Insulin-degrading Enzyme Activity Research Articles

P 162 - Impact of the age-related protein aggregate lipofuscin on β-cell functionality

The formation of cross-linked protein aggregates such as lipofuscin is a typical hallmark of cellular aging and especially affects long-living and postmitotic cells such as neurons and skeletal muscle cells. Lipofuscin can neither be degraded nor exocytosed, it is a prominent source of oxidants and able to inhibit proteolytic systems such as the 20 S proteasome and the autophagy-lysosomal pathway. Adult pancreatic β-cells are also considered to be long-living cells which may last for years or even a life-time. It was shown, that these cells are also characterized by an age-dependent increase in lipofuscin content, but little is known about the process of accumulating protein aggregates and their impact on β-cell functionality. We used the pancreatic β-cell line MIN6 with a well-established artificial lipofuscin to investigate different parameters of β-cell functionality and the impact of lipofuscin on the insulin-degrading enzyme (IDE). Interestingly, we observed both, a significantly higher insulin amount and secretion in the aggregate-fed MIN6 cells. While the expression of insulin does not seem to be affected by lipofuscin treatment, the presence of lipofuscin is able to decrease the activity of IDE by an unknown mechanism.

Interleukin-6 increases the expression and activity of insulin-degrading enzyme

Impairment of the insulin-degrading enzyme (IDE) is associated with obesity and type 2 diabetes mellitus (T2DM). Here, we used 4-mo-old male C57BL/6 interleukin-6 (IL-6) knockout mice (KO) to investigate the role of this cytokine on IDE expression and activity. IL-6 KO mice displayed lower insulin clearance in the liver and skeletal muscle, compared with wild type (WT), due to reduced IDE expression and activity. We also observed that after 3-h incubation, IL-6, 50 and 100 ng ml−1, increased the expression of IDE in HEPG2 and C2C12 cells, respectively. In addition, during acute exercise, the inhibition of IL-6 prevented an increase in insulin clearance and IDE expression and activity, mainly in the skeletal muscle. Finally, IL-6 and IDE concentrations were significantly increased in plasma from humans, after an acute exercise, compared to pre-exercise values. Although the increase in plasma IDE activity was only marginal, a positive correlation between IL-6 and IDE activity, and between IL-6 and IDE protein expression, was observed. Our outcomes indicate a novel function of IL-6 on the insulin metabolism expanding the possibilities for new potential therapeutic strategies, focused on insulin degradation, for the treatment and/or prevention of diseases related to hyperinsulinemia, such as obesity and T2DM.

Inositol phosphates and phosphoinositides activate insulin-degrading enzyme, while phosphoinositides also mediate binding to endosomes

Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, including insulin, amylin, and the amyloid β peptides. Polyanions activate IDE toward some substrates, yet an endogenous polyanion activator has not yet been identified. Here we report that inositol phosphates (InsPs) and phosphatdidylinositol phosphates (PtdInsPs) serve as activators of IDE. InsPs and PtdInsPs interact with the polyanion-binding site located on an inner chamber wall of the enzyme. InsPs activate IDE by up to ∼95-fold, affecting primarily Vmax The extent of activation and binding affinity correlate with the number of phosphate groups on the inositol ring, with phosphate positional effects observed. IDE binds PtdInsPs from solution, immobilized on membranes, or presented in liposomes. Interaction with PtdInsPs, likely PtdIns(3)P, plays a role in localizing IDE to endosomes, where the enzyme reportedly encounters physiological substrates. Thus, InsPs and PtdInsPs can serve as endogenous modulators of IDE activity, as well as regulators of its intracellular spatial distribution.

New Medications for the Treatment of Diabetes.

New Medications for the Treatment of Diabetes.

Toward Allosterically Increased Catalytic Activity of Insulin-Degrading Enzyme against Amyloid Peptides.

