PHD Thesis LenaLeder PDF

Healthy dietary patterns, lipids and
inflammation in human
randomized controlled trials
Lena Leder
Dissertation for the degree of Philosophiae Doctor (Ph.D.)
Department of Nutrition
Institute of Basic Medical Sciences
University of Oslo
2016
© Lena Leder, 2017
Series of dissertations submitted to the

Faculty of Medicine, University of Oslo
ISBN 978-82-8377-000-1
All rights reserved. No part of this publication may be

reproduced or transmitted, in any form or by any means, without permission.
Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.
Acknowledgements
This work was carried out at the Department of Nutrition, Institute of Basic
Medical Sciences at the University of Oslo, Norway from 2012 until 2016. The
primary financial support was a four-year doctoral fellowship from the Institute
of Basic Medical Sciences, University of Oslo, Norway.
Prof. Dr. Kirsten Holven was my principal supervisor. Thank you for pro-
viding an incredible environment for conducting my PhD in your expert su-
pervision. The scientific guidance, critical questions, support, sharing extensive
knowledge and never-ending optimism were greatly appreciated. Your positive
and kind attitude was inspiring and always helped me to get back on track.
With the same gratitude I want to thank my co-supervisor Prof. Dr. Stine
Ulven for sharing your invaluable knowledge, your patience, and encourage-
ment means a lot to me. Thank you for taking this role and supporting me
wherever and whenever you could.
Thank you to the SYSDIET consortium for allowing access to the study
material and to all the people participating in the SYSDIET study. My special
gratitude I want to express to Marjukka Kolehmainen for her tremendous help
with the SYSDIET paper. Thank you for your inspiring suggestions, great ideas
and very fruit-full discussions.
Thank you to the NoMa study team at UiO, HiOA and Mills DA for mak-
ing this study possible. I express my gratitude to all people participating in the
NoMa study.
All my colleagues formed an extremely friendly work environment that al-
lowed to discuss ideas as well as critical issues. I am very grateful to Inger Ottes-
tad, Gyrd Omholt Gjevestad, Ingunn Narverud, Jacob Juel Christensen, Patrik
Hansson, Amanda Rundblad, Mari Myhrstad and Vibeke Telle-Hansen for the
i
good and productive times in seminars, in our “Kollokvie”, in the lunch breaks,
at conferences, at dinners, while running and cycling. A great thank you to
Kristin Eckardt, Christin Zwafink and Rikke Nørgaard. Our discussions about
lab methods, teaching and life in general were highly appreciated. Thank you
to all who proofread my thesis and gave invaluable comments.
Thank you to Marit Sandvik, Navida Akhter Sheikh and Ellen Raael for
their expertise and technical support in the lab.
Thank you to Anine Medin and Susanne Strohmaier for our master-mind
group. It was extremely inspiring, funny, crazy, critical and knowledgeable. I
hope we keep in touch and keep it running in one way or the other!
I want to express my gratitude to Carina Knudsen for her creative support
with the figures in my thesis and to Magne Thoresen for all the biostatistical
support during my PhD.
Deep gratitude goes to my dear family. My parents-in-law Beatrix and Rein-
hard Leder always made it possible to help out even though there are 1500km
between us. You are incredible. A huge thank you for in-depth revision of the
language in my thesis. My parents Beate and Oskar Lückel have always encour-
aged and believed in me whatever I have been up to; no matter if it was crazy,
well-considered or just for fun. Thank you for your unfailing love and your
never-ending support.
My deepest thank you I want to express to my beloved husband, Felix Leder,
and my children, Finus and Nuka. I am deeply thankful for your motivation
and inspiration, and that you always believed in me. Finus and Nuka are my
sunshines showing me what really matters in life. I would not have made it as
far without the three of you. You guys rock!
ii
Abstract
Healthy dietary patterns have been subject of considerable attention in recent

years. A healthy Nordic diet may improve cardiovascular risk factors and thereby
prevent cardiovascular diseases. In order to reduce plasma cholesterol and dis-
ease risk, one central aspect of the healthy Nordic diet is the combination of
reduced dietary intake of saturated fatty acids and increased dietary intake of
polyunsaturated fatty acids. However, although the effect of dietary fat quality
on plasma cholesterol concentration is well established, the effects and mech-
anisms of whole diets on plasma lipids and inflammation are less examined.
Therefore, we aimed to investigate the role of healthy dietary patterns on lipids
and inflammation with special focus on fat quality in populations with car-
diometabolic risk.
Two randomized controlled dietary intervention studies were included in
this thesis. In an eight-week double-blinded study, healthy adults aged 25-70
years with moderate hypercholesterolemia were assigned to an experimental
diet or a control diet. The experimental diet group received commercially avail-
able food items in which saturated fat was replaced by vegetable sunflower and
rapeseed oil. The control diet group received similar commercial food items
with a higher content of saturated fat and lower content of polyunsaturated fat.
In an 18-24 week, Nordic multi-center study, subjects between 30-65 years with
features of metabolic syndrome were assigned to follow a healthy Nordic diet
or an isocaloric control diet. An oral glucose tolerance test was performed at
baseline and at the end of the study.
Exchanging food products with improved fat quality reduced total- and LDL-
cholesterol by 9% and 11%, respectively, and increased the serum levels of
bile acid, but we did not detect an effect on circulating inflammatory markers.
iii
The cholesterol-lowering effect observed seemed to be induced by a change in
mRNA expression of the LDL receptor, potentially leading to increased choles-
terol in the cell, increasing mRNA expression of liver X receptor alpha (LXRA)
and LXRA target genes in peripheral blood mononuclear cells (PBMCs). The
increase in serum bile acid may reflect increased LXRA activity in liver, and
thus our data confirm that changes in gene expression in PBMCs reflect changes
in hepatic lipid metabolism, as has been shown by others. A long-term healthy
Nordic diet modified the expression of genes involved in inflammation and lipid
metabolism in PBMCs after a 2h oral glucose tolerance test (OGTT) in individ-
uals at risk of metabolic diseases.
In conclusion, a healthy Nordic diet and an improvement of the fat quality,
as part of a healthy dietary pattern, has positive effects on a variety of markers
of cardiovascular diseases and on the transcription of genes involved in lipid
metabolism and inflammation.
iv
List of papers
Paper I
Ulven SM, Leder L, Elind E, Ottestad I, Christensen JJ, Telle-Hansen VH,
Skjetne AJ, Raael E, Sheikh NA, Holck M, Torvik K, Lamglait A, Thyholt
K, Byfuglien MG, Granlund L, Andersen LF and Holven KB: Exchanging few
commercially regular-consumed food items with improved fat quality reduces total
and LDL cholesterol– a double-blind randomized controlled trial. British Journal
of Nutrition. In press.
Paper II
Leder L, Ulven SM,Ottestad I, Christensen JJ, Telle-Hansen VH, Granlund L,
Andersen LF and Holven KB: Replacement of SFAs with PUFAs increases the ex-
cretion of bile acids and up-regulates the mRNA expression level of the LDL receptor
and LXR alpha in peripheral blood mononuclear cells: a double-blind randomized
controlled trial. Submitted.
Paper III
Leder L, Kolehmainen M, Narverud I, Dahlman I, Myhrstad MCW, de Mello
VD, Paananen J, Carlberg C, Schwab U, Herzig K-H, Cloetens L, Ulmius Storm
M, Hukkanen J, Savolainen MJ, Rosqvist F, Hermansen K, Dragsted LO, Gun-
narsdottir I, Thorsdottir I, Risérus U, Åkesson B, Thoresen M, Arner P, Pouta-
nen KS, Uusitupa M, Holven KB and Ulven SM: Effects of a healthy Nordic diet
on gene expression changes in peripheral blood mononuclear cells in response to an
oral glucose tolerance test in subjects with metabolic syndrome: a SYSDIET sub-
study. Genes & Nutrition 2016 Mar 17;11:3.
v
Abbreviations
AA arachidonic acid cDNA complementary DNA

ABCA1 ATP binding cassette subfamily CE cholesteryl ester
A member 1
CHD coronary heart disease
ABCG1 ATP binding cassette subfamily
G member 1 ChREBP carbohydrate regulatory
element-binding protein
ACAT acyl CoA cholesterol
acyltransferase CPT1A carnitine palmitoyltransferase
1A
ADRB2 adrenoceptor beta 2
CPT1B carnitine palmitoyltransferase 1B
ADRB2 adrenoceptor beta 2
CRAT carnitine O-acetyltransferase
ALA α-linolenic acid
CRP C-reactive protein
ALOX5AP arachidonate 5-lipoxygenase
activating protein CVD cardiovascular disease
CXCR2 C-X-C motif chemokine
apoB100 apolipoprotein B100 receptor 2
ARHGAP15 Rho GTPase activating protein CYP27A1 cytochrome P450 family 27
15 subfamily A member 1
BMI body mass index Cyp7a1 cholesterol 7 alpha-hydroxylase
CCL C-C motif chemokine ligand DGLA dihomo-c-linolenic acid
CCR2 C-C motif chemokine receptor 2 DHA docosahexaenoic acid
CD14 CD14 molecule DNA deoxyribonucleic acid
CD19 CD19 molecule DPA docosapentaenoic acid

CD36 CD36 molecule E% percent of energy
CD40LG CD40 ligand EPA eicosapentaenoic acid
CD72 CD72 molecule ER endoplasmatic reticulum
vii
ERK extracellular signal-regulated IL23A interleukin 23 subunit alpha
kinase
IL23R interleukin 23 receptor
FASN fatty acid synthase
IL7R interleukin 7 receptor
FXR farnesoid X receptor
INSIG insulin induced gene
GAPDH glyceraldehyde-3-phosphate
dehydrogenase L2HGDH L-2-hydroxyglutarate
dehydrogenase
GPR G protein-coupled receptor
LA linoleic acid
HBEGF heparin binding EGF like
growth factor LACTB lactamase beta
HDL-C HDL cholesterol LDL low density lipoprotein
HETE hydroxyeicosatetraenoic acid LDL-C LDL cholesterol
HIF1A hypoxia inducible factor 1 alpha LDLR LDL receptor

subunit
LPAR2 lysophosphatidic acid receptor 2
HMGCR HMG-CoA reductase
LPS lipopolysaccharide
HODE hydroxyoctadecadienoic acid
LTA4H leukotriene A4 hydrolase
HPRT1 hypoxanthine
phosphoribosyltransferase 1 LTB4 leukotriene B4
hs-CRP high-sensitive CRP LXR liver X receptor
HSPA5 heat shock protein family A LXRA liver X receptor alpha

(Hsp70) member 5
LXRE LXR response element
ICAM1 intercellular adhesion molecule 1
LY96 lymphocyte antigen 96
IFN interferon MAPK8 mitogen-activated protein kinase

8
IFNG interferon gamma
MetS metabolic syndrome
IGHD immunoglobulin heavy constant
delta MIF macrophage migration
inhibitory factor
IKBKB inhibitor of kappa light
polypeptide gene enhancer in MLX max-like factor X
B-cells, kinase beta
MMD monocyte to macrophage
IL interleukin differentiation associated
IL1B interleukin 1 beta MMP matrix metalloproteinase
IL1RN interleukin 1 receptor antagonist mRNA messenger RNA
viii
MUFA monounsaturated fatty acid PPARG peroxisome proliferator activated
receptor gamma
NAGPA N-acetylglucosamine-1-
phosphodiester PPRE PPAR response element
alpha-N-acetylglucosaminidase
PREDIMED Primary Prevention of
NFkB nuclear factor kappa B Cardiovascular Disease
NFKBIA NFkB inhibitor alpha PUFA polyunsaturated fatty acid
NK natural killer qPCR real-time quantitative

polymerase chain reaction
OGTT oral glucose tolerance test
RCT randomized controlled trial
OLR1 oxidized low density lipoprotein
receptor RELA RELA proto-oncogene, NF-kB
subunit
OLTT oral lipid tolerance test RIN RNA integrity number
PBMC peripheral blood mononuclear RIPK1 receptor interacting
cell serine/threonine kinase 1
PCSK9 proprotein convertase subtilisin RNA ribonucleic acid
kexin type 9
ROS reactive oxygen species
PDGFA platelet derived growth factor
subunit A RSAD2 radical S-adenosyl methionine
domain containing 2
PDGFB platelet derived growth factor
subunit B RXR retinoid X receptor
PDK4 pyruvate dehydrogenase kinase 4 SELP selectin P
PEAR1 platelet endothelial aggregation SFA saturated fatty acid
receptor 1
sICAM1 soluble intercellular adhesion
PGJ2 prostaglandin J2 molecule 1
PL phospholipid SLC22A5 solute carrier family 22 member

5
PLIN2 perilipin 2
SLC25A20 solute carrier family 25 member
20
POLK DNA polymerase kappa
SR scavenger receptor
PPAR peroxisome proliferator activated
receptor SREBP sterol regulatory binding protein
PPARA peroxisome proliferator activated sTNFR soluble tumor necrosis factor
receptor alpha receptor
PPARD peroxisome proliferator activated sVCAM1 soluble vascular cell adhesion
receptor delta molecule 1
ix
T2DM diabetes type 2 TNFRSF12A tumor necrosis factor receptor
superfamily member 12A
TAB2 TGF-beta activated kinase
1/MAP3K7 binding protein 2 TNFSF10 tumor necrosis factor
superfamily member 10
TBP TATA box binding protein
total-C total cholesterol
TG triglyceride
UCP2 uncoupling protein 2
TGFB2 transforming growth factor beta
2 VCAM1 vascular cell adhesion molecule 1
Th T helper VEGFB vascular endothelial growth

factor B
TLR Toll-like receptor
XBP1 X-box binding protein 1
TNF tumor necrosis factor
x
Contents
Acknowledgements i
Abstract iii
List of Papers v
Abbreviations vii
Contents xi
1 Introduction 1
1.1 Dietary patterns and cardiometabolic risk . . . . . . . . . . . . . . . 1
1.2 Fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 Dietary fat, lipids and cardiovascular risk . . . . . . . . . . . . . . . 5
1.4 LDL cholesterol metabolism . . . . . . . . . . . . . . . . . . . . . . . 7
1.5 Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.5.1 Atherosclerotic process . . . . . . . . . . . . . . . . . . . . . . 10
1.5.2 Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.6 Dietary fat, inflammation and cardiovascular risk . . . . . . . . . . 12
1.7 Fatty acids and gene regulation . . . . . . . . . . . . . . . . . . . . . . 13
1.8 Peripheral blood mononuclear cells . . . . . . . . . . . . . . . . . . . 16
1.9 Use of challenge tests in interventions . . . . . . . . . . . . . . . . . 17
2 Aims 21
xi
CONTENTS
3 Subjects and methods 23

3.1 The NoMa study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.2 The SYSDIET study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.3 Selection of candidate genes . . . . . . . . . . . . . . . . . . . . . . . . 27
4 Summary of results 31
5 Discussion 35
5.1 Methodological considerations . . . . . . . . . . . . . . . . . . . . . . 35
5.1.1 Study design of intervention studies . . . . . . . . . . . . . 35
5.1.2 Gene expression in peripheral blood mononuclear cells
as a model system in intervention studies . . . . . . . . . . 38
5.1.3 Quantitative real-time polymerase chain reaction . . . . . 39
5.1.4 Statistical considerations . . . . . . . . . . . . . . . . . . . . . 40
5.2 Discussion of results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
5.2.1 Healthy dietary patterns and lipids . . . . . . . . . . . . . . 41
5.2.2 Healthy dietary patterns and inflammation . . . . . . . . . 47
5.2.3 Oral glucose tolerance test as a tool in dietary interven-
tion studies to detect effects on inflammation . . . . . . . 49
5.3 Implication for public health . . . . . . . . . . . . . . . . . . . . . . . 51
6 Conclusions 53
Bibliography 55
xii
Chapter 1
Introduction
1.1 Dietary patterns and cardiometabolic risk

The four major noncommunicable diseases cardiovascular diseases (CVDs), can-
cer, chronic respiratory diseases and diabetes type 2 (T2DM) are responsible for
82% of noncommunicable disease deaths. CVDs are the leading cause of deaths
worldwide. In 2012, 17.5 million people died from CVDs representing 31% of
all global deaths [1, 2]. Lifestyle strategies are important for the reduction of
noncommunicable diseases, and especially diet plays a crucial role [3, 4, 5].
The term "cardiometabolic risk" may be considered to represent the com-
prehensive catalogue of factors that contribute to the development of both
CVDs and T2DM [6]. According to the World Health Organization, a risk
factor is "any attribute, characteristic or exposure of an individual that increases
the likelihood of developing a disease or injury". In general, several factors con-
tribute to cardiometabolic risk such as tobacco use, unhealthy diet and obesity,
physical inactivity and alcohol abuse, hypertension and dyslipidemia. However,
diet-related cardiometabolic risk factors are insulin resistance, obesity, dyslipi-
demia, hypertension and inflammation [7].
Nutrition research has traditionally focused on nutrients to identify the spe-
cific mechanisms and health impact of diet. However, associations between
single factors as nutrients as well as foods and chronic diseases can be difficult to
identify and to interpret. In contrast, studies of dietary patterns or whole diets
examine the association between the combinations of many foods and nutrients
1
CHAPTER 1. INTRODUCTION
and health. Therefore, more emphasis should be placed on the role of dietary
patterns in contributing to the prevention of the major diet-related chronic dis-
eases [8]. A very well-described dietary pattern is the Mediterranean-type of
diet. As early as mid last century, Ancel Keys started to investigate the role
of a diet and CVDs in the Seven Countries Study. The study has shown that
populations in different countries have widely diverse incidence and mortality
rates from coronary heart disease (CHD) as well as from other CVDs and over-
all mortality [9]. Higher rates were found in North America and Northern
Europe, and lower rates in Southern Europe - i.e. the Mediterranean coun-
tries - and Japan. These differences in CHD rates were strongly associated with
different levels of saturated fatty acid (SFA) consumption and average serum
cholesterol levels, with lowest rates in Greece and Japan where the total fat
intake was very different [10]. There is no standard definition of the term
"Mediterranean Diet", but the characteristics of healthy Mediterranean Diet are
high intake of fruits, vegetables, legumes, fish, whole grains, nuts, and olive oil.
Dairy products and wine are of moderate intake, and red and processed meats
as well as foods that contain high amounts of added sugar are of low intake
[11, 12]. There is very strong evidence from the Primary Prevention of Car-
diovascular Disease (PREDIMED) [13] and the Lyon Diet Heart Study [14]
showing that a Mediterranean-type diet is effective in primary and secondary
prevention of CVDs, respectively. In observational studies, the association be-
tween the Mediterranean diet and inflammatory markers in healthy persons
has been examined [15, 16, 17], and overall inverse correlations have been re-
ported. Moreover, intervention studies have been shown that consumption of
a Mediterranean diet resulted in a decline of inflammatory markers in healthy
subjects [18, 19] as well as in those with metabolic syndrome (MetS) [20] or
high risk of CVDs in the PREDIMED study [21]. Particularly, the results from
large intervention studies strongly suggest that a Mediterranean diet can lead to
reductions in chronic low-grade inflammation and improvements in endothelial
function, and thereby offering cardioprotective effects [22].
The idea of a "healthy Nordic diet" conceived of the Mediterranean diet,

which has long been related to improved health, but the acceptance of the
Mediterranean diet in the Nordic countries is challenging, probably because of
different food preferences and eating habits in these countries [23, 24]. Thus, a
2
Figure 1.1: Overview of a healthy Nordic diet and cardiometabolic health. A Healthy Nordic
dietary pattern or foods present in healthy Nordic diets may improve cardiometabolic risk fac-
tors, such as blood lipids, endothelial function, inflammation, glucose metabolism, insulin sensi-
tivity, blood pressure and obesity. Abbreviations: DHA, docosahexaenoic acid; EPA, eicosapen-
taenoic acid; LDL-C, low density lipoprotein cholesterol; MetS, metabolic syndrome; T2DM,
diabetes type 2.
3
healthy Nordic diet takes food culture, palatability and the environment into ac-
count [25, 26]. Healthy Nordic diets are characterized by fatty fish (e.g. salmon
and herring), whole grain cereals including rye, barley and oats, berries (e.g.
blueberries) and fruits (e.g. apples), vegetables, root vegetables and legumes
and rapeseed oil [27, 28]. In randomized controlled intervention studies con-
ducted in various Nordic populations it has been shown that healthy Nordic
diets or foods present in healthy Nordic diets and in accordance to the Nordic
Nutrition Recommendations improve key CVD risk factors (Figure 1.1), such
as blood lipid profiles [29, 28, 30, 31], endothelial function [32], inflammation
[32, 28, 33], glucose metabolism [34], insulin sensitivity [35, 36], and blood
pressure [37, 38]. Ad libitum consumption of a healthy Nordic diet in over-
weight and obese subjects resulted in a weight reduction [29, 37], which may
have an effect on cardiometabolic health [39].
In conclusion, improving fat quality, as part of a healthy dietary pattern
such as the Mediterranean diet or the healthy Nordic diet, has shown to im-
prove blood lipid profile and inflammation and subsequently decreasing car-
diometabolic risk.
1.2 Fatty acids

Fatty acids occur freely or as part of complex lipids such as triglycerides (TGs),
phospholipids (PLs) and cholesteryl esters (CEs), and play a central role in
energy metabolism, membrane formation, cell signaling, and as regulators of
gene expression (see section 1.7) [40]. TGs are the main contributors to dietary
fat in humans and are composed of one molecule of glycerol esterified with three
fatty acid molecules. Typical dietary fatty acids have between 6 and 24 carbons
and are either saturated, monounsaturated or polyunsaturated according to the
number of double bonds between the carbon atoms. SFAs, monounsaturated
fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) have none, one
or two or more double bonds, respectively [8, 40] (Figure 1.2).
Fatty acids can be obtained from the diet and several can also be produced
endogenously, either from glucose or protein sources by de novo lipogenesis
or from other fatty acids due to the activity of desaturases (addition of double
bonds) and elongases (addition of two carbon atoms) [41]. In the liver and
4
Figure 1.2: Structures of some common dietary fatty acids. Fatty acids consist of a chain of
carbon atoms with a carboxyl group (-COOH) at one end and a methyl group (-CH3 ) at the
other end. Dietary unsaturated fatty acids are classified as n-3, n-6 and n-9 specifying the first
position of the double bond by counting from the methyl end of the carbon chain.
in adipose tissue, even-numbered SFAs can be synthesized endogenously by de

novo lipogenesis with the main product being palmitic acid (16:0), which can
further elongated and/or desaturated into palmitoleic acid (16:1n-7), stearic acid
(18:0) and oleic acid (18:1n-9). The majority of dietary SFAs in a western diet
is palmitic acid (16:0), stearic acid (18:0) and myristic acid (14:0) [42]. In hu-
mans, linoleic acid (LA) (18:2n-6) and α-linolenic acid (ALA) (18:3n-3) cannot
be synthesized due to the lack of enzymes and therefore are called essential fatty
acids which require adequate dietary intake. The main sources for these fatty
acids are vegetable oils. Sunflower, rapeseed, soybean and corn oil are rich in
LA. The main sources for ALA are rapeseed and soybean oil, nuts as well as
flaxseed and flaxseed oil.
1.3 Dietary fat, lipids and cardiovascular risk

