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Endocrinology Vol. 143, No. 3 954-963
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Testosterone (T)-Induced Changes in Arcuate Nucleus Cocaine-Amphetamine-Regulated Transcript and NPY mRNA Are Attenuated in Old Compared to Young Male Brown Norway Rats: Contribution of T to Age-Related Changes in Cocaine-Amphetamine-Regulated Transcript and NPY Gene Expression

Elliott H. Sohn, Tami Wolden-Hanson and Alvin M. Matsumoto

Geriatric Research, Education, and Clinical Center, Veterans Administration Puget Sound Health Care System (E.H.S., T.W.-H., A.M.M.), and Division of Gerontology and Geriatric Medicine, Department of Medicine (E.H.S., T.W.-H., A.M.M.), and Population Center for Research in Reproduction (A.M.M.), University of Washington School of Medicine, Seattle, Washington 98108-1597

Address all correspondence and requests for reprints to: Alvin M. Matsumoto, M.D., Veterans Affairs Puget Sound Health Care System (S-182-GRECC), 1660 South Columbian Way, Seattle, Washington 98108-1597. E-mail: . alvin.matsumoto{at}med.va.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The age-related decrease in serum T levels is associated with impairments in food intake and weight regulation and alterations in brain peptides that regulate energy balance. To test the hypothesis that reduced T levels contribute to altered hypothalamic cocaine-amphetamine-regulated transcript (CART) and NPY gene expression, the mRNA content of these neuropeptides was measured by in situ hybridization in sham-operated (intact), castrated, and T-replaced castrated young and old male Brown Norway rats. T levels in T-replaced young and old rats were similar to those in intact young animals. Compared with castrated rats, arcuate nucleus CART mRNA was lower and NPY mRNA was higher in both young and old T-replaced castrated animals, suggesting reciprocal regulation of these peptides by T; these T-induced changes were localized primarily in the rostral arcuate and were markedly attenuated in old animals. Compared with intact animals, paraventricular nucleus CART mRNA was lower in castrated animals and similar in T-replaced young and old rats. We conclude that hypothalamic CART and NPY neurons remain responsive to T regulation in old rats, albeit less so than in young animals, suggesting that the age-related reduction of T contributes in part to altered brain neuropeptide gene expression favoring anorexia and wasting with aging.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
COCAINE-AMPHETAMINE REGULATED TRANSCRIPT (CART) and NPY are two highly abundant peptides that are widely distributed throughout the rat brain and found in substantial concentrations in the hypothalamus (1, 2, 3). CART was identified by differential display techniques as an mRNA transcript whose levels were elevated in rat striatum after cocaine or amphetamine administration (2). The CART peptide is novel, with no similarities to any known peptide, and its receptor has remained elusive (2, 4, 5). The distribution patterns of CART mRNA and peptide throughout the central and peripheral nervous systems suggest a role in reward and reinforcement, feeding, sensory processing, stress, and endocrine control (3, 5, 6). CART peptide and mRNA have been localized to several hypothalamic areas implicated in the central control of feeding behavior, including arcuate nucleus (ARC), paraventricular nucleus (PVN), dorsomedial and lateral hypothalamus, as well as in locus coeruleus, nucleus of the solitary tract, and parabrachial nuclei of the brain stem (2, 7, 8, 9). Afferent projections of these CART neurons have yet to be elucidated. CART is colocalized with other neuropeptides in most of these areas (8, 9). CART neurons are leptin sensitive; fasting decreases CART mRNA levels, and leptin administration induces Fos expression in CART neurons in some hypothalamic nuclei involved in the regulation of energy balance (4, 10, 11).

Hypothalamic NPY neurons are primarily located in the arcuate nucleus and project to a number of locations, including the paraventricular nucleus (12), the medial preoptic area, the lateral hypothalamus (13), and the median eminence (14). Among its neuromodulatory functions, NPY is a potent stimulator of food intake, modulator of hypothalamic GnRH secretion from the hypothalamus (15, 16, 17), and regulator of central autonomic functions (18). NPY neurons are leptin sensitive; fasting increases NPY mRNA levels, and leptin administration results in decreased NPY mRNA levels (10, 13, 19).

