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*
*
SCD1
SCD1
SCD1SCD1
SCD1
Fatty acids (FAs) are the main building blocks of the structurally very heterogeneous lipid compounds. e
diverse functions of these lipids include membrane formation, energy storage and signal transduction1. Excessive
FA overload leads to abnormal lipid accumulation, cell dysfunction, or even cell death in both adipose and non-
adipose tissues2–4, a phenomenon known as lipotoxicity. Several studies in various experimental settings suggest
that the saturated (SFAs) and the unsaturated FAs (UFAs) contribute dierently to lipotoxicity. e deleterious
eects of palmitate, which can be mitigated in the presence of oleate, have already been widely demonstrated5–8.
In contrast, very little is known about the cellular eects of dietary trans fatty acids (TFAs), despite their implica-
tion in type 2 diabetes mellitus (T2DM) and cardiovascular diseases9, systemic inammation10, dyslipidemia11,
endothelial dysfunction12, dierent types of cancer13 and neurodegenerative disorders14. TFAs are UFAs that
contain at least one double bond in trans conguration. e vast majority of TFAs are produced in industrial
processes (industrial or iTFAs). Elaidate is the primary iTFA, oen found in partially hydrogenated vegetable
oils. Ruminants’ milk and meat also contain small amounts of naturally occurring TFAs (ruminant or rTFAs),
mainly the trans isomer of vaccenate9.
Human de novo FA synthesis yields palmitate, which can be extended by two-carbon units to longer saturated
chains, thus, the balanced production of saturated (SFAs) and mono- (MUFAs) or endogenous polyunsaturated
FAs (PUFAs) is reliant on desaturation. Although cis double bonds can be formed at dierent positions up to 9,
the rst one must be created at Δ9 position by stearoyl-CoA desaturase-1 (SCD1), which makes the activity of
SCD1 crucial for the overall desaturation process15.
e desaturase activity currently available to the cell is dependent on the level of the SCD1 enzyme, which,
in turn, is determined by (i) the transcriptional and post-transcriptional regulation of the synthesis and (ii)
1Department of Molecular Biology, Semmelweis University, 1085 Budapest, Hungary. 2These authors contributed
equally: Kinga Tibori and Veronika Zámbó. *email: zambo.veronika@med.semmelweis-univ.hu; kereszturi.eva@
semmelweis.hu
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the regulation of the protein degradation. e gene contains an alternative polyadenylation site, which results
in two dierent 3’ untranslated regions (3’ UTRs) and consequently dierent mRNA transcripts with distinct
stability, which may allow a rapid and ecient regulation of protein levels16. SCD1 transcription is also con-
trolled by several activating (insulin, growth factors, glucose, sucrose and cholesterol) and inhibitory (leptin,
glucagon, docosahexaenoic acid and arachidonic acid) agents acting through various transcription factors (TFs)
(LXR, SREBP, PPAR, C/EBP, TR)17. It is evident that intracellular FA supply and composition also modulates
SCD1 expression as the transcription of SCD1 gene is eciently induced by saturated FAs such as palmitate or
stearate, whereas it is repressed by cis-MUFAs (e.g., oleate)18. In addition, a highly conserved PUFA-sensitive
region (PUFARE) has also been identied in the upstream regulatory region of SCD1, which signicantly down-
regulates the intracellular SCD1 mRNA pool in the presence of linoleate18–20. SCD1 is an enzyme with a short
half-life and its intracellular abundance is ne-tuned in the short term by the rapid degradation of the protein.
Its N-terminus contains a PEST degradation domain that is presumably involved in targeting the protein to
the proteasome during the ERAD process21. e stability of SCD1 protein in lung cancer cells is increased by
tyrosine phosphorylation22, and a UFA-induced degradation of stearoyl-CoA desaturase has been reported in
Drosophila, although the latter phenomenon has not been demonstrated in the case of the human orthologue23.
Natural genetic variations in SCD1 may also alter the above-described molecular mechanisms underly-
ing the control of SCD1 expression. A common missense single nucleotide polymorphism (SNP) (rs2234970,
M224L) was found to increase protein and mRNA stability, which could be further enhanced by dierent FAs,
and resulted in elevated intracellular UFA levels24. Furthermore, the GG haplotype of two intronic SCD1 vari-
ants (rs55710213 and rs56334587) signicantly reduced SCD1 expression by disrupting HNF4A TF binding25.
Conversely, a SNP (rs41290540) located in the 3’ UTR increased SCD1 expression in a luciferase reporter assay
by truncating a miR-498 target sequence26.
Changes in the intracellular level of SCD1 may represent risk factors for the development of various diseases.
However, despite their obvious health impact, the possible modulatory eects of either TFAs or natural human
polymorphisms in the SCD1 promoter have not been investigated. In the present study, we aimed to investigate
the eect of dierent saturated, cis- and trans-unsaturated fatty acids on the expression of SCD1 invitro at
mRNA and protein level. In addition, we planned to address the potential role of FAs, in particular elaidate and
vaccenate, and that of selected polymorphisms in the 5’ region of SCD1 in modulating promoter activity, both
separately and in combination, in a luciferase reporter system. We also aimed to analyze the potential impact
of functional promoter variants in silico and invitro on TF binding site modication and their correlation with
T2DM in an association study.
e modulating eect of the dietary SFAs, cis-MUFAs and PUFAs on the expression of the main desaturase
enzyme, SCD1 has been well characterized18–20, however the possible regulatory impact of the two major dietary
TFAs, i.e., elaidate (18:1 trans-Δ9) of industrial origin and the naturally occurring vaccenate (18:1 trans-Δ11)
remains to be elucidated. To compare the eects of TFAs with those of other FAs on the cellular level of the
SCD1 protein, HEK293T and HepG2 cells were treated with BSA-conjugated oleate, palmitate, stearate, linoleate,
vaccenate and elaidate at a nal concentration of 100µM for 24h and their SCD1 content was assessed by
immunoblotting (Fig.1A,C) then evaluated by densitometry (Fig.1B,D). Consistent with the literature, mono-
unsaturated oleate and polyunsaturated linoleate treatment resulted in signicantly lower intracellular protein
levels in HEK293T cells (Fig.1A). Oleate reduced the amount of the desaturase enzyme to less than a h of the
control level in untreated cells, and SCD1 level was barely detectable in the cells treated with linoleate (Fig.1B).
