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Endocrinology Vol. 141, No. 10 3623-3629
Copyright © 2000 by The Endocrine Society

ARTICLES

Nicotine Up-Regulates Expression of Orexin and Its Receptors in Rat Brain1

J. K. Kane, S. L. Parker, S. G. Matta, Y. Fu, B. M. Sharp and M. D. Li

Department of Pharmacology, University of Tennessee College of Medicine, Memphis, Tennessee 38163

Address all correspondence and requests for reprints to: Ming D. Li, Ph.D., Department of Pharmacology, University of Tennessee, 874 Union Avenue, Memphis, Tennessee 38163. E-mail: mdli{at}utmem.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Orexins are two recently discovered neuropeptides that can stimulate food intake. As the chronic use of tobacco typically leads to a reduction in body weight, it is of interest to determine whether nicotine, the major biologically active tobacco ingredient, has an effect on orexin metabolism in the brain. Using a semiquantitative RT-PCR technique, the levels of messenger RNA (mRNA) for prepro-orexin, orexin A (OX1-R) and orexin B (OX2-R) receptors were 20–50% higher in rats receiving nicotine for 14 days at the level of 2–4 mg/kg·day compared with rats receiving saline solvent alone. In animals treated with nicotine at 4 mg/kg·day, the expression levels of mRNA for prepro-orexin, OX1-R, and OX2-R were significantly higher compared with those in either the free-feeding control or pair-fed saline control rats. RIA data indicated that both orexin A and orexin B peptide levels were significantly elevated (45–54%; P < 0.01) in the dorsomedial nucleus (DMH) of the nicotine-treated rats compared with either solvent-only or pair-fed controls. Additionally, orexin B was significantly elevated (83%; P < 0.01), over levels in both types of the control animals, in the paraventricular nucleus (PVN) region. In summary, we demonstrated that an inverse association between nicotine and food intake as well as body weight held with doses comparable to those consumed by average human smokers. Moreover, our data indicated that chronic exposure to nicotine can induce a long-term increase in the expression levels of prepro-orexin and their receptor mRNA in the rat hypothalamus and in the levels of orexin A in the DMH and orexin B in the DMH and PVN among the six hypothalamic regions that we examined.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EPIDEMIOLOGICAL studies provide evidence for an inverse association between cigarette smoking and body weight (1). On the average, smokers weigh less than nonsmokers of the same age and sex (1); furthermore, cessation of smoking is generally accompanied by weight gain (2, 3, 4, 5). Many smokers conscious of their weight use this weight-reducing effect of nicotine as a reason to smoke or abstain from quitting. About 75% of the women and 35% of the men interviewed would not tolerate a quitting-related weight gain of more than 5 lb (6). A comprehensive understanding of the mechanisms underlying the effects of nicotine on body weight may help in the development of a new treatment(s) that would decrease the reluctance to quit smoking.

Two separate actions have been proposed to explain the inverse relationship between tobacco smoking and body weight: one is reduced food consumption, and the other is increased energy expenditure (7, 8). Studies looking into this inverse association also hold true in rodents (2). Several mechanisms regarding the effect of nicotine on body weight have been proposed (9, 10). Some studies have shown that nicotine suppresses food intake (11, 12), whereas other studies have demonstrated that nicotine increases metabolic rates (13, 14). Although there has been much research in studying the role of nicotine in body weight, the specific mechanisms underlying the anorexic effects of nicotine on body weight are still largely unknown. The overall objective of our studies is elucidation of neuronal molecular mechanisms that may underlie these physiological effects.

Our understanding of the neuronal circuitry governing ingestive behavior and energy regulation has grown substantially during the past several years (15). Identification of orexigenic molecules has shed new insights into the interaction of signals that function to maintain an ideal body weight through appetite regulation. Leptin, neuropeptide Y (NPY), orexin, galanin, and MSH are examples of molecules involved in a complex network of signals that regulate food intake and energy expenditure (for reviews, see Refs. 15, 16, 17). Orexins A and B [also called hypocretins (18)] are two novel orexigenic peptides derived from a single 131-residue precursor peptide named prepro-orexin by proteolytic processing, which could be produced exclusively in the lateral hypothalamic area (LHA) (19). Recent ingestive studies have demonstrated that orexins are involved in stimulation of food intake (19), with orexin A being more effective than orexin B (20). As we have recently observed a generalized increase in NPY levels in forebrain of rats chronically treated with nicotine (21), we hypothesized that orexins could also be chronically increased in nicotine-treated animals, contributing to a desensitization of overall feeding stimuli, and hence also to the well known effect of nicotine to reduce body weight. Very recently, orexins have been implicated in the regulation of sleep cycle and rapid eye movement (REM) sleep (22). Therefore, it could be that the effect of nicotine on sleeping pattern may be mediated in part through orexin neuropeptides.

