cytochrome-c-t and Starvation

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to be a "housekeeping" protein; studies in non-cardiomyocytic cells have shown that GAPDH plays pro-apoptotic role by translocating from cytoplasm to the nucleus or to the mitochondria. However, the cardiovascular roles of GAPDH are unknown. We observed that phenylephrine (PE) (100 µM) protected against serum and glucose starvation -induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and mitochondrial membrane potential depolarization. GAPDH glycolysis activity was positively correlated with the antiapoptotic action of PE. GAPDH activity inhibition blunted PE-induced protection of the mitochondrial membrane potential and cardiomyocytes. PE-induced Bcl-2 protein increase, Bax mitochondrial decrease and inhibition of cytochrome C release and Caspase 3 activation, as well as ROS production were blunted by GAPDH activity inhibition. Moreover, GAPDH overexpression provided protection against starvation-induced cardiomyocyte apoptosis in vitro and ischemia-induced cardiac infarction in vivo. Inhibition of Akt prevented PE-induced GAPDH activity increase and cardiomyocytes protection. In conclusion, the present study provides the first direct evidence of an antiapoptotic role of GAPDH in PE-induced cardiomyocytes protection; GAPDH activity elevation mainly affects the mitochondria-induced apoptosis.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cells, Cultured; Cytochromes c; DNA Nucleotidylexotransferase; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycolysis; In Situ Nick-End Labeling; Membrane Potential, Mitochondrial; Mitochondria; Myocytes, Cardiac; Phenylephrine; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species; Starvation

The aim of the present study was to investigate whether fasting for 24 and 48 h induces apoptosis of rat mesenteric lymph node lymphocytes similar to that observed previously in diabetic patients and alloxan-induced diabetic rats. Several features of lymphocyte death were evaluated by flow cytometry. Plasma levels of glucose, NEFAs (non-esterified fatty acids) and ketone bodies (acetoacetate and beta-hydroxybutyrate) were determined in rats fasted for 24 and 48 h. Lymphocytes obtained from fasted rats had an increase in DNA fragmentation and phosphatidylserine externalization after 48 h of culture, although there was no loss of membrane integrity in lymphocytes even after 48 h of culture. Cytochrome c release from the mitochondrial intermembrane space into the cytosol was increased significantly in lymphocytes from fasted rats cultured for 24 h, whereas the levels of bcl-2 and bax proteins were not affected. Activities of caspases 3, 6, 8 and 9 were increased significantly in lymphocytes from rats fasted for 24 h, whereas only an increase in caspase 3 and 9 activities were observed in rats fasted for 48 h. In conclusion, fasting for 24 and 48 h caused a significant increase in the proportion of lymphocytes undergoing apoptosis. The occurrence of apoptosis was observed by DNA fragmentation, phosphatidylserine externalization, cytochrome c release from the mitochondria and activation of the caspase cascade. These findings support the hypothesis that conditions that raise plasma fatty acids levels (e.g. diabetes and starvation) may impair immune function by causing lymphocyte death.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Biological Transport; Blotting, Western; Caspases; Cell Membrane; Cells, Cultured; Chromosomes; Cytochromes c; DNA Fragmentation; Lipid Metabolism; Lymphocytes; Male; Mitochondria; Phosphatidylserines; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Starvation

Prion diseases are transmissible neurodegenerative disorders that are invariably fatal in humans and animals. Although the nature of the infectious agent and pathogenic mechanisms of prion diseases are not clear, it has been reported that prion diseases may be associated with aberrant metabolism of cellular prion protein (PrP(C)). In various reports, it has been postulated that PrP(C) may be involved in one or more of the following: neurotransmitter metabolism, cell adhesion, signal transduction, copper metabolism, antioxidant activity or programmed cell death. Despite suggestive results supporting each of these mechanisms, the physiological function(s) of PrP(C) is not known. To investigate whether PrP(C) can prevent apoptotic cell death in prion diseases, we established the cell lines stably expressing PrP(C) from PrP knockout (PrP(-/-)) neuronal cells and examined the role of PrP(C) under apoptosis and/or serum-deprived condition. We found that PrP(-/-) cells were vulnerable to apoptotic cell death and that this vulnerability was rescued by the expression of PrP(C). The expression levels of apoptosis-related proteins including p53, Bax, caspase-3, poly(ADP-ribose) polymerase (PARP) and cytochrome c were significantly increased in PrP(-/-) cells. In addition, Ca(2+) levels of mitochondria were increased, whereas mitochondrial membrane potentials were decreased in PrP(-/-) cells. These results strongly suggest that PrP(C) may play a central role as an effective anti-apoptotic protein through caspase-dependent apoptotic pathways in mitochondria, supporting the concept that disruption of PrP(C) and consequent reduction of anti-apoptotic capacity of PrP(C) may be one of the pathogenic mechanisms of prion diseases.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Calcium; Caspase 3; Caspases; Cells, Cultured; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Fluoresceins; Hippocampus; Membrane Glycoproteins; Mice; Mice, Knockout; Microscopy, Fluorescence; Mitochondria; Neurons; Polymerase Chain Reaction; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; PrPC Proteins; Serum; Starvation; Time Factors; Transfection; Tumor Suppressor Protein p53

Hypoxia induces apoptosis in primary and transformed cells and in various tumor cell lines in vitro. In contrast, there is little apoptosis and predominant necrosis despite extensive hypoxia in human glioblastomas in vivo. We here characterize ultrastructural and biochemical features of cell death in LN-229, LN-18 and U87MG malignant glioma cells in a paradigm of hypoxia with partial glucose deprivation in vitro. Electron microscopic analysis of hypoxia-challenged glioma cells demonstrated early stages of apoptosis but predominant necrosis. ATP levels declined during hypoxia, but recovered with re-exposure to normoxic conditions unless hypoxia exceeded 8 h. Longer hypoxic exposure resulted in irreversible ATP depletion and delayed cell death. Hypoxia induced mitochondrial release of cytochrome c, but there was no cleavage of caspases 3, 7, 8 or 9, and no DNA fragmentation. Ectopic expression of BCL-XL conferred protection from hypoxia-induced cell death, whereas the overexpression of the antiapoptotic proteins X-linked-inhibitor-of-apoptosis-protein and cytokine response modifier-A had no effect. These findings suggest that glioma cells resist adverse effects of hypoxia until energy stores are depleted and then undergo necrosis rather than apoptosis because of energy deprivation.

Topics: Adenosine Triphosphate; bcl-X Protein; Brain Neoplasms; Caspases; Cell Death; Cell Hypoxia; Cell Nucleus; Cytochromes c; Cytoplasm; Energy Metabolism; Glioma; Glucose; Humans; Immunologic Factors; Microscopy, Electron; Mitochondria; Necrosis; Proteins; Proto-Oncogene Proteins c-bcl-2; Starvation; Tumor Cells, Cultured; X-Linked Inhibitor of Apoptosis Protein

Topics: Animals; Cytochromes c; Kidney; Lagomorpha; Liver; Oxidoreductases; Rabbits; Starvation

Topics: Animals; Cytochromes; Cytochromes c; Lagomorpha; Liver; Rabbits; Starvation