Article

Coppari, R. et al. The hypothalamic arcuate nucleus: a key site for mediating leptin's effects on glucose homeostasis and locomotor activity. Cell Metab. 1, 63-72

Department of Pathology & Cell Biology, Columbia University, New York, New York, United States
Cell Metabolism (Impact Factor: 17.57). 02/2005; 1(1):63-72. DOI: 10.1016/j.cmet.2004.12.004
Source: PubMed
ABSTRACT
Leptin is required for normal energy and glucose homeostasis. The hypothalamic arcuate nucleus (ARH) has been proposed as an important site of leptin action. To assess the physiological significance of leptin signaling in the ARH, we used mice homozygous for a FLPe-reactivatable, leptin receptor null allele (Lepr(neo/neo) mice). Similar to Lepr(db/db) mice, these mice are obese, hyperglycemic, hyperinsulinemic, infertile, and hypoactive. To selectively restore leptin signaling in the ARH, we generated an adeno-associated virus expressing FLPe-recombinase, which was delivered unilaterally into the hypothalamus using stereotaxic injections. We found that unilateral restoration of leptin signaling in the ARH of Lepr(neo/neo) mice leads to a modest decrease in body weight and food intake. In contrast, unilateral reactivation markedly improved hyperinsulinemia and normalized blood glucose levels and locomotor activity. These data demonstrate that leptin signaling in the ARH is sufficient for mediating leptin's effects on glucose homeostasis and locomotor activity.

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ARTICLE
The hypothalamic arcuate nucleus: A key site for mediating
leptin’s effects on glucose homeostasis and locomotor activity
Roberto Coppari,
1,6
Masumi Ichinose,
1,5,6
Charlotte E. Lee,
1
Abigail E. Pullen,
1
Christopher D. Kenny,
1
Robert A. McGovern,
1
Vinsee Tang,
1
Shun M. Liu,
3
Thomas Ludwig,
4
Streamson C. Chua, Jr.,
3,7
Bradford B. Lowell,
1,7,
* and Joel K. Elmquist
1,2,7,
*
1
Department of Medicine, Division of Endocrinology
2
Department of Neurology, Program in Neuroscience, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Avenue,
Boston, Massachusetts 02215
3
Department of Pediatrics
4
Department of Anatomy and Cell Biology, Columbia University, New York, New York 10032
5
Department of Anatomy, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan
6
These authors contributed equally to this work.
7
These authors contributed equally to this work.
*Correspondence: blowell@bidmc.harvard.edu (B.B.L.); jelmquis@bidmc.harvard.edu (J.K.E.)
Summary
Leptin is required for normal energy and glucose homeostasis. The hypothalamic arcuate nucleus (ARH) has been pro-
posed as an important site of leptin action. To assess the physiological significance of leptin signaling in the ARH, we
used mice homozygous for a FLPe-reactivatable, leptin receptor null allele (Lepr
neo/neo
mice). Similar to Lepr
db/db
mice,
these mice are obese, hyperglycemic, hyperinsulinemic, infertile, and hypoactive. To selectively restore leptin signaling in
the ARH, we generated an adeno-associated virus expressing FLPe-recombinase, which was delivered unilaterally into the
hypothalamus using stereotaxic injections. We found that unilateral restoration of leptin signaling in the ARH of Lepr
neo/neo
mice leads to a modest decrease in body weight and food intake. In contrast, unilateral reactivation markedly improved
hyperinsulinemia and normalized blood glucose levels and locomotor activity. These data demonstrate that leptin signaling
in the ARH is sufficient for mediating leptin’s effects on glucose homeostasis and locomotor activity.
Introduction these, the ARH has been proposed as an important site for
mediating leptin’s effect on energy homeostasis (Cowley et al.,
Leptin is secreted by adipocytes and signals to the brain the
2003; Schwartz et al., 2003; Zigman and Elmquist, 2003). In-
status of the body’s energy content (Spiegelman and Flier,
deed, several reports support this view: Takeda et al. (2002)
2001; Friedman, 2004). Mice lacking leptin (Lep
ob/ob
mice) or
demonstrated that icv leptin infusion failed to reduce body
leptin receptor signaling (Lepr
db/db
mice) are obese, diabetic,
weight in ARH-lesioned Lep
ob/ob
mice. Moreover, ARH-specific
infertile, and hypoactive (Chen et al., 1996; Chua et al., 1996;
LEPR-B gene therapy in rats lacking functional leptin receptor
Lee et al., 1996; Tartaglia et al., 1995; Zhang et al., 1994; Cole-
results in an amelioration of the obese phenotype (Morton et
man, 1978). Recently, it has also been shown that leptin plays
al., 2003). The ARH contains two populations of first-order, lep-
a critical role in neuronal plasticity (Pinto et al., 2004; Bouret et
tin-responsive neurons: The orexigenic NPY/AGRP and the
al., 2004). Substantial evidence suggests that the brain medi-
anorexigenic CART/POMC neurons (Spiegelman and Flier,
ates the majority of leptin’s action on energy homeostasis. For
2001; Saper et al., 2002). NPY/AGRP neurons are directly in-
example, deletion of leptin receptors (LEPRs) in neurons in-
hibited by leptin (van den Top et al., 2004), whereas CART/
duces obesity (Cohen et al., 2001), whereas expression of
LEPRs in neurons of Lepr
db/db
mice leads to an amelioration
POMC neurons are directly activated by leptin (Cowley et al.,
of their obesity (Kowalski et al., 2001). Moreover, intracerebro-
2001; Elias et al., 1999). Consistent with this, Lep
ob/ob
mice
ventricular (icv) administration of leptin in Lep
ob/ob
mice causes
have increased hypothalamic Agrp and Npy mRNA levels (Mi-
reduction of body weight and food intake (Campfield et al.,
zuno and Mobbs, 1999; Schwartz et al., 1996; Ahima et al.,
1995).