Central administration of CART peptide potently suppresses feeding (9, 20, 21, 22) and blocks the increase in food intake induced by NPY (4, 22, 23, 24). CART neurons are adjacent to but not colocalized with NPY neurons within the ARC (8, 22), and NPY-positive nerve endings are closely apposed to CART-positive cell bodies in the paraventricular nucleus (23, 25). The ability of CART to block NPY-induced feeding suggests that the actions of these neuropeptides are interrelated. Taken together, these findings strongly suggest functional interactions between NPY and CART in the control of food intake.

The regulation of food intake and body weight is impaired in the aging male Brown Norway rat (19). ARC NPY gene expression decreases with age and orchidectomy in male Brown Norway rats (26, 27). The age-related decrease in NPY gene expression is associated with impaired food intake and ability to regain body weight after a 72-h fast (28). Altered NPY gene expression may be related to serum T levels, which decline with aging in male Brown Norway rats as well as in humans (29, 30, 31). In young Brown Norway rats, orchidectomy decreases, and T replacement restores, ARC NPY mRNA content to that of intact young rats (32). In middle-aged Sprague Dawley rats, orchidectomy decreases, and T replacement increases, NPY peptide content in hypothalamic nuclei, including the ARC (33). The contribution of reduced T levels to the age-related decrease in NPY gene expression observed in old animals has not been investigated, nor has CART gene expression been investigated in the context of aging.

We hypothesize that age-related decreases in circulating T levels contribute to alterations in CART and NPY gene expression with aging. Given the contrast in feeding behavior induced by CART and NPY, we suspected a reciprocal relationship between these peptides with regard to aging and circulating levels of T. To test these hypotheses, we compared prepro-CART (ppCART) and prepro-NPY (ppNPY) mRNA contents, assessed by in situ hybridization histochemistry, within the ARC of intact (sham-operated), orchidectomized, and exogenously T-replaced orchidectomized young and old male Brown Norway rats. In addition, we considered the effects of aging, castration, and T replacement on CART and NPY gene expression in subpopulations of neurons by quantifying ppCART and ppNPY mRNA contents in rostral to caudal regions of the ARC as well as ppCART mRNA in the PVN.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental design
Adult male, inbred, specific-pathogen-free Brown Norway BN/Bi (BN) rats were purchased (Harlan Sprague Dawley, Inc., Indianapolis, IN) and housed in an American Association for Acceditation of Laboratory Animal Care-accredited facility at the V.A. Puget Sound Health Care System (Seattle, WA). Animals were individually housed in polycarbonate rat cages containing corncob bedding in a light- and temperature-controlled room on a 12-h light, 12-h dark cycle (lights off from 1800–0600 h). Animals had ad libitum access to Purina rodent chow (5001, Ralston Purina Co., St. Louis, MO) and tap water. Body weights were recorded twice weekly. Animals were allowed a 4-wk period of acclimatization before being used for these studies. All animal experiments were conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals, and were approved by the V.A. institutional animal care and use committee.

Experimental design
Young (4 months of age; 5–6/group) and old rats (23 months of age; 5–7/group) were randomly assigned to receive one of three aseptic surgeries under pentobarbital anesthesia (60 mg/kg body weight): bilateral orchidectomy with sc implantation of an empty (castrate) SILASTIC brand capsule (Dow Corning Corp., Midland, MI), bilateral orchidectomy with sc implantation of a SILASTIC brand capsule containing physiological levels of T (castrate + T), or sham operation consisting of 1- to 2-cm abdominal incision with sc implantation of an empty SILASTIC brand capsule (intact). SILASTIC capsules were made using medical grade SILASTIC brand tubing (Dow Corning Corp., no. 602-285; id, 0.062 in.; od, 0.125 in.), filled with crystalline T, plugged with silicone adhesive (Dow Corning Corp., no. 891), and incubated overnight in sterile saline at 4 C before implantation. Capsules were 30 mm in length for young rats and 40 mm in length for old rats to achieve similar levels of serum T in animals of different body size. Animals were killed by decapitation 21 d after surgery. Brains were quickly removed, frozen on dry ice, and stored intact at -70 C for sectioning later. Trunk blood was collected, and serum was separated for assay of leptin, insulin, and T. Visceral fat pads (epididymal, perirenal, and retroperitoneal) were dissected and weighed for assessment of changes in visceral body fat content.

