Insulin-degrading enzyme (IDE) as a modulator of microglial phenotypes in the context of Alzheimer's disease and brain aging.

The insulin-degrading enzyme (IDE) is an evolutionarily conserved zinc-dependent metallopeptidase highly expressed in the brain, where its specific functions remain poorly understood. Besides insulin, IDE is able to cleave many substrates in vitro, including amyloid beta peptides, making this enzyme a candidate pathophysiological link between Alzheimer's disease (AD) and type 2 diabetes (T2D). These antecedents led us to address the impact of IDE absence in hippocampus and olfactory bulb. A specific induction of microgliosis was found in the hippocampus of IDE knockout (IDE-KO) mice, without any effects in neither hippocampal volume nor astrogliosis. Performance on hippocampal-dependent memory tests is influenced by IDE gene dose in 12-month-old mice. Furthermore, a comprehensive characterization of the impact of IDE haploinsufficiency and total deletion in metabolic, behavioral, and molecular parameters in the olfactory bulb, a site of high insulin receptor levels, reveals an unambiguous barcode for IDE-KO mice at that age. Using wildtype and IDE-KO primary microglial cultures, we performed a functional analysis at the cellular level. IDE absence alters microglial responses to environmental signals, resulting in impaired modulation of phenotypic states, with only transitory effects on amyloid-ß management. Collectively, our results reveal previously unknown physiological functions for IDE in microglia that, due to cell-compartment topological reasons, cannot be explained by its enzymatic activity, but instead modulate their multidimensional response to various damaging conditions relevant to aging and AD conditions.

Pharmacological activation of insulin-degrading enzyme improves insulin secretion and glucose tolerance in diet-induced obese mice.

AIM: To investigate the use of synthetic preimplantation factor (sPIF) as a potential therapeutic tool for improving glucose-stimulated insulin secretion (GSIS), glucose tolerance and insulin sensitivity in the setting of diabetes. MATERIALS AND METHODS: We used a preclinical murine model of type 2 diabetes (T2D) induced by high-fat diet (HFD) feeding for 12 weeks. Saline or sPIF (1 mg/kg/day) was administered to mice by subcutaneously implanted osmotic mini-pumps for 25 days. Glucose tolerance, circulating insulin and C-peptide levels, and GSIS were assessed. In addition, ß-cells (Min-6) were used to test the effects of sPIF on GSIS and insulin-degrading enzyme (IDE) activity in vitro. The effect of sPIF on GSIS was also tested in human islets. RESULTS: GSIS was enhanced 2-fold by sPIF in human islets ex vivo. Furthermore, continuous administration of sPIF to HFD mice increased circulating levels of insulin and improved glucose tolerance, independently of hepatic insulin clearance. Of note, islets isolated from mice treated with sPIF exhibited restored ß-cell function. Finally, genetic (shRNA-IDE) or pharmacological (6bK) inactivation of IDE in Min-6 abolished sPIF-mediated effects on GSIS, showing that both the protein and its protease activity are required for its action. CONCLUSIONS: We conclude that sPIF is a promising secretagogue for the treatment of T2D.

Induction of ICAM1 in Brain Vessels is Implicated in an Early AD Pathogenesis by Modulating Neprilysin.

Intercellular adhesion molecule 1 (ICAM1) is a vessel adhesion protein induced during brain vascular inflammation, which could be closely linked with the development of Alzheimer's disease (AD). This study investigated the effect of ICAM1 on amyloid-degrading enzymes (ADEs) in endothelial cells and their potential involvement in inflammation and AD progression. TNF-α treatment increased ICAM1 in human brain microvascular endothelial cells (HBMVECs) but decreased the neprilysin (NEP) protein level. Knock-down of ICAM1 using siRNA enhanced NEP, which increased the degradation of amyloid-ß. In the brains of 4-month-old AD transgenic mice (APPswe/PSEN1dE9), there were significantly higher levels of ICAM1 expression and amyloid deposits but lower levels of NEP and insulin-degrading enzymes (IDE), demonstrating an inverse correlation of ICAM1 with NEP and IDE expression. Further studies demonstrated significantly increased GFAP protein levels in the brain, specifically localized near blood vessels, of both TNF-α-injected and 4-month-old AD transgenic mice. Taken together, the induction of ICAM1 in endothelial cells suppresses NEP expression, accelerating the accumulation of amyloid-ß in blood vessels. It also enhances leukocyte adhesion to blood vessels stimulating the migration of leukocytes into the brain, subsequently triggering brain inflammation.

