The Role Of NFE2L1 In Arsenic-induced Dysfunction Of Differentiation And Activation Of Brown Adipose Tissue

Objective: Arsenic,the 20 th most abundant element in the earth's crust,is ubiquitous in the environment in both inorganic and organic forms.Human exposure to inorganic arsenic(iAs)mainly occurs in environmental and occupational settings.According to the maximum allowable arsenic concentration of 10 ?g/L in the drinking water standard set by the World Health Organization,it is estimated that about 200 million people in the world are exposed to arsenic via drinking water.Chronic exposure of iAs is positively correlated with multiple organ cancers.iAs exposure is also associated wirh skin,endocrine,cardiovascular,nervous and immune system diseases.Epidemiological investigation shows that long-term drinking high concentration arsenic water(iAs > 150 ?g/L)is closely related to the occurrence of type 2 diabetes mellitus(T2D).A large number of animal experimental studies support that chronic arsenic exposure is a clear pathogenic factor of T2 D.In China,every 100 ?g/L increase in iAs concentration in drinking water increases the risk of T2 D by 13%.However,the specific mechanism of iAs induced diabetes is still unclear.Studies suggested that iAs may induce T2 D by impairing the glucose response of islet ? cells and reducing the insulin sensitivity of liver,adipose tissue and muscle.Dysfunctions of adipose tissue may lead to glucose and lipid metabolic diseases.Adipose tissue is involved in glucose and lipid metabolism and is one of the central organs of energy homeostasis regulation.Studies suggested that white adipocytes can isolate TG from liver,muscle and other tissues to limit its lipotoxicity.On the contrary,brown adipose tissue(BAT)plays an important role in the expression of ?-adrenergic receptors(?-AR).BAT may directly absorb glucose,reduce blood glucose concentration,increase energy consumption,improve metabolic rate and promote negative energy balance,which can reduce a variety of metabolic complications in patients with T2 D,including dyslipidemia,impaired insulin secretion and insulin resistance.NF-E2-related factor 1(NFE2L1)is widely expressed in a variety of tissues.It mediates antioxidant response,regulates physiological and biochemical processes,including lipid metabolism,oxidative stress,proteasome homeostasis,mitochondrial respiration,inflammatory response,cell proliferation and differentiation.With the deepening researches in recent years,the role of NFE2L1 in the regulation of inflammation and lipid metabolism has been investigated.Our research group used Nfe2l1-Flox to hybridize with Adipoq-Cre mice to obtain mature adipocyte specific knockout Nfe2l1(Nfe2l1(f)-KO)mice.The analysis showed that the subcutaneous adipose tissue of Nfe2l1(f)-KO mice was significantly diminished,adipocytes were abnormally hypertrophy,severe inflammation and insulin resistance.In vitro studies showed that L-NFE2L1 may negatively regulate the core transcription factor of adipocyte differentiation,peroxisome proliferators activated receptors ?(PPAR?),especially PPAR?2 subtype,thus inhibiting the terminal differentiation of adipocytes.It has been reported that the specific deletion of Nfe2l1 in BAC lead to decreasing of proteasome activity,endoplasmic reticulum stress,inflammatory reaction and mitochondrial dysfunction in BAT,resulting in BAT whitening.At present,there are few studies focused on the mechanism of environmental arsenic exposure on BAT.Previous studies of our group showed that subchronic drinking water arsenic exposure interfere with the energy consumption function of BAT by damaging mitochondrial oxidative phosphorylation,reducing the expression of thermogenic genes,inhibiting lipolysis and causing inflammatory reaction.However,the mechanism of Nfe2l1 gene regulation of BAT and whether arsenic impairs BAT thermogenesis through Nfe2l1 pathway are still unclear.This paper is divided into three parts.The first part is to study adipocytes specific Nfe2l1 knockout in the lipid metabolism of brown adipocytes.The second part is to study the effect of iAs