Adipocytes play pivotal roles in regulating metabolism through secretion of a variety of adipokines, which in turn is regulated by other metabolic factors (e.g., insulin). Understanding the regulations of adipokine secretion is important because adipokines are implicated with metabolic disorders, such as, obesity and diabetes mellitus. Here, we investigated the regulatory roles of angiotensin II (AngII) on the secretion of apelin in 3T3-L1 adipocytes, and distinct signaling pathways mediated by AngII receptor type 1 (AT₁) and type 2 (AT₂) were revealed. It was found that activation of AT₁ receptors stimulates apelin secretion in Ca²⁺, protein kinase C, and MAPK kinase dependent ways while activation of AT₂ receptors inhibits apelin secretion through cAMP and cGMP dependent pathways. Furthermore, we demonstrate that the expression of apelin receptor (APJ) is also similarly regulated by AT₁ and AT₂ receptors. Finally, a detailed AngII signaling map is proposed.

Apelin receptor(s) (APLNR) are suggested to mediate the actions of apelin and Elabela (ELA) peptides in many physiological processes, including cardiovascular development and food intake in vertebrates. However, the functionality of APLNR has not been examined in most vertebrate groups. Here, we characterized two APLNRs APLNR1, APLNR2) in chickens and red-eared sliders, and three APLNRs in zebrafish (APLNR2a, APLNR2b, APLNR3a), which are homologous to human APLNR. Using luciferase-reporter assays or Western blot, we demonstrated that in chickens, APLNR1 (not APLNR2) expressed in HEK293 cells was potently activated by chicken apelin-36 and ELA-32 and coupled to Gi-cAMP and MAPK/ERK signaling pathways, indicating a crucial role of APLNR1 in mediating apelin/ELA actions; in red-eared sliders, APLNR2 (not APLNR1) was potently activated by apelin-36/ELA-32, suggesting that APLNR2 may mediate apelin/ELA actions; in zebrafish, both APLNR2a and APLNR2b were potently activated by apelin-36/ELA-32 and coupled to Gi-cAMP signaling pathway, as previously proposed, whereas the novel APLNR3a was specifically and potently activated by apelin. Similarly, an apelin-specific receptor (APLNR3b) sharing 57% sequence identity with zebrafish APLNR3a was identified in Nile tilapia. Collectively, our data facilitates the uncovering of the roles of APLNR signaling in different vertebrate groups and suggests a key functional switch between APLNR1 and APLNR2/3 in mediating the actions of ELA and apelin during vertebrate evolution.

Apelin receptor (APJ) and kappa opioid receptor (KOR) are members of the family A of G protein-coupled receptors (GPCRs). These two receptors are involved in the central nervous system regulation of the cardiovascular system. Here, we explore the possibility of heterodimerization between APJ and KOR and investigate their novel signal transduction characteristics. Co-immunoprecipitation (Co-IP), co-localization and bioluminescence resonance energy transfer (BRET) assays confirmed the heterodimerization of APJ and KOR. In APJ and KOR stably transfected HEK293 cells, treatment with APJ ligand apelin-13 or KOR ligand dynorphinA (1-13) resulted in higher phosphorylation levels of extracellular-regulated kinases 1/2 (ERK1/2) compared to HEK293 cells transfected with either APJ or KOR alone. The siRNA knockdown of either APJ or KOR receptor in human umbilical vein endothelial cells (HUVECs) resulted in significant reduction of the apelin-13 induced ERK activation. Additionally both forskolin (FSK)-induced cAMP levels and cAMP response element reporter activities were significantly reduced, whereas the serum response element luciferase (SRE-luc) reporter activity was significantly upregulated. Moreover, the ERK phosphorylation and SRE-luc activity were abrogated by the protein kinase C (PKC) inhibitor. These results demonstrate for the first time that human APJ forms a heterodimer with KOR and leads to increased PKC and decreased protein kinase A activity leading to a significant increase in cell proliferation, which may translate to the regulation of diverse biological actions and offers the potential for the development of more selective and tissue specific drug therapies.

Maternal high-fat diet (HFD) consumption has been linked to metabolic disorders and reproductive dysfunctions in offspring. Troxerutin (TRO) has anti-hyperlipidemic, anti-oxidant, and anti-inflammatory effects. This study examined the effects of TRO on apelin-13, its receptors mRNA and ovarian histological changes in the offspring of HFD fed rats.

