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McGill University, Department of Psychiatry, Douglas Hospital Research Center, Montreal, Québec, Canada H4H 1R3; and Department of Physiology, Université Laval (S.C., D.R.), Ste-Foy, Québec, Canada G1K 7P4
Address all correspondence and requests for reprints to: Dr. Claire-Dominique Walker, Douglas Hospital Research Center, 6875 Lasalle Boulevard, Verdun, Québec, Canada H4H 1R3. E-mail: waldom{at}douglas.mcgill.ca
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although we are beginning to understand the interactions between leptin and the HPA axis in adults, virtually nothing is known about these in developing organisms. This is particularly important because neonates are exposed to high levels of leptin during the perinatal and suckling periods (6, 7, 21) and exhibit increased expression of leptin mRNA compared with the adult (22). This period of exposure to high circulating leptin concentrations coincides in neonatal rat pups with a period of relative adrenal quiescence between d 3–14 of life, often called the adrenal hyporesponsive period (23, 24). Interestingly, earlier studies found that the feeding component of maternal care is critical to maintain low adrenal sensitivity to ACTH (25), although the specific agents in maternal milk responsible for this effect have not yet been identified. During the neonatal period, a unique pattern of GR concentrations exists in the brain and pituitary (26, 27), which insures an efficient negative feedback action on the HPA axis (28, 29) and tight control over glucocorticoid production (30, 31). Efficient glucocorticoid negative feedback on the HPA axis is critical, because low stable corticosterone levels are optimal for neuronal development in glucocorticoid-sensitive brain regions (32). Through an action on glucocorticoid secretion and function, elevated levels of leptin in development might thus contribute to regulate HPA activity and favor neuronal development. Indeed, we previously documented that increasing leptin concentrations in neonates, either through changes in the maternal milk composition (6) or after exogenous leptin administration (8), significantly reduced the adrenocortical response to stressors in 10-d-old pups. Stress-induced, but not basal, CRF mRNA expression was diminished in leptin-injected pups, and the ACTH response to stress was significantly shortened, suggesting that changes in glucocorticoid feedback efficacy might mediate the actions of leptin on the HPA axis of the neonate. Therefore, we designed the present studies to determine 1) whether leptin and glucocorticoid secretion were related in the early neonatal period as in adults, and 2) whether leptin administration could modify the efficiency of glucocorticoid feedback on the HPA axis through changes in the expression of GR. We found that chronic neonatal leptin administration enhanced the ability of dexamethasone to suppress stress-induced ACTH secretion and that part of this effect can be explained by increased GR expression in the hippocampus and in the hypothalamic PVN.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Age-related changes in leptin and corticosterone levels and
determination of food intake
Trunk blood from fetuses on d 21 of gestation or neonates
(postnatal d 0–1, 2, 4, 6, 8, 10, 14, 18, 20, and 35) was collected
from undisturbed mothers and pups between 0900 and 1100 h. For fetuses
and young pups (postnatal d 0–1 and 2), blood from two pups was pooled
in one tube. Plasma was stored at -20 C until assayed for leptin and
corticosterone concentrations.
To determine whether chronic leptin affected food intake in pups, 8-d-old pups injected with either VEH or leptin (3 mg/kg BW) as described below were used. Pups were removed from the dam for 30 min, stimulated to urinate and defecate by stroking the anogenital region with an artist’s brush wetted with warm water, and weighed. During the intake test, pups were placed individually into plastic containers lined with paper towels wetted with a commercial half and half milk diet and kept on a warming pad warmed at 32–35 C. Pups were allowed to consume the diet for 30 min; the paper towels were rewetted with warm diet every 10 min during testing as necessary. Pups were then removed from the containers, dried carefully, and reweighed. Because pups at this age do not readily urinate and defecate spontaneously, particularly after being stimulated manually to do so, the amount of weight gained (expressed as a percentage of the pup’s predeprivation body weight) during the intake test was used as a reliable measure of intake (33).
Chronic leptin treatment and dexamethasone suppression
test
Murine leptin was obtained lyophilized from PeproTech, Inc.
