The development of the fetal rat intestine and its reaction to maternal diabetes

Diabetes Research and Clinical Practice, 6 ( 1989) 213-2 19

213

Elsevier DRC 0027 I

The development of the fetal rat intestine and its reaction to maternal diabetes II. Effect of mild and severe maternal diabetes B. Reusens-Billen’,

C. Remacle’

and J.J. Hoet2

‘Laboratory of Cell Bio1og.y. University of Louvain, Louvain-la-Neuve, Belgium and ‘Department of Medicine, University of Louvain, Brussels, Belgium

(Received 26 July 1988, accepted 26 October 1988)

Key words: Fetal rat intestine; Morphometry:

Maternal diabetes

Summary Diabetes during pregnancy induces specifically structural and functional changes in the fetal endocrine pancreas. Other organs are affected as well. In this study, the fetal intestinal tract which is in close connection with the endocrine pancreas was analysed during diabetic gestation. The disease was induced by two different doses of streptozotocin which led to a mild or severe diabetic state in the mother. In fetuses from mildly diabetic as well as from severely diabetic rats, the time sequence in the appearance of the differentiated cells was identical and similar to that of controls. However, morphometric analysis of the intestine of fetuses from severely diabetic rats revealed a decrease in each of the parameters measured which led to a general hypotrophy of the intestine. In the fetuses from mildly diabetic rats, the values of the morphometric parameters of the duodenal mucosa remained unchanged and comparable to those of the control group. The vascularisation of the duodenum is modified in these fetuses because the volume density of the blood vessels is significantly increased. In conclusion, both diabetic states of the mother induce various alterations in the fetal intestine, including the blood vessels. The nature of the structural changes observed in the intestine could lead to modifications in the function of the entero-pancreatic system in these fetuses.

Address for correspondence: toire

de Biologie

0168-8227/89/$03.50

cellulaire

Dr. B. Reusens-Billen,

(BANI/CELL),

Universiti:

IQ 1989 Elsevier Science Publishers

LaboraCath-

olique de Louvain, Neuve, Belgium.

B.V. (Biomedical

Division)

Place Croix du Sud, 5. B 1348. Louvain-la-

214 Introduction

characterisation

of the two diabetic

states is report-

ed in Table 1. In the severely diabetic

group, fetuses

Diabetes during pregnancy affects fetal development in the human as well as in animal models.

were removed on days 17.5, 18.5, 19.5, and 21.5 of gestation. In the mildly diabetic group, they were

Fetuses born from diabetic mothers show abnormal birthweight and disturbances in the development of

removed

the endocrine pancreas [14]. Other organs, such as lung, heart, kidney, and skeleton, could also be al-

Microscop_v and morphometrl For light and electron microscopy,

tered when the diabetes is not we!! controlled in the mother [557]. In this pathological condition, at least

tive tract was prepared as described elsewhere (companion paper). On paraffin and semi-thin sec-

glucose is increased in the maternal and fetal plasma, and also enhanced in the amniotic fluid [8,9] as well as in the fetal urine and gastric juice [9]. This is associated with a stimulated secretion and proliferation of the insulin cells. The development of the fetal intestine has never been analysed, and could be affected in this condition. In the present study, we describe and quantify by morphometric analysis the effects of mild and severe maternal diabetes on fetal intestinal maturation.

tions, the increment of absorptive area due to the villi were computed as described (see p. 199). To calculate the volume density and the size distribution of the blood vessels, three duodenal levels were analysed. On each section, four fields were randomly selected in the half of the duodenum opposite to the mesenteric connection. In each field, the surface area of all the individual blood vessels was measured in the subepithelial layers, as well as the total measured duodenal area. On electron micrographs, the increment of absorptive area due to the microvilli was also calculated as described (see p. 199).

on day 21.5.

the fetal diges-

Material and methods Results Animuls Wistar rats were used in these experiments. Mild or severe diabetes of the mother was induced by streptozotocin injection on the first day of gestation. The

TABLE

I

FETAL

WEIGHT

OF MlLDLY

AND GLUCOSE

OR SEVERELY

CONCENTRATION

DIABETIC

4.45 f 0.06 (8)

Glu~rsr concenrration Maternal

77 f

Fetal plasma Amniotic fluid

Mild diabetes

Severe diabetes

(30 mg streptozotocin/kg)

(50 mg streptozotocin/kg)

4.51 f- 0.08 (8)

3.16 * 0.16 (8)*

3.x (6)

345 * 20 (5)**

411 +

7 (5)**

336 i

I4 (S)**

32 * 3.2 (6)

