To investigate the consequences of enhanced ABCA1 expression on HDL metabolism, reverse cholesterol transport, and hepatic biliary excretion in vivo, we produced two independent lines of transgenic mice, which overexpress human ABCA1 under control of the apoE promoter. This promoter targeted expression of the human transgene primarily to the liver and macrophages of hABCA1-Tg mice, two tissues where ABCA1 is normally expressed (10
). Previous studies (12
) have indicated an important function for ABCA1 in the removal of cholesterol from macrophages. Foam cells, resulting from the accumulation of excess cholesterol in macrophages, lead to the formation of fatty streaks, complex lesions, and eventually plaque rupture (35
). Stimulation of macrophage cholesterol efflux by enhanced ABCA1 expression may inhibit foam cell formation and consequently reduce atherogenic risk. In contrast, the role of ABCA1 in hepatic cholesterol homeostasis is less clearly understood. Depending on its location, ABCA1 may enhance hepatic cholesterol efflux into plasma lipoproteins, facilitate biliary cholesterol excretion, or both. These two potential ABCA1-mediated hepatic processes could have opposite consequences on reverse cholesterol transport.
The present study establishes that enhanced ABCA1 expression in liver and macrophages has a significant impact on the plasma lipid profile, leading to elevated plasma cholesterol, HDL cholesterol, and apoA-I in a gene-related dose-response fashion. The HDL in hABCA1-Tg mice is cholesterol enriched, and apoE-rich HDL1 accumulates. Increased HDL1 has been previously reported in mice with SR-BI deficiency (36
) and LCAT overexpression (29
), metabolic conditions under which the cholesterol content of HDL is increased by either reduced HDL cholesterol selective uptake or enhanced HDL-C esterification. Interestingly, pre-β HDL, the subfraction of HDL, which is very effective in cellular cholesterol efflux (2
), were increased in hABCA1-Tg mice.
Cavelier et al. (38
) recently reported the generation of two different transgenic mouse lines from BACs containing the hABCA1 gene. In contrast to our findings, they reported that mice with increased hABCA1 expression in liver (twofold; determined by quantitative RT-PCR) and macrophages (twofold; determined by increase cholesterol efflux) or testis had no changes in the plasma lipids or in HDL-C levels. In the present study, hABCA1 expression was increased four- to ninefold in liver and three- and sixfold in macrophages in hABCA1-Tg mice. Thus, lower hABCA1 expression levels may account for the lack of increased plasma HDL concentrations in transgenic mice generated using hABCA1 BACs (38
). These authors have suggested (38
) that interaction between human ABCA1 and endogenous mouse ligands may be inefficient requiring higher levels of expression to alter the plasma lipid profile. Differences in in vivo function of LCAT have been reported in transgenic mice overexpressing the mouse versus the human enzyme (29
Kinetic studies using degradable and nondegradable protein labels in hABCA1-Tg showed that ABCA1 overexpression increased plasma apoA-I HDL levels by reducing the catabolism of apoA-I in both the liver and kidney of transgenic mice. Although the pool size of apoA-I HDL in hABCA1-Tg mice is larger than control mice, differences in HDL pool size have been previously shown not to have a major effect on FCR in apoA-I KO mice (33
), in patients with Tangier disease before and after HDL infusion (40
) or in a patient with marked hyperalphalipoproteinemia (41
). Several studies have demonstrated that one of the major determinants of HDL catabolism is HDL size (42
). Larger, lipid-rich HDL appears to be catabolized more slowly (42
). Lipid enrichment of HDL may alter the conformation and accessibility of surface apolipoproteins for interaction with cell-surface receptors responsible for HDL catabolism (43
). The enhanced lipidation of HDL facilitated by ABCA1-mediated cellular efflux in hABCA1-Tg mice, which leads to the formation of larger, CE-enriched HDL, may delay hABCA1-Tg HDL catabolism by a similar mechanism.
The metabolism of 3
H-CEt HDL was also altered by ABCA1 expression. Although the plasma and liver FCRs of 3
H-CEt HDL were similar in both study groups, when the increased plasma HDL-CE pool size was taken into account the absolute rate of hepatic HDL-CE delivery was increased by 750 μg/d in hABCA1-Tg, an amount equal to the entire CE pool size of control mice. These data indicate that increased ABCA1 expression in both macrophages and liver enhances the net hepatic delivery of exogenous HDL-CE in vivo. Of major interest is the finding that the cholesterol and PL content of bile was increased approximately 1.8-fold. Mdr2 deficiency in mice leads to defective biliary cholesterol and PL excretion (34
); however, mdr2 gene expression was unaltered in hABCA1-Tg mice. Increases in bile cholesterol but not PL content have been reported in mice overexpressing SR-BI (45
) and hABCG1 (47
). Our findings indicate that hepatic ABCA1 overexpression may directly facilitate the transport of both cholesterol and PL into bile. Further studies will be required to identify potential mechanisms by which ABCA1 overexpression modulates biliary lipid secretion. The lack of concomitant changes in bile acid concentrations in these three mouse models indicates independent regulation of the 17-α hydroxylase bile acid and cholesterol excretion pathways in bile.
The combined metabolic changes that result from ABCA1 overexpression reported in this study would be predicted to reduce atherogenic risk in hABCA1-Tg mice. In contrast, increased plasma apoB levels in hABCA1 Tg mice, should counter these potentially beneficial effects of ABCA1 overexpression. Decreased plasma apoB levels has been described in ABCA1-knockout mice (22
), but the underlying mechanism has not been reported. Here we demonstrate that the increase in plasma apoB levels in hABCA1-Tg mice is not due to increased apoB secretion, consistent with delayed catabolism.
Our combined studies show, for the first time to our knowledge, that ABCA1 overexpression in macrophages and liver alters the plasma lipid profile and modulates plasma HDL catabolism, hepatic reverse cholesterol transport, and biliary cholesterol excretion in vivo and thus may markedly alter atherogenic risk in humans.