Impaired adipogenesis and lipid storage capacity in subcutaneous adipose tissue of patients with PMOS

Figures

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  Figure 1. General design of the translational study and clinical characteristics of the 15 CTRL and 17 PMOS women. (A) General design of clinical and adipose tissue phenotyping studies. ABD-AT, s.c. abdominal adipose tissue; GF-AT, gluteofemoral adipose tissue; snRNA-seq, single-nuclei RNA-seq. (B) Insulin; glucose and free fatty acids (FFA) were quantified in serum samples collected during an OGTT test. n = 15 PMOS and 17 CTRL. *P < 0.05 Wilcoxon test between group at specific time point. AUC for each group have been reported as total area under the curve, plotted and compared using an unpaired 2-tailed t test. (C) IL-8 was measured in the serum samples of 15 PMOS and 17 CTRL women. *P < 0.05 unpaired 2-tailed t test. (D) Adipocyte size was determined by microscopy. Average of 100 adipocytes per sample is represented in ABD and GF depot. CTRL group samples are represented in green, and PMOS group samples are represented in pink. *P < 0.05 paired 2-tailed t test. (E) Correlation matrix of relevant clinical parameters and ABD and GF-AT adipocyte size. n = 15 PMOS and 17 CTRL. Only significant correlations (P < 0.05) are represented by a dot; negative correlations are depicted in brown and…

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  Figure 2. A single nuclei atlas of ABD and GF-AT in women with PMOS and BMI-matched women without PMOS. (A) UMAP projection of clusters formed by 11,638 human single nuclei isolated from ABD and GF-AT. n = 5 PMOS and 5 CTRL. The dotplot shows the marker genes used to identify each cluster. (B) Dotplot representation of relative expression of genes identified by Strieder-Barboza et al. (34) in each cell cluster. FA, Fibro Adipogenic Adipose Stem Cell; IML, Inflammatory Mesothelial like Adipose Stem Cell (34). (C) Stacked bar charts show the repartition of each cluster in CTRL and PMOS samples, split by depot (ABD and GF). (D) Intersection plots show the genes upregulated in CTRL and in PMOS that are common or unique to each cluster (MSC, preadipocytes, adipocytes). The analysis was performed independently in ABD (top graph) and GF-AT (lower graph). (E) Dotplot representation of functional enrichment analysis using Hallmark on the genes differentially regulated between CTRL and PMOS in each cell clusters. Pink indicates the pathways enriched in PMOS samples; green indicates the pathways enriched in CTRL samples. See also Supplemental Figure 1. Overlap represents the number of genes in…

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  Figure 3. Association between testosterone level and SAT gene expression and cell population proportion in ABD and GF-AT. (A) Heatmaps representation of the correlations between the gene modules identified by WGCNA analysis and relevant clinical parameters in ABD and GF-AT. (B) Intersection plot showing the number of common genes between the turquoise module (in ABD-AT) and the blue module (in GF-AT). Pathway analysis reveals transcription factor (TRRUST) and pathways enriched in these common genes. EMT, Epithelial Mesenchymal Transition. (C) Correlation matrix of cell proportions and testosterone (total, free and bioavailable) and SHBG level. Only significant correlations (P < 0.05) are represented by a dot; negative correlations are depicted in green and positive correlations are depicted in pink. The size of the dot indicates the R coefficient. (D) Spearman correlation plots between total testosterone and relevant cell clusters in ABD and GF-AT depot, in combined group. PMOS participants are represented in pink; CTRL participants are represented in green. See also Supplemental Figure 2. (E) Correlation matrix of ectopic fat accumulation markers and adipogenesis-related genes in ABD and…

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  Figure 4. Pseudotime analysis suggests block of differentiation in PMOS. (A) Distinct state of cells identified by pseudotime analysis in CTRL and in patients with PMOS using MSC, preadipocytes, AdipoLEP, and AdipoINS. (B) Ordering and quantification of cells from different clusters identified in A along the pseudotime trajectory and according to the condition (CTRL or PMOS). (C) Distinct state of cells identified by pseudotime analysis in CTRL and in patients with PMOS using MSC, preadipocytes and AdipoINS only.

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  Figure 5. A single-cell ATLAS of ABD and GF-stroma vascular fraction in women with PMOS and BMI-matched CTRL. (A) UMAP projection of clusters formed by 7,204 human single cells extracted from ABD and GF-SVF. n = 5 CTRL and 4 PMOS. The dotplot shows the marker genes used to identify each cluster. (B) Stacked bar plots showing the proportion of each cluster per subject and their average per group (CTRL versus PMOS) and per depot (ABD versus GF). (C) Heatmap showing the expression of the most DEG between the 3 populations of preadipocytes in each cell (Only Pread-1, Pread-2, and Pread-3 being included on the heatmap). (D) Distinct state of cells identified by pseudotime analysis in CTRL and in patients with PMOS. Ordering and quantification of cells from different clusters identified in A along the pseudotime trajectory and according to the condition (CTRL or PMOS). (E) Gene expression level during 12-days of adipogenic differentiation of ABD-MSC isolated from CTRL-SVF (n = 5) and PMOS-SVF (n = 5). (F) The degree of differentiation was measured by Oil red O in each group after 12 days (D12) and 16 days (D16) of differentiation. A representative picture of cells isolated from CTRL-SVF and from…

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  Figure 6. Potential interaction between MSC and Preadipocytes via SLIT2/ROBO pathway. (A) Level of differentiation of ABD-ADSCs after 12 days of incubation of adipogenic differentiation cocktail supplemented with testosterone (T). Oil red O was used to quantify the lipid accumulation at day 12. n = 10 ADSCs, isolated from 5 CTRL and 5 PMOS. Representative pictures of CTRL ADSCs at D12 of differentiation with or without testosterone in the media. Scale bars: 200 μm. (B) Circle plots of secreted signaling pathways coming from MSC population enriched in CTRL and in PMOS group. The colors of the circle specify which cell type receives the signal. (C) Circle plots of ECM-receptor signaling pathways enriched in MSC CTRL and in MSC PMOS group. The colors of the circle specify which cell type receives the signal. (D) Violin plots comparing the expression level of SLIT2, ROBO1, and ROBO2 in MSC, Pread-1, and Pread-2 nuclei between CTRL and PMOS group. (E) Immunofluorescence staining of s.c. adipose tissue using WGA (488 excitation length of the fluorochrome) and ROBO1 (647 excitation length of the fluorochrome) antibodies. Hoescht was used to stain the nuclei. Scale bar: 200 μm. (F)…