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Oleoyl-estrone does not alter hypothalamic neuropeptide Y in zucker lean and obese rats
Peptides, Vol. 19, No. 9, pp. 1631–1635, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00
PII S0196-9781(98)00104-1
BRIEF COMMUNICATION
Oleoyl-Estrone Does Not Alter Hypothalamic Neuropeptide Y in Zucker Lean and Obese Rats ´ N,*† JEU ´ S PE´REZ–CLAUSELL,‡ CRISTINA CABOT,*† MARIA DEL MAR GRASA,*† CRISTINA ADA ´ NDEZ–LO ¨ PEZ,* JORDI VIRGILI,* JORDI ESTRUCH,† JOSE´–ANTONIO FERNA 1 ` XAVIER REMESAR,* AND MARIA ALEMANY* *Departament de Bioquı´mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain †Laboratoris SALVAT, SA, Esplugues de Llobregat, 08950 Barcelona, Spain ‡Departament de Biologia Celzlular Animal i Vegetal, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain Received 27 February 1998; Accepted 15 June 1998 ´ N, J. PE´REZ–CLAUSELL, J. VIRGILI, J. ESTRUCH, J. A. CABOT, C., M. D. M. GRASA, C. ADA ´ NDEZ-LO ´ PEZ, X. REMESAR AND M. ALEMANY. Oleoyl-estrone does not alter hypothalamic FERNA neuropeptide Y in Zucker lean and obese rats. PEPTIDES 19(9) 1631–1635, 1998.—Female Zucker lean and obese rats were treated for 14 days with 3.5 mmol/kg oleoyl-estrone (OE) in liposomes (Merlin-2). After 0, 3, 6, 10, and 14 days of treatment, the rats were killed and hypothalamic nuclei (lateral preoptic, median preoptic, paraventricular, ventromedial and arcuate) were used for neuropeptide Y (NPY) radioimmunoassay. In 14 days, OE decreased food intake by 26% in lean and 38% in obese rats and energy expenditure by 6% in lean and 47% in obese rats; the body weight gap between controls and treated rats becoming 217.8% of initial b.wt. in the lean and 213.6% in the obese rats. Obese rats showed higher NPY levels in all the nuclei than the lean rats. Despite a negative energy balance and decreased food intake, there were practically no changes in NPY with OE treatment. The results indicate that oleoyl-estrone does not act through NPY in its control of either food intake or thermogenesis in lean and genetically obese rats. © 1998 Elsevier Science Inc. Neuropeptide Y Zucker obese rat
Oleoyl-estrone Merlin-2
Appetite
Hypothalamus
NEUROPEPTIDE Y (NPY) is a major factor in the control of appetite in mammals (19,26). High hypothalamic concentrations are associated with increased appetite (21) and low levels with situations in which intake is reduced (5). NPY is also involved in other physiological responses to feeding and overfeeding, such as its mediation in the thermogenic response to a meal (4). The direct intraventricular injection of NPY increases appetite (27). However, it has
Energy expenditure
Obesity
been found that total absence of NPY does not result in the complete loss of appetite in NPY knock-out mice (14). Thus, not all feeding responses are directly linked to NPY synthesis and release (13); nevertheless, it is widely accepted that NPY still plays a key role in the control of appetite and food consumption. The distribution of NPY in the hypothalamus is not uniform, and changes in the different hypothalamic nuclei
1 Requests for reprints should be addressed to Prof. Dr. Maria` Alemany, Departament de Bioquı´mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, 645, 08028 Barcelona, Spain. E-mail: [email protected]
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CABOT ET AL. TABLE 1
CHANGES IN BODY WEIGHT, FOOD INTAKE AND ENERGY AVAILABILITY OF ZUCKER LEAN AND OBESE RATS TREATED WITH LIPOSOMES LOADED WITH OLEOYL-ESTRONE (MERLIN-2) Parameter Boby weight Lean Obese Food intake Lean Obese Lean Obese Energy available Lean Obese Energy from internal sources Lean Obese
Units
Treated Day 0
Treated Day 3
Treated Day 6
Treated Day 10
Treated Day 14
Control Day 14
% of initial % of initial
100 100
96.3 6 0.5 98.8 6 0.7
93.5 6 0.9 97.6 6 1.0
92.2 6 2.5 96.7 6 1.4
91.8 6 1.4 96.4 6 1.8
109.6 6 1.2 110.0 6 0.7
g/day* g/day* g/day† g/day†
16.3 6 0.8 25.7 6 0.6 — —
9.4 6 0.4 14.8 6 1.5 8.1 6 1.3 14.8 6 0.8
8.1 6 0.7 15.2 6 1.2 8.8 6 0.6 14.3 6 0.7
9.4 6 0.8 15.2 6 1.0 9.1 6 0.6 14.2 6 0.6
10.7 6 0.5 15.0 6 1.4 11.1 6 1.1 14.8 6 0.6
14.5 6 0.4 24.2 6 2.0 14.2 6 1.4 23.9 6 6.9
W W
2.36 4.34
1.78 2.77
1.64 2.68
1.51 2.75
1.98 2.46
2.10 4.62
% of energy available‡ % of energy available‡
29.5§ 29.4§
9.5 9.9
22 12.7
5.5 21.8
29.5 29.4
23.1 9.6
The data are the mean 6 SEM of five to seven different animals. * Intake during the 24 hours preceding the day in which the animals were weighed. † Mean daily intake for the 3– 4-day period between the days marked for measurement in the Table. ‡ A positive value means a net withdrawal of substrates from body stores, a negative value represents net deposition of live matter. § This figure was not measured, but it is assumed to be the same as for Day 14. Statistical comparison between groups (ANOVA). Weight: obese vs. lean p 5 0.0001; effect of time: lean p 5 0.0051, obese p 5 0.0035. Intake: obese vs. lean p 5 0.0000; effect of time: lean p 5 0.0000, obese p 5 0.0004.
