Ethanol-Associated Alterations in the Kinetics of Putrescine Uptake and Metabolism by the Regenerating Liver

Biosynthesis of the polyamines, putrescine, spermidine, and spermine is required for DNA synthesis and liver regeneration after partial hepatectomy. We have previously reported that chronic ethanol consumption impairs polyamine synthesis and significantly retards liver regeneration after partial hepatectomy. In those studies, supplementation with putrescine restored hepatic DNA synthesis in ethanol-fed rats but exerted no effect in pair-fed controls. These differences in the response to putrescine treatment may have resulted from ethanol-associated differences in hepatic uptake, release, or metabolism of putrescine. To resolve these issues and define more completely how putrescine treatment affects DNA synthesis, we now assess the kinetics of putrescine uptake and metabolism after intraperitoneal or intravenous injection of radiolabeled putrescine (1.2 mmol/kg, specific activity 1 microCi/mmol) into rats fed 36% ethanol diets or isocaloric, nonethanol diets for 6 weeks prior to partial hepatectomy. After putrescine treatment, hepatic putrescine concentrations were greater in ethanol-fed rats than controls. Differences in post-treatment hepatic putrescine levels between ethanol and pair-fed groups could not be explained by differences in the rates of hepatic putrescine uptake or excretion into bile; residual de novo synthesis of putrescine from ornithine or metabolism of hepatic putrescine to its polyamine products, spermidine and spermine. Indeed, supplemental putrescine was not appreciably converted to spermidine or spermine in either ethanol or control rats. Hence, these latter polyamines are unlikely to be responsible for the treatment-associated improvement in DNA synthesis that has been noted in ethanol-fed rats. This suggests that putrescine itself acts to restore hepatic DNA synthesis in ethanol-fed rats.     

Inhibition of hepatic regeneration in rats by acute and chronic ethanol intoxication

We studied the effects of acute and chronic ethanol feeding on hepatic regeneration in rats after partial hepatectomy and toxic liver injury produced by D-galactosamine. Ethanol, when administered as a single dose (6 g/kg), inhibited 3H-thymidine incorporation into hepatic DNA; this effect depended in part on the time of ethanol feeding after partial hepatectomy. Multiple ethanol feedings produced an even greater inhibition, which persisted for at least 48 hr after partial hepatectomy. Rats chronically fed ethanol for 30 days also failed to achieve a hepatic proliferative response to either partial hepatectomy or D-galactosamine-induced hepatitis, comparable with isocaloric pair-fed controls. These investigations suggest that there may be a certain metabolic state in the hepatocyte cell cycle that is most susceptible to the action(s) of ethanol; inhibition of liver regeneration by acute or chronic ethanol consumption may result in delayed recovery from prior or coincident liver injury.     

Carbohydrate and oxygen metabolism during hepatocellular proliferation: a study in perfused livers from mirex-treated rats

Liver regeneration after partial hepatectomy is accompanied by altered hepatic intermediary metabolism. Because the organochlorine compound mirex also causes liver cell growth, the purpose of this study was to investigate hepatic carbohydrate and oxygen metabolism in perfused livers from mirex-treated rats and to localize cell proliferation in this model. Pretreatment with mirex (100 mg/kg, intragastrically) increased liver/body weight ratios and DNA synthesis in livers of fed rats, effects that were markedly diminished in livers of fasted rats. This finding shows that liver growth caused by mirex, as is the case after partial hepatectomy, is hindered when animals are deprived of food. Furthermore, perfused livers from mirex-treated rats had depleted glycogen stores but significantly elevated oxygen uptake compared with livers from control rats. Increases in oxygen uptake and hepatocellular proliferation were observed mostly in periportal regions of the liver lobule. In regenerating livers, most DNA synthesis was reported to also occur in these regions of the liver lobule. Taken together, these data show that liver cell growth caused by mirex is accompanied by changes in hepatic intermediary metabolism and sublobular proliferation similar to those observed after partial hepatectomy.     

Effects of malotilate on ethanol-inhibited hepatocyte regeneration in rats

The effects of malotilate, a hepatotrophic drug, on the inhibition of hepatocyte regeneration induced by ethanol were studied in rats. Following acute ethanol administration, both DNA synthesis and commencement of the S phase of the cell cycle in hepatocytes after partial hepatectomy were delayed. Malotilate prevents this ethanol-induced delay. Upon chronic ethanol administration, DNA synthesis after partial hepatectomy was reduced and commencement of the S phase was retarded. Treatment with malotilate clearly lessened the reduction in DNA synthesis in the resected liver, and retardation of the cell cycle was partially inhibited. These results indicate that malotilate prevents the ethanol-induced inhibition of hepatocyte regeneration at the dose tested and that this drug may be effective for the treatment of alcoholic liver disease.     

Ischemic preconditioning improves liver regeneration by sustaining energy metabolism after partial hepatectomy under ischemia in rats.

