The Maillard Reaction Product Nε-Carboxymethyl-L-Lysine Induces

Sinclair, D. A.; Smith-Sonneborn, J.; Snow, E. T.; Spear, L.; Stevenson, D. E.; Thomas, Y.; Tubiana, M.; Williams, G. M.; Mattson, M. P. Biological st...
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Chapter 7

The Maillard Reaction Product Nε-Carboxymethyl-L-Lysine Induces Heat Shock Proteins 72 and 90α via RAGE Interaction in HEK-293 Cells Sebastian Foth,1 Ann-Katrin Holik,2 and Veronika Somoza1,2,* 1German

Research Center for Food Chemistry, Lise-Meitner-Straße 34, Freising, Germany 2Department for Nutritional and Physiological Chemistry, Faculty of Chemistry, University of Vienna, Althanstraße 14, 1090 Vienna, Austria *E-mail: [email protected]

Thermal treatment of foods leads to browning through the generation of Maillard reaction products (MRPs). In addition to their generation in vivo, ingestion of foods high in MRPs may contribute to an accumulation of glycation products in tissues, named advanced glycation endproducts (AGEs). Increased plasma and tissue concentrations of AGEs have been associated with aging processes, and the progression of several diseases such as diabetes mellitus and Alzheimer’s disease. However, in non-pathological conditions, high AGE levels are not observed, indicating cellular mechanisms counteracting AGE accumulation. In this study, we investigated the effect of Nε-Carboxymethyl-L-lysine (CML), a well-characterized product of the Maillard reaction, on the protein expression of heat shock proteins 72 and 90α in HEK-293 cells and HEK-293 cells expressing only the extracellular domain of the receptor for AGEs (RAGE). In HEK-293 cells expressing full length RAGE, CML treatment resulted in an increase of heat shock protein 72 (Hsp72) and heat shock protein 90α (Hsp90α) expression in contrast to cells lacking the receptor’s cytosolic domain, indicating a RAGE-mediated mechanism. Furthermore, in HEK-293 cells exposed to repeated mild heat shocks (RMHS), high protein levels of Hsp72 and Hsp90α were © 2016 American Chemical Society Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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associated with protective effects against the accumulation of CML and its formation from glyoxal. In cells treated with RMHS after CML exposure, a reduction of the cellular CML concentration to 91.8 ± 0.7% in relation to CML treatment only (set to 100%) was observed. Inversion of the treatment order, i.e. RMHS prior to CML treatment, resulted in an even greater reduction of the cellular CML concentration to 75.2 ± 6.1% in relation to CML treatment only. Therefore, HSPs might counteract CML accumulation, and thereby help to prevent increasing CML tissue concentrations in physiological states.

