Carboxymethyl)lysine Accumulation Research Articles

Impact of Extrusion Parameters on the Formation of Nε-(Carboxymethyl)lysine, Nε-(Carboxyethyl)lysine and Acrylamide in Plant-Based Meat Analogues.

To investigate the impact of extrusion parameters on the formation of Nε-(carboxymethyl)lysine (CML), Nε-(carboxyethyl)lysine (CEL) and acrylamide in plant-based meat analogues (PBMAs), the content changes and the correlations of compounds related to their formation were studied. The extrusion promoted CML, CEL and acrylamide formation, with more CEL being formed than CML. Variations in the moisture level and barrel temperature exerted a greater influence on the CML, CEL, acrylamide and α-dicarbonyl compounds than the screw speed and the feed rate. An increase in the moisture content led to a decrease in the CEL content, whereas it enhanced CML formation. The impact of moisture on acrylamide formation varied depending on whether low- or high-moisture extrusion was applied. Elevated temperatures promoted the accumulation of CEL, methylglyoxal and 2,3-butanedione while diminishing the accumulation of CML, acrylamide, glyoxal and 3-deoxyglucosone. CML and CEL were positively correlated with glyoxal and methylglyoxal, respectively. CEL and methylglyoxal were negatively correlated with protein and water content, whereas CML, glyoxal and 3-deoxyglucosone displayed positive correlations. In summary, higher moisture levels and feed rates and lower screw speeds and barrel temperatures are advantageous for producing PBMAs with lower CEL and total advanced glycation end-products contents, while lower or higher moisture contents, a lower feed rate and a higher barrel temperature are beneficial to reducing the acrylamide content.

Lindera obtusiloba Blume Alleviates Non-Alcoholic Fatty Liver Disease Promoted by Nε-(carboxymethyl)lysine.

Non-alcoholic fatty liver disease (NAFLD) is a major issue because it is closely associated with metabolic diseases. Advanced glycation end products (AGEs) are implicated as risk factors for steatosis during NAFLD progression. AGEs influence NAFLD progression through a receptor-independent pathway involving AGE cross-link formation and a receptor-dependent pathway that binds to receptors like receptors for advanced glycation end products (RAGE). The objectives of this study are to examine the effect of Lindera obtusiloba Blume (LO) on NAFLD promoted by Nε-(carboxymethyl)lysine (CML), one of the most common dietary AGEs. The anti-glycation effects of LO were evaluated by inhibiting the AGEs formation and AGEs-collagen cross-links breaking. The efficacy of LO against NAFLD promoted by CML was assessed using both in vitro and in vivo models. NAFLD was induced in mice by feeding a high-fat diet and orally administering CML over a period of 12 weeks, and the effects of LO on lipid metabolism and its regulatory mechanisms were investigated. LO showed the effect of inhibited AGEs formation and breakage, and collagen cross-linking. Fed a high-fat diet with administered CML by gavage, LO administration resulted in a reduction in body weight, fat mass, serum triglycerides, total cholesterol, and low-density lipoprotein cholesterol levels. LO reduced hepatic CML accumulation and RAGE expression in mice fed a high-fat diet and orally administered CML. LO alleviated hepatic steatosis accompanied by lipid accumulation and histological damage by suppressing the expression of sterol regulatory element-binding protein 1c, carbohydrate response element binding protein, fatty acid synthase, stearoyl-CoA desaturase1, tumor necrosis factor-α, and interleukin-1β. LO alleviated the MAPK/NF-κB expression by attenuating CML and RAGE expression. Taken together, our results demonstrate that LO alleviates the progression of NAFLD by lowering the levels of AGEs by downregulating CML/RAGE expression.

