International Journal of Obesity (2011) 35, 226–235

& 2011 Macmillan Publishers Limited All rights reserved 0307-0565/11

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ORIGINAL ARTICLE
Spontaneous caloric restriction associated with
increased leptin levels in obesity-resistant aMUPA mice
O Froy1, H Sherman1, G Bhargava1, N Chapnik1, R Cohen2, R Gutman1, N Kronfeld-Schor2 and
R Miskin3
1
Robert H. Smith Faculty of Agriculture, Food and Environment, Institute of Biochemistry, Food Science and Nutrition,
The Hebrew University of Jerusalem, Rehovot, Israel; 2Faculty of Life Sciences, Department of Zoology, Tel Aviv University,
Tel Aviv, Israel and 3Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

Background: aMUPA mice carry as a transgene the cDNA encoding urokinase-type plasminogen activator, a member of the
plasminogen/plasmin system that functions in fibrinolysis and extracellular proteolysis. These mice spontaneously consume less
food when fed ad libitum and live longer compared with wild-type (WT) control mice. aMUPA mice are obesity resistant and
they share many similarities with calorically restricted animals. However, extensive metabolic characterization of this unique
transgenic model has never been performed.
Method: Metabolism of aMUPA mice was analyzed by measuring hormone, lipid and glucose levels in the serum, as well as
gene and protein expression levels in the liver, hypothalamus and brainstem.
Results: aMUPA mice were found to be leaner than WT mice mainly because of reduced fat depots. Serum analyses showed that
aMUPA mice have high levels of the anorexigenic hormones insulin and leptin, and low levels of the orexigenic hormone
ghrelin. Analyses of brain neuropeptides showed that the transcript of the anorexigenic neuropeptide Pomc is highly expressed
in the brainstem, whereas the expression of the orexigenic neuropeptides Npy, Orexin and Mch is blunted in the hypothalamus
of aMUPA mice. In addition, adenosine monophosphate (AMP)-activated protein kinase (AMPK) levels were higher in the liver
and lower in the hypothalamus, thus promoting simultaneously central reduction in appetite and peripheral loss of fat. The
levels of SIRT1 were low in the liver, but high in the hypothalamus, a feature that aMUPA mice share with calorically restricted
animals.
Conclusion: Taken together, aMUPA mice exhibit a unique metabolic phenotype of low-calorie intake and high leptin levels,
and could serve as a model for both spontaneous calorie restriction and resistance to obesity.
International Journal of Obesity (2011) 35, 226–235; doi:10.1038/ijo.2010.125; published online 15 June 2010

Keywords: aMUPA; satiety; calorie restriction; leptin; SIRT1

Introduction which subsequently activates the melanocortin receptor in the

The hypothalamus has a crucial role in determining energy
homeostasis.1 It is influenced by nutrients and hormones,
such as leptin, insulin, ghrelin and adiponectin. Leptin, a
satiety signal secreted from the adipose tissue, stimulates
anorexigenic neurons within the hypothalamic arcuate nu-
cleus to express pro-opiomelanocortin (POMC) and cocaine-
and amphetamine-regulated transcript (CART). POMC gives
rise posttranslationally to a-melanocyte-stimulating hormone,
Correspondence: Professor O Froy, Institute of Biochemistry, Food Sciences
and Nutrition, Hebrew University of Jerusalem, Rehovot 76100, Israel.
E-mail: froy@agri.huji.ac.il or Professor R Miskin, Department of Biological
Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
E-mail: ruth.miskin@weizmann.ac.il
Received 17 January 2010; revised 11 April 2010; accepted 12 May 2010;
published online 15 June 2010
paraventricular and lateral hypothalamus and results in
decreased food intake and increased energy expenditure.1 In
parallel, leptin suppresses and ghrelin activates a distinct set of
orexigenic neurons within the arcuate nucleus expressing
neuropeptide Y (NPY) and agouti-related protein (AgRP). These
peptides lead to the secretion of orexins and melanocyte-
concentrating hormone (MCH) from the lateral hypothalamus
and induce wakefulness and food-seeking behavior. Leptin and
ghrelin also affect orexin- and MCH-expressing neurons
directly.2,3 Thus, low levels of leptin and insulin during fasting
or starvation and high levels of ghrelin and adiponectin
activate orexigenic and inhibit anorexigenic neurons, leading
to food-seeking behavior and increased food intake.1
Leptin, ghrelin and insulin all affect adenosine mono-
phosphate (AMP)-activated protein kinase (AMPK) consi-
dered as the cellular ‘fuel gauge’, switching off anabolic

