Abstract
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, 3×/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 α, and adipose triglyceride lipase was decreased by smoke exposure, and even lower in pair-fed mice.
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.
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 α (TNF-α), and interleukin 6 (IL-6), which are induced by smoking and which are known procatabolic mediators of cachexia, and a novel enzyme that metabolizes triglyceride, adipose triglyceride lipase (ATGL). Some of the results of these studies have been previously reported in abstract form (29, 30).
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.
Mice were killed after anesthetic overdose (ketamine/xylazine, 180/32 mg/kg, intraperitoneally) by decapitation. Plasma was stored at −80°C for subsequent determination of plasma leptin and corticosterone concentrations. Brains were rapidly removed and microdissected on ice into regions containing PVN, Arc, anterior and posterior hypothalamus (AH, PH), and medulla, weighed and stored at −80°C for later determination of NPY. Body fat (interscapular brown adipose tissue [BAT], left retroperitoneal [Rp] white adipose tissue [WAT], testicular WAT) and liver were dissected and weighed. RpWAT and BAT were snap frozen for quantitative real-time polymerase chain reaction.
Endogenous brain NPY was extracted and NPY-like immunoreactivity was measured by a specific radioimmunoassay developed in our laboratory (33) using synthetic NPY as the standard (10–1,280 pg/tube; Auspep, Melbourne, Australia). The detection limit for the radioimmunoassay was routinely 2 pg NPY per tube, and the intra- and interassay coefficients of variation were 6 and 13%, respectively. NPY in each brain region was calculated as nanograms of NPY per milligram of tissue. Plasma leptin and corticosterone concentrations were measured using commercially available radioimmunoassay kits (Linco, St. Charles, MO, and MP Biomedicals, Irvine, CA, respectively).
Total RNA was isolated from 20 mg of both WAT and BAT using an RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The purified total RNA preparation was used as a template to generate first-strand cDNA synthesis using SuperScript II (Invitrogen, Carlsbad, CA) as previously described (34). Quantitative real-time polymerase chain reaction was performed (ABI 7900 HT Sequence Detection System; Applied Biosystems, Foster City, CA) using predeveloped primers from Applied Biosystems (34). Gene expression was quantified by multiplexing single reactions, where the gene of interest (UCP1, UCP3, TNF-α, IL-6, and ATGL) was standardized to 18s rRNA. An individual BAT sample from the control group was then arbitrarily assigned as a calibrator against which all other samples were expressed as fold difference.
Results are expressed as mean ± SEM. Body weight was analyzed using analysis of variance (ANOVA) with repeated measures, followed by a post hoc Fisher's protected least significant difference (LSD) test. Differences in average food intake, fat and organ mass, plasma leptin and corticosterone concentration, brain NPY concentration and content, and mRNA expression were analyzed using one-way ANOVA followed by a post hoc LSD test.
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.
Control | SE | PF | |
|---|---|---|---|
| Parameter | (n = 32) | (n = 36) | (n = 12) |
| Food intake, g/mouse/d (preexposure) | 2.90 ± 0.02 | 2.92 ± 0.04 | 2.87 ± 0.11 |
| Food intake, g/mouse/d (during exposure) | 2.85 ± 0.11 | 2.30 ± 0.05† | 2.32 ± 0.11† |
| Body weight, g | 25.0 ± 0.5 | 22.7 ± 0.5† | 23.2 ± 0.5† |
| BAT, mg | 93.5 ± 4.0 | 81.8 ± 2.4† | 97.2 ± 6.3‡ |
| RpWAT, mg | 155.2 ± 9.1 | 119.8 ± 9.0† | 150.7 ± 13.8 |
| Testicular WAT, mg | 675.7 ± 29.1 | 490.7 ± 24.5† | 652.0 ± 46.8‡ |
| Liver, mg | 1,161.0 ± 22.0 | 976.4 ± 13.6† | 1,006.0 ± 25.1† |
| Leptin, ng/ml* | 6.0 ± 0.3 | 4.9 ± 0.4† | 5.8 ± 0.5 |
| Corticosterone, ng/ml* | 204.4 ± 34.6 | 308.2 ± 33.0† | 372.9 ± 48.7† |
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.
