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From: Jian V. Zhang, Lei Li, Qingsheng Huang and Pei-Gen Ren, Obestatin Receptor
in Energy Homeostasis and Obesity Pathogenesis. In Ya-Xiong Tao, editor:
Progress in Molecular Biology and Translational Science, Vol. 114,
Burlington: Academic Press, 2013, pp. 89-107.
ISBN: 978-0-12-386933-3
© Copyright 2013 Elsevier Inc.
Academic Press
CHAPTER THREE
Obestatin Receptor in Energy
Homeostasis and Obesity
Pathogenesis
Jian V. Zhang*
,#
, Lei Li*
,1
, Qingsheng Huang*
,1
, Pei-Gen Ren
#
*Research Center for Gene and Cell Engineering, Institute of Biomedicine and Biotechnology, Shenzhen
Institute of Advance Technology, Chinese Academy of Sciences, Shenzhen, China
#
Innovative Pharmacology and Biotherapy Pre-clinical Test Public Service Platform of Shenzhen, China
1
These authors contributed equally to this work
Contents
1. Two Proposed Obestatin Receptors 90
1.1 Phylogenetic relationship of GPR39 and GLP-1R within their GPCR families 92
1.2 Gene, expression and structure of GPR39 and GLP-1R 93
1.3 Signal transduction across cell membrane for GPR39 and GLP-1R 96
2. Obestatin and its Receptor(s) in Energy Homeostasis 97
2.1 Food intake 98
2.2 Gastrointestinal motility 99
2.3 Growth hormone secretion 99
2.4 Lipid metabolism 99
2.5 GPR39 null mice 100
2.6 Obesity pathogenesis 100
Acknowledgments 102
References 102
Abstract
Based on the bioinformatic prediction, Zhang and colleagues discovered obestatin, a
new 23-amino acid hormone from rat stomach extract encoded by the ghrelin gene.
Obestatin is present not only in the gastrointestinal tract, but also in the spleen, mam-
mary gland, breast milk, and plasma. Obestatin appears to function as part of a complex
gut-brain network whereby hormones and substances from the stomach, intestine and
the brain about satiety or hunger. Given the current research regarding the effects of
obestatin and its possible cognate receptor(s), this chapter provides the latest review
of the physiological and pathological characteristics of this hormone and its possible
receptor(s) in energy homeostasis and obesity.
Progress in Molecular Biology and Translational Science, Volume 114 #2013 Elsevier Inc.
ISSN 1877-1173 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-386933-3.00003-0
89
Author's personal copy
Ghrelin stimulates food intake and adiposity and thereby increases body
weight in rodents after central as well as peripheral administration.
1
Based
on the bioinformatic prediction, Zhang and colleagues discovered that, ex-
cept ghrelin, the ghrelin gene encoded another secreted and bioactive pep-
tide. The precursor is encoded by the gene GHRL, which is located on
chromosome 3, band p25–26,
2
and is cleaved by protease into peptide seg-
ments. Zhang and colleagues isolated this new 23-amino acid hormone from
rat stomach, and it showed actions opposite to ghrelin on food intake, body
weight, and gastric emptying and named it “obestatin”.
3
In this chapter, we
summarize the possible physiological roles of obestatin and its receptor in
energy homeostasis and its possible pathological roles in obesity.
Accumulating publications have confirmed the role of obestatin in reg-
ulating energy homeostasis and its association with obesity, among many
other physiological functions. Its receptor(s) involved in these functions
remains, however, controversial. Two receptors for obestatin have been
proposed: GPR39 and GLP-1R.
3,4
Both G protein-coupled receptors
(GPCRs) are found to be correlated with the function of obestatin, especially
metabolism. Since less evidence demonstrated the exact mechanism, which
one is the innate receptor is still unknown.
5
1. TWO PROPOSED OBESTATIN RECEPTORS
GPR39 was first cloned from a fetal brain cDNA library, by hybrid-
ization at low stringency.
6
Due to the sequence similarity, GPR39, along
with another GPCR cloned at that time, GPR38, was considered related
to the growth hormone secretagogue receptor (GHS-R), whose agonists in-
cluded synthetic peptides and nonpeptide growth hormone (GH) secreta-
gogues.
6
Later the natural endogenous ligand for GHS-R was discovered,
named ghrelin,
7
and GPR39 was speculated to have a ligand of a peptide
regulating gastrointestinal functions, since all the other receptors homolo-
gous to GHS-R have such ligands.
