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Endocrinology Vol. 138, No. 3 1041-1047
Copyright © 1997 by The Endocrine Society

Articles

Elevated Corticosterone Is Not Required for the Rapid Induction of Neuropeptide Y Gene Expression by an Overnight Fast1

E. Simon Hanson, Nancy Levin and Mary F. Dallman

Department of Physiology (E.S.H., M.F.D.), University of California, San Francisco, California 94143-0444; and Genentech, Inc. (N.L.), South San Francisco, California 94080

Address all correspondence and requests for reprints to: E. Simon Hanson, Department of Physiology, University of California, San Francisco, San Francisco, California 94143-0444. E-mail: hanson{at}itsa.ucsf.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fasting stimulates corticosterone (B) secretion and the expression and secretion of hypothalamic neuropeptide Y in rats. These studies tested the hypothesis that the rapid and marked fasting-induced increases in plasma B are responsible for stimulation of neuropeptide Y (NPY) gene expression. Plasma leptin and insulin were measured because they are also signals known to affect NPY messenger RNA (mRNA). Intact or adrenalectomized rats given a low fixed level of corticosterone (B replaced) were fasted for 48 h. NPY mRNA in the mediobasal hypothalamus, measured by nuclease protection assay, was elevated similarly above ad lib-fed controls in both intact and B replaced groups at 15 and 48 h after the onset of fasting. NPY immunoreactivity in the mediobasal hypothalamus increased between 3 and 48 h after onset of the fast in intact but not in B replaced groups. The fasting-induced decreases in leptin observed in intact rats at 48 h did not occur in B replaced rats. Fasting-induced decreases in insulin occurred in B replaced rats but not in intact rats. We conclude that: 1) elevated B is not required for fasting-induced increases in hypothalamic NPY gene expression; and 2) decreases in neither leptin nor insulin alone signal the changes that occur in NPY mRNA in fasted rats.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROPEPTIDE Y (NPY) is a potent stimulator of food consumption when administered into the central nervous system (1, 2). One of the brain areas most sensitive to the orexigenic action of exogenous NPY is the paraventricular nuclei (PVN) of the hypothalamus (3). In rats, the PVN receive NPY innervation from two sources, the brainstem (4) and the arcuate nuclei of the hypothalamus (5). It is the arcuate-PVN connection that is putatively involved in the regulation of food intake (6). NPY in the arcuate and PVN is increased in states of increased metabolic demand (7) and is inhibited by insulin (8, 9) and leptin (10). Food deprivation for 48 h or more induces a rise in NPY immunoreactivity in the PVN (11, 12), as well as an increase in NPY gene expression in the arcuate nuclei (13, 14). In addition to its effect on food consumption, NPY injected into the PVN also stimulates the hypothalamic-pituitary-adrenal (HPA) axis (15, 16). This stimulation occurs at the level of the CRH neurons in the PVN (17), and is not dependent upon NPY induced feeding (18).

Jhanwar-Uniyal et al. (19) have reported a circadian rhythm in NPY content in the parvocellular PVN that is unimodal and peaks at the onset of darkness. This peak temporally corresponds to the period of greatest ad lib-food consumption and the peak in daily rhythm in B (20). It has been suggested that the diurnal rise in B is responsible for the circadian peaks in NPY gene expression and protein content in the arcuate and PVN, respectively (21). This model of glucocorticoid-stimulated feeding through NPY-containing cells in the arcuate that project to a site of secretion at the PVN is consistent with the histological findings of glucocorticoid receptors in NPY synthesizing cells in the arcuate (16), and glucocorticoid response elements located on the promotor of the NPY gene (22). In vitro, NPY messenger RNA (mRNA) expression is increased by glucocorticoids (22, 23). The effects of removing circulating B, however, are unclear. Adrenalectomy has been reported to decrease (24, 25) or have no effect (21, 26, 27, 28) on NPY gene expression in the arcuate nuclei. Adrenalectomized rats do, however, decrease their ad lib-food consumption (20) as well as their feeding response to intracerebroventricular NPY (29, 30).

Fasting has profound effects on both the HPA axis and hypothalamic NPY. Akana et al. (31) have shown that removal of food 1.5 h before the onset of darkness results in a rapid 3-fold increase in nocturnal B secretion above that of ad lib-fed controls. This nocturnal increase mimics the pattern of nocturnal food consumption and returns to normal ad lib-fed control values by the following morning. Ponsalle et al. (26) have shown that the increase in NPY gene expression that accompanies a 72-h fast requires the presence of glucocorticoids.

