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Division of Neuroscience (K.L.G., H.S.S., R.S.B., J.A.K., M.S.S., E.R.S.), Oregon Regional Primate Research Center, and Departments of Physiology and Pharmacology (M.S.S., E.R.S.) and Pathology (H.S.S.), Oregon Health & Science University, Beaverton, Oregon 97006
Address all correspondence and requests for reprints to: Kevin L. Grove, Ph.D., Oregon Regional Primate Research Center, Oregon Health & Science University, 505 NW 185th Avenue, Beaverton, Oregon 97006-5384. E-mail: grovek{at}ohsu.edu
Abstract
It is well known that maternal smoking during pregnancy can lead to low birth weight and low body fat in human newborns. The purpose of this study was to determine whether chronic maternal nicotine treatment alters levels of known regulators of energy balance in the newborn offspring. Pregnant rhesus monkeys were treated with nicotine tartrate (1.5 mg/kg·d) starting on d 26 of pregnancy and maintained through d 160 of gestation. Nicotine exposure had no significant effect on absolute birth weights of the neonatal monkeys, although there was a 10% reduction in birth weights with nicotine exposure when they were normalized to maternal weight. Postnatal d 1 plasma leptin levels were significantly reduced by about 50% in the nicotine treatment group compared with saline controls, suggesting that the infant monkeys exposed to nicotine may also have lower body fat levels. In situ hybridization studies demonstrated that chronic nicotine exposure resulted in a significant decrease in arcuate NPY mRNA expression in the neonatal monkeys. In addition, there was a 2-fold increase in POMC mRNA in the arcuate nucleus in the nicotine-exposed group. These data suggest that nicotine exposure during pregnancy may increase energy expenditure in the developing fetus through actions on hypothalamic systems, resulting in lower birth weights and body fat levels.
SMOKING DURING PREGNANCY poses a serious health concern to the developing fetus, because it has been directly associated with deficits in fetal growth and development. Some of the early characterized anthropometric effects associated with smoking during pregnancy include premature delivery, low birth weights, as well as a failure to thrive syndrome during the early postnatal period (1, 2, 3). Even more disconcerting is the report that low birth weights are evident with both maternal and paternal smoking (4), indicating that second hand smoke can also significantly affect fetal growth and development. More recently, it has been shown that these low birth weights associated with the effects of maternal and paternal smoking are at least partially due to low body fat levels (4, 5). However, the underlying pathophysiological mechanism(s) causing these problems remains unknown, but probably involve the effects of nicotine on both central and peripheral systems.
It is well accepted that smoking and/or nicotine can
decrease appetite and increase energy expenditure in humans as well as
rodent models, and that cessation of smoking or nicotine
treatment results in hyperphagia accompanied by weight gain
(6, 7, 8, 9, 10). The obvious central systems that may be involved
in these changes, due to their well characterized effects on appetite
or food intake and autonomic regulation of thermogenesis, are NPY and
MSH (see reviews in Refs. 11, 12, 13, 14). The hypothalamic NPY
system is considered one of most potent orexigenic circuits in the
brain as well as being a suppressor of energy expenditure. MSH,
which is cleaved from the precursor protein POMC, works in an opposing
manner to decrease appetite and increase energy expenditure. In the
rodent both of these hypothalamic systems have been shown to be
sensitive to changes in energy availability and to respond to
peripheral signals of energy balance, such as leptin and insulin
(11, 12, 13).
Previous studies from our group have shown that, as in the rodent, NPY
and MSH are major regulators of food intake in the adult nonhuman
primate (15, 16); however, the neuronal circuitry
involved, at least for the NPY system, is much more complex than that
in rodents (17). This increased complexity is evident by
the presence of NPY neurons in other areas of the hypothalamus [such
as the paraventricular nucleus of the hypothalamus (PVH) and
supraoptic nucleus (SON)], in addition to the species-conserved
population in the arcuate nucleus (ARH) (17). The overall
hypothesis of this project is that nicotine exposure
during the prenatal period may alter the development of hypothalamic
systems involved in the regulation of body weight homeostasis,
resulting in abnormal birth weights and body fat stores at birth as
well as causing long-term abnormalities in the central control of body
weight management. The purpose of the present study was to determine
whether chronic nicotine treatment during pregnancy
results in reduced fetal growth and development in the rhesus macaque,
similar to that reported in human clinical studies. Furthermore,
changes in NPY and POMC (precursor to MSH) gene expression were
investigated as possible mechanisms underlying the abnormal birth
weights and body fat levels.
