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.
- 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-γ, interferon-γ
- 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-α, tumor necrosis factor-α
- UAG, unacylated ghrelin
- TUNEL, TdT-mediated dUTP nick-end labeling
- Received August 15, 2007.
- Accepted December 13, 2007.