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Articles
in PresS. Am J Physiol Endocrinol Metab (January 2, 2008). doi:10.1152/ajpendo.00704.2007
Sexually different actions of leptin in proopiomelanocortin neurons to regulate
glucose homeostasis
Haifei Shi 1, April D. Strader 2, Joyce E. Sorrell 1, James B. Chambers 1, Stephen C.
Woods 1, Randy J. Seeley 1
1
University of Cincinnati, Cincinnati, Ohio
2
Southern Illinois University School of Medicine, Carbondale, Illinois
Corresponding Author:
Randy J. Seeley
2170 E.Galbraith Road,
Department of Psychiatry
University of Cincinnati
Cincinnati, Ohio 45237
513-558-6664 (Phone)
513-558-8990 (Fax)
[email protected]
Running head: Sex difference in leptin’s action in POMC neurons
Key Words: leptin receptor, energy balance; glucose tolerance; insulin sensitivity; fat
distribution
1
Copyright © 2008 by the American Physiological Society.
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Abstract
Leptin regulates energy balance and glucose homeostasis at least in part via
activation of receptors in the arcuate nucleus of the hypothalamus located in
proopiomelanocortin (POMC) neurons. Females have greater sensitivity to central leptin
than males, suggested by a greater anorexic effect of central leptin administration in
females. We hypothesized that the regulation of energy balance and peripheral glucose
homeostasis of female rodents would be affected to a greater extent than in males if the
action of leptin in POMC neurons is disturbed. Male and female mice lacking leptin
receptors only in POMC neurons gained significantly more body weight and accumulated
more body fat. However, female mice gained disproportionately more visceral adiposity
than males, and this appeared to be largely the result of differences in energy expenditure.
When maintained on a high-fat diet (HFD), both male and female mutants had higher
levels of insulin following exogenous glucose challenges. Chow- and HFD-fed males but
not females had abnormal glucose disappearance curves following insulin
administrations. Collectively these data indicate that the action of leptin in POMC
neurons is sexually different to influence the regulation of energy balance, fat
distribution, and glucose homeostasis.
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Introduction
Obesity is associated with adverse metabolic effects, including non-insulin
dependent diabetes mellitus, hyperlipidemia, certain cancers and cardiovascular disease
(16, 21). In addition to the detrimental effects of excess fat, where that fat is located is
critical and directly related to health risks of obesity. Specifically, excess fat in the
central region of the body carries a greater risk than does fat distributed subcutaneously
(7, 19, 42, 50). Men and women differ greatly with respect to where their fat is
distributed. On average, women carry more fat subcutaneously whereas men carry more
fat viscerally (31, 34, 50). Hence, there are sex-based differences with regard to obesityassociated health risks, with males being more likely to develop obesity-related
disturbance of glucose homeostasis (17, 32).
Leptin, a circulating adipocyte-derived signal secreted in direct proportion to
body fat, and especially to subcutaneous fat, exerts its effects on the regulation of energy
balance and glucose homeostasis through activation of the long form of the leptin
receptor and subsequent activation of the JAK-STAT3 pathway in specific hypothalamic
neurons (3, 9, 23, 49). Although leptin receptors are widely expressed in the brain (24,
27, 37, 43), previous studies have identified the arcuate nucleus of the hypothalamus
(ARC) as a critical site to mediate leptin’s anorexic action (2, 25, 45). The ARC contains
two distinct populations of leptin-responsive neurons: proopiomelanocortin (POMC) /
cocaine- and amphetamine-regulated transcript (CART) neurons and agouti-related
peptide (AgRP) / neuropeptide Y (NPY) neurons. Within the ARC, leptin directly
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inhibits orexigenic AgRP/NPY neurons and activates anorexigenic POMC/CART
neurons (15, 22, 44) leading to coordinated control of energy homeostasis (45, 46).
A requirement for normal leptin action is illustrated by the syndrome that results
from loss-of-function mutations in genes encoding leptin or the leptin receptor in rodents
or humans (8, 10, 13, 26, 38). For example, rodents that lack functional leptin receptors
are obese, hyperphagic, hypoactive, hyperglycemic and hyperinsulinemic, revealing
leptin’s role in regulating body fat, energy balance and glucose homeostasis (8, 10, 33).
Studies utilizing specific loss-of-function and return-of-function approaches have yielded
important information on the functional significance of specific populations of leptin
receptors. For example, mice lacking leptin receptors selectively in POMC neurons
display hyperleptinemia and modest obesity (1), implying that leptin receptor-expressing
POMC neurons are required for normal body weight homeostasis. Further, ARCselective restoration of leptin receptors in mice with a leptin receptor null background
causes a modest decrease in body weight but strikingly improved glucose homeostasis
and normalized locomotor activity (14), implying that in addition to their profound
effects on food intake and body weight, leptin receptors in the ARC are important for
glucose homeostasis and locomotor activity.
Leptin is mainly secreted from subcutaneous adipose tissue and the level of
circulating leptin correlates better with subcutaneous than with visceral fat (20, 35).
Consistent with differential fat distribution, plasma leptin is higher in women than in men
with the same body mass index (40). An important implication is that leptin signaling
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within the brain may differ in males and females, with females relying on leptin signaling
to a greater extent than males due to differential fat distribution and leptin signaling.
Consistent with this, female rats are more sensitive to the central anorexic effects of
leptin than their male counterparts (11, 12).
