RESUMO

Hyperinsulinemia is frequently associated with aging and may cause insulin resistance in elderly. Since insulin secretion and clearance decline with age, hyperinsulinemia seems to be maintained, primarily, due to a decrease in the insulin clearance. To investigate these aging effects, 3- and 18-month-old male C57BL/6 mice were subjected to intraperitoneal glucose and insulin tolerance tests (ipGTT and ipITT) and, during the ipGTT, plasma c-peptide and insulin were measure to evaluate in vivo insulin clearance. Glucose-stimulated insulin secretion in isolated pancreatic islets was also assessed, and liver samples were collected for molecular analyses (western blot). Although insulin sensitivity was not altered in the old mice, glucose tolerance, paradoxically, seems to be increased, accompanied by higher plasma insulin, during ipGTT. While insulin secretion did not increase, insulin clearance was reduced in the old mice, as suggested by the lower c-peptide:insulin ratio, observed during ipGTT. Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) and insulin-degrading enzyme (IDE), as well as the activity of this enzyme, were reduced in the liver of old mice, justifying the decreased insulin clearance observed in these mice. Therefore, loss of hepatic CEACAM1 and IDE function may be directly related to the decline in insulin clearance during aging.

Assuntos

Envelhecimento/metabolismo , Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Animais , Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Teste de Tolerância a Glucose , Insulina/sangue , Resistência à Insulina/fisiologia , Secreção de Insulina/fisiologia , Insulisina/metabolismo , Ilhotas Pancreáticas/metabolismo , Fígado/metabolismo , Masculino , Camundongos

RESUMO

SDF-1α (stromal cell-derived factor-1α) is a CXCR4-receptor agonist and DPP4 (dipeptidyl peptidase 4) substrate. SDF-1α, particularly when combined with sitagliptin to block the metabolism of SDF-1α by DPP4, stimulates proliferation of cardiac fibroblasts via the CXCR4 receptor; this effect is greater in cells from spontaneously hypertensive rats versus Wistar-Kyoto normotensive rats. Emerging evidence indicates that ubiquitin(1-76) exists in plasma and is a potent CXCR4-receptor agonist. Therefore, we hypothesized that ubiquitin(1-76), similar to SDF-1α, should increase proliferation of cardiac fibroblasts. Contrary to our working hypothesis, ubiquitin(1-76) did not stimulate cardiac fibroblast proliferation, yet unexpectedly antagonized the proproliferative effects of SDF-1α combined with sitagliptin. In this regard, ubiquitin(1-76) was more potent in spontaneously hypertensive versus Wistar-Kyoto cells. In the presence of 6bk (selective inhibitor of insulin-degrading enzyme [IDE]; an enzyme known to convert ubiquitin(1-76) to ubiquitin(1-74)), ubiquitin(1-76) no longer antagonized the proproliferative effects of SDF-1α/sitagliptin. Ubiquitin(1-74) also antagonized the proproliferative effects of SDF-1α/sitagliptin, and this effect of ubiquitin(1-74) was not blocked by 6bk and was >10-fold more potent compared with ubiquitin(1-76). Neither ubiquitin(1-76) nor ubiquitin(1-74) inhibited the proproliferative effects of the non-CXCR4 receptor agonist neuropeptide Y (activates Y1 receptors). Cardiac fibroblasts expressed IDE mRNA, protein, and activity and converted ubiquitin(1-76) to ubiquitin(1-74). Spontaneously hypertensive fibroblasts expressed greater IDE activity. Extracellular ubiquitin(1-76) blocks the proproliferative effects of SDF-1α/sitagliptin via its conversion by IDE to ubiquitin(1-74), a potent CXCR4 antagonist. Thus, IDE inhibitors, particularly when combined with DPP4 inhibitors or hypertension, could increase the risk of cardiac fibrosis.

