, fatty acidsynthase (FAS), NPY, and POMC detection (Supplementary Table 1). Anatom-ical regions were analyzed using the rat brain atlas (27). Sections were scanned,and the hybridization signal was quantified by densitometry using ImageJ-1.33(National Institutes of Health, Bethesda, MD) (8,18,26). We used between 16 and20 sections for each animal (4 to 5 slides with four sections per slide). The meanof these 16–20 values was used as the densitometry value for each animal.Enzymatic assays. FAS activity was performed as described previously (8,18).Western blotting. The hypothalamus, defined by the posterior margin of theoptic chiasm and the anterior margin of the mammillary bodies (to the depth of;2 mm), was dissected out and immediately homogenated on ice to preserve

phosphorylated protein levels (8,18,26). Hypothalamic total protein lysateswere subjected to SDS-PAGE, electrotransferred on a polyvinylidene fluoridemembrane, and probed with the following antibodies: ACC, pACC-Ser79,AMPKa1, and AMPKa2 (Upstate Biotechnology, Temecula, CA); FAS (BDBiosciences, Franklin Lakes, NJ), pAMPK-Thr172, and phospho-liver kinaseB1 (pLKB1)-Ser428 (Cell Signaling, Danvers, MA); PP2Ca and Ca2+/calmodulin-dependent protein kinase kinase a and b (CaMKKa and CaMKKb) (Santa CruzBiotechnology, Santa Cruz, CA); and b-actin (Sigma-Aldrich) as described pre-viously (8,18,26). Values were expressed in relation to b-actin levels.Real-time quantitative PCR.We performed real-time PCR (TaqMan; AppliedBiosystems, Carlsbad, CA) as described previously (8,26) using specific sets ofprimer probes (Supplementary Table 2). Values were expressed in relation tohypoxanthine-guanine phosphoribosyltransferase levels.EE, LA, respiratory quotient, and nuclear magnetic resonance analysis.

During the 48-h experiment and the 17-day experiment, rats were analyzed forEE, respiratory quotient (RQ), and LA using a calorimetric system (LabMaster;TSE Systems, Bad Homburg, Germany). This system is an open-circuit in-strument that determines O2 consumption, CO2 production, and RQ (VCO2/VO2)(12,28). Rats were placed for adaptation for 1 week before starting themeasurements. For the measurement of body composition, we used the nu-clear magnetic resonance imaging (Whole Body Composition Analyzer;EchoMRI, Houston, TX) (8,12,28).Statistical analysis. Data are expressed as mean 6 SEM. Statistical signifi-cance was determined by Student t tests or ANOVAs and post hoc two-tailedBonferroni test. P , 0.05 was considered significant.

RESULTS

Nicotine treatment induced negative energy balanceby decreasing feeding and increasing EE. Nicotine-treated rats exhibited a marked reduction in body weight(Fig. 1A) associated with reduced food intake over 24 and48 h of treatment (Fig. 1B). We evaluated whether theanorectic effect was a genuine action of nicotine or a sec-ondary effect of the drug. It has been reported that nico-tine induces RD in neonatal and anesthetized rats (29,30).Nicotine-treated rats showed a significant short-term RDafter the injection (RD-vehicle–treated: nondetected; RD-nicotine–treated: 5.0 6 1.4 min; P , 0.001). Ten minutesafter nicotine administration just 5% of the rats showedsymptoms of RD; 15 min after injection those symptomswere completely abolished (Fig. 1C). These data suggestthat an effect of RD on 12-h feeding patterns is unlikely.

We further evaluated whether the anorectic effect ofnicotine was associated with an aversive response by per-forming a CTA experiment. In this type of experiment asaccharin preference ratio ,50% was considered a markerof CTA, since it indicates that rats prefer to drink water afteran aversive response to saccharine (26). LiCl was used asa positive control, and as expected, LiCl-treated rats dranksignificantly less saccharin solution (LiCl paired flavor; Fig.1D), indicating that they had developed CTA (26). Vehicleand nicotine-treated rats had a similar saccharine pref-erence consumption ratio, and it is noteworthy that thiswas .50% (Fig. 1D). Supporting the absence of stress,illness, or malaise associated with the nicotine treatments,we did not observe reduced LA (Fig. 1G), changes in skinaspect, stool consistency, or obvious abnormal behaviorsuch as was evident in the LiCl-treated animals (data notshown) (26).

