Letizia Trovato

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.

Dual effects of IGFBP-3 on endothelial cell apoptosis and survival: Involvement of the sphingolipid signaling pathways

Insulin‐like growth factor binding protein (IGFBP)‐3 has both growth‐inhibiting and growth‐promoting effects at the cellular level. The cytotoxic action of several anticancer drugs is linked to increased ceramide generation through sphingomyelin hydrolysis or de novo biosynthesis. Herein, we investigated the role of IGFBP‐3 on apoptosis of human umbilical vein endothelial cells (HUVEC) and its relationship with ceramide levels. We report that IGFBP‐3 exerts dual effects on HUVEC, potentiating doxorubicin‐induced apoptosis but enhancing survival in serum‐starved conditions. Ceramide was increased by IGFBP‐3 in the presence of doxorubicin and decreased when IGFBP‐3 was added alone to cells cultured in serum‐free medium. The protection exerted by the ceramide synthase inhibitor fumonisin B1 over doxorubicin‐induced apoptosis was enhanced by IGFBP‐3 with concomitant reduction of ceramide levels. IGFBP‐3 alone activated sphingosine kinase (SK) and increased SK1 mRNA; the SK inhibitor N,N‐dimethylsphingosine (DMS) blocked IGFBP‐3 antiapoptotic effect. Moreover, IGFBP‐3 increased IGF‐I mRNA and dramatically enhanced IGF‐I release. IGF‐I receptor (IGF‐IR) and its downstream signaling pathways Akt and ERK were phosphorylated by IGFBP‐3, whereas inhibition of IGF‐IR phosphorylation with tyrphostin AG1024 suppressed the antiapopoptic effect of IGFBP‐3. Finally, IGFBP‐3 increased endothelial cell motility in all experimental conditions. These findings provide evidence that IGFBP‐3 differentially regulates endothelial cell apoptosis by involvement of the sphingolipid signaling pathways. Moreover, the survival effect of IGFBP‐3 seems to be mediated by the IGF‐IR.

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.

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.

A New Medical Device Rigeneracons Allows to Obtain Viable Micro‐Grafts From Mechanical Disaggregation of Human Tissues

Autologous graft is considered the gold standard of graft materials; however, this approach is still limited due to both small amount of tissue that can be collected and to reduced cell viability of cells that can be obtained. The aim of this preliminary study was to demonstrate the efficacy of an innovative medical device called Rigeneracons® (CE certified Class I) to provide autologous micro‐grafts immediately available to be used in the clinical practice. Moreover, Rigeneracons® is an instrument able to create micro‐grafts enriched of progenitors cells which maintain their regenerative and differentiation potential. We reported preliminary data about viability cell of samples derived from different kind of human tissues, such as periosteum, cardiac atrial appendage biopsy, and lateral rectus muscle of eyeball and disaggregated by Rigeneracons®. In all cases we observed that micro‐grafts obtained by Rigeneracons® displayed high cell viability. Furthermore, by cell characterization of periosteum samples, we also evidenced an high positivity to mesenchymal cell markers, suggesting an optimal regenerative potential. J. Cell. Physiol. 230: 2299–2303, 2015.

RFamide Peptides 43RFa and 26RFa Both Promote Survival of Pancreatic β-Cells and Human Pancreatic Islets but Exert Opposite Effects on Insulin Secretion

RFamide peptides 43RFa and 26RFa have been shown to promote food intake and to exert different peripheral actions through G-protein–coupled receptor 103 (GPR103) binding. Moreover, 26RFa was found to inhibit pancreatic insulin secretion, whereas the role of 43RFa on β-cell function is unknown, as well as the effects of both peptides on β-cell survival. Herein, we investigated the effects of 43RFa and 26RFa on survival and apoptosis of pancreatic β-cells and human pancreatic islets. In addition, we explored the role of these peptides on insulin secretion and the underlying signaling mechanisms. Our results show that in INS-1E β-cells and human pancreatic islets both 43RFa and 26RFa prevented cell death and apoptosis induced by serum starvation, cytokine synergism, and glucolipotoxicity, through phosphatidylinositol 3-kinase/Akt- and extracellular signal–related kinase 1/2-mediated signaling. Moreover, 43RFa promoted, whereas 26RFa inhibited, glucose- and exendin-4–induced insulin secretion, through Gαs and Gαi/o proteins, respectively. Inhibition of GPR103 expression by small interfering RNA blocked 43RFa insulinotropic effect, but not the insulinostatic action of 26RFa. Finally, 43RFa, but not 26RFa, induced cAMP increase and glucose uptake. In conclusion, because of their survival effects along with the effects on insulin secretion, these findings suggest potential for 43RFa and 26RFa as therapeutic targets in the treatment of diabetes.