Kappa Opioid receptors (KORs) change the impact of apelin on the phosphorylated ERK1/2 (pERK1/2). However, the role of interaction between KOR and apelin receptors (APJ) on the cardiac contractility effects of apelin and in regulation of pERK1/2 and PKC in the heart of renovascular hypertensive (2K1C) rats is unknown.

Apelin receptor (APJ) and bradykinin 1 receptor (B1R) are involved in a variety of important physiological processes, which share many similar characteristics in distribution and functions in the cardiovascular system. This study explored the possibility of heterodimerization between APJ and B1R, and investigated the impact of heterodimer on the signal transduction characteristics and the physiological functions in human endothelial cells after stimulation with their agonists. We first identified the endogenous expression of APJ and B1R in HUVECs and their co-localization on HEK293 membrane. The constitutive heterodimerization between the APJ and B1R was then demonstrated by BRET and FRET assays. Stimulation with Apelin-13 and des -Arg(9)-BK enhanced the phosphorylation of eNOS in HUVECs, which could be dampened by the knockdown of APJ or B1R, indicating the co-existence of APJ and B1R is critical for eNOS phosphorylation in HUVECs. Furthermore, APJ/B1R heterodimers were found to enhance the activity of PKC signaling pathway and increase intracellular Ca(2+) concentration in HEK293 cells, which might be the mechanism of APJ/B1R heterodimers promoting the phosphorylation of eNOS and leads to increased Gαq, PKC signal pathway activities and a significant increase in cell proliferation. The results provide a new theoretical and experimental base for revealed intracellular molecular mechanisms of physiological function involved in the APJ and B1R and provide potential new targets for the development of drugs and treating cardiovascular disease.

Angiogenesis is a necessary process for solid tumor growth. Cellular markers for endothelial cell proliferation are potential targets for identifying the vasculature of tumors in homeostasis. Here we customize the behaviors of engineered cells to recognize Apj, a surface marker of the neovascular endothelium, using synthetic Notch (synNotch) receptors. We designed apelin-based synNotch receptors (AsNRs) that can specifically interact with Apj and then stimulate synNotch pathways. Cells engineered with AsNRs have the ability to sense the proliferation of endothelial cells (ECs). Designed for different synNotch pathways, engineered cells express different proteins to respond to angiogenic signals; therefore, angiogenesis can be detected by cells engineered with AsNRs. Furthermore, T cells customized with AsNRs can sense the proliferation of vascular endothelial cells. As solid tumors generally require vascular support, AsNRs are potential tools for the detection and therapy of a variety of solid tumors in adults.

Dysfunctions in CNS regulation of arterial blood pressure lead to an increase in sympathetic nerve activity that participates in the pathogenesis of hypertension. The apelin-apelin receptor system affects arterial blood pressure homeostasis; however, the central mechanisms underlying apelin-mediated changes in sympathetic nerve activity and blood pressure have not been clarified. We explored the mechanisms involved in the regulation of [Pyr1 ]apelin-13-mediated cardiovascular control within the rostral ventrolateral medulla (RVLM) using selective receptor antagonists. We show that [Pyr1 ]apelin-13 acts as a modulating neurotransmitter in the normotensive RVLM to affect vascular tone through interaction with the vasopressin V1a receptor but that [Pyr1 ]apelin-13-induced sympathoexcitation is independent of angiotensin II receptor type 1, oxytocin, ionotropic glutamate and GABAA receptors. Our data confirm a role for the apelin peptide system in cardiovascular regulation at the level of the RVLM and highlight that this system is a possible potential therapeutic target for the treatment of hypertension.

Apelin receptor (APJR) is a key regulator of human cardiovascular function and is activated by two different endogenous peptide ligands, apelin and Elabela, each with different isoforms diversified by length and amino acid sequence. Here we report the 2.6-Å resolution crystal structure of human APJR in complex with a designed 17-amino-acid apelin mimetic peptide agonist. The structure reveals that the peptide agonist adopts a lactam constrained curved two-site ligand binding mode. Combined with mutation analysis and molecular dynamics simulations with apelin-13 binding to the wild-type APJR, this structure provides a mechanistic understanding of apelin recognition and binding specificity. Comparison of this structure with that of other peptide receptors suggests that endogenous peptide ligands with a high degree of conformational flexibility may bind and modulate the receptors via a similar two-site binding mechanism.

