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Pancreatic islets and blood sugars in prenatal and postnatal offspring from diabetic ratsBeta granulation and glycogen infiltration.

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Pancreatic Islets and Blood Sugars in Prenatal and
Postnatal Offspring from Diabetic Rats:
Beta Granulation and Glycogen Infiltration'
JAE NAM KIM,2 WALTER RUNGE, LEMEN J. WELLS
ARNOLD LAZAROW
DepaTtment of Anatomy, University of Minnesota,
Minneapolis 14, Minnesota
In a preliminary study we have reported
in abstract that the pancreatic islets of offspring from diabetic rats undergo several
cytological alterations (Kim, Runge, Wells
and Lazarow, '58). They include a marked
decrease in the number of granulated beta
cells and in the number of granules per
cell, hydropic changes usually present in
the centrally located islet cells, and hyperplasia of the islets. After birth, these alterations tend to disappear.
The present study extended the work by
giving insulin to some of the diabetic rats
and by increasing the number of observations in each age group. Histological and
histochemical findings in the islets were
correlated with the blood sugars.
The prenatal offspring include fetuses
which were obtained at daily intervals
from the 17th day of gestation to birth.
Postnatal offspring include young of one
to 10 days postpartum.
The present paper is one of our current
series on diabetes and pregnancy in the rat
(Kim, Runge, Wells and Lazarow, '60;
Lazarow, Kim and Wells, '60; Wells, Kim
and Lazarow, '60).
MATERIALS AND METHODS
Sprague-Dawley rats between 120 and
150 days of age were used. The animals
were fed on a standard diet of Purina fox
chow ad libitum.
The length of gestation, between the
time of witnessed mating and the time of
witnessed delivery of the first young, was
determined. Beginning at 21 days and 15
hours postcoitum, i.e., one hour prior to
the earliest time of delivery in normal
pregnancies (Wells, '50), the rats were
watched at 15- to 60-minute intervals.
AND
Diabetes was induced by two methods,
alloxan injection and pancreatectomy,
either before conception or on the 12th
day of pregnancy. Alloxan was administered intravenously in doses of 40 mg/
kg body weight, in accordance with the
method previously described (Lazarow and
Palay, '46). Subtotal pancreatectomy was
performed after the animals had been
anesthetized with ether. After pancreatectomy, the animals were given subcutaneous injections of 20% glucose for a period
of 4 days. Thereafter, they were force fed
by stomach tube for 6 to 10 days; this food
was a diet3 to which 3.46 gm of pancreatin
per 250 ml of diet was added. Finally,
the force feeding was stopped, and the
animals were permitted to eat a diet of
Purina fox chow. Blood sugar levels were
determined at intervals during each pregnancy.
In diabetic rats which were treated with
insulin, the diabetes was induced by injecting alloxan before conception; the dose
of alloxan was the same as that presented
above. All the diabetic rats were placed
in metabolism cages. Food was supplied
ad libitum; water was limited to 160 ml
per day. Some of the diabetic rats were
injected with 5 or 10 units of protamine
'Aided by grants from the Medical Research
Fund of the Graduate School and by grants A1224, A-1659 and A-1887 from the National Institute for Arthritis and Metabolic Disease, Public
Health Service.
2 Fellow of the International Cooperation Administration, 1955-59. Present address: Department of Anatomy, Seoul National University
Medical School, Seoul, Korea.
3High Carbohydrate Diet (Ingle, D. J. et al.,
Endocrinol., 39: 52, 1946); or Medium Carbohydrate Diet (Ingle, D. M., and R. C. Meeks, Am.
J. Physiol., 171: 600, 1952).
239
1
5
2
4
1
2
5
4
3
10
Each pancreas stained by both methods, aldehyde-fuchsin and periodic acid-Schiff.
