cytochrome-c-t and Hyperglycemia

Endothelial dysfunction is a driving force during the development and progression of cardiovascular complications in diabetes. Targeting endothelial injury may be an attractive avenue for the management of diabetic vascular disorders. Chicoric acid is reported to confer antioxidant and anti-inflammatory properties in various diseases including diabetes. However, the role and mechanism of chicoric acid in hyperglycemia-induced endothelial damage are not well understood.. In the present study, human umbilical vein endothelial cells (HUVECs) were incubated with high glucose/high fat (HG + HF) to induce endothelial cell injury.. We found that exposure of HUVECs to HG + HF medium promoted the release of cytochrome c (cytc) from mitochondrion into the cytoplasm, stimulated the cleavage of caspase-3 and poly ADP-ribose-polymerase (PARP), then inducing cell apoptosis, the effects that were prevented by administration of chicoric acid. Besides, we found that chicoric acid diminished HG + HF-induced phosphorylation and degradation of IκBα, and subsequent p65 NFκB nuclear translocation, thereby contributing to its anti-inflammatory effects in HUVECs. We also confirmed that chicoric acid mitigated oxidative/nitrative stresses under HG + HF conditions. Studies aimed at exploring the underlying mechanisms found that chicoric acid activated the AMP-activated protein kinase (AMPK) signaling pathway to attenuate HG + HF-triggered injury in HUVECs as AMPK inhibitor Compound C or silencing of AMPKα1 abolished the beneficial effects of chicoric acid in HUVECs.. Collectively, chicoric acid is likely protected against diabetes-induced endothelial dysfunction by activation of the AMPK signaling pathway. Chicoric acid could be a novel candidate for the treatment of the diabetes-associated vascular endothelial injury.

Topics: AMP-Activated Protein Kinases; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Caffeic Acids; Cell Survival; Cytochromes c; Cytoplasm; Endothelium, Vascular; Glucose; Human Umbilical Vein Endothelial Cells; Humans; Hyperglycemia; Inflammation; L-Lactate Dehydrogenase; Mitochondria; Nitrosative Stress; Oxidative Stress; Poly(ADP-ribose) Polymerases; RNA, Small Interfering; Succinates

Redox imbalance due to hyperglycemia is a causative factor for an increased generation of reactive oxygen species (ROS) that leads to mitochondrial dysfunction and the release of cytochrome-c. The aim of the present study is to elucidate the functional role of oxidative stress (OS) in the induction of apoptosis in H9c2 cells in the hyperglycemic state through glucose transporter-4 (GLUT-4) regulation and antioxidant status. H9c2 cells were incubated with 15, 24, and 33 mM glucose for 24, 48, and 72 hr to induce hyperglycemic stress. Hyperglycemic episodes have significantly influenced GLUT-4 mRNA regulation, depleted glutathione (GSH) and its associated enzymes, reduced cellular antioxidant enzymes (AOEs), caused nuclear condensation, and induced apoptosis by activating caspase-9 and 3 and annexin V binding in a concentration and duration-dependent manner. Trolox pretreatment significantly enhanced the GLUT-4 mRNA and antioxidant defense mechanism, suppressed nuclear condensation, and prevented cytochrome-c release, thereby reducing mitochondrial-dependent apoptosis. The present study shows that the toxic effect of high glucose is significantly regulated and that OS induction can be prevented through a water-soluble vitamin E analog "Trolox" treatment.

Topics: Animals; Antioxidants; Apoptosis; Chromans; Cytochromes c; Enzymes; Glucose; Glucose Transporter Type 4; Glutathione; Hyperglycemia; Myocytes, Cardiac; Oxidative Stress; Rats