1996; Stephens et al., 1995) and reduced Pomc mRNA levels
Among the five splice variants described in mice (Lee et al.,
(Schwartz et al., 1997; Thornton et al., 1997). Further support-
1996), the long form of the leptin receptor (LEPR-B) is required
ing the importance of the ARH in controlling leptin actions on
for normal body weight homeostasis (Chen et al., 1996; Lee et
energy homeostasis, we have recently shown that mice lacking
al., 1996). Within the brain, abundant expression of LEPR-B
LEPRs only in POMC neurons are mildly obese (Balthasar et
has been found in several sites including hypothalamic groups
al., 2004).
such as the arcuate (ARH), the ventromedial (VMH), the dor-
In addition to the well-documented effects on body weight,
somedial (DMH), and the ventral premammillary (PMV) nuclei
leptin signaling is required for normal glucose homeostasis as
(Mercer et al., 1996; Elmquist et al., 1998; Fei et al., 1997;
Schwartz et al., 1996; Thornton et al., 1997). Prominent among demonstrated by the fact that both Lep
ob/ob
and Lepr
db/db
CELL METABOLISM : JANUARY 2005 · VOL. 1 · COPYRIGHT © 2005 ELSEVIER INC. DOI:10.1016/j.cmet.2004.12.004 63
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ARTICLE
mice have impaired insulin/glucose homeostasis (Spiegelman Results
and Flier, 2001; Pelleymounter et al., 1995; Coleman, 1978).
Restoring leptin receptor expression
However, it is still unclear whether leptin regulates glucose ho-
in the arcuate nucleus
meostasis directly or indirectly through its action on body
In order to selectively express LEPRs in ARH neurons, we em-
weight. Several studies support the view that leptin does have
ployed Lepr
neo/neo
mice (McMinn et al., 2004). These mice are
an effect on glucose homeostasis independent of its effect on
homozygous for a FLPe-reactivatable, Lepr-null allele (Figure
body weight regulation. For example, Pelleymounter et al.
1B) and, as a result, are similar to Lepr
db/db
mice (McMinn et
(1995) reported that daily leptin administration in Lep
ob/ob
al., 2004). FLPe-mediated deletion of the FRT-flanked Neo cas-
mice, at doses that did not have an effect on body weight,
sette creates a normally functioning Lepr allele. Indeed, mice
normalized serum glucose level. Moreover, Schwartz et al.
homozygous for the FLPe-reactivated, Lepr allele (Lepr
flox/flox
(1996) showed that leptin-treated Lep
ob/ob
mice had 40%
mice, Figure 1C) had body weights indistinguishable from wild-
greater reduction in glucose level compared with pair-fed
type littermates (McMinn et al., 2004; Balthasar et al., 2004).
Lep
ob/ob
control mice. Furthermore, Shimomura et al. (1999)
Site-specific reactivation of the Lepr allele was achieved by
described that adipose-deficient, leptin-deficient, lipodystro-
stereotaxic delivery of FLPe-recombinase in Lepr
neo/neo
male
phic mice are insulin resistant and that insulin sensitivity can
mice. Due to the unavailability of antibodies for FLPe-recombi-
be restored in these mice by leptin infusion. This effect was
nase and in order to visualize FLPe-expressing cells in stereo-
also independent of leptin’s action on body weight. To date, it
taxically injected Lepr
neo/neo
mice, we engineered an adeno-
is unclear whether leptin’s effects on insulin-target tissues are
associated viral vector that would also express enhanced
mediated indirectly by the brain or directly by LEPRs in these
green fluorescent protein (eGFP) (Figure 1A). Therefore, we
were able to visualize FLPe-expressing cells by performing
tissues (Kamohara et al., 1997; Minokoshi et al., 2002).
immunohistochemistry for eGFP.
As previously stated, leptin directly acts on NPY/AGRP and
The AAV-FLPe-IRES-eGFP vector was stereotaxically in-
CART/POMC neurons. Thus, the NPY pathway and the mela-
jected into the hypothalamus with coordinates focused on the
nocortin pathway might be involved in leptin-mediated control
ARH of Lepr
neo/neo
mice. This procedure inherently resulted in
of glucose homeostasis. Indeed, Lep
ob/ob
mice lacking the Npy
a high percentage of injection sites that were centered outside
gene (Lep
ob/ob
; Npy
−/−
mice) have almost normal serum glu-
of the ARH as well as several injections that were centered in
cose levels and 50% reduced insulinemia compared to
the ARH. However, these missed injections served as impor-
Lep
ob/ob
mice (Erickson et al., 1996). Leptin activates the mela-
tant anatomic controls. Thus, we categorized the injections as
nocortin pathway by stimulating melanocortinergic POMC neu-
either ARH-misses (control group) or ARH-hits first by neuro-
rons and by inhibiting AGRP neurons (AGRP is the natural an-
anatomic inspection. In addition, as described in Experimental
tagonist at the melanocortin receptors) (Cowley et al., 2001;
procedures, we also categorized the injections by counting
Elias et al., 1999; Elmquist et al., 1999; Schwartz et al., 1996;
eGFP-positive cells within the hypothalamus. Briefly, brains
Roseberry et al., 2004). Recently, it has also been shown that
that had >100 eGFP-positive cells in the ARH (25 m sections
central melanocortin signaling regulates insulin action. For ex-
containing the ARH in a 1:5 series) were scored as ARH-hit.
ample, icv infusion of either the natural agonist (α-melanocyte
Those cases with <100 eGFP-positive cells in the ARH were
stimulating hormone [α-MSH]) or the synthetic antagonist
grouped as ARH-missed. Importantly, we also obtained cases
(SHU9119) of the melanocortin receptors 3 and 4 (MC3R and
that had >100 eGFP-positive cells in the ARH plus in other
MC4R) in rats has opposite effects. α-MSH-treated rats have
nuclei which are known to contain Lepr-b mRNA (e.g., the DMH
and the VMH). These cases with ARH rescue plus the addi-enhanced insulin action, whereas SHU9119-treated rats have
tional sites were not categorized in the ARH-hit group. Figures
diminished insulin action (Obici et al., 2001).
