Leptin deficiency and diet-induced obesity reduce hypothalamic kisspeptin expression in mice.

Abstract

The hormone leptin modulates a diverse range of biological functions, including energy homeostasis and reproduction. Leptin promotes GnRH function via an indirect action on forebrain neurons. We tested whether leptin deficiency or leptin resistance due to a high-fat diet (HFD) can regulate the potent reproductive neuropeptide kisspeptin. In mice with normalized levels of estradiol, leptin deficiency markedly reduced kisspeptin gene expression, particularly in the arcuate nucleus (ARC), and kisspeptin immunoreactive cell numbers in the rostral periventricular region of the third ventricle (RP3V). The HFD model was used to determine the effects of diet-induced obesity and central leptin resistance on kisspeptin cell number and gene expression. DBA/2J mice, which are prone to HFD-induced infertility, showed a marked decrease in kisspeptin expression in both the RP3V and ARC and cell numbers in the RP3V after HFD. This is the first evidence that kisspeptin can be regulated by HFD and/or increased body weight. Next we demonstrated that leptin does not signal (via signal transducer and activator of transcription 3 or 5, or mammalian target of rapamycin) directly on kisspeptin-expressing neurons in the RP3V. Lastly, in leptin receptor-deficient mice, neither GnRH nor kisspeptin neurons were activated during a preovulatory-like GnRH/LH surge induction regime, indicating that leptin's actions on GnRH may be upstream of kisspeptin neurons. These data provide evidence that leptin's effects on reproductive function are regulated by kisspeptin neurons in both the ARC and RP3V, although in the latter site the effects are likely to be indirect.

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Endocrinology 2011 152:1541-1550 originally published online Feb 15, 2011; , doi: 10.1210/en.2010-1100

Janette H. Quennell, Christopher S. Howell, Juan Roa, Rachael A. Augustine, David R. Grattan and Greg M. Anderson


Expression in Mice
Leptin Deficiency and Diet-Induced Obesity Reduce Hypothalamic Kisspeptin
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Leptin Deficiency and Diet-Induced Obesity Reduce
Hypothalamic Kisspeptin Expression in Mice
Janette H. Quennell, Christopher S. Howell, Juan Roa, Rachael A. Augustine,
David R. Grattan, and Greg M. Anderson
Centre for Neuroendocrinology and Departments of Anatomy and Structural Biology (J.H.Q., C.S.H.,
R.A.A., D.R.G., G.M.A.) and Physiology (J.R.), University of Otago School of Medical Sciences, Dunedin
9054, New Zealand
The hormone leptin modulates a diverse range of biological functions, including energy homeo-
stasis and reproduction. Leptin promotes GnRH function via an indirect action on forebrain neu-
rons. We tested whether leptin deficiency or leptin resistance due to a high-fat diet (HFD) can
regulate the potent reproductive neuropeptide kisspeptin. In mice with normalized levels of es-
tradiol, leptin deficiency markedly reduced kisspeptin gene expression, particularly in the arcuate
nucleus(ARC),andkisspeptinimmunoreactivecellnumbersintherostralperiventricularregionofthe
third ventricle (RP3V). The HFD model was used to determine the effects of diet-induced obesity and
centralleptinresistanceonkisspeptincellnumberandgeneexpression.DBA/2Jmice,whichareprone
to HFD-induced infertility, showed a marked decrease in kisspeptin expression in both the RP3V and
ARCandcellnumbersintheRP3VafterHFD.Thisisthefirstevidencethatkisspeptincanberegulated
by HFD and/or increased body weight. Next we demonstrated that leptin does not signal (via signal
transducer and activator of transcription 3 or 5, or mammalian target of rapamycin) directly on kiss-
peptin-expressing neurons in the RP3V. Lastly, in leptin receptor-deficient mice, neither GnRH nor
kisspeptinneuronswereactivatedduringapreovulatory-likeGnRH/LHsurgeinductionregime,indicating
that leptin’s actions on GnRH may be upstream of kisspeptin neurons. These data provide evidence that
leptin’s effects on reproductive function are regulated by kisspeptin neurons in both the ARC and RP3V,
although in the latter site the effects are likely to be indirect. (Endocrinology 152: 1541–1550, 2011)
F
lean body type can decrease GnRH production, resulting
in cessation of ovulation (1). Obesity can also have detri-
mentaleffectsonconceptionandimplantationrates(2,3).
Multiple metabolic signals (such as glucose and a raft of
metabolic hormones) connect nutritional status and re-
productive function (1, 4). Leptin is a metabolic hormone
secretedfromfatcells.Itsprimaryroleistoregulateenergy
intake and expenditure. However, animals with dysfunc-
tional leptin signaling also have marked suppression of
fertility (5, 6). Leptin replacement can overcome fasting-
induced suppression of LH pulses in both rodents and
primates (7, 8). The primary site of leptin’s effects on the
ertility and the hypothalamic-pituitary-gonadal axis
are closely linked to nutritional status. For women, a
reproductivesystemiscentral;however,werecentlydem-
onstrated that leptin does not directly act on GnRH neu-
rons (9). A number of neuropeptides could feasibly pro-
vide an interneuronal link between the leptin receptors
that sense an individual’s energy balance and the GnRH
system. Proopiomelanocortin, neuropeptide Y, and co-
caine- and amphetamine-regulated transcript (10–12)
have all been proposed as possible intermediaries. More
recently, kisspeptin joined this list as a potential interme-
diary neuropeptide linking metabolic status and repro-
ductive function (13).
Kisspeptinsactinthebraintocontrolfertility.Thekiss-
peptin peptides are an expression of the Kiss1 gene. Orig-
inally discovered as a 54-amino acid peptide, kisspep-
ISSN Print 0013-7227
Printed in U.S.A.
Copyright © 2011 by The Endocrine Society
doi: 10.1210/en.2010-1100 Received September 21, 2010. Accepted January 21, 2011.
First Published Online February 15, 2011
ISSN Online 1945-7170 Abbreviations: ARC, Arcuate nucleus; AVPV, anteroventral periventricular nucleus; DIO,
diet-induced obesity; HFD, high-fat diet; mTORC1, mammalian target of rapamycin com-
plex 1; OVX, ovariectomized; qRT-PCR, quantitative RT-PCR; RP3V, rostral periventricular
region of the third ventricle; STAT, signal transducer and activator of transcription.
N E U R O E N D O C R I N O L O G Y
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tin-54 or metastin was found to inhibit metastatic cancer
cells (14). Kisspeptin-54 or shorter products such as kiss-
peptin-10 have since been found to potently and directly
activate GnRH neurons (15). In rodents, kisspeptin-ex-
pressing neurons are predominately found in two discrete
populationswithinthehypothalamus:therostralperiven-
tricular region of the third ventricle [RP3V: composed of
the anteroventral periventricular nucleus (AVPV) and the
preoptic periventricular nucleus] and the arcuate nucleus
(ARC) (16).
