Placenta (2005), Vol. 26, Supplement A, Trophoblast Research, Vol. 19
doi:10.1016/j.placenta.2005.01.003
Fetoplacental Transport and Utilization
of Amino Acids in IUGR — A Review
T. R. H. Regnault*, J. E. Friedman, R. B. Wilkening, R. V. Anthony and W. W. Hay Jr.
Perinatal Research Center, Department of Pediatrics, Division of Perinatal Medicine, University of Colorado
Health Sciences Center, F441, Aurora, CO 80045, USA
Paper accepted 6 January 2005
Amino acids have multiple functions in fetoplacental development. The supply of amino acids to the fetus involves active
transport across and metabolism within the trophoblast. Transport occurs through various amino acid transport systems located on
both the maternal and fetal facing membranes, many of which have now been documented to be present in rat, sheep and human
placentas. The capacity of the placenta to supply amino acids to the fetus develops during pregnancy through alterations in such
factors as surface area and specific time-dependent transport system expression. In intrauterine growth restriction (IUGR),
placental surface area and amino acid uptakes are decreased in human and experimental animal models. In an ovine model of
IUGR, produced by hyperthermia-induced placental insufficiency (PI-IUGR), umbilical oxygen and essential amino acid uptake
rates are significantly reduced in the most severe cases in concert with decreased fetal growth. These changes indicate that severe
IUGR is likely associated with a shift in amino acid transport capacity and metabolic pathways within the fetoplacental unit. After
transport across the trophoblast in normal conditions, amino acids are actively incorporated into tissue proteins or oxidized. In the
sheep IUGR fetus, however, which is hypoxic, hypoglycemic and hypoinsulinemic, there appear to be net effluxes of amino acids
from the liver and skeletal muscle, suggesting changes in amino acid metabolism. Potential changes may be occurring in the
insulin/IGF-I signaling pathway that includes decreased production and/or activation of specific signaling proteins leading to
a reduced protein synthesis in fetal tissues. Such observations in the placental insufficiency model of IUGR indicate that the
combination of decreased fetoplacental amino acid uptake and disrupted insulin/IGF signaling in liver and muscle account for
decreased fetal growth in IUGR.
Placenta (2005), Vol. 26, Supplement A, Trophoblast Research, Vol. 19
Ó 2005 Published by IFPA and Elsevier Ltd.
Keywords: Amino acid; Placenta; Fetus; IUGR; Oxygen; Insulin; mTOR
INTRODUCTION
Amino acids and glucose constitute the carbon and nitrogen
requirements for the placenta and fetus. Recently, a greater
understanding of the fetal supply and metabolism of amino
acids by the placenta during pregnancy has led to the concept
that amino acids act as regulators of placental and fetal
development. In addition to being a source of protein building
blocks and oxidative fuel sources, amino acids influence
metabolic cycling pathways between the placenta and fetus.
They also act directly to regulate protein synthesis in various
fetal and placental tissues through interactions with the insulin
and IGF growth-promoting axis via regulation of the amount
and/or activity of a key signal transduction protein, mammalian
* Corresponding author. Perinatal Research Center, Department of
Pediatrics, Division of Perinatal Medicine, University of Colorado
Health Sciences Center, PO Box 6508, F441, Aurora, CO 80045,
USA. Tel.: C1 303 724 1603; fax: C1 303 724 0898.
E-mail address: tim.regnault@uchsc.edu (T.R.H. Regnault).
0143e4004/$esee front matter
target of rapamycin (mTOR). This review deals with aspects
of human and animal placental amino acid transport, fetal
utilization of amino acids, and aspects of the fetal sheep
insulin/IGF-I signaling pathway during normal pregnancy.
Additionally, we discuss changes in these aspects as they occur
in a sheep model of fetal growth restriction, and where possible
compare them with human as well as other animal models of
IUGR.