(Rocky Hill, NJ) and reconstituted in 10 mM Tris buffer, pH
9.5. After dissolution, the pH was readjusted to 7.4 by addition of
HCl. Dexamethasone was obtained from Sigma (St. Louis, MO)
and was first dissolved in a small volume of 100% ethanol, followed by
addition of 0.9% saline. The final concentration of ethanol in the
steroid solution was 1%. Leptin or VEH (10 mM Tris-HCl, pH
7.4) was injected ip in a volume of 50 µl, with all injections given
in the morning between 0800 and 1000 h. Litters were randomly assigned
to one of three groups receiving daily ip injections of either leptin
[1 mg/kg BW (L1) or 3 mg/kg BW (L3)] or VEH between d 2–9 of life.
At 0900 h on d 10 of life, pups from each chronic treatment group
(VEH, L1, and L3) were weighed and separated into four groups: those
receiving no injection and those receiving an ip injection of VEH
(0.9% saline and 1% ethanol), or dexamethasone at doses of 1 or 10
µg/kg BW. These doses of dexamethasone were chosen from previous
experiments where a dose-dependent effect of this steroid was
demonstrated on ACTH secretion in 10-d-old pups (29). Pups
were then returned to their mothers in a quiet room until tested for
their hormonal responses to ether stress. Three hours later (1200 h),
pups were separated from their mothers and exposed to ether vapor for 3
min, including 1 min in a glass jar saturated with ether vapors and 2
min under a nose cone containing cotton impregnated with ether. Pups
were then returned to clean cages and were killed 30 min after the
onset of stress. Control pups were killed without exposure to ether
stress. The time interval between stress onset and sacrifice (30 min)
was chosen according to our previous experiments on dexamethasone
suppression in neonates (29) and aimed to represent the
peak of ACTH secretion after this type of stress under our experimental
conditions (8). Trunk blood was collected in chilled
Eppendorf tubes containing 10 µl EDTA (60 mg/ml), and
plasma was kept frozen at -20 C until assayed for ACTH,
corticosterone, and leptin concentrations. Half of the brains were
rapidly collected and postfixed in a chilled solution of 4%
paraformaldehyde in phosphate buffer (0.05 M, pH 7.4, 4 C)
for 2 d, followed by immersion in a solution of 10% sucrose in
phosphate buffer for 2 d at 4 C. Brains were then frozen at -80 C
until processed for in situ hybridization. The other half of
the brains were collected, and the hippocampus was dissected and
rapidly frozen for Western blot analysis of GR protein levels.
Retroperitoneal fat pad and adrenal glands were dissected and
weighed.
In situ hybridization for MR and GR mRNA in brain and pituitary
tissues
Twenty-micron coronal brain and pituitary sections were
collected onto poly-L-lysine-coated slides, allowed to
dessicate overnight under vacuum at 4 C, and kept at -80 C until
processed for hybridization using cDNA fragments complementary to the
MR (34) and GR (35) as previously described.
The sections were fixed in paraformaldehyde (4%), digested for 30 min
at 37 C with proteinase K (10 µg/ml in 100 mM Tris-HCl
containing 50 mM EDTA, pH 8), acetylated with acetic
anhydride (0.25% in 0.1 M trietholamine, pH 8), and
dehydrated through graded concentrations (50%, 70%, 95%, and 100%)
of alcohol. After vacuum drying for at least 2 h, 90 µl
hybridization solution mixture, which contains an antisense
35S-labeled cRNA probe (10 million cpm/ml), were
spotted on each slide. The slides were sealed under a coverslip and
incubated overnight at 60 C in an hybridization oven. The next day, the
coverslips were removed, and the slides were rinsed four times in 4x
SSC (0.6 M NaCl and 60 mM sodium citrate
buffer, pH 7), digested 30 min at 37 C with ribonuclease A (20 µg/ml
in 10 mM Tris-500 mM NaCl containing 1
mM EDTA), washed in descending concentrations of SSC (2x,
10 min: 1x, 5 min: 0.5x, 5 min: 0.1x, 30 min at 60 C), and
dehydrated through graded concentrations of alcohol. After a 2-h period
of vacuum drying. The slides were exposed to x-ray film (Eastman Kodak Co., Rochester, NY) for 21–48 h. Radioactive standards
prepared from brain paste with high activity 3H
and 14C were exposed simultaneously.
