Neuron, Vol. 42, 983–991, June 24, 2004, Copyright 2004 by Cell Press
Leptin Receptor Signaling in POMC Neurons
Is Required for Normal Body Weight Homeostasis
plementation of the LEPR in neurons of Leprdb/dbmice
results in an amelioration of the obese phenotype (Ko-
walski et al., 2001). Additionally, central leptin adminis-
tration into the cerebral ventricles (icv) reduces body
weight and food intake in Lepob/oband normal mice
(Campfield et al., 1995). In the brain, the signaling form
of the leptin receptor (Lepr-b) is expressed in several
sites. This includes dense expression in the arcuate
nucleus (ARC) of the hypothalamus, which has been
proposed as an important site for the regulation of en-
ergy balance (Cowley et al., 2003; Schwartz et al., 2003;
Zigman and Elmquist, 2003). Indeed, ARC-specific le-
sions performed in rodents produce a profound obese
phenotype (Bergen et al., 1998; Meister et al., 1989).
Moreover, the ARC is required for leptin-induced anorexi-
genic responses, as icv leptin infusion in ARC-lesioned
Lepob/obmice do not cause body weight reduction
(Takeda et al., 2002).
genic CART/POMC neurons, which are direct leptin tar-
gets (Cowley et al., 2001; Elias et al., 1999; Elmquist
et al., 1999). Leptin activates CART/POMC-expressing
neurons, as demonstrated by electrophysiological re-
cordings that show that leptin depolarizes (i.e., acti-
fasted rodents (a condition of reduced leptinemia) and
Lepob/obmice both have decreased hypothalamic Pomc
mRNA content which can be normalized by exogenous
leptin administration (Mizuno et al., 1998; Schwartz et
NPY/AGRP-expressing neurons; Lepob/obmice as well as
fasted rodents have increased hypothalamic Agrp and
Npy mRNA levels which can be reduced by exogenous
leptin administration (Ahima et al., 1996; Schwartz et al.,
1996; Stephens et al., 1995). However, the physiological
relevance of leptin’s direct action on CART/POMC or
NPY/AGRP neurons has not yet been tested.
Although the importance of central and in particular
ARC leptin signaling has been demonstrated, it is not
clear which neuronal cell type or neurocircuits might be
the principal mediators of leptin actions. Given that ARC
lesions cause obesity, not leanness, and considering
the well-established role of ?-MSH (one of POMC’s en-
doproteolytic products) and its downstream receptors
in energy balance (Butler et al., 2001; Chen et al., 2000;
Huszar et al., 1997; Michaud et al., 1994; Yaswen et
al., 1999), POMC neurons have been proposed as the
primary cell type for mediating leptin’s anorexigenic ef-
fect (Cowley et al., 2001; Schwartz et al., 2003; Seeley
genic effects are not dependent on its regulation of
?-MSHsignaling (Bostonetal., 1997;Challiset al.,2004;
Marsh et al., 1999). In order to definitively test the hy-
pothesis that LEPRs on POMC neurons are required
to prevent excessive weight gain, we generated mice
lacking LEPRs specifically on these cells. This animal
model presents a unique opportunity to evaluate the
specific role of leptin signaling in POMC neurons.
Nina Balthasar,1,4Roberto Coppari,1,4
Julie McMinn,3Shun M. Liu,3
Charlotte E. Lee,1Vinsee Tang,1
Christopher D. Kenny,1Robert A. McGovern,1
Streamson C. Chua, Jr.,3,5,* Joel K. Elmquist,1,2,5,*
and Bradford B. Lowell1,5,*
1Department of Medicine
Division of Endocrinology
Beth Israel Deaconess Medical Center
Harvard Medical School
99 Brookline Avenue RN
Boston, Massachusetts 02215
2Department of Neurology
Beth Israel Deaconess Medical Center
Program in Neuroscience
Harvard Medical School
99 Brookline Avenue RN
Boston, Massachusetts 02215
3Department of Pediatrics
New York, New York 10032
Neuroanatomical and electrophysiological studies have
shown that hypothalamic POMC neurons are targets of
the adipostatic hormone leptin. However, the physio-
logical relevance of leptin signaling in these neurons
has not yet been directly tested. Here, using the Cre/
loxP system, we critically test the functional impor-
tance of leptin action on POMC neurons by deleting
leptin receptors specifically from these cells in mice.
