ORIGINAL ARTICLE
Nicotine Induces Negative Energy Balance Through
Hypothalamic AMP-Activated Protein Kinase
Pablo B. Martínez de Morentin,1,2 Andrew J. Whittle,3 Johan Fernø,4,5 Rubén Nogueiras,1,2
Carlos Diéguez,1,2 Antonio Vidal-Puig,3 and Miguel López1,2
Smokers around the world commonly report increased body
weight after smoking cessation as a major factor that interferes
with their attempts to quit. Numerous controlled studies in both
humans and rodents have reported that nicotine exerts a marked
anorectic action. The effects of nicotine on energy homeostasis
have been mostly pinpointed in the central nervous system, but
the molecular mechanisms controlling its action are still not
fully understood. The aim of this study was to investigate the
effect of nicotine on hypothalamic AMP-activated protein kinase
(AMPK) and its effect on energy balance. Here we demonstrate
that nicotine-induced weight loss is associated with inactivation
of hypothalamic AMPK, decreased orexigenic signaling in the
hypothalamus, increased energy expenditure as a result of increased locomotor activity, increased thermogenesis in brown
adipose tissue (BAT), and alterations in fuel substrate utilization.
Conversely, nicotine withdrawal or genetic activation of hypothalamic AMPK in the ventromedial nucleus of the hypothalamus reversed nicotine-induced negative energy balance. Overall these
data demonstrate that the effects of nicotine on energy balance
involve specific modulation of the hypothalamic AMPK-BAT axis.
These targets may be relevant for the development of new therapies for human obesity. Diabetes 61:807–817, 2012
N
icotine, the main addictive component of tobacco, shows anorexigenic properties and promotes body weight reduction in humans and
rodents (1–4). In keeping with these points,
smoking cessation is associated with hyperphagia and
weight gain in over 70% of people who attempt to do so. In
fact, in many cases, the prospect of weight gain itself has
acted as an incentive for persevering in the smoking habit
(1,2,4). Therefore, it is clear that smoking leads to a state
of negative energy balance and one that might be exploited
for therapeutic purposes if its molecular mechanisms were
better understood.
From the 1Department of Physiology, School of Medicine-CIMUS, University
of Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago de
Compostela, A Coruña, Spain; the 2CIBER Fisiopatología de la Obesidad y
Nutrición (CIBERobn), Santiago de Compostela, Spain; the 3Institute of
Metabolic Science, Metabolic Research Laboratories, NIHR Cambridge Biomedical Research Centre Addenbrooke’s Hospital, University of Cambridge,
Cambridge, U.K.; the 4Dr. Einar Martens’ Research Group for Biological
Psychiatry, 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].
Received 2 August 2011 and accepted 8 December 2011.
DOI: 10.2337/db11-1079
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1079/-/DC1.
Ó 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
Maintaining energy balance depends on the efficiency of
tightly regulated processes involved in 1) energy intake
(feeding); 2) energy expenditure (EE) comprising basal
metabolism, adaptive thermogenesis, and physical (locomotor) activity (LA); and 3) nutrient partitioning (5,6). The
hypothalamus is a region within the central nervous system that plays a major role in the modulation of energy
balance. The hypothalamic arcuate nucleus (ARC) expresses
the orexigenic (feeding-promoting) neuropeptides agoutirelated protein (AgRP) and neuropeptide Y (NPY) and the
anorexigenic precursors proopiomelanocortin (POMC) and
cocaine and amphetamine-regulated transcript (CART)
(5,6). In addition to controlling food intake, the hypothalamus modulates EE in brown adipose tissue (BAT) (7,8),
as well as glucose (9,10) and lipid metabolism in peripheral
tissues (11,12) through the autonomic nervous system
(7,8,12,13).