Assuntos

Proliferação de Células , Quimiocina CXCL12/metabolismo , Fibroblastos , Hipertensão/metabolismo , Insulisina , Miocárdio/patologia , Receptores CXCR4 , Animais , Pressão Sanguínea/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Insulisina/antagonistas & inibidores , Insulisina/metabolismo , Neuropeptídeo Y/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores CXCR4/agonistas , Receptores CXCR4/metabolismo , Transdução de Sinais , Fosfato de Sitagliptina/farmacologia , Ubiquitina/metabolismo

RESUMO

Disruption of insulin secretion and clearance both contribute to obesity-induced hyperinsulinemia, though reduced insulin clearance seems to be the main factor. The liver is the major site for insulin degradation, a process mainly coordinated by the insulin-degrading enzyme (IDE). The beneficial effects of taurine conjugated bile acid (TUDCA) on insulin secretion as well as insulin sensitivity have been recently described. However, the possible role of TUDCA in insulin clearance had not yet been explored. Here, we demonstrated that 15 days treatment with TUDCA reestablished plasma insulin to physiological concentrations in high fat diet (HFD) mice, a phenomenon associated with increased insulin clearance and liver IDE expression. TUDCA also increased IDE expression in human hepatic cell line HepG2. This effect was not observed in the presence of an inhibitor of the hepatic membrane bile acid receptor, S1PR2, nor when its downstream proteins were inhibited, including IR, PI3K and Akt. These results indicate that treatment with TUDCA may be helpful to counteract obesity-induced hyperinsulinemia through increasing insulin clearance, likely through enhanced liver IDE expression in a mechanism dependent on S1PR2-Insulin pathway activation.

Assuntos

Insulina/farmacocinética , Insulisina/efeitos dos fármacos , Fígado/enzimologia , Ácido Tauroquenodesoxicólico/farmacologia , Animais , Dieta Hiperlipídica , Células Hep G2 , Humanos , Hiperinsulinismo/tratamento farmacológico , Insulisina/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Obesos

RESUMO

The aim of this study was to investigate the effect of subdiaphragmatic vagotomy on insulin sensitivity, secretion, and degradation in metabolic programmed mice, induced by a low-protein diet early in life, followed by exposure to a high-fat diet in adulthood. Weaned 30-day-old C57Bl/6 mice were submitted to a low-protein diet (6% protein). After 4 weeks, the mice were distributed into three groups: LP group, which continued receiving a low-protein diet; LP + HF group, which started to receive a high-fat diet; and LP + HFvag group, which underwent vagotomy and also was kept at a high-fat diet. Glucose-stimulated insulin secretion (GSIS) in isolated islets, ipGTT, ipITT, in vivo insulin clearance, and liver expression of the insulin-degrading enzyme (IDE) was accessed. Vagotomy improved glucose tolerance and reduced insulin secretion but did not alter adiposity and insulin sensitivity in the LP + HFvag, compared with the LP + HF group. Improvement in glucose tolerance was accompanied by increased insulinemia, probably due to a diminished insulin clearance, as judged by the lower C-peptide : insulin ratio, during the ipGTT. Finally, vagotomy also reduced liver IDE expression in this group. In conclusion, when submitted to vagotomy, the metabolic programmed mice showed improved glucose tolerance, associated with an increase of plasma insulin concentration as a result of insulin clearance reduction, a phenomenon probably due to diminished liver IDE expression.

Assuntos

Resistência à Insulina/fisiologia , Insulina/metabolismo , Obesidade/cirurgia , Vagotomia/métodos , Animais , Dieta Hiperlipídica , Dieta com Restrição de Proteínas , Glucose/metabolismo , Insulisina/metabolismo , Fígado/metabolismo , Camundongos , Obesidade/metabolismo

RESUMO

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.

