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Towards novel strategies to improve lipid homeostasis - targeting the intestineWulp, Mariëtte Ymkje Maria van der

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Towards novel strategies to improve lipid homeostasis –

targeting the intestine

Mariëtte van der Wulp

Research on nutrition and health integrates laboratory and clinical studies to identify food The research described in this thesis was conducted at the Department of Pediatrics, Beatrix Children's Hospital, Center for Liver, Digestive and Metabolic Diseases, University of Groningen, University Medical Center Groningen, The Netherlands and was financially supported by Top Institute Food and Nutrition, Programme Nutrition and Health.

The author gratefully acknowledges the financial support for printing of this thesis by:

Top Institute Food and Nutrition (TIFN)

Rijksuniversiteit Groningen (University of Groningen)

University Medical Center Groningen (UMCG)

Groningen University Institute for Drug Exploration (GUIDE)

Nederlandse Vereniging voor Gastroenterologie (NVGE)

Sectie experimentele Gastroenterologie van de NVGE

Restaurant en feestgelegenheid Louis XV, Groningen

Cover design by: Hendrix Grafische Vormgeving (Groningen) en Mariëtte van der WulpLayout by: Nikki Vermeulen, Ridderprint BV, Ridderkerk, the NetherlandsPrinting by: Ridderprint BV, Ridderkerk, the Netherlands

ISBN 978-90-367-5797-3 (printed)978-90-367-5798-0 (digital)

© 2012 M.Y.M. van der WulpAll rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means without permission of the author and the publisher holding the copyrights of the articles.

NVGE

Towards novel strategies to improve lipid homeostasis –

targeting the intestine

Proefschrift

ter verkrijging van het doctoraat in deMedische Wetenschappen

aan de Rijksuniversiteit Groningenop gezag van de

Rector Magnificus, dr. E. Sterken,in het openbaar te verdedigen op

maandag 3 december 2012om 14.30 uur

door

Mariëtte Ymkje Maria van der Wulp

geboren op 14 maart 1981te Deventer

Promotores: Prof. dr. H.J. Verkade Prof. dr. A.K. Groen Prof. dr. E.H.H.M. Rings

Beoordelingscommissie: Prof. dr. R.P.J. Oude Elferink Prof. dr. K.N. Faber Prof. dr. G.P.A. Smit

Voor Nathanja,mijn lieve zusje

I carry your heart

I carry it in my heart

(E.E. Cummings - 95 Poems, 1958)

Paranimfen: Sjoerdtje Johanna van der Veen Marjan Wouthuyzen-Bakker

Table of Contents

Chapter 1 General introduction 9

Chapter 2 Laxative treatment with polyethylene glycol does 39 not affect lipid absorption in rats

Chapter 3 Laxative treatment with polyethylene glycol decreases 55 microbial sterol conversion in the intestine of rats

Chapter 4 Transintestinal cholesterol excretion can be 73 manipulated by dietary fat composition in mice

Chapter 5 Genetic inactivation of the bile salt export pump in 93 mice profoundly increases fecal cholesterol excretion

Chapter 6 Conclusion, discussion and future perspectives 109

Appendices Summary 123

Nederlandse samenvatting 125

Dankwoord 127

Biography/Biografie 132

List of publications 133

Chapter 1

General Introduction

Mariëtte Y.M. van der Wulp, Henkjan J. Verkade, Albert K. Groen

Adapted from: Regulation of cholesterol homeostasis. Mol Cell Endocrinol. 2012 Jun


11

Chapter

1Introduction

The intestinal epithelium forms a unique interface for interactions between the body and food

components. It allows selective passage of nutrients, while offering protection against harmful

substances. Under physiological conditions, the epithelium maintains a peaceful relationship with the

extremely large community of commensal bacteria that live in the intestine and influence our health. 1

The intrinsically dynamic epithelium with its ability to respond to luminal stimuli, provides ample

possibilities to modulate intestinal physiology by nutrition and microbiota.

Cholesterol is of vital importance for vertebrate cell membrane structure and function. 2 Metabolites of

cholesterol, such as bile salts (BS), steroid hormones and oxysterols, fulfill important biological functions. 3

Hypercholesterolemia however represents a major risk factor for cardiovascular disease. 4,5 Cholesterol

homeostasis is tightly regulated by its intestinal absorption, fecal excretion and de novo synthesis. 6

Classically, fecal cholesterol excretion was believed to be primarily driven by cholesterol secreted via

the hepatobiliary pathway. 7 However, it has very recently become apparent that direct secretion of

cholesterol from the blood compartment to the intestine, the process now adopted “TransIntestinal

Cholesterol Excretion” (TICE), plays a major role in fecal cholesterol disposal. 6,8 Reduction of cholesterol

absorption and induction of TICE represent attractive targets to facilitate cholesterol disposal. Ideally,

inhibition of cholesterol absorption and/or induction of TICE would be facilitated by simple dietary

intervention.

BS are important for absorption of cholesterol, fat and fat-soluble vitamins. On the other hand, elimination

of excess cholesterol from the body is partly facilitated by breakdown of cholesterol to BS in the liver and

their subsequent fecal excretion. 9

In this theses we assessed, in a quantitative manner, intestinal function with respect to its capacity to

digest and absorb lipids (dietary fats, cholesterol and BS), and its capacity to secrete cholesterol under

varying intestinal conditions.

In this general introduction, first of all a short overview of BS homeostasis [1] and dietary lipid (dietary fat

and cholesterol) absorption [2] will be provided. Subsequently, the introduction will cover cholesterol

homeostasis including the transport of cholesterol through plasma [3], the characteristics, analytical

possibilities and (pharmacological) inhibition of cholesterol synthesis [4] and absorption [5], and finally

pathways of cholesterol excretion [6].

1. Bile salt homeostasis

Under physiological pH (6-8) biliary and intestinal bile acids are present in the form of sodium salts 10 and

will therefore be referred to as BS. BS are amphiphatic molecules, which are produced by the liver from

cholesterol. BS stimulate bile flow and biliary phospholipid (PL) secretion, regulate their own synthesis,

and are indispensable for efficient lipid absorption (including dietary fats, cholesterol and fat-soluble

vitamins A,D,E and K). Chenodeoxycholate (CDC) and cholate are the major primary (produced by the

liver) BS. The rodent liver converts the majority of CDC to more hydrophilic α- and β-muricholic acid (α-/

β-MC). 11 Intrinsically, rodents show a more hydrophilic BS pool compared with humans. 12

Chapter 1

12

Nearly all BS are conjugated by liver peroxisomes, mainly with taurine in rodents and glycine in humans. 12,13 The liver secretes these BS conjugates across the canalicular membrane into the bile canaliculi.

This canalicular secretion occurs against a high concentration gradient and is facilitated by adenosine-

triphospate (ATP)-dependent transporters, the most important of which is the Bile Salt Export Pump

(BSEP or ABCB11). 14,15 Via the bile canaliculi, BS are finally transported to the duodenum where they

facilitate the solubilization of lipids.

BS can be reabsorbed passively, but are mainly reabsorbed actively by the Apical Sodium-dependent

BS Transporter (ASBT) 16 in the terminal ileum (overall ~95%). At the basolateral enterocyte membrane,

BS leave the cells via the organic solute transporter (OSTα/β). 17 They are transported back to the liver

through the portal system and are mostly taken up via the Na+-dependent Taurocholate Co-transporting

Polypeptide (NTCP) at the basolateral hepatocyte membrane. 18 This process is called enterohepatic

cycling (EHC). 9 Under physiological circumstances BS transport from hepatocyte into the bloodstream

is negligible. However, this may rapidly change under cholestatic conditions, where BS are delivered to

the blood via members of the Multi drug resistance Related Proteins (MRPs), such as MRP4/ABCC4. 19

The BS that escape absorption in the terminal ileum enter the colon. Primary BS can be deconjugated

and converted to secondary BS species by the intestinal microbiota. Bacteria can convert β-MC to

hyodeoxycholate (HDC) 20,21 and ω-MC. In addition, they are able to convert CDC to lithocholate (LC,

mainly in humans) 22 or ursodeoxycholate (UDC) 23, and cholate to deoxycholate (DC) 22. A part of colonic

BS is passively absorbed, while the remaining part is excreted with feces. Excretion of BS with feces

actually represents an important pathway for cholesterol disposal. Under steady state conditions, the

liver compensates for fecal BS loss by de novo BS synthesis.

BS regulate their own secretion and synthesis via an elaborate feedback pathway. 24 Hepatic BS activate

the nuclear receptor (NR) farnesoid X receptor (FXR), which induces BS secretion via BSEP and reduces

basolateral BS uptake via NTCP.

After uptake in the terminal ileum, BS activate intestinal FXR which activates OSTα/β-mediated export

of BS. FXR also induces the short heterodimer partner (SHP), resulting in release of fibroblast growth

factor (Fgf ) 15 (mice) or FGF19 (humans) into the portal blood. 24 Fgf15/FGF19 activates the fibroblast

growth factor receptor 4 (FGFR4) in the liver, which ultimately facilitates transcriptional inhibition of

cholesterol 7 alpha-hydroxylase (Cyp7a1, encoding the rate-limiting enzyme for BS synthesis). 25 Both

SHP and FXR lack a Cyp7a1 DNA binding site. Liver FXR is activated primarily by primary BS (mainly

CDC), which induces SHP. SHP can activate the liver-related-homolog-1 (LRH-1, rodents) or α-fetoprotein

transcription factor (FTF, humans). LRH-1/ FTF can bind the bile acid response element II (BARE-II) in the

Cyp7a1 gene, inhibiting its transcription. SHP blocks the interaction between hepatocyte nuclear factor

4α (HNF4α) and peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α), which also inhibits

Cyp7a1 transcription via BARE-II. Cyp7a1 transcription can in addition be inhibited via steroid hormones,

inflammatory cytokines, insulin, growth factors and NRs that operate independently of FXR. 26 LC is a

ligand for the pregnane X receptor (PXR), constitutive androstane receptor (CAR; indirectly) and vitamin

D receptor (VDR).


13

Chapter

1PXR and VDR bind the BARE-I and block recruitment of PGC1α, inhibiting Cyp7a1 transactivation by

HNF4α. CAR binds the BARE-II and competes with HNF4α for several coactivators, including PGC1α. 26

On the other hand, when the liver X receptor (LXR) is activated by oxysterols, transcription of Cyp7a1 is

induced via the BARE-I in rodents (not in humans due to an alteration in the BARE-I sequence), resulting

in production of BS from cholesterol. 24

It is becoming more and more clear that BS are not just simple detergents necessary for lipid absorption,

but are also involved in the regulation of glucose, lipid homeostasis as well as energy expenditure. 27

Recent data show that removal of BS from the intestine with sequestrants can decrease plasma low

density lipoprotein (LDL) levels and hyperglycemia in patients with type II diabetes. 28 The mechanisms

by which the total pool size and/or profile of BS influence different physiological processes are only

beginning to be understood.

2. Dietary lipid absorption

2.1 Dietary fat absorption

Lipid absorption in general involves emulsification, lipolysis, micellar solubilization, uptake by mucosal

epithelium, re-esterification, chylomicron (CM) formation and lipoprotein metabolism. 29 This paragraph

will focus on dietary fat, whereas specific aspects of cholesterol absorption will be discussed in paragraph

2.2. Triglycerides (also called triacylglycerols; TG) form the major lipid component in the human diet.

Lipids are dispersed by chewing, which increases the surface area. In the stomach, partial hydrolysis by

gastric and lingual lipase takes place (10-30% of TG) and diacylglycerol and free fatty acids (free FA) are

released. 30,31 Pancreatic lipase facilitates further hydrolysis, producing monoacylglycerol and free FA. 32

PL (from diet, bile and sloughed intestinal epithelial cells) are hydrolyzed by phospholipase A2, yielding

lyso-PL and free FA. 33 The products of pancreatic lipolysis have limited solublility and need subsequent

solubilization by BS and/or association with PL. 34,35

Adjacent to the luminal surface of enterocytes, micellar dissociation is promoted by decreased pH (5.3-

6.0) 36 in the so-called unstirred water layer 37, allowing for diffusion of FA across the cellular membrane.

FA can also be taken up by transporters such as FA transporter protein 4 (FATP4) and FA translocase (FAT/

CD36), but none of these transporters were shown to be critical for FA absorption. 38,39

In the enterocyte, FA may bind to the intestinal FA binding protein (IFABP) and diffuse into the

endoplasmatic reticulum. After activation acyl-CoAs are produced and re-esterified into TGs via

several steps. 40 Microsomal TG transfer protein (MTP) assembles TG, PL and cholesterol together with

apolipoprotein B48 (ApoB48) to form a CM particle or with ApoB100 (not in humans) to form a very low

density lipoprotein (VLDL) particle, both of which are secreted to the interstitium via the basolateral

membrane. 41 From the intestitium the particles enter lymphatic capillaries that drain into omental

lymphatic channels and eventually reach the systemic circulation via the thoracic duct. 40

Chapter 1

14

2.2 Cholesterol absorption

Cholesterol present in the intestinal lumen derives from several sources, including diet, bile, intestinal

secretion and desquamated epithelial cells. In humans consuming Western type diets, 300-500 mg

dietary cholesterol enters the intestinal lumen per day, whereas the contribution of biliary cholesterol

has been estimated to be approximately 800-1200 mg per day. 42 Cholesterol is a hydrophobic molecule,

and it intestinal absorption is facilitated via similar steps as described for FA (emulsification, hydrolysis of

dietary esterified cholesterol, micellar solubilization, and uptake within enterocytes). In healthy humans

normally approximately 50% of intestinal cholesterol is absorbed. 43 Micellar solubilization of cholesterol

is essential for cholesterol absorption. 44 The physical chemistry of biliary cholesterol may influence its

absorption in the intestine. Whereas micellar biliary cholesterol is readily available for absorption, dietary

cholesterol first has to be released from food oils or tissue membranes. Elegant isotope infusion studies

in rats, however, showed that only on high cholesterol diet dietary cholesterol is relatively malabsorbed

compared with biliary cholesterol.

On low and moderate cholesterol enriched diets micellized and non-micellized cholesterol appear

in lymph in similar amounts. 45 Biliary cholesterol is unesterified, whereas dietary cholesterol is partly

esterified. Dietary cholesterol esters (CE) thus must be hydrolyzed by pancreatic carboxyl ester lipase

(CEL) before cholesterol can be transported into enterocytes. Howles et al. showed that dietary esterified

cholesterol absorption was reduced by >60% in CEL-null mice, whereas absorption of free cholesterol

was normal. 46 Considering the low daily supply of intestinal CE 47, hydrolysis of CE by CEL may not be

critical for overall cholesterol absorption. CEL may however serve a function similar to phospholipase A2,

in providing sufficient hydrolysis of PL (mainly phosphatidylcholine (PC), which is required for adequate

micelle formation. For example, in rats drained of bile and pancreatic juice, administration of CEL

enhanced lymphatic recovery of cholesterol infused into the duodenum as a micellar, PC containing,

solution. 48 Moreover, CEL itself may be absorbed by enterocytes via endocytosis. 49 and the ceramidase

activity of CEL has been implicated in proper intracellular cholesterol trafficking and CM assembly. 50

In addition to cholesterol, a typical Western diet contains structurally similar phytosterols (including

plant sterols (~95%) and stanols (~5%)), in similar amounts as cholesterol, depending on the diet.

Plant sterols and stanols inhibit the absorption of cholesterol, however the mechanism(s) by which they

do so remain a matter of discussion (see paragraph 5.3.2). Uptake of cholesterol and plant sterols and

stanols is believed to be facilitated by the Niemann-Pick C1 Like 1 (NPC1L1) transporter 51, which is

located in the brush border membrane of enterocytes in the proximal (jejunum), but not the distal

(ileum) small intestine. Npc1l1 null mice showed 70% and 90% reduction in cholesterol and plant sterol/

stanol absorption, respectively, compared with control mice. 52,53

In addition, Npc1l1 null mice showed upregulation of intestinal and hepatic HMG-CoA synthase mRNA

and intestinal cholesterol synthesis. 53 In the proximal small intestine (and apical hepatocyte membrane),

NPC1L1 co-localizes with ABCG5 and ABCG8 (figure 1).


15

Chapter

1HDL

ApoA1

De novo synthesis

CM ApoB48

MTP

CE ACAT2

PS

Chol

LDL ApoB100

NPC1L1 ABCG5/G8

Chol PS Intestinal lumen

Lymph CM ApoB48

ABCA1 SR-‐B1 LDLR

A

LDL ApoB100

HDL ApoA1

De novo synthesis

IDL ApoB100

VLDL ApoB100 VLDL

Chol

ACAT2

MTP

CE

Apical membrane

Basolateral membrane

BSEP

BS

ABCG5/G8

NPC1L1

LDLR

ABCA1

ApoB100

SR-‐B1

LRP1 CR ApoE

B

Figure 1. Cholesterol transporters, converting enzymes and lipoproteins in liver and intestine. A: Cholesterol in enterocytes originates from absorption, synthesis and uptake from the circulation. Cholesterol and plant sterols/ stanols are absorbed from the intestinal lumen via NPC1L1 and secreted back to the lumen via ABCG5/G8. Intracellular cholesterol and plant sterols/ stanols can be esterifi ed by ACAT2, packaged into apoB48 containing CM by MTP, and secreted to the lymph. Alternatively, cholesterol can be secreted via ABCA1 in ApoA1 containing HDL particles. Uptake of cholesterol on the basolateral side occurs via SR-B1 (HDL-c) and LDLR (LDL-c), which recognizes ApoB100. B: The liver takes up HDL-c via SR-B1 and ApoE containing CR-c via LRP1 and the LDLR. In the hepatocyte ACAT2 esterifi es cholesterol and CE are reassembled into VLDL particles together with CR-derived products. VLDL particles contain apoB100 instead of apoB48. ApoB100 binding to MTP results in loading of lipids to the VLDL particle. The liver secretes the VLDL particles into the circulation for delivery of lipids to the periphery. VLDL is in part cleared by the hepatic LDLR, whereas the rest is transformed to IDL and LDL, which still contain apoB100 necessary for reuptake in the hepatocyte via the LDLR. The liver secretes cholesterol to bile via ABCG5/G8 and transforms cholesterol to BS, which are secreted via BSEP. Hepatic NPC1L1 can facilitate reuptake of biliary cholesterol. Abbreviations: ACAT2: acyl CoA:cholesterol acyltransferase 2, CM: chylomicrons, CR: chylomicron remnants, LRP1: LDLR-related protein 1, MTP: microsomal triglyceride transfer protein, CE: cholesteryl esters, BS: bile salts, BSEP: bile salt export pump.

Effl ux of unesterifi ed cholesterol and plant sterols and stanols from the enterocyte back to the intestinal

lumen, as well as biliary cholesterol secretion are facilitated by ABCG5/G8. 54,55 Intracellular cholesterol

that is not effl uxed by ABCG5/G8 travels to the endoplasmatic reticulum, where it is esterifi ed by acyl

CoA:cholesterol acyltransferase 2 (ACAT2). 56 When LXR is activated, it forms a heterodimer with the

Retinoid X receptor (RXR) and the complex induces transcription of target genes encoding proteins

involved in cholesterol disposal, including the two half-transporters ATP-binding cassette sub-family G

member 5 and 8 (Abcg5/ Abcg8). 57 Transcription of Npc1l1 on the other hand is downregulated upon

LXR activation. This likely repesents an indirect eff ect, since LXR is not known as a direct repressor. 24

Intracellular cholesterol is partly re-esterifi ed and fi nally delivered to the bloodstream, mainly as

component of CM, together with TG, PL and apoB48, for transport to the lymph. MTP regulates this

process, and also facilitates VLDL formation after cholesterol esterifi cation by ACAT2 in hepatocytes. 58

Chapter 1

16

In addition, enterocytic cholesterol can be transferred to apoAI high density lipoprotein (HDL) particles

via ABCA1 (basolaterally located in enterocytes; figure 1). 59

Up to now, only NPC1l1 seems to be critical for apical intestinal cholesterol import. 52,53 Studies, in which

proposed alternative candidates, such as scavenger receptor class B member 1 (SR-B1), CD36 and

caveolin-1, were eliminated, showed that none of these proteins was crucial for cholesterol uptake. 60-64

ABCA1 was implicated in cholesterol absorption in the past. However, unchanged fecal sterol excretion

in Abca1-/- mice indicated that ABCA1 plays no role in control of cholesterol absorption. 65

In vitro studies suggested a possible role for aminopeptidase N (CD13) in cholesterol absorption 66 which

has not yet been confirmed in in vivo studies. In contrast to ABCA1, ACAT2 and MTP do affect cholesterol

absorption (paragraph 5.3).

Cholesterol homeostasis

Disturbances in cholesterol homeostasis are associated with potential life-threatening consequences.

Hypercholesterolemia promotes atherosclerosis and thereby represents a major risk factor for

cardiovascular disease. 67,68 Pharmacological inhibition of cholesterol synthesis has been the most potent

treatment option for hypercholesterolemia during the last decades. Cholesterol synthesis inhibition by

itself however, can reduce the risk of cardiovascular disease by only a third. 69 This underlies the ongoing

search for alternative therapeutic modalities.

The liver has been considered the major site of control in maintenance of cholesterol homeostasis. 70 The

liver facilitates clearance of VLDL/ LDL particles and cholesterol-containing CM remnants, synthesizes

cholesterol, synthesizes and secretes (nascent) HDL particles, secretes cholesterol and BS to bile and is

involved in reverse cholesterol transport (RCT). 7

RCT is classically defined as the process by which cholesterol from peripheral tissues is transported to

the liver, followed by excretion via bile to feces in the form of neutral sterols and BS. In recent years,

however, the importance of the intestine in many aspects of cholesterol physiology is increasingly

recognized. The intestine has a major impact on cholesterol homeostasis at the level of cholesterol (re-)

absorption, fecal excretion and de novo synthesis. 71 It has become apparent that, at least in mice, direct

secretion of cholesterol from the blood compartment into the intestine, i.e. TICE, plays a major role in

disposal of cholesterol via the feces. 72

It would be desirable to induce fecal cholesterol loss via the intestine without increasing biliary

cholesterol secretion and thereby the risk of cholesterol gallstones. 73

3. Transport of cholesterol through plasma

A plethora of epidemiological studies have unequivocally shown that increased plasma cholesterol

levels are associated with cardiovascular disease risk. Interestingly this does not necessarily coincide

with increased tissue cholesterol, but is probably caused by changes in rates of secretion and uptake of

cholesterol. 74 Cholesterol is a lipophilic molecule which is transported through blood in lipoproteins.


17

Chapter

1The type of lipoprotein is determined by its buoyant density and apoprotein composition, which act as

emulsifying coating and target their metabolism. 75

There are marked differences in lipoprotein metabolism between humans and rodents. For example,

mice do not possess cholesterol-ester transport protein (CETP) and have an up to 40-fold higher LDL

clearance by the liver compared to humans. 70,76 Mice carry most of their plasma cholesterol in HDL

particles and as such are not a good model for human disease. 70,76

This is the reason why many studies on cholesterol metabolism have been performed in mice with

genetic deficiency of major determinators of plasma cholesterol metabolism such as the Ldl receptor

(Ldlr) 77 or ApoE 78. More recently “humanized” mice have become available in which human CETP 79 is expressed. On an Ldlr null or ApoE3 Leiden background these mice mimic many aspects of the

hyperlipidemic human phenotype. 80,81 Inhibition of CETP increases HDL and should ameliorate

atherosclerosis, but clinical trials with the first CETP inhibitor (torcetrapib) were terminated because of

adverse off-target effects (increased mortality and cardiovascular events).

Recently, a new CETP-related drug (dalcetrapib) showed lack of a clinically meaningful benefit in a clinical

trial, and further testing of the drug has been halted.82 Until now, inhibition of cholesterol synthesis has

remained the first line of therapy for hypercholesterolemia.

4. Cholesterol synthesis

4.1 Control of cholesterol synthesis

Cholesterol is synthesized from its precursor unit acetyl-CoA via a complex metabolic pathway

summarized in figure 2. 83,84 HMG-CoA reductase is the rate-limiting enzyme in cholesterol synthesis 85.

Recently squalene monooxygenase, which catalyzes the first oxygenation step in cholesterol synthesis,

was suggested to represent a possible second control point in cholesterol synthesis beyond HMG-

CoA reductase. 86 Cholesterol and fat biosynthesis are under control of a family of transcription factors

designated Sterol Regulatory Element Binding Proteins (SREBPs). Three isoforms of SREBP have been

described, i.e., SREBP1a, SREBP1c and SREBP2. Genes encoding enzymes and tranporters involved in

cholesterol absorption and efflux are under tight transcriptional control (reviewed recently by 57).

Cholesterol and its biologically active metabolites act as ligands for NR that regulate gene expression.

LXR is a major regulator of cholesterol metabolism. 57 When sterols are present in excess intracellularly,

LXR-mediated activation of SREBP1c transcription leads to induction of oleic acid synthesis. Oleic acid

is the preferred FA for the synthesis of cholesterol esters (CE), which can be stored intercellularly. LXR

activation thus protects cells from accumulation of excess free cholesterol, which is toxic and can result

in cell death.

Chapter 1

18

Figure 2. Cholesterol biosynthesis pathway. Cholesterol is synthesized from its precursor unit acetyl-CoA (Ac-CoA). Two acetyl-CoAs are condensed, forming acetoacetyl-CoA (AcAc-CoA). AcAc-CoA and a third acetyl-CoA are converted to 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) by the action of HMG-CoA synthase. HMG-CoA is converted to mevalonate by HMG-CoA reductase. Mevalonate is subsequently converted to an isoprenoid molecule, isopentenyl pyrophosphate (PP), with the concomitant loss of CO

2. Geranyl-PP and farnesyl-PP are produced from

isopentenyl-PP. Two farnesyl-PP subunits are combined to form squalene. Squalene is converted to lanosterol and subsequently cholesterol via many intermediates, including zymosterol, desmosterol and lathosterol. Solid line: direct step. Dashed line: product is formed via intermediate steps.

Cholesterol synthesis is tightly regulated by SREBPs (mainly type 2). When cellular cholesterol is high,

SREBP2 is located to the ER in a complex with SREBP2 cleavage–activating protein (SCAP). When cells

are depleted of sterols, SCAP escorts SREBP2 from the ER to the Golgi apparatus, where it is cleaved

in order to release part of the protein from the membrane. SREBP2 then can enter the nucleus, bind

to a sterol response element (SRE) in the enhancer/ promoter region of many target genes involved

in cholesterol synthesis, and activate their transcription. 87 The importance of cholesterol synthesis for

survival is illustrated by the fact that defects in the cholesterol synthesis pathway are generally lethal in

mice. Complete loss of function of early cholesterogenic enzymes is rarely described in humans and

deficiencies of these enzymes lead to severe malformations and disease.88

4.2 Measurement of cholesterol synthesis in vivo

The different methods available to determine cholesterol synthesis in vivo include sterol balance,

(plasma) cholesterol precursor measurement and tracer incorporation techniques [such as deuterium

incorporation (DI) and mass isotopomer distribution analysis (MIDA)], reviewed in ref. 89.


19

Chapter

1An early approach to estimate cholesterol synthesis has been cholesterol balance: measurement of

cholesterol and BS excretion in feces followed by subtraction of dietary cholesterol intake yields whole

body synthesis. Surrogate serum markers of cholesterol synthesis (and absorption) will be discussed

in section 3. Here we will briefly discuss the two more recently developed and refined methods:

DI and MIDA. The DI (2H) method has a similar background as the tritiated (3H) water method used

in experimental animals. The theory behind the method is described below. Deuterated water can

equilibrate in total body water and NADPH. Enrichment of deuterium in plasma water, representing

the precursor pool, and deuterium enrichment of cholesterol in either plasma or red blood cells can be

determined sensitively by isotope ratio mass spectrometry.

Eighteen acetyl CoA units containing 36 carbon (C) atoms are utilized to synthesize one molecule of

cholesterol, which contains 27 C atoms and 46 hydrogen (H) atoms. During synthesis of a cholesterol

molecule, 7 H atoms are incorporated originating directly from water, while another 15 H atoms are

inserted from nicotinamide adenine dinucleotide phosphate-oxidase (NADPH).

H atoms from water may also become incorporated into substrates that, later on, are used for the

generation of the cytosolic acetyl CoA pool that is used for cholesterol synthesis. If no labeled H from 2H

water were incorporated into these precursors of the cytosolic acetyl CoA pool, and if the reductive H of

the NADPH were derived entirely from unlabeled sources, only 7 labeled H atoms (those orginating from

the administered 2H water) could be found in each newly synthesized cholesterol molecule (minimal

theoretical labeled H/C incorporation ratio would be 7/27). Alternatively, if NADPH fully equilibrates with 2H labeled water, then a final labeled H/C incorporation ratio of 22/27 would be found. This is considered

the maximal labeled H/C ratio in short term measurements. 90 On the long run, an even greater value

might be obtained when there is significant incorporation of labeled H into an important acetyl-CoA

precursor, which by then has been used for cholesterol synthesis.

On a theoretical basis it is difficult to predict exactly how many labeled H atoms will be incorporated

into each cholesterol molecule. Labeled H/C ratios are not available for all organs. In short term in

vivo studies in mice and rats, 21-25 tritiated H atoms were incorporated into cholesterol molecules of

whole carcass, liver or brain per C atom entering the biosynthetic pathway as acetyl CoA 90 and these

values were subsequently used in other studies to calculate absolute cholesterol synthesis. However,

combining the DI method with MIDA (discussed below) in long term experiments (up to 8 weeks), the

maximum incorporation number in rat liver was found to be 30. 91 This could mean that earlier studies

have overestimated true cholesterol synthesis rates. Many human studies using the DI method have

used H incorporation values obtained from literature 92 and thus provide rough estimates of absolute

cholesterol synthesis rates.

With MIDA the precursor-pool enrichment, fractional synthesis, and absolute (whole body) synthesis

of cholesterol are calculated based on the pattern of excess enrichment among mass isotopomers of

cholesterol present in plasma after administration of stable isotope-labeled 13C-acetate. 93

Chapter 1

20

The distribution of abundances in newly formed cholesterol molecules, measured by gas-

chromatography-mass spectrometry, allows calculation of the abundance in the precursor. MIDA thus

eliminates the need to measure the precursor pool enrichment directly.

Knowing the true precursor-pool enrichment allows calculation of the fractional and absolute cholesterol

synthesis rates. MIDA reveals the weighted mean abundance of the precursor pools contributing to the

product mixture. 94

In the past, human as well as animal studies applying MIDA were performed in relatively short time

frames, which could underestimate whole body synthesis due to circadian effects and/or incomplete

equilibration of the free cholesterol pool. A human study showed that the DI method and MIDA yield

similar rates of fractional and absolute cholesterol synthesis when measured over at least 24h. 95

4.3 The body cholesterol pools

A complication when assessing de novo cholesterol synthesis is the existence of different pools of

cholesterol in the body with a differential rate of exchange. To incorporate this aspect, the turnover

of plasma cholesterol has been modeled by dividing total body cholesterol into 2 or 3 exchangeable

pools. 92,93

In the 3-pool model, pool 1 (rapidly miscible) is considered to consist of cholesterol in fairly rapid

equilibrium with plasma cholesterol (plasma, blood cells, liver and intestines), pool 2 consists of

cholesterol that equilibrates at an intermediate rate (visceral and peripheral tissues) and pool 3

represents the slowest cholesterol turnover compartment (adipose and connective tissue, skeletal

muscle and arterial walls).

The 3-pool model has been used for long term studies (up to 48 weeks). 92 Differentiation between pools

2 and 3 in short term experiments appeared not essential for accurate mathematical modeling. Pool 1 in

the 2-pool model represents the rapidly exchangeable pool similar to that in the 3-pool model, whereas

pool 2 of the 2-pool model represents a combination of pool 2 and 3 of the 3-pool model. 93

The central nervous system contains a major part of total body cholesterol (15% and 23% in mice and

humans, respectively). There is no detectable uptake of plasma cholesterol into the brain via the blood-

brain-barrier. 96 The central nervous system presumably excretes only a very small amount of cholesterol

to pool 3 under physiological conditions. Thus, it must be kept in mind that brain synthesis is not taken

into account when calculating whole body cholesterol synthesis.

In general, whole body cholesterol synthesis rates measured using either the DI method or MIDA were

shown to be comparable with those reported with the classical cholesterol balance (cholesterol intake

+ synthesis = fecal excretion of BS + neutral sterols). 93,97

4.4 Regional and whole body cholesterol synthesis

Virtually every mammalian cell synthesizes cholesterol, in most animals the main part being synthesized

in extrahepatic organs. 70,98,99 Whole body synthesis and the contribution of liver and intestine to whole

body synthesis have been determined for several species.


21

Chapter

1A complication in evaluating the value of these measurements is the circadian rhythm of cholesterol

synthesis, which has not always been taken into account (see below). It seems justified to conclude

that species such as hamsters, guinea pigs, rabbits and squirrel monkeys and humans have much lower

synthesis rates than rats and mice.

