Fig 2: Maternal obesity (MO) reduces T3 concentration and thyroid hormone signaling, linking to impaired brown adipogenesis with a fetal origin.a Fetal mass in control and obesity dam (MO) at 22 °C (n = 6). b Density of brown progenitors (solid circle) and preadipocytes (dash circle) in female offspring BAT at P0, 21 days (weaning), and 4 months of age. FACS sorted brown progenitors and preadipocytes in BAT-stromal vascular fractions using brown progenitor marker Lin: CD45−/PDGFRa+ (in black solid circle) and brown preadipocyte marker Lin: EBF2+/PDGFRa+ (dash black circle in right side quarter). c Quantified brown progenitors and preadipocytes in BAT of female offspring (n = 5). Whisker of box plots shows means and individual values from minimum to maximum. d Thyroid hormone (TH) thyroxine (T4) and triiodothyronine (T3) concentrations were analyzed in fetal and neonatal BAT at embryonic days E18.5 (n = 15) and P0 (n = 17). e, f mRNA expression of TH receptors TRα and TRβ (e), and TH responsive genes Ppargc1a and Ucp-1 (f) in fetal and neonatal BAT at E18.5 and P0 (n = 6). mRNA expression was normalized to 18 S rRNA. g Immunoblotting measurements of PGC-1a, UCP-1, and PRDM16 protein contents in fetal (E18.5) and neonatal BAT (n = 6). β-tubulin was used as a loading control. h Immunostaining of mitochondrial (mito-tracker) and UCP-1 protein in neonatal BAT (n = 4). Scale bar 100 µm. i Thermal imaging of female neonates born to control and MO dam (n = 14 in control; n = 11 in MO). Imaging was taken immediately after removing from cages to limit the effects of ambient temperature and heat loss. j Diagram illustrates TH uptake, T3 and T4, from blood circulation and bio-active conversion in brown adipocytes. TH membrane receptor MCP8 for TH uptake in brown adipocytes, TH responsive element (TRE), nuclear TH receptors (THRα and β), iodothyronine deiodinases Dio2 and Dio3, TH downstream thermogenic genes Ucp-1, Ppargc1a. k mRNA expression of Dio2 and Dio3 in fetal and female neonatal BAT at E18 and P0 (n = 6). mRNA expression was normalized to 18 S rRNA. l Protein contents of D2 and D3 were measured by immunoblotting; β-tubulin was used as a loading control (n = 6). For data analyses, each pregnancy (dam) was considered as a replicate unit. Data are presented as mean ± s.e.m. Unpaired Student’s t test with two-tailed distribution was used in data analyses.

Fig 3: FoxO1–Dio2 axis governs TH-induced hypertrophic growth of neonatal cardiomyocytes.a qRT-PCR analyses of mRNA levels of the indicated genes in NRVM transfected with control or two sequence-independent Dio2-specific siRNAs. Note that efficient knockdown of Dio2 did not affect Dio3 expression (n = 3 independent experiments). b Selective knockdown of Dio2 in NRVM abrogated the T4-induced, but not T3-induced, cellular growth response (n = 3 independent experiments). c Histology (left) (bar = 2 mm) and co-immunostaining with anti-pH3 (green) (middle) and anti-cTnT (red) (right) antibodies of P3 FoxO1-WT (WT) and FoxO1-cKO (cKO) heart. Proliferating cardiomyocytes in LV free wall are shown (arrowhead), and DAPI was used for the nuclear staining (bar = 10 μm). d Quantitation of proliferating cardiomyocytes in WT and cKO hearts (n = 4), where total number of proliferating cells in WT hearts was considered to be 100%. Note the significant decrease in proliferating cells in cKO heart. e qRT-PCR analyses of ventricular mRNA levels of the indicated genes in P3 WT and cKO mice, and mRNA levels of ribosomal 18S was used as control (n = 4). f qRT-PCR analyses of mRNA levels of the indicated genes in control- and si-FoxO1-transfected NRVM with or without T4 treatment (n = 3 independent experiments). In all panels, data are depicted as mean ± SEM. *p < 0.05 vs control; **p < 0.01 vs control; #p < 0.001 vs control. Statistical analyses were conducted using a two-tailed, unpaired Student’s t-test.

Fig 4: Activation of FoxO1 in early post-TAC heart is required for reciprocal regulation of Dio2 and Dio3 genes expression.a qRT-PCR analyses of mRNA levels of indicated genes in 4 day TAC- and sham-operated LVs (n = 4). b Immunoblotting (left) and quantitation (right) of total-FoxO1 (T-FoxO1) and phospho-FoxO1 (p-FoxO1) levels in 4 days TAC- and sham-operated LVs (n = 3). GAPDH was used as a loading control. Average of p-FoxO1/T-FoxO1 and p-FoxO1/GAPDH ratios were used to determine relative p-FoxO1 levels. c, d qRT-PCR analyses of mRNA levels of Dio2 (c) and Dio3 (d) in 4 days TAC- and sham-operated LVs of FoxO1-WT (WT, n = 12) and FoxO1-cKO (cKO, n = 12) mice. e Quantitative ChIP assays were conducted in 4 day sham- and TAC-operated ventricles of the WT mice (n = 3). Note the significant increase in FoxO1 occupancy at the Dio2 promoter in TAC hearts. Schematic of Dio2 illustrates that PCR amplification was performed using primers spanning the two FoxO-responsive elements (FREs) in the Dio2 promoter. f Schematic of Dio2 luciferase vector harboring two FREs. Note that co-transfection of a constitutively active FoxO1 (caFoxO1) elicited significant, concentration-dependent activation of reporter activity harboring WT, but not mutated, FREs. In all panels, data are depicted as mean ± SEM. *p < 0.05 vs sham/control; **p < 0.01 vs sham; ***p < 0.0001 vs sham, #p < 0.05 vs WT (f); #p < 0.001 vs control (c). Statistical analyses were conducted using a two-tailed, unpaired Student’s t-test.

Fig 5: A-FABP enhances energy expenditure and BAT recruitment in A-FABP KO mice.Male 4-week-old A-FABP KO mice fed with HFD for 4 weeks were infused with PBS (vehicle), recombinant A-FABP (rA-FABP, 1 μg h−1) or A-FABP mutant R126Q (1 μg h−1) for 14 days with or without subjected to cold exposure (6 °C). (a) Circulating rA-FABP level and (b) oxygen consumption (VO2) of A-FABP KO mice before or after infusion of recombinant proteins (n=6). (c) Mean VO2 of above A-FABP KO mice measured after infusion of recombinant proteins for 3 days (n=6). (d) Rectal temperature of above A-FABP KO mice infused with rA-FABP or R126Q during cold exposure (6 °C) for 8 h. (e) Haematoxylin and eosin staining and IHC staining of UCP-1 in BAT of mice after cold exposure for 8 h, scale bar, 20 μm; with magnification of 400 × . The right panel is the densitometry analysis for UCP-1. Representative images from three independent experiments are shown (n=6). (f) BAT isolated from above mice was subjected to immunoblotting using an antibody against UCP-1, β-tubulin as indicated. The right panel is the band intensity of UCP-1 relative to β-tubulin (n=6). (g) The mRNA abundance of the thermogenic genes PGC-1α, Cidea and Dio2 in BAT isolated from above mice (n=6). CPMA, count per minutes for beta particles. N.D., not detected. Uncropped western blot images are shown in Supplementary Fig. 13. Data are represented as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, $rA-FABP versus R126Q, $<0.05; #R126Q versus PBS, #P<0.05 (One-way analysis of variance with Bonferroni correction for multiple comparisons).