Protective effect of Inonotus obliquus polysaccharide on MGO-induced nonenzymatic glycation fibroblasts

BackgroundThe nonenzymatic glycation of fibroblasts causes functional downregulation and behavioral disorders in the skin. MethodsTo investigate the effect of Inonotus obliquus on the nonenzymatic glycation of skin, we examined the inhibition of advanced glycation end products (AGEs) using four extraction methods: n-butanol, ethyl acetate, n-hexane and aqueous alcohol precipitation. The physical properties and chemical structure of the most effective, purified, crude I. obliquus polysaccharide (IOP) were examined. The effects of IOP on carboxymethyl lysine (CML) accumulation, inflammatory factor release, reactive oxygen species (ROS) production, key extracellular matrix (ECM) protein (MMP 1, 2 and 9; FN-1, LM-5 and COL-1) mRNA expression, and cell survival, migration and adhesion were also examined via cellular assays. ResultsIOP is a polysaccharide with a molecular weight (Mw) of 2.396 × 104 (±6.626%) that is composed mainly of glucose, galactose, xylose, mannose and arabinose (29.094:21.705:14.857:9.375:7.709). In addition, a cellular antiglycation assay showed that IOP, which can promote ECM formation by inhibiting the accumulation of CML, inhibiting the release of inflammatory factors (IL-1β, IL-6, and TNF-α), inhibiting the production of reactive oxygen species (ROS), inhibiting the expression of matrix metalloproteinases (MMP-1\\-2\\-9), promoting the synthesis of ECMs (COL1, FN1, and LM5), and improving cellular dysfunction, had strong antiglycation activity at concentrations in the range of 6–24 μg/mL. ConclusionIOP effectively reduced the levels of inflammatory factors and reactive oxygen species produced by AGEs, further preventing the impairment of cell behavior (decreased migration and reduced cell adhesion) and preventing the downregulation of the expression of key extracellular matrix proteins induced by AGEs. The results indicate the potential application of IOP as an AGE inhibitor in skin care.

Rapid formation of Nε-(carboxymethyl)lysine (CML) from ribose depends on glyoxal production by oxidation

Ribose undergoes auto-oxidation and oxidative cleavage between C-2 and C-3 to generate GO and enhance CML accumulation.

Effect of edible oil type on the formation of protein-bound Nε-(carboxymethyl)lysine in roasted pork patties

Protein-bound Nε-(carboxymethyl)lysine (CML), an advanced glycation end product within meat products, poses a potential health risk to humans. The objective of this study was to explore the impact of various edible oils on the formation of protein-bound CML in roasted pork patties. Eleven commercially edible oils including lard oil, corn oil, palm oil, olive oil, flaxseed oil, blended oil, camellia oil, walnut oil, soybean oil, peanut oil, and colza oil were added to pork tenderloin mince, respectively, at a proportion of 4 % to prepare raw pork patties. The protein-bound CML contents in the pork patties were determined by HPLC-MS/MS before and after roasting at 200 °C for 20 min. The results indicated that walnut oil, flaxseed oil, colza oil, olive oil, lard oil, corn oil, blended oil, and palm oil significantly reduced the accumulation of protein-bound CML in pork patties, of which the inhibition rate was in the 24.43 %–37.96 % range. Moreover, the addition of edible oil contributed to a marginal reduction in the loss of lysine. Meanwhile, glyoxal contents in pork patties were reduced by 16.72 %–43.21 % after roasting. Other than blend oil, all the other edible oils restrained protein oxidation in pork patties to varying degrees (between 20.16 % and 61.26 %). In addition, camellia oil, walnut oil, and flaxseed oil increased TBARS values of pork patties by 2.2–8.6 times when compared to the CON group. After analyzing the fatty acid compositions of eleven edible oils, five main fatty acids (palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid) were selected to establish Myofibrillar protein-Glucose-fatty acids systems to simulate the roasting process. The results showed that palmitic acid, oleic acid, linoleic acid, and linolenic acid obviously mitigated the formation of myofibrillar protein-bound CML, exhibiting suppression rates ranging from 10.38 % to 40.32 %. In conclusion, the addition of specific edible oil may curb protein-bound CML production in roasted pork patty by restraining protein or lipid oxidation, reducing lysine loss, and suppressing glyoxal production, which may be attributed to the fatty acid compositions of edible oils. This finding provides valuable guidance for the selection of healthy roasting oils in the thermal processing of meat products.

