Early- and life-long intake of dietary advanced glycation end-products (dAGEs) leads to transient tissue accumulation, increased gut sensitivity to inflammation, and slight changes in gut microbial diversity, without causing overt disease.

Dietary advanced glycation end-products (dAGEs) accumulate in organs and are thought to initiate chronic low-grade inflammation (CLGI), induce glycoxidative stress, drive immunosenescence, and influence gut microbiota. Part of the toxicological interest in glycation products such as dietary carboxymethyl-lysine (dCML) relies on their interaction with receptor for advanced glycation end-products (RAGE). It remains uncertain whether early or lifelong exposure to dAGEs contributes physiological changes and whether such effects are reversible or permanent. Our objective was to examine the physiological changes in Wild-Type (WT) and RAGE KO mice that were fed either a standard diet (STD - 20.8 ± 5.1 µg dCML/g) or a diet enriched with dCML (255.2 ± 44.5 µg dCML/g) from the perinatal period for up to 70 weeks. Additionally, an early age (6 weeks) diet switch (dCML→STD) was explored to determine whether potential harmful effects of dCML could be reversed. Previous dCML accumulation patterns described by our group were confirmed here, with significant RAGE-independent accumulation of dCML in kidneys, ileum and colon over the 70-week dietary intervention (respectively 3-fold, 17-fold and 20-fold increases compared with controls). Diet switching returned tissue dCML concentrations to their baseline levels. The dCML-enriched diet had no significative effect on endogenous glycation, inflammation, oxidative stress or senescence parameters. The relative expression of TNFα, VCAM1, IL6, and P16 genes were all upregulated (∼2-fold) in an age-dependent manner, most notably in the kidneys of WT animals. RAGE knockout seemed protective in this regard, diminishing age-related renal expression of TNFα. Significant increases in TNFα expression were detectable in the intestinal tract of the Switch group (∼2-fold), suggesting a higher sensitivity to inflammation perhaps related to the timing of the diet change. Minor fluctuations were observed at family level within the caecal microbiota, including Eggerthellaceae, Anaerovoracaceae and Marinifilaceae communities, indicating slight changes in composition. Despite chronic dCML consumption resulting in higher free CML levels in tissues, there were no substantial increases in parameters related to inflammageing. Age was a more important factor in inflammation status, notably in the kidneys, while the early-life dietary switch may have influenced intestinal susceptibility to inflammation. This study affirms the therapeutic potential of RAGE modulation and corroborates evidence for the disruptive effect of dietary changes occurring too early in life. Future research should prioritize the potential influence of dAGEs on disease aetiology and development, notably any exacerbating effects they may have upon existing health conditions.

Glycated bovine serum albumin for use in feeding trials with animal models - In vitro methodology and characterization of a glycated substrate for modifying feed pellets.

This study investigated different methods to produce Nε-carboxymethyl-lysine (CML)-enriched bovine serum albumin (BSA) as alternatives to the classical approach using glyoxylic acid (GA) and sodium cyanoborohydride (NaBH3CN) which results in toxic hydrogen cyanide (HCN). The reaction of GA (6 mmol/L) and NaBH3CN (21 mmol/L) to produce CML remained the most effective with CML yields of 24-35%, followed by 13-24% using 300 mmol/L glyoxal (GO). GA promoted specific modification of lysine to CML, and fewer structural modifications of the BSA molecule compared with GO, as evidenced by fluorescence and proteomic analyses. GO promoted greater arginine modification compared with GA (76 vs 23%). Despite structural changes to BSA with GO, murine fecal clearance of CML was similar to literature values. Hence, BSA glycation with 300 mmol/L glyoxal is a suitable alternative to GA and NaBH3CN for generating CML-enriched protein free of HCN, but a CML-only fortification model remains to be described.

Carbamylation is a competitor of glycation for protein modification in vivo.

AIM: Chronic kidney disease (CKD) and diabetes mellitus are two diseases that accelerate protein molecular ageing through carbamylation and glycation reactions, characterized by the binding of urea-derived isocyanic acid and of sugars on proteins, respectively. These two reactions target the same protein amino groups and, thus, compete with each other. Such competition may arise especially in diabetic patients with nephropathy. This study aimed to evaluate their potential competitive effects in vitro and under conditions reproducing CKD and/or diabetes in vivo. METHODS: Albumin was incubated in vitro with glucose, urea or cyanate. Carbamylation in vivo was enhanced in normal and diabetic (db/db) mice by either subtotal nephrectomy or cyanate consumption. Homocitrulline, carbamylated haemoglobin and furosine were measured by LC-MS/MS, fructosamine by colorimetric assay and HbA1c by immunological assay. RESULTS: Reciprocal inhibition between carbamylation and glycation was observed during albumin incubations in vitro. Besides, 5 weeks after induction of CKD in vivo, plasma homocitrulline concentrations were similar in both diabetic and non-diabetic mice, whereas fructosamine and HbA1c were decreased (-23% and -42%, respectively) in diabetic mice with CKD compared with only diabetic ones. Fructosamine and HbA1c were also decreased in cyanate-spiked water-drinking mice compared with plain water-drinking diabetic mice. CONCLUSION: Carbamylation competes with glycation in vivo, especially under conditions of high glycation. Thus, the classic markers of glycaemic control should be interpreted with caution in diabetic patients with CKD because of this competitive effect.

A flavonoid fraction purified from Rutaceae aurantiae (Daflon(R)) inhibiting AGE formation, reduces urinary albumin clearance and corrects hypoalbuminemia in normotensive and hypertensive diabetic rats.

AIM: Advanced glycation endproducts (AGEs) have been shown to contribute to alteration of glomerular permselectivity to proteins in diabetes. Oxidative stress is required for AGE formation. Therefore we studied the effect of an antioxidant micronized purified flavonoid fraction (MPFF, Daflon(R) 500 mg), on urinary albumin clearance in diabetic rats. METHODS: Hyperglycaemia was induced by streptozotocin 55 mg/kg IM at days 0 and 7 in normotensive Wistar rats (NWR, diabetes duration 5 months) or hypertensive Wistar Kyoto rats (SHR, diabetes duration 2 months). MPFF was administered at 300 mg/kg/day, from day -2 until sacrifice. RESULTS: After 5 months of diabetes in NWR, MPFF reduced albumin clearance from 729±92 to 392±60 nl/min/kg, p<0.01, and restored albuminemia from 20.4±0.9 to 24.0±1 g/l, p<0.05; albumin fractional clearance was significantly diminished in the flavonoid-treated diabetic rats (0.360±0.037‰ versus 1.335±0.430‰ in the diabetic controls, p<0.001); MPFF did not significantly modify blood glucose and plasma fructosamine levels. After 2 months of diabetes in SHR, MPFF reduced albumin clearance from 243±121 to 101±47 nl/min/kg, p<0.05, and restored albuminemia from 21.1±1.6 to 26.7±2.2 g/l (p<0.05); MPFF also decreased plasma fluorescence characteristic of AGEs (p<0.02). Besides hesperetin, a main metabolite of MPFF recovered in plasma, inhibited in vitro the formation of the crosslinking AGE pentosidine in collagen incubated with high glucose (p<0.001). CONCLUSION: Our results confirm the role of glycoxidative stress in diabetic nephropathy. MPFF might be useful as complementary treatment for preventing diabetic microangiopathy.