Published ahead of print on March 10, 2006, doi:10.1164/rccm.200506-977OC
© 2006 American Thoracic Society doi: 10.1164/rccm.200506-977OC
Cigarette Smoke Exposure Reprograms the Hypothalamic Neuropeptide Y Axis to Promote Weight LossDepartment of Pharmacology, and CRC for Chronic Inflammatory Diseases; Department of Medicine, The University of Melbourne, Royal Melbourne Hospital, Victoria; and Department of Physiology and Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, Australia Correspondence and requests for reprints should be addressed to Professor Margaret J Morris, Ph.D., Department of Physiology and Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia. E-mail: m.morris{at}unsw.edu.au
Rationale: Despite irrefutable epidemiologic evidence, cigarette smoking remains the major preventable cause of lung disease morbidity worldwide. The appetite-suppressing effect of tobacco is a major behavioral determinant of smoking, but the underlying molecular and neuronal mechanisms are not understood. Neuropeptide Y (NPY) is an orexigenic neuropeptide, whose activity in the hypothalamic paraventricular nucleus governs appetite. Objectives: To compare the effects of smoke exposure and equivalent food restriction on body weight, organ mass, cytokines, and brain NPY in Balb/c mice. Methods: A pair-feeding study design compared smoke exposure (4 wk; 1 cigarette, 3x/d, 5 d/wk) to equivalent food restriction (pair-fed) and sham-exposed control mice.
Results: Smoke exposure rapidly induced mild anorexia. After 4 wk, smoke-exposed and pair-fed groups were lighter than control mice (22.0 ± 0.2, 23.2 ± 0.5, 24.9 ± 0.4 g, respectively; p < 0.05). Brown and white fat masses were only reduced by smoke exposure, relative to control mice. NPY concentration in the paraventricular nucleus was significantly and paradoxically reduced by smoke exposure, despite lower plasma leptin concentrations; this was not observed in the pair-fed group experiencing 19% food restriction. Adipose mRNA expression of uncoupling proteins, inflammatory cytokines interleukin 6 and tumor necrosis factor Conclusions: In contrast to food restriction, smoke exposure caused a reduction in hypothalamic NPY and fat mass, and regulated adipose cytokines. These findings may contribute to understanding weight loss in smoking-related lung disease and in the design of more effective smoking cessation strategies.
Key Words: anorexia • interleukin 6 • leptin • tumor necrosis factor Cigarette smoking is the leading preventable cause of death and disability from respiratory disease worldwide (1). The appetite-suppressing effect of tobacco is a major driver of smoking behavior, and potential weight gain on cessation may prevent people from quitting (2–4). The anorexigenic effect of cigarette smoke may also contribute to skeletal muscle wasting in long-term smokers who have developed chronic obstructive pulmonary disease (COPD). A negative correlation among smoking, body weight, and caloric intake has been well demonstrated across species (5, 6). Understanding the mechanisms of appetite regulation by tobacco could therefore contribute to the management of smoking-related disease. However, the neuromolecular basis of appetite regulation by cigarette smoke is unknown. Food intake is controlled by multiple signaling pathways (7), which match energy intake to energy expenditure over time. Several distinct hypothalamic neuropeptide-containing pathways regulate appetite in the central nervous system, including both orexigenic and anorexigenic pathways. Neuropeptide Y (NPY) is a 36-amino acid neurotransmitter with potent appetite stimulatory effects (8, 9). NPYergic projection from the arcuate nucleus (Arc) to the paraventricular nucleus (PVN) relays orexigenic signals (10, 11), and repeated central administration of NPY leads to obesity (12, 13). The physiologic response to reduced caloric intake is to increase NPY levels in brain appetite-regulating nuclei in part in response to withdrawal of circulating hormones such as leptin, a critical peripheral metabolic signal secreted by adipose tissue (14–17), whereas decreased hypothalamic NPY was observed in chronic diet-induced obesity (18). Leptin is also produced directly by inflamed lung tissue (19). Leptin was found to be reduced in patients with COPD (20), which might be important in signaling energy deficiency to the hypothalamic–pituitary axis (21). However, leptin was increased during acute exacerbations (22), which might enhance the catabolic effect of leptin to suppress appetite and heighten energy expenditure. Increased leptin in pulmonary cachexia may contribute to the poor response to nutritional support in some cachexic patients with COPD (23). Nicotine, the principal addictive constituent of tobacco, has been shown to suppress appetite in many studies (5, 6, 24–26). Nicotinic receptors are highly expressed in the hypothalamus and medulla (27). However, the effects of nicotine on brain NPY expression are controversial (24, 25). Moreover, the effects of cigarette smoke per se on appetite and hypothalamic NPY are unknown.
