Hormones and Behavior 64 (2013) 187–194

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Hormones and Behavior

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Review

Metabolic control of puberty: Roles of leptin and kisspeptins

Miguel A. Sanchez-Garrido, Manuel Tena-Sempere ⁎
Department of Cell Biology, Physiology and Immunology, University of Córdoba, CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, Spain
Instituto Maimónides de Investigaciones Biomédicas (IMIBIC)/Hospital Universitario Reina Sofía, 14004 Córdoba, Spain

a r t i c l e i n f o a b s t r a c t

Keywords: This article is part of a Special Issue “Puberty and Adolescence”.

Puberty
Leptin Reproduction is an energy-demanding function. Accordingly, puberty is metabolically gated, as a means to
Kisspeptins prevent fertility in conditions of energy insufficiency. In addition, obesity has been shown to impact the timing
Energy homeostasis of puberty and may be among the causes for the earlier trends of pubertal age reported in various countries.
Metabolism
The metabolic control of puberty in such a spectrum of situations, ranging from energy deficit to extreme
overweight, is the result of the concerted action of different peripheral hormones and central transmitters
that sense the metabolic state of the organism and transmit this information to the various elements of the
reproductive axis, mainly the GnRH neurons. Among the peripheral signals involved, the adipose hormone,
leptin, is known to play an essential role in the regulation of puberty, especially in females. Yet, although
it is clear that the effects of leptin on puberty onset are predominantly permissive and mainly conducted at
central (hypothalamic) levels, the primary sites and mechanisms of action of leptin within the reproductive
brain remain unsolved. In this context, neurons expressing kisspeptins, the products of the Kiss1 gene
that have emerged recently as essential upstream regulators of GnRH neurons, operate as key sensors of
the metabolic state and funnel of the reproductive effects of leptin. Yet, much debate has arisen recently on
whether the putative actions of leptin on the Kiss1 system are actually indirect and/or may primarily target
Kiss1-independent pathways, such as those originating from the ventral premmamilary nucleus. Moreover,
evidence has been presented for extra-hypothalamic or peripheral actions of leptin, including direct gonadal
effects, which may contribute to the metabolic control of reproduction in extreme body weight conditions.
In this work, we will critically review the experimental evidence supporting a role of leptin, kisspeptin and
putatively related pathways in the concerted control of puberty by energy balance and metabolism.
© 2013 Elsevier Inc. All rights reserved.

Contents

Introduction: Puberty is metabolically gated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Leptin as major determinant of mammalian puberty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
Kisspeptins as essential gatekeepers of mammalian puberty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Kisspeptins and the metabolic control of puberty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
Interplay of leptin and kisspeptins in the metabolic control of puberty: direct or indirect? . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Other sites of action of leptin in the metabolic control of puberty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Final remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
Disclosure statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Introduction: Puberty is metabolically gated

⁎ Corresponding author at: Department of Cell Biology, Physiology and Immunology,

Faculty of Medicine, University of Córdoba, Avda. Menéndez Pidal s/n, 14004 Córdoba,
Puberty is a complex developmental event within the continuum of
Spain. Fax: +34 957 218288. changes leading to sexual and somatic maturation of the individual, dur-
E-mail address: fi1tesem@uco.es (M. Tena-Sempere). ing which reproductive competence is achieved and multiple growth,