Consistent with previous studies, a slight increase in SCD1 protein levels was observed in response to saturated
palmitate and stearate, although the change was only statistically signicant in the latter (Fig.1B). Most impor-
tantly, both TFAs tested (elaidate and vaccenate) markedly aected SCD1 protein amounts in HEK293T cells,
but in opposite directions. Since SCD1 levels were approximately halved by vaccinate and almost doubled by
elaidate, the opposite impact of the two TFAs resulted in a signicant dierence of more than fourfold between
the SCD1 protein content detected in cells treated with the two TFAs (Fig.1A,B).
e impact of the same FAs on SCD1 protein levels was also tested in HepG2 cells, which are of hepatocyte
origin and thus more relevant for lipid metabolism. Although intracellular SCD1 content was not signicantly
altered upon administration of oleate or SFAs, the repression by linoleate, the induction by elaidate and the signif-
icant dierence between the two TFAs could also be demonstrated in HepG2 cells at the protein level (Fig.1C,D).
SCD1
e observed impact of the FAs tested on intracellular SCD1 protein levels may be attributed to changes in tran-
scriptional activity, mRNA stability or protein stability. To learn more about the underlying mechanism, SCD1
expression was also studied at mRNA levels in HEK293T and HepG2 cells. Aer FA treatment, the gDNA-free
total RNA extracts of the cells were reverse transcribed into cDNA and mRNA expression was assessed by qPCR
as described in Materials and Methods section. Consistent with the changes in protein expression, the SCD1
mRNA content showed a very similar pattern aer administration of dietary FAs (Fig.2A,B). In the HEK293T
cell line, oleate, linoleate and vaccenate signicantly decreased the expression of SCD1 mRNA, whereas palmitate,
stearate and elaidate did not cause any measurable change. Again, the two TFAs resulted in remarkably dier-
ent expression levels, i.e., the amount of SCD1 mRNA was approximately twice as high in elaidate-treated cells
than in vaccenate-treated ones (Fig.2A). e expression pattern of SCD1 mRNA in HepG2 cells also faithfully
reected the protein levels detected by immunoblotting, so both the down- and up-regulating eects of linoleate
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AC
B
D
SCD1
Actin
SCD1
Actin
40
40
55
kDa
40
40
55
kDa
OPSLV
100 µM for 24 hours
Ctrl EOPSLV
100 µM for 24 hours
Ctrl E
HEK293T HepG2
Relative density (SCD1 / Actin)
Relative density (SCD1 / Actin)
0
50
100
150
200
Ctrl OPSLVE
**
***
0
50
100
150
200
250
Ctrl OPSLVE
*
***
*
**
###
###
Figure1. Eect of various dietary FAs on the expression of SCD1 in HEK293T and HepG2 cells. Cells were
treated with BSA-conjugated oleate (O), palmitate (P), stearate (S), linoleate (L), vaccenate (V) or elaidate (E) at
a nal concentration of 100μM for 24h. Immunoblot analysis of cell lysates (20µg protein per lane) was carried
out using anti-SCD1 and anti-Actin antibodies. Representative results of four (HEK293T) or ve (HepG2)
independent experiments are shown (A, C). Uncropped versions of all parallel blot images are available in
the Supplementary Information le. e band intensities were determined by densitometry and SCD1/Actin
ratios are shown as bar graphs (B, D). Statistical analysis was performed with the Tukey–Kramer Multiple
Comparisons Test. Data are shown as mean values ± SD. Ctrl: control; *p < 0.05; **p < 0.01; *** and ###p < 0.001.
0
50
100
150
Ctrl OPSLVE
0
50
100
150
200
Ctrl OPSL
VE
AB
HEK293
TH
epG2
Relative mRNA expression
(SCD1 / GAPDH)
Relative mRNA expression
(SCD1 / GAPDH)
100 µM for 24 hours100 µM for 24 hours
******
***
###***
***
###
Figure2. SCD1 mRNA expression in FA-treated HEK293T and HepG2 cells. e levels of endogenous SCD1
mRNA were measured in HEK293T (A) and HepG2 (B) cells treated with dierent FAs. FA treatment and
sample preparation were performed as described in Materials and Methods. qPCR was carried out using SCD1
and GAPDH sequence specic primers as indicated in Materials and Methods. e diagram presented depicts
the results of six independent measurements. Statistical analysis was performed with the Tukey–Kramer
Multiple Comparisons Test. Data are shown as mean values ± S.D. *** or ###p < 0.001.
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and elaidate, respectively, were seen on SCD1 mRNA quantities. e mRNA levels in the elaidate-treated samples
exceeded those in the vaccenate-treated ones to a similar extent as observed in HEK293T cells (Fig.2B).
SCD1
It became evident that, similarly to oleate, linoleate, palmitate and stearate18–20, TFAs also remarkably aect the
expression of SCD1. We wanted to investigate whether this eect is based on transcriptional regulation and/or
RNA stabilization. To this end, the 1094 base pair long section of the 5’ regulatory region of SCD1 (Fig.4A) was
cloned into pGL3-Basic vector and used in a luciferase reporter system to assess the SCD1 promoter-dependent
AB
HEK293
TH
epG2
Relative luciferase activity
Relative luciferase activity
0
50
100
150
200
250
pGL3BCtrl OPSLVE
0
50
100
150
200
pGL3B Ctrl OPSL
VE
SCD1 promoter
pGL3B
OPSLVE
Ctrl
100 µM for 24 hours
SCD1 promoter
pGL3B
OPSL
VE
Ctrl
100 µM for 24 hours
***
***
*
**
****
*
***
###
##
Figure3. Eect of dietary FAs on SCD1 promoter activity. Transient transfection and FA treatment of
HEK293T (A) and HepG2 (B) cells were performed as described in Materials and Methods. pCMV-β-gal vector
served as transfection control. Luciferase and β-galactosidase enzyme activities were measured as indicated
in Materials and Methods and their relative ratios are shown as bar graphs. e diagram depicts the results of
three (HEK293T) or six (HepG2) independent measurements normalized to pGL3-SCD1 promoter vector.
Data are shown as mean values ± S.D. Statistical analysis was performed by using the Tukey–Kramer Multiple
Comparisons Test. Ctrl: control; O: oleate; P: palmitate; S: stearate; L: linoleate; V: vaccenate; E: elaidate;
*p < 0.05; **p < 0.01; *** and ###p < 0.001.
