Mechanism Of NFE2L1 In Lipid Catabolism Disorder Induced By Arsenic In Adipocyte

Object:Arsenic is a metal-like element widely found in natural environment.Long-term exposure to low concentrations of arsenic increases the risk of chronic diseases including diabetes,hypertension,and arteriosclerosis in humans.Studies have shown that arsenic exposure can increase the lipolytic activity in adipocytes,which in turn affects lipid metabolism.However,the exact mechanism is still not clear.The nuclear transcription factor NFE2L1 plays an important role in the regulation of lipid metabolism.At the same time,our research team has confirmed that iAs³⁺can strongly induce the expression of NFE2L1 in human skin keratinocytes and 3T3-L1 cells.Based on the above results,this study will focus on the effects of inorganic arsenic exposure on lipid catabolism and NFE2L1 expression in adipocytes.Subsequently,further analysis of NFE2L1 regulates the lipid catabolism mechanism of fat cells and its role in the arsenic-induced lipolysis disorder.Methods:1.Female C57BL/6 mice were exposed to drinking water containing arsenic,which wererandomly divided into control group and arsenic exposure group,and selected ternary iAs(iAs³⁺)and pentavalent iAs(iAs⁵⁺)1:1 mixed close to the actual exposure of the human population,drinking water Route exposure for 16 weeks;2.Male C57BL/6 mice with adipocyte-specific NFE2L1-knockout and control mice get acess to distilled water for 16 weeks;3.Glucose tolerance test and insulin tolerance test were performed to evaluate glucose metabolism and insulin sensitivity in mice;4.Calculate and evaluate the mouse organ coefficient;5.Using H&E staining of pathological section to assess the morphological changes and lipid accumulation of adipocytes after arsenic exposure in mice;6.Isolating primary adipose cells from white adipose tissue of NFE2L1 deficient mice,and treating cells with 10μM trivalent inorganic arsenic for 24 hours,confirming whether NFE2L1 deletion affects lipolytic enzyme expression under arsenic exposure conditions;7.Using qRT-PCR method to detect mRNA expression levels of NFE2L1 and lipolysis-related indicators in white adipose tissues of mice;8.Western Blot was used to detect expression levels of lipolysis-related protein in mice;9.Using chromatin immunoprecipitation assay to analyze the binding activity of NFE2L1 protein to the promoters of lipolytic genes;10.Using the reporter gene assay to clarify the transcriptional regulation of NFE2L1 on lipolytic enzymes.Results:1.Arsenic exposure impairs glucose and lipid metabolism in mice.Under the condition that there were no evident changes in other basic indicators,mice in the treatment group showed obvious glucose intolerance and insulin resistance;2.Arsenic exposure leads to a decrease in the volume of adipocytes in adult female mice and up-regulates lipases gene expression.The results indicated that the volume of adipocytes in the treated group shrank,and the expression levels of lipase mRNA increased,however,the protein expression did not change significantly.;3.Nfe2l1 deletion results in decreased expression of adipocyte lipases in adult male mice.Lipase expression decreased in bothe mRNA and protein levels in mice with Nfe2l1-deletion;4.Nfe2l1 plays a regulatory role in the increased expression of lipolytic enzymes induced by arsenicin mice.After treatment with arsenic in primary mouse fat cells,the level of lipases mRNA in Nfe2l1 deletion group were significantly lower than those in the control group,and the expression of RNA increased significantly after arsenic treatment in the control group;5.Multiple subtypes of NFE2L1 can bind to the lipase promoter region.Chromatin immunoprecipitation showed that long isoform（NFE2L1-741）and short isoforms（NFE2L1-453）may bound to the ARE region of the Lipe2 gene promoter;6.NFE2L1 regulates HSL transcription by binding to the ARE sequence.The reporter gene assay showed that the long isoform of NFE2L1（741bp）has a significant regulatory activity on the Lipe2 promoter containing the ARE sequence.The isoform NFE2L1-453also has obvious transcriptional regulation on the Lipe2 promoter containing the ARE sequence.When the ARE sequence is mutated or the ARE sequence is not included in the promoter,the transcriptional regulation is significantly attenuated.Conclusion:1.Long-term exposure to inorganic arsenic can cause increased expression of NFE2L1,increased expression of lipases,increased lipolysis,and weakened storage capacity of adipocytes,which may aggravate the disorder of glycolipid metabolism.2.Inorganic arsenic exposure causes increased expression of NFE2L1 in adipocytes and regulates the expression of lipolytic enzymes.The mechanism may be:arsenic stimulates NFE2L1 activation,enters the nucleus and binds to the sMAFs protein and the ARE sequence in the HSL-encoding gene Lipe2 promoter,and up-regulates the expression of the gene.HSL is expressed at high levels for a long time,and lipolytic enzymes such as ATGL,MGL and PLIN1 are also increased,which may lead to increased lipolysis of adipocytes.