Corrado Ghè

Cardiac effects of ghrelin and its endogenous derivatives des-octanoyl ghrelin and des-Gln14-ghrelin

The mechanisms underlying the cardiac activities of synthetic growth hormone secretagogues (GHS) are still unclear. The natural ligand of the GHS receptors, i.e. ghrelin, classically binds the GHS receptor and exerts endocrine actions in acylated forms only; its cardiovascular actions still need to be investigated further. In order to clarify these aspects, we studied the effects of either the synthetic peptidyl GHS hexarelin (1 microM), or the natural ghrelin (50 nM) and the endogenous ghrelin derivatives des-Gln14-ghrelin (1-100 nM) and des-octanoyl ghrelin (50 nM), on the tension developed by guinea pig papillary muscle and on L-type Ca2+ current (ICa) of isolated ventricular cells. The binding of these molecules to ventricular cell membrane homogenates was also studied. We observed that all peptides reduced the tension developed at low frequencies (60-120 beats/min) in a dose-dependent manner. No alteration in cardiac contractility was induced by des-Gln14-ghrelin or des-octanoylated ghrelin when the endocardial endothelium had been removed or after cyclooxygenase blockade. Pretreatment with tyramine (2 microM) had no effect on the inotropic response induced by des-Gln(14)-ghrelin. No significant effect on I(Ca) of isolated ventricular cells was observed in the presence of des-Gln14-ghrelin (100 nM). The order of potency on the tension of papillary muscle was: des-octanoyl ghrelin > ghrelin = des-Gln14-ghrelin > hexarelin. This gradient of potency was consistent with the binding experiments performed on ventricular membranes where either acylated or unacylated ghrelin forms, and hexarelin, recognized a common high-affinity binding site. In conclusion, ghrelin, des-Gln14-ghrelin and des-octanoyl ghrelin, show similar negative inotropic effect on papillary muscle; as des-octanoyl ghrelin is peculiarly devoid of any GH-releasing activity, the cardiotropic action of these molecules is independent of GH release. The binding studies and the experiments performed both on the isolated cells and on papillary muscle after endothelium removal or cyclooxygenase blockade indicate that the cardiotropic action of natural and synthetic ghrelin analogues reflects the interaction with a novel GHS receptor (peculiarly common for ghrelin and des-octanoyl ghrelin), leading to release of cyclooxygenase metabolites from endothelial cells, as indicated by direct measurement of prostacyclin metabolite 6-keto-PGF(1alpha).

OBESTATIN PROMOTES SURVIVAL OF PANCREATIC β-CELLS AND HUMAN ISLETS AND INDUCES EXPRESSION OF GENES INVOLVED IN THE REGULATION OF β-CELL MASS AND FUNCTION

OBJECTIVE—Obestatin is a newly discovered peptide encoded by the ghrelin gene whose biological functions are poorly understood. We investigated obestatin effect on survival of β-cells and human pancreatic islets and the underlying signaling pathways. RESEARCH DESIGN AND METHODS—β-Cells and human islets were used to assess obestatin effect on cell proliferation, survival, apoptosis, intracellular signaling, and gene expression. RESULTS—Obestatin showed specific binding on HIT-T15 and INS-1E β-cells, bound to glucagon-like peptide-1 receptor (GLP-1R), and recognized ghrelin binding sites. Obestatin exerted proliferative, survival, and antiapoptotic effects under serum-deprived conditions and interferon-γ/tumor necrosis factor-α/interleukin-1β treatment, particularly at pharmacological concentrations. Ghrelin receptor antagonist [D-Lys3]-growth hormone releasing peptide-6 and anti-ghrelin antibody prevented obestatin-induced survival in β-cells and human islets. β-Cells and islet cells released obestatin, and addition of anti-obestatin antibody reduced their viability. Obestatin increased β-cell cAMP and activated extracellular signal–related kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt; its antiapoptotic effect was blocked by inhibition of adenylyl cyclase/cAMP/protein kinase A (PKA), PI 3-kinase/Akt, and ERK1/2 signaling. Moreover, obestatin upregulated GLP-1R mRNA and insulin receptor substrate-2 (IRS-2) expression and phosphorylation. The GLP-1R antagonist exendin-(9-39) reduced obestatin effect on β-cell survival. In human islets, obestatin, whose immunoreactivity colocalized with that of ghrelin, promoted cell survival and blocked cytokine-induced apoptosis through cAMP increase and involvement of adenylyl cyclase/cAMP/PKA signaling. Moreover, obestatin 1) induced PI 3-kinase/Akt, ERK1/2, and also cAMP response element–binding protein phosphorylation; 2) stimulated insulin secretion and gene expression; and 3) upregulated GLP-1R, IRS-2, pancreatic and duodenal homeobox-1, and glucokinase mRNA. CONCLUSIONS—These results indicate that obestatin promotes β-cell and human islet cell survival and stimulates the expression of main regulatory β-cell genes, identifying a new role for this peptide within the endocrine pancreas.