0018-506X/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.yhbeh.2013.01.014
188 M.A. Sanchez-Garrido, M. Tena-Sempere / Hormones and Behavior 64 (2013) 187–194
behavioral and psychological changes occur (Ojeda and Skinner, 2006;
Ojeda et al., 2010; Parent et al., 2003). The timing of puberty is precisely
controlled by a plethora of endogenous signals and environmental cues
that impinge at different levels of the hypothalamic–pituitary–gonadal
(HPG or reproductive) axis (Parent et al., 2003). This phenomenon,
which ensures that the tempo of puberty is adjusted to the internal
and external conditions of the body, is also responsible for perturbations
of its timing when such conditions are not optimal. Accordingly, puberty
has been regarded not only as a fundamental developmental event, but
also as sensor of the proper interplay between genes and environment
along development (Tena-Sempere, 2010a). Hence, the recent trends
for the change (mainly, advance) in the age of puberty, as reported in
different populations, may be a sentinel for the deterioration of repro-
ductive and/or environmental health and, as such, have raised concerns
in the medical community and lay-public worldwide (Aksglaede et al.,
2009a, 2009b).
Among its different regulators, it is well established that metabolic
conditions and the amount of energy reserves of the organism play an
essential role in the modulation of the timing of puberty (Fernandez-
Fernandez et al., 2006; Martos-Moreno et al., 2010). This makes good
biological sense, especially in the female, so that the reproductive
capacity, which implies the potential metabolic drainage of pregnancy
and lactation, is only acquired when threshold energy stores and optimal
metabolic conditions are achieved. In any event, evidence for the influ-
ence of metabolic signals on puberty and fertility is also found in the
male (Castellano et al., 2009b), in which energy demands for proper re-
production are not so evident but probably required as well (e.g., for ter-
ritoriality and partner selection) (Elias, 2012; Elias and Purohit, 2013).
Moreover, not only conditions of metabolic distress associated with
energy insufficiency but also with its excess, such as morbid obesity, are
linked to reproductive perturbations, such as precocious puberty in the
female (Castellano et al., 2011) and trends for hypogonadism of central
origin in the male (Tajar et al., 2010). Moreover, early forms of nutritional
or metabolic challenge, such as under- or over-nutrition taking place dur-
ing the fetal and neonatal life, have been also shown to alter the timing of
puberty in rodents and humans (Caron et al., 2012; Castellano et al., 2011;
Ibanez and de Zegher, 2006). Altogether, these findings illustrate not only
the fact that the metabolic status has a clear impact on the timing of
puberty but also that the mechanisms underlying this phenomenon are
complex and likely involve different regulatory pathways (Roa and
Tena-Sempere, 2010; Roa et al., 2010).
Leptin as major determinant of mammalian puberty
Despite historical intuitions about the metabolic gating of puberty
that existed since old ages, the basis for such a close relationship between
the magnitude of energy (fat) stores and pubertal timing became scien-
tifically formulated only in the 1960's and 70's. This was mainly due to
the pioneering work of Kennedy and Mitra in rodents, and Frisch and
co-workers in humans (Fernandez-Fernandez et al., 2006; Pralong,
2010). This work resulted in the so-called critical fat mass hypothesis
that, although partially challenged later, set the scene for the search of
the factor(s) responsible for the integral control of energy metabolism,
puberty onset and fertility. The identification in 1994 of the adipose
hormone, leptin, as an essential signal in the control of body weight ho-
meostasis, whose levels in circulation are proportional to the size of fat
stores, led to specific analyses of the reproductive roles of this hormone.
These studies settled the contention that leptin plays a central function in
the metabolic control of puberty and fertility (Ahima et al., 2000;
Casanueva and Dieguez, 1999; Cheung et al., 1997; Fernandez-
Fernandez et al., 2006; Tena-Sempere, 2007). Nonetheless, recent exper-
imental evidence has cast doubts on the actual roles of leptin in some
particular aspects of the metabolic control of the gonadotropic axis. For
instance, normalization of mean leptin concentrations in conditions
of negative energy balance, such as acute fasting, failed to restore
gonadotropin levels in female rats (True et al., 2011). Yet, the physiolog-
ical relevance of this observation needs further evaluation.
While detailed description of the biological features of leptin is be-
yond the scope of this review, it is important to stress that leptin operates
as a major signal of energy sufficiency, thus allowing different physiolog-
ical systems (not only those controlling body weight) to sense the mag-
nitude of energy reserves. In fact, leptin is known to inform quite diverse
axes, from the immune system to the growth axis, of the existence of ad-
equate energy stores to face these various body functions (Casanueva
and Dieguez, 1999). Accordingly, conditions of leptin insufficiency, if per-
sistent in time, are coupled to perturbed function of numerous systems.
This is the case also of the reproductive axis, as illustrated in various an-
imal models and human pathologies associated with low or null leptin
levels, which are usually linked to a delay or absence of puberty onset
and perturbed fertility (Roa et al., 2010).
Although the important roles of leptin in the control of puberty were
well settled soon after its identification in the mid 90's, the precise na-
ture of leptin effects on pubertal control was the subject of some debate.
Thus, initial work in rodents suggested that leptin administration might
actually induce or advance the onset of female puberty, therefore im-
plying a net stimulatory action (Ahima et al., 1997; Chehab et al.,
1997). This possibility was considered relevant not only from a physio-
logic standpoint but also from a therapeutic perspective; if leptin was to
be used to treat forms of morbid obesity of early origin (especially in
leptin-null individuals), any potential action of leptin as trigger of
precocious puberty should be regarded as an important side-effect.
However, pharmacological studies in humans and rodents with leptin
deficiency clearly documented that, while leptin is indispensable for
puberty to proceed, leptin alone cannot trigger early puberty, therefore
supporting a major permissive role of this hormone in the metabolic
gating of pubertal maturation (Barash et al., 1996; Castellano et al.,
2009b; Cheung et al., 1997; Roa et al., 2010). This permissive function
has been better characterized in females, while leptin's role in male pu-
berty remains less clear. In fact, circulating leptin has shown to rise dur-
ing the pubertal transition in girls (Garcia-Mayor et al., 1997), while
puberty onset in boys and male monkeys does not seem to require a
similar increase in leptin levels (Garcia-Mayor et al., 1997; Plant and
Durrant, 1997).