Assuntos

Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Insulina/metabolismo , Insulisina/genética , Interleucina-6/farmacologia , Animais , Linhagem Celular , Células Hep G2 , Humanos , Insulisina/sangue , Insulisina/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Condicionamento Físico Animal

RESUMO

Our objective was to know how insulin is processing in mitochondria; if IDE is the only participant in mitochondrial insulin degradation and the role of insulin degradation on IDE accumulation in mitoplasts. Mitochondria and its fractions were isolated as described by Greenwalt. IDE was purified and detected in immunoblot with specific antibodies. High insulin degradation was obtained through addition to rat's diet of 25 g/rat of apple and 10 g/rat of hard-boiled eggs, 3 days a week. Mitochondrial insulin degradation was assayed with 5 % TCA, insulin antibody or Sephadex G50 chromatography. Degradation was also assayed 60 min at 37 °C in mitochondrial fractions (IMS and Mx) with diet or not and without IDE. Degradation in fractions precipitated with ammonium sulfates (60-80 %) were studied after mitochondrial insulin incubation (1 ng. insulin during 15 min, at 30 °C) or with addition of 2.5 mM ATP. Supplementary diet increased insulin degradation. High insulin did not increase mitoplasts accumulation and did not decrease mitochondrial degradation. High insulin and inhibition of degradation evidence insulin competition for a putative transport system. Mitochondrial incubation with insulin increased IDE in matrix as observed in immunoblot. ATP decreased degradation in Mx and increased it in IMS. Chromatography of IMS demonstrated an ATP-dependent protease that degraded insulin, similar to described by Sitte et al. Mitochondria participate in insulin degradation and the diet increased it. High insulin did not accomplish mitochondrial decrease of degradation or its accumulation in mitoplasts. Mitochondrial incubation with insulin increased IDE in matrix. ATP suggested being a regulator of mitochondrial insulin degradation.

Assuntos

Insulina/metabolismo , Insulisina/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Dietoterapia , Insulina/farmacologia , Mitofagia/efeitos dos fármacos , Ratos

RESUMO

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 subunitß(IRß), 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 IRß 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.

Assuntos

Insulina/metabolismo , Obesidade/metabolismo , Obesidade/terapia , Condicionamento Físico Animal/fisiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Peptídeo C/sangue , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Insulina/sangue , Insulisina/metabolismo , Fígado/metabolismo , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Músculo Esquelético/metabolismo , Obesidade/etiologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismo

RESUMO

OBJECTIVES: Glucocorticoid treatment induces insulin resistance (IR), which is counteracted by a compensatory hyperinsulinemia, due to increased pancreatic ß-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/METHODS: Adult male Swiss mice and Wistar rats were treated with the synthetic glucocorticoid dexamethasone intraperitoneally [1mg/kg body weight (b.w.)] for 5 consecutive days. RESULTS: Glucocorticoid 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. CONCLUSION: In 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.

Assuntos

Dexametasona/efeitos adversos , Hiperinsulinismo/induzido quimicamente , Insulina/metabolismo , Insulisina/metabolismo , Animais , Dexametasona/farmacologia , Glucocorticoides/efeitos adversos , Glucocorticoides/farmacologia , Teste de Tolerância a Glucose , Hiperinsulinismo/metabolismo , Resistência à Insulina , Insulisina/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Ratos Wistar

RESUMO

INTRODUCTION: Endurance training improves peripheral insulin sensitivity in the liver and the skeletal muscle, but the mechanism for this effect is poorly understood. Recently, it was proposed that insulin clearance plays a major role in both glucose homeostasis and insulin sensitivity. Therefore, our goal was to determine the mechanism by which endurance training improves insulin sensitivity and how it regulates insulin clearance in mice. METHODS: Mice were treadmill-trained for 4 weeks at 70-80% of maximal oxygen consumption (VO2 max) for 60 min, 5 days a week. The glucose tolerance and the insulin resistance were determined using an IPGTT and an IPITT, respectively, and the insulin decay rate was calculated from the insulin clearance. Protein expression and phosphorylation in the liver and the skeletal muscle were ascertained by Western blot. RESULTS: Trained mice exhibited an increased VO2 max, time to exhaustion, glucose tolerance and insulin sensitivity. They had smaller fat pads and lower plasma concentrations of insulin and glucose. Endurance training inhibited insulin clearance and reduced expression of IDE in the liver, while also inhibiting insulin secretion by pancreatic islets. There was increased phosphorylation of both the canonical (IR-AKT) and the non-canonical (CaMKII-AMPK-ACC) insulin pathways in the liver of trained mice, whereas only the CaMKII-AMPK pathway was increased in the skeletal muscle. CONCLUSION: Endurance training improved glucose homeostasis not only by increasing peripheral insulin sensitivity but also by decreasing insulin clearance and reducing IDE expression in the liver.