have been related to marked behavioral differences (9). Injection of NPY into the paraventricular nucleus (PVN) maximally enhances food intake, but injection into other hypothalamic areas also increases appetite (11). Increases in NPY content of PVN are associated with diet-induced thermogenesis (6). NPY is produced by the arcuate nucleus which projects a dense set of axons rich in NPY into the PVN nucleus (1). Oleoyl-estrone in liposomes (Merlin-2) induces rapid and significant losses in the fat stores of treated rats (23). Oleoyl-estrone slimming effects are found in Zucker obese rats (2) and dietary-obese animals (3). Since the slimming process is achieved by decreasing food intake and maintaining heat production (23,24), we devised this study to determine whether the oleoyl-estrone-induced loss of appetite is mediated by decreases in NPY concentrations in the hypothalamic areas related with NPY management and the control of appetite. METHOD Zucker lean (Fa/?) and obese (fa/fa) female rats weighing 170 –190 g (lean) or 370 – 410 g (obese) from Charles River (Aubin–les–Elbeuf, France) stock were used. They were maintained under standard conditions (21°C, 60 –70% relative humidity, lights on from 08:00 a.m. to 8:00 p.m.), and fed standard rat chow pellets (B&K, Sant Vicenc¸ dels Horts, Spain) ad libitum. The rats were handled following the guidelines established by the European Community and the Governments of Catalonia and Spain.
Oleoyl-estrone was synthesized and incorporated into liposomes (23) containing 20% lipid. This preparation was used to fill osmotic minipumps (Alzet 2ML-2 from Alza, Palo Alto, CA USA), which were inserted (under ethyl ether anesthesia) under the skin on the back of the rats, and connected via a short capillary tube (PE-10 polyethylene from Becton–Dickinson, Parsippanny, NJ USA) to the left jugular vein. The minipumps released 5 ml/h for 14 days at a constant rate; the dose administered to the rats was 3.5 mmol/dayzkg of b.wt. Control (lean and obese) rats were included in the study; they were implanted with minipumps containing only the liposome suspension, without oleoylestrone. Rat weight and food consumption was recorded daily. The energy output of each rat was calculated from the energy ingested (g of pellet food with a nominal energy of 14.6 kJ/g), and from the changes in body mass. It was assumed that the energy derived from loss of body weight under the effects of Merlin-2 was similar to that found under comparable circumstances (2), i.e., a mean value of 14.4 kJ per g of weight lost and 13.1 kJ per g of weight increase, since in the first case the loss is mainly fat, and in the second, protein accounts for half the energy deposited. Using these approximate values, the mean energy output of the rats was calculated and expressed in W. On Days 0 (intact rats), 3, 6, 10, and 14 after the implantation of the minipumps, groups of five treated rats (and liposome-receiving controls on Day 14) were killed by decapitation and their brain was carefully dissected, frozen
OLEOYL-ESTRONE EFFECT ON NPY
