Expression Analysis of Genes Involved in Fat Assimilation
in Human Monocytes
Maja Klapper1,*, Myriam Do ¨ pner1,*, Christina Vock1, Inke Nitz1, Ulf Helwig2, Ju ¨ rgen Schrezenmeir2
and Frank Do ¨ ring1
1Molecular Nutrition, Christian-Albrechts University, Kiel, Germany
2Institute for Physiology and Biochemistry of Nutrition, Federal Research Centre for Nutrition and Food, Kiel, Germany
The metabolic syndrome X is characterized by a group of risk
factors such as obesity, atherogenic dyslipidemia, hypertension, and
insulin resistance. To study the functional alterations resulting from
genetic variations, ex vivo studies are one option to be carried out.
Since it is not an easy procedure to obtain cells from the related
tissues ex vivo, the aim of the present study was to investigate
whether monocytes can serve as model cells. The purpose was to
check if monocytes are insulin target cells or not and to elucidate the
expression of genes involved in fat assimilation. Human monocytes
were drawn from venous blood of healthy donors, aged 25–30, using
density gradient separation and antibody-based magnetic cell
sorting of CD14-positive cells. An expression analysis of genes
was performed using RT-PCR and Western Blot. Transcripts of the
three splice-variants of the Acyl-CoA binding protein (ACBP), the
Medium-chain Acyl-CoA Synthetase 1 (MACS1), the Insulin
Receptor (INSR) and the Peroxisome Proliferator-activated
Receptor gamma (PPARc) are consistently expressed in monocytes
of all donors. Differences in gene expression between donors are
found for two other members of the MACS-family, the fatty acid
transport protein 3 (FATP3) and the FATP4. On protein level, we
tested for ACBP expression. The ACBP protein is consistently
expressed in monocytes of all donors. Human monocytes are insulin
target cells and capable of fatty acid metabolism to some extent.
Ex vivo-derived monocytes could be used in additional studies for
analyzing differences in genotype-dependent expression levels of
genes involved in fat assimilation such as ACBP, MACS1 or
IUBMB Life, 58: 435–440, 2006
Metabolic syndrome; monocytes; ex vivo; fat assimila-
tion; ACBP-splice variants.
The metabolic syndrome X is a constellation of metabolic
disorders that all result from the primary disorder of insulin
resistance. The characteristic disorders present in the meta-
bolic syndrome X include: hypertension, low HDL and high
LDL cholesterol levels and high triglyceride levels (1). The
chief abnormality present in the syndrome X is insulin
resistance. As a result, insulin levels become elevated in the
body’s attempt to overcome the resistance to insulin. The
elevated insulin levels lead, directly or indirectly, to the other
metabolic abnormalities seen in these patients. When diabetes
occurs, the high risk of cardiovascular complications grows
even higher. Worldwide, the prevalence for the metabolic
syndrome increases. In Germany for example 25% of the
population suffer from the disease as a result of the western
lifestyle, such as the aging population, high caloric nutrition
and less physical activity and genetic predisposition (2).
Concerning genetic predispositions, our current studies
(3, 4) focus on a series of genes which are involved in fat
assimilation, such as fatty acid transport proteins (FATP’s),
the family of fatty acid binding proteins (FABP’s), pancreatic
colipase, the acyl-CoA binding protein (ACBP) and the family
of medium-chain acyl-CoA synthetases (MACS). It is of great
interest what kind of alterations in the metabolism can be
observed in relation to a specific genotype of these genes.
Therefore different types of studies can be carried out: in vitro
studies using cell culture or promoter assays, in vivo studies
performing dietary interventions or tolerance-tests and ex-vivo
studies using isolated cells from genotyped subjects for
functional essays or just quantifications of mRNA levels in
purpose to identify biomarker molecules.
Ex vivo models currently in use concerning genetic analysis
of fatty acid metabolism are limited due to the different
relevance of the tissues for target pathways of metabolism and
the difficulties in the isolation of cells from some tissues for
daily use. Relevant tissues are adipose tissue and muscle tissue,
Received 30 March 2006; accepted 8 May 2006
Address correspondence to: Frank Do ¨ ring, Molecular Nutrition,
University of Kiel, Heinrich-Hecht-Platz 10, 24118 Kiel, Germany.
Tel: þ49 431 609 2469. E-mail: email@example.com
IUBMBLife, 58(7): 435–440, July 2006
ISSN 1521-6543 print/ISSN 1521-6551 online ? 2006 IUBMB
but the methods for isolating these cells require a lot of work
and are not suitable for common use. A way of obtaining cells
ex vivo easily is by their isolation from human salivary or
blood. In this study expression analysis of genes involved in fat
assimilation in freshly isolated human monocytes was
MATERIAL AND METHODS
Isolation and Purification of Human Monocytes
Peripheral blood mononuclear cell isolation was used and
described by various authors (5–9). Briefly, 25 ml of venous
blood was collected from each of the four female healthy
donors, aged 25–30, in tubes (1.6 mg EDTA/ml blood,
Sarstedt AG & Co, Nu ¨ mbrecht, Germany). Whole blood
was then diluted 1/1 with sterile 0.9% sodium acetate solution.
