Regulación de las gotas lipídicas en adipocitos: papel de RAB 18 y caracterización de su interactoma

El tejido adiposo (TA) juega un papel fundamental en la gestión de las reservas energéticas y como órgano endocrino productor de adipoquinas, que regulan la ingesta y la homeostasis energética, la inmunidad y la función cardiovascular (Frayn et al., 2013; Frühbeck et al., 2008). El exceso de TA en obesidad está comúnmente asociado a resistencia a insulina (IR), lo que constituye un factor de riesgo principal en el desarrollo de diabetes tipo 2, hipertensión, dislipemia y enfermedad cardiovascular, o cáncer (Kahn y Flier, 2000; Yusuf et al., 2005; Pischon et al., 2008). Los adipocitos constituyen el principal componente celular del TA, que también incluye la fracción de estroma vascular constituida por preadipocitos, células sanguíneas y tejido vascular (Frayn et al., 2003). Los adipocitos acumulan energía en forma de triglicéridos (TAGs) en el interior de la gran gota lipídica (LD) característica de este tipo celular (Reue, 2011; Walther y Farese, 2012). En ayuno, y bajo la influencia de las catecolaminas, los TAGs son hidrolizados por lipasas, liberando ácidos grasos que son transportados para su uso en otros tejidos como fuente de energía (Duncan et al., 2007; Kolditz y Langin, 2010). Por otra parte, en postprandio, los ácidos grasos procedentes de la dieta son internalizados por los adipocitos, esterificados y almacenados en las LDs. Este proceso está regulado por la insulina, que estimula la captación de ácidos grasos y de glucosa (utilizada para la producción del glicerol necesario para la síntesis de TAGs), y la expresión de enzimas lipogénicas (Kersten, 2001; Stahl et al., 2002; Ducharme y Bickel, 2008). En obesidad, se produce estrés oxidativo y del retículo endoplásmico (RE) en adipocitos, procesos que generan hipoxia y estrés mecánico como consecuencia del incremento del tamaño de los adipocitos, y la activación de rutas pro-inflamatorias, como JNK y NFκB (Hotamisligil, 2010; Bluher, 2013; Trayhurn, 2013). Esto provoca la liberación de factores pro-inflamatorios y quimioquinas (TNF-α, IL-6, MCP-1) que promueven la infiltración del TA por macrófagos y linfocitos, dando lugar al desarrollo del estado de inflamación crónica de bajo grado asociado a la obesidad (Guilherme et al., 2008; Ouchi et al., 2011; Mathis, 2013). Todos estos insultos bloquean la diferenciación de los preadipocitos a adipocitos (adipogénesis) e interfieren en la señalización de la insulina, provocando IR, así como la desregulación del almacenamiento de los TAGs y, en última instancia, la muerte celular (Guilherme et al., 2008; Ouchi et al., 2011; Mathis, 2013). En resumen, la regulación del metabolismo lipídico en los adipocitos es fundamental para el funcionamiento normal del TA y para...The adipose tissue (AT) plays a central role in managing the energy stores and as an endocrine organ by producing adipokines that regulate food intake and energy homeostasis, immunity and cardiovascular function (Frayn et al., 2003; Frühbeck, 2008). The excess in AT that occurs in obesity is commonly associated to insulin resistance (IR), which constitutes the main risk factor in the development of type 2 diabetes, hypertension, dyslipidemia and cardiovascular diseases (Kahn & Flier, 2000; Yusuf et al., 2005; Pischon et al., 2008). Adipocytes represent the main cellular constituent of AT, which also includes the so-called stroma-vascular fraction formed by preadipocytes, blood cells, and vascular tissue (Frayn et al., 2003). Adipocytes store energy in form of triglycerides (TAGs) within a large lipid droplet (LD) characteristic of this cell type (Reue, 2011; Walther & Farese, 2012). During fasting and under the influence of catecholamines, TAGs are hydrolyzed by lipases, releasing fatty acids (FAs) that are transported to other tissues for useas energy source (Duncan et al., 2007; Kolditz & Langin, 2010). On the other hand, in postprandio FAs from the diet are internalized within adipocytes, esterified and stored in LDs. This process is regulated by insulin, which stimulates uptake of FAs and glucose (used for production of glycerol needed for TAG synthesis), and the expression of lipogenic enzymes (Kersten, 2001; Stahl et al., 2002; Ducharme & Bickel, 2008). In obesity, oxidative stress and endoplasmic reticulum (ER) stress accumulate in adipocytes, processes that generate hypoxia and mechanical stress as a consequence of adipocyte enlargement, and activate pro-inflammatory pathways, such as JNK and NFkB (Hotamisligil, 2010; Bluher, 2013; Trayhurn, 2013). This provokes release of proinflammatory factors and chemokines (TNFα, IL-6, MCP-1) that promote macrophage and lymphocyte infiltration within the AT, leading to the low-grade, chronic inflammatory state associated to obesity (Guilherme et al., 2008; Ouchi et al., 2011; Mathis, 2013). Altogether, these insults block differentiation of preadipoytes into adipocytes (adipogenesis) and impair insulin signaling, leading to IR, as well as deregulation of TAG storing and, ultimately, cell death (Guilherme et al., 2008; Ouchi et al., 2011; Mathis, 2013). In summary, lipid metabolism regulation in adipocytes is..

