Peptide YY ablation in mice leads to the development of hyperinsulinaemia and obesity.

Abstract

Obese people exhibit reduced circulating peptide YY (PYY) levels, but it is unclear whether this is a consequence or cause of obesity. We therefore investigated the effect of Pyy ablation on energy homeostasis.
Body composition, i.p. glucose tolerance, food intake and hypothalamic neuropeptide expression were determined in Pyy knock-out and wild-type mice on a normal or high-fat diet.
Pyy knock-out significantly increased bodyweight and increased fat mass by 50% in aged females on a normal diet. Male chow-fed Pyy (-/-) mice were resistant to obesity but became significantly fatter and glucose-intolerant compared with wild-types when fed a high-fat diet. Pyy knock-out animals exhibited significantly elevated fasting or glucose-stimulated serum insulin concentrations vs wild-types, with no increase in basal or fasting-induced food intake. Pyy knock-out decreased or had no effect on neuropeptide Y expression in the arcuate nucleus of the hypothalamus, and significantly increased proopiomelanocortin expression in this region. Male but not female knock-outs exhibited significantly increased growth hormone-releasing hormone expression in the ventromedial hypothalamus and significantly elevated serum IGF-I and testosterone levels. This sex difference in activation of the hypothalamo-pituitary somatotrophic axis by Pyy ablation may contribute to the resistance of chow-fed male knock-outs to late-onset obesity.
PYY signalling is important in the regulation of energy balance and glucose homeostasis, possibly via regulation of insulin release. Therefore reduced PYY levels may predispose to the development of obesity, particularly with ageing or under conditions of high-fat feeding.

Full text

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Diabetologia (2006) 49: 1360–1370
DOI 10.1007/s00125-006-0237-0
ARTICLE
D. Boey.S. Lin.T. Karl.P. Baldock.N. Lee.
R. Enriquez.M. Couzens.K. Slack.R. Dallmann.
A. Sainsbury.H. Herzog
Peptide YY ablation in mice leads to the development
of hyperinsulinaemia and obesity
Received: 6 September 2005 / Accepted: 10 February 2006 / Published online: 21 April 2006
# Springer-Verlag 2006
Abstract Aims/hypothesis: Obese people exhibit reduced
circulating peptide YY (PYY) levels, but it is unclear
whether this is a consequence or cause of obesity. We
therefore investigated the effect of Pyy ablation on energy
homeostasis.Methods: Body composition, i.p. glucose
tolerance, food intake and hypothalamic neuropeptide
expression were determined in Pyy knock-out and wild-
type mice on a normal or high-fat diet.
knock-out significantly increased bodyweight and in-
creased fat mass by 50% in aged females on a normal
diet. Male chow-fed Pyy−/−mice were resistant to obesity
but became significantly fatter and glucose-intolerant
compared with wild-types when fed a high-fat diet. Pyy
knock-out animals exhibited significantly elevated fasting
or glucose-stimulated serum insulin concentrations vs
wild-types, with no increase in basal or fasting-induced
food intake. Pyy knock-out decreased or had no effect
on neuropeptide Yexpression in the arcuate nucleus of the
hypothalamus, and significantly increased proopiomelano-
cortin expression in this region. Male but not female
Results: Pyy
knock-outs exhibited significantly increased growth hor-
mone-releasing hormone expression in the ventromedial
hypothalamus and significantly elevated serum IGF-I and
testosterone levels. This sex difference in activation of the
hypothalamo–pituitary somatotrophic axis by Pyy ablation
may contribute to the resistance of chow-fed male knock-
outs to late-onset obesity. Conclusions/interpretation: PYY
signalling is important in the regulation of energy balance
and glucose homeostasis, possibly via regulation of insulin
release. Therefore reduced PYY levels may predispose to
the development of obesity, particularly with ageing or
under conditions of high-fat feeding.
Keywords Diabetes.Hyperinsulinaemia.Obesity.
Peptide YY
Abbreviations DXA: dual-energy X-ray absorptiometry.
GHRH: growth hormone-releasing hormone.NPY:
neuropeptide Y.POMC: proopiomelanocortin.PPY:
pancreatic polypeptide.PYY: peptide YY
Introduction
The gut hormone peptide YY (PYY) belongs to the
neuropeptide Y (NPY) family along with pancreatic
polypeptide (PPY) [1]. PYY, PPY and NPY mediate their
effects through Y receptors, of which there are several
types (Y1, Y2, Y4, Y5 and Y6) [2]. The endocrine L cells
of the lower gastrointestinal tract are the major source of
PYY; however, PYY is also produced in the alpha cells of
the islets of Langerhans as well as in the stomach and the
brainstem [3].
There are two endogenous forms of PYY: PYY1-36 and
PYY3-36. The latter is produced by the action of the cell-
surface enzyme dipeptidyl peptidase IVon secreted PYY1-
36 [4]. PYY1-36 binds to all known Y receptors, albeit
with differing affinities, whereas PYY3-36 is more selec-
tive for the Y2 receptor and to a lesser extent to the Y5
receptor [5]. Upon ingestion of a meal, PYY levels rise
within 15 min, peak at 60 min and remain elevated for up to
The last two authors named contributed equally to the work.
D. Boey.S. Lin.T. Karl.N. Lee.M. Couzens.K. Slack.
A. Sainsbury.H. Herzog (*)
Neurobiology Research Programme,
Garvan Institute of Medical Research, St Vincent’s Hospital,
384 Victoria Street, Darlinghust,
Sydney, NSW 2010, Australia
e-mail: h.herzog@garvan.org.au
Tel.: +61-29-2958296
Fax: +61-29-2958281
P. Baldock.R. Enriquez
Bone and Mineral Research Programme,
Garvan Institute of Medical Research, St Vincent’s Hospital,
Sydney, NSW, Australia
R. Dallmann
Department of Zoology, University of Toronto,
Toronto, ON, Canada
T. Karl
Neuroscience Institute of Schizophrenia and Allied Disorders,
Sydney, NSW, Australia

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6 h in humans [6]. The initial rise of PYYappears to be due
to an indirect neural reflex, whereas the sustained release of
PYY appears to be a direct effect of intraluminal contents
on endocrine L cells [7]. Dietary fat is the most potent
stimulant of PYY release [6].