Insulin degrading enzyme (IDE) expressed by Chinese hamster ovary (CHO) cells is responsible for degradation of insulin in culture media.

Chinese hamster ovary (CHO) cells cultured in serum-free chemically-defined media (CDM) are used for manufacturing of therapeutic proteins. Growth factors, such as insulin are commonly utilized in manufacturing platforms to enhance CHO cell viability and growth. Here we report that insulin is degraded in the culture media over time mainly due to the activity of the insulin degrading enzyme (IDE). Insulin degradation was faster in cell lines that released more IDE, which negatively impacted cell growth and in turn, production titers. Deletion of the IDE gene in a representative CHO cell line nearly abolished insulin degradation in seed train and end-of-production media. In summary, our data suggests that selecting cell lines that have lower IDE expression or targeted-deletion of the IDE gene can improve culture viability and growth for insulin-dependent CHO production platforms.

Restored degradation of the Alzheimer's amyloid-beta peptide by targeting amyloid formation.

Accumulation of neurotoxic amyloid-beta (Abeta) is central to the pathology of Alzheimer's disease (AD). Elucidating the mechanisms of Abeta accumulation will therefore expedite the development of Abeta-targeting AD therapeutics. We examined activity of an Abeta-degrading protease (matrix metalloprotease 2) to investigate whether biochemical factors consistent with conditions in the AD brain contribute to Abeta accumulation by altering Abeta sensitivity to proteolytic degradation. An Abeta amino acid mutation found in familial AD, Abeta interactions with zinc (Zn), and increased Abeta hydrophobicity all strongly prevented Abeta degradation. Consistent to all of these factors is the promotion of specific Abeta aggregates where the protease cleavage site, confirmed by mass spectrometry, is inaccessible within an amyloid structure. These data indicate decreased degradation due to amyloid formation initiates Abeta accumulation by preventing normal protease activity. Zn also prevented Abeta degradation by the proteases neprilysin and insulin degrading enzyme. Treating Zn-induced Abeta amyloid with the metal-protein attenuating compound clioquinol reversed amyloid formation and restored the peptide's sensitivity to degradation by matrix metalloprotease 2. This provides new data indicating that therapeutic compounds designed to modulate Abeta-metal interactions can inhibit Abeta accumulation by restoring the catalytic potential of Abeta-degrading proteases.

Effect of nelfinavir on insulin metabolism, proteasome activity and protein degradation in HepG2 cells.

HIV-1 protease inhibitors have revolutionized the treatment of HIV infection, but their use has been associated with lipodystrophy and insulin resistance. One suggestion for this has been the inhibition of insulin-degrading enzyme (IDE). We have previously demonstrated that insulin, through IDE, can inhibit the proteasome, thus decreasing cytosolic protein degradation. We examined whether the protease inhibitor nelfinavir inhibited IDE and its effect on protein degradation both in vitro and in whole cells. 125I-Insulin degradation was measured by trichloroacetic acid precipitation. Proteasome activities were measured using fluorogenic peptide substrates. Cellular protein degradation was measured by prelabelling cells with 3H-leucine and determining the release of TCA-soluble radioactivity. Nelfinavir inhibited IDE in a concentration-dependent manner with 50% inhibition at the maximal concentration tested, 100 microm. Similarly, the chymotrypsin-like and trypsin-like activities of the proteasome were decreased with an IC50 of approximately 3 microm. The ability of insulin to inhibit the proteasome was abrogated by nelfinavir. Treatment of HepG2 cells with 50 microm nelfinavir decreased 125I-insulin degradation and increased cell-associated radioactivity. Insulin alone maximally decreased protein degradation by 15%. Addition of 50 microm nelfinavir inhibited cellular protein degradation by 14% and blunted the effect of insulin. These data show that nelfinavir inhibits IDE, decreases insulin's ability to inhibit protein degradation via the proteasome and provides another possible mechanism for the insulin resistance seen in protease inhibitor-treated HIV patients.

Insulysin hydrolyzes amyloid beta peptides to products that are neither neurotoxic nor deposit on amyloid plaques.

Insulysin (EC. 3.4.22.11) has been implicated in the clearance of beta amyloid peptides through hydrolytic cleavage. To further study the action of insulysin on Abeta peptides recombinant rat insulysin was used. Cleavage of both Abeta(1-40) and Abeta(1-42) by the recombinant enzyme was shown to initially occur at the His(13)-His(14), His(14)-Gln(15), and Phe(19)-Phe(20) bonds. This was followed by a slower cleavage at the Lys(28)-Gly(29), Val(18)-Phe(19), and Phe(20)-Ala(21) positions. None of the products appeared to be further metabolized by insulysin. Using a rat cortical cell system, the action of insulysin on Abeta(1-40) and Abeta(1-42) was shown to eliminate the neurotoxic effects of these peptides. Insulysin was further shown to prevent the deposition of Abeta(1-40) onto a synthetic amyloid. Taken together these results suggest that the use of insulysin to hydrolyze Abeta peptides represents an alternative gene therapeutic approach to the treatment of Alzheimer's disease.

Differences in the cellular processing of AspB10 human insulin compared with human insulin and LysB28ProB29 human insulin.

Cellular metabolism studies were performed comparing human insulin with two rapid-acting analogs, LysB28ProB29 insulin (LysPro) and AspB10 insulin (B10-Asp). B10-Asp bound to isolated hepatocytes at 37 degrees C to a greater extent than LysPro or native insulin, which were equivalent. The rate of degradation was similar for the three materials, resulting in a significant reduction in the degraded/bound ratio for the B10 analog. The processing of membrane-bound material was examined by incubating cells with hormone at 4 degrees C, removing unbound insulin, and incubating the cells at 37 degrees C. Again, binding was greater for B10-Asp versus LysPro or native insulin, with a reduction in the degraded/bound ratio. Hormone internalization and processing was examined by an acid wash of cells incubated with 125I(A14)-labeled hormone to remove surface-bound materials. The processing rate was slower for B10-Asp versus LysPro or native insulin. Cell extraction and examination on molecular-sieve chromatography confirmed that B10-Asp was processed at a slower rate than either LysPro or native insulin. Intact B10-Asp was found in the cell after 4 hours, whereas all native insulin and LysPro were degraded by 90 to 120 minutes. B10-Asp also caused a greater incorporation of thymidine into DNA in cultured cells than LysPro or native insulin, which were similar. These data show that the cellular processing of LysPro is essentially identical to that of native insulin. However, B10-Asp has markedly different properties and is processed much more slowly. The prolonged cell residence time of B10-Asp could contribute to its greater effects on cell growth and mitogenesis.

Regulation of multicatalytic enzyme activity by insulin and the insulin-degrading enzyme.

The insulin-degrading enzyme (IDE) plays an important role in the cellular metabolism of insulin. Recent studies have also suggested a regulatory role for this protein in controlling the activity of cytoplasmic protein complexes, including the proteasome [multicatalytic proteinase (MCP)] and the glucocorticoid and androgen receptors. Binding of IDE to these complexes increases their activity, whereas the addition of substrates for IDE inhibits activity. This provides a potential mechanism of action for internalized insulin and other IDE substrates in the control of protein turnover. To examine further the interactions, partially purified IDE-MCP complex was treated with EDTA or EGTA, and activity was measured in the absence and presence of various divalent cations (Ca2+, Mn2+, Co2+, and Zn2+) and insulin. EDTA treatment reduced MCP activity and eliminated the effect of insulin on the complex. Divalent cations partially or completely restored MCP activity, but did not restore the effect of insulin. EGTA treatment had a lesser effect on MCP activity, but abolished insulin inhibition of activity. Divalent cations restored the insulin effect. Inhibitors of IDE also blocked the insulin effect on MCP activity, as did treatment with SDS. These findings suggest that conformational changes in the complex may play a role in the insulin control of MCP activity.

Mechanisms involved in degradation of human insulin by cytosolic fractions of human, monkey, and rat liver.

The degradation of native and 125I-labeled human insulin (HI) was examined in the cytosolic fraction of human, monkey, and rat liver. The purpose of these studies was to provide a species comparison of the interaction of insulin-degrading enzyme (IDE) and protein disulfide isomerase (PDI) in the degradation of HI. Western-blot analysis with monoclonal antibodies indicated the presence of both IDE and PDI in the cytosolic fraction of human and monkey liver. In contrast, rat liver cytosol contained, detectable levels of IDE only. A species comparison of metabolic profiles was performed by fractionating peptide products with reversed-phase high-performance liquid chromatography. After a 60-min incubation, human liver cytosol degraded unlabeled HI into three major products. Two of these peptides coeluted with the products of the incubation of HI with purified rat liver PDI. The three peptides were isolated and determined by NH2-terminal sequence analysis to be intact A chain, B chain, and des(Phe1)-B chain. Human liver cytosol also formed 125I-A chain and 125I-B chain as major products when specifically labeled 125I-HI isomers were used as substrate. Significant proteolytic degradation was observed only when reactions with human liver cytosol were supplemented with Mn2+. In contrast, monkey and rat liver cytosol proteolytically degraded 125I-HI isomers to small peptide fragments. The rat and monkey metabolic profiles were similar to each other and to that observed with Mn(2+)-supplemented human liver cytosol. Proteolysis in monkey and rat was sensitive to inhibition by EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)

The degradation of monoiodotyrosyl insulin isomers by insulin protease.

The four single-site monoiodotyrosyl insulin isomers were synthesized by lactoperoxidase-catalyzed iodination of porcine insulin and were separated from one another by high performance liquid chromatography. The susceptibility of the four isomers (A14-, A19-, B16-, and B26-monoiodotyrosyl insulin) to degradation by purified insulin protease was examined using several different assay methods, including trichloroacetic acid precipitation, immunoprecipitation, and Sephadex G-50 chromatograpy. Using trichloroacetic acid precipitation, isomer susceptibility, determined from the initial rate of hydrolysis, was highest with the A14 isomer, lowest with the A19 isomer, and intermediate and roughly equal with the two B-chain-labeled isomers. Based upon the initial rate of isomer hydrolysis, the Michaelis Menten constant (Km) of insulin protease was higher for the B16 isomer (55 nM) than for the other three isomers, whose Km values were not different from one another (A14 = 24 nM; A19 = 35 nM; B26 = 29 nM). In addition, the values for maximum velocity (Vmax) were higher for the A14 and B26 isomers than for the A19 and B16 isomers. However, during incubation, the order of isomer susceptibility to insulin protease changed to B26 greater than A14 greater than A19 greater than B16. This change in apparent isomer susceptibility was prevented by including in the incubation mixture a rat renal peptidase, which did not degrade the intact isomers, suggesting that insulin protease converted the isomers to trichloroacetic acid-soluble products via trichloroacetic acid-precipitable intermediates. Using the immunoprecipitation assay, the susceptibility of isomers to hydrolysis did not change during incubation, but remained highest with the A14 isomer, lowest with the A19 isomer, and intermediate with the two B-chain-labeled isomers, of which the B16 isomer was degraded more rapidly. Each isomer was converted more rapidly to nonimmunoprecipitable products than to trichloroacetic acid-soluble products, implying that insulin protease converted the isomers to trichloroacetic acid-precipitable, nonimmunoprecipitable intermediates, which it then converted to trichloroacetic acid-soluble form. Using Sephadex G-50 chromatography (SGC) assay, the susceptibility of isomers to hydrolysis did not change during incubation, but remained highest with the A14 isomer, lowest with the A19 isomer, and intermediate with the two B-chain-labeled isomers, of which the B16 isomer was hydrolyzed more rapidly. With the exception of the A19 isomer, isomer hydrolysis appeared faster with SGC assay than with either of the other two assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Degradation of insulin in serum by insulin-specific protease.

The effect of insulin specific protease (ISP) in degrading plasma insulin over a wide concentration range was studied. In sera containing more than 50 muU per milliliter, more than 85 per cent of the insulin was destroyed. However, a smaller percentage of the hormone was degraded in sera containing less than 20 muU per milliliter and the results were highly variable. The residual insulin-like immunoreactivity in plasma after exposure to ISP thus represents both undegraded insulin as well as proinsulin. These findings explain the higher plasma proinsulin values obtained by the enzyme method when compared to gel filtration.