Figure 1.
Serum and inguinal white adipose tissue levels of β-carotene, retinoids and apocarotenoids.
(A, B) β-carotene -15, 15′-monoxygenase (Bcmo1) and β-carotene 9′, 10′-dioxygenase (Bcdo2) mRNA levels in inguinal white adipose tissue (iWAT) of wild-type (WT) and Bcmo1-null (Bcmo1-/-) mice after 14 weeks on a control diet (open bars) or a β-carotene-enriched diet (black bars). Quantitative real-time PCR was used to determine normalized gene expression levels. (C-G) β-carotene, retinol and 10′ β-apocarotenol levels in serum and iWAT of WT and Bcmo1-/- mice after 14 weeks on a control diet (open bars) or a β-carotene-enriched diet (black bars). β-carotene, retinol and β-10′-apocarotenol were determined by HPLC (see Materials and Methods). Total retinol refers to free retinol plus retinyl esters. Data in (A to G) are the mean ± SEM of 6 animals per group; n.d. non-detectable; GxD, interaction between genotype and diet in two-way ANOVA analysis (p<0.05); G, genotype effect in two-way ANOVA analysis (p<0.05); *, p<0.05 in Student's t test, BC diet versus control diet.
Figure 2.
β-10′-apocarotenol is the major β-carotene cleavage product in Bcmo1-/- mice.
HPLC-profile at 420 nm of (A) the synthetic β-10′-apocarotenol standard and (B) a lipid extract of iWAT of BC supplemented Bcmo1-/- mice. Insets show the spectral characteristics of the β-10′-apocarotenol standard (peak 1) as compared to peak 1′. (C, D) Single extracted ion chromatograms for β-10′-apocarotenol standard (m/z = 361.42; peak 1 and 1′). (E, F) Fragmentation pattern of a parent ion selected at m/z = 361.42 are identical for the β-10′-apocarotenol standard (peak 1) and peak 1′.
Figure 3.
Supplemetation of β-carotene alters body adiposity in wild-type mice.
(A) Effects of dietary β-carotene supplementation on body weight gain, (B) cumulative energy intake, (C) energy efficiency, (D) adiposity index, (E) adipose depot weight, and (F) inguinal adipocyte mean sectional area, in wild-type (WT) and Bcmo1-null (Bcmo1-/-) mice. In (G), micrographs of sections of inguinal fat in the four groups are shown (The scale bar gives 100 µm). Data in (A to E) are the mean±SEM of 6 animals per group, and data in (F) of 3 animals per group Open bars, animals on the control diet; black bars, animals on the β-carotene-enriched diet. Body weight gain, cumulative energy intake and energy efficiency over the entire 14 week experimental period are shown. Adiposity index corresponds to the sum of all entirely dissected white adipose tissue depots (gonadal, Gon; inguinal, Ing; and retroperitoneal, Retr), expressed as percentage of the animal body weight (BW).. G, genotype effect in two-way ANOVA analysis (p<0.05); D, effect of diet in two-way ANOVA analysis (p<0.05);*, p<0.05 in Student's t test, BC diet versus control diet.
Table 1.
Fed blood parameters in wild-type and Bcmo1-/- mice following control and β-carotene enriched diet.
Figure 4.
Supplementation of β-carotene alters gene expression profiles in wild-type but not in Bcmo1-/- mice.
(A) Principal component analysis of transcriptional profiles in inguinal white adipose tissue of female wild-type (circles) and Bcmo1-/- (squares) mice fed the control diet (open symbols) or the β-carotene diet (closed symbols). Genome-wide expression profiles were obtained using the 4×44 k Agilent whole mouse genome microarrays for each animal in the experiment (n = 6 per group). (B) Venn diagram representing the number of significantly regulated genes (p<0.01) due to BC supplementation in wild-type (WT) and in Bcmo1-null (Bcmo1-/-) mice and the number of significantly regulated genes regardless of genotype (overlap). (C) Volcano plot representing the effect of BC supplementation on gene expression in WT mice, with the fold-change on the x-axis and the corresponding Student's t-test p-value on the y-axis. Every spot is a single gene and in black are all genes with a p-value <0.01. (D) Heat map representing the expression level of all genes regulated by BC with a Student's t-test p<0.01 in the WT mice in all four groups; WT control (Co) diet, WT BC diet, Bcmo1-/- control diet and Bcmo1-/- BC diet. Relative expression of every single gene was compared to gene expression in WT mice on the control diet and consequently, gene expression of every single gene in WT mice on the control diet was set to 1.0.
Figure 5.
β-carotene supplementation reduces PPARγ expression both on the mRNA and protein level.
(A) PPARγ mRNA levels, (B) RXRα mRNA levels, (C) lipoprotein lipase (LPL) mRNA levels, (D) PPARγ protein levels and (E) Cyp26a1 mRNA levels in inguinal white adipose tissue of wild-type (WT) and Bcmo1-null (Bcmo1-/-) mice after 14 weeks on a control diet (open bars) or a β-carotene -enriched diet (black bars). Quantitative real-time PCR was used to determine normalized gene expression levels as described in Materials and Methods. Immunoblotting was used to determine expression levels of PPARγ and β-actin, which was used as internal control for equal loading and blotting; shown at the bottom of (D) is a representative immunoblot for the two proteins. Data in (A to E) are the mean ± SEM of 6 animals per group. G, effect of genotype in two-way ANOVA analysis (p<0.05); *, p<0.05 in Student's t test, BC diet versus control diet.
Table 2.
Batch promoter analysis focused on peroxisome proliferator-activated receptor (PPAR) of the fifty top down-regulated genes in inguinal white adipose tissue of wild-type mice after β-carotene supplementation.
Table 3.
Composition of the control and beta-carotene (BC) diets.