pathways and turning on adenosine triphosphate-producing
pathways.4 AMPK has a key role in both the liver and the
hypothalamus. During fasting, AMPK activation in the liver
leads to its phosphorylation, and, as a result, inactivation of
acetyl CoA carboxylase (ACC), the rate-limiting enzyme in
fatty acid synthesis. Consequently, the levels of malonyl
CoA, the product of ACC catalysis, decrease leading to
increased import of fatty acids into the mitochondrial matrix
and their subsequent oxidation. In the hypothalamus, AMPK
has a key role in whole body energy homeostasis. Activation
of hypothalamic AMPK is mediated by ghrelin and adipo-
nectin leading to an increase in orexigenic neuropeptides
and stimulation of food intake, whereas inactivation of
AMPK by glucose, insulin and leptin leads to the inhibition
of food intake.5–8 Other key players in energy metabolism
are SIRT1 and peroxisome-proliferator activator receptor-g
(PPARg) coactivator 1a (PGC-1a). SIRT1 is a mammalian
homolog of the yeast Sir2 protein,9 which is a member of the
sirtuin enzyme family comprising NAD þ (nicotinamide
adenine dinucleotide)-dependent deacetylases.10 SIRT1 is a
metabolic sensor that can be modulated by AMPK.11 PGC-1a
is a transcriptional coactivator which is upregulated during
fasting and regulates energy metabolism in multiple
tissues,12 including the liver. It coactivates PPARa13,14 and
PPARg,15 both of which have a critical role in regulating lipid
metabolism.16,17 PGC-1a can be directly phosphorylated by
AMPK and deacetylated by SIRT1, leading to an increase in
its transcriptional activity.11
aMUPA mice carry as a transgene the cDNA encoding
urokinase-type plasminogen activator (uPA) fused to the
enhancer-promoter region of the aÁ-crystallin gene.18 uPA
is an extracellular serine protease that has a role in fibrino-
lysis and tissue remodeling19 and has been implicated in
brain development and plasticity.20–25 aMUPA mice show
transgenic expression in the ocular lens as expected
from the promoter specificity, however, and also show
ectopic expression specifically in the brain and developing
teeth.26,27 aMUPA mice have been used as a model for caloric
restriction (CR), as these mice spontaneously consume
20–30% less food when fed ad libitum and live longer
(B20%) compared with wild-type (WT) control FVB/N
mice.28,29 In addition, aMUPA mice are resistant to obesity
and remain lean throughout their lifetime.26,30 aMUPA mice
exhibit additional similarities with calorically restricted
mice, such as reduced body weight and body temperature,
reduced levels of serum insulin-like growth factor-1 or
glucose, increased levels of mitochondrion-mediated apop-
tosis and reduced incidence of spontaneous and induced
cancerous lesions.26,31,32 The spontaneously reduced calorie
intake seen in aMUPA mice indicates that these mice differ
from WT mice and CR-treated animals in some aspects of
energy metabolism. However, an extensive characterization
of aMUPA mice metabolism has never been performed.
Therefore, our aim was to characterize the metabolic state
of aMUPA mice in the serum, liver, hypothalamus and
brainstem. Our results show that aMUPA mice exhibit
Reduced fat tissue and high leptin in aMUPA mice
O Froy et al
227
a satiated phenotype with increased leptin levels, a state
distinct from that of WT mice and CR-treated animals.
Materials and methods
Animal treatments and tissues
Three-month-old female aMUPA (n ¼ 41) and FVB/N (WT)
mice (n ¼ 41) were obtained from the Weizmann Institute of
Science (Rehovot, Israel). Mice were housed in a facility with
controlled temperature (20–22 1C) and humidity (60%)
under a 12:12 h light:dark cycle with regular chow available
ad libitum. After 8 weeks, on the last day of the experi-
ment, mice were fasted for 12 h. Mice were anesthetized
by an intraperitoneal injection of ketamine/xylazine
(100/7.5 mg kg1) and blood, liver, hypothalamus and brain-
stem were collected at two time points, ZT8 (n ¼ 20 from
each mouse type) and ZT16 (n ¼ 20 from each mouse type)
(ZT0Fthe time when lights turn on). These time points were
selected as the mid-rest and mid-activity phases, respectively.
At the time of blood collection, blood glucose was
measured using a digital glucometer (Optium Xceed, Abbott
Laboratories, Alameda, CA, USA). Triglycerides, total choles-
terol, high-density lipoprotein and low-density lipoprotein
were analyzed using an enzymatic test according to the
manufacturer’s instructions (Roche, Basel, Switzerland).
Tissues were immediately frozen in liquid nitrogen and
stored at 80 1C for further analysis. The Institutional
Animal Care and Use Committee (IACUC) of the Hebrew
