cytochrome-c-t and Body-Weight

Exenatide could reduce blood glucose and alleviate cognitive dysfunction induced by diabetes mellitus (DM). In the present study, a diabetic model was established in Sprague‑Dawley rats to further explore the mechanism of exenatide on diabetes‑induced cognitive impairment. Notably, the model rats performed poorly in the Morris water maze test and had more apoptotic neurons compared with the control rats. By contrast, exenatide attenuated cognitive impairment and inhibited neuronal apoptosis in the DM rat model. To explore the neuroprotective mechanisms of exenatide, western blotting was performed to detect the expression levels of markers of endoplasmic reticulum stress, including cytochrome c (Cyt‑c), Caspase‑3, JNK and c‑JUN, in hippocampal tissue. Reverse transcription‑quantitative PCR was also performed to measure the mRNA expression levels of Cyt‑c and Caspase‑3. After 16 weeks of treatment, exenatide treatment downregulated Cyt‑c, Caspase‑3, phosphorylated (p)‑JNK and p‑c‑JUN expression in the hippocampal tissue of diabetic rats. Moreover, Cyt‑c, Caspase‑3, JNK and JUN expression levels were detected following treatment with a specific inhibitor of JNK (SP600125). The results revealed that SP600125 had similar inhibitory effects on the JNK pathway and ERS‑related protein expression (Cyt‑t, Caspase‑3, p‑JNK and p‑c‑JUN). These results suggested that exenatide improved cognitive dysfunction in DM rats and that the underlying mechanism may be associated with inhibiting apoptosis by suppressing the activation of JNK/c‑JUN.

Topics: Animals; Apoptosis; Blood Glucose; Body Weight; Caspase 3; Cognitive Dysfunction; Cytochromes c; Diabetes Mellitus, Experimental; Exenatide; Genes, jun; Hippocampus; Insulin; Learning; Male; MAP Kinase Signaling System; Memory; Neurons; Neuroprotective Agents; Rats, Sprague-Dawley

Doxorubicin (DOX) is one of the most widely used chemotherapeutic agents. However, it causes pulmonary toxicity which decreases its clinical use in human cancer therapy. The present study was undertaken to obtain an insight into the potential protective effect of hesperetin (HES) against doxorubicin-induced pulmonary toxicity in rats. The animals were divided into 4 groups with 7 rats per group. The experimental treatments were as follows: Control, DOX, DOX + HES, and HES groups. DOX was administered at the dosage of 15 mg/kg i.p for a single dose. HES was administered at the dosage of 50 mg/kg by oral gavage every other day. After 28 days, biochemical parameters, oxidative stress status, histopathological changes, apoptosis-related genes and apoptotic index (AI) were examined of lung tissue. Histopathological changes, Poly [ADP-ribose] polymerase 1 (PARP-1), Caspase-3 (Casp3), Cytochrome c (Cytc), apoptosis-related genes, and AI significantly increased in the DOX group relative to the control group. Malondialdehyde (MDA) significantly increased, while superoxide dismutase (SOD) and glutathione peroxidase (GPx) decreased in the DOX group relative to the control group. However, histopathological findings, MDA, AI, and PAPR1, Casp3 protein expression, mRNA expression of Cytc significantly decreased, while SOD, GPx increased in the DOX + HES group relative to the DOX group. These results attested HES might be a potential agent for the treatment of DOX-induced pulmonary toxicity.

Topics: Animals; Apoptosis; Body Weight; Caspase 3; Cytochromes c; Doxorubicin; Hesperidin; Lung; Male; Organ Size; Oxidative Stress; Poly(ADP-ribose) Polymerases; Rats, Sprague-Dawley; RNA, Messenger

Metabolic disorders often lead to cardiac complications. Metabolic deregulations during diabetic conditions are linked to mitochondrial dysfunctions, which are the key contributing factors in cardiac hypertrophy. However, the underlying mechanisms involved in diabetes-induced cardiac hypertrophy are poorly understood. In the current study, we initially established a diabetic rat model by alloxan-administration, which was validated by peripheral glucose measurement. Diabetic rats displayed myocardial stiffness and fibrosis, changes in heart weight/body weight, heart weight/tibia length ratios, and enhanced size of myocytes, which altogether demonstrated the establishment of diabetic cardiac hypertrophy (DCH). Furthermore, we examined the expression of genes associated with mitochondrial signaling impairment. Our data show that the expression of PGC-1α, cytochrome c, MFN-2, and Drp-1 was deregulated. Mitochondrial-signaling impairment was further validated by redox-system dysregulation, which showed a significant increase in ROS and thiobarbituric acid reactive substances, both in serum and heart tissue, whereas the superoxide dismutase, catalase, and glutathione levels were decreased. Additionally, the expression levels of pro-apoptotic gene PUMA and stress marker GATA-4 genes were elevated, whereas ARC, PPARα, and Bcl-2 expression levels were decreased in the heart tissues of diabetic rats. Importantly, these alloxan-induced impairments were rescued by N-acetyl cysteine, ascorbic acid, and selenium treatment. This was demonstrated by the amelioration of myocardial stiffness, fibrosis, mitochondrial gene expression, lipid profile, restoration of myocyte size, reduced oxidative stress, and the activation of enzymes associated with antioxidant activities. Altogether, these data indicate that the improvement of mitochondrial dysfunction by protective agents such as N-acetyl cysteine, selenium, and ascorbic acid could rescue diabetes-associated cardiac complications, including DCH.

Topics: Acetylcysteine; Animals; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Ascorbic Acid; Biomarkers; Blood Glucose; Body Weight; Calcium; Cardiomegaly; Cardiotonic Agents; Cytochromes c; Diabetic Cardiomyopathies; Disease Models, Animal; Down-Regulation; GATA4 Transcription Factor; Lipid Peroxidation; Lipids; Mitochondria, Heart; Myocardium; Oxidation-Reduction; Oxidative Stress; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; PPAR alpha; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA, Messenger; Selenium

Exercise training has a neuroprotective effect against ischaemic injury, but the underlying mechanism is not completely clear. This study explored the potential mechanisms underlying the protective effects of treadmill training and caveolin-1 regulation against mitochondrial dysfunction in cerebral ischaemic injury.. After middle cerebral artery occlusion (MCAO) surgery, rats were subjected to treadmill training and received daidzein injections and combined therapy. A series of analyses, including neurological function scoring; body weight measurement; Nissl, haematoxylin and eosin staining; cerebral infarction volume assessment; mitochondrial morphology examination; caveolin-1, cytoplasmic and mitochondrial cytochrome C (CytC), and translocase of outer membrane 20 (TOM20) expression analysis; apoptosis index analysis; and transmission electron microscopy were conducted.. Treadmill training increased caveolin-1 expression, reduced neurobehavioral scores and cerebral infarction volumes, improved tissue morphology, reduced neuronal loss, inhibited mitochondrial outer membrane permeabilization (MOMP) through the caveolin-1 pathway, prevented excessive Cyt-C release from mitochondria, and reduced the degrees of apoptosis and mitochondrial damage. In addition, treadmill training increased the expression of TOM20 through the caveolin-1 pathway and maintained import signal function, thereby protecting mitochondrial integrity.. Treadmill exercise protected mitochondrial integrity and inhibited the endogenous mitochondrial apoptosis pathway. The damage of cerebral ischaemia was alleviated in rats through enhancement of caveolin-1 by treadmill exercise.

Topics: Animals; Apoptosis; Body Weight; Brain Ischemia; Caveolin 1; Cytochromes c; Exercise Test; Isoflavones; Male; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Neuroprotective Agents; Physical Conditioning, Animal; Rats; Rats, Sprague-Dawley; Signal Transduction

To investigate the effects of Huaiqihuang Granules (, HQH), a mixture of Chinese herbs including Trametes robiniophila Murr, Fructus Lycii and Polygonatum sibiricum, on adriamycininduced nephropathy (ADRN) in rats and its underlying mechanisms.. Rats with ADRN were divided into four groups: the sham group, the model group (distilled water), the low-dose HQH-treated (2 g/kg) group, and the high-dose HQH-treated (4 g/kg) group. Body weight and 24-h urinary protein (Upro) were checked every week. After 5-week intervention, at the end of the study, the rats were sacrificed and blood samples were collected for examination of biochemical parameters, including glomerular morphological makers, podocyte shape, cellular apoptosis, expressions of nephrin, inflammatory and apoptosis markers.. HQH ameliorated the rat's general status, proteinuria, renal morphological appearance and glomerulosclerosis. The decreased expression of nephrin in ADRN rats was increased by HQH, as well as the impaired podocyte foot process fusion. Cytosolic levels of p65 and inhibitor of nuclear factor κBα (IκBα) were decreased in ADRN rats, and recovered by the treatment of HQH. Consistently, the induced expression of tumor necrosis factor α (TNF-α), phosphorylated nuclear factor κB p65 (p-NFκB p65) and IκBα in ADRN were markedly suppressed by HQH. In addition, induction of Bax, cleaved caspase-3 and cytochrome C in ADRN rats were suppressed by HQH, indicating the amelioration of apoptosis.. HQH could ameliorate renal impairments in ADRN rats by increasing nephrin expression, inhibiting NF-κB signaling pathway via the down-regulation of p-NF-κB p65 and p-IκBα, and suppression of glomerular and tubular apoptosis.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Body Weight; Caspase 3; Chromatography, High Pressure Liquid; Cytochromes c; Doxorubicin; Drugs, Chinese Herbal; Kidney; Kidney Diseases; Kidney Glomerulus; Kidney Tubules; Male; Membrane Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; Organ Size; Proteinuria; Rats, Sprague-Dawley; Signal Transduction; Transcription Factor RelA; Tumor Necrosis Factor-alpha

This study was conducted with objective of evaluating the toxic effects of nickel chloride (NiCl2) on development of bursa of Fabricius in broilers fed on diets supplemented with 0, 300, 600 and 900 mg/kg of NiCl2 for 42 days by using the methods of experimental pathology, flow cytometry (FCM), and quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that dietary NiCl2 in 300 mg/kg and over induced toxic suppression in the bursal development, which was characterized by decreasing lymphocytes histopathologically and relative weight, increasing G0/G1 phase (a prolonged nondividing state), reducing S phase (DNA replication) and proliferating index, and increasing percentages of apoptotic cells. Concurrently, the mRNA expression levels of bax, cytochrome c (cyt c), apoptotic peptidase activating factor 1 (Apaf-1), caspase-3, caspase-6, caspase-7 and caspase-9 were increased and the bcl-2 mRNA expression levels were decreased. The toxic suppression of bursal development finally impaired humoral immunity duo to the reduction of B lymphocyte population and B lymphocyte activity in the broiler chicken. This study provides new evidences for further studying the effect mechanism of Ni and Ni compoundson B-cell or bursa of Fabricius.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Avian Proteins; B-Lymphocytes; bcl-2-Associated X Protein; Body Weight; Bursa of Fabricius; Caspases; Cell Cycle; Cell Proliferation; Chickens; Cytochromes c; Gene Expression Regulation, Developmental; Lymphocyte Count; Nickel; Organ Size; Reverse Transcriptase Polymerase Chain Reaction

