Apelin Gene Research Articles

Tumour co-expression of apelin and its receptor is the basis of an autocrine loop involved in the growth of colon adenocarcinomas

Using a cancer profiling array, our laboratory has shown that apelin gene is up-regulated in half of colon adenocarcinomas. We have therefore postulated that apelin signalling might play a prominent role in the growth of colon tumours. We first confirmed by immunohistochemistry that apelin peptide is overexpressed in human colon adenomas and adenocarcinomas. We also observed a significant overexpression of apelin receptor (APJ) in adjacent sections. We then demonstrated that several colorectal cancer cell lines also expressed apelin and its receptor, the highest gene and peptide expression being detected in LoVo cells. In this cell line, the expression and functionality of apelin receptor were revealed by apelin-induced adenylyl cyclase inhibition and Akt phosphorylation. In addition, apelin clearly protected LoVo cells from apoptosis by inactivating a caspase-dependent pathway and decreasing the degradation of poly ADP ribose polymerase protein (PARP). Finally, treatment of these tumour cells by the (F13A)apelin13 receptor antagonist significantly reduced their proliferation rate. Altogether, these data suggest the existence of an autocrine loop by which constitutive activation of apelin signalling should participate in the growth of colon adenocarcinomas. Accordingly, apelin signalling is a promising pharmacological target for the treatment of human colon adenomas and adenocarcinomas.

Abstract 96: Sirt-3 is Essential for Apelin Gene Therapy Mediated Improvement of Angiogenesis and Cardiac Function in Stz Mice

Diabetic cardiomyopathy is a major diabetic complication featured by the impaired angiogenesis and cardiac dysfunction. Apelin (APLN) has been shown to prompt angiogenesis. Our recent study implicates a critical role of sirtuin 3 (SIRT-3) in the regulation of angiopoietins expression and angiogenesis. In the present study, we investigated the roles of SIRT-3 in APLN-induced angiogenesis in a diabetic cardiomyopathy model underwent ischemia. Male wild type (WT) mice and SIRT-3 knock-out (SIRT-3 KO) mice were induced into diabetic cardiomyopathy model by STZ injection and followed by left anterior descending artery ligation to induce myocardial infarction (MI). The experimental mice then underwent intra-myocardium injection with adenovirus Apelin (Ad -APLN) or β-gal adenovirus (Ad-β-gal) immediately after the ligation. Myocardial angiogenesis and cardiac function was measured 2 weeks after surgery. Our data revealed that the ejection fraction (EF) and fraction shorting (FS) were significantly improved in STZ mice received Ad-APLN treatment than in STZ mice received Ad-β-gal (EF: 64.1% ±3.2 vs 52.8%±5.6, P<0.001; FS: 34.3±2.2% vs, 26.6±3.5%, P<0.001). Ad-APLN treatment in STZ-SIRT3KO mice had little effects on EF and FS (EF: 47.9±5.2% vs, 49.7±8.2%, P=0.634; FS: 24.7±5.1% vs, 23.5±3.1%, P=0.728). The expressions of angiopoietins-1 and angiopoietins-2 were significantly increased in Ad-APLN treated STZ mice compared to Ad-β-gal treated STZ mice. These were accompanied by significant increases in myocardial arterioles and capillary densities. Ad-APLN mediated angiopoietins expressions and neovascularization were completely abolished in STZ-SIRT3 KO mice. In vitro, Ad-APLN significantly enhanced the proliferation and tube formation of endothelial cells derived from WT mice, but failed to do that in cells from SIRT 3 KO mice. Our data suggest that SIRT-3 is essential for the beneficial effect of Apelin on angiogenesis and heart function in diabetic cardiomyopathy.