9
5
10
1
5
5
5
12
3
5
10
5
8
21
1
3
3
4
2
5
5
3
2
1
1
1
1
2
2
2
8
20
4
5
6
3
3
8
8
6
2
7
3
Postnatal period
5
4
24
17
6
3
15
23
7
16
81
36
6
6
5
2
4
s
:
z
i
g
Blood sugar
Litters
9
3
4
4
1
2
27
18
42
33
12
12
4
1
spgg sEzsl
Pancreas
5
3
3
3
4
2
2
5
5
4
2
9
2
1
Litters
2
9
5
5
5
11
3
5
7
6
7
8
14
2
4
2
1
2
5
1
2
2
3
1
3
6
1
1
stains1
Two
s:zg
Pancreas
Insulin-treated diabetic rats
8
2
8
14
7
2
2
3
14
15
3
4
2
1
Litters
Prenatal period
sF$ig
Blood sugar
Litters
3
9
7
1
10
7
4
9
1
6
9
4
1
1
s2zsl
20
3
2
55
39
14
14
13
7
10
s:!g
Pancreas
Untreated diabetic rats
7
6
8
15
8
9
3
8
4
6
2
10
4
3
1
23
16
89
63
5
24
10
22
Birth
5
5
5
14
4
4
2
20
7
15
5
19
21
3
8
2
18
Litters
4
s:zig
Blood sugar
Litters
17
days
postnatal
Or
age
Normal rats
TABLE 1
Number of Tats used in studying blood sugar and pancreas
M
k
0
DIABETES : PANCREASES OF OFFSPRING
zinc insulin per kg of body weight per day,
beginning on the third day after alloxan
injection and continuing throughout the
period of the experiment. No attempt was
made to control the diabetes; insulin was
administered primarily to increase fertility
and the likelihood of pregnancy going to
term. Blood sugars and urine sugars were
determined at least twice per week.
Blood sugar was determined by the
micro method of Folin-Malmros ('29),
using a sample of 0.025 or 0.05 ml of
blood. Urine sugar was determined by
Somogyi's method ('41), using a 24-hour
sample of urine.
Pregnant rats were sacrificed at about
24-hour intervals from the 17th to the
22nd day of gestation (table 1). Maternal
and fetal blood sugars were determined,
the fetal blood having been obtained from
the neck after fetal decapitation. Fetal
pancreases were removed and fixed.
When pregnant rats delivered spontaneousIy, two to 4 newborns from each litter
were usually sacrificed in order to determine the blood sugars and to fix the pancreases. The remaining newborns were
nursed by their own mothers (normal rats)
or by foster mothers (diabetic rats), one
lactating mother nursing 6 newborns or
fewer. The young rats were sacrificed at
the following times after birth: 1, 2, 4, 8,
12, 16, 20, 24 and 48 hours; 3, 4, 5, 6, 7,
8, 9 and 10 days.
Two pancreases of littermates were
fixed, one in Bouin's fluid and another in
Gendre's fixative (Gendre, '37). They were
subsequently sectioned at 5 I.I and stained
by a modification of Gomori's aldehyde
fuchsin technique (Ferner and Runge,
'56) and by the periodic acid-Schiff method
(PAS, Lillie, '54), respectively. But, at
times, sections prepared from Bouin fixed
blocks were stained both by the aldehydefuchsin method and by the PAS method,
using adjacent sections. All the slides of
sections to be stained by the PAS method
were made in duplicate; one of the two
slides was stained by the PAS method; the
second slide (control) was subjected to
amylase digestion before staining in order
to distinguish glycogen from other substances that would also show a positive
reaction.
24 1
The size of the islets was determined by
measuring the long and short axes of the
islets. From each pancreas, 6 to 20 consecutive sections were mounted on microscope slides and stained by the aldehydefuchsin method. The slides were taken at
random in both experimental and control
groups, and the third section of each slide
was examined. The largest islet in this
section was measured in two dimensions at
a standard magniflcation by means of a
calibrated eye-piece micrometer.
OBSERVATIONS
Blood sugar during the fetal period.
The blood sugars of mothers and those of
fetuses and newborns are shown in figure
1 (table 1 shows the number of cases).
The 45" diagonal line in figure 1 was
drawn in order to assist readers in judging
the degree of correlation between the maternal and the fetal or neonatal blood sugars. The correlation was good in normal
rats, in untreated diabetic rats and in insulin treated diabetic rats.
Blood sugar during the postnatal period.
The blood sugars are presented in figure 2
(table 1 shows the number of cases).
Within 24 hours after birth, the blood
sugar of offspring from untreated diabetic
mothers dropped from a hyperglycemic
level to a normal level.
