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Pediatric Research Institute (B.S.S.), Cardinal Glennon Children’s Hospital (B.S.S.), and the Departments of Pediatrics (B.S.S.) and Pharmacological and Physiological Sciences (T.C.W.), St. Louis University School of Medicine, St. Louis, Missouri 63110; and the Division of Neonatology and Developmental Biology, Department of Pediatrics, University of Pittsburgh, Magee-Womens Research Institute (S.U.D.), Pittsburgh, Pennsylvania 15213
Address all correspondence and requests for reprints to: Dr. Sherin U. Devaskar, Department of Pediatrics, 300 Halket Street, Pittsburgh, Pennsylvania 15213-3180.
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Abstract |
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Introduction |
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Although ontogenic investigations have localized (2, 16) and demonstrated considerably lower levels of NPY in fetal rat brain (21), there are no studies characterizing the effect of an in utero diabetic environment on fetal whole brain NPY expression. Streptozotocin-induced maternal diabetes causes profound metabolic changes in the rat fetus (22, 23, 24), some of which have a long lasting postnatal effect on growth, peripheral insulin sensitivity, and acquisition of adult-onset obesity (25), not unlike those in the human (26, 27, 28, 29). To determine whether fetal whole brain NPY is amenable to the regulatory influences of in utero exposure to a maternal diabetic environment, we undertook the present study and examined the effect of streptozotocin-induced maternal diabetes on fetal brain NPY messenger RNA (mRNA) and peptide levels. In addition, to determine the isolated effect of hyperinsulinism, we examined the short and long term systemic and intracerebroventricular effects of insulin administration on fetal whole brain NPY synthesis and peptide concentrations.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Maternal diabetic model
On day 12 of gestation, rats received either 65 mg/kg freshly
reconstituted streptozotocin (Sigma Co., St. Louis, MO) in 0.5 ml
vehicle and maintained on ice or an equal volume of the vehicle as
described previously (30). Maternal urine was checked for ketonuria
using Ketostix strips (Ames Co., Indianapolis, IN). Maternal tail vein
glucose levels were monitored with a One Touch glucose analyzer
(Lifescan, Milpitas, CA), and the animals were divided into three
groups based on the treatment and glucose values. The vehicle-injected
animals demonstrated blood glucoses of less than 10 mM and
were assigned to the control group (CON; n = 8). The
streptozotocin-injected animals (n = 13) were further divided into
two groups. The animals that did not develop overt diabetes were
assigned to the streptozotocin nondiabetic group (STZ-ND; n = 7),
and had glucose levels below 10 mM. This group served as an
additional intermediate group that helped distinguish between the
effects of streptozotocin and those of overt maternal diabetes. The
rest of the animals that received streptozotocin had glucose levels
greater than 13.5 mM and were assigned to the severely
diabetic group (STZ; n = 6) (30). On day 20 of an expected 21-day
gestation, pregnant rats were anesthetized with pentobarbital and
subjected to hysterotomy, and the fetuses were delivered. This
experimental model is schematically represented in Fig. 1a
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Anthropometric and metabolic characteristics
Maternal body weights were recorded. Pooled fetal body and brain
weights from a single litter were recorded, and the mean fetal body and
brain weights were derived based on the number of pups per litter.
Maternal (intracardiac) and pooled fetal blood (jugular) samples were
collected; the plasma was separated by centrifugation at 4 C,
aliquoted, and stored at -20 C until further analyses. Maternal and
fetal plasma glucose levels were measured by the quantitative glucose
oxidase method (Sigma Diagnostics), and the insulin levels were
assessed by the RIA employing a guinea pig antirat insulin primary
antibody, 125I-labeled insulin as ligand, and rat insulin
standards (Linco Research, St. Louis, MO) (30).
Tissue preparation
Pooled fetal brains from a single litter were snap-frozen in
liquid nitrogen and stored at -70 C under ribonuclease-free
conditions. Polyadenylated enriched RNA fraction was extracted using
Fast Track kits (Invitrogen, San Diego, CA). RNA was quantitated
spectrophotometrically at the 260 nm wavelength. The purity of the RNA
samples was documented as a 260/280 nm wavelength optical density ratio
of 1.8 to 2, and the integrity of the samples was confirmed by gel
electrophoresis (32).
