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Pharmacological Effects of Nicotine on Norepinephrine Metabolism in Rat Brown Adipose Tissue: Relevance to Nicotinic Therapies for Smoking CessationPfizer Global Research and Development, Groton, Connecticut, USA Correspondence: Martin B. Finkelstein, Pfizer Global Research and Development, P.O. Box 8012, Eastern Point Road, Groton, CT 06340-8012, USA; e-mail:martin.b.finkelstein{at}pfizer.com.
In a two-year carcinogenicity study with administration of high doses of the partial nicotinic agonist varenicline (recently approved for smoking cessation), mediastinal hibernomas occurred in three male rats. To investigate potential mechanisms for partial and full nicotinic agonists to contribute to development of hibernomas, the effects of nicotine on rat brown adipose tissue (BAT) were studied. Male and female rats were administered nicotine at doses of 0, 0.3, and 1 mg/kg subcutaneously for fourteen days. Intrathoracic (mediastinal periaortic and mediastinal perithymic) BAT and interscapular BAT were examined microscopically, and determinations of uncoupling protein-1 (UCP-1) expression and norepinephrine (NE) content were made. Additionally, NE turnover was measured in mediastinal periaortic and perithymic BAT. Nicotine (1 mg/kg) administration resulted in decreased vacuolation only in mediastinal periaortic and mediastinal perithymic BAT of males and elevated UCP-1 in mediastinal periaortic BAT of males and females. Increased NE content occurred only in mediastinal periaortic BAT of males given 0.3 and 1 mg/kg doses, whereas NE turnover was decreased in both males and females given 1 mg/kg. Together, these data demonstrate that nicotine primarily affects mediastinal BAT in male rats, consistent with the gender and location of the hibernomas observed in the two-year carcinogenicity study.
Key Words: nicotine • nAChR • brown adipose tissue • rats • norepinephrine • hibernoma • varenicline Abbreviations: ANOVA, analysis of variance • Arbp, acidic ribosomal phosphoprotein P0 • BAT, brown adipose tissue • GAPDH, glyceraldehyde-3-phosphate dehydrogenase • HPLC, high performance liquid chromatography • IHC, immunohistochemistry • LSC, laser scanning cytometry • MHPG, 3-methoxy-4-hydroxyphenylglycol • mRNA, messenger ribonucleic acid • nAChR, nicotinic acetylcholine receptor • NE, norepinephrine • RT-PCR, reverse transcriptase polymerase chain reaction • UCP-1, uncoupling protein-1
The main function of brown adipose tissue (BAT) is to create heat via the mechanism of nonshivering thermogenesis. Norepinephrine (NE) is considered the most important factor in the regulation of BAT thermogenesis (Cannon and Nedergaard, 2004). NE stimulates the β3-adrenergic receptor on brown adipocytes leading to lipolysis and the β-oxidation of free fatty acids (Arch et al., 1984; Chaudhry and Granneman, 1999). The increase in β-oxidation generates electron donors in the BAT mitochondria, which results in the pumping of protons out of the mitochondrial matrix. In addition, NE induces the expression of uncoupling protein-1 (UCP-1), an essential component of the nonshivering thermogenic response that is uniquely expressed in BAT (Nedergaard et al., 2001). Activation of UCP-1 drives protons back into the mitochondria, and the energy released from this proton-motive force generates heat (Figure 1). At the same time, promotion of mitochondrial β-oxidation in the brown adipocytes also results in superoxide formation (Echtay et al., 2002), a major cause of intracellular oxidative damage, which may be partially countered by UCP-1.