2
GPR39 had been considered, however,
an orphan GPCR until 2005, when Zhang and colleagues discovered
obestatin encoded by the ghrelin gene. Based on the data of radiolabeled li-
gand binding assays, it was proposed that the new hormone was a cognate
ligand of GPR39.
3
The relationship of obestatin and GPR39 was later chal-
lenged by Chartrel, who stated that obestatin did not bind GPR39 and in-
duce any effects on GPR39-transfected cells.
8
In response, Zhang and
colleagues found out that the interaction of obestatin and GPR39 could
not be reproduced due to the instable bioactivity of iodinated obestatin.
9
Just
followed up their response about the bioactivity of obestatin, Zhang and
90 Jian V. Zhang et al.
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colleagues found out that mono-Iodinated obestatin can bind to GPR39 and
GPR39 SNP, but not over-iodinated obestatin
13
.Duringthattime,two
groups, Holst et al. and Lauwers et al., discovered that zinc ion (Zn
2þ
) is a phys-
iologically relevant agonist of GPR39.
10,11
Both groups emphasized that
obestatin did neither bind toGPR39-expressing cellsnor display any effect such
as cAMP production. Holst et al. stated that GPR39 might signal through Gs
pathway if Zn
2þ
presented in addition to Gq and G12/13 pathways that were
constitutively activated.
10
Lauwers et al. showedthat the Zn
2þ
responsewas not
affected by obestatin. But there was still some evidence for the role of obestatin
as a GPR39 agonist. Moechars et al. constructed GPR39-knockout mice and
partially confirmed the in vivo effects of obestatin dependingon GPR39.
12
The
knockout mice’s differences in energy homeostasis included: accelerated
gastric emptying, increased gastric secretion, higher mature body weights and
body fat compositions, reduced hyperphagia after fasting, and increased choles-
terol levels. Zhang et al. demonstrated that c-fos expression was induced by
obestatin in wild-type mice but not in GPR39 null mice, suggesting that acti-
vities of obestatin depended on GPR39.
13
In addition, various studies on bone
metabolism
14
and term pregnancy
15
have shown the correlation of expression
levels of obestatin and GPR39. Thus, the role of GPR39 remains unresolved.
The other candidate receptor GLP-1R has already gained attention before
the discovery of obestatin because it is the receptor for the glucagon-like
peptide-1 (GLP-1), which highly stimulates insulin secretion and results in
antidiabetogenic effects. The cDNA of GLP-1R was first isolated from a pan-
creatic islet cDNA library from rats, and its function as a receptor for GLP-1
was confirmed by binding experiments with radiolabeled GLP-1.
16
The
cDNA of GLP-1R of human was isolated by several groups in 1993.
17–19
At the same time, two exogenous ligands were identified, which were
exendin-4 (Ex-4) and exendin-(9–39).
19
The interation of GLP-1R to
obestatin was revealed by Granata et al.in2008.
4
They observed that obestatin
bound to GLP-1R and promoted b-cell and islet cell survival. The binding
of radiolabeled GLP-1 to the cell membrane was slightly inhibited by motilin,
GLP-1, acylated ghrelin, [D-Lys
3
]-GHRP-6, and Ex-9, and obestatin
dose-dependently inhibited the binding of GLP-1 to the cell membrane.
4
Thus, the binding assay indicated that obestatin and GLP-1 recognized the
same binding sites.
4
Furthermore, obestatin did not influence GLP-1 secre-
tion, thus the promotion of cell survival was independent of GLP-1.
4
Favaro
et al. later confirmed that the anti-apoptotic effects of obestatin and exendin-4
involved PI3K/Akt, ERK1/2, and cAMP/PKA pathways.
20
Astudyon
the metabolic effects of obestatin in rats yielded several negative results
that obestatin neither bound to cells overexpressing GLP-1R, nor altered
91Obestatin Receptor in Energy Homeostasis and Obesity Pathogenesis
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insulin release from islets, where both GLP-1R and GPR39 should be
expressed.
21
These controversies on both the obestatin receptor and its related
biological functions highlight the urgency of the introduction of study aspects
other than cell biology and physiology.
1.1. Phylogenetic relationship of GPR39 and GLP-1R
within their GPCR families
GPR39 and GLP-1R are both GPCRs. GPCR is a seven-transmembrane
protein, with a N-terminal extracellular region and three extracellular loops
recognizing a ligand molecule and a C-terminal intracellular region and
three intracellular loops interacting with a G protein. Although all GPCRs
possess this structural feature, they are results of convergent evolution.
