Localization, traffic and function of Rab34 in adipocyte lipid and endocrine functions.

BACKGROUND: Excessive lipid accumulation in the adipose tissue in obesity alters the endocrine and energy storage functions of adipocytes. Adipocyte lipid droplets represent key organelles coordinating lipid storage and mobilization in these cells. Recently, we identified the small GTPase, Rab34, in the lipid droplet proteome of adipocytes. Herein, we have characterized the distribution, intracellular transport, and potential contribution of this GTPase to adipocyte physiology and its regulation in obesity. METHODS: 3T3-L1 and human primary preadipocytes were differentiated in vitro and Rab34 distribution and trafficking were analyzed using markers of cellular compartments. 3T3-L1 adipocytes were transfected with expression vectors and/or Rab34 siRNA and assessed for secretory activity, lipid accumulation and expression of proteins regulating lipid metabolism. Proteomic and protein interaction analyses were employed for the identification of the Rab34 interactome. These studies were combined with functional analysis to unveil the role played by the GTPase in adipocytes, with a focus on the actions conveyed by Rab34 interacting proteins. Finally, Rab34 regulation in response to obesity was also evaluated. RESULTS: Our results show that Rab34 localizes at the Golgi apparatus in preadipocytes. During lipid droplet biogenesis, Rab34 translocates from the Golgi to endoplasmic reticulum-related compartments and then reaches the surface of adipocyte lipid droplets. Rab34 exerts distinct functions related to its intracellular location. Thus, at the Golgi, Rab34 regulates cisternae integrity as well as adiponectin trafficking and oligomerization. At the lipid droplets, this GTPase controls lipid accumulation and lipolysis through its interaction with the E1-ubiquitin ligase, UBA1, which induces the ubiquitination and proteasomal degradation of the fatty acid transporter and member of Rab34 interactome, FABP5. Finally, Rab34 levels in the adipose tissue and adipocytes are regulated in response to obesity and related pathogenic insults (i.e., fibrosis). CONCLUSIONS: Rab34 plays relevant roles during adipocyte differentiation, including from the regulation of the oligomerization (i.e., biological activity) and secretion of a major adipokine with insulin-sensitizing actions, adiponectin, to lipid storage and mobilization from lipid droplets. Rab34 dysregulation in obesity may contribute to the altered adipokine secretion and lipid metabolism that characterize adipocyte dysfunction in conditions of excess adiposity.

Plasma lipidic fingerprint associated with type 2 diabetes in patients with coronary heart disease: CORDIOPREV study.

OBJECTIVE: We aimed to identify a lipidic profile associated with type 2 diabetes mellitus (T2DM) development in coronary heart disease (CHD) patients, to provide a new, highly sensitive model which could be used in clinical practice to identify patients at T2DM risk. METHODS: This study considered the 462 patients of the CORDIOPREV study (CHD patients) who were not diabetic at the beginning of the intervention. In total, 107 of them developed T2DM after a median follow-up of 60 months. They were diagnosed using the American Diabetes Association criteria. A novel lipidomic methodology employing liquid chromatography (LC) separation followed by HESI, and detection by mass spectrometry (MS) was used to annotate the lipids at the isomer level. The patients were then classified into a Training and a Validation Set (60-40). Next, a Random Survival Forest (RSF) was carried out to detect the lipidic isomers with the lowest prediction error, these lipids were then used to build a Lipidomic Risk (LR) score which was evaluated through a Cox. Finally, a production model combining the clinical variables of interest, and the lipidic species was carried out. RESULTS: LC-tandem MS annotated 440 lipid species. From those, the RSF identified 15 lipid species with the lowest prediction error. These lipids were combined in an LR score which showed association with the development of T2DM. The LR hazard ratio per unit standard deviation was 2.87 and 1.43, in the Training and Validation Set respectively. Likewise, patients with higher LR Score values had lower insulin sensitivity (P = 0.006) and higher liver insulin resistance (P = 0.005). The receiver operating characteristic (ROC) curve obtained by combining clinical variables and the selected lipidic isomers using a generalised lineal model had an area under the curve (AUC) of 81.3%. CONCLUSION: Our study showed the potential of comprehensive lipidomic analysis in identifying patients at risk of developing T2DM. In addition, the lipid species combined with clinical variables provided a new, highly sensitive model which can be used in clinical practice to identify patients at T2DM risk. Moreover, these results also indicate that we need to look closely at isomers to understand the role of this specific compound in T2DM development. Trials registration NCT00924937.

