Type 2 diabetes (T2D) is posing a serious public health concern with a considerable impact on human life and health expenditures worldwide. The disease develops when insulin plasma level is insufficient for coping insulin resistance, caused by the decline of pancreatic β-cell function and mass. In β-cells, the lipotoxicity exerted by saturated free fatty acids in particular palmitate (PA), which is chronically elevated in T2D, plays a major role in β-cell dysfunction and mass. However, there is a lack of human relevant model to identify the underlying mechanism through which palmitate induces β-cell failure. In this frame, we have previously developed a cutting-edge 3D spheroid model of β-like cells derived from human induced pluripotent stem cells. In the present work, we investigated the signaling pathways modified by palmitate in β-like cells derived spheroids. When compared to the 2D monolayer cultures, the transcriptome analysis (FDR set at 0.1) revealed that the 3D spheroids upregulated the pancreatic markers (such as GCG, IAPP genes), lipids metabolism and transporters (CD36, HMGSC2 genes), glucose transporter (SLC2A6). Then, the 3D spheroids are exposed to PA 0.5 mM for 72 h. The differential analysis demonstrated that 32 transcription factors and 135 target genes were mainly modulated (FDR set at 0.1) including the upregulation of lipid and carbohydrates metabolism (HMGSC2, LDHA, GLUT3), fibrin metabolism (FGG, FGB), apoptosis (CASP7). The pathway analysis using the 135 selected targets extracted the fibrin related biological process and wound healing in 3D PA treated conditions. An overall pathway gene set enrichment analysis, performed on the overall gene set (with pathway significance cutoff at 0.2), highlighted that PA perturbs the citrate cycle, FOXO signaling and Hippo signaling as observed in human islets studies. Additional RT-PCR confirmed induction of inflammatory (IGFBP1, IGFBP3) and cell growth (CCND1, Ki67) pathways by PA. All these changes were associated with unaffected glucose-stimulated insulin secretion (GSIS), suggesting that they precede the defect of insulin secretion and death induced by PA. Overall, we believe that our data demonstrate the potential of our spheroid 3D islet-like cells to investigate the pancreatic-like response to diabetogenic environment.

中文翻译：

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棕榈酸对诱导多能干细胞衍生的 β 样细胞 3D 球体影响的转录组分析

2 型糖尿病 (T2D) 正在构成严重的公共卫生问题，对全世界人类的生命和健康支出产生相当大的影响。当血浆胰岛素水平不足以应对由胰腺 β 细胞功能和质量下降引起的胰岛素抵抗时，就会出现这种疾病。在 β 细胞中，饱和游离脂肪酸，特别是棕榈酸酯 (PA) 所产生的脂毒性在 T2D 中长期升高，在 β 细胞功能障碍和肿块中发挥着重要作用。然而，缺乏人类相关模型来确定棕榈酸酯诱导 β 细胞衰竭的潜在机制。在此框架中，我们之前开发了源自​​人类诱导多能干细胞的β样细胞的尖端3D球体模型。在目前的工作中，我们研究了棕榈酸酯在β样细胞衍生的球体中修饰的信号通路。与 2D 单层培养物相比，转录组分析（FDR 设置为 0.1）显示，3D 球体上调了胰腺标志物（如 GCG、IAPP 基因）、脂质代谢和转运蛋白（CD36、HMGSC2 基因）、葡萄糖转运蛋白（SLC2A6） ）。然后，将 3D 球体暴露于 PA 0.5 mM 72 小时。差异分析表明，主要调节32个转录因子和135个靶基因（FDR设置为0.1），包括脂质和碳水化合物代谢（HMGSC2、LDHA、GLUT3）、纤维蛋白代谢（FGG、FGB）、细胞凋亡（CASP7）的上调。使用 135 个选定目标进行的通路分析提取了 3D PA 处理条件下与纤维蛋白相关的生物过程和伤口愈合。对整个基因集进行的整体通路基因集富集分析（通路显着性截断值为 0.2）强调，PA 扰乱了柠檬酸循环、FOXO 信号传导和 Hippo 信号传导，正如在人类胰岛研究中观察到的那样。另外的 RT-PCR 证实 PA 诱导炎症（IGFBP1、IGFBP3）和细胞生长（CCND1、Ki67）途径。所有这些变化都与未受影响的葡萄糖刺激胰岛素分泌（GSIS）有关，表明它们先于胰岛素分泌缺陷和 PA 诱导的死亡。总的来说，我们相信我们的数据证明了我们的球体 3D 胰岛样细胞在研究对糖尿病环境的类胰腺反应方面的潜力。