Am J Physiol Endocrinol Metab 301: E1004–E1012, 2011.
First published August 9, 2011; doi:10.1152/ajpendo.00196.2011.
Circadian intervention of obesity development via resting-stage feeding
manipulation or oxytocin treatment
Guo Zhang and Dongsheng Cai
Department of Molecular Pharmacology and Diabetes Research Center, Albert Einstein College of Medicine, Bronx,
New York
Submitted 25 April 2011; accepted in final form 9 August 2011
oxytocin; obesity; feeding; circadian
OBESITY IS AN OUTCOME OF IMBALANCED interactions between
environmental factors and the body’s metabolic regulation (13,
38). Recent research has established that the hypothalamus in
the brain plays an essential role in the regulation of appetite
and body weight balance (9, 17, 37, 44, 57). The underlying
molecular basis involves hormone-dependent and nutrientdependent pathways in the mediobasal hypothalamus (1, 3, 4,
9, 12, 17, 25, 29, 37, 44). In addition to metabolic regulation,
the hypothalamus governs the circadian rhythms of various
physiological activities, primarily dictated by the circadian
pacemaker in the hypothalamic suprachiasmatic nucleus (14,
40, 51, 52). The circadian pace in this hypothalamic region is
predominantly exerted by the Clock-directed transcriptiontranslation oscillation loop (16, 20, 52), which can synchronize
the physiological activities of various organs, including periph-
Address for reprint requests and other correspondence: D. Cai, Dept. of
Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris
Park Ave., Bronx, NY 10461 (e-mail: [email protected]).
E1004
eral metabolic tissues (2, 53, 54). A recent study revealed that
knockout of the Clock gene led to the prominent metabolic
dysregulations, including overeating, obesity, and glucose intolerance (50). In the same direction, it was recently found that
mice fed on a high-fat diet (HFD) displayed altered diurnal
activities (26). However, despite these emerging observations,
it remains unclear how hypothalamic neuropeptide(s) are involved in the circadian control of feeding, energy expenditure,
and body weight balance and, moreover, if a brain target for
obesity intervention could be developed.
Physiological functions of the hypothalamus are ultimately
directed by hypothalamic neuropeptides and neurotransmitters.
Indeed, neuropeptide and neurotransmitter pathways in the
hypothalamic control of metabolic physiology have been depicted significantly. An important example is the hypothalamic
melanocortin system (9, 17, 37, 44, 57) in which neuropeptides, including ␣-melanocyte-stimulating hormone, agoutirelated peptide, and neuropeptide Y, are released by the arcuate
neurons to act on melanocortin 4 receptor (MC4R)-expressing
neurons in the paraventricular nucleus (PVN). Notably, one
group of MC4R-exprssing neurons in the PVN are oxytocin
(OXT) neurons, so named because they release neuropeptide
OXT (28, 47). Our recent study has revealed that the central
action of OXT in the brain is critical for feeding and body
weight balance, and dysfunction of OXT neurons can direct
obesity development (56). Here it should be pointed out that
the PVN receives afferent projections from both the arcuate
nucleus, which is the first-order metabolic regulator (3, 11, 18),
and the suprachiasmatic nucleus, which is the circadian pacemaker (23, 49). However, it remains unexplored whether and
how neuropeptide(s) released by PVN neurons might coordinate the circadian and metabolic systems to control whole body
metabolic physiology and disease. In this work, we focused on
PVN neurons and discovered that OXT critically integrates
circadian and metabolic controls of feeding. Our study provides a new mechanistic understanding for body weight control
and obesity development.
MATERIALS AND METHODS
Animals and metabolic and biochemical profiling. Adult male C57
BL/6 mice were obtained from the Jackson Laboratory. Mice were
housed under the diurnal cycle of 12 h light (resting stage) and 12 h
darkness (active stage) in standard conditions with free access to food
and water. HFD feeding was initiated when mice were 10 –12 wk old
for the indicated periods by individual experiments. Age-matched
mice maintained under chow feeding were used as dietary controls.
Food intake of mice was recorded for the indicated diurnal periods on
a daily basis. On each day at the same time, a laboratory technician
supplied individually housed mice with a preweighed amount of food,
and, at the same time on the following day, the food left over in each
cage was weighed. Extra attention was paid to collecting food left
0193-1849/11 Copyright © 2011 the American Physiological Society
http://www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on June 18, 2017
Zhang G, Cai D. Circadian intervention of obesity development
via resting-stage feeding manipulation or oxytocin treatment. Am J
Physiol Endocrinol Metab 301: E1004 –E1012, 2011. First published
August 9, 2011; doi:10.1152/ajpendo.00196.2011.—The obesity pandemic can be viewed as a result of an imbalanced reaction to changing
environmental factors. Recent research has linked circadian arrhythmicity to obesity and related diseases; however, the underlying mechanisms are still unclear. In this study, we found that high-fat diet
(HFD) feeding strikingly promoted daytime rather than nighttime
caloric intake in mice, leading to feeding circadian arrhythmicity.
Using scheduled feeding with a defined amount of daily HFD intake,
we found that an increase in the ratio of daytime to nighttime feeding
promoted weight gain, whereas a decrease of this ratio rebalanced
energy expenditure to counteract obesity. In identifying the underlying mechanism, we found that hypothalamic release of anorexigenic
neuropeptide oxytocin displayed a diurnal rhythm of daytime rise and
nighttime decline, which negatively correlated with the diurnal feeding activities of normal chow-fed mice. In contrast, chronic HFD
feeding abrogated oxytocin diurnal rhythmicity, primarily by suppressing daytime oxytocin rise. Using pharmacological experiments
with hypothalamic injection of oxytocin or oxytocin antagonist, we
showed that daytime manipulation of oxytocin can change feeding
circadian patterns to reprogram energy expenditure, leading to attenuation or induction of obesity independently of 24-h caloric intake.
Also importantly, we found that peripheral injection of oxytocin
activated hypothalamic oxytocin neurons to release oxytocin, and
exerted metabolic effects similar to central oxytocin injection, thus
offering a practical clinical avenue to use oxytocin in obesity control.
In conclusion, resting-stage oxytocin release and feeding activity
represent a critical circadian mechanism and therapeutic target for
obesity.
OXYTOCIN CIRCADIAN RHYTHM AND OBESITY
mouse that did not consume all supplied food for ⬎5% of experimental days were excluded from the final analysis.