Northern blot analysis
Ten micrograms of RNA were separated on a 1.2% agarose-2.2
M formaldehyde horizontal gel at 22 V for 16 h. The
uniformity of loading was assessed by ethidium bromide staining and
visualization of RNA under UV light. The RNA samples were transferred
to Nytran filters (Micron Separations, Westboro, MA) by capillary
action, and the RNA was cross-linked to the filters by exposure to UV
light at 1200 x 100 µJ for 45 sec (Stratalinker 1800, Stratagene, La
Jolla, CA). The Nytran filters were prehybridized for 2 h at 42 C
in 0.1% polyvinyl pyrolidone, 0.1% BSA, 0.1% Ficoll, 75
mM sodium chloride, 50 mM sodium phosphate
monobasic, 1.25 mM EDTA, 0.2% SDS, and 200 µg/ml
denatured salmon sperm DNA (32). Hybridization at 42 C for a period of
48 h was accomplished by the addition of 1 x 106
cpm/ml of a random primer 32P-labeled and denatured 511-bp
rat prepro-NPY complementary DNA (cDNA) (33). The filters were then
washed at room temperature in 5 x SSC (1 x SSC = 0.15
M sodium chloride and 0.015 M sodium citrate,
pH 7.0) and 0.5% SDS twice for 15 min each time, followed by two
washes in 1 x SSC and 1% SDS for 30 min each time at room
temperature, after which the filters were exposed to x-ray film at -70
C for various lengths of time until optimal resolution was achieved.
Interlane RNA loading was standardized by stripping the filters with 50
mM Tris (pH 8.0), 2 mM EDTA, 0.5% sodium
pyrophosphate, 0.002% polyvinyl pyrolidone, 0.002% BSA, and 0.002%
Ficoll, ensuring no signal on autoradiography, and then rehybridizing
the filters with a 1.0-kilobase (kb) rat 18S ribosomal RNA (rRNA) cDNA
probe (34). All autoradiographs with exposure times within a given
range of linearity were subjected to transmittance densitometry (Biomed
Instruments, Fullerton, CA), and the optical densities were used to
calculate the ratio between the NPY mRNA and 18S rRNA. The ratio was
then expressed as a percentage of the corresponding vehicle control
sample(s) per blot.
RIA
Brain tissue (100 mg) was extracted in a 10-fold volume of 0.1
N HCl (wt/vol). The fetal brain acid extracts were assayed
for NPY by RIA employing a rabbit antirat NPY antibody generated and
previously characterized by one of us (35). [125I]NPY
(Amersham, Arlington Heights, IL) and porcine NPY standards (Peninsula
Laboratories, Belmont, CA) were employed in a RIA using a goat
antirabbit serum (Linco Research, St. Louis, MO), and polyethylene
glycol was used for precipitation (Fisher, Fairlawn, NJ) (35). Specific
adult and fetal rat brain samples were employed as controls in all
assays. The sensitivity of the assay was 15 pg/ml, and the intraassay
variability ranged from 2–3%, whereas the interassay variability was
5–8%. The results were expressed per brain weight.
Data analysis
All results are expressed as the mean ± SEM.
Differences between the streptozotocin-treated severe diabetic,
nondiabetic, and vehicle control groups were determined by one-way
ANOVA, and the intergroup differences were validated by the
Newman-Keuls test. Additionally, nonparametric testing was also
conducted using Kruskal-Wallis with tied ranks followed by Dunn’s test
to ensure that the conclusions drawn were not due to either small
numbers or a heteroscedastic distribution of observations. Using both
parametric and nonparametric testing, statistically significant
differences between the groups were similar. When only two groups were
compared, as in the insulin-treated and respective control groups, the
Student’s t test was used at a P < 0.05
level of significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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demonstrates maternal and fetal plasma
glucose and insulin concentrations. As indicated, although maternal
insulin levels were significantly lower than those in the vehicle
control group, no difference between the maternal insulin
concentrations of the STZ-ND and CON groups was observed. These insulin
changes were associated with maternal glucose levels being 3-fold
higher in the STZ-D group, with no change in the STZ-ND group. In the
fetus, circulating glucose concentrations were 5-fold higher in the
STZ-D group compared to those in the STZ-ND and control groups, whereas
fetal insulin concentrations were not different among all three groups.
STZ-treated maternal diabetic and nondiabetic groups caused no change
in fetal body and brain weights. Further, there were no in
utero fetal losses.
