Degradation of insulin in isolated liver endosomes is functionally linked to ATP‐dependent endosomal acidification

Abstract

The degradation of insulin in isolated liver endosomes and the relationships of this process with ATP-dependent endosomal acidification have been studied. Incubation of endosomal fractions containing 1251-insulin in isotonic KCl at 30°C resulted in a rapid loss of insulin integrity as judged from trichloroacetic acid precipitability, Sephadex G-50 chromatography, immunoreactivity and receptor binding ability, with a maximum at pH 5 -6 ( t 1 , 2 : 10, 10, 6 and 6 min, respectively). On a log/log plot, the amount of acid-soluble products generated was linearly related to the amount of insulin associated with endosomes (slope, 0.80). Upon incubation, virtually all acid-soluble products diffused out of endosomes as judged from their solubility in aqueous poly(ethyleneglyco1). In permeabilized endosomes, intact insulin was also released in part extraluminally, but only when degradation was inhibited did this release increase with lowering pH. ATP shifted the pH for maximal insulin degradation to about 7.5 -8.5 and caused endosomal acidification as judged from the uptake of acridine orange and the fluorescence of internalized fluorescein-labeled dextran and galactosylated bovine serum albumin (dpH about 0.8 -0.9). GTP, ITP and UTP exerted comparable effects but with lower potencies. The ability of ATP to alter the pH dependence of insulin degradation was maximal in the presence of CI-, other anions being less effective (Br-> gluconate = SO:-> NO; = sucrose = mannitol) and/or inhibitory (NO;). Na', K + and Li' supported more effectively ATP-dependent insulin degradation than did choline. Divalent cations were required for the ATP effect (Mg2Little or no effects of ATP occurred in the presence of proton ionophores such as monensin and carbonyl cyanide chlorophenylhydrazone, and inhibitors of the proton ATPase such as N-ethylmaleimide. The abilities of nucleotides, ions and inhibitors to support or inhibit ATP-dependent insulin degradation were well correlated with their abilities to affect ATP-dependent acidification. The acidotropic agents chloroquine and quinacrine caused a leftward shift in the pH dependence of insulin degradation and a decrease in maximal degradation; in the presence of ATP, chloroquine almost completely inhibited degradation at pH 5 -9. It is concluded that ATP-dependent acidification, in part by enhancing the dissociation of the insulinreceptor complex, is required for optimum degradation of insulin within liver endosomes.Biochemical and morphological studies on intact liver and isolated hepatocytes have shown that, following binding to its receptor at the cell surface, insulin undergoes rapid endocytosis along with its receptor (reviewed in [l -31). Subsequently, the receptor is recycled in part to the cell surface, while most of the insulin is degraded within the cell. Several lines of evidence indicate that, although initially thought to occur in lysosomes, degradation of internalized insulin occurs mainly in endosomes. First, little association of insulin with authentical lysosomes is demonstr...

Negative Regulation of Epidermal Growth Factor Signaling by Selective Proteolytic Mechanisms in the Endosome Mediated by Cathepsin B

“…high (glucagon) (16,17), moderate (insulin) (4,30) and low (EGF and prolactin) (31). Using an alternate approach in which hydrolysis was measured by the generation of degradation products from unlabeled peptides, we have confirmed that EGF and insulin hydrolysis, but not glucagon hydrolysis, appears to be limited.…”

Endosomal proteolysis of internalised [ArgA0]-human insulin at neutral pH generates the mature insulin peptide in rat liver in vivo

“…Thus, the removal of Arg from Arg-extended peptides within the endosomal compartment may be highly selective. The present observation agrees well with our previous studies on the endosomal proteolysis of internalised human proinsulin (HPI) that show that rat liver endosomes produce selective cleavages in the Glu 33 -Lys 64 C-peptide without apparent modification of arginyl-peptide bonds [6]. Previously, other studies have reported that Triton X-100 solubilised rat liver endosomes [4] and rat hepatoma Fao cells stably transfected with HPI [5] processed HPI to des- [31,32]-HPI without conversion into mature HI.…”

“…While insulin was subject to rapid endosomal ligand dissociation and degradation [16,19,33], reduced in vivo and cell-free endosomal processing was observed with the H2-analogue [14,24,26] and proinsulin [6]. An altered IR phosphotyrosine profile was also observed at the endosomal locus, with this process being higher (H2-analogue) and of longer duration (H2-analogue and proinsulin) [6,14,24].…”

Endosomal Proteolysis of Internalized Insulin at the C-terminal Region of the B Chain by Cathepsin D

“…For some, degradation takes place early in the endocytic pathway. This is the case for polypeptide hormones such as insulin (2)(3)(4), glucagon (5), and parathyroid hormone (6) and growth factors such as the epidermal growth factor (7) and insulin-like growth factor-I (IGF-I) 1 (8) as well as endocytosed protein antigens for major histocompatibility class II presentation (9) and plant toxins (10).…”