The regulation of neuroendocrine electrical activity and gene expression by glucose is mediated through several distinct metabolic pathways. Many studies have implicated AMP and ATP as key metabolites mediating neuroendocrine responses to glucose, especially through their effects on AMP-activated protein kinase (AMPK), but other studies have suggested that glycolysis, and in particular the cytoplasmic conversion of NAD+ to NADH, may play a more important role than oxidative phosphorylation for some effects of glucose. To address these molecular mechanisms further, we have examined the regulation of agouti-related peptide (AgRP) in a clonal hypothalamic cell line, N-38. AgRP expression was induced monotonically as glucose concentrations decreased from 10 to 0.5 mM glucose, and with increasing concentrations of glycolytic inhibitors. However neither pyruvate nor 3-beta-hydroxybutyrate (3-HOB) mimicked the effect of glucose to reduce AgRP mRNA, but on the contrary produced the opposite effect of glucose and actually increased AgRP mRNA. Nevertheless, 3-HOB mimicked the effect of glucose to increase ATP and to decrease AMPK phosphorylation. Similarly, inhibition of AMPK by RNA interference increased, and activation of AMPK decreased, AgRP mRNA. Further studies demonstrated that neither the hexosamine nor the pentose/ChREBP pathways mediate the effects of glucose on AgRP expression. These studies do not support that either ATP or AMPK mediate effects of glucose on AgRP in this hypothalamic cell line, but support a role for glycolysis, and in particular NADH. These studies support that cytoplasmic or nuclear NADH, uniquely produced by glucose metabolism, mediate effects of glucose on AgRP expression.