目的:本研究旨在探讨糖尿病视网膜病变(DR)中支链氨基酸(BCAA)的分解代谢。
方法:野生型和db/db小鼠饲喂BCAA(5或10mg/kg/天)12周,和高血糖暴露的Müller细胞用BCAA(2或5mmol/L)处理24和48小时。使用MS/MS测量BCAA水平。进行蛋白质印迹以检测蛋白质。流式细胞术,耗氧率,和细胞计数试剂盒-8测定用于评估Müller细胞活力。每个实验至少进行三次。
结果:糖尿病小鼠视网膜和全身组织中的BCAAs和支链α-酮酸(BCKAs)增加,与野生型组相比,这些变化被额外的BCAA进一步增强至约2倍。体外,在高血糖Müller细胞中诱导BCAA和BCKAs,并补充BCAA。BCAA的异常分解代谢伴随mTORC1的激活,随后诱导TNF-α,VEGFA,GS,糖尿病条件下视网膜和Müller细胞中的GFAP。细胞凋亡率增加约50%,Müller细胞中的高血糖和BCAA抑制了线粒体呼吸。此外,mTORC1信号传导在Müller细胞中被亮氨酸激活。Sestrin2或LeuRS的敲除可显着消除亮氨酸诱导的mTORC1磷酸化并保护糖尿病条件下的Müller细胞活力。
结论:我们发现DR中的BCAA分解代谢通过mTORC1激活而受到阻碍。亮氨酸通过感应Müller细胞中的Sestrin2在诱导mTORC1中起关键作用。靶向Sestrin2可以改善BCAA积累对DR中Müller细胞的毒性作用。
OBJECTIVE: This study aimed to investigate branched-chain amino acid (BCAA) catabolism in diabetic retinopathy (DR).
METHODS: Wild-type and db/db mice were fed BCAAs (5 or 10 mg/kg/day) for 12 weeks, and hyperglycemia-exposed Müller cells were treated with BCAAs (2 or 5 mmol/L) for 24 and 48 h. BCAA levels were measured using MS/MS. Western blotting was performed to detect proteins. Flow cytometry, oxygen consumption rate, and Cell Counting Kit-8 assays were used to evaluate Müller cell viability. Each experiment was conducted at least thrice.
RESULTS: BCAAs and branched-chain α-keto acids (BCKAs) were increased in the retina and systemic tissues of diabetic mice, and these changes were further enhanced to approximately 2-fold by extra BCAAs compared to wild-type group. In vitro, BCAAs and BCKAs were induced in hyperglycemic Müller cells, and augmented by BCAA supplementation. The aberrant BCAA catabolism was accompanied by
mTORC1 activation and subsequently induced TNF-ɑ, VEGFA, GS, and GFAP in retinas and Müller cells under diabetic conditions. The cell apoptosis rate increased by approximately 50%, and mitochondrial respiration was inhibited by hyperglycemia and BCAA in Müller cells. Additionally,
mTORC1 signaling was activated by leucine in Müller cells. Knockdown of Sestrin2 or LeuRS significantly abolished the leucine-induced
mTORC1 phosphorylation and protected Müller cell viability under diabetic conditions.
CONCLUSIONS: We found that BCAA catabolism is hindered in DR through
mTORC1 activation. Leucine plays a key role in inducing
mTORC1 by sensing Sestrin2 in Müller cells. Targeting Sestrin2 may ameliorate the toxic effects of BCAA accumulation on Müller cells in DR.