Hormone assays
Serum was stored at -30 C until hormone assays were performed in duplicate. All samples were analyzed together for each assay. Leptin and insulin levels were determined by double antibody RIA kits (rat leptin, RL-83K; rat insulin, RI-13K, Linco Research, Inc., St. Louis, MO). The sensitivity of the leptin assay was 0.5 ng/ml, and intraassay variability was 7.2%. The limit of sensitivity of the rat insulin assay was 0.1 ng/ml, and intraassay variability was 3%. T was measured by fluoroimmunoassay (Delfia A050-101, Wallac, Inc., Turku, Finland). Assay sensitivity was 0.1 ng/ml, and intraassay variability was 3.1%.

Tissue processing
Frozen brains were blocked and mounted onto a cryostat chuck with OTC mounting compound (Miles Scientific, Naperville, IL). Serial 20-µm coronal sections (1:3 series) were obtained throughout the entire hypothalamus of each brain using a Reichert-Jung 2800 Frigocut cryostat (Vienna, Austria). Sections were thaw-mounted onto gelatin-subbed ribonuclease-free microscope slides (SuperFrost Plus, Fisher Scientific, Pittsburgh, PA) and stored at -70 C in sealed slide boxes until in situ hybridization histochemistry was performed. Thawed tissue sections were fixed in 4% paraformaldehyde (pH 7.4; 4 C) for 5 min, washed in PBS (2 min; 4 C), rinsed in 0.1 M triethanolamine buffer (pH 8.0), and treated with 0.25% acetic anhydride in triethanolamine buffer (10 min). Tissue was rinsed in 2x SSC, dehydrated through serial ethanol solutions, delipidated (10 min) in chloroform, passed through a second series of ethanol rinses, and air-dried.

CART in situ hybridization
DNA complimentary to nucleotides 20–409 of rat ppCART mRNA (accession no. U10071) was synthesized by PCR amplification (gift from T. M. Hahn and M. W. Schwartz, University of Washington, Seattle, WA) and used for riboprobe preparation. Purified linearized DNA (0.5 µg/µl) was transcribed (Riboprobe system T-7, P1440, Promega Corp., Madison, WI), and probe was 3'-end labeled with [35S]dUTP (NEN Life Science Products, Boston, MA). Labeled probe mixture (including yeast tRNA, Tris-EDTA-dithiothreitol (TED), and 50 mM dithiothreitol) was diluted in hybridization buffer to a concentration of 0.4 pmol/ml and applied to each slide. Slides were coverslipped and incubated overnight at 65 C. Coverslips were removed in 1x SSC; slides were loaded into slide racks and washed in 1x SSC for 30 min at room temperature, followed by a 30-min wash in ribonuclease buffer at 37 C and a further 30-min wash in 1x SSC at room temperature. Three 20-min stringent washes (0.1x SSC at 70 C) were followed by a final 30-min wash in 0.1x SSC at room temperature. Sections were dehydrated through a series of ethanol solutions and air-dried. When dried, slides were apposed to Hyperfilm-ßMax film for 26 h, and films were processed using Kodak Developer D19 and Rapid Fix (Eastman Kodak Co., Rochester, NY). CART mRNA levels were quantitated throughout the PVN and ARC of each brain using film autoradiography digitized with an image analysis system (MCID, Imaging Research, Inc., St. Catharines, Canada). Image analysis was performed by one operator, who was blinded to the age and condition of the subjects (T.W.-H. for CART and E.H.S. for NPY). For each brain section showing signal, hybridization area (square millimeters) was quantified by establishing a threshold value and determining the suprathreshold area (corrected for background labeling) with a predetermined template encompassing only the area of interest. The average OD and hybridization area for each section were determined by the image analysis software, and the product of the OD and hybridization area (expressed as hybridization units) was used as an index of the total amount of hybridization in the arcuate nucleus (28, 34). The patterns of CART gene expression in this study are consistent with those of previous studies demonstrating the distribution of CART mRNA in the hypothalamus (7, 9, 23, 25).

