Ascorbate reverses high glucose- and RAGE-induced leak of the endothelial permeability barrier

High glucose concentrations due to diabetes increase leakage of plasma constituents across the endothelial permeability barrier. We sought to determine whether vitamin C, or ascorbic acid (ascorbate), could reverse such high glucose-induced increases in endothelial barrier permeability. Human umbilical vein endothelial cells and two brain endothelial cell lines cultured at 25 mM glucose showed increases in endothelial barrier permeability to radiolabeled inulin compared to cells cultured at 5 mM glucose. Acute loading of the cells for 30–60 min with ascorbate before the permeability assay prevented the high glucose-induced increase in permeability and decreased basal permeability at 5 mM glucose. High glucose-induced barrier leakage was mediated largely by activation of the receptor for advanced glycation end products (RAGE), since it was prevented by RAGE blockade and mimicked by RAGE ligands. Intracellular ascorbate completely prevented RAGE ligand-induced increases in barrier permeability. The high glucose-induced increase in endothelial barrier permeability was also acutely decreased by several cell-penetrant antioxidants, suggesting that at least part of the ascorbate effect could be due to its ability to act as an antioxidant.     

High glucose-induced mitochondrial respiration and reactive oxygen species in mouse cerebral pericytes is reversed by pharmacological inhibition of mitochondrial carbonic anhydrases: Implications for cerebral microvascular disease in diabetes

Hyperglycemia-induced oxidative stress leads to diabetes-associated damage to the microvasculature of the brain. Pericytes in close proximity to endothelial cells in the brain microvessels are vital to the integrity of the blood–brain barrier and are especially susceptible to oxidative stress. According to our recently published results, streptozotocin-diabetic mouse brain exhibits oxidative stress and loose pericytes by twelve weeks of diabetes, and cerebral pericytes cultured in high glucose media suffer intracellular oxidative stress and apoptosis. Oxidative stress in diabetes is hypothesized to be caused by reactive oxygen species (ROS) produced during hyperglycemia-induced enhanced oxidative metabolism of glucose (respiration). To test this hypothesis, we investigated the effect of high glucose on respiration rate and ROS production in mouse cerebral pericytes. Previously, we showed that pharmacological inhibition of mitochondrial carbonic anhydrases protects the brain from oxidative stress and pericyte loss. The high glucose-induced intracellular oxidative stress and apoptosis of pericytes in culture were also reversed by inhibition of mitochondrial carbonic anhydrases. Therefore, we extended our current study to determine the effect of these inhibitors on high glucose-induced increases in pericyte respiration and ROS. We now report that both the respiration and ROS are significantly increased in pericytes challenged with high glucose. Furthermore, inhibition of mitochondrial carbonic anhydrases significantly slowed down both the rate of respiration and ROS production. These data provide new evidence that pharmacological inhibitors of mitochondrial carbonic anhydrases, already in clinical use, may prove beneficial in protecting the brain from oxidative stress caused by ROS produced as a consequence of hyperglycemia-induced enhanced respiration.     

Angiotensin II augments advanced glycation end product-induced pericyte apoptosis through RAGE overexpression

Advanced glycation end product (AGE)-their receptor (RAGE) and angiotensin II (AII) are implicated in diabetic retinopathy. However, a crosstalk between the two is not fully understood. In vivo, AGE injection stimulated RAGE expression in the eye of spontaneously hypertensive rats, which was blocked by an AII-type 1 receptor blocker, telmisartan. In vitro, AII-type 1 receptor-mediated reactive oxygen species generation elicited RAGE gene expression in pericytes through NF-kappaB activation. Further, AII augmented AGE-induced pericyte apoptosis, the earliest hallmark of diabetic retinopathy. Our present study may implicate a crosstalk between AGE-RAGE system and AII in diabetic retinopathy.     