Exploring the role of the inflammasomes on prostate cancer: Interplay with obesity.

Obesity is a weight-related disorder characterized by excessive adipose tissue growth and dysfunction which leads to the onset of a systemic chronic low-grade inflammatory state. Likewise, inflammation is considered a classic cancer hallmark affecting several steps of carcinogenesis and tumor progression. In this regard, novel molecular complexes termed inflammasomes have been identified which are able to react to a wide spectrum of insults, impacting several metabolic-related disorders, but their contribution to cancer biology remains unclear. In this context, prostate cancer (PCa) has a markedly inflammatory component, and patients frequently are elderly individuals who exhibit weight-related disorders, being obesity the most prevalent condition. Therefore, inflammation, and specifically, inflammasome complexes, could be crucial players in the interplay between PCa and metabolic disorders. In this review, we will: 1) discuss the potential role of each inflammasome component (sensor, molecular adaptor, and targets) in PCa pathophysiology, placing special emphasis on IL-1ß/NF-kB pathway and ROS and hypoxia influence; 2) explore the association between inflammasomes and obesity, and how these molecular complexes could act as the cornerstone between the obesity and PCa; and, 3) compile current clinical trials regarding inflammasome targeting, providing some insights about their potential use in the clinical practice.

Changes in quantity plant-based protein intake on type 2 diabetes remission in coronary heart disease patients: from the CORDIOPREV study.

PURPOSE: Diabetes remission is a phenomenon described in the context of drastic weight loss due to bariatric surgery or low-calorie diets. Evidence suggests that increasing the intake of plant protein could reduce the risk of type 2 diabetes. We sought for association between changes in plant protein intake in the context of 2 healthy diets without weight loss nor glucose-lowering medication, and diabetes remission in coronary heart disease patients from the CORDIOPREV study. METHODS: Newly diagnosed type 2 diabetes participants without glucose-lowering treatment were randomized to consume a Mediterranean or a low-fat diet. Type 2 diabetes remission was assessed with a median follow-up of 60 months according to the ADA recommendation. Information on patient's dietary intake was collected using food-frequency questionnaires. At first year of intervention, 177 patients were classified according to changes in plant protein consumption into those who increased or decreased its intake, in order to perform an observational analysis on the association between protein intake and diabetes remission. RESULTS: Cox regression showed that patients increasing plant protein intake were more likely to remit from diabetes than those who decreased its intake (HR = 1.71(1.05-2.77)). The remission occurred mainly at first and second year of follow-up with diminished number of patients achieving remission in the third year onwards. The increase in plant protein was associated with lower intake of animal protein, cholesterol, saturated fatty acids, and fat, and with higher intake of whole grains, fibre, carbohydrates, legumes, and tree nuts. CONCLUSION: These results support the need to increase protein intake of vegetal origin as dietary therapy to reverse type 2 diabetes in the context of healthy diets without weight loss.

Rab18 Drift in Lipid Droplet and Endoplasmic Reticulum Interactions of Adipocytes under Obesogenic Conditions.

The adipose tissue stores excess energy in the form of neutral lipids within adipocyte lipid droplets (LDs). The correct function of LDs requires the interaction with other organelles, such as the endoplasmic reticulum (ER) as well as with LD coat-associated proteins, including Rab18, a mediator of intracellular lipid trafficking and ER-LD interaction. Although perturbations of the inter-organelle contact sites have been linked to several diseases, such as cancer, no information regarding ER-LD contact sites in dysfunctional adipocytes from the obese adipose tissue has been published to date. Herein, the ER-LD connection and Rab18 distribution at ER-LD contact sites are examined in adipocytes challenged with fibrosis and inflammatory conditions, which represent known hallmarks of the adipose tissue in obesity. Our results show that adipocytes differentiated in fibrotic conditions caused ER fragmentation, the expansion of ER-LD contact sites, and modified Rab18 dynamics. Likewise, adipocytes exposed to inflammatory conditions favored ER-LD contact, Rab18 accumulation in the ER, and Rab18 redistribution to large LDs. Finally, our studies in human adipocytes supported the suggestion that Rab18 transitions to the LD coat from the ER. Taken together, our results suggest that obesity-related pathogenic processes alter the maintenance of ER-LD interactions and interfere with Rab18 trafficking through these contact sites.

