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 G-protein-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-protein-coupled 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 cell-specific 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 N-terminal 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 beta-cells 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 Akt-dependent 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.