Peripheral blood mononuclear cells (PBMCs) were obtained
by densitygradient centrifugation
(d¼1.077 g/ml, Nycomed Pharma AS, Oslo, Norway). The
samples were further centrifuged at 8006g for 20 min at room
temperature with no brakes. The interface, containing the
PBMCs, was collected and washed three times with sterile
washing buffer (10 mM phosphate-buffered saline (PBS)
þ10% NCS, purchased from Biochrom KG, Berlin, Ger-
many), 3006g, 10 min at 48C. PBMCs were then incubated
with anti-CD14 monoclonal antibody coated with microbeads
(Miltenyi Biotec, Bergisch Gladbach, Germany) and CD14 (þ)
cells (monocytes) were isolated by passing the PBMCs through
a magnetic cell separations system (MACS; Miltenyi Biotec)
with column type MS. Before passing the cells through the
column, they were first passed through a pre-separation filter
(mesh size: 30 mm, Miltenyi Biotec). The total number of
isolated monocytes ranged from 26106– 6.16106/ml, counted
in Coulter Multisizer II, Beckman Coulter GmbH, Krefeld,
Germany. About 95% of the cells were judged to be monocytes
by morphology and FITC-conjugated CD14 antibody staining
(FITC, Miltenyi Biotec). Viability was checked with tryphan
blue exclusion method.
Total RNA was obtained from freshly isolated, pelleted
CD14 (þ) cells (monocytes) and remaining CD14 (7) white
blood cells, using the RNeasy Mini Protocol (Qiagen GmbH,
Hilden, Germany). The optional on-column DNase Digestion
with the RNase-free DNase Set (Qiagen GmbH) was also
performed. The amount of total RNA obtained from CD14
(þ) cells ranged from 60–153 mg/ml and for CD14 (7) cell
from 55–117 mg/ml. The reverse transcription of the isolated
RNA was performed by using the Sensiscript Protocol for
Reverse Transcription of 550 ng RNA (Qiagen GmbH). For
first-strand cDNA synthesis Sensiscript Reverse Transcriptase
from Qiagen was used with 25 ng RNA of each of the four
donors. For RT-PCR the samples were incubated on thermo-
cyclers (Biometra T1 Thermocycler, Goettingen, Germany)
using the recommended master mix, containing Taq Poly-
merase, specific primers (c¼10 pmol) for 18 different genes
going to be tested and 2 ml cDNA in each sample. Primers
(purchased from MWG Biotech AG, Ebersberg, Germany)
were designed using the RNA/DNA primer analysis software
‘oligo’ version 4.0 (Molecular Biology Insights Inc., Cascade,
To check the quality of RNA, the samples were run on a
0.9% agarose gel at 80 V for 35 min. The amplified RT-PCR
products were run on a 2% agarose-gel at 90 V for 45 min,
band sizes were identified using a DNA marker, purchased
from New England Biolabs.
Lysis of pelleted monocytes and CD14 (7) cells was
performed in 150–650 ml NET Buffer, depending on the total
number of pelleted cells, containing 50 mM TRIS (pH 7.5),
150 mM NaCl, 1 mM EDTA (pH 8.0), 0.5% NP-40. Protease-
inhibitor (Roche, Mannheim, Germany) was added to the
buffer in a dilution of 1/10. After 10 min incubation on ice the
cell-lysates were treated with ultrasonic for 30 sec at 100 watt,
keeping the lysates on ice. Protein samples were analysed by
western blot technique according to the manufacturer’s
instructions (NuPage1, Invitrogen, Karlsbad, USA). The
blots were developed using an enhanced chemoluminescence
Bioscience, Buckinghamshire, UK).
Isolation of Human Monocytes
Blood was drawn from healthy female donors followed by
separation (density gradient) and purification (magnetic cell
labelling) procedure, for several times to obtain as many
monocytes (CD14 (þ)) as possible. The separation of CD14
(þ) and remaining CD14 (7) cells resulted in differing total
numbers of isolated cell fractions, depending on donor.
