Original Contribution
Cathepsins D and L reduce the toxicity of advanced glycation end products

https://doi.org/10.1016/j.freeradbiomed.2011.12.021Get rights and content

Abstract

Advanced glycation end product-modified proteins are known for accumulating during aging and in several pathological conditions such as diabetes, renal failure, and neurodegenerative disorders. There is little information about the intracellular fate of endocytosed advanced glycation end products (AGEs) and their influence on proteolytic systems. However, it is known that the lysosomal system is impaired during aging. Therefore, undegraded material may accumulate and play a considerable role in the development of diverse diseases. To investigate if AGEs can be degraded and to test whether they accumulate because of impaired lysosomal proteases we studied the effects of advanced glycation end products on the endosomal–lysosomal system. Five different types of AGEs were generated by bovine serum albumin incubation with glyoxal, methylglyoxal, glucose, fructose, and ribose. The first experiments revealed the uptake of AGEs by the macrophage cell line RAW 264.7. Further investigations demonstrated an increase in cathepsin D and L activity and an increase in mature cathepsins D and L. Increased activities were accompanied by the presence of more lysosomes, measured by staining with LysoTracker blue. To specify the roles of cathepsins D and L we used knockout cells to test the roles of both cathepsins on the toxicity of advanced glycation end products. In summary we conclude that both cathepsins are required for a reduction in advanced glycation end product-induced cytotoxicity.

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.

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