Although a role for kisspeptin in body weight regula-
tion is unlikely (17), kisspeptin may mediate the effects of
leptin on fertility. For example, lactating (a state of neg-
ativeenergybalance)ratshavedecreasedamountsofkiss-
peptin mRNA and protein in the ARC (18, 19). Smith et
al. (20) found approximately 40% of kisspeptin-express-
ing neurons in the ARC express the gene for leptin recep-
tor. Additionally, male mice lacking functional leptin dis-
playedlessKiss1mRNAintheARCasmeasuredbyinsitu
hybridization (20). In rats, mice, and sheep, food restric-
tion has been shown to reduce hypothalamic kisspeptin
mRNA levels (13, 21–23). These results provide evidence
for a leptin receptor 3 kisspeptin neuron 3 GnRH neu-
ron relay. Importantly, females have a much larger RP3V
kisspeptin population than males, and these neurons ex-
hibitapronouncedestrogen-inducedchangeinkisspeptin
expression (24). The effects of leptin deficiency (as op-
posed to negative energy balance) on kisspeptin expres-
sion have not previously been investigated in females. At
the other extreme, it is unknown how the kisspeptin sys-
tem responds to diet-induced obesity (DIO) and thus high
circulatinglevelsofleptin,whichultimatelyleadstoastate
of central leptin resistance.
Theaimofthispaperwastomeasuretheeffectofleptin
deficiency on kisspeptin mRNA levels and cell numbers
expressing kisspeptin protein in the RP3V and ARC of
female mice with different levels of circulating estrogens.
We also investigated the effect of DIO on kisspeptin cell
numbers and mRNA expression using a high-fat diet
(HFD) model. Additionally, we attempted to determine
whether leptin could regulate kisspeptin neurons directly
by assessing leptin-signaling pathways in kisspeptin neu-
rons using dual label immunohistochemistry. Three sig-
nalingpathwaysweremeasured:thesignaltransducerand
activatoroftranscription(STAT)3andSTAT5pathways,
and the mammalian target of rapamycin complex 1
(mTORC1)-dependant pathway. Whereas phosphoryla-
tion of STAT3 (pSTAT3) is widely used as a marker of
leptin signaling (25, 26), pSTAT5 and mTORC1 have
more recently been implicated in hypothalamic leptin sig-
naling (27, 28). Lastly, we investigated the effect of leptin
deficiency on kisspeptin neuronal activation during the
GnRH/LH surge.
Materials and Methods
Animals
All mice were obtained from the University of Otago animal
breeding facility. Mice were group housed (individual housing
wasusedaftersurgery)andmaintainedona12-hlight,12-hdark
cycle (lights on at 0600 h) at a constant temperature (22 ? 1 C).
Micehadfreeaccesstofoodandwater.TheUniversityofOtago
Animal Ethics Committee approved all animal protocols.
Experiment 1: effect of leptin deficiency on
kisspeptin mRNA levels and cell numbers
This study used adult female leptin-deficient homozygous
ob/ob mice (8–10 wk old; body weight ?40 g), with wild-type
littermates (body weight ?20 g) serving as controls. Three
different levels of circulating estradiol were used: ovariecto-
mized (OVX), OVX with low-dose replacement estradiol
(OVX?lowE; to mimic estrogen’s negative feedback effects),
andOVXwithlow-dosereplacementestradiolandtheninjected
with estradiol benzoate (OVX?surgeE; to mimic the estrogenic
state leading up to the preovulatory LH surge). Mice in the
OVX?lowEandOVX?surgeEgroupsweretreatedasdescribed
previously (9, 29). Briefly, mice were OVX and had a silicone
capsule (internal diameter, 1.0 mm; external diameter, 2.1 mm;
Dow Corning, Midland, MI) filled with silicone adhesive (Dow
Corning) containing 17-? estradiol (0.1 mg/ml adhesive; Sigma-
Aldrich, St Louis, MO) implanted sc. Each mouse was given a
1-cm capsule/20 g body weight. Six days after ovariectomy,
OVX?surgeEmicereceivedanscinjectionofestradiolbenzoate
(0.05 mg/kg; Sigma-Aldrich) at 0800 h. At 1800 h the following
day (the surge peak time), brains were collected from all three
groups for quantitative RT-PCR (qRT-PCR) analysis. Trunk
blood was collected from OVX?lowE and OVX?surgeE mice
for LH and/or estradiol assay.
Experiment 2: effect of HFD on kisspeptin mRNA
levels and cell numbers
Femalemice(DBA/2JandC57BL/6J)wereobtainedatwean-
ing and randomly assigned to receive either a low-fat standard
diet (4.8%; Specialty Feeds, Glen Forrest, Australia) or a diet
containing 23% fat (SF03-020, Specialty Feeds). In previous
work it has been shown that DBA/2J mice are prone to HFD-
induced infertility whereas C57BL/6J mice are not (Ref. 30 and
our own unpublished observations). All mice remained on their
respective diets until 24 wk of age. For mRNA analysis, mice
were weighed and OVX 7 d before being killed at the age of 24
wk. Brains were collected for qRT-PCR and trunk blood was
collected for leptin assay. A separate group of DBA/2J female
mice were raised on HFD or standard chow and then subjected
totheOVX?surgeEprotocolabove.At19wkofagebrainswere
collected and prepared for immunohistochemistry.
Experiment 3: measurement of leptin-induced
signaling in kisspeptin neurons
Mice(C57BL/6J)werefastedovernighttoreduceendogenous
circulating leptin concentrations, and 50 ?g of bromocriptine
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mesylate (Tocris Biosciences, Ellisville, MI) were given sc at
0900 h to reduce activation of the STAT5 pathway by prolactin
(31). Recombinant mouse leptin (5 mg/kg) (National Hormone
and Peptide Program, Torrance, CA) or vehicle (0.2 ml PBS, pH
7.8) was injected ip at 1200 h. Mice were perfused 2 h later via
the heart with 20 ml of 4% paraformaldehyde, and brains were
collected for immunohistochemistry.
Experiment 4: effect of deficient leptin signaling
on kisspeptin neuronal activation during a
preovulatory-like LH surge
This experiment used a forebrain neuron-specific leptin re-
ceptor knockout mouse that was part of another study (9). Neu-
ron-specific leptin receptor (Lepr) knockout mice were gener-
ated by crossing Lepr flox mice with CamKII?-iCre BAC mice
(Leprfl/fl, CamKII?-Cre) as described elsewhere (9). Littermates
homozygous for Lepr flox (Leprfl/fl), but with no CamKII?-iCre
expression, served as controls. Mice were subjected to an
OVX?surgeE protocol as described above. A group of control
mice (Leprfl/fl) subjected to an OVX?lowE protocol were in-
cluded to serve as nonsurging controls. Mice were killed and
tissue and blood were collected at 1800 h the following day.
Brains were processed for immunohistochemical detection of
kisspeptin and c-Fos (an early-intermediate gene used as a
markerofneuronalactivation),andbloodserawereprocessedto
assess LH concentration.
Kiss1 mRNA analysis by qRT-PCR
For measurement of Kiss1 mRNA, mice were decapitated,
and brains were immediately removed from the skull and frozen
at ?80 C for later use. Hypothalami were dissected along the
following boundaries: laterally 2 mm either side of the third
ventricle from the optic chiasm to the posterior border of the
mammillary bodies, and the thalamus dorsally. Hypothalami
were then bisected in the coronal plane immediately anterior to
the pituitary stalk. The anterior dissection contained all of the
RP3V, and the posterior dissection contained the entire ARC
(Fig.1).Tocontrolfortheamountoftissuedissectedpermouse,
and thus entering the reverse transcription reaction, hypotha-
lamic tissue blocks were weighed. No significant differences in
hypothalamic tissue block weight were noted between compa-
rable experimental groups (P ? 0.4).
