Crucial role of milk-borne insulin in the development
of pancreatic amylase at the onset of weaning in rats
TOSHI KINOUCHI, KYOKO KOIZUMI, TAMOTSU KUWATA, AND TAKAJI YAJIMA
Laboratory for Gastrointestinal Function, Department of Nutritional Research,
Nutrition Science Institute, Meiji Milk Products Company, Limited, Odawara 250-0862, Japan
dietary change; digestive function; milk formula; artificial
rearing
MAMMALS ENCOUNTER a significant change in the composition of their diet at the time of weaning, coinciding
with alterations in their digestive functions. Rat pups
start to chew a solid diet at 15 days of postnatal age,
and the gradual change from an exclusively milk diet to
a solid diet rich in carbohydrates begins at 17 days of
age (8). In addition to the changes in the proportions of
nutrients, the nature of the carbohydrates changes:
namely, milk has lactose as the principal carbohydrate,
whereas a solid diet has starch (5, 8). The activity of
pancreatic amylase, the main enzyme in complex carbohydrate digestion, is low in the preweaning rat pancreas and increases sharply at the time of weaning (8,
14, 16, 27). The factors that cause the development of
pancreatic amylase remain to be elucidated.
In adult rats, the pancreas adapts to individual
nutrients; e.g., a prolonged high-carbohydrate intake
leads to an increased concentration of amylase in the
pancreas (4). It is therefore possible that the development of pancreatic amylase during the 3rd wk of life in
rats is the result of dietary changes (14, 27). However,
there is growing evidence that the initiation of onto-
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R1958
genic changes in various gastrointestinal functions,
such as disaccharidases, may not be dependent on the
dietary change at the time of weaning (8), suggesting
that weaning may not be responsible for the development of amylase activity (3, 15). We are interested in
whether the dietary change in weaning rats influences
the developmental changes of pancreatic amylase.
Even if dietary changes are the cause of the increase
in pancreatic amylase during the weaning period,
hormonal regulation may also participate in the increase in amylase activity (1, 5, 17). It is well known
that insulin is the most important regulator of pancreatic amylase expression (13) and a potential mediator
of the pancreatic amylase adaptation to a high carbohydrate diet in adult rats (4). Because the plasma insulin
concentration is low in preweaning rats and begins to
increase in the third postnatal week (5), insulin may
play a key role in the ontogenic changes of pancreatic
amylase. The developmental changes in the rat plasma
insulin concentration during the third postnatal week
have not yet been compared with those of pancreatic
amylase.
A number of hormones and trophic factors have been
found at high concentrations in mammalian maternal
milk (11, 22, 31). In suckling rats, the permeability of
the gut to macromolecules is a recognized phenomenon
(6, 19, 20, 24, 28–30), and there is evidence that intact
large peptides in milk, such as insulin or epidermal
growth factor, pass across the intestinal epithelium
into the systemic circulation (11, 19). As shown in the
present study, rat milk also contains insulin at a high
concentration, suggesting that the milk may be a
source of insulin for suckling rats. It is not yet known
whether insulin in the milk is absorbed from the gut in
rat pups or whether it has any effect in peripheral
organs, including the pancreas.
The aim of the present study was to explore the
factors that regulate the developmental change of
pancreatic amylase in weaning rats. We examined the
association between the development of amylase activity and the dietary change and/or hormonal regulation.
We also investigated the potential role of insulin in
milk in the development of pancreatic amylase activity.
MATERIALS AND METHODS
Animals. Pregnant Sprague-Dawley rats were purchased
from Japan SLC (Shizuoka, Japan) and housed in separate
cages under controlled temperature (25 6 2°C), humidity
(55 6 2 % relative humidity), and light (lights on 0700–1900)
conditions. The day of birth is referred to as day 0. The litter
sizes were adjusted to 10 pups at 3 days of age to maintain a
standard nutritive status. During lactation, the dams were
fed ad libitum with powdered food (CA-1; Japan Clea, Tokyo,
0363-6119/98 $5.00 Copyright r 1998 the American Physiological Society
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Kinouchi, Toshi, Kyoko Koizumi, Tamotsu Kuwata,
and Takaji Yajima. Crucial role of milk-borne insulin in the
development of pancreatic amylase at the onset of weaning in
rats. Am. J. Physiol. 275 (Regulatory Integrative Comp.
Physiol. 44): R1958–R1967, 1998.—The development of pancreatic amylase activity was examined in rats fed in regular
cages or in special cages, designed so the pups could not reach
solid food to prevent weaning. In both groups, the amylase
activity in zymogen granules increased in rat pups aged 14
days, peaked at 18 days, and thereafter remained at a 1.6-fold
higher level than at 14 days of age. An increase in the plasma
concentration of immunoreactive insulin preceded the increase of amylase activity, whereas the plasma concentration
of C-peptide, indicating the secretion rate of endogenous
insulin, remained unchanged. The administration of insulin
at 20 ng/ml (the physiological concentration) in the milk
formula caused an increase in the plasma insulin concentration of 17-day-old pups. In addition, increased pancreatic
amylase activity was observed in 17-day-old rats raised on
milk formula to which insulin was added. We propose that the
increase of amylase activity at the beginning of weaning is
dependent on the milk-borne insulin and not on the dietary
change in rats.
