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. Author manuscript; available in PMC: 2025 Jan 1.
Published in final edited form as: Reprod Toxicol. 2023 Oct 28;123:108494. doi: 10.1016/j.reprotox.2023.108494

STZ-induced gestational diabetes exposure alters PTEN/AKT/mTOR-mediated autophagy signaling pathway leading to increase the risk of neonatal hypoxic-ischemic encephalopathy

Lei Gong 1,2,#, Siyi Jiang 1,3,#, Jia Tian 1, Yong Li 1, Wansu Yu 1, Lubo Zhang 1, Daliao Xiao 1
PMCID: PMC11068333  NIHMSID: NIHMS1944507  PMID: 38706688

Abstract

Exposure to gestational diabetes mellitus (GDM) during pregnancy has significant consequences for the unborn baby and newborn infant. However, whether and how GDM exposure induces the development of neonatal brain hypoxia/ischemia-sensitive phenotype and the underlying molecular mechanisms remain unclear. In this study, we used a late GDM rat model induced by administration of streptozotocin (STZ) on gestational day 12 and investigated its effects of GDM on neonatal brain development. The pregnant rats exhibited increased blood glucose levels in a dose-dependent manner after STZ administration. STZ-induced maternal hyperglycemia led to reduced blood glucose levels in neonatal offspring, resulting in growth restriction and an increased brain to body weight ratio. Importantly, GDM exposure increased susceptibility to hypoxia/ischemia (HI)-induced brain infarct sizes compared to the controls in both male and female neonatal offspring. Further molecular analysis revealed alterations in the PTEN/AKT/mTOR/autophagy signaling pathway in neonatal male offspring brains, along with increased ROS production and autophagy-related proteins (Atg5 and LC3-II). Treatment with the PTEN inhibitor bisperoxovanadate (BPV) eliminated the differences in HI-induced brain infarct sizes between the GDM-exposed and the control groups. These findings provide novel evidence of the development of a brain hypoxia/ischemia-sensitive phenotype in response to GDM exposure and highlight the role of the PTEN/AKT/mTOR/autophagy signaling pathway in this process.

Keywords: GDM, PTEN/AKT/mTOR, autophagy, neonatal brain ischemia-sensitive phenotype

1. Introduction

The perinatal period is a critical time for brain development, but it is also a period of vulnerability. Various injuries during this time can lead to severe consequences, including death or significant neurocognitive deficits. Among these injuries, neonatal hypoxic-ischemic encephalopathy (HIE) is a prominent cause of morbidity and mortality in term-born infants [1]. It is estimated that approximately 0.75 million babies worldwide experience moderate or severe HIE annually, resulting in approximately 400,000 infants with long-term neurodevelopmental impairments [2]. Unfortunately, the etiology and the options available for aiding recovery in infants with HIE are limited.

Recent evidence suggests that gestational diabetes mellitus (GDM) may be an important risk factor for neurodevelopmental disease and neonatal HIE. The prevalence of GDM has been increasing worldwide, and research indicates adverse effects on both mothers and their offspring [3]. Women with GDM are at a higher risk of pregnancy complications, metabolic disorders, and other cardiovascular diseases later in life. Furthermore, GDM has significant effects on the health of the offspring. Previous studies have reported that GDM has been associated with impaired fetal brain development and an increased risk of neurobehavior disorders in postnatal life [4,5]. However, the specific associations between GDM and adverse outcomes, such as fetal brain development and the risk of HIE, are not yet fully established. The precise molecular mechanisms underlying the development of a brain ischemia-sensitive phenotype in neonatal offspring due to maternal GDM are not fully understood. Further research is needed to unravel the complex molecular pathways involved and to identify potential therapeutic targets for intervention.

GDM-mediated developmental diseases involve complex and multifactorial mechanisms. Previous studies link GDM to alterations in the PTEN/AKT/mTOR-mediated autophagy signaling pathway [6,7]. AKT (Protein kinase B) is a key downstream mediator of insulin signaling and regulates glucose metabolism and cell survival, while PTEN (Phosphatase and tensin homolog) negatively regulates AKT signaling. mTOR (mammalian target of rapamycin) is a central regulator of cell growth and autophagy, responding to nutrient availability. Autophagy maintains cellular homeostasis and is modulated by PTEN/AKT/mTOR signaling. GDM-induced insulin resistance and hyperglycemia affect this pathway, leading to imbalances in glucose homeostasis and autophagy. Although evidence suggests that maternal GDM-induced adverse outcomes involve the PTEN/AKT/mTOR-mediated autophagy pathway [8,9], the molecular mechanisms underlying these associations and their impact on fetal/neonatal brain development are not fully understood. Furthermore, to our knowledge, there is lack of studies to investigate if GDM exposure specifically increases the risk of HIE in neonatal offspring. Moreover, it is unknown whether it is regulated by PTEN/AKT/mTOR-mediated autophagy signaling pathway.