In order to determine more precisely the
rate at which the blood sugar drops to normal, one rat with established diabetes was
made pregnant; the young were sacrificed
at 0, 1, 2, 4, 8, 12, 16 and 20 hours after
birth, and the blood sugars were determined (not shown in fig. 2). The blood
sugars at these times were: 282, 329, 273,
75, 73, 56, 46 and 51 mg%. This result
showed that the blood sugar, although elevated at one hour and at two hours, becomes normal at 4 hours after birth; it
remained at a level slightly lower than that
of normal young. The blood sugar in normal young at one hour after birth was likewise somewhat higher than that at birth
(174 mg% compared with 116 mg% , not
shown in figure 2). Between 4 and 20
hours after birth, normal young had a
blood sugar which ranged between 89 and
106 mg%.
242
JAE NAM KIM, WALTER RUNGE, LEMEN J. WELLS AND ARNOLD LAZAROW
Figure 2 shows that during fetal life
and at birth the blood sugar level of the
fetus appears to be higher in those fetuses
taken from diabetic mothers not treated
with insulin, intermediate in those from
treated diabetic mothers, and lower in
those from normal mothers. At 24 hours
after birth, however, the blood sugar level
of offspring from diabetic mothers which
were not treated with insulin tends to be
the lowest of the three groups.
It should be noted that newborns from
diabetic mothers which were not treated
with insulin were born 18 hours later than
those from the other two groups.
From 48 hours after birth to 10 days of
postnatal life, all blood sugars fluctuated
within the normal range.
J
/
Birth
z
!i3
hT
81
300-
200-
28
i
B
Y
Untreated diabetes
X
210
[o
100-
Normal control
Treated d i a b e t e s
No.= Days postcaitum
T
V
I
100
I
1
1
I
200
300
400
500
MEAN BLOOD SUGAR OF FETUSES (mg.
%I
Fig. 1 Correlation between maternal and fetal blood sugar. The plotted blood sugars of
offspring are means of the average value for the several litters. The neonatal blood sugars
(“Birth”) were usually determined within 5 to 30 minutes after birth.
\
3
500
0
Untreated diabetes
0
Treated diabetes
a Normal control
9,
I
RTH
--.,I
I
I
18
19 20 2 1
DAY O F
EMBRY 0 N I C L I FE
I
I
f
I
I
I
[
I
l
l
I
2
3
4
5
6
7
8
9
10
DAYS AFTER B I R T H
Fig. 2 Comparsion of blood sugar of offspring from normal and diabetic mothers. The
plotted blood sugars are means of the average values for the several litters.
243
DIABETES : PANCREASES O F OFFSPRING
Histological and histochemcial
observations and their
relation to blood
sugars
Pancreatic islets obtained from offspring
of normal and diabetic mothers were studied at various times, ranging from the 17th
day of gestation to the 10th day after birth.
In this study observations on the beta cell
granulation, hydropic change, and hyperplasia of the islets were made in the sections that were fixed in Bouin’s fluid and
stained by the aldehyde-fuchsin method;
the observations on glycogen infiltration
were made in sections that were stained by
the PAS method after they had been fixed
only for two or 4 hours in Bouin’s fluid or
in Gendre’s fixative (most often). The
Gendre’s fixative yielded the better preparations for glycogen, but Bouin’s fixative4
yielded satisfactory results when the period of fixation was short. Bouin’s fixation
permitted the staining of two adjacent sections differently, one by the aldehydefuchsin method and the other by the PAS
method.
Although undifferentiated islet cells
were present in the islets throughout the
prenatal period studied, no alpha granules
could be identified in the islets during this
period.
Glycogen infiltration was not found in
the islet cells nor in the ductules during
the prenatal period (figs. 15, 17). Occasionally, small particles of glycogen were
found in the lumina of capillaries.
Pancreases of the diabetic series. The
islets showed extensive hypertrophy and
hyperplasia, and were irregular in shape
(cf. figs. 5 , 6 ) . The hypertrophy was confirmed by measurements (table 2). The
data in table 2 indicated that the hypertrophy was largely due to an increased
length of islets.
The beta granulation was reduced markedly (cf. figs. 9, 10). Cellular counts
showed that in two experimental fetuses
which were obtained from untreated diabetic mothers on the 22nd day 0.17 and
0.16% of the islet cells were granulated
and that in one control fetus of the same
age 41.6% were granulated. These data
were obtained from stained serial sections
by counting all the cells in 30 or more isPrenatal and newborn offspring
Normal pancreases. The observations lets, a total of 3000 or more cells per panincluded those on the pancreases of 113 creas.