Introduction Advanced glycation endproducts (AGEs) are formed non-enzymatically by the reaction of amino acids and reducing carbohydrates during thermal treatment in processing of foods. In addition, formation in vivo (1) and accumulation in tissues with aging contribute to an increase in brown colour and fluorescence (2). The AGE Nε-carboxymethyl-L-lysine (CML), a lysine derivative and major AGE, has been detected in dairy products and numerous other foods (3, 4). CML has been shown to form under autoxidizing conditions from glucose via the key intermediate glyoxal (5). Investigations into the identification of key browning precursors, showed glyoxal to be formed at the beginning of thermal processing independently of the carbohydrate moiety (6). Ingestion of these browning indicators may influence the progression of pathological conditions of degenerative diseases as high tissue levels of CML have been described in Alzheimer’s disease (7), diabetic retinopathy (8), and diabetic nephropathy (9). Although no active transephitelial transport has been described for free AGEs so far (10), studies on Caco-2 monolayers indicated some dietary dipeptide-bound AGEs may be taken up by peptide transporter PEPT1, as shown for pyrraline dipeptides by Hellwig et al. (11, 12). Furthermore, several studies pointed to gastrointestinal resorption of dietary AGEs to some extend (13–15). AGEs likely provoke their adverse biological activity by interaction with various plasma membrane receptors, the best investigated being the interaction with the receptor for advanced glycation endproducts (RAGE) (16, 17). Activation of RAGE has been demonstrated to influence the concentration of reactive oxygen species (ROS) by activation of NADPH oxidase (18, 19) as well as activation of superoxide dismutase (SOD) (20). In addition, interaction of AGEs with RAGE has been demonstrated to result in induction of signal transduction pathways involved in a pro-inflammatory NF-kB response and the release of pro-inflammatory cytokines (16). Although free CML has been shown 82 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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to not stably bind to RAGE and thus needs to be part of a peptide structure to become a ligand (21–23), several studies also reported RAGE-related effects after incubation with free CML. Zill et al. reported an activation of p42/44 mitogen-activated protein (MAP) by CML as well as CML-enriched casein in Caco-2 cells (24). A RAGE-mediated activation of MAP kinases by free CML and casein-CML was further confirmed in HEK-293 cells, either expressing full length RAGE or RAGE lacking its cytosolic domain (25). Interaction of AGEs with RAGE may enhance further modification of proteins and may thereby contribute to subsequent intracellular accumulation of modified proteins characteristic of cellular aging and progression of several diseases. However, under physiological conditions, high CML levels are not observed, strongly indicating distinct cellular mechanisms may counteract AGE accumulation. Induction of hormesis, a phenomenon in which low levels of stress lead to functional improvements of cells, tissues and organisms, has been shown after repeated mild heat shocks (RMHS) at 41 °C (26–28). RMHS have been demonstrated to reduce age-related alternations in cell morphology, increase the replicative lifespan as well as the antioxidative defense of human fibroblasts, and keratinocytes (28–31). Similarly, a positive influence on AGE levels was observed after RMHS. Verbeke et al. reported 48 h incubation of human skin fibroblasts with 0.1 mM or 1 mM glyoxal to increase CML concentration 4- or 11-fold in untreated control cells while this increase was not observed in cells having received RMHS treatments 8-times (32). Furthermore, Verbeke et al. conducted experiments on human skin fibroblasts, showing consistent CML-rich protein levels up to a lifespan of 75% which then increased 3-fold in untreated control cells having completed a lifespan of over 90%. However, similar to the experiments with 8-times RMHS treatment and incubation with glyoxal, this increase was not observed in the RMHS group. In this study, the authors reported increased protein levels of a major inducible heat shock protein (HSP), HSP70, after RMHS in addition to an age-related rise in HSP70 protein levels in both control and RMHS cells (33). Heat shock proteins, expressed upon RMHS (29), have been associated with the refolding of denatured proteins, thus preventing protein aggregations from causing cellular damage (34, 35). A stable higher HSP expression level is believed to be involved in these anti-aging effects (29), in addition to increased proteasomal activity, which has been shown to be instrumental in the degradation of misfolded proteins (36, 37). However, HSPs and proteasomes are located intracellularly and little is known about the uptake of CML after interaction with the transmembrane receptor RAGE and the protective cellular systems counteracting CML accumulation. In this chapter, the effects of RMHS and thereby induced high expression levels of Hsp70 and Hsp90α on reduced CML accumulation in the human embryonic kidney cell line HEK-293 will be discussed. The involvement of RAGE was investigated using stably transfected cells expressing either full-length RAGE or a C-terminally truncated version of the receptor lacking the cytosolic domain. Furthermore, the uptake of free and casein-linked CML was quantified in both cell lines. 83 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Materials and Methods Materials All reagents were used in the highest purity available. CML was purchased from the PolyPeptide Group (PolyPeptide Group, Strasbourg, France). Casein-CML was prepared as described previously (38), resulting in a 72% modification of lysine. CasCML was fluorescence-labeled with DY-505 (Dyomics, Jena, Germany).

