Effects Of Conjugated Linoleic Acid On The Lipid Metabolism In HepG2 Cells And Its Mechanisms Research

Conjugated linoleic acid (CLA) refers to a group of polyunsaturated fatty acids that exist as positional and geometric isomers of linoleic acid (LA; 18:2). A lot of researches showed that CLA possessed many physiological functions such as anticarcinogenesis, antiatherosclerosis, antidiabetogenesis, stimulating immunity, increasing bone density, reducing body fat etc. In particular, the ability of CLA to reduce body fat had received considerable attention. However, some researches showed that CLA decreased body fat and caused hepatomegaly accompanying lipid deposition in liver; some other researches found that CLA increased catabolism and reduced fat in liver. The precise mechanisms by which CLA elicit its effects on liver are still largely unknown.The serine/threonine kinase-protein kinase B (PKB/Akt) has emerged as a critical signaling node within all cells of higher eukaryotes. As a pivotal downstream target of phosphatidylinositol 3-kinase (PI3K) after its phosphorylation, PKB/Akt is stimulated by cytokine and growth factors, and plays a vital role in diverse biological process including cell metabolism, cell survival, cell cycle regulation and transcriptional control. PI3K-PKB/Akt is the important signal pathway by which the insulin mediates to regulate the organism metabolism. After being stimulated by insulin, PI3K-PKB/Akt can regulate lipometabolism and glycometabolism in cells. Therefore, we hypothesized PI3K-PKB/Akt signal pathway influenced the effect of CLA on lipid metabolism in liver.In our experiments, HepG2 cells were adopted to study the possible mechanisms of CLA on lipometabolism in liver by examining lipid deposition, mRNA expression of acetyl-CoA carboxylase (ACC), Akt, p-Akt, AMPK, p-AMPK protein level. In addition, LY294002, an inhibitor of PI3K, was used to make further study to elucidate whether PI3K-PKB/Akt signal pathway influenced the effect of CLA on lipometabolism in HepG2 cells.MethodsIn vitro, HepG2 cells were incubated respectively with different concentration of CLA (10μmol/L, 50μmol/L, 100μmol/L) for 24 hours, and then were treated with insulin (100nmol/L) for 1 hour. Free fatty acid content was determined using colorimetric method; lipid deposition was observed by the use of oil red O stain; mRNA expression of acetyl-CoA carboxylase (ACC) was detected by reverse transcription polymerase chain reaction (RT-PCR); protein level of p-Akt, Akt, p-AMPK, AMPK were detected by Western-blot. LY294002 was used to make further study to observe the effect of CLA on lipid deposition in HepG2 cells. Results(1) CLA increased lipid deposition in cells. Red lipid droplets increased markedly in Group 50μmol/L and Group 100μmol/L of CLA compared with control group.(2) Free fatty acid in the medium decreased after treatment with CLA. Free fatty acid content in Group 50μmol/L and Group 100μmol/L of CLA decreased by 25 % and 50 % respectively compared with control group (P﹤0.05).(3) CLA increased the mRNA expression of ACC in HepG2 cells. Compared with control group, mRNA expression of ACC increased gradually with the increase of CLA concentration. Compared with insulin Group, mRNA expression of ACC in Group 50μmol/L and Group 100μmol/L of CLA increased by 1.38-fold and 1.53-fold respectively.(4) CLA increased phosphorylation level of Akt. Compared with insulin Group, phosphorylation level of Akt in Group 50μmol/L and Group 100μmol/L of CLA increased by 1.98-fold and 2.31-fold respectively. ( n=3, P﹤0.05).(5) Phosphorylation level of AMPK decreased along with the increase in CLA concentration. Compared with insulin Group, phosphorylation levels of AMPK in Group 50μmol/L and Group 100μmol/L of CLA decreased significantly( n=3, P﹤0.05). There was no significant difference between Group 10μmol/L of CLA and control group.(6) After pretreatment with LY294002, lipid deposition decreased in cells and red lipid droplets decreased markedly. Free fatty acid in the medium increased and the mRNA expression of ACC decreased.ConclusionsOur research showed that CLA increased lipid deposition in HepG2 cells. The study of the molecule mechanism suggested that CLA played a significant part in increasing the phosphorylation level of Akt protein, improving the mRNA expression of ACC and inhibiting the phosphorylation level of AMPK protein, all of which were involved in the effect of CLA on lipid deposition. Inhibition of PI3K by LY294002 exhibited inversion of lipid deposition and mRNA expression of ACC. The results suggested that PI3K-PKB/Akt signal pathway was associated with effect of CLA on lipid deposition. However, lipometabolism is a complex process in liver. Therefore, it is necessary to make further study to elucidate how does CLA regulate the PI3K-PKB/Akt signal pathway and if other mechanisms existence.