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Megestrol acetate stimulates food and water intake in the rat: effects on regional hypothalamic neuropeptide Y concentrations
ELSEVIER
European Journal of Pharmacology265 (1994) 99-102
eJp
Short communication
Megestrol acetate stimulates food and water intake in the rat: effects on regional hypothalamic neuropeptide Y concentrations H. David McCarthy *, Rachel E. Crowder, Simon Dryden, Gareth Williams Diabetes and Metabolism Research Group, Department of Medicine, University of Lit,erpool, P.O. Box 147, Licerpool, L69 3BX, UK
Received 7 September 1994; accepted 16 September 1994
Abstract
Megestrol acetate, a synthetic progestogen, stimulates appetite through an unknown mechanism. We tested the hypothesis that it might act, at least in part, by stimulating the activity of hypothalamic pathways containing neuropeptide Y, a potent central appetite stimulant in rats. Administration of megestrol acetate (50 mg/kg/day, n = 8) for 9 days significantly increased food and water intake compared with untreated controls (n = 8). Treated rats showed significant (90-140%) increases in neuropeptide Y concentrations in the arcuate nucleus (where neuropeptide Y is synthesized), in the lateral hypothalamic area (through which arcuate neurones project) and in the medial preoptic area, ventromedial nucleus and dorsomedial nucleus. The latter are sites of neuropeptide Y release and sensitive to neuropeptide Y-induced hyperphagia. Megestrol acetate may therefore stimulate neuropeptide Y synthesis, transport and release, and this could contribute to its appetite-stimulating effects. Keywords: Neuropeptide Y; Megestrol acetate; Appetite; Cancer anorexia; Cachexia; Hypothalamus
1. Introduction
Loss of appetite frequently accompanies terminal malignancy and severe infections including AIDS. It impairs quality of life, causes weight loss and wasting and may reduce the patient's capacity to respond to chemotherapy (Fearon, 1992). There is no effective treatment for 'cancer anorexia', although some centres are now studying the use of megestrol acetate in this context. Megestrol acetate (17a-acetoxy-6-methyl-4,6pregnandiene-3,20-dione) is a synthetic, orally active progestogen which is frequently used in the treatment of advanced breast cancer, and has been found to stimulate appetite and cause weight gain (Aisner et al., 1988). Recently, megestrol acetate was found to stimulate feeding and to inhibit weight loss in a murine model of cancer cachexia (Beck and Tisdale, 1990). The mechanism by which this drug stimulates feeding and weight gain is unknown and has not been systematically ex-
* Corresponding author. Present address: Department of Human Nutrition, University of Southampton, Bassett Crescent East, Southampton, SO9 3TU, UK. Tel. 0703 594223, fax 0703 594383. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00566-4
plored. A clearer understanding of this action is important, as it might lead to the development of more specific appetite stimulants suitable for use in man. Progestogens have previously been shown to stimulate feeding in gonadally intact female rats (Gray and Wade, 1981), but again their mode of action is unclear. Recent evidence raises the possibility that one mechanism may be by stimulating neuropeptide Yergic pathways in the hypothalamus, which are implicated in the regulation of both feeding and reproductive behaviours (Kalra et al., 1990). Neuropeptide Y is a 36-amino acid peptide highly concentrated in appetite-regulating areas of the hypothalamus. It is synthesized in neurones of the arcuate nucleus which project through the lateral hypothalamic area and end mainly in the paraventricular nucleus and also in the medial preoptic area and ventromedial and dorsomedial nuclei (Chronwall et al., 1985). Injection of neuropeptide Y into these areas induces a robust feeding response in the rat; indeed, neuropeptide Y is the most potent appetite stimulant known (see Leibowitz, 1991 for review). Steroid hormones interact closely with the hypothalamic neuropeptide Yergic system which carries various steroid receptors (Kalra et al., 1990). Progesterone
1O0
11.D. McCarthy et al. / European Journal o1"Pharmacology 265 (1994) 99-102
given acutely to oestradiol-primed, ovariectomized female rats transiently increases neuropeptide Y concentrations in the median eminence and arcuate nucleus (Crowley et al., 1985). This suggests that progesterone stimulates neuropeptide Y synthesis, and is supported by the fact that it also increases preproneuropeptide Y m R N A levels within the arcuate nucleus (Camp and White, 1990). As hypothalamic neuropeptide Yergic pathways influence both feeding and reproductive behaviours, and are stimulated by progesterone, we hypothesized that megestrol acetate could enhance appetite in the rat at the same time as activating hypothalamic neuropeptide Yergic neurones in critical hypothalamic appetite-regulating areas. Thus, the aims of the study were to determine whether megestrol acetate stimulated food intake in rats (whose feeding responses to megestrol acetate have not previously been reported) and whether it increased neuropeptide Y concentrations in the arcuate nucleus and its sites of projection.