in liquid nitrogen and kept at 280°C until further processing. Brains were placed in a freezing microtome and frontal sections were cut. The slices, 400 –500 mm thick were manipulated under a stereomicroscope in the cold (0 –2°C). The anatomical characteristics of hypothalamus were noted and the position of the five main nuclei and areas were identified and dissected through microdissection using fine scalpels and a sampling trochanter needle of 0.8 mm internal diameter, which was used to punch sampling holes for the smaller areas. Serial dissection of the 8 –9 slices containing the hypothalamus produced samples from lateral preoptic (LPO) and median preoptic (MPO) areas, and paraventricular (PVN), ventromedial (VMH) and arcuate (ARC) nuclei. The samples were immediately placed in 0.400 ml of chilled 0.5 M acetic acid for acidic NPY extraction; they were then heated at 100°C for 10 min. After cooling, the samples were sonicated in the cold for 15 s. A 0.100-ml aliquot was used for protein estimation (17). The remaining homogenate was centrifuged at 15,000 3 g for 15 min. The supernatant was concentrated and evaporated under vacuum, and kept at 220°C until further processing. The dry supernatants were diluted with 0.700 ml of buffer and used for the estimation of NPY by radioimmunoassay (10,20). The antibody used (cat N9528; Sigma, St Louis, MOUSA) was a polyclonal rabbit serum raised against porcine NPY. In order to determine the suitability of the radioimmunoassay for the measurement of rat NPY, we run HPLC separations of a whole hypothalamic extract of rat with or without added porcine NPY. We used a Kontron (Milano, Italy) HPLC connected to a Bio–Rad (mod. 2128, Hughes, CA USA) fraction collector. We used a Spherisorb ODS 5 mm (250 3 4.5 mm; Waters, Milford, MA USA) column with a C18 precolumn at 9,7 Mpa at 1 ml/min and a linear gradient elution system of 1.2 ml/l trifluoroacetic acid in water (A) and 1.0 ml/l of trifluoroacetic acid in acetonitrile (B) set at 65A/35B at t 5 0 min and 50A/50B at t 5 40 min (28). The eluates were recovered in 1 ml (1 min) fractions which were used for radioimmunoassay as indicated above. Hypothalamic extracts showed a single peak at 32 min that was coincident with that of porcine NPY. The suitability of the extraction and radioimmunoassay procedures for rat NPY was thus established. Groups were compared by ANOVA test, using a limit of statistical significance of 0.05. RESULTS Table 1 shows the changes in body weight, food intake and energy availability of the rats treated with oleoyl-estrone. All treated rats lost weight by 8.2% (lean) or 3.6% (obese) on Day 14. Controls receiving liposomes but not oleoylestrone increased in weight by a mean of 9.6% (lean) or 10.0% (obese), which sets the gap due to Merlin-2 treatment at 17.8% (lean) or 13.6% (obese) after 14 days of treatment.
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FIG. 1. Concentrations of NPY in the lateral preoptic (LPO), median preoptic (MPO) and ventromedial (VMH) areas, and paraventricular (PVN) and arcuate (ARC) nuclei of the hypothalamus from Zucker lean and obese rats treated with oleoyl-estrone in liposomes. The open symbols correspond to lean and the black symbols to obese rats. The data are the mean 6 SEM of four animals per group. Post-hoc Duncan test results for lean rats MPO: The 6-day value was significantly different from all other time points.
Mean energy expenditure for treated rats (14 days) was 94.3% of that of controls in lean rats and 53.3% in the obese. The corresponding figures for energy intake were 73.8% of controls in lean rats and 62.0% in the obese. Figure 1 shows the levels of NPY in the five selected hypothalamic locations during the treatment with Merlin-2. The changes in protein concentration were minimal (and non-significant) in the period studied. Obese rats (both controls and oleoyl-estrone-treated rats) showed higher NPY concentrations in all the areas and nuclei, and at practically all times studied, the differences being signifi-
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CABOT ET AL.