AMINO ACID TRANSPORT ACROSS
THE PLACENTA
In recent years, both in vivo and in vitro studies have
highlighted the complex characteristics of placental amino acid
transport and metabolism (see reviews, [1e4]). Unlike an
organ such as the liver, the placenta must carry out its own
metabolic functions while also operating as an organ of
transport, delivering amino acids from the maternal circulation
into the fetal circulation. The concentrations of most amino
Ó 2005 Published by IFPA and Elsevier Ltd.
Regnault et al.: Fetoplacental Amino Acid Metabolism
S53
acids, particularly some of the essential amino acids, are higher
in fetal than maternal plasma (noted in rat, sheep and human
pregnancies [5e9]), consistent with active transport of amino
acids across the placenta. An increased fetal-to-maternal ratio
of plasma amino acid concentrations has been documented for
many species, and although the fetal/maternal concentration
ratio is greater than 1.0 for most amino acids measured, there
are quantitative differences among species, as reviewed [4].
The active in vivo transport of amino acids across the
trophoblast involves three fundamental steps: (1) uptake from
the maternal circulation across the microvillous membrane; (2)
transport through the trophoblast cytoplasm; and (3) transport
out of the trophoblast across the basal membrane into the
umbilical circulation. This transport is regulated through
transporter protein systems on both membranes of the
trophoblast. Several mammalian amino acid transport systems
have been characterized over the years by such general
properties as ion-dependence, transport kinetics, substrate
specificity, light chain and heavy chain interactions (monomeric
or heterodimeric systems), and regulation of activity, which has
allowed the description of multiple transport systems for
neutral, anionic, and cationic amino acids [3,10e13]. Specific
studies in the human and rat trophoblast have yielded more
specific information concerning the location, direction of flux,
and functioning of amino acid systems in the placenta (see
reviews, [2,4]). Figure 1 represents a summary of selected
trophoblast amino acid transport system activity location, for
these two species, together with information detailing the
related cDNA. In addition to the wide number of systems
found to exist on the placental membranes, it is interesting to
note that while each system is distinct in its operational
requirements, many systems exhibit overlapping substrate
specificity. Several of these systems are under investigation in
the sheep placenta (GenBank Accession number: LAT-1,
AY162432; LAT-2, AY162433; EAAT-1, AY157709; EAAT-2,
AY157710; EAAT-3, AY157711) including examination of
changes in their expression and transport capacity in IUGR
and normal control conditions.
Heavy (4F2hc or rBAT)
and light chain associations
System substrates
Neutral AA
ALA, SER, PRO, GLN
MeAIB, AIB
A
ATA-1, -2 and –3
(SNAT-1, -2 and –4)
[21, 116-122]
ALA, SER, CYS,
Anionic AA
ASC
ASCT-1
[56, 118, 119, 123-125]
NEUTRAL AA,
BCH
Bo
ASCT-2
[126-129]
NEUTRAL AA,
LYS, ARG, LEU
bo,+
rBAT/bo,+
[23, 130-132]
LEU, ILE, VAL,
TRYP, PHE, MET,
TYR, GLN, BCH
L
4F2hc/LAT-1 and
4F2hc/LAT-2
[133-137]
Cationic AA
y +L
4F2hc/ y+LAT-1 and
4F2hc/ y+LAT -2
[23, 55, 131, 138-142]
Cationic AA
y+
CAT-1, -2B and -4
[22, 23, 138, 139, 143, 144, 145]
Anionic AA
GLU, ASP
XAG-
EAAT-1, -2 and -3
[146-150]
TAU
β
TAUT
[122, 151-153]
Apical membrane
Basal membrane
Figure 1. Schematic representation of selected sodium-dependent (C) and sodium-independent (B) amino acid transport system activity location for both the
apical (maternal) and basal (fetal) facing membranes. Amino acid substrates and the heavy and light chain proteins interactions for each of these placental-specific
systems are listed. References for each system’s trophoblast activity location and heavy and light chain interactions are also provided. Notes: substrates in bold
represent essential amino acids; MeAIB, methylaminoisobutyric acid; AIB, a-aminoisobutyric acid; BCH, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid.