Accumulation of fluorescent advanced glycation end products and carboxymethyl-lysine in human cortical and trabecular bone

Chemical crosslinks known as advanced glycation end-products (AGEs) are associated with increased bone fracture risk and deteriorated bone mechanical properties. However, measurement of bone AGEs via ex vivo and in vitro methods has been limited to quantification of bulk fluorescent AGEs (fAGEs) and pentosidine only, which is a crosslinking fluorescent AGE. However, a non-crosslinking and non-fluorescent AGE such as carboxymethyl-lysine (CML) is found to be 40–100 times higher in quantity than pentosidine, but only one previous study has reported it in cortical bone, and one study reported it in trabecular bone. In our study, we wanted to investigate if accumulation of CML differs in cortical and trabecular compartments and if they are more strongly associated with bone mechanical properties than with fAGEs. We hypothesized that CML and fAGEs level would be higher in the trabecular compartment and show negative correlations to mechanical properties in cortical and trabecular bone. We obtained human cadaveric cortical and trabecular bone specimens, induced the formation of AGEs via the established in vitro ribosylation method, imaged specimens by microcomputed tomography to assess specimen geometry and microarchitecture, and mechanically tested cortical specimens by cyclic reference point indentation and fracture toughness tests and trabecular specimens by compression tests, followed by measurement of fAGEs and CML. fAGEs were 22 % higher in cortical bone (687 ± 44.8 ng Q/mg collagen) compared to trabecular bone (859 ± 317.1 ng Q/mg collagen), whereas CML levels were found to be 148 % higher in trabecular bone (6189.9 ± 866 ng/mg of protein) compared to cortical bone (924.6 ± 576.3 ng/mg of protein). Pooling the specimens from both the control and ribose groups, Spearman correlation analysis indicated that CML levels, but not fAGEs, are moderately associated with cortical porosity (r = +0.505, p ≤ 0.05) and mechanical properties such indentation depth (r = +0.460, p ≤ 0.05), total indentation depth (r = +0.440, p ≤ 0.05), and average energy dissipated (r = +0.465, p ≤ 0.05) in cortical bone. fAGEs showed a trend towards negative association with crack propagation toughness in cortical bone (r = −0.365, p = 0.055). No significant correlations were observed between CML and microarchitecture or mechanical properties in trabecular bone. CML levels were also associated with fAGEs in cortical bone (r = +0.596, p ≤ 0.05) but not in trabecular bone. Our preliminary findings indicate that CML, a non-crosslinking AGE, may affect bone material and mechanical properties differently than bulk fluorescent AGEs, given the higher accumulation of CML in each bone compartment. This study provides direction to future studies to quantify crosslinking and non-crosslinking AGEs separately as their effect on material and mechanical properties may be different and it would help identify better biomarkers for bone strength prediction.

Liraglutide treatment attenuates dysfunction-related changes in the cardiac and cerebral microvasculature in streptozotocin-induced diabetic rats

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Novo Nordisk Background Diabetes mellitus (DM) induces cardiac and cerebral microvascular dysfunction via increased glycation, oxidative stress and endothelial activation. Liraglutide, a glucagon-like peptide-1 receptor analogue, inhibited the reactive oxygen species producing enzyme NOX2 and adhesion molecules in isolated endothelial cells. Here we have studied how Liraglutide affects advanced glycation, NOX expression and inflammatory status of the cardiac and cerebral microvasculature in vivo in diabetic rats. Methods DM was induced in Sprague Dawley rats (n=15) via intraperitoneal streptozotocin (STZ) injection (60 mg/kg body weight). 10 control rats remained non-diabetic. From day 9 post-STZ injection, Liraglutide (200 µg/kg bodyweight; n=7) or vehicle (n=8) was injected subcutaneously daily until termination on day 29. The advanced glycation end-product N-ε-(carboxymethyl)lysine (CML), NOX2, NOX4, ICAM-1 and VCAM-1 were subsequently analysed using immunohistochemistry and quantified to compare Liraglutide treatment to placebo. Results DM increased CML, NOX2, ICAM-1 and VCAM-1 in the intramyocardial vasculature, although not significant for NOX2. NOX4 was not significantly increased in the ventricles and absent in the atria. In the cerebral vasculature, CML, NOX2 and NOX4 were significantly increased, but not ICAM-1 and VCAM-1. Liraglutide significantly reduced the DM-associated CML accumulation and expression of NOX2, ICAM-1 and VCAM-1 in the intramyocardial, and CML, NOX2 and NOX4 in the cerebral vasculature. Liraglutide had these effects without changing blood glucose levels or body weight. Conclusions Our study implies that Liraglutide protects the cardiac and cerebral microvasculature against diabetes-induced dysfunction, independent of a blood glucose lowering effect in a type 1 diabetes rat model.

