ABSTRACT
AIMS/HYPOTHESIS: Phosphatidylinositol 3-OH kinases (PI3Ks) regulate beta cell mass, gene transcription, and function, although the contribution of the specific isoforms is unknown. As reduced type 1A PI3K signalling is thought to contribute to impaired insulin secretion, we investigated the role of the type 1A PI3K catalytic subunits α and ß (p110α and -ß) in insulin granule recruitment and exocytosis in rodent and human islets. METHODS: The p110α and p110ß subunits were inhibited pharmacologically or by small hairpin (sh)RNA-mediated knockdown, and were directly infused or overexpressed in mouse and human islets, beta cells and INS-1 832/13 cells. Glucose-stimulated insulin secretion (GSIS), single-cell exocytosis, Ca(2+) signalling, plasma membrane granule localisation, and actin density were monitored. RESULTS: Inhibition or knockdown of p110α increased GSIS. This was not due to altered Ca(2+) responses, depolymerisation of cortical actin or increased cortical granule density, but to enhanced Ca(2+)-dependent exocytosis. Intracellular infusion of recombinant PI3Kα (p110α/p85ß) blocked exocytosis. Conversely, knockdown (but not pharmacological inhibition) of p110ß blunted GSIS, reduced cortical granule density and impaired exocytosis. Exocytosis was rescued by direct intracellular infusion of recombinant PI3Kß (p110ß/p85ß) even when p110ß catalytic activity was inhibited. Conversely, both the wild-type p110ß and a catalytically inactive mutant directly facilitated exocytosis. CONCLUSIONS/INTERPRETATION: Type 1A PI3K isoforms have distinct and opposing roles in the acute regulation of insulin secretion. While p110α acts as a negative regulator of beta cell exocytosis and insulin secretion, p110ß is a positive regulator of insulin secretion through a mechanism separate from its catalytic activity.
Subject(s)
Class Ia Phosphatidylinositol 3-Kinase/metabolism , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Animals , Calcium Signaling , Catalytic Domain , Cell Membrane/metabolism , Enzyme Inhibitors/pharmacology , Exocytosis , Humans , Insulin Secretion , Male , Mice , Mice, Inbred C57BL , Middle Aged , Protein Isoforms/metabolism , RNA, Small Interfering/metabolism , Signal TransductionABSTRACT
AIMS/HYPOTHESIS: Activation of the G protein-coupled receptor (GPR)40 by long-chain fatty acids potentiates glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells, and GPR40 agonists are in clinical development for type 2 diabetes therapy. GPR40 couples to the G protein subunit Gα(q/11) but the signalling cascade activated downstream is unknown. This study aimed to determine the mechanisms of GPR40-dependent potentiation of GSIS by fatty acids. METHODS: Insulin secretion in response to glucose, oleate or diacylglycerol (DAG) was assessed in dynamic perifusions and static incubations in islets from wild-type (WT) and Gpr40 (-/-) mice. Depolymerisation of filamentous actin (F-actin) was visualised by phalloidin staining and epifluorescence. Pharmacological and molecular approaches were used to ascertain the roles of protein kinase D (PKD) and protein kinase C delta in GPR40-mediated potentiation of GSIS. RESULTS: Oleate potentiates the second phase of GSIS, and this effect is largely dependent upon GPR40. Accordingly, oleate induces rapid F-actin remodelling in WT but not in Gpr40 (-/-) islets. Exogenous DAG potentiates GSIS in both WT and Gpr40 (-/-) islets. Oleate induces PKD phosphorylation at residues Ser-744/748 and Ser-916 in WT but not Gpr40 (-/-) islets. Importantly, oleate-induced F-actin depolymerisation and potentiation of GSIS are lost upon pharmacological inhibition of PKD1 or deletion of Prkd1. CONCLUSIONS/INTERPRETATION: We conclude that the signalling cascade downstream of GPR40 activation by fatty acids involves activation of PKD1, F-actin depolymerisation and potentiation of second-phase insulin secretion. These results provide important information on the mechanisms of action of GPR40, a novel drug target for type 2 diabetes.
Subject(s)
Insulin/metabolism , Islets of Langerhans/metabolism , Protein Kinase C/physiology , Receptors, G-Protein-Coupled/physiology , Actins/metabolism , Animals , Cells, Cultured , Diglycerides/pharmacology , Glucose/pharmacology , Insulin Secretion , Islets of Langerhans/cytology , Islets of Langerhans/drug effects , Mice , Mice, Knockout , Models, Animal , Oleic Acid/pharmacology , Protein Kinase C-delta/deficiency , Protein Kinase C-delta/genetics , Protein Kinase C-delta/physiology , Receptors, G-Protein-Coupled/deficiency , Receptors, G-Protein-Coupled/genetics , Signal Transduction/physiologyABSTRACT
After routine measles-rubella-mumps (MRM) vaccine, seroconversion rate for measles heminhibiting (HI) antibodies in a group of 161 children was determined. Of the 154 children who had no HI antibodies in the first serum sample, 153 (99.3%) developed these antibodies in titres greater than or equal to 1:4 and 148 (96.1%) in titres greater than or equal to 1:8 at 6 weeks postvaccination. These results are in concord with the WHO standards. Another study was designed to evaluate persistence of HI antibodies to measles in a group of 123 children who were given MRM vaccine 1-6 years earlier. No significant decrease in HI antibody titers was recorded. It is concluded that immunity acquired through vaccination with the Edmonston-Zagreb measles virus strain in children aged 12 months to 3 years is satisfactory and that it does not decrease at least up to 6 years following vaccination.