Involvement of polyamines in apoptosis of cardiac myoblasts in a model of simulated ischemia

Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy, and heart failure. The polyamines putrescine, spermidine, and spermine are polycations absolutely required for cell growth and division. However, increasing evidence indicates that polyamines, cell growth, and cell death can be tightly connected. In this paper, we have studied the involvement of polyamines in apoptosis of H9c2 cardiomyoblasts in a model of simulated ischemia. H9c2 cells were exposed to a condition of simulated ischemia, consisting of hypoxia plus serum deprivation, that induces apoptosis. The activity of ornithine decarboxylase, the rate limiting enzyme of polyamine biosynthesis that synthesizes putrescine, is rapidly and transiently induced in ischemic cells, reaching a maximum after 3 h, and leading to increased polyamine levels. Pharmacological inhibition of ornithine decarboxylase by alpha-difluoromethylornithine (DFMO) depletes H9c2 cardiomyoblasts of polyamines and protects the cells against ischemia-induced apoptosis. DFMO inhibits several of the molecular events of apoptosis that follow simulated ischemia, such as the release of cytochrome c from mitochondria, caspase activation, downregulation of Bcl-xL, and DNA fragmentation. The protective effect of DFMO is lost when exogenous putrescine is provided to the cells, indicating a specific role of polyamine synthesis in the development of apoptosis in this model of simulated ischemia. In cardiomyocytes obtained from transgenic mice overexpressing ornithine decarboxylase in the heart, caspase activation is dramatically increased following induction of apoptosis, with respect to cardiomyocytes from control mice, confirming a proapoptotic effect of polyamines. It is presented for the first time evidence of the involvement of polyamines in apoptosis of ischemic cardiac cells and the beneficial effect of DFMO treatment. In conclusion, this finding may suggest novel pharmacological approaches for the protection of cardiomyocytes injury caused by ischemia.     

Effects of Ethanol on Rat Sertoli Cell Function: Studies In Vitro and In Vivo

Chronic alcohol administration to male animals is associated with testicular atrophy and gonadal failure. The Sertoli cell seems to be the first testicular cell injured as a result of alcohol exposure. To investigate the adverse effects of ethanol on testicular and particularly Sertoli cell function, the consequences of in vivo and in vitro ethanol exposure on rat Sertoli cell mRNA and protein levels of transferrin and ornithine decarboxylase were investigated. In vivo, ethanol exposure enhanced the levels of both hepatic and testicular (Sertoli cell) transferrin protein and mRNA. Ethanol exposure also enhanced testicular, but not hepatic, levels of ornithine decarboxylase protein and mRNA. These in vivo findings were confirmed when isolated Sertoli cells were studied in vitro. Specifically, ethanol exposure increased Sertoli cell transferrin protein and mRNA levels. Ethanol exposure increased Sertoli cell ornithine decarboxylase mRNA and protein when cultured in serum-free media, but not when cultured in the presence of serum. These studies demonstrate that ethanol exposure of rat Sertoli cells is associated with alterations in the levels of mRNA and protein that are known to be important in the process of spermatogenesis. These findings add to the body of evidence that suggests that, within the testes, the Sertoli cell may be an important target for ethanol-induced gonadal injury.     

Ethanol's Effect on Tissue Polyamines and Ornithine Decarboxylase Activity: A Concise Review

An extraordinarily diverse literature describes the cellular/tissue systems in which the molecular effects of both acute and chronic alcohol exposure seem to be mediated by changes in polyamine levels and/or ornithine decarboxylase (ODC) activity. The single unifying factor that links most of these studies is that they all, in some way, involve tissues that are undergoing relatively rapid cell division. Non-dividing cells expressing the NMDA receptor are a notable exception in that ethanol and the polyamines seem to act via discrete regions of that receptor. Under most cellular conditions, ODC activity is a reflection of the relative tissue polyamine content, and an increase in ODC activity and polyamine content seems to be one of the early events in the progression of quiescent cells toward cell division. Thus, it is not surprising that ethanol, which has been widely reported to delay cell division, should be found to interact with the ODC/ polyamine pathway. Perhaps the most unique aspect of these studies is the fact that, with rare exception, both acute and chronic ethanol exposure have been found to slow growth and to lower tissue polyamine (putrescine) content. Furthermore, in most studies, the ethanol-induced suppression of cell division could be overcome by the administration of exogenous putrescine. These data suggest that the ethanol-induced suppression of cell division resulted from the loss of putrescine. In addition, because the cells were able to respond to the exogenous putrescine, the studies suggest that the signaling pathway remained intact beyond the polyamine synthesis step. Increased ODC activity (and polyamines?) has been reported during the perinatal and postnatal periods in fetal animals exposed to ethanol during early development. Although not examined in all models, the perinatal/postnatal increase in fetal ODC activity may be a compensatory response to an initial loss of ODC activity, as the organism attempted to overcome the alcohol-induced growth suppression.     