Third-ventricle cannulation and pharmacological treatments. As
described previously (56, 57), we used a stereotaxic apparatus (David
Kopf Instruments) to place a guide cannula in the third ventricle of
anesthetized mice at the midline coordinates of 1.8 mm posterior to
the bregma and 5.0 mm below the skull surface of bregma. Mice were
allowed 2 wk for recovery and then verified for the success of surgery
using the ANG II-induced drink response. The injection of OXT or
OXT receptor antagonist [D-(CH2)5,Tyr(Me)2,Orn8]vasotocin (OVT)
was performed at the beginning of daytime vs. nighttime. For the
third-ventricle injection, individually housed mice were injected with
1 ␮g OXT (Bachem California) or 4 ␮g OVT (Bachem California)
over the period of 5 min. Artificial cerebrospinal fluid was used as
vehicle control. To evaluate the potential anti-obesity effect of OXT
via peripheral administration (7, 8, 31, 33, 43), which was shown to
partially penetrate across the blood-brain barrier in the literature (7, 8,
31, 33, 41, 43), mice with HFD-induced obesity received daily
intraperitoneal OXT injection at the beginning of daytime using the
dose (1 mg/kg) established in the literature. Similarly, to evaluate the
potential obesogenic effect of blocking systemic OXT, normal chowfed mice received daily intraperitoneal injection of OVT at the same
dose (1 mg/kg). Body weight-matched mice with intraperitoneal
injection of saline were used as controls.
Ex vivo OXT release assay. A detailed protocol has been described
previously (56). Briefly, PVN slices were dissected from the hypothalamus and incubated in the Locke solution supplied with 95% O2
and 5% CO2 at 37°C. Locke solution was changed every 5 min for 10
times. The 10th balancing solution was collected for the measurement
Fig. 1. Feeding circadian rhythm affects body weight independently of caloric intake. A: longitudinal monitoring of daytime (D) vs. nighttime (N) caloric intake
in C57BL/6 mice fed normal chow vs. a high-fat diet (HFD) starting at an adult age (⬃10 wk old). The data at each time point represent the average daily food
intake of each mouse during the week and then averaged per group. B–G: starting at an adult age (⬃10 wk), subgroups of male C57BL/6 mice received HFD
(H) feeding with daytime restriction (DR) vs. nighttime restriction (NR), whereas the 24-h HFD intake was controlled at the same level. Age-matched male mice
under ad libitum (AL) chow (C) feeding or HFD feeding were used as negative and positive controls, respectively. B–D: average daytime (B, left) vs. nighttime
(B, right) caloric intake, average total daily caloric intake (C, left), the diurnal ratio (daytime caloric intake divided by nighttime caloric intake) (C, right), and
longitudinal follow-up of body weight (D) during the 8-wk intervention period. E–G: mice were measured for oxygen consumption (E) and physical activities
(F) by metabolic chambers and lean vs. fat mass composition by magnetic resonance imaging (MRI, G) at 3– 4 wk following scheduled feeding. Data on oxygen
consumption shown in E were corrected based on the lean mass of mice. A–G: *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 10⫺4; n ⫽ 6 –11 (A–D) and n ⫽ 4 (E–G)
mice/group. Statistics in A indicate the comparisons between HFD-fed mice and chow-fed mice with matched diurnal cycles. Statistics in D indicate the
comparisons between HFD-fed mice under DR vs. NR. Error bars represent means ⫾ SE. H, HFD; C, chow.
AJP-Endocrinol Metab • VOL
301 • NOVEMBER 2011 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on June 18, 2017
over both on the cage frame as well as all visible food left in the
bedding of each cage. New clean cages were used on a daily basis for
each mouse. Body weight was measured at least two times a week.
Oxygen consumption was measured using an open-circuit indirect
calorimetry system (Columbus Instruments) and normalized by lean
body mass, which was determined by magnetic resonance imaging
(MRI). The calorimetry system also measured physical activities of
mice based on the infrared beam interruptions in both horizontal and
vertical directions. Glucose tolerance test was performed in overnightfasted mice by intraperitoneal injection of glucose (2 g/kg body wt)
and subsequent monitoring of tail vein glucose levels via a Glucometer (Bayer, Elkhart, IN) at the indicated time points. Serum OXT
levels were measured using the Oxytocin EIA kit (Assay Design).
HFD was purchased from Research Diets. All procedures were approved by the Institutional Animal Care and Use Committee at Albert
Einstein College of Medicine.
Scheduled feeding. A defined amount of food was provided to
individually housed mice immediately after the light was turned on or
right before the light was turned off and maintained for 12 h daytime
vs. nighttime. Two schemes of scheduled HFD feeding were simultaneously performed: 0.48 g for 12 h daytime and 1.92 g for 12 h
nighttime; and 0.8 g for 12 h daytime and 1.6 g for 12 h nighttime. At
the same time, matched mice under ad libitum HFD or chow feeding
were included as positive and negative controls, respectively. The
total, 24-h amount of HFD in the schedule feeding was 2.4 g, slightly
lower than the amount of ad libitum HFD feeding, to ensure complete
consumption of the designed HFD amount for the light vs. dark cycle.
New clean cages were used every 12-h diurnal cycle so that food that
mice had was accurate for both daytime and nighttime periods. Any
E1005
E1006
OXYTOCIN CIRCADIAN RHYTHM AND OBESITY
fat mass expansion following the first 3⬃4 wk of HFD feeding
(Fig. 1G). Thus, the diurnal pattern of caloric intake is critical for
body weight homeostasis, and this process can be dissociated
from total 24-h caloric intake, with the underlying physiology
involving reprogrammed energy expenditure homeostasis.
Diurnal OXT release correlates with feeding circadian patterns and body weight. We subsequently explored the potential
hypothalamic basis for the observations shown in Fig. 1. Our
attention was directed to the PVN because it represents the
converging site that integrates the metabolic regulatory neurons in the arcuate nucleus (3, 18) and the circadian pacemaker
neurons in the suprachiasmatic nucleus (23, 49). Although
diurnal rhythmicity of several PVN neuropeptides, such as
vasopressin, thyrotropin-releasing hormone, corticotropin-releasing factor, and somatostatin, were documented in the literature (5, 21, 22, 24), the primary actions of these neuropeptides are to control various physiological activities other than
feeding. Interestingly, we found that OXT, a PVN neuropeptide that we recently found to critically regulate feeding and
body weight (56), exhibited diurnal rhythmicity in mice. The
circulating concentrations of OXT in chow-fed mice displayed
a rapid daytime rise and a gradual nighttime decline (Fig. 2A).