284 * I5 (5)” 264 f 21 (5)**

32 f

247 * 20 (s)**

307 f

56 It 3

~~~ ~-

(6)

2.3 (6)

~~____

~._

Results are expressed as mean f SEM. Figures in parentheses give numbers of animals. For fetal weight, means were compared to control values using Student‘s r-test. l

P < 0.05.**

IN THE FETO-MATERNAL

(mgidlI

plasma

Fetal gastric juice __._

AT 21.5 DAYS OF GESTATION

RAT

Control

Fetal weight (g)

Development of’ the intestinal tract in ,fetuses ,from severely diabetic rats When analysing the morphological events specific

P < 001

392 & 28 (5)** 14 (5)**

UNIT

215

Fig.

I Light micrographs

of transverse

sections

in the duodenum diabetic

mothers

for each gestational day, no major differences appeared in the developmental time sequence between fetuses from control and severely diabetic mothers. In particular, the first villi as well as small microvilli and endocrine cells appeared on day 17.5, the goblet cells developed on day 18.5, and a duodenal structure similar to that observed in postnatal rats was reached on day 21.5 (Fig. 1).

of 21 .Sday-old (bottom

panel).

fetuses from control

mothers

(top panel) and severely

x 85.

The morphometric study was performed on paraffin sections of duodenum and jejunum of nine fetuses from three different severely diabetic mothers at 17.5, 19.5 and 21.5 days of gestation. Significant differences appeared when the parameters were compared to those of the control fetuses (Table 2). On day 17.5, the inner circumference and total length of the villi profiles were reduced in the duo-

216 TABLE

2

MORPHOMETRIC RATS

ANALYSIS

PERFORMED

ON PARAFFIN

SECTIONS

OF FETUSES

FROM

SEVERELY

DIABETIC

_~___ Duodenum Control

group

Diabetic

group

Control

group

Diabetic

group

17.5 JLI?:Y IC Wn)

715 f

2x

588 f

19*

667 f

20

502 *

Is*

LV (pm)

375 *

33

249 f

22*

331 *

31

218 f

Is*

1289 f

31*

19.5 dULY IC Wm)

1868 f

44

II50 I

27*

1436 f

25

LV (km0

x535 +

390

3774 f

l48*

5756 f

222

X

4.54 f 0.14

3.27 f 0.09*

4446 + 251*

3.97 f 0.1

3.34 f 0.1*

21.5 dqs

IC 0tm) LV (pm)

3040 f

X

2593 + 18227 i

6.7X * u.12* ~~ .~__~

___

16

2141 f

1000

51*

14515 f 62I*

x.1 f 0.18

6.71 f 0.1X* __.~__

LV, total length of the villi profile: X, LV/IC.

SEM of six fetuses from three different

Means were compared

66*

18045 + 769*

7.61 f 0.23

IC. inner circumference; X f

2708 l

100

23530 * 1454

by using Student’s

mothers,

four sections

per fetus.

r-test. * f < 0.005.

denum and jejunum. On days 19.5 and 21.5, the same parameters were also decreased in the severely diabetic group, as well as the increment in absorptive area. The height of the microvtlli calculated on electron

micrographs was reduced on day 19.5, albeit not significantly; the level of significance, however, was reached on day 21.5. The increment of the absorptive area due to the microvilli was significantly reduced at 19.5 as well as at 21.5 days (Table 3).

TABLE

TABLE

3

HEIGHT

OF THE

THE ABSORPTIVE

AREA

IN THE DUODENUM

4

INCREMENT

OF

MORPHOMETRIC

DUE TO THE MICROVILLI

(Yl

SEMI-THIN

MICROVILLI OF FETUS

AND FROM

SEVERELY

DIA-

FROM

NORMAL

OF

CALCULATED

DUODENUM

AND MILDLY

DIABETIC

OF

ON FETUS

RATS AT 21.5

DAYS

BETIC RATS -__ Control

group

Diabetic

0.68 f 0.016 4.98 f 0.23

.._ Control ~__~~..

group

group ~_

Diabetic

group

EC @ml

3449 +

73

3362 zt

0.66 zk 0.01

IC (mn)

3160 *

59

3028 zk 59

4.23 * 0.2*

LV (pm) X

IY.5 tfui:v height (pm) Y

PARAMETERS

SECTIONS

19872 zt 559 6.28 f 0.14

18679 f

48 733

6.12 + 0.2

21.5 rlars height (pm) Y X f

1.42 + 0.04 13.08 * 0.53

I.1

SEM of six fetuses from three different

surements. Means were compared

by using Student’s

* I’ < 0.025. ** P < 0.005.

f O.Oh**

10.48 zk 0.66** mothers,

I-test.

EC. external

circumference;

IC, inner circumference;

length of the villi profile: X, LVIIC. X + SEM of 15 fetuses from five different I8 mea-

per fetus. * P < 0.005.

mothers.

LV. total one section

217

Effect of milddiahetes in the mother on the duodenum of 21 S-day-old fetuses Semi-thin duodenal sections in 15 fetuses from five different mothers (three fetuses per mother) were observed in the control and mildly diabetic groups. Table 4 shows that the general morphometric parameters were similar in the two groups. By contrast, the volume density of the blood vessels was increased in the diabetic group, where it reached 5.22% vs. 3.69% in the control group. A two-way analysis of variance indicated that the difference was statistically significant (F = 69.33, P < 0.005). The size distributions of the vessels were then compared in both groups with the Kolmogorov-Smirnov test, and found to be similar.

Discussion