Effect of α-difluoromethylornithine on cardiac polyamine content and hypertrophy

Cardiac hypertrophy produced by treatment with thyroxine for 7 days was accompanied by a significant increase in polyamine content. Putrescine was elevated by 4- to 5-fold, spermidine by 1.6-fold and spermine 1.3-fold. Administration of α-difluoromethylornithine, a potent irreversible inhibitor of ornithine decarboxylase, prevented these increases provided that a high concentration of the drug was maintained by intraperitoneal injection of 200 mg/kg body wt every 12 h and supplementation of the drinking water with $\text{3 g of the drug}\text{100 ml}$. Such treatment with α-difluoromethylornithine prevented the rise in spermidine and spermine and reduced putrescine content to undetectable levels but did not prevent the increase in heart weight showing that the enhancement of polyamine concentration is not essential for thyroxine-induced cardiac hypertrophy.     

Ethanol and Polyamine Metabolism: Physiologic and Pathologic Implications: A Review

This review summarizes the effects of ethanol on the metabolism of polyamines (putrescine, spermidine, and spermine), which are endogenous polycations required for cell physiology, growth, and differentiation. Polyamines regulate DNA, RNA, and protein syntheses; stabilize ribosomes, membranes, and nucleic acids; and protect the cell against lipid peroxidation. Polyamine biosynthesis and catabolism may change after acute and chronic ethanol treatment, thereby playing a negative or positive role in the ethanol-induced injury on liver, extrahepatic tissues, embryo, and fetus.     

Impact of Chronic Alcoholism on the Aging Rat: Changes in Nutrition, Liver Composition, and Mortality

The adverse effects of chronic alcohol consumption (mean 6.68 g/kg/d) were assessed in 150 male Sprague-Dawley rats over their life span (25 months). Evaluations were performed at 2, 3, 8, 13, 19, and 25 months of age for changes in nutrition status, biochemical tests for liver injury, compositional changes in liver, and hepatic regenerative capacity. In spite of nearly identical caloric intake, alcohol treatment was associated with nutritional levels 10-30% lower than controls.
Maximal changes were observed at the two extremes of ages (2-3 months and 19-25 months). Hence, a nutritional contribution to other adverse changes could not be excluded. Fatty compositional increases (triglycerides) occurred early (5-fold increases after 1 month of treatment) then declined to levels only slightly above controls. Biochemical tests on sera for liver injury (AST and total bilirubin) were consistently higher with alcohol treatment. Regenerative capacity measured by [³H]thymidine uptake after partial hepatectomy was initially elevated in the alcoholic then rapidly declined beyond 7 months of age. In control animals, an age-related decline was also observed but occurred later beyond 12 months of age. Consistent with these adverse effects, ethanol diet survival was poorer than the pair-fed control groups by 15% (median survival for alcoholics, 17 months vs. 20 months in controls).     

肝脏天然免疫研究进展

从肠道至肝脏的血液中富含细菌产物、环境毒素和食物抗原。肝脏依靠强大的天然免疫系统能快速有效地清除外来物质,而不引起有害免疫应答。肝脏富含天然免疫细胞,包括巨噬细胞、自然杀伤细胞、自然杀伤T细胞和γδT细胞。肝细胞表达多种Toll样受体（TLRs）,其信号途径的激活在天然免疫应答中发挥重要作用。此外,肝脏通过合成大量可溶性模式识别受体和补体成分在宿主防御微生物入侵和肿瘤转化方面发挥重要作用。肝脏天然免疫系统还积极参与肝脏炎症、损伤和再生的过程。     

Effect of Vitamin E Deficiency on Inhibition of Liver Regeneration by Long-Term Administration of Alcohol

