Neuropeptide Y expression and function during osteoblast
differentiation – insights from transthyretin knockout
mice
Ana F. Nunes
1,2,
*, Ma
´
rcia A. Liz
1
, Filipa Franquinho
1
, Liliana Teixeira
3
, Vera Sousa
1
, Chantal
Chenu
4
, Meriem Lamghari
3,
 and Mo
´
nica M. Sousa
1,

1 Nerve Regeneration, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal
2 ICBAS, Universidade do Porto, Portugal
3 INEB – Instituto de Engenharia Biome
´
dica, Divisa˜o de Biomateriais, Universidade do Porto, Portugal
4 Department of Veterinary Basic Sciences, The Royal Veterinary College, London, UK

sis, as its function in the regulation of bone mass is unclear, we assessed
its expression in this tissue. By immunohistochemistry, we demonstrated,
both at embryonic stages and in the adult, that NPY is synthesized by
osteoblasts, osteocytes, and chondrocytes. Moreover, peptidylglycine a-am-
idating monooxygenase, the enzyme responsible for NPY activation by
amidation, was also expressed in these cell types. Using transthyretin
(TTR) KO mice as a model of augmented NPY levels, we showed that
this strain has increased NPY content in the bone, further validating the
expression of this neuropeptide by bone cells. Moreover, the higher ami-
dated neuropeptide levels in TTR KO mice were related to increased bone
mineral density and trabecular volume. Additionally, RT-PCR analysis
established that NPY is not only expressed in MC3T3-E1 osteoblastic cells
and bone marrow stromal cells (BMSCs), but is also detectable by RIA in
BMSCs undergoing osteoblastic differentiation. In agreement with our
in vivo observations, in vitro, TTR KO BMSCs differentiated in osteoblasts
had increased NPY levels and exhibited enhanced competence in undergo-
ing osteoblastic differentiation. In summary, this work contributes to a
better understanding of the role of NPY in the regulation of bone forma-
tion by showing that this neuropeptide is expressed in bone cells and that
increased amidated neuropeptide content is related to increased bone
mass.
Abbreviations
ALP, alkaline phosphatase; BMD, bone mineral density; BMSC, bone marrow stromal cell; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; KO, knockout; microCT, micro computed tomography; NF200,
neurofilament 200; NPY, neuropeptide Y; PAM, peptidylglycine a-amidating monooxygenase; PGP9.5, protein gene product 9.5; RANK,
receptor activator of nuclear factor-jB; T
4,
thyroxine; TTR, transthyretin.
FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS 263
receptors in bone [9]. Neuropeptide Y (NPY)-immuno-

levels in the hypothalamus, suggesting a commonality
of mechanism. However, it was recently shown that
leptin and Y2 receptor pathways independently modu-
late cancellous bone homeostasis [17]. With regard to
Y2 receptor-deficient mice, both germline and condi-
tional hypothalamic Y2 receptor KO mice share the
same high bone mass phenotype [5], demonstrating
that central hypothalamic Y2 receptors are crucial for
this process. Interestingly, although germline Y1 recep-
tor KO mice also display increased bone formation,
conditional deletion of hypothalamic Y1 receptors did
not alter bone homeostasis, suggesting a nonhypotha-
lamic control of bone mass [6]. The Y1 receptor being
the only NPY receptor identified in the bone, these
results suggest that absence of NPY signaling in the
bone (as occurs in Y1 receptor-deficient mice) results
in increased bone mass.
NPY effects in bone mass have been further inves-
tigated by exogenous administration. Whereas intra-
cerebroventricular infusion of NPY decreased bone
mass [7], vector-mediated overexpression of NPY in
the hypothalamus of wild-type mice resulted in no
alteration in cancellous bone volume, although osteo-
blast activity, estimated using osteoid width, was
markedly reduced following adeno-associated virus
NPY injection [17,18]. These results are not in accor-
dance with the cancellous bone phenotype of the
above-mentioned mouse models of elevated NPY lev-
els. All of these opposing results make necessary a
closer look at the role of NPY in the regulation of

