Maternal Retinoids Increase PDGFRα+ Progenitor Population and Beige Adipogenesis in Progeny by Stimulating Vascular Development

Figures

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  Fig. 1. Maternal vitamin A supplementation affects adipose tissue deposition and morphology. Adult C57BL/6 females during gestation and lactation were supplemented with 0 or 30 IU/ml vitamin A through water (designated as MVA experiment). (a) Body weight of offspring at birth and weaning. (b) Adipose tissue weight. (c) iWAT and BAT density. (d) Average number of nuclei per section. (e) Distribution of adipocytes size in iWAT. (f) Representative images of H&E stained iWAT. (g) Representative images of H&E stained BAT. (h) TA muscle weight. (i) Body length. Data presented are mean ± SEM, n = 6, unpaired two-tail t-test, *p < 0.05.

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  Fig. 2. Maternal vitamin A supplementation prevents offspring obesity induced by high fat diet. MVA offspring were fed a high fat diet (HFD, 45% energy from fat) from 30 days to 155 days old. (a) Growth curve of offspring fed with HFD. (b) Representative images of mice after HFD feeding. (c) Representative images of iWAT, eWAT and BAT tissues after HFD feeding. (d) Tissue weight/body weight ratio. (e) Representative images of fat tissue sections, stained by H&E. (f) Distribution of adipocyte sizes in iWAT. (g) Distribution of adipocyte sizes in eWAT. (h) Glucose tolerance test of offspring HFD feeding. (i–m) Oxygen consumption (i and j), CO2 production (k) and heat production (l and m) were measured in a metabolic chamber. Data presented are mean ± SEM, n = 6, unpaired two-tail t-test, *p < 0.05.

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  Fig. 3. Maternal vitamin A supplementation promotes brown/beige adipogenesis in both white and brown adipose tissues. MVA offspring were fed a high fat diet (HFD, 45% energy from fat) from 30 days to 155 days old. (a–b) Immunohistochemistry (IHC) images of iWAT (a) and BAT (b) using anti-UCP1 antibody. (c–e) Brown adipose protein contents in iWAT (c), eWAT (d) and BAT (e) analyzed by western blot. (f–h) Brown adipose gene mRNA levels in iWAT (f, eWAT (g) and BAT (h) analyzed by qRT-PCR. (i–j) Thermal images of MVA treated offspring at 9 days old were captured by a thermal camera (i), the average surface temperature was analyzed (j). (k) Core body temperature. (l) Relative oxygen consumption of SVCs isolated from iWAT of control and MVA offspring. Panel f–h and k–l: n = 6, panel j: n = 3. Data presented are mean ± SEM, unpaired two-tail t-test, *p < 0.05.

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  Fig. 4. Retinoic acid signaling is required for fetal adipose tissue development. (a) Retinoid levels in mother mice. (b) Retinoid levels in E18.5 fetuses (whole tissue lysate). (c) Retinoid levels in weanling offspring. (d–h) Pregnant mice were injected with BMS493 or RA at E10.5 and 13.5, one BMS493 treated group was supplemented with vitamin A during gestation. Samples were collected at weaning. Body weight (d) and adipose tissue weight (e) of offspring was measured at weaning. Adipose tissue sections were stained by H&E (f). Expression of brown adipose genes were analyzed by qRT-PCR (g) or western blot (h). (i–n) RARαDN expression was conditionally induced in PDGFRα+ cells at E10.5 by an injection of 20 mg/kg BW tamoxifen to the pregnant mothers (i). Offspring were euthanized at weaning, and body weight (j) and adipose tissue weight (k) were measured. iWAT and BAT tissue sections were stained by H&E (l) and IHC using anti-UCP1 antibody (m). Brown adipose genes expression were analyzed by qRT-PCR (n). Data presented are mean ± SEM, n = 6, unpaired two-tail t-test (a–c, j–k and n) *p < 0.05 or ANOVA (d–e and g), bars with different letters differ significantly.

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  Fig. 5. Maternal retinoic acid promotes vasculature in adipose tissues. (a) Blood vessels in iWAT and BAT stained by DiI. (b) mRNA levels of angiogenic genes in iWAT. (c) mRNA levels of angiogenic genes in BAT. (d) Representative images of E12.5 embryos. (e) mRNA levels of angiogenic genes in E12.5 embryos. (f) mRNA levels of angiogenic genes in iWAT. (g) Blood vessels in iWAT and BAT of RA or BMS493 treated offspring at weaning. Data presented are mean ± SEM, n = 6, unpaired two-tail t-test (b, c and e) *p < 0.05 or ANOVA (f), bars with different letters differ significantly.

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  Fig. 6. Maternal vitamin A increases PDGFRα+ population in adipose tissue. (a–d) For MVA weanling offspring. The population of PDGFRα+ cells in iWAT was analyzed by flow cytometry (a) (numbers in the quadrant indicate the percentages of cells) and IHC using anti-PDGFRα antibody (b). DiI labeled iWAT was further stained using anti-PDGFRα antibody (c). Brown adipose protein contents in iWAT-derived SVCs after 6 days of brown adipogenesis were analyzed (d). (e–f) PDGFRα+ cells in PDGFRα tracking mice were labeled by GFP in presence of tamoxifen (e). PDGFRα+ cells-derived adipocytes in iWAT after 1 week of cold stimulus were observed under confocal microscope (f).

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  Fig. 7. VEGF signaling plays an important role in RA regulation of adipogenesis. (a) Representative PDGFRα and VEGFR2 immunohistochemical staining images of E12.5 embryo treated with MVA. (b) FACS analysis of PDGFRα +/VEGFR2+ cells (CD45+ cells were excluded) in iWAT of maternal RA or BMS493 treated (at E10.5) fetuses at E12.5 (numbers in the circles indicate the percentages of cells). (c) FACS analysis of PDGFRα+/VEGFR2+ cells in iWAT of weanling MVA offspring. (d) VEGFR2 was knocked out in P19 cells, then embryo bodies were formed and pretreated with RA for 3 days and, then, induced brown adipogenesis for 4 days. Prdm16 expression was analyzed by qRT-PCR (n = 3). (e–g) Vegfr2 were knocked out in PDGFRα+ cells at E10.5 by an injection of 20 mg/kg BW tamoxifen to the pregnant mothers. Adipose tissue weight was measured at weaning (e, n = 6). Brown adipose gene expression in iWAT was analyzed by qRT-PCR (f, n = 6). Adipose tissue sections were stained by H&E or IHC using anti-UCP1 antibody (g). Data presented are mean ± SEM, unpaired two-tail t-test, *p < 0.05.