Acute Hypoglycemia Induces Retinal Cell Death in Mouse

BackgroundGlucose is the most important metabolic substrate of the retina and maintenance of normoglycemia is an essential challenge for diabetic patients. Glycemic excursions could lead to cardiovascular disease, nephropathy, neuropathy and retinopathy. A vast body of literature exists on hyperglycemia namely in the field of diabetic retinopathy, but very little is known about the deleterious effect of hypoglycemia. Therefore, we decided to study the role of acute hypoglycemia in mouse retina. Methodology/Principal FindingsTo test effects of hypoglycemia, we performed a 5-hour hyperinsulinemic/hypoglycemic clamp; to exclude an effect of insulin, we made a hyperinsulinemic/euglycemic clamp as control. We then isolated retinas from each group at different time-points after the clamp to analyze cells apoptosis and genes regulation. In parallel, we used 661W photoreceptor cells to confirm in vivo results. We showed herein that hypoglycemia induced retinal cell death in mouse via caspase 3 activation. We then tested the mRNA expression of glutathione transferase omega 1 (Gsto1) and glutathione peroxidase 3 (Gpx3), two genes involved in glutathione (GSH) homeostasis. The expression of both genes was up-regulated by low glucose, leading to a decrease of reduced glutathione (GSH). In vitro experiments confirmed the low-glucose induction of 661W cell death via superoxide production and activation of caspase 3, which was concomitant with a decrease of GSH content. Moreover, decrease of GSH content by inhibition with buthionine sulphoximine (BSO) at high glucose induced apoptosis, while complementation with extracellular glutathione ethyl ester (GSHee) at low glucose restored GSH level and reduced apoptosis. Conclusions/SignificanceWe showed, for the first time, that acute insulin-induced hypoglycemia leads to caspase 3-dependant retinal cell death with a predominant role of GSH content.

背景：葡萄糖是视网膜最重要的代谢底物，维持正常血糖水平是糖尿病患者面临的核心挑战。血糖波动可诱发心血管疾病、肾病、神经病变及视网膜病变。目前已有大量关于高血糖的研究文献，尤其是在糖尿病视网膜病变领域，但针对低血糖有害作用的研究却寥寥无几。因此，本研究旨在探讨急性低血糖对小鼠视网膜的作用。
方法与主要结果：为验证低血糖的生物学效应，我们开展了为期5小时的高胰岛素血症-低血糖钳夹实验；为排除胰岛素本身的干扰，我们设置高胰岛素血症-正常血糖钳夹实验作为对照。于钳夹术后不同时间点从各组小鼠体内分离视网膜组织，用于分析细胞凋亡情况及基因表达调控模式。同时，我们采用661W感光细胞系开展体外实验，以验证体内研究结果。本研究发现，低血糖可通过激活半胱天冬酶3（caspase 3）诱导小鼠视网膜细胞死亡。随后我们检测了谷胱甘肽转移酶ω1（glutathione transferase omega 1, Gsto1）与谷胱甘肽过氧化物酶3（glutathione peroxidase 3, Gpx3）的mRNA表达水平，这两种基因均参与谷胱甘肽（glutathione, GSH）稳态的调控。低葡萄糖环境可上调这两种基因的表达，进而导致还原型谷胱甘肽（reduced glutathione, GSH）含量降低。体外实验证实，低葡萄糖可通过超氧自由基产生与半胱天冬酶3激活诱导661W细胞死亡，该过程伴随还原型谷胱甘肽水平下降。此外，在高葡萄糖环境下使用丁胱亚磺酰亚胺（buthionine sulphoximine, BSO）抑制谷胱甘肽合成可诱发细胞凋亡；而在低葡萄糖环境下补充细胞外谷胱甘肽乙酯（glutathione ethyl ester, GSHee）则可恢复谷胱甘肽水平并减轻细胞凋亡。
结论与意义：本研究首次证实，急性胰岛素诱导的低血糖可引发半胱天冬酶3依赖性视网膜细胞死亡，其中谷胱甘肽含量发挥了主导调控作用。