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

Buetler, Timo M.; Latado, Hélia; Leclerc, Estelle; Weigle, Bernd; Baumeyer, Alexandra; Heizmann, Claus W.; Scholz, Gabriele

doi:10.1002/mnfr.201000140

Cited by 12 publications

(14 citation statements)

References 31 publications

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“…Despite the scientific need for LPS removal to establish the immunological activity of natively folded proteins, only few studies reported on the experimental conditions that affect the efficiency of endotoxin removal from protein extracts using TX-114. Reported protocols vary in incubation conditions and TX-114-removal strategies after LPS-extraction [11, 16, 17, 32], which we have shown to critically influence the application of the purified protein in in vitro assays [26]. Lopes et al .…”

Section: Discussionmentioning

confidence: 99%

“…Previously published procedures often disregard this phenomenon giving rise to conclusions based on TX-114 induced bias in concentration assays [11, 23, 26, 43]. Buetler and colleagues [11] showed that glycolaldehyde-modified BLG preparations were unable to induce inflammatory signaling in receptor for RAGE-expressing cells after TX-114 assisted extraction of LPS. The authors concluded that cell activation shown for non-treated preparations was connected to a LPS-like lipophilic contamination.…”

Section: Discussionmentioning

confidence: 99%

“…A vast array of LPS extraction procedures have been published over the years and these methods often include several chromatographic steps, such as ion exchange, hydrophobic interaction chromatography, and gel filtration [8–10]. These methods are laborious, and do not fully eliminate biological activity of residing LPS [11]. Polymyxin B (PMB) is an antibiotic that has been used as alternative strategy to eliminate LPS activity and even though PMB was shown to prevent TLR4 signaling [12] its presence does not prevent IL-1β secretion in monocyte cultures [12].…”

Section: Introductionmentioning

confidence: 99%

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Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins

et al. 2017

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ScopeInvestigations into the immunological response of proteins is often masked by lipopolysaccharide (LPS) contamination. We report an optimized Triton X-114 (TX-114) based LPS extraction method for β-lactoglobulin (BLG) and soy protein extract suitable for cell-based immunological assays.Methods and resultsOptimization of an existing TX-114 based phase LPS extraction method resulted in >99% reduction of LPS levels. However, remaining TX-114 was found to interfere with LPS and protein concentration assays and decreased viability of THP-1 macrophages and HEK-Blue 293 cells. Upon screening a range of TX-114 extraction procedures, TX-114-binding beads were found to most effectively lower TX-114 levels without affecting protein structural properties. LPS-purified proteins showed reduced capacity to activate TLR4 compared to non-treated proteins. LPS-purified BLG did not induce secretion of pro-inflammatory cytokines from THP-1 macrophages, as non-treated protein did, showing that LPS contamination masks the immunomodulatory effect of BLG. Both HEK293 cells expressing TLR4 and differentiated THP-1 macrophages were shown as a relevant model to screen the protein preparations for biological effects of LPS contamination.ConclusionThe reported TX-114 assisted LPS-removal from protein preparations followed by bead based removal of TX-114 allows evaluation of natively folded protein preparations for their immunological potential in cell-based studies.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins

et al. 2017

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“…Interaction of AGEs with their specific cell surface receptors RAGE may result, among others, in the production of reactive oxygen species and induction of micro-inflammation, which may in turn accelerate the formation of AGEs, and exacerbate insulin resistance [2], [22], [23]. However, some studies have disputed these findings: there are still controversies on the identity of the actual AGEs that initiate the RAGE-mediated reactions, and questions regarding evidence that endotoxin contamination of AGE protein preparations compromised the results of some studies [24], [25]. Experimental studies show that AGEs per se may initiate the insulin-resistant state in skeletal muscle and adipocytes [26]–[28], or decrease the insulin content and secretion in pancreatic islets [29]–[31].…”

