Fig. 4. Pio promotion of epididymal fat T_reg numbers and phenotype.

At 9 weeks of age, Pparg wt and T_reg-Pparg mut mice were fed HFD +/− Pio for 13 weeks. Cells from the spleen and epididymal fat SVF were stained and analyzed by flow cytometry. Numbers on dot plots indicate the percentage of cells in that gate for that particular experiment (representative of ≥3 experiments). (a) T_regs from Pparg wt mice on HFD +/− Pio. Left, representative dot plots; right, summary data. (b) Expression of VAT T_reg signature genes in Pparg wt mice on HFD+/−Pio. A volcano plot comparing P value versus FC for probes from VAT T_regs isolated from Pparg wt mice on HFD+Pio versus HFD alone. Genes from the VAT T_reg up- and down-signature are highlighted in pink and green, respectively. P determined by the chi-square test. (c) MFI of CD36 expression by T_regs. ΔMFI indicates, for gated CD3⁺CD4⁺Foxp3⁺ cells, the difference in CD36 expression for HFD-fed mice +/− Pio treatment. Epi-fat ΔMFI = 3828±1362 (*P=0.039); spleen ΔMFI = −182±597 (NS). (d) Cells were isolated from the spleen or epi-fat SVF of B6.Foxp3-(YFP−)Cre mice kept on HFD+/−Pio for 13 weeks, and stained for CD3, CD4 and Nile red. *P=0.01. (e) T_reg fraction. Cells from spleen or epi-fat SVF were stained and analyzed by flow cytometry. (f) Insulin sensitivity. Mice were assessed for blood fasting-glucose and fasting-insulin levels. These values were used to calculate the HOMA-IR. (g) Glucose tolerance. Left: intraperitoneal GTT on wt mice. Center: GTT on mutant mice. Right: area under the curve (AUC) calculations. n=13–14 mice per group. P values calculated using the Student’s T test. Unless otherwise specified: *P<0.05, **P<0.01, ***P< 0.001; NS=not significant. Error bars represent the mean ± SD for immunological parameters, mean ± SE for metabolic parameters, as is standard practice in the respective fields.