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1.  The association between skin collagen glucosepane and past progression of microvascular and neuropathic complications in type 1 diabetes 
PURPOSE
We determined the association between novel and acid-labile skin collagen-linked advanced glycation endproducts (AGEs) and the progression of microvascular and neuropathic complications from baseline to near study closeout in the Diabetes Control and Complications Trial (DCCT).
METHODS
From a skin biopsy obtained near the close of the DCCT, proteolytic collagen digests were analyzed by liquid chromatography/mass spectrometry (LC/MS/MS) for glucosepane (GSPNE), glyoxal and methylglyoxal hydroimidazolones (G-H1 and MG-H1) and the glycation product fructose-lysine(FL) using isotope dilution method.
RESULTS
GSPNE and MG-H1 correlated with age and diabetes duration (p<0.02), while GSPNE and FL correlated with the history of glycemia expressed as mean A1c(p≤0.003). Age and duration-adjusted GSPNE and FL levels were lower in intensive (INT) vs. conventional (CONV) treatment subjects in the primary prevention DCCT cohort (p < 0.0001), and FL lower in INT in the secondary intervention cohort (p < 0.0001). GSPNE was associated with increased incidence of retinopathy progression (odds ratio (OR)/unit increase in GSPNE: 2.5 for 3 step progression on the ETDRS scale, p=0.003) and sustained ≥ 3 microaneurysms (MA) (OR=4.8, p<0.0001) from DCCT baseline up to the time of the biopsy, and prevalence of microalbuminuria or AER>40 mg/24 hr (OR=5.3, P<0.0001), and confirmed clinical neuropathy (OR=3.4, p=0.015) at the time of the biopsy. GSPNE adjusted for mean A1c remained significant for ≥ 3 MA (p=0.0252) and AER (p=0.0006). The strong association of complications with A1c was reduced or eliminated when adjusted for GSPNE.
CONCLUSIONS
Glucosepane is a novel AGE marker of diabetic complications that is robustly associated with nephropathic, retinopathic and neuropathic outcomes despite adjustment for A1c, suggesting that it could be one mediator of these complications with possible diagnostic implications.
doi:10.1016/j.jdiacomp.2012.10.004
PMCID: PMC3577949  PMID: 23153673
Glycemia; retinopathy; nephropathy; collagen; methylglyoxal; advanced glycation endproducts (AGEs)
2.  Advanced Glycation End-Products Reduce Collagen Molecular Sliding to Affect Collagen Fibril Damage Mechanisms but Not Stiffness 
PLoS ONE  2014;9(11):e110948.
Advanced glycation end-products (AGE) contribute to age-related connective tissue damage and functional deficit. The documented association between AGE formation on collagens and the correlated progressive stiffening of tissues has widely been presumed causative, despite the lack of mechanistic understanding. The present study investigates precisely how AGEs affect mechanical function of the collagen fibril – the supramolecular functional load-bearing unit within most tissues. We employed synchrotron small-angle X-ray scattering (SAXS) and carefully controlled mechanical testing after introducing AGEs in explants of rat-tail tendon using the metabolite methylglyoxal (MGO). Mass spectrometry and collagen fluorescence verified substantial formation of AGEs by the treatment. Associated mechanical changes of the tissue (increased stiffness and failure strength, decreased stress relaxation) were consistent with reports from the literature. SAXS analysis revealed clear changes in molecular deformation within MGO treated fibrils. Underlying the associated increase in tissue strength, we infer from the data that MGO modified collagen fibrils supported higher loads to failure by maintaining an intact quarter-staggered conformation to nearly twice the level of fibril strain in controls. This apparent increase in fibril failure resistance was characterized by reduced side-by-side sliding of collagen molecules within fibrils, reflecting lateral molecular interconnectivity by AGEs. Surprisingly, no change in maximum fibril modulus (2.5 GPa) accompanied the changes in fibril failure behavior, strongly contradicting the widespread assumption that tissue stiffening in ageing and diabetes is directly related to AGE increased fibril stiffness. We conclude that AGEs can alter physiologically relevant failure behavior of collagen fibrils, but that tissue level changes in stiffness likely occur at higher levels of tissue architecture.
doi:10.1371/journal.pone.0110948
PMCID: PMC4217736  PMID: 25364829
3.  Small- and Large-Fiber Neuropathy After 40 Years of Type 1 Diabetes 
Diabetes Care  2013;36(11):3712-3717.
