Increased sphingomyelin content of plasma lipoproteins in apolipoprotein E knockout mice reflects combined production and catabolic defects and enhances reactivity with mammalian sphingomyelinase.

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Abstract
Apolipoprotein E knockout (apoE0) mice accumulate atherogenic remnant lipoproteins in plasma. We now provide evidence that these particles are enriched in sphingomyelin (SM), and explore the mechanisms and possible pathophysiological consequences of this finding. The phosphatidylcholine/sphingomyelin (PC/SM) ratio was reduced in all lipoproteins in apoE0 mice compared with wild-type (Wt) mice (2.0+/-0.2 vs. 4.7+/-0.5; 2.8+/-0.5 vs. 5.5+/-0.9; 1.9+/-0. 5 vs. 4.6+/-0.5 for VLDL, LDL, and HDL), reflecting 400 and 179% increases in plasma pools of SM and PC, respectively. Turnover studies using [14C]PC/[3H]SM VLDL or HDL showed that the fractional catabolic rate (FCR) of VLDL-SM and HDL-SM were markedly reduced in the apoE0 mice compared with Wt mice, while the FCRs of VLDL-PC and HDL-PC were similar. By contrast, the FCRs of [3H]PC ether and [14C]SM were identical in apoE0 and Wt mice. The production rates of VLDL-SM and HDL-SM in apoE0 mice were much higher than in Wt mice, while the production rates of lipoprotein PC were similar. To assess the underlying mechanisms, we also measured the PC/SM ratio in VLDL and LDL of LDL receptor knockout (LDLr0) and hepatic LDL receptor-related protein knockout/LDLr0 mice, but found no difference with Wt mice. Using S-sphingomyelinase, an enzyme secreted by macrophages and endothelial cells, we found that VLDL and LDL from apoE0, but not from Wt or LDLr0 mice, were significantly aggregated, and that aggregation was not prevented by adding back apoE. We then enriched the apoE0-VLDL and Wt-VLDL with different amounts of SM, and found that VLDL aggregation was enhanced. Thus, the increased SM content of lipoproteins in apoE0 mice is due to combined synthesis and clearance defects. Impaired SM clearance reflects resistance to intravascular enzymes and delayed removal by a non-LDLr, non-LDLr related protein pathway. The increased SM content in slowly cleared remnant lipoproteins may enhance their susceptibility to arterial wall SMase and increase their atherogenic potential.