The Regulatory Mechanism Of NFE2L1 On Adipocyte Homeostasis And Its Role In Adipocyte Dysfunction Induced By Cadmium Exposure

Objective:The worldwide prevalence of obesity has doubled since 1980 to an extent that over a third of the world’s population is now classified as overweight or obese.China has the largest number of obese people in the world.Obesity is the main pathological basis and an established risk factor of many chronic non-communicable diseases,which have a great negative impact on the quality of individual life and social healthcare costs.Meanwhile,with the acceleration of industrialization,the popularization of motor vehicles and the transformation of energy structure,heavy metals invade all aspects of life in the form of environmental pollutants,such as synthetic fertilizers,food packaging,automobile exhaust,electronic waste,etc.Their correlation with obesity and related metabolic disorders has become increasingly prominent.As one of the primary heavy metals officially approved in the 14th Five-Year Plan for the Comprehensive Prevention and Control of Heavy Metal Pollution,cadmium（Cd）is not only a class I carcinogen,but also associated with respiratory and nervous system,kidney,bone,cardiovascular and metabolic diseases.The mechanisms by which Cd elicits toxicity in specific organs are not entirely clear,although induction of oxidative stress,endoplasmic reticulum stress,inflammatory response,genotoxicity and interference with other essential trace elements have been implicated.Adipose tissue（AT）is a potential target for cadmium accumulation and an important biological matrix for the occurrence and development of obesity and related metabolic diseases.However,the current reports about the effects of Cd on AT are inconsistent.Further studies are needed to reveal the dose-dependent relationship between Cd and adipocyte differentiation and function,as well as to identify key regulatory factors in this process,so as to provide new insights into the prevention and treatment of metabolic disorders and related diseases related to Cd exposure.Nuclear factor erythroid-2 related factor 1（NFE2L1,also known as NRF1）is one of the vital defense factors in many pathological processes.It acts as a master regulator of antioxidant responses,proteasome homeostasis,genetic stability,inflammation,and response to the toxic effects of various harmful chemicals including Cd.Therefore,the NFE2L1-mediated transcriptional regulatory network might be an important protective mechanism in adipocyte damage induced by Cd.Although the novel functions of NFE2L1in adipocytes has been initially identified:NFE2L1 regulates adipogenesis in a subtype-specific manner;both brown AT（BAT）adaptive thermogenesis and white AT（WAT）lipolytic activity require the guardian and regulation of NFE2L1.However,underlying mechanisms still need to be further confirmed and elucidated.For instance,it’s necessary to investigate the function of NFE2L1 on adipogenesis at AT or the systemic level;to fully confirm the impaired lipolysis capacity of white adipocytes（WAC）caused by Nfe2l1deletion and thus further verify the regulation of NFE2L1 on WAT plasticity;as well as to analyze the role of NFE2L1 on BAT functional homeostasis at the cellular level from the perspective of adipocyte heterogeneity.In order to clarify the regulatory mechanism of NFE2L1 on adipocyte homeostasis under both normal and pathological conditions,we combined in vivo studies combined with in vitro experiments via constructing Nfe2l1 knockout/knockdown mouse models specific for preadipocytes[Nfe2l1（p）-KO/KD]and mature adipocytes[Nfe2l1（f）-KO]mice as well as cellular models.Based on our previous findings,we finely analyzed the metabolic phenotype and AT plasticity of Nfe2l1（p）-KO/KD mice（part I）as well as the histiocytic characteristics of WAT in Nfe2l1（f）-KO mice under the increased load of lipid metabolism induced by rosiglitazone（RGZ）intervention（part II）;combined with single nuclei RNA-sequencing of Nfe2l1（f）-KO mouse BAT（Part III）,and thus the important role of NFE2L1 in the maintenance of metabolic homeostasis in adipocytes was comprehensively and systematically clarified.Then,we screen and confirm the environmental heavy metals with strong activating effect on NFE2L1 in vitro,and focused on the effect of cadmium exposure on NFE2L1-lipid metabolism regulatory axis（PartⅣ）.The toxicity of cadmium on AT and the role of NFE2L1 were studied by whole animal experiments and in vitro cell to provide scientific support for the precise intervention of metabolic toxicity caused by environmental cadmium exposure.Methods:1.The mice carrying Nfe2l1^flox allele and Cre recombinase gene driven by the promoter of Pdgf Rαgene were crossed to establish Nfe2l1（p）-KO/KD mice.Flox/Flox and Flox/+mice were used as controls,respectively.The body weight of Nfe2l1（p）-KO and Flox/Flox mice was determined at 1 and 8 days of age,and the tissues were collected at the same time（8 mice in each group）.The food/water consumption,body weight,body composition,body temperature and blood glucose of Nfe2l1（p）-KD and Flox/+mice were determined under the condition of basic,high fat diet（HFD,60 kcal%Fat,10 wks）and cold exposure（8℃,14 d）.Glucose and insulin tolerance tests were performed.Harvest tissues or extract primary cells at the end of treatment,and the expression levels of genes related to adipogenesis,thermogenesis and lipid metabolism were also detected.Hematoxylin-eosin staining and immunohistochemistry were used to observe the morphological structure of AT or adipocytes,the distribution and expression of function-related proteins.2.Nfe2l1-Lox P mice were hybridized with Adipoq-Cre mice to obtain Nfe2l1（f）-KO and their littermate control