In addition to controlling BAT thermogenesis, NE controls the proliferation of BAT via β-adrenergic pathways (Himms-Hagen, 1985; Nagase et al., 1994; Tsukazaki et al., 1995). During adaptation to cold and increased thermogenesis, BAT undergoes hyperplasia (Bukowiecki et al., 1982). NE also suppresses apoptosis in BAT (Lindquist et al., 2000). Therefore, the combination of excessive oxidative stress associated with increased proliferation and decreased apoptosis may sensitize the BAT development of hibernomas. Nicotine, in addition to its well-known psychoactive properties, has a number of peripheral effects, one of which is increased thermogenesis in rodents (Lupien and Bray, 1988). The proposed mechanism is via sympathetic nervous system stimulation leading to an increase in NE. This stimulation could be a direct effect of nicotine on nicotinic acetylcholine receptors (nAChR) modulating sympathetic stimulation or an indirect response to the acute decrease in body temperature caused by nicotine (Rezvani and Levin, 2004). Whatever the mechanism, ultimately, nicotine increases both NE turnover and binding of guanosine 5'-diphosphate (a marker of thermogenesis) to mitochondria in BAT within three hours of treatment and increases expression of UCP-1 in BAT from the rodent (Arai et al., 2001; Lupien and Bray, 1988).
The relevance of the nicotinic role in BAT metabolism has been of interest for novel nicotinic agents in smoking cessation. Varenicline is a partial nicotinic agonist with high affinity for Based on the data summarized above, we hypothesized that nicotinic agents like varenicline, at exaggerated doses, could function similarly to nicotine and increase sympathetic stimulation and NE levels. This increase leads to elevated β-oxidation, proliferation, and increased UCP-1 expression with concurrent inhibition of apoptosis in BAT, which ultimately leads to the development of hibernomas. The objective of this study was to investigate the effect of high-dose nicotine on BAT of rats in an attempt to understand the site-specific location (mediastinum) and sex predilection (males only) for the occurrence of these tumors in the two-year varenicline study in rats. The parameters measured in this study were BAT vacuolation (an indicator of lipolysis), NE content and turnover, and UCP-1 messenger ribonucleic acid (mRNA) and protein expression in BAT as markers for the potential mechanism of hibernoma development in rats.
Animals Thirty male (150–200 g) and thirty female (125–175 g) Sprague-Dawley rats (Crl:CD), aged from six to seven weeks, were obtained from Charles River Laboratories, Kingston, NY, USA. Rats were individually housed in suspended, stainless-steel cages and exposed to a twelve-hour light (6 a.m. to 6 p.m.)–twelve-hour dark cycle. They received standard commercial laboratory rodent chow (certified rodent diet 5002, PMI Feeds, Inc.) and reverse-osmosis purified drinking water from a municipal source (regulated by the Environmental Protection Agency) ad libitum throughout the study.
Treatments
Rats (three/sex/group) were designated for measurement of BAT NE content and turnover. These rats were dosed acutely with a single injection (80 mg/kg at 1 mL/kg i.p.) of DL-
Evaluations
Histopathology and UCP-1 Immunohistochemical Quantitation Serial sections of mediastinal periaortic, mediastinal perithymic, and interscapular BAT were deparaffinized and dehydrated. For light microscopic histopathology, sections were stained with hematoxylin and eosin and examined for changes in the amount of cytoplasmic vacuolation. For UCP-1 IHC, the slides were stained with rabbit antibodies to UCP-1 followed by biotinylated anti-rabbit IgG and streptavidin AlexaFluor 488. Nuclear DNA was stained with DAPI. The amount of UCP-1 protein was quantified by analyzing cellular fluorescence using Laser Scanning Cytometry (LSC, CompuCyte Corp, Cambridge, MA, USA) and a quantitation method similar to that previously described by Pruimboom-Brees et al. (2005). Briefly, slides were visually inspected for positive staining under an epifluorescence microscope (Olympus BX51, Olympus America, Inc., Melville, NY, USA) using mercury lamp illumination. Then, using the iCyte model of the LSC system, AlexaFluor488 and DNA-associated DAPI fluorescence were excited with an argon ion laser (488 nm) and a violet diode laser (400 nm); emissions were detected and measured using standard band pass 515–545 nm and long pass 460–485 nm filters, respectively. Individual adipocytes were identified by DAPI fluorescence, and adipocyte perimeter was selected at 18 pixels outside the nuclear contour to include the cytoplasmic green fluorescence (contour analysis), to generate a mean integral fluorescence value of UCP-1/cell. Fluorescence of a minimum of 20,000 adipocytes per section of hibernoma was measured at 20X magnification. Negative control slides were used to determine the level of background fluorescence and to define gating prior to analysis.