Controlled Formation of Carboxymethyllysine in Bone Matrix through Designed Glycation Reaction.

ABSTRACTIt has been a challenge to establish a link between specific advanced glycation end products (AGEs) as causal agents of different pathologies and age‐related diseases, primarily because of the lack of suitable in vitro experimental strategies facilitating increased formation of a specific AGE, here carboxymethyllysine (CML), over other AGEs under controlled conditions. CML is of considerable importance to various oxidative stress–related diseases, because in vivo formation of this AGE is connected with cellular oxidative/carbonyl metabolism. The mechanistic implications of CML accumulation in bone remain to be elucidated. To facilitate such studies, we developed a new in vitro strategy that allows preferential generation of CML in bone matrix over other AGEs. Using bone samples from human donors of different age (young, middle‐age, and elderly), we show successful in vitro generation of the desired levels of CML and show that they mimic those observed in vivo in several bone disorders. Formation of such physiologically relevant CML levels was achieved by selecting two oxidative/carbonyl stress compounds naturally produced in the human body, glyoxal and glyoxylic acid. Kinetic studies using the two compounds revealed differences not only between their reaction rates but also in the progression and enhanced formation of CML over other AGEs (measured by their collective fluorescence as fluorescent AGEs [fAGEs]) Consequently, through the regulation of reaction time, the levels of CML and fAGEs could be controlled and separated. Given that the developed approach does not fully eliminate the formation of other uncharacterized glycation products, this could be considered as the study limitation. We expect that the concepts of our experimental approach can be used to develop diverse strategies facilitating production of the desired levels of selected AGEs in bone and other tissues, and thus, opens new avenues for investigating the role and mechanistic aspects of specific AGEs, here CML, in bone. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Preventive Effects of Aphanothece sacrum on Diabetic Cataracts

Approximately 20% of diabetic patients develop diabetic cataracts. As lens proteins are known to be only slightly metabolized during the lifetime, cataracts are difficult to recover from once they have progressed. Therefore, the daily intake of natural compounds would be an important strategy for the prevention of diabetic cataracts. Aphanothece sacrum Okada (Asa) is a freshwater blue-green algae endemic to Japan. It has been eaten since the Edo period in Kyushu. In this study, the inhibitory effects of Asa on the pathogenesis of diabetic cataracts were evaluated. Furthermore, the inhibitory effects of Asa on the formation of Nε-(carboxymethyl) lysine (CML), an oxidation-dependent advanced glycation end-product, were also measured. After 3-month administration, the CML contents in the lens were measured by liquid chromatography tandem mass spectrometry using an internal standard of CML or lysine. Asa significantly inhibited the progression of cataractogenesis and accumulation of CML in diabetic lens compared with the normal diet group. These results suggested that daily intake of Asa reduces oxidative stress and prevents the pathogenesis of cataracts.

Impairment of glyoxalase-1, an advanced glycation end-product detoxifying enzyme, induced by inflammation in age-related osteoarthritis