When mild diabetes is present in the mother, the vascularisation of the fetal duodenum is affected by the disturbance of the metabolic environment. The size distribution of the blood vessels is identical in the control and the diabetic group, but the volume density of these blood vessels is increased in the diabetic group. Therefore, the increase in volume density would result from an increased number of blood vessels. Hence mitogenic factors for the endothelial cell have to be evoked. Among these, insulin, insulin-like growth factors (IGFs), and even glucose could be responsible for the increased vascularisation in the duodenum. Fetuses from mildly diabetic mothers have elevated blood sugar and their amniotic fluid contains a high glucose concentration [8,9]. Glucose is permissive to mitosis. In addition these hyperglycaemic fetuses feature high plasma insulin levels. Bar [lo] has shown that endothelial cells have insulin receptors and King and Buzney [11] indicate that insulin has a growth-promoting action on the endothelial cells, at least in microvessels. Moreover, IGF levels are modulated by insulin levels [12]. IGF activity is higher in the fetuses from mildly diabetic rats than in the controls [13]. The proliferation of the endothelial cells of the calf retina as measured by thymidine incorporation in vitro is stimulated by IGF-I, IGF-II and insulin. This ef-

feet appears to be specific for endothelial cells because the vascular supporting cells are not stimulated by these factors [14]. The selective site of action of these growth-promoting factors is also apparent in our observations, where the enhanced vascularisation was not associated with an increased proliferation of the epithelial cells. We did not quantitate the endocrine cells in the intestinal mucosa. The effect of mild diabetes on the latter cannot be excluded, in particular on the gastric inhibitory peptide cells which could be challenged more in the hyperglycaemic environment. Mild hyperglycaemia of the mother did not seem to exert a trophic action on the fetal intestinal mucosa while this action might be seen specifically in the fetal pancreas, where islet size [2], B-cell proliferation [3,4] and B-cell function [15] are increased. Mild diabetes of the mother would therefore affect the duodenum and the pancreas but would not exert a trophic action on the enterocytes. In fetuses from severely diabetic mothers, the inner circumference, the length of the villi profiles, and the enlargement of the surface area due to the villi were significantly decreased in duodenum and jejunum. At the ultrastructural level as well, the length of the microvilli and their contribution to the increment in surface area were reduced. Moreover, the fetal endocrine pancreas shows decreased B-cell function [ 15-171, a reduced B-cell proliferation [ 181, and a reduced B-cell mass [2,19]. In this specific pathological condition of the mother, the fetuses are born small for date. In addition, in a situation where the maternal environment has been modified by protein malnutrition, the development of the fetal intestine is also altered in a manner comparable to our observations in fetuses of severely diabetic mothers [20]. In the former condition, the intestinal weight and the length are reduced as well as other parameters of intestinal growth. More specifically the number of villi per length of intestine, which reflects what we described as the increment of the absorption area due to the villi, is also decreased. In adult animals, the loss of intestinal tissue was more marked for the mucosa than for the intestinal muscle or matrix, when caloric deprivation was induced [21]. In severe diabetes as well as in malnutrition,

218 the growth retardation of the fetal intestinal mucosa could be part of a general hypotrophy, but the

( 1984) Cell proliferation

local changes that we observed could also be related to fewer nutrients present in the fetal digestive tract.

in vivo study.

Although

the lack of normal

may be related

nutrients

to the inhibition

in the gut

of the mucosal

growth, other growth factors such as insulin and IGF have to be taken into account. Fetal insulin plasma level is reduced in severe diabetes of the mother [l&17]. In conclusion, the time sequence in the appearance of differentiated cells was identical in the gut mucosa of control and diabetic groups. Growth retardation of the intestinal mucosa was detected in the pups of severely diabetic mothers and an enhanced vascularisation was demonstrated in the duodenum of pups from mildly diabetic mothers by our morphometric analysis. These data demonstrate changes in the entero-pancreatic system of pups born from mildly or severely diabetic mothers, and could contribute to the modifications observed in the fetal endocrine pancreas in these pathological conditions.

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