Download study of apelin and its effects

STUDY OF APELIN AND ITS EFFECTS
Abstract
Apelin is a newly identified bioactive peptide, which is secreted by the
adipocytes. It is the endogenous ligand of the G- protein coupled receptor
(APJ), which has been shown to be expressed in several human tissues
including the CNS, endothelium, lung, heart and pancreatic islets. Only a part
of the apelinergic pathway has been elucidated. The apelinergic system is
involved in the homeostasis of the cardiovascular system, especially in the
mechanisms of heart failure and vasorelaxation. It is associated with the
regulation of angiogenesis, body fluid homeostasis and gastric cell
proliferation and related to atherosclerosis, gastrointestinal and immune
system diseases, particularly HIV infection. Apelin secretion is found to be
upregulated directly by insulin and it is involved in the high prevalence of
morbidities such as obesity, diabetes mellitus type 2 and cardiovascular
diseases which constitute major risk in public health. The purpose of this
review was to present the possible role of the apelinergic system in the
pathophysiology of human disease with emphasis on the adipoinsular axis.
Introduction
Apelin was discovered years after its receptor was identified. A novel Gprotein-coupled receptor (GPCR) was identified by homology cloning in 1993
by O’Dowd and was named APJ.1 Due to the absence of an apparent
endogenous ligand, this receptor remained in the list of ‘orphan’ G-proteincoupled receptors. In 1998 Tatemoto et al purified the protein which was
bound to this receptor from bovine stomach extract.2 This protein was named
‘apelin’ and together with its receptor APJ , it constitutes the Apelinergic
System. Research has aimed at clarifying the role of this system in the
human. The apelinergic system may constitute a very important metabolic
system for further investigation as it may be a potential target for prospective
therapeutic applications on heart failure, diabetes mellitus type 2 and
hypertension.
The Apelin Receptor
G-protein-coupled receptors (GPCRs) belong to a large family of
transmembrane proteins, which recognize a variety of different ligands that
mediate the transduction of extracellular signals into activation of intracellular
cascades.3,4 Nuclear localization signals have also been identified in the
aminoacid sequences in some of the GPCRs.4 They are involved in
numerous physiologic functions in a wide range of different tissues and
therefore their signaling plays a crucial role in many pathophysiology
pathways.3
The human apelin receptor consists of 7 transmembrane domains and a total
number of 377 aminoacids. In the mouse and in the rat, the apelin receptor
also has 377 aminoacids and it shares 91% and 89% sequence homology,
respectively, with the human receptor.5 The gene of the human apelin
receptor is located on chromosome 11, locus 11q12, near the D2 and the D4
dopamine receptors.1 However, the apelin receptor has great sequence
homology (54% in the transmembrane regions) with the angiotensin II type 1
(AT1) receptor, another G-protein-coupled receptor. Despite this similarity,
angiotensin II is not able to interact with APJ, nor does apelin bind to AT1. 1
This indicates that apelin is the unique endogenous ligand for APJ.2
Given that the expression of a receptor correlates with its function,4 a
monoclonal antibody recognizing the apelin receptor could reveal the various
cell types, which express the apelin receptors and could clarify the apelin
signaling pathway. The apelin receptor was first detected in the endothelial
cells of large vessels during embryogenesis.6 The distribution of the apelin
receptor protein within the human brain has not been determined yet although
the mRNA encoding the apelin receptor is widely expressed in the human
central nervous system (CNS), in the spinal cord, corpus callosum, and in the
medulla.7-9 The apelin receptor is also abundantly expressed in the human
heart, adipose tissue, endothelium, lung, pancreatic islets, kidney, and the
stomach.4,8-10 The highest apelin receptor mRNA levels are found in the
human spleen and placenta.5 APJ is coupled to the Gi/o proteins; more
precisely desensitization patterns have been demonstrated between the
apelin receptor, Ga(i1) and Ga(i2) subunits.11
The intracellular cascades triggered by the interaction of apelin with its
receptor include the inhibition of cAMP accumulation induced by forskolin in
an apelin-concentration-dependent manner in Chinese hamster ovary cell
lines expressing APJ.12 The activation of the apelin receptor may also lead to
