α-Crystallin chaperone mimetic drugs inhibit lens γ-crystallin aggregation: Potential role for cataract prevention.

Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency-approved drugs and natural compounds for their ability to suppress or worsen H2O2 and/or heat-mediated aggregation of bovine γ-crystallins. The top two drugs, closantel (C), an antihelminthic drug, and gambogic acid (G), a xanthonoid, attenuated thermal-induced protein unfolding and aggregation as shown by turbidimetry fluorescence spectroscopy dynamic light scattering and electron microscopy of human or mouse recombinant crystallins. Furthermore, binding studies using fluorescence inhibition and hydrophobic pocket-binding molecule bis-8-anilino-1-naphthalene sulfonic acid revealed static binding of C and G to hydrophobic sites with medium-to-low affinity. Molecular docking to HγD and other γ-crystallins revealed two binding sites, one in the "NC pocket" (residues 50-150) of HγD and one spanning the "NC tail" (residues 56-61 to 168-174 in the C-terminal domain). Multiple binding sites overlap with those of the protective mini αA-crystallin chaperone MAC peptide. Mechanistic studies using bis-8-anilino-1-naphthalene sulfonic acid as a proxy drug showed that it bound to MAC sites, improved Tm of both H2O2 oxidized and native human gamma D, and suppressed turbidity of oxidized HγD, most likely by trapping exposed hydrophobic sites. The extent to which these drugs act as α-crystallin mimetics and reduce cataract progression remains to be demonstrated. This study provides initial insights into binding properties of C and G to γ-crystallins.

Plasma advanced glycation end products and the subsequent risk of microvascular complications in type 1 diabetes in the DCCT/EDIC.

INTRODUCTION: To assess impact of glycemic control on plasma protein-bound advanced glycation end products (pAGEs) and their association with subsequent microvascular disease. RESEARCH DESIGN AND METHODS: Eleven pAGEs were measured by liquid chromatography-mass spectrometry in banked plasma from 466 participants in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study at three time points (TPs): DCCT year 4 (TP1) and year 8 (TP2) and EDIC year 5/6 (TP3). Correlation coefficients assessed cross-sectional associations, and Cox proportional hazards models assessed associations with subsequent risk of microvascular complications through EDIC year 24. RESULTS: Glucose-derived glycation products fructose-lysine (FL), glucosepane (GSPN) and carboxymethyl-lysine (CML) decreased with intensive glycemic control at both TP1 and TP2 (p<0.0001) but were similar at TP3, and correlated with hemoglobin A1c (HbA1c). At TP1, the markers were associated with the subsequent risk of several microvascular outcomes. These associations did not remain significant after adjustment for HbA1c, except methionine sulfoxide (MetSOX), which remained associated with diabetic kidney disease. In unadjusted models using all 3 TPs, glucose-derived pAGEs were associated with subsequent risk of proliferative diabetic retinopathy (PDR, p<0.003), clinically significant macular edema (CSME, p<0.015) and confirmed clinical neuropathy (CCN, p<0.018, except CML, not significant (NS)). Adjusted for age, sex, body mass index, diabetes duration and mean updated HbA1c, the associations remained significant for PDR (FL: p<0.002, GSPN: p≤0.02, CML: p<0.003, pentosidine: p<0.02), CMSE (CML: p<0.03), albuminuria (FL: p<0.02, CML: p<0.03) and CCN (FL: p<0.005, GSPN : p<0.003). CONCLUSIONS: pAGEs at TP1 are not superior to HbA1c for risk prediction, but glucose-derived pAGEs at three TPs and MetSOX remain robustly associated with progression of microvascular complications in type 1 diabetes even after adjustment for HbA1c and other factors.

Vitamin C is a source of oxoaldehyde and glycative stress in age-related cataract and neurodegenerative diseases.