Assuntos

Resistência à Insulina , Insulina/sangue , Insulisina/metabolismo , Esforço Físico , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Glicemia/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Insulina/metabolismo , Insulisina/genética , Ilhotas Pancreáticas/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Músculo Esquelético/metabolismo , Consumo de Oxigênio , Receptor de Insulina/metabolismo , Transdução de Sinais

RESUMO

The aim of this study was to determine if insulin is transferred to mitoplasts by insulin-degrading enzyme (IDE).Hepatic mitochondria were isolated and controlled by electron microscopy. IDE was obtained from rats muscle by successive chromatography steps. Insulin accumulation in mitoplasts and outer membrane + intermembrane space (OM + IMS) was studied with (125)I-insulin. Mitochondrial insulin accumulation and degradation was assayed with Sephadex G50 chromatography, insulin antibody and 5 % TCA. Mitoplasts and OM + IMS were isolated with digitonin. Insulin accumulation was studied at 25 °C at different times, without or with IDE, Bacitracin, 2,4-dinitrophenol, apyrase or sodium succinate + adenosine diphosphate. Insulin accumulation in mitoplasts and OM + IMS after mitochondrial cross-linking was studied with electrophoresis in SDS-PAGE, immunoblots of IDE, insulin or TIM23 (inner mitochondrial transporter) and autoradiography.The studies showed that addition of IDE increased insulin transfer from OM + IMS to mitoplasts, and the insulin accumulation in mitoplast was IDE dependent. Bacitracin and 2,4-dinitrophenol decreased this transfer. The [Insulin-IDE] complex and [Mitoplasts] was studied as a bimolecular reaction following a second order reaction. The constant "k" (liter.mol⁻¹ s⁻¹) showed that IDE increased and Bacitracin or 2,4-dinitrophenol decreased the velocity of insulin transfer. SDS-PAGE and immunoblots studies showed bands and radioactivity coincident with IDE, insulin and TIM23. Non degraded insulin was demonstrated in immunoblot after IDE immunoprecipitation from mitoplasts. Confocal studies showed mitochondrial colocalization of IDE and insulin.The results showed that insulin at 25 °C were transferred from OM + IMS to mitoplasts by IDE or that the enzyme facilitates this transfer, and they reach the matrix together.

Assuntos

Insulina/metabolismo , Insulisina/metabolismo , Mitocôndrias Hepáticas/metabolismo , Animais , Masculino , Microscopia Confocal , Mitocôndrias Hepáticas/enzimologia , Ratos , Ratos Wistar

RESUMO

Nutrient restriction during the early stages of life usually leads to alterations in glucose homeostasis, mainly insulin secretion and sensitivity, increasing the risk of metabolic disorders in adulthood. Despite growing evidence regarding the importance of insulin clearance during glucose homeostasis in health and disease, no information exists about this process in malnourished animals. Thus, in the present study, we aimed to determine the effect of a nutrient-restricted diet on insulin clearance using a model in which 30-d-old C57BL/6 mice were exposed to a protein-restricted diet for 14 weeks. After this period, we evaluated many metabolic variables and extracted pancreatic islet, liver, gastrocnemius muscle (GCK) and white adipose tissue samples from the control (normal-protein diet) and restricted (low-protein diet, LP) mice. Insulin concentrations were determined using RIA and protein expression and phosphorylation by Western blot analysis. The LP mice exhibited lower body weight, glycaemia, and insulinaemia, increased glucose tolerance and altered insulin dynamics after the glucose challenge. The improved glucose tolerance could partially be explained by an increase in insulin sensitivity through the phosphorylation of the insulin receptor/protein kinase B and AMP-activated protein kinase/acetyl-CoA carboxylase in the liver, whereas the changes in insulin dynamics could be attributed to reduced insulin secretion coupled with reduced insulin clearance and lower insulin-degrading enzyme (IDE) expression in the liver and GCK. In summary, protein-restricted mice not only produce and secrete less insulin, but also remove and degrade less insulin. This phenomenon has the double benefit of sparing insulin while prolonging and potentiating its effects, probably due to the lower expression of IDE in the liver, possibly with long-term consequences.

