Site specificity of glycation and carboxymethylation of bovine serum albumin by fructose

We report an investigation of the site specificity, extent and nature of modification of bovine serum albumin (BSA) incubated with fructose or glucose at physiological temperature and pH. Sites of early glycation (Heyns rearrangement products (HRP) from fructose; fructoselysine (FL) from glucose) as well as advanced glycation (N-epsilon-(carboxymethyl)lysine; CML) wereanalyzed by liquid chromatography-mass spectrometry. The major site of modification by fructose, like glucose, is Lysine-524 and this results in, respectively, 31 and 76% loss of the corresponding unmodified tryptic peptide, Gln525-Lys533. In addition, total lysine, HRP, FL, CML and N-epsilon-(carboxyethyl)lysine in the incubations, was quantified. Almost all of the loss of lysine in the fructose-modified BSA was attributed to the formation of CML, with the yield of CML being up to 17-fold higher than glucose-modified BSA. A mechanism for the formation of CML from the HRP is proposed.

Glycation, carbonyl stress, EAGLEs, and the vascular complications of diabetes

The association between poor metabolic control and the microvascular complications of diabetes is now well established, but the relationship between long-term metabolic control and the accelerated atherosclerosis of diabetes is as yet poorly defined. Hyperglycemia is the standard benchmark by which metabolic control is assessed. One mechanism by which elevated glucose levels may mediate vascular injury is through early and advanced glycation reactions affecting a wide variety of target molecules. The "glycation hypothesis'' has developed over the past 30 years, evolving gradually into a "carbonyl stress hypothesis'' and taking into account not only the modification of proteins by glucose, but also the roles of oxidative stress, a wide range of reactive carbonyl-containing intermediates (derived not only from glucose but also from lipids), and a variety of extra- and intracellular target molecules. The final products of these reactions may now be termed "Either Advanced Glycation or Lipoxidation End-Products'' or "EAGLEs.'' The ubiquity of carbonyl stress within the body, the complexity of the reactions involved, the variety of potential carbonyl intermediates and target molecules and their differing half-lives, and the slow development of the complications of diabetes all pose major challenges in dissecting the significance of these processes. The extent of the reactions tends to correlate with overall metabolic control, creating pitfalls in the interpretation of associative data. Many animal and cell culture studies, while supporting the hypothesis, must be viewed with caution in terms of relevance to human diabetes. In this article, the development of the carbonyl stress hypothesis is reviewed, and implications for present and future treatments to prevent complications are discussed.

Prevention of retinal capillary basement membrane thickening in diabetic dogs by a non-steroidal anti-inflammatory drug

AIMS/HYPOTHESIS: To investigate the effect of treatment with the non-steroidal anti-inflammatory drug Sulindac on the early vascular pathology of diabetic retinopathy in the dog, and it's effect on recognised biochemical indices of hyperglycaemia-related pathophysiology. METHODS: Experimental diabetes (streptozotocin/alloxan) was induced in 22 male beagle dogs and 12 of the animals were assigned at random to receive oral Sulindac (10 mg/kg daily). Age- and sex-matched control animals were maintained as non-diabetic controls. After 4 years, several morphological parameters were quantified in the retinal microvasculature of each animal group using an established stereological method. Also, the following diabetes-associated biochemical parameters were analysed: accumulation of advanced glycation end products (AGEs), red blood cell polyol levels and antioxidant status. RESULTS: Diabetes increased red blood cell sorbitol levels when compared to non-diabetic controls (p<or =0.05), however, there was no difference in sorbitol levels between the untreated and the treated diabetic animals. No significant differences were found in red blood cell myoinositol levels between the three groups of animals. Pentosidine and other AGEs were increased two- to three-fold in the diabetic animals (p<or =0.001) although treatment with Sulindac did not affect their accumulation in diabetic skin collagen or alter diabetes-induced rises in plasma malondialdehyde. Retinal capillary basement membrane volume was significantly increased in the untreated diabetic dogs compared to non-diabetic controls or Sulindac-treated diabetic animals (p<or =0.0001). CONCLUSION/INTERPRETATION: This study has confirmed the beneficial effect of a non-steroidal anti-inflammatory drug on the early vascular pathology of diabetic retinopathy. However the treatment benefit was not dependent on inhibition of polyol pathway activity, advanced glycation, or oxidative stress.

