A. Nutritional regulation of Irf4 mRNA expression in murine white adipose tissue (WAT). Male FVB mice were sacrificed in the fed state, after a 24hr fast, or 24hr after refeeding (n=7-8). Data are normalized to 36B4 expression and are expressed as fold induction relative to Irf4 mRNA in the fed state. Results expressed as mean ± SD. B. Nutritional regulation of IRF4 protein expression in murine WAT, brown adipose tissue (BAT), and spleen. C. IRF4 mRNA expression in human WAT, sampled pre and post-fasting (n=7). *p<0.05 versus pre fasting. D. Regulation of Irf4 mRNA expression by insulin in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated in serum-free DMEM with insulin at the indicated doses for 8hrs (n=3). Data are normalized to 36B4 and presented as expression relative to 0nM insulin. *p<0.05 versus 0nM insulin. Results expressed as mean ± SD. E. Irf4 mRNA expression in WAT of streptozotocin (STZ)-treated mice. Irf4 mRNA expression was measured by Q-PCR in WAT of 10-week-old male mice given vehicle (control), STZ, or STZ followed by insulin replacement for 24h (STZ+Ins) (n=8-10/group). Data are normalized to 36B4 and presented as expression relative to control, *p<0.05. Results expressed as mean ± SD. F. Irf4 mRNA expression in WAT of Fat Insulin Receptor Knockout (FIRKO) mice (n=4/group). Data are normalized to 36B4 and presented as expression relative to control. Results expressed as mean ± SD. *p<0.05 versus control. G. Irf4 mRNA expression in 3T3-L1ΔCAR adipocytes transduced with adenovirus expressing EGFP, wild type (WT), constitutively active (CA), or dominant negative (DN) FoxO1. Data are normalized to 36B4 and presented as fold induction relative to EGFP cells. *p<0.05 versus EGFP cells. Results expressed as mean ± SD. H. Luciferase activity of Irf4 promoter constructs in 3T3-L1 adipocytes. Five days after adipogenic stimulation, 3T3-L1 cells were transfected with the indicated Irf4 promoter construct. Luciferase activity was measured 24 hr after transfection in the presence (filled bar) or absence (open bar) of insulin. Results are expressed as mean ± SD (n = 3). *p < 0.05 relative to the same construct without insulin. Results expressed as mean ± SD. I. ChIP assay of FoxO1 binding to Irf4 promoter constructs in 3T3-L1 adipocytes in the presence (filled bar) or absence (open bar) of insulin (n=3). All results normalized to IgG without insulin, and expressed as mean ± SD.

A. Basal and isoproterenol-stimulated glycerol release (6-hour stimulation) in WT and KO MEFs, with and without the addition of exogenous IRF4. *p<0.05 (n=6), Results expressed as mean ± SD. B. Pnpla2, Lipe, and Plin1 mRNA expression in WT and KO MEFs. *p<0.05 relative to WT control (n=3). Results expressed as mean ± SD. C. ATGL, HSL, and PLIN1 protein expression in WT and KO MEFs. D. Luciferase activity of Pnpla2 promoter reporter constructs transfected into 3T3-L1 adipocytes. Five days after adipogenic stimulation, 3T3-L1 cells were co-transfected with the indicated promoter construct and an IRF4 expression plasmid (or control). Luciferase activity was measured 24 hr after transfection. Results are expressed as mean ± SD (n = 6). *p < 0.05 relative to EGFP. E. ChIP analysis in 3T3-L1 adipocytes overexpressing IRF4. The signal in IgG is set as 1. Results expressed as mean ± SD.

A. Insulin (100nM) stimulation of ¹⁴C glucose incorporation into lipid was determined in WT and KO MEFs, with and without the addition of exogenous IRF4. *p<0.05 (n=6), Results expressed as mean ± SD. from three independent experiments. *p<0.05 relative to WT control under insulin stimulation, ^#p<0.05 relative to KO MEF under insulin stimulation. B. WT and KO MEFs were transduced with lentivirus expressing IRF4 or EGFP 5 days after differentiation induction. mRNA expression of lipogenesis genes was measured using Q-PCR 7 days after infection. Results expressed as mean ± SD, n=6.

A. Irf4 mRNA expression in WAT, BAT, spleen, and peritoneal macrophages of male Irf4^f/f (Flox) and FI4KO (KO) mice (n=3). Data are normalized to 36B4. *p<0.05 relative to Flox mice. Results are expressed as mean ± SD. B. IRF4 protein expression in WAT, BAT, spleen, and peritoneal macrophages of male Adipoq-Cre and FI4KO (KO) mice. C. Body weights of male WT, Adipoq-Cre, Flox, and FI4KO (KO) mice on high-fat diet (n=8-10). Results are expressed as mean ± SD. D. Body composition analysis in 14-week-old male mice (n=8-10) by MRI. Results are expressed as mean ± SD, *p<0.05 relative to Flox mice. E. Morphology of FI4KO (KO) mice (16 month old) and fat pads after high-fat feeding. F. Hematoxylin and eosin staining of paraffin-embedded epididymal WAT sections from 16-month-old mice. Scale bars = 50μm. G. Representative cell size distribution of adipocytes in epididymal WAT from Flox and FI4KO (KO) mice. H. Gross appearance and hematoxylin and eosin staining of paraffin-embedded BAT sections from 16-month-old mice. Scale bars = 50μm.

A. Lipolysis in Flox and FI4KO (KO) mice on chow diet. Glycerol and NEFA in plasma were measured in the absence and presence of 10μM of isoproterenol. Results are expressed as mean ± SD, * p<0.05, versus Flox mice (n=8). B, C. Rate of lipolysis in isolated adipocytes. Adipocytes were isolated from epididymal fat of male Flox and FI4KO (KO) mice. Glycerol and NEFA release were measured in the absence and presence of 10μM of isoproterenol. Results are expressed as mean ± SD, *p<0.05 relative to Flox mice (n=3). D. Q-PCR analysis of the indicated mRNAs in the adipocyte fraction of epididymal WAT from 12-week-old Flox or FI4KO (KO) mice on chow diet (n=5) under fed and 24h fasting conditions. All samples are normalized to 36B4. Results expressed as mean ± SD, * p<0.05, versus Flox mice.

A. Response of FI4KO mice to prolonged fasting. Results are expressed as mean ± SD, *p<0.05 relative to Flox mice (n=9). B. NEFA levels after 48hr of fasting (n=9 mice per group; *p<0.05). C. β-hydroxybutyrate levels after 48hr of fasting (n=9 mice per group; *p<0.05). Data are expressed as mean ± SD.

A. Rectal temperature of Flox and FI4KO (KO) mice during cold exposure (4°C). Results are expressed as mean ± SD, *p<0.05 relative to Flox mice (n=7 mice per group). B. Expression of Dio2, Cidea, Ucp1, and Pgc1α in BAT after cold exposure for 6 hr (n=7 mice per group; *p<0.05). Data are expressed as mean ± SD.