C
B
A
1094 bp
5’ 3’‒1089 +5
Translation
SCD1 promoter
rs2275656 G>C
rs2275657 G>C
rs670213 C>T rs1054411 C>G
0
50
100
150
0
50
100
150
Relative luciferase activity
HEK293
TH
epG2
Relative luciferase activity
Figure4. Position (A) and eect of SCD1 promoter SNPs on relative luciferase activity in HEK293T (B)
and HepG2 (C) cells. e subcloned region of SCD1 promoter, the translational start site and the location,
allelic options and ID number of the four selected polymorphisms are presented. Transfection was performed
as described in Materials and Methods. pCMV-β-gal vector served as transfection control. Luciferase and
β-galactosidase enzyme activities were measured as indicated in Materials and Methods and their relative ratios
are shown as bar graphs. e diagram depicts the results of three independent measurements normalized to the
wild type SCD1 promoter. Data are shown as mean values ± S.D. Statistical analysis was performed by using the
Tukey–Kramer Multiple Comparisons Test.
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transcriptional eects. HEK293T and HepG2 cells transfected with pGL3-SCD1 promoter construct were treated
with BSA-conjugated oleate, palmitate, stearate, linoleate, vaccenate or elaidate at 100µM concentration for 24h.
Aer harvesting the cells, relative luciferase and β-galactosidase activities were determined. e subcloned SCD1
5’ regulatory region worked as a potent promoter, increasing the relative luciferase activity in both cell lines by
approximately 20-fold compared to pGL3-B (Fig.3). As expected, a signicant suppression by linoleate on lucif-
erase activity was seen in both cell lines, but surprisingly, no such phenomenon was observed for oleate. e two
SFAs signicantly enhanced the relative luciferase activity in both HEK293T (Fig.3A) and HepG2 (Fig.3B) cells.
Of the two TFAs, elaidate caused an approximately 50% increase in SCD1 promoter activity, whereas vaccenate
was not eective in this assessment, thus the signicant dierence between the two TFAs was also revealed at
the transcriptional regulation of SCD1.
SCD1
Regulation of the transcription can be signicantly aected by genetic variations of the promoter region. ere-
fore, we tested the known SNPs in the SCD1 promoter with minor allele frequencies (MAFs) above 5% for their
potential impact on FA-sensitivity of SCD1 expression. Using the NCBI dbSNP and Ensembl databases, we
selected four polymorphisms that met the above criterion. Identication number, position, allelic variants and
MAF value of these variants are presented in Supplementary TableS1 and Fig.4A. All four SNPs were agged
as modiers by the VEP prediction program, indicating their potential functionality (Supplementary TableS1),
yet neither of them has been experimentally investigated. We generated both alleles of these genetic variants
in the pGL3-SCD1 promoter vector by site-directed mutagenesis and analyzed them by a luciferase reporter
assay in HEK293T and HepG2 cells aer transient transfection. is invitro approach revealed no signicant
modulation of basal promoter activity by any of the four polymorphisms, i.e., none of the minor allelic variants
altered the relative luciferase activity compared to the major variant (hereaer referred to as wild type) in either
of the two human cell lines (Fig.4B.C).
To further analyze the potential eect of these polymorphisms, the promoter activity of the allelic variants
was also tested in the presence of oleate, palmitate, stearate, linoleate, vaccenate or elaidate. HEK293T cells were
treated with 100µM BSA-conjugated FAs 5h aer transient transfection for 24h, and the relative luciferase
enzyme activities were measured in the collected samples as described in Materials and Methods. e G allele of
the rs1054411 SNP, which was shown to have the same basal promoter activity as the wild type (see Fig.4B,C),
resulted in a signicantly higher promoter activity than the wild type when exposed to any of the six FAs (Fig.5).
It is noteworthy that the most pronounced, almost threefold increase in the relative luciferase activity compared
to the wild type was observed upon elaidate treatment (Fig.5F). When the same allelic variant was tested in
HepG2 cells, the allele-specic impact of some other FAs was also observed, i.e., the relative luciferase activity
with the rs1054411_G promoter was increased not only by elaidate but also by linoleate and vaccenate in the
reporter system (Supplementary Fig.S1).
Beside the general FA-dependent enhancement of rs1054411_G promoter activity, it is also intriguing that
the rs2275656_C variant apparently lost its FA-sensitivity, as it did not respond to any FA treatments investigated
(Fig.5). e other two tested SCD1 promoter variants (rs670213_T and rs2275657_C) showed rather diverse
responsiveness to FAs. While neither of them modied the eect of the cis-unsaturated FAs (oleate and linoleate)
compared to the wild type (Fig.5A,D), rs2275657_C showed a signicantly increased promoter activity both
in the presence of SFAs (palmitate and stearate) (Fig.5B,C) and TFAs (vaccenate and elaidate) (Fig.5E,F), and
such modulation was only observed with palmitate in case of rs670213_T (Fig.5B).
SCD1
e possible impact of the four SNPs on TF binding sites in the promoter of the SCD1 gene was analyzed in silico
using the JASPAR transcription factor binding site prediction program. Specically, we addressed the question
whether the alteration of the four nucleotides aected by the polymorphisms could cause a predictable change
in the binding probability of any TFs in this region. e in silico TF selection protocol has been published
previously27. Briey, for each SNP, the two 41-nucleotide long DNA segments were compared, in which the poly-
morphic nucleotide was located at position 21. Hits were selected with a relative TF binding score greater than
80% for at least one allele and a relative score dierence of at least 15% between the two alleles. Five predicted
TF binding sites for rs1054411, two for rs670213 and rs2275657, and seven for rs2275656 met the above criteria,
their IDs and relative binding probabilities are summarized in Table1 and Supplementary TableS2. e predicted
eect of the rs1054411 polymorphism on ETS1 TF binding appeared to be the most probable hit in the screen.
ETS1 had the highest binding probability score of all hits for the wild type sequence (rs1054411_C: 98.14%),
and also the largest dierence between the two alleles, with the minor allele (rs1054411_G) 21.6% less likely to
form an ETS1 TF binding site (Table1). e predicted large dierence between the two alleles of this SNP is not
surprising, considering that the polymorphic nucleotide is located at the h, highly conserved position of the
six-nucleotide long consensus sequence of ETS1 response element (Fig.6A).
SCD1
In light of the FA sensitivity of the rs1054411_G allele variant observed in our previous experiments (see Fig.5),
and the highly diverse regulatory mechanism of ETS128, the question was raised whether the ETS1 expression
is also FA sensitive. To address this question, HEK293T cells were treated for 24h with 100µM BSA-conjugated
oleate, palmitate, stearate, linoleate, vaccenate or elaidate, and the endogenous gene expression of ETS1 was
assessed in the collected samples, but the ETS1 mRNA levels were not modied by any of the FAs tested (Sup-
plementary Fig.S2). e possible FA-dependent modication of endogenous ETS1 protein levels could not be
examined because the low sensitivity of the commercially available ETS1 antibodies did not allow for proper
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detection of the protein without overexpression. e amount of ETS1 protein overexpressed in transfected cells
was not aected by the presence of FAs (Supplementary Fig.S2).