Oxoaldehyde stress has recently emerged as a major source of tissue damage in aging and age-related diseases. The prevailing mechanism involves methylglyoxal production during glycolysis and modification of arginine residues through the formation of methylglyoxal hydroimidazolones (MG-H1). We now tested the hypothesis that oxidation of vitamin C (ascorbic acid or ASA) contributes to this damage when the homeostatic redox balance is disrupted especially in ASA-rich tissues such as the eye lens and brain. MG-H1 measured by liquid chromatography mass spectrometry is several fold increased in the lens and brain from transgenic mice expressing human vitamin C transporter 2 (hSVCT2). Similarly, MG-H1 levels are increased two- to fourfold in hippocampus extracts from individuals with Alzheimer's disease (AD), and significantly higher levels are present in sarkosyl-insoluble tissue fractions from AD brain proteins than in the soluble fractions. Moreover, immunostaining with antibodies against methylglyoxal hydroimidazolones reveals similar increase in substantia nigra neurons from individuals with Parkinson's disease. Results from an in vitro incubation experiment suggest that accumulated catalytic metal ions in the hippocampus during aging could readily accelerate ASA oxidation and such acceleration was significantly enhanced in AD. Modeling studies and intraventricular injection of 13 C-labeled ASA revealed that ASA backbone carbons 4-6 are incorporated into MG-H1 both in vitro and in vivo, likely via a glyceraldehyde precursor. We propose that drugs that prevent oxoaldehyde stress or excessive ASA oxidation may protect against age-related cataract and neurodegenerative diseases.

Collagen methionine sulfoxide and glucuronidine/LW-1 are markers of coronary artery disease in long-term survivors with type 1 diabetes. The Dialong study.

OBJECTIVES: Type 1 diabetes is a risk factor for coronary heart disease. The underlying mechanism behind the accelerated atherosclerosis formation is not fully understood but may be related to the formation of oxidation products and advanced glycation end-products (AGEs). We aimed to examine the associations between the collagen oxidation product methionine sulfoxide; the collagen AGEs methylglyoxal hydroimidazolone (MG-H1), glucosepane, pentosidine, glucuronidine/LW-1; and serum receptors for AGE (RAGE) with measures of coronary artery disease in patients with long-term type 1 diabetes. METHODS: In this cross-sectional study, 99 participants with type 1 diabetes of ≥ 45-year duration and 63 controls without diabetes had either established coronary heart disease (CHD) or underwent Computed Tomography Coronary Angiography (CTCA) measuring total, calcified and soft/mixed plaque volume. Skin collagen methionine sulfoxide and AGEs were measured by liquid chromatography-mass spectrometry and serum sRAGE/esRAGE by ELISA. RESULTS: In the diabetes group, low levels of methionine sulfoxide (adjusted for age, sex and mean HbA1c) were associated with normal coronary arteries, OR 0.48 (95% CI 0.27-0.88). Glucuronidine/LW-1 was associated with established CHD, OR 2.0 (1.16-3.49). MG-H1 and glucuronidine/LW-1 correlated with calcified plaque volume (r = 0.23-0.28, p<0.05), while pentosidine correlated with soft/mixed plaque volume (r = 0.29, p = 0.008), also in the adjusted analysis. CONCLUSIONS: Low levels of collagen-bound methionine sulfoxide were associated with normal coronary arteries while glucuronidine/LW-1 was positively associated with established CHD in long-term type 1 diabetes, suggesting a role for metabolic and oxidative stress in the formation of atherosclerosis in diabetes.

Evidence of glucuronidation of the glycation product LW-1: tentative structure and implications for the long-term complications of diabetes.