Doxycycline (DOX) exhibits anti-inflammatory, anti-tumor, and pro-apoptotic activity and is being tested in clinical trials as a chemotherapeutic agent for several cancers, including colon cancer.. In the current study, the chemotherapeutic activity of doxycycline was tested in a rat model of colon carcinogenesis, induced by colon specific cancer promoter, 1,2, dimethylhydrazine (DMH) as well as study the effect of DOX-alone on a separate group of rats.. Doxycycline administration in DMH-treated rats (DMH-DOX) unexpectedly increased tumor multiplicity, stimulated progression of colonic tumor growth from adenomas to carcinomas and revealed metastasis in small intestine as determined by macroscopic and histopathological analysis. DOX-alone treatment showed markedly enhanced chronic inflammation and reactive hyperplasia, which was dependent upon the dose of doxycycline administered. Moreover, immunohistochemical analysis revealed evidence of inflammation and anti-apoptotic action of DOX by deregulation of various biomarkers.. These results suggest that doxycycline caused chronic inflammation in colon, small intestine injury, enhanced the efficacy of DMH in tumor progression and provided a mechanistic link between doxycycline-induced chronic inflammation and tumorigenesis. Ongoing studies thus may need to focus on the molecular mechanisms of doxycycline action, which lead to its inflammatory and tumorigenic effects.

Topics: 1,2-Dimethylhydrazine; Animals; Body Weight; Carcinogenesis; Caspase 3; Caspase 9; Cell Proliferation; Chronic Disease; Colonic Neoplasms; Cytochromes c; Down-Regulation; Doxycycline; Immunohistochemistry; Inflammation; Intestine, Small; Male; Matrix Metalloproteinase 9; Neoplasm Metastasis; NF-kappa B; Rats, Sprague-Dawley; Tumor Suppressor Protein p53; Up-Regulation; Vascular Endothelial Growth Factor A

The oxidative phosphorylation (OxPhos) pathway is responsible for most aerobic ATP production and is the only metabolic pathway with proteins encoded by both nuclear and mitochondrial genomes. In studies examining mitonuclear interactions among distant populations within a species or across species, the interactions between these two genomes can affect metabolism, growth, and fitness, depending on the environment. However, there is little data on whether these interactions impact natural populations within a single species. In an admixed Fundulus heteroclitus population with northern and southern mitochondrial haplotypes, there are significant differences in allele frequencies associated with mitochondrial haplotype. In this study, we investigate how mitochondrial haplotype and any associated nuclear differences affect six OxPhos parameters within a population. The data demonstrate significant OxPhos functional differences between the two mitochondrial genotypes. These differences are most apparent when individuals are acclimated to high temperatures with the southern mitochondrial genotype having a large acute response and the northern mitochondrial genotype having little, if any acute response. Furthermore, acute temperature effects and the relative contribution of Complex I and II depend on acclimation temperature: when individuals are acclimated to 12°C, the relative contribution of Complex I increases with higher acute temperatures, whereas at 28°C acclimation, the relative contribution of Complex I is unaffected by acute temperature change. These data demonstrate a complex gene by environmental interaction affecting the OxPhos pathway.

Topics: Acclimatization; Alleles; Animals; Body Weight; Cytochromes c; Electron Transport Complex I; Electron Transport Complex II; Fundulidae; Gene Frequency; Gene-Environment Interaction; Genotype; Linear Models; Mitochondria; Oxidative Phosphorylation; Species Specificity; Temperature

While hippocampus is a brain region particularly susceptible to the effects of MDMA, the cellular and molecular changes induced by MDMA are still to be fully elucidated, being the dosage regimen, the species and the developmental stage under study great variables. This study compared the effects of one and four days of MDMA administration following a binge paradigm (3×5 mg/kg, i.p., every 2 h) on inducing hippocampal neurochemical changes in adolescent (PND 37) and young adult (PND 58) rats. The results showed that chronic MDMA caused hippocampal protein deficits in adolescent and young adult rats at different levels: (1) impaired serotonergic (5-HT2A and 5-HT2C post-synaptic receptors) and GABAergic (GAD2 enzyme) signaling, and (2) decreased structural cytoskeletal neurofilament proteins (NF-H, NF-M and NF-L). Interestingly, these effects were not accompanied by an increase in apoptotic markers. In fact, chronic MDMA inhibited proteins of the apoptotic pathway (i.e., pro-apoptotic FADD, Bax and cytochrome c) leading to an inhibition of cell death markers (i.e., p-JNK1/2, cleavage of PARP-1) and suggesting regulatory mechanisms in response to the neurochemical changes caused by the drug. The data, together with the observed lack of GFAP activation, support the view that chronic MDMA effects, regardless of the rat developmental age, extends beyond neurotransmitter systems to impair other hippocampal structural cell markers. Interestingly, inhibitory changes in proteins from the apoptotic pathway might be taking place to overcome the protein deficits caused by MDMA.

Topics: Aging; Animals; Animals, Newborn; Apoptosis; bcl-2-Associated X Protein; Body Temperature; Body Weight; Cytochromes c; Down-Regulation; Fatty Acid Desaturases; Glutamate Decarboxylase; Hallucinogens; Hippocampus; Male; N-Methyl-3,4-methylenedioxyamphetamine; Neurofilament Proteins; Rats; Rats, Sprague-Dawley; Receptor, Serotonin, 5-HT2A; Receptor, Serotonin, 5-HT2C; Signal Transduction

The cholinergic anti-inflammatory pathway is one of the putative biochemical pathways that link diabetes with Alzheimer disease. Hence, we aimed to verify the potential antidiabetic effect of galantamine, unveil the possible mechanisms and evaluate its interaction with vildagliptin. The n5-STZ rat model was adopted and the diabetic animals were treated with galantamine and/or vildagliptin for 4 weeks. Galantamine lowered the n5-STZ-induced elevation in body weight, food/water intake, serum levels of glucose, fructosamine, and ALT/AST, as well as AChE in the tested organs. Moreover, it modulated successfully the lipid profile assessed in serum, liver, and muscle, and increased serum insulin level, as well as % β-cell function, in a pattern similar to that of vildagliptin. Additionally, galantamine confirmed its antioxidant (Nrf2, TAC, MDA), anti-inflammatory (NF-κB, TNF-α, visfatin, adiponectin) and anti-apoptotic (caspase-3, cytochrome c) capabilities by altering the n5-STZ effect on all the aforementioned parameters. On the molecular level, galantamine/vildagliptin have improved the insulin (p-insulin receptor, p-Akt, GLUT4/GLUT2) and Wnt/β-catenin (p-GSK-3β, β-catenin) signaling pathways. On almost all parameters, the galantamine effects surpassed that of vildagliptin, while the combination regimen showed the best effects. The present results clearly proved that galantamine modulated glucose/lipid profile possibly through its anti-oxidant, -apoptotic, -inflammatory and -cholinesterase properties. These effects could be attributed partly to the enhancement of insulin and Wnt/β-catenin signaling pathways. Galantamine can be strongly considered as a potential antidiabetic agent and as an add-on therapy with other oral antidiabetics.

Topics: Adipokines; Animals; Apoptosis; Biomarkers; Blood Glucose; Body Weight; Brain; Caspase 3; Cholinesterase Inhibitors; Cytochromes c; Diabetes Mellitus, Experimental; Disease Models, Animal; Galantamine; Glucose; Glucose Tolerance Test; Hypoglycemic Agents; Inflammation Mediators; Insulin; Lipid Metabolism; Lipids; Liver; Muscle, Skeletal; Rats; Signal Transduction; Wnt Signaling Pathway

Androgen reduces fat mass, although the underlying mechanisms are unknown. Here, we examined the effect of testosterone on heat production and mitochondrial biogenesis. Testosterone-treated mice exhibited elevated heat production. Treatment with testosterone increased the expression level of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), ATP5B and Cox4 in skeletal muscle, but not that in brown adipose tissue and liver. mRNA levels of genes involved in mitochondrial biogenesis were elevated in skeletal muscle isolated from testosterone-treated male mice, but were down-regulated in androgen receptor deficient mice. These results demonstrated that the testosterone-induced increase in energy expenditure is derived from elevated mitochondrial biogenesis in skeletal muscle.

Topics: Androgens; Animals; Blood Glucose; Blotting, Western; Body Weight; Cell Line; Cytochromes c; DNA, Mitochondrial; Eating; Energy Metabolism; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria, Muscle; Muscle Fibers, Skeletal; Muscle, Skeletal; Myoglobin; Nuclear Respiratory Factor 1; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; Testosterone; Transcription Factors; Troponin; Weight Loss

An emerging literature suggests that bisphenol A (BPA), a widespread endocrine disrupting chemical, when exposure occurs in early life, may increase the risk of metabolic syndrome. In this study, we investigated the hypothesis that perinatal exposure to BPA predisposed offspring to fatty liver disease: the hepatic manifestation of metabolic syndrome, and its possible mechanism. Pregnant Wistar rats were administered with BPA (40μg/kg/day) or vehicle during gestation and lactation. Liver histology, biochemical analysis, transcriptome, and mitochondrial function were examined in male offspring at postnatal 3, 15 and 26 weeks. At 3 weeks of age, abnormal liver morphology and function were not observed in the BPA-exposed offspring, but a decrease in mitochondrial respiratory complex (MRC) activity (I and III) and significant changes in gene expression involved in mitochondrial fatty acid metabolism were observed compared with controls. At 15 weeks, micro-vesicular steatosis in liver, up-regulated genes involved in lipogenesis pathways, increased ROS generation and Cytc release were observed in the BPA-exposed offspring. Then, extensive fatty accumulation in liver and elevated serum ALT were observed in BPA-exposed offspring at 26 weeks. In the longitudinal observation, hepatic mitochondrial function including MRC activity, ATP production, ROS generation and mitochondrial membrane potential were progressively worsened in the BPA-exposed offspring. Perinatal BPA exposure contributes to the development of hepatic steatosis in the offspring of rats, which may be mediated through impaired hepatic mitochondrial function and up-regulated hepatic lipid metabolism.

