Karolinska institutet / Stockholm University
Department of medical nutrition
Nutritionslinjen, åk 3
Biochemical and toxicological nutrition, 10 credits
Vt-05
Name: Ylva Bonde
Date: 050525
Effects of a high carbohydrate diet
on bile acid metabolism – a study in mice
Supervisors:
Mats Rudling
Center for Metabolism &
Endocrinology (CME)
Department of Medicine,
Karolinska University Hospital,
141 86 Huddinge
Tel: 08-585 69 55
E-post: [email protected]
Thomas Lundåsen
Center for nutrition and toxicology
(CNT)
Karolinska Institutet, Novum
141 57 Huddinge
Telefon: 08-585 95 25
E-post: [email protected]
Contents
ABSTRACT
3
INTRODUCTION
3
Cholesterol
3
Lipoproteins
3
Hepatic cholesterol metabolism
3
Bile acid synthesis
4
The structure and function of bile acids
4
The enterohepatic circulation
4
PROJECT BACKGROUND
5
MATERIALS AND METHODS
5
RESULTS
8
DISCUSSION
10
REFERENCES
11
2
Effects of a high carbohydrate diet on bile acid metabolism
– a study in mice
Abstract
Increased levels of plasma cholesterol, especially within low density lipoproteins, is a risk
factor for atherosclerosis [1]. Bile acid synthesis provides a metabolic pathway through which
excessive cholesterol can be removed from the body by the conversion of cholesterol into bile
acids [2]. The aim of this study was to find out if a high carbohydrate diet could decrease bile
acid synthesis in regular C57BL mice when bile acid synthesis is stimulated by a classic resin
drug. Bile acid synthesis was measured by analysing the bile acid intermediate 7α-hydroxy-4cholesten-3-one C4 levels in pooled plasma samples, and by analysing mRNA levels of
cholesterol 7α-hydroxylase (cyp7a1), the rate-limiting enzyme of the neutral pathway. The
expected increase in bile acid synthesis failed to appear. Consequently, the results are very
difficult to interpret.
Introduction
Cholesterol
The major part of the body´s cholesterol pool is of endogenous origin. All cells can synthesize
cholesterol but the liver is responsible for most of the cholesterol synthesis. Cholesterol is
synthesized from acetate [3]. It is an energy demanding process, catalyzed by the rate-limiting
enzyme hydroxymethyl-glutaryl coenzyme A (HMG-CoA) reductase.
Cholesterol is the precursor of steroids such as bile acids, corticosteroids, sex hormones, and
vitamin D. It is also an important component of all membranes and lipoproteins [4].
Lipoproteins
To be transported in plasma, lipids such as cholesterol and triglycerides, must be packed into
water-soluble particles called lipoproteins [1]. Lipoproteins are divided into four major
classes depending on their size and density: chylomicrons, very low density lipoproteins
(VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL).
Chylomicrons are formed in the intestine and the chylomicron triglycerides are hydrolyzed
in peripheral tissues by the enzyme lipoprotein lipase (LPL), thereby providing muscle and
adipose tissue with free fatty acids. The remaining chylomicron is rapidly removed from the
circulation by the liver. VLDL particles are formed by the liver and secreted into the
circulation. VLDL particles are also hydrolyzed by lipoprotein lipase in peripheral tissues,
and the resulting remnants can be converted into LDL particles. In humans, most of the
cholesterol in the circulation is within LDL particles. Most LDL particles are taken up by the
liver [3]. High levels of LDL cholesterol in the blood are detrimental as they may lead to
excessive accumulation of cholesterol in vessel walls, a condition predisposing to the
development of atherosclerotic disease [5,6].
HDL particles are mainly formed in the blood from the remnants of hydrolyzed triglyceriderich lipoproteins. HDL transfers cholesterol from vessels and peripheral tissues back to the
liver. This process is called reverse cholesterol transport and is very important for
maintenance of cholesterol homeostasis [6].