The present studies were designed to test the hypothesis that the fasting induced rise in nocturnal B, which accompanies an overnight fast drives the fasting induced increases in NPY gene expression. The design, in addition to testing the hypothesis, provides useful information on the time course of fasting-induced changes in NPY gene expression and correlates these changes with fasting induced decreases in insulin and leptin, two well known anorexigenic signals. All of these variables are examined within the circadian context and in the presence and absence of a rhythm in circulating B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects and design
Young adult male Sprague Dawley rats weighing approximately 150 g at the time of experiment were obtained from Bantin and Kingman (Fremont, CA). Rats were housed individually in hanging wire cages in a temperature- and light-controlled room (12-h light, 12-h dark, lights on/off at 0700 h and 1900 h) in the University of California, San Francisco animal care facility. All rats were allowed ad lib access to rat chow (Ralston Purina, no. 5008, Ralston Purina, St. Louis, MO) before fasting. Water was supplied at all times. On the day of the experiment, food was removed from one half of the animals at 1730 h (1.5 h before lights out). A group of seven animals was killed at this time to establish a prefasting basal group. Groups of fed and fasted animals (6 per group) were then killed at 3, 15, and 48 h after food removal. These times correspond to clock times of 2030 h, 0830 h, and 1730 h, respectively. Rats were killed by decapitation within 30 sec of opening the cage. Trunk blood was collected into tubes containing 100 µl EDTA (60 mg/ml), and kept on ice until centrifugation. Aliquots of plasma were frozen at -20 C for subsequent determination of ACTH, corticosterone, leptin, insulin, and glucose.

To examine the effects of fasting in the absence of changes in circulating B, a second experiment identical to the first was performed except that five days before the experimental fast the rats were adrenalectomized by the dorsal approach under ether anesthesia, and implanted subcutaneously with a pellet containing 25% corticosterone in cholesterol. This percentage of corticosterone was chosen to replace normal daily corticosterone at a level of ~5 µg/dl (32). Pellets were placed under the skin caudal to the midline surgical incision which was then closed with wound clips. After adrenalectomy, all animals received 0.5% saline as their drinking fluid. In this second experiment, we used 6 rats/group with the exception of the 15 h point, which had seven rats/group. All experiments were approved by the UCSF Committee on Animal Research.

Microdissection
Immediately after decapitation, whole brains were rapidly removed and placed on ice. Brain removal took between 30–120 sec per brain. After all brains for a given collection time were on ice, a 3-mm coronal section of the brain was cut using a prechilled brain matrix (Harvard Apparatus). The rostral boundary of the section was defined by the caudal optic chiasm. The resulting section was then placed onto a prechilled glass plate with the rostral surface facing up, and the medial hypothalamus was dissected freehand, using the top of the third ventricle as the dorsal boundary and the lateral hypothalamic sulci as the lateral boundaries. This hypothalamic block was then cut in half horizontally, to produce a basal portion (medialbasal hypothalamus, MBH) which contained the arcuate nucleus, and a dorsal portion (dorsalmedial hypothalamus, DMH) which contained the PVN. Tissue sections were then frozen on dry ice and stored at -80 C until subsequent isolation of cytoplasmic RNA. The total time between decapitation and dry ice freezing of the last brain was not more than 20 min.

RNA purification and solution hybridization/nuclease protection assays
Tissue dissections were homogenized as previously described (21). One hundred microliters of the tissue homogenate was removed before RNA purification, acidified (1 µl of 10 M HCl) and frozen (-80 C) for subsequent determination of total protein content and NPY immunoreactivity by RIA as previously described (21, 33). The RIA for NPY has been previously described (34). Total protein to which NPY immunoreactivity was normalized was determined by the method of Bradford (35). NPY and cyclophilin gene expression were quantitated using a 511-bp segment of the rat NPY gene, a generous gift of Dr. Steven Sabol (22) and a 117-bp segment of the rat cyclophilin gene, a gift of Dr. James Douglas (36). The solution hybridization/nuclease protection assay employed was as previously described with minor modifications (21). For the second experiment, RNA probes were synthesized in the presence of [33P] UTP. Nuclease protected bands resulting from hybridization of cRNA to either mRNA or standard RNA synthesized in vitro were quantitated by phosphorimaging. Amounts of NPY and cyclophilin mRNA in each sample were determined by linear regression from NPY and cyclophilin standard curves. NPY mRNA data are expressed as pg NPY mRNA/pg cyclophilin mRNA.

