[Show abstract][Hide abstract] ABSTRACT: The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points.
Full-text · Article · Jun 2013 · International Journal of Molecular Sciences
[Show abstract][Hide abstract] ABSTRACT: Ponies suffering from recurrent episodes of laminitis when grazed at pasture (pasture-associated laminitis) exhibit phenotypes similar to those associated with human metabolic syndrome. In humans, evidence suggests that the obesity-related morbidities associated with metabolic syndrome, including diabetes and cardiovascular disease, are caused by an increase in the production of advanced glycoxidation end-products (AGEs). These end-products have been recognised as putative pro-inflammatory mediators and are considered a 'risk factor' for human health. However, the evaluation of AGEs in laminitic ponies has not been explored. The aim of this study was to compare plasma concentrations of the AGE pentosidine (PENT) in ponies presenting with clinical features of equine metabolic syndrome (EMS) with a history of recent laminitis and/or showing signs of laminitis at the time of sampling (LP) with those with no prior history of clinical laminitis (NL). Age, body condition score (BCS) and bodyweight were recorded and blood samples collected for the measurement of plasma concentrations of PENT, glucose, insulin, triglycerides (TG), non-esterified fatty acids (NEFA) and cortisol. Insulin sensitivity was assessed by the reciprocal of the square root of insulin (RISQI) and the insulin:glucose ratio. Plasma PENT concentrations were twofold higher (P<0.005) in LP than in NL ponies. Significant (P<0.05) correlations were also evident between PENT and insulin, RISQI, TG and age. These preliminary findings are consistent with the hypothesis that glycoxidation in laminitis is associated with EMS.
Full-text · Article · Nov 2012 · The Veterinary Journal
[Show abstract][Hide abstract] ABSTRACT: Matrix metalloproteinases (MMPs), their inhibitors (TIMPs) and inflammatory cytokines, such as interleukin-1 (IL-1), are considered markers of evolution and/or instability of atherosclerotic plaques. Accumulation of Advanced Glycation Endproducts (AGE) is a well known phenomenon in diabetes and has also been considered in the pathogenesis of atherosclerosis. Aim of the present study was to analyse the levels of pentosidine, a fluorescent AGE, and to evaluate the expression of MMP-2, TIMP-3, and IL-1 in an ex vivo model of human advanced atherosclerotic plaques. We intended to test the possible correlation between pentosidine and markers of ECM remodelling and inflammation in the atherosclerotic process, and to investigate if classic risk factors, such as diabetes and hypertension, influenced these biochemical parameters. We found that diabetic plaques showed higher level of pentosidine, as expected, but much lower, or even undetectable, expression levels of MMP-2 and TIMP-3; IL-1 expression was not different between diabetic and non diabetic plaques. Hypertension did not influence any of these parameters. Although the statistical correlations between the expression of the considered genes and pentosidine did not reach significance, slight negative trends were noted between TIMP-3 and IL-1 expression vs. pentosidine content. We suggest that in mature diabetic plaques AGE accumulation can exert stabilizing effects on matrix proteins, while scanty cell presence leads to poor capacity of reactive responses, such as remodelling and inflammation.
No preview · Article · Oct 2012 · Experimental and Clinical Endocrinology & Diabetes
[Show abstract][Hide abstract] ABSTRACT: Type 2 diabetes, which is the most common form of diabetes, is characterized by hyperglycemia and insulin resistance associated with a progressive deterioration of β-cell function and mass. High blood glucose plays a key role in the development of diabetic complications and may contribute to the progressive β-cell failure. Chronic exposure to high glucose levels increases non enzymatic reactions between aldehydic and amino group of molecules like sugars and proteins, lipids or nucleic acids leading to Advanced Glycation End-Products (AGEs) formation. Accumulation of AGEs is related to the aging process and is boosted by diabetes. Although the pathogenic role of AGEs in microvascular complications of diabetes has been widely investigated and recognized, their role in pancreatic β-cell dysfunction remains to be fully elucidated. Evidence of a direct role of AGEs on pancreatic β-cell dysfunction is discussed in this review. Findings show that exposure to high AGE concentration damages the β-cell functionality affecting insulin production and disturbing the insulin secretion machinery. The studies provide solid evidence that AGEs not only may play a causative role in diabetes complications but may be crucial in the onset and maintenance of it.