The diurnal pattern of OXT change was correlated with the
RESULTS
Feeding circadian rhythm affects body weight independently
of caloric intake. Total 24-h caloric intake is obviously relevant to body weight and obesity, but the effect of circadian
rhythm on 24-h caloric intake has received little attention. In
mice maintained on a standard chow diet, we confirmed that a
normal range of body weight was supported by a steady-state
caloric intake ratio of 0.2 ⫾ 0.05 between the daytime (resting)
stage and the nighttime (active) stage. In contrast, in mice with
HFD-induced obesity, we observed a striking and coursedependent increase in the daytime-to-nighttime caloric intake
ratio (Fig. 1A). However, the nighttime caloric intake between
HFD-fed and chow-fed mice differed only modestly, and the
progressive increase of daytime caloric intake primarily accounts
for the altered diurnal rhythm of caloric intake in HFD-fed mice
(Fig. 1A). To examine the significance of feeding circadian
rhythm in obesity, we performed scheduled HFD feeding to
artificially manipulate the diurnal ratio of caloric intake based on
a fixed amount of 24-h calorie intake. Over a 2-mo experimental
period, two groups of mice consumed the same daily amount of
calories, which is 14% higher than ad libitum chow feeding and
11% lower than ad libitum HFD feeding, but with a different
daytime-to-nighttime ratio of calorie intake at 0.50 and 0.25
(defined as the daytime caloric intake divided by nighttime caloric
intake), respectively (Fig. 1, B and C). We found that, despite the
equal amount of 24-h caloric intake, mice with a normal daytimeto-nighttime ratio of calorie intake were resistant to obesity development, whereas mice with an elevated daytime-to-nighttime
ratio of calorie intake were susceptible to obesity development
(Fig. 1D). To understand the underlying metabolic physiology, we
profiled energy expenditure levels of these mice using metabolic
chambers. We found that a normal daytime-to-nighttime ratio of
calorie intake is associated with elevated energy expenditure
levels during both daytime and nighttime periods (Fig. 1E);
however, the physical activities were not affected significantly
(Fig. 1F). MRI analysis further confirmed that mice with a normal
ratio of daytime to nighttime caloric intake were protected from
AJP-Endocrinol Metab • VOL
Fig. 2. Diurnal profiles of oxytocin (OXT) release in chow-fed and HFD-fed mice.
Adult male C57BL/6 mice received HFD vs. chow feeding from an adult age (⬃10
wk) for 12 wk and then were measured for 24-h profile of serum OXT levels (A) or
ex vivo OXT release under basal and depolarization (KCl) conditions as described in
MATERIALS AND METHODS (B). *P ⬍ 0.05; n ⫽ 8 –10 (A) and n ⫽ 6 –7 (B)
mice/group. Error bars represent means ⫾ SE. Statistics in A indicate the comparisons between HFD-fed mice and chow-fed mice at the matched time points.
301 • NOVEMBER 2011 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on June 18, 2017
of basal OXT release. The tissues were subsequently incubated in
Locke solution containing 70 mM KCl for 5 min. The solution was
collected and measured for the depolarization (KCl)-stimulated OXT
release. An OXT EIA kit (Assay Design) was used to determine OXT
concentrations in the solution. To evaluate the effect of intraperitoneal
OXT injection on hypothalamic OXT release, the PVN slices were
prepared at 30 min following intraperitoneal OXT injection and
analyzed using the protocol above.
Immunofluorescence. Chow-fed mice were injected OXT or saline
intraperitoneally, and, at 30 min postinjection, mice were anesthetized
and intracardially perfused with 4% paraformaldehyde (PFA). Brains
were then dissected, further fixed in 4% PFA for 4 h, and sequentially
incubated in 20 and 30% sucrose. Brains were sectioned under a
cryostat at 20-␮m thickness, and sections were rinsed, blocked, and
incubated with guinea pig antibody against OXT (Bachem California),
rabbit antibody against c-Fos (Santa Cruz Biotechnology), or monoclonal antibody against HuC/D (Invitrogen) overnight at 4°C. After
subsequent reaction with Alexa fluor 488, 555, or 633 secondary
antibodies (Invitrogen), sections were mounted with DAPI-containing
Vectashield medium. Image acquisitions were performed using a
confocal microscope.
Statistical analysis. Data are presented as means ⫾ SE. ANOVA
and post hoc Bonferroni test were performed for data involving more
than two groups, and Student’s t-test was used for data that involved
only two groups. P ⬍ 0.05 was considered significant.
OXYTOCIN CIRCADIAN RHYTHM AND OBESITY
injection of OXT at the beginning of daytime or nighttime. As
we previously observed (56), the effect of OXT injection on
food intake lasted ⬃6 h, so we were able to specifically control
daytime vs. nighttime feeding of mice by daytime vs. nighttime
OXT injection (Fig. 3, A and B). Although total 24-h caloric
intake was reduced similarly between daytime vs. nighttime
OXT treatment groups (Fig. 3C), the daytime-to-nighttime
ratio of feeding was affected differentially by different OXT
treatments, specifically, it was decreased by daytime OXT
treatment but increased by nighttime OXT treatment (Fig. 3D).
Such diurnal rhythmic change of feeding mimicked the model
of scheduled feeding shown in Fig. 1. Similar to the effect of
scheduled feeding, we found that daytime OXT administration
exerted stronger anti-obesity effect compared with nighttime
OXT administration (Fig. 3, E–G). We also observed that
energy expenditure of mice was increased by daytime but not
nighttime OXT treatment (Fig. 3, H and I), which further
explains the potentiated anti-obesity effect of daytime OXT
treatment. Taken together, OXT can act in the brain to affect
the diurnal ratio of caloric intake and thereby readjusts the set
point of energy expenditure to counteract against obesity.
