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 , CamundongosRESUMO
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/metabolismoRESUMO
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 ObesosRESUMO
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/metabolismoRESUMO
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 AnimalRESUMO
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 , RatosRESUMO
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/metabolismoRESUMO
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 WistarRESUMO
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 SinaisRESUMO
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⻹ s⻹) 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.