Another issue that has remained contentious for years is how and
where leptin conducts its reproductive actions (Elias, 2012). In one
hand, solid evidence has demonstrated that the permissive actions of
leptin are conducted primary at central (hypothalamic) levels, where
leptin is able to ultimately modulate the function of the GnRH neuronal
system (Cunningham et al., 1999). Notwithstanding this, evidence for
additional actions of leptin at other levels of the HPG axis has been
presented (Caprio et al., 2001; Tena-Sempere, 2007). Indeed, at high
concentrations, as those expected in obesity, leptin can directly inhibit
some gonadal functions, such as sex steroid secretion (Caprio et al.,
2001; Tena-Sempere et al., 1999). Although this was initially considered
counterintuitive given its primarily permissive/positive roles on the
HPG axis, such inhibitory actions of leptin may explain part of the
hypogonadal state frequently observed in morbid obese patients
(Caprio et al., 2001; Tena-Sempere and Barreiro, 2002). On the other
hand, even assuming a primary permissive action at central levels, lep-
tin effects on the GnRH system appear to be conducted indirectly, via
leptin-sensitive afferents that project to GnRH neurons, which are ap-
parently devoid of functional leptin receptors. This contention, which
was originally questioned by the detection of leptin receptors in immor-
talized GT1-7 cells, which are parentally derived from GnRH neurons, is
now solidly supported by data from brain expression studies in rodents
and primates (Finn et al., 1998; Hakansson et al., 1998), and recent
functional genomic approaches that showed that congenital elimina-
tion of putative leptin receptors from GnRH neurons was compatible
with preserved puberty or fertility (Quennell et al., 2009). Although
the occurrence of developmental compensation cannot be excluded in
the later model, the experimental evidence available strongly suggests
 M.A. Sanchez-Garrido, M. Tena-Sempere / Hormones and Behavior 64 (2013) 187–194
that the physiological actions of leptin on GnRH secretion and hence on
puberty onset are conducted indirectly (Quennell et al., 2009). This as-
sumption has led to the search for the primary targets of leptin within
the reproductive brain. Recent developments in this area will be
discussed in the following sections.
As final note to this section, the molecular mediators and signaling
pathways for the central reproductive actions of leptin have been also
the subject of active investigation. Based on the known properties of
functional leptin receptors (LepR-b or Ob-Rb), the role of leptin-
induced STAT3 signaling has been studied (Elias and Purohit, 2013). Ge-
netic models have demonstrated that, while elimination of STAT3 signal-
ing from the brain results in lack of pubertal maturation and infertility,
selective ablation of STAT3 from LepR-expressing neurons (hence, neu-
rons responsive to the actions of leptin) is compatible with proper devel-
opment of reproductive organs and cyclic ovarian function in mice (Bates
et al., 2003). This would suggest that STAT3-independent pathways are
recruited by leptin in the brain to conduct, at least part of, their effects
on the reproductive axis. These pathways may involve PI3K, a molecular
complex that is known to mediate part of the central effects of leptin in
terms of metabolic control (Elias and Purohit, 2013). In this sense, selec-
tive elimination of key catalytic subunits of PI3K from LepR-expressing
cells prevents leptin effects on key cellular functions in POMC and
AgRP neurons in the arcuate nucleus (ARC) (Al-Qassab et al., 2009), as
well as in the ventral premmamilary nucleus (PMV) (Williams et al.,
2011). However, whether blockade of leptin-induced activation of PI3K
has deleterious effects on puberty onset and reproductive function has
not been reported to date. Finally, central signaling pathways involving
the mammalian target of rapamycin (mTOR) have been also implicated
in mediating not only the metabolic actions of leptin (e.g., in terms of
food intake control), but also the permissive effects of leptin on puberty
onset (Roa and Tena-Sempere, 2010). Thus, central blockade of mTOR
blunted the ability of leptin to rescue puberty in female rats subjected
to chronic subnutrition (Roa et al., 2009). Despite this compelling evi-
dence, whether these effects of mTOR are conducted directly in Kiss1
neurons or in upstream afferents, sensitive to the regulatory actions of
leptin, remains unknown.
Kisspeptins as essential gatekeepers of mammalian puberty
Our knowledge of the neurobiological basis of puberty in general,
and of the mechanisms for its metabolic regulation in particular, has
substantially enlarged in recent years. Probably, the most important de-
velopment in this front was the recognition of the essential roles of
kisspeptins in the central control of different aspects of reproductive
function, including puberty onset (Clarkson et al., 2010; Oakley et al.,
2009; Pinilla et al., 2012; Roa et al., 2008a). Due to the astonishingly
rapid progress of this field, a detailed recapitulation of the major fea-
tures of the physiology of kisspeptins is beyond the scope of this review
and can be found elsewhere (Pinilla et al., 2012). Nonetheless, in order
to introduce later sections of this review, a brief account of the proposed
roles of kisspeptins as putative gatekeepers of puberty is presented
here.
Kisspeptins (Kp), encoded by the Kiss1 gene, are a family of
structurally-related peptides with the ability to activate the G protein-
coupled receptor, Gpr54 – or Kiss1R – (Oakley et al., 2009; Pinilla et
al., 2012; Roa et al., 2008a). Departing from the seminal finding of
forms of hypogonadotropic hypogonadism (HH) in patients with
inactivating mutations of GPR54 (de Roux et al., 2003; Seminara et al.,
2003), a growing number of studies, conducted in different model spe-
cies as well as in humans, have now set the contention that kisspeptins
are key transmitters in the reproductive brain. These originate from dis-
crete neuronal populations in the hypothalamus and are able to stimu-
late the secretory activity of GnRH neurons. The neuro-anatomy of such
populations has been well characterized in rodents, where two major
sets of Kiss1 neurons, located in the arcuate nucleus (ARC) and the
rostral periventricular area of the 3rd ventricle (RP3V), have been
189
described (Clarkson et al., 2009; Pinilla et al., 2012). The importance
of kisspeptins in reproductive function is not only illustrated by the
HH phenotypes of humans and mice with inactivating mutations of
the Kiss1 or Gpr54 genes, but also by solid neuroanatomical, electro-
physiological, pharmacological and hormonal data. Collectively, these
data have documented the involvement of the so-called Kiss1 system
in the regulation of virtually all aspects of reproductive maturation
and function, from brain sex differentiation to the neuroendocrine con-
trol of ovulation and sex steroid feedback.
In the above scenario, the possibility that the Kiss1 system may play a
physiological role in the control of puberty has drawn considerable
attention and has been the subject of thorough analyses in numerous
