Fabio Settanni

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.

Unraveling the role of the ghrelin gene peptides in the endocrine pancreas

The ghrelin gene peptides include acylated ghrelin (AG), unacylated ghrelin (UAG), and obestatin (Ob). AG, mainly produced by the stomach, exerts its central and peripheral effects through the GH secretagogue receptor type 1a (GHS-R1a). UAG, although devoid of GHS-R1a-binding affinity, is an active peptide, sharing with AG many effects through an unknown receptor. Ob was discovered as the G-protein-coupled receptor 39 (GPR39) ligand; however, its physiological actions remain unclear. The endocrine pancreas is necessary for glucose homeostasis maintenance. AG, UAG, and Ob are expressed in both human and rodent pancreatic islets from fetal to adult life, and the pancreas is the major source of ghrelin in the perinatal period. GHS-R1a and GPR39 expression has been shown in beta-cells and islets, as well as specific binding sites for AG, UAG, and Ob. Ghrelin colocalizes with glucagon in alpha-islet cells, but is also uniquely expressed in epsilon-islet cells, suggesting a role in islet function and development. Indeed, AG, UAG, and Ob regulate insulin secretion in beta-cells and isolated islets, promote beta-cell proliferation and survival, inhibit beta-cell and human islet cell apoptosis, and modulate the expression of genes that are essential in pancreatic islet cell biology. They even induce beta-cell regeneration and prevent diabetes in streptozotocin-treated neonatal rats. The receptor(s) mediating their effects are not fully characterized, and a signaling crosstalk has been suggested. The present review summarizes the newest findings on AG, UAG, and Ob expression in pancreatic islets and the role of these peptides on beta-cell development, survival, and function.

The ghrelin gene products and exendin-4 promote survival of human pancreatic islet endothelial cells in hyperglycaemic conditions, through phosphoinositide 3-kinase/Akt, extracellular signal-related kinase (ERK)1/2 and cAMP/protein kinase A (PKA) signalling pathways.

Aims/hypothesisPancreatic islet microendothelium exhibits unique features in interdependent relationship with beta cells. Gastrointestinal products of the ghrelin gene, acylated ghrelin (AG), unacylated ghrelin (UAG) and obestatin (Ob), and the incretin, glucagon-like peptide-1 (GLP-1), prevent apoptosis of pancreatic beta cells. We investigated whether the ghrelin gene products and the GLP-1 receptor agonist exendin-4 (Ex-4) display survival effects in human pancreatic islet microendothelial cells (MECs) exposed to chronic hyperglycaemia.MethodsIslet MECs were cultured in high glucose concentration and treated with AG, UAG, Ob or Ex-4. Apoptosis was assessed by DNA fragmentation, Hoechst staining of the nuclei and caspase-3 activity. Western blot analyses and pharmacological inhibition of protein kinase B (Akt) and extracellular signal-related kinase (ERK)1/2 pathways, detection of intracellular cAMP levels and blockade of adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) signalling were performed. Levels of NO, IL-1β and vascular endothelial growth factor (VEGF)-A in cell culture supernatant fractions were measured.ResultsIslet MECs express the ghrelin receptor GHS-R1A as well as GLP-1R. Treatment with AG, UAG, Ob and Ex-4 promoted cell survival and significantly inhibited glucose-induced apoptosis, through activation of PI3K/Akt, ERK1/2 phosphorylation and intracellular cAMP increase. Moreover, peptides upregulated B cell lymphoma 2 (BCL-2) and downregulated BCL-2-associated X protein (BAX) and CD40 ligand (CD40L) production, and significantly reduced the secretion of NO, IL-1β and VEGF-A.Conclusions/interpretationThe ghrelin gene-derived peptides and Ex-4 exert cytoprotective effects in islet MECs. The anti-apoptotic effects involve phosphoinositide 3-kinase (PI3K)/Akt, ERK1/2 and cAMP/PKA pathways. These peptides could therefore represent a potential tool to improve islet vascularisation and, indirectly, islet cell function.

