RESUMEN
Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4. The small GTPase Rac1 acts as a switch of signal transduction that regulates GLUT4 translocation to the plasma membrane following insulin stimulation. However, it remains obscure whether signaling cascades upstream and downstream of Rac1 in skeletal muscle are impaired by obesity that causes insulin resistance and type 2 diabetes. In an attempt to clarify this point, we investigated Rac1 signaling in the leptin-deficient (Lepob/ob) mouse model. Here, we show that insulin-stimulated GLUT4 translocation and Rac1 activation are almost completely abolished in Lepob/ob mouse skeletal muscle. Phosphorylation of the protein kinase Akt2 and plasma membrane translocation of the guanine nucleotide exchange factor FLJ00068 following insulin stimulation were also diminished in Lepob/ob mice. On the other hand, the activation of another small GTPase RalA, which acts downstream of Rac1, by the constitutively activated form of Akt2, FLJ00068, or Rac1, was partially abrogated in Lepob/ob mice. Taken together, we conclude that insulin-stimulated glucose uptake is impaired by two mechanisms in Lepob/ob mouse skeletal muscle: one is the complete inhibition of Akt2-mediated activation of Rac1, and the other is the partial inhibition of RalA activation downstream of Rac1.
Asunto(s)
Diabetes Mellitus Tipo 2 , Proteínas de Unión al GTP Monoméricas , Ratones , Animales , Insulina/metabolismo , Ratones Obesos , Proteínas de Unión al GTP Monoméricas/metabolismo , Leptina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Transducción de Señal , Músculo Esquelético/metabolismo , Insulina Regular Humana , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
White adipocytes act as lipid storage, and play an important role in energy homeostasis. The small GTPase Rac1 has been implicated in the regulation of insulin-stimulated glucose uptake in white adipocytes. Adipocyte-specific rac1-knockout (adipo-rac1-KO) mice exhibit atrophy of subcutaneous and epididymal white adipose tissue (WAT); white adipocytes in these mice are significantly smaller than controls. Here, we aimed to investigate the mechanisms underlying the aberrations in the development of Rac1-deficient white adipocytes by employing in vitro differentiation systems. Cell fractions containing adipose progenitor cells were obtained from WAT and subjected to treatments that induced differentiation into adipocytes. In concordance with observations in vivo, the generation of lipid droplets was significantly attenuated in Rac1-deficient adipocytes. Notably, the induction of various enzymes responsible for de novo synthesis of fatty acids and triacylglycerol in the late stage of adipogenic differentiation was almost completely suppressed in Rac1-deficient adipocytes. Furthermore, the expression and activation of transcription factors, such as the CCAAT/enhancer-binding protein (C/EBP) ß, which is required for the induction of lipogenic enzymes, were largely inhibited in Rac1-deficient cells in both early and late stages of differentiation. Altogether, Rac1 is responsible for adipogenic differentiation, including lipogenesis, through the regulation of differentiation-related transcription.
Asunto(s)
Lipogénesis , Proteínas de Unión al GTP Monoméricas , Ratones , Animales , Proteínas de Unión al GTP Monoméricas/metabolismo , Adipogénesis , Diferenciación Celular , Triglicéridos/metabolismo , Tejido Adiposo Blanco/metabolismo , Células Madre/metabolismo , Células 3T3-L1 , Tejido Adiposo/metabolismoRESUMEN
RATIONALE: Combining matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and Kendrick mass defect (KMD) analysis is a powerful tool for visualizing polymers in complex mass spectra. The identification of minor polymers by KMD analysis requires reduction of the broad noise peaks often observed in the low-mass region. METHODS: A machine-learning model was created using pix2pixHD. It converts an original mass spectrum into a pseudo-mass spectrum that contains only the original peaks at m/z positions that the model judges as sharp single-component peaks. It reduces noise by selecting only the m/z and intensity values from the original spectrum's peak list that correspond to peaks in the pseudo-mass spectrum. RESULTS: A machine-learning model was applied to a low-concentration polymer mass spectrum observed at m/z <2000. Extracting single-component peaks from the mass spectrum made the minor polymer series appear clearly in the KMD plot. The technique facilitated mass spectrometric imaging of the ultraviolet degradation of polyethylene terephthalate by plotting the polymers' spatial distributions. It could also distinguish between polymer series (before and after degradation) to identify their separate spatial distributions. CONCLUSIONS: A machine-learning method for peak extraction from high-resolution MALDI-TOFMS was developed. Single-component peaks of the mass spectrum were distinguished from noise peaks by their peak shapes. Combining with KMD analysis facilitated the identification of minor polymer series in complex mass spectra.