Hybridization signal on the autoradiograms was quantified from
pituitary and brain sections selected throughout the hippocampus and
including the medial portion of the PVN and using a computerized
densitometry by means of an MCID image analyzer system (Imaging Research, Inc., St. Catherine, Canada). For each experimental
group, 2–10 animals were analyzed with an average of 3–10
slides/animal. Once removed from the autoradiography cassettes, the
slides were defatted in xylene and dipped in NTB2 nuclear emulsion
(Eastman Kodak Co.) for 5 min. Finally, tissues were
rinsed in running distilled water for 1–2 h, counterstained with
thionine (0.25%), dehydrated through graded concentrations of alcohol,
cleared in xylene, and coverslipped with DPX.
Western blot analysis of GR protein levels in the
hippocampus
Frozen hippocampi from leptin- or VEH-treated 10-d-old pups were
rapidly weighed and homogenized (50 µg/100 µl, wt/vol) by
sonication in ice-cold extraction buffer containing 30 mM
Tris base, 1 mM EDTA, 0.4 M NaCl, 10%
glycerol, 2 mM dithiothreitol, 1 µg/ml aprotinin, 1
µg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride, 1 µg/ml
leupeptin, and 1 µg/ml pepstatin, pH 7.4. Samples were adjusted to a
final protein concentration of 60 µg/17 µl (Bio-Rad Laboratories, Inc., Mississauga, Ontario, Canada) by
diluting with 4x sample buffer and 10x DTT (Novex,
Helixx Technologies, Inc., Scarborough, Canada). Samples were denatured
at 70 C for 10 min, spun down at 15,000 rpm for 20 sec, and loaded onto
4–12% (3-(N-morpholino)propanesulfonic acid)
bis-Tris polyacrylamide gels (Novex). Proteins were
separated by SDS-PAGE (200 V, 30–40 min) using 1x
3-(N-morpholino)propanesulfonic acid electrophoresis buffer
containing 50 mM MOPS, 50 mM Tris-base, 3.5
mM SDS, and 1 mM EDTA, pH 7.7, and
Novex antioxidant. A high range protein molecular standard
(RPN 756, Amersham Pharmacia Biotech, Little Chalfont, UK)
was loaded at the same time as the samples. Separated proteins were
electrophoretically transferred (30 V, 60 min, on ice) from gels to
polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.) presoaked in transfer buffer containing 25 mM
Tris base, 25 mM bicine, 1 mM EDTA, 1
mM SDS, 20% methanol (pH 8.3), and Novex
antioxidant (1:1000). Polyvinylidene difluoride membranes were blocked
at room temperature for 1 h in 1x TBS (20 mM Tris
base and 136 mM NaCl, pH 7.6) containing 0.1% Tween 20
(TBST) and 5% skim milk powder before overnight incubation with the
specific monoclonal anti-GR antibody (BuGR2, Catalog No. MA1–510,
Affinity BioReagents, Inc., Golden, CO; 1:4000 final
dilution in 0.25 µg/ml TBST with 0.5% skim milk powder) at 4 C. The
antibody was raised against purified rat liver GR and was shown to
react with a single epitope in the DNA-binding domain of the GR.
Previous reports indicate that this antibody recognizes both the
activated and unactivated forms of the receptor as well as the
denatured receptor (36). Membranes were washed in TBST at
room temperature and incubated for 1 h with sheep antimouse Ig
antibody linked to horseradish peroxidase (Amersham Pharmacia Biotech; 1:3000 final dilution in TBST). After several washes in
TBST, immunopositive bands were visualized by chemiluminescence (ECL,
Amersham Pharmacia Biotech) using short exposure to
Hyperfilm ECL films (3–12 min). Exposure times were adjusted so that
the darkest bands did not saturate the film. Membranes were then
stripped for 30 min at 70 C with a solution containing 62.5
mM Tris-HCl, 100 mM mercaptoethanol, and 2%
SDS before overnight blocking in TBST with 5% skim milk powder at 4 C.
