ENDOSOME-LYSOSOME TRANSFER OF INSULIN AND GLUCAGON IN A LIVER CELL-FREE SYSTEM

The endosome-lysosome transfer of in vivo internalized insulin and glu cagon has been studied in a rat liver cell-free system and compared to that of galactosylated bovine serum albumin (GalBSA), a ligand of the asialoglycoprotein receptor. Density-gradient analysis of a postmitoc hondrial supernatant isolated 8 min after injection of [I-125]iodoinsu lin showed that the membrane-associated radioactivity (55 % of the tot al) migrated as a single peak at the position of galactosyltransferase , a Golgi marker (1.08-1.10 g/ml). After incubation at 37 degrees C in the presence of ATP, an additional peak of radioactivity (12%) was de tected at the position of acid phosphatase, a lysosomal marker (1.12-1 .14 g/ml). No such peak was observed in a lysosome-depleted fraction. An ATP-dependent conversion of [I-125]iodoinsulin to trichloroacetic-a cid-soluble products occurred during incubation (20 %) but this was un affected by lysosome depletion. Gel-filtration and HPLC analysis of ac id extracts of gradient fractions isolated after injection of [I-125]i odoinsulins selectively labeled at tyrosine residues A14 or B26 reveal ed the presence of components which differed from intact iodoinsulins by size and/or hydrophobicity. Low molecular-mass components were less abundant and, conversely, intact iodoinsulin and/or high molecular-ma ss components more abundant in lysosomal fractions than in endosomal f ractions. In vivo internalized [I-125]iodoglucagon and [I-125]iodogalB SA underwent a greater lysosomal transfer (17-21 %) and lesser degrada tion (8-11 %) than [I-125]iodoinsulin. Glycyl-L-phenylalanine 2-naphty lamide and methionine O-methyl ester, two lysosome-disrupting enzyme s ubstrates, partially released the radioactivity associated with lysoso mal fractions (GalBSA > insulin = glucagon) but caused little or no re lease of that associated with endosomal fractions. Analysis of the alp ha and beta subunits of the insulin receptor by cross-linking to [I-12 5]iodoinsulin and Western immunoblotting, respectively, revealed a par tial lysosomal transfer of these subunits during endosome-lysosome int eraction. We conclude that an endosome-lysosome transfer of insulin an d glucagon occurs in a liver cell-free system and suggest that the low recovery of these peptides in lysosomal fractions in vivo results fro m their rapid degradation within endosomes.