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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2008.00465.x |
Ghrelin and cell
differentiation
Geyang Xu1, Yin Li1, Wenjiao
An1, and Weizhen Zhang1,2*
1 Department of Physiology and
Pathophysiology, Peking University Health Science Center, Beijing 100191, China
2 Department of Surgery, University of Michigan
Medical Center, Ann Arbor, Michigan 48109-0346, USA
Received: June 25, 2008 Accepted: July 17, 2008
This work was supported by grants from the
National Natural Science Foundation of China (No. 30740096), and the “985”
Program at Peking University (No. 985-2-097-121)
*Corresponding author: Tel, 86-10-82802183;
Fax, 86-10-82802183; E-mail, [email protected]
Ghrelin, an
endogenous ligand for the growth hormone secretagogue receptor, is a gastric hormone
that has been found to have a wide variety of biological functions. This review
summarizes our current understanding of the effects of ghrelin on cell
differentiation and tissue development, with an emphasis on the lineage
determination of mesenchymal stem cells.
Keywords ghrelin; adipogenesis; myogenesis;
osteogenesis; neurogenesis; pancreas development
Ghrelin, a 28 amino acid gastric peptide, is identified as an
endogenous ligand for the 搊rphan receptor, growth hormone secretagogue receptor (GHSR), in a
reverse pharmacology paradigm. As ghre is the proto-Indo-European root
of the word growth, ghrelin is named for its original function, which
was to stimulate the release of growth hormone [1]. Three molecular forms of
ghrelin are found in the stomach: the 28 amino acid ghrelin having
n-octanoylated serine in position 3; des-acyl ghrelin, an identical peptide in
which the third amino acid serine is not acylated; and the 27 amino acid
des-glutamine14 ghrelin produced by alternative splicing of the ghrelin gene.
Acylation appears to be essential for ghrelin抯 capability to stimulate the release of growth hormone, as des-acyl
ghrelin does not demonstrate any endocrine functions. Once thought to be an
inactive form of ghrelin, des-acyl ghrelin has been recently reported to
exercise some biological activities such as acting as a survival factor for the
cardiomyocyte.
Ghrelin is synthesized mainly in neuroendocrine cells (X/A-like
cells in rodents and P/D1 cells in humans) of the gastric fundus, and secreted
into the circulation [2]. It is also synthesized in much smaller amounts in a
variety of human tissues including several areas of the brain (hypothalamus
[3], hippocampus, and cortex [4]), pituitary gland [5], small intestine
[1], and pancreas [6]. Ghrelin receptor GHSR is a classic seven-transmembrane G
protein-coupled receptor that is linked to multiple intracellular signaling
pathways, including the intracellular calcium signaling pathway. The gene
encoding the GHSR has two splice variants: the full-length GHSR-1a
and its truncated molecule GHSR-1b, which contains only five transmembrane
domains. GHSR-1a is the receptor to which ghrelin binds and through which
ghrelin exerts its effects on growth hormone release [7]; the physiological function
of GHSR-1b remains to be characterized.
Ever since the discovery of its first function in the stimulation
of growth hormone release [8,9], ghrelin has been reported to exercise a broad
array of functions including control of food intake, energy metabolism [10],
modulation of cardiovascular function [11], down-regulation of cell
differentiation antigen 40 expression in endothelial cells [12], regulation of
lymphocyte development and cytokine secretion [13], involvement in
pathoclinical profiles of digestive system cancer [14], and control of
reproduction [15]. Emerging evidence also suggests that ghrelin may play a
role in the regulation of cell differentiation during development. This review
will focus on our current understanding of ghrelin抯 effects on growth and
development.
Ghrelin and fetal/perinatal development
The behavior of ghrelin in fetal circulation and its gene expression
in placenta, fetal pancreas and neonatal pancreas imply that this hormone may
play an important role in the development of fetal tissue and perinatal growth.