LW-1 is a collagen-linked blue fluorophore whose skin levels increase with age, diabetes and end-stage renal disease (ESRD), and correlate with the long-term progression of microvascular disease and indices of subclinical cardiovascular disease in type 1 diabetes. The chemical structure of LW-1 is still elusive, but earlier NMR analyses showed it has a lysine residue in an aromatic ring coupled to a sugar molecule reminiscent of advanced glycation end-products (AGEs). We hypothesized and demonstrate here that the unknown sugar is a N-linked glucuronic acid. LW-1 was extracted and highly purified from ~99 g insoluble skin collagen obtained at autopsy from patients with diabetes/ESRD using multiple rounds of proteolytic digestion and purification by liquid chromatography (LC). Advanced NMR techniques (1H-NMR, 13C-NMR, 1H-13C HSQC, 1H-1H TOCSY, 1H-13C HMBC) together with LC-mass spectrometry (MS) revealed a loss of 176 amu (atomic mass unit) unequivocally point to the presence of a glucuronic acid moiety in LW-1. To confirm this data, LW-1 was incubated with ß-glycosidases (glucosidase, galactosidase, glucuronidase) and products were analyzed by LC-MS. Only glucuronidase could cleave the sugar from the parent molecule. These results establish LW-1 as a glucuronide, now named glucuronidine, and for the first time raise the possible existence of a "glucuronidation pathway of diabetic complications". Future research is needed to rigorously probe this concept and elucidate the molecular origin and biological source of a circulating glucuronidine aglycone.

Hand, shoulder and back stiffness in long-term type 1 diabetes; cross-sectional association with skin collagen advanced glycation end-products. The Dialong study.

AIMS: We aimed to: (i) estimate the prevalence of Dupuytren's disease, trigger finger, carpal tunnel syndrome and frozen shoulder; (ii) assess stiffness of the hand, shoulder and back; and (iii) explore the association of joint stiffness with both long-term HbA1c and collagen advanced glycation end-products (AGEs) in long-term type 1 diabetes mellitus (T1DM). METHODS: Patients with T1DM from 1970 or earlier attending a specialized diabetes center were included in this cross-sectional controlled study. We collected HbA1/HbA1c measurements from 1980 to 2015 and data on hand and shoulder diagnoses and joint stiffness through interviews, charts, and standardized examination. Skin biopsies were analyzed for collagen AGEs by liquid chromatography-mass spectrometry. RESULTS: Lifetime prevalence of hand and shoulder diagnoses in the diabetes group (n=102) ranged from 37%-76% (frozen shoulder) versus 11%-15% in controls (n=73) (p<0.001). There was an association between joint stiffness and long-term HbA1c (odds ratio 2.01 [95% CI 1.10-3.7]) and the AGEs methyl-glyoxal-lysine-dimer (odds ratio 1.68 [95% CI 1.03-2.73]) and pentosidine (odds ratio 1.81 [95% CI 1.04-3.16]). CONCLUSIONS: Patients with T1DM >45years had a very high prevalence of hand and shoulder diagnoses versus controls. Joint stiffness was associated with collagen AGEs. However, joint biopsies and prospective studies must explore this association further.

Evidence of Dual Mechanisms of Glutathione Uptake in the Rodent Lens: A Novel Role for Vitreous Humor in Lens Glutathione Homeostasis.

PURPOSE: Lens glutathione synthesis knockout (LEGSKO) mouse lenses lack de novo glutathione (GSH) synthesis but still maintain >1 mM GSH. We sought to determine the source of this residual GSH and the mechanism by which it accumulates in the lens. METHODS: Levels of GSH, glutathione disulfide (GSSG), and GSH-related compounds were measured in vitro and in vivo using isotope standards and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. RESULTS: Wild-type (WT) lenses could accumulate GSH from γ-glutamylcysteine and glycine or from intact GSH, but LEGSKO lenses could only accumulate GSH from intact GSH, indicating that LEGSKO lens GSH content is not due to synthesis by a salvage pathway. Uptake of GSH in cultured lenses occurred at the same rate for LEGSKO and WT lenses, could not be inhibited, and occurred primarily through cortical fiber cells. In contrast, uptake of GSH from aqueous humor could be competitively inhibited and showed an enhanced Km in LEGSKO lenses. Mouse vitreous had >1 mM GSH, whereas aqueous had <20 µM GSH. Testing physiologically relevant GSH concentrations for uptake in vivo, we found that both LEGSKO and WT lenses could obtain GSH from the vitreous but not from the aqueous. Vitreous rapidly accumulated GSH from the circulation, and depletion of circulating GSH reduced vitreous but not aqueous GSH. CONCLUSIONS: The above data provide, for the first time, evidence for the existence of dual mechanisms of GSH uptake into the lens, one mechanism being a passive, high-flux transport through the vitreous exposed side of the lens versus an active, carrier-mediated uptake mechanism at the anterior of the lens.