Since the ETS1 binding was predicted to be aected by the rs1054411 polymorphism, the allele-specic
eect of this TF was tested in a luciferase reporter system invitro (Fig.6). HEK293T cells were transiently co-
transfected with the pGL3-SCD1 promoter plasmid containing either the wild type or the rs1054411_G variant,
together with dierent amounts of ETS1 expression vector. 24h aer transfection, the cells were harvested, and
the relative luciferase activities were measured. e samples of the co-transfected cells were compared to the
corresponding wild type or rs1054411_G promoter activities without ETS1 overexpression. ETS1 overexpres-
sion resulted in enhanced activities of both versions of the promoter, and the eect was growing in parallel
with the increasing amounts of ETS1 expression construct applied (Fig.6B) and with the increasing amount of
ETS1 protein, as veried by immunoblotting (Fig.6C). However, there was a marked dierence between the
0
100
200
300
0
100
200
300
0
100
200
300
Relative luciferase activity
Relative luciferase activity
Relative luciferase activity
0
100
200
300
0
100
200
300
0
100
200
300
400
Relative luciferase activity
Relative luciferase activity
Relative luciferase activity
OleatePalmitate Stearate
Linoleate Vaccenat
eE
laidate
*
*** ***
** ** *
*** ***
***
*
ABC
DE
F
*
Figure5. Modulating eect of promoter polymorphisms on SCD1 promoter activity in the presence of various
FAs in HEK293T cells. Transient transfection and FA treatment of HEK293T cells were performed as described
in Materials and Methods. pCMV-β-gal vector served as transfection control. Luciferase and β-galactosidase
enzyme activities were measured as indicated in Materials and Methods and their relative ratios are shown
as bar graphs. e diagram depicts the results of at least three independent measurements normalized to the
relative luciferase activity of oleate- (A), palmitate- (B), stearate- (C), linoleate- (D), vaccenate- (E) or elaidate-
treated (F) wild type SCD1 promoter containing reporter vector. Data are shown as mean values ± S.D. Statistical
analysis was performed by using the Tukey–Kramer Multiple Comparisons Test. O: oleate; P: palmitate; S:
stearate; L: linoleate; V: vaccenate; E: elaidate; *p < 0.05; **p < 0.01; *** p < 0.001.
Table 1. List of transcription factors that are aected by rs1054411 polymorphism. Positive or negative values
of the relative score dierences indicate that the minor allele increases or decreases the probability of TF
binding, respectively.
Name TF ID Strand
Relative score (%)
C allele G allele Dierence
NFATC3 MA0625.2 + 61.45 80.39 18.94
SOX18 MA1563.1 ‒65.39 81.67 16.28
SPI1 MA0080.1 ‒81.85 66.40 ‒15.45
ETV5 MA0765.1 ‒85.96 69.13 ‒16.83
ETS1 MA0098.1 + 98.14 76.54 ‒21.60
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ETS1
Actin
55
40
55
kDa
40
0
50
100
150
200
250
300
Ctrl +10ng+25 ng +50ng+100ng+200ng
Relative luciferase activity
B
C
ETS1
Wild type
rs1054411_G
***
***
***
***
!!!
!!!
!!!
##
###
#
#
”
A
CATCCG
CATCGG
2
1.5
1
0.5
0
Bits
nt position
Wild type
rs1054411_G
5’ ‒
5’ ‒
‒ 3’
‒ 3’
Figure6. Eect of rs1054411 SNP on the ETS1-mediated stimulation of SCD1 promoter activity in a luciferase
reporter system. (A) Structure of the ETS1 TF binding sequence modied by rs1054411_G is illustrated. e
polymorphic site is highlighted in bold and red. (B) Transient co-transfection of HEK293T cells was performed
as described in Materials and Methods. pCMV-β-gal vector served as transfection control. Luciferase and
β-galactosidase enzyme activities were measured as indicated in Materials and Methods and their relative
ratios are shown as bar graphs. e diagram depicts the results of three to twelve independent measurements
normalized to ETS1-free wild type or rs1054411_G pGL3-SCD1 promoter vector, respectively. Data are shown
as mean values ± S.D. Statistical analysis was performed by using the Tukey–Kramer Multiple Comparisons
Test. Ctrl: control; #p < 0.05; ##p < 0.01; ***, ### or !!!p < 0.001. C e increasing amount of ETS1 protein expressed
in HEK293T cells co-transfected with an increasing amount of ETS1 plasmid (10, 25, 50, 100 or 200ng) was
veried by immunoblotting. Immunoblot analysis of cell lysates (20µg protein per lane) was carried out using
anti-ETS1 and anti-Actin antibodies as described in Materials and Methods. Uncropped versions of all parallel
blot images are available in the Supplementary Information le. Ctrl: control; ” indicates non-specic band on
ETS1 immunoblot; #p < 0.05; ##p < 0.01; ***, ### or !!!p < 0.001.
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extent of the ETS1-dependent enhancement between the two promoter versions tested. In case of the wild type
SCD1 promoter, even the small amount of ETS1 protein yielded by 25ng plasmid signicantly increased the
relative luciferase activity by almost one and a half times (Fig.6B). Furthermore, the largest amount of ETS1
plasmid applied (200ng) resulted in more than a twofold increase in the activity of the wild type SCD1 promoter.
Although the activity of the promoter holding the G allele of rs1054411 SNP showed an increasing trend with
increasing ETS1 levels, it remained below that of the wild type in all experimental conditions (Fig.6B), which
nding is in agreement with the in silico predicted reduction of the TF binding capacity (see Table1). e dif-
ference between the activity of the two alleles of rs1054411 aer co-transfection with 25, 50, 100 or 200ng ETS1
plasmid was signicant in favor of the wild type sequence (Fig.6B).
