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

Agouti-related protein (AgRP), expressed in both the periphery and the brai n, can result in obesity. Its active C-terminal fragment, AgRP(83-132), was recently reported to increase feeding and antagonize alpha-melanocyte-stim ulating hormone (alpha -MSH) and leptin. We used multiple-time regression a nalysis to show that the rate at which AgRP(83-132) crossed the blood-brain barrier (BBB) from the brood to the brain was very slow (K-i = 0.6 x 10(-4 ) mL/g.min). Entry was not self-inhibited by excess AgRP(83-132) after eith er intravenous (IV) injection or perfusion in brood-free medium, indicating the absence of a saturable transport system, and was not cross-inhibited b y alpha -MSH or leptin, Not only did AgRP(83-132) cross much slower than th e 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 depl etion showed that it entered the brain parenchyma rather than binding to ca pillary 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 tow octan ol/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 brood to the brain, its nonsaturable rate of entry is very slow, probably i nfluenced by aggregation, Copyright (C) 2000 by W.B. Saunders Company.