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Neuropeptide Y Gene Expression Is Increased in the Hypothalamus of Older Women

Departments of Pathology, Cell Biology and Anatomy, and Neurology (C.M.E., S.J.K., T.S.-G., N.E.R.), University of Arizona College of Medicine, Tucson, Arizona 85724; and Department of Pathology (M.L.V.), Section of Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

Address all correspondence and requests for reprints to: Naomi E. Rance, M.D., Ph.D., Department of Pathology, University of Arizona College of Medicine, 1501 North Campbell Avenue, Tucson, Arizona 85724. E-mail: nrance{at}u.arizona.edu.

	Abstract

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Neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the infundibular (arcuate) nucleus of the hypothalamus are part of a reciprocal circuit regulating reproduction and energy balance. Based on studies showing an age-related decrease in POMC mRNA, we hypothesized that NPY gene expression would increase in older women. In situ hybridization was used to compare NPY mRNA levels between young (premenopausal) and older (postmenopausal) women. We also measured NPY gene expression in intact and ovariectomized young cynomolgus monkeys. We report a significant increase (100%) in the numbers of autoradiographic grains/NPY neuron in the retrochiasmatic area and infundibular nucleus of older women. NPY mRNA was correlated with subject age and inversely proportional to the number of POMC neurons previously counted in the same subjects. In contrast, there was no difference in hypothalamic NPY mRNA in intact vs. ovariectomized monkeys. These data show that aging in women is associated with increased NPY gene expression and suggest that the functional relationship between NPY and POMC neurons demonstrated in other species also exists in the human. Our studies of intact and ovariectomized monkeys suggest that the increase in NPY mRNA in older women is due to factors other than the ovarian failure of menopause.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
AGING IS ACCOMPANIED by changes in endocrine function that have a significant impact on the quality of life (1). There are age-associated disruptions in circadian rhythms and altered regulation of the secretion of pancreatic, adrenal, thyroid, adipose tissue, and anterior pituitary hormones. In addition, gradual changes in body composition occur, characterized by an increase in fat mass, a reduction in muscle mass, and an overall gain in body weight (1). The most dramatic and consistent aging event in women, however, is the mid-life degeneration of ovarian follicles leading to the cessation of menstrual cycles (2). After menopause, a profound loss in estrogen secretion results in gonadotropin hypersecretion from the anterior pituitary gland (3). This hypoestrogenic state lasts for one third of an average woman’s life and has consequences throughout the body.

Marked changes in neuronal morphology and neuropeptide gene expression have been described in the medial basal hypothalamus of older women. Neurokinin B (NKB), substance P, and estrogen receptor mRNA-containing neurons in the infundibular nucleus become larger and express increased amounts of tachykinin gene transcripts (4, 5). GnRH gene expression also increases but in a separate subpopulation of neurons in the medial basal hypothalamus (6). Studies using pharmacological methods to estimate the amount of GnRH secretion provide additional evidence that this system is activated in older women (7). These studies show preservation of steroid negative feedback in postmenopausal women as well as an age-associated increase in GnRH secretion (7).

In contrast to the increase in NKB and GnRH gene expression, the detection of neurons expressing proopiomelanocortin (POMC) gene transcripts in the infundibular nucleus is significantly reduced in older women (8). The infundibular/arcuate nucleus of the hypothalamus is a key regulatory center for the control of reproduction and energy balance (9, 10). POMC and NPY neurons have been strongly implicated as essential components of this hypothalamic regulatory circuit (10, 11, 12). These neurons express leptin receptors and exert opposing actions on feeding behavior and energy metabolism (11, 13, 14). Furthermore, experimental paradigms that alter nutritional status and serum leptin levels produce reciprocal changes in POMC and NPY gene transcripts (9, 10, 14, 15, 16).