We next evaluated whether nicotine treatment induceda change in the other side of the energy balance equation.Nicotine-treated rats showed a significant increase in bodytemperature (Fig. 1E) associated with higher EE (Fig. 1F;area under the curve: vehicle: 1006 2.4; nicotine: 1326 9.1;P, 0.01), increased LA (Fig. 1G), and reduced RQ (Fig. 1H)compared with vehicle-treated rats. Overall, these dataindicate that nicotine treatment induced negative energybalance, including decreased feeding, increased EE, andincreased lipid oxidation.

NICOTINE MODULATES AMPK AND ENERGY BALANCE

808 DIABETES, VOL. 61, APRIL 2012 diabetes.diabetesjournals.org



FIG. 1. Effects of nicotine administration on energy balance. A: Body weight change. B: Food intake. C: RD. D: CTA. E: Body temperature change.F: EE (cumulative in left panel and total in right panel). G: LA (cumulative in left panel and total in right panel). H: RQ (cumulative in left paneland total in right panel) of vehicle (Veh) and nicotine-treated (Nic) rats for 48 h are shown. *P< 0.05, **P< 0.01, ***P< 0.001 vs. vehicle; ###P<0.001 nicotine vs. LiCl. In the right panels of F and G, the asterisks above the lines refer to the daily (diurnal + nocturnal) EE or LA. All data areexpressed as mean 6 SEM.

P.B. MARTÍNEZ DE MORENTIN AND ASSOCIATES


Nicotine treatment reduced AgRP and NPY andincreased POMC expression in the ARC and decreasedhypothalamic AMPK activation. Consistent with theirhypophagic state, expression of orexigenic neuropeptidesAgRP and NPY decreased and POMC expression in-creased (Fig. 2A and B) in the ARC of nicotine-treatedrats. No significant changes were detected in CARTmRNA expression in the ARC (Fig. 2B). Nicotine treat-ment also reduced FAS mRNA expression (Fig. 2C) andactivity (Fig. 2D) in the VMH. Recent data have demon-strated that inhibition of this enzyme in this hypothalamicnucleus induces anorexia (26). In keeping with the mRNAand activity data, FAS protein expression was reduced inthe hypothalamus of nicotine-treated rats (Fig. 2E).

We next focused on other central components thatcould mediate the effects of nicotine on energy balance.Nicotine-treated rats showed a marked decrease in phos-phorylation of hypothalamic AMPKa (pAMPKa) and itsmolecular target ACCa (pACCa) compared with vehicle-treated rats (Fig. 2E). The reduction in pAMPKa levels wasassociated with unaltered protein concentrations of thenonphosphorylated isoforms of AMPKa1 and AMPKa2.ACCa levels were reduced in the hypothalamus of nicotine-treated rats (Fig. 2E). AMPK is regulated in a tissue-specificfashion, normally opposite between brain and peripheraltissues. For example, AMPK activation promotes fatty acidoxidation and glucose uptake in skeletal muscle, whereasconversely, hepatic AMPK activity inhibits fatty acid andcholesterol synthesis. In the hypothalamus AMPK activationelicits feeding and inhibits EE (8,11,14–18). Thus, we an-alyzed the effect of nicotine on the AMPK pathway in liverand skeletal muscle. Our data show that 48-h nicotinetreatment did not induce changes in this metabolic path-way either in liver (Supplementary Fig. 1A) or muscle(Supplementary Fig. 1B).