Dyslipidemia is a major risk factor for CVDs and is defined as elevated blood
total cholesterol (total-C), LDL cholesterol (LDL-C) or TGs, or low levels
of HDL cholesterol (HDL-C). In Norway, the national guidelines for pri-
mary prevention of CVD recommend total-C < 5.0 mmol/L, LDL-C < 3.0
mmol/L, TGs ≤ 1.7 mmol/L and HDL-C ≥ 1.0 mmol/L (men) and ≥ 1.3
5
mg/dL mmol/L
2,4 0,06
2,0 0,05
1,6 0,04
1,2 0,03
0,8 0,02
0,4 0,01
0 0,00
-0,4 -0,01
-0,8 -0,02
12:0 14:0 16:0 18:0 18:1 18:2
n-9 n-6
Figure 1.3: Effects of dietary fatty acids on serum total-C (white bars), LDL-C (grey bars)
and HDL-C (black bars) when 1 E% from carbohydrates in the diet is replaced by 1 E% from
the fatty acid in question The values for lauric acid (12:0) are based on [48], for myristic acid
(14:0) on [49], for palmitic acid (16:0) on [48, 49, 50, 51], for stearic acid (18:0) on [51, 52],
and for oleic acid (18:1n-9) and LA (18:2n-6) on [46]. Abbreviations: HDL-C, high density
lipoprotein cholesterol; LA, linoleic acid; LDL-C, low density lipoprotein cholesterol; total-C,
total cholesterol. Adapted from [45] with permission.
mmol/L (women) [43]. Dietary fatty acid composition regulates lipoprotein

metabolism, which may affect plasma lipids and thereby potentially CVD risk
[44]. Several dietary SFAs such as lauric acid (12:0), myristic acid (14:0) and
palmitic acid (16:0) have an total-C and LDL-C raising effect. Stearic acid (18:0)
has a more neutral effect on LDL-C levels [45, 42, 41].
Human intervention studies that replace carbohydrates by SFAs, MUFAs
or PUFAs have shown that SFAs increase LDL-C and HDL-C level, but do
not change the total-C to HDL-C ratio compared with carbohydrates. The
replacement of carbohydrates by MUFAs or PUFAs resulted in a decrease in
the total-C to HDL-C ratio, a raise in total-C and LDL-C level and a slightly
increase in HDL-C. The replacement of SFAs by PUFAs may lead to an even
more favorable lipid profile [46, 45, 47] (Figure 1.3).
A pooled analysis of 11 cohort studies found that replacing 5 percent of
energy (E%) of SFAs with PUFAs was associated with a 13% lower risk of coro-
nary events and a 26% lower risk of coronary deaths [53]. In a prospective
cohort study, high circulating LA was inversely associated with total and CHD
6
mortality [54]. A meta-analysis of prospective cohort studies showed that di-

etary LA was inversely associated with CHD risk in a dose–response manner
[55]. The typical modern human diets contain more LA than ALA most likely
due to the increased use of vegetable oils rich in LA [56, 41]. LA accounts for
approximately 90% of the total dietary n-6 PUFA intake [57]. In 2012, a meta-
analysis of seven randomized controlled trials (RCTs) confirmed this beneficial
effect, with an estimated 10% reduction in CHD risk for each 5 E% increase
in PUFA consumption [58]. Another meta-analysis with RCTs concluded that
interventions with n-3 and n-6 PUFAs reduce the CHD risk whereas interven-
tions with n-6 PUFAs alone tend to increase the CHD risk [59]. However, the
overall evidence indicates that higher n-6 PUFA intake lowers the CHD risk
[60, 57].
It has been well demonstrated in RCTs that LA lowers serum total-C and
LDL-C concentrations, particularly when it replaces SFAs in the diet [46]. Also
in a more recent study it has been shown that replacing 9.5E% from SFAs
with MUFAs or n-6 PUFAs leads to a significant lower total-C and LDL-C and
total-C to HDL-C ratio after 4 months [61]. Schwab and co-workers included
45 RCTs in a systematic review investigating the effect of different fatty acids on
serum lipids and evaluated the reduction of total-C and LDL-C when SFAs are
replaced by cis-MUFAs or PUFAs as convincing [62]. These results have been
incorporated into the Nordic Nutrition Recommendations from 2012, in which
it is recommended to limit the intake of SFAs to <10 E%, whereas PUFAs (the
sum of n-6 and n-3 PUFAs) should contribute 5-10 E%, including at least 1 E%
from n-3 PUFAs. LA and ALA should contribute at least 3 E%, including at
least 0.5 E% from ALA. Likewise, because of the causal link between fat quality
and total-C and LDL-C concentrations and between increased LDL-C and in-
creased CVD risk, other countries and many authorities recommend restricting
the intake of SFAs while maintaining sufficient intake of MUFAs and PUFAs
[41].
1.4 LDL cholesterol metabolism

The low density lipoprotein (LDL) particles contain esterified cholesterol and
TGs surrounded by a shell of phospholipids, free cholesterol and apolipopro-
7
tein B100 (apoB100). They are the main carriers of cholesterol and can be taken
up by LDL receptors (LDLRs) or scavenger receptors (SRs) [63]. The LDLR
is the primary pathway for the removal of cholesterol from the circulation [64]
and LDLRs are mainly expressed in the liver, but also in smooth muscle cells, fi-
broblasts, and epithelial cells of the gastrointestinal tract and in blood cells such
as peripheral blood mononuclear cells (PBMCs). The LDLR takes up mainly
apoB100 but also apoE-containing lipoproteins and cluster in coated pits, the
portals by which many receptor-bound ligands enter the cells. The pits invagi-
nate to form coated endocytic vesicles and become endosomes. Within the en-
dosomes, the LDL particle separates from the receptor and therefore allows the
receptor to be recycled. Then, the endosome merges with the lysosome, where
CEs are hydrolyzed [65]. The lysosomes release unesterified cholesterol which
mediates cholesterol homeostasis in three ways: Inhibition of HMG-CoA re-
ductase (HMGCR); increase of acyl CoA cholesterol acyltransferase (ACAT)
activity; and decrease of the synthesis of LDLR [66]. Thus, the activity of the
LDLR and HMGCR is homeostatically regulated and the cells obtain choles-
terol either from exogenous lipoproteins or from endogenous synthesis (Figure
1.4).
LDL particles can also be taken up via SRs, which are mainly expressed by
macrophages. The SR-mediated uptake predominately occurs after LDL parti-
cles have been modified, particularly by oxidation [63, 68]. The accumulation
of oxidized LDL in macrophages results in their transformation into foam cells,
which are involved in the pathogenesis of atherosclerosis (see section 1.5). The
smallest, most dense LDLs are considered most vulnerable to oxidation and
thus most prone to be taken up via the SRs. Importantly, in contrast with the
LDLR, the uptake of LDL particles with the SRs is not rate-limited, as it is
proportional to the LDL-C concentration in circulation [66].
A milestone in the cholesterol-lowering therapy is the discovery of statins
which inhibit the HMGCR and therefore decrease the endogenous synthesis of
cholesterol. In response, the sterol regulatory binding protein (SREBP) cleav-
age is increased and the nuclear form of the SREBP2 activates the expression of
LDLR and HMGCR and other genes important for cholesterol uptake and syn-
thesis. However, as statins inhibit HMGCR (synthesis) but not LDLR (uptake),
the increased LDLRs lower LDL-C in plasma. A new cholesterol-lowering strat-
8
LDLR LDL plasma

PCSK9 LDLR
intracellular
recycling
SCAP
INSIG SREBP
endosome
endosome Cellular
1
degradation cholesterol
Cellular
2
cholesterol
LDLR
lysosome HMGCR SREBP-2
SRE
statin
nucleus
Figure 1.4: Mechanisms of LDLR regulation. (1) Cellular cholesterol low: Proteolytic cleavage
of SREBP2 is increased. The cleaved SREBP2 enters the nucleus to activate genes controlling
cholesterol synthesis (including HMGCR) and uptake (LDLR). (2) Cellular cholesterol high:
Proteolytic cleavage of SREBP2 is decreased, leading to decreased nuclear SREBP2 and decreased
activation of target genes. The decrease in LDLR leads to an increase LDL in plasma [67]. Ab-
breviations: HMGCR, HMG-CoA reductase; INSIG, insulin induced gene; LDL, low density
lipoprotein; LDLR, low density lipoprotein receptor; PCSK9, proprotein convertase subtilisin
kexin type 9; SCAP, SREBP cleavage activating protein; SREBP, sterol regulatory binding pro-
tein.
9
egy is the therapy with proprotein convertase subtilisin kexin type 9 (PCSK9)
inhibitors. PCSK9 is a protein secreted by hepatocytes that binds to an extracel-
lular pocket of the LDLR and targets it for lysosomal degradation in the cells,
effectively increasing plasma LDL-C. Therefore, antibodies that inhibit PCSK9
lead to reduced lysosomal degradation of LDLRs, increased expression on the
membrane surface and reduced LDL-C concentration in plasma [69, 70].
1.5 Atherosclerosis
1.5.1 Atherosclerotic process
Atherosclerosis is a chronic inflammatory disease of the blood vessels. The
initial step of the atherosclerotic process is the subendothelial retention of cir-
culating LDL particles and thus trapping of LDL particles in the intima of the
vessel wall [71]. In the intima, LDL particles are prone to oxidation forming
oxidized LDL. Moreover, endothelial cells may be activated by components
of oxidized LDL leading to the expression of cell adhesion molecules, such as
E-selectin and vascular cell adhesion molecule 1 (VCAM1) on the endothelial
surface of the artery [72]. Cell adhesion molecules induce arresting, rolling
and adherence of monocytes, dentritic cells and T cells onto the endothelial cell
surface resulting in migration of these cells into the intima [73]. Here, mono-
cytes respond to macrophage-colony stimulating factors and differentiate into
macrophages [74]. Macrophages express SRs on the surface which mediate the
uptake of oxidized LDL resulting in foam cell formation (Figure 1.5). The accu-
mulation of foam cells in the intima constitutes the nascent atherosclerotic le-
sion referred to as fatty streaks [75]. Moreover, several inflammatory mediators
are involved in the formation of the atherosclerotic plaque [76, 77]. In this pro-
cess, immune cells such as T cells, macrophages as well as foam cells express, re-
lease and respond to several growth factors, matrix metalloproteinases (MMPs),
chemokines and cytokines. In particular, macrophages release cytokines which
may increase the endothelial cell expression of cell adhesion molecules leading
to further migration of immune cells. T cells face antigens such as oxidized
LDL in the intima and may polarize into T helper (Th) cells. Th1 cells secrete
primarily pro-inflammatory cytokines (e.g. tumor necrosis factor (TNF)α, in-
10
Figure 1.5: Development of atherosclerotic lesions. Initial step is the sub-endothelial reten-
tion of LDL particles. Thus, LDL particles are trapped in the intima of the vessel wall, where
they are prone to oxidation forming oxLDL. Moreover, endothelial cells may be activated by
oxLDL leading to the expression of CAMs [72]. CAMs induce arresting, rolling and adherence
of immune cells onto the endothelial cell surface resulting in migration of these cells into the
intima [73]. Here, monocytes transform into macrophages [74] and express SRs on the surface
mediating the uptake of oxLDL. Cholesterol accumulation eventually turns these macrophages
into foam cells that are characteristic of the atherosclerotic lesion (fatty streaks). Macrophages
release cytokines that may increase the expression of CAMs on the endothelial cells, which
supports migration of immune cells in the intima. T cells face antigens in the intima and may
polarize into Th1 and Th2 cells [72]. Atherosclerosis is driven by the Th1 cell response. Ab-
breviations: CAM, cell adhesion molecule; DC, dentritic cell; IFN, interferon; IL, interleukin;
LDL, low density lipoprotein; MMP, matrix metalloproteinase; oxLDL, oxidized LDL; ROS,
reactive oxygen species; SR, scavenger receptor; Th, T helper; TNF, tumor necrosis factor.
Adapted from [72] with permission.
terleukin (IL)1 and IL6) and Th2 cells anti-inflammatory cytokines (e.g. IL4
and IL10) [74, 78, 75] (Figure 1.5). Thus, a chronic inflammation arises on top
of a lipid accumulation. The lesion progresses as the core grows by accumula-
tion of macrophages, endothelial cells and smooth muscle cells. These advanced
plaque filling can lead to fibrous cap thinning, plaque rupture or erosion, and
acute thrombotic vascular events such as myocardial infarction or stroke [71].
In summary, LDL particles and other apoB-containing lipoproteins can enter
the subendothelium and are tightly linked to the initiation of the atheroscle-
rotic process along with endothelial cell damage and inflammation [79, 80].
11
1.5.2 Inflammation
Inflammation is the immediate response of the body to infection or cellular in-
jury. Acute inflammatory reactions are usually self-limiting and resolve rapidly
due to negative feedback mechanisms such as secretion of anti-inflammatory cy-
tokines, inhibition of pro-inflammatory signaling cascades, loss of receptors for
inflammatory mediators and activation of regulatory cells. Thus, regulated in-
flammatory responses are essential to remain healthy and maintain homeostasis.
However, inflammatory responses that fail to regulate themselves can become
chronic and contribute to the maintenance and the progression of disease [78].
The characteristics of chronic inflammatory responses are loss of barrier func-
tion, responsiveness to a normally benign stimulus and increased production
of oxidants, cytokines, chemokines, eicosanoids and MMPs. Moreover, inflam-
matory cells massively infiltrate compartments in which they are found only
in low numbers in healthy conditions. Thus, chronic low-grade inflammation
is characterized by increased concentrations of inflammatory markers in the
systemic circulation and is a well recognized component of many diseases [81].
1.6 Dietary fat, inflammation and cardiovascular

risk
In a healthy dietary pattern, intake of vegetable oils with LA and ALA is of
high importance. In subgroup analysis of the Physicians’ Health Study and
the Nurses’ Health Study, dietary intake of LA was not associated with alter-
ations of C-reactive protein (CRP), IL6, soluble tumor necrosis factor recep-
tor (sTNFR)1, or sTNFR2 concentrations [82]. Moreover, the LA concen-
tration in blood lipids was not associated [83] or even inverse associated with
CRP or IL6 concentration [84, 85, 86, 87]. In 2012, a systematic review analysis
of RCTs concluded that there is no evidence for increased inflammation due
to dietary LA intake in healthy humans [88]. Increasing the amount of ALA
intake, either through the diet or as supplements, leads to proportional con-
version to eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA), but
not docosahexaenoic acid (DHA) [89, 90]. The conversion of ALA to DHA is
limited and in general human studies show that the body conversion is below
12
5% [90]. Thus, an important aspect of the anti-inflammatory action of ALA

is the reduced production of eicosanoids from arachidonic acid (AA) and the
increased production of EPA and DPA [41]. In controlled clinical trials, in-
creasing the intake of ALA has been shown to decrease serum concentrations
of CRP, which is strongly associated with reduced risk of cardiovascular events,
such as myocardial infarction and stroke [91, 92, 93].
Eicosanoids are key mediators and regulators of inflammation and are formed
from 20-carbon PUFAs. Inflammatory cells usually contain a high concentra-
tion of AA and therefore AA is typically the substrate for eicosanoid synthesis
[94]. Eicosanoids produced from AA play a role in inflammation [95, 96, 81].
Likewise, EPA is a precursor for eicosanoids but the inflammatory properties
of these eicosanoids are different compared to the AA-derived once [97].
In vitro and animal studies suggest a pro-inflammatory role for SFAs, in
particular of lauric acid (12:0) and palmitic acid (16:0) [41]. In a human obser-
vational study, the relationship between SFAs exposure and circulating inflam-
matory markers has been investigated reporting a positive association for CRP
and IL6 [98]. Moreover, the ratios of SFAs to n-6 PUFAs or SFAs to n-3 PUFAs
were positively associated with IL6 and CRP concentrations in overweight sub-
jects [99]. In a human intervention study, SFA intake increased levels of CRP,
fibrinogen, IL6, and soluble E-selectin compared with a diet enriched in oleic
acid (18:1n-9) [100]. Thus, dietary SFAs (12:0- 16:0) may increase inflammation
[41].
1.7 Fatty acids and gene regulation

Fatty acids are involved in gene regulation. This is achieved by direct fatty acid
binding to specific transcription factors, or by indirect mechanisms where fatty
acids regulate signaling pathways controlling the expression of transcription
factors (Figure 1.6) [101, 102, 103]. The latter can include phosphorylation,
ubiquitination, or proteolytic cleavage of transcription factors. In particular,
unsaturated fatty acids modulate gene transcription by regulating the activity of
numerous transcription factors, such as the nuclear receptors. Nuclear receptors
are a large subfamily within the group of transcription factors, with 48 mem-
bers [104]. The peroxisome proliferator activated receptors (PPARs), the liver
13
X receptors (LXRs) and the farnesoid X receptors (FXRs) are nuclear receptors
and function as ligand-activated transcription factors [105].
Fatty acid regulation of the PPAR family has been extensively studied. PPARs
bind mainly unsaturated fatty acids, such as LA, EPA and DHA, but may be also
regulated by enzymatically-modified fatty acids, such as eicosanoids. The latter
include for example leukotriene B4 (LTB4), prostaglandin J2 (PGJ2), hydrox-
yoctadecadienoic acids (HODEs) and hydroxyeicosatetraenoic acids (HETEs)
[107, 108, 106]. Three different PPAR isotypes have been identified: PPARα,
PPARβ/δ and PPARγ . PPARα is predominately expressed in the liver, heart
and brown adipose tissue. PPARδ (called PPARβ in rodents) is ubiquitously
expressed and has a most important function in skeletal muscle, liver and heart,
whereas PPARγ is highly expressed in white adipose tissue. PPARs heterodimer-
ize with the retinoid X receptor (RXR), which is another nuclear receptor. Lig-
ands, such as fatty acids and retinoic acid, enter the cell and bind PPARs and
RXR, respectively. The PPAR-RXR heterodimer binds to a specific genomic
binding site, PPAR response element (PPRE), which is present in or near the
promotor of the target genes. The ligand-activated nuclear receptors affect nu-
clear co-factors and the recruitment of additional proteins involved in gene tran-
scription, such as ribonucleic acid (RNA) polymerase II [109, 110].
PUFAs can also suppress the nuclear abundance of several
transcription factors, such as SREBP1, carbohydrate regulatory element-binding
protein (ChREBP), max-like factor X (MLX) and nuclear factor kappa B (NFkB).
The fatty acid regulation of these transcription factors, however, may not in-
volve direct fatty acid binding to the protein, but rather indirectly [111, 112,
113]. SREBPs are transcription factors and important regulators of choles-
terol and fatty acid metabolism. They are encoded by the two genes, SREBP1
and SREBP2, resulting in the three proteins SREBP1a, SREBP1c and SREBP2.
UBXD8, an endoplasmatic reticulum (ER)–bound protein, was identified as a
sensor for the unsaturated fatty acids. UBXD8 promotes the degradation of
insulin induced gene (INSIG)1, which in general holds the SCAP-SREBP com-
plex back in the ER and prevents its movement to the Golgi for cleavage and
maturation [114]. Thus, PUFAs inhibit the activity of UBXD8 with the result
that the SCAP-SREBP complex stays in the ER. Accordingly, the maturation
of precursor membrane-bound SREBP1 to the mature SREBP1 is inhibited and
14
FA SFA FA GPR40-43
TLR4 GPR120
precursor
SREBP-1
bHLH
inflammatory gene expression ?

gene
regulation
PUFA FA
LXR?
mature
ChREBP
bHLH
MLX
SREBP-1
PUFA
PUFA
ChREBP
PPAR
bHLH
MLX
RXR
Fatty acid & cholesterol

Glycolysis Fatty acid catabolism
synthesis
Figure 1.6: Mechanisms of gene regulation by FAs. 1. In hepatocytes, PUFAs may bind and in-
activate UBXD8, and thereby inhibit proteolytic processing of SREBP1 leading to an inhibition
of fatty acid and cholesterol synthesis. 2. PUFAs reduce expression of L-type pyruvate kinase
(glycolysis) in the liver, most likely by inhibiting nuclear translocation of MAX-like protein X
(MLX)–carbohydrate responsive element binding protein. 3. Activation of PPARα by PUFAs
in the liver may lead to an increase of FA catabolism. In particular, PUFAs act as ligands for
PPARs. DHA has been observed as ligand for RXR. GPR40–43 and GPR120 are membrane
receptors for various types of fatty acids, which are expressed by enterocytes and other cell
types. It is uncertain to what extent the activation of GPRs by fatty acids directly influences
gene transcription. TLR4 is expressed by macrophages and other cell types. SFAs may promote
inflammation by directly activating TLR4. The role of LXRs in mediating effects of PUFAs is
controversial. Abbreviations: bHLH, basic helix-loop-helix; ChREBP, carbohydrate-responsive
element binding protein; DHA, docosahexaenoic acid; FA, fatty acid; GPR, G protein-coupled
receptor; INSIG, insulin induced gene; LXR, liver X receptor; PPARα, peroxisome proliferator
activated receptor alpha; PUFA, polyunsaturated fatty acid; RXR, retinoid X receptor; SCAP,
SREBP cleavage activating protein; SFA, saturated fatty acid; SREBP, sterol regulatory binding
protein; TLR, Toll-like receptor. Adapted from [106] with permission.
15
fatty acid synthesis decreased [106].

Fatty acids may also regulate gene expression indirectly by binding to mem-
brane receptors such as Toll-like receptors (TLRs) and G protein-coupled recep-
tors (GPRs) [106]. TLR4 is one of several TLRs responsible for activating the
innate immune system. SFAs have been shown to active the TLR4 leading to
activation of the NFkB signaling pathway [115]. In contrast, n-3 PUFAs have
been shown to stimulate GPR120 in macrophages and adipocytes, and thereby
mediate an anti-inflammatory effect via inhibition of NFkB signaling [116, 117].
1.8 Peripheral blood mononuclear cells

To understand molecular mechanisms of food components and their effect on
health, the investigation of metabolic relevant tissues such as liver, adipose tis-
sue and skeletal muscle is of main interest. However, the availability of these
tissues obtained from healthy individuals is very limited and in particular liver
samples are almost impossible to access from healthy humans. Thus, easy ac-
cessible surrogate tissues, as for example PBMCs are of major importance to be
further investigated and may be used in human intervention studies. PBMCs
have been suggested as a surrogate tissue reflecting the hepatic regulation of
the cholesterol metabolism as well as metabolic and immune responses of hep-
atocytes and adipocytes [118]. In addition, PBMCs circulate in the body and
are exposed to nutrients and bioactive food components in the blood and in
metabolic tissues, and may therefore to a certain extent reflect systemic health
[119].
PBMCs belong to the innate and adaptive immune system and include lym-
phocytes and monocytes. Usually, 95% of the PBMCs are lymphocytes, of
which 75% are T cells, 15% B cells and 10% natural killer (NK) cells, and 5%
are monocytes (Figure 1.7). In humans, the occurrence of PBMCs varies across
individuals, and with age and infections and disease states [120].
16
Lymphocytes can be divided into

T lymphocytes, which originate in
the thymus, and B lymphocytes,
which are generated in the bone mar-
row. Whereas, T lymphocytes are
involved in cell-mediated immunity
by releasing cytokines and interacting
with pathogens directly. B lympho-
cytes are involved in humoral immu-
nity by releasing antigen-specific an- Figure 1.7: Composition of PBMCs. PBMCs
tibodies. T lymphoctes can be cat- consist to about 95% of lymphcytes and to
about 5% of monocytes. Of the lymphocytes,
egorized further into Th cells, also 75% are T cells, 15% B cells and 10% NK cells.
known as CD4+ lymphocytes, and Abbreviations: NK cell, natural killer cell;
cytotoxic T cells, also known as PBMC, peripheral blood mononuclear cell.
CD8+ lymphocytes. Th cells release
cytokines, which direct the immune response, and cytotoxic T cells release tox-
ics, which induce death of pathogen-infected cells [121]. Monocytes are cells of
the innate immune system and provide the first line of defense against bacterial
infections. They migrate from the blood stream to other tissues and differentiate
into tissue-specific macrophages [122].
1.9 Use of challenge tests in interventions

