Hepatic retinol secretion and storage are altered by dietary CLA: common and distinct actions of CLA c9,t11 and t10,c12 isomers

Abstract

Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid obtained from ruminant products. Previous studies in rats and pigs showed that a dietary equimolar mixture of c9,t11 and t10,c12 CLA isomers induces changes in serum and tissue levels of retinoids (vitamin A derivatives). However, the mechanism(s) responsible for these actions remain(s) unexplored. Given the numerous crucial biological functions regulated by retinoids, it is key to establish whether the perturbations in retinoid metabolism induced by dietary CLA mediate some of the beneficial effects associated with intake of this fatty acid or, rather, have adverse consequences on health. To address this important biological question, we began to explore the mechanisms through which dietary CLA alters retinoid metabolism. By using enriched preparations of CLA c9,t11 or CLA t10,c12, we uncoupled the effects of these two CLA isomers on retinoid metabolism. Specifically, we show that both isomers induce hepatic retinyl ester accumulation. However, only CLA t10,c12 enhances hepatic retinol secretion, resulting in increased serum levels of retinol and its specific carrier, retinol-binding protein (RBP). Dietary CLA t10,c12 also redistributes retinoids from the hepatic stores toward the adipose tissue and possibly stimulates hepatic retinoid oxidation. Using mice lacking RBP, we also demonstrate that this key protein in retinoid metabolism mediates hepatic retinol secretion and its redistribution toward fat tissue induced by CLA t10,c12 supplementation.

Fig. 1.

Effects of dietary CLA on body weight and composition in wild-type and RBP^−/− mice. A: Percent change in body weight (10 weeks vs. 14 weeks); n = 4 to 6 mice/group. B: Percent body fat mass. C: Percent body lean mass. Body composition was measured in three mice per group by a Lunar PIXImus Densitometer Dexa analysis at 14 weeks of age, prior to euthanasia. Statistical analysis was performed by ANOVA test. * indicates statistically significant differences between treatment and control groups within the wild-type strain (P < 0.05). # indicates statistically significant differences between treatment and control groups within the RBP^−/− strain (P < 0.05). Results are expressed as mean ± SE in panel A and as mean ± SD in panels B and C.

Fig. 2.

Serum RBP and TTR protein levels upon dietary CLA intake. A: A representative Western blot analysis of serum RBP and TTR levels in wild-type mice fed the control diet or the CLA isomers, as indicated at the bottom of the panels. The molecular weight of each detected protein is indicated on the left side. Quantification of (B) serum RBP levels in wild-type mice, (C) serum TTR levels in wild-type mice, and (D) serum TTR levels in RBP knockout mice (RBP^−/−). Quantification of Western blots was performed by densitometry of the bands detected upon Western analysis. Albumin (Alb) was used as a loading control, as described in Materials and Methods. A reference sample was loaded on each gel to allow for normalization between gels. Results (expressed as mean ± SD) are shown as a fold change in respect to wild-type mice on the control diet. Statistical analysis was performed by ANOVA. * indicates statistically significant differences between treatment and control group (P < 0.05). Five to six mice per group were analyzed at 14 weeks of age.

Fig. 3.

Effects of dietary CLA on tissue retinoid metabolism in wild-type mice. A: RBP and (B) TTR protein levels in the liver of wild-type mice. Analysis was performed by Western blot followed by densitometry. Albumin was used as a loading control. Results (expressed as mean ± SD) are shown as fold change from the reference (wild-type mice on the control diet). C: Retinol and (D) retinyl ester levels in the liver of wild-type mice. Retinoid concentrations were measured by reverse-phase HPLC and expressed as μg/g of tissue (mean ± SD). Hepatic mRNA levels of (E) LRAT, (F) Cyp26A1, and (G) Cyp2C39 in wild-type mice. Measurements were performed by RT real-time PCR analysis. Values are expressed as mean ± SE using the 2^−ΔΔCT. H: Retinol and (I) retinyl ester levels in the adipose tissue of wild-type mice. Retinoid concentrations were measured by reverse-phase HPLC and expressed as μg/g of tissue. J: RBP protein levels in adipose tissue of wild-type mice. Analysis was performed by Western blot followed by densitometry. Actin was used as a loading control. Results (expressed as mean ± SD) are shown as a fold change from the reference (wild-type mice on the control diet). Statistical analysis for all parameters was performed by ANOVA; n = 4 to 6 mice per group, except in (H) and (I) where n = 3. *P < 0.05 versus the wild-type on the control diet.

Fig. 4.

Effects of dietary CLA on retinoid metabolism in RBP knockout mice (RBP^−/−). A: Hepatic mRNA levels of LRAT measured by RT real-time PCR analysis. Values are expressed as mean ± SE using the 2^−ΔΔCT. B: Retinol and (C) retinyl ester levels in the liver of RBP^−/− mice. Retinoid concentrations were measured by reverse-phase HPLC and expressed as μg/g of tissue (mean ± SD). D: Hepatic mRNA levels of Cyp26A1 and Cyp2c39 in wild-type and RBP^−/− maintained on the control diet. Measurements were performed by RT real-time PCR analysis. Values are expressed as mean ± SE using the 2^−ΔΔCT. E: Cyp26A1 and (F) Cyp2c39 hepatic mRNA levels in RBP^−/− mice. Measurements were performed by RT real-time PCR analysis. Values are expressed as mean ± SE using the 2^−ΔΔCT. G: Retinol and (H) retinyl ester in adipose tissue of RBP^−/− mice. Retinoid concentrations were measured by reverse-phase HPLC and expressed as μg/g of tissue (mean ± SD). Statistical analysis for all parameters was performed by ANOVA. * P < 0.05 versus RBP^−/− mice on the control diet; ¶ P < 0.05 versus wild-type on control diet; and # P < 0.05 versus RBP^−/− mice on the CLA c9,t11 diet; n = 4 to 6 mice per group, except in (G) and (H) where n = 3.