RESUMEN
BACKGROUND: Metabolites of eicosapentaenoic acid exert various physiologic actions. 17,18-Epoxyeicosatetraenoic acid (17,18-EpETE) is a recently identified new class of antiallergic and anti-inflammatory lipid metabolite of eicosapentaenoic acid, but its effects on skin inflammation and the underlying mechanisms remain to be investigated. OBJECTIVE: We evaluated the effectiveness of 17,18-EpETE for control of contact hypersensitivity in mice and cynomolgus macaques. We further sought to reveal underlying mechanisms by identifying the responsible receptor and cellular target of 17,18-EpETE. METHODS: Contact hypersensitivity was induced by topical application of 2,4-dinitrofluorobenzene. Skin inflammation and immune cell populations were analyzed by using flow cytometric, immunohistologic, and quantitative RT-PCR analyses. Neutrophil mobility was examined by means of imaging analysis in vivo and neutrophil culture in vitro. The receptor for 17,18-EpETE was identified by using the TGF-α shedding assay, and the receptor's involvement in the anti-inflammatory effects of 17,18-EpETE was examined by using KO mice and specific inhibitor treatment. RESULTS: We found that preventive or therapeutic treatment with 17,18-EpETE ameliorated contact hypersensitivity by inhibiting neutrophil mobility in mice and cynomolgus macaques. 17,18-EpETE was recognized by G protein-coupled receptor (GPR) 40 (also known as free fatty acid receptor 1) and inhibited chemoattractant-induced Rac activation and pseudopod formation in neutrophils. Indeed, the antiallergic inflammatory effect of 17,18-EpETE was abolished in the absence or inhibition of GPR40. CONCLUSION: 17,18-EpETE inhibits neutrophil mobility through GPR40 activation, which is a potential therapeutic target to control allergic inflammatory diseases.
Asunto(s)
Antialérgicos/uso terapéutico , Antiinflamatorios/metabolismo , Ácidos Araquidónicos/metabolismo , Dermatitis por Contacto/tratamiento farmacológico , Neutrófilos/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Animales , Antialérgicos/farmacología , Ácidos Araquidónicos/farmacología , Ácidos Araquidónicos/uso terapéutico , Movimiento Celular , Células Cultivadas , Femenino , Macaca fascicularis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Seudópodos/patología , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Hepatosteatosis is associated with the development of both hepatic insulin resistance and Type 2 diabetes. Hepatic expression of Cd36, a fatty acid transporter, is enhanced in obese and diabetic murine models and human nonalcoholic fatty liver disease, and thus it correlates with hyperinsulinemia, steatosis, and insulin resistance. Here, we have explored the effect of hyperinsulinemia on hepatic Cd36 expression, development of hepatosteatosis, insulin resistance, and dysglycemia. A 3-week sucrose-enriched diet was sufficient to provoke hyperinsulinemia, hepatosteatosis, hepatic insulin resistance, and dysglycemia in CBA/J mice. The development of hepatic steatosis and insulin resistance in CBA/J mice on a sucrose-enriched diet was paralleled by increased hepatic expression of the transcription factor Pparγ and its target gene Cd36 whereas that of genes implicated in lipogenesis, fatty acid oxidation, and VLDL secretion was unaltered. Additionally, we demonstrate that insulin, in a Pparγ-dependent manner, is sufficient to directly increase Cd36 expression in perfused livers and isolated hepatocytes. Mouse strains that display low insulin levels, i.e. C57BL6/J, and/or lack hepatic Pparγ, i.e. C3H/HeN, do not develop hepatic steatosis, insulin resistance, or dysglycemia on a sucrose-enriched diet, suggesting that elevated insulin levels, via enhanced CD36 expression, provoke fatty liver development that in turn leads to hepatic insulin resistance and dysglycemia. Thus, our data provide evidence for a direct role for hyperinsulinemia in stimulating hepatic Cd36 expression and thus the development of hepatosteatosis, hepatic insulin resistance, and dysglycemia.