NPY in situ hybridization
A 36-base oligodeoxynucleotide probe complementary to the Asp40-Ser51 portion of rat ppNPY cDNA (35) was synthesized by the Molecular Biology Core Facility at the V.A. Puget Sound Health Care System. Probe was purified, 3'-end labeled with [35S]dATP, purified, and reconstituted in TED buffer, as described previously (36). Labeled probe mixture (including yeast tRNA and TED buffer) was diluted in hybridization buffer to a saturating concentration (26) and applied to each slide. Slides were coverslipped and incubated overnight at 30 C in moist chambers. Coverslips were removed in 1x SSC, and slides were passed through successive 1x SSC washes at 60 C, followed by two washes in 1x SSC at room temperature. Tissue sections were dehydrated through a series of ethanol solutions containing ammonium acetate and air-dried. When dried, slides were apposed to Hyperfilm-ßMax film for 72 h, and films were processed using Kodak Developer D19 and Rapid Fix. Levels of ppNPY mRNA were quantitated over the entire ARC of each rat brain using film autoradiography as described above.

Quantification of mRNA in hypothalamic regions
To quantify ppCART and ppNPY mRNA levels within regions of the ARC, coronal sections through the ARC of each rat were anatomically matched using the rat brain atlas of Paxinos and Watson as a guide (37). The ARC was divided into four regions of approximately equal length in a rostral to caudal direction, corresponding to regions defined previously (26, 38). The boundaries of region 1 (ARC I) were defined by the retrochiasmatic area rostrally (1.80 mm caudal to bregma) and elongation of the third ventricle caudally (2.3 mm caudal to bregma). Region 2 (ARC II) began 2.3 mm caudal to bregma and continued caudally to the rostral-most extent of the dorsomedial nucleus (DMN; 2.80 mm caudal to bregma). Region 3 (ARC III) contained the DMN (2.80–3.3 mm caudal to bregma), whereas region 4 (ARC IV) began with the caudal end of the DMN and continued to the end of the ARC (3.30–4.16 mm caudal to bregma). After anatomical matching, hybridization area and OD obtained from film autoradiographic analyses were determined for each animal and used as indexes of ppCART and ppNPY mRNA contents for each region of the ARC.

Quantification of ppCART mRNA levels in the PVN was accomplished by anatomically matching 18 coronal sections through the PVN of each rat (extending from 1.3 to 2.3 mm caudal to bregma). Hybridization area and OD obtained from film autoradiographic analyses were determined for each animal. PVN was not subdivided into regions for this analysis.

Statistical analyses
Leptin and other hormone levels were compared by two-way ANOVA, as were ppCART and ppNPY mRNA contents. Results are reported as the mean ± SEM, and the level of significance was set at P < 0.05. Post hoc testing by Fisher’s protected least significant difference test was performed for between-treatment differences separately for each age group. The statistical software package used was StatView version 4.57 for Windows (Abacus Concepts, Inc., Berkeley, CA; and SAS Institute, Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum hormone levels, body weight, and fat mass
Serum T levels (Table 1Go) were markedly lower in intact old compared with young rats and were below the assay limit of detectability in all (old and young) orchidectomized rats. T replacement in both old and young orchidectomized animals restored T to levels comparable to those in intact young BN rats.


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  		Table 1. Serum hormone levels, body weights, and adiposity of old and young BN rats

 
Serum leptin and insulin levels were higher in old compared with young rats (Table 1Go), but were similar among treatments (intact, castrate, and castrate+T) within each age group. Body weights were similar among treatments within each age group throughout the study (Fig. 1Go). At the time the rats were killed, young rats weighed significantly less than old rats (291.8 ± 10.5 and 448.2 ± 12.1 g, respectively). There was no effect of 21-d castration or T treatment on absolute or relative visceral fat mass (Table 1Go). Old animals had 3 times more fat in visceral depots, corresponding to twice the relative visceral adiposity (as a percentage of body weight), than young rats (P < 0.0001).



