Progress in Lipid Research 51 (2012) 6370

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Progress in Lipid Research

journal homepage: www.elsevier.com/locate/plipres

Review

Human nutrigenomics of gene regulation by dietary fatty acids

Lydia A. Afman , Michael Mller
Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, The Netherlands
Netherlands Nutrigenomics Centre, TI Food and Nutrition, Wageningen, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Nutrigenomics employs high-throughput genomics technologies to unravel how nutrients modulate gene
Available online 2 December 2011 and protein expression and ultimately inuence cellular and organism metabolism. The most often-
applied genomics technique so far is transcriptomics, which allows quantifying genome-wide changes
Keywords: in gene expression of thousands of genes at the same time in one sample. The performance of gene
Nutrigenomics expression quantication requires sufcient high-quality homogenous cellular material, therefore
Fatty acids research in healthy volunteers is restricted to biopsies from easy accessible tissues such as subcutaneous
Transcriptomics
adipose tissue, skeletal muscle and intestinal biopsies or even more easily accessible cells such as periph-
Gene expression
Peripheral blood mononuclear cells (PBMCs)
eral blood mononuclear cells from blood. There is now signicant evidence that fatty acids, in particular
unsaturated fatty acids, exert many of their effects through modulation of gene transcription by regulat-
ing the activity of numerous transcription factors, including nuclear receptors such as peroxisome prolif-
erator activated receptors, liver X receptor and sterol regulatory binding proteins. This review evaluates
the human nutrigenomics studies performed on dietary fat since the initiation of nutrigenomics research
around 10 years ago. Although the number of studies is still limited, all studies clearly suggest that
changes in dietary fatty acids intake and composition can have a signicant impact on cellular adaptive
response capacity by gene transcription changes in humans. This adds important knowledge to our
understanding of the strong effects that various fatty acids can have on numerous metabolic and inam-
matory pathways, signaling routes and homeostatic control in the cell and ultimately on whole body
health. It is important to use and integrate nutrigenomics in all future nutrition studies to build up the
necessary framework for evidence-based nutrition in near future.
2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2. Challenges in human nutrigenomics studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3. Transcriptional gene regulation by (dietary) fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
4. Application of transcriptomics in human intervention studies with dietary fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4.1. Postprandial intervention studies with dietary fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4.2. Longer-term intervention studies with dietary fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.3. Overlap between long term and postprandial effects of n 3 PUFA on PBMC transcriptome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
5. Future challenges in nutrigenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
6. Conclusions and perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

1. Introduction inappropriate dietary patterns. The last decade has revealed an

Human nutrition research is still largely focused on issues related
to nutrient deciencies and impairment of health and disease due to
Corresponding author. Address: Nutrition, Metabolism and Genomics Group,
Division of Human Nutrition, Wageningen University, Bomenweg 2, 6703 HD
Wageningen, The Netherlands. Tel.: +31 (0)317 485789; fax: +31 (0)317 483342.
E-mail addresses: lydia.afman@wur.nl (L.A. Afman), michael.muller@wur.nl
(M. Mller).
enormous wealth of insights in the complexity of human physiology
and in particular of pathophysiology of complex diseases. At the
same time we have to acknowledge a worldwide epidemic increase
in preventable diseases such as metabolic syndrome or diabetes
type 2 that are linked to obesity and largely related to higher energy
intake and reduced energy expenditure [1]. On the other hand new
high-throughput genomics technologies for the generation, process-
ing, and analysis of biological data about the composition and

0163-7827/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.plipres.2011.11.005
64 L.A. Afman, M. Mller / Progress in Lipid Research 51 (2012) 6370
functions of genomes has created unprecedented opportunities for
increasing our understanding of how nutrients modulate gene and
protein expression and ultimately inuence cellular and organ
metabolism. To overcome the signicant gap between molecular
biology, physiology and more traditional nutrition research, nutri-
genomics has been initiated as a new innovative strategy around
10 years ago when a working draft of the human genome was an-
nounced. There are several meanings of nutrigenomics as used in
the scientic literature mostly focusing on gene-environment inter-
actions and the relevance of genotype changes e.g. single nucleotide
polymorphism (SNPs) for individual variation in responses to foods,
dietary pattern and susceptibility to develop nutrition-related dis-
eases [2,3]. We have extent this denition of nutrigenomics by
including innovative combinations of molecular nutrition research
and genomics applications, largely performed in transgenic animal
models. The diverse tissue and organ-specic effects of bioactive
dietary components include gene expression patterns (transcrip-
tome), organization and modication of the chromatin structure
(epigenome), protein expression patterns including post-transla-
tional modications (proteome) as well as metabolite proles
(metabolome) [46]. Nutrigenomics promotes an increased under-
standing on how nutrition inuences metabolic pathways and
homeostatic control by providing mechanistic insights into how
nutrition works (evidence-based nutrition). Furthermore it reveals
how this regulation is disturbed in the early phases of diet-related
disease, and the extent to which individual sensitizing genotypes
contribute to such diseases by allowing comprehensive and deep
phenotyping and the identication of early biomarkers for pre-dis-
ease states [79]. Nutrigenomics is the approach that generates
the knowledge that allows development of new nutritional strate-
gies to improve peoples health and tness according to their indi-
vidual needs. This necessary evolution will eventually lead to
evidence-based dietary intervention strategies for restoring health
and tness and for preventing diet-related disease.
