Maternal diabetes on fetal endocrine pancreas development in rats

IESSI, ISABELA L.; DALLAQUA, BRUNA; SINZATO, YURI K.; GALLEGO, FRANCIANE Q.; NIELSEN, JENS H.; VOLPATO, GUSTAVO T.; CORRENTE, JOSÉ E.; DAMASCENO, DÉBORA C.

doi:10.1590/0001-3765202520240807

Abstract

To evaluate if an unfavorable maternal environment induced by hyperglycemia, hypoxia, and oxidative stress status impairs the morphological development of pancreatic islets in the fetuses on days 18 and 21 of pregnancy. Wistar rats were injected with streptozotocin for diabetes induction. At adulthood (3 months), all animals underwent an oral glucose tolerance test (OGTT) for glucose assessment as an inclusion criterion. Following, the animals were mated. On day 18 and 21 of pregnancy, the mothers were killed for blood biochemical data and, fetal pancreas was collected for immunohistochemical analysis. On the GD18/21, the diabetic (D) dams showed higher glycemia, erythropoietin and TBARS levels, and a disorganized cell distribution in fetal pancreatic islets compared to control (C) rat mothers. The fetal endocrine pancreas of D dams presented a higher ratio of insulin-stained cells on GD18. No difference in the immunostaining for PDX-1 was observed. Fetuses of D dams showed a lower ratio of cells immunostained for Ki-67 in GD18 and greater cell proliferation in GD21. At the GD21, the D group showed a higher ratio of cells undergoing apoptosis. Maternal hyperglycemia impairs fetal pancreatic islet structure, which causes functional changes contributing to fetal hyperglycemia at birth

Key words
Hyperglycemia; hypoxia; oxidative stress; pancreatic islets; rodents

INTRODUCTION

Diabetes mellitus (DM) is recognized as one of the major worldwide health problems and affects more than 10.5% of the adult population (20-79 years), with almost half uninformed that they are living with this condition. Around 2045, projections show that 1 in 8 adults, almost 783 million, will be living with diabetes, an increase of 46% (IDF 2021). Type 1 or type 2 DM diagnosed before a woman’s pregnancy is termed pregestational diabetes or preexisting diabetes (Deputy et al. 2018). 16.9% of pregnancies globally are affected by hyperglycemia in pregnancy (including pre-gestational and gestational diabetes) using the WHO criteria, equating to 21.4 million of 127.1 million live births to women (IDF 2021).

A high risk of adverse maternal and fetal consequences, such as congenital malformations, miscarriage, preterm delivery, preeclampsia, macrosomia, and perinatal mortality have been found in the presence of pregestational diabetes (ACOG 2022).

Hyperglycemia increases the intracellular oxygen consumption in mitochondria resulting in reduced oxygen tension in tissues, which leads to hypoxia (Ornoy et al. 2010). An increase in erythropoietin (EPO) production in the liver can be observed under hypoxic conditions (Eckardt & Kurtz 2005), which may be also driven by the high concentrations of reactive oxygen species (ROS) (Borysewicz-Sańczyk 2011). In addition, maternal diabetes leads to oxidative stress in the offspring and both may cause fetal disturbances (Clapés et al. 2013). Then, investigations focused on fetal development under a hyperglycemic environment have been encouraged. However, human studies are limited not only for ethical reasons but also due to uncontrollable variables that modify the intrauterine environment. Therefore, developing adequate experimental models is of ultimate importance (Lopez-Soldado & Herrera 2003). Several experimental models have been performed to reproduce a pregestational diabetes status, as an induction of mild diabetes (MD) using a beta (β)-cytotoxic drug (streptozotocin - STZ) (Bueno et al. 2020). In our laboratory, MD was induced in neonatal rats because it resembles to hyperglycemia status of the human pregestational diabetes (Type 2) (Bueno et al. 2020).

