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Nicotine Decreases Food Intake Through Activation of POMC Neurons

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Science  10 Jun 2011:
Vol. 332, Issue 6035, pp. 1330-1332
DOI: 10.1126/science.1201889

Abstract

Smoking decreases appetite, and smokers often report that they smoke to control their weight. Understanding the neurobiological mechanisms underlying the anorexic effects of smoking would facilitate the development of novel treatments to help with smoking cessation and to prevent or treat obesity. By using a combination of pharmacological, molecular genetic, electrophysiological, and feeding studies, we found that activation of hypothalamic α3β4 nicotinic acetylcholine receptors leads to activation of pro-opiomelanocortin (POMC) neurons. POMC neurons and subsequent activation of melanocortin 4 receptors were critical for nicotinic-induced decreases in food intake in mice. This study demonstrates that nicotine decreases food intake and body weight by influencing the hypothalamic melanocortin system and identifies critical molecular and synaptic mechanisms involved in nicotine-induced decreases in appetite.

Smoking remains the leading cause of preventable death in developed countries (1), and some smokers report that they smoke as a method of weight control (2, 3). Smokers have a notably lower body mass index than nonsmokers (4) and gain weight when they quit (5). These effects on body weight have been attributed to the nicotine in tobacco, because nicotine decreases feeding in animal models (6). Nicotine has some effects on peripheral energy metabolism (79), but little is known about potential central nervous system pathways mediating nicotine’s effects on food intake and body weight. Identifying these pathways could help to study potential cholinergic modulation of appetite and weight control and could also lead to the development of novel appetite suppressants that might aid in smoking cessation.

In a first step toward this goal, we determined that nicotine and the more selective drug cytisine [a full agonist at α3β4 nicotinic acetylcholine receptors (nAChRs), with weaker effects at other nAChRs (10)], could decrease weight gain over time [Fig. 1A; repeated-measures analysis of variance (ANOVA) F(72, 720) = 41.5, P < 0.0001], body fat mass by about 15 to 20% [Fig. 1B; ANOVA F2,26 = 7.7, P < 0.001], and food intake by up to 50% [Fig. 1C; F1,18 = 100.3, P < 0.001] in mice but did not affect water intake or tissue water content (fig. S1) (11). In addition, mecamylamine (a noncompetitive nicotinic antagonist) had no effects on its own but prevented acute and chronic cytisine-induced hypophagia (all F values < 1), whereas the non–brain-permeant nicotinic antagonist hexamethonium was ineffective in blocking these anorexic effects (Fig. 1C), suggesting that activation of central nAChRs was essential for reduced food intake.

Fig. 1

Change in weight, body fat content, and food intake after treatment with nicotinic drugs. Both nicotine (Nic) and cytisine (Cyt) dose-dependently limited weight gain in mice treated daily for 30 days, with more pronounced effects seen at higher doses (A) (all P values < 0.001). In contrast, the nonselective nicotinic antagonist mecamylamine had no significant effect (F < 1), suggesting that nAChR antagonism alone was not sufficient for the anorexic effects of nicotinic compounds. Error bars indicate SEM. Body fat measured by magnetic resonance imaging was also reduced in mice treated with cytisine [1.5 mg/kg; F1,23 = 13.7, P = 0.006] and nicotine [0.5 mg/kg; F1,23 = 5.08, P = 0.03] compared with saline (Sal) (B). Acute injection of cytisine decreased food intake after 2 hours [F1,18 = 100.3, P < 0.001], and this effect was still observed after 24 hours [F1,18 = 35.3, P < 0.001]. A.U., arbitrary units. The effect of cytisine was not blocked by the peripherally acting nAChR antagonist hexamethonium [2 hours, F1,18 = 121.5, P < 0.001; 24 hours, F1,18 = 37.9, P < 0.001] but was blocked by mecamylamine (F values < 1) (C), indicating cytisine acts at central nAChRs to exert its anorexic effects. *** indicates P < 0.001.

The pharmacological specificity of cytisine and the relatively low dose (1.5 mg/kg) needed to decrease food intake suggested that activation of central α3β4 nAChRs is essential for the anorexic effects of nicotinic compounds. We investigated this hypothesis by knocking down the expression of the β4 nAChR subunit with a neuron-specific adeno-associated virus (AAV-2) carrying specific short hairpin RNA molecules (shRNAs). Because the arcuate nucleus (ARC) is one of the most critical brain areas involved in feeding behavior and has been proposed as a potential site for the nicotinic modulation of appetite and food intake (8), we bilaterally infused 0.5 μl (1 μl per mouse) of high-titer AAV-shRNAs targeting the β4 nAChR subunit into the ventral hypothalamus (Fig. 2) and allowed at least 3 weeks for recovery (β4KD). We first quantified the efficacy of the knockdown [~55 ± 8.4% (SEM) in mRNA level compared to control (scrambled shRNA); fig. S2A] and did not detect nonspecific effects of the shRNA in other brain regions or at other nAChR subunits (fig. S2, B, C, and D). Mice with β4KD in ARC were resistant to the effects of cytisine on food intake [F1,14 = 0.01, P = 0.92], whereas β2KD did not change the anorexic effects of cytisine (Fig. 2 and fig. S3B, respectively).