2. Challenges in human nutrigenomics studies
Since the introduction of genomics techniques such as transcri-
ptomics many human studies have been performed to examine
phenotypic differences between diseased and healthy states with
the ultimate goal to improve early diagnostic and to increase our
understanding of underlying mechanisms of early disease pathol-
ogy or to investigate pharmacological efcacy to modulate disease
progression. Recently these techniques are also applied to achieve
more knowledge on metabolic, physiological or dietary changes on
alterations in transcriptional gene regulation in humans.
Several types of intervention studies can be performed in which
effects of specic nutrients (e.g. n 3 PUFA versus saturated fat) or
changes in more complex dietary patterns (Mediterranean versus
Western-style diets) can be examined on the levels of gene expres-
sion proles. The length of the study design can vary from a few
hours in postprandial studies, to weeks and years in respectively
completely controlled dietary intervention studies and long-term
interventions trials. It is expected that postprandial studies mirror
the more acute effects of daily consumption whereas long-term
studies will reect the extended inuence of nutrition on whole
body health. All types of studies, if well controlled, are of pivotal
importance to increase our knowledge and understanding on
how nutrients are sensed by our organ cells and how this translates
efciently into necessary adaptive physiological response mecha-
nism as essential for health, both on the short and long run.
The major bottleneck in studying these kind of often-subtle
changes is the limited accessibility of tissues as study populations
mostly consist of healthy volunteers. Whereas in ill subjects that
need to undergo surgery biopsies can be more easily obtained from
both pathological tissue and more normal surrounding tissue
areas, in patients without necessity surgery such as diabetes
patients and especially in healthy volunteers, collection of samples
from internal organs such as liver, pancreas and visceral adipose
tissue is impossible because of ethical reasons. Research in those
volunteers, is therefore restricted to biopsies from more easily
accessible tissues such as subcutaneous adipose tissue, muscle
and intestinal biopsies without a high health risk for the volunteer.
Although other metabolic organs such as liver and visceral fat, are
not assessable for invasive investigations, comprehensive pheno-
typing of these organs in healthy and early pathophysiological
stages in the development of diseases would be extremely valu-
able. These metabolic organs fulll key physiological functions in
the efcient regulation of homeostasis related to dietary fat metab-
olism and play a key role in the early development of nutrition-re-
lated diseases such as metabolic syndrome and type II diabetes as
shown to large extent in animal studies [1016]. The development
of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis
[15,16]; is a good example for this as a more recently frequently
observed human disease phenotype that is strongly linked to met-
abolic syndrome and both may even be two denitions of the same
problem (chronic metabolic stress) with ultimately the develop-
ment of diabetes type 2 and cardiovascular health problems as
consequences.
