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At least 55 records · Page 3Linked to original sources
PubMed1996

The expression of and insulin binding to cellular thyroid hormone binding protein, but not insulin degrading enzyme, is increased during 3T3-L1 adipocytes differentiation.

Specific insulin binding to several proteins (cytosolic insulin binding proteins; CIBPs) in the isolated cytoplasm of numerous cell types has been demonstrated. CIBPs include insulin degrading enzyme (IDE), CIBP p55 (identified as cellular thyroid hormone binding protein (CTHBP), which is also known as protein disulfide isomerase, or glutathione insulin transhydrogenase). To assess the possible role of CIBP p55/CTHBP in insulin action, we compared 125I-insulin binding to CIBP in cytosol isolated from 3T3-L1 cells at various time points during differentiation of the adipocytes. Insulin did not bind to CTHBP in fibroblasts, but the labeling was markedly increased during adipocyte differentiation. In contrast, insulin binding to IDE did not change during differentiation. Protein expression level of CTHBP in the cytosol fraction increased gradually during the differentiation of adipocytes, whereas that of IDE did not change throughout the period. These data indicate that CTHBP, but not IDE, was up-regulated during differentiation of the adipocytes, suggesting that CIBP p55/CTHBP may play a role in regulating some insulin action, especially the counter regulation between insulin and other hormones during adipocyte differentiation.

3T3 Cells↗
PubMed1998

Insulin acts intracellularly on proteasomes through insulin-degrading enzyme.

Insulin decreases cellular protein degradation, but the mechanism of this action is poorly understood. We have shown that insulin can have an inhibitory effect on the action of the proteasome in vitro, which requires the presence of insulin degrading enzyme (IDE). In this study we have used an antibody which inhibits the activity of IDE to show that IDE is required for insulin inhibition of protein degradation in intact cells. The anti-IDE antibody blocked the insulin effect on cellular degradation of proteins prelabeled with radioactive amino acids. The anti-IDE antibody also decreased insulin inhibition of proteasome degradation of a specific substrate in intact cells. These data suggest that insulin works intracellularly via IDE to inhibit protein degradation by the proteasome. Thus, IDE may function as an intracellular mediator for insulin effects on protein degradation. This is a novel signal transduction mechanism for peptide hormones.

Animals↗
PubMed1993

Purification of nonantibiotic insulinase inhibitors from bacitracin.

Bacitracin is commonly used in metabolic studies as an insulinase inhibitor. The many isoforms of the commercial preparation were fractionated by charge and size in order to find the most active rat-muscle insulinase inhibitors. CM-Sepharose chromatography revealed that most of the inhibitory activity was contained in a fraction (CM-Inh) that amounted to less than 5% of the mixture. The CM-Inh fraction could be further separated by size on Bio-Gel P4 columns. Six subgroups, each with characteristic specific activity, were isolated. The most potent inhibitor fractions have no antibiotic activity and have molecular weights about twice that of bacitracin A. The peaks isolated by means of Bio-Gel P4 chromatography can be further fractionated by reversed phase HPLC on a C8 column, and by electrophoresis on nonreducing acrylamide gels.

Amino Acids↗
PubMed2000

Functional human insulin-degrading enzyme can be expressed in bacteria.

Insulin-degrading enzyme (IDE) has been shown to degrade a number of biologically important peptides, including insulin and the amyloid-beta protein implicated in Alzheimer's disease. However, lack of a facile method to generate purified enzyme and related mutants has made it difficult to study the precise role of IDE in the clearance of these peptides. Therefore, we determined whether recombinant wild-type and mutant human IDEs can be overexpressed as functional enzymes in bacteria. Three vectors carrying cDNAs encoding N-terminally polyhistidine-tagged recombinant IDEs were constructed, and the proteins expressed in Escherichia coli were purified by metal affinity chromatography (final yield approximately 8 mg per liter of culture). The recombinant IDEs, like the endogenous mammalian enzyme, migrate with 110-kDa apparent molecular masses in SDS-polyacrylamide gels and as a approximately 200-kDa species in gel filtration. Further analysis by native PAGE indicates that IDE can form multimers of different complexities. The wild-type recombinant endopeptidase degrades insulin with an efficiency similar to that of the enzyme purified from mammalian tissues. Purified IDEs are stable at 4 degrees C for at least 1 month. Purified recombinant protein was used to raise specific polyclonal antibodies that can immunoprecipitate native mammalian IDE. Thus, the procedure described allows the rapid production of large amounts of purified IDE and demonstrates that IDE can be produced in an active form in the absence of other potential interacting mammalian proteins.

