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

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.

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

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.

Pregnant rats were administered with either saline or STZ on the 12^th 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.

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

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.

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

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.

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

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.

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

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.