Central delivery of OVT disrupts feeding circadian rhythm
to promote obesity. In addition to OXT gain-of-function experiments above, we tested whether blocking the brain action
of OXT could have opposite metabolic effects. To do this,
OVT, an effective antagonist of OXT (56), was delivered to the
brain of chow-fed mice via the third ventricle at the beginning
of daytime vs. nighttime. First, total caloric intake was similarly although slightly elevated by daytime vs. nighttime OVT
treatment (Fig. 4, A–C). Importantly, the daytime-to-nighttime
Fig. 3. Central OXT delivery normalizes feeding circadian rhythm to counteract obesity. Adult male C57BL/6 mice received HFD feeding from an adult age (⬃10
wk) for 12 wk. Subsequently, mice with matched body weight were subdivided into 3 groups to receive the following treatments. The group designated as “OXT,
day injection (inj)” received OXT injection at the beginning of daytime and vehicle injection at the beginning of nighttime (n). The group designated as “OXT,
night injection (inj)” received OXT injection at the beginning of nighttime and vehicle at the beginning of daytime. A matched control group received vehicle
injection at the beginning of both daytime and nighttime. Mice received these treatments on a daily basis for 1 wk. During these treatments, mice continued to
be maintained on HFD feeding. A–D: accumulated weekly daytime caloric intake (A), nighttime caloric intake (B), total caloric intake (C), and the diurnal ratio
of feeding (daytime caloric intake divided by nighttime caloric intake) (D) were obtained from these mouse groups. E–I: body wt (E), fat vs. lean mass measured
by MRI (F and G), and oxygen consumption measured by metabolic chambers (H and I) were obtained at the end of the 1-wk treatment. Data on oxygen
consumption shown in H and I were corrected based on the lean mass of mice. A–I: *P ⬍ 0.05 and **P ⬍ 0.01; n ⫽ 5– 6 (A–G) and n ⫽ 4 (H and I) mice/group.
Error bars represent means ⫾ SE.
AJP-Endocrinol Metab • VOL
301 • NOVEMBER 2011 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on June 18, 2017
low-level daytime feeding vs. the high-level nighttime feeding
activity in mice (Fig. 1A). This finding fits well with our recent
study showing that OXT acts in the brain to restrict food intake
and promote energy expenditure (56). In contrast, the diurnal
rhythmic change of OXT was abolished in mice with HFDinduced obesity (Fig. 2A). Specifically, chronic HFD feeding
abrogated the daytime rise of circulating OXT levels (Fig. 2A).
To determine whether the abrogation of circadian OXT fluctuation was caused by a defect of daytime OXT release or
nighttime OXT clearance, we examined ex vivo OXT release
from PVN slices prepared from mice killed at midday vs.
midnight. As shown in Fig. 2B, depolarization-induced hypothalamic OXT release was active during the daytime but
completely absent during the nighttime in chow-fed mice.
However, in mice with HFD-induced obesity, depolarization
was unable to stimulate either daytime or nighttime hypothalamic OXT release (Fig. 2B). Altogether, diurnal OXT release
is correlated with a feeding circadian pattern to control body
weight balance, and loss of the diurnal OXT release rhythm by
HFD feeding underlies feeding circadian arrhythmicity and
obesity development.
Central OXT delivery normalizes feeding circadian rhythm
to counteract obesity. Having shown that daytime OXT release
and the associated daytime caloric intake are critical factors in
preventing obesity in mice, we next investigated the brain
mechanism by which the daytime fraction of OXT contributes
to the circadian regulation of feeding and body weight. To
address this question, we first applied ⬃12 wk HFD feeding to
C57BL/6 mice to induce obesity. Following the development
of HFD-induced obesity, mice received intrathird ventricle
E1007
E1008
OXYTOCIN CIRCADIAN RHYTHM AND OBESITY
ratio of feeding was increased significantly by daytime but not
nighttime OVT treatment (Fig. 4D), partially mirroring the
observations from daytime vs. nighttime OXT treatment shown
in Fig. 3. Notably, daytime OVT treatment significantly increased body weight and fat mass; however, these effects were
not evident in the nighttime OVT treatment group (Fig. 4,
E–G). Also, while mice receiving daytime OVT injection
showed a reduction in energy expenditure, mice with nighttime
OVT injection did not (Fig. 4, H and I). Overall, these findings
further support the hypothesis that diurnal rhythmicity of
feeding is critical for maintenance of normal body weight
balance and prevention against obesity development.
Peripheral OXT administration stimulates OXT release in
the PVN. In conjunction with the studies employing brain OXT
injection, we examined whether alternative peripheral OXT
administration could have metabolic effects via the central
nervous system. To answer this question, we injected OXT vs.
control vehicle (saline) intraperitoneally in normal chow-fed
C57BL/6 mice. Using c-Fos induction as a neuronal activity
marker, we examined if peripheral OXT delivery could potentially activate some brain neurons. Data revealed that a single
intraperitoneal injection of OXT markedly induced c-Fos expression in magnocellular and parvocellular OXT neurons
within the PVN (Fig. 5, A–C), indicating that peripheral OXT
treatment can stimulate OXT neurons in the PVN. To test the
functional significance of this activation, we performed ex vivo
OXT release assay using PVN slices prepared from mice that
had received intraperitoneal injection of OXT vs. saline. Results showed that OXT release in the PVN was enhanced
significantly by peripheral OXT injection compared with
vehicle injection (Fig. 5D). To summarize, peripheral OXT
treatment can stimulate the neuronal activity and neuropepAJP-Endocrinol Metab • VOL
tide release of OXT neurons in the PVN. This finding can
suggest a useful strategy to manipulate central OXT actions
via peripheral OXT injection. Such a strategy may be
clinically significant since intranasal delivery of OXT has
been recently proven successful in treating neurological
diseases such as autism (27).
Peripheral OXT treatment activates brain neural pathway
downstream of OXT. Based on the results that peripheral OXT
can stimulate hypothalamic OXT release (Fig. 5), we further
asked whether peripheral OXT delivery can mimic the central
action of OXT to activate OXT-targeting neurons in the brain.
OXT-targeting neurons are widely distributed in the brain, and
two major regions that are directly innervated by OXT neurons
are the ventral medial nucleus in the hypothalamus (VMH) (48,
55) and the nucleus of solitary tract (NTS) in the brain stem
(32). Using c-Fos induction to report neuronal activation, we
found that a significant portion of neurons in the VMH and
NTS was activated by peripheral OXT injection (Fig. 6, A–C).
For control purposes, we also examined a few other brain
regions, such as the dorsal medial nucleus in the hypothalamus,
which has no neural connections with OXT neurons, and found
no induction of c-Fos in these regions by peripheral OXT
treatment (data not shown). Thus, the activation of OXTtargeting neurons was not a nonspecific reaction to peripheral
drug delivery. In summary, by enhancing central OXT release,
peripheral OXT delivery can reproduce, at least partially, the
biological effects of central OXT action.
Counteracting peripheral OXT distorts feeding circadian
rhythm to promote obesity. Subsequently, we asked if the
endogenous peripheral OXT has physiological significance in
maintaining diurnal feeding rhythm and body weight balance.