The gastrointestinal tract contributes around 15-35% to total cholesterol synthesis in experimental

animals. In rodents, the contribution of the liver to whole body synthesis at night has been shown to

vary from as low as ~15-20% in rabbits and guinea pigs to as high as 50% in rats. 70 In humans the liver

is thought to contribute only around 10% to whole body synthesis 70 (~10 mg/kg/d). Low rates of local

synthesis relative to the rates of uptake of newly synthesized cholesterol from blood in rats were found

in adrenal glands, spleen, lung and kidneys. These organs increase their cholesterol synthesis when

circulating levels of plasma cholesterol are decreased. 99

4.5 Influence of diet and time of day on cholesterol synthesis

Whole body as well as organ specific cholesterol synthesis rates vary depending on the presence of

cholesterol and other lipids in the diet. Dietary cholesterol suppresses hepatic cholesterol synthesis. In

rats fed no dietary fat, liver synthesis rates are decreased when feeding a 2% cholesterol containing diet,

while synthesis rates in the intestine and other extrahepatic tissue remain similar, suggesting that only

the liver senses the increase in cholesterol uptake. 100 Adding cholesterol to either a low or high fat diet

also leads to decreased hepatic cholesterol synthesis in hamsters. 101

It appears that dietary fat alone however does not affect hepatic and whole body cholesterol synthesis,

but can induce intestinal cholesterol synthesis. This was illustrated by several studies. First of all, on a high

fat compared with low fat diet, hepatic cholesterol synthesis is comparable in hamsters. 101 In addition,

it was shown in mice on a 0.2% cholesterol diet that dietary short, medium and long chain fatty acids

did not differentially affect extrahepatic cholesterol synthesis. 102 Infusion of corn oil in non-cholesterol,

non-fat fed rats increases cholesterol synthesis in the intestine, while liver synthesis remains the same. 100

A high fat diet in hamsters also increases cholesterol synthesis in the intestine in the presence of dietary

cholesterol, however whole body synthesis is not affected 103.

In addition to dietary fat and cholesterol content, the type of diet influences cholesterol synthesis

rates. These were shown to be lower on purified diets as compared to non-purified diets. 104,105 Energy

restriction per se seems to have the greatest (lowering) effect on cholesterol synthesis (reviewed by 89).

Both animals and humans display a circadian rhythm of cholesterol synthesis, with a peak in synthesis

several hours after feeding. Most studies on cholesterol turnover in experimental animals have been

performed during the dark phase of the light cycle. 106 It must be kept in mind that cholesterol synthesis

is 2-3 fold higher during the (end of the) dark phase compared to the light phase. 93,107,108 In extrahepatic

tissues the difference between day and night in cholesterol synthesis is much smaller compared with

the liver, at least in mice. 107 Circadian rhythm in the liver is controlled by different molecular mechanisms,

including cAMP-dependent and CLOCK/ BMAL1 regulation, which for example regulate expression of

cholesterol synthesis genes Hmgcr, Hmgcs, Sqs, Fpps and Cyp51. For details, see ref. 109-111.

Chapter 1

22

4.6 Targeting cholesterol synthesis in hypercholesterolemia

HMG-CoA reductase inhibitors (statins) have been used successfully to inhibit cholesterol synthesis and

reduce all-cause mortality since the late 1980’s. 112 Reduced hepatic cholesterol is compensated for by

synthesis of LDLR to draw cholesterol out of the circulation 113 leading to lowering of plasma cholesterol

levels. Some statins (pravastatin) seem to preferentially inhibit synthesis in liver and intestine (90%) 114,115 compared with synthesis in extrahepatic organs, such as kidneys (73%), testes (55%), lungs (53%),

spleen (53%) and adrenals (49%). 114 Other statins (lovastatin) exhibit widespread cholesterol synthesis

inhibition. 116 This may be mainly due to differences in cellular uptake 115, which can be caused by

differences in lipophilicity, pH-sensitivity, plasma protein binding, activity of metabolites and transporter

facilitated uptake and export. For example, simvastatin and lovastatin are administered as very lipophilic

(tissue penetrating) lactone prodrugs, whereas other statins are administered in their active form. 117

Different statins have different affinities for import proteins of hepatocytes such as organic anion

transporting polypeptides (OATP) and NTCP. 118-121 In addition, polymorphisms in these transporters

differentially affect statin uptake and (in vitro) affinity for transporters can differ between human and

experimental animal cells. 121

Although highly effective, statins do not produce the desired health effect in a significant group of

patients and can cause severe side effects such as myalgia and myopathies (rhabdomyolysis), 122 in

particular in case of polypharmacy. Statins decrease farnesyl and geranyl pyrophosphate (isoprenoids)

and dolichol synthesis, reducing essential post-translational prenylation and N-linked glycosylation of

proteins. This may result in myocyte and hepatocyte cell damage and even cell death. 123,124 Newer statins,

such as the recently approved pitavastatin, with a distinctive metabolic profile, may display a reduced

incidence of these adverse effects. 125 In addition, efforts are ongoing to produce new drugs targeting

cholesterol synthesis at different levels of the pathway. Squalene synthase inhibitors for example

increase farnesyl diphosphate while decreasing cholesterol synthesis. They are able to prevent statin

induced changes in protein farnesylation and cell death in vitro. 124 Since these inhibitors do not target

true rate-limiting steps in cholesterol synthesis, they may not effectively reduce cholesterol synthesis as

a monotherapy in tolerable dosages.

Variations in efficacy of statins can be caused by many factors, including race (genetics), body weight

and diet. Humans that inherently have lower cholesterol synthesis rates may respond less adequate to

statin treatment. LDL-cholesterol (LDL-c) response to statins is lower in black compared to white people,

which is associated with lower baseline LDL-c and variant haplotypes of the HMG-CoA reductase gene

in black people. 126 Statins may induce expression of proprotein convertase subtilisin-like kexin type

9 (PCSK9) and SREBP2. PCSK9 is a circulating protein that impairs LDL clearance by promoting LDLR

degradation. PCSK9 thus could diminish statin efficacy, hence PCSK9 inhibitors are currently tested in

clinical trials. 127

In general, cholesterol synthesis inhibition leads to a reciprocal increase in cholesterol absorption, 128

which has led to elaborate research to develop cholesterol absorption inhibitors (discussed below). With

growing knowledge on the determinants of response to cholesterol lowering therapy, in the future it

may become feasible to individually tailor treatment.


23

Chapter

15. Measurement and inhibition of cholesterol absorption

5.1 Measurement of cholesterol absorption in vivo

The available methods to measure cholesterol absorption in humans and mice include (radiolabeled or

stable) isotope based methods (fecal and plasma dual isotope method), intestinal perfusion and blood

levels of surrogate markers such as plant sterols and intermediates of cholesterol synthesis. Details can

be found in several reviews. 129,130 The intestinal perfusion method is the only method that can directly

quantitate absorption of intestinal cholesterol in humans. 131,132 Widespread use of this method is limited

by the need for intubation and radiation exposure.

The dual isotope ratio method has been optimized using stable instead of radioactive isotopes both in

the plasma (ratio of intravenously and orally administered labeled cholesterol) 133 and fecal (ratio of orally

administered labeled cholesterol and sitostanol in feces) dual isotope ratio method. 134 The fecal dual

isotope ratio method is limited by requirement of 72h feces collection. The plasma dual isotope ratio

method has been limited by measurement of single time point absorption, however this method can

nowadays be applied for longer periods, at least in mice. 135

The only approach available to easily estimate (relative) changes in cholesterol absorption in large scale

studies is to measure plasma surrogate markers. These include the ratio of plant sterols (campesterol

and sitosterol) or cholestanol (cholesterol metabolite) to cholesterol in plasma. 136 Surrogate makers

have shown to be valuable to answer questions related to changes in cholesterol absorption during

treatments. However, dietary factors (fat, cholesterol, plant sterols and stanols 137, drugs (statins) 138 and

metabolic diseases (diabetes mellitus) 139,140 can disturb correct interpretation of surrogate markers levels.

For example, the reduction in plasma LDL-c correlates poorly with baseline levels of noncholesterol sterol

markers of absorption (campesterol) and synthesis (lathosterol) in African- and European-American men

treated with ezetimibe (a cholesterol absorption inhibitor, discussed in paragraph 3.3.1), simvastatin or

a combination of the two drugs. 141 Similarly, although ezetimibe + simvastatin treatment significantly

reduces campesterol and sitosterol levels in patients with familial hypercholesterolemia, baseline

cholesterol absorption status does not determine LDL-c lowering response to ezetimibe + simvastatin

therapy. 142 It is currently recommended to use several rather than one serum marker, if possible in

addition to absolute measurements. 143

5.2 Targeting cholesterol absorption in hypercholesterolemia

Statin therapy is not sufficient to prevent cardiovascular disease risk in a substantial proportion

of individuals. 144 To increase treatment efficacy considerable interest has arisen in dietary and

pharmacological interventions that inhibit cholesterol absorption, possibly for combining this with

inhibition of cholesterol synthesis . Most agents are nonspecific and require consumption in high daily

quantities while modestly lowering plasma LDL-c. ACAT2 deficiency resulted in cholesterol malabsorption

in mice, however only in cholesterol fed and not in chow fed mice. 145 MTP inhibition normalized plasma

lipoprotein levels in a rabbit model for human homozygous familial hypercholesterolemia. 146

Chapter 1

24

However, this process is related to disturbed CM formation, not specific for absorption of cholesterol

and not intestine specific as it induces fat accummulation in the liver. 146 We will not discuss inhibition

of these intracellular factors. Ezetimibe represents an exception, as it relatively specifically decreases

cholesterol absorption. The impact of dietary plant sterols and stanols is discussed since it has become

apparent very recently that they might have an unexpected intestinal effect (see section 6). Other

cholesterol lowering food components are discussed elsewhere 147

5.2.1 Ezetimibe

Specific inhibition of cholesterol absorption, either from biliary or dietary origin, is accomplished after

binding of ezetimibe to NPC1L1. 148 The compound is first glucuronidated in the intestine before

travelling to the liver via the portal vein. Glucuronidated ezetimibe blocks the internalization of the

NPC1l1/ cholesterol complex 149 but does not block cholesterol absorption completely.

In the Npc1l1 null mouse there is residual cholesterol absorption, indicating that another pathway of

cholesterol absorption must exist. Ezetimibe does not inhibit this residual cholesterol absorption in

Npc1l1 null mice, indicating that this residual pathway is ezetimibe independent. 52

Studies in experimental animals indicated that ezetimibe may decrease absorption of vitamin E

(α-tocopherol), but not vitamin A and D. 150,151 However, ezetimibe did not seem to affect fat soluble

vitamin status in patients. 152,153 Ezetimibe can decrease plasma TG concentration. This does not seem to

be absorption dependent, but rather a secondary effect of ezetimibe induced hepatic LDLR expression

and subsequent increased clearance of ApoB100 and ApoB48 containing lipoproteins, particularly

during co-treatment with statins. 154

In humans, ezetimibe alone decreases cholesterol absorption by 54% and decreases plasma LDL-c

by ~19%, but increases whole body synthesis by 89%. 43 Combined treatment with statins for

hypercholesterolemia, should therefore be very effective. 155 Recently however, the effect of combination

therapy has become controversial. Some trials reported no additive effect of ezetimibe or even a worse

atherosclerosis outcome when ezetimibe is added to patients previously treated with statins. 156 Others

report increased efficacy of combination therapy over statin monotherapy, however data are limited

to the level of serum lipid profiles. 157-160 Due to the lack of well-executed long term trials it is presently

unclear whether combination therapy is better than increasing statin dosage. In the future, ezetimibe

may be prescribed in patients in whom statin therapy is inadequate or intolerated. As monotherapy

ezetimibe may improve postprandial hyperlipidemia and endothelial dysfunction. 161 In the presence of

pancreatic dysfunction, CEL inhibitors may provide additional inhibition of cholesterol absorption when

co-administered with ezetimibe. 150

5.2.2 Plant sterol/ stanol supplementation

In contrast to cholesterol, plant sterols and stanols can not be synthesized by the body and are poorly

absorbed (plant sterols 0.4-3.5% and plant stanols 0.02-0.3%). 162 Fractional absorption of different plant

sterols and stanols varies depending on their side chain length. 163


25

Chapter

1The role of ABCG5/G8 in intestinal and hepatobiliary efflux of plant sterols and stanols versus cholesterol

is not completely clear. In vitro studies showed comparable effectiveness of plant sterol and cholesterol

transport via ABCG5/G8. 164 However, loss of ABCG5/G8 function in mice results in hyperabsorption of

plant sterols and stanols, but not cholesterol, implying that ABCG5/G8 is more important for efflux of

plant sterols and stanols compared to cholesterol. 165 In contrast to cholesterol, plant sterols and stanols

are poor substrates for ACAT2 166, which seems to be the major factor limiting their absorption. Plant

sterols and stanols effectively lower plasma LDL-c levels in adults as well as children with (familial)

hypercholesterolemia. 167

Plant sterols and stanols naturally occur in foods such as oils, (wheat) cereals, nuts and seeds. There are

large differences in plant sterol/stanol consumption throughout the world, within the Western population

and particularly between vegetarians and meat consumers. 168 Plant sterols/stanols are added to food

products such as margarine, yoghurt and juices. To enable emulsification, added plant sterols/tanols are

often esterified and require hydrolysis before being able to compete with cholesterol for absorption.

It was recently suggested that the hydrolysis products of esterified plant sterols/ stanols (stigmasterol

and especially saturated stearic acid) together may be more effective at lowering cholesterol micellar

solubility than free plant sterols/stanols alone. 169

The mechanism via which plant sterols and stanols lower plasma cholesterol does however not seem

to be confined to decreasing cholesterol solubilization in the intestinal lumen. A recent meta-analysis

showed that despite differences in absorption, plant sterols and plant stanols have similar effects on

plasma lipid profiles. 170 Smaller repeated doses (3 times 0.6 g/d) of plant sterol/ stanol margarine as well

as a single high daily dose (1.8 g/d) are equally effective at lowering cholesterol absorption and plasma

LDL-c. 171

Given that plant sterols and stanols are taken up in enterocytes, it was hypothesized that cholesterol

absorption may be decreased by plant sterols/ stanols via increased cholesterol secretion to the

intestinal lumen, for example via ABCG5/G8 under transcriptional control of the Liver X Receptor (LXR).

However, Abcg5/g8 and Lxr knock-out mice did not reveal any change in cholesterol absorption 172-174,

implying that others factors are involved.

The combination of plant sterols with ezetimibe did not show an additive effect on (surrogate markers

of ) cholesterol absorption and plasma LDL-c in mildly hypercholesterolemic subjects in an open-label

study which was not controlled for dietary plant sterol and stanol content. 175 A recent randomized,

double-blind, placebo-controlled, triple cross-over study on the other hand showed that plant sterols

and stanols further reduced cholesterol absorption (fecal dual isotope method) and further increased

fecal sterol excretion compared with ezetimibe alone in mildly hypercholesterolemic subjects 176,177. This

study showed that the combination of statin and plant sterols and stanols exerted a minor additional

effect on plasma LDL-c levels. Further studies in severe hypercholesterolemic patients may provide

additional information on the usefulness of plant sterol/ stanol-ezetimibe combination therapy.

Chapter 1

26

An adverse effect of plant sterol and stanol consumption could be disturbed micellar incorporation

and absorption of fat-soluble vitamins.178,179 The long term potential adverse effects of plant sterols and

stanols, such as increased atherogenesis and tissue (liver, brain) accumulation of plant sterols and stanols

may seem negligible, but await further study. 168,180

6. Cholesterol excretion

6.1 Classical Reverse Cholesterol Transport (RCT)

The body can dispose itself from cholesterol predominantly in two ways: as neutral sterols (cholesterol

and its intestinal bacterial degradation metabolites) or as acidic sterols (BS). Cholesterol and BS are

excreted mostly with feces and only minimal amounts of cholesterol are additionally lost via the skin. The

classic view of RCT includes the flux of cholesterol from peripheral tissues to the liver mediated mainly

by HDL particles, and the subsequent secretion of this cholesterol by the liver in bile that is transported

to the intestinal lumen, leading to fecal excretion of cholesterol.

Hepatobiliary RCT for decades has been considered the main, if not the only, pathway for cholesterol

elimination. We will briefly discuss this pathway and then turn to other, more recently identified

pathway involved in cholesterol excretion.The liver plays an important role in cholesterol homeostasis

by regulating uptake of lipoproteins, cholesterol synthesis and cholesterol secretion (figure 1). Uptake of

cholesterol is facilitated primarily via basolateral LDLR (LDL and VLDL uptake) and SR-B1 (HDL uptake).

Cholesterol is basolaterally secreted from hepatocytes to the plasma compartment in VLDL and HDL

particles and apically from hepatocytes to bile either directly as free cholesterol (via ABCG5/G8) or after

conversion to BS (via the BS export pump (BSEP or ABCB11) 181 Biliary BS secretion is the main driving

force for secretion of cholesterol and PL. However, studies in mice in which the PL transporter multi-

drug resistance P-glycoprotein (Mdr2 or Abcb4) was eliminated 182, showed that PL secretion itself is also

required for cholesterol secretion.

Cholesterol as well as plant sterols and stanols are secreted into bile mainly via ABCG5/G8.

Recent studies of mice with altered hepatic expression of Niemann-Pick C2 (NPC2, a cholesterol-binding

protein that is involved in intracellular cholesterol trafficking in hepatocytes, but can also be secreted

to bile 183) revealed that NPC2 may positively regulate the biliary secretion of cholesterol, which was

supported by the correlation between levels of NPC2 protein and cholesterol in human bile. Secreted

NPC2 appears to specifically stimulate ABCG5/G8-mediated cholesterol efflux. It was suggested that

NPC2 binds cholesterol and thereby accelerates the transfer of cholesterol to micelles via ABCG5/G8. 183

In recent years it has become clear that the definition of RCT needs revision. 184 Elimination of cholesterol

via feces, at least in mice, mainly occurs not via the classical hepatobiliary route, but via an alternative

pathway, which is adopted Trans Intestinal Cholesterol Excretion (TICE).


27

Chapter

16.2 Transintestinal Cholesterol Excretion

6.2.1 The concept of TICE

The TICE concept has been reviewed recently. 8,73,184,185 Here we will briefly describe the pathway

and latest insights in its dynamics. It was shown in several mouse models with extremely low biliary

cholesterol secretion rates (inactivation of Abcg5, Abcg8 or Abcg5/g8 and Mdr2 or overexpression of

hepatic Npc1l1, see figure 3), that fecal neutral sterol excretion was unchanged or even increased 186-190.

This could not be explained by increased fecal loss of newly synthesized intestinal cholesterol. 191 At a

stable dietary cholesterol intake, this suggested that cholesterol could be excreted directly from blood

to feces via the intestinal mucosa.

Figure 3. Schematic presentation of cholesterol input and excretion in several mouse models. Mice deficient in Abcg8 or Mdr2 have extremely low biliary cholesterol secretion rates, however fecal neutral sterol excretion is relatively similar (Abcg8-/-) or even increased (Mdr2-/-). Similarly, in wildtype mice treated with an LXR-agonist the sum of dietary and biliary cholesterol secretion is outleveled by fecal cholesterol excretion. These models provided the first evidence for the existence of transintestinal cholesterol excretion in mice.

Definitive indications for the existence of TICE were obtained in intestinal perfusion studies in which the

bile duct was ligated and the absence of biliary components was compensated for by infusion of several

(cholesterol free) model bile solutions. Van der Velde et al. demonstrated that TICE occurs in the entire

small intestine, but particularly in the proximal part and plays quantitatively a more prominent role than

the hepatobiliary route in mice. Importantly, intestinal cell shedding and synthesis could not account for

the secreted cholesterol. 187,191 Brown et al. showed that mice with a targeted deletion of hepatic ACAT2

have normal biliary cholesterol secretion rates, in the presence of doubled fecal cholesterol excretion. The

authors determined the fate of newly secreted liver-derived cholesterol. For this purpose, hepatic sterol

pools were radiolabeled and nascent hepatic cholesterol-labeled lipoproteins were collected by isolated

liver perfusion. The liver-derived lipoproteins were then re-injected intravenously into hepatic ACAT2

deficient and control mice to examine the movement of liver-derived radiolabeled cholesterol. It was

found that radiolabeled cholesterol from perfusate of hepatic ACAT2 deficient mice was preferentially

delivered to the proximal small intestine of wild type mice. 192

Chapter 1

28

A similar phenomenon probably occurs in mice deficient in the rate-limiting enzyme for BS synthesis

(cholesterol 7α-hydroxylase, Cyp7a1). Cyp7a1 null mice have reduced BS synthesis and excretion and

a smaller intestinal BS pool, which is associated with a major impairment in cholesterol absorption.

Compared to wildtype mice, they have the same dietary intake and biliary cholesterol level. Nevertheless,

fecal neutral sterol excretion greatly exceeds the sum of total dietary and estimated biliary cholesterol

input to the intestine in these mice. 193

Our lab contributed to the evidence by quantification of in vivo cholesterol fluxes from plasma and bile

to feces by stable isotope methodology. 191 In these experiments, differentially stable isotope labeled

cholesterol was administered to mice intravenously and orally to determine cholesterol absorption and

label distribution over time in blood, bile and feces. In addition, cholesterol synthesis was measured

by MIDA of blood, bile and fecal samples collected over time during administration of stable isotope

labeled cholesterol precursor in drinking water. 191

6.2.2 Transport of cholesterol from blood to feces

The transport of cholesterol from the blood compartment to the enterocyte and the subsequent

excretion of cholesterol is probably facilitated by transport proteins. Sr-b1, being expressed on both

the apical and basolateral side of enterocytes, was considered a possible candidate. However TICE was

shown to be upregulated, rather than downregulated, in Sr-b1 knockout mice. 194 TICE was not altered by

the absence of either Npc1l1 or Ldlr. 192,195 The relation between TICE and cholesterol efflux via Abcg5/g8

is somewhat puzzling 135,185 Compared with wildtype mice, TICE was decreased in Abcg5 knockout mice

during LXR agonist treatment and plant sterol/stanol consumption. 135,191. Strikingly however, TICE was

not decreased by deletion of Abcg8 in mice. 187

By gene expression analyses, so far no apical or basolateral transporters have been identified that

regulate TICE. 8 At present it is unclear which lipoproteins are involved in donating the cholesterol to

the TICE pathway and how it is targeted to the enterocytes. TICE does not seem to be mediated by HDL

particles, based on unchanged fecal cholesterol excretion in mice deficient in HDL (Abca1 knockout

mice). 65 Some evidence points to VLDL as a possible candidate origin for cholesterol involved in TICE. 192

Mice with liver specific depletion of Acat2 showed normal HDL levels, but increased delivery of liver-

derived cholesterol to the lumen of the proximal small intestine as well as increased fecal cholesterol

excretion. 65,192 Alternatively, TICE may not be facilitated via lipoprotein transport, but for example via

increased delivery of erythrocyte cholesterol to the intestinal lumen. Additional research is required to

identify key players in the pathway.

6.2.3 Stimulation of TICE

It is currently unclear whether or not ezetimibe, via inhibition of cholesterol absorption, could stimulate

TICE. One study suggested pharmacological activation of TICE with ezetimibe. 196 However, this was not

confirmed in intestinal perfusion studies 195 indicating that several pathways may co-exist in TICE.


29

Chapter

1TICE can be stimulated with pharmaceuticals such as nuclear receptor agonists of LXR 189,191 and

peroxisome proliferator-activated receptor delta (PPARδ). 195 A high fat (low cholesterol) diet also induces

TICE. 194,197

Dietary plant sterols were shown to inhibit cholesterol absorption in mice. Brufau et al. recently found

that dietary plant sterols/ stanols dose dependently induce fecal neutral sterol excretion, without

affecting biliary cholesterol secretion. These data provide new insight into the cholesterol lowering

mechanism of action of plant sterols/ stanols. 135

A recent study showed that mice with acute biliary diversion are able to secrete labeled cholesterol

derived from intraperitoneally injected cholesterol-loaded macrophages to the intestinal lumen. 198

This study adds to the evidence that TICE may be quantitatively more important than hepatobiliary

cholesterol secretion in the elimination of cholesterol via fecal excretion (RCT).

6.2.4 TICE in humans?

Although the available evidence suggests that TICE may be present in humans 199,200, it is at present

unclear if TICE is present under healthy conditions and whether it can be stimulated pharmacologically

or, preferentially, by dietary means. It has been estimated that TICE may contribute to one-third of fecal

excretion in humans. 187 The possibility of TICE has never been tested in models for predicting plasma

cholesterol. In the future measurement of body cholesterol kinetics by stable isotope studies in humans

may provide new insight into the excretion and regulation of plasma cholesterol, and the dietary and

therapeutic strategies to decrease hypercholesterolemia and associated cardiovascular disease in man.

Scope of this thesis

The specific aim of the research described in this thesis was to determine the effect of manipulation

of intestinal function (by dietary, pharmacological and genetic intervention) on lipid homeostasis,

in particular cholesterol excretion. We hypothesized that acceleration of intestinal transit time could

induce fecal lipid excretion. This could be desirable in case of hypercholesterolemia. On the other hand,

many children nowadays suffer from constipation and are treated with oral laxatives. Administration of

these laxatives could negatively impact on their intestinal absorbtive function and lead to fecal lipid loss.

In chapter 2, we describe the effect of acceleration of intestinal transit on absorption of dietary fat

and cholesterol. In chapter 3, we studied the effect of accelerated intestinal transit on BS homeostasis

and intestinal microbiota. Changes in the composition of dietary fat have been shown to alter fecal fat

excretion. For example, lowering the ratio of polyunsaturated to saturated fatty acids (P/S ratio) was

shown to induce fecal fat excretion.In chapter 4, we studied the effect of two high fat diets with different

P/S ratios on cholesterol homeostasis. We used stable isotope methodologies to determine cholesterol

absorption, synthesis and we modified the method previously used 191 to determine the origin of fecal

sterols. Bsep-/- mice were previously shown to display defective biliary BS secretion. However, this was by

far not as prominent as in their human couterparts. 15 In chapter 5, we studied the effect of absence of

hepatic Bsep in mice on cholesterol homeostasis.

Chapter 1

30

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164. Wang J, Sun F, Zhang DW, et al. Sterol transfer by ABCG5 and ABCG8: in vitro assay and reconstitution. J Biol Chem. 2006 Sep;281(38):27894-904.

165. Yu L, Hammer RE, Li-Hawkins J, et al. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion. Proc Natl Acad Sci U S A. 2002 Dec;99(25):16237-42.

166. Temel RE, Gebre AK, Parks JS, et al. Compared with Acyl-CoA:cholesterol O-acyltransferase (ACAT) 1 and lecithin:cholesterol acyltransferase, ACAT2 displays the greatest capacity to differentiate cholesterol from sitosterol. J Biol Chem. 2003 Nov;278(48):47594-601.

167. Guardamagna O, Abello F, Baracco V, et al. Primary hyperlipidemias in children: effect of plant sterol supplementation on plasma lipids and markers of cholesterol synthesis and absorption. Acta Diabetol. 2011 Jun;48(2):127-33.

168. Marangoni F, Poli A. Phytosterols and cardiovascular health. Pharmacol Res. 2010 Mar;61(3):193-9.

169. Brown AW, Hang J, Dussault PH, et al. Phytosterol ester constituents affect micellar cholesterol solubility in model bile. Lipids. 2010 Sep;45(9):855-62.

170. Talati R, Sobieraj DM, Makanji SS, et al. The comparative efficacy of plant sterols and stanols on serum lipids: a systematic review and meta-analysis. J Am Diet Assoc. 2010 May;110(5):719-26.

171. AbuMweis SS, Vanstone CA, Lichtenstein AH, et al. Plant sterol consumption frequency affects plasma lipid levels and cholesterol kinetics in humans. Eur J Clin Nutr. 2009 Jun;63(6):747-55.

172. Calpe-Berdiel L, Escola-Gil JC, Blanco-Vaca F. Phytosterol-mediated inhibition of intestinal cholesterol absorption is independent of ATP-binding cassette transporter A1. Br J Nutr. 2006 Mar;95(3):618-22.

173. Calpe-Berdiel L, Escola-Gil JC, Blanco-Vaca F. Are LXR-regulated genes a major molecular target of plant sterols/stanols? Atherosclerosis. 2007 Nov;195(1):210-1.

174. Plosch T, Kruit JK, Bloks VW, et al. Reduction of cholesterol absorption by dietary plant sterols and stanols in mice is independent of the Abcg5/8 transporter. J Nutr. 2006 Aug;136(8):2135-40.

175. Jakulj L, Trip MD, Sudhop T, et al. Inhibition of cholesterol absorption by the combination of dietary plant sterols and ezetimibe: effects on plasma lipid levels. J Lipid Res. 2005 Dec;46(12):2692-8.

176. Lin X, Racette SB, Lefevre M, et al. Combined effects of ezetimibe and phytosterols on cholesterol metabolism: a randomized, controlled feeding study in humans. Circulation. 2011 Aug;124(5):596-601.


37

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1177. Scholle JM, Baker WL, Talati R, et al. The effect of adding plant sterols or stanols to statin therapy in

hypercholesterolemic patients: systematic review and meta-analysis. J Am Coll Nutr. 2009 Oct;28(5):517-24.

178. Richelle M, Enslen M, Hager C, et al. Both free and esterified plant sterols reduce cholesterol absorption and the bioavailability of beta-carotene and alpha-tocopherol in normocholesterolemic humans. Am J Clin Nutr. 2004 Jul;80(1):171-7.

179. Goncalves A, Gleize B, Bott R, et al. Phytosterols can impair vitamin D intestinal absorption in vitro and in mice. Mol Nutr Food Res. 2011 Sep;55 Suppl 2:S303-11.

180. Kreuzer J. Phytosterols and phytostanols: is it time to rethink that supplemented margarine? Cardiovasc Res. 2011 Jun;90(3):397-8.

181. Dikkers A, Tietge UJ. Biliary cholesterol secretion: more than a simple ABC. World J Gastroenterol. 2010 Dec;16(47):5936-45.

182. Oude Elferink RP, Ottenhoff R, van Wijland M, et al. Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse. J Clin Invest. 1995 Jan;95(1):31-8.

183. Yamanashi Y, Takada T, Yoshikado T, et al. NPC2 regulates biliary cholesterol secretion via stimulation of ABCG5/G8-mediated cholesterol transport. Gastroenterology. 2011 May;140(5):1664-74.

184. Brufau G, Groen AK, Kuipers F. Reverse cholesterol transport revisited: contribution of biliary versus intestinal cholesterol excretion. Arterioscler Thromb Vasc Biol. 2011 Aug;31(8):1726-33.

185. Vrins CL. From blood to gut: direct secretion of cholesterol via transintestinal cholesterol efflux. World J Gastroenterol. 2010 Dec;16(47):5953-7.

186. Plosch T, Bloks VW, Terasawa Y, et al. Sitosterolemia in ABC-transporter G5-deficient mice is aggravated on activation of the liver-X receptor. Gastroenterology. 2004 Jan;126(1):290-300.

187. van der Velde AE, Vrins CL, van den Oever K, et al. Direct intestinal cholesterol secretion contributes significantly to total fecal neutral sterol excretion in mice. Gastroenterology. 2007 Sep;133(3):967-75.

188. Tang W, Ma Y, Jia L, et al. Genetic inactivation of NPC1L1 protects against sitosterolemia in mice lacking ABCG5/ABCG8. J Lipid Res. 2009 Feb;50(2):293-300.

189. Kruit JK, Plosch T, Havinga R, et al. Increased fecal neutral sterol loss upon liver X receptor activation is independent of biliary sterol secretion in mice. Gastroenterology. 2005 Jan;128(1):147-56.

190. Temel RE, Tang W, Ma Y, et al. Hepatic Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe. J Clin Invest. 2007 Jul;117(7):1968-78.

191. van der Veen JN, van Dijk TH, Vrins CL, et al. Activation of the liver X receptor stimulates trans-intestinal excretion of plasma cholesterol. J Biol Chem. 2009 Jul;284:19211-9.

192. Brown JM, Bell TA,3rd, Alger HM, et al. Targeted depletion of hepatic ACAT2-driven cholesterol esterification reveals a non-biliary route for fecal neutral sterol loss. J Biol Chem. 2008 Apr;283(16):10522-34.

193. Schwarz M, Russell DW, Dietschy JM, et al. Marked reduction in bile acid synthesis in cholesterol 7alpha-hydroxylase-deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia. J Lipid Res. 1998 Sep;39(9):1833-43.

194. van der Velde AE, Vrins CL, van den Oever K, et al. Regulation of direct transintestinal cholesterol excretion in mice. Am J Physiol Gastrointest Liver Physiol. 2008 Jul;295(1):G203-8.

195. Vrins CL, van der Velde AE, van den Oever K, et al. Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux. J Lipid Res. 2009 Oct;50(10):2046-54.

196. Jakulj L, Vissers MN, van Roomen CP, et al. Ezetimibe stimulates faecal neutral sterol excretion depending on abcg8 function in mice. FEBS Lett. 2010 Aug;584(16):3625-8.

197. de Vogel-van den Bosch, H.M., de Wit NJ, Hooiveld GJ, et al. A cholesterol-free, high-fat diet suppresses gene expression of cholesterol transporters in murine small intestine. Am J Physiol Gastrointest Liver Physiol. 2008 May;294(5):G1171-80.

