Apo-14´-Carotenoic Acid Is a Novel Endogenous and Bioactive Apo-Carotenoid

Abstract

Carotenoids can be metabolized to various apo-carotenoids and retinoids. Apo-15´-carotenoic acid (retinoic acid, RA) is a potent activator of the retinoic acid receptor (RAR) in its all-trans- (ATRA) and 9-cis- (9CRA) forms. In this study we show firstly, that apo-14´-carotenoic acid (A14CA), besides retinoic acids, is present endogenously and with increased levels in the human organism after carrot juice supplementation rich in β-carotene. All-trans-A14C (ATA14CA) is just a moderate activator of RAR-transactivation in reporter cell lines but can potently activate retinoic acid response element (RARE)-mediated signalling in DR5/RARE-reporter mice and potently increase retinoid-reporter target gene expression in ATA14CA-supplemented mice and treated MM6 cells. Further metabolism to all-trans-13,14-dihydroretinoic acid (ATDHRA) may be the key for its potent effects on retinoid target gene activation in ATA14CA-treated MM6 cells and in liver of supplemented mice. We conclude that besides RAs, there are alternative ways to activate RAR-response pathways in the mammalian organism. ATA14CA alone and in combination with its metabolite ATDHRA may be an alternative pathway for potent RAR-mediated signalling.

Keywords: apo-carotenoids; carotene; retinoic acid; vitamin A.

Figure 1

Apo-14´-carotenoic acid (A14CA) is an endogenous occurring apo-carotenoid. (A) Representative structural formula of all-trans retinoic acid (ATRA) and all-trans-A14CA (ATA14CA). (B) Representative chromatograms showing a co-elution of the all-trans-isomer of a photo-isomerized standard solution from ATA14CA with a peak present in human serum after supplementation with carrot juice obtaining comparable retention time of 14.1 min marked by a dashed lined box, additional unknown peaks were marked with dashed line arrows. The peaks eluting before ATA14CA obtain similar UV spectra like ATA14CA and are likely geometric isomers of ATA14CA, while the unknown peak eluting after ATA14CA obtains an UVmax of 434 nM and is likely a hydrogenation metabolite of ATA14CA. (C) UV spectra of the peaks eluting at 14.1 min. originating from human serum after supplementation with carrot juice and ATA14CA standard compound obtaining an UVmax of 378 nm. (D) Representative chromatograms from human serum after supplementation of a carotenoid free diet and after supplementation with carrot juice focusing on ATA14CA eluting at 14.1 min and marked by a dashed line box.

Figure 2

All-trans-apo-14´-carotenoic acid (ATA14CA) induces retinoic acid response element (RARE)-mediated signalling and RAR-target gene expression in RARE/DR5-luciferase reporter animals. (A) RARE/DR5-luc bioimaging of liver, spleen, lung, white adipose tissue (WAT), and intestine from male and female mice. These animals were treated with CTRL (vehicle treatment), all-trans retinoic acid (ATRA) (50 mg/kg bw), or ATA14CA (equimolar to ATRA-treatment with 54.3 mg/kg bw). (B) Quantification of the RARE/DR5-luc bioimaging data. (C) Quantitative real time-polymerase chain reaction (QRT-PCR) based analysis of the retinoid target genes CYP26A1 and CYP26B1 in liver of male and female mice. Significant values were marked with a *, when p ≤ 0.05 vs. CTRL. (D) Representative chromatograms showing increased concentrations of ATDHRA (identified using MS-MS of 303–123 m/z and marked by a dashed lined box) in mouse serum after CTRL, ATRA, and ATA14CA treatments.

Figure 3

All-trans-apo-14´-carotenoic acid (ATA14CA) transactivates RAR- and RXR-mediated signalling and retinoid target gene expression. (A) Transcriptional activation of RAR-RXR heterodimer by ATRA and ATA14CA in RARα-RXRα reporter HEK cells. (B) RAR-antagonist AGN168109 (10⁻⁵ M) diminishes ATA14CA (10⁻⁶ M) induced RARα-mediated signalling (p ≤ 0.05 indicated by the black line over the bars). (C) Transcriptional activation of RXR heterodimer by 9CRA and ATA14CA in RXRα-reporter HEK cells. (D) RXR-antagonist LG100754 (10⁻⁵ M) diminishes ATA14CA (10⁻⁶ M) induced RXRα-mediated signalling (p ≤ 0.05 indicated by the black line over the bars). (E) Synergistic activation of ATDHRA (10⁻⁵ M) and ATA14CA (10⁻⁵ M) in RARα-reporter HEK cells (p ≤ 0.05 indicated by the black line over the bars). (F) ATRA and ATA14CA induce expression of the RAR target gene TG2 in treated MM6 cells. (G) ATRA and ATA14CA induce expression of the RXR/PPAR target gene ADRP in treated MM6 cells. # ATRA at concentrations of 10⁻⁵ M was cytotoxic. Significant values were marked with a *, when p ≤ 0.05 vs. CTRL.

Figure 4

Mechanisms of all-trans-apo-14´-carotenoic acid (ATA14CA) mediated RAR-signalling. Scheme of retinoid and β-carotene metabolism yielding bioactive RAR-activating retinoids. Black arrows represent metabolic pathways and dashed line interaction of ligands with the nuclear hormone receptor RAR. Abbreviations: All-trans-β-carotene (ATBC), all-trans retinoic acid (ATRA), retinyl-esters (RET-ester), retinol (ROL), retinoic acid receptor (RAR), all-trans-13,14-dihydroretinoic acid (ATDHRA), retinol dehydrogenase (RDH), retinaldehyde dehydrogenase (ALDH1A1/2/3), beta-carotene oxygenase 1 (BCO1), beta-carotene oxygenase 2 (BCO2).