An important mediator of all of these effects is hypothalamic AMP-activated protein kinase (AMPK), a cellular
gauge that controls whole-body energy balance and is activated in conditions of low energy (14,15). When activated,
AMPK optimizes energy utilization by increasing energyproducing and reducing energy-wasting processes at the
whole-body level. In the hypothalamus, AMPK activation
drives feeding by modulating mitochondrial fatty acid oxidation and intracellular levels of reactive oxygen species.
This untimely regulates neuropeptide expression in the
ARC (16–18) and also suppresses EE and thyroid-induced
thermogenesis in the BAT (7,8). Activated AMPK is also an
important regulator of fatty acid biosynthesis, suppressing
de novo lipogenesis by phosphorylation and inactivation of
acetyl-CoA carboxylase (ACC) (7,8,14,15,17,18).
Whether the effects of nicotine on energy balance are
mediated by modulation of hypothalamic AMPK is currently unknown. However, there are some clues that might
indicate the existence of such an action. Nicotine coregulates most of the processes controlled by AMPK. For
instance, chronic nicotine exposure in rats reduces the
expression of NPY (19) and consistently blunts NPYinduced hyperphagia (20). Nicotine withdrawal also increases hypothalamic NPY and AgRP expression, mechanisms
that contribute to increased feeding after nicotine cessation
(20,21). Current evidence has demonstrated that nicotine
also upregulates the expression of POMC (22) and decreases
feeding through activation of POMC neurons (3). Besides
its actions on hypothalamic feeding circuits nicotine also
modulates peripheral metabolism in rats, increasing BAT
thermogenesis through activation of the sympathetic nervous system (SNS) and increased expression of uncoupling
protein 1 (UCP1) (23,24).
Based on this evidence, we hypothesized that the effect
of nicotine on energy homeostasis could be mediated at
least in part by hypothalamic AMPK. The aim of this study
was to investigate the effect of nicotine on hypothalamic
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NICOTINE MODULATES AMPK AND ENERGY BALANCE
AMPK and its impact on feeding and physiological parameters modulating energy balance such as EE, LA, BAT
function, and nutrient partitioning. Our data confirm the
existence of a previously unknown link between nicotineinduced negative energy balance and hypothalamic AMPK.
RESEARCH DESIGN AND METHODS
Animals. We used adult male Sprague-Dawley rats (9–11 weeks old; AnimalarioGeneral USC). Rats were housed in a temperature-controlled (22°C) room, with
a 12-h light/dark cycle (7:00 to 19:00). The experiments were performed in
agreement with the International Law on Animal Experimentation and approved by the USC Bioethics Committee and the Ministry of Science and
Innovation of Spain (PS09/01880). For all of the experimental groups and
analytic 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 injection
every 12 h in both subchronic (48 h) and chronic (17 days) experiments. To
validate the EE data, all rats were matched for body weight before treatment
began. To control nicotine-induced respiratory depression (RD), rats were
treated with nicotine and symptoms of RD were visually controlled from 2 min
before to 12 h after treatment. For the antagonism of a3b4 nicotinic acetylcholine receptors, rats were treated previously (30 min) intraperitoneally with
mecamylamine (mecamylamine hydrochloride, 1 mg/kg; Sigma-Aldrich) as
described recently (3) and subcutaneously with nicotine during 48 h.
Intracerebroventricular treatment. Intracerebroventricular cannulae were
stereotaxically implanted under ketamine/xylazine anesthesia in the lateral
ventricle (coordinates 1.2 mm lateral to sagittal suture and 1.0 mm posterior to
bregma), as described previously (8,18,26), and demonstrated to be correctly
located by methylene blue staining. Rats received either a single administration of the AMPK activator AICAR (Sigma-Aldrich; 5 mg, dissolved in 5 mL of
saline) or vehicle (5 mL saline) (8) at 19:00.
Stereotaxic microinjection of adenoviral expression vectors. Rats were
placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) under
ketamine/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 coordinates 2.3/3.3 mm posterior to the bregma (two injections were performed in
each VMH), 60.6 mm lateral to midline and 10.2 mm below the surface of the
skull (8,18). Adenoviral vectors (green fluorescent protein [GFP] or AMPKaCA; Viraquest, North Liberty, IA) were delivered at a rate of 200 nL/min for
5 min (1 mL/injection site) (8,18).
