Adrenalectomy decreases neuropeptide Y mRNA levels in the arcuate nucleus

Adrenal~ctomy Decreases Neuropeptide Y mRNA Levels in the Arcuate Nucleus B. DOUGLAS

WHITE.*’

ROGER G. DEAN+ AND ROY J. MARTIN”

*Departmmt of Foods cmd Nutrition, and ~Depnrtment of Animal Science, l_Jniversi[~of Georgiu. Athens, GA 30602 Received

10 August 1990

WHITE. B. D., R. G. DEAN AND R. J. MARTIN. Adreizf~iei.f~J~~l~decreases n~ur~p~pt~d~ Y mRNA levels in ihe mwmte nucletrs. BRAIN RES BULL 25(5) 71 l-7 IS. 1990. -Recent studies suggest that glucocorticoids may increase NPY and NPY mRNA levels. To determine if endogenous corticosterone affects the level of NPY mRNA in areas that control NPY levels in the paraventricuiar nucleus. we examined the effects of adrenalectomy and corticosterone replacement on NPY mRNA levels in the arcuate nucleus and brainstem. Rats were either adrenalectomized, adrenalectomized and corticosterone replaced. or sham-operated. The arcuate nucleus, hypothalamus (excluding arcuate nucleus), and brainstem were collected and the RNA isolated. Dot blots were made of each tissue and the NPY mRNA quantitaied by densitometry. Adrenalectomy signiticantiy reduced NPY mRNA levels in the arcuate nucleus. while corticosterone replacement restored the NPY mRNA levels. NPY mRNA levels in the remainder of the hypothalamus were not affected by adrenalectomy. Adrenalectomy also had no affect on NPY mRNA levels in the brainstem. These data suggest that the paraventricular nucleus may be affected by glucocorticoids via an NPY pathway and that the two major afferent pathway\ of NPY-containing neurons to the paraventricular nucleus may be regulated by different mechanisms. N~ur(~pept~de Y mRNA

Corticosterone

Adrenalectomy

Arcuate nucleus

NEUROPEFTIDE Y (NPY) is widely distributed throughout the central and peripheral nervous system and is one of the most

for reprints should be addressed to B. Douglas

Dot blot

have previously examined the effect of adrenalectomy on the level of NPY mRNA in several brain regions (13). Adrenalectomy decreases NPY mRNA in the striatum and tends to decrease NPY mRNA levels in the whole hy~thalamus. Corticosterone replacement brings NPY mRNA levels toward control levels. The parvocellular division of the paraventricular nucleus (PVN) has a rich supply of NPY nerve terminals (9). The PVN is involved in both neuroendocrine and autonomic outputs from the hypothalamus (26) and is suggested as being a site of action of NPY’s effect on feeding (34, 37, 39). The cell bodies of these nerve terminals are located in the arcuate nucleus of the hypothalamus (5) and in various nuclei of the brainstem (36). These areas are thought to control NPY levels in the paraventricular nucleus. To determine if endo~enous corticosterone concentrations influence NPY mRNA levels in areas that regulate NPY levels in the paraventricular nucleus. we examined the effects of adrenalectomy and corticosterone replacement on the level of NPY mRNA in the arcuate nucleus and brainstem. Adrenalectomy significantly decreased NPY mRNA levels in the arcuate nucleus. Corticosterone replacement restored NPY mRNA levels to that of controls. Adrenalectomy had no effect on NPY mRNA levels in the brainstem.

abundant neuropeptides known (1.4). It appears to belong to the pancreatic polypeptide family and has considerable sequence homology with both pancreatic polypeptide and peptide YY (40). The amino acid sequence of NPY is highly conserved across species. Human and rat NPY are identical, while porcine NPY has only a single amino acid substitution (24). This implies that NPY has an impo~ant physiological function. NPY has been implicated in the control of blood pressure (16), pituitary hormone secretion (27), insulin secretion (29), the suppression of sexual behavior (IO) and the entrainment of circadian rhythms (2,8). One of the most impressive actions of NPY is its effect on food intake. NPY is the most potent inducer of food intake known (39). While much work has centered on the distribution and actions of NPY, little work has been done on its regulation. Recent research has suggested that glucocorticoids may have some control over NPY and NPY gene expression. Within specific nuclei of the brainstem and hypothalamus, all the cell bodies containing NPY show ~lucoco~icoid receptor immunoreactivity (20). This suggests that glucocorticoids may have a direct effect on the synthesis and/or secretion of NPY. Dexamethasone administration can increase the in vivo level of NPY in the basal medial hypothalamus (12). Dexamethasone also causes a dosedependent increase in the intracellular NPY level in cultured hypothalamic neurons (12). In several neural-derived cell lines. dexamethasone increases the amount of NPY mRNA (21). We

‘Requests GA 30602.