2A and 2B show representative photomicrographs of im-
Leptin signaling has also been shown to be a critical regula-
munohistochemistry for eGFP in ARH-missed and ARH-hit
tor of reproductive function. Indeed both Lep
ob/ob
and Lepr
db/db
Lepr
neo/neo
mice, respectively. The eGFP immunohistochem-
mice are infertile (Spiegelman and Flier, 2001; Bates et al.,
istry was used as an index to categorize the center of the injec-
2003; Coleman, 1978). The NPY pathway has been proposed
tion sites and, thus, the site containing the majority of FLPe-
to mediate leptin’s effect on reproductive function. Consistent
expressing cells. We also determined whether the delivery of
with this hypothesis, Lep
ob/ob
; Npy
−/−
mice have improved fer-
FLPe successfully restored leptin signaling in transduced neu-
tility (Erickson et al., 1996). Also, in agreement with this, mice
rons. To accomplish this, we assessed the rapid phosphoryla-
lacking leptin-mediated STAT3 activation, which are obese but
tion and nuclear translocation of signal transducer and activa-
have normal hypothalamic Npy gene expression, are fertile
tor of transcription 3 (STAT3) in response to leptin (Munzberg
(Bates et al., 2003).
et al., 2003; Li and Friedman, 1999; Baumann et al., 1996;
Finally, leptin exerts also a positive action on locomotor ac-
Hosoi et al., 2002). Therefore, 45 min before perfusion, mice
tivity as suggested by the fact that Lep
ob/ob
mice are hypo-
were injected intraperitoneally with leptin, and phospho-STAT3
active and that their locomotor activity can be normalized by
(P-STAT3) immunohistochemistry was performed. As can be
leptin treatment (Pelleymounter et al., 1995). To examine whether
seen in Figure 2C, ARH-missed cases displayed very modest
leptin signaling only in ARH neurons is sufficient to prevent obe-
leptin-induced P-STAT3 immunoreactivity in the ARH. In con-
sity, diabetes, infertility, and hypoactivity, we re-expressed LEPRs
trast, prominent P-STAT3 immunoreactivity was characteristi-
under the control of the endogenous leptin receptor promoter in
cally observed in mice with ARH-specific restoration of leptin
neurons in the ARH of mice otherwise deficient in leptin recep-
receptor signaling (Figure 2D). The hypothalamic distribution
of leptin-induced P-STAT3-positive neurons in these mice istor expression.
64 CELL METABOLISM : JANUARY 2005
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Leptin action in the hypothalamic arcuate nucleus
Figure 1. Schematic representation of adeno-asso-
ciated viral (AAV) vector and modified Lepr alleles
A) AAV-FLPe-IRES-eGFP was obtained by cloning
the FLPe gene into a transfer vector (pAAV-M2-
IGFP) such that FLPe and eGFP are driven by CMV
regulatory elements.
B) The FRT-Neo-FRT cassette is located upstream
of exon 17 of the Lepr allele. This allele was bred to
homozygosity to generate Lepr
neo/neo
mice.
C) The FLPe-mediated removal of the FRT-Neo-FRT
cassette produces the FRT-modified and loxP-
flanked Lepr allele which functions as a wild-type
allele. Lepr
flox/flox
mice are homozygous for this
allele.
D) The CRE-deleted Lepr allele lacks exon 17 and
is a null allele. Lepr
/
mice are homozygous for
this allele.
outlined in Table 1. These data demonstrate that leptin signal- gated the possibility that re-establishment, unilaterally, of leptin
signaling in ARH neurons would be sufficient to restore normaling was established in ARH-neurons of Lepr
neo/neo
mice.
We also attempted to rule out the possibility that the viral energy homeostasis in Lepr
neo/neo
mice. As shown in Figure
4A, ARH-hit Lepr
neo/neo
mice had significantly reduced bodyinjections and/or the expression of FLPe and eGFP by neurons
in the ARH per se had effects on body weight and glucose weight (starting at 7 weeks of age) when compared to ARH-
missed Lepr
neo/neo
mice. We found that the difference in bodyhomeostasis in obese and diabetic mice. We performed the
following experiment. The AAV-FLPe-IRES-eGFP vector was weight between 12-week-old ARH-hit Lepr
neo/neo
mice and
ARH-missed Lepr
neo/neo
mice was 5.2 g. This represented ap-stereotaxically injected into the hypothalamus with coordinates
focused on the ARH of mice homozygous for a Lepr-null allele proximately 22% of the total body weight difference between
mice with normal leptin signaling (not surgically treated Lepr
+/+
that cannot be reactivated by FLPe (Lepr
D/D
mice, Figure 2D).
Lepr
D/D
mice were categorized as ARH-hit and ARH-missed as mice) and ARH-missed Lepr
neo/neo
mice (Figure 4B). Body
composition analysis revealed that the reduction in bodydescribed above and in Experimental procedures. Figures 3A
and 3B show representative photomicrographs of immuno- weight observed in ARH-hit Lepr
neo/neo
mice was due to a re-
duction in fat mass (Figure 4D). Since ARH-hit Lepr
neo/neo
micehistochemistry for eGFP in ARH-missed and ARH-hit Lepr
D/D
mice, respectively. Both, ARH-missed and ARH-hit Lepr
D/D
had improved energy homeostasis, they must have either re-
duced food intake or increased energy expenditure or both.mice displayed no leptin-induced P-STAT3 immunoreactivity
(Figures 3C and 3D, respectively). Importantly, ARH-missed ARH-hit Lepr
neo/neo
mice had reduced cumulative food intake
(Figure 4E). However, energy expenditure was not elevated inand ARH-hit Lepr
D/D
mice had indistinguishable body weight
(12-week-old Lepr
D/D
mice: ARH-missed = 50.02 [g] ± 1.85 [n = these mice (Figure 4F). These data suggest that leptin signaling
in one side of the ARH is sufficient to mediate w20% of leptin’s6]; ARH-hit = 51.12 [g] ± 1.6 [n = 3], Figure 4C). These mice
also had indistinguishable serum insulin (12-week-old Lepr
D/D
action on body weight homeostasis.