Mice lacking leptin signaling in POMC neurons are
mildly obese, hyperleptinemic, and have altered ex-
pression of hypothalamic neuropeptides. In summary,
leptin receptors on POMC neurons are required but
not solely responsible for leptin’s regulation of body
Mice carrying mutations either in the adipocyte-derived
hormone leptin (Lepob/ob) or the leptin receptor (Leprdb/db)
genes have an array of abnormalities, including obesity,
diabetes, infertility, impaired growth, high bone mass,
and hypercorticosteronemia (Chen et al., 1996; Chua et
al., 1996; Lee et al., 1996; Takeda et al., 2002; Tartaglia
et al., 1995; Zhang et al., 1994). Leptin is secreted by
adipocytes and is thought to act mainly on the central
nervous system (CNS) (Spiegelman and Flier, 2001). In-
deed, the deletion of all forms of the leptin receptor
(LEPR) specifically in neurons leads to an obese pheno-
type (Cohen et al., 2001). Furthermore, transgenic com-
*Correspondence: firstname.lastname@example.org (B.B.L.); jelmquis@
bidmc.harvard.edu (J.K.E); email@example.com (S.C.C.Jr.)
4These authors are equally contributing first authors.
5These authors are joint senior authors.
Figure 1. Generation of Mice Lacking Leptin
Receptors on POMC Neurons
(A) Mice expressing Cre recombinase (Cre)
under Pomc promoter control were gener-
chromosome. Leprflox/floxmice are homozy-
gous for a loxP-flanked exon 17, i.e., JAK
docking site, of the leptin receptor allele.
(B) Pomc-Cre mice were mated with Z/EG
reporter mice and immunohistochemistry for
eGFP was performed in double transgenic
mice. Scale bar, 50 ?m.
(C) Double immunohistochemistry for ?-MSH
and eGFP was performed in Pomc-Cre,
POMC neurons in the ARC expressed functional Cre re-
In orderto generate micelacking LEPR onlyon POMC
neurons, mice homozygous for a loxP-modified Lepr
allele (Leprflox/flox, Figure 1A) were used. Cre-mediated
deletion of the loxP-modified Lepr allele is expected
to recapitulate the spontaneous db mutation. Leprdb/db
mice carry a point mutation which inserts a premature
STOP codon five amino acids downstream of the Lepr
exon 18b splice site. The resulting truncated LEPR-b
protein lacks the STAT3 motif required for leptin recep-
tor signaling (Bates et al., 2003). The Cre-deleted Lepr
allele (Lepr?) is expected to also generate a truncated
LEPR-b protein lacking both the STAT3 and also the
JAK (encoded by the loxP-flanked exon 17, Figure 1A)
motifs. Loss of the STAT3 motif is expected because,
if exon 16 were to splice to exon 18b, exon 18b would
be out of frame. Thus, the Lepr?allele is expected to
be null for Lepr-b. To assess whether the Lepr?allele
is indeed null for Lepr-b, we first generated mice that
are homozygous for the Cre-deleted Lepr allele (Lepr?/?
mice). This was achieved by taking advantage of Cre
being sporadically expressed in the germline of Pomc-
Cre, Leprflox/?mice before the first meiotic division,
thereby creating non-Cre-transgenic gametes bearing
Transgenic mice expressing Cre recombinase (Cre) in
POMC neurons were generated by engineering a Pomc
bacterial artificial chromosome (BAC) such that Cre is
driven by Pomc regulatory elements (Figure 1A). To as-
sess whether functional Cre protein was restricted to
areas known to contain POMC neurons, we crossed
Pomc-Cre mice with Z/EG reporter mice [Tg(ACTB-
which express enhanced green fluorescent protein
(eGFP) after Cre-mediated deletion of a loxP-flanked
lacZ gene (Novak et al., 2000). Double transgenic mice
expressed eGFP in the ARC and the nucleus of the
solitary tract (NTS) in the hindbrain (Figure 1B). Pomc
expression is reported to be limited to these two sites
in the rodent brain (Bronstein et al., 1992; Elias et al.,
1999). Scattered Cre activity was also noted in the den-
to ascertain that all ARC POMC neurons expressed Cre
recombinase, doubleimmunohistochemistry analysis of
?-MSH and eGFP were performed in double transgenic
Pomc-Cre, Z/EG mice (Figure 1C). Quantification analy-
sis indicated that ?90% of ?-MSH-positive neurons ex-
pressed eGFP, suggesting that the vast majority of
POMC Cell-Specific LEPR KO
involved in energy homeostasis, we explored the impor-
tance of LEPRs on POMC neurons in energy balance.
mice and Leprflox/floxmice have body weights identical to
wild-type littermates (data not shown). However, Pomc-
Cre, Leprflox/floxmice had significantly increased body
weights (Figures 3A and 3B). We compared the weights
of 10-week-old Leprflox/flox, Lepr?/?
Leprflox/floxmice and demonstrated that the increase in
body weight in Pomc-Cre, Leprflox/floxmice is 18.2% of
that in Lepr?/?mice (Figure 3C). To rule out that the
obesity phenotype in Pomc-Cre, Leprflox/floxmice may
Cre transgene, a second line of Pomc-Cre mice was
crossed with Leprflox/floxmice. A similar increase in body
weight was observed in this second line of Pomc-Cre,
Leprflox/floxmice as was demonstrated for the first line
To determine the origin of increased body weight in
Pomc-Cre, Leprflox/floxmice, body composition was as-
sessed using dual-energy X-ray absorptiometry (DEXA).
