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School of Dietetics and Human Nutrition, McGill University, Macdonald Campus, Ste. Anne de Bellevue, QC, Canada H9X 3V9
2To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • glycogen • phosphorylase • synthase • phosphoenolpyruvate carboxykinase (PEPCK) • neonatal rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Girard et al. 1992
). In
utero, the fetus benefits from a constant placental transfer of
glucose. At birth, this is interrupted and early postnatal hypoglycemia
is observed (Cuezva et al. 1985
, Girard et al. 1973
, Snell and Walker 1973
and 1978
). Failure
to correct postnatal hypoglycemia is lethal in neonatal rats
(Girard and Ferré 1982
) and dogs (Romsos et al. 1981
) and has been correlated with motor and intellectual
handicaps in newborn infants (Lucas et al. 1988
).
Maintenance of euglycemia during this vulnerable transition period,
between birth and commencement of feeding, is usually achieved through
mobilization of glycogen reserves and induction of gluconeogenesis.
Accumulation of perinatal tissue glycogen to concentrations
several-fold greater than in adult tissues is a key feature of late
gestation in most species (Girard 1989
, Girard et al. 1992
, Shelley 1961
). Until the onset of
gluconeogenesis, mobilization of these liver glycogen reserves plays a
critical role in maintaining neonatal plasma glucose (Girard 1990
, Girard et al. 1992
). The limiting
gluconeogenic enzyme, hepatic phosphoenolpyruvate carboxykinase
(PEPCK),3
is induced 4–6 h after birth (Cimbala et al. 1982
,
Lyonnet et al. 1988
)
Animal models that are designed to restrict glucose delivery to the
developing offspring such as maternal uterine artery ligation
(Bussey et al. 1985
, Marconi et al. 1993
,
Pollack et al. 1979
), maternal hyperinsulinemia
(James et al. 1990
, Ogata et al. 1987
)
and maternal fasting (Girard et al. 1973
,
Kliegman 1989
) have resulted in perturbations to one or
more of the glucoregulatory pathways, prolonged hypoglycemia and
increased perinatal mortality. Recently, we developed a dietary model
that impairs the availability of maternal dietary glucose directly and,
in contrast to more invasive models, provides sufficient energy to
maintain fetal growth by providing adequate nutrient delivery for all
nutrients except glucose (Koski and Hill 1986
, 1990
). We showed that the lack of maternal dietary
glucose limited liver glycogen accumulation in term rat fetuses
(Koski et al 1986
), significantly delayed renal PEPCK
gene expression in newborn rat pups (Liu and Koski 1997
), altered perinatal brain indoleamine profiles
(Koski et al. 1993
) and, most importantly,
increased neonatal mortality (Koski and Hill 1986
and 1990
). Inclusion of maternal dietary glucose prevented these
metabolic defects in a dose-dependent manner.
How maternal dietary glucose may modulate glucoregulatory pathways during perinatal development has not been investigated. The purpose of this study was to investigate the possibility that defects in glycogen accumulation and its mobilization or de novo synthesis of glucose lead to the poor postnatal prognosis, neonatal hypoglycemia and death. The specific objectives were to establish whether maternal dietary glucose restriction modified the pattern of glycogen mobilization by altering hepatic glycogen synthase and phosphorylase activities and/or disturbed the ontogeny of hepatic PECPK gene expression and the neonatal capacity for de novo synthesis of glucose.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Time-bred Sprague-Dawley rats (Charles River Canada, St-Constant,
Canada) were housed individually (12-h light:dark cycle, temperature,
22°C). From gestational day 2 to postnatal day 15, dams
(n = 18–22/diet) were fed semipurified
experimental diets containing graded levels of glucose [0, 12, 24 or
60% (control)] (Lanoue and Koski 1994
). The rationale
for choosing the specific macronutrient, vitamin and mineral levels has
been described (Koski et al. 1986
). Food intake and
maternal weight were measured daily. The length and time of parturition
were recorded with 0 h the birth time of the first-born pup.
Neonatal mortality was recorded. Surviving offspring were killed at
seven time periods: 0–2, 4–6, 12–16 and 24 h, and 3, 6 and
15 d postpartum. Comparable litter size among the four dietary
treatment groups was maintained at each time point.
