Receptor-mediated insulin degradation and insulin-stimulated glycogenesis in cultured foetal hepatocytes

Abstract

Insulin-stimulated glycogenesis and insulin degradation were studied simultaneously at 37 degrees C in cultured foetal hepatocytes grown for 2-3 days in the presence of cortisol. Degradation of cell-associated insulin, as measured by trichloroacetic acid precipitation, was significant after 4 min in the presence of 1-3 nM-125I-labelled insulin. This process became maximal (30% of insulin degraded) after 20 min, a time when binding-state conditions were achieved. No insulin-degradative activity was detected in a medium that had been exposed to cells. At steady-state, the appearance of insulin degradation products in the medium was linearly dependent on time (1.5 fmol/min per 10(6) cells at 1nM-125I-labelled insulin). Chloroquine (3-50 microM), bacitracin (0.1-10 mM) and NH4Cl (1-10 mM) inhibited insulin degradation as soon as this became detectable and caused an increase in the association of insulin to hepatocytes after 20 min. Lidocaine and dansylcadaverine had similar effects, whereas N-ethylmaleimide, aprotinin, phenylmethanesulphonyl fluoride and leupeptin were found to be ineffective. Chloroquine, and also bacitracin, at concentrations that inhibited insulin degradation, decreased the insulin-stimulated incorporation of [14C]glucose into glycogen over 2 h. This effect of chloroquine was specific, since it did not modify the basal glycogenesis, or the glycogenic effect of a glucose load in the absence of insulin. It therefore appears that the receptor-mediated insulin degradation (or some associated pathway) is functionally related to the glycogenic effect of insulin in foetal hepatocytes.

Hepatic glucagon metabolism. Correlation of hormone processing by isolated canine hepatocytes with glucagon metabolism in man and in the dog.

“…Although it is a matter of some debate whether peptide hormone metabolism plays a role in the mechanism of hormone action (32)(33)(34), it is at least clear that hormone metabolism by target tissues is a function, in large part, of ligand interaction with plasma membrane receptors and that it occurs in parallel with processes by which hormone-receptor interactions regulate cellular events (3)(4)(5)(6)16). By use of isolated hepatocytes, we have demonstrated that (a) a major peptide product of glucagon metabolism is glucagon(4-29), (b) the hormone fragment, rather than being stored or further degraded, is released into the cell incubation medium, and (c) the fragment arises from cell-mediated processes that require components in addition to highly purified plasma membranes per se.…”

Evidence of the lack of receptor-mediated insulin degradation in human cultured lymphocytes (RPMI-1788 line).

“…It has been hypothesized that the increase in insulin binding in the presence of chloroquine is due to the decrease in insulin degradation by the inhibition of lysosomal function (Marshall and Olefsky, 1979, Kobayashi et al, 1980, and Plas and Desbuquois, 1982. However, it was demonstrated that IM-9 lymphocytes lost receptor-mediated insulin degrading activity and that chloroquine did not decrease the insulin degradation (Baldwin et al , 1980, Misbin et al , 1980, Sonne and Gliemann, 1980, Saviolakis et al, 1981, and Iwamoto, et al, 1981.…”

“…Chloroquine, a lysosomotropic agent, increases cell-associated insulin, and decreases receptor-mediated insulin degradation in adipocytes Olefsky, 1979, Kobayashi et al 1980) and hepatocytes (Plas and Desbuquois, 1982). It has been hypothesized that the increase in insulin binding in the presence of chloroquine is due to the decrease in insulin degradation by the inhibition of lysosomal function (Marshall and Olefsky, 1979, Kobayashi et al, 1980, and Plas and Desbuquois, 1982.…”

“…The lysosomes are the main sites of receptor-mediated insulin degradation because chloroquine, an inhibitor of lysosomal proteolysis, inhibits insulin degradation and stimulates both an accumulation of intracellular insulin and insulin binding in several target cells (Marshall and Olefsky, 1979, Kobayashi et al, 1980, Plas and Desbuquois, 1982. On the other hand, insulin molecules are also degraded by other mechanism such as the non-receptor mediated pathway in several tissue (Carpentier et al, 1979).…”

Expression de la réponse glycogénique à l'insuline dans les hépatocytes fœtaux en culture

“…Le phénomène de diminution du nombre de récepteurs de l'insuline après exposition prolongée à l'hormone (« down regulation ») décrit dans les hépatocytes adultes en culture primaire (Blackard, Guzelian et Small, 1978 ;Caro et Amatruda, 1980) n'est pas détecta-ble dans les hépatocytes foetaux j menuelle et Plas, 19811. En présence de chloroquine qui modifie le devenir du complexe insuline-récepteur dans la cellule, on observe une perte spécifique de l'effet glycogénique de l'insuline sans modification de l'effet glycogénique d'une charge de glucose (Plas et Desbuquois, 1982). Cette drogue révèle donc une situation analogue à celle obtenue dans les hépa-tocytes désensibilisés à l'hormone.…”