Susceptibility to rotenone is increased in neurons from parkin null mice and is reduced by minocycline

Parkinson's disease is a neurodegenerative disorder which is in most cases of unknown etiology. Mutations of the Park-2 gene are the most frequent cause of familial parkinsonism and parkin knockout (PK-KO) mice have abnormalities that resemble the clinical syndrome. We investigated the interaction of genetic and environmental factors, treating midbrain neuronal cultures from PK-KO and wild-type (WT) mice with rotenone (ROT). ROT (0.025-0.1 microm) produced a dose-dependent selective reduction of tyrosine hydroxylase-immunoreactive cells and of other neurons, as shown by the immunoreactivity to microtubule-associated protein 2 in PK-KO cultures, suggesting that the toxic effect of ROT involved dopamine and other types of neurons. Neuronal death was mainly apoptotic and suppressible by the caspase inhibitor t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone (Boc-D-FMK). PK-KO cultures were more susceptible to apoptosis induced by low doses of ROT than those from WT. ROT increased the proportion of astroglia and microglia more in PK-KO than in WT cultures. Indomethacin, a cyclo-oxygenase inhibitor, worsened the effects of ROT on tyrosine hydroxylase cells, apoptosis and astroglial (glial fibrillary acidic protein) cells. N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, increased ROT-induced apoptosis but did not change tyrosine hydroxylase-immunoreactive or glial fibrillary acidic protein area. Neither indomethacin nor N-nitro-L-arginine methyl ester had any effect on the reduction by ROT of the mitochondrial potential as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Microglial NADPH oxidase inhibition, however, protected against ROT. The roles of p38 MAPK and extracellular signal-regulated kinase signaling pathways were tested by treatment with SB20358 and PD98059, respectively. These compounds were inactive in ROT-naive cultures but PD98059 slightly increased cellular necrosis, as measured by lactate dehydrogenase levels, caused by ROT, without changing mitochondrial activity. SB20358 increased the mitochondrial failure and lactate dehydrogenase elevation induced by ROT. Minocycline, an inhibitor of microglia, prevented the dropout of tyrosine hydroxylase and apoptosis by ROT; the addition of microglia from PK-KO to WT neuronal cultures increased the sensitivity of dopaminergic neurons to ROT. PK-KO mice were more susceptible than WT to ROT and the combined effects of Park-2 suppression and ROT reproduced the cellular events observed in Parkinson's disease. These events were prevented by minocycline.     

GDNF modulates HO-1 expression in substantia nigra postnatal cell cultures

Heme oxygenase-1 (HO-1) has been strongly highlighted because of its induction in many cell types by toxic stimuli, including oxidative stress. The intense HO-1 immunostaining in the substantia nigra of Parkinson disease (PD) patients suggests its involvement in the pathogenesis of this neurodegenerative disease. In this work we investigated HO-1 expression in rat substantia nigra postnatal cell cultures under conditions mimicking dopamine toxicity and its modulation by glial cell line-derived neurotrophic factor (GDNF), a potent neuroprotective factor for dopaminergic neurons. In neuron–glia cultures, we found that H₂O₂, a product of dopamine metabolism, or l-3,4-dihydroxyphenylalanine (l-DOPA), the dopamine precursor used in the therapy of PD, induced a fast up-regulation of HO-1 mRNA and protein levels, followed by a secondary down-regulation. H₂O₂ and l-DOPA also increased HO-1 expression in astrocyte cultures, but with a delayed time course in H₂O₂-treated cultures. HO-1 expression was decreased in neuron–glia cultures under conditions under which GDNF up-regulation was observed. Because exogenously applied GDNF prevented HO-1 up-regulation in cultures treated with H₂O₂ or l-DOPA, and antibody neutralization of GDNF prevented the secondary HO-1 down-regulation observed in neuron–glia cultures, we propose that GDNF negatively modulates HO-1 expression induced by oxidative stress. To our knowledge, this is the first report showing the modulation of HO-1 expression by GDNF.     