Assuntos

Dieta com Restrição de Proteínas/efeitos adversos , Regulação para Baixo , Metabolismo Energético , Regulação Enzimológica da Expressão Gênica , Insulina/metabolismo , Insulisina/metabolismo , Fígado/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Ingestão de Energia , Resistência à Insulina , Secreção de Insulina , Insulisina/genética , Ilhotas Pancreáticas/metabolismo , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Distribuição Aleatória , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transdução de Sinais , Desmame , Aumento de Peso

RESUMO

Mammalian insulin-degrading enzyme (IDE) cleaves insulin, among other peptidic substrates, but its function in insulin signaling is elusive. We use the Drosophila system to define the function of IDE in the regulation of growth and metabolism. We find that either loss or gain of function of Drosophila IDE (dIDE) can restrict growth in a cell-autonomous manner by affecting both cell size and cell number. dIDE can modulate Drosophila insulin-like peptide 2 levels, thereby restricting activation of the phosphatidylinositol-3-phosphate kinase pathway and promoting activation of Drosophila forkhead box, subgroup O transcription factor. Larvae reared in high sucrose exhibit delayed developmental timing due to insulin resistance. We find that dIDE loss of function exacerbates this phenotype and that mutants display increased levels of circulating sugar, along with augmented expression of a lipid biosynthesis marker. We propose that dIDE is a modulator of insulin signaling and that its loss of function favors insulin resistance, a hallmark of diabetes mellitus type II.

Assuntos

Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Insulisina/genética , Transdução de Sinais , Animais , Tamanho Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Insulisina/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Neuropeptídeos , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Asas de Animais/citologia , Asas de Animais/metabolismo

RESUMO

Insulin clearance plays a major role in glucose homeostasis and insulin sensitivity in physiological and/or pathological conditions, such as obesity-induced type 2 diabetes as well as diet-induced obesity. The aim of the present work was to evaluate cafeteria diet-induced obesity-induced changes in insulin clearance and to explain the mechanisms underlying these possible changes. Female Swiss mice were fed either a standard chow diet (CTL) or a cafeteria diet (CAF) for 8 weeks, after which we performed glucose tolerance tests, insulin tolerance tests, insulin dynamics, and insulin clearance tests. We then isolated pancreatic islets for ex vivo glucose-stimulated insulin secretion as well as liver, gastrocnemius, visceral adipose tissue, and hypothalamus for subsequent protein analysis by western blot and determination of mRNA levels by real-time RT-PCR. The cafeteria diet induced insulin resistance, glucose intolerance, and increased insulin secretion and total insulin content. More importantly, mice that were fed a cafeteria diet demonstrated reduced insulin clearance and decay rate as well as reduced insulin-degrading enzyme (IDE) protein and mRNA levels in liver and skeletal muscle compared with the control animals. Furthermore, the cafeteria diet reduced IDE expression and alternative splicing in the liver and skeletal muscle of mice. In conclusion, a cafeteria diet impairs glucose homeostasis by reducing insulin sensitivity, but it also reduces insulin clearance by reducing IDE expression and alternative splicing in mouse liver; however, whether this mechanism contributes to the glucose intolerance or helps to ameliorate it remains unclear.