Asunto(s)
Antígenos CD36/metabolismo , Hígado Graso/metabolismo , Resistencia a la Insulina , Hígado/metabolismo , Animales , Antígenos CD36/genética , Hígado Graso/etiología , Células Hep G2 , Humanos , Insulina/fisiología , Masculino , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , PPAR gamma/metabolismoRESUMEN
Aims: Individuals of African ancestry (AA) present with lower insulin sensitivity compared to their European counterparts (EA). Studies show ethnic differences in skeletal muscle fiber type (lower type I fibers in AA), muscle fat oxidation capacity (lower in AA), whilst no differences in total skeletal muscle lipids. However, skeletal muscle lipid subtypes have not been examined in this context. We hypothesize that lower insulin sensitivity in AA is due to a greater proportion of type II (non-oxidative) muscle fibers, and that this would result in an ancestry-specific association between muscle lipid subtypes and peripheral insulin sensitivity. To test this hypothesis, we examined the association between insulin sensitivity and muscle lipids in AA and EA adults, and in an animal model of insulin resistance with muscle-specific fiber types. Methods: In this cross-sectional study, muscle biopsies were obtained from individuals with a BMI ranging from normal to overweight with AA (N = 24) and EA (N = 19). Ancestry was assigned via genetic admixture analysis; peripheral insulin sensitivity via hyperinsulinaemic-euglycemic clamp; and myofiber content via myosin heavy chain immunohistochemistry. Further, muscle types with high (soleus) and low (vastus lateralis) type I fiber content were obtained from high-fat diet-induced insulin resistant F1 mice and littermate controls. Insulin sensitivity in mice was assessed via intraperitoneal glucose tolerance test. Mass spectrometry (MS)-based lipidomics was used to measure skeletal muscle lipid. Results: Compared to EA, AA had lower peripheral insulin sensitivity and lower oxidative type 1 myofiber content, with no differences in total skeletal muscle lipid content. Muscles with lower type I fiber content (AA and vastus from mice) showed lower levels of lipids associated with fat oxidation capacity, i.e., cardiolipins, triacylglycerols with low saturation degree and phospholipids, compared to muscles with a higher type 1 fiber content (EA and soleus from mice). Further, we found that muscle diacylglycerol content was inversely associated with insulin sensitivity in EA, who have more type I fiber, whereas no association was found in AA. Similarly, we found that insulin sensitivity in mice was associated with diacylglycerol content in the soleus (high in type I fiber), not in vastus (low in type I fiber).Conclusions; Our data suggest that the lipid contribution to altered insulin sensitivity differs by ethnicity due to myofiber composition, and that this needs to be considered to increase our understanding of underlying mechanisms of altered insulin sensitivity in different ethnic populations.
RESUMEN
Background: Individuals with type 2 diabetes (T2D) have an increased risk of cognitive symptoms and Alzheimer's disease (AD). Mis-metabolism with aggregation of amyloid-ß peptides (Aß) play a key role in AD pathophysiology. Therefore, human studies on Aß metabolism and T2D are warranted. Objective: The objective of this study was to examine whether acute hyperglycemia affects plasma Aß1-40 and Aß1-42 concentrations in individuals with T2D and matched controls. Methods: Ten participants with T2D and 11 controls (median age, 69 years; range, 66-72 years) underwent hyperglycemic clamp and placebo clamp (saline infusion) in a randomized order, each lasting 4 hours. Aß1-40, Aß1-42, and insulin-degrading enzyme (IDE) plasma concentrations were measured in blood samples taken at 0 and 4âhours of each clamp. Linear mixed-effect regression models were used to evaluate the 4-hour changes in Aß1-40 and Aß1-42 concentrations, adjusting for body mass index, estimated glomerular filtration rate, and 4-hour change in insulin concentration. Results: At baseline, Aß1-40 and Aß1-42 concentrations did not differ between the two groups. During the hyperglycemic clamp, Aß decreased in the control group, compared to the placebo clamp (Aß1-40: pâ=â0.034, Aß1-42: pâ=â0.020), IDE increased (pâ=â0.016) during the hyperglycemic clamp, whereas no significant changes in either Aß or IDE was noted in the T2D group. Conclusions: Clamp-induced hyperglycemia was associated with increased IDE levels and enhanced Aß40 and Aß42 clearance in controls, but not in individuals with T2D. We hypothesize that insulin-degrading enzyme was inhibited during hyperglycemic conditions in people with T2D.
