L⁵⁰⁰ and L⁵⁰³ are critical for the targeting of Glut4 to GSVs in the basal state.

<p>After co-expression of wild-type Glut4/eGFP and the various mutants within the IRM region of Glut4/Dsred by recombinant adenovirus infection, adipocytes were serum starved for 2 hours, washed with cold PBS, fixed with 4% paraformaldehyde, and then subjected to confocal microscopy analysis. The red color on the left panels represents mutants of Glut4/Dsred, the green color in the middle panels represents wild type Glut4/eGFP, and the yellow color in the right panels represents the colocalization of Glut4/eGFP and mutated Glut4/Dsred. The images were taken approximately through the middle of the cells and are representative of 4–5 independent experiments. The scale bar represents 10 μm.</p

Regulated energy metabolism proteins are CREB targets.

<p>The diagram depicts significantly regulated proteins in the glycolysis, TCA and electron transport pathways. The thirteen significantly regulated proteins are shown as large symbols. In addition, 12 of the 13 regulated proteins are from CREB target genes (shown as red symbols).</p

The expression of significantly regulated proteins from cytosolic extracts of rat NAcc (P<0.05) comparing EC vs. IC basal expression (blue), IC stress (green), and EC stress (orange), which were analyzed by Progenesis SameSpots in CBB stained gels.

<p>The expression of significantly regulated proteins from cytosolic extracts of rat NAcc (P<0.05) comparing EC vs. IC basal expression (blue), IC stress (green), and EC stress (orange), which were analyzed by Progenesis SameSpots in CBB stained gels.</p

Dynamic Proteomics of Nucleus Accumbens in Response to Acute Psychological Stress in Environmentally Enriched and Isolated Rats

<div><p>Our prior research has shown that environmental enrichment (<i>i.e.</i> rats reared in an environment with novel objects, social contact with conspecifics) produces a protective antidepressant-like phenotype in rats and decreases neurobiological effects of acute psychological stress. Although CREB activity has been identified as a major player, the downstream molecular mechanisms remain largely unexplored. Thus, the current study investigates proteomic differences in the accumbens of rats raised in an enriched condition (EC) versus those raised in an isolated control condition (IC) under basal conditions and after 30 min of acute restraint stress. Results showed that under basal conditions, EC rats generally expressed less mitochondria-related proteins, particularly those involved in TCA cycle and electron transport compared to IC rats. After 30 min of acute stress, EC rats displayed <b>increased</b> expression of energy metabolism enzymes (among others) while IC rats exhibited <b>decreased</b> expression of similar proteins. Further, network and pathway analyses also identified links to AKT signaling proteins, 14-3-3 family proteins, heat-shock proteins, and ubiquitin-interacting proteins. The protein ENO1 showed marked differential expression and regulation; EC rats expressed higher levels under basal conditions that increased subsequent to stress, while the basal IC expression was lower and decreased further still after stress. The results of this study define differential protein expression in a protective rat model for major depression and additionally identify a dynamic and coordinated differential response to acute stress between the two groups. These results provide new avenues for exploration of the molecular determinants of depression and the response to acute stress.</p></div

Canonical pathways identified <i>via</i> IPA analysis.

<p>Canonical pathways identified <i>via</i> IPA analysis.</p

L⁵⁰⁰ and L⁵⁰³ are critical for insulin-stimulated translocation of Glut4 to the cell periphery.

<p>After co-expression of wild-type Glut4/eGFP and the various mutants within the IRM region of Glut4/Dsred using recombinant adenovirus infection, adipocytes were serum starved for 2 hours, stimulated with 1 μM insulin for 30 minutes, washed with cold PBS, fixed with 4% paraformaldehyde, and then subjected to confocal microscopy analysis. The red color in the left panels represents mutants of Glut4-Dsred, the green color in the middle panels represents wild type Glut4/eGFP, and the yellow color in the right panels represents the colocalization of Glut4/eGFP and mutated Glut4/Dsred. The images were taken approximately through the middle of the cells and are representative of 4–5 independent experiments. The scale bar represents 10 μm.</p

Significant networks identified via IPA analysis of (a) basal EC vs. IC protein expression, (b and c) IC stress, and (d) EC stress.

<p>Red symbols represent upregulated and green denote downregulated proteins. Asterisks (*) denote multiple spots mapping to the same protein.</p

Immuno-adsorption of Glut4 and IRM mutant vesicles for Mass Spectrometry.

<p>RSV and SSV vesicles were pre-cleared with anti-rabbit IgG beads for 2 hours and the supernatant fractions were immuno-adsorbed with polyclonal anti-Glut4 magnetic beads. The supernatant fractions were subsequently subjected to immuno-adsorption with anti Dsred beads (to adsorb the mutant-containing vesicles). After washing, equal aliquots of the eluates from the beads were subjected to SDS PAGE and then subjected to immuno-blotting with monoclonal IF8 anti-Glut4 or anti-HA antibody (to detect the IRM mutant).</p

The IRM mutant was preferentially localized to the RSV fraction with a different sedimentation pattern.

<p>IRM/dsred mutant- (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068516#pone-0068516-g001" target="_blank">Figure 1</a>) infected adipocytes were serum starved overnight on day 8–9 post-differentiation and were then subjected to subcellular fractionation. The proteins from the RSV or SSV subcellular fractions were separated by SDS-PAGE, and then subjected to immunoblot analysis. (A). A representative western blot is shown on the left, and the quantification on the right shows the mean±SE from 3 independent experiments. “**” indicates P≤0.01 compared with control endogenous wild type Glut4. The isolated SSV (B) and RSV (C) subcellular fractions were subjected to sucrose velocity gradient analysis as described in “Experimental Procedures”. The fractions were collected from the bottom of the gradients and subjected to total protein quantification (left panels) or immunoblot analysis for endogenous wild-type Glut4 and the IRM mutant (upper pictures and lower quantifications). The data shown are representative of 2 independent experiments.</p

Proteins significantly regulated in the cytosolic fraction of rat NAcc in EC and IC rats.

<p>Proteins significantly regulated in the cytosolic fraction of rat NAcc in EC and IC rats.</p