We recently reported that mice exposed to three cigarettes, three times a day for 4 d displayed a marked decrease in food intake and body weight (28). Although plasma leptin concentration was significantly decreased in this acute model, no significant changes in hypothalamic NPY content were observed. The aim of the present study was to investigate NPY, appetite, and body composition changes during subchronic cigarette exposure. A pair-fed (PF) control study design matched food intake to that measured in smoke-exposed (SE) mice to determine whether the food restriction induced by cigarette smoke exposure is sufficient to change body weight, adipose tissue, and brain NPY. We measured adipose mRNA expression of uncoupling protein 1 (UCP1), UCP3, inflammatory cytokines, tumor necrosis factor
Animals Development of respiratory disease after cigarette smoke exposure is strain dependent (31, 32). Male Balb/c mice (aged 6 wk, n = 80) were chosen based on previous studies in our laboratory. Mice, obtained from the Animal Resource Centre Pty. Ltd. (Perth, Australia), were housed at 20 ± 2°C in microisolator cages, and maintained on a 12:12-h light/dark cycle, with ad libitum access to standard chow and water. After acclimatization, mice were randomly divided into the following three groups matched for body weight: cigarette smoke exposed (SE group), sham exposed (control group), and PF sham exposed (PF group). The animals exposed to cigarette smoke were placed inside a perspex chamber (18 L) and exposed to the smoke produced by one cigarette (Winfield Red, 16 mg or less of tar, 1.2 mg or less of nicotine, and 15 mg or less of CO; Philip Morris, Moorabbin, Australia), three times a day (09:00, 12:00, and 15:00 h), 5 d/wk for 4 wk. The control and PF groups were handled identically but were not exposed to smoke. Food intake of the SE group was measured daily and the PF group was given the same amount of chow eaten by the SE group. All mice were weighed twice weekly. All procedures were approved by the Animal Experimentation Ethics Committee of the University of Melbourne and conformed to international welfare standards.
Sample Collection
NPY, Plasma Leptin, and Corticosterone Radioimmunoassay
UCP1, UCP3, TNF-
Statistical Analysis
During the acclimatization period, food intake of the control and SE groups was similar (Table 1). Food intake of the SE group decreased by 31% after the first day of smoke exposure, and daily food intake during the 4-wk experimental period was significantly reduced by 19% compared with control mice (p < 0.05; Table 1). Each day, the mice in the PF group consumed all the chow provided. Thus, total chow intakes were 62.7 g per mouse in the control group, 50.5 g in the SE group, and 51.1 g in the PF group over 4 wk.
The body weights of control, SE, and PF groups were well matched at the beginning of this experiment (Figure 1). Shortly after the commencement of smoke exposure, body weight of the SE group dropped slightly compared with the preexposure level (21.7 ± 0.5 g vs. 22.1 ± 0.4 g; Figure 1); 4 d after smoke exposure, they were significantly lighter than control mice (21.7 ± 0.5 g vs. 23.4 ± 0.6 g, respectively; p < 0.05; Figure 1). Body weight of the SE group remained constant for nearly 2 wk, after which the mice began to gain weight; however, the body weight remained at a lower level compared with control mice, reflected by a significantly smaller body weight gain at the end of the experiment (0.6 ± 0.3 g in SE and 0.7 ± 0.2 g in PF groups vs. 2.6 ± 0.2 g in the control group, p < 0.05). The SE and PF groups at death were 9.2 and 7.2% lighter than the control group, respectively.