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Cell Culture Experiments were performed using two transfected cell-lines derived from the human embryonic kidney cells HEK-293: HEK-293 full length RAGE (FL) and HEK-293 δ-cyto RAGE (DC) cells expressing RAGE without its cytosolic domain (25). All cells were cultured to confluence under standard conditions (37 °C, 5% CO2 in a humidified incubator) in Dulbecco’s Modified Eagle medium supplemented with 10% fetal bovine serum, 2% L-glutamine, 2% penicillin/streptomycin and 0.2% geneticin for the selection of stably transfected cells. Cells were incubated with 1.1 mM (0.23 g/L) CML, 3.7 g/L CML-enriched casein (39) (CasCML; corresponding to 1.1 mM CML) or – to assess CML formation from glyoxal (32) - with glyoxal solutions of 2 mM or 4 mM for 10 min immediately after the last heat shock or prior to the first heat shock. Negative effects on cell viability of any of the treatments were excluded using the trypan blue exclusion assay. Quantification of Cellular CML-Concentrations by Stable Isotope Dilution Analysis (SIDA) HEK-293 FL and HEK-293 DC cells were incubated with free CML and CasCML before lysis with RIPA Buffer (Sigma, Steinheim, Germany). The lysate was reduced using NaBH4 and isotope-labeled internal standards of 100.7 nmol 13C6,15N2-lysine and 0.337 nmol 13C2-CML as well as 100 µL amino acid carrier (1 mg Ala, Leu, Val, Ser) were added to each standard and sample before acid hydrolysis at 110 °C for 24 h. The hydrolyzed samples were dried under nitrogen, dissolved in 1% trifluoroacetic acid and purified by solid phase extraction (SEP-PAK 18-C cartridges, Waters, Hertfordshire, UK) prior to derivatization with trifluoroacetic anhydride. The CML content was measured by GC-MS (6890 N Network GC system, Agilent Technologies, Waldbronn, Germany; column: J&W DB-5 30 m x 0.25 mm i.d., 0.25 µm film thickness; 1.5 mL He/min; temperature program: 90-140 °C 5°C/min ramp, 140-200 °C 20 °C/min ramp, 200-300 °C 10 °C/min ramp, 300 °C for 8 min) using an electron impact mass spectrometer (5973 Network, Agilent Technologies) set to an ionization energy of 70 eV. Isobutane was used as reactant gas and mass traces of m/z 320 (lysine), m/z 328 (13C6,15N2-lysine), m/z 392 (CML) and m/z 394 (13C2-CML) were recorded. The analyzed CML content was normalized to the determined lysine concentration (40). 84 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Confocal Microscopy

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For microscopic imaging, 1x103/well HEK-293 FL or HEK-293 DC cells were seeded into 96-well plates and cultivated over night. Thirty minutes prior to the analysis, culture media was replaced with serum-free media containing Hoechst 33342 (1 g/mL, Invitrogen, Darmstadt, Germany) and LysoTracker Red DND-99 (50 nM, Invitrogen). After 25 min, DY-505-CasCML (8 µg/mL) was added. The cells were washed with serum-free media three-times prior to confocal microscopy (BD Biosciences, Franklin Lakes, NJ) using a 40x objective (Olympus, Hamburg, Germany).

Protein Expression of HSPs Cells were exposed to different incubations prior to lysis and protein expression of Hsp72 and Hsp90α was analyzed by Western blot. Samples containing 15 µg of protein were mixed with loading buffer (New England Biolabs, Frankfurt/Main, Germany), heated to 95 °C for 5 min, spinned down and cooled on ice prior to SDS-PAGE. After electrophoresis, proteins were transferred to a methanol activated polyvinylidene difluoride membrane employing tank blotting (Mini-Trans-Blot, BioRad, Munich, Germany). Membranes were blocked over night, washed with Tris-bufferd saline – Tween buffer (TBS-T) and incubated with primary antibody for 1 h. After another TBS-T washing step, the membranes were incubated with secondary antibody for 1.5 h. After incubation with the secondary antibody, the membranes were washed with TBS-T buffer and the luminescence was measured for 20 min after addition of LumiGlow reagent (Stressgen, Ann Arbor, MI) using a Kodak Image Station 2000R. The signals were quantified with Kodak 1D software (version 3.6, Kodak, Stuttgart, Germany) and normalized to the signal recorded for α-tubulin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primary antibodies were purchased from assay designs/Stressgen (Lausen, Switzerland; Hsp90α, rabbit, polyclonal, used in 1:1000 dilution), Pineda antibody service (Berlin, Germany; Hsp72, rabbit, monoclonal, used in 1:2000 dilution) and Santa Cruz (Heidelberg, Germany; α-tubulin mouse, monoclonal, used in 1:1000 dilution). The secondary antibodies were obtained from Cell Signaling (Leiden, the Netherlands; HRP-labeled anti-rabbit, goat, polyclonal and HRP-labeled anti-mouse, goat, polyclonal, both used in 1:1000 dilutions).