Insulin-like growth factor binding protein-3 induces angiogenesis through IGF-I- and SphK1-dependent mechanisms

Angiogenesis is critical for development and repair, and is a prominent feature of many pathological conditions. Based on evidence that insulin‐like growth factor binding protein (IGFBP)‐3 enhances cell motility and activates sphingosine kinase (SphK) in human endothelial cells, we have investigated whether IGFBP‐3 plays a role in promoting angiogenesis. IGFBP‐3 potently induced network formation by human endothelial cells on Matrigel. Moreover, it up‐regulated proangiogenic genes, such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP)‐2 and ‐9. IGFBP‐3 even induced membrane‐type 1 MMP (MT1‐MMP), which regulates MMP‐2 activation. Decreasing SphK1 expression by small interfering RNA (siRNA), blocked IGFBP‐3‐induced network formation and inhibited VEGF, MT1‐MMP but not IGF‐I up‐regulation. IGF‐I activated SphK, leading to sphingosine‐1‐phosphate (S1P) formation. The IGF‐I effect on SphK activity was blocked by specific inhibitors of IGF‐IR, PI3K/Akt and ERK1/2 phosphorylation. The disruption of IGF‐I signaling prevented the IGFBP‐3 effect on tube formation, SphK activity and VEGF release. Blocking ERK1/2 signaling caused the loss of SphK activation and VEGF and IGF‐I up‐regulation. Finally, IGFBP‐3 dose‐dependently stimulated neovessel formation into subcutaneous implants of Matrigel in vivo. Thus, IGFBP‐3 positively regulates angiogenesis through involvement of IGF‐IR signaling and subsequent SphK/S1P activation.

Growth hormone-releasing hormone promotes survival of cardiac myocytes in vitro and protects against ischaemia–reperfusion injury in rat heart

AIMS The hypothalamic neuropeptide growth hormone-releasing hormone (GHRH) stimulates GH synthesis and release in the pituitary. GHRH also exerts proliferative effects in extrapituitary cells, whereas GHRH antagonists have been shown to suppress cancer cell proliferation. We investigated GHRH effects on cardiac myocyte cell survival and the underlying signalling mechanisms. METHODS AND RESULTS Reverse transcriptase-polymerase chain reaction analysis showed GHRH receptor (GHRH-R) mRNA in adult rat ventricular myocytes (ARVMs) and in rat heart H9c2 cells. In ARVMs, GHRH prevented cell death and caspase-3 activation induced by serum starvation and by the beta-adrenergic receptor agonist isoproterenol. The GHRH-R antagonist JV-1-36 abolished GHRH survival action under both experimental conditions. GHRH-induced cardiac cell protection required extracellular signal-regulated kinase (ERK)1/2 and phosphoinositide-3 kinase (PI3K)/Akt activation and adenylyl cyclase/cAMP/protein kinase A signalling. Isoproterenol strongly upregulated the mRNA and protein of the pro-apoptotic inducible cAMP early repressor, whereas GHRH completely blocked this effect. Similar to ARVMs, in H9c2 cardiac cells, GHRH inhibited serum starvation- and isoproterenol-induced cell death and apoptosis through the same signalling pathways. Finally, GHRH improved left ventricular recovery during reperfusion and reduced infarct size in Langendorff-perfused rat hearts, subjected to ischaemia-reperfusion (I/R) injury. These effects involved PI3K/Akt signalling and were inhibited by JV-1-36. CONCLUSION Our findings suggest that GHRH promotes cardiac myocyte survival through multiple signalling mechanisms and protects against I/R injury in isolated rat heart, indicating a novel cardioprotective role of this hormone.

Obestatin regulates adipocyte function and protects against diet-induced insulin resistance and inflammation

The metabolic actions of the ghrelin gene‐derived peptide obestatin are still unclear. We investigated obestatin effects in vitro, on adipocyte function, and in vivo, on insulin resistance and inflammation in mice fed a high‐fat diet (HFD). Obestatin effects on apoptosis, differentiation, lipolysis, and glucose uptake were determined in vitro in mouse 3T3‐L1 and in human subcutaneous (hSC) and omental (hOM) adipocytes. In vivo, the influence of obestatin on glucose metabolism was assessed in mice fed an HFD for 8 wk. 3T3‐L1, hSC, and hOM preadipocytes and adipocytes secreted obestatin and showed specific binding for the hormone. Obestatin prevented apoptosis in 3T3‐L1 preadipocytes by increasing phosphoinositide 3‐kinase (PI3K)/Akt and extracellular signal‐regulated kinase (ERK)1/2 signaling. In both mice and human adipocytes, obestatin inhibited isoproterenol‐induced lipolysis, promoted AMP‐activated protein kinase phosphorylation, induced adiponectin, and reduced leptin secretion. Obestatin also enhanced glucose uptake in either the absence or presence of insulin, promoted GLUT4 translocation, and increased Akt phosphorylation and sirtuin 1 (SIRT1) protein expression. Inhibition of SIRT1 by small interfering RNA reduced obestatin‐induced glucose uptake. In HFD‐fed mice, obestatin reduced insulin resistance, increased insulin secretion from pancreatic islets, and reduced adipocyte apoptosis and inflammation in metabolic tissues. These results provide evidence of a novel role for obestatin in adipocyte function and glucose metabolism and suggest potential therapeutic perspectives in insulin resistance and metabolic dysfunctions.—Granata, R., Gallo, D., Luque, R. M., Baragli, A., Scarlatti, F., Grande, C., Gesmundo, I., Córdoba‐Chacón, J., Bergandi, L., Settanni, F., Togliatto, G., Volante, M., Garetto, S., Annunziata, M., Chanclón, B., Gargantini, E., Rocchietto, S., Matera, L., Datta, G., Morino, M., Brizzi, M. F., Ong, H., Camussi, G., Castaño, J. P., Papotti, M., Ghigo, E. Obestatin regulates adipocyte function and protects against diet‐induced insulin resistance and inflammation. FASEB J. 26, 3393–3411 (2012). www.fasebj.org

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.

