PPAR agonist-induced reduction of Mcp1 in atherosclerotic plaques of obese, insulin-resistant mice depends on adiponectin-induced Irak3 expression

Abstract

Synthetic peroxisome proliferator-activated receptor (PPAR) agonists are used to treat dyslipidemia and insulin resistance. In this study, we examined molecular mechanisms that explain differential effects of a PPARα agonist (fenofibrate) and a PPARγ agonist (rosiglitazone) on macrophages during obesity-induced atherogenesis. Twelve-week-old mice with combined leptin and LDL-receptor deficiency (DKO) were treated with fenofibrate, rosiglitazone or placebo for 12 weeks. Only rosiglitazone improved adipocyte function, restored insulin sensitivity, and inhibited atherosclerosis by decreasing lipid-loaded macrophages. In addition, it increased interleukin-1 receptor-associated kinase-3 (Irak3) and decreased monocyte chemoattractant protein-1 (Mcp1) expressions, indicative of a switch from M1 to M2 macrophages. The differences between fenofibrate and rosiglitazone were independent of Pparγ expression. In bone marrow-derived macrophages (BMDM), we identified the rosiglitazone-associated increase in adiponectin as cause of the increase in Irak3. Interestingly, the deletion of Irak3 in BMDM (IRAK3(-/-) BMDM) resulted in activation of the canonical NFκB signaling pathway and increased Mcp1 protein secretion. Rosiglitazone could not decrease the elevated Mcp1 secretion in IRAK3(-/-) BMDM directly and fenofibrate even increased the secretion, possibly due to increased mitochondrial reactive oxygen species production. Furthermore, aortic extracts of high-fat insulin-resistant LDL-receptor deficient mice, with lower adiponectin and Irak3 and higher Mcp1, showed accelerated atherosclerosis. In aggregate, our results emphasize an interaction between PPAR agonist-mediated increase in adiponectin and macrophage-associated Irak3 in the protection against atherosclerosis by PPAR agonists.

Figure 1. Experimental protocol.

Twelve-week old DKO mice were treated for 12 weeks with fenofibrate (50 mg kg⁻¹ day⁻¹) or with rosiglitazone (10 mg kg⁻¹ day⁻¹) and were compared with placebo-treated DKO and C57BL/6 J background mice. LDL-receptor deficient (LDLR^−/−) mice, on the C57BL/6 J background, were fed for a period of 12 weeks with standard diet (SD) or with a high-fat diet (HFD). The mice were sacrificed at 24 weeks and total blood, the aortic arch, the abdominal aorta, and visceral adipose tissue were collected.

Figure 2. Rosiglitazone and not fenofibrate treatment reduces macrophage accumulation and improves adiponectin expression in visceral adipose tissue.

(A) Representative Mac-3 staining of visceral adipose tissue of placebo-, fenofibrate- and rosiglitazone-treated DKO mice at 24 weeks. (B) Relative RNA levels of Tnfα, IL6, Mcp1 and Adipoq as determined by qRT-PCR. Data are means ± SEM. Scale bar = 100 µm. *P<0.05 and ***P<0.001 DKO compared with C57BL/6 J mice; ^$$ P<0.01 and ^$$$ P<0.001 PPAR agonist-treated compared with placebo-treated DKO mice; ^££ P<0.01 and ^£££ P<0.001 rosiglitazone-treated compared with fenofibrate-treated DKO mice.

Figure 3. Rosiglitazone and not fenofibrate treatment decreases atherogenesis in obese, insulin resistant mice.

(A) Representative Mac-3 staining of aortic sinus plaques of placebo-, fenofibrate- and rosiglitazone-treated DKO mice at 24 weeks. (B) Gene expression in the aorta was analyzed by measuring relative RNA levels using qRT-PCR for Tnfα, IL6, Mcp1 and Irak3. Data are means ± SEM. Scale bar = 500 µm. *P<0.05, **P<0.01 and ***P<0.001 DKO compared with C57BL/6 J mice; ^$$ P<0.01 and ^$$$ P<0.001 PPAR agonist-treated compared with placebo-treated DKO mice; ^£ P<0.05, ^££ P<0.01 and ^£££ P<0.001 rosiglitazone-treated compared with fenofibrate-treated DKO mice.

Figure 4. Adiponectin-induced Irak3 plays an important role in rosiglitazone-mediated decrease of Mcp1.

(A) Soluble Mcp1 protein levels in DKO BMDM exposed to 50 µM fenofibrate, 10 µM rosiglitazone or 5 µM GW9662 for 24 hours as determined by ELISA. Data are means ± SEM; n = 16 from three different mice. ^$$ P<0.01 compared with fenofibrate-treated BMDM. (B) Irak3 RNA and protein levels of DKO BMDM exposed to 1 or 10 µg/mL globular adiponectin for 24 hours as determined by qRT-PCR and Western blotting. Data are means ± SEM; n = 6. ***P<0.001 compared with DKO BMDM; ^$ P<0.05 and ^$$ P<0.01 compared with DKO BMDM exposed to 1 µg/mL globular adiponectin. (C) Soluble Mcp1 protein levels (n = 18 from three different mice), NFκB p50 DNA binding activity (n = 8 from two different mice) and mROS production (n = 6) in IRAK3^−/− BMDM exposed to 50 µM fenofibrate or 10 µM rosiglitazone for 24 hours as determined by ELISA and flow cytometry. Data are means ± SEM. *P<0.05, **P<0.01 and ***P<0.001 compared with C57BL/6 J BMDM; ^$ P<0.05 and ^$$$ P<0.001 compared with IRAK3^−/− BMDM; ^£££ P<0.001 compared with fenofibrate-treated BMDM. Abbreviations: BMDM, bone marrow-derived macrophages; mROS, mitochondrial reactive oxygen species.

Figure 5. HFD-induced weight gain is associated with dyslipidemia, insulin resistance and hyperleptinemia in the presence of high blood adiponectin.

Data are means ± SEM. **P<0.01 and ***P<0.001 HFD-fed compared with SD-fed LDL-receptor deficient mice. Abbreviations: HFD, high fat diet; SD, standard diet.

Figure 6. HFD increases atherogenesis in insulin resistant mice.

Plaque volume was determined by measuring lipid (oil red O)-stained surfaces in subsequent sections; macrophages were stained with anti-Mac-3 antibody. Gene expression in the aorta was analyzed by measuring relative RNA levels using qRT-PCR for Pparγ, Mcp1, Irak3 and Adipoq. Data are means ± SEM. **P<0.01 and ***P<0.001 HFD-fed compared with SD-fed LDL-receptor deficient mice. Abbreviations: HFD, high fat diet; SD, standard diet.

Figure 7. Adiponectin and macrophage-associated Irak3 are indispensable molecules in the anti-atherosclerotic properties of PPAR agonists.

The schematic draw demonstrates the anti-atherosclerotic properties of the PPARγ agonist rosiglitazone. Treatment with rosiglitazone improves the adipocyte function characterized by a decrease in adipocyte size, a reduction in adipose tissue macrophages and an increased expression of anti-inflammatory adiponectin. The increase in blood adiponectin and de novo adiponectin production in atherosclerotic lesions is necessary for the upregulation of Irak3 in plaque macrophages, which is crucial for the indirect rosiglitazone-mediated decrease in Mcp1 secretion. Abbreviations: Mφ, macrophages; ROS, reactive oxygen species.