Brainstem

METHOD

Thirty male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing approximately 200 grams were ran-

White. Department

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of Foods and Nutrition,

University

of Georgia.

Dawson Hall. Athens,

712

domly divided into three groups: adrenalectomized, adrenalectomized with corticosterone replacement (Sigma, St. Louis, MO) or sham-operated. Rats were bilaterally adrenalectomized under ether anesthesia by a dorsal approach. The identical procedure was performed on sham rats except the adrenal glands were not removed, only located and manipulated. While still under anesthesia, an Alzet osmotic pump (Alza Corp., Palo Alto, CA) was placed subcutaneously between the scapulae of each rat. Rats in the adrenalectomized, corticosterone replaced group received corticosterone at a calculated dose of 38 pg/h. Rats in the other two groups received the vehicle, polyethylene glycol 300. After surgery, adrenalectomized rats (with and without corticosterone replacement) were given ad lib access to 0.9% saline. Sham rats received tap water. All rats were given ad lib access to Purina lab chow. Four days after surgery, rats were sacrificed by decapitation. Trunk blood was collected and the serum isolated for corticosterone concentration determination. Serum samples were stored at - 80°C. Whole brains were removed and placed on a cold glass plate. A section of the brainstem ranging from 1 mm rostra1 of the obex to the rostra1 end of the spinal cord was isolated. The remainder of the brain was placed in a cold brain block and coronal sections (approximately 800 p,m) were made through the hypothalamus. Sections were transferred to a cold glass plate and the area surrounding the arcuate nucleus from each section was isolated and combined (Fig. 1). The remainder of the hypothalamus (i.e., excluding arcuate nucleus) from each section was also isolated and combined (Fig. 1). To isolate total RNA, brain tissues from individual rats were homogenized in RNAzol (Cinna/Biotecx, Friendswood, TX) according to the manufacturer’s instructions. Briefly, after homogenizing the tissues, chloroform was added to the homogenate, vortexed and centrifuged for 1.5 minutes. The aqueous layer was transferred to a microfuge tube and RNA precipitated with isopropanol. Tubes were incubated at - 20°C for 45 minutes, centrifuged, and the supematant aspirated. The RNA pellet was washed and stored under 75% ethanol at - 80°C until the samples could be further processed. Samples were centrifuged and the ethanol aspirated. The pellet was dried under a vacuum for 10 minutes and resuspended in 40 p.1 of diethylpyrocarbonate-treated water. Total RNA was quantitated from a lo-p1 sample by UV spectrophotometry (wavelength 260). Dot blots of the three brain areas were made on separate nylon membranes (DuPont, Wilmington, DE) according to the manufacturers instructions. Additionally, a Northern blot of a test tissue (cortex) was made to insure the dot blot’s specificity for NPY mRNA. Membranes were prehybridized for 1 hour in a phosphate buffer (0.25 M NaPO,, 0.25 M NaCl, 1 mM EDTA) and 50% formamide with 7% SDS at 6263°C. A 32P-labeled riboprobe complementary to rat NPY mRNA was added and the membranes were hybridized overnight. [The NPY-5 cDNA insert from which the riboprobe was derived was kindly provided by Dr. Janet Allen. It consists of a 539 base pair fragment including a 5’ untranslated region, the translated region and a 3’ untranslated region of the mature rat NPY mRNA (3). J Membranes were washed and x-ray film exposed for 4-7 days. Levels of NPY mRNA were quantitated by densitometry and corrected for the total amount of RNA added to the membrane. Values were expressed relative to the mean density of the arcuate nucleus of sham-treated rats. Serum corticosterone concentrations were determined by RIA kits (Research Systems Labs, Carson, CA). Differences in NPY mRNA levels and serum corticosterone concentrations between the three treatment groups were determined by analysis of variance using PC SAS. Differences between individual groups were determined by Student-Newman-

WHITE. DEAN AND MARTIN

-2.12 mm BREGMA

2.80 mm BREOMA

- 3.80 mm BREGMA

ARCUATE

AREA

HYPOTHALAMIC

AREA

FIG. 1. Diagram of coronal sections through the hypothalamus. Drawings were derived from the computer program, Brain Browser, by F. E. Bloom, W. G. Young and Y. M. Kim; an electronic version of: The rat brain in stereotaxic coordinates, by G. Paxinos and C. Watson. Dark areas represent the tissue isolated to obtain the arcuate nucleus. Stippled areas represent the tissue isolated to obtain the remainder of the hypothalamus. Reprinted with permission. Keuls test and were deemed level.