To allow more efcient prevention of these diseases by evi-
dence-based nutritional and lifestyle intervention a comprehensive
understanding of the various phases (from organ dysfunction to
systemic pathophysiology) in the disease evolution is key. There-
fore the study of time-dependent transitions from the healthy to
disease state and the role that nutrition plays in these time-depen-
dent changes of organ phenotypes would be essential but is cur-
rently not feasible because of the invasive procedure, the low
tissue and RNA yield and the presence of numerous cell types in
the biopsy. A far less invasive method is the collection of blood cells
out of whole blood obtained via venipuncture. Blood cells travel
through the whole body, are exposed to all physiological changes
including changes in nutrient levels before and after meals or in
the fasting state. They are key cells in regulating organ-specic
and systemic immune function and inammatory responses. Non-
resolving inammation is a major driver of disease, e.g. blood cells
can attached to and invade in the vascular wall leading to foam cells
formation and ultimately atherosclerosis [17]. The most studied
blood cells are the peripheral blood mononuclear cells (PBMCs) that
consist of monocytes and lymphocytes. Whole genome transcrip-
tome analyzes of PBMCs have been applied successfully in discrim-
inating disease from healthy populations and in differentiating
several phases/diagnostics of disease [18,19]. It has also been
shown that variation in PBMC gene expression within persons is
smaller than the variation between persons [2022]. This smaller
within person variation combined with the usual applied measure-
ment of baseline and end samples within a person in nutritional
intervention studies enables potential successful application of
PBMC gene expression proling in nutritional intervention studies.
3. Transcriptional gene regulation by (dietary) fatty acids
Dietary fatty acids inuence human health in numerous ways
and inuence several indicators of health status. In recent years
much work has focused to elucidate more precisely the mode of ac-
tion how dietary fatty acids lead to short-and long-term changes in
cellular functions. There is now considerable evidence that fatty
acids, in particular unsaturated fatty acids, exert many of their bio-
logical effects through modulation of gene transcription by regu-
lating the activity of numerous transcription factors, including
sterol regulatory binding proteins (SREBPs) or nuclear receptors
such as peroxisome proliferator activated receptors (PPARs) and
liver X receptors (LXRs).
 L.A. Afman, M. Mller / Progress in Lipid Research 51 (2012) 6370
Three different highly conserved PPAR isotypes have been iden-
tied: PPARa (NR1C1), PPARb/r (NR1C2) and PPARc (NR1C3).
They act as nutrient sensors for fatty acids, but bind also numerous
fatty acid derivatives and compounds showing structural resem-
blance to fatty acids and inuence the expression of specic genes
[23,24] The PPARa isotype has been shown to govern expression of
numerous genes involved in fatty acid oxidation, ketogenesis,
gluconeogenesis, cholesterol catabolism and lipoprotein metabo-
lism as mainly studied using mouse models. More recent evidence
indicates that hepatic PPARa is not largely activated by plasma free
fatty acids as high during fasting, whereas it can be activated by
dietary fatty acids and fatty acids generated via de novo lipogene-
sis [25]. Recently, it was also shown that the effects of dietary
unsaturated fatty acids on hepatic gene expression are almost
exclusively mediated by PPARa and mimic the effect of synthetic
PPARa agonists [26]. Much less is known about the relevance of
these mainly mouse-based ndings for human physiology. A recent
analysis provided new insights into the mechanisms and impact of
transcriptional regulation by PPARa in human liver by performing
chromatin immunoprecipitation (ChIP)-chip in combination with
transcriptional proling on HepG2 human hepatoma cells treated
with the specic PPARa agonist GW7647. Several genes known
to be regulated by PPARa showed GW7647-induced PPARa bind-
ing to their promoter. Interestingly also certain SREBP-target genes
exhibited PPARa binding, suggesting cross-talk between PPARa
and SREBP signaling [27]. Another study compared the function
of PPARa in mouse and human hepatocytes via analysis of target
gene regulation. Minor overlap was observed between the
PPARa-regulated genes from mouse and human by a gene-by-gene
comparison, although more substantial overlap was observed at
the pathway level. This study clearly suggest that PPARa activation
has a major impact on gene regulation in human hepatocytes and
that the role of PPARa as master regulator of hepatic lipid metab-
olism is generally well-conserved between mouse and human [28].
More in depth studies are required to elucidate the specic role of
nutrient-sensing transcription factors in mediating the effects of
dietary fatty acids in other human cells including small intestinal
epithelial cells, macrophages or adipocytes.