As shown in Figure 4A, the increased body weight in
mice lacking leptin signaling in POMC neurons arises
mainly from an increase in fat mass. Indeed, dissection
of distinct fat pads confirmed that these were at least
twice as large as in Leprflox/floxcontrol mice (Figure 4B).
Consistent with the increased fat pads, serum leptin
levels were also increased (Figure 4C). These data dem-
onstrate that the absence of leptin signaling in POMC
neurons causes an impairment in energy balance which
leads to an increase in body fat. Lepob/oband Leprdb/db
mice have increased food intake and decreased energy
expenditure (Pelleymounter et al., 1995). However, nei-
ther of these parameters were significantly altered in
Pomc-Cre, Leprflox/floxmice (Figures 4D and 4E).
Leptin action has been shown to alter hypothalamic
neuropeptide expression levels. For example, both
Lepob/oband Leprdb/dbmice have decreased Pomc and
increased Agrp and Npy mRNA (Ahima et al., 1996; Mi-
zuno et al., 1998; Schwartz et al., 1996, 1997; Stephens
et al., 1995; Thornton et al., 1997). Consistent with a
direct action of LEPR signaling on Pomc gene expres-
sion, mice lacking LEPRs on POMC neurons had re-
duced hypothalamic Pomc mRNA content (Figure 5). In
addition, hypothalamic Agrp mRNA was also signifi-
cantly decreased, while Npy showed a similar trend as
Agrp mRNA in adult Pomc-Cre, Leprflox/floxmice (Figure
5). At first pass, a change in Npy/Agrp mRNA levels may
be unexpected since NPY/AGRP neurons in Pomc-Cre,
was able to induce Socs3 mRNA in ARC non-POMC
neurons, which presumably included NPY/AGRP neu-
rons. As mentioned previously, Pomc-Cre, Leprflox/flox
mice are significantly hyperleptinemic. We thus hypoth-
esize that the increased adiposity in Pomc-Cre,
Leprflox/floxmice activates compensatory mechanisms,
which may include increased inhibitory leptin action on
NPY/AGRP neurons, thereby reducing Npy/Agrp ex-
pression. In support of this, hypothalamic neuropeptide
levels were measured before the onset of obesity, and
Agrp mRNA content was found to be normal (measured
in 4-week-old females; Leprflox/flox? 2.28 ? 0.44 Agrp/
18S; Pomc-Cre, Leprflox/flox? 2.42 ? 0.30 Agrp/18S; n ?
Figure 2. Leptin-Induced Socs3 Activation Is Absent in POMC Neu-
rons of Pomc-Cre, Leprflox/floxMice
(A) Representative in situ hybridization for Pomc (DIG, brown stain)
and Socs3 (35S, silver grains) mRNA. Scale bar, 10 ?m.
(B) Socs3 mRNA induction was quantified by counting silver grains
decorating DIG-labeled POMC neurons (n ? 3–5, ***p ? 0.001).
Arrows denote neurons expressing both Pomc and Socs3 mRNA.
gosity. Lepr?/?mice were markedly obese, and their
body weight was identical to that of Leprdb/dbmice on a
similar genetic background (Figure 3C). Thus, because
the phenotype of Lepr?/?mice is indistinguishable from
that of Leprdb/dbmice, the Cre-deleted Lepr allele must
be null for Lepr-b.
Mice lacking leptin receptors only on POMC neurons
were obtained by crossing Pomc-Cre mice with
Leprflox/floxmice. To validate the loss of functional leptin
receptors exclusively on POMC neurons in mice that
are Pomc-Cre transgenic and homozygous for the loxP-
formed double in situ hybridization of Pomc mRNA and
leptin-induced Socs3 mRNA. It has previously been
shown that leptin binding to its receptor leads to induc-
tion of Socs3 mRNA (Bjorbaek et al., 1998; Elias et al.,
mic POMC neurons in Leprflox/floxmice (Figure 2). How-
ever, leptin was unable to induce Socs3 mRNA in hypo-
thalamic POMC neurons of Pomc-Cre, Leprflox/floxmice
(Figure 2). In addition to POMC neurons, the ARC also
contains NPY/AGRP and other leptin-responsive neu-
negative neurons in both Leprflox/floxand Pomc-Cre,
Leprflox/floxmice. Thus, Pomc-Cre, Leprflox/floxmice lack
functional leptin signaling only in POMC neurons.
Figure 3. Body Weight of Mice Fed a Normal-Fat Diet: 12.5% Kcal from Fat
(A) Body weight curve of male Leprflox/flox(?, n ? 13) and Pomc-Cre, Leprflox/flox(?, n ? 11) mice from line 1.
