University of Groningen

Cholesterol, bile acid and triglyceride metabolism intertwinedSchonewille, Marleen

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General Introduction

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Chapter 1 General Introduction

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General Introduction

Increased plasma lipid levels are risk factors for cardiovascular diseases

Hyperlipidemia is a risk factor for cardiovascular diseases and is characterized by disturbed plasma cholesterol and triglyceride levels.

Currently,thefirstlinetherapyincardiovasculardiseasesaimsatdecreasingplasma lipid levels. This thesis describes several experiments in which plasma levels of triglycerides and/or cholesterol have been decreased by using pharmacological interventions. These pharmacological interventions created opportunities to study bile acid and lipid metabolism, as well as the excretion of cholesterol to the feces. Cholesterol levels in the body can only be reduced by excretion to the feces, conversion into bile acids or steroids or inhibiting cholesterol synthesis. Excretion of cholesterol towards the feces can originate from twodifferent pathways, i.e. the classical hepatobiliarypathway or the newly discovered transintestinal cholesterol excretion (TICE) pathway. Cholesterol excretion via the feces, in particular via the TICE pathway, has been extensively studied in chapters 4-6. The conversion of cholesterol into bile acids is not the fastest way to eliminate cholesterol from the body since 95% of the bile acids are reabsorbed in the terminal ileum, whereas only 5% is removed via fecal excretion.1,2 However, removal of bile acids via fecal excretion with the use of bile acid sequestrants leads to increased bile acid synthesis and accordingly increased utilization of cholesterol, thereby decreasing plasma cholesterol levels.3 Apparently, bile acid and lipid metabolism are closely linked to one another and chapter 2 highlightstherecentdiscoveriesinthefieldofbileacidsandlipidmetabolism.Cholesterol and bile acid metabolism is explained in more detail below.

Cholesterol Metabolism

Cholesterol in the body can derive from either dietary intake or de novo synthesis. In the small intestine, absorption of dietary cholesterol as

well as reabsorption of biliary cholesterol takes place via a Niemann-Pick Disease, Type C1 Like-1 (NPC1L1)-mediated pathway.4 Cholesterol is poorly soluble in water and is therefore present in the intestinal lumen packed into mixed micelles.5 Via the apical transporter NPC1L1, cholesterol is absorbed intotheenterocyte,whereitisesterifiedbyacetyl-CoAacetyltransferase2(SOAT2/ACAT2).4 In the enterocyte, cholesterol can also be secreted back intotheintestinallumenviatheheterodimerATPbindingcassettesubfamilyG member 5/8 (ABCG5/G8) located on the apical membrane.6 Similar to cholesterol, plant sterols and plant stanols are also absorbed via NPC1L1, however they are a poor substrate for ACAT2 and therefore are excreted

back into the intestinal lumen by ABCG5/G8.7 Inhibition of cholesterol absorption via the NPC1L1 pathway by the use of plant sterols or ezetimibe are common strategies to reduce plasma cholesterol levels.8,9 New insights into the action of plants sterols and stanols or ezetimibe are described in chapter 3 and chapter 5 respectively.AftertheesterificationofcholesterolviaACAT2, cholesterol is incorporated into chylomicrons (CM) via a microsomal triglyceride transfer protein (MTTP)-mediated process and subsequently excreted via lymph into the blood circulation.10 In the circulation, CM are degraded via the enzymatic action of lipoprotein lipase (LPL) and the uptake of chylomicron remnants by the liver occurs by the LDL receptor (LDLR), VLDL receptor (VLDLR) or the LDL receptor-related protein (LRP) via receptor-mediated endocytosis.11 Besides uptake of cholesterol from the plasma, cholesterol in the liver can also be synthesized de novo. Cholesterol synthesis occurs mostly in the liver, but also in other organs like

Figure 1: Schematic overview of cholesterol metabolismCholesterol can enter the small intestine via the diet, biliary secretion or TICE. In the small intestine, cholesterol can either be excreted via the feces or be absorbed. After absorption of cholesterol, it is transported through the bloodstream as CM. Due to the action of lipoprotein lipase, CM are degraded to CM remnants and taken up by the liver. Cholesterol can be incorporated into VLDL and excreted into the bloodstream. VLDL can be degraded into IDL by the action of LPL. Subsequently, IDL can be degraded into LDL by the action of LPL and hepatic lipase. LDL can be taken up by the peripheral tissues or the liver. Cholesterol can be excreted from the peripheral tissues by HDL and HDL can subsequently be taken up by the liver. Hepatic cholesterol can be stored in the liver or used for bile acid synthesis. Hepatic cholesterol can also derive from de novo synthesis. Last but not least, hepatic cholesterol can be excretedintothebile,whereitfinallywillendupinthesmallintestine.

