| Objective: Obesity has been associated with a strong predisposition towards metabolic diseases and cancer.Thus,it constitutes a public health issue.Iodine,an important trace element,is directly involved in the synthesis of thyroid hormones.Notably,recent clinical studies have found that iodine intake status was negatively correlated with body weight.Excessive iodine status was a potential protective factor for central obesity.However,further mechanism studies are rare.The expansion of white adipose tissue(WAT)leads to overweight and obesity.Oxidative stress is closely related to obesity.Iodine is an inorganic antioxidant that scavenge reactive oxygen species(ROS).ROS removal from adipose tissue can promote the expansion of healthy fat,reduce ectopic lipid deposition,and improve insulin sensitivity.Nuclear factor E2-related factor 2(Nrf2),also called NFE2L2,is an important transcription factor which exert its transcriptional control on genes harboring antioxidant response elements(ARE).Kelch-like ECH-associated protein 1(Keap1)is a specific repressor of Nrf2.When Nrf2 dissociates from Keap1,it enters the nucleus from the cytoplasm,thereby activating the expression of downstream antioxidant enzyme genes,including heme oxygenase-1(HO-1).Intracellular ROS level is decreased and antioxidant protection is enhanced.Meanwhile,Nrf2-Keap1 signaling pathway also plays an important role in regulating adipogenesis and adipocyte browning.3T3-L1 preadipocytes are common models used in studying the phenotypes of adipogenesis,lipid metabolism and adipocyte browning.Once the cells are differentiated,they can express the characteristics of both white and brown adipocytes.Transcriptome sequencing(RNA-seq)was used to further explore the gene expression changes and underlying molecular mechanism of 3T3-L1 cells with iodine intervention.Immediate early response gene 3(IER3)is usually induced by various stimuli.Previous studies have shown that IER3 negatively regulates Nrf2.Low expression of IER3 can activate Nrf2 and enhance the ability of injury resistance.In addition,high expression of IER3 promoted the proliferation of human adipose progenitor cells(APC)and inhibited the differentiation of APC,which was associated with adipose tissue expansion and obesity.This study aimed to screen out differentially expressed genes in 3T3-L1 cells with iodine intervention by RNA-seq,and conducted in vitro and in vivo experiments using 3T3-L1 cell lines and Wistar rats to explore the function of iodine in regulationg of adipogenesis and adipocyte browning and the regulating mechanism of IER3/Nrf2 signaling pathway in the process.Methods:Part Ⅰ3T3-L1 cells were cultured and differentiated with different concentrations of iodine,divided into the control group(Con)with iodine concentration of 1μM and the high iodine group(HI)with iodine concentration of 10μM.RNA-seq was performed,and the differentially expressed genes(DEGs)obtained were further analyzed by GO and KEGG bioinformatics analysis.m RNA expression levels of obvious DEGs were verified by q RT-PCR.Part Ⅱ3T3-L1 cells were cultured and induced to differentiate under the intervention of different concentrations of iodine(0μM,1μM,5μM,10μM and 100μM).The effect of iodine on cell proliferation was detected by cell counting kit-8(CCK8)method.m RNA and protein expression levels of adipogenic marker genes were measured by q RT-PCR and Western blot method.Lipid droplets were measured by oil red O staining to observe the effect of iodine on adipogenesis.m RNA and protein expression levels of labeled molecules in brown adipocytes were measured,and the cell oxygen consumption rate(OCR)was measured by seahorse method to observe the effect of iodine on adipocyte browning.ROS was measured by fluorescent probe DCFH-DA.Protein expression levels of IER3/Nrf2 signaling pathway were measured by Western blot and immunofluorescence.After the IER3 overexpression plasmid or si-Nrf2 was transfected,protein expression levels of peroxisome proliferator activated receptor γ(PPARγ)and uncoupling protein 1(UCP1)were measured to further elucidate that iodine regulates adipogenesis and adipocyte browning through IER3/Nrf2 signaling pathway.Part ⅢSixty 4-week-old male Wistar rats were divided into normal iodine group(NI)and high iodine group(HI).Rats in HI group were fed with water containing 1200 μg/L potassium iodide(KI)for 24 weeks.Urinary iodine concentration(UIC)was measured by inductively coupled plasma-mass spectrometry(ICP-MS).Serum thyroid stimulatory hormone(TSH),total triiodothyronine(TT3)and total thyroxine(TT4)levels were measured by