Tatsuya Fujimiya

Phosphoinositide 3-kinase accelerates autophagic cell death during glucose deprivation in the rat cardiomyocyte-derived cell line H9c2

We investigated cell death during glucose deprivation in rat cardiomyocyte-derived H9c2 cells. Electron microscopic analysis revealed accumulation of autophagic vacuoles during glucose deprivation. The addition of 3-methyladenine or LY294002, which are known to inhibit autophagosome formation, reduced cell death while Z-VAD-FMK, a caspase inhibitor, slightly affected cell death. Thus, cell death during glucose deprivation is not type I programmed cell death (apoptotic cell death) but type II programmed cell death (autophagic cell death). Moreover, we found that both insulin-like growth factor-I and the adenovirus-mediated overexpression of wild-type class I PI 3-kinase accelerated cell death as well as accumulation of autophagic vacuoles during glucose deprivation while dominant-negative PI 3-kinase reduced these phenomena. The results indicate that IGF-I/PI 3-kinase accelerates the accumulation of autophagic vacuoles and subsequent autophagic cell death during glucose deprivation, revealing the opposing role of IGF-I/PI 3-kinase in two distinct types of programmed cell death (apoptotic and autophagic cell death).

The patient's right to information in Japan-legal rules and doctor's opinions

A questionnaire survey concerning informed consent was administered among Japanese physicians in Yamaguchi prefecture. The survey results showed that even though these Japanese physicians are willing to give their patients sufficient information to obtain informed consent, the discretion of the physician to provide information is still prevalent. The survey also revealed that Japanese physicians believe that information regarding the treatment to be administrated should be fully disclosed both in case when the treatment is still experimental and when it is established among specialists. Finally, the survey showed that despite the liberal attitude of the Japanese physicians toward informed consent, they are reluctant to make medical records accessible to the patients. It was found that when Japanese physicians were faced with special cases such as prior to administering high-risk diagnostic procedures, prior to disclosing the presence of cancer in their patients, or when faced with patients unwilling to receive treatment, the involvement of the patients family members in the decision-making process was preferred so as not to aggravate the patients emotional anxiety. With respect to cancer patients, the survey suggested that many Japanese physicians believe that cancer operations performed without informed consent from the patient should be legal. Finally, the survey concluded that, although the concept of individualized informed consent has been generally accepted among physicians, the involvement of family members in the decision-making process and a conservative attitude toward disclosure of information are still prevalent in Japan.

Phosphoinositide 3-kinase accelerates necrotic cell death during hypoxia.

Using H9c2 cells derived from rat cardiomyocytes, we investigated the mechanism of cell death during hypoxia in the presence of serum and glucose. Hypoxic cell death is by necrosis and is accompanied by metabolic acidosis. Moreover, hypoxic cell death is inhibited by Hepes buffer as well as by 2-deoxyglucose, an inhibitor of glycolysis, indicating that metabolic acidosis should play an essential role in hypoxic injury. The involvement of phosphoinositide 3-kinase (PI 3-kinase), which is known to activate glucose metabolism, was examined using its inhibitor, LY290042, or adenovirus-mediated gene transfer. Hypoxic cell death was inhibited by LY294002 in a dose-dependent manner. Overexpression of dominant negative PI 3-kinase was found to reduce cell death, whereas wild-type PI 3-kinase enhanced it. Dominant negative PI 3-kinase also reduced glucose consumption and acidosis, but this was stimulated by wild-type PI 3-kinase. The data indicate that PI 3-kinase stimulates cell death by enhancing metabolic acidosis. LY294002 significantly reduced glucose uptake, showing that PI 3-kinase regulates glycolysis at the step of glucose transport. These findings indicate the pivotal role of glucose metabolism in hypoxic cell death, and reveal a novel death-promoting effect of PI 3-kinase during hypoxia, despite this enzyme being considered to be a survival-promoting factor.