After washing, the membranes were incubated for 1 h at room
temperature with the monoclonal mouse anti 
-tubulin antibody
(Biodesign International, Kennebunkport, ME; final dilution, 1:4000 in
TBST). Proteins immunoreactive for tubulin were revealed as for the
specific anti-GR antibody using horseradish peroxidase-linked secondary
antibody (as above) and ECL. The OD of GR and tubulin immunoreactive
bands were measured using an MCID image analyzer system (Imaging Research, Inc., St. Catherine’s, Canada). Background OD levels
were taken for each blot and subtracted from the OD obtained for each
individual immunoreactive band. Results are expressed as the ratio of
GR-specific over tubulin-specific OD of the immunoreactive bands.
Hormone assays
Plasma ACTH levels were measured by specific RIA as described
previously (28). The limit of detection of the assay was
15.6 pg/ml, and the inter- and intraassay variability was 26% and 8%,
respectively. Plasma corticosterone concentrations were determined by
RIA using a kit from ICN Biomedicals, Inc. (Costa Mesa,
CA), with small modifications. The limit of detection was 0.2 µg/dl;
inter- and intraassay variability was 12% and 3%, respectively
(8). Plasma leptin levels were measured in basal samples
(0 min) by specific RIA using a kit from Linco Research, Inc. (St. Charles, MO). The limit of detection was 0.5 ng/ml,
and interassay variability was 9% (8). Plasma
corticosterone-binding globulin (CBG) concentrations were determined by
binding as described previously (37).
Statistical analysis
All results were analyzed using ANOVA, followed by
post-hoc Student-Newman-Keuls or Tukey’s highest
significant difference tests where appropriate. Significant
differences between two groups were determined by t test.
The level of significance was set at P < 0.05. All
values are expressed as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). As previously demonstrated
(8), daily leptin administration during the first 9 d
of life led to a significant and dose-related reduction in body weight
and fat pad weight (P < 0.01) in 10-d-old neonates.
Leptin-induced changes were specific to fat pad, as adrenal weight was
not altered by leptin treatment (P = 0.141). In
agreement with previous reports (38), we found that
despite a significant effect of leptin on body parameters, the high
dose of leptin (3 mg/kg BW) did not modify food intake as measured in
an acute test in 8-d-old pups [VEH, 0.33 ± 0.06 g/30 min (n
= 13); 3 mg leptin, 0.42 ± 0.05 (n = 13); not
significant].
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4250 fmol/mg protein or 750 nM)
(37). Circulating leptin concentrations at the time of
sacrifice, i.e. 28 h after the last treatment, were not
different between VEH- and leptin-injected pups [VEH, 3.15 ±
0.42 (n = 13); 1 mg leptin, 2.94 ± 0.25 (n = 17); 3 mg
leptin, 2.17 ± 0.24 (n = 16); P =
0.062].