S54
PLACENTAL AMINO ACID TRANSPORT
CAPACITY
An important aspect of placental transport capacity relates to
the total placental surface area across which transport can
occur. As pregnancy advances, the increasing nutrient
demands of the developing conceptus must be met through
an appropriate increase in placental nutrient transport
capacity. This enhanced performance is facilitated through
alterations in placental perfusion and changes in membrane
exchange surface area size and function. Fetal weight increases
approximately 20-fold between weeks 16 and 40 (term) of
gestation in the human, whereas the peripheral villous surface
area increases only ninefold [14e17]. Thus, the increased
microvillous density on trophoblast cells alone cannot explain
the exponential fetal growth occurring over this time period,
indicating that fetal growth is supported not only by changes
in villous surface area, but rather by the overall change in total
exchange capacity. The makeup of this capacity includes the
concentration and affinity characteristics of specific amino acid
carrier proteins on the cell membrane surface and circulating
amino acid concentrations.
Accompanying the maturational changes in trophoblast
surface area, the capacities of the amino acid transport systems
change with differing expression and transport parameters
throughout gestation [18e23]. For example, in the human firsttrimester microvillous membrane has increased transport
activity compared to term placental vesicles [20], and the
protein concentration of the heavy chain, 4F2hc (CD98), which
is associated with heterodimeric transport systems, differ
between early/mid pregnancy and term placenta [19,23]. Also
of interest is that in early gestation, the Km of the microvillous
high affinity System yCL is significantly less than in term
preparations [23]. It is also observed that the same amino acid
may be transported through different systems, depending upon
which membrane is being crossed. In term placenta, L-arginine
transport in microvillous membrane preparations is believed to
occur through both yC and yCL systems, while in the fetal
facing basal membrane, transport may be restricted to the yCL
system [23]. Such studies highlight the complex interactions
that occur between developing microvillous membrane and
basal membrane, within the trophoblast and between the two
circulations, to facilitate an increase in nutrient delivery to the
growing fetus as gestation advances.
Circulating maternal amino acid concentrations also play
a major role in influencing placental amino acid transport
capacity and ultimately determining fetal supply of amino
acids. In sheep studies, the simultaneous flux from maternal
to fetal circulations has been studied for the essential amino
acids [24e26]. These recent studies demonstrate striking
differences for rates of placental clearance of the essential
amino acids where clearance is most rapid for valine,
leucine, isoleucine, phenylalanine, and methionine, with
differences in their transplacental flux directly attributable to
the differences in their concentrations in maternal plasma
[25]. This observation among several amino acids studied
Placenta (2005), Vol. 26, Supplement A, Trophoblast Research, Vol. 19
simultaneously is supported by experiments in which the
concentration of individual amino acids is increased in the
maternal circulation by infusion, showing that net umbilical
uptake of the amino acid increases directly with its maternal
plasma concentration. This has been shown for alanine,
leucine, lysine, threonine, and glycine [27e31]. However,
because of competitive inhibition among multiple amino
acids for common transporters, when a large number of
amino acids are given to the mother simultaneously, the
umbilical uptake of some amino acids increases, but not that
of all amino acids [25,26,32]. These observations have direct
clinical relevance, as trials of infusions of mixed amino acid
solutions into pregnant women to improve amino acid
supply to the fetus might produce an imbalance in the
actual uptake of amino acids by the fetus, not necessarily
a potentially good therapeutic result. Such studies also have
shown that placental transport of a given amino acid is
a function of its concentration in the maternal plasma and
the concentration of inhibitory amino acids, and such
relationships are different for each amino acid. Studies in
human normal and IUGR pregnancies show similar
relationships among maternal plasma concentrations of
amino acids and net placental transport to the fetus [33,34].