In general, a healthy organism has an enormous capacity to maintain home-
ostasis. Challenge tests can be performed to temporarily disturb homeostasis in
the body and to investigate how flexible an organism can respond to stress, e.g.
nutritional stress. For this purpose glucose, lipid or protein tolerance tests, a
mixture of these macronutrients or whole diet challenges are applied. For ex-
ample, the oral lipid tolerance test (OLTT) can be used to detect impairments
in lipid handling, while the oral glucose tolerance test (OGTT) is used for ana-
lyzing glucose metabolism and hence mainly used for T2DM diagnosis [123].
The OGTT is the most standardized challenge test and widely used in both
research and clinical settings. The aim of an OGTT is to investigate the func-
tional flexibility of the glucose metabolism system. In a healthy subject, plasma
17
Figure 1.8: Challenge test during an intervention. On the left: a single marker-response profile
such as glucose response during homeostasis and after the challenge test (e.g. OGTT). The
OGTT evokes a response in glucose concentration which returns to homeostatic levels after a
period of time. On the right: single-marker response during homeostasis and after the challenge
test before and after the intervention should ideally lead to an improved challenge response in
terms of amplitude and duration. Abbreviations: OGTT, oral glucose tolerance test. Reprint
with permission [123].
glucose concentrations return to the basal level (homeostasis) within 2h after a

standardized dose of glucose (75g) given after an overnight fast, which is called
optimal flexibility. In subjects with impaired glucose tolerance, plasma glucose
levels are elevated for a longer period, which is called impaired flexibility [124].
After an OGTT, a modest increase in leukocyte numbers leading to a modest
increase in inflammatory responses has been found [125]. Additionally, due to
decrease in TG levels and blood pressure after glucose intake, oxidative stress
may decrease [126, 123]. The transition from fasting to feeding is associated
with changes in circulating metabolite concentration in order to achieve glu-
cose homeostasis. Hence, in healthy subjects the phenotypic flexibility mecha-
nisms that follow OGTTs prevent the onset of strong inflammatory reactions
[127]. On the other hand, high fat challenge tests are known to induce a tempo-
rary postprandial pro-inflammatory and atherogenic response [119, 22], which
has been shown as postprandial increase in plasma inflammatory markers and
temporary adverse effects on vascular function [128, 129, 130]. Therefore, in-
flammation is considered a normal physiological response to a high-fat challenge
test [78].
Postprandial effects of different fatty acids on inflammatory PBMC gene ex-
18
pression have been examined in some human studies [119]. In whole genome
analysis, postprandial inflammatory gene expression was significantly up-
regulated after n-3 PUFA intake [131] and several gene sets related to inflamma-
tion were more pronouncedly up-regulated after MUFA intake relative to SFA
intake [132]. Also, target gene approaches were used to examine inflammation-
related responses of n-3 PUFA and/or MUFA intake [133, 134, 135]. Other
studies reported modest inflammatory responses only after a mixed challenge
of glucose and fat, not with a fat challenge alone [125]. This effect occurs pos-
sibly because unsaturated fatty acids are more prone to oxidation than SFAs
which might lead to more oxidative stress and consequently to inflammation.
However, another explanation could be that palmitic acid induces less stress to
PBMCs because it is more regularly consumed and therefore the change in ex-
pression of inflammatory genes are lower compared to high doses of oleic acid
or DHA [133]. Nevertheless, it seems like that repeated moderate exposure
to stress-inducing fatty acids such as MUFAs or n-3 PUFAs will activate the
transcriptional response, which leads to an increased flexibility of the cell. An
improvement in cellular flexibility may lead to positive long-term health effects
[132]. Moreover, a challenge test can be used to assess effects of diet. A dietary
intervention should ideally lead to an improved challenge response with respect
to duration and magnitude compared to a control group (Figure 1.8). Thus,
findings about metabolic flexibility of an organism may be related to impaired
health and the development of future diseases. By using a stress test, small
differences in health status between subjects may be detected which cannot be
measured in fasting conditions. Only few studies have applied a nutritional chal-
lenge test in the context of a dietary intervention study to investigate circulating
markers [136, 137] or gene expression level of different targets [138, 139]. More-
over, it has been suggested that analyses performed in the postprandial state may
increase the sensitivity to detect alterations of gene expression in PBMCs [119].
In summary, an OGTT provides a powerful tool to detect subtle changes
in health status. This might be important to detect persons with a high risk
of developing a disease but also to detect effects of nutrition-related prevention
strategies [140].
19
Chapter 2
Aims
The aim of this project has been to study the role of healthy dietary patterns on
lipids and inflammation with special focus on fat quality in populations with
cardiometabolic risk.
The specific aims have been to:
• study the intake of commercially available food products, in which SFAs

have been replaced by mostly n-6 PUFAs, on lipid profile and inflamma-
tory markers in individuals with hypercholesterolemia (paper I)
• study the molecular mechanisms of the cholesterol-lowering effect of re-

placing SFAs with mostly n-6 PUFAs in humans using PBMC gene ex-
pression as a model system (paper II)
• study the effect of a healthy Nordic diet on PBMC gene expression after
an OGTT in individuals with MetS (paper III)
21
Chapter 3
Subjects and methods
3.1 The NoMa study
The NoMa ("Norsk Mat" - Norwegian Food) study is an eight-week double-

blinded randomized controlled parallel study conducted at the University of
Oslo and the Oslo and Akershus University College of Applied Sciences be-
tween 2012 and 2014. The experimental diet group consumed heart-friendly
food products of the series "Vita hjertego" in which part of saturated fat is
replaced with polyunsaturated fat (rapeseed and sunflower oil). These prod-
ucts also have a reduced content of sodium and a high content of dietary fiber.
The products are commercially available and produced by Mills DA. The con-
trol diet group consumed products among the most sold products of each food
group in the 2011. The amount of food was based on a national food survey
(Norkost 3). All participants included in the study had to eat the food products
of the control diet group for two weeks before the start of the intervention (run-
in period). At baseline, the participants (n=115) were randomized to either the
control diet group (n=59) or the experimental diet group (n=56) (Figure 3.1).
The study protocol was approved by the Regional Ethics Committee for Med-
ical Research in South East Norway and all participants provided their written
informed consent.
23
CHAPTER 3. SUBJECTS AND METHODS
Randomization
2 weeks with Control group

1-4 weeks control diet
Screening Run-in
Experimental group
Baseline End
(8 weeks)
4-day food diaries:
before week 2 and 8
Figure 3.1: Study design of the NoMa study. In the run-in period, all participants consumed the
food products of the control diet group for two weeks before they were randomized to either
the experimental diet group or the control diet group at baseline. The study duration was eight
weeks.
3.2 The SYSDIET study
The SYSDIET study is a randomized controlled multi-center study performed

in six study centers in the Nordic countries [Kuopio and Oulu (Finland), Lund
and Uppsala (Sweden), Aarhus (Denmark) and Reykjavik (Iceland)] for 18 or
24 weeks in the period of October 2009 until November 2010. For the gene ex-
pression analysis, PBMCs from the study centers Kuopio, Oulu and Lund were
used. After a screening visit the participants followed their habitual diet for four
weeks. During this period, the consumption of foods with plant sterols and fish
oil supplements was not allowed [28]. At baseline, the participants (n=213)
were randomized to either the control diet group or the healthy Nordic diet
group within each center (Figure 3.2). The key products were provided to the
participants in both groups. The participants in the healthy Nordic diet group
received whole-grain products (for example various kinds of bread), berry prod-
ucts as frozen berries (e.g. strawberries, black currants, bilberries) and dried
berry powder as well as dietary fats including rapeseed oil- and vegetable oil-
based spreads. Fish was either directly provided or the expenses for the fish
purchase were covered, because fish is an extremely perishable food. The par-
ticipants in the control diet group received low-fiber cereal products (for exam-
ple bread with a fiber content <6 g per 100 g) and dairy fat-based spread (for
example butter) [28]. The study protocol was approved by the local Ethical
24
Figure 3.2: Study design of the SYSDIET study. At baseline, participants were randomized to
the healthy Nordic diet group or the control diet group. The study duration was 18 to 24 weeks.
PBMCs were collected fasting and after the 2h OGTT at baseline and at the end of the study.
Abbreviations: OGTT, oral glucose tolerance test; PBMC, peripheral blood mononuclear cell.
Committees of all the participating centers and all participants provided their
written informed consent.
Table 3.1 gives an overview of study populations and study designs.
25
Table 3.1: Overview of the study populations and the study designs
Study Design Population Groups Duration Aim Paper

name
NoMa Randomized Healthy subjects with Paper I: 8 weeks To investigate the effect I and
controlled moderate experimental diet of replacing SFAs with II
double- hypercholesterolemia group (n=47) and PUFAs on total
blinded control diet group cholesterol and LDL-C
study (n=52), Paper II: and gene expression in
experimental diet PBMCs
group (n=45) and
26
control diet group
(n=50)
Paper I: n=99,
Paper II: n=95
SYSDIET Randomized Subjects with MetS or Healthy Nordic 18 or 24 To investigate the effect III
controlled features of MetS diet group (n=49) weeks of a healthy Nordic diet
multicenter and control diet on gene expression in
study group (n=40) PBMCs after 2h OGTT
n=89
Abbreviations: LDL-C, low density lipoprotein cholesterol; MetS, metabolic syndrome; OGTT, oral glucose tolerance test; PBMCs,
peripheral blood mononuclear cells; PUFAs, polyunsaturated fatty acids; SFAs, saturated fatty acids.
3.3 Selection of candidate genes

The selection of inflammatory and lipid metabolism-related target genes was
primarily based on a literature search. First, we searched for longer-term dietary
intervention studies with focus on fatty acids and polyphenols studying gene
expression in PBMCs. However, studies aiming for weight loss or not having a
control group were not included. Secondly, we searched for studies investigating
gene expression in PBMCs after a nutritional challenge test. An overview of the
studies considered to select the target genes is presented in Table 3.2.
TaqMan Array Micro Fluidic Cards (Applied Biosystems) were used for real-
time quantitative polymerase chain reaction (qPCR) analysis as they can be
customized with primers to detect the target gene of choice. For each card, we
have chosen a set-up of 48 target genes to be analyzed in eight samples.
27
Table 3.2: Overview of the studies considered to select our target genes
Studies Intervention Duration Population n= Candidate genes Reference

Longer-term studies
Kaminski 1993 n-3 PUFAs 6 weeks healthy men 14 PDGFA, PDGFB [141]
Baumann 1999 n-3, n-6 and n-9 enriched oil 4 weeks healthy men 28 PDGFA, PDGFB, IL10, [142]
HBEGF, CCL2
Mutungi 2007 dietary cholesterol 12 weeks overweight/ 28 HMGCR, LDLR [143]
obese
de Mello 2009 fatty and lean fish 8 weeks established 27 TNF, IL1B, CCL2, CCL5, [144]
CHD ICAM1, SELP
Bouwens 2009 fish oil 26 weeks healthy 111 CD36, PDK4, LTA4H, [145]
elderly PLIN2, CD14, HIF1A
Bakker 2010 "anti-inflammatory dietary 5 weeks healthy, 36 PEAR1, IGHD, LACTB, [146]
mix" supplements obese men LPAR2, RSAD2, NAGPA,
28
LDLR, L2HGDH
Konstantinidou 2010 virgin olive oil 3 months non obese, 56 ADRB2, ARHGAP15, [147]
healthy IFNG, IL7R, POLK

Radler 2011 polyphenols, fish oil, phos- 12 weeks moderately 42 PPARA, CPT1A, CPT1B, [148]
pholipids and L-carnithin hyperlipi- CRAT, SLC22A5
demic obese
Camargo 2012 Mediterranean diet with 3 weeks healthy 20 MMP9, MIF1, RELA, CCL2, [139]
MUFAs, SFA diet, low fat elderly TNF, IL6*
high carb enriched with n-3
PUFA
Castaner 2012 olive oil with polyphenols 3 weeks healthy men 18 CD40LG, IL23A, IL7R, [149]
CXCR2, ADRB2, OLR1,
VEGFB, IFNG, CCL2,
ICAM1, ALOX5AP,
TNFSF10
Studies Intervention Duration Population n= Candidate genes Reference
Cruz-Teno 2012 high SFA, high MUFAs, 12 weeks subjects with 75 TNF, MMP9, NFKBIA, [138]
low fat high complex carbo- MetS RELA, IL6, CCL2, MIF*
hydrates with long-chain n-
3 PUFAs, placebo
Kolehmainen 2012 Bilberries 8 weeks subjects with 27 RIPK1, LY96, TNFRSF12A, [150]
features of TAB2, CD19, CD72, CCR2,
MetS MMD
Yubero-Serrano 2012 Mediterranean diet with 4 weeks elderly 63 RELA, IKBKB, MMP9, [151]
and without antioxidant IL1B, MAPK8, XBP1,
(CoQ), SFA diet HSPA5*
Challenge tests
Kempf 2007 OGTT 2h subjects with 75 ICAM1, TNF, IL6 [152]
29
MetS
Bouwens 2010 n-3 PUFA, MUFA and SFA 6h young 21 PDK4, PLIN2, SLC25A20, [131]
healthy ABCA1, ABCG1, SREBP1,
men LXRA
Myhrstad 2011 cod-, linseed- or coconut oil 3 and 6 h young female 16 IL8, IL6, IL1B, PPARG, [134]
CPT1A
van Dijk 2012 n-3 PUFA, MUFA and SFA 2 and 4 h lean, obese or 42 CCL2, IL1B, IL8, NFkB1, [133]
diabetic men TNF, LDLR, ABCA1,
PDK4, SREBP1, LXRA,
CYP27A1
*in addition: postprandial (2 and 4 h) gene expression analysis was performed. Abbreviations: CHD, coronary heart disease; MetS,
metabolic syndrome; MUFAs, monounsaturated fatty acids; OGTT, oral glucose tolerance test; PUFAs, polyunsaturated fatty acids;
SFAs, saturated fatty acids. Abbreviations of the candidate genes are defined in the list of abbreviations.
Chapter 4
Summary of results
In paper I, commercially available and regularly consumed food items with dif-
ferent fat quality were incooperated in the diet and decreased total-C by 9% and
LDL-C by 11% when comparing the changes in the experimental diet group to
the changes in the control diet group. Total cholesterol decreased from 6.6. to
6.0 mmol/L in the experimental diet group (P<0.001) and LDL-C from 4.2
to 3.8 mmol/L in the experimental diet group (P<0.001). Circulating inflam-
matory markers as high-sensitive CRP (hs-CRP), IL6, interferon (IFN)γ and
sTNFR1 did not change significantly during the intervention (P >0.05). To-
tal plasma fatty acid composition (as % of total fat) was significantly different
between the experimental diet group and control diet group for the SFAs 12:0,
14:0 and 16:0, the n-6 PUFAs 18:2n-6 and 20:4n-6 and the n-3 PUFA 22:5n-3.
LA (18:2n-6) was significantly increased (P<0.001) and significantly decreased
in the control diet group (P=0.013). AA (20:4n-6) was significantly increased
in the experimental diet group (P<0.001), but there was no difference from
baseline to end of the study in the control diet group. DPA (22:5n-3) was signif-
icantly decreased in the experimental diet group (P<0.001), but there was no
difference from baseline to end of the study in the control diet group. However,
the total fat intake was not different between the experimental diet group and
the control diet group (42.9 E% and 42.8 E%, respectively, P=0.901) during
the intervention. Fat quality, however, was different with a decrease in 6.5 E%
in SFAs and an increase of 6.4 E% in PUFAs in the experimental diet group
compared to the control diet group. Protein intake was 1.5 E% higher and car-
31
CHAPTER 4. SUMMARY OF RESULTS
bohydrate intake 2.4 E% lower in the experimental diet group compared to the
control diet group. The experimental diet group consumed 11.5g (0.8 E%) more
fiber than the control diet group. The fatty acid composition in food showed
that the n-6 to n-3 PUFA ratio was 8.6 in the experimental diet group and 4.7
in the control diet group. The PUFA to SFA ratio was 2.5 in the experimental
diet group and 0.3 in the control diet group.
In conclusion, commercial products that are apparently quite the same for
the consumer have a clinical relevant impact on total-C and LDL-C reduction
without an effect on circulating inflammatory markers.
In paper II, we found that the gene expression of LDLR is upregulated

(1.15 fold change, P=0.044) in PBMCs when dietary saturated fat is replaced
by polyunsaturated fat potentially explaining the effect on plasma cholesterol
levels (see paper I). The gene expression level of the transcription factor liver
X receptor alpha (LXRA) was also increased (1.18 fold change, P<0.001) and a
positive correlation between the expression of the LDLR and LXRA was ob-
served (r=0.378, P<0.001). This results were accompanied by an increase in
gene expression of the LXRA target genes fatty acid synthase (FASN) and ATP
binding cassette subfamily G member 1 (ABCG1) (1.14 fold change, P=0.002 and
1.27 fold change, P<0.001, respectively) and a borderline significant increase in
the gene expression level of SREBP1 (1.15 fold change, P=0.059). The change
in plasma palmitic acid was negatively and plasma LA positively correlated with
the change in gene expression level of LXRA (r=-0.278, P=0.007 and r=0.410,
P<0.001, respectively). Only the change in plasma LA was positively corre-
lated (r=0.221, P=0.045) with the change in LDLR expression level. Further,
serum bile acid level was borderline increased in the experimental diet group
(P=0.051) and the changes from baseline to end of the study were significantly
different between the two groups (P= 0.019).
In conclusion, the cholesterol-lowering effect observed by replacing SFAs
with PUFAs seems to be induced by a change in the expression of the LDLR,
potentially leading to increased cholesterol in the cell, increasing the expression
of LXRA and LXRA target genes as well as increasing serum bile acid level.
32
CHAPTER 4. SUMMARY OF RESULTS
In paper III, the healthy Nordic diet significantly down-regulated the ex-
pression of TLR4 (β=-0.33, q = 0.042), IL18 (β =-0.73, q=0.042) and CD36
molecule (CD36) (β=-0.23, q=0.042) compared to the control diet after the
OGTT. In contrast, the healthy Nordic diet significantly up-regulated the ex-
pression of peroxisome proliferator activated receptor delta (PPARD) (β=0.21,
q=0.042) compared to the control diet after the OGTT. The messenger RNA
(mRNA) expression level of the control gene pyruvate dehydrogenase kinase
4 (PDK4) was significantly down-regulated from 0h (fasting) to 2h (after OGTT)
in the whole study population at baseline (q < 0.0001). The expression level of
several genes involved in inflammation and lipid metabolism was modified after
the 2h OGTT. However, the OGTT response after the intervention was not
different between the groups measuring glucose and insulin levels.
In conclusion, a long-term intake of a healthy Nordic diet regulates genes in-
volved in inflammation and lipid metabolism in individuals at risk of metabolic
diseases and thereby may reduce an unfavorable postprandial response.
33
Chapter 5
Discussion
5.1 Methodological considerations

5.1.1 Study design of intervention studies
The RCT is a rigorous study design to determine whether a cause-effect rela-
tion exists between the intervention and the outcome. In RCTs, subjects are
randomly assigned to one of the treatment groups. [153]. If well-conducted,
these types of studies offer the best evidence for changing clinical practice and
forming public health policy. Both intervention studies included in this thesis
are designed as parallel RCTs.
Randomization
The random assignment of the study participants to one of the treatment groups
is essential for a causal interpretation of an intervention [153]. The basic bene-
fits of randomization are the elimination of selection bias and the balancing of
the groups with respect to many known and unknown confounding variables
[154]. Several randomization methods exist, such as simple, block and stratified
randomization. The simple randomization approach may result in an unequal
number of participants among groups and an unequal distribution of important
aspects as gender, body mass index (BMI) and other characteristics, particularly
with small sample size. Block randomization, on the other hand, may be used
35
CHAPTER 5. DISCUSSION
to ensure a balance in sample size across groups. Finally, stratified randomiza-

tion addresses the need to control and balance the influence of covariates and is
often done in randomized blocks [155]. The participants in the NoMa study
(paper I and II) were randomized by using four strata, females younger than 50
years, females 50 years or older, males younger than 50 years and males 50 years
or older, and a block size of six to ensure equal amounts of participants in each
group. The participants in the SYSDIET study (paper III) were randomized
within each study center separately to ensure an equal number of participants
in each treatment group within a study center. Randomization was performed
by matching according to gender and medians of age, BMI and fasting plasma
glucose levels at screening [28].
Randomization ensures that each patient has an equal chance of receiving
any of the treatments and generates comparable intervention groups, which are
alike in all the important aspects except for the intervention each group receives.
Blinding
The assessment of study outcomes may be influenced by information bias, i.e.

to know about the group affiliation. Such bias can be avoided by blinding.
Single-blinded means that the researcher knows about the group affiliation but
the participant does not know (or vice versa). If neither the researcher nor the
participant knows, then the study is double-blinded. In intervention studies
with foods or whole diet approaches, blinding may be challenging because the
intervention is easily identifiable by both participants and researchers. How-
ever, neutral packaging of the food products can be used, as this may conceal the
test products to a level that the study design may considered as double-blinded
[156].
The strength of the NoMa study (paper I and II) is the double-blinded de-
sign. This was achieved by having the food items delivered in closed boxes so
that neither the participants nor the dietitians knew about the group affiliation.
This ensured that dietitians gave the same advise to all participants. A double-
blinded RCT is the gold standard of intervention studies [157, 158] and only
a few, if any double-blinded RCTs have investigated the effect of exchanging a
combination of few commercially available food items with similar food items
with improved fat quality on blood lipids and inflammatory markers. The SYS-
36
DIET study (paper III) did not have a blinded study design. In that study
the groups were identifiable by both participant and researcher. However, even
though it was not blinded, the participants were not actively informed about
the group affiliation.
Multi-center studies
RCTs can be performed in one study center or in several; the latter is accord-
ingly named multi-center studies. The advantage of multi-center trials includes
the access to a larger number of participants from different geographical, socioe-
conomic and ethnic groups. Multi-center trials may also increase generalization
of results by pooling and comparing results among centers. Further, they pro-
vide the chance for investigators with similar interests to cooperate for address-
ing a common problem [159]. The SYSDIET study (paper III) was designed as
a multi-center study investigating health effects of the healthy Nordic diet based
on the Nordic Nutrition Recommendations in the Nordic countries.
Challenges in dietary intervention studies
In controlled studies, the control group should not receive treatment. This is
important to eliminate and isolate confounding variables and bias. Thus, in di-
etary intervention studies the control group should receive foods or products
not providing the tested components. Ideally, the control product should be
matched for sensory characteristics and consumed in the same way as the inter-
vention product. However, it is very difficult to develop a control product or
diet that is identical in appearance, texture, taste and smell to the test product
apart from the tested components [156]. For example, there is no such thing as
a control egg.
In the NoMa study (paper I and II) the experimental diet group received
products from the food series "Vita hjertego", which contain high amounts of
vegetable PUFAs. These food products are commercially available in Norway.
The control products were similar products selected based on sales statistics and
being among the most sold products from each category in 2011. Regarding
taste and smell the control products were different from the "Vita hjertego"
products, a feature that is one of the most challenging parts of dietary inter-
37
vention studies. An important aspect of the NoMa study is the run-in period:
in the two weeks prior to baseline, all subjects were provided and ate the con-
trol products. This was implemented to ensure that participants were able to
include the food items in their daily diet and consume the products according
to the protocol by including the food items in their daily diet. Thus, providing
the control food items ensured that all participants had a lower variability in
dietary intake and plasma cholesterol prior to entering the study at baseline.
The control diet group in the SYSDIET study (paper III) ate foods based
on the national nutrition surveys in the Nordic countries. Prior to baseline, the
subjects ate a healthier habitual diet compared to the one designed for the con-
trol diet group. Indeed, experience shows that health-interested people more
often participate in dietary intervention studies, and that their habitual diet
might be healthier than the planned control diet. Consequently, participants
in the control diet group probably worsened their dietary quality. This may
explain why the drop-out rate was higher in the control diet group compared
to the healthy Nordic diet group. It may also explain why changes were mostly
observed in the control diet group, and not in the healthy Nordic diet group.
Ideally, in the control diet group no significant changes should be found. How-
ever, this is the challenge in dietary intervention studies since the control diet
group received active treatment. Using the habitual diet as a control is also
problematic with respect to large variations in the habitual diet often observed
between participants. Aspects related to the control diet group nicely illustrate
the difficulties of doing intervention studies in nutrition research.
5.1.2 Gene expression in peripheral blood mononuclear cells

as a model system in intervention studies
Human dietary intervention studies are known for their relatively small effect
sizes [160]. This is probably because biological systems are quite robust and
try to maintain homeostasis. Dietary interventions generally result in subtle
changes in gene expression, and, as expected, in both NoMa and SYSDIET
study we observed rather low fold changes in the gene expression in PBMCs of
selected target genes from the intervention. However, all these subtle changes
may lead to profound biological effects, especially in the long term. Thus, the
38
challenge is to determine which changes in gene expression may be of clinical

relevance or not.
One of the major challenges in human gene expression studies is the collec-
tion of appropriate analysis material. Often, because of ethical and practical rea-
sons, such studies are restricted to easily accessible samples of e.g. blood. Gene
expression in PBMCs changes in response to a dietary intervention, but often it
is not known whether these changes are comparable to or different in other cells
or tissues [161]. PBMCs and hepatic cells seem to share similarities in terms of
cholesterol homeostasis and may reflect hepatic regulation of the cholesterol
metabolism [162]. A validation study suggests that PBMCs can be used in hu-
mans as marker for hepatic cholesterol metabolism to assess dietary effects on
the expression of LDLR and HMGCR [163]. Thus, these immune cells may be
a useful model system for liver lipid metabolism [164, 165, 118]. It is well docu-
mented that dietary intervention studies change the gene expression of various
inflammatory genes and genes involved in lipid metabolism [145, 144, 166, 167]
and therefore, reflect nutritional changes at the level of gene expression. Thus,
PBMC gene expression can potentially be used as a biosensor for metabolic dys-
regulation [168] and might provide a good model system to identify early risk
markers [169, 102].
5.1.3 Quantitative real-time polymerase chain reaction

