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Departments of Heart and Lung Diseases (C.N., B.-M.L., P.B., A.H.), Histology (E.J.), and Pharmacology (E.E.) and the Wallenberg Laboratory (C.N., B.-M.L., P.B., A.H.), Goteborg University, S-413 45 Goteborg, Sweden; and Pennington Biomedical Research Center, Louisiana State University (D.A.Y.), Baton Rouge, Louisiana 70808
Address all correspondence and requests for reprints to: Dr. Cecilia Nilsson, Wallenberg Laboratory, Goteborg University, S-413 45 Goteborg, Sweden. E-mail: cecilia.nilsson{at}wlab.wall.gu.se
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Introduction |
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A systemic inflammatory response during pregnancy represents one form of stressful event for the fetus. Lipopolysaccharides (LPS) from Gram-negative bacteria act as an endotoxin and are a nonspecific immunostimulant. LPS has been shown to stimulate the hypothalamic-pituitary-adrenal (HPA) axis and inhibit the hypothalamic-pituitary-gonadal (HPG) axis via the release of cytokines (9, 10). Prenatal exposure to LPS results in increased basal plasma corticosterone levels and a reduction in the number of central glucocorticoid receptors (GR) regulating the HPA axis (11).
The present study was undertaken to explore to what extent maternal endotoxemia during the second trimester, a period of extensive fetal brain development, may influence body weight, the amount of fat tissue, the effect of insulin on glucose uptake, blood pressure, and lipid profile in adult offspring. These parameters are known to be aberrant in patients with the so-called metabolic syndrome, which includes insulin resistance, glucose intolerance, and/or manifest diabetes mellitus together with abdominal obesity, dyslipidemia, and/or elevated blood pressure (12). Prompted 1) by the fact that patients with metabolic syndrome often display increased serum levels of leptin and cortisol and changes in sex steroids (13, 14); 2) by the possible influence of leptin, glucocorticoids, and sex steroids on insulin resistance (13, 14); and 3) by the previously reported effects of maternal immune challenge or stress on the HPA and HPG axes in the offspring (8, 11), we also studied the effects of intrauterine endotoxin exposure on these endocrine parameters.
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Materials and Methods |
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Dams and litters
On gestational days 8, 10, and 12, dams (n = 8) received ip
injections of 0.79 mg/kg bacterial endotoxin (LPS). The LPS dose is
known to result in a low percentage of fetal anomalies, but not
abortion (15). Multiple injections were used to cover the
sensitive period during the second trimester when brain development is
most pronounced in the fetus (16). LPS (Escherichia
coli 026:B6, Sigma, St. Louis, MO) was dissolved in 1
ml sterile saline. Control dams (n = 17) received sterile saline
only. Dams weighed 191 ± 3 g on day 8. The rats were
lethargic and weak 2–3 h after each endotoxin injection, and the
effects lasted less than 24 h (15). Body temperature
was registered before and after injection. Gestation lasted for 20–22
days. Two treated dams and five control dams did not deliver any pups.
After birth, litters were counted and weighed. The pups were, after
recording of birth weight, randomly chosen to be included in this study
(males: controls, n = 11; LPS-treated, n = 8; females:
controls, n = 5; LPS-treated, n = 11) or in a study
addressing another question. They were left undisturbed until 4 weeks
of age, when they were weaned. From 4 weeks of age the offspring were
weighed regularly.
Food intake
When the rats were 10 weeks of age, food consumption for each
cage was recorded once a day (males: LPS-treated, n = 8, 2
rats/cage; controls, n = 11, 2–3 rats/cage; females: LPS-treated,
n = 11, 2–3 rats/cage; controls, n = 5, 2–3 rats/cage).
They were presented with the same amount of food, and their intake was
measured the following day by subtracting the uneaten food. This was
done during 1 week and was calculated as food intake in grams per rat
and per day.