University and Hadassah Medical Center approved the study
protocol for animal welfare. The Hebrew University is an
AAALAC (Association for Assessment and Accreditation of
Laboratory Animal Care) institute.
Body fat composition
Fat percentage was measured in 8-month-old female WT
and aMUPA mice (n ¼ 8 of each group) using double X-ray
absorption (GE Lunar PIXImus, Madison, WI, USA), as
described previously.33 During body composition measure-
ments, aMUPA and WT mice were anesthetized with
isoflurane using an anesthetic machine (Ohmeda Medical,
Laurel, MD, USA) with medical grade oxygen (2–3% vol).
Serum separation and enzyme-linked immunosorbent assay
Blood was kept at 4 1C for clotting and consequently
centrifuged at 8000  g for 15 min. Serum was collected and
stored at 20 1C for further analysis. Serum hormone levels
were determined for adiponectin (Linco Research Inc.,
St Charles, MO, USA), ghrelin (Linco Research Inc.), cortico-
sterone (AssayPro, St Charles, MO, USA), insulin (Mercodia,
Uppsala, Sweden), glucagon (Phoenix Pharmaceuticals Inc.,
Burlingame, CA, USA), cholecystokinin (Phoenix Pharmaceu-
ticals Inc.), peptide YY (PYY336) (Phoenix Pharmaceuticals
Inc.) and leptin (R&D Systems Inc., Minneapolis, MN,
International Journal of Obesity
 Reduced fat tissue and high leptin in aMUPA mice
O Froy et al
228
USA) using ELISA (enzyme-linked immunosorbent assay) kits.
All assays were performed according to the manufacturers’
instructions.
RNA extraction and quantitative real-time PCR
For gene expression analyses, RNA was extracted from
the hypothalamus, brainstem or liver using TRI Reagent
(Sigma, Rehovot, Israel) according to the manufacturer’s
instructions. RNA quality was validated using spectrometry
by calculating 260/280 nm and 260/230 nm ratios. Total RNA
was DNase I treated using RQ1 DNase (Promega, Madison,
WI, USA) for 2 h at 37 1C as previously described.34 In all, 2 mg
of DNase I-treated RNA was reverse transcribed using
MMuLV reverse transcriptase (Promega) and random
hexamers. Of the reaction, 1/20 was then subjected to
quantitative real-time PCR using the primers spanning
intron–exon boundaries and the ABI Prism 7300 Sequence
Detection System (Applied Biosystems, Foster City, CA, USA).
Primers for Pomc, Npy, Orexin, Mch, Agrp, Ppara, Pparg and
Pgc-1a were tested alongside the normalizing gene glyceral-
dehyde-3-phosphate dehydrogenase (Gapdh) (Supple-
mentary Table S1). Primers were designed with Primer
express v.2 (Applied Biosystems) and validated by a standard
curve and dissociation curve of the product. The fold change
in target gene expression was calculated by the 2DDCt
relative quantification method (Applied Biosystems). All
genes showed the same trend in both aMUPA and WT mice.
Some showed differences between ZT8 and ZT16 but with
the same trend in both mouse types. Although differences
between ZT8 and ZT16 in both aMUPA mice and WT mice
could suggest a circadian pattern, we could not rely on two
time points to determine circadian rhythmicity. ZT8 and
ZT16 for each gene were averaged for a better assessment of
transcript levels.
Western blot analysis
Tissue samples were homogenized in lysis buffer (20 mM Tris,
145 mM NaCl, 5% glycerol, 1% Triton, 100 mM phenylmethyl-
sulfonyl fluoride, 50 mM NaF, 1 mM sodium orthovanadate,
10 mg ml1 leupeptin, 10 mg ml1 aprotinin, 0.8 mg ml1 tryp-
sin inhibitors), as described previously.35 Samples were run
on an SDS-polyacrylamide gel (10% for AMPK and SIRT1,
7.5% for ACC) and subsequently proteins were transferred
into a nitrocellulose membrane. Blots were incubated with
mouse anti-AMPK/pAMPK/ACC/pACC (Cell Signaling Tech-
nology Inc., Danvers, MA, USA) or anti-SIRT1 (Millipore,
Billerica, MA, USA) antibodies and after several washes with
anti-rabbit IgG horseradish peroxidase-conjugated secondary
antibody (Cell Signaling Technology Inc.). b-Actin was used
as the loading control. The immune reaction was detected by
enhanced chemiluminescence. Bands were quantified by
scanning and densitometry and expressed as arbitrary units.
For a better assessment of protein levels, ZT8 and ZT16 were
averaged.
International Journal of Obesity
Statistics
Student’s t-test was used for comparison between mouse
strains. For all analyses, the significance level was set at
Po0.05.
Results
Body composition of aMUPA and WT mice
Analysis of body composition showed that aMUPA mice
weigh less than WT mice, their genetic background (aMUPA,
25.0±1 g; WT, 31.8±4.7 g; Po0.01) (Figure 1). In addition,
the fat percentage of aMUPA mice was significantly lower
than that of WT mice (aMUPA, 21.7±3.0%; WT, 34.6±3.1%;
Po0.01) (Figure 1). The 5.84 g difference in fat mass (aMUPA,
