The nonvesicular sterol transporter Aster-C plays a minor role in whole body cholesterol balance.

Introduction: The Aster-C protein (encoded by the Gramd1c gene) is an endoplasmic reticulum (ER) resident protein that has been reported to transport cholesterol from the plasma membrane to the ER. Although there is a clear role for the closely-related Aster-B protein in cholesterol transport and downstream esterification in the adrenal gland, the specific role for Aster-C in cholesterol homeostasis is not well understood. Here, we have examined whole body cholesterol balance in mice globally lacking Aster-C under low or high dietary cholesterol conditions. Method: Age-matched Gramd1c +/+ and Gramd1c -/- mice were fed either low (0.02%, wt/wt) or high (0.2%, wt/wt) dietarycholesterol and levels of sterol-derived metabolites were assessed in the feces, liver, and plasma. Results: Compared to wild type controls (Gramd1c +/+) mice, mice lackingGramd1c (Gramd1c -/-) have no significant alterations in fecal, liver, or plasma cholesterol. Given the potential role for Aster C in modulating cholesterol metabolism in diverse tissues, we quantified levels of cholesterol metabolites such as bile acids, oxysterols, and steroid hormones. Compared to Gramd1c +/+ controls, Gramd1c -/- mice had modestly reduced levels of select bile acid species and elevated cortisol levels, only under low dietary cholesterol conditions. However, the vast majority of bile acids, oxysterols, and steroid hormones were unaltered in Gramd1c -/- mice. Bulk RNA sequencing in the liver showed that Gramd1c -/- mice did not exhibit alterations in sterol-sensitive genes, but instead showed altered expression of genes in major urinary protein and cytochrome P450 (CYP) families only under low dietary cholesterol conditions. Discussion: Collectively, these data indicate nominal effects of Aster-C on whole body cholesterol transport and metabolism under divergent dietary cholesterol conditions. These results strongly suggest that Aster-C alone is not sufficient to control whole body cholesterol balance, but can modestly impact circulating cortisol and bile acid levels when dietary cholesterol is limited.

Duodenal Anaerobutyricum soehngenii infusion stimulates GLP-1 production, ameliorates glycaemic control and beneficially shapes the duodenal transcriptome in metabolic syndrome subjects: a randomised double-blind placebo-controlled cross-over study.

OBJECTIVE: Although gut dysbiosis is increasingly recognised as a pathophysiological component of metabolic syndrome (MetS), the role and mode of action of specific gut microbes in metabolic health remain elusive. Previously, we identified the commensal butyrogenic Anaerobutyricum soehngenii to be associated with improved insulin sensitivity in subjects with MetS. In this proof-of-concept study, we investigated the potential therapeutic effects of A. soehngenii L2-7 on systemic metabolic responses and duodenal transcriptome profiles in individuals with MetS. DESIGN: In this randomised double-blind placebo-controlled cross-over study, 12 male subjects with MetS received duodenal infusions of A. soehngenii/ placebo and underwent duodenal biopsies, mixed meal tests (6 hours postinfusion) and 24-hour continuous glucose monitoring. RESULTS: A. soehngenii treatment provoked a markedly increased postprandial excursion of the insulinotropic hormone glucagon-like peptide 1 (GLP-1) and an elevation of plasma secondary bile acids, which were positively associated with GLP-1 levels. Moreover, A. soehngenii treatment robustly shaped the duodenal expression of 73 genes, with the highest fold induction in the expression of regenerating islet-protein 1B (REG1B)-encoding gene. Strikingly, duodenal REG1B expression positively correlated with GLP-1 levels and negatively correlated with peripheral glucose variability, which was significantly diminished in the 24 hours following A. soehngenii intake. Mechanistically, Reg1B expression is induced upon sensing butyrate or bacterial peptidoglycan. Importantly, A. soehngenii duodenal administration was safe and well tolerated. CONCLUSIONS: A single dose of A. soehngenii improves peripheral glycaemic control within 24 hours; it specifically stimulates intestinal GLP-1 production and REG1B expression. Further studies are needed to delineate the specific pathways involved in REG1B induction and function in insulin sensitivity. TRIAL REGISTRATION NUMBER: NTR-NL6630.

Propionate attenuates atherosclerosis by immune-dependent regulation of intestinal cholesterol metabolism.