To further analyze the possible interaction between ETS1 TF and the presence of FAs, a combination of co-
transfection and FA treatment was performed. For this experiment elaidate was selected, as the rs1054411_G
allele in its presence was the most potent in enhancing SCD1 promoter activity. HEK293T cells were transiently
transfected with wild type or rs1054411_G allele-containing SCD1 promoter construct with or without 100ng
ETS1 expression vector and/or in the presence or absence of 100µM BSA-conjugated elaidate (Supplementary
Fig.S3). e appropriate amount of ETS1 vector and the optimal concentration of elaidate were chosen based
on our experiments shown in Figs.5 and 6. Samples were collected 24h aer FA treatment and relative lucif-
erase activities were measured. As expected, ETS1 inhibited and elaidate enhanced SCD1 promoter activity in
the presence of the rs1054411_G allele. When the two agents were administered together, however, the negative
eect of ETS1 could neither reverse nor neutralize the enhancing eect of elaidate on rs1054411_G allele-specic
promoter activity, but merely reduced it by about half (73% vs 37%, Supplementary Fig.S3).
e possible association between the rs1054411 polymorphism and T2DM was investigated in a case–control
setup. e results are summarized in Table2. e observed genotype distribution in the control group was in
agreement with the expected values based on the Hardy–Weinberg equilibrium (χ2-test p = 0.911). Allele frequen-
cies were in line with the European population data available in 1000Genomes (MAF: 41 vs. 40%), however, in
our control group the minor allele was slightly overrepresented compared to both ALFA (MAF: 35%) and global
frequencies (MAF: 28%). Association analyses were performed using both allele- and genotype-based approaches,
including the dominant model (i.e., genotype combination). As shown in Table2, the frequency of the G allele
was slightly but not signicantly lower in the T2DM group in all comparisons. Due to the limited number of
samples that could be included in the study, the power was as low as 35.6% suggesting that the lack of statistically
signicant result does not exclude the putative role of the SNP in the genetic risk of T2DM.
e role of SFAs and cis-unsaturated FAs in regulating the expression of SCD1, one of the key enzymes of lipid
metabolism, is a much-researched topic in the literature. SFAs, not surprisingly and in line with our results
(Figs.1, 2, 3), tend to increase the amount of SCD1 available in the cell, thus enhancing their own conversion to
unsaturated FAs, which favors their utilization in lipid synthesis and hence mitigates their own lipotoxic eects
throughout the body29,30. e attenuating eect of cis-unsaturated FAs on SCD1 expression is also well known20,31,
however, the mechanism of action of monounsaturated oleate and polyunsaturated linoleate may slightly dier.
Linoleate is thought to interfere with desaturation through the regulation of transcription, and it clearly repressed
SCD1 expression at all three levels we examined (promoter activity, mRNA and protein levels) in both cell lines.
is is in agreement with the fact that a PUFA-responsive element has been described and characterized in the
Table 2. Comparison of allele, genotype, and genotype combination frequencies of rs1054411 polymorphism
in control and T2DM groups.
Control
(N = 370) T2DM
(N = 282)
N % N %
Allele
C 437 59 351 62
G 303 41 213 38
χ2p = 0.2447
Genotype
C/C 127 34 107 38
C/G 183 49 137 49
G/G 60 16 38 13
χ2p = 0.4943
Genotype combination
C + 310 84 244 87
C‒60 16 38 13
χ2p = 0.3319
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upstream regulatory region of both human and mouse SCD1 genes18–20. While several studies have demonstrated
the SCD1-repressing eect of oleate in a variety of ways32, the exact mechanism is still unknown. Although oleate
clearly decreases the desaturase the level of the enzyme and thus the desaturase activity, there are inconsistent
ndings with respect to the eect at the mRNA level, and even more so with respect to the promoter activity,
suggesting that oleate acts through mRNA and/or protein stabilization rather than reducing transcription33. It
should be noted that the well-characterized reducing eect of oleate on SCD1 was not seen at all three regulatory
levels we examined in HepG2 cells, and the eect of other FAs was also rather modest in this cell line, probably
due to the relatively high FA tolerance of HepG2 cells34.
Human studies have reported a positive correlation between TFA intake and the development of lipid metab-
olism-related conditions such as the metabolic syndrome, T2DM, cardiovascular disease and cancer9, however,
the potential role of TFAs in the regulation of FA desaturation has been so far neglected. It has been reported
that TFAs may have similar protective eects against palmitate toxicity as cis-unsaturated oleate in cell cultures6.
ey promote inammation and ER stress to a much lesser extent than the most lipotoxicity-inducing SFAs,
so they are currently considered less harmful35–37. TFAs were also reported to stimulate cholesterol synthesisin
vitro38 and enhance hepatic fat accumulationin vivo39. It is therefore clear that the TFAs have special eects
on the metabolism and basic physiological processes of the human body, that are dierent from other dietary
FAs, and there is an ongoing scientic debate about the similarities and dierences between iTFAs and rTFAs in
terms of their health impacts. Several human studies have reported that the adverse eects of TFAs are limited to
iTFAs40,41, while rTFAs have been shown to be harmless or even benecial for metabolic health42,43. In contrast,
other epidemiological and clinical studies have shown that rTFAs are as culpable as iTFAs in the development
of metabolic and cardiovascular diseases44,45. In the light of the controversial data, we considered it important to
investigate and compare the eect of iTFAs and rTFAs on the expression of SCD1, as modulating the amount of
this key enzyme of lipid metabolism would provide an obvious means for the TFAs to inuence lipid homeostasis
in the human body. It was previously published that the administration of elaidate increased the desaturation
index in HASMC, HUVEC and HepG2 cells31,46, as well as the SCD1 mRNA expression in trophoblast and
HASMC cells31,47, while vaccenate did not seem to alter FA desaturation31,46,48. To rene the overall picture, we
systematically examined the eect of elaidate and vaccenate on SCD1 at the protein and mRNA levels, as well as
on the promoter activity in a luciferase reporter system in HEK293T and HepG2 cell lines. Consistent with the
limited scientic data summarized above, a signicant dierence was detected between the two types of TFAs, as
a marked inducing eect of elaidate was detected in all the three investigated parameters and in both cell lines,
which contrasted with the neutral nature of vaccenate (Figs.1, 2, 3).
Studying FA-dependent regulation of SCD1 expression is of particular interest since consequential alterations
in lipid desaturation have been implicated in a variety of diseases. Elevated cellular activity of SCD1, an enzyme
catalyzing the rate-determining step of FA desaturation, which in turn is essential for major synthetic pathways
of lipid metabolism, signicantly increases the likelihood of developing obesity and related conditions such as
the metabolic syndrome, diabetes, insulin resistance and hepatic steatosis17. SCD1 has also been identied as an
important modulator of cancer cell survival and progression49, and its expression is associated with poor progno-
sis in several cancer types50. Genetic polymorphisms may also regulate the intracellular availability of a gene or
protein in the context of gene-environment interactions, independently of, or possibly in combination with, the
eects of FAs51. As SNPs in the upstream regulatory region of SCD1 have not yet been functionally investigated,
and only the rs670213 polymorphism has been analyzed and found to be unrelated to metabolic risk52,53, in the
present study, we tested the promoter polymorphisms invitro in a luciferase reporter system both in the absence
(Fig.4) and presence (Fig.5, Supplementary Fig.S1) of various dietary FAs. e observed allele-specic inducing
properties of the FAs are not without precedent, as the elevated expression of the only common missense SCD1
variant (rs2234970) is also attributed partly to a FA-mediated and sequence-dependent protein stabilization24.