Mice exposed to cigarette smoke had significantly lower BAT, RpWAT, testicular WAT, and liver masses compared with control mice (Table 1; p < 0.05). When data were adjusted for body weight, the testicular WAT and liver remained smaller in the SE group (testicular WAT: control, 2.7 ± 0.1%, vs. SE, 2.2 ± 0.1%; p < 0.05; liver: control, 4.7 ± 0.1%, vs. SE, 4.4 ± 0.1%; p < 0.05). In the PF group, only liver mass was reduced relative to the control group (Table 1; p < 0.05). Plasma leptin concentration was significantly reduced in the SE group (p < 0.05), but was similar in both PF and control groups (Table 1). Plasma leptin concentration was positively correlated with RpWAT mass within each treatment group (p < 0.05; r = 0.79, 0.94, and 0.61; n = 21, 24, and 12 in control, SE, PF groups, respectively). The plasma corticosterone was increased by 51% in the SE group and 82% in the PF group compared with the control group (p < 0.05; Table 1). No statistical difference was observed between the SE and PF groups. NPY concentrations in the AH, PH, and Arc, as well as medulla, were not different among control, SE, and PF groups (Figure 2), whereas PVN NPY concentration was significantly decreased in the SE group compared with control group (p < 0.05).
UCP1 and UCP3 mRNA expression in WAT was decreased to a similar level in the SE and PF groups relative to the control group (Figures 3A and 3B). However, in BAT, pair feeding exerted greater effects than smoke exposure. Smoke exposure and food restriction (pair feeding) suppressed UCP1 mRNA expression by 50% (p < 0.05 vs. control) and 77% (p < 0.05 vs. control), respectively. Similarly, BAT UCP3 mRNA expression in the SE and PF groups was decreased by 30% (p < 0.05 vs. control) and 56% (p < 0.05 vs. control; Figure 3B). A similar pattern was evident in BAT UCP1 and UCP3 mRNA expression in both SE and PF groups, where the reduction in the PF group was significantly greater than in the SE group (p < 0.05).
In WAT, TNF-  mRNA expression was not altered by smoke exposure or pair feeding (Figure 4A). In BAT, TNF- was halved by smoke exposure (p < 0.05 vs. control group; Figure 4A), and food restriction had a bigger inhibitory effect on TNF- than did smoke exposure (p < 0.05 vs. control and SE groups). IL-6 mRNA expression was reduced to a similar level in WAT by both food restriction and smoke exposure with no effect in BAT (p < 0.05 vs. control group; Figure 4B).
The expression of ATGL mRNA in BAT was higher than in WAT in control animals. In WAT, the mRNA expression of ATGL was significantly reduced to a similar level by both smoke exposure and equivalent food restriction (p < 0.05 vs. control group; Figure 5). In BAT, the level of ATGL mRNA expression was halved by both smoke exposure and pair feeding (p < 0.05 vs. control; Figure 5).
The current study investigated, for the first time, the effects of subchronic cigarette smoke exposure on the regulation of appetite and body weight, brain NPY content, plasma hormones, and mRNA expression of UCPs, cytokines, and ATGL. Our results indicate that cigarette smoke fundamentally alters hypothalamic appetite regulation in the central nervous system. Cigarette smoke contains at least 6,000 components that may directly or indirectly affect energy expenditure. Many studies have investigated the mechanisms underlying the anorexia and weight loss accompanying smoking by administering nicotine (24–26). The determinants of smoking behavior that reinforce nicotine addiction in people are multifactorial. The use of smoking to regulate body mass has emerged as a principal determinant. In our study, the inclusion of the PF group enabled us to separate the contribution of food restriction from the catabolic effects of the multiple factors in cigarette smoke. The persistent decrease in daily food intake and body weight gain observed in mice exposed to cigarette smoke might reflect the anorexia and weight loss in human smokers. This was highlighted by the observations that anorexia nervosa alone is associated with emphysema (35, 36). Intriguingly, the delayed body weight change and lack of effects on WAT and BAT deposits in PF mice indicate reduced caloric intake is not the primary factor contributing to the fat loss induced by smoke exposure.