phosphorylation and activation of extracellular-regulated kinases via a
pertussis toxin sensitive G protein. 13
A rise in the intracellular calcium concentration was observed after stimulation
of the apelin receptor in NT2.N neurons.14 Subsequently, the study of the
potential inotropic effect of apelin on cardiac tissue revealed that the elevated
Na+/H+ exchange and the reverse-mode Na+/Ca+ exchange lead to
augmentation of the intracellular calcium concentration promoting myocardial
contraction.15
The activation of the p70S6 kinase by apelin via ERK-dependent
phosphorylation of the threonine residue and the PI3K-dependent
phosphorylation of the serine residue contribute significantly to endothelium
cell proliferation. Thus, activation of the apelinergic system has been
highlighted as mitogenic for the umbilical endothelial cells.16 Transduction
cascades of apelin signalling may also involve activation of the endothelial
nitric oxide (NO) Synthase, which may lead to NO release and peripheral
vasodilatation.17
A functional nuclear signal has been identified in the third intracellular loop of
the apelin receptor. This pattern enables the receptor to be identified in the
nucleus as well as the membrane. Its nuclear translocation seems to be cellspecific in the cerebellum and human hypothalamus.3,18 These data
reinforce the concept that apart from the role of the receptor at the membrane
level in extracellular and intracellular signaling, it may also play a role in the
regulation of gene transcription.
Apelin
Endogenous ligands for the orphan receptor, the apelin-36 and apelin-13
peptides were identified, isolated and purified from bovine stomach-tissue
extracts by Tatemoto et al in 1998, .2 After cloning the corresponding bovine
and human cDNA according to the amino acid sequences, they discovered a
77-amino acid polypeptide encoding gene. This polypeptide chain consisted
of a secretory signal sequence along with 36 residues of the C-terminal which
corresponded to apelin-36. Several mapping studies followed, until the apelin
gene was finally localized on the X-chromosome at the locus Xq25-q26.3.19
The gene encoding the 77 amino acid pre-proapelin peptide consisted of
1,726 base pairs, including 3 exons.
The 23 residues of the C-terminal of the pre-proapelin sequence are identical
in the rat, mouse, cattle and the human,19 .This means a considerable
homology across several species. The N-terminal sequence seems to be the
key region for the modulation of the ligand-receptor interaction,10 whereas
the 12 residues of the C-terminal fragment are thought to be indispensable for
the apelin binding to the receptor.17
Pre-proapelin is a high molecular weight peptide. It has a dimer form ,
disulfide stabilization linkages and many endopeptidase cleavage sites.20 All
apelin isoforms that have been described are C-terminal biological active
fragments of different length. Apelin-36, apelin-17, apelin-16, apelin-13 and
apelin-12 as well as the post-translationally modified (Pyr1)Apelin-13 are
known agonists of the apelin receptor . 2, 5, 9, 21, 22 The agonist
(Pyr1)Apelin-13 prevents enzymatic breakdown due to a conversion of the Nterminal glutamate to pyroglutamate and thus, preserves biological activity.
Data suggest that while shorter isoforms exert stronger cardiovascular action,
the longer apelin peptides present higher binding affinity to the receptor and
can inhibit HIV infection by blocking the HIV co-receptor APJ.15,23-25
The newly identified zinc-containing carboxypeptidase angiotensin converting
enzyme-2 (ACE-2), which metabolizes angiotensin I and II into Ang 1-9 and
Ang 1-7, respectively, has been found to cleave the C-terminal phenylalanine
from apelin-13 and apelin-36,26 without inactivating them. As regards to the
rat apelin receptor, which is expressed in Chinese hamster cells, the
phenylalanine lacking apelin fragments are shown to preserve their binding
ability along with their functional activity.27 However, they appear unable to
induce internalization of the receptor in vitro and a hypotensive action in vivo.
The above reports support the concept that the peptides produced by the
apelin ACE-2 metabolism induce a conformational state of the apelin
receptor.27 In accordance to this, Pitkin et al demonstrated in humans that
(Pyr1) apelin-13, which lacks the C-terminal phenylalanine, has comparable
binding affinity in vitro as well as functional activity in human tissues,5
providing evidence that the ACE2 cleavage of apelin does not constitute a
degradation pathway.