The Antiproliferative Effect of Synthetic Peptidyl GH Secretagogues in Human CALU-1 Lung Carcinoma Cells

The specific binding of [125I]Tyr-Ala-hexarelin, a radiolabeled peptidyl GH secretagogue (GHS), has been investigated in nontumoral and neoplastic human lung tissues. This binding was very marked in nonendocrine lung carcinomas with values that were greater than found in either normal lung or in endocrine lung neoplasms. Tyr-Ala-hexarelin binding was also present in a human lung carcinoma cell line (CALU-1). [125I]Tyr-Ala-hexarelin binding to tumor membranes was displaced by peptidyl GHS (GHRP-6, hexarelin) and EP-80317, an hexarelin analog devoid of GH-releasing activity in vivo. In contrast, no competition was observed in the presence of the nonpeptidyl GHS MK-0677 and the endogenous ligand of the GHS-R1a ghrelin. GHS-R1a mRNA expression was found in 50% of endocrine lung tumors but was never seen in other nontumoral and neoplastic lung tissues nor in CALU-1. In these cells, hexarelin and EP-80317, but not ghrelin or MK-0677, caused a dose-dependent inhibition of IGF-II-stimulated thymidine incorporatio...

Des-Acyl Ghrelin Has Specific Binding Sites and Different Metabolic Effects from Ghrelin in Cardiomyocytes

The current study aimed to compare the effects of the peptide hormone ghrelin and des-G, its unacylated isoform, on glucose and fatty acid uptake and to identify des-G-specific binding sites in cardiomyocytes. In the murine HL-1 adult cardiomyocyte line, ghrelin and des-G had opposing metabolic effects: des-G increased medium-chain fatty acid uptake (BODIPY fluorescence intensity), whereas neither ghrelin alone nor in combination with des-G did so. Ghrelin inhibited the increase in glucose uptake normally induced by insulin (rate of 2-[(3)H]deoxy-d-glucose incorporation), but des-G did not; des-G was also able to partially reverse the inhibitory effect of ghrelin. In HL-1 cells and primary cultures of neonatal rat cardiomyocytes, des-G but not ghrelin increased insulin-induced translocation of glucose transporter-4 from nuclear to cytoplasmic compartments (immunohistochemistry and quantitative confocal analysis). AKT was phosphorylated by insulin but not affected by ghrelin or des-G, whereas neither AMP-activated protein kinase nor phosphatase and tensin homolog deleted from chromosome 10 was phosphorylated by any treatments. HL-1 and primary-cultured mouse and rat cardiomyocytes each possessed two independent specific binding sites for des-G not recognized by ghrelin (radioreceptor assays). Neither ghrelin nor des-G affected viability (dimethylthiazol diphenyltetrazolium bromide assays), whereas both isoforms were equally protective against apoptosis. Therefore, in cardiomyocytes, des-G binds to specific receptors and has effects on glucose and medium-chain fatty acid uptake that are distinct from those of ghrelin. Real-time PCR indicated that expression levels of ghrelin O-acyltransferase RNA were comparable between HL-1 cells, human myocardial tissue, and human and murine stomach tissue, indicating the possibility of des-G conversion to ghrelin within our model.

