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Transcription
and
PEPCK
translation
promoter
PEPCK
Glucose




Hyperglycaemia

Figure 5.4 Liver programming by glucocorticoids. Prenatal dexamethasone permanently increases
PEPCK gene expression. This appears to be driven by increased GR and thus
increased sensitivity to glucocorticoid-mediated hyperglycaemia


to excess glucocorticoid in utero leads to offspring with permanent elevations in
PEPCK mRNA and enzyme activity from a few days postnatally to midlife. This
occurs selectively in the gluconeogenic periportal region of the hepatic acinus
(Nyirenda et al., 1998). This ¬nding, which appears speci¬c to PEPCK since other
hepatic enzymes examined were unaltered, may be of pathogenic importance (Figure
5.4). Thus, overexpression of PEPCK in a rat hepatoma cell line impairs suppression
of gluconeogenesis by insulin (Rosella et al., 1993) and transgenic mice with overex-
pression of hepatic PEPCK have impaired glucose tolerance (Valera et al., 1994). In
terms of molecular mechanisms, PEPCK is regulated by a host of transcription fac-
tors (Duong et al., 2002), but intriguingly, it is increased expression of GR itself that
occurs in the livers of prenatal dexamethasone-programmed rats (Nyirenda et al.,
1998; Cleasby et al., 2003b) in a pattern congruent with the periportal rise in PEPCK
gene expression (Nyirenda et al., 1998). This increase in GR may be crucial, since ani-
mals exposed to dexamethasone in utero have greater plasma glucose responses to
exogenous corticosterone suggesting a speci¬c increase in tissue sensitivity to the
glycemic effects of the steroid (Nyirenda et al., 1998). Thus, the observed glucose
intolerance in rats exposed to excessive glucocorticoids in utero may be explained in
part by programmed hepatic PEPCK overexpression leading to increased gluconeo-
genesis. Similar increases in hepatic GR are seen in the offspring of undernourished
ewes (Whorwood et al., 2001) suggesting the process is conserved.

Programming of hepatic 11 -HSD-1?
The 11 -HSD-1 is an NADPH-associated 11-keto reductase in intact cells and organs
such as liver (Jamieson et al., 1995; 2000). This functions to catalyse the reactivation
154 J. R. Seckl et al.


of cortisol (corticosterone in rodents) from inert circulating cortisone (11-dehydro-
corticosterone) (Seckl and Walker, 2001). The enzyme is highly expressed in liver and
adipose tissue (Seckl and Walker, 2001). 11 -HSD-1 often co-localizes with GR
(Whorwood et al., 1991) suggesting it may amplify intracellular ligands and their
access to the receptor (Seckl and Walker, 2001). Glucocorticoids in turn regulate 11 -
HSD-1 activity at least in liver cells in vitro (Voice et al., 1996), although this is less
certain in vivo (Jamieson et al., 1999). The activity of hepatic PEPCK is decreased in
the 11 -HSD-1 knockout mouse (Kotelevtsev et al., 1997). Intriguingly, both mater-
nal and fetal exposure to excess glucocorticoid increases hepatic 11 -HSD-1 mRNA
and protein in fetal sheep (Yang et al., 1995; Sloboda et al., 2002b). Such changes in
11 -HSD-1 increase the potential to regenerate active intrahepatic glucocorticoids,
and may further amplify the expression of glucocorticoid-dependent hepatic
enzymes of gluconeogenesis. In contrast to the ovine data, in the rat antenatal dexa-
methasone has no effect on adult liver 11 -HSD-1 (Nyirenda et al., 1998) and ante-
natal carbenoxolone actually reduces adult liver 11 -HSD-1 levels (Saegusa et al.,
1999). So the jury remains out on this issue.