Amino Acid Sequence↗
PubMed2005

Amyloid beta degradation: a challenging task for brain peptidases.

Amyloid beta (Abeta) accumulates in the neuropil and within the walls of cerebral vessels in association with normal aging, dementia or stroke. Abeta is released from its precursor protein as soluble monomeric species yet, under pathological conditions, it self-aggregates to form soluble oligomers or insoluble fibrils that may be toxic to neurons and vascular cells. Abeta levels could be lowered by inhibiting its generation or by promoting its clearance by transport or degradation. Here we will summarize recent findings on brain proteases capable of degrading Abeta, with a special focus on those enzymes for which there is genetic, transgenic or biochemical evidence supporting a role in the proteolysis of Abeta in vivo.

Alzheimer Disease↗
PubMed1982

125I-labelled insulin degradation by isolated rat hepatocytes: the roles of glutathione-insulin transhydrogenase and insulin-specific protease.

Isolated rat hepatocytes degraded 125I-insulin with a Km of 150 nmol/l. Degradation was stimulated by the addition of glutathione and dithiothreitol. In cells incubated with diamide, glutathione was oxidised to the disulphide. Regeneration of reduced glutathione commenced after a further 30 min incubation at 37 degrees C. Diamide (1 mmol/l) significantly inhibited insulin degradation by hepatocytes (p less than 0.001). The 'apparent Vmax' for insulin degradation was decreased tenfold and the Km decreased to 25 nmol/l. The diamide-insensitive degrading activity was cell-associated and produced an intermediate of hormone degradation that was apparently of a higher molecular weight than insulin A chain. The biological activity of the intermediate was 0.03% of that of insulin. The diamide-insensitive activity was not due to release of protease into the medium by cell lysis. We conclude that there are at least two pathways capable of degrading insulin existing in rat hepatocytes.

Animals↗
PubMed1984

The renal metabolism of insulin.

The kidney plays a pivotal role in the clearance and degradation of circulating insulin and is also an important site of insulin action. The kidney clears insulin via two distinct routes. The first route entails glomerular filtration and subsequent luminal reabsorption of insulin by proximal tubular cells by means of endocytosis. The second involves diffusion of insulin from peritubular capillaries and subsequent binding of insulin to the contraluminal membranes of tubular cells, especially those lining the distal half of the nephron. Insulin delivered to the latter sites stimulates several important processes, including reabsorption of sodium, phosphate, and glucose. In contrast, insulin delivered to proximal tubular cells is degraded to oligopeptides and amino-acids by one of two poorly delineated enzymatic pathways. One pathway probably involves the sequential action of insulin protease and either GIT or non-specific proteases; the other probably involves the sequential action of GIT and lysosomal proteases. The products of insulin degradation are reabsorbed into the peritubular capillaries, apparently via simple diffusion. Impairment of the renal clearance of insulin prolongs the half-life of circulating insulin by a number of mechanisms and often results in a decrease in the insulin requirement of diabetic patients. Much needs to be learned about these metabolic events at the subcellular level and how they are affected by disease states. Owing to the heterogeneity of cell types within the kidney and to their anatomical and functional polarity, investigation of these areas will be challenging indeed.

Animals↗
PubMed1994

Extracellular insulin degrading activity creates instability in a CHO-based batch-refeed continuous process.

In a batch-refeed continuous process involving a recombinant Chinese hamster ovary cell line, a brief upset was occasionally observed during which cell growth halted and cell viability dropped. This was found to be associated with depletion of insulin from the medium early during the affected passage. Insulin depletion was found to be primarily the result of insulin degrading activity released by the cells during the preceding passage.

