Adipocyte Gata3 Is a Diet-Inducible Driver of Pathological Adipose Remodeling and Systemic Metabolic Disease

Figures

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  Figure 1. Gata3 expression is depot-specific and regulated by nutritional status. Relative Gata3 mRNA expression across major adipose depots from chow-fed, designated as ND, and HFD-fed mice. qPCR was performed on total RNA from adipose tissue (n = 4–6 mice per condition) and normalized to Gapdh (glyceraldehyde 3-phosphate dehydrogenase). (A), Gata3. (B), Pparγ. Statistical significance was assessed using Welch’s corrected two-tailed t-test comparing ND versus HFD (GraphPad Prism v11.0.0).

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  Figure 2. Adipocyte-specific deletion of Gata3 does not affect adipogenesis or metabolic homeostasis under basal conditions. (A), Body weight of Adipoq-Cre controls and heterozygous and homozygous adipocyte-specific Gata3 knockout (aGata3KO) mice on chow diet (n = 9–12 mice per genotype). (B,C), Glucose tolerance and insulin tolerance tests showing comparable glucose handling and insulin sensitivity among genotypes (n = 5–7 mice per genotype). (D), Representative Oil Red O staining of SVF cells isolated from epididymal fat of the indicated genotypes after 16 weeks of HFD and differentiated ex vivo with adipogenic cocktail (IBMX, 0.5 mM; dexamethasone, 1 μM; insulin, 10 μg mL−1). (E), Quantification of lipid accumulation in differentiated adipocytes by dye extraction and absorbance at 492 nm. (F), qPCR analysis of adipogenic markers, including Pparγ and Cebpα, during differentiation. Total RNA was isolated from SVF-derived adipocytes at the indicated time points. Data are representative of three independent experiments from distinct cell isolates. Statistical significance was assessed using Welch’s corrected two-tailed t-test (GraphPad Prism v11.0.0). Statistically insignificant numbers are…

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  Figure 3. Adipocyte-specific Gata3 deficiency protects against diet-induced obesity and metabolic dysfunction. (A), Body weight trajectories during HFD feeding (n = 12–15 mice per genotype). (B), Representative photographs of whole mice, dissected animals, and epididymal fat pads after 16 weeks of HFD. (C), Weights of adipose depots and selected organs following HFD. (D), Food intake measurements. (E), qPCR analysis of adipocyte markers in isolated mature adipocytes and stromal vascular fraction (SVF). (F), Representative H&E-stained sections of epididymal adipose tissue after 16 weeks of HFD. (G,H), Quantification of adipocyte size and number from histological sections; values represent mean cell area and cell number averaged across four non-overlapping fields per sample. (I,J), Glucose tolerance and insulin tolerance tests after HFD feeding (n = 5 mice per genotype). (K–M), qPCR analysis of inflammatory mediator expression in epididymal adipose tissue after 16 weeks of HFD. (N), qPCR analysis of fibrosis-associated genes in epididymal adipose tissue. Statistical significance was assessed by two-tailed t-test.

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  Figure 4. Adipocyte-specific expression of human GATA3 restores obesity-associated phenotypes in Gata3-deficient mice. (A,B) Body-weight trajectories of the indicated genotypes during chow (ND) feeding (n = 8–12 mice per genotype) and high-fat diet (HFD) feeding (n = 12–15 mice per genotype). (C) Representative images of mice from each genotype and their corresponding dissected adipose depots. (D–G) Glucose tolerance tests and insulin tolerance tests performed in mice maintained on chow diet or HFD (n = 5 mice per genotype). (H,I) Circulating adiponectin and leptin concentrations, measured by ELISA (n = 4 mice per genotype).

