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Unexpected regulation of hypothalamic neuropeptide Y by food deprovation and refeeding in the Zucker rat.
Life Sciences, Vol. 50, pp. 923-930 Printed in the U S A
Pergamon P r e s s
UNEXPECTED R E G U L A T I O N OF H Y P O T H A L A M I C N E U R O P E P T I D E Y BY F O O D D E P R I V A T I O N A N D R E F E E D I N G IN T H E Z U C K E R RAT.
Bernard Beck, Arlette Burlet, Jean-Pierre Nicolas, Claude Burlet INSERM U. 308 - M~canismes de R~gulation du Comportement Alimentaire 38, rue Lionnois 54000 N A N C Y (FRANCE) (Received in final form January 21, 1992)
Summary Neuropeptide Y strongly stimulates food intake when it iS injected in the hypothalamic paraventricular (PVN) and ventromedian (VMN) nuclei, in Sprague-Dawley (SD) rats, NPY synthesis in the amuate nucleus (ARC) is increased by food deprivation and is normalized by refeeding. We have previously shown that the obese hyperphagic Zucker rat is characterized by higher NPY concentrations in this nucleus. NPY might therefore play an important role in the development of hyperphagia. The aim of the present study was to determine if the regulation by the feeding state works in the obese Zucker rat. For this purpose, 10 weeks-old male lean (n=30) and obese (n=30) Zucker rats were either fed ad libitum, either food-deprived (FD) for 48 hours or food-deprived for 48 h and refed (RF) for 6 hours. NPY was measured in several microdissected brain areas involved in the regulation of feeding behavior. NPY concentrations in the ARC was about 50 % greater in obese rats than in lean rats (p < 0.02) whatever the feeding state. In the VMN, NPY concentrations were higher in the lean FD rats than in the obese FD rat (p < 0.001). Food deprivation or refeeding did not modify NPY in the ARC, in the VMN or in the dorsomedian nucleus whatever the genotype considered. On the other hand, food deprivation induced a significant decrease in NPY concentrations in the PVN of lean rats. This decrease was localized in the parvocellular part of this nucleus (43.0 _+1.9 (FD) vs 54.2 _+2.1 (Ad lib) ng/mg protein ; p < 0.005). Ad lib levels were restored by 6 hours of refeeding. These variations were not observed in the obese rat. The regulation of NPY by the feeding state in the Zucker rat was therefore very different from that described in the SD rats. Strain or age of the animals used might explain these differences. High NPY levels and absence of regulation in obese Zucker rats could contribute to the abnormal feeding behavior of these rats.
Neuropeptide Y (NPY) strongly stimulates food intake when injected centrally in the third brain ventricle or in different hypothalamic nuclei (1-4). In Sprague Dawley rats, hypothalamic NPY is regulated by the feeding state of the animals since food deprivation during 2 or 3 days induces an increase in NPY concentrations in the arcuate and paraventricular nuclei (5-7) whereas a refeeding period of at least 6 to 24 hours normalizes these levels. We have previously shown that the obese hyperphagic Zucker rat is characterized by higher N P Y concentrations in different hypothalamic nuclei involved in the regulation of feeding behavior (8). This was particularly observed in the arcuate nucleus, the main site of NPY synthesis (9-10) and confirmed by increases of expression of NPY mRNA in this nucleus in the fatty rat (11). N P Y might
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press plc All rights reserved.
924
NPY and F e e d i n g S t a t e in t h e Z u c k e r Rat
Vol.
50, No.
13,
1992
play an important role in the development of hyperphagia and obesity since chronic continuous intracerebroventricular (ICV) infusion of NPY through Alzet minipumps perfectly mimicks the feeding behavior of the fatty rats (12). These effects are also observed with repeated PVN injection of NPY (13). From these results, we hypothesised that the regulatory mechanisms for the hypothalamic NPY could also be deficient in the obese Zucker and insensible to the feeding state. That is why we measured NPY in several microdissected hypothalamic nuclei after 2 days of food deprivation and after a period of 6 hours of refeeding in lean and obese Zucker rats.
Materials and Methods Animals Ten weeks-old lean Fa/Fa (n = 27) and obese fa/fa (n = 27) Zucker rats were housed in individual wire cages in a temperature controlled room with an automatic 12 h / 12 h light-dark (L/D) cycle with lights on at 2 P.M.. They were fed a standard chow diet (U.A.R. A04 - Villemoisson sur Orge - France) ad libitum. After 15 days of habituation to these conditions, lean and obese rats were randomly distributed in 3 groups of 9 animals. The first group continued to be fed on the same diet ad libitum, the second one was food-deprived (FD) for 48 hours and the third one was food-deprivated for 48 hours and refed (RF) for 6 hours. Tap water was always available to all rats ad libitum. All rats were killed by decapitation at the same time at the beginning of the light period (3.30 P.M.) because NPY follows a nycthemeral rhythm in several hypothalamic nuclei (14). Body weight and food intake were measured during the experiment.