Both PYY1-36 [8, 9] and PYY3-36 suppress appetite
and food intake in rodents [10–14]. However, one report
was unable to confirm this finding [15]. Most studies
investigating the role of PYY in energy balance have
focused primarily on its effects on feeding and bodyweight,
and less is known about the role of PYY in influencing
body composition and hormonal and metabolic parameters
that influence it, such as leptin etc. Indeed, many factors
that play a significant role in regulating energy homeostasis
do so in the absence of corresponding effects on food
intake or bodyweight [16, 17]. There is evidence that PYY
could influence energybalance via multiple pathways aside
from effects on food intake. Notably, PYY has been shown
to slow the transit of food through the gastrointestinal tract,
delay gastric and gallbladder emptying [6, 18] and delay
pancreatic and intestinal secretion [19] via direct actions on
target tissues or indirect actions via vagal pathways [7].
It has also been implied that PYY has an important
function in regulating insulin release and glucose homeo-
stasis. Previous in vitro studies demonstrate that PYYacts
to inhibit insulin secretion and glucose-stimulated insulin
release from isolated pancreatic islets [20, 21]. In obese
rodent models PYY3-36 reinforces insulin action on
glucose disposal independently of changes in food intake
and bodyweight [14, 22]. Since changes in gastrointestinal
function and circulating insulin concentrations per se have
been shown to have profound effects on energy balance
[23–25], it is possible that PYY may influence long-term
energy balance via these mechanisms.
The aim of this work was to elucidate the role of PYYin
the regulation of multiple hormonal and metabolic
determinants of lean and fat mass. To this end, we
generated Pyy knock-out mice and studied them under a
range of conditions that are known to influence energy
homeostasis, notably fasting, ageing and a high-fat diet.
Material and methods
Animal care
All research and animal care procedures were approved by
the Garvan Institute/St Vincent’s Hospital Animal Experi-
mentation Ethics Committee.
Pyy targeting vector construction and gene disruption
The 130 kb mouse genomic BAC clone was mapped and
various fragments were subcloned. A 10.5 kb SpeI fragment
containing 6 kb 5′-flanking sequence, the entire Pyy gene
and a 3 kb 3′-flanking sequence was chosen for the
construction of a Pyy-Cre knock-in construct. The linearised
version of that clone was transfected into ES cells. Two
positive clones were injected into C57BL/6 blastocysts and
chimericmicewerebredtogenerateheterozygous mice,and
subsequently homozygous Pyy-Cre knock-in mice were
bred. The genotype of the mice were determined by
Southern blot analysis of NheI-digested DNA employing
probes located outside the targeting sequence. Probe A and
B were generated by PCR (A for: 5′-AGTGATTTGCTCA
GAAGC-3′ and rev: 5′-CTAGTTCTATAGACCAGAC-3′)
and (B for: 5′-CTGCCATGGCTGACCATGC-3′ and B rev:
5′-TGGT GGTGGCATGCACAC-3′).The conditions for all
PCR reactions were 35 cycles of 94°C for 45 s, 58°C for
1 min and 72°C for 20 s.
Measurement of food intake and bodyweight
Wild-type and Pyy−/−animals originating from three or
four different breeding pairs were housed under conditions
of controlled temperature (22°C) and lighting (12-h light
cycle, lights on at 07.00 h). Male and female wild-type
and Pyy−/−mice had free access to a normal chow diet
(6% of calories from fat, 21% from protein, 71% from
carbohydrate; 2.6 kcal/g; Gordon’s Speciality Stock
Feeds, Yanderra, NSW, Australia). Separate groups of
wild-type and Pyy−/−mice were fed a high-fat diet (46%
of calories from fat, 21% from protein, 33% from
carbohydrate; 4.72 kcal/g) from 4 to 5 weeks of age
onwards. The high-fat diet, made in-house, was based on
the composition of Rodent Diet Catalogue Number
D12451 (Research Diets, New Brunswick, NJ, USA),
with the exception that safflower oil and copha were used
in place of soybean oil and lard, respectively. Bodyweight
was monitored weekly from 4 to 28 weeks of age for the
chow-fed animals and 4 to 14 weeks of age for the high-
fat-fed animals. At 11 to 12 weeks of age, basal and
fasting-induced food and water intake were measured.
Basal food and water intake were also determined at
24 weeks of age in male and female Pyy−/−and control
mice.
Glucose tolerance tests
At 13 weeks of age, chow-fed and high-fat-fed wild-type
and Pyy knock-out mice were fasted for 24 h prior to i.p.
glucose tolerance tests (1 g/kg). Serial blood samples were
collected from the tail for determination of serum glucose
and insulin levels. Glucose AUCs were calculated
between 0 and 120 min after glucose injection and
expressed as mmol·l−1·min−1. Insulin AUCs were calcu-
lated between 0 and 120 min after glucose injection and
expressed as pmol·l−1·min−1·g bodyweight−1.
Tissue collection and analysis
Wild-type and Pyy−/−mice at 14 or 28 weeks of age were
killed by cervical dislocation between 12.00 and 14.00 h
for collection of trunk blood. Brains were immediately
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removed and frozen on dry ice. Mice were scanned for
whole-body lean and fat mass using a dual-energy X-ray
absorptiometry (DXA) (Lunar PIXImus2 mouse densitom-
eter; GE Healthcare Technologies, Waukesha, WI, USA).
White adipose tissue depots (right inguinal, right epidid-
ymal or periovarian [gonadal], right retroperitoneal and
mesenteric) were removed and weighed. Serum glucagon
levels were measured by RIA kits from Linco Research (St
Louis, MO, USA), insulin levels were measured using an
ELISA kit from Mercodia (Uppsala, Sweden), serum-free
T4 and testosterone concentrations were measured with
kits from ICN Biomedicals (Costa Mesa, CA, USA), serum
IGF-I was determined using an RIA kit from Bioclone
(Marrickville, NSW, Australia), serum glucose was
determined with a glucose oxidase assay kit (Trace
Scientific, Melbourne, VIC, Australia), and serum triglyc-
eride and NEFAs were determined with kits from Roche
Diagnostics (Mannheim, Germany) and Wako (Osaka,
Japan), respectively.