In the present experiments we asked whether energy and glucose homeostasis, as
well as body fat distribution, are impacted to a greater extent in female than male mice
when leptin signaling in POMC neurons is uniquely disturbed. We used a Cre/loxP
mouse model with targeted disruption of the leptin receptor in POMC neurons as
described by Lowell and colleagues (1). Lack of leptin receptors in POMC neurons of
these Pomc-Cre, Leprflox/flox mice results in increased accumulation of total body fat in
both males and females relative to their Leprflox/flox counterparts (1). In the present
experiments we evaluated energy balance, body fat distribution, and glucose homeostasis
in male and female Pomc-Cre, Leprflox/flox mice.
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Methods
Animals
Mice lacking leptin receptors in POMC neurons (Pomc-Cre, Leprflox/flox mice)
were obtained by crossing Pomc-Cre mice (provided by Drs. Bradford B. Lowell and Joel
K. Elmquist, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston,
MA) with Leprflox/flox mice (provided by Dr. Streamson C. Chua, Jr., Albert Einstein
University, New York, NY). All mice were maintained and bred in our colony at the
University of Cincinnati. After weaning at 3 weeks of age, mice were housed
individually in micro-isolator cages in pathogen-free, temperature- and humiditycontrolled rooms with a 12/12 light/dark cycle with lights on at 0500 and off at 1700 hr.
RT-PCR analysis was used to genotype the mice.
Two cohorts of mice were used in the current study. Mice from the first cohort
were maintained on pelleted chow (Harlan Teklad, Madison, WI, rodent diet 8604; 12.8%
kcal% fat, 31.6% kcal% protein, 55.6% kcal% carbohydrates) until 16 wks of age when
they were changed to a high-fat butter-oil-based diet (HFD; Research Diets, Inc., New
Brunswick, NJ, D12451; 45% kcal% fat, 20% kcal% protein, 35% kcal% carbohydrates).
Mice from the second cohort were maintained on pelleted chow throughout. All mice
had ad libitum access to water. All animal procedures were approved by the University
of Cincinnati Institutional Animal Care and Use Committee.
Experimental design
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Experiment 1: Energy balance of chow-fed mice
While on the chow diet, male and female Leprflox/flox and Pomc-Cre, Leprflox/flox
mice from the first cohort were used to assess locomotor activity at 12 wks of age and
energy expenditure using indirect calorimetry at 14 wks of age.
Experiment 2: Body fat distribution of chow- and HFD-fed mice
Fat distribution was determined in the mice from the first cohort after they had
been on the HFD for over 8 weeks with age matched-, chow-fed mice in the second
cohort.
Experiment 3: Glucose homeostasis of chow- and HFD-fed mice
Glucose tolerance tests (ipGTT) were performed at 15 wks of age and insulin
tolerance tests (ipITT) at 16 wks of age in the mice from the first cohort while fed chow.
These mice were then placed on the HFD, and ipGTT were conducted at 22 wks of age
and ipITT at 23 wks of age.
Energy expenditure
Heat production, oxygen consumption and locomotor activity were measured in
males and in females at diestrous because the females in these experiments had 6-day
mean estrous cycles with diestrous lasting over 2 days. We opted to assess mice during
diestrous to reduce variance resulting from different phases of the cycles. Estrous
cyclicity was monitored by daily vaginal lavage. Vaginal smears were performed
between 1000 and 1100 hr each day in the middle of the light cycle and were stained with
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a DipQuick staining kit (Jorgensen Laboratories Inc., Loveland, CO) for the
determination of the phase of the estrous cycle in the females based on the pattern of cell
types of smear samples (4).
Heat production and oxygen consumption were measured using an Oxymax
System (Accuscan Instruments, Columbus, OH). Briefly, mice were placed into
individual metabolic chambers at the end of the light cycle, and remained in the chambers
for 24 hr with ad lib access to water and food. Whole-animal heat production and oxygen
consumption were determined as Kcal/hour and ml/kg/hour, respectively, and were
subsequently adjusted to kg of lean body mass. Locomotor activity was measured using
a running wheel apparatus (Lafayette Instrument Co., Lafayette, IN). Briefly, mice were
housed in cages (23.62 × 35.3 × 19.56 cm) fitted with an anodized aluminum wheel (12.7
cm in diameter) for 24 hr. Wheel revolutions were recorded and cumulative distance was
calculated using Animal Wheel Monitor Software (Lafayette Instrument Co., Lafayette,
IN).
Body composition and body fat distribution
A mouse-specific nuclear magnetic resonance (NMR) Echo MRI whole body
composition analyzer (EchoMedical Systems, Houston, TX) was used to assess body fat
and lean mass (48) in conscious mice, providing longitudinal data. After the mice were
sacrificed, the amounts of subcutaneous and visceral fat were assessed separately by
dividing the carcass into two portions and using the pelting method (11). In this
procedure, the skin with any attached fat and fat on the outer surface of any skeletal
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muscle was removed from the carcass. This pelt portion and the remaining carcass
portion including all muscle, skeleton, organs, intramyocellular and internal fat were then
assessed separately using the NMR.
Intraperitoneal glucose tolerance tests (ipGTT) and insulin sensitivity tests (ipITT)
ipGTT and ipITT were performed according to previously established procedures
(47). Mice were fasted for 16 hr and all blood samples were obtained from the tip of the
tail vein. For the ipGTT, after a baseline blood sample was taken (0 min), 1.5 g / kg body
weight (for chow-fed cohort) or 0.75 g / kg body weight (for HFD-fed cohort) of 20% Dglucose (Phoenix Pharmaceutical Inc., St. Joseph, MO) was injected intraperitoneally. A
lower dose of glucose was administered to the HFD-fed mice in order to ensure that the
resulting glucose levels were in the sensitivity range of the glucometers; i.e., 20 to 500
mg/dL. For the ipITT, 1U / kg body weight of insulin (for both chow- and HFD-fed
cohorts; Novolin, Novo Nordisk, Princeton, NJ) was injected intraperitoneally.