MILK-BORNE INSULIN REGULATION OF AMYLASE DEVELOPMENT
administration. In the artificial rearing (AR) experiments, an
intragastric cannula was implanted into rat pups at 12 days
of age, and the rat pups were given the milk formula with 0 or
20 ng/ml rat insulin until 17 days of age. The volume of the
formula given hourly to the pups was gradually increased
every day at noon from 0.22 ml on day 12 to 0.30 ml on day 16.
Preparations. All of the rat pups were killed by draining the
blood with a syringe from the abdominal aorta under anesthesia with ether within 30 min after separation from the dams
or the last administration of the milk formula in the morning
(0900–1100), except in the experiments on the time course
(0900–1500). The blood was collected in tubes containing
heparin. Plasma was separated and stored at 240°C until
assayed. For the preparation of zymogen granules, the pancreas was quickly removed and homogenized in an ice-cold
homogenization buffer: 0.25 M sucrose, 5 mM MOPS, and 0.1
mM MgSO4, pH 7.0. The homogenate was centrifuged at 150
g for 10 min to remove unbroken cells and nuclei. The
supernatant was recentrifuged at 1,300 g for 10 min. The
white layer at the bottom was suspended in 0.15 M Tris · HCl
(pH 8.2) containing 0.01 M CaCl2 and then briefly sonicated to
break the membranes of the zymogen granules. The suspension was centrifuged at 3,000 g for 5 min, and the resultant
supernatant was frozen at 240°C until used for the determination of the pancreatic enzyme activity. For the measurement of the insulin content, the pancreases were immediately
frozen after removal and stored at 280°C until assayed.
Assays. Blood glucose concentrations were measured with
a Glucose CII-Test Wako (Wako) based on a mutarotaseglucose oxidase method. Trypsin activity was determined
with benzoyl-arginine p-nitroanilide (Sigma, St. Louis, MO)
as a substrate, after activation with enterokinase at 37°C for
40 min. Crystalline porcine trypsin (Sigma) was used as a
standard. One unit of trypsin activity was defined as 1 µmol
p-nitroaniline liberated per minute at 37°C. Amylase activity
was determined with a Diacolor-Amy kit (Toyobo, Osaka,
Japan) based on a method involving 2,4-dichlorophenyl
b-maltopentoside as a substrate. One unit of amylase activity
was defined as 1 µmol dichlorophenol liberated per minute at
37°C. Crystalline porcine amylase (Wako) was used as a
standard. The protein concentration was determined with
Coomassie protein assay reagent (Pierce, Rockford, IL) with
bovine serum albumin as a standard. The amounts of insulin
in plasma, milk, and tissue were assayed with a rat insulin
125I assay system (Amersham, Buckinghamshire, UK). This
assay kit is based on a specific and sensitive radioimmunoassay and is recommended for blood and tissue samples.
Because the milk formula was shown to contain ,0.2 ng/ml of
insulin, which was possibly due to ingredients from the
bovine milk, and good recovery was obtained when rat insulin
was added to the milk formula and dams’ milk, the insulin
concentrations in the milk are precisely measured by this
assay system. The total plasma concentrations of corticosterone were determined with a rat corticosterone 125I assay
system (Amersham) based on a specific and sensitive radioimmunoassay. The amounts of C-peptide in plasma and milk
were assayed with a C-peptide radioimmunoassay kit (Linco
Research, St. Louis, MO) specific for rat C-peptide. Good
recovery was obtained when rat C-peptide was added to the
milk formula and dams’ milk, indicating that the C-peptide
concentrations in the milk are precisely measured by this
assay system.
Statistical analysis. Comparisons between groups were
carried out by means of an unpaired Student’s t-test or
analysis of variance followed by the Tukey test for multiple
comparisons, when appropriate.
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Japan). Because the suckling rats begin to nibble their
mothers’ food around 15 days of age, from 12 days of age the
pups, male and female, were randomly divided into two
groups of equal average body weight. The pups in one group
and their dams were placed in regular cages, and the pups in
the other group were placed in special cages designed so that
the rat pups had no access to the mothers’ food. The litter
sizes were maintained at 8–10 pups. The special cage consists
of a first floor where the pups are kept and a second floor
where the mother’s powdered food was placed. The structure
of the first floor of the special cage was identical to that of the
regular cage, and the second floor covered one-third of the
area of the first floor, at a height of 22 cm. The mothers could
freely go up and down between the floors, but the pups could
not climb to the second floor. From 14 days of age, a net was
laid on the first floor of each cage to prevent the pups from
eating the mothers’ feces. No visible sign of the powdered
food, such as a change in color or the presence of particles,
was found in the gastric contents of the rats reared in the
special cages until they were 23 days old. The pups’ body
weight curves were updated daily. The experimental protocol
was approved by our institute’s Committee for Research on
Experimental Animals and was conducted in accordance with
the National Research Council Guide for the Care and Use of
Laboratory Animals (1985).
