AgRP(83132) Acts as an Inverse Agonist on the Human-Melanocortin-4 Receptor
Wouter A. J. Nijenhuis,
Julia Oosterom and
Roger A. H. Adan
Molecular Neuroscience Rudolf Magnus Institute for
Neurosciences University Medical Center Utrecht Utrecht, the
Netherlands 3584 CG
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ABSTRACT
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The central melanocortin (MC) system has been
demonstrated to act downstream of leptin in the regulation of body
weight. The system comprises
-MSH, which acts as agonist, and
agouti-related protein (AgRP), which acts as antagonist at the MC3 and
MC4 receptors (MC3R and MC4R). This property suggests that MCR activity
is tightly regulated and that opposing signals are integrated at the
receptor level. We here propose another level of regulation within the
melanocortin system by showing that the human (h) MC4R displays
constitutive activity in vitro as assayed by adenylyl
cyclase (AC) activity. Furthermore, human AgRP(83132) acts as an
inverse agonist for the hMC4R since it was able to suppress
constitutive activity of the hMC4R both in intact B16/G4F melanoma
cells and membrane preparations. The effect of AgRP(83132) on the
hMC4R was blocked by the MC4R ligand SHU9119. Also the hMC3R and the
mouse(m)MC5R were shown to be constitutively active. AgRP(83132)
acted as an inverse agonist on the hMC3R but not on the mMC5R. Thus,
AgRP is able to regulate MCR activity independently of
-MSH. These
findings form a basis to further investigate the relevance of
constitutive activity of the MC4R and of inverse agonism of AgRP for
the regulation of body weight.
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INTRODUCTION
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Melanocortin receptors (MCRs) are G-protein coupled
receptors (GPCRs) that are positively coupled to the cAMP pathway. Of
the five MCRs that have been cloned, the MC1R is expressed on
melanocytes in the skin where it is involved in regulation of
pigmentation. The MC4R is expressed in the brain where it was shown to
be involved in regulating metabolism and food intake both in rodents
(1, 2, 3, 4) and humans (5, 6). The MC3R is expressed in the brain, placenta,
and gut. The exact role of the MC3R is still unclear, but it has been
proposed that in the brain the MC3R could have a regulatory role in
metabolism and food intake upstream of the MC4R (7).
In addition to the melanocortins (e.g. ACTH and
- and
-MSH), which act as agonists on MCRs, endogenous high-affinity
peptide ligands for MCRs have been found that act as antagonists,
namely agouti protein (agouti) (8, 9, 10) and agouti-related protein
(AgRP) (11, 12). The existence of different ligands with opposing
actions on the same receptor provides a mechanism to tightly regulate
MCR activity and to integrate opposing signals at the receptor
level.
Agouti is a paracrine factor expressed in dermal papilla in the
hair follicle, where it regulates the switch from eumelanin to
phaeomelanin synthesis by melanocytes in mice (13). In
vitro, agouti is an antagonist for the MC1R and MC4R (10), and it
has been suggested that agouti regulates pigmentation by blocking
-MSH binding to the MC1R (14). However, several effects of agouti
cannot be explained by competition with
-MSH only. For instance, it
has been reported that incubation of cells expressing the MC1R with
agouti in the absence of
-MSH resulted in lower cAMP formation (15, 16), decreased melanogenesis (16, 17, 18) and cell growth (19), and lower
tyrosinase expression (16). Furthermore, agouti can inhibit the
response to cholera toxin (17, 20). These effects could be explained by
inverse agonism of agouti.
AgRP, which shares sequence homology with agouti, is expressed in the
arcuate nucleus of the hypothalamus, subthalamic nucleus, and the
adrenal gland (11) and is a potent antagonist for the MC3R and MC4R,
and a weak antagonist for the MC5R (12, 21, 22). Considerable evidence
exists suggesting that AgRP is involved in body weight regulation (11, 12, 23, 24) by acting downstream of the adipose tissue-derived satiety
factor leptin (25, 26). AgRP reduces the maximal response of the
human-MC4R (hMC4R) to
-MSH, and evidence was provided that AgRP
reduces activation of the cAMP pathway in the absence of agonist, as
assayed by measurement of cAMP levels and reporter gene activity (12, 27).