Thesetissueswerethoroughlyhomogenizedwithsterileplas-
ticpestlesandsonicatedinTRIzolsolution(Invitrogen,Calsbad,
CA).TotalRNAwasseparatedfromexcessproteinandgenomic
DNA by chloroform phase separation and then further cleaned
using the standard protocol for RNeasy Mini Spin Columns
(QIAGEN, Valencia, CA).
RNAqualitywasassessedbyspectrophotometryandagarose
gelelectrophoresis.ThetotalamountofRNAineachsamplewas
quantified using an ND-1000 spectrophotometer (NanoDrop;
Thermo Scientific, Waltham, MA), and 500 ng of total RNA
were subjected to deoxyribonuclease I treatment (Ambion, Aus-
tin, TX) according to manufacturer’s instructions. Total RNA
was then reverse transcribed with SuperScript III reverse tran-
scriptase (Invitrogen) using random hexamers as primers.
Triplicate uniplex reactions for measurement of Kiss1 mRNA
levelswerecarriedoutoncDNAsamplesusingthefollowingprim-
ers: forward, 5?-CCTTCCTCCCAGAATGATCTC-3?; and re-
verse, 5?-GATCCAGGCTTCACTTTTGC-3? (NCBI reference se-
quence NM_178260.3). Primers specific for three reference genes
were tested over our experimental samples: ?-actin, polymerase
(RNA) II (DNA directed) polypeptide A (Polr2a), and tata-box
binding protein. The reference gene Polr2a was chosen in this ex-
periment due to its minimal variation across experimental groups:
forward, 5?-GCACCACGTCCAATGATAT-3? and reverse, 5?-
GTGCTGCTGCTTCCATAA-3?
NM_009089.2) (32). Reactions (20 ?l) were prepared in 96-well
opticalreactionplateswithopticaladhesivecovers(ABIPrism;Ap-
pliedBiosystems,FosterCity,CA)usingSYBRGreenPCRMaster
Mix (Applied Biosystems). Using an ABI PRISM 7300 qPCR ther-
mocycler (Applied Biosystems), samples were heated to 50 C for 2
min, then 95 C for 10 min before 40 cycles of 95 C for 15 sec and
60 C for 1 min as per the manufacturer’s instructions. Prior vali-
dation experiments were performed to demonstrate that amplifi-
cation efficiencies of Kiss1 (0.9) were approximately equal to that
of Polr2a (1.0).
Quantitative PCR data were analyzed using the comparative
Cqmethod in which Cqis the quantification cycle number at
which the fluorescence reading is first recorded above back-
groundlevels(33).TherangesofCqvaluesforPolr2aandKiss1
were similar (Cq ? 22–32 and 25–34, respectively). Subtracting
the average Cqvalue for the gene of interest from the average Cq
value for the reference gene gives the ?Cq. The comparative Cq
method is a relative measure of mRNA expression; thus a fixed
arbitrary number can be can be used as a calibrator. Subtracting
the calibrator from the ?Cqfor all samples gave the relative
change (??Cq). Finally the arithmetic formula 2???Cqwas used
toachieverelativequantitation(fordetailedinformationseeAp-
plied Biosystems’ User Bulletin 2).
(NCBIreferencesequence
Immunohistochemistry for kisspeptin- and
leptin-signaling molecules
Mice were perfused with 4% paraformaldehyde in 0.1 M
phosphatebufferforbraincollectionandimmunohistochemical
staining. Coronal (30 ?m thick) sections throughout the RP3V
and ARC were cut from each brain on a sliding microtome with
a freezing stage to provide three sets of consecutive sections (90
FIG. 1. Ventral view of mouse brain. The hypothalamus was dissected
out along the limits of the black square. Anterior and posterior
hypothalamic blocks were separated from each other (dotted line).
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?m apart). One set of sections was labeled for kisspeptin by first
incubating in polyclonal rabbit antikisspeptin primary antibody
(Chemicon AB9754; Millipore, Billerica, MA; 1:20,000 dilu-
tion), followed by biotinylated goat antirabbit IgG secondary
antibody (Vector Laboratories, Inc., Burlingame, CA; 1:500 di-
lution). The signal was amplified using avidin-biotin peroxidase
(Elite Vectorstain; Vector Laboratories) and then diaminoben-
zidine solution to visualize the kisspeptin immunoreactivity
(brown staining). Kisspeptin neurons in three sections through
theRP3V(Figs.26–32inRef.34).andthreesectionsthroughthe
ARC (Figs. 43–52 in Ref. 34) per animal were counted.
For dual-label immunohistochemistry, kisspeptin was first
stained as above, and then remaining peroxidase activity was
removed by washing with H2O2. Subsequently, sections were
incubated in polyclonal rabbit anti-pSTAT3 (Tyr750), anti-
pSTAT5 (Tyr694), anti-pS6 (Ser235/236; Cell Signaling Tech-
nology, Danvers, MA; 1:2000, 1:1000, and 1:100 dilution, re-
spectively),oranti-c-Fosprimaryantibodies(AB5;Calbiochem,
SanDiego,CA;1:20,000dilution).Phospho-S6isthephosphor-
ylated form of the ribosomal protein S6 kinase1, which is a
downstream target of mTORC1. Before incubation with the
pSTAT5 primary antibody, sections were heated for 5 min at 90
C in 0.01 M Tris (pH 10) to unmask antigens. For anti-pSTAT3,
5 and c-Fos, goat antirabbit horseradish peroxidase (Dako
P0449; Glostrup, Denmark; 1:500 dilution) was the secondary
antibodyused,andimmunoreactivitywasvisualizedusingnick-
el-enhanced diaminobenzidine to generate a blue-black nuclear
stain. Kisspeptin neurons were counted as pSTAT3-, pSTAT5-,
or c-Fos-positive if they had distinct blue-black stained nuclei
surrounded by a brown cytoplasmic stain.
Dual-labelimmunohistochemistryforkisspeptinandmTORC1
differed in that after incubating tissues with both antikisspeptin
(sheepantimousekindlyprovidedbyDr.A.Caraty)andanti-pS6
(rabbit antimouse), tissues were incubated in biotinylated don-
key antirabbit (Jackson ImmunoResearch Laboratories, Inc.,
West Grove, PA; 1:200 dilution) secondary. This was followed
by incubation in Texas Red donkey antisheep IgG (Jackson;
1:200 dilution) secondary and conjugated-Fluorescein green
strepavidin DCS (Cell sorting grade; Vector Laboratories Inc.;
1:200). Sections mounted with Vectashield Mounting Medium
(Vector Laboratories, Inc.) were visualized in the 540–580 and
460–490 excitation wavelength ranges, respectively.
An operator blind to the treatment groups performed all cell
counting. Omission of any of the primary anybodies resulted in
complete absence of staining.
