Effects of 13-oxo-ODA on PPARα activation determined by luciferase assay and using mouse primary hepatocytes.

<p>(A) Chemical structures of 13-oxo-ODA, 9-oxo-ODA, and CLA. (B) A reporter plasmid (p4xUASg-tk-luc) and an expression vector for a GAL4 PPARα chimeric protein (pM-hPPARα) were transfected into CV1 cells together with an internal control reporter plasmid (pRL-CMV). Twenty-four hours after the transfection, the cells were treated with synthesized CLA or 13-oxo-ODA for 24 h. GW7647 (5 nM), which is a PPARα-specific agonist, was used as a positive control. Luciferase activity was measured using a dual luciferase system. The activity of a vehicle control was set at 100%, and the relative luciferase activities are presented as fold induction with respect to that of the vehicle control. Data are presented as mean ± SEM (n = 4−5). **; <i>p</i><0.01 <i>versus</i> control. #; <i>p</i><0.05 compared between indicated groups.</p

Effects of 13-oxo-ODA on glucose tolerance in HFD-fed KK-Ay mice.

<p>OGTT was performed at 3 weeks period. For OGTT, glucose (1.5 g/kg body weight) was orally administered after an overnight fasting. Each bar represents the mean ± SEM (n = 6−8). ◊, vehicle; ▴,0.02% 13-oxo-ODA; •, 0.05% 13-oxo-ODA. *; <i>p</i><0.05, **; <i>p</i><0.01 <i>versus</i> control.</p

Effects of 13-oxo-ODA on mRNA expression levels of genes involved in lipid metabolism in the liver of HFD-fed KK-Ay mice.

<p>The mRNA expression levels of genes involved in fatty acid metabolism (<i>CPT1a</i>, <i>AOX</i>, <i>FAT</i>, <i>ACS</i>, and <i>UCP2</i>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031317#pone-0031317-g003" target="_blank">Fig. 3A</a>) and in TG synthesis (<i>SREBP1c</i>, <i>ABCA1</i>, <i>ABCG1</i>, and <i>FAS</i>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031317#pone-0031317-g003" target="_blank">Fig. 3B</a>) in the liver of control and 13-oxo-ODA-fed mice were quantified by real-time PCR. The relative amount of each transcript was normalized to the amount of the <i>36B4</i> transcript. The expression levels in the vehicle control are set at 100% and the relative expression levels are presented as fold induction with respect to that in the vehicle control. (C) Protein expression of AOX and β-actin in the liver of control and 13-oxo-ODA-fed mice were compared by immunoblotting assay. The same amounts of protein (25 µg/lane) were loaded and blotted onto PVDF membranes. The membranes were sequentially treated with primary antibodies as indicated and secondary antibodies conjugated with HRP. The enhanced chemiluminescence system was used for visualization of membranes. Data are presented as mean ± SEM (n = 6−8). *; <i>p</i><0.05 <i>versus</i> control.</p

Effects of 13-oxo-ODA on carbohydrate metabolism in HFD-fed KK-Ay mice.

<p>Fasting plasma glucose (A), insulin (B), and adiponectin (C) in the KK-Ay mice fed HFD with or without 13-oxo-ODA for 4 weeks. Each bar represents the mean ± SEM (n = 6−8). *; <i>p</i><0.05 <i>versus</i> control.</p

Effects of 13-oxo-ODA on rectal temperature in HFD-fed KK-Ay mice.

<p>At 4 weeks of the treatment period, the rectal temperature of all mice was measured using a thermometer probe. Data are presented as mean ± SEM (n = 6−8). *; <i>p</i><0.05 <i>versus</i> control.</p

Effects of 13-oxo-ODA on mRNA expression levels of genes involved in lipid metabolism in the skeletal muscle of HFD-fed KK-Ay mice.

<p>The mRNA expression levels of genes involved in fatty acid metabolism (<i>CPT1b</i>, <i>AOX</i>, <i>FAT</i>, <i>ACS</i>, and <i>UCP2</i>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031317#pone-0031317-g004" target="_blank">Fig. 4A</a>) and in TG synthesis (<i>SREBP1c</i>, <i>ABCA1</i>, <i>ABCG1</i>, and <i>FAS</i>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031317#pone-0031317-g004" target="_blank">Fig. 4B</a>) in the skeletal muscle of control and 13-oxo-ODA-fed mice were quantified as described before. Data are presented as mean ± SEM (n = 6−8). *; <i>p</i><0.05, **; <i>p</i><0.01 <i>versus</i> control.</p

Effects of 13-oxo-ODA on plasma and hepatic TG contents in HFD-fed KK-Ay mice.

<p>Plasma TG concentration (A) and hepatic TG content (B) at the end of treatment period. Data are presented as mean ± SEM (n = 4−5). *; <i>p</i><0.05 <i>versus</i> control. (C) Isolated livers were fixed in 10% formalin/PBS for more than 24 h and then embedded. Liver sections were cut into 10 µm sections. The liver sections were stained with Oil Red O and Mayer's hematoxylin. A bar shown in each photograph indicates 50 µm.</p