A significantly increased islet volume and mass have been confirmed in pregnant rats on day 18 of pregnancy, and a rapid return to pregestational values at the post-partum period (Pang et al. 1994). In the fetal rat pancreas, “islet-like” formations, composed mainly of insulin-containing cells, are present from day 18 of gestation (Pang et al. 1994). By day 20, the endocrine cells accumulate in clusters, which are organized into real “mantle-islets” with a core of insulin-producing cells (Aerts & Van Assche 1977). Maternal diabetes causes impaired fetal development (Jawerbaum & White 2017, Gallego et al. 2018, Sinzato et al. 2021), in addition, the hyperglycemia-induced oxidative stress in pancreatic islets of adult rats was confirmed by GSH-Px and MDA immunolabeling, and these findings were related to a reduced number of β-cell, higher number of alpha (α) and delta (δ) cell numbers. These morphological changes were associated with maternal impaired pancreatic function, hyperglycemia, and inadequate reproductive outcomes (Gallego et al. 2018).

Considering that, the adult female rats presenting mild diabetes reproduce human complications; we hypothesize that hyperglycemia generates maternal oxidative stress and hypoxia. Consequently, these inadequate conditions compromise fetal pancreatic development, as on the beginning (day 18 of pregnancy) as on the end (day 21) of pregnancy. The objective of the study was to evaluate how the inadequate intrauterine environment, caused by maternal hyperglycemia and consequently, hypoxia and oxidative stress status, impairs the morphological development of pancreatic islets in the fetuses on days 18 and 21 of pregnancy.

MATERIALS AND METHODS

Ethical Approval

The present study was approved by the institution’s Animal Research Ethics Committee (Protocol CEUA Number: 1218/ 2017) and carried out by the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978), and all efforts were made to minimize animal suffering.

Animals

Male and female Wistar rats (90 days of age), weighing approximately 220 grams (g) and 190 g, respectively, were obtained from CEMIB (Multidisciplinary Center for Biological Research - Campinas, São Paulo State). All animals were adapted and maintained in the Vivarium of the Laboratório de Pesquisa Experimental em Ginecologia e Obstetrícia (LAPGO), UNESP/Brasil. Water and food were given ad libitum in a controlled environment (room temperature: 22 ± 3°C, humidity: 50 ± 10%, and 12 hrs. light/dark cycle) during the whole experiment. Male and female normoglycemic (glycemia <140 mg/dL) rats were mated to obtain offspring for diabetes induction (Figure 1).

Figure 1
Illustrative scheme of the experimental design. *Male rats of same age were purchased for mating.

Diabetes induction and eligibility criteria

At birth (day 1 of life), a group of female offspring was subcutaneously injected with 100 mg streptozotocin/kg body weight (STZ – Sigma Chemical Company®, St. Louis, Millstone, USA) dissolved in citrate buffer (0.1 M, pH 4.5) to obtain rats with mild diabetes (Gallego et al. 2018) in a random way. Other female rats subcutaneously received only citrate buffer (nondiabetic rats, i.e., control group). In adulthood, the determination for inclusion and exclusion criteria was followed by Paula et al. (2022). For the control group (C), only rats with glycemia < 140 mg/dL at least three time points during the OGTT were included. The rats receiving STZ and presenting glycemia ≥ 200 mg/dL at least one time point during OGTT were included in the mild diabetic group (D). The female animals that did not present these characteristics for inclusion in the C or D group were excluded and euthanized. Due to partial pancreatic proliferation and/or neogenesis in the β-cells of the newborns exposed to STZ, the mild hyperglycemia existing in adulthood is similar to hyperglycemia of human Type 2 Diabetes mellitus (Thyssen et al. 2006, Gallego et al. 2018).