Samolinas Trunk blood was sampled in ice-cooled tubes containing EDTA and aprotinin (Iniprol - Laboratoires Choay - France). The brain were quickly removed, frozen on dry ice and stored at 80 °C. Serial sections of 300 I~m were cut and discrete hypothalamic and extrahypothalamic areas were microdissected. The landmarks for this dissection were derived from a standard atlas of the rat brain (15). The following areas were sampled : paraventricular (PVN ; magno and parvocellular parts), dorsomedian (DMN), ventromedian (VMN), arcuate (ARC), accumbens (ACC), supraoptic (SON) nuclei, median eminence (ME), lateral hypothalamus (LH) and parietal cortex (CX). The bilateral tissue samples were placed in 500 ILl of a solution of 0.2 N HCI aprotinin EDTA and stored at - 40 °C until extraction and assay. -
Assays Plasma glucose (BG), triglycerides (TG) and beta-hydroxybutyrate (OHB) were measured with classical enzymatic procedures using commercial kits (Boehringer Mannheim - Meylan (France) for TG and BG and Sigma Diagnostics - La Verpillii~re (France) for OHB). Plasma immunoreactive insulin (IRI) was measured with a single antibody-charcoal radioimmunoassay kit (CIS- Gif sur Yvette, France) and plasma corticosterone with a commercial kit ( HCORT kit, Biom~rieux, France). Neuropeptide Y was extracted and measured with a specific radioimmunoassay developed in our laboratory and previously described (8). For this experiment, the maximal binding was 41.6 _ 4.0 % and non specific binding averaged 5.3 + 0.5 %.
Vol.
50, No. 13, 1992
NPY and Feeding State in the Zucker Rat
925
Statistical analvsis The results are presented as mean + SEM. They were compared by two ways analysis of variance for main effects (treatment x genotype) and adequate multiple range (Fisher PLSD) tests. A probability of less than 5 % (two-tailed) was considered significant. Results Body weioht and food intake Body weight (BW) variations are shown in fig. 1. Initial body weight between the 3 lean groups or between the 3 obese groups were not significantly different but the obese rats were significantly heavier than the lean rats with an overweight reaching 60 % (p < 0.001). During food deprivation, both lean and obese rats regularly lose weight (p < 0.001). After 48 h without food, the weight loss of lean rats averaged 32.2 + 1.6 g (14.5 % of initial BW) and was significantly smaller than that of obese rats (42.0 + 1.2 g ; 12.0 % of initial BW p < 0.001). The obese rats refed for 6 h gained significantly more weight than the lean rats (22.3 + 0.8 vs 19.3 g ; p < 0.05).
380"
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Hours Fig.1 Body weight variations (mean + SEM in grams) in the obese (top) and lean (bottom) Zucker rats. • ..... • : Ad libitum fed rats; o ..... o "food-deprived rats; • ..... • : refed rats
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Obese rats fed ad libitum ate approximately 50 % more food than the lean rats during the 48 hours (55.9 + 1.8 vs 36.3 + 0.8 g ; p < 0.001). During the 6 hours of refeeding, food intake of both groups did not significantly differ [11.8 + 0.4 (obese) vs 10.5 + 0.8 g (lean)].
Plasma Plasma glucose, triglycerides, beta hydroxybutyrate, IRI and NPY concentrations are shown in table 1. Obese rats showed elevated levels of TG, IRI and CORT when compared with the lean rats (p < 0.05 or less). Food deprivation induced a decrease in plasma glucose, IRI and TG and an increase in beta hydroxybutyrate and CORT in both groups. Refeeding induced increases of plasma glucose over the ad libitum values in lean and obese rats and restored the C O R T levels in both groups, but IRI and OHB levels in the obese group only. Plasma NPY which was not different between lean and obese rats was not affected by the change of feeding state.
TABLE 1 Lean
Obese
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ad Lib
FD
. .
RF
. . . . . . . . .
. . . . . .
Ad Lib
.
. . . . . . . . . .
FD
. . . . . .
.
. . . . . . . . . .
. . .