198. Temel RE, Sawyer JK, Yu L, et al. Biliary sterol secretion is not required for macrophage reverse cholesterol transport. Cell Metab. 2010 Jul;12(1):96-102.

199. Cheng SH, Stanley MM. Secretion of cholesterol by intestinal mucosa in patients with complete common bile duct obstruction. Proc Soc Exp Biol Med. 1959 Jun;101(2):223-5.

200. DenBesten L, Reyna RH, Connor WE, et al. The different effects on the serum lipids and fecal steroids of high carbohydrate diets given orally or intravenously. J Clin Invest. 1973 Jun;52(6):1384-93.

Chapter 2

Laxative treatment with polyethylene glycol does not aff ect lipid absorption in rats

Mariëtte Y.M. van der Wulp 1, 2, Frans J.C. Cuperus 2, Frans Stellaard 2, Theo H. van Dijk 2, Jan Dekker 1,

Edmond H.H.M. Rings 1, 2, Albert K. Groen 1,2 and Henkjan J. Verkade 1, 2

1 Top Institute Food and Nutrition, Wageningen, Gelderland, The Netherlands2 Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Beatrix Children’s Hospital,

Groningen University Institute for Drug Exploration, Center for Liver, Digestive and Metabolic Diseases,

University of Groningen, University Medical Center Groningen, Groningen, Groningen, The Netherlands

J Pediatr Gastroenterol Nutr. 2012 Oct;55(4):457-62

Chapter 2

40

Abstract

Objectives Polyethylene glycol (PEG) is a frequently used laxative agent. It is unknown, however, if PEG

aff ects the absorptive capacity of the intestine. Reduced lipid (dietary fat and cholesterol) absorption

induced by long-term PEG treatment could negatively aff ect growth in children. We tested whether

PEG accelerates the gastrointestinal transit and alters lipid absorption and plasma lipid levels.

Methods Wistar rats were administered drinking water with or without PEG (7%) for two weeks. We

studied whole gut transit time by recording the fi rst appearance of red feces after intragastric carmine

red administration. We measured plasma concentrations of cholesterol and triglycerides, dietary fat

absorption by 48h fat balance and by plasma appearance of intragastrically administered stable-

isotope labeled fats, and cholesterol absorption with a dual stable isotope technique.

Results PEG decreased whole gut transit time by 20% (p = 0.028) without causing diarrhea. PEG

treatment did neither aff ect overall dietary fat balance, nor fat uptake kinetics, cholesterol absorption

or plasma lipid concentrations.

Conclusion PEG does not aff ect lipid absorption, nor steady state plasma lipid levels in rats, although

it accelerates the gastrointestinal transit.

Effect of PEG treatment on lipid absorption

41

Chapter

2

Introduction

Polyethylene glycol (PEG) is an inert polymer that originally was used to prepare bowels for endoscopy. 1

In the 1990’s PEG became available as osmotic laxative for the treatment of chronic constipation. 2

Currently it is one of the most widely prescribed laxative agents. Long-term treatment with PEG seems

safe and effective in adults 3 as well as children. 4,5 Recently it was stated that PEG shows better outcomes

than lactulose in terms of stool frequency per week, stool form, relief of abdominal pain and the need

for additional products. 6 However, it is not known whether PEG treatment affects intestinal absorptive

functions, for example by its effect on gastrointestinal transit.

Little attention has been given to the effect of PEG on gastrointestinal transit. Coremans et al. reported

acceleration of oro-cecal transit during PEG treatment as determined by breath tests in healthy

volunteers. 7 In constipated children, both high-dose (1.5 g. kg-1. d-1) and low dose (0.3 g. kg-1. d-1) PEG

were shown to accelerate total and segmental colonic transit after six and fourteen days of treatment,

respectively. 8,9 Only one study, performed in healthy adults, determined fecal excretion of nutrients

during PEG ingestion. 10 In that study, designed to study effects of diarrhea, PEG was shown to increase

fecal loss of fat and carbohydrates. 10 However, it has not been studied in detail in an appropriate animal

model whether long-term, non-diarrhea inducing, PEG dosages affect intestinal lipid (dietary fat and

cholesterol) absorption.

Several steps are required for adequate absorption of dietary lipids in the intestine, which in theory could

be affected by acceleration of intestinal transit, particularly of the small intestine. Triglycerides, the main

components of dietary fat, are partly lipolyzed in the stomach. 11 Emulsification in the stomach further

enhances lipolysis by pancreatic lipase in the intestinal lumen. 12 Lipolytic products and cholesterol are

solubilized by bile salts, providing efficient translocation into enterocytes. 13

The aim of this study was to test whether PEG, by acceleration of whole gut transit time (WGTT), alters

intestinal lipid absorption.

Previous studies on acceleration of intestinal transit were mainly executed with metabolized drugs. 14

Therefore, it is not clear whether the effects observed were directly related to acceleration of

gastrointestinal transit. PEG however, is minimally absorbed and not metabolized in the intestine. 15 We

attempted to accelerate WGTT with PEG without causing side-effects such as diarrhea and dehydration,

similar to its optimal clinical effect in patients.

We determined absorption and plasma levels of dietary fats and cholesterol. We studied fat absorption

with two independent methodologies, namely fat balance and stable isotope techniques. The

quantitative fat balance involves comparing the amount of fat intake and fecal output per time unit.

The fat balance technique can not discriminate between potential causes of fat malabsorption (e.g.

intraluminal versus intracellular processes). 16 We performed a qualitative analysis of fat absorption 16,17

by administering stable isotope labeled triglycerides and measuring plasma appearance of triglyceride-

derived fatty acids. This technique allows to assess differences in the rate of fat digestion and absorption.

We simultaneously studied the absorption of a stable isotope labeled free fatty acid (FFA) and triglyceride.

Chapter 2

42

By comparison of the plasma appearance of the two labels, the intestinal fatty acid absorption per se

can be discriminated from the process of digestion. 16 Cholesterol absorption was determined by a dual

stable isotope test. 18

Materials and Methods

Materials

Colofort® (polyethylene glycol + electrolytes) was obtained from Ipsen Farmaceutica B.V. (Hoofddorp,

The Netherlands). Colofort® contained per sachet (74 g): 64 g PEG, molecular weight 4 kDa, 5.7 g

sodium sulphate (anhydric), 1.68 g sodium bicarbonate, 1.46 g sodium chloride and 0.75 g potassium

chloride. Carmine was obtained from Macro-imPulse Saveur Ltd. (Stadtoldendorf, Germany). Intralipid®

(20%) was obtained from Fresenius Kabi, Den Bosch, The Netherlands. 2,2,4,4,6-Deuterium-cholesterol

(D5-cholesterol) was obtained from Medical Isotopes and 25,26,26,26,27,27,27-Deuterium-cholesterol

(D7-cholesterol) from Cambridge Isotope Laboratories Inc. 1-13C-stearate and tri-1-13C-palmitate were

obtained from Sigma Aldrich (St. Louis, MO). All isotopes were of 98-99% isotopic purity.

Animals

Growing male Wistar Unilever rats (150-174 g) were obtained from Harlan (Horst, The Netherlands). Rats

were housed individually in an environmentally controlled facility with diurnal (12/12h) light cycle. Food

and water were available ad libitum during the entire study period. The experiments were performed

in conformity with Public Health Service policy and in accordance with the national laws. The Ethics

Committee for Animal Experiments of the University Medical Center of Groningen approved the

experimental protocols.

Cholesterol absorption study

Rats were maintained on semisynthetic purified diet (supplementary table 1, code 4063.02, obtained from

Arie Blok, Woerden, The Netherlands). Upon arrival rats were randomly given a number from 1 to 14. After

a three week run-in period on semisynthetic diet, baseline parameters of all rats were obtained, including

WGTT. Subsequently, rats with an even number received PEG treatment (n= 7), whereas rats with an

uneven number were assigned to control group (no treatment; n= 7) for a total period of 16 days. PEG was

continuously available to the rats in their drinking water at a concentration (71g.l-1 PEG4000) previously

found to significantly accelerate intestinal transit. 19 Steady state conditions are expected to be reached

within five days of treatment. 15 Feces were collected during a period of 48h before the start of treatment and

again after one week of PEG treatment. WGTT was measured after intragastric administration of carmine

red inert dye 20 (1 ml 60 mg.ml-1 drinking water 19) under brief isoflurane/ oxygen inhalation anesthesia

(for handling) one day before, and during PEG treatment at day 14 at 9 A.M. during the light phase.

WGTT was defined as the time to appearance of the first red feces after administration of carmine.


43

Chapter

2

Food and fluids were available to the rats during the entire experiment. Food and fluid intake as well as

body weight (BW) were measured daily.

Plasma dual isotope ratio method

At day 10 after the start of PEG treatment, rats received an intravenous injection (tail) of 1.5 mg D7-

cholesterol dissolved in 500 μl intralipid and an oral dose of 3 mg D5-cholesterol dissolved in 1 ml

medium chain triglyceride (MCT) oil. At time points 0, 3, 6, 9, 12, 24, 48, 72, 96, 120, 144 and 168h after

administration, blood samples were obtained from the tail vein under isoflurane anesthesia.

Blood was collected in sodium-heparinized micro-hematocrit tubes (75 μl). Plasma was separated by

centrifugation (10 min, 2000 rpm, 4°C) and stored at -20°C until analyses. Feces were collected from day

three until day six after label administration.

At day 17, rats were anesthetized one by one by intraperitoneal injection of a mixture of Hypnorm

(fentanyl/ fluanisone 1 ml.kg-1) and diazepam (10 mg.kg-1). The common bile duct was cannulated for

bile collection. To ensure that hepatic production was accurately measured, bile produced during the

initial 5 min after cannulation was discarded, and bile was sampled for 30 min thereafter. During the

bile collection period, body temperature was maintained by keeping animals in a humidified incubator.

Blood was obtained by cardiac puncture and divided over lithium-heparin (for determination of renal

parameters) and EDTA-containing tubes. Rats were terminated by cervical dislocation.

Analytical procedures and calculations

Plasma parameters

Plasma cholesterol and triglyceride concentrations were determined using commercially available kits

(Roche Diagnostics, Mannheim, Germany). Renal functions (sodium, potassium, urea and creatinin) were

determined in plasma by routine spectrophotometry on a P800 unit of a modular analytics serum work

area from Roche Diagnostics Ltd. (Basel, Switzerland).

Feces

Fecal calcium and phosphate output were measured as described previously. 21 We measured calcium

concentration in 1.5 g aliquots of freeze-dried feces, and phosphate concentration in 1.0 g aliquots.

Fat balance

Fatty acid concentrations were determined in 50 mg aliquots of feces and of crushed food. 22 Fatty acid

methyl ester derivatives were measured by gas-chromatography (GC) on an HP-Ultra-1 column from

Hewlett-Packard (Palo-Alto, CA), using 100 μl heptadecanoic acid (C17:0, 50 mg.100 ml-1) as internal

standard. 22 Fat absorption was calculated for individual and total fatty acids as (intake - output)/ intake

*100%.

Chapter 2

44

Cholesterol absorption

Cholesterol was extracted from 10 μl plasma and isotope enrichments of sterols were determined in

the cholesterol fraction by gas chromatography-mass spectrometry (GC-MS). 18 Total fecal neutral sterols

(cholesterol plus bacterial metabolites) were separately determined by GC. 23 Biliary lipids were extracted

according to the method of Bligh and Dyer. 24 Total plasma and biliary cholesterol concentrations were

determined as described previously. 18 Fractional cholesterol absorption was determined as previously

described by Van der Veen et al. 18, modified for the influx of labeled cholesterol.

Fat absorption kinetic study

Theoretically, the composition of the diet could affect results. To evaluate this, we also performed

experiments with rats fed a different diet, containing for example less sugars and fiber (supplementary

table 1, AIN-93G, 25 Research Diet Services BV, Wijk bij Duurstede, The Netherlands). Rats served as their

own controls. We analyzed the effect of diet by repeating key measurements in rats before or during

PEG treatment. Body weight, fluid and food intake were determined daily. Feces (48h) and food were

collected. We administered stable isotope labeled saturated fat, in the form of FFA 1-13C-stearate and

triglyceride tri-1-13C-palmitate.

We administered, through oral gavage, a bolus of 500 μl oil (olive oil: MCT oil 1:3) per 300 g BW, which

represents 30% of daily fat intake. This bolus contained 10 mg of each labeled fat. Our rats consumed a

diet containing 7.2% fat. Daily average food intake was 23 g, containing 1.7 g of fat. The daily consumed

stearate and palmitate from food pellets was 4.6 mg (2.8 g. kg-1 in food) and 12.8 mg (7.7 g. kg-1 food)

of stearate and palmitate, respectively. At time points 0, 2, 4, 6, 8 and 10 h after administration blood

was drawn from the tail to determine label appearance. Baseline WGTT was determined two days later.

One day after WGTT measurement, PEG treatment was started as described above and continued until

termination. One week after the start of PEG treatment, all above measurements were repeated.

Analytical procedures

Plasma lipids were hydrolyzed with hydrogen chloride in acetonitrile. α-bromopentafluorotoluene

derivatives of fatty acids were extracted with hexane and analyzed by GC-MS. 26 Enrichment was defined

as the increase in M1/ M

0 fatty acid relative to baseline measurements. The ratio of plasma labeled fatty

acid and administered label (μmol) was calculated to obtain the % enrichment of label in plasma.

Palmitate % enrichment was corrected for molecular weight since we administered triglycerides, which

are detected in plasma as FFA.

Statistical Analyses

Normal distribution was examined by normal probability plots and Shapiro-Wilk tests. Depending on

normality of data, differences between two groups were determined by either Mann Whitney U or

Student’s t-test. The effect of PEG on WGTT was evaluated by a one way ANOVA repeated measurements

test, followed by post-hoc Wilcoxon Signed Rank tests to test within groups.


45

Chapter

2

Results are presented as means ± SD. The level of significance for all statistical analyses was set at

p < 0.05. Analyses were performed using SPSS version 16.0 for Windows (SPSS Inc., Chicago, IL).

Results

Animal characteristics

PEG-treated rats ingested similar amounts of food and water and showed similar body weights and

growth as control rats during the entire study period (table 1). PEG treatment did not affect biochemical

plasma parameters of renal function. PEG increased fecal output (dry weight, +42%; p= 0.001). No overt

diarrhea was noticed during PEG treatment (fecal pellets were formed). Because increased intestinal

calcium and phosphate availability can decrease fat absorption, we measured fecal concentrations and

calculated their fecal excretion. Both calcium and phosphate excretion were decreased in PEG-treated

vs. control rats (calcium -13% on average; p= 0.028; phosphate -19%; p= 0.022).

Table 1: Animal characteristics

Control PEG treated

Body weight (g) 398 ± 32 391 ± 44

Growth (g) 31 ± 4 26 ± 16

Intake and output

Food intake (g. kg-1.day-1) 58.7 ± 2.3 58.4 ± 4.0

Fluid intake (g. kg-1.day-1) 66.9 ± 13.1 79.2 ± 12.0

PEG intake (g. kg-1.day-1) 5.6 ± 0.6

Fecal dry weight (g. kg-1.day-1) 8.0 ± 0.8 11.4 ± 1.1*

Fecal calcium (mmol. kg-1.day-1) 4.8 ± 0.5 4.2 ± 0.4*

Fecal phosphate (mmol. kg-1.day-1) 3.8 ± 0.5 3.1 ± 0.5*

Plasma parameters

Cholesterol (mmol/ L) 2.3 ± 0.5 2.5 ± 0.3

Triglycerides (mmol/ L) 1.0 ± 0.4 0.9 ± 0.4

Creatinin (µmol/ L) 21.7 ± 2.3 20.8 ± 2.4

Ureum (mmol/ L) 8.5 ± 1.3 8.9 ± 1.0

Na (mmol/ L) 142.2 ± 2.1 140.1 ± 4.7

K (mmol/ L) 5.4 ± 0.4 5.3 ± 0.9

Body weight, growth, average daily intake and output, and plasma parameters in control rats and rats treated with 7% PEG in drinking water. Data are represented as mean ± SD, n=7 per group. Data of PEG treated rats were compared with those of control rats by unpaired two-sided Students’ t-tests. *P= <0.05

Chapter 2

46

PEG accelerates WGTT

PEG treatment decreased WGTT by 20% (from 10.1 ± 2.2h to 8.1 ± 2.7h; p= 0.028). Control rats showed

similar WGTT in both episodes (11.2 ± 2.3h and 11.3 ± 3.3h, figure 1).

Whole gut transit time

0 10

5

10

15

20

*

Control

PEG

Transit(h)

Figure 1. Effect of PEG on Whole gut transit time. WGTT was measured in rats fed a semisynthetic diet for 3 weeks. First a baseline measurement (0) was performed in all rats. Subsequently rats were randomly appointed to either control (white dots) or PEG treated (black dots) group and WGTT was measured in both groups (1). To test for differences in WGTT depending on PEG treatment, first a one way ANOVA repeated measurements test was executed. The error variance of the dependent variable (WGTT) was equal across groups (Levene’s test), indicating that it was correct to use this analysis. The repeated measurements test showed significant interaction (group*timepoint) (p<0.05). We used post-hoc Wilcoxon Signed rank tests to compare control WGTT at timepoints 0 and 1 (NS) and PEG treated WGTT (timepoint 1) compared to baseline WGTT in the same animals (timepoint 0; p=0.028). Data are represented as mean ± SD, n=7 per group. *P<0.05

Overall dietary fat balance is not affected by PEG treatment

We studied dietary fat absorption by subtracting fecal fat output from fat intake. Detailed analysis of fat

absorption using GC demonstrated that the absorption of unsaturated fatty acids (oleic, linoleic and

α-linolenic acid) was almost complete in control and PEG-treated animals (~93-100%). As described

previously 27, control rats showed relative malabsorption of the long chain saturated fatty acids palmitic

(~89%) and stearic acid (~66%) (figure 2).

PEG-treated animals absorbed significantly more saturated fatty acids than controls (palmitic acid 94

± 2% vs. 90 ± 2; p= 0.001; and stearic acid 76 ± 8 vs. 66 ± 7%; p= 0.03, respectively.) However, the

absorption of unsaturated fatty acids did not differ between both groups.

Total C16:0 C18:0 C18:1 C18:2 C18:30

25

50

75

100*

**

Fatt

y ac

id a

bsor

ptio

n (%

) Figure 2. 48h Fat balance of control (white bars) and PEG treated rats (black bars). Molar absorption percentage of total and separate dietary fatty acids. Major dietary fatty acids: palmitic acid (C16:0), linoleic acid (C18:2n6) and oleic acid (C18:1n9). Minor dietary fatty acids: stearic acid (C18:0) and α-linolenic acid (C18:3n3). Data are presented as mean + SD, n=7 per group. Data of PEG treated rats were compared with those of control rats by unpaired two-sided Students’ t-tests. *P<0.05, **p<0.01


47

Chapter

2

The overall percentage of lipid absorption did not differ between groups (96 ± 1 vs. 95 ± 2% in PEG-

treated vs. control animals), attributable to the minor relative contribution of the long-chain saturated

fatty acids to dietary fat.

In a separate experiment, we analyzed the effect of diet by repeating key measurements in rats on a

different semisynthetic diet (AIN-93G), again with or without PEG treatment. All relevant effects of PEG

treatment were virtually identical, including acceleration of WGTT by 27% and unchanged fat balance

(data not shown). This indicated that the PEG effect does not (strongly) depend on the type of diet.

0 2 4 6 8 100.00

0.05

0.10

0.15

0.20

Time after administration (h)

Plasma1-13C-palmitate

originating fromlabeled tripalmitin(%adm dose.ml -1)

A

0 2 4 6 8 100.00

0.05

0.10

0.15

Time after administration (h)

Plasma1-13C-stearate

(%adm dose.ml -1)

B

Figure 3. Fat uptake kinetics at baseline (white dots) and during PEG treatment (black dots). Panel A: Palmitate originating from tripalmitate was adequately absorbed during PEG-treatment, suggesting preserved digestion and absorption of saturated fat. Panel B: absorption of free stearate was likewise preserved during PEG-treatment (Area Under Curve). Plasma kinetics were studied during ten hours after administration of stable isotope labeled stearate and tripalmitate by oral gavage. Rats were used as their own control. Data are presented as mean + SD, n= 7 per group. *P(Wilcoxon Signed Rank test) <0.05

Chapter 2

48

PEG treatment does not affect the rate of fat digestion and absorption

Plasma 13C-fatty acid concentrations

Fat balance provides a quantitative analysis of fat absorption. It can not discriminate between potential

causes of differences, such as intraluminal factors (lipolysis of triglycerides, solubilization) or intracellular

factors (chylomicron formation). We used a different approach than fat balance to separately assess

whether PEG affected the rate of triglyceride lipolysis or the absorption of FFA. We administered stable

isotope labeled triglyceride (tri-1-13C-palmitate) and FFA (1-13C-stearate) intragastrically and determined

plasma appearance of 1-13C-palmitate and 1-13C-stearate, respectively (figure 3). Plasma 1-13C-stearate

was higher 2h after administration during PEG treatment (p= 0.028). A similar (non-significant) effect was

observed for 1-13C-palmitate (p= 0.063).

Maximum enrichment values of 1-13C-palmitate were reached after 2h in PEG-treated vs. 6h at baseline

conditions. Maximum values of 1-13C-stearate were reached after 6h in both conditions. The area under

the curves for both 1-13C-stearate and 1-13C-palmitate (0.81 ± 0.16 vs. 0.64 ± 0.11 and 0.91 ± 0.11 vs. 0.78

± 0.08 % of administered dose.ml plasma-1 under PEG treatment versus baseline conditions respectively

(not shown) did not differ, indicating that PEG did neither affect lipolysis nor FFA absorption.

PEG treatment does not affect cholesterol absorption

Similarly to the absorption of long-chain saturated fatty acids, cholesterol absorption is strongly bile

salt dependent. 13 We studied cholesterol absorption by means of a dual stable isotope technique. With

this technique we determine levels of stable isotope labeled cholesterols in plasma after prior i.v. and

oral administration of differentially labeled cholesterols. This well-known methodology 18,28 allows us to

calculate the fractional absorption of cholesterol from the intestine. Similarly to overall fat absorption,

fractional cholesterol absorption did not differ between PEG-treated and control rats (~54 and ~58%,

respectively, figure 4). PEG neither affected biliary cholesterol secretion, nor fecal sterol excretion (data

not shown).

Control PEG0

50

100

Frac

tiona

l cho

lest

erol

abso

rptio

n (%

)

Figure 4. Fractional cholesterol absorption in control rats (white bars) and in rats treated with PEG in drinking water (black bars). Absorption kinetics were calculated from labeled cholesterol in plasma collected after administration of i.v. D

7- and intragastric D

5-cholesterol.

Data are presented as mean + SD. Data were analyzed by unpaired two-sided Student’s t-test, n= 7 per group.


49

Chapter

2

Discussion

In the present study we determined in a rat model whether a frequently applied laxative, PEG, influences

intestinal lipid absorption via acceleration of the WGTT. Our data convincingly show that PEG treatment

does accelerate WGTT, but that it neither affects absorption of triglycerides, fatty acids or cholesterol,

nor plasma lipid levels.

We used PEG with added electrolytes because of its broad applicability with no net exchange of

electrolytes in the intestine. We considered this as a safe method during continuous administration

of PEG in drinking water. To our knowledge, there are no studies that directly compare effects of PEG

with or without added electrolytes in children. However, clinical experience and experimental data of

children taking PEG with or without added electrolytes show that both methods are highly effective and

safe. 29,30 PEG increased dry fecal output, which is mostly, if not entirely, attributable to appearance of PEG

itself in feces. 15 Unexpectedly, we found decreased fecal calcium and phosphate excretion. We cannot

exclude loss of these water soluble ions with fecal water in the bedding of PEG-treated rats.

Although probably underestimated due to evaporation and loss in bedding, we found increased water

content in PEG feces (up to 3-fold, data not shown) as determined by weight of fresh feces and that

after freeze-drying. We did not use metabolic cages to collect feces for our study, since the extra stress

would alter WGTT 31 and influence the effect of PEG. In addition, rats are able to consume feces directly

from their anus, which cannot be prevented in metabolic cages. It is known that coprophagia could

theoretically influence the absolute amounts of fat absorption. However, we do not have any indication

that PEG treatment selectively affects coprophagia and thereby relative fat absorption. We analyzed

the effects of PEG on dietary fat absorption with different, independent methodologies. 16,17 First, we

studied fat balance comparing the amounts of fat ingested over 48h via the food and the amount

excreted via feces. 16 PEG treatment did not affect the overall dietary fat absorption. PEG treatment

did slightly increase saturated fatty acid absorption, compared with control rats. This could be due to

increased intestinal bile salt availability as indicated by decreased fecal bile salt excretion during PEG

treatment. 19 Fat balance cannot discriminate between potential causes of differences in fat absorption,

such as intraluminal factors and intracellular factors. To further investigate fat uptake, we studied plasma

appearance of 13C-labeled fats, derived from intragastrically administered 13C-labeled FFA or triglycerides. 16 We found absorption kinetics to be comparable in PEG-treated versus baseline conditions. The bolus

of stable isotope labeled fat we administered contained 100-200% of the daily consumed saturated

fats by diet. Apparently, the difference in absorption during PEG treatment of specifically the saturated

fats (fat balance), when intake of those fats is minor, is abolished when we administer a single high

dose. Together, these results indicate that there are no relevant changes in fat absorption during PEG

treatment.

We cannot completely exclude that the amount of dietary fat may influence extrapolation of our results

towards the human situation, where a Western high-fat diet is often consumed. However, only one study

by Hammer et al. 10 in humans showed malabsorption of nutrients during PEG intake.

Chapter 2

50

This particular study was designed to study effects of osmotic diarrhea induced by high dose PEG. PEG

is nowadays most frequently used in lower dosages in humans to offer a long-term laxative treatment

by softening of stools and not to induce diarrhea. We mimicked this scenario in our study and did not

observe fat malabsorption.

We did not specifically measure small intestinal transit in this study, since it requires termination of the

rats. However, we previously showed that PEG induces a significant acceleration of small intestinal transit

in rats (+17%), comparable to the acceleration in total WGTT we found. 19

Whereas fat absorption percentage in healthy states is nearly complete 27, cholesterol absorption

is substantially lower. At least in part depending on the method applied 32, cholesterol absorption

percentage has been estimated between 30-70% in humans 33, in rodents 18,34,35 and in our present study

in rats. The substantially lower basal cholesterol absorption may provide increased sensitivity to detect

differences in cholesterol absorption as compared to fat absorption during an intervention. Our finding

that not only fat but also cholesterol absorption was not changed during PEG treatment supports the

concept that PEG treatment does not affect the absorptive capacity of the small intestine.

Together our data indicate that PEG treatment does not lead to relevant changes in lipid absorption

or plasma lipid levels in rats. These data suggest that in terms of lipid absorption, PEG can be safely

administered to children during growth and development.

Acknowledgements

The authors would like to thank Juul Baller and Rick Havinga for their help during animal experiments,

Renze Boverhof and Theo Boer for technical and Vincent Bloks for statistical assistance, respectively.

Conflicts of interest and source of funding

M.Y.M. van der Wulp is currently receiving an unrestricted research grant from Top Institute Food and

Nutrition via the University Medical Center Groningen. We have no conflicts of interest.


51

Chapter

2

References 1. Davis GR, Santa Ana CA, Morawski SG, Fordtran JS. Development of a lavage solution associated with minimal

water and electrolyte absorption or secretion. Gastroenterology. 1980 Feb;78(5 Pt 1):991-5.

2. Andorsky RI, Goldner F. Colonic lavage solution (polyethylene glycol electrolyte lavage solution) as a treatment for chronic constipation: a double-blind, placebo-controlled study. Am J Gastroenterol. 1990 Mar;85(3):261-5..

3. DiPalma JA, Cleveland MV, McGowan J, Herrera JL. A randomized, multicenter, placebo-controlled trial of polyethylene glycol laxative for chronic treatment of chronic constipation. Am J Gastroenterol. 2007 Jul;102(7):1436-41.

4. Pashankar DS, Loening-Baucke V, Bishop WP. Safety of polyethylene glycol 3350 for the treatment of chronic constipation in children. Arch Pediatr Adolesc Med. 2003 Jul;157(7):661-4.

5. Voskuijl W, de Lorijn F, Verwijs W, Hogeman P, Heijmans J, Makel W, et al. PEG 3350 (Transipeg) versus lactulose in the treatment of childhood functional constipation: a double blind, randomised, controlled, multicentre trial. Gut. 2004 Nov;53(11):1590-4.

6. Lee-Robichaud H, Thomas K, Morgan J, Nelson RL. Lactulose versus Polyethylene Glycol for Chronic Constipation. Cochrane Database Syst Rev. 2010 Jul;(7):CD007570.

7. Coremans G, Vos R, Margaritis V, Ghoos Y, Janssens J. Small doses of the unabsorbable substance polyethylene glycol 3350 accelerate oro-caecal transit, but slow gastric emptying in healthy subjects. Dig Liver Dis. 2005 Feb;37(2):97-101.

8. Bekkali NL, van den Berg MM, Dijkgraaf MG, van Wijk MP, Bongers ME, Liem O, et al. Rectal fecal impaction treatment in childhood constipation: enemas versus high doses oral PEG. Pediatrics. 2009 Dec;124(6):e1108-15.

9. Gremse DA, Hixon J, Crutchfield A. Comparison of polyethylene glycol 3350 and lactulose for treatment of chronic constipation in children. Clin Pediatr(Phila). 2002 May;41(4):225-9.

10. Hammer HF, Santa Ana CA, Schiller LR, Fordtran JS. Studies of osmotic diarrhea induced in normal subjects by ingestion of polyethylene glycol and lactulose. J Clin Invest. 1989 Oct;84(4):1056-62.

11. Moreau H, Laugier R, Gargouri Y, Ferrato F, Verger R. Human preduodenal lipase is entirely of gastric fundic origin. Gastroenterology. 1988 Nov;95(5):1221-6.

12. Liu CC, Carlson SE, Rhodes PG, Rao VS, Meydrech EF. Increase in plasma phospholipid docosahexaenoic and eicosapentaenoic acids as a reflection of their intake and mode of administration. Pediatr Res. 1987 Sep;22(3):292-6.

13. Westergaard H, Dietschy JM. The mechanism whereby bile acid micelles increase the rate of fatty acid and cholesterol uptake into the intestinal mucosal cell. J Clin Invest. 1976 Jul;58(1):97-108.

14. Holgate AM, Read NW. Relationship between small bowel transit time and absorption of a solid meal. Influence of metoclopramide, magnesium sulfate, and lactulose. Dig Dis Sci. 1983 Sep;28(9):812-9.

15. Pelham RW, Nix LC, Chavira RE, Cleveland MV, Stetson P. Clinical trial: single- and multiple-dose pharmacokinetics of polyethylene glycol (PEG-3350) in healthy young and elderly subjects. Aliment Pharmacol Ther. 2008 Jul;28(2):256-65.

16. Kalivianakis M, Minich DM, Havinga R, Kuipers F, Stellaard F, Vonk RJ, et al. Detection of impaired intestinal absorption of long-chain fatty acids: validation studies of a novel test in a rat model of fat malabsorption. Am J Clin Nutr. 2000 Jul;72(1):174-80.

17. Rings EH, Minich DM, Vonk RJ, Stellaard F, Fetter WP, Verkade HJ. Functional development of fat absorption in term and preterm neonates strongly correlates with ability to absorb long-chain Fatty acids from intestinal lumen. Pediatr Res. 2002 Jan;51(1):57-63.

18. van der Veen JN, van Dijk TH, Vrins CL, van Meer H, Havinga R, Bijsterveld K, et al. Activation of the liver X receptor stimulates trans-intestinal excretion of plasma cholesterol. J Biol Chem. 2009 Jul;284(29):19211-9.

19. Cuperus FJ, Iemhoff AA, van der Wulp MY, Havinga R, Verkade HJ. Acceleration of the Gastrointestinal Transit by Polyethylene Glycol Effectively Treats Unconjugated Hyperbilirubinemia in Gunn Rats. Gut. 2010 Mar;59(3):373-80.

20. Kotal P, Vitek L, Fevery J. Fasting-related hyperbilirubinemia in rats: the effect of decreased intestinal motility. Gastroenterology. 1996 Jul;111(1):217-23.

21. Cuperus FJ, Hafkamp AM, Havinga R, Vitek L, Zelenka J, Tiribelli C, et al. Effective treatment of unconjugated hyperbilirubinemia with oral bile salts in Gunn rats. Gastroenterology. 2009 Feb;136(2):673-82.

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22. Werner A, Minich DM, Havinga R, Bloks V, van Goor H, Kuipers F, et al. Fat malabsorption in essential fatty acid-deficient mice is not due to impaired bile formation. Am J Physiol Gastrointest Liver Physiol. 2002 Oct;283(4):G900-8.

23. van Meer H, Boehm G, Stellaard F, Vriesema A, Knol J, Havinga R, et al. Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats. Am J Physiol Gastrointest Liver Physiol. 2008 Feb;294(2):G540-7.

24. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911-7.