OBJECTIVE
To study large- and small-nerve fiber function in type 1 diabetes of long duration and associations with HbA1c and the advanced glycation end products (AGEs) N-ε-(carboxymethyl)lysine (CML) and methylglyoxal-derived hydroimidazolone.
RESEARCH DESIGN AND METHODS
In a long-term follow-up study, 27 persons with type 1 diabetes of 40 ± 3 years duration underwent large-nerve fiber examinations, with nerve conduction studies at baseline and years 8, 17, and 27. Small-fiber functions were assessed by quantitative sensory thresholds (QST) and intraepidermal nerve fiber density (IENFD) at year 27. HbA1c was measured prospectively through 27 years. Serum CML was measured at year 17 by immunoassay. Serum hydroimidazolone was measured at year 27 with liquid chromatography–mass spectrometry.
RESULTS
Sixteen patients (59%) had large-fiber neuropathy. Twenty-two (81%) had small-fiber dysfunction by QST. Heat pain thresholds in the foot were associated with hydroimidazolone and HbA1c. IENFD was abnormal in 19 (70%) and significantly lower in diabetic patients than in age-matched control subjects (4.3 ± 2.3 vs. 11.2 ± 3.5 mm, P < 0.001). IENFD correlated negatively with HbA1c over 27 years (r = −0.4, P = 0.04) and CML (r = −0.5, P = 0.01). After adjustment for age, height, and BMI in a multiple linear regression model, CML was still independently associated with IENFD.
CONCLUSIONS
Small-fiber sensory neuropathy is a major manifestation in type 1 diabetes of 40 years duration and more prevalent than large-fiber neuropathy. HbA1c and the AGEs CML and hydroimidazolone are important risk factors in the development of large- and small-fiber dysfunction in long-term type 1 diabetes.
doi:10.2337/dc13-0788
PMCID: PMC3816884  PMID: 24026557
4.  Green Tea Extract Suppresses the Age-Related Increase in Collagen Crosslinking and Fluorescent Products in C57BL/6 Mice 
Collagen crosslinking during aging in part results from Maillard reaction endproducts of glucose and oxoaldehydes. Because of the tight link between oxidative and carbonyl stress, we hypothesized that natural antioxidants and “nutriceuticals” could block collagen aging in C57BL/6 mice. Six groups of young and adult mice received vitamin C, vitamin E, vitamin C&E, blueberry, green tea extract (GTE), or no treatment for a period of 14 weeks. Body weights and collagen glycation were unaltered by the treatment. However, GTE or vitamin C&E combined blocked tendon crosslinking at 10 months of age (p < 0.05, adult group). GTE also blocked fluorescent products at 385 and 440 nm (p = 0.052 and < 0.05, respectively) and tended to decrease skin pentosidine levels. These results suggest that green tea is able to delay collagen aging by an antioxidant mechanism that is in part duplicated by the combination of vitamin C and E.
PMCID: PMC3561737  PMID: 14743550
Glucose; polyphenols; oxidant stress; vitamin C; vitamin E
5.  The Cation-π Interaction Between Lys53 and the Flavin of Fructosamine Oxidase (FAOX-II) is Critical for Activity† 
Biochemistry  2011;50(37):7977-7986.