Mating and experimental groups

On day 90 of life, C and MD adult female rats were mated overnight with nondiabetic males (90 days of life, obtained from CEMIB), and the next day when spermatozoa were found in the vagina smear was considered as gestational day zero (GD0) (Netto et al. 2018). After mating, the animals were randomly distributed in different experimental groups according to when they were euthanized and either the presence or absence of hyperglycemia. The two experimental groups (C and D) were composed of 10 animals/group, which were further subdivided based on their two different moments of euthanasia [on days 18 (n=5 rats/group) and 21 (n=5 rats/group) of pregnancy. The choice of day 18 of pregnancy represents the beginning of maximum fetal growth (Barr 1973).

Laparotomy

At day 17 of pregnancy the OGTT was evaluated. On days 18 or 21 of pregnancy, the rats (mothers and pups) were lethally anesthetized with sodium thiopental (Thiopentax® - intraperitoneal route - 120 mg/kg according to Ethical Committee’s protocols) and, after confirming the signs that showed the anesthetic procedure according to the instructions of the Institutional Veterinarian, the animals were decapitated.

Analysis of oxidative stress and hypoxia markers of the mothers

Following, blood samples were obtained for measurement of oxidative stress biomarkers in washed erythrocytes (thiobarbituric acid reactive substances – TBARS, superoxide dismutase - SOD, and reduced thiol groups – SH) and serum samples were used for determination of erythropoietin (a marker of hypoxia status). The blood samples were collected in tubes with anticoagulant and centrifuged at 90 × g for 10 minutes (min) at 4°C. The supernatants were discarded and erythrocytes were washed with phosphate buffer saline (0.01M, pH 7.4) followed by centrifugation at 263 × g for 1 min at 4°C. This procedure has repeated a total of three times for each sample after which the final cell pellets were then used for determination of SOD, GSH-Px activities, SH, and TBARS levels according to Sinzato et al. (2023) to confirm oxidative stress status. The EPO was measured by solid-phase, chemiluminescent immunometric assay Immulite 1000 (EPO1 kit, Catalog Number: LKEPZ) to confirm hypoxic status.

Immunohistochemical analysis of fetal pancreatic islets

Following maternal analysis procedures, the rats were submitted to an exploratory laparotomy under thiopental sodium thiopental (Thiopentax® - intraperitoneal route - 120 mg/kg) for fetal withdrawal, and glycemia was determined using blood obtained after decapitation using a conventional glucometer. Subsequently, the fetal pancreas was dissected and processed for immunohistochemical analyses. For IHC, a total of 5 pancreas were collected, from 5 litters per group/moment, one puppy/litter being standardized, with the first fetus on the right side of the uterine horn being selected, without distinction between males and females. The rest of the litter was anesthetized and decapitated, as previously mentioned. The immunohistochemical staining protocol was performed on paraffin sections for anti-insulin (Abcam®, Code: ab8304, dilution 1: 10.000, incubation for 2 h in an oven at 27 ° C), anti-glucagon (Abcam®, Code: ab8055, dilution 1:500, incubation for 1 h in an oven at 27 °C), anti-Ki-67 (proliferation marker) (anti-Ki-67_Spring®, dilution 1:100, overnight incubation in a refrigerator at 4 ° C), anti-caspase-3 cleaved (apoptosis marker) (Cell Signaling Technology®, USA, Code: 9661S, dilution 1:50, overnight incubation in a refrigerator at 4 °C), anti-pancreatic and duodenal homeobox 1 (PDX-1 – marker for beta-cell development) (Abcam®, Code ab47267, dilution 1:500, incubation for 2 h in an oven at 27 °C).

All morphometric and immunohistochemistry analyses were performed in an imaging computer system (KS-300 software, version 3.0, ZEISS® Germany), which receives images from a digital camera (CCD-IRIS/RGB, SONY®, China) coupled to a microscope (DMR, LEICA® type, Brazil). Five fetal pancreases were analyzed per group and from 8 to 10 pictures/photos were taken per section. Then, we could analyze a total of 40 to 50 photos for each stain/marker at the final of the experiment. The software “Image J” (NIH free access) was used to determine the total number of cells of fetal islets. The same software was used to count the number of stained cells of insulin and glucagon antibodies to analyze the synthesis and the number of stained cells for PDX-1, Ki-67, and cleaved caspase-3. Sequentially, the number of stained cells for hormones and other markers was calculated by the ratio of the number of stained cells of each hormone or marker/total number of cells per islet.

