ORIGINAL ARTICLE

Journal of

Insulin-Stimulated L-Arginine
Transport Requires SLC7A1 Gene
Expression and Is Associated
With Human Umbilical Vein
Relaxation

Cellular
Physiology

MARCELO GONZALEZ,1,2 VICTORIA GALLARDO,2 NATALIA RODRIGUEZ,2

N,1 FRANCISCO WESTERMEIER,1 ENRIQUE GUZMAN-GUTIERREZ,1
CARLOS SALOMO
A,1,3 ANDREA LEIVA,1 PAOLA CASANELLO,1 AND LUIS SOBREVIA1*
FERNANDO ABARZU
1

Cellular and Molecular Physiology Laboratory (CMPL) and Perinatology Research Laboratory (PRL), Division of Obstetrics

and Gynecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile
2

Vascular Physiology Laboratory, Department of Physiology, Faculty of Biological Sciences, Universidad de Concepcion,

Concepcion, Chile
3

Division of Obstetrics and Gynecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Catolica de Chile,

Santiago, Chile
Insulin causes endothelium-derived nitric oxide (NO)-dependent vascular relaxation, and increases L-arginine transport via cationic amino
acid transporter 1 (hCAT-1) and endothelial NO synthase (eNOS) expression and activity in human umbilical vein endothelium (HUVEC).
We studied insulin effect on SLC7A1 gene (hCAT-1) expression and hCAT-transport activity role in insulin-modulated human fetal
vascular reactivity. HUVEC were used for L-arginine transport and L-[3H]citrulline formation (NOS activity) assays in absence or presence
of N-ethylmaleimide (NEM) or L-lysine (L-arginine transport inhibitors). hCAT-1 protein abundance was estimated by Western blot,
mRNA quantification by real time PCR, and SLC7A1 promoter activity by Luciferase activity (1,606 and 650 bp promoter fragments
from ATG). Specific protein 1 (Sp1), and total or phosphorylated eNOS protein was determined by Western blot. Sp1 activity (at four sites
between 177 and 105 bp from ATG) was assayed by chromatin immunoprecipitation (ChIP) and vascular reactivity in umbilical vein
rings. Insulin increased hCATsL-arginine transport, maximal transport capacity (Vmax/Km), and hCAT-1 expression. NEM and L-lysine
blocked L-arginine transport. In addition, it was trans-stimulated (7.8-fold) by L-lysine in absence of insulin, but unaltered (1.4-fold) in
presence of insulin. Sp1 nuclear protein abundance and binding to DNA, and SLC7A1 promoter activity was increased by insulin. Insulin
increased NO synthesis and caused endothelium-dependent vessel relaxation and reduced U46619-induced contraction, effects blocked
by NEM and L-lysine, and dependent on extracellular L-arginine. We suggest that insulin induces human umbilical vein relaxation by
increasing HUVEC L-arginine transport via hCATs (likely hCAT-1) most likely requiring Sp1-activated SLC7A1 expression.
J. Cell. Physiol. 9999: 19, 2011. 2011 Wiley-Liss, Inc.

The reduced ability of the endothelium to cause nitric oxide

(NO)-mediated vasodilatation reflects endothelial dysfunction,
a phenomenon well-correlated with increased incidence of
cardiovascular diseases exhibited by patients with hypertension
(Wierzbicki et al., 2004) or diabetes mellitus (Casanello et al.,
2007; Hiden et al., 2009; Sobrevia et al., 2011), and in human
umbilical vein endothelium (HUVEC) isolated from pregnancies
with pre-eclampsia, gestational diabetes or intrauterine growth
restriction (IUGR) (Casanello et al., 2007, 2009; Sobrevia and
Gonzalez, 2009; Westermeier et al., 2009a,b; Sobrevia et al.,
2011). It is known that insulin increases leg blood flow in healthy
subjects via stimulation of endothelial NO synthase (eNOS)
(Steinberg and Baron, 2002). Even when this phenomenon has
been shown altered in type II diabetes mellitus (Steinberg and
Baron, 2002) or obese (Laakso et al., 1990) patients, associated
cell-signaling mechanisms behind this effect of insulin are not
reported in human endothelium (Casanello et al., 2007;
Sobrevia and Gonzalez, 2009; Sobrevia et al., 2011).
Insulin also increases NO synthesis and release in primary
cultures of HUVEC (Scherrer et al., 1994; Gonzalez et al.,
2004). In several cell types, NO synthesis requires uptake of the
semi-essential, cationic amino acid L-arginine, a phenomenon
that involves activity of several cationic amino acid membrane
2 0 1 1 W I L E Y - L I S S , I N C .

Additional Supporting Information may be found in the online

version of this article.
Contract grant sponsor: Fondo Nacional de Desarrollo Cientfico y
Tecnologico (FONDECYT), Chile;
Contract grant numbers: 1070865, 1080534, 11100192.
Contract grant sponsor: Programa de Investigacion Asociativa
(PIA) from Comision Nacional de Investigacion en Ciencia y
Tecnologa (CONICYT) (Anillos);
Contract grant number: ACT-73.
Contract grant sponsor: Direccion de Investigacion Universidad de
Concepcion (DIUC);
Contract grant number: 210.033.103-1.0.
Contract grant sponsor: Apoyo Realizacion de Tesis Doctoral from
CONICYT;
Contract grant numbers: AT-23070213, AT-24100210.
*Correspondence to: Luis Sobrevia, Cellular and Molecular
Physiology Laboratory (CMPL), Division of Obstetrics and
Gynecology, School of Medicine, Pontificia Universidad Catolica de
Chile, P.O. Box 114-D, Santiago, Chile.
E-mail: sobrevia@med.puc.cl
Received 22 November 2010; Accepted 3 January 2011
Published online in Wiley Online Library
(wileyonlinelibrary.com), 00 Month 2011.
DOI: 10.1002/jcp.22635