Assuntos

Carboidratos da Dieta/farmacologia , Gorduras na Dieta/farmacologia , Insulina/metabolismo , Insulisina/metabolismo , Obesidade/metabolismo , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Animais , Carboidratos da Dieta/efeitos adversos , Gorduras na Dieta/efeitos adversos , Modelos Animais de Doenças , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Resistência à Insulina/fisiologia , Fígado/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Obesidade/etiologia , Obesidade/fisiopatologia

RESUMO

Insulin-degrading enzyme (IDE) is a neutral Zn(2+) peptidase that degrades short peptides based on substrate conformation, size and charge. Some of these substrates, including amyloid ß (Aß) are capable of self-assembling into cytotoxic oligomers. Based on IDE recognition mechanism and our previous report of the formation of a stable complex between IDE and intact Aß in vitro and in vivo, we analyzed the possibility of a chaperone-like function of IDE. A proteolytically inactive recombinant IDE with Glu111 replaced by Gln (IDEQ) was used. IDEQ blocked the amyloidogenic pathway of Aß yielding non-fibrillar structures as assessed by electron microscopy. Measurements of the kinetics of Aß aggregation by light scattering showed that 1) IDEQ effect was promoted by ATP independent of its hydrolysis, 2) end products of Aß-IDEQ co-incubation were incapable of "seeding" the assembly of monomeric Aß and 3) IDEQ was ineffective in reversing Aß aggregation. Moreover, Aß aggregates formed in the presence of IDEQ were non-neurotoxic. IDEQ had no conformational effects upon insulin (a non-amyloidogenic protein under physiological conditions) and did not disturb insulin receptor activation in cultured cells. Our results suggest that IDE has a chaperone-like activity upon amyloid-forming peptides. It remains to be explored whether other highly conserved metallopeptidases have a dual protease-chaperone function to prevent the formation of toxic peptide oligomers from bacteria to mammals.

Assuntos

Peptídeos beta-Amiloides/biossíntese , Amiloide/antagonistas & inibidores , Insulisina/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Recombinantes/metabolismo , Amiloide/biossíntese , Peptídeos beta-Amiloides/ultraestrutura , Animais , Western Blotting , Dicroísmo Circular , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Insulina/química , Insulina/metabolismo , Cinética , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Mutagênese Sítio-Dirigida , Conformação Proteica , Ratos

RESUMO

Studies of post-mortem brains from Alzheimer disease patients suggest that oxidative damage induced by mitochondrial amyloid ß (mitAß) accumulation is associated with mitochondrial dysfunction. However, the regulation of mitAß metabolism is unknown. One of the proteases involved in mitAß catabolism is the long insulin-degrading enzyme (IDE) isoform (IDE-Met(1)). However, the mechanisms of its expression are unknown, and its presence in brain is uncertain. We detected IDE-Met(1) in brain and showed that its expression is regulated by the mitochondrial biogenesis pathway (PGC-1α/NRF-1). A strong positive correlation between PGC-1α or NRF-1 and long IDE isoform transcripts was found in non-demented brains. This correlation was weaker in Alzheimer disease. In vitro inhibition of IDE increased mitAß and impaired mitochondrial respiration. These changes were restored by inhibition of γ-secretase or promotion of mitochondrial biogenesis. Our results suggest that IDE-Met(1) links the mitochondrial biogenesis pathway with mitAß levels and organelle functionality.

Assuntos

Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Insulisina/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Animais , Encéfalo/patologia , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Insulisina/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Proteínas do Tecido Nervoso/genética , Fator 1 Nuclear Respiratório/genética , Fator 1 Nuclear Respiratório/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo

RESUMO

Cerebral amyloid ß (Aß) accumulation is pathogenically associated with sporadic Alzheimer's disease (SAD). BACE-1 is involved in Aß generation while insulin-degrading enzyme (IDE) partakes in Aß proteolytic clearance. Vulnerable regions in AD brains show increased BACE-1 protein levels and enzymatic activity while the opposite occurs with IDE. Another common feature in SAD brains is Notch1 overexpression. Here we demonstrate an increase in mRNA levels of Hey-1, a Notch target gene, and a decrease of IDE transcripts in the hippocampus of SAD brains as compared to controls. Transient transfection of Notch intracellular domain (NICD) in N2aSW cells, mouse neuroblastoma cells (N2a) stably expressing human amyloid precursor protein (APP) Swedish mutation, reduce IDE mRNA levels, promoting extracellular Aß accumulation. Also, NICD, HES-1 and Hey-1 overexpression result in decreased IDE proximal promoter activity. This effect was mediated by 2 functional sites located at -379/-372 and -310-303 from the first translation start site in the -575/-19 (556 bp) fragment of IDE proximal promoter. By site-directed mutagenesis of the IDE promoter region we reverted the inhibitory effect mediated by NICD transfection suggesting that these sites are indeed responsible for the Notch-mediated inhibition of the IDE gene expression. Intracranial injection of the Notch ligand JAG-1 in Tg2576 mice, expressing the Swedish mutation in human APP, induced overexpression of HES-1 and Hey-1 and reduction of IDE mRNA levels, respectively. Our results support our theory that a Notch-dependent IDE transcriptional modulation may impact on Aß metabolism providing a functional link between Notch signaling and the amyloidogenic pathway in SAD.

Assuntos

Precursor de Proteína beta-Amiloide/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Insulisina/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/fisiologia , Transcrição Gênica , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Hipocampo/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Insulisina/genética , Camundongos , Ligação Proteica , Receptores Notch/genética , Receptores Notch/metabolismo , Fatores de Transcrição HES-1

RESUMO

Insulin-degrading enzyme (IDE) has been shown to enhance the binding of androgen and glucocorticoid receptors to DNA in the nuclear compartment. Glucocorticoids cause hyperglycaemia, peripheral resistance to insulin and compensatory hyperinsulinaemia. The aim of the present study was to investigate the effect of dexamethasone (D), testosterone (T) and dexamethasone plus testosterone (D + T) on the regulation of IDE and on the remodelling of rat ventral prostate after castration (C). Castration led to a marked reduction in prostate weight (PW). Body weight was significantly decreased in the castrated animals treated with dexamethasone, and the relative PW was 2.6-fold (±0.2) higher in the D group, 2.8-fold (±0.3) higher in the T group and 6.6-fold (±0.6) higher in the D + T group in comparison with the castrated rats. Ultrastructural alterations in the ventral prostate in response to androgen deprivation were restored after testosterone and dexamethasone plus testosterone treatments and partially restored with dexamethasone alone. The nuclear IDE protein level indicated a 4.3-fold (±0.4) increase in castrated rats treated with D + T when compared with castration alone. Whole-cell IDE protein levels increased approximately 1.5-fold (±0.1), 1.5-fold (±0.1) and 2.9-fold (±0.2) in the D, T and D + T groups, respectively, when compared with castration alone. In conclusion, the present study reports that dexamethasone-induced hyperinsulinaemic condition plus exogenous testosterone treatment leads to synergistic effects of insulin and testosterone in the prostatic growth and in the amount of IDE in the nucleus and whole epithelial cell.

Assuntos

Castração , Dexametasona/farmacologia , Insulisina/metabolismo , Próstata/metabolismo , Próstata/patologia , Testosterona/farmacologia , Androgênios/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Dexametasona/efeitos adversos , Glucocorticoides/efeitos adversos , Glucocorticoides/farmacologia , Hiperinsulinismo/induzido quimicamente , Hiperinsulinismo/metabolismo , Insulisina/efeitos dos fármacos , Masculino , Modelos Animais , Próstata/efeitos dos fármacos , Ratos , Ratos Wistar

