Xingjun Fan

Vitamin C mediates chemical aging of lens crystallins by the Maillard reaction in a humanized mouse model

Senile cataracts are associated with progressive oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. We hypothesized that the Maillard reaction, which leads browning and aroma development during the baking of foods, would occur between the lens proteins and the highly reactive oxidation products of vitamin C. To test this hypothesis, we engineered a mouse that selectively overexpresses the human vitamin C transporter SVCT2 in the lens. Consequently, lenticular levels of vitamin C and its oxidation products were 5- to 15-fold elevated, resulting in a highly compressed aging process and accelerated formation of several protein-bound advanced Maillard reaction products identical with those of aging human lens proteins. These data strongly implicate vitamin C in lens crystallin aging and may serve as a model for protein aging in other tissues particularly rich in vitamin C, such as the hippocampal neurons and the adrenal gland. The hSVCT2 mouse is expected to facilitate the search for drugs that inhibit damage by vitamin C oxidation products.

Methylglyoxal–bovine serum albumin stimulates tumor necrosis factor alpha secretion in RAW 264.7 cells through activation of mitogen-activating protein kinase, nuclear factor κB and intracellular reactive oxygen species formation

Accumulating evidence suggests that the pathophysiology of diabetes is analogous to chronic inflammatory states. Circulating levels of inflammatory cytokines such as IL-6 and tumor necrosis factor alpha (TNFalpha) are increased in both type 1 and type 2 diabetes. TNFalpha plays an important role in the pathogenesis of insulin resistance in type 2 diabetes. However, the reason for this increase remains unclear. Levels of the dicarbonyl methylglyoxal (MGO) are elevated in diabetic plasma and MGO-modified bovine serum albumin (MGO-BSA) can trigger cellular uptake of TNF. Therefore we tested the hypothesis that MGO-modified proteins may cause TNFalpha secretion in macrophage-like RAW 264.7 cells. Treatment of cells with MGO-BSA induced TNFalpha release in a dose-dependent manner. MGO-modified ribonuclease A and chicken egg ovalbumin had similar effects. Cotreatment of cells with antioxidant reagent N-acetylcysteine (NAC) inhibited MGO-BSA-induced TNFalpha secretion. MGO-BSA stimulated the simultaneous activation of p44/42 and p38 mitogen-activated protein kinase. PD98059, a selective MEK inhibitor, inhibited MGO-BSA-induced TNFalpha release as well as ERK phosphorylation. Pretreatment of cells with NAC also resulted in inhibition of MGO-BSA-induced ERK phosphorylation. MGO-BSA induced dose-dependent NFkappaB activation as shown by electrophoresis mobility shift assay. The MGO-BSA-induced NFkappaB activation was prevented in the presence of PD98059, NAC, and parthenolide, a selective inhibitor of NFkappaB. Furthermore, the NFkappaB inhibitor parthenolide suppressed MGO-BSA-induced TNFalpha secretion. Confocal microscopy using dichlorofluorescein to demonstrate intracellular reactive oxygen species (ROS) showed that MGO-BSA produced more ROS compared with native BSA. MGO-BSA could also stimulate protein kinase C (PKC) translocation to the cell membrane, considered a key signaling pathway in diabetes. However, there was no evidence that PKC was involved in TNFalpha release based on inhibition by calphostin C and staurosporine. Our findings suggest that the presence of chronically elevated levels of MGO-modified bovine serum albumin may contribute to elevated levels of TNFalpha in diabetes.

Anaerobic vs aerobic pathways of carbonyl and oxidant stress in human lens and skin during aging and in diabetes: A comparative analysis

The effects of anaerobic (lens) vs aerobic (skin) environment on carbonyl and oxidant stress are compared using de novo and existing data on advanced glycation and oxidation products in human crystallins and collagen. Almost all modifications increase with age. Methylglyoxal hydroimidazolones, carboxymethyllysine, and carboxyethyllysine are severalfold higher in lens than in skin and markedly increase upon incubation of lens crystallins with 5mM ascorbic acid. In contrast, fructose-lysine, glucosepane crosslinks, glyoxal hydroimidazolones, metal-catalyzed oxidation (allysine), and H(2)O(2)-dependent modifications (2-aminoapidic acid and methionine sulfoxide) are markedly elevated in skin, but relatively suppressed in the aging lens. In both tissues ornithine is the dominant modification, implicating arginine residues as the principal target of the Maillard reaction in vivo. Diabetes (here mostly type 2 studied) increases significantly fructose-lysine and glucosepane in both tissues (P<0.001) but has surprisingly little effect on the absolute level of most other advanced glycation end products. However, diabetes strengthens the Spearman correlation coefficients for age-related accumulation of hydrogen peroxide-mediated modifications in the lens. Overall, the data suggest that oxoaldehyde stress involving methylglyoxal from either glucose or ascorbate is predominant in the aging noncataractous lens, whereas aging skin collagen undergoes combined attack by nonoxidative glucose-mediated modifications, as well as those from metal-catalyzed oxidation and H(2)O(2).