The pecking order of skin Advanced Glycation Endproducts (AGEs) as long-term markers of glycemic damage and risk factors for micro- and subclinical macrovascular disease progression in Type 1 diabetes.

To date more than 20 glycation products were identified, of which ~15 in the insoluble human skin collagen fraction. The goal of this review is to streamline 30 years of research and ask a set of important questions: in Type 1 diabetes which glycation products correlate best with 1) past mean glycemia 2) reversibility with improved glycemic control, 2) cross-sectional severity of retinopathy, nephropathy and neuropathy and 3) the future long-term risk of progression of micro- and subclinical macrovascular disease. The trio of glycemia related glycation markers furosine (FUR)/fructose-lysine (FL), glucosepane and methylglyoxal hydroimidazolone (MG-H1) emerges as extraordinarily strong predictors of existing and future microvascular disease progression risk despite adjustment for both past and prospective A1c levels. X(2) values are up to 25.1, p values generally less than 0.0001, and significance remains after adjustment for various factors such as A1c, former treatment group, log albumin excretion rate, abnormal autonomic nerve function and LDL levels at baseline. In contrast, subclinical cardiovascular progression is more weakly correlated with AGEs/glycemia with X(2) values < 5.0 and p values generally < 0.05 after all adjustments. Except for future carotid intima-media thickness, which correlates with total AGE burden (MG-H1, pentosidine, fluorophore LW-1 and decreased collagen solubility), adjusted FUR and Collagen Fluorescence (CLF) are the strongest markers for future coronary artery calcium deposition, while cardiac hypertrophy is associated with LW-1 and CLF adjusted for A1c. We conclude that a robust clinical skin biopsy AGE risk panel for microvascular disease should include at least FUR/FL, glucosepane and MG-H1, while a macrovascular disease risk panel should include at least FL/FUR, MG-H1, LW-1 and CLF.

New Locus for Skin Intrinsic Fluorescence in Type 1 Diabetes Also Associated With Blood and Skin Glycated Proteins.

Skin fluorescence (SF) noninvasively measures advanced glycation end products (AGEs) in the skin and is a risk indicator for diabetes complications. N-acetyltransferase 2 (NAT2) is the only known locus influencing SF. We aimed to identify additional genetic loci influencing SF in type 1 diabetes (T1D) through a meta-analysis of genome-wide association studies (N = 1,359) including Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) and Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR). A locus on chromosome 1, rs7533564 (P = 1.9 × 10(-9)), was associated with skin intrinsic fluorescence measured by SCOUT DS (excitation 375 nm, emission 435-655 nm), which remained significant after adjustment for time-weighted HbA1c (P = 1.7 × 10(-8)). rs7533564 was associated with mean HbA1c in meta-analysis (P = 0.0225), mean glycated albumin (P = 0.0029), and glyoxal hydroimidazolones (P = 0.049), an AGE measured in skin biopsy collagen, in DCCT. rs7533564 was not associated with diabetes complications in DCCT/EDIC or with SF in subjects without diabetes (nondiabetic [ND]) (N = 8,721). In conclusion, we identified a new locus associated with SF in T1D subjects that did not show similar effect in ND subjects, suggesting a diabetes-specific effect. This association needs to be investigated in type 2 diabetes.

Skin collagen fluorophore LW-1 versus skin fluorescence as markers for the long-term progression of subclinical macrovascular disease in type 1 diabetes.

