<i>In vivo</i> sodium tungstate effects.

<p>Mice were treated with 2 g/l of sodium tungstate in water for 14 days. Body weight gain (A) and food intake (B) were measured for the duration of treatment, and blood glucose (C) and TG (D) were measured at the beginning and at the end of treatment. Open squares correspond to untreated lean mice (UL) (n = 6), black squares to treated lean mice (TL) (n = 6), open circles to untreated obese mice (UO) (n = 6), and black circles to treated obese mice (TO) (n = 6). In the bar graph, white and black bars represent untreated and treated animals, respectively. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001 vs. lean mice. ⁺⁺⁺<i>p</i><0.001 treated lean vs. untreated lean mice. ^#<i>p</i><0.05; ###p<0.001 treated obese vs. untreated obese mice. Data are the mean±SD.</p

Sodium tungstate treatment significantly modulates expression of proteins involved in neuronal plasticity.

<p>Brains from mice fed with a standard diet were sliced following 14 days of tungstate treatment and were prepared for immunoreactivity assays. (A) c-fos antibody reveals an increase in c-fos immunoreactive neurons after tungstate treatment, (B) as does the GFAP antibody. (C) The β-tubulin antibody and (D) HPC-1 show a decrease in immunoreactive neurons for these proteins. (E) NeuroD-1 immunoreactivity and (F) SNAP25 immunoreactivity increase after treatment. Scale bar  = 50 µM. White and black bars represent untreated and treated animals, respectively. **<i>p</i><0.01; ***<i>p</i><0.001. Data are the mean±SD.</p

Tungstate targets identified by proteomics in different hypothalamic nuclei.

a<p>Spot numbers corresponding to 2D images for each type of nuclei (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039087#pone-0039087-g002" target="_blank">Figure 2</a>).</p>b<p>Mice phenotypes: L  =  lean. O  =  obese.</p>c<p>Ratios of protein expression levels calculated using DeCyder software as the fold change in normalized spot volume comparing tungstate treated and non treated animals (Student’s <i>t</i> test based on the log of the ratio of the treated group to the control group).</p

Sodium tungstate modifies neuronal microstructure.

<p>Brains from treated and untreated lean mice were analyzed by MRI. (A) Sodium tungstate treatment did not alter fractional anisotropy (FA), neither in the PVN nor in the ARC. In the PVN, treatment decreased: (B) Main diffusivity (MD); (C) axial diffusivity (AD); and (D) radial diffusivity (RD). White and black bars represent untreated and treated animals, respectively. *p<0.05; **<i>p</i><0.01. Data are the mean±SD.</p

Representative 2D gels from hypothalamic nuclei.

<p>Hypothalamic nuclei were isolated and subjected to 2D gel electrophoresis, with the first dimension being the pI from 3 to 10, and the second dimension being the MW resolved in a 10% acrylamide gel. Spot profile analysis was performed using DeCyder software. (A) Representative 2D gels from each hypothalamic nucleus. pI increased from left to right, and MW decreased from top to bottom. All spots exhibiting significant differences (numbered) were selected for protein digestion mass spectrometry, and protein identification summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039087#pone.0039087.s001" target="_blank">table S1</a>. As an example, a close-up image of differentially expressed proteins between untreated and treated obese mice is shown in (B).</p

Sodium tungstate has an effect on the CRMP2 protein.

<p>An important portion of all spots isolated were identified as CRMP2. (A) Representative ARC 2D gel from treated lean mouse. Insert image shows different CRMP2 proteins. Numbers correspond to identification of CRMP2 by mass spectrometry. (B) Protein identification using mass spectrometry was verified performing 2D-Western blot (insert image). Spots identified as CRMP2 by mass spectrometry co-localized perfectly with those identified by 2D-western blot, confirming that identification had been performed correctly. (C) p-CRMP2^T514 antibody reveals that a significant number of posttranslational changes of this protein involve phosphorylation, and the great pI variability indicates that multiple posttranslational changes occur all at once. (D) Representative close-up images of spots with different pI corresponding to CRMP2.</p