2. Materials and methods
Z 1. Animals and experimental procedure Sixteen female Wistar rats (Charles River UK, Margate, Kent, UK) weighing between 225-275 g were housed individually in wire-bottomed cages at an ambient temperature of 22 + I°C and maintained under a 12:12 h light:dark cycle (lights on at 07:00 h). Standard laboratory chow ( C R M Biosure, Cambridge, UK) and tap water were available ad libitum. Body weight and 24-h food and water intake were measured daily throughout the study. Eight rats were injected s.c. twice daily with megestrol acetate (25 m g / k g , in corn oil) and 8 controls received an equal volume of corn oil alone. T r e a t m e n t was continued for 9 days. On day 10, no drug was administered and the rats were killed by carbon dioxide inhalation.
2.2. Microdissection methods As previously described (Williams et al., 1989), the brain was rapidly removed, and 8 selected hypothalamic regions were microdissected from fresh brain slices using a vibrating microtome. The areas removed were: the medial preoptic area, lateral preoptic area, paraventricular nucleus, anterior hypothalamic area, ventromedial nucleus, lateral hypothalamic area, dorsomedial nucleus and a wedge of tissue at the base of the third ventricle containing the arcuate nucleus and median eminence. Pooled tissue from each area was boiled for 10 rain in 0,1 M HC1 to extract the peptide and sonicated to disperse the tissue. The extracts were
frozen at - 4 0 ° C until assayed for neuropeptide Y and protein content.
2.3. Assays Neuropeptide Y concentrations were measured by radioimmunoassay in 30-/.tl aliquots of hypothalamic extract employing J25I-labelled n e u r o p e p t i d e Y (Amersham International, Amersham, Bucks, UK) and porcine neuropeptide Y as standard (Bachem, Saffron Walden, UK). Rabbit anti-porcine neuropeptide Y antiserum raised in our laboratory was used at a final concentration of 1 : 30 000. Cross-reactivity was less than 5% with other related peptides (McKibbin et al,, 1991). The assay sensitivity was 1 f m o l / t u b e and the intra-assay coefficient of variation was < 4%. Protein content of the samples was measured using a modified Lowry technique (Lowry et al., 1951) after neutralization with 0.1 M N a O H , and neuropeptide Y concentrations in each region were expressed as fmol//x g protein.
2.4. Statistical analyses Values are expressed as mean _+ S.E.M. Food intakes and body weight were compared between groups using Student's t-test for unpaired data. For neuropeptide Y levels, differences between groups were tested by two-way analysis of variance ( A N O V A ) coupled to a post-hoc modified t-test (Bonferroni). As group differences were observed, individual nuclei were further compared using Student's t-test for unpaired data.
3. Results
3.1. Food and water intake and body weight These data are shown in Table 1. Megestrol acetate mildly enhanced food intake from the third day of treatment; over 9 days, cumulative food intake increased by 16% ( P < 0.001) above controls. Cumulative water intake was also significantly increased by 36% ( P < 0 . 0 0 1 ) in the megestrol acetate-treated group.