To assess the effects of Aleglitazar on hyperglycaemia-induced apoptosis.. We incubated human cardiomyocytes, cardiomyocytes from cardiac-specific peroxisome proliferator-activated receptor-γ knockout or wild-type mice in normoglycaemic or hyperglycaemic conditions (glucose 25 mM). Cells were treated with different concentrations of Aleglitazar for 48 h. We measured viability, apoptosis, caspase-3 activity, cytochrome-C release, total antioxidant capacity and reactive oxygen species formation in the treated cardiomyocytes. Human cardiomyocytes were transfected with short interfering RNA against peroxisome proliferator-activated receptor-α or peroxisome proliferator-activated receptor-γ.. Aleglitazar attenuated hyperglycaemia-induced apoptosis, caspase-3 activity and cytochrome-C release and increased viability in human cardiomyocyte, cardiomyocytes from cardiac-specific peroxisome proliferator-activated receptor-γ knockout and wild-type mice. Hyperglycaemia reduced the antioxidant capacity and Aleglitazar significantly blunted this effect. Hyperglycaemia-induced reactive oxygen species production was attenuated by Aleglitazar in both human cardiomyocyte and wild-type mice cardiomyocytes. Aleglitazar improved cell viability in cells exposed to hyperglycaemia. The protective effect was partially blocked by short interfering RNA against peroxisome proliferator-activated receptor-α alone and short interfering RNA against peroxisome proliferator-activated receptor-γ alone and completely blocked by short interfering RNA to both peroxisome proliferator-activated receptor-α and peroxisome proliferator-activated receptor-γ.. Aleglitazar protects cardiomyocytes against hyperglycaemia-induced apoptosis by combined activation of both peroxisome proliferator-activated receptor-α and peroxisome proliferator-activated receptor-γ in a short-term vitro model.

Topics: Animals; Apoptosis; Caspase 3; Cell Survival; Cells, Cultured; Cytochromes c; Cytoprotection; Dose-Response Relationship, Drug; Genotype; Hyperglycemia; Hypoglycemic Agents; Mice, Knockout; Myocytes, Cardiac; Oxazoles; Oxidative Stress; Phenotype; PPAR alpha; PPAR gamma; RNA Interference; Signal Transduction; Thiophenes; Time Factors; Transfection

Diabetes is a major driver of cardiovascular disease, but the underlying mechanisms remain elusive. Prolyl-isomerase Pin1 recognizes specific peptide bonds and modulates function of proteins altering cellular homoeostasis. The present study investigates Pin1 role in diabetes-induced vascular disease.. In human aortic endothelial cells (HAECs) exposed to high glucose, up-regulation of Pin1-induced mitochondrial translocation of pro-oxidant adaptor p66(Shc) and subsequent organelle disruption. In this setting, Pin1 recognizes Ser-116 inhibitory phosphorylation of endothelial nitric oxide synthase (eNOS) leading to eNOS-caveolin-1 interaction and reduced NO availability. Pin1 also mediates hyperglycaemia-induced nuclear translocation of NF-κB p65, triggering VCAM-1, ICAM-1, and MCP-1 expression. Indeed, gene silencing of Pin1 in HAECs suppressed p66(Shc)-dependent ROS production, restored NO release and blunted NF-kB p65 nuclear translocation. Consistently, diabetic Pin1(-/-) mice were protected against mitochondrial oxidative stress, endothelial dysfunction, and vascular inflammation. Increased expression and activity of Pin1 were also found in peripheral blood monocytes isolated from diabetic patients when compared with age-matched healthy controls. Interestingly, enough, Pin1 up-regulation was associated with impaired flow-mediated dilation, increased urinary 8-iso-prostaglandin F2α and plasma levels of adhesion molecules.. Pin1 drives diabetic vascular disease by causing mitochondrial oxidative stress, eNOS dysregulation as well as NF-kB-induced inflammation. These findings provide molecular insights for novel mechanism-based therapeutic strategies in patients with diabetes.

Topics: Analysis of Variance; Animals; Aorta; Case-Control Studies; Cells, Cultured; Chemokine CCL2; Cytochromes c; Diabetic Angiopathies; Endothelial Cells; Endothelium, Vascular; Gene Knockdown Techniques; Glucose; Humans; Hyperglycemia; Intercellular Adhesion Molecule-1; Male; Mice, Inbred C57BL; Mitochondrial Diseases; NF-kappa B; NIMA-Interacting Peptidylprolyl Isomerase; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Oxidative Stress; Peptidylprolyl Isomerase; Reactive Oxygen Species; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; Up-Regulation; Vascular Cell Adhesion Molecule-1; Vasculitis

