RESUMEN
Neutrophil elastase (NE) plays a pivotal role in inflammation. However, the mechanism underlying NE-mediated inflammation in obesity remains unclear. Here, we report that NE activates protease-activated receptor-2 (PAR2), stimulates actin filament (F-actin) formation, decreases intercellular junction molecule VE-cadherin expression, and increases the permeability of human arterial endothelial cells (hECs). NE also prompts degradation of VE-cadherin and its binding proteins p120- and ß-catenins via MG132-sensitive proteasomes. NE stimulates phosphorylation of myosin light-chain (MLC) and its regulator myosin phosphatase target subunit-1 (MYPT1), a target of Rho kinase (ROCK). Inhibitors of PAR2 and ROCK prohibit NE-induced F-actin formation, MLC phosphorylation, and VE-cadherin reduction in hECs, and impede monocyte transmigration through hEC monolayer pretreated with either neutrophils or NE. Further, administration of an NE inhibitor GW311616A significantly attenuates vascular leakage, leukocyte infiltration, and the expression of proinflammatory cytokines in the white adipose tissue from high-fat diet (HFD)-induced obese mice. Likewise, NE-deficient mice are resistant to HFD-induced vascular leakage in the heart. Together, NE regulates actomyosin cytoskeleton activity and VE-cadherin expression by activating PAR2 signaling in the endothelial cells, leading to increased vascular permeability and leukocyte extravasation. Hence, inhibition of NE is a potential approach to mitigate vascular injury and leukocyte infiltration in obesity-related systemic inflammation.
Asunto(s)
Permeabilidad Capilar , Elastasa de Leucocito , Actinas/metabolismo , Animales , Células Endoteliales/metabolismo , Humanos , Inflamación/metabolismo , Elastasa de Leucocito/metabolismo , Leucocitos/metabolismo , Ratones , Ratones Obesos , Obesidad/metabolismoRESUMEN
CDP138 is a calcium- and lipid-binding protein that is involved in membrane trafficking. Here, we report that mice without CDP138 develop obesity under normal chow diet (NCD) or high-fat diet (HFD) conditions. CDP138-/- mice have lower energy expenditure, oxygen consumption, and body temperature than wild-type (WT) mice. CDP138 is exclusively expressed in adrenal medulla and is colocalized with tyrosine hydroxylase (TH), a marker of sympathetic nervous terminals, in the inguinal fat. Compared with WT controls, CDP138-/- mice had altered catecholamine levels in circulation, adrenal gland, and inguinal fat. Adrenergic signaling on cyclic AMP (cAMP) formation and hormone-sensitive lipase (HSL) phosphorylation induced by cold challenge but not by an exogenous ß3 adrenoceptor against CL316243 were decreased in adipose tissues of CDP138-/- mice. Cold-induced beige fat browning, fatty acid oxidation, thermogenesis, and related gene expression were reduced in CDP138-/- mice. CDP138-/- mice are also prone to HFD-induced insulin resistance, as assessed by Akt phosphorylation and glucose transport in skeletal muscles. Our data indicate that CDP138 is a regulator of stress response and plays a significant role in adipose tissue browning, energy balance, and insulin sensitivity through regulating catecholamine secretion from the sympathetic nervous terminals and adrenal gland.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Catecolaminas/metabolismo , Proteínas de Homeodominio/metabolismo , Resistencia a la Insulina/fisiología , Proteínas de la Membrana/metabolismo , Transporte de Proteínas/fisiología , Glándulas Suprarrenales/metabolismo , Animales , Membrana Celular/metabolismo , Dieta Alta en Grasa/efectos adversos , Metabolismo Energético/fisiología , Expresión Génica/fisiología , Metabolismo de los Lípidos/fisiología , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Esterol Esterasa/metabolismo , Termogénesis/fisiologíaRESUMEN
The molecular mechanisms involved in the development of obesity and related complications remain unclear. Here, we report that obese mice and human subjects have increased activity of neutrophil elastase (NE) and decreased serum levels of the NE inhibitor α1-antitrypsin (A1AT, SerpinA1). NE null (Ela2(-/-)) mice and A1AT transgenic mice were resistant to high-fat diet (HFD)-induced body weight gain, insulin resistance, inflammation, and fatty liver. NE inhibitor GW311616A reversed insulin resistance and body weight gain in HFD-fed mice. Ela2(-/-) mice also augmented circulating high molecular weight (HMW) adiponectin levels, phosphorylation of AMP-activated protein kinase (AMPK), and fatty acid oxidation (FAO) in the liver and brown adipose tissue (BAT) and uncoupling protein (UCP1) levels in the BAT. These data suggest that the A1AT-NE system regulates AMPK signaling, FAO, and energy expenditure. The imbalance between A1AT and NE contributes to the development of obesity and related inflammation, insulin resistance, and liver steatosis.