Baseline hormone levels
At 7–10 weeks of age, blood samples were collected from a nick
in the tail after fasting overnight for determinations of glucose,
insulin (averaged from three sampling occasions), testosterone,
progesterone, 17ß-estradiol (10th week), FFA, glycerol, and leptin
(9th week). Samples were taken between 0700 and 0900 h. In female
rats, vaginal smears were obtained daily during 2 weeks to determine
stage of the estrous cycle (17). Blood samples for
progesterone, 17ß-estradiol, and testosterone were taken in the
diestrous phase of the cycle.
Stress test procedure
At 5 weeks of age, the acute corticosterone stress response of
the animals (males: LPS-treated, n = 8; controls, n = 11;
females: LPS-treated, n = 11; controls, n = 5) was tested by
novel environment stress according to a modified protocol described
previously (18). Before the test, the animals had a 4-week
rest period without injections, tests, or any other manipulations
except daily animal keeping. All tests started at 0700 h, taking
great care in keeping the rats undisturbed and fed the night before the
experiment. Tail blood was collected by a nick in the tail immediately
before the test for estimation of prestress levels of corticosterone
(30 µl). The rats were then transferred to a new cage in the
laboratory room. Blood for corticosterone determination (30 µl) was
taken from the tail 15, 30, 60, 90, and 120 min after transfer to the
new cage.
Euglycemic hyperinsulinemic clamp
At 12 weeks of age (males: LPS-treated, n = 7; controls,
n = 9) and 13–14 weeks of age (females: LPS-treated, n = 11;
controls, n = 5), the rats (fed) were subjected to a euglycemic
hyperinsulinemic clamp as described previously (19). The
animals were anesthetized with 125 mg/kg BW thiobutabarbital sodium
(Inactin, RBI, Natick, MA). Catheters were then inserted into the left
carotid artery for blood sampling and into the right jugular vein for
infusion of glucose and insulin. The body temperature was maintained at
37 C with a heating blanket. After a bolus injection, insulin (100
U/ml; human Actrapid, Novo Nordisk Pharma Ltd.,
Copenhagen, Denmark) was continuously infused at a rate of 5
mU/min·kg. A 10% glucose solution in physiological saline was
administered to maintain the plasma glucose concentration at 7
mmol/liter. Glucose was infused at a speed guided by glucose
concentration measurements in 30 µl blood at regular intervals (every
5 min during the first 40 min, then every 10 min). At 0, 40, 80, 120,
and 160 min of infusion, 250-µl blood samples were taken for
determination of insulin concentration. A total of less than 2 ml blood
were used for the determinations; this was compensated for by the
infusion volumes. Two male control rats and one LPS rat died during the
clamp.
Tissues
At the completion of the clamp (180 min), the rats were killed
with iv injection of KCl. The brain was quickly removed, and
hippocampus and hypothalamus were dissected, split into halves for RNA
and protein preparations, snap-frozen in liquid nitrogen, and stored at
-80 C. The adrenals, thymus, heart, spleen, and muscles of the hind
limb (extensor digitorum longus, plantaris, and tibialis anterior) were
rapidly excised and weighed. The epididymal, parametrial, mesenteric,
retroperitoneal, and inguinal adipose tissues were also dissected out
and weighed.
Isolation of RNA
RNA from hippocampus (males: LPS-treated, n = 8; controls,
n = 8) was isolated using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD) (20).
Northern blot analysis of GR messenger RNA (mRNA)
Total hippocampal RNA from each male individual (25 µg) was
denatured at 65 C for 5 min, separated on a 1% agarose formaldehyde
denaturing gel, and transferred to positively charged nylon membranes
(BrightStar-Plus, Ambion, Inc., Austin, TX). A 880-bp GR
complementary DNA (pGRII-122) HindIII fragment of pRBAL117
(provided by Dr. Sam Okret) containing part of the hormone-binding site
was used as a probe. A commercially available 1076-bp ß-actin
fragment from mouse was used as a control
(DECAtemplate-ß-actin-mouse, Ambion, Inc.). The probes
were labeled with a random priming kit (DECAprime II, Ambion, Inc.), using [
-32P]deoxy-CTP (10
mCi/ml; NEN Life Science Products, Boston, MA). Blots were
prehybridized overnight at 42 C in a solution containing 50%
formamide, 0.12 M
Na2HPO4 (pH 7.2), 0.25
M NaCl, and 7% SDS and then hybridized with the
probe at the same temperature for 16 h. Blots were washed with
2 x SSC (saline-sodium-citrate) buffer/0.1% SDS for 15 min at
room temperature, with 0.5 x SSC/0.1% SDS for 20 min at room
temperature, and finally with 0.1 x SSC/0.1% SDS for 15 min at
60 C. The intensities of the signals were quantified by PhosphorImager
analysis (Molecular Dynamics, Inc., Sunnyvale, CA) using
ImageQuant software.