The cytological alterations, including
prenatal offspring obtained from 47 nor- number
of granulated beta cells, hydropic
mal mothers and of 39 newborns from 16 change, glycogen
infiltration, and islet hynormal mothers (table 1) .
perplasia, were correlated with the materGranulation of the beta cells was not
blood sugars at autopsy or at delivery.
observed in the 17-dayislets, but beginning nalThe
relationship between the number of
granulation was observed in the 18-day granulated beta cells (number estimated
islets. At this period, the islets were small in a selected section of islet) and the maand usually associated with ductules. The ternal blood sugar is shown in figure 3.
size and number of the islets increased
4 Direct chemical determination of the glycowith increasing age; likewise the number
of recognized beta cells as well as granules gen content of Bouin fixed blocks of tissue (liver)
showed little loss of glycogen even after 24 hours
per beta cell increased progressively up to of
fixation (Arnold Lazarow and Paula Makinen,
birth (fig. 10).
unpublished observations).
TABLE 2
Mean size of islet o f Langerhans in fetuses f r o m normal mothers and untreated diabetic
mothers at 519 hours postcoitum
Dimensions
and area
of islets
Long axis
Short axis
Area
1P
Fetuses of normal mothers
No.
Mean and S.E.
(u2 or 1 ~ )
15
15
15
217f 2
1552 1.6
44,855-C-21
= probability of difference between means.
Fetuses of diabetic mothers
Mean and S.E.
No.
(us or L L )
15
15
15
335f 2.5
165e 1.3
62,299e17
P=
0.005
0.2
0.02
244
JAE NAM KIM, WALTER RUNGE, LEMEN J. WELLS AND ARNOLD LAZAROW
In the untreated diabetic group, the
number of granulated beta cells was decreased in all of the pancreases examined.
The maternal blood sugar was 226 mg%
or more.
In the treated diabetic group, 9 out of
11pancreases showed a normal number of
granulated beta cells (figs. 11, 10); the
maternal blood sugar was 200 mg% or
less. One pancreas which showed a decreased number of granulated beta cells
(open triangle above the upper dashed
line) was from a newborn of a mother
with a blood sugar of 455 mg% .
Maternal blood sugars of 240 mg% or
more were associated with a lack of granulation of the beta cells in the offspring
(cases above dashed line in figure 3). Maternal blood sugars of 175 mg% or less
were associated with normal granulation
(cases below dotted line, figure 3), as illustrated in figure 11.
There were 6 cases of maternal blood
sugars between 175 and 240 mg% (between dashed line and dotted line in figure
3). Three of the 6 cases showed a lack of
granulation in the islets (offspring of the
19th to 20th day). The other three cases
showed normal granulation (islets of older
off spring, newborns).
C y t o p 1a s m i c vacuolation (hydropic
change) was observed primarily in those
islet cells with few or no beta granules. It
was observed mainly in the centrally located islet cells. It was not seen in the
peripherally located islet cells and ductular
epithelial cells. In 57 pancreases in which
sections were stained by two methods,
aldehyde-fuchsin and PAS, all showed both
cytoplasmic vacuolation and glycogen infiltration (figs. 9, 18; cf. fig. 21). The degree of vacuolation was parallel to that of
glycogen infiltration. For the purpose of
examining individual cells for the presence
of vacuolation and glycogen infiltration,
one section of each of 10 pancreases was
stained with aldehyde-fuchsin and an adjacent section was stained by the PAS
method. It was found that both vacuolation and glycogen infiltration occurred
simultaneously in individual cells in each
of the 10 cases examined (see two illustrations of an older pancreas, figures 7, 8).
In those untreated or insulin-treated diabetic mothers whose blood sugars were
240 mg% or more, glycogen infiltration
was invariably observed in the islets of the
offspring (cases above dashed line in figure
3). In those mothers whose blood sugars
were 175 mg% or less, it was not observed
in the islets of the offspring (cases below
dotted line, figure 3), as illustrated in
figure 19.
There were 15 cases of maternal blood
sugars between 175 and 240 mg% (between dashed line and dotted line in figure 3). Eight of the 15 cases showed a
slight or a moderate degree of glycogen
infiltration in the islets (offspring of the
19th to 21st day). The other 7 cases showed
no glycogen in the islets (older offspring,
newborns).
