In vitro and in vivo characterization of retinoid synthesis from beta-carotene

Abstract

Retinoids are indispensable for the health of mammals, which cannot synthesize retinoids de novo. Retinoids are derived from dietary provitamin A carotenoids, like beta-carotene, through the actions of beta-carotene-15,15'-monooxygenase (BCMO1). As the substrates for retinoid-metabolizing enzymes are water insoluble, they must be transported intracellularly bound to cellular retinol-binding proteins. Our studies suggest that cellular retinol-binding protein, type I (RBP1) acts as an intracellular sensor of retinoid status that, when present as apo-RBP1, stimulates BCMO1 activity and the conversion of carotenoids to retinoids. Cellular retinol-binding protein, type II (RBP2), which is 56% identical to RBP1 does not influence BCMO1 activity. Studies of mice lacking BCMO1 demonstrate that BCMO1 is responsible for metabolically limiting the amount of intact beta-carotene that can be absorbed by mice from their diet. Our studies provide new insights into the regulation of BCMO1 activity and the physiological role of BCMO1 in living organisms.

Figure 1

Effects of cellular retinol binding proteins on BCMO1 activity. (A) Purified human BCMO1, RBP1 and RBP2 were analyzed on SDS-PAGE and stained with Coomassie. (B) Purified human BCMO1 (2.6 μg) was incubated with 15 μM β-carotene in our standard assay conditions in the presence of varying concentrations of RBP1, RBP2 or BSA as indicated. The production of all-trans-retinal from β-carotene was measured by HPLC.

Figure 2

Effects of pH on hRalR1 activity. Human RalR1 activity was measured at three different pHs as indicated. Microsomes (90 μg protein) prepared from RalR1/CHO cells were incubated with 5 μM retinal-RBP2 as a substrate and 2 mM NADPH as a cofactor in buffers of three different pH for 1 h at 37 °C, and the production of retinol was measured by HPLC.

Figure 3

Effects of RBP2 on human RalR1. (A) Microsomes (108 μg protein) prepared from RalR1/CHO cells were incubated at 37 °C for 1 h with various concentrations of either free- or RBP2 bound-retinal (holo-RBP2) as indicated. Production of retinol was measured by HPLC. (B) In the above assay conditions, increasing concentrations of apo-RBP2 was added to a constant level of free retinal (2 μM), and the production of retinol was measured by HPLC.

Figure 4

Effects of apo-RBP1 and apo-RBP2 on human LRAT activity. Microsomes (50–100 μg protein) prepared from LRAT/CHO cells were incubated with 2 μM holo-RBP1 (A) or holo-RBP2 (B) for 30 min in the absence or presence of apo-RBP 1 or 2 as indicated. X-axis represents the mole to mole ratio of apo− to holo-cellular retinol binding proteins at the start of the reaction (0 represents absence of apo-RBP 1 or 2). □, apo-RBP1; ■, apo-RBP2

Figure 5

Plasma retinol and liver retinyl esters for wild type, heterozygous and homozygous BCMO1-deficient mice. The bar graphs provide the mean ± SD from 4–5 animals/group. Baseline (0 weeks on diet) values are for 6 week-old mice. The β-carotene diet (100 μg β-carotene/g diet) was provided for 4 or 7 weeks starting at 6 weeks-of-age. One-way ANOVA followed by post-hoc analysis (Bonferoni) was performed to determine whether β-carotene diet changes retinoid levels significantly in different tissues for each genotype. *, p<0.05. Black bar, wild type mice (WT); gray bar, heterozygous mice; white bar, homozygous BCMO1-deficient mice (KO).

Figure 6

Lung and intestinal retinyl esters levels for wild type, heterozygous and homozygous BCMO1-deficient mice. The bar graphs provide the mean ± SD from 4–5 animals/group. Baseline (0 weeks on diet) values are from 6 week-old mice. The β-carotene diet (100 μg β-carotene/g diet) was provided for 4 or 7 weeks starting at 6 weeks-of-age. One-way ANOVA followed by post-hoc analysis (Bonferoni) was performed to determine whether β-carotene diet consumption changes significantly retinoid levels in different tissues for each genotype. *, P<0.05; **, P<0.01. Black bar, wild type mice (WT); gray bar, heterozygous mice; white bar, BCMO1-deficient mice (KO).

Figure 7

Retinyl ester concentrations in testes, ovaries, and perigonadal fat pads for wild type, heterozygous and homozygous BCMO1-deficient mice. The bar graphs provide the mean ± SD from 4 animals/group. Baseline (0 weeks on diet) values are from 6 week-old mice. β-carotene diet (100 μg β-carotene/g diet) was provided for 4 or 7 weeks starting at 6 weeks-of-age. One-way ANOVA followed by post-hoc analysis (Bonferoni) was performed to determine whether β-carotene diet changes significantly retinoid levels in different tissues for each genotype. **, P<0.01; ***, P<0.001. Black bar, wild type mice WT; gray bar, heterozygous mice; white bar, BCMO1-deficient mice (KO).

Figure 8

Retinyl ester concentrations in retroperitoneal and inguinal fat pads for wild type, heterozygous and homozygous BCMO1-deficient mice. The bar graphs provide mean ± SD for 4 animals/group. Baseline (0 weeks on diet) values are from 6 weeks-old mice. β-carotene diet (100 μg β-carotene/g diet) was provided for 4 or 7 weeks starting at 6 weeks-of-age. *, P<0.05; **, P<0.01. Black bar, wild type mice (WT); gray bar, heterozygous mice; white bar, BCMO1-deficient mice (KO).