Unacylated as well as acylated ghrelin promotes cell survival and inhibit apoptosis in HIT-T15 pancreatic β -cells

Ghrelin is mainly produced by the stomach, although it is expressed in other tissues, including the pancreas. Among its pleiotropic actions, ghrelin prevents the development of diabetes in rats and exerts mitogenic and antiapoptotic effects in different cell types. In addition, a ghrelin -producing ε-cell population has been demonstrated in rodent islets, suggesting a direct role in the control of islet cell survival. In this study, we investigated the effect of acylated ghrelin (AG) and unacylated ghrelin (UAG) on cell survival of HIT-T15 pancreatic β cells. We show that both AG and UAG equally prevented β cell death induced by serum withdrawal. In addition, both peptides inhibited serum starvation-induced apoptosis. These findings indicate that UAG and AG prevent cell death and apoptosis of pancreatic β cells. Since only AG, but not UAG, binds the GRLN receptor, a different and as yet unknown receptor is likely involved in these survival mechanisms.

Copeptin (CTproAVP), a new tool for understanding the role of vasopressin in pathophysiology

Abstract Arginine vasopressin (AVP) plays a key role in many physiologic and pathologic processes. The most important stimulus for AVP release is a change in plasma osmolality. AVP is also involved in the response and adaptation to stress. Reliable measurement of AVP is hindered by several factors. Over 90% of AVP is tightly bound to platelets, and its estimation is influenced by the number of platelets, incomplete removal of platelets or pre-analytical processing steps. Copeptin (CTproAVP), a 39-aminoacid glycopeptide, is a C-terminal part of the precursor pre-provasopressin (pre-proAVP). Activation of the AVP system stimulates CTproAVP secretion into the circulation from the posterior pituitary gland in equimolar amounts with AVP. Therefore CTproAVP directly reflects AVP concentration and can be used as a surrogate biomarker of AVP secretion. In many studies CTproAVP represents AVP levels and its behavior represents changes in plasma osmolality, stress and various disease states, and shows some of the various physiologic and pathophysiologic conditions associated with increased or decreased AVP. Increased CTproAVP concentration is described in several studies as a strong predictor of mortality in patients with chronic heart failure and acute heart failure. Autosomal polycystic kidney disease (ADPKD) patients have both central and nephrogenic defects in osmoregulation and CTproAVP balance. A possibility raised by these clinical observations is that CTproAVP may serve to identify patients who could benefit from an intervention aimed at countering AVP.

Obestatin in human neuroendocrine tissues and tumours: expression and effect on tumour growth.

The hormone obestatin, which is derived from the same precursor as ghrelin and whose receptor(s) is still unrecognized, possesses a variety of metabolic/modulatory functions mostly related to food intake suppression and reduction of gastrointestinal motility. The distribution of obestatin in normal and neoplastic human tissues is poorly understoood. We report that in fetal tissue samples, obestatin peptide was detected in the thyroid, pituitary, lung, pancreas and gastrointestinal tract, usually being co‐localized with chromogranin A. In adult tissues, obestatin protein expression was restricted to pituitary, lung, pancreas and gastrointestinal tract and was co‐localized strictly with ghrelin. By contrast, in endocrine tumours obestatin was expressed in a small fraction of thyroid, parathyroid, gastrointestinal and pancreatic neoplasms, in most cases with a focal immunoreactivity and co‐localized with ghrelin. Messenger RNA levels of the specific fragments of ghrelin and obestatin were comparable in both normal and tumour samples, confirming that post‐translational mechanisms rather than alternative splicing events lead to ghrelin/obestatin production. Finally, in TT and BON‐1 cell lines obestatin induced antiproliferative effects at pharmacological doses, opposite to those observed with ghrelin. In summary, our data demonstrate that obestatin is produced by the same endocrine cells that express ghrelin in normal tissues from fetal to adult life, whereas, as compared to ghrelin, in neoplastic conditions it is down‐regulated by post‐translational modulation and shows potential antiproliferative properties in vitro. Copyright