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  		Figure 1. Body weight curves of young (Y, 3-month-old) and old (O, 24-month-old) male BN rats over the 21-d experimental period. Animals were assigned randomly to treatment groups at the time of surgery. Values shown are the mean ± SEM. There were no differences in body weight or weight gain between treatment groups of the same age.

 
Effect of castration and T replacement on ppCART mRNA content in the ARC
Age effect: young vs. old intact animals.
ppCART mRNA content (hybridization units = OD x hybridization area) was greater in old compared with young intact rats in the entire ARC (Fig. 2Go). Regional analysis of the ARC indicated that the ppCART mRNA content was slightly greater in old compared with young intact animals in regions ARC I, ARC II, and ARC III (Fig. 3Go). ARC IV showed the most marked age-associated increase in ppCART mRNA content in old vs. young intact rats.



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  		Figure 2. A, Effect of 21-d castration and T replacement on ppCART mRNA in ARC of young (3-month-old) and old (24-month-old) BN rats. Hybridization units (OD x hybridization area) were used as the index of total mRNA content. Values shown are the mean ± SEM. *, P < 0.05 vs. both experimental groups of the same age; #, P < 0.05 vs. corresponding experimental group in young rats (by ANOVA and Fisher’s PLSD post hoc test). B, Digitized images of film autoradiograms of anatomically matched coronal sections in ARC II (at the level of bregma -2.60 mm) hybridized for ppCART mRNA of one representative animal from each age and experimental group.

 


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  		Figure 3. Effect of 21-d castration and T replacement on ppCART mRNA content by region in ARC of young (A; 3-month-old) and old (B; 24-month-old) BN rats. Hybridization units (OD x hybridization area) were used as the index of total arcuate ppCART mRNA content. Values shown are the mean ± SEM. *, P < 0.05 vs. both experimental groups of the same age; #, P < 0.05 vs. corresponding experimental group in young rats; {dagger}, P < 0.05 vs. intact old group (by ANOVA and Fisher’s protected least significant difference post hoc test).

 
Castration effect: castrated vs. intact animals.
In young animals, castration significantly increased the ppCART mRNA content in the entire ARC (Fig. 2Go) above that in the young intact group. This increase in ppCART mRNA content (Fig. 3AGo) was significant in ARC I and ARC II (rostral regions), but not in ARC III and ARC IV. In contrast, orchidectomy of old animals did not significantly alter ppCART mRNA content compared with that in intact animals either in the entire ARC (Fig. 2Go) or in individual regions (Fig. 3BGo).

Effect of T treatment in young and old animals.
In the young animals, ppCART mRNA content in the entire ARC (Fig. 2Go) of T-replaced rats was similar to levels in young intact rats and lower than levels in young castrated rats. Reductions of ppCART mRNA content by T replacement were most pronounced in the rostral ARC (Figs. 2BGo and 3AGo). The ppCART mRNA content in young T-replaced rats was similar to that in young intact rats in all regions of the ARC.

T replacement in old castrated animals reduced ppCART mRNA content in the entire ARC (Fig. 2AGo) compared with those in old intact and old castrated rats. Notably, ppCART mRNA content remained higher in old T-replaced compared with young intact rats. As in the young rats, the T-induced reduction of ppCART mRNA content was most pronounced in the rostral regions of the ARC (Figs. 2BGo and 3BGo).

Effect of castration and T replacement on ppCART mRNA content in the PVN
Age effect: young vs. old intact animals.
There was no effect of age on ppCART mRNA content in the PVN (Fig. 4Go). Intact young rats had levels identical to those in intact old animals.



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  		Figure 4. A, Effect of 21-d castration and T replacement on ppCART mRNA content in PVN of young (3 months old) and old (24 months old) BN rats. Hybridization units (OD x hybridization area) were used as the index of total ppCART mRNA content. Values shown are the mean ± SEM. *, P < 0.05 vs. both experimental groups of the same age (by ANOVA and Fisher’s PLSD post hoc test). B, Digitized images of film autoradiograms of anatomically matched coronal sections in PVN (at the level of bregma -1.80 mm), hybridized for ppCART mRNA of one representative animal from each age and experimental group.