(mammalian and nonmammalian) species (Pineda et al., 2010;
Tena-Sempere, 2010b). Indeed, the findings of the lack of pubertal mat-
urations in patients or mice with null mutations in Gpr54 or Kiss1 genes
suggest a prominent, indispensable role of kisspeptin signaling in the
control of puberty. Admittedly, however, the fact that congenital inacti-
vation of Gpr54 or Kiss1 perturbs puberty does not necessarily imply an
activational role of the Kiss1 system in puberty itself, as the above pheno-
type (lack of puberty) may derive from the alteration of early develop-
mental phenomena in the absence of kisspeptin signaling. In any event,
the available experimental evidence does suggest that kisspeptins
participate in the activational program responsible for puberty, as docu-
mented by a combination of neuroanatomical and functional analyses,
conducted mainly in rodent species, especially in the female (Roa and
Tena-Sempere, 2010; Tena-Sempere, 2010b).
Initial expression studies documented an increase in the hypotha-
lamic expression of the Kiss1 gene during pubertal maturation in the
rat and monkey, therefore suggesting a rise of the kisspeptin tone at
the hypothalamus during puberty (Navarro et al., 2004a; Shahab et al.,
2005). Of note, pharmacological analyses demonstrated the functional
relevance of such a phenomenon, since repeated administration of
kisspeptin-10 was sufficient to advance the occurrence of different indi-
ces of puberty onset in immature female rats (Navarro et al., 2004b).
Likewise, repeated kisspeptin administration to juvenile monkeys
evoked a pattern of GnRH secretion similar to that of (post)pubertal an-
imals (Plant et al., 2006). In addition, rodent studies have shown that
during puberty there is an increase in the sensitivity to the stimulatory
effects of kisspeptin in terms of GnRH/LH responses, as described in rats
and mice (Castellano et al., 2006a; Han et al., 2005). Likewise, in female
mice, the number of GnRH neurons expressing Gpr54 increases before
puberty (Herbison et al., 2010), as does the Gpr54 signaling efficiency
(Han et al., 2005). Importantly, Gpr54 seems to be less prone to
desensitization to the stimulatory effects of continuous kisspeptin stim-
ulation during the pubertal transition in female rats (Roa et al., 2008b).
This phenomenon may help to explain why at this period the rise of the
endogenous kisspeptin tone is compatible with the progressive activa-
tion of the gonadotropic axis, without any sign of receptor desensitiza-
tion. Finally, detailed neuro-anatomical studies in female mice have
demonstrated that during puberty there is a sharp rise in the number
of kisspeptin neurons, as documented by immunohistochemical
analyses (Clarkson et al., 2010). In addition, the number of projections/
appositions of Kiss1 neurons to GnRH neurons also increases during pu-
berty. This phenomenon seems to require, at least in female rodents, the
permissive action of some degree of estrogenic input of ovarian origin
(Clarkson et al., 2010). This would imply that the early stages of ovarian
activation during puberty take place before full activation of the hypo-
thalamic Kiss1 system, which would rather operate as an essential am-
plifier of the GnRH neurosecretory activity along pubertal maturation.
Altogether, the above experimental evidence demonstrates that
during puberty the hypothalamic Kiss1 system undergoes an extensive
and complex activational program that seems to be essential for proper
pubertal timing. In good agreement, studies from our group have dem-
onstrated that pharmacological blockade of Gpr54 is sufficient to delay
puberty onset in female rats (Pineda et al., 2010). Yet, in spite of the
above compelling evidence, recent data have challenged the view that
190 M.A. Sanchez-Garrido, M. Tena-Sempere / Hormones and Behavior 64 (2013) 187–194
intact kisspeptin signaling is indispensable for puberty onset in rodents,
as it has been shown, by the use of elegant genetically-modified mouse
models, that fertility can be apparently achieved even after the congen-
ital ablation of Kiss1 neurons in female mice (Mayer and Boehm, 2011).
Quite expectedly, this set of data has drawn enormous interest, as it ar-
gues against the consensus notion that kisspeptins are essential gate-
keepers of puberty. Some reconciliation with previous literature can
be obtained, however, by careful analysis of those results, as the same
publication clearly showed that timed elimination of Kiss1 neurons dur-
ing the early juvenile period (i.e., before puberty onset) did prevent
normal pubertal maturation and caused infertility (Mayer and Boehm,
2011). The most plausible explanation for these two, apparently contra-
dictory sets of data is that a residual group of Kiss1 neurons remaining
after congenital ablation would be sufficient to drive puberty onset,
due to developmental compensatory mechanisms. In fact, such a devel-
opmental compensation has been previously documented in models of
early, nearly complete depletion of GnRH neurons (Herbison et al.,
2008). In contrast, depletion of Kiss1 neurons at later stages of develop-
ment would not cause similar compensatory adaptation and, hence,
lowering of the endogenous kisspeptin tone would prevent the normal
timing of puberty. All in all, genomic, expression and functional data
jointly point out that, despite the potential existence of fail-safe mech-
anisms to compensate for the early loss of kisspeptin signaling, Kiss1
neurons do play an indispensable role in the activational mechanisms
leading to puberty onset.
Kisspeptins and the metabolic control of puberty
Considering the important roles of kisspeptin signaling in the con-
trol of puberty and reproduction, and the relevance of energy homeo-
stasis and metabolic cues in pubertal regulation, especially in females,
it is not surprising that the possibility of specific functions of Kiss1
neurons in the metabolic control of puberty and the gonadotropic
axis have been thoroughly analyzed in recent years using a number
of experimental paradigms. As a potential call of caution, the majority
of those studies have focused on the consequences, in terms of Kiss1
expression and/or function, of conditions of negative energy balance
that causes some degree of pubertal suppression and/or hypogonadism,
and hence have addressed rather extreme experimental conditions
(Castellano et al., 2010a). Notwithstanding this potential limitation,
the available evidence does suggest that Kiss1 neurons are sensitive to
the metabolic/energy state of the organism and likely participate in
the modulation of the reproductive axis by metabolic cues, at least
under certain circumstances. As side comment, most of the supportive
data has been obtained in adult models; hence, extrapolation of adult
results to puberty must be made with caution.
A wealth of experimental data has now documented that extreme
conditions of negative energy balance induce a suppression of the hypo-
thalamic Kiss1 system. This has been well characterized, at the mRNA
and protein levels, in pubertal rats, in which fasting decreased hypotha-
lamic Kiss1 expression and kisspeptin immunoreactivity, in association
with a significant lowering of circulating LH levels (Castellano et al.,