Reactive Oxygen Species-mediated TRPC6 Protein Activation in Vascular Myocytes,a Mechanism for Vasoconstrictor-regulated Vascular Tone

Both TRPC6 and reactive oxygen species (ROS) play an important role in regulating vascular function. However, their interplay has not been explored. The present study examined whether activation of TRPC6 in vascular smooth muscle cells (VSMCs) by ROS was a physiological mechanism for regulating vascular tone by vasoconstrictors. In A7r5 cells, arginine vasopressin (AVP) evoked a striking Ca²⁺ entry response that was significantly attenuated by either knocking down TRPC6 using siRNA or inhibition of NADPH oxidases with apocynin or diphenyleneiodonium. Inhibition of TRPC6 or ROS production also decreased AVP-stimulated membrane currents. In primary cultured aortic VSMCs, catalase and diphenyleneiodonium significantly suppressed AVP- and angiotensin II-induced whole cell currents and Ca²⁺ entry, respectively. In freshly isolated and endothelium-denuded thoracic aortas, hyperforin (an activator of TRPC6), but not its vehicle, induced dose- and time-dependent constriction in TRPC6 wide type (WT) mice. This response was not observed in TRPC6 knock-out (KO) mice. Consistent with the ex vivo study, hyperforin stimulated a robust Ca²⁺ entry in the aortic VSMCs from WT mice but not from KO mice. Phenylephrine induced a dose-dependent contraction of WT aortic segments, and this response was inhibited by catalase. Moreover, H₂O₂ itself evoked Ca²⁺ influx and inward currents in A7r5 cells, and these responses were significantly attenuated by either inhibition of TRPC6 or blocking vesicle trafficking. H₂O₂ also induced inward currents in primary VSMCs from WT but not from TRPC6 KO mice. Additionally, H₂O₂ stimulated a dose-dependent constriction of the aortas from WT mice but not from the vessels of KO mice. Furthermore, TIRFM showed that H₂O₂ triggered membrane trafficking of TRPC6 in A7r5 cells. These results suggest a new signaling pathway of ROS-TRPC6 in controlling vessel contraction by vasoconstrictors.     

Differential effects of l-DOPA on monoamine metabolism, cell survival and glutathione production in midbrain neuronal-enriched cultures from parkin knockout and wild-type mice

l-DOPA is the most effective treatment for Parkinson's disease but in isolated neuronal cultures it is neurotoxic for dopamine (DA) neurones. Experiments in vivo and clinical studies have failed to show toxicity of l-DOPA in animals or patients but that does not exclude the possibility of a toxic effect of l-DOPA on patients with certain genetic risk factors. Mutations of the parkin gene are the most frequent cause of hereditary parkinsonism. Parkin null mice have a mild phenotype that could be modified by different neurotoxins. The aim of this study was to investigate whether the toxic effects of l-DOPA on DA neurones are amplified in parkin null mice. We have measured the effects of l-DOPA on cell viability, tyrosine hydroxylase (TH) expression, DA metabolism and glutathione levels of parkin knockout (PK-KO) midbrain cultures. Neuronal-enriched cultures from PK-KO mice have similar proportions of the different cell types with the exception of a significant increment of microglial cells. l-DOPA (400 microm for 24 h) reduced the number of TH-immunoreactive cells to 50% of baseline and increased twofold the percentage of apoptotic cells in cultures of wild-type (WT) animals. The PK-KO mice, however, are not only resistant to the l-DOPA-induced pro-apoptotic effects but they have an increased number of TH-immunoreactive neurones after treatment with l-DOPA, suggesting that l-DOPA is toxic for neurones of WT mice but not those of parkin null mice. MAPK and phosphatidylinositol-3 kinase signalling pathways are not involved in the differential l-DOPA effects in WT and PK-KO cultures. Intracellular levels of l-DOPA were not different in WT and parkin null mice but the intracellular and extracellular levels of DA and 3-4-dihydroxyphenylacetic acid, however, were significantly increased in parkin null animals. Furthermore, monoamine oxidase activity was significantly increased in parkin null mice, suggesting that these animals have an increased metabolism of DA. The levels of glutathione were further increased in parkin null mice than in controls both with and without treatment with l-DOPA, suggesting that a compensatory mechanism may protect DA neurones from neuronal death. This study opens new avenues for understanding the mechanisms of action of l-DOPA on DA neurones in patients with Park-2 mutations.     