BackgroundAccumulation of advanced glycation end-products (AGEs) is involved in age-related osteoarthritis (OA). Glyoxalase (Glo)-1 is the main enzyme involved in the removal of AGE precursors, especially carboxymethyl-lysine (CML). We aimed to investigate the expression of several AGEs and Glo-1 in human OA cartilage and to study chondrocytic Glo-1 regulation by inflammation, mediated by interleukin (IL)-1β.MethodsEx vivo, we quantified AGEs (pentosidine, CML, methylglyoxal-hydroimidazolone-1) in knee cartilage from 30 OA patients. Explants were also incubated with and without IL-1β, and we assessed Glo-1 protein expression and enzymatic activity. In vitro, primary cultured murine chondrocytes were stimulated with increasing concentrations of IL-1β to assess Glo-1 enzymatic activity and expression. To investigate the role of oxidative stress in the IL-1β effect, cells were also treated with inhibitors of mitochondrial oxidative stress or nitric oxide synthase.ResultsEx vivo, only the human cartilage CML content was correlated with patient age (r = 0.78, p = 0.0031). No statistically significant correlation was found between Glo-1 protein expression and enzymatic activity in human cartilage and patient age. We observed that cartilage explant stimulation with IL-1β decreased Glo-1 protein expression and enzymatic activity. In vitro, we observed a dose-dependent decrease in Glo-1 mRNA, protein quantity, and enzymatic activity in response to IL-1β in murine chondrocytes. Inhibitors of oxidative stress blunted this downregulation.ConclusionGlo-1 is impaired by inflammation mediated by IL-1β in chondrocytes through oxidative stress pathways and may explain age-dependent accumulation of the AGE CML in OA cartilage.

Accumulation of carboxymethyl-lysine (CML) in human cortical bone.

Advanced glycation end-products (AGEs) are a category of post translational modification associated with the degradation of the structural properties of multiple different types of tissues. Typically, AGEs are the result of a series of post-translational modification reactions between sugars and proteins through a process known as non-enzymatic glycation (NEG). Increases in the rate of NEG of bone tissue are associated with type 2 diabetes and skeletal fragility. Current methods of assessing NEG and its impact on bone fracture risk involve measurement of pentosidine or total fluorescent AGEs (fAGEs). However, pentosidine represents only a small fraction of possible fAGEs present in bone, and neither pentosidine nor total fAGE measurement accounts for non-fluorescent AGEs, which are known to form in significant amounts in skin and other collagenous tissues. Carboxymethyl-lysine (CML) is a non-fluorescent AGE that is often measured and has been shown to accumulate in tissues such as skin, heart, arteries, and intervertebral disks, but is currently not assessed in bone. Here we show the localization of CML to collagen I using mass spectrometry for the first time in human bone. We then present a new method using demineralization followed by heating and trypsin digestion to measure CML content in human bone and demonstrate that CML in bone is 40-100 times greater than pentosidine (the current most commonly used marker of AGEs in bone). We then establish the viability of CML as a measurable AGE in bone by showing that levels of CML, obtained from bone using this technique, increase with age (p<0.05) and are correlated with previously reported measures of bone toughness. Thus, CML is a viable non-fluorescent AGE target to assess AGE accumulation and fragility in bone. The method developed here to extract and measure CML from human bone could facilitate the development of a new diagnostic assay to evaluate fracture risk and potentially lead to new therapeutic approaches to address bone fragility.

Sleep-disordered breathing is associated with higher carboxymethyllysine level in elderly women but not elderly men in the cardiovascular health study

Context: Carboxymethyl-lysine (CML) results from oxidative stress and has been linked to cardiovascular disease.Objective: The objective of this study is to investigate the association between sleep-disordered breathing (SDB) – a source of oxidative stress – and CML.Materials and methods: About 1002 participants in the Cardiovascular Health Study (CHS) were studied.Results: Women with SDB had significantly higher CML concentration compared with those without SDB (OR = 1.63, 95%CI = 1.03–2.58, p = 0.04). The association was not significant among men.Discussion: SDB was associated with CML concentration among elderly women but not men in the Cardiovascular Health Study.Conclusion: Accumulation of CML may be an adverse health consequence of SDB

Cytoprotective Mechanisms Mediated by Polyphenols from Chilean Native Berries against Free Radical-Induced Damage on AGS Cells.

