Effect Of Sulforaphane On The Glucose And Lipid Metabolism In Adipocytes

Objective: This study aimed to investigate the effect of sulforaphane on glucose and lipid metabolism of 3T3-L1 mature asipocytes,and explore the underling mechanism.Therefore,it can contribute to provide the evidence for effective prevention and treatment of obesity-related metabolic disorder.Methods: In this study,murine 3T3-L1 pre-adipocytes was differentiated into mature adipocytes by classic cocktail method.To select the optimal time of intervention,lipid accumulation was observed directly by the Oil Red O staining,and the glucose uptake was detected by the 2-（N-（7-Nitrobenz-2-oxa-1,3-diazol-4-yl）Amino）-2-Deoxyglucose（2-NBDG）staining in the differentiating 3T3-L1 pre-adipocytes differentiation.Then 3T3-L1 mature adipocytes were incubated with low dose of SFN（0 to 10.0μmol/L）for 48 h,the glucose uptake of the adipocyts after staining by 2-NBDG was qualitatively and quantitatively measured by confocal microscopy and multifunctional microplate reader.Furthermore,western blotting and real-time PCR were used to detected the protein and mRNA levels of glucose uptake and metabolism related genes including GLUT-4,GCK,CS and ACC,FAS in 3T3-L1 mature adipocytes treated with SFN for 48 h.In addition,the effect of SFN on lipid hydrolysis in 3T3-L1 adipocytes was evaluated by analysing glycerol content in medium using glycerol GPO-POD enzyme method.Finally,levels of lipid metabolism associated proteins(ATGL,HSL,p-HSL^Ser563,p-HSLSer565,p-HSL^Ser660,Perilipin,CPT1 A,CS,DGAT-1)in white adipocytes were detected by western blotting.All the above experiments were conducted to evaluated effects of SFN on glucose and lipid metabolism of 3T3-L1 mature adipocytes,and to clarify the molecular mechanism.Results: In the process of differentiating 3T3-L1 pre-adipocytes,lipid accumulation and glucose uptake increased gradually,and get to be stabilized on day 10.Thus,the mature adipocytes at day 10 of differentiation were chosen for this study.Compared with control,there is a significant increase of glucose uptake in 3T3-L1 mature adopocytes treated with SFN according to the data obtained through 2-NBDG staining.Besides,glycerol GPO-POD enzyme method showed that SFN remarkably increased the lipid hydrolysis in adipocytes.Meanwhile,western blotting analysis suggested that SFN was able to up-regulate the expression of glucose uptake and oxidation related protein GLUT-4,GCK,CS.Furthermore,real-time PCR results indicated that SFN downregulated the gene levels of ACC and FAS,both of them are important for endogenous fatty acid synthesis.The expression of proteins inhibiting lipid hydrolysis such as p-HSLSer565,Perilipin as well as DGAT-1,key enzyme associated with the de novo triglyceride synthesis was downregulated in adipocytes treated with SFN.In addition,SFN also increased significantly the levels of proteins,functioning to enhance the lipid hydrolysis like HSL,p-HSL^Ser563,p-HSL^Ser660,and the protein expression of CPT1 A and CS responsible for fatty acid beta-oxidation.Conclusion: Low dose of SFN（0-10.0μmol/L）could enhance glucose uptake and aerobic oxidation of 3T3-L1 mature adipocytes by up-regulating glucose uptake and metabolism related proteins GLUT-4,GCK,CS.Moreover,SFN was able to increase lipid metabolism in mature adipocytes by up-regulating the expression of protein related to lipid hydrolysis including HSL and its phosphorylated form p-HSL^Ser563,p-HSL^Ser660,and enhance the level of proteins pivotal for fatty acids beta-oxidation like CPT-1A and CS.On the contrary,SFN down-regulated the levels of ACC and FAS as well as DGAT-1,inhibiting the endogenous fatty acids and triglyceride synthesis.Above all,low concentration of SFN was capable of improving the glucose and lipid metabolism in white adipocytes,thereby could be a promising strategy to combat obesity-related metabolic disorder.