There are few reports pertaining to the effects of nicotine on these newly discovered genes participating in the regulation of feeding (11). In a recent study we found that nicotine greatly increased NPY expression at both messenger RNA (mRNA) and peptide levels (21). The complex signaling network involved in ingestive behavior would forecast involvement of many pathways that could compensate for each other, thus achieving similar physiological outputs via different neurochemical signaling pathways. In this communication, applying the treatment regimen used in our previous study (21), we indeed found that chronic nicotine administration increases orexin expression levels as well. To our knowledge, this represents the first report on the effect of nicotine on orexin expression at both RNA and peptide levels within the hypothalamus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, treatments, and tissue preparation
Male Holtzman or Sprague Dawley rats (250–350 g; Harlan Sprague Dawley, Inc., Madison, WI) were used in this study. Unless otherwise stated, male Holtzman rats were used exclusively in all experiments. Rats were housed in wire-bottomed cages at 22 C and maintained on a 12-h light, 12-h dark cycle. Standard laboratory chow and water were available ad libitum; however, available food was restricted in pair-fed animals, as explained later. Food intake was monitored daily. A fresh 5-mM solution of nicotine bitartrate (Sigma, St. Louis, MO) was prepared daily in 0.14 M NaCl-0.01 M sodium phosphate (pH 7.4). A total of 2–6 mg/kg·day of the alkaloid were administered in five equal ip doses at 2-h intervals from 0900–1700 h over 10 or 14 days (depending on experiment). All experiments were preceded by an initiation phase of 24 h, when sham injections were used to habituate the animals to the stress of the injection. Initially, animals were randomly assigned into each experimental group such that no significant difference in body weight was present among groups. At the completion of each experiment, rats were injected with a lethal dose of sodium pentobarbital (100 mg/ml, ip) and were decapitated after reaching full anesthesia. The whole brain was extirpated within 90 sec. Where needed, the whole hypothalamus was excised, frozen immediately in liquid nitrogen, and stored at -80 C before analysis. Alternatively, the brain was quickly frozen at 51 C by a flash-freezing spray (Freeze’It, Fisher Scientific, Philadelphia, PA) and stored for not more than 10 days at -80 C before slicing into 0.3-mm coronal sections using a Stoelting tissue slicer (Chicago, IL), starting at the inception of the optic chiasm, and ending in the middle of mammillary bodies, i.e. including the tissue located approximately 0.5–4.8 mm behind the position of the bregma landmark in the intact skull according to the atlas of Paxinos and Watson (23).

Data reported in this communication came from two independent experiments. Exp 1 included a group receiving saline solvent alone and groups receiving nicotine at 2, 4, or 6 mg/kg·day, respectively. Exp 2 included a saline solvent control group in which animals had unlimited access to food and water, a group treated with nicotine at 4 mg/kg·day, and a pair-fed saline control group in which animals were treated with saline solvent but had food supply limited each day to the average consumption found in the nicotine-treated group over the preceding 24 h.

RNA isolation and preparation
Only the brain tissues from male Holtzman rat experiments were used for the analysis of mRNA levels reported in this communication. Total RNA was isolated from individual frozen hypothalami by guanidine isothiocyanate extraction and sedimentation through CsCl (24). The integrity of RNA was ascertained through visualization of the ethidium bromide-stained 28S and 18S ribosomal RNA bands, and quantification was performed by measuring absorbance at 260 nm. The mRNA for the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene, which is constitutively expressed at appreciable levels in many mammalian tissues, including the brain, was used for normalization of RT-PCR values for each RNA sample as detailed below. Oligonucleotide sequences for sense and antisense rat G3PDH primers are given in TableGo 1.