Depending on the donor, the total number of isolated
monocytes ranged from 26106– 6.16106/ml. Staining with
a FITC-conjugated CD14 antibody was performed to check
the purity of the isolated CD14 (þ) blood monocytes. The
calculated purity was about 95%. As shown in Fig. 1, light
microscopy (a,b) shows CD14 (þ) cells after their isolation
from whole blood, whereas in (c,d) the same cells are imaged
by fluorescence microscopy. The comparison of (b) and (d),
being magnifications of the cells, shows the efficiency of the
monocytes isolation: 15 out of 16 cells in this example are
To evaluate the expression of genes involved in fat
assimilation, RT-PCR was performed in CD14 (þ) monocytes
436 KLAPPER ET AL.
and CD14 (7) cells of each of the 4 donors (Fig. 2). The
transcripts of the housekeeping gene GAPDH served as a
control (Fig. 3). As shown in Fig. 3, the expression level of
GAPDH-mRNA is very similar in donor A, C and D. In
donor B, we found a slight reduction of GAPDH-mRNA
expression. The following transcripts were not found in CD14
(þ) nor CD14 (7) blood cells (Fig. 2): MACS2, FABP1,
FABP2, Glut2, Glut4 and MTP. Transcripts detected with a
high reproducibility in 4 out of 4 donors and in both cells
fractions CD14 (þ) and CD14 (7) are: ACBP 1a, ACBP 1c
and MACS1. Coming to the differences between CD14 (þ)
and CD14 (7) cells and the different reproducibility of the
remaining genes, analysis showed that ACBP 1b, the insulin
receptor and PPARg are available in the CD14 (þ) cells but
not in CD14 (7) cells. ACBP1b was detected in 3 out of 4
donors and the insulin receptor gene in 2 of the 4 donors. The
CD14 (7) blood cells of all 4 donors lack transcripts of
FATP3, FATP4 and MACS3. In CD14 (þ) blood cells
FATP3 was detected in 1 out of 4 donors, FATP4 in 2 out of 4
donors and MACS3 in 1 out of 4 donors. In CD14 (7) cells
transcripts of SAH were found in each of the 4 donors,
compared to CD14 (þ) cells, in 3 out of 4 donors.
Protein Detection (Western Blot)
The CD14 (þ) and CD14 (7) blood cells were also analyzed
for expression of ACBP. As shown in Fig. 4, ACBP could be
detected in all donors. As ACBP variants 1a and 1c have a
very similar molecular weight, it cannot be distinguished if one
or both variants were detected.
To test the suitability of human monocytes for the use of
these cells for further ex vivo studies, the expression of those
genes, involved in fat assimilation that are associated with
diseases contributing to the metabolic syndrome X was
checked. Monocytes were isolated from venous blood of four
healthy female donors, aged 25–30, by density gradient
centrifugation and magnetic cell sorting, separating the
peripheral blood mononuclear cells (PBMCs) into the mono-
cyte-fraction CD14 (þ) and the remaining CD14 (7) cell
fraction (containing predominantly lymphocytes). Similar
methods are described by various authors and for the present
study this literature was consulted (5–9). ACBP was also
found at the protein level analyzed by western blotting.
The occurence of an insulin receptor proves that monocytes
are insulin sensitive cells, but the lack of GLUT4 gives
evidence that glucose uptake in monocytes is not insulin
mediated. Fu et al. (2004) showed that there are GLUT1 and
GLUT3 proteins are expressed in monocytes (10). Differentia-
tion of monocytes into macrophages was associated with
marked induction of GLUT3 and GLUT5 protein expression.
In agreement with our study, ACBP has been ubiquitously
found in all tissues (11) and it is also expressed in monocytes.
Monocytes were also analyzed for the PPARg2 subtype, since
a common polymorphism in this gene is strongly associated
with the metabolic syndrome. However, the PPARg2 tran-
script was not detected in monocytes.
Some gene transcripts were not found in all of the tested
subjects, some even appeared only in one subject, for example
Figure 1. Purity of isolated monocytes analyzed by staining with FITC. Light microscopy shows the cells after their isolation
from whole blood. Light microscopy (a,b) shows CD14(þ) cells after their isolation from whole blood, whereas in (c,d) the same
cells are imaged by fluorescence microscopy.
FAT ASSIMILATION GENES IN HUMAN MONOCYTES437
FATP3. The phenomenon could be due to various reasons. It
might be that in some subjects concentrations of these
transcripts were too low to detect them using the present
methods. It also can be supposed that the expression of some
genes is related with genotype, carriers of different variants
might have different expression levels of RNA transcripts. For
example, Iwai et al. (2002) reported an association between
expression level of mRNA of SAH and the A/G polymorph-
ism of this gene in vivo (12). An additional aspect which caused
concern was the ex vivo handling of blood (13) which identified
hundreds of genes in monocytes that are sensitive to ex vivo
handling prior to RNA extraction for gene expression. As
genes of fatty acid metabolism and transport are poorly
investigated in monocytes it is possible that for some genes this
is the case. Concerning the function of monocytes a reason for
reduced metabolic activity and thus low gene expression might
be that monocytes are only precursor cells of immunological
active macrophages which exhibit enhanced gene expression
(10). It is possible to differentiate ex vivo-derived monocytes
into macrophages in vitro (14) or even into specialized
dendritic cells (15). These types of protocols seem to be useful
for future studies.
