Effects of monensin on insulin interactions with isolated hepatocytes. Evidence for inhibition of receptor recycling and insulin degradation

Abstract

Recent evidence suggests that, during endocytosis, receptors for many polypeptide ligands are spared degradation and are recycled to the plasma membrane for re-utilization. The univalent ionophore monensin was shown to inhibit membrane recycling. We therefore examined its effects on insulin interactions with isolated rat hepatocytes to characterize further receptor endocytosis and recycling in these cells. At 10 degrees C, in the absence of endocytosis, no change in insulin binding was observed. However, at 37 degrees C a concentration-dependent decrease in 125I-insulin binding was seen in the presence of insulin; this reached a maximum of 60% at 1 nM-insulin. Competitive binding studies showed this to be due to a 50-60% decrease in cell-surface insulin-receptor concentration, although the total cellular receptor concentration remained unchanged, suggesting that monensin causes the intracellular sequestration of receptors. Time-course studies of the processing of 2.5 nM-insulin showed that monensin produced a 50-60% decrease in surface binding, accompanied by a similar decrease in internalization and total inhibition of insulin degradation. When hepatocytes with 125I-insulin prebound to their surface receptors at 10 degrees C were warmed to 37 degrees C, monensin had no effect on internalization, but caused marked impairment of intracellular insulin degradation. It is concluded that monensin inhibits receptor recycling and cellular insulin degradation.

Activation of Protein Kinase A Modulates Trafficking of the Human Cardiac Sodium Channel in Xenopus Oocytes

“…16 In addition, it increases lysosomal pH to interrupt recycling of cell surface proteins such as receptors for LDL, transferrin, and insulin. [17][18][19] A similar lysosomal effect is produced by chloroquine. 20 -22 To determine whether activation of PKA disrupts trafficking of the hH1 channel, cells were preincubated in either chloroquine (100 mol/L) or monensin (100 mol/L) before exposure to kinase activators.…”

“…These agents are known to perturb intracellular vesicle trafficking and have been used extensively to study recycling of membrane proteins. [17][18][19][20][21][22] As an important control for these experiments, chloroquine had no effect on the PKA response of K ϩ current derived from coexpression of Kv1.5 and Kv␤1.3, an effect mediated by direct phosphorylation of the K ϩ channel ␤ subunit. These results demonstrate that the PKA response in these cells was intact and that the mechanism of regulation for the 2 ion channel complexes must be different.…”

Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells

“…To test this idea, we used two recycling inhibitors: the carboxylic ionophore monensin and the calciumbinding protein calmodulin (CaM) inhibitor W-13, which we (44) previously used to study recombinant NBCe1. It is known that monovalent carboxylic ionophore monensin raises pH within endosomes and inhibits recycling of internalized proteins to the plasma membrane (5,58,65). It is also known that CaM controls the recycling step of the endocytic pathway of several membrane proteins, and its antagonist, W-13, blocks the exit of internalized cargo from early endosomes resulting in the formation of large endosomes (3,13,22,32,61).…”

Molecular mechanism of bis(maltolato)oxovanadium(IV)-induced insulin signaling in 3T3-L1 and IM9 cells: impact of dexamethasone

“…Rather, we considered it as a consequence of alteration in one or multiple steps of cellular processes that are associated with the packaging, release, trafficking, or the membrane flow of the receptor protein. In order to judge this more appropriately, we have exposed the BMOV-treated or -untreated IM9 cells for 2 h before insulin administration to monocarboxylic Na C /H C -antiport monensin, an antibiotic inhibitor of intracellular protein transport, which is also known to block the recycling of insulin receptor in hepatocytes (Whittaker et al 1986). …”

“…In order to focus on this issue more deeply, we have examined the effect of monocarboxylic ionophore monensin, an inhibitor of receptor recycling (Whittaker et al 1986), on the soluble IR pool of IM9 cells receiving an exposure to BMOV for 18 h and a brief insulin treatment for 15 min. Consequently, we have also checked whether treatment of dexamethasone which has been shown to increase the cellular content of IR in 3T3 fibroblasts and IM9 cells (Fantus et al 1982, Knutson et al 1982, McDonald & Goldfine 1988) can encounter the above negative impact of BMOV on IR level in both 3T3-L1 and IM9 cells and thereby resulting in an augmentation in the phosphorylation of IR with subsequent modulation in the downstream insulin signaling molecules, including Akt, GSK, and Raf.…”