The Role Of NRF2 In White Fat Browning And Its Mechanism

PART ONE NRF2 KNOCKOUT PROMOTES HIGH-FAT-DIET INDUCED OBESITY AND RELATED COMPLICATIONSObjective:To explore the relationship between NRF2 knockout and obesity and related complications in miceMethods:A total of 40 eight-week-old wild type?WT?and Nuclear Factor Erythroid 2-Related Factor 2?Nrf2?whole body knockout(Nrf2^-/-)male mice were randomly divided into the normal-diet-fed WT group?ND-WT?,normal-diet-fed Nrf2^-/-group(ND-Nrf2^-/-),high-fat-diet-fed WT group?HFD-WT?and high-fat-diet-fed Nrf2^-/-group(HFD-Nrf2^-/-),and 10 for each group,respectively.They were feed Normal-diet?ND?or high-fat-diet?HFD?for 12 weeks.The body weight and the ratio of fat to body weight were measured.The size of adipocytes was measured by HEstaining.The glucose tolerance and insulin tolerance were tested by IPGTT and IPITT.The food intake and rectal temperature were evaluated.The physical activity,O₂ consumption?VO₂?,CO₂ production?VCO₂?,energy expenditure?EE?and respiratory exchange ratio?RER?were determined by the Comprehensive Lab Animal Monitoring System?CLAMS?.The mRNA levels of Uncoupling Protein 1?Ucp1?in inguinal subcutaneous white fat tissue?iWAT?were measured by quantitative real-time PCR?qPCR?.In another in vivo study,20-week-old ND-WT?n=3?,HFD-WT?high-fat-diet fed for 12 weeks,n=3?and Normal-diet-fed db/db?ND-db/db,n=3?male mice were included.The mRNA levels of Nrf2 and Ucp1 in iWAT were measured by qPCR.The protein levels of NRF2 in iWAT were measured by western blotting?WB?.Results:Compared with HFD-WT mice,the body weight was significantly increased in HFD-Nrf2^-/-mice?p<0.01?.Compared with ND-WT mice,the mRNA levels of Nrf2 in iWAT were significantly decreased?P<0.05 or P<0.01?and the protein levels of NRF2 were also decreased in HFD-WT and ND-db/db mice.The ratio of weight of eWAT and iWATto body weight were significantly increased?p<0.01?and the size of adipocytes in iWAT was also increased in HFD-Nrf2^-/-mice when compared with HFD-WT mice.Compared with HFD-WT mice,the blood glucose levels during IPGTT and IPITT were significantly increased in HFD-Nrf2^-/-mice at multiple time points?p<0.01?.Compared with HFD-WT mice,the rectal temperature,VO₂,VCO₂ and EE were statistically decreased in HFD-Nrf2^-/-mice?p<0.05 or p<0.01?,while there were no significant differences in food intake,physical activity and RER.The mRNA level of Ucp1 in iWAT was significantly reduced in HFD-Nrf2^-/-mice when compared to HFD-WT mice?p<0.05?.Conclusion:NRF2 expression levels are inhibited in iWAT of HFD induced and db/dbobese mice.NRF2 null can exacerbate HFD induced abnormal glucose metabolism.NRF2 knockout exacerbates HFD induced obesity,duing to a decrease in energy consumption and inhibition of white fat browning.PART TWO NRF2 PROMOTES BROWNING OF WHITE FATObjective: To clarify the role of NRF2 in promoting white fat browning.Method:A total of 32eight-week-old WT and Nrf2^-/-male mice were randomly divided into the room-temperature WT group?RT-WT?,room-temperature Nrf2^-/-group(RT-Nrf2^-/-),cold-room-stimulated WT group?CR-WT?and cold-room-stimulated Nrf2^-/-group(CR-Nrf2^-/-),and 8 for each group,respectively.The body weight and the ratio of fat to body weight were measured.The size of fat cells was measured by HE staining.The food intake and rectal temperature were evaluated.The m RNA levels of Ucp1,Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-Alpha?Pgc-1??,PR Domain Zinc Finger Protein 16?Prdm16?and other genes related to white-brown switch in i WAT were measured by q PCR.In another study,a total of 20 eight-week-old WT and Nrf2^-/-male mice were randomly divided into the Saline-injected WT group?Saline-WT?,Saline-injected Nrf2^-/-group(Saline-Nrf2^-/-),ISO-injected WT group?ISO-WT?and ISO-injected Nrf2^-/-group(ISO-Nrf2^-/-),and 5 for each group,respectively.The size of fat cells was measured by HE staining.The m RNA levels of Ucp1,Pgc-1?,Prdm16 and other genes in i WAT were measured by q PCR.The m RNA levels of Ucp1,Pgc-1?,Prdm16 and other genes in fully brown differentiated i WAT SVF from eight-week-old WT and Nrf2^-/-male mice were measured by q PCR.Results:Both the body weight and the ratio of weight of e WAT and i WAT to body weight were significantly decreased in CR-WT mice when compared with CR-Nrf2^-/-mice?P < 0.05 or P < 0.01?.The size of adipocytes in i WAT was smaller in CR-WT mice than in CR-Nrf2^-/-mice.Compared with CR-WT mice,the rectal temperature was markedly reduced in CR-Nrf2^-/-mice?p<0.05 