Activation of ERK signalling by Src family kinases (SFKs) in DRG neurons contributes to hydrogen peroxide (H2O2)-induced thermal hyperalgesia

Concomitant generation of reactive oxygen species during tissue inflammation has been recognised as a major factor for the development and the maintenance of hyperalgesia, out of which H₂O₂ is the major player. However, molecular mechanism of H₂O₂ induced hyperalgesia is still obscure. The aim of present study is to analyse the mechanism of H₂O₂-induced hyperalgesia in rats. Intraplantar injection of H₂O₂ (5, 10 and 20 µmoles/paw) induced a significant thermal hyperalgesia in the hind paw, confirmed by increased c-Fos activity in dorsal horn of spinal cord. Onset of hyperalgesia was prior to development of oxidative stress and inflammation. Rapid increase in phosphorylation of extracellular signal regulated kinase (ERK) was observed in neurons of dorsal root ganglia after 20?min of H₂O₂ (10 µmoles/paw) administration, which gradually returned towards normal level within 24?h, following the pattern of thermal hyperalgesia. The expression of TNFR1 followed the same pattern and colocalised with pERK. ERK phosphorylation was observed in NF-200-positive and -negative neurons, indicating the involvement of ERK in C-fibres as well as in A-fibres. Intrathecal preadministration of Src family kinases (SFKs) inhibitor (PP1) and MEK inhibitor (PD98059) prevented H₂O₂ induced augmentation of ERK phosphorylation and thermal hyperalgesia. Pretreatment of protein tyrosine phosphatases (PTPs) inhibitor (sodium orthovanadate) also diminished hyperalgesia, although it further increased ERK phosphorylation. Combination of orthovanadate with PP1 or PD98059 did not exhibit synergistic antihyperalgesic effect. The results demonstrate SFKs-mediated ERK activation and increased TNFR1 expression in nociceptive neurons during H₂O₂ induced hyperalgesia. However, the role of PTPs in hyperalgesic behaviour needs further molecular analysis.     

H2O2-Induced Cell Death in Human Glioma Cells: Role of Lipid Peroxidation and PARP Activation

underlying mechanism of ROS-induced cell injury remains to be defined. This study was undertaken to examine the role of lipid peroxidation and poly (ADP-ribose) polymerase (PARP) activation in H₂O₂-induced cell death in A172 cells, a human glioma cell line. H₂O₂ induced a dose- and time-dependent cell death. The cell death was prevented by thiols (dithiothreitol and glutathione), iron chelators (deferoxamine and phenanthroline), H₂O₂ scavengers (catalase and pyruvate), and a hydroxyl radical scavenger (dimethylthiourea). Antioxidants N,N-diphenyl-p-phenylenediamine (DPPD) and Trolox had no effect on the H₂O₂-induced cell death. Lipid peroxidation did not increase in human glioma cells exposed to H₂O₂. The PARP inhibitor 3-aminobenzamide prevented the cell death induced by H₂O₂. The PARP activity was increased by H₂O₂ and the H₂O₂ effect was prevented by 3-aminobenzamide, dithiothreitol, and phenanthroline. The ATP depletion induced by H₂O₂ was prevented by catalase, dithiothreitol, phenanthroline, and 3-aminobenzamide, but not by DPPD. These results indicate that the H₂O₂-induced cell death is mediated by PARP activation but not by lipid peroxidation in human glioma cells.     

Inactivation of neuronal forebrain A2A receptors protects dopaminergic neurons in a mouse model of Parkinson’s disease

Adenosine A_2A receptors antagonists produce neuroprotective effects in animal models of Parkinson’s disease (PD). As neuroinflammation is involved in PD pathogenesis, both neuronal and glial A_2A receptors might participate to neuroprotection. We employed complementary pharmacologic and genetic approaches to A_2A receptor inactivation, in a multiple MPTP mouse model of PD, to investigate the cellular basis of neuroprotection by A_2A antagonism. MPTP·HCl (20 mg/kg daily for 4 days) was administered in mice treated with the A_2A antagonist SCH58261, or in conditional knockout mice lacking A_2A receptors on forebrain neurons (fbnA_2AKO mice). MPTP‐induced partial loss of dopamine neurons in substantia nigra pars compacta (SNc) and striatum (Str), associated with increased astroglial and microglial immunoreactivity in these areas. Astroglia was similarly activated 1, 3, and 7 days after MPTP administration, whereas maximal microglial reactivity was detected on day 1, returning to baseline 7 days after MPTP administration. SCH58261 attenuated dopamine cell loss and gliosis in SNc and Str. Selective depletion of A_2A receptors in fbnA_2AKO mice completely prevented MPTP‐induced dopamine neuron degeneration and gliosis in SNc, and partially counteracted gliosis in Str. Results provide evidence of a primary role played by neuronal A_2A receptors in neuroprotective effects of A_2A antagonists in a multiple MPTP injections model of PD. With the symptomatic antiparkinsonian potential of several A_2A receptor antagonists being pursued in clinical trials, this study adds to the rationale for broader clinical benefit and use of these drugs early in the treatment of PD.     

