ApoE is a major determinant of hepatic bile acid homeostasis in mice,☆☆

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Abstract

Apolipoprotein E (ApoE) plays a central role in lipid transport and cholesterol metabolism, with surplus cholesterol being removed from the liver through bile acid (BA) synthesis. Furthermore, BAs are of critical importance in fat absorption by forming intestinal lipid-bile salt mixed micelles. To define ApoE's role in BA homeostasis, the metabolism of cholesterol and BA was investigated in liver tissue and gallbladder bile of ApoE-deficient mice given a chow or high-cholesterol/high-fat diet (HCHF) diet for 6 months. When compared to wild-type mice, muricholic acid (MCA) and chenodeoxycholic acid (CDCA) increased approximately 15-, 82-, 22- and 38-fold, respectively, in hepatic tissue of ApoE-deficient mice given a chow or HCHF diet. Moreover, ApoE-deficient mice on an HCHF diet increased the amounts of hepatic free cholesterol, MCA and CDCA by 61%, 61% and 50% (P<.05). Conversely, total cholesterol and cholesterol esters were unchanged, and the bile acids taurohyodeoxycholic acid, taurodeoxycholic acid and hyodeoxycholic acid decreased to one third as compared to the chow diet (P<.05). Additionally, quantitative reverse-transcription polymerase chain reaction assays revealed induced expression of the bile acid receptor (Fxr) and associated transcription factors, i.e. Fxr, Lrh, Lxra and Srebp1c. Transcript expression of Cyp2a12, Cyp1b1, Cyp2e1, Cyp3a16 and Cyp4a10 was also induced. Note that Cyp4a10 catalyzes ω-hydroxylation of arachidonic acid to epoxy- and hydroxyeicosatrienoic acids to control vascular tone.

Altogether, MCA and CDCA synthesis is selectively induced in ApoE-deficient mice. These hydrophilic BAs alter micellar size and structure to lower intestinal cholesterol solubilization. Furthermore, CDCA and MCA are potent FXR agonist and antagonist, respectively, and function in a regulatory loop to mitigate impaired ApoE function.

Introduction

Apolipoprotein E (ApoE) is a glycoprotein of about 34 kDa that is predominantly synthesized in the liver, spleen (macrophages), neuronal cells and epidermis (keratinocytes) [1]. This lipoprotein is of critical importance in lipid transport and receptor-mediated lipid uptake. Its ability to bind to heparin and to interact with cell surface proteoglycans influences fat uptake as well. The roles of ApoE in cholesterol metabolism and lipid delivery are well described, and being a component of very low density lipoprotein, ApoE affects endogenous triglycerides and cholesterol metabolism by delivering it to extrahepatic tissue; however, surplus cholesterol is removed from the liver through bile acid synthesis or secreted into the bile.

Studies with ApoE-deficient mice reveal disrupted uptake of lipoproteins into tissues. This results in cholesterol accumulation in the systemic circulation, hypercholesterolemia and eventually atherosclerotic plaque formation [2], [3], [4]. Gradually, the lesions will grow in size to form fatty streaks, which are similar to the ones observed in humans [5]. Furthermore, a diet rich in cholesterol and fat accelerates plaque formation in ApoE-deficient mice [6].

There is overwhelming evidence for ApoE to protect against atherosclerosis by suppressing lipid accumulation in peripheral blood mononuclear cells and to reduce expression of inflammatory molecules on monocytes and vascular endothelium [7]. Definitive proof for ApoE's role in human lipid and cholesterol metabolism stems from genetic studies, and carriers of variant ApoE alleles develop hyperlipidemia and hypertriglyceridemia as well as atherosclerosis with risk for plaque rupture [8], [9], [10], [11]. There is also growing evidence for ApoE to play a role in neurodegenerative disorders, cognitive function and immunity [12], [13].