RESUMEN
Insulin stimulates glucose uptake in adipose tissue and skeletal muscle by inducing plasma membrane translocation of the glucose transporter GLUT4. Although the small GTPase Rac1 is a key regulator downstream of phosphoinositide 3-kinase (PI3K) and the protein kinase Akt2 in skeletal muscle, it remains unclear whether Rac1 also regulates glucose uptake in white adipocytes. Herein, we investigated the physiological role of Rac1 in white adipocytes by employing adipocyte-specific rac1 knockout (adipo-rac1-KO) mice. Subcutaneous and epididymal white adipose tissues (WATs) in adipo-rac1-KO mice showed significant reductions in size and weight. Actually, white adipocytes lacking Rac1 were smaller than controls. Insulin-stimulated glucose uptake and GLUT4 translocation were abrogated in rac1-KO white adipocytes. On the other hand, GLUT4 translocation was augmented by constitutively activated PI3K or Akt2 in control, but not in rac1-KO, white adipocytes. Similarly, to skeletal muscle, the involvement of another small GTPase RalA downstream of Rac1 was demonstrated. In addition, mRNA levels of various lipogenic enzymes were down-regulated in rac1-KO white adipocytes. Collectively, these results suggest that Rac1 is implicated in insulin-dependent glucose uptake and lipogenesis in white adipocytes, and reduced insulin responsiveness due to the deficiency of Rac1 may be a likely explanation for atrophy of WATs.
Asunto(s)
Tejido Adiposo Blanco/patología , Transportador de Glucosa de Tipo 4/metabolismo , Glucosa/metabolismo , Insulina/farmacología , Neuropéptidos/fisiología , Proteína de Unión al GTP rac1/fisiología , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Atrofia , Femenino , Transportador de Glucosa de Tipo 4/genética , Hipoglucemiantes/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Edulcorantes/farmacologíaRESUMEN
RATIONALE: Matrix-assisted laser desorption/ionization mass spectrometric imaging (MSI) is considered to be a powerful tool for visualizing the spatial distribution of synthetic polymers. However, a conventional method extracting an image of a specific m/z value is not suitable for polymers, which have a mass distribution. It is necessary to develop the visualization method to show the spatial distribution of entire polymer series. METHODS: The mass peaks included in polymer series were specified from the average mass spectrum of the entire MSI measurement region by using Kendrick mass defect analysis. The images of those mass peaks were extracted and the number average molecular weight (Mn ), the weight average molecular weight (Mw ) and dispersity (D) were calculated for each pixel. Finally, the spatial distribution of the polymer series was summarized to images using Mn , Mw and D as indices. RESULTS: The effects of the methods were investigated by (i) polymers with different mass distributions and (ii) polymers with different repeat units and end-groups. In both cases, the spatial distribution of specific polymer series including several dozens to hundreds of mass peaks was summarized into three images related to Mn , Mw and D, which are familiar indices in polymer analysis. The results are able to provide an overview of the spatial variation of each polymer more intuitively. CONCLUSIONS: The visualization of Mn , Mw and D will help provide an overview of the spatial distribution of polymer series combined with ion intensity distribution made by conventional methods. It can be also applied to other mass spectrometric imaging methods such as desorption electrospray ionization (DESI) or time-of-flight secondary ion mass spectrometry (TOF-SIMS).
RESUMEN
Diverse GTPases act as signal transducing enzymes in a variety of organisms and cell types [...].
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Proteínas de Unión al GTP Monoméricas/genética , Transducción de Señal/genética , Proteínas de Unión al GTP rho/genética , Animales , HumanosRESUMEN
Insulin-stimulated glucose uptake is mediated by translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes and skeletal muscle cells. In both types of cells, phosphoinositide 3-kinase and the protein kinase Akt2 have been implicated as critical regulators. In skeletal muscle, the small GTPase Rac1 plays an important role downstream of Akt2 in the regulation of insulin-stimulated glucose uptake. However, the role for Rac1 in adipocytes remains controversial. Here, we show that Rac1 is required for insulin-dependent GLUT4 translocation also in adipocytes. A Rac1-specific inhibitor almost completely suppressed GLUT4 translocation induced by insulin or a constitutively activated mutant of phosphoinositide 3-kinase or Akt2. Constitutively activated Rac1 also enhanced GLUT4 translocation. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was abrogated by inhibition of phosphoinositide 3-kinase or Akt2. On the other hand, constitutively activated Akt2 caused Rac1 activation, and insulin-induced Rac1 activation was suppressed by an Akt2-specific inhibitor. Moreover, GLUT4 translocation induced by a constitutively activated mutant of Akt2 or Rac1 was diminished by knockdown of another small GTPase RalA. RalA was activated by a constitutively activated mutant of Akt2 or Rac1, and insulin-induced RalA activation was suppressed by an Akt2- or Rac1-specific inhibitor. Collectively, these results suggest that Rac1 plays an important role in the regulation of insulin-dependent GLUT4 translocation downstream of Akt2, leading to RalA activation in adipocytes.