Topics: Adenosine Triphosphate; Animals; Benzhydryl Compounds; Blotting, Western; Body Weight; Cytochromes c; Cytosol; Fatty Acids, Nonesterified; Fatty Liver; Female; Hepatocytes; Lipids; Liver; Male; Membrane Potential, Mitochondrial; Mitochondrial Diseases; Phenols; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Wistar; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Triglycerides

Changes in the maternal nutritional environment during fetal development can influence offspring's metabolic risk in later life. Animal models have demonstrated that offspring of diet-induced obese dams develop metabolic complications, including nonalcoholic fatty liver disease. In this study we investigated the mechanisms in young offspring that lead to the development of nonalcoholic fatty liver disease (NAFLD). Female offspring of C57BL/6J dams fed either a control or obesogenic diet were studied at 8 wk of age. We investigated the roles of oxidative stress and lipid metabolism in contributing to fatty liver in offspring. There were no differences in body weight or adiposity at 8 wk of age; however, offspring of obese dams were hyperinsulinemic. Oxidative damage markers were significantly increased in their livers, with reduced levels of the antioxidant enzyme glutathione peroxidase-1. Mitochondrial complex I and II activities were elevated, while levels of mitochondrial cytochrome c were significantly reduced and glutamate dehydrogenase was significantly increased, suggesting mitochondrial dysfunction. Offspring of obese dams also had significantly greater hepatic lipid content, associated with increased levels of PPARγ and reduced triglyceride lipase. Liver glycogen and protein content were concomitantly reduced in offspring of obese dams. In conclusion, offspring of diet-induced obese dams have disrupted liver metabolism and develop NAFLD prior to any differences in body weight or body composition. Oxidative stress may play a mechanistic role in the progression of fatty liver in these offspring.

Topics: Adiposity; Age Factors; Animal Nutritional Physiological Phenomena; Animals; Body Weight; Cytochromes c; Electron Transport Complex I; Electron Transport Complex II; Fatty Liver; Female; Glutamate Dehydrogenase; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Glycogen; Homeostasis; Insulin; Lipase; Lipid Metabolism; Liver; Mice; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Obesity; Oxidative Stress; Phenotype; PPAR gamma; Pregnancy; Prenatal Exposure Delayed Effects; Prenatal Nutritional Physiological Phenomena; Risk Factors; Signal Transduction

The role of reactive oxygen species (ROS) in muscle protein hydrolysis and protein oxidation in thyrotoxicosis has not been explored. This study indicates that ROS play a role in skeletal muscle wasting pathways in thyrotoxicosis. Two experimental groups (rats) were treated for 5 days with either 3,3',5-triiodothyronine (HT) or HT with α-tocopherol (HT + αT). Two controls were used, vehicle (Control) and control treated with αT (Control + αT). Serum T3, peritoneal fat, serum glycerol, muscle and body weight, temperature, mitochondrial metabolism (cytochrome c oxidase activity), oxidative stress parameters and proteolytic activities were examined. High body temperature induced by HT returned to normal when animals were treated with αT, although total body and muscle weight did not. An increase in lipolysis was observed in the HT + αT group, as peritoneal fat decreased significantly together with an increase in serum glycerol. GSH, GSSG and total radical-trapping antioxidant parameter (TRAP) decreased and catalase activity increased in the HT group. The glutathione redox ratio was higher in HT + αT than in both HT and Control + αT groups. Carbonyl proteins, AOPP, mitochondrial and chymotrypsin-like proteolytic activities were higher in the HT group than in the Control. HT treatment with αT restored mitochondrial metabolism, TRAP, carbonyl protein, chymotrypsin-like activity and AOPP to the level as that of the Control + αT. Calpain activity was lower in the HT + αT group than in HT and Control + αT and superoxide dismutase (SOD) activity was higher in the HT + αT group than in the Control + αT. Although αT did not reverse muscle loss, ROS was involved in proteolysis to some degree.

Topics: alpha-Tocopherol; Animals; Antioxidants; Body Weight; Calpain; Cytochromes c; Male; Malondialdehyde; Muscles; Organ Size; Rats, Wistar; Reactive Oxygen Species; Thyrotoxicosis; Triiodothyronine; Wasting Syndrome

Increasing evidence has established causative links between obesity, chronic inflammation and insulin resistance; the core pathophysiological feature in type 2 diabetes mellitus. This study was designed to examine whether the combination of L-cysteine and metformin would provide additional benefits in reducing oxidative stress, inflammation and insulin resistance in streptozotocin-induced type 2 diabetes in rats. Male Wistar rats were fed a high-fat diet (HFD) for 8 weeks to induce insulin resistance after which they were rendered diabetic with low-dose streptozotocin. Diabetic rats were treated with metformin (300 mg/kg/day), L-cysteine (300 mg/kg/day) and their combination along with HFD for another 2 weeks. Control rats were fed normal rat chow throughout the experiment. At the end of treatment, fasting blood glucose, fasting serum insulin, homeostasis model assessment-insulin resistance index (HOMA-IR) and serum free fatty acids (FFAs) were measured. Serum levels of the inflammatory markers; monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP) and nitrite/nitrate were also determined. The liver was isolated and used for determination of malondialdehyde (MDA), reduced glutathione (GSH), caspase-3 and cytochrome c levels. The hypoglycemic effect of the combination therapy exceeded that of metformin and L-cysteine monotherapies with more improvement in insulin resistance. All treated groups exhibited significant reductions in serum FFAs, oxidative stress and inflammatory parameters, caspase-3 and cytochrome c levels compared to untreated diabetic rats with the highest improvement observed in the combination group. In conclusion, the present results clearly suggest that L-cysteine can be strongly considered as an adjunct to metformin in management of type 2 diabetes.

Topics: Animals; Body Weight; C-Reactive Protein; Caspase 3; Chemokine CCL2; Cysteine; Cytochromes c; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Drug Interactions; Fatty Acids, Nonesterified; Glutathione; Inflammation; Insulin Resistance; Liver; Male; Malondialdehyde; Metformin; Nitrates; Nitrites; Oxidative Stress; Rats; Rats, Wistar

Oxidative stress is associated with various diabetic complications and taurine plays an important role in ameliorating those difficulties. In the present study we, therefore, investigated whether taurine plays any beneficial role against diabetes induced liver dysfunction and if it does, what cellular mechanism it follows during protective action. Induction of diabetes by alloxan (ALX) (at a dose of 120mg/kg body weight, i.p., once) reduced body weight and plasma insulin level, enhanced blood glucose and serum markers related to hepatic injury, accelerated ROS production, disturbed the intra-cellular antioxidant machineries and disintegrated hepatic cells near central vein. This pathophysiology leads to apoptotic cell death as evidenced from DNA fragmentation and TUNEL aasay. Studies on the mechanism of apoptosis showed that ALX accelerated the markers of mitochondrial dependent apoptotic pathway (enhanced cytochrome C release in cytosol from mitochondria, altered the expression of Bax, Bcl-2, Apaf-1, caspase-9, caspase-3). Treatment with taurine (1% w/v for three weeks) post-hyperglycemia, however, could restore all the alteration caused by ALX. Moreover, taurine activates hepatic PI3Kinase, Akt, hexokinase and augments the translocation of GLUT 2 to hepatic membrane in diabetic rats. Combining all, as a potential therapeutic, taurine may normalize the complications of diabetic liver injury.

Topics: Alloxan; Animals; Apoptosis; Blood Glucose; Body Weight; Caspase 3; Caspase 9; Cytochromes c; Diabetes Mellitus, Experimental; Enzymes; Glucose Transporter Type 2; Lipid Peroxidation; Liver; Male; Mitochondria; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Taurine

Because the functional apoptosis-initiating protein, cytochrome C (CytC) is rapidly cleared from the circulation (t1/2 (half-life): 4 minutes), it cannot be used for in vivo therapy. We report herein on a hitherto unreported strategy for delivering exogenous CytC as a potential and safe antiobesity drug for preventing diet-induced obesity, the most common type of obesity in humans. The functional activity of CytC encapsulated in prohibitin (a white fat vessel-specific receptor)-targeted nanoparticles (PTNP) was evaluated quantitatively, as evidenced by the observations that CytC-loaded PTNP causes apoptosis in primary adipose endothelial cells in a dose-dependent manner, whereas CytC alone did not. The delivery of a single dose of CytC through PTNP into the circulation disrupted the vascular structure by the targeted apoptosis of adipose endothelial cells in vivo. Intravenous treatment of CytC-loaded PTNP resulted in a substantial reduction in obesity in high-fat diet (HFD) fed wild-type (wt) mice, as evidenced by the dose-dependent prevention of the percentage of increase in body weight and decrease in serum leptin levels. In addition, no detectable hepatotoxicity was found to be associated with this prevention. Thus, the finding highlights the promising potential of CytC for use as an antiobesity drug, when delivered through a nanosystem.

Topics: Adipose Tissue; Animals; Anti-Obesity Agents; Body Weight; Cholesterol; Cytochromes c; Diet, High-Fat; Drug Delivery Systems; Endothelial Cells; Immobilized Proteins; Leptin; Male; Mice; Mice, Inbred C57BL; Nanoparticles; Obesity; Prohibitins; Repressor Proteins; Triglycerides

Many diseases are associated with catabolic conditions that induce skeletal muscle wasting. These various catabolic states may have similar and distinct mechanisms for inducing muscle protein loss. Mechanisms related to muscle wasting may also be related to muscle metabolism since glycolytic muscle fibers have greater wasting susceptibility with several diseases. The purpose of this study was to determine the relationship between muscle oxidative capacity and muscle mass loss in red and white hindlimb muscles during cancer cachexia development in the Apc(Min/+) mouse. Gastrocnemius and soleus muscles were excised from Apc(Min/+) mice at 20 wk of age. The gastrocnemius muscle was partitioned into red and white portions. Body mass (-20%), gastrocnemius muscle mass (-41%), soleus muscle mass (-34%), and epididymal fat pad (-100%) were significantly reduced in severely cachectic mice (n = 8) compared with mildly cachectic mice (n = 6). Circulating IL-6 was fivefold higher in severely cachectic mice. Cachexia significantly reduced the mitochondrial DNA-to-nuclear DNA ratio in both red and white portions of the gastrocnemius. Cytochrome c and cytochrome-c oxidase complex subunit IV (Cox IV) protein were reduced in all three muscles with severe cachexia. Changes in muscle oxidative capacity were not associated with altered myosin heavy chain expression. PGC-1α expression was suppressed by cachexia in the red and white gastrocnemius and soleus muscles. Cachexia reduced Mfn1 and Mfn2 mRNA expression and markers of oxidative stress, while Fis1 mRNA was increased by cachexia in all muscle types. Muscle oxidative capacity, mitochondria dynamics, and markers of oxidative stress are reduced in both oxidative and glycolytic muscle with severe wasting that is associated with increased circulating IL-6 levels.

