Farnesoid X receptor and bile acids regulate vitamin A storage

Abstract

The nuclear receptor Farnesoid X Receptor (FXR) is activated by bile acids and controls multiple metabolic processes, including bile acid, lipid, carbohydrate, amino acid and energy metabolism. Vitamin A is needed for proper metabolic and immune control and requires bile acids for efficient intestinal absorption and storage in the liver. Here, we analyzed whether FXR regulates vitamin A metabolism. Compared to control animals, FXR-null mice showed strongly reduced (>90%) hepatic levels of retinol and retinyl palmitate and a significant reduction in lecithin retinol acyltransferase (LRAT), the enzyme responsible for hepatic vitamin A storage. Hepatic reintroduction of FXR in FXR-null mice induced vitamin A storage in the liver. Hepatic vitamin A levels were normal in intestine-specific FXR-null mice. Obeticholic acid (OCA, 3 weeks) treatment rapidly reduced (>60%) hepatic retinyl palmitate levels in mice, concurrent with strongly increased retinol levels (>5-fold). Similar, but milder effects were observed in cholic acid (12 weeks)-treated mice. OCA did not change hepatic LRAT protein levels, but strongly reduced all enzymes involved in hepatic retinyl ester hydrolysis, involving mostly post-transcriptional mechanisms. In conclusion, vitamin A metabolism in the mouse liver heavily depends on the FXR and FXR-targeted therapies may be prone to cause vitamin A-related pathologies.

Figure 1

FXR deficiency impairs hepatic retinoid storage. Whole body FXR-null mice and age-match wild-type control mice were analyzed for (A) H&E and oil red O staining, which revealed fat accumulation in livers of FXR-null mice as compared to control mice, while autofluorescence analysis revealed reduced vitamin A levels in livers of FXR-null mouse. (B) The body weight was not altered FXR-null mice, while the liver weight was increased in FXR-null mice. As expected, hepatic expression of Plin2, Fasn and Acc1 were increased in FXR-null mice vs wild-type mice. (C) hepatic retinyl palmitate and retinol levels were reduced in FXR-null mice, while plasma retinol levels were equal in both groups. Data are presented as Mean ± SEM and mRNA expression of genes is presented in 2^{−delta CT}, which was normalized to 36B4.

Figure 2

Hepatic and not intestinal FXR is essential for normal retinoid levels in the liver. (A) Hepatic retinyl palmitate and retinol were analyzed in intestinal specific FXR-null mice (iFXR-null) and wild-type littermates. Intestine-specific FXR-deficiency did not alter hepatic vitamin A levels. (B) Whole body FXR-null mice were transduced with ScAAV-produced FXRα2 or FXRα4 or GFP controls and after 4 weeks analyzed for hepatic retinyl palmitate and retinol levels. Both FXR-isoform enhanced hepatic retinoid levels, suggesting that hepatic FXR plays a crucial role in vitamin A storage and metabolism.

Figure 3

Post-transcriptional reduction of LRAT in FXR-null mice. Whole body FXR-null and wild type mice were analyzed for (A) hepatic mRNA expression of Lrat, Dgat1, Dgat2, Pnpla2 (Atgl), Pnpla3, Lipe (Hsl). Hepatic Lrat and Pnpla3 mRNA levels were increased in FXR-null mice, with no change in other vitamin A metabolizing enzymes as compared to control. Western blot analysis (B) and immunohistochemical staining (C) for LRAT revealed that hepatic protein levels of LRAT were significantly reduced in FXR-null mice as compared to wild-type control mice. Data are presented as Mean ± SEM and mRNA expression of genes is presented in 2^{−delta CT}, which was normalized to 36B4.

Figure 4

OCA treatment reduces hepatic retinyl palmitate, while increasing retinol levels in mouse livers. Control and OCA-treated mice were analyzed for hepatic vitamin A levels, mRNA and protein levels of FXR targets and vitamin A metabolizing factors. (A) The mRNA levels of FXR target genes Nr0b2 (encoding SHP) and Abcb11 (encoding BSEP) were strongly enhanced in livers of OCA-treated mice. (B) Hepatic retinyl palmitate levels were significantly reduced in OCA-treated mice, while hepatic retinol levels were significantly increased as compared to control mice. (C) Hepatic mRNA levels of Lrat, Rbp4 and Pnpla3 were not changed, while levels of Pnpla2 (ATGL) were decreased and Lipe (HSL) were increased in OCA-treated mice. (D) Protein levels of LRAT were not affected by OCA-treatment, while RBP4, ATGL, PNPLA3, HSL (and its active phosphorylated form pHSL), CYP7A1 and NTCP were all decreased and PEPCK was increased. GAPDH was used as loading control. (E) mRNA levels of genes involved in the conversion of retinol into retinoic acid (Raldh1, Raldh2, Raldh4) and of (F) retinoic acid-responsive genes (Cpt1a, Pck1, Ppargc1a, Cyp26a1, Ucp2, Fgf21) were enhanced in OCA-treated mice as compared to control animals. Data are presented as Mean ± SEM and mRNA expression of genes is presented in 2^{−delta CT}, which was normalized to 36B4.

Figure 5

Cholic acid (0.1%) treatment delays the accumulation of hepatic retinoids in mice. Mice were fed chow or a CA (0.1%)-containing diet for 4, 8 and 12 weeks. (A) Hepatic retinyl palmitate was significantly decreased CA-fed treated mice after 12 weeks as compared to control mice. (B) Hepatic and serum retinol levels did not differ at any time point between both groups of mice. (C) Hepatic mRNA levels FXR-responsive genes Nr0b2 (SHP) and Abcb11 (BSEP) were moderately increased in CA-treated animals compared to control mice. Data are presented as Mean ± SEM and mRNA expression of genes is presented in 2^{−delta CT}, which was normalized to 36B4.