The present study aimed to investigate the molecular mechanistic signaling pathway and potential therapeutic targets associated with GDM-induced abnormal development of a brain ischemia-sensitive phenotype in offspring. Using a pregnant rat model of gestational diabetes mellitus (GDM) exposure, we examined the effects of GDM on in utero growth restriction (IUGR), fetal brain development, plasma glucose levels, and the risk of neonatal hypoxic-ischemic encephalopathy (HIE). Subsequently, we explored the underlying molecular mechanisms and potential therapeutic targets to rescue the hypoxia/ischemia-sensitive phenotype in the brain. Our study assessed the levels and activities of AKT/mTOR in the brain tissues of neonatal offspring, measured ROS production and the protein levels of autophagy-related genes downstream, evaluated PTEN gene expression as an upstream signaling factor, and investigated whether inhibition of PTEN can rescue the hypoxia/ischemia-sensitive phenotype in neonatal offspring.

2. Materials and Methods

2.1. Experimental animal model of GDM

The time-dated pregnant Sprague-Dawley rats (day 10 of gestation) were obtained from Charles River Laboratories and housed individually in air-conditioned rooms with a 12-hour light-dark cycle. They had ad libitum access to pellet food and tap water. On day 12 of gestation, rats were randomly divided into two groups: 1) saline control and 2) streptozotocin (STZ) group. The pregnant rats received an intraperitoneal injection of either saline or STZ (25, 50 or 65 mg/kg, i.p.) (Sigma, USA) as described in previous studies by ours [10] and others [11,12]. STZ was chosen for its specific pancreatic β cell cytotoxic effect to induce maternal hyperglycemia without significantly affecting the fetuses [12]. Blood glucose levels in both dams and offspring were measured using the Germaine Laboratories AimStrip Plus Blood Glucose Testing System (Fisher Scientific, Pittsburgh PA. Cat# 23-111-275) via tailnicking following manufacturer’s instructions. Blood glucose levels were measured daily in the morning without fasting in the dams. There was a total of 39 pregnant rats (litters) used for this study, which were randomly divided into different groups (n=8 for control, n=10 for STZ-65mg/kg, n=16 for STZ-50mg/kg, and n=5 litters for STZ-25mg/kg). These pregnant rats produced approximately 390 offspring for this study. All animal-related experimental procedures followed the guidelines of the Institutional Animal Care and Use Committee of Loma Linda University and the NIH Guide for the Care and Use of Laboratory Animals.

2.2. Hypoxic-ischemic encephalopathy (HIE) model

A modified Rice-Vannuci model was employed to induce hypoxic-ischemic encephalopathy (HIE) on postnatal day 9 (P9) pups, following our previously described protocol [13,14]. P9 was chosen for HIE induction because the rat brain at this stage is particularly susceptible to hypoxic-ischemic injury. Briefly, pups from both GDM-exposed and control groups were anesthetized with isoflurane (4%–5% for induction, 2%–2.5% for maintenance). A neck incision was made slightly off the midline of the anterior neck after sanitization. The right common carotid artery was isolated and subjected to two ligations, followed by cutting of the artery. Pups were allowed to recover for 1 hour on a heating pad and then placed in a hypoxic chamber (8% oxygen) for 2 hours. The inhalation of 8% oxygen-balanced nitrogen induced systemic hypoxia while maintaining a survivable severity for later physiological measurement. After the hypoxic-ischemic procedure, pups were returned to their respective dams.

2.3. Measurement of brain infarct size

Forty-eight hours after the HIE procedures, neonatal pups were euthanized. Coronal slices of the brain (2 mm thick) were cut and immersed in a 2% solution of 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) at 37°C for 5 min and then fixed by 10% formaldehyde overnight for the measurement of infarct size. There were 5–6 slices cut per brain for measuring the size of the infarct. The stained red colors in the brain represented viable areas, and the white colors represented infarct areas. Each slice was weighed and photographed separately. The percentage of infarction area for each slice was analyzed by ImageJ software (v. 1.40; National Institutes of Health, Bethesda, MD), corrected by slice weight, summed for each brain, and expressed as a percentage of the whole brain.

2.4. Measurement of Reactive Oxygen Species (ROS)

Total reactive oxygen species (ROS) production levels in the right hemisphere brain tissue samples were measured with the Oxiselect in vitro reactive oxygen and/or nitrogen species (ROS/RNS) assay kit (Cell Biolabs Inc., San Diego, CA), following the manufacturer’s instructions. As previously described [15], brain tissues were homogenized at 50 mg/mL in phosphate-buffered saline (PBS) on ice and centrifuged at 10,000 rpm for 5 min at 4°C. Fifty microliters of supernatant from the brain homogenates or standard were added to a 96-well plate and mixed with 50 μL of catalyst and 100 μL of 2′,7′-dichlorodihydrofluorescein diacetate (DCF). After incubation at room temperature for 30 min, the fluorescence (Ex480nm/Em530nm) was measured using a Synergy HT Multi-Mode Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT).

2.5. Western blot analysis

Whole right hemisphere brain samples were collected from P9 rat pups and homogenized with a lysis buffer containing 150 mmol/L NaCl, 50 mmol/L Tris·HCl, 10 mmol/L EDTA, 0.1% Tween 20, 1% Triton, 0.1% β-mercaptoethanol, 0.1 mmol/L phenylmethylsulfonyl fluoride, 5 μg/mL leupeptin, and 5 μg/mL aprotinin, pH 7.4. The homogenates were centrifuged at 4°C for 15 min at 14,000 g, and the resulting supernatants were collected for further analysis. Protein concentrations were determined using a protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein (30 μg) were loaded onto a 10% polyacrylamide gel with 0.1% sodium dodecyl sulfate and separated by electrophoresis at 100 V for 90 to 120 min. Proteins were then transferred onto nitrocellulose membranes and blocked for 3 to 4 hours at room temperature with a Tris-buffered saline solution containing 5% dry milk to block nonspecific binding sites. The membranes were then probed with primary antibodies against ATG5 (Cell Signaling Technology, USA), LC3B (Cell Signaling Technology, USA), PTEN (Abcam Inc.), p-AKT (Cell Signaling Technology, USA), AKT (Cell Signaling Technology, USA), p-mTOR (Cell Signaling Technology, USA), mTOR (Cell Signaling Technology, USA), and GAPDH (Millipore Sigma, USA), respectively. After washing, the membranes were incubated with secondary horseradish peroxidase-conjugated antibodies. Protein bands were visualized using enhanced chemiluminescence reagents, and blots were exposed to Hyperfilm. The results were analyzed with Kodak ID image analysis software, and band intensities were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

2.6. Treatment with PTEN inhibitor BPV in vivo neonatal offspring.

Male neonatal offspring at the postnatal day 7 (P7) were subjected to repetitive intraperitoneal doses (200 μg/kg) of bisperoxovanadate (BPV) (Calbiochem, San Diego, CA) or vehicle (saline) every 4 hours as described previously [16,17]. After two days of BPV treatment, the offspring at postnatal day 9 (P9) underwent the hypoxic-ischemic encephalopathy (HIE) procedure, and the brain infarct size induced by hypoxia/ischemia (HI) was measured. Additionally, PTEN protein levels in the neonatal brains were determined using Western blotting analysis after BPV treatment.

2.7. Statistical analysis

All data are expressed as the mean ± SEM obtained from the number (n) of experimental animals given. Difference between the groups was compared by Student’s t-test or analysis of variance (ANOVA) using the Graph-Pad Prism software (GraphPad Software Version 9, San Diego, CA, USA), where appropriate. For all comparisons, P-values less than 0.05 indicated statistical significance.

3. Results

3.1. GDM exposure reduces blood glucose levels and induces growth restriction in the neonatal offspring.

To establish a pregnant rat model of gestational diabetes mellitus (GDM), we administered different doses of streptozotocin (STZ) on gestational day 12, as previously described by us [10] and others [11]. STZ treatment resulted in dose-dependent and time-dependent increases in maternal blood glucose levels, remaining consistently elevated from gestational day 12 to day 22. Figure 1A shows the time-dependent increase in maternal blood glucose levels after treatment with 50 mg/kg or 65 mg/kg of STZ, while 25 mg/kg of STZ did not significantly affect maternal blood glucose levels compared to the control group. In neonatal offspring at postnatal day 7, both male (Figure 1B) and female (Figure 1C) pups exhibited significantly reduced blood glucose levels in response to GDM exposure. Furthermore, Figure 2 demonstrates that STZ-induced GDM led to dose-dependent reductions in neonatal body weights in both male (Figure 2A) and female (Figure 2B) offspring. Neonatal brain weights were only reduced in male offspring at the dosage of 65 mg/kg (Figure 2C) but remained unchanged in female offspring (Figure 2D) compared to the control group. Interestingly, the ratios of brain to body weights were significantly increased in both neonatal male (Figure 1E) and female offspring (Figure 1F) in response to GDM exposure compared to the control offspring.

Figure 1. Effect of GDM on blood glucose in pregnant rats and their offspring.

Figure 1.

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Pregnant rats were administered with either saline or STZ on the 12th day of gestation. A) Daily blood glucose values in pregnant dams with diabetes induced by injection of STZ at dosages of 25 mg/kg (n=5), 50 mg/kg (n=12), 65 mg/kg (n=8), and sham control (n=8) are shown. B) Blood glucose levels in male neonatal offspring of STZ-treated groups at dosages of 50 mg/kg (n=44), 65 mg/kg (n=17), and control group (n=19) at postnatal day 7 (P7) are presented. C) Blood glucose levels in female neonatal offspring of STZ-treated groups at dosages of 50 mg/kg (n=41), 65 mg/kg (n=19), and control group (n=21) at postnatal day 7 (P7) are also presented. The values are expressed as means ± standard error (SE), with *p < 0.05 indicating significant differences compared to the control group. n represents animal number/group.

Figure 2. Effect of GDM on body weight and brain weight in the neonatal offspring.

Figure 2.

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Pregnant rats were administered with either saline or STZ on the 12th day of gestation. A) Male neonatal offspring’s body weights were measured at postnatal day 7 for the STZ-treated (50 mg/kg and 65 mg/kg) and saline control groups. B) Female neonatal offspring’s body weights were also measured at postnatal day 7 for the same groups. C) Male neonatal offspring’s brain weights were measured at postnatal day 7 for the STZ-treated (50 mg/kg and 65 mg/kg) and saline control groups. D) Female neonatal offspring’s brain weights were measured at postnatal day 7 for the same groups. E) Brain to body weight ratios were calculated for male neonatal offspring in both STZ-treated and control groups. F) Brain to body weight ratios were also calculated for female neonatal offspring in both groups. The values are presented as means ± standard error (SE), with *p < 0.05 indicating significant differences compared to the control group. In male offspring, n=12 for control, n=32 for STZ 50mg/kg, n=10 for STZ 65mg/kg; In female offspring, n=21 for control, n=32 for STZ 50mg/kg, n=15 animals for STZ 65mg/kg groups.

3.2. GDM exposure enhances hypoxia/ischemia (HI)-induced brain infarct size in the neonatal offspring.

In our well-established neonatal model of hypoxic-ischemic encephalopathy (HIE), we measured the brain infarction induced by hypoxia/ischemia (HI). The data showed that HI procedure resulted in increased infarct size in the neonatal brain (Figure 3). In male offspring (Upper panel of Figure 3), the HI-induced brain infarct sizes were significantly higher in the STZ-induced GDM groups at both 50 mg/kg and 65 mg/kg dosages compared to the control group. Similarly, in female offspring (Lower panel of Figure 3), STZ-induced GDM significantly enhanced HI-induced brain infarct sizes compared to the control group.

Figure 3. Effect of GDM exposure on hypoxia/ischemia (HI)-induced brain injury in neonatal offspring.

Figure 3.

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Male and female offspring pups on postnatal day 9 (P9) underwent HI procedures as described in the Materials and Methods Section. HI-induced brain infarct size in both control and STZ (50 mg/kg and 65 mg/kg) exposed groups was measured by TTC staining 48 hours after the HI procedures. Brian slice images represented the HI-induced brain infarct size, with stained red colors indicating viable areas and white colors representing infarct areas. The summed infarct sizes, expressed as a percentage of the total brain weight, are shown in the lower panel. The values are presented as means ± standard error (SE), with *p < 0.05 indicating significant differences compared to the control group. In male offspring, n=9 for control, n=6 for STZ 50mg/kg, n=6 for STZ 65mg/kg; In female offspring, n=11 for control, n=8 for STZ 50mg/kg, n=7 animals for STZ 65mg/kg groups.

3.3. GDM exposure attenuates the protein abundances of AKT/mTOR and their phosphorylation levels in neonatal male offspring brains.

Considering the absence of significant sex differences between male and female offspring in terms of GDM exposure-mediated brain infarction (Figure 3) and neonatal growth restriction (Figures 1 and 2), all subsequent molecular biology studies were conducted exclusively in male offspring from both control and GDM exposed groups. As shown in Figure 4A, GDM exposure led to a significant reduction in the protein abundances of the AKT gene and its phosphorylation (p-AKT) levels in the neonatal male offspring brain tissues, compared to the control group. Similarly, GDM exposure also significantly attenuated the protein abundances of mTOR gene and its phosphorylation (p-mTOR) levels in the neonatal male offspring brain tissues, compared to the control group (Figure 4B).

Figure 4. Effect of GDM on the protein expression of p-Akt/Akt and p-mTOR/mTOR in male neonatal offspring.

Figure 4.

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Brains were collected from both control (□) and STZ (50 mg/kg)-treated (■) groups of male neonatal offspring (P7). Western blot analysis was performed to determine the phosphorylation levels of Akt and its total protein abundances (PAkt & Akt) (A) and the phosphorylation levels of mTOR and its total protein abundances (pmTOR & mTOR) (B) in the brain tissue. The values are presented as means ± standard error (SE), with *p < 0.05 indicating significant differences compared to the control group. n=4 animals/group.

3.4. GDM exposure enhances autophagy-related protein abundances and ROS production in neonatal male offspring brains.

As shown in Figure 5A, in the neonatal male offspring brains, GDM exposure led to significant increases in autophagy-related protein Atg5 levels compared to the controls. Furthermore, there were significantly increased autophagy-related protein Atg5 levels of brain tissues in GDM exposed offspring as compared to the controls. Additionally, GDM exposure had no effect on LC3-I, but it significantly enhanced LC3-II protein abundances in the brain tissues of the GDM-exposed offspring compared to the controls (Figure 5B). Moreover, GDM exposure resulted in significantly elevated levels of reactive oxygen species (ROS) in neonatal brains of male offspring (Figure 5C) when compared to the control group.

Figure 5. Effect of GDM on autophagy-related protein expression and ROS level in male neonatal offspring.

Figure 5.

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Brains were collected from both control (□) and STZ (50 mg/kg)-treated (■) groups of male neonatal offspring (P7). Western blot analysis was performed to determine the protein abundances of Atg5 (A) and LC3-I/LC3-II (B) in the brain tissue. In addition, ROS levels in the brain tissues isolated from both control (□) and STZ-treated (■) groups were measured using in vitro ROS/RNS assay kit as described in the Materials and Methods Section. The values are presented as means ± standard error (SE), with *p < 0.05 indicating significant differences compared to the control group. n=4 animals/group for Western blot analysis, n=5 animals/group for ROS assay.

3.5. GDM exposure enhances PTEN protein abundances and inhibition of PTEN rescues GDM-mediated HI-induced brain injury in neonatal male offspring.

PTEN is a well-known upstream gene that negatively regulates the Akt/mTOR signaling pathway [17]. In this study, we measured the expression of the PTEN gene in neonatal male offspring brains and investigated its potential role in GDM-mediated brain ischemic injury. As shown in Figure 6A, GDM exposure significantly increased the abundance of the PTEN gene in the neonatal brains compared to the controls. Importantly, when we treated the GDM-exposed group and control group with the PTEN inhibitor BPV, it eliminated the differences in PTEN expression (Figure 6B) and HI-induced brain infarct size (Figure 6C) between the GDM-exposed group and the control group. These findings suggest that PTEN plays a crucial role in GDM-mediated brain ischemic injury in neonatal offspring.

Figure 6. Effect of PTEN inhibition on PTEN protein expression and HI-induced brain injury in male neonatal offspring.

Figure 6.

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Neonatal offspring from both saline control and GDM exposed groups were treated with PTEN inhibitor BPV on postnatal day 7. After 48 hours of BPV treatment, the protein abundances of PTEN in the absence (A) and presence (B) of BPV treatment were determined by Western blot analysis in the neonatal brains collected from both groups. Furthermore, their brain infarct sizes (C) in the presence of BPV treatment were measured as described in the Materials and Methods Section. (D) The proposed diagram illustrated that STZ-induced GDM enhanced ROS production and increased PTEN protein abundance in the developing neonatal brain. This elevated PTEN downregulated AKT/mTOR expression and inhibited their activities (phosphorylation), subsequently activated autophagy signaling, resulting in enhanced HI-induced brain injury in neonatal offspring. Furthermore, inhibition of PTEN using its inhibitor BPV rescued the GDM-mediated neonatal brain injury. The values are presented as means ± standard error (SE), with *p < 0.05 indicating significant differences compared to the control group. n=4 animals/group for Western blot analysis, n=8 animals/group for HI-induced brain injury experiments.

4. Discussion

Exposure to diabetes in utero is an important risk factor for developmental disorders in offspring [18,19]. In this study, we used a well-known chemical compound, streptozotocin (STZ), to induce diabetes in pregnant rats, resulting in maternal hyperglycemia limited to the last third of pregnancy [10,11]. Following STZ administration, the pregnant rats exhibited increased blood glucose levels in a dose-dependent manner, mimicking the hyperglycemic state observed in human GDM. Using this pregnant rat model of GDM exposure, we made the following major findings: 1) STZ-induced maternal hyperglycemia significantly reduced blood glucose levels in both male and female neonatal offspring; 2) GDM exposure in utero resulted in reduced neonatal body weights associated with an increased brain to body weight ratio in both male and female offspring; 3) GDM exposure enhanced hypoxia/ischemia (HI)-induced neonatal brain infarct sizes compared to the controls in both male and female offspring; 4) GDM exposure attenuated the protein abundances of AKT/mTOR and their phosphorylation levels, while increasing autophagy-related protein abundances (Atg5 and LC3-II) and ROS production in neonatal male offspring brains; 5) GDM exposure enhanced PTEN gene expression, and treatment with the PTEN inhibitor BPV eliminated the differences in HI-induced brain infarct sizes between the GDM-exposed group and the control group. These findings provide solid evidence that exposure to GDM in utero causes fetal and neonatal growth restriction and develops a brain hypoxia/ischemia-sensitive phenotype through alterations in the PTEN/AKT/mTOR/autophagy signaling pathway in offspring.

Given the fact that STZ is unable to substantially penetrate the placenta, STZ exposure in pregnant animals does not exert a direct influence on fetal blood glucose levels. However, in the present study, our data show a significant decrease in blood glucose levels in neonatal offspring in response to STZ exposure. This phenomenon aligns with previous findings that hypoglycemia is one of the major problems in new born babies following a pregnancy complicated by diabetes [20]. Based on our data and the previous findings, it appears that maternal hyperglycemia induced by STZ in pregnant animals leads to fetal hyperinsulinemia in utero. After birth, when maternal glucose supply ceases, the elevated fetal insulin levels persist, potentially causing neonatal hypoglycemia in the offspring. This is likely due to the neonatal pancreas’ adaptation to the higher insulin production during maternal hyperglycemia. Of interest, in the same rat model of STZ-induced GDM, our previous study demonstrated no significant differences in blood glucose levels in two-month-old offspring between GDM-exposed group and control group [10]. These observations suggest that neonatal hypoglycemia is a transient phenomenon. As the neonates grow and their pancreas adjusts to the postnatal glucose environment, their blood glucose levels normalize. This adaptative mechanism may explain the absence of significant differences in blood glucose levels in older offspring.

Fetal and neonatal macrosomia, an excessive increase in body size, is more likely to occur as a result of maternal diabetes. However, the effects of maternal diabetes on offspring body size are not always consistent, with studies reporting conflicting findings. Some studies have associated pre-gestational maternal diabetes and later gestational diabetes (developing during pregnancy) with large offspring, which aligns with the increased availability of glucose in the maternal bloodstream, leading to excessive fetal growth [21,22] Conversely, other studies that have reported smaller offspring associated with maternal diabetes [11,23]. In our present study, we observed that STZ-induced late maternal gestational hyperglycemia significantly decreased the body weights of both male and female offspring compared to the control group. The reasons for this discrepancy in the effects of maternal diabetes on offspring body size are not fully understood. Several factors may contribute to the inconsistent findings, including the timing and severity of diabetes, subtypes of diabetes, genetic and environmental factors, and more. The complex interplay of these factors makes it challenging to pinpoint a consistent effect of maternal diabetes on offspring body size. Further research is needed to better understand the underlying mechanisms and identify specific factors that contribute to the varied outcomes.

In epidemiological and pre-clinical animal studies, babies born to mothers with gestational diabetes have been found to exhibit smaller brain volumes, reduced brain function, and changes in brain structure compared to babies born to mothers without gestational diabetes [24,25,26]. The high levels of glucose in the mother’s blood during pregnancy can have an impact on the baby’s brain development, potentially leading to cognitive and behavioral problems later in life [21,27]. Consistent with previous findings, the present study indicated that STZ-induced GDM had no effect on neonatal brain weight in both male and female offspring at the dose of 50 mg/kg but reduced the brain weight in male offspring at the dose of 65 mg/kg. When the brain weights were normalized to their body weights, the ratios of brain/body weights in both male and female offspring were significantly higher in the STZ exposed group compared to the controls. These data suggest that GDM exposure could cause an asymmetrical developmental effect on the neonatal brain. Furthermore, the present study demonstrated that exposure to hyperglycemia in utero leads to impairment of brain functional response in postnatal life. Specifically, when the neonatal offspring were subjected to hypoxia/ischemia (HI) stimulation, the HI-induced brain infarct sizes were increased in the GDM exposed group compared to the control group in both male and female offspring. These findings suggest that in utero exposure to maternal diabetes could lead to the development of hypoxia/ischemia-sensitive phenotype in postnatal life.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a condition characterized by reduced oxygen and blood flow to the brain, resulting in brain injury. The underlying mechanisms that lead to enhanced susceptibility to ischemic insult in the GDM-exposed offspring are largely unknown. However, our current finding indicate that ROS production was significantly increased in the GDM-exposed neonatal male offspring. This suggests that increased oxidative stress may have implications for the developing brain of the offspring and could be a key mechanism contributing to the development of brain’s hypoxia/ischemia-sensitive phenotype. It is important to note that the mechanisms underlying GDM-mediated developmental diseases are likely complex and multifactorial. In addition to increased ROS production, our current data also showed significantly decreased AKT/mTOR abundances and their phosphorylation levels, but increased autophagy-related protein (Atg5 and LC3-II) levels in the neonatal offspring brains in response to GDM exposure. These findings are consistent with previous studies that have associated GDM with alterations in various cellular signaling pathways, including the AKT/mTOR-mediated autophagy signaling pathway [6,7]. AKT is a key downstream mediator of insulin signaling and plays a central role in regulating glucose metabolism and cell survival. mTOR is a protein kinase that acts as a central regulator of cell growth, metabolism, and autophagy. Autophagy is a cellular process involved in the degradation and recycling of damaged or unnecessary cellular components. Mammalian LC3B isoform is subject to a posttranslational ubiquitin-like conjugation pathway, by which the LC3s are covalently conjugated to phosphatidylethanolamine (PE) and thereby selectively localize to the autophagosomal membrane. This conversion of LC3 from an unconjugated form (LC3-I) to a PE-conjugated form (LC3-II) is pivotal for the formation of autophagosomes. Our present findings that GDM exposure selectively enhanced the expression of LC3-II suggest an increased autophagy pathway. Atg5 is a well-established key regulator involved in autophagosome formation, making it a prominent target of interest in autophagy studies. While an increase in Atg5 was observed in present study, it’s essential to acknowledge that autophagy is a complex process involving a network of genes and proteins. Therefore, further investigation into the changes of other autophagy-related genes is warranted to gain a comprehensive understanding of the regulatory mechanisms at play in the context of mTOR downregulation and autophagy activation.