It should be emphasized that beta granulation was not observed in the islets of normal offspring on the 17th day postcoitum,
whereas intracytoplasmic glycogen was observed on the 17th day in offspring from
untreated diabetic mothers (cf. figs. 15,
16). This suggests that glycogen infiltration can occur in beta cells at a stage of
differentiation prior to the appearance of
beta granulation.
At later stages, the absolute number of
islet cells which contain glycogen increased progressively with an increase in
the age of the offspring (fig. 18). Thus,
the number of islet cells showing glycogen
in the diabetic group approximated the
number of granulated beta cells in the
normal controls (cf. figs. 18, 10).
Fig. 3 Pancreatic islets in prenatal offspring
from diabetic mothers: granulated beta cells and
glycogen infiltration. A circular symbol is used
for the isIets of untreated animals and a triangular symbol for those in the insulin treated
group. Fully granulated beta cells (i.e., normal)
are designated by the solid symbol; moderately
granulated cells (i.e., partially degranulated) ,
by the half shaded symbol; cells with slight granulation (i.e., almost completely degranulated),
by the symbol with a dot in the center; cells with
no granulation (i.e., completely degranulated),
by the open symbol. In designating the degree of
glycogen infiltration, normal islets (i.e., no glycogen infiltration) are indicated by the open symbol; slight glycogen infiltration by the symbol with
a dot in the center; moderate infiltration by the
half shaded symbol; and marked glycogen infiltration by the solid symbol. Dashed lines and
dotted lines assist the reader in seeing where
most degranulation and most glycogen infiltration were observed.
245
DIABETES : PANCREASES O F OFFSPRING
600
500
0
00 0
1
~
0
~
6
0
00 0 0
0
00 0
00
-
-
loot
0
00 0
A
0 ~ 0 0 0
300
3
0
Granulated
0
oo
00
0 0
QOOQ
00000
0 0 0 0
00000
0
0
A
A A
Beta Cells
A
Untreated: o
Absent
Treated:
A
0
~
Figure 3
Slight
8
~
8
Moderate A Marked
246
JAE N A M KIM, WALTER RUNGE, L E M E N J. WELLS AND ARNOLD LAZAROW
Beta
Granulated
Cells
500
4 00
A
A
A
A
300
h
$200
A
m
E
v
L
A
A
A
I00
A
@ A A
A
0
0
cn
3
m
U
0
0
Glycogen lnfil tration
m 500
I
I
-
0
400
AA A0 AA
AA M AA
I
I
oq
00
8208a
AA
A
L
cu
c
0
300
200
I00
C
1
I
1
1
1
l
1
~
1
~
~
2 3 4 5 6 7 8 9 10
Age of
Offspring ( d a y s )
Untreated: 0
Absent
Treated:
A
Slight
0
A Moderate
0
A
Marked
Fig. 4 Pancreatic islets in postnatal offspring from diabetic mothers. Dashed lines assist
the reader in seeing where most degranulation and most glycogen infiltration were observed.
The symbols used are the same as those in figure 3.
DIABETES : PANCREASES O F OFFSPRING
Postnatal off spring
Normal pancreases. The number of
pancreases examined at each age is presented in table 1. The beta cells were intensely granulated, and usually occupied
the central portions of the islets (fig. 12).
Alpha granules were observed as early as
24 hours after birth in the cells located at
the periphery of the islets. Alpha granulation increased progressively through the
third day, at which time the alpha cells
were well differentiated. Glycogen was
usually not found in the islets (fig. 20).
Rarely a trace of it was found.
Pancreases of t h e diabetic series. Although the blood sugars of newborns
from diabetic mothers returned to normal
during the first 24 hours (as early as 4
hours), the islets continued to show cytological alterations for a number of days
after birth.
The offspring from diabetic mothers
whose blood sugars at parturition were
greater than 240 mg% showed less than
normal granulation of the beta cells during
the first three days after birth (figs. 4, 13).
The granulation increased progressively
with time. By the 5th day, all pancreases
showed normal beta cell granulation.
All except one of the offspring from
mothers whose blood sugars at parturition
were below 240 mg% showed normal beta
cell granulation (fig. 14). The one exceptional offspring was from a mother whose
average blood sugar during pregnancy was
266 mg% (average of 9 determinations)
but whose blood sugar on the day of parturition was only 160 mg% .
The offspring from mothers with blood
sugars greater than 240 mg% had glycogen deposits in the islets during the first
three days following birth (figs. 4, 21).