Asunto(s)
Péptidos beta-Amiloides , Diabetes Mellitus Tipo 2 , Técnica de Clampeo de la Glucosa , Hiperglucemia , Fragmentos de Péptidos , Humanos , Péptidos beta-Amiloides/sangre , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/sangre , Anciano , Masculino , Hiperglucemia/sangre , Femenino , Fragmentos de Péptidos/sangre , Glucemia/metabolismo , Insulisina/metabolismoRESUMEN
Age is associated with progressively impaired, metabolic, cardiac and vascular function, as well as reduced work/exercise capacity, mobility, and hence quality of life. Exercise exhibit positive effects on age-related dysfunctions and diseases. However, for a variety of reasons many aged individuals are unable to engage in regular physical activity, making the development of pharmacological treatments that mimics the beneficial effects of exercise highly desirable. Here we show that the pan-AMPK activator O304, which is well tolerated in humans, prevented and reverted age-associated hyperinsulinemia and insulin resistance, and improved cardiac function and exercise capacity in aged mice. These results provide preclinical evidence that O304 mimics the beneficial effects of exercise. Thus, as an exercise mimetic in clinical development, AMPK activator O304 holds great potential to mitigate metabolic dysfunction, and to improve cardiac function and exercise capacity, and hence quality of life in aged individuals.
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Proteínas Quinasas Activadas por AMP/genética , Tolerancia al Ejercicio/genética , Corazón/fisiología , Resistencia a la Insulina/genética , Ratones/fisiología , Proteínas Quinasas Activadas por AMP/metabolismo , Factores de Edad , Animales , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones/genética , Ratones/metabolismo , Condicionamiento Físico AnimalRESUMEN
AMP-activated protein kinase (AMPK) has an important role in cellular energy homeostasis and has emerged as a promising target for treatment of Type 2 Diabetes (T2D) due to its beneficial effects on insulin sensitivity and glucose homeostasis. O304 is a pan-AMPK activator that has been shown to improve glucose homeostasis in both mouse models of diabetes and in human T2D subjects. Here, we describe the genome-wide transcriptional profile and chromatin landscape of pancreatic islets following O304 treatment of mice fed high-fat diet (HFD). O304 largely prevented genome-wide gene expression changes associated with HFD feeding in CBA mice and these changes were associated with remodelling of active and repressive chromatin marks. In particular, the increased expression of the ß-cell stress marker Aldh1a3 in islets from HFD-mice is completely abrogated following O304 treatment, which is accompanied by loss of active chromatin marks in the promoter as well as distant non-coding regions upstream of the Aldh1a3 gene. Moreover, O304 treatment restored dysfunctional glucose homeostasis as well as expression of key markers associated with ß-cell function in mice with already established obesity. Our findings provide preclinical evidence that O304 is a promising therapeutic compound not only for T2D remission but also for restoration of ß-cell function following remission of T2D diabetes.