3
Hepatic cholesterol metabolism
The central organ for cholesterol homeostasis is the liver [3]. In the liver, levels of free
cholesterol are determined by: i) the regulation of the de novo synthesis of cholesterol, ii) the
uptake of low density lipoprotein (LDL) cholesterol, iii) the secretion of very low density
lipoprotein (VLDL) cholesterol, iv) the formation of cholesteryl esters, and v) the secretion of
cholesterol into bile either, as free cholesterol or after conversion into bile acids [4]. This
conversion of cholesterol into bile acids can be considered to be the last step in the reverse
cholesterol transport and it provides a metabolic pathway through which excessive cholesterol
can be removed from the body [6].
Bile acid synthesis
There are two distinct bile acid synthetic pathways. One is called the neutral (or classic)
pathway [2]. Metabolites of this pathway go through the first part mainly as neutral sterols,
hence the name “neutral pathway” [4]. It starts with the rate-limiting enzyme cholesterol 7αhydroxylase (cyp7a1) placing a hydroxyl group onto the 7α position of the cholesterol [2].
Cholesterol 7α-hydroxylase is a liver-specific member of the cytochrome P450 superfamily
[2,6], localized in the smooth endoplasmatic reticulum. This enzyme is regulated mainly at
the transcriptional level by bile acids returning to the liver via the enterohepatic circulation,
thus exerting a classical end-product feedback regulation [5].
In the second step, 7α-hydroxylcholesterol is converted into 7α-hydroxy-4-cholesten-3-one
[3]. Both 7α-hydroxycholesterol and 7α-hydroxy-4-cholesten-3-one can be measured in serum
and are used as markers of bile acid synthesis [4].
The other bile acid synthetic pathway is called the acidic (or alternative) pathway. The early
metabolites of this pathway are mainly acidic sterols, hence the name. In mice, the acidic
pathway may contribute by as much as 45% of the total bile acid production, while in humans
this pathway only contributes by approximately 10% of total bile acid production [2,3].
The bile acids synthesized in the liver are called primary bile acids. The human liver
synthesizes two primary bile acids: cholic acid and chenodeoxycholic acid. Cholic acid is the
main product of the neutral pathway and chenodeoxycholic acid is the main product of the
acidic pathway [4]. Mice also produce significant quantities of β-muricholic acid and
murideoxicholic acid [2]. The final step in bile acids synthesis is conjugation with either
glycine or taurine [2,3]. This converts the bile acid into a highly polar compound, which is
essential for solubility in the intestinal lumen [2,4].
The structure and function of bile acids
The most common bile acids are composed of 24 carbon atoms, two or three hydroxyl groups
and a side chain that ends in a carboxyl group that is ionised at pH 7.0 (hence the name bile
salt). The steroid skeleton is hydrophobic, while the carboxylate ion and the hydroxyl groups
are hydrophilic [4]. Bile acids are the main constituents of bile. Bile is concentrated and
stored in the gallbladder. During a meal, the gallbladder contracts and bile is released into the
proximal intestinal lumen [7]. Because of their amphipahtic structure, bile acids act as
detergents [4]. By emulsifying dietary lipids and forming micelles, bile acids facilitate the
absorption of dietary lipids (e.g. cholesterol and triglycerides) and fat-soluble vitamins [7].
The enterohepatic circulation
Of the bile acids excreted into the intestine, 95-99% are reabsorbed and returned to the liver
via the portal vein [4]. The majority of the reabsorption occurs in the distal part of ileum via
an active transporter called the intestinal bile acid transporter (ASBT or IBAT) [4] located at
the enterocyte [3]. The flow of bile acids between the liver and the intestine is called the
4
enterohepatic circulation [8]. The human bile salt pool (3-4g) circulates 6 to 10 times per 24
hours, leading to a loss of 0.5g per day through fecal excretion. This loss is continuously
compensated for by bile acid synthesis from cholesterol in the liver [3]. Lestid® is a resin
drug containing the active component kolestipol. Resins are serumlipid-lowering drugs that
binds bile acids and exert a local inhibitory effect on their reabsorption, leading to an
increased loss of bile acids [9], and consequently an increased bile acid synthesis.
