| Part Ⅰ Research of transformation of WAT to BAT and related factors of glucolipid and energy metabolism in ASAN miceObjective:Adipose tissue(AT),can be divided into white adipose tissue(WAT),and brown adipose tissue(BAT).WAT is mainly divided into subcutaneous adipose tissue(SAT)and visceral adipose tissue(VAT).In recent years,research has shown that great accumulation of adipose tissue has the close relation with vascular aging,occurrence of atherosclerosis and glucolipid metabolism in the body.Human aging is usually accompanied by a redistribution of body fat,but in the process,the specific conversion of AT is yet to be in-depth study.AT is closely related to oxidative stress.In the theoretical system of oxidative stress caused by aging,nuclear factor erythroid-2-related factor(Nrf2)plays a central role.At the same time,a lot of researches suggest thatNrf2 also plays an important role in glucose,fat and energy metabolism.While the liver is the main target of glucolipid metabolism.And liver cells play an important role in maintaining serum glucose steady state.But the study of the influence of glucolipid and energy metabolism after silence of Nrf2 in liver cells is relatively lacking.Therefore,this study was aimed to explore the Nrf2 and its regulation molecules of the downstream oxidation stress to transformation of WAT to BAT,and its effect on related cytokines expression of glucolipid metabolism in the transgenic mice model of specific knockout of Nrf2.Methods:Sixteen two and a half months male mice were selected and then they were divided into eight Control group(NF)and eight experimental group(ASAN).And at the same time,they were given a high-fat diet.Four months later,the method of cervical dislocation was taken respectively to be put to death in mice.And the mice were determined the IP GTT(0 h,1/2 h,1 h,2 h)before death.Then two groups of VAT,SAT,and liver tissue of mice were extracted.Using Western blot and qPCR techniques in the adipose tissue of mice to detect:1,the rise of BAT marker genes expression(pgc-1 alpha,PRDM16,Dio2);2,the production of mitochondria(UCP1,and Cycs);3,the expression of WAT marker gene(Bmp4,Rbl,and Resistin);4,related factors of FGF21 related metabolic signaling pathways(FGF21,AMPK alpha,Sirtl,pgc-1 alpha and UCP1);At the same time,using the methods of Western blot,and qPCR techniques in two groups of mice liver tissues to detect:1,endoplasmic reticulum and antioxidant stress related factors(p-EIF2 alpha,and NQO1,HO-1);2,related protein expression of glucose metabolism(Giut-4,IGF-1R,FOXO1,p-AKT and p-GSK3 alpha/beta).Results:Compared with the NF mice,the blood glucose of ASAN transgenic mice was elevated.And in AT of ASAN mice:1,the expression of BAT marker genes(pgc-1 alpha,Dio2 and PRDM16)was increased(P<0.05);2,the expression of mitochondria generate gene(UCP1 and Cycs)was increased significantly;3,the expression of WAT marker genes(Rb1,Bmp4 and Resistin was also increased;4,the expression of genes(AMPK alpha,Sirtl and pgc-1 alpha),participating the longevity genes,was also increased.5,the expression of related factors of FGF21 related metabolic signaling pathways(FGF21,AMPK alpha,Sirtl,pgc-1 alpha and UCP1)were increased.Meanwhile,compared with the NF mice,in the liver tissues of ASAN transgenic mice:1,intracellular endoplasmic reticulum(ER)stress levels were evaluated by measuring the activation of an ER stress reporter,a subunit of eukaryotic initiation factor(EIF2α),and the gross inflammatory status were evaluated by examining the protein expression of pro-inflammatory markers(IL-beta,TNF alpha,NF-kB,p-p65,MMP2 and MMP9);2,related protein expression of glucose metabolism(Giut-4,IGF-1R,FOXO1,p-AKT and p-GSK3 alpha/beta)was all significantly increased.Conclusion:Taken together,specific knockout of Nrf2 in ASAN transgenic mice,can make the blood glucose elevate and transformation of WAT to BAT.At the same time,negative regulating the signaling of FGF21,and