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Sezione di Farmacologia, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Università di Torino (F.Z., C.E.), Via Pietro Giuria 13, 10125 Torino, Italy and Sezione di Anatomia, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Università di Torino (G.C.P.), Corso Massimo d’Azeglio, 52, 10126 Torino, Italy
Address all correspondence and requests for reprints to: Prof. Carola Eva, Sezione di Farmacologia, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Via Pietro Giuria, 13, 10125 Torino, Italy. E-mail: carola.eva{at}unito.it
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Abstract |
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Food deprivation for 72 h decreased transgene expression in the paraventricular nucleus but not in the arcuate nucleus. Leptin treatment, that was per se ineffective, counteracted the decrease of transgene expression induced in the paraventricular nucleus by 72 h fasting. Supplementing the drinking water with 10% glucose increased ß-galactosidase expression both in the paraventricular nucleus and arcuate nucleus of control mice. Finally, none of the treatments altered transgene expression in the dorsomedial hyphothalamic, ventromedial, and amygdaloid nuclei. Results suggest that changes in energetic balance affect Y1 receptor expression in the paraventricular and arcuate nuclei and that leptin regulates the NPY-Y1 system in the paraventricular nucleus. Different regulatory signals might modulate the NPY-Y1 transmission in the dorsomedial hyphothalamic and ventromedial hyphothalamic nuclei.
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Introduction |
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Leptin, an adipocyte-derived hormone, inhibits food intake and may play a key role in regulating the daily pattern of food intake and energy homeostasis (17, 18, 19). An increase in body fat increases levels of leptin that, in turn, reduces food intake, whereas a decrease in body fat leads to a decreased level of the circulating hormone and to a stimulation of food intake (20). Mutations that result in leptin deficiency, or in leptin resistance, are associated with massive obesity in humans and rodents (21, 22).
Although NPY is not the only downstream regulator of body adiposity that responds to leptin, several lines of evidence indicate that NPY is a key component of leptin targets (3). NPY neurons within the ARC coexpress leptin receptor mRNA (23, 24, 25, 26). Leptin administration suppresses, in vitro and in vivo, NPY gene expression in the ARC and its release into the PVN, the most abundant projection region (20, 26, 27). In addition, leptin deficiency in the fasting state or in the ob/ob mice and leptin resistance in the db/db mice markedly elevates arcuate NPY mRNA, whereas leptin administration decreases NPY mRNA in ob/ob mice and fasted rats (20, 26, 27). Therefore, diminution of leptin feedback or leptin resistance may contribute to the hyperfagia and obesity through the modifications of the NPY orexigenic network.
The physiological actions of NPY in the central nervous system are mediated via, at least, six different receptor subtypes: Y1–Y6 (28). Both Y1 and Y5 receptors are expressed in the ARC, PVN, DMH, and VMH (29, 30, 31, 32). The Y5 receptor has been isolated as the receptor that has pharmacological properties most closely matching a proposed feeding receptor (33, 34, 35, 36, 37). However, the potent anorectic effect of selective Y1 receptor antagonists suggests that Y1 receptor is also involved in appetite regulation (38, 39, 40, 41). Recent studies, obtained from mutant mice lacking the Y1 or the Y5 receptor, demonstrated that, although both of the mutant mice feed and grow normally, the Y1 receptor knockout mice exhibit a marked reduced feeding response to fasting, whereas the feeding response of Y5 receptor knockout mice to NPY is significantly reduced, suggesting that both of the receptors are required for appetite regulation by NPY (35, 42, 43).
Despite the well recognized link between NPY and leptin and the complementary functions of both the Y1 and Y5 receptors in central feeding modulation, the specific role of the Y1 receptor subtype in mediating the function of leptin-responsive NPY neurons still remains little explored.
The aim of the present study was to investigate changes of Y1 receptor gene expression in the ARC, PVN, DMH, and VMH in response to food deprivation, leptin treatment, and glucose administration. At this purpose we used, as a model, a transgenic mouse line (Y1R/LacZ) carrying the 1.3 kb 5' flanking region of the mouse Y1 receptor promoter fused to the coding region of the Escherichia coli LacZ gene (44). We recently demonstrated that this construct contains sufficient information to replicate the expression pattern of the endogenous Y1 receptor gene in a central nervous system-restricted and developmental stage-specific manner in ten independent transgenic mouse lines (44). We also demonstrated that pharmacological treatments can modulate Y1R/LacZ transgene expression in a tissue-specific manner, suggesting that changes in the transgene expression may reflect changes of Y1 receptor steady-state and, therefore, they can be used as a marker of altered NPY-Y1 receptor signal transduction (45).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatments
To test the effect of food deprivation on
Y1R/LacZ mice, animals were randomly
divided into two groups of mice that were food deprived for 48 (FD48)
and 72 (FD72) h, respectively. Water was available ad
libitum to all mice.