 
Castration effect: castrated vs. intact animals.
Compared with intact animals of the same age, the PVN ppCART mRNA content was significantly lower in castrated young rats and slightly, but not significantly, lower in castrated old rats (Fig. 4Go). By visual inspection, this loss of hybridization signal appeared to be distributed across both medial and lateral areas of the nucleus (Fig. 4BGo).

Effect of T treatment in young and old animals.
T replacement to castrated young and old rats significantly increased PVN ppCART mRNA content (Fig. 4Go). T replacement to young castrated rats restored ppCART mRNA content to levels in young intact rats. In the old animals, T replacement significantly increased the ppCART mRNA content compared with intact as well as castrated rats.

Effect of castration and T replacement on ppNPY mRNA content in regions of ARC
Age effect: young vs. old intact animals.
The ppNPY mRNA content of intact rats was lower for old compared with young intact rats throughout the ARC (Fig. 5Go) as well as in each of the four individual regions of the ARC (Fig. 6Go).



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  		Figure 5. A, Effect of 21-d castration and T replacement on ppNPY mRNA in ARC of young (3-month-old) and old (24-month-old) BN rats where hybridization units (OD x hybridization area) were used as the index of total mRNA content. Values shown are the mean ± SEM. *, P < 0.05 vs. both experimental groups of the same age (by ANOVA and Fisher’s protected least significant difference post hoc test). B, Digitized images of film autoradiograms of anatomically matched coronal sections in ARC II (at the level of bregma -2.60 mm) hybridized for ppNPY mRNA of one representative animal from each age and experimental group.

 


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  		Figure 6. Effect of 21-d castration and T replacement on ppNPY mRNA content by region in ARC of young (A; 3-month-old) and old (B; 24-month-old) BN rats. Hybridization units (OD x hybridization area) were used as the index of total ARC ppNPY mRNA content. Values shown are the mean ± SEM. *, P < 0.05 vs. both experimental groups of the same age; {dagger}, P < 0.05 vs. intact old group (by ANOVA and Fisher’s protected least significant difference post hoc test). For each region, ARC I-IV, the ppNPY mRNA content of young animals (A) is significantly (P < 0.05) greater than that in the old intact group. For each region, ARC I-IV, the ppNPY mRNA content of old animals (B) is significantly (P < 0.05) less than that of the corresponding treatment group of young rats.

 
Castration effect: castrated vs. intact animals.
In the young rats there was a trend (P = 0.08) for reduced ppNPY mRNA content in the entire ARC of castrated compared with intact animals (Fig. 5Go). This overall trend was driven by a marked reduction in NPY hybridization in ARC II (P = 0.0008; Figs. 5BGo and 6AGo) in young castrated vs. intact rats. In old animals the ppNPY mRNA content of castrated animals was similar to that in intact old rats throughout the entire ARC (Fig. 5Go) and individual regions (Fig. 6BGo).

Effect of T treatment in young and old animals.
In young rats the ppNPY mRNA content in the entire ARC (Fig. 5Go) was virtually identical in intact and T-replaced rats. Regional analysis of the ppNPY mRNA content in young animals (Figs. 5BGo and 6AGo) showed that in ARC II, T replacement restored the ppNPY mRNA content to the level in young intact rats and above the levels found in young castrated animals; a similar pattern was observed in ARC I.

In the entire ARC (Fig. 5Go), T replacement in old castrated rats increased the amount of ppNPY mRNA compared with those in old intact and castrated rats. However, the ARC ppNPY mRNA content in young intact rats was about 3-fold greater than that in old T-treated rats. Regional analysis of the ppNPY mRNA content in old animals (Fig. 6BGo) demonstrated an increase in all areas of the ARC in T-replaced rats compared with intact and castrated rats, with significant increases in ARC II and ARC IV. Matched by region, the ppNPY mRNA content of T-replaced old was lower compared with that in young intact animals despite replacement of serum T to physiologically young levels.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found that T treatment of castrated rats decreased ppCART mRNA content and increased ppNPY mRNA content in the ARC of both young and old rats. This suggests that arcuate CART and NPY gene expressions are reciprocally regulated by T in both young and old animals, and remain responsive to T in old rats, although the effect of T on arcuate CART and NPY mRNA is markedly attenuated in old rats. Furthermore, we found evidence of regional specificity in the responses of CART and NPY mRNA to T administration; neurons with the highest sensitivity to T are localized within the rostral subregions of the ARC. Finally, we demonstrated that CART-producing neurons in the PVN are also regulated by T, although in an opposite direction than in the ARC.