The effects of vitamin E (VE) deficiency on liver regeneration suppressed by long-term administration of alcohol were studied. Male rats were divided into two groups: the alcohol group and the control group. In addition, each group was subdivided into two groups according to the presence or not of VE. Altogether, four groups were provided: a group maintained on the VE-deficient alcohol diet (group EA), a group maintained on the VE-deficient control diet (group EC), a group maintained on the ordinary alcohol diet (group A), and a group maintained on the ordinary control diet (group C). After pair-feeding for 6 weeks, partial hepatectomy was performed to determine the ornithine decarboxylase (ODC) activity, polyamine levels, lipid peroxide levels, and DNA synthesis. DNA synthesis at 24 hr after partial hepatectomy was suppressed significantly in the alcohol administration group, regardless of the presence or not of VE. DNA synthesis at 48 hr after partial hepatectomy tended to show low values in group EA, compared with group A. As for the hepatic ODC activity, group EA showed the lowest value at 4 hr after partial hepatectomy. Of polyamines, the putrescine level in group EA at 4 hr after partial hepatectomy was significantly low, compared with the other three groups. The levels of spermidine and spermine decreased by long-term administration of alcohol, but the effect of VE deficiency was not found. The lipid peroxide level increased significantly in the VE-deficient diet administration group, but the effect of alcohol administration was not found. These results suggested that the decrease in putrescine after ODC suppression by VE deficiency in addition to the decrease in spermidine and spermine caused by long-term alcohol administration were concerned with suppression of DNA synthesis later.     

Alterations of ATP Levels and of Energy Parameters in the Blood of Alcoholic and Nonalcoholic Patients with Liver Damage

Blood adenine nucleotides were determined in patients with alcoholic and non-alcoholic liver diseases. They included patients with alcoholic hepatitis (AH), alcoholic liver cirrhosis (ALC), non-alcoholic liver cirrhosis (NALC), and amoebic liver abscess (ALA) (28 patients). A decrease of 28% to 39% in blood ATP levels was observed among the patients with AH and the cirrhotic groups, respectively (p less than 0.05), whereas no significant changes in blood ATP levels were detected in the ALA group. Although total blood adenine nucleotides were significantly diminished in AH, ALC, and NALC groups, the AH patients retained their energy relationships within normal range. On the other hand, the cirrhotic groups, independently of their etiology, failed to maintain an adequate ATP/ADP ratio, energy charge, and phosphorylation potential in the blood, suggesting a decreased energy availability in their blood cells. Nevertheless, the mechanism involved in these effects remains to be elucidated, a failure of the damaged liver to supply purines to extra-hepatic tissues might be a major event altering the blood energy parameters.     

Effects of Bicyclol on Liver Regeneration After Partial Hepatectomy in Rats

Bicyclol is a synthetic antihepatitis drug with antioxidative property. The present study was performed to investigate the effect of bicyclol on liver regeneration after partial hepatectomy in rats. Bicyclol (300 mg/kg) was given to rats subjected to 70% hepatectomy three times before operation. At 6, 24, and 48 h after resection, samples were collected for the measurement of serum alanine aminotransferase (ALT), total bilirubin (TBil), hepatic glycogen, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH). Moreover, liver regeneration rate, proliferating cell nuclear antigen (PCNA) labeling, proliferation index, and histopathological examination were evaluated at 48 h after hepatectomy. As a result, bicyclol significantly increased regeneration rate, mitotic index (MI), PCNA labeling index, and proliferation index in PH rats. Additionally, bicyclol remarkably inhibited the elevation of serum ALT and TBil levels, alleviated the formation of liver MDA, restored impaired antioxidant SOD and GSH, increased hepatic glycogen content, and also attenuated hepatic vacuolar degeneration. These results suggested that bicyclol had a beneficial effect on liver regenerative capacity of the remnant liver tissue after hepatectomy, probably due to its antioxidative property.     

肝硬化及非肝消化病患者胆酸,透明质酸及层粘蛋白的变化

目的:研究血清胆酸(CG)、透明质酸(HA)及层粘蛋白(LN)对肝硬化及非肝消化病的诊断意义.方法:用放射免疫法检测血清CG、HA及LN,并计算它们之间的相关系数.结果:肝硬化患者血清CG、HA及LN明显高于健康人及非肝消化病组,差别有高度显著性,HA与LN存在正相关关系.结论:联合检测CC、HA及LN对肝硬化非盯消化病具有重要的意义.     