Adipokines influence energy metabolism and have effects on male reproduction, including spermatogenesis and/or Sertoli cell maturation; however, the relationship between these active proteins and androgens in testicular cells is limited. Here, we studied the impact of short-term exposure to flutamide (an anti-androgen that blocks androgen receptors) on the expression of chemerin, apelin, vaspin and their receptors (CCRL2, CMKLR1, GPR1, APLNR, GRP78, respectively) in adult rat testes. Moreover, the levels of expression of lipid metabolism-modulating proteins (PLIN1, perilipin1; TSPO, translocator protein) and intercellular adherens junction proteins (nectin-2 and afadin) were determined in testicular cells. Plasma levels of adipokines, testosterone and cholesterol were also evaluated. Gene expression techniques used included the quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB) and immunohistochemistry (IHC). The androgen-mediated effects observed post-flutamide treatment were found at the gonadal level as chemerin, apelin, and vaspin gene expression alterations at mRNA and protein levels were detected, whereas the cellular targets for these adipokines were recognised by localisation of respective receptors in testicular cells. Plasma concentrations of all adipokines were unchanged, whereas plasma cholesterol content and testosterone level increased after flutamide exposure. Differential distribution of adipokine receptors indicates potential para- or autocrine action of the adipokines within the rat testes. Additionally, changes in the expression of PLIN1 and TSPO, involved in the initial step of testosterone synthesis in Leydig cells, suggest that testicular cells represent a target of flutamide action. Increase in the gene expression of PLIN1 and TSPO and higher total plasma cholesterol content indicates enhanced availability of cholesterol in Leydig cells as a result of androgen-mediated effects of flutamide. Alterations in adherens junction protein expression in the testis confirm the flutamide efficacy in disruption of androgen signalling and presumably lead to impaired para- and autocrine communication, important for proper functioning of adipokines.

Dysfunction of the apelinergic system, comprised of the neuropeptide apelin mediating its effects via the G protein-coupled apelin receptor (APJ), may underlie the onset of cardiovascular disease such as hypertension. Apelin expression is increased in the rostral ventrolateral medulla (RVLM) in spontaneously hypertensive rats (SHRs) compared to Wistar-Kyoto (WKY) normotensive rats, however, evidence that the apelinergic system chronically influences mean arterial blood pressure (MABP) under pathophysiological conditions remains to be established. In this study we investigated, in conscious unrestrained rats, whether APJ contributes to MABP and sympathetic vasomotor tone in the progression of two models of hypertension - SHR and L-NAME-treated rats - and whether APJ contributes to the development of hypertension in pre-hypertensive SHR. In SHR we showed that APJ gene (aplnr) expression was elevated in the RVLM, and there was a greater MABP increase following microinjection of [Pyr1]apelin-13 to the RVLM of SHR compared to WKY rats. Bilateral microinjection of a lentiviral APJ-specific-shRNA construct into the RVLM of WKY, SHR, and L-NAME-treated rats, chronically implanted with radiotelemeters to measure MABP, decreased aplnr expression in the RVLM and abolished acute [Pyr1]apelin-13-induced increases in MABP. However, chronic knockdown of aplnr in the RVLM did not affect MABP in either SHR or L-NAME-treated rats. Moreover, knockdown of aplnr in the RVLM of prehypertensive SHR did not protect against the development of hypertension. These results show that endogenous apelin, acting via APJ, is not involved in the genesis or maintenance of hypertension in either animal model used in this study.

Apelin, a recently discovered peptide with wide tissue distribution, regulates feeding behavior, improves glucose utilization, and inhibits insulin secretion. We examined whether apelin is expressed in human islets, as well as in normal and type 2 diabetic (T2D) animal islets. Further, we studied islet apelin regulation and the effect of apelin on insulin secretion. Apelin expression and regulation was examined in human and animal specimens using immunocytochemistry, in situ hybridization, and real-time PCR. Insulin secretion was studied in INS-1 (832/13) clonal beta cells. APJ-receptor expression was studied using real-time PCR. In human and murine islets apelin was predominantly expressed in beta cells and alpha cells; a subpopulation of the PP cells in human islets also harbored apelin. In porcine and feline islets apelin was mainly expressed in beta cells. APJ-receptor expression was detected in INS-1 (832/13) cells, and in human and mouse islets. A high dose (1microM) of apelin-36 caused a moderate increase in glucose-stimulated insulin secretion (30%; p<0.001), while lower concentrations (10-100nM) of apelin robustly reduced insulin secretion by 50% (p<0.001). Apelin was upregulated in beta cells of T2D db/db mice (47% vs. controls; p<0.02) and GK-rats (74% vs. controls; p<0.002), but human islet apelin expression was unaffected by glucose. On the other hand, human islet apelin expression was diminished after culture in glucocorticoids (16% vs. controls; p<0.01). We conclude that apelin is a novel insulin-regulating islet peptide in humans and several laboratory animals. Islet apelin expression is negatively regulated by glucocorticoids, and upregulated in T2D animals. The presence of apelin receptors in islets suggests a role for apelin as a paracrine or autocrine messenger within the islets.