Mechanism of Lysine Oxidation in Human Lens Crystallins during Aging and in Diabetes

Oxidative mechanisms during nuclear sclerosis of the lens are poorly understood, in particular metal-catalyzed oxidation. The lysyl oxidation product adipic semialdehyde (allysine, ALL) and its oxidized end-product 2-aminoadipic acid (2-AAA) were determined as a function of age and presence of diabetes. Surprisingly, whereas both ALL and 2-AAA increased with age and strongly correlated with cataract grade and protein absorbance at 350 nm, only ALL formation but not 2-AAA was increased by diabetes. To clarify the mechanism of oxidation, rabbit lenses were treated with hyperbaric oxygen (HBO) for 48 h, and proteins were analyzed by gas and liquid chromatography mass spectrometry for ALL, 2-AAA, and multiple glycation products. Upon exposure to HBO, rabbit lenses were swollen, and nuclei were yellow. Protein-bound ALL increased 8-fold in the nuclear protein fractions versus controls. A dramatic increase in methyl-glyoxal hydroimidazolone and carboxyethyl-lysine but no increase of 2-AAA occurred, suggesting more drastic conditions are needed to oxidize ALL into 2-AAA. Indeed the latter formed only upon depletion of glutathione and was catalyzed by H2O2. Neither carboxymethyl-lysine nor glyoxal hydroimidazolone, two markers of glyco-/lipoxidation, nor markers of lenticular glycemia (fructose-lysine, glucospane) were elevated by HBO, excluding significant lipid peroxidation and glucose involvement. The findings strongly implicate dicarbonyl/metal catalyzed oxidation of lysine to allysine, whereby low GSH combined with ascorbate-derived H2O2 likely contributes toward 2-AAA formation, since virtually no 2-AAA formed in the presence of methylglyoxal instead of ascorbate. An important translational conclusion is that chelating agents might help delay nuclear sclerosis.

Glucosepane: a poorly understood advanced glycation end product of growing importance for diabetes and its complications

Abstract Advanced glycation end products (AGEs) represent a family of protein, peptide, amino acid, nucleic acid and lipid adducts formed by the reaction of carbonyl compounds derived directly or indirectly from glucose, ascorbic acid and other metabolites such as methylglyoxal. AGE formation in diabetes is of growing importance for their role as markers and potential culprits of diabetic complications, in particular retinopathy, nephropathy and neuropathy. Development of sensitive and specific assays utilizing liquid chromatography mass spectrometry with isotope dilution method has made it possible to detect and quantitate non-UV active AGEs such as carboxymethyl-lysine and glucosepane, the most prevalent AGE and protein crosslink of the extracellular matrix. Below we review studies on AGE formation in two skin biopsies obtained near the closeout of the Diabetes Control and Complications Trial (DCCT), one of which was processed in 2011 for assay of novel AGEs. The results of these analyses show that while several AGEs are associated and predict complication progression, the glucose/fructose-lysine/glucosepane AGE axis is one of the most robust markers for microvascular disease, especially retinopathy, in spite of adjustment for past or future average glycemia. Yet overall little biological and clinical information is available on glucosepane, making this review a call for data in a field of growing importance for diabetes and chronic metabolic diseases of aging.

UVA Light-excited Kynurenines Oxidize Ascorbate and Modify Lens Proteins through the Formation of Advanced Glycation End Products IMPLICATIONS FOR HUMAN LENS AGING AND CATARACT FORMATION

Background: Human lens proteins accumulate pigmented, protein-cross-linked AGE adducts during cataract formation, and the mechanisms of their formation are poorly understood. Results: UVA-excited kynurenines can oxidize ascorbate under anoxic conditions and promote synthesis of AGEs. Conclusion: UVA light-excited kynurenines promote AGE synthesis and contribute to cataract formation. Significance: This study provides a mechanism for UVA light-mediated damage to lens proteins during cataract formation. Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA light-excited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320–400 nm, 100 milliwatts/cm2, 45 min to 2 h), young human lenses (20–36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans.

Identification of glucose-derived cross-linking sites in ribonuclease A.

