An association among iron,copper, zinc,and selenium,and antioxidative status in dyslipidemic pediatric patients with glycogen storage disease types IA and III

Dyslipidemia in patients with glycogen storage disease types Ia (GSD Ia) and III (GSD III) does not lead to premature atherosclerosis. The aim of this study was to investigate the association among serum copper (Cu), zinc (Zn), iron (Fe), and selenium (Se) concentrations, and their carrier proteins: ceruloplasmin, albumin, and related antioxidant enzyme activities [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), paraoxonase (PON), and arylesterase (ARYL)] in 20 GSD Ia and 14 III patients compared to age and sex matched 20 healthy subjects. Erythrocyte oxidative stress was measured by erythrocyte thiobarbituric acid reactive substances (eTBARSs). Hypertriglyceridemia [333 (36–890) mg/dL] in GSD Ia and hypercholesterolemia with elevated LDL-cholesterol [188 (91–313) mg/dL] and decreased HDL-cholesterol [32(23–58) mg/dL] levels in GSD III were found. Serum Cu, Fe, and Zn showed no significant differences between groups. However, Se 60 (54–94), 81 (57–127) μg/L, ceruloplasmin 21 (10–90), 27 (23–65) μg/L, and albumin 2.4 (1.7–5.1), 2.8 (1.8–4.06) g/dL levels were decreased in GSD Ia and III groups, respectively, in comparison with the controls [Se 110 (60–136) μg/L, ceruloplasmin 72 (32–94) μg/L, and albumin 4.4 (4–4.8) g/dL)]. In spite of high oxidative stress in erythrocyte detected by elevated eTBARS/Hb levels in GSD group [674.8 (454.6–948.2) for GSD Ia, 636.3 (460.9–842.1) for GSD III, and 525.6 (449.2–612.6)], the activities of CAT, SOD, ARYL, and PON in GSD patients were not different from the controls. GPx activity was decreased in GSD Ia [3.7 (1.8–7.1) U/mL] and GSD III [4.2 (2.2–8.6) U/mL] compared with healthy controls [7.1 (2.9–16.2) U/mL].In conclusion, this study supplied the data for trace elements, their carrier, and antioxidative enzymes in the patients with GSD Ia and III. The trace elements and anti-oxidative enzyme levels in GSD patients failed to explain the atherosclerotic escape phenomenon reported in these patients.     

Proteolytic degradation by cathepsin D of glycated hemoglobin from diabetes patients gives rise to hemorphin-7 peptides

Previous studies showed a significantly reduced level of hemorphins in the serum of diabetes patients. In order to elucidate the biochemical mechanisms responsible for this anomaly, the influence of hemoglobin glycation on hemorphin generation was studied. The glycation of hemoglobin occurs in the blood of diabetes patients and this could modify its enzymatic digestion and the resulting proteolytic products. Several samples of hemoglobin were obtained from the blood of type 1 diabetes patients (n = 8) and normal healthy control subjects (n = 2). The glycated hemoglobin samples were classified on the basis of their HbA1c values expressed as a percentage of total hemoglobin. Four solutions of glycated hemoglobin characterized by HbA1c values of 6%, 9.1%, 10.7% and 12.1% were treated with cathepsin D and the hemorphins obtained following the proteolysis were compared to controls. It was found that hemorphins were produced whatever the level of glycation of hemoglobin and also that the degree of glycation had no effect on the quantity of hemorphins released. Thus the alteration of hemoglobin does not seem to be the essential reason for the decrease in hemorphin concentrations in the sera of diabetic patients.     

Decreased arylesterase activity of paraoxonase-1 (PON-1) might be a common denominator of neuroinflammatory and neurodegenerative diseases

High-density lipoprotein (HDL)-bound paraoxonase-1 (PON-1) is mechanistically related to oxidative stress, inflammation and atherosclerosis and this multirole nature positions the enzyme as potential pathogenic player and candidate biomarker for many diseases. Our previous work suggests that decline in serum PON-1 activities, i.e. arylesterase and paraoxonase, might be associated with the occurrence of mild cognitive impairment (MCI) to late onset Alzheimer’s disease (LOAD) or vascular dementia (VAD). The present study aimed to: (1) expand our previous findings in a larger and different population, including patients with LOAD-VAD mixed dementia (MD); (2) explore a possible association between PON-1 and multiple sclerosis (MS); (3) evaluate if cerebrospinal fluid (CSF) levels of PON-1 activities might be useful biomarkers for MS. We found that serum arylesterase, but not paraoxonase, levels of PON-1 were significantly lower in patients affected by MCI (n = 232), VAD (n = 65), LOAD (n = 175), MD (n = 88) as well as those with MS (n = 104) as compared to healthy controls. Notably, the most pronounced decline in this activity was shown by MD (−18%, p < 0.01) and MS (−23%, p < 0.001), while the lowest changes were detected in the MCI group (11%, p < 0.05). Only arylesterase was detectable in the CSF of MS patients and the levels were not significantly different from those detected in the other two neurological control groups. Overall our data suggest that a depressed arylesterase activity could be a common denominator of different neurological diseases which, independently of their peculiar ethiopathogenesis and pathophysiology, appear to be all characterized by an altered systemic redox balance.     

Trace elements,oxidative stress and glycemic control in young people with type 1 diabetes mellitus

Trace elements and oxidative stress are associated with glycemic control and diabetic complications in type 1 diabetes mellitus. In this study, we analyzed the levels of serum copper, zinc, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) and urinary MDA and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in 33 type 1 diabetic patients with optimal and suboptimal glycemic control (HbA_1C < 9.0%) and 40 patients with poor glycemic control (HbA_1C ≥ 9%) and 27 age- and sex-matched non-diabetic controls to evaluate the differences between these markers in different glycemic control states. Diabetic patients, especially poor-glycemic-control subjects (HbA_1C ≥ 9%), exhibited significantly lower levels of serum zinc and increased levels of serum copper (and, therefore, increased serum copper-to-zinc ratios), serum SOD, blood MDA, and urinary MDA and 8-OHdG, relative to non-diabetic subjects. Furthermore, significant correlations existed in these patients between the serum copper, serum copper-to-zinc ratio, and urinary MDA (all p < 0.001) and the levels of urinary 8-OHdG (p = 0.007) and HbA_1C. Our results suggest that high serum copper levels and oxidative stress correlate with glycemic control. Therefore, strict glycemic control, decreased oxidative stress, and a lower copper concentration might prevent diabetic complications in patients with type 1 diabetes mellitus.     

Methylglyoxal-induced modification of arginine residues decreases the activity of NADPH-generating enzymes

Inadequate control of plasma and cellular glucose and ketone levels in diabetes is associated with increased generation of reactive aldehydes, including methylglyoxal (MGO). These aldehydes react with protein side chains to form advanced glycation end-products (AGEs). Arg residues are particularly susceptible to MGO glycation and are essential for binding NADP⁺ in several enzymes that generate NADPH, a coenzyme for many critical metabolic and antioxidant enzymes. In most animal cells, NADPH is produced predominantly by glucose-6-phosphate dehydrogenase (G6PD) in the oxidative phase of the pentose phosphate pathway and, to a lesser extent, by isocitrate dehydrogenase (IDH) and malic enzyme (ME). In this study, the activities of isolated G6PD, IDH, and ME were inhibited by MGO (0–2.5 mM, 2–3 h, 37 °C), in a dose- and time-dependent manner, with G6PD and IDH more sensitive to modification than ME. Significant inhibition of these two enzymes occurred with MGO levels ≥500 μM. Incubation with radiolabeled MGO (0–500 µM, 0–3 h, 37 °C) demonstrated dose- and time-dependent adduction to G6PD and IDH. HPLC analysis provided evidence for AGE formation and particularly the hydroimidazolones MG-H1 and MG-H2 from Arg residues, with corresponding loss of parent Arg residues. Peptide mass mapping studies confirmed hydroimidazolone formation on multiple peptides in G6PD and IDH, including those critical for NADP⁺ binding, and substrate binding, in the case of IDH. These results suggest that modification of NADPH-producing enzymes by reactive aldehydes may result in alterations to the cellular redox environment, potentially predisposing cells to further damage by oxidants and reactive aldehydes.     