While earlier studies alluded to ApoE's role in lipid transport, dietary cholesterol absorption and its biliary excretions [4], [14], [15], [16], its role in bile acid homeostasis in hepatic tissue and gallbladder bile in ApoE-deficient mice given a high-cholesterol and high-fat diet (HCHF) is unknown. Furthermore, certain bile acids influence micellar size and structure and therefore fat and cholesterol absorption from the intestine [17], [18], [19], [20]. This prompted our interest to investigate bile acid metabolism in ApoE-deficient mice given a chow or HCHF diet for 6 months. By liquid chromatography (LC)–tandem mass spectrometry (MS/MS), the composition and concentration of individual bile acids in liver tissue and gallbladder bile of ApoE-deficient mice were determined. Moreover, expression of the bile acid receptor Fxr and associated nuclear transcription factors as well as CYP monooxygenases relevant for cholesterol and lipid metabolism was assayed by quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Lastly the findings for BA metabolism in ApoE mic on a low- and high-fat diet were compared to published BA profiles obtained for wild-type (WT) mice on a chow diet.

Our study highlights selective induction of muricholic (MCA) and chenodeoxycholic (CDCA) bile acid synthesis in ApoE-deficient mice given a chow diet, and the synthesis of these bile acids increased further on an HCHF diet. Importantly, earlier work had shown MCA to reduce cholesterol uptake from the intestine by altering micellar size and structure [17], and lowering its taurine conjugation additionally reduced cholesterol solubility in intestinal micelles [18]. Moreover, MCA inhibits intestinal bile salt receptor activity, and such inhibition improved metabolic parameters in obese mice [21]. Collectively, our studies point to a regulatory loop whereby ApoE-deficient mice adapt to an impaired ApoE function through the selective induction of MCA and CDCA bile acid synthesis, and next to cholesterol uptake, both BAs function as potent Fxr ligands to influence bile acid receptor signaling and cholesterol metabolism.

Section snippets

Animals and in-life observation

The study was performed in accordance with the American Association for Laboratory Animal Science Policy on the Human Care and Use of Laboratory Animals, and the study was approved by the local government (Aktenzeichen: 50.203.2-INBIFO 11/02.Bezirksregierung Köln).

Details regarding the original animal studies are given in von Holt et al. [6], and donated tissue samples from control (sham) animals were used to investigate cholesterol, bile acid and gene expression changes in ApoE-deficient mice

Bile acids in the liver tissue of ApoE-deficient mice

The composition and concentration of individual bile acids in liver tissue and gallbladder bile of ApoE-deficient mice given a chow diet with traces of 0.002% cholesterol and 4.5% fat or a milk-enriched diet containing 0.17% cholesterol and 18.7% fat (HCHF) were analyzed in groups consisting of 10 animals each. When compared to chow-fed animals, the mean body weight increased significantly by 15% and serum cholesterol levels were on average 2.3 times higher. In HCHF-fed animals, hepatic free

Discussion

As summarized in the seminal review of Mahley and Rall [12], ApoE is far more than just a lipid transport protein, and although ApoE's role in dietary cholesterol absorption and its biliary excretion have been the subject of previous investigations [14], [15] our study is the first report to highlight significant changes in BA homeostasis in ApoE-deficient mice given an HCHF diet. We examined bile acid homeostasis in hepatic tissue and gallbladder bile in ApoE-deficient mice and observed an

Acknowledgment

We are grateful for the statistical advice given by Dr. Hartmut Hecker, and we are thankful to Dr. Kong and Dr. Guo who shared the raw data of hepatic BAs in WT mice [28].

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      ApoE influences adipogenesis from triglyceride-rich lipoproteins.18 In mice, apoE adapts bile acid metabolism by upregulating sterol 6-β hydroxylase which converts chenodeoxycholic acid to the more hydrophilic muricholic acid and decreases fat absorption.19 Vascular function is affected in various ways by apoE: from maintaining blood-brain barrier integrity20 to inflammatory responses.21

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    Funding: The financial support from The Virtual Liver Network (grant 031 6154) of the German Federal Ministry of Education and Research to J.B. is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

    ☆☆

    Conflicts of interest: none.

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