Animals exposed to cigarette smoke had reduced body weight gain relative to control mice within the first week of the exposure. PF animals had a slower onset in body weight change compared with SE animals, suggesting the decreased weight gain in the SE mice was only partly due to food restriction per se, and other physiologic changes may be relevant. Over the 4-wk experimental period, the effects of food restriction on body weight became more marked, reflected by the similar body weight in the PF and the SE groups at the end of the experiment. Although WAT mass was decreased in mice exposed to cigarette smoke, it was not significantly altered by food restriction alone (PF group), suggesting the 19% reduction in food intake was not sufficient for the body to use WAT as an energy supply. In the studies of long-term diet-induced weight loss in humans, lean body mass was observed to be decreased together with fat mass when food intake was reduced (37). We have previously determined that the smoking protocol used in this study induced mild lung inflammation associated with an induction of the catabolic cytokines, such as TNF- Cigarette smoking is associated with sleep disturbance (38). A high dose of nicotine causes a transient (1 h) but not sustained increase in wakefulness in mice (39). It is therefore possible, but unlikely, that our protocol may have affected sleep–wake cycles, which affected activity levels; this possibility will be of interest to study in the future. NPY-expressing neurons in the rodent hypothalamus are largely confined to the Arc, lying close to the third ventricle in the medial part of the nucleus. These neurons send dense projections to other hypothalamic nuclei important for appetite regulation: the PVN, the dorsomedial hypothalamus, and the lateral hypothalamus (7, 40). The hypothalamus also receives NPY inputs that originate from outside the hypothalamus, notably cell groups in the medulla that receive peripheral nutrient signals (11). NPY is normally up-regulated during extreme energy deprivation. In previous studies, more than 30% restriction in energy intake for more than 14 d caused a significant increase in NPY in Arc (41, 42); however, in this study, a modest 19% restriction of energy intake for 4 wk (pair feeding) did not cause any change in NPY content in either the hypothalamus or medulla, suggesting decreasing food intake to that induced by smoke exposure was not sufficient to alter brain NPY levels. However, PVN NPY concentration was significantly decreased in the SE group, and this may contribute to the anorectic effect of cigarette smoking in mice. The Arc-PVN NPY projection is important in appetite regulation, and although a significant reduction in NPY was seen in the PVN, a similar trend was observed in the AH and Arc, suggesting an inhibitory effect of smoke exposure on hypothalamic NPY production. Because the time courses of previous chronic nicotine studies were less than 14 d (24, 25), it is difficult to draw comparisons between the effects of nicotine injection and our smoke exposure studies, particularly given that cigarette smoke contains more factors than just nicotine. Under normal physiologic conditions, Arc NPY gene expression is negatively regulated by leptin signaling (16, 43) and positively regulated by negative energy balance (9, 44). The decrease in plasma leptin concentration and WAT mass in the SE group would be expected to stimulate hypothalamic NPY production, in line with our previous observations of a negative correlation between leptin and hypothalamic NPY in the rat (18, 41, 45). However, in the current study, a reduction in NPY concentration was observed in the face of lower leptin level and fat mass, which may reflect inhibitory effects of smoke exposure on hypothalamic NPY levels. Previously, it was found that nicotine can blunt the hyperphagia produced by exogenous administration of NPY into the PVN (46), suggesting that it is possible that the effects of NPY can be offset by nicotine or other elements in cigarette smoke. In another study by Jang (47), the increased NPY expression in food-restricted rats was inhibited by further nicotine administration. Hypothalamic NPY Y1 receptor density was observed to be reduced by chronic nicotine treatment (48), which may result in reduced capacity of NPY to induce feeding. Therefore, NPY signaling might be inhibited by cigarette smoking, leading to an abnormal response to the declining adipose mass. Corticosterone is part of the adaptive physiologic response to environmental stressors. Although nicotine administration can activate the hypothalamic–pituitary–adrenocortical system and elevate plasma corticosterone concentrations in animals and humans, corticosterone returned to control levels after 7 consecutive d of injection (49, 50). Corticosterone is an important appetite stimulator that can be activated by food restriction (reviewed in Reference 51). Although plasma corticosterone was increased in the SE animals, this failed to override the anorexigenic effects of smoke exposure. Increased plasma corticosterone concentrations were also observed in the PF group, suggesting this was related to energy restriction rather than nonspecific