The physiological role of insulin-degrading enzyme (IDE) in the intracytosolic clearance of amyloid β (Aβ) and other amyloid-like peptides supports a hypothesis that human IDE hyperactivation could be therapeutically beneficial for the treatment of late-onset Alzheimer's disease (AD). The major challenge standing in the way of this goal is increasing the specific catalytic activity of IDE against the Aβ substrate. There were previous indications that the allosteric mode of IDE activity regulation could potentially provide a highly specific path toward degradation of amyloid-like peptides, while not dramatically affecting activity against other substrates. Recently developed theoretical concepts are used here to explore potential allosteric modulation of the IDE activity as a result of single-residue mutations. Five candidates are selected for experimental follow-up and allosteric free energy calculations: Ser137Ala, Lys396Ala, Asp426Ala, Phe807Ala, and Lys898Ala. Our experiments show that three mutations (Ser137Ala, Phe807Ala, and Lys898Ala) decrease the Km of the Aβ substrate. Mutation Lys898Ala results in increased catalytic activity of IDE; on the other hand, Lys364Ala does not change the activity and Asp426Ala diminishes it. Quantifying effects of mutations in terms of allosteric free energy, we show that favorable mutations lead to stabilization of the catalytic sites and other function-relevant distal sites as well as increased dynamics of the IDE-N and IDE-C halves that allow efficient substrate entrance and cleavage. A possibility for intramolecular upregulation of IDE activity against amyloid peptides via allosteric mutations calls for further investigations in this direction. Ultimately, we are hopeful it will lead to the development of IDE-based drugs for the treatment of the late-onset form of AD characterized by an overall impairment of Aβ clearance.

168 - Impact of Artificial Lipofuscin on β-Cell Functionality

Aging is a multi-causal irreversible process characterized by a progressive degeneration affecting all higher organisms. The formation of insoluble protein aggregates such as lipofuscin is one typical hallmark of aging. The accumulation of lipofuscin has already been shown in many different tissues, but especially long-living and postmitotic cells such as neurons and skeletal muscle cells are affected by the intralysosomal accumulation of this protein aggregate. Lipofuscin can neither be degraded by lysosomal enzymes nor exocytosed, furthermore postmitotic cells are not able to protect themselves from lipofuscin accumulation by cell division. Lipofuscin is a source of reactive oxygen species, able to inhibit proteolytic systems leading to a decline in proteasomal activity. Therefore, lipofuscin is an important contributor limiting the lifespan of especially postmitotic cells. Pancreatic β-cells are also considered to be long-living cells in adults and it was shown, that these cells are characterized by an age-dependent increase in lipofuscin content. However, even though β-cell replication is a rare event in aged humans and mice, little is known about the accumulation of protein aggregates and the intracellular effects of lipofuscin in β-cells. Therefore the pancreatic β-cell line MIN6 was treated with artificial lipofuscin to investigate the impact of this aggregate on the insulin-degrading enzyme (IDE) and different parameters of β-cell functionality. Interestingly, we observed a significant rise in insulin secretion and insulin content in the aggregate-fed MIN6 cells. While the expression of insulin does not seem to be affected by lipofuscin treatment, the prescence of lipofuscin is able to decrease the activity of IDE by an unknown mechanism.

Multiple allosteric sites are involved in the modulation of insulin-degrading-enzyme activity by somatostatin.

Somatostatin is a cyclic peptide, released in the gastrointestinal system and the central nervous system, where it is involved in the regulation of cognitive and sensory functions, motor activity and sleep. It is a substrate of insulin-degrading enzyme (IDE), as well as a modulator of its activity and expression. In the present study, we have investigated the modulatory role of somatostatin on IDE activity at 37 °C and pH 7.3 for various substrates [i.e. insulin, β-amyloid (Aβ)1-40 and bradykinin], aiming to quantitatively characterize the correlation between the specific features of the substrates and the regulatory mechanism. Functional data indicate that somatostatin, in addition to the catalytic site of IDE (being a substrate), is also able to bind to two additional exosites, which play different roles according to the size of the substrate and its binding mode to the IDE catalytic cleft. In particular, one exosite, which displays high affinity for somatostatin, regulates only the interaction of IDE with larger substrates (such as insulin and Aβ1-40 ) in a differing fashion according to their various modes of binding to the enzyme. A second exosite, which is involved in the regulation of enzymatic processing by IDE of all substrates investigated (including a 10-25 amino acid long amyloid-like peptide, bradykinin and somatostatin itself, which had been studied previously), probably acts through the alteration of an 'open-closed' equilibrium.