To answer this question, we performed intraperitoneal injec-
301 • NOVEMBER 2011 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on June 18, 2017
Fig. 4. Central delivery of [D-(CH2)5,Tyr(Me)2,Orn8]vasotocin (OVT) disrupts feeding circadian rhythm to promote obesity. Normal chow-fed, adult (⬃12 wk
old) male C57BL/6 mice with matched body weight were subdivided into the 3 groups described in the legend for Fig. 3. During these treatments, mice continued
to be maintained on chow feeding. A–D: accumulated weekly daytime caloric intake (A), nighttime caloric intake (B), total caloric intake (C), and the diurnal
ratio of feeding (daytime caloric intake divided by nighttime caloric intake) (D) were obtained from these mouse groups. E–I: body wt (E), fat vs. lean mass
measured by MRI (F and G), and oxygen consumption measured by metabolic chambers (H and I) were obtained at the end of the 1-wk treatment. Data on oxygen
consumption shown in H and I were corrected based on the lean mass of mice. A–I: *P ⬍ 0.05; n ⫽ 5–9 (A–G) and n ⫽ 4 (H and I) mice/group. Error bars
represent means ⫾ SE.
OXYTOCIN CIRCADIAN RHYTHM AND OBESITY
E1009
Fig. 5. Peripheral OXT injection activates OXT neurons in the paraventricular
nucleus (PVN) to release OXT. A: chow-fed adult male C57BL/6 mice
received a single intraperitoneal (IP) injection of OXT vs. control vehicle. At
30 min postinjection, mice were trans-heart perfused and fixed, and brains
were removed and sectioned for coimmunostaining of c-Fos (red) and OXT.
Merged micrographs demonstrate cytoplasmic OXT and nuclear c-Fos. Immunostaining for neuronal marker HuC/D (pink) demonstrates the overall anatomy of the PVN. Bar⫽20 ␮m. 3V, third ventricle. Insets: high-magnification
images of representative magnocellular (Mag) and parvocellular (Par) OXT
neurons; bar⫽5 ␮m. B and C: average nos. of c-Fos-positive cells in the PVN
(B) and averaged percentage of total, magnocellular (Mag), and parvocellular
(Par) populations of OXT neurons in the PVN sections that were positive for
c-Fos immunostaining (C). Data represent the analyses for median PVN
sections. *P ⬍ 0.05 and **P ⬍ 0.01; n ⫽ 3–5/group. Error bars represent
means ⫾ SE. D: chow-fed adult male C57BL/6 mice received a single ip
injection of OXT vs. control vehicle. At 30 min postinjection, mice were
killed, and PVN slices were prepared from the brain and subjected to ex vivo
OXT release assay as described in MATERIALS AND METHODS. *P ⬍ 0.05, n ⫽
6 –7/group. Error bars represent means ⫾ SE.
tion of OXT antagonist OVT or control vehicle to body
weight-matched, chow-fed C57BL/6 mice. Injection was given
at the beginning of the daytime period. As shown in Fig. 7, A
and B, OVT significantly increased daytime but not nighttime
caloric intake. The 24-h total caloric intake was only slightly
altered, but the ratio of daytime to nighttime food intake was
elevated significantly (Fig. 7C). Thus, peripheral OVT injection partially recapitulated the changes of feeding circadian
pattern in response to central OVT injection, as shown in Fig.
4. Similar to the obesogenic effect of central OVT injection
AJP-Endocrinol Metab • VOL
Fig. 6. Peripheral OXT injection activates the brain pathway of OXT neurons.
Chow-fed adult male C57BL/6 mice received a single ip injection of OXT vs.
control vehicle. At 30 min postinjection, mice were trans-heart perfused and
fixed, and the brains were removed and sectioned for immunostaining of c-Fos
(red) in the ventromedial hypothalamic (VMH) nucleus (A) and nucleus of the
solitary tract (NTS, B). Nuclear staining by DAPI reveals all cells in the
sections. Bar graphs (C) present the average nos. of c-Fos-positive cells in
the VMH and NTS. Data represent the cell nos. across the median section of
the VMH or NTS. *P ⬍ 0.05 and **P ⬍ 0.01; n ⫽ 3–5/group. Error bars
represent means ⫾ SE. ARC, arcuate nucleus; AP, area postrema; CC, central
canal; Veh, vehicle. Bar⫽20 ␮m.
301 • NOVEMBER 2011 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on June 18, 2017
(Fig. 4E), mice receiving peripheral OVT treatment gained
more body weight compared with vehicle treatment (Fig. 7D).
These data further confirmed that the resting-stage caloric
profile determines the circadian homeostasis of feeding and
body weight control, and the underlying regulatory mechanism
is the circadian programming of the central-peripheral OXT
axis.
Peripheral OXT treatment normalizes the feeding circadian
pattern to counteract obesity. Finally, we examined if peripheral administration of OXT can mimic central OXT treatment
to normalize the feeding circadian pattern and therefore counteract obesity. HFD-fed, adult C57BL/6 mice received intraperitoneal injection of OXT vs. control vehicle at the beginning
of daytime on a daily basis for 6 wk. Results showed that OXT
administration significantly reduced daytime but not nighttime
caloric intake (Fig. 8, A and B). Thus, despite the HFD feeding,
E1010
OXYTOCIN CIRCADIAN RHYTHM AND OBESITY
lend support to the metabolic benefit of OXT treatment in
obesity control.
DISCUSSION
peripheral OXT treatment normalized the feeding circadian
rhythm compared with vehicle treatment (Fig. 8C). Over a
6-wk follow-up period, OXT-injected mice showed a significant protection against HFD-induced obesity (Fig. 8D). In
addition to body weight monitoring, mice were subjected to a
glucose tolerance test at the end of the 6-wk treatment period.
As shown in Fig. 8E, control mice developed glucose intolerance, a major obesity comorbidity that often leads to the
development of type 2 diabetes. In contrast, OXT-treated mice
showed a decreased extent of glucose intolerance at 30 min
postglucose challenge in HFD-fed mice. This observation can
Fig. 8. Peripheral delivery of OXT normalizes feeding circadian pattern to counteract obesity. Adult male C57BL/6 mice received HFD feeding from an adult
age (⬃8 wk) for 6 wk. Subsequently, mice with matched body weight were divided into two groups to receive ip injection of OXT vs. vehicle at the beginning
of daytime on a daily basis for 6 wk. Mice continued to be maintained on HFD feeding during the entire treatment period. Data shown are accumulated weekly
daytime (A) vs. nighttime (B) caloric intake, the diurnal ratio of feeding (C) and longitudinal follow-up of body wt (D) and glucose tolerance test (GTT) (E) at
the completion of the 6-wk treatment. *P ⬍ 0.05; n ⫽ 5– 8/group. Error bars represent means ⫾ SE.