Asunto(s)
Metabolismo Energético , Resistencia a la Insulina , Elastasa de Leucocito/metabolismo , Obesidad/metabolismo , alfa 1-Antitripsina/sangre , Quinasas de la Proteína-Quinasa Activada por el AMP , Adiponectina/sangre , Tejido Adiposo Pardo/metabolismo , Animales , Dieta Alta en Grasa , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Hígado Graso/complicaciones , Hígado Graso/metabolismo , Hígado Graso/patología , Células Hep G2 , Humanos , Inflamación , Canales Iónicos/metabolismo , Leptina/metabolismo , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/sangre , Hígado/metabolismo , Metaboloma , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Ratones Transgénicos , Proteínas Mitocondriales/metabolismo , Obesidad/complicaciones , Obesidad/patología , Oxidación-Reducción , Fosforilación , Piperidinas/farmacología , Proteínas Quinasas/metabolismo , Proteína Desacopladora 1 , Aumento de Peso/efectos de los fármacos , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismoRESUMEN
The protein kinase B(ß) (Akt2) pathway is known to mediate insulin-stimulated glucose transport through increasing glucose transporter GLUT4 translocation from intracellular stores to the plasma membrane (PM). Combining quantitative phosphoproteomics with RNAi-based functional analyses, we show that a previously uncharacterized 138 kDa C2 domain-containing phosphoprotein (CDP138) is a substrate for Akt2, and is required for optimal insulin-stimulated glucose transport, GLUT4 translocation, and fusion of GLUT4 vesicles with the PM in live adipocytes. The purified C2 domain is capable of binding Ca(2+) and lipid membranes. CDP138 mutants lacking the Ca(2+)-binding sites in the C2 domain or Akt2 phosphorylation site S197 inhibit insulin-stimulated GLUT4 insertion into the PM, a rate-limiting step of GLUT4 translocation. Interestingly, CDP138 is dynamically associated with the PM and GLUT4-containing vesicles in response to insulin stimulation. Together, these results suggest that CDP138 is a key molecule linking the Akt2 pathway to the regulation of GLUT4 vesicle-PM fusion.
Asunto(s)
Adipocitos/metabolismo , Membrana Celular/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Fosfoproteínas , Proteómica/métodos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/genética , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Sitios de Unión , Calcio/metabolismo , Silenciador del Gen/efectos de los fármacos , Insulina/metabolismo , Masculino , Ratones , Péptidos/farmacología , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , ARN Interferente Pequeño/farmacología , Especificidad por Sustrato , Vesículas TransportadorasRESUMEN
Protein kinase B/Akt protein kinases control an array of diverse functions, including cell growth, survival, proliferation, and metabolism. We report here the identification of pleckstrin homology-like domain family B member 1 (PHLDB1) as an insulin-responsive protein that enhances Akt activation. PHLDB1 contains a pleckstrin homology domain, which we show binds phosphatidylinositol PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), as well as a Forkhead-associated domain and coiled coil regions. PHLDB1 expression is increased during adipocyte differentiation, and it is abundant in many mouse tissues. Both endogenous and HA- or GFP-tagged PHLDB1 displayed a cytoplasmic disposition in unstimulated cultured adipocytes but translocated to the plasma membrane in response to insulin. Depletion of PHLDB1 by siRNA inhibited insulin stimulation of Akt phosphorylation but not tyrosine phosphorylation of IRS-1. RNAi-based silencing of PHLDB1 in cultured adipocytes also attenuated insulin-stimulated deoxyglucose transport and Myc-GLUT4-EGFP translocation to the plasma membrane, whereas knockdown of the PHLDB1 isoform PHLDB2 failed to attenuate insulin-stimulated deoxyglucose transport. Furthermore, adenovirus-mediated expression of PHLDB1 in adipocytes enhanced insulin-stimulated Akt and p70 S6 kinase phosphorylation, as well as GLUT4 translocation. These results indicate that PHLDB1 is a novel modulator of Akt protein kinase activation by insulin.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células 3T3-L1 , Animales , Proteínas Sanguíneas/química , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Glucosa/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas/química , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Recent work has led to the identification of novel endocytic compartments with functional roles in both protein trafficking and growth factor signal transduction. The phosphatidylinositol 3-phosphate binding, FYVE domain-containing protein WDFY2 is localized to a distinct subset of early endosomes, which are localized close to the plasma membrane. Here, we find that the serine/threonine kinase Akt interacts with these endosomes in an isoform-specific manner. Using quantitative fluorescence microscopy we demonstrate specific co-localization of WDFY2 with endogenous Akt2, but not Akt1. Moreover, depletion of WDFY2 leads to impaired phosphorylation of Akt in response to insulin due to isoform specific reduction of Akt2, but not Akt1, protein levels, and to a marked reduction in the insulin-stimulated phosphorylation of numerous Akt substrates. This is accompanied by an impairment in insulin-stimulated glucose transport and, after prolonged silencing, a reduction in the level of expression of adipogenic genes. We propose that WDFY2-enriched endosomes serve as a scaffold that enables specificity of insulin signaling through Akt2.