RF
PG1
6.88 +_0.11
6.10 a +0.22
8.27 b + 0.27
7.10 +0.16
5.33 a _+0.22
8.32 b + 0.27
OHB 1
0.39 +0.03
0.97 a +- 0.08
0.77b + 0.08
0.70 +0.04
3.17 a _+0.32
0.55 +_0.05
TG1
0.49 _+0.04
0.31 a _+0.02
0.68b ~+ 0.06
1.89 + 0.11
1.38a _+0.11
1.24b _+0.07
IRI1
3.2 + 0.4
1.3a -+ 0.3
4.3b _+0.2
19.1 _+2.2
6.0a + 1.0
19.2 +- 2.3
NPY2
86.5 + 3.2
91.4 + 4.3
89.7 + 5.7
89.1 + 6.0
95.9 +_6.0
89.8 + 5.5
CORT2
108.0 + 20.2
322.6a _+23.0
128.1 _+25.7
176.3 -+ 17.8
330.6a + 24.4
183.7 _+28.4
Plasma concentrations of glucose (PG), hydroxybutyrate (OHB), triglycerides (TG), immunoreacUve insulin (IRI), neuropeptide Y (NPY) and corticosterone (CORT) in lean and obese Zucker rats either fed ad libitum (Ad lib), either food-deprived for 48 hours (FD) or food-deprived for 48 hours and refed for 6 hours (RF). 1 : mrnol/I;2 : ng/ml; a : significantly different from the two other groups with the same genotype with p < 0.002 or less; b : significantly different from ad lib values of the same genotype. Brain NPY
concentrations
in the
arcuate,
ventromedian,
paraventricular
and
Vol. 50, No. 13, 1992
1001
NPY and Feeding State in the Zucker Rat
1°°t 80]
ARC
8O z 60
0. 60 z
4O
40
PVN
20 0 Lean
z
Lean
Obese
100-
1001
80
80 t
eo
60
40
40
20
20
0
Obese
DMN
0 Lean
Obese
Lean
Obese
Fig. 2 Neuropeptide Y (NPY in ng/mg protein; mean _+ SEM) in the arcuate (ARC), paraventricular (PVN), ventromedian (VMN) and dorsomedian (DMN) nuclei of lean and obese Zucker rats either fed ad libitum (filled bars), either food-deprived for 48 hours (open bars) or food-deprived for 48 hours and refed for 6 hours (stripped
bars)
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dorsomedian nuclei are shown in fig. 2. There was a significant difference between the two genotypes in the arcuate and ventromedian nuclei. In the arcuate nucleus, obese rats had about 50 % greater NPY concentrations than the lean rats whatever the feeding state (ad lib 92.3 + 5.9 vs 66.2 + 7.2 ng/mg protein ; RF : 92.2 + 4.3 vs 67.3 + 4.4 ng/mg protein ; FD : 94.5 + 7.3 vs 63.1 + 5.5 ng/mg protein ; p < 0.02, p < 0.005 and p < 0.005 respectively). In the VMN, in the absence of food, obese rats had significantly smaller NPY concentrations than the lean rats (56.7 + 1.8 vs 70.7 + 2.0 ng/mg protein ; p < 0.001). In the other microdissected areas, there was not any significant variations except in the median eminence where the NPY concentrations were higher in the obese rats than in the lean rats (ad lib : 63.9 + 7.1 vs 40.4 + 4.9 ng/mg protein ; RF : 77.4 + 5.2 vs 33.7 + 2.5 ng/mg protein ; FD : 69.0 + 3.8 vs 40.5 + 3.9 ng/mg protein ; p< 0.02, p < 0.001 and p < 0.001 respectively).
Food deprivation or refeeding did not modify NPY concentrations either in the arcuate, ventromedian or dorsomedian nuclei but variations were noted in the paraventricuiar nucleus (p = 0.034). A significant decrease of NPY was measured in the PVN of lean food-deprived rats [58.4 + 1.5 vs 66.1 +%_2.4 (Ad lib) and vs 66.8 + 2.4 (RF) ; p < 0.05 and 0.025 respectively]. No variations were observed in the PVN of the obese rats. There was no treatment x genotype interaction (p=0.81). In the lean rats, the decrease was localized in the parvocellular part of the PVN [43.0 + 1.9 (FD) vs 54.2 + 2.1 (Ad libitum) and 57.8 + 1.8 (RF) ng/mg protein - p < 0.005 and 0.001 respectively]. No variations were noted in the magnocellular part of PVN of these rats. Refeeding restored NPY concentrations to levels comparable to ad lib levels in the PVN of lean rats.
Discussion In this experiment, we studied the effect of food deprivation and refeeding on plasma and hypothalamic NPY concentrations in lean and obese rats. Plasma NPY concentrations were not different between lean and obese Zucker rats and they were not modified by the feeding state unlike the case with plasma insulin and corticosterone concentrations. The classical effects of food deprivation (increase in ketone bodies and decrease in plasma glucose) were also observed. This confirms some of our previous results on the non-intervention of peripheral NPY in the regulation of feeding behavior (8,16). On the other hand, central and more precisely hypothalamic NPY varied.