G O N Z A L E Z E T A L .

transport systems (Sobrevia and Gonzalez, 2009). L-Arginine

uptake is mainly mediated by the Na-independent, high-affinity
cationic amino acid transporters 1 (hCAT-1) and 2 (hCAT-2),
and to a lesser extent via the very high affinity transport
system yL in HUVEC (Casanello et al., 2007; Sobrevia and
Gonzalez, 2009). Insulin also increases L-arginine transport via
hCAT-1 in this cell type (Sobrevia et al., 1996; Gonzalez et al.,
2004), a phenomenon proposed to be, at least in part,
responsible of the increased NO synthesis in response to this
hormone (Casanello et al., 2007). However, even when these
changes in the endothelial L-arginine membrane transport in
response to insulin are well characterized, potential
mechanisms at a transcriptional level in this phenomenon are
unknown (Mann et al., 2003; Casanello et al., 2007; Sobrevia and
Gonzalez, 2009; Sobrevia et al., 2011).
Biological effects of insulin involve activation of several
transcription factors, including the transcription factor specific
protein 1 (Sp1) in several cell types (Samson and Wong, 2002;
Solomon et al., 2008). Since SLC7A1 gene (encoding hCAT-1)
promoter lacks of TATA box (Hammermann et al., 2001;
Hatzoglou et al., 2004; Sobrevia and Gonzalez, 2009), we
hypothesize that insulin-increased L-arginine transport and
hCAT-1 expression result from higher SLC7A1 expression
involving Sp1 activation in HUVEC. Our results suggest that
insulin increased L-arginine transport mediated by hCAT-1,
most likely results from increased SLC7A1 expression, which
could require Sp1 activation in this cell type. In addition, insulin
causes endothelium-dependent vasorelaxation in umbilical
veins via a phenomenon that seems to involve hCAT-like
transport activity. These results could be important for a better
understanding of the reported endothelial dysfunction in
diseases or pregnancy where L-arginine/NO pathway in fetal
endothelium is reduced, such as pre-eclampsia, IUGR or fetal
hypoxia (Casanello et al., 2007).
Materials and Methods
Study participants

Written informed consents from pregnant women from maternity

of the Hospital Clnico Universidad Catolica, Santiago, Chile, were
obtained. Investigation follows the World Medical Association
Declaration of Helsinki, and approved protocols by the ethics
committee of Faculty of Medicine of the Pontificia Universidad
Catolica de Chile. An expanded Materials and Methods Section is
available in the Supplementary Online Material.
Cell culture

Endothelial cells were isolated by collagenase [0.25 mg/ml;

Collagenase Type II from Clostridium histolyticum (Boehringer,
Mannheim, FRG)] digestion from umbilical cord veins (HUVEC)
and cultured up to confluence (Gonzalez et al., 2004; Casanello
et al., 2009). Experiments were performed in absence or presence
(28 h) of insulin (0.001100 nM).
L-Arginine

transport

L-Arginine transport was measured in absence or presence (30 min

preincubation) of 200 mM N-ethylmaleimide (NEM, CATs

inhibitor) (Deves et al., 1993) or 1 mM L-lysine (competitive cisinhibitor of L-arginine transport) (Deves and Boyd, 1998; Mann
et al., 2003), as described (Gonzalez et al., 2004; Casanello et al.,
2009). The relative contribution of insulin to saturable L-arginine
transport kinetic parameters [maximal velocity (Vmax) and
apparent Km] was estimated from the maximal transport capacity
(Vmax/Km) values for L-arginine transport by:

1
C=Ins F

Km Ins Vmax
Vmax Ins Km

JOURNAL OF CELLULAR PHYSIOLOGY

where CVmax and CKm are the kinetics parameters for Larginine transport in control conditions, and InsVmax and InsKm
are kinetics parameters of L-arginine transport in HUVEC
exposed to insulin. In trans-stimulation experiments (Gonzalez
et al., 2004; Casanello et al., 2009), initial rates of L-arginine
transport were increased ( P < 0.05, n 4) by 7.8  0.3-fold by
L-lysine in control cells, but it was not significantly ( P > 0.05)
altered in the presence of insulin (1.4  0.5-fold).
3
L-[ H]Citrulline

assay

NOS activity was determined by incubation of cells with 100 mM Larginine and 4 mCi/ml L-[3H]arginine (30 min, 378C) in absence or
presence of 100 mM NG-nitro-L-arginine methyl ester (L-NAME).
Cells were exposed to 1 nM insulin for 8 h in absence or presence
of 200 mM NEM or 1 mM L-lysine. Digested cells (95% formic acid)
were passed through a cation ion-exchange resin Dowex-50W
(50X8-200) and L-[3H]citrulline determined in H2O eluate as
described (Gonzalez et al., 2004).
Sub-cellular fractionation

Cells scraped into ice-cold phosphate buffer saline (PBS)

supplemented with phenylmethylsulfonyl fluoride (1 M) were
centrifuged (5,400 rpm) and the pellet resuspended in cold lowsodium buffer, as described (Dignam et al., 1983). Mixture was then
passed through a 25-gauge syringe and centrifuged (14,000 rpm) to
collect the supernatant (cytoplasmic fraction). Pellet was
resuspended in cold low-potassium buffer, passed through a 1 ml
micropipette tip, incubated (48C, 20 min) and centrifuged
(14,000 rpm). Supernatant (nuclear fraction) was stored until use
(Dignam et al., 1983).
For plasma membrane fraction preparation, confluent cells were
washed twice with ice-cold PBS (mM: 130 NaCl, 2.7 KCl,
0.8 Na2HPO4, 1.4 KH2PO4, pH7.4) and harvested in 3-[Nmorpholino]propanesulfonic acidKOH (MOPS)KOH buffer
(20 mM MOPSKOH, 250 mM sucrose, pH 7.4). Cells were quickly
disrupted in liquid nitrogen and sonicated (two cycles, 15 sec,
100 W, 48C). Cytosol and plasma membrane fractions were
separated by differential centrifugation as reported (Aguayo et al.,
2005).
Western blotting

Proteins (70 mg) separated by polyacrylamide gel (810%)

electrophoresis were probed with primary polyclonal rabbit antihCAT-1 (1:250), anti-Sp1 (1:500), anti-eNOS (1:1,500), antiphosphorylated eNOS at serine1177 (PeNOS, 1:250), or
monoclonal mouse anti-b-actin (1:2,000) (Santa Cruz
Biotechnology, Santa Cruz, CA) antibodies (Gonzalez et al., 2004;
Casanello et al., 2009).
Isolation of total RNA and reverse transcription

Total RNA was isolated using the Quiagen RNAeasy kit (Quiagen,
Crawley, UK). RNA quality and integrity were insured by gel
visualization and spectrophotometric analysis (OD260/280),
quantified at 260 nm and precipitated to obtain 4 mg/ml. Aliquots
(1 mg) of total RNA were reversed transcribed into cDNA as
described (Gonzalez et al., 2004; Casanello et al., 2009).
Quantitative RT-PCR

Experiments were performed using a LightCyclerTM rapid thermal

cycler (Roche Diagnostics, Lewes, UK) in a reaction mix containing
0.5 mM primers, and dNTPs, Taq DNA polymerase and reaction
buffer provided in the QuantiTect SYBR Green PCR Master Mix
(Quiagen) as described (Gonzalez et al., 2004; Casanello et al.,
2009). HotStart Taq DNA polymerase was activated (15 min,
958C), and assays included a 958C denaturation (15 sec), annealing
(20 sec) at 548C (hCAT-1 and 28S), and extension (10 sec) at 728C
(hCAT-1 and 28S). Product melting temperature values were
79.18C (hCAT-1) and 86.78C (28S). Oligonucleotide primers:

INSULIN-INCREASED hCAT-1 EXPRESSION

hCAT-1 (sense) 50 -GAGTTAGATCCAGCAGACCA-30 , hCAT-1

(anti-sense) 50 -TGTTCACAATTAGCCCAGAG-30 , 28S (sense)
50 -TTGAAAATCCGGGGGAGAG-30 , 28S (anti-sense) 50 ACATTGTTCCAACATGCCAG-30 . The number of copies for
28S rRNA was not significantly altered ( P > 0.05, n 4) in all
experimental conditions used in this study (not shown).
Actinomycin D effect on hCAT-1 mRNA and protein

Total RNA and protein were isolated from HUVEC exposed (8 h)

to culture medium without or with 1.5 mM actinomycin D
(transcription inhibitor) (Puebla et al., 2008) in absence or
presence of 1 nM insulin. hCAT-1 and 28S mRNA were quantified
by real time RT-PCR, and hCAT-1 and b-actin proteins detected by
Western blot (see above).
hCAT1 promoter cloning

The upstream sequences 1,606 and 650 bp from the

transcription start codon of SLC7A1 gene (GenBank: AL596114)
were PCR-amplified and cloned into pGL3-basic reporter system
(Faras et al., 2010). The pGL3-hCAT1 reporter constructs
generated were pGL3-hCAT11,606 and pGL3-hCAT1650.
Transient transfection

Cell suspension (3.2  106 cells/ml) was mixed with pGL3-hCAT1

reporter constructs, pGL3-Basic (empty pGL3 vector), pGL3Control [Simian Virus 40 promoter (SV40) pGL3 vector], and
internal transfection control vector pRL-TK expressing Renilla
luciferase (Puebla et al., 2008; Faras et al., 2010). Cells were
electroporated and transfection efficiency estimated with pEGFPN3 vector (Clontech, Mountain View, CA) as described (Puebla
et al., 2008; Faras et al., 2010).
Luciferase assay

Electroporated cells were lysed in 200 ml passive lysis buffer

(Promega), and Firefly and Renilla luciferase activity was measured
using Dual-Luciferase1 Reporter Assay System (Promega) in a
Sirius luminometer (Berthold Detection System; Oak Ridge, TN)
as described (Puebla et al., 2008; Farias et al., 2010).
Chromatin immunoprecipitation (ChIP) assay

ChIP assay for Sp1 response elements identified in silico in the region
between 177 and 105 bp from ATG of the SLC7A1 promoter was
performed as described (Puebla et al., 2008; Faras et al., 2010). In
brief, fixed cells (1% paraformaldehyde) were incubated with 125 mM
glycine, rinsed with ice-cold PBS, scraped and centrifuged to obtain a
pellet which was resuspended in cell lysis buffer and incubated
(20 min, 48C) as described (Puebla et al., 2008; Farias et al., 2010). The
mix was then centrifuged and the obtained pellet resuspended in
nuclear lysis buffer containing protease inhibitors, incubated (20 min,
48C) and sonicated (20, 10-sec pulse). Supernatant (nuclear
fraction) was collected after centrifugation of sonicated samples
(100 ml aliquots of this fraction were saved as input).
Nuclear fractions (6 mg chromatin) were incubated with protein
G-agarose beads, incubated with 20 ml of salmon sperm DNA
(ssDNA) solution (10 mg/ml) and centrifuged. Supernatant (precleared nuclear fractions) aliquots were diluted to 1 ml in ChIP
dilution buffer and incubated with anti-Sp1 (2 mg/ml) antibody.
Aliquots were transferred into a tube containing 20 ml protein
G-agarose beads and incubated with BSA (1%) followed by a second
incubation with 20 ml of ssDNA solution (10 mg/ml). The mix was
further incubated for 4 h (DNA samples without antibody were
used as G-agarose drag-non-specific control). The
immunocomplex (protein G-agarose beads, anti-Sp1 antibody, Sp1
protein, and chromatin) was centrifuged, and pellets consecutively
rinsed with ChIP dilution buffer, dialysis buffer, TSE-500 buffer, and
LiCl detergent buffer, followed by two further rinses with TE buffer
JOURNAL OF CELLULAR PHYSIOLOGY

(Puebla et al., 2008; Faras et al., 2010). Pellets were incubated with
elution buffer and chromatin eluted from the immunocomplex by
centrifugation (supernatant fraction). Eluted (precipitated Sp1
DNA complex) and input (total sonicated DNA) DNA was
obtained by reversing the initial paraformaldehyde reaction by
incubation in 330 mM NaCl followed by precipitation with
isopropanol/glycogen. Resulting pellet was incubated in proteinase
K buffer and the DNA purified using phenol/chloroform (1:1, v/v),
precipitated with isopropanol/glycogen and then treated with
RNAse A (10 mg/ml). Aliquots (1 ml, 1:10 dilution) of DNA (input
and precipitate) were used for PCR assays. Cycling parameters for
amplifications (30 cycles) were 958C for 30 sec, 578C for 30 sec and
728C for 30 sec. Oligonucleotide primers: Sp1-hCAT-1 promotersense 50 -ATCCACCCCTCCAATCTTCT-30 and Sp1-hCAT-1
promoter-anti-sense 50 -GCAACCCAGATTCCAGTCTC-30
(product size 249 bp).
Umbilical vein reactivity

Ring segments (24 mm length) from the human umbilical veins

with and without endothelium were mounted in a myograph for
isometric force measurements with optimal diameter adjusted
from maximal active response to 62.5 mM KCl as described (Vega
et al., 2009). Response to insulin (0.001100 nM, 8 h) was
determined in 100 nM U466619-preconstricted vessels in absence
or presence of 200 mM NEM or 1 mM L-lysine. Vessel rings were
also incubated (378C) in Krebs for 2 or 8 h in absence or presence
of 1 nM insulin, followed by exposure to U46619 (0.11,000 nM).
Additional assays were performed in preparations incubated in
Krebs with or without 200 mM L-arginine.
Statistical analysis

Values are mean  SEM, where n indicates number of different cell

cultures (three to four replicates). Comparisons between two and
more groups were performed by means of Students unpaired ttest and analysis of variance (ANOVA), respectively. If the ANOVA
demonstrated a significant interaction between variables, post hoc
analyses were performed by the multiple-comparison Bonferroni
correction test. P < 0.05 was considered statistically significant.
Results
Effect of insulin on L-arginine transport

Uptake of 100 mM L-arginine was significantly increased after 4

and 8 h of exposure to insulin (Fig. 1A), an effect blocked by
NEM (94  4%) confirming our previous results in this cell type
(Gonzalez et al., 2004). We here extended these observations
showing that insulin effect was concentration-dependent with
similar ( P > 0.05) half-maximal stimulatory concentrations at
4 h (SC50 0.24  0.03 nM) and 8 h (SC50 0.29  0.04 nM)
of incubation (Fig. 1B). Insulin also increased the Vmax, with
no significant changes in the apparent Km of transport
(Fig. 2A, Table 1), in a concentration dependent manner
(SC50 0.21  0.06 nM; Fig. 2B). Eadie-Hofstee transformation
of transport data, an approach that allows estimating potential
involvement of more than one transport mechanism with
different affinities for a common substrate, or one transport
mechanism that could experience changes in its substrate
affinity, shows lineal regressions in all experimental conditions
(Fig. 2C). L-Arginine (100 mM) transport was also inhibited by Llysine in a same proportion as NEM, in absence or presence of
insulin (Fig. 2D).
Effect of insulin on L-[3H]citrulline formation

The fractions of L-[3H]citrulline formation inhibited by L-NAME

and eNOS phosphorylation at Ser1177 were increased by insulin
(Fig. 2E), confirming previous results in this cell type (Gonzalez
et al., 2004). Basal and insulin-increased L-[3H]citrulline

G O N Z A L E Z E T A L .

Actinomycin D effect on hCAT-1 protein and mRNA

expression

Incubation of cells with actinomycin D inhibited in a similar

proportion ( P < 0.05) the increase induced by insulin on
L-arginine uptake (91  2%; Fig. 4A), hCAT-1 protein
abundance (96  2%; Fig. 4B) and number of mRNA copies
(79  7%; Fig. 4C). However, actinomycin D did not significantly
alter these parameters in absence of insulin.
SLC7A1 transcriptional activity

Exposure of cells to 1 nM insulin for 8 h showed an increase in

the SLC7A1 promoter transcriptional activity when transfected
with pGL3-hCAT11,606 (1.3  0.09-fold) and pGL3hCAT1650 (1.4  0.03-fold) constructs, respectively,
compared with cells incubated in absence of insulin (Fig. 5).
Sp1 protein abundance and activity

Exposure of cells to insulin increased nuclear Sp1 protein

abundance compared with total Sp1 protein abundance
(Fig. 6A). Insulin also increased Sp1 binding to DNA in cells
transfected with the pGL3-hCAT1650 construct of SLC7A1
promoter (Fig. 6B).
Umbilical vein rings reactivity

Fig. 1. Effect of insulin on L-arginine transport. A: Overall L-arginine

transport (100 mM L-arginine, 2 mCi/ml L-[3H]arginine, 1 min, 37-C)
was measured in HUVEC monolayers in Krebs solution in absence
(Control) or presence of the indicated concentrations of insulin for
different time periods. Experiments were also performed in the
presence of 200 mM N-ethylmaleimide (NEM) in control (*), 0.1 nM
(&) or 1 nM (~) insulin. B: L-Arginine transport as in (A), in absence or
presence of the indicated concentrations of insulin for different
periods of time. Experiments were also performed in the presence of
200 mM NEM for 8 h (*), 4 h (~) or 2 h (&) of exposure of cells to
insulin. Values are means W SEM (n U 9). In (A), MP < 0.04 and yP < 0.05
versus corresponding value in absence of insulin. In (B), MP < 0.04
versus without insulin.

formation, but not eNOS phosphorylation was blocked by

NEM or L-lysine (Fig. 2F).
hCAT-1 expression

Insulin increased hCAT-1 protein abundance in a

concentration- (SC50 0.37  0.05 nM; Fig. 3A) and time- [halfmaximal stimulatory time (ST50) 3.13  0.5 h] dependent
manner (Fig. 3B). Insulin (1 nM)-induced increase of hCAT-1
protein abundance (2.8  0.4-fold) reached a maximal effect
after 4 h incubation, which was maintained for further 4 h
(3.2  0.1-fold). Incubation of cells with 1 nM insulin for 8 h
increased hCAT-1 protein abundance in plasma membrane, but
did not alter its abundance in cytosol fraction (Fig. 3C). Parallel
experiments confirmed previous results (Gonzalez et al., 2004)
showing that insulin increased the mRNA number of copies for
hCAT-1 (ST50 2.7  0.3 h), reaching a maximal effect at 4 h
(3.3  0.3-fold), declining by 8 h of incubation (2.2  0.2-fold)
(not shown).
JOURNAL OF CELLULAR PHYSIOLOGY

Insulin induced relaxation of endothelium-intact, but not in

endothelium-denuded human umbilical vein rings with a halfmaximal effect (EC50) 1.8  0.2 nM insulin (Fig. 7A). When
insulin (1 nM) effect was assayed in preparations preincubated
with NEM and L-lysine, insulin-induced relaxation was
abolished; however, these molecules did not significantly
alter KCl-induced vasoconstriction (Fig. 7B). Parallel
experiments show that U46619 induces vasoconstriction
(EC50 8.4  0.2 nM), an effect not significantly ( P > 0.05)
altered following 2 h of preincubation with insulin
(EC50 11.2  0.3 nM), but significantly reduced by this
hormone after 8 h of preincubation (EC50 20.1  0.4 nM,
P < 0.05; Fig. 7C). In addition, when vessel rings were incubated
in a L-arginine free solution, insulin did not induce
vasorelaxation neither altered U46619-induced contraction
(Fig. 7D).
Discussion

This study shows that insulin increases L-arginine transport

via a mechanism involving higher maximal transport capacity
associated with increased hCAT-1 expression in HUVEC.
Insulin-increased L-arginine transport results from activation of
SLC7A1 promoter via a mechanism that may involve Sp1 binding
to multiple consensus sequences between 177 and 105 bp
from ATG. In addition, insulin also induces relaxation of human
umbilical veins that depends on endothelium and hCATs-like
transport activity. Since insulin effects are seen at a physiological
plasma concentration, it is conceivable that SLC7A1 expression
and hCAT-1 activity are under tonic regulation by physiological
plasma insulin in HUVEC. These findings could be determinant
for fetal insulin modulation of endothelial-derived NO synthesis
in human umbilical vessels from pregnancy diseases associated
with hyperinsulinemia, such as gestational diabetes, and other
states of insulin resistance (Sobrevia and Gonzalez, 2009;
Westermeier et al., 2009a,b; Faras et al., 2010; Sobrevia et al.,
2011).
Insulin effect on overall and saturable L-arginine transport
was dependent on the concentration of this hormone leading to
higher Vmax, without significant changes in the apparent Km of
saturable L-arginine transport. Since basal insulin concentration
in human umbilical vein blood at term from healthy pregnancies
has been reported as 0.020.03 nM (Westgate et al., 2006;

INSULIN-INCREASED hCAT-1 EXPRESSION

Fig. 2. Insulin modulation of kinetic parameters for saturable L-arginine transport. A: Saturable L-arginine transport (01,000 mM L-arginine,
2 mCi/ml L-[3H]arginine, 1 min, 37-C) in HUVEC monolayers in Krebs solution in absence (Control) or presence (8 h) of the indicated
concentrations of insulin. B: Maximal velocity (Vmax) values for L-arginine transport calculated from data in A (see Materials and Methods Section)
against insulin concentration. C: Eadie-Hofstee plot of data in (A). D: Cells were cultured in absence (Control) or presence of insulin (8 h),
and L-arginine (100 mM) uptake was measured after preincubation (last 30 min of insulin incubation period) without () or with (R) 200 mM
N-ethylmaleimide (NEM) and/or 1 mM L-lysine. E: Cells as in (D) were used to measure L-[3H]citrulline formation from L-[3H]arginine (100 mM
3
G
L-arginine, 4 mCi/ml L-[ H]arginine, 30 min, 37-C) in absence or presence of 100 mM N -nitro-L-arginine methyl ester (L-NAME). Plotted data
correspond to the L-NAME inhibitable fraction of L-[3H]citrulline formation (see Materials and Methods Section). F: Western blots (representative
of other five different cell cultures) for total (eNOS) or Ser1177-phosphorylated eNOS (PeNOS) in whole cell extracts from cells in absence () or
presence (R) of insulin (1 nM, 8 h), 200 mM NEM or 1 mM L-lysine. In (B), MP < 0.05 versus values in absence of insulin. In (D) and (E), MP < 0.04 versus all
other values. Values are means W SEM (n U 512).

Lindsay et al., 2007), it is feasible that basal fetal plasma insulin

level will be tonically activating L-arginine transport in the
umbilical vein endothelium. Interestingly, the latter could be
important in gestational diabetes, since the half-maximal
stimulatory effect of insulin on L-arginine transport was reached
at 0.2 nM in HUVEC, a concentration close to values reported
for umbilical vein blood in newborns from pregnancies affected
by this syndrome (0.07 nM) (Westgate et al., 2006; Lindsay
et al., 2007). Our results show that HUVEC exposed to 0.1 nM
insulin exhibit 3-fold increase of the L-arginine transport
capacity (i.e., Vmax/Km), a finding 2-fold higher than in human
trophoblast-derived cells (Eaton and Sooranna, 1998),
suggesting differential modulation of L-arginine transport by
insulin in the human feto-placenta cell types. Since physiological
JOURNAL OF CELLULAR PHYSIOLOGY

concentrations of insulin (0.10.5 nM) selectively activate

insulin receptors (IRs), instead of insulin-like growth factor 1
(IGF-I) and hybrid IR/IGF-I receptors in the endothelium
(Li et al., 2005), it is likely that insulin effect was mainly due
to activation of insulin receptors in HUVEC. However, since
HUVEC expresses at least two isoforms of insulin receptors
(Westermeier et al., 2009a,b), differing by the absence (insulin
receptor A, IR-A) or presence (insulin receptor B, IR-B) of a 12
amino acid sequence in the COOH-terminus of the a-subunit
(Hiden et al., 2009), we cannot assign exclusive or preferential
involvement of IR-A or IR-B in the response to insulin.
Insulin causes vasorelaxation in the human vasculature via a
mechanism requiring endothelium-derived NO (Sobrevia and
Gonzalez, 2009), but nothing is known regarding insulin effect

G O N Z A L E Z E T A L .

TABLE 1. Effect of insulin on L-arginine transport kinetic parameters in HUVEC

Control
Insulin (nM)
0.01
0.1
1
10

Vmax (pmol/mg protein/min)

Km (mM)

Vmax/Km (pmol/mg protein/min/mM)

1/C/InsF

2.4  0.3

103  39

0.023  0.005

147  34
182  28
204  64
165  48

0.034  0.005
0.035  0.017
0.045  0.009
0.060  0.011

1.5  0.3
1.5  0.4
2.0  0.4
2.6  0.6

5.0  0.4
6.3  0.6
9.2  1.0
9.9  0.9

Kinetics of saturable L-arginine transport (01,000 mM L-arginine, 2 mCi/ml L-[3H]arginine, 1 min, 378C) was measured in primary cultures of HUVEC exposed (8 h) to Krebs solution without
(Control) or with insulin (see Materials and Methods Section). 1/C/InsF is the relative contribution of insulin (Ins) exposure compared with absence of this hormone (C) to changes in maximal
transport capacity (Vmax/Km) of L-arginine transport (see Materials and Methods Section). Values are means  SEM (n 1012).

P < 0.05 versus Control.

on human umbilical vein reactivity and its dependence on

L-arginine transport (Muniyappa et al., 2007; Sobrevia and
Gonzalez, 2009; Sobrevia et al., 2011). Our results show that
insulin causes endothelium-dependent vasorelaxation of human
umbilical vessel rings, which could depend on hCATs-like
transport activity since NEM and L-lysine, CATs-like transport
inhibitors (Deves et al., 1993; Deves and Boyd, 1998; Mann

et al., 2003), inhibited L-arginine transport in a similar

proportion (94%) in HUVEC and reversed insulin
vasodilatory effect. NEM and L-lysine also inhibited NOS activity
in HUVEC in absence or presence of insulin (most likely not
involving Ser1177-dephosphorylation of eNOS), which could
result from reduced disposal of extracellular L-arginine
(Moncada and Higgs, 2006; Casanello et al., 2009; Sobrevia and

Fig. 3. Effect of insulin on hCAT-1 protein abundance. A: Western blot (representative of other eight different cell cultures) for hCAT-1 protein
abundance and b-actin (internal reference) in HUVEC monolayers in absence (0 h, control) or presence (8 h) of indicated concentrations of insulin.
Lower part: hCAT-1/b-actin protein ratio densitometries normalized to 1 in control. B: Western blot for hCAT-1 protein abundance and b-actin
in absence (0, control) or presence of 1 nM insulin for the indicated time periods. Lower part: hCAT-1/b-actin protein ratio densitometries
normalized to 1 in control. C: Western blot for hCAT-1, NaR,KR-ATPase b-subunit (ATPase) and b-actin protein abundance at the cytosol (C) or
plasma membrane (PM) fractions from HUVEC cultured in absence (, control) or presence (R) of 1 nM insulin (8 h). Lower part: hCAT-1/b-actin
protein ratio densitometries normalized to 1 in control. D: hCAT-1/ATPase protein ratio densitometries normalized to 1 in plasma membrane
fractions in control. Values are means W SEM (n U 810). In (A), MP < 0.05 versus control and 0.1 nM insulin. In (B), MP < 0.05 versus control and 2 h.
In (C), MP < 0.05 versus control and insulin in cytosol fraction; yP < 0.05 versus all other values. In (D), MP < 0.05 versus control.

JOURNAL OF CELLULAR PHYSIOLOGY

INSULIN-INCREASED hCAT-1 EXPRESSION

Fig. 5. Effect of insulin in SLC7A1 (hCAT-1) promoter activity.

Luciferase (Luc) reporter constructs containing serial truncations of
SLC7A1 promoter were transfected in primary cultures of HUVEC
incubated (8 h) in absence (&) or presence (&) of 1 nM insulin, along
with Renilla reporter plasmid, and assayed for Firefly and Renilla
luciferase activity, respectively. Results depict ratio of Firefly/Renilla
luciferase activity in cells in absence or presence of insulin. Cells were
also transfected with the empty pGL3-basic vector or pGL3-control
vector (SV40 pGL3) as negative or positive controls, respectively.
Values are means W SEM (n U 8). MP < 0.05 versus corresponding
values in absence of insulin.

changes in L-arginine transport are crucial in insulin-modulated

vascular tone in endothelial dysfunction-associated diseases
such as hypertension (Yang and Kaye, 2009), intrauterine
growth restriction (Casanello et al., 2007, 2009; Myatt, 2010) or
preeclampsia (Casanello et al., 2007; Myatt, 2010).
Insulin increased hCAT-1 mRNA after 2 h, but hCAT-1
protein abundance and hCATs-like transport activity was
increased after 4 h of exposure to this hormone, suggesting a
temporal correlation in this phenomena. In addition, insulin
effects are paralleled by higher SLC7A1 gene transcriptional

Fig. 4. Effect of actinomycin D on hCAT-1 expression. A: Overall

3
L-arginine transport (100 mM L-arginine, 2 mCi/ml L-[ H]arginine,
1 min, 37-C) in HUVEC monolayers in Krebs solution in absence
(, control) or presence (R, 8 h) of 1 nM insulin and/or 1.5 mM
actinomycin D (see Materials and Methods Section). B: Western blot
(representative of other eight different cell cultures) for hCAT-1
protein abundance and b-actin (internal reference) in HUVEC
monolayers as in (A). Lower part: hCAT-1/b-actin protein ratio
densitometries normalized to 1 in control. C: Ratio for hCAT-1
mRNA/28S rRNA number of copies of hCAT-1 mRNA and 28S rRNA
in cells as in (A). Values are means W SEM (n U 812). MP < 0.05 versus
all other values. yP < 0.05 versus insulin.

Gonzalez, 2009; Sobrevia et al., 2011). Furthermore, NEM and

L-lysine blocked insulin-induced endothelium-dependent
relaxation in umbilical vein rings, suggesting that endothelial Larginine transport could limit NO synthesis in response to this
hormone. This is supported by the findings showing that insulininduced relaxation and insulin inhibition of U46619-induced
contraction are lost in absence of extracellular L-arginine. Thus,
JOURNAL OF CELLULAR PHYSIOLOGY

Fig. 6. Insulin effect on Sp1 nuclear protein abundance and activity.

A: Western blot (representative of other eight different cell cultures)
for nuclear and total Sp1 protein abundance (b-actin was internal
loading control) in HUVEC in absence (Control) or presence (8 h)
of insulin. Lower part: Sp1 nucleus/Sp1 total protein ratio
densitometries normalized to 1 in Control. B: Chromatin
immunoprecipitation assay for Sp1 binding to DNA in HUVEC. The
mRNA flanking Sp1 consensus sequences in SLC7A1 promoter region
(177 and 105 bp from ATG) was amplified by RT-PCR (see
Materials and Methods Section). Input is total DNA. Lower part: Sp1
consensus sequences over Input ratio densitometries normalized to 1
in Control. Values are means W SEM (n U 8). MP < 0.05 versus Control.

G O N Z A L E Z E T A L .

Fig. 7. Insulin effect on human umbilical vein rings reactivity. A: Endothelium-intact or endothelium-denuded human umbilical vein rings were
exposed to increasing concentrations of insulin. B: Endothelium-intact human umbilical vein rings in absence (, control) or presence (R) of 1 nM
insulin and/or 200 mM N-ethylmaleimide (NEM) or 1 mM L-lysine. C: Response of endothelium-intact human umbilical vein rings to U46619 in
absence (control) or presence of 1 nM insulin for the indicated time periods. D: Endothelium-intact human umbilical vein rings incubated in medium
with (R) or without () 200 mM L-arginine were exposed to 1 nM insulin (left part) and/or 100 nM U46619 (right part). Relative responses are given as
percentage fraction of the initial vessel response to KCl (see Materials and Methods Section). Values are mean W SEM (n U 47). In (A), MP < 0.05
versus corresponding values in endothelium-intact vessels. In (B), MP < 0.05 versus all other values. In (D), MP < 0.03 and yP < 0.05 versus all other
corresponding values.

activity. Thus, increased L-arginine transport may result from

increased hCAT-1 expression in HUVEC. Since inhibition of
transcription caused a comparable reduction (90%) in
L-arginine transport, hCAT-1 protein abundance and mRNA
expression in this cell type, the possibility that L-arginine
transport was higher due to insulin increased mRNA and/or
protein stability is unlikely, although this phenomenon cannot
definitively be ruled out. In addition, since hCAT-1 protein
abundance was increased at the plasma membrane, but it was
unaltered at the cytosol fraction in HUVEC exposed to insulin,
the feasibility of an internal pool of pre-existent hCAT-1
translocated to the plasma membrane is unlikely. Insulin also
increases CAT-1 protein abundance in rat hepatocytes (Wu
et al., 1994) and CAT-1 mRNA level in rat cardiomyocytes
(Simmons et al., 1996), thus changes in CAT-1 expression
limiting transport of cationic amino acids are not exclusive for
HUVEC. Since Eadie-Hofstee transformation of saturable
transport data was lineal, without significant changes in the
apparent Km of L-arginine transport, the possibility that insulinincreased transport was due to activation of additional
membrane transport systems is unlikely. Our results also show
that insulin blocked U46619-induced human umbilical vein
vasoconstriction only after 8 h of incubation, suggesting that a
time-dependent mechanism is also associated with the
reactivity of this tissue to insulin. Changes in hCAT-1 protein
abundance in HUVEC, paralleled by changes in L-arginine
transport, requires 4 h of incubation with insulin, and NEM
blocks insulin-induced vasodilatation in human umbilical vein
rings. Thus, insulin effect requires increased expression and/or
JOURNAL OF CELLULAR PHYSIOLOGY

availability of functional hCATs at the human umbilical vein

endothelium.
Insulin also increases Sp1 nuclear protein abundance and its
binding to a proximal region (177 and 105 bp from ATG)
of the SLC7A1 promoter containing at least four consensus
sequences for Sp1. Since increase in Sp1 protein abundance was
higher by 30% than its binding to DNA, increased SLC7A1
transcriptional activity may not only result from higher nuclear
Sp1 protein abundance, but most likely increased activity of this
transcription factor in one or more of the identified consensus
sequences within this promoter region. This is further
supported by findings showing that insulin-increased
transcriptional activity of constructs generated for SLC7A1
promoter region was similar to the increase in Sp1 binding.
Interestingly, CT polymorphism in the 30 -untranslated region
(30 -UTR) of SLC7A1, which contains Sp1 consensus sequences,
in patients with essential hypertension has been reported,
together with reduced SLC7A1 transcriptional activity due to
reduced Sp1 activity (Yang and Kaye, 2009), supporting Sp1
involvement in the response to insulin. However, since several
response elements to insulin have been identified in human
SLC7A1 promoter (Sobrevia and Gonzalez, 2009) as in other
mammalian cells (Hatzoglou et al., 2004), activation of response
elements within the cloned promoter region may explain in
part, but not the full insulin-induced increase in hCAT-1
transcript in HUVEC.
It has been reported that an enhancer element identified in
the first intron of SLC7A1 may play a bifunctional role regulating
SLC7A1 transcriptional activity, that is, increasing SLC7A1

INSULIN-INCREASED hCAT-1 EXPRESSION

transcriptional activity following binding of the purine-rich

element binding protein A (Pur alpha) under basal conditions
and activating transcription factor 4 (ATF4) in endoplasmic
reticulum stress (ERS), or decreasing SLC7A1 transcriptional
activity by the C/EBP homologous protein 10 (CHOP) binding
in ERS in C6 rat glioma cells (Huang et al., 2009). Thus, changes
in hCAT-1 protein not necessarily fit changes in SLC7A1
expression, opening the possibility that a transcriptional
regulation of SLC7A1 and/or post-transcriptional regulation of
SLC7A1 transcript as well as for the protein product expression
may occur. Thus, the findings of an insulin-increased transport
activity via hCAT-1 in HUVEC cannot exclude the possibility
that insulin induces post-translational modifications of hCAT-1.
Our results suggest that hCAT-1 expression and activity are
regulated by insulin in HUVEC, and suggest that insulin increases
expression of SLC7A1 gene due to increased transcriptional
activity most likely due to higher Sp1 activity. The fact that insulin
alters vascular human umbilical vein reactivity in an endotheliumand L-arginine transport (possibly hCAT-1)-dependent manner,
implies a physiological mechanism of relevance for modulation of
hCAT-1 expression and activity in this vessel type, particularly in
diseases associated with altered L-arginine and insulin
metabolism such as hypertension or diseases of pregnancy where
fetal vascular function is altered (Casanello et al., 2007; Sobrevia
and Gonzalez, 2009; Westermeier et al., 2009a,b; Sobrevia et al.,
2011).
Acknowledgments

Authors thank clinical staff from Hospital Clnico Pontificia

Universidad Catolica de Chile labor ward for supply of umbilical
cords. Supported by Fondo Nacional de Desarrollo Cientfico y
Tecnologico (FONDECYT 1070865, 1080534, 11100192),
Programa de Investigacion Asociativa (PIA) from Comision
Nacional de Investigacion en Ciencia y Tecnologa (CONICYT)
(Anillos ACT-73) and Direccion de Investigacion Universidad
de Concepcion (DIUC 210.033.103-1.0). Fellowship Apoyo
Realizacion de Tesis Doctoral from CONICYT AT-23070213
(MG) and AT-24100210 (FW), CONICYT-PhD (Chile)
fellowships (MG, EG-G, FW) and Faculty of Medicine, PUC-PhD
(Chile) fellowship (CS).
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