Diabetes Metab J.  2024 Sep;48(5):885-900. 10.4093/dmj.2023.0278.

Single-Cell Landscape and a Macrophage Subset Enhancing Brown Adipocyte Function in Diabetes

• Gu J
• Jin J
• Ren X
• Zhang X
• Li J
• Wang X
• Zhang S
• Yin X
• Zhang Q
• Wang Z

• KMID: 2558999
• DOI: http://doi.org/10.4093/dmj.2023.0278

Abstract

Background
Metabolic dysregulation is a hallmark of type 2 diabetes mellitus (T2DM), in which the abnormalities in brown adipose tissue (BAT) play important roles. However, the cellular composition and function of BAT as well as its pathological significance in diabetes remain incompletely understood. Our objective is to delineate the single-cell landscape of BAT-derived stromal vascular fraction (SVF) and their characteristic alterations in T2DM rats.
Methods
T2DM was induced in rats by intraperitoneal injection of low-dose streptozotocin and high-fat diet feeding. Single-cell mRNA sequencing was then performed on BAT samples and compared to normal rats to characterize changes in T2DM rats. Subsequently, the importance of key cell subsets in T2DM was elucidated using various functional studies.
Results
Almost all cell types in the BAT-derived SVF of T2DM rats exhibited enhanced inflammatory responses, increased angiogenesis, and disordered glucose and lipid metabolism. The multidirectional differentiation potential of adipose tissue-derived stem cells was also reduced. Moreover, macrophages played a pivotal role in intercellular crosstalk of BAT-derived SVF. A novel Rarres2+macrophage subset promoted the differentiation and metabolic function of brown adipocytes via adipose-immune crosstalk.
Conclusion
BAT SVF exhibited strong heterogeneity in cellular composition and function and contributed to T2DM as a significant inflammation source, in which a novel macrophage subset was identified that can promote brown adipocyte function.

Keyword

Adipocytes, brown; Adipose tissue, brown; Diabetes mellitus, type 2; Macrophages; Single-cell gene expression analysis

Figure

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  Fig. 1. Cell composition and function of brown adipose tissue (BAT) in normal rats (NC) and type 2 diabetes mellitus (T2DM) rats. (A) The t-distributed stochastic neighbor embedding (t-SNE) plot (left panel) and canonical correlation analysis (CCA) plot (right panel) of single-cell RNA sequencing data of the BAT stromal vascular fraction (SVF) in the NC and T2DM rats. (B, C) Violin plots of the relative expression levels of marker genes for non-immune (B) and immune (C) cell clusters in the BAT SVF. (D) Percentage of each cell cluster in non-immune cells (upper panel) or immune cells (lower panel) in BAT SVF of the NC rats or T2DM rats. (E, F) Functional enrichment analysis of non-immune cells (E) and immune cells (F) in BAT SVF. Fisher’s exact test with Benjamini-Hochberg false discovery rate multiple-test correction was used for the statistical analysis in E and F. ASPC, adipose stem/progenitor cell; FB, fibroblast; SMC, smooth muscle cell; EC, endothelial cell; MAC, macrophage; NK, natural killer; DC, dendritic cell; VSMC, vascular smooth muscle cell.