Bile acids that are not reabsorbed enters the large intestine where they are deconjugated and
metabolised by bacterial enzymes [4]. The bacterial enzyme 7α-dehyroxylase converts cholic
acid and chenodeoxycholic acid to the more hydrophobic forms deoxycholic acid and
lithocholic acid, respectively. These are called secondary bile acids [3,7], and because of their
increased hydrophobicity they are more readily excreted through feces [4].
Project background
Deletion of the ASBT gene in mice result in increased fecal bile acid excretion. To
compensate for the loss, bile acid synthesis increases [3]. In a recent study, it was found that a
high carbohydrate diet administered to ASBT knockout and wild type mice, reduces bile acid
synthesis. For example the mRNA levels of cyp7a1 was significantly decreased in the ASBT
k/o, and also in wild type animals, although the decrease was more pronounced in ASBT k/o
mice. There was no difference in C4 levels in plasma between the wt mice fed ordinary chow
and wt mice fed the high carbohydrate diet. However, in the group of ASBT k/o mice fed the
high carbohydrate diet the C4 level in plasma had decreased compared to ASBT k/o mice fed
the ordinary mouse chow. The question was now if a high carbohydrate diet could decrease
bile acid synthesis in regular C57BL mice when bile acid synthesis is stimulated by a classic
resin drug. The methods used are described below.
Materials and methods
Experimental setup
A total of 24 male C57BL wt mice (Bommice, Denmark), 7-8 weeks old were used. All
animals had free access to water and chow. Light-cycle hours were from 6:00 AM to 6:00
PM. The mice were divided into four separate groups (six/group) and were fed different diets
for ten days. Group 1 received standard mice chow and water. Group 2 received a high
carbohydrate diet (60% sucrose in chow and 10% fructose in water). Group 3 received chow
with 0,8% W/W of a common resin drug (Lestid®, Pfizer, Stockholm, Sweden). Group 4
received chow containing 0,8% W/W of Lestid® and the high carbohydrate diet.
The animals were fasted 3-4 hours before sacrifice to prevent emptying of gallbladders.
Animals were anesthetized by inhalation of isoflurane, bled by cardiac puncture, and killed by
cervical dislocation. Blood plasma was separated by centrifugation and stored at 4˚C. Livers
were frozen in liquid nitrogen and stored at -80˚C.
Total plasma cholesterol, triglyceride and glucose
Total plasma cholesterol, triglyceride and glucose were determined by using a Monarch
Automated Analyser with commercially available kits.
Lipid assay
To quantitate the lipoproteins, 25 µl of plasma from each mouse was transferred to vials with
25 µl of FPLC buffert. Lipoproteins were separated by fast performance liquid
chromatography (FPLC) and assayed for cholesterol and triglycerides on-line. Qantitation of
5
lipoproteins was calculated from the area percentage of the respective lipoprotein class and
the total lipid level of the sample.
Assay of 7α-hydroxy-4-cholestene-3-one (C4)
By taking 50 µl of plasma from each mouse in the respective group, four pools were made.
Samples were diluted with 1 ml of isotonic natrium chloride solution and 10 µl internal
standard 7β-hydroxy-4-cholestene-3-one was added. C8 Isolute® SPE columns (500 mg, 3
ml, International Sorbent Technology LTD) were prewashed with 2x2 ml 99% methanol and
with 2x2 ml of deionised water. The columns were then put in the Varian CEREX® SPE
processor equipped with a block heater (64.0˚C).
Samples were sonicated in water for 15 min and incubated at 37˚C for 15 min before being
loaded onto the preheated columns. After 5 min the column stoppers were removed and
samples were passed through the columns. Sample tubes were washed with 2 ml of 64˚C
deionised water, which was also loaded onto the columns. When all liquid had passed through
the columns, the heating block was removed and the columns cooled to room temperature.
