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INSERM U 457, Hôpital Robert Debré, Paris, France
1Correspondence: INSERM U 457, Hôpital Robert Debré, 48 boulevard Sérurier, 75019 Paris, France. E-mail: bbreant{at}infobiogen.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONANIMALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: malnutrition • ß-cell mass • proliferation • morphometry
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONANIMALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. The hypothesis of altered intrauterine pancreatic
ß-cell development has been suggested (5)
.
The formation of new ß cells in the pancreas is the result of a dual
process: differentiation of ductal precursor cells that further
assemble into mature islets, a process also called neogenesis, and
proliferation of preexisting differentiated ß cells (reviewed in refs 6
7
8
). It is generally admitted that neogenesis mostly takes place
during fetal and neonatal life, but neogenesis from the ductal
epithelium is feasible in the adult gland after partial pancreatectomy
(9)
, duct ligation (10)
, cellophane wrapping
(11)
, or in transgenic mice whose ß cells are induced to
express 
-interferon (12)
. In the absence of an external
stimulus, the low mitotic activity allows for ß-cell expansion after
weaning and until adult age (6
7
8)
.
We have recently shown that late fetal malnutrition in the rat
decreases ß-cell mass and number at birth (13)
. This
decrease could be attributed to a reduced islet number whereas ß-cell
proliferation rate remained normal, suggesting that general food
restriction impaired neogenesis rather than ß-cell proliferation.
Furthermore, when malnutrition was prolonged until weaning, ß-cell
mass was reduced by 70%, a decrease that was not fully recovered at 3
months of age, despite normal nutrition from weaning onwards
(14)
. This early abnormal development has dramatic
consequences later in life, because ß-cell mass cannot adapt to
situations of increased insulin demand like aging (15)
or
pregnancy (16)
and, as a consequence, glucose intolerance
develops.
To further investigate the early mechanisms responsible for abnormal
ß-cell development that could later compromise glucose homeostasis,
we have used the neonatal streptozotocin (STZ) model, appearing as the
most suitable experimental procedure to study ß-cell regenerative
capacity during the neonatal period. The injection of STZ at birth
destroys 80% of the ß cells thereby inducing a dramatic depletion of
pancreatic insulin stores and a severe hyperglycemic state
(17
18
19)
. However, this diabetic condition is transient.
Indeed, it has been demonstrated by various studies that ß-cell
regeneration that follows the STZ-induced ß-cell destruction involves
both increased ß-cell proliferation and neogenesis (18
, 20
21
22)
. This experimental procedure was applied at birth on
neonates born from food-restricted or normally fed rat dams and
maternal malnutrition was prolonged until weaning, as described
previously (23)
. Beta-cell mass, number, and proliferative
capacity, as well as islet number per tissue surface unit, were
morphometrically determined from birth to weaning in STZ-injected
normal and malnourished pups. Both groups were compared with their
respective noninjected animal group. The results sustain the notion
that malnutrition impairs the potential of ß cells to regenerate by
neogenesis, whereas ß-cell proliferation still occurs at a high rate.
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ANIMALS AND METHODS |
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TOPABSTRACTINTRODUCTIONANIMALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. At parturition, all pups were
immediately weighed, litter size equalized out to eight, and the
neonates with severe intrauterine growth retardation (body weight 
control weight - 2 SD) were selected.
Control pups were not selected (14
, 23)
.
Four groups of animals were studied. Two groups were injected i.p.
6–12 h after birth with a STZ-citrate solution (100 µg/g body
weight): animals born from and nursed by control mothers (C-STZ group)
and animals born from and nursed by food-restricted mothers (R-STZ
group). The animals included in the study showed at day 3 manifest
glycosuria (>300 mg/dl, 16.7 mmol/l) using urinary test strips
(Boehringer Mannheim, Meylan, France). These animals were compared with
noninjected animals from the same group (C and R groups). At each time
point studied (days 1, 4, 7, and 21), the rats from various litters
were randomly chosen. The number of lactating mothers was reduced to
keep nutrient availability constant to the remaining pups (eight per
litter), as described previously (23)
. The animals were
killed by decapitation. The laboratory has an agreement for laboratory
animal care facilities, delivered by the Agricultural Ministery
(authorization no. 7612).