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  		Table 1. Primer sequences and expected PCR product sizes (base pairs) for genes reported in this study

 
Semiquantitative RT-PCR
Optimal semiquantitative RT-PCR conditions for prepro-orexin, OX1-R, and OX2-R and G3PDH primers reported herein were determined by the strategy described previously (25). Briefly, 1.0 µg total RNA was reverse transcribed in a final volume of 20 µl containing 4 µl 5 x reverse transcriptase buffer [0.1 M Tris-HCl (pH 8.8), 0.5 M KCl, and 1% Triton X-100], 5 mM MgCl2, 10 mM dithiothreitol, 0.625 mM of each deoxy (d)-NTP, 20 U RNasin, 1 µl 50 µM random hexamers, and 200 U SuperScript II RNase H- reverse transcriptase (Life Technologies, Inc. Grand Island, NY). The RT mixtures were incubated at 42 C for 1 h and then heated at 95 C for 5 min to inactivate the reverse transcriptase. Amplification of 4 µl RT mixture (equivalent to 0.2 µg total RNA) was carried out with 5 µl 10 x PCR buffer (0.5 mM KCl and 0.1 mM Tris-HCl, pH 8.3), 4.0 µl 25 mM MgCl2, 1.0 µl 12.5 mM dNTP, 1 µCi [32P]dATP (Amersham Pharmacia Biotech, Arlington Heights, IL), 1 µl sense or antisense primers (0.1 µg/µl), and 2.5 U AmpliTaq DNA polymerase in a total volume of 50 µl. The RT-PCR reactions were initially denatured at 94 C for 3 min and then subjected to cycles of denaturation (94 C, 45 sec), annealing (60 C, 45 sec), and extension (72 C, 45 sec). The number of amplification cycles was 20 for prepro-orexin, 28 for OX1-R and OX2-R, and 25 for G3PDH. After the last cycle, the extension was continued for another 7 min at 72 C. A 15-µl sample was resolved on composite gels of 1.5% NuSieve GTG (FMC Bioproducts, Rockland, ME) and 1% agarose containing 25 µl ethidium bromide (500 µg/ml). The expression level of each gene of interest in samples was measured by scintillation counting of radioactivity incorporated into the amplified products using a liquid {gamma}-scintillation spectrometer (Beckman Coulter, Inc., Palo Alto, CA), or image-quantitative analysis of the amplified RT-PCR products. The primer sequences used for PCR amplification of prepro-orexin, OX1-R, and OX2-R genes and the expected respective PCR product sizes are shown in Table 1Go.

Extraction and measurement of orexin peptides
Slices 300 µm thick were cut as explained in the section on tissue preparation above. Each section was placed on a dish cooled to approximately -10 C, and specific brain regions were punched using a method previously described (26). Bilateral punches of a region were pooled for each rat and sonicated for 3–5 sec in 200 µl 0.05 M hydrochloric acid, followed by an extraction at 0–4 C over 60 min. After centrifugation (5 min at 6000 x g), 180 µl of each supernatant were neutralized with 18 µl 0.50 M NaOH and stored at -80 C until assayed for orexins A and B by RIA. Total protein concentration per sample was determined by the bicinchoninic acid assay, using the kit supplied by Pierce Chemical Co. (Rockford, IL). Amounts of orexins A and B were measured using the respective RIA kits purchased from Peninsula Laboratories, Inc. (Belmont, CA). The hypothalamic areas assayed for orexins A and B included the medial preoptic area, paraventricular nucleus (PVN), ventromedial nucleus, dorsomedial nucleus (DMH), arcuate nucleus, and LHA.

