Isolation and characterization of a novel proopiomelanocortin-derived peptide from hemofiltrate of chronic renal failure patients

We report the isolation of a novel human circulating proopiomelanocortin-derived peptide, VA- beta- MSH, from hemofiltrate and its pharmacological characterization. Screening for lipolytic activity in differentiated 3T3- L1 adipocytes led to the isolation from a hemofiltrate peptide library by alternating reverse phase and cation exchange chromatography. In the course of this isolation, we also identified human beta- MSH( 1 - 22). We synthesized VA- beta- MSH by the N-( 9- fluorenyl)methoxycarbonyl ( F- moc) solid phase method and used synthetic beta- MSH-( 1 - 22) to confirm that both isolated peptides are lipolytically active in a dose- dependent manner in differentiated 3T3- L1 adipocytes in the nanomolar range. Using cAMP ELISA, we demonstrate that stimulation with both peptides caused a strong cAMP elevation in this cell system. Furthermore, we show that the selective inhibitors of cAMP- dependent protein kinase, 8-( 4- Chlorophenylthio)adenosine- 3 ', 5 '- cyclic monophosphorothioate, Rp- isomer ( Rp- 8- CPT- cAMPS); N-[ 2-( p- Bromocinnamylamino) ethyl]- 5-isoquinolinesulfonamide ( H89), significantly reduce VA- beta-MSH- and beta- MSH-( 1 - 22)- mediated lipolysis. Although isolated after its lipolytic activity on 3T3- L1 cells, this newly identified circulating human melanocortin may serve other functions in human physiology. Moreover, the fact that these peptides have been identified after a functional assay, but have been overseen in large proteomic approaches, underscores the importance of such approaches in identifying previously undescribed circulating bioactive molecules.