Conditioned taste aversion. Five days before the test, rats were allowed 2-h
daytime access to water. On the day of the experiment, rats were given access
to 0.15% sodium saccharin rather than water for 30 min. Next, one experimental
group was injected intraperitoneally with 0.15 mol/L lithium chloride (LiCl) in
saline and subcutaneously with saline (vehicle). The second experimental
group was injected intraperitoneally with saline and subcutaneously with
nicotine. A third experimental group was injected intraperitoneally and subcutaneously with saline. Twenty-four hours later, rats were given a 2-h access to
a two-bottle choice test of 0.15% saccharin versus water. Conditioned taste
aversion (CTA) was expressed as percent saccharin preference ratio [100 3
saccharin intake/(saccharin intake + water intake)]. A saccharin preference
ratio ,50% was considered as a signal of CTA (26).
Temperature measurements. Body temperature was recorded with a rectal
probe connected to digital thermometer (BAT-12 Microprobe-Thermometer;
Physitemp, Clifton, NJ). Skin temperature surrounding BAT was recorded with
an infrared camera (E60bx: Compact-Infrared-Thermal-Imaging-Camera; FLIR,
West Malling, Kent, U.K.) and analyzed with a specific software package (FLIRTools-Software; FLIR). We used eight animals per group, and for each animal,
three or four pictures were analyzed. The skin temperature surrounding BAT for
one particular animal was calculated as the average temperature recorded by
analyzing those pictures.
In situ hybridization. Coronal brain sections (16 mm) were probed as published (8,18,26). We used specific oligonucleotides for AgRP, CART, fatty acid
synthase (FAS), NPY, and POMC detection (Supplementary Table 1). Anatomical 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 and
20 sections for each animal (4 to 5 slides with four sections per slide). The mean
of 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 the
optic 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
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phosphorylated protein levels (8,18,26). Hypothalamic total protein lysates
were subjected to SDS-PAGE, electrotransferred on a polyvinylidene fluoride
membrane, and probed with the following antibodies: ACC, pACC-Ser79,
AMPKa1, and AMPKa2 (Upstate Biotechnology, Temecula, CA); FAS (BD
Biosciences, Franklin Lakes, NJ), pAMPK-Thr172, and phospho-liver kinase
B1 (pLKB1)-Ser428 (Cell Signaling, Danvers, MA); PP2Ca and Ca2+/calmodulindependent protein kinase kinase a and b (CaMKKa and CaMKKb) (Santa Cruz
Biotechnology, Santa Cruz, CA); and b-actin (Sigma-Aldrich) as described previously (8,18,26). Values were expressed in relation to b-actin levels.
Real-time quantitative PCR. We performed real-time PCR (TaqMan; Applied
Biosystems, Carlsbad, CA) as described previously (8,26) using specific sets of
primer probes (Supplementary Table 2). Values were expressed in relation to
hypoxanthine-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 for
EE, respiratory quotient (RQ), and LA using a calorimetric system (LabMaster;
TSE Systems, Bad Homburg, Germany). This system is an open-circuit instrument that determines O2 consumption, CO2 production, and RQ (VCO2/VO2)
(12,28). Rats were placed for adaptation for 1 week before starting the
measurements. For the measurement of body composition, we used the nuclear magnetic resonance imaging (Whole Body Composition Analyzer;
EchoMRI, Houston, TX) (8,12,28).
Statistical analysis. Data are expressed as mean 6 SEM. Statistical significance was determined by Student t tests or ANOVAs and post hoc two-tailed
Bonferroni test. P , 0.05 was considered significant.