cant for all areas. NPY levels in the LPO, PVN, VMH, and ARC of lean and obese rats did not change significantly during oleoyl-estrone treatment. In the MPO area, again obese rats showed no change but lean rats decreased the NPY levels by mid-treatment. In all cases, the liposometreated controls showed values not different from those of Day 0 controls. DISCUSSION Treatment of lean and obese rats with oleoyl-estrone in liposomes results in the loss of energy stores, mainly in the form of fat, with a little loss of protein (23,24). This is achieved by a decrease in appetite and a relative maintenance of energy output (24). This applies both to lean and genetically obese Zucker rats (2). In the present study we observed that the loss of body weight was parallel to decreased food intake (appetite?) and maintained their energy output in lean rats and lowered it in the obese. The small changes in NPY levels—if any— observed in the different hypothalamic nuclei during oleoyl-estrone treatment contrast with the alterations observed during starvation (30) and modulation of appetite (4,21,26). The decreases in food intake induced by Merlin-2 were not paralleled by significant changes in NPY neither in obese nor in lean rats, which contrasts with the purported orexic effect of the peptide (5,19). The clear absence of decreases in NPY in either lean or obese rats in parallel to the decrease in food intake elicited by oleoyl-estrone suggests that this is not mediated by NPY. This does not seem to alter the basic status of Zucker obese rats, which maintain higher NPY levels (12) and also show a higher basal food intake than lean rats. Leptin has been postulated to decrease hypothalamic
NPY (25,29), but mice lacking NPY maintain food intake and their sensitivity to leptin control of food intake (13). Oleoyl-estrone decreases leptin synthesis in lean rats (22), and exerts effects similar to those of leptin on appetite and energy expenditure (15). Since oleoyl-estrone also affects fa/fa rats (2), in which leptin is not functional because of defective leptin receptors (18), it may be assumed that the point of action of oleoyl-estrone is downstream of the leptin-responding sites. In any case, these effects do not include modulation of food intake by means of NPY. The NPY levels in hypothalamic nuclei not only reflect changes in food intake or appetite modulation, since these levels are also related to the operation of the thermogenic apparatus (5), especially the PVN and arcuate nuclei (16). Increases in NPY levels in the PVN and arcuate nucleus lower body temperature (8), but this process is not achieved through direct effects on brown adipose tissue and uncoupling-protein expression (7). Again, the lack of changes in NPY run counter to the effects observed in the whole rat energy budget. This suggests that the oleoyl-estrone-induced changes in energy expenditure may be due to direct action on brown adipose tissue, or at least downstream of the hypothalamus. The data shown indicate that oleoyl-estrone does not act through NPY in its control of either food intake or thermogenesis. This is applicable both to lean and genetically obese rats in which the leptinergic pathway is not operative. ACKNOWLEDGEMENTS This work was fully financed by Laboratoris SALVAT, SA. Thanks are given to Robin Rycroft from the Language Advisory Service at the University of Barcelona for correction of the text. The work was carried out within the framework of the EC Network on Metabolic integration and energy control ERBCHRX-CT94 – 0490.
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Increased neuropeptide Y secretion in the hypothalamic paraventricular nucleus of obese (fa/fa) Zucker rats. Brain Res. 690:185–188; 1995. Erickson, J. C.; Clegg, K. E.; Palmiter, R. D. Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y. Nature (London). 381:415– 418; 1996. Erickson, J. C.; Hollopeter, G.; Palmiter, R. D. Attenuation of the obesity syndrome of ob/ob mice by the loss of neuropeptide Y. Science (Washington DC). 274:1704 –1707; 1996. Halaas, J. L.; Gajiwala, K. S.; Maffei, M.; Cohen, S. L.; Chait, B. T.; Rabinowitz, D.; Lallone, R. L.; Burley, S. K.; Friedman, J. M. Weight-reducing effects of the plasma protein encoded by the obese gene. Science (Washington DC). 269:543–546; 1995. Jolicoeur, F. B.; Bouali, S. M.; Fournier, A.; St. Pierre, S. Mapping Of hypothalamic sites involved in the effects of NPY on body-temperature and food-intake. Brain Res. Bull. 36: 125–129; 1995. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951. Phillips, M. S.; Liu, Q. Y.; Hammond, H. A.; Dugan, V.; Hey, P. J.; Caskey, C. T.; Hess, J. F. Leptin receptor missense mutation in the fatty Zucker rat. Nat. Genet. 13:18 –19; 1996. Sahu, A.; Kalra, S. P. Neuropeptidergic regulation of feedingbehavior—neuropeptide-Y. Tr. Endocrinol. Metab. 4:217–224; 1993. Sahu, A.; Kalra, S. P.; Crowley, W. R.; O’Donohue, T. L.; Kalra, P. S. Neuropeptide Y levels in microdissected regions of the hypothalamus and in vitro release in response to KCl and prostaglandin E2: effects of castration. Endocrinology. 120:1831–1836; 1987. Sahu, A.; Kalra, P. S.; Kalra, S. P. Food deprivation and ingestion induced reciprocal changes in neuropeptide Y concentrations in the paraventricular nucleus. Peptides. 9:83– 86; 1988. Sanchis, D.; Ada´n, C.; Arde´vol, A.; Grasa, M. M.; Cabot, C.; Balada, F.; Vila´, R.; Estruch, J.; Puerta, M. L.; Ferna´ndez– Lo´pez, J. A.; Remesar, X.; Alemany, M. Short-term treatment with oleoyl-estrone in liposomes (Merlin-2) strongly reduces the expression of the ob gene in young rats. Biochem. J. 326:357–360; 1997.