Statistical analysis

The data were presented as mean ± standard deviation (SD). The fetal data from the pancreatic parameters were performed using 5 litters per group/moment, one puppy/litter being standardized, with the first fetus on the right side of the uterine horn being selected, without distinction between males and females), with the litter as the unit of measure in the experimental groups. Student t-tes was used for data comparison between the groups D18 control versus D18 diabetic, and D21 control versus D21 diabetic, for biochemical data (mothers), ratio of immunostained cells (pups) for insulin, glucagon, Ki-67, cleaved caspase-3 and PDX-1. In this study, the comparison between moments (D18 versus D21) was not priority. P < 0.05 was applied and considered as the limit of statistical significance.

RESULTS

On days 18 and 21 of pregnancy (GD18 and 21), the diabetic (D) dams showed a higher glycemia compared to control (C) rat mothers (Figure 2a). The maternal blood glucose levels of D dams had a increase in time points 30 and 60 min of OGTT in relation to C rats (Figure 2b). No differences were found between the serum insulin levels of fetuses in the diabetic group and the control group on day 18 of pregnancy (Figure 2c). In relation to the erythropoietin (EPO) levels, the control, and D dams had increased levels from 18 to 21 days of pregnancy. Besides, the D dams presented higher EPO levels on GD18 and 21 compared to the control dams (Figure 2d). TBARS levels were higher in D dams in both gestational days compared to those of control dams (Figure 2e). The activities of superoxide dismutase (SOD) presented no changes in both days of pregnancy among different groups (Figure 2f). D rats had lower levels of the antioxidant (reduced thiol group - SH) on GD18 than control rats on the same day (Figure 2g).

Figure 2
Blood biochemical data of nondiabetic (control -C) and mild diabetic (D) rats on days 18 and 21 of pregnancy, and OGTT at day 17 of pregnancy. a- Fasting blood glucose levels; b- OGTT; c- Serum insulin levels; d- Erythropoietin (EPO) levels; e- Thiobarbituric reactive substance (TBARS) levels; f- Superoxide dismutase (SOD) activity; g- Reduced thiol group (-SH) levels. *p<0.05 – compared to the control group (Student t Test).

Figure 3 shows the immunohistochemical analyses related to insulin of fetuses from control and D dams on GD18 and 21. The ratio between the insulin-positive cells and total cell number in islets increased from GD18 to 21 in the fetuses of both groups. Additionally, the fetal endocrine pancreas of D dams presented a higher ratio of insulin-stained cells on GD18 than the fetuses of control dams (Figure 3a). On GD18, the cell distribution was more disorganized than on GD21 (Figure 3b-e).

Figure 3
a- Ratio of immunostained cells for insulin and total number of cells of fetuses. Photomicrograph of the endocrine pancreas for insulin-positive (Cytoplasmic immunostaining - red arrows) cells of fetuses of control (b, d) and mildly diabetic (c, e) dams on days 18 and 21 of pregnancy, respectively. Magnification: 40X.

Fetuses of the D dams presented glucagon-stained cells in the periphery of the islets, especially on GD21 (Figure 4a). All groups presented a disorganized cell distribution in fetal pancreatic islets of the D dams compared to those of the control group (Figure 4b-e).

Figure 4
a- Ratio of immunostained cells for glucagon and total number of cells of fetuses. Photomicrograph of endocrine pancreas for glucagon-positive cells (Cytoplasmic immunostaining - red arrows) of fetuses of control (b, d) and mildly diabetic (c, e) dams on days 18 and 21 of pregnancy, respectively. Magnification: 40X.