Pathological roles of bone marrow-derived stellate cells in a mouse model of alcohol-induced fatty liver

Chronic alcohol consumption activates hepatic stellate cells (HSCs) and causes fatty degeneration in the liver. However, the origin of HSCs and the mechanism of fatty changes of the liver have not been fully elucidated. Here, we examined the roles of bone marrow-derived cells (BMDCs) in a mouse model with chronic alcohol consumption. We performed bone marrow transplantation from transgenic mice expressing green fluorescence protein (GFP) to female wild-type and ROSA mice (B6.129S7-Gt 26Sor/J, transgenic mice expressing beta-galactosidase, beta-gal) and treated them with ethanol (EtOH) for 8 or 16 wk. GFP-expressing BMDCs increased in the liver with EtOH treatment in a time-dependent manner. In response to excess alcohol consumption, approximately 68% of the BMDCs became activated HSCs in that they expressed alpha-smooth muscle actin. Meanwhile, approximately 67% and approximately 66% of these BMDCs expressed Tnf-alpha and transforming growth factor (Tgf)-beta1, respectively, and the activities were further supported by the excessive mRNA expression of Tnf-alpha and Tgf-beta1 in RT-PCR, respectively. Cell fusion occurs between BMDCs and nonparenchymal cells but scarcely occurs between BMDCs and hepatocytes, demonstrated by double staining of beta-gal/GFP and further supported by the Y-chromosome staining. The EtOH withdrawal normalized most of the abnormalities produced by chronic alcohol consumption. These results indicate that excess alcohol consumption stimulates both the homing of HSCs from the bone marrow and their profibrogenic cytokine production in a mouse model of alcohol-induced fatty liver disease.

Sevoflurane Does Not Inhibit Human Platelet Aggregation Induced by Thrombin

Background Sevoflurane reportedly inhibits adenosine diphosphate–induced platelet aggregation by suppressing thromboxane A2 formation. The increase in intracellular calcium concentration that fosters platelet aggregation, however, is also induced by other cell signaling pathways, such as activation of the production of inositol 1,4,5-triphosphate by thrombin. The current study aimed to clarify the net influence of sevoflurane on thrombin-induced platelet aggregation. Methods Washed platelets were stimulated by thrombin after incubation with 0.5, 1.0, or 1.5 mM sevoflurane, halothane, or isoflurane. Aggregation curves were measured by an aggregometer. Intracellular calcium concentration was measured fluorometrically using fura-2. Calcium mobilization via plasma membrane calcium channels and the dense tubular system was assessed differentially. Intracellular inositol 1,4,5-triphosphate was measured by radioimmunoassay. Results Halothane significantly suppressed aggregation ratios at 5 min compared with those in controls (89 ± 7%) to 71 ± 10% (1.0 mM) and 60 ± 11% (1.5 mM) and the increase in intracellular calcium concentration (controls, 821 ± 95 nM vs. 440 ± 124 nM [1.0 mM] or 410 ± 74 nM [1.5 mM]). Halothane also significantly inhibited release of calcium from the dense tubular system (controls, 220 ± 48 nM vs. 142 ± 31 nM [1.0 mM]). Neither sevoflurane nor isoflurane produced a net change in aggregation ratios, intracellular calcium concentration, or calcium mobilization. Halothane (1 mM) significantly suppressed inositol 1,4,5-triphosphate concentrations, whereas neither 1 mM isoflurane nor 1 mM sevoflurane had any effect. Conclusions Although sevoflurane has been reported to inhibit human platelet aggregation induced by weak agonists such as adenosine diphosphate, it does not inhibit human platelet aggregation induced by strong agonists such as thrombin.

Carvedilol attenuates the progression of alcohol fatty liver disease in rats.