1635 23. Sanchis, D.; Balada, F.; Grasa, M. M.; Virgili, J.; Peinado, J.; Monserrat, C.; Ferna´ndez–Lo´pez, J. A.; Remesar, X.; Alemany, M. Oleoyl-estrone induces the loss of body fat in rats. Int. J. Obesity. 20:588 –594; 1996. 24. Sanchis, D.; Balada, F.; Pico´, C.; Grasa, M. M.; Virgili, J.; Farrerons, C.; Palou, A.; Ferna´ndez–Lo´pez, J. A.; Remesar, X.; Alemany, M. Rats receiving the slimming agent oleoyl-estrone in liposomes (Merlin-2) decrease food intake but maintain thermogenesis. Arch. Physiol. Biochem. 105:663–672; 1997. 25. Schwartz, M. W.; Baskin, D. G.; Bukowski, T. R.; Kuijper, J. L.; Foster, D.; Lasser, G.; Prunkard, D. E.; Porte, D.; Woods, S. C.; Seeley, R. J.; Weigle, D. S. Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes. 45:531–535; 1996. 26. Stanley, B. G. Neuropeptide Y in multiple hypothalamic sites controls eating behavior, endocrine, and autonomic systems for body energy balance. In: Colmers, W.; Wahlestedt, C., Eds. The biology of neuropeptide Y and related peptides. Totowa NJ: Humana; 1993:457–509. 27. Stephens, T. W.; Basinski, M.; Bristov, P. K.; Bue–Valleskey, J. M.; Burgett, S. G.; Craft, L.; Hale, J.; Hoffmann, J.; Hsiung, H. M.; Kriaucinas, A.; MacKellar, W.; Rosteck, P. R.; Schoner, B.; Smith, D.; Tinsley, F. C.; Zhang, X. -Y.; Helman, M. The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature (London). 377:530 –532; 1995. 28. Tatemoto, K.; Carlquist, M.; Mutt, V. Neuropeptide Y —a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature (London). 296:659–660; 1982. 29. Wang, Q.; Bing, C.; Al–Barazanji, K.; Mossakowaska, D. E.; Wang, X-M.; McBay, D. L.; Neville, W. A.; Taddayon, M.; Pickavance, L.; Dryden, S.; Thomas, M. E. A.; McHale, M. T.; Gloyer, I. S.; Wilson, S.; Buckingham, R.; Arch, J. R. S.; Trayhurn, P.; Williams, G. Interactions between leptin and hypothalamic Neuropeptide Y neurons in the control of food intake and energy homeostasis in the rat. Diabetes. 46: 335–341; 1997. 30. Yoshihara, T.; Honma, S.; Katsuno, Y.; Honma, K. Dissociation of paraventricular NPY release and plasma corticosterone levels in rats under food deprivation. Am. J. Physiol. 271:E239-E245; 1996.
PII S0196-9781(98)00104-1
BRIEF COMMUNICATION
Oleoyl-Estrone Does Not Alter Hypothalamic Neuropeptide Y in Zucker Lean and Obese Rats ´ N,*† JEU ´ S PE´REZ–CLAUSELL,‡ CRISTINA CABOT,*† MARIA DEL MAR GRASA,*† CRISTINA ADA ´ NDEZ–LO ¨ PEZ,* JORDI VIRGILI,* JORDI ESTRUCH,† JOSE´–ANTONIO FERNA 1 ` XAVIER REMESAR,* AND MARIA ALEMANY* *Departament de Bioquı´mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain †Laboratoris SALVAT, SA, Esplugues de Llobregat, 08950 Barcelona, Spain ‡Departament de Biologia Celzlular Animal i Vegetal, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain Received 27 February 1998; Accepted 15 June 1998 ´ N, J. PE´REZ–CLAUSELL, J. VIRGILI, J. ESTRUCH, J. A. CABOT, C., M. D. M. GRASA, C. ADA ´ NDEZ-LO ´ PEZ, X. REMESAR AND M. ALEMANY. Oleoyl-estrone does not alter hypothalamic FERNA neuropeptide Y in Zucker lean and obese rats. PEPTIDES 19(9) 1631–1635, 1998.