Figure 1. Body weight of control (white squares; n = 12), smoke-exposed (SE; solid squares; n = 12), and pair-fed (PF) groups (asterisks; n = 12) during the experimental period. Results are expressed as mean ± SEM. Data were analyzed by analysis of variance (ANOVA) with repeated measures. *SE group significantly different from control group, p < 0.05; #PF group significantly different from control group, p < 0.05.
[More] [Minimize]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).
Figure 2. Neuropeptide Y (NPY) concentrations of brain regions in control (open bars; n = 12), SE (striped bars; n = 12), and PF (checkered bars; n = 12) groups at 4 wk. Results are expressed as mean ± SEM. Data were analyzed by one-way ANOVA followed by a post hoc least significant difference (LSD) test. *Significantly different from control mice, p < 0.05. AH = anterior hypothalamus; Arc = arcuate nucleus; PH = posterior hypothalamus; PVN = paraventricular nucleus.
[More] [Minimize]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).
Figure 3. mRNA expression of uncoupling protein 1 (UCP1; A) and UCP3 (B) in white adipose tissue (WAT) and interscapular brown adipose tissue (BAT) in control (open bars; n = 12), SE (striped bars; n = 12), and PF (checkered bars; n = 12) groups at 4 wk. Results are expressed as fold difference relative to a control BAT sample. Data were analyzed by one-way ANOVA followed by a post hoc LSD test. * Significantly different from control group (p < 0.05); #significantly different from SE group (p < 0.05).
[More] [Minimize]In WAT, TNF-α mRNA expression was not altered by smoke exposure or pair feeding (Figure 4A). In BAT, TNF-α was halved by smoke exposure (p < 0.05 vs. control group; Figure 4A), and food restriction had a bigger inhibitory effect on TNF-α 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).
Figure 4. mRNA expression of tumor necrosis factor α (TNF-α; A) and interleukin 6 (IL-6; B) in WAT and BAT in control (open bars; n = 12), SE (striped bars; n = 12), and PF (checkered bars; n = 12) groups at 4 wk. Results are expressed as fold difference relative to a control BAT sample. Data were analyzed by one-way ANOVA followed by a post hoc LSD test. * Significantly different from control group (p < 0.05); #significantly different from SE group (p < 0.05).
[More] [Minimize]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).
Figure 5. mRNA expression of adipose triglyceride lipase (ATGL) in WAT and BAT in control (open bars; n = 12), SE (striped bars; n = 12), and PF (checkered bars; n = 12) groups at 4 wk. Results are expressed as fold difference relative to a control BAT sample. Data were analyzed by one-way ANOVA followed by a post hoc LSD test. * Significantly different from control group (p < 0.05).
[More] [Minimize]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-α and IL-6 (32). However, separate studies in our labs have demonstrated that loss of skeletal muscle mass is detectable only after 7 wk of smoke exposure (M.J.H., unpublished observations).
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-α and IL-6, which might contribute to the development of lung pathology (58). TNF-α was observed to contribute to the pathology of emphysema and pulmonary fibrosis in mice models (59, 60). Contradictory effects on either TNF-α concentration or mRNA expression were found in previous studies (58, 61), and the direct effect of smoke exposure on these cytokines in fat is unknown. Produced by both immune cells and adipocytes, TNF-α can regulate lipid metabolism, adipocyte differentiation, insulin resistance, and cachexia (62–64). TNF-α expression in human adipose tissue and skeletal muscle is positively correlated with body mass index, and inversely correlated with the activity of adipose tissue lipoprotein lipase (65). Little is known about the regulation of TNF-α in BAT, and it is possible that the regulation of TNF-α differs between WAT and BAT. The reduced TNF-α in PF and SE animals may reflect reduced adipocyte differentiation. The cytokine IL-6 elicits proinflammatory effects. IL-6 suppresses body fat, increases oxygen consumption and carbon dioxide production, reduces food intake (reviewed in Reference 66), and promotes insulin resistance by altering insulin signaling in hepatocytes (67). In this study, the down-regulation of IL-6 in WAT might potentially reduce energy expenditure and increase insulin sensitivity to increase glucose usage under negative energy intake. However, the impact of reduced TNF-α in BAT is not clear.
