This study was designed to assess the effect of maternal diabetes in rats on serum glucose and insulin concentrations, insulin resistance, histological architecture of pancreas and glycogen content in liver of offspring. The pregnant rat females were allocated into two main groups: normal control group and streptozotocin-induced diabetic group. After birth, the surviving offspring were subjected to biochemical and histological examination immediately after delivery and at the end of the 1st and 2nd postnatal weeks. In comparison with the offspring of normal control dams, the fasting serum glucose level of offspring of diabetic mothers was significantly increased at the end of the 1st and 2nd postnatal weeks. Serum insulin level of offspring of diabetic dams was significantly higher at birth and decreased significantly during the following 2 postnatal weeks, while in normal rat offspring, it was significantly increased with progress of time. HOMA Insulin Resistance (HOMA-IR) was significantly increased in the offspring of diabetic dams at birth and after 1 week than in normal rat offspring, while HOMA insulin sensitivity (HOMA-IS) was significantly decreased. HOMA beta-cell function was significantly decreased at all-time intervals in offspring of diabetic dams. At birth, islets of Langerhans as well as beta cells in offspring of diabetic dams were hypertrophied. The cells constituting islets seemed to have a high division rate. However, beta-cells were degenerated during the following 2 post-natal weeks and smaller insulin secreting cells predominated. Vacuolation and necrosis of the islets of Langerhans were also observed throughout the experimental period. The carbohydrate content in liver of offspring of diabetic dams was at all-time intervals lower than that in control. The granule distribution was more random. Overall, the preexisting maternal diabetes leads to glucose intolerance, insulin resistance, and impaired insulin sensitivity and
Maternal health plays a significant role in determining as well as predicating the health of the offspring later in their life [
Animal studies revealed that the offspring of diabetic rats have been shown to be insulin resistant [
A variety of diabetic animal models during pregnancy are used to assess long-term effects on the offspring. A concern of studies using STZ during pregnancy is the possibility that the toxin might cross the placenta and be directly harmful to the fetal pancreas and other fetal tissues, thus, making any analysis of the long term effects of hyperglycemia
In conduction with the previous studies, the current study aims to investigate the effect of preexisting experimentally induced diabetes mellitus in rat dams on the glycemic status, insulin resistance, and
Experiments were carried out on 65 white albino rats (
Adult rats were kept under observation for 2 weeks before experimentation to exclude any intercurrent infection and to acclimatize the animals to the new conditions. The selected animals were marked, housed in stainless steel cages with separate bottom, and kept at a temperature of
Diabetes mellitus was experimentally induced in female virgin animals fasted for 16 hours by intraperitoneal injection of 45 mg/kg b.wt. streptozotocin (Sigma-Aldrich Chemie GmbH, Germany) dissolved in citrate buffer (pH 4.5) [
To determine the estrus cycle, the vaginal smear of each virgin female was examined daily. Three types of cells, leukocytes and epithelial and cornified cells, were observed in photomicrographs of unstained vaginal smear. As reported by Marcondes et al. [
Proesterous normal and diabetic females were left for one night to copulate with the normal males (2 females with one male). Early next morning (before 7 am), copulation was checked by examining the outer surface of the vagina for the presence of a vaginal plug formed by coagulation of semen (white clotting, sperm clot). When such a grayish-white clot blocking the mouth of vagina was detected, this day was considered as the first day of gestation.
Each pregnant female was transferred into a separate cage and the weight gain was followed up throughout the pregnancy. Total numbers of 9 normal pregnant rats and 46 diabetic pregnant rats were included in the experiments.
After the birth, by the end of the 1st and 2nd postnatal weeks, blood and tissue samples of offspring of normal control and diabetic female dams were taken.
Blood samples of overnight fasted offspring of both groups were taken from jugular vein and centrifuged. The obtained sera from offspring were pooled within each litter and kept at −30°C until use.