To test the effect of leptin treatment to control or fasted mice, animals were divided into two groups: one group of mice had free access to food and water and received a single daily ip injection of 1 µg/g of murine recombinant leptin (LEP) (Sigma-Aldrich Corp., Milano, Italy). The other group was fasted for 72 h and treated for 3 d with leptin (1 µg/g, ip) (FD72 + LEP).
To study the effect of glucose administration on Y1R/LacZ expression, mice were divided into four groups: one group of mice was allowed to feed freely and received 10% glucose in the drinking water for 72 h (GLU). Two groups of mice were fasted for 48 or 72 h and received 10% glucose in the drinking water (FD48 + GLU, FD72 + GLU, respectively). One group of mice was fasted for 72 h, received a 10% glucose in the drinking water and was treated daily with leptin (1 µg/g, ip) (FD72 + GLU + LEP).
Finally, one group of mice with free access to food and water received a single daily ip injection of saline and served as a control group (VEH).
Mice were fed standard lab chow (Harlan Italy, S. Pietro al Natisone, Udine, Italy) providing 41.2% energy as amid, 4.9 as sugar, 18.9 as protein, and 5.7 as fat. The addition of 10% glucose to the drinking water did not induce a significant change in daily energy intake, because food consumption of mice receiving a 10% GLU was 66% lower than control mice. However, there was a significant difference in dietary composition, because supplementing of the drinking water with 10% glucose supplied approximately 53% energy as sugar.
All mice were weighted daily for 3 d, at 1600 h and were killed by cervical dislocation at the end of the d 2 or at the end of d 3 (for 48 h- or 72 h-fasted mice respectively).
Brains were quickly removed, placed in 10% embedding medium (Bio-optica, Milano, Italy) in PBS, frozen on crushed dry ice, and stored at -80 C until assayed.
ß-galactosidase staining
Y1R/LacZ expression was
determined by ß-galactosidase staining of brain coronal sections, as
previously described (44, 45). Briefly, frozen brains were
cut on a cryostat at -20 C. Twenty-five-µm-thick sections were
collected on clean slides starting from a level corresponding to the
end of the anterior commissure. Sections were dehydrated with
acetone-chloroform (1:1), air dried and shortly fixed in 2.5%
glutaraldehyde in PBS (each step for 5 min on ice), and incubated
overnight at 37 C in a solution containing 1 mg/ml of X-gal, 5
mM potassium ferrocyanide, 5
mM potassium ferrocyanide, 2
mM MgCl2, 0.01% Triton
X-100 in PBS. After washing in water, sections were counterstained with
nuclear fast red, dried and coverslipped with DPX mounting medium
(Fluka Chemical Co., Buchs, Switzerland).
Quantitation of transgene expression as determined by
ß-galactosidase histochemistry
Quantitation of the Y1R/LacZ
transgene expression was made by computer assisted morphometrical
analysis as previously described (45). To facilitate the
neuroanatomical identification of the regions, the sections were
counterstained with neutral fast red, and hypothalamic nuclei were
identified on the basis of the mouse brain atlas of Franklin and
Paxinos (46). The expression of the transgene appears as
medium-sized blue dots.
The ARC, DMH, and VMH nuclei were divided into a rostral, mid and
caudal levels (around bregma -1.34, -1.70, -2.06 mm for the ARC;
around bregma -1.46, -1.70, -2.06 mm for the DMH; around bregma
-1.22, -1.70, -2.06 mm for VMH). For each of these three levels, two
sections per animal were analyzed. Two standardized sections of
comparable levels of the PVN (around bregma -0.70/-0.82 mm) and of the
medial amygdaloid nucleus (AMY) (around bregma -1.70 mm) were examined
for each animal. Selected sections were placed on a Carl Zeiss Axioplan I microscope, observed by means of a x10
objective, and the corresponding image was transferred, via a black and
white CCD camera (PCO, VC44, Keilheim, Germany), to a digitizing board
(Scion LG-3, Scion Co, Frederick, MD) placed in a PowerPC 8200
Macintosh computer. Acquisition and analysis of the images were
performed using the software NIH-Image (version 1.62, a freeware by W.