LRP10, PGK1 and RPLP0: Best Reference Genes in Periprostatic Adipose Tissue under Obesity and Prostate Cancer Conditions.

Obesity (OB) is a metabolic disorder characterized by adipose tissue dysfunction that has emerged as a health problem of epidemic proportions in recent decades. OB is associated with multiple comorbidities, including some types of cancers. Specifically, prostate cancer (PCa) has been postulated as one of the tumors that could have a causal relationship with OB. Particularly, a specialized adipose tissue (AT) depot known as periprostatic adipose tissue (PPAT) has gained increasing attention over the last few years as it could be a key player in the pathophysiological interaction between PCa and OB. However, to date, no studies have defined the most appropriate internal reference genes (IRGs) to be used in gene expression studies in this AT depot. In this work, two independent cohorts of PPAT samples (n = 20/n = 48) were used to assess the validity of a battery of 15 literature-selected IRGs using two widely used techniques (reverse transcription quantitative PCR [RT-qPCR] and microfluidic-based qPCR array). For this purpose, ΔCt method, GeNorm (v3.5), BestKeeper (v1.0), NormFinder (v.20.0), and RefFinder software were employed to assess the overall trends of our analyses. LRP10, PGK1, and RPLP0 were identified as the best IRGs to be used for gene expression studies in human PPATs, specifically when considering PCa and OB conditions.

Metabolomics analysis of type 2 diabetes remission identifies 12 metabolites with predictive capacity: a CORDIOPREV clinical trial study.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is one of the most widely spread diseases, affecting around 90% of the patients with diabetes. Metabolomics has proven useful in diabetes research discovering new biomarkers to assist in therapeutical studies and elucidating pathways of interest. However, this technique has not yet been applied to a cohort of patients that have remitted from T2DM. METHODS: All patients with a newly diagnosed T2DM at baseline (n = 190) were included. An untargeted metabolomics approach was employed to identify metabolic differences between individuals who remitted (RE), and those who did not (non-RE) from T2DM, during a 5-year study of dietary intervention. The biostatistical pipeline consisted of an orthogonal projection on the latent structure discriminant analysis (O-PLS DA), a generalized linear model (GLM), a receiver operating characteristic (ROC), a DeLong test, a Cox regression, and pathway analyses. RESULTS: The model identified a significant increase in 12 metabolites in the non-RE group compared to the RE group. Cox proportional hazard models, calculated using these 12 metabolites, showed that patients in the high-score tercile had significantly (p-value < 0.001) higher remission probabilities (Hazard Ratio, HR, high versus low = 2.70) than those in the lowest tercile. The predictive power of these metabolites was further studied using GLMs and ROCs. The area under the curve (AUC) of the clinical variables alone is 0.61, but this increases up to 0.72 if the 12 metabolites are considered. A DeLong test shows that this difference is statistically significant (p-value = 0.01). CONCLUSIONS: Our study identified 12 endogenous metabolites with the potential to predict T2DM remission following a dietary intervention. These metabolites, combined with clinical variables, can be used to provide, in clinical practice, a more precise therapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00924937.

The clinical and molecular cardiometabolic fingerprint of an exploratory psoriatic arthritis cohort is associated with the disease activity and differentially modulated by methotrexate and apremilast.

OBJECTIVES: (1) To evaluate clinical and molecular cardiovascular disease (CVD) signs and their relationship with psoriatic arthritis (PsA) features and (2) to identify a clinical patient profile susceptible to benefit from methotrexate (MTX) and/or apremilast regarding CVD risk. METHODS: This cross-sectional study included 100 patients with PsA and 100 age-matched healthy donors. In addition, an exploratory cohort of 45 biologically naïve patients treated for 6 months with apremilast, MTX or combined therapy according to routine clinical practice was recruited. Extensive clinical and metabolic profiles were obtained. Ninety-nine surrogate CVD-related molecules were analysed in plasma and peripheral blood mononuclear cells (PBMCs). Hard cluster analysis was performed to identify the clinical and molecular phenotypes. Mechanistic studies were performed on adipocytes. RESULTS: Cardiometabolic comorbidities were associated with disease activity and long-term inflammatory status. Thirty-five CVD-related proteins were altered in the plasma and PBMCs of PsA patients and were associated with the key clinical features of the disease. Plasma levels of some of the CVD-related molecules might distinguish insulin-resistant patients (MMP-3, CD163, FABP-4), high disease activity (GAL-3 and FABP-4) and poor therapy outcomes (CD-163, LTBR and CNTN-1). Hard cluster analysis identified two phenotypes of patients according to the rates of cardiometabolic comorbidities with distinctive clinical and molecular responses to each treatment. CONCLUSIONS: (1) Novel CVD-related proteins associated with clinical features could be emerging therapeutic targets in the context of PsA and (2) the pleiotropic action of apremilast could make it an excellent choice for the management of PsA patients with high CVD risk, targeting metabolic alterations and CVD-related molecules.