AMINO ACID TRANSPORT IN ANIMAL
MODELS OF PLACENTAL INSUFFICENCY –
IUGR AND HUMAN IUGR
Human placental insufficiency is difficult to study, for ethical
and practical reasons, and is frequently limited to studies at
term. Thus, the mechanisms responsible for placental insufficiency and IUGR can be best investigated in animal
models. Several laboratories have used the pregnant sheep to
study early placental growth and development [35,36], and
placentalefetal physiological interactions [37e42]. Growth
restriction in fetal sheep can be induced by nutritional
restriction, placental infarction with microspheres, reduction
of placental implantation sites, uterine or umbilical artery
clamping or ligation, and exposure of the pregnant ewe to
elevated ambient temperatures (hyperthermia model;
[35,41,43e45]). The maternal hyperthermia model involves
prolonged increases in maternal core body temperature
(w0.6e0.8(C increase; [46]). It is a naturally occurring
condition common to sheep that live in warm climates that
have a ‘‘spring’’ estrus and carry the pregnancy through the
hotter summer months. This model produces placental
insufficiency (PI) resulting in IUGR that shares many of the
commonly measured characteristics of human IUGR that
results from placental insufficiency [47,48], in contrast to
mechanical models of placental growth inhibition that usually
are imposed late in gestation and involve either surgical
reductions in placental size or relatively severe reductions in
uterine and/or umbilical blood flow, none of which are
characteristic of the common form of human placental growth
restriction and nutrient transport insufficiency. Since the early
1980s, our efforts have focused on validating the maternal
Regnault et al.: Fetoplacental Amino Acid Metabolism
S55
Transplacental PO2 gradient
(mm Hg)
hyperthermia form of fetal growth restriction as a natural
experimental model of IUGR, and to compare it to similar
studies of human IUGR [27,29,35,37,41,42,46,49].
In the hyperthermia-induced IUGR model (PI-IUGR),
reduction in fetal oxygenation ranges from mild to severe, with
similar variable reductions in placental and fetal weights. In
the more severe cases, net umbilical oxygen uptake rate
(UO2U, mmol/min/kg fetus) and the ratio between oxygen
uptake and the transplacental arterial PaO2 gradient (UO2U/
DPO2, mmol/min/100 g placenta/mmHg) are both significantly reduced (Regnault et al., Unpublished data). The
reduced UO2U/DPO2 ratio specifically defines a decreased
placental oxygen diffusion capacity. More recent studies
examining severe IUGR fetuses have defined a significant
relationship between placental weight and the increasing
transplacental PaO2 gradient (Figure 2). In these IUGR fetus
the UO2U/DPO2 ratio is reduced by almost 35%. Such
observations indicate that the IUGR trophoblast, in this
model, has developed reductions in possibly two of the
primary factors associated with diffusing capacity, permeability and surface area, and therefore a reduced placental
functional capacity. Furthermore, another experimental model
of IUGR supports this observation. In transgenic mice with
a deletion of the placental-specific transcript (P0) of the Igf2
gene, placental surface area is reduced and placental thickness
increased, significantly reducing the theoretical diffusing
capacity of these placentae [50,51]. It is interesting to note
that in studies specifically concerned with abnormal placental
development near term in human IUGR, there is a reduction
in inter-microvillous space and absolute value for microvillous
and total trophoblastic surface area [52e54], supportive also of
a reduced placental functional capacity.
Actual absolute placental amino acid transport is reduced in
cases of placental and fetal growth restriction. Reduced uptake
of leucine and lysine in vesicles prepared from human IUGR
placentae has been described and indicates decreased transporter concentrations and/or activity for neutral and cationic
amino acids [55]. Also, in the maternal dietary protein
deprivation model of IUGR in the rat, there appears to be
a down regulation of placental amino acid transport in systems
60
50
40
R2 = 0.79
30
20
10
0
0
100
200
300
400
500
600
Placental weight (g)
Figure 2. Relationship between transplacental oxygen gradient (uterine
venous PO2 less umbilical venous PO2, mmHg) and placental weight (g) in
control (C) and IUGR (B) animals near term at 135 days gestational age.
