Bursell S E, Takagi C, Clermont A C, Takagi H, Mori F, Ishii H, King G L
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Invest Ophthalmol Vis Sci. 1997 Dec;38(13):2711-20.
Elevation of diacylglycerol (DAG) and protein kinase C (PKC) levels in diabetic vascular tissue is associated with abnormalities of retinal and renal hemodynamics. The object of this study was to determine whether direct elevation of retinal DAG levels, in the absence of diabetes or hyperglycemia, can mimic the hemodynamic abnormalities normally observed in diabetic rats. Retinal DAG levels were elevated using an inhibitor of DAG kinase that converts DAG to phosphatidic acid. The effectiveness of a specific PKC-beta isoform inhibitor introduced directly into the retinas of diabetic rats in reversing diabetes-related abnormal retinal hemodynamics was also investigated.
For retinal blood flow studies, diacylglycerol kinase (DGK) inhibitor R59949, at various concentrations, was injected into the vitreous of nondiabetic Sprague-Dawley rats (n = 33), and a PKC-beta isoform-selective inhibitor LY333531 was injected into the vitreous of rats with streptozotocin (STZ)-induced diabetes of 2 weeks' duration (n = 21). Retinal hemodynamic changes were quantitated using video-based fluorescein angiography. Total DAG levels were assayed from five nondiabetic rat retinas after DGK inhibition and retinal PKC activities were assayed from six diabetic rat retinas after PKC-beta inhibition.
DGK inhibitor R59949 injected into the vitreous dose dependently increased the mean circulation time (MCT) and decreased retinal blood flow (EC50 = 10(-8) M). After 30 minutes, 10(-5) M R59949 induced a 1.7-fold increase in total retinal DAG levels, compared with the levels in vehicle-injected eyes, an increase in MCT from 0.87 +/- 0.05 seconds to 1.44 +/- 0.12 seconds (P < 0.01) and a decrease in retinal blood flow from 105.3 +/- 6.5 pixel2/second to 64.1 +/- 5 pixel2/second (P < 0.01). The effect of R59949 was sustained for 60 minutes after injection. These retinal hemodynamic parameters after DGK inhibition were comparable to those measured at baseline in rats with STZ-induced diabetes of 2 weeks' duration (MCT = 1.38 +/- 0.20 seconds; retinal blood flow = 68 +/- 11.2 pixel2/second). Intravitreal injection of the PKC-beta inhibitor (LY333531) at 10(-5) M in diabetic rats decreased by a factor of 1.6 the diabetes-related increased PKC activation, decreased the prolonged MCT (0.98 +/- 0.13 seconds; P < 0.01) and increased retinal blood flow (93.4 +/- 14.2 pixel2/second; P < 0.01). The measured retinal circulatory parameters after PKC inhibition in the retina were comparable to those measured at baseline in the nondiabetic rats.
These results provide direct evidence that DAG elevation and subsequent PKC-beta isoform activation are the primary biochemical sequelae responsible for the development of the abnormal retinal hemodynamics observed in diabetic rats.
糖尿病血管组织中甘油二酯（DAG）和蛋白激酶C（PKC）水平升高与视网膜和肾脏血流动力学异常有关。本研究的目的是确定在无糖尿病或高血糖的情况下，直接升高视网膜DAG水平是否能模拟糖尿病大鼠中通常观察到的血流动力学异常。使用将DAG转化为磷脂酸的DAG激酶抑制剂来升高视网膜DAG水平。还研究了直接引入糖尿病大鼠视网膜的特异性PKC-β亚型抑制剂在逆转糖尿病相关的异常视网膜血流动力学方面的有效性。
对于视网膜血流研究，将不同浓度的DAG激酶（DGK）抑制剂R59949注入非糖尿病Sprague-Dawley大鼠（n = 33）的玻璃体中，并将PKC-β亚型选择性抑制剂LY333531注入链脲佐菌素（STZ）诱导的2周糖尿病大鼠（n = 21）的玻璃体中。使用基于视频的荧光素血管造影术对视网膜血流动力学变化进行定量分析。在DGK抑制后，从五只非糖尿病大鼠视网膜中测定总DAG水平，在PKC-β抑制后，从六只糖尿病大鼠视网膜中测定视网膜PKC活性。
注入玻璃体的DGK抑制剂R59949剂量依赖性地增加平均循环时间（MCT）并降低视网膜血流（半数有效浓度[EC50]=10^(-8)M）。30分钟后，与注射赋形剂的眼睛相比，10^(-5)M R59949使视网膜总DAG水平增加1.7倍，MCT从0.87±0.05秒增加到1.44±0.12秒（P<0.01），视网膜血流从105.3±6.5像素2/秒减少到64.1±5像素2/秒（P<0.01）。注射后R59949的作用持续60分钟。DGK抑制后的这些视网膜血流动力学参数与STZ诱导的2周糖尿病大鼠基线时测量的参数相当（MCT = 1.38±0.20秒；视网膜血流 = 68±11.2像素2/秒）。在糖尿病大鼠中玻璃体注射10^(-5)M的PKC-β抑制剂（LY333531）使糖尿病相关的PKC激活增加降低了1.6倍，使延长的MCT降低（0.98±0.13秒；P<0.01）并增加了视网膜血流（93.4±14.2像素2/秒；P<0.01）。视网膜中PKC抑制后测量的视网膜循环参数与非糖尿病大鼠基线时测量的参数相当。
这些结果提供了直接证据，表明DAG升高和随后的PKC-β亚型激活是导致糖尿病大鼠中观察到的异常视网膜血流动力学发展的主要生化后遗症。
