Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT)

Abstract

Lecithin:retinol acyltransferase (LRAT) is believed to be the predominant if not the sole enzyme in the body responsible for the physiologic esterification of retinol. We have studied Lrat-deficient (Lrat-/-) mice to gain a better understanding of how these mice take up and store dietary retinoids and to determine whether other enzymes may be responsible for retinol esterification in the body. Although the Lrat-/- mice possess only trace amounts of retinyl esters in liver, lung, and kidney, they possess elevated (by 2-3-fold) concentrations of retinyl esters in adipose tissue compared with wild type mice. These adipose retinyl ester depots are mobilized in times of dietary retinoid insufficiency. We further observed an up-regulation (3-4-fold) in the level of cytosolic retinol-binding protein type III (CRBPIII) in adipose tissue of Lrat-/- mice. Examination by electron microscopy reveals a striking total absence of large lipid-containing droplets that normally store hepatic retinoid within the hepatic stellate cells of Lrat-/- mice. Despite the absence of significant retinyl ester stores and stellate cell lipid droplets, the livers of Lrat-/- mice upon histologic analysis appear normal and show no histological signs of liver fibrosis. Lrat-/- mice absorb dietary retinol primarily as free retinol in chylomicrons; however, retinyl esters are also present within the chylomicron fraction obtained from Lrat-/- mice. The fatty acyl composition of these (chylomicron) retinyl esters suggests that they are synthesized via an acyl-CoA-dependent process suggesting the existence of a physiologically significant acyl-CoA:retinol acyltransferase.

FIGURE 1. CRBPIII but not CRBPI levels are elevated in adipose tissue from 3-month-old Lrat^−/− mice.

Western blots for CRBPI in the liver and epididymal adipose tissue and for CRBPIII in epididymal adipose tissue for two male wild type (+/+) and two male Lrat^−/− (−/−) mice are shown.

FIGURE 2. Effects of a totally retinoid-deficient diet on adipose tissue and liver levels of total retinol (retinol + retinyl ester) for wild type (+/+) and Lrat^−/− (−/−) mice

Panel A shows the levels of total retinol (retinol + retinyl esters) in perigonadal adipose tissue obtained from male and female wild type and Lrat^−/− mice. The groups receiving the control diet are indicated by light gray bars and the groups receiving the retinoid-deficient diet are indicated by dark gray bars. Panel B shows the levels of total retinol (retinol + retinyl esters) in the livers of male and female wild type and Lrat^−/− mice receiving a control diet (light gray bars) or a retinoid-deficient diet (dark gray bars). All mice were maintained on a chow diet through the first 3 months of age, and at this point they were then randomly assigned to groups and maintained for 4 additional weeks on either the same chow diet or a totally retinoid-deficient purified diet.

FIGURE 3. Reverse phase HPLC profiles showing the distribution of retinol and retinyl esters and ³H counts/min present in chylomicrons obtained from wild type (panel A) and Lrat^−/− (panel B) mice following administration of a gavage dose of retinol (6 μg containing 2 × 10^{6 3}H counts/min) in peanut oil

Panels A and B, the upper profiles show the distribution of [³H]retinoids and the lower profiles the UV absorbance of the retinoids. Note that for panels A and B the lower profiles are scaled to reflect at full scale the same absorbance units (AU). The extracted retinoids were separated on a 5-μm 4.6 × 250-mm Ultrasphere C₁₈ column preceded by a C₁₈ guard column, using 70% acetonitrile, 15% ethanol, 15% methylenechloride as the running solvent flowing at 1.8 ml/min. The numbers above the HPLC peaks indicate the following: 1, retinol; 2, retinyl linoleate; 3, retinyl oleate; 4, retinyl palmitate; and 5, retinyl stearate.

FIGURE 4. CRBPIII- but not CRBPI-bound retinol enables human DGAT1 catalysis of acyl-CoA-dependent esterification of retinol

Microsomes isolated from HEK293 cells transfected with a cDNA encoding human DGAT1 were incubated either in the presence of 16 μm free all-trans-retinol (panel A) or 16 μm all-trans-retinol bound to CRBPI (panel B) or 16 μm all-trans-retinol bound to CRBPIII (panel C) for 10 min. The HPLC peak identified with the arrow is that of all-trans-retinyl palmitate formed upon DGAT1 action.

FIGURE 5. The hepatic stellate cells of Lrat^−/− mice lack lipid droplets that are a morphologic hallmark of these cells

Panel A shows the immunohistochemical staining pattern for desmin, a marker for hepatic stellate cells, in liver sections from 3-month-old male wild type (+/+) and Lrat^−/− (−/−) mice. Panel B shows electron micrographs of liver sections prepared from 3-month-old male wild type and Lrat^−/− mice. The electron micrographs show the presence of characteristic retinyl ester-containing lipid droplets in hepatic stellate cells of wild type mice (−/−) and their absence in livers from Lrat^−/− mice (−/−). The arrows indicate the presence (+/+) or absence (−/−) of lipid droplets in hepatic stellate cells. The large adjoining cells are hepatocytes.

FIGURE 6. RBP levels are elevated in the livers of Lrat^−/− mice but not wild type mice fed a totally retinoid-deficient diet for 1 month

Panel A shows RBP concentrations in livers from male and female wild type (+/+) and Lrat^−/− (−/−) mice receiving either a nutritionally complete control diet (light gray bars) or a totally retinoid-deficient diet (dark gray bars). Panel B shows RBP concentrations in serum from male and female wild type (+/+) and Lrat^−/− (−/−) mice receiving either a nutritionally complete control diet (light gray bars) or a totally retinoid-deficient diet (dark gray bars). Panel C shows serum retinol concentrations for male and female wild type (+/+) and Lrat^−/− (−/−) mice receiving either a nutritionally complete control diet (light gray bars) or a totally retinoid-deficient diet (dark gray bars).