Table 1 The effect of megestrol acetate on body weight and food and watel intake in female Wistar rats
Control Megestrolacetate
Initial weight (g)
Weight change (g)
Total fl)od Total water consumed ~' consumed " (g) (ml)
25I+5 251+6
15+0.9 21+1.3b
176_+2 205±2'
269±5 367±7'
~' The food intake measurements and body weight changes are cumu. lative values for the 9-day treatment period, b p < 0.002. ~ P < 0.001 vs. control.
181
H.D. McCarthy et aL/ European Journal of Pharmacology 265 (1994) 99-102
16
C
,$
*
14
a.
lO
6 Z 2 0 MPO
LPO
PVN
AHA
VMN
LHA
DMN ARC
Hypothalamic area Fig. 1. Hypothalamic neuropeptide Y levels in 8 microdissected areas from control rats (open bars) and rats treated with megestrol acetate, 50 m g / k g / d a y (solid bars) for 9 days. Values = mean _+S.E.M., n = 8 per group. Key: MPO, medial preoptic area; LPO, lateral preoptic area; PVN, paraventricular nucleus; AHA, anterior hypothalamic area; VMN, ventromedial nucleus; LHA, lateral hypothalamic area; DMN, dorsomedial nucleus; ARC, arcuate nucleus/median eminence. • P < 0.05, ** P < 0.01.
Weight increase during the study was slightly but significantly increased in the treated group ( P < 0.002).
3.2. Regional hypothalamic neuropeptide Y concentrations Neuropeptide Y levels in the 8 hypothalamic regions are shown in Fig. 1. Two-way analysis of variance revealed a significant effect attributable t o group ( F I , l l l = 24.1, P < 0.0001) and region (F7,111 = 10.2, P < 0.0001). Regional neuropeptide Y concentrations were comparable between groups in the lateral preoptic area, anterior hypothalamic area and the paraventricular nucleus. However, major differences were observed between groups in the arcuate nucleus, which includes the main hypothalamic site of neuropeptide Y synthesis, and in the medial preoptic area, ventromedial nucleus, lateral hypothalamic area and dorsomedial nucleus, which are all sites of neuropeptide Y neuronal projections from the arcuate nucleus. In all these regions, neuropeptide Y concentrations were significantly higher in the megestrol acetate-treated group than in controls: medial preoptic area, a 93% rise ( P < 0.05); ventromedial nucleus,+ 130% ( P < 0.05); lateral hypothalamic area,+ 122% ( P < 0 . 0 1 ) ; dorsomedial nucleus,+ 92% ( P < 0.05) and the arcuate nucleus, + 143% ( P < 0.05).
4. Discussion
We found that megestrol acetate treatment significantly increased both food and water intake, therefore
101
confirming that it stimulates appetite in rats as well as in humans and mice. Body weight gain, although significantly higher in the treated rats, was modest and the nature of the weight gain is unknown. The study's main aim was to determine whether enhanced feeding resulting from megestroi acetate treatment was associated with changes in regional hypothalamic neuropeptide Y concentrations. It is evident from Fig. 1 that megestrol acetate treatment, or its metabolic or endocrine sequelae, caused widespread disturbances in hypothalamic neuropeptide Yergic pathways. Increased activity of this pathway is inferred to stimulate feeding and cause weight gain (Leibowitz, 1991; Dryden et al., 1994). Megestrol acetate increased neuropeptide Y concentrations in the arcuate nucleus, suggesting increased synthesis, which would be entirely consistent with the known effects of progesterone itself (Crowley et al., 1985; Camp and White, 1990). Increased neuropeptide Y levels along the projection in the lateral hypothalamic area and in sites rich in neuropeptide Y nerve terminals, particularly the dorsomedial nucleus and medial preoptic area, suggest increased transport of the peptide to its site of release. Here, increased release of neuropeptide Y could account for the appetite-stimulatory effect of megestrol acetate. It is interesting that neuropeptide Y levels were not also increased in the paraventricular nucleus, which is the main destination of the neuropeptide Yergic projections from the arcuate nucleus and of extrinsic fibres ascending from the medulla, and a site extremely sensitive to neuropeptide Y. The absence of an increase may be because release of the peptide from