5.47±0.8 g; WT, 11.3±1.51g; Po0.01) (Figure 1) explained
85.8% of the difference in body weight. Taken together,
these results show that aMUPA mice are leaner than WT
mice mainly because of reduced fat depots.
Levels of glucose, lipids and hormones in the serum
of aMUPA and WT mice
To characterize the metabolic status of aMUPA mice, we first
measured glucose and lipid levels in the serum and
compared them with those of WT mice. To better evaluate
the levels of these parameters, we measured them at two
time points, mid-rest phase (ZT8) and mid-activity phase
(ZT16). Glucose and triglyceride levels were not significantly
different in the two strains after a 12-h fasting period
(Table 1). However, cholesterol levels, total cholesterol, high-
density lipoprotein and low-density lipoprotein were signi-
ficantly higher (Po0.05) in aMUPA mice than in WT mice
(Table 1). Although cholesterol levels were slightly higher in
aMUPA mice, they were within the normal range.
We next measured the concentrations of several hormones
that regulate feeding, that is, insulin, glucagon, leptin,
ghrelin, adiponectin, PYY336, cholecystokinin and cortico-
sterone. Glucagon, adiponectin, cholecystokinin and cortico-
sterone did not show significant difference between aMUPA
and WT mice (Table 1). In contrast, PYY336 exhibited high
40
Body weight
* Body fat
30
Weight (g)
20
*
10
0
WT αMUPA
Figure 1 Body and fat weight of 8-month-old aMUPA and WT mice.
Fat weight was determined using double X-ray absorption (DEXA). *Po0.01,
t-test between lines of mice.
 Reduced fat tissue and high leptin in aMUPA mice
O Froy et al
229
levels in WT mice than in aMUPA mice (Po0.05) (Figure 2), hormones insulin and leptin, and low levels of the hunger
with expected higher levels during the feeding phase (data hormone ghrelin.
not shown). Insulin levels were B3.5-fold higher in aMUPA
mice than in WT mice (Po0.05) (Figure 2). Similarly, leptin
levels were B4.5-fold higher in aMUPA mice than in WT mice Levels of mRNA expression of PPARa, PPARg and PGC-1a
(Po0.05) (Figure 2). In aMUPA mice, the levels of leptin in the liver
inversely correlated with those of body fat (1739.2pg ml1 per As fat depots are the main difference between aMUPA and
g fat) compared with WT mice (274.9 pg ml1 per g fat), WT mice, we determined the expression level of several
yielding a 6.3-fold ratio difference (Po0.001, coefficient transcription factors involved in lipid metabolism. In
correlation of 0.73 for WT mice and 0.86 for aMUPA mice). aMUPA mice, Pgc-1a mRNA levels were decreased, whereas
In correlation with leptin and insulin, ghrelin exhibited lower those of Pparg were significantly higher than in WT mice
levels in aMUPA mice than in WT mice (Po0.05) (Figure 2). (Po0.05) (Figure 3). Ppara was not significantly different
As expected, in both strains, ghrelin showed a daily variation between aMUPA and WT mice (Figure 3).
being high during the rest period and low during the feeding
period (data not shown). Taken together, these results
show that aMUPA mice have high levels of the satiety Levels of brain neuropeptides in aMUPA and WT mice
We next examined the mRNA level of neuropeptides
involved in feeding behavior in the brainstem and hypo-
thalamus, the two regions in the brain involved in energy
Table 1 Glucose, lipid and hormone levels in the serum of WT and aMUPA homeostasis. We focused on the anorexigenic neuropeptide
mice
POMC and the orexigenic neuropeptides NPY, orexin, MCH
WT aMUPA and AgRP. In the hypothalamus, mRNA expression levels
of Pomc and Agrp did not differ between the two strains
Glucagon (ng ml1) 0.21±0.03 0.22±0.04
Glucose (mg per 100 ml) 64.71±2.11 66.32±4.71 (Figure 4). However, expression levels of the orexigenic
CCK (pg ml1) 90±30 70±10 neuropeptides Npy, Orexin and Mch were significantly
Corticosterone (ng ml1) 6.64±1.23 8.92±1.43 reduced in the hypothalamus of aMUPA mice by approxi-
Adiponectin (mg ml1) 17.56±0.80 16.06±0.60 mately fivefold (Po0.05) (Figure 4). In the brainstem, Pomc
Cholesterol (mg per 100 ml) 118.33±3.77 139.86±3.7*
LDL (mg per 100 ml) 8.33±0.5 11.71±0.9* exhibited B3-fold higher level of expression in aMUPA mice
HDL (mg per 100 ml) 92.7±1.9 113.03±2.91* (Po0.05) (Figure 4). As expected,36 Agrp expression could not
Triglycerides (mg per 100 ml) 125.7±12.02 123±9.2 be detected in the brainstem (Figure 4). Taken together, these
Abbreviations: CCK, cholecystokinin; HDL, high-density lipoprotein; LDL,
results show that the anorexigenic neuropeptide Pomc is
low-density lipoprotein; WT, wild type. Levels are expressed as mean ± s.e. highly expressed in the brainstem, whereas the expression of
*Po0.05. the orexigenic neuropeptides Npy, Orexin and Mch is