RESULTS
Nicotine treatment induced negative energy balance
by decreasing feeding and increasing EE. Nicotinetreated rats exhibited a marked reduction in body weight
(Fig. 1A) associated with reduced food intake over 24 and
48 h of treatment (Fig. 1B). We evaluated whether the
anorectic effect was a genuine action of nicotine or a secondary effect of the drug. It has been reported that nicotine induces RD in neonatal and anesthetized rats (29,30).
Nicotine-treated rats showed a significant short-term RD
after the injection (RD-vehicle–treated: nondetected; RDnicotine–treated: 5.0 6 1.4 min; P , 0.001). Ten minutes
after nicotine administration just 5% of the rats showed
symptoms of RD; 15 min after injection those symptoms
were completely abolished (Fig. 1C). These data suggest
that an effect of RD on 12-h feeding patterns is unlikely.
We further evaluated whether the anorectic effect of
nicotine was associated with an aversive response by performing a CTA experiment. In this type of experiment a
saccharin preference ratio ,50% was considered a marker
of CTA, since it indicates that rats prefer to drink water after
an aversive response to saccharine (26). LiCl was used as
a positive control, and as expected, LiCl-treated rats drank
significantly less saccharin solution (LiCl paired flavor; Fig.
1D), indicating that they had developed CTA (26). Vehicle
and nicotine-treated rats had a similar saccharine preference consumption ratio, and it is noteworthy that this
was .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 skin
aspect, stool consistency, or obvious abnormal behavior
such as was evident in the LiCl-treated animals (data not
shown) (26).
We next evaluated whether nicotine treatment induced
a change in the other side of the energy balance equation.
Nicotine-treated rats showed a significant increase in body
temperature (Fig. 1E) associated with higher EE (Fig. 1F;
area under the curve: vehicle: 100 6 2.4; nicotine: 132 6 9.1;
P , 0.01), increased LA (Fig. 1G), and reduced RQ (Fig. 1H)
compared with vehicle-treated rats. Overall, these data
indicate that nicotine treatment induced negative energy
balance, including decreased feeding, increased EE, and
increased lipid oxidation.
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P.B. MARTÍNEZ DE MORENTIN AND ASSOCIATES
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 panel
and 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 are
expressed as mean 6 SEM.
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Nicotine treatment reduced AgRP and NPY and
increased POMC expression in the ARC and decreased
hypothalamic AMPK activation. Consistent with their
hypophagic state, expression of orexigenic neuropeptides
AgRP and NPY decreased and POMC expression increased (Fig. 2A and B) in the ARC of nicotine-treated
rats. No significant changes were detected in CART
mRNA expression in the ARC (Fig. 2B). Nicotine treatment also reduced FAS mRNA expression (Fig. 2C) and
activity (Fig. 2D) in the VMH. Recent data have demonstrated that inhibition of this enzyme in this hypothalamic
nucleus induces anorexia (26). In keeping with the mRNA
and activity data, FAS protein expression was reduced in
the hypothalamus of nicotine-treated rats (Fig. 2E).
We next focused on other central components that
could mediate the effects of nicotine on energy balance.
Nicotine-treated rats showed a marked decrease in phosphorylation of hypothalamic AMPKa (pAMPKa) and its
molecular target ACCa (pACCa) compared with vehicletreated rats (Fig. 2E). The reduction in pAMPKa levels was
associated with unaltered protein concentrations of the
nonphosphorylated isoforms of AMPKa1 and AMPKa2.
ACCa levels were reduced in the hypothalamus of nicotinetreated rats (Fig. 2E). AMPK is regulated in a tissue-specific
fashion, normally opposite between brain and peripheral
tissues. For example, AMPK activation promotes fatty acid
oxidation and glucose uptake in skeletal muscle, whereas
conversely, hepatic AMPK activity inhibits fatty acid and
cholesterol synthesis. In the hypothalamus AMPK activation
elicits feeding and inhibits EE (8,11,14–18). Thus, we analyzed the effect of nicotine on the AMPK pathway in liver
and skeletal muscle. Our data show that 48-h nicotine
treatment did not induce changes in this metabolic pathway 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 suppressor liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent
protein kinase kinase a and b (CaMKKa and CaMKKb),
which phosphorylate AMPK at Thr172 (14,15). AMPK phosphorylation levels are also regulated by protein phosphatase2Ca (PP2Ca) (14,15). Our data show that CaMKKb (but
not CaMKKa) protein levels were reduced and PP2Ca increased in the hypothalamus of nicotine-treated rats (Fig. 2E),
whereas no changes were detected in the phosphorylation
levels of LKB1 (pLKB1; Fig. 2E).