RESUMO

The accumulation of amyloid-beta (Abeta) peptides in senile plaques is one of the hallmarks of Alzheimer's disease (AD) progression. The endocytic pathway has been proposed as a major subcellular site for Abeta generation while the compartments in which Abeta-degrading proteases interact with Abeta are still elusive. It was suggested that extracellular Abeta degradation may take place by plasma-membrane associated proteases or by extracellular proteases, among which insulin-degrading enzyme (IDE) is the most relevant. However, the mechanisms of IDE secretion are poorly understood. In the present study we used N2a cells to explore if IDE is indeed released through exosomes and the effect of exosomes release on extracellular levels of Abeta. We demonstrated that proteolytically-active plasma membrane associated-IDE is routed in living N2a cells to multivesicular bodies and subsequently, a major fraction is sorted to exosomes. We described that extracellular IDE levels decrease if the generation of multivesicular bodies is interfered and may be positively modulated by exosomes release under stress-induced conditions. Our results reinforce the relevance of functional IDE in the catabolism of extracellular Abeta.

Assuntos

Peptídeos beta-Amiloides/metabolismo , Encéfalo/enzimologia , Exossomos/metabolismo , Insulisina/metabolismo , Via Secretória/fisiologia , Vesículas Transportadoras/enzimologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Animais , Transporte Biológico/fisiologia , Exossomos/química , Camundongos , Neuroblastoma/enzimologia , Neuroblastoma/metabolismo , Vesículas Transportadoras/metabolismo , Células Tumorais Cultivadas

RESUMO

Alzheimer's disease (AD) is characterized by amyloid beta (A beta) accumulation in the brain and is classified as familial early-onset (FAD) or sporadic late-onset (SAD). Evidences suggest that deficits in the brain expression of insulin degrading enzyme (IDE) and neprilysin (NEP), both proteases involved in amyloid degradation, may promote A beta deposition in SAD. We studied by immunohistochemistry IDE and NEP cortical expression in SAD and FAD samples carrying the E280A presenilin-1 missense mutation. We showed that IDE, a soluble peptidase, is linked with aggregated A beta 40 isoform while NEP, a membrane-bound protease, negatively correlates with amyloid angiopathy and its expression in the senile plaques is independent of aggregated amyloid and restricted to SAD cases. NEP, but not IDE, is over-expressed in dystrophic neurites, both proteases are immunoreactive in activated astrocytes but not in microglia and IDE was the only one detected in astrocytes of white matter from FAD cases. Collectively, our results support the notion that gross conformational changes involved in the modification from "natively folded-active" to "aggregated-inactive" IDE and NEP may be a relevant pathogenic mechanism in SAD.

Assuntos

Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/metabolismo , Córtex Cerebral/enzimologia , Insulisina/metabolismo , Neprilisina/metabolismo , Fragmentos de Peptídeos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Astrócitos/enzimologia , Angiopatia Amiloide Cerebral/enzimologia , Córtex Cerebral/patologia , Feminino , Humanos , Insulisina/química , Masculino , Microglia/enzimologia , Pessoa de Meia-Idade , Neprilisina/química , Placa Amiloide/enzimologia , Presenilina-1/análise , Presenilina-1/genética , Conformação Proteica

RESUMO

Inulinase belongs to an important class of enzymes as it can be used to produce high-fructose syrups by enzymatic hydrolysis of inulin and fructooligosaccharides, which has been used as functional food. This work aimed to carry out a partial characterization of the crude enzymatic extract of two different inulinases, obtained by solid-state fermentation (SSF) and submerged fermentation (SmF), using agroindustrial residues as substrates. The crude enzymatic extract obtained by SmF showed an optimal pH and temperature for hydrolytic activity of 4.5 and 55 degrees Celsius, respectively; and that obtained by SSF conducted to optimal pH and temperature of 5.0 and 55 degrees Celsius, respectively. Both enzymes presented high thermostability, with a D value of 230.4 h and 123.1 h for SmF and SSF, respectively. The inulinase produced by SmF showed highest stability at pH 4.4, while inulinase obtained by SSF was more stable at pH 4.8. The results showed that inulinase obtained by SmF is less susceptible to pH effect and the inulinase obtained by SSF is more resistant to higher temperatures.

Assuntos

Fermentação , Indústria Alimentícia , Imersão , Insulisina/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Insulisina/química , Insulisina/isolamento & purificação , Cinética , Temperatura