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ing these associations in human populations are poorly understood, it is apparent
that elevated HPA activity later in life and a predisposition to later disease are
linked to alterations in fetal intrauterine growth and development.
Studies from our laboratory have shown that in the ovine fetus maternal
betamethasone administration results in HPA hyperactivity before birth (Sloboda
et al., 2000; Table 4.1) and early adulthood (Sloboda et al., 2002a; Figure 4.2), but
later in life the adrenal is incapable of sustaining cortisol output and relative adre-
nal insuf¬ciency develops (Sloboda et al., 2003). In these animals we observed a
dose dependant increase in basal ACTH levels in adulthood, associated with sig-
ni¬cant reductions in basal cortisol levels. Although alterations in HPA function
exist in offspring exposed in utero to either single or multiple doses of maternal
betamethasone, the changes were most pronounced in offspring that were exposed
to multiple doses. Our observations suggest that prenatal betamethasone exposure
125 Fetal HPA activation, preterm birth and postnatal programming

Table 4.1 Betamethasone administration in ewes at 3 weekly doses beginning at
104 days of gestation signi¬cantly alters cortisol-binding capacity (CBC) at 125 days of
gestation and increases fetal HPA activity at 146 days of gestation

Variables 125 days of gestation 146 days of gestation

Control Betamethasone Control Betamethasone
(n 5) (n 6) (n 7) (n 8)

ACTH (pg/ml) 29.5 3.0 36.0 10.1 54.6 5.2 82.1
Cortisol (ng/ml) 3.2 0.6 1.9 0.3 12.5 2.2 35.7 17.8
CBC (ng/ml) 17.3 3.2 47.9 57.3 17.7 54.3 14.4

All values are mean SEM, *P 0.05.
Source: Adapted from Sloboda et al. (2000).

in the sheep results in a dynamic sequence of altered adrenal function after birth
beginning with hyper- and ending with hypo-responsiveness by adulthood. A sin-
gle cross-sectional study after birth therefore, may provide a misleading impres-
sion of life-long consequences. The exact mechanisms regulating this evolution in
HPA responsiveness are unknown; however it seems likely that adrenal receptor
and/or steroidogenic enzyme expression or activity are likely to be altered in these
The potential impact of fetal glucocorticoid exposure on the developing HPA
axis may occur via the GR, which is expressed at every level of the axis. Synthetic
glucocorticoids can potentially impact at the level of the brain, hypothalamus,
pituitary and/or the adrenal. Remarkably little is known regarding the mechanisms
that regulate alterations in HPA function following maternal glucocorticoid admin-
istration and their relationship to postnatal disease. In most models, programming
of the HPA axis has been associated with alterations in hippocampal corticosteroid
receptor populations (Uno et al., 1994; Levitt et al., 1996; Dean and Matthews,
1999). Negative feedback at the level of the hippocampus results in an inhibition of
HPA activity, therefore reduced glucocorticoid feedback through alterations in
receptor number would elevate HPA activity (Jacobson and Sapolsky, 1991). HPA
hyperactivity has been demonstrated following prenatal undernutrition in the
guinea pig (Lingas et al., 1999), prenatal stress in the rat (Takahashi and Kalin,
1991; Weinstock et al., 1992) as well as maternal glucocorticoid administration in
the rat (Levitt et al., 1996), rhesus monkey (Uno et al., 1994) and sheep (Sloboda
et al., 2000).
Maternal administration of dexamethasone in the rhesus monkey at 132 and
133 days of gestation (term 165 days) results in signi¬cant alterations in the
cytoarchitechtural development of hippocampal neurons at 135 days of gestation
126 D. M. Sloboda et al.

(Uno et al., 1990). Degeneration of neurons and a signi¬cant reduction in the size
of the whole hippocampal formation were observed in dexamethasone-treated
fetuses at 135 and 162 days of gestation. Those fetuses that received multiple injec-
tions showed more severe damage, suggesting that these effects were dose dependant
(Uno et al., 1990). Furthermore, at 10 months of postnatal age, dexamethasone-
treated offspring demonstrated higher basal cortisol levels and higher plasma cor-
tisol levels following stress (Uno et al., 1994). In other experiments maternal dexa-
methasone treatment in the guinea pig on 50“51 days of gestation (term 70 days)
resulted in signi¬cant increases in basal cortisol levels in female fetuses but not
male fetuses. Furthermore, dexamethasone exposure resulted in signi¬cant increases
in MR and GR messenger ribonucleic acid (mRNA) in the hippocampus of female
fetuses but not in males (Dean and Matthews, 1999).