UCP-1 mRNA RT-PCR Analysis
Measurement of NE Content and Turnover
Statistical Analyses
No significant changes in body weight were observed, and the behavior and appearance of the treated rats were normal and not different from the control rats during the course of the study.
Histopathology
UCP-1 Protein Expression At 1 mg/kg, nicotine increased UCP-1 expression in the mediastinal periaortic BAT of both male and female rats. LSC analysis of interscapular and mediastinal BAT sections indicated significantly increased UCP-1 expression (p < .05) in the mediastinal periaortic but not in the mediastinal perithymic or interscapular BAT (Figure 3).
UCP-1 Gene Expression No significant changes in UCP-1 gene expression were measured in any of the three BAT areas sampled from male or female rats at either dose of nicotine, relative to endogenous reference gene expression (Figure 4).
NE Content of BAT In control animals, the NE content was significantly greater in female than in male rats for the mediastinal periaortic and mediastinal perithymic BAT groups (Figure 5). In treated animals, a significant increase in content of NE was observed only in the mediastinal periaortic BAT of male rats dosed at both 0.3 and 1 mg/kg nicotine (p < .05; Figure 5). No other statistically significant increases in BAT NE levels were observed in either males or females. Decreased NE levels (p < .05) (Figure 5) occurred in interscapular BAT from males and mediastinal periaortic BAT from females treated with nicotine 1 mg/kg.
NE Turnover in BAT Treatment of rats with nicotine resulted in a dose-related decrease in NE turnover (Figure 6). Compared with controls, NE turnover was reduced significantly in the mediastinal periaortic and mediastinal perithymic BAT of male rats administered the 1 mg/kg dose of nicotine (p < .05). Similarly, in female rats given the 1 mg/kg dose, NE turnover was reduced in mediastinal periaortic BAT (p < .05), but the decrease in the mediastinal perithymic BAT narrowly missed statistical significance. No decrease in NE turnover was observed in either sex from the 0.3 mg/kg dose group.
Nicotine and other nicotinic agonists (e.g., varenicline) stimulate release of NE, which plays a role in BAT-mediated thermogenesis. The objective of this study was to evaluate the potential pharmacological responses of different BAT depots in male and female rats administered a high dose of nicotine daily for fourteen days. The pharmacological response of BAT after nicotine treatment was assessed by NE content and turnover, the amount of cytoplasmic vacuolation (an indicator of lipolysis), and UCP-1 levels (a key protein for thermogenesis). In control animals, there was a sexual dimorphism in NE content, with females exhibiting greater NE content in both mediastinal periaortic and mediastinal perithymic BAT than males. Nicotine treatment increased NE content in BAT in a localized and gender-specific manner. Increases were observed only in the mediastinal periaortic BAT from males dosed with nicotine at 0.3 and 1 mg/kg. In contrast, NE content in interscapular BAT decreased at nicotine 1 mg/kg, consistent with previous findings (Lupien and Bray, 1988). In females, NE content was unchanged in interscapular and mediastinal perithymic BAT, but it was reduced in the mediastinal periaortic BAT at nicotine 1 mg/kg. This may be the result of a more intense and/or prolonged and gender-specific effect of nicotine in this BAT site compared with mediastinal perithymic and interscapular BAT. In control animals, sexual dimorphism was also observed in the decreased prominence of vacuolation of BAT at all sites in females as compared with males. Administration of nicotine (1 mg/kg) resulted in decreased vacuolation of BAT from all sites in males only, whereas no change in vacuolation occurred in females. In contrast, sexual dimorphism for NE turnover was not observed within the time frame of this study. Turnover was reduced in mediastinal periaortic and mediastinal perithymic BAT of males and in mediastinal periaortic BAT of females, with a trend toward decreased NE turnover in female mediastinal perithymic BAT, which narrowly missed significance. Similarly, a gender difference was not observed in UCP-1 protein levels, which were increased in the mediastinal periaortic BAT of both sexes with nicotine (1 mg/kg). The BAT changes observed in this study are consistent with nicotine-activated, NE-mediated lipolysis secondary to increased β-oxidation of the fatty acid that occurs to a greater extent in the mediastinal periaortic BAT of males. Although in normal circumstances the elevation of UCP-1 would be expected to limit superoxide