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.

Enhanced fatty acid oxidation in adipocytes and macrophages reduces lipid-induced triglyceride accumulation and inflammation.

Lipid overload in obesity and type 2 diabetes is associated with adipocyte dysfunction, inflammation, macrophage infiltration, and decreased fatty acid oxidation (FAO). Here, we report that the expression of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme in mitochondrial FAO, is higher in human adipose tissue macrophages than in adipocytes and that it is differentially expressed in visceral vs. subcutaneous adipose tissue in both an obese and a type 2 diabetes cohort. These observations led us to further investigate the potential role of CPT1A in adipocytes and macrophages. We expressed CPT1AM, a permanently active mutant form of CPT1A, in 3T3-L1 CARΔ1 adipocytes and RAW 264.7 macrophages through adenoviral infection. Enhanced FAO in palmitate-incubated adipocytes and macrophages reduced triglyceride content and inflammation, improved insulin sensitivity in adipocytes, and reduced endoplasmic reticulum stress and ROS damage in macrophages. We conclude that increasing FAO in adipocytes and macrophages improves palmitate-induced derangements. This indicates that enhancing FAO in metabolically relevant cells such as adipocytes and macrophages may be a promising strategy for the treatment of chronic inflammatory pathologies such as obesity and type 2 diabetes.

Improved integrative analysis of the thiol redox proteome using filter-aided sample preparation.

Changes in the oxidation state of protein Cys residues are involved in cell signalling and play a key role in a variety of pathophysiological states. We had previously developed GELSILOX, an in-gel method that enables the large-scale, parallel analysis of dynamic alterations to the redox state of Cys sites and protein abundance changes. Here we present FASILOX, a further development of the GELSILOX approach featuring: i) significantly increased peptide recovery, ii) enhanced sensitivity for the detection of Cys oxidative alterations, and iii) streamlined workflow that results in shortened assay duration. In mitochondria isolated from the adipose tissue of obese, diabetic patients, FASILOX revealed a sexually dimorphic trait of Cys oxidation involving mainly mitochondrial oxidative phosphorylation complexes. These results provide the first evidence for a decreased efficiency in the antioxidant response of men as compared to women.

Study of the proinflammatory role of human differentiated omental adipocytes.

Infiltration of monocyte-derived macrophages into adipose tissue has been associated with tissue and systemic inflammation. It has been suggested that macrophage infiltration affects fat expansion through a paracrine action on adipocyte differentiation. Our working hypothesis is that factors released by monocytes/macrophages may also affect mature adipocyte biology. Human differentiated omental adipocytes were incubated with LPS and conditioned media obtained from human macrophage-like cell line THP-1, previously activated or not with LPS. We show that LPS greatly increased the secretion levels of pro-inflammatory adipokines including IL-6, IL-8, GRO, and MCP-1. Macrophage-conditioned medium also upregulated IL-6, IL-8, GRO, and MCP-1 mRNA expression and protein levels and led to the novo secretion of ICAM-1, IL-1 beta, IP-10, MIP-1 alpha, MIP-1 beta, VEGF, and TNFalpha. Human differentiated adipocytes treated by macrophage-conditioned medium displayed marked reduction of adipocyte function as assessed by decreased phosphorylation levels of ERK1, ERK2, and p38 alpha and reduced gene expression of lipogenic markers including PPAR-gamma and fatty acid synthase. These data show that macrophage-secreted factors not only inhibit the formation of mature adipocytes but alter their function, suggesting that human differentiated omental adipocytes might also contribute to systemic chronic low-grade inflammation associated with human obesity.

Tackling the human adipose tissue proteome to gain insight into obesity and related pathologies.

Obesity is becoming an important public health problem given its strong association with insulin resistance and Type 2 diabetes. Previously considered an inert depot, fat is now regarded as a highly metabolically active tissue in many pathophysiological processes. In humans, the accumulation of omental rather than subcutaneous adipose tissue appears to be tightly linked to cardiovascular disease and other important comorbidities. Proteomics has emerged as a method for the large-scale study of proteins in biological samples, for instance, fluids, cells or tissues, which encompasses not only the identities of the proteins present, but also quantification and post-translational modification events. Human adipose tissue proteome analysis, still in its early stages, may help understand the molecular mechanisms of obesity and the role of omental fat in the pathogenesis of obesity-associated diseases. This review covers recent advances in human adipose tissue proteomics, focusing on the analysis of the omental and the subcutaneous fat.