Possible Involvement of Mitogen-Activated Protein Kinase in Phospholipase D Activation Induced by H2O2, but Not by Carbachol, in Rat Pheochromocytoma PC12 Cells

Abstract: We have previously reported that hydrogen peroxide (H₂O₂) induced a considerable increase of phospholipase D (PLD) activity and phosphorylation of mitogen-activated protein (MAP) kinase in PC12 cells. H₂O₂-induced PLD activation and MAP kinase phosphorylation were dose-dependently inhibited by a specific MAP kinase kinase inhibitor, PD 098059. In contrast, carbachol-mediated PLD activation was not inhibited by the PD 098059 pretreatment whereas MAP kinase phosphorylation was prevented. These findings indicated that MAP kinase is implicated in the PLD activation induced by H₂O₂, but not by carbachol. In the present study, H₂O₂ also caused a marked release of oleic acid (OA) from membrane phospholipids in PC12 cells. As we have previously shown that OA stimulates PLD activity in PC12 cells, the mechanism of H₂O₂-induced fatty acid liberation and its relation to PLD activation were investigated. Pretreatment of the cells with methylarachidonyl fluorophosphonate (MAFP), a phospholipase A₂ (PLA₂) inhibitor, almost completely prevented the release of [³H]OA by H₂O₂ treatment. From the preferential release of OA and sensitivity to other PLA₂ inhibitors, the involvement of a Ca²⁺-independent cytosolic PLA₂-type enzyme was suggested. In contrast, to OA release, MAFP did not inhibit PLD activation by H₂O₂. The inhibitory profile of the OA release by PD 098059 did not show any correlation with that of MAP kinase. These results lead us to suggest that H₂O₂-induced PLD activation may be mediated by MAP kinase and also that H₂O₂-mediated OA release, which would be catalyzed by a Ca²⁺-independent cytosolic PLA₂-like enzyme, is not linked to the PLD activation in PC12 cells.     

Adiponectin Modulates Oxidative Stress-Induced Autophagy in Cardiomyocytes

Diastolic heart failure (HF) i.e., “HF with preserved ejection fraction” (HF-preserved EF) accounts for up to 50% of all HF presentations; however there have been no therapeutic advances. This stems in part from an incomplete understanding about HF-preserved EF. Hypertension is the major cause of HF-preserved EF whilst HF-preserved EF is also highly associated with obesity. Similarly, excessive reactive oxygen species (ROS), i.e., oxidative stress occurs in hypertension and obesity, sensitizing the heart to the renin-angiotensin-aldosterone system, inducing autophagic type-II programmed cell death and accelerating the propensity to adverse cardiac remodeling, diastolic dysfunction and HF. Adiponectin (APN), an adipokine, mediates cardioprotective actions but it is unknown if APN modulates cardiomyocyte autophagy. We tested the hypothesis that APN ameliorates oxidative stress-induced autophagy in cardiomyocytes. Isolated adult rat ventricular myocytes were pretreated with recombinant APN (30µg/mL) followed by 1mM hydrogen peroxide (H₂O₂) exposure. Wild type (WT) and APN-deficient (APN-KO) mice were infused with angiotensin (Ang)-II (3.2mg/kg/d) for 14 days to induced oxidative stress. Autophagy-related proteins, mTOR, AMPK and ERK expression were measured. H₂O₂ induced LC3I to LC3II conversion by a factor of 3.4±1.0 which was abrogated by pre-treatment with APN by 44.5±10%. However, neither H₂O₂ nor APN affected ATG5, ATG7, or Beclin-1 expression. H₂O₂ increased phospho-AMPK by 49±6.0%, whilst pretreatment with APN decreased phospho-AMPK by 26±4%. H₂O₂ decreased phospho-mTOR by 36±13%, which was restored by APN. ERK inhibition demonstrated that the ERK-mTOR pathway is involved in H₂O₂-induced autophagy. Chronic Ang-II infusion significantly increased myocardial LC3II/I protein expression ratio in APN-KO vs. WT mice. These data suggest that excessive ROS caused cardiomyocyte autophagy which was ameliorated by APN by inhibiting an H₂O₂-induced AMPK/mTOR/ERK-dependent mechanism. These findings demonstrate the anti-oxidant potential of APN in oxidative stress-associated cardiovascular diseases, such as hypertension-induced HF-preserved EF.     