The prevalence of cytoprotective mechanisms induced by polyphenols such as activation of intracellular antioxidant responses (ICM) and direct free radical scavenging was investigated in native Chilean species of strawberries, raspberries, and currants. Human gastric epithelial cells were co- and preincubated with polyphenolic-enriched extracts (PEEs) from Chilean raspberries (Rubus geoides), strawberries (Fragaria chiloensis ssp. chiloensis f. chiloensis), and currants (Ribes magellanicum) and challenged with peroxyl and hydroxyl radicals. Cellular protection was determined in terms of cell viability, glyoxalase I and glutathione s-transferases activities, and carboxymethyl lysine (CML) and malondialdehyde levels. Our results indicate that cytoprotection induced by ICM was the prevalent mechanism for Rubus geoides and F. chiloensis. This agreed with increased levels of glyoxalase I and glutathione S-transferase activities in cells preincubated with PEEs. ORAC index indicated that F. chiloensis was the most efficient peroxyl radical scavenger. Moreover, ICM mediated by F. chiloensis was effective in protecting cells from CML accumulation in contrast to the protective effects induced by free radical scavenging. Our results indicate that although both polyphenol-mediated mechanisms can exert protective effects, ICM was the most prevalent in AGS cells. These results suggest a potential use of these native berries as functional food.

Aphanothece sacrum (Sur.) Okada Prevents Cataractogenesis in Type 1 Diabetic Mice.

Aphanothece sacrum (Sur.) Okada is a species of cyanobacteria found in Japan. Although it has been used in local cuisine in Kyushu, Japan, for 250 y, little is known about its beneficial effect as food. The daily intake of health beneficial phytochemicals is believed to be useful for preventing lifestyle-related diseases, such as diabetic cataracts. In this study, the inhibitory effect of freeze-dried A. sacrum (Asa) on the formation of diabetic cataracts (DCs) was evaluated. Type 1 diabetes was induced in mice using streptozotocin (STZ). The mice were divided into two groups: one was fed a normal diet (DM-control group) and the other was fed a diet containing 1% Asa (DM-Asa group). During the study, changes in blood glucose levels and the amount of food and water consumed were measured. After 3 mo, the amount of Nε-(carboxymethyl)lysine (CML), an oxidative stress marker, in the lens was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although the blood glucose levels (p=0.91) and food consumption did not significantly change in any group, the oral administration of Asa tended to suppress CML accumulation (p=0.15) and significantly inhibited the progression of cataractogenesis in the diabetic lens compared with that reported for the normal diet (p=0.009). These results suggested that the daily intake of A. sacrum prevents the pathogenesis of cataracts, and indicated that may reduce the number of DC patients.

High-fructose intake as risk factor for neurodegeneration: Key role for carboxy methyllysine accumulation in mice hippocampal neurons

Several studies indicate the involvement of advanced glycation end-products (AGEs) in neurodegenerative diseases. Moreover, the rising consumption of fructose in industrialized countries has been related to cognitive impairment, but the impact of fructose-derived AGEs on hippocampus has never been investigated.The present study aimed to evaluate in the hippocampus of C57Bl/6 mice fed a standard (SD) or a 60% fructose (HFRT) diet for 12weeks the production of the most studied AGEs, carboxy methyllysine (CML), focusing on the role of the glutathione-dependent enzyme glyoxalase (Glo-1), the main AGEs-detoxifying system, in relation to early signs of neuronal impairment.HFRT diet evoked CML accumulation in the cell body of pyramidal neurons, followed by RAGE/NFkB signaling activation. A widespread reactive gliosis and altered mitochondrial respiratory complexes activity have been evidenced in HFRT hippocampi, paralleled by oxidative stress increase due to impaired activity of Nrf2 signaling. In addition, a translocation of Glo-1 from axons toward cell body of pyramidal neurons has been observed in HFRT mice, in relation to CML accumulation. Despite increased expression of dimeric Glo-1, its enzymatic activity was not upregulated in HFRT hippocampi, due to reduced glutathione availability, thus failing to prevent CML accumulation. The prevention of CML production by administration of the specific inhibitor pyridoxamine was able to prevent all the fructose-induced hippocampal alterations. In conclusion, a high-fructose consumption, through CML accumulation and Glo-1 impairment, induces in the hippocampus the same molecular and metabolic alterations observed in early phases of neurodegenerative diseases, and can thus represent a risk factor for their onset.