statistically

significant

at the 0.05

RESULTS Analysis of Northern blots (data not shown) showed that the NPY mRNA probe bound to a single band with an approximate length of 800 base pairs. This is the approximate length of the mature mRNA species (24). Adrenalectomy significantly reduced serum corticosterone concentrations (Fig. 2). Corticosterone replacement increased serum corticosterone concentrations to that of sham-treated rats. Adrenalectomy decreased @<0.05) the amount of NPY mRNA in the arcuate nucleus by 50% as compared to sham-operated rats (Fig. 3). Corticosterone replacement of adren&etomized rats returned NPY mRNA levels to that of the controls. Adrenalectomized rats had significantly lower QdO.05) NPY mRNA levels than did adrenalectomized rats treated with corticosterone. In the remainder of the hypothalamus (i.e., excluding arcuate nucleus), adrenalectomy had no effect on NPY mRNA levels. Adrenal-

GLUCOCORTICOIDS

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AND NPY mRNA

ARCUATE

SERUM CORTICOSTERONE

NUCLEUS

1.2 B

1

0

-

1.0 0.8 0.6 0.4 0.2 0.0 ADX

ADX + COW.

SHAM

HYPOTHALAMUS 0.6

A -

ADX

ADX + CORT

SHAM

FIG. 2. Mean serum corticosterone

concentration in adrenalectomized (ADX), adrenalectomized-corticosterone replaced (ADX + CORT), and sham-operated rats. Means are shown t SE and represent 7-8 observations. Different letters signify significant (~K0.05) differences.

ectomy also brainstem.

was

without

effect

on NPY

mRNA

levels

ADX

in the

ADX + CORT.

SHAM

BRAIN STEM DISCUSSION

The significant decrease in NPY mRNA levels in the arcuate nucleus following adrenalectomy. coupled with the return to control levels following corticosterone replacement indicates that endogenous corticosterone had a stimulatory effect on NPY mRNA levels in the arcuate nucleus. This is consistent with and extends the finding that dexamethasone increases NPY levels in the basal medial hypothalamus, the site of the arcuate nucleus (12). It has not yet been determined if the reduction in NPY mRNA levels associated with adrenalectomy is due to decreased transcription of NPY mRNA or to increased NPY mRNA breakdown. We recently reported the presence of a putative glucocorticoid response element (GRE) sequence 5’ of the promoter and two putative GRE sequences within the first intron of the NPY gene (13). This suggests that glucocorticoids may increase the transcription rate of NPY mRNA. If NPY levels in the PVN are reflective of NPY mRNA levels in the arcuate nucleus, then the present finding may indicate an important pathway through which glucocorticoids could affect PVN function. One of the most noted affects of NPY at the PVN is its ability to induce feeding behavior (37,39). Adrenalectomy has been shown to decrease food intake, especially at the onset of the dark period (23,41). The decrease in food intake during the early dark period appears to be specific for the macronutrient carbohydrate (41). It is of interest that NPY-induced feeding specifically increases carbohydrate consumption (30,38). It is possible that adrenalectomy. through a reduction in arcuate nucleus NPY mRNA levels, may lead to a decreased release of NPY in the PVN and thus a reduction in food intake. The reduction in food intake associated with adrenalectomy has been suggested to be due to a decrease in norepinephrine-induced feeding brought about by a reduction in the alpha-2 adrenergic receptor binding capacity in the PVN (25). This proposal does not preclude a role of NPY in the reduction of food intake following adrenalectomy. Indeed, alpha-2 adrenergic receptors and NPY receptors interact in the nucleus tractus solitarius (17). Perhaps such interactions extend to the PVN as well, as suggested

3.0

A

2.5 4.0 1.5 1.0 0.5 0.0 ADX

ADX + CORT.

SHAM

FIG. 3. Neuropeptide Y mRNA levels in the arcuate nucleus (top panel), hypothalamus (excluding arcuate nucleus) (middle panel) and brainstem (bottom panel) of adrenalectomized (ADX). adrenalectomized-corticosterone replaced (ADX + CORT) and sham-operated rats. Dot blots were quantitated by densitometry and corrected for the amount of RNA loaded on the membrane. Values are expressed relative to the mean density of the arcuate nucleus of the sham-operated rats. Means are shown + SE and represent 7-8 observations. (Some observations were discarded due to incomplete adrenalectomy or specks in the dot blots.1 Within a panel, different letters signify significant (~~0.05) difference\.

