| Background:Liver fatty acid binding protein(L-FABP) which is a small cytosolic protein is a member of the superfamily of lipid-binding proteins. Liver fatty acid-binding protein (L-FABP) is a highly conserved key factor in lipid metabolism.which is involved in the transport and metabolism of intracellular FFA. A threonine to alanine substitution at codon 94 in the exon 3, might alter its function and thereby affectlipid metabolism in lipid-exposed subjects, as indicated by studies in L-FABP knockout mice.Objective:This study was undertaken to verify whether the T94A mutation in the L-FABP gene affects fatty acid uptake and intracellular esterification into specific lipid pools. Candidate SNPs were recreated using site-directed mutagenesis and tested for physical function in stably transfected Chang liver cell lines.Methods:L-FABP stably Transfected Chang Liver Cells was used which were divided into 4 groups:wild type, mutant group, empty vector group (transfected with wild-type L-FABP, mutant L-FABP and empty vector transfected Chang s liver cells) and normal control group (normal Chang liver cells).Protein determination by Western Blot. Localization of the recombinated protein in the cell by immunohistochemistry. Radiolabeled oleic acid uptake studies were performed on chang liver in different times. Then cells were lysed and the radioactivity taken up was determined by liquid scintillation counting. Efflux studies were performed on chang liver which loaded with [3H]-oleic acid for 15 min,the radioactivity was measured for different intervals of times. The in vitro models steatosis was established with the FFAs mixture (oleate:palmitate/2:1) which is associated with minor toxic and apoptotic effects, thus representing a cellular model of steatosis that mimics benign chronic steatosis. Intracellular Lipid deposition was determined by Oil Red O stainingTriglyceride and cholesterol levels were measured using a commercial kit and normalized to the protein content of the homogenate.Results:Immunohistochemistry identified the recombinated protein mainly expressed in endochylema which is coincident with the normal physiological expression.The function analysis identified L-FABP correlated with the FFA uptake and after a 60-min incubation, the oleic acid uptake value of wild-type-transfected cells was "double the values of the other three cell types (respectively,1.07±3.12,5.82±0.72,5.74±0.11 and 5.70±0.56 CMP 103/mg protein); but neither T allel nor A allel the efflux of FFA had no significant diference vs control (P>0.05). A time-dependent increase in the fat content of cells was observed, and the maximal TG (200.51±17.99 mg/g protein) was reached after 24 h of incubation with FFAs in these cell lines. The TG content was markedly higher in the wild-type-transfected (131.94±14.93 mg/g protein) cells than in the mutant-transfected cells when exposed to the FFA mixture for 24 h. By contrast, the wild-type-transfected cells accumulated significantly less cholesterol than mutant-transfexted cells (respectively, 15.35±1.65 mg/g protein and 20.53±2.67 mg/g protein), there had a significant difference (P<0.05).Conclusions:. We found that the T94A mutant of L-FABP lowered FFA uptake but had no effect on FFA efflux. L-FABP T94A-expressing cells showed decreased triglyceride content and increased cholesterol accumulation compared to the wild-type control for cells incubated with an FFA mixture (oleate:palmitate/2:1 ratio). In conclusion, our work provided additional indications of the functional relevance of the L-FABP T94A SNP in hepatic fatty acid and lipid metabolism in humans. |
PDF Full Text Request