Fructosamine oxidases (FAOXs) are flavin-containing enzymes that catalyze the oxidative deglycation of low molecular weight fructosamines or Amadori products. The fructosamine substrate is oxidized by the flavin in the reductive half-reaction, and the reduced flavin is then oxidized by molecular oxygen in the oxidative half-reaction. The crystal structure of FAOX-II from Aspergillus fumigatus reveals a unique interaction between Lys53 and the isoalloxazine. The ammonium nitrogen of the lysine is in contact with and nearly centered over the aromatic ring of the flavin on the si-face. Here, we investigate the importance of this unique interaction on the reactions catalyzed by FAOX by studying both half-reactions of the wild-type and Lys53 mutant enzymes. The positive charge of Lys53 is critical for flavin reduction, but plays very little role in the reaction with molecular oxygen. The conservative mutation of Lys53 to arginine had minor effects on catalysis. However, removing the charge by replacing Lys53 with methionine caused more than a million-fold decrease in flavin reduction, while only slowing the oxygen reaction by ~30-fold.
doi:10.1021/bi1020666
PMCID: PMC3557951  PMID: 21755947
6.  GWAS identifies an NAT2 acetylator status tag single nucleotide polymorphism to be a major locus for skin fluorescence 
Diabetologia  2014;57(8):1623-1634.
Aims/hypothesis
Skin fluorescence (SF) is a non-invasive marker of AGEs and is associated with the long-term complications of diabetes. SF increases with age and is also greater among individuals with diabetes. A familial correlation of SF suggests that genetics may play a role. We therefore performed parallel genome-wide association studies of SF in two cohorts.
Methods
Cohort 1 included 1,082 participants, 35–67 years of age with type 1 diabetes. Cohort 2 included 8,721 participants without diabetes, aged 18–90 years.
Results
rs1495741 was significantly associated with SF in Cohort 1 (p < 6 × 10−10), which is known to tag the NAT2 acetylator phenotype. The fast acetylator genotype was associated with lower SF, explaining up to 15% of the variance. In Cohort 2, the top signal associated with SF (p = 8.3 × 10−42) was rs4921914, also in NAT2, 440 bases upstream of rs1495741 (linkage disequilibrium r2 = 1.0 for rs4921914 with rs1495741). We replicated these results in two additional cohorts, one with and one without type 1 diabetes. Finally, to understand which compounds are contributing to the NAT2–SF signal, we examined 11 compounds assayed from skin biopsies (n = 198): the fast acetylator genotype was associated with lower levels of the AGEs hydroimidazolones of glyoxal (p = 0.017).
Conclusions/interpretation
We identified a robust association between NAT2 and SF in people with and without diabetes. Our findings provide proof of principle that genetic variation contributes to interindividual SF and that NAT2 acetylation status plays a major role.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-014-3286-9) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
doi:10.1007/s00125-014-3286-9
PMCID: PMC4079945  PMID: 24934506
Acetylation; Genome-wide association study; NAT2; Skin autofluorescence; Skin fluorescence; Skin intrinsic fluorescence
7.  Vitamin C–mediated Maillard Reaction in the Lens Probed in a Transgenic-mouse Model 
Aging human lens crystallins are progressively modified by yellow glycation, oxidation, and cross-linked carbonyl compounds that have deleterious properties on protein structure and stability. In order to test the hypothesis that some of these compounds originate from oxidized vitamin C, we have overexpressed the human vitamin C transporter 2 (hSCVT2) in the mouse lens. We find that levels of ascorbic and dehydroascorbic acid are highly elevated compared to the wild type and that the lenses have accumulated yellow color and advanced Maillard reaction products identical with those of the human lens. Treatment of the mice with nucleophilic inhibitors can slow down the process, opening new avenues for the pharmacological prevention of senile cataractogenesis.
doi:10.1196/annals.1433.064
PMCID: PMC3485640  PMID: 18448816
glycation; ascorbic acid; crystallin; cross-linking; aging
8.  ANAEROBIC VS. AEROBIC PATHWAYS OF CARBONYL AND OXIDANT STRESS IN HUMAN LENS AND SKIN DURING AGING AND IN DIABETES: A COMPARATIVE ANALYSIS 
Free radical biology & medicine  2010;49(5):847-856.