AIMS: Atherosclerotic cardiovascular disease (ACVD) is a major cause of mortality and morbidity worldwide, and increased low-density lipoproteins (LDLs) play a critical role in development and progression of atherosclerosis. Here, we examined for the first time gut immunomodulatory effects of the microbiota-derived metabolite propionic acid (PA) on intestinal cholesterol metabolism. METHODS AND RESULTS: Using both human and animal model studies, we demonstrate that treatment with PA reduces blood total and LDL cholesterol levels. In apolipoprotein E-/- (Apoe-/-) mice fed a high-fat diet (HFD), PA reduced intestinal cholesterol absorption and aortic atherosclerotic lesion area. Further, PA increased regulatory T-cell numbers and interleukin (IL)-10 levels in the intestinal microenvironment, which in turn suppressed the expression of Niemann-Pick C1-like 1 (Npc1l1), a major intestinal cholesterol transporter. Blockade of IL-10 receptor signalling attenuated the PA-related reduction in total and LDL cholesterol and augmented atherosclerotic lesion severity in the HFD-fed Apoe-/- mice. To translate these preclinical findings to humans, we conducted a randomized, double-blinded, placebo-controlled human study (clinical trial no. NCT03590496). Oral supplementation with 500 mg of PA twice daily over the course of 8 weeks significantly reduced LDL [-15.9 mg/dL (-8.1%) vs. -1.6 mg/dL (-0.5%), P = 0.016], total [-19.6 mg/dL (-7.3%) vs. -5.3 mg/dL (-1.7%), P = 0.014] and non-high-density lipoprotein cholesterol levels [PA vs. placebo: -18.9 mg/dL (-9.1%) vs. -0.6 mg/dL (-0.5%), P = 0.002] in subjects with elevated baseline LDL cholesterol levels. CONCLUSION: Our findings reveal a novel immune-mediated pathway linking the gut microbiota-derived metabolite PA with intestinal Npc1l1 expression and cholesterol homeostasis. The results highlight the gut immune system as a potential therapeutic target to control dyslipidaemia that may introduce a new avenue for prevention of ACVDs.

Maternal cecal microbiota transfer rescues early-life antibiotic-induced enhancement of type 1 diabetes in mice.

Early-life antibiotic exposure perturbs the intestinal microbiota and accelerates type 1 diabetes (T1D) development in the NOD mouse model. Here, we found that maternal cecal microbiota transfer (CMT) to NOD mice after early-life antibiotic perturbation largely rescued the induced T1D enhancement. Restoration of the intestinal microbiome was significant and persistent, remediating the antibiotic-depleted diversity, relative abundance of particular taxa, and metabolic pathways. CMT also protected against perturbed metabolites and normalized innate and adaptive immune effectors. CMT restored major patterns of ileal microRNA and histone regulation of gene expression. Further experiments suggest a gut-microbiota-regulated T1D protection mechanism centered on Reg3γ, in an innate intestinal immune network involving CD44, TLR2, and Reg3γ. This regulation affects downstream immunological tone, which may lead to protection against tissue-specific T1D injury.

Chronic opioid use modulates human enteric microbiota and intestinal barrier integrity.

Over the past three decades the United States has experienced a devastating opioid epidemic. One of the many debilitating side effects of chronic opioid use is opioid-induced bowel dysfunction. We investigated the impact of methadone maintenance treatment (MMT) on the gut microbiome, the gut bacterial metabolite profile, and intestinal barrier integrity. An imbalance in key bacterial communities required for production of short-chain fatty acids (SCFAs), mucus degradation, and maintenance of barrier integrity was identified. Consistent with dysbiosis, levels of fecal SCFAs were reduced in MMT. We demonstrated that metabolites synthesized by Akkermansia muciniphila modulate intestinal barrier integrity in vitro by strengthening the pore pathway and regulating tight junction protein expression. This study provides essential information about the therapeutic potential of A. muciniphila and warrants development of new clinical strategies that aim to normalize the gut microbiome in individuals affected by chronic opioid use.

Loss of HDAC6 alters gut microbiota and worsens obesity.