Growing evidence suggests that maternal GDM-induced adverse outcomes are associated with the change in the AKT/mTOR-mediated autophagy signaling pathway. However, the molecular link and the causal factors between GDM exposure and the development of a hypoxia/ischemia-sensitive phenotype are not fully understood. There is increasing support for the role of PTEN (Phosphatase and tensin homolog) as an upstream regulator of the AKT/mTOR pathway, and decreased PTEN activity can result in increased activation of the AKT pathway in neonatal brain tissues [16,17,28]. PTEN negatively regulates the AKT pathway by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate (PIP3) to phosphatidylinositol 4,5-bisphosphate (PIP2) [17]. Studies using conditional PTEN knockout models in mouse brains have shown that loss of PTEN leads to increased AKT activation, resulting in abnormal brain development and altered synaptic plasticity [29]. Additionally, alterations in the PTEN/AKT pathway during early brain development have been linked to neurodevelopmental disorders and behavioral abnormalities, including conditions like autism spectrum disorder (ASD) and epilepsy, underscoring its critical role in normal brain development and function [29]. Previous studies have also highlighted the significant role of PTEN/AKT signaling in the setting of brain ischemic injury [17]. Consistent with these previous studies, our present study demonstrated that GDM exposure enhanced PTEN protein expression in neonatal offspring. Moreover, treatment of PTEN inhibitor (BPV) eliminated the differences in PTEN protein expression and HI-induced brain injury between the GDM exposed group and the control group. Bisperoxovanadate (BPV) compounds are well-characterized potent PTEN inhibitors [30]. The effect of BPV on PTEN is primarily achieved by binding to the catalytic site of PTEN, inhibiting its phosphatase activity, thereby blocking PTEN’s negative regulatory effect on the PI3K/Akt signaling pathway. In addition to inhibiting the phosphorylation of PTEN, previous studies have also demonstrated that BPV is capable of decreasing PTEN expression [31, 32]. Indeed, our present study also showed that BPV attenuated the expression of PTEN and eliminated the difference of PTEN expression between the control and GDM-exposed groups. These findings suggest that the GDM-mediated overexpression of PTEN plays a causal role in the development of the neonatal brain hypoxia/ischemia-sensitive phenotype in offspring.

Study Limitations:

The STZ model is a well-established, reliable method for inducing hyperglycemia in rats to study GDM. Its advantage lies in the controlled dosage adjustment. Importantly, it can induce maternal hyperglycemia without penetrating the placenta and directly affecting the fetuses. However, there are some limitations for this model. For example, while STZ replicates beta cell death, not all cases of GDM are associated with this pathology. Therefore, it may not fully represent the entire spectrum of GDM pathophysiology, given that GDM is a complex condition with various contributing factors. To address these limitations, we plan to incorporate other GDM models (e.g., high-fat diet, genetic) in future research to explore various GDM factors, including insulin resistance, obesity, and inflammation. This will aid our investigation into GDM0027s potential impact on fetal neurodevelopmental disorders.

In conclusion, this study provides novel evidence that GDM exposure is a significant risk factor for fetal/neonatal growth restriction and the aberrant development of a neonatal brain with a hypoxia/ischemia-sensitive phenotype in offspring. As showing in Figure 6D, our data demonstrated that STZ-induced GDM caused oxidative stress and increased PTEN protein abundance in the developing neonatal brain. This elevated PTEN downregulated AKT/mTOR expression and inhibited their activities (phosphorylation), which subsequently activated autophagy signaling, resulting in enhanced HI-induced brain injury in neonatal offspring. Furthermore, inhibition of PTEN using its inhibitor BPV rescued the GDM-mediated neonatal brain injury. Our results strongly suggest that PTEN acts as a central regulator and plays causal role in the GDM-mediated development of the brain hypoxia/ischemia-sensitive phenotype. These findings hold promise as they could provide novel therapeutic strategies for targeting the PTEN signaling pathway to prevent or rescue the development of brain ischemic diseases in postnatal life. Understanding the intricate mechanisms involved in GDM-induced developmental disorders can lead to more effective interventions and improved outcomes for affected neonates.

Highlights.

  • STZ-induced maternal hyperglycemia (GDM) decreases neonatal offspring blood glucose levels.

  • Prenatal exposure to GDM produces a growth restriction and asymmetrical brain development.

  • GDM exposure causes alterations in the PTEN/AKT/mTOR/autophagy signaling pathway in neonatal offspring.

  • Prenatal GDM exposure programs a hypoxia/ischemia (HI)-sensitive phenotype of neonatal brain.

Acknowledgments

This work was supported by National Institutes of Health Grants HL135623, and HD088039 (to D. Xiao). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Footnotes

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Declaration of Competing Interest

The authors have declared that no known competing financial interests or personal relationships that could have appeared to influence the work reported in the paper.

Data availability

Data will be made available on request.

Reference

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Associated Data

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Data Availability Statement

Data will be made available on request.

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