Ghrelin directly regulates bone formation.
To clarify the role of ghrelin in bone metabolism, we examined the effect of ghrelin in vitro and in vivo. Ghrelin and its receptor, GHS-R1a, were identified in osteoblasts, and ghrelin promoted both proliferation and differentiation. Furthermore, ghrelin increased BMD in rats. Our results show that ghrelin directly affects bone formation.


BACKGROUND
Ghrelin is a gut peptide involved in growth hormone (GH) secretion and energy homeostasis. Recently, it has been reported that the adipocyte-derived hormone leptin, which also regulates energy homeostasis and opposes ghrelin's actions in energy homeostasis, plays a significant role in bone metabolism. This evidence implies that ghrelin may modulate bone metabolism; however, it has not been clarified. To study the role of ghrelin in skeletal integrity, we examined its effects on bone metabolism both in vitro and in vivo.


METHODS
We measured the expression of ghrelin and growth hormone secretagogue receptor 1a (GHS-R1a) in rat osteoblasts using RT-PCR and immunohistochemistry (IHC). The effect of ghrelin on primary osteoblast-like cell proliferation was examined by recording changes in cell number and the level of DNA synthesis. Osteoblast differentiation markers (Runx2, collagen alpha1 type I [COLI], alkaline phosphatase [ALP], osteocalcin [OCN]) were analyzed using quantitative RT-PCR. We also examined calcium accumulation and ALP activity in osteoblast-like cells induced by ghrelin. Finally, to address the in vivo effects of ghrelin on bone metabolism, we examined the BMD of Sprague-Dawley (SD) rats and genetically GH-deficient, spontaneous dwarf rats (SDR).


RESULTS
Ghrelin and GHS-R1a were identified in osteoblast-like cells. Ghrelin significantly increased osteoblast-like cell numbers and DNA synthesis in a dose-dependent manner. The proliferative effects of ghrelin were suppressed by [D-Lys(3)]-GHRP-6, an antagonist of GHS-R1a, in a dose-dependent manner. Furthermore, ghrelin increased the expression of osteoblast differentiation markers, ALP activity, and calcium accumulation in the matrix. Finally, ghrelin definitely increased BMD of both SD rats and SDRs.


CONCLUSIONS
These observations show that ghrelin directly stimulates bone formation.
Fukushima
Nobuhiro
N
Molecular Genetics, Institute of Life Science, Kurume University, Kurume, Fukuoka, Japan.
Hanada
Reiko
R
Teranishi
Hitoshi
H
Fukue
Yoshihiko
Y
Tachibana
Toshiaki
T
Ishikawa
Hiroshi
H
Takeda
Shu
S
Takeuchi
Yasuhiro
Y
Fukumoto
Seiji
S
Kangawa
Kenji
K
Nagata
Kensei
K
Kojima
Masayasu
M
D017667:Adipocytes / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015519:Bone Density; D001846:Bone Development; D001842:Bone and Bones / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis; D000090602:Collagen Type I, alpha 1 Chain; D050659:Core Binding Factor Alpha 1 Subunit; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D054439:Ghrelin; D007150:Immunohistochemistry; D020738:Leptin / Q000378:metabolism; D009363:Neoplasm Proteins / Q000096:biosynthesis; D010006:Osteoblasts / Q000378:metabolism; D015675:Osteocalcin / Q000378:metabolism; D036361:Peptide Hormones / Q000378:metabolism / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D054440:Receptors, Ghrelin; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis
D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D050659:Core Binding Factor Alpha 1 Subunit; D016895:Culture Media, Serum-Free; D054439:Ghrelin; D020738:Leptin; D009363:Neoplasm Proteins; D036361:Peptide Hormones; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D014157:Transcription Factors; D015675:Osteocalcin; D004247:DNA; D000469:Alkaline Phosphatase; D002118:Calcium