Alterations in gut microbiota are known to affect intestinal inflammation and obesity. Antibiotic treatment can affect weight gain by elimination of histone deacetylase (HDAC) inhibitor-producing microbes, which are anti-inflammatory by augmenting regulatory T (Treg) cells. We asked whether mice that lack HDAC6 and have potent suppressive Treg cells are protected from microbiota-induced accelerated weight gain. We crossed wild-type and HDAC6-deficient mice and subjected the offspring to perinatal penicillin, inducing weight gain via microbiota disturbance. We observed that male HDAC6-deficient mice were not protected and developed profoundly accelerated weight gain. The antibiotic-exposed HDAC6-deficient mice showed a mixed immune phenotype with increased CD4+ and CD8+ T-cell activation yet maintained the enhanced Treg cell-suppressive function phenotype characteristic of HDAC6-deficient mice. 16S rRNA sequencing of mouse fecal samples reveals that their microbiota diverged with time, with HDAC6 deletion altering microbiome composition. On a high-fat diet, HDAC6-deficient mice were depleted in representatives of the S24-7 family and Lactobacillus but enriched with Bacteroides and Parabacteroides; these changes are associated with obesity. Our findings further our understanding of the influence of HDACs on microbiome composition and are important for the development of HDAC6 inhibitors in the treatment of human diseases.-Lieber, A. D., Beier, U. H., Xiao, H., Wilkins, B. J., Jiao, J., Li, X. S., Schugar, R. C., Strauch, C. M., Wang, Z., Brown, J. M., Hazen, S. L., Bokulich, N. A., Ruggles, K. V., Akimova, T., Hancock, W. W., Blaser, M. J. Loss of HDAC6 alters gut microbiota and worsens obesity.

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.

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

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

An advanced glycation end product (AGE)-rich diet promotes Nε-carboxymethyl-lysine accumulation in the cardiac tissue and tendons of rats.

The purpose of this study was to investigate the intake, excretion, and tissue accumulation of carboxymethyl-lysine (CML), after feeding rats a diet containing advanced glycation end products (AGEs) from a glucose-lysine (GL) model system. Rats were distributed into two groups and assigned to a control diet or a diet including 3% heated GL (GL diet) for three months. Feces and urine were collected over the last week. After sacrifice, serum was obtained and some organs were removed for CML analysis. The percentage of fecal CML was 2.5-fold higher in the animals fed the GL diet (33.2 vs 76.5% for control and GL group), whereby total recovery was 91.8% compared with a level of 54.6% in the animals fed the control chow, evidencing the importance of the chemical form and the net quantity of dietary CML on its elimination. We suggest that dietary dicarbonyl compounds from GL diet or dietary CML itself are responsible for CML accumulation in hearts and tendons. The most significant result of the present study is that the regular consumption of dietary AGEs in healthy individuals promotes CML accumulation in some organs.

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

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

The impact of salsalate treatment on serum levels of advanced glycation end products in type 2 diabetes.

OBJECTIVE Salsalate is a nonacetylated salicylate that lowers glucose levels in people with type 2 diabetes (T2D). Here we examined whether salsalate also lowered serum-protein-bound levels of early and advanced glycation end products (AGEs) that have been implicated in diabetic vascular complications. RESEARCH DESIGN AND METHODS Participants were from the Targeting Inflammation Using Salsalate for Type 2 Diabetes (TINSAL-T2D) study, which examined the impact of salsalate treatment on hemoglobin A1c (HbA1c) and a wide variety of other parameters. One hundred eighteen participants received salsalate, 3.5 g/day for 48 weeks, and 109 received placebo. Early glycation product levels (HbA1c and fructoselysine [measured as furosine]) and AGE levels (glyoxal and methylglyoxal hydroimidazolones [G-(1)H, MG-(1)H], carboxymethyllysine [CML], carboxyethyllysine [CEL], pentosidine) were measured in patient serum samples. RESULTS Forty-eight weeks of salsalate treatment lowered levels of HbA1c and serum furosine (P < 0.001) and CML compared with placebo. The AGEs CEL and G-(1)H and MG-(1)H levels were unchanged, whereas pentosidine levels increased more than twofold (P < 0.001). Among salsalate users, increases in adiponectin levels were associated with lower HbA1c levels during follow-up (P < 0.001). Changes in renal and inflammation factor levels were not associated with changes in levels of early or late glycation factors. Pentosidine level changes were unrelated to changes in levels of renal function, inflammation, or cytokines. CONCLUSIONS Salsalate therapy was associated with a reduction in early but not late glycation end products. There was a paradoxical increase in serum pentosidine levels suggestive of an increase in oxidative stress or decreased clearance of pentosidine precursor.

Metabolic transit of N(ε)-carboxymethyl-lysine after consumption of AGEs from bread crust.