BACKGROUND: Skin collagen Long Wavelength Fluorescence (LWF) is widely used as a surrogate marker for accumulation of advanced glycation end-products. Here we determined the relationship of LWF with glycemia, skin fluorescence, and the progression of complications during EDIC in 216 participants from the DCCT. METHODS: LW-1 and collagen-linked fluorescence (CLF) were measured by either High Performance Liquid Chromatography (HPLC) with fluorescence detection (LW-1) or total fluorescence of collagenase digests (CLF) in insoluble skin collagen extracted from skin biopsies obtained at the end of the DCCT (1993). Skin intrinsic fluorescence (SIF) was noninvasively measured on volar forearm skin at EDIC year 16 by the SCOUT DS instrument. RESULTS: LW-1 levels significantly increased with age and diabetes duration (P < 0.0001) and significantly decreased by intensive vs. conventional glycemic therapy in both the primary (P < 0.0001) and secondary (P < 0.037) DCCT cohorts. Levels were associated with 13-16 year progression risk of retinopathy (>3 sustained microaneurysms, P = 0.0004) and albumin excretion rate (P = 0.0038), the latter despite adjustment for HbA1c. Comparative analysis for all three fluorescent measures for future risk of subclinical macrovascular disease revealed the following significant (P < 0.05) associations after adjusting for age, diabetes duration and HbA1c: coronary artery calcium with SIF and CLF; intima-media thickness with SIF and LW-1; and left ventricular mass with LW-1 and CLF. CONCLUSIONS: LW-1 is a novel risk marker that is robustly and independently associated with the future progression of microvascular disease, intima-media thickness and left ventricular mass in type 1 diabetes. Trial registration NCT00360815 and NCT00360893 at clinicaltrials.gov.

Skin collagen advanced glycation endproducts (AGEs) and the long-term progression of sub-clinical cardiovascular disease in type 1 diabetes.

BACKGROUND: We recently reported strong associations between eight skin collagen AGEs and two solubility markers from skin biopsies obtained at DCCT study closeout and the long-term progression of microvascular disease in EDIC, despite adjustment for mean glycemia. Herein we investigated the hypothesis that some of these AGEs (fluorescence to be reported elsewhere) correlate with long-term subclinical cardiovascular disease (CVD) measurements, i.e. coronary artery calcium score (CAC) at EDIC year 7-9 (n = 187), change of carotid intima-media thickness (IMT) from EDIC year 1 to year 6 and 12 (n = 127), and cardiac MRI outcomes at EDIC year 15-16 (n = 142). METHODS: Skin collagen AGE measurements obtained from stored specimens were related to clinical data from the DCCT/EDIC using Spearman correlations and multivariable logistic regression analyses. RESULTS: Spearman correlations showed furosine (early glycation) was associated with future mean CAC (p < 0.05) and CAC >0 (p = 0.039), [corrected] but not with CAC score <100 vs. >100. Glucosepane and pentosidine crosslinks, methylglyoxal hydroimidazolones (MG-H1) and pepsin solubility (inversely) correlated with IMT change from year 1 to 6(all P < 0.05). Left ventricular (LV) mass (cMRI) correlated with MG-H1, and inversely with pepsin solubility (both p < 0.05), while the ratio LV mass/end diastolic volume correlated with furosine and MG-H1 (both p < 0.05), and highly with CML (p < 0.01). In multivariate analysis only furosine (p = 0.01) was associated with CAC. In contrast IMT was inversely associated with lower collagen pepsin solubility and positively with glucosepane, CONCLUSIONS: In type 1 diabetes, multiple AGEs are associated with IMT progression in spite of adjustment for A1c implying a likely participatory role of glycation and AGE mediated crosslinking on matrix accumulation in coronary arteries. This may also apply to functional cardiac MRI outcomes, especially left ventricular mass. In contrast, early glycation measured by furosine, but not AGEs, was associated with CAC score, implying hyperglycemia as a risk factor in calcium deposition perhaps via processes independent of glycation. TRIAL REGISTRATION: Registered at Clinical trial reg. nos. NCT00360815 and NCT00360893, http://www.clinicaltrials.gov.

Biological Effects Induced by Specific Advanced Glycation End Products in the Reconstructed Skin Model of Aging.