Prenatal glucocorticoid exposure and postnatal metabolic function:
type 2 diabetes

Many studies have proposed that the increased incidence of glucose intolerance
and insulin resistance associated with type 2 diabetes later in life may be pro-
grammed in utero (Ravelli et al., 1998). In human beings, low birth weight has been
associated with a higher incidence of syndrome X; a cluster of risk factors including
insulin resistance, glucose intolerance, hyper-insulinemia, hyper-triglyceridemia,
decreased high-density lipoprotein cholesterol, and hypertension (syndrome X;
Reaven, 1988). This syndrome is accompanied by alterations in the HPA axis and
cortisol metabolism. Early studies found the risk of developing glucose intolerance
and diabetes later in life was double in men who had low birth weights (Ravelli
et al., 1998). Low birth weight in the rat has also been correlated with a reduction in
pancreatic function and impaired -cell growth and function (Berney et al., 1997).
Recent studies suggest that the effects of prenatal growth restriction in combi-
nation with accelerated postnatal growth may have important metabolic implica-
tions. Girls born within the lowest birth weight tertile that end up with a BMI in
the highest tertile have a 30% increased risk of developing syndrome X (Yarbrough
et al., 1998). The presence of prenatal growth restriction in combination with
increased postnatal weight gain or velocity has serious effects on carbohydrate
metabolism even in young children. Bavdekar et al. (1999) demonstrated that the
highest incidence of insulin resistance, high plasma total and low-density lipid
(LDL) cholesterol and fasting insulin levels, were observed in children who had
been of low birth weight and at 8 years of age were of high fat mass and height.
Others suggest that insulin resistance and pancreatic -cell activity/dysfunction
may be regulated by growth velocity between birth and 7 years of age (Crowther
et al., 2000). Small for gestational age neonates also exhibit insulin resistance,
127 Fetal HPA activation, preterm birth and postnatal programming

especially those children with catch-up growth and increases in BMI (Veening
et al., 2002).
The relationship between low birth weight and type 2 diabetes in adult life has
been described by the thrifty phenotype hypothesis (Hales et al., 1992). This hypoth-
esis proposes that the metabolic development of the fetus is programmed in utero
in a way that in¬‚uences postnatal metabolic responses. The developmental mech-
anisms that underpin the thrifty phenotype hypothesis are multifactorial. Although
much of the hypothesis was formulated around prenatal undernutrition, current
evidence points to prenatal exposure to elevated levels of glucocorticoids as a major
contributor in fetal programming. In the rat, maternal treatment with carben-
oxolone, a placental 11 -HSD2 inhibitor, allows increased passage of maternal
glucocorticoids to the fetus and resulted in reduced birth weight and glucose intol-
erance in offspring (Lindsay et al., 1996). We have shown previously that as little as
one dose of maternally administered betamethasone in the sheep results in insulin
responses to a glucose challenge that are similar to those seen in type 2 diabetes
(Moss et al., 2001; Figure 4.4).
Although the mechanisms are unclear, prenatal glucocorticoid overexposure
can potentially program a number of organ systems regulating glucose homeosta-
sis. Glucocorticoids regulate skeletal muscle glucose transporter expression (Coderre
et al., 1996), reduce basal and insulin stimulated glucose uptake and impair glucose
transporter recruitment (Weinstien et al., 1995; 1998). Glucocorticoids have been
shown to regulate insulin secretion (Lambillote et al., 1997) and the expression
of factors regulating pancreatic growth and remodelling, such as pancreatic duo-
denal homeobox-1 (Pdx-1) and insulin-like growth factor 2 (IGF2) (Sander
et al., 1997; Hill, 1999). Fetal rat corticosteroid concentrations are negatively corre-
lated with pancreatic insulin content, and -cell mass increased when fetal steroid
production was impaired (Blondeau et al., 2001). It is unknown if fetal
glucocorticoid exposure permanently alters fetal -cell development in a way that
alters life-long adult pancreatic morphology and function. It seems likely however,
that altered pancreatic function would impair postnatal metabolic function.
Expression of hepatic gluconeogenic enzymes (phosphophenolpyruvate carboxy-
kinase, PEPCK) in offspring of dexamethasone-treated pregnant rats is increased,
an effect that persists up to 8 months of postnatal age. These rats demonstrated a
signi¬cant reduction in birth weight as well as fasting hyperglycemia and elevated
glucose and insulin responses to glucose loading (Nyirenda et al., 1998). Such
observations may have important relevance in terms of hepatic insulin resistance,
since transgenic mice over-expressing PEPCK exhibit increases in hepatic glucose
output, increases in glucose-6 phosphatase levels, decreased insulin receptor
substrate 2 (IRS-2) levels and decreased phosphatidyl inositol 3 (PI3) kinase activ-
ity. Recent data suggest that programming of GR expression in rats prenatally
128 D. M. Sloboda et al.