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the colon, small intestine, skin and kidney.12 Cholesterol is synthesized from acetyl-CoA via a multistep enzymatic pathway with HMG-CoA reductase (HMGCR) being the rate-limiting enzyme.13 Notably, inhibition of HMGCR with the use of statins is the most frequently used therapy to reduce plasma cholesterol levels in hypercholesterolemic patients and chapter 4 describes some unexpected results after statin treatment in mice.14 Endogenous and plasmaderivedcholesterolcanbeesterifiedbyhepaticACAT2andstoredin the hepatocyte. Furthermore, hepatic cholesterol can also be excreted into the bloodstream as VLDL via an MTTP-mediated process.15 Via the action of LPL, triglycerides are removed from VLDL and thereby it will be converted into intermediate-density lipoprotein (IDL) followed by the conversion into LDL via the enzymatic action of both LPL and hepatic lipase.16 Finally, LDL is taken up by the liver or by peripheral tissues. An importantdifferencebetweenhumanandmouselipoproteinmetabolismisthat the cholesterylester transfer protein (CETP) is present in humans but not in mice.17,18 CETP transfers triglycerides from VLDL and LDL to HDL in exchange for cholesteryl esters, creating a LDL-dominated lipoprotein profileinhumansincomparisonwithaHDL-dominatedlipoproteinprofilein mice.18 In the liver, cholesterol can be converted into bile acids, steroid hormones or be secreted into the bile via ABCG5/G8 and end up in the small intestine.19,20 Interestingly, NPC1L1 is located on the canalicular site of the hepatocyte’s membrane, being able to transport cholesterol from the bile back into the liver, however this is only in the case in humans and not in mice.4 Another way for cholesterol to enter the small intestine is via transintestinal cholesterol excretion, which will be explained below. Finally, in the small intestine, cholesterol can be reabsorbed or excreted into the feces as neutral sterols, i.e. cholesterol and its bacterial metabolites. A schematic overview of cholesterol metabolism is presented in Figure 1.

Reverse Cholesterol Transport

Atherosclerosis is caused by excess cholesterol that has accumulated on the inside of blood vessels, forming an atherosclerotic plaque. Removal of

this cholesterol deposits is a major topic of research nowadays. Atherosclerotic plaques consist of cholesterol loaded macrophages called foam cells.21 Reverse cholesterol transport (RCT) refers to the removal of cholesterol from these macrophages towards excretion via the feces.22 However, the discussion remains whether the hepatobiliary pathway or TICE is responsible for the excretion of cholesterol from macrophages in the atherosclerotic plaques to the feces, called macrophage-to-feces RCT. In chapter 6, we investigated which pathway is involved in macrophage-to-feces RCT. Much is known about cholesterol excretion via the classical hepatobiliary pathway, however the mechanism behind the newly discovered TICE pathway remains elusive. The TICE pathway concerns the excretion of cholesterol from the blood directly into the intestinal lumen thus avoiding the hepatobiliary route.23 Under standard conditions, the TICE pathway contributes to around 30% of

the total fecal neutral sterols excretion.24,25 To date, some clues have emerged designating a possible mechanism of the TICE pathway. Stimulation of the TICE pathway can highly increase the excretion of fecal neutral sterols, and is thereby a promising target for cholesterol elimination from the body.23 Currently, several compounds were shown to act as stimulators of theTICEpathwayand severalgeneswere identified toplaya role in theTICE pathway.26 Therefore, TICE-stimulating compounds as well as several knockout animal models are used to investigate the mechanism underlying TICE. Inhibition of cholesterol absorption, with the use of plant sterols or ezetimibe, is shown to be involved in the TICE pathway and thereby an importantroleforthecholesterolinfluxtransporterNPC1-Like1(NPC1L1)in the regulation of this pathway was revealed.27,28 Further investigation on theinfluenceofezetimibeonTICEisdescribedinchapter 5. To date, it is not known which lipoprotein is the donor for the cholesterol excreted via TICE. Knockdown of hepatic ACAT2 increases excretion of cholesterol via the TICE pathway by increased mobilization of cholesterol onto apolipoprotein B (apoB) and E (apoE)-containing lipoproteins that deliver the cholesterol fortheTICEflux.29 Moreover, hepatic knockdown of MTTP led to a decrease in TICE, designating apoB-containing lipoproteins, such as VLDL and LDL, as possible contributors to the TICE pathway.30 However, more research is needed to prove which lipoproteins are responsible for cholesterol excretion via TICE. Furthermore, some past and recent studies imply a role of the cholesteroleffluxheterodimerABCG5/G8forTICEandthishasbeenfurtherinvestigated in chapter 5.27 Additionally, data obtained from knockout modelsoftheABCG5/G8heterodimerimplicatethatothercholesteroleffluxtransporters must be involved in TICE. Le May et al. have shown that the transporterATPbindingcassettesubfamilyBmember1(ABCB1)isinvolvedin TICE.31 However, further investigation is necessary to elucidate the role of theseABC-transporters,andpossibleothers,intheeffluxofTICEfromtheenterocyte into the intestinal lumen.