Promising effects of the 4HPR-BSO combination in neuroblastoma monolayers and spheroids

To enhance the efficacy of fenretinide (4HPR)-induced reactive oxygen species (ROS) in neuroblastoma, 4HPR was combined with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, in neuroblastoma cell lines and spheroids, the latter being a three-dimensional tumor model. 4HPR exposure (2.5-10 μM, 24 h) resulted in ROS induction (114-633%) and increased GSH levels (68-120%). A GSH depletion of 80% of basal levels was observed in the presence of BSO (25-100 μM, 24 h). The 4HPR-BSO combination resulted in slightly increased ROS levels (1.1- to 1.3-fold) accompanied by an increase in cytotoxicity (110-150%) compared to 4HPR treatment alone. A correlation was observed between the ROS-inducing capacity of each cell line and the increase in cytotoxicity induced by 4HPR-BSO compared to 4HPR. No significant correlation between baseline antioxidant levels and sensitivity to 4HPR or BSO was observed. In spheroids, 4HPR-BSO induced a strong synergistic growth retardation and induction of apoptosis. Our data show that BSO increased the cytotoxic effects of 4HPR in neuroblastoma monolayers and spheroids in ROS-producing cell lines. This indicates that the 4HPR-BSO combination might be a promising new strategy in the treatment of neuroblastoma.     

Modulation of advanced glycation endproduct synthesis by kynurenines in human lens proteins

Human lens proteins (HLP) become chemically modified by kynurenines and advanced glycation end products (AGEs) during aging and cataractogenesis. We investigated the effects of kynurenines on AGE synthesis in HLP. We found that incubation with 5 mM ribose or 5 mM ascorbate produced significant quantities of pentosidine, and this was further enhanced in the presence of two different kynurenines (200–500 µM): N-formylkynurenine (Nfk) and kynurenine (Kyn). Another related compound, 3-hydroxykynurenine (3OH-Kyn), had disparate effects; low concentrations (10–200 µM) promoted pentosidine synthesis, but high concentrations (200–500 µM) inhibited it. 3OH-Kyn showed similar effects on pentosidine synthesis from Amadori-enriched HLP or ribated lysine. Chelex-100 treatment of phosphate buffer reduced pentosidine synthesis from Amadori-enriched HLP by ∼ 90%, but it did not inhibit the stimulating effect of 3OH-Kyn and EDTA. 3OH-Kyn (100–500 μM) spontaneously produced copious amounts of H₂O₂ (10–25 μM), but externally added H₂O₂ had only a mild stimulating effect on pentosidine but had no effect on N^ε-carboxymethyl lysine (CML) synthesis in HLP from ribose and ascorbate. Further, human lens epithelial cells incubated with ribose and 3OH-Kyn showed higher intracellular pentosidine than cells incubated with ribose alone. CML synthesis from glycating agents was inhibited 30 to 50% by 3OH-Kyn at concentrations of 100–500 μM. Argpyrimidine synthesis from 5 mM methylglyoxal was slightly inhibited by all kynurenines at concentrations of 100–500 μM. These results suggest that AGE synthesis in HLP is modulated by kynurenines, and such effects indicate a mode of interplay between kynurenines and carbohydrates important for AGE formation during lens aging and cataract formation.     

Anti-cancer effect of pharmacologic ascorbate and its interaction with supplementary parenteral glutathione in preclinical cancer models

Two popular complementary, alternative, and integrative medicine therapies, high-dose intravenous ascorbic acid (AA) and intravenous glutathione (GSH), are often coadministered to cancer patients with unclear efficacy and drug-drug interaction. In this study we provide the first survey evidence for clinical use of iv GSH with iv AA. To address questions of efficacy and drug-drug interaction, we tested 10 cancer cell lines with AA, GSH, and their combination. The results showed that pharmacologic AA induced cytotoxicity in all tested cancer cells, with IC₅₀ less than 4 mM, a concentration easily achievable in humans. GSH reduced cytotoxicity by 10-95% by attenuating AA-induced H₂O₂ production. Treatment in mouse pancreatic cancer xenografts showed that intraperitoneal AA at 4 g/kg daily reduced tumor volume by 42%. Addition of intraperitoneal GSH inhibited the AA-induced tumor volume reduction. Although all treatments (AA, GSH, and AA + GSH) improved survival rate, AA + GSH inhibited the cytotoxic effect of AA alone and failed to provide further survival benefit. These data confirm the pro-oxidative anti-cancer mechanism of pharmacologic AA and suggest that AA and GSH administered together provide no additional benefit compared with AA alone. There is an antagonism between ascorbate and glutathione in treating cancer, and therefore iv AA and iv GSH should not be coadministered to cancer patients on the same day.     