AMPK is activated by phosphorylation of upstream kinases.The main upstream AMPK kinases are the tumor suppres-sor liver kinase B1 (LKB1) and Ca2+/calmodulin-dependentprotein kinase kinase a and b (CaMKKa and CaMKKb),which phosphorylate AMPK at Thr172 (14,15). AMPK phos-phorylation levels are also regulated by protein phosphatase-2Ca (PP2Ca) (14,15). Our data show that CaMKKb (butnot CaMKKa) protein levels were reduced and PP2Ca in-creased in the hypothalamus of nicotine-treated rats (Fig. 2E),whereas no changes were detected in the phosphorylationlevels of LKB1 (pLKB1; Fig. 2E).Nicotine administration increased BAT temperatureand the expression of thermogenic markers in BAT.Recent evidence has linked inhibition of hypothalamicAMPK to activation of BAT thermogenesis (7,8). To furthercharacterize the mechanisms mediating nicotine-inducednegative energy balance, we examined the effects of nic-otine on BAT temperature. Our data showed that nicotine-treated rats displayed a marked and significant increase inskin temperature surrounding interscapular BAT (Fig. 2F).In keeping with this effect, nicotine treatment for 48 hincreased the expression of thermogenic markers such asUCP1 and peroxisome proliferator–activated receptor gcoactivator 1a and -b in BAT (Fig. 2G).Nicotine action on hypothalamic AMPK pathway ismediated by a3b4-nicotinic acetylcholine receptors.Current data have demonstrated that nicotine-inducedactivation of hypothalamic a3b4-nicotinic acetylcholinereceptors leads to activation of POMC neurons and de-creased feeding (3). Therefore, we aimed to investigatewhether these receptors were also mediating the actions

of nicotine on AMPK. Our data show that cotreatment withmecamylamine, an antagonist of a3b4 receptors (3,30,31),blunted nicotine-induced weight loss (Fig. 3A) anorexia(Fig. 3B) and inhibition of AMPK (Fig. 3C). Overall, theseresults indicated that a3b4 receptors mediated the effectsof nicotine on hypothalamic AMPK.Nicotine withdrawal restored energy balance, hy-pothalamic AMPK pathway, and the thermogenic pro-gram in BAT. We next investigated the effect of nicotinewithdrawal on energy balance. First, we confirmed thatchronic nicotine administration for 8 days resulted in markednegative energy balance, as demonstrated by reduced bodyweight (Fig. 4A), decreased feeding (Fig. 4B), and loweredfat mass (Fig. 4C; without affecting lean mass). At the sametime we observed increased EE (Fig. 4D; area under thecurve: vehicle: 100 6 4.1; nicotine: 121 6 6.2; withdrawal:103.9 6 2.1; P , 0.05 vehicle vs. nicotine; P , 0.05 nicotinevs. withdrawal), elevated LA (Fig. 4E), and decreased RQ(Fig. 4F). Withdrawal of nicotine treatment shifted all of theabove parameters toward the level of vehicle-treated rats.

In keeping with restoration of normal feeding and EE,nicotine withdrawal also elevated the decreased hypotha-lamic protein levels of the AMPK pathway (Fig. 5A) andreversed the elevated mRNA expression of BAT thermo-genic markers (Fig. 5B). Overall, these data suggest thatnicotine withdrawal restored energy balance by nor-malizing feeding, EE/thermogenesis, and lipid mobilization,potentially via normalization of AMPK activity.Activation of hypothalamic AMPK reversed nicotine-induced negative energy balance. To establish the de-pendence of the actions of nicotine on AMPK, we investigatedwhether pharmacological or genetic activation of hypotha-lamic AMPK could reverse nicotine-induced effects on foodintake and EE. First, intracerebroventricular administrationof the AMPK activator AICAR reversed the hypophagiaobserved in rats treated with nicotine for 48 h (Fig. 6A).Second, we aimed to identify the hypothalamic nucleuswhere nicotine exerted its actions on AMPK. To elucidatethe contribution of AMPK in the VMH to the effects ofnicotine on neuropeptide expression and the thermogen-esis in BAT, adenoviruses encoding either constitutivelyactive AMPKa (AMPKa-CA) with GFP, or control virusexpressing GFP alone (8,18), were injected stereotaxicallyinto the VMH of nicotine-treated rats. Infection efficiencywas assessed by 1) expression of GFP in the VMH, 2) in-creased hypothalamic protein concentration of pACCa,and 3) decreased hypothalamic malonyl-CoA levels (datanot shown) (8,18). Overexpression of AMPKa-CA in theVMH was accompanied by increased body weight (Fig. 6B)and food intake (Fig. 6C) in nicotine-treated rats. AMPKa-CA administration also restored expression levels of AgRP,NPY, and POMC in the ARC (Fig. 6D and E) and mRNAexpression of BAT thermogenic markers (Fig. 6F). Theseresults are in agreement with the initial observation thatnicotine impairs the function of AMPK and that this isassociated with decreased orexigenic signaling (AgRP andNPY) and increased anorexigenic signaling (POMC) in thehypothalamus and reduced activation of the thermogenicprogram in BAT.