We hypothesized that hypothalamic NPY gene expression would be altered in older women, based on our previous study showing an age-associated decrease in the number of neurons expressing POMC mRNA (8). To test this hypothesis, we compared the gene expression of NPY neurons in the medial basal hypothalamus of the same group of subjects as our previous study. In a second experiment, we examined the effects of ovariectomy on NPY gene expression in young cynomolgus monkeys. This intact and ovariectomized cynomolgus monkey model mimics the endocrine status of pre- and postmenopausal women while controlling for age. If the changes in NPY gene expression in older women were due to the ovarian failure of menopause, we predicted that we would be able to replicate these findings by ovariectomy of young monkeys.

	Materials and Methods

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Human tissue collection

Sections were selected from serially sectioned hypothalami used in previous studies of neuropeptide gene expression in the human hypothalamus (6, 8). The hypothalami were from women who had died from sudden unexpected causes (trauma, acute myocardial infarct, cardiac arrhythmia, seizure disorder, or pulmonary embolus) with no history of estrogen replacement therapy. There was no history of drug abuse or chronic systemic illness (other than arteriosclerosis) in any of the subjects before death. These pathological specimens were collected and recorded in such a manner that subjects could not be identified, in accordance with the guidelines set forth in Federal Register 46.101 and the Human Subjects Committee at the University of Arizona. Two subjects (41 and 54 yr of age) from the original study (6) were not evaluated because of sectioning or processing artifact. The subjects in the present study consisted of young (premenopausal, age from 21–39 yr; mean = 30.7; n = 7) and older (postmenopausal, age from 59–86 yr; mean = 72.6; n = 8) women. There was no significant difference in the mean postmortem interval between the young and older groups. For further clinical information on the subjects please see Ref. 6 .

At autopsy, each brain was bisected in the midsagittal plane. Hypothalamic blocks were dissected and snap-frozen in isopentane at –30 C. Sagittal sections were serially sectioned at 20 µm in a cryostat, thaw-mounted onto gelatinized slides, and stored at –80 C until selected for this study. In situ hybridization was performed on every 20th section throughout the medial hypothalamus. After hybridization, one section from each subject was matched to Fig. 4-4 from Nauta and Haymaker (17) at a level where the infundibular nucleus has a well-defined border that is clearly delineated from the ventromedial nucleus.

Monkey tissue collection

Hypothalamic sections were selected from 18 adult female cynomolgus macaques (Macaca fascicularis) from a previous study on the effects of ovariectomy on hypothalamic gene expression (18). The monkeys (8–10 yr of age) were obtained from Primate Products Inc. (Miami, FL) and housed in the Comparative Medicine Clinical Research Center at the Wake Forest University School of Medicine. Animal treatments were carried out in compliance with state and federal laws, standards of the Department of Health and Human Services, and the guidelines of the Institutional Animal Care and Use Committee at the Wake Forest School of Medicine and the University of Arizona. After quarantine for 3 months, the monkeys were assigned to two groups: intact (INTACT, n = 9) and ovariectomized (OVX, n = 9). The OVX animals were ovariectomized under ketamine and xylazine anesthesia. The animals were housed individually and fed an ad libitum diet that was devoid of phytoestrogens. For additional information on the animal treatments see Ref. 18 .

To allow matching of an OVX monkey with INTACT animals in the midfollicular phase of the menstrual cycle, the animals were killed from 10–16 months after group assignment. The necropsies were conducted from 0900–1300 h but were not restricted to a particular season. Animals were sedated with ketamine (15 mg/kg, im), deeply anesthetized with sodium pentobarbital (35 mg/kg, iv), and perfused transcardially with cold, 0.1 M PBS (pH 7.4). The brains were rapidly removed and sliced into 1.0-cm slabs with the aid of a monkey brain matrix. The hypothalami were snap-frozen, packed in dry ice, and shipped to the University of Arizona, where they were stored at –80 C until sectioning. The tissue blocks were coronally sectioned in a cryostat (12-µm thickness), and sections were mounted on gelatinized slides and stored at –80 C until hybridization. Two sequential sections were matched to plate 840 of a monkey hypothalamic atlas (19) for in situ hybridization. At this level, the infundibular nucleus is a well-defined arcuate shape and is clearly delineated from the ventromedial nucleus.

