AMPK is a metabolic master switch activated by multiple stimuli (e.g. hypoglycaemia, muscle contraction and hypoxia) that increase the cellular AMP: ATP ratio (Rutter et al. 2003). Once activated AMPK inhibits ATP consuming processes and enhances ATP production partly, at least, by changes in fuel selection. Pancreatic β-cells however, display unique properties with regard to fuel sensing and energy maintenance. The β-cell isoform of hexokinase (hexokinase IV; also known as glucokinase) is only basally active at normal glucose levels, and is not negatively regulated by glucose-6-phosphate, in contrast to the liver isoform. Since β-cells are essentially nutrient deprived at normal glucose levels, insulin is secreted at a basal level. Upon raised extracellular glucose, glucokinase becomes more active, increasing ATP levels leading to closure of the ATP-sensitive K⁺-channel. This leads to membrane depolarisation, activation of voltage dependent Ca²⁺-channels, influx of Ca²⁺ and induction of insulin secretion. To examine the influence of AMPK activity on glucose sensing, the glucose-insensitive insulin-secreting cell line, CRI-G1, was used. Using patch-clamp electrophysiology, AMPK activation by acute exposure to AICAR (1 mM; 30-40 minutes) did not affect the membrane potential of CRI-G1 cells. However, acute removal of glucose induced a hyperpolarisation (n = 9), significantly greater in magnitude than in cells not exposed to AICAR (n = 14). To determine if this was due to an effect on glucose uptake, the uptake of the fluorescent glucose analogue, 2-NBDG was measured using confocal microscopy. AICAR did not significantly alter glucose uptake for exposure times up to 40 minutes An in vitro enzyme assay for glucokinase revealed that AICAR (40 minutes) reduced glucokinase activity, as measured by the production of NADPH. Confocal immunofluorescence demonstrated that neither the total glucokinase expression nor the nuclear/cytoplasmic ratio of glucokinase changed during the time course of the experiment. Thus, in CRI-G1 cells, AMPK activation sensitises cells to changes in glucose by limiting glucokinase activity when glucose levels are low. This contrasts with other tissues, where activation of AMPK leads to maintenance of ATP levels. This is surprising given that β-cells exhibit differential glucose sensing and that large changes in ATP are required as the primary regulatory mechanism for insulin secretion. Additional studies are underway to define further the role of AMPK in regulating glucokinase activity and glucose sensing in pancreatic β-cells.