Nicotine administration increased BAT temperature
and the expression of thermogenic markers in BAT.
Recent evidence has linked inhibition of hypothalamic
AMPK to activation of BAT thermogenesis (7,8). To further
characterize the mechanisms mediating nicotine-induced
negative energy balance, we examined the effects of nicotine on BAT temperature. Our data showed that nicotinetreated rats displayed a marked and significant increase in
skin temperature surrounding interscapular BAT (Fig. 2F).
In keeping with this effect, nicotine treatment for 48 h
increased the expression of thermogenic markers such as
UCP1 and peroxisome proliferator–activated receptor g
coactivator 1a and -b in BAT (Fig. 2G).
Nicotine action on hypothalamic AMPK pathway is
mediated by a3b4-nicotinic acetylcholine receptors.
Current data have demonstrated that nicotine-induced
activation of hypothalamic a3b4-nicotinic acetylcholine
receptors leads to activation of POMC neurons and decreased feeding (3). Therefore, we aimed to investigate
whether these receptors were also mediating the actions
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of nicotine on AMPK. Our data show that cotreatment with
mecamylamine, 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, these
results indicated that a3b4 receptors mediated the effects
of nicotine on hypothalamic AMPK.
Nicotine withdrawal restored energy balance, hypothalamic AMPK pathway, and the thermogenic program in BAT. We next investigated the effect of nicotine
withdrawal on energy balance. First, we confirmed that
chronic nicotine administration for 8 days resulted in marked
negative energy balance, as demonstrated by reduced body
weight (Fig. 4A), decreased feeding (Fig. 4B), and lowered
fat mass (Fig. 4C; without affecting lean mass). At the same
time we observed increased EE (Fig. 4D; area under the
curve: 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 nicotine
vs. withdrawal), elevated LA (Fig. 4E), and decreased RQ
(Fig. 4F). Withdrawal of nicotine treatment shifted all of the
above parameters toward the level of vehicle-treated rats.
In keeping with restoration of normal feeding and EE,
nicotine withdrawal also elevated the decreased hypothalamic protein levels of the AMPK pathway (Fig. 5A) and
reversed the elevated mRNA expression of BAT thermogenic markers (Fig. 5B). Overall, these data suggest that
nicotine withdrawal restored energy balance by normalizing feeding, EE/thermogenesis, and lipid mobilization,
potentially via normalization of AMPK activity.
Activation of hypothalamic AMPK reversed nicotineinduced negative energy balance. To establish the dependence of the actions of nicotine on AMPK, we investigated
whether pharmacological or genetic activation of hypothalamic AMPK could reverse nicotine-induced effects on food
intake and EE. First, intracerebroventricular administration
of the AMPK activator AICAR reversed the hypophagia
observed in rats treated with nicotine for 48 h (Fig. 6A).
