For protein analysis, hypothalamic samples were collected 48 h post-adenoviral injection. Rat hypothalamus was homogenized in buffer A (0.3 mM sucrose, 3 mM DTT, 1 mM EDTA, 100 μg/mL PMSF, 2 μg/mL pepstatin A, 2 μg/mL leupeptin and 2 μg/mL aprotinin) and sonicated three times on ice at 300 W (Sonics & Material INC, VCF1, Connecticut, USA) for 10 s. After centrifugation at 4,000× g for 10 min, the proteins were resolved by SDS-PAGE (50 µg/lane) in a 12% (w/v) polyacrylamide gel, transferred to PVDF membranes (0.45 µm pore, Amersham Pharmacia Biotech., Piscataway, NJ, USA), and probed with rabbit anti-GK (1:100, Santa Cruz Biotechnology, CA, USA), rabbit anti-GKRP (Santa Cruz Biotechnology), rabbit anti-GLUT2 (Alpha Diagnostics, 1:1000), rabbit anti-EGFP and anti-β-actin (1:1000) antibodies. After extensive washing, the PVDF membranes were incubated for 1 h at 4 °C with peroxidase-labeled anti-rabbit IgG (1:5000; Jackson ImmunoResearch). The reaction was developed using an enhanced chemiluminescence (ECL) Western blotting analysis system (Amersham Biosciences). Negative controls consisted of incubating the membrane in the absence of anti-GK. The images shown are representative of at least three analyses performed on samples from at least three separate experiments. β-actin expression levels were used as a loading control, and GFP expression levels were used as a transduction control.