RESUMEN
Liraglutide, a human long-lasting GLP-1 analogue, is currently regarded as a powerful treatment option for type 2 diabetes. Apart from glucoregulatory and insulinotropic actions, liraglutide increases ß-cell mass through stimulation of ß-cell proliferation and islet neogenesis, as well as inhibition of ß-cell apoptosis. However, the underline molecular mechanisms have not been fully characterized. In this study, we investigated the mechanism by which liraglutide preserves islet ß-cells in an animal model of overt diabetes, the obese db/db mice, and protects a mouse pancreatic ß-cell line (ßTC-6 cells) against apoptosis. Treatment of 12-week-old diabetic mice with liraglutide for 2 weeks had no appreciable effects on blood non-fasting glucose concentration, islet insulin content and body weight. However, morphological and biochemical examination of diabetic mouse pancreatic islets demonstrated that liraglutide restores islet size, reduces islet ß-cell apoptosis and improves nephrin expression, a protein involved in ß-cell survival signalling. Our results indicated that liraglutide protects ßTC-6 cells from serum withdrawal-induced apoptosis through inhibition of caspase-3 activation. The molecular mechanism of the anti-apoptotic action of liraglutide in ßTC-6-cells comprises stimulation of PI3-kinase-dependent AKT phosphorylation leading to the phosphorylation, hence inactivation of the pro-apoptotic protein BAD and inhibition of FoxO1 transcription factor. In conclusion, we provided evidence that the GLP-1 analogue liraglutide exerts important beneficial effects on pancreatic islet architecture and ß-cell survival by protecting cells against apoptosis. These findings extend our understanding of the actions of liraglutide and further support the use of GLP-1R agonists in the treatment of patients with type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Péptido 1 Similar al Glucagón/genética , Células Secretoras de Insulina/efectos de los fármacos , Liraglutida/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Proteína Forkhead Box O1/genética , Péptido 1 Similar al Glucagón/análogos & derivados , Péptido 1 Similar al Glucagón/farmacología , Humanos , Hipoglucemiantes/farmacología , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Liraglutida/farmacología , Ratones , Ratones Obesos , Fosfatidilinositol 3-Quinasas/genética , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: Chronic hyperglycaemia, as seen in type II diabetes, results in both morphological and functional impairments of podocytes in the kidney. We investigated the effects of high glucose (HG) on the insulin signaling pathway, focusing on cell survival and apoptotic markers, in immortalized human glomerular cells (HGEC; podocytes) and isolated glomeruli from healthy rats. METHODS AND FINDINGS: HGEC and isolated glomeruli were cultured for various time intervals under HG concentrations in the presence or absence of insulin. Our findings indicated that exposure of HGEC to HG led to downregulation of all insulin signaling markers tested (IR, p-IR, IRS-1, p-Akt, p-Fox01,03), as well as to increased sensitivity to apoptosis (as seen by increased PARP cleavage, Casp3 activation and DNA fragmentation). Short insulin pulse caused upregulation of insulin signaling markers (IR, p-IR, p-Akt, p-Fox01,03) in a greater extent in normoglycaemic cells compared to hyperglycaemic cells and for the case of p-Akt, in a PI3K-dependent manner. IRS-1 phosphorylation of HG-treated podocytes was negatively regulated, favoring serine versus tyrosine residues. Prolonged insulin treatment caused a significant decrease of IR levels, while alterations in glucose concentrations for various time intervals demonstrated changes of IR, p-IR and p-Akt levels, suggesting that the IR signaling pathway is regulated by glucose levels. Finally, HG exerted similar effects in isolated glomeruli. CONCLUSIONS: These results suggest that HG compromises the insulin signaling pathway in the glomerulus, promoting a proapoptotic environment, with a possible critical step for this malfunction lying at the level of IRS-1 phosphorylation; thus we herein demonstrate glomerular insulin signaling as another target for investigation for the prevention and/ or treatment of diabetic nephropathy.