Sample collection.
Pups killed at 0–2, 4–6, 12–16 and 24 h postpartum were
exsanguinated from severed axillary vessels and blood was collected
into heparinized capillary tubes. Pups killed on d 3, 6 and 15
postpartum were anesthetized with ketamine-HCl (Rogarsetic, 30 mg/kg,
Rogar/STB, London, Canada) and blood was collected via heart puncture.
Blood was collected into tubes containing one of the following:
1) heparin for insulin and glucose determinations;
2) heparin with trasylol (100 g/L, Miles Laboratories,
Rexdale, Canada) for glucagon analyses; or 3) heparin
with 0.59 mol/L perchloric acid (Anachemia, Montreal, Canada) for
lactate and ß-hydroxybutyrate (ß-OHB) analyses. Lungs, hearts and
livers were rapidly excised from the neonates and frozen in liquid
nitrogen. Samples were stored at -80°C until analyzed.
All animal procedures were approved by the animal care committee of
McGill University according to the guidelines of the Canadian Council for Animal Care (1984)
.
Plasma and tissue analyses.
Plasma insulin and glucagon were determined in duplicate by RIA
(Biodata, NCS Diagnostics, Mississauga, Canada); the detection limit of
insulin and glucagon was 36 pmol/L and 15 ng/L, respectively. Glucose,
lactate and ß-OHB plasma concentrations were determined by enzymatic
colorimetric methods on a VP Super System Analyzer (Abbott, Irving,
TX), using Sigma kits (Sigma 16-UV, 826-UV and 310-UV; Sigma Chemical,
St. Louis, MO). Neonatal liver, lung and heart glycogen was measured on
individual tissue samples as described by Lo et al. (1970)
.
Enzyme analyses.
Hepatic glycogen synthase and phosphorylase activities were determined
on individual tissue samples homogenized in a sucrose buffer [500
mmol/L sucrose, 62.5 mmol/L glycylglycine, 50 mmol/L sodium fluoride
and 5 mmol/L EDTA (pH 7.8)]. Glycogen synthase activity represented
the incorporation of UDP-[U-14C] glucose (Amersham,
Toronto, Canada) into glycogen, in the presence (total synthase) or
absence (synthase a) of 6.7 mmol/L glucose-6-phosphate according to the
procedure of Thomas et al. (1968)
. Glycogen
phosphorylase activity was determined on homogenates diluted in a
buffer containing 50 mmol/L
2-N-morpholino-ethane-sulfonic acid and 57.2 mmol/L
mercaptoethanol as described by Margolis (1993)
.
Phosphorylase activity was measured by the incorporation of
[U-14C]glucose-1-phosphate (Amersham) in the presence
(total phosphorylase) or absence (phosphorylase a) of 5 mmol/L AMP as
described by Tan and Nutall (1975)
. A unit of glycogen
phosphorylase or synthase activity was defined as 1 µmol of substrate
incorporated into glycogen per gram wet weight of liver per minute at
30°C.
PEPCK mRNA analysis.
Total RNA was isolated from the liver of the neonates
(n = 3 in each diet group per day) by precipitation
with guanidinium thiocyanate-phenol-chloroform (Chomczynski and Sacchi 1987
). Total RNA (15 µg) was separated
on a 1% agarose gel containing 6.6% formaldehyde. The RNA was
transferred to nylon membrane (Hybond, Amersham) and cross-linked by
UV-cross-linker Stratalinker 2400. Membranes were prehybridized
overnight at 42°C (35% formamide, 5X Denhardt's, 5X
SSPE, 0.5% SDS and 100 mg/L salmon sperm DNA). Hybridization was done
using the same conditions with the addition of a 1.5-kb PEPCK cDNA
probe kindly provided by Dr. Richard W. Hanson (Case Western Reserve
University, Cleveland, OH). The 32P-labeled cDNA probe was
labeled using random prime labeling. Following hybridization, the
membranes were washed three times for 15 min at 42°C (2X
SSC, 0.1% SDS) and once at 68°C for 15 min (0.1X SSC,
0.1% SDS) (Sambrook et al. 1989
). After
autoradiography, blots were stripped and rehybridized with a
radiolabeled DNA probe corresponding to rat glyceraldehyde-3-phosphate
dehydrogenase (GADPH), kindly provided by Dr. R. E. Mackenzie
(McGill University, Montreal, Canada). Quantitation of PEPCK mRNA was
done by densitometric scanning of the autoradiograms (Macintosh
Photoshop with NIH 1.60 software). All Northern blots were additionally
probed with GAPDH digoxigenin-labeled cDNA probe. Because liver GAPDH
is constitutively expressed, at least in adult tissues, and there is no
evidence that postnatal hepatic GAPDH is developmentally regulated, the
PEPCK/GAPDH ratio was used to correct for relative loading differences.
