Ruan Hong, Zarnowski Mary Jane, Cushman Samuel W, Lodish Harvey F
Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.
J Biol Chem. 2003 Nov 28;278(48):47585-93. doi: 10.1074/jbc.M305257200. Epub 2003 Sep 15.
Isolation and subsequent in vitro culture of primary adipose cells are associated with down-regulation of GLUT4 mRNA and simultaneous induction of GLUT1 gene expression. Progressive loss of insulin-responsive GLUT4 contributes to the decrease in insulin-mediated glucose uptake in these cells when cultured in vitro. The mechanisms underlying these alterations are unknown. Here, we report that the standard procedure for isolating primary adipose cells from mouse adipose tissue triggers induction of many genes encoding inflammatory mediators including TNF-alpha, interleukin (IL)-1 alpha, IL-6, multiple chemokines, cell adhesion molecules, acute-phase proteins, type I IL-1 receptor, and multiple transcription factors implicated in the cellular inflammatory response. Secretion of TNF-alpha protein was also significantly induced during the 2-h collagenase digestion of adipose tissue. Isolated primary adipose cells exhibit dramatic changes in expression of multiple mRNAs that are characteristic of TNF-alpha-treated 3T3-L1 adipocytes including down-regulation of many genes important for insulin action and triglyceride synthesis. Addition of TNF-alpha to primary adipose cells in culture did not change the kinetics or the extent of the repression of adipose cell-abundant genes. Moreover, TNF-alpha-neutralizing antibody failed to block the changes in gene transcription in isolated primary adipose cells. Also, the standard isolation procedure induced the expression of NF-kappa B family members and their target genes in primary adipose cells prepared from TNF-alpha-/- mice to the same extent as in cells isolated from wild-type mice and resulted in almost identical changes in global gene expression when these cells were cultured in vitro. Thus, these data suggest that the standard isolation procedure-triggered reprogramming of gene expression in primary adipose cells that results in decreased insulin sensitivity does not require TNF-alpha, at least in this in vitro model system, but may be dependent on other inflammatory cytokines produced by these cells.
原代脂肪细胞的分离及随后的体外培养与GLUT4 mRNA的下调以及GLUT1基因表达的同时诱导有关。在体外培养时，胰岛素反应性GLUT4的逐渐丧失导致这些细胞中胰岛素介导的葡萄糖摄取减少。这些改变背后的机制尚不清楚。在此，我们报告，从小鼠脂肪组织中分离原代脂肪细胞的标准程序会触发许多编码炎症介质的基因的诱导，这些炎症介质包括肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1α、IL-6、多种趋化因子、细胞黏附分子、急性期蛋白、I型IL-1受体以及多种参与细胞炎症反应的转录因子。在脂肪组织2小时胶原酶消化过程中，TNF-α蛋白的分泌也被显著诱导。分离的原代脂肪细胞在多种mRNA的表达上表现出显著变化，这些变化是TNF-α处理的3T3-L1脂肪细胞所特有的，包括许多对胰岛素作用和甘油三酯合成重要的基因的下调。在培养的原代脂肪细胞中添加TNF-α并没有改变脂肪细胞丰富基因抑制的动力学或程度。此外，TNF-α中和抗体未能阻断分离的原代脂肪细胞中基因转录的变化。同样，标准分离程序在从TNF-α基因敲除小鼠制备的原代脂肪细胞中诱导核因子-κB（NF-κB）家族成员及其靶基因的表达，其程度与从野生型小鼠分离的细胞相同，并且当这些细胞在体外培养时，在整体基因表达上产生几乎相同的变化。因此，这些数据表明，标准分离程序触发原代脂肪细胞中基因表达的重编程，导致胰岛素敏感性降低，至少在这个体外模型系统中不需要TNF-α，但可能依赖于这些细胞产生的其他炎症细胞因子。
