Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Institut für Experimentelle Endokrinologie, 13353 Berlin, Germany.
Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Institut für Experimentelle Endokrinologie, 13353 Berlin, Germany.
Mol Cell Endocrinol. 2018 Jan 15;460:219-228. doi: 10.1016/j.mce.2017.07.026. Epub 2017 Jul 25.
3-iodothyronamine (3-TAM), a decarboxylated and deiodinated thyroid hormone metabolite, leads at pharmacological doses to hypoinsulinemia, hyperglucagonemia and hyperglycemia in vivo. As the pancreatic Langerhans islets express thyroid hormone transmembrane transporters (THTT), we tested the hypothesis that not only plasma membrane-mediated 3-TAM binding to and activation of G-protein coupled receptors, but also 3-TAM metabolite(s) generated by 3-TAM uptake and metabolism might decrease glucose-stimulated insulin secretion (GSIS).
Murine pancreatic β-cells MIN6 were characterized for gene expression of THTT, deiodinases and monoamine oxidases. 3-TAM uptake and intracellular metabolism to the corresponding 3-iodothyroacetic acid were analysed by liquid-chromatography tandem mass spectrometry (LC-MS/MS) at different time points in cells as well as the conditioned medium. Mitochondrial activity, especially ATP-production, was monitored real-time after 3-TAM application using Seahorse Bioanalyzer technique. Effect of 3-TAM on GSIS into the culture medium was assayed by ELISA.
MIN6 cells express classical THTT, proposed to transport 3-TAM, as well as 3-TAM metabolizing enzymes comparable to murine primary pancreatic islets. 3-TAM accumulates in MIN6 cells and is metabolized by intracellular MaoB to 3-iodothyroacetic, which in turn is rapidly exported. 3-TAM decreases mitochondrial ATP-production concentration dependently. GSIS is diminished by 3-TAM treatment. Using LC-MS/MS, no further 3-TAM metabolites except 3-iodothyroacetic were detectable.
This data provides a first link between cellular 3-TAM uptake and regulation of mitochondrial energy metabolism in ß-cells, resulting in reduced insulin secretion. We conclude that MIN6 is an appropriate cell model to study 3-TAM-dependent (intra-)cellular biochemical mechanisms affecting insulin production in vitro.
3-碘甲状腺原氨酸（3-TAM）是一种脱羧和脱碘的甲状腺激素代谢物，在药理学剂量下导致体内胰岛素水平降低、胰高血糖素水平升高和血糖水平升高。由于胰腺胰岛表达甲状腺激素跨膜转运体（THTT），我们检验了这样一个假设，即不仅是质膜介导的 3-TAM 与 G 蛋白偶联受体结合和激活，而且 3-TAM 摄取和代谢产生的 3-TAM 代谢物也可能降低葡萄糖刺激的胰岛素分泌（GSIS）。
MIN6 是一种来源于小鼠胰腺的β细胞系，我们通过基因表达分析、细胞摄取和代谢实验来鉴定其是否表达 THTT、脱碘酶和单胺氧化酶。我们通过液相色谱-串联质谱法（LC-MS/MS）在不同时间点分析 3-TAM 在细胞内以及条件培养基中的摄取和代谢产物。应用 Seahorse Bioanalyzer 技术实时监测 3-TAM 应用后线粒体活性，特别是 ATP 的产生。通过 ELISA 测定 3-TAM 对细胞培养基中 GSIS 的影响。
MIN6 细胞表达经典的 THTT，推测其可以转运 3-TAM，同时还表达类似于小鼠原代胰腺胰岛的 3-TAM 代谢酶。3-TAM 在 MIN6 细胞内积聚并被细胞内 MaoB 代谢为 3-碘甲状腺乙酸，后者又迅速被排出。3-TAM 浓度依赖性地降低线粒体 ATP 的产生。3-TAM 处理可减少 GSIS。通过 LC-MS/MS 检测，除 3-碘甲状腺乙酸外，未检测到其他 3-TAM 代谢物。
本研究首次证明了 MIN6 细胞内的 3-TAM 摄取与β细胞中线粒体能量代谢的调节之间存在联系，从而导致胰岛素分泌减少。我们得出结论，MIN6 是研究 3-TAM 依赖性（细胞内）生化机制影响胰岛素产生的体外合适细胞模型。
