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.

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.

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 was 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>40mg/24h (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.

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.

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 (chi2 = 59.4, P < 0.0001) and nephropathy (chi2 = 18.2, P = 0.0001), even after adjustment for mean HbA(1c) (A1C) (chi2 = 32.7, P < 0.0001 for retinopathy) and (chi2 = 12.8, P = 0.0016 for nephropathy). The predictive effect of A1C vanished after adjustment for furosine and CML (chi2 = 0.0002, P = 0.987 for retinopathy and chi2 = 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.