High density lipoprotein metabolism in low density lipoprotein receptor-deficient mice

Abstract

The LDL receptor (LDLR) and scavenger receptor class B type I (SR-BI) play physiological roles in LDL and HDL metabolism in vivo. In this study, we explored HDL metabolism in LDLR-deficient mice in comparison with WT littermates. Murine HDL was radiolabeled in the protein ((125)I) and in the cholesteryl ester (CE) moiety ([(3)H]). The metabolism of (125)I-/[(3)H]HDL was investigated in plasma and in tissues of mice and in murine hepatocytes. In WT mice, liver and adrenals selectively take up HDL-associated CE ([(3)H]). In contrast, in LDLR(-/-) mice, selective HDL CE uptake is significantly reduced in liver and adrenals. In hepatocytes isolated from LDLR(-/-) mice, selective HDL CE uptake is substantially diminished compared with WT liver cells. Hepatic and adrenal protein expression of lipoprotein receptors SR-BI, cluster of differentiation 36 (CD36), and LDL receptor-related protein 1 (LRP1) was analyzed by immunoblots. The respective protein levels were identical both in hepatic and adrenal membranes prepared from WT or from LDLR(-/-) mice. In summary, an LDLR deficiency substantially decreases selective HDL CE uptake by liver and adrenals. This decrease is independent from regulation of receptor proteins like SR-BI, CD36, and LRP1. Thus, LDLR expression has a substantial impact on both HDL and LDL metabolism in mice.

Keywords: cholesteryl ester; scavenger receptor class B type I; selective uptake.

Fig. 1.

FPLC analysis of plasma cholesterol from WT or from LDLR^−/− mice. After fasting for 4 h, blood was harvested from 4 WT or from 4 LDLR^−/− male mice. Finally, the pooled plasma was subjected to FPLC, and cholesterol was analyzed in each fraction as outlined in Materials and Methods. Shown is a representative experiment from a total of n = 2.

Fig. 2.

Plasma decay kinetics of ¹²⁵I-TC-/[³H]CEt-WT-HDL in WT mice or in LDLR^−/− mice. ¹²⁵I-TC-/[³H]CEt-WT-HDL was injected intravenously in a WT or LDLR^−/− male mouse. Thereafter, during a 24 h interval, periodic blood samples were harvested, and plasma was analyzed for ¹²⁵I-TC (¹²⁵I) and [³H]CEt ([³H]). The y-axis represents the fraction of the tracer in plasma (%). Shown are typical experiments of n = 13 WT mice and n = 9 LDLR^−/− mice.

Fig. 3.

Plasma-FCRs and tissue tracer uptake rates for ¹²⁵I-TC-/[³H]CEt-WT-HDL in WT mice or LDLR^−/− mice. ¹²⁵I-TC-/[³H]CEt-WT-HDL was injected intravenously in WT or LDLR^−/− male mice. A: During the subsequent 24 h interval, blood was harvested periodically to determine the plasma decay of both tracers. ¹²⁵I-TC (¹²⁵I) and [³H]CEt ([³H]) were analyzed, and plasma-FCRs for ¹²⁵I-TC (¹²⁵I) and [³H]CEt ([³H]) were calculated. The difference in plasma-FCRs between [³H]CEt and ¹²⁵I-TC was calculated. Twenty-four hours after tracer injection, the animals were euthanized, and tissues were analyzed for both tracers. Liver (B), adrenal (C), and kidney (D) organ-FCRs for ¹²⁵I-TC (¹²⁵I) and [³H]CEt ([³H]) and the difference in organ-FCRs between [³H]CEt and ¹²⁵I-TC were calculated. All calculations were done as described in Materials and Methods. A: Values are means ± SEM of n = 13 (WT) or n = 9 (LDLR^−/−) mice. B–D: Values are means ± SEM of n = 7 (WT) or n = 5 (LDLR^−/−) mice. An independent experiment yielded qualitatively identical results.

Fig. 4.

Plasma-FCRs and liver tracer uptake rates for ¹²⁵I-TC-/[³H]CEt-LDLR^−/−HDL in WT mice or in LDLR^−/− mice. ¹²⁵I-TC-/[³H]CEt-LDLR^−/−HDL was injected intravenously in WT or in LDLR^−/− male mice. During the subsequent 24 h interval, blood was drawn periodically, and 24 h after tracer injection, the animals were euthanized and tissues were harvested. Plasma-FCRs and liver organ-FCRs for ¹²⁵I-TC (¹²⁵I), for [³H]CEt ([³H]), and for selective uptake ([³H]CEt − ¹²⁵I-TC) were analyzed and calculated as outlined in Fig. 3. All values are means ± SEM of n = 5 (WT) or n = 5 (LDLR^−/−) mice. Where no error bars are shown, the SEM is on the respective line.

Fig. 5.

Uptake of ¹²⁵I-TC-/[³H]CEt-WT-HDL by hepatocytes isolated from WT or LDLR^−/− mice. Hepatocytes were isolated from a WT or an LDLR^−/− male mouse. These cells were incubated (37°C, 2.0 h) in medium containing ¹²⁵I-TC-/[³H]CEt-WT-HDL, and the respective concentrations are given in the abscissae. Finally, cells were harvested, and apparent HDL particle uptake was analyzed as outlined in Materials and Methods. Values are means of n = 3 (WT) or n = 2 (LDLR^−/−) independent experiments; within each experiment, incubations were done in triplicates. Comparing all data from WT and LDLR^−/− hepatocytes, P < 0.05; two (WT) and two (LDLR^−/−) independent similar experiments yielded qualitatively identical results.

Fig. 6.

SR-BI, LDLR, LRP1, and CD36 expression in liver membranes prepared from WT or LDLR^−/− mice. Membrane fractions were isolated from livers originating from WT or LDLR^−/− male mice. The indicated mass of protein was subjected to electrophoresis and transfer to a membrane. Finally, the proteins were immunoblotted using SR-BI- or LDLR-specific (A), LRP1-specific (B), and CD36- or β-actin-specific (C) antibodies. β-actin was used as loading control. Representative blots are shown. D: Three independent blots were quantified by densitometric scanning; P < 0.05 for all blots.

Fig. 7.

ABCA1 expression in liver membranes prepared from WT or LDLR^−/− mice. Membrane fractions were isolated from livers originating from WT or LDLR^−/− male mice. The indicated mass of protein was subjected to electrophoresis and transfer to a membrane. Finally, the proteins were immunoblotted using ABCA1- or β-actin-specific antibodies. β-actin was used as loading control. A: A typical blot is shown; three independent blots yielded qualitatively identical results. B: Densitometric scanning of three blots, P < 0.05.