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Proteínas Quinasas Activadas por AMP/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Dieta Alta en Grasa/efectos adversos , Activadores de Enzimas/farmacología , Expresión Génica/efectos de los fármacos , Código de Histonas/efectos de los fármacos , Código de Histonas/genética , Histonas/metabolismo , Islotes Pancreáticos/metabolismo , Obesidad/metabolismo , Tiadiazoles/farmacología , Proteínas Quinasas Activadas por AMP/fisiología , Familia de Aldehído Deshidrogenasa 1/genética , Familia de Aldehído Deshidrogenasa 1/metabolismo , Animales , Diabetes Mellitus Tipo 2/fisiopatología , Modelos Animales de Enfermedad , Glucosa/metabolismo , Homeostasis/efectos de los fármacos , Células Secretoras de Insulina/fisiología , Ratones , Ratones Endogámicos CBA , Obesidad/etiología , Retinal-Deshidrogenasa/genética , Retinal-Deshidrogenasa/metabolismoRESUMEN
Obesity is typically associated with elevated levels of free fatty acids (FFAs) and is linked to glucose intolerance and type 2 diabetes. FFAs exert divergent effects on insulin secretion from beta cells: acute exposure to FFAs stimulates insulin secretion, whereas chronic exposure impairs insulin secretion. The G protein-coupled receptor GPR40 is selectively expressed in beta cells and is activated by FFAs. We show here that GPR40 mediates both acute and chronic effects of FFAs on insulin secretion and that GPR40 signaling is linked to impaired glucose homeostasis. GPR40-deficient beta cells secrete less insulin in response to FFAs, and loss of GPR40 protects mice from obesity-induced hyperinsulinemia, hepatic steatosis, hypertriglyceridemia, increased hepatic glucose output, hyperglycemia, and glucose intolerance. Conversely, overexpression of GPR40 in beta cells of mice leads to impaired beta cell function, hypoinsulinemia, and diabetes. These results suggest that GPR40 plays an important role in the chain of events linking obesity and type 2 diabetes.
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Diabetes Mellitus Tipo 2/metabolismo , Hígado Graso/metabolismo , Glucosa/fisiología , Homeostasis/fisiología , Hiperinsulinismo/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatología , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/metabolismo , Hipertrigliceridemia/genética , Hipertrigliceridemia/metabolismo , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Ratones , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Type 2 diabetes (T2D), alike Parkinson's disease (PD), belongs to the group of protein misfolding diseases (PMDs), which share aggregation of misfolded proteins as a hallmark. Although the major aggregating peptide in ß-cells of T2D patients is Islet Amyloid Polypeptide (IAPP), alpha-synuclein (αSyn), the aggregating peptide in substantia nigra neurons of PD patients, is expressed also in ß-cells. Here we show that αSyn, encoded by Snca, is a component of amyloid extracted from pancreas of transgenic mice overexpressing human IAPP (denoted hIAPPtg mice) and from islets of T2D individuals. Notably, αSyn dose-dependently promoted IAPP fibril formation in vitro and tail-vein injection of αSyn in hIAPPtg mice enhanced ß-cell amyloid formation in vivo whereas ß-cell amyloid formation was reduced in hIAPPtg mice on a Snca -/- background. Taken together, our findings provide evidence that αSyn and IAPP co-aggregate both in vitro and in vivo, suggesting a role for αSyn in ß-cell amyloid formation.
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Amiloide/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/genética , alfa-Sinucleína/genética , Animales , Diabetes Mellitus Tipo 2/genética , Modelos Animales de Enfermedad , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Ratones , Ratones Transgénicos , Agregado de Proteínas , alfa-Sinucleína/metabolismoRESUMEN
AMPK activated protein kinase (AMPK), a master regulator of energy homeostasis, is activated in response to an energy shortage imposed by physical activity and caloric restriction. We here report on the identification of PAN-AMPK activator O304, which - in diet-induced obese mice - increased glucose uptake in skeletal muscle, reduced ß cell stress, and promoted ß cell rest. Accordingly, O304 reduced fasting plasma glucose levels and homeostasis model assessment of insulin resistance (HOMA-IR) in a proof-of-concept phase IIa clinical trial in type 2 diabetes (T2D) patients on Metformin. T2D is associated with devastating micro- and macrovascular complications, and O304 improved peripheral microvascular perfusion and reduced blood pressure both in animals and T2D patients. Moreover, like exercise, O304 activated AMPK in the heart, increased cardiac glucose uptake, reduced cardiac glycogen levels, and improved left ventricular stroke volume in mice, but it did not increase heart weight in mice or rats. Thus, O304 exhibits a great potential as a novel drug to treat T2D and associated cardiovascular complications.