LXRs, closely PPAR-related members of the nuclear receptor fam-
ily, are important regulators of cholesterol, fatty acid, glucose
homeostasis and play an essential role at the interface between
metabolism and inammation [29]. Their endogenous activators
are oxysterols and other derivatives of cholesterol metabolism
(22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 24(S), 25-
epoxycholesterol). LXRa is highly expressed in the liver but is also
found in kidney, intestine, adipose tissue, and macrophages. LXRb
is expressed ubiquitously[30]. Nutritional or pharmacological mod-
ulation of the activity of these ligand-activated transcription factors
have been shown to inuence the development of metabolic disor-
ders such as hyperlipidemia and atherosclerosis [30]. Mice without
functional LXRa e.g. are healthy when fed with a low-cholesterol
diet but when fed a high-cholesterol diet these LXRa knockout mice
develop enlarged fatty livers, less functional hepatocytes, high cho-
lesterol levels in liver, and impaired liver function [31]. LXR is a ma-
jor regulator of fatty acid synthesis and the expression of other
lipogenic genes through direct interaction with their promoters as
well as inducing sterol regulatory element binding protein-1c
(SREBP-1c) gene transcription. Activation of LXR by oxysterols (re-
lated to high dietary cholesterol) or other agonists elevates nuclear
content of SREBP-1 and increases expression of the enzymes in-
volved in de novo lipogenesis [32].
SREBPs are transcription factors of the helix-loop-helix family
and important regulators of cholesterol and fatty acid metabolism.
SREBPs are encoded by two genes, SREBP-1 and SREBP-2 resulting
in 3 proteins SREBP-1a, SREBP-1c and SREBP-2 (for more informa-
tion in particular on the complex mode of activation of these
65
transcription factors see Desvergne et al. [30,33]. Insulin induces
SREBP-1 nuclear which induces lipogenesis, MUFA and triglyceride
synthesis and storage [34]. The n 3 PUFA docosahexaneoic acid
(DHA) is a very strong nutritional suppressor of nuclear SREBP-1
expression of genes involved in lipogenesis and MUFA synthesis
in liver [35].
4. Application of transcriptomics in human intervention studies
with dietary fatty acids
4.1. Postprandial intervention studies with dietary fatty acids
Due to an easy accessible surplus of large processed food (rich in
isolated sugars and saturated fats) we are in the postprandial state
the greater part of the day in particular if increased energy
consumption by e.g. exercise rarely allows to compensate. A better
understanding of meal effects on body function and health are of
critical importance. It is known that a meal can provoke a transient
inammatory response which is low compared to a general inam-
matory response [36]. In addition, postprandial effects of high fat
meals have been observed on vascular reactivity [37] and on blood
cell activation [38]. The arrival of comprehensive genomics tech-
niques such as transcriptomics enables the unbiased measurement
of total postprandial genome-wide effects on the cellular level. Re-
cently, we assessed postprandial transcriptional responses to high
fat shakes varying in types of fatty acids [39]. Twenty healthy sub-
jects consumed a butterfat-enriched saturated fatty acid (SFA)
shake and a DHA-enriched polyunsaturated fatty acid (PUFA) shake
in random order and whole genome PBMCs gene expression pro-
les were assessed. Postprandial gene expression responses were
distinctly different between shakes as illustrated by the higher
number of genes changed upon PUFA shake consumption. It is ex-
pected that adaptive cellular response by means of gene expression
changes will only occur when cellular capacity is insufcient to deal
efciently with the high load of intracellular free fatty acids. High
concentrations of n 3 PUFA are more stress-inducing molecules
than SFA for blood cells that need to activate adaptive gene tran-
scription program to be able to cope with the high levels of this
stressors by rapid processing of n 3 PUFA and increased cellular
protection against e.g. oxidative stress. The increased expression
of genes involved in cellular stress response upon n 3 PUFA shake
consumption points clearly in this direction. Other differences be-
tween the SFA and n 3 PUFA shake could mainly be attributed
to expression alterations of genes involved in lipid metabolism with
opposite effects for the two shakes. SFA caused a postprandial up-
regulation in transcription of ABCA1, ABCG1, and SREBP1, whereas
an overload of n 3 PUFA resulted in a down-regulation in expres-
sion of the same genes. Both effects were conrmed by ex vivo incu-
bation of PBMCs with palmitic acid and the DHA, pointing to an
effect likely induced by the fatty acids itself. Interestingly, a whole
genome gene expression study in a population of around 1500 indi-
viduals showed that gene expression of ABCA1 and, ABCG1 was neg-
atively associated with TG levels and, SCD gene expression was
found to be negatively associated with HDL-cholesterol [40]. An-
other study also observed a postprandial increase in expression of
LXR-related genes in PBMCs after a high fat load. In this study the
postprandial response upon a high fat load consisting of a mix of
fatty acids in 22 patients with metabolic syndrome was examined