participant the metabolism of glucose,fat and energy.Part Ⅱ Role of specific knockdown of Nrf2 in related factors of transformation of adipose tissue and glucolipid and energy metabolism in 3T3-L1 and AML12 cellsObjective:In cellular level,the influences of related factors of transformation of adipose tissue and glucolipid metabolism by specific knockdown of nuclear factor E2-related factor 2(Nrf2)in 3T3-L1 and AML12 cells.Methods:① Primary 3T3-L1 cells were induced into adipose cells.In cells,Nrf2 siRNA was transfected to obtain the cells of specific knockdown of Nrf2.In the cells,changes caused by expression levels of Nrf2 were detected,such as:1,the expression of marker genes of WAT,BAT and mitochondria generates,and UCP1 and Sirtl involved in the signaling pathways of longevity genes;2,the glucose uptake capacity of adipose cells was detected by glucose detection kit.②The same method was used to obtain the AML 12 cells of specific knockdown of Nrf2.The glucose uptake capacity of adipose cells was also detected.In addition,AML12 cells were treated with the high-glucose(33mmol/L).The cells were divided into four groups,including Control + Low Glucose(Con + LG),Control+ High Glucose(Con + HG),Nrf2-knock down + Low Glucose(NK + LG),and Nrf2-knock down + High Glucose(NK + HG).Testing the following contents:1,endoplasmic reticulum and antioxidant stress related factors(p-EIF2 alpha,and NQO1,HO-1);2,the total state of inflammation related factors(IL-beta,TNF alpha,NF-kB,p-p65,MMP2 and MMP9);3,related factors of FGF21 related metabolic signaling pathways(FGF21,AMPK alpha,Sirtl,pgc-1 alpha and UCP1);4,related protein expression of glucose metabolism(Giut-4,IGF-1R,FOXO1,p-AKT and p-GSK3 alpha/beta).Results:①In 3T3-L1 adipose cells,when compared with Control group,the mRNA and protein expression of Nrf2 were about 80%and 70%lower.The expression of Nrf2 downstream antioxidant enzymes(NQOl and HO-1)was also decreased significantly.At the same time:1,the expression of BAT marker genes(pgc-1 alpha,Dio2 and Cox7)was increased(P<0.05);2,the expression of mitochondria generate gene(UCP1 and Cycs)was increased significantly;3,the expression of WAT marker genes(Rb1,Bmp4 and Resistin was also increased;4,the expression of genes(AMPK alpha,Sirtl and pgc-1 alpha),participating the longevity genes,was also increased.At the same time,in the Nrf2 knockdown group,the glucose absorption was increased.②In AML12 cells,compared with control group,Nrf2 mRNA and protein levels decreased about 90%and 70%respectively.The expression of Nrf2 downstream antioxidant enzymes(NQO1 and HO-1)was also decreased significantly.Meanwhile:1,intracellular endoplasmic reticulum(ER)stress levels were evaluated by measuring the activation of an ER stress reporter,a subunit of eukaryotic initiation factor(EIF2a),and antioxidant stress related factors(NQO1,and HO-1)were decreased;2,the gross inflammatory status were evaluated by examining the protein expression of pro-inflammatory markers(IL-beta,TNF alpha,NF-kB,p-p65,MMP2 and MMP9);3,the expression of related factors of FGF21-related metabolic signaling pathways(FGF21,AMPK alpha,Sirtl,pgc-1 alpha and UCP1)was all significantly elevated;4,related protein expression of glucose metabolism(Giut-4,IGF-1R,FOXO1,p-AKT and p-GSK3 alpha/beta)was all significantly increased,too;5,the ability of glucose uptake of cells was significantly increased.Conclusion:In vitro,in adipose cells,under the condition of Nrf2 silence makes the expression of BAT genes increase,WAT genes reduce,increases the generation of mitochondria and expression of signaling pathways involved in longevity genes(UCP1 and Sirtl);Promote the ability of adipose cells to glucose uptake.In AML 12 cells,after the silence of Nrf2,the endoplasmic reticulum stress,the total state of inflammation,FGF21 related to metabolism and sugar metabolism increased,and oxidative stress resistance decreased;Increase the ability of glucose uptake. |