stress due to smoke exposure. The relative lack of contribution of fat loss to the fall in body weight observed in PF mice was striking and unexpected. We therefore studied the contribution of UCPs. BAT UCP1 activity is responsible for nonshivering thermogenesis, a major component of thermogenesis in newborn humans and in small mammals. However, UCP1 is expressed in human retroperitoneal fat (52). Due to the sensitivity of the technique we used, UCP1 was detectable in WAT with low expression compared with BAT. Fasting and chronic food deprivation can down-regulate UCP1 expression in BAT (53, 54). In this study, UCP1 mRNA expression in WAT was down-regulated to similar levels by both smoke exposure and pair feeding, suggesting an inhibitory effect of food restriction on UCP1 expression in WAT; however, the contribution to energy expenditure is not clear. Reduced UCP1 expression in BAT in pair feeding suggests an attempt to reserve energy expenditure during negative energy intake by reducing thermogenesis. However, the less marked change in SE animals suggests that smoke exposure might interfere with the signaling induced by decreased food intake (pair feeding) that is responsible for reducing thermogenesis. Previously, UCP1 mRNA expression was induced in nicotine-treated animals compared with PF animals, consistent with our observation (55). The changes we observed in UCP3 were similar to those in UCP1, and opposite from our previous results using short-term smoke exposure (28). UCP3 is expressed in BAT and skeletal muscle in both rodents and humans and is implicated in mitochondrial fatty acid transport, and influences basal metabolic rate (54, 56). Significantly increased UCP3 expression was observed in fasting animals, in which fatty acid oxidation was high (52, 57). However, in this study, chronic food restriction reduced UCP3 expression, suggesting fatty acid metabolism and basal metabolic rate were decreased in response to reduced caloric intake. The changes in BAT UCP3 were less obvious in the SE group, suggesting an inducing effect of cigarette smoke on top of food restriction. We propose that the smaller inhibition of UCP1 and UCP3 mRNA expression by smoke exposure compared with food restriction alone may contribute to the weight loss and decreased fat deposits observed in SE mice.
Cigarette smoking alters the production of many inflammatory cytokines from macrophages, such as TNF- In mammals, triglycerides are stored in adipose tissue to provide the primary source of energy during negative energy balance. This is the first study reporting the effects of long-term, moderate food restriction on the lipolysis marker ATGL in adipose tissue. ATGL is highly expressed in both WAT and BAT of humans and mice (68). ATGL catalyzes the initial step in triglyceride hydrolysis (68), and contributes to basal lipolysis (69). In contrast to an earlier study where ATGL expression was increased by 24-h fasting (70), we report that moderate, long-term food restriction induced by smoke exposure led to a reduction in ATGL mRNA expression in both WAT and BAT. Nicotine infusion has been demonstrated to stimulate lipolysis (71, 72), and there was fat loss in the SE group, which was absent in the PF group. The reduction in ATGL mRNA expression level in both the SE and PF groups was similar, suggesting the long-term reduction in caloric intake caused by smoke exposure, but not fat loss per se, played a key role in the regulation of adipose ATGL mRNA expression. In summary, 4 wk of cigarette smoke exposure significantly decreased body weight, food intake, fat mass, as well as plasma leptin concentration, whereas equivalent food restriction only decreased body weight. Hypothalamic energy balance circuits were disturbed by cigarette smoke exposure, as evidenced by the altered NPY concentration in the PVN, suggesting NPY signaling is involved in the appetite-suppressive effects of cigarette smoking. The lack of change in fat mass and hypothalamic NPY concentration by food restriction alone indicates other metabolic pathways may contribute to the fat loss seen in SE animals. The relative preservation of UCPs in smoke exposure, compared with the larger impact of modest caloric restriction alone, may contribute to the weight and fat loss seen with smoking. Understanding the neuromolecular processes that lead to loss of body weight in smokers may provide novel intervention points for improving cessation therapy and may also contribute to understanding wasting syndromes in smoking-related lung diseases.
This work was supported by the National Health and Medical Research Council of Australia and the CRC for Chronic Inflammatory Diseases. Originally Published in Press as DOI: 10.1164/rccm.200506-977OC on March 10, 2006 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form June 24, 2005; accepted in final form March 6, 2006
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B induction profiles during lung inflammation induced by subchronic cigarette smoke exposure in mice. Am J Physiol Lung Cell Mol Physiol (In press)
2-Containing nicotinic receptors contribute to the organization of sleep and regulate putative micro-arousals in mice. J Neurosci 2004;24:5711–5718.