Bile acids

Bileacidsaresynthesizedfromcholesterol in the livervia twodifferentenzymatic pathways, i.e. the classical (neutral pathway) or the alternative

(acidic) pathway (Figure 2). In the classical pathway, the conversion of cholesterol into the bile acid cholic acid (CA) is initiated via cytochrome P450, Family 7, Subfamily A, Polypeptide 1 (CYP7A1), while the alternative pathway which primarily produces chenodeoxycholic acid (CDCA) starts with the enzymatic action of CYP27A1.20 Furthermore, the enzymatic action of CYP8B1 is required for the synthesis of cholic acid (CA).32

In humans, the primary bile acids that are synthesized in the liver are CDCA andCA.Inrodents,CDCAisconvertedtotheα-muricholicacid(α-MCA)andβ-muricholicacid(β-MCA),creatingadifferentbileacidcomposition

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in rodents compared to humans.33 The gut microbiota is responsible for the formation of secondary bile acids such as lithocholic acid (LCA), hyocholic acid (HCA), hyodeoxcycholic acid (HDCA), deoxycholic acid (DCA), ursodeoxycholicacid(UDCA)andω-muricholicacid(ω-MCA).34 Bile acids cyclethroughtheenterohepaticcirculationveryefficiently;only5%ofthebile acids in the intestine are lost via fecal excretion. Bile acid are reabsorbed in the terminal ileum into the enterocytes via the transporter apical sodium-dependent bile acid transporter (SLC10A2/ASBT).35 Bile acids are exported from the enterocyte to the portal system by the receptor solute carrier family 51,alphasubunit(SLC51A/OSTα)andbetasubunit(SLC51B/OSTβ).36 In the liver, bile acids are taken up by solute carrier family 10 (sodium/bile acid cotransporter), member 1 (SLC10A1/NTCP) and solute carrier organic anion transporterfamily,member1A2(SLCO1A2/OATP),andfinallysecretedbackinto the bile via the bile salt export pump (ABCB11/BSEP).37 Interestingly, bile acids can regulate their own synthesis via binding to the farnesoid X receptor (NR1H4/FXR) and thereby inhibiting bile acid synthesis via a multistep pathway.2 In the enterocyte, activation of FXR by bile acids leads tothesecretionoffibroblastgrowthfactor15(FGF15)orfibroblastgrowthfactor 19 (FGF19) in mice or humans respectively.38 FGF15/19 binds to the FGFR4/β-Klothoreceptorcomplexofthehepatocyteresultingininhibitionof Cyp7a1 gene expression.39 Besides intestinal FXR, bile acids can also activate hepatic FXR, likewise leading to repression of Cyp7a1. Activation of hepatic FXR will lead to inhibition of Cyp7a1 via a small heterodimer partner (NR0B2/SHP) and liver receptor homolog-1 (NR5A2 /LRH1) dependent signaling pathway.40 Besides regulation of Cyp7a1, hepatic FXR is also able to regulate the expression of Cyp8b1 and Cyp27a1, by SHP and hepatocyte nuclear factor 4, Alpha (HNF4α)-dependent pathway.41,42 In addition, FXR was shown to be involved in other metabolic pathways like, glucose metabolism, energy expenditure and lipid metabolism.2 Therefore, targeting FXR by pharmaceutical compounds which act as FXR agonists are popular tools to investigate FXR signaling nowadays.43,44 Further information about bile acid in control of lipid metabolism can be found in chapter 2, where the recent discoveries in thisfieldaresummarized.

Figure 2: The classical versus the alternative pathway in bile acid synthesisIn the liver, primary bile acids are synthesized from cholesterol via the classical or the alternative pathway. The classical pathway describes the production of CA and CDCA and is initiated by the enzymatic action of CYP7A1. CDCA can also be produced via the alternative pathway, which starts with the enzymatic action of CYP27A1.