Subsequent blood samples were taken at 15, 30, 45, 60 and 120 min after glucose
administration, and at 15, 30, 45 and 60 min after insulin administration. Glucose was
measured on duplicate samples using FreeStyle™ glucometers and test strips (FreeStyle,
Alameda, CA). During the ipGTT, an additional blood sample was taken from the tail
vein 13-15 min after the glucose administration for measurement of plasma insulin using
the rat insulin enzyme-linked immunosorbent assay (ELISA) kits (Crystal Chem Inc.,
Downers Grove, IL). The coefficients of variation of intra-assay and inter-assay were
5.5% and 6.1%, respectively. To evaluate glucose tolerance, calculations of the area
under the glucose curves (AUC) were made based on the glucose baseline levels at 0 min,
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and glucose clearance rates were calculated between the 15-min peak level and the 30min value following glucose administration. To assess insulin sensitivity, the slope of the
glucose decrease between 0 and 60 min was calculated.
Statistical analysis
Data are expressed as mean ± standard error mean (SEM). Comparisons among
multiple groups were made using appropriate one-way or two-way analysis of variance
(ANOVA). Post-hoc tests of individual groups were made using Tukey’s tests
(SigmaStat 3.1, San Rafael, CA). Significance was set at P < 0.05. Exact probabilities
and test values were omitted for simplicity and clarity of the presentation of the results.
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Results
Male and female Leprflox/flox or Pomc-Cre, Leprflox/flox mice gained body weight
and adiposity after being switched to the HFD, with Pomc-Cre, Leprflox/flox mice
accumulating more body fat than Leprflox/flox controls (Fig. 1A and B). Body weights and
total body fat masses were significantly greater in Pomc-Cre, Leprflox/flox mice compared
with their same-sex counterparts (Fig. 1 and Table 1), whereas total lean body masses
were not different (Fig. 1C).
Experiment 1: Energy balance of chow-fed mice
Consistent with a previous report (1), daily food intake was not changed (data not
shown); thus, Pomc-Cre, Leprflox/flox mice have greater weight gain with no alteration in
feeding. In contrast to the previous report of energy expenditure at 8-10 wks of age (1),
total heat production was greater over a 24-hr period in 14-wk old chow-fed Pomc-Cre,
Leprflox/flox males compared to Leprflox/flox control males. This was true for absolute 24-hr
total heat production (Fig. 2A) as well as after adjustment for lean body mass (Fig. 2B).
The opposite was observed in females, with less heat production in Pomc-Cre, Leprflox/flox
females than controls (Fig. 2A and 2B). Oxygen consumption (VO2) had a similar trend
as heat production, although absolute VO2 of male mice was not different between PomcCre, Leprflox/flox and their controls (Fig. 2C and 2D).
Control Leprflox/flox females ran a significantly longer distance than Pomc-Cre,
Leprflox/flox and Leprflox/flox males (Fig. 3). Leprflox/flox females had a tendency to run farther
than Pomc-Cre, Leprflox/flox females, suggesting a slight decrease of locomotor activity and
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thus energy conservation of Pomc-Cre, Leprflox/flox females (Fig. 3). The difference of the
cumulative running distance, however, did not reach statistical significance between
genotypes of the same sex.
Experiment 2: Body fat distribution of chow- and HFD-fed mice
Female Pomc-Cre, Lepr flox/flox mice accumulated more body fat than Pomc-Cre,
Lepr flox/flox males (Table 1). The difference between males and females was exacerbated
on a HFD. After being placed on the HFD, Pomc-Cre, Lepr flox/flox females accumulated
nearly seven times the total body fat as controls whereas male Pomc-Cre,Lepr flox/flox mice
only doubled their body fat relative to their male controls (Table 1). The difference of
total fat mass between female Pomc-Cre, Lepr flox/flox and Lepr flox/flox mice was greater
than the difference between male Pomc-Cre, Lepr flox/flox and Lepr flox/flox mice under the
same dietary conditions (Table 1). Specifically, when fed with chow diet, total fat mass
was 2.12 g greater in female Pomc-Cre, Lepr flox/flox mice than in female Lepr flox/flox mice,
whereas total fat mass was 1.67 g greater in male Pomc-Cre, Lepr flox/flox mice than in
male Lepr flox/flox controls; when fed with a HFD, total fat mass was 8.06 g greater in
female Pomc-Cre, Lepr flox/flox mice than Lepr flox/flox females, whereas total fat mass was
6.33 g greater in male Pomc-Cre, Lepr flox/flox mice compared to male Lepr flox/flox controls.
In addition to the differences in total body fat accumulation between male and
female mice, there were also important differences as to where that body fat was
accumulated. In order to compare the amount of fat added in the subcutaneous and
visceral compartments in male and female mice by disruption of leptin signaling in
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POMC neurons, we used the following calculations: % increase in visceral fat =
(visceral fat of each Pomc-Cre, Lepr flox/flox mouse – average visceral fat of Lepr flox/flox
mice) / (total body fat of each Pomc-Cre, Lepr flox/flox mouse – average total body fat of
Lepr flox/flox mice) × 100%. % increase in subcutaneous fat = (subcutaneous fat of each
Pomc-Cre, Lepr flox/flox mouse – average subcutaneous fat of Lepr flox/flox mice) / (total fat
of each Pomc-Cre, Lepr flox/flox mouse – average total body fat of Lepr flox/flox mice) ×
100%. Using these calculations, female Pomc-Cre, Lepr flox/flox mice had a greater
increase in visceral fat and a smaller increase in subcutaneous fat than Pomc-Cre, Lepr
flox/flox
males (Table 2). Male Pomc-Cre, Lepr flox/flox mice accumulated similar
percentages of fat at both visceral and subcutaneous depots; whereas female Pomc-Cre,
Lepr flox/flox mice accumulated a greater amount of their additional body fat in the form of
visceral fat when they were fed with either chow or HFD (Table 2). Female Pomc-Cre,
Lepr flox/flox mice accumulated more visceral fat than males on either chow or HFD,
suggesting that the lack of functional leptin signaling in the POMC neurons affected
female fat distribution more than males by shifting fat accumulation to the visceral
compartment.