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.
| 1. | World Health Organization. The World Health report 2002. Chapter 4 [cited 6 March 2005]. Available from: http://www.who.int/whr/2002/ chapter4/en/index6.html. |
| 2. | Filozof C, Fernandez Pinilla MC, Fernandez-Cruz A. Smoking cessation and weight gain. Obes Rev 2004;5:95–103. |
| 3. | Stamford BA, Matter S, Fell RD, Papanek P. Effects of smoking cessation on weight gain, metabolic rate, caloric consumption, and blood lipids. Am J Clin Nutr 1986;43:486–494. |
| 4. | Fulkerson JA, French SA. Cigarette smoking for weight loss or control among adolescents: gender and racial/ethnic differences. J Adolesc Health 2003;32:306–313. |
| 5. | Wager-Srdar SA, Levine AS, Morley JE, Hoidal JR, Niewoehner DE. Effects of cigarette smoke and nicotine on feeding and energy. Physiol Behav 1984;32:389–395. |
| 6. | Hajek P, Jackson P, Belcher M. Long-term use of nicotine chewing gum: occurrence, determinants, and effect on weight gain. JAMA 1988;260:1593–1596. |
| 7. | Schwartz MW, Woods SC, Porte DJ, Seeley RJ, Baskin DG. Central nervous system control of food intake. Nature 2000;404:661–671. |
| 8. | Billington CJ, Briggs JE, Grace M, Levine AS. Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism. Am J Physiol Regul Integr Comp Physiol 1991;260:R321–R327. |
| 9. | Kalra SP, Dube MG, Sahu A, Phelps CP, Kalra PS. Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food. Proc Natl Acad Sci USA 1991;88:10931–10935. |
| 10. | Devaskar SU. Neurohumoral regulation of body weight gain. Pediatr Diabetes 2001;2:131–144. |
| 11. | Williams G, Harrold JA, Cutler DJ. The hypothalamus and the regulation of energy homeostasis: lifting the lid on a black box. Proc Nutr Soc 2000;59:385–396. |
| 12. | Stanley BG, Kyrkouli SE, Lampert S, Leibowitz SF. Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity. Peptides 1986;7:1189–1192. |
| 13. | Zarjevski N, Cusin I, Vettor R, Rohner-Jeanrenaud F, Jeanrenaud B. Chronic intracerebroventricular neuropeptide-Y administration to normal rats mimics hormonal and metabolic changes of obesity. Endocrinology 1993;133:1753–1758. |
| 14. | Beck B, Jhanwar-Uniyal M, Burlet A, Chapleur-Chateau M, Leibowitz SF, Burlet C. Rapid and localized alterations of neuropeptide Y in discrete hypothalamic nuclei with feeding status. Brain Res 1990;528:245–249. |
| 15. | Brady LS, Smith MA, Gold PW, Herkenham M. Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats. Neuroendocrinology 1990;52:441–447. |
| 16. | Stephens TW, Basinski M, Bristow PK, Bue-Valleskey JM, Burgett SG, Craft L, Hale J, Hoffmann J, Hsiung HM, Kriauciunas A, et al. The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 1995;377:530–532. |
| 17. | Woods SC, Seeley RJ, Porte DJ, Schwartz MW. Signals that regulate food intake and energy homeostasis. Science 1998;280:1378–1382. |
| 18. | Hansen MJ, Jovanovska V, Morris MJ. Adaptive responses in hypothalamic neuropeptide Y in the face of prolonged high-fat feeding in the rat. J Neurochem 2004;88:909–916. |
| 19. | Broekhuizen R, Vernooy JH, Schols AM, Dentener MA, Wouters EF. Leptin as local inflammatory marker in COPD. Respir Med 2005;99:70–74. |
| 20. | Takabatake N, Nakamura H, Abe S, Hino T, Saito H, Yuki H, Kato S, Tomoike H. Circulating leptin in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;159:1215–1219. |