Rasband, NIH, Bethesda, MD). Sections were observed and digitized first
by using a built-in green filter to better identify the nuclei
extension (Fig. 1
). A line, drawn
following the boundaries of the selected nuclei, defined the area of
interest (AOI, Figs. 3 and 4). The same section was then digitized
using a built-in red filter obtaining a strong enhancement of the
histochemical signal, but losing the definition of the nuclear
boundaries. The AOI selected on the first image was finally
superimposed on the second image to delimit the region in which dots
should be counted. Using a manual thresholding method, dots were
selected and the corresponding image was binarized. For each animal and
nucleus, the cumulative number of dots and the cumulative areas of the
analyzed sections were considered to obtain the density expression of
the transgene expressed as dots per µm2. The
ARC, DMH, and VMH hypothalamic nuclei exhibited low to moderate
histochemical staining, whereas a the highest level of expression was
observed in the PVN, as previously described (44).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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).
Effect of fasting, leptin treatment, and supplementing of drinking
water with 10% glucose on Y1R/LacZ transgene expression in
the PVN
Y1R/LacZ transgene expression
was determined by histochemical ß-galactosidase staining of brain
coronal sections of the PVN using the chromogenic substrate X-gal.
Fasting for 72 h decreased ß-galactosidase staining in PVN
compared with normally fed mice (Fig. 3, A and C), and leptin treatment (1 µg/g for 3 d) abrogates this
effect (Fig. 3D). Conversely, an increased ß-galactosidase staining
was observed in the PVN of normally fed mice with free access to a 10%
glucose solution (Fig. 3B). Quantitative analysis (summarized in Fig. 5) demonstrated that 72 h fasting decreased ß-galactosidase
expression in the PVN by 50% compared with normally fed mice (Fig. 5A). Leptin treatment of fasted animals abrogates this effect but
failed to modify Y1R/LacZ expression
in normally fed mice. Conversely, supplementing the drinking water with
10% glucose significantly increased ß-galactosidase expression in
fed mice and prevented the decrease of transgene expression induced by
food deprivation for 72 h (Fig. 5A). No significant changes in
ß-galactosidase expression were observed in the PVN of mice
food-deprived for 48 h with free access to normal drinking water
or to drinking water supplemented with 10% glucose, compared with
normally fed mice (Fig. 5B).
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shows that ß-galactosidase staining
in the rostral level of the ARC of 72 h fasted mice (Fig. 4C) and
of 72 h-fasted mice treated for 3 d with 1 µg/g of murine
recombinant leptin (Fig. 4D) was not different from staining in the
corresponding level of the ARC of normally fed mice (Fig. 4A).
Conversely, supplementing the drinking water with 10% glucose
increased ß-galactosidase staining in the rostral level of the ARC of
normally fed mice (Fig. 4B). Quantitative analysis (illustrated in Fig. 6 )
demonstrated that fasting and leptin treatment did not affect
significantly Y1R/LacZ transgene
expression in the rostral, mid and caudal levels of the ARC (Fig. 6A).
Glucose administration significantly increased transgene expression in
the rostral and mid levels of the ARC of normally fed mice (by 78 and
36%, respectively) but failed to affect transgene expression in the
caudal level of the ARC (Fig. 6A). Furthermore, supplementing the
drinking water with 10% glucose increased ß-galactosidase expression
in the rostral and mid levels of the ARC of mice fasted for 48 h
by 46% and 40%, respectively (Fig. 6B). Leptin treatment or glucose
administration did not affect transgene expression in the rostral, mid,
or caudal levels of the ARC of mice fasted for 72 h (Fig. 6A).
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). Leptin treatment and glucose
administration also failed to affect ß-galactosidase expression in
the VHM and DMH of 72-h fasted mice (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A goal of our studies was to identify the hypothalamic nuclei where changes in Y1 receptor gene expression correlate with altered feeding behavior and leptin treatment. In this regard, we demonstrated that fasting, leptin treatment, and glucose administration differentially affect Y1R/LacZ in the hypothalamic sites involved in the daily regulation of ingesting behavior and energy balance (ARC, PVN, DMH, and VMH). In particular, fasting decreased transgene expression in PVN but not in ARC, leptin treatment counteracted the fasting-induced decrease in transgene expression in the PVN, and supplementing the drinking water with glucose increased transgene expression in both the PVN and the ARC. Finally, none of the treatments altered transgene expression in DMH, VMH, and AMY.
Fasting for 72 h induces a marked decrease in the transgene expression only in the PVN. Leptin, completely ineffective on its own, abolishes this effect. This observation suggests that Y1 receptor in PVN participate in the regulation of energy homeostasis and that the target cells mediating the restrain by leptin on NPY-induced feeding response (26, 27, 48) resides in this nucleus. We could speculate that, in a state of negative energetic balance, when the orexigenic NPYergic pathway projecting from ARC to PVN is activated and the NPY release in the PVN increased (49, 50), Y1 receptor is down-regulated in the PVN. Leptin, which blunts the effect of fasting to increase NPY mRNA (27), prevents the food deprivation-induced reduction of Y1 receptor gene expression. These results are in line with previous studies showing that a subset of Y1 receptor-containing neurons in PVN may be the site of interplay between leptin and NPY in regulation of feeding and that this interplay is apparent during fasting (48, 51).