Plasma RIAs, food consumption, and statistics
Corticosterone and ACTH were measured as previously described (31). Insulin was measured using a commercially available kit (Diagnostic Systems Laboratories, Webster TX). Plasma leptin levels were measured at AMGEN, Inc. in the lab of Dr. Margery Nicolson, by a solid phase sandwich enzyme immunoassay (EIA), utilizing an affinity purified polyclonal antibody immobilized in microtiter wells. Bound leptin was detected with affinity purified antibody conjugated to horseradish peroxidase, and quantitated with a chromogenic substrate (TMB/peroxide). Leptin concentrations were calculated from standard curves generated for each assay using recombinant mouse leptin. The minimal leptin detection limit was 70 pg/ml. The inter- and intraassay coefficients of variation were 9.2% and 6.5% respectively. Leptin values which were over three standard deviations away from their group mean were not included in our analysis. Glucose was measured by the glucose oxidase technique on a Beckman glucose analyzer 2 (Palo Alto, CA). In nonfasted groups, cumulative ad lib-food consumption was measured during the fasting periods. At the time of fasting onset (5:30 pm) animals in the ad lib-fed groups were given a preweighed amount of fresh rat chow. When the experiment was over, uneaten food was collected and weighed. Cumulative food consumption for the 3, 15, and 48 h time periods was determined by subtracting the amount of uneaten rat chow from the initial amount provided.

Data were analyzed by two-way ANOVA. Each animal was treated as an independent determination. When appropriate, post hoc Newman-Keuls tests were performed. A P < 0.05 was considered significant for all tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of fasting in intact rats
Figure 1Go shows the effects of 0, 3, 15, and 48 h of fasting on plasma ACTH and B in intact rats. The light-dark bar at top shows the relative circadian time, at which the samples were taken. All subsequent figures retain this same format. Removal of food 1.5 h before lights out resulted in a significant increase in plasma B above ad lib-fed controls at all times measured (F = 24.12 P < 0.001; Fig. 1BGo). Fasted ACTH was also elevated above ad lib-fed groups (F = 10.53 P < 0.003); however, this increase only reached significance in the morning, 15 h after food removal (N.K. P < 0.005; Fig. 1AGo). Table 1Go shows the fasting induced changes in body weight for both experiments. In intact rats, fasting significantly decreased body weight by 15 h after food removal. The difference between ad lib-fed and fasted body weights was greatest after 48 h of fasting.



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  		Figure 1. Plasma ACTH (A) and corticosterone (B) values in intact rats either ad lib-fed or fasted. Stippled bars represent basal values of these hormones taken immediately before removal of food at 1730 h (time 0). Light-dark bar at top indicates relative phase of the light cycle. Data are expressed as mean ± SEM. *, Post hoc Newman-Keuls comparison of P < 0.05. All subsequent figures follow the same format as Fig. 1Go.

 

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  		Table 1. Body weight with time after food removal or continued food availability in Exp 1 (intact) and 2 (adrenalectomized, B-replaced)

 
NPY mRNA levels within the MBH are shown in Fig. 2Go. ANOVA revealed significant effects of time, treatment and a significant interaction. Compared with the ad lib-fed groups, fasting increased NPY mRNA in the MBH by 15 h after food removal (P < 0.001) and was further elevated by 48 h (P < 0.001). There were no changes in MBH NPY mRNA levels in ad lib-fed animals across time.



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  		Figure 2. NPY mRNA as measured by solution hybridization assay for fasted and ad lib-fed intact rats. Closed, fasted; open, ad lib-fed; stippled, basal. Light-dark bar at top indicates relative phase of the light cycle. Data are expressed as mean ± SEM. *, Post hoc Newman-Keuls comparison of P < 0.05.

 
NPY immunoreactivity in both the MBH and DMH dissection of the hypothalamus is shown in Table 2Go. In the MBH, there was a significant change over time (F = 5.177 P < 0.004) in intact rats, with increased immunoreactivity in the MBH between 3 and 48 h after the onset of the fast (P < 0.03). NPY immunoreactivity did not change over time or with treatment condition in the DMH.