Programming the pancreas
In utero undernutrition impairs rat -cell development (Garofano et al., 1997;
1998), resulting in reduced -cell mass and subsequent glucose intolerance. Recent
evidence suggests that glucocorticoids may play an important part in this (Blondeau
et al., 2001). In rats with normal nutrition, fetal pancreatic-insulin content is nega-
tively correlated with fetal corticosterone levels, and -cell mass increases when fetal
steroid production is impaired (Blondeau et al., 2001). Maternal malnutrition in the
rat is associated with elevated maternal and fetal corticosterone levels in addition to
decreased fetal pancreatic-insulin content and -cell mass. Preventing the cortico-
sterone increase in food-restricted dams by adrenalectomy with corticosterone
replacement restores -cell mass (Blondeau et al., 2001). The mechanisms by which
glucocorticoids modulate pancreatic development are not fully discerned, but dexa-
methasone downregulates -cell Pdx-1 and induces C/EBP , key factors in the
induction and repression (respectively) of insulin gene expression (Shen et al., 2003).
Further, glucocorticoids in¬‚uence the expression of IGF-2, a key peptide growth
factor in pancreatic development, in addition to the IGF receptor and several IGF-
binding proteins (Hill and Duvillie, 2000).

Adipose tissue and programming: an emerging area?
Muscle and fat
Exposure to antenatal dexamethasone in rats is also associated with programming
of fat and muscle metabolism (Cleasby et al., 2003a). In skeletal muscle the pheno-
type is subtle; prenatal glucocorticoid exposure decreases GR selectively in the type
155 Prenatal glucocorticoids and the programming of adult disease


2 ¬bre-enriched soleus muscle, but not in muscles rich in type 1 or glycolytic ¬bres.
In contrast, prenatal glucocorticoid exposure causes a striking increase of GR expres-
sion in visceral but not peripheral adipose tissue in adult rats (Cleasby et al., 2003a)
and sheep (Whorwood et al., 2001). Elevated GR expression in visceral adipose tissue
in the presence of circulating hypercorticosteronaemia suggests increased glucocorti-
coid action in visceral fat. This may contribute to both adipose and hepatic-insulin
resistance. These changes in GR expression do not appear to be the result of meta-
bolic derangement in the adult animal, correction of the hypercorticosteronaemia
and insulin sensitization are not suf¬cient to normalize the programmed changes in
GR (Cleasby et al., 2003b). However, intriguingly, metformin selectively normalized
the elevated GR in dexamethasone-programmed liver, an effect apparently distinct
from insulin sensitization since a thiazolidinedione (PPAR agonist) did not exert
the selective effect.

Leptin
Leptin, an adipose gene product that signals both centrally and peripherally, where it
plays a role in insulin sensitivity, is present in the circulation of human and porcine
fetuses from midgestation (Jaquet et al., 1998; Chen et al., 2000), and in adipose tis-
sue of human fetuses by 20 weeks gestation (Lepercq et al., 2001). Additionally,
mRNA for leptin and leptin receptors, have been detected in the fetal tissues of many
other species (Yuen et al., 1999; Hoggard et al., 2000; Lepercq et al., 2001; Mostyn
et al., 2001; Thomas et al., 2001). In the human fetus, circulating leptin levels increase
towards term, associated with a signi¬cant increase in body fat after 34 weeks of ges-
tation (Jaquet et al., 1998; Geary et al., 1999; Cetin et al., 2000) and in fetal sheep, lep-
tin mRNA increases in adipose tissue with increasing gestational age (Yuen et al.,
1999). Intriguingly, leptin concentrations in human fetal cord blood correlate directly
with body weight and adiposity at birth (Koistinen et al., 1997; Schubring et al., 1997;
Jaquet et al., 1998; Ong et al., 1999; Lepercq et al., 2001) indicating a potential role for
leptin in linking fetal growth and metabolic programming.
In rats, antenatal treatment of the pregnant mother with dexamethasone
reduces fetal plasma and placental levels of leptin, while maternal plasma leptin
levels remain unchanged or increase (Sugden et al., 2001; Smith and Waddell,
2002). Dexamethasone also reduces placental expression of the leptin receptor iso-
form Ob-Rb, which mediates leptin action (Smith and Waddell, 2002), while levels
of the isoform ObR-S (the proposed transport form of the receptor) are modestly
increased (Sugden et al., 2001). In the adult offspring, antenatal glucocorticoids lead to
increased leptin levels (Sugden et al., 2001), which may contribute to the cardio-
metabolic phenotype produced. Of course, the glucocorticoid-sensitive leptin tran-
script (Slieker et al., 1996) may in part be driven by the higher expression of adipose
tissue GR in adult prenatal glucocorticoid-programmed rats, though leptin is mainly
156 J. R. Seckl et al.