| 21. | Chan JL, Mantzoros CS. Role of leptin in energy-deprivation states: normal human physiology and clinical implications for hypothalamic amenorrhoea and anorexia nervosa. Lancet 2005;366:74–85. |
| 22. | Creutzberg EC, Wouters EF, Vanderhoven-Augustin IM, Dentener MA, Schols AM. Disturbances in leptin metabolism are related to energy imbalance during acute exacerbations of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000;162:1239–1245. |
| 23. | Schols AM, Creutzberg EC, Buurman WA, Campfield L, Saris WH, Wouters EF. Plasma leptin is related to proinflammatory status and dietary intake in patients with chronic obstructive oulmonary disease. Am J Respir Crit Care Med 1999;160:1220–1226. |
| 24. | Frankish HM, Dryden S, Wang Q, Bing C, MacFarlane IA, Williams G. Nicotine administration reduces neuropeptide Y and neuropeptide Y mRNA concentrations in the rat hypothalamus: NPY may mediate nicotine's effects on energy balance. Brain Res 1995;694:139–146. |
| 25. | Li MD, Kane JK, Parker SL, McAllen K, Matta SG, Sharp BM. Nicotine administration enhances NPY expression in the rat hypothalamus. Brain Res 2000;867:157–164. |
| 26. | Frankham P, Cabanac M. Nicotine lowers the body-weight set-point in male rats. Appetite 2003;41:1–5. |
| 27. | Jo YH, Talmage DA, Role LW. Nicotinic receptor-mediated effects on appetite and food intake. J Neurobiol 2002;53:618–632. |
| 28. | Chen H, Vlahos R, Bozinovski S, Jones J, Anderson GP, Morris MJ. Effect of short-term cigarette smoke exposure on body weight, appetite and brain neuropeptide y in mice. Neuropsychopharmacology 2005;30:713–719. |
| 29. | Chen H, Vlahos R, Hansen M, Jones J, Bozinovski S, Anderson G, Morris MJ. One month cigarette smoke exposure reduces body weight, appetite and hypothalamic neuropeptide Y in mice [abstract]. Appetite 2004;42:347. |
| 30. | Chen H, Jones J, Hansen M, Vlahos R, Bozinovski S, Anderson GP, Morris MJ. Orexigen signal processing is reprogrammed in the brains of mice with COPD-associated weight loss and wasting [abstract]. Presented at the American Thoracic Society 2005 International Conference, San Diego, CA; 2005. |
| 31. | Guerassimov A, Hoshino Y, Takubo Y, Turcotte A, Yamamoto M, Ghezzo H, Triantafillopoulos A, Whittaker K, Hoidal JR, Cosio MG. The development of emphysema in cigarette smoke exposed mice is strain dependent. Am J Respir Crit Care Med 2004;170:974–980. |
| 32. | Vlahos R, Bozinovski S, Jones JE, Powell J, Gras J, Lilja A, Hansen MJ, Gualano RC, Irving L, Anderson GP. Differential protease, innate immunity and NFκB induction profiles during lung inflammation induced by subchronic cigarette smoke exposure in mice. Am J Physiol Lung Cell Mol Physiol (In press) |
| 33. | Morris MJ, Russell AE, Kapoor V, Cain MD, Elliott JM, West MJ, Wing LM, Chalmers JP. Increases in plasma neuropeptide Y concentrations during sympathetic activation in man. J Auton Nerv Syst 1986;17:143–149. |
| 34. | Bozinovski S, Jones J, Beavitt SJ, Cook AD, Hamilton JA, Anderson GP. Innate immune responses to LPS in mouse lung are suppressed and reversed by neutralization of GM-CSF via repression of TLR-4. Am J Physiol Lung Cell Mol Physiol 2004;286:L877–L885. |
| 35. | Coxson HO, Chan IH, Mayo JR, Hlynsky J, Nakano Y, Birmingham CL. Early emphysema in patients with anorexia nervosa. Am J Respir Crit Care Med 2004;170:748–752. |
| 36. | Coxson HO, Mayo JR, Birmingham CL. Anorexia nervosa and emphysema. Am J Respir Crit Care Med 2005;172:398–399. |
