Plasma S1P concentrations are elevated in obesity.

<p>Plasma S1P concentrations in chow (N = 13) and HFD (N = 14) fed mice (A); plasma S1P levels in lean (N = 6) and <i>ob/ob</i> (N = 8) mice (B) and circulating S1P in lean (N = 15) and obese (N = 10) humans (C). Data are mean ± SEM. **P&lt;0.01.</p

Plasma S1P concentrations in mice are elevated in response to fasting.

<p>Changes in body mass (A), blood glucose (B), plasma NEFA (C) and plasma S1P concentrations (D) in response to fasting. Data are mean ± SEM. *P&lt;0.05; **P&lt;0.01, N = 11–12.</p

Plasma S1P concentrations correlate with clinical indices of metabolic dysfunction in humans.

<p>Plasma S1P concentrations correlate with percent body fat (A), BMI (B), waist circumference (C), fasting plasma insulin (D), HOMA-IR (E), HbA1c (F), total (G) and LDL (H) cholesterol.</p

Myotube Akt phosphorylation, human primary macrophage cholesterol content and THP-1 macrophage JNK phosphorylation. (a)

<p>Phosphorylation of Akt at Serine-473 in human primary skeletal myotubes treated as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056601#pone-0056601-g001" target="_blank">Figure 1</a>, n = 6/group. <b>(b)</b> Total intracellular cholesterol (free cholesterol and cholesteryl esters) in human primary macrophages after treatment with vehicle (PBS) or 75 µg/ml acLDL/10 µg/ml Sandoz with or without 50 µg/ml HDL for 18 hours, n = 6/group. <b>(c)</b> Phosphorylation of JNK in THP-1 macrophages treated with vehicle or 75 µg/ml acLDL/10 µg/ml Sandoz with or without 50 µg/ml HDL for 1, 2 and 4 hours. n = 9/group; data are presented as mean ± SEM; *indicates significantly different from Con at the corresponding time point, P<0.05.</p

Basal and insulin-stimulated glucose uptake in cultured skeletal myotubes.

<p>Conditioned media (2% in final media volume) from <b>(a,</b> basal state; <b>b</b> insulin-stimulated<b>)</b> primary human and <b>(c,</b> basal state; <b>d</b> insulin-stimulated<b>)</b> THP-1 macrophages treated with vehicle (PBS), 75 µg/ml acLDL/10 µg/ml Sandoz compound with or without 50 µg/ml HDL for 18 hours was placed on human primary skeletal myotubes for 24 hours. Cells were treated with insulin (100 nM) or vehicle for 30 minutes before glucose uptake was measured. n = 6/group; data are presented as mean ± SEM; *indicates significantly different from Con and acLDL+HDL, P<0.05.</p

Fold-change in UCP1 protein content post-differentiation compared with pre-differentiation (n = 4, L; Lean, O; Obese; *P<0.05 vs pre-differentiation, † P<0.05 vs Obese post-differentiation, pre-differentiation normalized to 1.0).

<p>Fold-change in UCP1 protein content post-differentiation compared with pre-differentiation (n = 4, L; Lean, O; Obese; *P<0.05 vs pre-differentiation, † P<0.05 vs Obese post-differentiation, pre-differentiation normalized to 1.0).</p

Primers and probes.

<p>Primers and probes used in real time PCR.</p

UCP1 and PGC-1α mRNA content in iWAT.

<p>UCP1 and PGC-1α mRNA in iWAT after 7 days of daily injection of IL-6 or PBS in C57BL/6 mice (A and B), in response to 3 days of cold exposure in WT and IL-6 KO mice (C and D) and in untrained and trained WT and IL-6 KO mice (E and F). The target mRNA is normalized to single stranded (ss) DNA or TBP mRNA content. Values are mean ± SE, (n = 6–12). *: significantly different from PBS or 22°C (p<0.05).</p

UCP1 protein Content in iWAT.

<p>iWAT UCP1 protein content after 7 days of daily injection of IL-6 or PBS in C57BL/6 mice (A), in untrained and trained WT and IL-6 KO mice (B), and in response to 3 days of cold exposure in WT and IL-6 KO mice (C). Representative blots from 7 days of daily injection of IL-6 or PBS in C57BL/6 mice (D), from untrained and trained WT and IL-6 KO mice (E) and from WT and IL-6 KO mice after 3 days of cold exposure (F). UCP1 antibody was verified with lysate from retinoblastoma deficient (RB −/−) differentiated to brown adipocytes cells and mouse iWAT (G). UCP1 protein content is normalized to β-actin protein content. Values are mean ± SE, (n = 6–12). #: significantly different from WT (p<0.05).*: significantly different from 22°C (p<0.05). A horizontal line indicates a main effect.</p

Cidea and Elovl3 mRNA content in iWAT.

<p>iWAT Cidea and Elovl3 mRNA content after 7 days of daily injection of IL-6 or PBS in C57BL/6 mice (A and B), in response to 3 days of cold exposure in WT and IL-6 KO mice (C and D) and in untrained and trained WT and IL-6 KO mice (E and F). The target mRNA is normalized to single stranded (ss) DNA or TBP mRNA content. Values are mean ± SE, (n = 6–12).</p