A. van der Zee

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.

Domains of apolipoprotein E contributing to triglyceride and cholesterol homeostasis in vivo. Carboxyl-terminal region 203-299 promotes hepatic very low density lipoprotein-triglyceride secretion.

Apolipoprotein (apo) E has been implicated in cholesterol and triglyceride homeostasis in humans. At physiological concentration apoE promotes efficient clearance of apoE-containing lipoprotein remnants. However, high apoE plasma levels correlate with high plasma triglyceride levels. We have used adenovirus-mediated gene transfer in apoE-deficient mice (E− /−) to define the domains of apoE required for cholesterol and triglyceride homeostasis in vivo. A dose of 2 × 109 plaque-forming units of apoE4-expressing adenovirus reduced slightly the cholesterol levels of E− /− mice and resulted in severe hypertriglyceridemia, due to accumulation of cholesterol and triglyceride-rich very low density lipoprotein particles in plasma. In contrast, the truncated form apoE4–202 resulted in a 90% reduction in the plasma cholesterol levels but did not alter plasma triglyceride levels in the E− /− mice. ApoE secretion by cell cultures, as well as the steady-state hepatic mRNA levels in individual mice expressing apoE4 or apoE4–202, were similar. In contrast, very low density lipoprotein-triglyceride secretion in mice expressing apoE4, but not apoE4–202, was increased 10-fold, as compared with mice infected with a control adenovirus. The findings suggest that the amino-terminal 1–202 region of apoE4 contains the domains required for the in vivo clearance of lipoprotein remnants. Furthermore, the carboxyl-terminal 203–299 residues of apoE promote hepatic very low density lipoprotein-triglyceride secretion and contribute to apoE-induced hypertriglyceridemia.

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.

Reversal of Hypercholesterolemia in Apolipoprotein E2 and Apolipoprotein E3-Leiden Transgenic Mice by Adenovirus-Mediated Gene Transfer of the VLDL Receptor

We have investigated the interaction of apolipoprotein E2(Arg158-Cys) (apoE2) and apolipoprotein E3-Leiden (apoE3-Leiden) with the very low density lipoprotein (VLDL) receptor in vivo and in vitro to define the possible role of this receptor in lipoprotein metabolism and atherosclerosis. The in vivo binding specificity of the VLDL receptor for apoE2 and apoE3-Leiden was investigated by adenovirus-mediated gene transfer of the VLDL receptor in apoE2 and apoE3-Leiden transgenic mice lacking endogenous mouse apoE (Apoe-/-). Ectopic overexpression of the VLDL receptor gene in the liver resulted in a >50% decrease of plasma cholesterol levels in both apoE2 and apoE3-Leiden transgenic mice compared with liver expression of the beta-galactosidase gene. This reduction in plasma cholesterol was mainly due to a reduction in the VLDL level. Overexpression of the VLDL receptor did not affect the hepatic VLDL triglyceride production, indicating that the hypocholesterolemic effect is due to an increased level of plasma clearance mediated by the VLDL receptor. In vitro binding analysis showed that both apoE2 and apoE3-Leiden VLDL compete efficiently with rabbit beta-VLDL for binding to the VLDL receptor expressed on LDL receptor-deficient Chinese hamster ovary cells. We conclude from these data that both apoE2 and apoE3-Leiden function as proper ligands for the VLDL receptor in vitro and in vivo. This finding substantiates a possible role for the VLDL receptor in atherosclerosis in hyperlipidemic subjects homozygous for apoE2 or carrying apoE3-Leiden and indicates that the VLDL receptor expressed on the liver has therapeutic potential as an alternative route for clearance of binding-defective lipoproteins.

Apolipoprotein E2 (Lys146→Gln) Causes Hypertriglyceridemia due to an Apolipoprotein E Variant–Specific Inhibition of Lipolysis of Very Low Density Lipoproteins–Triglycerides

The apolipoprotein E2 (Lys146-->Gln) variant is associated with a dominant form of familial dysbetalipoproteinemia. Heterozygous carriers of this variant have elevated levels of plasma triglycerides, cholesterol, and apolipoprotein E (apoE). It was hypothesized that the high amounts of triglycerides in the very low density lipoprotein (VLDL) fraction are due to a disturbed lipolysis of VLDL. To test this hypothesis, apoE knockout mice were injected with an adenovirus containing the human APOE*2 (Lys146-->Gln) gene, Ad-E2(146), under the control of the cytomegalovirus promoter. ApoE knockout mice injected with an adenovirus vector encoding human apoE3 (Ad-E3) were used as controls. Five days after adenovirus injection, plasma cholesterol levels of mice injected with a high dose of Ad-E2(146) (2x10(9) plaque-forming units) were not changed compared with preinjection levels, whereas in the group who received a low dose of Ad-E2(146) (5x10(8) plaque-forming units) and in the groups injected with a low or a high dose of Ad-E3, plasma cholesterol levels were decreased 5-, 6-, and 12-fold, respectively. Plasma triglycerides were not affected in mice injected with Ad-E3. In contrast, a 7-fold increase in plasma triglycerides was observed in mice injected with the low dose of Ad-E2(146) compared with mice injected with Ad-E3. Injection with the high dose of Ad-E2(146) resulted in a dramatic increase of plasma triglycerides (50-fold compared with Ad-E3 injection). In vitro lipolysis experiments showed that the lipolysis rate of VLDLs containing normal amounts of apoE2 (Lys146-->Gln) was decreased by 54% compared with that of VLDLs containing comparable amounts of apoE3. The in vivo VLDL-triglyceride production rate of Ad-E2(146)-injected mice was not significantly different from that of Ad-E3-injected mice. These results demonstrate that expression of apoE2 (Lys146-->Gln) causes hypertriglyceridemia due to an apoE variant-specific inhibition of the hydrolysis of VLDL-triglycerides.