Vasopressin and apelin are reciprocally regulated hormones which are implicated in the pathophysiology of heart failure and the regulation of metabolism; however, little is known about their interactions under pathological conditions. In this study, we determined how post-infarct heart failure (HF) and a high fat diet (HFD) affect expression of the apelin APJ receptor (APJR) and the V1a (V1aR) and V1b (V1bR) vasopressin receptors in the hypothalamus, the heart, and the retroperitoneal adipose tissue. We performed experiments in male 4-week-old Sprague Dawley rats. The animals received either a normal fat diet (NFD) or a HFD for 8 weeks, then they underwent left coronary artery ligation to induce HF or sham surgery (SO), followed by 4 weeks of NFD or HFD. The HF rats showed higher plasma concentration of NT-proBNP and copeptin. The HF reduced the APJR mRNA expression in the hypothalamus. The APJR and V1aR protein levels in the hypothalamus were regulated both by HF and HFD, while the V1bR protein level in the hypothalamus was mainly influenced by HF. APJR mRNA expression in the heart was significantly higher in rats on HFD, and HFD affected the reduction of the APJR protein level in the right ventricle. The regulation of APJR, V1aR and V1bR expression in the heart and the retroperitoneal adipose tissue were affected by both HF and HFD. Our study demonstrates that HF and HFD cause significant changes in the expression of APJR, V1aR and V1bR, which may have an important influence on the cardiovascular system and metabolism.

Apelin/APJ system plays an important role in the regulation of myocardial contractility (MC) and blood pressure. Opioid receptors (OPRs) are also important cardiovascular regulators and exert many of their effects through modulating the function of other systems. This study analyzed the interaction between APJ and kappa OPRs (KOR) in cardiac responsiveness to apelin in acute reno-vascular hypertension (2K1C).

(-)-Epicatechin (EC) is part of a large family of biomolecules called flavonoids and is widely distributed in the plant kingdom. Several studies have shown the beneficial effects of EC consumption. Many of these reported effects are exerted by activating the signaling pathways associated with the activation of two specific receptors: the G protein-coupled estrogen receptor (GPER), a transmembrane receptor, and the pregnane X receptor (PXR), which is a nuclear receptor. However, the effects of EC are so diverse that these two receptors cannot describe the complete phenomenon. The apelin receptor or APLNR is classified within the G protein-coupled receptor (GPCR) family, and is capable of activating the G protein canonical pathways and the β-arrestin transducer, which participates in the phenomenon of receptor desensitization and internalization. β-arrestin gained interest in selective pharmacology and mediators of the so-called "biased agonism". With molecular dynamics (MD) and in vitro assays, we demonstrate how EC can recruit the β-arrestin in the active conformation of the APLN receptor acting as a biased agonist.

Apelin participates in cardiovascular functions, metabolic disease, and homeostasis disorder. However, the biological function of apelin in liver diseases, especially liver fibrosis is still under investigation. The present study aimed to investigate the expression of apelin in nonalcoholic fatty liver disease (NAFLD) and the mechanism of apelin promoting hepatic fibrosis through ERK signaling in hepatic stellate LX-2 cells. The results showed that the ALT and AST levels in serum were increased in the mice fed HFC. The histological staining revealed that hepatocellular steatosis and ballooning degeneration was severe, and fibrogenesis appeared as increased pericellular collagen deposition along with pericentral (lobular) collagen deposition in the mice fed HFC. Immunochemistry and qRT-PCR results showed that the expression of apelin and profibrotic genes was higher as compared to the control group. The in vitro experiments demonstrated that apelin-13 upregulated the transcription and translation levels of collagen type I (collagen-I) and α-smooth muscle actin (α-SMA) in LX-2 cells. The immunofluorescent staining, qRT-PCR, and Western blot results showed that the overexpression of apelin markedly increased the expression of α-SMA and cyclinD1. The LX-2 cells treated with apelin-13 displayed an increased expression of pERK1/2 in a time-dependent manner, while the pretreatment with PD98059 abolished the apelin-induced expression of α-SMA and cyclinD1. Furthermore, the in vivo and in vitro assays suggested a key role of apelin in promoting liver fibrosis, and the underlying mechanism might be ascribed to the apelin expression of profibrotic genes via ERK signaling pathway.