Proteomic analysis of human omental adipose tissue in the polycystic ovary syndrome using two-dimensional difference gel electrophoresis and mass spectrometry.

BACKGROUND: Our aim was to study the protein expression profiles of omental adipose tissue biopsies obtained from morbidly obese women with or without polycystic ovary syndrome (PCOS) at the time of bariatric surgery to evaluate the possible involvement of visceral adiposity in the development of PCOS. METHODS: Ten PCOS patients and nine control samples were included. We used two-dimensional difference gel electrophoresis (2D-DIGE) followed by in-gel digestion, and mass spectrometry (MS) of selected protein spots. RESULTS: The 2D-DIGE technology allowed the analysis of approximately 1840 protein spots in the comparative study of control and patient proteomes, revealing 15 statistically significant spot changes (>2-fold, P < 0.05). Unambiguous protein identification was achieved for 9 of these 15 spots by MS. This preliminary study revealed differences in expression of proteins that may be involved in lipid and glucose metabolism, oxidative stress processes and adipocyte differentiation; they include proapolipoprotein Apo-A1, annexin V, glutathione S-transferase M3 (GSTM3), triosephosphate isomerase, peroxiredoxin 2 isoform a, actin and adipocyte plasma membrane-associated protein. The most relevant finding was an increase of GSTM3 in the omental fat of PCOS patients confirming previous studies conducted by our group. CONCLUSIONS: Proteomic analysis of omental fat reveals differential expression of several proteins in PCOS patients and non-hyperandrogenic women presenting with morbid obesity. The application of this novel methodology adds further evidence to support the role of visceral adiposity in the pathogenesis of PCOS.

Mitoproteomics: Tackling Mitochondrial Dysfunction in Human Disease.

Mitochondria are highly dynamic and regulated organelles that historically have been defined based on their crucial role in cell metabolism. However, they are implicated in a variety of other important functions, making mitochondrial dysfunction an important axis in several pathological contexts. Despite that conventional biochemical and molecular biology approaches have provided significant insight into mitochondrial functionality, innovative techniques that provide a global view of the mitochondrion are still necessary. Proteomics fulfils this need by enabling accurate, systems-wide quantitative analysis of protein abundance. More importantly, redox proteomics approaches offer unique opportunities to tackle oxidative stress, a phenomenon that is intimately linked to aging, cardiovascular disease, and cancer. In addition, cutting-edge proteomics approaches reveal how proteins exert their functions in complex interaction networks where even subtle alterations stemming from early pathological states can be monitored. Here, we describe the proteomics approaches that will help to deepen the role of mitochondria in health and disease by assessing not only changes to mitochondrial protein composition but also alterations to their redox state and how protein interaction networks regulate mitochondrial function and dynamics. This review is aimed at showing the reader how the application of proteomics approaches during the last 20 years has revealed crucial mitochondrial roles in the context of aging, neurodegenerative disorders, metabolic disease, and cancer.

Differential gene expression profile in omental adipose tissue in women with polycystic ovary syndrome.

CONTEXT: The polycystic ovary syndrome (PCOS) is frequently associated with visceral obesity, suggesting that omental adipose tissue might play an important role in the pathogenesis of the syndrome. OBJECTIVE: The objective was to study the expression profiles of omental fat biopsy samples obtained from morbidly obese women with or without PCOS at the time of bariatric surgery. DESIGN: This was a case-control study. SETTINGS: We conducted the study in an academic hospital. PATIENTS: Eight PCOS patients and seven nonhyperandrogenic women submitted to bariatric surgery because of morbid obesity. INTERVENTIONS: Biopsy samples of omental fat were obtained during bariatric surgery. MAIN OUTCOME MEASURE: The main outcome measure was high-density oligonucleotide arrays. RESULTS: After statistical analysis, we identified changes in the expression patterns of 63 genes between PCOS and control samples. Gene classification was assessed through data mining of Gene Ontology annotations and cluster analysis of dysregulated genes between both groups. These methods highlighted abnormal expression of genes encoding certain components of several biological pathways related to insulin signaling and Wnt signaling, oxidative stress, inflammation, immune function, and lipid metabolism, as well as other genes previously related to PCOS or to the metabolic syndrome. CONCLUSION: The differences in the gene expression profiles in visceral adipose tissue of PCOS patients compared with nonhyperandrogenic women involve multiple genes related to several biological pathways, suggesting that the involvement of abdominal obesity in the pathogenesis of PCOS is more ample than previously thought and is not restricted to the induction of insulin resistance.