Although the rs1054411 SNP, which was found to be functional in the presence of FAs (Fig.5), did not show
signicant association with T2DM in our study (Table2), its role in the development of metabolic conditions
cannot be ruled out completely. In light of the results of our in silico analysis and invitro experiments, its possible
correlation with diabetes should be assessed in larger samples, complemented with other phenotypic and clinical
data (e.g., dietary intake composition and serum FA prole). e NCBI LDmatrix tool indicates complete linkage
disequilibrium for rs2275656 and rs2275657 SNPs, while the other two loci are not or only partially linked. In
line with this, the NCBI LDhap predicts the presence of ve haplotypes out of 16 possible combinations of the
four SNPs in the European population. is suggests that the rs670213 and rs1054411 SNPs are evolutionally
younger, their polymorphic alleles are likely to have arisen and combined with the GG haplotype of rs2275656
and rs2275657, whereas the CC haplotype of rs2275656 and rs2275657 is only found with the ancestral alleles
(both C) of rs670213 and rs1054411. Taking these together, it may be worthwhile to analyze the haplotypes of
the four SCD1 promoter SNPs from both a functional and an association perspective in the future.
ETS1, a member of the ETS protein family of TFs, regulates the expression of a diverse set of proteins through
its interaction with specic consensus sequences upstream of target genes. Increased expression of ETS1 has
been detected in a wide variety of cancers and associated particularly with tumor progression and invasion, and
there is also increasing interest in its role in basic metabolic processes54, as it has been revealed to up-regulate
key enzymes in FA metabolism55. Although the highly diversied transcriptional, post-transcriptional and post-
translational control of ETS1 has been thoroughly characterized28, the possible role of FAs in this regulation has
not been investigated. Although ETS1 expression itself was not found to be FA-sensitive in our experimental
setup (Supplementary Fig.S2), our in silico analysis identied ETS1 as a TF with allele-specic binding to the
SCD1 promoter region carrying the rs1054411 SNP (Table1). Moreover, the predicted allele-specic binding of
ETS1 was also veried invitro (Fig.6).
In summary, our results indicate that the two most common TFAs, industrial elaidate and natural vaccenate,
have signicantly dierent eects on SCD1 expression, as the induction by elaidate manifested invitro not only
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at the protein and mRNA levels of the endogenous expression but also at the promoter activity assessed in a
reporter gene model. Among the investigated promoter polymorphisms, the rs1054411, which did not modify
basal SCD1 expression, largely aected SCD1 promoter activity in the presence of dierent dietary FAs or under
the inuence of ETS1 TF, as measured by using a luciferase reporter assay.
Elevation of SCD1 expression in various health conditions is of utmost importance, whether as a cause or a
consequence17,32. e enzyme is a promising target for the treatment of metabolic diseases, and eorts have been
made to develop liver-targeted SCD1 inhibitors56. Since genetic variations have a major impact on the ecacy of
therapy57,58, SCD1 variants, including functional promoter polymorphisms, such as rs1054411, are likely to alter
the eectiveness or even the need for medical treatment with SCD1 inhibitors. e development of individual-
ized therapeutic protocols based on genetic proling seems a reasonable future goal in the treatment of lipid
metabolism-related diseases. However, this goal can only be achieved if the pathomechanisms are understood at
the level of gene-environment interaction, which requires both detailed functional characterization of disease-
associated gene polymorphisms and thorough mapping of environmental risk factors.
e main strength of this work lies in its diversity, as the opposing eects of the two trans-monounsaturated
FAs on SCD1 have been successfully demonstrated at multiple levels and in dierent cell lines. Furthermore, a
unique FA-dependent transcriptional modulation mechanism of the rs1054411 SNP in the SCD1 promoter has
been identied, which may be further ne-tuned in an allele-specic manner by the ETS1 proto-oncogene TF.
However, the association study performed is of limited value as it has very low statistical power due to the rather
small sample size. In addition, functional analysis of the four promoter polymorphisms in haplotypes and exten-
sion of the invitro studies to animal models could further increase the reliability of the present work in the future.
Culture medium and supplements were purchased from ermo Fisher Scientic (Waltham, MA, USA). Oleate,
palmitate, stearate, linoleate, elaidate, vaccenate, bovine serum albumin, HEK293T and HepG2 cells were pur-
chased from Sigma-Aldrich (St. Louis, MO, USA). All chemicals used in the study were of analytical grade. All
experiments and measurements were performed using Millipore ultrapure water.
Based on the NCBI and Ensembl databases, SCD1 promoter SNPs with MAF above 5% and heterozygosity
above 0.095 were selected. e JASPAR (http:// jaspar. gener eg. net/, accessed on 30 June 2022) open-access, non-
redundant TF biding prole database was used to predict the potential eect of rs1054411, rs670213, rs2275657
and rs2275656 polymorphisms on TF binding to the SCD1 promoter59. e allele-specic eect on TF binding
was analyzed as previously described27. Briey, both allelic variants of each SNP were compared pairwise. TFs
showing a score dierence of at least 15% between the two variations of the given polymorphism, and a rela-
tive score above 80% for at least one of the alleles, were retained for further analysis. e impact of the selected
sequence variants was predicted in silico using the Variant Eect Predictor (https:// www. ensem bl. org/ Homo_
sapie ns/ Tools/ VEP/, accessed on 12 January 2023)60.