Gene expression in human monocytes/macrophages has
already been analyzed by various authors mainly focussing
on questions of immunological aspects due to the natural
Figure 2. RT-PCR-products detected in CD14 (þ) cells
(monocytes). Different pcr-programs were used to obtain the
different products (see Table 1). Product-bands have to appear
at 475 bp for FABP2, 528 bp for insulin receptor, 357 bp for
PPARg, 619 bp for FATP3, 420 bp for FATP4, 440 bp for
Glut4, 643 bp for MTP, 213 bp for FABP1, 503 for MACS1,
546 bp for MACS2, 530 bp for MACS3, 499 bp for SAH,
411 bp for ACBP 1a, 421 bp for ACBP 1b and 600 bp for
ACBP 1c. The upper band appearing in the ACBP 1c sample is
due to unspecific binding. . to check efficiency of the designed
primers, controls (CaCo cells for MTP and FABP1; and
muscle cells for Glut4) were also analysed.
Figure 3. RT-PCR-products of the housekeeping gene
GAPDH in CD14 (þ) cells (monocytes) of different donors.
RT-PCR-products appear at 508 bp. For PCR-conditions see
Figure 4. Western blot analysis of ACBP expression. ACBP
variants 1a and/or 1c (similar molecular weight of 10 kDa)
were detected in CD14 (þ) (monocytes) and CD14 (7) cells.
438KLAPPER ET AL.
function of monocytes/macrophages (16). But there are also
already studies using human monocytes/macrophages in the
context of fatty acid metabolism and diabetes type 2. Kerkhoff
et al. (1997) used macrophages to prove that the mRNA level
of ACBP is not altered by cholesterol loading in vitro (17). The
insulin receptor of human monocytes was also already used to
evaluate the association between insulin resistance and insulin
receptor tyrosine kinase activity (18).
In conclusion, human monocytes are insulin target cells and
capable of fatty acid metabolism to some extent. Ex vivo-
derived monocytes could be used in further studies for
analyzing differences in genotype-dependent expression levels
Primers used for RT-PCR
GenePrimer sequence Product length PCR-conditions
FABP2 Forward 50-gacagcacttggaaggtagac-30
Reverse 50-acgataccaaagttgtcatggatg - 30
GLUT2 484 bpA
GAPDH 508 bpA
FATP3 619 bpA
GLUT4 440 bpA
MTP 643 bpB
FABP1 213 bpB
ACBP 1a 411 bpC
ACBP 1b 421 bpC
ACBP 1c600 bpD
A: 16948C, 3 min.; 356948C, 30 sec.; 578C, 30 sec.; 728C, 50 sec.; 16728C, 10 min.
B: 16948C, 3 min.; 356948C, 30 sec.; 548C, 30 sec.; 728C, 50 sec.; 16728C, 10 min.
C: 16948C, 60 sec.; 56948C, 15 sec.; 668C, 15 sec.; 728C, 50 sec.; 66948C, 15 sec.; 648C, 15 sec.; 728C, 50 sec.; 336948C, 15 sec.;628C, 15 sec.; 728C,
50 sec.;16728C, 6 min.
D: 16948C, 5 min.; 56948C, 30 sec.; 668C, 30 sec.; 728C, 50 sec.; 66948C, 30 sec.; 648C, 30 sec.; 728C, 50 sec.; 336948C, 30 sec.; 628C, 30 sec.; 728C,
50 sec.; 16758C, 6 min.
E: 16948C, 3 min.; 56958C, 20 sec.; 568C, 20 sec.; 728C, 30 sec.; 56948C, 20 sec.; 548C, 20 sec.; 728C, 30 sec.; 256948C, 20 sec.; 528C, 20 sec.; 728C,
30 sec.; 16728C, 10 min.
F: 16948C, 3 min.; 56948C, 20 sec.; 588C, 20 sec.; 728C, 30 sec.; 56948C, 20 sec.; 568C, 20 sec.; 728C, 30 sec.; 256948C, 20 sec.; 548C, 20 sec.; 728C,
30 sec.; 16728C, 10 min.
FAT ASSIMILATION GENES IN HUMAN MONOCYTES439
of genes involved in fat assimilation such as ACBP, MACS1 Download full-text
This work was financially supported by the BMBF-Project
‘Fat and metabolism – gene variation, gene regulation and
gene function’ (AZ 0312823A/B). We thank Y. Dignal,
D. Stengel, and M. Steinke for excellent technical assistance.
We thank N. J. Faergeman for the ACBP antibody.
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