Hormone assays
Trunk blood was obtained at death and allowed to clot at
room temperature for 30 min and then centrifuged at 2500 ? g
for 15 min at 4 C. Blood sera were then transferred into clean
tubes and stored at ?20 C until required. An ELISA was used to
measure mouse serum leptin (EZML-82K, Millipore) as per
manufacturer’s instructions. Serum LH concentrations were
measured by RIA. Values are expressed in terms of the rat stan-
dard National Institute of Diabetes and Digestive and Kidney
Diseases(NIDDK)-ratLH-RP-3.Thetracer,iodinatedhormone
(NIDDK-ratLH-I-10)wasused,andtheprimaryantiserumwas
NIDDK-rabbitantiratLH-S11(finaldilution1:500,000).Serum
estradiol levels were quantified using an estradiol RIA (DSL-
4800,DiagnosticSystemsLaboratories,Inc.Webster,TX)asper
manufacturer’s instructions except an overnight incubation was
used after addition of [125I]estradiol. Intraassay coefficient of
variationfortheLHassayswas16%;thoseforleptinELISAand
estradiol RIA were less than 5%, and the sensitivities of the
assayswere0.2ng/ml,0.2ng/ml,and0.8pg/ml,respectively.All
samples were analyzed in single assays. LH levels from the mice
used in experiment 4 have been reported previously (9).
Statistical analysis
AlldatawereanalyzedbyStudent’sttestorone-wayANOVA
followed by Bonferroni post hoc analysis where appropriate.
The threshold level for statistical significance was set at 5%
(P ? 0.05).
Results
Experiment 1a: levels of Kiss1 mRNA in the ARC
are reduced during leptin deficiency
The replacement estradiol protocol produced circulat-
ing estradiol concentrations that were only slightly above
assay detection levels in OVX?lowE animals but mark-
edly elevated in OVX?surgeE animals (OVX?lowE:
4.1 ? 0.9 pg/ml; n ? 24 compared with OVX?surgeE:
63.7.5 ? 24.4 pg/ml; n ? 22; P ? 0.05). There were no
significant differences in circulating estradiol levels be-
tweenfatandthinanimalstreatedwiththesameestradiol
regime. Wild-type control animals (n ? 15) that under-
went OVX?surgeE had LH levels significantly higher
than ob/ob mice (n ? 13; 4.4 ? 1.0 ng/ml vs. 0.6 ? 0.1
ng/ml, respectively; P ? 0.01), indicating that leptin-de-
ficientmicewereunabletomountanLHsurgeeveninthe
presence of high levels of estrogens.
Kiss1mRNAlevelsweremeasuredintheRP3Vandthe
ARC under three estradiol conditions in ob/ob mice and
wild-typelittermates.Kiss1mRNAincreasedintheRP3V
with increasing levels of estradiol in both wild-type and
ob/ob mice under all conditions. There was a nonsignifi-
cant decrease in the amount of Kiss1 mRNA found in
leptin-deficientob/obmiceinallgroups(Fig.2,toppanel).
As expected there was more Kiss1 mRNA in the ARC of
OVX animals than in those with low-dose estradiol re-
placement (Fig. 2, bottom panel). The amount of Kiss1
mRNA under surge conditions was between those found
in OVX and OVX?lowE treated animals. The amount of
Kiss1mRNAdetectedintheARCofleptin-deficientob/ob
mice was significantly less than wild-type mice in all es-
trogenic states (P ? 0.05; Fig. 2, bottom panel).
Experiment 1b: kisspeptin-immunopositive
neurons in the RP3V are fewer in leptin-deficient
mice
Similarly to Kiss1 mRNA levels in the RP3V, the num-
ber of kisspeptin-immunoreactive cell bodies in the RP3V
of wild-type mice increased with the amount of estradiol
exposure (Fig. 3A). However, ob/ob mice had markedly
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less kisspeptin-immunoreactive cells under all estrogenic
conditions tested (P ? 0.057 for OVX and P ? 0.05 for
other groups; Fig. 3, A–C).
As previously reported by others (35), it was very dif-
ficulttovisualizekisspeptin-immunoreactivecellbodiesin
the ARC due to the dense immunopositive fiber network.
On average less than one cell was identified per section;
thussignificantconclusionscouldnotbedrawnfromthese
data.
Experiment 2a: hypothalamic Kiss1 mRNA
decreases in DBA/2J mice after DIO
FemaleDBA/2Jmice(and,toalesserextent,C57BL/6J
mice) showed significant weight gain after 21 wk on a
HFD (Fig. 4A). This weight gain was proportional to cir-
culating serum leptin levels recorded at death (Fig. 4B).
Kiss1mRNAlevelsweremeasuredintheRP3Vandthe
ARC in standard chow and HFD animals in both strains.
ItisofnotethatDBA/2Jmice,whicharehighlysusceptible
to obesity-induced infertility (30), had 10-fold less Kiss1
mRNA compared with their C57BL/6J counterparts (Fig.
4C). Kiss1 mRNA was markedly decreased in both the
RP3V and ARC of DBA/2J mice fed a HFD (Fig. 4C, left).
Incontrast,theamountofKiss1mRNAdidnotchangein
either the RP3V or the ARC of C57BL/6J mice fed a HFD
(Fig. 4C, right).
Experiment 2b: kisspeptin-immunopositive
neurons in the RP3V decrease in DBA/2J mice
after DIO
Female DBA/2J mice subjected to an OVX?surgeE
protocol showed a 35% decrease in the number of kiss-
peptin-expressing cells in the RP3V in response to 16 wk
of HFD exposure (Fig. 4D).
Experiment 3: kisspeptin neurons of the RP3V do
not colocalize with leptin-induced pSTAT signaling
Leptin administered ip induced a marked increase in
nuclear pSTAT3 and pSTAT5 immunoreactivity in the
RP3V and other hypothalamic regions, particularly the
ventromedial hypothalamus and the ARC. There was a
striking absence of pSTAT colocalization within kisspep-
tin neurons in the RP3V (0/385 kisspeptin neurons colo-
calized with pSTAT3, and 1/280 kisspeptin neurons co-
localized with pSTAT5; n ? 7; Fig. 5, A–D). Leptin also
inducedmTORC1pathwaysignalingasevidencedbypS6
immunoreactivity with a similar distribution to pSTAT3
and 5. Colocalization of pS6 with kisspeptin-expressing
neuronsintheRP3Vwasalsocompletelyabsent(Fig.5,E
and F).
Asnotedabove,kisspeptin-expressingcellbodiesinthe
ARC were very difficult to distinguish among the dense
kisspeptin-positivefibernetwork.Definitionofthesecells
FIG. 3. Effects of leptin deficiency on numbers of immunoreactive
kisspeptin neurons in the RP3V in female mice with normalized
estrogen levels (A). Representative examples of immunoreactive
kisspeptin neurons in the RP3V (B and C) in wild-type (B) and leptin-
deficient (C) mice are shown. Examples shown are from OVX?lowE
mice. The number of animals per group is given at the base of each
column. *, P ? 0.05.
FIG. 2. Effects of leptin deficiency on Kiss1 mRNA in the RP3V (top)
and ARC (bottom) in female mice with normalized estrogen levels. The
number of animals per group is given at the base of each column.
*, P ? 0.05.
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was even more difficult after leptin treatment and pSTAT
immunostaining.
