Insulin-like growth factor I reduces lipid oxidation and foam cell formation via downregulation of 12/15-lipoxygenase

Abstract

Objective: We have shown that insulin-like growth factor I (IGF-1) infusion in Apoe(-/-) mice decreased atherosclerotic plaque size and plaque macrophage and lipid content suggesting that IGF-1 suppressed formation of macrophage-derived foam cells. Since 12/15-lipoxygenase (12/15-LOX) plays an important role in OxLDL and foam cell formation, we hypothesized that IGF-1 downregulates 12/15-LOX, thereby suppressing lipid oxidation and foam cell formation.

Approach and results: We found that IGF-1 decreased 12/15-LOX plaque immunopositivity and serum OxLDL levels in Apoe(-/-) mice. IGF-1 reduced 12/15-LOX protein and mRNA levels in cultured THP-1 macrophages and IGF-1 also decreased expression of STAT6 transcription factor. IGF-1 reduction in macrophage 12/15-LOX was mediated in part via a PI3 kinase- and STAT6-dependent transcriptional mechanism. IGF-1 suppressed THP-1 macrophage ability to oxidize lipids and form foam cells. IGF-1 downregulated 12/15-LOX in human blood-derived primary macrophages and IGF-1 decreased LDL oxidation induced by these cells. IGF-1 reduced LDL oxidation and formation of foam cells by wild type murine peritoneal macrophages, however these effects were completely blocked in 12/15-LOX-null macrophages suggesting that the ability of IGF-1 to reduce LDL oxidation and foam cells formation is dependent on its ability to downregulate 12/15-LOX.

Conclusions: Overall our data demonstrate that IGF-1 reduces lipid oxidation and foam cell formation via downregulation of 12/15-LOX and this mechanism may play a major role in the anti-atherosclerotic effects of IGF-1.

Keywords: Atherosclerosis; Foam cells; LDL/Oxidation/antioxidants; Lipoxygenase; Macrophages/monocytes; Oxidized lipids.

Figure 1. IGF-1 downregulated 12/15-LOX in atherosclerotic plaque

Apoe−/− mice were infused with human IGF-1 or with saline (control) and fed with Western type diet for 12 weeks. Serial cross-sections were obtained through aortic valves, stained with rabbit 12/15-LOX antibody (or with “normal” rabbit IgG, negative control), developed with a/rabbit-biotin/streptavidin-horseradish peroxidase system and counterstained with hematoxylin. A, Representative images. B, Quantative data.

Figure 2. IGF-1 downregulated 12/15-LOX and reduced lipid oxidation in Apoe^−/− mice

Mice were fed with Western type diet and infused with human IGF-1 or with saline (control). A, Total (mouse+human) serum IGF-1 levels. B, 12/15-LOX mRNA levels in atherosclerotic aortas. C, Serum OxLDL levels. D, Serum ApoB levels. E, Serum levels of malondialdehyde (MDA), an index of lipid oxidation.

Figure 3. IGF-1 decreased 12/15-LOX expression in THP-1 macrophages and in human PBMC-derived macrophages. IGF-1 decreased 12/15-LOX in THP-1 cells via PI3 kinase-dependent mechanism

THP-1 macrophages were treated with IGF-1 and 12/15-LOX mRNA levels were measured with real time-RT-PCR (A) and protein expression with immunoblotting (B, C). IGF-1 downregulates 12/15-LOX in human PBMC-derived macrophages (D, E). THP-1 pre-treatment with PI3 kinase inhibitor (LY2940032) partially prevented 12/15-LOX downregulation, however pre-treatment with ERK inhibitor (PD98059) had no effect (F,G).

Figure 4. STAT6 transcription factor is involved in IGF-1-induced 12/15-LOX downregulation

A, THP-1 macrophages were treated with IGF-1 and STAT6 protein expression was measured with immunoblotting. B, THP-1 macrophages were transfected with STAT6-targeted siRNA (or with scrambled siRNA, scr siRNA, negative control) and STAT6 and 12/15-LOX protein expression were measured with immunoblotting.

Figure 5. IGF-1 decreased lipid oxidation in THP-1 macrophages and in PBMC-derived macrophages. IGF-1 suppressed lipid uptake in THP-1 macrophages

THP-1 macrophages (A) or PBMC-derived macrophages (B) were treated with IGF-1 followed by incubation with LDL and lipid oxidation was assessed by TBARS assay. C, LDL-treated THP-1 macrophages were stained with Oil Red, imaged with a microscope and lipid uptake was quantified after extraction of Oil Red and measurement of absorbance at 571 nm. D, representative images.

Figure 6. IGF-1 suppressed lipid oxidation and foam cell formation via its ability to downregulate 12/15-LOX

Peritoneal macrophages were isolated from 12/15 LOX-null mice or from wild-type mice (WT) and treated with IGF-1 followed by incubation with LDL. A, Lipid oxidation (TBARS assay), B, Lipid uptake (Oil Red assay), C, representative images.