CB₁ receptor activation inhibits neuronal and astrocytic intermediary metabolism in the rat hippocampus

Cannabinoid CB₁ receptor (CB₁R) activation decreases synaptic GABAergic and glutamatergic transmission and it also controls peripheral metabolism. Here we aimed at testing with ¹³C NMR isotopomer analysis whether CB₁Rs could have a local metabolic role in brain areas having high CB₁R density, such as the hippocampus. We labelled hippocampal slices with the tracers [2-¹³C]acetate, which is oxidized in glial cells, and [U-¹³C]glucose, which is metabolized both in glia and neurons, to evaluate metabolic compartmentation between glia and neurons. The synthetic CB₁R agonist WIN55212-2 (1 μM) significantly decreased the metabolism of both [2-¹³C]acetate (−11.6 ± 2.0%) and [U-¹³C]glucose (−11.2 ± 3.4%) in the tricarboxylic acid cycle that contributes to the glutamate pool. WIN55212-2 also significantly decreased the metabolism of [U-¹³C]glucose (−11.7 ± 4.0%) but not that of [2-¹³C]acetate contributing to the pool of GABA. These effects of WIN55212-2 were prevented by the CB₁R antagonist AM251 (500 nM). These results thus suggest that CB₁Rs might be present also in hippocampal astrocytes besides their well-known neuronal localization. Indeed, confocal microscopy analysis revealed the presence of specific CB₁R immunoreactivity in astrocytes and pericytes throughout the hippocampus.In conclusion, CB₁Rs are able to control hippocampal intermediary metabolism in both neuronal and glial compartments, which suggests new alternative mechanisms by which CB₁Rs control cell physiology and afford neuroprotection.     

Oxidized lipoprotein induces the macrophage ascorbate transporter (SVCT2): Protection by intracellular ascorbate against oxidant stress and apoptosis

To assess whether ascorbic acid decreases the cytotoxicity of oxidized human low density lipoprotein (oxLDL) in cells involved in atherosclerosis, its interaction with oxLDL was studied in murine RAW264.7 macrophages. Macrophages took up ascorbate to millimolar intracellular concentrations and retained it with little loss over 18 h in culture. Culture of the macrophages with oxLDL enhanced ascorbate uptake. This was associated with increased expression of the ascorbate transporter (SVCT2), which was prevented by ascorbate and by inhibiting the NF-κB pathway. Culture of RAW264.7 macrophages with oxLDL increased intracellular dihydrofluorescein oxidation and lipid peroxidation, both of which were decreased by intracellular ascorbate. Ascorbate also protected the cells against oxLDL-induced cytotoxicity and apoptosis, but it did not affect macrophage accumulation of lipid from oxLDL or oxLDL-induced increases in macrophage cytokine secretion. These results suggest that ascorbate protects macrophages against oxLDL-induced oxidant stress and subsequent apoptotic death without impairing their function.     

Identification of a novel GPR119 agonist, AS1269574, with in vitro and in vivo glucose-stimulated insulin secretion

The G protein-coupled receptor 119 (GPR119) is highly expressed in pancreatic β-cells. On activation, this receptor enhances the effect of glucose-stimulated insulin secretion (GSIS) via the elevation of intracellular cAMP concentrations. Although GPR119 agonists represent promising oral antidiabetic agents for the treatment of type 2 diabetes therapy, they suffer from the inability to adequately directly preserve β-cell function. To identify a new structural class of small-molecule GPR119 agonists with both GSIS and the potential to preserve β-cell function, we screened a library of synthetic compounds and identified a candidate molecule, AS1269574, with a 2,4,6-tri-substituted pyrimidine core. Here, we examined the preliminary in vitro and in vivo effects of AS1269574 on insulin secretion and glucose tolerance. AS1269574 had an EC₅₀ value of 2.5 μM in HEK293 cells transiently expressing human GPR119 and enhanced insulin secretion in the mouse pancreatic β-cell line MIN-6 only under high-glucose (16.8 mM) conditions. This contrasted with the action of the sulfonylurea glibenclamide, which also induced insulin secretion under low-glucose conditions (2.8 mM). In in vivo studies, a single administration of AS1269574 to normal mice reduced blood glucose levels after oral glucose loading based on the observed insulin secretion profiles. Significantly, AS1269574 did not affect fed and fasting plasma glucose levels in normal mice. Taken together, these results suggest that AS1269574 represents a novel structural class of small molecule, orally administrable GPR119 agonists with GSIS and promising potential for the treatment of type 2 diabetes.     

Arsenate accumulation and arsenate-induced glutathione export in astrocyte-rich primary cultures

Arsenate is a toxic compound that has been connected with neuropathies and impaired cognitive functions. To test whether arsenate affects the viability and the GSH metabolism of brain astrocytes, we have used primary astrocyte cultures as model system. Incubation of astrocytes for 2 h with arsenate in concentrations of up to 10 mM caused an almost linear increase in the cellular arsenic content, but did not acutely compromise cell viability. The presence of moderate concentrations of arsenate caused a time- and concentration-dependent loss of GSH from viable astrocytes which was accompanied by a matching increase in the extracellular GSH content. Half-maximal effects were observed for arsenate in a concentration of about 0.3 mM. The arsenate-induced stimulated GSH export from astrocytes was prevented by MK571, an inhibitor of the multidrug resistance protein 1. Exposure of astrocytes to arsenite increased the specific cellular arsenic content and stimulated GSH export to values that were similar to those observed for arsenate-treated cells, while dimethylarsinic acid was less efficiently accumulated by the cells and did not modulate cellular and extracellular GSH levels. The observed strong stimulation of GSH export from astrocytes by arsenate suggests that disturbances of the astrocytic GSH metabolism may contribute to the observed arsenic-induced neurotoxicity.     

Atorvastatin exerts its anti-atherosclerotic effects by targeting the receptor for advanced glycation end products

Recent studies demonstrated the beneficial role of atorvastatin in reducing the risk of cardiovascular morbidity and mortality in patients with diabetes mellitus and/or metabolic syndrome. To investigate the mechanisms underlying the anti-atheroscleroic action of atorvastatin, we examined the expression of the receptor for advanced glycation end products (RAGE) and its downstream target gene, monocyte chemoattractant protein-1 (MCP-1) using real-time PCR. In in vitro studies, exposure to high glucose or AGE induced oxidative stress and activation of the AGE/RAGE system in human umbilical vein endothelial cells. Treatment of the cells with atorvastatin significantly released the oxidative stress by restoring the levels of glutathione and inhibited the RAGE upregulation. In diabetic Goto Kakisaki (GK) rats fed with a high-fat diet for 12 weeks, RAGE and MCP-1 were upregulated in the aortas, and there was a significant correlation between RAGE and MCP-1 mRNA abundance (r = 0.482, P = 0.031). Treatment with atorvastatin (20 mg/kg qd) significantly downregulated the expression of RAGE and MCP-1. These data thus demonstrate a novel “pleiotropic” activity of atorvastatin in reducing the risk of cardiovascular diseases by targeting RAGE expression.     