Both acylated and des-acyl ghrelin are present in fetal rat plasma at 20 d of
gestation [16]. In human beings, ghrelin immunoreactivity is present in umbilical
cord blood samples as early as week 20 of gestation. Even though maternal
ghrelin is reportedly transported from maternal blood to the fetus, total
ghrelin concentrations in umbilical cord veins are higher than that in maternal
blood [17,18], suggesting that ghrelin is produced in the fetus. In rats,
ghrelin is present in the whole fetus as early as 12 d of pregnancy [19].
Ghrelin gene levels are low in the fetal stomach of rats by 18 d of gestation
[20]. Both the number of ghrelin-immunoreactive cells and plasma ghrelin
levels increase significantly in the early postnatal period and reaches adult
levels by 3–5 weeks. These observations suggest that the stomach may contribute
negligible amounts to circulating fetal ghrelin.
In contrast, placenta may be the major source of circulating fetal
ghrelin, especially in the first trimester. Expression of ghrelin is mainly
contained in cytotrophoblast cells and, to a lesser extent, in
syncytiotrophoblast cells. Both ghrelin mRNA and immunoreactivity have been
detected in the human placenta. Expression of ghrelin in placenta decreases
significantly during the course of a pregnancy and becomes undetectable at
full term.
Fetal circulating ghrelin may also derive from pancreas. High levels
of ghrelin gene expression are present in a fifth islet cell-e cell of fetal
pancreas, suggesting that it may be a major source of circulating fetal ghrelin
[6,21]. Ghrelin-expressing cells can be detected as early as embryonic 10.5 d
in the pancreases of mouse embryos. The number of these ghrelin-producing cells
increases as development proceeds. Close to birth, these ghrelin-producing
cells begin to localize at the periphery of developing islets and remain
visible in marginal areas of the islets in the pancreases of neonates and
adults [22].
The abundance of ghrelin in the fetal endocrine pancreas suggests
that ghrelin may regulate the development of pancreatic b cells. This
notion is supported by two genetic studies demonstrating that expansion of
ghrelin-producing cells leads to the loss of insulin-producing cells during
the development of pancreatic islets [21]. Reciprocal gene expression changes
of insulin and ghrelin, specifically the repression of insulin and activation
of ghrelin, have been observed in Nkx2.2 mutant and Pax 4 mutant mice.
Within the pancreatic islet, homeodomain protein Nkx2.2 is essential for the
differentiation of all insulin-producing b cells and a subset of
glucagon-producing a cells. Mice lacking Nkx2.2 have relatively normal-sized islets, but
a large number of cells within the mutant islet fail to produce any of the
four major islet hormones. Instead, they produce ghrelin. These observations
suggest that an early block in the differentiation of insulin-producing b cells in
Nkx2.2 mutant mice leads to the expansion of cells that produce
ghrelin, perhaps by a cell fate switch. Pax4 mutant mice also display a similar
phenotype, showing the expansion of ghrelin-producing cells at the expense of b cells. Taken
together, these studies suggest that insulin and ghrelin cells share a common
progenitor and that Nkx2.2 and Pax4 are required for the determination and
differentiation of b cells. However, it is worth noting that these studies do not
demonstrate that ghrelin directly suppresses the differentiation of b cells. Indeed,
ghrelin may act to promote the regeneration of b cells in
streptozocin-treated newborn rats. Early administration of ghrelin may prevent
the development of diabetes in disease-prone subjects after beta cell
destruction [23].
Effect of ghrelin on the differentiation of mesenchymal
stem cells
Emerging evidence has indicated that ghrelin may modulate cell
proliferation and differentiation. Depending on cell type, ghrelin either
stimulates or inhibits cell proliferation. Mitotic effects of ghrelin have
been demonstrated in preosteoblasts [24,25], neuronal precursors [26,27],
preadipocytes [28,29], cardiomyocytes [30], and the rat GH3 pituitary cell line
[31]. In cell lines derived from carcinomas of the prostate [32], thyroid
[33], mammary gland [34] and lung, ghrelin acts to inhibit proliferation [35]. In
vitro studies suggest that ghrelin induces the differentiation of several
types of cells, including osteoblasts, adipocytes and neurons. Ghrelin also
stimulates proliferating myoblast cells to differentiate and fuse into
multinucleated myotubes. Since osteoblasts, adipocytes and myocytes are all
derived from common precursor cells, the mesenchymal stem cells, it is likely
that ghrelin acts to determine the linage differentiation of these cells (Fig.