Paraoxonase 1 genetic polymorphisms and susceptibility to breast cancer: a meta-analysis

AimThe paraoxonase 1 gene (PON1, MIN: 168820) is a member of the multifactorial antioxidant enzyme paraoxonase family (EC 3.1.1.2). Two common functional single-nucleotide polymorphisms L55M (dbSNP: rs854560) and Q192R (dbSNP: rs662) have been identified in the coding region of PON1. Several studies have investigated the associations between polymorphisms of PON1 and susceptibility to breast cancer, but have yielded apparently conflicting results. We therefore carried out a meta-analysis of published studies to clarify this inconsistency and to establish a comprehensive picture of the relationship between PON1 gene variants and breast cancer risk. Method: Overall six eligible studies were identified. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were obtained using fixed and random-effect models. Results: In our meta-analysis, the presence of the R allele was associated with decreased risk of breast cancer (QR + RR compared to QQ genotype, summary OR = 0.57, 95% CI: 0.49–0.67, P < 0.001). Both heterozygosity (OR = 1.32, 95% CI: 1.10–1.58, P = 0.002) and homozygosity (OR = 2.16, 95% CI: 1.75–2.68, P < 0.001) for the 55M allele were associated with increased risk of breast cancer. Also there was a significant linear trend in risk associated with zero, one, and two 55M alleles (χ² = 54.2, P < 0.001).ConclusionThe present study showed that PON1 M and Q alleles are associated with a higher risk of breast cancer. Individuals having MM and QQ genotypes have a lower level and lower detoxification activity of the PON1 enzyme, which may increase the vulnerability of the breast to genetic damage by reducing the ability to detoxify inflammatory oxidants, as well as dietary carcinogens.     

Methylglyoxal-modified collagen promotes myofibroblast differentiation

Fibrosis is a frequent complication of diabetes mellitus in many organs and tissues but the mechanism of how diabetes-induced glycation of extracellular matrix proteins impacts the formation of fibrotic lesions is not defined. As fibrosis is mediated by myofibroblasts, we investigated the effect of collagen glycation on the conversion of human cardiac fibroblasts to myofibroblasts. Collagen glycation was modeled by the glucose metabolite, methylglyoxal (MGO). Cells cultured on MGO-treated collagen exhibited increased activity of the α-smooth muscle actin promoter and enhanced expression of α-smooth muscle actin, ED-A fibronectin and cadherin, which are markers for myofibroblasts. In cells remodeling floating or stress-relaxed collagen gels, MGO treatment promoted more contraction (p < 0.025) than vehicle controls, which was MGO dose-dependent. Transwell assays showed that cell migration was increased by MGO-treated collagen (p < 0.025). In shear-force detachment assays, cells on MGO-treated collagen were less adherent than untreated collagen, and the formation of high affinity, β1 integrin-dependent adhesions was inhibited. MGO-collagen-induced expression of SMA was dependent on TGF-β but not on Rho kinase. We conclude that collagen glycation augments the formation and migration of myofibroblasts, critical processes in the development of fibrosis in diabetes.     

Selenite cataracts: Activation of endoplasmic reticulum stress and loss of Nrf2/Keap1-dependent stress protection