BACKGROUND: The protective effect of ischemic preconditioning (IPC) has been reported on improvement of survival, reduction of liver necrosis and enhancement of the regenerative capacity of hepatocytes after partial hepatectomy. This study was undertaken to confirm that IPC has a significant impact on regeneration of hepatocytes after partial hepatectomy in ischemically damaged liver. In addition, we sought to examine the role of adenine nucleotides in this process. METHODS: Wistar rats were subjected to 60 min of total hepatic ischemia, followed by 70% hepatectomy. The animals were subdivided into an IPC (10/15 min) group and a non-IPC (control) group. Liver function tests and arginase activity were analyzed. Hepatic adenosine triphosphate (ATP), adenosine diphosphate and adenosine monophosphate were measured using gradient high-performance liquid chromatography. The liver regeneration was identified using relative liver weight and proliferating cell nuclear antigen (PCNA) labeling index. RESULTS: IPC treatment improved serum liver enzymes and tissue arginase activity (P<0.05) when compared with the control group. The preconditioned livers were associated with upregulation of ATP expression and also increased tissue energy charge. Regenerated liver weight in the IPC group was significantly higher than in the control group (P<0.05). The PCNA labeling index in the remnant livers in the IPC group was also significantly increased at 24 and 48 h after partial hepatectomy (P<0.05). CONCLUSION: These results suggest that IPC-augmented liver regeneration after hepatectomy, probably due to the stabilization of energy metabolism in rats.     

Relationship between expression of cyclin D1 and impaired liver regeneration observed in fibrotic or cirrhotic rats

BACKGROUND AND AIM: The mechanisms responsible for impaired regenerative ability after hepatic resection observed in chronic liver disease are not fully understood. We have examined the relationships between an altered expression of cell cycle-related proteins in regenerating liver after partial hepatectomy and the impaired regenerative process observed in fibrotic and cirrhotic rats. METHODS: We performed 70% partial hepatectomy in both control and porcine serum-induced fibrotic rats, and 45% partial hepatectomy in thioacetamide-induced cirrhotic rats because of the high mortality associated with 70% partial hepatectomy. Liver regeneration was monitored by proliferating cell nuclear antigen labeling index and the expression of G1 regulatory cell cycle-related proteins was determined by immunoblot analysis. RESULTS: Compared with controls, hepatocyte DNA synthesis, and induction of cyclin D1 and p21(CIP1) proteins were delayed but not suppressed in porcine serum-induced fibrotic rats and markedly inhibited in thioacetamide-induced cirrhotic rats. p27(KIP1) protein levels were unaffected by partial hepatectomy and did not differ among all three groups. CONCLUSION: Two distinct rat models of liver fibrosis and cirrhosis showed markedly different proliferative responses after partial hepatectomy. The delay or failure of cyclin D1 induction, but not the increase of p21(CIP1) or p27(KIP1) might be responsible for their impaired liver regeneration.     

The Hepatoprotective Effect of Hepatic Stimulator Substance (HSS) Against Liver Regeneration Arrest Induced by Acute Ethanol Intoxication

Male Wistar rats were randomized to receive ethanol (2.5 ml/kg by gastric intubation every 8 hr; group I), equal volumes of isocaloric to ethanol sucrose solution (group II), or ethanol and HSS (100 mg/kg intraperitoneally 10 and 16 hr after partial hepatectomy; groups III and IV, respectively) for up to 96 hr after partial hepatectomy, with ethanol administration starting 1 hr prior to partial hepatectomy. Animals were killed at 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 60, and 96 hr after partial hepatectomy. The rate of liver regeneration was evaluated by the mitotic index in H&E-stained sections, immunochemical detection of Ki67 nuclear antigen, rate of [³H]thymidine incorporation into hepatic DNA, and liver thymidine kinase enzymatic activity. The biological activity of HSS in groups I and II rats was evaluated using a bioassay. Ethanol administration arrested liver regeneration during the first 32 hr after partial hepatectomy and suppressed HSS activity throughout the period examined. Liver regeneration progressed after 32 hr despite the low levels of HSS activity. HSS administration at 10 and 16 hr reversed liver regeneration arrest induced by ethanol. Acute ethanol administration induces cell cycle arrest during the first 32 hr after partial hepatectomy and suppression of HSS biological activity seems to contribute to this effect. HSS administration reversed the inhibitory effect of ethanol on liver regeneration and caused synchronized entrance of hepatocytes in the S phase of the cell cycle. HSS seems to participate in the network of growth factors controlling the G1/S cell cycle checkpoint.     