First, we measured very high NPY concentrations in the arcuate nucleus of the obese rat. This increase in NPY concentrations in the fatty rat has been previously reported (8) and is related to an increase in NPY synthesis since expression of NPY mRNA is increased in the arcuate nucleus of obese rats (11). NPY is mainly synthesized in this nucleus and in the brainstem (9-10). NPY neurons of the ARC projects to the VMN, DMN and PVN to form a dense NPY network (17) and food intake is strongly stimulated when NPY is injected in two of these nuclei namely PVN and VMN (3-4). The paraventricular nucleus also receives NPY efferents from the brainstem (18). A study of Sahu and coworkers has recently shown that food intake is not modified when these
Vol.
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NPY and Feeding State in t h e Z u c k e r R a t
929
efferents and all connections from the brainstem are cut (19) and others reported that NPY mRNA content in the brainstem did not vary with food deprivation (20, 21). NPY originating from the ARC seems therefore to play a major role in the regulation of feeding behavior. Moreover, we demonstrated with different experimental manipulations that the arcuate-paraventricular nucleus axis plays a preponderant role in the NPY network (22). In the present study, NPY in the ARC was not modified by food deprivation either in the lean or in the obese rats. As Sanacora and coworkers have shown that a period of 72 hours of food deprivation induces a two-fold increase in the preproNPY mRA in 14 weeks-old lean Zucker rats (11), the absence of effect on NPY concentrations in our experiment might result from an increased transport from the synthesis site to the target nuclei. However, in two of these nuclei (DMN and VMN), there was no significant variations whereas in the PVN, there was a significant NPY decrease. This decrease might reflect an intense liberation of the peptide. This phenomenom was slowed down by refeeding since NPY levels in lean RF rats were comparable to those of ad lib lean rats.
This regulatory mechanism is very different from that described in Sprague-Dawley rats. Indeed, we and others (5,6) have reported an increase in NPY concentrations in the arcuate nucleus following a fasting period of 48 or 72 hours. This increase was associated with an increase in NPY mRNA expression (23, 24). A concomittant NPY increase was observed in the PVN (5-7). This strain difference is perhaps also explained by the age or body weight of our lean animals which are younger and lighter than the Sprague-Dawley rats used in the previous experiments.
The second information of this experiment is that the mechanism existing in the lean rats does not work in the obese rat. It is possible that the NPY release has already reached maximum levels because of the higher NPY levels in the ARC. Only in vivo studies on NPY release through push-pull or microdialysis techniques could confirm this deficiency as well as the mechanism in the lean rat. The absence of NPY regulation by the feeding state in the Zucker fatty rat is an additional argument supporting an important role of this peptide in the development of its obesity and hyperphagia.
Ackn0wlQdgements The authors thank Ms F. Bergerot and Mr. F. Giannangeli for their excellent technical assistance and Ms A. Turenne for preparing the manuscript.
References 1. 2. 3. 4. 5.
6.
A.S. LEVINE, and J.E MORLEY. Peptides 5 1025-1029 (1984). J.T. CLARK, P.S. KALRA, W.R. CROWLEY and S.P KALRA. Endocrinology 115 427-429 (1984). B.G.STANLEY,A.S. CHIN and S.F. LEIBOWlTZ. Brain Res. Bull. 14 521-524 (1985). J.E. MORLEY, A.S. LEVINE, B.A. GOSNELL, J. KNEIP and M. GRACE. Am. J. Physiol. 252 R599R609, 1987. A. SAHU, P.S. KALRA and S.P. KALRA. Peptides 9 83-86 (1988) B. BECK, M. JHANWAR-UNIYAL, A. BURLET, M. CHAPLEUR-CHATEAU, S.F. LEIBOWlTZ and C. BURLET. Brain Res. 528 245-249 (1990).