Our aim was to investigate carboxymethyl-lysine (CML) intake and excretion after feeding rats with diets containing advanced glycation end-products (AGEs) from bread crust (BC) or its soluble or insoluble fractions, and to identify the factors responsible for the effects observed. CML in serum and different tissues was measured to detect possible accumulations. For 88 days, weanling rats were fed with either a control diet or one containing BC, or its soluble low molecular weight (LMW), soluble high molecular weight (HMW) or insoluble fractions. In the last week of the assay, faeces and urine were collected daily and stored as a 1 week pool. After sacrifice, blood was drawn to obtain serum and some organs were removed. CML analysis was performed by HPLC/MS/MS in diets, faeces, urines, serum and tissues. Faecal excretion of CML was strongly influenced by dietary CML levels and represents the major route of excretion (i.e. 33.2%). However, the urinary elimination of CML was probably limited or saturated, especially when more complex compounds were present in the diet. BC consumption increased CML in the cardiac tissue (170 ± 18 vs. 97 ± 3 µmol per mol lysine for BC and control groups), which correlated with the CML intake. The levels of this AGE in bone were unaffected by the dietary treatment, but in tail tendons CML was greatly increased in the animals that consumed the BC diet (102 ± 13 vs. 51 ± 8 µmol per mol lysine for BC and control groups, P = 0.006), which was associated with the intake of soluble LMW compounds present in BC. Despite the CML accumulation detected in different tissues, serum levels of protein-bound CML were unchanged, indicating the importance of measuring the free CML in this fluid as a real index of dietary CML.

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.

Advanced glycation end-products as markers of aging and longevity in the long-lived Ansell's mole-rat (Fukomys anselli).

Mole-rat of the genus Fukomys are mammals whose life span is strongly influenced by reproductive status with breeders far outliving nonbreeders. This raises the important question of whether increased longevity of the breeders is reflected in atypical expression of biochemical markers of aging. Here, we measured markers of glycation and advanced glycation end-products formed in insoluble skin collagen of Ansell's mole-rat Fukomys anselli as a function of age and breeding status. Glucosepane, pentosidine, and total advanced glycation end-product content significantly increased with age after correction for breeder status and sex. Unexpectedly, total advanced glycation end-products, glucosepane, and carboxymethyl-lysine (CML) were significantly higher in breeders versus nonbreeders suggesting that breeders have evolved powerful defenses against combined oxidant and carbonyl stress compared with nonbreeders. Most interestingly, when compared with other mammals, pentosidine formation rate was lower in mole-rat compared with other short-lived rodents confirming previous observations of an inverse relationship between longevity and pentosidine formation rates in skin collagen.

Topical application of L-arginine blocks advanced glycation by ascorbic acid in the lens of hSVCT2 transgenic mice.

PURPOSE: Previous experiments from our laboratory showed that the oral intake of selected guanidino compounds could block the formation of crystallin-bound advanced ascorbylation products. Here we tested whether these were also active when applied as eye drops. METHODS: Two month old hSVCT2 transgenic mice (n=10) were treated twice daily with one drop of 0.1% L-arginine, γ-guanidinobutyric acid (GBA), penicillamine (PA) or N-acetylcysteine (NAC) in one eye and vehicle only in the other eye. After seven months, lens crystallins were isolated, dialyzed, and proteolytically digested to determine the protein-bound fluorescence at 335/385 and 370/440 nm excitation/emission and the advanced glycation/ascorbylation endproducts carboxymethyl-lysine (CML), carboxyethyl-lysine (CEL), glucosepane, glyoxal, and methylglyoxal hydroimidazolones G-H1 and MG-H1. The topical uptake of L-arginine and NAC was also evaluated in vitro and in vivo in rabbit lens. RESULTS: In hSVCT2 mice, L-arginine decreased 335/385 and 370/440 nm fluorescence by 40% (p<0.001), CML, CEL, and glucosepane crystallin crosslinks by 35% (p<0.05), 30% (p<0.05), and 37% (p<0.05), respectively, without affecting MG-H1 and G-H1. NAC decreased 335/385 nm fluorescence by 50% (p<0.001) but, like PA and GBA, had no effect on other modifications. L-Arginine uptake into rabbit eyes treated topically reached identical lenticular plateau levels (~400 nmol/g wet weight) at 0.5% and 2.0% but levels remained three times higher at 5 h at 2% versus 0.5% concentration, respectively. In vitro studies showed a 100 fold higher L-arginine level than NAC levels, implicating high affinity uptake of the former. CONCLUSIONS: L-Arginine when applied both orally and topically is a potent and broad suppressor of advanced ascorbylation in the lens. Its uptake in rabbit lens upon topical application suggests transcorneal uptake into the human lens should be feasible for testing its potential anticataract properties in clinical trials.

