I hypothesised that mouse models could be used to investigate the molecular, hormonal and symptomatic responses to acute and recurrent hypoglycaemia. To test this hypothesis, I used hyperinsulinaemic clamps to create carefully controlled experimental manipulations of blood glucose.

It is well established that the hypothalamus plays a fundamental role in glucose sensing and coordination of the concerted effort to maintain blood glucose within a tight homeostatic range. However, little is known about the molecular changes that take place in the hypothalamus under conditions of hypoglycaemia or hyperglycaemia. Therefore, I aimed to use single-nucleus RNA sequencing (snRNAseq) to reveal the hypothalamic molecular signatures and gene expression profiles of different blood glucose levels. Hyperinsulinaemic clamps were used to create carefully controlled hypoglycaemic, euglycaemic and hyperglycaemic experimental interventions. Upon sequencing of the hypothalami, two thirds of nuclei were identified as neuronal. Across the dataset, there was relatively low expression of immediate early genes (IEGs), which could evidence support for the successful minimisation of experimentally induced stress during the hyperinsulinaemic clamp technique. Numerous genes that were differentially expressed under hypoglycaemic or hyperglycaemic conditions were identified. Increased understanding of the glucose responsive transcriptome in health will provide the basis for subsequent investigations into changes that take place in pathophysiological states, such as impaired awareness of hypoglycaemia (IAH).

IAH resulting from recurrent exposure to hypoglycaemia (RH) remains a major obstacle when intensive insulin therapy is used to treat diabetes. RH can cause attenuation of the usual protective physiological (hormonal and behavioural) responses, termed hypoglycaemia associated autonomic failure (HAAF). HAAF has been reproduced experimentally in both humans and rats, however a mouse model of HAAF has not yet been comprehensively validated. In this thesis I found that four consecutive days of hypoglycaemia in mice was sufficient to significantly reduce hormonal responses to a subsequent hypoglycaemic episode, and therefore create a murine model of HAAF.

Using this 4-day HAAF model I examined glucoprivic feeding to assess hunger, as an important symptom of hypoglycaemia awareness, and found a non-significant trend towards attenuation of the feeding response following HAAF induction. I also began to probe the neurocircuitry of IAH through designing and piloting an experimental paradigm to investigate restoration of HAAF. Validation of a murine HAAF model is essential to ensure future basic research is reproducible and accurately reflects the clinical condition. This will allow application of powerful modern molecular techniques to investigate neurocircuitry implicated in HAAF development. Improved mechanistic understanding of HAAF will facilitate the identification of potential therapeutic targets to either prevent the loss of, or restore, awareness to hypoglycaemia in the clinic.