The obtained serum of offspring of normal and diabetic dams was used for determination of glucose concentration according to the method of Trinder [
Serum insulin concentration of offspring of normal and diabetic dams was determined with radioimmunoassay kits of DPC (Diagnostic Products Corporation, Los Angeles, CA, USA) (Coat-A-Count) according to the method of Marschner et al. [
Insulin resistance (HOMA-IR), insulin sensitivity (HOMA-IS), and beta-cell function (HOMA-
At specific time intervals (zero time, after one and 2 weeks), offspring of both normal and diabetic dams were sacrificed and liver and pancreas were immediately excised. Small tissue blocks were fixed in 10% neutral buffered formalin, embedded in paraffin wax, and cut serially at 5 µm thickness. Pancreas sections were stained with modified aldehyde fuchsin stain according to Bancroft and Stevens [
The data are analyzed by one-way analysis of variance (ANOVA) using PC-STAT, University of Georgia, followed by LSD analysis to discern the main effects and to compare various groups with each other [
To investigate the effect of preexisting maternal diabetes on development of newborn, offspring of normal control and diabetic females were examined at birth, after one and two weeks after delivery.
Serum glucose concentration of offspring of normal and diabetic dams at different experimental periods.
One-way ANOVA
Groups | Periods | ||
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0 week | 1 week | 2 weeks | |
offspring of normal dams |
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offspring of diabetic dams |
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% Difference | +27% | +23.8% | +37.8% |
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>0.05 | <0.001 | <0.001 |
LSD at 5% level | — | 20.96 | 16.64 |
LSD at 1% level | — |
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Two-way ANOVA
Effect of time | Effect of diabetes | Time-diabetes interaction |
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Data are given as mean ± SE; means with the same superscript are not significantly different.
0 week = at birth; 1 week = end of the 1st postnatal week; 2 weeks = end of the 2nd postnatal week; number of observations; % difference: difference between offspring of normal and diabetic dams.
At birth, the serum glucose of normal control offspring showed a mean of
The serum glucose concentration of diabetic dam offspring was at birth
The increases in glucose levels of diabetic dam offspring were 39.9 mg/dL (+48.8%) from birth to 1st week and 10.2 mg/dL (+8.4%) from 1st to 2nd week.
Two-way ANOVA revealed that while the effect of time or diabetes is very highly significant (
Serum insulin concentration (
One-way ANOVA
Groups | Periods | ||
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0 week | 1 week | 2 weeks | |
offspring of normal dams |
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offspring of diabetic dams |
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% Difference | +37.1% | −39.7% | −54.8% |
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<0.001 | <0.001 | <0.001 |
LSD at 5% level | 0.14 | 0.14 | 0.14 |
LSD at 1% level |
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Two-way ANOVA
Effect of time | Effect of diabetes | Time-diabetes interaction |
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Data are given as mean ± SE; means with the same superscript are not significantly different.
0 week = at birth; 1 week = end of the 1st postnatal week; 2 weeks = end of the 2nd postnatal week; number of observations; % difference: difference between offspring of normal and diabetic dams.
At birth, the insulin concentration of normal control dam offspring showed a mean of
The insulin concentration of diabetic group offspring was at all-time intervals significantly different at a 1.0% level from that of control neonates. At birth, the hormone level was
The analysis of the two-way ANOVA revealed that effect of time, diabetes, and their interaction is very highly significant throughout the experiment.
This model was used to estimate insulin resistance (IR), insulin sensitivity (IS), and the
HOMA-IR of offspring of normal and diabetic dams at different experimental periods.
One-way ANOVA
Groups | Periods | ||
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0 week | 1 week | 2 weeks | |
offspring of normal dams |
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offspring of diabetic dams |
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% Difference | +162% | +36.9% | +11.1% |
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<0.001 | <0.001 | >0.05 |
LSD at 5% level | 0.14 | 0.08 | — |
LSD at 1% level |
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— |
Two-way ANOVA
Effect of time | Effect of diabetes | Time-diabetes interaction |
---|---|---|
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Data are given as mean ± SE; means with the same superscript are not significantly different.
0 week = at birth; 1 week = end of the 1st postnatal week; 2 weeks = end of the 2nd postnatal week; number of observations; % difference: difference between offspring of normal and diabetic dams; HOMA-IR: homeostasis model assessment for insulin resistance.
The HOMA-IR of diabetic dam offspring was at birth and after one week significantly higher than that of control, while after the second week, the mean of both groups differed insignificantly. The differences between groups were +162%, +36.9%, and +11.1%, respectively. The decreases of HOMA-IR of diabetic dam offspring were −0.13
The analysis of the two-way ANOVA showed that while the effect of diabetes or its interaction with time was very highly significant (
HOMA-IS of offspring of normal and diabetic dams at different experimental periods.