Preparation of protein extracts
Frozen tissue (hypothalamus and hippocampus; males: LPS-treated,
n = 8; controls, n = 8; females: LPS-treated, n = 11;
controls, n = 5) was placed into an ultracentrifuge
Eppendorf tube containing 5 vol ice-cold TEGMD buffer (20
mM Tris, 1 mM EDTA, 10 mM sodium
molybdate, 10% glycerol, and 1 mM dithiothreitol) with
protease inhibitors. Tissue and cells were disrupted with an Ultrasonic
homogenizer sonicator (Cole Parmer Instruments, Chicago, IL). Complete
homogenization was confirmed by light microscopy. After centrifugation
at 2 C for 45 min at 105,000 x g (TL-100
Ultracentrifuge, Beckman Coulter, Inc., Palo Alto,
CA), the supernatant was collected, aliquoted, and stored at -80 C.
Protein content was determined using the bicinchoninic acid protein
assay (Pierce Chemical Co., Rockford, IL), and 20 µg
total protein were used in the Western blot analysis.
Western blot analysis of GR, leptin receptor (ObR), and ObRb
protein
Sample from each individual (20 µg total protein from
hippocampus or hypothalamus) was mixed with 4 x SDS sample
buffer, boiled for 5 min, and resolved by electrophoresis in 8%
SDS-PAGE gels in Tris-glycine-SDS buffer. Protein was
electrophoretically transferred to polyvinylidene difluoride Western
blotting membranes (Roche Molecular Biochemicals,
Mannheim, Germany) in Tris-glycine-methanol buffer (overnight, 4 C, 200
mA) using a Transblot Electrophoresis Transfer Cell (Bio-Rad Laboratories, Inc., Hercules, CA). The membranes were blocked
for 1 h at room temperature with 5% nonfat dry milk in
Tris-buffered saline-Tween (TBS-T), washed briefly in TBS-T, and
incubated for 1 h (anti-GR and antiactin) or 2 h (anti-ObR
and anti-ObRb) at room temperature in TBS-T with 1% nonfat dry milk
containing the appropriate dilution of antibody [1:4000 mouse
monoclonal antiactin (clone AC-40, Sigma, St Louis, MO),
1:1000 rabbit polyclonal anti-GR (M-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1:1000 goat polyclonal anti-ObR (Research
Diagnostics, Flanders, NJ), and 1:500 rabbit polyclonal anti-ObRb
(Alpha Diagnostic International, San Antonio, TX)]. Membranes were
washed in TBS-T and placed in TBS-T with 1% nonfat dry milk containing
a 1:2000 dilution of peroxidase-conjugated secondary antirabbit,
antigoat, and antimouse antibodies, respectively (Amersham Pharmacia Biotech, Arlington Heights, IL) for 1 h at room
temperature. Blots were washed in TBS-T and detected with Western blot
Chemiluminescence Reagent Plus (NEN Life Science Products), exposed to ECL Hyperfilm (Amersham Pharmacia Biotech), and quantified on an Alphainnotech Chemi-Imaging
system (San Leandro, CA).