Male Infertility: A Clinical Reflection

The introduction of intracytoplasmic sperm injection (ICSI) has led to an inappropriate decrease in interest in male fertility. It is apparent that light microscopy provides limited information and molecular techniques show that DNA abnormalities need to be considered further. Abnormalities include not only Yq11 deletions but also DNA strand breaks. Increases in advanced glycation end-products in sperm from well controlled diabetics may provide a mechanism for this damage in non-diabetics. In addition, much publicity is given to decreased male fertility: this is NOT confirmed as technical variations and differences in study populations make it difficult to draw conclusions. The generation of stem cell derived germ cells provides hope for men without germ cells but this is currently only experimental.

Upregulation of Oxidative Stress Markers in Human Microvascular Endothelial Cells by Complexes of Serum Albumin and Digestion Products of Glycated Casein

The extent of absorption of dietary advanced glycation end products (AGEs) is not fully known. The possible physiological impact of these absorbed components on inflammatory processes has been studied little and was the aim of this investigation. Aqueous solutions of bovine casein and glucose were heated at 95 degrees C for 5 h to give AGE-casein (AGE-Cas). Simulated stomach and small intestine digestion of AGE-Cas and dialysis (molecular mass cutoff of membrane = 1 kDa) resulted in a low molecular mass (LMM) fraction of digestion products, which was used to prepare bovine serum albumin (BSA)-LMM-AGE-Cas complexes. Stimulation of human microvascular endothelial cells with BSA-LMM-AGE-Cas complexes significantly increased mRNA expression of the receptor of AGE (RAGE), galectin-3 (AGE-113), tumor necrosis factor alpha, and a marker of the mitogen-activated protein kinase pathway (MAPK-1), as well as p65NF-kappa B activation. Cells treated with LMM digestion products of AGE-Cas significantly increased AGE-R3 mRNA expression. Intracellular reactive oxygen species production increased significantly in cells challenged with BSA-LMM-AGE-Cas and LMM-AGE-Cas. In conclusion, in an in vitro cell system, digested dietary AGEs complexed with serum albumin play a role in the regulation of RAGE and down-stream inflammatory pathways. AGE-R3 may protect against these effects.

AGEs and Diabetic Retinopathy

The pathogenesis of diabetic retinopathy is multifactorial, and a range of hyperglycemia-linked pathways have been implicated in the initiation and progression of this condition. All cells in the retina are affected by the diabetic milieu, and in view of such disease and tissue complexity, it is unlikely that any single process is solely responsible for retinal pathophysiology. Nevertheless, establishing causal mechanisms remains an important research goal. This review concentrates on the formation of advanced glycation end products (AGEs) and the role they play in diabetic retinopathy. Perspective is provided on advanced glycation in the retina, the impact that this process has on retinal cell function, and how it relates to other pathogenic pathways. Emphasis is also placed the modulatory role of the receptor for AGEs (RAGE) and how its activation could evoke retinal inflammatory disease. Further research is needed to achieve a clear understanding of the cellular and molecular processes that underpin diabetic retinopathy's initiation and progression. Such advances in basic mechanisms may lead to effective treatments that can prevent progression of retinopathy from the point of the diagnosis of diabetes to sight-threatening proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME).

Multiplex analysis of age-related protein and lipid modifications in human Bruch's membrane

Aging of the human retina is characterized by progressive pathology, which can lead to vision loss. This progression is believed to involve reactive metabolic intermediates reacting with constituents of Bruch's membrane, significantly altering its physiochemical nature and function. We aimed to replace a myriad of techniques following these changes with one, Raman spectroscopy. We used multiplexed Raman spectroscopy to analyze the age-related changes in 7 proteins, 3 lipids, and 8 advanced glycation/lipoxidation endproducts (AGEs/ALEs) in 63 postmortem human donors. We provided an important database for Raman spectra from a broad range of AGEs and ALEs, each with a characteristic fingerprint. Many of these adducts were shown for the first time in human Bruch's membrane and are significantly associated with aging. The study also introduced the previously unreported up-regulation of heme during aging of Bruch's membrane, which is associated with AGE/ALE formation. Selection of donors ranged from ages 32 to 92 yr. We demonstrated that Raman spectroscopy can identify and quantify age-related changes in a single nondestructive measurement, with potential to measure age-related changes in vivo. We present the first directly recorded evidence of the key role of heme in AGE/ALE formation.