mTORC1 activity is supported by spatial association with focal adhesions

The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery. Here, we show that translocation of lysosomes toward the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, contribute to both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli

Genomic targets and selective inhibition of DNA methyltransferase isoforms

Background: DNA methylation in the human genome is established and maintained by DNA methyltransferases (DNMTs). DNMT isoforms show differential expression by cell lineage and during development, but much remains to be elucidated about their shared and unique genomic targets. Results: We examined changes in the epigenome following overexpression of 13 DNMT isoforms in HEK293T cells. We observed increased methylation (Δβ > 0.2) at 43,405 CpG sites, with expression of DNMT3A2, DNMTΔ3B4 and DNMTΔ3B2 associated with the greatest impact. De novo methylation occurred primarily within open sea regions and at loci with intermediate methylation levels (β: 0.2-0.6). 53% of differentially methylated loci showed specificity towards a single DNMT subfamily, primarily DNMTΔ3B and DNMT3A and 39% towards a single isoform. These loci were significantly enriched for pathways related to neuronal development (DNMTΔ3B4), calcium homeostasis (DNMTΔ3B3) and ion transport (DNMT3L). Repetitive elements did not display differential sensitivity to overexpressed DNMTs, but hypermethylation of Alu elements was associated with their evolutionary age following overexpression of DNMT3A2, DNMT3B1, DNMT3B2 and DNMT3L. Differential methylation (Δβ > 0.1) was observed at 121 of the 353 loci associated with the Horvath 'epigenetic clock' model of ageing, with 51 showing isoform specificity, and was associated with reduction of epigenetic age by 5-15 years following overexpression of seven isoforms. Finally, we demonstrate the potential for dietary constituents to modify epigenetic marks through isoform-specific inhibition of methylation activity. Conclusions: Our results provide insight into regions of the genome methylated uniquely by specific DNMT isoforms and demonstrate the potential for dietary intervention to modify the epigenome

The caveolae‐associated coiled‐coil protein, NECC2, regulates insulin signalling in Adipocytes