Materials and Methods
Animals
Pregnant monkeys were maintained in single animal cages in a room with a controlled lighting schedule (lights on, 0700–1900 h) and had food and water ad libitum, consisting of high protein monkey chow biscuits (Ralston Purina Co., St. Louis, MO) plus half an apple per d. All animal procedures were approved by the Oregon Regional Primate Research Center Institutional Animal Care and Use Committee.
Female and male monkeys were paired for 3 d, and females with missed menstruation cycles were submitted to an ultrasound examination on d 23–24 after mating to determine pregnancy status. The pregnant monkeys were randomly assigned to a control or a nicotine-treated group. On d 26 of gestation, pregnancy was again confirmed by ultrasound, and the animals were implanted with sc Alzet osmotic minipumps (2ML4, Alza Corp., Palo Alto, CA) containing saline or nicotine tartrate (Sigma, St. Louis, MO) in normal saline to deliver 1.5 mg/kg·d. The pumps were replaced every 3 wk so as to maintain a constant delivery of nicotine throughout the pregnancy. During replacement of osmotic minipumps on gestational d 118 and 139, amniotic fluid samples (by amniocentesis) and maternal blood samples were obtained for nicotine and hormone measurements. Near term (gestational d 160) the infant monkeys were delivered by cesarean section (considered postnatal d 0), and amniotic fluid and maternal blood samples were again obtained for nicotine and hormone measurements. Animals received cefazolin (150 mg/twice daily) for 3 d after pump insertion and/or amniocentesis.
After delivery, the infants were placed in a nursery and were fed every 2 h, according to standard nursery protocol, up until the time of death at 1300 h on postnatal d 1. On the day of death the animals were sedated at approximately 1230 h with ketamine HCl (10 mg/kg, im), and deeply anesthetized with sodium pentobarbital (>30 mg/kg, iv). The chest cavity was opened, the abdominal aorta was bisected, and the cardiovascular system was flushed with 0.9% NaCl containing 2% sodium nitrite (800–1000 ml) by intracardiac perfusion, followed by transcarotid infusion of the head with 4% paraformaldehyde in 0.1 M sodium phosphate buffer solution (pH 7.4). The brain was removed, and hypothalamic tissue blocks were cut and immersed in 4% paraformaldehyde overnight at 4 C. The tissue blocks were then immersed in 10% glycerol solution for 24 h at 4 C and in a 20% glycerol solution for an additional 48 h at 4 C. The tissue was frozen and stored at -80 C until sectioned.
RIA
Serum leptin levels were assessed using a human-specific leptin RIA kit (Diagnostics Systems Laboratories, Inc., Webster, TX). For the assay, 100 µl sample were used, and data are expressed as nanograms per ml. The sensitivity of the human leptin assay is 0.18 ng/ml, and the ED50 is 4.90 ng/ml.
Amniotic fluid cortisol levels were determined using an electrochemiluminescence immunoassay (Elecsys 2010, Roche Molecular Biochemicals, Indianapolis, IN). The assay range was 0.2–600 ng/ml.
The nicotine content in amniotic fluid was determined using gas chromatography-mass spectrometry at the Benowitz laboratory as previously described (18).
In situ hybridization
Coronal hypothalamic sections (25 µm) were collected in a 1 in 4 series using a freezing microtome. The sections were stored in cryoprotectant (30% sucrose and 30% polyethylene glycol, buffered with NaPO4, pH 7.2) at -20 C until processed for in situ hybridization. For in situ hybridization one series of sections was mounted on slides (SuperFrost Plus, Fisher Scientific, Pittsburgh, PA) in ribonuclease-free potassium phosphate buffer (pH 7.4) and dried overnight. The sections were then postfixed in 4% paraformaldehyde (pH 7.4 in 0.1 M NaPO4), rinsed in phosphate buffer, incubated with proteinase K (5 µg/ml) for 30 min at 37 C, and then treated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). The sections were washed in 2 x SSC, dehydrated through a graded series of alcohols, delipidated in chloroform, rehydrated through a second series of alcohols, and air-dried.