Asunto(s)
Glucosa/farmacología , Proteínas Sustrato del Receptor de Insulina/genética , Insulina/farmacología , Podocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Línea Celular Transformada , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Regulación de la Expresión Génica , Glucosa/metabolismo , Humanos , Hiperglucemia/genética , Hiperglucemia/metabolismo , Hiperglucemia/patología , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Podocitos/citología , Podocitos/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Serina/metabolismo , Técnicas de Cultivo de Tejidos , Tirosina/metabolismoRESUMEN
PURPOSE: The use of nanoparticles has seen exponential growth in the area of health care, due to the unique physicochemical properties of nanomaterials that make them desirable for medical applications. The aim of this study was to examine the effects of crystal phase-nanostructured titanium dioxide particles on bioactivity/cytotoxicity in breast cancer epithelial cells. MATERIALS AND METHODS: Cultured Michigan Cancer Foundation (MCF)-7 and human breast adenocarcinoma (MDA-MB-468) breast cancer epithelial cells were exposed to ultraviolet A light (wavelength 350 nm) for 20 minutes in the presence of aqueous dispersions of two different nanostructured titanium dioxide (TiO2) crystal phases: anatase and an anatase-rutile mixture. Detailed characterization of each titanium dispersion was performed by dynamic light scattering. A 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) colorimetric assay was employed to estimate the percentage of viable cells after each treatment. Western blot analysis of protein expression and characterization, as well as a deoxyribonucleic acid (DNA)-laddering assay, were used to detect cell apoptosis. RESULTS: Our results documented that 100% anatase TiO2 nanoparticles (110-130 nm) exhibited significantly higher cytotoxicity in the highly malignant MDA-MB-468 cancer cells than anatase- rutile mixtures (75%/25%) with the same size. On the contrary, MCF-7 cells (characterized by low invasive properties) were not considerably affected. Exposure of MDA-MB-468 cells to pure anatase nanoparticles or anatase-rutile mixtures for 48 hours resulted in increased proapoptotic Bax expression, caspase-mediated poly(adenosine diphosphate ribose) polymerase (PARP) cleavage, DNA fragmentation, and programmed cell death/apoptosis. CONCLUSION: The obtained results indicated that pure anatase TiO2 nanoparticles exhibit superior cytotoxic effects compared to anatase-rutile mixtures of the same size. The molecular mechanism of TiO2 nanoparticle cytotoxicity involved increased Bax expression and caspase-mediated PARP inactivation, thus resulting in DNA fragmentation and cell apoptosis.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Neoplasias de la Mama/metabolismo , Nanoestructuras/química , Titanio/farmacología , Antineoplásicos/química , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Titanio/química , Rayos UltravioletaRESUMEN
BACKGROUND: Renal podocytes form the main filtration barrier possessing a unique phenotype maintained by proteins including podocalyxin and nephrin, the expression of which is suppressed in pathological conditions. We used an in vitro model of human glomerular epithelial cells (HGEC) to investigate the role of high glucose in dysregulating the podocytic epithelial phenotype and determined the time needed for this change to occur. RESULTS: In our in vitro podocyte system changes indicating podocyte dedifferentiation in the prolonged presence of high glucose included loss of podocalyxin, nephrin and CD10/CALLA concomitant with upregulation of mesenchymal vimentin. Our study demonstrates for the first time that podocyte-specific markers undergo changes of expression at different time intervals, since glucose-mediated podocalyxin downregulation is a progressive process that precedes downregulation of nephrin expression. Finally we demonstrate that high glucose permanently impaired WT1 binding to the podocalyxin gene promoter region but did not affect WT1 binding on the nephrin gene promoter region. CONCLUSION: The presence of high glucose induced a phenotypic conversion of podocytes resembling partial dedifferentiation. Our study demonstrates that dysregulation of the normal podocytic phenotype is an event differentially affecting the expression of function-specific podocytic markers, exhibiting downregulation of the epithelial marker CD10/CALLA and PC first, followed by stably downregulated nephrin. Furthermore, it is herein suggested that WT1 may not be directly involved with upregulation of previously reduced PC and nephrin expression.