International Journal of Obesity (1998) 22, 678±683
ß 1998 Stockton Press All rights reserved 0307±0565/98 $12.00
http://www.stockton-press.co.uk/ijo
Effects of a potent melanocortin agonist on the
diabetic=obese phenotype in yellow mice
MB Zemel1, JW Moore1, N Moustaid1, JH Kim1,2, JS Nichols3, SG Blanchard3, DJ Parks3, C Harris3, FW Lee4 ± 6,
M Grizzle4,5, M James4,5,7, WO Wilkison3
1
4
Department of Nutrition, University of Tennessee, Knoxville, TN; 2Jackson Laboratories, Bar Harbor, ME; 3Molecular Biochemistry,
Pharmacology, 5BioMet, GlaxoWellcome Inc, NC; 6Dupont Merck, Philadelphia, PA; and 7Inspire Pharmaceuticals, RTP, NC, USA
OBJECTIVE: To test the hypothesis that a melanocortin agonist can reverse obesity and insulin resistance in mice
overexpressing the agouti protein.
EXPERIMENTAL MODEL: Mice overexpressing the agouti protein either by transgene introduction (b
b-actin promotor)
or by mutation (Ay ).
DESIGN: NDPMSH was tested for pharmacokinetic suitability. NDPMSH at various doses was administered subcutaneously twice a day for 2 ± 3 weeks.
MEASUREMENTS: Fur pigmentation, various fatness parameters (core temperature, fat pad weight and body weight),
blood glucose and hormones, fatty acid synthase measurement.
RESULTS: NDPMSH caused fur pigmentation and core temperature changes, but failed to affect any metabolic
parameters in agouti-dependent manner.
CONCLUSION: NDPMSH, as a representation melanocortin agonist, does not compete with agouti in reversing agoutidependent metabolic effects. This suggests that 1) agouti works via a receptor other than a melanocortin receptor to
mediate its metabolic effects, 2) agouti-dependent metabolic effects are mediated through melanocortin receptors but
not via antagonism of these receptors, or 3) NDPMSH is pharmacodynamically an inappropriate molecule for these
types of studies.
Keywords: agouti; melanocortin receptors; NDPMSH; obesity
Introduction
Dominant mutations at the agouti locus results in mice
with yellow coat color that become obese and hyperinsulinaemic.1,2 The murine agouti gene encodes for a
131 amino acid secreted protein, normally expressed
in the skin during hair growth to regulate pigmentation.3 Abnormal expression of the agouti protein
causes the obese and diabetics, as well as the yellow
coat phenotypes, as these are reproduced when the
murine agouti cDNA is placed under the transcriptional control of an ubiquitous promoter in transgenic
mice.4,5
The murine agouti protein normally functions in a
paracrine manner to regulate coat colour through
competitive antagonism of alpha-melanocyte stimulating hormone (aMSH) binding to the aMSH receptor
(melanocortin receptor 1 or MC1R). Agouti protein
antagonism prevents the increase in intracellular
cAMP produced upon ligand binding.6,7 This increase
in cAMP leads to the switching of the molecular
pathway of pigment production from phaeomelanin
to eumelanin. It is possible that the obese=diabetic
phenotype induced by agouti overexpression may
occur via altered signalling of other melanocortin
receptor (MCR) family members, as they are
expressed in energy relevant tissues such as adipose, muscle and brain.8,10 Recently, we have shown
that agouti antagonizes the MC4R and MC3R receptors with Ki's closer than originally described.11
In order to test the hypothesis that agouti antagonism of MCRs leads to these phenotypes we introduced
norleucine,4 D-phenylalanine7 aMSH (NDPMSH), a
potent ligand and agonist of all the MCRs,11,12 into
transgenic mice ubiquitously overexpressing the wild
type agouti protein. We report here that while the
NDPMSH showed good systemic effects on coat
coloration and core temperature regulation, it failed
to have any effect on insulin, glucose levels and
weight. This suggests that agouti may have effects
on a different receptor type than MCRs, or it may
mediate its effects on MCRs other than through ligand
antagonism.
Methods and materials
Materials
Correspondence: William O. Wilkison, Zen-Bio, Inc., 3200 Chapel
Hill, Nelson Blvd, Suite 102, PO Box 12503, RTP, NC 27709.