Distribution and Characterization of Prolactin Binding Sites in the Male and Female Rat Brain: Effects of Hypophysectomy and Ovariectomy

The binding of 125I-labeled rat prolactin (125I-rat PRL) to membranes from different regions of the rat brain was studied. Among these regions the hypothalamus showed the highest specific binding. Clearly detectable specific binding was also observed in substantia nigra, whereas it was very scanty in other brain regions. No significant sex differences in PRL binding to various brain regions were observed, except for hypothalamus where a higher binding was observed in female rats. The binding of 125I-rat PRL to hypothalamus from female rats was inhibited in a dose-dependent manner by both unlabeled rat and ovine PRL but not by several other polypeptide hormones. Scatchard analysis of the binding revealed the presence of the binding sites with low capacity and high affinity for rat ligand. Ovariectomy markedly decreased PRL binding in the hypothalamus; an even more pronounced decrease was found after hypophysectomy of female animals. A treatment with estradiol restored the PRL binding in the ovariectomized rats to above normal levels. These results of in vitro biochemical analysis together with the experimental modulation of hormonal status provide strong preliminary evidence for the presence of PRL binding sites in rat brain.

Unacylated ghrelin and obestatin increase islet cell mass and prevent diabetes in streptozotocin-treated newborn rats

The ghrelin gene products, namely acylated ghrelin (AG), unacylated ghrelin (UAG), and obestatin (Ob), were shown to prevent pancreatic beta-cell death and to improve beta-cell function under treatment with cytokines, which are major cause of beta-cell destruction in diabetes. Moreover, AG had been described previously to prevent streptozotocin (STZ)-induced diabetes in rats; however, the effect of either UAG or Ob has never been examined in this context. In the present study, we investigated the potential of UAG and Ob to increase islet beta-cell mass and to reduce diabetes at adult age in STZ-treated neonatal rats. One-day-old rats were injected with STZ and subsequently administered with either AG, UAG or Ob for 7 days. On day 70, plasma glucose levels, plasma and pancreatic insulin levels, pancreatic islet area and number, insulin and pancreatic/duodenal homeobox-1 (Pdx1) gene expression, and antiapoptotic BCL2 protein expression were determined. Similarly to AG, both UAG and Ob counteracted STZ-induced high glucose levels and improved plasma and pancreatic insulin levels, which were reduced by the diabetogenic compound. UAG and Ob increased islet area, islet number, and beta-cell mass with respect to STZ treatment alone. Finally, in STZ-treated animals, UAG and Ob up-regulated insulin and Pdx1 mRNA and increased the expression of BCL2 similarly to AG. Taken together, our results suggest that in STZ-treated newborn rats, UAG and Ob improve glucose metabolism and preserve islet cell mass, granting a therapeutic potential in medical conditions associated with impaired beta-cell function.

Proliferative and protective effects of growth hormone secretagogues on adult rat hippocampal progenitor cells.

Progenitor cells in the subgranular zone of the hippocampus may be of significance for functional recovery after various injuries because they have a regenerative potential to form new neuronal cells. The hippocampus has been shown to express the GH secretagogue (GHS) receptor 1a, and recent studies suggest GHS to both promote neurogenesis and have neuroprotective effects. The aim of the present study was to investigate whether GHS could stimulate cellular proliferation and exert cell protective effects in adult rat hippocampal progenitor (AHP) cells. Both hexarelin and ghrelin stimulated increased incorporation of (3)H-thymidine, indicating an increased cell proliferation. Furthermore, hexarelin, but not ghrelin, showed protection against growth factor deprivation-induced apoptosis, as measured by annexin V binding and caspase-3 activity and also against necrosis, as measured by lactate dehydrogenase release. Hexarelin activated the MAPK and the phosphatidylinositol 3-kinase/Akt pathways, whereas ghrelin activated only the MAPK pathway. AHP cells did not express the GHS receptor 1a, but binding studies could show specific binding of both hexarelin and ghrelin, suggesting effects to be mediated by an alternative GHS receptor subtype. In conclusion, our results suggest a differential effect of hexarelin and ghrelin in AHP cells. We have demonstrated stimulation of (3)H-thymidine incorporation with both hexarelin and ghrelin. Hexarelin, but not ghrelin, also showed a significant inhibition of apoptosis and necrosis. These results suggest a novel cell protective and proliferative role for GHS in the central nervous system.