Effect of endurance training on skeletal muscle myokine expression in obese men: identification of apelin as a novel myokine

It has been suggested that the metabolic benefits of physical exercise could be mediated by myokines. We examined here the effect of exercise training on skeletal muscle expression of a panel of myokines in humans. Pathways regulating myokine expression were investigated in human myotubes. Eleven obese non-diabetic male subjects were enrolled in an 8-week endurance training program. Insulin sensitivity was assessed by an oral glucose tolerance test. Subcutaneous adipose tissue and Vastus lateralis muscle biopsy samples were collected before and after training. RNAs were prepared from adipose tissue and skeletal muscle. Primary culture of myoblasts was established. As expected, exercise training improved aerobic capacity and decreased fat mass. No significant change in interleukin 6, fibroblast growth factor 21, myostatin (MSTN) or irisin mRNA level was found in muscle after training. A twofold increase in apelin mRNA level was found in muscle but not in adipose tissue. No change in circulating myokine and adipokine plasma levels was observed in the resting state in response to training. Interestingly, apelin was significantly expressed and secreted in primary human myotubes. Apelin gene expression was upregulated by cyclic AMP and calcium, unlike the other myokines investigated. Importantly, changes in muscle apelin mRNA levels were positively related to whole-body insulin sensitivity improvement. Collectively, our data show that exercise training upregulates muscle apelin expression in obese subjects. Apelin expression is induced by exercise signaling pathways and secreted in vitro in human primary myotubes, and may behave as a novel exercise-regulated myokine with autocrine/paracrine action.

Hemodynamic shear stress regulates the apelin/APJ system in human endothelial cells

Introduction: The apelin/APJ system is being considered to play a crucial role in cardiovascular function. Namely, the isoforms apelin-12 and apelin-13 seem to be most active in the cardio vasculature. Application of fluid shear stress to endothelial cells elicits the formation of NO via PI3K/AKT signaling and phosphorylation of the endothelial NO synthase (eNOS). We suppose that the apelin/APJ system is involved in the transduction of mechanical forces into intracellular signaling. Methods: We applied hemodynamic shear stress (1.5 dyne/cm2 for venous, 20 dyne/cm2 for arterial conditions) on HUVEC, HCAEC, and HCASMC using a parallel plate flow chamber. Cells were stimulated with apelin-12 and apelin-13. Static controls were kept under similar conditions. After shear stress exposure cells were harvested and prepared for real-time qPCR and Western Blot analysis or fixed and stained for anti-APJ (IF). The supernatant was collected for nitrite measurements. Additionally, HUVEC were transfected with APJ siRNA. Results: After application of shear stress, apelin gene and protein expression were regulated in ECs only. In HUVEC, expression of apelin was increased by 40% under low shear stress, whereas under high shear stress conditions apelin expression decreased by 50% (p<0.01). Interestingly, HCAEC showed an opposite behaviour (p<0.001). Furthermore, in ECs APJ gene and protein expression increased after exposure to shear stress (p<0.05), which could be confirmed by IF. Incubation of HUVEC with apelin-12 and -13 revealed a dose-dependent increase of NO production only after incubation with apelin-12 (P<0.01). Western Blot Analysis underlined apelin isoform dependent cellular signaling. Incubation with apelin-12 for up to 15 min lead to a significant increase in PI3 kinase and Akt phosphorylation under static and dynamic conditions (p=0.02), whereas incubation with apelin-13 had no significant influence on PI3k/Akt signalling and lead to phosphorylation of Erk 1/2 instead. In order to demonstrate shear stress dependency of the apelin/APJ system, we transfected HUVEC with APJ siRNA and exposed them to our standard flow conditions. Transfection with APJ siRNA lead to a loss of shear stress dependent eNOS up-regulation and adhesion. Conclusion: Our results show for the first time a shear stress regulated adjustment of the Apelin/APJ system in human endothelial cells depending on arterial or venous flow conditions. This is confirmed by an enhanced eNOS expression and NO production. Interestingly, apelin-12 and apelin-13 activate different signaling pathways in HUVEC and give rise for various biological responses.

GW24-e0002 Interactive association of five candidate polymorphisms in apelin/APJ pathway with coronary artery disease among Chinese hypertensive patients

ObjectivesThe apelin/angiotensin receptor-like 1 (apelin/APJ) pathway has emerged as an essential novel mediator of cardiovascular disease. Via sequencing the genes of apelin/APJ pathway, we have recently identified and validated four...

