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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

¹ Laboratory of Molecular and Cellular Endocrinology, Department of Internal Medicine, University of Turin, Turin, Italy
² Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Turin, Turin, Italy
³ Department of Medicine, Transplant Unit, Scientific Institute San Raffaele, Vita-Salute University, Milan, Italy
⁴ Department of Pharmaceutical Sciences, University of Salerno, Fisciano (Salerno), Italy
⁵ Department of Anatomy, Pharmacology, and Forensic Medicine, University of Turin, Turin, Italy
⁶ Department of Clinical and Biological Sciences and San Luigi Hospital, University of Turin, Turin, Italy

Address correspondence and reprint requests to Riccarda Granata, PhD, Laboratory of Molecular and Cellular Endocrinology, Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Turin, Corso Dogliotti, 14-10126 Turin, Italy. E-mail: riccarda.granata{at}unito.it

Abbreviations: BrdU, 5-bromo-2-deoxyuridine; CREB, cAMP response element–binding protein; [D-Lys³]-GHRP-6, [D-Lys³]-growth hormone releasing peptide-6; EIA, enzyme immunoassay; ERK, extracellular signal–related kinase; Ex-4, exendin-4; Ex-9, exendin-(9-39); FBS, fetal bovine serum; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor; GPR39, G-protein–coupled receptor 39; GRLN-R, ghrelin receptor; IBMX, 3-isobutyl-1-methylxanthine; IL-1β, interleukin-1β; IFN-, interferon-; IRS-2, insulin receptor substrate-2; KRBH, Krebs-Ringer bicarbonate HEPES buffer; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; PDX-1, pancreatic and duodenal homeobox-1; PI 3-kinase, phosphatidylinositol 3-kinase; PKA, protein kinase A; RIA, radioimmunoassay; TNF-, tumor necrosis factor-; UAG, unacylated ghrelin; TUNEL, TdT-mediated dUTP nick-end labeling

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-/tumor necrosis factor-/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.

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