2005, 2010a). A roughly similar response to fasting has been also
reported in adult female rats (Brown et al., 2008; Kalamatianos et al.,
2008) and adult male mice, in which a decrease in hypothalamic Kiss1
mRNA was detectable as soon as 12-h after beginning of food deprivation
(Luque et al., 2007). Other conditions of negative energy balance and
metabolic distress causing suppression of gonadotropic function display
also analogous inhibitory responses in terms of hypothalamic Kiss1
expression. For instance, 50% caloric restriction induced a significant
suppression of Kiss1 mRNA expression in the ARC and RP3V in adult
female rats (True et al., 2011). Similarly, uncontrolled experimental dia-
betes in rats, which is associated to hypogonadotropic hypogonadism,
causes also a lowering of Kiss1 mRNA levels at the hypothalamus in
both males and females (Castellano et al., 2006b, 2009a). Likewise,
acute inflammatory challenges, such as those induced by bacterial
lipopolysaccharide S (LPS) injection, not only provoked metabolic stress
and hypogonadotropism but also reduced hypothalamic Kiss1 mRNA ex-
pression in male rats (Castellano et al., 2010b).
Evidence for the metabolic control of the Kiss1 system does not only
come from studies in rodent species under negative energy balance
conditions. A recent study also documented that hypogonadotropic
lean ewes display lower Kiss1 mRNA levels in the ARC than their corre-
sponding normal weight counterparts (Backholer et al., 2010). This
finding suggests that a reduced kisspeptin tone in the hypothalamus
is common to conditions of energy deficit in various mammalian
species. The functional relevance of this phenomenon is confirmed by
experimental studies in rodents, where repeated administration of
kisspeptin in models of negative energy balance was sufficient to ame-
liorate the state of reproductive failure. This was the case in pubertal fe-
male rats submitted to chronic sub-nutrition (30% reduction in daily
calorie intake from day 23 onwards that prevented normal puberty
onset), in which repeated central injections of kisspeptin-10 rescued
vaginal opening as an external sign of puberty, in a notable proportion
of cases and evoked very potent gonadotropic and estrogenic responses,
despite the persistent reduction in body weights (Castellano et al.,
2005). Analogous observations have been obtained in other models of
metabolic stress in adulthood, such as uncontrolled diabetic male and
female rats. In these animals, kisspeptin-10 induced robust gonadotrop-
ic responses in spite of the lowering of the prevailing gonadotropin
levels due to the diabetic state (Castellano et al., 2009a, 2006b), and
its repeated administration substantially improved different indices of
gonadotropic function (e.g., testicular and prostate weights) and testos-
terone levels (Castellano et al., 2006b). All in all, these data indirectly
support the involvement of suppressed kisspeptin tone in the genera-
tion of the state of hypo-gonadotropism associated with conditions
of energy insufficiency, and emphasize the functional relevance of cen-
tral kisspeptin pathways in the metabolic control of puberty and
reproduction.
In addition, very recent studies in models of metabolic stress in pu-
bertal female rats have demonstrated that the hypothalamic neurokinin
B (NKB) system is also sensitive to conditions of negative energy bal-
ance. NKB is co-expressed with kisspeptins in ARC Kiss1 neurons and
defects of NKB signaling are incompatible with normal puberty onset
and fertility, as is also the case for inactivation of kisspeptin signaling
(Navarro and Tena-Sempere, 2012). For instance, by using acute fasting
as a model of metabolic stress, it has been recently shown that condi-
tions of energy deficit coupled to hypogonadotropism do not only sup-
press Kiss1 mRNA expression in the ARC and RP3V, but inhibited also
the hypothalamic expression of the genes encoding NKB and its recep-
tor (NK3R) in pubertal female rats (Navarro et al., 2012). Importantly,
the delay of female puberty caused by 30% under-nutrition during the
juvenile period was partially prevented by repeated administration of
an NKB agonist (Navarro et al., 2012). This response is very similar to
that obtained after kisspeptin-10 injection in pubertal female rats sub-
mitted to similar degree of sub-nutrition (see above and Castellano et
al., 2005). As a whole, these observations support the view that NKB sig-
naling may cooperate with kisspeptins in the metabolic control of pu-
berty, at least in the female (Navarro et al., 2012). In good agreement,
50% caloric restriction in adult rats has been shown to suppress NKB
mRNA expression in the ARC (True et al., 2011).
As final note in this section, the impact upon the hypothalamic Kiss1
system of other forms of metabolic stress due to persistent energy ex-
cess, such as in morbid obesity, remains less well characterized. Yet,
the translational relevance of such a putative influence is high, due to
(a) the escalating prevalence of obesity worldwide; and (b) the fre-
quent reproductive co-morbidities associated to obesity, which include
male and female sub/infertility, as well as altered timing of puberty
(Ahmed et al., 2009; Biro and Wien, 2010; Loret de Mola, 2009;
Rachon and Teede, 2010). Although the available data in this front are
still limited, there is indirect evidence supporting the possibility that
obesity has an impact on the Kiss1 system. First, while initial studies
 M.A. Sanchez-Garrido, M. Tena-Sempere / Hormones and Behavior 64 (2013) 187–194
in diet-induced obese male mice of the C57/B6 strain did not show sig-
nificant changes in relative Kiss1 mRNA levels in whole hypothalamic
fragments (Luque et al., 2007), these animals had dramatically
suppressed testosterone levels, and hence their hypothalamic Kiss1 ex-
pression should be considered inappropriately low (Luque et al., 2007).
In good agreement, subsequent analyses in other mouse strains, such as
the DBA/2J, more prone to develop obesity-induced infertility demon-
strated a significant suppression of kisspeptin expression in the ARC
and RP3V of females in conditions of diet-induced obesity (Quennell
et al., 2011). On the other hand, we have recently shown that early
onset overweight in female rats, which is linked to precocious occur-
rence of puberty, is associated with detectable changes in the hypotha-
lamic expression of Kiss1 and a trend for increased numbers of
kisspeptin fibers in the RP3V immediately prior the onset of puberty
(Castellano et al., 2011). The latter observations may be of help to ex-
plain the underlying mechanisms for the association between child
obesity and earlier puberty. In addition, they stress the importance of
early developmental/metabolic challenges in the timing of puberty
onset, which manifests not only in conditions of overfeeding but also
of early malnutrition. In fact, our studies in undernourished rats during
the postnatal period evidenced a suppression of the hypothalamic Kiss1
system coupled to delayed puberty (Castellano et al., 2011). This con-
tention has been recently confirmed in mice (Caron et al., 2012).
Interplay of leptin and kisspeptins in the metabolic control of