Protection of erythrocytes by the macrophage synthesized antioxidant 7,8 dihydroneopterin

Neopterin and the reduced form, 7,8-dihydroneopterin (78NP), are pteridines released from macrophages when stimulated with γ-interferon in vivo. The role of 78NP in inflammatory response is unknown though neopterin has been used clinically as a marker of immune cell activation, due to its very fluorescent nature. Using red blood cells as a cellular model, we demonstrated that micromolar concentrations of 78NP can inhibit or reduce red blood cell haemolysis induced by 2,2′-azobis(amidinopropane)dihydrochloride (AAPH), hydrogen peroxide, or hypochlorite. One hundred μM 78NP prevented HOCl haemolysis using a high HOCl concentration of 5 μmole HOCl/10⁷ RBC. Fifty μM 78NP reduced the haemolysis caused by 2 mM hydrogen peroxide by 39% while the same 78NP concentration completely inhibited haemolysis induced by 2.5 mM AAPH. Lipid peroxidation levels measured as HPLC-TBARS were not affected by addition of 78NP. There was no correlation between lipid oxidation and cell haemolysis suggesting that lipid peroxidation is not essential for haemolysis. Conjugated diene measurements taken after 6 and 12 hour exposure to hydrogen peroxide support the TBARS data. Gel electrophoresis of cell membrane proteins indicated 78NP might inhibit protein damage. Using dityrosine as an indicator of protein damage, we demonstrated 200 μM 78NP reduced dityrosine formation in H₂O₂/Fe⁺⁺ treated red blood cell ghosts by 30%. HPLC analysis demonstrated a direct reaction between 78NP and all three oxidants. Two mM hydrogen peroxide oxidised 119 nM of 78NP per min while 1 mM AAPH only oxidised 50 nM 78NP/min suggesting that 78NP inhibition of haemolysis is not due to 78NP scavenging the primary initiating reactants. In contrast, the reaction between HOCl and 78NP was near instant. AAPH and hydrogen peroxide oxidised 78NP to 7,8-dihydroxanthopterin while hypochlorite oxidation produced neopterin. The cellular antioxidant properties of 78NP suggest it may have a role in protecting immune cells from free radical damage during inflammation.     

Nobiletin protects human retinal pigment epithelial cells from hydrogen peroxide–induced oxidative damage

Nobiletin (3′,4′,5,6,7,8‐hexamethoxyflavone), a dietary polymethoxylated flavonoid found in Citrus fruits, has been reported to have antioxidant effect. However, the effect of nobiletin on human retinal pigment epithelium (RPE) cells induced by hydrogen peroxide (H₂O₂) is still unclear. Therefore, we investigated the protective effect of nobiletin against H₂O₂‐induced cell death in RPE cells. Our results demonstrated that nobiletin significantly increased cell viability from oxidative stress. Nobiletin inhibited H₂O₂‐induced ROS production and caspase‐3/7 activity in ARPE‐19 cells. Furthermore, nobiletin significantly increased Akt phosphorylation in ARPE‐19 cells exposed to H₂O₂. Meanwhile, LY294002, an inhibitor of PI3K/Akt, abolished the protective effect of nobiletin against H₂O₂‐induced decreased cell viability and increased caspase‐3/7 activity in ARPE‐19 cells. In summary, these data show that nobiletin protects RPE cells against oxidative stress through activation of the Akt‐signaling pathway. Thus, nobiletin should be an oxidant that attenuates the development of age‐related macular degeneration.     