Outline

Understanding the underlying mechanisms of lipid metabolism opens doors to possible new therapies in the treatment of cardiovascular

diseases. However, the metabolic effects of many existing therapies isnot always completely understood. For example, plant sterols and plant stanols are used to decrease plasma cholesterol levels. Plant sterols and stanols are compounds which are structurally similar to cholesterol and they inhibit cholesterol absorption in the small intestine.45 A daily intake of 2 grams of plant sterols or stanols decreases LDL cholesterol up to 10%.46 Interestingly, plant sterols and stanols are apparently also able to reduce plasma triglyceride levels in hyperlipidemic patients.47,48 The underlying mechanism of the triglyceride-lowering properties of plant sterols and stanols has been investigated in a high fat diet mouse model and is described in chapter 3. Another way to reduce plasma cholesterol levels is with the use of statins, which are the most prescribed drugs to reduce plasma cholesterol levels worldwide. Statins constitute a class of drugs that inhibit cholesterol synthesis.49 Cholesterol is synthesized via a multistep pathway, and statins inhibit the rate limiting enzyme HMGCR.50 Although statins are widely used, the underlying mechanisms are not completely understood. Remarkably, the effectsofstatinsoncholesterolsynthesisrateshavebarelybeenexaminedin vivo, and the literature describes some contradicting results. Therefore, we have measured cholesterol synthesis rates in vivo in statin-treated mice and Chapter 4 describes that statins paradoxically increase cholesterol synthesis.

Furthermore,weassessedtheeffectsofthenovelFXRagonistPX20606oncholesterol and bile acid metabolism. Chapter 5 describes the extensive effectofFXRagonismontheTICEpathway.Inthisstudy,wetreatedmicewith PX20606 combined with the cholesterol absorption inhibitor ezetimibe, which resulted in an even more pronounced increase of TICE. Mice treated with PX20606 in combination with ezetimibe excreted up to 80 % of their daily circulating pool of cholesterol into the feces. Since TICE was enhanced very extensively in this study, we tried to elucidate the underlying mechanism. Our research revealed that complex interactions between intestinal NPC1L1, intestinal ABCG5/G8 and the hydrophilicity of bile acids in the intestinal lumen are involved in the regulation of TICE.

It is established that cholesterol excretion towards the feces can occur via the hepatobiliary or TICE pathway, however which pathway is important in macrophage-to-feces RCT remains a point of discussion. Recently, a study reported that macrophage-to-feces RCT in hamsters treated with ezetimibe occurs only via the hepatobiliary pathway.51 However, the bile ducts of these hamsters were ligated and thereby removing bile acids and phospholipids from the small intestine, which has previously been shown to be necessary for functional TICE.24 In contrast, another study reported that macrophage-to-feces RCT is mainly mediated by TICE.52 In chapter 6,we tried tofindout which cholesterol excretion pathway is more dominant in macrophage-

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to-feces RCT in a rat model with undisturbed enterohepatic circulation. We have set up a complex experiment in which bile is exchanged between two rats (Figure 3). The bile exchange has been made possible by bile diversion surgery, which creates an extended bile duct. In this experimental setup, bile ofthefirstratwastransferredtotheduodenumofthesecondratandviceversa. Reabsorption of cholesterol in the small intestine was not possible due totreatmentwiththecholesterolabsorptioninhibitorezetimibe.Thefirstratwas injected with macrophages containing radioactive labeled cholesterol. The injected radioactively labeled cholesterol could be excreted to the feces via either the hepatobiliary pathway or TICE. In this experimental setup, radiolabeled cholesterol derived from the hepatobiliary pathway ended up in the feces of the second rat and TICE derived cholesterol ended up in the feces ofthefirstrat.Thankstothisexperimentalsetup,wewereabletodistinguishwhich route is the most important in macrophage-to-feces RCT under natural conditions without disrupting the hepatobiliary system. Results from this experiment showed that both pathways contribute to macrophage-to-feces RCT, however the contribution of the hepatobiliary route is stronger.

Figure 3: Schematic overview of the rat diversion experiment Radioactive labeled cholesterol was injected into the first rat on the left side. The radioactive labeledcholesterolcanbeexcretedtothefecesviatwodifferentroutes,i.e.theclassicalhepatobiliaryrouteorTICE.If the radiolabeled cholesterol was excreted via the hepatobiliary route, it was transferred via the cannula fromthebileofthefirstratintotheduodenumofthesecondratontherightsideandfinallyendedinthefeces of the second rat. If the radiolabeled cholesterol was excreted via TICE, it ended up in the feces of the firstrat.Notethattreatmentwithezetimibepreventedreabsorptionofcholesterolinthesmallintestine.