Statistical analyses.
Reproductive data at birth were analyzed for the effect of dietary
glucose by one-way ANOVA. Food intake was included as a covariate in
the statistical model because maternal food intake was significantly
lower in dams fed the glucose-free diet for the prenatal but not the
postnatal time points (Lanoue and Koski 1994
). Postnatal
mortality was analyzed using GLIM, a statistical program that analyzes
nonnormally distributed data by analysis of deviance (Baker and Nelder 1978
). The predicted probability of mortality was given
by the equation: p = ex/(l +
ex), where x represents the
number of dead pups, calculated as the total number of live pups minus
the number of pups killed, and fitted on the underlying scale;
p is the predicted proportion. When all pups in any
group died, mortality rates were compared among the remaining dietary
treatment groups. When a significant F-ratio was
generated by analysis of deviance, group comparisons of mortality rates
were done using linear contrasts. All other variables including plasma
metabolites, tissue glycogen, hepatic enzyme activities and mRNA of
PEPCK (expressed as the PEPCK/GAPDH ratio) were analyzed by two-way
ANOVA. Group differences were determined by least-square means and
considered significant at P < 0.05. When no
differences were calculated among the 12, 24 and 60% glucose diets,
these groups were pooled and compared with the 0% glucose diet. If
pups died, statistical analyses were performed on the remaining groups.
All statistics were performed using SAS for personal computers version
6.0 (SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Maternal dietary glucose restriction significantly perturbed
reproductive performance as shown by differences in length of gestation
(Table 1), pup weight at birth (Table 1)
and postnatal mortality
(Table 2). Feeding a glucose-free diet throughout pregnancy prolonged the
length of gestation by 1 d. This longer gestation was not
associated with greater pup weight because birth weights of pups from
dams fed the glucose-free diet were significantly lower than those of
pups from dams fed glucose-supplemented diets (Table 1)
. The results
from analysis of deviance showed that the differences in mortality at
0–2, 4–6, 12–16 and 24 h and at 3 and 15 d resulted from
differences in maternal dietary glucose. Three periods of high neonatal
mortality were observed, i.e., 0–16 h, d 1 and d 3. During the first
16 h postpartum, high neonatal mortality was observed in pups born
to dams fed a glucose-free diet, whereas pups born to dams fed diets
containing as little as 12% glucose successfully survived the first
24-h transition period. On d 1, all newborns from the 0% glucose
dietary group were dead, and a higher mortality rate (10–11 vs. 4%)
was observed in the 12 and 24% glucose groups than in the (60%)
control group. A third critical period of neonatal mortality was
observed on d 3, when significantly more deaths occurred in the 12 and
24% glucose groups, even though pup weights were comparable to those
of controls (60% glucose). Taken together, these results suggested
that although glucose-restricted diets enabled pups to survive the
first 24 h postpartum,12 and 24% glucose did not constitute
sufficient dietary levels to guarantee neonatal growth and survival.
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At birth, neonatal hypoglycemia was observed in all dietary groups
(Fig. 1A
). However, this hypoglycemia was only transient in pups from dams
fed glucose-containing diets because significant increases in plasma
glucose were observed in the 12, 24 and 60% glucose diets within
4 h postpartum. This was not the case for the glucose-deprived
pups (0% glucose) who did not have normal plasma glucose at any time
during the next 24 h and died. In the 12 and 24% glucose diet
groups, the transient rise in plasma glucose during the first 4 h
was followed by its subsequent decline, with values reaching a
comparable nadir at 24 h. In contrast, in the control pups, the
lowest glucose measurement occurred 12 h earlier.
|
B. As expected, the I:G was significantly
lower in the 0% glucose group for the first 16 h; this lower
ratio was driven by markedly reduced plasma insulin and higher glucagon
concentrations in these pups. Increasing dietary glucose to 12% was
associated with an increased I:G, but this value remained lower than
those of the 24 and 60% glucose diet groups until 12–16 h.
Thereafter, there were no significant differences among the 12, 24 and
60% groups. By the third postpartum day, I:G in all three groups (12,
24 and 60%) was significantly higher than the values calculated at
12 h, suggesting that pups had switched by this time from a
glucagon-driven state to one in favor of insulin. Tissue glycogen.
Maternal dietary glucose significantly modified perinatal tissue
glycogen reserves at parturition (Fig. 2
). Pups (0% glucose) were born with 50% less lung and liver
glycogen and 35% lower heart glycogen reserves compared with controls.
Glycogen reserves among the 12, 24 and 60% glucose diet groups were
comparable or exceeded those of controls at birth, indicating that if
12% glucose was included in the maternal diet during pregnancy,
accumulation of glycogen occurs in sufficient amounts in the perinatal
heart and lungs, whereas the liver required 24% glucose in the
maternal diet. However, these differences in maternal dietary glucose
and glycogen reserves did not modify the developmental pattern of
perinatal tissue glycogen metabolism. The first 16 h postpartum
were characterized by declines in lung, liver and heart glycogen in all
dietary groups. There was a tendency toward higher rates of hepatic
glycogenolysis in the 12 and 24% glucose diet groups as suggested by
the developmental profile plotted in Figure 2
. By 16 h, lung and
liver glycogen reserves were at their lowest levels. Thereafter, liver
and lung glycogen stores were progressively replenished, whereas heart
glycogen continued to decline, reaching its lowest level at d 15. The
depletion of all of these glycogen reserves simultaneously was
associated with 100% mortality.
|
). As shown in Figure 2
B, pups of the 12, 24 and 60%
glucose diet groups mobilized 42 mg of liver glycogen per gram of
tissue between birth and 16 h postpartum, whereas pups of the 0%
glucose diet-fed dams mobilized only 14 mg/g during the same period.
Contrary to what might have been expected from the lower I:G ratio
(Fig. 1
B), liver glycogen mobilization did not occur at a
faster rate in the 0% glucose group, despite the greater metabolic
distress observed in these pups. This could be explained by the absence
of an increase in glycogen phosphorylase a activity in pups in the 0
and 12% glucose diet groups; this increase was observed in pups of the
24 and 60% glucose diet groups (Fig. 3)
. Although the postnatal rise
in phosphorylase a activity was significantly delayed by 6 h in
pups of dams fed the 12% glucose diet, the activity of phosphorylase a
in pups from dams fed all glucose-containing diets was significantly
higher by 12 h postpartum compared with that of pups from dams fed
the glucose-free diet. The subsequent postnatal accumulation of liver
glycogen between 12 and 16 h and the third postnatal day was
consistent with a significant increase in hepatic synthase a activity
such that significantly higher synthase a activities were observed in
the control (60% glucose) diet group compared with either the 12 or
24% glucose-restricted diet groups. However, significant differences
in glycogen reserves were not detected at d 15 (Fig. 2)
.
|
In neonates, the onset of gluconeogenesis, which occurs concomitant
with the depletion of liver glycogen stores, is an important
glucoregulatory mechanism. PEPCK mRNA levels were measured at 12–16 h
and 3, 6 and 15 d postpartum; qualitative and quantitative results
are presented in Figures 4
A
and B, respectively. PEPCK mRNA could not be detected
before birth, even in the most compromised fetuses (data not shown).
PEPCK mRNA was detected at 12–16 h postpartum (Fig. 4
A), and there was a significant difference among
dietary groups (Fig. 4
B). At that time, the levels of
PEPCK mRNA in the pups of dams fed 0 and 60% glucose were not
different, but were significantly lower (P < 0.05)
than those levels measured in the pups of dams fed 12% glucose. Pups
from the 24% glucose group had intermediate values. Interestingly, the
lower I:G ratio in neonates from the 0% glucose diet group was not
associated with higher PEPCK mRNA, whereas in the 12% glucose group,
the lower I:G ratio was associated with PEPCK mRNA levels that were
significantly greater than in controls.
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Plasma lactate and ß-OHB profiles are shown in Figures 5A
and B. At birth, pups from the 0% glucose diet-fed
dams had significantly higher circulating plasma lactate and ß-OHB
levels. Plasma lactate decreased progressively during the first 16 h postpartum in all dietary groups. This represented a 95% reduction
in the 0% glucose group pups and only a 50% drop in the other dietary
treatments. On postnatal d 1, 3, 6 and 15, there was no significant
change in plasma lactate levels and no significant difference among the
dietary groups.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 1990
) and in dogs (Romsos et al. 1981
), showing
that dietary carbohydrate restriction during pregnancy is incompatible
with postnatal survival, but importantly demonstrated that inadequate
maternal dietary glucose interfered with the normal development of
neonatal glucoregulatory pathways. Birth to 16 h mortality.
Transient hypoglycemia without mortality was observed in all pups born
to dams fed the glucose-containing diets. In sharp contrast, pups from
dams consuming the glucose-free diet had persistent hypoglycemia, and
neonatal mortality occurred in all pups born to dams deprived of
dietary glucose. In part, we attributed the hypoglycemia and early
death to the neonate's insufficient accumulation of tissue glycogen
reserves, particularly liver glycogen, whose importance to neonatal
glucose homeostasis has been largely demonstrated (Girard et al. 1992
, Shelley 1961
, Snell and Walker 1973
). The lack of sufficient glycogen reserves in the
glucose-deprived pups interfered with their ability to withstand this
first transition period until de novo glucose and/or feeding was
initiated.
Neonates normally adjust to postpartum hypoglycemia with an overall
decrease of glucose utilization by switching to alternate substrates.
The acute and marked decline in this study of the high circulating
levels of plasma lactate and ketones during the first 6 h
postpartum supported this observation. The rapid postnatal removal of
lactate shown here and by others (Juanes et al. 1986
,
Medina 1985
) suggested that lactate utilization by
neonatal rat lung (Patterson et al. 1986
) and brain
(Arizmendi and Medina 1983
), before the onset of
glycogenolysis, were contributing, as expected, to glucose sparing. In
vitro studies have shown preferential utilization of lactate as a
substrate over glucose and ketones by brain (Fernandez and Medina 1986
, Shambaugh et al. 1977
) and liver
(Almeida et al. 1992
) slices. However, despite higher
postpartum concentrations and plasma extraction of these substrates,
our data indicated that neither lactate nor ß-OHB sufficiently spared
glucose and overcame the hypoglycemia and mortality generated by the
0% glucose diet. Thus, for immediate postpartum survival, fetal liver
glycogen reserves may still be the most critically important factor.
It is possible that other glycogen reserves could play an unrecognized
role in the early postpartum period. Not unlike liver glycogen, lung
and cardiac glycogen concentrations of our newborns were also depressed
as a result of complete maternal dietary glucose restriction.
Massaro et al. (1986)
reported delayed glycogen
mobilization in type II alveolar cells of lungs of newborn rat pups
from dams with severely restricted access to food during the last
5 d of pregnancy. Lung glycogen may provide energy to support cell
division (Sorokin 1961
) and surfactant synthesis
(Bourbon et al. 1982
) required for postnatal survival.
Additionally, the neonatal heart requires a transition period from a
predominantly glucose-dependent organ in utero to that of an adult,
which relies on fatty acid oxidation (Riva and Hearse 1991
). Rat fetuses are more resistant to hypoxia than newborn
rats, which in turn are more resistant than 7-d-old newborns as a
result of higher heart glycogen (Hoerter and Opie 1978
).
In our study, heart glycogen levels of pups from dams fed 12, 24 and
60% glucose dropped during the first 6 h and remained stable
thereafter, whereas the large and prolonged heart glycogen mobilization
observed in pups from dams fed 0% glucose implied that the myocardium
remained dependent on the diminishing supply of glucose as energy
substrate. This may represent an example of developmental delay
associated with the growth retardation induced by maternal glucose
restriction.
In contrast to other models that disturb glucose delivery such as
maternal uterine artery ligation (Bussey et al. 1985
,
Marconi et al. 1993
, Pollack et al. 1979
), maternal hyerinsulinemia (James et al. 1990
, Ogata et al. 1987
) and maternal fasting
(Girard et al. 1973
, Kliegman 1989
), our
data showed that pups deprived of dietary glucose had limited ability
to counteract the severe hypoglycemia despite appropriate changes in
the insulin to glucagon ratio. In vivo and in vitro studies have
previously shown that the abrupt fall of insulin concomitant with an
increase in plasma glucagon was responsible for the stimulation of
hepatic phosphorylase activity (Biondi and Viola-Magni 1977
, Kawai and Arinze 1981
, Margolis 1983
) and the induction of PEPCK (Girard et al. 1992
) after increased intracellular cAMP concentrations
(Girard 1990
, Lyonnet et al. 1988
,
Mayor and Cuezva 1985
). For glycogenolysis, the onset
reportedly does not occur until 3 h postpartum (Cuezva et al. 1985
, Kawai and Arinze 1981
, Snell and Walker 1973
). This delay has been attributed to the
transient resistance by neonatal liver to the effects of glucagon
(Blazquez et al. 1976
, Kawai and Arinze 1981
, Snell and Walker 1978
). Similarly, liver
gluconeogenesis is initiated a few hours after birth, reaches adult
values within 12 h postpartum and is the dominant factor required
to maintain normal blood glucose levels at this time (Girard et al. 1992
, Mayor and Cuezva 1985
, Nehlig and Pereira de Vasconcelos 1993
, Pearce et al. 1974
). It is promoted by changes in the insulin to glucagon
ratio. In our study we found no increase in hepatic glycogen
phosphorylase activity in the most severely glucose-deprived pups
during the first 16 h postpartum, whereas increases in
phosphorylase a activity were observed in the offspring from the
glucose-containing (12, 24 or 60%) dietary groups. Additionally,
although PEPCK mRNA was detected at 12–16 h in all dietary groups,
significantly higher amounts were observed in the 12% dietary group,
but not the 0% dietary group, which also had a low I:G ratio compared
with the 12% dietary group. Thus, the increase in liver glycogen
phosphorylase activity required for liver glycogen mobilization in the
hours after birth (Bashan et al. 1979
, Margolis 1983
) and the induction of cytosolic PEPCK, both of which are
key factors to sustain glucose production in the newborn (Girard 1990
, Girard et al. 1992
), did not occur in our
glucose-deprived pups. We suggest that this failure might represent a
maturational delay of the glucoregulatory mechanisms in response to
dietary glucose deficiency, but additional experimentation is required.
During the first neonatal day, surviving pups whose dams had been fed 12 and 24% glucose showed signs of metabolic adaptations to the reduced availability of glucose with more active hepatic glycogenolysis as suggested by the steeper slope between birth and 16 h. During this time, which corresponded to the nadir hepatic glycogen concentration, hepatic PEPCK mRNA expression was greater in the pups from dams fed 12 and 24% glucose. This up-regulation of gluconeogenesis, suggested by the higher PEPCK mRNA expression, may explain why these pups survived their first day of life without mortality and strongly suggests that dietary glucose is critically important for PEPCK induction.
Why the normal hormonal signals failed to induce the appropriate
regulatory mechanisms is open to speculation. In our study, complete
dietary deficiency of glucose resulted in intrauterine growth
retardation (IUGR). Small-for-gestational-age (SGA) newborn infants
have an increased incidence of hypoglycemia (Lubchenco and Bard 1971
) and elevated lactate and alanine plasma levels,
suggesting a developmental delay in the induction of postnatal
gluconeogenesis (Haymond et al. 1974
). IUGR rat pups
also have diminished liver glycogen concentration, but our study is the
first to report that glycogen phosphorylase a activity is also reduced
in IUGR pups born to dams fed a glucose-free diet and to suggest that
the decreased body weight may have delayed the postnatal induction of
hepatic phosphorylase a and produced low levels of steady-state PEPCK
mRNA. Previously, IUGR had been associated with delayed or no hepatic
PEPCK activity, which had been attributed to a diminished hepatic
sensitivity to glucagon stimulation (Bussey et al. 1985
). Other researchers have described the failure of IUGR
newborn rats to maintain euglycemia, despite markedly elevated glucagon
and depressed insulin concentrations, after uterine artery ligation
(Bussey et al. 1985
) and insulin-induced maternal
hypoglycemia (Ogata et al. 1987
). Hence, the
significantly reduced birth weight, the lack of responsiveness to
insulin and glucagon, the delayed induction of hepatic phosphorylase a
and low levels of PEPCK mRNA observed in the neonates whose dams were
fed the 0% glucose diet suggested that a glucose-free diet, despite
adequate energy and protein intake, induces severe metabolic
disturbances similar to those induced by complete nutrient deprivation
such as maternal food deprivation or uterine artery ligation.
Day 1 and 3 mortality.
The elevated mortality observed on d 1 and 3 in neonates from dams of
the 12 and 24% glucose diet groups demonstrated that not all postnatal
mortality can be explained by the effect of dietary glucose on glycogen
accumulation and/or fetal growth because there were no significant
difference from controls in weight or glycogen reserves. Moreover, none
of the other metabolic indices showed any difference from controls
except for plasma glucose, which was lower on d 1 and 3 postpartum in
the pups from dams fed 12 and 24% glucose diets and were days on which
we observed mortality. Hence, it is possible that the lower plasma
glucose concentration, although not significant, was insufficient to
meet the brain glucose requirements. The rate of brain glucose
utilization is very low at birth (3–5% of adult) and increases
steadily to 42–48% of adult by d 5 (Nehlig and Pereira de Vasconcelos 1993
). However, during suckling, ketones also
become an important substrate to sustain brain energy and lipid
synthesis demands (Girard et al. 1992
, Nehlig and Pereira de Vasconcelos 1993
, Williamson 1992
).
In our study, there was no difference in the circulating levels of
ß-OHB among the three dietary groups on d 1 and 3. Because the rate
of ketone utilization is largely determined by its concentration in
plasma (Williamson 1992
), differences in circulating
ß-OHB cannot explain the differences in mortality. On the other hand,
we have previously shown that dams fed 12 and 24% glucose diets have
significantly lower milk fat concentration compared with control dams
(Lanoue and Koski 1994
). Therefore, these pups not only
ingested less metabolizable energy, but most importantly, had lower
fatty acid intake. Fatty acid oxidation stimulates gluconeogenesis in
newborn rats by supplying acetyl-CoA and reducing equivalents
(Ferré et al. 1979
). Suckling rats are critically
dependent on milk fat intake to maintain their gluconeogenesis because
they have little adipose tissue at birth (Girard et al. 1992
). Thus, an effect of diet in maternal lactational
performance is likely to explain some of the later mortality.
In summary, our study demonstrated that complete dietary glucose
deprivation during pregnancy was incompatible with postnatal life and
showed that > 24% glucose is required to ensure neonatal
survival to weaning. Our newborns, in addition to depleted glycogen
reserves, did not demonstrate the appropriate glucoregulatory
mechanisms required after birth. Glycogen phosphorylase a activity did
not occur in the absence of maternal dietary glucose intake and was
delayed by several hours in the glucose-restricted pups. Interestingly,
the lower I:G ratio in neonates deprived of dietary glucose (0%
glucose) was not associated with higher and/or earlier PEPCK mRNA
expression, whereas in the most glucose-restricted (12%) group, the
lowered I:G ratio was associated with significantly higher PEPCK mRNA
expression compared with controls. Taken together, these latter
observations support the recent suggestion that dietary carbohydrate
may have a role independent of insulin on the transcription of the
PEPCK gene in neonatal liver (Hanson and Reshef 1997
).
|
FOOTNOTES |
|---|
2 Abbreviations used: ß-OHB,
ß-hydroxybutyrate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
I:G, insulin to glucagon molar ratio; IUGR, intrauterine growth
retardation; PEPCK, phosphoenolpyruvate carboxykinase; SGA, small for
gestational age.
Manuscript received July 9, 1998. Initial review completed September 8, 1998. Revision accepted January 5, 1999.
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REFERENCES |
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|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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