Differences in mouse models of diabetes mellitus in studies of male reproduction

Diabetes Mellitus (DM) has been found to have subtle yet profound effects on the metabolic status of the testis, the expression of numerous spermatogenic genes and is associated with increased numbers of sperm with nuclear DNA damage. The precise mechanism causing these detrimental effects remains unknown. The presence of increased levels of the most prominent member (carboxymethyllysine - CML) of the advanced glycation end product adducts and their receptor (RAGE) in the reproductive tract of DM men has provided a new avenue for research. As there are suspicions that the antibiotic (streptozotocin - STZ) employed to induce DM is also capable of causing oxidative stress and DNA damage, we compared CML and RAGE levels in the reproductive tract and sperm nDNA status of STZ mice with the levels in the Ins(2Akita) mouse to determine which more closely mimics the situation described in the human diabetic. CML was observed in the testes, epididymes and sperm of all animals. Sperm from DM mice showed particularly strong CML immunolocalization in the acrosomal cap, the equatorial region and whenever present, cytoplasmic droplets. Although increased, the level of CML on the sperm of the STZ and Ins(2Akita) DM mice did not reach statistical significance. RAGE was present on the developing acrosome and epididymal sperm of all animals and in discrete regions of the epididymes of the DM models. Only the epididymal sperm of the Ins(2Akita) mice were found to have significantly increased (p < 0.0001) nDNA damage. The Ins(2Akita) mouse therefore appears to more accurately reflect the conditions found in the human and, as such, is a more representative model for the study of diabetes and glycation's influence on male fertility.

The AGE inhibitor pyridoxamine inhibits development of retinopathy in experimental diabetes

We examined the ability of pyridoxamine (PM), an inhibitor of formation of advanced glycation end products (AGEs) and lipoxidation end products (ALEs), to protect against diabetes-induced retinal vascular lesions. The effects of PM were compared with the antioxidants vitamin E (VE) and R-alpha-lipoic acid (LA) in streptozotocin-induced diabetic rats. Animals were given either PM (1 g/l drinking water), VE (2,000 IU/kg diet), or LA (0.05%/kg diet). After 29 weeks of diabetes, retinas were examined for pathogenic changes, alterations in extracellular matrix (ECM) gene expression, and accumulation of the immunoreactive AGE/ALE N-epsilon-(carboxymethyl)lysine (CML). Acellular capillaries were increased more than threefold, accompanied by significant upregulation of laminin immunoreactivity in the retinal microvasculature. Diabetes also increased mRNA expression for fibronectin (2-fold), collagen IV (1.6-fold), and laminin beta chain (2.6-fold) in untreated diabetic rats compared with nondiabetic rats. PM treatment protected against capillary drop-out and limited laminin protein upregulation and ECM mRNA expression and the increase in CML in the retinal vasculature. VE and LA failed to protect against retinal capillary closure and had inconsistent effects on diabetes-related upregulation of ECM mRNAs. These results indicate that the AGE/ALE inhibitor PM protected against a range of pathological changes in the diabetic retina and may be useful for treating diabetic retinopathy.

Elevated AGE-modified ApoB in sera of euglycemic, normolipidemic patients with atherosclerosis: relationship to tissue AGEs.:relationship to tissue AGEs

BACKGROUND: Advanced glycation endproducts (AGEs) are implicated in the pathogenesis of atherosclerotic vascular disease of diabetic and nondiabetic etiology. Recent research suggests that advanced glycation of ApoB contributes to the development of hyperlipidemia. AGE-specific receptors, expressed on vascular endothelium and mononuclear cells, may be involved in both the clearance of, and the inflammatory responses to AGEs. The aim of this study was to examine whether there is a relationship between serum AGE-ApoB and AGEs in arterial tissue of older normolipidemic nondiabetic patients with occlusive atherosclerotic disease, compared with age-matched and younger asymptomatic persons.

MATERIALS AND METHODS: Serum AGE-ApoB was measured by ELISA in 21 cardiac bypass patients. Furthermore, an AGE-specific monoclonal antibody, and polyclonal antibodies against anti-AGE-receptor (anti-AGE-R) 1 and 2 were used to explore the localization and distribution of AGEs and AGE-R immunoreactivity (IR) in arterial segments excised from these patients.