25. Reeves PG, Nielsen FH, Fahey GC,Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993 Nov;123(11):1939-51.

26. Oosterveer MH, van Dijk TH, Tietge UJ, Boer T, Havinga R, Stellaard F, et al. High fat feeding induces hepatic fatty acid elongation in mice. PLoS One. 2009 Jun;4(6):e6066.

27. Minich DM, Havinga R, Stellaard F, Vonk RJ, Kuipers F, Verkade HJ. Intestinal absorption and postabsorptive metabolism of linoleic acid in rats with short-term bile duct ligation. Am J Physiol Gastrointest Liver Physiol. 2000 Dec;279(6):G1242-8.

28. Turley SD, Herndon MW, Dietschy JM. Reevaluation and application of the dual-isotope plasma ratio method for the measurement of intestinal cholesterol absorption in the hamster. J Lipid Res. 1994 Feb;35(2):328-39.

29. Candy D, Belsey J. Macrogol (polyethylene glycol) laxatives in children with functional constipation and faecal impaction: a systematic review. Arch Dis Child. 2009 Feb;94(2):156-60.

30. Tabbers MM, Boluyt N, Berger MY, Benninga MA. Clinical practice : diagnosis and treatment of functional constipation. Eur J Pediatr. 2011 Aug;170(8):955-63.

31. Enck P, Merlin V, Erckenbrecht JF, Wienbeck M. Stress effects on gastrointestinal transit in the rat. Gut. 1989 Apr;30(4):455-9.

32. Matthan NR, Lichtenstein AH. Approaches to measuring cholesterol absorption in humans. Atherosclerosis. 2004 Jun;174(2):197-205.

33. Grundy SM. Absorption and metabolism of dietary cholesterol. Annu Rev Nutr. 1983;3:71-96.

34. Dietschy JM, Turley SD. Control of cholesterol turnover in the mouse. J Biol Chem. 2002 Feb;277(6):3801-4.

35. Voshol PJ, Schwarz M, Rigotti A, Krieger M, Groen AK, Kuipers F. Down-regulation of intestinal scavenger receptor class B, type I (SR-BI) expression in rodents under conditions of deficient bile delivery to the intestine. Biochem J. 2001 Jun;356(Pt 2):317-25.


53

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Supplementary table 1. Composition of semisynthetic diets

% by weight 4063.02 AIN93G

Protein 17.6 17.7

Carbohydrate 53.8 60.1

Fat 5.2 7.2

g.kg-1

Casein 200 200

Corn-starch 100 397

Maltodextrin 132

Sucrose 100

Cerelose/ dextrose 500

Cellulose 50

DICACEL2+4/ cellulose 100

Soybean oil 50 70

Calcium 5 5

Phosphate 4 3

During the cholesterol absorption study, rats were maintained on semisynthetic purified diet code 4063.02. The fat absorption experiment was performed with rats fed AIN-93G diet.

Chapter 3

Laxative treatment with polyethylene glycol decreases microbial sterol conversion

in the intestine of rats

Mariëtte Y.M. van der Wulp 1, 2, Muriel Derrien 1,3, Frans Stellaard 2, Henk Wolters 2, Michiel Kleerebezem 1,3,

Jan Dekker 1, Edmond H.H.M. Rings 1, 2, Albert K. Groen 1,2 and Henkjan J. Verkade 1, 2

1 Top Institute Food and Nutrition, Wageningen, The Netherlands2 Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Beatrix Children’s Hospital,

Groningen University Institute for Drug Exploration, Center for Liver, Digestive and Metabolic Diseases,

University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 3 Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands

Submitted

Chapter 3

56

Abstract

Objectives Polyethylene glycol (PEG) is a frequently used osmotic laxative that accelerates

gastrointestinal transit. It has remained unclear however, if PEG aff ects intestinal functions. We aimed

to determine the eff ect of PEG treatment on intestinal sterol metabolism.

Methods Rats were treated with PEG in drinking water (7%) for two weeks or left untreated (controls).

We studied the enterohepatic circulation of the major bile salt (BS) cholate with a plasma stable isotope

dilution technique and determined BS profi les and concentrations in bile, intestinal lumen contents

and feces. We determined the fecal excretion of cholesterol plus its intestinally formed metabolites.

Finally, we determined the cytolytic activity of fecal water (a surrogate marker of colorectal cancer risk)

and the amount and composition of fecal microbiota.

Results Compared with control rats, PEG treatment increased the pool size (+51%; p<0.01) and

decreased the fractional turnover of cholate (-32%; p<0.01). PEG did not aff ect the cholate synthesis

rate, corresponding with an unaff ected fecal primary BS excretion. PEG reduced fecal excretion of

secondary BS and of cholesterol metabolites (each p<0.01). PEG decreased the cytolytic activity of

fecal water (54 [46-62] vs. 87 [85-92] % erythrocyte potassium release in PEG treated and control rats,

respectively; p<0.01). PEG treatment increased the contribution of Verrucomicrobia (p<0.01) and

decreased that of Firmicutes (p<0.01) in fecal fl ora.

Conclusion PEG treatment increases the pool size of the primary BS cholate and decreases the

bacterial conversion of BS and cholesterol in rats. PEG decreases cytolytic activity of fecal water, which

could improve intestinal health.

PEG treatment affects enterohepatic circulation of bile salts

57

Chapter

3

Introduction

Polyethylene glycol (PEG) currently is one of the most widely prescribed laxatives. Long-term treatment

with PEG is believed to be safe and highly effective. 1,2 However, it is not known whether or not long-term

PEG treatment affects specific intestinal metabolic functions. PEG may change the intestinal milieu by

accelerating the passage of luminal contents and/or by increasing the luminal water content, possibly

leading to a change in the intestinal microflora. 3

We have previously shown that PEG treatment accelerates the transit through the small intestine as

well as the whole gut in rats. 3 PEG decreases fecal excretion of bile salts (BS) in rats. 3 BS facilitate the

solubilization of lipids (dietary fats, cholesterol and fat-soluble vitamins) in the small intestine, which

is required for efficient lipid absorption. 4 It has recently become clear that BS are not only detergents

necessary for lipid absorption, but are also involved in the regulation of glucose and lipid homeostasis,

and energy expenditure. 5,6 This is illustrated for example by the fact that removal of BS from the intestine

with sequestrants improves plasma low density lipoprotein levels and hyperglycemia in patients with

type II diabetes. 7,8 The mechanisms by which the total pool size and/or profile of BS influence different

physiological processes are just beginning to be understood. 5,6

BS are amphiphatic molecules, produced in the liver (primary BS) by catabolism of cholesterol. 9 The

liver conjugates BS with taurine or glycine and secretes BS into bile, which is transported to the small

intestine. 10 Lipid absorption mainly takes place in the proximal small intestine whereas BS are actively

reabsorbed with high efficiency (~95%) in the terminal ileum. 10 From there, they are transported back

to the liver through the portal system, i.e. completing the enterohepatic cycle (EHC). Under steady state

conditions, the liver compensates for fecal BS loss by synthesis of new BS which then are secreted into

bile.

The small percentage of BS that escapes absorption in the terminal ileum enters the colon. Intestinal

microbiota deconjugate (through BS hydrolases) and dehydroxylate (7α-dehydroxylase) primary BS to

so called secondary BS species 10 A part of colonic BS is passively absorbed, while the remaining part

is lost with feces. Secondary BS such as deoxycholate (DC) and litocholate (LC) are highly hydrophobic.

It is generally thought that these hydrophobic BS can damage colonic mucosal cells and play a role in

initiating or propagating the formation of gastrointestinal malignancies. 10,11 In addition, an increased

hydrophobic BS pool size is associated with the formation of cholesterol gallstones. 12-14

Based on our previous studies 3, we hypothesized that PEG disrupts EHC of BS by changing the intestinal

milieu. We determined relevant parameters of the EHC of cholate (quantitatively the major BS in humans

and rodents) during PEG treatment by an isotope dilution technique. 15,16 We determined the effects of

PEG on BS profiles and concentrations in different compartments of the EHC, on the cytotoxic activity

of fecal water, and on fecal microbiota. This study shows that PEG increases the cholate pool size and

changes the microbial composition, decreases secondary BS formation, and finally decreases fecal water

cytotoxicity.

Chapter 3

58


Materials

Colofort® (polyethylene glycol + electrolytes) was obtained from Ipsen Farmaceutica B.V. (Hoofddorp,

The Netherlands). Colofort® contained per sachet (74 g): 64 g PEG, molecular weight 4 kDa, 5.7 g sodium

sulphate (anhydric), 1.68 g sodium bicarbonate, 1.46 g sodium chloride and 0.75 g potassium chloride.

24-13C-cholate was obtained from Cambridge Isotope Laboratories Inc and was of 98-99% isotopic purity.

Complete protease inhibitor was obtained from Roche. Deuterium labeled D7-7α-hydroxycholesterol

(7α-hydroxycholesterol-25,26,26,26,27,27,27-D7) was obtained from CDN isotopes inc. (product nr.

D-4064).

Animals

Outbred male Wistar Unilever rats (150-174 g) were obtained from Harlan (Horst, the Netherlands). Rats

were housed individually in an environmentally controlled facility with diurnal (12/ 12 h) light cycle.

They were maintained on semisynthetic purified diet (code 4063.02 low fat normal calcium food, Arie

Blok BV, Woerden, the Netherlands). Before any intervention, rats were accustomed to the diet during a

three week run-in period. Food and water were available ad libitum during the entire study period. The

experiments were performed in conformity with Public Health Service policy and with national laws. The

Ethics Committee for Animal Experiments of the University Medical Center of Groningen approved the

experimental protocols.

Cholate kinetic study

After the run-in period on semisynthetic diet, rats were randomly assigned to either control group (no

treatment; n=7) or intervention group (71 g/l PEG4000 via drinking water; n=7) for a total period of

sixteen days. Feces were collected during a period of 48 h before the start of treatment and again after

one week of PEG treatment. Food and fluid intake and body weight were measured daily.

Cholate kinetics

Intravenous 24-13C-cholate (3 mg per rat in a solution of 250 μl 0.5% NaHCO3 in phosphate-buffered

saline (PBS), pH 7.4) was administered at day 10 after the start of PEG treatment. Blood was drawn from

the tail vein at 0, 12, 24, 36, 48, and 60 h after administration under isoflurane anesthesia. At day 17, rats

were anesthetized by intraperitoneal injection of a mixture of Hypnorm (fentanyl/ fluanisone 1ml/ kg)

and diazepam (10 mg/ kg). The common bile duct was cannulated for bile collection. To ensure that

hepatic production was accurately measured, bile produced during the initial 5 min after cannulation

was discarded, and bile was sampled for 30 min thereafter. During the bile collection period, body

temperature was maintained by keeping animals in a humidified incubator. Blood was obtained by

cardiac puncture and stored (-20°C). Rats were terminated by cervical dislocation.


59

Chapter

3

Analytical procedures and calculations

Feces

BS and neutral sterol composition and concentration in feces were determined by gas chromatography

(GC) to calculate daily excretion. 17

Cholate kinetics

Plasma and bile samples were prepared for isotopic analysis of cholate α-bromopentafluorotoluene

(PFB)- trimethylsilyl (TMS) derivatives by GC-mass spectrometry (MS). 15 The ions monitored were 623

and 624 corresponding to cholate (M0) and 24-13C-cholate (M

1). Enrichment (increase in M

1-cholate/ M

0-

cholate relative to baseline measurements) was expressed as the natural logarithm of the atom percent

excess (ln APE) value. The decay of ln APE over time was calculated by linear regression analysis. The

fractional turnover rate (FTR) per day equals the slope of the regression line. Pool size (μmol.100g BW-1)

and cholate synthesis rate (μmol.100 g BW-1.day-1) were calculated as described previously. 15

Enterohepatic Cycling of Cholate

The cholate biliary secretion rate was calculated by multiplying the bile flow (ml.100 g BW-1.h-1) by

the biliary cholate concentration (mM) as determined by GC analysis. 17 Bile flow was determined

gravimetrically, assuming a density of 1 g.ml-1 for bile. The percentage of cholate reabsorbed per day was

calculated as follows: 100% * (biliary secretion – synthesis) / biliary secretion. The percentage of cholate

lost in feces per enterohepatic cycle was calculated by 100% minus the calculated % reabsorption. 16

Hepatic cholesterol 7α-hydroxylase (cytochrome P450 7a1 or cyp7a1) activity

The activity of microsomal cyp7a1 was assayed essentially as described 18 with some modifications. In

short, microsomes (200 µg protein) in 100 mM K-phosphate buffer, pH 7.4 containing 1 mM EDTA ,

1 mM NADPH and complete protease inhibitor were incubated at 370C (total volume 1 ml). After 4

min the enzymatic reaction was stopped by addition of 8 ml of chloroform/ methanol (2:1, v/v). D7-7α-

hydroxycholesterol was added as internal standard (40 ng). After adding 1 ml 0.9% NaCl and vigorous

shaking, the samples were centrifuged. The chloroform phase was removed and evaporated under a

stream of nitrogen. Calibration standards with unlabeled D0-7α-hydroxycholesterol were treated in a

similar way. Samples were converted to TMS ether by overnight treatment with a mixture of N,O-bis

{TMS} trifluoroacetamide, pyridine and trimethylchlorosilane (50:50:1, v/v). Samples were dried under

a stream of nitrogen, resuspended in hexane, and analyzed on GC-MS (Agilent 9575C inert MSD,

Agilent Technologies, Amstelveen). The column used was J+W Scientific DB-17MS ; 20m; 0.18mm; film

0.18 µm. Operating parameters: helium flow 0.7 ml/min; temperature: source 2200C; column: 1500C

for 0.5 min-600C/min up to 300C-3000C for 5 min. The amount of 7α-hydroxycholesterol produced

from the endogenous microsomal cholesterol was calculated from the ratio labeled: unlabeled

7α-hydroxycholesterol using the calibration standards.

Chapter 3

60

Cytotoxic activity of fecal water

Fecal water was prepared by reconstitution of lyophilized feces. 19 Potassium release resulting from lysis

of human erythrocytes incubated in 154 mM NaCl was set as 0% and distilled water represented 100%

(maximal) potassium release. Subsequently, fecal water was incubated with erythrocytes and potassium

release as a measure of cell lysis was expressed as percentage of maximal potassium release. 20

Intestinal bile salt contents

BS in intestinal contents were measured in a separate group of rats, treated and handled identical

to described above: rats were randomly assigned to control group or PEG-treated group (n=7 each

group). At day 16 after the start of PEG treatment, rats were terminated under anesthesia and the entire

intestinal tract from duodenum until anus was removed. Small intestines were divided into proximal,

mid and distal segments of equal length. Cecum was separated from remaining colon and its contents

were collected directly in pre-weighed cups. Small intestine and remaining colon were flushed with 5

ml PBS to collect contents. Samples were lyophilized overnight and weighed again to calculate total

dry content weight. Aliquots of 10 (proximal and middle part of small intestine) to 50 (distal small

intestine, cecum and colon) mg were used to determine BS composition by GC. BS were first isolated

by reversed-phase solid-phase extraction. 17 The eluate was evaporated to dryness under a stream of

nitrogen and dry material was redissolved in hexane. The solution was divided over two glass tubes, after

which samples were dried once more. The first part was converted to TMS derivatives and free cholate

was determined as described previously. 17 The remaining part was mixed in 500 μl DEMI water, 500 μl

sodium acetate (0.2 M; pH 5.6) and 12 U choloyl glycine hydrolase (0.6 U/ μl) to hydrolyze BS for 15 h

at 37°C. Afterwards, BS were isolated and derivatized as described. This way, we obtained the total and

free cholate fractions. Free cholate was subtracted from total cholate to obtain the mass of conjugated

cholate in different compartments.

Bacterial DNA extraction

Fecal bacterial genomic DNA was isolated using the Fast DNA Spin kit (Qbiogene, Inc, Carlsbad, CA, USA)

using 0.1 g of fecal sample and eluted in 100 μl DES. Purity and amount of DNA was measured using

Nanodrop-1000 spectrophotometer (NanoDrop® Technologies, Wilmington, DE).

Quantitative PCR

Quantitative PCR (qPCR) was performed with an IQ5 Cycler apparatus (Bio-Rad, Veenendaal, The

Netherlands). All reactions were performed in triplicate in one run. Samples were analysed in a 25-μl

reaction mix consisting of 12.5 μl Bio-Rad master mix SYBR Green (50 mM KCl, 20 mM Tris-HCl, pH 8.4, 0.2

mM of each dNTP, 0.625 U iTaq DNA polymerase, 3 mM MgCl2, 10 nM fluorescein), 0.1 µM of each primer

Bact-1369F (5’ CGGTGAATACGTTC-3’) and Prok-1492R (5’-GGWTACCTTGTTAC-3’) and 5 μl of template

fecal DNA diluted 1:100 or 1:1000.


61

Chapter

3

Standard curves of 16S rRNA PCR product from Lactobacillus casei were created using serial 10-

fold dilution of the purified PCR product corresponding to 108 to 100 16S rRNA copies. The following

conditions of qPCR used were: 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 15

sec, annealing temperature of 60°C for 20 sec, extension at 72°C for 30 sec and a final extension step at

72°C for 5 min. A melting curve was performed at the end of each run to verify the specificity of the PCR

amplicons by slowly heating the final reaction mix to 95°C (0.5°C per cycle). Data analysis was performed

using the Bio-Rad software.

Microbial fermentation product analysis

Fresh large bowel content samples were analyzed for short chain fatty acid (SCFA) profiles, including

quantitative detection of acetate, butyrate, propionate and lactate using HPLC (Spectra System, RI-150).

Samples of intestinal content (approximately 0.1 g) were thoroughly mixed with four volumes of distilled

water. Insoluble residue was removed by centrifugation (15 min at 13.000 g, 4°C). The subsequent

supernatant was mixed with the same volume of 1M HCLO4 and mixed organic acid was analyzed by

HPLC as previously described. 21

16S rRNA gene amplicon pyrosequencing

Amplicons from the V1-V3 region of 16S rRNA genes were generated by PCR using 27F-DegS

(5’-GTTYGATYMTGGCTCAG-3’) in combination with 520R-Deg for 14 fecal samples. To facilitate

pyrosequencing using titanium chemistry, each forward primer was appended with the titanium adaptor

A (5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3’) and a ‘NNNN’ barcode sequence on the 5’ end, where

NNNN is a sequence of four nucleotides that was unique for each sample. The reverse primer carried the

titanium adaptor B (5’-BioTEG/CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-3’) on the 5’ end. Sequencing

was performed from adaptor A. Adaptor and barcode sequences were provided by GATC Biotech (www.

GATC-Biotech.com). PCRs were performed in a total volume of 50 μl containing 1× PCR buffer, 1 μl PCR

Grade Nucleotide Mix, 0.4 μl of Faststart Taq DNA polymerase (Roche, Diagnostics GmbH, Mannheim,

Germany), 200 nM of a forward and the reverse primer (Biolegio BV, Nijmegen, The Netherlands), and 20

of template DNA. The amplification program consisted of an initial denaturation step at 95°C for 5 min,

35 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 40 s and elongation at 72°C for 70 s and a

final extension step at 72°C for 10 min. The size of the PCR products was confirmed by gel electrophoresis

using 1 μl of the reaction mixture on a 1% (w/v) agarose gel containing ethidium bromide. DNA yield

from purified PCR products were measured by NanoDrop ND-1000 spectrophotometer. Pooled purified

PCR (final DNA concentration of 100 ng/μl) was subsequently sent to GATC-Biotech for pyrosequencing

using a Genome Sequencer FLX in combination with titanium chemistry.

16S rRNA gene sequence analysis 16S rRNA sequences generated from pyrosequencing were quality

filtered. Sequences were removed if they were shorter than 200 nucleotide, longer than 1,000 nucleotide,

contained primer mismatches or ambiguous bases.

Chapter 3

62

The remaining sequences were analysed using the open source software package Quantitative Insights

Into Microbial Ecology (QIIME). 22 16S rRNA gene sequences were assigned to operational taxonomic

units (OTUs) using UCLUST with a threshold of 97% pair-wise identity, then classified taxonomically using

the Ribosomal Database Project (RDP) classifier 2.0.1. Results were displayed as relative abundance of

bacterial taxa and richness.

Statistical AnalysesNormal distribution was examined by Kolmogorov-Smirnov and Shapiro-Wilk tests and normal

probability plots. Nonparametrically distributed data were tested for significant differences by Mann

Whitney U test (values represent median and interquartile range) and parametrically distibuted data by

Student’s unpaired t-test (values represent mean ± SD). Since some of the data regarding BS kinetics and

sterols in intestinal lumen contents and feces were clearly nonparametrically distributed, we decided

to test all these data nonparametrically to allow for comparison between graphs. Correlations are

expressed as nonparametric Spearman correlation coefficient. Statistical analyses were performed using

SPSS 18.0 for Windows, Chicago, IL and GraphPad Prism. Differences between groups were considered

statistically significant at p< 0.05.

Results

PEG treatment results in major changes of the EHC of cholateTo determine the effects of PEG on the EHC of BS, we studied the kinetics of cholate, representing the

major BS in human as well as rodent BS pools. PEG-treated rats had a similar bile flow and total biliary BS

concentration and secretion rate compared to control rats (table 1).

Table 1. Bile flow and biliary output parameters

Control PEG

Bile flow (ml.day-1.100g BW-1) 6.5 [5.7-6.9] 6.2 [5.5-6.5]

BS concentration (mM) 22.7 [19.3-25.0] 19.8 [16.2-23.3]

Cholate concentration (mM) 8.3 [6.3-12.7] 10.2 [6.9-13.0]

Biliary BS secretion rate (µmol.day-1.100 g BW-1)

133.0 [112.1-172.5] 117.5 [101.7-132.0]

Bile flow was measured during a 30 min. collection period. BS secretion rate was calculated from total biliary BS concentration as determined by GC. Data are presented as median and interquartile range, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by Mann Whitney U tests.

Cholate kinetic parameters are schematically summarized in figure 1A and 1B. PEG did not change biliary

secretion rate or intestinal reabsorption (~96%) of cholate, nor the cholate synthesis rate. The latter

finding corresponded with similar Cyp7a1 (rate-limiting enzyme for BS synthesis) activity in livers of PEG

treated and control rats (figure 1C). PEG treatment increased the cholate pool size (+51%; p=0.008) and

decreased the Fractional Turnover Rate (FTR) by 32% (p=0.003).


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μ

μ

μ

C

Figure 1. Cholate kinetics (A and B). Biliary secretion, intestinal reabsorption, fecal excretion and synthesis of cholate did not differ between control (A) and PEG-treated (B) rats. Cholate pool size on the other hand was increased during PEG-treatment, in the presence of reduced fractional turnover rate (FTR). Plasma cholate kinetics were studied during 60h after intravenous administration of stable isotope labeled 13C-cholate. If not indicated, values are expressed in µmol. 100g BW-1.day-1. Data are presented as median and interquartile range, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by Mann Whitney U tests. **P<0.01. As an indicator of total BS synthesis, Cyp7a1 activity was measured in liver microsomes. (C) Data are presented as mean ± SD, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by unpaired Student’s t-test.

Chapter 3

64

PEG did not change fecal primary BS excretion, but decreased fecal secondary BS excretion by 46% (2.0

[1.6-2.6] vs. controls 3.6 [3.1-4.1] µmol.100 g BW-1.day-1, respectively. P=0.001; figure 2A and 2B).

A B

Figure 2. Excretion of individual BS (A) and primary versus secondary BS (B) in feces. Primary BS include chenodeoxycholate (CDC), cholate, allo-cholate (allo-C), α- and β-muricholate (MC). Secondary BS include ursodeoxycholate (UDC), deoxycholate (DCA), δ22-β-MC, hyodeoxycholate (HDC) and ω-MC. Fecal BS excretion was determined in 48h collected feces on day 8 and 9 after the start of PEG treatment. Data are presented as median and interquartile range, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by Mann Whitney U tests. *P<0.05, **p<0.01

PEG treatment alters intestinal BS composition

Considering the kinetics of the enterohepatic circulation, we hypothesized that the increased cholate

pool size would be reflected by higher CA amounts in the intestinal luminal content. Indeed, the total

CA content in the intestinal lumen was similarly increased in PEG-treated rats (figure 3) as was the CA

pool size measured by the stable isotope dilution technique (+60% and +51%, respectively). However,

the variation in the total CA content in the intestinal lumen was considerable and the difference did not

reach statistical significance (figure 3; p=0.051).

Figure 3. Cholate amount in different intestinal compartments. The small intestine (SI) was divided in three segments of equal size, colon was divided in cecum and remaining colon. SIP= Small Intestine Proximal segment, SIM= Small Intestine Middle segment, SID= Small Intestine Distal segment, Cec= cecum, Col= remaining colon. Data are presented as median and interquartile range, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by Mann Whitney U tests. *P<0.05


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PEG treatment decreases lipid metabolites

The unaffected primary BS and decreased secondary BS excretion via the feces could be due to altered

bacterial deconjugation and conversion of primary BS. In agreement with this, PEG treated rats had

higher amounts of primary BS in cecum and total intestinal lumen contents (the latter not statistically

significant; figure 4A).

A

B

C

D

E

F

Figure 4. Bile salts (BS), neutral sterols (NS) and short chain fatty acids (SCFA). Distribution of primary (A) and secondary (B) BS in intestinal lumen. Secretion of individual BS (C) and primary versus secondary BS (D) in bile. Fecal NS excretion; Copr= coprostanol, Chol= cholesterol, DiH-Chol= dihydrocholesterol. (E) Fecal SCFA concentration; A (Acetate), P (Propionate), B (Butyrate), L (Lactate). (F) Data are presented as median and interquartile range, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by Mann Whitney U tests. *P<0.05, **p<0.01.

Chapter 3

66

On the other hand, PEG treated rats had lower amounts of secondary BS (figure 4B) in their intestinal

lumen contents. The data corresponded with those found in bile, in which we also found decreased

secondary BS (figure 4C and 4D). To determine whether the decreased microbial conversion upon PEG

treatment was specific for BS metabolism, we analyzed fecal neutral sterols and SCFA. In accordance

with the BS data, we found almost no cholesterol metabolites (figure 4E) or SCFA (figure 4F) in feces.

PEG treatment decreases fecal water cytotoxic activity

The aqueous, rather than the solid, phase of feces is considered to contain the compounds that interact

with (large) intestinal mucosal cells. 23 In our study, the cytotoxic activity induced by fecal water was

significantly decreased in PEG treated compared with control rats (54 [46-62] vs. 87 [85-91] % erythrocyte

K release, respectively. Figure 5A; p=0.001). The fecal water cytotoxicity was related to both the total fecal

BS excretion (figure 5B) and the hydrophobic DC excretion (figure 5C).

A

B

C

Figure 5. (A) Cytotoxic activity of fecal water as determined by potassium (K) release of human erythrocytes after incubation with fecal water. Data are presented as mean ± SD, n= 6-7 per group. Data of PEG treated rats were compared with those of control rats by unpaired Student’s t-test. **P<0.01. (B) Correlation between fecal water cytotoxicity and BS excretion. Spearman correlation (r) and p value are indicated. (C) Correlation between fecal water cytotoxicity and DC excretion. Spearman correlation (r) and p value are indicated.

PEG treatment changes intestinal microbiota profile

The PEG-induced differences in intestinal BS and sterol metabolite contents suggested alterations in

the amount and/or activity of the intestinal microflora. Quantitative PCR of 16S amplicon showed that

PEG-fed rats harbored a non-significantly lower bacterial load expressed as Log10 16SrRNA copies.g

wet feces-1 (p=0.053, figure 6A; 11.4 ± 0.1 versus 11.0 ± 0.10 in control and PEG-fed rats, respectively).


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Microbiota composition of both groups was investigated by 454-pyrosequencing of the V1-V2 variable

region. A total of 192 158 sequences were obtained.

After quality filtering, the average of sequences size was 314.6 (min: 200, max: 551.0). The number of

reads per sample was on average 13725. PEG treatment increased Verrucomicrobia (p=0.006), but did

not change other phyla significantly (Figure 6B). Looking at lower taxonomical level, multivariate analysis

on relative abundance of bacterial taxa (genus level) showed that microbiota from PEG-treated rats differ

significantly from that of control rats (Monte Carlo permutation test, p=0.004, figure 6C).

Figure 6. Microbiota. (A) Quantification of total bacteria in control and PEG-fed rats, expressed as log10 number of 16S copies.g wet feces-1. Data of PEG treated rats were compared with those of control rats by Mann Whitney U tests. (B) Relative abundance (% of sequences) of bacterial phyla detected in control and PEG-fed rats. (C) Redundancy analysis on relative abundance of bacteria taxa (genus level) in control (grey circles) and PEG-fed rats (black circles). (D) Relative abundance of major bacterial taxa (>5%) that were significantly different between control and PEG-fed rats. (E) Relative abundance of bacterial taxa that were significantly different between control and PEG-fed rats and that account for <5% of the total sequences. Data of PEG treated rats were compared with those of control

rats by Mann Whitney U tests. *P<0.05, **p<0.01.

A

C

E

B

D

Chapter 3

68

Significant changes of genus level included mostly increased relative abundance of known mucus

associated bacteria (Akkermansia, Bacteroides, Ruminococcus), members of Proteobacteria and

concomitant decreased relative abundance of Clostridia (Firmicutes) (Figure 6D and 6E). Moreover, the

total species richness, as estimated by Chao1 index was 1573 ± 209 and 1639 ± 135 OTUs for control and

PEG-fed rats respectively, showed no significant difference between both groups. Overall, microbiota

analysis indicated a trend towards reduced number of fecal bacteria, with no change of richness, but

changes in microbiota composition with a relative increase of mucus-associated bacteria in PEG treated

rats.

Discussion

Our study shows that PEG treatment increases the pool size of the primary bile salt cholate and decreases

its fractional turnover rate in rats. PEG changes the microbiota composition and decreases intestinal

metabolism of bile salts and cholesterol. Finally, PEG treatment decreases the cytotoxicity of fecal water,

a surrogate marker of colon cancer risk.

PEG increased the amount of cholate in the cecum contents of rats. Theoretically, this could be caused

by decreased ileal cholate absorption or decreased bacterial conversion of cholate. The re-absorption of

cholate in quantitative terms did not differ between PEG treated and control rats. Our data do indicate

decreased bacterial conversion of primary BS to secondary BS. PEG also increases the cecum content

weight (+97%, p<0.01). It seems likely that the increased poolsize is at least partly attributable to an

increased volume of luminal contents, rather than to increased BS concentrations per volume unit. In

control rats, we found a sharp decrease in cholate in cecum contents compared with small intestinal

contents, and a concomitant increase in DC. These effects are expected, since most of the cholate will

be actively absorbed in the distal small intestine 24, and the part that is not absorbed will be mostly

converted to DC by the microbiota of the cecum. 10 However, in PEG treated rats cholate remained

higher in the cecum compared to controls and DC appeared minimally (data not shown). Together, these

findings indicate decreased microbial conversion of primary BS in the cecum during PEG treatment. This

was consistent with the significant relative decrease of Clostridia related bacteria, that were previously

identified as major bacteria involved in conversion of primary to secondary BS. 25,26

We have previously shown that PEG accelerates whole gut transit time (WGTT) and small intestinal

transit in rats. 3 In the current study PEG treated rats received the same diet and PEG dosage, and WGTT

was similarly accelerated. Marcus and Heaton have shown significant correlations between acceleration

of WGTT with senna (by 62%) and decreased DC pool (-30%) and between prolongation of WGTT

with loperamide (by 115%) and increased DC pool (+43%). 14 Veysey et al. have also found significant

correlations between prolonged colonic transit and increased DC pool size in humans. 27

Serum DC % was related to microbial BS converting enzyme activity and colonic pH. 13,28 It was found that

cholate pool size was reciprocally decreased during prolonged colonic transit. 27 Berr et al. found that

increased activity of microbial BS converting enzymes in patients with cholesterol gallstones enhances

DC pool size in the absence of changes in WGTT. 12


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Together, the data show that changes in BS pool size may be due to alterations in WGTT, but could also

be facilitated via other mechanisms.

Many bacterial species are able to deconjugate BS, whereas the conversion of primary to secondary BS

seems to be restricted to few gram-positive anaerobes. 10 A lower bacterial load, with a shift in intestinal

microbial profile, leading to decreased amount of luminal bacteria, such as Clostridia, that were able

to convert cholate (and HDC) into other BS, seems to have primarily influenced cholate kinetics. The

exact mechanism inducing these microbial changes is currently unknown, but may include increased

intestinal water content and changes in luminal pH induced by PEG.

The cytotoxic effect of fecal water is primarily enhanced by BS and fatty acids. 23 Fatty acids are not

metabolized in the intestinal lumen and we previously showed that the absorption and excretion of fatty

acids is not changed by PEG 29, implying that fatty acids would not differentially influence fecal water

cytotoxicity in PEG treated rats. The decrease in fecal water cytotoxicity during PEG treatment correlated

with decreased (secondary) BS excretion. Previous studies reported decreased tumor development

during PEG treatment in different mouse and rat models of colon cancer. 30-32 A population-based study

in patients undergoing colonoscopy showed a lower prevalence of colorectal tumors in PEG4000 users.33

An increased primary BS (cholate) pool may prevent BS damage in colonocytes. 11 It is tempting to

speculate that PEG positively influences gut health by decreasing the production of secondary BS in

the colon. Moreover, we found a relative increase of mucus associated bacteria (Akkermansia spp. 34),

shown to be important for a healthy mucus layer in the human gut with respect to mucus production

and thickness.