Animals↗
PubMed1987

Possible role of cell surface insulin degrading enzyme in cultured human lymphocytes.

The kinetic changes of insulin receptors and cell surface insulin degrading enzyme were examined in Bri-7 cultured human lymphocytes after preincubation with or without insulin. The concentration of cell surface insulin degrading enzyme was determined by immunoenzymatic labeling method using a polyclonal antiserum to insulin degrading enzyme. In Bri-7 cells preincubated with 10(-10) to 10(-5) mol/l insulin for 18 h, the surface insulin receptors and insulin degrading enzyme decreased progressively as a function of the concentration of insulin in the preincubation medium. The surface insulin receptors and insulin degrading enzyme of cells preincubated with 10(-6) mol/l insulin were decreased to 25 and 35% of the control respectively. In Bri-7 cells preincubated with 10(-6) mol/l insulin for 30 min to 18 h, the loss of surface insulin degrading enzyme was slightly slower than that of the receptors; however, the curves were essentially parallel to each other. Thus, the treatment of Bri-7 cells with insulin caused down-regulation of insulin receptors in a dose- and time-dependent manner. Cell surface insulin degrading enzyme also decreased simultaneously. A combination of several insulin degradation assays (trichloroacetic acid precipitation, gel filtration and receptor rebinding) demonstrated that cell surface bound insulin remained intact, and that the degradation in Bri-7 cells seemed to be a limiting proteolysis of insulin. Furthermore, by the receptor rebinding method insulin degrading activity in cells after preincubation with 10(-6) mol/l insulin (19.6 +/- 4.6%) was decreased, although not significantly, as compared with cells after preincubation without insulin (24.6 +/- 4.8%).(ABSTRACT TRUNCATED AT 250 WORDS)

B-Lymphocytes↗
PubMed1999

Mechanisms of nuclear translocation of insulin.

Insulin (Ins) and various other hormones and growth factors have been shown to be rapidly internalized and translocated to the cell nucleus. This review summarizes the mechanisms that are involved in the translocation of Ins to the nucleus, and discusses its possible role in Ins action, based on observations by the authors and others. Ins is internalized to endosomes by both receptor-mediated and fluid-phase endocytosis, the latter occurring only at high Ins concentrations. The authors recently demonstrated the caveolae are the primary cell membrane locations responsible for initiating the signal transduction cascade induced by Ins. Once Ins is internalized, Ins dissociates from the Ins receptor in the endosome, and is translocated to the cytoplasm, where most Ins is degraded by Ins-degrading enzyme (IDE), although how the polypeptides cross the lipid bilayer is unknown. Some Ins escapes the degradation and binds to cytosolic Ins-binding proteins (CIBPs), in addition to IDE. IDE and some CIBPs are known to be binding proteins for other hormones or their receptors, and are involved in gene regulation, suggesting physiological relevance of CIBPs in the signaling of Ins and other hormones. Ins is eventually translocated through the nuclear pore to the nucleus, where Ins tightly associates with nuclear matrix. The role of Ins internalization and translocation to the nucleus is still controversial, although there is substantial evidence to support its role in cellular responses caused by Ins. Many studies indicate that nuclear translocation of various growth factors and hormones plays an important role in cell proliferation or DNA synthesis. It would be reasonable to suggest that Ins internalization, its association with CIBPs, and its translocation to the nucleus may be essential for the regulation of nuclear events by Ins.

Animals↗
PubMed1984

Insulin degradation in hepatic cirrhosis.

Hepatic insulinase activity was studied in cirrhosis in an attempt to explain the hyperinsulinemia known to occur in this disease. Liver tissue was obtained during laparotomy in seven patients with cirrhosis of the liver and five patients without liver disease. Insulin degradation was significantly decreased in the cirrhotic liver at each time interval measured (p less than 0.05). Insulinase inhibitor activity was measured in the plasma of 12 patients with cirrhosis of the liver and six controls. There was no significant difference between the two groups. Decreased insulinase activity in cirrhosis may account, in part, for the hyperinsulinemia seen in this disease.

Animals↗
PubMed2002

The brain insulin signal transduction system and sporadic (type II) Alzheimer disease: an update.