The aim of this study was to determine the in vitro effect of glutamine and insulin on apoptosis, mitochondrial membrane potential, cell permeability, and inflammatory cytokines in hyperglycemic umbilical vein endothelial cells.. Human umbilical vein endothelial cells were grown and subjected to glutamine and insulin to examine the effects of these agents on the hyperglycemic state. Mitochondrial function and the production of inflammatory cytokines were assessed using fluorescence analysis and multiple cytotoxicity assays. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay.. Glutamine maintains the integrity of the mitochondria by reducing the cell permeability and cytochrome c levels and increasing the mitochondrial membrane potential. The cytochrome c level was significantly (p<0.005) reduced when the cells were treated with glutamine. An apoptosis assay revealed significantly reduced apoptosis (p<0.005) in the glutamine-treated cells. Moreover, glutamine alone or in combination with insulin modulated inflammatory cytokine levels. Interleukin-10, interleukin-6, and vascular endothelial growth factor were up-regulated while tumor necrosis factor-α was down-regulated after treatment with glutamine.. Glutamine, either alone or in combination with insulin, can positively modulate the mitochondrial stress and cell permeability in umbilical vein endothelial cells. Glutamine regulates the expression of inflammatory cytokines and maintains the balance of the mitochondria in a cytoprotective manner.

Topics: Apoptosis; Cell Membrane Permeability; Cells, Cultured; Cytochromes c; Cytokines; Drug Combinations; Glutamine; Human Umbilical Vein Endothelial Cells; Humans; Hyperglycemia; Hypoglycemic Agents; Insulin; Mitochondria; Oxidative Stress

Since Thymus caramanicus Jalas is used as a folk medicine for the treatment of rheumatism, skin disorders, bacterial infections and diabetes and it contain antioxidant agents, we decided to investigate the possible effects of Thymus caramanicus Jalas (TCJ) extract on in vitro and in vivo models of diabetic neuropathy.. The high glucose-induced cell injury in Pheochromocytoma (PC12) cells and streptozotocin-induced diabetic rats were used. Tail-flick and rotarod treadmill assessments were used to determine nociceptive threshold and motor coordination. Cell viability was determined by MTT assay test. Western blotting was performed to measurement of apoptosis markers.. The data showed that elevation of glucose consecutively increases functional cell injury and apoptosis. Furthermore, diabetic rats developed thermal hyperalgesia and motor deficit. Activated caspase 3, cytochrome c release and Bax/Bcl-2 ratio were significantly increased in high glucose-treated PC12 cells and in spinal cord of diabetic animals. TCJ extract (60 and 80 µg/ml) attenuates high glucose-induced PC12 cells damage and apoptosis. In diabetic animals, TCJ extract at daily doses of 100 and 150 mg/kg ameliorated hyperalgesia and suppressed spinal apoptosis.. The data indicate that TCJ extract has neuroprotective effects against high glucose-induced neural damage. These protective effects are mediated, at least in part, through attenuation of neural apoptosis and suggest therapeutic potential of TCJ extract in amelioration of diabetic neuropathy.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blood Glucose; Caspase 3; Cell Survival; Cytochromes c; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Hyperglycemia; Male; Neuralgia; Pain Measurement; PC12 Cells; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Thymus Plant

Previous studies on isolated islets have demonstrated tight coupling between calcium (Ca(2+)) influx and oxygen consumption rate (OCR) that is correlated with insulin secretion rate (ISR). To explain these observations, we have proposed a mechanism whereby the activation of a highly energetic process (Ca(2+)/metabolic coupling process [CMCP]) by Ca(2+) mediates the stimulation of ISR. The aim of the study was to test whether impairment of the CMCP could play a role in the development of type 2 diabetes.. Glucose- and Ca(2+)-mediated changes in OCR and ISR in isolated islets were compared with the time course of changes of plasma insulin concentrations observed during the progression to hyperglycaemia in a rat model of type-2 diabetes (the University of California at Davis type 2 diabetes mellitus [UCD-T2DM] rat). Islets were isolated from UCD-T2DM rats before, 1 week, and 3 weeks after the onset of hyperglycaemia.. Glucose stimulation of cytosolic Ca(2+) and OCR was similar for islets harvested before and 1 week after the onset of hyperglycaemia. In contrast, a loss of decrement in islet OCR and ISR in response to Ca(2+) channel blockade coincided with decreased fasting plasma insulin concentrations observed in rats 3 weeks after the onset of hyperglycaemia.. These results suggest that phenotypic impairment of diabetic islets in the UCD-T2DM rat is downstream of Ca(2+) influx and involves unregulated stimulation of the CMCP. The continuously elevated levels of CMCP induced by chronic hyperglycaemia in these islets may mediate the loss of islet function.