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  Fig. 2. Enhanced intercellular crosstalk in brown adipose tissue (BAT) stromal vascular fraction (SVF) of type 2 diabetes mellitus (T2DM) rats. (A) Crosstalk numbers between each cell type in BAT SVF of normal rats (NC) and T2DM rats. Cells emitting arrows (indicated by arrows) express ligands (receptors). Edge width: crosstalk numbers. (B) Heatmap of the differential number of cell–cell interactions/crosstalks between distinct cell populations in BAT SVF of T2DM rats compared to NC rats. The top (right) bar shows the sum number of the differential interactions/crosstalks of T2DM rats compared to NC rats in corresponding column (row) in the heatmap. (C) Bar graph of the relative (left) and overall (right) information flow, which is defined by total communication probability among all pairs of cell populations in BAT SVF. (D, E) Chord diagram of the angiopoietin-like protein (ANGPTL) (D) and macrophage inhibitory factor (MIF) (E) signaling network between various cell types in BAT SVF. (F, G) Heatmap of the interaction/crosstalks strength of outgoing (F) and incoming (G) signaling pathways among various cell types. The top (right) bar presents the sum of the relative interaction/crosstalks strength of all (a) signaling pathway(s) in a (all) cell population(s). Relative strength: the contribution level of certain signal to outgoing or incoming signaling in a (all) cell population(s) calculated by Cellchat. ASPC, adipose stem/progenitor cell; FB, fibroblast; SMC, smooth muscle cell; EC, endothelial cell; MAC, macrophage; NK, natural killer; DC, dendritic cell; NT, neurotransmitter; CCL, chemokine C-C motif ligand; GAS, growth arrest-specific; APRIL, a proliferation-inducing ligand; IL, interleukin; MK, mitogen-activated protein kinase; PROS, protein S; PDGF, platelet-derived growth factor; CXCL, C-X-C motif chemokine ligand; BAFF, b-cell activating factor; VEGF, vascular endothelial growth factor; CSF, colony stimulating factor.

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  Fig. 3. Subclusters and functions of adipose stem/progenitor cells (ASPCs) in brown adipose tissue (BAT). (A) The canonical correlation analysis (CCA) plot of ASPC subsets in BAT. (B) Distribution of ASPC1 and ASPC2 subsets on the pseudotime trajectory. (C, H) The t-distributed stochastic neighbor embedding (t-SNE) plot of the subclusters of ASPC1 (C) or ASPC2 (H) in BAT. (D, I) Violin plots of the relative expression levels of marker genes in each subpopulation of ASPC1 (D) or ASPC2 subcluster (I). (E, J) Functional enrichment analysis of the four subclusters of ASPC1 (E) or the five subclusters of ASPC2 (J). Circle size represents the number of related genes of each item. (F) Heatmap of relative expression levels and functional enrichment of representative differentially expressed genes in the ASPC1-s1 subcluster of ASPC1 in BAT of type 2 diabetes mellitus (T2DM) rats compared to normal rats (NC). (G) Distribution of ASPC1-s1 and ASPC1-s2 subclusters of ASPC1 on the pseudotime trajectory. (K) Distribution of ASPC2-s0, ASPC2-s1 and ASPC2-s3 subclusters of ASPC2 on the pseudotime trajectory. Fisher’s exact test with Benjamini-Hochberg false discovery rate multiple-test correction was used for the statistical analysis in E and J. The Mann-Whitney U test was performed for the statistical analysis in F. TNF, tumor necrosis factor; TGF-β, PDGF, platelet-derived growth factor.

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  Fig. 4. Subclusters and functions of endothelial cells (ECs) in brown adipose tissue (BAT). (A) The canonical correlation analysis (CCA; left panel) and t-distributed stochastic neighbor embedding (t-SNE; right panel) plots of EC subclusters in BAT. (B) Violin plots of relative expression levels of marker genes in each EC subcluster. (C) Functional enrichment analysis of each EC subcluster. (D) Representative immunofluorescence images of leucine rich repeat containing 8 VRAC subunit C (Lrrc8c)+cadherin 5 (Cdh5)+ECs in rat BAT. (E, F) Heatmap of relative expression levels (E) and functional enrichment analysis (F) of the significantly regulated genes in each EC subcluster in BAT of type 2 diabetes mellitus (T2DM) rats compared to those of normal rats (NC). Fisher’s exact test with Benjamini-Hochberg false discovery rate multiple-test correction was used for the statistical analysis in C and F. Mann-Whitney U test was performed for the statistical analysis in E. VEGF, vascular endothelial growth factor; SMC, smooth muscle cell; HDL, high density lipoprotein; DAPI, 4´,6-diamidino-2-phenylindole; TGF-β, transforming growth factor-β; ROS, reactive oxygen species; LDL, low density lipoprotein.