Cataract-induced by sodium selenite in suckling rats is one of the suitable animal models to study the basic mechanism of human cataract formation. The aim of this present investigation is to study the endoplasmic reticulum (ER) stress-mediated activation of unfolded protein response (UPR), overproduction of reactive oxygen species (ROS), and suppression of Nrf2/Keap1-dependent antioxidant protection through endoplasmic reticulum-associated degradation (ERAD) pathway and Keap1 promoter DNA demethylation in human lens epithelial cells (HLECs) treated with sodium selenite. Lenses enucleated from sodium selenite injected rats generated overproduction of ROS in lens epithelial cells and newly formed lens fiber cells resulting in massive lens epithelial cells death after 1–5 days. All these lenses developed nuclear cataracts after 4–5 days. Sodium selenite treated HLECs induced ER stress and activated the UPR leading to release of Ca^2 + from ER, ROS overproduction and finally HLECs death. Sodium selenite also activated the mRNA expressions of passive DNA demethylation pathway enzymes such as Dnmt1, Dnmt3a, and Dnmt3b, and active DNA demethylation pathway enzyme, Tet1 leading to DNA demethylation in the Keap1 promoter of HLECs. This demethylated Keap1 promoter results in overexpression of Keap1 mRNA and protein. Overexpression Keap1 protein suppresses the Nrf2 protein through ERAD leading to suppression of Nrf2/Keap1 dependent antioxidant protection in the HLECs treated with sodium selenite. As an outcome, the cellular redox status is altered towards lens oxidation and results in cataract formation.     

Genotoxicity assessment in iron deficiency anemia patients using sister chromatid exchanges and chromosomal aberrations assays

Previous studies have shown that iron deficiency anemia is associated with oxidative stress produced by a decrease in antioxidant enzyme activities and/or a high level of oxidants. Because oxidative stress induces DNA damage, we investigated genotoxicity in lymphocytes from patients with iron deficiency anemia (IDA) using chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) assays. Eighteen IDA subjects and a similar number of age-matched healthy controls were included in the study. The results demonstrated that IDA was associated with a slight increase in the frequency of spontaneous CAs and a decrease in the frequency of SCEs (P < 0.05). In addition, the level of SCEs was positively correlated with both the ferritin concentration (r = 0.485, P < 0.05) and hemoglobin content (r = 0.514, P < 0.05) in subjects. Moreover, vitamin E treatment reduced the frequency of SCEs in IDA patients and control subjects by the same percentage (～30%) without affecting the magnitude of the difference in the levels of SCEs between the two groups. In conclusion, our results demonstrated that IDA has a differential effect on the frequency of spontaneous CAs and SCEs.     

Increased proton leak and SOD2 expression in myotubes from obese non-diabetic subjects with a family history of type 2 diabetes

Muscle insulin resistance is linked to oxidative stress and decreased mitochondrial function. However, the exact cause of muscle insulin resistance is still unknown. Since offspring of patients with type 2 diabetes mellitus (T2DM) are susceptible to developing insulin resistance, they are ideal for studying the early development of insulin resistance. By using primary muscle cells derived from obese non-diabetic subjects with (FH +) or without (FH ?) a family history of T2DM, we aimed to better understand the link between mitochondrial function, oxidative stress, and muscle insulin resistance. Insulin-stimulated glucose uptake and glycogen synthesis were normal in FH + myotubes. Resting oxygen consumption rate was not different between groups. However, proton leak was higher in FH + myotubes. This was associated with lower ATP content and decreased mitochondrial membrane potential in FH + myotubes. Surprisingly, mtDNA content was higher in FH + myotubes. Oxidative stress level was not different between FH + and FH ? groups. Reactive oxygen species content was lower in FH + myotubes when differentiated in high glucose/insulin (25 mM/150 pM), which could be due to higher oxidative stress defenses (SOD2 expression and uncoupled respiration). The increased antioxidant defenses and mtDNA content in FH + myotubes suggest the existence of compensatory mechanisms, which may provisionally prevent the development of insulin resistance.     

l-Lysine supplementation does not affect the bioavailability of copper or iron in rats

l-lysine (Lys) is an essential amino acid that is added to foods and dietary supplements. Lys may interact with mineral nutrients and affect their metabolism. This study examined the effect of dietary Lys supplementation on the bioavailability of copper (Cu) and iron (Fe). Weanling male Sprague-Dawley rats were fed one of five diets (20% casein) for 4 weeks containing normal Cu and Fe (control) or low Cu or Fe without (LCu, LFe) or with (LCu + Lys, LFe + Lys) addition of 1.5% Lys. Final body weights, body weight gains and food consumption of the rats did not differ (P ≥ 0.05) among diet groups. Rats fed the low Cu or Fe diets showed changes in nutritional biomarkers compared to control rats, demonstrating reduced Cu and Fe status, respectively. Hematological parameters, serum ceruloplasmin activity and Cu and Fe concentrations in serum, liver, kidney and intestinal mucosa were unaffected (P ≥ 0.05) by Lys supplementation. These results indicate that in the context of an adequate protein diet, Lys supplementation at a relatively high level does not affect Cu or Fe bioavailability in rats.     

Lack of an Effect of Pioglitazone or Glipizide on Lipoprotein-Associated Phospholipase A2 in Type 2 Diabetes

ObjectiveTo study the effects of pioglitazone, a peroxisome proliferator-activated receptor-γ agonist with vascular beneficial effects, and glipizide, an insulin secretagogue, on novel inflammatory vascular risk markers in subjects with and without type 2 diabetes.MethodsWe studied 11 subjects without diabetes and 19 matched subjects with diabetes. The subjects with diabetes were randomly assigned to receive either 45 mg daily of pioglitazone (N = 8) or 10 mg daily of glipizide (N = 11) (median dose) for 12 weeks. Lipoprotein-associated phospholipase A₂ (LpPLA2), vascular cell adhesion molecule (VCAM-1), intracellular adhesion molecule (ICAM-1), and e-selectin were measured by established techniques before and after therapy with either agent. The subjects without diabetes were studied only once.ResultsThe study subjects with diabetes had higher (P < 0.05) LpPLA2, e-selectin, and VCAM-1 levels than did those without diabetes. ICAM-1 levels tended to be higher (P = 0.07) in the study subjects with than in those without diabetes. Neither pioglitazone nor glipizide therapy significantly altered LpPLA2 or VCAM-1 concentrations. While pioglitazone therapy reduced (P < 0.05) e-selectin concentrations, glipizide therapy reduced (P < 0.03) ICAM-1 concentrations.ConclusionType 2 diabetes is associated with elevated concentrations of the novel vascular risk marker LpPLA2 and inflammatory risk markers e-selectin and VCAM-1. Neither pioglitazone nor glipizide significantly altered LpPLA2, VCAM-1, or highly sensitive C-reactive protein levels after 12 weeks of therapy. In study subjects with type 2 diabetes, e-selectin concentrations declined significantly with pioglitazone therapy, whereas ICAM-1 concentrations decreased significantly with glipizide therapy. (Endocr Pract. 2007;13:147-152)     

Elevated Fructosamine Levelsin Acquired Immunodeficiency Syndrome

ObjectiveTo investigate whether the mechanism of increased glycation in acquired immunodeficiency syndrome (AIDS) is due to an alteration in a circulatory plasma enhancer.MethodsWe assessed glycation of serum protein and hemoglobin in patients with AIDS without altered carbohydrate metabolism. Fasting concentrations of glucose, ethanol, vitamin E, fructosamine, hemoglobin, hemoglobin A1c (A1C), and partial pressure of alveolar oxygen (Pao₂) were determined in 50 men with AIDS and in 25 age-matched healthy men in whom normal glucose tolerance was established by oral glucose tolerance tests.ResultsFasting serum glucose was not significantly different between the men with AIDS (87 ± 4 mg/dL) and the healthy male volunteers (84 ± 6 mg/dL); however, A1C (6.9 ± 0.2%) and serum fructosamine levels (288 ± 15 μmol/L) were significantly higher (P < .01) in the patients with AIDS than in the normal subjects (A1C, 5.6 ± 0.1%; fructosamine, 204 ± 14 μmol/L). Moreover, both A1C and fructosamine concentrations were significantly higher (P < .01) in the patients with AIDS than in the normal subjects divided into subgroups on the basis of fasting plasma glucose concentrations (70 to 79 mg/dL, 80 to 89 mg/dL, and 90 to 99 mg/dL). None of the study participants had anemia (hemoglobin < 12 g/dL) or hypoxia (Pao₂ < 95 mm Hg), and serum ethanol was undetectable. Furthermore, vitamin E concentrations were not significantly different between the patients with AIDS (25 ± 3 mg/L) and the normal subjects (22 ± 4 mg/L).ConclusionOn the basis of this study, glycation of some circulating proteins appears to be enhanced in AIDS and may be induced by an undetermined plasma enhancer, inasmuch as known circulating factors promoting glycation were absent. (Endocr Pract. 2008;14:686-690)     

Effect of Bromocriptine-Qr On Glycemic Control in Subjects with Uncontrolled Hyperglycemia On One or Two Oral Anti-Diabetes Agents

ObjectiveTo investigate the effect of Bromocriptine QR on glycemic control in patients with type 2 diabetes whose glycemia is poorly controlled on one or two oral anti-diabetes agents.MethodsFive hundred fifteen Type 2 Diabetes Mellitus (T2DM) subjects (ages 18 to 80 and average body mass index [BMI] of 32.7) with baseline HbA1c ≥ 7.5 and on one or two oral anti-diabetes (OAD) medications (metformin, sulfonylurea, and/or thiazolidinediones) were randomized 2:1 to bromocriptine-QR (1.6 to 4.8 mg/day) or placebo for a 24 week treatment period. Study investigators were allowed to adjust, if necessary, subject anti diabetes medications during the study to attempt to achieve glycemic control in case of glycemic deterioration. The impact of bromocriptine-QR treatment intervention on glycemic control was assessed in subjects on any one or two OADs (ALL treatment category) (N = 515), or on metformin with or without another OAD (Met/OAD treatment category) (N = 356), or on metformin plus a sulfonylurea (Met/SU treatment category) (N = 245) 1) by examining the between group difference in change from baseline a) concomitant OAD medication changes during the study, and b) HbA1c and 2) by determining the odds of reaching HbA1c of ≤ 7.0% on bromocriptine-QR versus placebo.ResultsSignificantly more patients (approximately 1.5 to 2-fold more; P < .05) intensified concomitant anti diabetes medication therapy during the study in the placebo versus the bromocriptine-QR arm. In subjects that did not change the intensity of the baseline diabetes therapy (72%), and that were on any one or two OADs (ALL), or on metformin with or without another OAD (Met/OAD), or on metformin plus sulfonylurea (Met/SU), the HbA1c change for bromocriptine-QR versus placebo was − 0.47 versus + 0.22 (between group delta of − 0.69, P < .0001), − 0.55 versus + 0.26 (between group delta of − 0.81, P < .0001) and − 0.63 versus + 0.20 (between group delta of − 0.83, P < .0001) respectively, after 24 weeks on therapy. The odds ratio of reaching HbA1c of ≤ 7.0% was 6.50, 12.03 and 11.45 (P < .0002) for these three groups, respectively.ConclusionIn T2DM subjects whose hyperglycemia is poorly controlled on one or two oral agents, bromocrip tine-QR therapy for 24 weeks can provide significant added improvement in glycemic control relative to adding placebo. (Endocr Pract. 2012;18:931-943)     

