Agouti-related protein(83-132) aggregates and crosses the blood-brain barrier slowly

Agouti-related protein (AgRP), expressed in both the periphery and the brain, can result in obesity. Its active C-terminal fragment, AgRP(83-132), was recently reported to increase feeding and antagonize alpha-melanocyte-stimulating hormone (alpha-MSH) and leptin. We used multiple-time regression analysis to show that the rate at which AgRP(83-132) crossed the blood-brain barrier (BBB) from the blood to the brain was very slow (Ki = 0.6 x 10(-4) mL/g x min). Entry was not self-inhibited by excess AgRP(83-132) after either intravenous (i.v.) injection or perfusion in blood-free medium, indicating the absence of a saturable transport system, and was not cross-inhibited by alpha-MSH or leptin. Not only did AgRP(83-132) cross much slower than the saturably entering leptin, but the entry was slower than almost all other non-saturably entering endogenous peptides or neurotrophins. Nevertheless, high-performance liquid chromatography (HPLC) showed that the small amount of AgRP(83-132) crossing the BBB did so in intact form, and capillary depletion showed that it entered the brain parenchyma rather than binding to capillary endothelial cells or adhering to vascular components. There was no rapid efflux system out of the brain that might have misleadingly appeared as slow entry for AgRP(83-132). Poor lipophilicity was shown by a low octanol/buffer partition coefficient. By size-exclusion chromatography, AgRP(83-132) appeared as a 17-kd substance in both blood and buffer. Since protein was absent from the buffer, the 17-kd peak probably represented a trimer of the 5.7-kd AgRP(83-132). Capillary electrophoresis confirmed that most of the AgRP(83-132) existed as a trimer, with much smaller amounts as a dimer and monomer. Thus, although intact AgRP(83-132) can cross the BBB from the blood to the brain, its nonsaturable rate of entry is very slow, probably influenced by aggregation.