New N-acyl-d-glucosamine 2-epimerases from cyanobacteria with high activity in the absence of ATP and low inhibition by pyruvate

N-Acetylneuraminic acid, an important component of glycoconjugates with various biological functions, can be produced from N-acetyl-d-glucosamine (GlcNAc) and pyruvate using a one-pot, two-enzyme system consisting of N-acyl-d-glucosamine 2-epimerase (AGE) and N-acetylneuraminate lyase (NAL). In this system, the epimerase catalyzes the conversion of GlcNAc into N-acetyl-d-mannosamine (ManNAc). However, all currently known AGEs have one or more disadvantages, such as a low specific activity, substantial inhibition by pyruvate and strong dependence on allosteric activation by ATP. Therefore, four novel AGEs from the cyanobacteria Acaryochloris marina MBIC 11017, Anabaena variabilis ATCC 29413, Nostoc sp. PCC 7120, and Nostoc punctiforme PCC 73102 were characterized. Among these enzymes, the AGE from the Anabaena strain showed the most beneficial characteristics. It had a high specific activity of 117 ± 2 U mg⁻¹ at 37 °C (pH 7.5) and an up to 10-fold higher inhibition constant for pyruvate as compared to other AGEs indicating a much weaker inhibitory effect. The investigation of the influence of ATP revealed that the nucleotide has a more pronounced effect on the K_m for the substrate than on the enzyme activity. At high substrate concentrations (≥200 mM) and without ATP, the enzyme reached up to 32% of the activity measured with ATP in excess.     

The effect of testosterone on ascorbic acid synthesis in rooster comb

The level of “total” ascorbic acid (ascorbate+dehydroascorbate) has been measured in the mucoid layer of combs from normal roosters, capons and capons treated with testosterone. The “total” ascorbate level in capon comb was lower than the value obtained from combs from normal roosters. This value returned towards normal in combs from capons treated with testosterone. The specific activity of L-gulonate-NADP⁺ oxidoreductase, an enzyme in the pathway of ascorbate biosynthesis, also was measured. The specific activity levels followed a pattern similar to the ascorbate levels in the three types of combs utilized. The results are consistent with the possible role of L-ascorbic acid as a cofactor in the synthesis of collagen, a process which also appears to be dependent on the level of testosterone in the comb mucoid layer.     

Dopamine but not l-dopa stimulates neural glutathione metabolism. Potential implications for Parkinson’s and other dopamine deficiency states

Dopamine is produced first by hydroxylalation of l-tyrosine to l-dihydroxyphenylalanine (l-dopa) and subsequently by the decarboxylation of l-dopa to dopamine catalysed by the enzymes tyrosine hydroxylase and aromatic l-amino acid decarboxylase (AADC) respectively. Reduced glutathione (GSH) acts as a major cellular antioxidant. We have investigated the role of dopamine in the control of GSH homeostasis in brain cells. The SH-SY5Y human neuroblastoma cell line was found to increase intracellular GSH levels in response to 50 μM dopamine treatment. Similarly the 1321N1 human astrocytoma cell line was found to increase GSH release in response to 50 μM dopamine. The same concentration of l-dopa was also found to increase intracellular GSH in SH-SY5Y cells, however when AADC was inhibited this affect was abolished. Furthermore 1321N1 cells which were found to have almost undetectable levels of AADC activity did not increase GSH release in response to 50 μM l-dopa. These results suggest that at these concentrations dopamine has the potential to act as a signal for the upregulation of GSH synthesis within neuronal-like cells and for the increased trafficking of GSH from astrocytes to neurons. This effect could potentially relate to the activation of antioxidant response elements leading to the induction of phase II detoxifying enzymes including those involved in GSH synthesis and release. The inability of l-dopa to produce a similar effect when AADC was inhibited or when AADC activity was absent indicates that these effects are relatively specific to dopamine. Additionally dopamine but not l-dopa treatment led in an increase in complex I activity of the respiratory chain in SH-SY5Y cells which may be related to the effect of dopamine on GSH levels.     

Functional relevance of the cannabinoid receptor 2 — heme oxygenase pathway: A novel target for the attenuation of portal hypertension

Aims

In liver cirrhosis, inflammation triggers portal hypertension. Kupffer cells (KC) produce vasoconstrictors upon activation by bacterial constituents. Here, we hypothesize that the anti-inflammatory action of the cannabinoid receptor 2 (CB₂) agonists JWH-133 and GP 1a attenuate portal hypertension.