Age-related and reciprocal changes in plasma corticosterone and
leptin in neonates
Age-related changes in basal plasma concentrations of
corticosterone and leptin are shown in Fig. 1 (left). Significant age
effects were observed for both leptin and corticosterone secretion
(P < 0.001). Plasma corticosterone concentrations were
high perinatally and decreased during the first 2 wk of life as
demonstrated previously (31). Plasma levels of leptin were
elevated in suckling rats compared with concentrations reported in
adults (13, 22), and the peak of secretion appeared to be
on d 10 of life. Between d 2 and 6, changes in corticosterone secretion
paralleled those in leptin, but starting on d 6 of age, increases in
leptin secretion were exactly correlated with reductions in
corticosterone concentrations, suggesting a marked inverse relationship
between total plasma corticosterone and leptin concentrations (Fig. 1, right). We next examined whether a linear relationship might
exist between leptin and corticosterone secretion in neonates. Because
of the dramatic changes in circulating levels of both hormones
perinatally and to prevent interference from the maternal transfer and
clearance of both hormones, d 2 was the first age considered. We
initially restricted our linear regression of the effects of leptin on
corticosterone secretion to ages 2–14, because CBG levels are known to
increase significantly after the first 2 wk of life. With these ages
(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14), the linear regression of leptin on corticosterone
secretion under basal conditions was not quite significant [Pearson
correlation coefficient (Rval) = 0.702; r2
= 0.493; P > 0.05]. However, the regression was
significant when all ages between d 6–20 (Rval = 0.967;
r2 =0.935; P < 0.01) or 6–35
(Rval = 0.817; r2 =0.0.667;
P < 0.05) were considered.
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(top), plasma ACTH secretion
was increased 30 min after ether stress in all treatment groups
(uninjected vs. saline groups, P < 0.01, no
effect of chronic treatment group). The effect of acute treatment on
ACTH secretion was highly significant (P < 0.0001),
and the interaction between acute (saline or dexamethasone) and chronic
(VEH or leptin) treatment was modestly significant (P =
0.0559). Pretreatment with the low dose of dexamethasone (1 µg/kg BW)
produced significant reductions in ACTH secretion only in pups
chronically injected with the high dose of leptin (3 mg/kg BW). In
contrast, the high dose of dexamethasone (10 µg/kg BW) was able to
suppress stress-induced ACTH release in all groups of pups
(P < 0.01). The enhanced ability of dexamethasone to
suppress stimulated ACTH secretion in pups exposed to high circulating
leptin levels is further illustrated when the data are expressed as the
change in ACTH secretion over that in the saline-injected groups (Fig. 2, bottom). For the pups receiving the high dose of leptin,
both doses of dexamethasone significantly reduced ACTH secretion,
whereas only the highest dose of dexamethasone was able to do so in
other groups. In contrast to our previous experiments, we found that
the high dose of leptin significantly reduced basal (uninjected) ACTH
secretion in pups (VEH vs. 3 mg/kg BW leptin,
P < 0.05). The effect of leptin on basal ACTH was not
observed in pups receiving the lowest dose of leptin.
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. When
all groups were considered, there was a significant effect of the acute
treatment (saline, dexamethasone, P < 0.001), but no
effect of the chronic treatment (VEH, saline, P =
0.127) or interaction between acute and chronic treatments
(P = 0.135). Stress-induced significant corticosterone
secretion 30 min after its onset in VEH- and 3 mg leptin-injected pups.
Dexamethasone suppressed stimulated corticosterone secretion in a
dose-dependent manner, with significant reductions observed for all
chronic treatment groups.
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(top)
and 4, chronic leptin administration (3
mg/kg BW) in pups significantly increased GR mRNA levels in the
hypothalamic PVN (P < 0.05) and in the dentate gyrus
(DG) of the hippocampus (P < 0.05 for the high leptin
dose). There was a trend for GR expression to be increased in other
regions of the hippocampus (CA1 and CA3) after the high leptin dose,
but these changes did not reach significance. In contrast, leptin
significantly reduced GR mRNA levels in the CA2 region of the
hippocampus (P < 0.01 with the low dose of leptin).
For all chronic treatment groups, the highest expression of GR mRNA
levels was found in the CA1 region of the hippocampus, followed by the
CA2 region and the PVN. Expression of GR mRNA in the CA3, and the DG
was the lowest of all regions examined. In the hypothalamic PVN,
leptin-induced increases in the expression of GR mRNA levels appeared
to span both the parvocellular and magnocellular portions of the
nucleus (Fig. 4, bottom panels), although we did not analyze
these two subregions separately in these experiments. Leptin-induced
changes in GR were specific to the GR, as no significant variations in
the expression of MR mRNA levels were found in the hippocampal
formation of neonates (Fig. 3, bottom). In the hippocampus,
expression of MR mRNA was low compared with that of GR mRNA levels in
all experimental groups.