Protection against oxidative stress by vitamin D in cone cells

Photoreceptor degeneration (PD) refers to a group of heterogeneous outer retinal dystrophies characterized by the death of photoreceptors. Both oxidative stress and inflammation are involved in the pathogenesis of PD. We investigate whether vitamin D has a potential for the treatment of PD by evaluating the anti‐oxidative stress and anti‐inflammatory properties of the active form of vitamin D₃, 1,α, 25‐dihydroxyvitamin D₃, in a mouse cone cell line, 661W. Mouse cone cells were treated with H₂O₂ or a mixture of H₂O₂ and vitamin D; cell viability was determined. The production of reactive oxygen species (ROS) in treated and untreated cells was measured. The expression of key anti‐oxidative stress and inflammatory genes in treated and untreated cells was determined. Treatment with vitamin D significantly increased cell viability and decreased ROS production in 661W cells under oxidative stress induced by H₂O₂. H₂O₂ treatment in 661W cells can significantly down‐regulate the expression of antioxidant genes and up‐regulate the expression of neurotoxic cytokines. Vitamin D treatment significantly reversed these effects and restored the expression of antioxidant genes. Vitamin D treatment also can block H₂O₂ induced oxidative damages. The data suggested that vitamin D may offer a therapeutic potential for patients with PD.     

Redox Signal-mediated Enhancement of the Temperature Sensitivity of Transient Receptor Potential Melastatin 2 (TRPM2) Elevates Glucose-induced Insulin Secretion from Pancreatic Islets

Transient receptor potential melastatin 2 (TRPM2) is a thermosensitive Ca²⁺-permeable cation channel expressed by pancreatic β cells where channel function is constantly affected by body temperature. We focused on the physiological functions of redox signal-mediated TRPM2 activity at body temperature. H₂O₂, an important molecule in redox signaling, reduced the temperature threshold for TRPM2 activation in pancreatic β cells of WT mice but not in TRPM2KO cells. TRPM2-mediated [Ca²⁺]_i increases were likely caused by Ca²⁺ influx through the plasma membrane because the responses were abolished in the absence of extracellular Ca²⁺. In addition, TRPM2 activation downstream from the redox signal plus glucose stimulation enhanced glucose-induced insulin secretion. H₂O₂ application at 37 °C induced [Ca²⁺]_i increases not only in WT but also in TRPM2KO β cells. This was likely due to the effect of H₂O₂ on K_ATP channel activity. However, the N-acetylcysteine-sensitive fraction of insulin secretion by WT islets was increased by temperature elevation, and this temperature-dependent enhancement was diminished significantly in TRPM2KO islets. These data suggest that endogenous redox signals in pancreatic β cells elevate insulin secretion via TRPM2 sensitization and activity at body temperature. The results in this study could provide new therapeutic approaches for the regulation of diabetic conditions by focusing on the physiological function of TRPM2 and redox signals.     

MEK/ERK pathway mediates cytoprotection of salvianolic acid B against oxidative stress‐induced apoptosis in rat bone marrow stem cells

To improve the survival and/or differentiation of grafted BMSCs (bone marrow stem cells) represents one of the challenges for the promising cell‐based therapy. Considerable reports have implicated Sal B (salvianolic acid B), a potent aqueous extract of Salvia miltiorrhiza, in enhancing the survival of cells under various conditions. In this study, we investigated the effect of Sal B on H₂O₂‐induced apoptosis in rat BMSCs, focusing on the survival signalling pathways. Results indicated that the MEK [MAPK (mitogen‐activated protein kinase)/ERK (extracellular‐signal‐regulated kinase) kinase] inhibitor (PD98059) and 10 μM Sal B remarkably prevented BMSCs from H₂O₂‐induced apoptosis through attenuating caspase‐3 activation, which is accompanied by the significant up‐regulation of Bcl‐2. In addition, the ROS (reactive oxygen species) accumulation was also reduced after Sal B treatment. Furthermore, Sal B inhibited the ERK1/2 phosphorylations stimulated by H₂O₂. Taken together, our results showed that H₂O₂‐induced apoptosis in BMSCs via the ROS/MEK/ERK1/2 pathway and Sal B may exert its cytoprotection through mediating the pathway.     

