Characterization of transgenic mice constitutively expressing dominant negative form of BMAL1.

BMal1，也称为MOP3或Arnt3，是一种已知的调节昼夜节律的转录因子。在这里，我们确定它参与了成熟脂肪细胞的成脂和脂代谢活性的调控。在3T3-L1细胞脂肪分化过程中，BMAL1基因的表达水平在诱导后4d开始升高，并在分化后的细胞中高表达。在分离自C57BL/6J小鼠的白色脂肪组织中，BMAL1主要在含有脂肪细胞的部分表达，而间质-血管部分则主要表达。BMal1基因敲除小鼠胚胎成纤维细胞(MEF)未能分化为脂肪细胞。重要的是，通过腺病毒基因转移重新加入BMAL1，恢复了BMAL1基因敲除的MEF分化的能力。通过RNAi技术下调3T3-L1细胞中BMAL1的表达，使细胞只在细胞中积累最少量的脂滴。腺病毒介导的BMAL1在3T3-L1脂肪细胞中的表达可诱导多种与脂肪生成有关的因子的产生。这些基因的启动子活性是以BMAL1依赖的方式被刺激的。有趣的是，这些因子在小鼠脂肪组织中的表达显示出明显的昼夜节律。此外，BMAL1在脂肪细胞中的过表达增加了脂肪细胞的脂质合成活性。为了了解BMAL1在体内脂肪细胞中的作用，我们建立了脂联素启动子控制下在脂肪细胞中组成性表达BMAL1的转基因小鼠。这些小鼠将成为分析BMAL1在成熟脂肪细胞中功能的有用工具。这些结果表明，BMAL1是昼夜节律的主要调节者，也在成熟脂肪细胞的脂肪分化和脂肪生成的调节中发挥重要作用。

BMAL1, also termed as MOP3 or Arnt3, is a transcription factor known to regulate circadian rhythm. Here, we established its involvement in the control of adipogenesis and lipid metabolism activity in mature adipocytes. During adipose differentiation in 3T3-L1 cells, the level of BMAL1 mRNA began to increase 4 days after induction and was highly expressed in differentiated cells. In white adipose tissues isolated from C57BL/6J mice, BMAL1 was predominantly expressed in a fraction containing adipocytes, as compared with the stromal-vascular fraction. BMAL1 knockout mice embryonic fibroblast cells (MEFs) failed to be differentiated into adipocytes. Importantly, adding BMAL1 back by adenovirus gene transfer restored the ability of BMAL1 knockout MEFs to differentiate. Knock-down of BMAL1 expression in 3T3-L1 cells by an RNAi technique allowed the cells to accumulate only minimum amounts of lipid droplets in the cells. Adenovirus-mediated expression of BMAL1 in 3T3-L1 adipocytes resulted in induction of several factors involved in lipogenesis. The promoter activity of these genes was stimulated in a BMAL1-dependent manner. Interestingly, expression of these factors showed clear circadian rhythm in mice adipose tissue. Also, overexpression of BMAL1 in adipocytes increased lipid synthesis activity. To understand the roles of BMAL1 in adipocytes in vivo, we established transgenic mice constitutively expressing BMAL1 in adipocytes under the control of adiponectin promoter. These mice will be the useful tools to analyze the function of BMAL1 in mature adipocytes. These results indicate that BMAL1, a master regulator of circadian rhythm, plays important roles also in the regulation of adipose differentiation and lipogenesis in mature adipocytes.