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

Abstract

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A 1-21 -B 1-24 insulin resulting from a major cleavage at residues Phe B24 -Phe B25 . This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulindegrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SW XL high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and ␤-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe B24 -Phe B25 bond, generating the inactive A 1-21 -B 1-24 insulin intermediate.Proteins entering the endocytic pathway encounter an increasingly hydrolytic environment imposed by a progressive decrease in pH and an increase in protease concentrations (reviewed in Ref. 1). Ultimately, most are degraded in lysosomes to small peptides and free amino acids. For some, degradation takes place early in the endocytic pathway. This is the case for polypeptide hormones such as insulin (2-4), 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).In liver parenchyma the endosomal degradation of internalized insulin is thought to occur after acidification of the endosomal lumen by a soluble enzyme termed endosomal acidic insulinase (EAI) (2, 3, 11), although this protease has yet to be identified. EAI, which was easily extracted by hypotonic shock from hepatic endosomes, displayed an acidic pH opti...

Function of a genetically modified human liver cell line that stores, processes and secretes insulin

“…This is consistent with there being some constitutive release of insulin as well as degradation of insulin by the liver cells. 16,17 Neither of these features occurs in a pancreatic b cell, but insulin degradation does occur in a normal liver cell. Constitutive release is not a feature of a pancreatic b cell, but a small amount of insulin is degraded in these cells, within the secretory granules.…”

“…By comparison the amount of insulin degraded by NIH 3T3 cells (negative control) was negligible. It has been known for some time that the endosomal compartment of hepatocytes contains an acidic endopeptidase or endosomal acidic insulinase, which has recently been confirmed to be cathepsin D. 17 The activity of cathepsin D is almost completely inactivated by protease inhibitors, such as pepstatin A. 17,18 We incubated Huh7 cells over a 24-h period in DMEM medium and a 6-h period in basal medium, in the presence of pepstatin A, and confirmed that insulin degradation was negligible in the presence of the protease inhibitor.…”

“…The pH of the collected supernatants was neutralized with NaOH before the samples were placed in the RIA. Insulin degradation was also measured in Huh7 cells following 24 h incubation with pepstatin A (Sigma) (0.1-5 mg/ml) 17 in DMEM medium and a 6 h incubation in basal medium.…”

Metabolic relationship between diabetes and Alzheimer's Disease affected by Cyclo(His-Pro) plus zinc treatment

“…To reset the system, the inactive insulin is separated from the receptor, and the receptor is either recycled to the cell surface or degraded in the cytosol. In the meantime, insulin is degraded by IDE in the endosome [106], [107] and then completely digested into amino acids by lysosomal peptidases. If insulin is incompletely degraded in the cytosol, insulin peptide fragments in the cytosol can interfere with insulin-mediated signal transduction mechanisms resulting in insulin resistance and diabetes.…”

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

“…Polyclonal antibody against growth factor receptor-bound protein 14 (GRB14) has been previously described [15]. Rabbit antimouse cathepsin D R291 [16,17], sheep anti-human cathepsin D M8147 [16,17] and rabbit anti-rat cathepsin B 7183 [18] were obtained from J. S. Mort (Shriners Hospital for Crippled Children, Montreal, Canada). Monoclonal antibody directed against insulin-degrading enzyme (IDE) [19,20] was obtained from R. A. Roth (Stanford University, Stanford, CA, USA).…”