Statistical analysis
Data are presented as the mean ± SEM. Regression coefficient of body weight on the days of treatment was obtained using least square linear fits (S-Plus, MathSoft, Inc., Seattle, WA) for each animal followed by two-sample t test for each strain separately. Two-way ANOVA (model = strain + treatment + strain x treatment) was used to assess the effect of nicotine on body weight. In the case of positive ANOVA, the Bonferroni t test procedure was used for the post-hoc multiple comparisons. Comparisons yielding more than 95% statistical confidence (P < 0.05) were considered significantly different.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nicotine decreases body weight in a dose-dependent manner
High doses of nicotine are known to decrease body weight in rodents. However, it is not known whether this would hold at the dose levels internalized by chronic human smokers, which are typically in the range of 2–4 mg/kg·day. As indicated in Table 2Go, there were no significant differences in body weight among the four experimental groups on day 0. By day 3 of nicotine treatment, a significant reduction (P < 0.05) in body weight was detected in 6 mg/kg·day nicotine-treated group. Thereafter, highly significant reductions (P < 0.01) in body weight over that in saline rats were also found in groups receiving nicotine at 2 and 4 mg/kg·day. Thus, nicotine treatment reduced body weight in a dose-dependent manner. Relative to saline-solvent controls or groups receiving 2–4 mg/kg·day nicotine, increased incidence of adverse behavioral effects (i.e. repetitive front paw movements and enhanced locomotion) was found in the group receiving nicotine at 6 mg/kg·day. This nicotine dose thus appears to be too high to model the effects of nicotine on body weight and food consumption in humans. Given the adverse behavioral effects observed in the 6 mg/kg·day nicotine group, the dose of 4 mg/kg·day was employed in all subsequent studies reported herein.


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  		Table 2. Mean and SE of the body weight among nicotine and saline control groups after 3, 7, or 10 days of treatment

 
Effects of nicotine on body weight and food intake in two rat strains
To determine whether the inverse association between the level of nicotine treatment and body weight is dependent on the rat strain used, a second strain of rats, Sprague Dawley, was included in our study. Also, we determined the importance of the level of food intake in the inverse association between nicotine treatment and body weight. Two-sample t tests that were performed separately on the regression coefficient of each individual for each strain (Holtzman and Sprague Dawley) indicated that the nicotine administration results in a significant reduction in body weight (Holtzman: t = 4.21; P = 0.0003; Sprague Dawley: t = 8.91; P < 10-5). Furthermore, the two-way ANOVA tests demonstrated that both rat strains respond negatively, but consistently, to the nicotine administration (F = 68.04; P = 10-5), and no interactions between the nicotine treatment and rat strain were detected (F = 1.17; P = 0.284).

The overall average daily food consumption for Holtzman rats was 28.7 ± 0.7 g for saline controls and 24.5 ± 0.7 g for nicotine-treated animals, whereas for Sprague Dawley rats it was 24.1 ± 0.3 g for controls and 19.3 ± 0.5 g for treated animals. The average food consumption by the nicotine-treated rats was thus 14.6% and 20.0% lower than that in the respective controls in Holtzman and Sprague Dawley rats, respectively (Fig. 1BGo).



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  		Figure 1. Body weight differences between saline- and nicotine-treated (4 mg/kg·day) Holtzman and Sprague Dawley rats (A). Rats received five injections of saline alone or 4 mg/kg·day nicotine. Values are expressed as the percent difference relative to day 0 (representing the first day of nicotine treatment). Daily food intake was determined throughout the 10 days of treatment (B). At least eight rats were included in each treatment group.

 
Nicotine dose dependently enhances orexin mRNA levels in hypothalamus
Given the physiological effect of orexins in the regulation of ingestive behavior (18, 19, 20), it was of interest to examine whether the level of mRNA for the precursor of these peptides is changed with nicotine treatment. Expression of prepro-orexin mRNA in nicotine-treated and solvent-control rats was measured by semiquantitative RT-PCR with gene-specific primers (see Table 1Go for primer sequences). The groups receiving nicotine at 2, 4, and 6 mg/kg·day showed respective 14% (P < 0.10), 47% (P < 0.01), and 31% (P < 0.05) increases in orexin mRNA expression levels compared with that in saline rats. The highest expression level of prepro-orexin mRNA was observed at the nicotine dose of 4 mg/kg·day, and then it tapered off at the highest dose of 6 mg/kg·day (Fig. 2Go). These findings indicated that the dose of 6 mg/kg·day was too high, which also agreed with our behavioral observations.



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  		Figure 2. Effects of nicotine on prepro-orexin mRNA expression levels in the median basal hypothalamus (MBH). Holtzman rats received 2, 4, or 6 mg/kg·day nicotine by ip injection for 10 days. Total RNA was isolated from the MBH region as described in Materials and Methods. Prepro-orexin and G3PDH mRNA expression levels were measured by semiquantitative RT-PCR with gene-specific primers. PCR products were separated by electrophoresis on agarose gels (A). B, The mean hypothalamic orexin mRNA (±SEM) levels relative to that in the saline control group at three doses of nicotine. Amplification cycles were 22 for orexin and 25 for G3PDH. The amounts of complementary DNA amplified from G3PDH mRNA were used to normalize the orexin results. In this and other figures, the nicotine groups designated b or c are significantly different (P < 0.05 or <0.01) from each other or the saline control (a).