Analytical methods
Blood was collected in heparinized microtubes and centrifuged
immediately in a microcentrifuge. Plasma concentrations of glucose and
lactate were simultaneously enzymatically determined in 15-µl samples
on a 2700 SELECT biochemical analyzer (YSI, Inc., Yellow
Springs, OH). Plasma insulin was analyzed with a rat insulin RIA kit
(Linco Research, Inc., St. Charles, MO). Glycerol and FFA
were measured using enzymatic colorimetric methods (CMA 600, CMA Microdialysis, Stockholm, Sweden; and NEFA C, Wako Chemicals,
Neuss, Germany, respectively). Testosterone was measured with a solid
phase RIA (Coat-A-Count Total Testosterone, Diagnostic Products, Los Angeles, CA). 17ß-Estradiol and progesterone
were assayed with commercially available enzyme immunoassays
(progesterone and estradiol enzyme-linked immunosorbent assays; Biomar
Diagostics Systems Laboratories, Inc., Marburg, Germany).
Corticosterone was determined by RIA (RSL 125I
corticosterone RIA, ICN Biomedicals, Inc., Costa Mesa,
CA). Leptin was determined by RIA (Rat Leptin RIA kit, Linco Research, Inc.). Insulin collected during the clamp measurements
was analyzed with a double antibody RIA (Pharmacia Biotech, Uppsala, Sweden).
Hemodynamic measurements
Systemic arterial pressure and heart rate in conscious rats were
measured on three different occasions, once a week, between 9 and 11
weeks of age by a tail cuff method that was previously shown to give
similar results as direct arterial cannulation (21). An
instrument with a light-emitting diode with a photoresistor connected
to a dual channel recorder was used. Rats were warmed at 38 C for 10
min before measuring. Three stable consecutive measurements were
averaged.
Histology
Cryostat sections, 8 µm thick, were prepared from the
fresh-frozen ovaries. The sections were fixed in 4% buffered
formaldehyde, stained with hematoxylin and eosin, dehydrated, and
mounted.
Statistical analysis
All results are presented as the mean ± SE;
the range is shown in parentheses. The statistical methods
used were Student’s t test and Mann-Whitney nonparametric U
test, from the StatView program (SAS Institute Inc., Cary, NC)
in the Macintosh system.
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Results |
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Male rats
Food intake, body composition, and hormones. Food intake
was increased in 11-week-old male LPS offspring compared with control
offspring [22.6 ± 0.5 (21.6–23.9) and 19.4 ± 0.4
(18.2–21.0) g/day, respectively; P = 0.012]. Figure 1
shows total body weight from weaning to
12 weeks of age. LPS offspring had significantly elevated body weight
from 4 weeks onward. Table 1 shows
weights of various tissues at 12 weeks of age. Epididymal and
retroperitoneal fat depots were significantly heavier in LPS offspring,
whereas mesenteric and inguinal fat depots did not differ between the
groups. No significant differences were found in the weights of muscles
between the groups. There were no differences in weights of spleen,
adrenals, thymus, and heart (data not shown).
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depicts overnight fasting plasma
concentrations of leptin, 17ß-estradiol, progesterone, testosterone,
glucose, and insulin in the adult offspring. The plasma concentrations
of 17ß-estradiol and progesterone were significantly higher in LPS
offspring than in control offspring. LPS offspring also exhibited
significantly elevated leptin levels. There were no significant changes
between the groups in the other hormones measured. Plasma FFA
[LPS-treated, 0.82 ± 0.08 (0.56–1.15) mmol/liter (n = 8);
controls, 0.75 ± 0.08 (0.51–1.22) mmol/liter (n = 11);
P = 0.53] and glycerol levels [LPS-treated, 0.38
± 0.03 (0.24–0.45) mmol/liter (n = 8); controls, 0.38 ±
0.04 (0.22–0.53) mmol/liter (n = 11); P = 0.98]
did not differ between the groups.
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depicts the glucose infusion rate
during euglycemic hyperinsulinemic clamp. LPS offspring needed a
significantly lower glucose infusion to maintain euglycemia during
steady state (60–180 min) than controls [15.5 ± 1.2
(12.3–19.8) and 20.1 ± 1.7 (13.4–25.2) mg/kg·min,
respectively; P = 0.05]. The mean plasma insulin
levels during steady state (60–180 min) were not significantly
different between the groups [173 ± 8 (150–191) and 206 ±
50 (165–288) mU/liter in LPS-treated rats and controls, respectively;
P = 0.088]. Plasma glucose did not differ between the
groups throughout the insulin clamp [LPS-treated, 6.8 ± 0.1
(6.5–7.0); controls, 6.9 ±0.1 (6.6–7.7) mmol/liter;
P = 0.45].