—Female Zucker lean and obese rats were treated for 14 days with 3.5 mmol/kg oleoyl-estrone (OE) in liposomes (Merlin-2). After 0, 3, 6, 10, and 14 days of treatment, the rats were killed and hypothalamic nuclei (lateral preoptic, median preoptic, paraventricular, ventromedial and arcuate) were used for neuropeptide Y (NPY) radioimmunoassay. In 14 days, OE decreased food intake by 26% in lean and 38% in obese rats and energy expenditure by 6% in lean and 47% in obese rats; the body weight gap between controls and treated rats becoming 217.8% of initial b.wt. in the lean and 213.6% in the obese rats. Obese rats showed higher NPY levels in all the nuclei than the lean rats. Despite a negative energy balance and decreased food intake, there were practically no changes in NPY with OE treatment. The results indicate that oleoyl-estrone does not act through NPY in its control of either food intake or thermogenesis in lean and genetically obese rats. © 1998 Elsevier Science Inc. Neuropeptide Y Zucker obese rat
Oleoyl-estrone Merlin-2
Appetite
Hypothalamus
NEUROPEPTIDE Y (NPY) is a major factor in the control of appetite in mammals (19,26). High hypothalamic concentrations are associated with increased appetite (21) and low levels with situations in which intake is reduced (5). NPY is also involved in other physiological responses to feeding and overfeeding, such as its mediation in the thermogenic response to a meal (4). The direct intraventricular injection of NPY increases appetite (27). However, it has
Energy expenditure
Obesity
been found that total absence of NPY does not result in the complete loss of appetite in NPY knock-out mice (14). Thus, not all feeding responses are directly linked to NPY synthesis and release (13); nevertheless, it is widely accepted that NPY still plays a key role in the control of appetite and food consumption. The distribution of NPY in the hypothalamus is not uniform, and changes in the different hypothalamic nuclei
1 Requests for reprints should be addressed to Prof. Dr. Maria` Alemany, Departament de Bioquı´mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, 645, 08028 Barcelona, Spain. E-mail: [email protected]
1631
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CABOT ET AL. TABLE 1
CHANGES IN BODY WEIGHT, FOOD INTAKE AND ENERGY AVAILABILITY OF ZUCKER LEAN AND OBESE RATS TREATED WITH LIPOSOMES LOADED WITH OLEOYL-ESTRONE (MERLIN-2) Parameter Boby weight Lean Obese Food intake Lean Obese Lean Obese Energy available Lean Obese Energy from internal sources Lean Obese
Units
Treated Day 0
Treated Day 3
Treated Day 6
Treated Day 10
Treated Day 14
Control Day 14
% of initial % of initial
100 100
96.3 6 0.5 98.8 6 0.7
93.5 6 0.9 97.6 6 1.0
92.2 6 2.5 96.7 6 1.4
91.8 6 1.4 96.4 6 1.8
109.6 6 1.2 110.0 6 0.7
g/day* g/day* g/day† g/day†
16.3 6 0.8 25.7 6 0.6 — —
9.4 6 0.4 14.8 6 1.5 8.1 6 1.3 14.8 6 0.8
8.1 6 0.7 15.2 6 1.2 8.8 6 0.6 14.3 6 0.7
9.4 6 0.8 15.2 6 1.0 9.1 6 0.6 14.2 6 0.6
10.7 6 0.5 15.0 6 1.4 11.1 6 1.1 14.8 6 0.6
14.5 6 0.4 24.2 6 2.0 14.2 6 1.4 23.9 6 6.9
W W
2.36 4.34
1.78 2.77
1.64 2.68
1.51 2.75
1.98 2.46
2.10 4.62
% of energy available‡ % of energy available‡
29.5§ 29.4§
9.5 9.9
22 12.7
5.5 21.8
29.5 29.4
23.1 9.6
The data are the mean 6 SEM of five to seven different animals. * Intake during the 24 hours preceding the day in which the animals were weighed. † Mean daily intake for the 3– 4-day period between the days marked for measurement in the Table. ‡ A positive value means a net withdrawal of substrates from body stores, a negative value represents net deposition of live matter. § This figure was not measured, but it is assumed to be the same as for Day 14. Statistical comparison between groups (ANOVA). Weight: obese vs. lean p 5 0.0001; effect of time: lean p 5 0.0051, obese p 5 0.0035. Intake: obese vs. lean p 5 0.0000; effect of time: lean p 5 0.0000, obese p 5 0.0004.