N-acetylcysteine inhibits kinase phosphorylation during 3T3-L1 adipocyte differentiation.

OBJECTIVES: Reports investigating the effects of antioxidants on obesity have provided contradictory results. We have previously demonstrated that treatment with the antioxidant N-acetylcysteine (NAC) inhibits cellular triglyceride (Tg) accumulation as well as total cellular monoamine oxidase A (MAOA) expression in 3T3-L1 mature adipocytes (Calzadilla et al., Redox Rep. 2013;210-218). Here we analyzed the role of NAC on adipogenic differentiation pathway. METHODS: Assays were conducted using 3T3-L1 preadipocytes (undifferentiated cells: CC), which are capable of differentiating into mature adipocytes (differentiated cells: DC). We studied the effects of different doses of NAC (0.01 or 1 mM) on DC, to evaluate cellular expression of phospho-JNK½ (pJNK½), phospho-ERK½ (pERK½) and, mitochondrial expression of citrate synthase, fumarate hydratase and MAOA. RESULTS: Following the differentiation of preadipocytes, an increase in the expression levels of pJNK½ and pERK½ was observed, together with mitotic clonal expansion (MCE). We found that both doses of NAC decreased the expression of pJNK½ and pERK½. Consistent with these results, NAC significantly inhibited MCE and modified the expression of different mitochondrial proteins. DISCUSSION: Our results suggested that NAC could inhibit Tg and mitochondrial protein expression by preventing both MCE and kinase phosphorylation.

Differential proteomic and oxidative profiles unveil dysfunctional protein import to adipocyte mitochondria in obesity-associated aging and diabetes.

Human age-related diseases, including obesity and type 2 diabetes (T2DM), have long been associated to mitochondrial dysfunction; however, the role for adipose tissue mitochondria in these conditions remains unknown. We have tackled the impact of aging and T2DM on adipocyte mitochondria from obese patients by quantitating not only the corresponding abundance changes of proteins, but also the redox alterations undergone by Cys residues thereof. For that, we have resorted to a high-throughput proteomic approach based on isobaric labeling, liquid chromatography and mass spectrometry. The alterations undergone by the mitochondrial proteome revealed aging- and T2DM-specific hallmarks. Thus, while a global decrease of oxidative phosphorylation (OXPHOS) subunits was found in aging, the diabetic patients exhibited a reduction of specific OXPHOS complexes as well as an up-regulation of the anti-oxidant response. Under both conditions, evidence is shown for the first time of a link between increased thiol protein oxidation and decreased protein abundance in adipose tissue mitochondria. This association was stronger in T2DM, where OXPHOS mitochondrial- vs. nuclear-encoded protein modules were found altered, suggesting impaired mitochondrial protein translocation and complex assembly. The marked down-regulation of OXPHOS oxidized proteins and the alteration of oxidized Cys residues related to protein import through the redox-active MIA (Mitochondrial Intermembrane space Assembly) pathway support that defects in protein translocation to the mitochondria may be an important underlying mechanism for mitochondrial dysfunction in T2DM and physiological aging. The present draft of redox targets together with the quantification of protein and oxidative changes may help to better understand the role of oxidative stress in both a physiological process like aging and a pathological condition like T2DM.

Proteome-wide alterations on adipose tissue from obese patients as age-, diabetes- and gender-specific hallmarks.