Understanding the mechanisms by which melanocortin ligands
regulate their receptors is essential for unraveling the physiological
role of the melanocortin system. Therefore, we tested directly whether
the human (h) MC4R displays constitutive activity and whether AgRP is
able to suppress this constitutive activity. We used synthetic
AgRP(83132) (28) and B16/G4F (29) cell lines stably expressing
different levels of the hMC4R and assayed for adenylyl cyclase
activity.
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RESULTS
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Constitutive Activity of the hMC4R
No specific [125I]NDP-MSH
([Nle4-D-Phe7]-
-MSH) binding
was detected in wild-type B16/G4F cells (data not shown). Also,
incubation of this cell line with up to 1 µM
-MSH did
not induce adenylyl cyclase (AC) activity (Table 1
), indicating that there is no
expression of endogenous MC receptors in this cell line.
Four clones of B16/G4F cells were generated, each with a
different expression level of functional hMC4R as measured by
[125I]NDP-MSH binding and response to
-MSH (Fig. 1
). The cell lines were
tested for basal (unstimulated) and forskolin-induced AC activity. The
response to forskolin was determined because it has been shown to
correlate with the amount of constitutive activity of receptors
expressed in cell lines (30). Basal AC activity was not significantly
different (0.54, 0.51, and 0.41% cAMP) in three clones expressing 1.6
x104, 5.0 x104, and
5.6 x 104 receptors per cell (clones
MC41, MC42, and MC43, respectively). However, significantly
higher (1.26% cAMP, P < 0.05) AC activity was
detected in clone MC44, which expresses 23 x
104 receptors per cell (Fig. 1
). The
forskolin-induced AC activity correlated with the expression level of
the clones (Fig. 1
, r = 0.97 and r =
0.93 respectively, P < 0.001).
Suppression of Constitutive Activity of the hMC4R by
AgRP(83132)
The effect of 100 nM human AgRP(83132) on
basal and forskolin-induced AC activity of hMC4R-expressing cells was
determined (Fig. 2
). AgRP(83132)
suppressed basal AC activity up to 60% in cells expressing the hMC4R.
This effect, however, was not seen in clone MC41, which displayed the
lowest level of hMC4R expression. Similarly, AgRP(83132) suppressed
forskolin-induced AC activity only in the three cell lines with the
highest expression of the hMC4R. Suppression of both basal and
forskolin-induced AC activity was most profound in cells with high
expression levels of the hMC4R. AgRP(83132) did not alter basal and
forskolin-induced AC activity in wild-type B16/G4F cells (Fig. 2
).

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Figure 2. Suppression of AC Activity by AgRP(83132) in
Cells Expressing the hMC4R
Effect of 100 nM AgRP(83132) on basal (A) and forskolin
(1 µM)-induced (B) AC activity of wild-type (wt) B16/G4F
cells and B16/G4F cells with different expression levels of the hMC4R.
AC activities of untreated (black bars) cells were set
at 100% for each cell line. Relative AC activities of cells treated
with AgRP(83132) are given in gray bars. Data are
shown as mean ± SEM (n = 3 or 4). *,
Statistically significant different (P < 0.01).
Replicated four times.
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AgRP(83132) suppressed basal and forskolin-induced AC activity in a
dose-dependent manner (Fig. 3
). The
EC50 values were 21 and 37 nM,
respectively; 1 µM SHU9119, originally identified as
antagonist for the hMC4R (31), blocked the effect of
-MSH on AC
activity in a cell line expressing the hMC4R at 3.6 x
104 receptors per cell (Fig. 4A
). In this cell line, SHU9119 also
blocked the effect of AgRP(83132) on both basal and forskolin-induced
AC activity when coadministered (Fig. 4
, B and C). SHU9119 by itself
did not influence AC activity in these cells. Also in clone MC44,
which expresses 23 x 104 receptors per cell
and shows a larger response to AgRP(83132), SHU9119 was able to block
the effect of AgRP(83132) on basal AC activity (Fig. 4D
). However, in
this clone SHU9119 displayed weak partial agonism.