Protection from retinopathy and other complications in patients with type 1 diabetes of extreme duration: the joslin 50-year medalist study.

OBJECTIVE: To assess complication prevalence and identify protective factors in patients with diabetes duration of ≥50 years. Characterization of a complication-free subgroup in this cohort would suggest that some individuals are protected from diabetes complications and allow identification of endogenous protective factors. RESEARCH DESIGN AND METHODS: Cross-sectional, observational study of 351 U.S. residents who have survived with type 1 diabetes for ≥50 years (Medalists). Retinopathy, nephropathy, neuropathy, and cardiovascular disease were assessed in relation to HbA(1c), lipids, and advanced glycation end products (AGEs). Retrospective chart review provided longitudinal ophthalmic data for a subgroup. RESULTS: A high proportion of Medalists remain free from proliferative diabetic retinopathy (PDR) (42.6%), nephropathy (86.9%), neuropathy (39.4%), or cardiovascular disease (51.5%). Current and longitudinal (the past 15 years) glycemic control were unrelated to complications. Subjects with high plasma carboxyethyl-lysine and pentosidine were 7.2-fold more likely to have any complication. Of Medalists without PDR, 96% with no retinopathy progression over the first 17 years of follow-up did not experience retinopathy worsening thereafter. CONCLUSIONS: The Medalist population is likely enriched for protective factors against complications. These factors might prove useful to the general population with diabetes if they can be used to induce protection against long-term complications. Specific AGE combinations were strongly associated with complications, indicating a link between AGE formation or processing with development of diabetic vasculopathy.

Favored and disfavored pathways of protein crosslinking by glucose: glucose lysine dimer (GLUCOLD) and crossline versus glucosepane.

We describe the isolation and molecular characterization of a novel glucose-lysine dimer crosslink 1,3-bis-(5-amino-5-carboxypentyl)-4-(1',2',3',4'-tetrahydroxybutyl)-3H-imidazolium salt, named GLUCOLD. GLUCOLD was easily formed from the Amadori product (fructose-lysine). However, when BSA was incubated with 100 mM glucose for 25 days, the levels of the lysine-lysine glucose crosslinks GLUCOLD and CROSSLINE were only 21 and <1 pmol/mg, respectively, compared to 611 pmol/mg protein for the lysine-arginine GLUCOSEPANE crosslink, in spite of more than 20 potential lysine-lysine crosslinking sites in the protein. Mechanistic investigation revealed that metal-free phosphate ions catalyzed formation of fructose-lysine and all three crosslinks from amino acids, while cationic MOPS buffer had an opposite effect. This together with the rapid formation of N (6)-1,4-dideoxy-5,6-dioxoglucosone derivatives by dicarbonyl trapping agents, such as 1,2-diaminobenzene or γ-guanidinobutyric acid, strongly suggests that enolization of the Amadori product and trapping of the 5,6-dioxo derivative by arginine residues constitutes the major pathway for glucose-mediated crosslinking in proteins.

Pudendal nerve injury reduces urethral outlet resistance in diabetic rats.

Diabetics have voiding and continence dysfunction to which elevated levels of advanced glycation end products (AGE) may contribute. In addition, pudendal nerve injury is correlated with voiding dysfunction and stress incontinence in rats. The aim of this study was to investigate whether pudendal nerve crush (PNC) in diabetic rats alters urinary function. Female virgin Sprague-Dawley rats (144) were divided equally into diabetic, diuretic, and control groups. Half of the animals in each group were subjected to PNC, and the other half to sham PNC. Diabetes was induced 8 wk before PNC or sham PNC by streptozotocin injection (35 mg/kg). Animals underwent conscious cystometry and leak point pressure (LPP) testing 4 or 13 days after PNC or sham PNC. Tissues of half the animals were tested for levels of AGEs. Qualitative histological assessment was performed in the remaining animals. Diabetic rats 4 days after PNC voided significantly greater volume in a shorter time and with significantly less pressure than after sham PNC, suggesting that diabetic rats have a functional outlet obstruction that is relieved by PNC. LPP was significantly reduced 4 days after PNC in diabetic and diuretic animals and returned to normal 13 days after PNC. Diabetic rats with PNC demonstrated increased muscle fiber disruption and atrophy of the external urethral sphincter. AGEs were significantly elevated in diabetic rats. PNC relieves a functional outlet obstruction in diabetic rats. AGEs are elevated in diabetic rats and could play a role in urinary dysfunction and recovery from PNC.

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

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

Mechanism of lysine oxidation in human lens crystallins during aging and in diabetes.

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