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 (MG-H1), carboxymethyl-lysine (CML), and carboxyethyl-lysine (CEL) are several folds higher in lens than skin, and markedly increase upon incubation of lens crystallins with 5 mM ascorbic acid. Vice-versa, fructose-lysine, glucosepane crosslinks, glyoxal hydroimidazolones (G-H1), metal catalyzed oxidation (allysine) and H2O2 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 (AGEs) . However, diabetes strengthens the Spearman correlation coefficients for age-related accumulation of hydrogen peroxide mediated modifications in the lens. Overall, the data suggest oxoaldehyde stress involving methylglyoxal from either glucose or ascorbate is predominant in the aging non-cataractous lens, while aging skin collagen undergoes combined attack by non-oxidative glucose mediated modifications, as well as those from metal catalyzed oxidation and H2O2.
doi:10.1016/j.freeradbiomed.2010.06.003
PMCID: PMC2910832  PMID: 20541005
crystallins; collagen; glycation; oxidative stress; methylglyoxal; metals
9.  Topical application of L-arginine blocks advanced glycation by ascorbic acid in the lens of hSVCT2 transgenic mice 
Molecular Vision  2011;17:2221-2227.
Purpose
Previous experiments from our laboratory showed that the oral intake of selected guanidino compounds could block the formation of crystallin-bound advanced ascorbylation products. Here we tested whether these were also active when applied as eye drops.
Methods
Two month old hSVCT2 transgenic mice (n=10) were treated twice daily with one drop of 0.1% L-arginine, γ-guanidinobutyric acid (GBA), penicillamine (PA) or N-acetylcysteine (NAC) in one eye and vehicle only in the other eye. After seven months, lens crystallins were isolated, dialyzed, and proteolytically digested to determine the protein-bound fluorescence at 335/385 and 370/440 nm excitation/emission and the advanced glycation/ascorbylation endproducts carboxymethyl-lysine (CML), carboxyethyl-lysine (CEL), glucosepane, glyoxal, and methylglyoxal hydroimidazolones G-H1 and MG-H1. The topical uptake of L-arginine and NAC was also evaluated in vitro and in vivo in rabbit lens.
Results
In hSVCT2 mice, L-arginine decreased 335/385 and 370/440 nm fluorescence by 40% (p<0.001), CML, CEL, and glucosepane crystallin crosslinks by 35% (p<0.05), 30% (p<0.05), and 37% (p<0.05), respectively, without affecting MG-H1 and G-H1. NAC decreased 335/385 nm fluorescence by 50% (p<0.001) but, like PA and GBA, had no effect on other modifications. L-Arginine uptake into rabbit eyes treated topically reached identical lenticular plateau levels (~400 nmol/g wet weight) at 0.5% and 2.0% but levels remained three times higher at 5 h at 2% versus 0.5% concentration, respectively. In vitro studies showed a 100 fold higher L-arginine level than NAC levels, implicating high affinity uptake of the former.
Conclusions
L-Arginine when applied both orally and topically is a potent and broad suppressor of advanced ascorbylation in the lens. Its uptake in rabbit lens upon topical application suggests transcorneal uptake into the human lens should be feasible for testing its potential anticataract properties in clinical trials.
PMCID: PMC3164690  PMID: 21897744
10.  Skin Collagen Glycation, Glycoxidation, and Crosslinking Are Lower in Subjects With Long-Term Intensive Versus Conventional Therapy of Type 1 Diabetes 
Diabetes  1999;48(4):870-880.
The relationships between long-term intensive control of glycemia and indicators of skin collagen glycation (furosine), glycoxidation (pentosidine and N∊-[carboxymethyl]-lysine [CML]), and crosslinking (acid and pepsin solubility) were examined in 216 patients with type 1 diabetes from the primary prevention and secondary intervention cohorts of the Diabetes Control and Complications Trial. By comparison with conventional treatment, 5 years of intensive treatment was associated with 30–32% lower furosine, 9% lower pentosidine, 9–13% lower CML, 24% higher acid-soluble collagen, and 50% higher pepsin-soluble collagen. All of these differences were statistically significant in the subjects of the primary prevention cohort (P < 0 .006–0.001) and also of the secondary intervention cohort (P < 0.015–0.001) with the exception of CML and acid-soluble collagen. Age- and duration-adjusted collagen variables were significantly associated with the HbA1c value nearest the biopsy and with cumulative prior HbA1c values. Multiple logistic regression analyses with six nonredundant collagen parameters as independent variables and various expressions of retinopathy, nephropathy, and neuropathy outcomes as dependent variables showed that the complications were significantly associated with the full set of collagen variables. Surprisingly, the percentage of total variance (R2) in complications explained by the collagen variables ranged from 19 to 36% with the intensive treatment and from 14 to 51% with conventional treatment. These associations generally remained significant even after adjustment for HbA1c, and, most unexpectedly, in conventionally treated subjects, glycated collagen was the parameter most consistently associated with diabetic complications. Continued monitoring of these subjects may determine whether glycation products in the skin, and especially the early Amadori product (furosine), have the potential to be predictors of the future risk of developing complications, and perhaps be even better predictors than glycated hemoglobin (HbA1c).
