An improved <i>in vitro</i> 3T3-L1 adipocyte model of inflammation and insulin resistance

Tumor necrosis factor alpha (TNF-α)/hypoxia-treated 3T3-L1 adipocytes have been used to model inflamed and insulin-resistant adipose tissue: this study examines gaps in the model. We tested whether modulating TNF-α/hypoxia treatment time could reduce cell death while still inducing inflammation and insulin resistance. Adipocytes were treated with TNF-α (12 h or 24 h) and incubated in a hypoxic chamber for 24 h. To examine maintenance of the phenotype over time, glucose and FBS were added at 24 h post initiation of treatment, and the cells were maintained for an additional 48 h. Untreated adipocytes were used as a control. Viability, insulin resistance, and inflammation were assessed using Live/Dead staining, RT-qPCR, ELISA, and glucose uptake assays. Treatment for 12 h with TNF-α in the presence of hypoxia resulted in an increase in the percentage of live cells compared to 24 h treated cells. Importantly, insulin resistance and inflammation were still induced in the 12 h treated adipocytes: the expression of the insulin sensitive and inflammatory genes was decreased and increased, respectively. In 72 h treated adipocytes, no significant differences were found in the viability, glucose uptake or insulin-sensitive and inflammatory gene expression. This study provides a modified approach to in vitro odeling adipocyte inflammation and insulin resistance.本研究以经肿瘤坏死因子α（Tumor necrosis factor alpha, TNF-α）联合低氧处理的3T3-L1脂肪细胞作为炎症性与胰岛素抵抗性脂肪组织的体外模型，针对该模型尚存的研究空白展开探究。本研究旨在验证调整TNF-α联合低氧的处理时长，是否可在仍诱导炎症与胰岛素抵抗的同时降低细胞死亡水平。实验中将脂肪细胞以TNF-α分别处理12小时或24小时，随后置于低氧培养箱中孵育24小时。为探究该细胞表型随时间的维持情况，我们于处理启动后的24小时向培养基中添加葡萄糖与胎牛血清（Fetal Bovine Serum, FBS），并将细胞继续培养48小时。以未做任何处理的脂肪细胞作为空白对照。采用死活染色法（Live/Dead staining）、实时定量聚合酶链反应（Real-time quantitative polymerase chain reaction, RT-qPCR）、酶联免疫吸附实验（Enzyme-Linked Immunosorbent Assay, ELISA）以及葡萄糖摄取实验，分别对细胞活力、胰岛素抵抗水平与炎症状态进行评估。与经24小时TNF-α联合低氧处理的细胞相比，经12小时TNF-α联合低氧处理的细胞存活比例更高。值得注意的是，经12小时处理的脂肪细胞仍可诱导出胰岛素抵抗与炎症表型：胰岛素敏感基因的表达水平显著下调，而炎症相关基因的表达水平则显著上调。经72小时处理的脂肪细胞中，细胞活力、葡萄糖摄取能力以及胰岛素敏感与炎症相关基因的表达水平均未出现显著差异。本研究为脂肪细胞炎症与胰岛素抵抗的体外建模提供了一种改良方案。