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Division of Biology of Growth and Reproduction, Department of Pediatrics, University of Geneva School of Medicine (P.D.R., V.D., P.B., M.L.A.), 1211 Geneva 14, Switzerland; and Division of Endocrinology and Metabolism, Centre Hospitalier Universidaire Vaudois, University of Lausanne School of Medicine (E.C., F.P.P.), 1011 Lausanne, Switzerland
Address all correspondence and requests for reprints to: Dr. M. L. Aubert, Hôpital des Enfants, HUGs, 6 rue Willy-Donzé, 1211 Geneva 14, Switzerland. E-mail: aubert{at}cmu.unige.ch
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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MSH is a
melanocortin (MC) peptide that induces satiety by activating the MC4
receptor subtype. Genetic models with disruption of MC4 receptor
signaling are associated with obesity. In the present study, a 7-day
intracerebroventricular infusion to male rats of either the MC receptor
antagonist SHU9119 or porcine NPY (10 nmol/day) was shown to strongly
stimulate food and water intake and to markedly increase fat pad mass.
Very high plasma leptin levels were found in NPY-treated rats
(27.1 ± 1.8 ng/ml compared with 9.9 ± 0.9 ng/ml in
SHU9119-treated animals and 2.1 ± 0.2 ng/ml in controls). As
expected, NPY infusion induced hypogonadism, characterized by an
impressive decrease in seminal vesicle and prostate weights. No such
effects were seen with the SHU9119 infusion. Similarly, whereas the
somatotropic axis of NPY-treated rats was fully inhibited, this axis
was normally activated in the obese SHU9119-treated rats. Chronic
infusion of SHU9119 strikingly reduced hypothalamic gene expression for
NPY (65.2 ± 3.6% of controls), whereas gene expression for POMC
was increased (170 ± 19%). NPY infusion decreased hypothalamic
gene expression for both POMC and NPY (70 ± 9% and 75.4 ±
9.5%, respectively). In summary, blockade of the MC4 receptor subtype
by SHU9119 was able to generate an obesity syndrome with no apparent
side-effects on the reproductive and somatotropic axes. In this
situation, it is unlikely that hyperphagia was driven by increased NPY
release, because hypothalamic NPY gene expression was markedly reduced,
suggesting that hyperphagia mainly resulted from loss of the satiety
signal driven by MC peptides. NPY infusion produced hypogonadism and
hyposomatotropism in the face of markedly elevated plasma leptin levels
and an important reduction in hypothalamic POMC synthesis. In this
situation NPY probably acted both by exacerbating food intake through Y
receptors and by reducing the satiety signal driven by MC peptides. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MSH, that
derive from the large precursor POMC, were recognized to exert
anorectic effects more than 15 yr ago (4). The different
actions of POMC peptides are mediated by five different melanocortin
receptors (MC1-R to MC-5-R) (5). It was found later that
the Agouti protein, which regulates pigmentation by antagonism of the
MC1-R on melanocytes, is also a MC4-R antagonist (6).
Subsequently, a 150-amino acid polypeptide produced in the
hypothalamus, Agouti-related transcript (7) or
Agouti-related peptide (AGRP) (8), was shown to be another
naturally occurring MC-R antagonist acting mostly on MC4-Rs. Ectopic
expression of either Agouti (AY/a) (9, 10) or AGRP (8, 11) or targeted null mutation of
the MC4-R (12) all result in hyperphagia, maturity-onset
obesity, hyperinsulinemia, and hyperglycemia. These observations
established that the MC4-R pathway is involved in the regulation of
feeding and energy balance (2, 13). This concept was
further substantiated by the use of the MC4-R agonist melanotan-II
(MT-II) and antagonist SHU9119 that respectively inhibit and stimulate
food intake in both mice (14) and rats (15).