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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
N P Y and F e e d i n g
S t a t e in t h e Z u c k e r Rat
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L. CALZA, L. GIARDINO, N BA'I-FISTINI., M. ZANNI, S. GALENI, F. PROTOPAPA and A. VELARDO. Neurosci. Lett. 104 99-104 (1989) B. BECK, A. BURLET, J.P. NICOLAS and C. BURLET. Physiol. Behav. 47 449-453 (1990) B.M. CHRONWALL, D.A. DI MAGGIO, V.J. MASSARI, V.M. PICKELS, D.A. RUGGIERO and T.L. O'DONOHUE. Neuroscience 15 1159-1181 (1985). B.J. EVERITT, T. HOKFELT, L. TERENIUS, K. TATEMOTO, V. MUTT and M. GOLDSTEIN Neuroscience 11 443-462 (1984) G. SANACORA, M. KERSHAW, J.A. FINKELSTEIN and J.D. WHITE. Endocrinology 1~7 730-737 (1990) B. BECK, A. STRICKER-KRONGRAD, J.P. NICOLAS and C. BURLET Int. J. Obesity (in press) B.G. STANLEY, S.E. KYRKOULI, S. LAMPERT and S.F. LEIBOWlTZ. Peptides 71189-1192 (1986). M. JHANWAR-UNIYAL, B. BECK, C. BURLET, S.F. LEIBOWlTZ. Brain Res. 536 331-334 (1990) G. PAXINOS and C. WATSON The rat brain in stereotaxic coordinates. New York : Academic Press, 1982 B. BECK, A. STRICKER-KRONGRAD, A. BURLET, J.P. NICOLAS and C.BURLET. Neuropeptides 17 197-203 (1990) F.L. BAI, M. YAMANO, Y. SHIOTANI, P.C. EMSON, A.D. SMITH, J.F.POWELL and M. TOYAMA. Brain Res. 331 172-175 (1985). A. SAHU, S.P. KALRA, W.R. CROWLEY, and P.S. KALRA. Brain Res. 457 376-378 (1988). A. SAHU, M.G.DUBE, S.P. KALRA and P.S. SAHU. Peptides 91269 -1273 (1989) S.C. CHUA, R.L. LEIBEL and J. HIRSCH. Mol. Brain Res. 995-101 (1991). R.D. O'SHEA and A.L. GUNDLACH. J. Neuroendocrinol. 3 11-14 (1991) B. BECK, A. STRICKER-KRONGRAD, A. BURLET, M. JHANWAR-UNIYAL and C. BURLET. Neuroendocrinology 52 (SuDDI.1) 45 (1990) J.D. WHITE, D. OLCHOVSKY and M. KERSHAW. Mol. Cell. Neurosci. 1 41-48 (1989) L.S. BRADY, M.A. SMITH, GOLD, P.W. and M. HERKENHAM. Neuroendocrinology 52 441-447 (1990)
Pergamon P r e s s
UNEXPECTED R E G U L A T I O N OF H Y P O T H A L A M I C N E U R O P E P T I D E Y BY F O O D D E P R I V A T I O N A N D R E F E E D I N G IN T H E Z U C K E R RAT.
Bernard Beck, Arlette Burlet, Jean-Pierre Nicolas, Claude Burlet INSERM U. 308 - M~canismes de R~gulation du Comportement Alimentaire 38, rue Lionnois 54000 N A N C Y (FRANCE) (Received in final form January 21, 1992)
Summary Neuropeptide Y strongly stimulates food intake when it iS injected in the hypothalamic paraventricular (PVN) and ventromedian (VMN) nuclei, in Sprague-Dawley (SD) rats, NPY synthesis in the amuate nucleus (ARC) is increased by food deprivation and is normalized by refeeding. We have previously shown that the obese hyperphagic Zucker rat is characterized by higher NPY concentrations in this nucleus. NPY might therefore play an important role in the development of hyperphagia. The aim of the present study was to determine if the regulation by the feeding state works in the obese Zucker rat. For this purpose, 10 weeks-old male lean (n=30) and obese (n=30) Zucker rats were either fed ad libitum, either food-deprived (FD) for 48 hours or food-deprived for 48 h and refed (RF) for 6 hours. NPY was measured in several microdissected brain areas involved in the regulation of feeding behavior. NPY concentrations in the ARC was about 50 % greater in obese rats than in lean rats (p < 0.02) whatever the feeding state. In the VMN, NPY concentrations were higher in the lean FD rats than in the obese FD rat (p < 0.001). Food deprivation or refeeding did not modify NPY in the ARC, in the VMN or in the dorsomedian nucleus whatever the genotype considered. On the other hand, food deprivation induced a significant decrease in NPY concentrations in the PVN of lean rats. This decrease was localized in the parvocellular part of this nucleus (43.0 _+1.9 (FD) vs 54.2 _+2.1 (Ad lib) ng/mg protein ; p < 0.005). Ad lib levels were restored by 6 hours of refeeding. These variations were not observed in the obese rat. The regulation of NPY by the feeding state in the Zucker rat was therefore very different from that described in the SD rats. Strain or age of the animals used might explain these differences. High NPY levels and absence of regulation in obese Zucker rats could contribute to the abnormal feeding behavior of these rats.
Neuropeptide Y (NPY) strongly stimulates food intake when injected centrally in the third brain ventricle or in different hypothalamic nuclei (1-4). In Sprague Dawley rats, hypothalamic NPY is regulated by the feeding state of the animals since food deprivation during 2 or 3 days induces an increase in NPY concentrations in the arcuate and paraventricular nuclei (5-7) whereas a refeeding period of at least 6 to 24 hours normalizes these levels. We have previously shown that the obese hyperphagic Zucker rat is characterized by higher N P Y concentrations in different hypothalamic nuclei involved in the regulation of feeding behavior (8). This was particularly observed in the arcuate nucleus, the main site of NPY synthesis (9-10) and confirmed by increases of expression of NPY mRNA in this nucleus in the fatty rat (11). N P Y might
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press plc All rights reserved.
924
NPY and F e e d i n g S t a t e in t h e Z u c k e r Rat
Vol.
50, No.
