The increasing worldwide incidence of obesity and the limitations of current treatments raise the need for finding novel therapeutic approaches to treat this disease. The purpose of the current study was first to investigate the effects of tungstate on body weight and insulin sensitivity in a rat model of diet-induced obesity. Second, we aimed to gain insight into the molecular mechanisms underlying its action. Oral administration of tungstate significantly decreased body weight gain and adiposity without modifying caloric intake, intestinal fat absorption, or growth rate in obese rats. Moreover, the treatment ameliorated dislipemia and insulin resistance of obese rats. These effects were mediated by an increase in whole-body energy dissipation and by changes in the expression of genes involved in the oxidation of fatty acids and mitochondrial uncoupling in adipose tissue. Furthermore, treatment increased the number of small adipocytes with a concomitant induction of apoptosis. Our results indicate that tungstate treatment may provide the basis for a promising novel therapy for obesity.

Aims/hypothesis. Sodium tungstate has recently emerged as an effective oral treatment for diabetes. We examined the effects of tungstate administration in the beta-cell mass of the pancreas as well as its therapeutic potential. Methods. Sodium tungstate was administered via drinking water to healthy and neonatal streptozotocin (nSTZ)-diabetic rats for one month. The pancreas from each rat was removed and morphometric and immunocytochemical studies were carried out. The molecular mechanism of tungstate's action was also studied. Results. In nSTZ rats administration of this compound normalised glycaemia, and increased insulinaemia and islet insulin content. Blood glucose concentrations were normalised as early as on day 4 of treatment, and tungstate treatment produced a partial recovery of beta-cell mass. The rats remained normoglycaemic after tungstate withdrawal. Morphometric studies showed that the increase in beta-cell mass was not due to beta-cell hypertrophy but to hyperplasia, with an increase in islet density in treated diabetic rats. Tungstate treatment increased extra-islet beta-cell replication without modifying intra-islet beta-cell replication rates. Moreover, the treatment induced increases in insulin-positive cells located close to ducts; and in PDX-1 positive cells scattered in the exocrine tissue, suggesting active neogenesis. In islets from treated diabetic rats, tungstate is able to increase the phosphorylation state of PDX-1 through the activation of p38. Conclusion/interpretation. These observations indicate that tungstate treatment is able to regenerate a stable, functional pancreatic beta-cell population which leads to and maintains normoglycaemia. [Diabetologia (2004)

A role for cytokine regulated proteins in epithelial cells has been suggested in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to identify such cytokine regulated targets using a proteomic functional approach. Protein patterns from (35)S-radiolabeled homogenates of cultured colon epithelial cells were compared before and after exposure to interferon-gamma, interleukin-1beta and interleukin-6. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Both autoradiographies and silver stained gels were analyzed. Proteins showing differential expression were identified by tryptic in-gel digestion and mass spectrometry. Metabolism related proteins were also investigated by Western blot analysis. Tryptophanyl-tRNA synthetase, indoleamine-2,3-dioxygenase, heterogeneous nuclear ribonucleoprotein JKTBP, interferon-induced 35kDa protein, proteasome subunit LMP2 and arginosuccinate synthetase were identified as cytokine modulated proteins in vitro. Using purified epithelial cells from patients, overexpression of indoleamine-2,3-dioxygenase, an enzyme involved in tryptophan metabolism, was confirmed in Crohn's disease as well as in ulcerative colitis, as compared to normal mucosa. No such difference was found in diverticulitis. Potentially, this observation opens new avenues in the treatment of IBD.

Recent efforts to sequence human cancer genomes have highlighted that point mutations in genes involved in the epigenetic setting occur in tumor cells. Small cell lung cancer (SCLC) is an aggressive tumor with poor prognosis, where little is known about the genetic events related to its development. Herein, we have identified the presence of homozygous deletions of the candidate histone acetyltransferase KAT6B, and the loss of the corresponding transcript, in SCLC cell lines and primary tumors. Furthermore, we show, in vitro and in vivo, that the depletion of KAT6B expression enhances cancer growth, while its restoration induces tumor suppressor-like features. Most importantly, we demonstrate that KAT6B exerts its tumor-inhibitory role through a newly defined type of histone H3 Lys23 acetyltransferase activity.