GPR39 and GLP-1R belong to two different families sharing little sequence
similarity.
GPR39 is characterized as a member of family A, or rhodopsin-like
GPCR. The sequence of GPR39 is most related to the ghrelin receptor
(or GHS-R), the neurotensin receptors, the motilin receptor (GPR38),
and neuromedin-U receptors.
6
The ghrelin receptor is the cognitive recep-
tor for the peptide hormone ghrelin, which is encoded by the same gene of
obestatin but with opposite roles to obestatin in the control of food intake.
22
The two neurotensin receptors, NTS-R1 and NTS-R2, are receptors of
a tridecapeptide neurotensin.
23,24
Neurotensin is a cleavage segment of a
17 kDa precursor protein, which is expressed in the brain and gut.
24
Neu-
rotensin and its two receptors are correlated to the regulation of dopaminer-
gic neurotransmissions.
24
The motilin receptor was once known as GPR38,
cloned at the same time of GPR39.
6,25
Its ligand, the motilin peptide,
regulates interdigestive gastrointestinal motility by stimulating rhythmic
contraction of the smooth muscle.
25
The two neuromedin-U receptors
are NMU-R1 and NMU-R2. Neuromedin-U, similar to motilin, induces
smooth muscle contraction. NUM-R1 is expressed in greatest abundance
in the gastrointestinal and urogenital systems but at a much lower
level within the central nervous system, and NUM-R2 is expressed in a
complementary manner, lower in peripheral tissues but higher in the central
nervous system.
26
GLP-1R belongs to GPCR family B, or the secretin-like family, charac-
terized by the three disulfide bonds in the N-terminal domain.
27
Ligands for
receptors in this family are large peptides with paracrine activities.
28
By
sequence similarity, GLP-1R forms a subgroup with GLP-2R, GCGR
92 Jian V. Zhang et al.
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(the glucagon receptor), and GIPR (the gastric inhibitory polypeptide
receptor).
28
These receptors, together with their ligands, are all essential in
maintaining glucose homeostasis. GLP-2R is stimulated by GLP-2, a
33-amino acid peptide cleaved from proglucagon,
29
which also encodes gluca-
gon and GLP-1. GLP-2R does not respond to GLP-1.
29
GCGR recognizes
glucagon and plays a role in controlling the production of hepatic glucose
and insulin secretion, so as to regulate the level of blood glucose.
30
GIPR is a
receptor for GIP,
31
a hormone that stimulates insulin secretion and thus is a
potential drug treating type 2 diabetes.
32
1.2. Gene, expression and structure of GPR39 and GLP-1R
The human GPR39 gene is located in chromosome 2, band q21–q22,
6
con-
sisting of two exons.
33
The first exon encodes residues 1 to 285, corres-
ponding to the N-terminal region and the first five transmembrane
helices, and the second exon encodes residues 286 to 435, corresponding
to the last two transmembrane helices and the C-terminal region.
34
Between
the two exons there is an intron as large as 200 kb, whose analogue appears in
the genes of the ghrelin receptor and the motilin receptor.
34
The second
exon of GPR39 shares a segment of DNA with LYPD1 gene
34
: GPR39
is encoded by the forward strand of DNA, while LYPD1 is transcribed from
the reverse strand. The two-exon structure and LYPD1 gene-overlapping
pattern are conserved among several species.
34
Beside the canonical form
of GPR39 (named GPR39-1a), which is 435-amino acid long and has
seven-transmembrane helices,
6
a splice variant GPR39-1b has been identi-
fied, which is encoded by the first exon and thus truncated after the fifth
transmembrane helix.
34
The maximal promoter activity resides in the 50-UTR region. Such a re-
gion in U-138 cell lines was reported to be from 673 to 14 from the trans-
lation initiation codon, while in HepG2 cell from 573 to 14.
34
Although
the negative regulatory elements have not yet been determined, there was
such an element from 673 to 573 in HepG2 cells. The translation initia-
tion codon is at 469 nucleotides downstream of the transcriptional start site.
34
As a product of transcription, alternative splicing, and translation, full-
length GPR39-1a is a protein with a molecular weight of 52 kDa
15
; another
product, the GPR39-1b protein, with a molecular weight of 32 kDa, is
identical in sequence to GPR39-1a for residues 1 to 285, and after residue
285, its sequence is Ser-Glu-Ser and end, which is different from the
corresponding sequence of GPR39-1a.