RESULTS: Serum AGE-ApoB levels were significantly elevated in the asymptomatic, older population, compared with those in young healthy persons (259 +/- 24 versus 180 +/- 21 AGE U/mg of ApoB, p < 0.01). Higher AGE-ApoB levels were observed in those patients with atherosclerosis (329 +/- 23 versus 259 +/- 24 AGE U/mg ApoB, p < 0.05). Comparisons of tissue AGE-collagen with serum AGE-ApoB levels showed a significant correlation (r = 0.707, p < 0.01). In early lesions, AGE-IR occurred mostly extracellularly. In fatty streaks and dense, cellular atheromatous lesions, AGE-IR was visible within lipid-containing smooth muscle cells and macrophages, while in late-stage, acellular plaques, AGE-IR occurred mostly extracellularly. AGE-R1 and -R2 were observed on vascular endothelial and smooth-muscle cells and on infiltrating mononuclear cells in the early-stage lesions, whereas in dense, late-stage plaques, they colocalized mostly with lipid-laden macrophages. On tissue sections, scoring of AGE-immunofluorescence correlated with tissue AGE and plasma AGE-ApoB.

CONCLUSIONS: (1) The correlation between arterial tissue AGEs and circulating AGE-ApoB suggests a causal link between AGE modification of lipoproteins and atherosclerosis. AGE-specific receptors may contribute to this process. (2) Serum AGE-ApoB may serve to predict atherosclerosis in asymptomatic patients.

AGEs, RAGE, and Diabetic Retinopathy

Diabetic retinopathy is a major diabetic complication with a highly complex etiology. Although there are many pathways involved, it has become established that chronic exposure of the retina to hyperglycemia gives rise to accumulation of advanced glycation end products (AGEs) that play an important role in retinopathy. In addition, the receptor for AGEs (RAGE) is ubiquitously expressed in various retinal cells and is upregulated in the retinas of diabetic patients, resulting in activation of pro-oxidant and proinflammatory signaling pathways. This AGE-RAGE axis appears to play a central role in the sustained inflammation, neurodegeneration, and retinal microvascular dysfunction occurring during diabetic retinopathy. The nature of AGE formation and RAGE signaling bring forward possibilities for therapeutic intervention. The multiple components of the AGE-RAGE axis, including signal transduction, formation of ligands, and the end-point effectors, may be promising targets for strategies to treat diabetic retinopathy.

Diabetes-related adduct formation and retinopathy

The pathogenesis of diabetic retinopathy is complex, reflecting the array of systemic and tissue-specific metabolic abnormalities. A range of pathogenic pathways are directly linked to hyperglycaemia and dyslipidaemia, and the retina appears to be exquisitely sensitive to damage. Establishing the biochemical and molecular basis for this pathology remains an important research focus. This review concentrates on the formation of a range of protein adducts that form after exposure to modifying intermediates known to be elevated during diabetes. These so-called advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs) are thought to play an important role in the initiation and progression of diabetic retinopathy, and mechanisms leading to dysfunction and death of various retinal cells are becoming understood. Perspective is provided on AGE/ALE formation in the retina and the impact that such adducts have on retinal cell function. There will be emphasis placed on the role of the receptor for AGEs and how this may modulate retinal pathology, especially in relation to oxidative stress and inflammation. The review will conclude by discussion of strategies to inhibit AGE/ALE formation or harmful receptor interactions in order to prevent disease progression from the point of diabetes diagnosis to sight-threatening proliferative diabetic retinopathy and diabetic macular oedema.

Effect of Inhibitor Compounds on N-epsilon-(Carboxymethyl)lysine (CML) and N-epsilon-(Carboxyethyl)lysine (CEL) Formation in Model Foods

The possible adverse effects on health of diet-derived advanced glycation endproducts (AGES) and advanced lipoxidation endproducts (ALES) is of current interest. This study had the objective of determining the effects of the addition of AGE/ALE inhibitors and different types of sugar and cooking oil on N-epsilon-(carboxymethyl)lysine (CML) and N-epsilon-(carboxyethyl)lysine (CEL) formation in model foods (sponge cakes). The cake baked using glucose produced the highest level of CML (2.07 +/- 0.24 mmol/mol lysine), whereas the cake baked using fructose produced the highest concentration of CEL (25.1 +/- 0.15 mmol/mol lysine). There were no significant differences between CML concentrations formed in the cakes prepared using different types of cooking oil, but significant differences (P