Physiological parameters.

<p>(A) Body weight (BW) and (B) adipose tissues weights of T_reg cell-proficient (PBS) and T_reg cell-deficient (DT) mice after cold exposure. BAT, brown adipose tissue; scWAT, subcutaneous white adipose tissue, aWAT, abdominal white adipose tissue. (C) Blood glucose, (D) serum non-estherified fatty acids (NEFA) and (E) serum triglycerides in PBS and DT mice. Values are mean ± SD (n = 9–10); *P<0.05 (Student’s t-test).</p

Inflammatory status of adipose tissue.

<p>Real-time RT-PCR analysis of (A) brown adipose tissue (BAT) and (B) subcutaneous white adipose tissue of T_reg cell-proficient (PBS) and T_reg cell-deficient (DT) mice after cold exposure. Ucp1, uncoupling protein 1; Cidea,cell death-inducing DNA fragmentation factor, alpha subunit-like effector A; Dio2,deiodinase, iodothyronine, type II; Pparg, peroxisome proliferator-activated receptor gamma; Prdm16, PR domain containing 16; Cd68, Cd68 antigen; Ccl2,chemokine (C-C motif) ligand 2; Tnfa, tumor necrosis factor alpha; Ifng, interferon, gamma; Mrc1, mannose receptor, C type 1; Mgl1, macrophage galactose-type C-type lectin 1; Arg1,arginase 1; Il-10, interleukin 10; Il-4, interleukin 4. Data are mean ± SD (n = 9–10); *p<0.05 (Student’s t-test). (C) Representative hematoxylin and eosin (H&E) staining (left) and immunohistochemical anti-MAC-2 staining (right; brown color) in BAT from PBS and DT mice. Scale bar 100 μm. Quantification of MAC-2 positive area (panel below MAC-2 staining) as a percentage of total area.</p

Genotypical comparison of T_reg and T_conv cells isolated from brown adipose tissue (BAT) and spleen tissue (SPL) in cold- and warm-conditioned animals generated with an Illumina Mouse Expression Array.

<p>(A) Gene expression profiles comparing T_reg (top) or T_conv (bottom) cell populations between spleen and adipose tissue samples isolated from warm-conditioned animals (left) or between cells isolated from cold vs warm-conditioned animals (right). Numbers indicate genes either up- or downregulated more than 2-fold (cut-off: dotted line), with the number of significantly different (p<0.05) genes shown in brackets with an asterisk. (B) Volcano plot comparing gene expression and significance values between T_reg and T_conv genes isolated from BAT in warm-conditioned animals. Key up- or downregulated genes in T_reg cells are annotated (Foxp3, Il10, Cxcl1/2, Tcf7, Ifng) and serve as quality control to the published consensus T_reg-cell signature. (C) Hierarchical clustering of the top-30 upregulated genes and the top-10 downregulated genes in warm-conditioned brown adipose tissue T_reg cells versus spleen T_reg cells. (D) Comparison of BAT-T_reg-specific genes with visceral adipose tissue (VAT)-specific genes. We first determined 430 genes to upregulated in BAT warm-conditioned T_reg cells, with 222 genes being significantly altered (p<0.05). We then overlaid BAT T_reg-upregulated genes with VAT T_reg tissue specific expression gene data. 181 genes were matched between both microarary chips, with 169 genes also upregulated in VAT, and only 12 genes specific for BAT (left). The corresponding analysis of the 516 genes upregulated in cold BAT T_reg cells versus warm spleen T_reg cells revealed 194 genes to be significantly upregulated. 158 could be matched to VAT T_reg-specific genes, of which 148 were VAT-specific, whereas only 10 were specific for BAT. P-values indicate the significance of overrepresentation of BAT T_reg-specific genes in the VAT T_reg signature. (E) Comparison of VAT-T_reg specific genes on BAT warm (left) or BAT cold (right) gene signatures. Of 1839 genes specifically overexpressed in VAT T_reg cells, 1059 were statistically significantly (p<0.05) upregulated. Of these 1059 genes, 829 were also detectable in the BAT T_reg microarray. When comparing the VAT T_reg signature to warm BAT T_reg cells, 660 genes were overrepresented in VAT, whereas cold BAT T_reg cells show 685 genes to be overrepresented in VAT.</p

Principal biological processes affected by T_reg cell depletion in BAT.

<p>Principal biological processes affected by T_reg cell depletion in BAT.</p

Indirect calorimetry.

<p>At 6 weeks after the initiation of the experiment, oxygen consumption (<i>V</i>O₂) and carbon dioxide production were recorded every 2 min using indirect calorimetry. The measurements were performed following the diet-switch protocol in individual mice (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-g001" target="_blank">Fig. 1B</a>). During the first part of the measurements (between 6.00 p.m. and 8.00 a.m.), animals had <i>ad libitum</i> access to water and various cHF-based diets. After that period, the animals were fasted for 10 hours. At the beginning of the dark cycle at 6.00 p.m., all subgroups were switched to Chow diet, and the measurements continued for 20 more hours (‘Re-feeding Chow ‘). The measurements were performed under the 12-hour light-dark cycle (lights on from 6∶00 a.m.) at ambient temperature of 22°C. Data are means±SE (<i>n</i> = 5; mice randomly chosen from each subgroup, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-t001" target="_blank">Table 1</a>) expressed for the following three time-periods (i) from 0.00 p.m. to 8.00 a.m., feeding various cHF-based diets; (ii) from 9.00 a.m. to 5 p.m., fasting; and (iii) from 0.00 p.m. to 8.00 a.m., re-feeding Chow. ΔRER, the difference in RER between mice re-fed Chow diet and fasted mice.</p>a<p>Significantly different from cHF diet;</p>b<p>significantly different from cHF+ROSI diet (ANOVA).</p

Content of PDK4 protein in gastrocnemius muscle.