A new type of neuron-specific aminopeptidase NAP-2 in rat brain synaptosomes

A novel neutral aminopeptidase (NAP-2) was found exclusively in the rat central nervous system (CNS). It was separated from the ubiquitous puromycin-sensitive aminopeptidase (PSA) and the neuron-specific aminopeptidase (NAP) by an automated FPLC-aminopeptidase analyzer. The activity of the neuronal aminopeptidase enriched in the synaptosomes is different from NAP and PSA in distribution and during brain development. The enzyme was purified 2230-fold to apparent homogeneity from rat brain cytosol with 4% recovery by ammonium sulfate fractionation, followed by column chromatography successively on Phenyl-Sepharose, Q-Sepharose, Sephadex G-200, and Mono Q. The single-chain enzyme with a molecular mass of 110 kDa has an optimal pH of 7.0 and a pI of 5.6. It splits β-naphthylamides of amino acid with aliphatic, polar uncharged, positively charged, and aromatic side chain. Leucyl β-naphthylamide (Leu βNA) is the best substrate with the highest hydrolytic coefficiency followed by Met βNA = Arg βNA = Lys βNA > Ala βNA > Tyr βNA > Phe βNA. The cysteine-, metallo-, glyco-aminopeptidase releases the N-terminal Tyr from Leu-enkephalin with a K_m 82 μM and a k_cat of 1.08 s⁻¹, and Met-enkephalin with a K_m of 106 μM and a k_cat of 2.6 s⁻¹. The puromycin-sensitive enzyme is most susceptible to amastatin with an IC₅₀ of 0.05 μM. The data indicate that the enzyme is a new type of NAP found in rodent. Its possible function in neuron growth, neurodegeneration, and carcinomas is discussed.     

Uptake of advanced glycation end products by proximal tubule epithelial cells via macropinocytosis

Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. We describe AGE uptake in LLC-PK1 and HK2 proximal tubule cell lines by macropinocytosis, a non-specific, endocytic mechanism. AGE–BSA induced dorsal circular actin ruffles and amiloride-sensitive dextran–TRITC uptake, significantly increased AGE–BSA–FITC uptake (167 ± 20% vs BSA control, p < 0.01) and was ezrin-dependent. AGE–BSA–FITC uptake was significantly inhibited by amiloride and inhibitors of Arf6, Rac1, racGEF Tiam1, PAK1 and actin polymerisation. AGE–BSA–FITC, Arf6 and PIP2 co-localised within dorsal circular actin ruffles. AGE–BSA increased PAK1 kinase activity (212 ± 41% vs control, p < 0.05) and protein levels of Tiam1, a Rac1 activator. AGE–BSA significantly increased TGF-β₁ protein levels (160 ± 6%, p < 0.001 vs BSA), which were significantly inhibited by inhibitors of Arf6 (82 ± 19%, p < 0.001 vs AGE) and actin polymerisation (107 ± 11%, p < 0.001 vs AGE), suggesting AGEs partially exert their profibrotic effects via macropinocytosis. PAK1 and PIP5Kγ siRNA significantly decreased AGE–BSA–FITC uptake (81 ± 6% and 64 ± 7%, respectively, p < 0.05 vs control for both), and AGE-stimulated TGF-β₁ protein release (99 ± 15% and 49 ± 8% of control, p < 0.05 and p < 0.001, respectively). Inhibition of AGE uptake by macropinocytosis inhibitors and a neutralising TGF-β antibody, reversed the AGE-induced decrease in surface Na⁺K⁺ATPase, suggesting AGE uptake by macropinocytosis may contribute to diabetic kidney fibrosis and/or EMT by modulating this pump. Understanding methods of cellular uptake and signalling by AGEs may lead to novel therapies for diabetic nephropathy.     

Soluble receptor for advanced glycation end products as a potential biomarker to predict weight loss and improvement of insulin sensitivity by a very low calorie diet of obese human subjects