Experiment 3: Glucose homeostasis of chow- and HFD-fed mice
At 15 wks of age, chow-fed male Pomc-Cre, Leprflox/flox mice had higher fasting
glucose and higher glucose levels 45 and 60 min following glucose administration than
Leprflox/flox males (Fig. 4A). In addition, glucose clearance between the 15-min peak level
and 30-min post glucose injection was significantly lower in male Pomc-Cre, Leprflox/flox
than male Leprflox/flox controls (Table 3), suggesting impaired glucose clearance by Pomc-
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Cre, Leprflox/flox males following a glucose challenge. The 15-min insulin levels during
ipGTT were not different between chow-fed male Pomc-Cre, Leprflox/flox and Leprflox/flox
mice (male Leprflox/flox: 0.65 ± 0.11; male Pomc-Cre, Leprflox/flox: 1.01 ± 0.22; Fig. 4A).
Chow-fed females had comparable fasting glucose levels and comparable glucose levels
at all time points, and their 15-30 min glucose clearance constants were also comparable
(Fig. 4B; Table 3). However, the 15-min insulin level during ipGTT of female PomcCre, Leprflox/flox mice was significantly higher than that of female Leprflox/flox mice (female
Leprflox/flox: 0.83 ± 0.17; female Pomc-Cre, Leprflox/flox: 1.36 ± 0.21; Fig. 4B), implying
that the female Pomc-Cre, Leprflox/flox mice increased their insulin secretion to maintain
normal glucose tolerance.
In the insulin sensitivity tests, although glucose disappearance rates following
insulin administrations were similar in males between 0 and 60 min (male Leprflox/flox:
2.07 ± 0.17; male Pomc-Cre, Leprflox/flox: 1.56 ± 0.28; Fig. 5A), chow-fed male Pomc-Cre,
Leprflox/flox mice had higher fasting glucose levels and higher glucose levels at all time
points after insulin administration (Fig. 5A), suggesting that although hyperglycemia
occurred, the animals were comparably insulin sensitive as chow-fed Leprflox/flox males.
Chow-fed female Pomc-Cre, Leprflox/flox mice, on the other hand, had comparable glucose
levels at all time points following insulin injections (Fig. 5B) and similar glucose
disappearance rates as their female Leprflox/flox counterparts (female Leprflox/flox: 1.41 ±
0.38; female Pomc-Cre, Leprflox/flox: 1.80 ± 0.20; Fig. 5B).
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After consuming the HFD, glucose levels across ipGTT were comparable
between male Leprflox/flox and Pomc-Cre, Leprflox/flox mice (Fig. 6A). In addition, their
average 15-30 min glucose clearance constants were not significantly different (Table 3).
Although glucose excursion curves were similar between male Leprflox/flox and Pomc-Cre,
Leprflox/flox mice, the 15-min insulin level during ipGTTs of male Pomc-Cre, Leprflox/flox
mice was significantly higher than that of Leprflox/flox males (male Leprflox/flox: 1.62 ± 0.22;
male Pomc-Cre, Leprflox/flox: 3.03 ± 0.31; Fig. 6A), suggesting that HFD-fed Pomc-Cre,
Leprflox/flox males secreted more insulin to achieve similar glucose levels as Leprflox/flox
males, and thus Pomc-Cre, Leprflox/flox males were relatively insulin insensitive compared
to male Leprflox/flox controls. This was confirmed by ipITT which revealed significantly
higher glucose levels at 30, 45 and 60 min following insulin injections in Pomc-Cre,
Leprflox/flox males than in Leprflox/flox males (Fig. 7A). ipITT also indicated significantly
lower glucose disappearance rates for male Pomc-Cre, Leprflox/flox mice than that of
Leprflox/flox males (male Leprflox/flox: 1.52 ± 0.27; male Pomc-Cre, Leprflox/flox: 0.54 ± 0.20;
Fig. 7A), implying a relative insulin resistance in Pomc-Cre, Leprflox/flox males compared
with their Leprflox/flox male counterparts in the context of a HFD.
Similarly to the chow-fed female Pomc-Cre, Leprflox/flox mice, HFD-fed female
Pomc-Cre, Leprflox/flox mice also had a significantly higher level of 15-min insulin levels
during ipGTT than Leprflox/flox females (female Leprflox/flox: 0.79 ± 0.11; female Pomc-Cre,
Leprflox/flox: 2.17 ± 0.18; Fig. 6B), suggesting insulin insensitivity of Pomc-Cre, Leprflox/flox
females comparing to female Leprflox/flox controls. However, this was not confirmed by
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ipITT where glucose disappearance rates were similar during ipITT (female Leprflox/flox:
2.03 ± 0.50; female Pomc-Cre, Leprflox/flox: 1.41 ± 0.29; Fig 7B).
Under both chow and HFD feeding, females of both genotypes had lower fasting
glucose levels, lower AUC, and faster glucose disappearance rates during ipGTTs than
males (Table 3), suggesting that overall, female mice were more glucose tolerant than
males within the same genotype and under the same dietary condition.