Asunto(s)
Adipocitos/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Ratones , Microscopía Fluorescente , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , Proteína de Unión al GTP rac1/genética , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismoRESUMEN
Its recent adaptation to low-resolution mass spectra of polymers using fractional base units raises the question of the minimal resolution needed for a Kendrick mass defect (KMD) analysis. Intuiting an oligomeric resolution since the mass of a repeat unit is the sole value to be known, it is challenged by the relative failure of the KMD plots computed from an isotopically resolved matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrum to display clear alignments in the high mass range. Another procedure based on the remainders of Kendrick mass (RKMs) overcomes this pitfall with oligomers perfectly aligned in a new RKM plot. Despite a concomitant degradation of the resolving power and accuracy, with the example of MALDI-TOF/TOF mass spectra of a variety of homo- and copolymer ions, the RKM procedure still allows a rapid enumeration, assignment, and any further manipulation of all the product ion series in visual RKM plots. Successfully extended to the critical case of a MALDI mass spectrum recorded with a linear TOF analyzer allowing a bare oligomeric resolution, the RKM plot turns the distributions differing by their end-groups or adducted ion into clear horizontal lines. It eventually gives intuition its due by answering the original question: the minimal resolution required for a mass defect analysis can be as low as oligomeric with the appropriate formulas.
RESUMEN
RATIONALE: Bi cluster secondary ion mass spectrometry (SIMS) is one of the most promising tools for precise analysis of synthetic polymers. However, the sensitivity in the high-mass region is still insufficient compared with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Accordingly, the effects of metal assistance (cationization agents) were investigated in this study. METHODS: To investigate the effects caused by varying the ionization agent, three different polyethylene glycol (PEG) samples were prepared, one with an Ag-deposited film, and two others mixed with Ag and Na, respectively. The measurements were performed by using a commercial Bi cluster SIMS and MALDI-TOFMS systems. The mass spectrum obtained with MALDI-TOFMS was used as a reference molecular weight distribution to evaluate the effects of molecular weight and primary ion species (Bi+ , Bi3+ , Bi32+ ) on the sensitivity of Bi cluster SIMS. RESULTS: The intensity of each secondary ion was the highest in Bi32+ irradiation, and the lowest in Bi+ irradiation. Regarding the cationization agents, the secondary ion yield was the highest for the sample mixed with Ag, while the degree of decay of sensitivity along with the increase in molecular weight was the smallest for the sample mixed with Na. CONCLUSIONS: It was suggested that the cationization mechanism consists of pre-formed ionization and gas-phase ionization processes. The sensitivity of Bi cluster SIMS decreases to approximately one-fiftieth in every 1000 u. These results might help in understanding the mechanism of cationization and further enhancement of secondary ion yields of polymers. Copyright © 2016 John Wiley & Sons, Ltd.
RESUMEN
Insulin-stimulated glucose uptake in skeletal muscle is mediated by the translocation of the glucose transporter GLUT4 from intracellular storage sites to the plasma membrane. The small GTPase Rac1 has been implicated in this insulin signalling, but the mechanism whereby Rac1 stimulates GLUT4 translocation remains obscure. In the present study, we examined the role of the small GTPase RalA downstream of Rac1 in skeletal muscle fibres isolated from genetically modified mice. A dominant-negative mutant of RalA, when ectopically overexpressed, significantly reduced GLUT4 translocation in response to insulin or either one of constitutively activated mutants of Rac1 and its upstream regulators, including the guanine-nucleotide-exchange factor FLJ00068, the protein kinase Akt2 and phosphoinositide 3-kinase. Constitutively activated Rac1 also failed to induce GLUT4 translocation in mouse skeletal muscle fibres in which the expression of RalA was abrogated by specific siRNA molecules. Furthermore, we applied a novel approach to detect the activated form of RalA in situ by immunofluorescence microscopy of mouse skeletal muscle fibres, demonstrating that constitutively activated mutants of Rac1 and its upstream regulators as well as insulin indeed cause the activation of RalA. Notably, this RalA activation was remarkably impaired in rac1-deficient skeletal muscle fibres. Taken together, these results provide evidence that RalA is indeed activated and involved in the regulation of GLUT4 translocation in response to insulin downstream of Rac1 in mouse skeletal muscle.