produced in peripheral depots whereas GR expression was mainly elevated in vis-
ceral fat (Cleasby et al., 2003a). Most intriguingly, concomitant treatment of mal-
nourished pregnant and lactating rats with leptin appears to reverse, in part, the
adult metabolic effects of antenatal challenge, at least for maternal malnutrition
(Stocker et al., 2003).
However, the same may not pertain in the sheep, in which exogenous cortisol or
dexamethasone administration directly to the fetus increases rather than reduces
plasma leptin concentrations, albeit transiently (Forhead et al., 2002; Mostyn et al.,
2003). The differences between these studies may re¬‚ect the route of glucocorti-
coid administration, species, fetal body fat levels and/or maternal nutrient intake.

Adiponectin
Adiponectin (acrp30, adipoQ) is an abundant, adipose-speci¬c protein which is
secreted into the blood. Adiponectin is negatively associated with fat mass (Hu et al.,
1996) and positively associated with insulin sensitivity (Weyer et al., 2001) and
may mediate obesity-related resistance to insulin. Lower plasma adiponectin levels
appear to predict the later occurrence of type 2 diabetes (Lindsay et al., 2002).
Adiponectin is strikingly regulated by hormones and other factors during postnatal
development (Combs et al., 2003). Given the emerging biology of the adipocyte and
its important role in some programming phenomena (Cleasby et al., 2003a), it is
likely to be of interest. However, a recent study in humans found no association
between birth weight and adiponectin levels (Lindsay et al., 2003).


Glucocorticoid programming of the brain

˜We all are born mad. Some remain so™, Samuel Beckett, Waiting for Godot (1955). The
CNS has long been subject to scrutiny for organizational in¬‚uences in early life upon
adult function and the pathogenesis of neuropsychiatric disorders. Many studies have
exploited maternal and/or fetal stressors to alter developmental trajectories of spe-
ci¬c CNS structures or gene products and reported persistent effects (Weinstock,
2001). While the effects of stress are in part mediated by glucocorticoid secretion,
steroids are by no means the only efferent effector pathway of the stress response and
other hormones (catecholamines are obvious candidates), neurotransmitters, vascu-
lar and metabolic systems are altered as well in a stressor and strain-speci¬c manner.
Nonetheless, studies of antenatal stress in animals have clearly documented long-
term effects upon a host of CNS functions (reviewed in Weinstock, 2001; Welberg
and Seckl, 2001) and have laid the foundations for studies of more speci¬c program-
ming agents including glucocorticoids.
Glucocorticoids are important for normal brain maturation, exerting a range of
effects in most regions of the developing CNS (Meaney et al., 1996; Korte, 2001;
157 Prenatal glucocorticoids and the programming of adult disease


Weinstock, 2001; Welberg and Seckl, 2001) including the initiation of post-mitotic ter-
minal maturation, axo-dendritic remodelling and the modulation of neonatal brain
cell death (Meyer, 1983). Prenatal glucocorticoid administration retards brain weight
at birth in sheep, with a suggestion of dose dependency (Huang et al., 1999). This is
associated with delays in the cellular maturation of neurones, glia and cerebral
vasculature (Huang et al., 2001a) and retarded CNS myelination (Huang et al.,
2001b). Given such widespread effects of glucocorticoids it is unsurprising that GR
and MR are highly expressed in the developing brain with complex locus-speci¬c
ontogenies to allow selectivity of effects (Fuxe et al., 1985; Diaz et al., 1996; Kitraki
et al., 1997).
However, whether these receptors are occupied by endogenous glucocorticoids
until late gestation is not clear, because there is also plentiful 11 -HSD-2 in the CNS
at midgestation (Brown et al., 1996a; Diaz et al., 1996; Robson et al., 1998). This pre-
sumably functions to ˜protect™ vulnerable developing cells from premature glucocor-
ticoid actions. Strikingly, 11 -HSD-2 expression is dramatically switched-off in a CNS
locus-speci¬c manner, mainly at the end of midgestation in the rat and mouse brain.
Possibly this widespread gene silencing in the CNS coincides with the terminal stage
of brain nucleus development (Brown et al., 1996a; Diaz et al., 1998). At birth in the
rat the main areas of residual 11 -HSD-2 expression are in the thalamus and
cerebellum, areas exhibiting substantial postnatal development. At least in the
cerebellum this is highly sensitive to glucocorticoids (Bohn and Lauder, 1978; 1980).
By weaning at postnatal day 21, CNS 11 -HSD-2 expression is con¬ned to those
few areas seen in the adult (Robson et al., 1998). Similarly, in human fetal brain
11 -HSD-2 appears to be silenced between gestational weeks 19 and 26 (Stewart et al.,
1994; Brown et al., 1996b). So, there appears to be an exquisitely timed system of pro-
tection and then exposure of developing brain regions to circulating glucocorticoids.
The HPA axis and its limbic system connections (hippocampus, amygdala) are
also particularly sensitive to endogenous and exogenous glucocorticoids during
perinatal development (Bohn, 1980; Gould et al., 1991a, b) and indeed to perinatal
glucocorticoids or stress programme-speci¬c effects in these regions of the brain
(Welberg and Seckl, 2001). Programming of neuroendocrine and limbic systems
appears conserved and is observed across a range of experimental species and, less
certainly, in humans.

