Abstract
Adipose tissue, as the main energy storage organ and through its endocrine activity, is interconnected with all physiological functions. It plays a fundamental role in energy homeostasis and in the development of metabolic disorders. Up to now, this tissue has been analysed as a pool of different cell types with very little attention paid to the organization and putative partitioning of cells. Considering the absence of a complete picture of the intimate architecture of this large soft tissue, we developed a method that combines tissue clearing, acquisition of autofluorescence or lectin signals by confocal microscopy, segmentation procedures based on contrast enhancement, and a new semi-automatic image analysis process, allowing accurate and quantitative characterization of the whole 3D fat pad organization. This approach revealed the unexpected anatomic complexity of the murine subcutaneous fat pad. Although the classical picture of adipose tissue corresponds to a superposition of simple and small ellipsoidal lobules of adipose cells separated by mesenchymal spans, our results show that segmented lobules display complex 3D poly-lobular shapes. Despite differences in shape and size, the number of these poly-lobular subunits is similar from one fat pad to another. Finally, investigation of the relationships of these subunits between each other revealed a never-described organization in two clusters with distinct molecular signatures and specific vascular and sympathetic nerve densities correlating with different browning abilities. This innovative procedure reveals that subcutaneous adipose tissue exhibits a subtle functional heterogeneity with partitioned areas, and opens new perspectives towards understanding its functioning and plasticity.
Highlights
A small but growing amount of data suggests that tissues/organs should be seen as sophisticated ecosystems depending on highly complex but structured interactions between different, organized sets of cells and their micro-environments[1]
Volume quantification of both regions of the fat pad revealed that the proportion corresponding to the core was similar in all samples (20.1 ± 1.9% of the total volume of the fat pad) with a mean volume of 19.5 ± 8 mm[3] (Table 1) (n = 3)
Quantification of lectin labelling revealed an increase of vascular density in areas 1 and 2 after cold exposure without changing the differential pattern between areas 1, 2 and 3 observed at 22 °C (Fig. 6D). We demonstrated that this depot was spatially organized in two large regions with sub-domains associated with a compartmentalization of browning ability[13]
Summary
A small but growing amount of data suggests that tissues/organs should be seen as sophisticated ecosystems depending on highly complex but structured interactions between different, organized sets of cells and their micro-environments[1]. Since Wassermann’s work in 1960, which described the “lobular” structure of adipose tissue using classical 2D photonic microscopy images[10], very few studies have investigated the architecture of adipose tissues in depth[11,12,13,14]. A large number of studies have demonstrated that adipose depots differ from each other in terms of “adipocyte biology” including adipokine secretion and/or rate of lipolysis and triglyceride synthesis and that a complex cellular heterogeneity exists within a single fat pad[6,15,16,17,18,19,20,21]. In this study, automatic 3D segmentation of lobules using commercially available softwares was unsuccessful and systematic analysis of several samples was not possible This prevented us from studying 3D structures of the subunits and their interconnectivity
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