Villa Marika, Parravano Mariacristina, Micheli Arianna, Gaddini Lucia, Matteucci Andrea, Mallozzi Cinzia, Facchiano Francesco, Malchiodi-Albedi Fiorella, Pricci Flavia
Dept of Cardiovascular, Dysmetabolic and Aging-associated diseases.
Dept of Ophthalmology, G.B. Bietti Eye Foundation-IRCCS, Rome, Italy.
Metabolism. 2017 Jun;71:64-69. doi: 10.1016/j.metabol.2017.03.004. Epub 2017 Mar 9.
Advanced glycation end-products (AGEs) constitute a highly heterogeneous family of compounds, relevant in the pathogenesis of diabetic complications, which could represent efficient biomarkers of disease progression and drug response. Unfortunately, due to their chemical heterogeneity, no method has been validated to faithfully monitor their levels in the course of the disease. In this study, we refine a procedure to quantitatively analyze fluorescent AGEs (fAGEs), a subset considered remarkably representative of the entire AGE family, and measure them in in vitro glycated BSA (gBSA) and in plasma and vitreous of diabetic rats, for testing its use to possibly quantify circulating AGEs in patients, as markers of metabolic control.
fAGE levels were evaluated by spectrofluorimetric analysis in in vitro and in vivo experimental models. BSA was glycated in vitro with increasing D-glucose concentrations for a fixed time or with a fixed D-glucose concentration for increasing time. In in vivo experiments, streptozotocin-induced diabetic rats were studied at 1, 3, 6 and 12weeks to analyze plasma and vitreous. To confirm the presence of AGEs in our models, non-diabetic rat retinal explants were exposed to high glucose (HG), to reproduce short-term effects, or in vitro gBSA, to reproduce long-term effects of elevated glucose concentrations. Rat retinal explants and diabetic retinal tissues were evaluated for the receptor for advanced glycation end-product (RAGE) by Western blot analysis.
In in vitro experiments, fluorescence emission showed glucose concentration- and time-dependent increase of fAGEs in gBSA (p≤0.05). In streptozotocin-induced diabetic rats, fAGE in plasma and vitrei showed an increase at 6 (p≤0.005) and 12 (p≤0.05) weeks of diabetes, with respect to control. RAGE was time-dependently upregulated in retinas incubated with gBSA, but not with HG, and in diabetic retinal tissue, substantiating exposure to AGEs.
Applying the proposed technique, we could show that fAGEs levels increase with glucose concentration and time of exposure in vitro. Furthermore, in diabetic rats, it showed that circulating fAGEs are similarly upregulated as those in vitreous, suggesting a correlation between circulating and tissue AGEs. These results support the use of this method as a simple and reliable test to measure circulating fAGEs and monitor diabetes progression.
晚期糖基化终产物（AGEs）是一类高度异质性的化合物家族，与糖尿病并发症的发病机制相关，可能是疾病进展和药物反应的有效生物标志物。不幸的是，由于其化学异质性，尚无经过验证的方法可在疾病过程中准确监测其水平。在本研究中，我们完善了一种程序，用于定量分析荧光AGEs（fAGEs），这是整个AGE家族中一个具有显著代表性的子集，并在体外糖基化牛血清白蛋白（gBSA）以及糖尿病大鼠的血浆和玻璃体中对其进行测量，以测试其用于量化患者循环中AGEs作为代谢控制标志物的用途。
通过荧光分光光度分析在体外和体内实验模型中评估fAGE水平。在体外，使牛血清白蛋白与不断增加的D - 葡萄糖浓度在固定时间内进行糖基化反应，或与固定的D - 葡萄糖浓度在不断增加的时间内进行糖基化反应。在体内实验中，对链脲佐菌素诱导的糖尿病大鼠在第1、3、6和12周进行研究，以分析血浆和玻璃体。为了证实我们模型中AGEs的存在，将非糖尿病大鼠视网膜外植体暴露于高糖（HG）以重现短期效应，或暴露于体外糖基化牛血清白蛋白以重现高糖浓度的长期效应。通过蛋白质免疫印迹分析评估大鼠视网膜外植体和糖尿病视网膜组织中的晚期糖基化终产物受体（RAGE）。
在体外实验中，荧光发射显示gBSA中fAGEs的荧光强度随葡萄糖浓度和时间呈依赖性增加（p≤0.05）。在链脲佐菌素诱导的糖尿病大鼠中，与对照组相比，糖尿病第6周（p≤0.005）和第12周（p≤0.05）时血浆和玻璃体中的fAGE增加。在与gBSA孵育的视网膜中，RAGE随时间上调，但与HG孵育时未上调，并且在糖尿病视网膜组织中也上调，证实了AGEs的暴露。
应用所提出的技术，我们可以证明体外fAGEs水平随葡萄糖浓度和暴露时间增加。此外，在糖尿病大鼠中，结果表明循环中的fAGEs与玻璃体中的fAGEs同样上调，提示循环和组织AGEs之间存在相关性。这些结果支持将该方法用作测量循环fAGEs和监测糖尿病进展的简单可靠测试方法。