or p<0.01?,while there were no statistically differences in food intake.The m RNA levels of Ucp1,Pgc-1? and Prdm16 in i WAT was significantlyhigher in CR-WT mice than in CR-Nrf2^-/-mice?p<0.01?.In another study,The size of adipocytes was smaller and the m RNA levels of Ucp1,Pgc-1? and Prdm16 were significantly higher?P < 0.05 or P < 0.01?in i WAT of ISO-WT mice than that of ISO-Nrf2^-/-mice.Compared with Nrf2^-/-adipocytes,the m RNA levels of Ucp1,Pgc-1?,Prdm16 and other genes were significantly increased in WT adipocytes?p<0.05 or p<0.01?.Conclusion:In vivo study,NRF2 supressedweight gain in cold environment and promoted the cold-inducedand ISO-induced white-to-brown fat switch.In vitro study,NRF2 accelerated browning of white fat cells.PART THREE THE MECHANISM OF WHITE FAT BROWNING BY NRF2Objective:To explore the mechanism by which NRF2 promotes browning of white fatMethods: The size of lipid droplet,the m RNA and protein levels of Nrf2 and Ucp1 genes in fully differentiated i WAT SVF from eight-week-old WT and Nrf2^-/-mice after brown adipocyte differentiation and palmitate?PA?intervention were evaluated by Oil red O staining,q PCR,WB and immunofluorescence staining?IF?,respectively.The size of lipid droplet in fully differentiated i WAT SVF from eight-week-old WT and Ucp1 whole body knockout(Ucp1^-/-)mice after brown adipocyte differentiation and PA intervention were detected by Oil red O staining.The size of lipid droplet,the m RNA and protein levels of Nrf2 and Ucp1 genes in fully differentiated i WAT SVF from WT and Ucp1^-/-mice after brown adipocyte differentiation and PA intervention with Nrf2 overexpression lentivirus?Lv-Nrf2?were measured by Oil red O staining,q PCR,WB and IF,respectively.The m RNA and protein levels of Nrf2,Sirt3 and Ucp1 genes in i WAT of WT and Nrf2^-/-mice intervened with cold stimulation or ISO injection were assessed by q PCR and WB.The m RNA and protein levels of Nrf2,Sirt3 and Ucp1 genes in fully differentiated i WAT SVF from eight-week-old WT and Sirtuin 3?Sirt3?whole body knockout(Sirt3^-/-)mice after brown adipocyte differentiation with Lv-Nrf2 transfection were detected with q PCR,WB and IF,respectively.The m RNA and protein levels of Nrf2,Sirt3 and Ucp1 genes in fully differentiated i WAT SVF from WT and Nrf2^-/-mice after brown adipocyte differentiation with Sirt3 overexpression lentivirus?Lv-Sirt3?transfectionwere evaluated using q PCR,WB and IF,respectively.Results:PA intervention inhibited Nrf2 and Ucp1 m RNA?p<0.05 or p<0.01?and protein expression levels in WT adipocytes.NRF2 deletion aggravated PA-induced lipid accumulation in adipocytes,and inhibited the m RNA?p<0.01?and protein expression levels of Ucp1 and other genes.UCP1 knockout exacerbated PA-induced lipid accumulation in adipocytes.NRF2 overexpression in adipocytes inhibited PA-induced lipid accumulation in WT group,but this effect was not present in the Ucp1^-/-group.Cold and ISO stimulation promoted the m RNA?p<0.01?and protein expression of Nrf2,Sirt3 and Ucp1 genes in i WAT of WT mice.Compared with WT mice,the m RNA?p<0.01?and protein levels of Sirt3 and Ucp1 genes in i WAT were decreased in Nrf2^-/-mice after cold and ISO stimulation.Compared with WT+Con adipocytes,the m RNA levels of Nrf2,Sirt3 and Ucp1 genes was significantly increased?p<0.01?and the protein levels of Nrf2,Sirt3 and Ucp1 genes were also increased in WT+Lv-Nrf2 adipocytes.Compared with Sirt3^-/-+Conadipocytes,the m RNA level?p<0.01?and protein level of Nrf2 were higher,the m RNA and protein levels of Sirt3 were difficult to detect and the m RNA level?p<0.01?of Ucp1 was mildly increased in Sirt3^-/-+Lv-Nrf2 adipocytes,the m RNA level?p<0.01?and protein level of Ucp1 were significantly decreased.Compared with WT+Con adipocytes,the m RNA levels of Sirt3 and Ucp1 were significantly increased ane the protein levels of Sirt3 and Ucp1 genes were also increased in WT+Lv-Sirt3 adipocytes.The m RNA levels of Sirt3 and Ucp1?p<0.01?and protein levels of Sirt3 and Ucp1 genes were significantly higher in Nrf2^-/-+Lv-Sirt3 adipocytes than in Nrf2^-/-+Conadipocytes.Conclusion : PA intervention inhibited NRF2 expression in WT adipocytes.NRF2 knockdown aggravated PA-induced lipid accumulation in adipocytes.The presence of UCP1 is required for NRF2 to inhibit PA-induced lipid accumulation in adipocytes.NRF2 can regulate white fat browning partly through the SIRT3 pathway,which in turn affects the accumulation of lipids in fat cells,but there are still other signaling pathways.