Experiment 4: kisspeptin neurons are not activated
by a preovulatory-like LH surge in leptin
receptor-deficient mice
Asdescribedelsewhere,neuron-specificLeprknockout
mice were significantly heavier and infertile compared
with control mice (9). After wild-type mice were exposed
to preovulatory surge-like levels of estradiol LH surge-
like levels (OVX?surgeE ? 63.8 pg/ml compared with
OVX?lowE?6.8pg/ml),andGnRHneuronalactivation
wasobservedinwild-typebutnotLeprknockoutmice(9).
Inwild-typemice,thepercentageofRP3Vkisspeptincells
expressing c-Fos was significantly increased compared
with mice receiving only low-level estradiol treatment
(37 ? 6.5% vs. 11 ? 3.8%; P ? 0.01). Significantly, this
increase was not observed in Lepr knockout mice receiv-
ing preovulatory surge-like levels of estradiol (5 ? 6%;
Fig. 6).
Discussion
The present study aimed to determine whether kisspeptin
gene expression is sensitive to changes in the amount of
circulating leptin and DIO in female mice. Additionally,
leptin signaling was examined in kisspeptin-expressing
neurons in the RP3V and ARC. We have shown here that
leptindeficiencydecreaseskisspeptingeneexpressionpre-
dominantly in the ARC, whereas there are fewer kisspep-
tin-expressing neurons in the RP3V of leptin-deficient
mice. Moreover, DBA/2J mice, which
arepronetoobesity-inducedinfertility,
showed a marked decrease in kisspep-
tin gene expression in both the RP3V
andARCandadecreaseinthenumber
of kisspeptin-immunoreactive neurons
in the RP3V after DIO. This is the first
evidence that kisspeptin can be regu-
lated by HFD and/or increases in body
weight.
Due to leptin’s profound effect on
the reproductive system, much interest
hasfocusedonfindingthelinkbetween
leptinsignalingandfemalefertility.Re-
cently we showed that this link was
central, occurring via an indirect neu-
ronal pathway to activate GnRH neu-
rons (9). A number of lines of evidence
suggest that kisspeptin could be the in-
termediary linking leptin signaling to
GnRH neurons. It is well known that
estrogens regulate kisspeptin gene expression (36); hence,
we used three experimental groups with differing circu-
lating levels of estradiol. Kiss1 mRNA and kisspeptin-ex-
pressing neurons increased in the RP3V in response to
increasingestradiollevelsinwild-typemice,asinprevious
studies(37,38).Interestingly,ob/obKiss1mRNAlevelsin
the RP3V also increased from a state of low estrogen to
surge-like levels of estrogen, indicating that estradiol can
promote at least some transcription of Kiss1 even in the
absence of leptin.
Previously, female OVX rats with low-dose estrogen
replacement fasted for 48 h displayed less Kiss1 mRNA
than fed controls in the AVPV (a subregion of the RP3V)
but not in the ARC (22). Another report found that pre-
pubertal rats treated with rapamycin (which blocks
mTORC1 signaling and suppresses appetite) showed de-
creased Kiss1 mRNA in both the AVPV and ARC regions
of the hypothalamus (28). We report here that Kiss1
mRNA levels decreased in both the RP3V and ARC in
responsetoleptindeficiency,althoughnotsignificantlyin
the former region.
Consistentwiththeliterature,Kiss1mRNAintheARC
was highest under OVX conditions. However, unlike
otherstudies(16,37),whereasARCKiss1mRNAexpres-
sion dropped under conditions of low estradiol replace-
ment,itwasonlyslightlyreducedunderpreovulatory-like
surgeprotocolconditions.Thisdiscrepancymaybedueto
differentsurgeprotocols;themiceinthestudybySmithet
al.(16)studyandtheratsinthestudybyAdashietal.(37)
had chronically (?7 d) elevated circulating estradiol con-
centrationsof200–500pg/ml,whereasourOVX?surgeE
FIG. 4. Effects of HFD on body weight (A), serum leptin concentration (B) Kiss1 mRNA levels
(C), and RP3V kisspeptin-immunoreactive cell numbers (D) in adult female OVX mice. The
number of animals per group is given at the base of each column. *, P ? 0.05.
1546
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Page 8

mice had circulating estradiol concentrations of approx-
imately 60 pg/ml for only 33 h.
The present qRT-PCR results suggest leptin deficiency
impairs Kiss1 gene expression in the ARC under all estro-
genic states. Changes in ARC Kiss1 mRNA expression in
responsetoleptindeficiencyagreewithpreviousliterature
that used fasted prepubertal female rats or male mice (13,
23). The work presented here is consistent with data from
male ob/ob mice that also display less Kiss1 mRNA in the
ARC, as measured by in situ hybridization (20). Impor-
tantly, the work of Smith et al. (20) showed that Lepr and
Kiss1 mRNA were coexpressed in the ARC (at least in
male mice). The physiological role of the kisspeptin-ex-
pressingARCneuronsisstillunclear,but
they have been proposed to mediate the
negative feedback effects of sex steroids
ongonadotropinsecretion(16,39),pos-
sibly by regulating GnRH pulses at or
near GnRH nerve terminals (40). Neu-
ronal tract-tracing studies have shown
that unidentified cells in the ARC send
projections forward directly to GnRH
neurons (29). More recently, it has been
shown that colocalized kisspeptin/neu-
rokinin B neurons from the ARC project
not only to the GnRH fibers in median
eminence but also forward to the
periventricularareaofthethirdventricle
(40). However, very few of these rostral
projections extend to the preoptic area
where the GnRH neurons reside (40).
Taken together with the present results,
itislikelythatleptinactsdirectlyonARC
kisspeptin neurons and thus may con-
tributepositivelytoGnRHneuronalout-
putatthemedianeminence,butwhether
this is essential for fertility remains to be
proven.
Instudyingkisspeptinproteinexpres-
sion, we found that kisspeptin-immuno-
reactive cell numbers were markedly de-
creased in leptin-deficient mice in the
RP3V under all three estrogenic states
tested. This effect was most marked in
the OVX?surgeE group of mice. Kiss1
expression in these mice, although tend-
ingtobelowerinob/obanimals,wasnot
significantly reduced. This apparently
conflicting result with the mRNA data
could be due to posttranscriptional def-
icitsinleptin-deficientmiceorduetothe
high degree of variability in the control
group (Fig. 2, top panel). These RP3V
kisspeptin neurons probably mediate the preovulatory
GnRH/LH surge (41), and thus an impairment of this
function would be predicted in response to leptin defi-
ciency. Accordingly, we showed that mice with deficient
leptin signaling were unable to generate a preovulatory-
likeGnRH/LHsurge(9)ortheassociatedincreaseinkiss-
peptinactivationintheRP3V(currentdata)inresponseto
exogenous estrogen.
