Myeloid lineage cell-restricted insulin resistance protects apolipoproteinE-deficient mice against atherosclerosis

Abstract

Inflammatory processes play an important role in the pathogenesis of vascular diseases, and insulin-resistant diabetes mellitus type 2 represents an important risk factor for the development of atherosclerosis. To directly address the role of insulin resistance in myeloid lineage cells in the development of atherosclerosis, we have created mice with myeloid lineage-specific inactivation of the insulin receptor gene. On an ApoE-deficient background, MphIRKO mice developed smaller atherosclerotic lesions. There was a dramatic decrease in LPS-stimulated IL-6 and IL-1beta expression in the presence of macrophage autonomous insulin resistance. Consistently, while insulin-resistant IRS-2-deficient mice on an ApoE-deficient background display aggravated atherosclerosis, fetal liver cell transplantation of IRS-2(-/-) ApoE(-/-) cells ameliorated atherosclerosis in Apo-E-deficient mice. Thus, systemic versus myeloid cell-restricted insulin resistance has opposing effects on the development of atherosclerosis, providing direct evidence that myeloid lineage autonomous insulin signaling provides proinflammatory signals predisposing to the development of atherosclerosis.

Figure. 1. Effective and selective myeloid lineage-specific inactivation of the insulin receptor gene in MphIRKO–mice

A) PCR analysis of Cre-mediated recombination in thioglycollate-elicited macrophages from IR^flox/flox (WT) and IR^flox/floxLysMCre^+/– (KO) mice. The upper-right panel shows the percentage of macrophages (MAC1 positive, CD19 negative) present in peritoneal lavage after thioglycollate treatment by FACS analysis using CD19 and MAC1-antibodies. The lower panel shows the expression of the insulin receptor b subunit in thioglycollate-elicited macrophages from four individual wild-type and knockout mice. The lower panel shows a Western blot analysis against the chemokine receptor CCR-1, serving as a loading control. B) Western blot analysis of the insulin receptor b subunit in white adipose tissue (WAT), liver, brain, skeletal muscle, and kidney of IR^flox/flox (WT) and IR^flox/flox LysMCre^+/– (KO) mice.

Figure 2. Reduced atherosclerosis in ApoE-deficient MphIRKO mice

A) Representative Sudan IV stainings of male ApoE^–/– IR^flox/flox and ApoE^–/–IR^flox/floxLysMCre^+/– mice under high-cholesterol diet and quantitation of lesion size in the descending aorta of male ApoE^–/–IR^flox/flox (control) and ApoE^–/–IR^flox/floxLysMCre^+/– (MphIRKO) mice. Data represent the mean of six to nine animals of each genotype (*p < 0.05 in unpaired Student's t test). B) Plasma glucose, insulin, and cholesterol concentrations were determined in male ApoE^–/–IR^flox/flox (control) and ApoE^–/–IR^flox/floxLysMCre^+/– (MphIRKO) mice exposed to high-cholesterol diet at the age of 5 months after overnight fasting. Data represent the mean ± SEM of six to nine animals of each genotype. C) Lipoprotein profiles as determined by FPLC analysis from pooled sera of male ApoE^–/–IR^flox/flox (red line) and ApoE^–/–IR^flox/flox LysMCre ^+/– (green line) mice.

Figure 3. Unaltered metabolism of modified LDL particles in insulin-resistant macrophages

A) Northern blot analysis of CD36 in IR^flox/flox (WT) and Cre-transduced IR^flox/flox (KO) immortalized macrophage cell lines. Cells had been left either untreated or were stimulated with 100 nM insulin for 24 hr. The upper-right panel shows a Western blot analysis of CD36 expression in thioglycollate-elicited macrophages of four individual ApoE^–/–IR^flox/flox (WT) and IR^flox/floxLysMCre^+/– (KO) mice. The lower panel shows Western blot analysis of CD36 expression in IR^flox/flox (WT) and Cre-transduced IR^flox/flox (KO) immortalized macrophage cell lines. B) FACS analysis of CD36 surface expression in control and IR-deficient immortalized macrophage cell lines. C) Specific uptake of oxidized LDL cholesterol in thioglycollate-elicited macrophages from IR^flox/flox (WT) and IR^flox/floxLysMCre^+/– (KO) mice. Data represent the mean ± SEM of four to six animals of each genotype. D) Cholesterol efflux in thioglycollate-elicited macrophages from control and IR-deficient mice. Cholesterol efflux was determined in the absence (open bars) and in the presence of 10 μg/ml ApoA1 (closed bars). Data represent the mean ± SEM of four to six animals of each genotype.