Obesity is a main global health issue and an outstanding cause of morbidity and mortality predisposing to type 2 diabetes (T2DM) and cardiovascular diseases. Huge research efforts focused on gene expression, cellular signalling and metabolism in obesity have improved our understanding of these disorders; nevertheless, to bridge the gap between the regulation of gene expression and changes in signalling/metabolism, protein levels must be assessed. We have extensively analysed visceral adipose tissue from age-, T2DM- and gender-matched obese patients using high-throughput proteomics and systems biology methods to identify new biomarkers for the onset of T2DM in obesity, as well as to gain insight into the influence of aging and gender in these disorders. About 250 proteins showed significant abundance differences in the age, T2DM and gender comparisons. In diabetic patients, remarkable gender-specific hallmarks were discovered regarding redox status, immune response and adipose tissue accumulation. Both aging and T2DM processes were associated with mitochondrial remodelling, albeit through well-differentiated proteome changes. Systems biology analysis highlighted mitochondrial proteins that could play a key role in the age-dependent pathophysiology of T2DM. Our findings could serve as a framework for future research in Translational Medicine directed at improving the quality of life of obese patients.

Transducin-like enhancer of split 3 (TLE3) in adipose tissue is increased in situations characterized by decreased PPARγ gene expression.

UNLABELLED: Transgenic overexpression of adipose tissue (AT) transducin-like enhancer of split 3 (TLE3) mimicked peroxisome proliferator-activated receptor gamma (PPARγ) agonists, improving insulin resistance in mice. This study aimed to investigate TLE3 gene expression (qRT-PCR) and protein (Western blot) in subjects with a wide spectrum of obesity and insulin sensitivity and in an independent cohort of obese subjects following surgery-induced weight loss. TLE3 was analyzed in human adipocytes and after treatment with rosiglitazone. Given the findings in humans, TLE3 was also investigated in mice after a high-fat diet (HFD) and in PPARγ knockout mice. Subcutaneous (SC) AT TLE3 was increased in subjects with type 2 diabetes (T2D). In fact, SC TLE3 was associated with increased fasting glucose (r = 0.25, p = 0.015) and S6K1 activity (r = 0.671, p = 0.003), and with decreased Glut4 (r = -0.426, p = 0.006) and IRS-1 expression (-31 %, p = 0.007) and activation (P-IRS-1/IRS-1, -17 %, p = 0.024). TLE3 was preferentially expressed in mature adipocytes and increased during in vitro differentiation in parallel to PPARγ. Weight loss led to improved insulin sensitivity, increased AT PPARγ and decreased TLE3 (-24 %, p = 0.0002), while rosiglitazone administration downregulated TLE3 gene expression in fully differentiated adipocytes (-45 %, p < 0.0001). The concept that TLE3 may act as a homeostatic linchpin in AT was also supported by its increased expression in HFD-fed mice (39 %, p = 0.013) and PPARγ knockout (74 %, p = 0.001). In summary, increased AT TLE3 in subjects with T2D and in AT from HFD-fed and PPARγ knockout mice suggest that TLE3 may play an adaptive regulatory role that improves AT function under decreased PPARγ expression. KEY MESSAGE: TLE3 is expressed in mature adipocytes concomitantly with PPARγ. Subcutaneous adipose TLE3 is increased in T2D patients. Adipose TLE3 is upregulated in genetically ablated PPARγ and HFD-fed mice. TLE3 may be a homeostatic linchpin in insulin resistance and defective PPARγ.

Parathyroid Hormone-Related Protein, Human Adipose-Derived Stem Cells Adipogenic Capacity and Healthy Obesity.