Columns were then washed with 2x2 ml of 65 % methanol and dried by a stream of nitrogen
applied for 30 sec. To elute product, 2x2 ml of hexan:chloroform (95:5) was loaded onto the
columns. The eluted product was dried under a stream of nitrogen and dissolved in 100 µl
acetonitrile. The samples were sonicated for 3 min, vortexed, and transferred to HPLC vials
100 µl.
At last, 50 µl was separated by HPLC (HP 1100 series, Hewlett Packard GmbH) at 20˚C
(mobile phase: acetonitril:water 95:5, 1 ml/min) using a Nova-Pak C18 steel column 300x3,9
mm I.D., 4 µm particle size (Waters Corporation). The wavelength was 241 nm. C4 was
quantified from the areas of the respective peaks using the internal standard.
Assay of hepatic lipids
Frozen liver samples (~50 mg) were homogenized for 5 min in 4 ml Folch (CHCl3:MeOH
2:1) using an Kinematica Polytron (Kriens). Samples were spun down at 3500 rpm for 10 min
and 3 ml supernate was transferred to new tubes. After adding 0.8 ml 50 mM NaCl, samples
were mixed by a vortex and spun down at 3500 rpm for 10 min. From the lower phase, 2x100
µl was transferred to new tubes and a 100 µl of 75 mg/ml TritonX-100 (375 mg TritonX100:5ml Folch) was added to samples prior to evaporation. TG-reagent was added to tubes
which were left at room temperature for 20 min before mixing by vortex. The amount of
triglycerides was determined by measuring the absorbance at 500 nm using a DU® 640
Specctrophotometer (Beckman Coulter™) and comparing the data to an external standard.
Quantitation of mRNA
Total RNA extraction
Frozen liver samples (~50 mg) were homogenized in 1 ml of TRIZOL® reagent (Life
Technologies) using a Polytron. The samples were incubated for 5 minutes at room
temperature, adding 0,2 ml of chloroform and shaking the tubes vigorously for 15 seconds
before incubating them again for 3 minutes. Following centrifugation (8000 or 14 000 rpm,
4˚C, 20 min), phase separation was achieved with the RNA in the upper, aqueous phase. The
aqueous phase was transferred to fresh tubes and total RNA precipitated by adding 0,5 ml of
isopropyl alcohol. Samples were incubated at room temperature for 10 minutes and
centrifuged (14 000 rpm, 4˚C, 20 min). The supernate was removed and the RNA pellet
washed with 0,75 ml 75% ethanol and centrifuged (14 000 rpm, 4˚C, 10 min), the ethanol was
removed and the pellet air-dried for 10-15 min. The pellet was redissolved in 150 µl
deionised water. The samples were stored at -80˚C.
6
Determination of total RNA concentration
Samples were diluted 50 times with deionised water before measuring the concentration using
a spectrophotometer (Ultraspec 3000) and assuming that 1 A260 unit = 40 µg of RNA/ml.
DNase treatment of RNA
From each mouse, 30 µg of total RNA was subjected to 10 µl of RQ1 DNase (Promega), 5 µl
RQ1 buffer (Promega) and deionised water in a total volume of 50 µl at 37˚C for 20 minutes
and 20˚C for 15 min. At this temperature 1,2 µl of DNase Stop Solution (Pomega) was added
before the temperature finally reached 70˚C for 10 min. Samples were stored at -80˚C.
cDNA synthesis
From each mouse, 6 µl of DNase-treated RNA was taken and used for cDNA synthesis
together with 2 µl random hexamer primers (Promega), 1 µl 10 mM dNTP (Promega) and
deionised water in a total volume of 12 µl. Samples were incubated at 65˚C for 5 min, 25˚C
for 10 min and 42˚C for 2 min. After that, 2 µl of 0,10 M DTT (Invitrogen),1 µl of RNasin
(Promega), 4 µl of 5x first strand buffer (Invitrogen) and 1 µl of Superscript II were added.