A plasma fatty acid profile associated to type 2 diabetes development: from the CORDIOPREV study.

PURPOSE: The prevalence of type 2 diabetes mellitus (T2DM) is increasing worldwide. For this reason, it is essential to identify biomarkers for the early detection of T2DM risk and/or for a better prognosis of T2DM. We aimed to identify a plasma fatty acid (FA) profile associated with T2DM development. METHODS: We included 462 coronary heart disease patients from the CORDIOPREV study without T2DM at baseline. Of these, 107 patients developed T2DM according to the American Diabetes Association (ADA) diagnosis criteria after a median follow-up of 60 months. We performed a random classification of patients in a training set, used to build a FA Score, and a Validation set, in which we tested the FA Score. RESULTS: FA selection with the highest prediction power was performed by random survival forest in the Training set, which yielded 4 out of the 24 FA: myristic, petroselinic, α-linolenic and arachidonic acids. We built a FA Score with the selected FA and observed that patients with a higher score presented a greater risk of T2DM development, with an HR of 3.15 (95% CI 2.04-3.37) in the Training set, and an HR of 2.14 (95% CI 1.50-2.84) in the Validation set, per standard deviation (SD) increase. Moreover, patients with a higher FA Score presented lower insulin sensitivity and higher hepatic insulin resistance (p < 0.05). CONCLUSION: Our results suggest that a detrimental FA plasma profile precedes the development of T2DM in patients with coronary heart disease, and that this FA profile can, therefore, be used as a predictive biomarker. CLINICAL TRIALS.GOV. IDENTIFIER: NCT00924937.

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.

Crohn's Disease Increases the Mesothelial Properties of Adipocyte Progenitors in the Creeping Fat.

Our understanding of the interplay between human adipose tissue and the immune system is limited. The mesothelium, an immunologically active structure, emerged as a source of visceral adipose tissue. After investigating the mesothelial properties of human visceral and subcutaneous adipose tissue and their progenitors, we explored whether the dysfunctional obese and Crohn's disease environments influence the mesothelial/mesenchymal properties of their adipocyte precursors, as well as their ability to mount an immune response. Using a tandem transcriptomic/proteomic approach, we evaluated the mesothelial and mesenchymal expression profiles in adipose tissue, both in subjects covering a wide range of body-mass indexes and in Crohn's disease patients. We also isolated adipose tissue precursors (adipose-derived stem cells, ASCs) to assess their mesothelial/mesenchymal properties, as well as their antigen-presenting features. Human visceral tissue presented a mesothelial phenotype not detected in the subcutaneous fat. Only ASCs from mesenteric adipose tissue, named creeping fat, had a significantly higher expression of the hallmark mesothelial genes mesothelin (MSLN) and Wilms' tumor suppressor gene 1 (WT1), supporting a mesothelial nature of these cells. Both lean and Crohn's disease visceral ASCs expressed equivalent surface percentages of the antigen-presenting molecules human leucocyte antigen-DR isotype (HLA-DR) and CD86. However, lean-derived ASCs were predominantly HLA-DR dim, whereas in Crohn's disease, the HLA-DR bright subpopulation was increased 3.2-fold. Importantly, the mesothelial-enriched Crohn's disease precursors activated CD4+ T-lymphocytes. Our study evidences a mesothelial signature in the creeping fat of Crohn's disease patients and its progenitor cells, the latter being able to present antigens and orchestrate an immune response.

Biological senescence risk score. A practical tool to predict biological senescence status.