Regulation of oxidative stress and somatostatin, cholecystokinin, apelin gene expressions by ghrelin in stomach of newborn diabetic rats

The aim of the study was to determine whether ghrelin treatment has a protective effect on gene expression and biochemical changes in the stomach of newborn streptozotocin (STZ) induced diabetic rats. In this study, four groups of Wistar rats were used: control, ghrelin control, diabetic and diabetic+ghrelin. The rats were sacrificed after four weeks of treatment for diabetes. The gene expressions of: somatostatin, cholecystokinin, apelin and the altered active caspase-3, active caspase-8, proliferating cell nuclear antigen, were investigated in the pyloric region of the stomach and antioxidant parameters were measured in all the stomach. Although ghrelin treatment to diabetic rats lowered the stomach lipid peroxidation levels, the stomach glutathione levels were increased. Exogenous ghrelin caused an increased activities of stomach catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase in diabetic rats. Numbers of somatostatin, cholecystokinin and proliferating cell nuclear antigen immunoreactive cells decreased in the diabetic+ghrelin group compared to the diabetic group. Apelin mRNA expressions were remarkably less in the diabetic+ghrelin rats than in diabetic rats. The results may indicate that ghrelin treatment has a protective effect to some extent on the diabetic rats. This protection is possibly accomplished through the antioxidant activity of ghrelin observed in type 2 diabetes. Consequently exogenous ghrelin may be a candidate for therapeutic treatment of diabetes.

Interactive Association of Five Candidate Polymorphisms in Apelin/APJ Pathway with Coronary Artery Disease among Chinese Hypertensive Patients

BackgroundVia sequencing the genes of apelin/angiotensin receptor-like 1 (apelin/APJ) pathway, we have recently identified and validated four common polymorphisms (rs3761581, rs56204867, rs7119375, and rs10501367) implicated in the development of hypertension. Extending these findings, we, in Chinese hypertensive patients, sought to investigate the association of these four polymorphisms and one additional promising candidate (rs9943582) from this pathway with the risk of developing coronary artery disease (CAD).Methodology/Principal FindingsGenotypes were obtained from 994 sporadic CAD patients and 708 age- and sex-matched controls. All participants were hypertensives and angiographically-confirmed. Data were analyzed by Haplo.Stats and multifactor dimensionality reduction (MDR) softwares. Genotype distributions of five examined polymorphisms satisfied Hardy-Weinberg equilibrium in controls of both genders. Single-locus analyses exhibited no significant differences in the genotype/allele frequencies of examined polymorphisms between CAD patients and controls (P>0.05), even after controlling traditional cardiovascular confounders. In haplotype analyses, low-penetrance haplotype G-A (in order of rs56204867 and rs3761581 from apelin gene) was significantly overrepresented in controls (1.73%) relative to in CAD patients (0.4%) in males (P = 0.047). Further interaction analyses suggested an overall best MDR model including rs3761581 in males (P = 0.0408) and including rs7119375 and rs9943582 in females (P<0.0001), which were further substantiated in the classical logistical regression model.ConclusionsOur findings demonstrated a contributive role of low-penetrance haplotype in apelin gene on CAD in males, and more importantly, interactive effects of genetic defects in apelin/APJ pathway might confer a potential risk in Chinese hypertensive patients.

Associations of Common Variants atAPLNand Hypertension in Chinese Subjects with and without Diabetes

Background. Apelin, the endogenous ligand for the APJ receptor, has a potent hypotensive effect via a nitric oxide-dependent mechanism in vivo. The aim of the study was to investigate the association between the common variants of apelin gene (APLN) and hypertension, which was reported recently in a Chinese Han population with and without diabetes. Methods. Three single nucleotide polymorphisms (SNPs) on APLN were genotyped in 3156 diabetic patients and 3736 nondiabetic individuals. For non-diabetic subjects, 1779 were enrolled in stage 1 and 1757 were recruited for validation. A meta-analysis combining the two stages was carried out to obtain the overall effect. Results. In diabetic patients, no significant associations of the three SNPs with hypertension were observed. In contrast, we found that rs2235306 was associated with hypertension in non-diabetic males after adjusting for covariates (OR = 1.19, P = 0.039) while rs2235307 and rs3115759 displayed no evidence of association in both genders. One haplotype, C-C-A, also showed an association with hypertension (OR = 1.47, P = 0.032) only in men. However, analysis in stage 2 and meta-analysis did not support these findings. Conclusions. We conclude that common variants on APLN are not associated with the prevalence of hypertension in the Chinese.

