Effect of fasting and leptin deficiency on hypothalamic neuropeptide Y gene transcription in vivo revealed by expression of a lacZ reporter gene.

Neuropeptide Y (NPY), a peptide synthesized in the hypothalamic arcuate nucleus, is implicated in the physiologic control of food intake and body weight. Because both genetic (e.g. in obese ob/ob mice) and acquired leptin deficiency (e.g. fasting in normal mice) increase hypothalamic NPY accumulation, and as leptin administration reverses this effect, we hypothesized that leptin inhibits transcription of the NPY gene by arcuate nucleus neurons. To test this hypothesis, we studied mice with a targeted mutation of the NPY gene (NPY knockout mice), in which the lacZ reporter gene was inserted into the first exon of the NPY gene. As a result, these mice express beta-galactosidase (beta gal; the enzyme encoded by lacZ) in neurons that normally express the NPY gene. To determine whether beta gal staining provides a valid measure of lacZ expression, we used a histochemical method to count the number of beta gal+ neurons in coronal sections of brain tissue from mice bearing either one (NPY+/-) or two (NPY-/-) mutant alleles. In both the arcuate nucleus and the thalamic reticular nucleus, beta gal+ cell number was 260% higher in NPY-/- than in NPY+/- mice (P < 0.05). Fasting for 48 h also increased the mean beta gal+ cell number in the arcuate nucleus of NPY+/- mice by 260% (P < 0.001), but had no effect in the thalamic reticular nucleus. Similarly, obese leptin-deficient ob/ob, NPY+/- mice had a 67.3% increase in arcuate nucleus beta gal+ cell number compared with lean ob/+, NPY+/- littermates (P < 0.05), and this effect was attenuated 36.6% (P < 0.05) by leptin administration (70 microg/day, i.p., for 4 days). Based on the results of this novel method for measuring NPY gene transcription in vivo, we conclude that both fasting and genetic leptin deficiency increase NPY gene transcription in the arcuate nucleus and that this transcriptional response is attenuated by leptin administration in ob/ob, NPY+/- mice.