Fig. 2

Knockdown of β4 nAChRs in the ventral hypothalamus. We used AAV to deliver shRNAs to knock down the β4 subunit in the ventral hypothalamus. Sites of infusion were verified by GFP detection (photograph; 3V, third ventricle). After knockdown of β4 nAChRs and recovery, cytisine (1.5 mg/kg) was unable to decrease food intake, contrary to what was seen in the control group of animals. ***P < 0.001. Error bars indicate SEM.

The ability of β4 nAChR knockdown in the ARC to abolish the anorexic effect of cytisine suggested the involvement of the hypothalamic melanocortin system, an essential brain pathway involved in the regulation of energy balance and food intake (12), as a target for nicotinic drugs. In particular, activation of pro-opiomelanocortin (POMC) cells in the ARC decreases food intake and increases energy expenditure (13), and loss of function of the POMC gene leads to obesity in humans and animals (14, 15). We therefore hypothesized that activation of α3β4 nAChRs may induce POMC neuron firing, leading to the anorexogenic effects of nicotinic agonists. We first confirmed that the β4 nAChR is expressed in POMC neurons by using laser-capture microdissection of neurons from transgenic mice expressing green fluorescent protein (GFP) under control of the POMC promoter (fig. S4). We then performed double-labeling immunohistochemical experiments for Fos-like immunoreactivity (FOS-IR) and POMC-IR (fig. S5A) as a measure of neuronal activation in the ARC of mice treated with nicotinic drugs. There was a significant difference across treatments [F3,12 = 5.85, P = 0.0106], and we found that chronic nicotine and cytisine treatment, unlike mecamylamine, increased FOS-IR selectively in POMC neurons of the ARC by about 50% [Fig. 3A; nicotine F1,6 = 8.60, P = 0.026; cytisine F1,6 = 7.68, P = 0.032] with no detectable effects on other neuronal subtypes in this region (fig. S5B) and that the effect was even greater immediately after an acute injection [about 80%, F1,14 = 20.73, P < 0.001; fig. S5C]. In contrast, there was no significant effect of mecamylamine treatment on FOS- or POMC-IR in the ARC overall [saline versus mecamylamine, F3,12 = 0.619, P = 0.46; for FOS-IR, F3,12 = 1.39, P = 0.29; for POMC-IR F3,12 = 0.405, P = 0.75]. We then identified direct electrophysiological effects of nicotinic drugs on POMC neurons by recording excitatory postsynaptic potentials in the presence of tetrodotoxin (TTX) (0.5 μM) and the γ-aminobutyric acid type A (GABAA) receptor antagonist picrotoxin (50 μM) from identified, GFP-labeled POMC neurons in slices from POMC-GFP transgenic mice. Application of nicotine (0.5, 5, 50 μM) for 1 to 2 min depolarized the membrane potential and strongly increased the spontaneous firing of POMC neurons to 289.0 ± 82.8% of baseline [Fig. 3B; F2,2 = 4.25, P < 0.05]. The nicotine effects were dose-dependent (Fig. 3B, top right). Both 0.5 and 50 μM nicotine increased the spike frequency to 173.4 ± 27.5% of baseline [F2,17 = 4.1, P < 0.05] and 456.3 ± 53.8% of baseline [F2,17 = 33.65, P < 0.001], respectively. Similarly, cytisine (10 μM) increased the firing rate of POMC neurons (Fig. 3C), with an average increase of action potential frequency of 186.2 ± 23.6% of baseline compared with that of baseline [F2,29 = 11.3, P < 0.01]. Conversely, the nicotinic antagonist mecamylamine did not induce significant changes in the frequency of action potentials in POMC neurons (fig. S6), and, in the presence of TTX (0.5 μM) and picrotoxin (50 μM), neither nicotine nor cytisine had a significant effect on the frequency and amplitude of mEPSCs (fig. S6, B and C, respectively).

Fig. 3

POMC neuron activation by nicotinic drugs. Administration of nicotine (0.1 mg/kg) or cytisine (1.5 mg/kg), unlike mecamylamine (1 mg/kg), resulted in activation of POMC cells in the ARC as measured by c-fos immunoreactivity (A). Electrophysiological studies further demonstrate a dose-dependent [see (B) inset], reversible increase in the firing rate of identified POMC neurons in response to nicotine (B) or cytisine (C) application. *P < 0.05; **P < 0.01. Food intake was not significantly affected by nicotine or cytisine in POMC KO mice at two different doses (D). Furthermore, knockdown of Mc4r by AAV-shRNAs in the PVN [detected by GFP fluorescence (fig. S7)] significantly blunted the hypophagic response to nicotine over time compared with the response of mice injected with control virus and treated with nicotine. No signs of tolerance to nicotine were observed over 30 days, consistent with a role for MC4R signaling in the anorexic effects of nicotinic agents (E). ***P < 0.001. Error bars indicate SEM.

