Puri Vishwajeet, Chakladar Abhijit, Virbasius Joseph V, Konda Silvana, Powelka Aimee M, Chouinard My, Hagan G Nana, Perugini Richard, Czech Michael P
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
J Lipid Res. 2007 Feb;48(2):465-71. doi: 10.1194/jlr.D600033-JLR200. Epub 2006 Nov 8.
Cultured adipocyte cell lines are a model system widely used to study adipose function, but they exhibit significant physiological differences compared with primary cells from adipose tissue. Here we report short interfering RNA-based methodology to selectively attenuate gene expression in mouse and human primary adipose tissues as a means of rapidly validating findings made in cultured adipocyte cell lines. The method is exemplified by depletion of the PTEN phosphatase in white adipose tissue (WAT) from mouse and humans, which increases Akt phosphorylation as expected. This technology is also shown to silence genes in mouse brown adipose tissue. Previous work revealed upregulation of the mitochondrial protein UCP1 in adipose cells from mice lacking the gene for the transcriptional corepressor RIP140, whereas in cultured adipocytes, loss of RIP140 has a little effect on UCP1 expression. Application of our method to deplete RIP140 in primary mouse WAT elicited markedly increased oxygen consumption and expression of UCP1 that exactly mimics the phenotype observed in RIP140-null mice. This ex-vivo method of gene silencing should be useful in rapid validation studies as well as in addressing the depot- and species-specific functions of genes in adipose biology.
培养的脂肪细胞系是广泛用于研究脂肪功能的模型系统，但与来自脂肪组织的原代细胞相比，它们表现出显著的生理差异。在此，我们报告了一种基于短干扰RNA的方法，用于选择性地减弱小鼠和人类原代脂肪组织中的基因表达，以此作为快速验证在培养的脂肪细胞系中所获发现的一种手段。该方法通过从小鼠和人类的白色脂肪组织（WAT）中敲除PTEN磷酸酶得以例证，正如预期的那样，这会增加Akt磷酸化。该技术还显示可使小鼠棕色脂肪组织中的基因沉默。先前的研究表明，在缺乏转录共抑制因子RIP140基因的小鼠的脂肪细胞中，线粒体蛋白UCP1上调，而在培养的脂肪细胞中，RIP140的缺失对UCP1表达影响甚微。应用我们的方法在小鼠原代WAT中敲除RIP140，引发了耗氧量显著增加以及UCP1表达上调，这与在RIP140基因敲除小鼠中观察到的表型完全一致。这种基因沉默的离体方法在快速验证研究以及探讨脂肪生物学中基因的储存库和物种特异性功能方面应该会很有用。