35
93Obestatin Receptor in Energy Homeostasis and Obesity Pathogenesis
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GPR39 is expressed in many organs and various tissues, especially in
stomach, white adipose tissue, and hypothalamus.
6,34,36
GPR39-1b is
widely expressed but may be nonfunctional. Egerod et al. pointed out that
some studies before might have been misled by the unawareness of GPR39-
1b and LYPD1. Both have a region of sequence overlapping with GPR39-
1a, the canonical and functional receptor.
34
The level of constitutive activity
of GPR39-1a could be affected by the splicing producing GPR39-1b,
which may be an implication of the conservation of the two-exon structure
and LYPD1 gene-overlapping pattern.
34
Furthermore, there are GPCRs
that have splice variants like GPR39. For the luteinizing hormone receptor,
the calcitonin receptor, and a1A-adrenoceptor, the variants with shorter
sequences interact, perhaps by forming heterodimers, with the full-length
receptors and alter their functions.
35
Because GPR39, similar to related
GPCRs such as the ghrelin receptor, keeps a rather high constitutive,
ligand-independent activity, the expression of GPR39-1b may be a way
to change the signaling of GPR39-1a.
The human GPL-1R gene has been mapped to chromosome 6, band
p21.1,
37
including 13 exons.
38
Along with the gene, there are two regions
with highly polymorphic simple sequence repeat, named CA1 and CA3.
39
The simple tandem repeat DNA polymorphism adopts the form of (TG)
n
37
and discriminates many alleles. When these two repeats were identified,
14 alleles for CA1 were observed in African-Americans and six in Cauca-
sians, and 16 alleles for CA3 were observed in African-Americans and eight
in Caucasians.
39
Various analyses of allelic frequencies in noninsulin-
dependent diabetes mellitus patients negate the hypothesis that inherited
defects in GLP-1R in African-Americans and Caucasians determine the
genetic susceptibility to noninsulin-dependent diabetes mellitus.
39
In the 50-UTR of the GLP-1R gene, three binding sites for transcript
factor Sp1 have been localized.
40
By experiments with constructed mutants,
mutation of the two proximal sites, 123 and 58 nucleotides upstream from
the transcription start site, were shown to be relevant to the reduction of
activity, while the distal one, 339 nucleotides upstream from the transcrip-
tion start site, to be a cis-regulatory-element as a repressor. Both Sp1 and Sp3
could bind to these sites, involved in the regulation of the expression.
40
Be-
sides, there is yet another distal cell-specific silencer element. This element
lies between 2285 to 2235 in the 50-flanking region of the GLP-1R
gene. The silencer element may function for tissue- and cell-specific expres-
sion of GLP-1R, because Sp1 is a ubiquitous transcription factor
maintaining basal promoter activity. It was reported that the transcription
94 Jian V. Zhang et al.
Author's personal copy
of the human GLP-1R gene was repressed in fibroblasts and pancreatic
A-cells, and not pancreatic B- and D-cells.
41
Structural study on GPCR family B is less bustling than that on GPCR
family A. There are no experimentally determined structures of full-length
receptors of family B, but structures of the N-terminal extracellular domains
of several receptors, including GLP-1R and GIPR. These structures reveal
detailed insights of the receptors, for example, the structural mechanism of
the recognition between ligands and receptors. In the crystal structure of
GPL-1R extracellular domain bound with an antagonist exendin-4
(9–39),
27
the N-terminal domain of GLP-1R embraces the antagonist pep-
tide, forming an amphipathic a-helix, by both hydrophobic and hydrophilic
interactions. Similar to other solved structures of GPCR family B, the core of
the N-terminal domain of GPL-1R possesses three conserved disulfide bonds
and two regions of antiparallel b-sheets.
27
The hydrophobic binding site is
defined by several a-helices and a loop linking two antiparallel b-strands.
In contrast to a hypothesis
42
suggested by experiments with truncated ana-
logues of exendin-4 and explaining the superior affinity of exendin-4, the
Trp cage of exendin-4, which is a segment of nine amino acid residues in
the C-terminus of exendin-4, does not participate the in interaction with
GLP-1R.
27
The binding pattern of exendin-4 with GLP-1R is similar to that
of other GPCRs of family B with their ligands, for example, GIPR and GIP.