and similar to our SFA shake the gene expression of SREBP1 and
LXRa was found to be increased. [41]. Several mice and in vitro stud-
ies have examined the effects on n 3 PUFAs on hepatic gene reg-
ulation and showed that DHA is the most potent hepatic fatty acid
suppressor of SREBP1 gene expression [35]. Less studies have
explored the effects of n 3 PUFAs on monocytes/macrophages
but in vitro studies in macrophage cell lines showed down-regula-
tion of the LXR target genes ABCA1 and ABCG1 upon n 3 PUFA
66 L.A. Afman, M. Mller / Progress in Lipid Research 51 (2012) 6370
incubation[4244]. As these LXR-target genes play a major role in
modulating cholesterol efux from macrophages, the n 3 PUFA-
reduced postprandial expression of these genes in PBMCs in
humans may lead to an inhibition of cellular cholesterol efux. Con-
versely, the increased expression of LXR and its target genes after
SFA or mixed fatty acid consumption might result in an increased
postprandial cholesterol efux. Interestingly, daily consumption
of a lower dose of n 3 PUFAs for a longer period did not result
in a reduced expression of these genes when determined in the fast-
ing state [45]. This data combined with the nding that levels of
LXR-related gene expression where heading back to baseline 8 h
postprandially, suggests that the effects observed in PBMCs might
be temporarily and specic for the postprandial situation when
blood lipid concentrations are elevated [39]. Still, the presence of
the postprandial state the greater part of the day may lead to an
continuous trigger of which the consequences on gene expression
and cholesterol efux are yet unknown.
4.2. Longer-term intervention studies with dietary fatty acids
Daily changes in postprandial exposure will ultimately lead to
longer-term adaptations in the whole body. Daily exposure to spe-
cic fatty acids will activate adaptive transcriptional responses fre-
quently, ultimately leading to functional alterations in activity of
pathways and networks in organs such as the small intestine, liver,
heart, skeletal muscle and adipose tissue. Longer-term consump-
tion will in addition lead to incorporation of fatty acids into mem-
branes of practical every cell in the body, which will have their
consequences as well.
To determine longer-term effects of fatty acids on the cellular
transcriptome we assessed changes in whole genome transcrip-
tional proles in PBMCs and adipose tissue after long-term con-
sumption of different fatty acids. The study comprised a 26 weeks
intervention with daily supplementation of 0.4 g EPA/DHA, 1.8 g
EPA/DHA or a control oil in an elderly population. Daily consumption
of 1.8 g n 3 PUFA resulted in changes in expression of 1000 genes
while expression of only 300 genes was changed in the control
group that consumed high oleic sunower oil. The larger part of
the changes in the n 3 PUFA could be attributed to a down-regula-
tion in expression of immune related genes and genes known to be
involved in development of atherosclerosis. Additional changes
were related to an increased expression of cell-cycle genes and of
genes involved in transcriptional and translational regulation [45].
Another nutrigenomics study [46] explored the effects of two
months supplementation with 3 g EPA/DHA daily in 10 male volun-
teers on gene expression using pooled RNA of lymphocytes on a
macroarray with a selected gene set. Parallel to our results they ob-
served a decrease in expression of cytokine-related genes, but con-
trary to our data a decrease in expression of cell cycle and
transcriptional regulation genes was observed. However, it must
be noted that this study used pooled samples for macroarray ana-
lyzes in which signicant inter-individual differences are impossi-
ble to determine and one must rely on fold change cut offs.
Furthermore, PBMCs consist of a combination of lymphocytes and
monocytes which likely will have inuenced the effects on tran-
scription as well. Besides the use of arrays to study whole genome
gene transcription or a large set of genes simultaneously one study
examined the expression of a selection of genes in PBMCs in 27
healthy volunteers and found a reduction in gene expression of some
cytokines after 4 weeks supplementation with 775 mg EPA and bor-
age oil (831 mg GLA) daily [47]. Although no control group consum-
ing placebo oil with the same energy content was included in the
study and the changes in gene expression cannot solely be attributed
to EPA, the results point in the same direction as the previous two
studies. The question remains what a down-regulation in transcrip-
tion of inammatory genes means for cellular functionality and
what the systemic implications are. In the study of Gorjao [46] be-
sides pooled macroarrays, stimulation assays were performed on
the lymphocytes showing an increased production of IL-10, IFN-
gamma and TNFa and a decreased production of IL2. However, most
other studies observed a decrease in production of cytokines after
sh oil supplementation, which is more in line with the observed ef-
fect on transcriptional regulation [4851]. In addition, Meydani ob-
served a higher reduction in cytokine response upon stimulation in
older compared to young women [50] after n 3 PUFA supplemen-
tation, underlining the potential age-dependency of responses.