Potential efficacy of Lactobacillus casei IBRC_M10711 on expression and activity of insulin degrading enzyme but not insulin degradation.

Type 2 diabetes (T2D) is a condition with insufficient insulin production or in the setting of insulin resistance with many origins including intestinal microbiota-related molecular mechanism. Insulin-degrading enzyme (IDE) is responsible for insulin breakdown in various tissues and is known as a potential drug target for T2D. Here, we assessed the effects of cell-free supernatant (CFS) and UV-killed Lactobacillus casei IBRC_M10711 on IDE expression, IDE activity, and insulin degradation in Caco-2 cell line. It was found that CFS and UV-killed L. casei IBRC_M10711 led to lower expression of IDE. UV-killed L. casei IBRC_M10711 significantly inhibited IDE activity but CFS did not. Insulin degradation was affected with none of them. In conclusion, L. casei IBRC_M10711 is effective on IDE expression and its activity, but not on insulin degradation. Future studies are recommended to explore the effect of this probiotic on other substrates of IDE.

Acute exercise restores insulin clearance in diet-induced obese mice.

The aim of this study was to investigate the insulin clearance in diet-induced obese (DIO) mice submitted to acute endurance exercise (3h of treadmill exercise at 60-70% VO2max). Glucose-stimulated insulin secretion in isolated islets; ipGTT; ipITT; ipPTT; in vivo insulin clearance; protein expression in liver, skeletal muscle, and adipose tissue (insulin degrading enzyme (IDE), insulin receptor subunitβ(IRβ), phospho-Akt (p-Akt) and phospho-AMPK (p-AMPK)), and the activity of IDE in the liver and skeletal muscle were accessed. In DIO mice, acute exercise reduced fasting glycemia and insulinemia, improved glucose and insulin tolerance, reduced hepatic glucose production, and increased p-Akt protein levels in liver and skeletal muscle and p-AMPK protein levels in skeletal muscle. In addition, insulin secretion was reduced, whereas insulin clearance and the expression of IDE and IRβ were increased in liver and skeletal muscle. Finally, IDE activity was increased only in skeletal muscle. In conclusion, we propose that the increased insulin clearance and IDE expression and activity, primarily, in skeletal muscle, constitute an additional mechanism, whereby physical exercise reduces insulinemia in DIO mice.

Characterization of insulin-degrading enzyme-mediated cleavage of Aβ in distinct aggregation states

To enhance our understanding of the potential therapeutic utility of insulin-degrading enzyme (IDE) in Alzheimer's disease (AD), we studied in vitro IDE-mediated degradation of different amyloid-beta (Aβ) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aβ monomers and higher ordered aggregates. We identify Met35–Val36 as a novel IDE cleavage site in the Aβ sequence and show that Aβ fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aβ clearance in AD patients by modulating IDE activity.

Mechanisms by which the thiazolidinedione troglitazone protects against sucrose‐induced hepatic fat accumulation and hyperinsulinaemia

Thiazolidinediones (TZD) are known to ameliorate fatty liver in type 2 diabetes. To date, the underlying mechanisms of their hepatic actions remain unclear. Hepatic triglyceride content and export rates were assessed in 2 week high-sucrose-fed Wistar rats treated with troglitazone and compared with untreated high-sucrose rodent controls. Fractional de novo lipogenesis (DNL) contributions to hepatic triglyceride were quantified by analysis of triglyceride enrichment from deuterated water. Hepatic insulin clearance and NO status during a meal tolerance test were also evaluated. TZD significantly reduced hepatic triglyceride (P < 0.01) by 48%, decreased DNL contribution to hepatic triglyceride (P < 0.01) and increased postprandial non-esterified fatty acids clearance rates (P < 0.01) in comparison with the high-sucrose rodent control group. During a meal tolerance test, plasma insulin AUC was significantly lower (P < 0.01), while blood glucose and plasma C-peptide levels were not different. Insulin clearance was increased (P < 0.001) by 24% and was associated with a 22% augmentation of hepatic insulin-degrading enzyme activity (P < 0.05). Finally, hepatic NO was decreased by 24% (P < 0.05). Overall, TZD show direct actions on liver by reducing hepatic DNL and increasing hepatic insulin clearance. The alterations in hepatic insulin clearance were associated with changes in insulin-degrading enzyme activity, with possible modulation of NO levels.

