Impaired tricarboxylic acid cycle activity in mouse livers lacking cytosolic phosphoenolpyruvate carboxykinase

Liver-specific phosphoenolpyruvate carboxykinase (PEPCK) null mice, when fasted, maintain normal whole body glucose kinetics but develop dramatic hepatic steatosis. To identify the abnormalities of hepatic energy generation that lead to steatosis during fasting, we studied metabolic fluxes in livers lacking hepatic cytosolic PEPCK by NMR using 2H and 13C tracers. After a 4-h fast, glucose production from glycogenolysis and conversion of glycerol to glucose remains normal, whereas gluconeogenesis from tricarboxylic acid (TCA) cycle intermediates was nearly absent. Upon an extended 24-h fast, livers that lack PEPCK exhibit both 2-fold lower glucose production and oxygen consumption, compared with the controls, with all glucose production being derived only from glycerol. The mitochondrial reduction-oxidation (red-ox) state, as indicated by the NADH/NAD+ ratio, is 5-fold higher, and hepatic TCA cycle intermediate concentrations are dramatically increased in the PEPCK null livers. Consistent with this, flux through the TCA cycle and pyruvate cycling pathways is 10- and 40-fold lower, respectively. Disruption of hepatic cataplerosis due to loss of PEPCK leads to the accumulation of TCA cycle intermediates and a nearly complete blockage of gluconeogenesis from amino acids and lactate (an energy demanding process) but intact gluconeogenesis from glycerol (which contributes to net NADH production). Inhibition of the TCA cycle and fatty acid oxidation due to increased TCA cycle intermediate concentrations and reduced mitochondrial red-ox state lead to the development of steatosis.     

Cathepsin B inactivation attenuates hepatocyte apoptosis and liver damage in steatotic livers after cold ischemia-warm reperfusion injury

Hepatic steatosis predisposes the liver to cold ischemia-warm reperfusion (CI/WR) injury by unclear mechanisms. Because hepatic steatosis has recently been associated with a lysosomal pathway of apoptosis, our aim was to determine whether this cell-death pathway contributes to CI/WR injury of steatotic livers. Wild-type and cathepsin B-knockout (Ctsb(-/-)) mice were fed the methionine/choline-deficient (MCD) diet for 2 wk to induce hepatic steatosis. Mouse livers were stored in the University of Wisconsin solution for 24 h at 4 degrees C and reperfused for 1 h at 37 degrees C in vitro. Immunofluorescence analysis of the lysosomal enzymes cathepsin B and D showed a punctated intracellular pattern consistent with lysosomal localization in wild-type mice fed a standard diet after CI/WR injury. In contrast, cathepsin B and D fluorescence became diffuse in livers from wild-type mice fed MCD diet after CI/WR, indicating that lysosomal permeabilization had occurred. Hepatocyte apoptosis was rare in both normal and steatotic livers in the absence of CI/WR injury but increased in wild-type mice fed an MCD diet and subjected to CI/WR injury. In contrast, hepatocyte apoptosis and liver damage were reduced in Ctsb(-/-) and cathepsin B inhibitor-treated mice fed the MCD diet following CI/WR injury. In conclusion, these findings support a prominent role for the lysosomal pathway of apoptosis in steatotic livers following CI/WR injury.     

Augmented resistance to oxidative stress in fatty rat livers induced by a short-term sucrose-rich diet