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). This increase probably represents a
combination of occupied and unoccupied receptors, as the antibody used
for our Western blots does not discriminate between the two states of
the GR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present studies were designed to test the hypothesis that elevated leptin concentrations in the neonates might be beneficial to maintain low adrenocortical activity during development. Leptin’s effect could be to reduce glucocorticoid production under basal conditions as documented in adults (12) and/or to enhance glucocorticoid’s suppressive effects on the HPA axis. This hypothesis was developed based on our previous studies showing that chronic administration of leptin (8) or exposure to a high fat milk, which increased circulating concentrations of leptin (6), reduced the overall ACTH response to stress by promoting a faster return to baseline levels of ACTH (8). In several adult rat models, including aged (40) and handled rats (41), changes in the dynamics of ACTH secretion after stress are often associated with alterations in glucocorticoid feedback mechanisms. Thus, one of the major findings of our study is that chronic leptin administration during the first 9 d of life had a significant effect on the ability of dexamethasone to suppress ACTH secretion in 10-d-old pups. Leptin’s effect was dose dependent and related to the dose of dexamethasone injected 3 h before the stressor. The lowest dose of dexamethasone was discriminant for the leptin’s effect, as it significantly reduced ACTH secretion in pups receiving 3 mg/kg leptin, but the reduction was not significant in the group receiving 1 mg/kg leptin. No reduction in stimulated ACTH secretion was observed in the VEH group with the low dose of dexamethasone. Surprisingly, however, we did not observe a clear relationship between ACTH and corticosterone secretion with the low dose of dexamethasone. This could be due to the unique time point that we used in our experiments (30 min post stress), which might not optimally reflect differences in the time course of ACTH and corticosterone secretion.
We previously demonstrated that the adrenocortical axis of neonatal rats is exquisitely sensitive to the suppressive effect of glucocorticoids (28, 29) in part because of the low corticosterone-binding globulin (CBG) concentrations, and consequently, the high percentage of free circulating corticosterone that is observed at this age (42). In our experiments we observed that chronic leptin administration tended to increase plasma CBG concentrations, and therefore, to avoid the confounding effect of CBG variations, we chose to use dexamethasone rather than the natural steroid corticosterone. The increase in CBG production in leptin-treated pups could be a direct consequence of the effect of leptin at reducing plasma corticosterone. In adults, CBG secretion is inhibited by increased corticosterone secretion and stimulated by thyroid hormones. In neonates, we failed to see a significant increase in T4 concentrations after leptin administration (Walker, C.-D., K. Kudreikis, and B. Kerman, unpublished); therefore, one possible explanation for the increase in CBG after chronic leptin treatment is that the reduction in basal corticosterone induced by leptin might allow for a concomitant small increase in CBG levels. If significant, leptin-induced increases in plasma CBG would decrease the MCR of corticosterone (43) and reduce, rather than enhance, glucocorticoid feedback action on the HPA axis. However, this is not what we observed in our experiments. Thus, we speculated that the increase in glucocorticoid feedback efficiency obtained here would result from the increased expression of GR in brain areas implicated in the regulation of the HPA axis and also possibly in the pituitary. In neonates, efficient glucocorticoid feedback is favored by a unique developmental pattern of GR in the brain, whereas the concentration of GR is increased during the first 3 wk of life, and the affinity of GR for CORT is higher in neonates compared with adults (44, 45). In agreement with other studies (46) we found that expression of MR in the hippocampus is very low compared with that of GR in this same structure. Interestingly, leptin treatment did not affect hippocampal MR expression, but significantly increased GR expression in the hypothalamic PVN and in the DG of the hippocampus in 10-d-old rats. Other regions, such as the pituitary or hippocampal CA1 and CA3 subfields, exhibited a nonsignificant trend toward increased GR expression in leptin-treated pups. In contrast, we found a significant reduction in GR expression in the CA2 subfield with the low dose of leptin, although this result was not confirmed with the highest dose of leptin. When GR protein levels were determined, the leptin-induced increase in GR expression was obvious in the hippocampus of neonates. Both CA1 pyramidal cells and DG neurons are thought to be closely linked to the activity of the HPA axis in rodents (41). Moreover, neurogenesis and synaptogenesis are active in these regions during the early period of neonatal development (47), and these processes are highly dependent on adequate amounts of glucocorticoids and GR (2). The effect of leptin on hippocampal GR expression (in particular in the DG) could be mediated by a reduction in endogenous corticosterone secretion or by accelerated neurogenesis in this particular structure. Indeed, our preliminary studies using DNA microarrays have shown that leptin injection in developing pups increases the expression of several proteins implicated in neurogenesis and synaptogenesis in the hippocampus (48). It is possible therefore that leptin acts as a facilitatory factor in neurogenesis and GR expression on newly formed neurons.