Samples were then incubatet at 42˚C for 50 min and 70˚C for 10 min, and stored at -80˚C.
Real time PCR – cyp7a1
From each mouse, 3 µl of 20x diluted cDNA was used for amplification by real time PCR
together with 12,5 µl 2x Cybergreen Master Mix, 1 µl 5 µM reversed primer (5´GTCCGGATATTCAAGGATGCA-3´),
1
µl
5
µM
forward
primer
(5´AGCAACTAAACAACCTGCCAGTACTA-3´), and deionised water in a total volume of 25
µl. The assay was performed by an ABI Prism® 7700 Sequence detector. The samples were
qantitated by the delta Ct method, using GAPDH as an endogenous control.
Real time PCR – scd1
From each mouse, 3 µl of 20x diluted cDNA was used for amplification by real time PCR
together with 12,5 µl 2x PCR Master Mix, 0,5 µl 10 µM reversed primer (5´TAGCCTGTAAAAGATTTCTGCAAACC-3´), 0,5 µl 10 µM forward primer (5´CCGGAGACCCCTTAGATCGA-3´), and 0,5 µl 20 µM scd1 probe and deionised water in a
total volume of 25 µl. The TaqMan probe was labelled with FAM and Tamra. For
endogenous control we used VIC labelled GAPDH TaqMan assay (Applied Biosystems). The
assay was performed and analysed using an ABI Prism® 7700 Sequence detector. The
samples were qantitated by the delta Ct method, using GAPDH as an internal standard.
Statistical analyses
Data are presented as means ± SEM.
7
Results
cyp7a1 mRNA level
Quantitative real time PCR analysis of cyp7a1 mRNA is shown in figure 1.
No clear increase in cyp7a1 mRNA was seen in the group fed lestid.
cyp7a1 enzyme activity
C4 levels in pooled plasma samples are shown in figure 2. There was no clear increase in C4
in the group fed lestid.
Cyp7a1mRNA
20
18
2,5
ng C4/ml plasma
16
2
1,5
1
14
12
10
8
6
4
0,5
2
0
0
ct rl
ct rl+HC
Lest id
ctrl
Lest id+HC
Fig 1. Levels of cyp7a1 mRNA in the different
groups measured by quantitative real time PCR. Ctrl
= control, HC = high carbohydrate diet
ctrl+HC
Lestid
Lestid+HC
Fig 2. Plasma C4 levels (ng/ml) after separation
of pooled plasma samples by HPLC. Ctrl =
control, HC = high carbohydrate diet
Total plasma cholesterol, triglyceride and glucose
Figures show the results from analysis of total plasma cholesterol, triglyceride and glucose.
There is no change in total plasma cholesterol, triglyceride or glucose between the different
groups.
Hepatic triglyceride content
The results from the analysis of hepatic triglyceride content are shown in figure 4. There is no
difference between the groups concerning the hepatic triglyceride content.
Cholesterol
Triglyceride
Glucose
14
30
µg triglyceride/mg liver tissue
16
12
mmol/L
10
8
6
4
2
25
20
15
10
5
0
ctrl
ctrl+HC
Lestid
0
Lestid+HC
Fig 3. Total plasma cholesterol, triglyceride and
glucose (mmol/L serum).
ct r l
ct rl + HC
Lest id
Lest id + HC
Fig 4. Triglyceride content in liver (µg/mg liver tissue)
8
Cholesterol content (arbitrary units)
Lipoprotein profiles
The results from the determination of cholesterol content in different lipoproteins are shown
in figure 5. The peak to the left represents the cholesterol content in very low density
lipoproteins (VLDLs). The peak in the middle represents the cholesterol content in low
density lipoproteins (LDLs) and the peak to the right represents the cholesterol content in high
density lipoproteins (HDLs). The cholesterol content of LDL is increased in the group fed
lestid and the high carbohydrate diet but decreased in the group fed lestid, compared to the
group fed ordinary chow. The cholesterol content of HDL is decreased in the group fed lestid
and the high carbohydrate diet but increased in the group fed lestid, compared to the group
fed ordinary chow. The cholesterol content of VLDL is decreased in the group fed lestid and
the group fed a high carbohydrate diet, compared to the group fed ordinary chow.