1). This concept is supported by studies from our laboratory and others
[36–48].
Ghrelin and Adipogenesis
While the chronic administration of ghrelin in adult animals
reportedly increases body weight by reducing metabolic rate and fat catabolism
via a central mechanism [36], the direct effect of ghrelin on adipogenesis
during development is less clear and controversial. Choi et al [37]
have shown that exogenous ghrelin stimulates adipogenesis in primary cultures
of adult rat preadipocytes, whereas Ott et al [38] reported that chronic
treatment of SV40 large T antigen-immortalized brown adipocytes with ghrelin
had no effect on adipogenesis. Using primary cultured bone marrow stromal
cells, Thompson et al [39] reported that ghrelin and des-acyl ghrelin
acts to stimulate the differentiation of adipocytes, an effect likely mediated
by a yet-to-be-identified subtype of ghrelin receptor. Reasons underlying
these contradictions are still unclear, but the different models used may have
contributed to these conflicting observations.
To investigate the direct effect of ghrelin on adipogenesis during
development, we established a stable 3T3-L1 cell line overexpressing ghrelin.
Cells overexpressing ghrelin demonstrate significantly attenuated
differentiation of adipocytes [28]. Expression of peroxisome
proliferator-activated receptor g (PPAR g), which is
commonly used as a marker of adipocyte differentiation, is significantly inhibited
at both mRNA and protein levels. The ghrelin-mediated inhibition of
adipogenesis is likely mediated by mitogen-activated protein kinase, an enzyme
previously reported to regulate PPAR g and, therefore, adipocyte differentiation [40]; both ghrelin
overexpression and exogenous ghrelin stimulate the phosphorylation of mitogen-activated
protein kinase. The receptor-mediated effect of ghrelin on adipogenesis appears
to be an unidentified subtype. Reverse transcription-polymerase chain reaction
with the primer sequence of the previously identified ghrelin receptor subtypes
did not detect a signal even though ghrelin binding activity is demonstrated in
both native 3T3-L1 cells and cells overexpressing ghrelin [28]. To further
explore the effect of ghrelin on the development of adipose tissue, we
generated a transgenic mouse that, driven by the fatty acid binding protein 4 (FABP
4) promoter, overexpressed ghrelin in adipose tissue. FABP4 promoter
has been reported to direct the specific expression of Wnt 10b in
adipose tissue [41]. Our study also confirmed the specific expression of
ghrelin in adipose tissue under the control of FABP4 promoter [42]. FABP4-ghrelin
transgenic mice demonstrate a significant decrease in the amount of adipose
tissue and are resistant to obesity induced by high fat diet. These in vivo
studies suggest that ghrelin may impair the development of adipose tissue [42].
Ghrelin and Myogenesis
Since myocytes and adipocytes are derived from a common progenitor
cell, and development of muscle and adipose tissues often has a reciprocal
relationship, it is likely that ghrelin has the potential to regulate the
differentiation of myocytes. Studies from our laboratory and others [43,44]
provide clear evidence in support of this concept. In C2C12 cells, a mouse
premyocyte cell line, overexpression of ghrelin significantly increases the
differentiation of premyocytes into myocytes. Cells overexpressing ghrelin
demonstrate increased myogenesis relative to control cells, as indicated by an
increment in myogenic index. Expression of both Myo D, an early marker, and
myosin heavy chain protein, a late marker of skeletal muscle differentiation,
is elevated in cells overexpressing ghrelin compared to control cells [43].
Similar results have also been reported by Filigheddu et al [44].
Exogenous ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal
myoblasts to differentiate and fuse into multinucleated myotubes by activating
p38. Ghrelin抯
stimulatory effect on myogenesis is likely mediated by an unidentified subtype
of ghrelin receptor because no signal of GHSR-1a mRNA has been detected
in C2C12 cells, though they contain a common high-affinity binding site
recognized by ghrelin.