Maternal rat milk and milk formula for rats. On the day
when all pups were removed for experiments, the dams were
intraperitoneally given 1.5 U/animal of oxytocin (Wako Pure
Chemicals, Osaka, Japan), and then milk was collected by
manual expression. The milk samples were centrifuged at
1,500 g for 10 min to remove fat and then frozen at 240°C
until assayed. The milk formulas were prepared according to
the previously described procedure (10). The osmolarity and
pH of the formulas were 349 and 6.28, respectively. The
concentration of insulin in the milk formula, due to ingredients from bovine milk, was at a level ,0.2 ng/ml. Rat insulin
(Cosmo Bio, Tokyo, Japan) was added to the milk formulas to
a final concentration of 5, 20, or 80 ng/ml. The complete milk
was divided into 50-ml sterilized bottles and then stored
frozen at 240°C.
Intragastric administration of insulin and artificial rearing. In the experiment on the effect of insulin administration
on the plasma insulin concentration, an intragastric cannula
was implanted into rat pups on day 13 or day 16 by the Hall
method (7). The rat pups were given milk formula with 0 or 20
ng/ml of rat insulin at 0.24 ml/h on day 13 or 0.30 ml/h on day
16 by an intermittent gastric infusion (15-min infusion every
1 h). The administration of the formula was performed
according to the previously described procedure (10). Briefly,
the intragastric cannula was joined to lead tubing (PE-50)
that was connected to a syringe mounted on an infusion pump
(model pump 44; Harvard Apparatus, South Natick, MA). The
syringe on the pump was placed in a refrigerator (5°C) to
prevent bacterial growth in the milk formula. One meter of
the lead tubing was extended outside the refrigerator so that
the pups received the formula at room temperature. To
prevent the effect of the insulin in the maternal milk, the rat
pups were administered the milk formula overnight, and
blood samples were collected from the pups from the abdominal aorta under ether anesthesia. In the experiments on the
time course and dose dependency of the increase in insulin
concentration in plasma, the rat pups were administered
overnight a milk formula to which insulin was not added, at
the rate of 0.30 ml/h. From the morning of the experimental
day, the pups were administered a milk formula with 0, 5, 20,
or 80 ng/ml of rat insulin at the rate of 0.30 ml/h. Blood
samples were collected at 0, 1, 3, and 6 h after the first
R1959
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MILK-BORNE INSULIN REGULATION OF AMYLASE DEVELOPMENT
RESULTS
Fig. 2. Developmental patterns of the activities of amylase (A) and
trypsin (B) in pancreatic zymogen granules and the effects of dietary
change on the enzyme activities. Pancreases from normal weaning
and nonweaning rat pups were used for the preparation of zymogen
granules. Activities of the enzymes were determined as described in
MATERIALS AND METHODS. Results are means 6 SE for 4 animals at
each time point.
Fig. 1. A: effect of dietary change at the time of weaning on the body
growth of suckling rats. Rat pups were mother reared in regular
cages (normal weaning) or cages designed so that the pups had no
access to the mothers’ food (nonweaning). Number of pups per mother
rat was 8–10. Results are means 6 SE for 8 animals originating from
4 different litters at each time point. B: effect of the dietary change at
the time of weaning on the growth of the pancreas. Results are means
6 SE for 4 animals at each time point.
groups, suggesting that the dietary change at the time
of weaning does not affect the increase in pancreas
mass. We determined the developmental pattern of
amylase activity in zymogen granules in both groups
during the third postnatal week. The amylase activity,
at a low level during the first two postnatal weeks (data
not shown), increased constantly after 14 days of age
(Fig. 2A) and peaked at 18 days of age (P , 0.05,
compared with 14-day-old rats). Thereafter, the amylase activity in pancreatic zymogen granules decreased,
but remained constant at a level 1.6-fold higher than
that at 14 days. After 22 days of age, the amylase
activity increased again (44.7 6 3.8 U/mg protein,
normal weaning rats at 22 days old). During the third
week of life, the amylase development showed no
significant difference between the normal weaning
group and the nonweaning group. These results suggested that the ontogenic changes of amylase activity in
pancreatic zymogen granules during the early part of
weaning are not dependent on the dietary change, i.e.,
the weaning itself. In other words, these data indicated
that there are other regulatory factors that cause the
ontogenic increase of amylase activity in preparation
for weaning. In contrast, the trypsin activity in the
zymogen granules reached the adult level (0.689 6
0.088 U/mg protein in 2-mo-old rats) at 14 days of age
and then remained at a high level (Fig. 2B). Addition-
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Growth. In the present study, rat pups were raised in
regular cages (normal weaning group) or in special
cages so that they had no access to the mothers’ food
(nonweaned group). The dams and normal weaning rat
pups were fed a commercial powdered diet that was
57% carbohydrate. No difference in the growth rates of
dams was observed between the two groups. There was
no significant difference in the growth rates between
the nonweaning pups and the normal weaning pups
(Fig. 1A). These results indicated that the pups and
their dams were not stressed by the special cages; the
special cage therefore appears to be an appropriate
system for investigating the effects of the dietary
change at the time of weaning. These cages were thus
used to investigate the development of pancreatic amylase during the third postnatal week and its correlation
with the dietary change.