For detection of POMC gene expression, a monkey-specific cRNA probe for
POMC was transcribed from a 1115-bp cDNA clone in a pGEM4 vector [a
gift from R. A. Steiner (19)] with 40% of the UTP
being labeled with 35S (NEN Life Science Products, Boston, MA). For NPY gene expression a human-specific
cRNA probe was transcribed from a 500-bp cDNA clone in a pGEM4 vector
with 25% of the UTP being labeled with 35S
(NEN Life Science Products). The specific activity of the
probes ranged from 6–7 x 108 dpm/µg. A
saturating concentration of 0.3 µg/ml·kb of the probe was used,
diluted in hybridization buffer (50% formamide, 6.25% dextran
sulfate, 0.7% Ficoll, and 0.7% polyvinyl pyrolidone). The sections
were hybridized with the cRNA probes overnight (
16 h) in a moist
chamber at 55 C. After incubation the slides were washed in 4 x
SSC/ribonuclease A at 37 C and in 0.1 x SSC at 60 C. Slides were
then dehydrated through a graded series of alcohols and dried. Slides
were then dipped in Kodak NBT2 emulsion (Eastman Kodak Co., Rochester, NY) diluted 1:1 in 600 mM
ammonium acetate, placed in light-tight boxes containing desiccant, and
stored at 4 C for an appropriate period. The slides were developed and
counterstained with cresyl violet, and the distribution of silver
grains was analyzed by darkfield microscopy.
Semiquantitative image analysis
Slides were analyzed for silver grain density using Optimus Imaging software (Media Cybernetics, Silver Springs, MD). An individual brain section was captured by a CCD camera (Cohu High Performance CCD camera, San Diego, CA) and displayed on a computer monitor. The silver grain density (representing [35S]POMC or [35S]NPY cRNA labeling) was measured using a sampling box that encompassed the entire region of interest. This sampling box was held constant for all sections and animals. Measurements were taken bilaterally through the complete rostral-caudal extent of the ARH (ranging from 15–25 sections, resulting in a total of 30–50 densitometric values/brain), as determined by histological analysis of the sections. Some sections were not measured due to tears or folds in the region of interest. Background labeling was determined by placing the sampling box over the ventromedial hypothalamic nucleus, a region known not to contain NPY or POMC gene expression. Background labeling was then subtracted from densitometric values of labeling in the ARH. To best represent the relative level of cRNA labeling, the top 15 densitometric values from each brain were averaged to give the mean value for that individual animal.
Statistical analysis
Differences in body weight, crown-rump length, brain weight, adrenal weight, and pancreas weight between the means of control and treatment groups were determined by t test. P < 0.05 was considered significant.
For amniotic fluid leptin and cortisol levels, a two-way ANOVA was used to analyze gestational age x treatment effects. A post-hoc analysis was performed using Fisher’s least significant difference (LSD) test. A t test was used to compare postnatal d 1 plasma leptin and cortisol levels. P < 0.05 was considered significant.
For the in situ hybridization studies the detection of each gene (NPY and POMC) was divided into two assays due to the number of slides analyzed. Therefore, to eliminate the assay to assay variability, the data were normalized to the average value of the control animals for each individual assay. The data are represented as the mean ± SEM. Differences between the groups was tested using a t test, with significance at P < 0.05.
Results
The nicotine treatment regimen had no effect on the
progression of the pregnancy and did not cause any obvious changes in
behavior in the mothers. Amniotic fluid nicotine levels on
the day of delivery (13.8 ± 4 ng/ml amniotic fluid) were well
within physiological range of those observed in amniotic fluid of
pregnant women who are heavy smokers (20). Fetal body
weight, fetal body weight corrected for maternal weight, and crown-rump
length of nicotine-treated newborn monkeys were only
slightly decreased (3%, 10%, and 4%, respectively) compared with
control values (Table 1
). These
differences, although not statistically significantly, are consistent
with those we reported in our previous study (21) and are
consistent with the magnitude of changes seen in offspring of human
smokers (22, 23). There were also significant reductions
in adrenal and pancreas weights (P = 0.008 and 0.03,
respectively) in the nicotine-exposed infant monkeys
(Table 1).
|
). Furthermore,
postnatal (P) d 1 serum levels were also not significantly altered
(P = 0.10; Fig. 1). Maternal serum cortisol levels were
also not altered by the nicotine treatment at any time
during pregnancy (data not shown).
|
); however, there was an overall
significant treatment effect (P = 0.046). In contrast,
P1 serum leptin levels were significantly decreased by almost 50%
(P = 0.026) in the nicotine-exposed infant
monkeys (Fig. 2). Maternal leptin levels were not altered by the
nicotine treatment (data not shown).
|
). In addition, a low level of NPY mRNA
was detected in the lateral hypothalamic area of several of the infant
monkeys (not shown). However, unlike the adult rhesus macaque
(17), no NPY mRNA was detected in the PVH or SON of the
infant monkeys. Nicotine exposure resulted in a
significant 44% reduction (P < 0.001) in NPY mRNA in
the ARH of the infant monkeys (Fig. 4A).