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  Figure 5. Global proteomic profiling identifies inflammatory and metabolic pathways regulated by adipocyte Gata3. (A) Pearson correlation matrix of epididymal white adipose tissue (eWAT) proteomes from high-fat diet (HFD)-fed Adipoq-Cre control mice (Con; n = 5) and adipocyte-specific Gata3 knockout mice (aGata3KO; KO; n = 5). (B,C) Proteomic quality-control metrics, including coefficients of variation and proteome coverage. (D) Heat map of differentially abundant proteins in eWAT from control and aGata3KO mice after 16 weeks of HFD feeding. (E) Volcano plot showing proteins differentially abundant following adipocyte-specific Gata3 deletion. (F,G) Enrichment analyses of protein classes and associated functional pathways among proteins enriched in control eWAT, including Reactome pathway annotations. (H) Protein–protein interaction network and hub-protein analysis of the control-enriched protein module. (I) Transcription-factor enrichment analysis of proteins enriched in control mice, highlighting STAT3-associated inflammatory signaling. (J) Quantitative PCR analysis of Stat3 mRNA expression in the indicated adipose depots after 16 weeks of HFD feeding, showing reduced Stat3 expression in…

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  Figure 6. Adipocyte Gata3 deficiency promotes a metabolically adaptive adipose state. (A), Heat map of differentially abundant metabolites in visceral adipose tissue from control and aGata3KO mice. Metabolite abundances were normalized and row-standardized (Z score) before unsupervised hierarchical clustering. Rows represent metabolites and columns represent biological samples (control, n = 3; aGata3KO, n = 3). Warmer colors indicate higher relative abundance and cooler colors indicate lower relative abundance. Metabolites are annotated by biochemical class, highlighting coordinated, genotype-dependent remodeling of central carbon metabolism. (B), Volcano plot of differential metabolite abundance between control and aGata3KO mice. The x-axis shows log2 fold change (aGata3KO/control) and the y-axis shows −log10 (p value). Significantly increased metabolites are shown in red, significantly decreased metabolites in blue, and non-significant metabolites in gray. (C), Top enriched metabolite sets identified by mapping significantly altered metabolites to curated metabolic pathways. (D), Directionality of enriched metabolite sets, shown as the mean log2 fold change of significant metabolites…

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  Figure 7. Mass cytometry reveals broad immune remodeling following adipocyte Gata3 deletion. Inguinal (iWAT) and epididymal (eWAT) adipose depots were isolated from Adipoq-Cre and aGata3KO mice after 16 weeks of HFD. Depots were digested, SVF cells were prepared, and cells from each genotype were pooled (n = 3 mice per genotype) for CyTOF analysis with the indicated antibody panel. (A), Macrophages (CD45+CD11b+CD64+). (B), Neutrophils (CD11b+Ly6G+). (C), Monocytes (CD11b+CD64+Ly6C+). (D,E), M1-like and M2-like macrophage subsets. (F,G), Dendritic-cell populations, including CD11b− and CD11b+ subsets. (H), B cells (B220+CD19+). (I), T cells (TCRβ+CD3ε+). (J), Group 2 innate lymphoid cells (ILC2s). (K), Eosinophils (CD11b+Siglec-F+). Quantifications of CyTOF data were plotted for each fat depot and cell type.

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  Figure 8. GATA3 deficiency attenuates hepatic steatosis and remodels the circulating lipidome and chemokine pathways. (A), Representative H&E-stained liver sections from control and aGata3KO mice after HFD feeding, showing reduced hepatic steatosis in aGata3KO mice. (B), Serum cholesterol concentrations in control and aGata3KO mice during HFD feeding, measured by ELISA (n = 16 mice per genotype). (C), Hepatic Apoa1 and Pcsk9 mRNA expression after 16 weeks of HFD, quantified by qPCR from total liver RNA using gene-specific primers. (D), Principal component analysis (PCA) of the combined plasma lipidomic datasets from Experiments 2 and 7, performed on normalized abundances of all quantified lipid species. Each point represents one biological replicate (controls, light blue; knockouts, dark blue); ellipses denote 95% confidence intervals. (E), Volcano plot of all quantified plasma lipid species from the batch-adjusted linear model. The x-axis shows batch-adjusted log2 fold change (KO/control) and the y-axis shows −log10 (p value). Red points indicate lipid species significantly increased in knockout plasma (FDR q < 0.10), blue points indicate species significantly decreased (FDR q < 0.10),…