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Long-term hyperglycemia in patients with diabetes leads to human serum albumin (HSA) glycation, which may impair HSA function as a transport protein and affect the therapeutic efficacy of anticoagulants in patients with diabetes. In this study, a novel mass spectrometry approach was developed to reveal the differences in the profiles of HSA glycation sites between patients with diabetes and healthy subjects. K199 was the glycation site most significantly changed in patients with diabetes, contributing to different interactions of glycated HSA and normal HSA with two types of anticoagulant drugs, heparin and warfarin. An in vitro experiment showed that the binding affinity to warfarin became stronger when HSA was glycated, while HSA binding to heparin was not significantly influenced by glycation. A pharmacokinetic study showed a decreased level of free warfarin in the plasma of diabetic rats. A preliminary retrospective clinical study also revealed that there was a statistically significant difference in the anticoagulant efficacy between patients with diabetes and patients without diabetes who had been treated with warfarin. Our work suggests that larger studies are needed to provide additional specific guidance for patients with diabetes when they are administered anticoagulant drugs or drugs for treating other chronic diseases.

Purpose of the Study: Diabetes mellitus (DM) is characterized by chronic hyperglycemia and diabetic complications. Exacerbated cortical neuronal degeneration were observed in Alzheimer’s disease (AD) patients with DM. DM is considered a risk factor of AD, as DM-induced activation of stress responses in central nervous system (CNS) such as oxidative stress and neuroinflammation may lead to various neurodegenerative disorders. Abnormal accumulation of methylglyoxal (MG), one of the most reactive advanced glycation end-product (AGE) precursors, is found in the serum of diabetic patients. As MG is reported to induce brain cells impairment, like neuronal cell death, in CNS, and increased levels of AGEs have also been observed in brains of AD patients, hence the effect of MG leading to subsequent symptoms of AD was investigated. Methods: C57BL/6 mice were treated with MG solution (60mg/kg) or saline (100μl of 0.9% saline as sham control) by intraperitoneal injection for 9 weeks. The Morris water maze (MWM) was used to examine the spatial learning ability and cognition of mice. Footprint, escape latency and time spent in the target quadrant were measured and recorded. Summary of the Results: Significantly longer escape latency was observed in the MG-treated mice than that of the control group starting from Day 3. The mean escape latency of the MG-treated group was 13.41sec. while that of the control group was 5.450sec. in Day 5. It was also demonstrated in the probe trial that there was a significant reduction of the percentage time spent in the target quadrant of the MG-treated mice (44.07 ± 2.720) than that of the control group (58.29 ± 4.324). Conclusions: MG could induce AD symptoms causing spatial learning ability impairment and cognition decline in mice. MG may therefore play a role in the dysfunction of brain cells and dysregulation of CNS resulting in Alzheimer’s disease. Disclosure W. Li: None. S. Wu: None. F. Lee: None. K. Yue: None.

Advanced glycation end-products are proteins that become glycated after contact with sugars and are implicated in endothelial dysfunction and arterial stiffening. We hypothesized that advanced glycation end-products associate with vascular stiffness independent of glycemic status. Advanced glycation end-products were measured as skin autofluorescence, vascular stiffness was measured as pulse wave velocity, augmentation index and ankle-brachial index in 3,535 participants from the European Prospective Investigation into Cancer and Nutrition Potsdam cohort (median age 67 years, 60% women). A subset of 1,348 participants underwent an oral glucose tolerance test. Participants were sub-phenotyped into normoglycemic, prediabetes and diabetes groups. Skin autofluorescence associated with pulse wave velocity, augmentation index and ankle-brachial index, adjusted beta coefficients (95% CI) per unit skin autofluorescence increase: 0.38 (0.21; 0.55) for carotid-femoral pulse wave velocity, 0.25 (0.14; 0.37) for aortic pulse wave velocity, 1.00 (0.29; 1.70) for aortic augmentation index, 4.12 (2.24; 6.00) for brachial augmentation index and -0.04 (-0.05; -0.02) for ankle-brachial index. The associations were strongest in men, younger individuals and were consistent across all glycemic strata: for carotid-femoral pulse wave velocity 0.36 (0.12; 0.60) in normoglycemic, 0.33 (-0.01; 0.67) in prediabetes and 0.45 (0.09; 0.80) in diabetes groups; with similar estimates for aortic pulse wave velocity. Augmentation index was associated with skin autofluorescence only in normoglycemic and diabetes groups. Ankle-brachial index inversely associated with skin autofluorescence across all sex, age and glycemic strata. Our findings indicate that advanced glycation end-products are involved in vascular stiffening independent of glycemic status, age and other cardiometabolic risk factors. Disclosure A. Birukov: None. R. Cuadrat: None. E. Polemiti: None. F. Eichelmann: None. M. B. Schulze: None. Funding German Ministry of Education and Research; State of Brandenburg (82DZD00302)

Individual advanced glycation end products (AGEs) have been implicated in the development of chronic kidney disease (CKD). We evaluated the prognostic utility of a comprehensive multicomponent AGE panel in predicting long-term renal function loss and CKD when added to routine clinical measures in persons with type 2 diabetes (T2D) followed for 12.5 years (including risk factor intervention and observation phases). The five key AGEs (carboxymethyl and carboxyethyl lysine, and methylglyoxal, 3-deoxyglucosone and glyoxal hydroimidazolone) were measured via LC-MS/MS in baseline serum from 1,151 the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial participants. A composite AGE score was calculated as the mean of individual AGE z-scores. Outcomes were (1) renal function loss (≥40% decline in estimated glomerular filtration rate [eGFR]), (2) macroalbuminuria (MAU), and (3) high-risk CKD (hrCKD, KDIGO classification). Higher AGE score predicted greater risks of renal function loss (n=260 events, hazard ratio 1.49 [95%CI 1.24-1.79], p Disclosure J. Koska: None. H. C. Gerstein: Advisory Panel; Self; Novo Nordisk Inc., Pfizer Inc., Sanofi, Consultant; Self; Abbott, Covance Inc., Eli Lilly and Company, Kowa Company, Ltd., Sanofi, Other Relationship; Self; Boehringer Ingelheim (Canada) Ltd., DKSH, Eli Lilly and Company, Sanofi, Zuellig, Research Support; Self; AstraZeneca, Eli Lilly and Company, Merck & Co., Inc., Novo Nordisk Inc., Sanofi. P. J. Beisswenger: Stock/Shareholder; Self; PreventAGE Healthcare LLC. P. Reaven: Research Support; Self; AstraZeneca, Dexcom, Inc. Funding National Institutes of Health (R21HL150268)

Poor control of blood glucose could result in increase in advanced glycation end products (AGEs). AGEs cause various adverse vascular events such as atherosclerosis. Therefore, production of AGEs should be suppressed to prevent diabetic complications. In the present study, we evaluated effects of SGLT2 inhibitor (SGLT2i) and DPP-4 inhibitor (DPP-4i) on AGEs in patients with type 2 diabetes. The Japanese patients having poorly controlled blood glucose for 6 months were orally received SGLT2i (n=13; 2.5 mg/day luseogliflozin, 10 mg/day dapagliflozin, or 25 mg/day empagliflozin) or DPP-4i (n=11; 100 mg/day sitagliptin, 100 mg/day vildagliptin, or 25 mg/day alogliptin) for 3 months. Before and after the drug treatment, we measured HbA1c level and serum levels of lipids and hepatic function parameters. Besides, circulating methylglyoxal-derived hydroimidazolone-1 (MG-H1), one of AGEs, was determined optically. As shown in Table, SGLT2i significantly lowered HbA1c (P Disclosure M. Kusunoki: Other Relationship; Self; Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd. N. Wakazono: None. F. Hisano: None. T. Miyata: None.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive carcinoma with poor prognosis. Its poor prognosis is ascribed to the extensive fibrotic stroma. Pancreatic stellate cells (PSCs) are major contributors to PDAC fibrosis. Type 2 diabetes (T2D) is an established risk factor for PDAC. The common mechanistic link might be the accumulation of advanced glycation end-products (AGEs) and the signaling through receptor for advanced glycation end-products (RAGE), the receptor for AGEs. Nevertheless, the role of RAGE in PSCs activation was not evaluated in PDAC with T2D. First, PSCs were isolated from male obese T2D model, db/db (db) and db/m (C) mice at 12 weeks of age. mRNA expression of the makers for PSCs activation like TGF-β1, αSMA and collagen type1A1 were significantly increased in PSCs isolated from db compared to C (p Disclosure C. Uchida: None. H. Mizukami: None. K. Kudo: None. Y. Yamamoto: None. Funding Japan Society for the Promotion of Science (JP17K17579)

Transforming growth factor-β/Smad3 signaling plays an important role in diabetic nephropathy, but its underlying working mechanism remains largely unexplored. The current study uncovered the pathogenic role and underlying mechanism of a novel Smad3-dependent long noncoding RNA (lncRNA) (LRNA9884) in type 2 diabetic nephropathy (T2DN). We found that LRNA9884 was significantly upregulated in the diabetic kidney of db/db mice at the age of 8 weeks preceding the onset of microalbuminuria and was associated with the progression of diabetic renal injury. LRNA9884 was induced by advanced glycation end products and tightly regulated by Smad3, and its levels were significantly blunted in db/db mice and cells lacking Smad3. More importantly, kidney-specific silencing of LRNA9884 effectively attenuated diabetic kidney injury in db/db mice, as shown by the reduction of histological injury, albuminuria excretion, and serum creatinine. Mechanistically, we identified that LRNA9884 promoted renal inflammation-driven T2DN by triggering MCP-1 production at the transcriptional level, and its direct binding significantly enhanced the promoter activity of MCP-1. Thus, LRNA9884 is a novel Smad3-dependent lncRNA that is highly expressed in db/db mice associated with T2DN development. Targeting of LRNA9884 effectively blocked MCP-1–dependent renal inflammation, therefore suppressing the progressive diabetic renal injury in db/db mice. This study reveals that LRNA9884 may be a novel and precision therapeutic target for T2DN in the future.