Received 11 July 1997; revised 11 December 1997, 29 January
1998; accepted 6 February 1998
NDPMSH was purchased from Bachem (Philadelphia,
PA). Na125I was purchased from Amersham (Arlington Heights, IL). Enzymobeads were purchased from
BioRad (Hercules, CA).
Lack of NDPMSH effect on obesity
MB Zemel et al
Cold iodination of NDPMSH
Custom iodinated 10 mg of NDPMSH (Peninsula
Labs, Belmont, CA), yielded 8 mg of iodinated peptide. To dissolve the peptide 1 ml of 1 mM HCl was
added directly to the vial. All of the material was
puri®ed by HPLC (four separate runs). Brie¯y,
0.25 ml of the iodinated peptide solution was loaded
onto a Waters Sep-Pak C18 cartridge that had been
equilibrated in 50 mM ammonium acetate, pH 5.8 plus
15% acetonitrile. The material was eluted using a
peristaltic pump with a 40 ml gradient up to 40%
acetonitrile. The mono- and di-iodinated forms of the
peptide were collected separately, and vacuum evaporated to dryness. The monofractions from the four
runs were redissolved in 1 mM HCl and combined, as
were the difractions. Overall yield was 0.8 mg of each
of the two iodinated forms, with purity at > 98% as
assessed by HPLC. Peak identity was con®rmed by
HPLC=mass spectrometry (not shown).
Hot iodination of NDPMSH
NDPMSH was radiolabeled using Enzymobeads technology. Brie¯y, the following was placed in a microcentrifuge tube: 50 ml buffer (0.2 M sodium phosphate,
pH 7.3), 10 ml 0.6 mM NDPMSH (6 nmol), 50 ml
Enzymobeads and 20 ml Na125I (2 mCi; 1.2 nmol).
The reaction was started by the addition of 25 ml 1%
b-d-glucose, and allowed to proceed for 30 min at
room temperature. The reaction was stopped by
removal of the beads by centrifugation, followed by
the addition of 2 ml of 60 mM N-acetyl-tyrosine to the
supernatant. Puri®cation was by HPLC as described in
the preceding paragraph. The results of ®ve consecutive runs were combined to yield a total of 1.8 mCi of
125
I-NDPMSH. Prior to being concentrated to dryness
by vacuum evaporation, nonradioactive I-NDPMSH
was added to reduce the speci®c activity to
25 mCi=mmol.
Animals
C57Bl=6J-Ay and C57Bl=6J mice were purchased
from Charles River Laboratories Inc., Raleigh, NC.
Transgenic mice ubiquitously overexpressing agouti
were generated as previously described.4 Mice were
maintained on a powdered high-fat diet (mouse diet
5015, PMI Feeds, St Louis, MO). At 12 weeks of age,
male transgenic mice and littermate controls were
treated with NDPMSH or saline (control). Prior to
treatment, all mice were anesthetized under iso¯uorane and hair was plucked from a 1 cm2 area on the
ventral surface, in order to observe any subsequent
effects of NDPMSH administration on pigmentation
of newly grown hairs at a site distant from the
hormone administration site.
NDPMSH (1, 5 or 10 mg=kg body weight) was
dissolved in saline and administered subcutaneously
on the dorsal surface, between the scapulae. Saline
administration served as a control. NDPMSH or saline
was administered twice daily for 14 d or 21 d.
The animals were maintained on a 12 h light=dark
cycle (light from 07.00±19.00 h). Injections were
performed at approximately 18.00 h, and all core
temperature measurements were performed prior to
injection.
Physiological measurements
Core temperature was measured using a thermocouple
(Columbus Instruments, Columbus, OH). Probe insertion was performed on day 0, day 7 and day 14 at a
constant distance (1.8 cm) into the rectum. After
stabilization (10 s), the temperature was recorded
every 5 s for 30 s. All temperature measurements
were made between 09.00±10.30 h.
On day 14, animals were anesthetized with iso¯uorane following an overnight fast, and blood obtained via
cardiac puncture. Fat pads (epididymal, perirenal, retroperitoneal, inguinal and subscapular) were dissected,
immediately weighed, frozen in liquid nitrogen and
stored at 780 C for subsequent measure-ments of
fatty acid synthase activity, as described below.