Hepatic steatosis and the accompanying oxidative stress have been associated with a variety of liver diseases. It is not known if fat accumulation per se plays a direct role in the oxidative stress of the organ. This study tested if steatosis induced by a short-term carbohydrate-rich diet results in an increased hepatic sensitivity to oxidative stress. Antioxidant status was determined in a liver perfusion system and in isolated parenchymal, endothelial and Kupffer cells from rats kept on sucrose-rich diet or on regular diet for 48 h. t-Butyl hydroperoxide addition (2 mM) to the perfusion fluid resulted in a release of alanine aminotransferase (ALT) in livers from controls, whereas no ALT release was observed in fatty livers. After t-butyl hydroperoxide addition, oxidized glutathione release was 40% less in fatty than in control livers, whereas reduced glutathione (GSH) release was not different. Sinusoidal oxidant stress was mimicked by the addition of lipopolysaccharide (LPS) from Escherichia coli (10 microg/ml) followed by the addition of opsonized zymosan (8 mg/ml) to the perfusion medium. LPS plus zymosan treatments resulted in the release of ALT in control but not in fatty livers. At the end of perfusion, liver glutathione content was 3-fold elevated, and the tissue content of lipid peroxidation products was approx. 40% less in fatty livers compared to controls. GSH content was doubled and glucose-6-phosphate dehydrogenase (G6PD) expression was elevated by 3- and 10-fold in sinusoidal endothelial and parenchymal cells form fatty livers compared to cells from control animals. Following H(2)O(2) administration in vitro (0.2-1 mM), GSH remained elevated in endothelial and parenchymal cells from fatty livers compared to cells from controls. In contrast, G6PD activity and GSH content were similar in Kupffer cells isolated from fatty or control livers. The study shows that hepatic fat accumulation caused by a short-term sucrose diet is not accompanied by elevated hepatic lipid peroxidation, and an elevated hepatic antioxidant activity can be manifested in the presence of prominent steatosis. The diet-induced increase in G6PD expression and, thus, the efficient maintenance of reduced glutathione in endothelial and parenchymal cells are a supportive mechanism in the observed hepatic resistance against intracellular or sinusoidal oxidative stress.     

Chronic steatosis aggravates cold-storage induced acute ischemic injury in rat donor livers through the perturbation of lipophagy

In this study, we explored the effects of cold ischemia on chronic steatosis and lipid signaling in vivo. Sprague Dawley (SD) rat models of chronic steatosis were established. Pathological observations and liver indices were assessed through hematoxylin-eosin (HE)- and Oil Red O staining. Autophagy and metabolism in adipose tissue were analyzed under post-ischemia and hypoxic conditions via western blotting and immunofluorescent analysis.We found that cold ischemia treatment exacerbated hepatic steatosis and reduced lipid phagocytosis. This manifested as a loss of Microtubule-associated protein 1A/1B-light chain 3 (LC3) and Perilipin 2 (PLIN2), and lower levels of autophagy. Cold ischemia also inhibited lipophagy in transplanted rat livers, most notably in moderate-to-severe steatosis models. Ischemia and hypoxia inhibited lipid phagocytosis and increased lipid accumulation.Collectively, these data show that chronic steatosis aggravates cold storage induced acute ischemic injury in rat donor livers through the inhibition of lipophagy. Moderate-to-severe steatosis therefore influences the postoperative recovery of liver transplant recipients, which should be immediately transplanted to reduce the risk of cold ischemia.     

Insulin responsiveness of isolated perfused livers from rats with streptozotocin induced diabetes.

The ability of insulin to inhibit efflux of potassium (K) and amino acid nitrogen (AAN) from perfused livers of normal and insulin deficient rats was studied. Two groups of rats with different degrees of insulin deficiency were produced by injecting varying amounts of streptozotocin. One group, classified as being moderately diabetic (MD), had fasting plasma glucose levels between 235--425 mg%, while the other group, whose plasma glucose levels greater than 425 mg%, were considered to have severe diabetes (SD). Two other groups of rats were food restricted in order to attain body weights comparable to the two groups of diabetic rats, and livers from these animals were used for control perfusions. The results indicated that the ability of insulin to suppress efflux of K and AAN from perfused livers of rats with MD was comparable to that seen in control perfusions. On the other hand, insulin could not suppress the efflux of either K or AAN from perfused livers of rats with SD. These results indicate that normal hepatic responsiveness to insulin can be lost secondary to the production of insulin deficiency.     

Human livers with cirrhosis and hepatocellular carcinoma have less mitochondrial DNA deletion than normal human livers.

We measured the populations of mutated mitochondrial DNAs with the 7,436 bp or the 4,977 bp deletion from apparently normal human liver and human livers with chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The amount of the mutated mitochondrial DNA was at the same level between normal and chronically hepatitic livers but was significantly lower in human livers with cirrhosis and hepatocellular carcinoma, especially the latter, suggesting that the mutated mitochondrial DNAs may be decreased with the progress of liver disease from chronic hepatitis to cirrhosis and hepatocellular carcinoma. This phenomenon is opposite to that occuring in the ageing process.     

Ribonucleic acid labelling perfused livers of protein-fed and protein-deprived rats

Several observations suggest an increased RNA synthesis in livers of protein-deprived rats, though the RNA/DNA ratio is decreased. A number of hormones may be involved in these changes. Therefore, we studied in RNA metabolism in isolated perfused livers taken from protein-fed and protein-deprived rats. (3H)-orotic acid was given to the rats 2 h before liver explantation, and (14C)-orotic acid was added to the perfusate. Other rats, called controls in vivo, whose livers were not transplanted were also given (3H)-orotic acid followed by (14C)-orotic acid. The livers of these rats, which were not hormone supplemented, were labelled for the same length of times as the livers in vitro. The ratio specific RNA radioactivity/specific nucleotide radioactivity x RNA/DNA was determined and taken as a measure of the RNA synthesis per liver cell. In the controls in vivo, this ratio was significantly higher for protein-deprived than for protein-fed rats. In livers from the protein-fed rats, labelling in vitro increased significantly when growth hormone, hydrocortisone, insulin and tri-iodothyronine were added to the perfusate. Labelling was also significantly higher in these livers than in the controls in vivo. In livers from protein-deprived rats, the ratio in question was the same whether the hormones were added to the perfusate or not, and was significantly lower than in the controls in vivo. Differences in RNA labelling are thus obtained in our in vitro system. Gel electrophoresis of RNA demonstrated normal RNA labelling, showing that the system is suitable for studying liver RNA synthesis. Further refinement can be made by studying the labelling of UTP and CTP. The results might suggest that the liver from a protein-fed rat, explanted in vitro, may increase its RNA synthesis under the influence of the four hormones in question, and that the RNA synthesis of the liver of a protein-deprived rat is high in-vivo and that it might decrease, when it is explanted to in vitro conditions.     

Comparison of metabolism of free fatty acid by isolated perfused livers from male and female rats.

Livers from normal, fed male and female rats were perfused with different amounts of [1-14C]oleate under steady state conditions, and the rates of uptake and utilization of free fatty acid (FFA) were measured. The uptake of FFA by livers from either male or female rats was proportional to the concentration of FFA in the medium. The rate of uptake of FFA, per g of liver, by livers from female rats exceeded that of the males for the same amount of FFA infused. The incorporation by the liver of exogenous oleic acid into triglyceride, phospholipid, and oxidation products was proportional to the uptake of FFA. Livers from female rats incorporated more oleate into triglyceride (TG) and less into phospholipid (PL) and oxidation products than did livers from male animals. Livers from female rats secreted more TG than did livers from male animals when infused with equal quantities of oleate. The incorporation of endogenous fatty acid into TG of the perfusate was inhibite) by exogenous oleate. At low concentrations of perfusate FFA, however, endogenous fatty acids contributed substantially to the increased output of TG by livers from female animals. Production of 14CO2 and radioactive ketone bodies increased with increasing uptake of FFA. The partition of oleate between oxidative pathways (CO2 production and ketogenesis) was modified by the availability of the fatty acid substrate with livers from either sex. The percent incorporation of radioactivity into CO2 reached a maximum, whereas incorporation into ketone bodies continued to increase. The output of ketone bodies was dependent on the uptake of FFA, and output by livers from female animals was less than by livers from male rats. The increase in rate of ketogenesis was dependent on the influx of exogenous FFA, while ketogenesis from endogenous sources remained relatively stable. The output of glucose by the liver increased with the uptake of FFA, but no difference due to sex was observed. The output of urea by livers from male rats was unaffected by oleate, while the output of urea by livers from females decreased as the uptake of FFA increased. A major conclusion to be derived from this work is that oleate is not metabolized identically by livers from the two sexes, but rather, per gram of liver, livers from female rats take up and esterify more fatty acid to TG and oxidize less than do livers from male animals; livers from female animals synthesize and secrete more triglyceride than do livers from male animals when provided with equal quantities of free fatty acid.     

