Department of Ophthalmology, Fudan Eye & ENT Hospital, Shanghai, China.
Laboratory of Myopia, Chinese Academy of Medical Sciences, Shanghai, China.
J Mol Endocrinol. 2020 Nov;65(4):163-174. doi: 10.1530/JME-20-0143.
The study aimed to investigate the role of pyruvate dehydrogenase kinase (PDK) in regulating glycolysis and proliferation of perimysial orbital fibroblasts (pOFs) during the pathogenesis of thyroid-associated ophthalmopathy (TAO). EdU and BrdU incorporation assays were performed to examine cell proliferation. Lactate production and oxygen consumption assays were conducted to evaluate glycolysis. Real-time PCR was adapted to quantify PDK mRNA levels. Capillary Western immunoassay was adapted to quantify PDK2, Akt, pAkt308 and GAPDH in protein samples. The TAO pOFs exhibited stronger proliferation activity, higher intracellular lactate concentration, and lower oxygen consumption rate than the control pOFs. The PDK inhibitor dichloroacetic acid (DCA) dose-dependently suppressed the proliferation of both TAO and control pOFs. DCA reduced lactate production and promoted oxygen consumption in the TAO pOFs but showed no significant effects on glycolysis in the control pOFs. Among four PDK isotypes, PDK2 was overexpressed in the TAO pOFs. The potential PDK signaling mediator, cytoplasmic Akt, was more abundant in TAO pOFs than control pOFs. Knockdown of PDK2 resulted in lower lactate production, stronger oxygen consumption, weaker proliferation activity, and less cytoplasmic Akt in the TAO pOFs but showed no significant effects in the control pOFs. The Akt inhibitor MK2206 suppressed proliferation in both TAO and control pOFs, and lactate production was inhibited by MK2206 in the TAO OFs but not the control pOFs. To conclude, PDK2 overexpression enhances glycolysis and promotes proliferation via Akt signaling in the TAO pOFs. These findings yield insights that PDK2 is a potential therapeutic target for TAO treatment.
该研究旨在探讨丙酮酸脱氢酶激酶（PDK）在甲状腺相关眼病（TAO）发病过程中调节肌间膜 orbital 纤维母细胞（pOFs）糖酵解和增殖的作用。通过 EdU 和 BrdU 掺入实验检测细胞增殖，通过乳酸生成和耗氧实验评估糖酵解，采用实时 PCR 定量 PDK mRNA 水平，采用毛细管 Western 免疫印迹法定量蛋白样品中的 PDK2、Akt、pAkt308 和 GAPDH。与对照 pOFs 相比，TAO pOFs 表现出更强的增殖活性、更高的细胞内乳酸浓度和更低的耗氧率。PDK 抑制剂二氯乙酸（DCA）呈剂量依赖性抑制 TAO 和对照 pOFs 的增殖。DCA 降低 TAO pOFs 的乳酸生成并促进其氧耗，但对对照 pOFs 的糖酵解无显著影响。在四种 PDK 同工型中，PDK2 在 TAO pOFs 中过表达。潜在的 PDK 信号转导介质细胞质 Akt 在 TAO pOFs 中比对照 pOFs 更为丰富。PDK2 敲低导致 TAO pOFs 中的乳酸生成减少、氧耗增强、增殖活性减弱和细胞质 Akt 减少，但对对照 pOFs 无显著影响。Akt 抑制剂 MK2206 抑制 TAO 和对照 pOFs 的增殖，MK2206 抑制 TAO pOFs 中的乳酸生成，但不抑制对照 pOFs。综上所述，PDK2 过表达通过 Akt 信号增强 TAO pOFs 中的糖酵解并促进增殖。这些发现表明 PDK2 是 TAO 治疗的潜在治疗靶点。