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Discussion
A wealth of data has demonstrated that POMC neurons are important for
regulation of glucose homeostasis by responding to signals including leptin. Leptin
stimulates melanocortin POMC neurons within the ARC by acting through its specific
receptors (41) and regulates energy homeostasis through diverse effects on food intake
and metabolism (45, 46), a model implicating leptin signaling in the ARC POMC
neurons as a general integrator of glucose and energy homeostasis. Dysregulation of this
leptin-POMC regulatory system, either via the ability of POMC neurons to sense and
integrate coordinated responses to changing levels of leptin or defective leptin signaling,
causes pathophysiology of energy balance and thus predisposes to obesity, diabetes, and
other components of metabolic syndrome.
Mutations of the leptin receptor in mice (db/db) or in rats (fa/fa) cause disruption
in energy balance, progressive insulin resistance, hyperinsulinemia and hyperglycemia
(10, 30). Studies using db/db mice or fa/fa rats, however, give no insight into the role
played by specific populations of leptin receptors. Given that leptin receptors are found
in POMC neurons where they regulate both gene expression and neuronal firing, ablation
of leptin receptors specifically in POMC neurons may affect energy balance and glucose
homeostasis. The present study focused on male and female Pomc-Cre, Leprflox/flox mice
that have a targeted disruption of leptin action selectively in POMC neurons, and
compared them to Leprflox/flox where leptin receptor signaling should be unaffected. This
allows for the determination of the physiological role of leptin signaling in the POMC
neurons.
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The current data point to important sex differences in the role of leptin action in
POMC neurons on energy balance and glucose homeostasis. Lack of leptin receptors in
POMC neurons lead to conservation of energy expenditure in females but not in males.
Chow-fed Pomc-Cre, Leprflox/flox females accumulated greater adiposity by decreasing
heat production and oxygen consumption than control females. Pomc-Cre, Leprflox/flox
males on the other hand, had greater energy expenditure than male Leprflox/flox mice when
heat production and oxygen consumption were measured using indirect calorimetry at 14
wks of age, a time when adiposity and body weights were relatively stable and the mice
were not continuously growing. Energy expenditure is proportional to the body weight,
thus the greater energy expenditure of Pomc-Cre, Leprflox/flox males could be a possible
outcome of greater adiposity and body weights of male Pomc-Cre, Leprflox/flox mice than
Leprflox/flox males. Consistent with this sex difference in energy expenditure, female
Pomc-Cre, Lepr flox/flox mice accumulated more total and visceral adiposity than males.
Lack of leptin receptors in POMC neurons resulted in glucose intolerance and insulin
resistance in males. Chow- or HFD-fed Pomc-Cre, Lepr flox/flox females, however, had
glucose tolerance curves no different than their controls during ipGTT. Consistent with
this important sex difference, female Pomc-Cre, Lepr flox/flox mice on the HFD had normal
glucose disappearance after administration of peripheral insulin while male Pomc-Cre,
Lepr flox/flox on the HFD had an impaired glucose response to insulin.
The outcome is surprising in two ways. First, it might be expected that leptin
would exert a more powerful effect on glucose homeostasis in females since leptin more
potently reduces food intake after central administration in female as compared to male
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rats (11, 12). Second, the effect of disrupting leptin signaling in the POMC neurons had
different effects on body fat distribution. Consistent with a previous report (1), both male
and female Pomc-Cre, Lepr flox/flox mice had higher body fat content than their appropriate
controls on either chow or HFD. However, female Pomc-Cre, Leprflox/flox mice
accumulated a larger percentage of visceral fat and smaller percentage of subcutaneous
fat than Pomc-Cre, Leprflox/flox males when fed with either chow or HFD. The higher
amount of visceral fat might have been expected to produce both glucose intolerance and
relative insulin resistance in the female Pomc-Cre, Lepr flox/flox mice. Despite this body fat
distribution, it is clear that neither glucose nor insulin tolerance were impaired in the
female Pomc-Cre, Lepr flox/flox mice.
The female steroid hormone estrogen may contribute to the sex differences in the
action of leptin in POMC neurons. First, estrogen influences synaptic plasticity by
changing the axosomatic synapses in the ARC (39), increases the number of excitatory
inputs to POMC neurons, and enhances POMC tone in the ARC (28). In addition,
enhanced POMC tone in the ARC is a leptin independent effect that also occurs in leptindeficient (ob/ob) and leptin receptor–deficient (db/db) mice (28). Thus, estrogen
modulates POMC neuronal activity in the ARC. Thus, estrogen modulates POMC
neuronal activity in the ARC. Female Pomc-Cre, Leprflox/flox and Leprflox/flox mice have
similar circulating levels of estrogen (Shi & Seeley, unpublished finding), which may
influence glucose tolerance of Pomc-Cre, Leprflox/flox females independent of
dysfunctional leptin signaling in the POMC neurons.
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Second, estrogen modulates leptin signaling and leptin receptor expression. This
has been tested in intact and ovariectomized female rats. In intact female rats, estrogen
treatment down-regulates the leptin receptors in the hypothalamus (6). Estrogen levels
also regulate the expression of the leptin receptors during the estrous cycle. Specifically,
leptin receptor expression is lowest in proestrus when estrogen level is the highest, and
leptin receptor expression in the ARC is highest during metestrous when estrogen level
declines (6). Because circulating leptin levels do not change during the estrous cycle,
estrogen regulates leptin receptor expression independent of leptin levels. Ovariectomy
that leads to estrogen insufficiency, or treatment with estradiol or raloxifene (a selective
estrogen receptor modulator) that leads to increased estrogen level or enhanced estrogen
action, may modulate leptin receptor expression in the hypothalamus. Specifically,
expression of leptin receptor in the ARC is increased in ovariectomized rats (5, 36), and
these effects were reversed by either estradiol or raloxifene treatment (5, 36), suggesting
that central leptin receptor expression is modified by estrogen’s action. The potential
sites that would allow for the regulation of estrogen by leptin receptor are unknown but
could include nuclei that co-express the estrogen and leptin receptors, such as the ARC
as well as extra-ARC regions including the medial preoptic area, the parvicellular
paraventricular nucleus, and the ventromedial hypothalamic nucleus (18). Certainly the
ARC is not the only CNS regulator of glucose homeostasis. Certainly the ARC is not the
only CNS regulator of glucose homeostasis. Consequently, Pomc-Cre, Leprflox/flox
females with similar circulating estrogen levels as Leprflox/flox females may have
compensated leptin signaling on non-POMC neurons that work to maintain relatively
normal glucose tolerance.