OBJECTIVE: This study aimed to define the potential role of PTHrP on adipogenic regulation and to analyze its relationship with obesity and insulin resistance. DESIGN: This was a cross-sectional study in which visceral (VAT) and subcutaneous (SAT) adipose tissue were extracted from 19 morbidly obese, 10 obese, and 10 lean subjects. PTHrP mRNA levels were measured in VAT and SAT. VAT mesenchymal stem cells and 3T3-L1 cells were differentiated into adipocytes in presence or absence of PTHrP siRNA. PTHrP mRNA and protein levels as well as adipogenic markers were evaluated by Western blotting or qPCR. Immunohistochemistry and immunofluorescence procedures were used for PTHrP intracellular localization. RESULTS: Both human VAT and SAT express PTHrP protein mainly in the nucleolar compartment of stromal vascular fraction cells. The highest levels of PTHrP mRNA and protein expression were detected in undifferentiated mesenchymal cells and progressively decreased during adipogenesis. Remarkably, adipogenic differentiation in human mesenchymal stem cells (A-hMSC) was significantly impaired in a pthrp knockdown. PTHrP seems to be related to obesity-associated insulin resistance (IR), given that we found that PTHrP mRNA expression was higher in VAT from morbidly obese with a low IR degree (MO-L-IR) subjects than those from morbidly obese with a high IR degree (MO-H-IR) and lean subjects, and correlated positively with body mass index and hip circumference. We also found that A-hMSC from MO-L-IRs displayed higher adipogenic capacity than those from both MO-H-IRs and leans. In addition, adipogenesis was impaired in VAT from MO-H-IRs, given that mRNA expression levels of key adipogenic regulators were lower than those from MO-L-IR subjects. CONCLUSIONS: PTHrP could be a potential new therapeutic target for the reprograming of adipogenesis and adipose tissue expansion, thus possibly ameliorating the metabolic syndrome in obese subjects.

Association of the polycystic ovary syndrome with genomic variants related to insulin resistance, type 2 diabetes mellitus, and obesity.

We have evaluated the possible association of polycystic ovary syndrome (PCOS) with 15 genomic variants previously described to influence insulin resistance, obesity, and/or type 2 diabetes mellitus. Seventy-two PCOS patients and 42 healthy controls were genotyped for 15 variants in the genes encoding for paraoxonase (three variants), plasma cell differentiation antigen glycoprotein, human sorbin and SH3 domain containing 1, plasminogen activator inhibitor-1, peroxisome proliferator-activated receptor-gamma2, protein tyrosine phosphatase 1B (two variants), adiponectin (two variants), IGF1, IGF2, IGF1 receptor, and IGF2 receptor. Compared with controls, PCOS patients were more frequently homozygous for the -108T variant in paraoxonase (36.6% vs. 9.5%; P = 0.002) and homozygous for G alleles of the ApaI variant in IGF2 (62.9% vs. 38.1%; P = 0.018). Paraoxonase is a serum antioxidant enzyme and, because -108T alleles result in decreased paraoxonase expression, this increase in oxidative stress might result in insulin resistance. G alleles of the ApaI variant in IGF2 may increase IGF2 expression, and IGF2 stimulates adrenal and ovarian androgen secretion. In conclusion, the paraoxonase -108 C-->T variant and the ApaI polymorphism in the IGF2 gene are associated with PCOS and might contribute to increased oxidative stress, insulin resistance, and hyperandrogenism in this prevalent disorder.

Comment: the methionine 196 arginine polymorphism in exon 6 of the TNF receptor 2 gene (TNFRSF1B) is associated with the polycystic ovary syndrome and hyperandrogenism.

Inflammatory cytokines such as TNF alpha may play a role in the pathogenesis of common metabolic disorders, including hyperandrogenism and the polycystic ovary syndrome (PCOS). The TNF receptor 2 mediates most of the metabolic effects of TNF alpha. In the present study, we have evaluated serum soluble TNF receptor 2 levels, and several common polymorphisms in the TNF receptor 2 gene (TNFRSF1B), in women presenting with PCOS or hyperandrogenic disorders. Initial studies included 103 hyperandrogenic patients (42 presenting with PCOS) and 36 controls from Spain. The 196R alleles of the M196R (676 T-->G) variant in exon 6 of TNFRSF1B, which is in linkage disequilibrium with a CA-repeat microsatellite polymorphism in intron 4 of TNFRSF1B, tended to be more frequent in hyperandrogenic patients than in controls (P = 0.056), reaching statistical significance when the analysis was restricted to include only PCOS patients (P < 0.03). Extended analysis including another 11 hyperandrogenic patients from Spain and 64 patients and 29 controls from Italy confirmed the association between 196R alleles of the M196R variant and hyperandrogenic disorders (P < 0.05), which was maintained when restricting the analysis to PCOS patients (P < 0.02). On the contrary, the 3'-untranslated region (exon 10) variants 1663 G-->A, 1668 T-->G, and 1690 T-->C were not associated with hyperandrogenism. The soluble TNF receptor 2 levels were not different between patients and controls but were increased in obese subjects, compared with lean individuals, and were affected by the interaction between the 1663 G-->A and 1668 T-->G variants in the 3'-untranslated region of TNFRSF1B. The TNFRSF1B genotype did not influence any clinical or biochemical variable related to hyperandrogenism or insulin sensitivity and was not associated with obesity, both in hyperandrogenic patients and healthy controls considered separately. In conclusion, the M196R (676 T-->G) variant in exon 6 of TNFRSF1B is associated with hyperandrogenism and PCOS, further suggesting a role for inflammatory cytokines in the pathogenesis of these disorders.