Uptake of Extracellular, Dietary Putrescine Is an Important Regulatory Mechanism of Intracellular Polyamine Metabolism DuringCamostate-Induced Pancreatic Growth in Rats

Aim of the present study was to evaluate therole of cellular uptake of dietary [ H]putrescine forthe regulation of pancreatic, hepatic, and smallintestinal polyamine metabolism during normal andcamostate-induced pancreatic growth in rats in vivo. Initiallydose-response and time-course studies of[³H]putrescine uptake were performed. MaleWistar rats were either treated with the synthetictrypsin inhibitor camostate (200 mg/kg body wt orally twice daily),camostate plus the ornithine decarboxylase inhibitoralpha-difluoromethylornithine (DFMO) (2% in drinkingwater plus 3 × 300 mg/kg body wt intraperitoneallyduring daytime) or saline as controls. After 4, 8, 12,24, 36, 48, or 120 hr, five to seven animals per groupwere killed, respectively. Orally fed [ H]putrescine (10nmol/kg body wt. 2 hr prior to death) is rapidly taken up and further metabolized tospermidine in normal growing pancreas, liver, and smallintestine. Feeding of camostate significantly enhanceddietary [³H]putrescine uptake, whilesimultaneous inhibition of de novo synthesis ofintracellular polyamines by DFMO resulted in a highlysignificant further increase in cellular uptake oforally fed [³H]putrescine, which isimmediately metabolized to spermidine. The present in vivo data confirmthe important role of dietary putrescine uptake for themaintenance of intracellular polyamine pool in normaland stimulated pancreatic growth. Furthermore, dietary putrescine uptake is an importantregulatory mechanism to maintain the normal andgrowth-stimulated cellular polyamine pool in thepancreas after potent simultaneous inhibition ofintracellular de novo polyamine synthesis.     

Pharmacokinetics of the Ethanol Bioavailability in the Regenerating Rat Liver Induced by Partial Hepatectomy

It is well known that a single ethanol administration is capable of inhibiting the two-thirds partial hepatectomy (PH)-induced liver regeneration (LR); nonetheless, it has not been elucidated how ethanol metabolism by the remnant liver is exerting the deleterious ethanol actions on LR. Indeed, pharmacokinetics analysis of ethanol elimination is lacking in rats subjected to PH, which might extend our understanding in the mechanisms that account for the ethanol-induced inhibition on LR after PH in the rat. Therefore, the present study is a pharmacokinetics analysis comparing intragastric and in-traperitoneal administrations of ethanol to rats under PH, at several times after surgery (0 to 96 hr postsurgery). Our results show that PH rats had a much lower blood ethanol peak than sham-operated, when intragastrically administered during the first 4 hr after surgery that was transient and normalized at 6 hr post-PH. The area under the curve for blood ethanol was higher in PH animals, starting after 6 hr postsurgery and extended to the all replicative period, and returned within the control values thereafter. The quantity of ethanol absorbed after its intraperitoneal injection was essentially the same as the administered dose for all of the groups tested. Hence, ethanol bioavailability diminished due to an enhanced rate of the first-pass metabolism for ethanol in PH rats at the very early times post-PH. At later times of PH, ethanol bioavailability was practically normalized, and these effects were accompanied by a drastic increase in the liver capacity to metabolize ethanol, mainly at 48 to 96 hr after surgery, as calculated as ethanol elimination per gram of liver, as well as by total body weight. The very early changes in ethanol bioavailability in PH rats were not accounted for gastric ethanol retention in these animals. In conclusion, first-pass metabolism importantly participates in the modified ethanol bioavailability at very early times after PH, an event presumably attained to gastric catabolism of ethanol. However, the very enhanced metabolism of ethanol showed by the regenerating liver, particularly after the first 24 hr postsurgery, seems to be the main factor affecting ethanol pharmacokinetics in rats subjected to PH. The underlying mechanisms in this liver enhancement of ethanol oxidation by PH rats remains to be elucidated.     