PMCID: PMC2862597  PMID: 10102706
11.  Identification of Glucose-Derived Cross-Linking Sites in Ribonuclease A 
Journal of proteome research  2008;7(7):2756-2768.
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 (Δm/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.
doi:10.1021/pr700874a
PMCID: PMC2574603  PMID: 18500835
glycation; ribonuclease A; cross-linking; glycation sites; glucosepane; DODIC; AGEs
12.  Isolation, Purification and Characterization of Histidino-Threosidine, a Novel Maillard Reaction Protein Crosslink from Threose, Lysine and Histidine 
We isolated a novel acid-labile yellow chromophore from the incubation of lysine, histidine and D-threose and identified its chemical structure by one and two-dimensional 1H and DEPT NMR spectroscopy combined with LC-tandem mass spectrometry. This new cross-link exhibits a UV absorbance maximum at 305 nm and a molecular mass of 451 Da. The proposed structure is 2-amino-5-(3-((4-(2-amino-2-carboxyethyl)-1H-imidazol-1-yl)methyl)-4-(1,2-dihydroxyethyl)-2-formyl-1H-pyrrol-1-yl)pentatonic acid, a cross-link between lysine and histidine with addition of two threose molecules. It was in part deduced and confirmed through synthesis of the analogous compound from n-butylamine, imidazole and D-threose. We assigned the compound the trivial name histidino-threosidine. Systemic incubation revealed that histidino-threosidine can be formed in low amounts from fructose, glyceraldehyde, methylglyoxal, glycolaldehyde, ascorbic acid, and dehydroascorbic acid, but at a much higher yield with degradation products of ascorbic acid, i.e. threose, erythrose, and erythrulose. Bovine lens protein incubated with 10 and 50 mM threose for two weeks yielded 560 and 2840 pmol/mg histidino-threosidine. Histidino-threosidine is to our knowledge the first Maillard reaction product known to involve histidine in a crosslink.
doi:10.1016/j.abb.2007.03.007
PMCID: PMC1978223  PMID: 17466255
advanced glycation end-product; yellow chromophore; fluorophore; ascorbic acid; lens; glycation; aging; erythrulose
13.  Advanced Glycation End-Products as Markers of Aging and Longevity in the Long-Lived Ansell’s Mole-Rat (Fukomys anselli) 
Mole-rat of the genus Fukomys are mammals whose life span is strongly influenced by reproductive status with breeders far outliving nonbreeders. This raises the important question of whether increased longevity of the breeders is reflected in atypical expression of biochemical markers of aging. Here, we measured markers of glycation and advanced glycation end-products formed in insoluble skin collagen of Ansell’s mole-rat Fukomys anselli as a function of age and breeding status. Glucosepane, pentosidine, and total advanced glycation end-product content significantly increased with age after correction for breeder status and sex. Unexpectedly, total advanced glycation end-products, glucosepane, and carboxymethyl-lysine (CML) were significantly higher in breeders versus nonbreeders suggesting that breeders have evolved powerful defenses against combined oxidant and carbonyl stress compared with nonbreeders. Most interestingly, when compared with other mammals, pentosidine formation rate was lower in mole-rat compared with other short-lived rodents confirming previous observations of an inverse relationship between longevity and pentosidine formation rates in skin collagen.
doi:10.1093/gerona/glr208
PMCID: PMC3348492  PMID: 22156473
Longevity; Collagen; Glycemia; Markers; Oxidative stress
14.  The LEGSKO Mouse: A Mouse Model of Age-Related Nuclear Cataract Based on Genetic Suppression of Lens Glutathione Synthesis 
PLoS ONE  2012;7(11):e50832.