0,25
ctrl
ctrl+HC
Lestid
Lestid+HC
HDL
0,2
0,15
LDL
0,1
VLDL
0,05
0
0
19
37
56
75
-0,05
time (min)
Fig 5. Cholesterol profiles of lipoproteins after separation by FPLC. Control (mouse chow) (blue line),
ctrl + high carbohydrate diet (pink line), Lestid (yellow line), Lestid and high carbohydrate diet (light blue line).
The results from the determination of triglyceride content in different lipoproteins are shown
in figure 6. The peak to the left represents the triglyceride content in VLDL. The second peak
represents the triglyceride content in LDLs and the third peak represents the triglyceride
content in HDLs. The peak to the right represents free glycerol. There is a decrease in VLDL
triglyceride content in both groups fed the high carbohydrate diet, compared to those fed
lestid or ordinary chow.
0,25
Triglyceride content (arbitrary units)
free glycerol
0,2
ctrl
ctrl+HC
Lestid
Lestid+HC
0,15
VLDL
0,1
LDL
0,05
HDL
0
20
41
63
84
-0,05
time (min)
Fig 6. Triglyceride profiles of lipoproteins after separation by FPLC. Control (mouse chow) (blue line),
ctrl + high carbohydrate diet (pink line),. Lestid (yellow line), Lestid + high carbohydrate diet (light blue line).
9
scd1 mRNA level
The result from quantitative real time PCR analysis of scd1 mRNA can be seen in figure 7.
Stearoyl-CoA desaturase (scd) is the rate-limiting enzyme catalysing the biosynthesis of
monounsaturated fatty acids, mainly oleate (18:1) and palmitate (16:1). The monounsaturated
fatty acids synthesized by scd are used as major substrates for the synthesis of triglycerides
and cholesteryl esters [10].
Fig 7. Levels of scd1 mRNA in the different groups
measured by quantitative real time PCR.
scd1 mRNA
1,4
1,2
1
AU
0,8
0,6
0,4
0,2
0
ctrl
ctrl+HC
Lestid
Lestid+HC
Discussion
The aim of this study was to elucidate out if a high carbohydrate diet could decrease bile acid
synthesis in regular C57BL mice when bile acid synthesis is stimulated by a classic resin
drug. However, the results from the analyses of C4 and cyp7a1 mRNA showed no increased
bile acid synthesis in the group fed lestid. This indicates that the expected increase in bile acid
synthesis failed to appear. Consequently, the results are very difficult to interpret.
Resin treatment of rodents is a well established method to increase bile acid synthesis. Why
the increase of bile acid synthesis failed to appear in this case is a question difficult to answer.
One explanation could be that the mice did not eat the intended amount of resin. This could be
due to that lestid, like other resins, does not taste to good and therefore was rejected by the
mice. Also, it could be that the resin, due to its larger particle size, was not equally spread in
the chow or perhaps could redistribute in the grounded food with time . This thought can also
be applied when it comes to sugar. Looking back, we should have weighed the mice to see if
there were any differences in how much they had been eating. We did weigh their livers
which can be considered to be an indirect measure of body weight, and did not discover any
major differences between the groups.
Since the basic condition for being able to interpret the results from the analyses is not
fulfilled, it is not meaningful to do so.
An interesting thing to do in the future would be to perform this experiment again, this time
with an even greater effort given to the preparation of chow and in addition increase the resin
dose by 3-fold.
10
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