| 37. | Layman DK, Boileau RA, Erickson DJ, Painter JE, Shiue H, Sather C, Christou DD. A reduced ratio of dietary carbohydrate to protein improves body composition and blood lipid profiles during weight loss in adult women. J Nutr 2004;133:411–417. |
| 38. | Phillips BA, Danner FJ. Cigarette smoking and sleep disturbance. Arch Intern Med 1995;155:734–737. |
| 39. | Lena C, Popa D, Grailhe R, Escourrou P, Changeux JP, Adrien J. β2-Containing nicotinic receptors contribute to the organization of sleep and regulate putative micro-arousals in mice. J Neurosci 2004;24:5711–5718. |
| 40. | Morris BJ. Neuronal localisation of neuropeptide Y gene expression in rat brain. J Comp Neurol 1989;290:358–368. |
| 41. | Furness JB, Koopmans HS, Robbins HL, Clerc N, Tobin JM, Morris MJ. Effects of vagal and splanchnic section on food intake, weight, serum leptin and hypothalamic neuropeptide Y in rat. Auton Neurosci 2001;92:28–36. |
| 42. | Bi S, Robinson BM, Moran TH. Acute food deprivation and chronic food restriction differentially affect hypothalamic NPY mRNA expression. Am J Physiol Regul Integr Comp Physiol 2003;285:R1030–R1036. |
| 43. | Schwartz MW, Baskin DG, Bukowski TR, Kuijper JL, Foster D, Lasser G., Prunkard DE, Porte D Jr, Woods SC, Seeley RJ, et al. Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes 1996;45:531–535. |
| 44. | White JD, Kershaw M. Increased hypothalamic neuropeptide Y expression following food deprivation. Mol Cell Neurosci 1989;1:41–48. |
| 45. | Hansen MJ, Ball MJ, Morris MJ. Enhanced inhibitory feeding response to alpha-melanocyte stimulating hormone in the diet-induced obese rat. Brain Res 2001;892:130–137. |
| 46. | Bishop C, Parker GC, Coscina DV. Nicotine and its withdrawal alter feeding induced by paraventricular hypothalamic injections of neuropeptide Y in Sprague-Dawley rats. Psychopharmacology (Berl) 2002;162:265–272. |
| 47. | Jang MH. Nicotine administration decreases neuropeptide Y expression and increases leptin receptor expression in the hypothalamus of food-deprived rats. Brain Res 2003;964:311–315. |
| 48. | Kane JK, Parker SL, Li MD. Hypothalamic orexin-A binding sites are down-regulated by chronic nicotine treatment in the rat. Neurosci Lett 2001;298:1–4. |
| 49. | Caggiula AR, Donny EC, Epstein LH, Sved AF, Knopf S, Rose C, McAllister CG, Antelman SM, Perkins KA. The role of corticosteroids in nicotine's physiological and behavioral effects. Psychoneuroendocrinology 1998;23:143–159. |
| 50. | Rasmussen DD. Effects of chronic nicotine treatment and withdrawal on hypothalamic proopiomelanocortin gene expression and neuroendocrine regulation. Psychoneuroendocrinology 1998;23:245–259. |
| 51. | Dallman MF, la Fleur SE, Pecoraro NC, Gomez F, Houshyar H, Akana SF. Minireview: glucocorticoids–food intake, abdominal obesity, and wealthy nations in 2004. Endocrinology 2004;145:2633–2638. |
| 52. | Argyropoulos G, Harper ME. Uncoupling proteins and thermoregulation. J Appl Physiol 2002;92:2187–2198. |
| 53. | Champigny O, Ricquier D. Effects of fasting and refeeding on the level of uncoupling protein mRNA in rat brown adipose tissue: evidence for diet-induced and cold-induced responses. J Nutr 1990;120:1730–1736. |
| 54. | Samec S, Seydoux J, Dulloo AG. Role of UCP homologues in skeletal muscles and brown adipose tissue: mediators of thermogenesis or regulators of lipids as fuel substrate? FASEB J 1998;12:715–724. |
| 55. | Arai K, Kim K, Kaneko K, Iketani M, Otagiri A, Yamauchi N, Shibasaki T. Nicotine infusion alters leptin and uncoupling protein 1 mRNA expression in adipose tissues of rats. Am J Physiol Endocrinol Metab 2001;280:E867–E876. |