Topics: Adipose Tissue; Aldehydes; Animals; Body Weight; Cachexia; Catalase; Colonic Neoplasms; Cytochromes c; DNA, Mitochondrial; Electron Transport Complex IV; Gene Expression; Genes, APC; GTP Phosphohydrolases; Hindlimb; Interleukin-6; Ion Channels; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria, Muscle; Mitochondrial Proteins; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Oxidative Phosphorylation; Oxidative Stress; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Sirtuin 1; STAT3 Transcription Factor; Succinate Dehydrogenase; Superoxide Dismutase; Trans-Activators; Transcription Factors; Uncoupling Protein 3

CD47, a receptor for thrombospondin-1, limits two important regulatory axes: nitric oxide-cGMP signaling and cAMP signaling, both of which can promote mitochondrial biogenesis. Electron microscopy revealed increased mitochondrial densities in skeletal muscle from both CD47 null and thrombospondin-1 null mice. We further assessed the mitochondria status of CD47-null vs WT mice. Quantitative RT-PCR of RNA extracted from tissues of 3 month old mice revealed dramatically elevated expression of mRNAs encoding mitochondrial proteins and PGC-1α in both fast and slow-twitch skeletal muscle from CD47-null mice, but modest to no elevation in other tissues. These observations were confirmed by Western blotting of mitochondrial proteins. Relative amounts of electron transport enzymes and ATP/O(2) ratios of isolated mitochondria were not different between mitochondria from CD47-null and WT cells. Young CD47-null mice displayed enhanced treadmill endurance relative to WTs and CD47-null gastrocnemius had undergone fiber type switching to a slow-twitch pattern of myoglobin and myosin heavy chain expression. In 12 month old mice, both skeletal muscle mitochondrial volume density and endurance had decreased to wild type levels. Expression of myosin heavy chain isoforms and myoglobin also reverted to a fast twitch pattern in gastrocnemius. Both CD47 and TSP1 null mice are leaner than WTs, use less oxygen and produce less heat than WT mice. CD47-null cells produce substantially less reactive oxygen species than WT cells. These data indicate that loss of signaling from the TSP1-CD47 system promotes accumulation of normally functioning mitochondria in a tissue-specific and age-dependent fashion leading to enhanced physical performance, lower reactive oxygen species production and more efficient metabolism.

Topics: Aging; Animals; Body Composition; Body Weight; CD47 Antigen; Cytochromes b; Cytochromes c; Energy Metabolism; Female; Gene Expression; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Myocytes, Smooth Muscle; Myosin Heavy Chains; Nuclear Respiratory Factor 1; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Physical Endurance; Protein Isoforms; Reactive Oxygen Species; Superoxides; Thrombospondin 1; Trans-Activators; Transcription Factors; Voltage-Dependent Anion Channel 1

Doxorubicin (DOX) is an anthracycline drug with a wide spectrum of clinical antineoplastic activity, but increased apoptosis has been implicated in its cardiotoxicity. Resveratrol (RES) was shown to harbour major health benefits in diseases associated with oxidative stress. In this study, we aimed to determine the effect of RES on DOX-induced myocardial apoptosis in mice.. Male Balb/c mice were randomized to one of the following four treatments: saline, RES, DOX, or RES plus DOX (10 mice in each group). DOX treatment markedly depressed cardiac function, decreased the heart weight, the body weight, and the ratio of heart weight to body weight, but inversely increased the level of protein carbonyl, malondialdehyde, and serum lactate dehydrogenase, and induced mitochondrial cytochrome c release and cardiomyocyte apoptosis. However, these effects of DOX were ameliorated by its combination with RES. Further studies with a co-immunoprecipitation assay revealed an interaction between p53 and Sirtuin 1 (SIRT1). It was found by western blot and electrophoretic mobility shift assay that DOX treatment increased p53 protein acetylation and cytochrome c release from mitochondria, activated p53 binding at the Bax promoter, and up-regulated Bax expression, but supplementation with RES could weaken all these effects.. The protective effect of RES against DOX-induced cardiomyocyte apoptosis is associated with the up-regulation of SIRT1-mediated p53 deacetylation.

Topics: Acetylation; Animals; Antineoplastic Agents; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Body Weight; Cytochromes c; Doxorubicin; Gene Expression; Male; Mice; Mice, Inbred BALB C; Myocardium; Myocytes, Cardiac; Organ Size; Oxidative Stress; Resveratrol; Sirtuin 1; Stilbenes; Tumor Suppressor Protein p53; Ventricular Dysfunction, Left

The aim of the study was to investigate the role of mitochondrial apoptotic pathways in vascular endothelial injury in male rats with low androgen. 8 week-old adult male Sprague-Dawley (SD) rats were randomly divided into 3 groups (n=6/each group): control group, castrated group (low androgen), and replacement group (given androgen after castration). After 10 weeks, endothelial structure was observed by general light microscope and transmission electron microscope (TEM) respectively. Isolated mitochondria and mitochondrial membrane potential (MMP) were detected by fluorescence to access mitochondrial function. Chromatin degradation was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine-biotin nick end labeling (TUNEL) staining method. The mRNA and protein of bcl-2, cytochrome C (Cyt C), caspase-9, and caspase-3 were analyzed for apoptosis. Cell shrinkage and condensed chromatin, less mitochondria and a fall in MMP levels were observed in the castrated group, along with more apoptotic endothelial cells. Bcl-2 level was reduced and the expression of caspase-9, caspase-3 and Cyt C were elevated in the castrated group (p<0.05). But there was no significant difference between the replacement group and the control group (p>0.05). It was concluded that low androgen caused vascular endothelial damage. It may be, at least in part, related with the activating mitochondrial apoptotic pathways.

Topics: Androgens; Animals; Apoptosis; Arteries; Body Weight; Caspase 3; Caspase 9; Cytochromes c; Endothelial Cells; Endothelium, Vascular; Gene Expression Regulation; In Situ Nick-End Labeling; Male; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Testosterone

Previous research has demonstrated that diabetes induces learning and memory deficits. However, the mechanism of memory impairment induced by diabetes is poorly understood. The present study investigated the effect of streptozotocin (STZ)-induced diabetes on spatial learning and memory using the Morris Water Maze. The effects of diabetes on CA1 pyramidal neurons in hippocampus were also examined. Diabetes impaired spatial learning and memory of rats. Diabetes induced the apoptosis of neurons and translocation of Bax from cytoplasm to mitochondria. On the contrary, diabetes induced cytochrome c release into the cytoplasm from mitochondria. Release of Cyt-c from mitochondria into cytoplasm may play a role in apoptosis of the CA1 pyramidal neurons, which resulted in a decrease of the number of neurons in hippocampus and impaired the performance function. These results partially explain the mechanism of the effect of diabetes on learning and memory. To protect mitochondrial function is possible candidate for preventing the impairments of diabetes on hippocampal function.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blood Glucose; Blotting, Western; Body Weight; CA1 Region, Hippocampal; Cytochromes c; Diabetes Mellitus, Experimental; Hippocampus; Immunohistochemistry; Male; Maze Learning; Memory; Microscopy, Electron, Transmission; Mitochondria; Psychomotor Performance; Pyramidal Cells; Rats; Rats, Sprague-Dawley

Pancreatic adenocarcinoma is one of the most common malignancies worldwide. Gemcitabine is currently the standard first-line chemotherapeutic agent for pancreatic cancer. However, gemcitabine can induce activation of Akt and nuclear factor-κB (NF-κB), which is associated with its chemoresistance. It has been reported that gemcitabine combination therapies result in improved survival outcomes in pancreatic cancer. Therefore, agents that can either enhance the effects of gemcitabine or overcome chemoresistance to the drug are needed for the treatment of pancreatic cancer. Emodin is an active component of Chinese medicinal herbs and can inhibit the activation of Akt and NF-κB. In this study, we investigated whether emodin could enhance the anticancer effect of gemcitabine on pancreatic cancer in vivo. We demonstrated that treatment of gemcitabine combined with emodin efficiently suppressed tumor growth in mice inoculated with pancreatic tumor cells. This treatment paradigm promoted apoptotic cell death and mitochondrial fragmentation. Furthermore, it reduced phosphorylated-Akt (p-Akt) level, NF-κB activation and Bcl-2/Bax ratio, increased caspase-9 and -3 activation, Cytochrome C (CytC) release occurred in combination therapy. Collectively, emodin enhanced the activity of gemcitabine in tumor growth suppression via inhibition of Akt and NF-κB activation, thus promoting the mitochondrial-dependent apoptotic pathway. Therefore, our findings may provide new insights into understanding the pharmacological regulation of emodin on gemcitabine-mediated proapoptosis in pancreatic cancer and may aid in the design of new therapeutic strategies for the intervention of human pancreatic cancers.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Body Weight; Caspase 3; Cell Line, Tumor; Cytochromes c; Emodin; Female; Humans; Mice; NF-kappa B; Pancreatic Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Rheum; Tumor Burden; Xenograft Model Antitumor Assays

Breathing-disordered states, such as in obstructive sleep apnea, which are cyclical in nature, have been postulated to induce neurocognitive morbidity in both pediatric and adult populations. The oscillatory nature of intermittent hypoxia, especially when chronic, may mimic the paradigm of ischemia-reperfusion in that tissues and cells are exposed to episodes of low and high O(2) and this may lead to oxidant stress. Therefore, we decided to explore the potential contribution of oxidant stress in our intermittent hypoxia/hypercapnia animal model and the role that mitochondria might play in this stress. Neonatal mice were exposed to intermittent hypoxia/hypercapnia for 10 days and 2 wk. Combined intermittent hypoxia/hypercapnia led to a marked increase in apoptotic cell death in the cerebral cortex. Oxygen consumption studies in isolated mitochondria from intermittent hypoxia/hypercapnia-exposed brains demonstrated significant reductions in both state 4 and state 3 respiratory activities by approximately 60% and 75%, respectively. Electron paramagnetic resonance spectroscopy registered a significant increase in superoxide production during nonphosphorylating state 4 by 37%, although superoxide leakage during state 3 did not increase upon treatment. Neuronal superoxide-specific dihydroethidium oxidation was also greater in exposed animals. These studies indicate that intermittent hypoxia/hypercapnia leads to oxidative stress due to mitochondrial response within the mouse central nervous system.