The immunostaining for PDX-1 is represented in Figure 5. There was no difference in the immunostaining for beta cell development between the groups (control and D) at the two moments studied (GD18 and 21) (Figure 5a).

Figure 5
a- Ratio of immunostained cells for PDX-1 and total number of cells of fetuses. Photomicrograph of endocrine pancreas for PDX-1-positive cells (Nuclear immunostaining - red arrows) of fetuses of control (b, d) and mildly diabetic (c, e) dams on days 18 and 21 of pregnancy, respectively. Magnification: 40X.

Figure 6 represents the ratio of cell proliferation (Ki-67) in pancreatic islets. Fetuses of the D dams showed a decreased ratio of cells immunostained for Ki-67 in GD18 (Figure 6a-c) and increased cell proliferation in GD21 (Figure 6a-e) compared to those of the control group. The ratio of cell death by apoptosis (cleaved caspase-3) is represented in Figure 7. In GD18, fetuses of the D mothers showed a lower ratio of cells immunostained for cleaved caspase-3 when compared to G18 of the control group (Figure 7a-c). At the end of pregnancy (GD21), the D group showed a higher ratio of cells undergoing apoptosis when compared to their respective control (Figure 7a-e).

Figure 6
a- Ratio of immunostained cells for Ki-67 and total number of cells of fetuses. Photomicrograph of endocrine pancreas for Ki-67-positive cells (Nuclear immunostaining - red arrows) of fetuses of control (b, d) and mildly diabetic (c, e) dams on days 18 and 21 of pregnancy, respectively. Magnification: 40X. *p<0.05 – compared to the control group (Student t Test).

Figure 7
a- Ratio of immunostained cells for cleaved caspase-3 and the total number of cells of fetuses. Photomicrograph of the endocrine pancreas for cleaved caspase-3-positive cells (Perinuclear immunostaining - red arrows) of fetuses of control (b, d) and mildly diabetic (c, e) dams on days 18 and 21 of pregnancy, respectively. Magnification: 40X. *p<0.05 – compared to the control group (Student t Test).

DISCUSSION

In the present study, the impaired embryo-fetal growth and adverse pancreatic development were caused by maternal hyperglycemia associated with maternal oxidative stress and hypoxia. The maternal hyperglycemia caused an abnormal intrauterine environment and an impaired fetal metabolism as we observed in the fetuses of diabetic dams, which presented higher blood glucose levels on GD18 (data not shown).

Considering the relationship between unfavorable maternal intrauterine environment and adverse fetal pancreatic development, our study showed that the diabetic dams presented oxidative stress status, as confirmed by high thiobarbituric reactive substances (TBARS) concentrations and abnormal GSH-Px activities in the blood. Diabetic dams showed a two-fold increase in TBARS concentration on GD18. Although the antioxidant enzymatic system did not present an evident reduction in the diabetic dams, the higher lipoperoxidation in these dams demonstrated an insufficient antioxidant defense to prevent this condition. We also observed maternal hypoxia levels as identified by increased erythropoietin concentrations in diabetic rats on GD21. These data indicate that hyperglycemia-induced hypoxia contributed to the oxidative stress status.

Hypoxia is essential in early embryonic development (Dunwoodie 2009), however, controlled oxygen tension is necessary for maintaining normal fetal growth (Burton 2009). Moreover, the partial oxygen tension regulates the development of several embryo organs, such as the pancreas (Fraker et al. 2007, Heinis et al. 2010). In addition, hypoxia may be enhanced by embryonic oxidative stress. Therefore, in hyperglycemia and hypoxia, the status of oxidative stress may be a common pathogenic mechanism that damages the developing embryo and fetus (Ornoy et al. 2010). Thus, the intrauterine environment in which the fetus develops may contribute to physiological and metabolic changes throughout life (Plagemann 2011), suggesting that these changes can cause damage to the organism, including the endocrine pancreas.