Basal glycemia
After decapitation, blood flowing from opened necks was
collected and basal glycemia was immediately determined with the
glucose oxidase technique using a glucometer One Touch II (Lifescan,
Roissy, France).
Pancreatic insulin contents
Six to seven animals of each group were analyzed. Pancreases
were dissected out, and insulin was extracted with an acid-alcohol
solution and measured by radioimmunoassay as described previously
(23)
.
Tissue processing
The animals (four to five in each group) were injected i.p. with
a dose of 50 µ g/g body weight of 5-bromo-2'-deoxyuridine (BrdU;
Sigma, La Verpillère, France) 1 h before killing. The whole
pancreas was excised and its weight determined. The organ was fixed for
2 h in 4% paraformaldehyde-PBS solution and frozen as described
previously (14)
. Each pancreas was sectioned (6-µm
thick) throughout its length.
Double immunohistochemistry and morphometry
Every 30th section was analyzed (yielding four to five sections
from each pancreas) and double immunostained for insulin and BrdU.
Proliferating cells were detected with a mouse monoclonal anti-BrdU
antibody (Sigma) using the avidin-biotin-peroxidase complex (Amersham,
Les Ulis, France) and visualized in brown with 3, 3'-Diaminobenzidine
(DAB; Sigma) as chromogen. Beta cells were detected with a guinea pig
polyclonal anti-insulin antibody (Dako, Trappes, France) followed by
incubation with an alkaline phosphatase anti-rabbit antibody (Promega,
Lyon, France) and visualized in blue with nitroblue tetrazolium (NBT;
Vector, Compiègne, France). Previous experiments had shown that
these conditions allowed the detection of all insulin-positive cells
(14)
. Controls for immuno-staining consisted in the
omission of the primary antibodies, and these tests resulted in
negative staining reactions.
Pancreatic tissue area, insulin-positive-cell area, individual ß-cell
size, and islet number were determined by computer-assisted
measurements using Leica microscope and software, as described
previously (14)
. Beta-cell fraction in the pancreas was
calculated as the ratio of insulin-positive area to the total area of
the tissue section. Absolute ß-cell mass per pancreas was calculated
as the product of mean ß-cell fraction by the corresponding pancreas
weight. Aggregates composed of more than five immunoreactive insulin
cells (equivalent diameter >25 µm) were counted on each section and
expressed as islet number/cm2. Individual
ß-cell area was manually measured after light counterstaining in at
least 100 insulin-positive cells per group and age. The number of ß
cells in a section was deduced as the ratio of total ß-cell area to
the individual ß-cell area. Beta-cell proliferation was determined as
the ratio of the ß-cell BrdU-labeled nuclei to the total ß-cell
nuclei (BrdU labeling index, LI). Values of ß-cell mass and
pancreatic density (measured as described in ref 14
) were used to
calculate total ß-cell volume. The calculation of ß-cell number per
pancreas was the ratio of total ß-cell volume to the volume of an
individual ß cell, assuming it to a sphere (14
, 24)
.
Evaluation of growth parameters
Based on measured BrdU LI and an estimated S phase duration
(TS) of 6.4 h (25)
, the cell
birth rate (CBR) (i.e., the production of new cells per 24 h) can
be calculated with the equation CBR =
(LI/TS) x 24 h. When BrdU LI was
different between the two time points considered, the mean value of LI
was used for the calculation of CBR (D. Finegood, personal
communication). Using the ß-cell number per pancreas and CBR, the
predicted ß-cell growth by means of proliferation can be calculated
with the following equation: ßCN2 =
[ßCN1 x CBR x
(t2-t1)] +
ßCN1, where ßCN1 is the
number of ß cells at a time before ßCN2 and
t2-t1 is the time (in
days) between the two points, assuming that no cells die during the
period in which the calculation is applied (7
, 26)
. An
observed ß-cell number higher from the one predicted by proliferation
suggests net neogenesis, an observed ß-cell number lower than the
predicted one would suggest net cell death. Identical calculated and
observed values suggest either a defect in the neogenesis process or
that neogenesis occurs but is compensated by cell death.