Earlier reports showed that 48 h fasting reduces the number of Y1-receptor immunoreactive cells and Y1 receptor mRNA levels in the ARC (52). It is possible that the fasting-induced activation of the ARC NPY system may change the steady-state of ARC Y1 receptor by other mechanisms, such as alteration in the mRNA stability or degradation. Alternatively, the time course of shifts of Y1 receptor transcription and translation might be different.
Another interesting finding of this study, which is consistent with other reports (53) and requires comments, is that leptin reverses the fasting-induced decrease in Y1R/LacZ expression in PVN but fails to decrease mice body weight. These results show that the inhibition of arcuate NPY neurons and the weight reduction are triggered by leptin at different sensitivities and that these two events can become dissociated. These data also provide further evidence that NPY is not the only downstream regulator of adiposity that responds to leptin.
Conversely, supplementing the drinking water with 10% glucose increases Y1R/LacZ transgene expression both in ARC and PVN of normally fed mice, suggesting that a change in the diet composition, with an increase in sugar intake, elicits the up-regulation of Y1 receptors in these nuclei. Furthermore, glucose administration to 48 h-fasted mice increases in Y1R/LacZ expression in the rostral and mid levels of the ARC but not in the PVN, suggesting that the arcuate NPY-Y1 system is more sensitive to positive changes in energy balance.
Collectively, these observations suggest a different functional role for the Y1 receptor in the ARC. Within the ARC, local circuit NPY neurons innervate a group of POMC neurons, projecting to the PVN and DMH, that also express Y1 receptor mRNA (54, 55). Recent studies indicate that arcuate NPY and POMC population antagonistically interact and that NPY inhibits the anorexigenic ARC/DMH pathway by activating the inhibitory Y1 receptor located on the POMC cell body (54, 55, 56). We can speculate that, in a state of positive energetic balance, when POMC product and others anorectic signals must be activated, the NPY inhibitory signal to POMC is also inhibited, triggering the up-regulation of Y1 receptor in ARC. This hypothesis is supported by the observation that glucose administration increases Y1R/LacZ transgene expression in the rostral and mid levels of the ARC, where the percentage of Y1 receptor-like immunoreactive neurons that coexpress ACTH/POMC immunoreactivity is highest, but it fails to affect transgene expression in more caudal regions where number of Y1 receptor/ACTH positive neurons decreases significantly (54).
On the other hand, the effect of glucose on Y1 receptor gene expression draws in another negative feedback signal beside leptin that regulates the synthesis and release of effector molecules such as NPY. This might correspond to insulin, which is secreted in response to meals (57). The possibility that insulin may play a role in the glucose-induced development of ARC Y1 receptor up-regulation is currently under investigation.
Finally, in the present study we demonstrated that the expression of Y1R-LacZ in the DMH and VMH was not altered to significant extent by any of the treatments. It is possible that, in VMH and DMH, starvation or leptin and glucose administration might induce changes in the expression of other NPY receptor subtypes, such as the Y5 receptor. Alternatively, other factors, such as glucocorticoids, might be required to modulate the NPY-Y1 signaling within the VMH. In line with this possibility, Wisialowsky and co-workers (58) recently reported that adrenalectomy induces the down-regulation of Y1 receptor mRNA selectively in the VMH but not in the ARC or in the PVN.
In conclusion, our data suggest that the decrease in the energetic balance, induced by fasting, down-regulates Y1 receptor gene expression only in PVN and that leptin treatment abrogates this effect. Conversely, an increase in sugar intake, induced by glucose administration, up-regulates Y1 receptor gene expression both in PVN and ARC. These results are consistent with the hypothesis that the ARC and PVN Y1 receptor participate in the regulation of feeding behavior and that the NPY-Y1 system projecting to PVN is under the control of leptin.
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Acknowledgments |
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Footnotes |
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Abbreviations: AMY, Amygdaloid nucleus; ARC, arcuate nucleus; AOI, area of interest; DMH, dorsomedial hyphothalamic nucleus; FD48, mice food deprived for 48 h; FD72, mice food deprived for 72 h; GLU, glucose in the drinking water; LEP, leptin; PVN, paraventricular nucleus; VEH, vehicle; VMH ventromedial hyphothalamic nucleus.
Received January 24, 2001.
Accepted for publication June 1, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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