The Spatiotemporal Regulation Of N-3 Polyunsaturated Fatty Acids On Metabolome Of 3T3-L1 Adipocytes And Molecular Mechanism On Promotion Of Beiging Process

Beige adipocytes are a noval class of adipocytes,similar to brown adipocytes,which would produce heat via adaptive non-shivering thermogenesis in response to various stimuli and could be a potential theapy of obesity.So increasing attention was gained recently.n-3 polyunsaturated fatty acids(n-3 PUFAs)show benefits on growth and development,cell membrane function stablation,immune regulation and antiinflammatory.But the congnitions of adipocytes metabolomics and regulation mechanism of beiging process by n-3 PUFA are still insufficient and unclear.Therefore,setting 3T3-L1 cells as the research model,current dissertation work took the representative n-3 PUFA monomers eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA)as objectives and explored their effect on the metabolome of adipocytes and molecular mechanism of beiging process.Moreover,current work explored the possibility of using n-3 PUFA as a peroxisome proliferators-activated receptors(PPAR?)agonist to promote the beiging process.The details of research plans and outcomes are shown as follows:1.Cell metabolomics study of EPA and DHA on intervention of 3T3-L1 preadipocyes and mature adipocytesThe concentrations of EPA and DHA were determined by MTT colorimetric method so 10 ?M and 100 ?M were chosen to be the proper interventional concentration.The endometabolomes and exometabolomes in different periods were collected.Through the UHPLC-Q-Exactive-HRMS and established statistical models,the potential differentially expressed metabolitea were screened in positive/negative ion models and compared in the Human Metabolome Database(HMDB)and the MZcloud database.The possible metabolic pathways were analyzed to further explore the metabolite content changes.After intervention of EPA and DHA on preadipocytes for48 h,the metabolites in cell and supernatant did not significantly change.After intervention of EPA and DHA on mature adipocytes for 24 h,they significantly increased the succinic acid content in tricarboxylic acid cycle(TCA).In the negative ion mode,two unknown compounds were found,of which concentrations were correlated with the type and dose of n-3 PUFA intervened.The mass differences between unknown compounds and corresponding n-3 PUFA were similar(<5 ppm)and their structures might be similar.2.Cell metabolomics study of the effect of EPA and DHA during 3T3-L1differentiationThe concentrations of EPA and DHA were determined by MTT colorimetric method so 10 and 100 ?M were chosen to be the proper interventional concentration.The established cell metabolomics analysis strategy was used to futher explore the effet of EPA and DHA on adipocytes during differentiation.After intervention during the whole differentiation stage,the whole types of metabolites were huger than these of preadipocyes and mature adipocytes.EPA significantly affected metabolites related to TCA cycle and glycerophospholipid metabolism while DHA also significantly influenced metabolites related to glycerophospholipid metabolism,which might produce beiging process.In the negative ion mode,four unknown compounds were found while two of them were the same with the former.Their concentrations were also significantly correlated with the type of n-3 PUFA.The mass differences between two new unknown compounds and corresponding n-3 PUFA were also similar(<5 ppm)and their structures might be similar.3.The effect of EPA and DHA on beige adipocyte phenotype and genes expressionConsidering on the metabolome results of preadipocytes and mature adipocytes,intervention during differentiation stage would significantly influence energy metablosim and lipid metabolism in cell.Thus,intervention during differentiation stage was chosen to further analysis the effect of n-3 PUFA on molecular mechanism of beiging process.The results showed that 10?M and 100?M DHA and EPA significantly increased the glucose consumption of adipocytes(all p < 0.001)while 10?M and100?M DHA significantly decreased the intracellular triglyceride content 37% and 40%(both p < 0.05).DHA and EPA could upregulate the expression of mitochondrial subproteins(succinate dehydrogenase complex flavoprotein subunit A,Sdh A and ATP synthase,H+ transporting,mitochondrial F1 complex,alpha subunit 1,ATP5A1)and promote the energy metabolism of mitochondrias.DHA and EPA upregulated the uncoupling protein 1(UCP1)gene and marker genes of beige adipocyte tissue(CD40,CD137 and CITED1)in adipocytes.100?M DHA and EPA could increase the expression of UCP1 and CD137 proteins.So n-3 PUFA could promote the beige adipocyte phenotype and genes expression in 3T3-L1 adipocytes.4.Molecular mechanism of regulatory effects of EPA and DHA on beiging processFurther analysed the molecular mechanism of n-3 PUFA on beige adipocytes development based on PPAR? and PRDM16 regulation pathway.100?M DHA and EPA could increase PRDM16 and PPAR? protein expression in adipocytes through promoting PRDM16 stabilization as well as PPAR? deacetylation.10?M and 100?M EPA and DHA would stabilize the PRDM16 protein synthesis while 100?M EPA was superior than 100?M DHA.Further,the protein expression of UCP1 and CD137 didn't change on the intervention of EPA and DHA in PRDM16 knock-out 3T3-L1 cell.Moreover,miRNAs array results deeply showed that 100 ?M EPA chould significantly downregulated miR-378 a and miR-34 a expression and significantly upregulated miR-26 a expression,which were reported to regulate beige adipocytes development in previous studies.Moreover,miR-362 and miR-486 a/b might be potential sites for EPA to affect the beiging process.Based on this,q PCR results confirmed that100?M DHA and EPA could reduce the expression of miR-378 a and miR-34 a significantly.In summary,current dissertation work comprehensively explored the effect of EPA and DHA on the cell metabolism of adipocytes and the regulation on molecular mechanism of the beiging process.Cell metabolics provided guidance for the subsequent beige adipocyte regulation research.DHA and EPA could promote beigeing process and phenotype of adipocytes via upregulating protein expression of PRDM16 and PPAR?,upregulating beige marker genes(CD40,CD137 and CITED1)and so on.These research outcomes make up the research gap of n-3 PUFA spatiotemporal regulation on adipocyte metabolics,deepen the scientific understanding of the molecular mechanism of n-3 PUFA regulating beigeing process and provide support on n-3 PUFA as an PPAR? agonist of beige adipocytes to be a potential therpy on obesity prevention and treatment.