Proteomic analysis of livers from a transgenic mouse line with activated polyamine catabolism

We have generated a transgenic mouse line that over expresses the rate-controlling enzyme of the polyamine catabolism, spermidine/spermine N ¹-acetyltransferase, under the control of a heavy metal inducible promoter. This line is characterized by a notable increase in SSAT activity in liver, pancreas and kidneys and a moderate increase in the rest of the tissues. SSAT induction results in an enhanced polyamine catabolism manifested as a depletion of spermidine and spermine and an overaccumulation of putrescine in all tissues. To study how the activation of polyamine catabolism affects other metabolic pathways, protein expression pattern of the livers of transgenic animals was analyzed by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. A total of 23 proteins were shown to be differentially expressed in the transgenic from the wild-type animals. Many of the identified proteins showed expression patterns associated with polyamine catabolism activation. However, the expression pattern of other proteins, such as repression of GST pi and selenium-binding protein 2 and 60 kDa heat-shock protein, could be explained by the overexpression of peroxisome proliferator-activated receptor γ co-activator 1α in response to depleted ATP pools. The activation of the latter proteins is thought to lead to the improved insulin sensitivity seen in the MT-SSAT animals.     

Oxidative stress and inflammation in the pathogenesis of activated polyamine catabolism-induced acute pancreatitis

Summary. The markers of oxidative stress and inflammation were studied in acute pancreatitis in transgenic rats exhibiting activated polyamine catabolism. In addition, the effect of bismethylspermine (Me₂Spm) pretreatment, preventing pancreatitis in this model, on these mediators was investigated. Lipid peroxidation was increased at 6 and 24 h after induction of pancreatitis. These changes as well as the markedly decreased superoxide dismutase activity at 24 h were abolished by Me₂Spm pretreatment. Glutathione level and catalase activity changed transiently, and the effect of Me₂Spm was clear at 24 h. Serum inflammatory cytokine levels increased already at 4 h whereas NF-κB was distinctly activated only at 24 h. Me₂Spm prevented the increase in TNF-α and IL-6 while it had no effect on NF-κB activation. These results show that typical inflammatory and, to a lesser degree, some oxidative stress mediators are involved and beneficially affected by the disease-ameliorating polyamine analogue in our pancreatitis model.     

Ebselen prevents early alcohol-induced liver injury in rats

Oxidants have been shown to be involved in alcohol-induced liver injury. Moreover, 2-phenyl-1,2-benzisoselenazole-3(2H)-one (ebselen), an organoselenium compound and glutathione peroxidase mimic, decreases oxidative stress and protects against stroke clinically. This study was designed to test the hypothesis that ebselen protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-16 g/kg/d) continuously for up to 4 weeks using the intragastric enteral feeding protocol developed by Tsukamoto and French. Ebselen (50 mg/kg twice daily, intragastrically) or vehicle (1% tylose) was administered throughout the experiment. Mean urine ethanol concentrations were not significantly different between treatment groups, and ebselen did not affect body weight gains or cyclic patterns of ethanol concentrations in urine. After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (37 +/- 5 IU/l) by enteral ethanol (112 +/- 7 IU/l); ebselen blunted this increase significantly (61 +/- 8 IU/l). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver (pathology score: 4.3 +/- 0.3). In contrast, these pathological changes were blunted significantly by ebselen (pathology score: 2.5 +/- 0.4). While there were no significant effects of either ethanol or ebselen on glutathione peroxidase activity in serum or liver tissue, ebselen blocked the increase in serum nitrate/nitrite caused by ethanol. Furthermore, ethanol increased the activity of NF-kappaB over 5-fold, the number of infiltrating neutrophils 4-fold, and the accumulation of 4-hydroxynonenal over 5-fold. Ebselen blunted all of these effects significantly. These results indicate that ebselen prevents early alcohol-induced liver injury, most likely by preventing oxidative stress, which decreases inflammation.     