Given the frequent and long-term prescriptions of PEG in clinical practice, we believe that human

studies are warranted to further delineate the effect of PEG on intestinal microbiota and BS metabolism.

AcknowledgementsThe authors would like to thank Juul Baller and Rick Havinga for their help during animal experiments

and Theo van Dijk for contributing to the analyses of cholate kinetics. In addition, we would like to thank

Renze Boverhof and Theo Boer for technical and Vincent Bloks for statistical assistance.



Nutrition via the University Medical Center Groningen. None of the authors has a conflict of interest to

declare.

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70

References1. DiPalma JA, Cleveland MV, McGowan J, et al. A randomized, multicenter, placebo-controlled trial of polyethylene

glycol laxative for chronic treatment of chronic constipation. Am J Gastroenterol. 2007 Jul;102(7):1436-41.

2. Pashankar DS, Loening-Baucke V, Bishop WP. Safety of polyethylene glycol 3350 for the treatment of chronic constipation in children. Arch Pediatr Adolesc Med. 2003 Jul;157(7):661-4.

3. Cuperus FJ, Iemhoff AA, van der Wulp MY, et al. Acceleration of the Gastrointestinal Transit by Polyethylene Glycol Effectively Treats Unconjugated Hyperbilirubinemia in Gunn Rats. Gut. 2010 Mar;59(3):373-80.


5. Li T, Chiang JY. Bile Acid signaling in liver metabolism and diseases. J Lipids. 2012;2012:754067.

6. Out C, Groen AK, Brufau G. Bile acid sequestrants: more than simple resins. Curr Opin Lipidol. 2012 Feb;23(1):43-55.

7. Beysen C, Murphy EJ, Deines K, et al. Effect of bile acid sequestrants on glucose metabolism, hepatic de novo lipogenesis, and cholesterol and bile acid kinetics in type 2 diabetes: a randomised controlled study. Diabetologia. 2012 Feb;55(2):432-42.

8. Brufau G, Stellaard F, Prado K, et al. Improved glycemic control with colesevelam treatment in patients with type 2 diabetes is not directly associated with changes in bile acid metabolism. Hepatology. 2010 Oct;52(4):1455-64.

9. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and functions. Front Biosci. 2009;14:2584-98.

10. Ridlon JM, Kang DJ, Hylemon PB. Bile salt biotransformations by human intestinal bacteria. J Lipid Res. 2006 Feb;47(2):241-59.

11. Bernstein H, Bernstein C, Payne CM, et al. Bile acids as endogenous etiologic agents in gastrointestinal cancer. World J Gastroenterol. 2009 Jul;15(27):3329-40.

12. Berr F, Kullak-Ublick GA, Paumgartner G, et al. 7 Alpha-Dehydroxylating Bacteria Enhance Deoxycholic Acid Input and Cholesterol Saturation of Bile in Patients with Gallstones. Gastroenterology. 1996 Dec;111(6):1611-20.

13. Thomas LA, Veysey MJ, Bathgate T, et al. Mechanism for the transit-induced increase in colonic deoxycholic acid formation in cholesterol cholelithiasis. Gastroenterology. 2000 Sep;119(3):806-15.

14. Marcus SN, Heaton KW. Intestinal transit, deoxycholic acid and the cholesterol saturation of bile--three inter-related factors. Gut. 1986 May;27(5):550-8.

15. Hulzebos CV, Renfurm L, Bandsma RH, et al. Measurement of parameters of cholic acid kinetics in plasma using a microscale stable isotope dilution technique: application to rodents and humans. J Lipid Res. 2001 Nov;42(11):1923-9.

16. Hulzebos CV, Bijleveld CM, Stellaard F, et al. Cyclosporine A-induced reduction of bile salt synthesis associated with increased plasma lipids in children after liver transplantation. Liver Transpl. 2004 Jul;10(7):872-80.

17. van Meer H, Boehm G, Stellaard F, et al. Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats. Am J Physiol Gastrointest Liver Physiol. 2008 Feb;294(2):G540-7.

18. Einarsson K, Angelin B, Ewerth S, et al. Bile acid synthesis in man: assay of hepatic microsomal cholesterol 7 alpha-hydroxylase activity by isotope dilution-mass spectrometry. J Lipid Res. 1986 Jan;27(1):82-8.

19. Sesink AL, Termont DS, Kleibeuker JH, et al. Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme. Cancer Res. 1999 Nov;59(22):5704-9.

20. Govers MJ, Termont DS, Lapre JA, et al. Calcium in milk products precipitates intestinal fatty acids and secondary bile acids and thus inhibits colonic cytotoxicity in humans. Cancer Res. 1996 Jul;56(14):3270-5.

21. Starrenburg MJ, Hugenholtz J. Citrate Fermentation by Lactococcus and Leuconostoc spp. Appl Environ Microbiol. 1991 Dec;57(12):3535-40.

22. Caporaso JG, Kuczynski J, Stombaugh J, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010 May;7(5):335-6.

23. Pearson JR, Gill CI, Rowland IR. Diet, fecal water, and colon cancer--development of a biomarker. Nutr Rev. 2009 Sep;67(9):509-26.

24. Craddock AL, Love MW, Daniel RW, et al. Expression and transport properties of the human ileal and renal sodium-dependent bile acid transporter. Am J Physiol. 1998 Jan;274(1 Pt 1):G157-69.


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25. Ridlon JM, Kang DJ, Hylemon PB. Isolation and characterization of a bile acid inducible 7alpha-dehydroxylating operon in Clostridium hylemonae TN271. Anaerobe. 2010 Apr;16(2):137-46.

26. Ridlon JM, Hylemon PB. Identification and characterization of two bile acid coenzyme A transferases from Clostridium scindens, a bile acid 7alpha-dehydroxylating intestinal bacterium. J Lipid Res. 2012 Jan;53(1):66-76.

27. Veysey MJ, Thomas LA, Mallet AI, et al. Colonic transit influences deoxycholic acid kinetics. Gastroenterology. 2001 Oct;121(4):812-22.

28. Thomas LA, Veysey MJ, Murphy GM, et al. Octreotide induced prolongation of colonic transit increases faecal anaerobic bacteria, bile acid metabolising enzymes, and serum deoxycholic acid in patients with acromegaly. Gut. 2005 May;54(5):630-5.

29. van der Wulp MY, Cuperus FJ, Stellaard F, et al. Laxative treatment with polyethylene glycol does not affect lipid absorption in rats. J Pediatr Gastroenterol Nutr. 2012 Oct;55(4):457-62.

30. Roy HK, Gulizia J, DiBaise JK, et al. Polyethylene glycol inhibits intestinal neoplasia and induces epithelial apoptosis in Apc(min) mice. Cancer Lett. 2004 Nov;215(1):35-42.

31. Do K, Barnard GF. Effect of concomitant polyethylene glycol and celecoxib on colonic aberrant crypt foci and tumors in F344 rats. Dig Dis Sci. 2005 Jul;50(7):1304-11.

32. Corpet DE, Parnaud G, Delverdier M, et al. Consistent and fast inhibition of colon carcinogenesis by polyethylene glycol in mice and rats given various carcinogens. Cancer Res. 2000 Jun;60(12):3160-4.

33. Dorval E, Jankowski JM, Barbieux JP, et al. Polyethylene glycol and prevalence of colorectal adenomas. Gastroenterol Clin Biol. 2006 Oct;30(10):1196-9.

34. Belzer C, de Vos WM. Microbes inside-from diversity to function: the case of Akkermansia. ISME J. 2012 Mar;6:1449-58.

Chapter 4

Transintestinal cholesterol excretioncan be manipulated by dietary fat

composition in mice

Mariëtte Y.M. van der Wulp 1, 2, Sabina Lukovac 2, Theo H. van Dijk 2, Vincent W. Bloks 2,

Mark V. Boekschoten 1,3, Jan Dekker 1,4, Edmond H.H.M. Rings 1, 2, Albert K. Groen 1,2

and Henkjan J. Verkade 1, 2

1 Top Institute Food and Nutrition, Wageningen, The Netherlands2 Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Beatrix Children’s Hospital,

Groningen University Institute for Drug Exploration, University of Groningen, University Medical Center

Groningen, Groningen, The Netherlands

3 Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands4 Department of Animal Sciences, Wageningen University, Wageningen, The Netherlands

Submitted

Chapter 4

74

Abstract

Introduction Cholesterol can be excreted from the body with feces via a hepatobiliary or a

transintestinal route. We investigated the quantitative eff ects of the degree of dietary fatty acid

saturation on the two origins of fecal cholesterol.

Methods Mice were fed either a standard high fat diet (controls) or a high fat diet with a low

polyunsaturated to saturated fatty acid (P/S) ratio. We determined parameters of cholesterol

homeostasis and performed RNA-microarray analysis on jejunal mucosa.

Results Intake and absorption of cholesterol were unchanged during low P/S ratio diet feeding,

but fecal cholesterol excretion was increased compared with control diet (+68%; p<0.01). The low

P/S ratio diet did not aff ect biliary cholesterol secretion but increased transintestinal cholesterol

excretion (TICE; +137%; p<0.01). The low P/S ratio diet increased jejunal expression of genes involved

in cholesterol synthesis (Srebf2 and target genes), however, whole body de novo cholesterol synthesis

was quantitatively unaltered. Feeding a low P/S ratio diet resulted in a net negative body cholesterol

balance (p<0.01).

Conclusion A low P/S ratio diet induces TICE and a net negative cholesterol balance. Dietary

manipulation in general could represent an attractive strategy to induce TICE in prevention and

treatment of hypercholesterolemia and cardiovascular disease.

Dietary fat induced fecal cholesterol disposal

75

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Introduction

Disturbances in cholesterol homeostasis are associated with potential life-threatening consequences.

For example, hypercholesterolemia represents a major risk factor for cardiovascular disease. 1,2 Cholesterol

homeostasis is mainly regulated by intestinal absorption, fecal excretion and de novo synthesis of

cholesterol. 3 Fecal excretion of cholesterol was classically believed to be primarily driven by cholesterol

secreted via the hepatobiliary pathway. Recently, however, it has become apparent that direct secretion

of cholesterol from the blood compartment to the intestine, or so-called “transintestinal cholesterol

excretion” (TICE), plays a major role in disposal of cholesterol in feces, at least in mice. 3,4

Inhibition of cholesterol synthesis has been the most potent therapy for hypercholesterolemia for

decades. 5 However, inhibition of cholesterol synthesis alone reduces risk of cardiovascular disease by

only 30% and alternative therapeutic modalities are needed. 6,7 In recent years, reduction of intestinal

cholesterol absorption has become increasingly important as a target to decrease plasma cholesterol

levels. 8 A new focus in this field may be induction of TICE, which was shown to be regulated by nuclear

receptor agonists and dietary fat content. 9-12 Recently, Lo Sasso et al. demonstrated that selective

upregulation of intestinal Liver X receptor (Nr1h3 or Lxr) increased fecal sterol output and decreased

atherosclerosis in apo E knock-out mice, suggesting that upregulation of TICE may have beneficial effects

in atherosclerosis. 13 The mechanism involved in cholesterol transport from the blood compartment to

the intestinal lumen remains enigmatic and more studies are needed to clarify the conditions under

which TICE can be stimulated.

We previously induced essential fatty acid deficiency (EFAD) in mice to study its effects on intestinal

function. EFAD was induced by replacing dietary polyunsaturated fatty acids with saturated fatty acids

in a high fat diet, thus highly decreasing the polyunsaturated to saturated fatty acid ratio (P/S ratio). We

found that fecal fat excretion was increased in EFAD mice. 14 A detailed analysis of the development of

fecal lipid (dietary fat and cholesterol) excretion indicated that it increased already before mice became

essential fatty acid deficient (EFAD is defined by plasma triene (20:3n-9)-to-tetraene (20:4n-6) ratio >0.2,

see supplementary figure 1 and ref. 14). This observation suggested that the low P/S ratio, rather than

the EFAD itself, increased the lipid excretion.

Recently, it was shown that, compared with a low fat diet, a high fat diet decreased cholesterol absorption

and increased HMG-CoA reductase activity in the small intestine of mice. 15 In hamsters, it was previously

shown that dietary saturated stearic acid can increase fecal cholesterol excretion. 16,17 Schneider et al.

showed decreased cholesterol absorption and suggested increased cholesterol synthesis in hamsters

fed a high stearic acid diet. 16

The aim of the current study was to test whether cholesterol excretion could be manipulated by feeding

mice a high fat diet with a low P/S ratio as compared with a high fat control diet. We hypothesized that

administration of a low P/S ratio diet would affect total body cholesterol homeostasis. We determined

the consequences of a low P/S ratio diet on cholesterol absorption and synthesis, the origin of fecal

sterols, and total body cholesterol balance.

Chapter 4

76

The stable isotope methodology to obtain quantitative information on these relevant parameters of

cholesterol homeostasis had previously been developed and validated in our laboratory. 18 In order to

characterize the effects of the low P/S ratio diet on the metabolic pathways involved in cholesterol

homeostasis in the small intestinal mucosa, we analyzed the transcriptional response to the low P/S ratio

diet in mice. Our data show that the low P/S ratio diet does not significantly affect de novo cholesterol

synthesis rate or cholesterol absorption, but that it increases cholesterol output via increased TICE.

Collectively, the low P/S diet induced a negative cholesterol balance, indicative of net loss of cholesterol

from the body.


MaterialsIntralipid® (20%) was obtained from Fresenius Kabi, Den Bosch, The Netherlands. 2,2,4,4,6-Deuterium-

cholesterol (D5-cholesterol) was obtained from Medical Isotopes and 25,26,26,26,27,27,27-Deuterium-

cholesterol (D7-cholesterol) from Cambridge Isotope Laboratories Inc. 1-13C-acetate was obtained from

Sigma Aldrich. All isotopes were of 98-99% isotopic purity.

Mice and dietFriend Virus B (FVB type NHanHsd) male mice of 8 weeks old were purchased from Harlan (Horst, The

Netherlands) and were housed in a light- and temperature-controlled facility. Tap water and food were

allowed ad libitum. Mice were maintained on either control (#4141.07; 0.20 μmol cholesterol.g food-1)

or low P/S ratio (#4141.08; 0.18 μmol cholesterol.g food-1) diet for 8 weeks (n= 6-7 per group), which

were both custom synthesized by Arie Blok BV (Woerden, The Netherlands). Fatty acid composition

and polyunsaturated to saturated fatty acid (P/S) ratio of the diets are indicated in table 1. We studied

cholesterol homeostasis and in a separate experiment, we performed microarray analyses on small

intestinal RNA. The experiments were performed in conformity with Public Health Service policy and

in accordance with the national laws. The Ethics Committee for Animal Experiments of the University

Medical Center of Groningen approved the experimental protocols.

Table 1. Fatty acid composition and P/S ratio of high fat control and intervention diet

Mol% Control diet (4141.07) Low P/S ratio diet (4141.08)

Myristic acid (14:0)Palmitic acid (16:0)Stearic acid (18:0)Oleic acid (18:1n-9)Vaccenic acid (18:1n-7)Linoleic acid (18:2n-6)α-Linolenic acid (18:3n-3)

0.933.54.6

32.51.1

26.90.6

1.460.132.94.30.21.00.0

P/S ratio 0.7 0.01

Concentrations are indicated in mol% of total fatty acid concentrations determined by GC.


77

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4

Cholesterol kinetic study

After 8 weeks of diet, baseline bloodspots were collected on filter paper from the tail vein and feces were

collected during a 24h period (day -1). Food pellet weight was determined before and after the 24h

feces collection period and pellets were collected.

Subsequently, mice were switched to tap water containing 2% stable isotope labeled 1-13C-acetate at

9 A.M. (day 1) for 3 days. Bloodspots were collected every morning at 9 o’clock and body weight and

food intake were determined daily for the remainder of the experiment. Day 4-6 represented a wash-

out period for labeled acetate. At day 7 mice received an intravenous (retro-orbital) injection of 1.5

mg D7-cholesterol dissolved in 500 μl intralipid and an oral dose of 3 mg D

5-cholesterol dissolved in 1

ml medium chain triglyceride (MCT) oil. At time points 3, 6, 12, 24, 48, 72, 96, 120, 144 and 168h after

labeled cholesterol administration, bloodspots were obtained. Feces were collected daily from label

administration on until the end of the experiment. At day 14, mice were anesthetized and the common

bile duct was cannulated for bile collection during 30 minutes as previously described. 19 Mice were

sacrificed by cardiac puncture and cervical dislocation. The small intestine was divided into three equal

parts, which were rinsed with phosphate-buffered saline (PBS). Livers were snap frozen in liquid nitrogen

and stored at -80°C.

Analytical methods Indirect cholesterol balance

Biliary lipids were extracted 20 and total plasma and biliary cholesterol concentrations were determined. 18

Food pellets and fecal samples were ground and 50 mg was prepared for neutral sterol (cholesterol plus

bacterial metabolites coprostanol and dehydrocholesterol) and bile salt analysis by gas chromatography

(GC) as described previously. 21 Indirect cholesterol balance was determined by subtraction of dietary

cholesterol intake and hepatobiliary secretion from fecal output of neutral sterols (all calculated in μmol

cholesterol.day-1.100 g of body weight-1). 10

Neutral sterol content in intestinal lumen

Small intestines were divided into three parts of equal size. Cecum was separated from remaining colon.

Small intestine and remaining colon were flushed with 5 ml PBS. Samples were stored at -20°C. Before

analyses, tubes were lyophilized overnight. Aliquots of 10 (proximal and middle part of small intestine)

to 50 (distal small intestine, cecum and colon) mg were used to determine neutral sterols by GC as

described above.

Fractional cholesterol absorption

The procedure for this study, as described in detail by van der Veen et al. 18, was modified for the influx

of labeled cholesterol. Briefly, cholesterol was extracted overnight from bloodspots with 1 ml 100%

ethanol/ acetone (1:1 v/ v). Unesterified cholesterol was derivatized and enrichments of sterols were

determined by GC-mass spectrometry (GC-MS).

Chapter 4

78

The ions monitored were m/z 458-465, corresponding to the M0-M

7 mass isotopomers of cholesterol-

methyl ester/ trimethylsilyl (TMS) derivatives. Fractional cholesterol absorption (F(a)) was calculated as

the ratio between fraction (area under the curve (AUC) of 7 days following label administration) of orally

administered D5-cholesterol and IV administered D

7-cholesterol, after correction for their administered

doses: F(a)= (AUC oral

/ AUCIV) x (Dose

IV / Dose

oral).

Cholesterol synthesis

Fractional cholesterol synthesis was determined by mass isotopomer distribution analysis (MIDA). 22 With

MIDA we could determine the enrichment of acetyl-CoA precursor units (precursor pool enrichment)

that entered newly synthesized cholesterol during 1-13C-acetate administration by analysis of the mass

isotopomer pattern of cholesterol molecules according to a probability model of cholesterolgenesis. In

short, labeled and unlabeled monomeric acetyl-CoA combine in newly formed polymeric cholesterol

molecules. The mass isotopomer pattern of M1 and M

3 cholesterol-TMS derivatives as quantified

by GC-MS was compared to theoretical patterns calculated for cholesterol over a range of precursor

enrichments. 18 A match between experimental and theoretical combinatorial patterns established the

isotopic enrichment of the precursor pool. Knowing the precursor pool enrichment, it was possible

to calculate the expected frequency of M1 and M

3 isotopomers in newly synthesized cholesterol. The

fractional cholesterol synthesis was then determined by comparing the actual M1 and M

3 enrichment

during the experiment with the expected frequencies.

Origin of fecal sterols

In order to determine the origin of fecal sterols, we modified the method described by van der Veen et

al. 18 In physiological terms, fecal sterols can originate from either diet, or de novo synthesis via various

routes, e.g. biliary secretion, transintestinal cholesterol excretion (TICE) or shedding of enterocytes.

Stable isotope studies in combination with mathematical modeling allow quantification of the different

fluxes. Assuming a similar absorption of dietary and biliary derived cholesterol 23 the different fluxes can

be modeled as follows:

Fecal sterols excreted (totalE) were calculated as the average over the time period 24-144h.

The dietary contribution to fecal neutral sterol loss can be calculated as:

ED→F

= (1-F(a)) * D

In which E D→F

is the cholesterol intake calculated in the diet (D) corrected for fractional cholesterol

absorption.

The fecal output of biliary excreted cholesterol (totalEbile) was calculated as bile flow (in µl.100g BW-1.day-1)

times cholesterol concentration in bile, and corrected for cholesterol absorption:totalEbile = flow * [chol] * (1-F(a))

TICE was calculated as: TICE= totalE- totalEbile -ED→F


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This method does not allow discrimination of blood-derived TICE and fecal cholesterol derived from

the intestinal mucosa itself (i.e. epithelial shedding is included in the TICE fraction). Previous intestinal

perfusion studies showed that epithelial shedding contributes no more than 15% to intestinal cholesterol

secretion. 9,24

Total body cholesterol balance

Total body cholesterol balance was calculated as follows: (cholesterol intake + cholesterol synthesis) –

(fecal neutral sterol + bile salt excretion).

Microarray analyses

Mice were maintained on semisynthetic diets in two groups as described above (6 mice per group).

Body weight and food intake were determined weekly. After 8 weeks of diet, mice were anesthetized

and sacrificed by cervical dislocation. Small intestines were rinsed with PBS, divided into three equal

parts, and the middle parts were sliced and immediately frozen in liquid nitrogen. Subsequently, small

intestinal tissue was stored at -80°C for RNA isolation. Livers were snap frozen in liquid nitrogen and

stored at -80°C.

Analytical methods RNA isolation and measurement of RNA expression levels by microarray analysis and quantitative PCR

Total RNA was prepared from the middle third of the small intestinal tissue and liver using TRIzol reagent

(Invitrogen, Breda, The Netherlands). Subsequently, cDNA synthesis and quantitative PCR (qPCR) analysis

were performed as described by Grefhorst et al. 25 qPCR results were normalized to mRNA expression

of the housekeeping gene 18S. Primer and probe sequences for all tested genes have been deposited

at the RTprimerDB. 26 For microarray analysis, the Affymetrix platform was employed. After RNA

isolation with TRIzol reagent, RNA was used individually and further purified using RNeasy MinElute

micro columns (Qiagen, Venlo, The Netherlands). RNA integrity was checked on an Agilent 2100 bio-

analyzer (Agilent Technologies, Amsterdam, The Netherlands) using 6000 Nano Chips according to

the manufacturer’s instructions. RNA was judged as suitable for array hybridization only if samples

exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no

chromosomal peaks or RNA degradation products (RNA Integrity Number.7.0-8.5). Five hundred ng of

RNA were used for one cycle cRNA synthesis (Affymetrix, Santa Clara, CA). Hybridization, washing and

scanning of Affymetrix Gene chip mouse 1.0 ST arrays were performed according to standard Affymetrix

protocols. Scans of the Affymetrix arrays were processed using packages from the Bioconductor

project. 27 Quality control of microarray data (using simpleaffy and affyplm packages), normalization

and differential expression analysis were performed through the Management and Analysis

Database for MicroArray eXperiments (MADMAX 28) analysis pipeline (Wageningen, The Netherlands).

Expression levels of probe sets were calculated using regular normalization strategies: VSN in small

intestine followed by identification of differentially expressed probe sets using Limma. 29

Chapter 4

80

Comparisons were made between treated (low P/S ratio diet) and untreated (control) groups

(Limma package, applying linear models and moderated t-statistics that implement empirical Bayes

regularization of standard errors. 30 False discovery rate (FDR) of 1% (p-value <0.01) was used as a

threshold for significance of differential expression. Identification of overrepresented functional

categories among responsive genes and their grouping into functionally related clusters (Biological

Processes:BP-4) was performed using DAVID Functional Annotation Clustering tool. 31 All microarray data

reported in the manuscript are described in accordance with MIAME guidelines 32 and are available in

the Gene Expression Omnibus (GEO) databank (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token

=nrkjxeokqomuwxe&acc=GSE37097).

Determination of cholesterol concentration in small intestinal mucosa

Thirty mg of mucosal scrapings of the mid part of the small intestine was homogenized in 200 μl of 0.9%

NaCl. Lipids were extracted from the homogenate 20 and cholesterol concentrations were determined

with a commercially available kit (DiaSys Diagnostic Systems, Holzheim, Germany).

Statistical analysis

Using SPSS version 16 statistical software (Chicago, IL, USA), we calculated significance of differences

between mice on low P/S ratio versus control diet with Mann-Whitney U-tests. P-values <0.05 were

considered statistically significant. Data represent median values and interquartile range.

Results

Mice on a low P/S ratio diet have an increased fecal cholesterol excretion (indirect cholesterol

balance)

In order to investigate the effect of the dietary fat composition on fecal sterol excretion, FVB mice were

fed for 8 weeks with either a control high fat diet (polyunsaturated/saturated fat or P/S ratio 0.7) or a

high fat diet with a very low P/S ratio (P/S ratio 0.01) (table 2). Feeding the low P/S ratio diet nearly

doubled daily fecal cholesterol excretion compared with control diet (table 2). Interestingly, the low P/S

ratio diet did not affect dietary cholesterol intake nor biliary cholesterol secretion. (table 2). Net fecal

cholesterol excretion thus was higher than the sum of cholesterol derived from intake and cholesterol of

hepatobiliary origin (table 2). Note that this contrasts to the control situation where net fecal cholesterol

excretion is lower than the sum of intake and hepatobiliary flux, indicating net (re)absorption.


81

Chapter

4

Table 2. Indirect cholesterol balance

μmol.day-1.100g body weight-1 Control Low P/S ratio

Dietary cholesterol intake

Biliary cholesterol secretion

Fecal neutral sterol excretion

2.1 [2.1-2.2]

2.5 [1.6-4.1]

4.7 [3.2-5.3]

2.3 [2.1-2.4]

3.5 [2.5-4.0]

7.9 [6.8-9.8]**

Net non-hepatobiliary cholesterol excretion(<0 = net absorption; >0 = net excretion)

~ -0.5 ~ 2.5*

Data represent medians and interquartile ranges of 6-7 mice per group. *p<0.05, **p<0.01 indicate significant differences between low P/S ratio diet and control mice after 8 weeks of diet, respectively.

To gain more insight in the origin of the fecal cholesterol, we determined neutral sterol content in

the intestinal tract. Neutral sterols recovered from the intestinal lumen during low P/S ratio diet were

significantly increased in the middle part of the small intestine (0.08 [0.07-0.10] versus 0.03 [0.02-0.07]

μmol; p= 0.01) and in the total intestinal tract (0.4 [0.4-0.5] versus 0.3 [0.3-0.4] μmol; p<0.05), compared

with controls (figure 1).

Figure 1. Neutral sterol content in intestinal lumen of low P/S ratio fed (black bars) and control (white bars) mice. SIP= small intestine proximal part, M= middle part, D= distal part, Cec= cecum, Col= remaining part of colon. Values represent medians and interquartile ranges for n=6-7 mice per group. *p<0.05 indicates significant difference between the two groups.

Low P/S ratio diet does not change cholesterol absorption or synthesis, but induces transintestinal

cholesterol excretion

To be able to estimate the quantitatively most important cholesterol fluxes in the body we determined,

in addition to dietary intake and biliary flux, cholesterol absorption and whole body de novo synthesis.

Using stable isotope labeled tracers the contribution of dietary, biliary and intestinal cholesterol to

fecal sterols was calculated. 18 Fractional cholesterol absorption was measured by a dual stable isotope

method. Although fractional cholesterol absorption on average was lower in low P/S ratio diet fed

mice versus controls (49 [39-57] versus 65 [46-71]), the difference did not reach statistical significance

(p>0.1). In order to assess whole body cholesterol synthesis, mice were given drinking water containing

1-13C-acetate.

Chapter 4

82

Subsequently cholesterol synthesis was determined by analysis of cholesterol mass-isotopomer

distributions due to incorporation of the precursor 1-13C-acetate in cholesterol in bloodspots over

3 consecutive days. Mice on a low P/S ratio diet tended to have a lower cholesterol synthesis rate

compared to controls (figure 2, table 3) but the difference was not significant (p>0.07). As absorption

and biliary cholesterol secretion were not altered by the low P/S diet, it was not surprising that fecal

cholesterol originating from diet and bile did not differ between dietary groups (figure 3). The low P/S

ratio diet strongly increased the amount of cholesterol derived from non-hepatobiliary origin, i.e. TICE,

compared with control diet (5.0 [3.7-7.0] versus 2.2 [1.5-3.3] μmol.100g BW-1.day-1; p<0.01, figure 3).

Table 3. Total body cholesterol balance

μmol.day-1.100g body weight-1 Control Low P/S ratio




Fecal bile salt excretion

2.4 [2.3-2.5]

6.1 [3.3-7.4]

4.7 [3.2-5.3]

4.7 [4.4-7.3]

2.6 2.7-2.8]

2.9 [2.5-4.3]

7.9 [6.8-9.8]**

8.0 [6.0-10.8]

Total body cholesterol balance -3.5 [-4.5-0.8] -9.7 [-14.0-(-7.8)]**

Data represent medians and interquartile ranges of 6-7 mice per group. **P<0.01 indicates significant differences between low P/S ratio diet and control mice, respectively.

Figure 2. Cholesterol synthesis in low P/S ratio fed (black bar) and control (white bar) mice. Values represent medians and interquartile ranges for n=6-7 per group.

Figure 3. Origin of fecal sterols in low P/S ratio fed and control mice. Values represent means for n=6-7 per group. **p<0.01 indicates significant difference between the two groups.

Low P/S ratio diet induces a negative total body cholesterol balance

Bile salts are produced in the liver by breakdown of cholesterol and secreted to the intestine with bile.

Fecal bile salt excretion is a major route of disposal of body cholesterol. 33 A total body cholesterol

balance can be calculated by taking the sum of whole body cholesterol synthesis and dietary intake

minus the excreted fecal neutral sterols and bile salts.


83

Chapter

4

On a low P/S ratio diet the total body cholesterol balance was negative suggesting lack of compensation

of fecal cholesterol loss by synthesis (table 3).

Low P/S ratio diet induces expression of jejunal genes involved in cholesterol metabolism

To investigate the effect of a low P/S ratio diet on gene expression microarray analysis was carried

out on samples from the middle part of the small intestine (jejunum). A large number of in total 962

genes were differentially regulated during low P/S ratio diet in mice, using a false discovery rate (FDR)

of 1% as a threshold for significance of differential expression. 565 genes were upregulated and 397

were downregulated. Based on the Gene Ontology categorization, low P/S ratio diet mainly affected

metabolic processes (table 4). In the top 10 of the processes most significantly enriched during low P/S

ratio diet, steroid biosynthetic processes and processes involved in steroid and fatty acid metabolism

were listed (table 4, indicated in bold font).

Table 4. Biological processes enriched during low P/S ratio diet in murine jejunum – microarray analysis (GOTERM_BP_4). False discovery rate (FDR) <1%.

GOTERM BP 4: Biological processes Number of genes p-value Fold enrichment

Cellular lipid metabolic process Translation

Lipid biosynthetic process Oxoacid metabolic process

Fatty acid metabolic process Sterol biosynthetic processCholesterol metabolic processSteroid biosynthetic processTransmembrane transport

Steroid metabolic process

69

52

44

60

3414181851

26

1.2E-15 3.7E-14

2.9E-113.4E-116.0E-114.2E-102.7E-83.4E-8

7.4E-8

2.4E-7

2.9 3.4

3.22.63.89.65.35.2

2.3

3.3

Top 10-scoring list of processes enriched in jejunum upon low P/S ratio diet feeding in mice.

Low P/S ratio diet alters expression of genes encoding jejunal cholesterol transporters

Over 20 genes involved in sterol metabolic processes were enriched during low P/S ratio diet, including

several genes involved in cholesterol absorption and efflux (table 5). Transcription of the Liver X receptors

(Lxrs) Lxrα (nr1h3) and Lxrβ (nr1h2), important transcriptional regulators of cholesterol metabolism, was

not significantly higher in jejunum during low P/S ratio diet (table 5). Expression of Lxr targets such

as Abcg5/g8 (cholesterol efflux) and ATP-binding cassette (Abc) A1 (table 5) was also unaltered or

even decreased. Verification by qPCR showed no change in expression for either Abca1 or Abcg5/g8.

Expression of Niemann-Pick c1-like 1 gene (Npc1l1, encoding the small intestinal cholesterol importer),

was decreased in jejunum of mice fed the low P/S ratio diet. Gene expression of the Ldl receptor (Ldlr),

implied in basolateral cholesterol uptake into enterocytes, was increased during low P/S ratio diet. qPCR

analysis confirmed the microarray results for Npc1l1 and Ldlr (figure 4).

Chapter 4

84

Table 5. Fold changes of genes involved in intestinal cholesterol transport and synthesis – microarray analysis.