Nox2 Mediates Skeletal Muscle Insulin Resistance Induced by a High Fat Diet

Inflammation and oxidative stress through the production of reactive oxygen species (ROS) are consistently associated with metabolic syndrome/type 2 diabetes. Although the role of Nox2, a major ROS-generating enzyme, is well described in host defense and inflammation, little is known about its potential role in insulin resistance in skeletal muscle. Insulin resistance induced by a high fat diet was mitigated in Nox2-null mice compared with wild-type mice after 3 or 9 months on the diet. High fat feeding increased Nox2 expression, superoxide production, and impaired insulin signaling in skeletal muscle tissue of wild-type mice but not in Nox2-null mice. Exposure of C2C12 cultured myotubes to either high glucose concentration, palmitate, or H₂O₂ decreases insulin-induced Akt phosphorylation and glucose uptake. Pretreatment with catalase abrogated these effects, indicating a key role for H₂O₂ in mediating insulin resistance. Down-regulation of Nox2 in C2C12 cells by shRNA prevented insulin resistance induced by high glucose or palmitate but not H₂O₂. These data indicate that increased production of ROS in insulin resistance induced by high glucose in skeletal muscle cells is a consequence of Nox2 activation. This is the first report to show that Nox2 is a key mediator of insulin resistance in skeletal muscle.     

Enhanced chilling tolerance at the booting stage in rice by transgenic overexpression of the ascorbate peroxidase gene, OsAPXa

Low temperatures during the booting stage reduce rice yields by causing cold-induced male sterility. To determine whether antioxidant capacity affects the ability of rice plants to withstand chilling at the booting stage, we produced transgenic rice plants that overexpress OsAPXa and have increased APX activity. The effect of increased APX activity on the levels of H₂O₂ and lipid peroxidation were determined by measuring H₂O₂ levels and malondialdehyde (MDA) contents in spikelets during cold treatments at the booting stage. The levels of H₂O₂ and the MDA content increased by 1.5-fold and twofold, respectively in WT plants subjected to a 12°C treatment for 6 days. In contrast, transgenic lines showed small changes in H₂O₂ levels and MDA content under cold stress, and H₂O₂ levels and MDA content were significantly lower than in WT plants. APX activity showed negative correlations with levels of H₂O₂ and MDA content, which increased during cold treatment. Cold tolerance at the booting stage in transgenic lines and WT plants was evaluated. Spikelet fertility was significantly higher in transgenic lines than in WT plants after a 12°C treatment for 6 days. These results indicate that higher APX activity enhances H₂O₂-scavenging capacity and protects spikelets from lipid peroxidation, thereby increasing spikelet fertility under cold stress.     

Hepatocyte or serum albumin protein carbonylation by oxidized fructose metabolites: Glyceraldehyde or glycolaldehyde as endogenous toxins?

Excessive sugar intake in animal models may cause tissue damage associated with oxidative and carbonyl stress cytotoxicity as well as inflammation. Fructose became a 100-fold more cytotoxic if hepatocytes were exposed to a non-toxic infusion of H₂O₂ so as to simulate H₂O₂ released by Kupffer cells or infiltrating immune cells. In order to determine the molecular mechanisms involved, protein carbonylation of fructose and its metabolites were determined using the 2,4-dinitrophenylhydrazine method. In a cell-free system, fructose was found to carbonylate bovine serum albumin (BSA) only if low concentrations of FeII/H₂O₂ were added. Protein carbonylation by the fructose metabolites glyceraldehyde or glycolaldehyde was also markedly increased by FeII/H₂O₂. The protein carbonylation may be attributed to glyoxal formation by hydroxyl radicals as the glyoxal trapping agent aminoguanidine or hydroxyl radical scavengers prevented protein carbonylation. Glyoxal was also much more effective than other carbonyls at causing protein carbonylation. When BSA was replaced by isolated rat hepatocytes, fructose metabolite glyceraldehyde in the presence of non-toxic 2 μM FeII:8-hydroxyquinoline (HQ) and a H₂O₂ generating system (glucose/glucose oxidase) markedly increased cytotoxicity, protein carbonylation and reactive oxygen species (ROS)/H₂O₂ formation. Furthermore this was prevented by hydroxyl radical scavengers or aminoguanidine, a glyoxal scavenger. CuII: 8-hydroxyquinoline increased H₂O₂ induced hepatocyte protein carbonylation less but was prevented by aminoguanidine. However, cytotoxicity and protein carbonylation induced by glyceraldehyde/CuII:HQ/H₂O₂ were not affected by hydroxyl radical scavengers. Although fatty liver induced by an excessive sugar diet in animal models has been proposed as the first hit for non-alcoholic steatohepatitis (NASH) we propose that oxidative stress induced by the oxidation of fructose or fructose metabolites catalysed by Fenton FeII/H₂O₂ could be a ‘second hit’. A perpetual cycle of oxidative stress in hepatocytes could lead to cytotoxicity and contribute to NASH development.     