Asunto(s)
Proteínas Portadoras/fisiología , Endosomas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Células 3T3-L1 , Animales , Transporte Biológico , Western Blotting , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Insulina/farmacología , Ratones , Microscopía Fluorescente , Fosforilación , Isoformas de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos HíbridosRESUMEN
Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr(590). RNAi (RNA interference)-mediated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxyglucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr(389), a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway.
Asunto(s)
Regulación de la Expresión Génica , Transportador de Glucosa de Tipo 1/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Células 3T3-L1 , Adipocitos , Animales , Anticuerpos/inmunología , Cricetinae , Proteínas Activadoras de GTPasa/metabolismo , Transportador de Glucosa de Tipo 1/genética , Insulina/farmacología , Ratones , Mutación/genética , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Fosforilación/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato , Serina-Treonina Quinasas TORRESUMEN
3-Azioctanol is a photoactivatable analogue of octanol that noncompetitively inhibits nicotinic acetylcholine receptors (nAChRs). Photolabeling studies using [3H]-3-azioctanol in Torpedo nAChR identified alphaE262 as a site of desensitization-dependent incorporation. However, it is unknown whether photolabeling of alphaE262 causes functional effects in nAChRs and what other roles this residue plays in gating, desensitization, and channel block. We used ultrafast patch-perfusion electrophysiology and ultraviolet (UV) irradiation to investigate the state-dependence of both reversible nAChR inhibition by 3-azioctanol and the irreversible effects of photoactivated 3-azioctanol. Channels with mutations at alphaE262 were studied to determine ACh EC50s, desensitization rates, and sensitivities to reversible and photoirreversible 3-azioctanol inhibition. Exposure to 3-azioctanol in the presence of 365 nm UV light produced irreversible inhibition of wild-type nAChRs. Desensitization with ACh dramatically increased the degree of irreversible inhibition by photoactivated 3-azioctanol. Mutations at alphaE262 that reduce diazirine photomodification decreased the irreversible inhibition induced by photoactivated 3-azioctanol. Hydrophobic mutations at alphaE262 significantly slowed rapid ACh-induced desensitization and dramatically slowed fast resensitization. In contrast, alphaE262 mutations minimally affected 3-azioctanol channel block, and a half blocking concentration of 3-azioctanol did not alter the rate of ACh-induced fast desensitization. Our results indicate that position alphaE262 on muscle nAChRs contributes to an allosteric modulator site that is strongly coupled to desensitization. Occupation of this pocket by hydrophobic molecules stabilizes a desensitized state by slowing resensitization.
