Original ContributionCathepsins D and L reduce the toxicity of advanced glycation end products
Graphical abstract
Highlights
► Increase of amount and activity of cathepsin D/L after AGE treatment in macrophages. ► Reduced viability in cathepsin D and L deficient cells after AGE treatment. ► Enhanced reactive oxygen species in cathepsin D/L deficiency after AGE treatment.
Section snippets
Materials
RPMI medium 1640, Dulbecco's modified Eagle's medium (DMEM), penicillin (10,000 E)/streptomycin (10,000μg/ml), and fetal bovine serum were purchased from Biochrom (Berlin, Germany). LysoTracker Blue DND-22 was purchased from Molecular Probes Invitrogen (Karlsruhe, Germany). Pepstatin A was bought from Enzo Life Sciences (Loerrach, Germany). d-[1-14C]Glucose (250μCi) was purchased from PerkinElmer (Rodgau, Germany). Other chemicals were of the best grade available from Sigma–Aldrich
Uptake of advanced glycation end products in RAW 264.7 macrophages
As described above, results from in vivo and in vitro studies demonstrate a role for AGEs in modulation of immune response, especially during aging. To further explore the effects of AGEs on the proteolytic mechanisms, we first investigated the uptake of these aggregates into the macrophage cell line RAW 264.7 with different methods. We prepared radioactively labeled glucose–AGEs as described under Materials and methods and incubated RAW cells with 25mM [14C]glucose-modified albumin for 1, 24,
Discussion
AGEs are formed at high rates in the metabolic syndrome and in many other diseases [5], [6], [7], [8], [9]. We produced five different AGE types, because these molecules constitute a broad family of glycated proteins, showing different responses [42]. AGEs produce oxygen free radicals by binding to receptors or by their internalization by inflammatory cells. So recently Guimaraes et al. showed that AGEs induce the production of ROS by the activation of NADPH oxidase [43]. In glyoxal-modified
Acknowledgment
This work was supported by the Deutsche Forschungsgemeinschaft and the Bundesministerium für Bildung und Forschung.
References (73)
Advanced glycation endproducts—role in pathology of diabetic complications
Diabetes Res. Clin. Pract.
(2005)Glycation in diabetic neuropathy: characteristics, consequences, causes, and therapeutic options
Int. Rev. Neurobiol.
(2002)- et al.
Advanced glycation end products in Alzheimer's disease and other neurodegenerative diseases
Am. J. Pathol.
(1998) - et al.
Molecular mechanisms of late endosome morphology, identity and sorting
Curr. Opin. Cell Biol.
(2006) - et al.
Clarifying lysosomal storage diseases
Trends Neurosci.
(2011) - et al.
Proteases and bone remodelling
Cytokine Growth Factor Rev.
(2009) - et al.
Lysosomal cysteine proteases: more than scavengers
Biochim. Biophys. Acta
(2000) - et al.
Dual contrasting roles of cysteine cathepsins in cancer progression: apoptosis versus tumour invasion
Biochimie
(2008) - et al.
Degradation of glycated bovine serum albumin in microglial cells
Free Radic. Biol. Med.
(2006) - et al.
Non-enzymatic posttranslational modifications of bovine serum albumin by oxo-compounds investigated by chromatographic and electrophoretic methods
J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
(2005)