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Proteínas Quinasas Activadas por AMP/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Compuestos Heterocíclicos/farmacología , Homeostasis , Animales , Glucemia/efectos de los fármacos , Presión Sanguínea , Cardiomegalia , Enfermedades Cardiovasculares , Glucógeno/metabolismo , Corazón , Holoprosencefalia/prevención & control , Humanos , Resistencia a la Insulina , Células Secretoras de Insulina , Anomalías Maxilomandibulares/prevención & control , Metformina/uso terapéutico , Ratones , Ratones Obesos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Ratas , Volumen SistólicoRESUMEN
The insulin-degrading enzyme (IDE) degrades amyloidogenic proteins such as Amyloid ß (Αß) and Islet Amyloid Polypeptide (IAPP), i.e. peptides associated with Alzheimer's disease and type 2 diabetes, respectively. In addition to the protease activity normally associated with IDE function an additional activity involving the formation of stable, irreversible complexes with both Αß and α-synuclein, an amyloidogenic protein involved in Parkinson's disease, was recently proposed. Here, we have investigated the functional consequences of IDE-α-synuclein interactions in vitro. We demonstrate that IDE in a nonproteolytic manner and at sub-stoichiometric ratios efficiently inhibits α-synuclein fibril formation by binding to α-synuclein oligomers making them inert to amyloid formation. Moreover, we show that, within a defined range of α-synuclein concentrations, interaction with α-synuclein oligomers increases IDE's proteolytic activity on a fluorogenic substrate. We propose that the outcomes of IDE-α-synuclein interactions, i.e. protection against α-synuclein amyloid formation and stimulated IDE protease activity, may be protective in vivo.
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Insulisina/química , alfa-Sinucleína/química , Amiloide/química , Benzotiazoles , Calorimetría/métodos , Microscopía de Fuerza Atómica , Multimerización de Proteína , Tiazoles/químicaRESUMEN
Genome-wide association studies have identified several type 2 diabetes (T2D) risk loci linked to impaired ß-cell function. The identity and function of the causal genes in these susceptibility loci remain, however, elusive. The HHEX/IDE T2D locus is associated with decreased insulin secretion in response to oral glucose stimulation in humans. Here we have assessed ß-cell function in Ide knockout (KO) mice. We find that glucose-stimulated insulin secretion (GSIS) is decreased in Ide KO mice due to impaired replenishment of the releasable pool of granules and that the Ide gene is haploinsufficient. We also show that autophagic flux and microtubule content are reduced in ß-cells of Ide KO mice. One important cellular role for IDE involves the neutralization of amyloidogenic proteins, and we find that α-synuclein and IDE levels are inversely correlated in ß-cells of Ide KO mice and T2D patients. Moreover, we provide evidence that both gain and loss of function of α-synuclein in ß-cells in vivo impair not only GSIS but also autophagy. Together, these data identify the Ide gene as a regulator of GSIS, suggest a molecular mechanism for ß-cell degeneration as a consequence of Ide deficiency, and corroborate and extend a previously established important role for α-synuclein in ß-cell function.
