Lin Dingbo, Harris Richie, Stutzman Rachael, Zampighi Guido A, Davidson Harriett, Takemoto Dolores J
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
Curr Eye Res. 2007 Jun;32(6):523-32. doi: 10.1080/02713680701418124.
The purpose of this study is to demonstrate the early activation of the protein kinase C-gamma (PKC-gamma) pathway in the streptozotocin (STZ)-induced diabetic rat lens.
Twelve-week-old male and female Sprague-Dawley rats were injected with 80 mg/kg (body weight) of STZ (N-[methylnitrosocarbamoyl]-D-glucosamine) intraperitoneally. Very high glucose (VHG) diabetes was defined as a nonfasting blood glucose level of at least 450 mg/dl, confirmed by daily monitoring with Accu-Check Advantage test strips, and occurred about 2 weeks after STZ administration. All assayed lenses were from VHG or age-matched control rats, harvested within 24 hr of VHG detection. PKC-gamma activation was measured by enzyme activity assay and by Western blotting to show autophosphorylation on Thr514. Cellular insulin-like growth factor-1 (IGF-1), PKC-gamma phosphorylation of Cx43 on Ser368, and activation of phospholipase C-gamma 1 (PLC-gamma 1), extracellular signal-regulated kinase (ERK1/2), and caspase-3 were determined by Western blotting. Endogenous diacylglycerol (DAG) levels were measured with a DAG assay kit. Lens gap junction activity was determined by the microinjection/Lucifer yellow dye transfer assay. Electron microscopy was applied to affirm fiber cell damage in the VHG diabetic lenses.
In the lenses of VHG diabetic rats, PKC-gamma enzyme was activated. PKC-gamma could be further activated by 400 nM phorbol-12-myristate-13-acetate (PMA), but the PKC-gamma protein levels remained constant. No elevation of IGF-1 level was observed. Western blots showed that activation of PKC-gamma may be due to activation of PLC-gamma 1, which synthesized endogenous DAG, a native PKC activator. The level of PKC-gamma -catalyzed phosphorylation of Cx43 on Ser368 and resulting inhibition of lens gap junction dye transfer activity was increased in the VHG diabetic lenses. At this early time period, the diabetic lens showed no activation of either caspase-3 or ERK1/2. Only a single fiber cell layer deep within the cortex (approximately 90 cell layers from capsule surface) showed vacuoles and damaged cell connections.
Early activation of PLC-gamma 1 and elevated DAG were observed within VHG diabetic lenses. These were correlated with activation of PKC-gamma, phosphorylation of Cx43 on Ser368, and inhibition of dye transfer. Abnormal signaling from PKC-gamma to Cx43 in the epithelial cells/early fiber cells, observed within VHG diabetic lenses, may be responsible for fiber cell damage deeper in the lens cortex.
本研究旨在证明链脲佐菌素（STZ）诱导的糖尿病大鼠晶状体中蛋白激酶C-γ（PKC-γ）通路的早期激活。
12周龄雄性和雌性Sprague-Dawley大鼠腹腔注射80mg/kg（体重）的STZ（N-甲基亚硝基脲基-D-葡萄糖胺）。极高血糖（VHG）糖尿病定义为非空腹血糖水平至少为450mg/dl，通过使用Accu-Check Advantage试纸条每日监测得以确认，且在STZ给药后约2周出现。所有检测的晶状体均来自VHG或年龄匹配的对照大鼠，在检测到VHG后的24小时内采集。通过酶活性测定和蛋白质印迹法测量PKC-γ的激活，以显示苏氨酸514位点的自磷酸化。通过蛋白质印迹法测定细胞胰岛素样生长因子-1（IGF-1）、PKC-γ对Cx43丝氨酸368位点的磷酸化以及磷脂酶C-γ1（PLC-γ1）、细胞外信号调节激酶（ERK1/2）和半胱天冬酶-3的激活。使用二酰基甘油（DAG）检测试剂盒测量内源性DAG水平。通过显微注射/荧光素黄染料转移试验测定晶状体缝隙连接活性。应用电子显微镜确认VHG糖尿病晶状体中的纤维细胞损伤。
在VHG糖尿病大鼠的晶状体中，PKC-γ酶被激活。400nM佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）可进一步激活PKC-γ，但PKC-γ蛋白水平保持恒定。未观察到IGF-1水平升高。蛋白质印迹显示，PKC-γ的激活可能是由于PLC-γ1的激活，PLC-γ1合成内源性DAG，DAG是一种天然的PKC激活剂。在VHG糖尿病晶状体中，PKC-γ催化的Cx43丝氨酸368位点的磷酸化水平以及由此导致的晶状体缝隙连接染料转移活性的抑制增加。在此早期阶段，糖尿病晶状体未显示半胱天冬酶-3或ERK1/2的激活。仅皮质内深层的单个纤维细胞层（距囊膜表面约90个细胞层）显示有空泡和受损的细胞连接。
在VHG糖尿病晶状体中观察到PLC-γ1的早期激活和DAG升高。这些与PKC-γ的激活、Cx43丝氨酸368位点的磷酸化以及染料转移的抑制相关。在VHG糖尿病晶状体中观察到的上皮细胞/早期纤维细胞中从PKC-γ到Cx43的异常信号传导可能是晶状体皮质中更深层纤维细胞损伤的原因。