on the expression of Nfe2l1 gene.The third part is to study the role of Nfe2l1 in arsenic induced differentiation and activation disorder of brown adipocytes.The purpose of this study is to systematically study the mechanism of iAs impairing the thermogenic function of BAT through Nfe2l1 pathway,providing a new theoretical basis for the prevention and treatment of T2 D and other glucose and lipid metabolic diseases.Methods: 1.Treat adipocyte specific Nfe2l1 knockout mice with acute or chronic cold exposure Nfe2l1-Flox was hybridized with Adipoq-Cre tool mice to obtain Nfe2l1(f)-KO mice and control(Nfe2l1-Flox)mice.Male and female mice at 16-week-old were treated with 4? acute cold stimulation for 4 hours and 8? chronic cold exposure for 14 days,12-15 mice in each group.During acute cold stimulation,the core body temperature of mice was detected;during chronic cold exposure,the basic vital signs of mice were observed and recorded at any time.2.Treatment of experimental samples of adipocyte specific Nfe2l1 knockout mice The morphology of BAT mitochondria was observed by electron microscopy.The lipid droplets,cell morphology and protein expression position and quantity of BAT were observed by oil red staining,H&E staining and immunohistochemical staining.The total RNA of BAT was extracted,and the thermogenic gene,mitochondrial oxidative phosphorylation,?-oxidation,adipocyte differentiation and lipolysis function were detected.The transcription level of related genes,lipid metabolism and inflammatory response related genes were detected.The total BAT protein was extracted to detect the above key protein levels.3.Treat normal and differentiated HIB1 B cells with iAs Normal and differentiated HIB1 B cells were treated with gradient iAs to study the dose-response relationship between iAs and transcription factor NFE2L1 expression.The normal and differentiated HIB1 B cells were treated with iAs at different times to study the time-effect relationship between iAs and transcription factor NFE2L1 expression.4.Induction differentiation of Nfe2l1 silened and overexpressed HIB1 B cells Inducing differentiation of Nfe2l1 silenced and overexpression HIB1 B cells to investigate the effect of transcription factor NFE2L1 on differentiation of brown adipocytes.5.Treat differentiated of Nfe2l1 silened and overexpressed HIB1 B cells with iAs The effect of Nfe2l1 on the differentiation of brown adipocytes and its mechanisms were studied by Nfe2l1 silented and overexpressed HIB1 B cell lines.The mechanism of Nfe2l1 on differentiation dysfunction of brown adipocytes induced by iAs was studied by HIB1 B cells in the basal and differentiation process treated by iAs.Results: 1.Adipocyte specific Nfe2l1 knockout causes whitening phenotype of BAT in mice Acute cold exposure induce the expression of transcription factor NFE2L1 protein in BAT(P < 0.05).Nfe2l1(f)-KO mice showed significantly lower body temperature under acute 4? cold exposure and fasted for 16 h,increased mortality under chronic 8? cold exposure and decreased basal metabolic rate at night(P < 0.05).In vitro,the size and color of BAT of Nfe2l1(f)-KO mice were reduced.H&E and oil red staining showed obvious lipid accumulation in BAT of Nfe2l1(f)-KO mice.The content of TG in BAT in Nfe2l1(f)-KO mice was significantly higher than that in control mice.m RNA and protein expression of core markers related to heat generation in Nfe2l1(f)-KO mice,such as Uncouple protein 1(Ucp1),Peroxisome proliferator-activated receptor-? coactivator 1-?(Ppargc1?),Iodothyronine Deiodinase 2(Dio2)were significantly lower compared to the control mice(P < 0.05).Mitochondrial morphology abnormalities,decreased number,mitochondrial respiration and ?