In the NoMa study (paper II) and in the SYSDIET study (paper III), we used
qPCR to study the expression of single genes. Therefore, some methodological
considerations ensuring the quality of this approach have to be discussed. Iso-
lated RNA needs to be of high purity (absence of protein and deoxyribonucleic
acid (DNA) contamination as well as inhibitors) and high integrity (not de-
graded) which is of high importance for downstream analyses during the qPCR
process [170]. Particularly, contaminations may lead to an inhibition of the
reverse transcription process (complementary DNA (cDNA) synthesis) result-
ing in biased data. To prevent contamination of genomic DNA, we incubated
the samples with DNAse during the RNA isolation procedure which digests
genomic DNA. The concentration and purity of the samples were assessed
by spectrophotometry (using a Nanodrop ND-1000 Thermo Fisher Scientific),
39
which automatically performs measurements of different wave lengths: 260nm

is the absorption maximum of nucleic acids, 280nm is the absorption maxi-
mum of proteins. The 260 to 280 ratio is commonly used as an indicator of
RNA purity and a value of about 2.0 is considered as pure RNA [171]. RNA
integrity was assessed with the Bioanalyzer (Agilent Technologies) which cal-
culates a quality index representing the level of degradation. With an Agilent
RNA integrity number (RIN) value between 8 and 10, we ensured intact RNA
for downstream application.
The qPCR analysis is a process in three steps. First, RNA is reverse tran-
scribed into cDNA. Secondly, the specific sequence of interest in the cDNA
is amplified using qPCR and thirdly, amplification products are detected and
quantified [172, 173, 170]. The qPCR process has a wide dynamic range, low
quantification limits and the required amount of starting material is low. Ran-
dom primers were used in the reverse transcription step for all samples. These
primers can hybridize anywhere on the RNA and provide multiple starting
points for cDNA synthesis by the enzyme reverse transcriptase. Hence, even
if some RNA molecules are degraded the complete mRNA will be transcribed
[174].
For the analysis of the gene expression, we used relative quantification. An
important requirement for this type of analysis is the selection of appropri-
ate endogenous controls. An endogenous control is a gene whose expression
ideally does not change across the different samples [175]. TaqMan Human
Endogenous Control Assays (Applied Biosystems) were used to select endoge-
nous controls. Of the 16 possible candidates, we selected TATA box binding
protein (TBP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hy-
poxanthine phosphoribosyltransferase 1 (HPRT1) as the best candidates.
5.1.4 Statistical considerations

When multiple outcome comparisons are presented without adjustment of the
p-value, the question of type I errors arises (i.e. the chance of introducing inef-
fective treatments). However, whilst p-value adjustments reduce the chance of
making a type I error, they also increase the chance of making a type II error (i.e.
the chance that effective treatments are not discovered) [176]. In exploratory
40
studies, in which data are collected without a pre-specified key hypothesis, mul-
tiple test adjustments may not be strictly required [177]. Therefore, in the
NoMa study (paper II), we decided not to adjust for multiple testing as the
number of tests with 15 candidate genes was limited and the aim of our study
was not confirmative but rather explorative.
5.2 Discussion of results

5.2.1 Healthy dietary patterns and lipids
Healthy dietary patterns have been reported as a protective factor for CVDs in
both observational studies [178] and intervention studies [13, 14]. The Mediter-
ranean dietary pattern is classified as a healthy dietary pattern, is the most in-
vestigated and established dietary pattern [179, 180] and has been suggested to
be an effective and feasible tool for the prevention of CVDs [179]. In addition,
two prospective cohort studies conducted in Denmark and Sweden investigating
a healthy Nordic diet showed an association between people with high adher-
ence to a self-reported Healthy Nordic Food Index and reduced risk of all-cause
mortality [181, 182]. Adherence to a healthy Nordic Food Index was inversely
associated with the risk of T2DM in the Danish cohort [183]. However, no
association between the Healthy Nordic Food Index and overall CVD risk was
found in the Swedish cohort [184]. RCTs investigating the effect of healthy
Nordic dietary patterns on hard endpoints have not been performed yet.
LDL-C is a surrogate endpoint that generally reflects CVD risk and national
guidelines recommend lowering LDL-C in order to decrease cardiovascular risk
[185]. Generally, when biomarkers are used as proxies for clinical end points,
they are referred to as surrogate end points [186]. A biomarker is defined as
"a characteristic that is objectively measured and evaluated as an indicator of
biological processes, pathogenic processes or pharmacologic responses to a ther-
apeutic intervention" [187, 188].
Healthy Nordic diets improve blood lipids mainly because of changes in
dietary fat quality [189]. For example, rapeseed oil has distinct beneficial ef-
fects on blood lipid profile when compared with butter [190]. Rapeseed-oil-
based margarine was also the main food product provided in the Lyon heart
41
study which showed a ∼70% reduced risk of CVD mortality in patients with
mycardial infarction [14]. However, it is unclear which amount of food with
improved fat quality is needed to examine an effect on lipid metabolism and in-
flammation. It has been shown that replacing a usual breakfast with a prudent
breakfast after the Nordic Nutrition Recommendations for 12 weeks does not
influence blood lipids, body weight or glucose metabolism, but reduces visceral
fat and inflammation [191]. Individuals may achieve a healthy diet in multiple
ways and the quality of fat seems to be a very important aspect of a healthy
dietary pattern. Importantly, however, for long-term maintenance of a healthy
dietary pattern individual preferences and easy accessibility should be consid-
ered [192]. The knowledge of the effect of exchanging regular-consumed com-
mercially available food products with similar products of improved fat quality
on lipid metabolism and inflammation is limited.
In general, the effect of dietary fat quality on plasma cholesterol concentra-

tion is well established. However, the quality of dietary fat in the prevention of
CVD seems to be a never ending debate [193, 194, 195]. In a meta-analysis of
prospective cohort studies, dietary SFAs are not associated with an increased
risk of CHD [193]. Further, a meta-analysis of prospective, cohort studies
and RCTs reported no cardiovascular benefit related to high PUFA and low
SFA consumption [194]. These results received much publicity and aroused the
question if the dietary recommendations are still credible. Observational stud-
ies have several weaknesses, which question the validity of their conclusions.
For example, the accuracy of dietary data, the adjustment for serum cholesterol
concentrations and the disregard of all existing evidence were major concerns
[196, 197, 198]. In general, the role of observational studies in nutrition is
to document associations and generate hypotheses regarding heath effects of
diet. Hence, the interpretation of associations should be very cautiously made
and even strong associations need to be confirmed by well controlled interven-
tion studies. Accordingly, all available, existing evidences need to be considered
when discussing new findings [199]. Besides these discussions on the controver-
sial role of dietary SFAs, the optimal amount of total fat, individual fatty acids
and food sources of fatty acids for CHD prevention are also still under debate
[200, 199]. However, there is good evidence that the type of dietary fat plays a
far more pivotal role in health than absolute amounts [201].
42
In the NoMa study (paper I), we intervened with commercially available

and regularly consumed food products as bread, baguettes, buns and cereals as
well as spread, oil, liver pate, cheese, cream and mayonnaise. All of those prod-
ucts had an improved fat quality, either because of the raw material (e.g. oats)
or the direct exchange of fatty acids in the products by replacing some of the
saturated fat with sunflower and rapeseed oil. We found a significant reduc-
tion of total-C and LDL-C by 9% and 11%, respectively. It has been estimated
that a 10% ( 0.6 mmol/L) reduction in LDL-C is associated with a 27% reduc-
tion in CVD risk [202], suggesting that the observed LDL-C lowering effect
of 11% by improving fat quality is clinically relevant. Additionally, the in-
take of fiber was significantly different between the groups with 25.7g (1.8 E%)
in the control diet group and 37.2g (2.6 E%) in the experimental diet group
(P<0.001). An intake of ≥ 3g of β-glucan per day has been shown to reduce
LDL-C and total-C by 0.25 mmol/L and 0.30 mmol/L relative to the controls,
respectively [203, 204]. In our study, the amount of β-glucan was estimated to
be 1.83 g/day in the experimental diet based on the minimum intake of bread,
baguettes, buns and cereals suggesting a LDL-C and total-C lowering of 0.15
mmol/L and 0.18 mmol/L, respectively. Thus, fiber additionally enhanced the
cholesterol-lowering effect of the total diet, but the main effect seems to be at-
tributed to improved fat quality.
We aimed for achieving a clinically relevant cholesterol-lowering effect with-
out significantly interfering with the daily life of the participants. One impor-
tant aspect for long-term maintenance may be that the dietary changes are rela-
tively easy to implement in the daily diet. If we could have prolonged the NoMa
study where the subject continued the replacement of regularly consumed com-
mercially available key food items with similar food products of improved fat
quality, we might have been able to investigate if these changes were possible
to maintain for a long-term period. Our results support the Nordic Nutrition
Recommendations advocating to improve dietary fat quality by balancing the
fatty acid proportions to reduce the risk for chronic diseases. These results were
achieved by replacing various food products by others with improved fat qual-
ity, which is part of a healthy dietary pattern.
To identify the molecular mechanism of how replacing dietary SFAs with n-
43
6 PUFAs reduces total-C and LDL-C, we performed gene expression analysis on

RNA isolated from PBMCs. We particularly focused on genes involved in lipid
metabolism (paper II). The novel and important finding is that replacement of
SFAs with n-6 PUFAs increases LDLR mRNA expression which in turn was
positively correlated with LXRA mRNA expression and negatively correlated
with the percentage change in serum LDL-C. One explanation for these find-
ings is that the increased expression of LDLR resulted in increased intracellular
cholesterol concentrations, which subsequently increased mRNA expression of
LXRA and LXRA target genes (Figure 5.1).
LXRs are nuclear receptors regulating cholesterol metabolism but also have
a central role in the integration of sterol, fatty acid, and glucose metabolism.
LXRα is highly expressed in the liver but can be also detected in kidney, intes-
tine, adipose tissue, and macrophages, whereas LXRβ is expressed ubiquitously
[101]. LXRs bind to LXR response elements (LXREs) as heterodimers with
RXR and regulate the expression of target genes [110]. Cholesterol is not a
direct activating ligand of LXRs. The cells have to generate an indirect signal
of cholesterol excess to activate LXRs. The enzymatic conversion of choles-
terol to specific oxysterols indicates a physiologically important mechanism for
the regulation of LXR functions in many cell types, including cells of the im-
mune system [205, 206]. Thus, oxysterols are the natural endogenous ligands
of LXRs.
Serum bile acid levels were increased by exchanging SFAs with PUFAs in the
diet. In the hepatocytes, the bile acid synthesis regulates cholesterol homeosta-
sis. The cholesterol 7 alpha-hydroxylase (Cyp7a1) was the first LXRA target gene
identified [206]. In mice, LXRA induces Cyp7a1 gene transcription to stimu-
late bile acid synthesis. However, in humans, it is not well documented if LXR
activation leads to increased bile acid synthesis from cholesterol [207]. The in-
crease in serum bile acid may reflect increased LXRA activity in liver, and thus
our data clearly show that hepatic lipid metabolism is reflected in PBMCs gene
expression, as shown by others [164, 165].
If intracellular cholesterol levels are high, the LXR family of transcription
factors is activated, which subsequently decreases cellular cholesterol by up-
regulating efflux and by decreasing endogenous synthesis and uptake. For exam-
ple, HMGCR activity is inhibited and LDLR synthesis is down-regulated [208].
44
LDL ABCG1 Ĺ
Cholesterol
Efflux (FC) SREBP1
Cholesterol ester (CE)
pool Cytosol
LDLR Ĺ
FASN Ĺ
In lysosomes Free cholesterol (FC)

CE
Lipogenesis Ĺ (?)
ȕ-oxidation Ļ (?)
Oxysterols
UCP2 Ļ
LXRA Ĺ
Nucleus
LDLR SREBP1 FASN ABCG1 UCP2
ϬϱͬϭϬͬϮϬϭϱ ^&ĂŶĚĐŚŽůĞƐƚĞƌŽůͲŵĞƚĂďŽůƐŝŵ
Figure 5.1: Possible mechanism when SFAs are exchanged by n-6 PUFAs. Plasma total-C and
LDL-C have been reduced which may be mediated by an increase in LDLR mRNA expres-
sion. Excess cellular cholesterol may lead to an increase in LXRA mRNA expression as well
as LXRA target genes ABCG1 and FASN. ABCG1 may enhance cholesterol efflux and FASN
may increase lipogenesis that some fatty acids can be used for cholesterol-esterification. This
might also explain the down-regulation of UCP2 mRNA expression, potentially leading to a re-
duction in β-oxidation and therefore, conserving some fatty acids for cholesterol-esterification.
Abbreviations: ABCG1, ATP binding cassette subfamily G member 1; CE, cholesterol ester;
FASN, fatty acid synthase; FC, free cholesterol; LDL-C, low density lipoprotein cholesterol;
LDLR, LDL receptor; LXRA, liver X receptor alpha; PUFA, polyunsaturated fatty acid; SFA,
saturated fatty acid; SREBP, sterol regulatory binding protein; total-C, total cholesterol; UCP2,
uncoupling protein 2.
45
If intracellular cholesterol levels are low, the transcription factor SREBP2 is ac-
tivated and acts to increase cellular cholesterol levels by facilitating its synthesis
and uptake as well as decreasing efflux. In that case, HMGCR activity is in-
creased and LDLR synthesis is enhanced [111]. Our results indeed show an
up-regulation of LXRA but no down-regulation of the LDLR, which may be
explained of the enhanced cholesterol efflux via ABCG1 in PBMCs. The ma-
jor regulator of LDLR synthesis is the amount of cholesterol within the cell
[67]. In human hepatic cells, LA has been shown to upregulate hepatic LDLR
gene and protein expression [209]. It is suggested that this effect is probably the
main mechanism by which an increased intake of LA lowers blood cholesterol
concentrations [41].
Further, LXRs and SREBPs have functions related to the fatty-acid
metabolism. A major function of LXR in the liver is the elevation of de novo
biosynthesis of fatty acids through the stimulation of SREBP1c and its target
genes (e.g. FASN). Some of these fatty acids may be used for cholesterol-
esterification and storage in cholesterol-pools in order to avoid toxic levels of
free cholesterol [110]. Thus, SREBP1c is a form of SREBP1 that mostly acti-
vates genes involved in de novo lipogenesis, whereas SREBP2 has a preference to
regulate genes involved in cholesterol synthesis and uptake [205, 67].
In the SYSDIET study (paper III), we observed that a healthy Nordic

diet compared to a control diet up-regulated the mRNA expression of PPARD
after an OGTT in subjects with MetS. The fasting PPARD mRNA expres-
sion was not different between the healthy Nordic diet group and the con-
trol diet group (data not shown). Studies have shown that PPARδ regulates
lipid metabolism, glucose homeostasis, and fatty acid oxidation [210, 211]. In
a human study, a two-week PPARδ activation resulted in a reduction of fast-
ing and postprandial plasma TGs, LDL-C, apoB and liver fat content [212].
Thus, the activation of PPARδ is possibly beneficial and might lower the risk
for CHD [213]. Moreover, PPARδ may improve insulin sensitivity [110].
In macrophages, PPARδ polarizes macrophages toward the alternatively acti-
vated, anti-inflammatory M2 phenotype, improving insulin sensitivity in adi-
pose tissue. In adipocytes, PPARδ activation inhibits the lipopolysaccharide
(LPS)-induced NFkB activation by inhibiting extracellular signal-regulated ki-
46
nase (ERK)1/2, thereby reducing the production of pro-inflammatory cytokines

involved in the development of insulin resistance [214]. In the SYSDIET main
study, no effect on insulin sensitivity and glucose metabolism has been detected
on circulating markers after an OGTT [28].
In conclusion, we have shown that improving the fat quality, as is part
of a healthy dietary pattern, has beneficial and clinically relevant cholesterol-
lowering effects, which are associated with reduced CVD risk. Moreover, we
suggested a molecular mechanism of how the LDL-C concentration decreases
when exchanging fatty acids. According to our results in the SYSDIET study,
PBMC gene expression after a glucose load, such as an OGTT, may provide
additional information about the effect of diet on human health.
5.2.2 Healthy dietary patterns and inflammation

In a recent meta-analysis with RCTs, the consumption of a healthy dietary pat-
tern has been shown to decrease CRP levels and to attenuate the inflammatory
state [215]. The Mediterranean dietary pattern has been associated with a re-
duced incidence of MetS [216, 217] and meta-analyses have shown a decrease in
inflammation markers and an improvement in endothelial function [218, 219].
In the SYSDIET study, the change in interleukin 1 receptor antagonist (IL1RN)
levels during the intervention was significantly different between the healthy
Nordic diet and the control diet group. The IL1RN level increased markedly in
the control diet group probably as a result of worsening dietary quality in this
group. The increase of IL1RN in the control diet group has been suggested as a
compensatory appearance to a more atherogenic and pro-inflammatory compo-
sition of the control diet. The change in other inflammatory markers, including
hs-CRP, adiponectin, IL1β, IL6, IL10 and sTNFR2, was not different between
the groups [28]. The IL1RN mRNA expression in PBMCs was down-regulated
in the healthy Nordic diet group compared to the control diet group (P=0.068,
data not shown), but in line with the result seen for plasma IL1RN.
In the SYSDIET study (paper III), mRNA expression of TLR4, IL18 and
CD36 was down-regulated in the healthy Nordic diet group compared to the
control diet group after an OGTT in subjects with MetS. TLRs are key regula-
tors of immune responses. They activate NFkB followed by the production of
47
cytokines as TNFα and IL1β [220]. IL18 is a pro-inflammatory cytokine. The

down-regulation in TLR4 and IL18 after an OGTT in the healthy Nordic diet
group may indicate less inflammation. CD36 is a scavenger receptor involved
in lipid uptake and foam cell formation in macrophages [221]. A blockage of
TLR4 and CD36 in human macrophages reduced the secretion of IL1β, IL6
and IL8 and the subsequent foam cell formation [222]. Thus, the lower mRNA
transcript level of CD36 after an OGTT in the healthy Nordic diet group may
reduce foam cell formation and inflammation.
In the NoMa study (paper I), we investigated the effect of replacing SFAs
with mostly n-6 PUFAs on markers of inflammation in serum (hs-CRP, IL6,
sTNFR1 and IFNγ ). These inflammatory markers did neither change signif-
icantly within the experimental diet group nor between the groups. In sev-
eral RCTs, intake of n-6 PUFAs was not related to inflammation, oxidative
stress or endothelial activation, which is an early step in the development of
atherosclerosis [61, 223, 224]. In the randomized, controlled, 10-week HEP-
FAT study investigating the effect of PUFAs on liver fat, systemic inflammation
and metabolic disorders, the authors describe n-6 PUFAs as potentially anti-
inflammatory since sTNFR2 and IL1RN concentrations were reduced [223].
In the NoMa study (paper I), plasma LA and AA were increased and plasma
EPA, DPA and DHA were decreased in the experimental diet group from base-
line to the end of the study. Between-group differences were found for LA,
AA, and DPA. LA can be converted into AA (20:4n-6) with dihomo-c-linolenic
acid (DGLA) (20:3n-6) as an important intermediate. ALA can be converted
into EPA (20:5n-3), DPA (22:5n-3) and DHA (22:6n-3). LA and ALA are me-
tabolized by the same enzymes and they compete for the same desaturation and
elongation enzymes in the synthesis of longer PUFAs [225, 226, 40]. Thus,
the increased intake of LA in the experimental diet group may have increased
the desaturation and elongation enzyme activity leading to an increase in AA.
Therefore, the synthesis of EPA from ALA is reduced, which may lead to re-
duced levels of DPA and DHA in plasma. There has been concern that di-
etary n-6 PUFAs, especially LA, may contribute to excess inflammation by pro-
moting the synthesis of pro-inflammatory eicosanoids from AA as well as that
the synthesis of anti-inflammatory eicosanoids from EPA and/or DHA may
be inhibited [227, 228, 229, 230]. Due to the competition for the Δ-6 desat-
48
urase, a high intake of LA may lead to reduced elongation from ALA to EPA
and/or DHA, and subsequently reduce the formation of anti-inflammatory
eicosanoids.However, the experimental evidence supporting this concern orig-
inated mainly from rodent and cell culture studies [96]. Human studies have
shown that LA and AA intake is reflected by plasma fatty acids. However, di-
etary LA does not always increase circulating AA concentrations [231, 232] and,
likewise, AA has not always an impact on EPA and/or DHA concentrations
[88]. Some studies show that dietary LA decreases circulating EPA concentra-
tions, but the evidence that DHA is affected is not convincing [232], and other
studies show no change with LA intake on EPA or DHA [142, 233]. Thus,
dietary intake of LA and ALA may have effects on the conversion of ALA to
EPA and even DHA, but whether it results in increased inflammatory markers
and chronic disease outcomes remains unknown.
In conclusion, improving fat quality as part of a healthy dietary pattern had
no unfavorable effect on serum inflammatory markers. A healthy Nordic diet
when compared to a control diet may be less inflammatory and may lead to an
improvement of insulin sensitivity via TLR4, IL18 and CD36 down-regulation.
5.2.3 Oral glucose tolerance test as a tool in dietary interven-

tion studies to detect effects on inflammation
Nutritional challenge tests are used to stress the biological system by temporar-
ily disturbing homeostasis in the body and investigating how flexible an or-
ganism can respond to stress. Thus, health may be measured by the ability to
adapt to conditions of temporary stress. An OGTT is such a stress test which
monitors the ability of the body to respond to glucose intake, and is primarily
used for addressing the degree of glucose tolerance and insulin resistance [140].
A nutritional challenge test can for example be used to investigate differences
in response between different phenotypes (e.g. healthy subjects versus subjects
with metabolic risk) or to investigate differences in response to different diets.
In the SYSDIET study, an OGTT was included at the beginning and at the
end of the study, since two of the main outcomes were glucose tolerance and in-
sulin sensitivity in subjects with MetS. However, no differences between groups
were observed in glucose and insulin metabolism comparing the healthy Nordic
49
diet group with the control diet group [28]. One possible explanation for this
finding is that the participants maintained a stable body weight during the inter-
vention period. Therefore, we used PBMC gene expression as a tool to investi-
gate the effect of diet on OGTT response. As changes in glucose homeostasis are
linked to inflammation, we examined the gene expression of inflammation and
lipid metabolism-related genes (paper III). Changes in PBMC gene expression
may act as biomarkers of metabolic health not only in the fasting state but also
in the postprandial state [234]. Thus, it was interesting to investigate whether
a dietary intervention changes the OGTT response rather on the transcription
level than on the plasma level.
A glucose load can induce the production of reactive oxygen species (ROS)
which can trigger a biochemical cascade resulting in inflammation and endothe-
lial dysfunction. If these postprandial changes are repeated multiple times per
day they may eventually lead to an increase in CVD risk factors [235]. An
OGTT increases biomarkers of systemic low-grade inflammation and endothe-
lial dysfunction such as hs-CRP, IL6, TNFα, soluble intercellular adhesion
molecule 1 (sICAM1), soluble vascular cell adhesion molecule 1 (sVCAM1),
and soluble E-selectin in healthy and T2DM subjects [236]. Other studies have
found a moderate temporary increase in circulating leukocytes, suggesting an
inflammatory response [237, 125]. This increase in leukocytes seems mainly
due to the increased numbers of neutrophils [237, 125], which have been linked
to changes in endothelial function. However, the OGTT response compared
to the response of a water control did not show an increase in classical pro-
inflammatory markers such as IL1β, IL6, IL8, IL10, TNFα and IFN-γ [125].
The OGTT response can be detected at gene expression level in PBMCs
[238, 152]. In the SYSDIET study (paper III), we examined the PBMC mRNA
expression before and after an OGTT in subjects with MetS. Several pro-
inflammatory genes as C-C motif chemokine receptor 2 (CCR2), CD40 ligand
(CD40LG), C-X-C motif chemokine receptor 2 (CXCR2), IL1RN, interleukin 23
receptor (IL23R), MMP9, TNF and transforming growth factor beta 2 (TGFB2)
were up-regulated. Acute glucose intake has been shown to be able to acti-
vate the transcription factor NFkB, which induces the mRNA expression of
TNF in PBMCs in healthy subjects after an OGTT [239]. Previous studies
have also shown an up-regulation in mRNA expression of MMP9 after a glucose
50
load [240]. These effects are potentially relevant to atherosclerotic plaque rup-
ture [73]. In contrast, the expression of IL6 and intercellular adhesion molecule
1 (ICAM1) was significantly down-regulated by an OGTT in the present study.
Both genes are associated with pro-inflammatory pathways, but for IL6 also
beneficial effects have been shown [241].
In conclusion, inflammatory changes in plasma after a glucose load may con-
tribute to endothelial dysfunction and are possibly of relevance for the patho-
genesis of atherosclerosis in the long term. Our results show that glucose uptake
leads to immune activation and increased expression of pro-inflammatory genes
in PBMCs of metabolically dysregulated individuals. It can be speculated that
the glucose-mediated effect might be modulated by other metabolic factors such
as TGs and oxidative stress. Unfortunately, we did not have a healthy control
group to be able to compare the effect of a glucose load between a healthy and
a metabolically dysregulated group.
5.3 Implication for public health