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It is interesting to note that chow- and HFD-fed female Pomc-Cre, Lepr flox/flox
mice displayed significantly elevated levels of insulin during an ipGTT compared to their
controls. Although the female Pomc-Cre, Lepr flox/flox mice and controls had identical
glucose tolerance curves, the Pomc-Cre, Lepr flox/flox females required greater insulin
secretion for lowering plasma glucose, suggestive of insulin resistance. The increase in
insulin in the female Pomc-Cre, Lepr flox/flox mice compared to the controls is consistent
with the disproportionate increase in visceral adipose tissue, as circulating insulin is
closely associated with visceral adiposity. These data are consistent with a role for
leptin’s action in POMC neurons in the regulation of glucose homeostasis and body
weight gain. Opposite to these findings, but conceptually consistent with our current data
is the inhibitory role of SOCS-3 in POMC neurons within the ARC on glucose regulation
and body weight (29). Mice lacking SOCS-3 within POMC neurons in the ARC are
functionally more sensitive to leptin’s actions since the endogenous suppressor is not
present. These mice show enhanced glucose tolerance on both a chow and HFD
compared to their wildtype or SOCS-3 intact controls. Interestingly, these mice also
showed resistance to the obesogenic effects of a HFD that was attributed to increased
energy expenditure. Food intake in mice with SOCS-3 deficiency in POMC neurons was
identical to wild type controls (29).
A major theme of contemporary research is to use genetic mouse models to assess
the role of specific populations of leptin receptors to regulate energy balance and glucose
homeostasis. The current results indicate that leptin receptors in POMC neurons
21
Page 22 of 49
influence energy balance and glucose homeostasis in mice. In females, leptin receptors
in POMC neurons have a greater influence over energy balance and fat distribution than
glucose homeostasis; whereas in males, leptin action in the POMC neurons has a greater
influence over glucose homeostasis than fat distribution. Such results contribute to a
growing literature indicating important sexual difference involved in the circuitry that
regulates crucial metabolic processes. Current outcomes highlight the possibility that
treatment strategies for metabolic disorders may need to be very different for males and
females and the crucial need for detailed comparisons between males and females in
metabolic research.
22
Page 23 of 49
Acknowledgements
We thank Drs Bradford B. Lowell, Joel K. Elmquist and Streamson Chua Jr. for
making the mouse models available to us. This work was supported by NIDDK grants
DK56863, DK 17844 to SC Woods, DK54080, DK73505 to RJ Seeley, and National
Research Service Award DK75255 to H Shi.
23
Page 24 of 49
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Tables:
Table 1: Body masses and fat distribution (including total fat, visceral and
subcutaneous fat masses) of chow- or HFD-fed male and female mice.
Values are mean ± SEM. n = 10 from each group. † denotes statistically significant
difference between genotypes within sex (P < 0.05).
Table 2: Percentages of gained visceral and subcutaneous fat of Pomc-Cre,
Leprflox/flox mice compared to their same sex Leprflox/flox mice.
Values are mean ± SEM. n = 10 from each group. * denotes statistically significant
difference between sexes.
Table 3: Baseline glucose, glucose disappearance rates, and area under the glucose
curves of chow- or HFD-fed male or female Leprflox/flox or Pomc-Cre, Leprflox/flox mice
during an ipGTT.
Values are mean ± SEM. n = 10 from each group. † denotes statistically significant
difference between genotypes within sex (P < 0.05). * denotes statistically significant
difference between sexes within genotype (P < 0.05).
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Figure legends:
Figure 1: Weekly body mass, total body fat and lean tissue of age-matched, chow- or
HFD-fed mice.
Body mass (A), fat tissue mass (B), and lean tissue mass (C) of mice.
indicates the
beginning of HFD feeding of the first cohort. Data were expressed as means ± SEM. *
denotes statistically significant difference between male Leprflox/flox and Pomc-Cre,
Leprflox/flox mice, and between female Leprflox/flox and Pomc-Cre, Leprflox/flox mice (P <
0.05).
Figure 2: Energy expenditure of chow-fed mice.
Total heat production (2A), heat production per lean mass (2B), total oxygen
consumption (2C), and oxygen consumption per lean mass (2D) of male and female
mice. Data were expressed as means ± SEM. * denotes statistically significant
difference between genotypes within sex (P < 0.05).
Fig. 3 Locomotor activity of 12-wk old chow-fed female and male mice.
Data were expressed as means ± SEM. * denotes statistically significant difference
between groups (P < 0.05).
Fig. 4: Glucose levels and 13-15 min insulin levels during glucose tolerance tests in
chow-fed male (4A) and female mice (4B).
Data were expressed as means ± SEM. * denotes statistically significant difference
between genotypes within sex (P < 0.05).
32
Page 33 of 49
Fig. 5: Glucose levels and glucose disappearance rates during insulin sensitivity tests
in chow-fed male (5A) and female mice (5B).
