Human visceral and subcutaneous adipose stem and progenitor cells retain depot-specific adipogenic properties during obesity

Abstract

Abdominal obesity associates with cardiometabolic disease and an accumulation of lipids in the visceral adipose depot, whereas lipid accumulation in the subcutaneous depot is more benign. We aimed to further investigate whether the adipogenic properties where cell-intrinsic, or dependent on a depot-specific or obesity-produced microenvironment. We obtained visceral and subcutaneous biopsies from non-obese women (n = 14) or women living with morbid obesity (n = 14) and isolated adipose stem and progenitor cells (ASPCs) from the stromal vascular fraction of non-obese (n = 13) and obese (n = 13). Following in vitro differentiation into mature adipocytes, we observed a contrasting pattern with a lower gene expression of adipogenic markers and a higher gene expression of immunogenic markers in the visceral compared to the subcutaneous adipocytes. We identified the immunogenic factor BST2 as a marker for visceral ASPCs. The effect of obesity and insulin resistance on adipogenic and immunogenic markers in the in vitro differentiated cells was minor. In contrast, differentiation with exogenous Tumor necrosis factor resulted in increased immunogenic signatures, including increased expression of BST2, and decreased adipogenic signatures in cells from both depots. Our data, from 26 women, underscore the intrinsic differences between human visceral and subcutaneous adipose stem and progenitor cells, suggest that dysregulation of adipocytes in obesity mainly occurs at a post-progenitor stage, and highlight an inflammatory microenvironment as a major constraint of human adipogenesis.

Keywords: adipogenesis; human adipocytes; immunogenic adipocytes; obesity; subcutaneous adipocytes; visceral adipocytes.

FIGURE 1

Gene expression of immunogenic markers in adipose tissue of non-obese women and women living with obesity. Adipose tissue biopsies from subcutaneous and visceral adipose regions were collected from gallstone patients (non-obese, n = 14) and gastric surgery patients (obese, n = 14). (A) Cartoon of adipose tissue depots and isolated cells. (B–E) qPCR analysis of the immunogenic markers TNF, IL6, CCL2 and TP53 in paired samples of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) obtained from non-obese women and women living with obesity. Differences between groups and adipose depots were assessed using two-way anova with subsequent post-tests. Data are presented as violin plots with thick dotted line showing median and thin dotted lines showing quartiles. Two-way anova assessed difference between groups and depots. Results from significant post-tests are shown as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2

Adipogenic and immunogenic gene expression in APSCs from SAT and VAT of non-obese women and women living with obesity. ASPCs were isolated from the subcutaneous and visceral adipose biopsies from women living with obesity (n = 13) and non-obese women (n = 13). (A) qPCR analysis was used to measure the relative expression of the adipogenic markers PPARG, LPL, ADIPOQ and FABP4. QPCR analysis was used to measure the relative expression of the immunogenic markers TNF, IL6 and CCL2 in (B) Differentiated adipocytes and (C) Proliferating APSCs. Data are presented as violin plots with thick dotted line showing median and thin dotted lines showing quartiles. Two-way anova assessed difference between groups and depots. Results from significant post-tests are shown as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3

Characterization of APSCs derived from human SAT and VAT. (A) APSCs derived from SAT and VAT were cultured and characterized for surface expression of CD90, CD166 and CD56 using flow cytometry. Granularity and relative size were evaluated by side- and forward scatter area (SSC-A and FSC-A) to select the preadipocyte population for analysis. From these cells, single cells were selected by evaluation of forward scatter height and width (FSC-h and FSC-W). Within this population, cells positive for CD166, CD90 and CD55 were selected. Gating strategy: preadipocytes/singlets/CD166+CD90+/CD56+. Flow cytometry illustrated a smeared DIM CD56⁺ population within the preadipocyte population in both visceral and subcutaneous adipocytes. Right: summary graph of CD56⁺ APSCs (n = 3) (B) Sub-cultured APSCs harvested at 80% confluency was analyzed using GenEx qPCR array. Heatmap shows top 10 genes with lowest p-values from pairwise comparisons among the four groups. All data is shown in (Supplementary Material S1) (C) Data derived from the qPCR array on APSCs showing BST2 mRNA levels. (D) Data derived from the qPCR array on APSCs showing BST2 mRNA levels when samples were reorganized based on HOMA-IR. As clinical data were not available for all subjects, the sample groups were slightly reduced compared to the non-obese vs obese groups. HOMA high represents HOMA-IR values over 2 and HOMA low represents HOMA-IR values under or equal to 2. SAT HOMA low: n = 11 (7 non-obese, 4 obese); SAT HOMA high: n = 10 (4 non-obese, 6 obese); VAT HOMA low: n = 12 (8 non-obese, 4 obese); VAT HOMA high: n = 9 (4 non-obese, 6 obese). Data are presented as violin plots with thick dotted line showing median and thin dotted lines showing quartiles. Two-way anova assessed difference between groups and depots. Significant post-test is shown as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4

Adipogenic and immunogenic gene expression in APSCs stimulated with TNF during differentiation. APSCs derived from SAT and VAT of non-obese women (n = 13) were differentiated in media supplemented with TNF or saline control. (A) Cartoon showing experimental set-up. (B) Flow cytometry measuring CD56⁺ positive cells in APSCs exposed to 10 ng/ml TNF or saline control for 48 h (n = 3/condition). (C) qPCR analysis was used to measure the relative expression of the adipogenic markers PPARG, LPL, FABP4 and ADIPOQ. Adiponectin protein levels was measured in the cell media using ELISA. (D) qPCR analysis was used to measure the relative expression of the immunogenic markers BST2, IL6 and CCL2. IL-6 and C-C motif chemokine-2 protein levels were measured in the cell media using ELISA. Data are presented as violin plots with thick dotted line showing median and thin dotted lines showing quartiles. Two-way anova assessed difference between groups and depots. Results from significant post-tests are shown as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.