1 Department of Internal Medicine, Institute of Endocrine Research, Yonsei University College of Medicine , Seoul, Korea.
2 Brain Korea 21 Plus Project for Medical Science, Yonsei University , Seoul, Korea.
Thyroid. 2018 Apr;28(4):528-536. doi: 10.1089/thy.2017.0338. Epub 2018 Mar 28.
It was hypothesized that the biguanides metformin and phenformin, which are anti-hyperglycemic drugs used for diabetes mellitus, would have therapeutic effects in an in vitro model of Graves' orbitopathy (GO). Because adipogenesis, hyaluronan production, and inflammation are considered important in the pathogenesis of GO, this study aimed to determine the therapeutic effects and underlying mechanisms of biguanides on these parameters.
In vitro experiments were performed using primary cultured orbital fibroblasts from patients with GO. Orbital preadipocyte fibroblasts were allowed to differentiate into adipocytes and were treated with various concentrations of metformin or phenformin. Oil Red O staining was performed to evaluate lipid accumulation within the cells. Western blot analysis was used to measure the expression of adipogenic transcription factors and the phosphorylation of AMP-activated protein kinase and mitogen-activated protein kinase signaling proteins. Hyaluronan production was measured using enzyme-linked immunosorbent assay, and mRNA levels of proinflammatory molecules were determined using real-time polymerase chain reaction after interleukin (IL)-1β stimulation with or without biguanide treatment.
Lipid accumulation during adipogenesis in GO orbital fibroblasts was dose-dependently suppressed by both metformin and phenformin. Adipocyte differentiation was attenuated, and the adipogenic transcription factors peroxisome proliferator-activated receptor γ and CCAAT-enhancer-binding proteins-α/β were downregulated. Furthermore, metformin and phenformin increased the phosphorylation of AMP-activated protein kinase and suppressed extracellular-regulated kinase activation. The IL-1β-induced hyaluronan production and mRNA expression of IL-6, cyclooxygenase-2, and intercellular adhesion molecule-1 were also significantly suppressed after metformin or phenformin co-treatment.
The present study indicates that the biguanides metformin and phenformin exert an anti-adipogenic and inhibitory effect on hyaluronan production and expression of pro-inflammatory molecules in GO orbital fibroblasts, suggesting that they could potentially be used for the treatment of GO.
二甲双胍和苯乙双胍是用于治疗糖尿病的抗高血糖药物，有人假设它们在格雷夫斯眼病（GO）的体外模型中具有治疗作用。因为脂肪生成、透明质酸产生和炎症被认为在 GO 的发病机制中很重要，所以本研究旨在确定二甲双胍和苯乙双胍对这些参数的治疗作用和潜在机制。
使用来自 GO 患者的原代培养眼眶成纤维细胞进行体外实验。眼眶前脂肪细胞被允许分化为脂肪细胞，并以不同浓度的二甲双胍或苯乙双胍处理。用油红 O 染色法评估细胞内脂质积累。Western blot 分析用于测量脂肪生成转录因子的表达和 AMP 激活蛋白激酶和丝裂原激活蛋白激酶信号蛋白的磷酸化。通过酶联免疫吸附试验测量透明质酸产生，并用实时聚合酶链反应测定白细胞介素（IL）-1β刺激前后促炎分子的 mRNA 水平，并用或不用二甲双胍处理。
GO 眼眶成纤维细胞脂肪生成过程中的脂质积累被二甲双胍和苯乙双胍剂量依赖性地抑制。脂肪细胞分化减弱，脂肪生成转录因子过氧化物酶体增殖物激活受体γ和 CCAAT 增强子结合蛋白-α/β下调。此外，二甲双胍和苯乙双胍增加了 AMP 激活蛋白激酶的磷酸化并抑制了细胞外调节激酶的激活。IL-1β诱导的透明质酸产生和 IL-6、环氧化酶-2 和细胞间黏附分子-1 的 mRNA 表达在二甲双胍或苯乙双胍共同处理后也显著受到抑制。
本研究表明，二甲双胍和苯乙双胍在 GO 眼眶成纤维细胞中发挥抗脂肪生成和抑制透明质酸产生以及促炎分子表达的作用，表明它们可能可用于治疗 GO。