Data were expressed as means ± SEM. * denotes statistically significant difference
between genotypes within sex (P < 0.05).
Fig. 6: Glucose levels and 13-15 min insulin levels during glucose tolerance tests in
HFD-fed male (6A) and female mice (6B).
Data were expressed as means ± SEM. * denotes statistically significant difference
between genotypes within sex (P < 0.05).
Fig. 7: Glucose levels and glucose disappearance rates during insulin sensitivity tests
in HFD-fed male (7A) and female mice (7B).
Data were expressed as means ± SEM. * denotes statistically significant difference
between genotypes within sex (P < 0.05).
33
Page 34 of 49
Table 1: Body masses and fat distribution (including total fat, visceral and subcutaneous fat masses) of chow- or HFD-fed
male and female mice.
Leprflox/flox
Cohort 1: HFD-fed (between 16wk- and 24wk-old)
Male
Female
Body Mass (g)
29.73 ± 1.24
19.65 ± 0.95
Total Fat (g)
4.61 ± 0.76
1.43 ± 0.38
Visceral Fat (g)
3.12 ± 0.44
0.72 ± 0.23
Subcutaneous Fat (g)
1.42 ± 0.26
0.47 ± 0.13
Pomc-Cre, Leprflox/flox
Male
37.85 ± 1.29 †
10.94 ± 1.15 †
6.44 ± 0.53 †
4.45 ± 0.65 †
Female
30.66 ± 2.26 †
9.49 ± 1.79 †
5.53 ± 1.14 †
3.39 ± 0.66 †
Male
31.16 ± 0.54 †
4.16 ± 0.69 †
3.02 ± 0.31 †
1.14 ± 0.38 †
Female
23.94 ± 0.65 †
3.09 ± 0.40 †
2.13 ± 0.24 †
0.96 ± 0.16 †
Cohort 2: Chow-fed
Body Mass (g)
Total Fat (g)
Visceral Fat (g)
Subcutaneous Fat (g)
Male
27.90 ± 0.87
2.49 ± 0.19
2.22 ± 0.06
0.28 ± 0.14
Female
21.55 ± 0.50
0.97 ± 0.28
0.79 ± 0.17
0.18 ± 0.12
Values are mean ± SEM. n = 10 from each group.
† denotes statistically significant difference between genotypes within sex (P < 0.05).
Page 35 of 49
Table 2: Percentages of gained visceral and subcutaneous fat of Pomc-Cre, Leprflox/flox mice compared to their same sex
Leprflox/flox mice.
visceral fat increase (%)
subcutaneous fat increase (%)
Chow-fed intact
male
female
52.39 ± 3.31
63.06 ± 1.77 *
47.61 ± 3.31
36.94 ± 1.77 *
HFD-fed intact
male
female
54.01 ± 2.27
61.97 ± 1.52 *
45.99 ± 2.27
38.03 ± 1.52 *
Values are mean ± SEM. n = 10 from each group.
* denotes statistically significant difference between sexes.
Page 36 of 49
Table 3: Baseline glucose, glucose disappearance rates, and area under the glucose curves of chow- or HFD-fed male or female
Leprflox/flox or Pomc-Cre, Leprflox/flox mice during an ipGTT.
Baseline glucose
(mg/dL)
Chow
Male
Female
HFD
Male
Female
128.3 ± 6.2
102.3 ± 5.4 *
15-30 min glucose
disappearance rate
Leprflox/flox mice
1.84 ± 0.61
2.87 ± 0.47
145.0 ± 7.3
95.8 ± 5.5 *
Leprflox/flox mice
0.81 ± 0.27
2.07 ± 0.34 *
0-120 min AUC
(mg/dL/min)
8729.25 ± 1385.01
5691.00 ± 782.51
11313.75 ± 2119.84
3878.75 ± 1008.17 *
Baseline glucose
(mg/dL)
15-30 min glucose
0-120 min AUC
disappearance rate
(mg/dL/min)
flox/flox
Pomc-Cre, Lepr
mice
153.3 ± 10.2 †
0.65 ± 0.35 †
11990.25 ± 2200.66
95.8 ± 6.4 *
2.56 ± 0.57 *
6438.00 ± 886.61 *
140.8 ± 6.2
109.7 ± 8.5 *
Values are mean ± SEM. n = 10 from each group.
† denotes statistically significant difference between genotypes within sex (P < 0.05).
* denotes statistically significant difference between sexes within genotype (P < 0.05).
Pomc-Cre, Leprflox/flox mice
0.12 ± 0.21
15020.83 ± 2194.01
2.89 ± 0.33 *
6081.38 ± 1297.75 *
Page 37 of 49
Figure 1: Weekly body mass, total body fat and lean tissue of age-matched, chow- or HFD-fed mice.
Fig. 1A Weekly body mass
45
HFD
40
Body Mass (g)
35
*
30
*
*
*
*
*
*
*
*
20
21
25
20
15
male Lepr flox/flox
male Pomc-Cre, Lepr flox/flox
female Lepr flox/flox
female Pomc-Cre, Lepr flox/flox
10
5
0
12
13
14
15
16
17
Age (Weeks)
18
19
Page 38 of 49
Fig. 1B Body fat tissue mass
Total Body Fat Tissue Mass (g)
flox/flox
12
Lepr
Pomc-Cre, Lepr flox/flox
30
*
*
10
8
6
*
4
*
2
0
Male
Female
Chow
Male
Female
HFD
Total Body Lean Tissue Mass (g)
14
Fig. 1C Body lean tissue mass
Lepr flox/flox
Pomc-Cre, Lepr flox/flox
25
20
15
10
5
0
Male
Female
Chow
Male
Female
HFD
Page 39 of 49
Figure 2: Energy expenditure of chow-fed mice.