Advanced glycation end products (AGEs) accumulate in the aging skin. To understand the biological effects of individual AGEs, skin reconstructed with collagen selectively enriched with N(ɛ)-(carboxymethyl)-lysine (CML), N(ɛ)-(carboxyethyl)-lysine (CEL), methylglyoxal hydroimidazolone (MG-H1), or pentosidine was studied. Immunohistochemistry revealed increased expression of α6 integrin at the dermal epidermal junction by CEL and CML (p<0.01). Laminin 5 was diminished by CEL and MG-H1 (p<0.05). Both CML and CEL induced a robust increase (p<0.01) in procollagen I. In the culture medium, IL-6, VEGF, and MMP1 secretion were significantly decreased (p<0.05) by MG-H1. While both CEL and CML decreased MMP3, only CEL decreased IL-6 and TIMP1, while CML stimulated TIMP1 synthesis significantly (p<0.05). mRNA expression studies using qPCR in the epidermis layer showed that CEL increased type 7 collagen (COL7A1), ß1, and α6 integrin, while CML increased only COL7A1 (p<0.05). MG-H1-modified collagen had no effect. Importantly, in the dermis layer, MMP3 mRNA expression was increased by both CML and MG-H1. CML also significantly increased the mRNAs of MMP1, TIMP1, keratinocyte growth factor (KGF), IL-6, and monocyte chemoattractant protein 1 (MCP1) (p<0.05). Mixed effects were present in CEL-rich matrix. Minimally glycoxidized pentosidine-rich collagen suppressed most mRNAs of the genes studied (p<0.05) and decreased VEGF and increased MCP1 protein expression. Taken together, this model of the aging skin suggests that a combination of AGEs tends to counterbalance and thus minimizes the detrimental biological effects of individual AGEs.

Glucosepane and oxidative markers in skin collagen correlate with intima media thickness and arterial stiffness in long-term type 1 diabetes.

AIMS: To study intima media thickness (cIMT) and arterial stiffness in type 1 diabetes of long duration, and their associations with the collagen cross-linker glucosepane and inflammatory and oxidative markers. METHODS: Twenty-seven individuals with type 1 diabetes mellitus of 40 years duration from the Oslo Study cohort and 24 age-matched controls were included. cIMT measurements of the carotid artery were performed longitudinally. Pulse wave velocity (PWV), augmentation index (AIx) and augmentation pressure (AP) were assessed cross-sectionally. Glucosepane and the oxidative product methionine sulfoxide (MetSO) were determined in skin collagen by liquid chromatography-mass spectrometry. Circulating inflammatory markers were determined by ELISAs. RESULTS: The diabetes patients had significantly increased cIMT and arterial stiffness compared to controls. Significant correlations were noted for skin glucosepane with cIMT (r=0.41) and PWV (r=0.44). Skin MetSO and monocyte chemoattractant protein-1 (MCP-1) correlated significantly with AIx and AP. After correcting for age and mean arterial pressure in multiple linear regression analysis, MetSO and MCP-1 were both independently associated with AIx and AP. CONCLUSIONS: These results suggest more premature atherosclerosis and arterial pathology in individuals with diabetes compared to age-matched controls. They also suggest an association between the arterial pathology and markers of collagen crosslinking, oxidative damage and inflammation in type 1 diabetes patients of forty years disease duration.

Skin advanced glycation end products glucosepane and methylglyoxal hydroimidazolone are independently associated with long-term microvascular complication progression of type 1 diabetes.