Second, we aimed to identify the hypothalamic nucleus
where nicotine exerted its actions on AMPK. To elucidate
the contribution of AMPK in the VMH to the effects of
nicotine on neuropeptide expression and the thermogenesis in BAT, adenoviruses encoding either constitutively
active AMPKa (AMPKa-CA) with GFP, or control virus
expressing GFP alone (8,18), were injected stereotaxically
into the VMH of nicotine-treated rats. Infection efficiency
was assessed by 1) expression of GFP in the VMH, 2) increased hypothalamic protein concentration of pACCa,
and 3) decreased hypothalamic malonyl-CoA levels (data
not shown) (8,18). Overexpression of AMPKa-CA in the
VMH was accompanied by increased body weight (Fig. 6B)
and food intake (Fig. 6C) in nicotine-treated rats. AMPKaCA administration also restored expression levels of AgRP,
NPY, and POMC in the ARC (Fig. 6D and E) and mRNA
expression of BAT thermogenic markers (Fig. 6F). These
results are in agreement with the initial observation that
nicotine impairs the function of AMPK and that this is
associated with decreased orexigenic signaling (AgRP and
NPY) and increased anorexigenic signaling (POMC) in the
hypothalamus and reduced activation of the thermogenic
program in BAT.
DISCUSSION
This study identifies a previously unknown link between
nicotine-induced negative energy balance and hypothalamic
AMPK. More specifically, the inactivation of hypothalamic
AMPK caused by nicotine alters the physiological parameters
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FIG. 2. Effects of nicotine administration on hypothalamic neuropeptides, AMPK, and BAT thermogenic program. In situ hybridization autoradiographic images (A) and AgRP, NPY, POMC, and CART mRNA levels in the ARC (B), FAS mRNA levels in the VMH (C), hypothalamic FAS
activity (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, third
ventricle. PGC1, peroxisome proliferator–activated receptor g coactivator 1. All data are expressed as mean 6 SEM. (A high-quality digital representation of this figure is available in the online issue.)
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FIG. 3. Effect of antagonism of a3b4-nicotinic acetylcholine receptors on nicotine-induced inhibition of hypothalamic AMPK. Body weight
change (A), food intake (B), and Western blot autoradiographic images (left panel) and hypothalamic protein levels of the different proteins
of the AMPK pathway (right panel) (C) of rats treated with vehicle (Veh), nicotine (Nic), and mecamylamine (Mec). Dividing lines show spliced
bands. *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 as
mean 6 SEM.
of energy balance. This ultimately decreases feeding behavior, stimulates EE (by triggering both physical activity
and thermogenesis in BAT), and drives an increase in
lipid oxidation.
The current therapeutic arsenal to treat obesity is very
limited. Besides lifestyle interventions focusing on diet,
exercise, or behavioral changes (32,33) there is a paucity
of available pharmacological treatments (34). For this
reason, and given the urgency of the problem, we decided
to focus on current situations where there is evidence of
interventions leading to weight loss. One of these is smoking,
such that there is no doubt that nicotine promotes weight
loss and, conversely, those individuals who stop smoking
gain weight. Extensive epidemiological studies have shown
a 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 replacement
therapy and in particular the nicotine gum, has proved to be
effective in delaying postcessation weight gain (1,37).
It is well known that nicotine, the main addictive constituent of tobacco, is the major component linking smoking
to negative energy balance. Nicotine exerts anorectic
actions 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 administration
leads to increased expression of UCP1 in BAT (and white
adipose tissue) were interpreted as a mechanism by which
nicotine influences body weight homeostasis by acting at
peripheral level in nonneural tissues (23,38). However, it is
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generally accepted that despite the many effects exerted by
nicotine at peripheral level, the main effect of nicotine on
feeding homeostasis is exerted at a central level. Data
gleaned in this regard have led to the assumption that the
main hypothalamic effects of nicotine are exerted on NPY
(19–21,39,40) and POMC neurons (3,22) in the ARC. In
light of the aforementioned findings we decided to focus
our attention in the VMH, since data have involved this
nucleus in the context of AMPK-driven biological effects
(7,8,18).
AMPK controls feeding by directly regulating neuropeptide expression in the ARC (16–18) and also regulates
EE by modulating thermogenesis in BAT via the SNS
(7,8,41). In this study we aimed to investigate whether
nicotine-induced negative energy balance was mediated
by specific modulation of the hypothalamic AMPK pathway.
