From the Section on Integrative Biophysics and.
Section on Growth and Obesity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 2018 Sep 14;293(37):14249-14259. doi: 10.1074/jbc.RA118.003751. Epub 2018 Jul 13.
In adipose tissue, resistance to insulin's ability to increase glucose uptake can be induced by multiple factors, including obesity. Impaired insulin action may take place at different spatial loci at the cellular or subcellular level. To begin to understand the spatial response to insulin in human subcutaneous adipose tissue (hSAT), we developed a quantitative imaging method for activation of a major signaling node in the glucoregulatory insulin signaling pathway. After treatment with insulin or control media, biopsied tissues were immunostained for Akt phosphorylation at Thr-308/9 (pAkt) and then imaged by confocal fluorescence microscopy automated to collect a large grid of high resolution fields. In hSAT from 40 men and women with obesity, substantial heterogeneity of pAkt densities in adipocyte membranes were quantified in each image mosaic using a spatial unit of at least twice the size of the point spread function. Statistical analysis of the distribution of pAkt spatial units was best fit as the weighted sum of two separate distributions, corresponding to either a low or high pAkt density. A "high pAkt fraction" metric was calculated from the fraction of high pAkt distributed units over the total units. Importantly, upon insulin stimulation, tissues from the same biopsy showed either a minimal or a substantial change in the high pAkt fraction. Further supporting a two-state response to insulin stimulation, subjects with similar insulin sensitivity indices are also segregated into either of two clusters identified by the amount of membrane-localized pAkt.
在脂肪组织中，多种因素都可能导致胰岛素增加葡萄糖摄取能力的作用受阻，肥胖就是其中之一。胰岛素作用的受损可能发生在细胞或亚细胞水平的不同空间位置。为了初步了解胰岛素在人体皮下脂肪组织（hSAT）中的空间反应，我们开发了一种定量成像方法，用于激活葡萄糖调节胰岛素信号通路中的主要信号节点。在接受胰岛素或对照培养基处理后，对活检组织进行 Akt 磷酸化 Thr-308/9（pAkt）的免疫染色，然后通过共聚焦荧光显微镜自动进行成像，以收集大量高分辨率场的网格。在来自 40 名肥胖男性和女性的 hSAT 中，使用至少是点扩散函数两倍大小的空间单位，对每个图像镶嵌图中的脂肪细胞膜中 pAkt 密度的异质性进行了量化。使用两个独立分布的加权和对 pAkt 空间单位分布进行统计学分析，分别对应低或高 pAkt 密度。从高 pAkt 分布单位占总单位的分数计算出“高 pAkt 分数”指标。重要的是，在胰岛素刺激下，来自同一活检的组织表现出高 pAkt 分数的微小或实质性变化。进一步支持胰岛素刺激的两态反应，具有相似胰岛素敏感性指数的受试者也被分为两个聚类，由膜定位的 pAkt 量确定。