Effective treatment of diabetic kidney disease (DKD) remains a large unmet medical need. Within the disease’s complicated pathogenic mechanism, activation of the advanced glycation end-products (AGEs)–receptor for AGEs (RAGE) axis plays a pivotal role in the development and progression of DKD. To provide a new therapeutic strategy against DKD progression, we developed a vaccine against RAGE. Three rounds of immunization of mice with the RAGE vaccine successfully induced antigen-specific serum IgG antibody titers, and elevated antibody titers were sustained for at least 38 weeks. In addition, RAGE vaccination significantly attenuated the increase in urinary albumin excretion in streptozotocin-induced diabetic mice (type 1 diabetes model) and leptin-receptor–deficient db/db mice (type 2 diabetes model). In microscopic analyses, RAGE vaccination suppressed glomerular hypertrophy and mesangial expansion in both diabetic models and significantly reduced glomerular basement membrane thickness in streptozotocin-induced diabetic mice. Results of an in vitro study indicated that the serum IgG antibody elicited by RAGE vaccination suppressed the expression of AGE-induced vascular cell adhesion molecule 1 and intracellular adhesion molecule 1 in endothelial cells. Thus, our newly developed RAGE vaccine attenuated the progression of DKD in mice and is a promising potential therapeutic strategy for patients with DKD.

Objective: Chronic hyperglycemia increases glycation as well as oxidization, and, thus, facilitates production of advanced glycation end products (AGEs), whose accumulation is involved in the pathophysiology of diabetic complications. Although association between AGE and insulin resistance has been reported thoroughly, association between AGE and insulin secretion has not been well examined, especially in relation with age. Research Design: Among participants of the population-based Iwaki study of Japanese held in 2014, those who attended the Iwaki study consecutively in 2014 and 2015 were enrolled in this study (n=518; model A), and we excluded fasting blood glucose levels 140 to calculate HOMA-β and IR (n=489; model B). We used skin autofluorescence as AGE, and also measured blood levels of Pentosidine and urine albumin-to-creatinine ratio. Results: 1. AGE levels significantly increased from 1.93±0.52 to 1.97±0.44 (p=0.014) in the 1 year period. 2. Regression analysis showed correlations between ∆AGE (change in AGE in the 1 year period) and age, BUN, FPG and log10uACR in model A, and, age, log10uACR, and log10HOMA-ß in model B. However, these correlations became none-significant after adjustment for multiple factors mentioned above as well as factors representing glucose control levels such as HbA1c, except for age in models A and B, respectively. 3. Subjects were stratified into tertiles (mildly, moderately, rapidly progress) based on their ∆AGE, Logistic analysis revealed the aging is a significant risk for the accumulation of AGE (rapidly progress) (OR: 1.02. 95%CI: 1.01-1.03). ROC analysis showed the age of 60 as an optimal cut-off value for determining the risk subjects for the increase in AGE (OR: 1.74, 95%CI: 1.20-2.51). Conclusions: The accumulation of AGEs may increase with increasing speed, which becomes rapid over the age of 60 independent of glycemic levels, uACR and HOMA-ß, indicating that not glycemic control levels but aging per se is a major contributor for AGE accumulation. Disclosure S. Mizushiri: None. M. Daimon: None. H. Murakami: None. A. Kamba: None. M. Murabayashi: None. Y. Nishiya: None.

Advanced glycation end-products (AGEs) are known to be produced by the Maillard reaction, ingested through food. AGEs are actively generated and accumulated in the circulating blood and various tissues in diabetes mellitus patients by chronic hyperglycemia. AGEs play an important role in the development of diabetic vascular complications. The aim of this study was to assess levels and related factors of AGEs in Korean patients with type 2 diabetes mellitus. A total of 310 Korean men (n=177) and women (n=133) with T2DM subjects were participated. This study was measured and analyzed anthrometric data, body composition, and biochemical parameters. Tissue accumulated AGEs can be evaluated a non-invasive methodology by skin autofluorescence reader. The means age of total subjects was 58.9 ± 11.0 years, BMI was 25.8 ± 4.5 kg/m2. There is no difference between male and female patients in levels of AGEs, pulse wave velocity (PWV) and vein age. There is a significant correlation among AGEs, height, weight, body mass index, skeletal muscle mass, diastolic blood pressure and HDL cholesterol for male patients and among fasting blood sugar, glycated hemoglobin A1c, total cholesterol, LDL cholesterol, and systolic blood pressure for female patients. The correlation of AGEs for both male and female subjects is highly associated with age, duration of diabetes and PWV. This study shows possibility of using AGEs as an index for clinical diagnosis, complications prediction, and diabetes management in Korean patients with type 2 diabetes mellitus. However, it is considered that a change in AGEs may be influenced by gender, age, and duration of diabetes. Disclosure J. Yim: None. Y. Kim: None. J. Park: None.

HGI measures how far a person’s HbA1c lies above or below average compared to other people with similar blood glucose concentrations. Multiple studies confirm that high HGI is associated with greater risk for iatrogenic hypoglycemia in diabetes patients and chronic vascular disease in diabetic and nondiabetic populations. However, significant differences in how blood glucose is assessed and in the demographic composition of different study populations have resulted in marked interstudy differences in the slopes and intercepts of regression equations used to calculate HGI. The purpose of the present study was to develop a standard linear regression equation that could be widely used to calculate HGI in diverse human populations. To accomplish this, we used data from the demographically diverse 1999-2016 National Health and Nutrition Examination Survey (NHANES) to identify 18,675 participants age ≥20 y without self-reported diabetes. The regression equation developed from this treatment naïve adult reference population was HbA1c = 0.024 FPG + 3.1. HGI was calculated for reference population participants who were then subdivided into 1) low, moderate, and high subgroups, and 2) normal, prediabetic and diabetic subgroups based on OGTT results. Novel observations include lack of association between HGI and hemoglobin concentration, plasma insulin or HOMA-IR. Consistent with previous reports, high HGI was associated with 1) black race regardless of diabetes status, 2) older age and higher BMI in nondiabetic but not diabetic participants, and 3) no difference in 2 h OGTT results in normal, prediabetic or treatment naïve diabetic participants. Standardizing how HGI is calculated across studies will facilitate clinical and basic research to better understand 1) why some people have lower or higher than average HbA1c levels independent of blood glucose concentration, 2) why high HGI is associated with greater risk for chronic vascular disease in diabetic and nondiabetic people, and 3) what can be done to reduce risk in high HGI people. Disclosure D.S. Hsia: None. S. Yang: None. S. Liu: None. J.M. Hempe: None. Funding National Institute of General Medical Sciences (U54GM104940)

Glycated hemoglobin (A1c) is the gold-standard metric of average glycemia and quality of glycemic control, and a key predictor of long-term diabetes complications. We present a method for tracking A1c from continuous glucose monitoring (CGM)-derived time-in-range (TIR), which provides daily estimates of A1c (eA1c). The method uses a dynamical model of hemoglobin glycation/clearance and is calibrated with a single lab A1c value. CGM data collected by the DIAMOND study from 304 individuals with type 1 and type 2 diabetes (T1D/T2D) over 24 weeks were used in the analysis. Lab A1c samples were collected at weeks 12 and 24. Three population parameters were estimated by training the model on data from 50/50 randomly selected T1D/T2D subjects; the remaining 103/101 subjects were used for model validation. One individual glycation parameter was estimated from the week 12 lab A1c for each subject (model calibration), and was fixed thereafter. The week 24 lab A1c samples were used to test model performance (Table 1). The TIR-A1c relationship is mediated by glycation parameters, 3 population and 1 personal, the latter estimated using a single lab A1c value. Following this personalization, TIR provides accurate estimates of A1c in both T1D and T2D for (at least) 3 months post calibration. Thus, CGM-derived TIR can be a viable clinical alternative to A1c, allowing daily tracking of changes in average glycemia via a dynamical model of hemoglobin glycation. Disclosure C. Fabris: Consultant; Self; Abbott. R. Beck: None. B. Kovatchev: Advisory Panel; Self; Dexcom, Inc. Research Support; Self; Dexcom, Inc., Tandem Diabetes Care. Speaker’s Bureau; Self; Dexcom, Inc., Sanofi, Tandem Diabetes Care. Other Relationship; Self; Dexcom, Inc., Johnson & Johnson, Sanofi. Funding University of Virginia Strategic Investment in Diabetes (88); Dexcom, Inc.