BACKGROUND: Ageing and biological senescence, both related to cardiovascular disease, are mediated by oxidative stress and inflammation. We aim to develop a predictive tool to evaluate the degree of biological senescence in coronary patients. METHODS: Relative telomere length (RTL) of 1002 coronary patients from the CORDIOPREV study (NCT00924937) was determined at baseline in addition to markers of inflammatory response (hs-C-Reactive Protein, monocyte chemoattractant protein-1, IL-6, IL-1ß, TNF-α, adiponectin, resistin and leptin) and oxidative stress (nitric oxide, lipid peroxidation products, carbonylated proteins, catalase, total glutathione, reduced glutathione, oxidized glutathione, superoxide dismutase and peroxidated glutathione). Biological senescence was defined using the cut-off value defined by the lower quintile of relative telomere length in our population (RTL = 0.7629). We generated and tested different predictive models based on logistic regression analysis to identify biological senescence. Three models were designed to be used with different sets of information. RESULTS: We selected those patients with all the variables proposed to develop the predictive models (n = 353). Statistically significant differences between both groups (Biological senescence vs. Nonbiological senescence) were found for total cholesterol, catalase, superoxide dismutase, IL-1ß, resistin and leptin. The area under the curve of receiver-operating characteristic to predict biological senescence for our models was 0.65, 0.75 and 0.72. CONCLUSIONS: These predictive models allow us to calculate the degree of biological senescence in coronary patients, identifying a subgroup of patients at higher risk and who may require more intensive treatment.

Cytoskeletal transgelin 2 contributes to gender-dependent adipose tissue expandability and immune function.

During adipogenesis, preadipocytes' cytoskeleton reorganizes in parallel with lipid accumulation. Failure to do so may impact the ability of adipose tissue (AT) to shift between lipid storage and mobilization. Here, we identify cytoskeletal transgelin 2 (TAGLN2) as a protein expressed in AT and associated with obesity and inflammation, being normalized upon weight loss. TAGLN2 was primarily found in the adipose stromovascular cell fraction, but inflammation, TGF-ß, and estradiol also prompted increased expression in human adipocytes. Tagln2 knockdown revealed a key functional role, being required for proliferation and differentiation of fat cells, whereas transgenic mice overexpressing Tagln2 using the adipocyte protein 2 promoter disclosed remarkable sex-dependent variations, in which females displayed "healthy" obesity and hypertrophied adipocytes but preserved insulin sensitivity, and males exhibited physiologic changes suggestive of defective AT expandability, including increased number of small adipocytes, activation of immune cells, mitochondrial dysfunction, and impaired metabolism together with decreased insulin sensitivity. The metabolic relevance and sexual dimorphism of TAGLN2 was also outlined by genetic variants that may modulate its expression and are associated with obesity and the risk of ischemic heart disease in men. Collectively, current findings highlight the contribution of cytoskeletal TAGLN2 to the obese phenotype in a gender-dependent manner.-Ortega, F. J., Moreno-Navarrete, J. M., Mercader, J. M., Gómez-Serrano, M., García-Santos, E., Latorre, J., Lluch, A., Sabater, M., Caballano-Infantes, E., Guzmán, R., Macías-González, M., Buxo, M., Gironés, J., Vilallonga, R., Naon, D., Botas, P., Delgado, E., Corella, D., Burcelin, R., Frühbeck, G., Ricart, W., Simó, R., Castrillon-Rodríguez, I., Tinahones, F. J., Bosch, F., Vidal-Puig, A., Malagón, M. M., Peral, B., Zorzano, A., Fernández-Real, J. M. Cytoskeletal transgelin 2 contributes to gender-dependent adipose tissue expandability and immune function.

High-fat diet promotes adrenaline production by visceral adipocytes.

PURPOSE: Catecholamines (CA) play a major role in metabolism and immune response. Recent reports showing adipose tissue can synthetize CA enlighten new roles for these amines in obesity. This study aimed to evaluate the expression of both tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) and CA content along preadipocytes differentiation, under normal and obesigenic conditions. METHODS: 8-9 week-old male C57BL/6 mice were divided in two groups: one fed with a high-fat diet (HFD) and other with a standard diet (SD) for 20 weeks. Afterwards, both TH and PNMT expression, localization, and CA content in adipocytes, were evaluated. RESULTS: qPCR results showed no changes for TH and PNMT expression during the differentiation process for visceral and subcutaneous preadipocytes from mice fed with SD. Comparing to SD, HFD increased TH gene expression of subcutaneous preadipocytes and PNMT gene expression of both visceral preadipocytes and adipocytes. HPLC-ED analyses revealed HFD increased visceral adipocytes noradrenaline intracellular content comparing with preadipocytes (p = 0.037). When compared with SD, HFD raised and decreased noradrenaline content, respectively, in visceral adipocytes (p = 0.004) and subcutaneous preadipocytes (p = 0.001). Along the differentiation process, HFD increased visceral adrenaline intracellular content comparing with SD (p < 0.001). HFD increased visceral comparing to subcutaneous adrenaline content for both preadipocytes (p = 0.004) and adipocytes (p = 0.001). CONCLUSIONS: TH and PNMT expression in adipose tissue is differently modulated in visceral and subcutaneous adipose depots, and seems to depend on diet. Differences observed in visceral adipose CA handling in HFD-fed mice might uncover novel pharmacological/nutritional strategies against obesity and cardiovascular risk.