Apelin gene polymorphism influences apelin expression and obesity phenotypes in Chinese women

Apelin, which is a newly identified adipokine, is related to obesity and insulin resistance. A positive correlation between plasma apelin concentrations and obesity traits was reported. We tested associations between apelin gene (APLN) polymorphisms, BMI, and waist circumference (WC) and compared APLN expression levels in cells of different genotypes. Four tagging single nucleotide polymorphisms (SNPs) and one promoter SNP were genotyped in 1627 Chinese subjects. Because APLN was located on the chromosome X, statistical analyses were conducted in a sex-specific manner. Adipocytes of different genotypes were derived from the omental fat tissue of 10 women. We treated the primary adipocytes with high glucose plus insulin because of a close relation between insulin resistance and obesity. SNP rs3115757 was significantly associated with BMI and WC in women. Compared with the CG or GG genotype, the CC genotype had an OR of 2.07 (95% CI: 1.23, 3.49) for having a high WC (P = 0.006) and an OR of 2.29 (95% CI: 1.25, 4.19) for having a BMI (in kg/m(2)) ≥27 (P = 0.007). None of the SNPs was associated with BMI or WC in men. In adipocytes that carried the CC genotype of rs3115757, APLN messenger RNA levels and protein concentrations were higher in cells treated with high glucose plus insulin than in those with normal glucose. There was no difference between the 2 conditions in adipocytes of the CG or GG genotype. Both association and functional studies suggested that APLN polymorphisms were associated with risks of obesity phenotypes.

Vitamin C modulates the interaction between adipocytes and macrophages

Increased adiposity is related with monocyte infiltration into the adipose tissue that accentuates inflammation. Antioxidant treatments emerge as approaches to counteract this phenomenon. Cocultures of differentiated 3T3-L1 adipocytes and RAW264.7 macrophages were incubated for 24-72 h with/without 100 nM insulin and/or 200 μM vitamin C (VC). Nitric oxide (NO) secretion (24 h) was measured. Also, expression (24 h) and secretion (72 h) of MCP-1, leptin and apelin were analyzed. NO secretion was significantly inhibited by insulin and VC only in cocultures. MCP-1 expression/secretion was enhanced in cocultures. Insulin incubation reduced MCP-1 expression in both cultures and VC only in controls. Both treatments inhibited MCP-1 secretion in cocultures. Apelin gene expression was induced in cocultures. Insulin induced apelin mRNA expression, but VC inhibited its expression in cocultures under insulin treatment. Apelin secretion was notably induced by insulin and inhibited by VC in cocultures. Leptin expression was decreased in coculture, while presented no effects by VC. VC importantly modulates the established pro-inflammatory state in the interaction between adipocytes and macrophages.

Effects of lipoic acid on apelin in 3T3-L1 adipocytes and in high-fat fed rats

Lipoic acid (LA) is an antioxidant with therapeutic properties on several diseases like diabetes and obesity. Apelin is a novel adipokine with potential beneficial actions on glucose metabolism and insulin resistance. The aim of this study was to examine in 3T3-L1 adipocytes the effects of LA on apelin gene expression and secretion, as well as elucidate the signaling pathways involved. We also tested the regulation of adipose apelin gene expression by LA supplementation in a model of high-fat diet-induced obesity. LA increased apelin secretion but not apelin gene expression in 3T3-L1 adipocytes. The AMPK inhibitor Compound C induced an increase in LA-stimulated apelin production, and, on the contrary, the AMPK activator AICAR completely reversed the LA stimulatory effects on apelin secretion, also inducing a significant reduction in apelin mRNA levels in this in vitro model. Apelin mRNA levels were increased in those animals fed with the high-fat diet, while the caloric restriction decreased apelin mRNA to control levels. However, apelin gene expression was not significantly modified in rats treated with LA compared with the obese group. The current data suggest the ability of LA to modulate apelin secretion by adipocytes. However the insulin-sensitizing effect of LA in vivo is not related to changes in apelin gene expression in our model of diet-induced obesity.

