Regulation Of FABP4on Fatty Acid Metabolism And Mitochondrial Function In3T3-L1Adipocytes

Fatty acid binding protein4(FABP4), a member of fatty acid binding protein family,was identified as peripheral membrane protein combined with a variety of hydrophobiccompounds, such as long chain fatty acids, various cyclooxygenase and lipoxygenasemetabolites, and involved in the metabolism and signal transduction at the same time, playinga key role in the formation of diabetes, obesity and lipid metabolism disorders and otherdiseases. Mitochondrial fatty acid oxidation (FAO) will cause body energy supply disorderedand insufficient if the abnormality occurs in any one of the metabolic pathways, which wasinvolved in many fields of energy metabolism regulation. This study aimed to investigate theeffect of the FABP4on fatty acid metabolism, mitochondrial function and related signalingpathways in adipocyte. These results will provide theoretical basis for study systematicallythe function and mechanism of mitochondrial regulation in animal lipolysis process.To explore the role of FABP4on fatty acid metabolism and mitochondrial function, theexperiment constructed the eukaryotic expression vector for FABP4firstly and obtainedinterference vectors of FABP4using RNA silencing technology. Through transfecting andinducting3T3-L1preadipocytes, we used real-time quantitative PCR, Western blot, ELISA,flow cytometry and other experimental techniques to detect FABP4impact on the expressionof critical factors in fatty acid metabolism and transportation, mitochondrial function, theactivity of related signaling molecules and the phosphorylation level of signaling pathways in3T3-L1cells, and discussed the regulation of FABP4on fatty acid metabolism andmitochondrial function in3T3-L1cells. The main results were summarized as following:1. Taking RNA template which extracted from mouse adipose tissue, we obtainedFABP4gene including CDS region cloned by PCR and successfully constructed recombinantplasmid vector named pcDNA3.1-Fabp4that comfirmed by enzyme digestion and sequenceidentification.2. Transfecting overexpression and interference vectors of FABP4by liposome, we usedRT-PCR and Western blotting to detect the expression of mRNA and protein of FABP4. Theresults showed that compared with the control group, FABP4mRNA and protein levels weresignificantly elevated in pcDNA3.1-Fabp4transfected group (P <0.01) but reduced in the four groups transfected with interference vectors conversely (P <0.01), and the forth group was thelowest.3. FABP4overexpression played a positive role in regulation of3T3-L1adipocytedifferentiation and lipid deposition, and significantly promoted the expression of PPARγ,CEBPα, FAS, ACC, FATP1, ACC, ACS and the release of FFA in medium(P<0.01), butdownregulated LPL, ATGL, FAT, HSL and CPT-1expression (P<0.01), while FABP4interference inhibitted FFA release and PPARγ CEBPα, FAS ACC, FATP1and LPLexpression and upregulated the expression of ATGL and FAT (P <0.01).4. The expression levels of PGC-1α, NRF-1, TFAM, CPT-1, MDH-1and UCP-1weredecreased significantly in FABP4overexpression group (P<0.01) but increased in FABP4interference group (P<0.01). Testing permeability of mitochondrial membrane by flowcytometry, the results showed that the mitochondrial membrane potential was reduced inFABP4overexpression group but raised in FABP4interference group (P<0.01). FABP4overexpression could significantly decreased the expression levels of mRNA and protein ofCytc compared with the control group, as well as the fluorescence intensity (P<0.01). On thecontrary, in FABP4interference group the fluorescence intensity was higher than the controlgroup, and the the expression of Cytc was also increased significantly (P<0.01).5. High expression of FABP4significantly inhibited the protein phosphorylation level ofp38MAPK and ERK but improved TOR in adipocyte (P<0.01). However the levels of proteinphosphorylation in p38MAPK and ERK were increased and TOR decreased significantlyafter reducing FABP4expression in the interference group (P<0.01).In summary, the study indicated that FABP4overexpression significantly promotedadipocyte differentiation and lipid deposition, but FABP4interference inhibited adipocytedifferentiation. Overexpressing FABP4decreased the expression of oxidation and respirationfactors in adipocytes and reduced mitochondrial membrane potential causing mitochondrialfunction inhibitted, whereas FABP4gene deletion could maintain a high mitochondrialmembrane potential to enhance mitochondrial function. p38MAPK, ERK and TOR signalingpathways involved in the regulation of FABP4on mitochondrial function.