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, a and b). This decline in
mRNA amounts was associated with a 40% decrease in fetal brain
immunoreactive NPY levels (Fig. 2c). The NPY mRNA and peptide levels in
the STZ-ND group were not different from those in the vehicle control
group.
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demonstrates the fetal plasma glucose and
insulin concentrations. As noted, in the short term study, although no
changes in circulating glucose concentrations were observed, a 400-fold
increase in plasma insulin concentrations was noted in the ip
insulin-treated group. In contrast, the intracerebroventricularly
insulin-treated group demonstrated only a 10-fold increase in
circulating insulin concentrations. In the long term insulin-treated
groups, a 5-fold increase in plasma insulin levels was noted only in
the ip treated group; the intracerebroventricularly treated group
demonstrated no difference compared to the respective control. No
change in circulating glucose levels was observed in the long term
insulin-treated groups compared to the respective controls. Comparison
of fetal glucose levels between the short and long term studies
revealed higher levels in the former secondary to the proximity of
blood sampling to the laparotomy procedure. At the doses and timing of
insulin administration, despite significant hyperinsulinemia in some
treatment groups, there were no in utero fetal losses due to
the presence of fetal normoglycemia.
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). NPY mRNA levels were not different between the
insulin-treated groups and the respective control groups in the short
term (4-h) studies. Similarly, in the long term (24-h) systemic
hyperinsulinemia group, there was no difference in NPY mRNA levels
compared to those in the vehicle control. However, a minimal,
statistically insignificant decrease in NPY mRNA levels was noted in
the long term (24-h) intracerebrally treated insulin group. In
contrast, a 50–60% decline in the immunoreactive NPY levels was
observed in both the short term (4-h) and long term (24-h)
intracerebrally treated insulin groups compared to those in the
respective vehicle-treated control groups. A relatively smaller
insignificant trend toward a decline (20–30%) in NPY levels was also
noted in both the 4- and 24-h ip insulin-treated groups compared to
those in the respective controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to this orexigenic effect, NPY administration profoundly inhibits both the gonadotropic and somatotropic axes (42) as well as promotes anxiolytic behavior in adult rats (6). These effects have not been studied in utero or in the immediate postnatal period.
Although all these biological functions have been ascribed to NPY, the factors that regulate the synthesis and release of this peptide are currently being explored. In the adult rat, streptozotocin-induced diabetes associated with hyperglycemia and hypoinsulinemia caused an increase in hypothalamic NPY levels (11). This increase is similar to that observed in a hypoinsulinemic, hypoglycemic, and energy-depleted state of starvation (14, 18). Further systemic administration of insulin was noted to reverse the changes in adult rat hypothalamic NPY concentrations that were induced by the diabetic state (7, 18, 19). These adult studies collectively demonstrated a predominant role for circulating insulin rather than glucose in regulating NPY synthesis. Recent studies in which insulin was administered intracerebroventricularly, leading to a suppression of hypothalamic NPY levels in both adult control and diabetic rats lend further support to this contention. This decrease in NPY concentrations led to a decline in food intake and body weight (43). Similar to the adult, whether insulin rather than glucose is involved in regulating fetal brain NPY synthesis remained unknown until the present investigation.
The fetus of a diabetic rat is generally growth retarded due to insufficient placental blood flow (44). In the present study care was taken to ensure that maternal diabetes was not prolonged or severe enough to interfere with fetal growth. Based on the model employed, there was no major compromise to fetal body and brain weights. Employing this model, our present studies demonstrate that the maternal diabetic state associated with fetal hyperglycemia in the absence of fetal insulin changes led to a decline in fetal brain NPY mRNA and protein levels. This suggests that hyperglycemia alone, in the absence of insulin perturbations, can suppress fetal brain NPY synthesis. The present in utero results contrast the observations of previous studies conducted in the postnatal rat which demonstrated that the progeny of a streptozotocin-induced diabetic pregnant rat develop insulin resistance and obesity during adult life (25). This change occurs despite postnatal normalization of circulating glucose and insulin concentrations. Thus, the suppression of fetal brain NPY synthesis and concentrations must have an immediate effect on fetal phagic behavior, perhaps resulting in sluggish ingestion of amniotic fluid, thereby contributing to the development of polyhydramnios of a diabetic pregnancy (45). In addition to its effect on ingestion, the suppression of fetal brain NPY levels in utero may predispose the progeny to altered sexual behavior and a heightened level of anxiety. All of these functional responses to suppressed fetal NPY levels need future investigations.