IntroductionObesity is associated with low-grade systemic inflammation which is thought to trigger the development of comorbidities such as type 2 diabetes. The soluble receptor for advanced glycation end products (sRAGE) belongs to the innate immune system and has been linked to obesity, recently. The aim of the present study was to examine whether serum sRAGE concentrations are related to the grade of weight loss and improvement of insulin resistance due to a very low calorie diet (VLCD).Methods22 severe obese subjects (Median Body Mass Index (BMI): 44.5 kg/m²) were included in a dietary intervention study of 6 month, consisting of a very low calorie formula diet phase (VLCD: 800 kcal/d) for 12 weeks and a following 12 week weight maintenance phase. Fasting glucose, fasting insulin, adiponectin, leptin and sRAGE were determined from sera. Insulin sensitivity was estimated by Homeostasis Model Assessment (HOMA) index and leptin-to-adiponectin-ratio (LAR).ResultsMean body weight reduction by VLCD accounted to 21.7 kg with a significant improvement of insulin resistance. At baseline, sRAGE serum levels were significantly inversely related to BMI (r_S = −0.642, p = 0.001) and HOMA (r_S = −0.419, p = 0.041). Of interest, sRAGE serum levels at baseline were significantly lower in study subjects with greater reduction of BMI (p = 0.017). In addition, a significantly greater HOMA reduction was observed in subjects with lower sRAGE serum levels at baseline (p = 0.006). Finally, correlation analysis revealed, that changes of sRAGE serum levels were significantly correlated to changes of BMI (r_S = −0.650, p = 0.022) during intervention.ConclusionAnti-inflammatory sRAGE might be a potential future biomarker to predict weight loss and improvement of insulin resistance by a VLCD whereby lower baseline sRAGE serum levels indicate a better outcome of the dietary intervention.     

Substrate specificity and inhibitory study of human airway trypsin-like protease

Human airway trypsin-like protease (HAT), also referred to as TMPRSS11D, is an important physiological enzyme with the main activity pronounced in an airway. In this work we have described the substrate specificity and selectivity study of the protease, performed by the combinatorial approach. Fluorogenic/chromogenic tetrapeptide library was used for this purpose. The most efficiently hydrolyzed substrates’ sequences that we selected were ABZ-Arg-Gln-Asp-Arg(Lys)-ANB-NH₂. The most active inhibitor with C-terminal Arg residue underwent detectable proteolysis action in the presence of 35 pM of HAT. Based on the selected sequences the two peptide aldehydes were synthesized and (Abz-Arg-Gln-Asp-Arg(Lys)-H) were found to be an effective HAT inhibitor, working in nanomolar range with inhibition constant 54 nM and 112 nM, respectively.     

Fasting plasma levels of nesfatin-1 in patients with type 1 and type 2 diabetes mellitus and the nutrient-related fluctuation of nesfatin-1 level in normal humans

The novel satiety factor nesfatin-1 has been shown to decrease food intake and body weight in rodents after i.c.v. injection. However, no further developments regarding the true patho-physiological relevance of nesfatin-1 in obesity and type 1 diabetes mellitus (T1 DM) and type 2 diabetes mellitus (T2 DM) have been reported. A recent study by Stengel et al. demonstrated that a down-regulation of NUCB2 mRNA in gastric endocrine cells was observed after 24-h fasting. They raised the possibility that nesfatin/NUCB2 gene expression may be regulated by nutritional status, suggesting that nesfatin-1 in the stomach might play a role in satiety. In the present study, fasting levels in plasma nesfatin-1, insulin and glucose were measured and analyzed in healthy subjects and in patients with T1 DM and T2 DM. Plasma nesfatin-1 levels were measured 6 times before and after oral glucose ingestion in healthy subjects. No sex differences in plasma nesfatin-1 were found. The mean fasting plasma nesfatin-1 levels were slightly but not significantly higher in T1 DM patients compared to healthy subjects. However, fasting plasma nesfatin-1 levels were significantly lower in T2 DM patients compared to healthy subjects and T1 DM patients. Plasma nesfatin-1 did not change acutely, although a small rise in circulating nesfatin-1 occurred within 30 min after the beginning of an oral glucose ingestion (from a mean basal value of 0.99 ± 0.23 ng/ml to a maximum of 1.08 ± 0.24 ng/ml). No significant difference in plasma nesfatin-1 before and after an oral glucose was observed. In conclusion, we showed that fasting nesfatin-1 was significantly lower in T2 DM patients compared to healthy subjects and T1 DM patients. The significance of this result is unclear but the reduction in fasting nesfatin-1 may be one of the appetite-related hormones involved in diabetic hyperphagia. In addition, neither glucose nor saline ingestions affected plasma nesfatin-1, suggesting that gastric chemosensation is not sufficient for the nesfatin-1 response under the present conditions.