Receptor-independent intracellular radical scavenging activity of an angiotensin II receptor blocker

OBJECTIVES: Angiotensin II plays a crucial role in the induction of oxidative stress and the pathogenesis of cardiovascular and renal diseases, and the beneficial mechanisms of angiotensin II receptor 1 blockers (ARBs) are multifactorial. We investigated the receptor-independent protective role of an ARB using primary-cultured mesangial cells from angiotensin II receptor 1 knockout or wild-type mice and a highly lipophilic ARB, telmisartan. METHODS AND RESULTS: Intracellular reactive oxygen species were estimated using a fluorogenic probe, CM-H2DCFDA. Non-angiotensin II-induced reactive oxygen species production was generated by exposing cells to hydrogen peroxide alone or after treatment with telmisartan. Flow cytometry analysis showed that angiotensin II induced an increase in oxidant production in a dose-dependent manner in wild-type cells, but not in knockout cells. In contrast, hydrogen peroxide induced oxidative stress in both wild-type and knockout cells. Interestingly, telmisartan attenuated the oxidative stress induced by hydrogen peroxide in both cells, suggesting that it acted via a receptor-independent antioxidant effect. Intracellular concentrations of telmisartan were confirmed by high-performance liquid chromatography analysis. Expression of plasminogen activator inhibitor 1, which is stimulated by oxidative stress, was also attenuated by telmisartan in a receptor-independent as well as receptor-dependent manner. Telmisartan did not change expression levels of antioxidative enzymes such as catalase or glutathione peroxidase. Furthermore, the amelioration of oxidative stress by telmisartan did not involve the peroxisome proliferator-activated receptor-gamma pathway. CONCLUSIONS: Telmisartan inhibits intracellular oxidative stress, at least in part, in a receptor-independent manner, possibly owing to its lipophilic and antioxidant structure.     

Superoxide dismutase, catalase and glutathione peroxidase in the spontaneously hypertensive rat kidney: effect of antioxidant-rich diet

BACKGROUND AND OBJECTIVE: Earlier studies have shown increased production of reactive oxygen species (ROS) and upregulation of ROS-generating enzyme, nicotinamide adenine dinucleotide (phosphate) oxidase, in the kidney of spontaneously hypertensive rats (SHR). This study aimed to examine the activities and protein abundance of the main antioxidant enzymes [i.e. superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX)] in the kidney of SHR fed a regular or an antioxidant-rich diet. METHODS: Pregnant SHR and their offspring were fed either a regular diet or an antioxidant-rich diet (alpha-tocopherol, ascorbic acid, zinc and selenium) and observed for 6 months. Wistar-Kyoto (WKY) rats fed a regular or antioxidant-fortified diet served as controls. RESULTS: The untreated SHR showed severe hypertension and significant increases in plasma hydrogen peroxide and renal tissue nitrotyrosine abundance, indicating the presence of oxidative/nitrosative stress. Despite oxidative stress, Cu Zn SOD, CAT and GPX activities were unchanged in the cortex and medulla of untreated SHR. Immunodetectable Mn SOD was reduced in the medulla and elevated in the cortex, whereas, Cu Zn SOD protein was unchanged in the cortex and reduced in the medulla. By contrast, CAT protein abundance was increased in both cortex and medulla while GPX protein was elevated in the cortex and unchanged in the medulla. Comparison of protein abundance and activities of the antioxidant enzymes revealed significant discordance in the untreated SHR. Lifelong antioxidant therapy diminished the severity of hypertension, improved oxidative stress and ameliorated or reversed abnormalities of antioxidant enzyme expressions and activities. By contrast, antioxidant therapy had no effect on the measured parameters in the WKY rat controls. CONCLUSIONS: Oxidative stress in SHR was associated with a lack of coordinate upregulation of the antioxidant enzymes and discordance between their protein abundance and enzymatic activity. These findings suggest an impaired antioxidant defense system and the presence of functionally abnormal enzymes in the SHR kidney. Lifelong antioxidant therapy improved expression, activity and activity-to-mass relationship of the measured enzymes. The latter suggests oxidative and nitrosative modification of these molecules in the SHR kidney.     