endings in the paraventricular nucleus increased in step with the input from the arcuate nucleus; alternatively, the extrinsic medullary input may have confounded the issue. This uncertainty will only be resolved by further studies of in-vivo release. This study was able to demonstrate that megestro[ acetate treatment both increases hypothalamic neuropeptide Y levels and enhanced feeding behaviour. Two other well-documented models of hyperphagia the fatty Zucker rat and the streptozotocin-diabetic rat - also display increases in regional hypothalamic neuropeptide Y concentrations including the arcuate nucleus (McKibbin et al., 1991; Williams et al., 1989), with concomitant increases in preproneuropeptide Y m R N A concentrations (Sanacora et al., 1990; White et al., 1990). Taken together, these findings suggest that neuropeptide Y could mediate hyperphagia due to various different causes, although this hypothesis can only be tested rigorously using appropriate antagonists or antiserum to block the actions of neuropeptide Y released in the paraventricular nucleus and elsewhere. Neuropeptide Y is undoubtedly not the only peptide involved in regulating feeding, or in driving hyperphagia. In addition, high doses of megestrol acetate re-
1112
H.D. McCarthy et al. / European Journal of Pharmacology 265 (19941 99-102
quired to stimulate feeding might be pharmacological rather than representing physiological activation of hypothalamic progesterone receptors. In view of the effect of megestrol acetate on appetite and body weight in cachectic patients, and its interesting association with enhanced hypothalamic levels of neuropeptide Y, research into future anticachectic drugs and appetite stimulants could focus more closely on neuropeptide Y analogues or secretagogues.
Acknowledgements This work was funded by the Cancer Research Campaign. Megestrol acetate was generously donated by Bristol-Myers Squibb. The authors wish to thank Mrs. Anne Kilpatrick for excellent technical assistance and the staff of the Royal Liverpool University Hospital animal unit for the care of the animals.
References Aisner, J., S. Tchekmedyian, N. Tait, H. Parnes and M. Novak, 1988, Studies of high dose megestrol acetate: potential applications in cachexia, Semin. Oncol. 15 (Suppl. 1), 68. Beck, S.A. and M.J. Tisdale, 1990, Effect of megestrol acetate on weight loss induced by t u m o u r necrosis factor alpha and a cachexia-inducing t u m o u r (MACI6) in N M R I mice, Br. J. Cancer 62, 420. Camp, P. and J.D. White, 1990, Steroid hormone regulation of brain neuropeptide Y m R N A levels, Ann. NY Acad. Sci. 611,478. Chronwall, B.M., D.A. Dimaggio, V.J. Massari, V.M. Pickel, D. Ruggiero and T.L. O ' D o n o h u e , 1985, The anatomy of the neu-
ropeptide Y-containing neurones in rat brain, Neuroscience 15, 1159. Crowley, W.R., R.E. Tessel, T.L. O ' D o n o h u e . B.A. Adler and S.P. Kalra, 1985, Effects of ovarian hormones on the concentrations of immunoreactive neuropeptide Y in discrete brain regions of the female rat: correlation with serum luteinizing hormone (LH) and median eminence LH-releasing hormone, Endocrinology 117, 1151. Dryden, S., H. Frankish, Q. Wang and G. Williams, 1994, Neuropeptide Y and energy balance: one way ahead for the treatment ol obesity?, Eur. J. Clin. Invest. 24, 293. Fearon, K.C.H., 1992, The mechanisms and treatment of weight loss in cancer, Proc. Nutr. Soc. 51,251. Gray, J.M. and G.N. Wade, 1981, Food intake, body weight and adiposity in female rats: actions and interactions of progestins and antiestrogens, Am. J. Physiol. 