Endosome-lysosome transfer of insulin and glucagon in a liver cell-free system.
The endosome-lysosome transfer of in vivo internalized insulin and glucagon 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 postmitochondrial supernatant isolated 8 min after injection of [125I]iodoinsulin showed that the membrane-associated radioactivity (55% of the total) 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 detected 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 [125I]iodoinsulin to trichloroacetic-acid-soluble products occurred during incubation (20%) but this was unaffected by lysosome depletion. Gel-filtration and HPLC analysis of acid extracts of gradient fractions isolated after injection of [125I]iodoinsulins selectively labeled at tyrosine residues A14 or B26 revealed 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-mass components more abundant in lysosomal fractions than in endosomal fractions. In vivo internalized [125I]iodoglucagon and [125I]iodogalBSA underwent a greater lysosomal transfer (17-21%) and lesser degradation (8-11%) than [125I]iodoinsulin. Glycyl-L-phenylalanine 2-naphtylamide and methionine O-methyl ester, two lysosome-disrupting enzyme substrates, partially released the radioactivity associated with lysosomal fractions (GalBSA > insulin = glucagon) but caused little or no release of that associated with endosomal fractions. Analysis of the alpha and beta subunits of the insulin receptor by cross-linking to [125I]iodoinsulin and Western immunoblotting, respectively, revealed a partial lysosomal transfer of these subunits during endosome-lysosome interaction. We conclude that an endosome-lysosome transfer of insulin and glucagon occurs in a liver cell-free system and suggest that the low recovery of these peptides in lysosomal fractions in vivo results from their rapid degradation within endosomes.
Chauvet
G
G
INSERM Unité 30, Groupe Hospitalier Necker Enfants Malades, Paris, France.
Tahiri
K
K
Authier
F
F
Desbuquois
B
B
D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002474:Cell-Free System; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D005934:Glucagon / Q000378:metabolism*; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000378:metabolism
D007328:Insulin; D007457:Iodine Radioisotopes; D005934:Glucagon; D011972:Receptor, Insulin