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  		Table 2. Neuropeptide Y (NPY) content in the medial basal hypothalamic (MBH) and dorsomedial hypothalamic (DMH) fragments with time after food removal or continued food availability in Exp 1 (intact) and 2 (adrenalectomized, B-replaced)

 
Effects of fasting in rats with fixed corticosterone
In rats that were adrenalectomized and given a sc B pellet 5 days before fasting, neither fasting nor time of day altered circulating plasma B concentrations (Fig. 3BGo). ACTH, however, was markedly elevated in corticosterone replaced rats above the levels in intact rats (Fig. 3AGo; note scale change, compare with Fig. 1AGo). The only exception to this was in the B replaced ad lib-fed rats killed in the morning, 15 h after fasting onset (Fig. 3AGo). At this time there was also a significant difference in ACTH between ad lib-fed and fasted B replaced rats (P < 0.001). Body weight changes with fasting are shown in Table 1Go.



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  		Figure 3. Plasma ACTH (A) and corticosterone (B) values in adrenalectomized rats replaced with a corticosterone-containing pellet 5 days before fasting. Closed, fasted; open, ad lib-fed; stippled, basal). Note that the scale for ACTH is different from Fig. 1Go. Light-dark bar at top indicates relative phase of the light cycle. Data are expressed as mean ± SEM. *, Post hoc Newman-Keuls comparison of P < 0.05.

 
NPY mRNA in the MBH increased with duration of fasting in rats with fixed corticosterone in a similar pattern to that seen in intact rats (Fig. 4Go: compare with Fig. 2Go). There were significant effects of time, treatment, and interaction by ANOVA. Significant increases in NPY mRNA occurred in fasted rats, compared with ad lib-fed controls at 15 and 48 h (P < 0.04 and 0.001, respectively). In the steroid-replaced animals, there were no significant differences in NPY immunoreactivity between ad lib-fed and fasted rats in either the MBH or DMH dissection (Table 2Go).



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  		Figure 4. NPY mRNA as measured by solution hybridization assay for fasted and ad lib-fed adrenalectomized corticosterone replaced rats. Closed, fasted; open, ad lib-fed; stippled, basal). Light-dark bar at top indicates relative phase of the light cycle. Data are expressed as mean ± SEM. *, Post hoc Newman-Keuls comparison of P < 0.05.

 
Leptin, insulin, and glucose
Plasma leptin, insulin, and glucose levels from all groups are shown in Fig. 5Go. The top row of graphs (Fig. 5Go, A–C) shows the effect of fasting on these variables in intact rats, whereas the corresponding bottom graphs (Fig. 5Go, D–F) shows fasting effects in B replaced rats. Leptin, a hormone that has been correlated with changes in adipose mass, is shown in Fig. 5Go, A and D. Leptin levels in intact rats (Fig. 5AGo) were significantly lower in fasted than ad lib-fed controls only at 48 h after onset of fasting (N.K. P < 0.05). There was a significant change in plasma leptin in intact rats with time of day. Plasma leptin was increased in the morning (15 h after the onset of the fast) above prefasting evening levels in both fed and fasted conditions (P < 0.001). In B replaced rats (Fig. 5DGo), there was a significant decrease in leptin in fasted rats at 15 h after fasting onset (P < 0.05), but not at any other time. The circadian increase in morning (15 h) plasma leptin seen in intact rats was not seen in B replaced groups. Initial prefasting leptin was, however, higher in the B replaced group than in the intact group (compare 0 h time between Fig. 5Go, A and D; P < 0.05).



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  		Figure 5. Plasma leptin (A, D), insulin (B, E), and glucose (C, F) values for both adrenal intact (top row) and adrenalectomized corticosterone-replaced (bottom row) rats. Closed, fasted; open, ad lib-fed; stippled, basal. Light-dark bar at top indicates relative phase of the light cycle. See text for statistics.

 
The effect of fasting on plasma insulin concentrations in intact rats is shown in Fig. 5BGo. Fasting did not change plasma insulin concentrations from prefasting basal levels in intact rats. As we have previously shown, nocturnal plasma insulin was significantly increased above evening basal levels in ad lib-fed rats (P < 0.05). Due to this feeding-associated rise in insulin levels in the fed condition, insulin in intact fasted groups was significantly lower than in ad lib-fed groups at all times after onset of the fast (P < 0.01). In rats with fixed corticosterone (Fig. 5EGo), fasting did decrease plasma insulin below prefasting basal levels at 15 and 48 h after onset of the fast (P < 0.02). Insulin concentrations in fasted rats were also lower than in ad lib-fed rats at all times (P < 0.001). In the corticosterone replaced ad lib-fed rats, there were no changes in insulin over time. The initial insulin levels were, however, higher in the B replaced animals than in intact rats (compare 0 h time between Fig. 5Go, B and E; P < 0.05).