Repeated Mild Heat Shock Treatment A mild heat shock of 41 °C was applied to HEK-293 FL cells either once for time periods of 2 min to 2 h or for 10 min repeated 1-, 5- or 10-times within two days. Individual treatments are specified in the results section. The expression of HSPs was evaluated by Western blot as described above (Figure 1). 85 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 1. Western blot of Hsp90α, Hsp72 and GAPDH. Lanes from left to right: 10x heat treatment (1), 5x heat treatment (2), 1x heat treatment (3), control (4) Statistics In bar diagrams, mean values and SEM of three to eight replicates are shown, each consisting of at least three independent biological replicates. Outliers were excluded using the Nalimov outlier test. All data are shown in relation to nontreated control cells (set to 100%), denominated as T/C (treated vs. control) in the figures. Statistical significances between control vs. treated cells were calculated by Student’s t-test, and a p ≤ 0.05 was considered as statistically significant (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001). Time dependent effects were tested by ANOVA followed by Dunn’s post hoc test. Statistical analyses were carried out using either Microsoft Excel 2003 (Microsoft, Redmond, WA) or SigmaStat (Systat Software, Erkrath, Germany).

Results Cellular Uptake of CML The cellular uptake of CML, either free or protein-bound, was demonstrated by a combination of two independent methods: First, the concentration of cellular CML after incubation with free CML or CasCML was determined by stable isotope dilution analysis (SIDA). The intracellular CML-levels of untreated HEK-293 FL (0.033 mmol/mol lysine) and HEK-293 DC cells (0.027 mmol/mol lysine) differed only slightly (Table 1), though statistically significant (p < 0.01). This may be explained by differential uptake of minute concentrations of CML in the cell culture medium used, originating from the reaction of amino acids and glucose present in Dulbecco’s Modified Eagle medium. Incubation with CML increased the cellular CML-content significantly in both, HEK-293 FL (0.189 mmol/mol lysine, p < 0.001) and HEK-293 DC cells (0.108 mmol/mol lysine, p < 0.001). After incubation with CasCML, even higher concentrations of CML were measured in HEK-293 DC (0.197 mmol/mol lysine , p < 0.001) and HEK-293 FL (0.292 mmol/mol lysine, p < 0.001), resulting in an increase of over 750% in the latter. In all cases, CML-levels in HEK-293 FL were significantly 86 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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higher than in HEK-293 DC (p < 0.01). Although cells were thoroughly washed after incubation, CML might have attached to extracellular structures on the cell surface (like RAGE) and been carried into the lysate. Thus, confocal fluorescence microscopy was applied as a second means of determination of the cellular CML uptake to verify the CML-concentrations measured by SIDA in fact originated from within the cells. Figure 2 shows an overlay of the bright field and three confocal fluorescence microscopic images. Nuclei (shown in blue) and lysosomes (red) are clearly visible in all cells. Uptake of DY-505-CasCML is shown by the green fluorescence signal of the DY-505 label. This indicates uptake of DY-505-CasCML by HEK-293 FL cells. Also, the overlay shows yellow signals due to an overlap of green (DY505-CasCML) and red (lysosome staining) indicating a translocation of the CMLenriched protein to the lysosomes. In HEK-293 DC cells, only little CML-enriched protein was taken up as no green fluorescence of the DY-505 tag was observed. However, CasCML was taken up to some extend as indicated by the yellow overlay signal, originating from translocation of CasCML to the lysosomes as seen in HEK-293 FL cells.

Effect of CML and Casein-CML on the Protein Expression of HSPs Increased protein expression of two major inducible Hsps, Hsp72 and Hsp90α, by free CML and protein-bound CML in HEK-293 cells was determined by Western blot. The results are shown in Table 2.

Table 1. Concentrations of CML in HEK-293 full length RAGE cells or HEK-293 δ-cyto RAGE cells after 1.1 mM CML/CasCML (10 min) calculated as mmol CML/mol lysine; data are displayed as average ± SEM; statistics: Student’s t-test: each substance vs. control; b= p