Adipocyte dysfunction in obesity is commonly associated with impaired insulin signalling in adipocytes and insulin resistance. Insulin signalling has been associated with caveolae, which are coated by large complexes of caveolin and cavin proteins, along with proteins with membrane‐binding and remodelling properties. Here, we analysed the regulation and function of a component of caveolae involved in growth factor signalling in neuroendocrine cells, neuroendocrine long coiled‐coil protein‐2 (NECC2), in adipocytes. Studies in 3T3‐L1 cells showed that NECC2 expression increased during adipogenesis. Furthermore, NECC2 co‐immunoprecipitated with caveolin‐1 (CAV1) and exhibited a distribution pattern similar to that of the components of adipocyte caveolae, CAV1, Cavin1, the insulin receptor and cortical actin. Interestingly, NECC2 overexpression enhanced insulin‐activated Akt phosphorylation, whereas NECC2 downregulation impaired insulin‐induced phosphorylation of Akt and ERK2. Finally, an up‐regulation of NECC2 in subcutaneous and omental adipose tissue was found in association with human obesity and insulin resistance. This effect was also observed in 3T3‐L1 adipocytes exposed to hyperglycaemia/hyperinsulinemia. Overall, the present study identifies NECC2 as a component of adipocyte caveolae that is regulated in response to obesity and associated metabolic complications, and supports the contribution of this protein as a molecular scaffold modulating insulin signal transduction at these membrane microdomains.La disfunción de los adipocitos en la obesidad se asocia comúnmente con la alteración de la señalización de la insulina en los adipocitos y la resistencia a la insulina. La señalización de la insulina se ha asociado con las caveolas, que están recubiertas por grandes complejos de proteínas de caveolina y cavina, junto con proteínas con propiedades de remodelación y unión a la membrana. Aquí, analizamos la regulación y la función de un componente de las caveolas involucrado en la señalización del factor de crecimiento en las células neuroendocrinas, la proteína 2 neuroendocrina de espiral larga (NECC 2 ) , en los adipocitos. Los estudios en células 3T3‐L1 mostraron que la expresión de NECC 2 aumentó durante la adipogénesis. Además, NECC 2 co‐inmunoprecipitado con caveolina‐1 ( CAV1) y mostró un patrón de distribución similar al de los componentes de las caveolas adipocitarias, CAV 1, Cavin1, el receptor de insulina y la actina cortical. Curiosamente, la sobreexpresión de NECC 2 mejoró la fosforilación de Akt activada por insulina, mientras que la regulación negativa de NECC 2 perjudicó la fosforilación de Akt y ERK 2 inducida por insulina . resistencia a la insulina. Este efecto también se observó en adipocitos 3T3‐L1 expuestos a hiperglucemia/hiperinsulinemia. En general, el presente estudio identifica NECC2 como un componente de las caveolas de los adipocitos que se regula en respuesta a la obesidad y las complicaciones metabólicas asociadas, y respalda la contribución de esta proteína como un andamio molecular que modula la transducción de señales de insulina en estos microdominios de membrana

Adipose tissue depot-specific intracellular and extracellular cues contributing to insulin resistance in obese individuals.

Adipose tissue dysregulation in obesity strongly influences systemic metabolic homeostasis and is often linked to insulin resistance (IR). However, the molecular mechanisms underlying adipose tissue dysfunction in obesity are not fully understood. Herein, a proteomic analysis of subcutaneous (SC) and omental (OM) fat from lean subjects and obese individuals with different degrees of insulin sensitivity was performed to identify adipose tissue biomarkers related to obesity-associated metabolic disease. Our results suggest that dysregulation of both adipose tissue extracellular matrix (ECM) organization and intracellular trafficking processes may be associated with IR in obesity. Thus, abnormal accumulation of the small leucine-rich proteoglycan, lumican, as observed in SC fat of IR obese individuals, modifies collagen I organization, impairs adipogenesis and activates stress processes [endoplasmic reticulum and oxidative stress] in adipocytes. In OM fat, IR is associated with increased levels of the negative regulator of the Rab family of small GTPases, GDI2, which alters lipid storage in adipocytes by inhibiting insulin-stimulated binding of the Rab protein, Rab18, to lipid droplets. Together, these results indicate that lumican and GDI2 might play depot-dependent, pathogenic roles in obesity-associated IR. Our findings provide novel insights into the differential maladaptive responses of SC and OM adipose tissue linking obesity to IR

A comprehensive systematic review of CSF proteins and peptides that defne Alzheimer’s disease