Improved aerobic exercise capacity (VO2max) has emerged as one of the most important health benefits of aerobic exercise training. However, hyperglycemia is associated with low VO2max in humans, even when physical activity levels are matched. To determine how glycemia influences aerobic adaptations with exercise training, we induced moderate hyperglycemia (+50-70 mg/dL vs. control) in mouse models that mimic the pathologies of type 1 or type 2 diabetes: 1) Low-dose Streptozotocin treatment (STZ), or 2) Western diet (WD) feeding. Normoglycemic mice acted as controls (CON). All mice completed ∼500 km of voluntary wheel running over an 8-wk aerobic training period, and achieved the expected metabolic benefits from exercise training. However, metabolic improvements were uncoupled from aerobic adaptation in hyperglycemic WD and STZ mice, who failed to improve VO2max beyond the level of sedentary controls. In contrast, CON mice had a 2-fold increase in VO2max in response to the same training intervention. In hyperglycemic models, blunted improvements in VO2max were associated with structural changes to muscle, including glucose-induced modifications to the extracellular matrix (e.g., glycation and collagen accretion), and reduced exercise-induced angiogenesis and oxidative fiber-type shifts. Thus, our data provide evidence that aerobic muscle remodeling, which is critical for augmenting VO2max, is impaired by hyperglycemia. In addition, we show that chronic hyperglycemia causes hyper-activation of a JNK/SMAD2 signaling network in muscle with acute aerobic exercise, which is a known molecular mechanism that inhibits aerobic adaptation. Accordingly, in human subjects, we show that impaired glucose tolerance strongly predicts hyper-activation of this inhibitory signaling network in conjunction with low VO2max. Taken together, we demonstrate that hyperglycemia, regardless of etiology, may interfere with fundamental adaptations to aerobic exercise training and negatively regulate improvements in VO2max. Disclosure T. MacDonald: None. P. Pathak: None. N.M. Fernandez: None. E.C. Freitas: None. S. Hafida: None. J. Mitri: Consultant; Spouse/Partner; Janssen Pharmaceuticals, Inc., kymera. Consultant; Self; national dairy council/local dairy council. Research Support; Spouse/Partner; AbbVie Inc., beigene, Janssen Pharmaceuticals, Inc. Research Support; Self; Kowa Pharmaceuticals America, Inc., national dairy council. Research Support; Spouse/Partner; pharma cyclic, TG therapeutics. S.J. Lessard: None. Funding American Heart Association (19POST34381036)

Impaired wound healing is one of the main causes of diabetic foot ulcerations. However, the exact mechanism of delayed wound healing in diabetes is not fully understood. Long noncoding RNAs (lncRNAs) are widely involved in a variety of biological processes and diseases, including diabetes and its associated complications. In this study, we identified a novel lncRNA, MRAK052872, named lncRNA UpRegulated in Diabetic Skin (lnc-URIDS), which regulates wound healing in diabetes. lnc-URIDS was highly expressed in diabetic skin and dermal fibroblasts treated with advanced glycation end products (AGEs). lnc-URIDS knockdown promoted migration of dermal fibroblasts under AGEs treatment in vitro and accelerated diabetic wound healing in vivo. Mechanistically, lnc-URIDS interacts with procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plod1), a critical enzyme responsible for collagen cross-linking. The binding of lnc-URIDS to Plod1 results in a decreased protein stability of Plod1, which ultimately leads to the dysregulation of collagen production and deposition and delays wound healing. Collectively, this study identifies a novel lncRNA that regulates diabetic wound healing by targeting Plod1. The findings of the current study offer some insight into the potential mechanism for the delayed wound healing in diabetes and provide a potential therapeutic target for diabetic foot.

The phenomenon of a discrepancy between glycated hemoglobin levels and other indicators of average glycemia may be due to many factors but can be measured as the glycation gap (GGap). This GGap is associated with differences in complications in patients with diabetes and may possibly be explained by dissimilarities in deglycation in turn leading to altered production of advanced glycation end products (AGEs). We hypothesized that variations in the level of the deglycating enzyme fructosamine-3-kinase (FN3K) might be associated with the GGap. We measured erythrocyte FN3K concentrations and enzyme activity in a population dichotomized for a large positive or negative GGap. FN3K protein was higher and we found a striking threefold greater activity (323%) at any given FN3K protein level in the erythrocytes of the negative-GGap group compared with the positive-GGap group. This was associated with lower AGE levels in the negative-GGap group (79%), lower proinflammatory adipokines (leptin-to-adiponectin ratio) (73%), and much lower prothrombotic PAI-1 levels (19%). We conclude that FN3K may play a key role in the GGap and thus diabetes complications such that FN3K may be a potential predictor of the risk of diabetes complications. Pharmacological modifications of its activity may provide a novel approach to their prevention.

Advanced glycation end products (AGEs) are involved in the progression of diabetic nephropathy. AGEs filtered by glomeruli or delivered from the circulation are endocytosed and degraded in the lysosomes of kidney proximal tubular epithelial cells (PTECs). Autophagy is a highly conserved degradation system that regulates intracellular homeostasis by engulfing cytoplasmic components. We have recently demonstrated that autophagic degradation of damaged lysosomes is indispensable for cellular homeostasis in some settings. In this study, we tested the hypothesis that autophagy could contribute to the degradation of AGEs in the diabetic kidney by modulating lysosomal biogenesis. Both a high-glucose and exogenous AGE overload gradually blunted autophagic flux in the cultured PTECs. AGE overload upregulated lysosomal biogenesis and function in vitro, which was inhibited in autophagy-deficient PTECs because of the impaired nuclear translocation of transcription factor EB. Consistently, streptozotocin-treated, PTEC-specific, autophagy-deficient mice failed to upregulate lysosomal biogenesis and exhibited the accumulation of AGEs in the glomeruli and renal vasculature as well as in the PTECs, along with worsened inflammation and fibrosis. These results indicate that autophagy contributes to the degradation of AGEs by the upregulation of lysosomal biogenesis and function in diabetic nephropathy. Strategies aimed at promoting lysosomal function hold promise for treating diabetic nephropathy.

We examined associations of advanced glycation end products (AGEs) with renal function loss (RFL) and its structural determinants in American Indians with type 2 diabetes. Data were from a 6-year clinical trial that assessed renoprotective efficacy of losartan. Participants remained under observation after the trial concluded. Glomerular filtration rate (GFR) was measured annually. Kidney biopsies were performed at the end of the trial. Five AGEs were measured in serum collected at enrollment and at kidney biopsy. RFL was defined as ≥40% decline of measured GFR from baseline. Of 168 participants (mean baseline age 41 years, HbA1c 9.2%, GFR 164 mL/min, and albumin-to-creatinine ratio 31 mg/g), 104 reached the RFL end point during median follow-up of 8.0 years. After multivariable adjustment, each doubling of carboxyethyl lysine (hazard ratio [HR] 1.60 [95% CI 1.08–2.37]) or methylglyoxal hydroimidazolone (HR 1.30 [95% CI 1.02–1.65]) concentration was associated with RFL. Carboxyethyl lysine, carboxymethyl lysine, and methylglyoxal hydroimidazolone correlated positively with cortical interstitial fractional volume (partial r = 0.23, P = 0.03; partial r = 0.25, P = 0.02; and partial r = 0.31, P = 0.003, respectively). Glyoxyl hydroimidazolone and methylglyoxal hydroimidazolone correlated negatively with total filtration surface per glomerulus (partial r = −0.26, P = 0.01; and partial r = −0.21, P = 0.046, respectively). AGEs improve prediction of RFL and its major structural correlates.