Common single nucleotide polymorphisms in intron 3 of the calpain-10 gene influence hirsutism.

OBJECTIVE: To study three common polymorphisms in intron 3 of the calpain-10 gene (CAPN10) in hyperandrogenic patients. DESIGN: Case-control study. SETTING: Academic hospital. PATIENT(S): Ninety-seven hyperandrogenic patients and 37 healthy controls. INTERVENTION(S): Basal and adrenocorticotropin-stimulated serum samples and genomic DNA samples were obtained during the follicular phase of the menstrual cycle. MAIN OUTCOME MEASURE(S): Genotyping of the UCSNP43, UCSNP44, and UCSNP45 polymorphisms in CAPN10 and serum androgen levels. RESULT(S): Sixteen patients had idiopathic hirsutism, defined as normal serum androgen levels and regular menstrual cycles. Eighty-one hyperandrogenic patients (those presenting with hyperandrogenemic hirsutism or the polycystic ovary syndrome) were analyzed further. UCSNP45 alleles were distributed differently among the study groups. Heterozygosity for the uncommon C allele was increased in patients with idiopathic hirsutism (31.3%) and reduced in hyperandrogenic patients (7.4%) compared with controls (16.2%). The UCSNP44 and UCSNP43 alleles were in linkage disequilibrium, and were distributed equally among patients with idiopathic hirsutism, hyperandrogenism, and controls. However, the uncommon A allele at UCSNP43 was associated with higher hirsutism score (mean [+/- SD], 9.9 +/- 6.8, 12.7 +/- 7.7, and 14.6 +/- 8.2 in GG, GA, and AA participants, respectively). No other differences were observed in clinical and biochemical characteristics, including insulin sensitivity, by CAPN10 variant. CONCLUSION(S): The C allele at the UCSNP45 locus in CAPN10 is associated with idiopathic hirsutism, and UCSNP43 influences the hirsutism score.

Characterization of a 6p21 translocation breakpoint in a family with idiopathic generalized epilepsy.

The idiopathic generalized epilepsies (IGE), for which a genetic cause is widely accepted, account for 20-30% of all epilepsies. Mapping these epilepsies is difficult, but progress in the positional cloning of idiopathic epilepsy genes responsible for monogenic forms provide emerging evidence that many idiopathic epilepsies are caused by mutations in genes coding for ion channels. Here, we show the characterization of a balanced translocation present in three members of a nuclear family, two of them affected with IGE. The translocation involved chromosome 6p21 [t(4;6) (q35;p21)], a region in which a susceptibility locus for IGE (EJM1) has been reported. Fluorescence in situ hybridization analysis with YACs and PACs resulted in the identification of a PAC clone that included the 6p21 translocation breakpoint. The genomic sequence of this PAC clone contains two 2-pore potassium channel genes, TALK-1 and TALK-2. We characterized the genomic organization of both genes, including three different isoforms of TALK-1, and investigated them in IGE patients, finding some polymorphisms in the coding sequence of TALK-1A.

ITCH deficiency protects from diet-induced obesity.