Hybridization histochemistry

Both human and monkey sections were hybridized with a synthetic ³⁵S 48-base radiolabeled cDNA probe complementary to bases 171–218 of human NPY (20). The in situ hybridization methodology has been previously described in detail (5, 21). All slides for each species were processed within the same hybridization procedure. After stringent washes, the slides were dried, dipped in Kodak NTB-3 nuclear emulsion (diluted 1:1 with water), and stored in the dark at 4 C. After 12 d of exposure, slides were developed and counterstained with toluidine blue.

Control sections were hybridized using the same experimental protocol, either with radiolabeled NPY sense probes or in buffer without probe. These treatments yielded no labeling in both human and monkey sections. In addition, Northern analysis of human tissues with the NPY probe revealed the transcripts of the appropriate size (5).

Computer microscopy and image analysis

To prevent bias, slides were analyzed without knowledge of the experimental group. Sections were systematically scanned using an image-combining computer microscope equipped with a motorized stage, a Lucivid miniature CRT and Neurolucida software (Microbrightfield, Baltimore, MD). All labeled neurons (defined as exhibiting silver grains greater than five times background) within the slides of the medial basal hypothalamus of each subject were mapped and counted. The human medial basal hypothalamus was divided into two regions: the retrochiasmatic region (anterior third) and the infundibular nucleus (posterior two thirds).

A systematic, random-sampling design was used to select neurons for quantitative analysis of autoradiographic grains. A Lucivid attachment and StereoInvestigator Software (Microbrightfield, Colchester, VT) were used to superimpose a computer-generated sampling grid over the microscopic image. For the human sections, the grid was used to select approximately 20 labeled neurons from the retrochasmatic region and 40 neurons from the infundibular nucleus. For the monkey sections, approximately 20 labeled neurons from the infundibular nucleus were selected. This sampling scheme was designed so that the variance within subjects contributed only a minor amount to the total variance within groups (22, 23). Images of selected neurons were acquired with a Dage-MTI CCD-100 camera (Michigan City, IN) attached to a Nikon Optiphot microscope with a x60 Nikon oil immersion objective. The number of autoradiographic grains associated with each labeled neuron was quantified using Simple PCI software (Compix Inc., Cranberry Township, PA). Background grain counts were subtracted to determine the number of grains per cell. The cell perimeters were also manually digitized for subsequent calculations of cell profile area.

Statistical analysis

The autoradiographic grains per neuron, the numbers of labeled cells, and neuronal cell profile areas were calculated for individual human or monkey subjects by hypothalamic region, and these values were used to compute group means. The differences between groups were analyzed by t tests. In the human study, the Pearson product-moment correlation coefficient was calculated to determine the relationship between the indices of NPY gene expression (grains per neuron, number of labeled cells, or cell area) and subject age, postmortem interval, or the numbers of infundibular POMC neurons (8).

	Results

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Hypothalamic NPY gene expression in the infundibular nucleus of young and older women

In the human sections, the greatest number of neurons expressing NPY mRNA was located in the retrochiasmatic region and infundibular nucleus (Fig. 1). These neurons were small, round, or oval and without definite orientation (Fig. 2). NPY neurons were also localized in the pituitary stalk, anterior hypothalamic area, and suprachiasmatic nucleus (Fig. 1). Variable numbers of parvocellular and magnocellular NPY neurons were identified within the paraventricular nucleus. Small, scattered neurons expressing NPY mRNA were also identified in the bed nucleus of the stria terminalis and the posterior hypothalamus. No qualitative difference was appreciated in the distribution of neurons between the young and older women (Fig. 1).

