The TMAO-Generating Enzyme Flavin Monooxygenase 3 Is a Central Regulator of Cholesterol Balance

Abstract

Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) have recently been linked to cardiovascular disease (CVD) risk. Here, we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT) and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic endoplasmic reticulum (ER) stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation.

Conflict of interest statement

CONFLICTS OF INTEREST

M.W., D.M.S., A.C.B., D.F., A.D.G., A.L.B., S.M., A.M., R.C.S., J.S., X.R., T.d.A.V., J.C., P.T.I., D.S.M, H.A.B., X.L., P.P., P.T., A.J.L., R.E.T., and J.M.B. have no conflicts of interest to declare. S.L.H. and Z.W. are named as co-inventors on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics, and have the rights to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics. S.L.H. reports he has been paid as a consultant by the following companies: Cleveland Heart Lab, Inc., Esperion, Liposciences Inc., and Procter & Gamble. S.L.H. also reports he has received research funds from Cleveland Heart Lab, Esperion, Liposciences Inc., Proctor & Gamble, Roche, and Takeda. Richard Lee, Rosanne Crooke, and Mark Graham are employees at Isis Pharmaceuticals, Inc. (Carlsbad, CA).

Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Transcriptional Profiling Defines FMO3 as a Regulator of RCT

(A) Microarray analysis in acute TICE mouse model. Female C57BL/6 mice were treated with a non-targeting control ASO (Con. ASO) or an ASO targeting ACAT2 mRNA (A2 ASO) as previously described for 1 week (Marshall et al., 2014). Differentially expressed genes (DEGs) are shown as a heatmap (all genes shown have pTg; Temel et al., 2010) were maintained on a high cholesterol diet (0.2%, wt/wt) for 8 weeks. Differentially expressed genes (DEGs) are shown as a heatmap (all genes shown have p<0.001, n = 5). (C) Venn Diagram showing coordinately upregulated genes in both microarray datasets. The shared gene indicated is Gm10567, and is of unknown function. (D) Venn Diagram showing coordinately downregulated genes in both microarray datasets. The shared gene is FMO3. (E) qPCR quantification of hepatic FMO3 mRNA levels in female control and ACAT2 ASO-treated mice (n=5). (F) qPCR quantification of hepatic FMO3 mRNA levels in female WT and NPC1L1-liver transgenic mice (NPC1L1Tg) (n=5).

Figure 2. ASO-Mediated Knockdown of FMO3 Reorganizes Whole Body Cholesterol Balance

Female C57BL/6 mice were fed either a low (0.02%, wt/wt) or high (0.2%, wt/wt) cholesterol diet and treated with either a control (non-targeting) ASO or an ASO targeting FMO3 mRNA for 6 weeks. (A) qPCR quantification of hepatic FMO3 mRNA levels. (B) Western blot determination of FMO3 protein levels. (C) Circulating levels of the FMO3 substrate trimethylamine (TMA). (D) Circulating levels of the FMO3 product trimethylamine-N-oxide (TMAO). (E) Fractional cholesterol absorption was determined using the dual fecal isotope method. (F) Fecal neutral sterol excretion was determined by gas liquid chromatography. (G) Hepatic cholesteryl ester (CE) levels. (H) Hepatic free cholesterol (FC) levels. (I) Biliary cholesterol secretion rate was measured following common bile duct cannulation. (J) Total plasma cholesterol (TPC) levels. (K) Cholesterol distribution of pooled plasma in mice fed a low cholesterol diet (n=5 per pool). (L) Cholesterol distribution of pooled plasma in mice fed a high cholesterol diet (n=5 per pool). (M) Very low density lipoprotein cholesterol (VLDLc) levels. (N) Low density lipoprotein cholesterol (LDLc) levels. (O) High density lipoprotein cholesterol (HDLc) levels. (P) Total plasma triglyceride (TG) levels. All data represent the mean ± S.E.M. from 5–10 mice per group, * = significantly different from the control ASO group within each diet group (p

Figure 3. FMO3 Coordinates Sterol-Sensing Transcription Factor Activation in the Liver

Female C57BL/6 mice were fed either a low (0.02%, wt/wt) or high (0.2%, wt/wt) cholesterol diet and treated with either a control (non-targeting) ASO or an ASO targeting FMO3 mRNA for 6 weeks. (A–H) Expression of LXR and downstreatm target genes in the liver. (I–L) Expression of SREBP2 and downstream target genes in the liver. (M–R) Hepatic levels of oxysterols All data represent the mean ± S.E.M. from 4–5 mice per group, * = significantly different from the control ASO group within each diet group (p

Figure 4. FMO3 Knockdown Stimulates Non-Biliary Macrophage RCT

Female C57BL/6 mice were fed a low (0.02%, wt/wt) cholesterol diet and treated with either a control (non-targeting) ASO or an ASO targeting FMO3 mRNA for 6 weeks. During the last week of treatment, mice also were orally gavaged with either a vehicle or an exogenous LXR agonist (T0901317). During the last 48 hours, mice were singly housed, and macrophage RCT into different compartments was measured as described in the Experimental Procedures. (A) Total plasma cholesterol (TPC) levels. (B) Time course of [3H]cholesterol accumulation in plasma. (C) [3H]cholesterol distribution of pooled plasma (n = 5 per pool). (D) [3H]cholesterol recovery in the feces. (E) [3H]bile acids recovery in the feces. (F) Mass fecal neutral sterol excretion. (G) [3H]cholesterol recovery in gall bladder bile. (H) [3H]bile acids recovery in gall bladder bile. (I) [3H]cholesterol recovery in the liver. (J) [3H]bile acids recovery in the liver. (K) [3H]cholesterol recovery in the small intestine (SI) wall. (L) [3H]bile acids recovery in the small intestine (SI) wall. (M) qPCR quantification of hepatic FMO3 mRNA levels. (N) Western blot analysis of hepatic FMO3 protein levels. (O) qPCR quantification of NPC1L1 mRNA levels in the duodenum. All data represent the mean ± S.E.M. from 6–10 mice per group, and means not sharing a common superscript differ significantly, (p

Figure 5. FMO3 Knockdown Promotes Hepatic Inflammation and ER Stress by Dampening LXR Activation

For dietary cholesterol-induced LXR activation (panels A–C), female C57BL/6 mice were fed either a low (0.02%, wt/wt) or high (0.2%, wt/wt) cholesterol diet and treated with either a control (non-targeting) ASO or an ASO targeting knockdown of FMO3 for 6 weeks. For exogenous ligand-induced LXR activation (panels D–E), female C57BL/6 mice were fed a low (0.02%, wt/wt) cholesterol diet and treated with either a control (non-targeting) ASO or an ASO targeting FMO3 mRNA for 6 weeks. During the last week of treatment, mice also were orally gavaged with either a vehicle or an exogenous LXR agonist (T0901317) as described in the macrophage RCT experiments in the Experimental Procedures. (A) H&E stained liver sections (200× magnification) from female C57BL/6 mice fed a low (0.02%, wt/wt) cholesterol diet and treated with either a control ASO or FMO3 ASO for 6 weeks. Arrows indicate areas of localized immune cell infiltration. (B) Hepatic lobular inflammation score from pathological report; Data represent the mean ± S.E.M. from 4 mice per group, * = significantly different from the control ASO group within each diet group (pTyr416); n=3 individual animals shown per group Data represent the mean ± S.E.M. from 4–8 mice per group, and means not sharing a common superscript differ significantly, (p