Plasma glucose in intact rats is shown in Fig. 5CGo. Fasting lowered plasma glucose levels below prefasting basal levels at all times measured (P < 0.001). This same pattern of fasting-induced decreases in plasma glucose was seen in B replaced groups (Fig. 5FGo; P < 0.001). Comparing between intact and B replaced conditions, plasma glucose was significantly higher in B replaced ad lib-fed rats than in intact ad lib-fed rats at all times measured including the prefasting basal time point (compare between Fig. 5Go, C and F; P < 0.01).

Cumulative ad lib food consumption was measured in the rats allowed to eat. In the first 3 h of nocturnal feeding, intact animals ate 4.33 ± 0.41 g of rat chow, and B-replaced rats ate 3.40 ± 0.22 g. By 15 h, intact rats had eaten significantly more food than the B replaced group (18.72 ± 0.19 g vs. 17.41 ± 0.28 g, respectively; P < 0.05). However, by 48 h, total food eaten by B-replaced rats was greater than the amount eaten by the intact rats (42.17 ± 1.29 vs. 36.37 ± 1.36 g, respectively; P < 0.01).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of these studies demonstrate that elevated B is not required for fasting-induced increases in NPY gene expression. By 15 h after food removal, NPY mRNA is elevated similarly in both intact and B replaced rats. Elimination of the normal circadian and fasting-induced changes in plasma B did not alter the responses of insulin or glucose to fasting, although the initial levels of both were higher in the B replaced group, suggesting that the daily rhythm in B may be important in maintaining the diurnal rhythm in food consumption. It appears from these results that singular changes in leptin and insulin are not required to stimulate the fasting-induced increase in NPY mRNA.

Previous studies examining the effect of fasting on NPY gene expression have reported increases in NPY mRNA after fasts of 48 (14) to 96 (13) h. We have found a rapid increase in NPY mRNA that occurs in the morning, 15 h after the onset of an overnight fast. A fast initiated just before lights out may be considered analogous to a 24-h fast initiated in the morning because its onset was at a time when rats are in a relatively fasted state due to their nocturnal rhythm in ad lib-feeding (37a). Schwartz et al. (38) examined a 24-h fasting period and did not find a significant increase in NPY mRNA in the arcuate nuclei, as measured by in situ hybridization. The discrepancies between the present study and that of Schwartz et al. may be due to the sensitivity of the methods used to detect changes in NPY mRNA. The rats studied here were also smaller than those used in (38). Rats in the weight range of this study, 120–180 g, are still growing rapidly and may have been more responsive than larger rats to the removal of food.

Because rats feed primarily at night, our studies were designed to examine the effects of fasting and hormone manipulation within a circadian context. As expected, intact ad lib-fed animals maintained their circadian rhythmicity in B, whereas fasting increased nocturnal plasma B 3-fold over intact control values. This fasting induced B increase, and the circadian rhythm in B was successfully abolished in B-replaced animals. In agreement with previously published studies on feedback sensitivity of the hypothalamic-pituitary-adrenal axis (39), B-replaced animals had increased ACTH values at all evening time points. In the morning, a time when there is no secretory drive to the hypothalamic-pituitary-adrenal axis, ACTH values were lowest and not significantly different from each other in intact and B replaced ad lib-fed animals, indicating that our replacement paradigm was adequate.

The main purpose of the present study was to test the physiological importance of this nocturnal increase in B that accompanies an overnight fast. Ponsalle et al. (26) found that adrenalectomy without B replacement prevented the increase in NPY gene expression following a 72-h fast and that provision of B restored the response. However, in the study of Ponsalle et al., the circulating levels of B provided to adrenalectomized rats were two to three times higher than the normal mean daily B levels that are required to replace steroid-dependent variables to normal in adrenalectomized rats and were similar to the elevated levels seen in intact rats that are fasted overnight (31; this study). To ascertain whether in the normal rat these high levels of B serve the physiological function of inducing NPY gene expression, the rats in the present study were replaced with an amount of B that resulted in plasma corticosterone levels that equals the mean daily level in intact rats (40); this level of B has previously been shown to normalize numerous variables affected by adrenalectomy (41). In the present study, B replacement resulted in identical temporal characteristics and similar magnitudes of changes in NPY gene expression in intact and B replaced rats, indicating that a nocturnal increase in B is not required for increased NPY mRNA in fasted rats.