As has been noted by others (35), accurate quantifica-
tion of kisspeptin-immunoreactive neurons in the ARC of
mice is not possible because positive cells are obscured
among a dense kisspeptin-positive fiber network that ex-
AB
CD
80 µm
30 µm
80 µm30 µm
EF
80 µm
20 µm
FIG. 5. Leptin signaling in kisspeptin-immunopositive neurons in response to an acute
injection of leptin (5 mg/kg, ip). Phosphorylated STAT3 (A and B), pSTAT5 (C and D), or pS6
(E and F) were not colocalized with kisspeptin-expressing neurons in the RP3V of adult
female mice. The dotted square on the left is magnified on the right. Arrows indicate a lack
of pSTAT3/5 or pS6 immunoreactivity in kisspeptin-expressing cells. Arrowheads indicate
pSTAT3/5 or pS6 (green) immunoreactivity in unidentified cells.
Endocrinology, April 2011, 152(4):1541–1550endo.endojournals.org
1547

Page 9

ists in the ARC. Extending the period of OVX or using col-
chicinepretreatment(adrugthatleadstoanaccumulationof
secretory material within the perikarya of neurosecretory
neurons)didnotincreasedefinitionofkisspeptin-expressing
neurons (data not shown). It may be that kisspeptin protein
expressionintheARCisverydynamic,withahighturnover
and/orrapidtransportationoutofthecells.ThusintheARC,
mRNA measurements may be a more relevant measure for
this neurosecretory product.
Our data do not allow us to distinguish as to whether
the reduction in kisspeptin expression is a direct conse-
quenceofleptindeficiency,orsimplyasideeffectoffailure
to initiate puberty in ob/ob mice. To clarify this it would
be necessary to replace leptin daily in ob/ob mice until
pubertaldevelopmentisachieved,andthen,afterallowing
leptin levels to subside, repeat the kisspeptin measure-
ments. However, leptin-deficient animals that were stim-
ulated to go through puberty in this way were unable to
reproduce after leptin withdrawal, suggesting that adult
fertility is indeed reliant on continuous leptin availability
(42). Additionally, food restricted postpubertal sheep
have reduced kisspeptin gene expression, and leptin infu-
sion can partially reverse this (21), suggesting the effects
are not simply a reflection of prepubertal state.
DIO due to HFD exposure for 21 wk was most notice-
ableintheDBA/2Jstrainofmice,withanaverageincrease
in body weight of about 11 g compared with an approx-
imately 3 g increase for C57BL/6J mice. It has been pre-
viously noted that C57BL/6J female mice are resistant to
DIO compared with DBA/2J mice (30, 43). The DBA/2J
female mouse is sensitive to HFD-induced hypothalamic
hypogonadism, showing a decreased GnRH gene expres-
sion, a 60% reduction in pregnancy rate, and relatively
inactive ovaries (30). We have observed that the diet-in-
duced obese DBA/2J mouse exhibits central resistance to
leptin signaling in both the RP3V and ARC and an im-
paired ability to generate a preovulatory-like GnRH/LH
surge(ourunpublisheddata).Thedataherearethefirstto
show that kisspeptin cell numbers and mRNA expression
are decreased by exposure to a HFD in female DBA/2J
mice.Luqueetal.(23)usedmaleC57BL/6JmiceonaHFD
for 16 wk; these mice did become approximately 10 g
heavierbutdidnotdisplayanychangesinhypothalamic
Kiss1 gene expression. This was similar to our findings
in C57BL/6J HFD-fed female mice (23). Because the
most marked effects were seen in female DBA/2J mice,
female C57BL/6J mice may not be susceptible to leptin
resistance(30)andthereforetheassociatedfertilityper-
turbations. Because our ob/ob mice, which are on a
C57BL/6J background strain, had reduced kisspeptin
gene expression and were infertile, HFD-induced infer-
tility may occur by different mechanisms than inferti-
lity caused by leptin deficiency. Our data reinforce the
use of the HFD-fed DBA/2J mouse model to study
the neuropeptide changes underpinning DIO-related
infertility.
Anatomically, dual label in situ hybridization has been
used to show Lepr and Kiss1 colocalization only in the
ARC of male mice, and additionally in the preoptic area
and ARC of female sheep (20, 21). In our study we ob-
served no evidence for leptin signaling in the RP3V kiss-
peptin-expressingneurons.Thepossibilitythatleptinacts
directly on RP3V kisspeptin neurons in rodents to drive
the preovulatory GnRH/LH surge has not been investi-
gateduntilnow.Usingafunctionalimmunohistochemical
approach, leptin-induced pSTAT3, pSTAT5, and pS6
were investigated in kisspeptin-expressing neurons. Vir-
tuallynocolocalizationofanyofthetheseleptin-signaling
molecules occurred in kisspeptin-expressing cells within
the RP3V, although nearby unidentified cells showed
STAT3, STAT5, and S6 activation. Although leptin’s ac-
tions via other signaling pathways remain possible (44),
the STAT3 pathway has generally been thought to be
FIG. 6. Kisspeptin-immunoreactive neurons within the preoptic
periventricular nucleus showing c-Fos signaling in response to a
preovulatory-like surge induction protocol compared with control
littermates (A). Representative examples show kisspeptin-
immunoreactive neurons (brown cytoplasmic staining) and cFos (black
nuclear staining) in neuron-specific Lepr knockout (B) and control (C)
mice. Solid arrows indicate examples of colocalized neurons; open
arrows indicate examples of noncolocalized neurons. The number of
animals per group is given at the base of each column. *, P ? 0.05 vs.
both other groups.
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Page 10

dominantbecauseneuronalSTAT3deletionrecapitulates
theinfertility,obesity,andthermaldysregulationofglobal
leptin and leptin receptor knockout mice (45). However,
othershavedescribedaleptinreceptorknockintransgenic
mouse strain that is unable to specifically activate STAT3
signaling; in these mice 43% of the females were fertile
(46). One alternative signaling pathway leptin may use to
affect fertility is the mTORC1 pathway (27), because the
blockadeofthispathwaywithrapamycininhibitstheabil-
ity of leptin to stimulate puberty onset (28). Indeed, we
observed leptin-induced pS6 (evidence of mTORC1 sig-
naling)intheRP3VwithasimilardistributiontopSTAT3,
but no colocalization with kisspeptin immunoreactivity.
Our data therefore strongly indicate that RP3V kisspep-
tin-expressing cells are not directly activated by leptin.
Futurestudiesarerequiredtodeterminetheleptin-respon-
sive inputs to these cells that cause them to become im-
paired in states of leptin deficiency. In this regard, it has
recently been shown that cells from the ventral premam-
millary nucleus (a region that appears to be critical for
leptin’s effects on puberty initiation) project to Kiss1 cells
in the AVPV (47).
In summary, both leptin deficiency and DIO impair kiss-
peptin expression. This impairment extends to kisspeptin
neuronal activation in the RP3V during a preovulatory-like
surge. The work here suggests that whereas the effects of
leptin on the ARC kisspeptin neurons may be direct, its ef-
fects in the RP3V appear to be indirectly mediated by un-
known upstream leptin-sensitive neurons. The identity and
locationoftheseleptin-sensitiveneurons,whichfeedintothe
kisspeptin/GnRHsystem,areunknownandwarrantsystem-
atic investigation.
Acknowledgments
We thank Hayden McEwen (University of Otago, Dunedin,
New Zealand) for providing HFD animal tissues for
immunohistochemistry.
Address all correspondence and requests for reprints to: Dr.