Other MC4-R antagonists, such as HS014 and HS024, enhance feeding in
satiated rats (16, 17, 18). A large body of knowledge indicates that neuropeptide Y (NPY) (19) is important for the regulation of normal feeding (20, 21, 22, 23) and energy metabolism (24). When released in excess, NPY causes hyperphagia and other metabolic dysregulations (25, 26). Several lines of evidence suggest that MC peptides and NPY interact in the control of feeding. Firstly, populations of both MC3-R and MC4-R are present in areas of the brain where NPY-containing neurons are located (2). Next, NPY messenger RNA (mRNA) was found to be increased in the dorsomedial hypothalamus (DMH) in association with obesity in both the AY/a and MC4R knockout mice, suggesting that NPY could be one of the downstream effectors in these obesity syndromes (27). Finally, MT-II is able to prevent the increase in food intake induced by NPY in both rats and mice (14, 15), and increased feeding induced by HS014 can be partially reversed by NPY Y1 receptor antagonists such as 1229U91, BIBP3226, and BIBO 3304 (28, 29). Therefore, it is possible that in the genetic models of obesity, in food-deprived and food-restricted rats suffering from energy deficiency, and in normal nighttime feeding, the increased NPYergic signaling is responsible for curtailing the restraint by MC signaling on feeding (1, 2, 3).
In addition to its powerful orexigenic effect, NPY is involved in the neuroendocrine modulation of several pituitary secretions. Whereas central administration of NPY stimulates LH release in sex steroid-primed, ovariectomized rats (for review, see Ref. 30), it inhibits LH secretion in castrated animals (31, 32, 33, 34). Furthermore, in both male and female intact rats, central infusion of NPY into the lateral ventricle leads to a profound inhibition of both the gonadotropic and somatotropic axes (35, 36) and prolongation of sexual immaturity in young rats (37). We recently demonstrated that the inhibitory action of NPY on the gonadotropic axis is predominantly mediated by the Y5 receptor subtype (38).
Leptin, the ob/ob gene product (39), was shown to specifically modulate the different neuronal systems involved in the control of feeding, including NPY and POMC neurons (3, 40, 41, 42). The absence of leptin action in leptin-deficient ob/ob mice (39, 43, 44) or leptin-resistant fa/fa Zucker rats (45) results not only in morbid obesity, but also in other alterations, such as decreased thermogenesis and dysregulations of neuroendocrine axes leading to hypogonadism, hypothyroidism, and hypercorticism (45).
The difference in the level of endocrine deficiencies seen between obese phenotypes resulting from either leptin deficiency associated with elevated hypothalamic NPY or disruption of the melanocortin signaling system prompted us to compare the effects of chronic administration of a MC4-R antagonist with the model of chronic NPY infusion known to associate obesity, hypogonadism, hypercorticism, and hyposomatotropism (21, 35, 36, 46). It was anticipated that chronic MC4-R blockade would produce an obesity syndrome exempt of endocrine side-effects. We demonstrate that the obesity syndromes induced by central infusion of either NPY or MC4R antagonist reproduce the corresponding phenotype observed in genetically deficient animals. Furthermore, chronic blockade of the MC4-R was not accompanied by increased NPY synthesis in the arcuate nucleus, suggesting that hyperphagia in this case is mainly driven by the absence of POMC-originating satiety signal.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides
Porcine NPY and SHU9119 (Neosystem, Strasbourg, France) were
dissolved in 0.04 M phosphate buffer containing 0.15
M NaCl, 0.01% ascorbic acid, and 0.1% BSA, adjusted to pH
7.4. The phosphate buffer used as solvent was sterilized and filtered
using a 0.2-µm pore size Nalgene filter (Rochester, NY).
Chronic NPY and SHU9119 infusion in intact male rats
A stainless steel cannula (id, 0.5 mm) was implanted
stereotaxically into the right lateral ventricle of the brain under
ketalar/xylazin anesthesia as previously described (35).
