RAT FOETAL ENDOCRINE PANCREAS IN EXPERIMENTAL DIABETES L. AERTS

AND

F. A. VAN ASSCHE

Laboratory of Gynecological Physiopathology, Academisch Ziekenhuis (KUL), Leuven, Belgium

(Received 2 August 1976) SUMMARY

The endocrine pancreas of foetuses and newborn rats of experimental diabetic mothers showed morphological and ultrastructural changes. Islet hypertrophy and \g=b\cell hyperplasia were constantly present, but the \g=b\ cells of foetuses of severely diabetic mothers were degranulated. The ultrastructural changes indicated hyperfunction in the \g=b\cells of foetuses of experimental diabetic mothers. The morphological changes mentioned were similar to those seen in human diabetic pregnancy.

INTRODUCTION

have shown that infants of diabetic mothers show morphological and functional features of hyperinsulinism (Van Assche, 1970; Van Assche & Gepts, 1971). Work on the rat and rabbit has shown that offspring of experimental diabetic mothers have hyperfunction of the endocrine pancreas (Gepts, 1957; Kim, Runge, Wells & Lazarow, 1960; Rishi, Golob, Becker & Shah, 1969; Wellman, Wölk, Lazarus & Branceto, 1969; Golob, 1970; Pitkin & Van Orden, 1974). The aims of our study were to investigate the morphological and functional behaviour of the rat foetal and neonatal endocrine pancreas in experimental diabetes and to correlate these findings with our data in man. In

man we

MATERIALS AND METHODS

The animals studied comprised foetuses and newborn pups of normal pregnant albino Wistar rats (Group I) and pregnant rats injected with a single dose of 30 (Group II), 40 (Group III) or 50 mg (Group IV) streptozotocin/kg body weight into the tail vein on day 1 of gestation (day of mating). There were 30 pregnant animals in each group, since the final purpose was a follow-up of the offspring until 60 days afterbirth. The endocrine pancreas of the offspring was studied at days 20 and 22 in utero and between 3 and 10 h on the day of birth. Blood from non-fasting mothers was collected from the tail vein for glucose deter¬ mination using a method adapted from Hoffman (1937). Twenty-four-hour urine collections on days 17 and 22 were performed for estimation of glucosuria (Clinitest, Ames), acetonuria (Acetest, Ames) and proteinuria (BM test, Boehringen). Pooling of foetal and neonatal blood per litter was performed for the determination of blood glucose and serum insulin concentration (Morgan & Lazarow, 1963) for five litters in each group. From at least ten litters of each group, pancreases were carefully dissected, weighed and fixed in Bouin's solution. Staining was performed using haemalum eosin and Ivic's (1959) method. For electron optical examination pancreases from four animals from each group

studied. The tissue was fixed for 2 h in 25 % glutaraldehyde and 4 % paraformaldehyde (6:10, v/v) in cacodylate buffer (pH 7-2). Post-fixation was done in 1 % Os04 in phosphate buffer (pH 7-4) for 1 h. The tissues were embedded in Epon and sectioned with glass knives on an LKB ultramicrotome. The sections (±700 Â) were placed on 200 mesh copper grids, stained with uranyl acetate and lead citrate and examined in a Zeiss Em 9S2 electron were

microscope. For the quantitative study we used the standard stereological procedure of Weibel (1969). At the light optical level the volume density of the islets in the pancreatic tissue was deter¬ mined by counting at least 2000 points (real distance of 004 mm) on each of the pancreases studied per group (between 14 and 23 pancreases, Table 1). For quantitation at the ultra-

structural level between 15 and 20 islets from four different foetal or neonatal pancreases used from each group. From each islet studied three micrographs (final magnification 28 500) of ß cells were taken at random and morphometric data were collected using a test grid with 1024 points (real distance 0-175 µ ) with a Kontron MOP analysing system. The following measurements were performed on the ß cells : numerical density of light (NVL) and dark (NVD) granules, volume density of light (VVL) and dark (VVD) granules, volume density of mitochondria (VVM) and volume density of the rough endoplasmic reticulum (VVR). The results of the different groups were compared using the statistical method of Kruskal-Wallis. This method is used in groups of different number and distribution were

(Siegel, 1956). RESULTS

General features The litter sizes were equal in the various groups. Increased neonatal loss was observed in the severely diabetic groups between days 1 and 4 after birth. As shown in Table 1, the 30 mg streptozotocin group (Group II) had significantly higher maternal mean blood glucose levels than the control group (Group I) and can therefore be considered as having mild diabetes (P < 005). By the same criterion, the 40 mg streptozotocin group (Group III) (P < 0-01) and Table 1. Blood glucose, various

body and pancreatic weights experimental groups (means±s.o.)

in the

Foetal and neonatal : Maternal blood

Day 20: Group 1 C Group II S30 Group III S40 Group IV S50 Day 22: Group I Group II Group III Group IV

