RESUMO
Insulin stimulates glucose uptake by promoting translocation of the Glut4 glucose transporter from intracellular storage compartments to the plasma membrane. In the absence of insulin, Glut4 is retained intracellularly; the mechanism underlying this process remains uncertain. Using the TC10-interacting protein CIP4 as bait in a yeast two-hybrid screen, we cloned a RasGAP and VPS9 domain-containing protein, Gapex-5/RME-6. The VPS9 domain is a guanine nucleotide exchange factor for Rab31, a Rab5 subfamily GTPase implicated in trans-Golgi network (TGN)-to-endosome trafficking. Overexpression of Rab31 blocks insulin-stimulated Glut4 translocation, whereas knockdown of Rab31 potentiates insulin-stimulated Glut4 translocation and glucose uptake. Gapex-5 is predominantly cytosolic in untreated cells; its overexpression promotes intracellular retention of Glut4 in adipocytes. Insulin recruits the CIP4/Gapex-5 complex to the plasma membrane, thus reducing Rab31 activity and permitting Glut4 vesicles to translocate to the cell surface, where Glut4 docks and fuses to transport glucose into the cell.
Assuntos
Adipócitos/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Insulina/metabolismo , Camundongos , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
MicroRNAs (miRNAs) are involved in the regulation of immunity via targeting of mRNA encoding immune response elements. In this report, alterations in the expression of microRNAs as autoantibody levels increase was investigated. The (NZB×NZW)F1 or B/W mouse model of systemic lupus erythematosus (SLE) naturally has increased autoantibodies with aging. IFNα (type I IFN) accelerates B/W disease, however, the effects of a related IFN, IFNλ, which is a type III IFN, is largely unknown. The purpose of the study was to investigate the relationship between IFN-accelerated disease, microRNAs, immunoregulatory B cell subsets and autoantibody production in the autoimmune-prone environment in vivo. B/W mice received osmotic pumps to chronically deliver IFNα and IFNλ for up to 16 weeks. Urine protein level was monitored weekly by urine strips and proteinuria was used as the disease marker. Splenic cells were taken for flow analysis of B cell subsets and levels of microRNAs determined. Plasma were analyzed for autoantibodies and microRNA levels. As a result of treatment, IFNα accelerated proteinuria in a dose dependent manner, while IFNλ single treatment did not show a significant effect, but greatly enhanced low dose IFNα effects in the combination treatment. Both the splenic cellular and plasma miR-15a were elevated in diseased compared to pre-diseased mice as well as autoantibody levels. Autoantibodies and miR-15a levels were significantly correlated. The immunosuppressive B subpopulation, B-10, was reduced following IFNα treatment. In addition in diseased mice, B-10 versus B-2 ratios were reduced in IFN-treated B/W compared to the control PBS treated group. In all B/W the miR-15a was highly expressed in the B-10 subset and this increased with disease development, suggesting that miR-15a has a specific negative effect on the B-10 subpopulation. In conclusion, our data support the involvement of elevated miR-15a in autoimmune disease development in B/W mice and suggest that decreasing this microRNA might be beneficial in B/W mice.
Assuntos
Autoanticorpos/biossíntese , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , MicroRNAs/genética , MicroRNAs/imunologia , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Sequência de Bases , Primers do DNA/genética , Modelos Animais de Doenças , Feminino , Interferon-alfa/administração & dosagem , Interferons/administração & dosagem , Interferons/classificação , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Camundongos Endogâmicos NZB , MicroRNAs/sangue , MicroRNAs/metabolismo , Proteínas Recombinantes/administração & dosagem , Baço/imunologia , Baço/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
Insulin-stimulated glucose uptake requires the fusion of GLUT4 transporter-containing vesicles with the plasma membrane, a process that depends on the SNARE (soluble N-ethylmaleimide-sensitive fusion factor attachment receptor) proteins VAMP2 (vesicle-associated membrane protein 2) and syntaxin 4 (Stx4)/SNAP23 (soluble N-ethylmaleimide-sensitive fusion factor attachment protein 23). Efficient SNARE-dependent fusion has been shown in many settings in vivo to require the generation of both phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidic acid (PA). Addition of PA to Stx4/SNAP23 vesicles markedly enhanced the fusion rate, whereas its addition to VAMP2 vesicles was inhibitory. In contrast, addition of PIP2 to Stx4/SNAP23 vesicles inhibited the fusion reaction, and its addition to VAMP2 vesicles was stimulatory. The optimal distribution of phospholipids was found to trigger the progression from the hemifused state to full fusion. These findings reveal an unanticipated dependence of SNARE complex-mediated fusion on asymmetrically distributed acidic phospholipids and provide mechanistic insights into the roles of phospholipase D and PIP kinases in the late stages of regulated exocytosis.