Ghrelin and Osteogenesis
Both in vivo and in vitro studies have demonstrated
that ghrelin is a potent stimulator for osteogenesis. Intraperitoneal
injections of ghrelin increase bone mineral density (BMD) of the femur [45].
This effect is independent of the growth hormone because similar results have
been observed in growth hormone-deficient rats. Clinical studies by Misra et
al showed that ghrelin secretion strongly predicts BMD in healthy
adolescents [46]. Our studies also showed that ghrelin promotes osteogenesis of intramembranous bone and improves the repair of calvarial bone defect in rats [47]. In vitro, both
osteoblast cell lines and primary cultured osteoblasts respond to ghrelin with
an increase in cell proliferation and differentiation [45,48]. There is still
controversy about the receptor mediating the effect of ghrelin on osteogenesis.
Studies by Fukushima et al [45] demonstrated the expression of GHSR-1a
mRNA and immunoreactivity in osteoblast cells in culture and in situ. In
contrast, human bone does not express GHSR-1a. Only the GHSR
splice variant 1b is detected in femoral bone [48]. Since the functionality and
signaling pathway for GHSR-1b is unknown, the receptor mediating the
ghrelin-induced osteogenesis remains to be identified.
In summary, data supporting the notion that ghrelin may be involved
in the cell fate determination of mesenchymal stem cells are emerging. It is
worth noting that all these data are based on the studies of precursor cells
instead of the mesenchymal stem cells. Future studies should reveal the direct
mechanism involved in the ghrelin-induced lineage determination of mesenchymal
stem cells during development.
Ghrelin and neurogenesis
Neurogenesis, a process through which precursor cells differentiate
into a mature neuronal phenotype, persists in the circumventricular regions of
the adult brain. Neurogenesis in the circumventricular region and hippocampal
dentate gyrus of the adult rat nervous system has been demonstrated either
under physiological conditions involving in the brain adaptation to learning,
exercise and dietary restriction, or subsequent to ischemic brain injury.
Global or focal cerebral ischemia has been reported to stimulate neurogenesis,
typically defined by increased incorporation of bromodeoxyuridine (BrdU) into
cells that express neuronal marker proteins in the subventricular zone,
subgranular zone of the hippocampal dentate gyrus or cerebral cortex. Although
the molecular mechanisms remain unknown, neurogenesis has been demonstrated to
involve proliferation of radial cells located in the floor of cerebral
ventricles [49,50].
Neurogenesis in embryos and in adult neural stem cells is regulated
by a group of growth factors including stem cell factor, vascular endothelial
growth factor, fibroblast growth factor, insulin-like growth factor and bone
morphogenetic proteins. Our studies and others have [26,51–59] demonstrated
that ghrelin has the potential to promote neuronal development and
regeneration.
Ghrelin
stimulates neurogenesis in the dorsal motor nucleus of the vagus
The dorsal motor nucleus of the vagus (DMNV) contains neurons that
provide the parasympathetic efferent outflow to the gastrointestinal system
[60]. Both the organization and phenotype of DMNV neurons projecting into the
gastrointestinal tract undergo extensive specification and reorganization in
the perinatal period. The molecular mechanisms that control alteration in
vagal innervations are not clear. Our studies [26] have demonstrated that
ghrelin may function to control neuronal proliferation and regeneration in
DMNV. Both mRNA and immunoreactivity of ghrelin receptor are detected in rat
DMNV tissues. In vivo study demonstrates that neurogenesis exists in
adult brain nuclei surrounding the fourth cerebral ventricle, including the
DMNV. Although lacking active proliferation under basal conditions, the DMNV
responds to perturbation induced by vagotomy with an increase in cell
proliferation. Systemic administration of ghrelin significantly increases BrdU
incorporation in the DMNV of adult rats with cervical vagotomy. In vitro
exposure of cultured DMNV neurons to ghrelin significantly increases the
percentage of BrdU incorporation into cells in both dose-dependent and
time-dependent manners. All these data suggest that ghrelin acts
directly on DMNV neurons to stimulate neurogenesis. Since the dorsal
vagal complex is devoid of a brain blood barrier, the source of ghrelin
responsible for stimulation of neurogenesis in the DMNV may come from the
circulating blood.