C S30 S40 S50

Day of birth : Group I C Group II S30 Group III S40 Group IV S50

glucose (mg/100 ml)

Blood glucose

132+11 231 ±17 482 ±20

92±6(18) (19) (20) (17)

32 ±9 (4) 22 ±3 (5) 294 ±84 (5) 287 ±54 (4)

203±011 (18)

78 ±9 (51) 96+11 (41) 282 ±45 (38)

107 ±20 (4) 95 ± 15 (5) 299 ±50 (5)

4-70 ±0-33

(36)

324±52(5)

3-89±0-52 (32)

(33)

2005±4-58 (29) 20-93 ±4-18 (28)

83 ±14 (24) 108 ±7 (32) 435 ±73 (34) 461 ±74 (44)

84±17(5)

4-64 ±0-56 (36) 5-44 ±0-57 (32) 4-62 ±0-68 (34) 4-91 ±0-49 (44)

23-99±5-98(36) 30-71 ±7-29 (28) 30-40±505 (30) 30-31 ±7-43 (36)

361 ±41

(mg/100 ml)

94±15 (16) 231 ±57 (34) 203 ± 32

(33)

Body wt (g)

Pancreatic wt (mg)

5-70±l-70(18)

l-97±0-18(19) 1-88 ±0-35 (20) 1-59±018(17)

710±110(19) 5-70±l-80(20) 4-30±l-00(17)

(47) 4-90±0-85(36) 3-84 ±0-41

Number of observations is given in parentheses. C, Control; S30, S40, S50: 30, 40 and 50 mg streptozotocin/kg body wt

injected

26-28 ±5-36 (43) 27-31 ±405 (32)

on

day 1 of gestation.

the 50 mg

streptozotocin

group

(Group IV) (P < 0001) had severe diabetes. Mild streptozotocin group. In the two severely diabetic

present in the 30 mg

glucosuria groups glucosuria was more pronounced, albuminuria and acetonuria were also found in the severest diabetic rats (Group IV). Table 1 shows that at day 20 foetal blood glucose was low, about 40 %of that of the mother in Groups I and II. In Groups III and IV the foetal blood glucose was higher, at about 75 % of that found in the mother. At day 22, a marked increase was found in the foetal blood glucose in Groups I and II (P < 0001) but no significant changes were present in Groups III and IV compared with day 20. In Groups I and II the foetal blood glucose level was very close to that of the mother at day 22. On the day of birth, no change was observed in the neonatal blood glucose in Groups I and II but there was a significant fall in Groups III and IV (P < 0005) compared with day 22. This was also reflected in the ratio between foetal and maternal glycaemia. At day 20 (Table 1) the foetal weight was significantly (P < 0-05) lower in Group IV than in the controls. From days 20 to 22 foetal weight increased in all groups (P < 0-001); by day 22 the greatest increase was in Group II and the smallest increases were in Groups III and IV. At day 22 the foetal weight was significantly lower in the severely diabetic groups (III and IV) compared with the controls (I) and the mildly diabetic group (II). By parturition the weight of the newborn pup was further significantly increased (P < 0001) in the streptozotocin groups but the weight of the controls was the same as that on day 22. At day 20 the pancreatic weight was greater in Group II (P < 005) but less in Group IV (P < 005) than in the controls. For all groups there was a four to five times increase of the pancreatic weight between days 20 and 22 at which time the weight in Groups III and IV was less than in Groups I and II. At the first postnatal day the pancreatic weight was increased further in all streptozotocin groups (P < 005) (Table 1). The insulin level in foetal blood increased in all Groups from days 20 to 22, but decreased at birth (Table 2). At day 22 there was a statistically significant decrease in foetal serum insulin between Group III and Groups I and II (P < 005). The apparent increase in foetal serum insulin in Group II as compared with Group I was not statistically significant but may prove to be so when larger numbers of animals are studied. was

Table 2. Insulin concentrations (ng/ml) in foetal and neonatal blood serum in the various groups (means ± 1 s.d.) Day 20: Group I Group II Group III Group IV

C S30 S40 S50

Day 22: Group I C Group II S30 Group III S40 Group IV S50 Day of birth: Group I C

Group II S30 Group III S40 Group IV S50

2-35±118(4) 1-65 ±0-45 (5) 1-72 ±0-37 (4) 1-56 ±0-46 (6) 5-45 ±0-81

(4) 806±312(5) 3-98 ±0-61 (5) 4-82 ± 0-59 (4) 1-98 ±0-45 (5) 1-39 ±0-24 (5) 1 -83 ± 0-44 (5) 1-38 ±0-23 (4)

Number of observations is given in parentheses. C, Control; S30, S40, S50: 30, 40 and 50 mg streptozotocin/kg body wt injected

on

day 1 of gestation.