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  Fig. 5. Subclusters and functions of macrophages (MACs) in brown adipose tissue (BAT). (A) The canonical correlation analysis (CCA; left panel) and t-distributed stochastic neighbor embedding (t-SNE; right panel) plots of MAC subclusters in BAT. (B) Violin plots of the relative expression levels of marker genes in each MAC subcluster. (C) Functional enrichment analysis of each MAC subcluster in BAT. (D) Proportion of each MAC subcluster in the total MACs in BAT of normal rats (NC) and type 2 diabetes mellitus (T2DM) rats. (E, F) Heatmap of relative expression levels (E) and functional enrichment analysis (F) of significantly regulated genes in each MAC subcluster of BAT in T2DM rats compared to those of NC rats. Fisher’s exact test with Benjamini-Hochberg false discovery rate multiple-test correction was used for the statistical analysis in C and F. Mann-Whitney U test was performed for the statistical analysis in E. ROS, reactive oxygen species; MHC, major histocompatibility complex; TNF, tumor necrosis factor; SMC, smooth muscle cell; TGF-β, transforming growth factor-β; PDGF, platelet derived growth factor.

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  Fig. 6. Retinoic acid receptor responder 2 (Rarres2)+macrophages (MACs) promote differentiation and function of brown adipocytes (BAs). (A) Representative immunofluorescence images of Rarres2+MACs in brown adipose tissue (BAT) of normal rats (NC). (B) Comparison of the percentage of Rarres2+MACs in white adipose tissue (WAT)-derived MACs and in BAT-derived MACs. (C) Comparison of the relative expression levels of Rarres2 mRNA in WAT-derived stromal vascular fraction (SVF), BA, and BAT-derived SVF. Fold change=The relative expression levels of each group compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/the mean of relative expression levels of WAT SVF compared to GAPDH. (D, E) Representative images of Oil Red O staining of brown adipocyte progenitor cells (BAPCs) with different treatments (D) and their quantitative comparison of the positive area of Oil Red O staining (E). Fold change=The positive area of Oil Red O staining of each group/the mean of positive area of Oil Red O staining of D0 BAPC. (F) Representative Western blot images of differentiation-related proteins and mitochondrial functional proteins in BAPCs with different treatments (left panel) and their quantitative comparison (right panel). (G) Quantitative comparison of the fluorescence intensity of mitochondria stained by Mitotracker Red in BAPCs with different treatments. Fold change=The fluorescence intensity of mitochondria stained of each group/the mean of fluorescence intensity of mitochondria stained of D0 BAPC. (H) Comparison of oxygen consumption rates (OCRs) at the plateau phase of BAPCs with different treatments. Fold change=The OCRs of each group/the mean of OCRs of D0 BAPC. (I) Representative Western blot images of phosphorylation levels of mammalian target of rapamycin (mTOR), AKT, and extracellular signal-regulated kinase (ERK) proteins, as well as expression levels of CCAAT enhancer binding protein beta (CEBPβ), CEBPα, and peroxisome proliferator-activated receptor γ (PPARγ) proteins in BAPCs with different treatments (left panel) and their quantitative comparison (right panel). D8 BAPC & Rarres2-MAC: BAPC were cocultured with Rarres2-MACs and differentiated on day 8; D8 BAPC & Rarres2+ MAC: BAPC were cocultured with Rarres2+MACs and differentiated on day 8. In F and I, fold change=The relative expression levels of each group compared to GAPDH/the mean of relative expression levels of D0 BAPC compared to GAPDH. Two-tailed Student’s t-test (B) and one-way analysis of variance (ANOVA) test with appropriate correction were used for statistical analysis (C, E-I). DAPI, 4´,6-diamidino-2-phenylindole; PRDM16, PR domain-containing 16; UCP1, uncoupling protein 1; COX7RP, cytochrome c oxidase subunit 7A2 like; TFAM, transcription factor A, mitochondrial. aP<0.05, bP<0.01, cP<0.001, dP<0.0001.

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