Gene Description and protein IDFold change low P/S ratio fed versus control mice

Cholesterol transport

Lxrα (Nr1h3) Nuclear receptor subfamily 1, group H, member 3 No change

Lxrβ (Nr1h2) Nuclear receptor subfamily 1, group H, member 2 No change

Abcg5/g8 ATP-binding cassette sub-family G (White) member 5/8 No change*

Abca1 ATP-binding cassette, sub-family A (ABC1), member 1 ↓ 1.5 (NS in qPCR)

Npc1l1 Niemann-Pick C1-like protein 1 ↓ 1.8*

Ldlr Low density lipoprotein receptor ↑ 2.0*


Mvk Mevalonate kinase ↑ 1.2

Pmvk Phosphomevalonate kinase ↑ 2.3

Mvd Mevalonatediphosphodecarboxylase ↑ 1.6

Fdps Farnesyldiphosphate synthase ↑ 2.0

Fdft1 Farnesyldiphosphate farnesyl transferase 1 (squalene synthase) ↑ 1.6

Sqle Squalene epoxidase (squalene monooxygenase) ↑ 1.8

Lss Lanosterol synthase ↑ 1.9

Cyp51 Cytochrome P450, family 51 (lanosterol 14-alpha demethylase) ↑ 2.4

Dhcr7 7-dehydrocholesterol reductase ↑ 1.4

Srebf1 Sterol regulatory element binding transcription factor 1 No change

Srebf2 Sterol regulatory element binding factor 2 ↑ 1.3*

Hmgcr 3-hydroxy-3-methylglutaryl-Coenzyme A reductase ↑ 1.4*

Hmgcs1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (cytoplasmic) ↑ 1.8*

Fold changes are indicated by the arrows in the direction of up- (↑) or downregulation (↓) of gene expression. False discovery rate (FDR) <1%. mRNA expression of genes indicated with an asterisk (*) has been confirmed by qPCR analysis (p<0.05).

Figure 4. Small intestinal expression of genes involved in cholesterol import and efflux in low P/S ratio fed (black bars) versus control (white bars) mice. Data represent mRNA expression relative to the ribosomal RNA expression of the housekeeping gene 18S. Values represent medians and interquartile ranges for n=6 mice per group. *p<0.05 indicates significant difference between the two groups.


85

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4

Low P/S ratio diet is associated with enhanced expression of genes involved in cholesterol synthesis in jejunum and liver

Microarray analysis of jejunal samples revealed that expression of 32 genes involved in steroid

biosynthesis was enriched during low P/S ratio diet (table 4), including mRNA expression of the rate

limiting enzyme in cholesterol synthesis, HMG-CoA reductase (Hmgcr, table 5). mRNA expression of

other genes relevant in the cholesterol biosynthesis pathway were also increased, namely soluble HMG-

CoA synthase 1 (Hmgcs1, cytoplasmic), mevalonate kinase (Mvk), phosphomevalonate kinase (Pmvk),

mevalonatediphosphodecarboxylase (Mvd), farnesyldiphosphate synthase (Fdps), squalene synthase

(Fdft1), squalene monooxygenase (Sqle), lanosterol synthase (Lss), Cytochrome P450, family 51 (Cyp51)

and 7-dehydrocholesterol reductase (Dhcr7) (table 5). Transcription of all of these genes is regulated by

the sterol regulatory element binding protein 2 (Srebp2). 34 Microarray analysis showed induction of

the gene encoding Srebp2 (Srebf2) in low P/S ratio fed mice (table 5). Results were validated with qPCR

analysis of mRNA expression of Hmgcr, Hmgcs1 and Srebf2 (figure 5A).

A

C D

B

Figure 5. (A) Small intestinal expression of genes involved in cholesterol synthesis in low P/S ratio fed (black bars) versus control (white bars) mice. Data represent mRNA expression relative to the ribosomal RNA expression of the housekeeping gene 18S. (B) Cholesterol concentrations in small intestinal mucosa of low P/S ratio fed (black bars) and control (white bars) mice. (C) Hepatic Hmgcr expression and (D) Total cholesterol concentration in liver. Values represent medians and interquartile ranges for n=6 mice per group. *p<0.05 and **p<0.01 indicate significant difference between the two groups.

Chapter 4

86

Increased expression of Hmgcr was also detected in liver (figure 5C). We did not detect significant

changes in whole body cholesterol synthesis in our stable isotope study. The low P/S ratio diet did not

affect cholesterol concentration in jejunal mucosa (3.2 [2.8-3.5] versus 3.0 [2.1-3.4] μmol.mg mucosa-1),

and only slightly affected it in liver (13.5 [12.9-14.5] versus 11.1 [9.6-12.2] μmol.mg liver-1; p= 0.01,

respectively) (figure 5B and 5D).

Discussion

The major finding in our study is that a high fat diet containing predominantly saturated fatty acids

induces a major increase in net sterol excretion from the body via induction of TICE. The low P/S ratio diet

had no effect on dietary cholesterol intake, biliary cholesterol secretion or overall cholesterol absorption.

Since de novo cholesterol synthesis did not compensate for the sterol loss, the low P/S diet induced a net

negative total body cholesterol balance after 8 weeks of dietary supplementation.

By performing intestinal perfusion experiments in mice, it was previously shown that a high fat diet (P/S

ratio ~4) increased TICE in the proximal part of the intestine compared with a low fat diet. 10 Van der Velde

et al. suggested that stimulation of TICE induced the observed increase in neutral sterol excretion. 10

The experimental set up of van der Velde et al. did not allow actual quantification of the contribution of

TICE to total cholesterol excretion. We modified the method described by van der Veen et al. 18 which

allows quantification of the net contribution of the major body fluxes of cholesterol to feces. In the

present study we found that the degree of fatty acid saturation in a high fat diet influences cholesterol

excretion. A low P/S ratio diet with high amounts of saturated stearic and palmitic acid and low amounts

of unsaturated oleic and linoleic acid compared with a higher P/S ratio diet turned out to be very

efficient in increasing neutral sterol excretion and this effect was mainly accounted for by increased TICE.

Reduced mRNA expression of the apical uptake transporter Npc1l1 35 in the small intestine suggested

that the increase in fecal neutral sterol excretion could, at least in part, be caused by cholesterol

malabsorption. However, we did not find significantly decreased cholesterol absorption during low

P/S ratio diet feeding using a dual stable isotope test. It was shown previously that induction of TICE

by peroxisome proliferator-activated receptor delta (PPARδ, Nr1c2) activation did not correlate with

decreased Npc1l1 mRNA 11. Stimulation of Lxr has been shown to increase TICE in mice. 12,18 In low P/S

ratio fed mice we did not find changed expression levels of Lxr nor its target genes Abcg5 and Abcg8.

In contrast, expression of Cyp27a1, producing 27OH-cholesterol, a major Lxrα ligand in enterocytes 36,

was significantly downregulated during low P/S ratio diet (not shown). These data suggest that TICE is

induced independently of Lxr activation in our model.

The low P/S ratio diet increased mRNA expression of the transcriptional regulator of cholesterol

biosynthesis Srepf2 37 and its target genes in jejunum, whereas cholesterol concentrations in the jejunal

mucosa were similar compared to mice on control diet. It is possible that intestinal cholesterol synthesis

is indeed increased during low P/S ratio diet to compensate for cholesterol loss via TICE. Our method

does not allow measurement of cholesterol synthesis in individual organs.


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Yet, whole body synthesis was not increased by the low P/S diet. Compared with previous studies in mice,

we found relatively low whole body cholesterol synthesis rates. 18,38-40 Several factors can account for the

differences, including the method applied, the time point, time frame and differences in cholesterol

absorption between different mouse strains. Earlier studies were performed with the tritiated water

incorporation method in the dark phase of the light cycle, when synthesis is known to be higher than

during the light phase. In addition, mice were terminated one hour after label administration. 38-40 Based

on previous studies, it is thought that measurement of cholesterol synthesis with MIDA over at least 24h

yields accurate whole body cholesterol synthesis rates. 22,41 Depending on the mouse strain, cholesterol

absorption and thus synthesis differ. 6 Compared with previous studies (in C57BL/6J 18,38, 129Sv mice 39

and mice with mixed backgrounds 40) we found a relatively high cholesterol absorption in FVB mice,

and therefore not surprisingly a relatively low cholesterol synthesis rate. Since fecal sterol excretion

increased, the mice developed a negative total body cholesterol balance on the low P/S diet. Carcass

analyses should be performed in future studies to determine cholesterol concentration and synthesis

in individual organs.

The question arises where exactly the extra fecal cholesterol excretion originated from. Theoretically,

increased fecal cholesterol excretion on the low P/S ratio diet could be due to increased desquamation of

small intestinal enterocytes. During intestinal perfusions studies in mice, cholesterol from desquamated

cells was estimated to contribute ~15% to fecal sterols. 9 Our group recently demonstrated that most

of the TICE flux induced by plant sterols, measured via the stable isotope method, is abrogated in the

absence of Abcg5/g8 suggesting that these transporters play an important role in the pathway. 42 The

dominant role of Abcg5/g8 also confirms the relatively minor contribution of intestinal cell shedding

to TICE. Previous studies showed that there were no significant differences in proliferative capacity of

enterocytes between low P/S ratio and control diet. 19 Taking these considerations into account, we

suggest hat (most of ) the increased neutral sterol output on a low P/S ratio diet originates from the blood

compartment. In early human studies the estimated contribution of cholesterol from desquamated cells

to the intestinal lumen was at most ~10-20%. 43-45 However, the existence of a transport route from

blood to feces via the intestine was not taken into account in these studies. The turnover of intestinal

epithelial cells in humans (3-5 days) is slower than that in mice. It is tempting to speculate that part of the

suggested desquamation-derived fecal cholesterol in humans represents blood derived TICE. Studies

are warranted to clarify the origin of fecal sterols in humans.

Mice on low P/S ratio diet were not ill and their intake and body weights were normal. We calculated

the loss of body cholesterol with the help of literature values 46 of total body cholesterol. In contrast

to control mice that were balanced, mice on a low P/S ratio diet were estimated to lose a small but

significant fraction (0.5%; p<0.01) of their total body cholesterol per day. This indicates that activation of

TICE may actually lead to net cholesterol disposal from the body. Additional studies (of longer duration)

are necessary to investigate this further. Our model allows for quantification of the net TICE derived

cholesterol in feces, however actual TICE flux cannot be measured. Since part of the secreted cholesterol

is likely reabsorbed in the intestine, the actual flux may in reality be much higher.

Chapter 4

88

TICE might thus become much more prominent under conditions of impaired cholesterol absorption. 42

Altogether, our data clearly show that a low P/S ratio diet induces cholesterol excretion via the

transintestinal route. Transcriptional activation of cholesterol synthesis fails to compensate for fecal loss of

cholesterol. Induction of TICE by dietary manipulation (for example addition of long chain unabsorbable

fatty acids to avoid the negative side effects of saturated fatty acids) could represent an attractive target

in prevention and treatment of hypercholesterolemia and cardiovascular disease.

Acknowledgements

The authors would like to thank Juul Baller and Rick Havinga for their help during animal experiments,

and Mechteld Grootte Bromhaar, Renze Boverhof and Theo Boer for technical assistance.




declare.


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17. Imaizumi K, Abe K, Kuroiwa C, et al. Fat containing stearic acid increases fecal neutral steroid excretion and catabolism of low density lipoproteins without affecting plasma cholesterol concentration in hamsters fed a cholesterol-containing diet. J Nutr. 1993 Oct;123(10):1693-702.


19. Lukovac S, Los EL, Stellaard F, et al. Essential fatty acid deficiency in mice impairs lactose digestion. Am J Physiol Gastrointest Liver Physiol. 2008 Sep;295(3):G605-13.

20. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37:911-7.

21. van Meer H, Boehm G, Stellaard F, et al. Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats. Am J Physiol Gastrointest Liver Physiol. 2008 Feb;294:G540-7.


23. Wilson MD, Rudel LL. Review of cholesterol absorption with emphasis on dietary and biliary cholesterol. J Lipid Res. 1994 Jun;35(6):943-55.

24. Ferezou J, Coste T, Chevallier F. Origins of neutral sterols in human feces studied by stable isotope labeling (D and 13C). Existence of an external secretion of cholesterol. Digestion. 1981;21(5):232-43.

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25. Grefhorst A, Elzinga BM, Voshol PJ, et al. Stimulation of lipogenesis by pharmacological activation of the liver X receptor leads to production of large, triglyceride-rich very low density lipoprotein particles. J Biol Chem. 2002 Sep;277(37):34182-90.

26. Lefever S, Hellemans J, Pattyn F, et al. RDML: structured language and reporting guidelines for real-time quantitative PCR data. Nucleic Acids Res. 2009 Apr;37(7):2065-9.

27. Gentleman RC, Carey VJ, Bates DM, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004;5(10):R80.

28. Lin K, Kools H, de Groot PJ, et al. MADMAX - Management and analysis database for multiple ~omics experiments. J Integr Bioinform. 2011 Jul;8(2):160.

29. Irizarry RA, Wu Z, Jaffee HA. Comparison of Affymetrix GeneChip expression measures. Bioinformatics. 2006 Apr;22(7):789-94.

30. Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004;3:Article3.

31. Dennis G,Jr., Sherman BT, Hosack DA, et al. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 2003;4(5):3.

32. Brazma A, Hingamp P, Quackenbush J, et al. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet. 2001 Dec;29(4):365-71.

33. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and functions. Front Biosci. 2009 Jan;14:2584-98.



36. Li T, Chen W, Chiang JY. PXR induces CYP27A1 and regulates cholesterol metabolism in the intestine. J Lipid Res. 2007 Feb;48(2):373-84.

37. Hua X, Yokoyama C, Wu J, et al. SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element. Proc Natl Acad Sci U S A. 1993 Dec;90(24):11603-7.

38. Jolley CD, Dietschy JM, Turley SD. Genetic differences in cholesterol absorption in 129/Sv and C57BL/6 mice: effect on cholesterol responsiveness. Am J Physiol. 1999 May;276(5 Pt 1):G1117-24.

39. Osono Y, Woollett LA, Herz J, et al. Role of the low density lipoprotein receptor in the flux of cholesterol through the plasma and across the tissues of the mouse. J Clin Invest. 1995 Mar;95(3):1124-32.

40. Woollett LA, Osono Y, Herz J, et al. Apolipoprotein E competitively inhibits receptor-dependent low density lipoprotein uptake by the liver but has no effect on cholesterol absorption or synthesis in the mouse. Proc Natl Acad Sci U S A. 1995 Dec;92(26):12500-4.

41. Di Buono M, Jones PJ, Beaumier L, et al. Comparison of deuterium incorporation and mass isotopomer distribution analysis for measurement of human cholesterol biosynthesis. J Lipid Res. 2000 Sep;41(9):1516-23.

42. Brufau G, Kuipers F, Lin Y, et al. A reappraisal of the mechanism by which plant sterols promote neutral sterol loss in mice. PLoS One. 2011;6(6):e21576.

43. DenBesten L, Reyna RH, Connor WE, et al. The different effects on the serum lipids and fecal steroids of high carbohydrate diets given orally or intravenously. J Clin Invest. 1973 Jun;52(6):1384-93.

44. Cheng SH, Stanley MM. Secretion of cholesterol by intestinal mucosa in patients with complete common bile duct obstruction. Proc Soc Exp Biol Med. 1959 Jun;101(2):223-5.

45. Grundy SM, Metzger AL. A physiological method for estimation of hepatic secretion of biliary lipids in man. Gastroenterology. 1972 Jun;62(6):1200-17.

46. Hirsch RL, Kellner A. The pathogenesis of hyperlipemia induced by means of surface-active agents. I. Increased total body cholesterol in mice given triton WR 1339 parenterally. J Exp Med. 1956 Jul;104(1):1-13.


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Supplementary figure 1. Fecal fatty acid (FA) excretion in low P/S ratio fed (black bars) and control (white bars) mice after 4 weeks of diet (A). FA concentrations were determined in feces by gas chromatography and normalized to daily fecal output and body weight. Fecal neutral sterol excretion (B) and erythrocyte triene/ tetraene (TT) ratio (C) in low P/S ratio fed (black bars) and control (white bars) mice after 4 weeks of diet. TT ratio represents the ratio between eicosatrienoic (mead) acid (C20:3n-9) and arachidonic acid (C20:4n-6). Essential fatty acid deficiency is present when the TT ratio is increased above a threshold value of 0.2. Values represent medians and interquartile ranges for n=6 mice per group. **p<0.01 indicates significant difference between the two groups.

Chapter 5

Genetic inactivation of the bile salt export pump in mice profoundly increases fecal

cholesterol excretion

Mariëtte Y.M. van der Wulp 1,2, Theo H. van Dijk 2, Vincent W. Bloks 2,

Albert K. Groen 1,2, Henkjan J. Verkade 1,2

1 Top Institute Food and Nutrition, Wageningen, The Netherlands2 Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Beatrix

Children’s Hospital, Groningen University Institute for Drug Exploration, University of Groningen,

University Medical Center Groningen, Groningen, The Netherlands

In preparation

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Abstract

Objectives The bile salt export pump (Bsep) is the major hepatobiliary bile salt (BS) transporter,

facilitating transfer of BS from hepatocytes into the bile canaliculus. Bsep-/- mice were previously

shown to escape severe cholestasis by increasing biliary BS hydrophilicity, resulting in activation of

alternative transporters. Bsep-/- mice surprisingly displayed increased biliary cholesterol secretion.

The consequence of Bsep defi ciency and increased biliary cholesterol secretion for intestinal sterol

handling is unknown. To determine these downstream eff ects of Bsep defi ciency we determined BS

and whole body cholesterol fl uxes.

Methods Mice were fed either a low or a high fat diet (LFD or HFD, respectively), since it was shown

previously that a HFD can induce cholesterol excretion. We determined cholesterol intake, biliary

secretion, absorption and synthesis, output (feces), and the origin of fecal sterols.

Results Results were essentially the same in Bsep-/- mice fed a LFD or a HFD versus control mice.

Here, only results of LFD are presented. Biliary BS secretion was decreased in Bsep-/- compared

with Bsep+/+ mice (-40%; p<0.01). Biliary cholate secretion was decreased (-85%; p<0.001), whereas

beta-muricholate secretion was increased (+55%; p<0.01) in Bsep-/- mice. Cholesterol intake (+25%;

p<0.001), biliary secretion (4 fold; p<0.001) and synthesis (11 fold; p<0.001) were increased, whereas

cholesterol absorption was decreased (-90%; p<0.001) in Bsep-/- mice. Bsep-/- mice showed increased

fecal cholesterol excretion (8 fold; p<0.001), mostly via the transintestinal pathway (22 fold increase;

p<0.001).

Conclusion Bsep-/- mice show increased (transintestinal) cholesterol excretion, which appears to be

induced by severely impaired cholesterol absorption in the presence of a hydrophilic BS pool. Potential

health benefi ts of an increasingly hydrophilic BS pool in terms of intestinal cholesterol excretion require

further research.

Bsep deficiency induced fecal cholesterol disposal

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Introduction

Bile salts (BS) are produced by enzymatic modification of cholesterol in liver hepatocytes. They are

secreted by hepatocytes into bile canaliculi, to end up with bile in the small intestine. In the small

intestine, BS are indispensable for adequate absorption of lipids (dietary fat and cholesterol) and fat-

soluble vitamins. In the terminal ileum, BS are efficiently reclaimed by absorption (~95%) to continue

their enterohepatic circulation. 1

The main transporter facilitating transfer of BS from hepatocyte to bile canaliculus is the adenosine

triphosphate-dependent binding cassette transporter B11 (ABCB11), or bile salt export pump (BSEP). 2,3

Mutations in the Bsep gene can result in several forms of intrahepatic cholestasis. 4,5 Heterozygous Bsep

mutations have been identified in patients with intrahepatic cholestasis of pregnancy and drug-induced

cholestasis. 5 Missense Bsep mutations predominate in a relatively mild form of cholestatic disease

termed benign recurrent intrahepatic cholestasis type 2 (BRIC-2). 6 Frequently, Bsep mutations result in

the absence of canalicular BSEP protein 7, and are associated with biliary BS concentrations of less than

1% of normal. 3 Absence of functional BSEP results in progressive familial intrahepatic cholestasis type

2 (PFIC-2), a disease characterized by early onset of severe intrahepatic cholestasis, often requiring liver

transplantation within the first decade of life. 3,8

Murine Bsep and human BSEP were previously shown to possess comparable affinities for different

BS. 9 Bsep mainly transports conjugated di- and tri-hydroxylated BS, such as cholate (the main BS of

the human and rodent body BS pool) and chenodeoxycholate. 10,11 Bsep-/- mice were created to study

the effects of Bsep deficiency in detail in vivo. 12 An unexpected finding was that these mice displayed

a preserved basal bile flow rate. 12-15 Whereas the secretion of hydrophobic BS (cholate, deoxycholate

and chenodeoxycholate) was severely impaired, secretion of more hydrophilic BS (ursodeoxycholate

and muricholates) remained relatively unchanged. 12,15 Interestingly, Bsep-/- mice secreted substantial

amounts of tetra-hydroxylated BS in their bile, which were not found in Bsep+/+ mice. 12,14-16

It was shown that the multidrug resistance 1 protein could serve as a salvage (low affinity / high

capacity) pathway for BS secretion in Bsep-/- mice. 13,15 Bsep-/- mice showed uncoupling of secretion of

cholesterol and phospholipids with BS. In Bsep-/-, both biliary cholesterol and phospholipid secretion

were significantly increased. 12

Cholesterol homeostasis is mainly regulated by its intestinal absorption, fecal excretion and de novo

synthesis. 17 The influence of increased biliary cholesterol secretion for its intestinal handling are unknown.

The aim of the current study was to determine parameters of total body cholesterol homeostasis in Bsep-/-

mice and their wildtype littermates (Bsep+/+). Our data show that increased hydrophilic BS secretion in

Bsep-/- mice is associated with severely impaired cholesterol absorption and increased fecal cholesterol

output, mainly via the transintestinal pathway.

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Materials

Intralipid® (20%) was obtained from Fresenius Kabi, Den Bosch, The Netherlands. 2,2,4,4,6-Deuterium-

cholesterol (D5-cholesterol) was obtained from Medical Isotopes and 25,26,26,26,27,27,27-Deuterium-

cholesterol (D7-cholesterol) from Cambridge Isotope Laboratories Inc. 1-13C-acetate was obtained from

Sigma Aldrich (St. Louis, MO). All isotopes were of 98-99% isotopic purity. Sucrose and Trizma® base

were obtained from Sigma Aldrich (St. Louis, MO). Trimethylchlorosilane was obtained from Thermo

Scientific, Rockford, IL. Hydrochloric acid 37%, methanol, hexane and pyridine were obtained from

Merck, Darmstadt, Germany. Heptane was obtained from Rathburn chemicals ltd, Walkerburn, Scotland

and N,O-bis-(trimethyl)trifluoroacetamide (BSTFA) from Supelco, Bellefonte, PA.

Mice and diet

Mice were housed in a light- and temperature-controlled facility. Tap water and food were available ad

libitum. Mice were maintained on standard low fat diet (LFD) (RMH-B, 5 wt% fat, 0.66 µmol cholesterol.g-1)

or high fat diet (HFD) (#4141.07, 16 wt% (34 energy%) fat, 0.19 µmol cholesterol.g-1), both obtained from

Arie Blok BV (Woerden, The Netherlands). Bsep-/- mice on a mixed background 12 had been backcrossed

with C57BL/6 mice for at least 10 generations. Due to the low birth rate of Bsep-/- mice, it was inevitable

to use mice of different ages. Every mouse in the Bsep-/- group of a particular age was paired with a

wildtype (Bsep+/+) littermate of the same age. For the cholesterol kinetic study, male Bsep-/- and Bsep+/+

mice 4-15 months of age (LFD, n= 6 per group), and male Bsep-/- and Bsep+/+ mice 3-5 months of age

(HFD, n= 5 per group) were used. The experiments were performed in conformity with Public Health

Service policy and in accordance with the national laws. The Ethics Committee for Animal Experiments

of the University Medical Center of Groningen approved the experimental protocols.

Cholesterol kinetic study

The LFD was supplied to the mice from weaning until the experiment. The HFD was fed for 10 weeks

before the start of the experiment. Before any intervention, baseline bloodspots were collected on filter

paper from the tail vein and feces were collected during a 24h period (day -1). Food pellet weight was

determined before and after the 24h feces collection period and pellets were collected for quantification

of cholesterol content.

At day 1 mice received an intravenous (retro-orbital) injection of 1.5 mg D7-cholesterol dissolved in 500

μl intralipid and an oral dose of 3 mg D5-cholesterol dissolved in 1 ml medium chain triglyceride oil. At

time points 3, 6, 12, 24, 48, 72, 96, 120, 144 and 168h (day 8) after labeled cholesterol administration,

bloodspots were obtained. After taking the bloodspot at time point 168h, mice were switched to tap

water containing 2% stable isotope labeled 1-13C-acetate for 72h. Bloodspots were collected 12, 24, 32,

48, 56 and 72h (day 11, low fat diet) and 24, 32, 48, 56, 72, 80, 96h (day 12, HFD experiment) after the

start of 1-13C-acetate.


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Body weight and food intake were determined and feces were collected daily during the entire

experiment. At day 11 or 12 mice were anesthetized and the common bile duct was cannulated for bile

collection during 30 minutes as previously described. 18 Mice were sacrificed by cardiac puncture and

cervical dislocation. The small intestine was divided into three equal segments, which were rinsed with

phosphate-buffered saline (PBS 5 ml, low fat diet) and in addition with 5 ml of DEMI water containing

protease inhibitor (HFD experiments, 1 tablet per 50 ml of DEMI). Livers and intestines per segment were

snap frozen in liquid nitrogen and stored at -80°C.

Analytical methods

BS and indirect cholesterol balance

Biliary lipids were extracted 19 and total plasma cholesterol and biliary cholesterol and BS concentrations

were determined. 20,21 Food pellets and fecal samples were ground and 50 mg was prepared for neutral

sterol (cholesterol plus bacterial metabolites coprostanol and dehydrocholesterol) and BS analysis by

gas chromatography (GC) as described previously. 21 Indirect cholesterol balance was determined by

subtraction of dietary cholesterol intake and hepatobiliary secretion from fecal output of neutral sterols

(all calculated in μmol cholesterol.day-1). 22 Biliary BS hydrophobicity was calculated according to the

method of Heuman. 23

Fractional cholesterol absorption

The procedure for this study 20, was modified for the influx of labeled cholesterol. Fractional cholesterol

absorption (F(a)) was calculated as the ratio between fraction (area under the curve (AUC) of 7 days

following label administration) of orally administered D5-cholesterol and IV administered D

7-cholesterol,

after correction for their administered doses: F(a)= (AUC oral

/ AUCIV) x (Dose

IV / Dose

oral).


Fractional cholesterol synthesis was determined by mass isotopomer distribution analysis (MIDA) 24, of

the M1 and M

3 mass isotopomers. 20

Origin of fecal sterols

In order to determine the origin of fecal sterols, we modified the method described by van der Veen et

al. 20

Neutral sterol content in intestinal lumen

Before analyses, tubes containing flushed intestinal lumen contents were lyophilized for 48h. Aliquots of

10 (proximal and middle part of small intestine) to 50 (distal small intestine, cecum and colon) mg were

used to determine neutral sterols by GC as described above.

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98

Preparation of intestinal mucosa homogenates

Intestinal sections (proximal small intestine (SIP), middle segment of small intestine (SIM) and distal

segment of small intestine (SID) were thawed (and kept) on ice and cut open. Mucosa was scraped off

the interior with an object glass, transferred to a pre-weighed potter glass (2 ml) and the potter glass was

weighed. One ml of ice-cold sucrose buffer (250 mM sucrose in 10 mM Trizma® base, pH 7.4) was added

and the solution was homogenized by pottering (10 strokes). The solution was further homogenized

by putting it through 1 ml syringes with needles of 20 and 26 G, respectively, 5-10 times with each

needle. The homogenate was thoroughly vortexed and divided over 3 eppendorf cups (2 ml). Ten µl of

homogenate was transferred to 2 10 ml glass tubes (tube 1 one for GC 21 and tube 2 for GC-MS 20 analyses

of cholesterol (M0-M

7). Glass tubes and remaining homogenate were kept at -20°C until further analyses.

Determination of cholesterol concentration in intestinal mucosa

The glass tubes containing homogenate were thawed and internal standard (5 nmol 5α-cholestane)

for neutral sterol quantification by GC was added to tube 1. Tube 2 was worked up for cholesterol label

analyses (GC-MS). Lipids were extracted from all tubes as described previously. 19 Cholesterol esters

were hydrolyzed with 2 ml of a mixture containing 37% hydrochloric acid (15 ml), DEMI water (10 ml)

and methanol (125 ml) (1h at 90°C). The mixture was evaporated under a stream of nitrogen at 55°C.

After addition of 2 ml DEMI, lipids were extracted (2 times) by adding 3 ml of hexane, vortexing (30

seconds per tube), centrifuging (5 min at 2500 rpm) and transferring the top (hexane) layer to a new

glass tube. The hexane was evaporated under a stream of nitrogen at 55°C. Unesterified cholesterol

was derivatized using BSTFA/ pyridine (1:1 v/v) with 1% trimethylchlorosilane at RT overnight. The

mixture was evaporated under a stream of nitrogen at RT and samples were redissolved in 1 ml heptane

containing 5% BSTFA (GC) or 150 µl heptane containing 5% BSTFA (GC-MS).

Statistical analysis

Using Brightstat, we calculated the significance of differences between groups (Bsep-/- and Bsep+/+ mice

on a LFD and HFD) with Kruskal-Wallis rank tests (and in case this test indicated significant differences

between groups) with Conover multiple comparisons tests. Data that were only generated in HFD fed

Bsep+/+ and Bsep-/- mice were compared with Mann Whitney U tests. P-values <0.05 were considered

statistically significant. Data represent median and interquartile range.

Results

Characteristics of Bsep-/- compared with Bsep+/+ mice

We compared characteristics of Bsep-/- and Bsep+/+ mice on both a LFD and a HFD (table 1). Mice

consumed a slightly smaller amount of food per day on HFD compared with LFD (p<0.05), but Bsep-/-

mice consumed more than Bsep+/+ mice on either diet (p<0.001). Bsep+/+ and Bsep-/- mice had similar

body weights on LFD at the start and end of the experiment (only final body weight is shown).


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Although the body weights on HFD did not significantly differ between genotypes at the start or at the

end of the experiment (due to one relatively small Bsep+/+ mouse), Bsep-/- mice gained less weight on

HFD compared with Bsep+/+ mice (p<0.01). Bsep-/- mice showed a higher fecal output compared with

Bsep+/+ mice on either diet (p<0.001).

Both Bsep+/+ and Bsep-/- mice showed increased plasma cholesterol levels on HFD compared with LFD

(p<0.001). Bsep-/- mice however had significantly lower plasma cholesterol levels than Bsep+/+ mice on

LFD (p<0.01) and HFD (p<0.05).

Table 1. Characteristics of Bsep-/- and Bsep+/+ mice on low and high fat diet

Bsep+/+ Bsep-/- Bsep+/+ Bsep-/-

Diet LFD HFD

Basal body weight (g) 30.8 [23.0-32.0] 32.6 [31.7-34.2]

Body weight (g) at termination 35.8 [28.6-36.9]

31.9 [29.0-34.9]

40.9 [30.7-42.3]

36.8 [36.4-38.5]

Growth (g) 10.2 [7.5-11.2] a 4.4 [4.0-5.0] b

Bile flow 3.1 [2.4-3.6] a 4.3 [4.1-4.6] b 1.7 [1.3-2.5] c 3.6 [3.1-4.1] a

Small intestine (cm) 32.5 [31.0-34.0] a

37.0 [33.5-38.0] b

31.5 [29.5-34.0] c

33.5 [30.0-36.0] ab

Colon (cm) 6.0 [5.5-6.5] 7.0 [6-8] 7.0 [6.5-7] 6.5 [6.5-8]

Cecum (g) 0.6 [0.4-0.6] a 0.6 [0.4-0.8] a 0.2 [0.2-0.3] b 0.3 [0.2-0.3] b

Intake and output

Food intake (g.day-1) 4.0 [3.7-4.2] a 4.6 [4.5-4.9] b 3.5 [3.4-3.7] c 4.3 [4.0-4.6] d

Fecal output (g.day-1) 0.6 [0.6-0.7] a 1.0 [0.9-1.2] b 0.4 [0.4-0.4] c 0.7 [0.6-0.7] a

Plasma parameters

Cholesterol (mmol.L-1) 2.4 [2.1-2.7] a 0.7 [0.7-0.9] b 4.8 [3.4-5.0] c 2.5 [2.4-3.0] a

Data represent median and interquartile range, n= 5-6 mice per group. Different letters indicate significant differences between groups, one or more letters in common indicate nonsignificant differences between groups.