8 4.5
7 * 4 *
Leptin (ng/ml)

6 3.5
Insulin (I.U.)

5 3
2.5
4 2
3 1.5
2 1
1 0.5
0 0
WT αMUPA WT αMUPA

2.5 70
60
PYY3-36 (pg/ml)
Ghrelin (ng/ml)

2
50
1.5 40 *
*
1 30
20
0.5
10
0 0
WT αMUPA WT αMUPA
Figure 2 Hormone levels of WT and aMUPA mice. Blood was collected from WT or aMUPA mice. ELISA was used to determine insulin (a), leptin (b), ghrelin (c) and
PYY336 (d) levels in the serum. Levels of ZT8 and ZT18 were averaged. Levels are mean±s.e.m., n ¼ 16 for each strain, *Po0.05.

International Journal of Obesity

 Reduced fat tissue and high leptin in aMUPA mice
O Froy et al
230
were significantly higher (Po0.05) (Figure 5). In the hypo-
1.4
Relative Pgc-1mRNA levels thalamus, total AMPK protein levels were significantly
1.2 higher in aMUPA mice than in WT mice (Po0.05) (Figure 5).
1
However, hypothalamic pAMPK levels were significantly
lower in aMUPA mice compared with those of WT mice
0.8 (Po0.05) (Figure 5). In the brainstem, neither average AMPK
0.6 *
nor pAMPK levels differed between the mouse strains
(Figure 5). As AMPK phosphorylates and, hence, inactivates
0.4
ACC, we determined total ACC protein levels and its
0.2 phosphorylated fraction. In both the liver and the hypo-
0 thalamus, we found significantly higher levels of ACC and
WT αMUPA pACC in aMUPA mice compared with WT mice (Po0.05)
(Figure 6).
2.5 As aMUPA mice live longer and share similar features with
Relative PparmRNA levels

* animals under CR,26 we evaluated SIRT1 levels, a key

2 metabolic regulator that has been implicated in the in vivo
response to CR37 and CR-mediated life span extension.38 In
1.5 the liver, levels of the SIRT1 protein were significantly lower
in aMUPA mice than in WT mice (Po0.05) (Figure 7). In
1 contrast, hypothalamic SIRT1 protein levels were signi-
ficantly higher (Po0.05) in aMUPA mice than in WT mice
0.5 (Figure 7).