Advanced glycation end-products (AGEs) are endogenously produced or exogenously derived glycated proteins and lipoproteins. The accumulation of AGEs may have a variety of adverse effects since they cause oxidative stress and thus may act as contributing factors to the development of disease states such as, for example, complications of diabetes mellitus. The aim of this study was to investigate whether the levels of three AGEs (pentosidine, N(6)-carboxymethyllysine [CML], and methylglyoxal [MGO]) and the soluble form of the AGE receptor (sRAGE) may be used as markers for the presence and extent of coronary artery disease (CAD). We recruited 363 consecutive patients with suspected CAD who presented for cardiac catheterization at Lenox Hill Hospital in New York City (54% of patients were male, 48% of the patients had diabetes). At the time of this abstract submission 169 patients had circulating pentosidine levels measured (74 of these patients had diabetes). We examined relations between pentosidine levels, HbA1c, LDL, and the extend of CAD (measured by SYNTAX score). Serum pentosidine levels did not significantly correlate with any of other parameters. There was, however, a positive correlation between HbA1c levels and the SYNTAX score (Spearman correlation coefficients of 0.26, ρ = 0.0001 for the entire group; 0.18, ρ = 0.01 for patients without diabetes; and 0.29, ρ= 0.0001 for patients with diabetes). In conclusion, our preliminary results demonstrate that in patients with suspected CAD undergoing cardiac catheterization, SYNTAX score does not correlate with pentosidine levels but correlates positively with HbA1c. The data on all three AGEs and sRAGE in the entire cohort of 363 patients will be presented at the meeting. Disclosure C. Basman: None. D. Avtanski: None. K. Ziskovich: None. R. Jonas: None. S. Fishman: None. H. Sonmez: None. U. Rashid: None. A. Kodra: None. G. Stoffels: None. M. Lesser: None. V. Singh: None. L. Poretsky: None. Funding The Gerald J. and Dorothy R. Friedman New York Foundation for Medical Research

We previously showed that skin collagen AGEs at DCCT trial closeout were strongly associated with progression of microvascular disease even after adjustment for A1c (Glyconj. J. 33: 569, 2016). We now tested whether plasma protein AGEs are associated with subsequent progression of retinopathy, nephropathy and neuropathy. Methods: Fourteen AGEs were measured by LC/MS/MS in banked plasmas from 466 DCCT participants at three time points (TPs): DCCT year 4 (TP1), year 8 (TP2) and EDIC year 5/6 (TP3). Data were analyzed using correlation coefficients, while associations with risk of complications used Cox proportional hazards models were determined without adjustment for multiplicity. Results: Fructose-lysine (FL), glucosepane (GSPN) and carboxymethyl-lysine (CML) were lower in the former intensive glycemic control group at both TP1 and TP2 (P In conclusion, after adjustment for A1C, FL, GSPN, CML and MG-H1 are the plasma AGEs most strongly associated with progression of microvascular complications in type 1 diabetes. Disclosure V.M. Monnier: Consultant; Self; Juvenescence Ltd. D. Sell: None. J. Lachin: Board Member; Self; Tolerion, Inc. I. Bebu: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DP3DK101123-01)

Previous studies have shown that diabetes mellitus increases the risk of sarcopenia. We previously reported that higher serum advanced glycation end product (AGE) and lower serum insulin-like growth factor-I (IGF-I) were independently associated with muscle mass reduction in patients with type 2 diabetes. The present study examined effects of AGEs and IGF-I on myogenic differentiation and apoptosis in C2C12 cells. Real-time PCR and western blot analyses showed that AGE3 significantly decreased the mRNA and protein expressions of MyoD and Myogenin, whereas IGF-I significantly increased them and reversed the effects of AGE3. AGE3 significantly decreased endogenous IGF-I expression and suppressed IGF-I-induced Akt activation. High glucose (22 mM), but not AGE3, significantly increased expression of receptor for AGEs (RAGE), while IGF-I significantly decreased it. Moreover, DNA fragment ELISA showed that AGE2 and AGE3 significantly increased apoptosis of C2C12 cells, whereas IGF-I significantly suppressed the AGE2- and AGE3-induced apoptosis. To investigate whether high glucose enhances the AGE3’ effects, the effects of co-treatment with high glucose and AGE3 were examined. Although high glucose alone did not affect the apoptosis of C2C12 cells, it enhanced AGE3-induced apoptosis. IGF-I significantly reversed the effects of co-incubation of high glucose and AGEs on the expression of MyoD and Myogenin as well as the apoptosis. These findings indicate that AGE3 inhibits myogenic differentiation and increases apoptosis in C2C12 cells, and that high glucose increases RAGE and enhances the AGE3-induced apoptosis, suggesting that AGEs and high glucose might contribute to the reduction of muscle mass and function. Moreover, IGF-I reversed the detrimental effects of AGEs and high glucose in myoblastic cells; thus, IGF-I-Akt signal could be a therapeutic target of DM-induced sarcopenia. Disclosure I. Kanazawa: None. N. Adachi: None. K. Tanaka: None. A. Takeno: None. M. Notsu: None. S. Tanaka: None. T. Sugimoto: None.

Aim: Prescription eicosapentaenoic acid (EPA) reduced cardiovascular events in the REDUCE-IT study population, including patients with diabetes. As EPA has been previously shown to reduce LDL oxidation, we investigated if it may prevent HDL oxidation at a pharmacologic dose. Atheroprotective properties of HDL are compromised in patients with diabetes due to oxidative damage and glycation. Methods: Isolated human HDL at 200 µg/mL was incubated with vehicle or EPA at levels that match plasma concentrations (∼10 µM) at 4 g/d. All samples were subjected to oxidation over 6 hour under low and high glucose (200 mg/dL) conditions. Results: EPA inhibited HDL oxidation in a highly dose- and time-dependent manner. At lower concentrations (≤ 3.5 µM), EPA inhibited oxidation until 4 hour or less. At higher concentrations (>7.0 µM), EPA inhibited HDL oxidation 6 hour. At 10.0 µM, EPA inhibited HDL oxidation by 80% (p Conclusions: These data support concentration-dependent antioxidant effects for EPA that require pharmacologic concentrations for sustained activity. These effects of EPA may provide one potential mechanism for cardiovascular benefits in patients with diabetes. Disclosure S. Sherratt: None. R. Mason: Consultant; Self; Amarin Corporation. Research Support; Self; Amarin Corporation, Amgen Inc., Novartis AG, Pfizer Inc. Speaker's Bureau; Self; Pfizer Inc.

The accumulation of advanced glycation end products (AGEs) in the body contributes to pathogenesis of many diseases including complications of diabetes including retinopathy and neuropathy. Proteins in the lens of the eye do not turnover and therefore give an indication of the average glucose levels over a very long period of time. AGEs have a unique measurable fluorescence, however non-invasive measurement of AGEs was not available until recently with introducing a new scanning confocal biomicroscope. The main aim of the present study was to investigate if measurement of lens Auto fluorescence (FL) can distinguish subjects with impaired Glucose Tolerance (IGT) and type 2 diabetes (T2DM) from Healthy control subjects and to investigate the relationship between levels of FL. ratio, and corneal nerves morphology. 20 T2DM, 20 IGT and 20 Healthy aged matched control subjects underwent comprehensive medical and neurological assessments including corneal confocal microscopy and measurement of Crystalline Lens Autofluorescence by using a confocal bio microscope. There was a significant difference at the level of fluorescence ratios in Control subjects, IGT patient and T2DM patients. There was a significant correlation between FL. Ratios and level of HbA1C, and CNFL. The results of this study showed the Lens fluorescence is significantly greater in patients with IGT and T2DM. The level of AGEs products was correlated with HbA1C and alterations in corneal nerves morphology. However, the relationship with HbA1c was rather poor since HbA1c cannot completely reflect long-term glycation process. Lens autofluorescence could be a robust marker of long-term diabetes control predicting future complication risks. This supports the feasibility of lens autofluorescence to screen subjects for undiagnosed type 2 diabetes. However, confirmation of such hypothesis will need larger and long-term clinical studies. Disclosure M. Tavakoli: None.

Using banked urines from 156 DCCT participants of the former skin biopsy cohort (Diabetes 48:870,1999) we tested whether free AGEs measured at DCCT year 4 (Timepoint TP1), year 8(TP2) and EDIC year 17 (TP3) are associated with risk of microvascular complications progression. Methods: Fructose-lysine(FL), glucosepane(GSPN), carboxymethyl-lysine(CML), methylglyoxal (MG-H1) and 10 other markers were measured by LC/MS/MS. Data were analyzed using correlation coefficients, while associations with risk of complications using Cox proportional hazards (PH) models without adjustment for multiplicity. Results: FL (P Conclusions: FL, GSPN, and CML in urine consistently emerge as the AGEs most significantly associated with risk of subsequent microvascular complications. Disclosure D. Sell: None. J. Lachin: Board Member; Self; Tolerion, Inc. I. Bebu: None. V.M. Monnier: Consultant; Self; Juvenescence Ltd. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DP3DK101123)

Glucosepane is a member of the class of advanced glycation end-products (AGEs), which form non-enzymatically in the human body. Glucosepane, is the most abundant AGE found in human collagen and has been implicated in the pathophysiology of various conditions ranging from diabetes to normal human aging. Glucosepane crosslinks impact the structural and mechanical properties of collagen and contribute to stiffening of collagenous tissues and vascular dysfunction. We have previously demonstrated the total synthesis of glucosepane, enabling methods for detecting and targeting glucosepane. The present study seeks to identify enzymes that are capable of catalyzing the decomposition of synthetic glucosepane crosslinks. To identify glucosepane crosslink breaking enzymes, we developed a novel screening technology based on metagenomics. Using this strategy, four enzymes were identified for in vitro validation of glucosepane degradation activity. Thus far, our efforts have focused on identifying metabolites of one class I-like enzyme in particular, since it is heterologously expressed at high levels and does not require any unusual cofactors. In vitro enzymatic assays showed that incubation of glucosepane with the class I-like enzyme led to a product consistent with citrulline. We are currently in the process of evaluating the enzyme’s mechanisms of action and identifying other metabolites generated. This is the first demonstration that glucosepane can be broken down enzymatically. Our findings may provide new insight into the role of glucosepane in aging and disease and aid in the development of novel therapeutic strategies for targeting glucosepane. Disclosure M. Streeter: None. T.N. Goddard: None. J.M. Crawford: None. D.A. Spiegel: None.