240, E474. Kalra, S.P., A. Sahu, P.S. Kalra and W.R. Crowley, 1990, Hypothalamic neuropeptide Y: a circuit in the regulation of gonadotropin secretion and feeding behavior, Ann. NY Acad. Sci. 611, 273. Leibowitz, S.F., 1991, Brain neuropeptide Y: an integrator of endocrine, metabolic and behavioral processes, Brain Res. Bull. 27, 333. Lowry, O., N. Rosebrough, A. Farr and R. Randall, 1951, Protein m e a s u r e m e n t with the Folin phenol reagent, J. Biol. Chem. 193, 265. McKibbin, P.E., S.J. Cotton, S. MacMillan, B. Holloway, R. Mayers, H.D. McCarthy and G. Williams, 1991, Altered neuropeptide Y concentrations in specific hypothalamic regions of obese ( f a / f a ) Zucker rats: possible relationship to obesity and neuroendocrine disturbances, Diabetes 40, 1423. Sanacora, G., M. Kershaw, J.A. Finkelstein and J.D. White, 1991t, Increased hypothalamic content of preproneuropeptide Y messenger ribonucleic acid in genetically obese Zucker rats and its regulation by food deprivation, Endocrinology 127, 731/. White, J.D., D. Olchovsky, M. Kershaw and M. Berelowitz, 1990, Increased hypothalamic content of preproneuropeptide Y messenger ribonucleic acid in streptozocin-diabetic rats, Endocrinology 126, 765. Williams, G., J.S. Gill. Y.C. Lee, H.M. Cardoso, B.E. Okpere and S.R. Bloom, 1989, Increased neuropeptide Y concentrations in specific hypothalamic regions of streptozocin-induced diabetic rats, Diabetes 38, 321.
European Journal of Pharmacology265 (1994) 99-102
eJp
Short communication
Megestrol acetate stimulates food and water intake in the rat: effects on regional hypothalamic neuropeptide Y concentrations H. David McCarthy *, Rachel E. Crowder, Simon Dryden, Gareth Williams Diabetes and Metabolism Research Group, Department of Medicine, University of Lit,erpool, P.O. Box 147, Licerpool, L69 3BX, UK
Received 7 September 1994; accepted 16 September 1994
Abstract
Megestrol acetate, a synthetic progestogen, stimulates appetite through an unknown mechanism. We tested the hypothesis that it might act, at least in part, by stimulating the activity of hypothalamic pathways containing neuropeptide Y, a potent central appetite stimulant in rats. Administration of megestrol acetate (50 mg/kg/day, n = 8) for 9 days significantly increased food and water intake compared with untreated controls (n = 8). Treated rats showed significant (90-140%) increases in neuropeptide Y concentrations in the arcuate nucleus (where neuropeptide Y is synthesized), in the lateral hypothalamic area (through which arcuate neurones project) and in the medial preoptic area, ventromedial nucleus and dorsomedial nucleus. The latter are sites of neuropeptide Y release and sensitive to neuropeptide Y-induced hyperphagia. Megestrol acetate may therefore stimulate neuropeptide Y synthesis, transport and release, and this could contribute to its appetite-stimulating effects. Keywords: Neuropeptide Y; Megestrol acetate; Appetite; Cancer anorexia; Cachexia; Hypothalamus
1. Introduction
Loss of appetite frequently accompanies terminal malignancy and severe infections including AIDS. It impairs quality of life, causes weight loss and wasting and may reduce the patient's capacity to respond to chemotherapy (Fearon, 1992). There is no effective treatment for 'cancer anorexia', although some centres are now studying the use of megestrol acetate in this context. Megestrol acetate (17a-acetoxy-6-methyl-4,6pregnandiene-3,20-dione) is a synthetic, orally active progestogen which is frequently used in the treatment of advanced breast cancer, and has been found to stimulate appetite and cause weight gain (Aisner et al., 1988). Recently, megestrol acetate was found to stimulate feeding and to inhibit weight loss in a murine model of cancer cachexia (Beck and Tisdale, 1990). The mechanism by which this drug stimulates feeding and weight gain is unknown and has not been systematically ex-
* Corresponding author. Present address: Department of Human Nutrition, University of Southampton, Bassett Crescent East, Southampton, SO9 3TU, UK. Tel. 0703 594223, fax 0703 594383. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00566-4