|
|
). There was a nearly 2-fold increase
in POMC mRNA in the ARH of the nicotine-exposed infant
monkeys (Fig. 4B); however, due to variability the difference was not
significant. One of the five nicotine-exposed animals had
low (compared with the saline-treated animals) POMC gene expression,
and one of the four saline-treated animals had elevated POMC gene
expression. As these data points were 2 SD from the means
of the groups, elimination of these animals from the means resulted in
a highly significant increase in POMC mRNA in the ARH of the
nicotine-exposed infant monkeys (P =
0.004). Discussion
It is well recognized that smoking during pregnancy is associated
with lower birth weights (compared with nonexposed infants born at
similar gestational ages) and body fat levels at birth
(1, 2, 3). In the present study we observed small changes in
absolute birth weight, birth weight normalized to maternal weight, and
crown-rump length between nicotine- and saline-exposed
monkey neonates; however, the differences did not reach significance
(Table 1). Despite the lack of significance, the magnitude of the
reductions observed were similar to those reported in studies of human
infants whose mothers smoked during pregnancy (4, 5, 22, 23). The lack of statistically significant changes in the
studies reported here as opposed to the human studies probably reflects
both our small sample size (n = 6–7) compared with the large
sample size of epidemiological studies and the fact that
nicotine is only one of the toxic components of cigarette
smoke that affects human fetal development. The effects of
nicotine on birth weight and composition are probably the
result of many mechanisms, involving both peripheral and central
systems, that reduce placental nutrient uptake and/or increase energy
expenditure.
The significantly smaller adrenal and pancreas weights in the nicotine-exposed infant monkeys are suggestive of changes in cortisol and/or insulin levels, respectively. Indeed, prenatal cortisol levels in amniotic fluid, at least on G118 and G160, were significantly suppressed in the nicotine-exposed group compared with controls. As expected, there was a dramatic increase in serum cortisol levels in both groups of newborn monkeys. This stress-induced increase in cortisol at the time of birth is well characterized in many species and was not significantly different between the two groups, indicating that the responsivity of the adrenal gland was not affected by nicotine exposure. Although insulin levels were not measured in the present study, it has been shown in rodents that chronic nicotine treatment decreases insulin levels (24, 25), and there is also a decrease in insulin sensitivity associated with long-term nicotine consumption in humans (26). Changes in both cortisol and pancreatic function would probably have actions on central and peripheral management of food intake and energy balance. In addition, there would probably be long-term consequences on energy homeostasis and body weight management; however, further study is needed to fully investigate the changes in these systems in this model.
Another important peripheral system involved in the regulation of food intake and energy balance, as well as being an excellent marker of adiposity, is leptin. An abundance of human clinical studies have shown that there is a strong positive correlation between cord leptin levels and fetal fat mass and body mass index (27, 28, 29, 30). A clinical study also showed that babies born of mothers who smoked had lower cord leptin levels compared with nonexposed infants, and that the lower leptin levels correlated with lower birth weights (31). This is in contrast to adult humans, in whom chronic smoking or nicotine treatment is associated with higher leptin levels, and was hypothesized to be one of the mechanisms by which nicotine suppressed appetite (26). Therefore, the significant treatment effect that we reported on prenatal leptin levels in the amniotic fluid might reflect direct stimulatory effects of nicotine on leptin-producing cells in the placenta (32, 33), whereas the low serum leptin levels on postnatal d 1 may reflect lower body fat levels in the nicotine-exposed animals. These data suggest that although there are some differences between our results and those obtained in the human clinical studies (namely the lack of a significant difference in absolute birth weight), there are a number of similarities that make the rhesus macaque an excellent model for studying the mechanisms involved in the changes in fetal growth and development in response to prenatal nicotine exposure.