PYY, glucagon and PPY staining
Pancreas and segments of the small intestine and colon
obtained from three wild-type and three knock-out animals
were fixed in 4% paraformaldehyde in PBS, processed,
embedded in paraffin and cut at 7 μm sections. Sections
were deparaffinised, rehydrated and incubated in 1% H2O2
in methanol for 20 min. Sections were then rinsed in PBS
and blocked with either 20% normal goat serum or donkey
serum in PBS for 20 min. Rabbit anti-human PYY
antiserum (1:1,000) (Peninsula Laboratories, CA, USA),
rabbit anti-glucagon antibody (1:500) (ICN), or guinea pig
anti-rat PPY serum (Linco Research) (1:1,000) was applied
for 1 h at room temperature. For the PYY antibody, cross-
reactivity was minimised by incubating the antiserum with
protein extracted from brain of Pyy−/−mice. Slides were
rinsed in PBS before incubation with a peroxidase-
conjugated goat anti-rabbit IgG (H+L) antibody (1:1,000)
(Zymed Laboratories, San Francisco, CA, USA) or a
biotinylateddonkeyanti-guinea-pig
(1:5,000) (Jackson ImmunoResearch Laboratories, West
Grove, PA, USA) for 30 min at room temperature. The
sections incubated with the biotinylated antibody were
rinsed in PBS and incubated with Avidin–Biotin–Peroxi-
dase Vectastain (Vector Laboratories, Burlingame, CA,
USA) for 30 min at room temperature. Sections were
washed in PBS and treated with diaminobenzidine from
DAKO (Carpinteria, CA, USA) for 5 min. Slides were
rinsed in water, counterstained with haematoxylin and
dehydrated through to xylene before coverslipping.
IgG antibody
Measurement of islet cell size and number
Six random pancreatic sections were collected from five
wild-type and five Pyy−/−male mice. H&E stained sections
were observed using a ProgRes 3008 camera (Zeiss, Jena,
Germany) mounted on a Zeiss Axiophot microscope. The
average number and size of islet cells per section were
quantified using the ×20 objective and Leica IM 1000
version 1.20 software (Leica Microsystems, Heerbrig,
Switzerland).
In situ hybridisation
Coronal brain sections (20 μm) were cut on a cryostat and
thaw-mounted on Superfrost slides (Menzel-Glaser, Bruns-
wick, Germany). Matching sections from the same coronal
brain level of Pyy knock-out and wild-type mice (n=5 mice/
group) were assayed together using DNA oligonucleotides
complementary to mouse Npy (5′-GAGGGTCAGTCCA
CACAGCCCCATTCGCTTGTTACCTAGCAT-3′); mouse
proopiomelanocortin (Pomc) (5′-TGGCTGCTCTCCAGG
CACCAGCTCCACACATCTATGGAGG-3′); and mouse
growth hormone-releasing hormone (Ghrh) (5′-GCTTGT
CCTCTGTCCACATGCTGTCTTCCTGGCGGCTYGAG
CCTGG-3′) as described previously [26].
For evaluation of mRNA levels in scattered neurons,
images from dipped sections were digitised using a
ProgRes 3008 camera (Zeiss) mounted on a Zeiss Axiophot
microscope. Silver grain density over single neurons was
evaluated using NIH-Image 1.61 software (written by
Wayne Rasband and available from anonymous FTP at
zippy.nimh.nih.gov). Background labelling was uniform
and never exceeded 5% of specific signal.
Activity measurements
The total cage activity was recorded using a passive
infrared detector (PID) (Conrad Electronics, Hirschau,
Germany) on top of the cage lid of single-housed 16-week-
old chow-fed animals. The signals of the PIDs were
detected by an I/O interface card (PIO48 II; Dr Schetter
BMC Ing, Puchheim, Germany) and stored using custom-
made software. To show the activity pattern of the mice,
data were plotted so that each cycle’s activity is shown both
to the right and below that of the previous cycle, a so-called
double-plotted actogram with the x-axis showing 48 h
starting on the left with 00.00 h (ZT 6). In order to test for
differences in total activity between the genotypes, the
activity counts were summed up to 30-min bins and a
mean/day was calculated by averaging corresponding bins
of seven consecutive days.
Statistical analyses
Results for bodyweight, lean and fat mass, food intake,
activity measurements and differences in serum glucose
and insulin levels during glucose tolerance tests were
compared among groups using repeated-measures ANOVA
followed by Fisher’s post hoc tests. Differences in serum
hormone and metabolite concentrations between Pyy−/−
and wild-type mice were assessed by ANOVA with
subsequent Fisher’s post hoc tests. Alterations in neuro-
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peptide mRNA expression and pancreatic islet parameters
between knock-out and wild-type mice were assessed by
ANOVA. StatView version 4.5 (Abacus Concepts, Berke-
ley, CA, USA) was used for all statistical analyses and
p<0.05 was accepted as being statistically significant.
Results
Generation of Pyy knock-out mice
A knock-out-targeting vector for the Pyy gene was
designed which also allows the Pyy promoter-driven
expression of the Cre-recombinase gene under control of
the inducible tetracycline promoter (Fig. 1a). Two positive
ES cell clones for the Pyy construct were used to obtain
chimeric mice, heterozygotes, and subsequently homozy-
gous knock-out mice. DNA isolated from tail tips was used
in Southern blot analysis (NheI) to confirm correct
integration and modification of the targeted allele
(Fig. 1b,c).
Breeding of heterozygous germline knock-out animals
produced a slight deviation from the expected Mendelian
ratio of genotypes of 25:50:25 to 43:38:19 (wild-types:
heterozygotes:homozygous
pairs). However, Pyy−/−mice breed normally with an
average litter size of 7.15±0.37 pups/litter, which was not
significantly different from wild-type litter sizes of 6.57±
0.97 pups (n=7–13 breeding pairs). The gender ratio in
Pyy−/−offspring shows a trend towards a greater propor-
tion of females (57.1%, n=112).
knock-outs,n=7 breeding
Unaltered tissue morphology in Pyy−/−mice
Successful deletion of the Pyy gene was confirmed by the
absence of staining for PYY in the pancreas and colon of
Pyy−/−mice (Fig. 2a–d). The morphology of these PYY-
expressing tissues did not show any obvious changes when
examined using H&E staining. Moreover, the average size
of the islets of Langerhans in knock-out mice (0.013±
0.0002 mm2) did not differ significantly from that of wild-
type mice (0.020±0.0005 mm2) (n=5 mice/genotype). The
same is true for the number of islets per section (8.2±1.5 in
wild-type mice vs 6.6±1.3 in Pyy−/−mice, six sections from
five mice of each genotype). Importantly, the expression of
the Ppy gene, which is located only 7 kb downstream of the
Pyy gene locus, was not influenced by the removal of the
Pyy gene (Fig. 2e,f), nor was the expression of glucagon
(Fig. 2g,h) or insulin (data not shown) in the islets of
Langerhans.