43
The crystal structure displaying the binding pattern confirms a two-step model
of the binding mechanism
44
: The first step is the C-terminal region of the pep-
tide ligand interacts with the N-terminal extracellular domain of the GPCR,
adjusting the orientation and may be secondary structure; the second step is
the N-terminal region of the ligand touches the other part of the GPCR, es-
pecially the transmembrane helices, leading to signal transduction. Structure of
the other portion of GLP-1R is unknown and believed similar to GPCR of
family A.
43
There is still the forth disulfide bond formed by residue Cys226
and Cys296, in which Cys296 belongs to the third extracellular helix and
is close to the activation pocket because mutation of Cys226 to alanine de-
creases GLP-1 potency while mutation of Cys296 to alanine in addition re-
stores the lost potency.
45
In the 463-amino acid-long sequence, residues
Cys341 and Arg348 are conserved and could be sites of mono-ADP-
ribosylation.
46
GLP-1R was first known as a regulator of insulin secretion and mainly
expressed in pancreatic b-cells. In other tissue, GLP-1R mRNA can be
identified in lung, stomach, kidney, hypothalamus, brain, and heart, but
not in skeletal muscle, liver, and adipose.
47,48
95Obestatin Receptor in Energy Homeostasis and Obesity Pathogenesis
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1.3. Signal transduction across cell membrane for GPR39
and GLP-1R
GPCR mediates signaling by association with G proteins and involves sec-
ond messengers such as cAMP and calcium ions (Ca
2þ
). Some signaling pro-
cess of GPR39 might be triggered by obestatin or zinc ions, beside pathways
that are thought to be constitutively activated.
10
The obestatin-binding sites
have not yet been identified, although a Gs-mediated signaling pathway has
been proposed.
3
In cultured preadipocytes, obestatin bound to GPR39 is
reported to stimulate the phosphorylation of ERK1/2 and potentially
Akt, the downstream signaling partners.
4,13
Obestatin induces the
expression of c-fos, a transcription factor as a marker of activity in gastroin-
testinal and other tissues. If tested with GPR39-null mice, the obestatin-
induced c-fos expression was abolished, confirming the role of GPR39 in
inducing the action of obestatin.
13
The zinc ion-binding sites are residues
His17 and His19 according to mutagenesis experiments.
49
If Zn
2þ
is present,
GPR39 might also signal through Gs pathway, in addition to Gq and
G12/13 pathways that are constitutively activated.
10
It remains unknown,
however, if Zn
2þ
is only an allosteric modulator enhancing the function
of another hormone.
10
GLP-1R is a GPCR for GLP-1,
17
a hormone playing a key role in
incretin effect. The term “incretin” is used to denote the fact that a plasma
insulin response can be greater to oral glucose load than to an isoglycemic
intravenous glucose infusion.
50
Incretin effect is impeded in patients with
type 2 diabetes, thus incretin-based therapies, for example, with GLP-1R
agonists such as Liraglutide (approved by Europe in 2009), seem promising
for the treatment of the disease.
51
By analysis of mice with null mutations,
it was demonstrated that loss of GLP-1R action results in impaired insulin
response to oral glucose.
52
GLP-1R couples to Gs, Gq/11, and Gi1/2.
53
Via the coupling, it can trigger the activation of phospholipase C
54
and
MAPK,
53
as well as adenylyl cyclase,
55
leading to increases of cytosolic cal-
cium. The Lys334-Leu335-Lys336 sequence within the third intracellular
loop is a putative site for G protein coupling, for deletion of the sequence
abolishes the activation of adenylyl cyclase and insulin secretion.
56–58
In
this loop, residues V327, I328, V331, and K334 may be also essential in
interaction with G protein and stimulation of cAMP production, as shown
in an alanine scanning experiment.
59
These residues were located in one
face of an a-helix in a helical wheel model, suggesting that the secondary
structure of the third intracellular region consists of an a-helix.
59
In studies
96 Jian V. Zhang et al.
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with synthetic peptides analogous to this region, the peptide of the entire
loop strongly activated pertussis toxin- and cholera toxin-sensitive G pro-
teins.
58
By measurements of GTPase activity stimulated by these ana-
logues, the effect of the third intracellular loop was further divided into
two parts: the N-terminal half for activating cholera toxin-sensitive G pro-
teins (Gs), and the C-terminal half for activating pertussis toxin-sensitive G
proteins (Gi).
58
This division provides an explanation of the existence of
several G protein partners of GLP-1R.
