Branched-chain fatty acids affect the expression of fatty acid synthase and C-reactive protein genes in the hepatocyte cell line

Abstract

Fatty acids (FAs) are known to play an important role in human metabolism; however, still little is known about the functions of certain FA classes present in blood at relatively low concentrations. Examples of such compounds include branched-chain fatty acids (BCFAs). Recently, lowered BCFAs blood concentration was noticed in obese patients. An inverse correlation was found between serum concentrations of BCFAs and triglyceride levels, as well as C-reactive protein (CRP) concentration. Obesity is the most frequently observed component of metabolic syndrome and both disorders are accompanied by the dysregulation of FAs metabolism. However, not all of them are well understood. Our study is the first attempt at presenting the opposite effects of an iso-BCFA (14-methylpentadecanoic acid, 14-MPA) and an anteiso-BCFA (12-methyltetradecanoic acid, 12-MTA) on selected genes related to fatty acid synthesis and inflammation: FASN, SREBP1, CRP, and IL-6 in the HepG2 cell line. We observed lowered expression of FASN, SREBP1, CRP, and IL-6 in cells treated with 14-MPA in comparison with control cells. In contrast, supplementation with 12-MTA caused opposite effects: increased mRNA levels of FASN, CRP, and IL-6. 12-MTA did not influence SREBP1 expression. The results of our preliminary study may suggest potential benefits of the supplementation of iso-BCFAs in obese patients, for inflammation and hypertriglyceridemia prevention.

Keywords: branched-chain fatty acids; hepatocytes; inflammation; nutrition; obesity; triglycerides.

Figure 1. HepG2 cells morphology (magnification: 400×) and viability

(A) HepG2 cells non-treated with BCFA. (B) HepG2 cells after treatment with 10 µM 14-MPA. (C) HepG2 cells after treatment with 10 µM 12-MTA. (D) Cell viability after treatment with 14-MPA. (E) Cell viability after treatment with 12-MTA. ○ - individual data points. Three independent experiments were performed and a total number of repetitions (n), summed from all experiments, is indicated in the bars.

Figure 2. Uptake of BCFAs by HepG2 cells from the experimental media

Concentration of BCFAs in HepG2 cells collected after 48 h of incubation with 10 µM 14-MPA or 10 µM 12-MTA. The analysis conducted in the samples prepared by pooling three individual cell cultures from a single experiment.

Figure 3. The mRNA levels of FASN and SREBP1 in HepG2 cells treated with 14-MPA and 12-MTA

(A) Cells treated with 14-MPA. (B) Cells treated with 12-MTA. Data in (A,B) are presented as means ± SEM; statistical test used: single-factor ANOVA with Tukey post-hoc test for unequal group size; *P-value<0.05 in comparison with control cells, ♦P-value<0.05 in comparison with cells treated with 1 μM BCFA; *P-value<0.05 in comparison with cells treated with 2 μM BCFA; ○ - individual data points. Three independent experiments were performed and a total number of repetitions (n), summed from all experiments, is indicated in the bars. Concentrations 1 and 2 μM of 14-MPA were used in two of the independent experiments only, with one repetition per experiment.

Figure 4. The mRNA levels of genes related to inflammation in HepG2 cells treated with 14-MPA and 12-MTA

(A) CRP mRNA levels. (B) IL-6 mRNA levels. Data in (A,B) are presented as means ± SEM; statistical test used: single-factor ANOVA with Tukey post-hoc test for unequal group size; *P-value<0.05 in comparison with control cells, ♦P-value<0.05 in comparison with cells treated with 1 μM BCFA; *P-value<0.05 in comparison with cells treated with 2 μM BCFA. Three independent experiments were performed and a total number of repetitions (n), summed from all experiments, is indicated in the bars. For IL-6 gene expression analysis, one repetition for 1 μM of 14-MTA was excluded (outlier), so the total number of repetitions in this case is two.