13,
1992
play an important role in the development of hyperphagia and obesity since chronic continuous intracerebroventricular (ICV) infusion of NPY through Alzet minipumps perfectly mimicks the feeding behavior of the fatty rats (12). These effects are also observed with repeated PVN injection of NPY (13). From these results, we hypothesised that the regulatory mechanisms for the hypothalamic NPY could also be deficient in the obese Zucker and insensible to the feeding state. That is why we measured NPY in several microdissected hypothalamic nuclei after 2 days of food deprivation and after a period of 6 hours of refeeding in lean and obese Zucker rats.
Materials and Methods Animals Ten weeks-old lean Fa/Fa (n = 27) and obese fa/fa (n = 27) Zucker rats were housed in individual wire cages in a temperature controlled room with an automatic 12 h / 12 h light-dark (L/D) cycle with lights on at 2 P.M.. They were fed a standard chow diet (U.A.R. A04 - Villemoisson sur Orge - France) ad libitum. After 15 days of habituation to these conditions, lean and obese rats were randomly distributed in 3 groups of 9 animals. The first group continued to be fed on the same diet ad libitum, the second one was food-deprived (FD) for 48 hours and the third one was food-deprivated for 48 hours and refed (RF) for 6 hours. Tap water was always available to all rats ad libitum. All rats were killed by decapitation at the same time at the beginning of the light period (3.30 P.M.) because NPY follows a nycthemeral rhythm in several hypothalamic nuclei (14). Body weight and food intake were measured during the experiment.
Samolinas Trunk blood was sampled in ice-cooled tubes containing EDTA and aprotinin (Iniprol - Laboratoires Choay - France). The brain were quickly removed, frozen on dry ice and stored at 80 °C. Serial sections of 300 I~m were cut and discrete hypothalamic and extrahypothalamic areas were microdissected. The landmarks for this dissection were derived from a standard atlas of the rat brain (15). The following areas were sampled : paraventricular (PVN ; magno and parvocellular parts), dorsomedian (DMN), ventromedian (VMN), arcuate (ARC), accumbens (ACC), supraoptic (SON) nuclei, median eminence (ME), lateral hypothalamus (LH) and parietal cortex (CX). The bilateral tissue samples were placed in 500 ILl of a solution of 0.2 N HCI aprotinin EDTA and stored at - 40 °C until extraction and assay. -
Assays Plasma glucose (BG), triglycerides (TG) and beta-hydroxybutyrate (OHB) were measured with classical enzymatic procedures using commercial kits (Boehringer Mannheim - Meylan (France) for TG and BG and Sigma Diagnostics - La Verpillii~re (France) for OHB). Plasma immunoreactive insulin (IRI) was measured with a single antibody-charcoal radioimmunoassay kit (CIS- Gif sur Yvette, France) and plasma corticosterone with a commercial kit ( HCORT kit, Biom~rieux, France). Neuropeptide Y was extracted and measured with a specific radioimmunoassay developed in our laboratory and previously described (8). For this experiment, the maximal binding was 41.6 _ 4.0 % and non specific binding averaged 5.3 + 0.5 %.
Vol.
50, No. 13, 1992
NPY and Feeding State in the Zucker Rat
925
Statistical analvsis The results are presented as mean + SEM. They were compared by two ways analysis of variance for main effects (treatment x genotype) and adequate multiple range (Fisher PLSD) tests. A probability of less than 5 % (two-tailed) was considered significant. Results Body weioht and food intake Body weight (BW) variations are shown in fig. 1. Initial body weight between the 3 lean groups or between the 3 obese groups were not significantly different but the obese rats were significantly heavier than the lean rats with an overweight reaching 60 % (p < 0.001). During food deprivation, both lean and obese rats regularly lose weight (p < 0.001). After 48 h without food, the weight loss of lean rats averaged 32.2 + 1.6 g (14.5 % of initial BW) and was significantly smaller than that of obese rats (42.0 + 1.2 g ; 12.0 % of initial BW p < 0.001). The obese rats refed for 6 h gained significantly more weight than the lean rats (22.3 + 0.8 vs 19.3 g ; p < 0.05).
380"
Q
2800 m
180
I
0
24
!
48 54
Hours Fig.1 Body weight variations (mean + SEM in grams) in the obese (top) and lean (bottom) Zucker rats. • ..... • : Ad libitum fed rats; o ..... o "food-deprived rats; • ..... • : refed rats
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Obese rats fed ad libitum ate approximately 50 % more food than the lean rats during the 48 hours (55.9 + 1.8 vs 36.3 + 0.8 g ; p < 0.001). During the 6 hours of refeeding, food intake of both groups did not significantly differ [11.8 + 0.4 (obese) vs 10.5 + 0.8 g (lean)].