Area (ng/min/ml)



Insulin (ng/ml)

Saline M1 M4




0 20 40 60 80 100 120 140 160 180
Time (minutes)
Figure 4.4 Maternal betamethasone administered at 104 days of gestation (M1) or on 4 occasions
at weekly intervals (M4) resulted in signi¬cant increases in insulin responsiveness to a
glucose challenge at 6 months of postnatal age, compared with saline administration
(MS). M1 (shaded squares) represents animals that received one single dose of
betamethasone at 104 days of gestation followed by 3 weekly injections of saline. M4
(black diamonds) represents animals that received 4 weekly doses of maternal
betamethasone beginning at 104 days of gestation. MS (open triangles) represents
animals that received 4 doses of saline at weekly intervals starting at 104 days of
gestation. Histograms represent the area under the insulin response curves. Values are
expressed as mean 0.05. Adapted from Moss et al. (2001)

overexposed to glucocorticoids may be a primary mechanism of insulin resistance
(Cleasby et al., 2003). We have previously shown that repeated exposure of the fetal
sheep to glucocorticoids results in signi¬cant increases in hepatic 11 -HSD1 and
CBG levels (Sloboda et al., 2002a) (Figures 4.5 and 4.6). These data suggest that
glucocorticoids may not only directly program metabolic enzymes, but also pro-
gram intra-hepatic levels of glucocorticoids, thus providing a feed-forward loop of
glucocorticoid effects. The gluconeogenic responses of 11 -HSD1 knockout mice
were attenuated after stress and these animals resist hyperglycemia induced by
chronic high-fat feeding. These observations support 11 -HSD1 as an important
ampli¬er of intra-hepatic glucocorticoid action in vivo (Kotelevtsev et al., 1997).
129 Fetal HPA activation, preterm birth and postnatal programming

Control Betamethasone

1.8 kb
Control Betamethasone

34 kDa

* 3.0 *

11 -HSD1

11 -HSD1

(a) (b) 0.0

Figure 4.5 Repeated doses of maternally administered betamethasone signi¬cantly increased fetal
sheep hepatic 11 -HSD1 mRNA and protein levels at 125 days of gestation. Fetal sheep
hepatic 11 -HSD1 mRNA (a) and protein (b) levels following either saline (open bar) or
maternal betamethasone (shaded bars) administration are represented in histograms.
The relative optical density (ROD) of 11 -HSD1 mRNA was expressed as a ratio 11 -HSD1
ROD:18S ROD. 11 -HSD1 protein levels are expressed as arbitrary optical density
(AOD) units. Values presented as mean 0.05. Adapted from Sloboda
et al. (2002a)

Glucocorticoids may program postnatal metabolism through an increase in
postnatal accumulation of visceral fat. The risk factors for diabetes rise as body fat
content increases and visceral fat depots are strongly linked to insulin resistance
and syndrome X (Kahn et al., 2000). In cases of glucocorticoid excess, such as
Cushing™s syndrome, visceral adiposity is increased (Wolf, 2002) and tissue speci¬c


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