The glycogen infiltration decreased progressively with time. By the 7th day, all
but one of the pancreases lacked glycogen.
By the 9th day, all lacked it.
All except three of the offspring from
mothers with blood sugars below 240 mg%
at parturition lacked glycogen deposits
(fig. 22). The three exceptional offspring
were from mothers in which the blood
sugars ranged from 285 to 385 mg% during the period of one to two days before
parturition, but in which the parturition
blood sugars were below 240 mg% .
247
DISCUSSION
In utero and at birth, the blood sugars
of fetuses from diabetic mothers approximated those of their mothers. This is in
agreement with the results reported by
others (Frye, '57; Scow, '58).
Our findings on the differentiation of
islet tissue of the rat are similar to those
recorded by Bensley ('11), Hard ('44) and
Frye ('57).
In the diabetic series, the beta cells were
almost completely degranulated, whereas
their cytoplasm showed glycogen infiltration. These changes were observed in all
offspring in which the maternal blood sugars were greater than 240 mg%. During
the period between the 19th and 21st days,
degranulation and hydropic degeneration
likewise occurred when the blood sugar
was greater than 175 mg% ; at birth, however, these two changes were not observed
unless the maternal blood sugar was
greater than 240 mg%. It has been reported that in the pancreases of adult diabetic rats treated with insulin, glycogen
infiltration was observed in the islets only
when the average blood sugar was greater
than 325 mg% (Kim, '59). These findings
suggest that the maternal hyperglycemia
leads to degranulation, hydropic change,
and glycogen infiltration in the islets of
the offspring and that relatively smaller increases in the maternal blood sugar level
are more effective in inducing these
changes in young fetuses (19th to 21st
day) than in older offspring (birth).
During the postnatal period, glycogen
infiltration usually disappeared by the 7th
day. The beta granulation was usually restored to normal by the 5th day.
Our observations pointed to the conclusion that the pancreatic islets in the fetal
rat start functioning at the 17th or 18th
day of gestation. The beta cell granules
were observed in the fetal islets at the 18th
day, and, in view of the studies of Wrenshall, Hartroft and Best ('54), it might be
assumed that these granules represent
stored insulin. The beta cells responded to
hyperglycemia in two ways: they lacked
granules (became degranulated ? ) and
they showed glycogen infiltration when the
maternal blood sugar exceeded a threshold
value. The glycogen infiltration was found
in those islet cells which are centrally lo-
248
JAE NAM KIM, WALTER RUNGE, LEMEN J. WELLS AND ARNOLD LAZAROW
cated and which contain a diminished
number of granules. In other words, it was
found in the beta cells, both completely
degranulated and partially degranulated
beta cells. It was not found in the alpha
cells of the pancreases in the postnatal offspring. These findings led us to believe
that in the fetal islets of the diabetic series beta cells are already differentiated
even though they lack the granulation observed in normal islets. Even in the islets
showing only a very small number of
granulated beta cells (less than 1% of
total number of islet cells), the actual
number of beta cells appeared to be much
larger. In the offspring of diabetic rats,
the number of islet cells with glycogen deposits appeared to be at least as large as
the number of granulated beta cells in
normal islets of the same age; in normal
fetuses of the 22nd day, 42% of the total
islet cells were granulated beta cells. Thus,
the decreased number of granulated beta
cells in the fetuses of the diabetic series
might be due to a physiological response to
a threshold level of hyperglycemia. It is
probable that the cells showing glycogen
Mltration in the fetal islets of a 17-day
fetus from a diabetic mother are actually
beta cells even though they do not show
beta granulation.
SUMMARY AND CONCLUSIONS
Experimental diabetes was induced,
either before or after conception, by alloxan injection or by pancreatectomy. Diabetic pregnant rats were either treated with
insulin or not treated.
Fetal blood sugars are approximately the
same as the maternal blood sugars in pregnant normal rats and in pregnant diabetic
rats.
The blood sugar levels of the neonatal
offspring born to diabetic mothers drop to
normal levels within less than 24 hours.
Beta granules are first observed in the
islets of normal fetuses at the 18th day of
gestation.
Glycogen infiltration in the islet cells of
the diabetic series is first observed at the
17th day of gestation.