Black youth with T1D have been noted to have higher HbA1c’s, less frequent monitoring of capillary glucose and higher reported occurrence of hypoglycemia compared to white patients. We hypothesized that this combination of factors would be associated with higher prescribed doses of insulin in black patients compared to whites. A retrospective analysis was performed on data collected from patients at the Children’s Diabetes Center, New Orleans, LA who participated in a study of racial differences in hemoglobin glycation. Patients self-identified as either black or white and were under the clinical care of several different attending diabetologists. The influence of race on total daily prescribed dose of insulin per kg body weight (TDDI), was evaluated in a general linear model adjusted for HbA1c, insulin delivery method, gender, and chronologic age as well as the interactive effect of race and HbA1c. Hemoglobin Glycation Index (HGI) was also calculated and substituted in the model for HbA1c. The population studied consisted of 33 (19F, 14M) black patients and 52 (22F,30 M)whites. Average age was 14.0±3.6 years and duration of diabetes 5.7±3.9 years. As previously reported from this population blacks had higher HbA1c even after adjustment for age and mean blood glucose level (MBG). Mean TDDI was 0.89±0.33 units/kg/d. TDDI was highly correlated with both HbA1c (r=0.40,p Disclosure S. Chalew: None. R. Gomez: None. J.M. Hempe: None.

Introduction: Early diagnostic measures that predict development of complications in individuals with type 1 diabetes would be clinically relevant. Our goal was to determine if glycative and oxidative stress biomarker levels are independent risk factors in the development of advanced diabetic kidney disease (DKD) prior to detection of overt disease in the DCCT and follow-up EDIC. Methods: Using a case-cohort design, we measured plasma and urine levels of 5 Advanced Glycation (AGEs) and 2 oxidation end-products by LC-MS/MS in 109 DKD cases (>40% eGFR decline from baseline confirmed on two consecutive visits) and 350 non-progressor controls. Sampling, done at study baseline and three other time points during DCCT and early EDIC prior to later GFR decline, was analyzed by Cox models adjusted for risk covariates (Age, HbA1c, BMI, HDL, LDL, Sex, SBP, and DBP) to assess the association between the AGEs and risk of DKD. Results: Significant associations between GFR decline and plasma biomarkers occurred for three specific AGEs: Carboxymethyllysine (CML, p=0.003), 3-deoxyglucosone hydroimidazolone (3DG-H, p=0.0007), and carboxyethyllysine (CEL, p=0.021) using multi-variate adjusted analyses. Prediction by AGEs was independent of HbA1c at the same time points. Urinary AGEs were not predictive. Calculation of c-statistics also showed that plasma 3DG-H significantly increased predictive value for progression of DKD above traditional risk factors. Conclusions: Two predictive AGEs, CML and CEL, previously shown to predict DKD (Diabetes Care, 36, 3234-9, 2013) were found to independently identify progression in DCCT/EDIC. DKD prediction by 3DG-H also confirms prior data showing that DKD is associated with increases in its precursor, 3-deoxyglucosone. Early identification of those at high risk of progression to DKD by measuring specific AGEs may provide predictive power above traditional risk factors and allow the institution of preventive therapies. Disclosure P.J. Beisswenger: Employee; Self; PreventAGE Health Care, LLC.. I. Bebu: None. S. Howell: None. J. Lachin: Board Member; Self; AbbVie Inc., Celgene Corporation.

Context and Objectives: Advanced glycation end products (AGEs) promote the development of diabetic complications through activation of their receptor (RAGE). Isoforms of soluble RAGE (sRAGE) sequester AGEs and thus protect against RAGE-mediated diabetic complications. Low cellular energy, such as during fasting, activates sRAGE production. We investigated the effect of an overnight fast on circulating substrates, hormones, AGEs, and sRAGE isoforms in 26 individuals with type 1 diabetes (T1DM). Participants and Methods: Blood was collected from young (18-30 years) T1DM patients on insulin pumps before and after an overnight fast. Circulating AGEs were measured via LC-MS/MS and sRAGE isoforms were analyzed via ELISA. eGFRcystatin-c was calculated based on circulating cystatin-c measured via ELISA. Results: Glucose, insulin, glucagon, and eGFRcystatin-c decreased while cortisol increased following the overnight fast (p0.05). AGEs (CML, CEL, 3DG-H, MG-H1, and G-H1) decreased (21-58%, p Conclusions: Following an overnight fast, sRAGE isoforms were increased and sequestered MG-H1. Sequestration of AGEs by sRAGE has been shown to attenuate AGE/RAGE-mediated complications. Energy restriction appears to be important for protection against AGE/RAGE-mediated complications. Disclosure E.R. Miranda: None. K.N. Fuller: None. R. Perkins: None. P.J. Beisswenger: Employee; Self; PreventAGE Health Care, LLC.. S.S. Farabi: None. L.T. Quinn: None. J.M. Haus: None.

Age-related macular degeneration (AMD) is one of the most important causes of blindness in the elderly patients. Statins, inhibitors for HMG-CoA reductase, are recently reported to be useful for decreasing the risk of wet-type AMD. However, the molecular mechanisms how statins reduce the risk of AMD remain unclear. In this study, we examined the effects of statins (atorvastatin and lovastatin) on the expression of VEGF and RAGE in human retinal pigment epithelial (RPE) cells. We used h1RPE7, human RPE cells, damaged by 20 µM hydroquinone (HQ)+300 µg/mL advanced glycation end-product (AGE) as an experimental AMD model. These cells were treated with atorvastatin (100 nM) or lovastatin (2 µM) for 24 hours. After the treatment, the expression of VEGF and RAGE was evaluated by real-time RT-PCR, and we found that the mRNA levels of VEGF and RAGE in the HQ+AGE treated cells were increased. The additions of statins significantly attenuated the HQ+AGE-induced VEGF and RAGE mRNA expression. We also measured the VEGF concentration in the cell culture medium by ELISA and found that it was markedly decreased by the addition of statins. In order to know the impact of RAGE on the VEGF expression, small interfering RNA (siRNA) for RAGE (siRAGE) was introduced into h1RPE7 cells by lipofection method and the VEGF expression was measured by real-time RT-PCR. The RT-PCR results show that siRAGE attenuated the up-regulation of VEGF in the HQ+AGE treated cells. The luciferase reporter assays for RAGE and VEGF transcription showed that the RAGE expression in the statin-treated cells was regulated at the transcriptional level, and that the VEGF expression in these cells was regulated by a post-transcriptional mechanism. These results indicate that statins attenuated the HQ+AGE-induced VEGF expression via decreasing RAGE expression. As VEGF is an important factor for the development of wet-type AMD, statins could decrease the risk of wet-type AMD and be a preventive medicine for diabetic patients. Disclosure H. Tsujinaka: None. A. Itaya-Hironaka: None. A. Yamauchi: None. S. Sakuramoto-Tsuchida: None. M. Makino: None. R. Shobatake: None. N. Masuda: None. H. Hirai: None. N. Ogata: Research Support; Self; Alcon Japan, Bayer Yakuhin, Ltd., Santen Seyaku Co., Ltd., AMO, Otsuka Holdings Co., Ltd., HOYA Co., Ltd.. S. Takasawa: None.

Background and Aims: Galectin-3, a member of the multifunctional galectin family, is ubiquitously expressed and acts a broad-spectrum biological response modifier. It is involved in tissue fibrosis, tumorigenesis, immunity and inflammatory response. It can also act as a receptor for advanced glycation end products. Recent evidence suggests that galectin-3 is associated with heart failure and with progression of nephropathy in type 2 diabetes. We have therefore evaluated whether serum galectin-3 level is associated with all-cause mortality in patients with type 2 diabetes. Materials and Methods: Galectin-3 was measured in the baseline serum samples by ELISA in 1495 type 2 diabetic patients followed up in a teaching hospital specialist diabetes clinic. All-cause mortality was ascertained from hospital electronic medical records. Results: The mean age of the subjects was 55.3 ± 9.9 years and 54% were male. During a median follow-up of 11 years, 169 subjects died from all causes, and baseline serum galectin-3 levels were significantly higher in subjects with a fatal outcome (9.86 ± 3.11 ng/ml vs. 8.15 ± 2.73, p Conclusion: Elevated serum galectin-3 level was independently associated with all-cause mortality in patients with type 2 diabetes. Galectin-3 may potentially be useful as a predictor of adverse outcome. Disclosure K.C.B. Tan: None. S. Shiu: None. J.K.Y. Lam: None. A.C. Lee: None. Y. Wong: None.

Diabetes is associated with HDL dysfunction and perturbed iron metabolism. ApoAI, transferrin (Trf) and ceruloplasmin (Cp) are the key HDL proteins involved in cholesterol efflux and iron metabolism. We tested the hypothesis that hyperglycemia-induced glycation contributes to HDL dysfunction and altered iron metabolism in diet-controlled patients with T2D. HDL from patients with T2D and matched healthy controls (n=9/group) was isolated and it’s anti-oxidant and cholesterol efflux properties were quantified. HDL proteome composition and post-translational modification of proteins were quantified by proteomics aproach. Metabolic 2H2O-labeling was applied to quantify HDL proteome dynamics. Patients with T2D and controls had similar lipid (triglycerides, total cholesterol, and HDL cholesterol) profile. HDL from T2D patients had reduced anti-oxidant (PON1 activity) and macrophage-cholesterol efflux capacity (P Disclosure T. Kasumov: None. M. Golizeh: None. S. Kashyap: None.