One of the salient observations derived from the present studies is that of a significant relationship between leptin and corticosterone starting on d 6 of age and maintained throughout the suckling period. Studies in adult rodents and in humans have similarly documented an inverse relationship between these two hormones (2, 4) but no study to date has focused on the existence of such a relationship in neonatal rats. Due to maternal transfer at the time of delivery, plasma corticosterone levels were high perinatally, declined dramatically over the first few days of life (49), and remained low until the third week of life as described previously (50). In contrast, plasma leptin concentrations declined briefly at birth and then increased to levels higher than those in the adult rat throughout the suckling period. The high levels of circulating leptin reported in human neonates (51) and in developing rodents (7, 52) have been attributed to increased leptin expression in adipose tissue (53) and pituitary (54) as well as to maternal leptin transfer through the milk (55). The inverse relationship between corticosterone and leptin found from d 6 on suggests that leptin might suppress basal adrenal production of corticosterone starting as early as the first week of life in rat pups. Hence, in agreement with earlier studies on the role of feeding on HPA function (25), it is tempting to speculate that naturally occurring high concentrations of leptin in developing pups are critical to maintain blunted adrenal glucocorticoid secretion during this period.
The functional relationship between leptin and corticosterone has been investigated in adult rodents, primarily on metabolic endpoints such as regulation of food intake, body weight gain and thermogenesis (18, 19, 20). Some reports showed that glucocorticoids limit the action of leptin on food intake in rats (19) and on body weight gain and fat stores in normal mice (20), whereas others failed to report a significant interaction between the two hormones on food intake and thermogenesis in genetically obese ob/ob mice (18). In developing rats a significant functional relationship between the two hormones might be difficult to demonstrate on food intake, because hypothalamic centers controlling food intake are not sensitive to leptin’s action in the early postnatal weeks. However, based on the present data, we hypothesize that a functional relationship between leptin and corticosterone exists in the regulation of the neonatal HPA axis. How it is occurring exactly is presently under investigation in our laboratory.
Contrary to our previous study (8), we did not observe significant changes in plasma leptin concentrations between groups in the present experiments. This difference could be attributed to the interval between the last injection of leptin and sacrifice, which was longer in the present experiments (28 vs. 24 h). Acute changes in circulating leptin concentrations do not represent the sole indicator of the effectiveness of chronic leptin treatment in pups. Indeed, the significant lower body weight and fat pad weight observed in the leptin-treated pups clearly demonstrated that chronic leptin treatment had a physiological effect in the present study. However, as demonstrated in other experiments (38), the high dose of leptin did not alter food intake in 8-d-old pups. This result should not be interpreted as an argument against a physiological effectiveness of leptin in pups, but, rather, as an additional fact supporting the hypothesis that leptin may serve other purpose than regulating food intake during the neonatal period (7).