Six skin collagen advanced glycation end products (AGEs) originally measured near to the time of the Diabetes Control and Complications Trial (DCCT) closeout in 1993 may contribute to the "metabolic memory" phenomenon reported in the follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) study. We have now investigated whether the addition of four originally unavailable AGEs (i.e., glucosepane [GSPNE], hydroimidazolones of methylglyoxal [MG-H1] and glyoxal, and carboxyethyl-lysine) improves associations with incident retinopathy, nephropathy, and neuropathy events during 13-17 years after DCCT. The complete 10-AGE panel is associated with three-step Early Treatment of Diabetic Retinopathy Study scale worsening of retinopathy (P ≤ 0.002), independent of either mean DCCT or EDIC study A1C level. GSPNE and fructose-lysine (furosine [FUR]) correlate with retinopathy progression, independently of A1C level. The complete panel also correlates with microalbuminuria (P = 0.008) and FUR with nephropathy independently of A1C level (P ≤ 0.02). Neuropathy correlates with the complete panel despite adjustment for A1C level (P ≤ 0.005). MG-H1 and FUR are dominant, independent of A1C level (P < 0.0001), whereas A1C loses significance after adjustment for the AGEs. Overall, the added set of four AGEs enhances the association of the original panel with progression risk of retinopathy and neuropathy (P < 0.04) but not nephropathy, while GSPNE and MG-H1 emerge as the principal new risk factors. Skin AGEs are robust long-term markers of microvascular disease progression, emphasizing the importance of early and sustained implementation of intensive therapy.

Advanced glycation end-products reduce collagen molecular sliding to affect collagen fibril damage mechanisms but not stiffness.

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.

GWAS identifies an NAT2 acetylator status tag single nucleotide polymorphism to be a major locus for skin fluorescence.

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 r (2) = 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.

Impaired left ventricular function and myocardial blood flow reserve in patients with long-term type 1 diabetes and no significant coronary artery disease: associations with protein glycation.

Our aims were to study left ventricular (LV) function and myocardial blood flow reserve (MBFR) in long-term type 1 diabetes and associations with advanced glycation end products (AGEs). A total of 20 type 1 diabetes patients from the Oslo Study without significant stenosis on coronary angiography were compared with 26 controls. LV systolic and diastolic functions were assessed by two-dimensional strain and the ratio between pulsed Doppler transmitral early (E) velocity and tissue Doppler velocity (E'), respectively. MBFR was evaluated by contrast echocardiography. The AGE methylglyoxal-derived hydroimidazolone was analysed in serum. Glyoxal hydroimidazolone in skin collagen was determined by liquid chromatography-mass spectrometry. Strain was significantly reduced (-19.5% ± 1.9% vs -21.4% ± 3.5%, p < 0.05), and E/E' increased in the diabetes patients compared to controls, 7.3 ± 2 versus 6.0 ± 1.5, p < 0.05. Significant lower MBFR was present in the diabetes patients, 3.4 (2.1, 5.3) versus 5.9 (3.9, 9.6), p < 0.01. Both AGEs correlated significantly with E/E'. The impaired LV function with correlation to AGEs in concert with reduced MBFR in diabetics without coronary artery disease may indicate possible mechanisms for diabetic cardiomyopathy.

Deletion of Mecom in mouse results in early-onset spinal deformity and osteopenia.

Recent studies have indicated a role for a MECOM allele in susceptibility to osteoporotic fractures in humans. We have generated a mutation in Mecom in mouse (termed ME(m1)) via lacZ knock-in into the upstream transcription start site for the gene, resulting in disruption of Mds1 and Mds1-Evi1 transcripts, but not of Evi1 transcripts. We demonstrate that ME(m1/m1) mice have severe kyphoscoliosis that is reminiscent of human congenital or primary kyphoscoliosis. ME(m1/m1) mice appear normal at birth, but by 2weeks, they exhibit a slight lumbar lordosis and narrowed intervertebral space. This progresses to severe lordosis with disc collapse and synostosis, together with kyphoscoliosis. Bone formation and strength testing show that ME(m1/m1) mice have normal bone formation and composition but are osteopenic. While endochondral bone development is normal, it is markedly dysplastic in its organization. Electron micrographs of the 1week postnatal intervertebral discs reveals marked disarray of collagen fibers, consistent with an inherent weakness in the non-osseous connective tissue associated with the spine. These findings indicate that lack of ME leads to a complex defect in both osseous and non-osseous musculoskeletal tissues, including a marked vertebral osteopenia, degeneration of the IVD, and disarray of connective tissues, which is likely due to an inherent inability to establish and/or maintain components of these tissues.

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

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.