On HFD both Bsep+/+ and Bsep-/- mice showed a lower bile flow rate than on LFD (p<0.001), but Bsep-/-

mice displayed a significantly higher flow rate than Bsep+/+ mice (p<0.01). Biliary BS secretion was 40%

lower in Bsep-/- compared with Bsep+/+ mice on LFD (p<0.001; figure 1A), decreased in both groups on

HFD (p<0.01) and was comparable in Bsep-/- and Bsep+/+ mice on HFD. On either diet, Bsep-/- mice showed

a decreased biliary secretion of cholate (the main BS in Bsep+/+ mice) compared with Bsep+/+ mice (LFD

-87%; p<0.001; HFD -82%; p<0.001; figure 1B). On the other hand Bsep-/- mice showed increased biliary

secretion of β-muricholate (BMC, the main BS in Bsep-/- mice) compared with Bsep+/+ mice (LFD +58%;

p<0.001; HFD +100%; p<0.01; figure 1C). In accordance with these data, Bsep-/- mice secreted a more

hydrophilic bile in general as shown by decreased BS hydrophobicity. (Heuman index on LFD (-0.66

[-0.69 to -0.62] vs. -0.29 [-0.32 to -0.25]; p<0.001 and on HFD (-0.69 [-0.71 to -0.67] vs. -0.36 [-0.42 to -0.24];

p<0.001; data not shown).

Chapter 5

100

Figure 1. Biliary bile salt secretion rate in Bsep-/-

and Bsep+/+ mice on LFD (white boxes) and HFD (grey boxes). A) Total biliary bile salt secretion rate (BSSR). B) Biliary cholate secretion rate (CA SR). C) Biliary beta-muricholate secretion rate (βMC SR). Data are represented as median and interquartile range, n= 5-6 mice per group. **p<0.01, ***p<0.001 represent significant differences between the indicated two groups.

Figure 2. Fecal bile salt excretion in Bsep-/- and Bsep+/+ mice on LFD (white boxes) and HFD (grey boxes). A) Total daily fecal bile salt excretion. B) Daily fecal cholate excretion. C) Daily β-muricholate excretion. Data are represented as median and interquartile range, n= 4-7 mice per group. *p<0.05, **p<0.01, ***p<0.001 represent significant differences between the indicated two groups.


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Bsep-/- mice excreted less fecal BS compared with Bsep+/+ mice on LFD (-80%; p<0.001) and on HFD

(-84%; p<0.001; figure 2A). On both diets, Bsep-/- excreted less cholate in their feces compared with

Bsep+/+ mice (LFD -86%; p<0.01; HFD -79%; p<0.05; figure 2B). On the other hand, Bsep-/- excreted a

similar amount of β-muricholate compared with Bsep+/+ mice on LFD and HFD; figure 2C).

Bsep-/- mice display increased non-hepatobiliary cholesterol excretion (indirect cholesterol

balance)

Compared with a LFD, a HFD may induce TICE. 22 In order to investigate the effect of Bsep deficiency

on cholesterol homeostasis, we fed mice both a LFD or a HFD for 10 weeks. Attrituble to the lower

cholesterol content of the HFD, both Bsep+/+ and Bsep-/- mice consumed less cholesterol per day on HFD

compared with LFD (p<0.001). Bsep-/- mice consumed 25% (LFD; p<0.001) to 35% (HFD; p<0.001) more

cholesterol than Bsep+/+ mice (table 2). Both Bsep+/+ and Bsep-/- mice secreted less cholesterol in bile

on HFD compared with LFD (p<0.001). In Bsep-/- mice biliary cholesterol secretion was increased ~4-6

fold on both diets compared with Bsep+/+ mice (table 2; p<0.001). The difference in biliary cholesterol

secretion could however not account for the ~8 fold (LFD; p<0.001) and ~15 fold (HFD; p<0.001)

increase in fecal cholesterol excretion that Bsep-/- displayed compared with Bsep+/+ mice. Altogether, the

data indicate that Bsep-/- mice excreted a higher amount of cholesterol in feces via a non-hepatobiliary

pathway than Bsep+/+ mice (table 2; p<0.001).

Table 2. Indirect cholesterol balance

μmol.day-1 Bsep+/+ Bsep-/- Bsep+/+ Bsep-/-

LFD HFD


Biliary cholesterol secretion


2.2 [2.2-2.4] a

2.5 [1.8-3.3] a

4.4 [3.5-5.8] a

2.8 [2.5-3.1] b

10.2 [9.0-10.6] b

34.6 [30.9-42.4] b

0.5 [0.5-0.6] c

0.6 [0.4-0.8] c

1.8 [1.8-3.2] c

0.7 [0.7-0.8] d

3.6 [3.2-4.3] d

27.9 [27.3-34.9] b

Net non-hepatobiliary cholesterol excretion(<0 = net absorption; >0 = net excretion)

-0.5 [-1.3-1.2] a 21.4 [19.0-29.1] b 0.9 [0.7-1.8] a 22.9 [22.7-31.0] b

Data represent median and interquartile range, n= 5-6 mice per group. Different letters indicate significant differences between groups (p<0.05).

Cholesterol absorption is impaired and synthesis is upregulated in Bsep-/- mice

We measured crucial parameters of cholesterol homeostasis, cholesterol absorption and synthesis, with

stable isotope methodology. Bsep-/- mice absorbed cholesterol to a much lower extent than Bsep+/+ mice

on a LFD (5 versus 43% (median); p<0.001; figure 3). On HFD, both Bsep-/- and Bsep+/+ mice absorbed

more cholesterol than on LFD (Bsep+/+ LFD vs. HFD p<0.05 and Bsep-/- mice LFD vs. HFD p<0.01), but

Bsep-/- mice again absorbed much less cholesterol than Bsep+/+ mice (18 versus 84% (median); p<0.001;

figure 3).

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102

Figure 3. Cholesterol absorption in Bsep-/- and Bsep+/+ mice on LFD (white boxes) and HFD (grey boxes). Data are represented as median and interquartile range, n= 5-6 mice per group. ***p<0.001 represents significant difference between the indicated two groups.

Bsep-/- mice synthesized more cholesterol compared with Bsep+/+ mice on both diets (LFD 13 fold;

p<0.001 and HFD 8 fold; p<0.001; figure 4A). In both Bsep-/- and Bsep+/+ mice, whole body cholesterol

synthesis was higher on HFD compared with LFD (Bsep+/+ LFD vs. HFD p<0.001 and Bsep-/- mice LFD

vs. HFD p<0.001). Bsep-/- mice showed a higher fraction of newly synthesized cholesterol in bile and

feces (both diets, table 3), and intestinal mucosa and intestinal lumen contents (measured on HFD, table

3). Similarly, compared with Bsep+/+ mice, Bsep-/- mice had an increased amount of (preformed and)

de novo synthesized cholesterol in bile (all p<0.001; figure 4B) and feces (all p<0.001; figure 4C), and

intestine luminal contents (all p<0.05,); figure 4D, measured on HFD). The amount of de novo synthesized

cholesterol in feces and bile was very small in Bsep+/+ mice. However, both Bsep-/- and Bsep+/+ mice

excreted lower amounts of de novo synthesized fecal (p<0.05) and biliary (p<0.001) cholesterol

on HFD compared with LFD (figure 4B and 4C). Bsep+/+ and Bsep-/- mice showed similar cholesterol

concentrations in small intestinal mucosa (figure 4E).

Table 3. Fractional cholesterol synthesis

% Bsep+/+ Bsep-/- Bsep+/+ Bsep-/-

LFD HFD

Bile 4.8 [3.6-6.1] a 26.6 [25.6-27.8] b 7.1 [5.9-8.7] c 23.2 [19.0-25.1] d

Feces 6.4 [6.1-6.9] a 21.5 [20.1-22.1] b 5.1 [5.1-7.7] a 12.5 [11.1-15.4] c

SIP mucosa 8.8 [7.6-10.7] a 27.4 [21.9-28.4] b

SIM mucosa 8.6 [7.5-10.8] a 23.3 [17.8-24.2] b

SID mucosa 10.3 [8.4-12.2] a 23.2 [17.6-23.7] b

SIP lumen contents 8.7 [7.6-9.7] a 23.6 [19.4-25.5] b

SIM lumen contents 7.8 [9.2-9.6] a 22.7 [18.2-24.2] b

SID lumen contents 9.3 [8.2-11.2] a 23.1 [17.5-23.8] b

Cecum lumen contents 10.6 [8.9-11.4] a 22.2 [17.3-22.9] b

Colon lumen contents 9.9 [8.6-10.6] a 21.9 [17.2-23.4] b

Data represent median and interquartile range, n= 4-6 mice per group. Different letters indicate significant differences (p <0.05) between groups.


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However, Bsep-/- mice displayed increased small intestinal length (table 1) and mucosal weight (data not

shown), which resulted in an increased total amount of cholesterol in small intestinal mucosa compared

with Bsep+/+ mice, in which preformed and de novo synthesized cholesterol were increased (figure 4F).

Figure 4. Cholesterol synthesis in Bsep-/- and Bsep+/+ mice. A) Total body synthesis on LFD (white boxes) and HFD (grey boxes). B) Preformed (black) and de novo synthesized (white) cholesterol in bile. C) Preformed (black) and de novo synthesized (white) neutral sterols in feces. D) Cholesterol concentration in different parts of the small intestine on HFD in Bsep+/+ (white) and Bsep-/- (grey) mice. E) Preformed (black) and de novo synthesized (white) cholesterol in small intestinal mucosa on HFD. F) Preformed (black) and de novo synthesized (white) neutral sterol content in intestine lumenal contents of Bsep-/- and Bsep+/+ mice on HFD. SIP= small intestine proximal segment, SIM= SI middle segment, SID= SI distal segment, Ce= cecum, Co= colon. Data are represented as medians (and interquartile range in E), n= 5-6 mice per group. *p<0.05, **p<0.01, ***p<0.001 represent significant differences between the indicated two groups. For significance of differences shown in figure B, C and D, see text.

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Transintestinal cholesterol excretion is induced in Bsep-/- mice

We used the data obtained during our stable isotope studies to model the net contribution of dietary,

biliary, and intestinal cholesterol to fecal sterols. 20

Bsep-/- mice excreted more dietary cholesterol compared with Bsep+/+ mice on either diet (LFD 2.2 fold;

p<0.001, HFD 4.5 fold; p<0.001; figure 5), due to decreased absorption as described above. Similarly,

Bsep-/- mice excreted more biliary cholesterol on both diets (LFD 4 fold; p<0.001, HFD 29 fold; p<0.001;

figure 5). Finally, Bsep-/- mice showed increased TICE (LFD 22 fold; p<0.001, HFD 14 fold; p<0.001; figure

5).

Figure 5. Origin of fecal sterols in Bsep-/-

and Bsep+/+ mice on LFD and HFD. N= 5-6 mice per group. For significance of differences shown in figure, see text.

Discussion

The major finding in our study is that Bsep-/- mice, that secrete more hydrophilic BS via bile, display

severely impaired cholesterol absorption, increased cholesterol synthesis and greatly induced (trans-

intestinal) cholesterol excretion either on a low or high fat diet.

Previous studies have shown that dietary hydrophilic BS (including β-muricholate) inhibit cholesterol

absorption. 25-27 In hamsters, it was shown that enrichment of bile with very hydrophilic 6-alpha-

hydroxylated BS induced a global hypocholesterolemic effect and enhanced 3-hydroxy-3-methylglutaryl-

Coenzyme A reductase (Hmgcr, the rate-limiting enzyme for cholesterol synthesis) activity and fecal

cholesterol excretion. 26 Cholesterol input into the intestine of Bsep-/- mice is increased, based on

slightly more food ingestion and on a higher biliary cholesterol secretion rate. However, the severely

impaired intestinal capacity to absorb cholesterol only explains ~10-40% of the amount of cholesterol

lost via the feces in Bsep-/- mice. Apparently, in Bsep-/- mice a non-dietary and non-biliary source of

cholesterol contributes to fecal cholesterol loss. Our data indicate that transintestinal cholesterol

excretion is responsible for ~60-90% of fecal cholesterol excretion in Bsep-/- mice. The induction of TICE

in the present model occurs together with the more hydrophilic biliary BS composition. Future studies

would have to be performed to determine whether the coincidence is “merely” an association or indeed

causally related.


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If the latter were the case, then strategies to enhance the hydrophilicity of the BS composition could

become of preventive and therapeutic potential for hypercholesterolemia. Increased biliary cholesterol

secretion does not seem to facilitate gallstone formation in the presence of a very hydrophilic BS

pool 26,28, which would otherwise be a drawback.

Overall, the differences between Bsep+/+ and Bsep-/- mice were highly consistent and mostly independent

of the diet. Bsep-/- mice displayed increased small intestinal length and mucosal weight, possibly as a

compensatory mechanism for the impaired absorption of cholesterol, and likely other nutrients. 29

Our previous studies showed that absorption of dietary fatty acids is decreased in Bsep-/- mice (~85%)

compared with Bsep+/+ mice (~95%, unpublished data). Another indicator of malabsorption is the

increased fecal output we found in Bsep-/- mice.

Bsep-/- showed a decrease in plasma cholesterol compared with Bsep+/+ mice on both diets, which is

likely due to several factors, including profoundly increased biliary cholesterol secretion, cholesterol

malabsorption and ineffective compensatory increased cholesterol synthesis. Cohen-Solal previously

showed increased biliary cholesterol secretion upon oral hydrophilic BS feeding 26, however, Wang et

al. in contrast showed the opposite. 25 In the presence of low intracellular cholesterol levels, activity

of the transcription factors sterol regulatory element-binding proteins (SREBPs) is increased and genes

involved in cholesterol synthesis (Hmgcr) and uptake (Low density lipoprotein receptor (Ldlr)) are induced. 30

Although hepatic Ldlr mRNA expression in Bsep-/- mice is not evidently increased 13,14, its activity

is unknown. On the other hand, the nuclear receptor Liver X receptor (Lxr) is a major transcriptional

regulator of cholesterol homeostasis, particularly in terms of cholesterol disposal from the body. 31 Lxr

activated transcription of the two half-transporters ATP-binding cassette sub-family G member 5 and

8 (Abcg5/g8), which facilitate cholesterol transfer from liver to bile and from enterocyte to intestinal

lumen 32, may play a role in Bsep-/- mice as well. Future studies are warranted to provide more insight

into the mechanism behind the profoundly decreased plasma cholesterol, increased biliary cholesterol

secretion, and increased TICE in Bsep-/- mice.

Altogether, our data clearly show that absence of Bsep in the mouse liver is associated with secretion of

hydrophilic BS and profound transintestinal cholesterol excretion. This provides a rationale for studying

the effect of hydrophilic BS feeding to hypercholesterolemic mice and perhaps humans on fecal disposal

of cholesterol.

AcknowledgementsThe authors would like to thank Angelika Jurdzinski, Juul Baller and Rick Havinga for their help during

animal experiments, and Renze Boverhof, Klaas Bijsterveld and Theo Boer for technical assistance.

Conflicts of interest and source of funding:



report.

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References1. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and functions. Front Biosci.

2009;14:2584-98.

2. Gerloff T, Stieger B, Hagenbuch B, et al. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem. 1998 Apr;273(16):10046-50.


4. Strautnieks SS, Bull LN, Knisely AS, et al. A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis. Nat Genet. 1998 Nov;20(3):233-8.

5. Stieger B. Recent insights into the function and regulation of the bile salt export pump (ABCB11). Curr Opin Lipidol. 2009 Jun;20(3):176-81.

6. van Mil SW, van der Woerd WL, van der Brugge G, et al. Benign recurrent intrahepatic cholestasis type 2 is caused by mutations in ABCB11. Gastroenterology. 2004 Aug;127(2):379-84.

7. Strautnieks SS, Byrne JA, Pawlikowska L, et al. Severe bile salt export pump deficiency: 82 different ABCB11 mutations in 109 families. Gastroenterology. 2008 Apr;134(4):1203-14.

8. Davit-Spraul A, Gonzales E, Baussan C, et al. Progressive familial intrahepatic cholestasis. Orphanet J Rare Dis. 2009 Jan;4:1.

9. Noe J, Stieger B, Meier PJ. Functional expression of the canalicular bile salt export pump of human liver. Gastroenterology. 2002 Nov;123(5):1659-66.

10. Green RM, Hoda F, Ward KL. Molecular cloning and characterization of the murine bile salt export pump. Gene. 2000 Jan;241(1):117-23.

11. Lecureur V, Sun D, Hargrove P, et al. Cloning and expression of murine sister of P-glycoprotein reveals a more discriminating transporter than MDR1/P-glycoprotein. Mol Pharmacol. 2000 Jan;57(1):24-35.

12. Wang R, Salem M, Yousef IM, et al. Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis. Proc Natl Acad Sci U S A. 2001 Feb;98(4):2011-6.

13. Wang R, Chen HL, Liu L, et al. Compensatory role of P-glycoproteins in knockout mice lacking the bile salt export pump. Hepatology. 2009 Sep;50(3):948-56.

14. Wang R, Lam P, Liu L, et al. Severe cholestasis induced by cholic acid feeding in knockout mice of sister of P-glycoprotein. Hepatology. 2003 Dec;38(6):1489-99.

15. Lam P, Wang R, Ling V. Bile acid transport in sister of P-glycoprotein (ABCB11) knockout mice. Biochemistry. 2005 Sep;44(37):12598-605.

16. Perwaiz S, Forrest D, Mignault D, et al. Appearance of atypical 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid in spgp knockout mice. J Lipid Res. 2003 Mar;44(3):494-502.


18. Lukovac S, Los EL, Stellaard F, et al. Essential fatty acid deficiency in mice impairs lactose digestion. Am J Physiol Gastrointest Liver Physiol. 2008 Sep;295(3):G605-13.

19. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911-7.


21. van Meer H, Boehm G, Stellaard F, et al. Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats. Am J Physiol Gastrointest Liver Physiol. 2008 Feb;294(2):G540-7.


23. Heuman DM. Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions. J Lipid Res. 1989 May;30(5):719-30.



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25. Wang DQ, Tazuma S, Cohen DE, et al. Feeding natural hydrophilic bile acids inhibits intestinal cholesterol absorption: studies in the gallstone-susceptible mouse. Am J Physiol Gastrointest Liver Physiol. 2003 Sep;285(3):G494-502.

26. Cohen-Solal C, Parquet M, Ferezou J, et al. Effects of hyodeoxycholic acid and alpha-hyocholic acid, two 6 alpha-hydroxylated bile acids, on cholesterol and bile acid metabolism in the hamster. Biochim Biophys Acta. 1995 Jul;1257(2):189-97.

27. Wang DQ, Tazuma S. Effect of beta-muricholic acid on the prevention and dissolution of cholesterol gallstones in C57L/J mice. J Lipid Res. 2002 Nov;43(11):1960-8.

28. Montet JC, Parquet M, Sacquet E, et al. beta-Muricholic acid; potentiometric and cholesterol-dissolving properties. Biochim Biophys Acta. 1987 Mar;918(1):1-10.

29. Ballatori N, Fang F, Christian WV, et al. Ostalpha-Ostbeta is required for bile acid and conjugated steroid disposition in the intestine, kidney, and liver. Am J Physiol Gastrointest Liver Physiol. 2008 Jul;295(1):G179-86.



32. Yu L, Li-Hawkins J, Hammer RE, et al. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol. J Clin Invest. 2002 Sep;110(5):671-80.

Chapter 6

Conclusion, discussion and future perspectives

Discussion

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Conclusion, discussion and future perspectives

Intestinal function in terms of its capacity to absorb lipids in general and to secrete acidic and neutrals

steroids under varying conditions is the main focus of this thesis. We showed in chapter 2 that significant

acceleration of whole gut transit with polyethylene glycol (PEG) laxative treatment did not affect the

absorption and excretion of dietary fat and cholesterol. Chapter 3 on the other hand showed that PEG

treatment did decrease conversion of intestinal sterols (i.e. bile salts (BS) and cholesterol) and changed

microbiota composition. Transintestinal cholesterol excretion (TICE) appears to be an important pathway

of reverse cholesterol transport (RCT), at least in mice. In chapter 4 we showed that a high fat diet with

extremely low amounts of polyunsaturated fatty acids (low P/S ratio) doubled total fecal neutral sterol

excretion and induced TICE compared with a high fat diet with a standard P/S ratio. Chapter 5 showed

that absence of the Bile salt export pump (Bsep) in the murine liver, which induces biliary secretion of

highly hydrophilic BS, severely affects cholesterol homeostasis. Bsep-/- mice display severely impaired

cholesterol absorption, increased cholesterol synthesis and greatly induced (trans-intestinal) cholesterol

excretion.

Laxative treatment with polyethylene glycol does not affect lipid absorption, but decreases intestinal sterol conversion in ratsUsing different methodologies, including fat balance and stable isotope techniques, we showed in

chapter 2 that significant acceleration of whole gut transit time (WGTT) with PEG does not affect lipid

absorption and secretion in rats. A reassuring result, since PEG is used worldwide by many constipated

children who need adequate nutrient absorption to maintain growth. On the other hand, if simple oral

treatment with inert PEG would induce lipid malabsorption and/ or TICE, this could provide a promising

strategy to decrease hyperlipidemia. At this point, it is not possible to exclude the possibility that more

pronounced acceleration of WGTT or small intestinal transit would have increased fecal lipid excretion in

rats. Moreover, our results in rats may not (completely) be translatable to the human situation. However,

it must be kept in mind that gastrointestinal transit in humans would have to be accelerated just to

the extent that it induces fecal lipid disposal, but not diarrhea. Likely, humans would not be willingly

to adhere to the drug if it induces diarrhea, and loss of (additional) important nutrients is undesirable.

Interestingly, PEG treatment led to major changes in enterohepatic circulation of BS, an effect which

was independent of its effect on WGTT (chapter 3). Our study showed that during PEG treatment, the

amount of secondary BS, which are produced by the intestinal microbiota, was significantly decreased in

intestinal contents, feces and bile. The pool of primary BS on the other hand was increased during PEG

treatment. Primary BS are cholesterol derivatives produced in the liver that are conjugated before biliary

secretion. Conjugated BS have many functions besides facilitating lipid absorption in the small intestine.

Via their receptors (farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (GPBAR1 or

TGR5) primary BS may positively influence triglyceride, cholesterol and glucose homeostasis. 1,2

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Moreover, they repress bacterial growth in the small intestine. 3 The human colon harbors a complex high

density microbial community, which can be seen as an ‘exteriorized organ’. 4 Bacteria in all major divisions

are capable of deconjugating BS via BS hydrolases (BSH; als referred to as choloylglycine hydrolase). 5 BSH

appear to promote bacterial survival and were proposed to facilitate colonization and development of

microbiota in the gut. 5,6 It is conceivable that local mucosal defense mechanisms act in synergy with BS

to prevent overgrowth of BSH producing bacteria in the small intestine.

Upon liberation of free primary BS, these are open to a wider pathway of modification (mainly 7α/β-

dehydroxylation) by a limited number of anaerobes (Clostridium) 6,7, producing secondary BS. 7α/β-

dehydroxylation is a multistep pathway that requires the activity of the gene products of the bile acid

inducible (bai) genes that are located to a bai operon identified in several Clostridium species. 6,8-11,11

We observed a decrease in Clostridium during PEG treatment in rats. Although the use of culture-

independent analysis of microbiota (pyrosequencing) allowed us to identify a large number of microbes,

it did not allow differentiation at the species level. Unfortunately, this made it impossible at this point

to direclty link decreased sterol conversion during PEG treatment to decreases in specific bai operon

carrying microbiota. However, PEG may not decrease intestinal sterol conversion by simply reducing

the number of specific microbes, but could also reduce activity of sterol converting enzymes. Future in

vitro studies (testing specific microbiota and their enzyme activities) might shed light on the mechanism

underlying our findings.

Unconjugated secondary BS, such as deoxycholate (DC), are more hydrophobic and have a higher

pKa, which permits their partial recovery via passive absorption in the colon. Whereas rodents are

capable of 7α-hydroxylation of DC in the liver, forming cholate, humans are not equipped to do so.

Thus, humans do not possess a metabolic pathway for removing DC under physiological conditions.

However, accumulation of DC, for example under conditions of slow gastrointestinal transit 12,13, has

been associated with gastrointestinal disease, ranging from cholesterol gallstones to gastrointestinal

malignancies. 14,15 Gut microbiota increase our capacity to harvest energy from the diet.

Germ-free mice fed a Western type diet are protected from obesity, insulin resistance and dyslipidemia. 16,17

Notably, the hypocholesterolemic effect in germfree mice has been attributed to decreased cholesterol

absorption due to decreased BS deconjugation. We did not observe differences in deconjugation of BS

in rats that we have treated with PEG and did not detect differences in cholesterol absorption. However,

we did find potentially beneficial changes in microbiota composition and showed decreased secondary

BS production during PEG treatment. Because of the link between secondary BS and gastrointestinal

disease, attempts are being made to reduce their production and/ or eliminate them from the

gastrointestinal tract. Several possible interventions with secondary BS production are summarized here.

Discussion

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· Probiotics: probiotics have potential health benefits via modulation of BS and cholesterol metabolism.

Orally administered probiotics first have to survive passage through gastric juice and bile. The finding

that microencapsulated BSH-active Lacobacillus plantarum in rats are excreted alive in feces in

this sense is promising. 18 Increased BS deconjugation in the small intestine could lead to BS and

cholesterol malabsorption, lowering plasma cholesterol levels. 19 Jones at al. recently showed that

microencapsulated BSH active Lactobacillus reuteri can decrease plasma cholesterol levels in adults

with hypercholesterolemia. 20 Many Lactobacilli are able to assimilate cholate. 21 Since probiotics do

no produce secondary BS themselves, this could protect BS from being converted to hydrophobic

BS. Thus, if the capacity to assimilate cholate is not sufficient, an increased amount of unconjugated

cholate will become available to the resident colon microbiota 18 during BSH-active probiotic

treatment, which could counteract the positive probiotic effects or even worsening the situation in

terms of increased 7α/β-dehydroxylation. Rigorous human studies would be required to determine

the benefits and possible adverse effects of probiotic treatment.

· Antibiotics: Long term use of antibiotics to decrease secondary BS formation by 7α/β-dehydroxylation

would be impractical since it would induce side effects such as diarrhea and antibiotic resistance.

Furthermore, although antibiotic treatment in mice led to reduction of DC below detection limit, it

disturbed BS signaling in general as evidenced by increased BS synthesis. 22 This option at this point

does not seem favorable.

· Pharmaceuticals that inhibit microbial 7α/β-dehydroxylating enzymes: these compounds would also

be subject to drug resistance, similar to antibiotics.

PEG induced a decrease in the intestinal (microbial) conversion of BS and cholesterol. The decrease in

secondary BS during PEG treatment was related to decreased cytotoxic activity of fecal water, which

represents a surrogate marker of colon cancer risk. PEG induced a (non-significant) decrease in bacterial

load, decreased Clostridium (Firmicutes) and increased mucus-associated bacteria such as Akkermansia

(Verrucomicrobia) and Bacteroides (Bacteroidetes). It was previously shown that the ratio between

Bacteroidetes and Firmicutes is altered in response to dietary changes. High fat diets increase Firmicutes

and decrease Bacteroidetes in mice, a similar effect was shown in one study in which obese subjects

were put on a low calorie diet. 23 Although the value of the Bacteroidetes/ Firmicutes ratio in humans is

at present unclear, our results in PEG treated rats may indicate a positive effect on gut health.

Altogether, our studies provide a rationale to study the effect of PEG treatment on BS metabolism and

microbiota composition in humans. PEG may be beneficial for gastrointestinal health in humans with a

tendency to produce increased amounts of secondary BS.

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Transintestinal cholesterol excretion in mice can be manipulated by dietary fat compositionCardiovascular disease represents the second cause of death in our society. Hypercholesterolemia, a

major risk factor for cardiovascular disease, requires adequate monitoring and treatment. Although many

people can benefit from the therapeutic effect of cholesterol synthesis inhibitors (statins), these drug

can have severe side effects and do not produce the necessary decrease in plasma cholesterol levels

in a substantial amount of patients 24,25, which has led to an intense search for alternative therapeutic

modalities. Cholesterol can be excreted with feces via bile (hepatobiliary pathway) or directly via the

intestine (i.e. TICE). 26 Removal of cholesterol via increased secretion into bile seems unpractical, since

it could increase the risk of cholesterol gall stone formation. TICE is an alternative pathway for body

cholesterol removal. Although the steps in the pathway need to be unraveled in detail, TICE may

represent an attractive target for treatment of hypercholesterolemia in humans. Induction of TICE via

simple dietary intervention would be an attractive strategy to decrease plasma cholesterol in a simple

manner with low risk of side effects, increasing compliance.

In chapter 4 and 5, we studied the effects of dietary intervention and defective BS secretion (discussed

below) on TICE and cholesterol homeostasis in general. Chapter 4 shows that it is indeed possible to

induce TICE by dietary means, without an adequate compensatory increase in cholesterol synthesis.

A high fat diet with a very low ratio of polyunsaturated to saturated fatty acids (P/S ratio) doubled the

neutral sterols in feces originating from TICE, compared with a standard high fat diet.

It was previously shown that dietary saturated fatty acids can increase fecal cholesterol excretion in rats 27 and mice 28. Human data on this subject have been conflicting 29; most studies reported increased fecal

neutral sterols by high dietary P/S ratio, whereas one study showed increased fecal neutral sterols by a

low P/S ratio diet (P/S ratio 0.2 vs. 1.9). 30 It has been suggested that the increased fecal neutral sterol

secretion during high P/S ratio diet feeding was transient 27,31 and/or that part of the increase may have

been due to differences in dietary plant sterols that were not adequately separated from the neutral

sterol fraction before the advent of gas-chromotography analysis. 30

The question remains which dietary component was exactly responsible for the induction of fecal

neutral sterol excretion in our experiments. The low P/S diet contained almost no linoleic acid (essential

fatty acid), which was replaced by high amounts of palmitic and stearic acid. Previous studies had shown

that the effect of the low P/S ratio diet on cholesterol excretion was not due to essential fatty acid

deficiency (chapter 4). It is possible that (part of ) the effect we showed of the low P/S ratio diet is due to

the increased dietary stearic acid intake. The possible effects of stearic acid, a long chain fatty acid, are

often overlooked 32 since it belongs to the group of saturated fatty acids, which are generally considered

as plasma (LDL) cholesterol raising. Stearic acid is however considered a biologically neutral fatty acid,

especially with respect to the regulation of LDL-c concentration. 33-35 It is not entirely clear why stearic acid

does not raise plasma cholesterol, but contributing factors may be the lower absorption rate of stearic

acid compared with other (unsaturated) fatty acids 36 and rapid conversion of stearic to oleic acid. 35

Discussion

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Schneider et al. showed decreased cholesterol absorption and increased fecal cholesterol excretion in

hamsters fed a high stearic acid diet 28, but no studies have been conducted on the effect of high dietary

stearic acid on fecal cholesterol excretion/ TICE in humans. Dietary stearic acid may however improve

the atherogenic risk factor profile in humans. 32,37,38 In chapter 4 we did not see an effect of the low P/S

ratio diet on cholesterol absorption or plasma cholesterol concentration. However, wildtype mice do not

represent a good model to study effects of different dietary fat compositions on plasma (LDL) cholesterol

levels, since they carry most of their plasma cholesterol in HDL particles. 39 Future studies could include

specific stearic acid enriched diets in other animal models. In addition, it would be interesting to reveal

the effect of non-absorbed long chain fatty acids on fecal cholesterol excretion and TICE.

In addition, dietary omega-3 fatty acids have been suggested to be beneficial in lowering cardiovascular

disease risk. They however mainly lower plasma trigycerides and not cholesterol. 40 Currently, there

is no firm trial evidence that clinical outcomes (in terms of myocardial infarction, stroke or mortality)

improve more with omega-3 fatty acids added to statin therapy as compared to high dose statin

monotherapy. 40,41

The search for compounds that decrease plasma cholesterol levels in humans while inducing

minimal side effects is ongoing. Ezetimibe is the well known pharmaceutical that blocks cholesterol

absorption 42,43 and it may induce TICE. 44 It can be hypothesized that inhibition of cholesterol absorption

in general induces secretion of cholesterol by the intestine. Compounds that create a ‘hydrophobic

sink’ in the intestine, impairing cholesterol absorption, might increase fecal cholesterol excretion by

decreased absorption as well as induced TICE. The indigestible sucrose fatty acid polyester olestra dose-

dependently decreases cholesterol absorption. 45 Olestra does not change fecal BS excretion 46, but

decreases intestinal cholesterol conversion and increases fecal neutral sterol excretion. 47 Caution should

be undertaken however, since olestra decreases plasma fat-soluble vitamins as well. 48 It is unknown

whether cholesterol lowering compounds such as olestra, and perhaps stearic acid, can induce TICE. If

so, these oral compounds, in combination with other cholesterol lowering therapies, may reduce the

need for (high dose) pharmaceuticals such as statins and thereby may reduce treatment burden and

increase compliance.