In recent decades, the content of fat and fatty acids in the diet has changed
significantly in the Nordic countries. The intake of SFAs has decreased from the
1970s to the 1990s, then leveled off and now increased in some Nordic countries
in the recent years [242, 8]. A similar trend has been seen for the intake of
total fat. According to recent surveys, the consumption of SFAs is above the
recommendations and the ratio of unsaturated fat to saturated fat is below the
recommendations in Nordic countries. Therefore, current guidelines emphasize
the need to further lower SFA intake to reduce the risk for CHD [8].
In the two dietary intervention studies included in this thesis, we have
shown that a healthy Nordic diet as well as a replacement of SFAs with mostly
n-6 PUFAs in the diet lead to an improvement in lipid profile which is associated
with reduced CHD risk. In the healthy Nordic diet improving the fat quality is
of great importance, which may be achieved with an increased consumption of
vegetable oils, low-fat dairy products and less meat. However, other food items,
such as fish and berries, seem to play an important role as well.
Practical issues might withhold people from adapting to a healthy Nordic
diet. A healthy Nordic diet contains many fresh products (fruits, vegetables
51
and fish) which need more preparation time and might also be more expensive
compared to the often consumed energy dense pre-packaged and (semi-) pre-
pared foods. The replacement of SFAs by PUFAs might be easier to implement
in daily life as the products can be replaced one by one. The products we used in
paper I and II are commercially available and therefore easily accessible and ap-
plicable to the daily diet, which is an important aspect for public health. Food
producers have an important role in this process to develop tasteful, health pro-
moting products and to implement them on the market. New products need a
good marketing and a clear messaging with health claims to point out beneficial
health effects.
52
Chapter 6
Conclusions
• Improving fat quality by replacing SFAs with mostly n-6 PUFAs with
commercially available and regular-consumed food products, lowers serum
cholesterol levels in individuals with hypercholesterolaemia.
• The replacement of SFAs with mostly n-6 PUFAs did not have an effect
on serum inflammatory markers.
• The cholesterol-lowering effect observed by replacing SFAs with mostly

n-6 PUFAs seems to be induced by a change in the expression of the
LDLR, potentially leading to increased cholesterol in the cell, increasing
the expression of LXRA and LXRA target genes as well as serum bile acid
levels.
• A long-term healthy Nordic diet modified the expression of inflammatory

and lipid metabolism-related genes in PBMCs after a 2hOGTT in individ-
uals with MetS. PBMC gene expression was used as a tool to examine that
a healthy Nordic diet can modify the OGTT response.
53
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75
I
Exchanging few commercially regular-consumed food items with
improved fat quality reduces total and LDL cholesterol– a double-blind
randomised controlled trial
Stine M. Ulven1,2, Lena Leder2, Elisabeth Elind1, Inger Ottestad1,2, Jacob J. Christensen1,2,
Vibeke H. Telle-Hansen3, Anne J. Skjetne1, Ellen Raael1, Navida A. Sheikh1, Marianne
Holck1, Kristin Torvik1,2, Amandine Lamglait3, Kari Thyholt3, Marte G. Byfuglien3,
Linda Granlund3, Lene F. Andersen2, and Kirsten B. Holven2,4
1
Department of Health, Nutrition and Management, Faculty of Health Sciences, Oslo and
Akershus University College of Applied Sciences, P.O Box 4, St.Olavsplass, 0130 Oslo,
Norway
2
Department of Nutrition, Institute for Basic Medical Sciences, University of Oslo, P.O.
Box 1046, 0317 Blindern, Norway
3
Mills DA, P.O Box 4644 Sofienberg, 0506 Oslo, Norway
4
Norwegian National Advisory Unit on Familial Hypercholesterolemia, Department of
Endocrinology, Morbid Obesity and Preventive Medicine, Oslo University Hospital,
Rikshospitalet, P.O Box 4950 Nydalen, Norway
Address correspondence to: Stine Marie Ulven, Department of Nutrition, Institute for
Basic Medical Sciences, University of Oslo, P.O. Box 1046, 0317 Blindern, Norway
smulven@medisin.uio.no, +47 851102.
Reprints will not be available from the author.

Short running head: Dietary fat quality and LDL cholesterol
Key words: Cardiovascular risk factors, fatty acids, food intake, inflammation,
lipoproteins, nutrition
List of abbreviations: ALAT; Alanine aminotransaminase, ASAT; aspartate

aminotransferase, C-diet group; control diet group, CHD; coronary heart disease CVD;
cardiovascular disease, Ex-diet group; experimental diet group, MUFA; monounsaturated
fatty acid, PUFA; polyunsaturated fatty acids, SFA; saturated fatty acids, years; y.
The study was registered at ClinicalTrials.gov (ClinicalTrials.gov Identifier: NCT

01679496).
1
ABSTRACT
Healthy Nordic diet has previously been shown to have health beneficial effects among
subjects at risk of cardiovascular disease (CVD). However the extent of food changes
needed to achieve these effects is less explored. The aim of the study was to investigate
the effects of exchanging few commercially regularly-consumed key food items (e.g.
spread on bread, fat for cooking, cheese, bread and cereals) with improved fat quality on
total cholesterol, LDL-C and inflammatory markers in a double-blind randomised,
controlled trial. In total 115 moderately hypercholesterolemic non-statin treated adults
(25-70 y) were randomly assigned to an experimental diet group (Ex-diet group) or
control diet group (C-diet group) for eight weeks with commercially available food items
with different fatty acid composition (replacing SFA with mostly n-6 PUFA). In the Ex-
diet group, serum total cholesterol (P<0.001) and LDL-C (P<0.001) were reduced after
eight weeks, compared to the C-diet group. The difference in change between the two
groups at the end of the study was -9 % and -11 % in total cholesterol and LDL-C,
respectively. No difference in change in plasma levels of inflammatory markers (high
sensitive C reactive protein, interleukin-6, soluble TNF receptor 1 and interferon γ) was
observed between the groups. In conclusion, exchanging few regularly-consumed food
items with improved fat quality reduces total cholesterol, with no negative effect on
levels of inflammatory markers. This shows that an exchange of few commercially
available food items was easy and manageable and leads to clinically relevant cholesterol
reduction potentially affecting future CVD risk.
2
INTRODUCTION
Cardiovascular disease (CVD) still remains the major contributor to the global burden
(1)
of disease worldwide . Even though there has been substantial reduction in CVD mortality
over the last 30 years, new reports show an increase in acute myocardial infarction among the
younger population in Norway, and similar observations have been reported also from other
countries (2; 3). Elevated plasma low-density lipoprotein cholesterol (LDL-C) is an established
(4)
risk factor of CVD , and dietary fatty acids play a significant role in modulating plasma
LDL-C and thereby influencing the risk of CVD (5; 6; 7; 8). In particular there is strong evidence
that replacing saturated fatty acids (SFA) with polyunsaturated fatty acids (PUFA) will reduce
(9; 10)
the risk of CVD . However, controversy still exist about beneficial versus potential
harmful effects of n-6 PUFA since n-6 PUFA has been suggested to promote inflammation (11;
12)
.
Adherence to a healthy Nordic diet based on the Nordic nutrition recommendations
has previously been shown to have beneficial effect on blood lipids among subjects at risk of
(13; 14; 15)
CVD . However the extent of food changes needed to achieve these effects is less
explored. In order to increase compliance to dietary fat intake recommendations in the general
population it is important that one can achieve this with relatively small dietary changes,
leading to improved lipid profile. Few, if any double-blind randomised controlled trials have
investigated the effect of exchanging a combination of few commercially available food items
with similar food items with improved fat quality on blood lipids and inflammatory markers.
The aim of the present study was therefore to investigate the effect of exchanging regularly-
consumed commercially available key food items with similar food items with improved fat
quality (replacing SFA with PUFA, mostly n-6 fatty acids but also n-3 fatty acids) in the daily
diet for eight weeks in a double-blind randomised controlled trial, on serum total cholesterol,
LDL-C and inflammatory markers among non-statin treated healthy subjects with moderate
hypercholesterolemia.
EXPERIMENTAL METHODS
Subjects
Healthy adults aged 25-70 years (y) were recruited by advertisements in local
newspapers and local area community flyer postings. Inclusion criteria were high sensitive C
reactive protein (hs-CRP) < 10 mg/L, serum total cholesterol 5-7.8 mmol/L (for those
3
between 50-70 y), 5.0-6.9 mmol/L (for those between 30-49 y) and 5.0-6.1 mmol/L (for those
between 25-29 y). Since we wanted to include subjects with cholesterol values at the upper
range of normal plasma cholesterol values, and these ranges varies among age groups, we
used the age-specific cholesterol concentrations that were set by the commercial laboratory
we used for analysis to define the normal range. Furthermore all subjects should have LDL-C
≥ 3.5 mmol/L and fasting triglycerides ≤ 2.6 mmol/L, a stable body weight during the last
three months (± 5 %), BMI between 20-35 kg/m2, willing to eat all the study products daily
during the study and not taking fish oil or other dietary supplements the last three weeks
before study start and during the study. Stable use of medications against high blood pressure
was allowed. Exclusion criteria were use of lipid-lowering, anti-inflammatory (except drugs
against allergy), anti-diabetes or weight reduction drugs or planned weight reduction. In
addition, subjects with chronic illness such as kidney, liver and other endocrine diseases, and
coronary heart disease (CHD) during the last three months were excluded. Further exclusion
criteria were increased thyroid hormones (T3 > 6.5 pmol/L and T4 > 23 pmol/L) or thyroid-
stimulating hormone (TSH) (TSH > 4 mU/L), fasting blood glucose > 6.0 mmol/L, increased
alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) (> 3 x upper
reference value), hormonal treatment (except stable dose of thyroxin and oral contraceptives),
pregnancy and lactation, use of plant sterols within three weeks prior to study start and during
the intervention, and high alcohol intake (> 40 g/d).
All subjects were advised to maintain their usual lifestyle habits throughout the study, without
changing their physical activity habits, alcohol consumption, and dietary habits except the
inclusion of study products or other lifestyle factors besides adhering to the study products
during the study. All subjects were weighed during the study (every second week) and if there
was a change, a registered dietitian gave advice to maintain weight. The study was conducted
according to the guidelines laid down in the Declaration of Helsinki. All subjects gave written
informed consent, and the Regional Ethics Committee for Medical Research in South East
Norway approved the study. The study was registered at ClinicalTrials.gov
(ClinicalTrials.gov Identifier: NCT 01679496).
Study design
An eight-week double-blind randomised, controlled parallel designed trial was
conducted at the Oslo and Akershus University College of Applied Sciences and the
University of Oslo, Norway from July 2012 until April 2014. A telephone interview and a
screening visit were performed to check the eligibility criteria of the participants entering the
4
study. After screening 701 subjects, 115 were randomised, 108 received allocated
interventions, 100 completed the study and 99 were analysed. Subjects lost during follow-up
and the numbers of subjects included in the statistical analysis are outlined in the flowchart
(Figure 1). The 15 drop-outs did not differ to the subjects completing the study with respect to
age and lipid profile, however BMI was slightly higher among the subjects who dropped-out
(27.1 kg/m2 and 25.0 kg/m2, respectively) and more men than women dropped out (9 and 6,
respectively).
Before the baseline visit, all subjects performed a two week run-in period (starting one
to four weeks after the screening visit) in which all subjects had to include the control food
items in their daily diet. A registered dietician delivered the food items and gave them
instructions how to introduce the food items in their regular diet, and reminded them to eat at
least the minimum amount of each product (Table 1). At baseline, subjects were randomly
assigned and stratified by gender and age into one of two intervention groups (1:1 ratio),
where the control diet group (C-diet group) continued with the control food items and the
Experimental diet group (Ex-diet group) received experimental food items. The experimental
food items were the same type of food products as the control food items but with a different
fat composition (SFA replaced with mostly n-6 PUFA) (Table 1). Clinical and blood
laboratory assessments were performed at baseline and after eight weeks follow-up. Every
second week the participants received food items at the study centre and body weight was
recorded.
Study products
In the present study, we wanted to investigate the effect of consumption of
commercially available food items with different fat quality by exchanging regular food items
used habitually by the population in Norway. The control food items were chosen based on
Norwegian sales statistics and were among the most sold products within each food category
(Nielsen market trends 2011, Norway). The minimum amount of intake of each food item to
(16)
be consumed was based on data from the National nutrition survey in Norway .The
experimental food items were similar, commercially available food items (Mills DA, Oslo,
Norway) with an improved fatty acid composition (17). In these products, part of the saturated
fat was replaced by sunflower and rapeseed oils. Rapeseed oil is rich in n-6 PUFA, n-3 PUFA
and monounsaturated fatty acids (MUFA) and is a typical Nordic product. In combination
with sunflower oil, rapeseed oil is known to have an effect in cholesterol based on the
(17)
equation developed by Muller et al . The experimental food items were therefore
5
particularly rich in n-6 PUFA, but contained also n-3 PUFA and MUFA, thereby increasing
the total unsaturated fatty acid content. The study products included liquid margarine,
margarine-based spread, oil, liver pate, cheese, bread, bun or baguette, muesli cereals or
flakes, cream and crème fraîche and mayonnaise. The cereals and bread/baguettes/buns were
included as they also had an improved fatty acid composition (higher amount of n-6 PUFAs)
due to the content of added plant oil and ingredients (e.g. oat and sunflower seeds). In general,
the products were mainly used for breakfast, lunch and supper, and oils, butter/margarine,
cream and crème fraîche were used for cooking. An overview of the products, the SFA and
PUFA content and the daily minimum amount of each product that the participants were
instructed to consume is shown in Table 1. In addition, in the food items of the C-diet group,
the fibre content in the bread, buns and baguettes was 5.1 g/100 g and in the muesli cereals
and flakes, the fibre content was 7 g/100 g and 3 g/100 g, respectively. In the food items of
the Ex-diet group, the fibre content of the bread, bun and baguettes was 10 g /100 g and the
cereals were 18 g/100 g. In addition, the amount of E-glucan was estimated to be 1.83 g/day
in the Ex-diet group based on minimum intake of bread/baguettes/buns and cereals.
The fatty acid composition in the control food items and the experimental food items
differed particularly with respect to the PUFA and SFA content (Table 2). The total content of
PUFA was 5.1 g/d and 14.4 g/d, in the control food items and in the experimental food items
respectively, based on the minimum intake described in Table 1. In the control food items, the
total amount of n-6 fatty acids was 4.2 g/d, while the content in the experimental food was
12.9 g/d. The total content of SFA in the control food items and the experimental food items
were 19.2 g/d and 5.7 g/d, respectively.
Blinding and randomisation

The randomisation list was created by an external statistician (LINK Medical,
Norway), using four strata, female younger than 50 y, female 50 y or older, males younger
than 50 y and male 50 y or older, and a block size of 6. The SAS system (R) was used to
generate the list. The randomisation allocations, selected consecutively, were sent to the food
packaging personnel on demand, according to strata information of newly recruited subjects.
All food items were packed in boxes outside the study centre and only the people
packing the boxes knew who would be allocated to which group. Each box was labelled with
an ID number, and the closed boxes with the food items were delivered to the study centre. At
the study centre, the subjects received the ID-labelled boxes. Thus, the study was double-
6
blinded since neither the subjects nor the nutritionist who handed out the food boxes knew
which group the subjects were assigned to.
Dietary assessments and compliance

All subjects who received allocated intervention completed a four day pre-coded food
diary in the beginning and at the end of the intervention period. The diary used was originally
(18; 19; 20)
developed for use among Norwegian children and adolescents . The diary included
more than 270 food items grouped together according to the typical Norwegian meal pattern
(18; 19; 20)
. Each food group was supplemented with open-ended alternatives. Along with the
food diary, each participant received a validated photography-booklet that contained 13 series
of colored photographs, each with four different portion sizes ranging from small to large.
Food amounts were estimated in predefined household units (e.g. glasses, pieces or
tablespoons) or from photographs. We included specific pre-coded questions about the
control/experimental food items used in the intervention and the participants had to mark
when they used the control/experimental food items and how much was eaten.
The diaries were scanned using the Teleform program, version 6.0 (InfoShare
Solutions AS, Oslo, Norway). Daily intake of energy was computed using the food database
and software system KBS (KBS, version 7), developed at the Department of Nutrition,
University of Oslo(21). Compliance was assessed by using a checklist where all the products
consumed each week were registered. Each food group was ranked equally and the percent
(%) of intake based on the minimum amounts to be consumed per day were calculated during
the whole study period. A person was considered compliant to the protocol if the average of
the percent compliance of all food items eaten during the study was 80 %. Only three of the
subjects were not compliant to the protocol. The overall compliance in both groups was
similar (123 % and 112 %; Ex-diet group and C-diet group, respectively). The compliance to
the different food items was also quite similar in all food categories between the groups and
described in detail below; bread/baguettes/buns (114 % and 110 %), muesli cereals or flakes
(102 % and 78 %), cheese (107 % in both groups), liver pate (220 % and 197 %), butter-
based spread (104 % and 100 %), frying fat (104 % and 90 %), oil (95 % and 93 %) ,
mayonnaise (129 % and 108 %), cream (124 % and 110 %) and crème fraiche (131 % and
129 %), Ex-diet group and C-diet group, respectively.
7
Blood sampling and laboratory analysis
The day before blood sampling, the subjects were told to refrain from alcohol
consumption and vigorous physical activity. Venous blood samples were drawn after an
overnight fast (≥ 12 h). Serum was obtained from silica gel tubes (Becton Dickinson
Vacutainer Systems, Plymouth, UK) and kept at room temperature for at least 30 min, until
centrifugation (1500g, 15 min). Plasma was obtained from EDTA tubes (BD vacutainer,
Plymouth, UK), immediately placed on ice and centrifuged within 10 minutes (2000g, 4 °C,
15 min.). Fasting serum hs-CRP, total cholesterol, LDL-C, HDL-C, lipoprotein (a) [Lp (a)],
apolipoprotein (apo) A and apoB, triglycerides, glucose, insulin, ALAT and ASAT were
measured by standard methods at a routine laboratory (Fürst Medical Laboratory, Oslo,
Norway). Serum interleukin (IL)-6 was measured by Quantikine high sensitivity and soluble
Tumor Necrosis Factor (sTNF) receptor 1 by Quantikine ELISA kit from R&D Systems
(Minneapolis, MN, USA) according the manufacturer’s instructions.. Interferon (IFN) γ was
measured by high sensitivity ELISA from eBioscience (San Diego, CA, USA) according the
manufacturer’s instructions.
Total plasma fatty acid analysis

40 μL EDTA plasma was added 100μl internal standard (1,2-diheptadecanoyl-sn-
glycero-3-Phosphatidylcholine) and 800μl 3N Methanolic HCl, mixed and heated at 80 °C for
120 minutes. The resulting fatty acid methyl esters (FAME) were added 500μl hexane and
300μl 3M KOH in water. GC analysis of the hexane phase was performed directly from this
solution according to the method described below. Analyses were performed with a 7890N
GC with a split/splitless injector, a 7683B automatic liquid sampler, and flame ionization
detection (Agilent Technologies, Palo Alto, CA). Separations were performed with a SP-2380
(30 m × 0.20 mm i.d. × 0.25 μm film thickness) column from Supelco. The concentration of
the individual fatty acids was measured as Pg fatty acid/mL plasma (Vitas Analytical Service,
Oslo, Norway) and presented as % of total fatty acids.
Clinical assessment
Body weight (kg) was measured on a digital scale in light clothing without shoes.
Blood pressure was measured in a sitting position on the right arm after a 15 min rest. Two
measurements were performed with a minimum of 3 min interval, and the average value was
8
calculated. If the two values differed more than 5 mmHg, a third measurement was performed
and the average of the two most similar values was used.
Fatty acid analysis in food items

A duplicate of all test food items distributed to the participants was collected at three
time points during the study. All duplicates were kept at -20ºC until the end of the study, and
fatty acid composition was measured at a routine food analysis laboratory (Eurofins, Oslo,
Norway). The mean value of two measurements (start and end of study) was calculated.
Statistical analysis
Power calculations estimated that 180 subjects (including a 20 % drop-out rate) were
required for obtaining 80 % power with a type I error of 5 % to detect a difference between
the two groups of 8 % in LDL-C at the end of the study. Data are presented as mean ± SD if
normally distributed or median (25th to 75th percentile) if not normally distributed. Paired t-
test was used to assess change within groups and independent t-tests to compare changes
between groups when the data was normally distributed. Wilcoxon signed rank test was used
to assess change within groups and Mann Whitney U test was used to compare changes
between groups when the data was not normally distributed for continuous variables. Chi-
square test and Fischers’ exact test were used for categorical variables depending on the
expected cell frequencies. P< 0.05 was regarded as significant. IBM SPSS Statistics v 20
(Armonk, NY, USA) was used for statistical analysis.
RESULTS
Of 115 randomised subjects, 108 subjects received allocated intervention, and eight
subjects [five in the Ex-diet group and three in the C-diet group] were lost to follow-up. One
subject was excluded from the analysis in the Ex-diet group because of missing blood
analysis at end of study, providing 99 subjects for analysis (41 men and 58 women) (Figure
1). There were no significant differences between the C-diet group and the Ex-diet group at
baseline (Table 3).
9
Dietary changes
The dietary registrations showed no change in dietary intake within each group during
the intervention period, except for the fibre intake, which increased in the Ex-diet group from
first to last week (data not shown). The mean dietary intake during the intervention for each
group showed that 6.5 E% of SFA was replaced by PUFA in the Ex-diet group compared to
the C-diet group (Table 4). The E % from fat was similar in both group, however there was a
difference in the E % of protein and carbohydrates between the groups. The E % from protein
was slightly higher and the E % from carbohydrates was slightly lower in the Ex-diet group.
Total plasma fatty acids

After eight weeks intervention, the inclusion of Ex-diet food items caused a significant
change in total plasma fatty acids compared to C-diet group (Table 5). In particular the C12:0,
C14:0 and C16:0 fatty acids were significantly reduced in the Ex-diet group, compared to the
C-diet group (P<0.001 for all). In addition, the n-6 PUFAs, linoleic acid (LA, C18:2, n-6) and
arachidonic acid (AA, 20:4, n-6) significantly increased in the Ex-diet group compared to the
C-diet group (P<0.001 for LA and P=0.001 for AA). Oleic acid (C18:1, n-9) and
docosapentaenoic acid (DPA, C22:5, n-3) were significantly reduced in the Ex-diet group
compared to the C-diet (P<0.001 for both).
Effects on total cholesterol, LDL cholesterol

The inclusion of food items with improved fat quality in the Ex-diet group caused
significant lowering of serum total cholesterol (P<0.001), LDL-C (P<0.001), HDL-C
(P=0.006), and apoB (P<0.001) after eight weeks, compared to the C-diet group (Table 6).
The percentage change in total cholesterol and LDL-C within the Ex-diet group was -8 % and
-9 %, receptively, and the percentage change between the two groups was -9 % and -11 % in
total cholesterol and LDL-C, respectively. No significant changes in serum total cholesterol,
LDL-C, HDL-C, apoA and apoB were observed within the C-diet group. The intervention did
not affect plasma levels of lipoprotein (a) [Lp(a)].
Effects on inflammatory markers