A 1094 base pair fragment of the upstream regulatory region of SCD1 was amplied from human genomic DNA
template by iProof™ High-Fidelity DNA Polymerase (Bio-Rad, Hercules, CA, USA) and cloned into the pGL3-
Basic plasmid (pGL3B, Promega, Madison, WI, USA) between the Xho I and Hind III restriction endonuclease
recognition sites with 5’‒AAA TTT CTC GAG CAA AAC ATC CCG CAC GCA T–3’ sense and 5’‒AAA TTT
AAG CTT GGC ATC TTG GCT CTC GGA TG –3’ antisense primers. Bold letters indicate the recognition sites
of the two endonucleases, respectively. Aer purication and restriction endonuclease (ermo Fisher Scientic,
Waltham, MA, USA) digestion, the amplicons were ligated (T4 Ligase, ermo Fisher Scientic, Waltham, MA,
USA) into pGL3B vector (Promega, Madison, WI, USA) upstream the luciferase reporter gene. e natural
variants were generated using Q5® Site-Directed Mutagenesis Kit (New England BioLabs, Ipswich, MA, USA)
following the manufacturer’s instruction. Mutagenic primers were designed using the online NEB primer design
soware, NEBaseChanger™. Aer digestion of the original non-mutated and methylated plasmid by KLD reac-
tion, an aliquot of the constructs was transformed into XL10-Gold® Ultracompetent Cells (Agilent, Santa Clara,
CA, USA), which were then screened for positive colonies by PCR. e cloning and mutagenic primers are listed
in Supplementary TableS3. e ETS1 expression plasmid was purchased from BioCat (Heidelberg, Germany)
with pcDNA3.1(‒) vectorial background. All constructs were veried by Sanger sequencing.
Human embryonic kidney (HEK293T) and hepatocellular carcinoma (HepG2) cells were cultured in 12-well
plates (1 × 106 cells per well) in Dulbecco’s modied Eagle medium (DMEM) supplemented with 10% fetal bovine
serum and 1% penicillin/streptomycin solution at 37°C in a humidied atmosphere containing 5% CO2. Cells
were transfected with 0.5μg pGL3B-SCD1 promoter constructs using 3 µL Lipofectamine 3000 that was sup-
plemented with 2 µL P3000 (Invitrogen, Carlsbad, CA, USA) in 1mL DMEM. As a transfection control, 0.5µg
pCMV-β-gal plasmid was co-transfected. Cells were harvested and processed 24–30h aer transfection.
Oleate, palmitate, stearate, linoleate, elaidate, and vaccenate were diluted in ethanol (Molar Chemicals, Halász-
telek, Hungary) to a nal concentration of 50mM and conjugated with 20% FA-free BSA in 1:4 ratio at 50°C
for 1h. e working solution for FA treatments was prepared freshly in FBS-free and antibiotic-free medium at
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100µM nal concentration. e FA treatment was carried out for 24h in 12-well plates. For luciferase assay, the
culture medium was replaced 5h aer transfection and the cells were incubated for a further 24h.
Cell lysates were prepared for immunoblot analysis by removing the medium and washing the cells twice with
PBS. 100 µL RIPA lysis buer (0.1% SDS, 5mM EDTA, 150mM NaCl, 50mM Tris, 1% Tween 20, 1mM Na3VO4,
1mM PMSF, 10mM benzamidine, 20mM NaF, 1mM pNPP, and protease inhibitor cocktail) was added to each
well and the cells were scraped and briey vortexed. Aer 15min incubation at room temperature, the lysates
were centrifuged for 5min at maximum speed in a benchtop centrifuge at 4°C to remove cell debris. Protein
concentration of the supernatant was measured with Pierce® BCA Protein Assay Kit (ermo Fisher Scientic,
Waltham, MA, USA) and the samples were stored at ‒20°C until further analysis.
For the luciferase reporter assay, cells were washed twice with PBS and then scraped in 100 µL reporter lysis
buer (Promega, Madison, WI, USA) and vortexed briey. A single freeze–thaw cycle was followed by centrifug-
ing in a benchtop centrifuge (5min, max speed, 4°C). Supernatants were used for enzyme activity determination.
For total RNA isolation, cells were washed twice with PBS and collected in 350 µL RLT buer (Qiagen, Hilden,
Germany) supplemented with 1% β-mercaptoethanol according to manufacturer’s protocol. Samples were stored
at ‒80°C until further analysis.
Aliquots of cell lysates (20µg protein per lane) were analyzed by SDS-PAGE on 12% Tris–glycine minigels, and
transferred onto Immobilon-P membranes (Millipore, Billerica, MA, USA). Primary and secondary antibodies
were applied overnight at 4°C and for 1h at room temperature, respectively. Horseradish peroxidase (HRP)-
conjugated goat polyclonal anti-Actin (Cell Signaling, Danvers, MA, USA, sc-1616) antibodies were used at
1:2000 dilution. SCD1 was detected with a rabbit polyclonal antibody (Cell Signaling, Danvers, MA, USA,
2438S), used at a dilution of 1:2000, followed by HRP-conjugated goat polyclonal anti-rabbit IgG (Cell Signaling,
Danvers, MA, USA, 7074S) at a dilution of 1:2000. ETS1 was detected with a goat polyclonal antibody (Bethyl
Laboratories, A190-110A), used at a dilution of 1:2000, followed by HRP conjugated mouse monoclonal anti-goat
IgG (Cell Signaling, Danvers, MA, USA, sc-2354) at a dilution of 1:2000. HRP was detected by C-DiGit® Blot
Scanner (LI-COR, Lincoln, NE, USA) using the SuperSignalWest Pico Chemiluminescent Substrate (ermo
Fisher Scientic,Waltham, MA, USA). As the edges of the membranes can blend into the background due to
digital imaging, a protein marker is run on each side of the sample sets to clearly dene them. Uncropped ver-
sions of all parallel blot images are available in the Supplementary Information le.
Luciferase activity was detected using the Luciferase Assay System kit (Promega, Madison, WI, USA) by adding
15 µL Luciferin reagent to 5 µL of al cell extracts. β-galactosidase activity of 20 µL cell lysates was measured by
determining the o-nitrophenyl-β-D-galactopyranoside (at a nal concentration of 3mM) cleavage rate. Lumi-
nescence was detected using a Varioskan multi-well plate reader (ermo Fisher Scientic, Waltham, Massa-
chusetts, USA). Values for luciferase activity were normalized to β-galactosidase activity (measured by standard
protocol using the same Varioskan plate reader in photometry mode). Each experiment was repeated three times
independently, and each sample was analyzed in triplicate.