AJP-Endocrinol Metab • VOL
301 • NOVEMBER 2011 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on June 18, 2017
Fig. 7. Peripheral delivery of OVT distorts feeding circadian rhythm to
promote obesity. Chow-fed adult male C57BL/6 mice with matched body
weight were divided into two groups to receive ip injection of OVT vs. vehicle
at the beginning of daytime on a daily basis for 1 wk. Data shown are
accumulated weekly daytime (A) vs. nighttime (B) caloric intake and diurnal
ratio of feeding (C) during the 1-wk treatment and body weight (D) following
the completion of the 1-wk treatment. *P ⬍ 0.05; n ⫽ 5–7/group. Error bars
represent means ⫾ SE.
OXT-directed resting-stage feeding mediates circadian control of body weight balance. The 24-h rotation of the earth
generates daily circadian rhythms. To maintain a healthy body
weight, living organisms have evolutionally developed a strategy to synchronize levels of caloric intake with the day-night
cycle and the concurrent physical activity-resting cycle. In this
study, we discovered that circadian release of OXT from the
hypothalamic PVN directs hypothalamic regulation of feeding
circadian rhythms to maintain body weight homeostasis. Disturbance of this control system underlies obesity development.
Indeed, environmental overnutrition, such as chronic fat-enriched diet, is a major contributor to an impaired circadian
release pattern of OXT, leading to altered feeding rhythms and
body weight imbalance. However, using pharmacological or
nutritional (scheduled feeding) models, we were able to treat
HFD-induced body weight changes roughly by 50% through
normalizing feeding circadian rhythm even without much
change in the total, 24-h caloric intake. More importantly,
advancing from recent observations that both caloric excess
(26) and caloric shortage (19) impact feeding circadian
rhythms, we further proved that the resting stage (daytime for
mice, nighttime for humans), the period that is characterized by
limited caloric intake and physical activity, is the critical
component for controlling feeding circadian rhythms. Our
finding is corroborated by previous research showing that a
phase delay in eating is associated with obesity despite the
relatively normal amount of total 24-h caloric intake (39).
From a medical viewpoint, such phase delay of eating is most
often seen in patients with night-eating syndrome, who typically develop morbid obesity and need medicinal or surgical
interventions to lose weight (10). Another human subjectbased study showed that scheduled nighttime meal intake
resulted in reduced diet-induced thermogenesis compared with
morning meal intake (42), indicating that energy expenditure
status can be entrained by the feeding circadian pattern to cause
or prevent obesity. We found that resting-stage release of
hypothalamic OXT underlies diurnal feeding rhythmicity to
OXYTOCIN CIRCADIAN RHYTHM AND OBESITY
AJP-Endocrinol Metab • VOL
the literature (36, 46). Second, peripherally injected OXT
might employ afferent neural pathway(s) to activate various
brain regions, including the hypothalamic OXT neurons, resulting in elevated OXT release from OXT neurons. Regardless of the underlying mechanisms, our data showed that
peripherally delivered OXT indeed upregulated OXT release
by OXT neurons in the PVN in mice. Overall, while native
OXT does have limitations for direct use in patients with
obesity, such as the short half-life and possible off-target
effects, our study can provide a preclinical basis for next-step
development of OXT analogs with optimized pharmacological
properties and target actions as an approach for treating human
obesity or related diseases.
ACKNOWLEDGMENTS
We thank D. Cai’s laboratory members for technical assistance.
GRANTS
This study was supported by the internal support of Albert Einstein College
of Medicine, National Institutes of Health Grants R01 DK-078750 and R01
AG-031774 (all to D. Cai).
DISCLOSURES
The authors state that they have no competing financial interests.
REFERENCES
1. Balthasar N, Dalgaard LT, Lee CE, Yu J, Funahashi H, Williams T,
Ferreira M, Tang V, McGovern RA, Kenny CD, Christiansen LM,
Edelstein E, Choi B, Boss O, Aschkenasi C, Zhang CY, Mountjoy K,
Kishi T, Elmquist JK, Lowell BB. Divergence of melanocortin pathways
in the control of food intake and energy expenditure. Cell 123: 493–505,
2005.
2. Bass J, Takahashi JS. Circadian integration of metabolism and energetics. Science 330: 1349 –1354, 2010.
3. Bouret SG, Draper SJ, Simerly RB. Trophic action of leptin on hypothalamic neurons that regulate feeding. Science 304: 108 –110, 2004.
4. Bruning JC, Gautam D, Burks DJ, Gillette J, Schubert M, Orban PC,
Klein R, Krone W, Muller-Wieland D, Kahn CR. Role of brain insulin
receptor in control of body weight and reproduction. Science 289: 2122–
2125, 2000.
5. Burhol PG, Lygren I, Jenssen TG, Florholmen J, Jorde R. Somatostatin release and plasma molecular somatostatin components in man. Acta
Physiol Scand 121: 223–228, 1984.
6. Camerino C. Low sympathetic tone and obese phenotype in oxytocindeficient mice. Obesity (Silver Spring) 2009.
7. Carson DS, Cornish JL, Guastella AJ, Hunt GE, McGregor IS.
Oxytocin decreases methamphetamine self-administration, methamphetamine hyperactivity, and relapse to methamphetamine-seeking behaviour
in rats. Neuropharmacology 58: 38 –43, 2010.
8. Carson DS, Hunt GE, Guastella AJ, Barber L, Cornish JL, Arnold
JC, Boucher AA, McGregor IS. Systemically administered oxytocin
decreases methamphetamine activation of the subthalamic nucleus and
accumbens core and stimulates oxytocinergic neurons in the hypothalamus. Addict Biol 15: 448 –463, 2010.
9. Coll AP, Farooqi IS, O’Rahilly S. The hormonal control of food intake.
Cell 129: 251–262, 2007.
10. Colles SL, Dixon JB, O’Brien PE. Night eating syndrome and nocturnal
snacking: association with obesity, binge eating and psychological distress. Int J Obes (Lond) 31: 1722–1730, 2007.
11. Cone RD. Anatomy and regulation of the central melanocortin system.
Nat Neurosci 8: 571–578, 2005.