(B) Body weight curve of female Leprflox/flox(?, n ? 13) and Pomc-Cre, Leprflox/flox(?, n ? 16) mice from line 1.
(C) Body weight of male Leprflox/flox(n ? 13), Pomc-Cre, Leprflox/flox(n ? 11), Lepr?/?(n ? 8), Leprdb/db(genetic background C57Bl6/J ? FVB N2,
n ? 5) mice at 10 weeks of age from line 1.
(D) Body weight curve of male Leprflox/flox(?, n ? 8) and Pomc-Cre, Leprflox/flox(?, n ? 9) mice from line 2. (*p ? 0.05; **p ? 0.01; ***p ? 0.001
6). Importantly, Pomc mRNA was significanlty reduced
even before the onset of obesity (measured in 4-week-
old females; Leprflox/flox? 2.85 ? 0.39 Pomc/18S; Pomc-
Cre, Leprflox/flox? 1.86 ? 0.15 Pomc/18S; n ? 6, p ?
0.05). The reduced Npy/Agrp expression levels in adult
Pomc-Cre, Leprflox/floxmice could ultimately be limiting
the degree of obesity.
In addition to regulating energy balance, leptin is also
Figure 4. Body Composition, Serum Leptin
Levels, Food Intake, and Oxygen Consump-
tion in Mice LackingLEPR on POMC Neurons
(A) Fat and lean mass in male 8- to 10-week-
old Leprflox/floxand Pomc-Cre, Leprflox/floxmice
were analyzed by DEXA measurement (n ?
(B) Distinct fat pads were dissected and
weighed in male Leprflox/floxand Pomc-Cre,
Leprflox/floxmice (n ? 6–7).
(C) Serum leptin levels were assessed by
ELISA in male 8- to 10-week-old Leprflox/flox
and Pomc-Cre, Leprflox/floxmice (n ? 6–7).
(D) Food intake was measured from week
3–14 in male Leprflox/flox
Leprflox/floxmice (n ? 10–12) and is presented
here as food intake/day per mouse.
(E) Oxygen consumption was measured and
averaged over a 48 hr time period in male
8- to 10-week-old Leprflox/floxand Pomc-Cre,
try and is presented here as ml/min per
mouse. (**p ? 0.01; ***p ? 0.001).
POMC Cell-Specific LEPR KO
question in the field is the identity of first-order, leptin-
responsive neurons that relay this leptin signal from the
circulation to downstream neural circuits regulating en-
ergy balance. Numerous lines of evidence have led to
a model in which NPY/AGRP neurons and CART/POMC
neurons, both located in the arcuate nucleus of the hy-
pothalamus, are critical first-order, leptin-responsive
neurons (Cowley et al., 2003; Schwartz et al., 2003; Zig-
man and Elmquist, 2003). While supported by indirect
evidence, the importance of these first-order neurons
has not yet been directly tested. To determine whether
LEPRs on POMC neurons are critical mediators of lep-
from these neurons in mice.
Our data demonstrate that LEPRs on POMC neurons,
a subpopulation of only ?3000 neurons in mice (Cowley
stasis. However, while an increase in body weight and
body fat is clearly seen in Pomc-Cre, Leprflox/floxmice,
it is markedly smaller than that caused by complete
deficiency of LEPRs (i.e., body weight increase of only
18% of that observed in mice with complete deficiency
of LEPRs). This demonstrates that LEPRs on POMC
neuronsare notsolely responsiblefor leptin’sregulation
of body weight homeostasis and that LEPRs on other
neuronsare alsoimportant.Indeed, aswill bediscussed
later, leptin-sensitive neurons have been demonstrated
in numerous hypothalamic (Elmquist et al., 1999) and
extrahypothalamic CNS sites (Elmquist et al., 1998; Grill
et al., 2002).
Given the established role of leptin in controlling
POMC neuron activity and neuropeptide expression, as
well as the important role of ?-MSH and its downstream
receptors inenergy homeostasis,the degreeof disregu-
lation of energy balance seen in Pomc-Cre, Leprflox/flox
mice is less than expected. It is formally possible that
the absence of a more dramatic obesity phenotype in
these mice may be due to incomplete deletion of Lepr
onPOMCneurons. Thus,itiscritical todemonstrateCre
expression in all POMC neurons and more importantly
to validate the lack of functional LEPRs on all POMC
neurons in Pomc-Cre, Leprflox/floxmice. Double immuno-
Figure 5. Hypothalamic Neuropeptide Expression Levels
Neuropeptide expression levels in female 8- to 10-week-old
Leprflox/floxand Pomc-Cre, Leprflox/floxmice were measured in hypo-
thalamic RNAextracts byTaqMan quantitativeRT-PCR andnormal-
ized to 18S ribosomal RNA content. (n ? 8, **p ? 0.01, *p ? 0.05).
known to affect fertility, bone mineral density, body
length, glucose, insulin, corticosterone, and thyroxine
(T4) serum levels (Ahima et al., 1996; Coleman, 1978,
and insulin levels were normal, as shown in Table 1.
