Timo Buetler

Dietary factors and low-grade inflammation in relation to overweight and obesity

Low-grade inflammation is a characteristic of the obese state, and adipose tissue releases many inflammatory mediators. The source of these mediators within adipose tissue is not clear, but infiltrating macrophages seem to be especially important, although adipocytes themselves play a role. Obese people have higher circulating concentrations of many inflammatory markers than lean people do, and these are believed to play a role in causing insulin resistance and other metabolic disturbances. Blood concentrations of inflammatory markers are lowered following weight loss. In the hours following the consumption of a meal, there is an elevation in the concentrations of inflammatory mediators in the bloodstream, which is exaggerated in obese subjects and in type 2 diabetics. Both high-glucose and high-fat meals may induce postprandial inflammation, and this is exaggerated by a high meal content of advanced glycation end products (AGE) and partly ablated by inclusion of certain antioxidants or antioxidant-containing foods within the meal. Healthy eating patterns are associated with lower circulating concentrations of inflammatory markers. Among the components of a healthy diet, whole grains, vegetables and fruits, and fish are all associated with lower inflammation. AGE are associated with enhanced oxidative stress and inflammation. SFA and trans-MUFA are pro-inflammatory, while PUFA, especially long-chain n-3 PUFA, are anti-inflammatory. Hyperglycaemia induces both postprandial and chronic low-grade inflammation. Vitamin C, vitamin E and carotenoids decrease the circulating concentrations of inflammatory markers. Potential mechanisms are described and research gaps, which limit our understanding of the interaction between diet and postprandial and chronic low-grade inflammation, are identified.

Nε-carboxymethyllysine-modified proteins are unable to bind to RAGE and activate an inflammatory response

Advanced glycation endproducts (AGEs) containing carboxymethyllysine (CML) modifications are generally thought to be ligands of the receptor for AGEs, RAGEs. It has been argued that this results in the activation of pro-inflammatory pathways and diseases. However, it has not been shown conclusively that a CML-modified protein can interact directly with RAGE. Here, we have analyzed whether beta-lactoglobulin (bLG) or human serum albumin (HSA) modified chemically to contain only CML (10-40% lysine modification) can (i) interact with RAGE in vitro and (ii) interact with and activate RAGE in lung epithelial cells. Our results show that CML-modified bLG or HSA are unable to bind to RAGE in a cell-free assay system (Biacore). Furthermore, they are unable to activate pro-inflammatory signaling in the cellular system. Thus, CML probably does not form the necessary structure(s) to interact with RAGE and activate an inflammatory signaling cascade in RAGE-expressing cells.

Comparative LC–MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N(epsilon)-fructoselysine (FL), N(epsilon)-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34+/-3.81 nmol CML per micromol of free Lys (Lys(free)) and 81.5+/-87.8 nmol Pyr micromol(-1) Lys(free)(-1) vs. 3.72+/-1.29 nmol FL micromol(-1) Lys(free)(-1). In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47+/-0.08 nmol FL micromol(-1) of protein-bound Lys (Lys(p-b)), 0.04+/-0.03 nmol CML micromol(-1) Lys(p-b)(-1) and 0.06+/-0.02 nmol Pyr micromol(-1)Lys(p-b)(-1). It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.

Synthesis, tandem MS- and NMR-based characterization, and quantification of the carbon 13-labeled advanced glycation endproduct, 6-N-carboxymethyllysine

Summary.6-N-carboxymethyllysine (CML), generated by the glycation and/or oxidation of lysine residues, has been measured in biological materials and food products using techniques such as ELISA, HPLC with fluorescence detection and mass spectrometry methods. Only limited information has been reported regarding the preparation of standards labeled with either deuterium, 13C or 15N atoms to be used as internal standards. In the present paper, a synthesis of carbon-13 labeled CML is described using l,2-13C2-glyoxylic acid and 2-N-acetyllysine as starting materials. The resulting labeled 2-N-acetyl-CML was purified by HPLC-UV as a dibutyl ester. After a deprotection step, the yield was evaluated to be 53% when the reaction was conducted 17 h at 37°C. CML was extensively studied by 1H- and 13C-NMR and the fragments observed in the collision induced dissociation (CID) spectrum were also assigned. Finally, the standards of CML and carbon-13 labeled CML were accurately quantified based on 1H-NMR and tandem MS using lysine as an internal reference.