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Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Insulisina/metabolismo , alfa-Sinucleína/metabolismo , Animales , Western Blotting , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Humanos , Inmunohistoquímica , Técnicas In Vitro , Insulisina/genética , Ratones , Ratones Noqueados , alfa-Sinucleína/genéticaRESUMEN
OBJECTIVE: The G-protein-coupled receptor Gpr40 is expressed in beta-cells where it contributes to free fatty acid (FFA) enhancement of glucose-stimulated insulin secretion. However, other sites of Gpr40 expression, including the intestine, have been suggested. The transcription factor IPF1/PDX1 was recently shown to bind to an enhancer element within the 5'-flanking region of Gpr40, implying that IPF1/PDX1 might regulate Gpr40 expression. Here, we addressed whether 1) Gpr40 is expressed in the intestine and 2) Ipf1/Pdx1 function is required for Gpr40 expression. RESEARCH DESIGN AND METHODS: In the present study, Gpr40 expression was monitored by X-gal staining using Gpr40 reporter mice and by in situ hybridization. Ipf1/Pdx1-null and beta-cell specific mutants were used to investigate whether Ipf1/Pdx1 controls Gpr40 expression. Plasma insulin, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and glucose levels in response to acute oral fat diet were determined in Gpr40 mutant and control mice. RESULTS: Here, we show that Gpr40 is expressed in endocrine cells of the gastrointestinal tract, including cells expressing the incretin hormones GLP-1 and GIP, and that Gpr40 mediates FFA-stimulated incretin secretion. We also show that Ipf1/Pdx1 is required for expression of Gpr40 in beta-cells and endocrine cells of the anterior gastrointestinal tract. CONCLUSIONS: Together, our data provide evidence that Gpr40 modulates FFA-stimulated insulin secretion from beta-cells not only directly but also indirectly via regulation of incretin secretion. Moreover, our data suggest a conserved role for Ipf1/Pdx1 and Gpr40 in FFA-mediated secretion of hormones that regulate glucose and overall energy homeostasis.
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Células Enteroendocrinas/fisiología , Ácidos Grasos no Esterificados/metabolismo , Polipéptido Inhibidor Gástrico/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Animales , Duodeno/citología , Duodeno/fisiología , Células Enteroendocrinas/metabolismo , Expresión Génica/fisiología , Genes Reporteros , Proteínas de Homeodominio/metabolismo , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Ratones , Ratones Mutantes , Píloro/citología , Píloro/fisiología , Transactivadores/metabolismoRESUMEN
Oxygen delivery in many animals is enabled by the formation of unicellular capillary tubes that penetrate target tissues to facilitate gas exchange. We show that the tortuous outgrowth of tracheal unicellular branches towards their target tissues is controlled by complex local interactions with target cells. Slit, a phylogenetically conserved axonal guidance signal, is expressed in several tracheal targets and is required both for attraction and repulsion of tracheal branches. Robo and Robo2 are expressed in different branches, and are both necessary for the correct orientation of branch outgrowth. At the CNS midline, Slit functions as a repellent for tracheal branches and this function is mediated primarily by Robo. Robo2 is necessary for the tracheal response to the attractive Slit signal and its function is antagonized by Robo. We propose that the attractive and repulsive tracheal responses to Slit are mediated by different combinations of Robo and Robo2 receptors on the cell surface.
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Proteínas de Drosophila , Drosophila/embriología , Drosophila/fisiología , Proteínas del Tejido Nervioso/fisiología , Tráquea/embriología , Tráquea/fisiología , Animales , Animales Modificados Genéticamente , Sistema Nervioso Central/embriología , Drosophila/genética , Ganglios/embriología , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Operón Lac , Proteínas del Tejido Nervioso/genética , Neuroglía/citología , Fenotipo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología , Receptores Inmunológicos/genética , Receptores Inmunológicos/fisiología , Transducción de Señal , Proteínas RoundaboutRESUMEN
Slit proteins steer the migration of many cell types through their binding to Robo receptors, but how Robo controls cell motility is not clear. We describe the functional analysis of vilse, a Drosophila gene required for Robo repulsion in epithelial cells and axons. Vilse defines a conserved family of RhoGAPs (Rho GTPase-activating proteins), with representatives in flies and vertebrates. The phenotypes of vilse mutants resemble the tracheal and axonal phenotypes of Slit and Robo mutants at the CNS midline. Dosage-sensitive genetic interactions between vilse, slit, and robo mutants suggest that vilse is a component of robo signaling. Moreover, overexpression of Vilse in the trachea of robo mutants ameliorates the phenotypes of robo, indicating that Vilse acts downstream of Robo to mediate midline repulsion. Vilse and its human homolog bind directly to the intracellular domains of the corresponding Robo receptors and promote the hydrolysis of RacGTP and, less efficiently, of Cdc42GTP. These results together with genetic interaction experiments with robo, vilse, and rac mutants suggest a mechanism whereby Robo repulsion is mediated by the localized inactivation of Rac through Vilse.