In LDL receptor-deficient mice, catabolism of remnant lipoproteins requires a high level of apoE but is inhibited by excess apoE.

To investigate the quantitative requirement for apolipoprotein (apo) E in the clearance of lipoproteins via the non-low density lipoprotein (LDL) receptor mediated pathway, human APOE was overexpressed at various levels in the livers of mice deficient for both the endogenous Apoe and Ldlr genes (Apoe -/-. Ldlr -/-) using adenovirus-mediated gene transfer. We found that a low level of APOE expression, that was capable of reducing the hyperlipidemia in Apoe -/- mice, did not result in a reduction of the hyperlipidemia in Apoe -/-. Ldlr -/- mice. Surpisingly, a very high level of APOE expression also did not result in a reduction of hypercholesterolemia in Apoe -/-. Ldlr -/- mice, despite very high levels of circulating apoE (>160 mg/dl). Only a moderately high level of APOE expression resulted in a reduction of serum cholesterol level (from 35.2 +/- 6.7 to 14.6 +/- 2.3 mmol/l) and the disappearance of VLDL from the serum. Moreover, the very high level of APOE expression resulted in a severe hypertriglyceridemia in Apoe -/-. Ldlr -/- mice and not Apoe -/- mice (25.7 +/- 8.9 and 2.2 +/- 1.8 mmol/l, respectively). This hypertriglyceridemia was associated with an APOE-induced increase in the VLDL triglyceride production rate and an inhibition of VLDL-triglyceride lipolysis. We conclude from these data that, for efficient clearance, the non-LDL receptor-mediated pathway requires a higher level of APOE expression as compared to the LDL receptor, but is more sensitive to an APOE-induced increase in VLDL production and inhibition of VLDL-triglyceride lipolysis.-van Dijk, K. W., B. J. M. Van Vlijmen, H. B. van't Hof, A. van der Zee, S. Santamarina-Fojo, T. J. C. van Berkel, L. M. Havekes, and M. H. Hofker. In LDL receptor-deficient mice, catabolism of remnant lipoproteins requires a high level of apoE but is inhibited by excess APOE.

Effects of dietary fish oil on serum lipids and VLDL kinetics in hyperlipidemic apolipoprotein E*3-Leiden transgenic mice.

Studying the effects of dietary fish oil on VLDL metabolism in humans is subject to both large intra- and interindividual variability. In the present study we therefore used hyperlipidemic apolipoprotein (APO) E*3-Leiden mice, which have impaired chylomicron and very low density lipoprotein (VLDL) remnant metabolism, to study the effects of dietary fish oil on serum lipids and VLDL kinetics under highly standardized conditions. For this, female APOE*3-Leiden mice were fed a fat- and cholesterol-containing diet supplemented with either 0, 3 or 6% w/w (i.e. 0, 6, or 12% of total energy) of fish oil. Fish oil-fed mice showed a significant dose-dependent decrease in serum cholesterol (up to -43%) and triglyceride levels (up to -60%), mainly due to a reduction of VLDL (-80%). LDL and HDL cholesterol levels were not affected by fish oil feeding. VLDL-apoB kinetic studies showed that fish oil feeding resulted in a significant 2-fold increase in VLDL-apoB fractional catabolic rate (FCR). Hepatic VLDL-apoB production was, however, not affected by fish oil feeding. VLDL-triglyceride turnover studies revealed that fish oil significantly decreased hepatic VLDL-triglyceride production rate (-60%). A significant increase in VLDL-triglyceride FCR was observed (+70%), which was not related to increased lipolytic activity. We conclude that APOE*3-Leiden mice are highly responsive to dietary fish oil. The observed strong reduction in serum very low density lipoprotein (VLDL) is primarily due to an effect of fish oil to decrease hepatic VLDL triglyceride production rate and to increase VLDL-apoB fractional catabolic rate.

In the absence of endogenous mouse apolipoprotein E, apolipoprotein E*2(Arg-158 --> Cys) transgenic mice develop more severe hyperlipoproteinemia than apolipoprotein E*3-Leiden transgenic mice.