Xÿ
AG
A, y and
[56,57]. In the PI-IUGR model there is
a reduction of maternal leucine flux into the placenta and into
the fetus. In contrast to these changes in placental leucine flux,
the uteroplacental oxygen and glucose consumption rates are
similar to control animals on a per gram basis, indicating that
the decrease in leucine metabolism is not a result of reduced
placental metabolic rate [29]. The reduction of leucine flux into
the placenta is believed to be the result of a deficiency in the
number and/or activity of amino acid transporters in the IUGR
placenta. Both fetal leucine concentration and the flux of leucine
from the fetus back to the placenta are reduced, and fetal
oxidation of leucine is decreased [29]. These findings are similar
to those reported to occur in human IUGR [58,59]. Specifically,
the fetal/maternal ratio of leucine has been correlated with the
severity of growth restriction, and as in sheep IUGR studies,
demonstrates a decreased transplacental leucine flux and/or
increased protein breakdown within the IUGR fetoplacental
compartments [58]. More recent studies in the PI-IUGR sheep
model have highlighted other specific defects in the placental
neutral and anionic amino acid transport systems [49,60].
Current research in the sheep PI-IUGR model is attempting to
determine the exact nature of these changes through examination of System L and Xÿ
AG transport isoform expression and
surface area calculations. These human and animal studies
clearly demonstrate that a number of important amino acid
transport systems on both membranes of the trophoblast are
impaired in experimental models and natural conditions of
placental and fetal growth restriction.
Earlier studies in humans showed reduced concentrations of
essential amino acids such as leucine and lysine in growthrestricted fetuses [61e63], indicating possible alterations in
placental amino acid transporter systems. Recently, however, in
situations of well-defined IUGR, but with significantly lower
than normal umbilical venous oxygen saturation, oxygen
content, and pH, umbilical venous concentrations of leucine
and phenylalanine were not significantly different from the
AGA pregnancies [59]. Interestingly in the sheep model of
placental insufficiency induced IUGR, we also observe that
while there are significant reductions in the placental flux of the
essential amino acids leucine and threonine [27,29], amino acid
concentrations are not necessarily different from normal
concentrations. For example in the earlier leucine flux studies,
fetal arterial leucine concentration was significantly reduced
(w30%) [29], while in later studies, concentrations of threonine
were not significantly different between the two study groups
[27], yet in both studies, significant reductions in specific amino
acid flux per unit placenta were observed. Furthermore, in past
and current studies of severely hypoxic and hypoglycemic sheep
fetuses in which umbilical uptake of the essential amino acids is
significantly reduced, fetal essential amino acid concentrations
are unaltered compared to control animals [64e67], similar to
observations in humans and guinea pigs [68e70]. It is possible
that in the presence of relatively more hypoxic and hypoglycemic conditions, a hypometabolic state within the IUGR fetus
contributes to the maintenance of essential amino acid
concentrations, despite reduced umbilical uptakes. Despite
C
S56
these observations of highly variable differences in fetal amino
acid concentrations among conditions and models, both human
and animal studies emphasize that placental amino acid
transport is reduced in cases of placental growth restriction.
This reduction is likely a combination of many factors including
changes in trophoblast exchange capacity as well as uteroplacental oxygenation and fetoplacental metabolism.
FETAL HEPATIC AND SKELETAL MUSCLE
UTILIZATION OF AMINO ACIDS
During normal fetal development there is no appreciable rate
of fetal hepatic gluconeogenesis. Fetal hepatic glucose production would be counterproductive since an increased glucose
concentration would reduce the transplacental glucose gradient, leading to decreased umbilical glucose uptake from the
mother [71,72]. Although gluconeogensis in the adult liver
consumes amino acids, there is no measurable fetal gluconeogenesis, despite the higher concentration of amino acids in the
fetal rather than the maternal circulations. However, there is in
fact a high rate of hepatic uptake of all of the essential and
most of the nonessential amino acids by both lobes of the fetal
liver [7,73], including all of the gluconeogenic amino acids. In
fetal life, the carbon from these amino acids is released
primarily as glutamate and pyruvate with a smaller contribution coming from the hepatic release of serine, ornithine, and
aspartate [7,74e76], in contrast to postnatal life, where the
carbon from these amino acids is released from the liver solely
as glucose. Studies concerned with hepatic function in the
model of PI-IUGR are currently being conducted. However, it
is interesting to note that in the situation of experimentally
elevated fetal dexamethasone levels, analogous to elevated
cortisol concentrations as in other models of IUGR, hepatic
glutamine and alanine uptakes and hepatic glutamate output
are reduced [77]. This might suggest that in the hypoxic,
hypoglycemic IUGR fetus, hepatic glutamine and alanine
uptakes may also be reduced.