Genetic inactivation of the bile salt export pump in mice leads to massive cholesterol excretionThe bile salt export pump (BSEP) is the main transporter facilitating transfer of BS from hepatocyte to bile

canaliculus. 49,50 Mutations in the Bsep gene can result in several forms of intrahepatic cholestasis. 51,52 For

example, heterozygous Bsep mutations have been identified in patients with intrahepatic cholestasis of

pregnancy and drug-induced cholestasis. 51 Missense Bsep mutations on the other hand predominate

in a relatively mild form of cholestatic disease termed benign recurrent intrahepatic cholestasis type 2

(BRIC-2). 53 Frequently however, Bsep mutations result in the absence of canalicular BSEP protein 54, and

are associated with biliary BS concentrations of less than 1% of normal in humans. 50

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Absence of functional BSEP results in progressive familial intrahepatic cholestasis type 2 (PFIC-2), a disease

characterized by severe intrahepatic cholestasis in young children, often requiring liver transplantation

within the first decade of life. 50,55 In contrast to these patients, mice in which Bsep is eliminated from

the liver, are able to convert their hepatic BS to more hydrophilic forms and secrete them via alternative

transporters. 56

In chapter 5 we showed that Bsep-/- mice have severely disturbed intestinal function and absorb minimal

amounts of cholesterol. Fecal neutral sterol excretion was increased dramatically, at least in part via

the transintestinal pathway. Previously, Wang et al. fed gallstone-susceptible C57L/J mice with a range

of BS and showed a positive correlation between hydrophobicity indices of the BS pool and percent

cholesterol absorption. 57 In these mice both beta-muricholate (βMC) and ursodeoxycholate (UDC)

reduced cholesterol absorption and the biliary secretion rate of cholesterol. However, βMC was even

more effective in prevention and dissolution of cholesterol gallstones than UDC. 58

Others 59 have shown a global hypocholesterolemic effect of 6-alpha-hydroxylated BS, with concomitant

increased cholesterol synthesis in hamsters. Cholesterol absorption was reduced by 59% and fecal

neutral sterol excretion was increased up to 10-fold. 59 Administration of the 6-alpha-hydroxylated BS led

to increased biliary cholesterol secretion in hamsters 59,60,60, mice 61 and prairie dogs 62. Gallstones were

however not detected; apparently as a result of the weak capacity of very hydrophilic BS (as compared

to UDC and chenodeoxycholate) to form micelles. 63 A defect in biliary micelle formation can also explain

the reduction in cholesterol absorption by hydrophilic BS. 64 UDCA (ursodeoxycholic acid) is used for

treatment of cholesterol gall stones in humans and can decrease cholesterol absorption. 65,66 In patients

with primary biliary cirrhosis, UDCA lowers LDL-c. 67

Potentially, bacterial transformation of UDCA into the highly hydrophobic lithocholate (LC) could be a

drawback for UDCA use in humans. 6,68 However, the evidence is limited (LC is unsolvable) and it was

shown that LC can activate the vitamin D receptor in intestinal epithelial cells 69, which leads to induction

of genes encoding proteins that metabolize LC. 70 This in turn may limit LC toxicity to the intestinal

mucosa. At this point it remains unclear if the therapeutic potential for administration of hydrophilic BS

such as hyodeoxycholate eventually will be greater. Human studies that delineate the effect of feeding

these BS on TICE and cholesterol homeostasis in general are awaited.

Conlusion

This thesis shows that several minor interventions with intestinal function may provide health benefits.

Oral laxative treatment did not affect intestinal lipid absorption, but decreased intestinal acidic and

neutral sterol metabolism. The decrease in secondary bile salt formation observed during treatment

with polyethylene glycol may improve intestinal function and health in humans as well. We showed that

TICE can be induced by dietary means. This provides a rationale for human studies with oral hydrophilic

bile salt ingestion and for example non-absorbable long-chain fatty acids to quantitate the effect of

dietary interventions on TICE and potential health benefits in terms of decreasing hypercholesterolemia

and ultimately cardiovascular disease.

Discussion

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Chapter

6

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Appendices

Summary

Nederlandse samenvatting

Dankwoord

Biography / Biografi e

List of publications

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123

Summary

The intestinal epithelium forms a dynamic interface for interactions between the body and food

components. Under physiological conditions, the intestinal epithelium maintains a peaceful equilibrium

with an extremely large community of commensal bacteria that live in the intestinal lumen and regulates

absorption of nutrients and excretion of waste.

Lipids (fat and cholesterol) are of vital importance for vertebrate cell membrane structure and function.

Cholesterol plays a central role in maintaining membrane fluidity, but shows ‘Dr. Jekyll and Mr. Hyde’ type

of characteristics. Its sterol nucleus is so resistant to breakdown that the body can almost not metabolize

cholesterol, leading to accumulation and induction of vascular disease when cholesterol is present in

excess. The only way to dispose of cholesterol is direct excretion with feces or conversion to bile salts

(BS). Besides mediating excretion of the sterol nucleus BS also fulfil important biological functions. BS are

important for efficient absorption of cholesterol, fat and fat-soluble vitamins.

Hypercholesterolemia is common in our Western society and represents a major risk factor for

cardiovascular disease. Classically, fecal cholesterol excretion was believed to be primarily driven by

cholesterol secreted via the hepatobiliary pathway (either in the form of cholesterol, or after breakdown

to BS). However, it has recently become apparent that direct secretion of cholesterol from the blood

compartment to the intestinal lumen, the process now adopted “TransIntestinal Cholesterol Excretion”

(TICE), plays a major role in fecal cholesterol disposal. Reduction of cholesterol absorption and induction

of TICE represent attractive therapeutic targets. Ideally, inhibition of cholesterol absorption and/or

induction of TICE would be facilitated by simple oral (dietary) intervention.

In this thesis we assessed, in a quantitative manner, intestinal function with respect to its capacity to

digest and absorb lipids (dietary fats, cholesterol and BS), and its capacity to secrete cholesterol under

varying intestinal conditions. In chapter 1, we reviewed current insights into the regulation of lipid

homeostasis. In chapter 2 we studied the effect of significant acceleration of whole gut transit (the time

for substances to travel from mouth to anus) with polyethylene glycol (PEG). PEG is a non-absorbed, non-

metabolized drug that is widely used around the world as a laxative agent to treat constipation in adults

and children. We found that PEG treatment did not affect the absorption and excretion of dietary fat

and cholesterol. In chapter 3 on the other hand we showed that PEG treatment did decrease microbial

conversion of intestinal sterols (i.e. BS and cholesterol) and changed fecal microbiota composition

(increased Verrucomicrobia and decreased Firmicutes in PEG treated versus control rats, respectively),

which can possibly be health-promoting. In chapter 4 we showed that a high fat diet with extremely low

amounts of polyunsaturated fatty acids (low polyunsaturated to saturated fatty acid (P/S) ratio) doubled

total fecal neutral sterol (cholesterol and its metabolites formed in the intestinal lumen) excretion and

induced TICE compared with a high fat diet with a standard P/S ratio. This research provides a proof-

of-concept; fecal cholesterol excretion via TICE can be induced by dietary manipulation. Additional

studies are needed to obtain the desired effect (inducing TICE), without inducing well known negative

(hyperlipidemic) side effects of saturated fatty acids.

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124

The major hepatic transporter facilitating transfer of BS from liver to bile is called the Bile salt export

pump (Bsep). Chapter 5 showed that the absence of Bsep in the murine liver, which induces biliary

secretion of highly hydrophilic BS, severely affects cholesterol homeostasis. Bsep-/- mice display severely

impaired cholesterol absorption, increased cholesterol synthesis and greatly induced (trans-intestinal)

cholesterol excretion.

Together this thesis shows that simple oral interventions, at least in laboratory animals, appear to have

health-promoting effects in terms of lipid homeostasis. The studies described provide a rationale for

human studies to test the effect of for example dietary non-absorbed long chain fatty acids and dietary

hydrophilic BS on TICE. Moreover, a relatively simple human study could provide more insight into

the possible health promoting effects of PEG treatment (via its effect on intestinal microbial flora) of

(apparently) healthy humans and constipated patients.

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125

Nederlandse samenvatting

Het darmepitheel (binnenste slijmvlieslaag) vormt een dynamische scheidslijn die interactie tussen

voeding en het lichaam toestaat. Onder natuurlijke/ gezonde omstandigheden verkeert het

darmepitheel in evenwicht met een enorme gemeenschap van bacteriën die in de darmholte leeft en

de opname (absorptie) van voedingsstoffen en uitscheiding van afvalstoffen reguleert.

Lipiden (vetten en cholesterol) zijn van vitaal belang voor de structuur en functie van celmembranen.

Cholesterol speelt een centrale rol in het behoud van vloeibaarheid van celmembranen, maar vertoont

‘Dr. Jekyll and Mr. Hyde’ karakteristieken. De zogenaamde sterolenkern van cholesterol is dermate

resistent tegen afbraak dat het lichaam nauwelijks in staat is cholesterol af te breken, wat leidt tot

stapeling en het ontstaan van vaatziekten wanneer er teveel cholesterol aanwezig is. Het lichaam kan

zich alleen van cholesterol ontdoen door het of direct uit te scheiden met ontlasting, of door het om

te zetten in galzouten (GZ) in de lever, waarna deze GZ naar de darm kunnen worden uitgescheiden

met gal en uiteindelijk ook met de ontlasting het lichaam kunnen verlaten. Naast het mediëren van de

uitscheiding van de sterolenkern, vervullen GZ belangrijke biologische functies. De aanwezigheid van

GZ in de dunne darm is essentieel om de opname van cholesterol, (voedings-)vetten en vetoplosbare

vitamines efficiënt te laten verlopen.

Hypercholesterolemie (teveel cholesterol in het bloed) komt zeer veel voor in onze Westerse

samenleving en is een grote risicofactor voor hart- en vaatzieken. In het verleden werd gedacht dat

cholesteroluitscheiding voornamelijk gedreven werd via de lever-gal (hepatobiliaire) route. Echter,

recent is duidelijk geworden dat uitscheiding van cholesterol via de bloedbaan naar de darmholte en

vervolgens de ontlasting, het proces wat “TransIntestinale Cholesterol Excretie” (TICE) gedoopt is, een grote

rol kan spelen in de cholesteroluitscheiding. Het remmen van cholesterolopname en het stimuleren

van TICE kunnen tot twee aantrekkelijke therapeutische doelen verworden. Idealiter zou remming van

cholesterolopname en/of stimulatie van TICE tot stand gebracht worden door simpele dieet (orale)

aanpassingen.

In dit proefschrift hebben wij de darmfunctie bepaald in termen van de capaciteit om lipiden te verteren

en op te nemen, en de capaciteit om cholesterol uit te scheiden onder wisselende omstandigheden in

de darm. Hoofdstuk 1 van dit proefschrift geeft een overzicht van de huidige inzichten in de regulatie

van lipidenhuishouding. In hoofdstuk 2 hebben we het effect bestudeerd van versnelling van de

darmpassage (de zogenaamde “whole gut transit”, ofwel passagetijd van mond tot anus) met het

laxeermiddel polyethyleen glycol (PEG). PEG is een polymeer die in de darm niet opgenomen en niet

omgezet wordt. PEG wordt wereldwijd veel gebruikt bij de behandeling van obstipatie van volwassenen

en kinderen. We zagen dat PEG geen invloed had op de opname en uitscheiding van voedingsvetten en

cholesterol. In hoofdstuk 3 daarentegen, hebben we laten zien dat behandeling van ratten met PEG leidt

tot verminderde bacteriële omzetting van sterolen (GZ en cholesterol) in de darm en een veranderde

samenstelling van de darmbacteriën (zich uitend in een toename in Verrucomicrobia en een afname

in Firmicutes in ontlasting van PEG behandelde versus onbehandelde controle ratten, respectievelijk),

welke mogelijk gezondheidsbevorderend is.

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126

In hoofdstuk 4 hebben we laten zien dat een hoogvet dieet met extreem lage hoeveelheden

meervoudig onverzadigde vetzuren (lage ‘polyunsaturated to saturated’ vetzuur (P/S) ratio) de

totale neutrale sterolen (cholesterol en stofwisselingsproducten hiervan die door de darmbacteriën

geproduceerd worden) uitscheiding verdubbelt en de TICE stimuleert in vergelijking met een hoogvet

dieet met een standaard P/S ratio. Deze studie geeft een zogenaamd ‘bewijs-van-concept”; namelijk

dat cholesteroluitscheiding wel degelijk gestimuleerd kan worden door een simpele aanpassing van

de dieetsamenstelling. Toekomstige dieetinterventies moeten de focus leggen op stimulatie van

TICE, zonder negatieve effecten (hyperlipidemie; verhoogd vetgehalte in het bloed) van verzadigde

voedingsvetten te veroorzaken.

Het belangrijkste transporteiwit in de lever dat transport van GZ vanuit de levercel naar de gal verzorgt,

wordt de GZ export pomp (Bile salt export pump (Bsep) in het Engels) genoemd. In hoofdstuk 5 hebben

wij laten zien dat afwezigheid (knock-out) van Bsep in de muizenlever, hetgeen uitscheiding van zeer

hydrofiele (‘waterminnende’) GZ veroorzaakt, grote veranderingen in de cholesterolhuishouding teweeg

brengt. Bsep-/- muizen hebben een zeer gestoorde cholesterolopname in de darm, een verhoogde

cholesterolaanmaak, en scheiden zeer veel cholesterol uit in de ontlasting, voornamelijk via TICE.

Samenvattend laat dit proefschrift zien dat simpele (dieet-)aanpassingen, althans in proefdieren,

gezondheidsbevorderende effecten kunnen hebben in termen van GZ- en cholesterolhuishouding

in het lichaam. De beschreven studies bieden perspectief voor vervolgstudies in mensen om te

onderzoeken wat het effect is van de inname van bijvoorbeeld zogenaamde lange keten vetzuren, die

niet door de darm opgenomen kunnen worden, en hydrofiele GZ op TICE. Daarnaast kan een relatief

eenvoudige studie gestart worden om het mogelijk gezondheidsbevorderende effect van PEG (via het

effect op de darmbacteriën) van (ogenschijnlijk) gezonde mensen en mensen met obstipatie is.

Appendices

127

Dankwoord

Elke reis begint met de eerste stap

De afgelopen jaren ben ik volgens mij een echte ‘poepdokter’-maar-dan-anders geworden. Ik heb het

allemaal gezien: (karmijn-) rode poep, bruine (chow) poep, witte (semisynthetisch dieet) poep, droge

poep, natte (PEG) poep, rattenpoep, muizenpoep, mij niks te gek. (Misschien is dit wel de reden dat

ons huis vol Rituals spullen staat?) Laten we het netjes zeggen vanaf nu: geen poep, maar feces. Wie wil

er in mijn Fecesbook? (Concilium Hilaricum Pediatricum 2011.) Darmpassagetijden meten door vijf uur

voor muizenkooien langs te lopen en te noteren wanneer ze eindelijk eens rode feces produceren na

toediening van een rode vloeistofbolus in de maag. En goed opletten, want voor je het weet eten ze

het direct bij het naar buiten komen op, en dan heb jij ‘je moment’ gemist. Met ratten is het ook leuk,

dan mag je tot een uur of drie ‘s nachts door en hoop je dat ze tegen die tijd allemaal aan de beurt

geweest zijn. Gelukkig had ik Sonja, die na het stappen langs kwam om samen chippies te eten en

op de laptop ‘uitzending gemist’ te kijken. Leg maar eens aan je ouders uit: Pap, mam, ik doe nu écht

wetenschappelijk onderzoek! Feces verzamelen, sorteren, wegen, crushen (Doe de poo-jito, met dank

aan Maxi.) en opwerken. Bloed, zweet en tranen, voor het verzamelen van bloed, gal, feces en meer. De

meesten van ons weten het wel: onderzoek doen gaat met pieken en dalen. De dalen meestal vooral

in de eerste helft van het traject, de pieken meestal en gelukkig vooral in de tweede helft (Dankje Karin

L., je had gelijk!). Gelukkig doe je het nooit alleen en staan er veel mensen klaar om je te helpen. Samen

is het zoveel leuker! Ik wil dan ook iedereen ontzettend bedanken die mij op de een of andere manier

bijgestaan heeft in dit traject.

Geachte Prof. dr. H.J. Verkade, beste Henkjan, ik ben heel dankbaar dat ik in jouw groep mocht beginnen

met mijn afstudeeronderzoek voor Biologie, naar leverziekte bij Cystic Fibrosis. Ik had het ontzettend naar

mijn zin en was dan ook vereerd dat je mij halverwege dit project vroeg om ook mijn promotietraject

hier te doen. Jouw scherpe blik en kritische vragen doen mensen soms enigszins sidderen, maar ik weet

zeker dat je mensen gewoon probeert te stimuleren om het beste uit zichzelf te halen. Door alle drukte

(volgens onze e-mail correspondentie slaap je hooguit tussen 01.00 en 06.00 uur) was er soms weinig

tijd voor overleg, maar jouw one-liners (‘Don’t make the reader think’, ‘less is more’, ‘meaner and leaner’

etc.) waren altijd raak en ik zal ze nooit vergeten. De manier waarop jij dingen uitlegt (met prachtige

tekeningen en verhalen over winkelwagentjes in de rij bij de supermarkt) werkt zeer verhelderend en

motiverend. Ik hoop dat onze samenwerking hier niet eindigt.

Geachte Prof. dr. E.H.H.M. Rings, beste Edmond, Henkjan en jij vullen elkaar zo mooi aan. Jij en jouw

zware, kalme stem brengen altijd rust. Je was niet alleen geïnteresseerd in het onderzoek, maar

ook in de persoon erachter en je vroeg altijd even hoe het was en hoe het ervoor stond met mijn

toekomstplannen. Bedankt voor je luisterend oor, je adviezen en al je hulp. Ik hoop dat we elkaar snel

weer zien in het UMCG.

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128

Geachte Prof. dr. A.K. Groen, beste Bert, bedankt voor de tijd die je in mijn project hebt willen steken en

je aanwezigheid en steun bij de TIFN-meetings. Bedankt dat ik zomaar bij je kon binnenvallen met een

‘acute’ vraag. Cholesterol-onderzoek is leuk, maar soms ook zo frustrerend! Ik hoop dat ‘ons model’ (met

dank aan velen die mij voor gingen, zoals Jelske van der Veen) de komende tijd verder uitgewerkt kan

worden en we nog veel meer informatie uit onze experimenten (o.a. hoofdstuk 4 en 5 van dit boekje)

kunnen halen. Bedankt ook voor je persoonlijke interesse toen plotseling niets meer was zoals het moest

zijn.

Graag wil ik de leden van de leescommissie, bestaande uit Prof. dr. Oude Elferink, Prof. dr. Faber en Prof.

dr. Smit, heel hartelijk bedanken voor het beoordelen (en het goedkeuren!) van mijn proefschrift.

Ook wil ik de leden van ‘mijn’ TIFN project bedanken, met name Jan Dekker, mijn projectleider (Bedankt

voor het meedenken en je vele bezoekjes aan het Verre Groningen.) en Roelof van der Meer (Als niemand

een vraag stelt, doe jij het. En anders ook. Heel motiverend!).

Lieve Els, mijn Groningse mama. Bedankt voor je steun en de goede gesprekken, ik hoop dat je in de

buurt blijft. Astrid, bedankt voor je hulp, je hebt de touwtjes strak in handen! Hilde R. en Gea, bedankt

voor de hulp bij het maken van (bijna onmogelijke) afspraken met alle Profs. tegelijk en bij het faxen naar

het buitenland. ;)

Ik wil graag al mijn collega’s van het lab Kindergeneeskunde bedanken voor de goede samenwerking en

de gezellige buitenschoolse activiteiten. Mijn roomies in de twee kamers naast elkaar op de 3e: Golnar,

Sabina, Annelies, Maxi, Jan Freark (Nu ik ga, moet jij ook! Dat pak stond je goed, dus zet ‘em op. Maar

wel even een van je paranimfen vragen op je fiets en jas te passen ;) ), Krystof, Nienke, Matthijs, Wei-lin,

Marjolein en Shiva. Tim, Agnes, Jolita en Elodi. Margot, Jaana, Wytske, Maurien, Maaike, Jelske, Arne, Niels,

Gijs en Karen. Jurre, Hilde H., Anke (Respect, jij bent zo sterk!), Marijke, Brenda, Carolien, Marije, Jelena

(Jammer dat je niet ’mijn’ co-assistent kon worden, veel succes in Leiden!), Marleen & Gemma (Please

find the key to TICE!) en Harmen (In herinnering). Frans C., Karin G., Marjan, Willemien en Andrea. Janine,

Torsten, Frans S. (’Ik kom even een attachment brengen.’), Mark D., Jaap en Anniek. Dolf, Klary, Barbara,

Rebecca (Zie je in Meerstad?), Hans (Ik zie graag binnenkort je trouwfoto’s! Kunnen we meteen even

bijpraten over het CF onderzoek?), Dirk-Jan, Uwe (Thanks for being my favourite blood-draw-patient

and funniest colleague).

Alle analisten van ons lab wil ik bedanken voor hun inzet en bereidheid tot meedenken: Renze (voor de

hoge tonen) en Henk (voor de lage tonen. Never a dull moment als je tussen jullie in zit op het lab.), Aycha

(Lief!), Juul (Je was overgelopen naar het CDP, maar toch hielp je mij met mijn laatste experiment, dank!),

Angelika (Regel jij nu de Ladies Nights?), Niels, Danny, Nicolette, Trijnie en Wytse. Vincent: dankjewel voor

al je spontane en gevraagde hulp. Jij hebt mijn statistiek significant verbeterd! Dr. Theo, bedankt voor al

je (reken-) hulp, zelfs als je geen tijd had.

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Als we dat TICE model nou maar gewoon uit ons hoofd gezet hadden, was hoofdstuk 4 véél eerder af

geweest en hadden we samen een halfjaar op wereldreis gekund. ;) (Ach, ik zal niet klagen. Ondanks

mijn omzwervingen door de enterohepatische kringloop, heb ik El Fin del Mundo (Argentina!) toch

maar mooi gevonden.) Ik hoop toch dat er nog meer mooie dingen uit ons model voort zullen komen.

P.S.: Nummer 25 nog niet verplaatsen naar de vergane-glorie-map hoor! V. en T.: bedankt voor jullie

heerlijke nuchterheid en flauwe grappen! En snoepjes.

Gerard (Geen Rhesus-aap, maar een Feces-aap!), bedankt voor je hulp, foto’s en humor! Ook dank aan

de collega’s van het metabole lab en LHVG, in het bijzonder Theo B., Ingrid en Klaas. Jullie stonden altijd

open voor vragen en overleg, ik heb dat ontzettend gewaardeerd!

Daarnaast dank aan alle collega’s van de Maag-, Darm en Leverziekten op ons shared lab: Anouk, Golnar,

Mark, Manon, Tjasso, Haukeline, Bojana, Elise, Janette, Martijn, Mariska, Sandra, Floris, Atta, Han en Klaas-

Nico.

Dank aan alle mensen op het dierenlab (CDP) die mijn proefdieren goed verzorgd hebben en mij

geholpen hebben rondom de planning van mijn experimenten, in het bijzonder: Flip, Alex, Hester

(Zonnetje van het CDP), Sylvia, Diana, Ar, Harm, Maurice, Andrea, Angela, Ralph, Ramon, Marcia, Annet,

Arie, Wiebe, Miriam, en Lucas (In herinnering). Rick Havinga (‘Een beetje van ons en een beetje van het

CDP’), bedankt voor alle galcannulaties, zonder gal kom je nergens!

Wim Avis: bij ons kwam de kinderarts gewoon aan huis (en wij bij hem), hoe bijzonder! En hij kwam niet

alleen als dokter, maar ook voor de gezelligheid en voor het klussen met pa. Een posterprijs winnen op

het NVK congres was leuk, maar na al die jaren jou vinden, mijn naambordje omhoog houden en de

blik in je ogen te zien, dat was onbetaalbaar! Bedankt voor de goede zorg voor mijn zus. Je bent een

voorbeeld voor mij. Frank Bodewes, bedankt voor de samenwerking, hopelijk komt er nog een mooi stuk

uit voort en wie weet kunnen we nog eens samenwerken. Gieneke Gonera, Marlon Wilsterman en Linda

Leer: bedankt voor jullie hulp bij het verzamelen van kinder-feces, ik hoop dat we er nog iets moois uit

kunnen halen. Ik wil ook graag de kinderartsen en arts-assistenten van de afdeling Kindergeneeskunde

in het Martini Ziekenhuis bedanken voor alle steun die ik heb ontvangen, ook al voordat ik ‘echt’ begon.

De afgelopen jaren mocht ik deel uitmaken van ‘de Meisjes van Verkade’, a.k.a. Mjan, Mien, Mgot, Mjet

en An. Wat hebben wij genoten van de voorbereiding op het optreden van de ‘Spijsgirls’ (uw zang-

en dansgroep) op het oratie-feest van ‘Dear Prof. Rings’ (You are very smart!). Samen oefenen in het

appartement dat Margot en ik toen deelden. Samen make-uppen voor de start. En toen moesten we

ook nog daadwerkelijk de bühne op...oeps, waar waren we aan begonnen?! Of heb ik juist daardoor een

baan in het Martini Ziekenhuis gekregen? Meiden, bedankt voor alle hysterische lol!

Lieve Karin en Pieter, nu beiden ‘van der Wulp’! Eigenlijk zijn we écht familie. ;)

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Bedankt voor de gezelligheid en goede diners. Lieve Mjannie en Wimpie, bedankt voor jullie steun, jullie

zijn zo lief voor mij geweest.

Willem, dankjewel voor je Meesterlijke Maaltijden, waarvan ik zoveel eet dat ik daarna bijna niet meer

met jullie kan praten. Ik heb zin in een reis met z’n vieren! Lieve Marije (‘Ik ben blij dat jij ook van hard

lachen houdt!’) en Anouk (Almeloooo, brooood), bedankt voor alle gesprekken, etentjes en filmavondjes.

Marije, Anouk en Aycha, bedankt voor jullie steun toen het mis ging met mijn zus, maar ook daarna.

Lieve Anouk en Niek, bedankt voor jullie warmte en gezelligheid, ik wens jullie heel veel geluk straks

met z’n drietjes! Lieve Lisette, ik hoop dat de lijn blijft stijgen, je verdient het! Lieve Margot, vanaf dag

1 van ons promotietraject in hetzelfde schuitje: jonge dokters, samenwonen op afstand, studiootje in

Groningen, vrijdags haasten naar de andere kant van het land, maandagmorgen om 05.00 uur op en

weer terug. (Even kort samengevat.) Alle pieken, dalen, twijfels en successen hebben we gedeeld. Wij

hebben zoveel ‘Weet je nog, die keer...’ momenten. Ik ben heel blij dat ik je heb leren kennen, je bent een

grote steun voor mij geweest de afgelopen jaren, zowel op het werk als privé. Bedankt voor de mooie

tijd in New York (met onze mannen) en veel succes daar de komende tijd, topper! Ondanks de afstand

weten wij elkaar wel te vinden.

Mijn lieve paranimfen, ik ben jullie veel dank verschuldigd. Bedankt dat jullie ook nu voor mij klaarstaan.

Marjan, je bent een schat. Ik begrijp niet hoe jij altijd (ogenschijnlijk?) zo rustig kan blijven. Die rust breng

je over op anderen, een gave. Ik weet zeker dat jou een mooie toekomst als internist-infectioloog te

wachten staat. Lieve Sonja (Sonnie), waar moet ik beginnen? Mijn leven in Groningen zou niet hetzelfde

geweest zijn zonder jou. Samen biologie studeren, allebei geneeskunde studeren, samen eten, sporten

(We DID it! 4 Mijl Groningen 2012), stappen (Antwerpen is niet veilig!), shoppen, vrouwenseries kijken

etc. etc. For better & for worse. Ons leven staat dit jaar op zijn kop. Hopelijk kan ik net zo goed klaar staan

voor jou als jij voor mij. Je hebt een groot hart! Ik hoop dat ik voor altijd jouw vriendinnetje mag zijn.

Home is wherever I’m with you...(Edward Sharpe & The Magnetic Zeros, Home)

Lieve Ilona, Leo en Rico, lieve Remco en papa en mama. Wie zou ik geworden zijn zonder jullie? Ik ben

trots dat ik uit zo’n liefdevol gezin kom. Rico, je gaat al bijna naar school! Tante Mariëtte is heel trots op

je! De wereld ligt aan je voeten.

Papa en mama, mijn helden. Jullie leven heeft altijd in het teken van ons gestaan. Ons verlies, zo kort

geleden, kon niet groter zijn. Ik zal altijd voor jullie klaarstaan, vergeet dat nooit. Als er iets is wat ik van

jullie geleerd heb, is het dat ik ervoor moet gaan als ik iets écht graag wil. Gewoon, op eigen kracht. Ik

hoop dat ik jullie trots maak. Jullie zijn goud.

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Lieve Nathanja, lief zusje. Mijn voorbeeld, mijn geweten, mijn soulmate. Ik heb zo vaak nagedacht over

het moment dat we je zouden verliezen, maar dit scenario had ik nooit kunnen bedenken. Ik kan me

niet herinneren dat ik je 1 keer in mijn leven heb horen klagen. Nooit zei je dat je er genoeg van had,

dat het niet eerlijk was. Waarom jij? Waarom deze rotziekte? Nooit. Je vocht met alles wat je had om de

betere periodes zo goed mogelijk te benutten. Altijd was er jouw lieve, mooie lach. Alle kansen die je

kreeg, greep je met beide handen aan. We hebben gewandeld in Zuid-Europa, gefietst in NL, geskied

in Oostenrijk en gesnorkeld in de Rode Zee. Je vroeg maar 1 ding en dat was een kans op een tweede

kans, met nieuwe longen. Als iemand het verdiende, was jij het wel. Helaas mocht het niet zo zijn. Jou

verliezen was het ergste wat mij kon gebeuren. Ik weet niet hoe ik de rest van mijn leven zonder jou

moet. Ik zou je zo graag zien als mijn paranimf, mijn bruidsmeisje, als tante van mijn kinderen...

Toch weet ik dat het zal lukken. Jouw kracht is mijn kracht en niets komt daar tussen. Ik hou me vast aan

de gedachte dat ik je ooit, ergens, weer zal vinden. Is dat naïef? Laat mij dan maar naïef zijn. Ik hou van

je zusje. Jij was de mooiste.

“Did you ever know that you’re my hero?

You’re everything I wish I could be.

I could fly higher than an eagle,

‘cause you are the wind beneath my wings.”

(Bette Midler, Wind beneath my wings)

Lieve Stef, ondanks alles wat we hebben meegemaakt in slechts een paar jaar tijd, kunnen we nog

steeds samen lachen. Daar gaat het om, toch?

Je weet hoe gek ik op Ilse ben, deze is voor jou:

“You will always be my beautiful distraction

One part sweetness two parts passion

You asked for my heart not perfection

The first one to lead me in the right direction

This crazy world won’t change the same old me

Who loves the same old you”

(Ilse de Lange, Beautiful distraction)

Lieve mensen, vergeet nooit te genieten.

Later is nu!!!

Mariëtte

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Biography

Mariëtte van der Wulp was born on March 14th 1981 in Deventer. In 1999 she graduated from high school

(Alexander Hegius Lyceum) in Deventer. After obtaining her diploma, she continued her education at the

University of Groningen where she studied Biology (Master’s degree Medical Biology received in 2008)

and Medicine (MD degree received in 2007). Research projects performed during her studies involved

Pediatric Oncology and Cystic Fibrosis related liver disease. In 2008, Mariëtte started her PhD project

at the Department of Pediatric Gastroenterology and Hepatology at the University Medical Center

Groningen, under supervision of Prof. dr. H.J. Verkade, Prof. dr. A.K. Groen and Prof. E.H.H.M. Rings. Results

obtained from her PhD-studies are described in this dissertation and were part of the Top Institute Food

and Nutrition project ‘Nutrition and Health’. Since August 2012, Mariëtte is employed by the Martini

Hospital in Groningen, where se works as a physician at the Department of Pediatrics.

Biografie

Mariëtte van der Wulp werd op 14 maart 1981 geboren te Deventer. In 1999 behaalde zij haar

Atheneumdiploma aan het Alexander Hegius Lyceum te Deventer. In datzelfde jaar begon zij aan

haar studie Biologie aan de Rijksuniversiteit Groningen en in 2001 startte zij daar tevens haar studie

Geneeskunde. Zij behaalde haar artsdiploma in 2007 en haar doctorandus titel in de Medische Biologie

in 2008. De wetenschappelijke onderzoeken tijdens haar studies richtten zich op kinderoncologie en

leverziekte bij Cystic Fibrosis. In 2008 startte Mariëtte met haar promotieonderzoek bij de afdeling

Kinder-maag-darm-leverziekten in het Universitair Medisch Centrum Groningen onder begeleiding

van Prof. dr. H.J. Verkade, Prof. dr. A.K. Groen en Prof. E.H.H.M. Rings. De resultaten van haar onderzoek,

gefinancieerd door Top Institute Food and Nutrition, project ‘Nutrition and Health’, worden in dit

proefschrift beschreven. Sinds augustus 2012 is Mariëtte werkzaam als arts-assistent Kindergeneeskunde

in het Martini Ziekenhuis Groningen.

Appendices

133

List of publications

van der Wulp MY, Cuperus FJ, Stellaard F, van Dijk TH, Dekker J, Rings EH, Groen AK, Verkade HJ. Laxative

treatment with polyethylene glycol does not affect lipid absorption in rats. J Pediatr Gastroenterol Nutr.

2012 Oct;55(4):457-62.

van der Wulp MY, Verkade HJ, Groen AK. Regulation of cholesterol homeostasis. Mol Cell Endocrinol.

2012 Jun.

Cuperus FJ, Iemhoff AA, van der Wulp M, Havinga R, Verkade HJ. Acceleration of the gastrointestinal

transit by polyethylene glycol effectively treats unconjugated hyperbilirubinaemia in Gunn rats. Gut.

2010 Mar;59(3):373-80.

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