12. Cota D, Proulx K, Smith KA, Kozma SC, Thomas G, Woods SC,
Seeley RJ. Hypothalamic mTOR signaling regulates food intake. Science
312: 927–930, 2006.
13. Cummings DE, Schwartz MW. Genetics and pathophysiology of human
obesity. Annu Rev Med 54: 453–471, 2003.
14. Dibner C, Schibler U, Albrecht U. The mammalian circadian timing
system: organization and coordination of central and peripheral clocks.
Annu Rev Physiol 72: 517–549, 2010.
301 • NOVEMBER 2011 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on June 18, 2017
maintain normal body weight. Conversely, impairment of resting-stage hypothalamic OXT release mediates diurnal feeding
arrhythmicity to cause obesity. We recognize that several other
PVN neuropeptides, such as vasopressin, thyrotropin-releasing
hormone, corticotropin-releasing factor, and somatostatin,
were known to have diurnal rhythmicity (5, 21, 22, 24). While
the primary actions of these neuropeptides are to control
various physiological activities other than feeding, they may
secondarily contribute to the action of OXT in the regulation of
metabolic rhythmicity. Overall, our data suggest that deviation
from the normal circadian eating pattern, a phenomenon prevalent with modern society’s lifestyle, is an important risk factor
for obesity independent of total daily caloric intake, and the
underlying neurobiological basis is disrupted hypothalamic
OXT rhythmicity.
Therapeutic potentials of OXT mimetics for obesity and
related diseases. OXT is well known for its actions in mediating female reproductive activities during parturition and
lactation. However, OXT is similarly abundant in the brain and
circulation of males and females, suggesting that OXT has
functions beyond female reproduction. Recent literature has
linked OXT to promoting social behaviors (15) and anxiolytic
effects (30). More recently, we have reported that OXT can act
in the brain to restrict food intake and promote energy expenditure in mice (56). Importantly, these metabolic effects of
central OXT treatment did not cause adverse behavioral
changes, rationalizing OXT as an ideal candidate as an antiobesity therapeutic (56). In this context, the current research
aimed to gain further mechanistic understanding of central
OXT actions in metabolic regulation. We discovered that brain
OXT employs a circadian system to control the diurnal feeding
pattern and body weight balance. This metabolic regulation by
OXT is in line with the recent appreciation that obesity can
result from circadian arrhythmicity (50). It is also well supported by the obesity phenotype of OXT (6) or OXT receptor
(45) knockout mice. As a matter of fact, circadian rhythmicity
is a shared feature of many social behaviors recently found to
be regulated by OXT (15), further supporting the circadian
component of OXT actions. Our current study demonstrated
that central OXT treatment can counteract obesity by normalizing diurnal feeding rhythmicity. This finding can inspire
future studies regarding OXT and diseases associated with
circadian arrhythmicity. One limitation of our study was that
we have not been able to test the potential effects of multiple
OXT injections within 24 h or continuous OXT delivery
through osmotic minipump. In addition to central administration, this study demonstrated that the peripheral OXT delivery
can effectively mimic the central OXT delivery to affect
feeding diurnal rhythm, body weight, and obesity. The underlying processes might involve several mechanisms. First, a
portion of the peripherally delivered OXT may cross the
blood-brain barrier. According to the literature (33), peripheral
OXT injection at pharmacological doses can deliver a small
fraction of OXT in the central nervous system across the
blood-brain barrier, and, even though the absolute transferred
amount was small (⬃0.002%), it transiently elevated OXT
concentrations in the cerebrospinal fluid by approximately
fivefold. Also importantly, we found in the current study that
exogenously provided OXT further promoted hypothalamic
OXT release presumably via the autocrine feedforward system
of OXT neurons, which was also indicated by observations in
E1011
E1012
OXYTOCIN CIRCADIAN RHYTHM AND OBESITY
AJP-Endocrinol Metab • VOL
39. O’Reardon JP, Ringel BL, Dinges DF, Allison KC, Rogers NL,
Martino NS, Stunkard AJ. Circadian eating and sleeping patterns in the
night eating syndrome. Obes Res 12: 1789 –1796, 2004.
40. Reppert SM, Weaver DR. Coordination of circadian timing in mammals.
Nature 418: 935–941, 2002.
41. Ring RH, Malberg JE, Potestio L, Ping J, Boikess S, Luo B, Schechter
LE, Rizzo S, Rahman Z, Rosenzweig-Lipson S. Anxiolytic-like activity
of oxytocin in male mice: behavioral and autonomic evidence, therapeutic
implications. Psychopharmacology (Berl) 185: 218 –225, 2006.
42. Romon M, Edme JL, Boulenguez C, Lescroart JL, Frimat P. Circadian
variation of diet-induced thermogenesis. Am J Clin Nutr 57: 476 –480,
1993.
43. Schorscher-Petcu A, Sotocinal S, Ciura S, Dupre A, Ritchie J, Sorge
RE, Crawley JN, Hu SB, Nishimori K, Young LJ, Tribollet E, Quirion
R, Mogil JS. Oxytocin-induced analgesia and scratching are mediated by
the vasopressin-1A receptor in the mouse. J Neurosci 30: 8274 –8284,
2010.
44. Schwartz MW, Porte D Jr. Diabetes, obesity, and the brain. Science 307:
375–379, 2005.
45. Takayanagi Y, Kasahara Y, Onaka T, Takahashi N, Kawada T,
Nishimori K. Oxytocin receptor-deficient mice developed late-onset obesity. Neuroreport 19: 951–955, 2008.
46. Theodosis DT. Oxytocin-immunoreactive terminals synapse on oxytocin
neurones in the supraoptic nucleus. Nature 313: 682–684, 1985.
47. Tolson KP, Gemelli T, Gautron L, Elmquist JK, Zinn AR, Kublaoui
BM. Postnatal Sim1 deficiency causes hyperphagic obesity and reduced
Mc4r and oxytocin expression. J Neurosci 30: 3803–3812, 2010.
48. Tomizawa K, Iga N, Lu YF, Moriwaki A, Matsushita M, Li ST,
Miyamoto O, Itano T, Matsui H. Oxytocin improves long-lasting spatial
memory during motherhood through MAP kinase cascade. Nat Neurosci
6: 384 –390, 2003.
49. Tousson E, Meissl H. Suprachiasmatic nuclei grafts restore the circadian
rhythm in the paraventricular nucleus of the hypothalamus. J Neurosci 24:
2983–2988, 2004.