production, the apparent predisposition to increased NE content in mediastinal periaortic BAT of males could potentially overcome any protective effect of increased UCP-1, which could result in mediastinal periaortic BAT of males being more susceptible to oxidative radical injury and subsequent hibernoma formation than mediastinal periaortic BAT of females. The reason for the sexual dimorphism in BAT pharmacology and hibernoma susceptibility in the rat is not fully understood. However, it is known that sexual dimorphism exists in the adrenergic control of brown adipocytes in the rat. Studies have shown that overfeeding in males causes the release of NE and subsequent activation of the β3-adrenergic receptor on brown adipocytes. In females, overfeeding results in body weight excess but a lower activation of thermogenesis (Rodriguez et al., 2001). This may be a result of a higher level of β3-adrenergic receptors in BAT of males leading to greater thermogenic capacity. In female rats, estradiol and progesterone reduce the density of the β3-adrenergic receptor and inhibit BAT thermogenesis (Malo and Puerta, 2001), which could be an explanation for the absence of hibernomas in female rats. The reason for the differences in nicotinic pharmacology in the different BAT depots is also not clear. It is possible that cholinergic innervation is present in rat mediastinal BAT but not in BAT from the interscapular, cervical, or perirenal areas in this species (Giordano et al., 2004). This difference in innervation of various BAT depots could explain the discrete localization in the present study of the nicotine-mediated increase in NE content in male rat mediastinal periaortic BAT and potential for hibernoma observed at that BAT site in the varenicline study. BAT, although present in most mammals, varies across species in its time of development, quantity, and function. In smaller mammals, such as rodents, BAT is present at birth, develops rapidly after birth, and is important in thermogenesis (Cannon and Nedergaard, 2004). In larger mammals, BAT depots are evident at birth but diminish rapidly afterward. In humans, BAT is present in the fetus, with the maximal amount (1% of body weight) present at birth (Lean and James, 1986). Unlike rodents, BAT in humans becomes devoid of mitochondria and loses its thermogenic capacity soon after birth (Sell et al., 2004). The rats in the varenicline study in which hibernomas were observed were exposed to high doses, far in excess of the therapeutic dose used for smoking cessation therapy in humans. The rare occurrence of hibernoma in rats is likely secondary to high-dose pharmacological stimulation of BAT via a nicotinic pathway and the result of a mechanism specific to this rodent species with thermogenically active brown fat. Spontaneous hibernomas are a rare occurrence in both research and clinical settings, and their etiology is not well understood. Hibernomas arising in the thoracic cavity of rats have been reported (Coleman, 1980; Stefanski et al., 1987). In humans there are only 170 case reports of hibernomas described (Furlong et al., 2001). Clinical evidence suggests hibernomas in humans are slow growing and associated with sites of persistent brown fat in adults. Hibernomas in humans were considered benign and were almost always subcutaneous in location (Furlong et al., 2001). In conclusion, these data are consistent with the hypothesis that during adrenergic-mediated thermogenesis, the mediastinal periaortic BAT of male rats is more susceptible to oxidative radical injury than in females, and chronic, high-level stimulation of NE release by excessive activation of nAChRs with nicotinic agonists in male rats may lead to development of hibernoma in the mediastinal periaortic BAT.
The authors gratefully thank Gwen Curry, Jeanne Wolfgang, Germaine Boucher, James Yan, Raoul Jamon, Kristine Hibbard, and Alane Kennedy (all of Pfizer Global Research and Development, Groton, Connecticut, USA) for their technical assistance in these studies. This study was sponsored by Pfizer Inc. Editorial support was provided by Christopher Grantham, PhD, of Envision Pharma Ltd, Horsham, UK, and was funded by Pfizer Inc.
Current address for Dominique J. Brees: Glaxo SmithKline, Ware, UK Current address for Steven B. Sands: Johnson & Johnson Pharmaceutical Research and Development, San Diego, CA, USA Current address for Michael R. Elwell: Covance Laboratories, Vienna, VA, USA
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Abstract
Introduction
4β2 nAChR (
Significant differences between male and female controls; p < .05, N = five or six rats/group for both comparisons.