mice: ARH-missed = 89.22 ng/ml ± 18.21 [n = 4]; ARH-hit = 80.88
ng/ml ± 27.58 [n = 3], Figure 5B) and glucose levels (12-week- Restoration of leptin receptors in the arcuate nucleus
dramatically improves glucose homeostasisold Lepr
D/D
mice: ARH-missed = 492.5 mg/dl ± 34.17 [n = 4];
ARH-hit = 562 mg/dl ± 81.6 [n = 3], Figure 5D). These data As noted, leptin signaling is required for normal blood glucose
and insulin levels. In fact, Lep
ob/ob
mice and Lepr
db/db
micedemonstrate that the viral injections and/or the expression of
FLPe and eGFP in neurons in the ARH had no effects on body develop overt diabetes (Spiegelman and Flier, 2001; Coleman,
1978). We found that unilateral restoration of leptin signaling inweight and glucose homeostasis in obese and diabetic mice.
the ARH was sufficient to remarkably improve glucose homeo-
stasis in Lepr
neo/neo
mice. Indeed, insulinemia was greatly re-Leptin action in the arcuate nucleus reduces body weight
Mice lacking leptin signaling have excessive body and fat duced at both 4 and 8 weeks after FLPe-recombinase was de-
livered into the ARH of Lepr
neo/neo
mice (Figure 5A). Notably, 4mass, increased food intake, and reduced energy expenditure
(Spiegelman and Flier, 2001; Friedman, 2004). Thus, we investi- weeks after surgery, the blood glucose levels in ARH-hit
CELL METABOLISM : JANUARY 2005 65
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Figure 2. Unilateral Lepr gene reactivation in the
ARH of Lepr
neo/neo
mice
Photomi crograp h of immunohistoc hem is try for eGFP
in ARH-missed (A; Case 17 in Table 1) and ARH-hit
(B;Case9inTable 1) Lepr
neo/neo
mice. Photomicro-
graph of immunohistochemistry for leptin-induced
phospho-STAT3 in ARH-missed (C; Case 17) and
ARH-hit (D; Case 9) Lepr
neo/neo
mice. Median emi-
nence (ME), third ventricle (3V), hypothalamic arcu-
ate nucleus (ARH). Scale bar = 100 m.
Lepr
neo/neo
mice were not statistically reduced compared to are able to mediate leptin’s effect on locomotor activity, we
recorded ambulatory movements of ARH-hit Lepr
neo/neo
andthat of ARH-missed Lepr
neo/neo
mice (although a trend toward
lower glycemia was seen). However, at 8 weeks after surgery, ARH-missed Lepr
neo/neo
mice. As shown in Figure 6A, ARH-hit
Lepr
neo/neo
mice had significantly increased 24 hr locomotorblood glucose levels were normalized. Indeed, we found that
12-week-old ARH-hit Lepr
neo/neo
mice had blood glucose activity when compared to ARH-missed Lepr
neo/neo
mice. No-
tably, the majority of the increase in activity occurred during thelevels indistinguishable to age-matched Lepr
+/+
mice (Figure
5C). These data strongly suggest that insulin action was greatly dark cycle (Figures 6B and 6C). Since the ambulatory activity of
ARH-hit Lepr
neo/neo
mice was not statistically different to thatimproved in ARH-hit Lepr
neo/neo
mice.
of wild-type control mice (Figure 6A), we conclude that
leptin-sensitive, ARH neurons are sufficient for mediating theArcuate nucleus leptin receptors and reproduction
Leptin is also known to affect fertility. In fact, mice lacking lep- majority, if not all of leptin’s action on locomotor activity.
tin signaling are infertile (Spiegelman and Flier, 2001; Coleman,
1978; Bates et al., 2003). Therefore, we tested whether ARH-hit Discussion
Lepr
neo/neo
male mice were able to produce offspring by breed-
ing these male mice for one week with adult females. The The incidence of obesity and diabetes continues to rise in in-
dustrialized countries (Flier, 2004; Friedman, 2004; Barsh et al.,one-week breeding period was chosen because in similar
housing conditions, adult wild-type male mice were able to im- 2000). In order to prevent and/or treat these conditions it is
critical to understand the cellular and neuroanatomic pathwayspregnate adult wild-type female mice in this window of time.
All male mice (n = 4) that were bred with female mice for 6–7 that control energy and glucose homeostasis. The hormone
leptin is required for normal body weight and glucose homeo-days generated offspring. In contrast, both ARH-hit and
ARH-missed Lepr
neo/neo
mice were unable to produce off- stasis and is key in governing these biological programs (Flier,
2004; Friedman, 2004; Barsh et al., 2000). During the last de-spring. Indeed, none of the females were found to be pregnant
after the one-week breeding period. These data indicate that cade it has become evident that leptin’s primary site of action
is the central nervous system (CNS). However, leptin receptorsunilateral leptin signaling in the ARH is insufficient to restore
the capacity of Lepr
neo/neo
male mice to generate offspring. are expressed in several CNS sites and relatively little is known
about which neuronal groups mediate each of the specific ac-
tions of leptin. The arcuate nucleus in the hypothalamus hasLeptin action in the arcuate nucleus
and locomotor activity been proposed as one important site for mediating leptin’s ef-
fect on energy homeostasis (Cowley et al., 2003; Schwartz etLeptin positively regulates locomotor activity as supported by
the fact that Lep
ob/ob
mice are hypoactive and their total activ- al., 2003; Zigman and Elmquist, 2003). Supporting this view,
we have shown that deletion of LEPRs only in POMC cellsity can be normalized by leptin administration (Pelleymounter
et al., 1995). To date, it is unknown which sites in the brain leads to mild obesity (Balthasar et al., 2004). In addition,
Morton et al. (2003) performed ARH-specific Lepr-b gene ther-mediate this effect of leptin. In order to assess if ARH neurons
66 CELL METABOLISM : JANUARY 2005
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Leptin action in the hypothalamic arcuate nucleus
Table 1. Hypothalamic distribution of leptin-induced P-STAT3 positive neurons in ARH-missed and ARH-hit Lepr
neo/neo
mice
ARH RCA VMH DMH LH Sch
Lt Rt Lt Rt Lt Rt Lt Rt Lt Rt Lt Rt
ARH-hit Lepr
neo/neo
mice
1 531 441691401 00000
2 525 96 20 9 8 0 152 0 25 0 0 0
3 213 254 20 00 00000
4 364 430 00 00 20000
5 606 72304107500000
6 411 7715167 20 00040
7 432 13626113 00 00000
8 325 317 32 00 00000
9 360 631002301101000
ARH-missed Lepr
neo/neo
mice
10 25 1822231 10 00003
11 16 40 00 00 00000
12 6 21 00 00 00000
13 62 3217140 00 000312
14 74 71921201700000
15 7 40 00 00 00000
16 1 60 00 00 00000
17 81 5117117 02 00000
18 2 511120 00 00004
P-STAT3-positive neurons were estimated in hypothalamic nuclei using a camera lucida device (all sections; 1:5 series). Arcuate nucleus (ARH); retrochiasmatic area
(RCA); ventromedial nucleus (VMH); dorsomedial nucleus (DMH); lateral hypothalamic area (LH); suprachiasmatic nucleus (Sch). Lt = left side; Rt = right side.