Prenatal offspring and newborns from
mothers with blood sugars greater than
240 mg% invariably show glycogen infil-
tration and decreased beta granulation
(degranulation) in the islets.
Fetuses and newborns from diabetic
mothers with blood sugars less than 175
mg% show normal beta cell granulation
and an absence of glycogen infiltration.
By day 4 and day 6 , respectively (following parturition), beta granulation returns
to normal.
Hydropic change or glycogen infiltration
occurs in the beta cells of islets from offspring of the diabetic series but not in the
alpha cells. Glycogen infiltration seems
to be a sensitive indicator of beta cells
even before beta granulation appears.
The fetal pancreases of the untreated
diabetic series show hyperplasia and hypertrophy of the islets.
These observations suggest that the beta
cells begin to function at the 17th or 18th
day of gestation.
LITERATURE CITED
Bensley, R. R. 1911 Studies on the pancreas of
the guinea pig. Am. J. Anat., 12: 297-388.
Ferner, H., and W. Runge 1956 Morphologische
Untersuchungen iiber die Wirkung des NIsulf anilyl-Nz-n-butylcarbamid auf die Insellen
von Kaninchen und Ratten. Arzneimittel-Forschung, 6: 256-260.
Folin, O., and H. Malmros 1929 Improved form
of Folin's micro method for blood sugar determination. J. Biol. Chem., 83: 115-120.
Frye, B. E. 1957 Differentiation of the endocrine pancreas in fetuses of alloxan diabetic and
insulin treated rats. J. Morph., 101: 325-357.
Gendre, H. 1937 A propos des procbd6s de fixation et de dbtection histologique du glycogene.
Bull. d'Histol., 14: 262-264.
Hard, W. L. 1944 The origin and differentiation of the alpha and beta cells in the pancreatic islets of the rat. Am. J. Anat., 75: 369403.
Kim, J. N. 1959 Threshold of blood glucose i n
glycogen infiltration of pancreas and kidney
in diabetic rats. Anat. Rec., 133: 399.
Kim, J. N., W. Runge, L. J. Wells and A. Lazarow 1958 Pancreatic islets in fetuses and
offspring of diabetic rats. The Physiologist, 1:
40-41.
1960 The effects of experimental diabetes on the offspring of the rat: fetal growth,
birth weight, gestation period and fetal mortality. Diabetes, in press.
Lazarow, A,, J. N. Kim and L. J. Wells 1960
Birth weight and fetal mortality in pregnant
subdiabetic rats. Ibid., 9: 114-117.
Lazarow, A., and S. L. Palay 1946 Production
and course of alloxan diabetes in the rat. J.
Lab. Clin. Med., 31: 1004-1015.
249
DIABETES : PANCREASES O F OFFSPRING
Lillie, R. D. 1954 Histopathologic Technic and
Practical Histochemistry. Blakiston Co., Philadelphia.
Scow, R. O., S. S. Chernick and B. Smith 1958
Ketosis in rat fetus. proc. sot. E ~ ~ ~i ~&fed,,
. l ,
98: 833-835.
SomogYi, M. 1940-41 A rapid method for the
estimation of urine sugar. J. Lab. Clin. Med.,
26: 1220-1223.
Wells, L. J. 1950 Subjection of fetal rats to
surgery and repeated subcutaneous injections:
method and survival. Anat. Rec., 108: 309432.
lg6'
L* J . 7 J* N' Kim and
Effects of insulin on the ComPfications of diabetes and pregnancy i n the rat. Diabetes, in
press.
Wrenshall, G. A., W. S. Hartroft and C. H. Best
1954 Insulin extractable from the pancreas
and islet cell histology. Ibid., 3: 444-452.
**
PLATE 1
EXPLANATION OF FIGURES
5
Section of pancreas from a normal newborn, showing the islet (I) and acinar ( A ) tissues. 5 p, Bouin’s solution, aldehyde fuchsin. X 95.
6
Section of pancreas of newborn from an untreated diabetic mother. Note extensive area
of islet tissue ( I ) and close association of islets with exocrine ducts ( D ) . A, acini. 5 p,
Bouin’s solution, aldehyde fuchsin. X 95.
7 and 8 Adjacent sections of the pancreas of a three-day postnatal rat from a n untreated
diabetic mother.
7 The arrow marks a beta cell which contains granules and a vacuole (hydropic change).
5 p, Bouin’s solution, aldehyde fuchsin. x 1125.