T replacement in young and old Brown Norway rats revealed marked differences in the responses of hypothalamic NPY and CART neurons, which may have depended on the baseline level of endogenous T. Orchidectomy of young animals with initially high baseline levels of T resulted in increased ppCART mRNA and decreased ppNPY mRNA content in the ARC. Replacement of physiological levels of serum T in young castrated rats normalized ppCART and ppNPY in the ARC to levels similar to those in young intact animals. In contrast, castration of old animals with initially low baseline levels of T had no effect on ppCART or ppNPY mRNA levels, but replacing T to youthful levels resulted in decreased ppCART mRNA content and increased ppNPY mRNA content.

To our knowledge, this is the first report investigating the effects of age and T on CART gene expression. Relative to young intact rats, old animals demonstrate increased ppCART mRNA in all regions of the ARC, especially the most caudal region, ARC IV. However, there was no detectable response of CART gene expression to orchidectomy or T replacement in this region. Higher ppCART mRNA in old rats may be due to the increased leptin levels observed in old animals. The role of leptin as a stimulator of CART gene expression is well established (4, 9, 22). Although there have been no published reports describing the rostral-caudal distribution of leptin receptors in the ARC, we might speculate that differences in abundance of leptin receptors may account for the predominance of age (and not androgen)-related effects in the caudal arcuate.

As leptin levels were similar in intact, castrated, and T-replaced animals of the same age, it appears that changes in CART gene expression in rostral arcuate neurons induced by alterations in T levels are not mediated by leptin. The CART-producing neurons in the rostral ARC appear to be the most sensitive to alterations in levels of circulating T. This further supports the idea that neurons in the ARC that produce CART are distributed in a regionally heterogeneous manner. Arcuate CART neurons coexpress another neuropeptide, POMC (11), and castration has been shown to decrease POMC gene expression only in rostral regions of the ARC (38). CART and {alpha}MSH (a product of POMC) are both anorectic neuropeptides that are stimulated by leptin (10). Thus, CART and POMC neurons are not only anatomically colocalized in the ARC, but our results suggest that this regional specificity has a physiological and functional basis that is T dependent. Further studies would be required to clarify this concept.

Another population of CART-producing neurons that may not be sensitive to leptin is in the PVN. These neurons appear to be sensitive to androgen, but interestingly, the response was in the opposite direction from that observed in the ARC. Because there were no age-related differences in ppCART mRNA content, it may be that the low levels of serum T in old animals are sufficient to maintain basal PVN CART gene expression at levels comparable to those in young rats. CART is colocalized with a number of neuropeptides in the PVN, including TRH, vasopressin, and oxytocin, all of which are altered with aging (39, 40, 41), in contrast to our results with CART gene expression. Vasopressin mRNA content has recently shown to be affected by castration in young rats (42), although T replacement in old BN rats (40) did not normalize vasopressin levels. The effect of T in the PVN appears to depend on the physiological state of the animal, making comparisons between studies difficult. What is clear from our results is that CART neurons in the PVN do not respond to removal of T in the same way that arcuate CART neurons respond.

We confirmed our previous findings (26) that ARC ppNPY mRNA content is comparable between intact and castrated old rats. As serum T levels of intact rats are relatively low in the old animals, removal of the testes of old animals had minimal effects on arcuate NPY gene expression. It is plausible that the profound aging effect limited our ability to detect subtle changes due to T. The novel finding in this study is the markedly attenuated increase in NPY gene expression in T-replaced old vs. young rats, suggesting that T is only partially responsible for the age-related decline in NPY mRNA levels. However, it is possible that longer-term T treatment may have resulted in a greater increase in ppNPY mRNA in older rats, suggesting a greater role of low T levels in the age-related decrease in NPY gene expression.