The similarity of the increases in NPY mRNA in fasted intact and B replaced rats is of further interest in light of the differences observed between the groups in plasma leptin, insulin, and glucose. Differences between fed and fasted leptin values occurred in intact rats only at the 48 h time after the onset of fasting, well after the increases in NPY mRNA had begun. Moreover, leptin levels were not persistently decreased in fasted, B-replaced rats, suggesting that circadian and fasting-induced changes in plasma B may be involved in fasting induced changes in leptin signal at 48 h. The slow time course of fasting induced changes in leptin suggests that changes in leptin may be a consequence of decreased fat stores (see body weight data). Mobilization of fat occurs through activation of hormone-sensitive lipase which is sensitive to corticosterone (42). The fact that initial leptin levels were three times higher in the B-replaced rats, and that they did not change with time, as in intact rats, may reflect the positive effect of B on leptin mRNA in fat (43). However, recent data from our lab on adrenalectomized rats indicates that B is not required for fasting induced decreases in plasma leptin (unpublished observation). This, coupled with recent in vitro findings that insulin-stimulated leptin release is inhibited by ß3-adrenergic agonists (44), suggests that sympathetic activation may be a more important mediator of leptin levels than circulating B in fasting.

Previously, Schwartz et al. (45) reported that insulin infused intracerebroventricularly inhibits fasting-induced increases in hypothalamic NPY mRNA and suggested that fasting-induced increases in NPY biosynthesis are dependent on low insulin levels. Our results show that B replaced rats had elevated initial insulin levels that decreased with the onset of fasting, but plasma insulin did not decrease with the onset of fasting in intact rats. The latter finding is probably due to the lack of daytime feeding in intact rats. Because both groups had similar increases in hypothalamic NPY mRNA content, we conclude that decreases in insulin are not required for the fasting-induced increase in NPY mRNA. However, the increase in insulin that occurs with feeding may normally serve to inhibit NPY mRNA synthesis. It is also likely that one or more inputs other than changes in leptin, insulin, or glucocorticoids are, in part, responsible for signaling the onset of a fast to the NPY-synthesizing neurons in the arcuate nuclei. We speculate this may involve neural signals transmitted from the periphery via vagal afferents.

Previous studies have shown, by both tissue punch (11, 46) and push pull canulae (7) techniques, that NPY increases in the PVN with 48 h or more of fasting. In these studies, we did not observe fasting-induced changes in NPY immunoreactivity in the dorsomedial hypothalamic dissection that contained the PVN. Dissecting the hypothalamus into only two parts may not have been sensitive enough to detect changes in NPY in the individual areas of interest due to the inclusion of nuclei containing NPY fibers that may not be modulated by food intake. In the mediobasal hypothalamus containing the arcuate nuclei, where the relative concentration of NPY is much higher (47), NPY was decreased in intact rats 3 h after food removal. We suggest that this decrease may have been a result of increased axonal transport and/or release of NPY in fasted rats. By the morning after food removal, NPY peptide content is normal; however, at this time mRNA levels are increased. This sequence of changes suggests that increased NPY synthesis compensates for increased release of NPY within 15 h.

Fixed B levels in adrenalectomized rats abolish both fasting and circadian changes in the steroid. The effect of removing the circadian rhythm in B, while not the primary purpose of this study, provides insight into a role of the hormone on food ingestion. Prefasting initial levels of leptin and insulin were elevated in the B replaced group, compared with the intact group. Although B does have a stimulatory effect on leptin mRNA (43) and plasma insulin (28), because glucose levels were also elevated in B replaced rats, it is likely that the onset of food consumption was earlier in these rats than in the intact controls. This is supported by comparison of the amounts of food consumed by the ad lib-fed groups. Overnight food consumption (15 h) was significantly decreased in the B-replaced rats compared with intact controls; in contrast, cumulative food consumption at 48 h, which included two periods of daytime feeding, was significantly increased in B-replaced rats above that in intact controls. The possibility that B-replaced rats eat more during the day than intact rats is supported by the results of studies examining the effects of restricted feeding and circadian rhythms (48, 49, 50).

In summary, we have found that, while plasma levels of B and insulin move in opposite directions with the onset of fasting, and that leptin is decreased by 48 h, these changes individually are not required for fasting-induced induction of NPY mRNA which occurs within 15 h after food removal. These studies provide new information about the temporal characteristics of fasting-induced increases in NPY gene expression and begin to examine the roles of B, leptin and insulin in the NPY response to fasting.


   Acknowledgments
 
We thank Dr. Steven Sabol for the NPY probe, Dr. Margery Nicolson and her lab for the measurement of leptin, and Cydney Horsley for her technical assistance.


   Footnotes
 
1 This work was supported, in part, by NIH Grant DK-28172. Back

Received September 10, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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