apy in leptin receptor-deficient rats and showed that this ame- us to express endogenous LEPRs at physiological levels only
in neurons that would normally express LEPRs.liorated obesity. This study used adenoviral vectors to trans-
genically express LEPR-B under the control of CMV regulatory Collectively, our data suggest that restoring physiological
leptin signaling in ARH neurons is sufficient to prevent the fullelements. In contrast, our approach used the delivery of
FLPe-recombinase in young FLPe-reactivatalbe, Lepr-null mice obesity syndrome seen in leptin receptor-deficient mice. How-
ever, the reduction in body weight is relatively modest (w20%)(Lepr
neo/neo
mice) (McMinn et al., 2004). This approach allowed
Figure 3. Delivery of AAV-FLPe-IRES-eGFP in the
ARH of Lepr
/
mice does not lead to Lepr gene re-
activation
Photomi crograp h of immunohistoc hem is try for eGFP
in ARH-missed (A) and ARH-hit (B) Lepr
/
mice.
Photomicrograph of immunohistochemistry for lep-
tin-induced phospho-STAT3 in ARH-missed (C) and
ARH-hit (D) Lepr
/
mice. Median eminence (ME),
third ventricle (3V), hypothalamic arcuate nucleus
(ARH). Scale bar = 100 m.
CELL METABOLISM : JANUARY 2005 67
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ARTICLE
Figure 4. Unilateral reactivation of the Lepr gene in the hypothalamic arcuate nucleus of Lepr
neo/neo
mice leads to reduced body weight, fat mass, and food intake
A) Body weight curves of ARH-missed (n = 9) and ARH-hit (n = 9) Lepr
neo/neo
mice.
B) Body weight of 12-week-old ARH-missed (n = 9), ARH-hit (n = 9) Lepr
neo/neo
and Lepr
+/+
(n = 10) mice.
C) Body weight of 12-week-old ARH-missed (n = 6), ARH-hit (n = 3) Lepr
/
mice. Please note that the body weights are displayed separately from the mice in (B)
since the mice have dissimilar genetic backgrounds (C57Bl6/J and 129 in the case of Lepr
neo/neo
mice and C57Bl6/J, 129 and FVB in the case of Lepr
/
mice).
D) Fat and lean mass in 12-week-old ARH-missed (n = 9), ARH-hit (n = 6) Lepr
neo/neo
and Lepr
+/+
(n = 4) mice were measured by DEXA.
E) Cumulative food intake in ARH-missed (n = 9) and ARH-hit (n = 9) Lepr
neo/neo
mice was collected between ages 5 and 12 weeks.
F) Oxygen consumption was measured with CLAMS in 13-week-old ARH-missed (n = 9) and ARH-hit (n = 5) Lepr
neo/neo
mice. *p < 0.05; **p < 0.01; ***p < 0.001 versus
ARH-missed Lepr
neo/neo
mice.
compared to mice with normal leptin signaling. Given the fact al., 2000). Since the ARH contains first-order, leptin-responsive
that leptin signaling was restored only in one side of the ARH,
neurons that secrete either the agonist (α-MSH) or the antago-
it remains unknown whether bilateral reactivation would lead
nist (AGRP) of the melanocortin receptors (Schwartz et al.,
to normal body weight homeostasis. However, it is notable that
1996, 1997; Cowley et al., 2001; van den Top et al., 2004),
hypothalamic lesions (including the ARH) in rodents cause sig-
mice with restored leptin signaling in ARH neurons would be
nificant obesity only when performed bilaterally (Elmquist et al.,
expected to have improved melanocortin signaling and per-
1999). The fact that unilateral lesions fail to produce obesity
haps insulin action. Moreover, it has been shown that NPY is
suggests that unilateral ARH function should be sufficient for
required for the development of the full diabetes syndrome in
normal body weight regulation.
leptin-deficient mice (Erickson et al., 1996), suggesting that
Interestingly, despite the modest reduction in body weight,
elevated NPY (as found in Lep
ob/ob
) positively contributes to
unilateral reactivation markedly improved hyperinsulinemia and
their diabetes. Since leptin inhibits NPY-secreting ARH neurons
normalized blood glucose levels. Although our study demon-
(van den Top et al., 2004; Roseberry et al., 2004), restoration
strates that LEPRs expression by ARH neurons is sufficient to
of leptin signaling in the ARH is expected to reduce NPY and
mediate leptin’s effects on glucose homeostasis, the down-
therefore to ameliorate glucose homeostasis. Alternatively, it is
stream pathways mediating these effects are still unknown. We
possible that the improvement of glucose/insulin homeostasis
propose that melanocortin r eceptor- and NPY receptor -expr ess-
is secondary to the reduction in body weight. However, it is
ing neurons are downstream targets of leptin-responsive ARH
unlikely that this is the sole mechanism underlying the improve-
neurons in the leptin-mediated control of glucose homeostasis.