One-way ANOVA
Groups | Periods | ||
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0 week | 1 week | 2 weeks | |
offspring of normal dams |
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offspring of diabetic dams |
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% Difference | −68.9% | −22.5% | −12.9% |
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<0.001 | <0.01 | >0.05 |
LSD at 5% level | 26.8 | 7.03 | — |
LSD at 1% level |
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— |
Two-way ANOVA
Effect of time | Effect of diabetes | Time-diabetes interaction |
---|---|---|
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Data are given as mean ± SE; means with the same superscript are not significantly different.
0 week = at birth; 1 week = end of the 1st postnatal week; 2 weeks = end of the 2nd postnatal week; number of observations; % difference: difference offspring of normal and diabetic dams; HOMA-IS: homeostasis model assessment for insulin sensitivity.
The insulin sensitivity of diabetic dam offspring was at birth significantly lower (−68.9%) than that of control showing a mean of
The analysis of the two-way ANOVA showed that the effect of time, diabetes, and their interaction is very highly significant throughout the experiment.
HO1MA
One-way ANOVA
Groups | Periods | ||
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0 week | 1 week | 2 weeks | |
Normal offspring |
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Diabetic offspring |
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% Difference | −45.6% | −75.3% | −81.4% |
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<0.001 | <0.001 | <0.001 |
LSD at 5% level | 0.21 | 0.14 | 0.25 |
LSD at 1% level |
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Two-way ANOVA
Effect of time | Effect of diabetes | Time-diabetes interaction |
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Data are given as mean ± SE; means with the same superscript are not significantly different.
0 week = at birth; 1 week = end of the 1st postnatal week; 2 weeks = end of the 2nd postnatal week; number of observations; % difference: difference between offspring of normal and diabetic dams; HOMA-beta-cell function: homeostasis model assessment for
The calculated beta-cell function of diabetic dam offspring was at all-time intervals significantly lower than that of control. Furthermore, the values decreased with time showing, by the end of the experiment, a difference of
The analysis of the two-way ANOVA showed that the effect of time, diabetes, and their interaction is significant (
The offspring of normal dams showed after birth (Figure
Photomicrograph of pancreas section of normal rat offspring after birth. The parenchyma is divided by septa (s) into pancreatic acini (pa) and pancreatic duct (pd). The endocrine portion of the pancreas consists of the islets of Langerhans (IL). Alpha (a), beta (b), and delta (d) cells are apparent (×400).
In offspring of diabetic dam at birth, the islets of Langerhans were hypertrophied and incompletely delineated by reticular fibers. The different cell types showed a disturbed arrangement. A large number of normally sized alpha-cells were found at the periphery of the islet. Beta-cells were hypertrophied. Vacuolation and necrosis were also observed within the endocrine portion of the tissue (Figure
Photomicrograph of pancreas section of diabetic rat offspring just after birth showing disturbed islets of Langerhans (IL) with necrosis (nc) and vacuolations (v). Beta cells (b) are hypertrophied, but low in number. Alpha (a) cells are large in number compared to their corresponding control, (pa) pancreatic acini (×400).
Photomicrograph of pancreas section of normal rat offspring after one week of birth. Islets are intact and larger than after birth with higher numbers of beta (b) cells and fewer numbers of alpha (a) cells. Pd: pancreatic ductile; IL: islets of Langerhans; pa: pancreatic acini (×400).
After one week, the islets of Langerhans seemed to be even smaller than at birth (Figure
Photomicrograph of pancreas section of diabetic rat offspring after one week of birth showing very small islets of Langerhans (IL) with many necrotic foci (nc) and vacuolations (v) (×400).
Photomicrograph of pancreas section of normal rat offspring after two weeks of birth illustrating intact islets of Langerhans (IL) with many beta cells (b). Alpha (a) cell, and delta (d) cells are clearly observed (×400).
Photomicrograph of pancreas section of diabetic rat offspring after two weeks of birth showing severe degenerative changes. Many necrotic foci (nc) vacuolations (v) are noticed in the islets (IL); the number of beta-cells (b) is reduced and the hypertrophied ones are degenerating. Alpha-cells, delta-cells, and pancreatic acini (pa) are indicated by symbols a, d, and pa, respectively, (×400).