puberty: direct or indirect?
Considering the individual roles of leptin and kisspeptin pathways
in the metabolic control of puberty, it is not surprising that it was ini-
tially hypothesized that one of the major mechanisms whereby leptin
influences the timing of puberty is via its ability to regulate the hypo-
thalamic Kiss1 system. Such a potential regulatory mechanism,
which has been experimentally tested using different paradigms, is
supported by a wealth of complementary data, which can be summa-
rized as follows: (a) the mRNA encoding the functional leptin recep-
tor, namely LepR or Ob-Rb, has been detected in ARC Kiss1 neurons in
the mouse and sheep (Backholer et al., 2010; Smith et al., 2006), al-
though only a relatively modest sub-population of Kiss1 neurons
(ranging from ~ 40 to b10%, depending on the studies) does apparent-
ly express LepR in the mouse (Cravo et al., 2011); (b) leptin can di-
rectly excite ARC Kiss1 neurons in the guinea pig by activation of
TRPC channels (Qiu et al., 2011); (c) models of leptin deficiency dis-
play lower Kiss1 mRNA expression, especially in the ARC (Quennell
et al., 2011; Smith et al., 2006), and decreased numbers of kisspeptin
neurons in the RP3V (Quennell et al., 2011); and (d) leptin adminis-
tration in rodent models of leptin deficiency, due to genetic inactiva-
tion (as the ob/ob) or metabolic stress (as the uncontrolled diabetic
rats), was capable to increase the hypothalamic expression of Kiss1
gene (Backholer et al., 2010; Castellano et al., 2006b; Luque et al.,
2007; Smith et al., 2006). The later phenomenon has been also observed
after leptin injection in a sheep model of central hypogonadism
associated to leanness, as well as in murine and human neuronal cell
lines endogenously expressing Kiss1 following leptin stimulation
(Backholer et al., 2010; Luque et al., 2007; Morelli et al., 2008). Altogeth-
er, these data strongly suggested a direct mode of action of leptin in the
control of Kiss1 neurons; a view that has dominated the field until very
recently (see below). It is stressed, though, that most of the above
experimental evidence has not been obtained in specific models of pu-
berty. Yet, our data correlating the timing of puberty, circulating levels
of leptin and hypothalamic expression of the Kiss1 system indirectly
suggest a close association between higher leptin levels, increased
Kiss1/kisspeptin expression and earlier puberty onset (or vice versa)
in the female rat (Castellano et al., 2011). As a note of caution, recent
data from adult models of negative energy balance suggested that
while supra-physiological levels of leptin can prevent the state of
hypogonadotropism associated to fasting, leptin replacement to
191
physiological levels would be insufficient to normalize Kiss1 expression
and LH levels in situations of energy deficit (True et al., 2011). Whether
the above protocols of leptin replacement fully mimicked physiological
conditions and, if so, whether the same phenomenon applies to puberty
is yet to be defined.
In spite of the compelling evidence summarized above, recent ex-
perimental data have questioned the consensus of a predominant direct
mode of action of leptin on Kiss1 neurons. Thus, by the use of functional
genomics, Donato and co-workers have recently reported that congen-
ital selective elimination of LepR from Kiss1-expressing cells seems to
be compatible with preserved puberty onset and fertility (Donato et
al., 2011). It must be stressed, however, that the Kiss1 gene appears to
be widely expressed in other brain and other peripheral tissues at
early stages of development. Thus, it is possible that the selectivity of
LepR elimination in postnatal Kiss1 neurons in the hypothalamus is
compromised in that model. Moreover, it is also plausible that congen-
ital ablation of LepR from Kiss1 cells may have caused compensatory
changes that would obscure the impact of elimination of leptin signal-
ing in this neuronal population. In any event, the possibility of a pre-
dominantly indirect mode of action of leptin in Kiss1 neurons has
been reinforced by a recent independent study mapping the distribu-
tion of the functional LepRb in various hypothalamic areas. That study
could not find evidence for expression of functional LepR in GnRH or
Kiss1 neurons, except for a small population of Kiss1 cells in the ARC
(Louis et al., 2011). Of note, in the same work, an uncharacterized pop-
ulation of LepRb-expressing neurons was identified in the ARC and
RP3V, in the close vicinity of Kiss1 neurons (Louis et al., 2011). All in
all, these data suggest that a (substantial) part of the effects of leptin
on the hypothalamic expression of Kiss1/kisspeptin may be conducted
indirectly and transmitted via intermediate pathways of as yet un-
known nature (Louis et al., 2011). The physiological significance of
such an intricate, indirect mode of regulation of Kiss1/GnRH neurons
by leptin is yet to be deciphered.
Other sites of action of leptin in the metabolic control of puberty
Despite the central roles of kisspeptin pathways in the control of pu-
berty and their interplay with leptin (see above), experimental evi-
dence has been presented in recent years to document that the
reproductive effects of leptin are, at least partially, mediated by primary
actions conducted in hypothalamic areas that do not apparently contain
Kiss1 neurons. Importantly, this evidence does not refute a critical role
of kisspeptins in the metabolic control of reproduction, neither does it
rule out the possibility of interactions between these alternative circuits
and Kiss1 pathways. In fact, the latter possibility has gained momentum
given the assumption of the indirect mode of action of leptin upon Kiss1
neurons. Nonetheless, recognition and detailed characterization of such
alternative leptin-sensitive circuits are essential to obtain a complete
view of how leptin modulates the reproductive brain.
A paradigmatic example of a target area for the reproductive effects
of leptin outside Kiss1-enriched hypothalamic nuclei is the PMV. In-
deed, this nucleus had been pointed out as node for the transmission
of environmental cues to the reproductive centers well before the iden-
tification of the roles of leptin and kisspeptins (Elias and Purohit, 2013).
Recently, the PMV has emerged as an important primary target and
transmitting hypothalamic area for the permissive/stimulatory effects
of leptin on puberty and fertility (Donato et al., 2009, 2011). The main
experimental evidence supporting this role can be summarized as fol-
lows: (a) a subset of neurons in the PMV express LepR and are
depolarized (excited) after leptin stimulation; (b) bilateral excitotoxic
lesions of PMV lowered LH and estradiol levels, and prevented the
positive effects of leptin on the gonadotropic axis in fasted animals;
and (c) rescue of expression of LepR selectively in PMV neurons in a
LepR-null background was sufficient to induce puberty and improve
fertility, selectively in female mice (Elias, 2012; Elias and Purohit,
2013). Intriguingly, a similar procedure of LepR rescue in male mice
192 M.A. Sanchez-Garrido, M. Tena-Sempere / Hormones and Behavior 64 (2013) 187–194

