Neuronal metabolic adaptations in response to recurrent hypoglycaemia

Background and Aims: Hypoglycaemia remains an all-too-common side-effect of insulin treatment in type 1 diabetes (T1D). It is well appreciated that recurrent hypoglycaemia (RH) results in a progressive reduction in the counterregulatory response (CRR) to hypoglycaemia, increasing the risk of severe hypoglycaemia and development of impaired awareness of hypoglycaemia (IAH). The hypothalamus is involved in glucose-sensing, maintenance of whole-body glucose homeostasis, and is known to have a crucial role in the CRR. A specialised subset of hypothalamic neurons alters their activity in response to glucose concentrations. Glucose use and ATP generation for subsequent channel modulation are critical determinants of these cells’ activity. As the mitochondria is the main site of ATP generation, it is likely that mitochondrial function has an impact on cell function and activity. As such, this project aimed to investigate the impact of RH on mitochondrial function and metabolism which in turn may cause cellular adaptations contributing to a defective CRR. Methods: RH was induced in vitro in the hypothalamic cell line, mHypoA-2/21, and in primary hypothalamic cultures. A Seahorse XFe24 Analyser was used to investigate cellular metabolism, bulk RNA-seq to quantify gene expression changes, and metabolomics to further investigate the effect of RH on glucose metabolism. A range of additional techniques were also employed. Results: Six episodes of RH significantly increased basal and maximal OCR 24-hours after the final hypoglycaemia event but not after 3-hours. No effect of treatment was reported with three episodes of RH. RNA-seq analysis highlighted DEGs relating to the ETC, protein synthesis, along with aspects of dendritic and synaptic structure and function. Metabolomic analysis was underpowered; however, an emerging effect of RH was identified and is worth pursuing. Conclusions: The experiments in this thesis have identified metabolic adaptations, potential alterations to protein synthesis, and probable structural cellular changes occurring in response to RH in mHypoA-2/21 cells. Further work is required to recapitulate these findings in a primary culture setting. Altogether, this thesis has given an indication of the metabolic and cellular adaptations occurring in neuronal cells in response to RH which may contribute to further impairment of the CRR.