In rodents, the cytoarchitecture of the pancreatic islets is a structure known as mantle-core, that is, beta cells are located in the center, while non-beta cells are located on the periphery of the pancreatic islets (Bonner-Weir et al. 2015). In our study, the fetuses from diabetic dams presented an abnormal organization of the endocrine cells, furthermore, a substantial reduction in the number of islets, and the number of cells per islet as well as abnormal number and ratio of cellular death and proliferation were observed.

On GD21, the fetuses of diabetic dams presented an increase of proliferating cells, but there was also a higher ratio of cells in apoptosis (cleaved caspase-3-positive cells). Casasnovas et al. (2019) evaluated RNA sequencing in islets from hyperglycemic fetuses on GD22. The authors verified that hyperglycemic fetuses presented up-regulated genes for inflammation and cell death pathways, demonstrating that hyperglycemia at the end of pregnancy alters the transcriptome of fetal pancreatic islets and increases the susceptibility of offspring to the development of pancreatic islet dysfunction later in adult life. Our previous data also revealed that adult offspring of diabetic mother rats have more apoptotic β-cells than controls (Casasnovas et al. 2019). Epigenetic factors, such as DNA methylation, may be associated with beta cell apoptosis in the offspring of diabetic rats in adulthood, including hypomethylation of the CDKN2A/B promoter (Nazari et al. 2017). In this study, a greater number of cells immunostained for cleaved caspase-3 was observed in the periphery of the pancreatic islet. Takeda et al. (2012) have demonstrated that the distribution of caspase-3 positive cells in islets of non-diabetic rats is similar to our findings even in adult animals, suggesting that the non-β-cells are more prone to cell death than β-cells. Nevertheless, further studies are required to identify the cell types involved in this process since we did not observe significant changes in the number and ratio of non-β-cells. However, additional studies are needed to verify the mechanism by which apoptosis of pancreatic islet cells occurs in fetuses that developed in the hyperglycemic intrauterine environment.

A study conducted by Barco et al. (2022a) demonstrated that adult daughters of diabetic rats have an imbalance between cell proliferation and death in the pancreatic islets. In addition, these females present hyperglycemia in adulthood, suggesting that the imbalance between cell proliferation and death since the fetal period may remain at adulthood and cause damage to the endocrine pancreas, leading to diabetes.

The fetal endocrine pancreatic hormones were analyzed by the ratio between the number of positively staining cells and the total cell number of the islets. Recent studies have focused on morphological and functional adaptations of maternal pancreatic α-cells in rodents during normal pregnancy (Quesada-Candela et al. 2019) and diabetic pregnancy (Gallego et al. 2018). The studies reveal an increased α-cell mass and secretion of glucagon on GD18. This suggests that α-cell adaptation is crucial for the maintenance of fetal metabolism during pregnancy. In our study, both diabetic groups displayed no changes in the number of glucagon-stained cells, but the ratio of α-cells per total islet cells was reduced on GD18. In contrast, this ratio was increased in the MD group on GD21. Therefore, the lower glucagon ratio on GD18 could be an adaptation towards preventing a potential hyperglycemic state. Indeed, these findings might suggest a sensitive crucial role of fetal pancreatic α-cells for an adaptation to the intrauterine environment. About the β-cell findings, even with the increased insulin content in the fetuses from D dams on GD21 as compared to the GD18, the insulin was lower in comparison with fetuses of nondiabetic dams. The elucidation of the mechanisms responsible for fetal adaptation during pregnancy disturbed by maternal hyperglycemia is essential to comprehend the transgenerational repercussions of diabetes (Page 2019). The streptozotocin-induced diabetic females (induction during the neonatal period) presented higher blood glucose levels (above 200 mg/dL) at two-time points in the OGTT and increased area under the curve (AUC) in day 17 of pregnancy. These results corroborate previous studies conducted at our laboratory (Gallego et al. 2018, Barco et al. 2022b) as well as other reports in the literature (Adamo et al. 2012, Jawerbaum & White 2017).