Pulmonary function changes in rats with taurocholate‐induced pancreatitis are attenuated by pretreatment with melatonin

Melatonin is a free radical scavenger and broad‐spectrum antioxidant with immunomodulatory effects. We studied the effects of melatonin on changes in lung function, oxidative/nitrosative stress, and inflammatory cell sequestration in an acute pancreatitis (AP)‐associated lung inflammation model. Acute pancreatitis was induced by injection of 5% sodium taurocholate into the pancreatic duct of rats. Animals were randomized into control, AP, and a melatonin pretreatment (10 mg/kg)/AP group. Functional residual capacity (FRC), lung compliance (Cchord), expiratory flow rate at 50% (FEF50), airway resistance index (RI), and peak expiratory flow rate (PEF) were evaluated. White blood cell count (WBC) and hydrogen peroxide, lung lavage fluid WBC, methylguanidine, protein, lactic dehydrogenase (LDH), nitric oxide (NO), and leukotriene B4 (LTB4) levels were determined. Lung wet‐to‐dry weight ratio, peroxynitrite, and inducible nitric oxide synthase (NOS) mRNA and protein were measured. AP induction resulted in reductions in FRC, Cchord, FEF50, and PEF, and increase in RI and lung wet‐to‐dry weight ratio. Blood and lung lavage fluid WBC, lavage fluid LDH, protein, and blood hydrogen peroxide also increased. Levels of hydroxyl radicals, nitric oxide, and LTB4 in lung lavage fluid, inducible NOS mRNA, protein expression, and peroxynitrite in lung tissue also were significantly elevated. Pretreatment with melatonin attenuated obstructive and restrictive ventilatory insufficiency induced by AP. Blood and lavage WBC, lavage LDH and protein, lung edema, oxidative/nitrosative stress, and lipoxygenase pathway derivatives were also significantly attenuated by melatonin. We conclude that melatonin decreases AP‐induced obstructive and restrictive lung function changes via its antioxidant and anti‐inflammatory properties.     

The combined inducible nitric oxide synthase inhibitor and free radical scavenger guanidinoethyldisulfide prevents multiple low-dose streptozotocin-induced diabetes in vivo and interleukin-1beta-induced suppression of islet insulin secretion in vitro

Inhibition of inducible nitric oxide synthase has been shown to be antiinflammatory in a variety of disease states. Type I diabetes is an autoimmune disease resulting from the specific destruction of the insulin-producing pancreatic beta cells. Here we demonstrate that guanidinoethyldisulfide (GED), a combined inducible nitric oxide synthase inhibitor and peroxynitrite/reactive oxygen species scavenger reduces the hyperglycemia and incidence of type I diabetes induced in mice by multiple low-dose streptozotocin treatment. GED treatment (10 and 30 mg/kg/d) protected against the decrease in pancreatic insulin content as well as completely attenuating the increased pancreatic oxidative stress as determined by tissue levels of malondialdehyde. GED treatment also decreased neutrophil infiltration into the pancreas and reduced pancreatic levels of the chemokine MIP-1alpha and the proinflammatory cytokines IL-1 and IL-12. We hypothesize that GED exerts these latter effects by protecting beta cells from destruction reducing autoantigen release and decreasing the autoimmune response. In vitro GED treatment of isolated rat islets of Langerhans protected glucose-stimulated insulin secretion from inhibition by IL-1beta. In conclusion, inhibiting formation and/or scavenging reactive nitrogen or oxygen species with GED protects against development of diabetes in vivo and isolated pancreatic islets of Langerhans from cytokine inhibitory effects in vitro.     