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.
doi:10.1371/journal.pone.0050832
PMCID: PMC3511334  PMID: 23226398
15.  Oxygen Reactivity in Flavoenzymes: Context Matters 
Journal of the American Chemical Society  2011;133(42):16809-16811.
Many flavoenzymes – oxidases and monooxygenases – react faster with oxygen than free flavins do. There are many ideas on how enzymes cause this. Recent work has focused on the importance of a positive charge near N5 of the reduced flavin. Fructosamine oxidase has a lysine near N5 of its flavin. We measured a rate constant of 1.6 × 105 M−1s−1 for its reaction with oxygen. The Lys276Met mutant reacted with a rate constant of 291 M−1s−1, suggesting an important role for this lysine in oxygen activation. The dihydroorotate dehydrogenases from E. coli and L. lactis also have a lysine near N5 of the flavin. They react with O2 with rate constants of 6.2 × 104 M−1s−1 and 3.0 × 103 M−1s−1, respectively. The Lys66Met and Lys43Met mutant enzymes react with rate constants that are nearly the same as the wild-type enzymes, demonstrating that simply placing a positive charge near N5 of the flavin does not guarantee increased oxygen reactivity. Our results show that the lysine near N5 does not exert an effect without an appropriate context; evolution did not find only one mechanism for activating the reaction of flavins with O2.
doi:10.1021/ja2081873
PMCID: PMC3203534  PMID: 21958058
Dihydroorotate dehydrogenase; Fructosamine oxidase; Oxygen reactivity; Oxygen activation; Flavin
17.  Protection From Retinopathy and Other Complications in Patients With Type 1 Diabetes of Extreme Duration 
Diabetes Care  2011;34(4):968-974.
OBJECTIVE
To assess complication prevalence and identify protective factors in patients with diabetes duration of ≥50 years. Characterization of a complication-free subgroup in this cohort would suggest that some individuals are protected from diabetes complications and allow identification of endogenous protective factors.
RESEARCH DESIGN AND METHODS
Cross-sectional, observational study of 351 U.S. residents who have survived with type 1 diabetes for ≥50 years (Medalists). Retinopathy, nephropathy, neuropathy, and cardiovascular disease were assessed in relation to HbA1c, lipids, and advanced glycation end products (AGEs). Retrospective chart review provided longitudinal ophthalmic data for a subgroup.
RESULTS
A high proportion of Medalists remain free from proliferative diabetic retinopathy (PDR) (42.6%), nephropathy (86.9%), neuropathy (39.4%), or cardiovascular disease (51.5%). Current and longitudinal (the past 15 years) glycemic control were unrelated to complications. Subjects with high plasma carboxyethyl-lysine and pentosidine were 7.2-fold more likely to have any complication. Of Medalists without PDR, 96% with no retinopathy progression over the first 17 years of follow-up did not experience retinopathy worsening thereafter.
CONCLUSIONS
The Medalist population is likely enriched for protective factors against complications. These factors might prove useful to the general population with diabetes if they can be used to induce protection against long-term complications. Specific AGE combinations were strongly associated with complications, indicating a link between AGE formation or processing with development of diabetic vasculopathy.
doi:10.2337/dc10-1675
PMCID: PMC3064059  PMID: 21447665
18.  Partial Characterization of the Molecular Nature of Collagen-Linked Fluorescence: Role of Diabetes and End-Stage Renal Disease 
Collagen-linked fluorescence at excitation/emission 370/440 nm has widely been used as a marker for advanced glycation in studies of aging, diabetic complications and end-stage renal disease (ESRD). Diagnostic devices measuring skin autofluorescence at this wavelength revealed an association between fluorescence and cardiovascular morbidity and mortality. We now report the presence of a major fluorophore (LW-1) in human skin collagen which increases with age, diabetes and ESRD. It has a molecular weight of 623.2 Da, a UV maximum at 348 nm, and involves a lysine residue in an aromatic ring. LW-1 could not be synthesized using traditional glycation chemistry suggesting a complex mechanism of formation, perhaps related to hypoxia since elevated levels were also found in nondiabetic individuals with chronic lung disease.