Overall, long-term sh oil supplementation reduces expression
of leukocyte inammatory genes and decreases cytokine produc-
tion upon leukocyte stimulation. The relevance for health outcome
of this reduced immune response has been postulated to be bene-
cial in progression and treatment of inammatory diseases such
as rheumatoid arthritis [52,53].
Besides effects of consumption of fatty acids on transcriptional
activity in blood cells dietary fatty acid effects have also been deter-
mined on whole genome gene expression of adipose tissue. Within a
completely controlled randomized dietary intervention trial of
8 weeks the effects of different types of fatty acids was studied on
adipose tissue transcriptome. Participants either consumed a but-
ter-derived saturated fatty acids (SFA)-rich diet or a rened olive
oilderived monounsaturated fatty acids (MUFA)-rich diet and adi-
pose tissue biopsies were taken before and after intervention. Whole
genome microarray analyses of these samples revealed that the SFA-
diet resulted in the most distinct changes in gene expression. Where
SFA-diet effected expression of around 1500 genes, MUFA consump-
tion effected expression of 600 genes. The most remarkable obser-
vation was the increased transcription of genes involved in immune
function after consumption of the SFA-rich diet, which was
distinctly different from the effects on the MUFA diet, after which
a much smaller and more or less opposite effect was observed on
expression of immune-related genes. Interestingly, the SFA-induced
changes in gene expression were similar to differences observed in
gene expression in adipose tissue of obese versus lean subject
[54,55] and opposite to the changes observed after weight loss in ob-
ese subjects [56]. Keeping in mind that no weight changes were
present in the participants, consumption of the more healthy type
of dietary fat (unsaturated) may be of great relevance for healthy
functional adipose tissue in obesity. Although no changes in macro-
phage inltration were observed, prolonged consumption of a SFA-
rich diet may result in inltration of macrophages as frequently per-
ceived in obesity [57,58]. Some studies have shown that SFA may
activate immune related genes by binding and activation of Toll-like
receptor 4 [59,60]. In addition, saturated FA are poor ligands for
PPARy [61], and as PPARy activation result in a reduced inamma-
tion, a decreased activation of PPARy by SFA might lead to increased
inammation. PPARy gene expression in adipose tissue and expres-
sion of some target genes was reduced upon SFA consumption.
PPARc also plays an important role in alternative M2 macrophage
activation that is of key importance for adipose tissue remodeling,
inammatory status and health [62] as shown in numerous mouse
studies [63] and increasingly also in human studies [62]. Interest-
ingly, in a recently performed mouse study we found that adipose
tissue function is essential for the prevention of non-alcoholic fatty
liver disease [12]. This again highlights the importance of the com-
prehensive understanding of nutrition-relevant inter-organ depen-
dency and crosstalk as essential basis for a healthy body function.
Future nutrigenomics research should be leading in this effort.
4.3. Overlap between long term and postprandial effects of n 3 PUFA
on PBMC transcriptome
The above described studies show that fatty acids can not only
affect gene transcription directly after consumption but will also
 L.A. Afman, M. Mller / Progress in Lipid Research 51 (2012) 6370
induce long-term changes. Based on the pathway analyses it
appears that depending on the length of the intervention expres-
sion of other genes and pathways are affected. To reveal whether
overlapping genes in PBMCs were changed between postprandial
and long-term n 3 PUFA consumption we compared datasets of
the 26 weeks intervention and the postprandial study. The overlap
was relatively low; of the 900 genes specically changed by n 3
PUFA in the long-term study and the 291 genes in the postprandial
study, 29 genes were changed upon n 3 PUFA in both studies
(Fig. 1). The expression of the larger part of the genes i.e. 19 genes
are changed in the same direction and may reect the overall ef-
fects of sh oil consumption. The expression of the remaining 10
genes is changed in the opposite direction. One of the genes up-
regulated postprandial and down-regulated after long-term inter-
vention was ADFP, a known PPARa target gene [64]. Other PPARa
target genes were down-regulated in the long-term intervention.