Validation of genetic association in apelin–AGTRL1 system with hypertension in a larger Han Chinese population

We have recently resequenced apelin and AGTRL1 genes to identify candidate polymorphisms in family-based association with hypertension and related phenotypes. In this study, we aimed to determine and replicate these polymorphisms via a larger, independent case-control study in Shanghai Han Chinese. Two polymorphisms [rs3761581 (A/C) and T-1860C] in apelin gene and two [rs7119375 (G/A), rs10501367 (G/A)] in AGTRL1 gene were genotyped using the TaqMan assay among 969 patients diagnosed with essential hypertension and 980 age and sex-matched controls. Data were analyzed using Haplo.stats program. In single-locus analysis, significant differences were observed in allele distribution of rs3761581 (P = 0.0156) in men and in rs7119375 (P = 0.0488) genotype distribution in women between patients and controls. Haplotype analysis indicated that haplotypes C-C-G-G (in order of T-1860C, rs3761581, rs7119375 and rs10501367) [adjusted odds ratio (ORadjusted) = 1.67, P = 0.0061] and T-A-A-A (ORadjusted = 1.62, P = 0.0008) conferred an increased risk for hypertension after adjustment for age, onset age, body mass index (BMI) and waist-to-hip ratio, whereas haplotype C-C-A-A (ORadjusted = 0.33, P = 0.0048) conferred a protective effect. In women, increased risk for hypertension was seen for haplotypes T-A-G-G (ORadjusted = 1.30, P = 0.0051), C-C-G-G (ORadjusted = 2.86, P < 0.0001), T-A-A-A (ORadjusted = 1.66, P = 0.0003) and C-C-A-A (ORadjusted = 2.65, P < 0.0001), whereas decreased risk was seen for haplotypes C-A-G-G (ORadjusted = 0.48, P < 0.0001) and T-C-G-G (ORadjusted = 0.40, P < 0.0001). Further haplotype-phenotype analyses indicated the robustness of these associations. For example, haplotype T-A-A-A was significantly associated with obesity index (BMI and waist-to-hip ratio) in both sexes and blood pressures in men (P-sim < 0.05). In this exploratory pilot case-control study, we found robust haplotype-based and haplotype-phenotype associations of four well characterized polymorphisms in apelin-AGTRL1 system with hypertension and related phenotypes.

Maternal Apelin Physiology during Rat Pregnancy: The Role of the Placenta

Objective Apelin is a multifunctional peptide which is catabolized by the angiotensin-converting enzyme-related carboxypeptidase-2 (ACE2). The peptide is well known for its hemodynamic effects and its role in energy and fluid homeostasis. Pregnancy is a state of dramatically altered maternal hemodynamics and metabolism, but the role of apelin is unknown. To gain further insight in apelin physiology, we investigated relative tissue expression, plasma clearance and metabolic pathways of apelin in pregnant rats. Methods We measured maternal plasma apelin levels throughout normal rat gestation and examined relative apelin gene expression in several tissues, including the placenta. We documented apelin clearance using radiolabeled apelin and assessed maternal plasma levels in rats that underwent surgical reduction of the fetoplacental mass, thereby further examining the role of the placenta in apelin clearance. Finally, we localized apelin and ACE2 in the placenta and mesometrial triangle using immunohistochemistry. Results Maternal apelin plasma concentrations dropped by 50% between mid- and late gestation. Apelin expression was comparable between non-pregnant and late-pregnant rats in non-reproductive tissues. The placenta showed low apelin gene expression compared to brain tissue. Apelin clearance was enhanced in term gestation as evidenced by a steeper decline of the slow phase of the elimination curve of radiolabeled apelin. Compared to sham-operated dams, maternal plasma apelin was raised by 23% in late-pregnant rats in which half of the fetoplacental units were removed at day 16 of gestation. ACE2 mRNA expression was detectable in late- but not mid-pregnancy placental tissue; immunohistochemically, ACE2 was primarily localized in the smooth muscle layer of fetal arterioles in the labyrinth. Conclusion Maternal circulating apelin drops considerably between mid- and late- pregnancy owing to faster clearance. The current data suggest a role for placental ACE2 in the accelerated apelin metabolism.