Diabetic Retinopathy (DR) is the leading cause of blindness in the United States corresponding to 20,000 new diagnoses annually (cite this). Hyperglycemia is a major risk factor of type 2 diabetes and leads to the formation of plasma proteins known as Advanced Glycation End Products (AGEs). In the eye, AGES’s bind to the receptor of the Advanced Glycation End Products (RAGEs) eventually leading to DR. The following study analyzed drug targets (single drugs and combination of drugs) that are currently marketed for heart disease, diabetes, and hypertension to understand how they interact with RAGE with the goal of identifying compounds that are predicted to inhibit the ligand-receptor interaction. Docking programs, AutoDock Tools and AutoDock Vina, were used to determine the binding affinity and visual orientation of the drug receptor binding. Cholesterol lowering drugs such as Atorvastatin and Fenofibrate were able to bind to RAGE with a high hydrophobicity and great negative affinity (-7.5 and -7.7). In combination, the hypertensive drugs such as Telmisartan (Angiotensin II receptor blocker) and Amlodipine (calcium channel blocker) bind with the RAGE receptor with a high hydrophobicity and high negative affinity (-9.7). Metformin administered alone or in combination was not shown to bind to RAGE. Overall this analysis shows that a combination of hypertensive drugs and cholesterol lowering drugs may decrease the progression of DR in type II diabetes. Disclosure S. Sriramoju: None. K. Goetz: None.

The mechanisms for the development of diabetic cardiomyopathy remain largely unknown. Methylglyoxal (MG) can accumulate and promote inflammation and vascular damage in diabetes. We examined if overexpression of the MG-metabolizing enzyme glyoxalase 1 (GLO1) in macrophages and the vasculature could reduce MG-induced inflammation and prevent ventricular dysfunction in diabetes. Hyperglycemia increased circulating inflammatory markers in wild-type (WT) but not in GLO1-overexpressing mice. Endothelial cell number was reduced in WT-diabetic hearts compared with nondiabetic controls, whereas GLO1 overexpression preserved capillary density. Neuregulin production, endothelial nitric oxide synthase dimerization, and Bcl-2 expression in endothelial cells was maintained in the hearts of GLO1-diabetic mice and corresponded to less myocardial cell death compared with the WT-diabetic group. Lower receptor for advanced glycation end products and tumor necrosis factor-α (TNF-α) levels were also observed in GLO1-diabetic versus WT-diabetic mice. Over a period of 8 weeks of hyperglycemia, GLO1 overexpression delayed and limited the loss of cardiac function. In vitro, MG and TNF-α were shown to synergize in promoting endothelial cell death, which was associated with increased angiopoietin 2 expression and reduced Bcl-2 expression. These results suggest that MG in diabetes increases inflammation, leading to endothelial cell loss. This contributes to the development of diabetic cardiomyopathy and identifies MG-induced endothelial inflammation as a target for therapy.

In diabetes, toxic oxidative pathways are triggered by persistent hyperglycemia and contribute to diabetes complications. A major proposed pathogenic mechanism is the accumulation of protein modifications that are called advanced glycation end products. However, other nonenzymatic post-translational modifications may also contribute to pathogenic protein damage in diabetes. We demonstrate that hypohalous acid–derived modifications of renal tissues and extracellular matrix (ECM) proteins are significantly elevated in experimental diabetic nephropathy. Moreover, diabetic renal ECM shows diminished binding of α1β1 integrin consistent with the modification of collagen IV by hypochlorous (HOCl) and hypobromous acids. Noncollagenous (NC1) hexamers, key connection modules of collagen IV networks, are modified via oxidation and chlorination of tryptophan and bromination of tyrosine residues. Chlorotryptophan, a relatively minor modification, has not been previously found in proteins. In the NC1 hexamers isolated from diabetic kidneys, levels of HOCl-derived oxidized and chlorinated tryptophan residues W28 and W192 are significantly elevated compared with nondiabetic controls. Molecular dynamics simulations predicted a more relaxed NC1 hexamer tertiary structure and diminished assembly competence in diabetes; this was confirmed using limited proteolysis and denaturation/refolding. Our results suggest that hypohalous acid–derived modifications of renal ECM, and specifically collagen IV networks, contribute to functional protein damage in diabetes.

The deleterious effects of high glucose levels and enhanced metabolic flux on the vasculature are thought to be mediated by the generation of toxic metabolites, including reactive dicarbonyls like methylglyoxal (MG). In this article, we demonstrate that increasing plasma MG to levels observed in diabetic mice either using an exogenous source (1% in drinking water) or generated following inhibition, its primary clearance enzyme, glyoxalase-1 (with 50 mg/kg IP bromobenzyl-glutathione cyclopentyl diester every second day), was able to increase vascular adhesion and augment atherogenesis in euglycemic apolipoprotein E knockout mice to a similar magnitude as that observed in hyperglycemic mice with diabetes. The effects of MG appear partly mediated by activation of the receptor for advanced glycation end products (RAGE), as deletion of RAGE was able to reduce inflammation and atherogenesis associated with MG exposure. However, RAGE deletion did not completely prevent inflammation or vascular damage, possibly because the induction of mitochondrial oxidative stress by dicarbonyls also contributes to inflammation and atherogenesis. Such data would suggest that a synergistic combination of RAGE antagonism and antioxidants may offer the greatest utility for the prevention and management of diabetic vascular complications.

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.

In mammals, changes in the metabolic state, including obesity, fasting, cold challenge, and high-fat diets (HFDs), activate complex immune responses. In many strains of rodents, HFDs induce a rapid systemic inflammatory response and lead to obesity. Little is known about the molecular signals required for HFD-induced phenotypes. We studied the function of the receptor for advanced glycation end products (RAGE) in the development of phenotypes associated with high-fat feeding in mice. RAGE is highly expressed on immune cells, including macrophages. We found that high-fat feeding induced expression of RAGE ligand HMGB1 and carboxymethyllysine-advanced glycation end product epitopes in liver and adipose tissue. Genetic deficiency of RAGE prevented the effects of HFD on energy expenditure, weight gain, adipose tissue inflammation, and insulin resistance. RAGE deficiency had no effect on genetic forms of obesity caused by impaired melanocortin signaling. Hematopoietic deficiency of RAGE or treatment with soluble RAGE partially protected against peripheral HFD-induced inflammation and weight gain. These findings demonstrate that high-fat feeding induces peripheral inflammation and weight gain in a RAGE-dependent manner, providing a foothold in the pathways that regulate diet-induced obesity and offering the potential for therapeutic intervention.

The interaction of advanced glycation end products (AGEs) and their receptor (RAGE) plays a central role in diabetic nephropathy. We screened DNA aptamers directed against RAGE (RAGE-aptamers) in vitro and examined the effects on the development and progression of diabetic nephropathy in streptozotocin-induced diabetic rats. RAGE-aptamer bound to RAGE with a Kd of 5.68 nmol/L and resultantly blocked the binding of AGEs to RAGE. When diabetic rats received continuous intraperitoneal injection of RAGE-aptamer from week 7 to 11 of diabetes, the increases in renal NADPH oxidase activity, oxidative stress generation, AGE, RAGE, inflammatory and fibrotic gene and protein levels, macrophage and extracellular matrix accumulation, and albuminuria were significantly suppressed, which were associated with improvement of podocyte damage. Two-week infusion of RAGE-aptamer just after the induction of diabetes also inhibited the AGE-RAGE-oxidative stress system and MCP-1 levels in the kidneys of 8-week-old diabetic rats and simultaneously ameliorated podocyte injury and albuminuria. Moreover, RAGE-aptamer significantly suppressed the AGE-induced oxidative stress generation and inflammatory and fibrotic reactions in human cultured mesangial cells. The findings suggest that continuous infusion of RAGE-aptamer could attenuate the development and progression of experimental diabetic nephropathy by blocking the AGE-RAGE axis.

Efficient biomarkers for diabetic nephropathy (DN) have not been established. Using ELISA, we found previously that urinary levels of full-length megalin (C-megalin), a multiligand endocytic receptor in proximal tubules, was positively correlated with DN progression in patients with type 2 diabetes mellitus (T2DM). Here, we found that urinary extracellular vesicle (UEV) excretion and C-megalin content in UEVs or in their exosomal fraction increased along with the progression of the albuminuric stages in patients with T2DM. Cultured immortalized rat proximal tubule cells (IRPTCs) treated with fatty acid–free BSA or advanced glycation end product–modified BSA (AGE-BSA), endocytic ligands of megalin, increased EV excretion, and their C-megalin content. C-megalin excretion from IRPTCs via extracellular vesicles was significantly blocked by an exosome-specific inhibitor, GW4869, indicating that this excretion is mainly exocytosis-mediated. AGE-BSA treatment of IRPTCs caused apparent lysosomal dysfunction, which stimulated multivesicular body formation, resulting in increased exosomal C-megalin excretion. In a high-fat diet–induced, megalin-mediated kidney injury model in mice, urinary C-megalin excretion also increased via UEVs. Collectively, exocytosis-mediated urinary C-megalin excretion is associated with the development and progression of DN in patients with T2DM, particularly due to megalin-mediated lysosomal dysfunction in proximal tubules, and hence it could be a candidate biomarker linked with DN pathogenesis.

