Multiple pathways ensure retinoid delivery to milk: studies in genetically modified mice

Abstract

Retinoids are absolutely required for normal growth and development during the postnatal period. We studied the delivery of retinoids to milk, availing of mouse models modified for proteins thought to be essential for this process. Milk retinyl esters were markedly altered in mice lacking the enzyme lecithin:retinol acyltransferase (Lrat(-/-)), indicating that this enzyme is normally responsible for the majority of retinyl esters incorporated into milk and not an acyl-CoA dependent enzyme, as proposed in the literature. Unlike wild-type milk, much of the retinoid in Lrat(-/-) milk is unesterified retinol, not retinyl ester. The composition of the residual retinyl ester present in Lrat(-/-) milk was altered from predominantly retinyl palmitate and stearate to retinyl oleate and medium chain retinyl esters. This was accompanied by increased palmitate and decreased oleate in Lrat(-/-) milk triglycerides. In other studies, we investigated the role of retinol-binding protein in retinoid delivery for milk formation. We found that Rbp(-/-) mice maintain milk retinoid concentrations similar to those in matched wild-type mice. This appears to arise due to greater postprandial delivery of retinoid, a lipoprotein lipase (LPL)-dependent pathway. Importantly, LPL also acts to assure delivery of long-chain fatty acids (LCFA) to milk. The fatty acid transporter CD36 also facilitated LCFA but not retinoid incorporation into milk. Our data show that compensatory pathways for the delivery of retinoids ensure their optimal delivery and that LRAT is the most important enzyme for milk retinyl ester formation.

Fig. 1.

Lecithin:retinol acyltransferase (LRAT) is normally the predominant enzyme responsible for the formation of milk retinyl esters. Retinoid concentrations are shown for wild-type (n = 9; black bars) and Lrat^−/− (n = 9; gray bars) mice. The figure shows the levels of all-trans retinol; peak 3, retinyl linoleate; peak 4, retinyl oleate; peak 5, retinyl palmitate; peak 6, retinyl stearate. Also shown are peaks a, b, and c, which are unknown retinyl esters found only in milk from Lrat^−/− dams. These peak designations are identical to the ones shown in Fig. 2. All values are shown as the mean ± 1 SD. Statistical significances were determined by t-tests: ^aP < 0.00001, ^bP < 0.001, ^cP < 0.005, ^dP < 0.01, and ^eP < 0.05 compared with wild-type mice.

Fig. 2.

Retinyl esters are the predominant retinoid in milk of wild-type but not Lrat^−/− mice, which contains considerable unesterified retinol. Typical high-performance liquid chromatography (HPLC) profile for 30 μl of milk from wild-type (A) and Lrat^−/− (B) dams maintained on a chow diet was collected at 10–15 days postpartum. HPLC peaks were identified on the basis of retention times and UV-Vis spectra compared with those of known standards. Peak 1, all-trans retinol; peak 2, retinyl acetate (internal standard); peak 3, retinyl linoleate; peak 4, retinyl oleate; peak 5, retinyl palmitate; peak 6, retinyl stearate; peaks a, b, and c are unknowns. Peaks a, b, and c are found only in milk from Lrat^−/− dams. We conclude on the basis of their spectra (see Supplemental Fig. S1) and retention times that these peaks are retinyl esters, probably consisting of medium-chain fatty acyl groups.

Fig. 3.

Lipoprotein lipase and CD36 contribute significantly to the fatty acid (FA) composition of milk triglycerides (TG). Milk was collected from wild-type (black bars; n = 4), CD36^−/− (light gray bars; n = 5), and MCK-LpL0 (dark gray bars; n = 5) mice, and the FA composition of the TG was analyzed. Values represent means ± 1 SD. A: levels of the relatively abundant myristic (14:0), palmitic (16:0), and oleic acids (18:1). B: levels of the less abundant linoleic (18:2), arachidonic (20:4), and docosahexaenoic acids (22:6). Statistical significance: ^aP < 0.05 compared with wild type.

Fig. 4.

Alternate pathways act to maximize retinoid delivery and incorporation into milk. A: results of a diet study where both wild-type (black bars) and retinol-binding protein (Rbp)^−/− (gray bars) dams were placed, at the time of birth of their litters, on either a retinoid-sufficient diet containing 22 IU retinol/g (control), a retinoid-deficient diet (VAD), or a diet containing 220 IU retinol/g (excess). The concentration of retinoid was measured in milk at 10–15 days postpartum (n = 10–18 lactating dams for each diet group and genotype). B: pup hepatic retinoid levels for the different diets. Retinol and retinyl ester levels were determined in the livers of wild-type (black bars) or Rbp^−/− (gray bars) pups at weaning (21 days; n = 10–15 pups/group). Data are provided as means ± 1 SD. Statistical significance: ^aP < 0.00005 compared with all other diet groups; ^bP < 0.0005 compared with wild-type control; ^cP < 0.05 compared with wild-type control diet.

Fig. 5.

Lipase action is needed to assure retinoid incorporation into milk. ³H-cpm/μl milk is given for wild-type (dark bars; n = 6) and Rbp^−/− mice (light bars; n = 5) 2, 4, and 6 h after administration of a gavage dose of [³H]retinol in 50 μl of peanut oil, with (+) and without (−) P-407 administration. Data are shown as means ± 1 SD. Statistical significance: ^aP < 0.005, untreated Rbp^−/− mice different from all other treatment groups at 2 h; ^bP < 0.00001, untreated Rbp^−/− mice different from all other treatment groups at 4 h; ^cP < 0.05 different from wild-type treated; ^dP < 0.05, untreated Rbp^−/− mice different from all other treatment groups at 6 h.