Topics: Animals; Calcium; Cytochromes c; Cytosol; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Glucose; Hyperglycemia; Insulin; Insulin Secretion; Islets of Langerhans; Male; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Time Factors

Many individuals with abnormalities of mitochondrial respiratory chain complex III remain genetically undefined. Here, we report mutations (c.288G>T [p.Trp96Cys] and c.643C>T [p.Leu215Phe]) in CYC1, encoding the cytochrome c1 subunit of complex III, in two unrelated children presenting with recurrent episodes of ketoacidosis and insulin-responsive hyperglycemia. Cytochrome c1, the heme-containing component of complex III, mediates the transfer of electrons from the Rieske iron-sulfur protein to cytochrome c. Cytochrome c1 is present at reduced levels in the skeletal muscle and skin fibroblasts of affected individuals. Moreover, studies on yeast mutants and affected individuals' fibroblasts have shown that exogenous expression of wild-type CYC1 rescues complex III activity, demonstrating the deleterious effect of each mutation on cytochrome c1 stability and complex III activity.

Topics: Amino Acid Sequence; Child, Preschool; Consanguinity; Cytochromes c; Cytochromes c1; Electron Transport; Female; Fibroblasts; Genetic Complementation Test; Humans; Hyperglycemia; Insulin; Iron-Sulfur Proteins; Ketosis; Male; Mitochondria; Models, Molecular; Molecular Sequence Data; Mutation; Protein Subunits; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Skin

Increased expression of COX-2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX-2 protects hepatocytes from several pro-apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX-2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain wild type (Wt) and transgenic in COX-2 (hCOX-2 Tg) were separated into Control (vehicle) and SID (streptozotocin induced diabetes, 200 mg/kg body weight, i.p.). Seven days post-injection, Wt diabetic animals showed a decrease in PI3K activity and P-Akt levels, an increase of P-JNK, P-p38, pro-apoptotic Bad and Bax, release of cytochrome c and activities of caspases-3 and -9, leading to an increased apoptotic index. This situation was improved in diabetic COX-2 Tg. In addition, SID COX-2 Tg showed increased expression of anti-apoptotic Mcl-1 and XIAP. Pro-apoptotic state in the liver of diabetic animals was improved by over-expression of COX-2. We also analyzed the roles of high glucose-induced apoptosis and hCOX-2 in vitro. Non-transfected and hCOX-2-transfected cells were cultured at 5 and 25 mM of glucose by 72 h. At 25 mM there was an increase in apoptosis in non-transfected cells versus those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX-2-transfected cells was suppressed by addition of DFU (COX-2 selective inhibitor), and mimicked by addition of PGE(2) in non-transfected cells. Taken together, these results demonstrate that hyperglycemia-induced hepatic apoptosis is protected by hCOX-2 expression.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; Caspase 3; Caspase 9; Cell Line; Cyclooxygenase 2; Cytochromes c; Diabetes Mellitus, Experimental; Glucose; Humans; Hyperglycemia; JNK Mitogen-Activated Protein Kinases; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myeloid Cell Leukemia Sequence 1 Protein; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Streptozocin; X-Linked Inhibitor of Apoptosis Protein

Helonias dioica (HD) is a threatened species of herb growing in North America. It is used as a traditional medicine for treating various ailments particularly related to reproductive issues. The root is reported to contain approximately 10% of a saponin (chamaelirin; C(36)H(62)O(18)) apart from certain other fatty acids. As saponins are known to have hypoglycemic effects, we suspected its possible antihyperglycemic potentials. We injected intraperitoneally alloxan (ALX) at the dose of 200 mg/kg body weight (bw) to induce hyperglycemia in mice and tested possible hypoglycemic effects of HD in vivo by deploying two doses (100 and 200 mg/kg bw, respectively). We also tested its effects on the isolated pancreatic islets cells in vitro. We used various standard protocols like reactive oxygen species (ROS) generation and DNA damage, activities of biomarkers like catalase (CAT), superoxide dismutase (SOD), lipid peroxidase (LPO), reduced glutathione (GSH) of the pancreas tissue and glucokinase and glycogen content of the liver of hyperglycemic mice. With a mechanistic approach, we also tracked down the possible signaling pathway involved. We found an elevated level of ROS generation, LPO and overexpression of inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), p38 Map kinase (p38 MAPK), nuclear factor (NF)-κβ, interferon gamma (IFN-γ), cytochrome c, caspase 3, poly [ADP ribose] polymerase (PARP) and cyclo oxygenase 2 (COX2) in ALX-induced diabetic mouse. Treatment of hyperglycemic mice with both the doses of HD showed a significant decrease with respect to all these parameters of study. Thus, our results suggest that HD prevents ALX-induced islet cell damage and possesses antihyperglycemic and antioxidative potentials.