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Figure 3. Dose-Dependent Suppression of hMC4R Activity by
AgRP(83132)
Effect of AgRP(83132) on basal (A) and forskolin (1
µM)-induced (B) AC activity in cells expressing the hMC4R
(23 x 104 receptors per cell). Data are shown as
mean ± SEM (n = 3). Replicated three times.
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Figure 4. SHU9119 Blocks the Effect of AgRP(83132) and
-MSH on the hMC4R
A, Effect of 1 µM SHU9119 on 1 µM
-MSH-induced AC activity in cells expressing the hMC4R at 3.9
x 104 receptors per cell. B, Effect of 1 µM
SHU9119 on inhibition of AC activity by 200 nM
AgRP(83132) in the same cell line as in panel A. C, Effect of 1
µM SHU9119 on the inhibition of forskolin-induced AC
activity by 200 nM AgRP(83132) in the cells mentioned in
panel A. D, Effect of 1 µM SHU9119 on the inhibition of
AC activity by AgRP(83132) in clone MC44. Basal (A, B, and D) and
forskolin-treated (C) samples were set at 100%. Where multiple
compounds were used in one sample, compounds were added simultaneously.
Abbreviations: MSH, -MSH; S, SHU9119; F, forskolin; and A,
AgRP(83132). Data are expressed as mean ± SEM
(n = 3 for A, C, and D; n = 4 for B). *, Statistically
significant different from basal (A, B, and D) or forskolin-treated (C)
(P < 0.05). Replicated two (A, C, and D) and three
(B) times.
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To investigate the influence of receptor internalization, AgRP(83132)
was also tested in a cell-free AC activity assay using membrane
preparations instead of intact cells. Incubation of membranes prepared
from clone MC44 with AgRP(83132) reduced basal and
forskolin-induced AC activity, but did not affect AC activity in
membranes from B16/G4F cells (Fig. 5
).

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Figure 5. AgRP(83132) on hMC4R-Containing Membranes
Effect of 200 nM AgRP(83132) on basal (A) and forskolin
(1 µM)-induced (B) AC activity in membranes from clone
MC44 and B16/G4F cells. *, Statistically significant different
(P < 0.05). Replicated three times.
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The Effect of AgRP(83132) on the hMC3R and the mMC5R
Cells expressing the hMC3R (190 x
104 receptors per cell) and the mouse (mMC5R, 54
x104 receptors per cell) showed increased basal
and forskolin-induced AC activity compared with wild-type cells (Table 1
). Both cell lines responded to
-MSH (Table 1
). The
hMC3R-expressing cells and membrane preparations from these cells
showed 2035% reduction in basal and forskolin-induced AC activity
upon incubation with 200 nM AgRP(83132) (Fig. 6
). There was no effect of 200
nM AgRP(83132) on basal and forskolin-induced AC activity
in cells expressing the mMC5R (data not shown).

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Figure 6. Effect of AgRP(83132) on the hMC3R
Effect of 200 nM AgRP(83132) treatment (gray
bars) on basal (A), and forskolin (1 µM)-induced
(B) AC activity of cells expressing the hMC3R and membranes of these
cells. Bars indicate mean ± SEM (n = 4). *,
Statistically significant different (P < 0.01).
Replicated two (membranes) and three (cells) times.
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DISCUSSION
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In this study, we show that the hMC4R displays constitutive
activity in vitro and that AgRP(83132) acts as an inverse
agonist for this receptor. These findings represent a new mechanism by
which MCR activity may be regulated in vivo.
In cells expressing the hMC4R, AgRP(83132) lowered both basal and
forskolin-induced AC activity in a dose-dependent manner. The data
suggest that the effect of AgRP(83132) occurs via direct interaction
with the hMC4R since this effect was not seen in wild-type B16/G4F
cells and was blocked by 1 µM of the MC4R antagonist
SHU9119. Although at this high dose of SHU9119 there was a partial
agonistic effect of SHU9119 on cells expressing the highest number of
hMC4R (clone MC44), this was only a small effect compared with the
effect of AgRP(83132). In any case, SHU9119 was able to fully block
the effect of AgRP(83132) on this clone. This is most likely via
competition for binding to the hMC4R since SHU9119 has nanomolar
affinity for the hMC4R (31) and at 1 µM it is thus
expected that all receptors are occupied.
