Aims Glucokinase (GK) serves as a glucose sensor in several tissues including glucose‐sensitive neurons of the arcuate nucleus within the hypothalamus. We have previously demonstrated a role for arcuate GK in the regulation of food and glucose intake. However, its role in the regulation of glucose homeostasis is less clear. We therefore sought to investigate the role of arcuate GK in the regulation of glucose homeostasis. Materials and Methods Recombinant adeno‐associated virus expressing either GK or an antisense GK construct was used to alter GK activity specifically in the hypothalamic arcuate nucleus. GK activity in this nucleus was also increased by stereotactic injection of the GK activator, compound A. The effect of altered arcuate nucleus GK activity on glucose homeostasis was subsequently investigated using glucose and insulin tolerance tests. Results Increased GK activity specifically within the arcuate nucleus increased insulin secretion and improved glucose tolerance in rats during oral glucose tolerance tests. Decreased GK activity in this nucleus reduced insulin secretion and increased glucose levels during the same tests. Insulin sensitivity was not affected in either case. The effect of arcuate nucleus glucokinase was maintained in a model of type 2 diabetes. Conclusions These results demonstrate a role for arcuate nucleus GK in systemic glucose homeostasis