plored. A clearer understanding of this action is important, as it might lead to the development of more specific appetite stimulants suitable for use in man. Progestogens have previously been shown to stimulate feeding in gonadally intact female rats (Gray and Wade, 1981), but again their mode of action is unclear. Recent evidence raises the possibility that one mechanism may be by stimulating neuropeptide Yergic pathways in the hypothalamus, which are implicated in the regulation of both feeding and reproductive behaviours (Kalra et al., 1990). Neuropeptide Y is a 36-amino acid peptide highly concentrated in appetite-regulating areas of the hypothalamus. It is synthesized in neurones of the arcuate nucleus which project through the lateral hypothalamic area and end mainly in the paraventricular nucleus and also in the medial preoptic area and ventromedial and dorsomedial nuclei (Chronwall et al., 1985). Injection of neuropeptide Y into these areas induces a robust feeding response in the rat; indeed, neuropeptide Y is the most potent appetite stimulant known (see Leibowitz, 1991 for review). Steroid hormones interact closely with the hypothalamic neuropeptide Yergic system which carries various steroid receptors (Kalra et al., 1990). Progesterone
1O0
11.D. McCarthy et al. / European Journal o1"Pharmacology 265 (1994) 99-102
given acutely to oestradiol-primed, ovariectomized female rats transiently increases neuropeptide Y concentrations in the median eminence and arcuate nucleus (Crowley et al., 1985). This suggests that progesterone stimulates neuropeptide Y synthesis, and is supported by the fact that it also increases preproneuropeptide Y m R N A levels within the arcuate nucleus (Camp and White, 1990). As hypothalamic neuropeptide Yergic pathways influence both feeding and reproductive behaviours, and are stimulated by progesterone, we hypothesized that megestrol acetate could enhance appetite in the rat at the same time as activating hypothalamic neuropeptide Yergic neurones in critical hypothalamic appetite-regulating areas. Thus, the aims of the study were to determine whether megestrol acetate stimulated food intake in rats (whose feeding responses to megestrol acetate have not previously been reported) and whether it increased neuropeptide Y concentrations in the arcuate nucleus and its sites of projection.
2. Materials and methods
Z 1. Animals and experimental procedure Sixteen female Wistar rats (Charles River UK, Margate, Kent, UK) weighing between 225-275 g were housed individually in wire-bottomed cages at an ambient temperature of 22 + I°C and maintained under a 12:12 h light:dark cycle (lights on at 07:00 h). Standard laboratory chow ( C R M Biosure, Cambridge, UK) and tap water were available ad libitum. Body weight and 24-h food and water intake were measured daily throughout the study. Eight rats were injected s.c. twice daily with megestrol acetate (25 m g / k g , in corn oil) and 8 controls received an equal volume of corn oil alone. T r e a t m e n t was continued for 9 days. On day 10, no drug was administered and the rats were killed by carbon dioxide inhalation.
2.2. Microdissection methods As previously described (Williams et al., 1989), the brain was rapidly removed, and 8 selected hypothalamic regions were microdissected from fresh brain slices using a vibrating microtome. The areas removed were: the medial preoptic area, lateral preoptic area, paraventricular nucleus, anterior hypothalamic area, ventromedial nucleus, lateral hypothalamic area, dorsomedial nucleus and a wedge of tissue at the base of the third ventricle containing the arcuate nucleus and median eminence. Pooled tissue from each area was boiled for 10 rain in 0,1 M HC1 to extract the peptide and sonicated to disperse the tissue. The extracts were
frozen at - 4 0 ° C until assayed for neuropeptide Y and protein content.
2.3. Assays Neuropeptide Y concentrations were measured by radioimmunoassay in 30-/.tl aliquots of hypothalamic extract employing J25I-labelled n e u r o p e p t i d e Y (Amersham International, Amersham, Bucks, UK) and porcine neuropeptide Y as standard (Bachem, Saffron Walden, UK). Rabbit anti-porcine neuropeptide Y antiserum raised in our laboratory was used at a final concentration of 1 : 30 000. Cross-reactivity was less than 5% with other related peptides (McKibbin et al,, 1991). The assay sensitivity was 1 f m o l / t u b e and the intra-assay coefficient of variation was < 4%. Protein content of the samples was measured using a modified Lowry technique (Lowry et al., 1951) after neutralization with 0.1 M N a O H , and neuropeptide Y concentrations in each region were expressed as fmol//x g protein.