ment in glucose homeostasis. First, blood glucose levels, while
Consistent with this hypothesis, it has been shown that activa-
normalized at 8 weeks after FLPe-recombinase was delivered
tion of the central melanocortin pathway leads to incr eased insu-
lin action (Obici et al., 2001) and decreased insulin levels (Fan et into the ARH of Lepr
neo/neo
mice, were not normalized at 4
68 CELL METABOLISM : JANUARY 2005
Page 6
Leptin action in the hypothalamic arcuate nucleus
Figure 5. Unilateral reactivation of the Lepr gene in
the hypothalamic arcuate nucleus of Lepr
neo/neo
mice leads to improved glucose homeostasis
(A) Serum insulin and (C) blood glucose levels in fed
ARH-missed (n = 9), ARH-hit (n = 6) Lepr
neo/neo
and
Lepr
+/+
(n = 3–9) mice. Note that the AAV injections
do not affect glucose homeostasis. Also note that
panels (B) and (D) are separated from (A) and (C)
since the mice have dissimilar genetic backgrounds
(as noted in the legend for Figure 4). (B) Serum
insulin and (D) serum glucose levels in fed ARH-
missed (n = 4), ARH-hit (n = 3) Lepr
/
mice. *p <
0.05; **p < 0.01; ***p < 0.001 versus ARH-missed
Lepr
neo/neo
mice.
weeks after surgery. Since body weight was similarly reduced homeostasis. This concept would be consistent with other
claims that hypothalamic neurons regulate glucose homeosta-at both the 4 and 8 week points, it is unlikely to be the cause
of the normal glycemia at the 8 week point. Moreover, food sis (Obici et al., 2001; Fan et al., 2000).
Leptin also exerts a positive action on locomotor activity asrestriction experiments in obese mice, which led to a similar
reduction of body weight as that seen in ARH-hit Lepr
neo/neo
suggested by the fact that Lep
ob/ob
mice are hypoactive and
that their locomotor activity can be normalized by leptin treat-mice, were not able to normalize blood glucose levels
(Schwartz et al., 1996; Yamamoto et al., 2000). In addition, as ment (Pelleymounter et al., 1995). However, the neuronal
groups that mediate this effect of leptin are unknown. Ourdetailed below, reactivation of LEPRs in both the VMH and the
lateral hypothalamic area (LH) of Lepr
neo/neo
mice resulted in a study shows that restoring leptin signaling in ARH neurons is
sufficient to normalize locomotor activity. Therefore, this findingreduction in body weight similar to ARH-hit Lepr
neo/neo
mice.
However, this body weight reduction was not associated with establishes the ARH as a key site for mediating leptin’s effect
on locomotor activity. We suggest the existence of a novel,improved glucose homeostasis. Thus, we conclude that the
improvements in glucose homeostasis are likely to be indepen- yet undefined, neuronal pathway connecting ARH neurons to
cortical and/or subcortical areas regulating voluntary locomo-dent of a body weight reduction. This suggests that leptin sig-
naling in ARH neurons exerts direct control over insulin/glucose tion. Additional studies will be required to reveal the impor-
Figure 6. Unilateral reactivation of the Lepr gene in the hypothalamic arcuate nucleus of Lepr
neo/neo
mice leads to increased locomotor activity
(A)24hr(B) nocturnal and (C) diurnal locomotor activity were measured with CLAMS in 13-week-old ARH-missed (n = 9), ARH-hit (n = 5) Lepr
neo/neo
and Lepr
+/+
(n =
5) mice. *p < 0.05; **p < 0.01; ***p < 0.001 versus ARH-missed Lepr
neo/neo
mice.
CELL METABOLISM : JANUARY 2005 69
Page 7
ARTICLE
293A cells and purified by heparin column. The eluted virus was dialyzed
tance of this hypothesized neurocircuit mediating leptin’s effect
against PBS and the titer was assessed by dot blot hybridization. All these
on ambulatory movements.
procedures were performed by the Harvard Gene Therapy Initiative core
Further supporting our conclusions that leptin signaling
facility (http://hgti.med.harvard.edu).
specifically in the ARH is a major feeding-independent regula-
Four-week-old Lepr
neo/neo
and Lepr
D/D
male mice were stereotaxically in-
tor of glucose homeostasis and locomotor activity were physi-
jected with AAV-FLPe-IRES-eGFP into the ARH with a glass micropipette
ological data on four Lepr
neo/neo
mice that had brains with
and air pressure injector system (Chamberlin et al., 1998).
>100 eGFP-positive cells in both the LH and the VMH and
Body and blood composition
<100 eGFP-positive cells in any other nucleus known to con-
Tail vein blood was collected at noon ± 2 hr from fed 8- and 12-week-old
tain Lepr-b mRNA. Like ARH-hit Lepr
neo/neo
mice, these LH +
mice. Blood was assayed for glucose level (Fisher Scientific, Morrison
VMH-hit Lepr
neo/neo
mice had w20% reduction in body weight
Plains, New Jersey) and successively serum was collected by centrifugation
(12-week-old Lepr
neo/neo
mice: ARH-missed = 47.44 [g] ± 0.56
and assayed for insulin levels using commercially available kits (Crystal
[n = 9]; ARH-hit = **42.23 [g] ± 1.6 [n = 9]; LH + VMH-hit =
Chem. Inc., Downers Grove, Illinois). Serum was assayed for glucose levels
*43.74 [g] ± 2.09 [n = 4]; *p < 0.05, **p < 0.01 versus
using an enzymatic glucose oxidase method (Thermo Electron, Victoria,
ARH-missed). Importantly, despite the reduced body weight,
Australia). After blood was collected, mice were ketamine anesthetized for
dual-energy X-ray absorptiometry (MEC Lunar Corp., Minster, Ohio)
blood glucose levels and locomotor activity were not normal-
analysis.
ized in LH + VMH-hit Lepr
neo/neo
mice (blood glucose levels in
12-week-old Lepr
neo/neo
mice: ARH-missed = 300 [mg/dl] ± 48
Oxygen consumption, locomotor activity, and fertility test
[9]; LH + VMH-hit = 231 [mg/dl] ± 76 [n = 4]; locomotor activity
Metabolic rate and physical activity were measured in 13-week-old mice
in 12-week-old Lepr
neo/neo
mice: ARH-missed = 4447 [counts/
using a comprehensive lab animal monitoring system (CLAMS, Columbia
day] ± 631 [n=9]; LH+VMH-hit = 4646 [counts/day] ± 923 [n =
Instruments, Columbus, Ohio). Mice were acclimated in the monitoring
4]). Further analysis, which is in progress in our laboratories, is
chambers for 2 days then data were collected for 3 days. Data analysis was
needed to definitely assess the physiological importance of
performed only in mice that did not lose weight during the experiment. After
CLAMS analysis, every mouse was housed with two 7- to 12-week-old FVB
leptin signaling specifically in the LH or the VMH. However,
female mice for a period of 6–7 days. Female mice were monitored for the
we predict that leptin’s effects on glucose homeostasis and
following 4 weeks and the presence of the offspring was counted as event
locomotor activity are not mediated by LEPRs on neurons con-
of pregnancy. Female control mice were tested for their fertility by breeding
tained within these two hypothalamic areas.