enzyme-linked immunosorbent assay(ELISA).The body weight and perirenal WAT(pr WAT)weight of Wistar rats were monitored,and the ratio of pr WAT weight to body weight was calculated.pr WAT was collected for HE staining to measure the adipocyte morphology.Protein expression levels of PPARγ、UCP1 and IER3/Nrf2 signaling pathway in pr WAT were measured by Western blot method.Results:Part ⅠA total of 2193 genes were differentially expressed between Con group and HI group(p-adjust <0.05).Among them,IER3,DUSP6,DUSP1,PHLDA1 and BTG2 expressed significantly decreasing.GO results showed that the DEGs were mainly enriched in cellular process,biological regulation and metabolic process in biological process;in organelle and cell part in cellular component;and in binding and catalytic activity in molecular function.KEGG results showed that the DEGs were mainly enriched in MAPK and cancer related signaling pathways.Part Ⅱ3T3-L1 cells expressed sodium iodine transporter(NIS)and had the ability of iodine uptake.The CCK8 results of 72 hours showed that,compared with 0μM group,the cell proliferation rate was higher in 10μM group(P <0.01).There was no significant difference of cell proliferation rate between 1μM group and the other iodine intervention groups.The oil red O straining results showed that,compared with 1μM group,adipogenesis was more significant and adipocytes were smaller in 10μM group.Semi-quantitative analysis showed that lipid accumulation was more in 5μM and 10μM groups(P < 0.05).q RT-PCR results showed that the m RNA expression of adipogenic marker genes PPARγ,CCAAT/enhancer-binding protein β(C/EBP β),fatty acid binding protein 4(FABP4)and fatty acid synthase(FASN)were all increased in 10μM group(P <0.05).Western blot results showed that the protein expression of PPARγ was also increased in 10μM group(P < 0.05).The seahorse results showed that the basal respiration,ATP production and maximum respiration in iodine intervention groups(5μM and 10μM)were significantly higher than those in 1μM group(P all < 0.01),the space capacity was slightly higher than that in 1μM group(P < 0.05).The m RNA and protein expression of UCP1 and type 2 deiodinase(Dio2)in 10μM group were higher than those in 1μM group(P < 0.05).DCFA-DA results showed that compared with 1μM group,intracellular ROS levels in 5μM and 10μM groups were decreased(P < 0.05).Furthermore,the IER3/Nrf2 antioxidant signaling pathway was activated in 10μM group,which showed decreased IER3,increased Nrf2,decreased Keap1,and increased HO-1m RNA and protein expression levels.The immunofluorescence results showed that,compared with 1μM group,the green fluorescence intensity in 5μM and 10μM groups were stronger(P <0.05),and the protein expression of Nrf2 in the nucleus were significantly increased.IER3 overexpression or Nrf2 knockdown hindered adipogenesis and adipocyte browning,and the expression of PPARγ and UCP1 could not be increased with 10μM iodine intervention.Part ⅢThe UIC in HI group was higher than that in NI group at each time(4w,8w,12 w,18w and 24w)(P < 0.01),and the mean value of UIC in HI group was about 5-6 times of that in NI group.There were no significant differences in serum TSH,TT3 and TT4 levels between the two groups(P > 0.05),which proved that the animal models we re successfully established.After 24 weeks feeding,the body weight and pr WAT weight/body weight in HI group were decreased compared with those in NI group(P <0.01).HE staining of pr WAT showed that adipogenesis was more significant with more and smaller adipocytes in HI group.Semi-quantitative analysis showed that the average adipocyte area in HI group was significantly smaller than that in NI group(P < 0.01).The protein expression of PPARγ and UCP1 in HI group were higher than those in NI group(P < 0.05).The protein expression of Nrf2 and HO-1 were higher and the expression of IER3 and Keap1 were lower in HI group(P < 0.05).The IER3/Nrf2 antioxidant signaling pathway was activated in HI group.Conclusion:1.mRNA of functional genes were abnormally expressed in 3T3-L1 cells with low concentration iodine intervention.Bioinformatics analysis showed that DEGs were mainly enriched in regulating the structure and function of adipocytes.2.Low concentration of iodine increase the antioxidant effect through the IER3/Nrf2 signaling pathway to promote adipogenesis and adipocyte browning of 3T3-L1 cells.3.Long-term low concentration iodine excess can reduce the body weight and pr WAT weight/body weight of Wistar rats and participate in the process of resisting obesity. |
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