| 56. | Schrauwen P, Hesselink M. Uncoupling protein 3 and physical activity: the role of uncoupling protein 3 in energy metabolism revisited. Proc Nutr Soc 2003;62:635–643. |
| 57. | Boss O, Samec S, Kuhne F, Bijlenga P, Assimacopoulos-Jeannet F, Seydoux J, Giacobino JP, Muzzin P. Uncoupling protein-3 expression in rodent skeletal muscle is modulated by food intake but not by changes in environmental temperature. J Biol Chem 1998;273:5–8. |
| 58. | Fernandez-Real JM, Broch M, Vendrell J, Ricart W. Smoking, fat mass and activation of the tumor necrosis factor-alpha pathway. Int J Obes Relat Metab Disord 2003;27:1552–1556. |
| 59. | Churg A, Wang RD, Tai H, Wang X, Xie C, Wright JL. Tumor necrosis factor-alpha drives 70% of cigarette smoke-induced emphysema in the mouse. Am J Respir Crit Care Med 2004;170:492–498. |
| 60. | Lundblad LK, Thompson-Figueroa J, Leclair T, Sullivan MJ, Poynter ME, Irvin CG, Bates JH. Tumor necrosis factor-alpha overexpression in lung disease: a single cause behind a complex phenotype. Am J Respir Crit Care Med 2005;171:1363–1370. |
| 61. | Ouyang Y, Virasch N, Hao P, Aubrey MT, Mukerjee N, Bierer BE, Freed BM. Suppression of human IL-1beta, IL-2, IFN-gamma, and TNF-alpha production by cigarette smoke extracts. J Allergy Clin Immunol 2002;106:280–287. |
| 62. | Torti FM, Torti SV, Larrick JW, Ringold GM. Modulation of adipocyte differentiation by tumor necrosis factor and transforming growth factor beta. J Cell Biol 1989;108:1105–1113. |
| 63. | Bullo-Bonet M, Garcia-Lorda P, Lopez-Soriano FJ, Argiles JM, Salas-Salvado J. Tumour necrosis factor, a key role in obesity? FEBS Lett 1999;451:215–219. |
| 64. | Langhans W, Hrupka B. Interleukins and tumor necrosis factor as inhibitors of food intake. Neuropeptides 1999;33:415–424. |
| 65. | Rosenson RS, Tangney CC, Levine DM, Parker TS, Gordon BR. Elevated soluble tumor necrosis factor receptor levels in non-obese adults with the atherogenic dyslipoproteinemia. Atherosclerosis 2004;177:77–81. |
| 66. | Jansson JO, Wallenius K, Wernstedt I, Ohlsson C, Dickson SL, Wallenius V. On the site and mechanism of action of the anti-obesity effects of interleukin-6. Growth Horm IGF Res 2003;13(Suppl A):S28–S32. |
| 67. | Guerre-Millo M. Adipose tissue and adipokines: for better or worse. Diabetes Metab 2004;30:13–19. |
| 68. | Zimmermann R, Strauss JG, Haemmerle G, Schoiswohl G, Birner-Gruenberger R, Riederer M, Lass A, Neuberger G, Eisenhaber F, Hermetter A, et al. Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science 2004;306:1383–1386. |
| 69. | Langin D, Dicker A, Tavernier G, Hoffstedt J, Mairal A, Ryden M, Arner E, Sicard A, Jenkins CM, Viguerie N, et al. Adipocyte lipases and defect of lipolysis in human obesity. Diabetes 2005;54:3190–3197. |
| 70. | Kershaw EE, Hamm JK, Verhagen LAW, Peroni O, Katic M, Flier JS. Adipose triglyceride lipase: function, regulation by insulin, and comparison with adiponutrin. Diabetes 2006;55:148–157. |
| 71. | Sztalryd C, Hamilton J, Horwitz BA, Johnson P, Kraemer FB. Alterations of lipolysis and lipoprotein lipase in chronically nicotine-treated rats. Am J Physiol 1996;270:E215–E223. |
| 72. | Andersson K, Arner P. Systemic nicotine stimulates human adipose tissue lipolysis through local cholinergic and catecholaminergic receptors. Int J Obes Relat Metab Disord 2001;25:1225–1232. |