Metabolic and antiproliferative consequences of activated polyamine catabolism in LNCaP prostate carcinoma cells

Depletion of intracellular polyamine pools invariably inhibits cell growth. Although this is usually accomplished by inhibiting polyamine biosynthesis, we reasoned that this might be more effectively achieved by activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase (SSAT); a strategy first validated in MCF-7 breast carcinoma cells. We now examine the possibility that, due to unique aspects of polyamine homeostasis in the prostate gland, tumor cells derived from it may be particularly sensitive to activated polyamine catabolism. Thus, SSAT was conditionally overexpressed in LNCaP prostate carcinoma cells via a tetracycline-regulatable (Tet-off) system. Tetracycline removal resulted in a rapid approximately 10-fold increase in SSAT mRNA and an increase of approximately 20-fold in enzyme activity. SSAT products N(1)-acetylspermidine, N(1)-acetylspermine, and N(1),N(12)-diacetylspermine accumulated intracellularly and extracellularly. SSAT induction also led to a growth inhibition that was not accompanied by polyamine pool depletion as it was in MCF-7 cells. Rather, intracellular spermidine and spermine pools were maintained at or above control levels by a robust compensatory increase in ornithine decarboxylase and S-adenosylmethionine decarboxylase activities. This, in turn, gave rise to a high rate of metabolic flux through both the biosynthetic and catabolic arms of polyamine metabolism. Treatment with the biosynthesis inhibitor alpha-difluoromethylornithine during tetracycline removal interrupted flux and prevented growth inhibition. Thus, flux-induced growth inhibition appears to derive from overaccumulation of metabolic products and/or from depletion of metabolic precursors. Metabolic effects that were not excluded as possible contributing factors include high levels of putrescine and acetylated polyamines, a 50% reduction in S-adenosylmethionine, and a 45% decline in the SSAT cofactor acetyl-CoA. Overall, the study demonstrates that activation of polyamine catabolism in LNCaP cells elicits a compensatory increase in polyamine biosynthesis and downstream metabolic events that culminate in growth inhibition.     

Moringa oleifera Lam. leaf extract prevents early liver injury and restores antioxidant status in mice fed with high-fat diet

Consumption of high-fat diet (HFD) induces nonalcoholic fatty liver disease (NAFLD) and may lead to multiple complications affecting human health. In the present study, effect of Moringa oleifera leaf extract (MoLE) in alleviating HFD induced liver injury in mice has been reported. Liver histology and serum activity of hepatic marker enzymes i.e. aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) have been studied. Lipid peroxidation (LPO), ferric reducing antioxidant power (FRAP) and reduced glutathione (GSH) were also estimated using liver homogenate. Results of the study suggested that MoLE treatment protected HFD-induced liver damage as indicated by histopathology and liver enzyme activity compared to only-HFD fed group (P < 0.05). Interestingly, early signs of HFD-induced fatty liver were also alleviated by MoLE. Moreover, significant increase in endogenous antioxidant parameters and lower lipid peroxidation were found in liver of all MoLE treated groups. Results of the study indicated that MoLE has both preventive as also curative hepatoprotective activity.     

S-allyl cysteine prevents CCl4-induced acute liver injury in rats

Aged garlic extract (AGE) possesses multiple biological activities. We evaluated the protective effect of S-allyl cysteine (SAC), one of the organosulfur compounds of AGE, against carbon tetrachloride (CCl₄)-induced acute liver injury in rats. SAC was administrated intraperitoneally (50–200 mg/kg). SAC significantly suppressed the increases of plasma ALT and LDH levels. SAC also attenuated histological liver damage. CCl₄ administration induced lipid peroxidation accompanied by increases in the plasma malondialdehyde and hepatic 4-hydroxy-2-nonenal levels, and SAC dose-dependently attenuated these increases. The hepatic total level of hydroxyoctadecadienoic acid (HODE), a new oxidative stress biomarker, was closely correlated with the amount of liver damage. These results suggest that SAC decreased CCl₄-induced liver injury by attenuation of oxidative stress, and may be a better therapeutic tool for chronic liver disease.     

Pancreatic enzyme activity in early phases of acute experimental pancreatitis in rats

Experiments were performed on laboratory rats with acute pancreatitis caused by local freezing the pancreas with chlorethyl. An active action of enzymes alpha-amilase, lipase, phospholipase A2, was revealed. During the first hours, an increase in action of all three enzymes, particularly that of phospholipase A2, was found. It was established that the lipid spectrum of pancreas had changed. It shows that cell membranes were destroyed. Experiments revealed an activating role of Ca2+ ions for all the enzymes and a correcting action of chlorpromasine.     

Glucose homeostasis during the early stages of liver regeneration in fasted rats

Kinetic studies with [2-3H]glucose in vivo and gluconeogenic activity measurements in vivo and in vitro were performed in 70% hepatectomized rats submitted to fasting, which represents an extra burden for glucose synthesis but does not impair liver regeneration. Rates of glucose replacement, under steady-state conditions, 14 and 24 h postoperatively, did not differ in partially hepatectomized fasted rats and sham-operated controls. Phosphoenolpyruvate carboxykinase activities increased more rapidly during fasting in remnant livers than in intact livers from controls. Rates of incorporation of 14C from alanine into circulating glucose in hepatectomized rats were already maximal 14 h after surgery, whereas in controls they continued to augment. The maximal rates after partial hepatectomy could not be surpassed by performing the operation in diabetic animals. It is concluded that the relatively high blood sugar levels during fasting in hepatectomized rats do not depend on a reduced peripheral utilization of glucose, but only on a rapid increase in the gluconeogenic activity. The data suggest that hepatocytes in remnant liver can proliferate under conditions of maximal gluconeogenic and low glycolytic activities.     

Ultrastructural changes in rat liver in early stages of experimental acute pancreatitis]

Morphologic disorders in the liver during the acute pancreatitis are an important aspect of multiorgan disturbances seen in this disease. The study aimed at evaluating ultrastructural disorders in the of the experimental pancreatitis. The study involved 24 male Wistar rats. The acute experimental pancreatitis has been produced by the injection of a 5% sodium taurocholate during sterile laparotomy. Samples for examination have been taken after 1, 3, 6, and 12 hours. Collected samples have been examined systematically. Mitochondrial pleomorphism, partial RER degranulation, decrease in glycogen content, and autophagocytosis have been noted already within 1 and 3 hours. The degree of these disorders has increased within 6 hours, reaching its peak after 12 hours. Marked degeneration of mitochondria, high autophagocytosis, nearly complete disappearance of glycogen, impaired sinusal endothelium, and Kupffer's cells stimulation, in which increased phagocytic activity has been noted made a complete picture of the ultrastructural disorders. Such morphologic changes in the liver have indicated its damage in the early stages of the acute pancreatitis. It may be of importance for the development of multiorgan complications of this disease.     

S-allyl cysteine prevents CCl(4)-induced acute liver injury in rats

Aged garlic extract (AGE) possesses multiple biological activities. We evaluated the protective effect of S-allyl cysteine (SAC), one of the organosulfur compounds of AGE, against carbon tetrachloride (CCl₄)-induced acute liver injury in rats. SAC was administrated intraperitoneally (50-200 mg/kg). SAC significantly suppressed the increases of plasma ALT and LDH levels. SAC also attenuated histological liver damage. CCl₄ administration induced lipid peroxidation accompanied by increases in the plasma malondialdehyde and hepatic 4-hydroxy-2-nonenal levels, and SAC dose-dependently attenuated these increases. The hepatic total level of hydroxyoctadecadienoic acid (HODE), a new oxidative stress biomarker, was closely correlated with the amount of liver damage. These results suggest that SAC decreased CCl₄-induced liver injury by attenuation of oxidative stress, and may be a better therapeutic tool for chronic liver disease.     