Section: Introductionmentioning

confidence: 99%

Advanced Glycation End Products in Infant Formulas Do Not Contribute to Insulin Resistance Associated with Their Consumption

et al. 2013

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IntroductionInfant formula-feeding is associated with reduced insulin sensitivity. In rodents and healthy humans, advanced glycation end product (AGE)-rich diets exert diabetogenic effects. In comparison with human breast-milk, infant formulas contain high amounts of AGEs. We assessed the role of AGEs in infant-formula-consumption-associated insulin resistance.MethodsTotal plasma levels of Nε-(carboxymethyl)lysine (CML), AGEs-associated fluorescence (λex = 370 nm/λem = 445 nm), soluble adhesion molecules, markers of micro- binflammation (hsCRP), oxidative stress (malondialdehyde, 8-isoprostanes) and leptinemia were determined, and correlated with insulin sensitivity in a cross-sectional study in 166 healthy term infants aged 3-to-14 months, subdivided according to feeding regimen (breast-milk- vs. infant formula-fed) and age (3-to-6-month-olds, 7-to-10-month-olds, and 11-to-14-month-old infants). Effects of the consumption of low- vs. high-CML-containing formulas were assessed. 36 infants aged 5.8±0.3 months were followed-up 7.5±0.3 months later.ResultsCross-sectional study: 3-to-6-month-olds and 7-to-10-month-old formula-fed infants presented higher total plasma CML levels and AGEs-associated fluorescence (p<0.01, both), while only the 3-to-6-month-olds displayed lower insulin sensitivity (p<0.01) than their breast-milk-fed counterparts. 3-to-6-month-olds fed low-CML-containing formulas presented lower total plasma CML levels (p<0.01), but similar insulin sensitivity compared to those on high-CML-containing formulas. Markers of oxidative stress and inflammation, levels of leptin and adhesion molecules did not differ significantly between the groups. Follow-up study: at initial investigation, the breast-milk-consuming infants displayed lower total plasma CML levels (p<0.01) and AGEs-associated fluorescence (p<0.05), but higher insulin sensitivity (p<0.05) than the formulas-consuming infants. At follow-up, the groups did not differ significantly in either determined parameter.ConclusionsIn healthy term infants, high dietary load with CML does not play a pathophysiological role in the induction of infant formula-associated insulin resistance. Whether a high load of AGEs in early childhood affects postnatal programming remains to be elucidated.

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“…An interaction between endotoxins and the AGE receptor system exists (28) and endotoxin-like products might contaminate AGE preparations (29). While intravenous administration of endotoxins induces severe toxicity in mammals, oral administration of a high dose of lipopolysaccharides caused no toxicity or systemic inflammation (30), probably due to poor endotoxin absorption (31).…”

Section: Discussionmentioning

confidence: 99%

Acute Macrovascular Dysfunction in Patients With Type 2 Diabetes Induced by Ingestion of Advanced Glycated β-Lactoglobulins

et al. 2013

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OBJECTIVERecent evidence indicates that heat-enhanced food advanced glycation end products (AGEs) adversely affect vascular function. The aim of this study was to examine the acute effects of an oral load of heat-treated, AGE-modified β-lactoglobulins (AGE-BLG) compared with heat-treated, nonglycated BLG (C-BLG) on vascular function in patients with type 2 diabetes mellitus (T2DM).RESEARCH DESIGN AND METHODSIn a double-blind, controlled, randomized, crossover study, 19 patients with T2DM received, on two different occasions, beverages containing either AGE-BLG or C-BLG. We measured macrovascular [brachial ultrasound of flow-mediated dilatation (FMD)] and microvascular (laser-Doppler measurements of reactive hyperemia in the hand) functions at baseline (T0), 90 (T90), and 180 (T180) min.RESULTSFollowing the AGE-BLG, FMD decreased at T90 by 80% from baseline and remained decreased by 42% at T180 (P < 0.05 vs. baseline, P < 0.05 vs. C-BLG at T90). By comparison, following C-BLG, FMD decreased by 27% at T90 and 51% at T180 (P < 0.05 vs. baseline at T180). A significant decrease in nitrite (T180) and nitrate (T90 and T180), as well as a significant increase in Nε-carboxymethyllisine, accompanied intake of AGE-BLG. There was no change in microvascular function caused by either beverage.CONCLUSIONSIn patients with T2DM, acute oral administration of a single AGE-modified protein class significantly though transiently impaired macrovascular function in concert with decreased nitric oxide bioavailability. These AGE-related changes were independent of heat treatment.

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

Cited by 12 publications

References 31 publications

Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins

Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins

Advanced Glycation End Products in Infant Formulas Do Not Contribute to Insulin Resistance Associated with Their Consumption

Acute Macrovascular Dysfunction in Patients With Type 2 Diabetes Induced by Ingestion of Advanced Glycated β-Lactoglobulins

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