RESUMEN
AIMS/INTRODUCTION: Insulin stimulates translocation of glucose transporter 4 (GLUT4) from the perinuclear location to the plasma membrane. In the unstimulated state, intracellular vesicles containing GLUT4 are sequestered into specialized storage vesicles that have come to be known as the insulin-responsive compartment (IRC). The IRC is a functional compartment in the perinuclear region that is a target of the insulin signaling cascade, although its precise nature is unclear. Here, we report a novel molecular mechanism facilitating formation of the IRC. MATERIALS AND METHODS: We determined synaptosomal-associated protein of 29 kDa (SNAP29) by mass spectrometry to be an EH domain-containing protein 1 (EHD1)-binding protein. Then, its expression was confirmed by western blotting. Subcellular localization of SNAP29 was determined by immunofluorescent microscopy. Interactions between SNAP29 and syntaxins were determined by immunoprecipitation. We measured glucose uptake and GLUT4 translocation in 3T3-L1 adipocyte expressing SNAP29 or silencing SNAP29. RESULTS: We found SNAP29 to be localized in the perinuclear region and to show partial co-localization with GLUT4 under basal conditions. We also found that SNAP29 binds to syntaxin6, a Qc-SNARE, in adipocytes. In SNAP29-expressing cells, vesicles containing GLUT4 were observed to aggregate around the perinuclear region. In contrast, when SNAP29 was silenced, perinuclear GLUT4 vesicles were dispersed throughout the cytosol. Insulin-stimulated glucose uptake was inhibited in both SNAP29-expressing and SNAP29-silenced cells. CONCLUSIONS: These data suggest that SNAP29 sequesters and anchors GLUT4-containing vesicles in the perinuclear region, and might have a role in the biogenesis of the perinuclear IRC.
Asunto(s)
Proteínas de Transporte de Monosacáridos , Proteínas SNARE , Humanos , Proteínas SNARE/metabolismo , Adipocitos/metabolismo , Insulina/metabolismo , Glucosa/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Transporte de Proteínas/fisiología , Proteínas de Transporte Vesicular/metabolismo , Proteínas Qb-SNARE/metabolismoRESUMEN
The liver is the major organ maintaining metabolic homeostasis in animals during shifts between fed and fasted states. Circadian oscillations in peripheral tissues including the liver are connected with feeding-fasting cycles. We generated transgenic mice with hepatocyte specific E4BP4, D-box negative regulator, overexpression. Liver-specific E4BP4 overexpression was also achieved by adenoviral gene transfer. Interestingly, hepatic E4BP4 overexpression induced marked insulin resistance, that was rescued by DBP, a competing D-box positive regulator, overexpression. At basal conditions hepatocyte E4BP4 transgenic mice exhibited increased gluconeogenesis with reduced AKT phosphorylation in liver. In muscle, AKT phosphorylation was impaired after insulin stimulation. Such muscle insulin resistance was associated with elevated free fatty acid flux from the liver and reduced fatty acid utilization as an energy source during the inactive phase. E4BP4, one of the clock-controlled output genes, are key metabolic regulators in liver adjusting liver and muscle metabolism and insulin sensitivity in the feeding-fasting cycles. Its tuning is critical for preventing metabolic disorders.