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.

The cytoskeletal protein septin 11 is associated with human obesity and is involved in adipocyte lipid storage and metabolism.

AIMS/HYPOTHESIS: Septins are newly identified members of the cytoskeleton that have been proposed as biomarkers of a number of diseases. However, septins have not been characterised in adipose tissue and their relationship with obesity and insulin resistance remains unknown. Herein, we characterised a member of this family, septin 11 (SEPT11), in human adipose tissue and analysed its potential involvement in the regulation of adipocyte metabolism. METHODS: Gene and protein expression levels of SEPT11 were analysed in human adipose tissue. SEPT11 distribution was evaluated by immunocytochemistry, electron microscopy and subcellular fractionation techniques. Glutathione S-transferase (GST) pull-down, immunoprecipitation and yeast two-hybrid screening were used to identify the SEPT11 interactome. Gene silencing was used to assess the role of SEPT11 in the regulation of insulin signalling and lipid metabolism in adipocytes. RESULTS: We demonstrate the expression of SEPT11 in human adipocytes and its upregulation in obese individuals, with SEPT11 mRNA content positively correlating with variables of insulin resistance in subcutaneous adipose tissue. SEPT11 content was regulated by lipogenic, lipolytic and proinflammatory stimuli in human adipocytes. SEPT11 associated with caveolae in mature adipocytes and interacted with both caveolin-1 and the intracellular fatty acid chaperone, fatty acid binding protein 5 (FABP5). Lipid loading of adipocytes caused the association of the three proteins with the surface of lipid droplets. SEPT11 silencing impaired insulin signalling and insulin-induced lipid accumulation in adipocytes. CONCLUSIONS/INTERPRETATION: Our findings support a role for SEPT11 in lipid traffic and metabolism in adipocytes and open new avenues for research on the control of lipid storage in obesity and insulin resistance.

Virgin olive oil rich in phenolic compounds modulates the expression of atherosclerosis-related genes in vascular endothelium.

PURPOSE: Previous studies have shown the anti-inflammatory and antioxidant properties of phenolic compounds of virgin olive oil (VOO). However, the effect of bioavailable phenolic compounds on the vascular endothelium is unknown. We aimed to evaluate the effect of the consumption of virgin olive oil rich in phenolic compounds on the vascular endothelium. METHODS: We treated HUVEC with human serum obtained in fasting state and after the intake of a breakfast prepared with VOO with a high or low content of phenolic compounds. RESULTS: Treatment of HUVEC with serum obtained 2 h after the intake of the high-phenol VOO-based breakfast decreased p65 and MCP-1 gene expression (p < 0.001 and p = 0.002, respectively) and increased MT-CYB, SDHA and SOD1 gene expression (p = 0.004, p = 0.012 and p = 0.001, respectively), as compared with the treatment of HUVEC with the serum obtained 2 h after the intake of the low-phenol VOO-based breakfast. The treatment with serum obtained 4 h after the intake of the high-phenol VOO-based breakfast decreased MCP-1 and CAT gene expression (p < 0.001 and p = 0.003, respectively) and increased MT-CYB gene expression (p < 0.001), as compared to the treatment with serum obtained 4 h after the intake of the low-phenol VOO-based breakfast. CONCLUSION: Our results suggest that the consumption of virgin olive oil rich in phenolic compounds may reduce the risk of atherosclerosis development by decreasing inflammation and improving the antioxidant profile in the vascular endothelium.

Proteome from patients with metabolic syndrome is regulated by quantity and quality of dietary lipids.

