Abstract

Aims/hypothesisRecurrent hypoglycaemia (RH) is a major side-effect of intensive insulin therapy for people with diabetes. Changes in hypoglycaemia sensing by the brain contribute to the development of impaired counterregulatory responses to and awareness of hypoglycaemia. Little is known about the intrinsic changes in human astrocytes in response to acute and recurrent low glucose (RLG) exposure.MethodsHuman primary astrocytes (HPA) were exposed to zero, one, three or four bouts of low glucose (0.1 mmol/l) for three hours per day for four days to mimic RH. On the fourth day, DNA and RNA were collected. Differential gene expression and ontology analyses were performed using DESeq2 and GOseq, respectively. DNA methylation was assessed using the Infinium MethylationEPIC BeadChip platform.Results24 differentially expressed genes (DEGs) were detected (after correction for multiple comparisons). One bout of low glucose exposure had the largest effect on gene expression. Pathway analyses revealed that endoplasmic-reticulum (ER) stress-related genes such as HSPA5, XBP1, and MANF, involved in the unfolded protein response (UPR), were all significantly increased following low glucose (LG) exposure, which was diminished following RLG. There was little correlation between differentially methylated positions and changes in gene expression yet the number of bouts of LG exposure produced distinct methylation signatures.Conclusions/interpretationThese data suggest that exposure of human astrocytes to transient LG triggers activation of genes involved in the UPR linked to endoplasmic reticulum (ER) stress. Following RLG, the activation of UPR related genes was diminished, suggesting attenuated ER stress. This may be a consequence of a successful metabolic adaptation, as previously reported, that better preserves intracellular energy levels and a reduced necessity for the UPR.

Highlights

  • Iatrogenic hypoglycaemia is a limiting factor to optimal glycaemic control in people with type 1 (T1D) and insulin/ sulphonylurea-treated type 2 diabetes [T2D [1]]

  • Astrocytic glutamate uptake is impaired following RH, contributing to counterregulatory failure [16]. Together these data suggest an active role of astrocytes in glucose detection, despite this evidence little is known about the intrinsic changes within astrocytes, especially human astrocytes, following recurrent low glucose (RLG)

  • Volcano plots displaying the pairwise comparisons of each treatment group versus control shows that LG (Figure 2Ai) produced the largest effect on gene expression, whereas changes induced by antecedent RLG (aRLG) (Figure 2Aii) and RLG (Figure 2Aiii) were more modest

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Summary

Methods

Human primary astrocytes (HPA) were exposed to zero, one, three or four bouts of low glucose (0.1 mmol/l) for three hours per day for four days to mimic RH. Differential gene expression and ontology analyses were performed using DESeq and GOseq, respectively. DNA methylation was assessed using the Infinium MethylationEPIC BeadChip platform

Results
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