In conclusion, the present studies demonstrate that under basal conditions, the high circulating leptin concentrations found in developing pups might play a critical role in regulating glucocorticoid production and the response of the adrenocortical axis to stress. Pharmacological manipulations of leptin in neonates allowed us to unravel the possible actions of leptin at maintaining GR in brain regions that are critical for HPA axis regulation. We propose that the ability of leptin to enhance the efficiency of glucocorticoid feedback in young neonates is mediated by these changes in GR, rather than by variations in the plasma distribution of free corticosterone. The effect of leptin on brain GR has important physiological implications for the maturation of hippocampal functions critically dependent upon glucocorticoids. These effects occur during a period of intense neurogenesis and synaptogenesis in the developing rat, and leptin might provide a critical factor mediating the maternal and environmental effects on neonatal physiology.
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Acknowledgments |
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Footnotes |
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Abbreviations: CBG, Corticosterone-binding globulin; CRF, corticotropin-releasing factor; DG, dentate gyrus; HPA, hypothalamic-pituitary-adrenal; PVN, paraventricular nucleus; TBST, 1x TBS (20 mM Tris base and 136 mM NaCl, pH 7.6) containing 0.1% Tween 20; VEH, vehicle.
Received March 16, 2001.
Accepted for publication August 3, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 S. Boullu-Ciocca, A. Dutour, V. Guillaume, V. Achard, C. Oliver, and M. Grino Postnatal Diet-Induced Obesity in Rats Upregulates Systemic and Adipose Tissue Glucocorticoid Metabolism During Development and in Adulthood: Its Relationship With the Metabolic Syndrome Diabetes, January 1, 2005; 54(1): 197 - 203. [Abstract] [Full Text] [PDF]
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 A. Varma, J. He, B.-C. Shin, L. A. Weissfeld, and S. U. Devaskar Postnatal intracerebroventricular exposure to leptin causes an altered adult female phenotype Am J Physiol Endocrinol Metab, December 1, 2004; 287(6): E1132 - E1141. [Abstract] [Full Text] [PDF]
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S. G. Bouret and R. B. Simerly Minireview: Leptin and Development of Hypothalamic Feeding Circuits Endocrinology, June 1, 2004; 145(6): 2621 - 2626. [Abstract] [Full Text] [PDF]
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C. Salzmann, M. Otis, H. Long, C. Roberge, N. Gallo-Payet, and C.-D. Walker Inhibition of Steroidogenic Response to Adrenocorticotropin by Leptin: Implications for the Adrenal Response to Maternal Separation in Neonatal Rats Endocrinology, April 1, 2004; 145(4): 1810 - 1822. [Abstract] [Full Text] [PDF]
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 Y. Ueta, Y. Ozaki, J. Saito, and T. Onaka Involvement of Novel Feeding-Related Peptides in Neuroendocrine Response to Stress Experimental Biology and Medicine, November 1, 2003; 228(10): 1168 - 1174. [Abstract] [Full Text] [PDF]
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K. Proulx, D. Richard, and C.-D. Walker Leptin Regulates Appetite-Related Neuropeptides in the Hypothalamus of Developing Rats without Affecting Food Intake Endocrinology, December 1, 2002; 143(12): 4683 - 4692. [Abstract] [Full Text] [PDF]
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) and corticosterone (
) during the
perinatal and neonatal periods of rat pups. On gestation d 21 (GD21),
an average of five or six fetuses per mother constituted one
determination, and the mean was obtained from six pregnant mothers. At
all other ages, values represent the mean ± SEM of
five (d 0) to seven pups per age. Right, Linear
regression analysis of the effects of leptin on corticosterone
secretion between 6–20 d of age. Values are taken from the left
panel. The calculated t value was 9.17 (df
= 4) with the Bonferroni test. Because opposite variations in
hormone levels were not observed before d 6, linear regression with
earlier ages did not reach the significance level.
) or dexamethasone (
, 1 µg;
, 10 µg/kg BW) was injected ip 3 h before exposure to ether
stress. Pups were killed 30 min after the onset of stress. Control pups
were not exposed to stress (
) and were killed at the end of the
experiment. Values are pooled from two separate experiments and
represent the mean ± SEM of 6 (control) to 15
pups/group. Bottom, Maximum change (
) in ACTH
secretion from stress-induced ACTH release (saline group) in the two
dexamethasone doses as a function of neonatal treatment. 