0
WT αMUPA
Discussion
Relative mPpar mRNA levels

1.4
1.2 The results of this study indicate that aMUPA mice have a
unique metabolic phenotype of reduced calorie intake
1 accompanied with satiety. Accordingly, aMUPA mice have
0.8 significantly reduced fat depots in their body compared with
their genetic background WT mice. Hormonal analyses
0.6
reflect quite clearly that aMUPA mice exhibit a satiated state
0.4 compared with WT mice, as they have high serum levels of
0.2 leptin and insulin, which induce satiety, and low levels of
ghrelin, a hunger-inducing peptide.8,39 These results are
0
WT αMUPA compelling, as the animals were fasted 12 h before all
analyses and they have less leptin-producing fat tissue.
Figure 3 Pgc-1a, Pparg and Ppara mRNA expression levels in the liver. Liver
tissue was collected from WT or aMUPA mice and total RNA was extracted. Ghrelin has been shown in some studies to have an
Real-time PCR analyses were performed to determine mRNA level of Pgc-1a inhibitory effect on insulin secretion,40 explaining, at least
(a), Pparg (b) and Ppara (c). Levels are mean±s.e.m., n ¼ 16 for each strain, in part, the higher levels of insulin in aMUPA mice. The
*Po0.05.
levels of PYY336, which is secreted in the small intestine
after the meal, are known to correlate with the amount of
blunted in the hypothalamus of aMUPA mice, leading to digested calories and inhibit food intake for several hours.41
overall satiety. As aMUPA mice consume fewer calories than do WT mice,
the low levels found in aMUPA are expected.
Reduced food intake, which occurred unexpectedly in
Levels of AMPK, ACC and SIRT1 in aMUPA and WT mice aMUPA mice, was detected in two transgenic lines26,31 thus
As insulin, leptin and ghrelin affect the activation of AMPK, pointing to uPA, the product of transgenic expression, as the
the energy ‘gauge’ in the cell, which has a critical role in primary causative factor. The transgenic effect is likely to be
energy homeostasis, we evaluated its levels in the liver, developmental, similarly to the impressive remodeling effect
hypothalamus and brainstem. Ampk mRNA was significantly recently detected in aMUPA developing incisor teeth.27 More
(Po0.05) lower in the liver, brainstem and hypothalamus in specifically, the two transgenic lines show ectopic expression
aMUPA mice than in WT mice (Figure 5). At the protein of uPA in the trigeminal nucleus of the brain stem, a region
level, AMPK levels were lower in aMUPA mice in the devoid of uPA expression in the WT brain.26 Thus, although
liver, but its phosphorylated active form (pAMPK) levels this region has not been associated so far with food intake, it

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 Reduced fat tissue and high leptin in aMUPA mice
O Froy et al
231
Hypothalamus Brainstem

Relative Pomc mRNA levels

2 3.5 *
1.8
3
1.6
1.4 2.5
1.2 2
1
0.8 1.5
0.6 1
0.4
0.2 0.5
0 0
WT αMUPA WT αMUPA
Relative Npy mRNA levels

1.4 1.6
1.2 1.4
1 1.2
1
0.8
0.8
0.6
* 0.6
0.4
0.4
0.2 0.2
0 0
WT αMUPA WT αMUPA
Relative Mch mRNA levels

1.4 1.4
1.2 1.2
1 1
0.8 0.8
0.6 * 0.6
0.4 0.4
0.2 0.2
0 0
WT αMUPA WT α-MUPA
Relative Agrp mRNA levels Relative Orexin mRNA levels

1.4
1.2
1
0.8
0.6
0.4 *
0.2
0
WT αMUPA
1.4
1.2
1
0.8
0.6
0.4
0.2
0
WT αMUPA
Figure 4 Neuropeptide mRNA expression levels in the hypothalamus and brainstem. Tissues were collected from WT and aMUPA mice. Real-time PCR analyses
were performed to determine mRNA levels of Pomc, Npy, Mch and Agrp in hypothalamus and brainstem. Data were normalized to WT. Levels are mean±s.e.m.,
n ¼ 16 for each strain, *Po0.05.

