Barceló-Batllori Sílvia, Kalko Susana G, Esteban Yaiza, Moreno Sílvia, Carmona María C, Gomis Ramon
Laboratory of Experimental Diabetes and Obesity, Endocrinology and Diabetes Unit, Hospital Clínic de Barcelona, E-08036 Barcelona, Catalonia, Spain.
Mol Cell Proteomics. 2008 Feb;7(2):378-93. doi: 10.1074/mcp.M700198-MCP200. Epub 2007 Nov 5.
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.
我们之前的研究结果表明，钨酸盐可通过增加产热和脂质氧化来降低肥胖大鼠的体重增加和肥胖程度，这表明棕色脂肪组织是其抗肥胖作用的靶点之一。为了确定钨酸盐的潜在靶点，我们使用差异凝胶电泳（DIGE）比较了以下实验组棕色脂肪组织的蛋白质提取物：未处理的瘦鼠、钨酸盐处理的瘦鼠、未处理的肥胖鼠和钨酸盐处理的肥胖鼠。为了区分钨酸盐作用的直接靶点与那些继发于体重减轻的靶点，我们在分析中还纳入了另一组通过热量限制减肥的肥胖大鼠。方差分析和t检验对比的层次聚类清楚地分离了不同的实验组。DIGE分析确定了20种蛋白质为钨酸盐肥胖直接靶点，这些靶点参与三羧酸循环、糖酵解、脂肪分解和脂肪酸氧化、电子传递以及氧化还原过程。钨酸盐处理可降低蛋白质氧化，证实其在氧化还原过程中的作用；然而，作为脂肪酸β氧化指标的棕榈酸氧化并未因钨酸盐而改变，因此对其在脂肪酸氧化中的假定功能提出了质疑。使用Ingenuity Pathways Analysis进行的蛋白质网络分析突出显示过氧化物酶体增殖物激活受体γ共激活因子1α（PGC-1α）为潜在靶点。我们通过实时PCR证实，钨酸盐确实上调了PGC-1α，并且如蛋白质印迹所示，其主要靶点解偶联蛋白1也增加了。这些结果说明了蛋白质组学和生物信息学方法在确定肥胖治疗靶点方面 的实用性，并表明在棕色脂肪组织中，钨酸盐可调节氧化还原过程，并通过解偶联和上调PGC-1α增加能量消耗，从而有助于其整体抗肥胖作用。