<p>Mice were killed either without any additional manipulations, that is, while offered the ‘original’ cHF-based diet in fed state (crossed bars), or following the diet-switch protocol when re-fed Chow diet (full bars). <b>A</b>. Representative Western blot analysis. <b>B</b>. Quantification of PDK4 protein in skeletal muscle. Values are means±S.E. (<i>n</i> = 5–8).^aSignificantly different from mice offered the cHF+F+ROSI diet (ANOVA).</p

Expression of selected genes in gastrocnemius muscle.

<p>Mice were killed either without any additional manipulations, that is, while offered various ‘original’ cHF-based diets (<b>OrD</b>; crossed bars), or following the diet-switch protocol when re-fed Chow diet (full bars); see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-g001" target="_blank">Fig. 1</a>. <b>A</b>.Genes involved in carbohydrate metabolism: pyruvate dehydrogenase kinase isozyme 4 (<i>Pdk4</i>); fructose-1,6-bisphosphatase isoenzyme 2 (<i>Fbp2</i>); and glucose transporter type 4 (<i>Glut4</i>). <b>B</b>. Genes involved in lipid metabolism: acyl-CoA thioesterase 1 (<i>Acot1</i>); carnitine palmitoyltransferase 1b (<i>Cpt1b</i>); and CD36 antigen (<i>Cd36</i>). <b>C</b>. Slow muscle (oxidative) fiber genes: myosin, heavy polypeptide 6 (<i>Myh6</i>); myosin, heavy polypeptide 7 (<i>Myh7</i>); and troponin C type 1 (<i>Tnnc1</i>). <b>D. </b><i>Pgc1α</i>. <b>E.</b> Cytochrome P450, family 1, subfamily a, polypeptide 1 (<i>Cyp1a1</i>). Data are means±SE (<i>n</i> = 7–8). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone.0043764.s007" target="_blank">Table S6</a>. ^aSignificantly different from cHF, OrD; ^bsignificantly different from cHF+F, OrD; ^csignificantly different from cHF+ROSI, OrD; ^dsignificantly different from cHF+F+ROSI, OrD; ^esignificantly different from cHF+F+ROSI, re-fed Chow (ANOVA).</p

Concentrations of selected metabolites in gastrocnemius muscle extracts.

<p>Analysis was performed in mice re-fed Chow diet (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-g003" target="_blank">Fig. 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-t001" target="_blank">Table 1</a>). <b>A.</b> Carnitines: propionyl-L-carnitine and isovalerlycarnitine (C3+C5); malonyl-L-carnitine (C4-OH); and various even-chain monounsaturated acylcarnitines (C10∶1, C14∶1, C16∶1, and C18∶1; individual acylcarnitines are denoted by their side chain; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone.0043764.s002" target="_blank">Table S1</a>). <b>B</b>. Amino acids. <b>C.</b> Lysophosphatidylcholines: stearoyl lysophasphatidylcholine (lysoPC C18∶0); linoleoyl lysophosphatidylcholine (lysoPC C18∶2); and arachidonoyl lysophasphatidylcholine (lysoPC C20∶4). <b>D.</b> Sphingolipids: palmitoyl sphingomyeline (SM C16∶0); stearoyl sphingomyeline (SM C18∶0); and hydroxysphingomyeline [SM(OH) C24∶1]. <b>D.</b> Data are means±SE (<i>n</i> = 7–8). Dietary groups are: cHF (black bars), cHF+F (blue bars); cHF+ROSI (green bars) and cHF+F+ROSI (violet bars). ^aSignificantly different from cHF; ^bsignificantly different from cHF+F; ^csignificantly different from cHF+ROSI (ANOVA).</p

Heatmap analysis of the effects of various interventions on selected analytes in the muscle.

<p>Analysis was performed in mice re-fed Chow diet (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-g001" target="_blank">Fig. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-g003" target="_blank">Fig. 3</a>). Heatmap representations of the pairwise correlation matrix were generated using selected muscle metabolites in mice fed cHF (<b>A</b>), cHF+F (<b>B</b>), cHF+ROSI (<b>C</b>), and cHF+F+ROSI (<b>D</b>) diets. Each square represents Pearson correlation coefficient between the metabolite in the row with that in the column. The strength of correlation (red, positive; green, negative) is expressed as a color intensity, see the color scale bar. CX, acylcarnitine with the chain of X carbons; SHORT, sum of acylcarnitines C3-C7; MEDIUM, sum of carnitines C8-C13; LONG, sum of acylcarnitines C14–18; TOTAL, sum of all acylcarnitines.</p

Growth characteristics and plasma parameters.

<p>Three-month-old mice were placed on various diets and killed 8 weeks thereafter. Plasma parameters were followed as described in Methods, either in mice with free access to various cHF-based diets, or when mice were re-fed Chow (using the diet-switch protocol; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-g001" target="_blank">Fig. 1C</a>). BHB, β-hydroxybutyrate in the animals re-fed Chow.</p>a<p>Significantly different from cHF;</p>b<p>significantly different from cHF+F;</p>c<p>significantly different from cHF+ROSI;</p>d<p>significantly different from cHF+F+ROSI (ANOVA).</p>f<p>Significantly different from Chow (<i>t</i>-test).</p