The effect of AgRP(83132) on AC activity was most profound in the
cell line with the highest hMC4R expression level. The effect was less
in cells expressing an intermediate level of the hMC4R and not
detectable in cells expressing very low amounts of hMC4R. Thus, the
efficacy of AgRP(83132) to suppress basal and forskolin-induced AC
activity correlates with the expression level of the receptor.
Inverse agonism is defined as the ability of a ligand to stabilize the
inactive conformation of a receptor (32). The results discussed above
suggest that AgRP(83132) is an inverse agonist for the hMC4R.
However, the decreases in AC activity observed here could also be due
to hMC4R internalization induced by AgRP(83132), since AC activity
correlates with receptor density of a constitutively active receptor
(33). To investigate the role of endocytosis in the suppression of AC
activity by AgRP(83132), AC activity was measured in cell membranes.
During homogenization, the actin cytoskeleton, which is necessary for
endocytosis (34), is disrupted mechanically. Additionally, endocytosis
needs cytosolic components, which are washed away during membrane
preparation. Therefore it is expected that endocytosis is severely
impaired or absent in isolated membranes. Because AgRP(83132)
suppressed constitutive activity of the hMC4R in membrane preparations
to the same extent as in intact cells, it is reasonable to conclude
that internalization is not the underlying mechanism for the effects of
AgRP(83132) on the hMC4R. Therefore, based on the results that 1)
AgRP(83132) suppressed AC activity via direct interaction with the
hMC4R, 2) this suppression depended on the expression level of the
hMC4R, and 3) this suppression occurred in intact cells as well as in
membrane preparations, we conclude that AgRP(83132) is an inverse
agonist for the hMC4R.
Because inverse agonism can only be measured in the presence of
constitutive activity, it is to be expected that in our test system the
hMC4R displays constitutive activity. Indeed, the response to 1
µM forskolin correlated with the expression levels of the
hMC4R. This was shown before to be characteristic for constitutively
active receptors (30). Also, compared with the other hMC4R-expressing
clones, the clone with the highest hMC4R expression level showed
elevated basal AC activity. These data strongly support the notion that
the hMC4R is constitutively active. However, basal AC activity in the
clones with lower hMC4R expression did not correlate with the
expression level of the receptor. This is not due to limited
sensitivity of the assay because, upon incubation with AgRP(83132),
lower AC activity could be measured in these clones. One obvious
explanation is that intracellular compensatory mechanisms exist that
are able to counteract basal constitutive activity only at low
expression levels of the receptor. At higher expression levels of the
receptor (as in clone MC44) or in the presence of forskolin, these
mechanisms may not be able to compensate for constitutive activity.
Thus, in these experiments the use of forskolin allows a more sensitive
detection of constitutive activity, as has been described previously
(30).
Expressing the hMC3R or the mMC5R in B16/G4F cells increased basal and
forskolin-induced AC activity as compared with wild-type cells. This
indicates that both receptors are constitutively active. As is shown in
Fig. 6
, AgRP(83132) is an inverse agonist for the hMC3R. In contrast,
although the mMC5R has constitutive activity, AgRP(83132) did not
affect AC activity in cells with this receptor. This is in agreement
with the low affinity of AgRP for the MC5R (12, 21, 22).
Several lines of evidence suggest that activation of central MCR
inhibits food intake (1, 3). If the hMC4R exhibits constitutive
activity in vivo, this would contribute to the tonic
inhibition of the melanocortin system on feeding. The observed
haploinsufficiency of obese human subjects with hMC4R gene variants
reported in several studies (5, 6, 35) and the intermediate obese
phenotype of mice heterozygous for MC4R deletion (1) may therefore be
the result of a gene dosage effect resulting in decreased constitutive
activity. However, it cannot be excluded that an impaired response to
-MSH in the heterozygous mutants causes the haploinsufficiency.