Topics: Alloxan; Animals; Caspase 3; Catalase; Cells, Cultured; Cyclooxygenase 2; Cytochromes c; DNA Damage; Endangered Species; Glucokinase; Glutathione; Hyperglycemia; Hypoglycemic Agents; Islets of Langerhans; Lipid Peroxides; Liver Glycogen; Magnoliopsida; Mice; NF-kappa B; Nitric Oxide Synthase Type II; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Pancreas; Phytotherapy; Plant Extracts; Plant Roots; Plants, Medicinal; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Saponins; Signal Transduction; Superoxide Dismutase; Tumor Necrosis Factor-alpha

To investigate the effect of mitochondria-targeted antioxidant peptide SS31 on prevention of high glucose-induced injury on human retinal endothelial cells (HRECs).. Cultured P3-P5 HRECs were divided into three groups: 5 mM glucose group, 30 mM glucose group and 30 mM glucose co-treated with 100 nM SS31 group. 24 and 48 h after treatment, Annexin V-FITC/PI staining was used to evaluate the survival of HRECs. Overproduction of ROS was assessed by MitoSOX staining under confocal microscope. Change of mitochondrial potential (ΔΨ(m)) of HRECs was measured by flow cytometry after JC-1 fluorescent probe staining. Release of cytochrome c was assessed by confocal microscopy and western blot. Expression of caspase-3 and thioredoxin-2 (Trx-2) were measured by western blot and real-time PCR.. Compared to the high glucose group, co-treatment with 100 nM SS31 significantly protected HRECs from high glucose-induced injury, reduced the production of ROS in mitochondria, stabilized ΔΨ(m), decreased the release of cytochrome c from mitochondria to cytoplasm, decreased the expression of caspase-3 and increased the expression of Trx-2 in high glucose-treated HRECs.. SS31 attenuates the high glucose-induced injuries on HRECs by stabilizing ΔΨ(m), decreasing ROS production, preventing the release of cytochrome c from mitochondria, decreasing the expression of caspase-3 and increasing the expression of Trx-2. Our study suggests that SS31 may be as a potential new treatment for diabetic retinopathy and other oxidative stress-related diseases.

Topics: Antioxidants; Caspase 3; Caspase Inhibitors; Cells, Cultured; Cytochromes c; Diabetic Retinopathy; Endothelial Cells; Glucose; Humans; Hyperglycemia; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Proteins; Oligopeptides; Retina; Thioredoxins

To establish the role of the transcription factor Pax4 in pancreatic islet expansion and survival in response to physiological stress and its impact on glucose metabolism, we generated transgenic mice conditionally and selectively overexpressing Pax4 or a diabetes-linked mutant variant (Pax4R129W) in β-cells.. Glucose homeostasis and β-cell death and proliferation were assessed in Pax4- or Pax4R129W-overexpressing transgenic animals challenged with or without streptozotocin. Isolated transgenic islets were also exposed to cytokines, and apoptosis was evaluated by DNA fragmentation or cytochrome C release. The expression profiles of proliferation and apoptotic genes and β-cell markers were studied by immunohistochemistry and quantitative RT-PCR.. Pax4 but not Pax4R129W protected animals against streptozotocin-induced hyperglycemia and isolated islets from cytokine-mediated β-cell apoptosis. Cytochrome C release was abrogated in Pax4 islets treated with cytokines. Interleukin-1β transcript levels were suppressed in Pax4 islets, whereas they were increased along with NOS2 in Pax4R129W islets. Bcl-2, Cdk4, and c-myc expression levels were increased in Pax4 islets while MafA, insulin, and GLUT2 transcript levels were suppressed in both animal models. Long-term Pax4 expression promoted proliferation of a Pdx1-positive cell subpopulation while impeding insulin secretion. Suppression of Pax4 rescued this defect with a concomitant increase in pancreatic insulin content.. Pax4 protects adult islets from stress-induced apoptosis by suppressing selective nuclear factor-κB target genes while increasing Bcl-2 levels. Furthermore, it promotes dedifferentiation and proliferation of β-cells through MafA repression, with a concomitant increase in Cdk4 and c-myc expression.