Topics: Animals; Animals, Newborn; Apoptosis; Body Weight; Cell Death; Cerebral Cortex; Cytochromes c; Disease Models, Animal; Electron Spin Resonance Spectroscopy; Hematocrit; Hypercapnia; Hypoxia; Mice; Mitochondria; Nerve Degeneration; Neurons; Oxidation-Reduction; Oxidative Phosphorylation; Oxidative Stress; Oxygen Consumption; Superoxides; Time Factors

We investigated whether early combined autologous adipose-derived mesenchymal stem cell (ADMSC) and sildenafil therapy offers an additive benefit in preserving heart function in rat dilated cardiomyopathy (DCM).. Adult Lewis rats (n = 8 per group) were divided into group 1 (normal control), group 2 (saline-treated DCM rats), group 3 [2.0 × 106 ADMSC implanted into left ventricular (LV) myocardium of DCM rats], group 4 (DCM rats with sildenafil 30 mg/kg/day, orally), and group 5 (DCM rats with combined ADMSC-sildenafil). Treatment was started 1 week after DCM induction and the rats were sacrificed on day 90.. The results showed that mitochondrial protein expressions of connexin43 and cytochrome-C were lowest in group 2, and lower in groups 3 and 4 than in group 5 (p < 0.002). Conversely, oxidative index was highest in group 2, and also higher in groups 3 and 4 than in group 5 (p < 0.0003). The mRNA expressions of interleukin (IL)-10, Gro/IL-8, endothelial nitric oxide synthase, and Bcl-2 were lowest in group 2, and lower in groups 3 and 4 compared with group 5 (p < 0.0001). The mRNA expressions of matrix metalloproteinase-9, Bax, caspase 3, and stromal-cell derived factor-1α were highest in group 2, and higher in groups 3 and 4 than in group 5 (p < 0.0004). Apoptosis and fibrosis in LV myocardium were most prominent in group 2 and higher in groups 3 and 4 than in group 5, whereas angiogenesis and LV ejection fraction were lowest in group 2 and lower in groups 3 and 4 than in group 5 (p < 0.003).. Early combined ADMSC/sildenafil is superior to either treatment alone in preserving LV function.

Topics: Adipose Tissue; Animals; Apoptosis; Body Weight; Cardiomyopathy, Dilated; Connexin 43; Cytochromes c; Flow Cytometry; Immunohistochemistry; Male; Mesenchymal Stem Cells; Organ Size; Oxidative Stress; Phosphodiesterase Inhibitors; Piperazines; Purines; Rats; Rats, Inbred Lew; Reverse Transcriptase Polymerase Chain Reaction; Sildenafil Citrate; Sulfones; Ultrasonography

Flavonoids, polyphenol derivatives of plant origin, possess a broad range of pharmacological properties. A number of studies have found both pro/anti-apoptotic effects for many of these compounds. For these reasons we investigated whether Provinols flavonoids obtained from red wine, have anti-apoptotic properties. The investigations have been carried out in rats treated with Cyclosporine A (CsA). In particular, four groups of rats have been treated for 21 days with either olive oil (control group), with CsA, with Provinols, or with CsA and Provinols simultaneously. Oxidative stress, systolic blood pressure, body weight, biochemical parameters and different markers of pro/anti-apoptotic pathway were measured. CsA produced an increase of systolic blood pressure, a decrease in body weight, serum creatinine levels, urinary total protein concentration and creatinine clearance. Moreover, CsA induced renal alterations and the translocation of Bax and cytochrome c from cytoplasm to mitochondria and vice versa. These changes activated the caspase cascade pathway, that leads to morphological and biochemical features of apoptosis. Provinols restored morphological and biochemical alterations and prevented nephrotoxicity. In conclusion, this study may augment our current understanding of the controversial pro-/anti-apoptotic properties of flavonoids and their molecular mechanisms.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blood Pressure; Body Weight; Cyclosporine; Cytochromes c; Flavonoids; Kidney; Kidney Function Tests; Male; Oxidative Stress; Phenols; Polyphenols; Rats; Rats, Wistar; Wine

Increased myocyte apoptosis in diabetic hearts has been previously reported. Therefore, the purpose of this study was to evaluate the effects of insulin on cardiac apoptotic, hypertrophic, and survival pathways in streptozotocin (STZ)-induced diabetic rats. Forty-eight male Wistar rats at 8 weeks of age were randomly divided into control group (Control), STZ-induced (65 mg/kg STZ i.v.) Type 1-like diabetic rats (DM), and DM rats with 4 IU insulin replacement (DI) for 4 and 8 weeks, respectively. The levels of protein involved in cardiac apoptotic, hypertrophic, and survival pathways were measured by Western blotting. Cardiac mitochondrial-dependent apoptotic pathways, such as Bad, cytosolic cytochrome c, activated caspase 9 and 3, and calcineurin-nuclear factor activation transcription 3 (NFAT3) hypertrophic pathway in DM were increased compared to Control and attenuated in DI group after 8 weeks whereas those were not found after 4 weeks. Cardiac anti-apoptotic Bcl2 and phosphorylated-Bad were significantly decreased in DM group but not in DI group after 8 weeks. Insulin-like growth factor-I receptor (IGFIR), phosphatidylinositol 3'-kinase (PI3K), and the protein kinase B (Akt) were significantly decreased in DM relative to Control and DI after 8 weeks whereas those were not found after 4 weeks. Insulin replacement not only prevents activation of the cardiac mitochondrial-dependent apoptotic pathway and calcineurin-related NFAT3 hypertrophic pathway in diabetes but it also enhances the cardiac insulin/IGFIR-PI3K-Akt survival pathway, all of which are attenuated with insulin therapeutic duration-dependent manners. The findings may provide possible diabetes-related apoptotic, hypertrophic, and survival pathways for potentially preventing cardiac abnormality in diabetes.

Topics: Animals; Apoptosis; bcl-Associated Death Protein; Body Weight; Calcineurin; Cardiomegaly; Caspase 3; Caspase 9; Cell Survival; Cytochromes c; Diabetes Mellitus, Experimental; Enzyme Activation; Insulin; Mitochondria; Models, Biological; Myocardium; NFATC Transcription Factors; Organ Size; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Receptor, IGF Type 1; Streptozocin

Even though exhaustive exercise-induced oxidative stress increases the risk of tissue damage, regular endurance training is widely assumed to improve cardiac function and protects against heart disease. We tested the hypothesis that an endurance training program prevents exhaustive exercise-induced increases in cardiac dysfunction and apoptosis in left ventricle (LV). Thirty-two male Sprague-Dawley rats were randomly divided into four groups: sedentary control (C), trained (T), exhaustively exercised (E), and trained plus exhaustively exercised (TE). Rats in T and TE groups ran on a motorized treadmill for 12 weeks. Rats in groups E and TE performed an exhaustive running test on a treadmill. The main effects of training were indicated by increased running time to exhaustion (80 +/- 5 and 151 +/- 13 min for groups E and TE, respectively, P = 0.0001), myocardial hypertrophy (0.38% and 0.47% for untrained and trained rats, respectively, P = 0.0002), decreased LV ejection fraction (88% and 71% for untrained and trained rats, respectively, P < 0.0001), accelerated mitochondrial DNA 4834-bp large deletion (mtDNA4834 deletion), and up-regulated protein levels of heat shock protein-70, cytochrome C, cleaved capsase-3, and cleaved PARP in LV following a bout of exhaustive exercise. Contrary to our hypothesis, these results suggest that endurance training induced significant impairment of regional systolic and diastolic LV myocardial function and ejection fraction in rats. Our findings show that endurance training accelerates exhaustive exercise-induced mtDNA4834 deletion and apoptosis in the LV.

Topics: Animals; Apoptosis; Body Weight; Caspase 3; Cytochromes c; DNA, Mitochondrial; HSP70 Heat-Shock Proteins; Male; Matrix Metalloproteinase 9; Myocardium; Physical Conditioning, Animal; Physical Endurance; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Sequence Deletion; Ventricular Dysfunction, Left

Previous research has demonstrated that paradoxical sleep has a key role in learning and memory, and sleep deprivation interferes with learning and memory. However, the mechanism of memory impairment induced by sleep deprivation is poorly understood. The present study investigated the effect of paradoxical sleep deprivation (PSD) on spatial learning and memory using the Morris Water Maze. Effects of PSD on CA1 pyramidal neurons in hippocampus were also examined. PSD impaired spatial learning of rats. PSD induced translocation of Bax to mitochondria and cytochrome c release into the cytoplasm, and decreased the membrane excitability of CA1 pyramidal neurons, effects which may contribute to the deficits in learning behavior. These results may partially explain the mechanism of the effect of PSD on learning. Modulating the excitability of hippocampal neurons and protecting mitochondrial function are possible targets for preventing the effects of paradoxical sleep deprivation.

Topics: Animals; bcl-2-Associated X Protein; Blotting, Western; Body Weight; Cytochromes c; Cytoplasm; Data Interpretation, Statistical; Electroencephalography; Hippocampus; Male; Maze Learning; Memory; Mitochondrial Membranes; Mitochondrial Proteins; Organ Size; Protein Transport; Psychomotor Performance; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Sleep Deprivation; Sleep, REM

The aim of the present study was to test the hypothesis that peroxisome proliferator activated receptor-gamma coactivator (PGC) 1alpha is required for exercise-induced adaptive gene responses in skeletal muscle. Whole body PGC-1alpha knockout (KO) and littermate wild-type (WT) mice performed a single treadmill-running exercise bout. Soleus and white gastrocnemius (WG) were obtained immediately, 2 h, or 6 h after exercise. Another group of PGC-1alpha KO and WT mice performed 5-wk exercise training. Soleus, WG, and quadriceps were obtained approximately 37 h after the last training session. Resting muscles of the PGC-1alpha KO mice had lower ( approximately 20%) cytochrome c (cyt c), cytochrome oxidase (COX) I, and aminolevulinate synthase (ALAS) 1 mRNA and protein levels than WT, but similar levels of AMP-activated protein kinase (AMPK) alpha1, AMPKalpha2, and hexokinase (HK) II compared with WT mice. A single exercise bout increased phosphorylation of AMPK and acetyl-CoA carboxylase-beta and the level of HKII mRNA similarly in WG of KO and WT. In contrast, cyt c mRNA in soleus was upregulated in WT muscles only. Exercise training increased cyt c, COXI, ALAS1, and HKII mRNA and protein levels equally in WT and KO animals, but cyt c, COXI, and ALAS1 expression remained approximately 20% lower in KO animals. In conclusion, lack of PGC-1alpha reduced resting expression of cyt c, COXI, and ALAS1 and exercise-induced cyt c mRNA expression. However, PGC-1alpha is not mandatory for training-induced increases in ALAS1, COXI, and cyt c expression, showing that factors other than PGC-1alpha can exert these adaptations.