Developmental patterns of pancreas weight, amylase,
and trypsin activity in pancreatic zymogen granules. As
shown in Fig. 1B, the wet weight of the pancreas
increased gradually in the two groups until 18 days of
age and thereafter the growth rate of the pancreas
weight increased more sharply. No significant difference was observed in this parameter between the two
MILK-BORNE INSULIN REGULATION OF AMYLASE DEVELOPMENT
R1961
Fig. 3. Developmental patterns of the circulating concentrations of
corticosterone (A) and insulin (B) in normal weaning rats and
nonweaning rats. Blood was collected from the abdominal aorta
within 30 min after separation from the dams. Plasma concentrations of the hormones were determined with specific radioimmunoassays. Results are means 6 SE for 4 animals at each time point.
ally, in the pancreatic homogenates, the developmental
patterns of amylase and trypsin activity in the two
groups were similar to those in the zymogen granules
(data not shown).
Plasma concentrations of hormones. It has been well
documented that the expression of pancreatic amylase
is affected mainly by glucocorticoid and insulin (13, 18).
We quantified plasma corticosterone, the main secreted
glucocorticoid in rats, by a radioimmunoassay method.
As shown in Fig. 3A, the circulating concentrations of
corticosterone increased gradually throughout the third
week of life, but the increase in corticosterone was not
in parallel with the change in amylase activity in either
group.
Insulin is another possible regulatory factor. Previous investigations suggested the involvement of insulin
as a mediator of pancreatic development in early
weaning (1, 5). As shown in Fig. 3B, the plasma insulin
concentration rose constantly from a level of ,0.7
ng/ml after 14 days of age, peaked at a level of ,1.5
ng/ml at 17 days (P , 0.05, compared with 14-day-old
rats), and decreased abruptly at 18 days. The plasma
insulin level in 19-day-old rats returned to the level
seen in 14-day-old rats and then increased gradually.
Thus the plasma insulin level in preweaning rats
changed in parallel with the amount of amylase activ-
Fig. 4. A: amounts of pancreatic insulin per body weight in normal
weaning rats and nonweaning rats. B: concentrations of glucose in
the plasma in normal weaning rats and nonweaning rats. Blood was
collected from the abdominal aorta within 30 min after separation
from the dams. C: plasma concentrations of C-peptide in normal
weaning rats and nonweaning rats. Results are means 6 SE for 4
animals at each time point.
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ity, and the developmental curve of the plasma insulin
level shifted to the left 1 day before the amylase curve.
These results suggest that the development of amylase
activity may be influenced mainly by insulin rather
than glucocorticoid. Additionally, the prevention of
weaning did not modify the plasma concentrations of
corticosterone or insulin, as well as the enzyme activity
of amylase.
Endogenous insulin. We then explored the factors
that regulated the developmental change of plasma
insulin. We speculated that the developmental increase
in plasma insulin could be due to the developmental
increase in insulin in the pancreas, the increase in a
stimulator of insulin release, or the increase of pancreatic insulin release. As shown in Fig. 4A, the amount of
pancreatic insulin per body weight increased gradually
during the 3rd wk, but the transient increase that
peaks at 17 days of age was not observed in the amount
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MILK-BORNE INSULIN REGULATION OF AMYLASE DEVELOPMENT
Fig. 5. Serial changes in the insulin content of maternal milk.
Concentration of insulin was determined by means of a specific
radioimmunoassay. Results are means 6 SE for 4 animals at each
time point.
Fig. 6. Effect of insulin administration on the plasma insulin concentration (A) and plasma C-peptide concentration (B) in rat pups. Rat
pups at 14 or 17 days of age were administered milk formula with 20
ng/ml of rat insulin at 0.30 ml/h, and blood samples were collected 6 h
after the first administration. Results are means 6 SE for 8 animals.