One week after implantation of the cannula, Alzet osmotic minipumps
(Alza Corp., Charles River Laboratories, Inc., Saint-Aubin-les-Elbeuf, France) were filled with
solutions of NPY or SHU9119 calculated to deliver 10 nmol/day. A vinyl
medical tube (size V73, Bolab, Scientific Marketing Associates, Barnet,
UK) was connected to the osmotic minipumps and introduced into the
intracerebroventricular (icv) cannula. The pumps were implanted
dorsally under the skin under light ether anesthesia. Body weight (BW)
and food and water intake were monitored daily before and during the
treatment. After 7 days of infusion, rats were killed by decapitation.
Tissues of interest (pituitary, prostate, testes, seminal vesicles, and
inguinal and retroperitoneal fat pads) were dissected and weighed.
Trunk blood was collected in heparinized tubes, and plasma was stored
until LH, FSH, testosterone, leptin, insulin, corticosterone, GH, and
insulin-like growth factor I (IGF-I) determinations. Hypothalamus and
liver were frozen and stored at -80 C until extraction for mRNA
quantification.
Hormone RIA assays
Plasma IGF-I was determined using antiserum UB3–189 provided by
Drs. L.E. Underwood and J. J. Van Wyk and a biosynthetic IGF-I
preparation received from Dr. L. Fryklund (Pharmacia Biotech, Stockholm, Sweden). Serum extraction was performed
according to the method described by Breier et al.
(47), and within- and between-assay variances were 8% and
17%, respectively. Plasma GH concentrations were analyzed using NIDDK
antirat GH S5 and the RP-2 reference preparation, as described
previously (48). Within- and between-assay variations did
not exceed 5% and 17%, respectively. Plasma LH and FSH were
determined using reagents prepared by Dr. A. F. Parlow and
provided by the NIDDK (Bethesda, MD). Antirat FSH S11 and LH S11 sera
were used, and plasma LH and FSH concentrations were expressed in terms
of the NIDDK RP-1 reference preparation. Within- and between-assay
variations for LH and FSH assays did not exceed 5% and 15%,
respectively. Plasma testosterone was determined with a kit (DSL-4100)
from Diagnostics Systems Laboratories, Inc. (Webster, TX).
Plasma leptin and insulin were determined using Linco Research, Inc. kits (RL-83K and RI-13K; St. Charles, MO). Plasma
corticosterone levels were determined according to a previously
described RIA method (49).
Determination of pituitary GnRH receptor content
The pituitary content of GnRH receptors was assayed by
saturation analysis using the stable superactive GnRH analog
[D-Trp6,(N-Et)Pro9,Des-Gly10]GnRH
as radioiodinated tracer and standard (50). To obtain a
precise determination of the GnRH receptor content of single pituitary
glands, a simplified saturation technique was used, as described
previously (35). Briefly, individual pituitary glands were
homogenized in 1 ml 10 mM Tris-HCl and 1
mM MgCl2, buffer, pH 7.4.
Three 100-µl aliquots of the homogenate were incubated with a mixture
of 50,000 cpm [125I]GnRH analog (
20 pg) and
400 pg unlabeled analog (saturating mixture), and three other aliquots
of pituitary homogenate received the same mixture plus an excess of
unlabeled GnRH analog (100 ng). Incubation tubes were centrifuged after
16-h incubation at 4 C, and binding capacity to pituitary membrane
debris was calculated directly from the amount of radioactivity present
in the pellet after deduction of nonspecific binding and expressed as
femtomoles per pituitary.
Ribonuclease (RNase) protection assay
The hepatic IGF-I mRNA was determined by RNase protection assay.
The hepatic tissue was homogenized in TriPure solution (TriPure Reagent
kit, Roche Molecular Biochemicals, Indianapolis, IN) using
a Polytron homogenizer (Brinkmann Instruments, Inc.,
Westbury, NY). After organic extraction with chloroform, the aqueous
phase was separated and precipitated with isopropanol, and the RNA
pellet was rinsed with 75% ethanol. RNA was then dried, resuspended in
sterile water, checked on agarose minigel, and quantified by absorbency
at 260 nm.