Apolipoprotein E*2(Arg-158 --> Cys) (APOE*2) transgenic mice were generated and compared to the previously generated apolipoprotein E*3-Leiden (APOE*3-Leiden) transgenic mice to study the variable expression of hyperlipoproteinemia associated with these two APOE variants. In the presence of the endogenous mouse Apoe gene, the expression of the APOE*3-Leiden gene resulted in slightly elevated levels of serum cholesterol as compared with control mice (2.7 +/- 0. 5 versus 2.1 +/- 0.2 mmol/liter, respectively), whereas the expression of the APOE*2(Arg-158 --> Cys) gene did not affect serum cholesterol levels, even after high/fat cholesterol feeding. The extreme cholesterol level usually found in apoE-deficient mice (Apoe-/- mice; 23.6 +/- 5.0 mmol/liter) could be rescued by introducing the APOE*3-Leiden gene (APOE*3-Leiden.Apoe-/-; 3.6 +/- 1. 5 mmol/liter), whereas the expression of the APOE*2(Arg-158 --> Cys) gene in Apoe-/- mice minimally reduced serum cholesterol levels (APOE*2.Apoe-/-; 16.6 +/- 2.9 mmol/liter). In vivo very low density lipoprotein (VLDL) turnover studies revealed that APOE*2.Apoe-/- VLDL and APOE*3-Leiden.Apoe-/- VLDL display strongly reduced fractional catabolic rates as compared with control mouse VLDL (4.0 and 6.1 versus 22.1 pools/h). In vitro low density lipoprotein (LDL) receptor binding studies using HepG2 and J774 cells showed that APOE*2. Apoe-/- VLDL is completely defective in binding to the LDL receptor, whereas APOE*3-Leiden.Apoe-/- VLDL still displayed a considerable binding activity to the LDL receptor. After transfection of APOE*2.Apoe-/- and APOE*3-Leiden.Apoe-/- mice with adenovirus carrying the gene for the receptor-associated protein (AdCMV-RAP), serum lipid levels strongly increased (15.3 to 42.8 and 1.4 to 15.3 mmol/liter for cholesterol and 5.0 to 35.7 and 0.3 to 20. 7 mmol/liter for triglycerides, respectively). This indicates that RAP-sensitive receptors, possibly the LDL receptor-related protein (LRP), mediate the plasma clearance of both APOE*2.Apoe-/- and APOE*3-Leiden. Apoe-/- VLDL. We conclude that in vivo the APOE*2 variant is completely defective in LDL receptor binding but not in binding to LRP, whereas for the APOE*3-Leiden mutant both LRP and LDL receptor binding activity are only mildly affected. As a consequence of this difference, APOE*2.Apoe-/- develop more severe hypercholesterolemia than APOE*3-Leiden.Apoe-/- mice.

Modulation of very low density lipoprotein production and clearance contributes to age- and gender- dependent hyperlipoproteinemia in apolipoprotein E3-Leiden transgenic mice.

Apolipoprotein E3-Leiden (APOE*3-Leiden) transgenic mice have been studied to identify factors modulating chylomicron and VLDL remnant lipoprotein metabolism. Transient elevated levels of VLDL/LDL-sized lipoproteins occurred in these mice with maximal levels during the period of rapid growth (optimum at 45 d of age). After about 100 d of age, serum cholesterol and triglyceride levels stabilized to slightly elevated levels as compared to control mice. The expression of the APOE*3-Leiden transgene was not age-dependent. In young mice the in vivo hepatic production of VLDL-triglycerides was 50% increased as compared to older mice. This is sustained by in vivo VLDL-apo B turnover studies showing increased (75%) VLDL-apo B secretion rates in young mice, whereas the VLDL-apo B clearance rate appeared not to be age dependent. On a high fat/cholesterol diet, females displayed significantly higher cholesterol levels than males (10 versus 7.0 mmol/liter, respectively). Serum levels of VLDL/LDL sized lipoproteins increased upon administration of estrogens, whereas administration of testosterone gave the opposite result. As compared to male mice, in female mice the hepatic VLDL-triglyceride production rate was significantly elevated. Injection of estrogen in males also resulted in increased VLDL-triglyceride production, although not statistically significant. In vivo VLDL-apo B turnover experiments showed that the VLDL secretion rate tended to be higher in females. Although, the fractional catabolic rate of VLDL-apo B is not different between males and females, administration of estrogens in males resulted in a decreased clearance rate of VLDL, whereas administration of testosterone in females resulted in an increased clearance rate of VLDL. The latter presumably due to an inhibiting effect of testosterone on the expression of the APOE*3-Leiden transgene. We conclude that hyperlipidemia in APOE*3-Leiden transgenic mice is strongly affected by age via its effect on hepatic VLDL production rate, whereas gender influences hyperlipidemia by modulating both hepatic VLDL production and clearance rate.