Different modulation of dipeptidyl peptidase-4 activity between microvascular and macrovascular human endothelial cells.

Dipeptidyl peptidase 4 (DPP-4) is an enzyme that is produced by endothelial cells in different districts and circulates in plasma. Patients with type 2 diabetes show a reduction in active Glucagon-Like Peptide-1 (GLP-1) that could be due to impairment of secretion or its degradation or both. GLP-1 is rapidly inactivated in vivo, mainly by the DPP-4. Some authors suggest that Metformin has no direct inhibitory effect on DPP-4 activity and that Metformin and the other biguanides enhance GLP-1 secretion; others suggest a possible role of Metformin in the inhibition of the DPP-4 activity. In order to better elucidate the role of insulin sensitizers on the modulation of GLP-1 circulating levels, DPP-4 activity and mRNA expression were measured in cultured human aortic endothelial cells (HAEC) and human microvascular dermal endothelial cells (HMVEC) exposed to high glucose, Metformin and Rosiglitazone. Present data show that hyperglycemia is capable of increasing in a significant manner the DPP-4 activity only in microvascular endothelial cells. Rosiglitazone is able to modulate in a negative manner the expression of DPP-4 but not its activity in macrovascular endothelial cells, while at 24 h of exposure it is able to increase significantly DPP-4 activity but not its expression in microvascular endothelial cells. Metformin at 48 h only in microvascular endothelial cells is able to reduce in a significant manner (p = 0.01) the activity of DPP-4 but not its expression. The modulation of DPP-4 is site specific.

Adipokines as possible new predictors of cardiovascular diseases: a case control study.

Background and Aims. The secretion of several adipocytokines, such as adiponectin, retinol-binding protein 4 (RBP4), adipocyte fatty acid binding protein (aFABP), and visfatin, is altered in subjects with abdominal adiposity; these endocrine alterations could contribute to increased cardiovascular risk. The aim of the study was to assess the relationship among adiponectin, RBP4, aFABP, and visfatin, and incident cardiovascular disease. Methods and Results. A case-control study, nested within a prospective cohort, on 2945 subjects enrolled for a diabetes screening program was performed. We studied 18 patients with incident fatal or nonfatal IHD (Ischemic Heart Disease) or CVD (Cerebrovascular Disease), compared with 18 matched control subjects. Circulating adiponectin levels were significantly lower in cases of IHD with respect to controls. Circulating RBP4 levels were significantly increased in CVD and decreased in IHD with respect to controls. Circulating aFABP4 levels were significantly increased in CVD, while no difference was associated with IHD. Circulating visfatin levels were significantly lower in cases of both CVD and IHD with respect to controls, while no difference was associated with CVD. Conclusions. The present study confirms that low adiponectin is associated with increased incidents of IHD, but not CVD, and suggests, for the first time, a major effect of visfatin, aFABP, and RBP4 in the development of cardiovascular disease.

Sex steroids and leptin regulate the "first Kiss" (KiSS 1/G-protein-coupled receptor 54 system) in human gonadotropin-releasing-hormone-secreting neuroblasts.

INTRODUCTION: The G-protein-coupled receptor 54 (GPR54) and its ligand kisspeptin, encoded by the KiSS-1 gene, have been involved in the molecular mechanisms underlying the reawakening of gonadotropin-releasing hormone (GnRH) neurons at puberty. GPR54 mutations cause hypogonadotropic hypogonadism in human and mice. Aim. Our aim was to study regulation of the KiSS-1/GPR54 system using a previously characterized primary culture of human fetal GnRH-secreting neuroblasts, FNC-B4. METHODS: KiSS-1/GPR54 gene and protein expressions in FNC-B4 were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunocytochemistry, and Western blot. Expression of kisspeptin and GPR54 in fetal olfactory mucosa (OM), from which FNC-B4 cells were derived, was analyzed with confocal microscopy. MAIN OUTCOME MEASURES: Regulation of KiSS-1/GPR54 expression in FNC-B4 was evaluated in response to sexual steroids and leptin. Effect of kisspeptin on GnRH secretion and migration in FNC-B4 was also investigated. RESULTS: Kisspeptin and GPR54 were immunolocalized and co-expressed with GnRH in OM and FNC-B4 cells. Kisspeptin (1 microM, 24 hours) induced GnRH secretion, but not gene expression, and inhibited migration (IC(50) = 6.28 +/- 3.71 nM) in FNC-B4. The 24-hour exposure to increasing concentrations of 17-beta-estradiol (0.01-1 nM) significantly and dose-dependently decreased, whereas androgens (dihydrotestosterone [DHT], 0.01-1 nM) significantly stimulated KiSS-1/GPR54 mRNA. Testosterone (1 nM) showed a stimulatory effect only after blocking its aromatization with letrozole. In addition, leptin (1 nM, 24 hours), an adipocyte-derived hormone acting on the reproductive axis, significantly increased KiSS-1/GPR54 expression in FNC-B4. Immunocytochemistry and Western blot analysis confirmed the regulatory effects found with qRT-PCR. Interestingly, leptin (1 nM, 24 hours) also significantly increased both leptin receptor (LEPR) and androgen receptor (AR) mRNA. DHT (0.01-1 nM) also up-regulated LEPR and AR genes, suggesting a synergistic action between leptin and androgens aimed to up-regulate the KiSS-1/GPR54 system, which, in contrast, was inhibited by estrogens. CONCLUSION: Our results indicate that an interplay between metabolic and sexual hormones may trigger the KiSS-1/GPR54 signaling to GnRH neurons suggesting new mechanisms which regulate puberty onset.