 
Effects of nicotine on OX1-R and OX2-R mRNA levels in hypothalamus
In view of the observed increase in prepro-orexin mRNA in the hypothalamus of nicotine-treated rats (see the preceding section), it was also of interest to determine the expression levels for orexin receptor mRNA in our animal model. We measured OX1-R and OX2-R mRNA levels using semiquantitative RT-PCR with orexin receptor-specific primers (see Table 1Go for primer sequences). Surprisingly, enhanced expression patterns for OX1-R and OX2-R mRNA levels by nicotine differed between these two orexin receptors across the doses of nicotine that we examined (Fig. 3Go). Expression level of OX1-R mRNA was approximately 19% (P< 0.01) and 15% (P < 0.05) higher in the 2 and 4 mg/kg·day nicotine groups, respectively, than in the solvent controls. On the other hand, almost no changes in OX1-R mRNA level were detected in the 6 mg/kg·day group. With OX2-R, a significant increase in mRNA expression level (22%; P < 0.01) was obtained at the dose of 4 mg/kg·day, but no significant differences were detected for the 2 and 6 mg/kg·day groups compared with saline controls. These data suggest that the expression of OX1-R and OX2-R has different sensitivity to nicotine stimulation, and the enhanced expression pattern of OX2-R mRNA by nicotine is more similar to that for prepro-orexin mRNA.



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  		Figure 3. Effects of nicotine on OX1-R and OX2-R mRNA expression levels in the median basal hypothalamus. The mean hypothalamic OX1-R (A) and OX2-R (B) mRNA levels relative to that in the saline control group at nicotine doses of 2, 4, and 6 mg/kg·day, respectively.

 
Increased expression levels of prepro-orexin, OX1-R, and OX2-R mRNA are due to the effects of nicotine
To determine whether the changes in prepro-orexin, OX1-R, and OX2-R mRNA could be linked to a level of nutrition independent of nicotine administration, another experiment was initiated. This experiment consisted of three groups: saline solvent alone, nicotine at 4 mg/kg·day, and pair-fed rats. Using the semiquantitative RT-PCR conditions employed above, we detected significantly higher expression levels of prepro-orexin (52%; P < 0.01), OX1-R (43%; P < 0.01), and OX2-R (54%; P < 0.01) in the nicotine-treated group compared with those in both saline and pair-fed control groups. No significant differences were detected between the solvent-only and pair-fed groups for any of the genes of interest (Fig. 4Go). This indicated that the increases in prepro-orexin, OX1-R, and OX2-R mRNA levels were due to the effects of nicotine per se, not to other factors, such as differences in body weight and food intake among these animals.



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  		Figure 4. Increases in orexin, OX1-R, and OX2-R mRNA levels are due to nicotine and not to other factors, such as the decrease in body weight or food intake. Orexin, OX1-R, and OX2-R mRNA expression levels in the medial basal hypothalamus of rats receiving saline alone or nicotine (4 mg/kg·day) or in rats pair-fed with the nicotine group for 14 days were determined by semiquantitative RT-PCR. G3PDH mRNA expression levels were used to normalize the results. The mean levels (±SEM) in each group were expressed as a percentage of the saline control value.

 
Nicotine increases orexin A level in DMH and orexin B level in DMH and PVN regions
RIAs of orexins A and B were performed separately to determine whether enhanced prepro-orexin mRNA expression by nicotine is associated with an increase in orexin peptide levels and to further determine which orexin peptide is affected by nicotine in which hypothalamic area. Both orexin A and orexin B concentrations were measured by RIA in six hypothalamic areas, including LHA, medial preoptic area, ventromedial nucleus, DMH, PVN, and arcuate nucleus. As shown in Fig. 5Go, a significant increase (45%; P < 0.05) in the orexin A peptide level was found only in the DMH region among the six hypothalamic regions examined. With orexin B peptide, significantly higher concentrations were detected in the PVN (83%; P < 0.01) and DMH (54%; P < 0.01) regions (see Fig. 6Go). No differences were detected in the other four hypothalamic regions over values in the saline or pair-fed groups.



