The major limiting factor in the successful management of type 1 diabetes is Recurrent Hypoglycaemia (RH), resulting from supraphysiological insulin supplementation, which attenuates the Counterregulatory Response (CRR) to future hypoglycaemic episodes. It is now widely accepted that the hypothalamus contributes to hypoglycaemia detection and activation of the CRR (1). Within the hypothalamus, glucose-sensing (GS) neurons exist which are thought to contain the same glucose sensing cellular machinery as pancreatic β-cells, such as ATP-sensitive K+ channels (KATP), glucokinase, glucose transporter proteins and AMPK (2). Therefore, we hypothesise that disruption of normal function in these specialised neurons may occur following RH. We have previously shown that mouse hypothalamic GT1-7 cells are GS neurons (3) and display impaired electrical activity to low glucose following RH, independent of changes in glucose uptake/oxidation (4). Here we have investigated AMPK activity and mitochondrial function as possible mediators of altered electrical activity following RH. GT1-7 cells were exposed to 3 hours of low (0.1 mM) glucose for 3 days to model RH, with cells being assayed on day 4. AMPK activity was measured by radiolabelled kinase activity assay. Cellular Oxygen Consumption Rate (OCR) was measured under euglycaemic conditions using an XF24 extracellular flux analyser (Seahorse Bioscience, Inc.). All data sets are expressed as means ± standard error of the mean. Statistical analysis was performed using either Student’s t-test or ANOVA. Following RH of GT1-7 cells, the normal increment in α1AMPK activity to 0.1 mM glucose stimulus was significantly attenuated after 15 minute (RH = 0.97 fold ± 0.31 vs. Cont = 1.48 fold ± 0.09, p<0.01; n = 7) and 3 hour incubations (RH = 1.07 fold ± 0.20 vs. Cont = 3.98 fold ± 0.67, p<0.001; n=7). α2AMPK activity was not significantly perturbed and no change in AMPK subunit expression was observed following RH. Additionally there were no significant changes in total or phosphorylated protein levels of the upstream AMPK activating kinases, TAK1 or LKB1. Examination of mitochondrial function following RH showed no change in spare respiratory capacity or the proportion of OCR contributing to ATP synthesis. However, RH significantly reduced basal OCR of GT1-7 cells (RH = 4.85 ± 0.87 vs. Cont = 8.10 ± 0.60 pmoles O2/min/μg, p<0.05; n=5). We conclude that blunted α1AMPK activation to low glucose following RH reduces substrate flux through the oxidative phosphorylation pathway in association with defective GS of hypothalamic neurons.