Classically activated macrophages (M1) secrete proinflammatory cytokine and are predominant in obese adipose tissue. M2 macrophages, prevalent in lean adipose tissue, are induced by IL-13 and IL-4, mainly secreted by Th2 lymphocytes, and produce the anti-inflammatory cytokine IL-10. ITCH is a ubiquitously expressed E3 ubiquitin ligase involved in T-cell differentiation and in a wide range of inflammatory pathways. ITCH downregulation in lymphocytes causes aberrant Th2 differentiation. To investigate the role of Th2/M2 polarization in obesity-related inflammation and insulin resistance, we compared wild-type and Itch(-/-) mice in a context of diet-induced obesity (high-fat diet [HFD]). When subjected to HFD, Itch(-/-) mice did not show an increase in body weight or insulin resistance; calorimetric analysis suggested an accelerated metabolism. The molecular analysis of metabolically active tissue revealed increased levels of M2 markers and genes involved in fatty acid oxidation. Histological examination of livers from Itch(-/-) mice suggested that ITCH deficiency protects mice from obesity-related nonalcoholic fatty liver disease. We also found a negative correlation between ITCH and M2 marker expression in human adipose tissues. Taken together, our data indicate that ITCH E3 ubiquitin ligase deficiency protects from the metabolic disorder caused by obesity.

The MRC1/CD68 ratio is positively associated with adipose tissue lipogenesis and with muscle mitochondrial gene expression in humans.

BACKGROUND: Alternative macrophages (M2) express the cluster differentiation (CD) 206 (MCR1) at high levels. Decreased M2 in adipose tissue is known to be associated with obesity and inflammation-related metabolic disturbances. Here we aimed to investigate MCR1 relative to CD68 (total macrophages) gene expression in association with adipogenic and mitochondrial genes, which were measured in human visceral [VWAT, n = 147] and subcutaneous adipose tissue [SWAT, n = 76] and in rectus abdominis muscle (n = 23). The effects of surgery-induced weight loss were also longitudinally evaluated (n = 6). RESULTS: MCR1 and CD68 gene expression levels were similar in VWAT and SWAT. A higher proportion of CD206 relative to total CD68 was present in subjects with less body fat and lower fasting glucose concentrations. The ratio MCR1/CD68was positively associated with IRS1gene expression and with the expression of lipogenic genes such as ACACA, FASN and THRSP, even after adjusting for BMI. The ratio MCR1/CD68 in SWAT increased significantly after the surgery-induced weight loss (+44.7%; p = 0.005) in parallel to the expression of adipogenic genes. In addition, SWAT MCR1/CD68ratio was significantly associated with muscle mitochondrial gene expression (PPARGC1A, TFAM and MT-CO3). AT CD206 was confirmed by immunohistochemistry to be specific of macrophages, especially abundant in crown-like structures. CONCLUSION: A decreased ratio MCR1/CD68 is linked to adipose tissue and muscle mitochondrial dysfunction at least at the level of expression of adipogenic and mitochondrial genes.

Attenuated metabolism is a hallmark of obesity as revealed by comparative proteomic analysis of human omental adipose tissue.

Obesity is recognized as an epidemic health problem worldwide. In humans, the accumulation of omental rather than subcutaneous fat appears to be tightly linked to insulin resistance, type 2 diabetes and cardiovascular disease. Differences in gene expression profiles in the adipose tissue comparing non-obese and obese subjects have been well documented. However, to date, no comparative proteomic studies based on omental fat have investigated the influence of obesity in protein expression. In this work, we searched for proteins differentially expressed in the omental fat of non-obese and obese subjects using 2D-DIGE and MS. Forty-four proteins, several of which were further studied by immunoblotting and immunostaining analyses, showed significant differences in the expression levels in the two groups of subjects. Our findings reveal a clearly distinctive proteomic profile between obese and non-obese subjects which emphasizes: i) reduced metabolic activity in the obese fat, since most down-regulated proteins were engaged in metabolic pathways; and ii) morphological and structural cell changes in the obese fat, as revealed by the functions exerted by most up-regulated proteins. Interestingly, transketolase and aminoacylase-1 represent newly described molecules involved in the pathophysiology of obesity, thus opening up new possibilities in the study of obesity.