The interaction of obestatin and GLP-1R was first identified by the
transduction of survival signals.
4
In the study, obestatin showed specific
binding on HIT-T15 cells and INS-1E b-cells, both of which were from
islet b-cells and induced the proliferation and survival of serum-starved
and cytokine-treated cells. With the treatment of obestatin, intracellular
cAMP concentrations in INS-1E cell increased in 30 min and then decreased
but still were higher than control at 60 and 90 min.
4
Any one of the antag-
onist of the Gs protein, inhibitor of adenylyl cyclase, and inhibitor of protein
kinase A blocked the anti-apoptotic effect induced by obestatin. In addition,
obestatin promoted ERK1/2 and Akt phosphorylation but not Src kinase,
suggesting that the anti-apoptotic effect of obestatin on b-cells depended on
adenylyl cyclase/cAMP/PKA, PI3K/Akt, and ERK1/2.
4
2. OBESTATIN AND ITS RECEPTOR(S) IN ENERGY
HOMEOSTASIS
Two independent studies revealed obestatin immunoreactivity in cells of
the gastric mucosa, myenteric plexus, and in Leydig cells of thetestis
60
and in the
stomach,duodenum, jejunum, colon, and pancreas
61
of Sprague–Dawley rats,
respectively. The obestatin immunoreactivity in human tissue was localized to
the mucosa of the gastrointestinal tract, the pancreatic islets, and the epithelial
cells in the ducts of mammary glands.
62
The GPR39 immunoreactivity was
localized to gastric mucosa, intestinal villi, white adipose tissues, hepatic cords,
and kidney tubules of adult mice.
13
The colocalization of immunoreactive
obestatin and its receptor GPR39 in the gastric mucosa and intestinal tissues
implies its involvement in energy homeostasis.
Obestatin may play a role in energy homeostasis through different phys-
iological processes, like modulating food intake and gastrointestinal motility,
GH secretion, and lipid metabolism.
97Obestatin Receptor in Energy Homeostasis and Obesity Pathogenesis
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2.1. Food intake
In the first report of obestatin, Zhang et al. showed that the intraperitoneal
injection (125 and 1000 nmol/kg) and intracerebroventricular treatment
(8 nmol/kg) of obestatin in adult male mice decreased food intake.
3
Re-
sults also showed that treatment with obestatin for 5, 6, 7, and 8 days
(1000 nmol/kg, three times a day) in adult male rats resulted in suppressed
body weight gain.
3
Then many groups repeated its anorectic effects in a
number of experimental paradigms after both peripheral and central ad-
ministration of obestatin. Bresciani et al. showed that obestatin inhibited
feeding after short-term starvation in young adult male rats.
63
Green
et al. reported a 43% reduction in food intake after intraperitoneal obestatin
injection 4 h prior to an allowed 15 min period of feeding in mice.
64
Bru-
netti et al. also repeated its anorectic effect by intraperitoneal daily injection
of obestatin for 12 days. The decrease in food intake and weight gain be-
came evident on day 6 and day 8, respectively, and maintained through to
day 12.
65
Sibilia et al. reported that acute intracerebroventricular admin-
istration of obestatin in 16 h-starved rats induced a trend toward a reduc-
tion of food intake but not statistically significant. Chronic
intracerebroventricular administration of obestatin for 1 day significantly
decreased food intake but this effect vanished thereafter for the next
2–28 days.
66
Based on their study, Sibilia et al. concluded that the effect
of obestatin on food intake tends to be short-term instead of long-term
influence.
66
Since administration of ghrelin induces potent stimulating effects on
food intake, the relation between obestatin and ghrelin in food intake
was also studied. Though Zizzari et al. found no difference in the food intake
at 1, 3, 5, and 18 h postintraperitoneal injection of 1000 nmol/kg obestatin
in either 24 h fasted mice or normal diet mice, they showed that obestatin
inhibited ghrelin-induced orexigenic effect in normal diet mice.
67
On the other hand, several researchers were not able to confirm the effect
of obestatin on food intake,
21,68–71
thus the role of obestatin as an anorectic
hormone was challenged.
72
The reason for the discrepancies is unclear. The
different experimental paradigms such as different animal species, methods
of administration, and the duration of drug treatment, might have influenced
the outcome of the effect of obestatin on food intake in some way. Also, a
recent study questioning the purity of synthetic peptides including obestatin
might shed a clue on these discrepancies.