Furthermore, Pomc-Cre, Leprflox/floxare fertile and able
to lactate. Bone mineral density is unaltered (measured
by DEXA in 10-week-old males; Leprflox/flox? 0.0513 ?
0.0006 g/cm2; Pomc-Cre, Leprflox/flox? 0.0506 ? 0.0014
g/cm2; n ? 6–7). Pomc-Cre, Leprflox/floxmice have the
corticosterone and T4 levels were also normal (Table 1).
These data demonstrate that LEPRs on POMC neurons
are not required for the aforementioned effects of leptin
and that these must be regulated by LEPRs on other
Leptin is secreted by adipocytes, and its level in the
blood reflects the status of fat stores. Leptin’s primary
site of action is the brain, where it promotes decreased
food intake and increased energy expenditure, hence
reducing adiposity (Spiegelman and Flier, 2001). A key
Table 1. Body Length and Blood Composition of Mice Lacking LEPRs on POMC Neurons
Nose/anus length (cm)
Femur length (mm)
10.12 ? 0.04 (12)
9.69 ? 0.08 (8)
10.16 ? 0.07 (10)
9.86 ? 0.06 (9)
14.3 ? 0.1 (10)
14.0 ? 0.5 (6)
14.7 ? 0.2 (6)
14.2 ? 0.2 (10)
0.74 ? 0.15 (9)
0.69 ? 0.15 (5)
1.49 ? 0.47 (8)
0.98 ? 0.18 (6)
165.21 ? 3.83 (14)
151.00 ? 4.82 (10)
173.42 ? 12.06 (12)
148.12 ? 4.35 (16)
9.06 ? 1.77 (7) 6.86 ? 1.33 (6)
2.08 ? 0.18 (6)
2.11 ? 0.26 (6)
2.24 ? 0.07 (5)
2.07 ? 0.14 (6)
Mice at 8–10 weeks of age. All data represent the mean ? SEM. The number of mice per group is shown in parentheses.
histochemistry analysis of ?-MSH and eGFP in double
transgenic Pomc-Cre, Z/EG mice showed that ?90% of
ARC POMC neurons expressed eGFP, indicating that
Cre was active in most if not all POMC neurons. To date,
antibodiessuitable forLEPR detectionare notavailable,
and we were unable to generate an in situ hybridization
probe capable of detecting the “floxed” exon 17 se-
quence. Thus, to demonstrate the absence of functional
LEPRs on POMC neurons, a well-characterized assay
for testing direct leptin signaling, i.e., leptin’s ability to
induce Socs3 mRNA, was chosen (Bjorbaek et al., 1998;
Elias et al., 1999). Indeed, we demonstrated absence
of LEPR signaling in POMC neurons of Pomc-Cre,
Leprflox/floxmice by showing that leptin was unable to
significantly induce Socs3 mRNA specifically in these
neurons. However, although not statistically different
from Socs3 mRNA activation in saline control animals,
we did observe 7% of Socs3-positive POMC neurons
in Pomc-Cre, Leprflox/floxmice. Whether these very few
potentially leptin-responsive POMC neurons, if they do
indeed exist, would be able to prevent a more drastic
obese phenotype in Pomc-Cre, Leprflox/floxmice is un-
known but seems unlikely.
Alternatively, we suggest that the smaller than ex-
pected degree of obesity observed in mice lacking
LEPRs on POMC neurons is related to one of the follow-
ing two possibilities. Either leptin regulation of POMC
neurons can occur indirectly, for example, through
LEPRs on NPY/AGRP neurons and their collateral pro-
jections to POMC neurons, or leptin regulation of POMC
neurons, either directly or indirectly, plays only a small
With regard to indirect regulation of POMC neurons by
leptin, it has been shown that NPY/AGRP neurons,
which are themselves inhibited by leptin, synapse onto
hypothalamic POMC neurons, inhibiting these POMC
neurons by release of GABA and NPY (Cowley et al.,
2001; Roseberry et al., 2004). With this in mind, it is of
reduced Npy/Agrp mRNA content. This suggests that
NPY/AGRP neurons in adult obese mice are receiving
of POMC neurons.
As suggested above, it is also possible that leptin
plays only a small role in controlling body weight. In
contrast to this view, Seeley et al. (1997) suggested that
MC4R signaling is important in mediating leptin’s acute
have shown that leptin’s anorexigenic effects are inde-
pendent of melanocortin receptor signaling (Boston et
al., 1997; Challis et al., 2004; Marsh et al., 1999). These
melanocortin receptor-independent actions of leptin
could be mediated by additional factors released by
POMC neurons, for example, the neuropeptide CART
(Elias et al., 1998a) or the neurotransmitter glutamate
(Collin et al., 2003), or could be mediated by other first-
deleted leptin receptors from POMC neurons, strongly
supports the latter possibility. These non-POMC, leptin-
responsive neurons could be within the arcuate nucleus
(NPY/AGRP neurons, for example) or could be located
in a number of other hypothalamic nuclei, such as the
dorsomedial, ventral medial, and premammilary nuclei.