The developmental origin of adult health and disease (DOHaD) hypothesis postulates maternal insults might change the central regulatory mechanisms of developing fetuses, mostly in the organogenesis period (Adamo et al. 2012, Briana & Malamitsi-Puchner 2017). Alterations in maternal metabolic conditions impair fetal pancreatic development. On GD18, the ratio between the Ki-67-stained cells (cell proliferation marker) and the total number of cells was lower in the fetal pancreas of the diabetic dams, which indicates that mild hyperglycemia damaged the proliferation of pancreatic islet cells. There is evidence that pancreatic beta cells are the main cells of the endocrine pancreas that proliferate and reach functional maturation at the end of pregnancy. These cells are probably the most sensitive to excessive glucose supply during this period (Freinkel 1980, 1986). In the present study, it is not possible to confirm which cell had an impaired proliferation since double labeling was not performed for Ki-67 and pancreatic endocrine cells. However, we suggest that the decrease in cell proliferation is not occurring in pancreatic beta cells as there was an increase in insulin-positive cells on GD18.

There is evidence that the fetal period is critical for the endocrine pancreatic development in rodents and humans. Then, an inadequate formation of beta cells at this time could favor the development of Type 2 Diabetes in adulthood (Floréz 2008). Another relevant factor for pancreatic development is the PDX-1 (homeodomain-containing transcription factor), this factor is expressed early as E8.5 in the dorsal and ventral endoderm (Kordowich et al. 2010) and is essential in the embryonic development of the duodenum and the α-cells and some δ-cells (Haber et al. 2006). In mature beta cells, PDX-1 transactivates the insulin gene and other genes involved in glucose sensing and metabolism, such as GLUT2 and glucokinase (Kaneto et al. 2007). Furthermore, PDX-1 mainly controls the proliferation of insulin-producing cells in the fetal period, at the end of pregnancy (Gannon et al. 2008). In our study, we have verified an unchanged PDX-1 ratio in the fetal islets from the diabetic rats, and there was no change in the ratio of cells immunostained for insulin, suggesting that the maternal hyperglycemia found in the model used did not cause changes in these parameters.

CONCLUSIONS

The relationship between the number of islets and cells per islets in fetuses of the dams with hyperglycemia, oxidative stress, and hypoxia was pronounced. Thus, our findings confirmed that an unfavorable intrauterine environment (hyperglycemic, hypoxemic, and oxidative stress status) compromises fetal development, regardless of the gestational day studied, leading to intrauterine growth restriction (IUGR) and impaired pancreatic development. Although this paper uses rats as an experimental model and pancreatic development differs between humans and rodents, it is necessary to emphasize the importance of strict glycemic control before and into pregnancy to avoid adverse embryo-fetal and perinatal complications, which can lead to harm at adulthood of the offspring. In conclusion, oxidative stress and hypoxia induced by maternal hyperglycemia lead to impaired fetal pancreatic development at different times of pregnancy.

ACKNOWLEDGMENTS

The authors thank Mr. Danilo Chaguri, Mr. Jurandir Antonio, and Mr. Carlos Roberto G. Lima (Academic Support Assistant – ASA, Unidade de Pesquisa Experimental - UNIPEX) for the care and maintenance of animals in vivariums.

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PANG K, MUKONOWESHURO C & WONG GG. 1994. Beta cells arise from glucose transporter type 2 (Glut2)-expressing epithelial cells of the developing rat pancreas. Proc Natl Acad Sci USA 91: 9559-9563.
PAULA VG, VESENTINI G, SINZATO YK, MORAES-SOUZA RQ, VOLPATO GT & DAMASCENO DC. 2022. Intergenerational high-fat diet impairs ovarian follicular development in rodents: a systematic review and meta-analysis. Nutr Rev 10: 889-903.
PLAGEMANN A. 2011. Maternal diabetes and perinatal programming. Early Hum Dev 87: 743-747.
QUESADA-CANDELA C, TUDURÍ E, MARROQUÍ L, ALONSO-MAGDALENA P, QUESADA I & NADAL Á. 2019. Morphological and functional adaptations of pancreatic alpha-cells during late pregnancy in the mouse. Metabolism 5: 153963.
SINZATO YK ET AL. 2021. Comparison of streptozotocin-induced diabetes at different moments of the life of female rats for translational studies. Lab Anim 55: 329-340.
SINZATO YK, RODRIGUES T, CRUZ LL, BARCO VS, SOUZA MR, VOLPATO GT & DAMASCENO DC. 2023. Assessment of Oxidative Stress Biomarkers in Rat Blood. Bio Protoc 13: e4626.
TAKEDA Y, FUJITA Y, HONJO J, YANAGIMACHI T, SAKAGAMI H, TAKIYAMA Y, MAKINO Y, ABIKO A, KIEFFER TJ & HANEDA M. 2012. Reduction of both beta cell death and alpha cell proliferation by dipeptidyl peptidase-4 inhibition in a streptozotocin-induced model of diabetes in mice. Diabetologia 55: 404-412.
THYSSEN S, ARANY E & HILL DJ. 2006. Ontogeny of regeneration of beta-cells in the neonatal rat after treatment with streptozotocin. Endocrinology 147: 2346-56.

Publication Dates

Publication in this collection
21 Mar 2025
Date of issue
2025

History

Received
24 July 2024
Accepted
27 Oct 2024

This is an open-access article distributed under the terms of the Creative Commons Attribution License

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[33] PAGE KA. 2019. The transgenerational cycle of diabetes. Sci Transl Med 11: eaaw5327.

[34] PANG K, MUKONOWESHURO C & WONG GG. 1994. Beta cells arise from glucose transporter type 2 (Glut2)-expressing epithelial cells of the developing rat pancreas. Proc Natl Acad Sci USA 91: 9559-9563.

[35] PAULA VG, VESENTINI G, SINZATO YK, MORAES-SOUZA RQ, VOLPATO GT & DAMASCENO DC. 2022. Intergenerational high-fat diet impairs ovarian follicular development in rodents: a systematic review and meta-analysis. Nutr Rev 10: 889-903.

[36] PLAGEMANN A. 2011. Maternal diabetes and perinatal programming. Early Hum Dev 87: 743-747.

[37] QUESADA-CANDELA C, TUDURÍ E, MARROQUÍ L, ALONSO-MAGDALENA P, QUESADA I & NADAL Á. 2019. Morphological and functional adaptations of pancreatic alpha-cells during late pregnancy in the mouse. Metabolism 5: 153963.

[38] SINZATO YK ET AL. 2021. Comparison of streptozotocin-induced diabetes at different moments of the life of female rats for translational studies. Lab Anim 55: 329-340.

[39] SINZATO YK, RODRIGUES T, CRUZ LL, BARCO VS, SOUZA MR, VOLPATO GT & DAMASCENO DC. 2023. Assessment of Oxidative Stress Biomarkers in Rat Blood. Bio Protoc 13: e4626.

[40] TAKEDA Y, FUJITA Y, HONJO J, YANAGIMACHI T, SAKAGAMI H, TAKIYAMA Y, MAKINO Y, ABIKO A, KIEFFER TJ & HANEDA M. 2012. Reduction of both beta cell death and alpha cell proliferation by dipeptidyl peptidase-4 inhibition in a streptozotocin-induced model of diabetes in mice. Diabetologia 55: 404-412.

[41] THYSSEN S, ARANY E & HILL DJ. 2006. Ontogeny of regeneration of beta-cells in the neonatal rat after treatment with streptozotocin. Endocrinology 147: 2346-56.