Effects of Pyy deletion on bodyweight, adiposity
and food intake
No change in bodyweight was observed in male Pyy−/−
mice between 4 and 28 weeks of age under chow-fed
conditions (Fig. 3a,b), which was also confirmed by the
comparison of body-weight AUC measurements (wild-
type 628.5±7.8 g/24 weeks vs Pyy−/−657.1±21.3 g/24
weeks) (p=0.2). However, female Pyy−/−mice had a sig-
nificantly higher bodyweight than controls under chow-
fed conditions from 4 to 28 weeks of age (Fig. 3a) (AUC:
Fig. 1 Generation of Pyy−/−
mice. a Targeting vector design
and screening strategy. The bold
horizontal bars indicate the po-
sition of probes used for South-
ern analysis of genomic DNA
from targeted ES cells as well as
knock-out animals.b,cSouthern
analysis of genomic DNA iso-
lated from wild-type (+/+), het-
erozygous (+/−) and knock-out
(−/−) mice cut with NheI or
BamHI, using probes A and B
(see Material and methods, Pyy
targeting vector construction…),
respectively
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wild-type 505.0±8.9 g/24 weeks; Pyy−/−554.0±14.5 g/24
weeks) (p<0.01). Both female and male 14-week-old
chow-fed Pyy−/−mice had a significant increase in total
body lean mass (Fig. 3d,f) with the females also showing a
significant decrease in whole body fat mass at 14 weeks as
determined by DXA (Fig. 2e). However, female Pyy−/−
mice exhibited a marked increase in bodyweight gain from
15 to 28 weeks (Fig. 3a) (AUC females: wild-type 307.6±
7.3 g/15–28 weeks; Pyy−/−344.0± 10.4 g/15–28 weeks)
(p<0.02), which was not observed in the male knock-outs
(Fig. 3b) (AUC males: wild-type 381.8±6.g/15–28 weeks;
Pyy−/−401.0±16.3g/15–28weeks)(p=0.23).Consistentwith
the increased bodyweight gain, chow-fed female Pyy−/−
mice showed a significant 50% increase in fat mass at
28 weeks of age as determined by DXA (Fig. 3e). This was
also confirmed by marked and significant increases in the
weight of all white adipose tissue depots measured infemale
chow-fed Pyy−/−mice at 28 weeks of age (Table 1).
These findings show that female but not male Pyy−/−
mice develop late-onset obesity, as indicated by significant
increases in bodyweight andfat mass when fed a chow diet.
It is well documented that long-term exposure to high-fat
diet causes obesity in a number of species [27]. Moreover,
PYY release from the gut is strongly stimulated by dietary
fat [7]. Considering male knock-outs did not develop late-
onset obesity, we were interested in determining whether
high-fat feeding would induce obesity similarly to that
observed in female knock-outs. Therefore we investigated
the effect of a high-fat diet on bodyweight and energy
homeostasis in male Pyy−/−and wild-type mice. When fed
a high-fat diet from 4 weeks of age onwards, male Pyy−/−
mice showed no significant change in bodyweight when
compared with wild-type mice (Fig. 3c). However, these
high-fat-fed Pyy−/−mice showed a significant increase in
total body fat mass when compared with wild-types, as
determined by DXA (Fig. 3g), as well as a significant
increase in the weight of the gonadal white adipose tissue
depot (Table 2).
In young chow-fed female mice, the observed changes in
body composition (increased lean mass and decreased fat
mass) were associated with a significant decrease in non-
fasted food and water intake (Table 1). Interestingly,
however, the increases in bodyweight and/or fat mass
observed in older female and high-fat-fed male Pyy−/−mice
were not associated with any significant changes in non-
fasted food or water intake (Tables 1 and 2). Additionally,
there was no significant difference between 14-week-old
chow- or fat-fed Pyy−/−and wild-type mice with respect to
food intake at 1, 2, 8, 24, 48 or 72 h after a 24-h fast (data
not shown).
Effect of Pyy deficiency on glucose homeostasis
There was no significant difference between wild-type
and Pyy−/−mice with respect to fed serum glucose levels
(data not shown). When animals were fasted for 24 h
prior to the glucose tolerance test at 13 weeks of age,
there was no significant difference between wild-type
and knock-out mice with regards to basal serum glucose
levels, independently of dietary condition (Fig. 4a–c).
There was no effect of Pyy knock-out on glucose
tolerance in female or male chow-fed mice at 13 weeks
of age (Fig. 4a,d and b,e). Interestingly, both male and
female chow-fed Pyy−/−
mice showed significantly
higher serum insulin levels than controls throughout
the course of the glucose tolerance tests (Fig. 4g,j and h,
k). As weight gain is known to drive insulin resistance,
insulin levels during the glucose tolerance test and the
insulin AUC were calculated by normalising for body-
weight. This adjustment of the data did not change the
shape of the curves shown in Fig. 4g,h (data not shown),
nor did it change the AUC for insulin for females (wild-
type 458.7±68.1 pmol·l−1·min−1·g−1vs Pyy−/−659.8±
62.8 pmol·l−1·min−1·g−1) (p<0.05) and males (wild-
type 338.7±32.6 pmol·l−1·min−1·g−1vs Pyy−/−442.4±
27.7 pmol·l−1·min−1·g−1) (p<0.04).
Fig. 2 Immunostaining for PYY, PPY and glucagon. PYY staining
in the islets of Langerhans and colon of wild-type mice (a, c) and
Pyy−/−mice (b, d). e, f PPY staining in pancreas of Pyy−/−and wild-
type mice, respectively. g, h Glucagon staining in pancreas of Pyy−/−
and wild-type mice, respectively. Arrows indicate the positively
stained cells. Scale bar=10 μm. Pictures are representative of
staining observed in tissues obtained from 14-week-old animals
(n=3 mice/group)
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Under conditions of high-fat feeding, male knock-outs
were less glucose-tolerant than wild-type mice (p<0.05),
shown by a significant increase in the AUC (Fig. 4c,f).
However, this difference did not persist when the data were
normalised for bodyweight (wild-type 54.5±2.2 mmol·l−1·
min−1·g−1;Pyy−/−57.4±1.8mmol·l−1·min−1·g−1)(p=0.33)
Fasting and glucose-induced serum insulin levels are
significantly elevated in Pyy knock-outs compared with
wild-type mice fed a high-fat diet, whether expressed as
absolute values (Fig. 4i,l) or normalised for bodyweight
(wild-type 612.3±30.8 pmol·l−1·min−1·g−1; Pyy−/−782.7±
59.3 pmol·l−1·min−1·g−1) (p<0.02).