Gene expression was determined as follows. Ten micrograms of total
hepatic RNA and uridine-5'--32P-labeled
complementary RNA (IGF-I probe provided by Dr. P. Rotwein, St. Louis,
MO) were allowed to hybridize at 45 C overnight, followed by combined
RNase A and T1 digestion of nonhybridized probe at 45 C for 15 min.
Stable hybrids were treated with proteinase K for 15 min at 37 C, then
phenol/chloroform extracted, ethanol precipitated, denatured, and
separated on a 8% polyacrylamide/8 M urea gel. The dried
gel was exposed in a phosphorimager cassette. ImageQuant (version
3.2, Molecular Dynamics, Inc., Sunnyvale, CA) was used to
quantify the signals obtained. The IGF-I mRNA values were normalized
according to the ribosomal RNA 18S (cohybridized for each sample;
Ambion, Inc., Austin, TX).
Northern blot analysis
The hypothalamus was dissected from the brain immediately after
death by cutting with a razor blade anteriorly at the level of the
optic chiasm, posteriorly at the level of the mammillary bodies, and
laterally along the hypothalamic sulci. A dorsal cut was then performed
at a depth of 2–3 mm to isolate a tissue fragment corresponding to the
mediobasal hypothalamus and containing the arcuate nucleus. Total
hypothalamic RNA was prepared using the TriPure reagent kit as
described above for hepatic RNA. Gene expression of POMC, NPY, and
GH-releasing hormone (GHRH) was then evaluated by Northern blot
analysis and quantification as previously described (51).
Briefly, total RNA was size-fractionated on a 1% agarose gel
containing 8% formaldehyde and transferred to GeneScreen membranes
(DuPont/NEN, Boston, MA) by capillary blotting.
Membranes were UV cross-linked and stored at -20 C until use. Specific
DNA probes for NPY and glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
provided by J.A. Haefliger), for POMC (provided by I. de Keyser) and
for GHRH (provided by R. Steiner) were labeled with
[-32P]deoxy-CTP (Amersham Pharmacia Biotech) by the random priming method. Hybridization was
performed overnight at 42 C under stringent conditions. Densitometric
analysis of specific mRNA signals on autoradiograms was performed with
a scanner (Molecular Dynamics, Inc.) and ImageQuant
software. To correct for variations in RNA loading, the signals
measured were expressed relative to GAPDH, a ubiquitously expressed
gene.
Statistical analysis
First, ANOVA was performed to evaluate the overall variation due
to peptide treatment, then individual variations were analyzed by the
Student-Newman-Keuls test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. After
Alzet pump implantation under ether anesthesia on day 0, a decrease in
food intake was observed during the first 24 h in the
vehicle-injected rats (-8.1 g) and to a lesser extent in
SHU9119-treated rats (-1.5 g). No initial decrease was observed with
the NPY infusion. In contrast, NPY immediately elicited a strong
stimulation of food intake. After 2 days, a highly significant increase
in food intake was seen for both NPY and SHU9119. Mean values of daily
food intake were 45.1 ± 2.7 and 41.4 ± 1.0 g for NPY
and SHU9119, compared with 30.0 ± 0.5 g for controls. Both
peptides also produced a significant increase in water intake for the
last 6 days of infusion [51.8 ± 2.4 ml for NPY
(P < 0.05) and 67.2 ± 2.4 ml for SHU9119
(P < 0.01) compared with 45.1 ± 1.7 ml for
controls]. Both NPY and SHU9119 induced a highly significant
(P < 0.001) increase in inguinal fat pad mass, from
8.8 ± 0.3 mg/g BW in controls to 13.6 ± 1.0 and 11.2
± 0.3 mg/g BW in NPY and SHU9119 groups, respectively (Fig. 2). The increase in fat pad mass for the
NPY treatment was significantly higher (P = 0.019) than
that for the other treatment. Equally important were the increases in
retroperitoneal fat pad from 5.2 ± 0.5 mg/g BW in controls to
12.0 ± 0.8 and 8.7 ± 0.3 mg/g BW for NPY and SHU9119
groups, respectively (P < 0.001). Again, the increase
induced by NPY was significantly larger (P = 0.0007).