was ineffective in terms of pubertal restoration, thus suggesting a
marked sexual dimorphism in the role of PMV in mediating the repro-
ductive actions of leptin. As a call of caution, only a fraction of PMV
neurons do express LepR, so it remains possible that part of the
reproductive phenotypes of the excitotoxic lesions described above
may derive from the damage of leptin-independent pathways. In any
event, the above observations would suggest the existence of Kiss1-
independent circuits for the transmission of leptin effects onto GnRH
neurons. Notwithstanding, neuroanatomical data suggest that neurons
from the PMV innervate Kiss1 and GnRH neurons in the preoptic area;
therefore, it is highly plausible that leptin-sensitive pathways origi-
nating from the PMV may impinge onto Kiss1 circuits for transmitting
part of the reproductive actions of leptin. A tentative model illustrating
the putative direct, indirect and independent actions of leptin on Kiss1
circuits, and thereby GnRH neurons, is provided in Fig. 1.
As additional note to this section, it is stressed that the experimental
evidence available suggests that leptin can act at levels of the gonado-
tropic axis other than the hypothalamus, including the pituitary and
the gonads. At the pituitary level, leptin has been shown to modestly
stimulate gonadotropin secretion. In the gonads, both direct stimulato-
ry and inhibitory actions of leptin on different aspects of gonadal func-
tion have been reported (Tena-Sempere, 2007; Tena-Sempere and
Barreiro, 2002). As noted earlier in this review, at high concentrations,
leptin has been documented to (paradoxically) inhibit testicular and
ovarian steroidogenesis. This phenomenon may contribute to the state
of hypogonadism frequently associated to conditions of persistent
hyperleptinemia, such as morbid obesity (Tena-Sempere, 2007). In
any event, pituitary and gonadal effects of leptin have been character-
ized mainly in adulthood; hence, the contribution of such peripheral ac-
tions to the metabolic control of puberty is yet to be defined.
Final remarks
Puberty is a complex developmental phenomenon that is sensitive to
the regulatory actions of many endogenous and environmental signals.
Among those, puberty is influenced by the metabolic and energy status
of the organism, so that deregulation of energy homeostasis and metab-
olism affects puberty, while puberty itself, and its deviations, may have
an impact of different metabolic indices and body weight. Such a tight
connection is conveyed by the concerted actions of numerous neurohor-
monal signals. In this work, we have highlighted the important contribu-
tion of the adipose hormone, leptin, and the central (hypothalamic)
circuits involving kisspeptins in the metabolic control of puberty onset.
In doing so, we have intended not only to critically review the available
evidence suggesting an important role of leptin and kisspeptins in this
phenomenon, but also to stress the unsolved questions and the experi-
mental data challenging to some extent this consensus view. Certainly,
the basis for the metabolic regulation of puberty is much more complex
than merely leptin–kisspeptin interactions, and involves the important
contribution of additional peripheral signals (e.g., ghrelin, insulin) and
central neuropeptides (e.g., NPY, melanocortins). Notwithstanding this,
it is clear that identification of the essential reproductive roles of leptin
and kisspeptins, and of their ways of interaction, has revolutionized our
understanding of how reproductive maturity is attained and of the
mechanisms whereby this complex phenomenon is finely regulated by
(and tightly coupled to) the metabolic/energy state of the organism.
Disclosure statement
The authors have nothing to disclose.
Acknowledgments
The authors are indebted with the members of the research team at
the Physiology Section of the University of Cordoba, and especially
with J.M. Castellano, V.M. Navarro, J. Roa and L. Pinilla, who actively par-
ticipated in the generation of experimental data discussed herein. The
work from the authors' laboratory summarized in this article was
supported by grants BFU 2008-00984 and BFU2011-25021 (Ministerio
de Economía y Competitividad, Spain; co-funded with EU FEDER