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  		Figure 5. Effects of 10 days of nicotine treatment (4 mg/kg·day, ip) on orexin A levels in six microdissected hypothalamic regions compared with those in saline and pair-fed controls. A significantly higher level (P < 0.05) of orexin A was detected only in the DMH region among the six regions examined. Orexin A concentrations were determined by RIA, as described in Materials and Methods. Values are the mean ± SEM. *, P < 0.05.

 


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  		Figure 6. Effects of 10 days of nicotine treatment (4 mg/kg·day, ip) on orexin B levels in six microdissected hypothalamic regions compared with those in saline and pair-fed controls. A significantly higher level (P < 0.05) of orexin B was detected only in the DMH and PVN regions among the six regions examined. Orexin B concentrations were determined by RIA kit, as described in Materials and Methods. Values are the mean ± SEM. *, P < 0.05.

 
Distribution and levels of orexin A and orexin B within the hypothalamus
Relative neuropeptide levels of orexin A and orexin B are summarized as ratios of these two peptides in Table 3Go. The measured orexin A levels indicated a greater abundance of this peptide over orexin B in five of the six hypothalamic nuclei studied. In the LHA, however, the concentration of orexin B appeared to be slightly higher than that of orexin A. These data suggest that orexins A and B are probably equally translated from mRNA and processed in the LHA region. However, orexin A is preferentially detected in projections from the LHA to the other nuclei in the hypothalamus, possibly also in connection with a lower degradation of orexin A relative to orexin B (27).


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  		Table 3. Ratios of orexin-A to orexin-B peptides in six hypothalamic regions

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Consistent with other reports in rodents (28), we demonstrated that an inverse association between nicotine and food intake as well as body weight held with doses comparable to those consumed by average human smokers. In addition, this inverse association was observed for two rat strains that we examined, Holtzman and Sprague Dawley, indicating that the effects of nicotine on food intake and body weight are independent of the rat strains used in the study. The food intake data are in agreement with the results of Grunberg et al. (29) and Frankish et al. (11), but not with some earlier studies (13, 30). This could be due to differences in the administration and doses of nicotine used in each study (31).

The primary objective of our studies was to understand the mechanism underlying the inverse association between nicotine and body weight, using an animal model that mimics as closely as possible the intake of nicotine found in humans. Administration of nicotine in this study (at 2-h intervals between 0900–1700 h) was intended to mimic the multiple discontinuous intakes of nicotine that smokers have during the day, producing a wave-like plasma nicotine level over the day while leaving a 12-h interval when plasma nicotine concentrations were allowed to drop completely. A discontinuous treatment schedule was found to be necessary in another study that indicated a significant activity of nicotine in neuropeptide release (32). In an attempt to approximate the nicotine intake in average human smokers, the dose of nicotine used in most experiments reported in this study was 4 mg/kg·day, which we have shown to increase NPY levels in several hypothalamic areas (21). This dose is about 3 times lower than that used in another study on the effect of nicotine upon brain NPY levels in the rat (11). This regimen served to induce a significant decrease in food intake and body weight despite the fact that rats are mainly night-feeding animals.

Prepro-orexin mRNA is produced in the lateral hypothalamus (19), and the processed orexin peptides are released in many important feeding hypothalamic regions of the brain, where they are involved in ingestive behaviors (19, 20, 33, 34). To determine whether the effects of nicotine on food intake and body weight can be explained at least partly by changing the orexin expression levels in this orexigenic pathway, a series of studies was conducted in our laboratory. Our data indicated that nicotine enhances prepro-orexin mRNA expression in a dose-dependent manner, with an apex at 4 mg/kg·day and subsequent dampening of mRNA levels at the higher dose of 6 mg/kg·day found in the hypothalamus. Furthermore, our data indicated that the increase in orexin mRNA level was due to the effects of nicotine per se and not to indirect effects related to a decrease in food intake and/or body weight. This was derived from a comparison of the orexin mRNA expression levels between the nicotine-treated and pair-fed saline control rats, with no significant differences in food intake or body weight detected between the two groups. In another study using the same model, we have shown the effects of nicotine on the mRNA expression of the neuropeptide Y gene to have a similar positive inducement (21).