Advanced glycation end products (AGEs) and their receptor (RAGE) play a role in diabetic nephropathy. We screened DNA aptamer directed against AGEs (AGEs-aptamer) in vitro and examined its effects on renal injury in KKAy/Ta mice, an animal model of type 2 diabetes. Eight-week-old male KKAy/Ta or C57BL/6J mice received continuous intraperitoneal infusion of AGEs- or control-aptamer for 8 weeks. AGEs-aptamer was detected and its level was increased in the kidney for at least 7 days. The elimination half-lives of AGEs-aptamer in the kidney were about 7 days. Compared with those in C57BL/6J mice, glomerular AGEs levels were significantly increased in KKAy/Ta mice, which were blocked by AGEs-aptamer. Urinary albumin and 8-hydroxy-2′-deoxy-guanosine levels were increased, and glomerular hypertrophy and enhanced extracellular matrix accumulation were observed in KKAy/Ta mice, all of which were prevented by AGEs-aptamer. Moreover, AGEs-aptamer significantly reduced gene expression of RAGE, monocyte chemoattractant protein-1, connective tissue growth factor, and type IV collagen both in the kidney of KKAy/Ta mice and in AGE-exposed human cultured mesangial cells. Our present data suggest that continuous administration of AGEs-aptamer could protect against experimental diabetic nephropathy by blocking the AGEs-RAGE axis and may be a feasible and promising therapeutic strategy for the treatment of diabetic nephropathy.

Atherosclerotic vascular disease is a major cause of cardiovascular (CV) morbidity and mortality and a significant driver of health care costs in patients with diabetes. Systemic abnormalities, including dyslipidemia, insulin resistance, hyperinsulinemia, hyperglycemia, oxidative stress, accentuated renin-angiotensin system, and tissue inflammation, have been proposed to play a role in mediating accelerated atherosclerosis in diabetes (1). Hyperglycemia not only predicts CV disease risk in individuals without known CV disease or diabetes (2), but atherosclerosis is also quite prevalent in prediabetes (3). Although difficult to achieve, near-normal glycemic control is not typically associated with further reduction in macrovascular events in patients with type 2 diabetes (4). Consequently, elucidating mechanisms by which hyperglycemia affects atherosclerosis is necessary to identify novel therapeutic targets. Increased mitochondrial reactive oxygen species (ROS) has been identified as a common upstream event that mediates the atherogenic effects of hyperglycemia (5). ROS increases the formation of advanced glycation end products (AGEs), augments the expression of the receptor for AGEs (RAGE) and its ligands, activates protein kinase C (PKC) isoforms (primarily PKCβ isoform), and accentuates flux through the hexosamine and polyol pathways (5). Among these, the ligand-RAGE axis appears to play a major role in vascular dysfunction in diabetes. AGEs, S100/calgranulins, and high-mobility group box 1 are principal endogenous ligands for RAGE (6). Persistent activation of RAGE promotes atherosclerosis by activating a diverse array of intracellular signaling pathways that stimulate expression of cytokines, cellular adhesion molecules, growth factors, ROS, and vascular matrix metalloproteinases (6). Diabetes-induced atherosclerosis …

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.

OBJECTIVE Skeletal muscle myopathy is a common diabetes complication. One possible cause of myopathy is myocyte failure to repair contraction-generated plasma membrane injuries. Here, we test the hypothesis that diabetes induces a repair defect in skeletal muscle myocytes. RESEARCH DESIGN AND METHODS Myocytes in intact muscle from type 1 (INS2Akita+/−) and type 2 (db/db) diabetic mice were injured with a laser and dye uptake imaged confocally to test repair efficiency. Membrane repair defects were also assessed in diabetic mice after downhill running, which induces myocyte plasma membrane disruption injuries in vivo. A cell culture model was used to investigate the role of advanced glycation end products (AGEs) and the receptor for AGE (RAGE) in development of this repair defect. RESULTS Diabetic myocytes displayed significantly more dye influx after laser injury than controls, indicating a repair deficiency. Downhill running also resulted in a higher level of repair failure in diabetic mice. This repair defect was mimicked in cultured cells by prolonged exposure to high glucose. Inhibition of the formation of AGE eliminated this glucose-induced repair defect. However, a repair defect could be induced, in the absence of high glucose, by enhancing AGE binding to RAGE, or simply by increasing cell exposure to AGE. CONCLUSIONS Because one consequence of repair failure is rapid cell death (via necrosis), our demonstration that repair fails in diabetes suggests a new mechanism by which myopathy develops in diabetes.

OBJECTIVE Excess accumulation of advanced glycation end products (AGEs) contributes to aging and chronic diseases. We aimed to obtain evidence that exposure to AGEs plays a role in the development of type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS The effect of AGEs was examined on insulin secretion by MIN6N8 cells and mouse islets and in vivo in three separate rodent models: AGE-injected or high AGE–fed Sprague-Dawley rats and nonobese diabetic (NODLt) mice. Rodents were also treated with the AGE-lowering agent alagebrium. RESULTS β-Cells exposed to AGEs displayed acute glucose-stimulated insulin secretory defects, mitochondrial abnormalities including excess superoxide generation, a decline in ATP content, loss of MnSOD activity, reduced calcium flux, and increased glucose uptake, all of which were improved with alagebrium treatment or with MnSOD adenoviral overexpression. Isolated mouse islets exposed to AGEs had decreased glucose-stimulated insulin secretion, increased mitochondrial superoxide production, and depletion of ATP content, which were improved with alagebrium or with MnTBAP, an SOD mimetic. In rats, transient or chronic exposure to AGEs caused progressive insulin secretory defects, superoxide generation, and β-cell death, ameliorated with alagebrium. NODLt mice had increased circulating AGEs in association with an increase in islet mitochondrial superoxide generation, which was prevented by alagebrium, which also reduced the incidence of autoimmune diabetes. Finally, at-risk children who progressed to T1D had higher AGE concentrations than matched nonprogressors. CONCLUSIONS These findings demonstrate that AGEs directly cause insulin secretory defects, most likely by impairing mitochondrial function, which may contribute to the development of T1D.

OBJECTIVE Circulating levels of soluble receptor for advanced glycation end products (sRAGE) likely comprise both a secreted isoform (esRAGE) and wild-type RAGE cleaved from the cell membrane. Both sRAGE and esRAGE have been proposed as biomarkers of cardiovascular disease (CVD), but prospective data are limited. We examined the relationship of sRAGE and esRAGE to incident coronary heart disease (CHD) and stroke in type 2 diabetic patients followed for 3.9 years in a trial of atorvastatin: the Collaborative Atorvastatin Diabetes Study (CARDS). RESEARCH DESIGN AND METHODS We used a nested case-control design sampling all incident cases of CVD with available plasma and randomly selecting three control subjects, who were free of CVD throughout follow-up, per case. Analysis was by Cox regression with adjustment for treatment allocation and relevant covariates. RESULTS sRAGE and esRAGE were strongly correlated (ρ = 0.88) and were both higher in those with lower BMI (P < 0.001), higher adiponectin (P < 0.001), lower estimated glomerular filtration rate (P = 0.009), and white ethnicity (P < 0.001). Both sRAGE and esRAGE were associated with incident CHD events, independently of treatment allocation and the above factors; hazard ratio (HR) = 1.74 (95% CI 1.25–2.41; P = 0.002) for a doubling of the sRAGE level; HR = 1.45 (1.11–1.89; P = 0.006) for a doubling of the esRAGE level. There was no significant association with stroke; HR for sRAGE = 0.66 (0.38–1.14). Atorvastatin, 10 mg daily, did not alter sRAGE. CONCLUSIONS Higher levels of sRAGE and esRAGE are associated with incident CHD but not stroke in type 2 diabetes.

OBJECTIVE To study whether modification of LDL by methylglyoxal (MG), a potent arginine-directed glycating agent that is increased in diabetes, is associated with increased atherogenicity. RESEARCH DESIGN AND METHODS Human LDL was isolated and modified by MG in vitro to minimal extent (MGmin-LDL) as occurs in vivo. Atherogenic characteristics of MGmin-LDL were characterized: particle size, proteoglycan-binding, susceptibility to aggregation, LDL and non-LDL receptor–binding, and aortal deposition. The major site of modification of apolipoprotein B100 (apoB100) modification was investigated by mass spectrometric peptide mapping. RESULTS MGmin-LDL contained 1.6 molar equivalents of MG modification—mostly hydroimidazolone—as found in vivo. MGmin-LDL had decreased particle size, increased binding to proteoglycans, and increased aggregation in vitro. Cell culture studies showed that MGmin-LDL was bound by the LDL receptor but not by the scavenger receptor and had increased binding affinity for cell surface heparan sulfate–containing proteoglycan. Radiotracer studies in rats showed that MGmin-LDL had a similar fractional clearance rate in plasma to unmodified LDL but increased partitioning onto the aortal wall. Mass spectrometry peptide mapping identified arginine-18 as the hotspot site of apoB100 modification in MGmin-LDL. A computed structural model predicted that MG modification of apoB100 induces distortion, increasing exposure of the N-terminal proteoglycan–binding domain on the surface of LDL. This likely mediates particle remodeling and increases proteoglycan binding. CONCLUSIONS MG modification of LDL forms small, dense LDL with increased atherogenicity that provides a new route to atherogenic LDL and may explain the escalation of cardiovascular risk in diabetes and the cardioprotective effect of metformin.