Effect of Pyy knock-out on serum hormone
concentrations
Since Pyy deletion significantly increases lean mass in
young, chow-fed animals (Fig. 3d,f), we investigated
serum concentrations of IGF-I, the main mediator of the
growth effects of growth hormone. No changes were
observed in IGF-I levels in the female knock-outs
(Table 1). However, serum IGF-I levels were significantly
increased in malePyy−/−mice under chow-fed butnot high-
fat-fed conditions (Tables 1 and 2), consistent with the
pattern of change in total body lean mass (Fig. 3f).
Bodyweight (g)
c
b
Bodyweight (g)
10
15
20
25
30
35
468 10 12 14
Weeks of age
16 18 2022 24
26
28
Lean mass (g)
14 weeks 28 weeks 14 weeks
High-Fat
14 weeks 28 weeks 14 weeks
High-Fat
0
5
10
15
20
25
30
Whole body fat
mass (% BW)
0
5
10
15
20
25
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35
10
15
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456789 1011 12 13
g
f
Weeks of age
a
10
15
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4681012 14
Weeks of age
16 18202224
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0
4
8
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14 weeks 28 weeks14 weeks 28 weeks
0
5
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25
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Bodyweight (g)
Lean mass (g)
Whole body fat
mass (% BW)
d
e
*
**
**
*
*
*
***
Fig. 3 EffectofPYYdeficiencyonbodyweight(a–c),leanmass(d,f)
and adiposity(e, g).a–c Bodyweightcurves from 4 to28 weeks of age
forafemale(wild-typen=37;Pyy−/−n=35)andbmale(wild-typen=32;
Pyy−/−n=33) wild-type (filled squares) and Pyy−/−(filled circles) mice
fed a chow diet. c Bodyweight curves for high-fat-fed male wild-type
(filled squares) (n=8) and Pyy−/−(filled circles) (n=8) mice from 4 to
13 weeks of age. d, e Whole-body lean and fat mass obtained using
DXA forfemalewild-type (open bars) andPyy−/−(filledbars)miceon
a chow diet at 14 weeks (wild-type n=7; Pyy−/−n=8) and 28 weeks of
age (wild-type n=6; Pyy−/−n=8). f, g DXA results for male mice at
14 weeks (wild-type n=8; Pyy−/−n=8) and 28 weeks of age (wild-type
n=7;Pyy−/−n=7)onachowdietandat14weeksofageonahigh-fatdiet
(wild-type n=8; Pyy−/−n=9). Data are means±SEM of the number of
mice shown in parentheses. *p<0.05, **p<0.01, ***p<0.001 vs wild-
type mice. BW, bodyweight
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Table 1 The effect of PYY deficiency on food and water intake, white adipose tissue (WAT) weights and serum parameters of 14- and
28-week-old chow-fed wild-type (WT) and Pyy−/−mice
WT Pyy−/−
Food intake (g/24 h) (11 weeks)
Female
Male
Water intake (g/24 h) (11 weeks)
Female
Male
WAT inguinal (g) (14 weeks)
Female
Male
WAT gonadal (g) (14 weeks)
Female
Male
WAT mesenteric (g) (14 weeks)
Female
Male
WAT retroperitoneal (g) (14 weeks)
Female
Male
Food intake (g/24 h) (24 weeks)
Female
Male
Water intake (g/24 h) (24 weeks)
Female
Male
WAT inguinal (g) (28 weeks)
Female
Male
WAT gonadal (g) (28 weeks)
Female
Male
WAT mesenteric (g) (28 weeks)
Female
Male
WAT retroperitoneal (g) (28 weeks)
Female
Male
IGF-I (nmol/l)
Female
Male
Testosterone (nmol/l) (male)
Glucagon (pmol/l)
Female
Male
Free T4 (pmol/l)
Female
Male
NEFA (mmol/l)
Female
Male
Triglycerides (mmol/l)
Female
Male
7.71±0.22 (12)
7.61±0.82 (10)
4.73±0.43 (9)*
6.86±0.77 (10)
4.66±0.34 (12)
5.03±0.41 (10)
3.76±0.17 (9)*
3.75±0.10 (10)*
0.14±0.01 (22)
0.13±0.01 (18)
0.11±0.005 (22)**
0.14±0.01 (22)
0.13±0.02 (22)
0.16±0.01 (18)
0.12±0.01 (22)
0.15±0.01 (22)
0.16±0.01 (22)
0.18±0.02 (18)
0.15±0.01 (22)
0.20±0.01 (22)
0.02±0.003 (22)
0.04±0.004 (18)
0.03±0.002 (22)
0.04±0.004 (22)
4.10±0.475 (5)
4.43±0.18 (5)
3.73±0.30 (5)
4.61±0.25 (5)
4.27±0.34 (5)
4.03±0.24 (5)
4.34±0.31 (5)
3.92±0.23 (5)
0.16±0.01 (15)
0.22±0.01 (14)
0.30±0.04 (13)**
0.24±0.05 (11)
0.20±0.04 (15)
0.29±0.03 (14)
0.52±0.07 (13)**
0.30±0.05 (11)
0.18±0.02 (15)
0.29±0.02 (14)
0.37±0.05 (13)**
0.30±0.05 (11)
0.03±0.01 (15)
0.08±0.01 (14)
0.08±0.01 (13)**
0.08±0.02 (11)
37.9±2.88 (10)
24.0±3.78 (16)
1.96±0.75 (11)
39.76±1.58 (23)
33.19±2.29 (24)*
20.80±6.86 (13)*
1.9±0.3 (8)
9.3±2.3 (10)
2.6±0.5 (12)
7.3±3.4 (13)
23.87±1.24 (10)
18.04±1.18 (11)
26.94±1.77 (14)
19.46±1.68 (13)
1.04±0.031 (9)
1.36±0.10 (15)
1.23±0.06 (14)*
1.37±0.1 (13)
0.67±0.1 (8)
1.0±0.1 (15)
1.02±0.1 (12)*
1.26±0.1 (13)
Data are means±SEM (number of mice in parentheses)
*p<0.05, **p<0.01 vs WT mice
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Testosterone has been shown not only to increase lean
mass, but also to decrease fat mass [28]. The serum
concentrations of testosterone were increased in male
Pyy−/−mice, under both chow-fed and fat-fed conditions,
and the difference attained statistical significance in the
chow-fed animals (Tables 1 and 2). There was no
