Plasma levels of carnitine derivatives and carnitine precursors in male Wistar rats after three weeks of treatment.

<p><b>A.</b> Plasma L-carnitine; <b>B.</b> Plasma acetylcarnitine; <b>C.</b> Plasma γ-butyrobetaine; <b>D.</b> Plasma trimethyllysine; <b>E.</b> Protein expression of carnitine translocase (CACT). Values are shown as mean ± SD (n = 6–8). One-Way ANOVA with p<0.05 was followed by Fisher LSD to determine significant difference between all three groups. Different letters above bars indicate significant difference between group mean values, p<0.05; same letters above bars indicate no significant difference between group mean values p>0.05. C–Control, M–Mildronate (550 mg/kg body weight), MT–combination of Mildronate (550 mg/kg body weight) and 1-triple TTA (100 mg/ kg body weight).</p

Hepatic enzyme activities involved in lipid anabolism in male Wistar rats after three weeks of treatment.

<p>(A) Enzyme activity of acetyl-CoA carboxylase (ACC). (B) Enzyme activity of fatty acid synthase (FAS). (C) Enzyme activity of citrate-ATP lyase. (D) Enzyme activity of glycerol-3-phosphate transferase (GPAT). Values are shown as mean± SD (n = 6–8). One-Way ANOVA with p<0.05 was followed by Fisher LSD to determine significant difference between all three groups: Different letters above bars indicate significant difference between group mean values, p<0.05; same letters above bars indicate no significant difference between group mean values p>0.05. C–Control, M–Mildronate (550 mg/kg body weight), MT–combination of Mildronate (550 mg/kg body weight) and 1-triple TTA (100 mg/ kg body weight).</p

Hepatic β-oxidation and enzyme activities involved in lipid catabolism in male Wistar rats after three weeks of treatment.

<p>(A) Total β-oxidation of palmitoyl-CoA in liver; (B) Total β-oxidation of palmitoyl-CoA with addition of malonyl-CoA in liver; (C) Enzyme activity of acyl-CoA synthetase; (D) Enzyme activity of carnitine palmitoyltransferase (CPT) 2; (E) Enzyme activity of 3-ketothiolase; (F) Enzyme activity of malonyl-CoA decarboxylase (MCD); (G) Enzyme activity of acyl-CoA oxidase (ACOX); (H) Enzyme activity of citrate synthase. Values are shown as means ± SD (n = 6–8). One-Way ANOVA with p<0.05 was followed by Fisher LSD to determine significant difference between all three groups: Different letters above bars indicate significant difference between group mean values, p<0.05; same letters above bars indicate no significant difference between group mean values p>0.05. C–Control, M–Mildronate (550 mg/kg body weight), MT–combination of Mildronate (550 mg/kg body weight) and 1-triple TTA (100 mg/ kg body weight).</p

Hepatic gene expression in Wistar male rats after three weeks of treatment.

<p>Hepatic gene expression in Wistar male rats after three weeks of treatment.</p

Hepatic energy parameters in male Wistar rats after three weeks of treatment.

<p>(A) Energy charge (ATP + 0.5 ADP)/(AMP + ADP + ATP). (B) Ratio of AMP and ATP. Values are shown as mean ± SD (n = 6–8). One-Way ANOVA with p<0.05 was followed by Fisher LSD to determine significant difference between all three groups: Different letters above bars indicate significant difference between group mean values, p<0.05; same letters above bars indicate no significant difference between group mean values p>0.05. C–Control, M–Mildronate (550 mg/kg body weight), MT–combination of Mildronate (550 mg/kg body weight) and 1-triple TTA (100 mg/ kg body weight).</p

Plasma and liver lipids in male Wistar rats after three weeks of treatment.

<p>Plasma and liver lipids in male Wistar rats after three weeks of treatment.</p

ROS are induced by LPC and contribute to LPC-induced impairment of relaxation.

<p>A) The rings were equilibrated in 100 µl PSS buffer containing 10 µM DETCA and 10 µM lucigenin at 37°C for 30 minutes, followed by addition of PSS (control) or LPC (10 µM). Emitted light (RLU) was recorded every 10 seconds for 30 seconds. The RLU were normalised to protein content of respective aortic rings and expressed as percentage of control set to 100%. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065155#s3" target="_blank">Results</a> are means ± SEM of three separate experiments, each performed with three rings for each LPC. The rings were preincubated without (no LPC) or with 10 µM LPC 18:1 (B), 18:2 (C), 20:4 (D) or 16:0 (E) in the absence or presence of 200 µM Tempol for 30 minutes, followed by precontraction with NE and cumulative addition of ACh. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065155#s3" target="_blank">Results</a> for each condition are mean ± SEM of 12 rings from 6 mice. *P<0.05, **P<0.01.</p

ACh-induced relaxation of mouse aortic rings is attenuated by LPC.

<p>The rings were preincubated without (no LPC) or with 10 µM LPC 16:0 (A), 18:1 (B), 18:2 (C) or 20:4 (D) for 30 minutes, followed by precontraction with NE and cumulative addition of ACh. Relaxation values were expressed as a percentage of the NE-induced contraction. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065155#s3" target="_blank">Results</a> of each experimental condition are mean ± SEM of 24 rings for each case from 6 mice. *P<0.05, **P<0.01***P<0.001.</p

Inhibition of TXA₂- and PGI₂- synthase improves relaxation attenuated by LPC 20:4.

<p>The rings were preincubated without (no LPC) or with LPC 20:4 in the absence or presence of 10 µM furegrelate, a TXA₂ synthase inhibitor (A) or 10 µM tranylcypromine, a PGI₂ synthase inhibitor (B) or a combination of both (C) for 30 minutes, followed by precontraction with NE and cumulative addition of ACh. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065155#s3" target="_blank">Results</a> are mean ± SEM of 12 rings from 6 mice. *P<0.05, **P<0.01.</p

Prostanoid release from LPC-treated aortic rings.

<p>The rings were incubated in 200 µl aerated PSS under cell culture conditions at 37°C for 1 h. Thereafter, buffer was replaced with fresh PSS supplemented with PSS (control) or 10 µM LPC followed by further incubation under cell culture conditions at 37°C for 1 h. (A) 6-keto PGF_1α, a stable degradation product of PGI₂ (B), TXB₂, a stable degradation product of TXA₂, (C) PGE₂ and (D) PGF_2α were quantified by EIA assays and rings were solubilized for protein quantification. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065155#s3" target="_blank">Results</a> shown in A and B are means ± SD of four experiments and those in C and D of three experiments, done in triplicates. *P<0.05, **P<0.01***P<0.001.</p