The brain contains a subpopulation of glucosensing neurons that alter their firing rate in response to elevated glucose concentrations. In pancreatic -cells, glucokinase (GK), the rate-limiting enzyme in glycolysis, mediates glucose-induced insulin release by regulating intracellular ATP production. A similar role for GK is proposed to underlie neuronal glucosensing. Via in situ hybridization, GK mRNA was localized to hypothalamic areas that are thought to contain relatively large populations of glucosensing neurons (the arcuate, ventromedial, dorsomedial, and paraventricular nuclei and the lateral area). GK also was found in brain areas without known glucosensing neurons (the lateral habenula, the bed nucleus stria terminalis, the inferior olive, the retrochiasmatic and medial preoptic areas, and the thalamic posterior paraventricular, interpeduncular, oculomotor, and anterior olfactory nuclei). Conversely, GK message was not found in the nucleus tractus solitarius, which contains glucosensing neurons, or in ependymal cells lining the third ventricle, where others have described its presence. In the arcuate nucleus, >75% of neuropeptide Y-positive neurons also expressed GK, and most GK + neurons also expressed KIR6.2 (the pore-forming subunit of the ATP-sensitive K + channel). The anatomic distribution of GK mRNA was confirmed in micropunch samples of hypothalamus via reverse transcription-polymerase chain reaction (RT-PCR). Nucleotide sequencing of the recovered PCR product indicated identity with nucleotides 1092-1411 (within exon 9 and 10) of hepatic and -cell GK. The specific anatomic localization of GK mRNA in hypothalamic areas known to contain glucosensing neurons and the coexpression of KIR6.2 and NPY in GK + neurons support a role for GK as a primary determinant of glucosensing in neuropeptide neurons that integrate multiple signals relating to peripheral energy metabolism. Diabetes 49:693-700, 2000 M ammalian feeding behavior and general energy homeostasis appear to be regulated by circulating levels of nutrients (glucose) and peptides (e.g., leptin, insulin). Sensors to detect levels of these factors have been found to reside within specific nuclei of the hypothalamus (1-8), where central regulation of energy homeostasis is believed to be coordinated. For example, large changes in blood glucose are correlated with centrally mediated responses such as thermogenesis through activation of the sympathetic nervous system. These changes are monitored by the brain (9-11), and such responses are altered in obesity-prone animals (11-13). Moreover, lesions of the ventromedial hypothalamus (VMH) prevent the hypoglycemic activation of the sympathetic response (14). Thus, available data indicate that glucose detection by hypothalamic neurons may play an important role in regulating energy homeostasis.Glucosensing neurons are among the best characterized of such metabolic sensors. Unlike most neurons, they use glucose as a signaling molecule to alter their firing rate in response to changes in ambient glucose le...