In parallel to its receptor, human apelin shares a wide tissue expression in the
CNS and in the periphery. Apelin mRNA has been detected in every tested
part of the CNS. The highest expression has been documented in the spinal
cord, corpus callosum, amygdala, substantia nigra and pituitary gland.9 In the
periphery, apelin is expressed in the placenta, lung,21 adipose tissue,28
kidney, gastrointestinal tract,29 magnocellular neurons,30 mammary glands,2
vascular and in the endocardial endothelial cells.31
Many apelin isoforms have been detected in the human. (Pyr1)apelin-13,
apelin-13 and apelin-17 are the predominant circulating plasma apelin
peptides. 30,32-33(Pyr1)apelin-13 is the major isoform in the cardiac
tissue.34
The apelinergic system in the adipoinsular axis
Adipose tissue is considered as an endocrine organ and a plethora of
adipocyte-secreted bioactive peptides, the adipokines, have been
identified.35 Adipokines have paracrine and endocrine actions,. They regulate
the adipose tissue physiology (fat development, energy storage and
metabolism) locally, and act as circulating hormones at remote cells.36 In
obesity, the increased number of adipocytes along with the increased per unit
synthesis of the adipose tissue mass result in the upregulation of
adipokines.37 This highlights the quantitative importance of the adipokines in
all biological functions, particularly in the development of complications in
obesity.
The presence of apelin in the adipose tissue was first described by Tatemoto
et al,17 but Boucher et al demonstrated that apelin is expressed and released
by human adipocytes and identified it as the most recently described
adipokine.28 The increased apelin expression during adipocyte
differentiation,28 in parallel with apelin secretion by differentiated adipocytes
in vitro, reinforce the adipokine identity of apelin.11 Presently, the adipose
tissue presents a possible source of apelin detected in the plasma. 5,37
A strong relationship between the apelinergic system and insulin status has
been reported.28 In the mouse, plasma apelin levels and adipocytes apelin
expression have been found to be regulated by the nutritional status. Fasting
decreases and refeeding restores apelin secretion and fat cell apelin gene
expression.28 Indeed, plasma and adipocyte apelin levels are increased in all
obesity mice models when hyperinsulinemia is present. This indicates that
neither obesity nor high-fat feeding are the main determinants of apelin
upregulation.28 In the same study, obese patients had significantly high
plasma insulin and apelin levels. This finding indicates the presence of an
impaired apelin homeostasis in obesity according to which raised insulin
levels promote the rise in plasma apelin levels. Similarly, plasma apelin levels
measured in patients with morbid obesity were found elevated only in type 2
diabetics. This means that obesity is not the main cause of the apelin rise.38
Boucher et al reported that adipocytes of insulin-deficient mice were deficient
in apelin expression in parallel with low insulin production. This is the first
evidence that insulin exerts a direct regulation on the apelin gene expression
and secretion in the adipose tissue, through the activation of both
phosphatidyIinositol 3-kinase and protein kinase C pathways28.