Fig. 2A total heat production
24hr Heat Production
(Kcal/hr)
0.6
*
0.5
35
Lepr flox/flox
POMC-Cre, Lepr flox/flox
*
0.4
0.3
0.2
0.1
0.0
Males
Females
24hr Heat Production Per Lean Mass
(Kcal/Kg/hr)
0.7
Fig. 2B heat production per lean mass
30
Lepr flox/flox
*
25
POMC-Cre, Lepr flox/flox
*
20
15
10
5
0
Males
Females
Page 40 of 49
Fig. 2C total VO2 consumption
VO2 Consumption
(ml/Kg/Hr)
6000
8000
Lepr flox/flox
POMC-Cre, Lepr flox/flox
*
5000
4000
3000
2000
1000
0
Males
Females
VO2 Consumption per Lean Mass
(ml/Kg/Hr)
7000
Fig. 2D VO2 consumption per lean mass
Lepr flox/flox
POMC-Cre, Lepr flox/flox
6000
*
*
4000
2000
0
Males
Females
Page 41 of 49
12000
Cumulative Distance (m)
10000
8000
6000
Cumulative Distance (m)
Figure 3: Locomotor activity of chow-fed female and male mice.
*
12000
10000
8000
6000
4000
2000
0
Male
Female
4000
2000
male Lepr flox/flox
male Pomc-Cre, Lepr flox/flox
female Lepr flox/flox
female Pomc-Cre, Lepr flox/flox
0
0
20
40
60
80
100
Time (every 10 min)
120
140
Page 42 of 49
Figure 4: Glucose levels and 13-15 min insulin levels during glucose tolerance tests in chow-fed male (4A) and female mice (4B).
Fig. 4A
Blood Glucose Levels (mg/dL)
350
*
300
Plasma insulin (ng/ml)
2.0
*
250
1.5
1.0
0.5
0.0
Lepr
200
flox/flox
*
150
chow-fed male Lepr flox/flox
100
chow-fed male Pomc-Cre, Lepr flox/flox
50
0
15
30
45
60
Time (min)
75
90
105
120
Pomc-Cre,
flox/flox
Lepr
Page 43 of 49
Fig. 4B
Plasma insulin (ng/ml)
Blood Glucose Levels (mg/dL)
350
300
250
2.0
*
1.5
1.0
0.5
0.0
Lepr
200
flox/flox
Pomc-Cre,
flox/flox
Lepr
150
100
chow-fed female Lepr flox/flox
chow-fed female Pomc-Cre, Lepr flox/flox
50
0
15
30
45
60
Time (min)
75
90
105
120
Page 44 of 49
Figure 5: Glucose levels and glucose disappearance rates during insulin sensitivity tests in chow-fed male (5A) and female mice (5B).
Fig. 5A
2.5
Glucose Disappearance Rate
2.0
1.5
Blood Glucose Levels (mg/dL)
200
*
1.0
*
0.5
*
150
0.0
Lepr
flox/flox
*
Pomc-Cre,
flox/flox
Lepr
100
50
*
chow-fed male Lepr flox/flox
chow-fed male Pomc-Cre, Lepr flox/flox
0
0
15
30
Time (min)
45
60
Page 45 of 49
Fig. 5B
2.5
Blood Glucose Levels (mg/dL)
200
Glucose Disappearance Rate
2.0
180
1.5
160
1.0
140
0.5
0.0
120
Lepr
flox/flox
100
Pomc-Cre,
flox/flox
Lepr
80
60
chow-fed female Lepr flox/flox
40
chow-fed female Pomc-Cre, Lepr flox/flox
20
0
15
30
Time (min)
45
60
Page 46 of 49
Plasma insulin (ng/ml)
Figure 6: Glucose levels and 13-15 min insulin levels during glucose tolerance tests in HFD-fed male (6A) and female mice (6B).
Fig. 6A
Blood Glucose Levels (mg/dL)
350
300
250
4
*
3
2
1
0
Lepr
flox/flox
200
150
HFD-fed male Lepr flox/flox
100
HFD-fed male Pomc-Cre, Lepr flox/flox
50
0
15
30
45
60
Time (min)
75
90
105
120
Pomc-Cre,
flox/flox
Lepr
Page 47 of 49
Fig. 6B
4
Plasma insulin (ng/ml)
Blood Glucose Levels (mg/dL)
350
300
250
3
*
2
1
0
200
Lepr
flox/flox
150
100
HFD-fed female Lepr flox/flox
HFD-fed female Pomc-Cre, Lepr flox/flox
50
0
15
30
45
60
Time (min)
75
90
105
120
Pomc-Cre,
flox/flox
Lepr
Page 48 of 49
Figure 7: Glucose levels and glucose disappearance rates during insulin sensitivity tests in HFD-fed male (7A) and female mice (7B).
Fig. 7A
2.0
Glucose Disappearance Rate
1.5
1.0
Blood Glucose Levels (mg/dL)
200
*
0.5
0.0
Lepr
150
*
flox/flox
*
*
100
50
HFD-fed male Lepr flox/flox
HFD-fed male Pomc-Cre, Lepr flox/flox
0
0
15
30
Time (min)
45
60
Pomc-Cre,
flox/flox
Lepr
Page 49 of 49
Fig. 7B
Glucose Disappearance Rate
3.0
2.5
Blood Glucose Levels (mg/dL)
200
2.0
1.5
1.0
150
0.5
0.0
Lepr
flox/flox
100
Pomc-Cre,
flox/flox
Lepr
50
HFD-fed female Lepr flox/flox
HFD-fed female Pomc-Cre, Lepr flox/flox
0
0
15
30
Time (Min)
45
60