Myeloid lineage (CD45+ CD11b+ cells) analysis sorted from stromal vascular fraction of visceral and subcutaneous adiposetissue in mice in different diet conditions or after rosiglitazone treatment

Unlike visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT) can play a protective role against the development of insulin resistance and metabolic dysfunction in obesity. These changes are associated with higher induction of pro-inflammatory programs and macrophage infiltration in VAT compared with SAT in obesity. Moreover, our results suggest that subcutaneous adipose tissue macrophages (ATMs) release small extracellular vesicles (sEVs) that can improve insulin sensitivity, opposite to the effect of visceral ATM sEVs in obesity. This functional difference was associated with changes in the proportion of resident ATMs, as well as proinflammatory, recruited ATMs. To study the mechanism by which adipose tissue inflammation and macrophage infiltration are differentially regulated in SAT vs VAT, we performed single cell RNA sequencing (scRNA-seq) analysis in sorted live CD45+ CD11b+ myeloid cells from stromal vascular fraction (SVCs) of SAT and VAT of mice fed normal chow diet, high fat diet, or SVCs of HFD mice after the diet switch from high fat diet to normal chow diet, or treatment with insulin sensitizing, rosiglitazone. Overall design: Four different groups of C57BL6 mice were used. Group 1 mice fed normal chow diet for 15 weeks. Group2 mice were fed a high fat diet (HFD) for 15 weeks. Group3 mice were fed a HFD for 12 weeks, followed by the diet switch from HFD to normal chow diet for additional 3 weeks. Group4 mice were fed a HFD for 12 weeks, followed by switch diet to rosiglitazone-containing HFD for additional 3 weeks. At sacrifice, all mice were in the same age. Subcutaneous (inguinal) and visceral (epididymal) adipose tissues taken from these mice were chopped and digested in collagenase buffer. Stromal vascular cells (SVCs) were separated from adipocytes using centrifugation, and pelleted SVCs were used to sort live CD45+ CD11b+ myeloid lineage cells. Sorted cells were, then, subjected to scRNA-seq.