Eﬀects of ghrelin and des-acyl ghrelin on neurogenesis
of the rat fetal spinal cord
, Keiko Nakahara
, Shintaro Goto
, Hiroyuki Kaiya
, Mikiya Miyazato
, Masamitsu Nakazato
, Kenji Kangawa
, Noboru Murakami
Department of Veterinary Physiology, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2155, Japan
National Cardiovascular Center Research Institute, Osaka 565-8565, Japan
Third Department of Internal Medicine, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan
Received 6 September 2006
Available online 26 September 2006
Expressions of the growth hormone secretagogue receptor (GHS-R) mRNA and its protein were conﬁrmed in rat fetal spinal cord
tissues by RT-PCR and immunohistochemistry. In vitro, over 3 nM ghrelin and des-acyl ghrelin induced signiﬁcant proliferation of pri-
mary cultured cells from the fetal spinal cord. The proliferating cells were then double-stained using antibodies against the neuronal pre-
cursor marker, nestin, and the cell proliferation marker, 5-bromo-2
-deoxyuridine (BrdU), and the nestin-positive cells were also found to
be co-stained with antibody against GHS-R. Furthermore, binding studies using [
I]des-acyl ghrelin indicated the presence of a speciﬁc
binding site for des-acyl ghrelin, and conﬁrmed that the binding was displaced with unlabeled des-acyl ghrelin or ghrelin. These results
indicate that ghrelin and des-acyl ghrelin induce proliferation of neuronal precursor cells that is both dependent and independent of
GHS-R, suggesting that both ghrelin and des-acyl ghrelin are involved in neurogenesis of the fetal spinal cord.
Ó 2006 Elsevier Inc. All rights reserved.
Keywords: Ghrelin; Des-acyl ghrelin; GHS-R; Neurogenesis; Spinal cord; Fetal development
Ghrelin, a peptide hormone secreted from the stomach,
has been identiﬁed as the endogenous ligand for the growth
hormone secretagogue receptor (GHS-R), through which
ghrelin stimulates GH release in the pituitary . Two
types of GHS-R, type1a and 1b (GHS-R1a and 1b, respec-
tively), have so far been found, and only the former is able
to activate signal transduction of the receptor downstream
linking to phospholipase C, resulting in an increase of
intracellular calcium . Ghrelin consists of 28 amino acids
and is characterized by esteriﬁed modiﬁcation with octano-
ic acid on serine 3, which is essential for activation of
GHSR-1a, although the modiﬁcation mechanism remains
unknown. On the other hand, the level of des-acyl ghrelin,
which is inactive on GHS-R1a because of a lack of octanoic
acid, is 4 times as high as that of ghrelin in the blood .
Many studies have reported that ghrelin has multiple
eﬀects other than GH secretion, including regulation of
food intake  and energy metabolism , and gastrointes-
tinal coordination [6,7], as well as facilitation of cell surviv-
al, and/or inhibition of apoptosis [8–15]. Although these
multiple functions of ghrelin would account for the very
wide distribution of GHS-R1a, it is debatable whether
GHS-R1a contributes to all of the actions of ghrelin, i.e.
that ghrelin may act as a ligand for other types of receptors
. So far, however, this possibility remains uninvestigat-
ed, and no such alternative receptor has been identiﬁed.
We have previously demonstrated that rat fetal growth
was increased by treatment of the mother with exogenous
ghrelin, and that the eﬀect of ghrelin on fetal growth is
diminished by immunization against ghrelin in vivo .
In addition, we have found that amniotic ﬂuid contains a
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Biochemical and Biophysical Research Communications 350 (2006) 598–603