doi:10.1016/j.abb.2009.10.013
PMCID: PMC2812593  PMID: 19879855
aging; glycation; skin; structure; marker
19.  Favored and disfavored pathways of protein crosslinking by glucose: glucose lysine dimer (GLUCOLD) and crossline versus glucosepane 
Amino acids  2010;40(1):167-181.
We describe the isolation and molecular characterization of a novel glucose-lysine dimer crosslink 1,3-bis-(5-amino-5-carboxypentyl)-4-(1′,2′,3′,4′-tetrahydroxybutyl)-3H-imidazolium salt, named GLUCOLD. GLUCOLD was easily formed from the Amadori product (fructose–lysine). However, when BSA was incubated with 100 mM glucose for 25 days, the levels of the lysine-lysine glucose crosslinks GLUCOLD and CROSSLINE were only 21 and <1 pmol/mg, respectively, compared to 611 pmol/mg protein for the lysine-arginine GLUCOSEPANE crosslink, in spite of more than 20 potential lysine-lysine crosslinking sites in the protein. Mechanistic investigation revealed that metal-free phosphate ions catalyzed formation of fructose–lysine and all three crosslinks from amino acids, while cationic MOPS buffer had an opposite effect. This together with the rapid formation of N6-1,4-dideoxy-5,6-dioxoglucosone derivatives by dicarbonyl trapping agents, such as 1,2-diaminobenzene or γ-guanidinobutyric acid, strongly suggests that enolization of the Amadori product and trapping of the 5,6-dioxo derivative by arginine residues constitutes the major pathway for glucose-mediated crosslinking in proteins.
doi:10.1007/s00726-010-0631-2
PMCID: PMC2972412  PMID: 20607325
Advanced glycation end-product; Maillard reaction; Glycation; Crosslink; Diabetes
20.  RAGE and Modulation of Ischemic Injury in the Diabetic Myocardium 
Diabetes  2008;57(7):1941-1951.
OBJECTIVE—Subjects with diabetes experience an increased risk of myocardial infarction and cardiac failure compared with nondiabetic age-matched individuals. The receptor for advanced glycation end products (RAGE) is upregulated in diabetic tissues. In this study, we tested the hypothesis that RAGE affected ischemia/reperfusion (I/R) injury in the diabetic myocardium. In diabetic rat hearts, expression of RAGE and its ligands was enhanced and localized particularly to both endothelial cells and mononuclear phagocytes.
RESEARCH DESIGN AND METHODS—To specifically dissect the impact of RAGE, homozygous RAGE-null mice and transgenic (Tg) mice expressing cytoplasmic domain-deleted RAGE (DN RAGE), in which RAGE-dependent signal transduction was deficient in endothelial cells or mononuclear phagocytes, were rendered diabetic with streptozotocin. Isolated perfused hearts were subjected to I/R.
RESULTS—Diabetic RAGE-null mice were significantly protected from the adverse impact of I/R injury in the heart, as indicated by decreased release of LDH and lower glycoxidation products carboxymethyl-lysine (CML) and pentosidine, improved functional recovery, and increased ATP. In diabetic Tg mice expressing DN RAGE in endothelial cells or mononuclear phagocytes, markers of ischemic injury and CML were significantly reduced, and levels of ATP were increased in heart tissue compared with littermate diabetic controls. Furthermore, key markers of apoptosis, caspase-3 activity and cytochrome c release, were reduced in the hearts of diabetic RAGE-modified mice compared with wild-type diabetic littermates in I/R.