DISCUSSION

This study identifies a previously unknown link betweennicotine-induced negative energy balance and hypothalamicAMPK. More specifically, the inactivation of hypothalamicAMPK caused by nicotine alters the physiological parameters





FIG. 2. Effects of nicotine administration on hypothalamic neuropeptides, AMPK, and BAT thermogenic program. In situ hybridization autora-diographic images (A) and AgRP, NPY, POMC, and CART mRNA levels in the ARC (B), FAS mRNA levels in the VMH (C), hypothalamic FASactivity (D), Western blot autoradiographic images (left panel) and hypothalamic protein levels of the different proteins of the AMPK pathway(right panel) (E), and infrared thermal images (left panel) with quantification of temperature (Temp; right panel) (F) and thermogenic markers(G) in the BAT of vehicle (Veh) and nicotine-treated (Nic) rats for 48 h are shown. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle. 3V, thirdventricle. PGC1, peroxisome proliferator–activated receptor g coactivator 1. All data are expressed as mean 6 SEM. (A high-quality digital repre-sentation of this figure is available in the online issue.)



of energy balance. This ultimately decreases feeding be-havior, stimulates EE (by triggering both physical activityand thermogenesis in BAT), and drives an increase inlipid oxidation.

The current therapeutic arsenal to treat obesity is verylimited. Besides lifestyle interventions focusing on diet,exercise, or behavioral changes (32,33) there is a paucityof available pharmacological treatments (34). For thisreason, and given the urgency of the problem, we decidedto focus on current situations where there is evidence ofinterventions leading to weight loss. One of these is smoking,such that there is no doubt that nicotine promotes weightloss and, conversely, those individuals who stop smokinggain weight. Extensive epidemiological studies have showna strong relationship between smoking and body weight,with nonsmokers weighing more than smokers at any age(1,4,35,36). In keeping with this, nicotine replacementtherapy and in particular the nicotine gum, has proved to beeffective in delaying postcessation weight gain (1,37).

It is well known that nicotine, the main addictive con-stituent of tobacco, is the major component linking smokingto negative energy balance. Nicotine exerts anorecticactions and elicits weight loss in humans and rodents(1,2,4,35,36). Several hypotheses have been put forward.Data showing that the peripheral nicotine administrationleads to increased expression of UCP1 in BAT (and whiteadipose tissue) were interpreted as a mechanism by whichnicotine influences body weight homeostasis by acting atperipheral level in nonneural tissues (23,38). However, it is

generally accepted that despite the many effects exerted bynicotine at peripheral level, the main effect of nicotine onfeeding homeostasis is exerted at a central level. Datagleaned in this regard have led to the assumption that themain hypothalamic effects of nicotine are exerted on NPY(19–21,39,40) and POMC neurons (3,22) in the ARC. Inlight of the aforementioned findings we decided to focusour attention in the VMH, since data have involved thisnucleus in the context of AMPK-driven biological effects(7,8,18).

AMPK controls feeding by directly regulating neuro-peptide expression in the ARC (16–18) and also regulatesEE by modulating thermogenesis in BAT via the SNS(7,8,41). In this study we aimed to investigate whethernicotine-induced negative energy balance was mediatedby specific modulation of the hypothalamic AMPK pathway.Our findings show that nicotine decreases body weightthrough its combined effects of hypophagia and increasedthermogenesis in BAT, in addition to driving elevated lo-comotor activity and increased lipid mobilization (demon-strated by a lower RQ). In keeping with former reports(3,19–22,39,40), nicotine also decreased the expression ofARC orexigenic neuropeptides such as AgRP and NPY andincreased POMC expression. Our data indicate that at thesame dose that caused anorexia, nicotine inhibited thehypothalamic AMPK pathway. This effect, as well as theaforementioned actions, was reversed by nicotine cessationand antagonism of a3b4 nicotinic acetylcholine receptors,which have recently been demonstrated to be involved in

FIG. 3. Effect of antagonism of a3b4-nicotinic acetylcholine receptors on nicotine-induced inhibition of hypothalamic AMPK. Body weightchange (A), food intake (B), and Western blot autoradiographic images (left panel) and hypothalamic protein levels of the different proteinsof the AMPK pathway (right panel) (C) of rats treated with vehicle (Veh), nicotine (Nic), and mecamylamine (Mec). Dividing lines show splicedbands. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle; #P < 0.05, ###P < 0.001 nicotine vehicle vs. nicotine mecamylamine. All data are expressed asmean 6 SEM.