Asunto(s)
Relojes Circadianos , Metabolismo Energético , Hígado/metabolismo , Músculo Esquelético/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Grasas/metabolismo , Gluconeogénesis , Resistencia a la Insulina , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Regulación hacia ArribaRESUMEN
AIMS/INTRODUCTION: Double C2 domain protein b (DOC2b), one of the synaptotagmins, has been shown to translocate to the plasma membrane, and to initiate membrane-fusion processes of vesicles containing glucose transporter 4 proteins on insulin stimulation. However, the mechanism by which DOC2b is regulated remains unclear. Herein, we identified the upstream regulatory factors of DOC2b in insulin signal transduction. We also examined the role of DOC2b on systemic homeostasis using DOC2b knockout (KO) mice. MATERIALS AND METHODS: We first identified DOC2b binding proteins by immunoprecipitation and mutagenesis experiments. Then, DOC2b KO mice were generated by disrupting the first exon of the DOC2b gene. In addition to the histological examination, glucose metabolism was assessed by measuring parameters on glucose/insulin tolerance tests. Insulin-stimulated glucose uptake was also measured using isolated soleus muscle and epididymal adipose tissue. RESULTS: We identified an isoform of atypical protein kinase C (protein kinase C iota) that can bind to DOC2b and phosphorylates one of the serine residues of DOC2b (S34). This phosphorylation is essential for DOC2b translocation. DOC2b KO mice showed insulin resistance and impaired oral glucose tolerance on insulin and glucose tolerance tests, respectively. Insulin-stimulated glucose uptake was impaired in isolated soleus muscle and epididymal adipose tissues from DOC2b KO mice. CONCLUSIONS: We propose a novel insulin signaling mechanism by which protein kinase C iota phosphorylates DOC2b, leading to glucose transporter 4 vesicle translocation, fusion and facilitation of glucose uptake in response to insulin. The present results also showed DOC2b to play important roles in systemic glucose homeostasis.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Intolerancia a la Glucosa/patología , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Resistencia a la Insulina , Insulina/farmacología , Isoenzimas/metabolismo , Proteínas del Tejido Nervioso/fisiología , Proteína Quinasa C/metabolismo , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Células Cultivadas , Intolerancia a la Glucosa/inducido químicamente , Intolerancia a la Glucosa/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , FosforilaciónAsunto(s)
Fluorodesoxiglucosa F18 , Hígado/patología , Linfohistiocitosis Hemofagocítica/complicaciones , Linfohistiocitosis Hemofagocítica/diagnóstico , Linfoma de Células T/complicaciones , Linfoma de Células T/diagnóstico , Anciano de 80 o más Años , Femenino , Humanos , Hígado/diagnóstico por imagen , Linfohistiocitosis Hemofagocítica/diagnóstico por imagen , Linfoma de Células T/diagnóstico por imagen , Tomografía de Emisión de Positrones , Tomografía Computarizada por Rayos XRESUMEN
Insulin secretion is precisely regulated by blood glucose with unique biphasic pattern. The regulatory mechanism of the second-phase insulin release is unclear. In this study, we report that DOC2b (double C2 domain protein isoform b), a SNARE related protein, was associated with insulin vesicles and translocated to plasma membrane within several minutes upon high-glucose stimulation followed by an interaction with syntaxin4, but not syntaxin1. This binding specificity and the time course of DOC2b translocation were suitable for the regulation of second-phase insulin release. Increased DOC2b expression enhanced glucose-stimulated insulin secretion. In contrast, silencing DOC2b inhibited delayed release of insulin, without affecting rapid (approximately 7min) phase secretion. Interestingly, DOC2b had no effects on KCl-triggered insulin release. These data suggest that DOC2b may be a regulator for delayed (second-phase) insulin secretion in MIN6 cells.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas SNARE/metabolismo , Animales , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/genética , Línea Celular Tumoral , Membrana Celular/metabolismo , Glucosa/farmacología , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/ultraestructura , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Vesículas Secretoras/metabolismoRESUMEN
OBJECTIVE: Insulin stimulates glucose uptake in skeletal muscle and adipose tissues primarily by stimulating the translocation of vesicles containing a facilitative glucose transporter, GLUT4, from intracellular compartments to the plasma membrane. The formation of stable soluble N-ethyl-maleimide-sensitive fusion protein [NSF] attachment protein receptor (SNARE) complexes between vesicle-associated membrane protein-2 (VAMP-2) and syntaxin-4 initiates GLUT4 vesicle docking and fusion processes. Additional factors such as munc18c and tomosyn were reported to be negative regulators of the SNARE complex assembly involved in GLUT4 vesicle fusion. However, despite numerous investigations, the positive regulators have not been adequately clarified. RESEARCH DESIGN AND METHODS: We determined the intracellular localization of DOC2b by confocal immunoflorescent microscopy in 3T3-L1 adipocytes. Interaction between DOC2b and syntaxin-4 was assessed by the yeast two-hybrid screening system, immunoprecipitation, and in vitro glutathione S-transferase (GST) pull-down experiments. Cell surface externalization of GLUT4 and glucose uptake were measured in the cells expressing DOC2b constructs or silencing DOC2b. RESULTS: Herein, we show that DOC2b, a SNARE-related protein containing double C2 domains but lacking a transmembrane region, is translocated to the plasma membrane upon insulin stimulation and directly associates with syntaxin-4 in an intracellular Ca(2+)-dependent manner. Furthermore, this process is essential for triggering GLUT4 vesicle fusion. Expression of DOC2b in cultured adipocytes enhanced, while expression of the Ca(2+)-interacting domain mutant DCO2b or knockdown of DOC2b inhibited, insulin-stimulated glucose uptake. CONCLUSIONS: These findings indicate that DOC2b is a positive SNARE regulator for GLUT4 vesicle fusion and mediates insulin-stimulated glucose transport in adipocytes.