Most likely, full-length AgRP also acts as an inverse agonist because
the C terminus of AgRP has been shown to possess the same
pharmacological properties in vitro and in vivo
as full-length AgRP (22, 24, 27). Furthermore, other studies already
showed effects of nearly full-length AgRP that do not fit with neutral
antagonism (12, 27).
This indicates that AgRP may reduce the activity of the melanocortin
system independently of
-MSH in the brain. POMC and AgRP are
expressed in different neurons (36, 37). If AgRP(83132) only blocks
-MSH-induced activation, the presence of
-MSH (i.e.
activation of POMC neurons) is necessary for AgRP to function. However,
if AgRP acts independently of
-MSH, AgRP-containing neurons can act
independently of POMC-containing neurons, thereby adding a new level of
regulation to the melanocortin system. Indeed, in rat brain, AgRP and
-MSH production seem to be counterregulated since there is increased
AgRP mRNA and decreased POMC mRNA expression in response to fasting
(38, 39). The opposite effect is seen in response to leptin (26, 36, 38, 39, 40). Interestingly, the changes in AgRP mRNA levels are larger than
those measured for POMC (25). Thus, for the regulation of body weight
the melanocortin system may be controlled more by AgRP than
-MSH.
Similarly, for the MC1R it is the expression level of agouti rather
than that of
-MSH that determines the coat color (13).
The data presented here form a basis to further investigate the
relevance of constitutive activity of the MC4R and of inverse agonism
of AgRP for the regulation of body weight.
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MATERIALS AND METHODS
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Chemicals and Ligands
Forskolin was purchased from Sigma (Steinheim,
Germany), and synthetic human AgRP(83132) was obtained from
Phoenix Pharmaceuticals, Inc. (Mountain View, CA).
-MSH
and NDP-MSH were purchased from Bachem (Bubendorf,
Switzerland). SHU9119 was synthesized as described previously (41).
Generation of Cell Lines
B16/G4F cells (29) were grown in RPMI 1640 medium (Life Technologies, Inc., Paisley, U.K.) supplemented with 10% FCS
(Integro, Zaandam, The Netherlands) and 15 mM sodium
hydrogen carbonate (NaHCO3). Cells were
transfected with hMC3R (42), hMC4R (43), or mMC5R (44) cDNA cloned into
pcDNA3 (Invitrogen, Carlsbad, CA) using calcium phosphate
precipitation. Clones, stably expressing MCRs, were selected in medium
containing the neomycin analog G418 (800 µg/ml, Life Technologies, Inc.).
AC Activity
AC activity was determined using a modified method of Salomon
(45).
Intact-Cell Assay
Cells were grown in 24-well plates and incubated for 2 h with 2
µCi/ml [3,8-3H]-adenine (21.7 Ci/mmol,
NEN Life Science Products, Boston, NA) in DMEM (Life Technologies, Inc.) containing 0.2% BSAe (BSA, ICN Biomedicals, Inc., Aurora, OH), 2 mM
L-glutamine (Life Technologies, Inc.) and
nonessential amino acids. Subsequently, the cells were washed with PBS
(Life Technologies, Inc.) containing 0.25 mM
isobutylmethylxanthine (IBMX, Sigma) (PBS/IBMX) and
incubated for 20 min with compounds diluted in PBS/IBMX. Then 1 ml of
cold stop solution (5% trichloric acid (Merck & Co. Inc.,
West Point, PA), 1 mM cAMP (Roche Molecular Biochemicals, Indianapolis, IN), 1 mM ATP
(Roche Molecular Biochemicals)] per well was added and
the plates were centrifuged at 250 x g. Finally, ATP
and cAMP fractions were separated on Dowex (AG-50W-X4, Bio-Rad Laboratories, Inc. Hercules, CA) and alumina (WN-3,
Sigma) columns, respectively. ATP and cAMP fractions were
dissolved in scintillation cocktail (Ultima Gold, Packard, Meriden, CT)
and counted in a ß-counter.