-oxidative function were observed in BAT of KO mice,such as Cytochrome C oxidase IV(COX IV),NADH: ubiquitquinone oxidoredoreductase subunit S4(NDUFS4),Oxidative phosphorylation(OXPHOS)and other proteins were significantly decreased(P < 0.05).2.Nfe2l1 deficiency attenuated lipolysis function and severe inflammation of BAT High-volume RNA-seq results showed that the transcription factor NFE2L1 in adipocytes was deleted,and the signaling pathways related to inflammatory response in BAT were significantly altered(P < 0.05).Immunohistochemical staining of macrophage marker F4/80 showed that the staining of Nfe2l1(f)-KO was significantly deeper compared to control mice.Mechanism analysis found that the m RNA and protein expression of lipophylase related genes,such as adipose triglyceride lipase(ATGL),hormone-sensitive lipase(HSL),monoacyllipase(MGL)were significantly reduced in Nfe2l1(f)-KO mice(P < 0.05).The lipolysis function of KO mice was weakened,resulting in lipid accumulation in the cells,causing abnormal hypertrophy of adipocytes that exceeded the capacity limit.The m RNA expression of inflammatory response related genes in Nfe2l1(f)-KO mice was significantly increased(P < 0.05).3.Nfe2l1 deficiency in adipocytes causes thermogenic dysfunction in BAT under acute and chronic cold exposure After 4 h of acute cold stimulation at 4?,the oxygen consumption and carbon dioxide production of Nfe2l1(f)-KO mice were significantly lower compared to control mice(P < 0.05).The expression of classical ?-AR-c AMP-PKA pathway and its downstream protein in cold-induced activation of BAT were significantly decreased in Nfe2l1(f)-KO mice(P < 0.05).Compared with the same genotype at room temperature,the lipid droplets in the Nfe2l1(f)-KO mice decreased in area,but still larger than that in the control mice under cold exposure.Immunohistochemical of UCP1 protein results showed that Nfe2l1(f)-KO was significantly lower than the control mice.Protein and m RNA expression of UCP1,PGC1?,Di O2 and Cell Death Appraisement of DFFA Like Effector A(CIDEA)in Nfe2l1(f)-KO mice were significantly lower in Nfe2l1(f)-KO mice(P < 0.05).4.Inorganic arsenic exposure activates the expression of NFE2L1 subtypes in HIB1 B cells Inorganic arsenic exposure induced expression of NFE2L1 subtypes in HIB1 B cells,including 741,742,583 and 453 subtypes.It also induced m RNA expression of Nfe2l1 downstream genes,such as Glutamate-cysteine ligase catalytic subunit(Gclc),?-glutamate cysteine ligase regulation subunit(Gclm),NAD(P)H:quinone oxidoreductase 1(Nqo1),Proteasome Subunit Beta Type 6(Psmb6),Proteasome Subunit Beta Type 4(Psmb4),and showed dose-response and time-response relationships.5.Transcriptional factor NFE2L1 negatively regulates the thermogenic dysfunction of HIB1 B cells induced by inorganic arsenic exposure The m RNA and protein expression levels of Nfe2l1 gene increased during the differentiation of HIB1 B cells(P < 0.05).Inorganic arsenic treatment inhibited HIB1 B cells differentiation(P < 0.05).The m RNA expression level of differentiation-related genes,including Ucp1,Peroxisome proliferator-activated receptor-?(Ppar?),PRD1-BF-1-RIZ1 homologous domain-containing protein 16(Prdm16)were higher in Nfe2l1 silenced cells under iAs treatment(P < 0.05).The m RNA expression level of differentiation-related gene Ucp1,Ppar?,Prdm16 were lower in Nfe2l1 overexpressed cells under iAs treatment(P < 0.05).Conclusion: 1.The activation ability of BAT decreased under acute cold exposure and the proliferation of BAT slowdown under long-term chronic cold exposure in Nfe2l1(f)-KO mice.The mechanism may be that Nfe2l1 deletion causes the decrease of mitochondrial number and ?-oxidation function in BAT,inhibits the expression of lipolytic protein,damages the function of lipolysis,causes abnormal accumulation of lipid and causes obvious inflammatory reaction,resulting weakened of heat production function of BAT in Nfe2l1(f)-KO mice.2.Inorganic arsenic exposure induced expression of NFE2L1,especially L-NFE2L1 expression in HIB1 B cells,and increases expression of its downstream genes.3.Nfe2l1 gene silence rescued iAs induced HIB1 B cell differentiation inhibition and up-regulation of thermogenic genes expression,such as Ucp1.Overexpression of Nfe2l1 gene aggravated iAs induced HIB1 B cell differentiation inhibition and induce decreased m RNA expression of Ucp1.These results indicated that transcription factor NFE2L1 plays a negative regulatory role in the inhibition of HIB1 B cell differentiation induced by iAs.