Asunto(s)
Adipocitos/metabolismo , Proteínas de Unión al Calcio/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Qa-SNARE/metabolismo , Células 3T3-L1 , Adenoviridae/genética , Secuencia de Aminoácidos , Análisis de Varianza , Animales , Transporte Biológico/efectos de los fármacos , Northern Blotting , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Desoxiglucosa/metabolismo , Transportador de Glucosa de Tipo 4/genética , Immunoblotting , Inmunoprecipitación , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Unión Proteica , Proteínas Qa-SNARE/genética , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
Insulin stimulates glucose uptake in fat and muscle primarily by stimulating the translocation of vesicles containing facilitative glucose transporters, GLUT4, from intracellular compartments to the plasma membrane. Although cell surface externalization of GLUT4 is critical for glucose transport, the mechanism regulating cell surface GLUT4 remains unknown. Using a yeast two-hybrid screening system, we have screened GLUT4-binding proteins, and identified a novel glycosyl phosphatidyl inositol (GPI)-linked proteoglycan, Glypican3 (GPC3). We confirmed their interaction using immunoprecipitation and a GST pull-down assay. We also revealed that GPC3 and GLUT4 to co-localized at the plasma membrane, using immunofluorescent microscopy. Furthermore, we observed that glucose uptake in GPC3-overexpressing adipocytes was increased by 30% as compared to control cells. These findings suggest that GPC3 may play roles in glucose transport through GLUT4.
Asunto(s)
Transportador de Glucosa de Tipo 4/metabolismo , Glucosa/metabolismo , Glipicanos/metabolismo , Animales , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Transportador de Glucosa de Tipo 4/análisis , Transportador de Glucosa de Tipo 4/genética , Glipicanos/análisis , Glipicanos/aislamiento & purificación , Humanos , Inmunoprecipitación , Insulina/metabolismo , Insulina/farmacología , Ratones , Ratas , Técnicas del Sistema de Dos HíbridosRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) signaling through the IkappaB kinase (IKK) complex attenuates insulin action via the phosphorylation of insulin receptor substrate 1 (IRS-1) at Ser307. However, the precise molecular mechanism by which the IKK complex phosphorylates IRS-1 is unknown. In this study, we report nuclear factor kappaB essential modulator (NEMO)/IKK-gamma subunit accumulation in membrane ruffles followed by an interaction with IRS-1. This intracellular trafficking of NEMO requires insulin, an intact actin cytoskeletal network, and the motor protein Myo1c. Increased Myo1c expression enhanced the NEMO-IRS-1 interaction, which is essential for TNF-alpha- induced phosphorylation of Ser307-IRS-1. In contrast, dominant inhibitory Myo1c cargo domain expression diminished this interaction and inhibited IRS-1 phosphorylation. NEMO expression also enhanced TNF-alpha-induced Ser307-IRS-1 phosphorylation and inhibited glucose uptake. In contrast, a deletion mutant of NEMO lacking the IKK-beta-binding domain or silencing NEMO blocked the TNF-alpha signal. Thus, motor protein Myo1c and its receptor protein NEMO act cooperatively to form the IKK-IRS-1 complex and function in TNF-alpha-induced insulin resistance.