The link between apelin and insulin has also been confirmed by Sorhede,
Winzell et al, who demonstrated that the apelin receptor is expressed in
pancreatic islets and that apelin-36 administration inhibits glucose-stimulated
insulin secretion in mice.39 Whereas apelin decreases insulin secretion,
insulin stimulates adipose apelin expression, representing a complex
interaction between the two systems.5 Recently islet cell derived apelin has
been reported to be regulated by glucocorticoids and not by glucose.40 The
islet cell apelin production combined with the apelin receptor expression in
the islets, support the concept of an apelinergic regulatory pancreatic system
together with an autocrine/paracrine insulin regulatory mechanism.40
According to the above mentioned data, apelin-36 exerts a dual
concentration-dependent effect on insulin secretion from b-cells,40 with
insulin inhibition being the predominant effect on the islet. Similarly, when rat
insulinoma INS-1 cells were incubated in order to examine the effects of
apelin on insulin secretion, apelin exerted a direct inhibitory action on betacells via the activation of phosphoinositide 3-kinase dependent
phosphodiesterase 3B and therefore suppression of cAMP levels.41
Ringstrom et al hypothesized that islet apelin acts as a negative feedback
signal aiming at the inhibition of insulin secretion in hyperinsulinemic
conditions . This hypothesis was based on the reported positive regulation of
the adipocyte apelin expression by insulin 28 and the increased apelin
expression in islets of hyperinsulinemic T2D animal models.40 However,
there is a lack of agreement regarding the plasma apelin levels in humans
with abnormal glucose homeostasis. Elevated plasma apelin levels were
reported in individuals with impaired glucose tolerance and diabetes mellitus
type 2,42- 43 Lower plasma apelin levels have been found in newly
diagnosed non-treated patients with diabetes mellitus type 2 as compared to
the control group. 44-45 The control group in the latter study presented higher
BMI than in the majority of the performed studies. This may present a
possible explanation for the observed difference.44 Moreover, it has been
recently demonstrated that antidiabetic therapy (metformin or add-on
rosiglitazone) may result in a remarkable increase in baseline plasma apelin
concentration in diabetic patients.46
In normal and obese mice, Dray et al demonstrated a glucose-decrease
effect associated with enhanced glucose utilization in the skeletal muscle and
adipose tissue after an acute intravenous administration of apelin through the
involvement of the eNOs, AMP activated protein kinase (AMPK) and Aktdependent pathways.47 Enhanced glucose uptake via the AMPK-dependent
pathway has also been described when apelin was delivered to cultured
C2C12 myotubes.48 Given the fact that apelin is able to activate AMPK, it
has been proposed recently that insulin-sensitizers may trigger apelin
secretion through AMPK activation, leading to the alleviation of insulin
resistance.46
Higuchi et al investigated the effects of apelin on body adiposity in order to
further explore the role of apelin pathophysiology by studying the uncoupling
protein expression (UCP).49 The repeated intraperitoneal administration of
apelin for 14 days leaded to reduced body adiposity (without the influence of
food intake). It also reduced leptin, insulin and triglyceride serum levels and
increased adiponectin serum levels in normal and obese mice. Increased
mRNA expression of UCP1 (a marker of peripheral energy expenditure) in
brown adipose tissue was observed in normal mice. Thus, apelin appears to
regulate body adiposity and lipid metabolism in lean and obese mice.49
These data suggest that apelin may be a beneficial peptide whose
overproduction is probably an adaptive response or a last protective
mechanism before obesity-derived comorbidities arise.37
Several reports described positive association of plasma apelin levels with
body mass index28 as well as TNF-a levels.38 The direct positive effect of
TNF-a, which upregulates apelin secretion in mice adipocytes, indicated a
possible link between obesity and inflammation.50
Apelin and Cardiovascular system
The binomial Apelin/ APJ system has been described as an important
modulator of the development of the cardiovascular system since it is
expressed in embryonic and adult tissues.51 Animal model studies have
demonstrated the necessity of the system for the normal development of the
cardiovascular system through the regulation of the differentiation of the
embryonic stem cells, the discrete cell population movement and
migration.52-55 Studies on APJ knock-out mice reported retardation of
vascular development during embryogenesis.56-57 Other researchers did not
confirm any embryonic or other histological abnormalities. Heart deterioration
occurred with ageing and chronic pressure overload.58 D’ Aniello et al
recognized apelin and APJ as down streamers of mammalian
cardiomyogenesis.59
Despite reported data regarding the role of apelin in pressure/volume
homeostasis, the exact mechanisms involved still remain unclear. The
presence of the apelin receptor in vascular endothelium as well as smooth
muscle is suggestive of a role in endothelium-dependent and independent