T replacement in old rats increased NPY gene expression compared with that in old castrate and intact rats in all ARC regions, especially in the rostral region ARC II and the caudal region ARC IV. The minimal effect of T in other regions of the arcuate suggests that there are specific subpopulations of NPY-containing neurons that are more responsive to T, possibly due to differences in concentrations of androgen, estrogen, or leptin receptors. NPY neurons, either possessing AR or modulated by other neurons that are T sensitive, may be located mostly in ARC II and ARC IV. It has been suggested that specific subsets of NPY-producing neurons in the ARC concentrate T (33), but this remains unconfirmed. NPY-producing neurons also possess ER (43), and it is possible that T is aromatized to E2, and that the effect of T on NPY-producing neurons is estrogenic, rather than androgenic. Both AR and ER mRNA are present in the ARC, although ER mRNA-containing neurons are reportedly more abundant (44). Alternatively, it is possible that ARC regions that are not responsive to T contain a smaller percentage of T-sensitive NPY neurons, so that changes in ppNPY mRNA content in the few T-responsive neurons becomes obscured by a larger number of cells responding to other signals such as leptin.

Castration would be expected to result in increased fat mass, and therefore increased leptin levels (45, 46, 47). In our 21-d study, no changes in adiposity, body weight, leptin, or insulin levels were observed between experimental groups of the same age. In old weight-stable animals compared with young rats, higher percentages of body fat result in higher leptin levels (48), which are associated with increased CART and decreased NPY gene expression. However, numerous studies have demonstrated central and peripheral leptin resistance in aging male rats (49, 50) or rats made hyperleptinemic by constant leptin infusion (10), resulting in resistance to the anorexic and metabolic effects of leptin and to leptin effects on neuropeptide gene expression. Therefore, the attenuated responsiveness of neuropeptide gene expression that we observed in old rats may be explained partially by, but cannot be attributed entirely to, high circulating leptin levels. Our studies suggest that independent of leptin levels, T and/or its active metabolites (E2 or DHT) may also contribute to alterations in CART and NPY gene expression with aging in the male BN rat.

In summary, our findings suggest that ARC CART and NPY neurons have a reciprocal relationship to each other, at least with respect to the effects of both age and T levels. The age-related increase in gene expression of the anorexigenic peptide CART, in conjunction with the reduction in gene expression of the orexigenic peptide NPY, may contribute to reduced relative food intake and impaired food intake and weight gain after fasting in old compared with young rats (28). The regional specificity and degree of the effects of T on CART and NPY gene expression indicate the presence of more than one mechanism responsible for the age-related changes in these peptides, most likely involving increased leptin and decreased basal T in older animals. We conclude that certain arcuate neurons producing CART and NPY in old rats are responsive to short-term T administration, suggesting that the age-related reduction of circulating T levels contributes at least in part to the altered pattern of neuropeptide gene expression that impairs energy balance and favors anorexia and wasting with aging.


   Acknowledgments
 
We are grateful to Brett T. Marck for excellent assistance with surgeries and hormone assays, and to Lesley Leong for animal care. We thank Drs. Robert Steiner and Andrea Gore for their comments on an earlier version of the manuscript.


   Footnotes
 
This work was supported by an American Federation for Aging Research Summer Research Fellowship grant (to E.H.S.), V.A. Medical Research Funds, and NIH Grant P50-HD-12629. Work in this report has been presented and published in part at the 28th Annual Western Student’s Medical Research Forum, Carmel, CA, 2000, and the 83rd Annual Meeting of The Endocrine Society, Denver, CO, 2001.

Abbreviations: ARC, Arcuate nucleus; BN, Brown Norway BN/Bi; CART, cocaine-amphetamine-regulated transcript; DMN, dorsomedial nucleus; prepro-CART, prepro-cocaine-amphetamine-regulated transcript; ppNPY, prepro-NPY; PVN, paraventricular nucleus; TED, Tris-EDTA dithiothreitol.

Received July 11, 2001.

Accepted for publication November 2, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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