CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India; Department of Experimental Biology, Wrocław University of Environmental and Life Sciences, Wroclaw, Poland.
CSIR- Indian Institute of Chemical Technology, Hyderabad, India.
Biomed Pharmacother. 2021 Jan;133:111032. doi: 10.1016/j.biopha.2020.111032. Epub 2020 Dec 1.
Insulin resistance is associated with obesity and can lead to several metabolic disorders including type II diabetes, nonalcoholic fatty liver disease and cardiovascular problems. Search for the small molecules which can either induce or mimic the insulin action are of great interest and can be utilized to manage insulin resistance. There are several dietary phytochemicals which can potentially have insulinomimetic action. Nevertheless, high throughput screening methods to test efficiency of small molecules to act as an insulinomimetic are not fully established. In this paper we have performed chemical screen analysis based on GLUT4 translocation using a cell line CHO-HIRC-myc-GLUT4 eGFP that expresses GLUT4-GFP in association with human Insulin receptor. We have established a high content screening-based method which can track and quantify the GLUT4 translocation from perinuclear area to the cell membrane. The assay involves measuring fluorescence intensity in a defined perinuclear area and a defined area along the cell membrane; and the results are expressed as the ratio of fluorescence intensity in the perinuclear to membrane area. The assay could collect real time data of GLUT4 translocation from thousand of cells/ sample and from many such samples in one experiment. We validated the assay using Insulin, insulin mimics/sensitizers and insulin inhibitors. The agonist or antagonists were analyzed for their ability to enhance or block the GLUT4 translocation independent of insulin. The outcome of the assay was correlated by performing glucose uptake assay using differentiated 3T3L1 cells. Using this platform we further identified several plant extracts which had the insulin mimetic action. We confirmed that these plant extracts were non-toxic to the beta cells using RIN mf5cells and 3T3L1 cells. We have identified plant extracts with the potential insulinomimetic action using novel high-content screening approach; these can be further tested for their efficiency in-vivo in pre-clinical trials.
胰岛素抵抗与肥胖有关，并可能导致多种代谢紊乱，包括 2 型糖尿病、非酒精性脂肪肝和心血管问题。寻找既能诱导又能模拟胰岛素作用的小分子具有重要意义，可用于治疗胰岛素抵抗。有几种膳食植物化学物质可能具有胰岛素模拟作用。然而，用于测试小分子作为胰岛素模拟物效率的高通量筛选方法尚未完全建立。在本文中，我们使用表达与人类胰岛素受体结合的 GLUT4-GFP 的 CHO-HIRC-myc-GLUT4 eGFP 细胞系进行了基于 GLUT4 易位的化学筛选分析。我们建立了一种基于高内涵筛选的方法，可以跟踪和量化 GLUT4 从核周区到细胞膜的易位。该测定法涉及测量核周区和细胞膜上的定义区域的荧光强度；并将结果表示为核周区与膜区荧光强度的比值。该测定法可以从数千个细胞/样本中实时收集 GLUT4 易位数据，并且可以在一次实验中从多个样本中收集。我们使用胰岛素、胰岛素模拟物/敏化剂和胰岛素抑制剂验证了该测定法。分析激动剂或拮抗剂是否能够独立于胰岛素增强或阻断 GLUT4 易位。使用分化的 3T3L1 细胞进行葡萄糖摄取测定来对测定结果进行相关性分析。使用该平台，我们进一步鉴定了几种具有胰岛素模拟作用的植物提取物。我们使用 RIN mf5 细胞和 3T3L1 细胞确认这些植物提取物对β细胞没有毒性。我们使用新型高内涵筛选方法鉴定了具有潜在胰岛素模拟作用的植物提取物；这些可以在临床前试验中进一步测试其体内效率。
