The Mechanism And Multiple Functions Of Nrf2 In Regulating Metabolic Homeostasis And Inflammatory Factor Secretion In Mice

Part1 Adipose-specific ablation of Nrf2 transiently delayed high-fat diet-induced obesity by altering glucose,lipid and energy metabolism of male miceBackground and objective: Nuclear factor E2-related factor 2(NRF2)is a well-known master controller of the cellular adaptive antioxidant and detoxification response.White adipose tissue(WAT)has been classically defined as a reservoir for the storage and release of energy.WAT is also the largest endocrine organ and performs a variety of additional functions including secretion of numerous immune and inflammatory mediators responsible for cardiovascular disease,insulin resistance,type 2 diabetes,brain dysfunctions,breast cancer,and other effects.Recent studies demonstrated altered glucose,lipid and energy metabolism in mice with a global Nrf2 knockout.In the present study,we aim to determine the effects of an adipose-specific ablation of Nrf2(ASAN)on diet-induced obesity(DIO)in male mice.Methods: We have successfully made the adipose-specific Nrf2 knockout(ASAN/NK)mouse model.The 6-week-old NK and its Nrf2 control(NC)mice were fed with either control diet(CD)or high-fat diet(HFD)for 14weeks.Changes in body weight were measured during the CD and HFD feeding period.Metabolic measurement was performed for 1 week using mice at week 8 of the CD/HFD feeding using an Oxymax indirect calorimetry system.After overnight fasting,the animals received blood glucose tolerance test.At 20 weeks of age,the mice were fasted overnight,euthanized by isoflurane anesthesia,exsanguinated via cardiac puncture.VF(epididymal fat pads)and SF(inguinal fat pads)were collected,weighed.The plasma was isolated and analyzed using ELISA according to the manufacturer's instructions for free fatty acid,total cholesterol,LEPTIN and ADIPONECTIN.The SF and VF samples were stained with hematoxylin & eosin(H&E).This study also explored the metabolism related m RNA and protein expression in SF and VF samples by q PCR and Western Blot.Results: NK mice exhibited transiently delayed body weight(BW)growth from week 5 to week 11 of HFD feeding,higher daily physical activity levels and preferential use of fat over carbohydrates as a source of energy at week 8 of the CD-feeding period.After 14 weeks of feeding,NK mice showed comparable results with NC mice with respect to the overall BW and body fat content,but exhibited reduced blood glucose,reduced number but increased size of adipocytes,accompanied with elevated expression of many genes and proteins in the visceral fat related to glucose,lipid and energy metabolism(e.g.Fgf21,Pgc1a).Conclusion: These results indicated that NRF2 was an important mediator for glucose,lipid and energy metabolism in adipose tissue,and ASAN could have beneficial effect for prevention of DIO during the early development of mice.Part2 Nrf2 exerts mixed inflammation and glucose metabolism regulatory effects on murine RAW264.7 macrophagesBackground and objective: The results in part1 indicated that NRF2 was an important mediator for glucose,lipid and energy metabolism in adipose tissue,and ASAN(Nrf2flox/flox,a P2-Cre+/-)could have beneficial effect for prevention of DIO during the early development of mice.Adipose tissue mainly contains adipocytes and macrophages,while a P2-Cre gene is regulated by a P2 promoter and expresses in both adipocytes and macrophages.Therefore,the phenotype of ASAN mice should be regulated by Nrf2 in adipocytes and macrophages.The cytokines secreted by macrophages affect the surrounding tissue homeostasis and function through endocrine and paracrine effects,and the inflammatory mediators produced by macrophages are the etiology of obesity-related metabolic disorders.In order to better subdivide the role of Nrf2 in adipocytes and macrophages,part2 of the present study investigated the effects of Nrf2 on the regulation of inflammation and metabolism on murine macrophage.Methods: We specifically knocked down the Nrf2 gene by Nrf2 retrovirus in RAW264.7 macrophages to construct a Nrf2 knockdown(NK)cell model.Macrophages were then stimulated with 100 ng/ml lipopolysaccharide(LPS) for 24 hours to induce it into M1 activation state(M1).q PCR,Western Blot and ELISA were used to detect the effects of Nrf2 knockdown on macrophage inflammation and glucose metabolism.Four groups of macrophage(Con,NK,M1,NK-M1)conditioned medium(CM)were collected and used to induce the primary endothelial cells.The effects of CM on endothelial cell inflammation and aging markers were detected by q PCR and Western Blot.Results: The q PCR results indicated that Nrf2 knockdown significantly enhanced expression of the inflammatory genes Il1?and Il? in unstimulated macrophages and increased expression of the inflammatory genes Il1 a,Il1b,Il6,Il10,Ccl2,Ccl22 and CD38 but decreased that of Tnfa and Tgfb1 in M1 macrophages.Nrf2 knockdown also significantly elevated IL6 and IL10 secretion by M1 macrophages.Western blotting showed that Nrf2 knockdown reduced i NOS protein levels in resting macrophages and enhanced CD38 protein levels in both resting and M1 macrophages.The differential regulation of these macrophage inflammation and polarization markers by Nrf2 reveals multiple roles for Nrf2 in regulating inflammation in macrophages.Moreover,Nrf2 knockdown increased the Glut4 protein level and decreased AKT and GSK3? protein phosphorylation in M1 macrophages,suggesting multiple roles for Nrf2 in regulating glucose metabolism in macrophages.In addition,HUVECs in Con-CM and NK-CM groups still maintained in relatively low inflammatory condition and the endothelial cell inflammatory and senescent markers in M1-CM and NK-M1-CM groups were significantly elevated.Comparing to M1-CM group,NK-M1-CM group showed higher inflammatory level and higher protein expression of senescent markers of P53 and P21,indicating that the endocrine function regulated by Nrf2 in macrophage could influence endothelial cell inflammation and aging.Conclusion: Overall,our results are the first to demonstrate mixed inflammation and glucose metabolism regulatory effects of Nrf2 in macrophages that may occur independent of its classic function in redox regulation.The endocrine function regulated by Nrf2 in macrophage could influence endothelial cell inflammation and aging.These findings support the potential of Nrf2 as a therapeutic target for the prevention and treatment of inflammation-and obesity-associated syndromes,including diabetes and atherosclerosis.