Whether the brain synthesizes insulin is currently debated. Two clonal, immortalized mouse hypothalamic cell lines from e17, mHypoE-39 and mHypoE-46, express insulin 2 (Ins2), but not Ins1. We analyzed regions necessary for basal gene activity and found that the mouse Ins2 region -110/+183 bp stimulates promoter activity in hypothalamic neurons. The rat Ins2 showed moderate activity, whereas the human promoter construct is repressed below basal levels. In MIN6 pancreatic beta-cells, all of the Ins1 and Ins2 promoter constructs display high levels of transcriptional activity. The cell lines also express components of glucose-sensing machinery and the endogenous glucagon-like peptide 1 receptor (Glp-1R). We observed that 16.7 mM glucose induces Ins2 mRNA, while forskolin and a Glp-1 agonist, exendin-4, induce a biphasic Ins2 mRNA response in mHypoE-39 neurons. The insulin cis-regulatory regions differ between the pancreas and the hypothalamus, and glucose and Glp-1 regulate the expression of hypothalamic insulin.