BACKGROUND Hepatosteatosis is an essential step in liver disease progression. However, the mechanisms that mediate the progression of hepatosteatosis and the optimal inhibitor of them remain largely unclear. The sympathetic nervous system (SNS) is responsible for the lipid metabolism and the accumulation of collagen that occurs in an injured liver. Medicines that inhibit this pathway may be a relevant treatment for the hepatosteatosis, and then reduce the liver injury that progresses through the stage of steatosis to fibrosis. METHODS Using an ethanol-liquid-diet-fed rat model of alcohol fatty liver disease (AFLD), we studied the effects of carvedilol, which can block the SNS completely via β1, β2, and α1 adrenergic receptors, on the sympathetic tone, hepatosteatosis, and fibrosis based on histological, immunohistochemical, Western blot, and reverse transcriptase polymerase chain reaction analyses. RESULTS Carvedilol inhibited the ethanol-induced whole-body and hepatic sympathetic activities based on the serum 3-methoxy-4-hydroxyphenylglycol level and hepatic tyrosine hydroxylase expression. Carvedilol attenuated the hepatosteatosis, as evidenced by reduced hepatic triglyceride level and the accumulation of fatty droplets within hepatocytes, down-regulated fatty acid synthase and sterol regulatory element binding protein-1, and up-regulated peroxisome proliferator-activated receptor-α. No fibrosis signs were shown in our rat model. Carvedilol inhibited ethanol-induced the thickening of zone 3 vessel walls, reduced the activation of hepatic stellate cells (HSCs), and decreased the induction of collagen, transforming growth factor β1, and tissue inhibitor of metalloproteinases-1. Tumor necrosis factor α (TNF-α) was expressed on the activated HSCs and inhibited by carvedilol based on the immunohistochemical double staining analysis. CONCLUSIONS Ethanol metabolism-induced lipogenesis may trigger the SNS-activated HSCs feedback loop, and then induct the activated HSCs and the activated HSCs-derived TNF-α, the mediator of lipogenesis, overproduction. Carvedilol may block this feedback loop via antisympathetic activity and demonstrate its preventive role on the development of hepatosteatosis in rat with AFLD.

Abrupt termination of an ethanol regimen provokes ventricular arrhythmia and enhances susceptibility to the arrhythmogenic effects of epinephrine in rats.

BACKGROUND Pathologists examining victims of sudden unexpected death encounter alcoholics more often than expected; alcohol may play a role in sudden arrhythmic death. Here we determine whether a pattern of alcohol consumption, chronic ethanol intake, and withdrawal increases the incidence of malignant ventricular arrhythmia and modulates susceptibility to the arrhythmogenic potential of sympathetic stimulation from an epinephrine test in rats. METHODS Male Wistar rats were treated with a continuous ethanol liquid diet for 7 weeks, and then subjected to 1-day withdrawal or 21-day abstinence. Ventricular ectopy was evaluated by 24-hour electrocardiographic telemetry recording; whole-body sympathetic activation, cardiac sympathovagal balance, and susceptibility to ventricular arrhythmia induced by sympathetic stimulation were evaluated based on blood noradrenalin metabolite concentrations, heart rate variability, and a 3-step epinephrine test. RESULTS Ventricular arrhythmia and related death were observed only in rats at 1 day of withdrawal, but not in nonalcoholic, continuous ethanol intake or 21-day abstinence rats. One-day withdrawal after a 7-week continuous ethanol regimen elevated circulating noradrenalin metabolite levels and induced cardiac sympathovagal imbalance. Deaths related to the epinephrine test and ventricular arrhythmia induced by low doses of epinephrine were observed only in 1-day withdrawal rats. However, all anomalies were normalized by 21-day abstinence. CONCLUSIONS Abrupt termination of a 7-week continuous ethanol regimen is sufficient to enhance the whole-body sympathetic activation and cardiac sympathovagal imbalance that contribute to ventricular arrhythmia and sudden death in alcoholic rats. Those providing medical care for alcoholics, including in cases of legal imprisonment, should be aware of the possibility of enhanced susceptibility to sudden arrhythmic death due to the abrupt termination of a chronic ethanol regimen.