Janette Quennell, Centre for Neuroendocrinology and Depart-
ment of Anatomy and Structural Biology, University of Otago
School of Medical Sciences, PO Box 913, Dunedin 9054, New
Zealand. E-mail: janette.quennell@anatomy.otago.ac.nz.
ThisworkwassupportedbyTheRoyalSocietyofNewZealand
FastStartprogram(toJ.H.Q.),andTheHealthResearchCouncilof
New Zealand (to G.M.A and D.R.G.). J.H.Q. was supported by
The Health Sciences Career Development Program Postdoctoral
Fellowship from the University of Otago. Grant 1PO1DK26687
fromTheNewYorkObesityResearchCenter(toS.C.)fundedthe
development of the floxed leptin receptor mouse.
Disclosure Statement: The authors have nothing to disclose.
References
1. Schneider JE 2004 Energy balance and reproduction. Physiol Behav
81:289–317
2. Brewer CJ, Balen AH 2010 The adverse effects of obesity on con-
ception and implantation. Reproduction 140:347–364
3. Marsh EE, Barnes RB 2008 Obesity and conception. In: Rees M,
KaroshiM,KeithL,eds.Obesityandpregnancy.London:TheRoyal
Society of Medicine Press Limited; 78–88
4. I’Anson H, Sundling LA, Roland SM, Ritter S 2003 Immunotoxic
destruction of distinct catecholaminergic neuron populations dis-
rupts the reproductive response to glucoprivation in female rats.
Endocrinology 144:4325–4331
5. BarashIA,CheungCC,WeigleDS,RenH,KabigtingEB,KuijperJL,
Clifton DK, Steiner RA 1996 Leptin is a metabolic signal to the
reproductive system. Endocrinology 137:3144–3147
6. Todd BJ, Ladyman SR, Grattan DR 2003 Suppression of pulsatile
luteinizing hormone secretion but not luteinizing hormone surge in
leptin resistant obese Zucker rats. J Neuroendocrinol 15:61–68
7. Finn PD, Cunningham MJ, Pau KY, Spies HG, Clifton DK, Steiner
RA 1998 The stimulatory effect of leptin on the neuroendocrine
reproductive axis of the monkey. Endocrinology 139:4652–4662
8. Nagatani S, Guthikonda P, Thompson RC, Tsukamura H, Maeda
KI, Foster DL 1998 Evidence for GnRH regulation by leptin: leptin
administration prevents reduced pulsatile LH secretion during fast-
ing. Neuroendocrinology 67:370–376
9. Quennell JH, Mulligan AC, Tups A, Liu X, Phipps SJ, Kemp CJ,
Herbison AE, Grattan DR, Anderson GM 2009 Leptin indirectly
regulates gonadotropin-releasing hormone neuronal function. En-
docrinology 150:2805–2812
10. ConeRD2005Anatomyandregulationofthecentralmelanocortin
system. Nat Neurosci 8:571–578
11. Rondini TA, Baddini SP, Sousa LF, Bittencourt JC, Elias CF 2004
Hypothalamiccocaine-andamphetamine-regulatedtranscriptneu-
rons project to areas expressing gonadotropin releasing hormone
immunoreactivity and to the anteroventral periventricular nucleus
in male and female rats. Neuroscience 125:735–748
12. Schioth HB, Kakizaki Y, Kohsaka A, Suda T, Watanobe H 2001
Agouti-related peptide prevents steroid-induced luteinizing hor-
moneandprolactinsurgesinfemalerats.Neuroreport12:687–690
13. Castellano JM, Navarro VM, Ferna ´ndez-Ferna ´ndez R, Nogueiras
R, Tovar S, Roa J, Vazquez MJ, Vigo E, Casanueva FF, Aguilar E,
Pinilla L, Dieguez C, Tena-Sempere M 2005 Changes in hypotha-
lamic KiSS-1 system and restoration of pubertal activation of the
reproductive axis by kisspeptin in undernutrition. Endocrinology
146:3917–3925
14. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi
K, Terao Y, Kumano S, Takatsu Y, Masuda Y, Ishibashi Y, Wa-
tanabe T, Asada M, Yamada T, Suenaga M, Kitada C, Usuki S,
Kurokawa T, Onda H, Nishimura O, Fujino M 2001 Metastasis
suppressor gene KiSS-1 encodes peptide ligand of a G-protein-cou-
pled receptor. Nature 411:613–617
15. Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J,
Thresher RR, Malinge I, Lomet D, Carlton MB, Colledge WH,
Caraty A, Aparicio SA 2005 Kisspeptin directly stimulates gonad-
otropin-releasing hormone release via G protein-coupled receptor
54. Proc Natl Acad Sci U S A 102:1761–1766
16. Smith JT, Cunningham MJ, Rissman EF, Clifton DK, Steiner RA
2005 Regulation of Kiss1 gene expression in the brain of the female
mouse. Endocrinology 146:3686–3692
17. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno Jr
JS,ShagouryJK,Bo-AbbasY,KuohungW,SchwinofKM,Hendrick
AG, Zahn D, Dixon J, Kaiser UB, Slaugenhaupt SA, Gusella JF,
O’Rahilly S, Carlton MB, Crowley Jr WF, Aparicio SA, Colledge
WH 2003 The GPR54 gene as a regulator of puberty. N Engl J Med
349:1614–1627
18. Xu J, Kirigiti MA, Grove KL, Smith MS 2009 Regulation of food
intake and gonadotropin-releasing hormone/luteinizing hormone
Endocrinology, April 2011, 152(4):1541–1550endo.endojournals.org
1549

Page 11

during lactation: role of insulin and leptin. Endocrinology 150:
4231–4240
19. Yamada S, Uenoyama Y, Kinoshita M, Iwata K, Takase K, Matsui
H, Adachi S, Inoue K, Maeda KI, Tsukamura H 2007 Inhibition of
metastin (kisspeptin-54)-GPR54 signaling in the arcuate nucleus-
median eminence region during lactation in rats. Endocrinology
148:2226–2232
20. Smith JT, Acohido BV, Clifton DK, Steiner RA 2006 KiSS-1 neu-
rones are direct targets for leptin in the ob/ob mouse. J Neuroen-
docrinol 18:298–303
21. Backholer K, Smith JT, Rao A, Pereira A, Iqbal J, Ogawa S, Li Q,
ClarkeIJ2010Kisspeptincellsintheewebrainrespondtoleptinand
communicate with neuropeptide y and proopiomelanocortin cells.