* P , 0.05, compared with control rats by an unpaired Student’s
t-test.
insulin level in the pups administered the milk formula
without insulin was not different from that observed in
mother-reared (MR) rats and no elevation of plasma
insulin by the administration of 20 ng/ml of insulin was
observed. In the 17-day-old rats administered the milk
formula without rat insulin, the plasma level of insulin
was as low as that in 14-day-old rats. Moreover, the
administration of 20 ng/ml of insulin in the milk
formula caused a significant increase in the plasma
insulin concentration at 17 days old. No difference in
the plasma C-peptide level between the cannulated
groups and the MR group at 14 and 17 days of age was
observed (Fig. 6B), suggesting that the insulin release
from the pancreas was not altered by the milk-borne
insulin or by the surgical manipulation. The plasma
concentrations of glucose in the rats administered the
milk formula with or without insulin (151 6 6, 154 6 2
mg/ml plasma, respectively) did not differ significantly
from that of 17-day-old MR rats (Fig. 4B).
The elevation of plasma insulin by the intragastric
administration of insulin was examined further in
17-day-old rats. Figure 7A shows the time course of the
increase in the insulin concentration in plasma. The
experiments were performed after the overnight administration of a milk formula to which insulin was not
added to prevent the effect of the insulin in the maternal milk. The plasma level of insulin in the control rats
that were intragastrically administered the milk formula was lower than that in the MR rats throughout
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of pancreatic insulin. The change in the amount of
plasma glucose, which is the main regulator of pancreatic insulin release, during the experimental period
was determined. An age-dependent change in the
plasma glucose level was not observed (Fig. 4B), suggesting that the change in the plasma insulin level in
weaning rats (Fig. 3B) is not dependent on the plasma
glucose. We then determined the plasma levels of
C-peptide, the production of which would be accompanied by the endogenous insulin production. The plasma
C-peptide concentrations were maintained at a constant level from 14 to 21 days of age (Fig. 4C). These
results implied that the increase in plasma insulin,
which peaked at 17 days of age, could not be attributed
to the endogenous insulin. In addition, the plasma
glucose level, the insulin concentration in the pancreas,
and the plasma C-peptide concentration causing the
change of amylase activity were not affected by the
dietary change during the 3rd wk of life. Therefore, the
participation of milk-borne insulin, the exogenous insulin for pups, was assessed.
Milk-borne insulin and plasma insulin concentrations. High concentrations of insulin are found in the
colostrum and milk of several species (11, 21, 31),
suggesting that milk may be a source of insulin for
sucklings. In the present study, the insulin concentration in the dams’ milk was first measured because there
are no available data on the insulin levels in rat milk.
Figure 5 shows the ontogenic changes in the insulin
content of the dams’ milk. The insulin concentration
was increased at 13 days postpartum and then plateaued at a level of ,15 ng/ml.
It is possible that the insulin in the mothers’ milk is
responsible for the increase and maintenance of amylase activity in rat pups. However, it is not yet known
whether insulin in milk is absorbed from the gut in rat
pups or whether the absorbed insulin affects neonatal
development. For the determination of whether insulin
in milk elevates the pups’ plasma concentration of
insulin, rat pups were intragastrically administered
overnight the milk formula with or without rat insulin.
As shown in Fig. 6A, at 14 days of age, the plasma
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MILK-BORNE INSULIN REGULATION OF AMYLASE DEVELOPMENT
the experimental period. The administration of 20
ng/ml of insulin in the milk formula caused a significant increase in the plasma insulin concentration (1.5fold increase over the control, P , 0.05) at 3 h after the
first administration, and then the level of insulin
remained unchanged up to 6 h. Next, the dose dependency of the increase in the plasma insulin level by
insulin administration was estimated. Blood samples
Table 1. Effect of insulin contained in milk on the pancreatic amylase activity in rat pups at the time of weaning
Weight
Animals
MR rats
AR rats
Insulin 2
Insulin 1
Plasma
Pancreatic Enzymes
Body,
g
Pancreas,
mg
Glucose,
mg/dl
Insulin,
ng/ml
C-peptide,
ng/ml
Corticosterone,
ng/ml
Amylase,
U/mg protein
Trypsin,
U/mg protein
31.9 6 0.7
72.3 6 7.5
152 6 2
1.63 6 0.30
1.66 6 0.17
84.9 6 21.5
34.0 6 3.5
0.69 6 0.02
30.5 6 1.7
30.5 6 0.2
78.3 6 4.7
74.0 6 2.1
146 6 3
151 6 7
0.71 6 0.04
1.11 6 0.13*
1.66 6 0.33
1.59 6 0.21
90.7 6 18.5
84.7 6 24.9
19.5 6 1.6
28.8 6 2.6*
0.71 6 0.07
0.66 6 0.07
Results are means 6 SE for 4 animals. An intragastric cannula was implanted into rat pups in the artificially reared (AR) groups at 12 days
old, and the rat pups were given milk formula with (insulin 1) or without (insulin 2) 20 ng/ml rat insulin until 17 days of age. MR, mother
reared. * P , 0.05, compared with the insulin 2 group by an unpaired Student’s t-test.