Salacia chinensis L. extract ameliorates abnormal glucose metabolism and improves the bone strength and accumulation of AGEs in type 1 diabetic rats.

Although extracts of the roots and stems of Salacia chinensis have been used in folk medicines for chronic diseases such as rheumatism, irregular menstruation, asthma and diabetes mellitus, little is known about the mechanism by which Salacia chinensis extract (SCE) ameliorates these diseases. To clarify whether SCE ameliorates the progression of lifestyle-related diseases, the inhibitory effect of SCE on the formation of advanced glycation end products (AGEs) was analyzed in a rat model of streptozotocin-induced diabetes. Although the oral administration of SCE did not ameliorate the diabetes-induced decrease in body weight, it ameliorated the increase in glycoalbumin levels in diabetic rats. An analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) demonstrated that the levels of N(ε)-(carboxymethyl)lysine (CML) were highest in the femurs and that they increased by the induction of diabetes. The administration of SCE also ameliorated the decreased femur strength and the accumulation of CML. Furthermore, when all of the carbohydrates in the chow of diabetic rats were replaced with free glucose, the administration of SCE significantly ameliorated a diabetes-induced increase in glycoalbumin and decrease in serum creatinine level and body weight. This study provides evidence to support that SCE ameliorates diabetes-induced abnormalities by improving the uptake of glucose by various organs.

Protein-Bound Plasma Nε-(Carboxymethyl)lysine Is Inversely Associated With Central Obesity and Inflammation and Significantly Explain a Part of the Central Obesity-Related Increase in Inflammation: The Hoorn and CODAM Studies.

Adipose tissue inflammation contributes to the development of complications, such as insulin resistance and type 2 diabetes mellitus. We previously reported that plasma levels of N(ε)-(carboxymethyl)lysine (CML) were decreased in obese subjects resulting from CML accumulation in adipose tissue and that this CML accumulation plays an important role in adipose tissue inflammation. The objective of this study is to investigate associations between obesity (body mass index, waist circumference, and trunk fat mass), plasma CML (as an inversely correlated marker of CML accumulation in adipose tissue), and low-grade inflammation (LGI) in a large sample of individuals whose weight status ranged from normal to morbid obesity. We studied 1270 individuals of the Cohort on Diabetes and Atherosclerosis Maastricht Study and Hoorn Study, in whom protein-bound CML levels were measured by UPLC-Tandem MS (ultra performance liquid chromatography-tandem mass spectrometry), and 6 inflammatory markers were measured with multiarrays. These inflammatory markers were compiled into an LGI score. Multiple linear regression, adjusted for covariates, showed that (1) waist circumference was inversely associated with protein-bound CML plasma levels (standardized regression coefficient [β]=-0.357 [95% confidence interval: -0.414; -0.301]); (2) protein-bound CML was inversely associated with LGI score (β=-0.073 [-0.130;-0.015]); and (3) the association between waist circumference and LGI (β=0.262 [0.203;0.321]) was attenuated after adjustment for protein-bound CML plasma levels and other potential mediators (to β=0.202 [0.138;0.266]), with CML explaining the greatest portion of the attenuation (≈12%). Further analysis with dual-energy X-ray absorptiometry measures of body composition confirmed a strong inverse association of fat mass preferentially accumulated in the trunk with protein-bound CML plasma levels, significantly explaining ≈21% of the trunk fat-LGI association. Obesity, in particular central obesity, is characterized by greater levels of LGI but by lower levels of circulating CML; the latter significantly explaining a portion of the positive association between central obesity and inflammation.