In addition,LEPRs are found inextrahypothalamic sites,
including the brain stem (Elmquist et al., 1998). With
respect to the latter, the nucleus of the solitary tract
(NTS) has been suggested to be an important site of
leptin’s anorexigenic action (Grillet al., 2002). Additional
studies will be required to evaluate the importance of
these other candidate, first-order, leptin-responsive
As reviewed above, our study strongly suggests the
existence of other first-order, leptin-responsive neurons
Given this, the body weight curves of Pomc-Cre,
Leprflox/floxmice are worthy of further comment. The ma-
jority of the increase in body weight, in comparison to
controls, occurs between the age of 4 and 6 weeks.
After that, the rate of increase in weight, in comparison
to controls, is greatly reduced. This pattern of weight
gain is uncommon and suggests that the Pomc-Cre,
Leprflox/floxmice, after the age of 6 weeks, have reached
a new set point which they are then able to defend. It
is interesting to speculate whether this attenuation in
weight gain after the age of 6 weeks is the result of
compensatory mechanisms activated by the increased
adiposity and their action on neurons, for example NPY/
AGRP neurons, as supported by the neuropeptide
mRNA data. These compensatory mechanisms may in-
The increased weight in Pomc-Cre, Leprflox/floxmice is
mainly due to increased fat mass, suggesting that
LEPRs on POMC neurons are important regulators of
body fat content. Indeed the melanocortins have been
proposed as important regulators in lipid metabolism
(Albarado et al., 2004; Richter and Schwandt, 1987).
Neither food intake nor energy expenditure is statisti-
cally significantly affected by the loss of LEPRs on
POMC neurons. Since the body weight increase in
Pomc-Cre, Leprflox/floxmice is small, any differences in
food intake or energy expenditure would also be ex-
pected to be small and may thus be difficult to detect.
As mentioned earlier, leptin is known to control glu-
cose homeostasis and reproductive function. Pomc-
Cre, Leprflox/floxmice are fertile and not diabetic. Consis-
tent with this finding, both reproductive function and
glucose homeostasis have previously been suggested
to be regulated through NPY and not POMC pathways
(Hohmann et al., 2000). For example improved diabetes
and reproductive function were noted in Lepob/obmice
lacking the Npy gene, and NPY infusion in rodents leads
to reduced reproductive function (Catzeflis et al., 1993;
Erickson et al., 1996). Furthermore, improved reproduc-
tive function and glycemic control were also observed
in micelacking leptin-mediatedSTAT3 activation,which
has been attributed to their normal Npy expression lev-
els (Bates et al., 2003), even in the presence of reduced
hypothalamic Pomc mRNA contents. The generation of
mice lacking LEPRs specifically on NPY neurons will
critically test the hypothesis that reproductive function
and glucose homeostasis are regulated through leptin-
Leptin signaling has been shown to be a critical regu-
lator of growth and bone formation (Ducy et al., 2000).
Our data suggest that leptin signaling in POMC neurons
is not involved in this process. Indeed, Takeda et al.
POMC Cell-Specific LEPR KO
a commercially available kit (Quiagen, Valencia, CA) and microin-
jected circular into pronuclei of fertilized one-cell stage embryos of
FVB mice (Jackson Laboratories) using standard methods (Hogan
et al., 1986). Ten founders were obtained. Genotyping of Pomc-
Cre transgenic mice was performed by PCR. Endogenous Pomc
sequences were amplified with two primers (1, 5?-TGG CTC AAT
GTC CTT CCT GG; 2, 5?-CAC ATA AGC TGC ATC GTT AAG), while
a third primer (3, 5?-GAG ATA TCT TTA ACC CTG ATC) in combina-
tion with (1) generated a transgene-specific amplicon.
with arcuate MSG-induced damage not having an effect
on leptin’s antiosteogenic function (Takeda et al., 2002).
In summary, lack of leptin signaling only in POMC
neurons leads to impaired energy homeostasis. How-
ever, given the established role of leptin in controlling
POMC neurons, as well as the important role of ?-MSH
and its downstream receptors in energy homeostasis,
the small increase in adiposity is less than expected.