Topics: Animals; Apoptosis; Cyclin-Dependent Kinase 4; Cytochromes c; Glucose Transporter Type 2; Homeodomain Proteins; Hyperglycemia; Immunoblotting; Immunohistochemistry; Insulin; Insulin-Secreting Cells; Maf Transcription Factors, Large; Mice; Mice, Transgenic; Paired Box Transcription Factors; Polymerase Chain Reaction; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Streptozocin; Stress, Physiological

Apoptosis and inflammatory/oxidative stress have been associated with hyperglycemia in human peritoneal mesothelial cells (HPMCs) and other cell types. We and others have highlighted the role of early products of non-enzymatic protein glycation in inducing proinflammatory conditions and increasing apoptotic rates in HPMCs. Loss of HPMCs seems to be a hallmark of complications associated with peritoneal membrane dysfunction. The aim of this work is to elucidate the mechanisms by which Amadori adducts may act upon HPMC apoptosis.. HPMCs isolated from different patients were exposed to different Amadori adducts, i.e. highly glycated hemoglobin (10 nM) and glycated bovine serum albumin (250 μg/ml), to study cell death and several proapoptotic markers by different experimental approaches.. Amadori adducts, but not their respective controls, impaired cell proliferation and cell viability by means of apoptosis in a time-dependent manner. They regulated the intrinsic mitochondrial cell death signaling pathway and modulated activation of caspases, Bax, iNOS, p53, NF-κB, and mitogen-activated protein kinases (p38 and JNK) through different reactive oxygen and nitrosative species.. Our data strongly support the idea that long-term hyperglycemia could act as an inducer of apoptosis in HPMCs through Amadori adducts, involving different oxidative and nitrosative reactive species.

Topics: Animals; Apoptosis; Cattle; Cell Death; Cytochromes c; Epithelium; Glycolipids; Humans; Hyperglycemia; Inflammation; L-Lactate Dehydrogenase; MAP Kinase Signaling System; Nitrogen; Oxidative Stress; Phosphatidylethanolamines; Proto-Oncogene Proteins c-jun; Signal Transduction

In this study, we analyzed the contribution of hydroxyl radical in the liver apoptosis mediated by hyperglycemia through the Bax-caspase pathway and the effects of insulin protection against the apoptosis induced by hyperglycemia. Male adult Wistar rats were randomized in three groups: control (C) (sodium citrate buffer, i.p.), streptozotocin (STZ)-induced diabetic (SID) (STZ 60 mg/kg body weight, i.p.), and insulin-treated SID (SID+I; 15 days post STZ injection, SID received insulin s.c., twice a day, 15 days). Rats were autopsied on day 30. In liver tissue, diabetes promoted a significant increase in hydroxyl radical production which correlated with lipid peroxidation (LPO) levels. Besides, hyperglycemia significantly increased mitochondrial BAX protein expression, cytosolic cytochrome c levels, and caspase-3 activity leading to an increase in apoptotic index. Interestingly, the treatment of diabetic rats with desferoxamine or tempol (antioxidants/hydroxyl radical scavengers) significantly attenuated the increase in both hydroxyl radical production and in LPO produced by hyperglycemia, preventing apoptosis by reduction of mitochondrial BAX and cytosolic cytochrome c levels. Insulin treatment showed similar results. The finding that co-administration of antioxidants/hydroxyl radical scavengers together with insulin did not provide any additional benefit compared with those obtained using either inhibitors or insulin alone shows that it is likely that insulin prevents oxidative stress by reducing the effects of hydroxyl radicals. Importantly, insulin significantly increased apoptosis inhibitor protein expression by induction of its mRNA. Taken together, our studies support that, at least in part, the hydroxyl radical acts as a reactive intermediate, which leads to liver apoptosis in a model of STZ-mediated hyperglycemia. A new anti-apoptosis signal for insulin is shown, given by an increase of apoptosis inhibitor protein.