OBJECTIVE High cholesterol levels in circulating immune complexes (IC), surrogate markers of modified LDL, are associated with increased carotid intima-media thickness (IMT) and cardiovascular events in type 1 diabetes. Different modifications of LDL are involved in IC formation, but which of these are predictive of vascular events is not known. Therefore, we measured oxidized LDL (oxLDL), advanced glycation end products–modified LDL (AGE-LDL), and malondialdehyde-modified LDL (MDA-LDL) in IC and determined their relationship with increased carotid IMT and compared the strength of the association with that observed with conventional risk factors. RESEARCH DESIGN AND METHODS Levels of oxLDL, AGE-LDL, and MDA-LDL were measured in circulating IC isolated from sera of 479 patients of the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) cohort, collected at baseline. Internal and common carotid IMT were measured 8 and 14 years later by DCCT/EDIC. RESULTS OxLDL, AGE-LDL, and MDA-LDL levels in circulating IC were significantly correlated with diabetes duration, BMI, and lipid and blood pressure, but not with age. Multivariate logistic regression models indicated that individuals in the highest versus lowest quartile of oxLDL and AGE-LDL in IC had a 6.11-fold [confidence interval (CI) 2.51–14.8] and a 6.4-fold (CI 2.53–16.2) increase in the odds of having high carotid IMT, respectively, after adjusting for conventional risk factors. Parallel analyses resulted in odds ratios of 2.62 (CI 1.24, 5.55) for LDL-C, 1.45 (CI 0.69, 3.03) for diastolic blood pressure, and 2.33 (CI 1.09, 4.99) for A1C. CONCLUSIONS OxLDL and AGE-LDL in circulating IC were significantly associated with progression and increased levels of carotid IMT in type 1 diabetes.

OBJECTIVE RAGE interacts with the endogenous ligands S100 calgranulins and high mobility group box 1 (HMGB1) to induce inflammation. Since hyperglycemia-induced reactive oxygen species (ROS) activate many pathways of diabetic tissue damage, the effect of these ROS on RAGE and RAGE ligand expression was evaluated. RESEARCH DESIGN AND METHODS Expression of RAGE, S100A8, S100A12, and HMGB1 was evaluated in human aortic endothelial cells (HAECs) incubated in normal glucose, high glucose, and high glucose after overexpression of either uncoupling protein 1 (UCP1), superoxide dismutase 2 (SOD2), or glyoxalase 1 (GLO1). Expression was also evaluated in normal glucose after knockdown of GLO1. Expression was next evaluated in high glucose after knockdown of nuclear factor (NF)-κB p65 (RAGE) and after knockdown of activated protein-1 (AP-1) (S100A8, S100A12, and HMGB1), and chromatin immunoprecipitation (ChIP) was performed ± GLO1 overexpression for NFκB p65 (RAGE promoter) and AP-1 (S100A8, S100A12, and HMGB1 promoters). Finally, endothelial cells from nondiabetic mice, STZ diabetic mice, and STZ diabetic mice treated with the superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) were evaluated. RESULTS High glucose increased RAGE, S100A8, S100A12, and HMGB1 expression, which was normalized by overexpression of UCP1, SOD2, or GLO1. GLO1 knockdown mimicked the effect of high glucose, and in high glucose, overexpression of GLO1 normalized increased binding of NFκB p65 and AP-1. Diabetes increased RAGE, S100A8, and HMGB1 expression, and MnTBAP treatment normalized this. CONCLUSIONS These results show that hyperglycemia-induced ROS production increases expression of RAGE and RAGE ligands. This effect is mediated by ROS-induced methylglyoxal, the major substrate of glyoxalase 1.

OBJECTIVE The goal of this study was to characterize glycation adducts formed in both in vivo extracellular matrix (ECM) proteins of endoneurium from streptozotocin (STZ)-induced diabetic rats and in vitro by glycation of laminin and fibronectin with methylglyoxal and glucose. We also investigated the impact of advanced glycation end product (AGE) residue content of ECM on neurite outgrowth from sensory neurons. RESEARCH DESIGN AND METHODS Glycation, oxidation, and nitration adducts of ECM proteins extracted from the endoneurium of control and STZ-induced diabetic rat sciatic nerve (3–24 weeks post-STZ) and of laminin and fibronectin that had been glycated using glucose or methylglyoxal were examined by liquid chromatography with tandem mass spectrometry. Methylglyoxal-glycated or unmodified ECM proteins were used as substrata for dissociated rat sensory neurons as in vitro models of regeneration. RESULTS STZ-induced diabetes produced a significant increase in early glycation Nε-fructosyl-lysine and AGE residue contents of endoneurial ECM. Glycation of laminin and fibronectin by methylglyoxal and glucose increased glycation adduct residue contents with methylglyoxal-derived hydroimidazolone and Nε-fructosyl-lysine, respectively, of greatest quantitative importance. Glycation of laminin caused a significant decrease in both neurotrophin-stimulated and preconditioned sensory neurite outgrowth. This decrease was prevented by aminoguanidine. Glycation of fibronectin also decreased preconditioned neurite outgrowth, which was prevented by aminoguanidine and nerve growth factor. CONCLUSIONS Early glycation and AGE residue content of endoneurial ECM proteins increase markedly in STZ-induced diabetes. Glycation of laminin and fibronectin causes a reduction in neurotrophin-stimulated neurite outgrowth and preconditioned neurite outgrowth. This may provide a mechanism for the failure of collateral sprouting and axonal regeneration in diabetic neuropathy.

OBJECTIVE—Mitochondrial superoxide levels are elevated in the retina in diabetes, and manganese superoxide dismutase overexpression prevents the development of retinopathy. Superoxide inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which activates major pathways implicated in diabetic complications, including advanced glycation end products (AGEs), protein kinase C, and hexosamine pathway. Our aim is to investigate the role of GAPDH in the development and progression of diabetic retinopathy and to elucidate the mechanism. RESEARCH DESIGN AND METHODS—Rats with streptozotocin-induced diabetes were in a state of poor control (GHb >11%) for 12 months, good control (GHb RESULTS—In rats with poor control, retinal GAPDH activity and expressions were subnormal with increased ribosylation and nitration (25–30%). GAPDH activity was subnormal in both cytosol and nuclear fractions, but its protein expression and nitration were significantly elevated in nuclear fraction. Reinstitution of good control failed to protect inactivation of GAPDH, its covalent modification, and translocation to the nucleus. PKC, AGEs, and hexosamine pathways remained activated, and microvascular histopathology was unchanged. However, GAPDH and its translocation in good control rats were similar to those in normal rats. CONCLUSIONS—GAPDH plays a significant role in the development of diabetic retinopathy and its progression after cessation of hyperglycemia. Thus, therapies targeted toward preventing its inhibition may inhibit development of diabetic retinopathy and arrest its progression.

OBJECTIVE Embryos exposed to a diabetic environment in utero have an increased risk to develop congenital heart malformations. The mechanism behind the teratogenicity of diabetes still remains enigmatic. Detrimental effects of glycation products in diabetic patients have been well documented. We therefore studied a possible link between glycation products and the development of congenital cardiovascular malformations. Furthermore, we investigated other possible mechanisms involved in this pathogenesis: alterations in the levels of vascular endothelial growth factor (VEGF) or phosphorylated Smad2 (the latter can be induced by both glycation products and VEGF). RESEARCH DESIGN AND METHODS We examined the temporal spatial patterning of the glycation products Nε(carboxymethyl)lysine (CML) and methylglyoxal (MG) adducts, VEGF expression, and phosphorylated Smad2 during cardiovascular development in embryos from normal and diabetic rats. RESULTS Maternal diabetes increased the CML accumulation in the areas susceptible to diabetes-induced congenital heart disease, including the outflow tract of the heart and the aortic arch. No MG adducts could be detected, suggesting that CML is more likely to be indicative for increased oxidative stress than for glycation. An increase of CML in the outflow tract of the heart was accompanied by an increase in phosphorylated Smad2, unrelated to VEGF. VEGF showed a time-specific decrease in the outflow tract of embryos from diabetic dams. CONCLUSIONS From our results, we can conclude that maternal diabetes results in transient and localized alterations in CML, VEGF expression, and Smad2 phosphorylation overlapping with those regions of the developing heart that are most sensitive to diabetes-induced congenital heart disease.