by the finding that alpha-2 receptor blockade decreases NPY-induced feeding (11). Additionally, NPY has been shown to have a biphasic effect on catecholamine utilization in the PVN (19). Depending on the kinetic relationships between the levels of corticosterone. NPY mRNA and NPY, the finding that corticosterone can increase NPY mRNA levels implies that NPY might have a role in the circadian rhythm of feeding. In rats. food intake increases greatly during the first hours after the onset of the dark period. Serum corticosterone concentrations peak prior to the onset of dark. If the half-life of NPY mRNA and NPY is fairly short, higher serum corticosterone concentrations prior to the onset of dark may induce significantly greater NPY mRNA levels in the arcuate nucleus, resulting in greater NPY release in the PVN. Greater NPY release in the PVN may contribute to the

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WHITE. DtAN

increased feeding at the onset of the dark period. Kalra et al. (22) have previously postulated that an increase in NPY secretion during the dark phase may be responsible for the nocturnal feeding pattern of rats. Obese Zucker rats lack a circadian rhythm of feeding and eat a relatively large percent of their daily food intake during the day (7, 1.5, 31). Obese Zucker rats also lack a circadian rhythm of serum corticosterone, maintaining relatively high concentrations throughout the day (14,28). From the results of the present study it might be expected that relatively high serum corticosterone concentrations in the day would increase NPY mRNA levels in the arcuate nucleus, leading to increased NPY release in the PVN and increased daytime feeding. Elevated levels of NPY in the PVN (6) as well as elevated levels of NPY mRNA in the arcuate nucleus (35) have been reported in obese rats. NPY mRNA levels in the remainder of the hypothalamus were not effected by adrenalectomy. This difference with the arcuate nucleus suggests specificity within the hypothalamus. Previously, when we examined the whole hypothalamus, adrenalectomy only tended @ =0.08) to reduce NPY mRNA levels C13). The present study suggests that the relative lack of response of the whole hypothalamus to adrenalectomy may have been due to dilution with hypothalamic areas not responsive to adrenalectomy. Levels of NPY mRNA in the brainstem were also unaffected by adrenalectomy. This finding was surprising considering that NPY-producing cell bodies in the brainstem show immunoreactivity for glucocorticoid receptors (20). Other factors may be involved in glucocorticoid regulation of NPY mRNA levels. Often, a combination of nuclear transcriptional factors must work in concert to significantly affect gene expression. Synergism between glucocorticoids, forskolin, phorbol ester and calcium ionophores in the control of NPY mRNA levels has been described (21). Perhaps in the brainstem, the NPY-containing cell bodies lack some other factor(s) necessary for glucocorticoids to affect NPY mRNA. It is possible that NPY mRNA in some brainstem regions may be responsive to adrenalectomy, but could not be detected because of dilution with nonresponsive areas. Some controversy exists over the degree of control the brainstem nuclei have over NPY levels in the PVN. It has been dem-

AND MARTIN

onstrated that destruction of the arcuate nucleus significantly decreases the number of NPY fibers in the PVN, but that neural transection between the brainstem and PVN did not affect NPY fibers in the PVN (5). This suggests that NPY innervation from the brainstem has little effect on NPY in the PVN. This is sup ported by the finding that destruction of the catecholaminergic nerve terminals in the PVN did not affect NPY immunoreactivity (18). On the other hand, other studies have shown neural transection between the brainstem and PVN to significantly lower NPY levels in the PVN (32) and to increase the sensitivity to exogenous NPY (33). This suggests a significant involvement of the brainstem in the control of NPY levels of the PVN. While more work needs to be done to elucidate the relationship between the arcuate nucleus and the brainstem nuclei in the control of NPY levels in the PVN, the present study suggests that NPY mRNA in these two areas may be regulated by different mechanisms. NPY mRNA in cell bodies from the arcuate nucleus appeared responsive to endogenous circulating corticosterone concentrations. while NPY mRNA in cell bodies from brainstem nuclei was unaffected by the serum corticosterone concentration. In summary, the present study found that adrenalectomy significantly decreased the level of NPY mRNA in the arcuate nucleus. Corticosterone replacement reversed the effect of adrenalectomy, suggesting a role of endogenous corticosterone in the control of in vivo NPY mRNA levels in the arcuate nucleus. Within the hypothalamus, the effect of adrenalectomy appeared to be specific for the arcuate nucleus. Brainstem NPY mRNA levels were not affected by adrenalectomy. The present results may indicate a glucocorticoid-NPY pathway which could possibly influence the feeding, autonomic and neuroendocrine functions of the PVN. ACKNOWLEDGEMENTS

This research was supported by the National Institutes of Health, Grant HD22226, and the University of Georgia Experiment Station, Project 527. We would like to express our appreciation to Vema Burden, Cathy Carver and Barbara Rose for their technical participation. We are additionally indebted to Dr. Janet Allen for providing us with the NPY-S cDNA.

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