Research Institute for Medicines (imed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal.
Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
Front Endocrinol (Lausanne). 2023 Jan 13;13:1043543. doi: 10.3389/fendo.2022.1043543. eCollection 2022.
The development of reliable hepatic models may provide insights into disease mechanisms, linking hepatocyte dysmetabolism and related pathologies. However, several of the existing models depend on using high concentrations of hepatocyte differentiation-promoting compounds, namely glucose, insulin, and dexamethasone, which is among the reasons that have hampered their use for modeling metabolism-related diseases. This work focused on modulating glucose homeostasis and glucocorticoid concentration to improve the suitability of a mesenchymal stem-cell (MSC)-derived hepatocyte-like cell (HLC) human model for studying hepatic insulin action and disease modeling.
We have investigated the role of insulin, glucose and dexamethasone on mitochondrial function, insulin signaling and carbohydrate metabolism, namely AKT phosphorylation, glycogen storage ability, glycolysis and gluconeogenesis, as well as fatty acid oxidation and bile acid metabolism gene expression in HLCs. In addition, we evaluated cell morphological features, albumin and urea production, the presence of hepatic-specific markers, biotransformation ability and mitochondrial function.
Using glucose, insulin and dexamethasone levels close to physiological concentrations improved insulin responsiveness in HLCs, as demonstrated by AKT phosphorylation, upregulation of glycolysis and downregulation of and gluconeogenesis and fatty acid oxidation pathways. Ammonia detoxification, EROD and UGT activities and sensitivity to paracetamol cytotoxicity were also enhanced under more physiologically relevant conditions.
HLCs kept under reduced concentrations of glucose, insulin and dexamethasone presented an improved hepatic phenotype and insulin sensitivity demonstrating superior potential as an platform for modeling energy metabolism-related disorders, namely for the investigation of the insulin signaling pathway.
开发可靠的肝模型可以深入了解疾病机制，将肝细胞代谢失调与相关病理联系起来。然而，现有的一些模型依赖于使用高浓度的肝细胞分化促进化合物，即葡萄糖、胰岛素和地塞米松，这也是阻碍其用于模拟代谢相关疾病的原因之一。本研究专注于调节葡萄糖稳态和糖皮质激素浓度，以提高间充质干细胞（MSC）衍生的肝细胞样细胞（HLC）人类模型用于研究肝胰岛素作用和疾病建模的适用性。
我们研究了胰岛素、葡萄糖和地塞米松对线粒体功能、胰岛素信号和碳水化合物代谢（即 AKT 磷酸化、糖原储存能力、糖酵解和糖异生以及脂肪酸氧化和胆汁酸代谢基因表达）的作用。此外，我们还评估了细胞形态特征、白蛋白和尿素的产生、肝特异性标志物的存在、生物转化能力和线粒体功能。
使用接近生理浓度的葡萄糖、胰岛素和地塞米松水平改善了 HLC 的胰岛素反应性，表现在 AKT 磷酸化、糖酵解上调和下调以及脂肪酸氧化和糖异生途径。在更接近生理的条件下，氨解毒、EROD 和 UGT 活性以及对扑热息痛细胞毒性的敏感性也得到了增强。
在较低浓度的葡萄糖、胰岛素和地塞米松下培养的 HLC 表现出改善的肝表型和胰岛素敏感性，显示出作为模拟能量代谢相关疾病的平台的优越潜力，特别是用于研究胰岛素信号通路。