Increased intake of n-6 PUFA in the diet had no negative effect on plasma levels of
the inflammatory markers hs-CRP, IL-6, sTNFR1 and IFNγ neither within nor between
groups (Table 6).
10
Effects on other cardiovascular risk factors
No changes in fasting serum glucose, insulin, HbA1c, and blood pressure were
observed either within group or between groups (Table 6). We did however detect a small but
significant increase in body weight of 0.4 kg within the C-diet group (P=0.006) leading to
significant difference between the two group (P=0.044) even though no effect on body weight
was seen in the Ex-group (P=0.885). In addition, we observed a slight increase in fasting
serum triglycerides in the C-diet group (P=0.004) leading to a significant difference in the
change in serum triglycerides between the groups (P=0.002).
DISCUSSION
In the present study, we show that by exchanging a combination of few commercially

available regularly-consumed key food items in the daily diet, by increasing mostly n-6
PUFA intake and reducing the SFA intake, serum total cholesterol and LDL-C are reduced by
9 % and 11 %, respectively compared to a control diet. Importantly, no detectable negative
effect was observed on circulating inflammatory markers.
In order to achieve long-lasting effects of dietary changes, the dietary changes have to
be easy to implement. In a recent study, it has been shown that replacing only food items
included to obtain a prudent breakfast based on Nordic foods for 12 weeks was not enough to
(22)
have any effect on plasma total cholesterol and LDL-C . In contrast, large reductions in
LDL-C were observed in two other studies where all foods were prepared and supplied
(15; 23)
throughout the study . In the SYSDIET study, which was a Nordic multi-centre study
focusing on a healthy Nordic diet based on the Nordic Nutrition recommendations; non-HDL
cholesterol was significantly reduced in the healthy Nordic diet group compared to the control
(13)
group . In the present study, the participants were supplied with few regular-consumed
commercially available key food items used in a Norwegian diet, primarily used for breakfast,
lunch and supper, in addition to oil, cream/ crème fraîche and butter/margarine for use in
dinner meals. No specific advices were given according to dinner choices. All the participants
in the present study had a high adherence to the intervention with 97 % of all subjects
complying to the intervention with respect to intake of test products. This suggests that the
amount of the products used in the present study is manageable to eat on a daily base and easy
to implement. These food exchanges should therefore be possible to achieve in the general
population and shows that it is possible to make beneficial replacements of dietary fat sources
11
in the diet without interfering too much with dietary habits. Our results are in line with the
(24; 25)
results observed in the DIVAS study , where successful implementation of a food-
exchange model achieved the dietary target intake for exchanging SFAs with MUFAs or n–6
PUFAs leading to reduced plasma total and LDL cholesterol in a free-living population (24; 25).
The E % from fat was similar in both group, however there was a difference in the E % of
protein and carbohydrates between the groups. The E % from protein was slightly higher and
the E % from carbohydrates was slightly lower in the Ex-diet group. The test products in the
Ex-diet group contained in gram more protein and carbohydrates, in particular the bread,
which contained twice the amount of protein in the Ex-diet group compared to the C-diet
group. This may partly explain the differences observed in protein intake given in E %. Since
total fat intake (E %) was similar, this will lead to an increase in E % from carbohydrates in
the C-diet group compared to Ex-diet group.
Even though there is strong evidence that replacing SFA with PUFA will reduce the
(9; 10) (26; 27)
risk of CHD , recent data have questioned this association and the intake of SFA
(28)
has increased in the general population . In the present study, we particularly focused on
increasing the intake of n-6 PUFA by substituting SFA in food items. The largest difference
in the two groups was an increased intake of PUFA by 6.5 E % with a subsequent 6.0 E %
reduction of SFA in the Ex-diet group compared to the C-diet group, respectively. These
changes were also reflected in the plasma fatty acid profile after the intervention. Previously it
has been estimated that a 10 % (~0.6 mmol/L) reduction in LDL-C is associated with a 27 %
(29)
reduction in CVD risk , suggesting that the observed LDL-C lowering effect of 11 %
achieved after consuming the Ex-diet in the present study is clinically relevant. If dietary
changes to lower LDL-C are successfully implemented earlier in life, the CVD risk reduction
(29)
is even greater than this estimate . In a recent drug trial (Improve-it study) including more
than 18 000 patients after acute coronary syndromes, where Ezetimibe treatment was added
on to statin treatment, the difference in plasma LDL-C between the statin and statin +
Ezetimibe group was 0.4 mmol/L (1.8 vs 1.4 mmol/L) leading to a significant lowered CVD
event rate (30). In the present dietary study, we find that by exchanging few regular-consumed
key food items with improved fatty acid composition, one can obtain similar reduction in
plasma cholesterol indicating that dietary changes may be as effective and easy to implement
as add-on statin therapy and may be an alternative for hypercholesterolemic subjects.
In the present study, we included food items rich in rapeseed oil and sunflower oil.
These oils are particularly rich in LA but contain also n-3 fatty acids and MUFA. Despite the
increased intake of n-6 PUFA, we did not observe any increase in plasma levels of well-
12
established inflammatory markers such as hs-CRP, sTNFR1, IFNγ or IL-6 in the Ex-diet
group, and our data therefore do not support evidence for any potential harmful effects of
(11; 12; 31)
increased intake of n-6 PUFA with regard to inflammation . Our data are in line with
two recent RCTs which did not observe any change in inflammatory markers when intake of
n-6 PUFA replaced SFA (25; 32). Moreover, a high intake of LA (8 % of total energy intake) are
(33)
inversely associated with total and CHD mortality and a recent systematic review and
meta-analysis of cohort studies showed that increased LA consumption were associated with
reduced risks of CHD events and CHD-related deaths, independently from other dietary
(34)
factors . Our data are thus in line with these cohort studies, showing a beneficial effect of
replacing SFA with PUFA and in particular LA in the diet.
In the present study, the major changes in plasma fatty acid composition were an
increase in LA and a decrease in palmitic acid. However, we also observed an increase in AA,
and a decrease in the plasma eicosapentaenoic acid (EPA, C20:5, n-3), DPA and
docosahexaenoic acid (DHA, C22:6, n-3) in the Ex-diet group (Table 5). The increased intake
of LA in the Ex-diet group may have increased the desaturase and elongase enzyme activity
leading to a higher synthesis of AA. Since LA and D-linolenic acid (ALA, C18:3, n-3) is
competing for the same enzymes, a possible explanation could be that increased synthesis of
AA from LA would lead to reduced synthesis of EPA from ALA subsequently leading to
reduced plasma levels of DPA and DHA. Indeed, the same observation with reduced DPA
(24)
levels as in the present study, were also reported in a recent paper by Weech et al who
developed a flexible food-exchange model to investigate the effects of substituting SFA with
n-6 PUFA on CVD risk thus supporting the finding in the present study.
The observed total cholesterol-lowering effect corresponded with the predicted total
(17)
cholesterol-lowering effect calculated using an equation developed by Muller H et al to
calculate the expected change in total serum cholesterol based on the changes of fatty acids in
products in the Ex-diet food group. By using this equation, an expected change in total serum
cholesterol relative to control was calculated to be 0.43 mmol/L. In the present study, we
observed a reduction of total cholesterol of 0.6 mmol/L in the Ex-diet group, and no change in
serum cholesterol in the C-diet group. This indicates that approximately 72 % of the change
in total cholesterol observed in the study may be due to the changes in fat intake. The food
items in the two groups contained different amount of fibre. The amount of β-glucan was
estimated to be 1.83 g/day in the Ex-diet group based on minimum intake of
bread/baguettes/buns and cereals. It has previously been shown that intake of oat β-glucan ≥ 3
g/d reduces LDL cholesterol and total cholesterol relative to control by 0.25 mmol/L and 0.30
13
mmol/L, respectively (35), which is in line with our results showing a possible additional fibre
effect on plasma cholesterol (0.17 mmol/L) considering the lower intake of oat β-glucan in
the present study (1.83 g/d). Unfortunately, we do not have information about the β-glucan
content in the control food items. However, by using a linear multivariable regression model,
changes in plasma fatty acid composition (16:0 and 18:2n-6) between baseline and end of the
intervention correlated with changes in LDL-C. The regression models were adjusted for
mean fibre intake during the intervention, however, adjustments for fibre intake did not
change the model, suggesting that the main effect was mediated by the change in fatty acid
composition (data not shown).
There was a significant increase in body weight in the control group during the
intervention leading to a significant difference in body weight change between the groups.
Because the only change in lipid profile in the control group during the intervention, was an
increase in plasma triglycerides, we performed linear regression analysis to test if the change
of serum triglycerides in the control group was due to body weight changes in this group.
However, we observed that the weight change in the C-group did not make a unique statistical
contribution to the prediction of the change in triglycerides (Beta=0.066 and P=0.653).
The strengths of our study are the randomised and double-blind study design to avoid
biases and obtain a high rate of compliance. Few, if any randomised dietary studies including
commercially available food items have been performed in a double-blind manner.
Furthermore, the fact that the daily minimum amounts of the food items to be consumed
during the intervention were based on intake data from the Norwegian dietary survey among
adults (16) suggests that consumption of these regular food items were manageable and seemed
to be easy to include in the daily diet. The food items in both intervention groups were
supplied to the participants to increase compliance. By using the equation developed by
(17)
Muller H et al to calculate the expected change in total serum cholesterol based on the
changes of fatty acids in products, the equation explained 72 % of the change in total
cholesterol, and the main effect therefore seems to be mediated by the change in fatty acid
composition and not fibre intake. However, since the equation was developed for margarine
products and the results from this equation has been extrapolated to other products, we cannot
rule out that the expected change in total cholesterol may have been underestimated since we
also included other food products in the present study which have been developed based on
this equation. Another limitation in the study was the dietary registrations of ingredients in
recipes of oils, butter, cream and crème fraîche in the food diaries, which may have resulted
in overestimation of the amounts of fat used for cooking in both groups. Finally, a limitation
14
was that we did not manage to include the sufficient number of subjects according to the
power calculations, however, post-hoc analyses showed that the number of subjects recruited
were in accordance with the observed 10 % change in LDL-C between the groups (n=47
subjects in each group).
In conclusion, increasing mostly the n-6 PUFA intake and reducing SFA intake by
exchanging few regular-consumed commercially available, key food items with similar food
items with improved fat quality, reduces serum total cholesterol and LDL-C in healthy adults
with moderate hypercholesterolemia by 9 % and 11 %, respectively with no negative effects
on plasma inflammatory markers. The present study shows that a daily exchange of few
commercially available food items with improved fatty acid composition lead to clinically
relevant cholesterol reduction potentially affecting future CVD risk.
ACKNOWLEDGEMENTS
We would like to thank Professor Emeritus Jan I Pedersen, Department of Nutrition,
University of Oslo, Norway, for valuable discussions and reading the manuscript, researcher
Jurate Saltyte-Benth at the Division of Health Science and Research and Psychiatry, Akershus
University Hospital, the University of Oslo, Norway, with help to the power calculations. In
addition, we would like to thank Marit Sandvik, Ingunn Narverud, Anne Marte Wetting
Johansen, all from the University of Oslo, Department for Nutrition, for technical help with
blood sampling, preparation of blood samples and food diary calculations. We would also like
to thank all the participants in the study.
FINANCIAL SUPPORT
This study was supported by Akershus University College of Applied Sciences, Norway, the
University of Oslo, Norway, the Throne-Holst Foundation for Nutrition Research, Oslo,
Norway, Nordplus Horizontal (NPHZ-2013/10151) and Mills DA, Oslo, Norway. Mills DA
was involved in the design of the study, but Ulven and Holven had the final responsibility of
the design. Mills DA was involved in conducting the trial and provided logistical support
during the trial. None of the employees at Mills DA were involved in the statistical analysis.
CONFLICT OF INTEREST
Mills DA partially funded the study and Vibeke H. Telle-Hansen, Amandine Lamglait, Kari
Thyholt, Marte G. Byfuglien and Linda Granlund are all employed at Mills DA. None of
15
them owns any stocks in the company. Ulven and Holven have received research funding
from TINE SA and Olympic seafood not related to the current project. No other conflicts of
interest.
AUTHORSHIP
SMU, LG, LFA and KBH designed research; LL, EE, IO, JJC, AJS, ER, NAS, KTO,
conducted research; VHTH, AL, MGB, KTH, LG provided essential material; SMU, LL and
KBH performed statistical analysis; SMU, LL, VHTH, LFA and KBH wrote paper; SMU and
KBH had the responsibility for final content. All authors read and approved the final
manuscript.
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19
TABLE 1. Minimum daily intake of food items and the PUFA and SFA content
Minimum
amount C-diet group PUFA SFA Ex-diet group PUFA SFA
g/day g/100g g/100g g/100g g/100g
15 g/day Butter based spread 10.4 34.8 Margarine based spread 24 15

7 g /day Butter 2.1 51.5 Liquid margarine 34.5 8.8
8 g/day Olive oil 6.6 14 Rapeseed and sunflower oil 23 8.9
5 g/day Liver pate# 5 2.3 Liver pate 9 2.1
30 g/day Cheese 0.7 17.8 Cheese** 6.8 1.5
* *
120 g/day Bread, bun or baguette 1.3/1.0/0.9 0.4/0.5/0.4 Bread, bun or baguette 3.4/3.7/3.8 1.0/1.1/1.1
* # *
30 g/day Muesli cereals or flakes 1.4/0.5 2.9/0.2 Muesli cereals or flakes 1.5/2.1 0.7/0.8
9 g/day Cream 0.6 13.5 Cream 5.2 1.7
4 g/day Mayonnaise 23.5 5.9 Mayonnaise light 16.5 3.6
9 g/day Crème fraiche 0.8 19.3 Crème fraîche** 5.1 4.2
C-diet group, control diet group; Ex-diet group, experimental diet group.
#
Two types of food items were delivered to the participants, and they could choose which of them they wanted to eat daily.
*The participants could include either of these products in their daily diet, in total 120g.
**The products have exchanged the dairy fat with vegetable oils (sunflower and rapeseed).
20
TABLE 2. Fatty acid composition in food items delivered to the subjects based on
minimum daily intake#
C-diet group Ex-diet group

(g/day) (g/day)
Total fat 45.7 39.5

Linoleic acid 18:2n-6 4.1 12.9
Sum n-6 fatty acids 4.2 12.9
Sum n-3 fatty acids 0.9 1.5
Ratio n-6/n-3 4.7 8.6
Myristic acid 14:0 2.9 0.2
Palmitic acid 16:0 9.2 2.7
Stearic acid 18:0 3.4 2.0
Sum SFA 19.2 5.7
Sum MUFA 16.9 17.0
Sum PUFA 5.1 14.4
Ratio PUFA/SFA 0.3 2.5
C-diet group, control diet group; Ex-diet group, experimental diet group.
#
The data are given as mean value of the two analysis of each food product and added
together with all food items in each food group based on the minimum daily intake of each
product.
21
TABLE 3. Baseline characteristics after randomisation to diets of those who
completed the study
n=52 n=47
Age, y 55.2 ± 9.8 53.6 ± 9.7
Female, n (%) 31 (59.6) 27 (57.4)
Smokers, n (%) 9 (17.6) 4 (8.5)
BMI 24.6 ± 3.0 25.4 ± 2.9
Systolic Blood pressure, mmHg 128 ± 14 128 ± 12
Diastolic Blood pressure, mmHg 76 ± 9 78 ± 8
Hypertensive medication, n (%) 2 (3.8) 3 (6.4)
Total cholesterol, mmol/L 6.6 ± 0.8 6.6 ± 0.8
LDL-C, mmol/L 4.1 ± 0.6 4.2 ± 0.6
HDL-C, mmol/L 1.7 ± 0.4 1.7 ± 0.5
ApoA-1, g/L 1.6 ± 0.2 1.6 ± 0.2
ApoB, g/L 1.3 ± 0.2 1.3 ± 0.2
Triglycerides, mmol/L 1.20 (0.8 - 1.5) 1.31 (0.9 - 1.6)
Lp(a), pmol/L 181 (81 - 459) 116 (63 - 560)
Glucose, mmol/L 5.2 (4.9 - 5.4) 5.3 (5.0 - 5.6)
Insulin, pmol/L 43 (31 - 60) 52 (36 - 79)
HbA1c, % 5.4 ± 0.2 5.3 ± 0.2
hs-CRP, mg/L 1.00 (0.5 - 2.0) 1.20 (0.6 - 2.0)
Creatinine, mmol/L 75 ± 12 74 ± 12
ASAT, U/L 22 (18 - 26) 20 (16 - 28)
ALAT, U/L 24 (21 - 28) 23 (19 - 34)
C-diet group, control diet group; Ex-diet group, experimental diet group; Lp(a),
lipoprotein (a); hs-CRP, high-sensitive C-reactive protein; ASAT, aspartate aminotransferase;
ALAT, alanine aminotransferase. Data are given as mean ± SD, numbers (percentages),
or median (25th–75th percentile).
apoB; n=46 (Ex-diet group); hs-CRP; n=51 (C-diet group).
22
TABLE 4. Dietary intake during the intervention
C-diet group Ex-diet group Difference P
n=52 n=47
Energy, KJ 11263 ± 2673 11460 ± 2524 197 0.707

Protein, E % 15.0 ± 1.5 16.5 ± 2.0 1.5 <0.001
Fat, E % 42.8 ± 4.0 42.9 ± 3.3 0.1 0.901
SFA, E % 18.0 ± 2.1 11.5 ± 1.6 -6.5 <0.001
MUFA, E % 15.4 ± 1.9 15.7 ± 1.4 -0.3 0.396
PUFA, E % 5.6 ± 0.8 12.0 ± 1.6 6.4 <0.001
Carbohydrates, E % 36.6 ± 4.3 34.2 ± 3.8 -2.4 0.003
Fiber, E % 1.8 ± 0.3 2.6 ± 0.4 0.8 <0.001
Fiber, g 25.7 ± 7.6 37.2 ± 8.2 11.5 <0.001
Sugar, E % 7.4 ± 2.0 6.7 ± 2.8 0.7 0.160
Alcohol, E %# 3.5 (1.4-4.6) 3.4 (1.2-5.3) 0.1 0.905
Data are given as mean ± SD, average of two dietary registrations; or median (25th–75th
percentile). The bold values indicate significant P values.
Independent t-test was used for normally distributed variables.
#
Mann-Whitney U test was used for not normally distributed variables.
23
TABLE 5. Proportion of plasma fatty acids (% of total fatty acids) at baseline and end of the intervention

Baseline 8 weeks Baseline 8 weeks
1
n=52 n=52 P n=47 n=47 P2 P3
SFA
12:0#£ 0.07 ± 0.03 0.07 ± 0.03 0.518 0.08 ± 0.06 0.06 ± 0.03 0.001 0.015
14:0# 0.89 ± 0.26 0.94 ± 0.30 0.193 0.97 ± 0.27 0.69 ± 0.17 <0.001 <0.001
16:0 20.0 ± 1.42 20.2 ± 1.30 0.194 20.0 ± 1.28 18.6 ± 1.09 <0.001 <0.001
18:0 6.5 ± 0.46 6.5 ± 0.52 0.415 6.6 ± 0.52 6.6 ± 0.55 0.636 0.916
MUFA
18:1n-9 19.0 ± 2.09 19.3 ± 2.21 0.076 19.1 ± 2.34 18.3 ± 2.73 0.002 <0.001
n-6 PUFA
18:2n-6 27.1 ± 3.00 26.5 ± 3.16 0.013 27.5 ± 2.93 30. 4 ± 2.84 <0.001 <0.001
18:3n-6 0.35 ± 0.12 0.37 ± 0.15 0.189 0.37 ± 0.15 0.37 ± 0.13 0.886 0.321
20:3n-6 1.21 ± 0.28 1.26 ± 0.28 0.187 1.33 ± 0.26 1.30 ± 0.30 0.286 0.092
20:4n-6 5.16 ± 0.72 5.18 ± 0.73 0.779 5.18 ± 0.91 5.57 ± 1.12 <0.001 0.001
n-3 PUFA
18:3n-3 0.64 ± 0.14 0.70 ± 0.15 0.010 0.66 ± 0.17 0.69 ± 0.21 0.158 0.560
20:5n-3 1.58 ± 0.71 1.59 ± 0.77 0.932 1.31 ± 0.55 1.16 ± 0.57 0.021 0.113
22:5n-3 0.59 ± 0.12 0.59 ± 0.11 0.905 0.57 ± 0.10 0.52 ± 0.09 <0.001 <0.001
22:6n-3 2.86 ± 0.79 2.82 ± 0.82 0.504 2.68 ± 0.67 2.53 ± 0.63 0.035 0.243
C-diet group, control diet group; Ex-diet group, experimental diet group. Data are given as mean ± SD.
P1: baseline v. end of study C-diet group; paired t-test.
P2: baseline vs end of study Ex-diet group; paired t-test.
P3: between group change; independent t-test.
#
Wilcoxon`s signed-rank test was used as the data were not normally distributed.
£
n=41 in the Ex-diet group and n=48 in C-diet group due to samples below detection limit.
24
TABLE 6. Clinical and biochemical values at baseline and at the end of the study
Baseline 8 weeks Baseline 8 weeks
n=52 n=52 P1 n=47 n=47 P2 P3
Body weight, kg 74.1 ± 13.1 74.5 ± 13.2 0.006 75.7 ± 12.7 75.7 ± 12.7 0.885 0.044
Total cholesterol, mmol/L 6.6 ± 0.8 6.6 ± 0.8 0.627 6.6 ± 0.8 6.0 ± 0.7 <0.001 <0.001
LDL-C, mmol/L 4.1 ± 0.6 4.2 ± 0.6 0.517 4.2 ± 0.6 3.8 ± 0.5 <0.001 <0.001
HDL-C, mmol/L 1.7 ± 0.4 1.7 ± 0.5 0.683 1.7 ± 0.5 1.6 ± 0.4 <0.001 0.006
ApoA-1, g/L 1.6 ± 0.2 1.6 ± 0.2 0.922 1.6 ± 0.2 1.5 ± 0.2 0.005 0.079
ApoB, g/L 1.3 ± 0.2 1.3 ± 0.2 0.358 1.3 ± 0.2 1.2 ± 0.2 <0.001 <0.001
Triglycerides, mmol/L 1.20 (0.92 - 1.48) 1.24 (1.01 - 1.60) 0.004 1.31 (0.87-1.56) 1.19 (0.77 - 1.46) 0.151 0.002
Lp(a), pmol/L 181 (81 - 459) 188 (85 - 428) 0.548 116 (63 - 560) 110 (66 - 516) 0.655 0.949
Glucose, mmol/L 5.2 (4.9-5.4) 5.1 (4.9 - 5.4) 0.202 5.3 (5.0 - 5.6) 5.3 (5.0 - 5.6) 1.00 0.366
Insulin, pmol/L 43 (31 -60) 43 (33 - 72) 0.397 52 (36 - 79) 58 (42 - 73) 0.676 0.961
HbA1c, % 5.3 (5.2 - 5.5) 5.4 (5.2 - 5.6) 0.377 5.3 (5.2 - 5.5) 5.3 (5.2 - 5.4) 0.839 0.724
hs-CRP, mg/L 1.0 (0.5 - 2.0) 1.0 (0.6 - 1.6) 0.544 1.2 (0.6 - 2.0) 0.90 (0.5 - 1.9) 0.315 0.241
IL-6, pg/mL 1.05 ± 0.77 1.09 ± 1.02 0.682 1.12 ± 1.12 1.09 ± 0.81 0.732 0.930
sTNFR1, pg/ml 999.18 ± 184.19 1013.66 ±188.23 0.272 1009.97 ±185.97 1019.94 ± 189.72 0.463 0.810
#
IFNg pg/ml 5.71 ± 10.1 5.22 ± 8.9 0.272 9.55 ± 10.4 9.71 ± 9.1 0.946 0.604
Systolic BP, mmHg 128 ± 14 125 ± 12 0.117 128 ± 12 125 ± 12 0.148 0.927
Diastolic BP, mmHg 76 ± 9 76 ± 8 0.595 78 ± 8 79 ± 9 0.323 0.304
C-diet group, control diet group; Ex-diet group, experimental diet group; Lp(a), lipoprotein (a); hs-CRP, high-sensitive C-reactive protein;
sTNFR1, soluble TNF receptor 1; IFN, interferon; BP, blood pressure. Data are given as mean ± SD, or median (25th-75th percentile). The bold
values indicate significant P values.
P1: baseline v. end of study C-diet group; paired t-test or Wilcoxon`s signed-rank test.
P2: baseline v. end of study Ex-diet group; paired t-test or Wilcoxon`s signed-rank test.
P3: between group changes; independent t-test or Mann-Whitney U test.
#n=31 in C-diet group and n=25 in Ex-diet group.
25
FIGURE LEGENDS
Figure 1. Flow chart of the participants. C-group, control diet group; Ex-group, experimental
diet group.
26
Figure 1
Enrolment Assessed for eligibility (n=701)
Excluded (n=586)
Not meeting inclusion criteria
(n=309)
Declined to participate (n=229)
Other reasons (n=48)
Randomised (n=115)
Allocation
Ex-group: Allocated to intervention (n=56) C-group: Allocated to intervention (n=59)
Received allocated intervention (n=53) Received allocated intervention (n=55)
Did not receive allocated intervention Did not receive allocated intervention
(n=3) Should not have been included (n=3) (n=4)
Should not have been included (n=1)
Discontinued during run in period (n=3)
Follow-up
Lost to follow-up (n=5) Lost to follow-up (n= 3)
Discontinued intervention (n=5) Discontinued intervention (n=3)
No reason (n=2), wanted weight reduction No reason (n=1), took too much time
(n=1), did not like the food (n=1), took too (n=2)
much time (n=1)
Analysis
Analysed (n=47) Analysed (n=52)
Excluded from analysis (n=1 ) Excluded from analysis (n=0)
Analysis at end of study not performed
27
III
Leder et al. Genes & Nutrition (2016) 11:3
DOI 10.1186/s12263-016-0521-4
RESEARCH Open Access
Effects of a healthy Nordic diet on gene