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shows the plasma corticosterone
response to a novel environment stress test. There were no differences
in prestress levels of corticosterone between the groups. LPS offspring
showed a significantly lower response in terms of plasma corticosterone
than control offspring throughout the experiment. Both groups exhibited
apparently the same time kinetics in corticosterone response to
stress.
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, A and B, shows the expression of GR
mRNA in hippocampus as a ratio to ß-actin. There were no differences
in GR/ß-actin mRNA ratios between the groups. However, as depicted in
Fig. 5, A and B, LPS offspring had a
significantly higher level of GR protein expression in hippocampus,
expressed as a ratio to actin, compared with controls
(P = 0.042). There were no statistically significant
differences in hypothalamic GR protein expression between the groups
(data not shown).
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Hemodynamic measurements. There were no significant differences in either blood pressure or heart rate between the two groups (data not shown).
Female rats
LPS exposure in female offspring resulted in few significant
changes in adulthood. There was no significant difference in food
intake between the groups [LPS-treated, 14.9 ± 0.4 (13.3–16.0)
g/day; controls, 14.9 ± 0.3 (14.9–15.0) g/day; P
= 0.49]. Table 3 depicts body weight and
weights of skeletal muscle and adipose tissue at 14 weeks. No
significant differences were seen between the groups. Significant
enlargements of adrenals [0.42 ± 0.03 (0.31–0.70) and 0.32
± 0.03 (0.24–0.39) g/kg BW; P = 0.020] and heart
[3.14 ± 0.09 (2.83–3.84) and 2.81 ± 0.07 (2.59–2.99)
g/kg BW; P = 0.010] were present in LPS-treated female
offspring compared with control offspring. There were no difference in
weights of spleen and thymus (data not shown). Testosterone was
significantly elevated (P = 0.050), which, together
with fasting plasma concentrations of progesterone, 17ß-estradiol,
leptin, glucose, and insulin, is summarized in Table 4. There were no significant differences
in glucose infusion rate at steady state (60–180 min) during the
euglycemic hyperinsulinemic clamp measurement (5 mU/min·kg) between
the groups [LPS-treated, 21.4 ± 1.5 (13.0–28.4) mg/kg·min;
controls, 19.2 ± 1.1 (16.6–21.6) mg/kg·min; P
= 0.24], as shown in Fig. 6. Figure 7 shows the novel environment stress test
in female LPS offspring and control rats, respectively. No significant
difference in stress-induced corticosterone secretion was seen between
the groups, however, prestress corticosterone levels were significantly
higher in LPS offspring than in controls (P = 0.036).
Despite significantly elevated testosterone levels, the ovaries from
female LPS offspring showed normal histology. They had similar relative
proportions of stroma and gamete-producing structures in the cortex as
ovaries from control animals. Furthermore, all stages of developing
follicles as well as corpora lutea could be identified. There were no
differences in hemodynamic measurements (data not shown). No
significant differences were detected between the groups in
hypothalamic levels, expressed as a ratio to actin, of either GR
[LPS-treated, 0.88 ± 0.08 (0.15–1.26; n = 11); controls,
0.81 ± 0.14 (0.27–1.09; n = 5); P = 0.71],
ObR [LPS, 0.52 ± 0.07 (0.21–0.92; n = 11; controls,
0.49 ± 0.13 (0.22–0.89; n = 5); P = 0.81]
or ObRb [LPS-treated, 1.82 ± 0.13 (1.08–2.59; n = 11);
controls, 1.60 ± 0.31 (0.72–2.53; n = 5); P
= 0.40].