Asunto(s)
Músculos/metabolismo , Antagonistas Nicotínicos/química , Octanoles/química , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/efectos de la radiación , Sustitución de Aminoácidos , Animales , Electrofisiología , Femenino , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Activación del Canal Iónico/genética , Ratones , Microinyecciones , Músculos/química , Antagonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/farmacología , Octanoles/metabolismo , Octanoles/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Estructura Secundaria de Proteína , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Rayos Ultravioleta , Xenopus laevisRESUMEN
Insulin signaling through protein kinase Akt/protein kinase B (PKB), a downstream element of the phosphatidylinositol 3-kinase (PI3K) pathway, regulates diverse cellular functions including metabolic pathways, apoptosis, mitogenesis, and membrane trafficking. To identify Akt/PKB substrates that mediate these effects, we used antibodies that recognize phosphopeptide sites containing the Akt/PKB substrate motif (RXRXX(p)S/T) to immunoprecipitate proteins from insulin-stimulated adipocytes. Tryptic peptides from a 250-kDa immunoprecipitated protein were identified as the protein kinase WNK1 (with no lysine) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, consistent with a recent report that WNK1 is phosphorylated on Thr60 in response to insulin-like growth factor I. Insulin treatment of 3T3-L1 adipocytes stimulated WNK1 phosphorylation, as detected by immunoprecipitation with antibody against WNK1 followed by immunoblotting with the anti-phosphoAkt substrate antibody. WNK1 phosphorylation induced by insulin was unaffected by rapamycin, an inhibitor of p70 S6 kinase pathway but abolished by the PI3K inhibitor wortmannin. RNA interference-directed depletion of Akt1/PKB alpha and Akt2/PKB beta attenuated insulin-stimulated WNK1 phosphorylation, but depletion of protein kinase C lambda did not. Whereas small interfering RNA-induced loss of WNK1 protein did not significantly affect insulin-stimulated glucose transport in 3T3-L1 adipocytes, it significantly enhanced insulin-stimulated thymidine incorporation by about 2-fold. Furthermore, depletion of WNK1 promoted serum-stimulated cell proliferation of 3T3-L1 preadipocytes, as evidenced by a 36% increase in cell number after 48 h in culture. These data suggest that WNK1 is a physiologically relevant target of insulin signaling through PI3K and Akt/PKB and functions as a negative regulator of insulin-stimulated mitogenesis.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Insulina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Androstadienos/farmacología , Animales , Apoptosis , Sitios de Unión , Transporte Biológico , Membrana Celular/metabolismo , Proliferación Celular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Regulación de la Expresión Génica , Glucosa/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/metabolismo , Espectrometría de Masas , Ratones , Antígenos de Histocompatibilidad Menor , Péptidos/química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor de Insulina/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Sirolimus/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Timidina/metabolismo , Factores de Tiempo , Transfección , Tripsina/farmacología , Proteína Quinasa Deficiente en Lisina WNK 1 , WortmaninaRESUMEN
Glucose homeostasis is controlled by insulin in part through the translocation of intracellular glucose transporter 4 to the plasma membrane in muscle and fat cells. Akt/protein kinase B downstream of phosphatidylinositol 3-kinase has been implicated in this insulin-signaling pathway, but results with a variety of reagents including Akt1-/- and Akt2-/- mice have been equivocal. Here we report the application of small interfering RNA-directed gene silencing to deplete both Akt1 and Akt2 in cultured 3T3-L1 adipocytes. Loss of Akt1 alone slightly impaired insulin-mediated hexose transport activity but had no detectable effect on glycogen synthase kinase (GSK)-3 phosphorylation. In contrast, depletion of Akt2 alone by 70% inhibited approximately half of the insulin responsiveness. Combined depletions of Akt1 plus Akt2 in these cells even more markedly attenuated insulin action on glucose transporter 4 movements, hexose transport activity, and GSK-3 phosphorylation. These data demonstrate a primary role of Akt2 in insulin signaling, significant functional redundancy of Akt1 and Akt2 isoforms in this pathway, and an absolute requirement of Akt protein kinases for regulation of glucose transport and GSK-3 in cultured adipocytes.
Asunto(s)
Silenciador del Gen , Insulina/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Células 3T3 , Adipocitos/metabolismo , Animales , Transporte Biológico , Western Blotting , Células Cultivadas , Desoxiglucosa/farmacocinética , Electroporación , Fibroblastos/metabolismo , Glucosa/metabolismo , Humanos , Ratones , Microscopía Fluorescente , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Plásmidos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Factores de TiempoRESUMEN
Insulin stimulates glucose uptake in muscle and adipocytes by signalling the translocation of GLUT4 glucose transporters from intracellular membranes to the cell surface. The translocation of GLUT4 may involve signalling pathways that are both independent of and dependent on phosphatidylinositol-3-OH kinase (PI(3)K). This translocation also requires the actin cytoskeleton, and the rapid movement of GLUT4 along linear tracks may be mediated by molecular motors. Here we report that the unconventional myosin Myo1c is present in GLUT4-containing vesicles purified from 3T3-L1 adipocytes. Myo1c, which contains a motor domain, three IQ motifs and a carboxy-terminal cargo domain, is highly expressed in primary and cultured adipocytes. Insulin enhances the localization of Myo1c with GLUT4 in cortical tubulovesicular structures associated with actin filaments, and this colocalization is insensitive to wortmannin. Insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane is augmented by the expression of wild-type Myo1c and inhibited by a dominant-negative cargo domain of Myo1c. A decrease in the expression of endogenous Myo1c mediated by small interfering RNAs inhibits insulin-stimulated uptake of 2-deoxyglucose. Thus, myosin Myo1c functions in a PI(3)K-independent insulin signalling pathway that controls the movement of intracellular GLUT4-containing vesicles to the plasma membrane.