AC activity was calculated as the percentage of
[3H]ATP that is converted to
[3H]cAMP using the equation
[3H]cAMP/([3H]cAMP +
[3H]-ATP). In a typical experiment, from one
well containing approximately 2 x·105 cells,
1.2 pmol (equivalent to 60,000 dpm) of tritiated cAMP and ATP were
counted.
Membrane Assay
Two days after plating into 10-cm dishes, approximately 1.5 x
109 cells were scraped and suspended in PBS.
After centrifugation (15 min; 500 x g) the cells were
homogenized in 10 ml solution containing 1 mM
NaHCO3, 1 mM dithiothreitol
(DTT, Sigma), 0,2 mM magnesium
acetate (MgAc), 200 µg/ml DNaseI (Roche Molecular Biochemicals), and protease inhibitors (Complete, EDTA-free,
Roche Molecular Biochemicals) at 4 C, using a Teflon on
glass homogenizer. Homogenates were centrifuged for 15 min at
1,500 x g to remove intact cells and cell debris. The
supernatant was centrifuged for 45 min at 40,000 x g,
and the final pellet was resuspended in 1 mM
NaHCO3 + 1 mM DTT. Total
protein content was determined using Bradford reagents with BSA as
standard. Assay mixtures contained 25 mM Tris
acetate (pH 7.5), 5 mM MgAc, 0.5
mM ATP, 1 mM DTT, 0.1
mM IBMX, 0.1 g/l BSA, 10
µM GTP (Roche Molecular Biochemicals), 50 µM cAMP, 1 µCi/ml
[2,8-3H]-ATP (34.5 Ci/mmol, NEN Life Science Products, Boston, NA), 5 mM
phosphocreatine (Sigma) and 50 U/ml creatine kinase
(Sigma). The assay was started by adding 20 µg of total
membrane protein (contained in 60 µl) to 40 µl of assay mixtures
and compounds at the appropriate concentrations. Incubations were
performed at 30 C for 30 min. After incubation, samples were treated
exactly as described above for the intact cell assay, except that 0.9
ml of stop solution was added. Typically, from one sample a total
amount of 2.3 pmol (equivalent to 180,000 dpm) of
[3H]cAMP and [3H]ATP
were counted.
Receptor Expression
Bmax of the cell lines was determined in
saturation experiments with [125I]NDP-MSH as
tracer. NDP-MSH was iodinated using bovine lactoperoxidase
(Calbiochem, La Jolla, CA) and
[125I]Na (ICN Biochemicals, Inc.)
according to Oosterom et al. (46) and subsequently HPLC
purified on a C18 column (µBondapak 3.9 x 300 mm, Waters Corp., Milford, MA). Cells were washed with Tris-buffered saline
(TBS) supplemented with 2.5 mM calcium chloride
and incubated for 30 min at room temperature with tracer diluted in
Hams F10 medium (Life Technologies, Inc.) supplemented
with 2.5 mM calcium chloride, 0.25% BSA, and 200
KIU/ml aprotinin (Sigma). After two washes with ice-cold
TBS (+ 2.5 mM calcium chloride) the cells were
lysed in 1 M sodium hydroxide and samples were
counted in a
-counter.
Data Analysis
The Spearman correlation coefficient was determined to assess
correlation between receptor expression level and AC activity response
to forskolin or
-MSH. Differences in basal AC activity between the
hMC4R-expressing cell lines were evaluated using the
Student-Newman-Keuls test. Students t test was used to
analyze the effect of AgRP(83132) treatment on basal and
forskolin-induced AC activity. EC50 values were
calculated with curve fitting (nonlinear, variable slope) using Prism
software (GraphPad Software, Inc., San Diego, CA).
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ACKNOWLEDGMENTS
|
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We thank D. H. Vrinten M.D. for her help with the
statistical analysis of the data.
 |
FOOTNOTES
|
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Address requests for reprints to: Dr. R. A. H Adan, Rudolf Magnus Institute for Neurosciences, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands. E-mail: A.H.Adan{at}med.uu.nl
J. Oosterom was supported by Netherlands Organisation for Scientific
Research (NWO) Grant 90349-162.
Received for publication April 11, 2000.
Revision received August 14, 2000.
Accepted for publication October 3, 2000.
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