RFamides are a family of peptides containing arginine-phenylalanine-amide at their C terminus. A novel 43-amino acid RFamide, named QRFP43, and a shorter endogenous peptide 26RFa, were discovered and identified as ligands of the G protein-coupled receptor GPR103. Different studies have shown that RFamides, such as neuropeptide FF and QRFP43 and 26RFa, play a role in food intake, thermogenesis and energy homeostasis. Here, we investigated the effects of QRFP peptides on survival, proliferation, apoptosis, insulin secretion and glucose uptake of INS-1E pancreatic β-cells and human pancreatic islets. The signaling pathways involved in these actions were also determined. GPR103 expression was evaluated by immunofluorescence and RT-PCR; cell survival by MTT and proliferation by BrdU incorporation; apoptosis was assessed by Hoechst 33258 nuclear staining and caspase-3 activity; insulin secretion by RIA; activation of signaling pathways by Western blot analysis; intracellular cAMP production by ELISA; glucose uptake by [3H]2-deoxyglucose incorporation. The results showed that both INS-1E β-cells and human pancreatic islets express GPR103. Moreover, either QRFP43 or 26RFa promoted survival and proliferation and reduced apoptosis, in both serum-free conditions and under treatment with the cytokines TNF-, IFN-γ and IL-1β. QRFP43 also stimulated both basal and glucose-induced insulin secretion in β-cells and human islets, enhanced glucose uptake, increased intracellular cAMP levels and ERK1/2 and PI3K/Akt phosphorylation. Conversely, 26RFa reduced insulin secretion and cAMP levels, inhibited glucose uptake and only increased ERK1/2 phosphorylation. The opposite metabolic effects of QRFP43 and 26RFa involved activation of the G proteins Gαs and Gαi respectively, as demonstrated by the use of specific inhibitors. In all, these findings indicate that both QRFP43 and 26RFa promote β-cell survival; however, they display opposite effects on β-cell function, suggesting a novel and differential role in glucose metabolism.
QRFP43 and its fragment 26RFa both promote β-cell survival but differently regulate insulin secretion and glucose uptake in pancreatic β-cells and human pancreatic islets
TROVATO, Letizia;SETTANNI, Fabio;GALLO, DAVIDE;GARGANTINI, ELEONORA;BERGANDI, Loredana;GHIGO, Ezio;GRANATA, Riccarda
2012-01-01
Abstract
RFamides are a family of peptides containing arginine-phenylalanine-amide at their C terminus. A novel 43-amino acid RFamide, named QRFP43, and a shorter endogenous peptide 26RFa, were discovered and identified as ligands of the G protein-coupled receptor GPR103. Different studies have shown that RFamides, such as neuropeptide FF and QRFP43 and 26RFa, play a role in food intake, thermogenesis and energy homeostasis. Here, we investigated the effects of QRFP peptides on survival, proliferation, apoptosis, insulin secretion and glucose uptake of INS-1E pancreatic β-cells and human pancreatic islets. The signaling pathways involved in these actions were also determined. GPR103 expression was evaluated by immunofluorescence and RT-PCR; cell survival by MTT and proliferation by BrdU incorporation; apoptosis was assessed by Hoechst 33258 nuclear staining and caspase-3 activity; insulin secretion by RIA; activation of signaling pathways by Western blot analysis; intracellular cAMP production by ELISA; glucose uptake by [3H]2-deoxyglucose incorporation. The results showed that both INS-1E β-cells and human pancreatic islets express GPR103. Moreover, either QRFP43 or 26RFa promoted survival and proliferation and reduced apoptosis, in both serum-free conditions and under treatment with the cytokines TNF-, IFN-γ and IL-1β. QRFP43 also stimulated both basal and glucose-induced insulin secretion in β-cells and human islets, enhanced glucose uptake, increased intracellular cAMP levels and ERK1/2 and PI3K/Akt phosphorylation. Conversely, 26RFa reduced insulin secretion and cAMP levels, inhibited glucose uptake and only increased ERK1/2 phosphorylation. The opposite metabolic effects of QRFP43 and 26RFa involved activation of the G proteins Gαs and Gαi respectively, as demonstrated by the use of specific inhibitors. In all, these findings indicate that both QRFP43 and 26RFa promote β-cell survival; however, they display opposite effects on β-cell function, suggesting a novel and differential role in glucose metabolism.
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