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Fig. 7. A: time course of the increase in insulin concentration in
plasma after insulin administration in rat pups at 17 days of age. Rat
pups were administered milk formula with 0 or 20 ng/ml of rat
insulin at the speed of 0.30 ml/h, and blood samples were collected at
0, 1, 3, and 6 h after the first administration. B: concentrationdependent effect of insulin administration on the plasma insulin
concentration. Rat pups at 17 days of age were administered milk
formula with 0, 5, 20, or 80 ng/ml of rat insulin at 0.30 ml/h, and blood
samples were collected at 6 h after the first administration. Results
are means 6 SE for 8 animals. * P , 0.05, compared with control rats
by an unpaired Student’s t-test (A) or analysis of variance followed by
the Tukey test for multiple comparisons (B).
were collected 6 h after the first administration. When
5 ng/ml of insulin was administered intragastrically to
suckling rats, an elevation of the insulin concentration
in plasma occurred, but the elevation was not significant (Fig. 7B). The administration of 20 ng/ml of
insulin, corresponding to the level in the dams’ milk, to
suckling rats led to a significant increase in the plasma
insulin level compared with that in the control rats
(P , 0.05). When 80 ng/ml of insulin was administered,
the plasma insulin level increased even more (1.6-fold
higher compared with the control), although the administration of insulin failed to increase the plasma insulin
level to that in the 17-day-old MR rats. These findings
indicate that milk-borne insulin induces an elevation of
the plasma insulin level in preweaning rats.
Effect of milk-borne insulin on pancreatic amylase
activity. Finally, we investigated whether milk-borne
insulin is a modulator of amylase activity in the third
postnatal week with the use of a rat artificial rearing
(AR) technique (10). An intragastric cannula was implanted in rat pups at 12 days of age, and the pups were
given milk formula with (insulin-plus group) or without
(insulin-minus group) 20 ng/ml of rat insulin until 17
days of age. As shown in Table 1, the body and pancreas
weights in the two groups were not significantly different from those of the MR rats. In the insulin-minus
group, the plasma level of insulin was as low as that in
the 14-day-old MR rats, and the plasma insulin level in
the insulin-plus group was 1.6-fold higher compared
with the insulin-minus group, corresponding to the
results in the rats administered insulin overnight (Fig.
7). The developmental increase in amylase activity was
abolished in the insulin-minus group; the amylase
activity in their zymogen granules was as low as that in
the 14-day-old MR rats. Furthermore, in the AR rats
raised on the milk formula with insulin, the amylase
activity was significantly higher (1.5-fold) than that of
the insulin-minus group. There was no significant
difference in the concentrations of the plasma glucose;
C-peptide; or corticosterone, a stress-responsive hormone; the daily growth rates after 13 days of age; or
pancreatic trypsin activity between the two AR groups
and the MR rats. These results indicated that the
effects of stresses attributable to the infusion system
and the operation on the AR rats were kept at a
minimum and showed that the development of the AR
rats was not delayed at all. The results suggested that
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MILK-BORNE INSULIN REGULATION OF AMYLASE DEVELOPMENT
milk-borne insulin is crucial in the development of
pancreatic amylase in naturally weaned rats.
DISCUSSION
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Developmental alterations in the digestive functions
are essential for the dietary change at the time of
weaning. The functional development of the exocrine
pancreas results in an increase in an animal’s abilities
to synthesize digestive enzymes and secrete them.
Because the enzyme secretion by the rat pancreas
increases markedly during the third postnatal week (3,
27), the changes in the pancreatic contents of digestive
enzymes during this period are of significance. In
particular, the increase in amylase activity is important in rats because the most significant dietary change
at the time of weaning is the increase in complex
carbohydrate intake. The results of the present study
showed the development of amylase activity in pancreatic zymogen granules, which was characterized by the
finding that the amylase activity increased from 14
days, peaked at 18 days, and then decreased slowly
(Fig. 2A). No visible sign of solid food was found in the
gastric contents of the nonweaned pups at 18 days of
age, whereas in the rats raised in the regular cages, the
major part of the gastrointestinal contents consisted of
solid food at 19 days of age. It stands to reason that the
amylase activity increased before the age of 18 days,
when the rapid dietary transition began. Although
several studies have demonstrated the development of
pancreatic amylase during the rat weaning period (3, 8,
27), the mechanisms underlying the initiation of the
ontogenic changes of amylase are unknown.