Blood glucose was measured using a blood glucose
monitoring system (Accu-check, Milpitas, CA).
Plasma insulin and leptin levels were measured via
radioimmunoassay using commercial kits (Linco, St
Louis, MO). Fatty acid synthase activity was measured in the subcutaneous and visceral fat depots by a
as
previously
spectrophotometric
method13
14
described. Protein was determined using a modi®ed
Lowry method.15
All data are presented as mean s.e.m. for four
groups of mice: Control mice on saline, control mice
on NDPMSH, transgenic mice on saline and transgenic mice on NDPMSH. Data were analyzed via
two-way analysis of variance or, in the case where
only two groups were being compared to characterize
transgenic vs control, by Student's t-test.
Pharmacokinetics of
125
I-NDPMSH in mice
Male mice of C57B16 strain with body weight in
range 25±30 g were supplied by Charles River
Laboratories. Mice were divided into two groups of
28 each. The ®rst group received an intravenous bolus
dose of 125I-NDPMSH via a tail vein. The second
group received a subcutaneous dose of 125I-NDPMSH.
The dose was 0.08 mg=kg (50 nmol=kg, 1000 mCi=kg)
in all studies. Food and water were provided through
the entire experiment. Two mice were randomly
assigned to each time point except the last time
point consisting of four mice. At each time point,
except at 72 h, mice (n ˆ 2) were killed to only collect
blood samples. Blood, urine and feces were collected
from the 72 h time point group (n ˆ 4). The 125INDPMSH dose was prepared in normal saline at
approximately 0.04 mg=ml. Blood samples (approx
0.3 ml) were withdrawn via cardiac puncture at predose, 5, 15, 30, 45 min and 1, 1.5, 2.5, 4, 6, 8, 24 and
72 h following dose administration and transferred
into heparinized microfuge tubes. The blood samples
679
Lack of NDPMSH effect on obesity
MB Zemel et al
680
were centrifuged to separate the plasma. The plasma
samples were stored in a freezer at 775 C. Red blood
cells were discarded. Predose, 0±24, 24±48, 48±72 h
urine and fecal samples were collected from the 72 h
time point group (n ˆ 4) and stored in a freezer at
775 C. Samples were extracted with acetonitrile and
were analyzed as essentially described for the puri®cation of the NDPMSH iodinated forms.
Results
Rationale for using NDPMSH
While previous experiments have been performed
using the natural ligands, aMSH and des-acetyl
MSH,16,17 it is also clear that these ligands have
extremely short half-lives in serum and are labile to
a number of proteases.18 To overcome these issues,
we used a stable, potent analog, NDPMSH.12
To determine the various pharmacokinetic parameters of this compound, the iodinated compound
was ®rst examined with respect to structure and
ef®cacy. Puri®cation of commercially prepared cold
iodinated NDPMSH was performed to separate the
mono-iodinated form from the di-iodinated and uniodinated NDPMSH. These forms were then examined for their ability to antagonize 125I-NDPMSH
binding to B16F10 cells. These cells predominantly
express the MC1R receptor.19,20 All forms of
NDPMSH were equipotent in their ability to antagonize 125I-NDPMSH binding to the cells (not shown).
We then examined whether iodination affected the
agonist effect on MC1R with respect to cAMP accumulation. All forms of the ligand were equipotent in
their ability to stimulate cAMP production in B16F10
cells (EC50 ˆ 0.6 nM). Finally, we examined the ability of agouti to antagonize these ligands (Table 1). As
shown, agouti was equally effective in antagonizing
all forms of the NDPMSH ligands' ability to increase
intracellular cAMP levels.
Pharmacokinetics
Following the intravenous administration of 125INDPMSH (0.028 mg=kg) the parent drug was rapidly
eliminated with a half-life of 0.7 h (Figure 1). The
majority of the radioactivity dosed was recovered in
urine (> 80%). The plasma level was below the
Table 1 Iodinated forms of NDPMSH are equally antagonized
by agouti
1 nM
aMSH
NDPMSH
I-NDPMSH
I2-NDPMSH
94
97
95
96
B16F10 cells were incubated with the indicated concentration
of peptides along with 100 nM agouti protein. cAMP
concentrations were equally stimulated by the different ligands
(Vmax ˆ 2000 pmol=106 cells) in the absence of agouti and
determined as described.5 Agouti alone had no effect (not
shown). Values are percent inhibition of cAMP level increase.