We hypothesize that daily intake of higher amounts of n 3 PUFA
as mentioned above induces whole body adaptation in particular in
metabolic organs such as the small intestine, liver or adipose tissue
with the consequence of lower FFA levels and consequently differ-
ent changes in gene expression levels in PBMCs compared to acute
studies. The same may hold for JUNB, a stressor/inammatory gene
and the other genes in the list. The function of the overlap in genes
varies, but several genes are immune-related such as CLEC4E and
Il13RA1 or lipid-related such as SCD. G-protein coupled receptor
18 (GPR18) is one of the genes with the highest signicantly aver-
age increase upon both long-term and acute consumption. GPR18
is know to be highly expressed in lymphocytes and N-arachidonyl-
glycine has been suggested to be an endogenous ligand of GPR18
[65], but further research concerning its function and the relation-
ship to n 3 PUFA is needed. The analysis also reveals that tran-
scriptional changes of a large number of genes are very specic
for either long-term or acute consumption of n 3 PUFA.
In conclusion, acute postprandial studies lead to short term di-
rect effects of fatty acids on transcription whereas long-term inter-
vention with daily consumption will lead to more systemic effects
that still can be monitored in PBMCs.
Fig. 1. Average gene expression changes of genes signicantly changed upon long term and acute consumption of omega3-FA. Each row represents a single gene; the rst
column represents the long-term study, the second column represents the acute study. The expression scale is log2-based and ranges from 6 0.3 (green) to P0.3 (red).
67
5. Future challenges in nutrigenomics
It is evident from the rst 10 years of nutrigenomics research and
applying comprehensive genomics techniques such as transcripto-
mics that researchers have to acknowledge relatively small changes
in gene expression induced by nutrients as compared to drugs
[26,66] with large inter-individual variations. This is not unexpected
considering the different ways drugs and nutrients work [4]. Where
drugs are designed to specically and constantly activate certain
receptors with the goal to strongly activate specic pathways, nutri-
ents will induce a much milder activation of specic receptors and
consequently of specic pathways. Instead nutrients will lead to a
more balanced cellular response by activation of multiple control
systems with the main purpose to metabolize the nutrient and to
regulate metabolism to rapidly reach homeostatic balance. If studies
are performed under physiological conditions it consequently
means that rather low amounts of bioactive compounds are applied
and low numbers of signicantly changed genes and an lower induc-
tion in changes in gene expression can be expected as there is no
need for a stronger transcriptional response. On average fatty acids
induce effect sizes ranging from a few percent till 100% with individ-
ual outliers to 300% or 400% often for immune-related genes
[39,45,67]. Both points, the low fold changes and low proportion
of genes signicantly changed require a rigorous high-quality con-
trol system coupled to an array analysis platform with high quality
arrays, strict quality control criteria and the need for integrated bio-
informatics and statistical procedures optimized for studies with
small effect sizes [68]. Moreover, it emphasizes the importance of
use of whole genome data and not only a biased selection and the
application of most recent well annotated pathway analyzes tools,
enabling the uncovering or enrichment of certain pathways or
signaling networks [69,70].
Another challenge is the great individual variations in gene
expression response to nutritional interventions largely due to
age, sex and genotype differences. As with other metabolites in plas-
ma responders and non-responders to a dietary intervention can be
identied [64]. The challenge lies in the identication of subgroups
68 L.A. Afman, M. Mller / Progress in Lipid Research 51 (2012) 6370
of responder-genes which is still complicated in the small popula-
tions studied, so far. This variance in response also emphasizes the
need for within person measurements and the relevance of cross-
over interventions or large intervention groups when using parallel
designs. The underlying reason for this response variance can be sev-
eral and may vary from genetic and epigenetic variation to nutri-
tional and life style factors. For example, imprinting during
pregnancy or during early years in life may affect phenotypic
responses at older age with the consequence of increased risk to de-
velop nutrition-related diseases [71]. Nutritional habits may also be
a reason why persons will or will not respond to a change in a diet.