Plasma Plasma glucose, triglycerides, beta hydroxybutyrate, IRI and NPY concentrations are shown in table 1. Obese rats showed elevated levels of TG, IRI and CORT when compared with the lean rats (p < 0.05 or less). Food deprivation induced a decrease in plasma glucose, IRI and TG and an increase in beta hydroxybutyrate and CORT in both groups. Refeeding induced increases of plasma glucose over the ad libitum values in lean and obese rats and restored the C O R T levels in both groups, but IRI and OHB levels in the obese group only. Plasma NPY which was not different between lean and obese rats was not affected by the change of feeding state.
TABLE 1 Lean
Obese
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ad Lib
FD
. .
RF
. . . . . . . . .
. . . . . .
Ad Lib
.
. . . . . . . . . .
FD
. . . . . .
.
. . . . . . . . . .
. . .
RF
PG1
6.88 +_0.11
6.10 a +0.22
8.27 b + 0.27
7.10 +0.16
5.33 a _+0.22
8.32 b + 0.27
OHB 1
0.39 +0.03
0.97 a +- 0.08
0.77b + 0.08
0.70 +0.04
3.17 a _+0.32
0.55 +_0.05
TG1
0.49 _+0.04
0.31 a _+0.02
0.68b ~+ 0.06
1.89 + 0.11
1.38a _+0.11
1.24b _+0.07
IRI1
3.2 + 0.4
1.3a -+ 0.3
4.3b _+0.2
19.1 _+2.2
6.0a + 1.0
19.2 +- 2.3
NPY2
86.5 + 3.2
91.4 + 4.3
89.7 + 5.7
89.1 + 6.0
95.9 +_6.0
89.8 + 5.5
CORT2
108.0 + 20.2
322.6a _+23.0
128.1 _+25.7
176.3 -+ 17.8
330.6a + 24.4
183.7 _+28.4
Plasma concentrations of glucose (PG), hydroxybutyrate (OHB), triglycerides (TG), immunoreacUve insulin (IRI), neuropeptide Y (NPY) and corticosterone (CORT) in lean and obese Zucker rats either fed ad libitum (Ad lib), either food-deprived for 48 hours (FD) or food-deprived for 48 hours and refed for 6 hours (RF). 1 : mrnol/I;2 : ng/ml; a : significantly different from the two other groups with the same genotype with p < 0.002 or less; b : significantly different from ad lib values of the same genotype. Brain NPY
concentrations
in the
arcuate,
ventromedian,
paraventricular
and
Vol. 50, No. 13, 1992
1001
NPY and Feeding State in the Zucker Rat
1°°t 80]
ARC
8O z 60
0. 60 z
4O
40
PVN
20 0 Lean
z
Lean
Obese
100-
1001
80
80 t
eo
60
40
40
20
20
0
Obese
DMN
0 Lean
Obese
Lean
Obese
Fig. 2 Neuropeptide Y (NPY in ng/mg protein; mean _+ SEM) in the arcuate (ARC), paraventricular (PVN), ventromedian (VMN) and dorsomedian (DMN) nuclei of lean and obese Zucker rats either fed ad libitum (filled bars), either food-deprived for 48 hours (open bars) or food-deprived for 48 hours and refed for 6 hours (stripped
bars)
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928
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dorsomedian nuclei are shown in fig. 2. There was a significant difference between the two genotypes in the arcuate and ventromedian nuclei. In the arcuate nucleus, obese rats had about 50 % greater NPY concentrations than the lean rats whatever the feeding state (ad lib 92.3 + 5.9 vs 66.2 + 7.2 ng/mg protein ; RF : 92.2 + 4.3 vs 67.3 + 4.4 ng/mg protein ; FD : 94.5 + 7.3 vs 63.1 + 5.5 ng/mg protein ; p < 0.02, p < 0.005 and p < 0.005 respectively). In the VMN, in the absence of food, obese rats had significantly smaller NPY concentrations than the lean rats (56.7 + 1.8 vs 70.7 + 2.0 ng/mg protein ; p < 0.001). In the other microdissected areas, there was not any significant variations except in the median eminence where the NPY concentrations were higher in the obese rats than in the lean rats (ad lib : 63.9 + 7.1 vs 40.4 + 4.9 ng/mg protein ; RF : 77.4 + 5.2 vs 33.7 + 2.5 ng/mg protein ; FD : 69.0 + 3.8 vs 40.5 + 3.9 ng/mg protein ; p< 0.02, p < 0.001 and p < 0.001 respectively).