Thus, other sites of leptin action must also be important
in leptin’s regulation of energy homeostasis. Delivery of
Cre either by stereotaxic injections of AAV-Cre or by
transgenesis, using neuron-specific
Leprflox/floxmice, will be powerful tools for surveying the
physiologically important neurocircuits mediating lep-
Generation of Pomc-Cre, Leprflox/floxMice
Pomc-Cre mice were mated with Leprflox/flox(129-C57Bl6/J ? FVB
N2) mice (supplied by S. Chua Jr., New York), and a breeding colony
was maintained by mating Leprflox/floxand Pomc-Cre, Leprflox/floxmice.
Only animals from the same mixed background strain generation
were compared to each other. Leprflox/floxanimals were genotyped
by PCR with primers crossing the loxP site: (4, 5?-AAT GAA AAA
GTT GTT TTG GGA CGA, and 5, 5?-CAG GCT TGA GAA CAT GAA
CAC AAC AAC).
Generation of Lepr?/?Mice
Pomc-Cre, Leprflox/?mice were mated with Leprflox/?mice, and
Lepr?/?mice were obtained sporadically through expression of Cre
mated with Lepr?/?and Lepr?/?were obtained. Lepr?/?were geno-
typed by PCR across the floxed exon 17 using primer 4 and 6, 5?-
CTG ATT TGA TAG ATG GTC TTG AG).
Care of all mice was within institutional Institutional Animal Care
and Use Committee (IACUC) guidelines, and all procedures were
approved by the Beth Israel Deaconess Medical Center IACUCC.
Mice were housed in groups of two to four at 22?C–24?C using a 14
hr light/10 hr dark cycle with chow food (Teklad F6 Rodent Diet
8664, 4.05 kcal/g, 3.3 kcal/g metabolizable energy, 12.5% kcal from
fat, Harlan Teklad, Madison, WI, www.harlan.com) and water pro-
vided ad libitum. Body weight was measured once a week. For food
intake studies, male mice were housed individually. Large, intact
pellets of food were provided every 7 days in order to reduce spill-
age, and cages were changed every time that food weight was
measured. Mice were killed by CO2narcosis.
Generation of Leprdb/dbMice
Leprdb/db(C57Bl6/J) mice were purchased from Jackson Labora-
tories (stock# 00697) and mated with FVB mice for two generations.
?-MSH and eGFP Immunohistochemistry
Pomc-Cremicewere matedwithZ/EGreporter mice(JacksonLabs,
stock# 003920 [Novak et al., 2000]). Pomc-Cre, Z/EG double trans-
genic mice were perfused with 10% formalin, the brains sectioned
on a microtome, and eGFP immunohistochemistry was performed
as previously described (Liu et al., 2003). Pomc-Cre, Z/EG double
transgenic mice used for eGFP and ?-MSH double staining were
colchicine treated and immunohistochemistry performed as de-
scribed previously (Elias et al., 1998b). eGFP and ?-MSH double
labeled neurons were counted in three arcuate nucleus sections per
mouse (n ? 2).
Generation of Pomc-Cre BAC Transgenic Mice
The FRT-Kan-FRT cassette from the plasmid pSV-FLP (a generous
gift fromDr. F. Stewart,Heidelberg, Germany) was amplifiedby PCR
and cloned into pGEM-T-Easy (Promega, Madison, WI), generating
the vector here called pGEM-FRT-Kan-FRT. The Cre gene was am-
plified by PCR from the plasmid pMC-Cre (a generous gift from
Dr. K. Rajewsky, Boston, MA) using the following primer set: N09
5?-ATCGGGCCCATGCCCAAGAAGAAGAGGAAG-3? and N10 5?-ATC
then cloned into pTOPO (Invitrogen, Carlsbad, CA), and the vector
here called pTOPO-Cre was obtained. pTOPO-Cre was cut with
ApaI, the Cre-containing fragment was then cloned into the ApaI
site of pGEM-FRT-Kan-FRT, and the vector here called pGEM-Cre-
FRT-Kan-FRT was generated. A mouse genomic bacterial artificial
chromosome DNA library was screened for the Pomc gene (In-
vitrogen). DNA from the BAC clone containing at least 45 kb of 5?
and 70 kb of 3? Pomc flanking sequences was transformed into
the recombinogenic EL250 bacteria cells (Lee et al., 2001), and
homologous recombination was performed as described by Lee et.