View larger version (19K): [in this window] [in a new window]   FIG. 1. Computer-assisted maps showing the distribution of neurons expressing NPY mRNA in representative sections from a young (A) and an older (B) woman. No qualitative difference was observed in the distribution of NPY neurons between young and older subjects. The open arrows mark the approximate division between the retrochiasmatic region and the infundibular nucleus. Each symbol represents one labeled neuron. Abbreviations: ac, anterior commissure; AH, anterior hypothalamus; fx, fornix; INF, infundibular nucleus; MB, mammillary body; MPO, medial preoptic area; mt, mamillothalamic tract; oc, optic chiasm; PH, posterior hypothalamus; RCA, retrochiasmatic area.

View larger version (63K): [in this window] [in a new window]   FIG. 2. Representative photomicrographs of NPY neurons in the infundibular nucleus of young (A) and older (B) women. The grains mark the location of NPY mRNA, and the sections are counterstained with toluidine blue. There are increased numbers of autoradiographic grains in the older group without an increase in somatic area. The arrow shows an unlabeled hypertrophied neuron in the older subject. The hypertrophied neurons have previously been shown to express NKB, SP, and estrogen receptor mRNA (4 5 ). Bar, 25 µm.

View larger version (8K): [in this window] [in a new window]   FIG. 3. Number of autoradiographic grains per NPY neuron in the infundibular nucleus of young and older women (A) and INTACT and OVX cynomolgus monkeys (B). The mean number of autoradiographic grains was significantly increased in the older women, but there was no difference detected in the numbers of grains per NPY neuron between INTACT and OVX monkeys.

Hypothalamic NPY gene expression in INTACT and OVX cynomolgus monkeys

NPY neurons were largely restricted to the infundibular nucleus at the level of the hypothalamus corresponding to plate 840 of the monkey hypothalamic atlas (19) (Fig. 4). These neurons were small and round and not specifically oriented relative to the pial surface. There was no qualitative morphological difference in the distribution of hypothalamic NPY neurons between INTACT and OVX monkeys.

View larger version (13K): [in this window] [in a new window]   FIG. 4. Computer-assisted maps showing the distribution of NPY neurons in representative sections from an INTACT (A) and an OVX (B) monkey. There was no difference between groups in the distribution and number of NPY neurons in the infundibular nucleus. Each symbol represents one labeled neuron. 3V, Third ventricle; ot, optic tract; me, median eminence.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study shows a remarkable increase in the cellular levels of NPY gene expression in the medial basal hypothalamus of older women. The elevation in the number of autoradiographic grains per neuron occurred in both the retrochiasmatic area and infundibular nucleus and was strongly correlated with subject age. Comparison with our previous studies of young and older women showed that NPY gene expression was inversely correlated with the number of POMC mRNA-expressing neurons in sections of the infundibular nucleus (8). These data provide evidence that the close relationship between NPY and POMC neurons demonstrated in experimental animals may also exist in the human (10, 12, 14, 15).

NPY neurons in the infundibular/arcuate nucleus project to the median eminence, a circumventricular organ that lacks a blood-brain barrier (24, 25). These neurons respond to a wide array of circulating hormones and nutrients, including leptin, ghrelin, insulin, peptide YY, and glucose (9, 26). Arcuate NPY neurons also have extensive efferent projections to various hypothalamic regions including the paraventricular nucleus, medial preoptic area, dorsomedial hypothalamus, perifornical region, lateral hypothalamus, and the brain stem (11, 27, 28, 29). Through these interactions, NPY neurons influence numerous physiological functions including food intake, energy expenditure, reproduction, thermoregulation, and the regulation of adrenal and thyroid hormones (30, 31, 32). Thus, a change in the function of NPY neurons could have a profound impact on the physiology of older women.

In a second experiment, we compared NPY gene expression in a primate model that simulates the ovarian status of pre- and postmenopausal women while controlling for age. Interestingly, we found no effect of ovariectomy on NPY gene transcripts in young cynomolgus monkeys. These data are in agreement with a previous study showing no change in hypothalamic NPY mRNA in rhesus monkeys after orchidectomy (33). Our findings appear to be relevant to the human because a previous study showed no difference in NPY gene expression in intact vs. castrated men (34). Thus, these data do not support the hypothesis that ovarian failure is the cause of the increase in NPY gene expression in older women.

