Human blood-brain barrier leptin receptor - Binding and endocytosis in isolated human brain microvessels

The peripheral production of leptin by adipose tissue and its putative effect as a signal of satiety in the central nervous system suggest that leptin gains access to the regions of the brain regulating energy balance by crossing the brain capillary endothelium, which constitutes the blood-brain barrier in vivo. The present experiments characterize the binding and internalization of mouse recombinant leptin in isolated human brain capillaries, an in vitro model of the human blood-brain barrier. Incubation of I-125-leptin with isolated human brain capillaries resulted in temperature-dependent binding: at 37 degrees C, similar to 65% of radiolabeled leptin was bound per milligram of capillary protein. Two-thirds of the bound radioactivity was resistant to removal by acid wash, demonstrating endocytosis of I-125-leptin into capillary cells, At 4 degrees C, binding to isolated capillaries was reduced to similar to 239%/mg of protein, the majority of which was acid wash resistant, Binding of I-125-leptin to brain capillary endothelial plasma membranes was saturable, described by a two-site binding model with a high-affinity dissociation constant of 5.1 +/- 2.8 nhl and maximal binding capacity of 0.34 +/- 0.16 pmol/mg of membrane protein, Addition of porcine insulin or insulinlike growth factor at a final concentration of 100 nM had a negligible effect on leptin binding. These results provide evidence for a leptin receptor that mediates saturable, specific, temperature-dependent binding and endocytosis of leptin at the human blood-brain barrier.