Four- to 24-h intracerebral exposure to insulin led to a decline in NPY protein concentrations without a similar decline in corresponding NPY mRNA concentrations. These fetal findings are not similar to those noted in the adult rat (18, 43). This discrepancy may be related to the fact that in the adult, the studies were of longer duration and were carried out over a period of 7–17 days, whereas in the fetus our studies were limited to a maximum of 24 h due to the inability to chronically deliver insulin in vivo to the intact rat fetus. Thus, it appears that over this limited period insulin either suppresses translation or leads to an acute depletion of the secreted NPY peptide. However, our present relatively short term studies do not rule out an added pretranslational suppressive effect on NPY synthesis, which may manifest in future longer term studies, that could be feasible in a larger animal model.
Intraperitoneal insulin administration, which led to pharmacological levels of circulating insulin concentrations without a change in circulating glucose levels, did not suppress the fetal brain radioimmunoassayable NPY amounts to the same extent as direct administration of the hormone to the brain. Nevertheless, a trend toward a decline in NPY peptide levels was noted in both the 4- and 24-h studies. This suggests that insulin suppresses fetal brain NPY concentrations by a direct action. Thus, high levels of circulating insulin concentrations can only render this effect by crossing the blood-brain barrier and acting upon the NPY-producing cells. It appears that circulating fetal insulin does not acutely (within 24 h) cross the blood-brain barrier in sufficient amounts necessary to produce a 50% suppression of fetal brain NPY concentrations. Chronic fetal hyperinsulinemia, as observed in the human fetus of a diabetic mother, could cumulatively lead to sufficient intracerebroventricular insulin levels capable of suppressing NPY concentrations.
Recent studies in adult animals have demonstrated systemic hyperinsulinemia to increase adipocytic secretion of circulating leptin (46), which, in turn, binds to leptin receptors in both the choroid plexus and the hypothalamic paraventricular nucleus, leading to a suppression of neuropeptide mRNA levels (47). In addition to a direct suppressive action of insulin on hypothalamic neuropeptide synthesis (18, 43), it is feasible that leptin acts as the intermediary for systemic insulin’s suppressive effect on hypothalamic NPY. Thus, it appears that both of these peptides influence adult hypothalamic NPY synthesis. In the present study, the measured fetal brain NPY concentrations reflect NPY present in areas of the brain other than the hypothalamus alone. However, the fact that ip insulin administration in the fetus did not have a similar effect as intracerebroventricular administration suggests that pharmacological levels of fetal hyperinsulinemia as achieved in the present study may either not evoke an increase in circulating leptin levels similar to that in the adult or fail to express an adult type leptin-induced suppression of whole brain NPY synthesis. Future studies are required to examine the direct effect of insulin separate from that of leptin on fetal brain NPY concentrations.
We conclude that in the fetus, before the establishment of cyclical postnatal feeding behavior, sexual function, and development of anxiety, hyperglycemia of 6-day duration in the absence of insulin perturbations leads to a pretranslational suppression of fetal rat brain NPY synthesis and levels. In contrast, acute fetal hyperinsulinemia of significant proportion independent of changes in circulating fetal glucose levels fails to alter fetal brain NPY mRNA and peptide concentrations to the same extent. Intracerebroventricular hyperinsulinism independent of hyperglycemia, however, suppresses fetal brain NPY levels, perhaps secondary to either translational or posttranslational effects or an acute depletion of secreted NPY. Thus, in the fetus, hyperglycemia rather than acute hyperinsulinemia appears to have a dominant suppressive effect on fetal brain NPY concentrations. The combined effect of hyperglycemia and chronic hyperinsulinemia leading to intracerebroventricular hyperinsulinism as in the human fetus of a diabetic pregnancy could additively inhibit fetal brain NPY synthesis leading to lowered NPY levels and depressed function. Based on these observations we speculate that this in utero suppression of NPY may perturb the fetal ingestive pattern of amniotic fluid, contributing to the development of polyhydramnios and/or predispose the fetus to altered immediate postnatal feeding behavior, body weight gain pattern, vasomotor stability, sexual function, and level of anxiety.
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Acknowledgments |
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Footnotes |
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Received July 8, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-globulin into the hypothalamic
paraventricular nucleus decreases food intake in rats. Brain Res 601:313–316[CrossRef][Medline]
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