Obestatin affords cardioprotection to the ischemic-reperfused isolated rat heart and inhibits apoptosis in cultures of similarly stressed cardiomyocytes

Obestatin, a newly discovered peptide encoded by the ghrelin gene, induces the expression of genes regulating pancreatic beta-cell differentiation, insulin biosynthesis, and glucose metabolism. It also activates antiapoptotic signaling pathways such as phosphoinositide 3-kinase (PI3K) and ERK1/2 in pancreatic beta-cells and human islets. Since these kinases have been shown to protect against myocardial injury, we sought to investigate whether obestatin would exert cardioprotective effects. Both isolated perfused rat heart and cultured cardiomyocyte models of ischemia-reperfusion (I/R) were used to measure infarct size and cell apoptosis as end points of injury. The presence of specific obestatin receptors on cardiac cells as well as the signaling pathways underlying the obestatin effect were also studied. In the isolated heart, the addition of rat obestatin-(1-23) before ischemia reduced infarct size and contractile dysfunction in a concentration-dependent manner, whereas obestatin-(23-1), a synthetic analog with an inverse aminoacid sequence, was ineffective. The cardioprotective effect of obestatin-(1-23) was observed at concentrations of 10-50 nmol/l and was abolished by inhibiting PI3K or PKC by the addition of wortmannin (100 nmol/l) or chelerythrine, (5 micromol/l), respectively. In rat H9c2 cardiac cells or isolated ventricular myocytes subjected to I/R, 50 nmol/l obestatin-(1-23) reduced cardiomyocyte apoptosis and reduced caspase-3 activation; the antiapoptotic effect was blocked by the inhibition of PKC, PI3K, or ERK1/2 pathways. In keeping with these functional findings, radioreceptor binding results revealed the presence of specific high-affinity obestatin-binding sites, mainly localized on membranes of the ventricular myocardium and cardiomyocytes. Our data suggest that, by acting on specific receptors, obestatin-(1-23) activates PI3K, PKC-epsilon, PKC-delta, and ERK1/2 signaling and protects cardiac cells against myocardial injury and apoptosis induced by I/R.

Prolactin binding sites in human erythrocytes and lymphocytes.

Specific binding sites for prolactin (PRL) have been studied in human peripheral lymphocytes and erythrocytes of normal adult volunteers and of term cord bloods. In erythrocytes from healthy adult subjects of both sexes a very low specific binding of 125I-human PRL was found (0.24%), whereas a higher binding was found in term cord blood (1.1%). The binding was hormone specific, the binding capacity was 2.6 fmol/4 X 10(9) cells and the Kd was 3.4 X 10(-10) M. In lymphocytes of both adults and term cord bloods an evident specific binding was observed (male adults: 1.6%; female adults: 1.7%; cord blood: 1.8%). The binding was specific for lactogenic hormones and the binding capacity was 3.7 fmol/2 X 10(6) cells and the Kd was 3.9 X 10(-10) M. The presence of specific binding sites for PRL on human erythrocytes and lymphocytes could be used to study PRL binding on blood cells of patients in different physiological or pathological situations.

Obestatin induced recovery of myocardial dysfunction in type 1 diabetic rats: underlying mechanisms

BackgroundThe aim of this study was to investigate whether obestatin (OB), a peptide mediator encoded by the ghrelin gene exerting a protective effect in ischemic reperfused heart, is able to reduce cardiac dysfunctions in adult diabetic rats.MethodsDiabetes was induced by STZ injection (50 mg/kg) in Wistar rats (DM). OB was administered (25 μg/kg) twice a day for 6 weeks. Non-diabetic (ND) rats and DM rats were distributed into four groups: untreated ND, OB-treated ND, untreated DM, OB-treated DM. Cardiac contractility and ß-adrenergic response were studied on isolated papillary muscles. Phosphorylation of AMPK, Akt, ERK1/2 and GSK3ß as well ß-1 adrenoreceptors levels were detected by western blot, while α-MHC was measured by RT-PCR.ResultsOB preserved papillary muscle contractility (85 vs 27% of ND), ß-adrenergic response (103 vs 65% of ND), as well ß1-adrenoreceptors and α-MHC levels in diabetic myocardial tissue. Moreover, OB up-regulated the survival kinases Akt and ERK1/2, and enhanced AMPK and GSK3ß phosphorylation. OB corrected oxidative unbalance, reduced pro-inflammatory cytokine TNF-α plasma levels, NFkB translocation and pro-fibrogenic factors expression in diabetic myocardium.ConclusionsOB displays a significant beneficial effect against the alterations of contractility and ß-adrenergic response in the heart of STZ-treated diabetic rats, which was mainly associated with the ability of OB to up-regulate the transcription of ß1-adrenergic receptors and α-MHC; this protective effect was accompanied by the ability to restore oxidative balance and to promote phosphorylation/modulation of AMPK and pro-survival kinases such as Akt, ERK1/2 and GSK3ß.