73
98 Jian V. Zhang et al.
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2.2. Gastrointestinal motility
Not only food intake Zhang et al. also showed that jejunal contraction was
inhibited and obestatin treatment resulted in a sustained suppression of gas-
tric emptying activity.
3
Ataka et al. showed that intravenous injection of
obestatin inhibited motor activity in the stomach antrum and duodenum
of freely moving conscious rats and that CRF type 1 and type 2 receptors
in the brain might be involved in these effects of obestatin on gastroduodenal
motility.
74
These results were reproduced in another study using freely
moving conscious rat models.
75
Some other studies also addressed the effect of obestatin on ghrelin-
induced gastrointestinal motility. Depoortere et al. reported that peripheral
administration of obestatin showed no effect on fundic strip contractions but
cotreatment of obestatin tended to reduce the excitatory effect of ghrelin in
mice.
76
However, another study reported that obestatin failed to antagonize
ghrelin-induced stimulation of gastroduodenal motility.
74
In summary, the effect of obestatin on gastrointestinal motility remains
controversial. The effects of obestatin in gastric emptying and the contrac-
tility of the strips from fundus and small intestine of mice and rats were not
reproduced by De Smet et al..
77
2.3. Growth hormone secretion
Zhang et al. showed that unlike ghrelin, obestatin did not increase in vitro GH
secretion by cultured rat pituitary cells,
3
which was confirmed by other
groups. Bresciani et al. showed that obestatin had no effect on GH secretion
or GH-stimulated hexarelin production or in spontaneous corticosterone se-
cretion in 10-day-old rats.
63
Nogueiras et al. reported no effect of obestatin
treatment not only on GH release but also some hypothalamic neuropeptides
involved in energy balance regulation.
68
However, Zizzari et al. reported
obestatin (10 mg/rat)-affected ghrelin-induced GH secretion in vivo though
obestatin did not affect spontaneous or ghrelin-induced GH release from
superfused pituitaries ex vivo or basal GH levels in vivo.
67
2.4. Lipid metabolism
Another physiological role of obestatin might be as a regulator of adipocyte
metabolism and adipogenesis. Zhang and colleagues showed that treatment
of preadipocytes 3T3-L1 cells with obestatin induced an early-response
gene c-fos and stimulated ERK1/2 phosphorylation.
13
Then the study by
99Obestatin Receptor in Energy Homeostasis and Obesity Pathogenesis
Author's personal copy
Gurriara
´n-Rodrı
´guez et al. supported a role of obestatin in promoting
adipogenesis and adipocyte metabolism regulation.
78
Obestatin activated Akt
and its downstream targets while it inactivated AMPK and decreased ACC
phosphorylation in 3T3-L1 cells. Obestatin treatment together with IBMX
and DEX was shown to regulate the expression of C/EBPa,C/EBPb,C/EBPd,
and PPARgpromoting adipogenesis. The involvement of obestatin in cell
metabolism and adipogenesis was confirmed in vivo in the white adipose tissue
obtained after 24 and 72 h continuous subcutaneous infusion of obestatin.
78
Miegueu et al. reported that obestatin stimulated cell differentiation and increased
fatty acid uptake in a dose-dependent manner while obestatin decreased lipolysis
in 3T3-L1 cells.
79
in vivo study showed that plasma triglyceride levels were
significantly reduced by a chronic 14-day treatment with a stable obestatin
analog, N-terminally PEGylated obestatin.
80
Cholesterol levels were increased
significantly in the putative obestatin receptor GPR39 null mice compared with
wild-type mice.
12
These results support a role of obestatin in lipid metabolism.
2.5. GPR39 null mice
GPR39 knockout mice may reveal the physiological roles of obestatin/
GPR39. Moechars et al. showed that GPR39 null mice showed accelerated
gastric emptying, increased volume of gastric secretion, higher mature body
weight and body fat composition, and increased cholesterol levels compared
with wild type.
12
The report also showed that food intake during day 1 was
not different between the two genotypes while food intake after fasting was
decreased in GPR39 null mice.
12
Then Zhang and colleagues further
showed that treatment with obestatin induced an early-response gene c-fos
expression in gastric mucosa of wild-type, but not GPR39 null, mice, und-
erscoring a mediating role of this receptor in obestatin actions.
13
The altered
gastric activity, body weight, body fat composition, and cholesterol levels in
GPR39 null mice might be due to the lost of obestatin biological function
due to its missing receptor. All these supported a role of obestatin-GPR39
system in energy homeostasis.