The accumulation of glycation derived cross-links has been widely implicated in extracellular matrix damage in aging and diabetes, yet little information is available on the cross-linking sites in proteins and the intra- versus intermolecular character of cross-linking. Recently, glucosepane, a 7-membered heterocycle formed between lysine and arginine residues, has been found to be the single major cross-link known so far to accumulate during aging. As an approach toward identification of glucose derived cross-linking sites, we have preglycated ribonuclease A first for for 14 days with 500 mM glucose, followed by a 4-week incubation in absence of glucose. MALDI-TOF analysis of tryptic digests revealed the presence of Amadori products (Delta m/ z = 162) at K1, K7, K37 and K41, in accordance with previous studies. In addition, K66, K98 and K104 were also modified by Amadori products. Intramolecular glucosepane cross-links were observed at K41-R39 and K98-R85. Surprisingly, the only intermolecular cross-link observed was the 3-deoxyglucosone-derived DODIC at K1-R39. The identity of cross-linked peptides was confirmed by sequencing with tandem mass spectrometry. Recombinant ribonuclease A mutants R39A, R85A, and K91A were produced, purified, and glycated to further confirm the importance of these sites on protein cross-linking. These data provide the first documentation that both intramolecular and intermolecular cross-links form in glucose-incubated proteins.

The LEGSKO mouse: a mouse model of age-related nuclear cataract based on genetic suppression of lens glutathione synthesis.

Age-related nuclear cataracts are associated with progressive post-synthetic modifications of crystallins from various physical chemical and metabolic insults, of which oxidative stress is a major factor. The latter is normally suppressed by high concentrations of glutathione (GSH), which however are very low in the nucleus of the old lens. Here we generated a mouse model of oxidant stress by knocking out glutathione synthesis in the mouse in the hope of recapitulating some of the changes observed in human age-related nuclear cataract (ARNC). A floxed Gclc mouse was generated and crossed with a transgenic mouse expressing Cre in the lens to generate the LEGSKO mouse in which de novo GSH synthesis was completely abolished in the lens. Lens GSH levels were reduced up to 60% in homozygous LEGSKO mice, and a decreasing GSH gradient was noticed from cortical to nuclear region at 4 months of age. Oxidation of crystallin methionine and sulfhydryls into sulfoxides was dramatically increased, but methylglyoxal hydroimidazolones levels that are GSH/glyoxalase dependent were surprisingly normal. Homozygous LEGSKO mice developed nuclear opacities starting at 4 months that progressed into severe nuclear cataract by 9 months. We conclude that the LEGSKO mouse lens mimics several features of human ARNC and is thus expected to be a useful model for the development of anti-cataract agents.

Effects of advanced glycation end product modification on proximal tubule epithelial cell processing of albumin.

Aim: The goal of this work is to understand the cellular effects of advanced glycation end product (AGE)-modified protein on renal proximal tubule cells. Background: A major function of the proximal tubule is to reabsorb and process filtered proteins. Diabetes is characterized by increased quantities of tissue and circulating proteins modified by AGEs. Therefore in diabetes, plasma proteins filtered at the glomerulus and presented to the renal proximal tubule are likely to be highly modified by AGEs. Methods: The model system was electrically resistant polarized renal proximal tubular epithelial cells in monolayer culture. The model proteins comprise a well-characterized AGE, methylglyoxal-modified bovine serum albumin (MGO-BSA), and unmodified BSA. Results: Renal proximal tubular cells handle MGO-BSA and native BSA in markedly disparate ways, including differences in: (1) kinetics of binding, uptake, and intracellular accumulation, (2) processing and fragmentation, and (3) patterns of electrical conductance paralleling temporal changes in binding, uptake and processing. Conclusion: These differences support the idea that abnormal protein processing by the renal tubule can be caused by abnormal proteins, thereby forging a conceptual link between the pathogenic role of AGEs and early changes in tubular function that can lead to hypertrophy and nephropathy in diabetes.

Binding of recombinant but not endogenous prion protein to DNA causes DNA internalization and expression in mammalian cells.

Recombinant prion protein, rPrP, binds DNA. Both the KKRPK motif and the octapeptide repeat region of rPrP are essential for maximal binding. rPrP with pathogenic insertional mutations binds more DNA than wild-type rPrP. DNA promotes the aggregation of rPrP and protects its N terminus from proteinase K digestion. When rPrP is mixed with an expression plasmid and Ca2+, the rPrP·DNA complex is taken up by mammalian cells leading to gene expression. In the presence of Ca2+, rPrP by itself is also taken up by cells in a temperature- and pinocytosis-dependent manner. Cells do not take up rPrPΔKKRPK, which lacks the KKRPK motif. Thus, rPrP is the carrier for DNA and the KKRPK motif is essential for its uptake. When mixed with DNA, a pentapeptide KKRPK, but not KKKKK, is sufficient for DNA internalization and expression. In contrast, whereas the normal cellular prion protein, PrPC, on the cell surface can also internalize DNA, the imported DNA is not expressed. These findings may have relevance to the normal functions of PrPC and the pathogenic mechanisms of human prion disease.