modulation of vascular tone.5 Lee et al first demonstrated that apelin
administration decreased arterial blood pressure in rats, 60 Other
investigators confirmed it.17,27,61-63 Immediately after the discovery of the
antihypertensive activity, NO Synthase was found to block the above effect,17
providing evidence for an NO-dependent underlying route. Apelin
administration was proved to be a stimulus to increase blood pressure. A
vasoconstrictive role for apelin in human saphenous vein64 and in mammary
arteries in vitro34 has been described. The vascular smooth muscle cells
which express APJ may be involved. Apelin infusion into the human forearm
results in vasodilatation.65 Furthermore, a vasodilatation of the human
splachnic artery has also been described after apelin administration.66 The
concept that the apelinergic system exerts a hypotensive effect by activating
NO Synthase and by promoting endothelium-dependent vasodilatation
predominates. By contrast, in endothelium dysfunction, apelin action on the
smooth muscle cells leads to a vasoconstrictive effect.5,67
Physical exercise exerts a beneficial effect on blood pressure reduction
probably triggered through the apelinergic system. Long term exercise was
found to increase the apelin/APJ expression in the cardiovascular system in
hypertensive rats.68
Szokodi et al demonstrated that apelin plays a major role in myocardial
contractility by acting as a potent dose-dependent- inotropic agent directly on
the isolated perfused rat heart.15,69-71 Maguire et al confirmed these results
in human cardiac tissue supporting that apelin is among the most potent
endogenous inotropic agents yet reported in isolated human cardiac tissue.34
Hemodynamic models in mice indicated that apelin increased cardiac output
by reducing the left ventricle preload and afterload without the interference of
a hypertrophy mechanism.72 Regarding cell signaling, the inotropic action of
apelin appears to be mediated by the activation of PLC, PKC and
sarcolemmal Na+/H+ exchanger and the Na+/Ca++ exchanger.15 Apelin is
also involved, by a mechanism not clearly defined, in the pathophysiology of
heart failure. In the early stages of heart failure both plasma apelin levels and
APJ cardiac density are reported to be increased. 73-74 There was
diminished apelin production in the progressed heart failure, which may be
explained probably by the concurrent endothelial dysfunction.75
Various isolated reported properties of the
Apelinergic system
The apelin receptor has been reported to be highly expressed in the
endothelium of embryonic and retinal vessels.6,76 The fact that the
apelinergic system regulates vessel diameter during angiogenesis supported
apelin as a potent angiogenic factor.6,57, 77
Apelin is expressed in specific human tumors. A unique study in vitro has
demonstrated that apelin expression promotes tumor growth.78 The
apelin/APJ system in oxidative-linked atherosclerosis has been investigated
in APJ and apolipoprotein E double knock-out mice.79 It is reported that the
double knock-out mice that were fed a high cholesterol diet manifested a
dramatic reduction in their atherosclerotic lesions. The APJ deficient mice
showed decreased oxidative stress in their vascular smooth muscle cells
suggesting that the apelinergic system may be a critical factor in oxidative
stress mediated atherosclerosis.79
Concerning the immune system, the apelin receptor has been identified as
co-receptor for the Human Immunodeficiency Virus.25 Apelin/APJ interaction
in vivo blocks virus invasion in human cell lines, co-expressing CD4 and
APJ.25
In the gastrointestinal system, apelin was first isolated from stomach extracts.
Currently, it is known that apelin is expressed in gastric mucosa cells and the
stomach fundus.80 Known effects of the apelinergic system activation is the
modulation of cholecystokinin secretion from a murine enteroendocrine cell
line via a MAPK cascade, as well as endothelial cell proliferation .80 There
may be a possible role of apelin in the regulation of appetite. It has been
reported that intracerebroventricular infusion of apelin inhibits food intake.81
Inflammatory bowel disease is reported to be associated with increased
apelin immunostaining probably due to hypoxia and inflammation.82
There is wide distribution of APJ expression in the brain. The apelinergic
system may be involved in fluid homeostasis. The co-localization of the apelin
peptide and its receptor with the angiotensinogen and the arginine
vasopressin (AVP) expression reinforced this concept.67 Controversial
findings have been reported regarding the possible role of apelin as an
antidiuretic agent.19,83-84 The different results may be explained by the
dose and mode of apelin administration, (intraperitoneal or
intracerebroventricular).19,83-84
Conclusion
Current research indicates a clear involvement of the apelinergic system in
the adipoinsular axis as well as in the modulation of the cardiovascular
system. The relationship between the adipoinsular axis and obesity
emphasizes the importance of this novel system in the maintenance of
homeostasis and the prevention of obesity derived co-morbidities.
Acknowledgement
The authors express their gratitude to Professor S Nousia-Arvanitakis and
Professor Bessie Spiliotis for the critical review of the paper.