Determination of Glut2 immunoreactivity at birth
Pancreatic sections were double-immunostained with rabbit
anti-Glut2 (Valbiotech, Paris, France) and mouse monoclonal
anti-insulin antibodies (Sigma), which were respectively revealed with
FITC-labeled anti-rabbit (Immunotech, Marseille, France) and Texas-red
anti-mouse secondary antibodies (Jackson, Marseille, France).
Approximately 350 insulin-positive cells (70 islets) were counted per
animal (n=3), in which the coexpression of Glut2 was
investigated. Cells were classified positive regardless of the
intensity of Glut2 staining (faint staining was quoted positive). Glut2
immunoreactivity was investigated in insulin cells organized in an
islet structure and the results were expressed as number of
immunopositive cells per islet. In the case of islet clusters, defined
as groups composed of less than five insulin-positive cells, we
arbitrarily considered that the cluster was Glut2-positive when half
the cells plus one were positively stained (i.e., greater than or equal
to three in a cluster of five cells).
Statistical analysis
Values are expressed as means ± SD.
Differences between groups were analyzed with StatView 4.5 software,
using unpaired Student’s t test or one-way analysis of
variance when multiple comparison was required.
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RESULTS |
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TOPABSTRACTINTRODUCTIONANIMALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
|
Regeneration of ß-cell mass after destruction by STZ
When compared with the respective noninjected group, 20% of the
ß-cell mass remained at day 4 in C-STZ animals (Fig. 1A
). Beta-cell mass regenerated rapidly to 55% that of the
noninjected controls at day 7, with no further increase until day 21.
In contrast, ß-cell mass from R-STZ animals was 40% that of R
animals at day 4 and regenerated poorly until day 7 and thereafter
(Fig. 1B
).
|
To determine whether fewer ß cells would express the glucose
transporter Glut2 and hence be less susceptible to be destroyed by STZ,
an immunostaining for Glut2 was performed at birth before STZ
injection. The majority of insulin cells organized in islets were
Glut2-positive, both in control and in malnourished neonates
(Fig. 2A
) but the staining was weaker in the malnourished,
suggesting less Glut2 protein per ß cell (refer to the micrographs in
Figure 2
). Furthermore, only 30% of insulin cell clusters, likely to
result from recent neogenesis, were positive for Glut2 in the
malnourished neonates, opposed to 57% in controls (P<0.01,
Fig. 2B
). These observations suggest that malnourished
animals possess more immature Glut2-negative beta cells, not likely to
be destroyed by STZ.
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Individual ß-cell size and total ß-cell number
At day 14, mean individual ß-cell size was reduced in all animal
groups, when compared with the preceding or following time points, and
more so in malnourished animals (Table 2
). STZ treatment significantly diminished ß-cell size by 20% from day
7 onward, both in control and malnourished groups (Table 2)
. As a
consequence, ß-cell number evolved similarly to ß-cell mass in
C-STZ and R-STZ rats (Fig. 1C
, 1D
, respectively).
However, in noninjected R animals, ß-cell number increased twofold
from birth to day 14 and decreased at day 21 (P<0.01, Fig. 1D
). This decrease also occurred in C animals, although it
did not reach statistical significance (P=0.07, Fig. 1C
). The evolution of pancreatic insulin contents paralleled
that of ß-cell numbers in C and R animals. STZ treatment induced a
dramatic exhaustion of pancreatic insulin at day 4 both in C-STZ and
R-STZ rats. From day 7 onward, insulin content increased markedly,
albeit at a slower rate in the R-STZ animals (Fig. 3A
, B
).