A relationship between the development of digestive
enzymes and the changes in the diet composition has
been documented (3, 14, 15, 17). These studies obtained
conflicting results regarding the effect of weaning on
the development of digestive functions. This may be
because there has been no appropriate model system
for the assessment of natural weaning with which
suckling rats can grow without any stress (such as
early forced weaning, the feeding of abnormal milk, and
intermittent feeding). In this respect, the present system using the bilevel cage is suitable for examining the
developmental process of the digestive functions on
natural weaning. In the present study, the effects of the
deprivation of solid food on the development of pancreatic exocrine enzymes and of plasma hormones that
regulate this development were investigated using the
special cages. The results resolved the controversy
regarding the presence or absence of feeding controls of
the digestive functions in weaning rats; that is, the
amylase development during the third postnatal week
is not under the control of nutritional changes, and
there are other mechanisms by which pancreatic amylase increases with the dietary alteration of natural
weaning.
Possible roles of insulin and corticosterone as mediators of the maturation of pancreatic amylase have been
proposed (5, 8, 17), but the relationship between the
development of pancreatic amylase during natural
weaning and the changes of these hormone concentra-
tions in plasma was by no means clear. In the present
experiments, the close relationship between the plasma
insulin level and pancreatic enzyme activities suggested the involvement of insulin as a stimulatory
factor of pancreatic development in the early stage of
weaning. Interestingly, the increased plasma insulin
did not affect the plasma glucose level during the 3rd
wk. Issad et al. (9) reported that an insulin-resistant
state in plasma glucose metabolism was present in
suckling rats that disappeared after weaning. The
elevation of the plasma insulin level at the onset of
weaning would be important as a stimulant of developmental processes, such as pancreatic amylase development, rather than the regulator of the plasma glucose
level. Accumulating evidence indicates that the intestinal maturation at the beginning of the weaning period
is controlled by plasma corticosterone (8, 31), a possible
regulatory factor for pancreatic amylase (17, 18). Although corticosterone may participate in amylase development, the decrease in amylase activity after 19 days
(Fig. 2A) and the present AR experiments (Table 1)
indicated that amylase development during the third
postnatal week may be mediated mainly by plasma
insulin. Despite the marked decrease in the plasma
insulin concentration at 18 days, amylase activity was
maintained at a relatively high level, probably due to
the activated mechanism of amylase synthesis and the
slow second rise in the plasma insulin. The second
increase in plasma insulin could also be important for
the acute increase in amylase activity after 22 days.
The present data indicated that the secretion of
endogenous insulin by the endocrine pancreas might
remain unchanged during the 3rd wk of life in MR rats
(Fig. 4). In addition, the release of endogenous insulin
in 14- or 17-day-old rats administered milk formula
with or without insulin might not differ from that in
MR rats (Fig. 6). These results suggested that the
increase in the plasma insulin level, which peaked at 17
days of age, may be attributable to exogenous insulin,
i.e., milk-borne insulin, rather than the endogenous
insulin secreted by the endocrine pancreas. Nevertheless, the endogenous insulin may also be important for
the pancreatic amylase development. Lee et al. (16)
showed that streptozotocin treatment of neonatal rats
led to an impairment of their pancreatic amylase
development. The increased plasma insulin based on
both the constant secretion of endogenous insulin and
the additional exogenous insulin may induce amylase
development at weaning. Alterations of plasma insulin
concentration could also occur as a result of altered
rates of insulin clearance. In the present study, however, in rats administered insulin-deficient milk formula, the plasma insulin concentrations at 17 days of
age were as low as those at 14 days of age in contrast
with the increased values of the MR rats, suggesting
that the age-dependent decrease in the clearance rates
of plasma insulin did not occur from 14 to 17 days of
age. The gradual increase in the plasma insulin concentration after 19 days could possibly be attributed to the
decreasing rate of insulin clearance, because the plasma
C-peptide level remained unchanged at the same time
MILK-BORNE INSULIN REGULATION OF AMYLASE DEVELOPMENT
activity in zymogen granules as a result. A possible
explanation is that rat milk contains protease inhibitors, important factors in the effective transport of
large peptides from the milk to the blood in suckling
rats (25, 29). Other studies showed that milk-borne
peptides such as IGF-I remained stable in the gastrointestinal tract (26) and were protected against endoluminal degradation by several milk antiproteases (25).
Similarly, it is possible that milk-borne insulin is
protected by protease inhibitors contained in milk in
MR rats. After 18 days of age, endoluminal insulin
could be expected to be digested, owing to the increase
in pancreatic enzyme activities (27). Further experiments are necessary to clarify the putative actions
of protease inhibitors for the effective absorption of
insulin.
It is well known that some bioactive substances in
milk are selectively transported intact across the gastrointestinal epithelium (6, 28) and that other proteins are
taken up through ileal nonspecific endocytosis in suckling rats (6, 11, 28). In the present study, we did not
examine whether insulin is selectively or nonselectively absorbed, or the sites of absorption of insulin.