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 Reduced fat tissue and high leptin in aMUPA mice
O Froy et al
232
Liver Hypothalamus Brainstem
Relative Ampk mRNA levels 1.2 1.2 1.4
1 1 1.2
*
0.8 1
0.8 *
* 0.8
0.6 0.6
0.6
0.4 0.4 0.4
0.2 0.2 0.2
0 0 0
WT αMUPA WT αMUPA WT αMUPA
Relative AMPK protein levels

1.2 2.5 1.4

*
1 1.2
2
0.8 1
* 1.5 0.8
0.6
1 0.6
0.4 0.4
0.2 0.5
0.2
0 0 0
WT αMUPA WT αMUPA WT αMUPA
Relative pAMPK protein levels

2.5 * 1.2 2.5

2 1 2
0.8 *
1.5 1.5
0.6
1 1
0.4
0.5 0.2 0.5
0 0 0
WT αMUPA WT αMUPA WT αMUPA

Figure 5 Ampk mRNA and AMPK and pAMPK protein levels in the liver, hypothalamus and brainstem. Tissues were collected from WT and aMUPA mice. Real-time
PCR analyses were performed to determine Ampk mRNA levels and western blots to determine AMPK and pAMPK. Levels are mean±s.e.m., n ¼ 16 for each strain,
*Po0.05.
Relative pACC protein levels Relative ACC protein levels

Liver Hypothalamus
2.5 2.5 *
*
2 2
1.5 1.5
1 1
0.5 0.5
0 0
WT αMUPA WT αMUPA

2.5 * 2.5 *
2 2
1.5 1.5
1 1
0.5 0.5
0 0
WT αMUPA WT αMUPA

Figure 6 ACC and pACC protein levels in the liver and hypothalamus. Tissues were collected from WT and aMUPA mice. ACC and pACC protein levels were
determined by western blots. Levels are mean±s.e.m., n ¼ 16 for each strain, *Po0.05.

is plausible that the transgenic expression in the trigeminal nucleus tractus solitarius and dorsal motor of the vagus, two
nucleus is responsible for the changes detected in aMUPA. brain areas that regulate food intake,42 may contribute to the
The close proximity of the trigeminal nucleus with the enervation of adipose tissue through the autonomic nervous

