2H2O-based high-density lipoprotein turnover method for the assessment of dynamic high-density lipoprotein function in mice

Abstract

Objective: High-density lipoprotein (HDL) promotes reverse cholesterol transport from peripheral tissues to the liver for clearance. Reduced HDL-cholesterol (HDLc) is associated with atherosclerosis; however, as a predictor of cardiovascular disease, HDLc has limitations because it is not a direct marker of HDL functionality. Our objective was to develop a mass spectrometry-based method for the simultaneous measurement of HDLc and ApoAI kinetics in mice, using a single (2)H2O tracer, and use it to examine genetic and drug perturbations on HDL turnover in vivo.

Approach and results: Mice were given (2)H2O in the drinking water, and serial blood samples were collected at different time points. HDLc and ApoAI gradually incorporated (2)H, allowing experimental measurement of fractional catabolic rates and production rates for HDLc and ApoAI. ApoE(-/-) mice displayed increased fractional catabolic rates (P<0.01) and reduced production rates of both HDLc and ApoAI (P<0.05) compared with controls. In human ApoAI transgenic mice, levels and production rates of HDLc and human ApoAI were strikingly higher than in wild-type mice. Myriocin, an inhibitor of sphingolipid synthesis, significantly increased both HDL flux and macrophage-to-feces reverse cholesterol transport, indicating compatibility of this HDL turnover method with the macrophage-specific reverse cholesterol transport assay.

Conclusions: (2)H2O-labeling can be used to measure HDLc and ApoAI flux in vivo, and to assess the role of genetic and pharmacological interventions on HDL turnover in mice. Safety, simplicity, and low cost of the (2)H2O-based HDL turnover approach suggest that this assay can be scaled for human use to study effects of HDL targeted therapies on dynamic HDL function.

Keywords: apolipoprotein A-I; cholesterol; deuterium oxide; lipoproteins, HDL; mass spectrometry; protein biosynthesis.

Figure 1

HDL turnover in ApoE-deficient (triangular symbols) and wild type (square symbols) mice assessed with ²H₂O-metabolic labeling technique. Intraperitoneal bolus loading followed by free access to drinking water enriched with ²H₂O (6%) led to a steady state body water labeling of ~3.4%. Time course enrichment of ²H incorporation into HDLc (A) and ApoA1 (B). Data show mean ± SD, n=6 per group.

Figure 2

Effect of ApoE deletion on hepatic SR-B1 expression. A. RT-PCR of hepatic SR-B1 mRNA (means ± SD; *, p<0.05; N=6 per group). B. Western blot of hepatic SR-B1 from wild type and ApoE-deficient mice, with actin used as a loading control.

Figure 3

HDL turnover in ApoAI transgenic mice liver. A. Time course of ²H incorporation into HDLc. B. Time course of ²H incorporation into human (triangular symbols) and mouse (square symbols) ApoAI. To account for any variations in the total body water labeling, the net labeling of ApoAI at each time point was normalized to water labeling. Insets give the corresponding levels, FCR, and PR for HDL-c and human and mouse ApoAI (mean ± SD, N=6 per group).

Figure 4

Effect of myriocin on HDL turnover in wild type mice. Time course of ²H incorporation into HDLc (A) and ApoA1 (B) for control (square symbols) and myriocin treated (triangular symbols) mice (mean ± SD, N=5 control group and N=4 myriocin group).

Figure 5

Effect of myriocin on macrophage-specific RCT. A. Time course of RCT to the plasma (% of injected [³H]cholesterol) after subcutaneous injection of cholesterol-labeled macrophages. B. Daily and cumulative fecal RCT. C. Fecal [³H] dpm in neutral sterols. D. Fecal [³H] dpm in bile acids. For all panels, open symbols and bars represent control group and closed symbols and bars for myriocin group, mean ± SD, N=5 control group and N=3 myriocin group (one mouse eliminated from myriocin group due to loss of labeled donor foam cells); *. p<0.05; **, p<0.01; ***, p<0.001.

Figure 6

Effect of myriocin on hepatic RNA expression of ATP-binding cassette transporters involved in bilary cholesterol excretion (mean ± SD, N=5 control group and N=4 myriocin group); *p<0.001.

Figure 7

Effect of myriocin on hepatic cholesterol metabolism. A. Hepatic cholesterol content. B. Hepatic cholesterol FCR. C. Hepatic cholesterol PR. For all panels, open bars represent control group and closed bars for myriocin group, mean ± SD, N=5 control group and N=4 myriocin group; *. p<0.01.

Figure 8

The relationships between concentration and turnover of HDLc (A and B) and ApoAI (C and D). The data are derived from Tables 1 and 2 (means ± SD). The levels of ApoAI were directly related to PR (D). Similar, but not significant association was observed between HDLc levels and its PR (B). There were not significant inverse relationships between HDLc and ApoAI levels and their FCR (A and C).