The effect of chronic ethanol feeding on ornithine decarboxylase activity and liver regeneration

The effects of ethanol on liver regeneration are poorly understood. Acute and chronic exposure to ethanol have been found to exert opposite effects on the induction of ornithine decarboxylase, the rate-limiting enzyme for polyamine biosynthesis. Polyamines are necessary for DNA synthesis and liver regeneration after chemical or surgical liver injury. Short-term exposure to ethanol, which inhibits ornithine decarboxylase has been shown to inhibit DNA synthesis and liver regeneration, whereas more chronic exposure to ethanol increases ornithine decarboxylase activity and therefore could conceivably stimulate DNA synthesis and regeneration. To explore this later possibility, the effects of chronic ethanol consumption on ornithine decarboxylase activity, DNA synthesis and liver regeneration were studied in rats after sham laparotomy and partial hepatectomy. Chronic ethanol feeding failed to inhibit the induction of ornithine decarboxylase that occurred after partial hepatectomy and yet significantly inhibited posthepatectomy DNA synthesis and restitution of liver mass. These data suggest that the induction of hepatic polyamine biosynthesis is dissociated from DNA synthesis and liver regeneration after chronic consumption of ethanol.     

Decreased expression of peroxisome proliferator-activated receptor alpha and liver fatty acid binding protein after partial hepatectomy of rats and mice.

BACKGROUND AIMS: Marked changes in metabolism, including liver steatosis and hypoglycemia, occur after partial hepatectomy. Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a nuclear hormone receptor that is activated by fatty acids and involved in hepatic fatty acid metabolism and regeneration. Liver fatty acid binding protein (LFABP) is an abundant protein in liver cytosol whose expression is regulated by PPAR alpha. It is involved in fatty acid uptake and diffusion and in PPAR alpha signaling. The aim of this study was to investigate the expression of PPAR alpha and LFABP during liver regeneration. METHODS: Male Sprague-Dawley rats and male C57 Bl/6 mice were subjected to 2/3 hepatectomy and LFABP and PPAR alpha mRNA and protein levels were measured at different time points after surgery. The effect of partial hepatectomy was followed during 48 h in rats and 72 h in mice. RESULTS: PPAR alpha mRNA and protein levels were decreased 26 h after hepatectomy of rats. The LFABP mRNA and protein levels paralleled those of PPAR alpha and were also decreased 26 h after hepatectomy. In mice, the mRNA level was decreased after 36 and 72 h after hepatectomy. In this case, LFABP mRNA levels decreased more slowly after partial hepatectomy than in rats. CONCLUSIONS: A marked decrease in PPAR alpha expression may be important for changed gene expression, e.g. LFABP, and metabolic changes, such as hypoglycemia, during liver regeneration.     

Heparin accelerates liver regeneration following portal branch ligation in normal and cirrhotic rats with increased plasma hepatocyte growth factor levels

BACKGROUND/AIMS: Heparin is widely used as a general anticoagulant, and has been recently reported to elevate plasma hepatocyte growth factor (HGF) levels by releasing HGF sequestrated in the extracellular matrix. Therefore, we investigated the effects of heparin administration on liver regeneration following portal branch ligation (PBL) in normal and cirrhotic rats. METHODS: Dimethylnitrosamine-induced cirrhotic rats and control rats underwent portal ligation of the left lateral and median branches, followed by intraperitoneal heparin injections, every 12 h. To examine the feasibility of an extensive hepatectomy in the cirrhotic livers, cirrhotic rats with or without heparin treatment underwent resection of occluded lobes at 72 h after the PBL. RESULTS: Heparin injections significantly augmented liver regeneration after PBL in both normal and cirrhotic rats, following an increase in hepatocellular DNA synthesis at 24 h after the PBL. The plasma HGF concentrations were elevated by heparin treatment in both groups. In addition, heparin administration dramatically improved the survival rate after an extensive hepatectomy in the cirrhotic rats. CONCLUSIONS: Heparin treatment significantly accelerated liver regeneration following the PBL, with an increase in the plasma HGF levels in both normal and cirrhotic rats. Heparin administration may make an extensive hepatectomy clinically feasible even for cirrhotic livers.     

Transforming growth factor beta mRNA increases during liver regeneration: a possible paracrine mechanism of growth regulation.

Transforming growth factor beta (TGF-beta) is a growth factor with multiple biological properties including stimulation and inhibition of cell proliferation. To determine whether TGF-beta is involved in hepatocyte growth responses in vivo, we measured the levels of TGF-beta mRNA in normal liver and during liver regeneration after partial hepatectomy in rats. TGF-beta mRNA increases in the regenerating liver and reaches a peak (about 8 times higher than basal levels) after the major wave of hepatocyte cell division and mitosis have taken place and after the peak expression of the ras protooncogenes. Although hepatocytes from normal and regenerating liver respond to TGF-beta, they do not synthesize TGF-beta mRNA. Instead, the message is present in liver nonparenchymal cells and is particularly abundant in cell fractions enriched for endothelial cells. TGF-beta inhibits epidermal growth factor-induced DNA synthesis in vitro in hepatocytes from normal or regenerating liver, although the dose-response curves vary according to the culture medium used. We conclude that TGF-beta may function as the effector of an inhibitory paracrine loop that is activated during liver regeneration, perhaps to prevent uncontrolled hepatocyte proliferation.