expression changes in peripheral blood
mononuclear cells in response to an oral
glucose tolerance test in subjects with
metabolic syndrome: a SYSDIET sub-study
Lena Leder1, Marjukka Kolehmainen2, Ingunn Narverud1, Ingrid Dahlman3, Mari C. W. Myhrstad4,
Vanessa D. de Mello2, Jussi Paananen2, Carsten Carlberg5, Ursula Schwab2,6, Karl-Heinz Herzig7,8,
Lieselotte Cloetens9, Matilda Ulmius Storm9, Janne Hukkanen10,11,12, Markku J. Savolainen10,11,12,
Fredrik Rosqvist13, Kjeld Hermansen14, Lars O. Dragsted15, Ingibjörg Gunnarsdottir16, Inga Thorsdottir16,
Ulf Risérus13, Björn Åkesson9,17, Magne Thoresen18, Peter Arner3, Kaisa S. Poutanen2, Matti Uusitupa2,19,
Kirsten B. Holven1,20 and Stine M. Ulven1,4*
Abstract
Background: Diet has a great impact on the risk of developing features of metabolic syndrome (MetS), type 2
diabetes mellitus (T2DM), and cardiovascular diseases (CVD). We evaluated whether a long-term healthy Nordic diet
(ND) can modify the expression of inflammation and lipid metabolism-related genes in peripheral blood
mononuclear cells (PBMCs) during a 2-h oral glucose tolerance test (OGTT) in individuals with MetS.
Methods: A Nordic multicenter randomized dietary study included subjects (n = 213) with MetS, randomized to a ND
group or a control diet (CD) group applying an isocaloric study protocol. In this sub-study, we included subjects (n = 89)
from three Nordic centers: Kuopio (n = 26), Lund (n = 30), and Oulu (n = 33) with a maximum weight change of ±4 kg,
high-sensitivity C-reactive protein concentration ≤10 mg L−1, and baseline body mass index <39 kg m−2. PBMCs were
isolated, and the mRNA gene expression analysis was measured by quantitative real-time polymerase chain reaction
(qPCR). We analyzed the mRNA expression changes of 44 genes before and after a 2hOGTT at the beginning and the
end of the intervention.
Results: The healthy ND significantly down-regulated the expression of toll-like receptor 4 (TLR4), interleukin 18 (IL18), and
thrombospondin receptor (CD36) mRNA transcripts and significantly up-regulated the expression of peroxisome
proliferator-activated receptor delta (PPARD) mRNA transcript after the 2hOGTT compared to the CD.
Conclusions: A healthy ND is able to modify the gene expression in PBMCs after a 2hOGTT. However, more studies are
needed to clarify the biological and clinical relevance of these findings.
Keywords: mRNA gene expression, Metabolic syndrome, PBMCs, Nordic diet, OGTT
* Correspondence: smulven@medisin.uio.no
1
Department of Nutrition, Institute of Basic Medical Sciences, University of
Oslo, P.O. Box 1046, Blindern 0317 Oslo, Norway
4
Department of Health, Nutrition and Management, Faculty of Health
Sciences, Oslo and Akershus University College of Applied Sciences, Oslo,
Norway
Full list of author information is available at the end of the article
© 2016 Leder et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Leder et al. Genes & Nutrition (2016) 11:3 Page 2 of 13
Background (Bouwens et al. 2010; Cruz-Teno et al. 2012; van Dijk

The metabolic syndrome (MetS) is a cluster of risk fac- et al. 2012b; Myhrstad et al. 2011) modulate the gene ex-
tors increasing the risk of type 2 diabetes mellitus pression of inflammatory genes in leucocytes and mono-
(T2DM) and cardiovascular diseases (CVD) (Alberti nuclear cells. PBMCs also reflect the immune
et al. 2009). Obesity, insulin resistance (IR), and T2DM component of the white adipose tissue transcriptome
are associated with chronic low-grade inflammation after OGTT and after oral lipid tolerance test (O’Grada
(Bastard et al. 2006; Wellen and Hotamisligil 2005), et al. 2014), and thus, changes in PBMC gene expression
which plays a pivotal role in all phases of atherosclerosis may act as biomarkers of metabolic health not only in
(Libby et al. 2009). Diet has a great impact on the risk of the fasting state but also in the postprandial state
MetS, T2DM, and CVD (Alberti et al. 2009; Mozaffarian (O’Grada et al. 2014).
et al. 2011). Thus, it is crucial to understand the role of Changes in glucose and lipid homeostasis by acute
diet and dietary compounds on inflammation in the de- challenge tests are linked to inflammation. It has been
velopment of these diseases. shown in dietary intervention studies that the quality of
A healthy Nordic diet (ND) has been shown to im- diet affects OGTT response and improves insulin sensi-
prove lipid profile among hyper-cholesterolemic subjects tivity and glucose tolerance in individuals with MetS
(Adamsson et al. 2011). The Systems Biology in Con- (Laaksonen et al. 2005; Paniagua et al. 2007). However,
trolled Dietary Interventions and Cohort Studies (SYS- no previous study has examined the long-term effect of
DIET) study was a multicenter randomized dietary study a dietary intervention on the OGTT response using
in individuals with features of MetS. A healthy ND with PBMCs and gene expression analysis.
whole-grain products, berries, fruits and vegetables, The main aim of this sub-population of the SYSDIET
rapeseed oil, three fish meals per week, and low-fat dairy study was to investigate if a long-term (18–24 weeks)
products was compared to an average Nordic diet served healthy ND could modify the expression of inflamma-
as control diet (CD) (Uusitupa et al. 2013). In the SYS- tion and lipid metabolism-related genes in PBMCs dur-
DIET study, we showed that an isocaloric healthy ND ing 2hOGTT in individuals with MetS.
improved the lipid profile, low-grade inflammation, and
ambulatory blood pressure among subjects with MetS Results
(Uusitupa et al. 2013; Brader et al. 2014). No changes in Characteristics of the subjects
glucose metabolism were observed since it may be diffi- At baseline, no differences were observed between the
cult to improve glucose metabolism in established MetS CD and ND groups related to age, BMI, serum lipids,
without attendant weight loss and very distinct changes glucose, insulin, circulating inflammation markers, lipid-
in the diet (Uusitupa et al. 2013). lowering drugs, antihypertensive drugs, smoking, and
The peripheral blood mononuclear cells (PBMCs) in- MetS (Table 1). The change in glucose, insulin, triglycer-
clude monocytes and lymphocytes, which are cells cen- ides, and free fatty acids from 0h (fasting) to 2h (after
tral in inflammation. These cells circulate in the body OGTT) was not significantly different between the CD
and are exposed to nutrients, bioactive food compo- and ND groups (P = 0.330, P = 0.845, P = 0.196, and P =
nents, and metabolic tissues. Alterations in gene expres- 0.681, respectively) (Fig. 1).
sion levels in these cells may therefore reflect systemic
health (Afman et al. 2014). It has been shown that long- Dietary data
term dietary intervention studies change the gene ex- The dietary intake of this sub-population is shown in
pression of inflammatory genes and genes involved in Table 2. The results are in line with the original analysis
lipid metabolism (Myhrstad et al. 2014; van Dijk et al. with the whole SYSDIET study population (Uusitupa
2012a; De Mello et al. 2009; Bouwens et al. 2009), sug- et al. 2013). The intake of polyunsaturated fatty acids
gesting that PMBCs are a good model system identifying was higher and of saturated fatty acids lower in the ND
early risk markers (Visvikis-Siest et al. 2007) and are compared to the CD group. Further, α-linolenic acid,
sensitive to dietary changes. fiber, β-carotene, vitamin C, vitamin E, folate, potassium,
Stress responses can be more informative than static and magnesium intake were higher in the ND versus the
homeostasis on nutrition-related health. An acute glu- CD group.
cose load of a 2-h oral glucose tolerance test (OGTT) is
such a stress response, monitors the ability of the body Changes in 2hOGTT gene expression response at baseline
to respond to glucose intake, and is primarily used for Since it is well known that glucose uptake inhibits pyru-
addressing the degree of glucose tolerance and insulin vate dehydrogenase kinase, isozyme 4 (PDK4), PDK4
resistance (van Ommen et al. 2009). Several studies have mRNA expression in PBMCs was used as a positive con-
shown that an OGTT (Choi et al. 2012; Kempf et al. trol for the 2hOGTT response. A significant down-
2007; Aljada et al. 2006) as well as fat challenge tests regulation of PDK4 mRNA expression from 0h (fasting)
Table 1 Baseline characteristics of the participants

Number CD Number ND P
Sex (female) 40 25 (63 %) 49 34 (69 %) 0.51
Age (year) 40 55.8 ± 7.8 49 54.4 ± 8.3 0.43
BMI (kg m−2) 40 31.9 ± 2.7 49 31.8 ± 3.1 0.90
Total cholesterol (mM) 40 5.3 ± 1.0 49 5.3 ± 1.0 0.91
LDL cholesterol (mM) 40 3.3 ± 0.9 49 3.2 ± 0.9 0.93
HDL cholesterol (mM) 40 1.3 ± 0.5 49 1.4 ± 0.3 0.51
Fasting triglycerides (mM) 40 1.5 ± 0.5 49 1.5 ± 0.7 0.84
Fasting glucose (mM) 40 5.8 ± 0.6 49 5.8 ± 0.6 0.46
Fasting insulin (pM) 40 59.5 (47.0–82.3) 49 55.0 (41.0–75.5) 0.43
IL1Ra (ng L−1) 40 308.7 (233.4–465.6) 49 203.8 (220.0–502.0) 0.96
IL1β (ng L−1) 39 0.12 (0.12–0.21) 49 0.12 (0.12–0.14) 0.44
−1
IL6 (ng L ) 40 1.3 (1.1–1.8) 49 1.3 (1.0–1.9) 0.76
IL10 (ng L−1) 39 0.9 (0.8–1.5) 49 0.8 (0.8–1.5) 0.32
−1
sTNFRII (ng L ) 40 1899.6 ± 415.4 49 1954.5 ± 461.1 0.56
hs-CRP (mg L−1) 40 1.5 (0.9–3.7) 49 1.5 (0.8–2.9) 0.68
HMW adiponectin (μg L−1) 40 3.6 (2.2–6.7) 49 4.0 (2.8–6.5) 0.36
Lipid-lowering drugs 40 13 (33 %) 49 12 (25 %) 0.48
Antihypertensive drugs 40 20 (50 %) 49 31 (63 %) 0.28
Smoking 40 6 (15 %) 49 4 (8 %) 0.34
Metabolic syndrome 40 34 (85 %) 49 42 (86 %) 1.00
Values are expressed as means ± SDs, medians (25th–75th percentiles), or numbers (%)
CD control diet, ND healthy Nordic diet, BMI body mass index, LDL low density lipoprotein, HDL high-density lipoprotein, IL1Ra interleukin-1 receptor antagonist,
IL1β interleukin-1 beta, IL6 interleukin 6, IL10 interleukin 10, sTNFRII tumor necrosis factor receptor 2, hs-CRP high-sensitivity C-reactive protein, HMW adiponectin
human high molecular weight adiponectin
to 2h (after OGTT) was observed in the whole study Discussion

population (q < 0.0001) (Additional file 1). Transcript In the present study, we found that the healthy ND
levels of several inflammatory and lipid metabolism- modulated the mRNA levels of TLR4, IL18, CD36, and
related genes were regulated after the OGTT (up- PPARD differently after the OGTT compared to the CD.
regulation with fold changes between 1.11 and 1.36 We also showed that several genes related to inflamma-
and down-regulation with fold changes between 0.72 tion and lipid metabolism were significantly modulated
and 0.93 (q < 0.05)) (Additional file 1). by an OGTT in PBMCs of subjects with MetS at
baseline.
Immune response and lipid metabolism are closely
Changes in 2hOGTT gene expression response after linked in metabolic diseases, and alterations in these re-
dietary intervention sponses after a food challenge may play an important
To study if the healthy ND could change the 2hOGTT role in the prevention and early detection of diseases
gene expression response in PBMCs, we conducted a (van Ommen et al. 2009). The healthy ND down-
linear multiple regression analysis adjusting for changes regulated the expression of TLR4 compared to the CD
at baseline and differences in the study centers. Among group after the OGTT. TLR4 is involved in the pro-
the 44 genes, the healthy ND significantly down- inflammatory response by regulating nuclear factor
regulated the expressions of toll-like receptor 4 (TLR4) kappa B (NFκB) activity (Doyle and O’Neill 2006) and is
(β = −0.33, q = 0.042), interleukin 18 (IL18) (β = −0.73, q a key regulator of immune response. Previously, it has
= 0.042), and CD36 (β = −0.23, q = 0.042) compared to been reported that an increased TLR4 mRNA expression
the CD after the OGTT (Table 3 and Fig. 2a–c). In con- in monocytes in individuals with MetS compared to
trast, a healthy ND significantly up-regulated the expres- healthy controls (Hardy et al. 2013) and down-regulation
sion of peroxisome proliferator-activated receptor delta of the TLR4 mRNA expression by weight loss are associ-
(PPARD) (β = 0.21, q = 0.042) compared to the CD after ated with improvement of insulin sensitivity in the indi-
the OGTT (Table 3 and Fig. 2d). viduals with MetS (De Mello et al. 2008). This would
Fig. 1 Changes in glucose, insulin, triglycerides, and free fatty acids

from 0h to 2hOGTT after intervention. The effect of the healthy ND
compared to the CD on changes in glucose (a), insulin (b),
triglycerides (c), and free fatty acids (d) from 0 h to 2hOGTT after
intervention. The effect of the independent variable study group is
adjusted for changes in glucose, insulin, triglycerides, or free fatty
acids at baseline, study centers, gender, age (log10 transformed),
and body weight at the end of the study. Box plots show the
medians with 25th and 75th percentiles. Whiskers express the
1.5 × interquartile range
indicate that the ND compared to the CD may be less

inflammatory and may lead to improvement of insulin
sensitivity via TLR4 down-regulation in the present
study. We were, however, not able to show any improve-
ment in insulin sensitivity or fasting glucose or insulin
concentrations in the main SYSDIET trial (Uusitupa
et al. 2013), suggesting that the number of subjects in
the original study was too low to see an effect. Anyhow,
it may be speculated that the use of PBMC gene expres-
sion analysis could serve as a more sensitive model sys-
tem than measurement of circulating markers of glucose
metabolism.
Interestingly, we observed that the healthy ND re-
duced the IL18 mRNA expression after the OGTT com-
pared to CD. IL18 is a pro-inflammatory cytokine shown
to be a strong predictor of cardiovascular events in eld-
erly men with MetS, and the effect is stronger with ele-
vated fasting glucose (Troseid et al. 2009). The mRNA
expression of IL18 is also increased in obese individuals,
and this increase is correlated with insulin resistance
(Ahmad et al. 2013). Thus, the down-regulation of IL18
mRNA after the OGTT by healthy ND in the present
study may indicate the impact of the ND on disease de-
velopment among the individuals with MetS.
CD36 is a scavenger receptor involved in lipid uptake and
foam cell formation in macrophages (Silverstein 2009).
Intracellular lipids taken up by CD36 activate TLR4, which
generate pro-inflammatory signals by activating NFκB
(Fessler et al. 2009). A blockage of TLR4 and CD36 in hu-
man macrophages reduced secretion of IL1β, IL6, and IL8
and the subsequent foam cell formation (Chavez-Sanchez
et al. 2014). We found a down-regulation of CD36 mRNA
transcript level after the OGTT in the healthy ND group
compared to the CD group. We could speculate that the
beneficial effects of ND may be executed also via reduction
of the foam cell formation and inflammation indicated by
down-regulation of the CD36 mRNA expression in the
postprandial state. In contrast, the PPARD gene transcript
was increased after the OGTT challenge in the ND group
compared to the CD group. PPARD is expressed in several
tissues in the body, including macrophages. The expression
of PPARD genes regulates lipid metabolism and glucose
homeostasis, increases fatty acid oxidation, and decreases
inflammation as well as platelet activation (Monsalve et al.
Table 2 Dietary intake in the CD and healthy ND group at baseline and end of intervention
CD (n = 40) ND (n = 47) Regression coefficient β (95 % CI)
Baseline End Baseline End Unadjusted Adjusted Pa
Energy, kJ 8074 ± 2173 8301 ± 1542 8077 ± 1757 8537 ± 1791 235 (−484 to 954) 297 (−218 to 813) 0.254
Protein, E% 17.2 ± 2.4 17.0 ± 2.3 16.7 ± 2.7 16.8 ± 2.3 −0.1 (−1.1 to 0.9) 0.1 (−0.8 to 1.0) 0.772
Carbohydrate, E% 46.4 ± 2.3 43.2 ± 7.0 45.1 ± 5.8 45.5 ± 5.2 2.3 (0.6 to 4.5) 2.5 (0.5 to 4.4) 0.013
Sucrose, g 40.1 ± 17.3 34.8 ± 15.4 41.2 ± 15.2 37.6 ± 15.6 2.8 (−3.9 to 9.4) 1.8 (−4.0 to 7.6) 0.541
Fat, E% 32.1 ± 6.2 35.7 ± 5.1 32.5 ± 7.3 32.9 ± 5.1 −2.8 (−4.9 to − 0.6) −2.9 (−5.0 to − 0.9) 0.005
SFA, E% 13.0 ± 3.2 15.3 ± 2.9 13.2 ± 3.5 10.8 ± 2.3 −4.5 (−5.6 to − 3.4) −4.8 (−5.8 to − 3.7) <0.001
MUFA, E% 11.5 ± 2.3 12.8 ± 2.0 11.6 ± 2.8 12.7 ± 2.2 −0.1 (−1.0 to 0.8) −0.1 (−0.9 to 0.7) 0.864
PUFA, E% 4.7 ± 1.6 4.5 ± 1.1 4.9 ± 1.4 6.9 ± 1.5 2.5 (1.9 to 3.0) 2.5 (1.9 to 3.0) <0.001
Linoleic acid, g 7.3 ± 2.4 8.2 ± 2.8 8.0 ± 3.0 8.7 ± 3.9 0.5 (−0.9 to 2.0) 0.5 (−0.9 to 1.9) 0.517
α-Linolenic acid, g 1.2 ± 0.6 1.4 ± 0.7 1.2 ± 0.6 2.0 ± 1.4 0.7 (0.2 to 1.2) 0.7 (0.4 to 1.1) <0.001
Fiber, g 21.2 ± 6.5 16.4 ± 4.9 21.7 ± 7.2 36 ± 10.1 19.6 (16.2 to 23.1) 19.5 (16.2 to 22.8) <0.001
Cholesterol, mg 268 ± 127 283 ± 118 254 ± 102 214 ± 74 −69 (−111 to − 28) −58 (−90 to − 25) 0.001
Salt, g 7.2 ± 2.9 7.0 ± 2.0 7.1 ± 2.2 6.5 ± 2.4 −0.4 (−1.4 to 0.5) −0.3 (−1.1 to 0.5) 0.478
β-Carotene, mg 2561 ± 2090 1733 ± 1173 2449 ± 1891 2987 ± 1857 1254 (578 to 1930) 1213 (543 to 1884) 0.001
Vitamin C, mg 120 ± 100 66 ± 32 112 ± 59 138 ± 50 72 (54 to 90) 72 (54 to 91) <0.001
Vitamin E, mg 8.9 ± 3.4 8.2 ± 2.2 9.5 ± 3.4 13.6 ± 3.0 5.3 (4.2 to 6.5) 5.2 (4.1 to 6.2) <0.001
Folate, mg 251 ± 76 226 ± 63 271 ± 83 343 ± 172 117 (60 to 174) 104 (51 to 158) <0.001
Sodium, mg 2855 ± 1151 2722 ± 78 2813 ± 878 2654 ± 980 −68 (−451 to 314) −27 (−337 to 284) 0.865
Potassium, mg 3711 ± 1175 3276 ± 933 3626 ± 975 4017 ± 910 742 (348 to 1135) 767 (482 to 1053) <0.001
Magnesium, mg 359 ± 111 309 ± 82 370 ± 101 421 ± 101 112 (73 to 152) 106 (79 to 132) <0.001
Calcium, mg 1006 ± 411 951 ± 383 967 ± 370 997 ± 310 46 (−102 to 193) 60 (−43 to 163) 0.250
Alcohol, E% 2.1 ± 2.8 3.0 ± 3.3 3.0 ± 4.1 1.8 ± 2.9 −1.2 (−2.5 to 0.1) −1.2 (−2.3 to − 0.1) 0.036
Values are means ± SDs
CD control diet, ND healthy Nordic diet, E % percentage of energy, SFA saturated fatty acids, MUFA monounsaturated fatty acids, PUFA polyunsaturated fatty acids
a
With linear multivariable regression analysis, the effect of the independent variable “study group” adjusted for dietary data at baseline, study center, gender,
log10-transformed age, and body weight at the end of the intervention was assessed. The regression coefficient expresses the mean difference between the
groups, unadjusted and adjusted. The CD and the ND groups did not differ from each other at baseline (P > 0.05)
2013). We have previously shown that obese subjects at risk explained by increased glucose oxidation and decreased
had reduced PBMC gene expression of PPARD compared fatty acid oxidation.
with metabolically healthy obese and control subjects The strength of this study is the relatively high number
(Telle-Hansen et al. 2013), and PPARD activation improves of subjects, and to the best of our knowledge, the current
multiple metabolic disorders (especially blood lipids) in dietary intervention study is the first one to use PBMC
obese subjects (Riserus et al. 2008). gene expression as a tool to examine if diet can modify
In accordance to other studies, the expression of PDK4 the OGTT response. We used a well-characterized
was down-regulated (Zhang et al. 2014), and several pro- glucose-regulated gene as a positive control to ensure that
inflammatory genes (TNF, TGFB2, CXCR2, CD40LG, changes in mRNA level could be measured 2h after
IL1RN, CCR2, IL23R, and MMP9) and lipid metabolism re- OGTT. The limitation of the study is that we cannot dif-
lated genes (CD36, ABCG1 and ABCA1) were up- ferentiate any specific food components responsible for
regulated, after the OGTT in the whole study population, the effect on the change in 2hOGTT response since we
confirming the use of PBMC gene expression analysis as a did not focus the intervention on single nutrients but on
model system to detect metabolic responses after an OGTT the whole diet. Our primary aim was however to study the
(Aljada et al. 2006; Aljada et al. 2004; Griffin et al. 2001). effects of the whole diet, since this approach is closer to
The mRNA level of CPT1A was down-regulated during real-life situations.
2hOGTT. Since CPT1 is involved in oxidation of fatty
acids, and the oxidation is suppressed in the presence of an Conclusions
adequate glucose supply (Bonnefont et al. 2004), the re- We show that the long-term intake of a healthy ND
duced expression of CPT1 during 2hOGTT may be down-regulates genes involved in inflammation and lipid
Table 3 Effect of the healthy ND compared to the CD on gene expression changes after 2hOGTT
Number Group effect (regression coefficient β) 95 % CI for β q values
Inflammatory genes
CCL2
Unadjusted 78 0.12 −0.19–0.43 0.64
Adjusted 78 0.12 −0.20–0.42 0.69
CCL5
Unadjusted 85 −0.002 −0.14–0.14 0.98
Adjusted 85 −0.002 −0.14–0.14 0.98
CCR2
Unadjusted 76 −0.06 −0.27–0.15 0.72
Adjusted 76 −0.07 −0.28–0.14 0.69
CCR4
Unadjusted 77 −0.07 −0.23–0.10 0.64
Adjusted 77 −0.06 −0.22–0.11 0.69
CD40
Unadjusted 87 −0.14 −0.37–0.10 0.46
Adjusted 87 −0.03 −0.25–0.19 0.88
CD40LG
Unadjusted 87 −0.09 −0.22–0.04 0.42
Adjusted 87 −0.08 −0.21–0.06 0.47
CXCR2
Unadjusted 86 −0.27 −0.47–(−0.07) 0.05
Adjusted 86 −0.25 −0.45–(−0.05) 0.09
ICAM1
Unadjusted 84 −0.02 −0.22–0.19 0.89
Adjusted 84 0.02 −0.19–0.22 0.93
IFNG
Unadjusted 86 −0.29 −0.53–(−0.05) 0.09
Adjusted 86 −0.27 −0.51–(−0.02) 0.13
IKBKB
Unadjusted 83 −0.24 −0.41–(−0.06) 0.05
Adjusted 83 −0.24 −0.42–(−0.06) 0.08
IL18
Unadjusted 72 −0.60 −1.01–(−0.18) 0.05
Adjusted 72 −0.73 −1.20–(−0.26) 0.042
IL1B
Unadjusted 85 −0.17 −0.43–0.10 0.42
Adjusted 85 −0.16 −0.41–0.09 0.45
IL1RN
Unadjusted 87 −0.10 −0.26–0.06 0.42
Adjusted 87 −0.10 −0.26–0.06 0.47
IL23A
Unadjusted 87 0.15 0.00–0.31 0.17
Adjusted 87 0.14 −0.01–0.30 0.20
Leder et al. Genes & Nutrition _#####################_ Page 7 of 13
Table 3 Effect of the healthy ND compared to the CD on gene expression changes after 2hOGTT (Continued)
IL23R
Unadjusted 88 −0.06 −0.36–0.24 0.78
Adjusted 88 −0.05 −0.34–0.25 0.88
IL6
Unadjusted 86 −0.18 −0.45–0.09 0.42
Adjusted 86 −0.19 −0.46–0.09 0.43
IL8
Unadjusted 83 −0.50 −1.09–0.09 0.30
Adjusted 83 −0.53 −1.12–0.05 0.22
MMP9
Unadjusted 71 −0.09 −0.52–0.34 0.78
Adjusted 71 −0.12 −0.57–0.33 0.74
NFKBIA
Unadjusted 87 0.09 −0.03–0.22 0.37
Adjusted 87 0.10 −0.03–0.22 0.34
OLR1
Unadjusted ND ND ND ND
Adjusted ND ND ND ND
PDGFA
Unadjusted 64 0.21 −0.06–0.48 0.37
Adjusted 64 0.29 0.00–0.57 0.17
PDGFB
Unadjusted 77 0.24 0.03–0.45 0.12
Adjusted 77 0.24 0.03–0.45 0.11
PDK4
Unadjusted 88 −0.14 −0.41–0.13 0.55
Adjusted 88 −0.08 −0.34–0.18 0.70
RELA
Unadjusted 83 −0.06 −0.21–0.10 0.64
Adjusted 83 −0.10 −0.25–0.06 0.45
TGFB2
Unadjusted 75 0.12 −0.15–0.39 0.64
Adjusted 75 0.10 −0.18–0.38 0.69
TLR4
Unadjusted 85 −0.36 −0.54–(−0.18) 0.008
Adjusted 85 −0.33 −0.52–(−0.15) 0.042
TNF
Unadjusted 86 −0.09 −0.23–0.05 0.42
Adjusted 86 −0.09 −0.23–0.05 0.45
TNFRSF1A
Unadjusted 88 −0.24 −0.41–(−0.07) 0.05
Adjusted 88 −0.22 −0.40–(−0.04) 0.09
TNFRSF1B
Unadjusted 88 −0.11 −0.29–0.08 0.46
Adjusted 88 −0.10 −0.28–0.08 0.47
Lipid metabolism-related genes
ABCA1
Unadjusted 72 −0.12 −0.39–0.16 0.64
Adjusted 72 −0.12 −0.39–0.15 0.64
ABCG1
Unadjusted 81 0.09 −0.14–0.32 0.64
Adjusted 81 0.11 −0.12–0.34 0.60
CD36
Unadjusted 85 −0.24 −0.40–(−0.07) 0.05
Adjusted 85 −0.23 −0.39–(−0.08) 0.042
CPT1A
Unadjusted 78 0.05 −0.17–0.26 0.78
Adjusted 78 0.05 −0.27–0.27 0.80
CPT1B
Unadjusted 82 −0.26 −0.49–(−0.02) 0.12
Adjusted 82 −0.26 −0.49–(−0.03) 0.13
CRAT
Unadjusted 84 0.01 −0.13–0.16 0.89
Adjusted 84 0.02 −0.13–0.17 0.88
HMGCR
Unadjusted 86 0.04 −0.12–0.19 0.78
Adjusted 86 0.04 −0.12–0.20 0.74
LDLR
Unadjusted 73 0.24 0.04–0.45 0.10
Adjusted 73 0.25 0.04–0.46 0.09
LIPE
Unadjusted ND ND ND ND
Adjusted ND ND ND ND
NAMPT
Unadjusted 80 −0.07 −0.25–0.12 0.64
Adjusted 80 −0.06 −0.25–0.12 0.69
PLIN2
Unadjusted 80 0.02 −0.18–0.21 0.89
Adjusted 80 0.004 −0.20–0.20 0.98
PPARA
Unadjusted 87 0.03 −0.15–0.21 0.81
Adjusted 87 0.02 −0.16–0.20 0.88
PPARD
Unadjusted 87 0.22 0.09–0.36 0.021
Adjusted 87 0.21 0.08–0.35 0.042
SREBF1
Unadjusted 81 −0.15 −0.69–0.39 0.74
Adjusted 81 −0.24 −0.77–0.28 0.61
UCP2
Unadjusted 84 0.16 −0.04–0.36 0.32
Adjusted 84 0.17 −0.02–0.36 0.23
Adjusted models: The effect of the independent variable “study group” is adjusted for fold change at baseline (log2 transformed) and study center. It should be
noted that the regression coefficient expresses the mean difference between the groups, unadjusted and adjusted. A q value <0.05 (FDR < 5 %) were
considered significant
ND not detected
metabolism in individuals at risk for metabolic diseases Methods