Asunto(s)
Insulina/metabolismo , Músculo Esquelético/enzimología , Neuropéptidos/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Animales , Activación Enzimática , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/enzimología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Neuropéptidos/genética , Proteína de Unión al GTP rac1/genética , Proteínas de Unión al GTP ral/genéticaRESUMEN
The small GTPase Rac1 acts as a molecular switch of intracellular signaling in mammals. For understanding the regulatory mechanism, it is important to identify subcellular locations in which Rac1 is activated following multiple extracellular stimuli. However, it is difficult to detect Rac1 activation in situ in animal tissues, and thus a novel method is highly desirable. Here, we report a simple method to visualize the activation of endogenous Rac1 in mouse skeletal muscle fibers. In this assay, specific interaction between activated Rac1 and a binding polypeptide is detected by immunofluorescent microscopy. This approach is readily applicable to other small GTPases.
Asunto(s)
Proteínas de Unión al GTP Monoméricas/metabolismo , Músculo Esquelético/enzimología , Animales , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Insulin is a hormone that regulates the blood glucose level by stimulating various physiological responses in its target tissues. In skeletal muscle and adipose tissue, insulin promotes membrane trafficking of the glucose transporter GLUT4 from GLUT4 storage vesicles to the plasma membrane, thereby facilitating the uptake of glucose from the circulation. Detailed mechanisms underlying insulin-dependent intracellular signal transduction for glucose uptake remain largely unknown. In this article, I give an overview on the recently identified signaling network involving Rab, Ras, and Rho family small guanosine triphosphatases (GTPases) that regulates glucose uptake in insulin-responsive tissues. In particular, the regulatory mechanisms for these small GTPases and the cross-talk between protein kinase and small GTPase cascades are highlighted.
Asunto(s)
Adipocitos/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas ras/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Transporte Biológico , Humanos , Transducción de SeñalRESUMEN
Cluster of differentiation 36 (CD36) is a scavenger receptor (SR), recognizing diverse extracellular ligands in various types of mammalian cells. Long-chain fatty acids (FAs), which are important constituents of phospholipids and triglycerides, also utilize CD36 as a predominant membrane transporter, being incorporated from the circulation across the plasma membrane in several cell types, including cardiac and skeletal myocytes and adipocytes. CD36 is localized in intracellular vesicles as well as the plasma membrane, and its distribution is modulated by extracellular stimuli. Herein, we aimed to clarify the molecular basis of insulin-stimulated translocation of CD36, which leads to the enhanced uptake of long-chain FAs, in adipocytes. To this end, we developed a novel exofacial epitope-tagged reporter to specifically detect cell surface-localized CD36. By employing this reporter, we demonstrate that the small GTPase Rac1 plays a pivotal role in insulin-stimulated translocation of CD36 to the plasma membrane in 3T3-L1 adipocytes. Additionally, phosphoinositide 3-kinase and the protein kinase Akt2 are shown to be involved in the regulation of Rac1. Downstream of Rac1, another small GTPase RalA directs CD36 translocation. Collectively, these results suggest that CD36 is translocated to the plasma membrane by insulin through mechanisms similar to those for the glucose transporter GLUT4 in adipocytes.