Figure 1 I125-NDPMSH Pharmacokinetics. NDPMSH was radiolabeled and puri®ed as described in the `Material and Methods'
section. The radiolabeled material was administered intravenously (i.v.) or subcutaneously (s.c.) in a saline solution.
Blood was drawn at the indicated time points and parent compound (I125-NDPMSH) concentrations were determined using the
HPLC method described.
detection limit 4 h after dosing. The parent compound
level was 0.12 ng=ml at 4 h. The overall compound
exposure based on a 0 h to in®nity area under the
plasma concentration curve, was 18.6 h=ng=ml. The
total body clearance and volume of distribution were
25 ml=min=kg and 0.6 L, respectively. An elimination
half-life of 1.4 h was observed after subcutaneous
administration of 125I-NDPMSH at a dose of
0.044 mg=kg (Figure 1). The mean plasma concentration of 125I-NDPMSH at 4 h after dosing was
0.17 ng=ml, a concentration approximately equal to
the Kd for binding of 125I-NDPMSH to the MC1R. As
compared to an equivalent intravenous dose, administration via the subcutaneous route seems to involve
some degree of depot formation as evidenced by: 1) a
longer half-life (1.4 vs 0.66 h); 2) a lower area under
the curve (11 h*ng=ml vs 18.6 h*ng=ml); and 3) a
lower initial plasma concentration (26 vs 39 ng=ml).
Importantly, the maximal concentration in the
serum observed with this compound, was as high as
100 nM. This indicated that adequate circulating
NDPMSH was available to overcome the competitive
inhibition seen by agouti on the MCRs.
Yellow mouse studies
Given the parameters for half-life in mice determined,
we initiated a dose-response study in the C57Bl=6J-Ay
(yellow obese) or C57Bl=6J (control) mice. NDPMSH
was administered subcutaneously twice daily in 15week-old animals for 3 weeks. Interestingly, the
animals' fur turned black in various areas (not just
the dorsal injection site, Figure 2). There was no
apparent effect of NDPMSH on body weight, blood
glucose or blood insulin (not shown) at either the
1 mg=kg or 5 mg=kg dose. These doses are predicted
to have serum concentrations no lower than 8 nM and
40 nM, respectively.
Follow-up studies were then performed, to determine if this is speci®c for the animal strain background. A b-actin agouti transgenic line (BAP20) was
Lack of NDPMSH effect on obesity
MB Zemel et al
weights in all depots of the transgenics was greater
than the controls, along with a 3-fold increase in
adipocyte fatty acid synthase activity. However,
none of these changes appeared to be signi®cantly
in¯uenced by NDPMSH (10 mg=kg) administration in
either control or transgenics after 14 d.
Although a number of criteria were examined, only
core temperature was signi®cantly effected by injection of NDPMSH and this effect was observed in both
control and agouti expressing lines. Basal core temperature for the agouti expressing line was signi®cantly lower than that of the background control strain
(Figure 3). NDPMSH administration caused a marked
increase in core temperature. However, this effect was
of a similar magnitude in both strains of mice.
Figure 2 Systemic availability of NDPMSH as evidenced by coat
coloration. Agouti transgenic mice4 were subcutaneously
injected twice a day with either 10 mg NDPMSH=kg body
weight or saline (control) for 14 d prior to being photographed.
After two weeks, the mice exhibit black fur in areas not localized
to the dorsal injection site.
used for the same study. Clear differences between
transgenic and control mice were observed with
respect to body weight increases, hyperinsulinaemia
and hyperglycaemia (Table 2 and Table 3) at 12
weeks of age. In addition, leptin levels were highly
elevated (5±10-fold) in the trangenics. Fat pad
Discussion
We have shown that introduction of NDPMSH to
mice overexpressing the agouti protein, can cause
reversal of hair pigmentation from yellow to black,
suggesting systemic availability of this compound.