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Beta-cell proliferation, neogenesis, and calculation of growth
parameters
To further analyze the mechanisms by which ß-cell recovery was
impaired in R-STZ animals, ß-cell proliferation was measured, as well
as the number of islets/cm2, a reflection of
neogenesis.
In noninjected animals, the BrdU-labeling index in the ß cells
(ß-cell LI) was 3% at day 4 and dropped to 1.5% at day 14. It did
not differ in control and malnourished animals (Fig. 4A
, B
). In STZ-injected animals, ß-cell LI was enhanced when
compared with nontreated animals. In C-STZ group, ß-cell LI increased
slightly and gradually to a maximum at day 7 (5.7±1.2% in C-STZ vs.
2.7±0.5% in C animals, P<0.01, Fig. 5A
). In R-STZ animals, a huge increase was reached at day 4
(11.1±3.6% in R-STZ vs. 3.0±0.5% in R rats, P<0.01),
followed by a modest increase (1.4-fold) at day 7 (Fig. 4B
).
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When ß-cell regeneration reaches its maximal level at day 7,
the number of islet/cm2 of C-STZ rats was similar
to that of C animals, suggesting a significant capacity to regenerate
by neogenesis (Fig. 5A
). In contrast, a 50% diminution of
the islets number/cm2 was observed in R-STZ rats
when compared with R, C, or C-STZ animals (Fig. 5A
, 5P
<0.01), suggesting an impaired capacity of the ß cells
from malnourished animals to regenerate by neogenesis. This finding was
confirmed by calculations of ß-cell growth parameters. In C-STZ
animals, the ß-cell number observed at day 7 was 2.7-fold higher than
that predicted by proliferation, suggesting net neogenesis, whereas in
the R-STZ group the values observed at day 7 were identical to those
predicted by proliferation, suggesting either a lack of neogenesis or
that it was compensated by cell death (Fig. 5B
). In
noninjected C and R animals, the ß-cell numbers predicted by
proliferation were similar to the values observed in the respective
groups at day 7, again suggesting that if neogenesis occurred during
this time period, it was likely to be compensated by cell death.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONANIMALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mortality rate was similar in R-STZ and C-STZ animals over the entire period studied. However, R-STZ neonates died mostly during the 1st wk, whereas C-STZ pups died all along the lactating period. It is possible that the earlier death of R-STZ neonates is a reflection of a higher severity of diabetes, incompatible with survival. Because this study was performed on the animals who did survive and hence partly regenerate their ß-cell mass, it may contribute to some selection bias.
Control STZ-injected animals showed 80% destruction of the ß cells 4
days after the injection, when compared with age-matched noninjected
animals, similar to the decrease already described by others (18
, 20
, 22)
and corresponding to the maximal fall of insulin content
and hyperglycemic state (ref 27
and this study). A rapid but partial
regeneration of ß-cell mass and number was observed at day 7 with no
further increase until weaning, in good agreement with the literature
and explaining the transient character of this experimental diabetes
(20
21
22
, 26
27
28)
. In contrast, 4 days after STZ injection,
malnourished animals still display 40% of the ß-cell mass of
noninjected malnourished pups. Furthermore, it regenerated poorly. In
both malnourished and control animals, the normalization of glycemia
was a result of a partial regeneration of the ß cells from days 4–7,
followed by a phase of stationary cell growth with intense insulin
synthesis from days 7–21. The observation that malnourished neonates
display more Glut2-negative insulin-positive cells and thus a smaller
number of STZ-sensitive ß cells (29
, 30)
is a plausible
explanation for the relatively high ß-cell number remaining at day 4
in R-STZ survivors.