Under normal physiologic conditions, the hind limb of
a growing ovine fetus shows net uptake of both essential and
nonessential amino acids from the fetal circulation, presumably
contributing to the relatively high nitrogen accretion rate of
the fetus [78]. The fetal hind limb also has no net release of
alanine [73], in contrast to the adult hind limb that displays
a large output of alanine and glutamine [79]. The normal ovine
fetal hind limb also consumes the two amino acids that are not
supplied by the placenta: glutamate and serine, which are
produced from the fetal liver in amounts that could account for
the combined uptake of glutamate by both placenta and hind
limb [75,76,78,80]. With reduced placental supply of glucose
to the growth-restricted fetus, there is likely an increased
efflux of the gluconeogenic amino acids, glutamine and
alanine, and the Branched Chain Amino Acids (BCAA) from
the fetal hind limb, possibly from increased rates of protein
breakdown, as has been demonstrated to occur in fetal
hypoglycemic studies [65,66,81].
Placenta (2005), Vol. 26, Supplement A, Trophoblast Research, Vol. 19
CHANGES IN INSULIN, IGF-I AND AMINO
ACID REGULATION OF THE INSULIN/IGF-I
SIGNALING PATHWAY IN IUGR
In the hyperthermia-induced ovine model of placental insufficiency, the fetuses are hypoxic, hypoglycemic [41,42], and
hypoinsulinemic and have reduced circulating IGF-I concentrations (Regnault et al., unpublished data). Importantly, with
respect to the growth-restricted fetus, insulin and, to a lesser
extent, the IGFs activate protein synthesis via the PI 3-kinase/
Akt/mTOR/p70S6k/4E-BP-1/eIF4E signaling pathway, as
illustrated schematically in Figure 3. Recent, studies have
established the initiation phase of mRNA translation as
a pivotal site of regulation for global rates of protein synthesis,
as well as a site through which the synthesis of specific proteins
is controlled [82]. Activation of the insulin and IGF-1 receptor
triggers docking of the insulin receptor substrate-1 (muscle),
INSULIN
IGF-I
α
S S
β
α
β
p
p
IRS-1,2
p
p85
p
p110
PI 3
Kinase
p
Akt-1,2
Hypoxia
p
mTOR
Amino Acids
GSK-3b
p
Glycogen
p
p
P70 S6K
4E-BP1
p
Ribosomal
protein
GS
S6
eIF4E
Complex
Translation
initiation
GDP
eIF2B
GTP
Protein Synthesis
Figure 3. A schematic representation of control of mRNA translation
initiation by growth factors insulin and IGF-1. The signaling pathway from
tyrosine kinase receptors activates insulin receptor substrates (IRS)-1,2, p85
phosphatidylinositol-3-kinase, Akt, and mTOR. Glycogen Synthase Kinase-3b
(GSK-3b) is a major negative regulator of Glycogen Synthase (GS) enzyme.
Insulin-mediated phosphorylation of GSK-3 via Akt relieves this inhibition.
Insulin and amino acids stimulate, while hypoxia inhibits the phosphorylation
of mTOR to control downstream signaling of protein synthesis. Activation of
mTOR increases phosphorylation of the suppressor 4E-BP-1, thereby
reducing its affinity for eIF4E. Release of eIF4E enables formation of eIF4E
complex, allowing eIF2B to bind GTP and interact with Met-tRNA and the
40S ribosomal subunit of mRNA, thereby promoting protein synthesis. The
p70S6 kinase is directly phosphorylated by mTOR and triggers phosphorylation of ribosomal protein S6 at multiple sites. S6 kinases are required for
promoting the translation of mRNAs.
