Subcutaneous adipose tissue imaging of human obesity reveals two types of adipocyte membranes: insulin responsive and non-responsive. In adipose tissue, resistance to insulins 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 continued to develop our 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 Thr308/9 (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.
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