CONCLUSIONS—These findings demonstrate novel and key roles for RAGE in I/R injury in the diabetic heart.
doi:10.2337/db07-0326
PMCID: PMC2453611  PMID: 18420491
21.  Glycation and Carboxymethyllysine Levels in Skin Collagen Predict the Risk of Future 10-Year Progression of Diabetic Retinopathy and Nephropathy in the Diabetes Control and Complications Trial and Epidemiology of Diabetes Interventions and Complications Participants With Type 1 Diabetes 
Diabetes  2005;54(11):3103-3111.
Several mechanistic pathways linking hyperglycemia to diabetes complications, including glycation of proteins and formation of advanced glycation end products (AGEs), have been proposed. We investigated the hypothesis that skin collagen glycation and AGEs predict the risk of progression of microvascular disease. We measured glycation products in the skin collagen of 211 Diabetes Control and Complications Trial (DCCT) volunteers in 1992 who continued to be followed in the Epidemiology of Diabetes Interventions and Complications study for 10 years. We determined whether the earlier measurements of glycated collagen and AGE levels correlated with the risk of progression of retinopathy and nephropathy from the end of the DCCT to 10 years later. In multivariate analyses, the combination of furosine (glycated collagen) and carboxymethyllysine (CML) predicted the progression of retinopathy (χ2 = 59.4, P < 0.0001) and nephropathy (χ2 = 18.2, P = 0.0001), even after adjustment for mean HbA1c (A1C) (χ2 = 32.7, P < 0.0001 for retinopathy) and (χ2 = 12.8, P = 0.0016 for nephropathy). The predictive effect of A1C vanished after adjustment for furosine and CML (χ2 = 0.0002, P = 0.987 for retinopathy and χ2 = 0.0002, P = 0.964 for nephropathy). Furosine explained more of the variation in the 10-year progression of retinopathy and nephropathy than did CML. These results strengthen the role of glycation of proteins and AGE formation in the pathogenesis of retinopathy and nephropathy. Glycation and subsequent AGE formation may explain the risk of these complications associated with prior A1C and provide a rational basis for the phenomenon of “metabolic memory” in the pathogenesis of these diabetes complications.
PMCID: PMC2622724  PMID: 16249432
22.  Nucleophilic compounds decrease advanced glycation end products (AGEs) from ascorbic acid in the hSVCT2 transgenic mouse model of lenticular aging 
Purpose
Senile cataracts are associated with oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. This process is partially explained by advanced glycation endproducts (AGEs) from ascorbic acid (ASA), as unequivocally demonstrated in our hSVCT2 transgenic mouse(PNAS 103:16912, 2006). We now present the first pharmacological intervention study against ascorbylation in these mice.
Methods
Five groups of mice (10 mice/group) were fed from two to nine months a diet containing 0.1% (wt/wt) aminoguanidine (AG), pyridoxamine (PM), penicillamine (PA), and nucleophilic compounds NC-I and NC-II. AGEs were determined in crystallin digests using HPLC, LC-MS or GC-MS. In vitro incubations of lens protein extract with ASA or dehydroascorbic aicd (DHA) were also performed.
Results
ASA level increased ~10 fold in all groups and was unaffected by treatment. AGEs were several fold increased in transgenic compared to control lenses. Body weight, food intake, lenticular glutathione and glycated lysine level were unaltered. In vitro, all compounds inhibited AGE formation. In vivo, NC-I and NC-II significantly decreased protein fluorescence at λex335/em385 (p=0.045, 0.017, respectively) and λex370/em440 (p=0.029, 0.007, respectively). Other inhibitors had no effect. After 7 months, only NC-1 and NC-2 induced a 50 % reduction in pentosidine (n.s, p=0.035 respectively). NC-1 also decreased carboxymethyllysine (CML) (p=0.032) and carboxyethyllysine (CEL) (p= n.s). Fluorescent crosslink K2P was decreased by NC-1, NC-2, AG and PM (p= n.s).
Conclusions
Pharmacologically blocking protein ascorbylation with absorbable guanidino compounds is feasible and may represent a new strategy for the delay of age-related nuclear sclerosis of the lens.
doi:10.1167/iovs.08-1813
PMCID: PMC2576497  PMID: 18421088

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