Background: During the last two decades, over 100 proteomics studies have identifed a variety of potential bio‑ markers in CSF of Alzheimer’s (AD) patients. Although several reviews have proposed specifc biomarkers, to date, the statistical relevance of these proteins has not been investigated and no peptidomic analyses have been generated on the basis of specifc up- or down- regulation. Herein, we perform an analysis of all unbiased explorative proteom‑ ics studies of CSF biomarkers in AD to critically evaluate whether proteins and peptides identifed in each study are consistent in distribution; direction change; and signifcance, which would strengthen their potential use in studies of AD pathology and progression. Methods: We generated a database containing all CSF proteins whose levels are known to be signifcantly altered in human AD from 47 independent, validated, proteomics studies. Using this database, which contains 2022 AD and 2562 control human samples, we examined whether each protein is consistently present on the basis of reliable statistical studies; and if so, whether it is over- or under-represented in AD. Additionally, we performed a direct analysis of available mass spectrometric data of these proteins to generate an AD CSF peptide database with 3221 peptides for further analysis. Results: Of the 162 proteins that were identifed in 2 or more studies, we investigated their enrichment or depletion in AD CSF. This allowed us to identify 23 proteins which were increased and 50 proteins which were decreased in AD, some of which have never been revealed as consistent AD biomarkers (i.e. SPRC or MUC18). Regarding the analysis of the tryptic peptide database, we identifed 87 peptides corresponding to 13 proteins as the most highly consistently altered peptides in AD. Analysis of tryptic peptide fngerprinting revealed specifc peptides encoded by CH3L1, VGF, SCG2, PCSK1N, FBLN3 and APOC2 with the highest probability of detection in AD. Conclusions: Our study reveals a panel of 27 proteins and 21 peptides highly altered in AD with consistent statistical signifcance; this panel constitutes a potent tool for the classifcation and diagnosis of AD

The long coiled-coil protein NECC2 is associated to caveolae and modulates NGF/TrkA signaling in PC12 cells [corrected].

TrkA-mediated NGF signaling in PC12 cells has been shown to be compartimentalized in specialized microdomains of the plasma membrane, the caveolae, which are organized by scaffold proteins including the member of the caveolin family of proteins, caveolin-1. Here, we characterize the intracellular distribution as well as the biochemical and functional properties of the neuroendocrine long coiled-coil protein 2 (NECC2), a novel long coiled-coil protein selectively expressed in neuroendocrine tissues that contains a predicted caveolin-binding domain and displays structural characteristics of a scaffolding factor. NECC2 distributes in caveolae, wherein it colocalizes with the TrkA receptor, and behaves as a caveolae-associated protein in neuroendocrine PC12 cells. In addition, stimulation of PC12 cells with nerve growth factor (NGF) increased the expression and regulated the distribution of NECC2. Interestingly, knockdown as well as overexpression of NECC2 resulted in a reduction of NGF-induced phosphorylation of the TrkA downstream effector extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) but not of Akt. Altogether, our results identify NECC2 as a novel component of caveolae in PC12 cells and support the contribution of this protein in the maintenance of TrkA-mediated NGF signaling.journal articleresearch support, non-u.s. gov't20132013 09 06importe

Spatial and Temporal Protein Modules Signatures Associated with Alzheimer Disease in 3xTg-AD Mice Are Restored by Early Ubiquinol Supplementation