Our findings show that nicotine decreases body weight
through its combined effects of hypophagia and increased
thermogenesis in BAT, in addition to driving elevated locomotor activity and increased lipid mobilization (demonstrated by a lower RQ). In keeping with former reports
(3,19–22,39,40), nicotine also decreased the expression of
ARC orexigenic neuropeptides such as AgRP and NPY and
increased POMC expression. Our data indicate that at the
same dose that caused anorexia, nicotine inhibited the
hypothalamic AMPK pathway. This effect, as well as the
aforementioned actions, was reversed by nicotine cessation
and antagonism of a3b4 nicotinic acetylcholine receptors,
which have recently been demonstrated to be involved in
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P.B. MARTÍNEZ DE MORENTIN AND ASSOCIATES
FIG. 4. Effects of nicotine withdrawal on energy balance. Body weight (BW) change (A), food intake (B), fat and lean mass (C), EE (D; cumulative
in 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 right
panel) of vehicle (Veh), nicotine (Nic), and nicotine withdrawal (With) rats are shown. EE, LA, and RQ data are from nicotine and nicotine withdrawal 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 as
mean 6 SEM.
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FIG. 5. Effects of nicotine withdrawal hypothalamic AMPK pathway and BAT thermogenic program. Western blot autoradiographic images (left
panel) and hypothalamic protein levels of the different proteins of the AMPK pathway (right panel) (A) and thermogenic markers (B) in the BAT
of 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 receptor
g coactivator 1. All data are expressed as mean 6 SEM.
the action of nicotine in the hypothalamus (3). Together,
these results suggest the existence of a mechanistic link
between hypothalamic AMPK function and nicotineinduced changes in energy homeostasis. To specifically
address this hypothesis we investigated whether activation
of hypothalamic AMPK could prevent the anorexigenic
and thermogenic effects of nicotine. We first showed that
intracerebroventricular administration of the AMPK activator AICAR reversed nicotine-induced anorexia. Secondly,
overexpression of AMPKa-CA in the VMH reversed the
anorectic and thermogenic effects observed in nicotinetreated rats. The VMH is considered as a key hypothalamic
nucleus controlling both sides of the energy balance equation (feeding and EE) (41–43), and recent evidence has
demonstrated that AMPK in this nucleus plays a major role
in its ability to do so (8,18,41). In addition, our data
demonstrate that genetic overactivation of AMPK in the
VMH totally reversed the nicotine-induced anorexia and
weight loss. AMPK-mediated normalization of energy balance was associated with normalized mRNA levels of AgRP,
NPY, and POMC in the ARC, as well as reduced or restored
levels of thermogenic markers in BAT increased previously
by nicotine treatment. Overall, our results indicate that
nicotine impairs AMPK function in the hypothalamus and
that this effect is essential for the weight loss, hypophagia,
and increased BAT-mediated energy dissipation. The mechanism connecting the nicotine-induced changes in the hypothalamus that stimulates the thermogenic program in the
BAT is likely mediated via activation of the a3b4-nicotinic
acetylcholine receptors (3) and through the SNS. This is
supported by recent evidence showing that AMPK inhibition stimulates thermogenic program in the BAT through
the SNS (7,8,41) and that nicotine increases BAT thermogenesis
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DIABETES, VOL. 61, APRIL 2012
and UCP1 expression in rats through SNS activation
(7,8,23,24,38,41).
Our data support the pathophysiological relevance of
the hypothalamic AMPK-BAT axis in the control of energy
balance (7,8,41) and demonstrate that the well-established
effects of nicotine on body weight are mediated through
this mechanism. These results also raise the question of its
suitability as a therapeutic target for the treatment of human obesity. Up to now this was considered unlikely because of the well-known addictive actions of nicotine. Our
data, however, show that its effects on increasing EE are
mediated by a specific hypothalamic nuclei, the VMH (8).
VMH neurons play a minor role in the rewarding properties
of nicotine (31), indicating that by targeting this nicotineactivated pathway it may be feasible to preserve nicotine’s
effect on body weight without developing addiction.