When the mass of both fat pads was added, NPY infusion produced a 184%
increase in adiposity, whereas SHU9119 induced a 142% increase over
control values. Thus, the increase in fat pad mass was significantly
more important in the case of NPY infusion. Pituitary weight was
dramatically decreased in the NPY group, from 10.3 ± 0.4 mg in
controls to 6.8 ± 0.4 mg (P < 0.01). No such
effect was seen in the SHU9119-infused rats. BW gain in the SHU9119
group (9.5 ± 0.9 g/day) was clearly enhanced compared with that
in control rats (6.8 ± 0.6 g/day). In contrast, BW gain in the
NPY-infused rats was unchanged (6.3 ± 0.6 g/day; Fig. 2).
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and Table 1). As observed in a previous study
(36), chronic icv administration of NPY (10 nmol) strongly
inhibited the gonadotropic axis, with highly significant decreases in
all relevant parameters studied (testis, seminal vesicle, and prostate
weights; plasma LH, FSH, and testosterone levels; and pituitary GnRH
receptor content). The reductions in seminal vesicles and prostate
weight (72% and 56%, respectively) were most impressive (Fig. 3),
consistent with the reduced testosterone secretion (0.26 ± 0.13,
vs. 2.20 ± 0.40 ng/ml for controls; Table 1). In sharp
contrast, SHU9119 infusion affected neither seminal vesicle and
prostate weights nor plasma LH, FSH, and testosterone levels (Fig. 3
and Table 1). Even if a significant, but slight, reduction in testis
weight was observed with the MC4-R antagonist (6% decrease), the mean
value for this group (3.25 ± 0.04 g) was still highly
significantly elevated over the lower testis weight of NPY-treated rats
(2.83 ± 0.07 g).
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). Plasma GH levels were extremely low
in NPY-infused rats (1.8 ± 0.1 ng/ml with no peaks) compared with
control levels (275 ± 92 ng/ml). Plasma IGF-I secretion was
markedly decreased (63% reduction) in NPY-infused rats, and
consistently, the hepatic IGF-I mRNA concentration was significantly
decreased, representing only 47% of control values. Finally, gene
expression for hypothalamic GHRH was almost abolished (4.9% of
controls) in the NPY group. In sharp contrast, the chronic central
infusion of SHU9119 did not affect any of the parameters of the
somatotropic axis (Fig. 4).
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).
NPY and SHU9119 infusions also produced highly significant increases
(P < 0.01) in plasma insulin levels, from 1.7 ±
0.1 ng/ml in controls to 5.1 ± 0.6 ng/ml in SHU9119-treated rats
and 10.8 ± 1.2 ng/ml in NPY-treated rats (Table 1). Furthermore,
as expected, the NPY infusion produced a marked increase in
corticosterone secretion, with a mean level at sacrifice of 327 ±
52 ng/ml that was highly significantly different from controls (50
± 11 ng/ml; P < 0.01). The SHU9119 infusion also
induced an increase in corticosterone secretion, with a mean value of
155 ± 48 ng/ml at death (P < 0.05).
Hypothalamic NPY gene expression was significantly reduced in
NPY-treated rats (75.4 ± 9.5% of controls; P <
0.05) and was further reduced in the SHU9119 group (65.2 ± 3.6%
of controls; P < 0.01; Fig. 5). Highly significant changes in
hypothalamic POMC gene expression were also observed. A 70% increase
in POMC gene expression was observed with the SHU9119 infusion compared
with controls (P < 0.01), whereas a significant
reduction in POMC gene expression was observed with the NPY infusion
(70 ± 9% of controls; P < 0.05; Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It was already known that acute injection(s) of the MC-R antagonist SHU9119 would promptly increase feeding (13, 14, 15, 53), but no data were available on the chronic effects of such antagonism. SHU9119 is a nonspecific MC-R antagonist, binding to all MC-R with almost the same activity, but its ability to modify food intake is attributed to its action on the MC4-R subtype (16). We show here that a 7-day infusion of the MC4-R antagonist SHU9119 at 10 nmol/day induced unabated hyperphagia and polydypsia, resulting in a clear obesity syndrome characterized by increases in BW gain and weight of fat pads. At the same dosage, central infusion of NPY produced a significantly larger increase in fat pad weight, but no increase in BW gain. For both treatments, an impressive increase in plasma leptin levels was seen that was proportional to the increase in fat pad weight.