Asunto(s)
Quinasa I-kappa B/metabolismo , Miosinas/metabolismo , FN-kappa B/metabolismo , Fosfoproteínas/efectos de los fármacos , Serina/efectos de los fármacos , Factor de Necrosis Tumoral alfa/fisiología , Células 3T3-L1 , Animales , Glucosa/metabolismo , Técnicas In Vitro , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Ratones , Proteínas Motoras Moleculares , Miosina Tipo I , FN-kappa B/efectos de los fármacos , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Serina/biosíntesis , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/farmacologíaAsunto(s)
Coma/inducido químicamente , Hipoglucemia/inducido químicamente , Hipoglucemiantes/efectos adversos , Examen de Aptitud para la Conducción de Vehículos/legislación & jurisprudencia , Sistema Nervioso Autónomo/fisiología , Biomarcadores/sangre , Glucemia/análisis , Edema Encefálico/diagnóstico , Edema Encefálico/etiología , Edema Encefálico/terapia , Coma/diagnóstico , Coma/prevención & control , Coma/terapia , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/fisiopatología , Glucagón/administración & dosificación , Glucagón/fisiología , Glucosa/administración & dosificación , Humanos , Hipoglucemia/diagnóstico , Hipoglucemia/prevención & control , Hipoglucemia/terapia , Manitol/administración & dosificación , Pronóstico , Prevención SecundariaRESUMEN
We describe a case of thoracic aortic aneurysm complicated by chronic disseminated intravascular coagulation (DIC). Initially the DIC was controlled successfully by administration of gabexate mesilate and dalteparin. However, because these drugs were given intravenously, the patient could not be discharged. Subsequently, the DIC was treated successfully by changing to orally administered camostat mesilate, warfarin and aspirin, which allowed the patient to leave hospital.
Asunto(s)
Anticoagulantes/administración & dosificación , Aneurisma de la Aorta Torácica/tratamiento farmacológico , Aspirina/administración & dosificación , Coagulación Intravascular Diseminada/tratamiento farmacológico , Gabexato/análogos & derivados , Guanidinas/administración & dosificación , Warfarina/administración & dosificación , Administración Oral , Anciano , Aneurisma de la Aorta Torácica/complicaciones , Enfermedad Crónica , Coagulación Intravascular Diseminada/complicaciones , Quimioterapia Combinada , Ésteres , Humanos , MasculinoRESUMEN
We report a 43-year-old Japanese woman with microcytic and hypochromic anemia, who had been erroneously diagnosed as having iron deficiency anemia 20 years previously at the time of her first labor, and treated with iron and blood transfusion. At the present visit to our clinic, she was found to have an increased HbA2 level and prolonged glycerol lysis time. Genetic analysis of the beta-globin gene revealed deletion of 3 bases at codons 127/128 (CAG/GCT-->CCT). A genetic study of the patient's family showed that two of her four children possessed the same mutation. The patient had mild anemia, her first son had very mild anemia, and her second daughter had moderate anemia with hemolysis. These affected family members were diagnosed as having dominant-phenotype beta-thalassemia.
Asunto(s)
Genes Dominantes/genética , Talasemia beta/genética , Adulto , Femenino , Humanos , Linaje , FenotipoAsunto(s)
Medios de Contraste/efectos adversos , Paro Cardíaco/inducido químicamente , Yohexol/efectos adversos , Trombocitopenia , Medios de Contraste/administración & dosificación , Paro Cardíaco/etiología , Humanos , Infusiones Intravenosas , Yohexol/administración & dosificación , Masculino , Persona de Mediana Edad , Trastornos MieloproliferativosRESUMEN
We report a case of therapy-related myelodysplastic syndrome (t-MDS) in adult T-cell lymphoma. A 69-year-old man suffered from cutaneous adult T-cell lymphoma, which was treated with radiation to the skin and combination chemotherapy of CHOP-V-MMV and VEPA-B. After 14 months of these therapies, anemia and thrombocytopenia appeared, and bone marrow aspiration smears showed immature myeloblasts, dysplastic erythroblasts, and micromegakaryocytes. Therapy-related MDS of refractory anemia with an excess of blasts was diagnosed. Cytogenetic study of the bone marrow cells showed 5q- and additional abnormalities. Rearrangement of the MLL gene was observed in the bone marrow cells. Mutations of N-ras codons at 12,13, and 61, p53 tumor suppressor gene, and monoclonal integration of human T-lymphotrophic virus -1 provirus DNA were not observed in the bone marrow cells. The patient died of pneumonia 21 months after diagnosis of cutaneous adult T-cell lymphoma.