Morphological aspects the endocrine tissue was higher than the controls (P < 0001) in all percentage day 20, At groups streptozotocin (Table 3). day 22 the percentage endocrine tissue was only signifi¬ increased in cantly Group I (the controls) (P < 0005) compared with day 20; there was even a decrease in Groups III and IV (P < 0-001). At day 22, the percentage endocrine tissue was not significantly different in the streptozotocin groups compared with the controls. At birth the percentage endocrine tissue increased significantly in all groups compared with the corresponding groups at day 22, and at birth there was a significant difference in the percen¬ tage of endocrine tissue between the control group and the streptozotocin groups (P < 0-05). The same trend was found in the weight of the total endocrine pancreatic tissue. In all streptozotocin groups, marked ß cell hyperplasia was found at all stages but in Groups III and IV the vast majority of the ß cells were degranulated. The granulated ß cells of Groups I (controls) and II (30 mg streptozotocin) were quantitated (Table 3). The percentage of granulated ß cells increased in both groups at the same rate but the percentage of granu¬ lated ß cells was less in Group II than in Group ( < 005). At

Table 3.

Quantitation of endocrine tissue in the pancreas in the groups (means ± 1 s.d.)

Day 20: Groupl C Group II S30

Group III S40 Group IV S50 Day 22: Groupl C Group II S30

Group III S40 Group IV S50 Day of birth: Groupl C Group II S30 Group III S40 Group IV S50

Endocrine tissue in pancreas (%)

2-0±0-7(18) 4-0±0-6(19) 5-4±0-9(20) 5-4+1-1 (19)

Granulated

ß cells in islets (%) 32-2 ±41 (10) 26-3 ±4-4 (10) —

various

Weight of endocrine tissue (mg/pancreas) 0-13 ±0-07

(18) 0-27±0-05 (19) 0-30±0-10 (20) 0-21 ±008 (19)



3-8±l-7 (18) 4-6±2-l (21) 3-3± 1-4 (14)

550±4-2(10) 47-6±2-7(10)

3-9±l-4(17)

0-96±0-45 (18) l-33±0-60 (21) 0-63 ±0-25 (14) 0-89±0-37 (17)

5-4±l-8 (17)

59-4 ±40 (10) 1-27 ±0-50 (17) 2-54± 1-26(16) 8-6±2-6(16) 54-7±4-7(10) 7-9±21 (16) 2-41 ±0-67 (16) 7-0±2-5(23) 204±0-76(23) Number of observations is given in parentheses. C, Control; S30, S40, S50: 30, 40 and 50 mg streptozotocin/kg body wt injected on day 1 of gestation. —



Ultrastructural aspects As in the pancreas of the adult rat, in the pancreas of the foetus and newborn pup dark and light (pale) granules in the ß cells could be distinguished. In Groups III and IV most of the ß cells were degranulated and the results from Group IV will be considered in this section as representing both groups (PI. 1, fig. 1). Distended rough endoplasmic reticulum (RER) with sacculation in the streptozotocin groups was present at all ages; this feature was also found in the controls at day 22 and at birth, but to a lesser extent. In the ß cells of foetuses and newborn pups of experimental diabetic mothers enlarged and swollen mitochondria were observed close to the distended RER (PI. 1, fig. 2; PI. 2, fig. 4). Glycogen deposition was observed in the 50 mg streptozotocin group (Group IV), seldom at day 20, but more frequently at day 22 and at birth in the degranulated ß cells (PI. 2, fig. 3). The volume density of the dark granules (VVD) increased with age (Table 4). Compared

with controls the volume density of the dark granules (VVD) was higher in the 30 mg streptozotocin group (P < 0-001 at birth), and lower in the 50 mg streptozotocin group (P< 0-01 at birth). Changes in the light (pale) granules were less pronounced. At day 22 the volume density of the light granules (VVL) was greater in the streptozotocin groups than in the controls (P < 0001). The volume density of the mitochondria (VVM) was not signifi¬ cantly different in the three groups at day 20 (Table 4). The two streptozotocin groups had a significantly higher volume density of the mitochondria at day 22 (P < 0005) and at birth (P < 005). The volume density of the RER increased with foetal age for Groups I (jP < 0-05) and II (P < 0001) but was unchanged for Group IV. At day 20 the volume density of the RER was not significantly different in the three groups. At day 22 the VVR in the 30 mg streptozotocin group was higher than in the control and in the 50 mg streptozoto¬ cin group (P < 0-0005). At birth only the difference between Groups II and IV was statisti¬

cally significant (P

Rat foetal endocrine pancreas in experimental diabetes.

RAT FOETAL ENDOCRINE PANCREAS IN EXPERIMENTAL DIABETES L. AERTS AND F. A. VAN ASSCHE Laboratory of Gynecological Physiopathology, Academisch Zieken...
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