BACKGROUND: Metabolic syndrome is a multi-component disorder associated to a high risk of cardiovascular disease. Its etiology is the result of a complex interaction between genetic and environmental factors, including dietary habits. We aimed to identify the target proteins modulated by the long-term consumption of four diets differing in the quality and quantity of lipids in the whole proteome of peripheral blood mononuclear cells (PBMC). RESULTS: A randomized, controlled trial conducted within the LIPGENE study assigned 24 MetS patients for 12 weeks each to 1 of 4 diets: a) high-saturated fatty acid (HSFA), b) high-monounsaturated fatty acid (HMUFA), c) low-fat, high-complex carbohydrate diets supplemented with placebo (LFHCC) and d) low-fat, high-complex carbohydrate diets supplemented with long chain (LC) n-3 polyunsaturated fatty acids (PUFA) (LFHCC n-3). We analyzed the changes induced in the proteome of both nuclear and cytoplasmic fractions of PBMC using 2-D proteomic analysis. Sixty-seven proteins were differentially expressed after the long-term consumption of the four diets. The HSFA diet induced the expression of proteins responding to oxidative stress, degradation of ubiquitinated proteins and DNA repair. However, HMUFA, LFHCC and LFHCC n-3 diets down-regulated pro-inflammatory and oxidative stress-related proteins and DNA repairing proteins. CONCLUSION: The long-term consumption of HSFA, compared to HMUFA, LFHCC and LFHCC n-3, seems to increase the cardiovascular disease (CVD) risk factors associated with metabolic syndrome, such as inflammation and oxidative stress, and seem lead to DNA damage as a consequence of high oxidative stress.

Mitochondria in metabolic disease: getting clues from proteomic studies.

Mitochondria play a key role as major regulators of cellular energy homeostasis, but in the context of mitochondrial dysfunction, mitochondria may generate reactive oxidative species and induce cellular apoptosis. Indeed, altered mitochondrial status has been linked to the pathogenesis of several metabolic disorders and specially disorders related to insulin resistance, such as obesity, type 2 diabetes, and other comorbidities comprising the metabolic syndrome. In the present review, we summarize information from various mitochondrial proteomic studies of insulin-sensitive tissues under different metabolic states. To that end, we first focus our attention on the pancreas, as mitochondrial malfunction has been shown to contribute to beta cell failure and impaired insulin release. Furthermore, proteomic studies of mitochondria obtained from liver, muscle, and adipose tissue are summarized, as these tissues constitute the primary insulin target metabolic tissues. Since recent advances in proteomic techniques have exposed the importance of PTMs in the development of metabolic disease, we also present information on specific PTMs that may directly affect mitochondria during the pathogenesis of metabolic disease. Specifically, mitochondrial protein acetylation, phosphorylation, and other PTMs related to oxidative damage, such as nitrosylation and carbonylation, are discussed.

Adiponectin receptors form homomers and heteromers exhibiting distinct ligand binding and intracellular signaling properties.

Adiponectin binds to two widely expressed receptors (AdipoR1 and AdipoR2) that contain seven transmembrane domains but, unlike G-protein coupled receptors, present an extracellular C terminus and a cytosolic N terminus. Recently, AdipoR1 was found to associate in high order complexes. However, it is still unknown whether AdipoR2 may also form homomers or heteromers with AdipoR1 or if such interactions may be functionally relevant. Herein, we have analyzed the oligomerization pattern of AdipoRs by FRET and immunoprecipitation and evaluated both the internalization of AdipoRs in response to various adiponectin isoforms and the effect of adiponectin binding to different AdipoR combinations on AMP-activated protein kinase phosphorylation and peroxisome proliferator-activated receptor α activation. Transfection of HEK293AD cells with AdipoR1 and AdipoR2 showed that both receptors colocalize at both the plasma membrane and the endoplasmic reticulum. Co-transfection with the different AdipoR pairs yielded high FRET efficiencies in non-stimulated cells, which indicates that AdipoR1 and AdipoR2 form homo- and heteromeric complexes under resting conditions. Live FRET imaging suggested that both homo- and heteromeric AdipoR complexes dissociate in response to adiponectin, but heteromers separate faster than homomers. Finally, phosphorylation of AMP-activated protein kinase in response to adiponectin was delayed in cells wherein heteromer formation was favored. In sum, our findings indicate that AdipoR1 and AdipoR2 form homo- and heteromers that present unique interaction behaviors and signaling properties. This raises the possibility that the pleiotropic, tissue-dependent functions of adiponectin depend on the expression levels of AdipoR1 and AdipoR2 and, therefore, on the steady-state proportion of homo- and heteromeric complexes.