To determine whether POMC neurons and melanocortin pathways were necessary for nicotinic-induced hypophagia, we treated POMC knockout (KO) mice with different doses of nicotine or cytisine and measured food intake over 24 hours (Fig. 3D). POMC KO mice showed no significant difference in food intake in response to nicotine [F2,16 = 0.56, P = 0.58] or cytisine [F2,20 = 0.78, P = 0.46] treatments, whereas cytisine-treated wild-type mice showed a decrease in food intake at each of the concentrations tested [at 1.5 mg/kg, F1,8 = 82.5, P < 0.001; at 3 mg/kg, F1,8 = 57.1, P < 0.01]. Lastly, we confirmed that release of melanocortin is critical for nicotinic-induced hypophagia by using AAV-shRNA delivery to knock down expression of the widely expressed melanocortin 4 receptor (Mc4r; fig. S7) in the paraventricular nucleus (PVN), where efferents of POMC neurons are present (16). Chronic treatment induced a blunting of nicotine-induced hypophagia in mice with Mc4R knockdown in the PVN [knockdown versus control: F10,180 = 5.54, P < 0.0001; Fig. 3E]; a similar pattern was observed in response to acute cytisine (1.5 mg/kg) [fig. S7; F1,18 = 10.05, P = 0.005].

Previous reports demonstrated that Mc4r activation by melanocortins is critical for the regulation of food intake and energy expenditure (17), as confirmed by a trend for increased feeding at baseline after Mc4rKD in PVN. These data demonstrate that nicotinic drugs decrease food intake primarily through β4* (where * represents other nAChR subunits still to be identified) nAChR–dependent activation of POMC neurons and melanocortin pathways. It has been demonstrated that POMC neurons express cholinergic markers (18) and that the naturally obese Tub/Tub strain of mice displays a decrease of perivascular cholinergic innervation in the ARC (19). These observations underscore a possible role for acetylcholine in metabolic regulation through POMC neurons (Fig. 4). It has also been suggested that cholinergic projections to the ventral hypothalamus could be provided by very localized groups of neurons found in the median eminence (20), a region harboring cells secreting hypophysiotropic hormones, including corticotropin-releasing hormone, all known to affect metabolism. Postsynaptic modulation of POMC neurons could also occur through cholinergic projections emanating from the pedunculopontine tegmental and lateradorsal tegmental nuclei (Ch5 and Ch6), regions that can adapt rapidly to metabolic stimulation (21, 22) and that are also involved in feeding behavior (23). All these mechanisms could therefore alter activity of POMC neurons and neurotransmitter release from presynaptic terminals that could, in turn, affect energy expenditure and feeding patterns. Our results further suggest that β4* nAChRs are critical receptors mediating these effects. α3β4 agonists may therefore be useful for limiting weight gain after smoking cessation, and nicotinic drugs could also be useful to control obesity and related metabolic disorders.

Fig. 4

Hypothetical model of the anorexogenic effect of nicotine in the ARC. POMC neurons express nAChRs and therefore respond to nicotinic drugs. In the basal state (i.e., in the absence of nicotine), POMC neurons project to second order neurons that decrease food intake. When nicotine reaches the ARC (facilitated by its proximity to the third ventricle), activity of POMC neurons is increased (as measured by c-fos expression and neuronal activity measured in slices) through activation of α3β4 nAChRs and subsequent activation of MC4 receptors in the PVN of the hypothalamus (nicotine-activated state).

Supporting Online Material

www.sciencemag.org/cgi/content/full/332/6035/1330/DC1

Materials and Methods

Figs. S1 to S7

References (2435)

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Acknowledgments: These studies were supported by grants DA14241, DA00436, and AA15632 from the NIH. X.-B.G. was supported by DK070723. Y.M. was supported by a Transdisciplinary Tobacco Use Research Center young investigator pilot grant. Alfonso Abizaid was supported by a postdoctoral fellowship from the Natural Science and Engineering Research Council of Canada. D.G. was supported by RR016467. Y.R., X.-B.G., and S.D. were supported by American Diabetes Association grants 1-08-RA-36 and DK070039. M.D. was supported by DA017173. T.L.H. was supported by OD006850 and DK080000. The authors gratefully acknowledge U. Hochgeschwender (Oklahoma Medical Research Foundation) for providing heterozygous POMC breeding pairs.
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