A redox-sensitive peroxiredoxin that is important for longevity has tissue- and stress-specific roles in stress resistance

Oxidative damage caused by reactive oxygen species (ROS) is implicated in many diseases and in aging. Removal of ROS by antioxidant enzymes plays an important part in limiting this damage. For instance, peroxiredoxins (Prx) are conserved, abundant, thioredoxin peroxidase enzymes that function as tumor suppressors. In addition to detoxifying peroxides, studies in single-cell systems have revealed that Prx act as chaperones and redox sensors. However, it is unknown in what manner the different activities of Prx influence stress resistance or longevity in the context of whole animals. Here, we reveal three distinct roles for the 2-Cys Prx, PRDX-2, in the stress resistance of the nematode worm Caenorhabditis elegans. (i) The thioredoxin peroxidase activity of PRDX-2 protects against hydrogen peroxide. (ii) Consistent with a chaperone activity for hyperoxidized PRDX-2, peroxide-induced oxidation of PRDX-2 increases resistance to heat stress. (iii) Unexpectedly, loss of PRDX-2 increases the resistance of C. elegans to some oxidative stress-causing agents, such as arsenite, apparently through a signaling mechanism that increases the levels of other antioxidants and phase II detoxification enzymes. Despite their increased resistance to some forms of oxidative stress, prdx-2 mutants are short-lived. Moreover, intestinal expression of PRDX-2 accounts for its role in detoxification of exogenous peroxide, but not its influence on either arsenite resistance or longevity, suggesting that PRDX-2 may promote longevity and protect against environmental stress through different mechanisms. Together the data reveal that in metazoans Prx act through multiple biochemical activities, and have tissue-specific functions in stress resistance and longevity.     

The role of CHOP messenger RNA expression in the link between oxidative stress and apoptosis

Low expression of antioxidant enzymes makes pancreatic β-cells susceptible to cell damage by oxidative stress. Pancreatic β-cell loss caused by endoplasmic reticulum stress is associated with the onset of diabetes mellitus. The present studies were undertaken to investigate a possible involvement of proapoptotic gene CHOP in pancreatic β-cells damage by oxidative stress. The induction of CHOP messenger RNA and apoptosis were investigated in βHC-9 cells after the oxidative stress by hydrogen peroxide and ribose. Latter was examined after the suppression of CHOP by small interfering RNA. For in vivo study, the pancreatic β-cells were examined in CHOP-knockout (KO) mice after multiple low-dose streptozotocin (MLDS) administration. In βHC-9 cells, both hydrogen peroxide and ribose obviously increased apoptotic cells, accompanied with enhanced CHOP messenger RNA expression. However, the number of apoptotic cells by those stimulations was significantly reduced by the addition of small interfering RNA against CHOP. In vivo study also showed that CHOP-KO mice were less susceptible to diabetes after MLDS administration. Although the oxidative stress marker level was similar to that of MLDS-treated wild type, the pancreatic β-cell area was maintained in CHOP-KO mice. The present studies showed that CHOP should be important in pancreatic β-cell injury by oxidative stress and indicate that CHOP may play a role in the development of pancreatic β-cell damage on the onset of diabetes mellitus.     

Control of hyperglycemia significantly improves oxidative stress profile of pancreatic islets

Pancreatic islets are known to express low levels of antioxidant enzymes compared to other tissues and are therefore vulnerable to oxidative stress. Enhancing antioxidant defense mechanisms in pancreatic islets help them to cope better with oxidative stress. Persistent hyperglycemia under diabetic condition leads to continuous generation of reactive oxygen species, and different tissues exposed to this are oxidatively damaged depending on their antioxidant defense. Since islet cells are very poor in their antioxidant defense, our interest was to assess their antioxidant profile under normal, diabetic, insulin treated diabetic and untreated diabetic condition. On one hand, antioxidant defense was measured in terms of antioxidant enzymes and antioxidant molecules while on the other, damage caused to biomolecules was estimated. Our data demonstrate that oxidative damage to all biomolecules increased in islets cultured from diabetic animals, which enhanced further in islets from untreated diabetic animals. Insulin treatment significantly improved oxidative stress profile of islets indicating that the control of hyperglycemia leads to improvement in oxidative stress profile.     