Table 2 The effect of PYY deficiency on food and water intake, white adipose tissue (WAT) weights and serum parameters of 14-week-old
high-fat-fed wild-type (WT) and Pyy−/−mice
WTPyy−/−
Food intake (g/24 h)
Water intake (g/24 h)
WAT inguinal (g)
WAT gonadal (g)
WAT mesenteric (g)
WAT retroperitoneal (g)
IGF-I (nmol/l)
Testosterone (nmol/l)
Free T4 (pmol/l)
NEFA (mmol/l)
Triglycerides (mmol/l)
2.97±0.16 (8)
3.35±0.13 (8)
0.27±0.03 (8)
0.37±0.04 (8)
0.32±0.02 (8)
0.12±0.02 (8)
33.31±1.96 (8)
3.47±1.07 (13)
16.18±1.32 (7)
1.64±0.11 (7)
1.56±0.18 (8)
2.97±0.50 (8)
3.38±0.31 (8)
0.33±0.02 (8)
0.53±0.05 (8)**
0.43±0.04 (8)
0.14±0.01 (8)
29.47±5.36 (9)
18.40±7.15 (8)
16.21±1.43 (12)
1.30±0.11 (9)
1.44±0.14 (9)
Data are means±SEM (number of mice in parentheses)
**p<0.01 vs WT mice
0
5
10
15
20
0 20 40
Time (min)
60 80100 120
Time (min)
0
50
100
150
200
250
020406080100120
Insulin (pmol/l)
Glucose area
(mmol/l/min)
Glucose (mmol/l)
adg
0
50
100
150
200
250
020406080 100120
Insulin (pmol/l)
Glucose area
(mmol/l/min)
Glucose (mmol/l)
b
e
h
c
fi
0
50
100
150
200
250
0 20 4060 80100120
Insulin (pmol/l)
Glucose area
(mmol/l/min)
Glucose (mmol/l)
Time (min)
Time (min)
0
5
10
15
20
0 20 4060 80100 120
0
200
400
600
800
1,000
1,200
1,400
1,600
1,800
0
200
400
600
800
1,000
1,200
1,400
1,600
1,800
0
200
400
600
800
1,000
1,200
1,400
1,600
1,800
WTPYY-/-
WT
PYY-/-
WT
PYY-/-
0
5
10
15
20
020406080 100120
0
5,000
10,000
15,000
20,000
25,000
Insulin area
(pmol/l/min)
WT
PYY-/-
0
5,000
10,000
15,000
20,000
25,000
Insulin area
(pmol/l/min)
0
5,000
10,000
15,000
20,000
25,000
Insulin area
(pmol/l/min)
WTPYY-/-
WT
PYY-/-
j
k
l
*
**
*
*
Fig. 4 Effect of PYY deficiency on glucose homeostasis. Changes
in serum glucose levels after i.p. glucose injection (1.0 mg/kg) in 24-
h-fasted chow-fed female (a) wild-type (filled squares) (n=7) and
Pyy−/−(filled circles) (n=8) mice and in (b) male wild-type (filled
squares) (n=6) and Pyy−/−(filled circles) (n=7) mice at 13 weeks of
age. c Changes in serum glucose levels after i.p. glucose injection
(1.0 mg/kg) in 24-h-fasted high-fat-fed male wild-type (filled
squares) (n=7) and Pyy−/−(filled circles) mice (n=8) at 13 weeks
of age. d–f Glucose response AUCs. g–i Changes in insulin levels
after i.p. glucose injection (1.0 mg/kg) in 24-h-fasted chow or high-
fat-fed female and male wild-type (filled squares) and Pyy−/−(filled
circles) mice. j–l Insulin response AUCs. Data are means±SEM of
the number of mice shown in parentheses. *p<0.05, **p<0.01 vs the
comparison shown by horizontal bars. WT, wild-type
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significant effect of Pyy deletion on serum glucagon
concentrations in male or female animals (Table 1),
consistent with the observation that our Pyy knock-out
strategy did not alter the morphology or the number of
glucagon-staining alpha cells in the islets of Langerhans
(Fig. 2g,h).
As an index of hypothalamo–pituitary–thyroid function,
an important determinant of resting metabolic rate [29],
serum-free T4 levels, were measured in Pyy−/−and wild-
type mice. There was no significant effect of Pyy ablation
on serum-free T4 levels in mice of either gender or dietary
condition (Tables 1 and 2).
Unaltered motor activity in Pyy−/−mice
Mice were tested for general activity as well as for different
motor activity tasks over a period of 14 days. Male and
female Pyy−/−and wild-type mice at 14–16 weeks of age
were indistinguishable in the amount and pattern of activity
(Fig. 5). Furthermore, motor activity assessed in the open-
field test was not different between male and female Pyy−/−
and wild-type mice (data not shown), suggesting that
locomotion is not altered in these mice.
Hypothalamic changes in neuropeptide mRNA
expression in Pyy−/−mice
To assess possible central mechanisms for changes in
energy homeostasis in Pyy−/−mice, we analysed the
expression of hypothalamic neuropeptides known to
regulate energy balance such as NPY, POMC and GHRH.
Fourteen-week-old female Pyy knock-outs did not show
any significant changes in Npy mRNA expression in the
arcuate nucleus of the hypothalamus, but exhibited a 51%
increase in Pomc mRNA expression compared with
controls (Fig. 6a–d), which was consistent with the
significant reduction in food intake (Table 1) and fat
mass (Fig. 3e) observed in these animals. Fourteen-week-
old male Pyy−/−mice showed a significant 21% reduction
in Npy and a significant 25% increase in Pomc mRNA
expression levels in the arcuate nucleus compared with
wild-type mice (Fig. 6e–h). While these changes in male
mice were associated with increased lean mass and
increased serum concentrations of IGF-I and testosterone,
consistent with the expected effects of reduced central
NPY-ergic expression [30], they were not associated with
reductions in food intake.