Despite its robust proteopathic nature, the spatiotemporal signature of disrupted protein modules in sporadic Alzheimer's disease (AD) brains remains poorly understood. This considered oxidative stress contributes to AD progression and early intervention with coenzyme Q10 or its reduced form, ubiquinol, delays the progression of the disease. Using MALDI-MSI and functional bioinformatic analysis, we have developed a protocol to express how deregulated protein modules arise from hippocampus and cortex in the AD mice model 3xTG-AD in an age-dependent manner. This strategy allowed us to identify which modules can be efficiently restored to a non-pathological condition by early intervention with ubiquinol. Indeed, an early deregulation of proteostasis-related protein modules, oxidative stress and metabolism has been observed in the hippocampus of 6-month mice (early AD) and the mirrored in cortical regions of 12-month mice (middle/late AD). This observation has been validated by IHC using mouse and human brain sections, suggesting that these protein modules are also affected in humans. The emergence of disrupted protein modules with AD signature can be prevented by early dietary intervention with ubiquinol in the 3xTG-AD mice model.A pesar de su robusta naturaleza proteopática, la firma espaciotemporal de los módulos de proteínas interrumpidos en los cerebros de la enfermedad de Alzheimer (EA) esporádica sigue siendo poco conocida. Este considerado estrés oxidativo contribuye a la progresión de la EA y la intervención precoz con coenzima Q10 o su forma reducida, el ubiquinol, retrasa la progresión de la enfermedad. Usando MALDI-MSI y análisis bioinformático funcional, hemos desarrollado un protocolo para expresar cómo surgen módulos de proteína desregulados del hipocampo y la corteza en el modelo de ratones AD 3xTG-AD de una manera dependiente de la edad. Esta estrategia nos permitió identificar qué módulos se pueden restaurar de manera eficiente a una condición no patológica mediante una intervención temprana con ubiquinol. De hecho, una desregulación temprana de los módulos proteicos relacionados con la proteostasis, Se ha observado estrés oxidativo y metabolismo en el hipocampo de ratones de 6 meses (EA temprana) y se refleja en regiones corticales de ratones de 12 meses (EA media/tardía). Esta observación ha sido validada por IHC utilizando secciones de cerebro humano y de ratón, lo que sugiere que estos módulos de proteína también se ven afectados en humanos. La aparición de módulos de proteínas interrumpidos con la firma AD puede prevenirse mediante una intervención dietética temprana con ubiquinol en el modelo de ratones 3xTG-AD

mTORC1 and Nutrient Homeostasis: The Central Role of the Lysosome

The mechanistic target of rapamycin complex 1 (mTORC1) coordinates cellular growth and metabolism with environmental inputs to ensure that cells grow only under favourable conditions. When active, mTORC1 stimulates biosynthetic pathways including protein, lipid and nucleotide synthesis and inhibits cellular catabolism through repression of the autophagic pathway, thereby promoting cell growth and proliferation. The recruitment of mTORC1 to the lysosomal surface has been shown to be essential for its activation. This finding has significantly enhanced our knowledge of mTORC1 regulation and has focused the attention of the field on the lysosome as a signalling hub which coordinates several homeostatic pathways. The intriguing localisation of mTORC1 to the cellular organelle that plays a crucial role in catabolism enables mTORC1 to feedback to autophagy and lysosomal biogenesis, thus leading mTORC1 to enact precise spatial and temporal control of cell growth. This review will cover the signalling interactions which take place on the surface of lysosomes and the cross-talk which exists between mTORC1 activity and lysosomal function

mTORC1 and Nutrient Homeostasis: The Central Role of the Lysosome

The mechanistic target of rapamycin complex 1 (mTORC1) coordinates cellular growth and metabolism with environmental inputs to ensure that cells grow only under favourable conditions. When active, mTORC1 stimulates biosynthetic pathways including protein, lipid and nucleotide synthesis and inhibits cellular catabolism through repression of the autophagic pathway, thereby promoting cell growth and proliferation. The recruitment of mTORC1 to the lysosomal surface has been shown to be essential for its activation. This finding has significantly enhanced our knowledge of mTORC1 regulation and has focused the attention of the field on the lysosome as a signalling hub which coordinates several homeostatic pathways. The intriguing localisation of mTORC1 to the cellular organelle that plays a crucial role in catabolism enables mTORC1 to feedback to autophagy and lysosomal biogenesis, thus leading mTORC1 to enact precise spatial and temporal control of cell growth. This review will cover the signalling interactions which take place on the surface of lysosomes and the cross-talk which exists between mTORC1 activity and lysosomal function