Programming the HPA axis
Studies in animal models indicate that the HPA axis is an important target for gluco-
corticoid programming. The HPA axis is controlled by a negative feedback system.
Glucocorticoids from the adrenal cortex activate GR in the pituitary and paraven-
tricular nucleus (PVN) of the hypothalamus as well as to extrahypothalamic CNS
feedback sites. The latter include the hippocampus (Jacobson and Sapolsky, 1991)
158 J. R. Seckl et al.


which highly expresses both GR and the higher-af¬nity MR in rodents (Reul and
de Kloet, 1985) and probably humans (Seckl et al., 1991). Overactivity at any
point along the pathway results in negative feedback to decrease the amount of
corticotrophin-releasing hormone (CRH) released from the PVN, and thus decrease
adrenocorticotrophic hormone (ACTH) release from the pituitary and hence the
synthesis and secretion of glucocorticoids.
Prenatal dexamethasone exposure or 11 -HSD-2 inhibition permanently increases
basal plasma corticosterone levels in adult rats (Levitt et al., 1996; Welberg et al.,
2001). Whilst the mechanism of hypercorticosteronaemia is not fully understood,
the density of GR and MR in the hippocampus are reduced in this model. This
would be anticipated to attenuate feedback sensitivity which may well explain
basal hypercorticosteronaemia. Moreover, the glucocorticoid excess may drive, at
least in part, the hypertension and hyperglycaemia observed in this and other pre-
natal environmental programming models (Langley-Evans, 1997). Of course, this
will be ampli¬ed by the documented increase in hepatic (and presumably visceral
adipose tissue) glucocorticoid sensitivity (Nyirenda et al., 1998; Cleasby et al., 2003a).
Similarly, in sheep, exposure to betamethasone in utero alters HPA responsiveness in
the offspring at up to 1 year of age, though earlier exposure to dexamethasone has no
persisting HPA effects in this species (Dodic et al., 2002c). Intriguingly, the outcomes
vary according to the time of gestational exposure to steroid (Figure 5.5), and whether
it was administered to the mother or directly to the fetus (Sloboda et al., 2002a). Thus,
maternal administration of betamethasone elevates basal and stimulated cortisol
levels in the offspring, whereas betamethasone directly to the fetus attenuates offspring


Dex (early) Dex (late) CBX (mid)

HC MR, GR


Amyg Amyg
CRF CRF
GR GR




Plasma corticosterone

Figure 5.5 Timing of glucocorticoid exposure upon programming of the HPA axis. The same adult
phenotype can arise by distinct central mechanisms. HC: hippocampus; Amyg: amygdala;
CRF: corticotrophin-releasing factor; Dex: dexamethasone; CBX: carbenoxolone
159 Prenatal glucocorticoids and the programming of adult disease


ACTH responses to CRH with arginine vasopressin (AVP) (Sloboda et al., 2002a);
whether this discrepancy re¬‚ects relative dose, duration or perhaps indirect effects
of maternally administered steroids remains to be determined. Maternal undernutri-
tion in rats (Langley-Evans et al., 1996a) and sheep (Hawkins et al., 2000) also affects
adult HPA axis function, suggesting that HPA programming may be a common
outcome of prenatal environmental challenge, perhaps acting in part via alterations
in placental 11 -HSD-2 activity which is selectively downregulated by maternal

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