Asunto(s)
Insulina , Proteínas de Unión al GTP Monoméricas , Animales , Adipocitos/metabolismo , Antígenos CD36/metabolismo , Membrana Celular/metabolismo , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Insulina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transporte de Proteínas , Transducción de Señal , RatonesRESUMEN
Interferon-induced transmembrane protein 3 (IFITM3) iplays a crucial role in the antiviral responses of Type I interferons (IFNs). The role of IFITM3 in the central nervous system (CNS) is, however, largely unknown, despite the fact that its expression is increased in the brains of patients with neurologic and neuropsychiatric diseases. Here, we show the role of IFITM3 in long-lasting neuronal impairments in mice following polyriboinosinic-polyribocytidylic acid (polyI:C, a synthetic double-stranded RNA)-induced immune challenge during the early stages of development. We found that the induction of IFITM3 expression in the brain of mice treated with polyI:C was observed only in astrocytes. Cultured astrocytes were activated by polyI:C treatment, leading to an increase in the mRNA levels of inflammatory cytokines as well as Ifitm3. When cultured neurons were treated with the conditioned medium of polyI:C-treated astrocytes (polyI:C-ACM), neurite development was impaired. These polyI:C-ACM-induced neurodevelopmental abnormalities were alleviated by ifitm3(-/-) astrocyte-conditioned medium. Furthermore, decreases of MAP2 expression, spine density, and dendrite complexity in the frontal cortex as well as memory impairment were evident in polyI:C-treated wild-type mice, but such neuronal impairments were not observed in ifitm3(-) (/) (-) mice. We also found that IFITM3 proteins were localized to the early endosomes of astrocytes following polyI:C treatment and reduced endocytic activity. These findings suggest that the induction of IFITM3 expression in astrocytes by the activation of the innate immune system during the early stages of development has non-cell autonomous effects that affect subsequent neurodevelopment, leading to neuropathological impairments and brain dysfunction, by impairing endocytosis in astrocytes.
Asunto(s)
Astrocitos/patología , Proteínas de la Membrana/fisiología , Neuronas/patología , Animales , Animales Recién Nacidos , Astrocitos/inmunología , Células COS , Células Cultivadas , Chlorocebus aethiops , Citocinas/biosíntesis , Citocinas/genética , Endocitosis/efectos de los fármacos , Endocitosis/inmunología , Femenino , Inmunidad Innata/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Neuronas/inmunología , Poli I-C/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacosRESUMEN
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a suitable method for polymer analysis. MALDI is a soft ionization technique that can generate mainly singly charged ions. Therefore, the polymer's molecular weight distribution is easy to analyze, facilitating the calculation of the number average molecular weight and weight average molecular weight and polydispersity. However, there are polymers that are difficult to detect by MALDI-TOFMS. For example, polyacrylic acid includes carboxylic acid in the main chain, which is difficult to measure due to its low ionization efficiency. As a solution, the ionization efficiency was improved by methylation. In this technical report, we introduce a method to utilize derivatization to determine the degree of polymerization by accurate mass spectrometry (MS). Furthermore, the structures of both ends of the polymers were estimated by tandem time-of-flight MS.
RESUMEN
Electron ionization (EI) mass spectrum library searching is usually performed to identify a compound in gas chromatography/mass spectrometry. However, compounds whose EI mass spectra are registered in the library are still limited compared to the popular compound databases. This means that there are compounds that cannot be identified by conventional library searching but also may result in false positives. In this report, we report on the development of a machine learning model, which was trained using chemical formulae and EI mass spectra, that can predict the EI mass spectrum from the chemical structure. It allowed us to create a predicted EI mass spectrum database with predicted EI mass spectra for 100 million compounds in PubChem. We also propose a method for improving library searching time and accuracy that includes an extensive mass spectrum library.
RESUMEN
The Rho family GTPase Rac1 has been implicated in the regulation of glucose uptake in myoblast cell lines. However, no evidence for the role of Rac1 has been provided by a mouse model. The purpose of this study is to test the involvement of Rac1 in insulin action in mouse skeletal muscle. Intravenous administration of insulin indeed elicited Rac1 activation in gastrocnemius muscle, suggesting the involvement of Rac1 in this signaling pathway. We then examined whether insulin-stimulated translocation of the facilitative glucose transporter GLUT4 from its storage sites to the skeletal muscle sarcolemma depends on Rac1. We show that ectopic expression of constitutively activated Rac1, as well as intravenous administration of insulin, caused translocation of GLUT4 to the gastrocnemius muscle sarcolemma, as revealed by immunofluorescent staining of a transiently expressed exofacial epitope-tagged GLUT4 reporter. Of particular note, insulin-dependent, but not constitutively activated Rac1-induced, GLUT4 translocation was markedly suppressed in skeletal muscle-specific rac1-knockout mice compared to control mice. Immunogold electron microscopic analysis of endogenous GLUT4 gave similar results. Collectively, we propose a critical role of Rac1 in insulin-dependent GLUT4 translocation to the skeletal muscle sarcolemma, which has heretofore been predicted solely by cell culture studies.