However, only one physiological parameter examined, core temperature, was affected by the concentration of NDPMSH used in this study. This effect was
not in¯uenced by the presence of agouti.
Table 2 Effect of NDPMSH on body weight, weight gain, adipose depot mass and adipose fatty acid synthase activity in
control and agouti-transgenic mice
Strain
Treatment
Body weight (g)
Weight gain (g)
Fat pad weights (g)
Epidymal
Perirenal
Retroperitoneal
Inguinal
Subscapular
5 Fat pad sum
Fatty acid synthase
Activity (nmol=min=mg protein)
Abdominal
Subcutaneous
Control a
Saline
Control a
NDPMSH b
Transgenic a
Saline
31.6 1.2
1.3 0.4
31.9 1.2
0.3 0.4
39.6 1.7*
2.3 0.8*
2.35 0.31
0.37 0.07
0.91 0.23
0.94 0.18
0.61 0.15
5.20 0.88
2.36 0.49
0.37 0.11
0.82 0.17
0.91 0.23
0.56 0.08
5.03 1.07
4.01 0.30**
1.18 0.10**
1.53 0.10**
2.29 0.35**
1.33 0.14**
10.35 0.61*
22.9 3.5
38.5 6.2
26.8 6.5
50.8 9.3
41.8 9.1***
99.3 31.6***
Transgenic a
NDPMSH b
37.7 1.2*
2.0 0.7*
3.94 0.44**
0.92 0.15**
1.62 0.12**
1.80 0.28**
1.21 0.20**
9.48 1.14*
40.3 8.8***
95.0 25.2***
a
n ˆ 5=group. b 10 mg=kg NDPMSH.
* Signi®cant strain effect P < 0.0001; ** Signi®cant strain effect P < 0.001; *** Signi®cant strain effect P < 0.05.
Table 3 Effect of NDPMSH on circulating glucose, insulin and leptin in control and agouti transgenic mice
Strain
Treatment
Glucose (fasting)
(mg=dL)
Glucose (fed)
(mg=dL)
Insulin (fed)
(mg=mL)
Leptin (mg=mL)
Control a
Saline
Control a
NDPMSH b
Transgenic a
Saline
Transgenic a
NDPMSH b
118 7
119 15
124 4*
130 7*
163 21
165 13
201 12*
174 13*
3.63 1.53
9.00 3.06
2.17 0.65
6.55 1.99
7.16 2.09**
48.82 13.53***
7.28 1.76**
51.27 13.36***
a
n ˆ 10=group. b 10 mg=kg NDPMSH.
* Signi®cant strain effect P < 0.05; ** Signi®cant strain effect P < 0.01; *** Signi®cant strain effect P < 0.0005.
681
Lack of NDPMSH effect on obesity
MB Zemel et al
682
Figure 3 NDPMSH administration causes an elevation of core
temperature. Mice, transgenic (n ˆ 10) and non-transgenic littermates (n ˆ 10), were administered NDPMSH subcutaneously and
core temperature was measured as described (see `Methods and
Materials' section). There was a clear effect of compound on
elevation of core temperature.
The concept that agouti overexpression induces
diabetes=obesity via antagonism of melanocortin
receptors is testable. We tried to ensure that the
ligand, NDPMSH, was the best choice. NDPMSH is
relatively stable in mice, with a half-life of about an
hour. The molecule is a potent agonist of all members
of the MCR family, so selective competition of the
agouti from one MCR over another should not be a
major issue.11 Agouti and NDPMSH are competitive
with respect to their ability to bind the MC1R receptor, so increasing concentrations of either should be
suf®cient to counteract the opposing member. We
examined the pharmacokinetic parameters of subcutaneously injected 125I-NDPMSH (which behaves
similarly to NDPMSH with respect to MC1R kinetic
parameters). Our data indicates that the concentrations
and methods used to introduce NDPMSH allowed
NDPMSH concentrations in the blood suf®cient to
activate MCRs.
A positive control included in the study is the
production of black hair on the animal. Originally,
we had plucked hair from the ventral side of the
animal, while using dorsal injections of NDPMSH to
determine whether the compound was systematically
available. We found that black hair would appear in
multiple locations outside the injection site, regardless
of plucking. Thus, we can conclude that the compound was at least available to all portions of the skin.