with wild-type male mice. All female mice had events of pregnancy when
In summary, restoration of leptin signaling in ARH neurons
bred with wild-type male mice. Food and water were provided ad libitum
leads to improved, but not normal energy homeostasis. Thus,
during the entire period.
other leptin-responsive neuronal groups are likely also to be
important in mediating leptin’s effect on food intake and body
eGFP and P-STAT3 immunohistochemistry
Fed 14-week-old male mice were injected intraperitoneally with 100 gof
weight. In contrast, LEPRs expression by ARH neurons is suffi-
recombinant mouse leptin (A.F. Parlow, National Hormone and Peptide Pro-
cient to mediate the majority of leptin’s effects on glucose ho-
gram) and perfused with 10% formalin 45 min later. Either eGFP or P-STAT3
meostasis and locomotor activity. Delivery of FLPe in a
immunohistochemistry was performed on microtome cut 25 m brain sec-
neuron-specific fashion by the generation of neuron-specific
tions as described earlier (Liu et al., 2003; Elias et al., 1999; Munzberg et
FLPe-transgenic Lepr
neo/neo
mice (for example, Npy-FLPe,
al. 2003).
Pomc-FLPe, or Agrp-FLPe transgenic mice) will be critical to
reveal the relative contribution of leptin signaling in specific
Categorization of ARH-hit and ARH-missed Lepr
neo/neo
populations on neurons in the ARH in mediating the varied ef-
and Lepr
/
mice
fects of leptin.
Brain sections stained against eGFP by immunohistochemistry were used
for this categorization. The borders of the nuclei containing eGFP-positive
Experimental procedures
cells were drawn on a paper sheet using a camera lucida device using
darkfield optics and an atlas of the mouse brain (Franklin and Paxinos,
Animal care
1997). The eGFP-positive cells were plotted and counted such that the
Care of all mice was within the Institutional Animal Care and Use Committee
ARH-hit and the ARH-missed were grouped as follows. Brains that had
(IACUC) guidelines, and all the procedures were approved by the Beth Israel
>100 eGFP-positive cells in the arcuate nucleus of the hypothalamus (all
Deaconess Medical Center IACUC. Mice were housed individually at 22°C–
sections in a 1:5 series) and <100 eGFP-positive cells in any other nucleus
24°C using a 14 hr light/10 hr dark cycle with chow food (Tekland F6 Rodent
known to contain Lepr-b mRNA (all sections in a 1:5 series) were scored as
Diet8664, Harlan Tekland, Madison, Wisconsisn) and water provided ad li-
ARH-hit. Brains that had <100 eGFP-positive cells in any nucleus known to
bitum.
contain Lepr-b mRNA (all sections in a 1:5 series) were scored as
ARH-missed. These criteria were established a priori. Also, an individual
Experimental mice
blinded to the neuroanatomic categorizations and physiological responses
Lepr
neo/+
mice were provided by Dr. S. Chua, Jr. (McMinn et al., 2004) . The
of each case performed this analysis.
genetic background of Lepr
neo/+
mice is an admixture of C57Bl6/J and 129.
Lepr
neo/neo
and Lepr
+/+
male mice were obtained by mating Lepr
neo/+
mice
Statistical analysis
with Lepr
neo/+
mice. Mice were genotyped by PCR with primers (1 and 2)
Data sets were analyzed for statistical significance using PRISM 3.0
across the loxP site: 1, 5#-AAT GAA AAA GTT GTT TTG GGA CGA-3# and
(GraphPad, San Diego, California) for a two-tailed unpaired Student’s t test.
2, 5#-CAG GCT TGA GAA CAT GAA CAC AAC AAC-3#. Lepr
D/D
mice are
Statistical comparisons shown in Figures 4B, 4D, 5A, 5C, 6A, 6B, and 6C
homozygous for a null LEPR allele lacking exon 17 and were generated as
were made by using one-way ANOVA (Turkey’s post test). All parameters
described previously (Balthasar et al., 2004). The genetic background of
are expressed as mean ± SEM.
Lepr
D/D
mice is an admixture of C57Bl6/J, 129, and FVB.
AAV-FLPe-IRES-eGFP generation and microinjection
Acknowledgments
The FLPe gene was cloned into the transfer vector pAAV-M2-IGFP such
that the AAV vector plasmid called here pAAV-FLPe-M2-IGFP was gener-
The authors would like to thank Drs. Mineko Fujimiya, Clifford Saper and
ated. The virus was generated by tripartite transfection (AAV-rep/cap ex-
pression plasmid, adenovirus miniplasmid, and pAAV-FLPe-M2-IGFP) into Christian Bjorbaek for helpful advice. This work was supported by grants
70 CELL METABOLISM : JANUARY 2005
Page 8
Leptin action in the hypothalamic arcuate nucleus
Elmquist, J.K., Elias, C.F., and Saper, C.B. (1999). From lesions to leptin:
from the NIH (PO1 DK56116 to B.B.L. and J.K.E., DK57621 and DK26687
hypothalamic control of food intake and body weight. Neuron 22, 221–232.
to S.C.C., Jr., MH61583 and DK53301 to J.K.E.) and by Takeda Chemical
Industries, Ltd., Japan. We thank NIDDK’s National Hormone & Peptide Pro-
Erickson, J.C., Hollopeter, G., and Palmiter, R.D. (1996). Attenuation of the
gram and A.F. Parlow for providing recombinant mouse leptin.
obesity syndrome of ob/ob mice by the loss of neuropeptide Y. Science
274, 1704–1707.
Fan, W., Dinulescu, D.M., Butler, A.A., Zhou, J., Marks, D.L., and Cone, R.D.