50. Turek FW, Joshu C, Kohsaka A, Lin E, Ivanova G, McDearmon E,
Laposky A, Losee-Olson S, Easton A, Jensen DR, Eckel RH, Takahashi JS, Bass J. Obesity and metabolic syndrome in circadian Clock
mutant mice. Science 308: 1043–1045, 2005.
51. Weaver DR. The suprachiasmatic nucleus: a 25-year retrospective. J Biol
Rhythms 13: 100 –112, 1998.
52. Welsh DK, Takahashi JS, Kay SA. Suprachiasmatic nucleus: cell autonomy and network properties. Annu Rev Physiol 72: 551–577, 2010.
53. Yang X, Downes M, Yu RT, Bookout AL, He W, Straume M,
Mangelsdorf DJ, Evans RM. Nuclear receptor expression links the
circadian clock to metabolism. Cell 126: 801–810, 2006.
54. Yin L, Wu N, Curtin JC, Qatanani M, Szwergold NR, Reid RA, Waitt
GM, Parks DJ, Pearce KH, Wisely GB, Lazar MA. Rev-erbalpha, a
heme sensor that coordinates metabolic and circadian pathways. Science
318: 1786 –1789, 2007.
55. Yoshimura R, Kiyama H, Kimura T, Araki T, Maeno H, Tanizawa O,
Tohyama M. Localization of oxytocin receptor messenger ribonucleic
acid in the rat brain. Endocrinology 133: 1239 –1246, 1993.
56. Zhang G, Bai H, Zhang H, Dean C, Wu Q, Li J, Guariglia S, Meng Q,
Cai D. Neuropeptide exocytosis involving synaptotagmin-4 and oxytocin
in hypothalamic programming of body weight and energy balance. Neuron
69: 523–535, 2011.
57. Zhang X, Zhang G, Zhang H, Karin M, Bai H, Cai D. Hypothalamic
IKKbeta/NF-kappaB and ER stress link overnutrition to energy imbalance
and obesity. Cell 135: 61–73, 2008.
301 • NOVEMBER 2011 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on June 18, 2017
15. Droit-Volet S, Meck WH. How emotions colour our perception of time.
Trends Cogn Sci 11: 504 –513, 2007.
16. Dunlap JC. Molecular bases for circadian clocks. Cell 96: 271–290, 1999.
17. Elmquist JK, Flier JS. Neuroscience. The fat-brain axis enters a new
dimension. Science 304: 63–64, 2004.
18. Friedman JM. Modern science versus the stigma of obesity. Nat Med 10:
563–569, 2004.
19. Fuller PM, Lu J, Saper CB. Differential rescue of light- and foodentrainable circadian rhythms. Science 320: 1074 –1077, 2008.
20. Gallego M, Virshup DM. Post-translational modifications regulate the
ticking of the circadian clock. Nat Rev Mol Cell Biol 8: 139 –148, 2007.
21. George CP, Messerli FH, Genest J, Nowaczynski W, Boucher R,
Kuchel Orofo-Oftega M. Diurnal variation of plasma vasopressin in man.
J Clin Endocrinol Metab 41: 332–338, 1975.
22. Hiroshige T, Sakakura M. Circadian rhythm of corticotropin-releasing
activity in the hypothalamus of normal and adrenalectomized rats. Neuroendocrinology 7: 25–36, 1971.
23. Kalsbeek A, La FS, Van HC, Buijs RM. Suprachiasmatic GABAergic
inputs to the paraventricular nucleus control plasma glucose concentrations in the rat via sympathetic innervation of the liver. J Neurosci 24:
7604 –7613, 2004.
24. Kerdelhue B, Palkovits M, Karteszi M, Reinberg A. Circadian variations in substance P, luliberin (LH-RH) and thyroliberin (TRH) contents in
hypothalamic and extrahypothalamic brain nuclei of adult male rats. Brain
Res 206: 405–413, 1981.
25. Kitamura T, Feng Y, Kitamura YI, Chua SC Jr, Xu AW, Barsh GS,
Rossetti L, Accili D. Forkhead protein FoxO1 mediates Agrp-dependent
effects of leptin on food intake. Nat Med 12: 534 –540, 2006.
26. Kohsaka A, Laposky AD, Ramsey KM, Estrada C, Joshu C, Kobayashi Y, Turek FW, Bass J. High-fat diet disrupts behavioral and
molecular circadian rhythms in mice. Cell Metab 6: 414 –421, 2007.
27. Kosfeld M, Heinrichs M, Zak PJ, Fischbacher U, Fehr E. Oxytocin
increases trust in humans. Nature 435: 673–676, 2005.
28. Kublaoui BM, Holder JL Jr, Gemelli T, Zinn AR. Sim1 haploinsufficiency impairs melanocortin-mediated anorexia and activation of paraventricular nucleus neurons. Mol Endocrinol 20: 2483–2492, 2006.
29. Lam TK, Pocai A, Gutierrez-Juarez R, Obici S, Bryan J, guilar-Bryan
L, Schwartz GJ, Rossetti L. Hypothalamic sensing of circulating fatty
acids is required for glucose homeostasis. Nat Med 11: 320 –327, 2005.
30. Lee HJ, Macbeth AH, Pagani JH, Young WS, III. Oxytocin: the great
facilitator of life. Prog Neurobiol 88: 127–151, 2009.
31. Leuner B, Caponiti JM, Gould E. Oxytocin stimulates adult neurogenesis even under conditions of stress and elevated glucocorticoids. Hippocampus 2011.
32. McCann MJ, Rogers RC. Oxytocin excites gastric-related neurones in rat
dorsal vagal complex. J Physiol 428: 95–108, 1990.
33. Mens WB, Witter A, van Wimersma Greidanus TB. Penetration of
neurohypophyseal hormones from plasma into cerebrospinal fluid (CSF):
half-times of disappearance of these neuropeptides from CSF. Brain Res
262: 143–149, 1983.
36. Moos F, Freund-Mercier MJ, Guerne Y, Guerne JM, Stoeckel ME,
Richard P. Release of oxytocin and vasopressin by magnocellular nuclei
in vitro: specific facilitatory effect of oxytocin on its own release. J
Endocrinol 102: 63–72, 1984.
37. Murphy KG, Bloom SR. Gut hormones and the regulation of energy
homeostasis. Nature 444: 854 –859, 2006.
38. O’Rahilly S. Human genetics illuminates the paths to metabolic disease.
Nature 462: 307–314, 2009.