FIG. 4. Effects of nicotine withdrawal on energy balance. Body weight (BW) change (A), food intake (B), fat and lean mass (C), EE (D; cumulativein left panel and total in right panel), LA (E; cumulative in left panel and total in right panel), and RQ (F; cumulative in left panel and total in rightpanel) of vehicle (Veh), nicotine (Nic), and nicotine withdrawal (With) rats are shown. EE, LA, and RQ data are from nicotine and nicotine with-drawal rats during the last 72 h of nicotine cessation. *P< 0.05, **P< 0.01, ***P< 0.001 vs. vehicle; #P< 0.05, ##P< 0.01, ###P< 0.001 nicotine vs.withdrawal; in the right panels of D and E, the symbols above the lines refer to the daily (diurnal + nocturnal) EE or LA. All data are expressed asmean 6 SEM.



the action of nicotine in the hypothalamus (3). Together,these results suggest the existence of a mechanistic linkbetween hypothalamic AMPK function and nicotine-induced changes in energy homeostasis. To specificallyaddress this hypothesis we investigated whether activationof hypothalamic AMPK could prevent the anorexigenicand thermogenic effects of nicotine. We first showed thatintracerebroventricular administration of the AMPK acti-vator AICAR reversed nicotine-induced anorexia. Secondly,overexpression of AMPKa-CA in the VMH reversed theanorectic and thermogenic effects observed in nicotine-treated rats. The VMH is considered as a key hypothalamicnucleus controlling both sides of the energy balance equa-tion (feeding and EE) (41–43), and recent evidence hasdemonstrated that AMPK in this nucleus plays a major rolein its ability to do so (8,18,41). In addition, our datademonstrate that genetic overactivation of AMPK in theVMH totally reversed the nicotine-induced anorexia andweight loss. AMPK-mediated normalization of energy bal-ance was associated with normalized mRNA levels of AgRP,NPY, and POMC in the ARC, as well as reduced or restoredlevels of thermogenic markers in BAT increased previouslyby nicotine treatment. Overall, our results indicate thatnicotine impairs AMPK function in the hypothalamus andthat this effect is essential for the weight loss, hypophagia,and increased BAT-mediated energy dissipation. The mech-anism connecting the nicotine-induced changes in the hy-pothalamus that stimulates the thermogenic program in theBAT is likely mediated via activation of the a3b4-nicotinicacetylcholine receptors (3) and through the SNS. This issupported by recent evidence showing that AMPK in-hibition stimulates thermogenic program in the BAT throughthe SNS (7,8,41) and that nicotine increases BAT thermogenesis

and UCP1 expression in rats through SNS activation(7,8,23,24,38,41).

Our data support the pathophysiological relevance ofthe hypothalamic AMPK-BAT axis in the control of energybalance (7,8,41) and demonstrate that the well-establishedeffects of nicotine on body weight are mediated throughthis mechanism. These results also raise the question of itssuitability as a therapeutic target for the treatment of hu-man obesity. Up to now this was considered unlikely be-cause of the well-known addictive actions of nicotine. Ourdata, however, show that its effects on increasing EE aremediated by a specific hypothalamic nuclei, the VMH (8).VMH neurons play a minor role in the rewarding propertiesof nicotine (31), indicating that by targeting this nicotine-activated pathway it may be feasible to preserve nicotine’seffect on body weight without developing addiction.Further work addressing this issue is clearly merited, sinceit has also been demonstrated that peripheral AMPK isa suitable target for human disease using, for example,metformin and thiazolidinediones to improve insulin sen-sitivity (14,15). Current evidence has highlighted the im-portance of BAT in adult humans and its potential as analternative to the existing strategies to solely target foodintake (44–47), which have seen limited success (34).Taken together, our data support drug-induced BAT acti-vation as a robust strategy to promote weight loss.

