Figure 2.

Leptin stimulates PC1 and PC2 promoter activity via STAT3 in transfected cells and activates STAT3 in TRH neurons in vivo. 293T cells were transfected with ObRb cDNAs together with PC1 (A) or PC2 (B) promoter-luciferase reporter plasmids, and STAT3 expression vector or the corresponding empty vector was also cotransfected as indicated. A CMV-lacZ control plasmid was included in all transfections to correct for differences in transfection efficiencies. Serum-deprived cells were left unstimulated or treated with 20 nM leptin for 6 hours, and luciferase activities were measured in the cell lysates. Luciferase activities were normalized with β-galactosidase activities measured in the same samples. This experiment was performed 3 times, each one in triplicate. Shown are means ± SE. β-Galactosidase activities did not change with treatments. (C and D) Leptin-dependent STAT3 phosphorylation in TRH neurons in the PVN of rats. Animals were given a single i.v. injection of recombinant leptin (1.0 mg/kg) or vehicle (PBS) and killed 45 minutes later. Coronal brain sections were obtained and subjected to double IHC using anti–P-STAT3 nuclear (brown staining) and anti-proTRH cytoplasmic (green fluorescence) antiserum. Scale bar: 10 μm.