2.4. Statistical analyses Values are expressed as mean _+ S.E.M. Food intakes and body weight were compared between groups using Student's t-test for unpaired data. For neuropeptide Y levels, differences between groups were tested by two-way analysis of variance ( A N O V A ) coupled to a post-hoc modified t-test (Bonferroni). As group differences were observed, individual nuclei were further compared using Student's t-test for unpaired data.
3. Results
3.1. Food and water intake and body weight These data are shown in Table 1. Megestrol acetate mildly enhanced food intake from the third day of treatment; over 9 days, cumulative food intake increased by 16% ( P < 0.001) above controls. Cumulative water intake was also significantly increased by 36% ( P < 0 . 0 0 1 ) in the megestrol acetate-treated group.
Table 1 The effect of megestrol acetate on body weight and food and watel intake in female Wistar rats
Control Megestrolacetate
Initial weight (g)
Weight change (g)
Total fl)od Total water consumed ~' consumed " (g) (ml)
25I+5 251+6
15+0.9 21+1.3b
176_+2 205±2'
269±5 367±7'
~' The food intake measurements and body weight changes are cumu. lative values for the 9-day treatment period, b p < 0.002. ~ P < 0.001 vs. control.
181
H.D. McCarthy et aL/ European Journal of Pharmacology 265 (1994) 99-102
16
C
,$
*
14
a.
lO
6 Z 2 0 MPO
LPO
PVN
AHA
VMN
LHA
DMN ARC
Hypothalamic area Fig. 1. Hypothalamic neuropeptide Y levels in 8 microdissected areas from control rats (open bars) and rats treated with megestrol acetate, 50 m g / k g / d a y (solid bars) for 9 days. Values = mean _+S.E.M., n = 8 per group. Key: MPO, medial preoptic area; LPO, lateral preoptic area; PVN, paraventricular nucleus; AHA, anterior hypothalamic area; VMN, ventromedial nucleus; LHA, lateral hypothalamic area; DMN, dorsomedial nucleus; ARC, arcuate nucleus/median eminence. • P < 0.05, ** P < 0.01.
Weight increase during the study was slightly but significantly increased in the treated group ( P < 0.002).
3.2. Regional hypothalamic neuropeptide Y concentrations Neuropeptide Y levels in the 8 hypothalamic regions are shown in Fig. 1. Two-way analysis of variance revealed a significant effect attributable t o group ( F I , l l l = 24.1, P < 0.0001) and region (F7,111 = 10.2, P < 0.0001). Regional neuropeptide Y concentrations were comparable between groups in the lateral preoptic area, anterior hypothalamic area and the paraventricular nucleus. However, major differences were observed between groups in the arcuate nucleus, which includes the main hypothalamic site of neuropeptide Y synthesis, and in the medial preoptic area, ventromedial nucleus, lateral hypothalamic area and dorsomedial nucleus, which are all sites of neuropeptide Y neuronal projections from the arcuate nucleus. In all these regions, neuropeptide Y concentrations were significantly higher in the megestrol acetate-treated group than in controls: medial preoptic area, a 93% rise ( P < 0.05); ventromedial nucleus,+ 130% ( P < 0.05); lateral hypothalamic area,+ 122% ( P < 0 . 0 1 ) ; dorsomedial nucleus,+ 92% ( P < 0.05) and the arcuate nucleus, + 143% ( P < 0.05).