In contrast to the devastating effects of NPY infusion on the gonadotropic axis, no situation of hypogonadism was observed at the end of the 7-day infusion of SHU9119. The weights of androgen-dependent organs such as seminal vesicles and prostate were identical in SHU9119-treated rats and their controls, consistent with the normal plasma levels for testosterone and gonadotropins. Nevertheless, a small reduction in testis weight in parallel with a slight decrease in pituitary GnRH receptor content were noted. This could reflect a trend toward slow inactivation of the gonadotropic axis resulting from the ongoing obesity situation, unrelated to specific inhibition of MC4-Rs.
That chronic blockade of the MC4-Rs would not lead to major effects on the gonadotropic axis could be predicted from the observations made in the three genetic models with impaired MC4-R function. Indeed, no reduction in reproductive capacity was seen in overexpression of Agouti (Ay/a) (9, 10), AGRP (8, 11), or MC4-R knockout mice (12). Thus, as seen in the present study, inputs from the central nervous system to GnRH neurons do not appear to transit through the MC4-R. This concept is consistent with recently published data by Hohmann et al. (54), who addressed this question by using the otherwise sterile ob/ob mouse that becomes fertile upon leptin treatment (43, 44). Hohmann et al. administered SHU9119 together with leptin to ob/ob mice and demonstrated the same process of initiation of sexual function as with leptin alone, whereas leptin’s effects on feeding and BW gain were attenuated (54).
Another striking difference between the two models of induced obesity presented here is the status of the somatotropic axis. We previously demonstrated that chronic central NPY infusion fully inhibited pulsatile GH secretion, with a marked decrease in plasma IGF-I levels (36). In this study we extend this observation with the demonstration that hypothalamic gene expression for GHRH was abolished, confirming the central origin of this inhibition. We also show that gene expression for IGF-I at the hepatic level was markedly decreased. The level of inhibition of IGF-I secretion and hepatic IGF-I expression induced by central administration of NPY is equivalent to that obtained in our laboratory with complete blockade of GH secretion resulting from chronic administration of anti-GHRH serum (48). It is therefore possible that this absence of GH secretion was also responsible for a decrease in local IGF-I production in tissues other than the liver. This could explain both the major drop in pituitary weight and the absence of BW gain despite the sustained increase in food intake in NPY-treated animals. In our previous study (36) it was shown that the increase in BW gain was smallest with the highest dosage of NPY infused. The normal activation of the somatotropic axis of SHU9119-treated animals observed in our study is consistent with the observation of increased linear growth in genetic models with MC4-R signaling disruption (12, 55). It should be mentioned that the time frame of the present study did not allow us to observe an increase in linear growth. Specifically, monitoring of the 24-h GH secretory profile in these rats would be required to identify a possible enhancement of GH secretion.
The obesity induced by blockade of MC4-Rs by SHU9119 resulted in elevated insulin and leptin plasma levels. Increased leptin secretion probably reflects the increase in fat mass, although enhanced insulin action on leptin synthesis could also have played a role (56). These elevated plasma levels of insulin and leptin probably contributed to the up-regulation of hypothalamic POMC expression seen in our study (57, 58). Indeed, POMC neurons express the leptin receptor (59), POMC expression in the arcuate nucleus is increased by leptin (58, 60, 61), and the ability of leptin to acutely inhibit food intake is abolished when MC4-Rs are blocked by MC antagonists (62, 63). In addition, the fact that food intake was increased in rats treated with SHU9119 despite elevated plasma leptin levels further documents that blockade of MC4-Rs partly or fully prevents the downstream signaling of leptin to reduce feeding.