Asunto(s)
Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Sarcolema/metabolismo , Proteína de Unión al GTP rac1/fisiología , Animales , Ratones , Microscopía Electrónica , Proteínas de Unión al GTP Monoméricas , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestructura , Transporte de ProteínasRESUMEN
cAMP is well known to regulate exocytosis in various secretory cells, but the precise mechanism of its action remains unknown. Here, we examine the role of cAMP signaling in the exocytotic process of insulin granules in pancreatic beta cells. Although activation of cAMP signaling alone does not cause fusion of the granules to the plasma membrane, it clearly potentiates both the first phase (a prompt, marked, and transient increase) and the second phase (a moderate and sustained increase) of glucose-induced fusion events. Interestingly, all granules responsible for this potentiation are newly recruited and immediately fused to the plasma membrane without docking (restless newcomer). Importantly, cAMP-potentiated fusion events in the first phase of glucose-induced exocytosis are markedly reduced in mice lacking the cAMP-binding protein Epac2 (Epac2(ko/ko)). In addition, the small GTPase Rap1, which is activated by cAMP specifically through Epac2 in pancreatic beta cells, mediates cAMP-induced insulin secretion in a protein kinase A-independent manner. We also have developed a simulation model of insulin granule movement in which potentiation of the first phase is associated with an increase in the insulin granule density near the plasma membrane. Taken together, these data indicate that Epac2/Rap1 signaling is essential in regulation of insulin granule dynamics by cAMP, most likely by controlling granule density near the plasma membrane.
Asunto(s)
Proteínas Portadoras/fisiología , AMP Cíclico/fisiología , Gránulos Citoplasmáticos/metabolismo , Exocitosis , Factores de Intercambio de Guanina Nucleótido/fisiología , Insulina/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/efectos de los fármacos , Exocitosis/efectos de los fármacos , Glucosa/farmacología , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Insulina/análisis , Secreción de Insulina , Ratones , Ratones Noqueados , Potasio/farmacología , Transducción de Señal , Proteínas de Unión al GTP rap1/análisisRESUMEN
The Rap1 small GTPase has been implicated in regulation of integrin-mediated leukocyte adhesion downstream of various chemokines and cytokines in many aspects of inflammatory and immune responses. However, the mechanism for Rap1 regulation in the adhesion signaling remains unclear. RA-GEF-2 is a member of the multiple-member family of guanine nucleotide exchange factors (GEFs) for Rap1 and characterized by the possession of a Ras/Rap1-associating domain, interacting with M-Ras-GTP as an effector, in addition to the GEF catalytic domain. Here, we show that RA-GEF-2 is specifically responsible for the activation of Rap1 that mediates tumor necrosis factor-alpha (TNF-alpha)-triggered integrin activation. In BAF3 hematopoietic cells, activated M-Ras potently induced lymphocyte function-associated antigen 1 (LFA-1)-mediated cell aggregation. This activation was totally abrogated by knockdown of RA-GEF-2 or Rap1. TNF-alpha treatment activated LFA-1 in a manner dependent on M-Ras, RA-GEF-2, and Rap1 and induced activation of M-Ras and Rap1 in the plasma membrane, which was accompanied by recruitment of RA-GEF-2. Finally, we demonstrated that M-Ras and RA-GEF-2 were indeed involved in TNF-alpha-stimulated and Rap1-mediated LFA-1 activation in splenocytes by using mice deficient in RA-GEF-2. These findings proved a crucial role of the cross-talk between two Ras-family GTPases M-Ras and Rap1, mediated by RA-GEF-2, in adhesion signaling.
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Factores de Intercambio de Guanina Nucleótido/metabolismo , Integrinas/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Bazo/citología , Factor de Necrosis Tumoral alfa/farmacología , Proteínas de Unión al GTP rap1/metabolismo , Proteínas ras/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Agregación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Marcación de Gen , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Ratones , Modelos Biológicos , Bazo/efectos de los fármacosRESUMEN
Insulin stimulates glucose uptake via the translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes. Several lines of evidence suggest that the small GTPase Rac1 plays an important role in insulin-stimulated glucose uptake in skeletal muscle and adipocytes. The purpose of this study is to investigate the mechanisms whereby Rac1 is regulated in adipocyte insulin signaling. Here, we show that knockdown of the guanine nucleotide exchange factor FLJ00068 inhibits Rac1 activation and GLUT4 translocation by insulin and a constitutively activated form of the protein kinase Akt2. Furthermore, constitutively activated FLJ00068 induced Rac1 activation and Rac1-dependent GLUT4 translocation. Collectively, these results suggest the involvement of FLJ00068 downstream of Akt2 in insulin-stimulated glucose uptake signaling in adipocytes.