Additionally, we saw an NDPMSH-, but not agouti-,
dependent effect on elevation of basal core temperature (Figure 3). This effect has been observed by
others21 and indicates that the NDPMSH is available
at high enough levels to effect this physiological
parameter.
Therefore, the lack of effect of NDPMSH on
reversing=affecting insulin resistance and weight,
may be explained by at least three possibilities: 1)
Agouti mediates insulin sensitivity and weight gain
through a receptor other than one from the MCR
family; 2) Agouti mediates these effects through the
MCRs, but not by antagonism of melanocortin
ligands; or 3) NDPMSH may not be an appropriate
ligand for these studies.
With respect to the ®rst issue, it is unlikely that
agouti modulates energy homeostatis through receptors other than MCRs. Recently, it was shown that
homologous recombinant knockout mice of the MC4R
gene became obese and insulin resistant in a time
course similar to that seen for yellow mice.22 In
addition, analogues of NDPMSH relatively selective
for MC4R have been shown to reverse feeding behavior in mice upon intracerebroventricular introduction.23 Both reports support the hypothesis that
agouti-induced hyperphagia are mediated via MCRs,
particularly MC4R.
It is also possible that agouti mediates its effects via
MCRs, but not as a receptor antagonist. In fact, agouti
can mobilize intracellular calcium in the absence of
melanocortin ligands.24 This calcium mobilization is
dependent upon melanocortin receptors in HEK293
cells25 and leads to the accumulation of triglycerides
and elevation of fatty acid synthase activity in isolated
adipocytes.26 It is not known what the effect of the
various melanocortin analogues reported by Fan et al
would have an intracellular calcium. Interestingly, we
have demonstrated that site directed mutants of the
agouti polypeptide which fail to antagonize 125INDPMSH binding to the MCRs, also fail to stimulate
intracellular calcium (Zemel and Wilkison, unpublished data).
Finally, NDPMSH may not be the most appropriate
ligand for these studies. The compound appears to
display acceptable pharmacokinetics and access to
the central nervous system,27 albeit limited. However,
the compound may not couple MCRs ef®ciently to the
protein kinase A pathway.28 It is interesting to note
that NDPMSH administered intraperitoneally was
somewhat effective in decreasing serum insulin and
glucose.23 However, these were relatively short term
studies and the mode of administration was different
to the current study. We believe there may be a short
term effect of this peptide on glucose=insulin, but
longer term, this peptide does not show ef®cacy.
Conclusion
It is important to note that agouti induction of the
obese phenotype is likely to be mediated by both
central and peripheral mechanisms, which any be
differently affected by melanocortin receptor antagonism. The observation that homologous recombinant
knockout mice of the MC4R gene recapitulate several
characteristics of the agouti obesity syndrome,22
clearly implicates hypothalmic MC4R in the hyperphagia characteristic of agouti-induced obesity. The
®nding that intracerebroventricular administration of a
potent cyclic melanocortin agonist (MTII) inhibits
hyperphagia, while co-adminstration of a speci®c
MC4R antagonist (SHU9119) reverses this administration,23 reinforces this concept. On the other hand,
direct effects of agouti on adipocyte Ca‡2 signalling
Lack of NDPMSH effect on obesity
MB Zemel et al
and lipogenesis24 ± 26 that are not inhibited by
NDPMSH, argue for a peripheral effect of agouti
which is not dependent on melanocortin receptor
antagonism. In support of this concept, we have
recently reported that transgenic mice expression
agouti in adipose tissue under the control of the ap2
promoter, become obese in the presence of supplemental insulin, while insulin treated non-transgenic
littermates do not.29 This is consistent with our recent
observation of an agouti-insulin synergism in stimulating lipogenesis in isolated adipocytes.30 Thus, peripheral effects of agouti that are not predicated upon
melanocortin receptor antagonism, appear to contribute to the agouti-induced obesity syndrome.
Acknowledgements
The authors would like to acknowledge grant support
from GlaxoWellcome, Inc. (MBZ) and USDA
CSREES TEN 00093 (MBZ).
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