Regnault et al.: Fetoplacental Amino Acid Metabolism
-2 (liver), and the catalytic activation of phosphatidylinositol3-kinase (PI 3-kinase) mediated by binding and phosphorylation of the 85 kDa PI 3-kinase regulatory subunit. PI 3-kinase
activation triggers phosphorylation of the serine/threonine
kinases Akt-1, -2, -3 [83]. Akt phosphorylates and thereby
activates the protein kinase mammalian target of rapamycin
(mTOR) [84,85].
Mammalian target of rapamycin appears to be a key
regulator of cell growth by sensing upstream nutrient and
hormonal signals to coordinate gene activation, protein
synthesis, and cell growth [85,86]. Mammalian target of
rapamycin is a serine/threonine kinase that phosphorylates
and thereby coordinates the function of the translational
suppressor-eukaryotic translation initiation factor 4E binding
protein 1 (4E-BP1) and the 70 kDa small ribosomal subunit
kinase (p70S6k) [87]. Under conditions of substrate sufficiency
(amino acids and glucose) and hormonal stimulation, mTOR
phosphorylates the 4E-BP1 repressor on several sites,
triggering its dissociation from the inactive 4E-BP-1eIF4E
complex [88,89], thus making eIF4E available for binding to
eIF4G. The eIF4E binds to the mRNA cap structure present
at the 5#-end of all nuclear transcribed mRNAs to form an
eIF4Emethionyl-tRNAi complex. The eIF4EmRNA complex binds to eIF4G and eIF4A, forming the active eIF4F
complex, thereby allowing translation to proceed.
Similar to human IUGR, the ovine model of IUGR also
displays decreased fetal insulin and IGF-I circulating concentrations (Regnault et al., unpublished data). In association with
this, in the ovine IUGR, there appears to be up regulation of
the proximal signals for insulin action involved in control of
glucose metabolism (Regnault et al., unpublished data). This
up regulation includes an increase of the insulin receptor and
a suppression of glycogen synthase kinase-3-b, which normally
suppresses glycogen synthesis. Insulin normally inactivates
GSK-3b, which thereby dephosphorylates glycogen synthase
(GS), leading to the activation of GS [90,91]. Low levels of
GSK-3b would therefore tend to favor GS activity and
consequently an elevation in glycogen formation. In preliminary studies in our laboratory, fetal hepatic glycogen
concentrations in IUGR are indeed elevated, which is similar
to what has been previously observed in hyperthermic exposed
fetuses [92]. This elevation could be due to a higher GS
activity, and/or possibly less glycogen phosphorylase. Interestingly, while there is an up regulation at the proximal end
of the insulin signaling pathway, we have observed a concomitant decrease in protein expression more distally in the insulin
signaling cascade leading to protein translation. Initiation
factor eIF4E is significantly reduced by 63% in IUGR livers,
while the translational repressor 4E-BP1 is significantly
increased, 2.5-fold (Regnault et al., unpublished data). The
significant increase in the downstream translational repressor
4E-BP1, combined with a decrease in the limited amount of
eIF4E may be a key mechanism limiting protein translation in
IUGR livers.
There also appears to be an increase in proximal
insulin signaling in muscle despite reduced insulin/IGF-I
S57
concentrations in growth-restricted ovine fetuses. Insulin
receptor protein in skeletal muscle is increased 77%, while
there is a 36% decrease in the amount of the p85a subunit of
PI 3-kinase in skeletal muscle from the IUGR fetuses
(Regnault et al., unpublished data). It has recently been
demonstrated that reduced expression of the murine p85a
subunit of PI 3-kinase in heterozygous knockout mice actually
improves insulin signaling and ameliorates diabetes, indicating
that p85a expression is negatively correlated with insulin
sensitivity [93,94]. The mechanism for this effect involves
enhanced coupling between the p85a and p110 subunits and
increased PI 3-kinase production in mice in vivo. These
changes in insulin receptor and p85a may therefore be
compensatory responses to low insulin concentrations and/or
simultaneous hypoglycemia, and could favor increased responsiveness to insulin to facilitate increased glucose uptake.
