Wu Xiying, Zvonic Sanjin, Floyd Z Elizabeth, Kilroy Gail, Goh Brian C, Hernandez Teri L, Eckel Robert H, Mynatt Randall L, Gimble Jeffrey M
Stem Cell Laboratory, Pennington Biomedical Research Center, Baton Rouge, Louisiana 70808, USA.
Obesity (Silver Spring). 2007 Nov;15(11):2560-70. doi: 10.1038/oby.2007.308.
Genes encoding the circadian transcriptional apparatus exhibit robust oscillatory expression in murine adipose tissues. This study tests the hypothesis that human subcutaneous adipose-derived stem cells (ASCs) provide an in vitro model in which to monitor the activity of the core circadian transcriptional apparatus.
Primary cultures of undifferentiated or adipocyte-differentiated ASCs were treated with dexamethasone, rosiglitazone, or 30% fetal bovine serum. The response of undifferentiated ASCs to dexamethasone was further evaluated in the presence of lithium chloride. Lithium inhibits glycogen synthase kinase 3, a key component of the circadian apparatus. Total RNA was harvested at 4-hour intervals over 48 hours and examined by real-time reverse transcription polymerase chain reaction (RT-PCR).
Adipocyte-differentiated cells responded more rapidly to treatments than their donor-matched undifferentiated controls; however, the period of the circadian gene oscillation was longer in the adipocyte-differentiated cells. Dexamethasone generated circadian gene expression patterns with mean periods of 25.4 and 26.7 hours in undifferentiated and adipocyte-differentiated ASCs, respectively. Both rosiglitazone and serum shock generated a significantly longer period in adipocyte-differentiated ASCs relative to undifferentiated ASCs. The Bmal1 profile was phase-shifted by approximately 8 to 12 hours relative to Per1, Per3, and Cry2, consistent with their expression in vivo. Lithium chloride inhibited adipogenesis and significantly lengthened the period of Per3 and Rev-erbalpha gene expression profiles by >5 hours in dexamethasone-activated undifferentiated ASCs.
These results support the initial hypothesis and validate ASCs as an in vitro model for the analysis of circadian biology in human adipose tissue.
编码昼夜节律转录装置的基因在小鼠脂肪组织中呈现出强劲的振荡表达。本研究检验了这样一个假设，即人皮下脂肪来源干细胞（ASC）提供了一个体外模型，可用于监测核心昼夜节律转录装置的活性。
用 dexamethasone、罗格列酮或 30%胎牛血清处理未分化或已分化为脂肪细胞的 ASC 原代培养物。在存在氯化锂的情况下，进一步评估未分化 ASC 对 dexamethasone 的反应。锂抑制糖原合酶激酶 3，这是昼夜节律装置的一个关键组成部分。在 48 小时内每隔 4 小时收集总 RNA，并通过实时逆转录聚合酶链反应（RT-PCR）进行检测。
已分化为脂肪细胞的细胞对处理的反应比与其供体匹配的未分化对照细胞更快；然而，昼夜节律基因振荡的周期在已分化为脂肪细胞的细胞中更长。Dexamethasone 在未分化和已分化为脂肪细胞的 ASC 中分别产生了平均周期为 25.4 小时和 26.7 小时的昼夜节律基因表达模式。相对于未分化的 ASC，罗格列酮和血清休克在已分化为脂肪细胞的 ASC 中均产生了明显更长的周期。相对于 Per1、Per3 和 Cry2，Bmal1 的表达谱相位偏移了约 8 至 12 小时，这与其在体内的表达一致。在 dexamethasone 激活的未分化 ASC 中，氯化锂抑制脂肪生成，并使 Per3 和 Rev-erbalpha 基因表达谱的周期显著延长超过 5 小时。
这些结果支持了最初的假设，并验证了 ASC 作为分析人脂肪组织昼夜节律生物学的体外模型。