have been related to marked behavioral differences (9). Injection of NPY into the paraventricular nucleus (PVN) maximally enhances food intake, but injection into other hypothalamic areas also increases appetite (11). Increases in NPY content of PVN are associated with diet-induced thermogenesis (6). NPY is produced by the arcuate nucleus which projects a dense set of axons rich in NPY into the PVN nucleus (1). Oleoyl-estrone in liposomes (Merlin-2) induces rapid and significant losses in the fat stores of treated rats (23). Oleoyl-estrone slimming effects are found in Zucker obese rats (2) and dietary-obese animals (3). Since the slimming process is achieved by decreasing food intake and maintaining heat production (23,24), we devised this study to determine whether the oleoyl-estrone-induced loss of appetite is mediated by decreases in NPY concentrations in the hypothalamic areas related with NPY management and the control of appetite. METHOD Zucker lean (Fa/?) and obese (fa/fa) female rats weighing 170 –190 g (lean) or 370 – 410 g (obese) from Charles River (Aubin–les–Elbeuf, France) stock were used. They were maintained under standard conditions (21°C, 60 –70% relative humidity, lights on from 08:00 a.m. to 8:00 p.m.), and fed standard rat chow pellets (B&K, Sant Vicenc¸ dels Horts, Spain) ad libitum. The rats were handled following the guidelines established by the European Community and the Governments of Catalonia and Spain.
Oleoyl-estrone was synthesized and incorporated into liposomes (23) containing 20% lipid. This preparation was used to fill osmotic minipumps (Alzet 2ML-2 from Alza, Palo Alto, CA USA), which were inserted (under ethyl ether anesthesia) under the skin on the back of the rats, and connected via a short capillary tube (PE-10 polyethylene from Becton–Dickinson, Parsippanny, NJ USA) to the left jugular vein. The minipumps released 5 ml/h for 14 days at a constant rate; the dose administered to the rats was 3.5 mmol/dayzkg of b.wt. Control (lean and obese) rats were included in the study; they were implanted with minipumps containing only the liposome suspension, without oleoylestrone. Rat weight and food consumption was recorded daily. The energy output of each rat was calculated from the energy ingested (g of pellet food with a nominal energy of 14.6 kJ/g), and from the changes in body mass. It was assumed that the energy derived from loss of body weight under the effects of Merlin-2 was similar to that found under comparable circumstances (2), i.e., a mean value of 14.4 kJ per g of weight lost and 13.1 kJ per g of weight increase, since in the first case the loss is mainly fat, and in the second, protein accounts for half the energy deposited. Using these approximate values, the mean energy output of the rats was calculated and expressed in W. On Days 0 (intact rats), 3, 6, 10, and 14 after the implantation of the minipumps, groups of five treated rats (and liposome-receiving controls on Day 14) were killed by decapitation and their brain was carefully dissected, frozen
OLEOYL-ESTRONE EFFECT ON NPY
in liquid nitrogen and kept at 280°C until further processing. Brains were placed in a freezing microtome and frontal sections were cut. The slices, 400 –500 mm thick were manipulated under a stereomicroscope in the cold (0 –2°C). The anatomical characteristics of hypothalamus were noted and the position of the five main nuclei and areas were identified and dissected through microdissection using fine scalpels and a sampling trochanter needle of 0.8 mm internal diameter, which was used to punch sampling holes for the smaller areas. Serial dissection of the 8 –9 slices containing the hypothalamus produced samples from lateral preoptic (LPO) and median preoptic (MPO) areas, and paraventricular (PVN), ventromedial (VMH) and arcuate (ARC) nuclei. The samples were immediately placed in 0.400 ml of chilled 0.5 M acetic acid for acidic NPY extraction; they were then heated at 100°C for 10 min. After cooling, the samples were sonicated in the cold for 15 s. A 0.100-ml aliquot was used for protein estimation (17). The remaining homogenate was centrifuged at 15,000 3 g for 15 min. The supernatant was concentrated and evaporated under vacuum, and kept at 220°C until further processing. The dry supernatants were diluted with 0.700 ml of buffer and used for the estimation of NPY by radioimmunoassay (10,20). The antibody used (cat N9528; Sigma, St Louis, MOUSA) was a polyclonal rabbit serum raised against porcine NPY. In order to determine the suitability of the radioimmunoassay for the measurement of rat NPY, we run HPLC separations of a whole hypothalamic extract of rat with or without added porcine NPY. We used a Kontron (Milano, Italy) HPLC connected to a Bio–Rad (mod. 2128, Hughes, CA USA) fraction collector. We used a Spherisorb ODS 5 mm (250 3 4.5 mm; Waters, Milford, MA USA) column with a C18 precolumn at 9,7 Mpa at 1 ml/min and a linear gradient elution system of 1.2 ml/l trifluoroacetic acid in water (A) and 1.0 ml/l of trifluoroacetic acid in acetonitrile (B) set at 65A/35B at t 5 0 min and 50A/50B at t 5 40 min (28). The eluates were recovered in 1 ml (1 min) fractions which were used for radioimmunoassay as indicated above. Hypothalamic extracts showed a single peak at 32 min that was coincident with that of porcine NPY. The suitability of the extraction and radioimmunoassay procedures for rat NPY was thus established. Groups were compared by ANOVA test, using a limit of statistical significance of 0.05. RESULTS Table 1 shows the changes in body weight, food intake and energy availability of the rats treated with oleoyl-estrone. All treated rats lost weight by 8.2% (lean) or 3.6% (obese) on Day 14. Controls receiving liposomes but not oleoylestrone increased in weight by a mean of 9.6% (lean) or 10.0% (obese), which sets the gap due to Merlin-2 treatment at 17.8% (lean) or 13.6% (obese) after 14 days of treatment.