International Journal of Obesity


Relative SIRT1 protein levels
Liver
1.4
1.2
1 1.2
0.8
0.6
0.4
0.2
0 0
WT
Figure 7 SIRT1 protein levels in the liver and hypothalamus. Tissues were collected from WT and aMUPA mice. SIRT1 protein levels were determined by western
blots. Levels are mean±s.e.m., n ¼ 16 for each strain, *Po0.05.
system resulting in oversecretion of leptin in aMUPA mice
notwithstanding low fat body levels. The satiety exhibited
in aMUPA mice, as detected in this study, the capacity of
aMUPA mice to accomplish their daily dose within a 3-h
period under restricted feeding43 and the finding that
aMUPA mice ate less even when fed pulverized food26 all
reiterate the fact that the superficial enamel alterations
previously reported in aMUPA incisors27 do not affect
feeding in these transgenic mice.
The higher levels of leptin and insulin and lower levels of
ghrelin in aMUPA could be the key to their altered energy
homeostasis. High levels of leptin and insulin lead to inhi-
bition of NPY/AgRP neurons and decreased hypothalamic
AMPK activation, thus, leading to overall satiety.4,44 In
contrast, high ghrelin levels lead to activation of NPY/AgRP
neurons and AMPK, thus leading to increased appetite.39,44
Indeed, pAMPK levels, the activated form of AMPK, were
lower in the hypothalamus in aMUPA mice. In the lateral
hypothalamus, inhibition of NPY/AgRP by leptin also leads
to decreased expression of orexin and MCH, peptides which
induce wakefulness and food-seeking behavior.45 Indeed, in
aMUPA mice, the levels of these orexigenic neuropeptides
were lower in the hypothalamus compared with WT mice.
These findings were supported by increased levels of Pomc
expression in their brainstem, as POMC neurons in the
nucleus tractus solitarius have been shown to relay satiety
signals from the periphery to the hypothalamus.46 In
parallel, in the liver, leptin and insulin lead to AMPK
activation and ghrelin to AMPK suppression.39 Activation
of AMPK in the liver leads to inhibition of fatty acid
synthesis and increased fatty acid oxidation.4 Indeed,
pAMPK levels were higher in the liver in aMUPA mice. The
opposite effects on AMPK in aMUPA mice, inhibition in the
hypothalamus alongside activation in the liver, promote
simultaneously central reduction in appetite and peripheral
loss of fat. It is also noted that although pAMPK levels were
lower in the hypothalamus, the levels of pACC were higher
in aMUPA mice. These results may indicate that ACC could
be phosphorylated by other kinases or that its phosphatase
activities are reduced, at least in this brain area.
As PGC-1a is activated under fasting in the liver,12 the
reduction in its mRNA expression levels supports the satiated
Reduced fat tissue and high leptin in aMUPA mice
O Froy et al
233
Hypothalamus
1.6 *
1.4
* 1
0.8
0.6
0.4
0.2
αMUPA WT αMUPA
state of aMUPA. In contrast, Pparg expression levels were
higher than those of WT mice. As PPARg is involved in fatty
acid import into the cell,16 the increase in Pparg expression
could stem from the high fatty acid oxidation in the liver,
as a result of AMPK activation. As PPARg requires PGC-1a
as a transcription coactivator, and PGC-1a remained low,
this could explain why lipogenic and adipogenic pathways
in aMUPA were not activated. The low levels of the SIRT1
protein correlate with the low Pgc-1a and high Pparg
expression levels, as SIRT1 represses PPARg and activates
PGC-1a.47
aMUPA mice share some similarities with those of Lou/C,
both obesity-resistant long-lived rodents. However, there are
some fundamental differences in Lou/C rats compared with
aMUPA mice, such as increased levels of PGC-1a and SIRT1
in the liver, increased levels of ghrelin and reduced levels
of leptin and insulin in the serum, although with some
improved sensitivity for the latter two hormones.48,49 Over-
all, it seems that aMUPA mice are metabolically different
from Lou/C rats.
Although aMUPA mice exhibit reduced calorie intake and
body fat, they show remarkable differences in energy
metabolism compared with CR-treated animals. In parti-
cular, calorically restricted animals exhibit high levels of
ghrelin and low levels of leptin50 and insulin.51 In addition,
CR-treated mice exhibit high expression levels of PGC-1a
and no change in Pparg levels in the liver.50,52 These findings
are in sharp contrast with those found in aMUPA mice.
Despite these differences, both calorically restricted ani-
mals51 and aMUPA mice26 benefit from the reduced calories
as both show improved health and extended life span.
Interestingly, aMUPA mice showed low SIRT1 expression in
the liver similar to CR-treated animals53 and high levels in
the hypothalamus similar to starved animals.54 Results
obtained with SIRT1-null mice have recently suggested that
this enzyme is required for in vivo response to CR.37 It seems
that SIRT1 levels could be elevated in the hypothalamus
through AMPK activation in calorically restricted mice,55
and through leptin signaling54 in aMUPA mice.
Recent studies have shown that SIRT1 interacts directly with
CLOCK and deacetylates BMAL1 and PER2, proteins of the
core mechanism of the circadian clock.56,57 In addition, SIRT1
International Journal of Obesity
 Reduced fat tissue and high leptin in aMUPA mice
O Froy et al
234
mRNA was found to be abundant in the suprachiasmatic
nucleus,54 a hypothalamic area comprising the central
circadian clock. The suprachiasmatic nucleus expresses leptin
receptors58 and could respond to direct exposure to leptin.59,60
Therefore, we suggest that in aMUPA mice, the high leptin
levels could facilitate SIRT1 activation in the suprachiasmatic
nucleus, which, in turn, leads to robust circadian rhythms, as
we have shown.30,34 As robust circadian rhythms can extend
the life span and CR is capable of entraining the suprachias-
matic nucleus clock,30 the increased hypothalamic SIRT1
levels in aMUPA mice could be a key mediator through which
their circadian clock is synchronized, thus improving health
and extending longevity.
In summary, unlike CR-treated animals and Lou/C rats,
aMUPA mice show an unusual combination of low-calorie
intake and metabolism of satiated animals with high levels
of leptin. Increased leptin levels lead, most likely, to the
reduced hypothalamic expression of orexigenic neuropep-
tides, which, in turn, reduces appetite. Although the
mechanism by which aMUPA mice exhibit such features
remains to be elucidated, aMUPA mice could be a model for
both spontaneous calorie restriction and resistance to
obesity.
Conflict of interest
The authors declare no conflict of interest.
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