Department of Medicine, Boston University School of Medicine, 650 Albany Street, Boston 02118, USA.
Am J Pathol. 2010 Jul;177(1):447-55. doi: 10.2353/ajpath.2010.091029. Epub 2010 Jun 3.
Mitochondrial dysfunction has been implicated in diabetic complications; however, it is unknown whether hyperglycemia affects mitochondrial morphology and metabolic capacity during development of diabetic retinopathy. We investigated high glucose (HG) effects on mitochondrial morphology, membrane potential heterogeneity, cellular oxygen consumption, extracellular acidification, cytochrome c release, and apoptosis in retinal endothelial cells. Rat retinal endothelial cells grown in normal (5 mmol/L) or HG (30 mmol/L) medium and double-stained with MitoTracker Green and tetramethylrhodamine-ethyl-ester-perchlorate were examined live with confocal microscopy. Images were analyzed for mitochondrial shape change using Form Factor and Aspect Ratio values, and membrane potential heterogeneity, using deviation of fluorescence intensity values. Rat retinal endothelial cells grown in normal or HG medium were analyzed for transient changes in oxygen consumption and extracellular acidification using an XF-24 flux analyzer, cytochrome c release by Western blot, and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Rat retinal endothelial cells grown in HG medium exhibited increased mitochondrial fragmentation concurrent with membrane potential heterogeneity. Metabolic analysis showed increased extracellular acidification in HG with reduced steady state/maximal oxygen consumption. Cytochrome c and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were also increased in HG. Thus, HG-induced mitochondrial fragmentation with concomitant increase in membrane potential heterogeneity, reduced oxygen consumption, and cytochrome c release may underlie apoptosis of retinal endothelial cells as seen in diabetic retinopathy.
线粒体功能障碍与糖尿病并发症有关；然而，高血糖是否会影响糖尿病性视网膜病变发展过程中的线粒体形态和代谢能力尚不清楚。我们研究了高葡萄糖（HG）对视网膜内皮细胞中线粒体形态、膜电位异质性、细胞耗氧量、细胞外酸化、细胞色素 c 释放和细胞凋亡的影响。将大鼠视网膜内皮细胞在正常（5mmol/L）或高葡萄糖（30mmol/L）培养基中培养，并与 MitoTracker Green 和 tetramethylrhodamine-ethyl-ester-perchlorate 双重染色，然后使用共聚焦显微镜进行活细胞观察。使用形态因子和纵横比值分析线粒体形状变化，使用荧光强度值的偏差分析膜电位异质性。使用 XF-24 通量分析仪分析正常或高葡萄糖培养基中生长的大鼠视网膜内皮细胞的氧耗和细胞外酸化的瞬时变化，使用 Western blot 分析细胞色素 c 释放，使用末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记法分析细胞凋亡。在高葡萄糖培养基中培养的大鼠视网膜内皮细胞表现出线粒体碎片化增加，同时伴有膜电位异质性。代谢分析显示，高葡萄糖导致细胞外酸化增加，稳态/最大耗氧量减少。高葡萄糖中细胞色素 c 和末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记阳性细胞也增加。因此，高葡萄糖诱导的线粒体碎片化伴膜电位异质性增加、耗氧量减少和细胞色素 c 释放可能是糖尿病性视网膜病变中视网膜内皮细胞凋亡的基础。