Topics: 5-Aminolevulinate Synthetase; Adaptation, Physiological; Animals; Blotting, Western; Body Weight; Cyclic AMP-Dependent Protein Kinases; Cyclooxygenase 1; Cytochromes c; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation; Glycogen; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria, Muscle; Muscle Proteins; Muscle, Skeletal; Myoglobin; Nucleotides; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Physical Conditioning, Animal; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trans-Activators; Transcription Factors

nocturnal sustained hypoxia during sleeping time has been reported in severe obesity, but no information regarding the cardiac molecular mechanism in the coexistence of nocturnal sustained hypoxia and obesity is available. This study evaluates whether the coexistence of nocturnal sustained hypoxia and obesity will increase cardiac Fas death receptor and mitochondrial-dependent apoptotic pathway.. 32 lean and 32 obese 5- to 6-mo-old rats with or without nocturnal sustained hypoxia were studied and assigned to one of four subgroups: normoxia lean (NL), normoxia obese (NO), hypoxia lean (HL, 12% O(2) for 8 h and 21% O(2) 16 h/day, 1 wk), and hypoxia obese (HO). The heart weight index, tail cuff plethysmography, echocardiography, hematoxylin-eosin staining, TUNEL assays, Western blotting, and RT-PCR were performed.. systolic and diastolic blood pressures in HO were higher than those in NL, and fractional shortening in HO was reduced compared with others. The whole heart weight, the left ventricular weight, the abnormal myocardial architecture, and TUNEL-positive apoptotic cells, as well as the activity of cardiac Fas-dependent and mitochondrial-dependent apoptotic pathway, were significantly increased in obese group or nocturnal sustained hypoxia group and were further increased when obesity and nocturnal sustained hypoxia coexisted, the evidence for which is based on decreases in an anti-apoptotic protein Bcl2 level and Bid and increases in Fas, FADD, pro-apoptotic Bad, BNIP3, cytosolic cytochrome c, activated caspase-8, activated caspase-9, and activated caspase-3.. The cardiac Fas receptor- and mitochondrial-dependent apoptotic pathways were more activated in obesity with coexistent nocturnal sustained hypoxia, which may represent one possible apoptotic mechanism for the development of heart failure in obesity with nocturnal sustained hypoxia.

Topics: Animals; Apoptosis; Blood Pressure; Blotting, Western; Body Weight; Caspases; Cytochromes c; Cytosol; Echocardiography; Electrophoresis, Polyacrylamide Gel; fas Receptor; Hypoxia; In Situ Nick-End Labeling; Membrane Proteins; Mitochondria, Heart; Mitochondrial Proteins; Myocardium; Obesity; Organ Size; Plethysmography; Proto-Oncogene Proteins; Rats; Rats, Zucker; Reverse Transcriptase Polymerase Chain Reaction; RNA; Sleep Apnea, Obstructive; Ventricular Function, Left

The mechanisms underlying the age-dependent reversal of female cardioprotection are poorly understood and complicated by findings that estrogen replacement is ineffective at reducing cardiovascular mortality in postmenopausal women. Although several protective signals have been identified in young animals, including PKC and Akt, how these signals are affected by age, estrogen deficiency, and ischemia-reperfusion (I/R) remains unknown. To determine the independent and combined effects of age and estrogen deficiency on I/R injury and downstream PKC-Akt signaling, adult and aged female F344 rats (n = 12/age) with ovaries intact or ovariectomy (Ovx) were subjected to I/R using Langendorff perfusion (31-min global-ischemia). Changes in cytosolic (s), nuclear (n), mitochondrial (m) PKC (delta, epsilon) levels, and changes in total Akt and mGSK-3beta phosphorylation after I/R were assessed by Western blot analysis. Senescence increased infarct size 50% in ovary-intact females (P < 0.05), whereas no differences in LV functional recovery or estradiol levels were observed. Ovx reduced functional recovery to a greater extent in aged compared with adult rats (P < 0.05). In aged (vs. adult), levels of m- and nPKC(-delta, -epsilon) were markedly decreased, whereas mGSK3beta levels were increased (P < 0.05). Ovx led to greater levels of sPKC(-delta, -epsilon) independent of age (P < 0.05). I/R reduced p-Akt(Ser473) levels by 57% and increased mGSK-3beta accumulation 1.77-fold (P < 0.05) in aged, ovary-intact females. These data suggest, for the first time, that estrogen alone cannot protect the aged female myocardium from I/R damage and that age- and estrogen-dependent alterations in PKC, Akt, and GSK-3beta signaling may contribute to loss of ischemic tolerance.

Topics: Aging; Animals; Blotting, Western; Body Weight; Coloring Agents; Coronary Circulation; Cytochromes c; Estrogens; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; In Vitro Techniques; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Oncogene Protein v-akt; Ovariectomy; Protein Kinase C-delta; Protein Kinase C-epsilon; Rats; Rats, Inbred F344; Tetrazolium Salts

Ciliary neurotrophic factor (CNTF) can lead to weight loss by up-regulating energy metabolism and the expression of UCP-1 in mitochondria. To investigate the up-stream regulators of the expression of UCP-1, recombinant human CNTF (rhCNTF) (0.1, 0.3, 0.9 mg/kg/day s.c.) administered to KK-Ay mice for 30 days resulting in reductions in body weight and perirenal fat mass. In brown adipose tissues, the gene expressions of nuclear respiratory factor (NRF)-1, mitochondrial transcription factor A (TFam) and uncoupling protein (UCP)-1 were found up-regulated by rhCNTF. To the best of our knowledge, these effects represent new insights on the mechanisms of action of weight loss by rhCNTF. In addition, we also found that rhCNTF increased the activity of mitochondrial complex IV. The stimulation of NRF-1, TFam, UCP-1 and the enhanced activity of mitochondrial complex IV may be associated with remedying obesity. The result indicates that rhCNTF can enhance the expressions of NRF-1 and TFam, both of which can up-regulate the expression of UCP-1.

Topics: Adipose Tissue, Brown; Animals; Anti-Obesity Agents; Body Weight; Ciliary Neurotrophic Factor; Cytochromes c; DNA-Binding Proteins; Dose-Response Relationship, Drug; Electron Transport Complex IV; Humans; Ion Channels; Membrane Potential, Mitochondrial; Mice; Mice, Obese; Mitochondria; Mitochondrial Proteins; Nuclear Respiratory Factor 1; RNA, Messenger; Time Factors; Transcription Factors; Uncoupling Protein 1; Up-Regulation

Spermatogenic cells constitute one of the body tissues that are susceptible to doxorubicin-induced oxidative stress and apoptosis. To explore whether doxorubicin toxicity to these male germ cells could be prevented by adjuvant medication, this study was designed to examine the possible ameliorating action of doxycycline, an antibiotic with anti-oxidant property, on doxorubicin-induced oxidative and apoptotic effects in mouse testes. Male mice at 5-week of age were treated with vehicles, doxorubicin alone (3 mg/kg, i.p. every other day for 3 doses), doxycycline alone (2.5 mg/kg, i.p. every other day for 3 doses), or doxycycline plus doxorubicin (each dose given 1 day post-doxycycline). After 28 days, mice treated with doxorubicin alone displayed smaller body and testicular weights, reduced sperm counts, impaired spermatogenic capability (scarcer spermatids and spermatocytes), increased oxidative stress (malondialdehyde levels), decreased anti-oxidant activity (superoxide dismutase and glutathione peroxidase), and elevated apoptotic indexes (upregulation of Bax and Bad, downregulation of Bcl-2 and Bcl-xL, release of cytochrome c from mitochondria to cytosol, activation of caspase-3, and increase of cleaved caspase-3 abundance and TUNEL positive cells), while doxycycline pretreatment could effectively prevent nearly all of these abnormalities. These results provide firm evidence that doxycycline pretreatment would offset the oxidative and apoptotic impact imposed by doxorubicin, and imply doxycycline to be a promising adjuvant agent that may attenuate the toxicity of doxorubicin on testicular tissues in clinical practice.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Body Weight; Caspase 3; Cytochromes c; Down-Regulation; Doxorubicin; Doxycycline; Male; Mice; Mice, Inbred ICR; Organ Size; Oxidative Stress; Spermatogenesis; Testis; Up-Regulation

Obesity is often associated with the development of heart failure, but the precise mechanisms remain uncertain. The purpose of this study was to evaluate the key components of the mitochondrial-dependent apoptotic pathway in excised heart from obese Zucker rats.. Twelve obese Zucker rats were studied at 5 to 6 months of age, and 12 age-matched lean Zucker rats served as control. The myocardial architecture and key components of the mitochondrial-dependent apoptotic pathway in the excised left ventricle from rats were measured by histopathological analysis, Western blotting, and reverse transcription polymerase chain reaction (RT-PCR).. The ratios of whole heart weight to tibia length were significantly increased in the obese group. Cardiomyocyte disarray, the increased interstitial space, and minor cardiac fibrosis were observed in obese rat hearts. Pro-apoptotic Bcl2 family members, Bcl-2/adenovirus E1B 19 kDa interacting protein (BNIP3) and Bad levels, were significantly increased in obese rat hearts, whereas anti-apoptotic Bcl2 family member, Bcl2 level, was significantly decreased. Cytosolic cytochrome c indicating cytochrome c release from mitochondria was significantly increased in obese rat heart. In addition, upstream pro-caspase-9 and pro-caspase-3 were significantly decreased, whereas activated caspase-9 and activated caspase-3 were significantly increased in obese rat hearts, compared with lean rat heart, implying that pro-forms of caspase-9 and caspase-3 were cleaved into active-forms caspase-9 and caspase-3.. The cardiac mitochondrial-dependent apoptotic pathway was more activated in obese Zucker rats than in lean rats, which may provide one possible apoptotic mechanism for developing heart failure in obesity.