Enhanced Mitochondrial Superoxide Scavenging Does Not Improve Muscle Insulin Action in the High Fat-Fed Mouse

Improving mitochondrial oxidant scavenging may be a viable strategy for the treatment of insulin resistance and diabetes. Mice overexpressing the mitochondrial matrix isoform of superoxide dismutase (sod2^tg mice) and/or transgenically expressing catalase within the mitochondrial matrix (mcat^tg mice) have increased scavenging of O₂˙ˉ and H₂O₂, respectively. Furthermore, muscle insulin action is partially preserved in high fat (HF)-fed mcat^tg mice. The goal of the current study was to test the hypothesis that increased O₂˙ˉ scavenging alone or in combination with increased H₂O₂ scavenging (mtAO mice) enhances in vivo muscle insulin action in the HF-fed mouse. Insulin action was examined in conscious, unrestrained and unstressed wild type (WT), sod2^tg, mcat^tg and mtAO mice using hyperinsulinemic-euglycemic clamps (insulin clamps) combined with radioactive glucose tracers following sixteen weeks of normal chow or HF (60% calories from fat) feeding. Glucose infusion rates, whole body glucose disappearance, and muscle glucose uptake during the insulin clamp were similar in chow- and HF-fed WT and sod2^tg mice. Consistent with our previous work, HF-fed mcat^tg mice had improved muscle insulin action, however, an additive effect was not seen in mtAO mice. Insulin-stimulated Akt phosphorylation in muscle from clamped mice was consistent with glucose flux measurements. These results demonstrate that increased O₂˙ˉ scavenging does not improve muscle insulin action in the HF-fed mouse alone or when coupled to increased H₂O₂ scavenging.     

CD36 and Fyn Kinase Mediate Malaria-Induced Lung Endothelial Barrier Dysfunction in Mice Infected with Plasmodium berghei

Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT) and CD36−/− mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σ_alb) compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36−/− mice had near normal fluid conductance but a similar reduction in σ_alb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS). To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC) from WT and CD36−/− mice were exposed to H₂O₂. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER) from H₂O₂ indicating loss of barrier function, CD36−/− MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36−/− endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36−/− lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn−/− mice, which were also protected from H₂O₂- and malaria-induced lung endothelial leak, respectively. Our results demonstrate that CD36 and Fyn kinase are critical mediators of the increased lung endothelial fluid conductance caused by malaria infection.     

Overexpression of a peroxiredoxin gene from <Emphasis Type="Italic">Tamarix hispida</Emphasis>, <Emphasis Type="Italic">ThPrx1</Emphasis>, confers tolerance to oxidative stress in yeast and Arabidopsis

Peroxiredoxins (Prxs) are ubiquitous thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative Type II Prx (ThPrx1) was identified and characterized from Tamarix hispida. The expression of ThPrx1 is highly induced in response to hydrogen peroxide (H₂O₂) and methyl viologen (MV) stresses. When expressed ectopically, ThPrx1 showed enhanced tolerance against oxidative stress in yeast and Arabidopsis. In addition, transgenic Arabidopsis plants overexpressing ThPrx1 displayed improved seedling survival rates and increased root growth and fresh weight gain under H₂O₂ and MV treatments. Moreover, transgenic Arabidopsis plants showed decreased accumulation of H₂O₂, superoxide (O₂^??) and malondialdehyde (MDA), increased superoxide dismutase (SOD) activity compared to wild-type (WT) plants under oxidative stress. Moreover, transgenic plants maintained higher photosynthesis efficiency and lower electrolyte leakage rates than that of WT plants under stress conditions. These results clearly indicated that ThPrx1 plays an important role in cellular redox homeostasis under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress.