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.
INSERM Unité 30, Groupe Hospitalier Necker Enfants Malades, Paris, France.
G
G
Chauvet
K
K
Tahiri
F
F
Authier
B
B
Desbuquois
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