Nfe2l1-floxed mice.Male mice of two genotypes were fed normally until 13 weeks and then randomly divided into two treatment groups.They were given RGZ（10 mg/kg/d）or Vehicle（Veh,2%Dimethyl sulfoxide）by oral gavage once daily for 3 weeks（n=5–8 per group）.Intakes of food and water,body weight,blood glucose and body composition were dynamically monitored.AT was collected for pathological staining and expression analyses of gene related to adipogenesis,lipid metabolism,inflammation using q PCR and Western blot.3.The morphological structure of BAT in 4-and 8-week-old Nfe2l1（f）-KO mice was observed by pathological staining.Single nuclei RNA-sequencing of BAT was performed at their age of 4 weeks.4.The model of long-term Cd exposure was established by giving 14-week-old male wild-type C57BL/6 mice water containing 100 ppm or 200 ppm Cd Cl₂ for 24 weeks,with5 mice in each group.The basic physiological indexes were monitored during the treatment.The tissues were collected for determining the effects of Cd exposure on the structure as well as transcriptome of AT.5.Cell culture,differentiation and heavy metal treatments:Mouse white preadipocytes line 3T3-L1 and brown preadipocytes line HIB 1B were used to study the effects of acute heavy metal exposure on cell viability,NFE2L1 isoform expressions and transcriptional activities.3T3-L1 cells were exposed to Cd Cl₂ at different doses or in various periods during differentiation,the expression levels of adipogenesis-related genes were detected and oil red O staining was performed.Fully differentiated 3T3-L1 cells were exposed to cadmium for 48 h,and the cell response to insulin signal was measured.We used sh RNA to specifically silence Nfe2l1 in 3T3-L1 preadipocytes（termed as Nfe2l1-KD）and defined the cells transduced with the non-target sh RNA control vector as Scramble cells.The cell viability was compared between Nfe2l1-KD and Scramble cells treated with Cd Cl₂ before and after differentiation.6.Statistical analyses:The experimental data were statistically analyzed by TWO-WAY ANOVA,ONE-WAY ANOVA and t-test using Graph Pad Prism 8.0 software.Oil red O staining of 3T3-L1 cells and Western blot data were quantitatively analyzed via Image Pro Plus 6.0 and Image J 1.51j8 software.All experimental data are expressed as mean±SD,with P<0.05 taken as significant.Results:1.Nfe2l1（p）-KO mice shown growth retardation and early death.Abnormal genes expression related to BAT differentiation and function,increased lipid accumulation in brown adipocytes（BAC）were observed in young mice,and the phenotype was aggravated with the aging of mice.Preadipocyte-specific Nfe2l1 knockdown did not affect the metabolic capacity and AT mass under the basic condition,but promoted adipogenesis.Compared with Flox/+mice,Nfe2l1（p）-KD mice fed with HFD exhibited decreased glucose tolerance,more significant intracellular lipid accumulation and hypertrophy,and BAT whitening.Responding to chronic cold stress,Nfe2l1（p）-KD mice had lower core body temperature,less activation of BAT,and lower expression levels of thermogenesis-related genes.2.Nfe2l1（f）-KO mice displayed down-regulated the m RNA levels of lipolysis-related genes,WAC hypertrophy,severe inflammation and deteriorative glucose intolerance.RGZ,which increased fatty acid intake and synthesis,aggravated the above metabolic phenotypes in Nfe2l1（f）-KO mice.3.Adipocyte-specific Nfe2l1 knockout results in the proportion disorder and dysfunction of various BAC subpopulations:the extinction of the A3 subgroup with high expression of guanylate binding proteins,and the emergence of a novel A4 subpopulation with strong metabolic capacity,were accompanied by the declined proteasome level and thermogenesis ability as well as up-regulated inflammatory response and regulated cell death of the remaining original subsets overall.When the A4 subgroup was unable to compensate the loss,BAT showed an age-dependent progressive BAT whitening and dysfunction.4.Among heavy metals,Cd²⁺,Pb²⁺,Cr⁶⁺,Sb³⁺have relatively strong toxicity on adipocytes.NFE2L1 reacts to various heavy metals-induced stress in preadipocytes and especially shows stable reaction to Cd.Long-term Cd exposure caused abnormal AT structure and disturb gene expression of AT related to differentiation and function in vivo,and the toxic effects of Cd depended on the fat depots.In vitro,Cd²⁺had a significant effect on the early differentiation of 3T3-L1 cells,inhibiting lipid accumulation and decreasing the expression level of adipogenesis-related genes during the differentiation.In mature3T3-L1 adipocytes,Cd²⁺interfered with insulin-stimulated phosphorylation of key functional proteins.Loss of Nfe2l1 increased the susceptibility of preadipocytes and mature adipocytes to Cd Cl₂ toxicity.Conclusions:NFE2L1 is involved in sound regulation of adipocyte homeostasis and metabolic plasticity through negative regulation of adipogenesis and positive regulation of lipolysis.Reactive oxygen species induced by cadmium exposure can either act as a second messenger or cause oxidative damage.In addition,it causes adverse effects on the body’s glucose and lipid metabolism by inhibiting adipocyte differentiation,limiting the formation of intracellular lipids,impairing the response of mature adipocytes to insulin signals and the expression of various functional genes,and increase the risk of related diseases.NFE2L1 mediates a variety of acute stress responses predominantly to protect adipocytes from cytotoxicity induced by acute cadmium exposure.Meanwhile,continuous activation of NFE2L1 may also disturb the function and plasticity of AT by affecting the differentiation and fate of adipocytes,thereby damaging the homeostasis of glucose and lipid metabolism.