Figure 4. Impaired inflammatory response in insulin-resistant macrophages

A) Northern blot analysis of MCP1, TNF-α, IL-1β, and IL-6 expression in IR^flox/flox immortalized macrophage cell lines treated without (WT) or with HNTC-Cre (KO), which have been cultured either in the absence or presence of 10 ng/ml LPS for 24 hr. The lower panel shows the densitometric quantitation of mRNA-expression obtained in three independent experiments. Signal intensity detected in unstimulated WT cells was set as 1. Data represent the mean ± SEM (**p < 0.01 in unpaired Student's t test). B) Amount of the indicated cytokines released into the culture medium by control or IR-deficient immortalized macrophages, which had been cultured either in the absence or presence of 10 ng/ml LPS for 3 days. Data represent the mean ± SEM obtained in three independent experiments (**p < 0.01 in unpaired Student's t test). C) Amount of the indicated cytokines released into the culture medium by TG-elicited macrophages from control (WT) and MphIRKO-(KO) mice. Cells have been cultured in the absence or presence of 10 ng/ml LPS for 3 days. Data represent the mean ± SEM obtained from cells of four mice of each genotype, each cell line assayed in triplicate (*p < 0.05 in unpaired Student's t test).

Figure 5. Metabolic characterization of IRS-2-deficient mice on an ApoE-deficient background

A) Glucose-tolerance tests were performed at the age of 12 weeks in IRS-2^+/+ApoE^–/– (square) and IRS-2^–/–ApoE^–/– (diamond) male mice. Data represent the mean ± SEM of 10–14 animals of each genotype. B) Glucose-tolerance tests in male ApoE-deficient mice which have been reconstituted after lethal irradiation with either IRS-2^+/+ApoE^–/– (square) or IRS-2^–/–ApoE^–/– (diamond) fetal liver cells. Glucose-tolerance test was performed 10 weeks after transplantation. Data represent the mean ± SEM of 12–13 animals in each group. C) Plasma triglyceride and cholesterol concentrations in the different mutant mice. Concentrations of triglyceride and cholesterol were determined in serum obtained from IRS-2^+/+ApoE^–/– and IRS-2^–/–ApoE^–/– mice at the age of 12 weeks, after 9 weeks of exposure to Western diet. Plasma cholesterol and triglyceride concentrations in irradiated and reconstituted IRS/ApoE-deficient mice were determined 11 weeks after transfer, having been exposed for 8 weeks to high-cholesterol diet. Data represent the mean ± SEM of 8–12 mice in each group.

Figure 6. Aggravated atherosclerosis in IRS-2^–/–ApoE^–/– mice

A) Lesion surface area in the abdominal aorta was determined in 4-month-old IRS-2^+/+ApoE^–/– and IRS-2^–/–ApoE^–/– mice after 12 weeks of exposure to high-cholesterol diet. The left panel demonstrates an example of the lipid-stained lesions, the graph demonstrates a quantitative assessment of lesion size in 12–22 animals in each group. (p < 0.05 in unpaired Student's t test). B) Quantification of lipid-positive lesions of the aortic arch as percentage of surface. The left panel demonstrates exemplary pictures, the graph on the right side presents the quantitative assessment of lesion size in 12–20 mice of each genotype. (p < 0.05 in unpaired Student's t test). C) Cross-section analysis of atherosclerotic lesions in the aortic root. Left panel demonstrates a representative result obtained in IRS-2^+/+ApoE^–/– and IRS-2^–/–ApoE^–/– mice. The graph shows the quantitative assessment of lesion area in these animals for 10–15 animals in each genotype. (p < 0.05 in unpaired Student's t test).

Figure 7. Adoptive transfer of IRS-2^+/+ApoE^–/– and IRS-2^–/–ApoE^–/– fetal liver cells protects against atherosclerosis

Male ApoE-deficient mice were lethally irradiated at the age of 6–8 weeks and reconstituted the following day by iv injection of fetal liver cells obtained from male IRS-2^+/+ApoE^–/– and IRS-2^–/–ApoE^–/– embryos. Mice were allowed to recover under normal chow diet for 2 weeks and then exposed to high-cholesterol diet for 12 weeks before analysis of atherosclerosis. A) Quantification of lipid-positive lesions of the aortic arch as percentage of surface. The left panel demonstrates exemplary pictures, the graph on the right side presents the quantitative assessment of lesion size in 17–22 mice of each genotype. (p < 0.05 in unpaired Student's t test). B) Cross-section analysis of atherosclerotic lesions in the aortic root. Left panel demonstrates a representative result, the graph shows the quantitative assessment of lesion area in these animals for 14–15 animals in each genotype. (p < 0.05 in unpaired Student's t test).