In contrast to the liver, the levels of 4E-BP1 and eIF4E
examined in fetal skeletal muscle from IUGR animals are
unchanged, indicating that fetal growth restriction may have
tissue-specific effects on mechanisms for reduced organ
growth. However, mTOR was reduced significantly by 38%
in hind limb skeletal muscle from IUGR fetuses (Regnault
et al., unpublished data). A decrease in mTOR expression
potentially has multiple downstream consequences that could
lead to a decrease in fetal growth. The mTOR-dependent
p70S6K and 4E-BP1/eIF4E pathways are activated independently during muscle growth in vivo [84,95] and these
pathways cooperate with each other to promote cell growth
and increased cell size. mTOR also coordinates the cellular
responses to extracellular nutrient concentrations by modulating nutrient transporter gene expression [85,89,96,97]. The
reasons for the reduced mTOR in muscle cells from growthrestricted fetuses are not known. Amino acids, particularly the
BCAA, leucine, stimulate mTOR in a number of tissues in the
adult [88,98e100], and reduced levels of mTOR may be
associated with reduced intracellular amino acid concentrations. As previously mentioned, under hypoxic conditions
amino acids leave the cell, including the BCAAs from the fetal
muscle [70], which is the only site of BCAA utilization besides
the placenta [101,102], and this associated change in mTOR
may potentially represent a direct site of protein synthesis
control in the growth-restricted fetus. Other factors that
impact mTOR signaling include reduced cellular energy
(ATP/AMPK levels) [103e105], and DNA damage through
changes in the transcription factor p53 [106,107]. The
transcription factor p53 is an important regulator of cell cycle
progression and apoptosis and has been associated with
apoptosis in IUGR fetuses [108e111]. It negatively regulates
the expression of Bcl-2 and up regulates Bax expression
[112,113], and these two cell transcription factors play major
roles in activating the caspase-3 pathway which is necessary for
the chromatin condensation and DNA fragmentation that
characterize apoptosis [114].
mTOR is also directly regulated by decreased oxygen partial
pressure [115]. The reversible inhibition of protein synthesis
by hypoxia is thought to be an important aspect of energy
S58
conservation in an oxygen deficient environment. Arterial PO2
in growth-restricted fetuses may be as low as 12 mmHg (w2%
oxygen), which is similar to the oxygen partial pressures in cell
culture conditions in which reduced oxygen levels have been
suggested to initiate cellular energy conservation strategies,
including the hypophosphorylation of mTOR, independent of
other commonly associated hypoxia changes, such as ATP
levels, HIF-1 and Akt/protein kinase B and AMP-activated
protein kinase phosphorylation [115].
CONCLUSIONS
In severe cases of placental and fetal growth restriction,
particularly as shown in a well established ovine model of
Placenta (2005), Vol. 26, Supplement A, Trophoblast Research, Vol. 19
IUGR with placental insufficiency, the increased transplacental gradients for oxygen is not large enough to overcome the
reduced diffusion capacities of the growth-restricted placenta.
Both limited oxygen and nutrient substrate supplies, therefore,
limit the capacity for fetal protein accretion and may augment
mechanisms leading to protein breakdown. Furthermore,
hypoxia, reduced nutrient supplies and intracellular concentrations of nutrient substrates may initiate differential cellular
energy conservation strategies, some of which may not be fully
reversible. The complete mechanisms by which oxygen,
nutrient substrates, and related anabolic hormones and growth
factors regulate placental amino acid uptake, fetal utilization
and metabolism, and cellular growth remain to be a field of
active investigation.
ACKNOWLEDGMENTS
The authors wish to sincerely thank our colleagues, past and present, and all the staff of the Perinatal Research Center who over the many years, have helped
contribute to improving our understanding of the placental transport and the fetoplacental metabolism of amino acids. We are grateful to the National Institute of
Child Health and Human Development for the funding that has supported this work.
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