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Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Cientificas-Universidad Complutense de Madrid), Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain; and Laboratoire de Physiopathologie de la Nutrition-Centre National de la Recherche Scientifique URA 307 (E.B., B.P.), F-75251 Paris Cedex 05, France
Address all correspondence and requests for reprints to: Dr. A. M. Pascual-Leone, Instituto de Bioquímica (Consejo Superior de Investigaciones Cientificas-Universidad Complutense de Madrid), Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain. address: apascual{at}eucmvx.sim.ucm.es
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Introduction |
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Materials and Methods |
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On day 21.5 of pregnancy, mothers were put under pentobarbital anesthesia (4 mg/100 g BW), and fetal blood was obtained after axillary artery incision of fetuses when they were still connected to the maternal circulation; serum was separated and stored frozen at -20 C until analyzed. Fetuses were individually weighed, and pancreases were dissected, weighed, and either fixed for histological examination or extracted for determination of insulin content (see below).
All studies were conducted according to the principles and procedures outlined in the NIH Guidelines for the Care and Use of Experimental Animals.
Glucose tolerance test
Glucose tolerance tests were performed in 14–21 U mothers and
in control pregnant mothers fed ad libitum under
pentobarbital anesthesia (4 mg/100 g BW) on day 21.5 of gestation.
The animals were weighed, and glucose was injected in the saphenous vein at a dose of 2 g/kg BW. Blood was withdrawn from the tail vein before and 10, 15, 20, 30, 60, and 120 min after the glucose injection. Serum glucose and insulin were determined in 150-µl aliquots taken at each time point, representing a total of 1050 µl blood removed from the animal throughout the test. Also calculated were the integrated glucose and insulin responses, which are the incremental values above basal levels of their respective concentrations over a period of 120 min after glucose injection. Serum was separated and stored frozen at -20 C until analyzed.
Isolated islets of pregnant rats
Islets were isolated by the collagenase digestion procedure of
Malaisse-Lagae and Malaisse (9). Briefly, islets were isolated
from the pancreases of two or three rats and subsequently separated
from the remaining exocrine tissue by hand-picking under a dissecting
microscope. The islets were immediately used for experiments.
Collagenase from Clostridium histolyticum was purchased from
Boehringer Mannheim (Mannheim, Germany). Hanks’ Balanced Salt Solution
saturated with 95% O2-5% CO2 was used during
the isolation procedure.
Islet culture and isolation
Islets from fetal rats (21.5 days gestation) were obtained
according to the method of Hellerström et al. (10).
Briefly, 20–24 pancreases were minced in sterile Hanks’ Balanced Salt
Solution. The fragments were transferred into a sterile vial containing
Hanks’ Balanced Salt Solution supplemented with 5–7 mg collagenase
(Boehringer Mannheim). The vial was shaken for 10 min at 37 C, and the
tissue digest was washed three times with Hanks’ Balanced Salt
Solution. The pellet was resuspended in culture medium and transferred
to 4–6 plastic dishes containing the same medium. The culture medium
consisted of RPMI 1640 (ICN, Nuclear Iberica, Spain) with 200
mM L-glutamine, penicillin, and streptomycin
and 10% FBS (ICN). The islets were maintained at 37 C in an atmosphere
of 5% CO2, and the medium was renewed every 48 h.
After 7–8 days in culture, the islets were gently detached from the
plates, and clean islets were individually transferred, under a
dissecting microscope, to the incubation vials. Reversal effects in the
islets after 8 days of culture can be disregarded because all
conditions were compared to control plates cultured for the same
period.
Insulin content and insulin release from isolated pancreatic
islets
For the study of ß-cell secretory function, islets in groups
of seven were incubated for 90 min at 37 C in plastic microbeakers
placed in sealed glass vials and containing 1.0 ml Krebs-Ringer
bicarbonate medium (10) equilibrated against a mixture of 95%
O2-5% CO2 and supplemented with 5.0 mg/ml BSA
(fraction V, Sigma Chemical Co., St. Louis, MO) and one of the
following: 2.8 mM glucose, 2.8 mM glucose plus
19 mM arginine, 16.7 mM glucose, 16.7
mM glucose plus 19 mM arginine, or 10
mM leucine. At the end of the incubation period, aliquots
of the medium were stored at -20 C until assayed for insulin using the
RIA described below. Supraphysiological doses of arginine were used in
islets from both control and undernourished rats to prove unmistakably
the differences between the groups.
Groups of 20 freshly isolated islets were sonicated in acid-ethanol (1.5 ml 12 M HCl/100 ml ethanol) and stored at -20 C for determination of insulin content, which was measured by RIA as described below.