The mRNA expression of the growth-promoting neuro-
peptide, Ghrh,wasunaltered in the arcuate nucleus (Fig. 6i,
j), but showed a significant 32% increase in the
ventromedial hypothalamic nucleus of Pyy−/−
(Fig. 6k,l), consistent with activation of the somatotrophic
axis and the significantly higher serum levels of IGF-I
(Table 1) and increased lean mass (Fig. 3f) in the chow-fed
male Pyy−/−mice.
mice
Discussion
This study demonstrates that PYY-mediated pathways are
important for the regulation of body composition, serum
insulin levels and glucose homeostasis. Deficiency of Pyy
resulted in late-onset obesity—indicated by significant
increases in bodyweight and fat mass—in female mice on a
chow diet. Male Pyy−/−mice were resistant to late-onset
obesity on a chow diet, but became significantly fatter than
wild-types when fed a high-fat diet. These effects of Pyy
ablation were associated with significant increases in basal
and/or glucose-induced serum insulin levels in both male
and female Pyy−/−mice, in the absence of increases in basal
or fasting-induced food intake. Moreover, these increases
in serum insulin levels were clearly apparent in chow-fed
Pyy−/−mice at 13 weeks of age. Since hyperinsulinaemia is
causally linked to the aetiology of obese states [23, 24], it is
possible that hyperinsulinaemia contributes to the devel-
opment of increased adiposity in Pyy−/−mice. The elevated
basal and glucose-induced serum insulin levels seen in our
Pyy−/−mice are most probably due to the lack of direct
PYYinhibitory action on Y1 receptors in the pancreas [31,
32], as well as indirectly via a lack of PYY stimulation of
Y2 receptors in the brainstem, which is known to alter
vagal output [33].
Fig. 5 Effect of PYY deficiency on motor activity: 24-h activity
pattern of 16-week-old chow-fed female (a) and male (b) wild-type
(filled squares) and Pyy−/−mice (filled circles). Dark bar, night
phase. Data are means±SEM of eight mice per group
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PYY and its processed form, PYY3-36, are known to
activate Y-receptors in the arcuate nucleus of the hypo-
thalamus and brainstem, thereby regulating the expression
of NPYand POMC. Selective activation of Y2 receptors by
PYY3-36 administration has been shown to downregulate
NPY mRNA and upregulate POMC mRNA expression in
the arcuate nucleus [12]. Interestingly, removal of the PYY
gene and therefore removal of the ability to activate Y1, Y2
or Y5 receptors either decreased or had no effect on the
mRNA expression of NPY in the arcuate nucleus. More-
over, in both male and female mice, Pyy ablation resulted in
a significant increase of Pomc expression in the hypotha-
lamic arcuate nucleus. These changes in hypothalamic
peptide expression may be caused by the high serum
insulin levels observed in Pyy−/−mice, since peripheral
insulin administration is known to inhibit hypothalamic
Npy and stimulate Pomc mRNA expression [34]. The
observed changes in hypothalamic Pomc expression are in
keeping with the significant reduction in food intake
observed in the young (but not old) female mice. In male
Pyy−/−mice, the increase in lean mass and resistance to
late-onset obesity on a chow diet may be further attributed
to stimulation of the hypothalamo-pituitary somatotrophic
axis via increased Ghrh mRNA expression in the
ventromedial hypothalamic nucleus and elevated IGF-I
levels, as well as the increased serum testosterone levels
[28, 35].
Recently, another report of a Pyy knock-out mouse was
published [36]. In that publication, fasting-induced food
intake and bodyweight of male and female chow-fed Pyy
knock-out animals was no different from that of wild-types,
albeit basal food intake, body composition, glucose-
induced glycaemia and insulinaemia, and ageing- and
diet-induced obesity were not investigated. It is important
to differentiate that PPY expression was also absent in the
previously published Pyy−/−mouse model effectively
making it a double Pyy/Ppy knock-out mouse.
In summary, we describe that the absence of PYY has a
significant effect on body composition and on basal and
glucose-induced serum insulin levels. Since circulating
PYY levels are decreased in obese, hyperinsulinaemic
people [37], our findings suggest that low circulating PYY
concentrations may causally contribute to the development
of hyperinsulinaemia and obesity, during ageing or under
conditions of long-term consumption of a high-fat diet,
respectively.
Acknowledgements
Batterham (UCL, London, UK) for providing the mouse Pyy gene.
We thank J. Ferguson (Garvan Institute) for veterinary advice and the
staff of the Garvan Institute Biological Testing Facility. We thank D.
James, T. Kraegen and L. Heilbronn (Garvan Institute) for critical
review of the manuscript. This research was supported by the
National Health and Medical Research Council (Project Grant
#230816), a Grant by the Prader Willi Foundation and NHMRC
Fellowships to A. Sainsbury and H. Herzog.
We are grateful to D. Withers and R.
Fig. 6 Effect of PYY deletion on Npy, Pomc and Ghrh mRNA
levels in the hypothalamus of 14-week-old female and male wild-
type and Pyy−/−mice. High-power bright-field photomicrographs of
dipped sections obtained from wild-type and Pyy−/−mice after in
situ hybridisation. Npy (a, b, e, f) in the arcuate hypothalamic
nucleus (Arc); Pomc (c, d, g, h); Ghrh in the arcuate (i, j) and
ventromedial hypothalamic (VMH) nucleus (k, l). Scale bar=10 μm.
**p<0.01, ***p<0.001 vs wild-type mice. Pictures are representa-
tive of mRNA expression from five mice per group. 3V, 3rd ventricle
1369

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References
1. Larhammar D (1996) Evolution of neuropeptide Y, peptide YY
and pancreatic polypeptide. Regul Pept 62:1–11
2. Herzog H (2003) Neuropeptide Y and energy homeostasis:
insights from Y receptor knockout models. Eur J Pharmacol
480:21–29
3. Pieribone VA, Brodin L, Friberg K et al (1992) Differential
expression of mRNAs for neuropeptide Y-related peptides in rat
nervous tissues: possible evolutionary conservation. J Neurosci
12:3361–3371
4. Lundberg JM, Terenius L, Hökfelt T, Tatemoto K (1984)
Comparative immunohistochemical and biochemical analysis
of pancreatic polypeptide-like peptides with special reference to
presence of neuropeptide Y in central and peripheral neurons.