Obestatin: is it really doing something?

Obestatin was identified in 2005 by Zhang and colleagues as a ghrelin-associated peptide, derived from posttranslational processing of the prepro-ghrelin gene. Initially, obestatin was reported to activate the G-protein-coupled receptor GPR39 and to reduce food intake and gastric emptying. However, obestatin remains a controversial peptide, as these findings have been questioned and its receptor is still a matter of debate, as well as its effects on feeding behavior. Recently, interaction with the glucagon-like peptide 1 receptor has been suggested, in line with obestatin-positive effects on glucose and lipid metabolism. In addition, obestatin displays a variety of cellular effects, by regulating metabolic cell functions, increasing cell survival and proliferation, and inhibiting apoptosis and inflammation in different cell types. Finally, like ghrelin, obestatin is produced in the gastrointestinal tract, including the pancreas and adipose tissue, and exerts both local actions in peripheral tissues, and distant effects at the central level. Therefore, obestatin may indeed be considered a hormone, although additional studies are required to clarify its physiopathological role and to definitely identify its receptor.

GH-Releasing Hormone Induces Cardioprotection in Isolated Male Rat Heart via Activation of RISK and SAFE Pathways

GHRH stimulates GH synthesis and release from the pituitary and exerts direct effects in extrapituitary tissues. We have previously shown that pretreatment with GHRH reduces cardiomyocyte apoptosis and improves heart function in isolated rat hearts subjected to ischemia/reperfusion (I/R). Here, we determined whether GHRH given at reperfusion reduces myocardial reperfusion injury and investigated the molecular mechanisms involved in GHRH effects. Isolated rat hearts subjected to I/R were treated at the onset of reperfusion with: 1) GHRH; 2) GHRH+GHRH antagonist JV-1-36; 3) GHRH+mitochondrial ATP-dependent potassium channel inhibitor 5-hydroxydecanoate; 4) GHRH+mitochondrial permeability transition pore opener atractyloside; 5) GHRH+ phosphoinositide 3-kinase/Akt inhibitor Wortmannin (WM); and 6) GHRH+signal transducer and activator of transcription-3 inhibitor tyrphostin-AG490 (AG490). GHRH reduced infarct size at the end of reperfusion and reverted contractility dysfunction in I/R hearts. These effects were inhibited by either JV-1-36, 5-hydroxydecanoate, atractylosid, WM, or AG490. Western blot analysis on left ventricles showed GHRH-induced phosphorylation of either the reperfusion injury salvage kinases (RISK), phosphoinositide 3-kinase/Akt, ERK1/2, and glycogen synthase kinase-3β or signal transducer and activator of transcription-3, as part of the survivor activating factor enhancement (SAFE) pathway. GHRH-induced activation of RISK and SAFE pathways was blocked by JV-1-36, WM, and AG490. Furthermore, GHRH increased the phosphorylation of endothelial nitric oxide synthase and AMP-activated protein kinase and preserved postischemic nicotinamide adenine dinucleotide (NAD(+)) levels. These results suggest that GHRH protects the heart from I/R injury through receptor-mediated mechanisms, leading to activation of RISK and SAFE pathways, which converge on mitochondria and possibly on AMP-activated protein kinase.

Pluripotent stem cells isolated from human amniotic fluid and differentiation into pancreatic β-cells

Human amniotic fluid (HAF) contains multipotent stem cells [amniotic fluid-derived stem cells (AFSC)] which can differentiate into a variety of different cell types. Recently, we demonstrated that obestatin, a peptide encoded by the ghrelin gene, exerts anti-apoptotic effects in pancreatic β-cells and human islets and increases the expression of genes involved in β-cells differentiation. We investigated whether: 1) AFSC would differentiate into pancreatic β-cells and 2) obestatin would increase β-cells differentiation from AFSC. Fluorescence-activated cell sorting analysis and immunocytochemical staining showed the presence of mesenchymal and endothelial markers in AFSC. Real-time PCR evidenced the expression of Octamer binding transcription factor 4 (OCT-4), a marker of pluripotency, during the early differentiation phase. However, the β-cells differentiation marker duodenal homeobox factor-1 (PDX-1) could not be detected. Obestatin increased OCT-4 expression but had no effect on β-cells differentiation. These results suggest that, at least under the experimental conditions used in this study, AFSC do not differentiate into β-cells and obestatin has no additional effect.