Eicosapentaenoic acid up‐regulates apelin secretion and gene expression in 3T3‐L1 adipocytes

Recent studies have shown the ability of apelin to restore glucose tolerance in obese and insulin-resistant mice. Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid (PUFA) from the omega-3 family that has many beneficial effects in obesity-linked disorders. The aim of this study was to examine in vitro the effects of EPA on apelin secretion and gene expression in mature 3T3-L1 adipocytes. Treatment with EPA (100 and 200 microM) significantly increased basal (p<0.01) and insulin-stimulated (p<0.001) apelin secretion and gene expression in adipocytes. EPA also stimulated Akt phosphorylation, a down-stream target of phosphatidylinositol 3-kinase (PI3K), in 3T3-L1 adipocytes. Moreover, treatment with the PI3K inhibitor LY294002 completely blocked EPA-stimulatory action on apelin mRNA gene expression (p<0.001), but not modified the stimulatory effect of EPA on basal apelin secretion. Furthermore, the stimulatory effect of EPA on basal apelin release was also observed in the presence of Actinomycin D and Cycloheximide, suggesting that EPA might also regulate apelin secretion by via post-transcriptional mechanisms. These findings suggest that the mechanisms mediating EPA-induced apelin synthesis and/or secretion are complex, involving steps that are PI3K dependent and steps that are PI3K independent.

Abstract 5551: Apelin is Necessary for the Maintenance of Insulin Sensitivity

BACKGROUND: The recently discovered cardioactive peptide apelin has been shown to be involved in the maintenance of insulin sensitivity. However, questions persist regarding its precise role and molecular mechanism of action. METHODS: Insulin (0.75 U/kg) tolerance testing (ITT) was performed on mice with generalized deficiency of the apelin gene (APKO). Additionally, fasting glucose, insulin, free fatty acid (FFA), and adiponectin levels were determined. To assess the impact of exogenously delivered apelin on insulin sensitivity, osmotic pumps containing pyroglutamated apelin-13 or saline were implanted in APKO mice for 4 weeks. Following the infusion, ITTs were repeated and the animals sacrificed. In selected animals, insulin (0.75 U/kg) was delivered 30 minutes prior to sacrifice; soleus muscles were then harvested, homogenized in lysis buffer, and insulin-induced Akt phosphorylation determined by Western blotting. Apelin-13 infusion, ITTs, and serum insulin, glucose, FFA, and adiponectin measurements were also performed in obese diabetic db/db mice. To probe the underlying mechanism for apelin’s effects, apelin-13 (1 μ M) was also delivered to cultured C2C12 myotubes. 2-[ 3 H]-deoxyglucose uptake and Akt phosphorylation were assessed. RESULTS: APKO mice had significantly decreased insulin sensitivity by ITT, increased insulin (0.70±0.033 vs. 0.55±0.098 pg/mL; p &lt; 0.05) and FFA (231±11 vs. 172±14 μ mol/L; p &lt; 0.01) levels, and decreased adiponectin levels (64±3.3 vs. 80±8.0 μ g/mL; p &lt; 0.05). Soleus lysates had decreased insulin-induced Akt phosphorylation. Administration of apelin to both APKO and db/db mice resulted in improvements in insulin sensitivity. No changes in body weight were observed in either animal model with or without apelin. In C2C12 myotubes, apelin increased glucose uptake (10.6±0.70 vs. 6.01±0.18 pmol/mg protein/min; p &lt; 0.01) and Akt phosphorylation. These events were fully abrogated by inhibitors of G i (pertussis toxin) and AMPK (Compound C), but only partially by phosphoinositide 3-kinase inhibition (LY294002). CONCLUSIONS: Apelin is necessary for the maintenance of insulin sensitivity in vivo . Apelin’s effects on glucose uptake and Akt phosphorylation may be mediated by a G i and AMPK-dependent pathway.