Apelin receptor (APJ), a member of family A of the G protein-coupled receptors (GPCRs), is a potential pharmaceutical target for diseases of the nervous system. Our previous work revealed that human APJ can form a homodimer that has different functional characteristics than the monomer. To investigate the effects of APJ homodimers on neuroprotection in vascular dementia (VD), we established VD model in rats and treated the animals by injecting apelin-13 into the lateral ventricle. In addition, we established an oxygen-glucose deprivation/reoxygenation (OGD/R) model in SH-SY5Y cells treated with apelin-13. After apelin-13 stimulation in the VD rat, the level of APJ and APJ homodimer were elevated. Furthermore, APJ homodimer decreased the level of cleaved caspase-3 and cleaved caspase-9 via the Gαi3 and Gαq signaling pathway, thereby increasing the number of neurons and inhibiting apoptosis. Consequently, APJ homodimers may serve as a unique mechanism for neuroprotection against VD and provide new pharmaceutical targets for VD.

Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the β-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.

The hypothalamus is a key element of the neural circuits that control energy homeostasis. Specific neuronal populations within the hypothalamus are sensitive to a variety of homeostatic indicators such as circulating nutrient levels and hormones that signal circulating glucose and body fat content. Central injection of apelin secreted by adipose tissues regulates feeding and glucose homeostasis. However, the precise neuronal populations and cellular mechanisms involved in these physiological processes remain unclear. Here we examine the electrophysiological impact of apelin-13 on proopiomelanocortin (POMC) neuron activity. Approximately half of POMC neurons examined respond to apelin-13. Apelin-13 causes a dose-dependent depolarization. This effect is abolished by the apelin (APJ) receptor antagonist. POMC neurons from animals pre-treated with pertussis toxin still respond to apelin, whereas the Gβγ signaling inhibitor gallein blocks apelin-mediated depolarization. In addition, the effect of apelin is inhibited by the phospholipase C and protein kinase inhibitors. Furthermore, single-cell qPCR analysis shows that POMC neurons express the APJ receptor, PLC-β isoforms, and KCNQ subunits (2, 3 and 5) which contribute to M-type current. Apelin-13 inhibits M-current that is blocked by the KCNQ channel inhibitor. Therefore, our present data indicate that apelin activates APJ receptors, and the resultant dissociation of the Gαq heterotrimer triggers a Gβγ-dependent activation of PLC-β signaling that inhibits M-current.

Krüppel-like factor 4 (KLF4) is a key transcription factor that regulates genes involved in the proliferation or differentiation in different tissues. Apelin plays roles in cardiovascular functions, metabolic disease, and homeostatic disorder. However, the biological function of apelin in liver disease is still ongoing. In this study, we investigated the mechanism of KLF4-mediated protection against acute liver injury via the inhibition of the apelin signaling pathway. Mice were intraperitoneally injected with carbon tetrachloride (CCl4; 0.2 mL dissolved in 100 mL olive oil, 10 mL/kg) to establish an acute liver injury model. A KLF4 expression plasmid was injected through the tail vein 48 h before CCl4 treatment. In cultured LX-2 cells, pAd-KLF4 or siRNA KLF4 was overexpressed or knockdown, and the mRNA and protein levels of apelin were determined. The results showed that the apelin serum level in the CCl4-injected group was higher than that of control group, and the expression of apelin in the liver tissues was elevated while KLF4 expression was decreased in the CCl4-injected group compared to the KLF4-plasmid-injected group. HE staining revealed serious hepatocellular steatosis in the CCl4-injected mice, and KLF4 alleviated this steatosis in the mice injected with KLF4 plasmid. In vitro experiments showed that tumor necrosis factor-alpha (TNF-α) could downregulate the transcription and translation levels of apelin in LX-2 cells and also upregulate KLF4 mRNA and protein expression. RT-PCR and Western blotting showed that the overexpression of KLF4 markedly decreased basal apelin expression, but knockdown of KLF4 restored apelin expression in TNF-α-treated LX-2 cells. These in vivo and in vitro experiments suggest that KLF4 plays a key role in inhibiting hepatocellular steatosis in acute liver injury, and that its mechanism might be the inhibition of the apelin signaling pathway.