Total RNA was puried from transfected HEK293T and HepG2 cells by using RNeasy Plus Mini Kit (Qiagen,
Germantown, MD, USA) following the manufacturer’s instruction. Concentrations were measured using Nan-
oDrop1000 spectrophotometer. To assess the integrity and purity of the isolated total mRNA samples, the ratios
of their absorbance at 260/280 and 260/220nm were determined, and they were also analyzed by agarose gel
electrophoresis to visualize bands corresponding to 28S and 18S rRNAs, respectively. Possible DNA contamina-
tion was removed by DNase I treatment using RNAqueous®-4PCR Kit (Invitrogen, Carlsbad, CA, USA). cDNA
samples were produced by reverse transcription of 0.5µg DNA-free RNA, using the SensiFAST™ cDNA Synthesis
Kit (Meridian Bioscience, Memphis, TN, USA).
qPCR
Quantitative PCR assay was performed in 20 µL nal volume containing 5 µL 20 × diluted cDNA, 1 × PowerUp™
SYBR™ Green Master Mix, and 0.5µM forward and reverse primers using QuantStudio 12K Flex Real-Time PCR
System (ermo Fisher Scientic, Waltham, Massachusetts, USA). SCD1 and ETS1 sequences were amplied by
5’‒ CTG GCC TAT GAC CGG AAG AAA ‒3’ / 5’‒ GAC CCC AAA CTC ATT CCA TAG G ‒3’ and 5’ – AGA
TGA GGT GGC CAG GAG AT ‒ 3’ / 5’ – CTG CAG GTC ACA CAC AAA GC – 3’ primer pairs, respectively.
GAPDH cDNA was also amplied as an endogenous control using 5’ ‒ GTC CAC TGG CGT CTT CAC CA ‒
3’ / 5’ ‒ GTG GCA GTG ATG GCA TGG AC ‒ 3’ primer pair. e rst step of the thermocycle was an initial
denaturation and enzyme activation at 95°C for 2min. It was followed by 40 cycles of 95°C for 15s, 55°C for
15s, and 72°C for 1min; measurement of the uorescent signal was carried out during annealing. Reactions were
performed in triplicates, and a reaction mixture with RNase-free water instead of template cDNA was employed
as non-template control. Relative expression levels were calculated as 2‒ΔCT, where ΔCT values corresponded to
the dierence of the CT-values of the endogenous control and target genes.
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Subjects
282 patients diagnosed with T2DM in the 2nd Department of Internal Medicine, Semmelweis University (51.2%
female, 48.8% male, disease onset at the age of 62.4 ± 12.6 y) were recruited in the study. e control group con-
sisted of 370 volunteers with no medical history of any metabolic disease (61.4% female, 38.6% male, mean age:
33.1 ± 21.6 y). e diagnosis of diabetes was made based on fasting blood sugar values, oral glucose tolerance
test (OGTT), and HbA1C value according to WHO regulations. Individuals with autoimmune, infectious, or
metabolic disorders other than type 2 diabetes were excluded from the study. Genetic analysis of the participants
was approved by the Local Ethical Committee (ETTTUKEB ad.328/KO/2005, ad.323–86/2005-1018EKU from
the Scientic and Research Ethics Committee of the Medical Research Council). e study was conducted in
accordance with the principles of the Declaration of Helsinki. Participants signed a written informed consent
before sample collection for genetic analysis. To avoid the risk of spurious association caused by population
stratication, subjects of Hungarian origin were exclusively included to ensure the comparison of homogenous
populations. Buccal epithelial cells were collected by swabs. e rst step of DNA isolation was an incubation
of the buccal samples at 56°C overnight in 0.2mg/mL Proteinase K cell lysis buer. Subsequently, proteins were
denatured using a saturated NaCl solution. DNA was then precipitated by isopropanol and 70% ethanol. DNA
pellet was resuspended in 100 µL 0.5 × TE (1 × TE: 10mM Tris pH = 8.0; 1mM EDTA) buer. Concentration of
the samples was measured by NanoDrop1000 spectrophotometer.
Genotyping
Rs1054411 promoter polymorphism of the SCD1 gene was genotyped using pre-designed TaqMan assay
(C_34192814_10, ermo Fisher Scientic, Waltham, MA, USA). qPCR assay was performed in 5 µL nal vol-
ume containing approximately 4ng genomic DNA, 1 × TaqPath™ ProAmp™ Master Mix, and 1 × TaqMan® SNP
Genotyping Assay using QuantStudio 12K Flex Real-Time PCR System (ermo Fisher Scientic, Waltham,
MA, USA). ermocycle was started by activating the hot start DNA polymerase and denaturing genomic DNA
at 95°C for 10min. is was followed by 40 cycles of denaturation at 95°C for 15s, and combined annealing
and extension at 60°C for 1min. Real-time detection was carried out during the latter step to verify the results
of the subsequent post-PCR plate reads and automatic genotype calls.
Statistical analysis
Immunoblots were evaluated by densitometry using the Image Studio® 5.2 soware (LI-COR Biotechnology,
Lincoln, NE, USA), and are shown as relative band densities normalized to Actin as a reference. Relative band
densities, luciferase activities and mRNA levels are presented in the diagrams as mean values ± S.D. and were
compared by ANOVA with the Tukey’s multiple comparison post hoc test, using the GraphPad Prism 6.0 soware
(GraphPad Soware, Boston, MA, USA). Dierences with a p < 0.05 value were considered to be statistically
signicant. Genotype–phenotype association was assessed by χ2-test comparing the genotype distribution of the
patient and the control groups (i.e., additive model). Power of the genetic association study was assessed by the
GAS Power Calculator on line tool (https:// csg. sph. umich. edu/ abeca sis/ cats/ gas_ power_ calcu lator/) using the
additive disease model (prevalence of T2DM is 6.28%, genotype relative risk was 1.2.).
All data are available in the main text or in the supplementary material. e raw data and uncropped blot images
underlying the above presented results, as well as all Supplementary Figures and Tables are enclosed in the Sup-
plementary Information le. Any additional data from this study is available from the corresponding authors
(zambo.veronika@med.semmelweis-univ.hu and kereszturi.eva@semmelweis.hu) upon reasonable request.
Received: 28 August 2023; Accepted: 23 December 2023
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We thank Ms. Valéria Mile and Ms. Viktória Molnár for their skillful technical assistance.
K.T.: methodology, investigation, visualization, writing‒original dra preparation; V.Z.: methodology, investi-
gation, writing‒original dra preparation, funding acquisition; G.O.: validation; P.S.: visualization; F.S.: valida-
tion; V.T.: writing‒review and editing; Z.R.: soware, writing‒review and editing, funding acquisition; M.C.:
writing‒review and editing, funding acquisition; É.K.: conceptualization, writing‒original dra preparation,
writing‒review and editing, visualization, funding acquisition.
is work was supported by the Hungarian National Research, Development and Innovation Oce (NKFIH
grant numbers: FK138115, K131680 and PD142709). Project no. TKP2021-EGA-24 was implemented with
the support provided by the Ministry of Innovation and Technology of Hungary from the National Research,
Development and Innovation Fund, and nanced under the TKP2021-EGA funding scheme.
e authors declare no competing interests.
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 023- 50700-5.
Correspondence and requests for materials should be addressed to V.Z.orÉ.K.
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