al. The Cre-FRT-Kan-FRT cassette from the plasmid pGEM-Cre-
FRT-Kan-FRT was amplified by PCR using the following primer set:
CGCTTAGTT-3? and N18 5?-TCTGCTCCTTGCAGGGGTCCCTCCAA
CCCAAGAAGAAGAGGAAGGTGTC-3?. Use of these primers inserts
the Cre ATG exactly into the Pomc ATG and deletes the first 30
bp of the Pomc gene. Pomc BAC host EL250 cells were made
electrocompetent, and the homologous recombinases were in-
duced according to published protocols (Lee et al., 2001). The Cre-
FRT-Kan-FRT cassette was then transformed into the Pomc BAC
host EL250 cells and recombined. Pomc-Cre-FRT-Kan-FRT BAC
host EL250 clones were identified by PCR screening. The FRT-Kan-
FRT cassette was removed according to published protocols (Lee
et al., 2001), and a Pomc-Cre BAC host EL250 clone without muta-
tion in the Cre coding sequence was obtained. The loxP site present
in the vector sequence of the Pomc-Cre BAC was removed as
describedby Leeet al.The Pomc-CreBAC DNAwas preparedusing
Socs3 Induction in POMC Neurons
Fed male 10-week-old Leprflox/floxand Pomc-Cre, Leprflox/floxmice
were injected ip with 100 ?g recombinant mouse leptin (A.F. Parlow,
National Hormone and Peptide Program) and perfused with 10%
formalin 45 min later. In situ hybridization for Pomc (DIG) and Socs3
(35S) mRNA was performed on microtome cut 25 ?m brain sections
as described earlier (Elias et al., 1999). As described previously,
silver grains on POMC neurons were counted in three arcuate nu-
cleus sections per animal (n ? 3–5 per group), and neurons were
deemed responsive if silver grain counts were two times above
background (Elias et al., 1999).
Body and Blood Composition
Mice at 8–10 week of age and fed ad libitum were either ketamine
anesthetized for dual-energy X-ray absorptiometry (MEC Lunar
was collected by centrifugation and assayed for leptin (Crystal
Chem. Inc., Downers Grove, IL), insulin (Crystal Chem. Inc.), cortico-
sterone (ICN Biomedicals, Inc., CostaMesa, CA), and T4 (Diagnostic
Products Corporation, Los Angeles, CA) levels using commercially
available kits. Tail vein blood was assayed for glucose levels before
the sacrifice (Fisher Scientific, Morris Plains, NJ). The femur length
was measured using the DEXA image’s printout.
Oxygen consumption was measured by indirect calorimetry. Mice
were placed at room temperature (22?C–24?C) in 1.0 l chambers in
an OXYMAX system 4.93 (Columbus Instruments, Columbus, OH)
with a settling time of 100 s and a measuring time of 50 s with room
air as the reference. Food and water were provided ad libitum.
Mice were acclimatized in the chambers for 48 hr. Then oxygen
consumption was measured for 48 hr, and the average VO2is pre-
sented as ml/min.
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Hypothalamic Neuropeptide Expression
Neuropeptide mRNA was analyzed using quantitative PCR. RNA
was extracted from hypothalamic blocks using the Trizol Reagent
(Invitrogen, Life Technologies, Carlsbad, CA). Hypothalamic RNA
was reverse transcribed with RETROscript (Ambion, Inc., Austin,
TX) and amplified using Stratagene Brilliant QPCR Core reactions
(Stratagene, La Jolla, CA) with TaqMan probes and primers (Bio-
were as follows: POMC sense, 5?-GACACGTGGAAGATGCCGAG;
antisense, 5?-CAGCGAGAGGTCGAGTTTGC; probe sequence, 5?-
FAM-CAACCTGCTGGCTTGCATCCGG-TAMRA. AGRP sense, 5?-CTT
TGGCGGAGGTGCTAGA; antisense, 5?-GGACTCGTGCAGCCTTA
CACA; probe sequence, 5?-FAM-TCCACAGAACCGCGAGTCTCG
TTC-TAMRA. NPY sense, 5?-CACCAGACAGAGATATGGCAAGA;
antisense, 5?-TTTCATTTCCCATCACCACATG; probe sequence,
5?-FAM-CAGAAAACGCCCCCAGAACAAGGC-TAMRA. Relative ex-
pression of neuropeptide mRNA was determined using standard
curves based on hypothalamic cDNA, and samples were adjusted
for total RNA content by 18S ribosomal RNA quantitative PCR (Ap-
pliedBiosystems,Foster City,CA).QuantitativePCR wasperformed
on an Mx4000 instrument (Stratagene). Assays were linear over five
orders of magnitude.
Data sets were analyzed for statistical significance using PRISM
This work was supported by grants from the NIH (PO1 DK56116 to
B.B.L. and J.K.E., DK57621 and DK26687 to S.C.C.Jr., MH61583
and DK53301 to J.K.E.) and by Takeda Chemical Industries, Ltd.,
Japan. N.B was supported by The Wellcome Trust, UK, an EASD-
ADA and a BONRC grant. R.C. was supported by Universita’ Po-
litecnica delle Marche (previously Universita’ di Ancona), Italy. We
would like to thank Abby Pullen for expert technical assistance,
Satoshi Naganawa for the POMC BAC, T. Williams for colchicine
injections, and NIDDK’s National Hormone and Peptide Program
and A.F. Parlow for providing recombinant mouse leptin.
Received: March 10, 2004
Revised: May 3, 2004
Accepted: May 24, 2004
Published: June 23, 2004
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