Nutrigenomics can be used to asses this. For instance, people that
regularly consume sh rich in n 3 PUFA will likely exhibit a less
pronounced transcriptional response upon a sh oil challenge than
subjects that do not eat sh at all. This also may be the scientic basis
for the nutritional advice to enjoy a healthy diet consisting of a var-
ied food pattern as this will keep the homeostatic balance of our
organs most exible.
Many diseases are a result of disturbance of homeostasis or
homeostatic imbalance. As a hallmark of ageing every organism
will lose its efciency in its sensing and regulatory systems.
Diseases with a strong link to nutrition result from a homeostatic
imbalance such as obesity, metabolic syndrome or diabetes type
2. We increasingly understand the complex regulation of homeo-
static control mechanisms on cellular, organ and system level that
should prevent this imbalance from occurring. However, in some
people, these mechanisms do not work efciently enough because
of signicant genetic susceptibility [1] and or epigenetic mecha-
nism [71]. These people are much more vulnerable to develop a
disease in particular if the homeostatic control mechanisms are
chronically challenged by surplus of calories, non-ideal dietary pat-
terns and lifestyles [72]. Because we are still not able to detect the
early warning signals of such homeostatic imbalance efciently
most often medical intervention is necessary at a very late stage
of the disease development that is often unable however to restore
the homeostatic balance to regain normal health with irreversible
damage of organs as a consequence.
To change this inefcient concept in treatment of lifestyle
diseases we have to use interventions that allow the quantication
of the homeostatic balance or resilience by assessing it after chal-
lenging or perturbing a homeostatic situation. A well known exam-
ple for such as challenge is the oral glucose tolerance test (OGTT). A
standard 2 h OGTT is sufcient to diagnose or exclude forms of
diabetes mellitus at the various stages of development. Recently,
plasma metabolic proling combined with a glucose challenge
was used to differentiate between healthy individuals and individu-
als with an impaired glucose tolerance [73]. Application of meta-
bolic perturbation with comprehensive nutrigenomics-based
metabolic proling may improve the diagnostic sensitivity to help
discriminating between healthy, less-healthy and unhealthy people.
Furthermore, it may allow to predict differences in the responses be-
tween treatments [74]. In addition, combination of dietary interven-
tions with metabolic challenges magnies the difcult measurable
and relatively mild effects of nutritional alterations by testing the
cellular adaptive response capacity (for a more in depth discussion
[75,76]). This rather new concept of applying various challenges
on homeostatic control mechanisms in nutritional interventions is
still in its infancies and it is therefore necessary to carefully study
its value and limitations. One may expect important sex-dependent
differences in applying various homeostatic challenge-test as dem-
onstrated recently for OGTT [77]. But ultimately it will be the most
promising strategy to combine nutrigenomics-based comprehen-
sive phenotyping with systems perturbation interventions to allow
the precise assessment of individual health and nutritional needs.
6. Conclusions and perspective
In summary, well-controlled nutrigenomics studies on the reg-
ulation of gene transcription by dietary fatty acids in humans are
still limited partly because nutrigenomics is still a relative new
eld and not yet fully integrated in nutrition research and partly
because of still signicant costs of the genomics applications that
do not allow comprehensive phenotyping in large cohorts. The
studies that have been performed were largely of explorative nat-
ure with the purpose to identify whether transcriptional changes
can be detected after changes in dietary fatty acids consumption.
Although the number of studies is still limited, all studies clearly
suggest that changes in dietary fatty acids intake and composition
can have a signicant impact on cellular adaptive response capac-
ity by gene transcription changes in humans. This adds important
knowledge to our understanding of the strong effects that various
fatty acids can have on numerous metabolic and inammatory
pathways, signaling routes and homeostatic control in the cell
and ultimately on whole body health.
Nutritional science is entering a new era with a shift away from
little science towards big science. The major nutritional prob-
lems facing the world today (e.g. obesity, diabetes and malnutri-
tion) with huge impact on public and private parties cannot be
solved by little science, characterized by individualistic research,
lacking coordination and integration. Big science is needed, i.e.
multiple groups with complementary knowledge and expertise
working together in large national and international consortia sim-
ilar to consortia as currently working on the human microbiome
project [78] or genome-wide association studies [79]. This new
era takes full advantage of modern, high resolution, comprehensive
and unbiased Genomics technologies, bio-banking, sophisticated
and partly web-based database management, providing a new
platform for scientic dialogue and exchange [80]. We dene this
new Nutritional Science as Nutritional Science 2.0.
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