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In man, the metabolic syndrome is often associated with high serum levels of leptin (14). Other typical features of the metabolic syndrome are increased activity of the HPA axis, resulting in high levels of glucocorticoids, and decreased HPG axis activity, leading to a reduction in sex steroid secretion (13). To further evaluate the possible similarities between the metabolic syndrome in man and the combination of obesity and insulin resistance observed in prenatally LPS-exposed male rats, these parameters were also studied in the animals.
Leptin and leptin receptors
As in patients with the metabolic syndrome, plasma levels of
leptin were increased in the male LPS offspring (14).
Leptin is produced in adipose tissue and signals satiety via receptors
localized to the hypothalamic area in the brain (22).
Recently, several neuropeptides involved in leptin signaling and the
regulation of food intake have been identified, for example
neuropeptide Y and MSH (23, 24). In this study the male
LPS offspring exhibited increased food intake despite elevated levels
of leptin in serum, implying that the regulation of food intake by
leptin or other central effectors was inefficient after maternal
endotoxemia. A similar finding is common in obese humans, who display
both overeating and supranormal serum levels of leptin
(25).
Leptin is recognized by the ObR, which exists in several isoforms (26, 27). The long form (ObRb) is expressed at high levels in regions in the hypothalamus and seems to be important in mediating the biological effects of leptin. The functions of the other forms are unclear, but they may be involved in the transport of leptin across the blood-brain barrier. There were no changes in either the long or short forms of the leptin receptor in male LPS offspring, as confirmed with Western blot. However, as the protein was extracted from whole hypothalamus, this does not rule out changes in ObRb levels in specific hypothalamic regions involved in regulating feeding behavior and energy balance. Other potential mechanisms may include defects in leptin signal transduction or an impaired transport of leptin over the blood-brain barrier.
Sex steroids
The maternal systemic inflammatory response was also followed by
changes in serum levels of sex hormones in male offspring. Whereas the
levels of 17ß-estradiol and progesterone were significantly elevated,
testosterone levels tended to be decreased, but this difference between
groups was not statistically significant. Studies suggest that the
metabolic syndrome in men is associated with an increase in female sex
steroids and with a reduction in testosterone (28). Our
results regarding serum levels of sex steroids in male offspring of
LPS-exposed dams hence further reinforce the possible relevance of
these rats as an animal model of the metabolic syndrome.
Adipose tissue contains aromatase, the enzyme responsible for converting testosterone to estradiol, and in men adipose tissue mass and estradiol levels are positively correlated (29, 30). Thus, like the high levels of leptin, the increase in estradiol levels in male LPS offspring could be secondary to the increase in adipose tissue mass. However, the elevated progesterone levels and the modest reduction in testosterone suggest that the apparent feminization observed in the male LPS-treated offspring may be due to an influence on sexually dimorphic brain areas. As discussed above, prenatal stressors have previously been shown to induce feminization of the behavior of male rats as well as a change in the size of the sexually dimorphic nucleus of the preoptic area in brain (7, 31).
HPA axis
It has been suggested that stress-related glucocorticoid secretion
is associated with abdominal obesity in humans (32, 33).
Vicenatti et al. also showed that abdominally obese,
nondepressed women had decreased 24-h urinary free cortisol excretion
compared with women with peripheral obesity, characterizing both
central and peripheral alterations of the HPA axis
(33).