Analysis of insulin and glucose
The total insulin content of the pancreas was determined
according to the method of Best et al. (11). Whole glands
were minced and disrupted ultrasonically in acid-ethanol (1.5 ml 12
M HCl/100 ml ethanol) in a ratio of 10 ml/g pancreas for
pregnant animals and 0.5 ml/g total for fetuses. The disrupted glands
were extracted overnight at 4 C and centrifuged, and the supernatant
was stored at -20 C until analyzed.
Immunoreactive insulin in serum samples and pancreatic and islet extracts were measured with purified rat insulin as standard (Novo Nordisk, Bagsvaerd, Denmark), antibody to porcine insulin, and monoiodinated 125I-labeled human insulin. Charcoal was used to separate free from bound hormone, a method that allows the determination of 3 µU/ml (0.12 ng/ml) with within- and between-assay coefficients of variation of 10%.
Aliquots of 10 µl obtained from 30 µl Ba(OH)2-ZnSO4 deproteinized blood were used to determine glucose by a glucose oxidase method (Boehringer Mannheim).
Immunocytochemistry and morphometry
Pancreatic glands for light microscopic investigation were
obtained from pregnant rats on day 21.5 of pregnancy and from their
fetuses. The pancreatic glands were weighed and fixed by immersion in
Bouin’s fluid. The fixed tissue was embedded in paraffin and then
sectioned into 7-µm thick sections and mounted on glass slides.
Previous work had revealed that to prevent random differences due to
regional variation in islet cell distribution, 8–10 sections of each
gland from the pregnant mother and 6–8 sections of each gland from the
fetus should be analyzed. To obtain such samples, whole glands were
sectioned, and only 1 section of 100 (pregnant mothers) or 28 (fetuses)
was mounted and stained. This procedure yielded about 10 sections from
each adult pancreas and 8 sections from each fetal pancreas. Insulin
was stained in deparaffinized sections according to the methods of
Avrameas et al. (12) and Michel et al. (13). The
primary antibody was raised in guinea pigs against bovine insulin
(ICN). Secondary antibodies and peroxidase-antiperoxidase complexes
were purchased from Dako (Copenhagen, Denmark). The immunostained
sections were lightly counterstained with hematoxylin, dehydrated, and
mounted.
Pancreatic ß-cell volume density or the volumetric fraction of ß-cells in the pancreas was measured by planimetric analysis in an image analysis system (Imagenia 2000, BIOCOM, Les Ulis, France) fitted with an Olympus microscope (Olympus Corp., New Hyde Park, NY). The sections were first scanned at a magnification of x32 to determine the total area of pancreatic tissue and the area of nonpancreatic tissue. The sections were subsequently scanned at a magnification of x100, and the area of insulin-positive cells was measured.
Statistics
Values are given as the mean ± SEM for the
number of rats studied. Two-tailed t test for independent
observations was used for comparisons between two populations, and when
four populations were compared, one-way ANOVA followed by the protected
least significant difference test were used.
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Results |
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and 2)). An 8–10% increase in daily food intake was
observed in both 0–7 U and 7–14 U mothers after the onset of
refeeding.
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No changes in glycemia were found in fetuses from food-restricted
mothers, but a significant increase in plasma insulin was observed at
21.5 days of pregnancy in fetuses from 7–14 U and 14–21 U mothers
compared to control fetuses (P < 0.05). In accordance
with this increase in insulinemia, the pancreatic insulin concentration
was higher in fetuses from 14–21 U rats (Table 2
). Fetuses from 14–21
U mothers presented increased plasma insulin vs. fetuses
from 7–14 U mothers and increased pancreatic insulin concentration
vs. fetuses from both 0–7 U and 7–14 U animals.
Glucose tolerance and insulin secretory response to glucose in
pregnant rats in vivo (Fig. 1)
Basal blood glucose concentrations were similar in control
and 14–21 U mothers, and glucose injection caused an increase in blood
glucose levels of both groups, reaching a peak at 10 min. Blood glucose
was significantly higher in 14–21 U pregnant animals than in controls
15, 20, 30, 60, and 120 min after glucose injection. The integrated
glucose response was significantly greater in 14–21 U than in C rats
(Fig. 1A).
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In vitro insulin secretion from pregnant rat islets
In the islets of control and 14–21 U animals, a rise in the
glucose concentration to 16.7 mM caused an increase in
insulin output above the basal value (2.8 mM glucose), and
the association of arginine (19 mM) and glucose (2.8
mM or 16.7 mM) also stimulated insulin
secretion. Although the insulin content of the islets (4945 ± 241
µU insulin/islet in controls vs. 3177 ± 138 µU
insulin/islet in 14–21 U; P < 0.01) and the insulin
response to glucose in vivo (Fig. 1B) were significantly
decreased in the 14–21 U pregnant animals, the secretory responses
in vitro were not significantly lower in 14–21 U than in C
rats in all cases studied: 2.8 mM glucose (25.5 ± 1.3
µU insulin/islet in controls vs. 23.6 ± 2.8 µU
insulin/islet in 14–21 U), 2.8 mM glucose plus 19
mM arginine (140 ± 8.1 µU insulin/islet in controls
vs. 135 ± 13.2 µU insulin/islet in 14–21 U), 16.7
mM glucose (863 ± 176 µU insulin/islet in controls
vs. 630 ± 128 µU insulin/islet in 14–21 U), 16.7
mM glucose plus 19 mM arginine (1779 ± 95
µU insulin/islet in controls vs. 1532 ± 76 µU
insulin/islet in 14–21 U), and 10 mM leucine (76 ± 8
µU insulin/islet in controls vs. 78 ± 6 µU
insulin/islet in 14–21 U).
It should be pointed out that both the insulin response in vitro and the insulin content of the islets in all groups of pregnant rats were significantly higher than those in adult virgin control rats (14).
Insulin secretion by fetal islets in vitro (Fig. 2)
In agreement with the increase in insulinemia and total pancreatic
insulin concentration, the insulin content of the islets was higher in
fetuses from 14–21 U mothers than that in controls (Fig. 2A). Insulin
release in response to an increase in glucose concentration from 2.8 to
16.7 mM was significantly higher in fetal islets from
14–21 U than in fetal islets from control mothers (25.1 ± 2.3
µU/islet at 2.8 mM vs. 47.8 ± 9.1
µU/islet at 16.7 mM in fetuses from control rats;
43.3 ± 4.7 µU/islet at 2.8 mM vs.
113.8 ± 9.9 µU/islet at 16.7 mM in fetuses from
14–21 U rats; P < 0.01). As expected, when glucose
was combined with 19 mM arginine or the islets were
incubated in the presence of 10 mM leucine, insulin
secretion was significantly potentiated (P < 0.001) in
both groups of islets. Moreover, the output of insulin by fetal islets
from 14–21 U rats was again significantly increased under all
experimental conditions compared to that by fetal islets from control
rats (Fig. 2B).
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and Fig. 3)
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In contrast, ß-cell volume density and the ß-cell mass in fetuses
from 14–21 U mothers were significantly greater than those of fetuses
from control animals (
50% increase; Table 3 and Fig. 3).
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Discussion |
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It is worth considering that fetal pancreas cytodifferentiation occurs around days 14–15 (6, 7, 8), and undernutrition during this period leads to different important metabolic alterations of the normal adaptation of the mother to pregnancy, which takes place mostly during the third week of gestation. Pregnant rats undernourished during the third week of pregnancy show a reduced body weight and no changes in either basal glycemia or plasma insulin concentration vs. fed pregnant rats. However the tolerance to glucose was impaired, the integrated response of glucose was increased, and the response of insulin and islet insulin content were decreased in undernourished pregnant rats compared to those in fed pregnant rats. Thus, a functional diabetes-like situation seemed to be established in this group of undernourished pregnant rats, a possible explanation of which is offered in the following paragraph.
In normal pregnancy, especially in the third week of gestation, circulating substrate levels are kept high so that abundant supply is available to the growing fetus. Glucose tolerance is normal or only slightly impaired, but at the cost of increased insulin secretion (15, 16, 17, 18), which compensates for an increased peripheral resistance to the effects of insulin (16, 18, 19). To meet this increased demand for insulin production, an increased sensitivity of insulin biosynthesis and secretion to glucose is observed during the last stages of pregnancy (20, 21). Thus, pregnant rats, either undernourished during the third week or normally fed, showed increased insulin response and islet content in vitro compared to adult virgin rats (14). However, it has been previously shown that 70-day-old virgin rats, either undernourished from 16 days gestation (22) or food restricted for a limited period (23), present impairment of the in vivo insulin secretory response to glucose. This adaptation of the rat to undernutrition appears to be opposed to that described for rats during the last week of pregnancy; this could explain the results for insulin secretion and glucose homeostasis found in pregnant undernourished rats, which have to tolerate both conditions, pregnancy and undernutrition. However, islets from undernourished pregnant rats showed both an in vitro response upon stimulation with glucose and amino acids and a ß-cell mass similar to those of fed pregnant rats. Although the glucose intolerance observed in undernourished pregnant rats could be caused by a complex process involving multiple factors, some of which have not been studied in this work, the lack of functional adaptation of the ß-cells to such a complex situation, pregnancy and undernutrition, could be a decisive factor. In summary, pregnant rats undernourished during the third week of gestation present at the end of gestation not a true diabetic situation but, due to dual adaptation to food restriction and pregnancy, a transient functional diabetic-like situation. This situation may be caused by undernutrition-induced changes in estrogens, cortisol, and placental lactogen, which seem to mediate the elevated insulin secretory response and ß-cell hyperplasia in the islets of Langerhans described during the last week of normal pregnancy (24). As pregnancy is an adaptive process, different degrees of adaptation to pregnancy during the 3 weeks of gestation rather than the influence of refeeding, which is absent in the group undernourished during the third week, would probably explain the minor alteration in glucose homeostasis observed in pregnant rats undernourished during the first 2 weeks of gestation.
In the present report, fetuses from pregnant rats undernourished during the third week of gestation show at 21.5 days an increase in the plasma concentration and pancreatic content of insulin together with an increase in pancreatic ß-cell mass. Interestingly, the secretory function of ß-cells in these fetuses is also increased compared to that in fetuses from fed pregnant rats. This result differs from that previously described by other researchers (3, 4), who observed an impaired activity of pancreatic ß-cell in fetuses from pregnant rats submitted to a low protein diet. However, in most of these studies the effect of food restriction on insulin secretion in the pregnant rat was not investigated, and as food intake was reported to be isocaloric (3, 4) (contrary to the hypocaloric diet used in our present work), the transient functional diabetic-like situation described above might not be present in the pregnant rats. The effects of a hypocaloric food restriction milder than 65% of the diet on insulin secretion in pregnant rats remain the subject of further investigation.
The results obtained with our experimental approach show a greater
response to amino acids in islets of fetuses from undernourished
pregnant rats and, in agreement with previous experiments with fetuses
under normal conditions (25, 26), a reduced response of insulin to a
high concentration of glucose compared to that in normal adult animals.
Although several in vitro studies show that glucose is not
effective on insulin release from the fetal pancreas in humans and rats
(8), in the present study fetuses from protein-caloric undernourished
pregnant rats show a greater insulin response to glucose than those
from fed pregnant animals. Such an enhanced insulin response in islets
of fetuses from undernourished pregnant rats bears some similarity with
the report that glucose infusion to pregnant rats during the last third
of gestation increases the plasma concentration and pancreatic content
of insulin in the fetuses and induces an elevated response of fetal
islets to glucose (27). The transient functional diabetes-like
situation in the undernourished pregnant could generate postpandrial
hyperglycemic episodes despite basal normoglycemia, and such short
periods of hyperglycemia during the developmental period in the rat may
functionally disturb the ß-cell by speeding up fetal pancreas
sensitivity to glucose. Although the data from the glucose tolerance
test depicted in Table 1 cannot prove this hypothesis, studies to
confirm such a suggestion are currently in progress in our laboratory.
The results obtained in fetuses from the pregnant rats undernourished
during the third week of gestation show the importance of the changes
in maternal metabolism on the developmental period of the fetal
pancreas. These results show that transient metabolic alterations in
the mother may have profound effects on the insulin secretion of the
fetus at various stages of pancreatic development. Further studies are
needed on the consequences of such altered functional development in
adulthood, a fact recently described in the offspring from rats
undernourished during early stages of life (28).
It has been reported that severe diabetes of the mother provokes a poor development of the fetal endocrine pancreas (29, 30, 31, 32), and considerable hyperglycemia and wasting of the mother causes fetal growth retardation (33, 34, 35, 36, 37, 38, 39), but, according to our results, mild diabetes with little alteration of glucose tolerance in mothers leads to an increase in ß-cell mass and increased endocrine tissue (40). However, few studies have been published dealing with the fetal consequences of a mild disturbance of maternal glucose homeostasis. Development of the fetal pancreas depends on a balanced glucose homeostasis in the pregnant rat, which seems not to be directly related to structural changes of the maternal pancreas during gestation, but, rather, to the functional metabolic adaptation of the mother, a fact that should be considered to prevent fetal alterations in populations at risk.
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Acknowledgments |
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Footnotes |
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Received December 2, 1996.
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, 14–21 U mothers. A, Serum glucose and
mean incremental plasma glucose area (
G). B, Serum insulin and mean
incremental plasma insulin area (
, Control mothers; 
,
14–21 U mothers. Values are given as the mean ± SEM
from 12 rats/group. Significance of difference between C and 14–21 U
mothers (by two-tailed t test): *,P < 0.01; **, P < 0.001.
