敲低GPM6B促进白色脂肪细胞向棕色脂肪表型转变
Glycoprotein M6B knockdown promotes white adipocytes to brown adipocyte phenotype
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摘要:目的 研究糖蛋白M6B（Glycoprotein M6B，GPM6B）在白色脂肪细胞向棕色脂肪表型转变中的作用。方法 ① Western blot和qPCR检测小鼠白色脂肪组织（white adipose tissue, WAT）和棕色脂肪组织（brown adipose tissue, BAT）中GPM6B的表达。②分别用对照组（shScramble）和敲低GPM6B组（shGPM6B1和shGPM6B2）的慢病毒液感染C3H10T1/2 小鼠间充质细胞系, 荧光显微镜下观察病毒的转染效率。Western blot和qPCR检测GPM6B的干涉效率。③将shScramble, shGPM6B1和shGPM6B2的稳转细胞系分别给予白色脂肪诱导分化液8 d，油红O染色观察脂滴形态，Western blot检测棕色脂肪标记基因解偶联蛋白（uncoupling protein, UCP1）的表达，qPCR检测热生成相关基因的mRNA表达。结果 ①与WAT比，BAT中GPM6B的表达在蛋白和mRNA水平均显著降低（P<0.05）。②与shScramble组相比，shGPM6B1组和shGPM6B2组GPM6B的干涉效率在蛋白和mRNA水平均显著降低（P<0.05）。③敲低GPM6B后，白色脂肪细胞的脂滴变小，UCP1的表达和热生成相关基因的mRNA均显著升高（P<0.05）。结论 敲低GPM6B促进白色脂肪细胞向棕色脂肪表型转变。Abstract:AIM To investigate the role of glycoprotein M6B (GPM6B) in phenotypic transformation of white adipocytes to brown adipocytes.METHODS The expressions of GPM6B in white adipose tissue and brown adipose tissue were detected by Western Blot and qPCR. The C3H10T1/2 cells were infected with lentiviruses expressing two independent hairpin shRNA sequences for GPM6B (shGPM6B1 and shGPM6B2) or control shScramble sequence. The transfection efficiency of lentiviruses was observed by fluorescence microscope. The extent of the knockdown by the targeted shRNAs for GPM6B was confirmed by Western blot and qPCR analysis. The control (shScramble) and GPM6B knockdown (shGPM6B1 or shGPM6B2) stable cell lines were treated to induce adipogenesis for 8 days and examined for lipid accumulation by oil red O staining. The protein levels of UCP1 were tested by Western blot and the mRNA levels of the thermogenetic genes were tested by qPCR.RESULTS Compared with that in WAT, the expression of GPM6B was significantly down-regulated in BAT. Each of the shRNA constructs effectively knocked down the mRNA and protein expression of GPM6B (P<0.05). Remarkably smaller lipid droplets were observed in differentiated C3H10T1/2 cells when GPM6B was inhibited. Moreover, GPM6B knockdown significantly promoted the expression of UCP1 and the thermogenetic genes during this process (P<0.05).CONCLUSION GPM6B knockdown promotes phenotypic transformation of white adipocytes to brown adipocytes.
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