Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis

Abstract

Bile acid signaling is a critical regulator of glucose and energy metabolism, mainly through the nuclear receptor FXR and the G protein-coupled receptor TGR. The purpose of the present study was to investigate whether dual activation of FXR and TGR5 plays a significant role in the prevention of atherosclerosis progression. To evaluate the effects of bile acid signaling in atherogenesis, ApoE-/- mice and LDLR-/- mice were treated with an FXR/TGR5 dual agonist (INT-767). INT-767 treatment drastically reduced serum cholesterol levels. INT-767 treatment significantly reduced atherosclerotic plaque formation in both ApoE-/- and LDLR-/- mice. INT-767 decreased the expression of pro-inflammatory cytokines and chemokines in the aortas of ApoE-/- mice through the inactivation of NF-κB. In addition, J774 macrophages treated with INT-767 had significantly lower levels of active NF-κB, resulting in cytokine production in response to LPS through a PKA dependent mechanism. This study demonstrates that concurrent activation of FXR and TGR5 attenuates atherosclerosis by reducing both circulating lipids and inflammation.

Figure 1. INT-767 reduces high fat-induced adiposity and hyperlipidemia in ApoE^−/− mice.

Mice were treated with INT-767 as indicated in the Methods section. A) Body weight of ApoE^−/− mice treated with INT-767. B) White fat weight of ApoE^−/− mice. C) Cholesterol distribution in the lipoproteins of ApoE^−/− mice treated with INT-767. D) Triglyceride distribution in the lipoproteins of mice treated with INT-767. E) Body weight of LDLR^−/− mice treated with INT-767. F) Cholesterol distribution in the lipoproteins of LDLR^−/− mice treated with INT-767.

Figure 2. INT-767 reduces high fat-induced adiposity and hyperlipidemia in LDLR^−/− mice.

Mice were treated with INT-767 as indicated in the Methods section. A) Body weight of LDLR^−/− mice treated with INT-767. B) White fat weight of ApoE^−/− mice. C) Cholesterol distribution in the lipoproteins of LDLR^−/− mice treated with INT-767. D) Triglyceride distribution in the lipoproteins of mice treated with INT-767. **P<0.001 and ***P<0.001.

Figure 3. INT-767 reduces cholic acid and its metabolites but not chenodeoxycholic acid though the reduction of hepatic CYP7A1 and CYP8B1 expression in ApoE^−/− mice.

A) mRNA levels of hepatic FXR targets in ApoE^−/− mice treated with INT-767. B) Serum bile acid content in ApoE^−/− mice treated with INT-767. CA, cholic acid; MCA, muricholic acid; DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; T, tauro. *P<0.05 and ***P<0.001.

Figure 4. INT-767 inhibits the development of aortic lesions in ApoE^−/− and LDLR^−/− mice.

Atherosclerotic lesions were quantified by en face analysis. A) Representative picture of en face analysis of atherosclerosis in ApoE^−/− mice treated with INT-767. B) Quantification of en face analysis in ApoE^−/− mice treated with INT-767 for 12 weeks. C) Representative picture of en face analysis of atherosclerosis in LDLR^−/− mice treated with INT-767. D) Quantification of en face analysis in LDLR^−/− mice treated with INT-767 for 16 weeks. ***P<0.001.

Figure 5. INT-767 inhibits systemic and local inflammation in ApoE^−/− mice.

Serum A) IL-1β, B) IL-6, C) IL-8 and D) IL-12 levels in ApoE^−/− mice treated with INT-767. E) Representative picture of immunofluorescence analysis of CD68-positive macrophages and MCP-1 in the aortas of ApoE^−/− mice treated with INT-767. Quantification of immunofluorescence analysis of F) CD68 and G) MCP-1 in the aortas of ApoE^−/− mice treated with INT-767. H) mRNA levels of aortic cytokines and chemokines in ApoE^−/− mice treated with INT-767. I) NF-κB binding activity in the aortas of ApoE^−/− mice treated with INT-767. *P<0.05, **P<0.001 and ***P<0.001.

Figure 6. INT-767 inhibits activation of NF-κB and cytokine production through a TGR5-PKA-dependent mechanism.

A) TNFα and B) IL-1β mRNA levels, C) NF-κB binding activity by EMSA and D) the densitometric analysis of the NF-κB EMSA. J774.2 macrophages were pretreated with 10 µM INT-767 for 2 hours and treated with LPS (100 ng/ml) for 2 hours in the presence of a PKA inhibitor (1 mM, PKAI, Rp-8-Br-cAMPS). E) TGR5 or F) FXR was overexpressed in Raw294.7 macrophages, which express very low levels of TGR5 and FXR. G) INT-767 inhibits TNFα expression induced by LPS (100 ng/ml) in Raw294.7 macrophages overexpressing TGR5 but not FXR. * P<0.05, **P<0.01, and ***P<0.001.