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肥胖小鼠脂肪组织缺氧对脂联素表达的转录抑制作用

Transcriptional inhibition of adiponectin expression by adipose tissue hypoxia in obese mice

摘要:

目的 观察缺氧对脂肪细胞脂联素mRNA和蛋白表达的影响,探讨肥胖小鼠脂肪组织缺氧导致脂肪组织脂联素表达下降的机制.方法 采用实时定量聚合酶链反应(qRT-PCR)和蛋白免疫印迹法(Western blotting)检测遗传型肥胖小鼠(ob/ob,12周)和高脂饮食肥胖小鼠(HFD,53周)的附睾旁脂肪中脂联素mRNA和蛋白的表达;用小鼠3T3-L1脂肪细胞系为模型,采用RT-PCR和荧光素酶报告基因方法检测缺氧处理后脂联素和过氧化物酶体增殖物激活受体(PPAR)-γ mRNA的表达和稳定性、脂联素启动子的活性;用Western blotting和荧光素酶报告基因检测缺氧对PPAR-γ在核蛋白中集聚以及PPAR-γ转录因子活性的影响.组间数据比较采用t检验.结果 (1)缺氧时两种肥胖小鼠的脂肪组织中脂联素mRNA和蛋白的表达均显著下降(P<0.01);3T3-L1脂肪细胞系在缺氧8h和24 h后,脂联素mRNA表达量分别下降至0.65 ±0.05和0.29 ±0.05,较对照组(1.00±0.04)明显降低,差异有统计学意义(t=11.548、24.893,均P<0.01),但缺氧对脂联素mRNA的稳定性并没有影响;荧光素酶报告基因方法表明,脂联素启动子的活性受到缺氧的抑制.(2)在两种肥胖小鼠的脂肪组织中,PPAR-γ mRNA和蛋白的表达均明显下降(P<0.01);小鼠3T3-L1脂肪细胞系在缺氧8h和24 h后,PPAR-γ mRNA的表达量分别下降至0.72 ±0.09和0.54 ±0.07,与对照组(1.00±0.09)相比,差异有统计学意义(t=5.134、9.876,均P<0.01);PPAR-γ蛋白的核转位以及PPAR-γ转录因子活性也受到缺氧的抑制.结论 肥胖小鼠脂肪组织缺氧抑制了脂联素的表达,抑制作用可能发生在转录水平;其机制可能是通过抑制PPAR-γ mRNA的表达和PPAR-γ转录因子的活性而实现的.

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abstracts:

Objective To investigate the effects of hypoxia on adiponectin mRNA transcription and protein expression in adipose tissue of obese mice.Methods The mRNA and protein of adiponectin and peroxisome proliferator activated receptor-γ (PPAR-γ) in epididymal fat tissues of genetic obese mice (ob/ob) and high fat diet-induced (HFD) obese mice were examined through quantitative real-time PCR (qRT-PCR) and western blotting.mRNA expression and promoter activity of adiponectin was measured with real-time qRT-PCR and luciferase reporter assay in 3T3-L1 adipocytes.Adiponectin mRNA stability was checked with real-time RT-PCR.mRNA and mRNA stability of PPAR-γ were examined with qRT-PCR in 3T3-L1 adipocytes after hypoxia treatment and then nuclear accumulation of PPAR-γ and peroxisome proliferator response element (PPRE) transcriptional activity were determined in mature adipocytes.Comparison of data between groups was applied by using t test.Results (1) Adiponectin expression was inhibited in white adipose tissue of obese mice at both mRNA and protein levels(P < 0.01).In mature 3T3-L1 adipocytes,the qRT-PCR data showed that mRNA expression of adiponectin was reduced by hypoxia,compared with control group,especially after hypoxia for 8 and 24 hours,the level of adiponectin mRNA was reduced from 1.00 ± 0.04 to 0.65 ± 0.05 and 0.29 ± 0.05 (t =11.548,24.893,both P < 0.01).Half life of mRNA was not reduced by hypoxia.A luciferase reporter driven by the adiponectin gene promoter was inhibited by hypoxia in 3T3-L1 adipocytes.(2)Protein and mRNA expression of PPAR-γ were decreased in white adipose tissue of obese mice and in 3T3-L1 adipocytes after hypoxia treatment.After hypoxia for 8 and 24 hours,compared with control group,level of PPAR-γmRNA was significantly reduced from 1.00 ± 0.09 to 0.72 ± 0.09 and 0.54 ± 0.07 (t =5.134,9.876,both P < 0.01).PPAR-γ mRNA stability was reduced in 3T3-L1 adipocytes after hypoxia treatment.Nuclear translocation of PPAR-γ protein and transcriptional activity of PPAR-γwas also inhibited by hypoxia.Conclusion The adiponectin mRNA expression is reduced by hypoxia and the inhibition happened at the transcriptional level.It might be the result of suppression of PPAR-γ,an important transcription activator for adiponectin,at both mRNA expression level and its transcriptional activity.

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作者: 何庆 [1] 高占国 [2] 李晓通 [1] 叶建平 [2]
期刊: 《中华糖尿病杂志》2013年5卷8期 486-490页 ISTICPKUCSCD
栏目名称: 论著
DOI: 10.3760/cma.j.issn.1674-5809.2013.08.008
发布时间: 2013-10-12
基金项目:
美国国立卫生研究院(NIH)项目 美国糖尿病学会项目
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