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FIG. 1. Concentrations of NPY in the lateral preoptic (LPO), median preoptic (MPO) and ventromedial (VMH) areas, and paraventricular (PVN) and arcuate (ARC) nuclei of the hypothalamus from Zucker lean and obese rats treated with oleoyl-estrone in liposomes. The open symbols correspond to lean and the black symbols to obese rats. The data are the mean 6 SEM of four animals per group. Post-hoc Duncan test results for lean rats MPO: The 6-day value was significantly different from all other time points.
Mean energy expenditure for treated rats (14 days) was 94.3% of that of controls in lean rats and 53.3% in the obese. The corresponding figures for energy intake were 73.8% of controls in lean rats and 62.0% in the obese. Figure 1 shows the levels of NPY in the five selected hypothalamic locations during the treatment with Merlin-2. The changes in protein concentration were minimal (and non-significant) in the period studied. Obese rats (both controls and oleoyl-estrone-treated rats) showed higher NPY concentrations in all the areas and nuclei, and at practically all times studied, the differences being signifi-
1634
CABOT ET AL.
cant for all areas. NPY levels in the LPO, PVN, VMH, and ARC of lean and obese rats did not change significantly during oleoyl-estrone treatment. In the MPO area, again obese rats showed no change but lean rats decreased the NPY levels by mid-treatment. In all cases, the liposometreated controls showed values not different from those of Day 0 controls. DISCUSSION Treatment of lean and obese rats with oleoyl-estrone in liposomes results in the loss of energy stores, mainly in the form of fat, with a little loss of protein (23,24). This is achieved by a decrease in appetite and a relative maintenance of energy output (24). This applies both to lean and genetically obese Zucker rats (2). In the present study we observed that the loss of body weight was parallel to decreased food intake (appetite?) and maintained their energy output in lean rats and lowered it in the obese. The small changes in NPY levels—if any— observed in the different hypothalamic nuclei during oleoyl-estrone treatment contrast with the alterations observed during starvation (30) and modulation of appetite (4,21,26). The decreases in food intake induced by Merlin-2 were not paralleled by significant changes in NPY neither in obese nor in lean rats, which contrasts with the purported orexic effect of the peptide (5,19). The clear absence of decreases in NPY in either lean or obese rats in parallel to the decrease in food intake elicited by oleoyl-estrone suggests that this is not mediated by NPY. This does not seem to alter the basic status of Zucker obese rats, which maintain higher NPY levels (12) and also show a higher basal food intake than lean rats. Leptin has been postulated to decrease hypothalamic
NPY (25,29), but mice lacking NPY maintain food intake and their sensitivity to leptin control of food intake (13). Oleoyl-estrone decreases leptin synthesis in lean rats (22), and exerts effects similar to those of leptin on appetite and energy expenditure (15). Since oleoyl-estrone also affects fa/fa rats (2), in which leptin is not functional because of defective leptin receptors (18), it may be assumed that the point of action of oleoyl-estrone is downstream of the leptin-responding sites. In any case, these effects do not include modulation of food intake by means of NPY. The NPY levels in hypothalamic nuclei not only reflect changes in food intake or appetite modulation, since these levels are also related to the operation of the thermogenic apparatus (5), especially the PVN and arcuate nuclei (16). Increases in NPY levels in the PVN and arcuate nucleus lower body temperature (8), but this process is not achieved through direct effects on brown adipose tissue and uncoupling-protein expression (7). Again, the lack of changes in NPY run counter to the effects observed in the whole rat energy budget. This suggests that the oleoyl-estrone-induced changes in energy expenditure may be due to direct action on brown adipose tissue, or at least downstream of the hypothalamus. The data shown indicate that oleoyl-estrone does not act through NPY in its control of either food intake or thermogenesis. This is applicable both to lean and genetically obese rats in which the leptinergic pathway is not operative. ACKNOWLEDGEMENTS This work was fully financed by Laboratoris SALVAT, SA. Thanks are given to Robin Rycroft from the Language Advisory Service at the University of Barcelona for correction of the text. The work was carried out within the framework of the EC Network on Metabolic integration and energy control ERBCHRX-CT94 – 0490.
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