Food deprivation or refeeding did not modify NPY concentrations either in the arcuate, ventromedian or dorsomedian nuclei but variations were noted in the paraventricuiar nucleus (p = 0.034). A significant decrease of NPY was measured in the PVN of lean food-deprived rats [58.4 + 1.5 vs 66.1 +%_2.4 (Ad lib) and vs 66.8 + 2.4 (RF) ; p < 0.05 and 0.025 respectively]. No variations were observed in the PVN of the obese rats. There was no treatment x genotype interaction (p=0.81). In the lean rats, the decrease was localized in the parvocellular part of the PVN [43.0 + 1.9 (FD) vs 54.2 + 2.1 (Ad libitum) and 57.8 + 1.8 (RF) ng/mg protein - p < 0.005 and 0.001 respectively]. No variations were noted in the magnocellular part of PVN of these rats. Refeeding restored NPY concentrations to levels comparable to ad lib levels in the PVN of lean rats.
Discussion In this experiment, we studied the effect of food deprivation and refeeding on plasma and hypothalamic NPY concentrations in lean and obese rats. Plasma NPY concentrations were not different between lean and obese Zucker rats and they were not modified by the feeding state unlike the case with plasma insulin and corticosterone concentrations. The classical effects of food deprivation (increase in ketone bodies and decrease in plasma glucose) were also observed. This confirms some of our previous results on the non-intervention of peripheral NPY in the regulation of feeding behavior (8,16). On the other hand, central and more precisely hypothalamic NPY varied.
First, we measured very high NPY concentrations in the arcuate nucleus of the obese rat. This increase in NPY concentrations in the fatty rat has been previously reported (8) and is related to an increase in NPY synthesis since expression of NPY mRNA is increased in the arcuate nucleus of obese rats (11). NPY is mainly synthesized in this nucleus and in the brainstem (9-10). NPY neurons of the ARC projects to the VMN, DMN and PVN to form a dense NPY network (17) and food intake is strongly stimulated when NPY is injected in two of these nuclei namely PVN and VMN (3-4). The paraventricular nucleus also receives NPY efferents from the brainstem (18). A study of Sahu and coworkers has recently shown that food intake is not modified when these
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NPY and Feeding State in t h e Z u c k e r R a t
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efferents and all connections from the brainstem are cut (19) and others reported that NPY mRNA content in the brainstem did not vary with food deprivation (20, 21). NPY originating from the ARC seems therefore to play a major role in the regulation of feeding behavior. Moreover, we demonstrated with different experimental manipulations that the arcuate-paraventricular nucleus axis plays a preponderant role in the NPY network (22). In the present study, NPY in the ARC was not modified by food deprivation either in the lean or in the obese rats. As Sanacora and coworkers have shown that a period of 72 hours of food deprivation induces a two-fold increase in the preproNPY mRA in 14 weeks-old lean Zucker rats (11), the absence of effect on NPY concentrations in our experiment might result from an increased transport from the synthesis site to the target nuclei. However, in two of these nuclei (DMN and VMN), there was no significant variations whereas in the PVN, there was a significant NPY decrease. This decrease might reflect an intense liberation of the peptide. This phenomenom was slowed down by refeeding since NPY levels in lean RF rats were comparable to those of ad lib lean rats.
This regulatory mechanism is very different from that described in Sprague-Dawley rats. Indeed, we and others (5,6) have reported an increase in NPY concentrations in the arcuate nucleus following a fasting period of 48 or 72 hours. This increase was associated with an increase in NPY mRNA expression (23, 24). A concomittant NPY increase was observed in the PVN (5-7). This strain difference is perhaps also explained by the age or body weight of our lean animals which are younger and lighter than the Sprague-Dawley rats used in the previous experiments.
The second information of this experiment is that the mechanism existing in the lean rats does not work in the obese rat. It is possible that the NPY release has already reached maximum levels because of the higher NPY levels in the ARC. Only in vivo studies on NPY release through push-pull or microdialysis techniques could confirm this deficiency as well as the mechanism in the lean rat. The absence of NPY regulation by the feeding state in the Zucker fatty rat is an additional argument supporting an important role of this peptide in the development of its obesity and hyperphagia.
Ackn0wlQdgements The authors thank Ms F. Bergerot and Mr. F. Giannangeli for their excellent technical assistance and Ms A. Turenne for preparing the manuscript.
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A.S. LEVINE, and J.E MORLEY. Peptides 5 1025-1029 (1984). J.T. CLARK, P.S. KALRA, W.R. CROWLEY and S.P KALRA. Endocrinology 115 427-429 (1984). B.G.STANLEY,A.S. CHIN and S.F. LEIBOWlTZ. Brain Res. Bull. 14 521-524 (1985). J.E. MORLEY, A.S. LEVINE, B.A. GOSNELL, J. KNEIP and M. GRACE. Am. J. Physiol. 252 R599R609, 1987. A. SAHU, P.S. KALRA and S.P. KALRA. Peptides 9 83-86 (1988) B. BECK, M. JHANWAR-UNIYAL, A. BURLET, M. CHAPLEUR-CHATEAU, S.F. LEIBOWlTZ and C. BURLET. Brain Res. 528 245-249 (1990).
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