J Neurosci 4:2376–2386
5. Blomqvist AG, Herzog H (1997) Y-receptor subtypes—how
many more? Trends Neurosci 20:294–298
6. Adrian TE, Ferri GL, Bacarese-Hamilton AJ, Fuessl HS, Polak
JM, Bloom SR (1985) Human distribution and release of a
putative new gut hormone, peptide YY. Gastroenterology
89:1070–1077
7. Onaga T, Zabielski R, Kato S (2002) Multiple regulation of
peptide YY secretion in the digestive tract. Peptides 23:279–290
8. Chelikani PK, Haver AC, Reidelberger RD (2005) Intravenous
infusion of peptide YY(3–36) potently inhibits food intake in
rats. Endocrinology 146:879–888
9. Riediger T, Bothe C, Becskei C, Lutz TA (2004) Peptide YY
directly inhibits ghrelin-activated neurons of the arcuate
nucleus and reverses fasting-induced c-Fos expression. Neuro-
endocrinology 79:317–326
10. Batterham RL, Cowley MA, Small CJ et al (2002) Gut
hormone PYY3-36 physiologically inhibits food intake. Nature
418:650–654
11. Adams SH, Won WB, Schonhoff SE, Leiter AB, Paterniti JR Jr
(2004) Effects of peptide YY[3-36] on short-term food intake in
mice are not affected by prevailing plasma ghrelin levels.
Endocrinology 145:4967–4975
12. Challis BG, Pinnock SB, Coll AP, Carter RN, Dickson SL,
O’Rahilly S (2003) Acute effects of PYY3-36 on food intake
and hypothalamic neuropeptide expression in the mouse.
Biochem Biophys Res Commun 311:915–919
13. Halatchev IG, Ellacott KL, Fan W, Cone RD (2004) Peptide YY3-
36 inhibits food intake in mice through a melanocortin-4-receptor-
independent mechanism. Endocrinology 145:2585–2590
14. Pittner R, Moore C, Bhavsar S et al (2004) Effects of PYY[3-
36] in rodent models of diabetes and obesity. Int J Obes Relat
Metab Disord 28:963–971
15. Tschop M, Castaneda TR, Joost HG et al (2004) Physiology:
does gut hormone PYY3-36 decrease food intake in rodents?
Nature 430: 1 p following 165; discussion 2 pp following 165
16. Kushi A, Sasai H, Koizumi H, Takeda N, Yokoyama M,
Nakamura M (1998) Obesity and mild hyperinsulinemia found
in neuropeptide Y-Y1 receptor-deficient mice. Proc Natl Acad
Sci USA 95:15659–15664
17. Molero JC, Jensen TE, Withers PC et al (2004) c-Cbl-deficient
mice have reduced adiposity, higher energy expenditure, and
improvedperipheral insulin action. JClinInvest114:1326–1333
18. Hoentjen F, Hopman WP, Jansen JB (2001) Effect of
circulating peptide YY on gallbladder emptying in humans.
Scand J Gastroenterol 36:1086–1091
19. Tatemoto K (1982) Isolation and characterization of peptide
YY (PYY), a candidate gut hormone that inhibits pancreatic
exocrine secretion. Proc Natl Acad Sci USA 79:2514–2518
20. Bottcher G, Ahren B, Lundquist I, Sundler F (1989) Peptide
YY: intrapancreatic localization and effects on insulin and
glucagon secretion in the mouse. Pancreas 4:282–288
21. Karlsson S, Ahrén B (1996) A role for islet peptide YY in the
regulation of insulin secretion. Acta Physiol Scand157:305–306
22. van den Hoek AM, Heijboer AC, Corssmit EPM et al (2004)
PYY3-36 reinforces insulin action on glucose disposal in mice
fed a high-fat diet. Diabetes 53:1949–1952
23. Assimacopoulos-Jeannet F, Jeanrenaud B (1976) The hormonal
and metabolic basis of experimental obesity. Clin Endocrinol
Metab 5:337–365
24. Standridge M, Alemzadeh R, Zemel M, Koontz J, Moustaid-
Moussa N (2000) Diazoxide down-regulates leptin and lipid
metabolizing enzymes in adipose tissue of Zucker rats. FASEB
J 14:455–460
25. Furuse M, Mabayo RT, Okumura J (1997) Effects of dietary
cholestyramine on the utilisation of diets containing medium or
long chain triacylglycerols by chicks. Br Poult Sci 38:436–438
26. Sainsbury A, Schwarzer C, Couzens M et al (2002) Important
role of hypothalamic Y2 receptors in bodyweight regulation
revealed in conditional knockout mice. Proc Natl Acad Sci
USA 99:8938–8943
27. West DB, Boozer CN, Moody DL (1992) Dietary obesity in
nine inbred mouse strains. Am J Physiol 262:R1025–R1032
28. Mudali S, Dobs AS (2004) Effects of testosterone on body
composition of the aging male. Mech Ageing Dev 125:297–304
29. al-Adsani H, Hoffer LJ, Silva JE (1997) Resting energy
expenditure is sensitive to small dose changes in patients on
chronic thyroid hormone replacement. J Clin Endocrinol Metab
82:1118–1125
30. Pierroz DD, Catzeflis C, Aebi AC, Rivier JE, Aubert ML
(1996) Chronic administration of neuropeptide Yinto the lateral
ventricle inhibits both the pituitary–testicular axis and growth
hormone and insulin-like growth factor I secretion in intact
adult male rats. Endocrinology 137:3–12
31. Cho YR, Kim CW (2004) Neuropeptide Y promotes beta-cell
replication via extracellular signal-regulated kinase activation.
Biochem Biophys Res Commun 314:773–780
32. Morgan DG, Kulkarni RN, Hurley JD et al (1998) Inhibition of
glucose stimulated insulin secretion by neuropeptide Y is
mediated via the Y1 receptor and inhibition of adenylyl cyclase
in RIN 5AH rat insulinoma cells. Diabetologia 41:1482–1491
33. Chen CH, Rogers RC (1997) Peptide YY and the Y2 agonist
PYY-(13-36) inhibit neurons of the dorsal motor nucleus of the
vagus. Am J Physiol 273:R213–R218
34. Havel PJ, Hahn TM, Sindelar DK et al (2000) Effects of
streptozotocin-induced diabetes and insulin treatment on the
hypothalamic melanocortin system and muscle uncoupling
protein 3 expression in rats. Diabetes 49:244–252
35. Ho KK, O’Sullivan AJ, Hoffman DM (1996) Metabolic actions
of growth hormone in man. Endocr J 43:S57–S63
36. Schonhoff SE, Baggio L, Ratineau C et al (2005) Energy
homeostasis and gastrointestinal endocrine differentiation do
not require the anorectic hormone peptide YY. Mol Cell Biol
25:4189–4199
37. Batterham RL, Cohen MA, Ellis SM et al (2003) Inhibition of
food intake in obese subjects by peptide YY3-36. N Engl
J Med 349:941–948
1370