In summary, we show for the first time that nicotineinactivates hypothalamic AMPK and this effect mediatesdecreased food intake and BAT activation, likely via theSNS (23,24,38). Whether nicotine administration or smok-ing activates the AMPK-BAT axis in humans remains to bedetermined, but it is well known that EE is increased insmokers and that this effect may be mediated in part by

FIG. 5. Effects of nicotine withdrawal hypothalamic AMPK pathway and BAT thermogenic program. Western blot autoradiographic images (leftpanel) and hypothalamic protein levels of the different proteins of the AMPK pathway (right panel) (A) and thermogenic markers (B) in the BATof vehicle, nicotine, and nicotine withdrawal rats. Dividing lines show spliced bands. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle; ##P < 0.01,###P< 0.001 nicotine vs. withdrawal. HPRT, hypoxanthine-guanine phosphoribosyltransferase; PGC1, peroxisome proliferator–activated receptorg coactivator 1. All data are expressed as mean 6 SEM.



FIG. 6. Effects of hypothalamic AMPK activation on nicotine actions on energy balance, neuropeptides, and BAT thermogenic program. A: Foodintake in rats treated with nicotine and the AMPK activator AICAR. Body weight change (B); daily food intake (C); in situ hybridization auto-radiographic images (D); AgRP, NPY, and POMCmRNA levels in the ARC (E); and thermogenic markers (F) in the BAT of rats treated with vehicleor nicotine and stereotaxically treated with GFP-expressing adenoviruses or GFP plus AMPK constitutively active (AMPKa-CA) adenoviruses areshown. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle or vehicle GFP; #P < 0.05, ##P < 0.01, ###P < 0.001 nicotine vehicle vs. nicotine AICAR ornicotine GFP vs. nicotine AMPKa-CA. 3V, third ventricle; PGC1, peroxisome proliferator–activated receptor g coactivator 1; HPRT, hypoxanthine-guanine phosphoribosyltransferase. All data are expressed as mean 6 SEM.



the SNS (48). Thus, it is tempting to speculate that negativeenergy balance induced by cigarette smoking may be me-diated by the specific activation of the hypothalamic AMPK-BAT axis, a possibility that will require further investigation.Although nicotine is not considered the most harmfulcomponent of cigarette smoke, and in fact has beenreported to have potentially beneficial effects on mentalhealth (49,50), our results should not be construed assupporting continued tobacco use as a treatment option.It is noteworthy that our observation provides new insightsinto the molecular actions of nicotine and suggest thatevaluating candidate drugs modulating the hypothalamicAMPK-BAT axis (7,8,41) may provide a more targeted ap-proach to develop new therapeutic targets. These targetsmay be relevant not just for the treatment of obesity, butalso for smoking cessation in humans.

ACKNOWLEDGMENTS

The research leading to these results was funded by theEuropean Community’s Seventh Framework Programme(FP7/2007-2013) under Grant Agreements (No. 281854-theObERStress project [M.L.], No. 245009-the Neurofast pro-ject [R.N., C.D., and M.L.], and No. 018734-the Hepadipproject [A.V.-P.]); Dr. Einar Martens Fund (J.F.); NorwegianCouncil for Mental Health; ExtraStiftelsen Helse og Reha-bilitering (J.F.); Xunta de Galicia (M.L.: 10PXIB208164PR,R.N.: 2010/14); FIS (M.L.: PS09/01880); and MICINN (R.N.:RyC-2008-02219 and SAF2009-07049, C.D.: BFU2008-02001,M.L.: RyC-2007-00211). CIBER de Fisiopatología de laObesidad y Nutrición is an initiative of ISCIII. A.J.W. wassupported by the Biotechnology and Biological Sciences Re-search Council.

No potential conflicts of interest relevant to this articlewere reported.

P.B.M.M., A.J.W., and R.N. performed the in vivo experi-ments and analytical methods (in situ hybridization, Westernblotting); EE, RQ, LA, and nuclear magnetic resonanceanalyses; and collected the data. P.B.M.M., A.J.W., J.F., R.N.,C.D., A.V.-P., and M.L. designed the experiments; analyzedand interpreted the data; and discussed, reviewed, andedited the manuscript. P.B.M.M. and M.L. developed thehypothesis. M.L. wrote the manuscript. All authors had finalapproval of the submitted manuscript. M.L. is the guarantorof this work and, as such, had full access to all of the data inthe study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.

The authors thank David Carling (Imperial CollegeLondon) for reagents and advice.

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