Family-based analysis of apelin and AGTRL1 gene polymorphisms with hypertension in Han Chinese

Apelin and AGTRL1 (angiotensin receptor-like 1), elements of a newly identified pathway with a role in counter regulating the renin-angiotensin system, have been implicated in blood pressure regulation. This study aims to assess whether the apelin and AGTRL1 genetic polymorphisms might contribute to essential hypertension or its related phenotypes. We recruited 1015 Han Chinese from 248 families with essential hypertension. Each individual was genotyped for 6 single nucleotide polymorphisms (SNPs) in apelin and 6 SNPs in AGTRL1. Data were analyzed using the family-based association test (FBAT) and the haplotype-based association test (HBAT). FBAT analysis showed that two SNPs rs3761581 and T-1860C within apelin conferred significant association with hypertension and its related phenotypes even after correcting for age and gender. Three SNPs (rs7119375, rs10501367 and rs9943582) within AGTRL1 were found to be associated with hypertension, BMI and the onset age of hypertension, whereas after correction, only marginal associations were noted. Of the common haplotypes (with frequencies over 3%), haplotypes (A-T) and (C-C) comprising rs3761581 and T-1860C in apelin and haplotype (G-G) comprising rs10501367 and rs7119375 in AGTRL1 were shown to be significantly associated with hypertension, BMI and the onset age of hypertension, even after a permutation correction. Our study suggests that genetic variation within apelin and AGTRL1 likely contributes to essential hypertension, BMI and the onset age of hypertension. Future well designed epidemiological or functional studies would be warranted to validate this hypothesis.

Sequencing and cardiac expression of Apelin in Sus Scrofa

In humans, the Apelin gene is located on chromosome Xq25–26.1 and it encodes a 77-aminoacid prepropeptide. Considerable sequence homology exists across different mammalian species. Apelin is emerging as an important regulator of cardiovascular homeostasis but, at present, few data from humans are available and further studies are necessary to better define its role in cardiovascular pathophysiology. The role and function of Apelin in cardiovascular system could be reliably investigated in experimental models devoid of confounding effects reflecting only the natural history of the disease. The pig constitutes a model largely used in experimental pathology where it has a central role in “in vivo” clinical settings. Sus Scrofa genoma is not completely sequenced and Apelin gene is still lacking. Aim of this study was to sequence the Apelin in Sus Scrofa for future applications to molecular biology studies. Using the guanidinium thyocyanate–phenol–chloroform method, we extracted total RNA from samples obtained from heart of mouse and from atrium and ventricle of normal pigs. Pig Apelin mRNA was sequenced using polymerase chain reaction primers designed from mouse consensus sequences. A partial sequence of Sus Scrofa Apelin mRNA, 1–201 pb, was submitted to GenBank (accession number FJ362603). The bands obtained from pig cardiac tissue shared a 99% sequence identity with Mus musculus and 90% with Rattus norvegicus. The knowledge of Apelin sequence can be an useful starting point for future studies devoted to better understand the possible alterations of Apelin mRNA expression in different cardiac diseases.

Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary: Involvement of apoptosis and hormonal regulation

In the mammalian ovary, the microvasculature in the thecal layer of follicles is associated with follicular development. Apelin and its receptor, APJ, are expressed in the tissues and organs which include the vasculature. The aims of the present study were to examine the mRNA expression of apelin and the APJ receptor in granulosa cells and theca tissue of bovine follicles and the effects of steroid hormone and gonadotrophins on the expression of these genes in cultured granulosa cells and theca cells. The expression of apelin mRNA was not found in the granulosa cells of bovine follicles. The expression of the APJ gene was increased in granulosa cells of estrogen-inactive dominant follicles. The expression of apelin mRNA increased in theca tissues of estrogen-inactive dominant follicles. APJ expression in theca tissues increased with follicle growth. Progesterone stimulated the expression of APJ mRNA in the cultured granulosa cells. FSH stimulated the expression of APJ mRNA in the cultured granulosa cells. LH induced the expression of apelin and APJ receptor mRNAs in cultured theca cells. Taken together, our data indicate that the APJ receptor in granulosa cells and both apelin and the APJ receptor in theca tissues are expressed in bovine ovary, that APJ in granulosa cells may be involved in the appearance of the cell apoptosis, and that LH stimulates the expression of apelin and APJ genes in theca cells.

Signalisation apeline et physiopathologie vasculaire

The formation of the vascular system is an early step in organogenesis that involves the participation of various signalling pathways. Integration of the extracellular signals decoded by their cognate membrane receptors orchestrate the cell events, which act at different stages, from the primitive network formed by vasculogenesis to the arborescent network remodeled by angiogenesis. Our laboratory showed the participation of a new signalling pathway in physiological angiogenesis and tumour neovascularisation. This signalling pathway named apelin comprises a G protein-coupled receptor and a peptide ligand. Expression of apelin receptors is observed during the embryonic formation of blood vessels where it is localized in the endothelium. In HUVECs, which endogenously express apelin receptors, apelin promotes the phosphorylation of ERKs, Akt and p70 S6 Kinase. In addition, apelin increases in vitro the proliferation of these endothelial cells. Finally, injection of apelin in the vitreous induces in vivo the sprouting and the proliferation of endothelial cells from the retinal vascular network. Accordingly, all these results led us to study the role of apelin signalling in tumour neovascularisation. In two tumoral cell lines, we showed that hypoxia induces the expression of apelin gene. In addition, the overexpression of apelin gene resulting from stable transfection of these cell lines clearly accelerates in vivo tumour growth, as a consequence of an increased number of vessels irrigating these tumours. The pathological relevance of these data has been validated by the characterization of an overexpression of apelin gene in one third of human tumours. Taken together, apelin signalling is both involved in physiological angiogenesis and pathological neoangiogenesis, and therefore represents an interesting pharmacological target for anti-angiogenic therapies.

Effects of eicosapentaenoic acid ethyl ester on visfatin and apelin in lean and overweight (cafeteria diet-fed) rats

Previous studies have demonstrated that the n-3 fatty acid EPA improves insulin resistance induced by high-fat diets. The aim of the present study was to investigate the potential role of visfatin and apelin in the insulin-sensitising effects of EPA ethyl ester. The effects of EPA on muscle and adipose GLUT mRNA, as well as on liver glucokinase (GK) and glucose-6-phosphatase (G6Pase) activity, were investigated. Male Wistar rats fed on a standard diet or a high-fat cafeteria diet were daily treated by oral administration with EPA ethyl ester (1 g/kg) for 5 weeks. A significant decrease (P < 0.01) in white adipose tissue (WAT) visfatin mRNA levels was found in the cafeteria-fed rats, which was reversed by EPA administration (P < 0.05). Moreover, a negative relationship was observed between homeostatic model assessment (HOMA) and the visfatin:total WAT ratio. In contrast, cafeteria-diet feeding caused a significant increase (P < 0.01) in apelin mRNA in visceral WAT. EPA increased (P < 0.01) apelin gene expression, and a negative relationship between HOMA index with visceral apelin mRNA and serum apelin:total WAT ratio was also observed. EPA treatment did not induce changes in skeletal muscle GLUT1, GLUT4 or insulin receptor mRNA levels. Neither liver GK and G6Pase activity nor the GK:G6Pase ratio was modified by EPA. These data suggest that somehow the insulin-sensitising effects of EPA could be related to its stimulatory action on both visfatin and apelin gene expression in visceral fat, while changes in skeletal muscle GLUT, as well as in hepatic glucose production, are not likely to be the main contributing factors in the improvement in insulin resistance induced by EPA.