To begin to address some of the central mechanisms that may be involved in the decreased birth weights and body fat levels at birth associated with maternal smoking and nicotine exposure, the present study investigated the expression of NPY and POMC mRNA in the hypothalamus. These are two of the most characterized systems involved in the central control of food intake and energy expenditure in the rodent (11, 12, 13, 14) and rhesus macaque (15, 16). Although the reduction in growth and development in response to in utero nicotine exposure does not involve altered food intake in the developing fetus, changes in central control of energy expenditure are probably involved. In the present study NPY and POMC mRNA were detected in the ARH of the newborn monkey, with low levels of NPY mRNA present in cells in the lateral hypothalamus. However, there was no evidence of NPY mRNA expression in the PVH or SON. This is in contrast to the adult rhesus monkey, which displays NPY mRNA in both of these regions, in addition to expression in the ARH (17). The lack of expression of NPY mRNA in the PVH and SON could be due to several factors. 1) Expression of NPY mRNA in the PVH of the adult rhesus macaque was most abundant in animals that had been fasted for 48 h, with only low levels of expression in unfasted animals. Therefore, NPY mRNA in the PVH in these unfasted infant monkeys may be below detection levels. 2) NPY mRNA expression in the PVH and SON may not be activated until later stages of postnatal development. A developmental-specific expression pattern of NPY mRNA in the hypothalamus has been reported in the rat (34). Further study is needed to characterize the developmental expression of NPY in the hypothalamus of the nonhuman primate.
In the present study we also report a significant 44% reduction in NPY mRNA levels and a nearly 2-fold increase in POMC mRNA expression in the ARH of nicotine-treated animals. If these changes in NPY and POMC gene expression measured on postpartum d 1 represent the relative levels (taking into account that there are probably prenatal developmental changes) throughout the nicotine treatment (from G26 to G160), they have the potential of increasing basal energy expenditure during fetal development. The consequence of this increased energy expenditure would probably be decreased body fat at birth and subsequently lower birth weights. This possibility is supported by the relatively low birth weights (when normalized with maternal weights) and the low serum leptin levels at birth (an indicator of body fat levels) in the nicotine-exposed infant monkeys. The reduction in NPY mRNA and the increase in POMC mRNA resulting from the nicotine exposure that was observed in these studies are the opposite of the changes reported in the adult rat in response to chronic nicotine treatment (35, 36). However, changes in hypothalamic NPY and POMC mRNA in rat pups born to dams treated with nicotine throughout pregnancy have not been reported. It is unknown whether the difference in effects of chronic nicotine treatment on NPY and POMC gene expression are due to species or developmental differences in responsivity. In addition to the possible prenatal consequences, if these changes are maintained into the early postnatal period they could decrease the drive to eat and result in the failure to thrive syndrome described in the human clinical studies.
The mechanisms leading to the changes in NPY and POMC gene expression are unknown and may involve both direct and indirect signaling within the brain as well as peripheral signals. However, it seems unlikely that leptin levels, which were low, are playing a role in suppressing NPY mRNA levels, because leptin is known to inhibit NPY and stimulate POMC gene expression (13, 37, 38). Furthermore, although glucocorticoids are known to stimulate NPY gene expression (39), and amniotic fluid cortisol levels were lower during pregnancy, there was no difference between the two groups at the time of death. Therefore, it seems unlikely that glucocorticoids are responsible for the differences in NPY and POMC gene expression. There are nicotinic-cholinergic-type receptors present throughout the hypothalamus, including the ARH, although the levels are relatively low compared with those in other brain regions (40, 41). However, chronic nicotine treatment has been shown to increase nicotinic receptor expression in the brain, including the hypothalamus (42, 43). At present there are no reports of nicotinic receptors being expressed directly on NPY or POMC neurons in the ARH. Nicotine also has numerous physiological effects within the hypothalamus that could indirectly mediate the effects of nicotine on NPY and POMC gene expression, including modulation of the release of serotonin, dopamine, and norepinephrine (44, 45, 46, 47). Most notable is the serotonin system, which has been suggested to be involved in nicotine’s suppression of appetite (48, 49, 50) and is a known regulator of the NPY system (51, 52, 53).
The long-term consequences of these changes in hypothalamic feeding circuits in response to the in utero nicotine exposure remain unknown. It is well accepted that smoking during pregnancy can have long-term consequences on the stature, health, and intellectual capacity of the offspring (54, 55, 56). In addition, Vik et al. (5) demonstrated that children born to mothers who smoked during pregnancy, although having lower birth weights, actually were more obese at 5 yr of age than their unexposed counterparts. These data suggest that in utero nicotine exposure may have some long-term effects on the neuronal systems involved in body weight management.
Footnotes
This work was supported by NIH Grants HD-14643, HD-18185, HD/HL-37131, and DK-55819.
Abbreviations: ARH, Arcuate nucleus; G, gestational age (days); LSD, least significant difference; P, postnatal age (days); PVH, paraventricular nucleus of the hypothalamus; SON, supraoptic nucleus.
Received December 8, 2000.
Accepted August 6, 2001.
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