(2000). The central melanocortin system can directly regulate serum insulin
Received: November 10, 2004
levels. Endocrinology 141, 3072–3079.
Revised: December 22, 2004
Accepted: December 23, 2004
Fei, H., Okano, H.J., Li, C., Lee, G.H., Zhao, C., Darnell, R., and Friedman,
Published: January 19, 2005
J.M. (1997). Anatomic localization of alternatively spliced leptin receptors
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    • "PA is under biological control [50] mediated via complex interactions between regulators of energy balance, of which leptin and insulin play a major role5152535455. Leptin and insulin convey energy availability signals to the hypothalamus, a key site for the regulation of energy balance565758. The hypothalamus not only responds to hormonal signals, via leptin and insulin receptors (and others) [54, 59, 60] but also directly via nutrient related signals, such as glucose and fatty acids (see review [61]), conveying information on available and stored energy supplies. "
    [Show abstract] [Hide abstract] ABSTRACT: Calorie restriction (CR) delays the onset of age-related disease and extends lifespan in a number of species. When faced with reduced energy supply animals need to lower energy demands, which may be achieved in part by reducing physical activity (PA). We monitored changes in PA using implanted transmitters in male C57BL/6 mice in response to graded levels of CR (10 to 40%) or matched levels of graded protein restriction (PR) for 3 months. Mice were fed at lights out and ad libitum controls were limited to dark-phase feeding (12AL) or 24hr/day. Total daily PA declined in a non-linear manner over the first 30 days of CR or PR, remaining stable thereafter. Total daily PA was not related to the level of CR or PR. Total daily PA over the last 20 days of restriction was related to circulating leptin, insulin, tumor necrosis factor-α (TNF- α) and insulin-like growth factor (IGF)-1 levels, measured after 3 months. Mice under restriction showed a high level of activity in the 2hrs before feeding (food anticipatory activity: FAA). FAA followed a complex pattern, peaking around day 20, falling on ~day 37 then increasing again. FAA was also positively related to the level of restriction and inversely to leptin, insulin, TNF-α and IGF-1. Non-FAA, in contrast, declined over the period of restriction, generally more so in mice under greater restriction, thereby offsetting to some extent the increase in FAA. Mice under PR displayed no changes in PA over time or in comparison to 12AL, and showed no increase in FAA.
    Full-text · Article · Feb 2016 · Oncotarget
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    • "Few studies have investigated the mechanisms underlying the effects of leptin on physical activity. The hypoactivity of obese, LepR-deficient mice is corrected by restoration of LepR to the mediobasal hypothalamus (Coppari et al., 2005) or proopiomelanocortin neurons (Huo et al., 2009 ), underscoring the role of the hypothalamus in the actions of leptin to stimulate locomotor behavior. However, leptin suppression of physical activity when food is limited suggests the presence of distinct neural mechanisms. "
    [Show abstract] [Hide abstract] ABSTRACT: The adipose hormone leptin potently influences physical activity. Leptin can decrease locomotion and running, yet the mechanisms involved and the influence of leptin on the rewarding effects of running ("runner's high") are unknown. Leptin receptor (LepR) signaling involves activation of signal transducer and activator of transcription-3 (STAT3), including in dopamine neurons of the ventral tegmental area (VTA) that are essential for reward-relevant behavior. We found that mice lacking STAT3 in dopamine neurons exhibit greater voluntary running, an effect reversed by viral-mediated STAT3 restoration. STAT3 deletion increased the rewarding effects of running whereas intra-VTA leptin blocked it in a STAT3-dependent manner. Finally, STAT3 loss-of-function reduced mesolimbic dopamine overflow and function. Findings suggest that leptin influences the motivational effects of running via LepR-STAT3 modulation of dopamine tone. Falling leptin is hypothesized to increase stamina and the rewarding effects of running as an adaptive means to enhance the pursuit and procurement of food. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Aug 2015 · Cell metabolism
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    • "This would then allow for disinhibition of central melanocortin receptor action, ultimately affecting blood glucose levels via a CNS-peripheral pathway that is not yet fully understood [7]. GluN2B deletion thus mimics the anti-diabetic effect of exogenous leptin acting on leptin receptors expressed on AgRP neurons of Lep ob/ob mice [7,9,59]. These results bring forward the question of why loss of NR2B in AgRP neurons only improves glucose homeostasis in Lep ob/ob mice but is without effect in lean mice. "
    [Show abstract] [Hide abstract] ABSTRACT: Objective: Hypothalamic agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) expressing neurons play critical roles in control of energy balance. Glutamatergic input via n-methyl-d-aspartate receptors (NMDARs) is pivotal for regulation of neuronal activity and is required in AgRP neurons for normal body weight homeostasis. NMDARs typically consist of the obligatory GluN1 subunit and different GluN2 subunits, the latter exerting crucial differential effects on channel activity and neuronal function. Currently, the role of specific GluN2 subunits in AgRP and POMC neurons on whole body energy and glucose balance is unknown. Methods: We used the cre-lox system to genetically delete GluN2A or GluN2B only from AgRP or POMC neurons in mice. Mice were then subjected to metabolic analyses and assessment of AgRP and POMC neuronal function through morphological studies. Results: We show that loss of GluN2B from AgRP neurons reduces body weight, fat mass, and food intake, whereas GluN2B in POMC neurons is not required for normal energy balance control. GluN2A subunits in either AgRP or POMC neurons are not required for regulation of body weight. Deletion of GluN2B reduces the number of AgRP neurons and decreases their dendritic length. In addition, loss of GluN2B in AgRP neurons of the morbidly obese and severely diabetic leptin-deficient Lep (ob/ob) mice does not affect body weight and food intake but, remarkably, leads to full correction of hyperglycemia. Lep (ob/ob) mice lacking GluN2B in AgRP neurons are also more sensitive to leptin's anti-obesity actions. Conclusions: GluN2B-containing NMDA receptors in AgRP neurons play a critical role in central control of body weight homeostasis and blood glucose balance via mechanisms that likely involve regulation of AgRP neuronal survival and structure, and modulation of hypothalamic leptin action.
    Full-text · Article · Jul 2015 · Molecular Metabolism
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