Diabetes reduces the cholesterol exporter ABCA1 in mouse macrophages and kidneys

Abstract

Accumulation of cholesterol in arterial macrophages may contribute to diabetes-accelerated atherosclerotic cardiovascular disease. The ATP-binding cassette transporter ABCA1 is a cardioprotective membrane protein that mediates cholesterol export from macrophages. Factors elevated in diabetes, such as reactive carbonyls and free fatty acids, destabilize ABCA1 protein in cultured macrophages, raising the possibility that impaired ABCA1 plays an atherogenic role in diabetes. We therefore examined the modulation of ABCA1 in two mouse models of diabetes. We isolated peritoneal macrophages, livers, kidneys, and brains from type 1 non-obese diabetic (NOD) mice and mice made diabetic by viral-induced autoimmune destruction of pancreatic beta-cells, and we measured ABCA1 protein and mRNA levels and cholesterol contents. ABCA1 protein levels and cholesterol export activity were reduced by 40-44% (P<0.01) in peritoneal macrophages and protein levels by 48% (P<0.001) in kidneys in diabetic NOD mice compared with nondiabetic animals, even though ABCA1 mRNA levels were not significantly different. A similar selective reduction in ABCA1 protein was found in peritoneal macrophages (33%, P<0.05) and kidneys (35%, P<0.05) from the viral-induced diabetic mice. In liver and brain, however, diabetes had no effect or slightly increased ABCA1 protein and mRNA levels. The reduced ABCA1 in macrophages and kidneys was associated with increased cholesterol content. Impaired ABCA1-mediated cholesterol export could therefore contribute to the increased atherosclerosis and nephropathy associated with diabetes.

Fig. 1.

Diabetes-induced reduction in ABCA1 protein but not in mRNA levels in mouse macrophages. Resident (A, C, E) or thioglycollate-elicited (B, D, F) peritoneal macrophages were isolated from control and diabetic mice and assayed for ABCA1 protein and mRNA levels. A, B: Representative sets of ABCA1 and GAPDH immunoblots of the same amount of macrophage protein (50 μg/lane). C, D: Densitometric quantification of ABCA1 relative to GAPDH protein levels. E, F: Real-time RT-PCR quantification of ABCA1 mRNA expressed relative to GAPDH mRNA. Each value is the mean ± SD from seven pairs of mice (14 total) (C, E) or 5 individual mice (D, F). *P < 0.05, **P < 0.01 versus control (Student's t-test).

Fig. 2.

Diabetes-induced reduction in macrophage ABCA1 and ABCG1 but not in scavenger receptor A1 (SRA) and CD36. Thioglycolate-elicited peritoneal macrophages were isolated from control and diabetic NOD mice, (A) ABCA1, scavenger receptor A1 (SRA), CD36, (B) ABCG1, and (A, B) GAPDH protein levels were quantified from scans of immunoblots. B: ABCG1 mRNA was quantified by RT-PCR and expressed relative to GAPDH mRNA. Results are the mean ± SD of the ratio of protein to GAPDH for 5 (protein) or 10 (mRNA) animals per group. *P < 0.04, **P < 0.001 versus control (Student's t-test).

Fig. 3.

Diabetes-induced AGE modification of macrophage ABCA1 and a reduction in apoA-I-mediated cholesterol efflux from macrophages. A: Thioglycolate-elicited peritoneal macrophages were isolated from control and diabetic NOD mice, AGE proteins were immunoprecipitated (IP) from lysates, proteins were resolved by SDS PAGE and transferred to nitrocellulose, and ABCA1 was detected by immunoblot (IB) analysis. Results represent three separate experiments. B: Thioglycolate-elicited macrophages from NOD mice were plated for 1 h in acetylated LDL/[³H]cholesterol medium and incubated for 2.5 h minus or plus 10 μg/ml apoA-I or 50 μg/ml HDL, and [³H]cholesterol efflux was assayed. Results are the mean ± SD from four control and diabetic NOD mice performed in triplicate and normalized to control values for each mouse. *P < 0.001 versus control (Student's t-test).

Fig. 4.

Lack of a diabetes-induced reduction in ABCA1 in mouse livers. Each value is the mean ± SD from 13–15 NOD (C, E) and 5–8 LDLR^−/−; GP (D, F) mice. *P < 0.05, **P < 0.02 versus control (Student's t-test).

Fig. 5.

Diabetes-induced reduction in ABCA1 protein but not mRNA levels in mouse kidneys. Each value is the mean ± SD from 15 NOD (C, E) and 5 LDLR^−/−; GP (D, F) mice. *P < 0.02, **P < 0.001 versus control (Student's t-test).

Fig. 6.

Diabetes-induced increased neutral lipid droplets in macrophages from LDLR^−/−; GP mice. A: Lipid droplets in peritoneal macrophages from control and diabetic LDLR^−/−; GP mice visualized (red) by Oil Red O staining. B: The counted number of Oil Red O positive cells expressed as % of total cells. Results are mean ± SD of values for macrophages from five (non-diabetic) or nine (diabetic) mice plated in two different wells per animal. *P < 0.001 versus control (Student's t-test).

Fig. 7.

Diabetes-induced increased cholesterol content of mouse macrophages and kidneys. The free cholesterol (FC) and esterified cholesterol (EC) mass of macrophages (Mϕ), kidneys, and livers from control and diabetic NOD and LDLR^−/−; GP mice were measured and normalized to protein content. Each value is the mean ± SD from 10 NOD and 10 LDLR^−/−; GP mice per group. *P < 0.05, **P < 0.02 versus control (Student's t-test).