Control of hyperglycemia significantly improves oxidative stress profile of pancreatic islets

Pancreatic islets are known to express low levels of antioxidant enzymes compared to other tissues and are therefore vulnerable to oxidative stress. Enhancing antioxidant defense mechanisms in pancreatic islets help them to cope better with oxidative stress. Persistent hyperglycemia under diabetic condition leads to continuous generation of reactive oxygen species, and different tissues exposed to this are oxidatively damaged depending on their antioxidant defense. Since islet cells are very poor in their antioxidant defense, our interest was to assess their antioxidant profile under normal, diabetic, insulin treated diabetic and untreated diabetic condition. On one hand, antioxidant defense was measured in terms of antioxidant enzymes and antioxidant molecules while on the other, damage caused to biomolecules was estimated. Our data demonstrate that oxidative damage to all biomolecules increased in islets cultured from diabetic animals, which enhanced further in islets from untreated diabetic animals. Insulin treatment significantly improved oxidative stress profile of islets indicating that the control of hyperglycemia leads to improvement in oxidative stress profile.     

Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actin

Aims/hypothesis

The small G-protein ras-related C3 botulinum toxin substrate 1 (RAC1) plays various roles in mammalian cells, such as in the regulation of cytoskeletal organisation, cell adhesion, migration and morphological changes. The present study examines the effects of RAC1 ablation on pancreatic beta cell function.

Methods

Isolated islets from pancreatic beta cell-specific Rac1-knockout (betaRac1 ^?/?) mice and RAC1 knockdown INS-1 insulinoma cells treated with small interfering RNA were used to investigate insulin secretion and cytoskeletal organisation in pancreatic beta cells.

Results

BetaRac1 ^?/? mice showed decreased glucose-stimulated insulin secretion, while there were no apparent differences in islet morphology. Isolated islets from the mice had blunted insulin secretion in response to high glucose levels. In RAC1 knockdown INS-1 cells, insulin secretion was also decreased in response to high glucose levels, consistent with the phenotype of betaRac1 ^?/? mice. Even under high glucose levels, RAC1 knockdown INS-1 cells remained intact with F-actin, which inhibits the recruitment of the insulin granules, resulting in an inhibition of insulin secretion.

Conclusions/interpretation

In RAC1-deficient pancreatic beta cells, F-actin acts as a barrier for insulin granules and reduces glucose-stimulated insulin secretion.     

Interaction between pro-inflammatory and anti-inflammatory cytokines in insulin-producing cells

Pro-inflammatory cytokines cause beta-cell dysfunction and death. The aim of this study was to investigate the interactions between different pro- and anti-inflammatory cytokines and their effects on apoptotic beta-cell death pathways. Insulin-producing RINm5F cells were exposed to different combinations of cytokines. Gene expression analyses of manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) were performed by real-time RT-PCR. Cell viability was measured by the MTT assay, NFkappaB activation using a SEAP reporter gene assay, protein expression by western blotting and caspase-3 activity using the DEVD cleavage method. IL-1beta, tumour necrosis factor alpha (TNFalpha) and a combination of all three pro-inflammatory cytokines increased while IFNgamma alone did not affect NFkappaB activity and iNOS gene and protein expression. Interestingly, the anti-inflammatory cytokines IL-4, IL-13 and IL-10 decreased IL-1beta-stimulated NFkappaB activation and iNOS expression. IL-1beta, TNFalpha and the pro-inflammatory cytokine combination also increased MnSOD gene and protein expression. But IL-4, IL-13 and IL-10 did not affect MnSOD expression and did not modulate IL-1beta-stimulated MnSOD expression. Caspase-3 activity was increased by IL-1beta and the pro-inflammatory cytokine combination, and to a lesser extent by TNFalpha. In contrast, IFNgamma had no effect on caspase-3 activity. IL-4, IL-13 and IL-10 decreased caspase-3 activity and increased viability of insulin-producing cells treated with pro-inflammatory cytokines. The anti-inflammatory cytokines counteracted the cytotoxic effects of pro-inflammatory cytokines in insulin-producing cells. This was achieved through the reduction of nitrosative stress. Thus, a balance between the anti-inflammatory and the pro-inflammatory cytokines is of crucial importance for the prevention of pancreatic beta-cell destruction.