Hyperinsulinemia caused by dexamethasone treatment is associated with reduced insulin clearance and lower hepatic activity of insulin-degrading enzyme

ObjectivesGlucocorticoid treatment induces insulin resistance (IR), which is counteracted by a compensatory hyperinsulinemia, due to increased pancreatic β-cell function. There is evidence for also reduced hepatic insulin clearance, but whether this correlates with altered activity of insulin-degrading enzyme (IDE) in the liver, is not fully understood. Here, we investigated whether hyperinsulinemia, in glucocorticoid-treated rodents, is associated with any alteration in the insulin clearance and activity of the IDE in the liver. Materials/methodsAdult male Swiss mice and Wistar rats were treated with the synthetic glucocorticoid dexamethasone intraperitoneally [1mg/kg body weight (b.w.)] for 5 consecutive days. ResultsGlucocorticoid treatment induced IR and hyperinsulinemia in both species, but was more impactful in rats that also displayed glucose intolerance and hyperglycemia. Insulin clearance was reduced in glucocorticoid-treated rats and mice, as judged by the reduction of insulin decay rate and increased insulin area-under-the-curve (47% and 87%, respectively). These results were associated with reduced activity (35%) of hepatic IDE in rats and a tendency to reduction (p=0.068) in mice, without alteration in hepatic IDE mRNA content, in both species. ConclusionIn conclusion, the reduced insulin clearance in glucocorticoid-treated rodents was due to the reduction of hepatic IDE activity, at least in rats, which may contributes to the compensatory hyperinsulinemia. These findings corroborate the idea that short-term and/or partial inhibition of IDE activity in the liver could be beneficial for the glycemic control.

Insulin-degrading enzyme is activated by the C-terminus of α-synuclein

The insulin-degrading enzyme (IDE) plays a key role in type-2 diabetes and typically degrades small peptides such as insulin, amyloid β and islet amyloid polypeptide. We recently reported a novel non-proteolytical interaction in vitro between IDE and the Parkinson's disease 140-residue protein α-synuclein that resulted in dual effects: arrested α-synuclein oligomers and, simultaneously, increased IDE proteolysis activity. Here we demonstrate that these outcomes arise due to IDE interactions with the C-terminus of α-synuclein. Whereas a peptide containing the first 97 residues of α-synuclein did not improve IDE activity and its aggregation was not blocked by IDE, a peptide with the C-terminal 44 residues of α-synuclein increased IDE proteolysis to the same degree as full-length α-synuclein. Because the α-synuclein C-terminus is acidic, the interaction appears to involve electrostatic attraction with IDE's basic exosite, known to be involved in activation.

Abstract P197: Extracellular Ubiquitin Blocks CXCL12-Induced Proliferation of Cardiac Fibroblasts

CXCR4 receptors mediate in part hypertension-induced cardiac fibrosis. This suggests that endogenous CXCR4-receptor agonists stimulate cardiac fibroblast (CF) proliferation; however, this possibility is unexplored. Although CXCL12 (aka SDF-1α) is the best described endogenous CXCR4 agonist, ubiquitin 1-76 (U-76) exists in the extracellular compartment (10 to 100 nM) and is reported to be a CXCR4 agonist. Therefore, we investigated the ability of both CXCL12 and U-76 to stimulate growth of rat CFs. Low concentrations of CXCL12 (10 nM x 4 days) increased CF proliferation (from 38,973 ± 384 to 44,429 ± 774 cells/well, n=6, p&lt;0.0001), and this effect was augmented by sitagliptin, a dipeptidyl peptidase 4 (DPP4) inhibitor (% increase by CXCL12: without sitagliptin, 14 ± 2 (n=6); with 1 μM of sitagliptin, 38 ± 9, n=6, p&lt;0.0001). This finding is consistent with the known ability of DPP4 to metabolize CXCL12, i.e., sitagliptin inhibits the metabolism of CXCL12 and enhances CXCL12-induced effects. Not only did U-76 at low physiological concentrations (10 nM) not stimulate CF proliferation, U-76 surprisingly nearly abolished CF proliferation induced by CXCL12 + sitagliptin (% increase by CXCL12 + sitagliptin: without U-76, 58 ± 5, n=12; with U-76, 10 ± 7, n=12, p&lt;0.0001). Ubiquitin 1-74 (U-74), a metabolite of U-76, inhibited the pro-growth effects of CXCL12 + sitagliptin 10-fold more potently than did U-76. CFs expressed insulin degrading enzyme (IDE) mRNA (qRT-PCR), protein (western blot), and activity (IDE activity assay) and inhibition of IDE with the newly discovered potent IDE inhibitor 6bK (1 μM) prevented U-76 from blocking the growth effects of CXCL12 + sitagliptin. Analysis by mass spectrometry (selected ion monitoring) demonstrated that CFs converted U-76 to U-74 and this conversion was attenuated by 6bK. Conclusions: CFs express IDE and therefore convert U-76 to U-74; U-74 blocks CXCR4 receptors thus protecting against CXCL12 + DPP4 inhibitor induced CF proliferation. Implications: In patients with low IDE activity (due to genes or drugs) who are treated with DPP4 inhibitors, U-74 (CXCR4 antagonist) levels would be low and CXCL12 (CXCR4 agonist) levels would be high, thus possibly creating a “perfect storm” for CF over-proliferation and cardiac fibrosis.

An Extended Polyanion Activation Surface in Insulin Degrading Enzyme.

Insulin degrading enzyme (IDE) is believed to be the major enzyme that metabolizes insulin and has been implicated in the degradation of a number of other bioactive peptides, including amyloid beta peptide (Aβ), glucagon, amylin, and atrial natriuretic peptide. IDE is activated toward some substrates by both peptides and polyanions/anions, possibly representing an important control mechanism and a potential therapeutic target. A binding site for the polyanion ATP has previously been defined crystallographically, but mutagenesis studies suggest that other polyanion binding modes likely exist on the same extended surface that forms one wall of the substrate-binding chamber. Here we use a computational approach to define three potential ATP binding sites and mutagenesis and kinetic studies to confirm the relevance of these sites. Mutations were made at four positively charged residues (Arg 429, Arg 431, Arg 847, Lys 898) within the polyanion-binding region, converting them to polar or hydrophobic residues. We find that mutations in all three ATP binding sites strongly decrease the degree of activation by ATP and can lower basal activity and cooperativity. Computational analysis suggests conformational changes that result from polyanion binding as well as from mutating residues involved in polyanion binding. These findings indicate the presence of multiple polyanion binding modes and suggest the anion-binding surface plays an important conformational role in controlling IDE activity.

Mechanisms linking brain insulin resistance to Alzheimer's disease.

Several studies have indicated that Diabetes Mellitus (DM) can increase the risk of developing Alzheimer's disease (AD). This review briefly describes current concepts in mechanisms linking DM and insulin resistance/deficiency to AD. Insulin/insulin-like growth factor (IGF) resistance can contribute to neurodegeneration by several mechanisms which involve: energy and metabolism deficits, impairment of Glucose transporter-4 function, oxidative and endoplasmic reticulum stress, mitochondrial dysfunction, accumulation of AGEs, ROS and RNS with increased production of neuro-inflammation and activation of pro-apoptosis cascade. Impairment in insulin receptor function and increased expression and activation of insulin-degrading enzyme (IDE) have also been described. These processes compromise neuronal and glial function, with a reduction in neurotransmitter homeostasis. Insulin/IGF resistance causes the accumulation of AβPP-Aβ oligomeric fibrils or insoluble larger aggregated fibrils in the form of plaques that are neurotoxic. Additionally, there is production and accumulation of hyper-phosphorylated insoluble fibrillar tau which can exacerbate cytoskeletal collapse and synaptic disconnection.

A neglected modulator of insulin-degrading enzyme activity and conformation: The pH

Insulin-degrading enzyme (IDE), a ubiquitously expressed zinc metalloprotease, has multiple activities in addition to insulin degradation and its malfunction is believed to connect type 2 diabetes with Alzheimer's disease. IDE has been found in many different cellular compartments, where it may experience significant physio-pathological pH variations. However, the exact role of pH variations on the interplay between enzyme conformations, stability, oligomerization state and catalysis is not understood.Here, we use ESI mass spectrometry, atomic force microscopy, surface plasmon resonance and circular dichroism to investigate the structure–activity relationship of IDE at different pH values. We show that acidic pH affects the ability of the enzyme to bind the substrate and decrease the stability of the protein by inducing an α-helical bundle conformation with a concomitant dissociation of multi-subunit IDE assemblies into monomeric units and loss of activity. These effects suggest a major role played by electrostatic forces in regulating multi-subunit enzyme assembly and function. Our results clearly indicate a pH dependent coupling among enzyme conformation, assembly and stability and suggest that cellular acidosis can have a large effect on IDE oligomerization state, inducing an enzyme inactivation and an altered insulin degradation that could have an impact on insulin signaling.

Partial Loss of the Glutamate Transporter GLT-1 Alters Brain Akt and Insulin Signaling in a Mouse Model of Alzheimer's Disease.

The glutamate transporter GLT-1 (also called EAAT2 in humans) plays a critical role in regulating extracellular glutamate levels in the central nervous system (CNS). In Alzheimer's disease (AD), EAAT2 loss is associated with neuropathology and cognitive impairment. In keeping with this, we have reported that partial GLT-1 loss (GLT-1+/-) causes early-occurring cognitive deficits in mice harboring familial AD AβPPswe/PS1ΔE9 mutations. GLT-1 plays important roles in several molecular pathways that regulate brain metabolism, including Akt and insulin signaling in astrocytes. Significantly, AD pathogenesis also involves chronic Akt activation and reduced insulin signaling in the CNS. In this report we tested the hypothesis that GLT-1 heterozygosity (which reduces GLT-1 to levels that are comparable to losses in AD patients) in AβPPswe/PS1ΔE9 mice would induce sustained activation of Akt and disturb components of the CNS insulin signaling cascade. We found that partial GLT-1 loss chronically increased Akt activation (reflected by increased phosphorylation at serine 473), impaired insulin signaling (reflected by decreased IRβ phosphorylation of tyrosines 1150/1151 and increased IRS-1 phosphorylation at serines 632/635 - denoted as 636/639 in humans), and reduced insulin degrading enzyme (IDE) activity in brains of mice expressing familial AβPPswe/PS1ΔE9 AD mutations. GLT-1 loss also caused an apparent compensatory increase in IDE activity in the liver, an organ that has been shown to regulate peripheral amyloid-β levels and expresses GLT-1. Taken together, these findings demonstrate that partial GLT-1 loss can cause insulin/Akt signaling abnormalities that are in keeping with those observed in AD.

Modulation of insulin degrading enzyme activity: A link between T2DM and liver cancer?

Modulation of insulin degrading enzyme activity: A link between T2DM and liver cancer?

Accumulation of BRI2-BRICHOS ectodomain correlates with a decreased clearance of Aβ by insulin degrading enzyme (IDE) in Alzheimer’s disease

The precursor protein BRI2 that in its mutated form is associated with British and Danish dementia, can regulate critical processes involved in AD pathogenesis including not only the metabolism of amyloid precursor protein (APP) and formation of Aβ, but also the levels of secreted insulin degrading enzyme (IDE), an enzyme involved in Aβ clearance. We recently observed increased levels of a 45kDa BRI2 form as well as BRI2 ectodomain deposits in Aβ plaques in human AD hippocampus, which may affect BRI2 functional activity. Since BRI2 regulated the levels of secreted IDE and subsequent degradation of Aβ in human cell culture models, we explored if BRI2 changes could affect the Aβ degradation capacity of IDE in human hippocampus (n=28). We observed that IDE is the main enzyme involved in Aβ degradation, and both IDE levels as well as Aβ degradation tend to be decreased in AD. Interestingly, the levels of the 45kDa BRI2 form and BRI2 deposits in hippocampal tissue were inversely correlated with IDE protein levels (r=−0.52, p=0.005; r=−0.4, p=0.045) and IDE activity (r=−0.5935, p=0.0004; r=−0.4, p=0.03). Taken together, the current results suggest a relationship between BRI2 protein changes, IDE activity and Aβ levels in human hippocampus. Thus, the formation and accumulation high of molecular weight BRI2 forms observed in AD may impair IDE functioning and consequently lead to impaired Aβ clearance and to the accumulation of Aβ.