Main methods

In vivo measurements of portal pressures and non-recirculating liver perfusions were performed in rats 4 weeks after bile duct ligation (BDL). Zymosan (150 μg/ml, isolated liver perfusion) or LPS (4 mg/kg b.w., in vivo) was infused to activate the KC in the absence or presence of JWH-133 (10 mg/kg b.w.), GP 1a (2.5 mg/kg b.w.) or ZnPP IX (1 μM). Isolated KC were treated with Zymosan (0.5 mg/ml) in addition to JWH-133 (5 μM). The thromboxane (TX) B₂ levels in the perfusate and KC media were determined by ELISA. Heme oxygenase-1 (HO-1) and CB₂ were analyzed by Western blot or confocal microscopy.

Key findings

JWH-133 or GP 1a pre-treatment attenuated portal pressures following KC activation in all experimental settings. In parallel, HO-1 expression increased with JWH-133 pre-treatment. However, the inhibition of HO-1 enhanced portal hypertension, indicating the functional role of this novel pathway. In isolated KC, the expression of CB₂ and HO-1 increased with Zymosan, LPS and JWH-133 treatment while TXB₂ production following KC activation was attenuated by JWH-133 pre-treatment.

Significance

JWH-133 or GP 1a treatment attenuates portal hypertension. HO-1 induction by JWH-133 plays a functional role. Therefore, the administration of JWH-133 or GP 1a represents a promising new treatment option for portal hypertension triggered by microbiological products.     

Plumbagin and juglone induce caspase-3-dependent apoptosis involving the mitochondria through ROS generation in human peripheral blood lymphocytes

The phytochemicals plumbagin and juglone have recently been gaining importance because of their various pharmacological activities. In this study, these compounds are shown to induce concentration- and time-dependent toxicity in human peripheral blood lymphocytes via the apoptotic pathway. Flow cytometry data revealed the occurrence of about 28% early apoptotic cells after 6 h exposure to 10 μM plumbagin and 35% late apoptotic cells and about 43% sub-G1 population after 24 h. The cytotoxic effect of plumbagin was at least twofold higher than that of juglone as evidenced by the IC₅₀ value for cytotoxicity. Characteristic apoptotic features such as chromatin condensation and apoptotic body formation were observed through TEM, and membrane blebbing and cell surface smoothening were seen in SEM studies. Generation of ROS was evidenced through the HPLC analysis of superoxide-specific 2-OH-E+ formation. In addition, a decrease in GSH levels parallel to ROS production was observed. Reversal of apoptosis in both NAC- and Tempol-pretreated cells indicates the involvement of both ROS generation and GSH depletion in plumbagin- and juglone-induced apoptosis. The mechanistic pathway involves a decrease in MMP; alterations in the levels of Bcl-2, Bax, and cytosolic cytochrome c; and PARP-1 cleavage subsequent to caspase-3 activation.     

Occurrence of D-serine in rice and characterization of rice serine racemase

Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative amount of D-serine (D/(D + L)%) reached approximately 10% six days after germination. A putative serine racemase gene (serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic DNA of Oryza sativa L. ssp. Japonica cv. Nipponbare. This was expressed as serr in Escherichia coli and its gene product (SerR) was purified to apparent homogeneity. SerR is a homodimer with a subunit molecular mass of 34.5 kDa, and is highly specific for serine. In addition to a serine racemase reaction, SerR catalyzes D- and L-serine dehydratase reactions, for which the specific activities were determined to be 2.73 and 1.42 nkatal/mg, respectively. The optimum temperature and pH were respectively determined for the racemase reaction (35 °C and pH 9.0) and for the dehydratase reaction (35 °C and pH 9.5). SerR was inhibited by PLP-enzyme inhibitors. ATP decreased the serine racemase activity of SerR but increased the serine dehydratase activity. Kinetic analysis showed that Mg²⁺ increases the catalytic efficiency of the serine racemase activity of SerR and decreases that of the serine dehydratase activity. Fluorescence-quenching analysis of the tryptophan residues in SerR indicated that the structure of SerR is distorted by the addition of Mg²⁺, and this structural change probably regulates the two enzymatic activities.