A fast and sensitive high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC-APCI-MS/MS) method to identify and quantify oxysterols, phytosterols and non-cholesterol sterols at the pico-molar concentration level in human serum in only one run was developed in this study. This method allows the simultaneous separation and quantitation of individual phytosterols, cholesterol precursors and oxidized derivatives of cholesterol without a derivatization step in a single run, thus providing a more confident quantitation of sterols in serum. After saponification, solid-phase extraction (SPE) used as a clean-up step and HPLC separation, detection by MS was developed using APCI and multiple ion monitoring modes. This method employs reversed-phase C 18 SPE cartridges and serum calibrators, as well as isotopically labelled cholesterol as an internal standard added before sample processing. The time consumed for a single sample is reduced from the 4 hours of conventional sterol analysis to 1 hour including the chromatographic run time. The method has been evaluated by analyzing a certified cholesterol sample as well as by comparison to other two methods used as reference, based on GC and enzymatic reaction, respectively. Serum from 14 individuals was successfully analyzed. Detection limits for oxysterols, phytosterols and non-cholesterol sterols, all determined in a single run in small serum volumes, were between 0.47 and 1.69 pM. Intra-day precision was <7% for all sterols in inhouse-made lipoprotein-deficient serum. The limits of detection showed high sensitivity with very good intraday-and interday-precision for all sterols. The recoveries of the phytosterols, cholesterol precursors and cholestanol ranged from 77% to 92%. These analytical parameters provide a reliable and reproducible method for the identification and quantitation of human sterols.

Adipose tissue plays an active role in the development of obesity, and thus characterization of the molecular changes related to obesity in this tissue is a priority. Recently, we identified tungstate as a potent body weight reducing agent in obese animals, adipose tissue being one of the targets of its action. In this study a proteomics approach combining 2-DE and MS was used to identify proteins associated with obesity and targets of tungstate in white adipose tissue. Twenty-nine proteins were found differentially expressed between lean and diet-induced obese rats. Expression changes in transferrin, vimentin, vinculin, peroxiredoxins, Rho-GTP dissociation inhibitor, grifin, guanine deaminase and 3-phosphoglycerate dehydrogenase were associated here for the first time with obesity. Furthermore, tungstate treatment of obese rats reverted expression changes of 70% of the proteins modulated by obesity and another ten proteins were regulated by tungstate independently of the body weight reduction. The results suggest that the tungstate antiobesity effect can be mediated by the modulation of cellular structure, metabolism, redox state and signalling processes in adipose tissue. These findings open new avenues for the study of the aetiology of obesity and its treatment.

Our previous results demonstrated that tungstate decreased weight gain and adiposity in obese rats through increased thermogenesis and lipid oxidation, suggesting that brown adipose tissue was one of the targets of its antiobesity effect. To identify potential targets of tungstate, we used DIGE to compare brown adipose tissue protein extracts from the following experimental groups: untreated lean, tungstate-treated lean, untreated obese, and tungstate-treated obese rats. To distinguish direct targets of tungstate action from those that are secondary to body weight loss, we also included in the analysis an additional group consisting of obese rats that lose weight by caloric restriction. Hierarchical clustering of analysis of variance and t test contrasts clearly separated the different experimental groups. DIGE analysis identified 20 proteins as tungstate obesity direct targets involved in Krebs cycle, glycolysis, lipolysis and fatty acid oxidation, electron transport, and redox. Protein oxidation was decreased by tungstate treatment, confirming a role in redox processes; however, palmitate oxidation, as a measure of fatty acid ␤-oxidation, was not altered by tungstate, thus questioning its putative function in fatty acid oxidation. Protein network analyses using Ingenuity Pathways Analysis highlighted peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC-1␣) as a potential target. We confirmed by real time PCR that indeed tungstate up-regulates PGC-1␣, and its major target, uncoupling protein 1, was also increased as shown by Western blot. These results illustrate the utility of proteomics and bioinformatics approaches to identify targets of obesity therapies and suggest that in brown adipose tissue tungstate modulates redox processes and increases energy dissipation through uncoupling and PGC-1␣ up-regulation, thus contributing to its overall antiobesity effect.

BackgroundProtein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast; however, few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A-related processes in mitotic arrested cells.ResultsWe identified 62 statistically significant PP2ACdc55 substrates involved mainly in actin-cytoskeleton organization. In addition, we validated new PP2ACdc55 substrates such as Slk19 and Lte1, involved in early and late anaphase pathways, and Zeo1, a component of the cell wall integrity pathway. Finally, we constructed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84–90, thus highlighting the relevance of these aminoacids for substrate interaction.ConclusionsWe used phosphoproteomics of Cdc55-deficient cells to uncover new PP2ACdc55 substrates and functions in mitosis. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases’ consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases distinct from Cdk1. Indeed, Pkc1 emerged as a novel node of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in actin cytoskeleton and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome.