Sobrevia & Gonzalez 2009

8/10/2019 Sobrevia & Gonzalez 2009

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A Role for Insulin on L-Arginine Transport in Fetal Endothelial Dysfunc-tion in Hyperglycaemia

I Cellular and Molecular Physiology LaboratOl:r (CI"fPL) and Perinatology Research LaboratOlY (PRL), Department of

Obstetrics and Gynaecology, Medical Research Centre (Cn1), School o f M edicine, Faculty of Medicine, Pontificia Uni-

versidad Catolica de Chile, P.O. Box 114-D, Santiago, Chile; 1Department of Physiology, Faculty of Biological Sci-

ences, Universidad de Concepcion, Chile

Abstract: Endothelial cells are keyin the regulationofvascular tone throughthe release of vasoactive molecules,includ-

ingnitric oxide (NO). NO is a gassynthesized from thecationic amino acid L-arginine via the endothelial NO synthase

(eNOS). The semi-essentialamino acid L-arginine is a taken up byendothelial cells via systems y+ and y+L in primary

cultures of human umbilical vein endothelialcells (HUVEC). Systemy+isa familyof membranetransportersincluding at

least five transport systems for cationic amino acids (CAT) of which HUVEC express human CAT-I (hCAT-I) and

hCAT-2B. Exposure ofHUVECtohigh extracellularconcentrationsofD-glucose increasesL-argininetransport,hCAT-lmRNA expressionand eNOS activity.These phenomena are also relatedwith increased production of reactive oxygen

species (ROS), thus supporting the possibility that changes in L-argininelNO signalling pathway result from elevated

ROS. It has been shown that insulin blocks D-glucose-increased L-arginine transport and cGMP accumulation in

HUVEC,whereasin this cell type insulin alsomodulateshigh D-glucoseeffectsby activatingthetranscriptional factors

Spl andNFKB.Thesetranscriptionfactorshaveresponse elements in SLC7A] (for hCAT-I) genepromoterregion,thus

representing2 possible targetsforregulationoftheexpressionof thistransporter byD-glucose and/or insulin inthis cell

type. Recent evidencessuggest that insulin blocksthestimulatoryeffectof D-gJucoseon L-arginine transport byreducing

thetranscriptionalactivity ofSLC7A] via Spl-, NFKB-and ROS-dependent mechanisms. Thus, a role for these transcrip-

tionfactorsin responseto insulin is proposed infetalendothelialcellsexposed tohyperglycaemia.

Keywords: Glucose, hyperglycaemia, diabetes, L-arginine, transport, human, endothelium.

Endothelial cells are of mesenchymal ongm forming a

lane epithelium named endothelium. For decades the endo-

elium was considered as a simply barrier between the

lood and other body tissues. However, this early vision has

now radically changed and currently holds the endothelium

a real organ, made up of approximately ten trillion cells,

,-hich fulfils multiple roles in the physiology and patho-

.hysiology of vascular tone, with autocrine, paracrine and

c:J.docrine actions and involved in the processes of coagula-

:::on and fibrinolysis [1, 2]. Endothelial cells are involved in

:-egulation of vascular tone through the release of a number

f vasoactive substances, such as prostacyclin (PGlz) [3],

dotelin-l [4] and nitric oxide (NO) [3, 5]. Endothelium-

-=crived NO spreads to the underlying smooth muscle cell

~yer in vascular vessels where increases cGMP levels to

bsequently induce relaxation of the muscle leading to

odilatation [3,5,6]. Both in vivo and in vitro experiments

-c:w demonstrated that NO synthesis in endothelial cells is a

__cess that could be stimulated by various molecules, in-

~ding insulin [7-9], D-glucose [10] and adenosine [6].

~ilarly, NO synthesis and/or its bioavailability are lower

.".: ress correspondence to this author at the Cellular and M olecular

_"iology Laboratory (CMPL), Department of Obstetrics and Gynaecol

:..' 'vIedical Research Centre (CIM), School of Medicine, Faculty of Medi-

Pontificia Universidad Cat6lica de Chile, P.O. Box 114-D, Santiago,

- ~: Tel: 562-3548116; Fax: 562-6321924; E-mail: [email protected]

in pathological conditions such as intrauterine growth restric-

tion (IUGR) [11], diabetes mellitus [9,12] or atherosclerosis

[13].

The signalling mechanisms involved in NO synthesis

have been studied in several cell types including human um-

bilical vein endothelial cells (HUVEC). The gas NO is syn-

thesized from the cationic, semi-essential amino acid L-

arginine in a metabolic reaction leading to equimolar forma-

tion of L-citrulline and NO [6, 14]. This reaction requires the

activity of endothelial NO synthases (NOS), a group of en-

zymes conformed by at least three isoforms i.e. neuronal

NOS (nNOS or Type I), inducible NOS (iNOS or type II)

and endothelial NOS (eNOS or Type III) [15]. In fact, there

is evidence that NOS activity may depend on the ability of

endothelial cells to take up its specific substrate L-arginine

via a variety of membrane transport systems [6, 10, 11, 14-

16]. L-Arginine is taken up by endothelial cells via mem-

brane transport systems grouped as systems l,y+L, bO,+andBO.+[6, 16-18]. It is well known that system y+ conforms a

family of proteins known as cationic amino acid transporters

(CATs) family (hereafter referred as 'CATs family'), con-

formed by CAT-I, CAT-2A, CAT-2B, CAT-3 and CAT-4

[17, 19]. CAT-l is ubiquitously expressed, while CAT-2A

and CAT -3 are constitutively expressed in liver and brain,

respectively, and CAT-2B is induced in a variety of celltypes in response to bacterial endotoxins and pro-

inflammatory cytokines [6,17, 19-21]. CAT-4 derives from


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a sequence of cDNA that is41-42% identical to other mem-

bers of CATs family, but no transport activity has been yet

described [6,17]. CAT-I, CAT-2B and CAT-3 are function-

ally characterized as high affinity (Km ~100-400 f.lM),Na+..

independent transporters, while CAT-2A has arelatively low

affinity for cationic amino acids (Km ~2-5 mM). Interest-

ingly, only 2 members of the CATs family have been func-tionally characterized in primary cultures of HUVEC i.e.

human CAT-l (hCAT-l) and hCAT-2B, while hCAT-2A

seems not to be expressed inthis cell type [6, 10, 11, 17, 18].

In addition, hCAT-l protein and mRNA, and only hCAT-2B

mRNA have been detected in this cell type [17].

Phylogenetically, the SLC7 ('solute carrier') family con-

sists of 2 subfamilies formed by CATs and the amino acid

transporters associated to glycoproteins (HATs, heterodi-

meric amino acid transporters). CATs family members are

encoded by genes SLC7Ai, 2, 3 and 4, whose protein prod-

ucts contain 14 tr ansmembrane domains [19]. Specifically,

the gene encoding hCAT-1 protein i.e. SLC7 Ai (located onchromosome 13q 12-q 14), consists of an open reading fr ame

with 11 exons and 10 introns. However, towards the 5'-end

two additional untr anslatable exons (-2 and -1) have been

described r..-:;.Th -.. ~. now suggested that SLC7Ai couldbe formed by I xo -. \yhere transcription start site (ATG)

is located in exon -_ [_ ]. \- e have recently cloned a fr ag-

ment of -6"0' u ~ the ATG of SLC7Ai in primary

cultures of HC\"EC and detected that this fragment of the

potential promo er of SCL Al contains several consensus

sequences for transcription factors that may be critical for theregulation of its expression by insulin and/or D-glucose [23,

24] (Fig. 1).

INSULIN AND D-GL 'COSE EFFECT ON ENDOTHE-

LIAL L-ARGININE/NO PATHWAY

L-Arginine Transport

Incubation of HUVEC primary cultures in an euglycae-

mic culture medium (i.e. containing -5 m M D-glucose) with

physiological r esting concentrations of insulin (-0.1 nM)

increased the maximal transport velocity (Vmax) without sig-

nificant changes in the apparent Km for L-arginine transport

[23]. This phenomenon was seen as a higher maximal tr ans-

port capacity (i.e. ~naJKm) [17, 20, 25, 26] of the involved

membrane transporters for cationic amino acids [23]. The

-650 GTTAAGGTGAACTGCGCTCCAGCCTGGGCAATAGAGTAACACCCTGTCTCTAAAATGAAAAGAAAACAG- - .-581 TTTAAACCTTTTAAGTGCATACCAAATCTTTTATTTTGGAGAAGGAAAACTGGTCTCGAGTTCCGTGTG

P53

-512 AGCTCCCTGGGGCCCGCCGGGAGGGGGTTGGCACGGCCGGACCTGCAGCACTAGTTCTGGCCAGGGCGC

-443 TGTGGGATCTGCAGGGGACCACAGGATGCTGTGGCGCGGTGCGCTCAGATTGGCGGAGAAACGGCCACA

EGR-1 NFKB (p50)

-374 CGCCTACGGAGCTACTGAGAAGGCGAGCGGAGGCGCAGCCCGCCCGCCCGCCGCGGGAACCCCAGGTTG

-305 GGGCGCTGGGCGCGCGAAGACTCAGCCGCCCCGCCCACCAAGGGCGCGTCGGTCCCCGGCCGCAGCCTC

CREB Sp1

-236 TGGGCTGGCAGCCGCCGCCGCGCCGCGCTCCCATTGGTGCCCGGCGGTGACGCGGCCGAGCGGGCCGGG

Sp1 Sp1 Sp1

-167 GCTGCCTGGTCCGGGGGCGGGCGTGGGGCGCGGGGCGCGGAGCGCGAGGGGCGGGGGCCGGGCGCACTG

Elk- 1

-98 CTGATGAAACCTGGCGCCGGAACCCGCCAGCCCTCGGCGCCCATTCAGTCCGCGCAGGCAGGTGTGAGC

*

+109 GAGCGCGTCCGACAGTCTGTCTGTTCGCGATCCTGCCGGAGCCCCGCCGCCGCCGGCTTG~

Fig. (1). Scheme of the -650 bp region from proposed transcription start site at SLC7AI. Thesequenceofthe region -650 bpfromthe

proposed transcriptionstartsite(*)ofthe potential promoter ofthe geneS LC7 A I (forhuman cationic amino acid transporter 1, hCAT-1) that

hasbeencloned is shown.Proposed firstexon ofthetranscript extendsbetween -650to+ 142bp. Thelocation ofprimersusedtoamplifYthi

region of thepromoter isindicatedbythe arrowsandcorrespondedto:sense ACGCGTTAA GGTGAA CTGCGC TCC, antisense CCATGGATC GCG AAC AGA CAG ACT.Underlinedare bindingsitesfor transcriptionfactors such asstimulatoryprotein 1(Spl), nuclear

factorkappaB (NFKB), p53 tumor repressor factor,early growth responsefactor1 (EGR-l), cyclic AMP responseelement-binding (CREB)

and the transcriptionfactorwith ETS domain (Elk- J) .


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(NT1 ONOO.

L-Arginine

hCAT-1 protein

" heAT' mRNA

Insulin ~

! -- /..ROSj~.....

SplNF,B

Fig. (2). Proposed regulatory mechanisms of hCAT-I expression and activity by high D-glucose and insulin in HUVEC. Exposure of

HUVEC to an elevated extracellular level of D-glucose (High D-glucose) increases the intracellular level of reactive oxygen species (ROS)

likely derived from NAD(P)H oxidase. ROS formation may stimulate via Sp I and NFkB transcription factors the SLC7AI gene promoter

activity leading to increased hCAT-1 mRNA expression and protein abundance. These events result in an increased L-arginine tr ansport via

heAT-I and nitric oxide (NO) and L-citrulline synthesis via the endothelial NO synthase (eNOS). The NO could rapidly react with ROS to

fonn peroxynitrite (ONOO) contributing to endothelial dysfunction. Insulin isproposed to protect the endothelial cell by blocking the effect

of D-glucose on the generation of intracellular ROS, reducing the effect of these molecules on SLC7Al expression and L-arginine transport.

Insulin under hyperglycaemia could also act as a repressor of D-glucose activated ROS generation protecting the endothelial cells from this

abnormal environment.

reported magnitude of the insulin stimulatory effect on L-

arginine transport (~5 fold) was comparable to the trans-

-rimulatory effect of L-lysine on L-arginine transport meas-

ured under zero-trans conditions for L-lysine in this cell type

[II, 23] (see Fig. (2. These results are complemented and

supported by parallel studies showing that insulin increased

dlehCAT-I mRNA number of copies [23] and protein abun-

ance (Gonzalez M, Sobrevia L, unpublished data). These

-rudies suggest that insulin-induced L-arginine transport is

most likely due to increased expression and activity of

,CAT-! membrane transporters in HUVEC. In addition,

:l1Sulineffect on L-arginine transport was proposed as a phe-

:lOmenon resulting from a sequential activation of cell sig-

:1alling molecules, including phosphatidylinositol 3 kinase

PI3k), protein kinase C (PKC), NOS and mitogen-activated

rotein kinases p42 and p44 (p42/44mapk), which are known

to be involved also in the modulation of several other mem-

rane transport systems in mammalian cells [6, 17].

It has been reported that incubation ofprimary cultures of

~VEC with increasing concentrations of extracellular D-

glucose for a period of 2 minutes (acute effect) increased L-

c:rginine transport, reaching a maximal effect at 25 mM D-

_ucose [10]. The increase in L-arginine transport induced by

ute exposure of HUVEC to high D-glucose has been ex-

lained as an increase in the activity ofhCAT-! transporters.-;tis phenomenon is more likely due to plasma membrane

yperpolarization due to increased K+ efflux through ATP-

: nsitive K+ channels ([K +]ATP)[10]. It has also been re-

ported that longer incubation periods (24 h) with high con-

centrations of extracellular D-glucose (~25 mM) increases

the ~nax of the L-arginine transport, an effect blocked by

inhibition of protein synthesis or when cells are pre-

incubated with 1 nM insulin for 8h [16]. The increase of L-

arginine transport induced by chronic incubation with D-

glucose was also associated with increased hCAT-l mRNA

level and L_[3H] citrulline synthesis from L-eH] arginine

[24]. In preliminary assays, we have been able to detect a

potential effect of D-glucose at a transcriptional level modu-

lating hCAT-! expression, a phenomenon that is also

blocked by i nsulin (Gonzalez M, Sobrevia L, unpublished

data). Thus, it is feasible that insulin could be triggering sig-

nalling pathways that will potentially interfere with an in-

creased SLC7Ai transcriptional activity in response to a high

D-glucose environment.

eNOS Expression and Activity

In endothelial cells from human aorta, long incubation

periods (7 days) with 25 mM D-glucose decreases eNOS

activity, protein abundance and mRNA level, effects that

was associated with r educed activity of eNOS promoter [27].

In HUVEC, chronic incubation with 25 m M D-glucose (up

to 24 h) increases eNOS protein abundance, an effect that

seems involve P13k and protein kinase B (PKB)/Akt activity[24, 28]. Several studies report that in an early hyperglycae-

mic state, activation of these signalling molecules would lead

to cell survival, but after 2 4 h it will increase cell death by


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apoptosis, 'an effect regulated by activation of the nuclear

factor kappa B (NFKB) [28]. This differential response of

cells to states of hyperglycaemia could result f rom accumu-

lation of reactive oxygen species (ROS), whose generation is

elevated in cells exposed to high extracellular concentrations

of D-glucose [28, 29]. On the other hand, in endothelial cells

from mouse lungs it has been reported that insulin could pro-tect in a NO-dependent manner against endothelial dysfunc-

tion (estimated by variations of the electrical trans-

endothelial resistance) induced by hydrogen peroxide (HzOz)

[30]. However, endothelial cells from bovine aorta (BAEC)

exhibit less NO synthesis induced by insulin when cells were

incubated with high concentrations of extracellular D-

glucose, an effect that appears to depend on a signalling

pathway involving insulin receptor type 1 (lR-I), PI3k and

the inhibitor of the kinase subunit of nuclear factor kappa 13

(IKKB) [31]. In studies performed in primary cultures of

HUVEC, high D-glucose (25 mM, 24 h) increased cGMP

synthesis, an effect blocked by 1 nM insulin (8 h) [17]. In the

same cell type it has been shown that at this concentrationand time of exposure to insulin is enough to block the high

D-glucose-dependent inhibition of the membrane transport

of adenosine, an endogenous nucleoside that induces NO-

depehdent vasodilatation in most vascular beds [32, 33]. All

together these results indicate that deleterious effects of

chronic exposure to high concentrations of extracellular D-

glucose could be mediated by increased NO synthesis in

human umbilical vein endothelium. Whether the effect of

high D-glucose and the potential role of insulin as a protec-

tive factor in this phenomenon is either at a transcriptional

and/or post-transcriptional level has not yet been described[9, 17,33].

INSULIN AND D-GLUCOSE EFFECT ON ROS

SYNTESIS

Cell death by apoptosis induced by high concentrations

of extracellular D-glucose has been linked to the increased

production of ROS. This is reflected as increased nitroty-

rosine formation and PKC activity in HUVEC incubated in

the presence of high concentrations of extracellular D-

glucose, either under permanent (20 mM, 14 days) or inter-

mittent (repeated periods of 24 hours with concentrations of

5 or 20 mM D-glucose for 14 days) incubation periods [34].

In BAEC, ithas been shown that incubation with 25 mM D-

glucose for 3 h increases ROS synthesis dependent of

NAD(P)H oxidase [35]. This study also shows that incuba-

tion with physiological concentrations of insulin for 3 h in-

duces an even greater increase in the synthesis of ROS, al-

though during the first hour of incubation with this hormone

is possible to detect a decline in the synthesis of ROS in-

duced by D-glucose [35]. Thus, it is likely that an acute ef-

fect of insulin could be acting to protect from the damage

induced by overproduction of ROS in response to high con-

centrations of extracellular D-glucose. This effect of insulin

could probably be due to activation of NOS and increased

NO bioavailability in the endothelium. The mechanisms by

which ROS induces its effect include regulation of gene ex-

pression and post-translational mod ifications [36]. One of

the most studied transcription factor regulated by ROS is

NFKB, which is found in most cells at rest, and that rapidly

translocates to the nucleus inducing gene transcription after

its a 'y ::0 ::1 . _'hBis also involved in the d evelopment of

insulin res' .one of the first events that occur before the

establ"-hm t 0 diabetes mellitus [37]. Although we know

that imer D-glu 0e or insulin increase L-arginine transport

and:\O . 'llth -is. a haracterization of the mechanisms be-

hind the r ulting in reased ROS production and activation

of transcription fac ors, including d1e zinc finger promoter-selectiYe transcription factor specific protein 1(Spl), in re-

sponse to these molecules is not well established. Unveiling

the intrinsic mechanisms behind modulation of gene tran-

scriptional activity will give us clues to understand a poten-

tial protective effect of insulin before endothelial damage is

triggered bypathological states of hyperglycaemia.

D-GLUCOSE AND I SULIN EFFECT ON Spl ACTI-

VI TY

Within aregion of~4000 bp upstream from the transcrip-

tion start site of SLC7AI, multiple consensus sites of various

transcription factors have been identified by insilica analysis

of sequence. Within d1ese factors, it is possible to identify

consensus sites for Smad-4, NFKB and Spl, among others,

which are regulated by insulin or D-glucose. Within this re-

gion d1ere are at least four consensus sites for Spl close to

the transcription start site ofSLC7 AI. These sites could play

key roles as regulators of the transcription of this gene since

it lacks of TATA box [22]. Also, there are 2 consensus sites

for NFKB around -3200 and -600 bp r egion from the tran-

scription start site, which are potentially regulated by ROS.

Since there is experimental evidence suggesting that Spl is

involved in gene-specific responses to a variety of cellular

signals independent of the interaction with other inducible

transcription factors, and since this transcription factor is

apparently involved in the regulation of membrane transport-

ers expression, such as SLC29AI for the human equilibrative

nucleoside transporters type 1(hENTl) in HUVEC [38], in

preliminary experiments we have studied a region of 650 bp

upstream of the transcription start site in SLC7AI [39]. Spl

is activated by phosphorylation of serine and threonine resi-

dues, a phenomenon altered in response to various stimuli

that activate different signalling pathways. The amino acid

sequence of Spl contains consensus phosphorylation sites

for numerous protein kinases, including calmodulin kinases

(CamKs), casein kinases (CK) 1 and 2, protein kinase A

(PKA), PKC, and p42/44mapk [40]. Interestingly, it has been

reported that increase in the tr anscriptional activity of plas-

minogen activator inhibitor-l (PAl-I) induced by insulin

(100 nM, 16 hours) in HepG2 cells transfected with PAI-l

promoter, involves activity of PI3k, PKC and p42/44map\

among other signalling molecules [41]. In PAI-I gene at

least three consensus sites for transcription factors induced

by insulin has been identified, all of which co-localize with

putative consensus sites for SpI [41]. Thus, a potential role

for Sp 1 as modulator of PAl-1 gene expression is suggested.

In addition, in the same cell type, D-glucose incubation (~23

mM, 24 and 48 h) decreases the level of apolipoprotein A 1

(apoA-I) mRNA, an effect that was blocked by insulin. This

phenomenon was dependent on a region of the promoter

where an insulin response core element (IRCE) is present

[42]. SpI binds to this element and the signallinp pathwaytriggered by insulin appears to involve p42/44map and PKC

activity, and Spl phosphorylation [43].


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I

There is also evidence that SpI is essential for basal tran-

scription of calmodulin gene and for the increase of tran-

scriptional activity of this gene induced by insulin [44]. In

addition, insulin increases the o-glycosylation and phos-

phorylation of SpI in the HAllE hepatoma cell line, which

is reduced when cells are from streptozotocin-induced dia-betic rats [45]. Recently, it has been shown that insulin (100

) increases PKC8 expression, aphenomenon that is under

::cgulation by PKCa in the skeletal muscle cell line L6 via a

:;:nechanismrequiring Sp 1and NFKB activation [46]. Since in

rimary cultures of HUVEC it has been reported that insulin

srimulation of hCAT- I -mediated L-arginine transport de-

_ nds on PI3k, PKC and p42/44mapk activity [23], it is likely

:hat SpI would be acting as akey transcription factor modu-

~ -ng SLC7Ai expression by insulin or high extracellular

:uncentrations of D-glucose in HUVEC (Table 1).

Preliminary experiments performed inHUVEC incubated

'm 25 mM D-glucose (24 hours) and I nM insulin (for last

h of the 24 h period of incubation with high D-glucose)-' ow increased SpI protein abundance in nuclear protein

_ :::-acts(Gonzalez M, Sobrevia L, unpublished data). Co-

_ -ubation of HUVEC monolayers with D-glucose and insu-

lin produced a greater increase of S p I n uclear abundance,

which would possibly indicates that both insulin and D-

glucose activated diff erent signalling pathways to increase

Sp I activity in this cell type. Although ithas not been possi-

ble to determine whether it also leads to an increase in phos-

phorylation or glycosy lation of Sp 1,we found increased S p 1

binding to a region of the SLC7 Ai promoter containing four

consensus sites for SpI in tandem. This finding indicates that

at least part of the D-glucose- and insulin-increased hCAT-I

mRNA level could be due to increased expression and activ-

ity of Sp I on SLC7 Ai. Sp I activity seems to regulate not

only hCA T-1 expression, but could also participate in the

transcriptional regulation of eNOS. The promoter region of

eNOS has been characterized and is known to exhibit con-

sensus sites for transcription factors such as activator pro-

tein-I (AP-I), GATA binding protein 2 (GATA-2), ETS

binding domain (Ets) family members, and members of the

Sp family (Spl and Sp3), among others [47]. On this back-

ground it is tempting to propose that the signalling pathwaysinduced by D-glucose and insulin might include activation of

Spl inducing expression of genes relevant to the endothelial

L-arginine/NO pathway.

I I Stimuli Gene Blank Cell Type Promoter Mechanism ReferencesActivity

Insulin PAl-l Human hepatoblastoma Increased Increased Sp1binding to DNA [41]

HepG2

Insulin Apo-Al Human hepatoblastoma Increased Increased Spl binding to IRCE motifs [42]

HepG2

Insulin Apo-Al Human hepatoblastoma Increased Phpsphorylation of Sp 1 a nd increase of [43]

HepG2 IRCE binding

Insulin Calmodulin Rat hepatoma H-4 lIE Increased Promotor without Sp I binding sites, cannot [44]

I I be stimulated by insulin

Insulin PKC15 Skeletal muscle L6 Increased Inhibition of expression and activity of Sp I [46]

blocked the effect of insulin on PKCo

promoter

J-Glucose TGF-j3l PAl-l Rat mesangial cells Increased Inhibition of expression and activity of SpI [61]

blocked the effect of insulin on TGF-fil

and PAI-I promoter

J-Glucose lR-l Rat hepatocytes Increased Effect ofD-glucose on promoter was [62]

blocked when 3 or 4 binding sites of SpI

were mutated

._ ose Insulin Leptin Adipocytes and hepa- Increased Mutation ofSpl response element, blocked [63]

tocytes from rat the D-glucose/insulin effect

=>-Glucose Acetyl-CoA SL2 from Drosophila Increased Mutation of Sp 1binding sites, blocked the [64]

effect of D-glucose

H,O, Spl Nuclear extracts f rom n.d. H,O, decreased the Spl binding to DNA [65]

rat kidney

H,O, Aldolase Pyruvate Rat timocytes n.d. H,O, decreased the Sp I binding to DNA [66]

kinase


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INSULIN ~D D-GLUCOSE EFFECT ON NFKB

Since it was described in 1986, the transcription f actor

NFKB has been the subject of many studies due to its impor-

tant role in inflammatory processes and other pathologies. In

the beginning NFKB was described as a specific protein of B

cells, but it is now known to be an ubiquitous transcription

factor [48]. NFKB activation is induced and independent of

protein synthesis, requiring post-translational changes for its

migration to the nucleus. Initially, NFKB was described as a

transcription factor that could be stimulated by several im-

munological stimuli, such as tumour necrosis factor (TN F),

lipopolysaccharide (LPS) [49, 50], interleukin-I (IL-I) [51],

or as a f actor that activates T cells [52]. However, more re-

cently it has been shown that the transcriptional activity of

several genes is increased via NFKB in response to a series of

stimuli that are unrelated with the immune response, such as

ultraviolet radiation, growth factors [53] or viral infections

[54]. The latent nature of NFKB is in relation with it associa-

tion with the inhibitory protein kappa B inhibitor (IKB). The

release of this complex allows the migration of NFKB to the

nucleus [55]. Interestingly, it has been reported that incuba-

tion of BAEC in culture medium containing 30 mM D-

glucose increases NFKB translocation toward the nucleus

[56]. In addition, in HUVEC it has been shown that hyper-

glycaemia also increased NFKB protein abundance in the

nucleus, and that this increase depends on the activity of

PI3kJAkt [57], signalling molecules that ar e determinant for

L-arginine transport and NO synthesis in response to insulin

in this cell type [17, 23]. Other studies also show that incuba-

tion of BAEC and human aortic endothelial cells (HAEC) in

30 mM D-glucose (16 hours) increases NFKB binding to the

DNA [58]. Thus, it seems clear that high D-glucose effect on

endothelial cells involves NFKB activation in a variety of

endothelia indicating that this phenomenon is not limited to a

specific type of endothelium. On the other side, insulin has

been shOKl1 to either reduce NFKB binding to the DNA in

HAEC [59] or increase )!FKB-induced transcriptional activ-

ity of genes in '-ascular smooth muscle cells from bovine

aorta (YSMC) transfected with a vector containing response

elements to NFKB [60]. Thus, literature regarding the poten-

tial involvement of NFKB in cell response to insulin is wide

open and specific mechanisms are not well understood (Ta-

ble 2).

CONCLUDING REMARKS

It is proposed that insulin modulates L-arginine transport

and NO synthesis in human fetal endothelium via cell signal-

ling mechanisms that respond to increased activity of Sp I

andNFKB transcription factors. The gene SLC7 Al coding for

hCAT -I membrane transporters express consensus se-

quences for binding of t hese transcription factors, particu-

larly S p I, suggesting that increased expression of hCA T-I in

response to elevated D-glucose and insulin could result from

increased expression ofSLC7 AI. Nothing is known regard-

ing ROS and the transcriptional activity of SLC7AI in re-sponse to elevated D-glucose or in response to insulin, even

when it is accepted that the pathological condition of hyper-

glycaemia leads to generation of abnormal levels of ROS in

HUVEC. These fmdings could be d eterminant in the dy-

namic of the regulation of expression and activity of L-

arginine membrane transporters (particularly hCA T-I) and

eNOS in human fetal endothelium d erived from pregnancies

where abnormally elevated plasma D-glucose levels could be

found such as in gestational diabetes.

Supported by Fondo Nacional de Desarrollo Cientifico y

Tecnol6gico (FONDECYT 1070865, 1080534, 7070249)

Stimuli Gene blank Cell type Effect on Mechanism References

mRNA level

D-Glucose NFJd3 BAEC n.d. Increased nuclear localization ofNFK.B [56]

D-Glucose COX-2 HUVEC n.d. Increased NFKB DNA binding [57]

D-Glucose NFKB HAEC n.d. Increased NFKB DNA binding [58]

D-Glucose Fibronectin HUVEC Increased Increased NFKB DNA binding [67]

D-Glucose VCAM-l HAEC Increased Increased NFKB DNA binding [68]

D-Glucose Fibronectin HUVEC Increased Increased NFKB DNA binding [69]

Insulin ?KCa L6 skeletal muscle Increased Inhibition ofNFKB activity blocked the [46]

increase ofmRNA

Insulin MC?-! HAEC Decreased Decreased NfKB DNA binding [59]

ROS NFKB HUVEC n.d. Inhibition ofROS synthesis blocked ROS- [70]

induced NFKB activity

ROS NFKB HMEC.l n.d. LPS-increased ROS increased NfKB DNA [71]

binding

BAEC: bovine artery endothelialcells, HUVEC: humanumbilicalvein endothelialcells, HAEC: human arteryendotbelialcells, HMEC.I: human dermal microvascularendotheli2l

cells,LPS: lipopolysaccharide,ROS: reactive oxygen species,n.d.: not determined.


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#

dComisi6n Nacional de Ciencia y Tecnologia (CONICYT

-~070213), Chile. M. Gonzalez holds a CONICYT-PhD

~!Jowship (Chile). We thank the researchers at the Cellular

Molecular Physiology Laboratory (CMPL) and Perina-

_ogyResearch Laboratory (PRL) of the Pontificia Univer-

d Cat6lica de Chile (puq for their contribution in the__ duction of the experimental data that has been cited

:=oughout the text. Authors also thank the personnel of the

o pital Clinico Pontificia Universidad Cat6lica de Chile-,' urward for supply of umbilical cords.

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Sobrevia & Gonzalez 2009