Depletion of activated hepatic stellate cell correlates with severe liver damage and abnormal liver regeneration in acetaminophen-induced liver injury

Hepatic stellate cells (HSCs) are important part of the local 'stem cell niche' for hepatic progenitor cells (HPCs) and hepatocytes. However, it is unclear as to whether the products of activated HSCs are required to attenuate hepatocyte injury, enhance liver regeneration, or both. In this study, we performed 'loss of function' studies by depleting activated HSCs with gliotoxin. It was demonstrated that a significantly severe liver damage and declined survival rate were correlated with depletion of activated HSCs. Furthermore, diminishing HSC activation resulted in a 3-fold increase in hepatocyte apoptosis and a 66% decrease in the number of proliferating hepatocytes. This was accompanied by a dramatic decrease in the expression levels of five genes known to be up-regulated during hepatocyte replication. In particular, it was found that depletion of activated HSCs inhibited oval cell reaction that was confirmed by decreased numbers of Pank-positive cells around the portal tracts and lowered gene expression level of cytokeratin 19 (CK19) in gliotoxin-treated liver. These data provide clear evidence that the activated HSCs are involved in both hepatocyte death and proliferation of hepatocytes and HPCs in acetaminophen (APAP)-induced acute liver injury.     

Rho-kinase inhibitor prevents hepatocyte damage in acute liver injury induced by carbon tetrachloride in rats

A protective effect of Rho-kinase inhibitor on various organ injuries is gaining attention. Regarding liver injury, Rho-kinase inhibitor is reported to prevent carbon tetrachloride (CCl4)- or dimethylnitrosamine-induced liver fibrosis and hepatic ischemia-reperfusion injury in rats. Because Rho-kinase inhibitor not only improved liver fibrosis but also reduced serum alanine aminotransferase (ALT) level in CCl4-induced liver fibrosis, we wondered whether Rho-kinase inhibitor might exert a direct hepatocyte-protective effect. We examined this possibility in acute CCl4 intoxication in rats. Rho-kinase inhibitor, HA-1077, reduced serum alanine ALT level in rats with acute liver injury induced by CCl4 with the improvement of histological damage and the reduction of the number of apoptotic cells. In cultured rat hepatocytes in serum-free condition, HA-1077 reduced apoptosis evaluated by quantitative determination of cytoplasmic histone-associated DNA oligonucleosome fragments with the reduction of caspase-3 activity and the enhancement of Bcl-2 expression. HA-1077 stimulated phosphorylation of Akt, and wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, abrogated the reduction of hepatocyte apoptosis by HA-1077 in vitro. Furthermore, wortmannin abrogated the reduction of serum ALT level by HA-1077 in rats with acute liver injury induced by CCl4, suggesting that the activation of PI3-kinase/Akt pathway may be involved in the hepatocyte-protective effect by Rho-kinase inhibitor in vivo. In conclusion, Rho-kinase inhibitor prevented hepatocyte damage in acute liver injury induced by CCl4 in rats and merits consideration as a hepatocyte-protective agent in liver injury, considering its direct antiapoptotic effect on hepatocytes in vitro.     

Role of reactive oxygen species in the early stages of liver regeneration in streptozotocin-induced diabetic rats

Diabetes mellitus is a risk factor for prognosis after liver resection. In previous work, we found a pro-apoptotic state in the diabetic rat liver. In this work, this was also observed 1 hour post-partial hepatectomy (PH) and resulted in a deficient regenerative response 24 hours post-PH. Treatment with insulin and/or Desferoxamine (DES) (iron chelator) or Tempol (TEM) (free radicals scavenger) was effective in preventing the liver reactive oxygen species (ROS) production induced by diabetic state. High levels of ROS play a role in hepatic lipid peroxidation in diabetes before and after PH, and lead to increased pro-apoptotic events, which contribute to a reduced regenerative response. This becomes of relevance for the potential use of antioxidants/free radical scavengers plus insulin for improvement of post-surgical recovery of diabetic patients subjected to a PH.