Nosologically, Alzheimer disease may not be considered to be a single disorder in spite of a common clinical phenotype. Only a small proportion of about 5% to 10% of all Alzheimer cases is due to genetic mutations (type I) whereas the great majority of patients was found to be sporadic in origin. It may be assumed that susceptibility genes along with lifestyle risk factors contribute to the causation of the age-related sporadic Alzheimer disease (type II). In this context, the desensitization of the neuronal insulin receptor similar to not-insulin dependent diabetes mellitus may be of pivotal significance. This abnormality along with a reduction in brain insulin concentration is assumed to induce a cascade-like process of disturbances including cellular glucose, acetylcholine, cholesterol, and ATP associated with abnormalities in membrane pathology and the formation of both amyloidogenic derivatives and hyperphosphorylated tau protein. Sporadic Alzheimer disease may, thus, be considered to be the brain type of diabetes mellitus II. Experimental evidence is provided and discussed.

ATP-Binding Cassette Transporters↗
PubMed2004

Sequence variation in the proximity of IDE may impact age at onset of both Parkinson disease and Alzheimer disease.

We recently reported that a linkage disequilibrium (LD) block on chromosome 10q encompassing the gene encoding insulin-degrading enzyme ( IDE) harbors sequence variants that associate with Alzheimer disease (AD). Evidence also indicated effects upon a number of quantitative indices of AD severity, including age-at-onset (AAO). Since linkage of this immediate region to AAO has been shown in both AD and Parkinson disease (PD), we have explored the possibility that polymorphism within this LD block might also influence PD. Utilizing single nucleotide polymorphisms that delineate common haplotypes from this region, we observed significant evidence of association with AAO in an Australian PD case-control sample. Analyses were complemented with AAO data from two independent Swedish AD case samples, for which previously reported findings were replicated. Results were consistent between AD and PD, suggesting the presence of equivalent detrimental and protective alleles. These data highlight a genomic region in the proximity of IDE that may contribute to AD and PD in a similar manner.

Age of Onset↗
PubMed2005

Aging-related correlation of insulin-degrading enzyme with gamma-secretase-generated products involving insulin and glucose levels in transgenic mice.

Insulin-degrading enzyme (IDE) is a 110-kDa thiol zinc-methalloendopeptidase that can cleave small Abeta peptides and the APP intracellular domain (AICD). The aim of this study was to examine aging-related correlation of IDE with gamma-secretase-generated products involving insulin and glucose levels in transgenic brains expressing neuron-specific enolase (NSE)-controlled human mutant presenilin-2 (hPS2m). Herein, we concluded that the levels of IDE expression in transgenic brains were decreased relative to those of control mice at 15 months of age. In parallel, inhibition in the IDE expression at this age underlies to the levels-up of Abeta-42, AICD, gamma-secretase, and glucose with a level-down of insulin. Thus, IDE expression is critical target for the therapeutic trials.

Aging↗
PubMed2006

Novel mucosal insulin delivery systems based on fusogenic liposomes.

PURPOSE: Fusogenic liposomes (FLs) are unique delivery vehicles capable of introducing their contents directly into the cytoplasm with the aid of envelope glycoproteins of Sendai virus (SeV). The objective of this study was to evaluate the potential of FL to improve the mucosal absorption of insulin from rat intestinal membranes. METHOD: The FLs containing insulin were prepared by fusing insulin-loaded liposomes with inactivated SeV particles and were administered directly into the ileal, the colonic, and the rectal loops (10 IU/kg). RESULTS: The FL successfully enhanced the insulin absorption and induced a significant hypoglycemic effect following the colonic and the rectal administration without detectable mucosal damage. This enhancing effect of insulin absorption was further improved by increasing the amount of insulin loaded in the FL and by coencapsulating insulin-degrading enzyme inhibitor. In contrast, the insulin absorption was not increased by the ileal administration of FL because the mucous/glycocalyx layers overlaid on the ileal epithelium impede the fusion of FL to the intestinal mucosa. CONCLUSION: Our results indicated that FL is a useful carrier for improving the absorption of poorly absorbable drugs, such as insulin, via the intestinal tract.

Animals↗