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Fate of injected glucagon taken up by rat liver in vivo. Degradation of internalized ligand in the endosomal compartment.

ABSTRACT: The uptake and processing of glucagon into liver endosomes were studied in vivo by subcellular fractionation. After injection of [[125I]iodo-Tyr10]glucagon and [[125I]iodo-Tyr13]glucagon to rats, the uptake of radioactivity into the liver was maximum at 2 min (6% of the dose/g of tissue). On differential centrifugation, the radioactivity in the homogenate was recovered mainly in the nuclear (N), microsomal (P) and supernatant (S) fractions, with maxima at 5, 10 and 40 min, respectively; recovery of radioactivity in the mitochondrial-lysosomal (ML) fraction did not exceed 6% and was maximal at 20 min. On density-gradient centrifugation, the radioactivity associated first (2-10 min) with plasma membranes and then (10-40 min) with Golgi-endosomal (GE) fractions, with 2-5-fold and 20-150-fold enrichments respectively. Subfractionation of the GE fractions showed that, unlike the Golgi marker galactosyltransferase, the radioactivity was density-shifted by diaminobenzidine cytochemistry. Subfractionation of the ML fraction isolated at 40 min showed that more than half of the radioactivity was recovered at lower densities than the lysosomal marker acid phosphatase. Throughout the time of study, the [125I]iodoglucagon associated with the P, PM and GE fractions remained at least 80-90% trichloroacetic acid (TCA)-precipitable, whereas that associated with other fractions, especially the S fraction, became progressively TCA-soluble. On gel filtration and h.p.l.c., the small amount of degraded [125I]iodoglucagon associated with GE fractions was found to consist of monoiodotyrosine. Chloroquine treatment of [125I]iodoglucagon-injected rats caused a moderate but significant increase in the late recovery of radioactivity in the ML, P and GE fractions, but had little effect on the association of the ML radioactivity with acid-phosphatase-containing structures. Chloroquine treatment also led to a paradoxical decrease in the TCA-precipitability of the radioactivity associated with the P and GE fractions. Upon h.p.l.c. analysis of GE extracts of chloroquine-treated rats, at least four degradation products less hydrophobic than intact [125I]iodoglucagon were identified. Radio-sequence analysis of four of these products revealed three cleavages, affecting bonds Ser2-Gln3, Thr5-Phe6 and Phe6-Thr7. When GE fractions containing internalized [125I]iodoglucagon were incubated in iso-osmotic KCl at 30 degrees C, a rapid generation of TCA-soluble products was observed, with a maximum at pH 4. We conclude that endosomes are a major site at which internalized glucagon is degraded, endosomal acidification being required for optimum degradation.

SUBMITTER: Authier F

PROVIDER: S-EPMC1149766 | biostudies-other | 1990 Dec

REPOSITORIES: biostudies-other

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Project description:Subcellular-fractionation techniques were used to characterize the endocytic pathway followed by ricin in rat liver in vivo and tentatively identify the site(s) at which the ricin interchain disulphide bridge is split. After injection of 125I-ricin, hepatic uptake of radioactivity was maximum at 30 min (40% of injected dose). At 5 min, about 80% of the radioactivity in the homogenate was recovered in the microsomal (P) fraction, but later on the recovery of the radioactivity in the mitochondrial-lysosomal (ML) fractions progressively increased (50% at 30 min) at the expense of that in the P fraction. Subfractionation of the P and ML fractions on analytical sucrose-density gradients revealed a time-dependent translocation of the radioactivity from low- to high-density endocytic structures, with median relative densities at 5 and 60 min of about 1.15 and 1.16 (P fraction) and 1.19 and 1.22 (ML fraction) respectively. The late distribution of the radioactivity in the ML fraction was similar to that of the lysosomal marker acid phosphatase. Studies with co-injected lactose and mannan showed that ricin was internalized mainly via the mannose receptor. In the presence of mannan, the late recovery of radioactivity in the ML fraction was decreased, and the distribution of the radioactivity associated with the P fraction was shifted toward lower densities (median relative density 1.13), indicating a different pathway of endocytosis. Analysis of the radioactivity associated with the ML and S fractions by SDS/PAGE revealed a time-dependent increase in the amount of intact A- and B-chains and low-molecular-mass products. When ML fractions containing partially processed ricin were incubated at 37 degrees C at pH 5 or at pH 7.2 in the presence of ATP, only low-molecular-mass products were generated. We conclude that internalized ricin associates with endocytic structures whose size and density of equilibration increase with time, and that, although detectable in these structures, reduction of the ricin interchain disulphide bridge occurs to a large extent in the cytosol.

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Fate of injected glucagon taken up by rat liver in vivo. Degradation of internalized ligand in the endosomal compartment.

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