Topics: Animals; Apoptosis; Caspase 3; Cytochromes c; Diabetes Mellitus; Disease Models, Animal; Gene Expression Regulation; Humans; Hydroxyl Radical; Hyperglycemia; Insulin; Liver; Male; Random Allocation; Rats; Rats, Wistar

High-glucose (HG)-induced mesangial apoptosis and fibrogenesis possibly involves reactive oxygen species (ROS) formation and activated mitochondrial stress. We investigated the therapeutic effect of the antioxidant N-acetylcysteine (NAC) on cellular apoptosis and matrix accumulation in HG-treated rat mesangial cells (RMCs).. RMCs were cultured in media containing 5 (control) or 35 mM (HG) glucose. Cellular apoptosis was assayed by TdT-mediated dUTP nick-end labeling staining. Collagen and transforming growth factor-1 gene expression were measured by reverse transcriptase-polymerase chain reaction or Northern blotting. Mitochondrial capacity and intracellular ROS generation was assayed by fluorescence microscopy and flow cytometry, respectively. Cellular ATP production and malondialdehyde (MDA) formation were determined by a luciferin-luciferase reaction and high-performance liquid chromatography, respectively. Cytochrome c release, caspase activation and poly(ADP)ribose polymerase cleavage were assayed by Western blotting.. HG-treated RMCs displayed enhanced cellular apoptosis (65%) and collagen gene expression (1.8-fold increase); these reactions could be significantly suppressed by 1 mM NAC (p < 0.05). Intracellular ROS generation, production of ATP and MDA, and caspase-3, -8 and -9 activities were significantly increased in HG-treated RMCs, and were effectively attenuated by addition of NAC.. It is concluded that NAC prevents HG-induced mesangial apoptosis and fibrogenesis pathways by the reduction of oxidative stress.

Topics: Acetylcysteine; Adenosine Triphosphate; Animals; Apoptosis; Caspases; Cells, Cultured; Collagen; Cytochromes c; Enzyme Activation; Extracellular Matrix; Free Radical Scavengers; Gene Expression; Hyperglycemia; Malondialdehyde; Mesangial Cells; Poly(ADP-ribose) Polymerases; Rats; Reactive Oxygen Species; Transforming Growth Factor beta1

Apoptotic myocyte cell death, diastolic dysfunction, and progressive deterioration in left ventricular pump function characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG) transmits danger signals in cardiac muscle cells. The growth factor adapter protein p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here we demonstrate that interventions which attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser36) inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes obtained by enzymatic dissociation were transduced with mutant-36 p66ShcA (mu-36) dominant-negative expression vector and plated in serum-free media containing 5 or 25 mM glucose. At HG, adult rat ventricular myocytes exhibit a marked increase in reactive oxygen species production, upregulation of phospho-Ser36, collapse of mitochondrial transmembrane potential, and increased formation of p66ShcA/cytochrome-c complexes. These indexes of oxidative stress were accompanied by a 40% increase in apoptosis and the upregulation of cleaved caspase-3 and the apoptosis-related proteins p53 and Bax. To test whether p66ShcA functions as a redox-sensitive molecular switch in vivo, we examined the hearts of male Akita diabetic nonobese (C57BL/6J) mice. Western blot analysis detected the upregulation of phospho-Ser36, the translocation of p66ShcA to mitochondria, and the formation of p66ShcA/cytochrome-c complexes. Conversely, the correction of HG by recombinant adeno-associated viral delivery of leptin reversed these alterations. We conclude that p66ShcA is a molecular switch whose redox function is turned on by phospho-Ser36 and turned off by interventions that prevent this modification.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cardiomyopathies; Caspase 3; Catalase; Cells, Cultured; Cytochromes c; Disease Models, Animal; Genetic Therapy; Hyperglycemia; Leptin; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mitochondria, Heart; Mutation; Myocytes, Cardiac; Oxidation-Reduction; Oxidative Stress; Phosphorylation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; Superoxide Dismutase; Transduction, Genetic; Tumor Suppressor Protein p53

High blood glucose may auto-oxidize and generate free radicals, which are proposed to induce apoptosis in cardiac cells. The aim of the present study was to investigate the cell damage induced by glucose/glucose oxidase-dependent oxidative stress and the protective effect of N-acetylcysteine (NAC) on H9c2 cardiac muscle cells.. H9c2 cells were exposed to 33 mM glucose (G)+1.6 milliunits (mU) of glucose oxidase (GO) and termed G/GO. Cell apoptosis, generation of reactive oxygen species (ROS-super oxide anion and hydrogen peroxide) and reactive nitrogen species (RNS-peroxinitrite), and the change in mitochondrial membrane potential (DeltaPsim) was studied using flow cytometry and confocal microscopy, and cytochrome c release was measured using confocal microscopy. The expression of Bcl-2, Bax and the activation of procaspase-9 was studied by western blot.. Exposure of H9c2 cells to G/GO resulted in a significant increase in cellular apoptosis (P<0.05) and the generation of ROS and RNS (P<0.001). Further, G/GO treatment led to a decrease in DeltaPsim, release of cytochrome c, decrease in Bcl-2, increase in Bax expression and the activation of procaspase-9. Treatment with NAC significantly decreased apoptosis (P<0.05) and reduced the levels of ROS and RNS (P<0.001). NAC was also able to normalize DeltaPsim, inhibit cytochrome c release, increase Bcl-2 and decrease Bax expression and procaspase-9 activation.. Our studies suggest that NAC has antioxidative and antiapoptotic activity against G/GO-induced oxidative stress through the inhibition of mitochondrial damage in H9c2 cells.