Topics: Animals; Apoptosis; bcl-Associated Death Protein; Body Weight; Caspase 3; Caspase 9; Cytochromes c; Disease Models, Animal; Male; Membrane Proteins; Mitochondria, Heart; Mitochondrial Proteins; Myocardium; Obesity; Organ Size; Proto-Oncogene Proteins; Rats; Rats, Zucker

In spite of opposing changes in rates of adenosine triphosphate turnover, hypertrophy and atrophy of the heart are accompanied by the same changes in gene expression, resembling a fetal genotype. Fetal hearts are characterized by increased ischemia tolerance. We assessed respiratory capacity of mitochondrial subpopulations from unloaded and pressure-overloaded hearts before and after 15 minutes of normothermic ischemia. Unloading was achieved by heterotopic rat heart transplantation and overloading by aortic banding. Respiratory chain gene expression (NADH dehydrogenase, cytochrome c oxidase [COX]) were analyzed by reverse transcriptase-polymerase chain reaction. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated by differential centrifugation. Citrate synthase was used as mitochondrial marker enzyme. Adenosine diphosphate-stimulated oxygen consumption (state 3) was measured with a Clark-type electrode. Unloading resulted in atrophy, overloading in hypertrophy. State 3 was reduced in atrophied hearts both in SSM and IFM (SSM: 204 +/- 79 vs 804 +/- 147 natoms oxygen min(-1) mL(-1), P < .001; IFM: 468 +/- 158 vs 1141 +/- 296 natoms oxygen min(-1) mL(-1), P < .05), but was unchanged in hypertrophied hearts. NADH dehydrogenase and COX expression was also decreased with atrophy and was unchanged with hypertrophy. Ischemia caused decreased recovery of citrate synthase in isolates of SSM (P < .05) but not of IFM. State 3 in control hearts was reduced in IFM (-41%, P < .01) and SSM (-19%, not significant). This ischemia-induced decrease was less pronounced in SSM (-2%) and IFM (-22%) of atrophied and IFM (-23%) of hypertrophied hearts. Subsarcolemmal mitochondria of hypertrophied hearts displayed the greatest ischemia-induced decrease of state 3 (-32%, P < .05). In conclusion, (1) long-term changes in workload differentially affect maximal respiratory capacity and ischemia tolerance of isolated mitochondria. The changes are not parallel to the changes in energy requirements. (2) Mitochondria of atrophied hearts appear to be more resistant against ischemia than controls.

Topics: Adenosine Diphosphate; Animals; Atrophy; Body Weight; Cardiomegaly; Citrate (si)-Synthase; Cytochromes c; Electron Transport; Gene Expression Regulation, Enzymologic; Heart Diseases; In Vitro Techniques; Male; Mitochondria, Heart; Muscle Proteins; Myocardial Ischemia; Myocardial Reperfusion Injury; NADH Dehydrogenase; Organ Size; Oxygen Consumption; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction

The aim of this study was to characterize the response to exercise training in several mouse strains and estimate the genetic contribution to phenotypic variation in the responses to exercise training. Male mice from three inbred strains [C57Bl/6J (BL6), FVB/NJ (FVB), and Balb/cJ (Balb/c)] and three hybrid F(1) strains [CB6F1/J (CB6 = female Balb/c x male BL6), B6F F(1) (female BL6 x male FVB), and FB6 F(1) (female FVB x male BL6)] completed an exercise performance test before and after a 4-wk treadmill running program. Distance was used as the primary estimate of endurance exercise performance. FVB mice showed the greatest response to training, with five- to sevenfold greater increases in distance run compared with BL6 and Balb/c strains. Specifically, BL6, FVB, and Balb/c strains increased distance by 33, 172, and 23%, respectively. A similar pattern of changes across strains was observed for run time (17, 87, and 11%) and work (99, 287, and 57%). As a group, F(1) hybrid mice derived from BL6 and FVB strains showed an intermediate response to training (61%). However, further analysis indicated that training responses in FB6 F(1) mice (80%) were approximately 2.5-fold greater than responses in B6F F(1) mice (33%, P = 0.08). A similar pattern of changes between FB6 and B6F F(1) mice was observed for run time (44.5 and 17%) and work (141 and 59%). These data demonstrate that there are large strain-dependent differences in training responses among inbred mouse strains, suggesting that genetic background contributes significantly to adaptation to exercise. Furthermore, the contrasting responses in B6F and FB6 F(1) strains show that a maternal component strongly influences strain-dependent differences in training responses.

Topics: Animals; Blotting, Western; Body Composition; Body Weight; Cytochromes c; Female; Hybridization, Genetic; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Muscle, Skeletal; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Physical Conditioning, Animal; Physical Endurance; Physical Exertion; Pregnancy; Running; Species Specificity

Iron deficiency is associated with multiple health problems, including the cardiovascular system. However, the mechanism of action of iron-deficiency-induced cardiovascular damage is unclear. The aim of the present study was to examine the effect of dietary iron deficiency on cardiac ultrastructure, mitochondrial cytochrome c release, NOS (nitric oxide synthase) and several stress-related protein molecules, including protein nitrotyrosine, the p47phox subunit of NADPH oxidase, caveolin-1 and RhoA. Male weanling rats were fed with either control or iron-deficient diets for 12 weeks. Cardiac ultrastructure was examined by transmission electron microscopy. Western blot analysis was used to evaluate cytochrome c, endothelial and inducible NOS, NADPH oxidase, caveolin-1 and RhoA. Protein nitrotyrosine formation was measured by ELISA. Rats fed an iron-deficient diet exhibited increased heart weight and size compared with the control group. Heart width, length and ventricular free wall thickness were similar between the two groups. However, the left ventricular dimension and chamber volume were significantly enhanced in the iron-deficient group compared with controls. Ultrastructural examination revealed mitochondrial swelling and abnormal sarcomere structure in iron-deficient ventricular tissues. Cytochrome c release was significantly enhanced in iron-deficient rats. Protein expression of eNOS (endothelial NOS) and iNOS (inducible NOS), and protein nitrotyrosine formation were significantly elevated in cardiac tissue or mitochondrial extraction from the iron-deficient group. Significantly up-regulated NADPH oxidase, caveolin-1 and RhoA expression were also detected in ventricular tissue of the iron-deficient group. Taken together, these results suggest that dietary iron deficiency may have induced cardiac hypertrophy characterized by aberrant mitochondrial and irregular sarcomere organization, which was accompanied by increased reactive nitrogen species and RhoA expression.

Topics: Animals; Body Weight; Cardiomegaly; Cytochromes c; Heart Ventricles; Heat-Shock Proteins; Iron Deficiencies; Male; Microscopy, Electron; Mitochondria, Heart; Myocardium; Myocytes, Cardiac; Nitric Oxide Synthase; Organ Size; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Tyrosine

Muscle atrophy is a determinant of exercise capacity in heart failure (CHF). Myocyte apoptosis, triggered by tumor necrosis factor-alpha (TNF-alpha) or its second messenger sphingosine (SPH), is one of the causes of atrophy. Growth hormone (GH) improves hemodynamic and cardiac trophism in several experimental models of CHF, but its effect on skeletal muscle in CHF is not yet clear. We tested the hypothesis that GH can prevent skeletal muscle apoptosis in rats with CHF. CHF was induced by injecting monocrotaline. After 2 wk, 2 groups of rats were treated with GH (0.2 mg.kg(-1).day(-1) and 1.0 mg.kg(-1).day(-1)) subcutaneously. A third group of controls had saline. After 2 additional weeks, rats were killed. Tibialis anterior cross-sectional area, myosin heavy chain (MHC) composition, and a study on myocyte apoptosis and serum levels of TNF-alpha and SPH were carried out. The number of apoptotic nuclei, muscle atrophy, and serum levels of TNF-alpha and SPH were decreased with GH at high but not at low doses compared with CHF rats. Bcl-2 was increased, whereas activated caspases and bax were decreased. The MHC pattern in GH-treated animals was similar to that of controls. Monocrotaline slowed down both contraction and relaxation but did not affect specific tetanic force, whereas absolute force was decreased. GH treatment restored contraction and relaxation to control values and brought muscle mass and absolute twitch and tetanic tension to normal levels. These findings may provide an insight into the therapeutic strategy of GH given to patients with CHF to improve exercise capacity.

Topics: Angiotensin II; Animals; Apoptosis; Body Weight; Cardiac Output, Low; Caspases; Cytochromes c; Human Growth Hormone; In Situ Nick-End Labeling; Insulin-Like Growth Factor I; Isometric Contraction; Male; Monocrotaline; Muscle, Skeletal; Muscular Atrophy; Myosin Heavy Chains; Physical Endurance; Rats; Rats, Sprague-Dawley; Sphingosine; Tumor Necrosis Factor-alpha

The mechanisms of apoptosis in the loss of myocytes in skeletal muscle with age and the role of mitochondrial and sarcoplasmic reticulum-mediated pathways of apoptosis are unknown. Moreover, it is unknown whether lifelong calorie restriction prevents apoptosis in skeletal muscle and reverses age-related alterations in apoptosis signaling. We investigated key apoptotic regulatory proteins in the gastrocnemius muscle of 12 and 26 month old ad libitum fed and 26 month old calorie-restricted male Fischer-344 rats. We found that apoptosis increased with age and that calorie-restricted rats showed less apoptosis compared with their age-matched cohorts. Moreover, pro- and cleaved caspase-3 levels increased significantly with age and calorie-restricted rats had significantly lower levels than the aged ad libitum group. Neither age nor calorie restriction had any effect on muscle caspase-3 enzyme activity, but the levels of X-linked inhibitor of apoptosis, particularly an inhibitor of caspase-3, increased with age and were reduced significantly in the 26 month old calorie-restricted cohort. The apoptotic inhibitor apoptosis repressor with a caspase recruitment domain (ARC), which inhibits cytochrome c release, underwent an age-associated decline in the cytosol but increased with calorie restriction. In contrast, mitochondrial ARC levels increased with age and were lower in calorie-restricted rats than in age-matched controls, suggesting a translocation of this protein to attenuate oxidative stress. The translocation of ARC may explain the reduction in cytosolic cytochrome c levels observed with age and calorie restriction. Moreover, we found a striking approximately 350% increase in the expression of procaspase-12 (caspase located at the sarcoplasmic reticulum) with age which was significantly lower in the 26 month old calorie-restricted group. The total protein level of apoptosis-inducing factor in the plantaris muscle increased with age and was reduced calorie-restricted rats compared with age-matched controls, but there were no significant changes in this pro-apoptotic protein in the isolated nuclei. Calorie restriction is able to lower the apoptotic potential in aged skeletal muscle by altering several key apoptotic proteins toward cellular survival, thereby reducing the potential for sarcopenia.

Topics: Aging; Animals; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Apoptotic Protease-Activating Factor 1; Body Weight; Caloric Restriction; Caspase 12; Caspase 3; Caspase 9; Caspases; Cytochromes c; Flavoproteins; Inhibitor of Apoptosis Proteins; Male; Membrane Proteins; Mitochondria; Muscle Proteins; Muscle, Skeletal; Organ Size; Protein Precursors; Proteins; Rats; Rats, Inbred F344; X-Linked Inhibitor of Apoptosis Protein

We investigated whether permeability transition-mediated release of mitochondrial cytochrome c is a potential therapeutic target for treating acute spinal cord injury (SCI). Based on previous reports, minocycline, a second-generation tetracycline, exerts neuroprotection partially by inhibiting mitochondrial cytochrome c release and reactive microgliosis. We first evaluated cytochrome c release at the injury epicenter after a T10 contusive SCI in rats. Cytochrome c release peaked at approximately 4-8 h postinjury. A dose-response study generated a safe pharmacological regimen that enabled i.p. minocycline to significantly lower cytosolic cytochrome c at the epicenter 4 h after SCI. In the long-term study, i.p. minocycline (90 mg/kg administered 1 h after SCI followed by 45 mg/kg administered every 12 h for 5 days) markedly enhanced long-term hind limb locomotion relative to that of controls. Coordinated motor function and hind limb reflex recoveries also were improved significantly. Histopathology suggested that minocycline treatment alleviated later-phase tissue loss, with significant sparing of white matter and ventral horn motoneurons at levels adjacent to the epicenter. Furthermore, glial fibrillary acidic protein and 2',3' cyclic nucleotide 3' phosphodiesterase immunocytochemistry showed an evident reduction in astrogliosis and enhanced survival of oligodendrocytes. Therefore, release of mitochondrial cytochrome c is an important secondary injury mechanism in SCI. Drugs with multifaceted effects in antagonizing this process and microgliosis may protect a proportion of spinal cord tissue that is clinically significant for functional recovery. Minocycline, with its proven clinical safety, capability to cross the blood-brain barrier, and demonstrated efficacy during a clinically relevant therapeutic window, may become an effective therapy for acute SCI.

Topics: Animals; Astrocytes; Body Weight; Cytochromes c; Disease Models, Animal; Female; Kinetics; Minocycline; Mitochondria; Oligodendroglia; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries

Nonalcoholic fatty liver disease, frequently associated with obesity, can lead to nonalcoholic steatohepatitis (NASH) and cirrhosis. The pathophysiology of NASH is poorly understood, and no effective treatment is available. In view of a potential deleterious role for reactive oxygen species (ROS), we investigated the origin of ROS overproduction in NASH. Mitochondrial production of ROS and its alterations in the presence of antioxidant molecules were studied in livers from ob/ob mice that bear a mutation of the leptin gene and develop experimental NASH. N-acetyl-cysteine and the superoxide dismutase (SOD) mimics ambroxol, manganese [III] tetrakis (5,10,15,20 benzoic acid) (MnTBAP), and copper [II] diisopropyl salicylate (CuDIPS) were used to target different checkpoints of the oxidative cascade to determine the pathways involved in ROS production. Liver mitochondria from ob/ob mice generated more O(2)*- than those of lean littermates (P <.01). Ex vivo, all three SOD mimics decreased O(2)*- generation (P <.001) and totally inhibited lipid peroxidation (P <.001) versus untreated ob/ob mice. Those modifications were associated with in vivo improvements: MnTBAP and CuDIPS reduced weight (P <.02) and limited the extension of histological liver steatosis by 30% and 52%, respectively, versus untreated ob/ob mice. In conclusion, these data demonstrate deleterious effects of superoxide anions in NASH and point at the potential interest of nonpeptidyl mimics of SOD in the treatment of NASH in humans.

Topics: Animals; Antioxidants; Biomarkers; Body Weight; Caspase 3; Caspases; Cytochromes c; Fatty Liver; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Mitochondria, Liver; Nitric Oxide; Superoxide Dismutase; Superoxides

Previous studies found that repeated application of smoke condensate from tobacco-burning reference cigarettes to chemically initiated SENCAR mouse skin promoted the development of tumors in a statistically significant and dose-dependent manner, while condensate from prototype cigarettes that primarily heat tobacco promoted statistically fewer tumors. Based on the recognized correlation between sustained, potentiated epidermal hyperplasia and tumor promotion, we conducted tests to examine the utility of selected short-term analyses for discriminating between condensates exhibiting significantly different promotion activities. In vitro analyses assessing the potential for inducing cytotoxicity (ATP bioluminescence) or free radical production (cytochrome c reduction, salicylate trapping) demonstrated significant reductions when comparing condensate collected from prototype cigarettes to reference condensate. Short-term in vivo analyses conducted within the context of a mouse skin, tumor-promotion protocol (i.e., comparative measures of epidermal thickness, proliferative index, myeloperoxidase activity, leukocyte invasion, mutation of Ha-ras, and formation of modified DNA bases) provided similar results. Reference condensate induced statistically significant and dose-dependent increases (relative to vehicle control) for nearly all indices examined, while prototype condensate possessed a significantly reduced potential for inducing changes that we regarded as consistent with sustained epidermal hyperplasia and/or inflammation. Collectively, these data support the contention that selected short-term analyses associated with sustained hyperplasia and/or inflammation are capable of discriminating between smoke condensates with dissimilar tumor-promotion potentials. Moreover, our results suggest that comparative measures of proliferative index and myeloperoxidase activity, both possessing favorable correlation coefficients relative to tumor formation (i.e., > or = 0.95 after 8 or 12 weeks of promotion), may constitute reasonable end points for further investigation.

Topics: Adenosine Triphosphate; Animals; Body Weight; Carcinogenicity Tests; Carcinogens; Cell Proliferation; Cytochromes c; DNA Adducts; Genes, ras; Hydroxyl Radical; Hyperplasia; Inflammation; Leukocytes; Luminescent Measurements; Mice; Mice, Inbred SENCAR; Oxidation-Reduction; Oxidative Stress; Peroxidase; Salicylates; Skin; Skin Neoplasms; Smoke; Superoxides

We investigated in vivo the chemotherapeutic anthracycline agents doxorubicin and its ability to activate mitochondrial-mediated, receptor-mediated and endoplasmic/sarcoplasmic reticulum-mediated apoptosis transduction pathways in cardiac tissue from male and female rats. We administered a single low dose of doxorubicin (10 mg/kg of body weight, i.p.) and then isolated mitochondrial and cytosolic proteins one and four days later from the heart. Caspase-3 protein content and caspase-3 activity were significantly increased after day four of doxorubicin treatment in both male and female rats. However, while males had DNA fragmentation at day one but not day four following doxorubicin administration, females showed no significant increase in DNA fragmentation at either time. Caspase-12, localized in the SR, is considered a central caspase, and its activation by cleavage via calpain indicates activation of the SR-mediated pathway of apoptosis. Cleaved caspase-12 content and calpain activity significantly increased after day four of doxorubicin treatment in both sexes. In the mitochondrial-mediated pathway, there were no significant treatment effects observed in cytosolic cytochrome c and cleaved (active) caspase-9 in either sex. In control rats (saline injection), glutathione peroxidase (GPX) activity and hydrogen peroxide (H2O2) production were lower in females compared to males. Doxorubicin treatment did not significantly affect H2O2, GPX activity or ATP production in isolated mitochondria in either sex. Female rats produced significantly lower levels of H2O2 production one day after doxorubicin treatment, whereas male rats produced significantly less mitochondrial H2O2 four days after doxorubicin treatment. The receptor-mediated pathway (caspase-8 and c-FLIP) showed no evidence of being significantly activated by doxorubicin treatment. Hence, doxorubicin-induced apoptosis in vivo is mediated by the SR to a greater extent than other apoptotic pathways and should therefore be considered for targeted therapeutic interventions. Moreover, no major sex differences exist in apoptosis signaling transduction cascade due to doxorubicin treatment.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Body Weight; Calpain; Caspase 12; Caspases; Cytochromes c; Cytosol; Doxorubicin; Endoplasmic Reticulum; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Estradiol; Female; Glutathione Peroxidase; Heart Ventricles; Male; Mitochondria, Heart; Models, Biological; Organ Size; Rats; Rats, Inbred F344; Sarcoplasmic Reticulum; Sex Factors; Signal Transduction; Time Factors

Reductions in copper due to dietary restriction or transporter deficiency in brindled mice or humans with Menkes disease lead to reduced cuproenzyme activities, mitochondrial abnormalities, neurodegeneration and early mortality. The mechanisms for observed neuropathology remain unknown. Some researchers studying mutant mice suggest brain apoptosis as a possible factor based on changes in transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and increased cytosolic cytochrome c and decreased Bcl-2 levels. Perinatal copper deficiency was induced in Holtzman rats during late gestation and lactation to investigate the role of apoptosis in the developing brain. Analysis of 13- and 24-d-old (P13 and P24) brains from male copper-deficient and copper-adequate rats revealed no difference in cytosolic cytochrome c or total Bcl-2 levels. Cerebellar TUNEL staining and caspase-3 activity were higher in the P12 copper-deficient than in the copper-adequate pups. However, TUNEL staining decreased and caspase-3 activity was not detected at P24 even though pups were more copper deficient based on cortex copper, Cu, Zn-superoxide dismutase and cytochrome c oxidase activities. This suggests that neuronal apoptosis is not enhanced by dietary copper deficiency in the brain. Lower Bcl-2 levels were detected in the copper-deficient rat hearts, consistent with apoptotic processes in mice reported by others. A robust enhancement of cytochrome c was observed in the total brain extracts and purified brain mitochondria of copper-deficient pups. Higher cytochrome c appeared to be correlated with the degree of copper deficiency and seemed to be associated with increased mitochondrial mass, because higher levels of voltage-dependent anion channel and mitochondrial complex I were also detected. The biochemical mechanisms for elevated cytochrome c are not known nor are the physiological consequences.

Topics: Animals; Animals, Newborn; Apoptosis; Body Weight; Brain; Cerebral Cortex; Copper; Cytochromes c; Female; Liver; Mitochondria; Organ Size; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Sprague-Dawley