Abstract
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 beta-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 gamma coactivator 1alpha (PGC-1alpha) as a potential target. We confirmed by real time PCR that indeed tungstate up-regulates PGC-1alpha, 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-1alpha up-regulation, thus contributing to its overall antiobesity effect.
Highlights
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 specific targets of tungstate related to its antiobesity effect, we originally designed a strategy consisting of finding proteins that were modified by tungstate treatment in obese rats and that were not altered when lean rats were treated, selecting tungstate obesity targets
Proteomics analyses have been proven useful in the characterization of the adipocyte proteome [23], in the identification of obesity targets in different models of experimental obesity, and to characterize targets of several agents such as the insulin sensitizer rosiglitazone (24 –26) or our recently discovered antiobesity agent tungstate [6]
Summary
All animal procedures were conducted in accordance with principles of laboratory animal care (European Community and local government guidelines) and approved by the Animal Research Committee of the University of Barcelona. Proteins were considered identified when (a) the first hit was the same as that identified by Aldente and had a Mascot score above 25, (b) the peptide MS/MS had a ion score of at least 5, (c) a minimum of 10 ions were matched to the precursor ion from the candidate protein, and the highest peaks of the MS/MS spectra were assigned, and (d) the protein had a molecular weight/pI similar to the experimental values found from the 2D gels This threshold was selected and used as a confirmation of the previous identification with Aldente when the score was p Ͻ 1eϪ06 and the following non-homologous protein had a score of at least 1 order lower than the first hit. Results were analyzed using SDS2.1 software (Applied Biosystems) and normalized to 18S as the housekeeping gene (18 S rRNA predesigned TaqMan probe and primers Applied Biosystems), and statistics were analyzed by Student’s t test (p Ͻ 0.05)
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