However, we found that the insulin concentration in
milk remained unchanged after 13 days (Fig. 5), although the plasma insulin level increased constantly
from 14 to 17 days (Fig. 3B); in addition, insulin
absorption was observed at 17 days but not at 14 days
(Fig. 6). These results may indicate that a specific
pathway for insulin appears at the onset of weaning.
Because an excess uptake of insulin would have a
hypoglycemic effect on pups and because insulin would
be precious for the mothers, insulin absorption could be
efficiently and strictly controlled via such a specific
pathway. The presence of insulin receptors on the
intestinal epithelium during the suckling period was
recently demonstrated (2, 21), raising the possibility
that the receptor is correlated with the transport of
insulin across the gastrointestinal epithelium.
Taken together, the present results suggested that
milk-borne insulin plays a crucial role in the development of pancreatic amylase. The increase in plasma
insulin concentration attributable to endogenous insulin may begin before or after 20 days of age, although
the beginning of weaning occurs before then. Therefore,
the exogenous milk-borne insulin, as a substitute for
endogenous insulin, might stimulate the amylase development of the exocrine pancreas. Pancreatic amylase
activity decreased in response to the decrease in plasma
insulin after 18 days, but was maintained at a relatively high level. This series of processes is most likely
essential for a smooth weaning process. After 21 days of
age, the insulin release from the pancreas could increase and concomitantly pancreatic amylase could be
expected to begin to change adaptively in response to
dietary carbohydrate (4, 27).
Perspectives
Milk contains a significant number of substances in
addition to insulin that are known to possess biological
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(Fig. 4C). Additionally, C-peptide was detected at ,0.38
ng/ml in rat’s milk, whereas it was undetectable in the
milk formula. However, the level of the plasma Cpeptide in rats administered milk formula corresponded to that in MR rats, suggesting that milk-borne
C-peptide would not result in the increase in the
plasma C-peptide level.
The level of insulin in the rat milk from 10 to 21 days
postpartum was determined. It is known that there is a
high amount of insulin in the colostrum, which decreases rapidly to the low level present in mature milk
in several species (11, 21, 31). In rats, in contrast, a
high level of insulin is detected in mature milk. Interestingly, the insulin in rat milk in the present study was
relatively low at 10 days postpartum and then increased just before 15 days when the plasma concentration of insulin began to increase. The present study
showed that 5 ng/ml of insulin did not sufficiently
increase the plasma insulin level, whereas 20 ng/ml of
insulin, corresponding to the level in rat milk, significantly increased the plasma insulin (Fig. 7B). These
results might indicate that the elevation of the milkborne insulin level at 13 days postpartum is important
for the increase in plasma insulin in rat pups. The
insulin concentration in milk remained unchanged
thereafter, in contrast to insulin-like growth factor
(IGF)-I, the level of which in rat milk was reported to
decrease sharply at 20 days postpartum (11).
The results of the present study clearly demonstrated that intragastrically administered insulin
caused an increase in the plasma insulin concentration
in weaning rats. The absorption of intact large peptides, such as epidermal growth factor (6, 12, 30), IGF-I
(12, 23, 32), immunoglobulin G (29), and nerve growth
factor (28), from the gut during the suckling period is a
widely recognized phenomenon. Indeed, it has been
demonstrated by others that intragastrically administered insulin may be absorbed from the gut in several
species, including rats (19, 20, 24), but the doses of
administered insulin in these experiments were over
100-fold higher than that found in rat milk. Attributable to the difficulty in removing specific substances
from milk, pharmacological doses of each factor, whose
effect exceeds that of the factor contained in milk, were
usually needed to examine the functional role of milkborne substances in suckling rats. In this respect, the
AR technique would be suitable and useful (10), because the artificial milk formula prepared in our laboratory contains scarcely any hormones and its composition can be controlled. The present data obtained with
the AR technique showed that the oral intake of a
physiological dose of insulin in milk elevates the plasma
insulin level during the beginning of the weaning
period in rats and can account for the characteristic
change in plasma insulin in weaning rats. In regard to
the present results with C-peptide, intragastrically
administered insulin might be absorbed across the
gastrointestinal epithelium in weaning rats.
In the present experiments, the intragastric administration of insulin failed to increase the insulin level in
plasma to that in MR rats or the pancreatic amylase
R1965
R1966
MILK-BORNE INSULIN REGULATION OF AMYLASE DEVELOPMENT
We thank the late Professor Otakar Koldovsky (University of
Arizona) for critical review of the manuscript. We also thank Makiko
Inoue, Masako Yajima, and Minako Akao for designing a cage to
prevent weaning and all members of our laboratory group for skillful
technical assistance.
Address for reprint requests: T. Yajima, Dept. of Nutritional
Research, Nutrition Science Institute, Meiji Milk Products Co., Ltd.,
540 Naruda, Odawara 250-0862, Japan.
Received 12 March 1998; accepted in final form 10 August 1998.
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