It is known that LPS leads to activation of the HPA axis via production of endogenous cytokines (10). Cytokines act both at the hypothalamic level, by stimulating neurons releasing CRH, and at the adrenal level, by enhancing corticosterone secretion (34, 35). In this study baseline levels of corticosterone were not significantly different in male LPS offspring compared with those in controls. The glucocorticoid response to stress was reduced rather than enhanced, and hippocampal GR protein was up-regulated. A higher density of hippocampal GR probably contributes to a more efficient inhibitory feedback control and hence reduced stress-related glucocorticoid secretion. The results obtained are in contrast to previous observations of increased basal plasma corticosterone levels and down-regulation of hippocampal GR levels after prenatal immune challenge with LPS (11). In those experiments, however, the dose of LPS was lower, and the injections were not repeated. Previous studies have shown that chronic stress can eventually lead to blunted stress activation of the HPA axis, with reduced corticosterone secretion in response to stress (36). It might be considered that the rather extensive, repeated LPS exposure in this study caused a more severe effect on HPA axis regulation, similar to that seen after chronic stress. Hypocortisolism is reported in several human states connected to chronic stress, such as posttraumatic stress disorder, fibromyalgia, and chronic fatigue syndrome (37). The underlying mechanisms behind this hypocortisolism in combination with repeated stress are not clear, but dysregulation of several levels of the HPA axis may be involved. Atrophy of hippocampal neurons has been shown to be associated with long-lasting stress and stress-related disorders in both primates and humans (38, 39). Chronic intensive challenge of the HPA axis not only affects the glucocorticoid secretion in response to stress, but also the diurnal secretion pattern of ACTH and corticosterone (40). In this study repeated measurements of baseline ACTH and corticosterone were not undertaken, and abnormalities in the diurnal secretion pattern cannot be excluded. In accordance with the results presented here, prenatal or early postnatal exposure to interleukin-1ß resulted in a reduced corticosterone response to stress (41, 42). This was observed in combination with a reduction of the numerical density of neurons in the paraventricular nucleus in the hypothalamus and a clear enlargement of the nuclei. In contrast to male offspring, female offspring exhibited increased baseline levels of corticosterone and adrenal enlargement, but no effect on stress-induced corticosterone secretion. However, due to the small number of female controls, it is difficult to discern whether it is an arbitrary or a true finding.
To conclude, LPS exposure of rat dams during the second trimester of pregnancy resulted in male offspring displaying obesity, overeating, and insulin resistance. As in men with metabolic syndrome, these characteristics were associated with high serum levels of leptin, a feminized pattern of serum sex steroids, and a dysregulated HPA axis. Interestingly, the effects seem to be sex specific. Gender-specific effects were also reported after undernutrition during the first 2 weeks of gestation, which resulted in obese hyperphagic male offspring, but no effects on female offspring (43).
The mechanisms underlying the development of obesity and insulin resistance in male LPS offspring remain unclear, and this study cannot further clarify whether it is due to central or peripheral effects. Prenatal protein restriction has been reported to affect pancreatic development (44). Furthermore, glucocorticoid exposure during late gestation leads to overexpression of hepatic GR and a gluconeogenetic enzyme, PEPCK (45). This may lead to consequences such as insulin resistance and glucose intolerance in adulthood. In this study, however, it seems likely that it is a consequence of programming during the development of brain structures. The overeating despite high serum levels of leptin, the sex discrepancy, and the changes in regulation of the HPA and HPG axes seem to place the main focus on hippocampal and hypothalamic centers as the locus for the damaging effects of LPS exposure in utero. Previous studies have indicated the brain as an important target for fetal programming (11, 41). The lack of elevation of blood pressure suggests, however, that the central hypothalamic regulation of the sympathetic nervous system was not affected. The possible causal relationship between the different endocrine changes observed is unclear, but there are multiple possibilities, including, for example, a stimulating effect of estradiol on leptin production (46), an inhibitory effect of HPA axis activity on sex steroid secretion (47), and an inhibitory role of glucocorticoids on leptin responsiveness (48).
In this study a novel animal model of obesity with features of the metabolic syndrome is presented. The relative significance of the various metabolic and endocrine factors characterizing adult male offspring of LPS-exposed dams and the mechanisms behind this kind of programming need to be elucidated in further studies. This study supports the previous idea that interference in utero is followed by perturbations in the regulation of energy intake and metabolism in adulthood.
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Footnotes |
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Received December 14, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L. Chen and B. L. G. Nyomba Glucose Intolerance and Resistin Expression in Rat Offspring Exposed to Ethanol in Utero: Modulation by Postnatal High-Fat Diet Endocrinology, February 1, 2003; 144(2): 500 - 508. [Abstract] [Full Text] [PDF]
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) and control
offspring (n = 11; 
). Data are the mean ± SE.
*, P < 0.05; **, P < 0.01;
***, P < 0.001 (by Student’s t
test).
95
kDa) and actin (