Glycolaldehyde-modified β-lactoglobulin AGEs are unable to stimulate inflammatory signaling pathways in RAGE-expressing human cell lines

SCOPE Advanced glycation endproducts (AGEs) are suspected to stimulate inflammatory signaling pathways in target tissues via activation of the receptor for AGEs. Endotoxins are generally recognized as potential contamination of AGE preparations and stimulate biological actions that are very similar as or identical to those induced by AGEs. METHODS AND RESULTS In our study, we used glycolaldehyde-modified β-lactoglobulin preparations as model AGEs and employed two methods to remove endotoxin using either affinity columns or extraction with Triton X-114 (TX-114). Affinity column-purified AGEs retained their ability to stimulate inflammatory signaling as measured by mRNA expression of inflammatory cytokines in the human lung epithelial cell line Beas2b. However, glycolaldehyde-modified AGEs purified by extraction with TX-114 did not show any stimulation of mRNA expression of inflammatory cytokines. The presence of a cell stimulating endotoxin-like activity was demonstrated in the detergent phase after extraction with TX-114, thus indicating that not AGEs but a lipophilic contamination was responsible for the stimulation of inflammatory signaling. CONCLUSION Our results demonstrate that glycolaldehyde-modified AGEs are unable to induce inflammatory signaling in receptor for AGE-expressing cells. The observed cell-activating activity can be ascribed to an endotoxin-like lipophilic contamination present in AGE preparations and affinity column purification was insufficient to remove this contamination.

Dicarbonyls Stimulate Cellular Protection Systems in Primary Rat Hepatocytes and Show Anti‐inflammatory Properties

Advanced glycation endproducts (AGEs) and their precursor dicarbonyls are generally perceived as having adverse health effects. They are also considered to be initiators and promoters of disease and aging. However, proof for a causal relationship is lacking. On the other hand, it is known that AGEs and melanoidins possess beneficial properties, such as antioxidant and metal‐chelating activities. Furthermore, some AGEs may stimulate the cellular detoxification system, generally known as the phase II drug metabolizing system. We show here that several reactive dicarbonyl intermediates have the capability to stimulate the cellular phase II detoxification systems in both a reporter cell line and primary rat hepatocytes. In addition, we demonstrate that dicarbonyls can attenuate the inflammatory signaling induced by tumor necrosis factor‐α in a reporter cell system.

The effects of AGEing on diet.

This Correspondence relates to “Oral Glycotoxins Determine the Effects of Calorie Restriction on Oxidant Stress, Age-Related Diseases, and Lifespan” (Am J Pathol 2008, 173:327–336).

No Evidence for Advanced Glycation Endproducts in Cancer

cancer development. To date, there is no clinical or epidemiological evidence that dicarbonyls could be involved in cancer development. ESRD is marked by decreased elimination of many toxins besides AGEs and several of these could probably be linked to cancer. In addition, diseases such as ESRD are accompanied by inflammation [6, 7] and increased generation of free radicals [8, 9] and it is likely that these alone could account for the increased cancer incidence in ESRD. It is probably impossible to dissociate any hypothetical carcinogenic effect of AGEs/dicarbonyls from oxidative stress, inflammation and other toxins in ESRD. However, the question may be allowed, why the tissues with the highest AGE levels (skin, brain, hemopoietic system) are not predominantly touched by tumors if AGEs are considered to be responsible for tumor formation? If one considers overall mortality in hemodialysis patients, it should be noted that there are also studies showing an inverse association between AGEs and mortality in hemodialysis patients [10] . Thus, there is no scientific evidence that would single out AGEs or their precursors as cancer-causing agents. It is well accepted that AGE formation is increased under conditions of oxidative stress, and Dear Sir, In a series of recent articles, Šebeková et al. [1] , Schupp et al. [2] , Stopper et al. [3] , Fragedaki et al. [4] and Wagner et al. [5] have tried to link advanced glycation end products (AGEs) to genome damage and malignancies in end-stage renal disease (ESRD) patients. They base their arguments mainly on the fact that, in general, ESRD patients present increased incidences of several types of cancers and link this to the increased circulating and tissue levels of AGEs and dicarbonyls in ESRD patients. However, these arguments are only correlative and there is no solid evidence for such a link. It is unfortunate that the authors were not more critical in the choice of their arguments and only present circumstantial evidence on this very delicate topic, cancer. The only data that could possibly link AGEs/dicarbonyls to cancer come from a few studies showing that under specific experimental conditions some dicarbonyls are mutagenic in the Ames test. However, toxicologists know that it is a long way from a positive Ames test to actually prove that an agent is causing cancer. It is regrettable that the authors did not discuss this and that they intermingle AGEs and dicarbonyls as if they were the same. It is clear that carcinogenesis is a multistage process and multiple factors play a role in Published online: August 14, 2007