Gestational diabetes induces behavioral and brain gene transcription dysregulation in adult offspring

Abstract

The etiology of Autism Spectrum Disorders (ASD) includes a strong genetic component and a complicated environmental component. Recent evidence indicates that maternal diabetes, including gestational diabetes, is associated with an increased prevalence of ASD. While previous studies have looked into possible roles for maternal diabetes in neurodevelopment, there are few studies into how gestational diabetes, with no previous diabetic or metabolic phenotype, may affect neurodevelopment. In this study, we have specifically induced gestational diabetes in mice, followed by behavioral and molecular phenotyping of the mice offspring. Pregnant mice were injected with STZ a day after initiation of pregnancy. Glucose levels increased to diabetic levels between E7 and E14 in pregnancy in a subset of the pregnant animals. Male offspring of Gestational Diabetic mothers displayed increased repetitive behaviors with no dysregulation in the three-chambered social interaction test. RNA-seq analysis revealed a dysregulation in genes related to forebrain development in the frontal cortex and a dysregulation of a network of neurodevelopment and immune related genes in the striatum. Together, these results give evidence that gestational diabetes can induce changes in adulthood behavior and gene transcription in the brain.

Fig. 1. STZ-induced gestational diabetes.

A Blood Glucose levels at 2 weeks of pregnancy (14 days) in pregnant female mice that were injected with STZ at E0.5. STZ-treated mice either develop gestational diabetes by 2 weeks of age (STZ + D) or show very little increase in blood glucose levels (STZ − D), which are similar to pbs-streated mice. B Blood glucose level analysis of pregnant mothers at 3 days, 7 days, and 14 days post conception. Differences between STZ + D and STZ − D groups are already present at 3 days of pregnancy, but reach hyperglycemia only at 14 days of pregnancy. C Number of pups per litter in each experimental group. n = 8 litters per experimental group. Data are presented as mean ± standard error of the mean.

Fig. 2. Effects of STZ treatment on pancreas of offspring.

A Immunofluorescence staining of the STZ + D, STZ − D and Control offspring’s pancreata. Insulin in red, Glucagon in green and DAPI in blue. scale bar = 50 µm. B Comparing the percentage of islet cells in all groups. n = 30 STZ + D, n = 22 STZ − D and n = 16 control. *p < 0.05. C Blood glucose levels of all groups’ offspring. n = 23 STZ + D, n = 22 STZ − D and n = 22 control.

Fig. 3. Effects of Gestational diabetes on behavior.

Total distance traveled in the open field test in male (A) and female (B) mice shows less distance traveled specifically in the male STZ + D mice. Rotarod tests shows no differences in locomotor abilities in all experimental groups in both male (C) and female (D) mice. Grooming time was calculated in all experimental groups during a 10-minute period in the open field maze. There is a significant increase in grooming time in the STZ + D group of both male (E) and female (F) mice. Mice were subjected to a 30-min period in open chamber with 20 marbles. Number of buried marbles were counted. STZ + D group showed significant increase in buried marbles, compared to control group in the male mice (G). No differences are seen in the female mice (H). Data are presented as mean ± standard error of the mean. I, J Mice were subjected to the three chambered social interaction test. All experimental groups displayed normal preference toward stranger mice compared to an empty chamber in both male (I) and female (J) mice. K, L Cue Fear conditioning test was carried out in all experimental groups. In male mice (K), STZ + D mice display significantly more freezing, compared to control mice. There were no differences between groups in female mice. Data are presented as mean ± standard error of the mean. One way Anova. *p < 0.05 n = 10–13.

Fig. 4. Effects of gestational diabetes on cortical gene transcription.

RNA-seq analysis was performed on frontal cortex and striatum samples from all experimental groups. A List of genes that were differentially expressed in the STZ + D frontal cortex. B Gene ontology analysis of the differentially expressed genes from the frontal cortex shows enrichment of genes involved in forebrain development. C, D Real-Time PCR analysis of genes bhlhe22 and Neurod1 in all three experimental groups. Data are presented as mean ± standard error of the mean. One way Anova. *p < 0.05 n = 7 per experimental group.

Fig. 5. Effects of gestational diabetes on striatal gene transcription.

A WGCNA analysis of striatal RNA-seq data. Modules were correlated to maternal diabetes (STZ + D group), amount of marbles buried, and STZ treatment. B, C Scatter plots indicate the correlation (Pearson’s r correlation coefficient) and its significance (p value) between module membership and gene significance (gene correlation to diabetes state). The positive correlation gives further evidence that genes in these modules are associated with the state of gestational diabetes in the mother. D, E Gene ontology analysis of genes in the dark-red and turquoise modules.