[Show abstract][Hide abstract] ABSTRACT: Advanced Glycation End-Products (AGEs), a group of compounds resulting from the non-enzymatic reaction of reducing sugars with the free amino group of proteins, are implicated in diabetic complications. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T 15 to high concentrations of AGEs significantly decreases cell proliferation and insulin secretion, and affects transcription factors regulating insulin gene transcription. The glucagon-like peptide-1 (GLP-1) is an incretin hormone that increases proinsulin biosynthesis, stimulates insulin secretion, and improves pancreatic beta-cell viability. The aim of this work was to investigate the effects of GLP-1 on the function and viability of HIT-T 15 cells cultured with AGEs. HIT-T 15 cells were cultured for 5days in presence of AGEs alone, or supplemented with 10nmol/l GLP-1. Cell viability, insulin secretion, redox balance, and expression of the AGEs receptor (RAGE) were then determined. The results showed that GLP-1 protected beta cell against AGEs-induced cell death preventing both apoptosis and necrosis. Moreover, addition of GLP-1 to the AGEs culture medium restored the redox balance, improved the responsiveness to glucose, and attenuated AGEs-induced RAGE expression. These findings provide evidence that GLP-1 protects beta cells from the dangerous effects of AGEs.
No preview · Article · Jul 2010 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: To develop a blood-based test for screening populations at risk for Alzheimer disease.
Case-control study. Subjects A total of 180 patients with mild cognitive impairment (MCI) and 105 age-matched, cognitively normal controls.
The titer of beta-amyloid 1-42 autoantibodies in the plasma was obtained at the time of diagnosis and evaluated by enzyme-linked immunosorbent assay before and after dissociation of the antigen-antibody complexes. A total of 107 patients with MCI were followed up for 36 months; 70 of the 107 cases progressed to Alzheimer disease.
The average level of beta-amyloid 1-42 plasma autoantibodies in patients with MCI that progressed to Alzheimer disease, but not that of the stable cases, was significantly higher than in cognitively normal controls (P < .001).
The results suggest that the plasma beta-amyloid 1-42 autoantibodies parallel beta-amyloid 42 deposition in the brain, which is known to precede by several years the clinical onset of Alzheimer disease. The evaluation of beta-amyloid 1-42 autoantibodies after dissociation of the complexes is a simple and inexpensive method that can be used to predict the occurrence of Alzheimer disease.
No preview · Article · Jul 2010 · Archives of neurology
[Show abstract][Hide abstract] ABSTRACT: Advanced Glycation End-Products (AGEs) are generated by the covalent interaction of reducing sugars with proteins, lipids or nucleic acids. AGEs are implicated in diabetic complications and pancreatic beta-cell dysfunction. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T15 to high concentrations of AGEs leads to a significant decrease of insulin secretion and content. Insulin gene transcription is positively regulated by the beta cell specific transcription factor PDX-1 (Pancreatic and Duodenal Homeobox-1). On the contrary, the forkhead transcription factor FoxO1 inhibits PDX-1 gene transcription. Activity of FoxO1 is regulated by post-translational modifications: phosphorylation deactivates FoxO1, and acetylation prevents FoxO1 ubiquitination. In this work we investigated whether AGEs affect expression and subcellular localization of PDX-1 and FoxO1. HIT-T15 cells were cultured for 5 days in presence of AGEs. Cells were then lysed and processed for subcellular fractionation. We determined intracellular insulin content, then we assessed the expression and subcellular localization of PDX-1, FoxO1, phosphoFoxO1 and acetylFoxO1. As expected intracellular insulin content was lower in HIT-T15 cells cultured with AGEs. The results showed that AGEs decreased expression and nuclear localization of PDX-1, reduced phosphorylation of FoxO1, and increased expression and acetylation of FoxO1. These results suggest that AGEs decrease insulin content unbalancing transcription factors regulating insulin gene expression.
No preview · Article · Mar 2010 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Osteoporosis, a multifactorial and progressive skeletal metabolic disease, is characterized by low-mass density and structural deterioration of bone micro-architecture that leads to enhanced bone fragility and increased susceptibility to fractures. Recently, it has been proposed that age-related bone loss could be correlated with the glycoxidative process. The aim of the present study was to investigate the in vitro effects of pentosidine, a glycoxidative end product, on human osteoblasts (HOb). The mineralization rate, the specific bone markers (alkaline phosphatase [ALP], collagen Ialpha1 [COL Ialpha1], osteocalcin [BGP]), and the human receptor for advanced glycation end products (RAGE) gene expression have been evaluated. Pentosidine incubation of HOb caused a significant decrease in ALP, Col Ialpha1, and RAGE mRNA levels, but only the RAGE gene expression decreased with no dose dependency. Moreover, pentosidine incubation of osteoblasts hampered the formation of bone nodules. No effect was observed on BGP gene expression under all experimental conditions. Our data gives further support to a detrimental effect of AGEs on bone that leads to functional alterations of osteoblasts. This study addresses a crucial role of protein glycoxidation in the bone mineralization process. AGEs formation and accumulation in bone may be one of the first pathogenetic steps of bone remodeling in aging and in age-related diseases, leading to enhanced bone mass loss.
Full-text · Article · May 2008 · Annals of the New York Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The purpose of the present study was to evaluate the direct effects of advanced glycation end products (AGEs) on beta-cells by their exposure to a glycated serum to estimate the cellular viability and the related insulin secretion. Glycation of fetal calf serum was obtained by incubation with 50 mol/L ribose at 37 degrees C for 7 days; at the end of this incubation period, the pentosidine content ranged between 15 and 16 x 10(5) pmol/L. HIT-T15 cells, a pancreatic islet cell line, were grown and cultured for 5 days in Roswell Park Memorial Institute (RPMI) medium containing either not glycated (NGS) or glycated (GS) fetal calf serum. Cellular oxidative stress (ie, thiobarbituric acid-reactive substances) was assessed by high-performance liquid chromatography. Cellular viability was evaluated by detection of proliferation, cell necrosis, and cell apoptosis rate. The insulin secretion and the related intracellular content were evaluated by enzyme-linked immunosorbent assay. The present study reported, after 5 days of exposure to the glycation environment, a moderately reduced cellular proliferation (-20.44% +/- 2.92%) with a corresponding increase of cell necrosis (+67.7% +/- 1.56%) and cell apoptosis (+39.83% +/- 2.92%) rate in comparison with the untreated cells. Oxidative intracellular stress was higher in GS conditions compared with the NGS ones (+293.3% +/- 87.53%). Insulin release from GS-treated HIT-T15 cells was lower than that of NGS-treated cells both when cells were stimulated with low glucose concentration (2.8 mmol/L, -30.3% +/- 4.91%) or when they were challenged with high glucose concentration (16.7 mmol/L, -29.2% +/- 5.82%). Incubation of HIT-T15 cells with glycated serum also caused a significant decrease of insulin intracellular content (-44.47% +/- 9.98%). Thus, AGEs were shown to exert toxic effects on insulin-secreting cells. Chronically high intracellular oxidative stress, due to accumulation of AGEs, affects the insulin secretion machinery. The present data suggest a pivotal role of the non-enzymatic glycation process in the onset and progression of diabetes during aging and a direct adverse effect of a glycated environment on the pancreatic islet cells.
[Show abstract][Hide abstract] ABSTRACT: Advanced glycation end product (AGE) accumulation in brain is believed to contribute to neuronal death in several neurodegenerative diseases. Neurons exposed to AGEs undergo oxidative stress, but the molecular mechanisms able to induce ROS generation and cell death are not yet clear. In this work, we exposed SH-SY5Y neuroblastoma cells to glycated albumin, as a model of AGE-modified protein, and we observed that cells differentiated by retinoic acid died after AGE exposure, through anion superoxide and peroxide generation, while undifferentiated cells resulted resistant. Retinoic acid induced marked increase in p47phox expression and in catalytic activity of PKC delta: the upregulation of a pathway involving NADPH oxidase and PKC delta is likely to be responsible for neuronal susceptibility to AGE. This hypothesis is confirmed by the fact that pre-treatments of differentiated cells with DPI, an inhibitor of NADPH oxidase, or with rottlerin, an inhibitor of PKC delta, were able to prevent AGE-induced neuronal death.
No preview · Article · May 2007 · Neuroscience Letters
[Show abstract][Hide abstract] ABSTRACT: Advanced glycation end-products (AGEs), which accumulate in the blood and tissues of patients with chronic renal failure (CRF) undergoing chronic hemodialysis, play an important role in the pathogenesis of uremic complications. Endothelin 1 (ET1), a 21-amino acid peptide with vasoconstricting and mitogenic properties, is an important factor in the endothelial dysfunction occurring in uremia. The circulating levels of both AGEs and ET1 have been reported to be increased in chronic renal failure. In the present study we evaluated the possible relationship between pentosidine and ET1 plasma levels in CRF patients undergoing chronic hemodialysis treatment. The plasma concentrations of "free" and bound pentosidine (HPLC methods) and endothelin-1 (RIA method) were measured before the hemodialysis session in 40 nondiabetic CRF patients (22 males and 18 females; 54+/-3 years) on chronic hemodialysis for at least 1 year. Forty age- and sex-matched normal subjects served as a control group. In hemodialyzed patients, the overall pentosidine residues and pentosidine-free adduct plus pentosidine-free adduct bound reversibly to protein levels (24.9+/-2.04 pmol/mg protein and 110.5+/-5.9 pmol/ml, respectively) were significantly higher than those recorded in normal subjects (2.0+/-0.2 pmol/mg protein and 0.7+/-0.2 pmol/ml, respectively ). Endothelin-1 was also significantly (p<0.01) increased in CRF patients (10.6+/-0.4 pmol/ml in CRF patients and 2.7+/-0.3 pmol/ml in normal subjects). A significant positive correlation (p<0.01) was seen between "total" pentosidine (pentosidine residues and pentosidine-free adduct plus pentosidine-free adduct bound reversibly to protein) levels and endothelin-1 plasma values. The correlation between pentosidine and endothelin-1 provides further evidence that some AGEs exert a detrimental effect on the vascular endothelium, thereby contributing to the hypertension and other cardiovascular damage seen in CRF patients.
No preview · Article · Jan 2007 · Hormone and Metabolic Research
[Show abstract][Hide abstract] ABSTRACT: Several lines of evidence suggest that both advanced glycation end products (AGEs) and oxidation processes play key roles in the physiology of aging and age-related pathologies, leading to irreversible proteins modifications in both tissues and the extracellular matrix. Such an accelerated accumulation of these modifications has been reported to be present in several age-related chronic diseases, such as atherosclerosis, diabetes, arthritis, and neurodegenerative diseases. The current literature reveals that the specific inhibition of AGEs may constitute an innovative therapeutic goal. In experimental animals, the use of sartans significantly reduces blood pressure and kidney pentosidine content, improving both histologic renal damage and proteinuria. In this study, 12 subjects who were affected by diabetes mellitus and hypertension were subjected to oral antihypertensive therapy with valsartan (class of sartans) with timed sampling of plasma and urine pentosidine, N(epsilon)-(carboxymethyl)lysine (CML), malondialdehyde, and isoprostanes levels, respectively, at baseline and after both 3 and 6 months, with parallel ongoing evaluation of glycemic control and blood pressure levels. Valsartan elicited a good antihypertensive effect with a 30% decrease in plasma pentosidine levels (P < .05) after 3 months of therapy, followed by a slight increase after 6 months. Urinary pentosidine concentrations exhibited a 40% decrease after 3 months (215 +/- 19 vs 129 +/- 23 nmol/24 h) and a further significant reduction after 6 months of therapy (105 +/- 24 nmol/24 h). Plasma CML levels showed a progressive decrease after 3 months (23.15 +/- 3.215 vs 19.88 +/- 1.684 micromol/mL) and achieved a further slight reduction after 6 months of therapy (19.48 +/- 1.339 micromol/mL); for urinary CML, a statistically significant reduction was gained after the sixth month of therapy (48.51 +/- 5.70 vs 30.30 +/- 2.77 micromol/24 h after 3 months and 27.02 +/- 4.13 micromol/24 h after 6 months; F = 7.62, P < .005). Plasma and urinary concentrations of malondialdehyde were slightly modified by valsartan treatment; the mean levels after both 3 and 6 months did not significantly differ from baseline. Urinary 15-F2t-isoprostanes (2.96 +/- 0.45 ng/24 h) levels displayed a progressive decrease after both 3 (2.27 +/- 0.31 ng/24 h) and 6 months (1.70 +/- 0.23 ng/24 h) with statistical significance achieved only at the end of the study (P < .05). The present data suggest interesting in vivo antiglycation and antioxidation effects of this angiotensin II receptor antagonist with reductions in plasma and urinary pentosidine, plasma CML, and urinary isoprostanes levels. The present study supports an antagonistic role of valsartan in the production of AGEs precursors through the chelation of transition metals and an antioxidant activity that scavenges reactive oxygen species. This property of valsartan may broaden the scope of newly developed pharmacologic inhibitors of advanced glycoxidation.
[Show abstract][Hide abstract] ABSTRACT: Somatosympathetic reflexes were studied in young hyperinsulinemic, insulin-resistant (Zucker fatty) rats (ZFR) and a related control (Zucker lean) strain (ZLR). Glucose metabolism was characterized by minimal model analysis of intravenous glucose tolerance test data. Seven-week-old ZFR (n=18) and ZLR (n=17) were studied under pentobarbital anesthesia. Mean body weight and plasma glucose and insulin concentration were significantly greater (P<0.05) in ZFR than in ZLR, whereas basal values of mean arterial pressure (MAP) and heart rate (HR) were not significantly different. Increments of MAP (DeltaMAP) and HR (DeltaHR) elicited by electrical stimulation of the sciatic nerve (5-s trains of 100 pulses, 0.5-ms pulse duration, 100- to 400-microA pulse intensity) were significantly higher (ANOVA, P<0.05) in ZFR at each level of stimulus intensity. Regression analysis showed a linear increase in DeltaMAP and DeltaHR with increasing sciatic nerve stimulus intensity. Pressor responses to phenylephrine after ganglionic blockade demonstrated that vascular reactivity to adrenergic stimulation is not increased in ZFR compared with ZLR. Thus this factor does not contribute to enhancement of somatosympathetic reflexes observed in this strain. Insulin sensitivity in ZFR was one-fourth (P<0.05) that in ZLR. These results suggest that stronger sympathetic nervous reactivity in ZFR is associated with a severe insulin-resistant state before the onset of hypertension and support the hypothesis that insulin-mediated stimulation of the sympathetic nervous system is involved in the development of cardiovascular diseases related to alterations of glucose metabolism.
No preview · Article · Aug 2006 · AJP Regulatory Integrative and Comparative Physiology
[Show abstract][Hide abstract] ABSTRACT: We evaluated expression, protein levels and activity of the Beta-site cleaving enzyme (BACE1) as well as the amount of products of lipid peroxidation in frontal cortex of three groups of cases: sporadic Alzheimer's disease (AD); control subjects (CTR); cognitively normal subjects with abundant amyloid plaques (NA). We found a significant increase of BACE1 activity and products of lipid peroxidation in brain tissue of AD cases, with normal gene expression, and non-significant elevation of protein levels. CTR and NA samples showed similar levels of BACE1 activity and oxidative products. BACE1 activity and the amount of oxidative products were significantly correlated in all cases.Moreover, both BACE1 activity and the level of 4-hydroxynonenal were correlated with the amount of Beta-amyloid pyroglutamated 3-42, the more toxic Beta-amyloid peptide that is characteristic of AD. These findings suggest that BACE1 activity reflects the type of ABeta species, rather than the Beta-amyloid plaques load. Hence, the increase of BACE1 activity occurring in sporadic AD is likely the effect, rather the cause, of ABeta accumulation and oxidative stress.
No preview · Article · Jul 2006 · Neurobiology of aging
[Show abstract][Hide abstract] ABSTRACT: Pringle's manoeuvre controls excessive bleeding, but results in ischaemia-reperfusion injury during liver surgery. Activation of the heat-shock protein system of cell defense has been demonstrated after ischaemia-reperfusion injury in animal tissues. The aim of the present study was to determine whether the ischaemia-reperfusion accompanying hepatic surgery induces heat-shock protein 70 (HSP70) in human liver and whether the induction of HSP70 is related to the recovery of liver function.
Heat-shock protein 70 and gamma-actin mRNAs were assayed in the liver biopsies of 10 subjects undergoing partial hepatectomy for localized lesions. Measurements were performed before the Pringle's manoeuvre and at the end of the surgery. Transaminases and fibrinogen were measured before and at 12, 24 and 36 h following hepatectomy.
After an average 40 +/- 8-min period of warm ischaemia, a significant increase of HSP70 mRNA (187 +/- 67%, 2P < 0.05) was observed. The acute increase of HSP70 mRNA correlates with the decrease of transaminases (AST: rs -0.964, ALT: rs -0.891, P < 0.002) and the increase of fibrinogen (rs -0.7, P < 0.02) observed between 12 and 24 h following surgery.
Heat-shock protein 70 is induced by ischaemia-reperfusion injury in human liver. Its induction seems to have beneficial effects, including a prompt reduction of transaminases and a rapid recovery of fibrinogen synthesis.
No preview · Article · Jun 2003 · European Journal of Clinical Investigation
[Show abstract][Hide abstract] ABSTRACT: It is currently under debate whether the pathogenesis of end-stage renal failure in non-insulin-dependent diabetes mellitus (NIDDM) is a consequence of microangiopathy alone. The aim of this study was to investigate intrarenal arteriosclerosis and its correlation with kidney function in NIDDM. In 36 diabetic subjects, and in 10 age- and sex-matched healthy control subjects we measured kidney volume and resistive index of the interlobar arteries by duplex Doppler ultrasonography. Clinical and metabolic parameters, renal function and vascular sequelae of the disease were also evaluated. In diabetic subjects resistive index (median 0.72, range 0.54-0.79) was higher than in control subjects (median 0.62, range 0.57-0.66) (2p < 0.002). Kidney volume and resistive index correlated with age (p < 0.004), body mass index (p < 0.001), mean blood pressure (p < 0.001), total and LDL cholesterol (p < 0.01) and creatinine clearance (p < 0.001 and < 0.01, respectively). Kidney volume also correlated with HbA1 (p < 0.01) and resistive index with uric acid (p < 0.01). Lower body macroangiopathy was associated with increased resistive index and reduced kidney volume (2p < 0.05), while upper body macroangiopathy and microangiopathy were not. Our data suggest that macroangiopathy rather than microangiopathy is mainly responsible for impairment of kidney function in NIDDM. The resistive index of interlobar arteries seems to be a reliable marker of intrarenal arteriosclerosis and can be used as a non-invasive, easily available parameter of its evolution.