肝硬化伴糖代谢异常的临床研究

目的 分析肝硬化并发糖尿病患者的临床特征,探讨肝源性糖尿病的发病机制及治疗方法.方法 对62例肝硬化并发糖尿病患者与同期住院56例肝硬化无糖尿病患者从临床及实验室表现、治疗效果进行对照研究.结果 肝硬化并发糖尿病者临床表现以消化道症状为主,糖尿病症状不典型.肝功能越差,糖尿病发生率越高,并发症越多,病死率高.结论 肝硬化并发糖尿病时糖尿病临床表现不明显,应常规检查尿糖和血糖,及时诊断和控制糖代谢异常,可以有效地改善肝硬化患者的预后.     

Vitamin K (Menaquinone-4) Metabolism in Liver Disease

We measured menaquinone-4 (MK-4) and MK-4 epoxide concentrations in plasma and liver tissue after intravenous injection of 200 μg/kg MK-4 in 42 patients who underwent hepatectomy. They were classified into normal (N; n = 10), chronic hepatitis (CH; n = 12), and liver cirrhosis (LC; n = 20) groups, on the basis of the diagnosis given by the pathologist after examining resected liver specimens. The plasma MK-4 epoxide concentration reached maximum level (C_max) 60 min after MK-4 injection. The C_max in groups LC and CH were 85.9 and 126.3 nmol/l, respectively, which is significantly reduced compared with that of group n (184.4 nmol/l) (p < 0.01 and p < 0.05, respectively). The MK-4 concentrations in liver tissues of 24 patients 60 min after MK-4 injection were 2.77 in group N, 3.79 in group CH, and 3.83 nmol/ g in group LC, and the MK-4 epoxide concentrations were 4.01, 3.09, and 2.62 nmol/g in the respective groups. Consequently, the ratio of MK-4 epoxide to total MK-4 (MK-4 + MK-4 epoxide) in groups CH and LC was significantly lower than in group n (p < 0.01). It is concluded that the C_max of MK-4 epoxide after MK-4 injection may serve as an indicator of liver function and that the low ratio of MK-4 epoxide to total MK-4 in the liver shows impairment in vitamin K metabolism.     

P-450 -Dependent Metabolism of Laurie Acid in Alcoholic Liver Disease: Comparison between Rat Liver and Kidney Microsomes

Monooxygenase enzymatic activities were measured in liver and kidney microsomes of control and ethanol-treated rats. Animals were administered alcohol by using a model for alcoholic liver injury. Several in vitro approaches were used to compare the laurate metabolism in liver and kidney microsomes: correlation studies between specific P-450 catalytic activities, immunoblot analysis, and chemical and immunoinhibitions. Ethanol treatment increased the liver and renal hydroxylations of chlorzoxazone and 4-nitrophenol. Moreover, lauric acid (ω-1)-hydroxylation was found to be significantly increased (～6-fold) after ethanol treatment in liver, but not in kidney microsomes. The laurate ω-1/ω ratio increased from 1.52 ± 0.49 to 4.11 ± 1.01 in liver microsomes of control and ethanol-treated rats, and from 0.29 ± 0.06 to 0.44 ± 0.07 in kidney microsomes. Immunoblot analysis using polyclonal anti-cytochrome P-450 (CYP) 2E1 or CYP4A antibodies showed an increase of CYP2E1 and CYP4A contents in both organs, but the increase was higher in liver than in kidney microsomes. Chemical inhibitions using CYP2E1 competitive inhibitors (such as chlorzoxazone and ethanol) led to a nonsignificant inhibition of the renal (ω-1)-hydroxylation of lauric acid. In contrast, 17-octadecynoic acid (a mechanism-based inhibitor of ω -hy-droxylase) was able to inhibit both to- and (ω -1)-hydroxylations of lauric acid in kidney microsomes. Immunoinhibitions specific to CYP2E1 significantly decreased the (ω -1)-hydroxylation of lauric acid in liver, but not in kidney microsomes, whereas the polyclonal anti-CYP4A1 antibody inhibited to- and (ω -1)-hydroxylations of lauric acid in kidney microsomes. All of these results show that lauric acid hydroxylations in liver and kidney respond in different manners to ethanol treatment. Lauric acid (ω -1)-hydroxylation, a highly specific probe for CYP2E1 in rat and human liver microsomes, is mediated by a CYP4A isoform in rat kidney microsomes.