2.6. Obesity pathogenesis
Obesity with its increasing prevalence becomes a global epidemic problem.
In the United States, more than 20% of adults are clinically obese as defined
by a body mass index of 30 kg/m
2
or higher, and an additional 30% are over-
weight.
81
As a new proposed anorectic peptide hormone, the association of
obestatin with obesity and obesity-related diseases draws great interest ever
100 Jian V. Zhang et al.
Author's personal copy
since its discovery. Considering the involvement of both ghrelin and
obestatin in energy homeostasis, both the plasma obestatin level and plasma
ghrelin/obestatin ratio were used to assess the possible involvement of
obestatin in obesity pathogenesis. Several groups reported decreased
plasma obestatin level in obese adults than normal weight control
15,82–86
among which Guo et al. detected an increased ghrelin to obestatin ratio
and Huda et al. detected no difference in ghrelin to obestatin ratio between
obese and lean subjects
1
accompanied by decreased obestatin level. On
the contrary, Vicennati et al. reported a higher obestatin level in obese
women compared to control together with a decreased ghrelin to obestatin
ratio.
87
Zou et al. reported no significant difference in plasma obestatin
level between obese and normal weight children with a decreased ghrelin
to obestatin ratio.
88
Due to the heterogeneity in the relationship between plasma obestatin
levels and obesity, Zhang et al. performed a meta-analysis on the related stud-
ies with obestatin hormone level with the published work before December
2009.
89
Concerning obestatin levels in the nine studies with 566 participants,
obestatin of the obese groups is lower than that of the normal weight groups
at 64.19 pg/mL [95% confidence interval (CI) ¼36.11–92.26, P<0.01].
Concerning the ghrelin/obestatin ratio in the five studies with 259 partici-
pants, the ratios of obese group were lower than those of the normal weight
group although not statistically significant.
89
Not only the plasma obestatin level was showed to be associated with
obesity, but gastric body mucosa obestatin expression was also reported to
decrease in overweight and obese patients by immunohistochemistry.
90
The association between obestatin levels (both circulating and gastric level)
with obesity all suggested a possible role for stable obestatin level or balanced
obestatin/ghrelin ratio in long-term body weight regulation.
The effects of weight loss surgery or exercise on obestatin level might
give us another perspective to decipher the relationship between obestatin
and obesity. Changes in plasma obestatin levels after weight loss through
either Roux-en-Y gastric bypass (RYGB) surgery or weight reduction in
summer camp were studied. Roth et al. reported that ghrelin concentrations
and ghrelin/obestatin ratios were lower in patients 2 years after RYGB
surgery than before, while obestatin levels did not change.
91
Martins et al.
reported that both ghrelin and obestatin plasma levels were significantly
elevated in the patients 3 years after the RYGB surgery as compared to the
control group and no statistically significant difference in ghrelin/obestatin ra-
tio was detected among the two groups.
92
In the study by Huda et al.,
101Obestatin Receptor in Energy Homeostasis and Obesity Pathogenesis
Author's personal copy
obestatin tended to be lower in gastrectomy subjects and their obestatin/
ghrelin ratio differed from healthy controls.
86
A 24 h to 7-day time-course
study on the effect of short-term anaerobic exercise training was performed
in college women though no effect of the exercise on the circulating obestatin
levels was observed.
93
Accumulating evidence piled up for the involvement of obestatin in
energy homeostasis and its association with obesity. But more investigation
is needed to decipher the detailed mechanism of its physiological and path-
ological roles including the innate receptor(s) for obestatin, from which
we can understand more about the regulation of energy homeostasis and
hopefully provide candidate(s) for obesity-related treatment.
ACKNOWLEDGMENTS
This work was supported by the following grants: One-hundred Talent Program of
Chinese Academy of Sciences (to J. V. Z.), The National Basic Research Program of
China (973 program 2013CB945503), Distinguished Young Scholars Program of The
National Natural Science Foundation of China (NSFC 31100850 to J. V. Z.), The
Distinguished Young Scholars of Shenzhen City (SW201110059 to J. V. Z.), Shenzhen
Engineering Laboratory of Single-molecule Detection and Instrument Development
and Innovative Pharmacology and Biotherapy Pre-clinical Test Public Service Platform,
Shenzhen.
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107Obestatin Receptor in Energy Homeostasis and Obesity Pathogenesis
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