4. Discussion
We found that megestrol acetate treatment significantly increased both food and water intake, therefore
101
confirming that it stimulates appetite in rats as well as in humans and mice. Body weight gain, although significantly higher in the treated rats, was modest and the nature of the weight gain is unknown. The study's main aim was to determine whether enhanced feeding resulting from megestroi acetate treatment was associated with changes in regional hypothalamic neuropeptide Y concentrations. It is evident from Fig. 1 that megestrol acetate treatment, or its metabolic or endocrine sequelae, caused widespread disturbances in hypothalamic neuropeptide Yergic pathways. Increased activity of this pathway is inferred to stimulate feeding and cause weight gain (Leibowitz, 1991; Dryden et al., 1994). Megestrol acetate increased neuropeptide Y concentrations in the arcuate nucleus, suggesting increased synthesis, which would be entirely consistent with the known effects of progesterone itself (Crowley et al., 1985; Camp and White, 1990). Increased neuropeptide Y levels along the projection in the lateral hypothalamic area and in sites rich in neuropeptide Y nerve terminals, particularly the dorsomedial nucleus and medial preoptic area, suggest increased transport of the peptide to its site of release. Here, increased release of neuropeptide Y could account for the appetite-stimulatory effect of megestrol acetate. It is interesting that neuropeptide Y levels were not also increased in the paraventricular nucleus, which is the main destination of the neuropeptide Yergic projections from the arcuate nucleus and of extrinsic fibres ascending from the medulla, and a site extremely sensitive to neuropeptide Y. The absence of an increase may be because release of the peptide from endings in the paraventricular nucleus increased in step with the input from the arcuate nucleus; alternatively, the extrinsic medullary input may have confounded the issue. This uncertainty will only be resolved by further studies of in-vivo release. This study was able to demonstrate that megestro[ acetate treatment both increases hypothalamic neuropeptide Y levels and enhanced feeding behaviour. Two other well-documented models of hyperphagia the fatty Zucker rat and the streptozotocin-diabetic rat - also display increases in regional hypothalamic neuropeptide Y concentrations including the arcuate nucleus (McKibbin et al., 1991; Williams et al., 1989), with concomitant increases in preproneuropeptide Y m R N A concentrations (Sanacora et al., 1990; White et al., 1990). Taken together, these findings suggest that neuropeptide Y could mediate hyperphagia due to various different causes, although this hypothesis can only be tested rigorously using appropriate antagonists or antiserum to block the actions of neuropeptide Y released in the paraventricular nucleus and elsewhere. Neuropeptide Y is undoubtedly not the only peptide involved in regulating feeding, or in driving hyperphagia. In addition, high doses of megestrol acetate re-
1112
H.D. McCarthy et al. / European Journal of Pharmacology 265 (19941 99-102
quired to stimulate feeding might be pharmacological rather than representing physiological activation of hypothalamic progesterone receptors. In view of the effect of megestrol acetate on appetite and body weight in cachectic patients, and its interesting association with enhanced hypothalamic levels of neuropeptide Y, research into future anticachectic drugs and appetite stimulants could focus more closely on neuropeptide Y analogues or secretagogues.
Acknowledgements This work was funded by the Cancer Research Campaign. Megestrol acetate was generously donated by Bristol-Myers Squibb. The authors wish to thank Mrs. Anne Kilpatrick for excellent technical assistance and the staff of the Royal Liverpool University Hospital animal unit for the care of the animals.
References Aisner, J., S. Tchekmedyian, N. Tait, H. Parnes and M. Novak, 1988, Studies of high dose megestrol acetate: potential applications in cachexia, Semin. Oncol. 15 (Suppl. 1), 68. Beck, S.A. and M.J. Tisdale, 1990, Effect of megestrol acetate on weight loss induced by t u m o u r necrosis factor alpha and a cachexia-inducing t u m o u r (MACI6) in N M R I mice, Br. J. Cancer 62, 420. Camp, P. and J.D. White, 1990, Steroid hormone regulation of brain neuropeptide Y m R N A levels, Ann. NY Acad. Sci. 611,478. Chronwall, B.M., D.A. Dimaggio, V.J. Massari, V.M. Pickel, D. Ruggiero and T.L. O ' D o n o h u e , 1985, The anatomy of the neu-
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