Our previous studies showed that the changes in NKB and GnRH gene expression in older women were simulated by ovariectomy of young monkeys, whereas the changes in POMC gene expression were not (18). Similarly, hormone replacement reduced NKB and GnRH gene expression in ovariectomized animals, but did not result in changes in POMC neurons (21, 35). Ovariectomy also did not alter body weight or serum leptin in young cynomolgus monkeys (18). These findings, combined with the absence of an effect of ovariectomy on NPY gene expression in cynomolgus monkeys, suggest that ovarian failure does not have a major impact on body weight regulation in this animal model. Extrapolation of these data to humans must be circumspect because of the possibility of species differences as well as potential interactions between age and hormone withdrawal. However, numerous studies have failed to show a significant impact of the menopausal transition or hormone replacement on body weight or serum leptin in women (36, 37, 38, 39, 40, 41, 42, 43), indicating that the lack of impact of ovarian failure on body weight regulation may be similar in women and cynomolgus monkeys. This impression is also supported by data from the Framingham study, which suggests that weight gain in women is associated with aging, rather than menopause per se (42, 44). Overall, these studies support the validity of the ovariectomized monkey as a model to study the repercussions of ovarian failure in women.

Increased hypothalamic NPY gene expression has also been described in human subjects with extended durations of chronic illness (34). Although the present study excluded subjects with histories of chronic systemic disease, it is possible that an increase in the level of subclinical illness contributed to the increase in NPY gene expression in our older group. Interestingly, the increase in hypothalamic NPY gene expression in older women stands in marked contrast to the decrease in NPY mRNA that has been described in association with age in rodents (45, 46). The direction of the change in aging humans suggests that this may represent a compensatory response to reduced feedback from somatic endocrine tissues. There is ample evidence in experimental animals that NPY gene expression will increase in response to diminished inhibitory feedback of circulating insulin or leptin (10, 14, 15, 47). Furthermore, the secretion of both leptin and insulin is diminished in aging women (1, 48, 49). Alternatively, the increase in NPY gene expression could represent an age-associated reduction in the ability of these hormones to suppress hypothalamic NPY mRNA (hormone resistance). For example, plasma leptin is less effective in suppressing hypothalamic NPY mRNA in aging rats (50). Either of these mechanisms (declining peripheral hormone levels or hormone resistance) could account for the increase in NPY gene expression as well as the reciprocal drop in POMC mRNA-expressing neurons observed in older women.

The finding of increased levels of NPY gene expression in the human medial basal hypothalamus adds another level of complexity to our understanding of the physiology of older women. Multiple neuropeptide systems are altered in postmenopausal women (5, 6, 8), consistent with current concepts of hypothalamic regulatory systems in which there are numerous converging afferents and redundant circuits (51, 52). Although the rise in NPY gene expression occurs in older women, the direction of this change is in concert with studies describing activation of the hypothalamus in aging humans (5, 6, 7, 46, 53, 54). Despite the decline in the secretion of many peripheral endocrine organs (1), considerable plasticity appears to remain in the aging human hypothalamus.

	Acknowledgments

 
We thank Stephanie Dance and Graciella Gutierrez for excellent technical support.

	Footnotes

 
This work was supported by NIH National Institute on Aging Grant AG-09214. C.M.E. was supported by a grant to the Undergraduate Biology Research Program from the Howard Hughes Medical Institute. T.S.-G. was a recipient of a predoctoral fellowship from the Robert S. Flinn Biomedical Research Initiative.

Abbreviations: NKB, Neurokinin B; NPY, neuropeptide Y; OVX, ovariectomized; POMC, proopiomelanocortin.

Received November 3, 2003.

Accepted February 3, 2004.

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