Role of endothelin-1 in the migration of human olfactory gonadotropin-releasing hormone-secreting neuroblasts.

FNC-B4 neuroblasts that express both neuronal and olfactory markers have been established and cloned. These cells express GnRH and both the endothelin-1 (ET-1) gene and protein and respond in a migratory manner to GnRH in a dose-dependent manner. Previous research has shown that FNC-B4 cells produce and respond to ET-1 by regulating the secretion of GnRH through endothelin type A receptors and by stimulating their proliferation through endothelin type B (ETB) receptors. In this study, we found that FNC-B4 cells are able to migrate in response to ET-1 through the involvement of ETB receptors. Combined immunohistochemical and biochemical analyses showed that ET-1 triggered actin cytoskeletal remodeling and a dose-dependent increase in migration (up to 6-fold). Whereas the ETB receptor antagonist (B-BQ788) blunted the ET-1-induced effects, the ETA receptor antagonist (A-BQ123) did not. Moreover, we observed that FNC-B4 cells were independently and selectively stimulated by ET-1 and GnRH. We suggest that ET-1, through ETB receptor activation, may be required to maintain an adequate proliferative stem cell pool in the developing olfactory epithelium and the subsequent commitment to GnRH neuronal migratory pattern. The coordinate interaction between ET receptors and GnRH receptor participates in the fully expressed GnRH-secreting neuron phenotype.

Expression and function of gonadotropin-releasing hormone (GnRH) receptor in human olfactory GnRH-secreting neurons: an autocrine GnRH loop underlies neuronal migration.

Olfactory neurons and gonadotropin-releasing hormone (GnRH) neurons share a common origin during organogenesis. Kallmann's syndrome, clinically characterized by anosmia and hypogonadotropic hypogonadism, is due to an abnormality in the migration of olfactory and GnRH neurons. We recently characterized the human FNC-B4 cell line, which retains properties present in vivo in both olfactory and GnRH neurons. In this study, we found that FNC-B4 neurons expressed GnRH receptor and responded to GnRH with time- and dose-dependent increases in GnRH gene expression and protein release (up to 5-fold). In addition, GnRH and its analogs stimulated cAMP production and calcium mobilization, although at different biological thresholds (nanomolar for cAMP and micromolar concentrations for calcium). We also observed that GnRH triggered axon growth, actin cytoskeleton remodeling, and a dose-dependent increase in migration (up to 3-4-fold), whereas it down-regulated nestin expression. All these effects were blocked by a specific GnRH receptor antagonist, cetrorelix. We suggest that GnRH, secreted by olfactory neuroblasts, acts in an autocrine pattern to promote differentiation and migration of those cells that diverge from the olfactory sensory lineage and are committed to becoming GnRH neurons.

Dipeptidyl peptidase-IV expression and activity in human glomerular endothelial cells.

Glucagon-like peptide-1 (GLP-1), a meal-stimulated gastrointestinal insulinotropic hormone inactivated by dipeptidyl peptidase-IV (DPP-IV), is reduced in type 2 diabetic patients. The present study shows that 2-week exposure of human glomerular endothelial cells to high glucose (22 mM) determines a highly significant increase in DPP-IV activity and mRNA expression, which cannot be entirely accounted for by hyperosmolarity. On the other hand, incubation of purified DPP-IV in a buffer solution added with high glucose does not affect enzyme activity. These results suggest that high glucose increases expression and activity of DPP-IV, possibly contributing to GLP-1 reduction in type 2 diabetic patients.