CNS

C PMV

A
Leptin

Kiss1
GnRH
WAT
T Neuron
Neuron
B
Intermed
Intermediate
diate
Neuron

Hypothalamus

Fig. 1. Tentative diagram for the potential mode of action of leptin on Kiss1 and/or GnRH neurons. Direct (A), indirect (B) and independent (C) actions of leptin on Kiss1 pathways,
as putative mechanism for transmission of the regulatory effects of the adipose hormone on GnRH neurons, are depicted. Note, however, that primary actions of leptin in hypotha-
lamic nuclei, such as the ventral premmamilary nucleus (PMV), initially considered as kisspeptin-independent, do not necessarily exclude the possibility of some interplay with
Kiss1 circuits, which is represented as a dotted line projection. For further details, see Interplay of leptin and kisspeptins in the metabolic control of puberty: direct or indirect?
and Other sites of action of leptin in the metabolic control of puberty sections. WAT: White adipose tissue; CNS: Central Nervous System.
 M.A. Sanchez-Garrido, M. Tena-Sempere / Hormones and Behavior 64 (2013) 187–194
funds), Project PI042082 (Ministerio de Sanidad, Spain), Project
P08-CVI-03788 (Junta de Andalucía, Spain), and EU research contract
DEER FP7-ENV-2007-1. CIBER is an initiative of Instituto de Salud Carlos
III (Ministerio de Sanidad, Spain).
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