Stimulation
of neurogenesis in the nucleus of the solitary tract by ghrelin
The nucleus of the solitary tract (NTS) borders the fourth ventricle
and possesses the characteristics of a circumventricular organ that may respond
to blood-borne and cerebrospinal fluid-borne factors [51,52]. Chemical injury,
such as hypoxia and hypoglycemia, results in multiple neuronal responses in
the NTS, including neuronal degeneration [53] and activation of c-fos
expression [54], suggesting neuronal plasticity in the NTS. Systemic
administration of ghrelin significantly increases BrdU incorporation in the
NTS in adult rats with cervical vagotomy [55]. Cultured NTS neurons contain
immature precursor cells as evidenced by the expression of Hu protein. Exposure
of cultured NTS neurons to ghrelin significantly increases the percentage of
BrdU incorporation into cells in both dose-dependent and time-dependent
manners. Co-localization of Hu immunoreactivity with BrdU labeling is
demonstrated by double fluorescent staining, suggesting that cells labeled with
BrdU are neuronal cells. Ghrelin receptor mRNA and immunoreactivity are
detected in tissues from the NTS. Treating cultured NTS neurons with ghrelin
receptor antagonist and calcium channel blocker abolishes the mitotic effect
of ghrelin. All these studies have demonstrated that ghrelin acts directly on
NTS neurons to stimulate neurogenesis via activation of GHSR-1a [55].
Ghrelin and
neuronal development in spinal cord
A study by Sato et al [56] found that both microtubule
associated protein 2 positive mature neurons and nestin positive precursor
cells in fetal spinal cord expressed GHSR mRNA and protein. Activation
of GHSR by ghrelin stimulates neuronal proliferation in cultured neurons
derived from fetal spinal cord tissues. Des-acyl ghrelin also induces a
significant increase in proliferation of the primary cultured neurons. Taken
together, these results suggest that both ghrelin and des-acyl ghrelin are
involved in neurogenesis of the fetal spinal cord [56] via both GHSR-dependent
and independent mechanisms.
Neuroprotection
Neuroprotection of ghrelin has been demonstrated in ischemia/reperfusion
injury [57]. Expression of GHSR-1a in rat cerebral cortex decreases
significantly as a result of ischemia/reperfusion injury. Intravenous
administration of ghrelin returns the GHSR mRNA to its normal level.
Neuronal apoptosis induced by ischemia/reperfusion injury is considerably
reduced by systemic administration of ghrelin. Expression of apoptosis-related
molecules caspases 3 and 9 are suppressed by ghrelin, while caspase 8 remains
unchanged. Neuronal damage induced by lipopolysaccharide (10 nM), glutamate
(100 mM), N-methyl-D-aspartate (100 mM) or hydrogen peroxide
(500 mM) is also attenuated by exogenous ghrelin [57]. All these studies
suggest that ghrelin may function as a survival factor to protect cortical
neurons in adult animals [57]. In the elegant study by Chung et al,
ghrelin was also reported to protect hypothalamic neurons [58] and cortical
neurons [59] from neuronal injury induced by ischemia. In addition, ghrelin
acts to reduce neuronal apoptosis provoked by oxygen-glucose deprivation in
cultured hypothalamic and cortical neurons. The anti-apoptotic effect of
ghrelin occurs through the preservation of mitochondrial integrity.
Conclusion
The characteristics of ghrelin outlined in this review raise fascinating
questions on the potential role of this hormone during cell differentiation and
tissue development. Ghrelin may function as an important signal in coordinating
the energy status and cell differentiation in processes such as b cell
development, neurogenesis and lineage determination of mesenchymal stem cells.
Future studies will soon unravel the mystery surrounding the mechanism linking
the metabolic function of ghrelin to growth and development.
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