Topics: Acetylcysteine; Animals; Apoptosis; Blotting, Western; Cell Line; Cytochromes c; Dose-Response Relationship, Drug; Glucose; Glucose Oxidase; Hyperglycemia; Membrane Potential, Mitochondrial; Mitochondria, Heart; Myocytes, Cardiac; Oxidative Stress; Peroxynitrous Acid; Reactive Oxygen Species

The objective of this study was to identify proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) in pancreatic beta-cells and to understand their roles in cell death under hyperglycemic conditions. Here we report that heat shock protein 60 (HSP60) is modified with O-GlcNAc. Levels of O-GlcNAcylated HSP60 increased twofold in response to hyperglycemic conditions. HSP60 is a chaperonin known to bind to Bax in the cytoplasm under normoglycemic conditions. Under hyperglycemic conditions, Bax detached from O-GlcNAcylated HSP60 and translocated to mitochondria. Hyperglycemic conditions were also associated with cytochrome c release, caspase-3 activation, and cell death, suggesting that elevated O-GlcNAcylation of HSP60 interferes with HSP60-Bax interactions, leading to pancreatic beta-cell death.

Topics: Acetylglucosamine; Animals; bcl-2-Associated X Protein; Caspase 3; Caspases; Cell Death; Cell Line; Chaperonin 60; Cytochromes c; Cytoplasm; Hyperglycemia; Insulin-Secreting Cells; Mitochondria; Protein Binding; Protein Processing, Post-Translational; Protein Transport; Rats

Diabetes mellitus is complicated by the development of a primary cardiomyopathy, which contributes to the excess morbidity and mortality of this disorder. The protein kinase C (PKC) family of isozymes plays a key role in the cardiac phenotype expressed during postnatal development and in response to pathological stimuli. Hyperglycemia is an activating signal for cardiac PKC isozymes that modulate a myriad of cell events including cell death and survival. The epsilon-isozyme of the PKC family transmits a powerful survival signal in cardiac muscle cells. Accordingly, to test the hypothesis that endogenous activation of cardiac PKC-epsilon will protect against hyperglycemic cell injury and left ventricular dysfunction, diabetes mellitus was induced using streptozotocin in genetically engineered mice with cardiac-specific expression of the PKC-epsilon translocation activator [psiepsilon-receptors for activated C kinase (psiepsilon-RACK)]. The results demonstrate a striking PKC-epsilon cardioprotective phenotype in diabetic psiepsilon-RACK (epsilon-agonist) mice that is characterized by inhibition of the hyperglycemia apoptosis signal, attenuation of hyperglycemia-mediated oxidative stress, and preservation of parameters of left ventricular pump function. Hearts of diabetic epsilon-agonist mice exhibited selective trafficking of PKC-epsilon to membrane and mitochondrial compartments, phosphorylation/inactivation of the mitochondrial Bad protein, and inhibition of cytochrome c release. We conclude that activation of endogenous PKC-epsilon in hearts of diabetic epsilon-agonist mice promotes the survival phenotype, attenuates markers of oxidative stress, and inhibits the negative inotropic properties of chronic hyperglycemia.

Topics: Animals; Apoptosis; Cardiomyopathies; Cytochromes c; Diabetes Mellitus, Experimental; Extracellular Signal-Regulated MAP Kinases; Hyperglycemia; Mice; Mice, Mutant Strains; Myocardium; Oxidative Stress; Phosphorylation; Protein Kinase C; Protein Kinase C-epsilon; Signal Transduction; Ventricular Function, Left

Topics: Cytochromes; Cytochromes c; Epinephrine; Hyperglycemia