Most studies using the neonatal-STZ model have demonstrated that
ß-cell regeneration involves both increased proliferation of
preexisting ß cells as well as neogenesis. However, the
quantification of the latter remains an open question. As a primary
approach, we have measured the number of islets per surface unit, as a
final reflection of the neogenesis process. This number was similar to
noninjected control animals at days 7 in C-STZ rats, suggesting that
neogenesis occurred normally. Calculations of ß-cell growth
parameters from ß-cell proliferation rates between days 4 and 7
confirmed previous studies attributing 40% of the regenerated ß
cells to increased ß-cell proliferation, indicating thereby that 60%
should be the product of neogenesis (21
, 26
, 28)
. In
contrast, a huge increase in ß-cell labeling index was observed at
days 4 and 7 in malnourished animals. The corresponding cell birth rate
that was calculated could explain 100% of the weak regeneration
observed in this time period, suggesting that there was no net
neogenesis. Two conclusions, albeit not exclusive from each other, can
be drawn. Either neogenesis does occur in the malnourished-STZ animals
but is compensated by cell death, or the animals show a defect in
regenerating by neogenesis. The observation of a 50% decrease in the
islet numerical density at day 7 favors the hypothesis that
regeneration by neogenesis is impaired, at least partly, in the
malnourished-STZ animals, but it cannot be excluded that cell death
could also contribute to some degree in the alteration. Interestingly,
similar abnormalities of ß-cell growth and regeneration after STZ
have been described in the Goto-Kakisaki rat, a model of genetic
diabetes (28)
. Moreover, the data presented in this study
confirm our previous observations that maternal general food
restriction does not reduce ß-cell proliferation rate in the
offspring (13
, 14)
, in contrast to the marked decrease
observed in rat pups whose mothers are fed a low-protein diet
(31
, 32)
. Interestingly, the timing of the huge increase
of ß-cell proliferation rate in malnourished-STZ rats exactly
coincided with the peak of hyperglycemia, suggesting that ß-cell
proliferative response to hyperglycemia would be more pronounced in
those animals, in an effort to compensate for impaired neogenesis.
Recently the new concept has emerged that the ß-cell mass is
dynamic and regulated, both positively and negatively, in a number of
physiological or pathophysiological situations, to maintain euglycemia
(7
, 24
, 33
34
35
36)
. Interestingly, it has been demonstrated
by several laboratories that a wave of apoptosis occurred in normal rat
pancreatic islet cells shortly before weaning (7
, 24
, 34)
,
concurrent with a period where ß-cell growth was stationary. Our
previous observation that ß-cell mass did not increase between birth
and weaning in malnourished R animals despite normal ß-cell
proliferation (14)
suggested that malnutrition could
increase the incidence of ß-cell death. Such an increase has recently
been described in a model of protein malnutrition (32)
but
has not been investigated in the present study. The juxtaposition in
the timing of the apoptotic wave described by others with the reduction
of ß-cell proliferation rate and size at day 14 postnatal suggests
that those cells could be new ß cells, actively synthesizing insulin.
Interestingly, similar observations have been described in another
situation where a remodeling of the ß-cell mass appears necessary,
during the involution of ß-cell mass in the postpartum rat pancreas
where increased apoptosis is associated to decreased ß-cell size and
proliferation rate (33)
.
In conclusion, fetal malnutrition decreases the number of Glut2-positive insulin cells at birth, suggesting that poor glucose supply to the fetus impairs the differentiation of precursor cells on their path to become fully mature ß cells. How malnutrition affects the differentiation of the endocrine pancreas remains to be elucidated. Early malnutrition decreases ß-cell neogenesis and is likely to impair the capacity to regenerate by neogenesis. It does not decrease ß-cell proliferation, which remains therefore the elective choice to increase ß-cell mass in malnourished animals. These early alterations are likely to be responsible for the previously described incapacity of ß-cell mass to adapt to later situations of increased insulin demand such as aging or pregnancy that aggravate glucose tolerance. Experimental research in animals supports the notion of causal link between undernutrition in utero, leading to alterations in fetal growth, and increased risk of chronic degenerative diseases such as diabetes later in life. These findings in the rat raise the question as to whether the programming of such a disease can also originate from early ß-cell defects in humans born with intrauterine growth retardation and at high risk of developing diabetes.
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ACKNOWLEDGMENTS |
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Received for publication January 27, 2000.
Revision received May 18, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONANIMALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-transgenic mice. Development 118,33-46[Abstract]
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