Further work addressing this issue is clearly merited, since
it has also been demonstrated that peripheral AMPK is
a suitable target for human disease using, for example,
metformin and thiazolidinediones to improve insulin sensitivity (14,15). Current evidence has highlighted the importance of BAT in adult humans and its potential as an
alternative to the existing strategies to solely target food
intake (44–47), which have seen limited success (34).
Taken together, our data support drug-induced BAT activation as a robust strategy to promote weight loss.
In summary, we show for the first time that nicotine
inactivates hypothalamic AMPK and this effect mediates
decreased food intake and BAT activation, likely via the
SNS (23,24,38). Whether nicotine administration or smoking activates the AMPK-BAT axis in humans remains to be
determined, but it is well known that EE is increased in
smokers and that this effect may be mediated in part by
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P.B. MARTÍNEZ DE MORENTIN AND ASSOCIATES
FIG. 6. Effects of hypothalamic AMPK activation on nicotine actions on energy balance, neuropeptides, and BAT thermogenic program. A: Food
intake in rats treated with nicotine and the AMPK activator AICAR. Body weight change (B); daily food intake (C); in situ hybridization autoradiographic images (D); AgRP, NPY, and POMC mRNA levels in the ARC (E); and thermogenic markers (F) in the BAT of rats treated with vehicle
or nicotine and stereotaxically treated with GFP-expressing adenoviruses or GFP plus AMPK constitutively active (AMPKa-CA) adenoviruses are
shown. *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 or
nicotine GFP vs. nicotine AMPKa-CA. 3V, third ventricle; PGC1, peroxisome proliferator–activated receptor g coactivator 1; HPRT, hypoxanthineguanine phosphoribosyltransferase. All data are expressed as mean 6 SEM.
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DIABETES, VOL. 61, APRIL 2012
815
NICOTINE MODULATES AMPK AND ENERGY BALANCE
the SNS (48). Thus, it is tempting to speculate that negative
energy balance induced by cigarette smoking may be mediated by the specific activation of the hypothalamic AMPKBAT axis, a possibility that will require further investigation.
Although nicotine is not considered the most harmful
component of cigarette smoke, and in fact has been
reported to have potentially beneficial effects on mental
health (49,50), our results should not be construed as
supporting continued tobacco use as a treatment option.
It is noteworthy that our observation provides new insights
into the molecular actions of nicotine and suggest that
evaluating candidate drugs modulating the hypothalamic
AMPK-BAT axis (7,8,41) may provide a more targeted approach to develop new therapeutic targets. These targets
may be relevant not just for the treatment of obesity, but
also for smoking cessation in humans.
ACKNOWLEDGMENTS
The research leading to these results was funded by the
European Community’s Seventh Framework Programme
(FP7/2007-2013) under Grant Agreements (No. 281854-the
ObERStress project [M.L.], No. 245009-the Neurofast project [R.N., C.D., and M.L.], and No. 018734-the Hepadip
project [A.V.-P.]); Dr. Einar Martens Fund (J.F.); Norwegian
Council for Mental Health; ExtraStiftelsen Helse og Rehabilitering (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 la
Obesidad y Nutrición is an initiative of ISCIII. A.J.W. was
supported by the Biotechnology and Biological Sciences Research Council.
No potential conflicts of interest relevant to this article
were reported.
P.B.M.M., A.J.W., and R.N. performed the in vivo experiments and analytical methods (in situ hybridization, Western
blotting); EE, RQ, LA, and nuclear magnetic resonance
analyses; 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; analyzed
and interpreted the data; and discussed, reviewed, and
edited the manuscript. P.B.M.M. and M.L. developed the
hypothesis. M.L. wrote the manuscript. All authors had final
approval of the submitted manuscript. M.L. is the guarantor
of this work and, as such, had full access to all of the data in
the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
The authors thank David Carling (Imperial College
London) for reagents and advice.
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