The amount and distribution of carbohydrates granules were estimated in offspring of both groups.
Normal rat offspring showed at birth a moderate amount of carbohydrate granules (Figure
Photomicrograph of liver section of normal rat offspring after birth showing a moderate amount of carbohydrate granules (×400).
Diabetic dam offspring revealed at after birth a much smaller amount of carbohydrate granules compared to control (Figure
Photomicrograph of liver section of diabetic rat offspring after birth. The stain is less intensive (×400).
Photomicrograph of liver section of normal rat offspring after one week of birth (×400).
Photomicrograph of liver section of diabetic rat offspring after one week. Notice the difference in distribution and intensity of stain (×400).
Photomicrograph of liver section of normal rat offspring after two weeks of birth. The stain intensity is much higher than after one week of birth (×400).
Photomicrograph of liver section of diabetic rat offspring after two weeks of birth. The stain is less intensive than after one week of birth (×400).
The worldwide increase in the incidence of diabetes in women at reproductive age is the bases for the growing interest in the use of experimental diabetic models in order to gain insight into the mechanisms of induction of developmental alterations in embryos and the effects on newborn. Using an appropriate animal model, several important aspects of human diabetic pregnancies such as the increased rates of spontaneous abortions, malformations, fetoplacental impairments, and offspring diseases in later life can be approached [
The present study indicated that the pancreatic islet architecture of normal offspring became more organized with the advanced age from the 1st postnatal day to the end of the 2nd week. This is in concurrent with Quinn et al. [
The histological examination of the pancreas in the present study also revealed significant changes in the endocrine tissue of offspring of diabetic dams. At birth, the islets of Langerhans were relatively increased in size and
In accordance with the present study, Aerts et al. [
In contrast to the present results, Rodríguez et al. [
Diabetic pregnancy results in several metabolic and hormonal disorders, both in the embryo and the fetus of different species, including humans [
The present biochemical investigation of diabetic female offspring revealed a steady and significant increased serum glucose concentration from birth to the end of the experimental period, associated with significantly elevated serum insulin levels at birth and a steadily and significantly decreasing hormone concentration thereafter. Concomitant with these changes in insulin, HOMA beta-cell function was significantly decreased in a time-dependent manner. In addition, the offspring of diabetic mothers revealed a state of tissue insulin resistance and decreased insulin sensitivity. This was indicated by the increase in HOMA-IR and the decrease in HOMA-IS as revealed in this study. HOMA-IR increased, HOMA-IS decreased slightly, and HOMA beta-cell function declined sharply.
These biochemical results are in parallel with the histological observations of the endocrine pancreas. The insulin producing beta-cells were hypertrophied at birth, degraded and reduced in number thereafter, and, finally, replaced by small beta-cells associated with the obtained decline in HOMA beta cell function. The decrease in glycogen content detected in the liver of offspring born from diabetic dams by periodic acid-Schiff (PAS) stain may be secondary to insulin resistance and impaired insulin secretion as indicated in the present study.
The present results are in accordance with observations of several research groups. Insulin secretion has been reported to be abnormal in islets from offspring of diabetic mothers [
Insulin resistance and beta-cell dysfunction are also described in first-degree offspring of type 2 diabetic patients in response to oral glucose challenge. Beta-cell impairment exists in insulin-sensitive offspring of patients with type 2 diabetes, suggesting beta-cell dysfunction to be a major defect determining diabetes development in offspring of diabetic mothers [
Studies on offspring of streptozotocin- (STZ-) induced diabetic rat mothers showed that insulin secretion was significantly impaired in offspring 15 weeks of age. Consistent with these changes, islet glucose metabolism and some important glucose metabolic enzyme activities were reduced; these deteriorations in glucose metabolism may be the cause of impaired beta-cell function in adult STZ offspring [
In conduction with the previous publication, many of the animal data on the long-term impact of maternal diabetes come from studies of two types of exposure to hyperglycemia
In conclusion, the preexisting diabetes mellitus and hyperglycemia before and during gestation may increase risks of glucose intolerance, insulin resistance, and impaired insulin sensitivity and
The authors confirm that there is no conflict of interests.