Activation of Mitogen Activated Protein Kinases and Apoptosis of Germ Cells After Vasectomy in The Rat

PURPOSE Vasectomy induces a large amount of germ cell apoptosis. We examined the activation of mitogen activated protein kinases (MAPKs) in association with the apoptosis and proliferation of germ cells after vasectomy in the rat. MATERIALS AND METHODS Eight-week-old Wistar rats underwent bilateral vasectomy and the testes were harvested 1 to 9 days after vasectomy. Germ cell apoptosis was evaluated by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling and electrophoretic assay of DNA fragmentation. Western blotting and immunohistochemistry were used to examine the temporal and spatial activation of signal regulated kinases 1/2, c-Jun-terminal kinases 1/2 and p38. Phospho-specific MAPK antibodies were used to examine their activations. Proliferation of germ cells was evaluated by proliferative nuclear cell antigen expression. RESULTS Germ cell apoptosis was detected predominantly in primary spermatocytes with a peak 7 days after vasectomy. Signal regulated kinases 1/2, c-Jun-terminal kinases 1/2 and p38 were constitutively expressed in the control testis. Western blotting and immunohistochemistry showed rapid activation of signal regulated kinases 1/2, followed by activation of c-Jun-terminal kinases 1/2 and p38. Immunohistochemical study demonstrated the temporal and spatial relationships of apoptosis and MAPK activation in primary spermatocytes. On the other hand, proliferating cell nuclear antigen expression was enhanced in tetraploid spermatocyte and spermatogonia maximally 5 days after vasectomy. CONCLUSIONS MAPKs were rapidly activated after vasectomy and germ cell apoptosis was observed after vasectomy. In contrast to the delayed phase up to 24 weeks after vasectomy, we observed hyperdynamic cellular turnover, spermatocyte loss through apoptosis and enhanced germ cell proliferation transiently at the early phase after vasectomy.

QT interval dispersion and cardiac sympathovagal balance shift in rats with acute ethanol withdrawal.

BACKGROUND Dysregulation of autonomic nervous system function and impaired homogeneity of myocardial repolarization are 2 important mechanisms for the genesis of ventricular arrhythmias in nonalcoholic subjects. Our previous study suggested that acute ethanol withdrawal promoted the shift of cardiac sympathovagal balance toward sympathetic predominance and reduced the vagal tone, which were related to a higher incidence of ventricular arrhythmia and related death. However, the homogeneity of myocardial repolarization and its relation with the cardiac sympathovagal balance are unknown, especially in alcoholic subjects. The aim of the present study was to clarify these points. METHODS Male Wistar rats were treated with a continuous ethanol liquid diet for 49 days, and then subjected to 1-day withdrawal and 1-day withdrawal with 7-day carvedilol (can block the sympathetic nervous system completely via beta1, beta2, and alpha adrenergic receptors) pretreatment. The cardiac sympathovagal balance and homogeneity of myocardial repolarization were evaluated based on the heart rate variability (HRV) and QT interval dispersion (QTd: dynamic changes in QT interval duration). RESULTS The increase in QTd was observed only in rats at 1-day withdrawal, but not in nonalcoholic, continuous ethanol intake, and 1-day withdrawal with 7-day carvedilol pretreatment rats. At 1-day withdrawal, the low-frequency power/high-frequency power (LF/HF) ratio in HRV was elevated and correlated with the QTd. The increased QTd and elevated LF/HF ratio were normalized by the 7-day carvedilol pretreatment in rats at 1-day ethanol withdrawal. CONCLUSIONS In rats with an abrupt termination of the chronic continuous ethanol intake, the homogeneity of myocardial repolarization impaired and correlated with the cardiac sympathovagal balance. Carvedilol pretreatment is associated with a reduction in both the QTd and LF/HF ratio, raising the possibility that the cardiac sympathovagal balance shift may be responsible for the impaired homogeneity of myocardial repolarization, and that beta-blocker pretreatment may decrease the mortality risk during alcoholic withdrawal.

Carvedilol improves ethanol-induced liver injury via modifying the interaction between oxidative stress and sympathetic hyperactivity in rats.

Oxidative stress is a major pathway mediating ethanol hepatotoxicity and liver injury. We previously found that carvedilol, which can block the sympathetic nervous system via β1‐, β2‐ and α1‐adrenoreceptors, modifies ethanol‐induced production of lipogenesis‐ and fibrogenesis‐related mediators from hepatic stellate cells (HSC). In the present study, we assessed the effects of carvedilol on ethanol‐induced liver injury, hepatic insulin resistance, and the interaction between oxidative stress and sympathetic hyperactivity in rats with alcoholic fatty liver disease (AFLD).