already described in the literature [14]. The periosteum
(Fig. 1Ab) also showed NPY immunoreactivity, as
already reported for mice and rats [10–12]. However,
we observed NPY immunostaining in chondrocytes,
osteoblasts, and osteocytes (Fig. 1Ac–f, respectively,
arrows). No NPY immunoreactivity was found in
osteoclasts (data not shown). Similar to our observa-
tions in the adult bone, NPY immunoreactivity was
detected starting at embryonic day 16 in megakaryo-
cytes, osteoblasts, and chondrocytes; this NPY detec-
tion pattern was maintained at embryonic day 18
(Fig. 1B). No immunoreactivity was detected when the
NPY is expressed in osteoblasts A. F. Nunes et al.
264 FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS
A F
CB
D
E
ab c
d
b
a
c
e f
a
b
c
d
e
BM

A. F. Nunes et al. NPY is expressed in osteoblasts
FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS 265
NPY antibody was replaced by mouse IgGs (Fig. 1C).
Moreover, in NPY KO mouse bone sections, none of
the different bone cells showed NPY immunostaining
(Fig. 1D), suggesting that the immunoreactivity
observed in WT and TTR KO bone tissue was NPY-
specific. To further demonstrate NPY synthesis in os-
teoblasts, osteoblast-specific staining was performed
with an antibody against osteocalcin (Fig. 1E, left
panel, arrow). The results obtained revealed that the
pattern of staining was comparable to that obtained
for NPY, as shown in the right panel of Fig. 1E, thus
confirming NPY expression in osteoblasts. To further
demonstrate that NPY is synthesized in these bone
cells, additional negative controls were performed.
Using antibody against neurofilament 200 (NF200) or
antibody against protein gene product 9.5 (PGP9.5),
two nerve fiber markers, no staining was observed in
chondrocytes, osteoblasts, or bone marrow cells
(Fig. 1Fa–d, respectively), whereas in the periosteum
typical nerve fiber labeling was detected (Fig. 1Fe).
TTR KO bone tissue has increased amidated NPY
levels
From the comparison between WT and TTR KO NPY
immunoreactivity in bone sections, we observed that
TTR KO bone tissue displayed increased amidated NPY
levels when compared to the wild type (Fig. 2A, arrows),
further demonstrating the expression of this neuropep-
tide by bone cells. NPY immunostaining was increased

in each panel, namely articular cartilage
chondrocytes (a), proliferating chondrocytes
(b), osteoblasts (c), osteocytes (d), bone
marrow cells (BM) and megakaryocytes (M)
(e). (B) PAM immunostaining in the bone
marrow (a; arrows indicate megakaryo-
cytes), osteocytes (b; arrows), osteoblasts
(b;p arrowheads), and chondrocytes (c).
(C) Quantification of the density of PAM
immunostaining in the bone marrow of WT
and TTR KO mice.
a
P < 0.05.
NPY is expressed in osteoblasts A. F. Nunes et al.
266 FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS
osteocytes (Fig. 2Bb, arrowheads and arrows, respec-
tively), as well as in chondrocytes (Fig. 2Bc). The major
difference in PAM expression among WT and TTR KO
bones was found in the bone marrow, where PAM
immunostaining was approximately two-fold higher in
TTR KO mice (Fig. 2C). Despite the fact that NPY and
PAM expression were not observed in osteoclasts, the
hypothesis that increased NPY levels in the bone of
TTR KO mice may have an indirect effect on osteoclasts
existed. To address this hypothesis, preosteoclasts and
mature osteoclasts in WT and TTR KO bones were
detected by OSCAR staining. Following quantification,
no differences in osteoclast number were detected
between strains (data not shown).
TTR KO mice have increased bone mineral

growth plates from WT and TTR KO mice.
NPY is expressed in osteoblasts
To further address NPY expression in bone cells,
namely in the osteoblastic cell line MC3T3-E1, and in
primary cultures of BMSCs throughout osteoblastic
differentiation, we performed RT-PCR analysis of
NPY expression. Using brain as the positive control of
NPY expression, we detected NPY in MC3T3-E1 cells
and in both WT and TTR KO BMSCs (Fig. 4A). Fur-
thermore, both WT and TTR KO BMSCs on days 3,
7 and 14 of culture in osteogenic differentiation media
showed NPY expression; no statistical differences were
observed between WT and TTR KO BMSC cultures
throughout the differentiation period (data not
shown). To determine whether TTR KO mice BMSCs
undergoing osteoblastic differentiation recapitulate our
findings in the nervous system, i.e. show increased
PAM transcription and increased levels of amidated
NPY, without increased NPY mRNA expression, we
quantified PAM expression and the levels of the bio-
logically active neuropeptide in differentiating WT and
TTR KO BMSC cultures. As expected, TTR KO mice
BMSCs displayed increased amidated NPY levels
(approximately 2.4-fold at day 3) when compared to
WT cells (Fig. 4B). Despite the fact that the NPY con-
tent decreased over the 14 days of differentiation, indi-
cating that undifferentiated BMSCs have higher levels
of NPY than differentiated osteoblasts, these still
expressed amidated neuropeptide. One should, how-
ever, note that in WT BMSCs, NPY levels were not

Fig. 3. MicroCT in WT and TTR KO mouse
femurs. (A) Bone microarchitecture in WT
and TTR KO mice. Left and middle panels:
2D microCT images of metaphyseal bone,
showing reconstructed longitudinal sections
(left panel) and transverse sections taken
 1 mm from the growth plate (middle
panel). The line crossing the transversal
sections indicates the orientation of the
longitudinal sections. Right panel: 3D mi-
croCT images of metaphyseal trabecular
bone in WT and TTR KO mice. (B) Quantifi-
cation of trabecular volume [bone vol-
ume ⁄ trabecular volume (BV ⁄ TV)] and BMD
in WT and TTR KO mice. Results are pre-
sented as average ± standard error of the
mean.
a
P < 0.05. (C) Hematoxylin ⁄ eosin
staining of the growth plate (femur) of WT
and TTR KO mice (3 months old). Scale
bars: 50 lm.
A B
C
Fig. 4. NPY and PAM expression in bone
cells from WT and TTR KO mice. (A) NPY
RT-PCR analysis in brain, MC3T3-E1 cells,
and BMSCs. (B) NPY quantification in
BMSCs from WT and TTR KO mice at
days 1, 3, 7 and 14 of differentiation into

transferase (HPRT)] as well as osteopontin, an extra
marker of osteoblastic differentiation. Day 3 of BMSC
differentiation was chosen for performance of the con-
firmation because, at this time point, not only ALP
activity but also osteocalcin expression are increased in
TTR KO BMSCs. The expression levels of both osteo-
calcin (Fig. 5C) and osteopontin (Fig. 5D) were always
increased in TTR KO BMSCs, irrespective of the
housekeeping gene used to perform the normalization.
Taken together, these data suggest that TTR KO
BMSCs show enhanced competence in undergoing
osteoblast differentiation in vitro.
Discussion
The data presented in this study demonstrate that
NPY is expressed in several types of bone cell, with
both in vitro and in vivo evidence. Moreover, we show
that increased NPY levels are related to increased bone
density, as well as to augmented competence in BMSC
differentiation into osteoblasts. In agreement with our
findings, a recent r eport further supports the contribution
18
16
14
12
10
8
ALP activity (nmolPNP·mg·h
–1
)
6

Day 3
Day 7
Day 7
c
b
b
TTR KO
TTR KO
WT
Day 14
Day 3 Day 7
Day 14
Day 14
WT
WT
TTR KO
b
b
WT
TTR KO
WT
osteocalcin
osteocalcin/actin
Day 3 osteocalcin/house keeping gene
Day 3 osteopontin/house keeping gene
β-actin
KO WT KO WT KO
B
A
C

receptor.
Until now, NPY expression has only been detected
in bone marrow cells, including megakaryocytes [14].
Here, we show for the first time that BMSCs also con-
tribute to NPY in the bone marrow, as NPY is
expressed both in BMSCs and in BMSCs undergoing
osteoblastic differentiation. Moreover, this article is
the first to report NPY expression in chondrocytes,
osteoblasts, osteocytes and the osteoblastic cell line
MC3T3-E1. In relation to chondrocytes, no studies
were performed regarding the role of NPY in the dif-
ferentiation of this cell type. This could probably be
the aim of a subsequent study, where possible differ-
ences in articular cartilage or growth plate between
WT and TTR KO bones should be addressed. In the
case of osteoclasts, although NPY expression was not
detected in this cell type, the elevated NPY levels in
TTR KO bones might have some indirect effect on
osteoclasts. In fact, we recently reported that NPY
modulates receptor activator of nuclear factor-jB
(RANK) ligand and osteoprotegerin, two key factors
regulating bone remodeling [23]. The inhibitory effect
of NPY on RANK ligand production by BMSCs was
also investigated by Amano et al. [24], who suggested
that the inhibitory effect of NPY on osteoclastogenesis
was caused by suppression of isoprenaline-induced
RANK ligand production by stromal cells, upstream
of RANK ligand mRNA expression.
It is known that central NPY regulates bone mass, as
conditional ablation of hypothalamic Y2 receptors

levels are not significantly different from those of WT
mice [31]. Moreover, retinoic acid plasma levels are two-
fold to three-fold higher in TTR KO mice, probably
compensating for their low retinol levels [31]. Taking
the above into account, it is highly unlikely that, with
normal retinol levels in tissues and increased retinoic
acid levels in the plasma, an impairment in retinol
homeostasis would be responsible for the increased
BMD in TTR KO mice. Regarding thyroid hormones,
it is well known that hyperthyroidism in adult patients
leads to decreased BMD [32]. As expected, both total T
4
and tri-iodothyronine serum levels are decreased in
TTR KO mice [32,33]. However, similar to what is
described above for retinol, this decrease is unrelated to
symptoms of hypothyroidism or thyroid gland abnor-
malities [34]. Again, in terms of tissue content, TTR KO
mice show no differences in T
4
levels from WT mice
[35,36]. This euthyroid status probably arises as a conse-
quence of the high free T
4
serum pool in the TTR KO
mice [34]. Such a euthyroid status is essential for normal
skeletal development and maintenance, and therefore it
is hard to see how the bone phenotype of TTR KO mice
could be related to thyroid hormones.
It is additionally possible that in TTR KO mice, as
a consequence of PAM overexpression, increased levels

that TTR KO osteoblasts have intrinsically augmented
PAM expression in relation to WT cells, as a conse-
quence of their physiological TTR-free environment.
A similar finding was reported for TTR KO neurons
(like BMSCs, neurons lack TTR expression), as these
cells were also shown to display intrinsically decreased
neurite outgrowth, as a consequence of their physio-
logical TTR-free environment [39].
NPY control of bone mass is still controversial. On
the one hand, there are two different mouse models
with increased NPY expression that show high cancel-
lous bone mass, the Y2 receptor KO mice [5] and mice
lacking leptin (ob ⁄ ob mice) [7,16]. Although sharing a
similar high cancellous bone phenotype, both models
differ in cortical bone regulation, with increased corti-
cal bone mass in Y2 receptor KO mice and decreased
cortical density in ob ⁄ ob mice [23]. On the other hand,
no NPY signaling in the bone, as is the case in Y1
receptor KO mice, leads to high bone mass [6], and
central NPY overexpression yields decreased osteoblast
activity [18] and bone mass [7], with no alteration in
cancellous bone volume [17,18]. With regard to this
central NPY overexpression, the consequential increase
in leptin levels [40,41] cannot be excluded as the cause
of the effects observed. Furthermore, the apparent dis-
crepancy between Y1 and Y2 receptor KO models
regarding NPY signaling and bone phenotype was
recently clarified by the hypothesis that the increased
central NPY levels observed in the Y2 receptor-defi-
cient mice lead to Y1 receptor downregulation on bone

in the bone regeneration process.
Experimental procedures
Animals
Mice were handled according to the European Communi-
ties Council Directive (86 ⁄ 609 ⁄ EEC) and national rules,
and all studies performed were approved by the Portuguese
General Veterinarian Board. Male WT and TTR KO [33]
littermate offspring of heterozygous breeding pairs, in the
129 ⁄ Sv background, were maintained at 24 ± 1 °C under a
12 h light ⁄ dark cycle and fed regular chow and tap water
ad libitum. Prior to all experimental procedures, animals
were anesthetized with ketamine (1 mgÆg
)1
body weight) ⁄ mede-
tomidine (0.02 lgÆg
)1
body weight). Animals were killed
with an overdose of anesthetic.
Immunohistochemistry
Femurs from 3 month old male WT (n = 6) and TTR KO
(n = 5) littermates were fixed in 4% paraformaldehyde
in NaCl ⁄ P
i
, decalcified in TBD-1 commercial solution
(Thermo Electron Corporation), and embedded in paraffin;
serial 4 lm thick longitudinal sections were then cut. For
studies during embryonic development, 16 day or 18 day
WT pregnant females were killed by cervical dislocation, and
the fetuses were collected by cesarian section. Sections were
then deparaffinized, dehydrated in a modified alcohol series,

for rabbit anti-NF200 IgG (Sigma), 1 : 4000 for rabbit anti-
PGP9.5 IgG (Serotec, Kidlington, UK), 1 : 500 for goat anti-
osteocalcin IgG (Biomedical Technologies Inc., Stoughton,
MA, USA), 1 : 500 for rabbit anti-PAM IgG (a kind gift
from R. Mains, University of Connecticut Health Center),
and 1 : 100 for mouse anti-OSCAR IgG (Santa Cruz Bio-
technology, Heidelberg, Germany). Antigen visualization
was performed with the biotin–extravidin–peroxidase kit
(Sigma), using 3-amino-9-ethylcarbazole (Sigma) as sub-
strate. On parallel control sections, the primary antibody was
replaced with blocking buffer. Immunohistochemical analy-
sis was performed independently by two observers. For
quantification of PAM immunohistochemistry, the number
of labeled cellsÆmm
)2
was scored in three nonoverlapping
micrographs with a magnification of · 40.
Bone histology
Femurs were harvested from 3 month old male WT
(n = 6) and TTR KO (n = 5) mice. After their length had
been measured, bones were fixed in 4% paraformaldehyde
in NaCl ⁄ P
i
, decalcified as described above, and embedded
in paraffin. Serial 10 lm thick longitudinal sections were
cut. Sections were then deparaffinized, dehydrated in a
modified alcohol series, and stained for hematoxylin ⁄ eosin.
MicroCT analysis
Dissected hindlimbs (femur plus tibia from WT and TTR
KO littermates, n = 9 and n = 10, respectively) were

gen, Carlsbad, CA, USA) supplemented with 10% (v ⁄ v) fetal
bovine serum (Invitrogen), 0.5% (v ⁄ v) gentamicin (Invitro-
gen), 1% (v ⁄ v) fungizone (Invitrogen), 50 lgÆmL
)1
vitamin C
(Sigma) and 10 mm b-glycerophosphate (Sigma) in a humidi-
fied 5% CO
2
incubator at 37 °C. The medium was changed
twice weekly. At confluence, the cells were trypsinized and
seeded in 24-well plates at a cell seeding density of
4 · 10
4
cells per well.
BMSC culture
Primary BMSCs were obtained according to the method
developed by Maniatopoulos et al. [43]. Briefly, femurs and
tibias from 1 month old male WT and TTR KO littermates
were aseptically excised from the hindlimbs, the epiphyses
were cut off, and the marrow was flushed with standard
culture medium, which consisted of alpha-MEM supple-
mented with 10% fetal bovine serum, 50 lgÆmL
)1
gentami-
cin sulfate, and 2.5 lgÆmL
)1
amphotericin B (Invitrogen).
Cells were seeded in 75 cm
2
plastic culture flasks, and incu-

ACTCACGGCAAATTC-3¢ and 5¢-CCTTCCACAATGC
CAAAGTT-3¢; for NPY, 5¢-TGGACTGACCCTCGCTC
TAT-3¢ and 5¢-GATGAGGGTGGAAACTTGGA-3¢; for
osteocalcin, 5¢-CTCTGTCTCTCTGACCTCACAG-3¢ and
5¢-CAGGTCCTAAATAGTGATACCG-3¢ [44]; for osteo
pontin, 5¢-TCTGATGAGACCGTCACTGC-3¢ and 5¢-TC
TCCTGGCTCTCTTTGGAA-3¢; for PAM, 5¢-CCTGG
GGTCACACCTAAAGA-3¢ and 5¢-TGTAAGGACACAC
CGGAACA-3¢; for Y1 receptor, 5¢-CTCGCTGGTTCTCA
TCGCTGTGGAACGG-3¢ and 5¢-GCGAATGTATATCT
TGAAGTAG-3¢ [15]; for Y2 receptor, 5¢-TCCTGGATTCC
TCATCTGAG-3¢ and 5 ¢-GGTCCAGAGCAATGACTG
TC-3¢ [15]; and for Y5 receptor, 5¢-CGCTTCCATCTCAA
GCAGA-3¢ and 5¢-AAGTCGTCTACGCTGCCTCT-3¢.
Unreferenced primers were designed using primer3 (http://
frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and the
sequence from the National Centre for Biotechnology Infor-
mation database. All primers used were located on two dif-
ferent exons to ensure that only properly spliced mRNA,
and not genomic DNA contaminants, was amplified. Ethid-
ium bromide-stained gels were scanned using a Typhoon
8600 (Amersham, Chalfont St Giles, UK), and amplified
bands were quantified using imagequant software (Amer-
sham). The fluorescence density of each PCR-amplified band
was normalized with the corresponding value of b-actin,
HPRT, and ⁄ or GAPDH. Experiments were performed in
triplicate, and a representative amplification is shown.
NPY quantification
NPY (amidated NPY
1–36

i
and lysed in 1% (v ⁄ v) Triton X-100 in
NaCl ⁄ P
i
. ALP activity was then measured by incubation of
cell lysates for 1 h at 37 °Cin0.1m NaHCO
3
⁄ Na
2
CO
3
buffer (pH 10), containing 0.1% Triton X-100, 2 mm
MgSO
4
, and 6 mm 4-nitrophenyl phosphate. The reaction
was stopped by adding 1 m NaOH, and absorbance was
measured at 405 nm. Enzyme activity was normalized for
cell protein content, measured using the bicinchoninic acid
assay (Pierce, Rockford, IL, USA).
Statistical analysis
Statistical analysis was performed using Student’s t-test.
Results are expressed as average ± standard error of the
mean. For all statistical analysis, P < 0.05 was accepted as
being statistically significant.
Acknowledgements
We thank R. Correia (IBMC) for tissue processing,
and P. Brites (IBMC) for thoughtful suggestions. This
work was supported by Association Franc¸ aise contre
les Myopathies, France, and Fundac¸ a
˜

During MJ et al. (2007) Novel role of Y1 receptors
in the coordinated regulation of bone and energy
homeostasis. J Biol Chem 282, 19092–19102.
7 Ducy P, Amling M, Takeda S, Priemel M, Schilling
AF, Beil FT, Shen J, Vinson C, Rueger JM & Karsenty
G (2000) Leptin inhibits bone formation through a
hypothalamic relay: a central control of bone mass. Cell
100, 197–207.
8 Elefteriou F, Takeda S, Liu X, Armstrong D & Karsen-
ty G (2003) Monosodium glutamate-sensitive hypotha-
lamic neurons contribute to the control of bone mass.
Endocrinology 144, 3842–3847.
9 Elefteriou F (2005) Neuronal signaling and the regula-
tion of bone remodeling. Cell Mol Life Sci 62, 2339–
2349.
10 Bjurholm A, Kreicbergs A, Terenius L, Goldstein M &
Schultzberg M (1988) Neuropeptide Y-, tyrosine
hydroxylase- and vasoactive intestinal polypeptide-
immunoreactive nerves in bone and surrounding tissues.
J Auton Nerv Syst 25, 119–125.
11 Hill EL & Elde R (1991) Distribution of CGRP-, VIP-,
D beta H-, SP-, and NPY-immunoreactive nerves in the
periosteum of the rat. Cell Tissue Res 264, 469–480.
12 Tabarowski Z, Gibson-Berry K & Felten SY (1996)
Noradrenergic and peptidergic innervation of the mouse
femur bone marrow. Acta Histochem 98, 453–457.
13 Ahmed M, Srinivasan GR, Theodorsson E, Bjurholm A
& Kreicbergs A (1994) Extraction and quantitation of
neuropeptides in bone by radioimmunoassay. Regul
Pept 51, 179–188.

role of secretory granules in peptide biosynthesis. Ann
NY Acad Sci 493, 278–291.
21 Prigge ST, Mains RE, Eipper BA & Amzel LM (2000)
New insights into copper monooxygenases and peptide
amidation: structure, mechanism and function. Cell Mol
Life Sci 57, 1236–1259.
22 Eipper BA, Stoffers DA & Mains RE (1992) The bio-
synthesis of neuropeptides: peptide alpha-amidation.
Annu Rev Neurosci 15, 57–85.
23 Teixeira L, Sousa DM, Nunes AF, Sousa MM, Herzog
H & Lamghari M (2009) NPY revealed as a critical
modulator of osteoblast function in vitro: new insights
into the role of Y1 and Y2 receptors. J Cell Biochem
107, 908–916.
24 Amano S, Arai M, Goto S & Togari A (2007) Inhibi-
tory effect of NPY on isoprenaline-induced osteoclasto-
genesis in mouse bone marrow cells. Biochim Biophys
Acta 1770, 966–973.
25 Baldock PA, Allison S, McDonald MM, Sainsbury A,
Enriquez RF, Little DG, Eisman JA, Gardiner EM &
Herzog H (2006) Hypothalamic regulation of corti-
cal bone mass: opposing activity of Y2 receptor
and leptin pathways. J Bone Miner Res 21, 1600–
1607.
26 Saper CB, Loewy AD, Swanson LW & Cowan WM
(1976) Direct hypothalamo-autonomic connections.
Brain Res 117 , 305–312.
NPY is expressed in osteoblasts A. F. Nunes et al.
274 FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS
27 Navia JM & Harris SS (1980) Vitamin A influence on

man ME & Saraiva MJ (1994) Thyroid hormone
metabolism in a transthyretin-null mouse strain. J Biol
Chem 269, 33135–33139.
35 Palha JA, Hays MT, Morreale de Escobar G, Episko-
pou V, Gottesman ME & Saraiva MJ (1997) Transthy-
retin is not essential for thyroxine to reach the brain
and other tissues in transthyretin-null mice. Am J
Physiol 272, E485–E493.
36 Palha JA, Fernandes R, de Escobar GM, Episkopou V,
Gottesman M & Saraiva MJ (2000) Transthyretin
regulates thyroid hormone levels in the choroid plexus,
but not in the brain parenchyma: study in a transthyre-
tin-null mouse model. Endocrinology 141 , 3267–3272.
37 Kingery WS, Offley SC, Guo TZ, Davies MF, Clark
JD & Jacobs CR (2003) A substance P receptor (NK1)
antagonist enhances the widespread osteoporotic effects
of sciatic nerve section. Bone 33, 927–936.
38 Hosaka H, Nagata A, Yoshida T, Shibata T, Nagao T,
Tanaka T, Saito Y & Tatsuno I (2008) Pancreatic poly-
peptide is secreted from and controls differentiation
through its specific receptors in osteoblastic MC3T3-E1
cells. Peptides 29, 1390–1395.
39 Fleming CE, Saraiva MJ & Sousa MM (2007) Trans-
thyretin enhances nerve regeneration. J Neurochem 103 ,
831–839.
40 Sainsbury A, Cusin I, Doyle P, Rohner-Jeanrenaud F
& Jeanrenaud B (1996) Intracerebroventricular adminis-
tration of neuropeptide Y to normal rats increases
obese gene expression in white adipose tissue. Diabetolo-
gia 39, 353–356.