Involvement of the receptor for advanced glycation end products in liver transplantation

Background and aim. Receptor for advanced glycation end products (RAGE) blockade by a soluble form of RAGE (sRAGE) appears to be protective against hepatocellular death and necrosis after I/R injury. Little is known about the role of the hepatic RAGE, its ligands, and the plasma levels of sRAGE in liver transplantation (LT).Material and methods. This was a prospective study on patients (n = 28) undergoing deceased donor LT. RAGE ligands [the N(epsilon)-carboxy-methyl-lysine (CML) adduct and the high-mobility group box 1 (HMGB1) protein] and sRAGE levels were measured in donors at the time of organ procurement, while in recipients they were tested before surgery (baseline), after graft reperfusion, and on day 1 and 7 posttransplantation. Donors and recipients liver biopsies were collected to assess the transcriptional expression of the full-length RAGE and of its truncated isoform, the endogenous secreted RAGE (esRAGE).Results. At baseline, CML levels were higher in LT recipients than in donors (p = 0.02), decreased immediately after graft reperfusion (p < 0.0001) and returned to baseline values on day 7. Baseline HMGB1 levels (3.8 ± 2.3 ng/mL) increased after graft reperfusion (39.9±18 ng/mL, p < 0.0001), and returned to baseline values within day 1, while circulating sRAGE decreased significantly on day 7 (p < 0.0001). The graft esRAGE mRNA expression was inversely associated with bilirubin on day 7 (β = -0.62, p = 0.005).Conclusions. Early on after LT, there is accumulation of CML and a rapid increase of HMGB1 concurrent with a remarkable decline in circulating sRAGE. The RAGE-ligand axis may also be involved in early graft dysfunction.

Increased renal semicarbazide-sensitive amine oxidase activity and methylglyoxal levels in aristolochic acid-induced nephrotoxicity

AimsAristolochic acid (AA) nephrotoxicity is related to accumulation of methylglyoxal (MGO) and Nε-(carboxymethyl)lysine (CML) in the mouse kidney. We studied the activity of renal semicarbazide-sensitive amine oxidase (SSAO), a key enzyme involved in MGO generation, in AA-treated mice, and investigated nephroprotective effects produced by metformin, a MGO scavenger. MethodsMice were orally administered water or metformin for 15days (12 or 24mgkg−1day−1), and injected AA (5mgkg−1day−1) intraperitoneally for 8days starting on day 8. Renal function was studied, and histopathological examination, determination of renal SSAO activity, and measurement of MGO levels were performed. Key findingsCompared to control mice, AA-injected mice showed significant renal damage and approximately 2.7-fold greater renal SSAO activity (p<0.05). Further, compared to control treatment, administration of 12mg/kg metformin inhibited formation of renal lesions, and significantly decreased renal MGO levels (37.33±9.78 vs. 5.89±2.64μg/mg of protein, respectively, p<0.01). In the AA-treated mice, metformin also inhibited the accumulation of CML in renal tubules, but did not affect SSAO activity. SignificanceThis study is the first to show elevated renal SSAO activity in AA-treated mice, which could be involved in MGO accumulation. Moreover, MGO scavenging by metformin reduces AA nephrotoxicity. These findings suggest that reducing MGO accumulation produces nephroprotection, revealing new therapeutic strategies for the management. SSAO is a key enzyme involved in MGO generation, and consequently, inhibition of renal SSAO activity is worth investigating in AA nephrotoxicity and other renal pathologies further.

An advanced glycation end product (AGE)-rich diet promotes Nε-carboxymethyl-lysine accumulation in the cardiac tissue and tendons of rats.

The purpose of this study was to investigate the intake, excretion, and tissue accumulation of carboxymethyl-lysine (CML), after feeding rats a diet containing advanced glycation end products (AGEs) from a glucose-lysine (GL) model system. Rats were distributed into two groups and assigned to a control diet or a diet including 3% heated GL (GL diet) for three months. Feces and urine were collected over the last week. After sacrifice, serum was obtained and some organs were removed for CML analysis. The percentage of fecal CML was 2.5-fold higher in the animals fed the GL diet (33.2 vs 76.5% for control and GL group), whereby total recovery was 91.8% compared with a level of 54.6% in the animals fed the control chow, evidencing the importance of the chemical form and the net quantity of dietary CML on its elimination. We suggest that dietary dicarbonyl compounds from GL diet or dietary CML itself are responsible for CML accumulation in hearts and tendons. The most significant result of the present study is that the regular consumption of dietary AGEs in healthy individuals promotes CML accumulation in some organs.