Supplementary Material for: Increased Release of Proinflammatory Proteins in Primary Human Adipocytes and Activation of the Inflammatory NFĸB, p38, and ERK Pathways upon Omentin Treatment

<b><i>Objectives:</i></b> To investigate the impact of omentin on the release of inflammation-related biomarkers and inflammatory pathways in primary human adipocytes. <b><i>Methods:</i></b> Adipocytes were treated with or without omentin (500 and 2,000 ng/mL), and the supernatants were analyzed for inflammation-related biomarkers using proximity extension assay technology. Potential upstream regulators of the omentin-stimulated proteins were identified using Ingenuity Pathway Analysis. Protein levels of components of inflammatory pathways were measured using Western blotting. <b><i>Results:</i></b> 2,000 ng/mL omentin induced the release of 30 biomarkers 97.1 ± 31.1-fold in the supernatants (all <i>p</i> &lt; 0.05). Most biomarkers were proin­flammatory chemokines and cytokines. We identified the transcription factor nuclear factor “kappa-light-chain-enhancer” of activated B cells (NFĸB) and the kinases p38 and extracellular signal-regulated kinase (ERK)1/2 as potential upstream regulators in silico. On the cellular level, treatment with 2,000 ng/mL omentin for 24 h enhanced the phosphorylation levels of NFĸB 2.1 ± 0.3-fold (<i>p</i> &lt; 0.05), of p38 2.6 ± 0.4-fold (<i>p</i> &lt; 0.05), and of ERK1/2 1.8 ± 0.2-fold (<i>p</i> &lt; 0.05). <b><i>Conclusions:</i></b> These data argue that omentin exerts proinflammatory effects through the activation of the inflammatory NFĸB, p38, and ERK1/2 pathways in cultured primary adipocytes.

<b><i>研究目的：</i></b> 探讨网膜素（omentin）对原代人脂肪细胞中炎症相关生物标志物释放及炎症通路的影响。 <b><i>研究方法：</i></b> 分别采用浓度为500 ng/mL与2000 ng/mL的网膜素处理脂肪细胞，设置空白对照组；采用邻近延伸测定技术（proximity extension assay）分析细胞上清液中的炎症相关生物标志物；通过Ingenuity通路分析（Ingenuity Pathway Analysis）鉴定网膜素调控蛋白的潜在上游调控因子；采用蛋白质免疫印迹（Western blotting）检测炎症通路组分的蛋白表达水平。 <b><i>研究结果：</i></b> 2000 ng/mL的网膜素可使细胞上清液中30种生物标志物的释放量上调97.1 ± 31.1倍（所有p值均<0.05）。其中绝大多数生物标志物为促炎性趋化因子与细胞因子。经计算机模拟分析，我们鉴定出激活B细胞的核因子κ轻链增强子（NFκB，全称nuclear factor "kappa-light-chain-enhancer" of activated B cells）以及激酶p38、细胞外调节蛋白激酶（ERK）1/2为潜在的上游调控因子。细胞层面实验显示，使用2000 ng/mL的网膜素处理24小时后，NFκB的磷酸化水平上调2.1 ± 0.3倍（p < 0.05），p38的磷酸化水平上调2.6 ± 0.4倍（p < 0.05），ERK1/2的磷酸化水平上调1.8 ± 0.2倍（p < 0.05）。 <b><i>研究结论：</i></b> 本研究数据表明，网膜素可通过激活原代培养人脂肪细胞中的炎症通路NFκB、p38及ERK1/2，发挥促炎效应。