It has been suggested that NPY could be a downstream effector responsible for the hyperphagia generated by MC4-R blockade (14, 28, 37). NPY gene expression is increased in the DMH of both obese Ay/a and MC4-R knockout mice, suggesting DMH as a possible target of POMC neurons in their effects on feeding and metabolism (27). However, gene expression for NPY was normal in the arcuate nucleus of these genetic models (27). This is a major difference from the important up-regulation of NPY activity observed in this key area of the hypothalamus in hyperphagic ob/ob mice (40), fa/fa Zucker rats (64), and several situations of food restriction or fasting (65). Whereas it is clear that increased gene expression for NPY in the arcuate nucleus is associated with increased feeding, the exact meaning for feeding behavior of increased NPY synthesis in the DMH is still unclear. In our study chronic infusion of SHU9119 clearly reduced NPY gene expression within the hypothalamic tissue collected, suggesting that the stimulatory effect on food intake by chronic blockade of MC4-Rs is independent of a NPY action originating from the arcuate nucleus. Seemingly, NPY synthesis could have been appropriately reduced by the elevated plasma leptin levels seen in this situation. Of note, the portion of hypothalamus collected in our study does not include the DMH, so there is no contradiction with the study by Kesterson et al. (27).
Large increases in plasma leptin and insulin were also observed in the
NPY-infused rats, but in contrast to the SHU9119 group, an important
reduction in POMC hypothalamic gene expression was observed. Thus, NPY
appears not only to induce hyperphagia by a specific action on NPY
receptors, but also to override the stimulatory effect of leptin on
POMC neurons for the production of MSH, thus reducing the satiety
signal driven by this MC peptide. The concept that NPY is able to
induce feeding independently of MC4-R signaling was already evident
from recent studies with
MC4-R-/- mice that
responded to the orexigenic effects of both NPY and the Y5 receptor
agonist peptide YY-(3–36) (66). Chronic infusion
of NPY into the lateral ventricle also reduced hypothalamic gene
expression for NPY, a reduction that could derive from a direct effect
of exogenous NPY on NPY neurons or could simply result from the highly
elevated leptin levels that would naturally suppress NPY synthesis.
A difference between NPY- and SHU9119-induced obesity could be linked
to differential effects of these treatments on the regulation of
glucocorticoid secretion in relation to central and peripheral
catecholaminergic activity. It is well known that chronic NPY infusion
to normal rats activates the corticotropic axis (24),
progressively mimicking the increased glucocorticoid tone described in
obese Zucker (fa/fa) rats (46). Furthermore, it
has been shown that the rise in norepinephrine levels within the
paraventricular nucleus after immobilization stress was significantly
lower in obese than in lean Zucker rats and paralleled the decreased
circulating catecholamine levels in obese animals (67).
This suggests that defective regulation of paraventricular nucleus
norepinephrine could reflect and contribute to the development and/or
maintenance by glucocorticoids of obesity in Zucker rats
(67). Thus, it could be reasoned that a key difference
between NPY vs. SHU9119 treatment in our study relies on the
effects of NPY on both appetite and fuel metabolism, whereas MSH
exerts its actions mostly on appetite. The effects of NPY on the
neuroendocrine axes, in particular, the induced hypogonadism, could be
explained by its inhibitory effects on the sympathetic system and
concomitant stimulation of CRH neurons; such effects are absent when
the MC4-R subtype is inhibited by an MSH antagonist.
A 7-day infusion of NPY into the lateral ventricle of normal rats resulted in an obesity phenotype that recapitulates both the metabolic and neuroendocrine consequences of the absence of leptin action observed in fa/fa Zucker rats (46), including hypogonadism and hypercorticism (35, 36). This is in contrast to the obesity models resulting from disruption of the melanocortin system (8, 9, 11, 12, 52), which leads to a milder obesity syndrome with maintenance of normal reproductive and adrenal axes (2). It could therefore be speculated that some of these differences are due to enhanced NPY action. Indeed, the observation in this study that SHU9119 infusion induced obesity independently of NPY, with no consequences on the gonadotropic and somatotropic axes, is consistent with the hypothesis that increased hypothalamic NPY release represents the vector responsible for induction of hypogonadism, hyposomatotropism, and hypercorticism.