An increase in prepro-orexin mRNA in nicotine-treated animals would be expected to result in an increase in orexin A and/or orexin B in either the LHA (the main area of their synthesis) and/or locations receiving the projections of orexin-producing neurons. Furthermore, as orexins A and B are translated from mRNA encoding their common precursor, it is also interesting to determine whether there is a preferential accumulation of either peptide resulting from this enhanced prepro-orexin mRNA expression by nicotine. Our RIA data indicate that the orexin A level is significantly higher in the DMH upon nicotine treatment compared with those in saline control and pair-fed saline control groups. Significantly higher levels of orexin B have also been found in the DMH and PVN regions of nicotine-treated rats. Both PVN and DMH regions are reported to be the action sites of orexin A, which are involved in the control of energy homeostasis (20). The nicotine treatment thus leads to increased levels of the processed orexin A neuropeptides in orexin-sensitive hypothalamic regions receiving projections from the LHA. This provides strong evidence implicating that the orexin A pathway may represent one of the targets of the effect of nicotine on the decrease in food intake.

A decrease in food intake by nicotine treatment would be expected to lead to a decrease in orexigenic signaling. However, our data demonstrate clearly that nicotine enhances orexin mRNA expression in the hypothalamus and the levels of orexin A in the DMH and orexin B in the PVN and DMH regions. There are many plausible explanations to explain this unexpected, but significant, finding. One explanation for this paradigm is that increases in prepro-orexin, OX1-R, and OX2-R mRNA and orexin A and B peptide levels are the result of nicotine positive feedback from lack of signaling at the orexin receptor levels, suggesting that nicotine may have an indirect effect on orexin signaling downstream from its transcription, translation, and secretion. This decrease in functional orexin receptors may result in increased levels of orexin peptides through a decreased sequestration and/or internalization. Multiple feedback signals may then be activated to compensate for the loss of orexin signaling, resulting in the increased transcription of prepro-orexin, OX1-R, and OX2-R as well as an increased translation and processing of these proteins. Alternatively, although it is very unlikely, it is possible that the elevated expression of orexin mRNA and peptides by nicotine may not be related to the reduced food intake and body weight observed in nicotine-treated animals.

Orexins A and B are produced in the LHA and released in many regions of the brain, which lends support to their many interactions physiologically (35, 36). Other than feeding, orexins have been demonstrated to be involved in the sleep cycle (37, 38) through increasing firing in the locus coeruleus and decreasing REM sleep (22). It has been reported that nicotine affects the sleep cycle and REM sleep (39, 40), but the mechanism underlying this pharmacological effect remains to be characterized. It is therefore likely that the effect of nicotine on sleep patterns may be mediated through its effect on orexin production. As indicated above, orexins A and B may be produced at equal levels in the LHA, and it is also known that orexin A is preferentially projected to the other nuclei of the hypothalamus; thus, we suspect that orexin B is more likely to be involved in the regulation of sleep. Closer examination of the relevant areas of the brain using a model system that better mimics the sleep-wake cycle is needed to answer this question more thoroughly.

In summary, we demonstrated that there exists an inverse association between body weight and the low doses of nicotine that are comparable to those experienced by average human smokers. Also, our data demonstrated for the first time that nicotine has a significant effect on prepro-orexin mRNA production and distribution within the hypothalamus of the brain. Taking into account other reports showing that orexin significantly influences ingestive behavior within the LHA, DMH, and PVN nucleus (20, 36), and our finding that nicotine affects the expression of orexin within these regions, we propose that orexins could be involved in mediation of the neuromolecular effects of nicotine on the observed decreases in food intake and body weight. Although an increase in the levels of orexigenic molecules is somewhat counterintuitive in explaining a decrease in food intake, we suspect that the chronically higher levels of orexins found in the nicotine-treated animals may result from decreased signaling at the orexin receptors, which remains to be characterized in future studies.


   Acknowledgments
 
The authors thank Dr. Ozlen Konu for data analysis.


   Footnotes
 
1 This work was supported in part by the American Heart Association, Southeast Affiliate (to M.D.L.), and NIH Grant DA-03977 (to B.M.S.). Back

Received March 2, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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