Decreased selective uptake of high density lipoprotein cholesteryl esters in apolipoprotein E knock-out mice

Abstract

Scavenger receptor BI (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters (CE) by cells, i.e., the uptake of CE without degradation of HDL protein. Mice with attenuated expression of SR-BI, because of targeted gene mutation (SR-BIatt mice), have increased plasma HDL levels as a result of decreased selective uptake in the liver. To further evaluate the role of SR-BI in lipoprotein metabolism, compound apolipoprotein E knock-out (apoE0)/SR-BIatt mice were bred. Hepatic SR-BI protein was increased (2.3-fold) in apoE0 mice compared with wild type (wt) and was reduced significantly in apoE0/SR-BIatt mice. However, the plasma lipoprotein profile of apoE0 and apoE0/SR-BIatt mice was identical. This was explained by HDL turnover studies that revealed that the selective clearance of HDL CE by the liver and adrenal was already profoundly impaired in apoE0 mice compared with wt (28% of wt in liver). A similar decrease in selective uptake was seen when apoE0 HDL was incubated with isolated apoE0 hepatocytes. The results suggest that apoE plays a major role in the selective clearance of HDL CE by the liver and adrenal gland, possibly by facilitating the presentation of HDL to SR-BI at the cell surface.

Figure 1

FPLC cholesterol profile. FPLC was performed with 200-μl pooled plasma samples obtained from five female mice. The cholesterol content of each fraction is indicated as the equivalent cholesterol concentration in plasma (mg/dl).

Figure 2

Western blot analysis of SR-BI expression in liver. (Upper) Immunoblots obtained for individual mice (30 μg of total protein per lane). (Lower) Arbitrary units of mass determined by densitometric scanning, relative to wt mice (100%). Statistical significance: ∗, P < 0.001 compared with wt; ∗∗, P < 0.05 compared with wt and apoE0 mice. Similar results were obtained after normalization of the SR-BI signal for tubulin expression.

Figure 3

Plasma decay curves for ¹²⁵I-labeled NMTC/[³H]cholesteryl oleyl ether-labeled HDL. Mice were injected with doubly labeled HDL and blood was collected periodically over 24 h. Autologous HDL was injected into apoE0 and apoE0/SR-BIatt mice. HDL from apoE0 mice was injected into wt mice. The values are means ± SD of four wt (C57BL/6), six apoE0, and seven apoE0/SR-BIatt mice.

Figure 4

Uptake of labeled HDL by the liver. Liver FCRs, calculated as described in Experimental Procedures, are shown for uptake of labeled protein (¹²⁵I, open bars) and cholesteryl ether (³H, solid bars). The hatched bars represent selective CE uptake (³H − ¹²⁵I). ∗, P < 0.05.

Figure 5

Uptake of labeled HDL by isolated hepatocytes. Hepatocytes were isolated from wt and apoE0 mice. Cells were incubated with doubly labeled HDL (0.5 μg/ml) for 1 h at 37°C. (A) Specific uptake from apoE0 HDL by wt and apoE0 hepatocytes. (B) Specific uptake from wt HDL by wt and apoE0 hepatocytes. Selective uptake represents the difference (³H − ¹²⁵I). Data were contained in four independent experiments in A and three in B, respectively. There are significant differences among apoE0 HDL/apoE0 hepatocyte vs. apoE0 HDL/wt hepatocyte (P < 0.05), wt HDL/apoE0 hepatocyte vs. apoE0 HDL/wt hepatocyte (P < 0.01), apoE0 HDL/apoE0 hepatocyte vs. wt HDL/wt hepatocyte (P < 0.05), and wt HDL/wt hepatocyte vs. wt HDL/apoE0 hepatocyte (P < 0.05). Inset shows the effect of human apoE cDNA transfection on selective uptake. Primary cultures of hepatocytes were transfected with human apoE3 expression plasmid or a mock plasmid by using Effectene (Qiagen). Uptake was measured after 4 h at 37°C.