and thereby may reduce this unfavorable postprandial Study design and subjects
response. The results need to be confirmed by further The study design and participants have been described
human intervention studies, preferably with meal chal- in detail elsewhere (Uusitupa et al. 2013). In short, this
lenges. In addition, experimental models (e.g., ex vivo study was a randomized controlled multicenter study
cell models or animal model) should be employed to ex- performed in six centers within the Nordic countries
tend our biological and clinical understanding of the [Kuopio and Oulu (Finland), Lund and Uppsala (Sweden),
data presented here. Aarhus (Denmark), and Reykjavik (Iceland)]. The
Fig. 2 Effect of ND compared to CD on gene expression changes after a 2hOGTT. The effect of the healthy ND compared to the CD on TLR4 mRNA
expression (a), IL18 mRNA expression (b), CD36 mRNA expression (c), and PPARD mRNA expression (d) presented as fold change (2−ΔCt of 2hOGTT/2
−ΔCt
of 0hOGTT) in the healthy ND group (n = 42–49) and in the CD group (n = 32–40) after intervention. Fold changes (2−ΔΔCt) are normalized for the
reference gene TBP and fasting values (0hOGTT). The box plots show the 2−ΔΔCt values at the end of the intervention. Box plots show the medians with
25th and 75th percentiles. Whiskers express the 1.5 × interquartile range. The number sign is a q value of 0.042. The q values indicate the effect of the
ND compared to the CD on gene expression changes after a 2hOGTT. The effect of the independent variable study group is adjusted for fold change
at baseline (log2 transformed) and study centers. A q value <0.05 (FDR < 5 %) was considered significant
participants were randomized after a 4-week run-in period

with habitual diet into a healthy ND group or a CD group
for 18–24 weeks. The composition of the diets has been
described in detail elsewhere (Uusitupa et al. 2013). The
main differences between the diets at the nutrient level
were the amount of dietary fiber and salt and the quality
of dietary fat. Both the ND and the CD were isocaloric
based on the evaluation of the habitual diet calculated
from a 4-day food record during the run-in period. The
Nordic Nutrition Recommendations (NNR) formed the
basis of the ND, and the main emphasis was on food items
such as whole-grain products, abundant use of berries,
fruit and vegetables, rapeseed oil, three fish meals per
week, low-fat dairy products, and avoidance of sugar-
sweetened products. The subjects in the CD consumed a
diet in accordance to the mean nutrient intake in the Nor-
dic countries. Key products were provided to the study
participants in both groups. The study participants were
advised to keep body weight and physical activity constant
and not to change their smoking and drinking habits or
drug treatment during the study. All study participants
provided their written informed consent, and local ethics
committees of all the participating centers approved the
study protocol.
Altogether, 309 individuals were originally contacted and
screened at the study clinics, and 213 were randomized as
described earlier (Uusitupa et al. 2013). Ninety-six individ-
uals in the ND group and 70 in the CD group completed
the trial (Uusitupa et al. 2013). The inclusion criteria were Fig. 3 Flow chart of the participants
age 30–65 years, BMI 27–38 kg m−2, and two other of the
International Diabetes Federation (IDF) criteria for MetS
(Alberti et al. 2009). Antihypertensive and lipid-lowering baseline or after the intervention (n = 6), baseline BMI
medication, as well as inhaled corticosteroids, were allowed above 39 kg m−2 (n = 3), and body weight change more
but without dosage changes during the trial. The main ex- than 4 kg during the intervention (n = 8). Since five sub-
clusion criteria included any chronic disease and condition, jects were excluded from the analysis due to low quan-
which could hamper the adherence to the dietary interven- tity of RNA (n = 3) or problems with the TaqMan Array
tion protocol, poor compliance, chronic liver, thyroid and Micro Fluidic Cards (n = 2), we analyzed data from 89
kidney diseases, alcohol abuse (>40 g per day), diabetes, subjects (n = 49 in ND and n = 40 in CD) (Fig. 3).
fasting triglycerides >3.0 mM, total cholesterol >6.5 mM,
and blood pressure >160/100 mmHg. A few study partici- Clinical and biochemical measurements
pants with triglycerides between 3 and 4 mM and with Procedures regarding the clinical and biochemical mea-
BMI between 38 and <40 kg m−2 were, however, included surements have been described previously (Uusitupa
in the main study population. et al. 2013). In short, subjects were examined in the
In this present sub-study of the SYSDIET trial, we in- morning after overnight fasting. Anthropometric mea-
cluded a total of 94 subjects (n = 54 in ND and n = 40 in surements were performed locally according to the
CD) out of the 166 subjects who completed the SYS- standard operational procedures. Concentrations of
DIET study (Fig. 3). We excluded subjects from Aarhus plasma glucose, cholesterol, and triglycerides were ana-
(n = 31), Uppsala (n = 9), and Reykjavik (n = 15), because lyzed locally in the centers using routine methodology.
the Aarhus study center did not collect PBMC samples, Blood samples to measure cytokines and adipokines
and the number of PBMC samples was limited from from all the study centers were analyzed in the Univer-
Uppsala (n = 9) and Reykjavik (n = 5). So, by excluding sity of Eastern Finland and Kuopio University Hospital,
these two centers, we reduced variance. We also ex- Finland. Plasma insulin was analyzed in the Aarhus Uni-
cluded subjects with high-sensitivity C-reactive protein versity Hospital, Denmark, using routine automated
(hs-CRP) concentration higher than 10 mg L−1 at clinical chemistry analyzers.
Standard 2hOGTT Statistical analysis

A standard 2hOGTT (75 g D-glucose) was performed For baseline characteristic comparisons, we used inde-
after an overnight fast at baseline and at the end of the pendent t test to test the difference between means,
intervention. Blood samples for PBMC isolation were Mann-Whitney U test to test the difference between me-
taken at the time points 0 and 120 min. dians and chi-square test to test the difference between
categorical variables. Power calculations (alpha <0.05,
beta >0.8) were carried out on serum cholesterol, fasting
PBMCs, RNA isolation, cDNA synthesis, and qPCR glucose, and insulin (Uusitupa et al. 2013).
After blood collection, PBMCs were isolated at base- Linear multivariable regression analyses were used to
line and at the end of the intervention at time points test the independent effect of the study groups on the
0 and 120 min by using the BD Vacutainer Cell Prep- change in glucose, insulin, triglycerides, and free fatty
aration tubes according to the manufacturer’s instruc- acids from 0h (fasting) to 2h (after OGTT) at the end of
tions (Becton, Dickinson San Jose, CA, USA) and the intervention as well as on the dependent dietary in-
stored as pellets at −80 °C for further analysis. Total take variables at the end of the intervention. The effect
RNA isolation was performed centrally at the Karo- of the independent variable study group was adjusted for
linska Institute (Stockholm, Sweden). The total RNA the corresponding baseline variable, study centers, gen-
was isolated using the RNeasy Mini Kit according to der, log10-transformed age, and body weight at the end
the manufacturer’s instructions (Qiagen, Valencia, CA, of the intervention. Changes in gene expression from 0h
USA). RNA quantity and quality measurements were per- to 2hOGTT within the whole study population were
formed using a Nanodrop ND-1000 Spectrophotometer tested with Wilcoxon signed rank test (2−ΔCt). Data are
(Thermo Fisher Scientific, Gothenburg, Sweden) and given as the median (25–75th percentiles). Linear
Agilent 2100 Bioanalyzer (Agilent Technologies, Santa multivariable regression analyses were also carried out
Clara, CA, USA), respectively. RNA from all samples was to test the independent effect of the study groups on
reverse transcribed by a high-capacity cDNA reverse tran- the dependent variable fold change at the end of the
scription kit (Applied Biosystems, Foster City, CA, USA). intervention. The analyses were adjusted for the inde-
Quantitative real-time polymerase chain reaction (qPCR) pendent variables fold change at baseline and study
was performed on an ABI PRISM 7900HT (Applied Bio- center. In the presentation, β denotes the regression
systems) using TaqMan Array Micro Fluidic Cards coefficient of the treatment group. Fold changes at
(Applied Biosystems). The target genes are shown in the baseline and at the end of the intervention (2−ΔΔCt)
Additional file 1. Primer sequences are commercially were log2 transformed before the analyses to improve nor-
available (Applied Biosystems) and can be provided upon mality. To account for multiple testing, we applied false
request. The selection of target genes was primarily discovery rate (FDR) analysis and q < 0.05 (FDR < 5 %) was
based on previous long-term and short-term dietary considered significant. Calculations were performed using
intervention studies where PBMC gene expression of IBM SPSS Statistics version 20 (Armonk, NY, USA) and R
inflammatory and lipid metabolism genes was modu- version 3.2.0.
lated or associated with features of MetS or meta-
bolic risk factors for T2DM and CVD (De Mello Additional file
et al. 2009; Bouwens et al. 2009; Jones et al. 2011;
Kaminski et al. 1993) and on results from our own Additional file 1: mRNA level at fasting (0hOGTT) and after 2hOGTT and
studies (Myhrstad et al. 2011; Telle-Hansen et al. fold change from fasting in the whole study population at baseline. Data
for 0hOGTT and 2hOGTT is given as 2−ΔCt (normalized for TBP). Data for fold
2013). change is given as 2−ΔΔCt (normalized for TBP and 0hOGTT values). All values
The relative mRNA level for each transcript was are presented as medians with 25th–75th percentiles. (DOCX 21.4 kb)
calculated by the ΔΔ cycle threshold (Ct) method
(Livak and Schmittgen 2001). TATA-box binding Abbreviations
protein (TBP) was used as reference gene for ABCA1: ATP-binding cassette, sub-family A (ABC1), member 1; ABCG1: ATP-
normalization. Briefly, the Ct values of each target binding cassette, sub-family G, member 1; CCL2: chemokine (C-C motif)
ligand 2; CCL5: chemokine (C-C motif) ligand 5; CCR2: chemokine (C-C motif)
gene were normalized to the Ct values of the TBP receptor 2; CCR4: chemokine (C-C motif) receptor 4; CD: control diet;
(=ΔCt). The fold change in mRNA gene expression CD36: CD36 molecule (thrombospondin receptor); CD40: CD40 molecule;
from TBP was calculated at fasting (0h) and after CD40LG: CD40 ligand; CPT1A: carnitine palmitoyltransferase 1A;
CPT1B: carnitine palmitoyltransferase 1B; CRAT: carnitine O-acetyltransferase;
(2h) OGTT (2−ΔCt) at baseline and at the end of the CXCR2: chemokine (C-X-C motif) receptor 2; CVD: cardiovascular diseases;
intervention. The fold change in mRNA gene expres- FDR: false discovery rate; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase;
sion from fasting was calculated as 2−ΔΔCt at baseline ICAM1: intercellular adhesion molecule 1; IFNG: interferon, gamma;
IKBKB: inhibitor of kappa light polypeptide gene enhancer in B cells, kinase
and at the end of the intervention, as 2−ΔCt 2hOGTT was beta; IL18: interleukin 18; IL1B: interleukin 1, beta; IL1RN: interleukin 1
divided by 2−ΔCt
0hOGTT. receptor antagonist; IL23A: interleukin 23, alpha subunit p19;
IL23R: interleukin 23, receptor; IL6: interleukin 6; IL8: interleukin 8; LDLR: and Caring Sciences, Clinical Nutrition and Metabolism, Uppsala University,
low-density lipoprotein receptor; LIPE: lipase, hormone-sensitive; Uppsala, Sweden. 14Department of Endocrinology and Internal Medicine,
MetS: metabolic syndrome; MMP9: matrix metallopeptidase 9; Aarhus University Hospital, Aarhus, Denmark. 15Department of Nutrition,
NAMPT: nicotinamide phosphoribosyltransferase; ND: Nordic diet; Exercise and Sport, University of Copenhagen, Copenhagen, Denmark. 16Unit
NFKBIA: nuclear factor of kappa light polypeptide gene enhancer in B cells for Nutrition Research, University of Iceland and Landspitali - The National
inhibitor, alpha; OLR1: oxidized low density lipoprotein (lectin-like) receptor 1; University Hospital of Iceland, Reykjavik, Iceland. 17Department of Clinical
PBMCs: peripheral blood mononuclear cells; PDGFA: platelet-derived growth Nutrition, Skåne University Hospital, Lund, Sweden. 18Department of
factor alpha polypeptide; PDGFB: platelet-derived growth factor beta Biostatistics, University of Oslo, Oslo, Norway. 19Research Unit, Kuopio
polypeptide; PDK4: pyruvate dehydrogenase kinase, isozyme 4; University Hospital, Kuopio, Finland. 20Norwegian National Advisory Unit on
PLIN2: perilipin 2; PPARA: peroxisome proliferator-activated receptor alpha; Familial Hypercholesterolemia, Department of Endocrinology, Morbid Obesity
PPARD: peroxisome proliferator-activated receptor delta; RELA: v-rel and Preventive Medicine, Oslo University Hospital, Oslo, Norway.
reticuloendotheliosis viral oncogene homologue A; SREBF1: sterol regulatory
element binding transcription factor 1; SYSDIET: Systems Biology in Received: 3 November 2015 Accepted: 15 January 2016
Controlled Dietary Interventions and Cohort Studies; T2DM: type 2 diabetes
mellitus; TGFB2: transforming growth factor beta 2; TLR4: toll-like receptor 4;
TNF: tumor necrosis factor; TNFRSF1A: tumor necrosis factor receptor
superfamily member 1A; TNFRSF1B: tumor necrosis factor receptor References
superfamily member 1B; UCP2: uncoupling protein 2. Adamsson V, Reumark A, Fredriksson IB, Hammarstrom E, Vessby B, Johansson G,
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Authors’ contributions inflammation in relation to health-insights from transcriptomic biomarkers in
MK, ID, MCWM, VdM, JP, CC, US, K-HH, LC, MUS, JH, MJS, FR, KH, LOD, IG, IT, PBMC of fatty acids and polyphenols. Molecular nutrition & food research
UR, BA, PA, KSP, MU, and SMU designed the research. LL, MK, IN, ID, US, K- Ahmad R, Al-Mass A, Al-Ghawas D, Shareif N, Zghoul N, Melhem M, Hasan A, Al-
HH, LC, MUS, JH, MJS, FR, KH, LOD, IG, IT, UR, BA, PA, MU, KBH, and SMU con- Ghimlas F, Dermime S, Behbehani K (2013) Interaction of osteopontin with
ducted the research. LL, MK, IN, MT, KBH, and SMU analyzed the data or per- IL-18 in obese individuals: implications for insulin resistance. PLoS One 8:
formed the statistical analysis. LL, MK, KBH, and SMU wrote the first draft of e63944
the manuscript and had primary responsibility for the final content. MK, KSP, Alberti KG, Eckel RH, Grundy SM, Zimmet PZ, Cleeman JI, Donato KA, Fruchart JC,
and MU were responsible for the coordination of the SYSDIET consortium. James WP, Loria CM, Smith SC Jr (2009) Harmonizing the metabolic
All authors have participated in and critically reviewed the manuscript and syndrome: a joint interim statement of the International Diabetes Federation
accepted it to be submitted. Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood
Institute; American Heart Association; World Heart Federation; International
Acknowledgements Atherosclerosis Society; and International Association for the Study of
We thank Maritta Siloaho (MS) for the excellent expertise and advice for Obesity. Circulation 120:1640–1645
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Funding matrix metalloproteinase concentrations. Am J Clin Nutr 80:51–57
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Dietary Interventions and Cohort Studies)] and further, by Oslo and Akershus kappaB, a fall in cellular inhibitor kappaB, and an increase in tumor necrosis
University College of Applied Sciences (Norway), Academy of Finland (131593 factor alpha messenger RNA by mononuclear cells in healthy human
to VDdM), University of Oslo (Norway), Swedish Research council, Svenska subjects. Metabolism 55:1177–1185
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Healthy dietary patterns, lipids and

Dissertation for the degree of Philosophiae Doctor (Ph.D.)

Institute of Basic Medical Sciences

Series of dissertations submitted to the

All rights reserved. No part of this publication may be

Cover: Hanne Baadsgaard Utigard.

Healthy dietary patterns have been subject of considerable attention in recent

AA arachidonic acid cDNA complementary DNA

CD19 CD19 molecule DPA docosapentaenoic acid

CD72 CD72 molecule ER endoplasmatic reticulum

HDL-C HDL cholesterol LDL low density lipoprotein

HETE hydroxyeicosatetraenoic acid LDL-C LDL cholesterol

HIF1A hypoxia inducible factor 1 alpha LDLR LDL receptor

hs-CRP high-sensitive CRP LXR liver X receptor

HSPA5 heat shock protein family A LXRA liver X receptor alpha

IFN interferon MAPK8 mitogen-activated protein kinase

IL1B interleukin 1 beta MMP matrix metalloproteinase

IL1RN interleukin 1 receptor antagonist mRNA messenger RNA

NFKBIA NFkB inhibitor alpha PUFA polyunsaturated fatty acid

NK natural killer qPCR real-time quantitative

PL phospholipid SLC22A5 solute carrier family 22 member

Th T helper VEGFB vascular endothelial growth

3 Subjects and methods 23

1.1 Dietary patterns and cardiometabolic risk

The idea of a "healthy Nordic diet" conceived of the Mediterranean diet,

1.2 Fatty acids

in adipose tissue, even-numbered SFAs can be synthesized endogenously by de

1.3 Dietary fat, lipids and cardiovascular risk

mmol/L (women) [43]. Dietary fatty acid composition regulates lipoprotein

mortality [54]. A meta-analysis of prospective cohort studies showed that di-

1.4 LDL cholesterol metabolism

LDLR LDL plasma

1.6 Dietary fat, inﬂammation and cardiovascular

5% [90]. Thus, an important aspect of the anti-inﬂammatory action of ALA

1.7 Fatty acids and gene regulation

inflammatory gene expression ?

Fatty acid & cholesterol

fatty acid synthesis decreased [106].

1.8 Peripheral blood mononuclear cells

Lymphocytes can be divided into

1.9 Use of challenge tests in interventions

glucose concentrations return to the basal level (homeostasis) within 2h after a

The speciﬁc aims have been to:

• study the intake of commercially available food products, in which SFAs

• study the molecular mechanisms of the cholesterol-lowering eﬀect of re-

Subjects and methods

3.1 The NoMa study

The NoMa ("Norsk Mat" - Norwegian Food) study is an eight-week double-

2 weeks with Control group

3.2 The SYSDIET study

The SYSDIET study is a randomized controlled multi-center study performed

Table 3.1 gives an overview of study populations and study designs.

Study Design Population Groups Duration Aim Paper

3.3 Selection of candidate genes

Studies Intervention Duration Population n= Candidate genes Reference

healthy IFNG, IL7R, POLK

In paper II, we found that the gene expression of LDLR is upregulated

5.1 Methodological considerations

to ensure a balance in sample size across groups. Finally, stratiﬁed randomiza-

The assessment of study outcomes may be inﬂuenced by information bias, i.e.

Challenges in dietary intervention studies

5.1.2 Gene expression in peripheral blood mononuclear cells

challenge is to determine which changes in gene expression may be of clinical