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Acknowledgments |
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Footnotes |
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2 Recipient of a Research Development Carrier Award from the Prof. Dr.
Max Cloëtta Foundation.
Received April 12, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-MSH antagonize feeding behavior stimulated by 
opiate agonists. Peptides 7:843–848[CrossRef][Medline]
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 E Kim, S Sohn, M Lee, J Jung, R D Kineman, and S Park Differential responses of the growth hormone axis in two rat models of streptozotocin-induced insulinopenic diabetes J. Endocrinol., February 1, 2006; 188(2): 263 - 270. [Abstract] [Full Text] [PDF]
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C. Morens, M. Keijzer, K. de Vries, A. Scheurink, and G. van Dijk Effects of high-fat diets with different carbohydrate-to-protein ratios on energy homeostasis in rats with impaired brain melanocortin receptor activity Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2005; 289(1): R156 - R163. [Abstract] [Full Text] [PDF]
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 N Hoggard, D V Rayner, S L Johnston, and J R Speakman Peripherally administered [Nle4,D-Phe7]-{alpha}-melanocyte stimulating hormone increases resting metabolic rate, while peripheral agouti-related protein has no effect, in wild type C57BL/6 and ob/ob mice J. Mol. Endocrinol., December 1, 2004; 33(3): 693 - 703. [Abstract] [Full Text] [PDF]
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 P. D. Raposinho, T. Pedrazzini, R. B. White, R. D. Palmiter, and M. L. Aubert Chronic Neuropeptide Y Infusion into the Lateral Ventricle Induces Sustained Feeding and Obesity in Mice Lacking Either Npy1r or Npy5r Expression Endocrinology, January 1, 2004; 145(1): 304 - 310. [Abstract] [Full Text] [PDF]
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 T. M. Mizuno, K. A. Kelley, G. M. Pasinetti, J. L. Roberts, and C. V. Mobbs Transgenic Neuronal Expression of Proopiomelanocortin Attenuates Hyperphagic Response to Fasting and Reverses Metabolic Impairments in Leptin-Deficient Obese Mice Diabetes, November 1, 2003; 52(11): 2675 - 2683. [Abstract] [Full Text] [PDF]
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 H. Watanobe Leptin directly acts within the hypothalamus to stimulate gonadotropin-releasing hormone secretion in vivo in rats J. Physiol., November 15, 2002; 545(1): 255 - 268. [Abstract] [Full Text] [PDF]
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 Y. Yang, M. Chen, Y. Lai, I. Gantz, K. E. Georgeson, and C. M. Harmon Molecular Determinants of Human Melanocortin-4 Receptor Responsible for Antagonist SHU9119 Selective Activity J. Biol. Chem., May 31, 2002; 277(23): 20328 - 20335. [Abstract] [Full Text] [PDF]
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D. K. Sindelar, P. Mystkowski, D. J. Marsh, R. D. Palmiter, and M. W. Schwartz Attenuation of Diabetic Hyperphagia in Neuropeptide Y-Deficient Mice Diabetes, March 1, 2002; 51(3): 778 - 783. [Abstract] [Full Text] [PDF]
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K. Baran, E. Preston, D. Wilks, G. J. Cooney, E. W. Kraegen, and A. Sainsbury Chronic Central Melanocortin-4 Receptor Antagonism and Central Neuropeptide-Y Infusion in Rats Produce Increased Adiposity by Divergent Pathways Diabetes, January 1, 2002; 51(1): 152 - 158. [Abstract] [Full Text] [PDF]
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 M. W. Schwartz Brain Pathways Controlling Food Intake and Body Weight Experimental Biology and Medicine, December 1, 2001; 226(11): 978 - 981. [Abstract] [Full Text] [PDF]
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