Endocrinology 151:2233–2243
22. Kalamatianos T, Grimshaw SE, Poorun R, Hahn JD, Coen CW
2008 Fasting reduces KiSS-1 expression in the anteroventral
periventricular nucleus (AVPV): effects of fasting on the expression
of KiSS-1 and neuropeptide Y in the AVPV or arcuate nucleus of
female rats. J Neuroendocrinol 20:1089–1097
23. Luque RM, Kineman RD, Tena-Sempere M 2007 Regulation of
hypothalamic expression of KiSS-1 and GPR54 genes by metabolic
factors: analyses using mouse models and a cell line. Endocrinology
148:4601–4611
24. ClarksonJ,HerbisonAE2006Postnataldevelopmentofkisspeptin
neurons in mouse hypothalamus; sexual dimorphism and projec-
tions to gonadotropin-releasing hormone neurons. Endocrinology
147:5817–5825
25. Anderson KD, Lambert PD, Corcoran TL, Murray JD, Thabet KE,
YancopoulosGD,WiegandSJ2003Activationofthehypothalamic
arcuatenucleuspredictstheanorecticactionsofciliaryneurotrophic
factorandleptininintactandgoldthioglucose-lesionedmice.JNeu-
roendocrinol 15:649–660
26. LadymanSR,GrattanDR2004Region-specificreductioninleptin-
inducedphosphorylationofsignaltransducerandactivatoroftran-
scription-3 (STAT3) in the rat hypothalamus is associated with lep-
tin resistance during pregnancy. Endocrinology 145:3704–3711
27. Cota D, Proulx K, Smith KA, Kozma SC, Thomas G, Woods SC,
Seeley RJ 2006 Hypothalamic mTOR signaling regulates food in-
take. Science 312:927–930
28. RoaJ,Garcia-GalianoD,VarelaL,Sa ´nchez-GarridoMA,PinedaR,
Castellano JM, Ruiz-Pino F, Romero M, Aguilar E, Lo ´pez M, Gay-
tan F, Die ´guez C, Pinilla L, Tena-Sempere M 2009 The mammalian
target of rapamycin as novel central regulator of puberty onset via
modulation of hypothalamic Kiss1 system. Endocrinology 150:
5016–5026
29. Wintermantel TM, Campbell RE, Porteous R, Bock D, Gro ¨ne HJ,
TodmanMG,KorachKS,GreinerE,Pe ´rezCA,Schu ¨tzG,Herbison
AE 2006 Definition of estrogen receptor pathway critical for estro-
gen positive feedback to gonadotropin-releasing hormone neurons
and fertility. Neuron 52:271–280
30. Tortoriello DV, McMinn J, Chua SC 2004 Dietary-induced obesity
andhypothalamicinfertilityinfemaleDBA/2Jmice.Endocrinology
145:1238–1247
31. Brown RS, Kokay IC, Herbison AE, Grattan DR 2010 Distribution
of prolactin-responsive neurons in the mouse forebrain. J Comp
Neurol 518:92–102
32. Greenwood AD, Horsch M, Stengel A, Vorberg I, Lutzny G, Maas
E,Scha ¨dlerS,ErfleV,BeckersJ,Scha ¨tzlH,Leib-Mo ¨schC2005Cell
line dependent RNA expression profiles of prion-infected mouse
neuronal cells. J Mol Biol 349:487–500
33. BustinSA,BenesV,GarsonJA,HellemansJ,HuggettJ,KubistaM,
Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wit-
twer CT 2009 The MIQE guidelines: minimum information for
publication of quantitative real-time PCR experiments. Clin Chem
55:611–622
34. Paxinos G, Franklin KBJ 2004 The mouse brain in stereotaxic co-
ordinates. San Diego: Elsevier Academic Press
35. Clarkson J, d’Anglemont de Tassigny X, Colledge WH, Caraty A,
Herbison AE 2009 Distribution of kisspeptin neurones in the adult
female mouse brain. J Neuroendocrinol 21:673–682
36. Navarro VM, Castellano JM, Ferna ´ndez-Ferna ´ndez R, Barreiro
ML, Roa J, Sanchez-Criado JE, Aguilar E, Dieguez C, Pinilla L,
Tena-Sempere M 2004 Developmental and hormonally regulated
messenger ribonucleic acid expression of KiSS-1 and its putative
receptor, GPR54, in rat hypothalamus and potent luteinizing hor-
mone-releasing activity of KiSS-1 peptide. Endocrinology 145:
4565–4574
37. AdachiS,YamadaS,TakatsuY,MatsuiH,KinoshitaM,TakaseK,
Sugiura H, Ohtaki T, Matsumoto H, Uenoyama Y, Tsukamura H,
Inoue K, Maeda K 2007 Involvement of anteroventral periventricu-
larmetastin/kisspeptinneuronsinestrogenpositivefeedbackaction
on luteinizing hormone release in female rats. J Reprod Dev 53:
367–378
38. Kinoshita M, Tsukamura H, Adachi S, Matsui H, Uenoyama Y,
Iwata K, Yamada S, Inoue K, Ohtaki T, Matsumoto H, Maeda K
2005 Involvement of central metastin in the regulation of preovu-
latoryluteinizinghormonesurgeandestrouscyclicityinfemalerats.
Endocrinology 146:4431–4436
39. Roseweir AK, Kauffman AS, Smith JT, Guerriero KA, Morgan K,
Pielecka-Fortuna J, Pineda R, Gottsch ML, Tena-Sempere M,
Moenter SM, Terasawa E, Clarke IJ, Steiner RA, Millar RP 2009
Discovery of potent kisspeptin antagonists delineate physiological
mechanismsofgonadotropinregulation.JNeurosci29:3920–3929
40. True C, Kirigiti M, Ciofi P, Grove KL, Smith MS 2011 Character-
isation of arcuate nucleus kisspeptin/neurokinin B neuronal projec-
tions and regulation during lactation in the rat. J Neuroendocrinol
23:52–64
41. ClarksonJ,d’AnglemontdeTassignyX,MorenoAS,ColledgeWH,
Herbison AE 2008 Kisspeptin-GPR54 signaling is essential for pre-
ovulatory gonadotropin-releasing hormone neuron activation and
the luteinizing hormone surge. J Neurosci 28:8691–8697
42. Chehab FF, Lim ME, Lu R 1996 Correction of the sterility defect in
homozygous obese female mice by treatment with the human re-
combinant leptin. Nat Genet 12:318–320
43. TortorielloDV,McMinnJE,ChuaSC2007Increasedexpressionof
hypothalamicleptinreceptorandadiponectinaccompanyresistance
to dietary-induced obesity and infertility in female C57BL/6J mice.
Int J Obes 31:395–402
44. Morris DL, Rui L 2009 Recent advances in understanding leptin
signalingandleptinresistance.AmJPhysiolEndocrinolMetab297:
E1247–E1259
45. GaoQ,WolfgangMJ,NeschenS,MorinoK,HorvathTL,Shulman
GI,FuXY2004Disruptionofneuralsignaltransducerandactivator
of transcription 3 causes obesity, diabetes, infertility, and thermal
dysregulation. Proc Natl Acad Sci U S A 101:4661–4666
46. Bates SH, Stearns WH, Dundon TA, Schubert M, Tso AW, Wang
Y, Banks AS, Lavery HJ, Haq AK, Maratos-Flier E, Neel BG,
SchwartzMW,MyersJrMG2003STAT3signallingisrequiredfor
leptin regulation of energy balance but not reproduction. Nature
421:856–859
47. Donato Jr J, Cravo RM, Frazao R, Gautron L, Scott MM, Lachey J,
Castro IA, Margatho LO, Lee S, Lee C, Richardson JA, Friedman J,
Chua Jr S, Coppari R, Zigman JM, Elmquist JK, Elias CF 2011
Leptin’s effect on puberty in mice is relayed by the ventral premam-
millary nucleus and does not require signaling in Kiss1 neurons.
J Clin Invest 121:355–368
1550
Quennell et al.Kisspeptin Regulation by Leptin and High-Fat Diet Endocrinology, April 2011, 152(4):1541–1550