8
250
The arrow marks the same beta cell which is marked by the arrow in figure 7. This
beta cell contains intracytoplasmic glycogen (black dots). Around it are other beta cells
which likewise show glycogen infiltration, even though they do show considerable beta
granulation (fig. 7). 5 p , Bouin’s solution, PAS. X 1125.
PANCREASES OF OFFSPRING
Jae N a m Kim, Walter Runge, Lemen J. Wells and Arnold Lazarow
PLATE 1
251
PLATE 2
EXPLANATION O F FIGURES
9 Section of pancreas of a newborn from a n untreated diabetic mother (enlargement of
part of fig. 6). The beta cells show no granules, but show hydropic change (vacuoles).
The arrow indicates one of the vacuolated beta cells. 5 p , Bouin’s solution, aldehyde
fuchsin. X 500.
10 Section of pancreas from a normal newborn. Note granulated beta cells i n the central
portion of the islet. 5 p, Bouin’s solution, aldehyde fuchsin. x 500.
11
252
Section of pancreas of newborn from a treated diabetic mother in which the blood sugar
was below 175 mg%. Islet shows normal beta granulation. 5 p, Bouin’s solution, aldehyde fuchsin. X 500.
PANCREASES OF OFFSPRING
Jae Nam Kim, Walter Runge, Lemen J. Wells and Arnold Lazsrow
PLATE 2
253
PLATE 3
EXPLANATION OF FIGURES
12 Section of gancreas of a three-day normal rat. The central portion of the islet is occupied by the beta cells ( B ) , the peripheral portion by the alpha cells ( A ) , A duct is
i n contact with the islet (left of center). 5 p, Bouin’s solution, aldehyde fuchsin. x 500.
13
Section of pancreas of a three-day rat from a n untreated diabetic mother. Occasional
beta cells have acquired granules after birth (arrows), but most of the islet cells lack
beta granulation. 5 p, Bouin’s solution, aldehyde fuchsin. x 500.
14 Section of pancreas of a three-day rat from a treated diabetic mother. The islet shows
normal beta granulation. 5 p, Bouin’s solution, aldehyde fuchsin.
254
x
500.
PANCREASES OF OFFSPRING
Jae Nam Kim, Walter Runge, Lemen J. Wells and Arnold Lazarow
PLATE 3
255
PLATE 4
EXPLANATION OF FIGURES
15
Section of pancreas from a 17-day normal fetus showing a small islet ( I ) . No glycogen
is seen i n the islet cells. 5 p, Gendre’s solution, PAS. x 1000.
16
Section of pancreas from a 17-day fetus from a n untreated diabetic mother. Note glycogen particles in the islet cells (black). 5 p, Gendre’s solution, PAS. x 1000.
17
Section of pancreas from a normal newborn. The islet (most of photo) shows no glycogen. 5 p , Gendre’s solution, PAS. X 1000.
18 Section of pancreas of newborn from an untreated diabetic mother. The centrally
located cells in the islet show glycogen infiltration (black). The ductular epithelial cells
( D ) and the peripherally located islet cells (site of future alpha cells) usually contain
n o glycogen. 5 p , Gendre’s solution, PAS. X 500.
256
PANCREASES OF OFFSPRING
Jae N a m Kim, Walter Runge, Lemen J. Wells and Arnold Lazarow
PLATE 4
257
PLATE 5
EXPLANATION O F FIGURES
19 Section of pancreas of newborn from a treated diabetic mother. The islet shows n o
glycogen. 5 p, Gendre’s solution, PAS. x 500.
258
20
Section of pancreas from a 3-day normal rat. Glycogen is absent in the islet cells ( 1 )
and in ductular cells ( D ) . 5 p , Gendre’s solution, PAS. x 500.
21
Section of pancreas of a three-dav rat from a n untreated diabetic mother. showing two
islets. The-centrally located cells -in both islets contain glycogen. 5 p, Gendre’s sol;tion,
PAS. x 500.
22
Section of pancreas of a three-day rat from a treated diabetic mother. Glycogen is not
present in the islet cells. In the lumina of the capillaries ( C ) fine particles of glycogen
are seen. 5 .u, Gendre’s solution, PAS. X 500.
PANCREASES O F OFFSPRING
Jde N a m Kim, Walter Runge, Lemen J. Wells and Arnold Lazarow
PLATE 5
259
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