Reduced inflammatory response and increased microcirculatory disturbances during hepatic ischemia-reperfusion injury in steatotic livers of ob/ob mice

Steatosis is a major risk factor for complications after liver surgery. Since neutrophil cytotoxicity is critical for ischemia-reperfusion injury in normal livers, the aim of the present study was to evaluate whether an exaggerated inflammatory response could cause the increased injury in steatotic livers. In C57Bl/6 mice, 60 min of warm hepatic ischemia triggered a gradual increase in hepatic neutrophil accumulation during reperfusion with peak levels of 100-fold over baseline at 12 h of reperfusion. Neutrophil extravasation and a specific neutrophil-induced oxidant stress (immunostaining for hypochlorous acid-modified epitopes) started at 6 h of reperfusion and peaked at 12-24 h. Ob/ob mice, which had a severe macrovesicular steatosis, suffered significantly higher injury (alanine transaminase activity: 18,000 +/- 2,100 U/l; 65% necrosis) compared with lean littermates (alanine transaminase activity: 4,900 +/- 720 U/l; 24% necrosis) at 6 h of reperfusion. However, 62% fewer neutrophils accumulated in steatotic livers. This correlated with an attenuated increase in mRNA levels of several proinflammatory genes in ob/ob mice during reperfusion. In contrast, sham-operated ob/ob mice had a 50% reduction in liver blood flow and 35% fewer functional sinusoids compared with lean littermates. These deficiencies in liver blood flow and the microcirculation were further aggravated only in ob/ob mice during reperfusion. The attenuated inflammatory response and reduced neutrophil-induced oxidant stress observed in steatotic livers during reperfusion cannot be responsible for the dramatically increased injury in ob/ob mice. In contrast, the aggravated injury appears to be mediated by ischemic necrosis due to massive impairment of blood and oxygen supply in the steatotic livers.     

Intereukin-10 and Kupffer cells protect steatotic mice livers from ischemia-reperfusion injury

Steatotic livers are more sensitive to ischemia/reperfusion (I/R) and are thus routinely rejected for transplantation because of their increased rate of primary nonfunction (PNF). Lean livers have less I/R-induced damage and inflammation due toKupffer cells (KC), which are protective after total, warm, hepatic I/R with associated bowel congestion. This protection has been linked to KC-dependent expression of the potent anti-inflammatory cytokine interleukin-10 (IL-10).We hypothesized that pretreatment with exogenous IL-10would protect the steatotic livers of genetically obese (ob/ob) mice from inflammation and injury induced by I/R. Lean and ob/ob mice were pretreated with either IL-10 or liposomally-encapsulated bisphosphonate clodronate (shown to deplete KC) prior to total, warm, hepatic I/R. IL-10 pretreatment increased survival of ob/ob animals at 24 hrs post-I/R from 30% to 100%, and significantly decreased serum ALT levels. At six hrs post-I/R, IL-10 pretreatment increased IL-10 mRNA expression, but suppressed up-regulation of the pro-inflammatory cytokine IL-1β mRNA. However, ALT levels were elevated at six hrs post-I/R in KC-depleted animals. These data reveal that pretreatment with IL-10 protects steatotic livers undergoing I/R, and that phagocytically active KC retain a hepatoprotective role in the steatotic environment.     

Influence of ethanol on the metabolism of perfused normal, fatty and cirrhotic rat livers

1. The influence of ethanol on the metabolism of perfused livers from normal rats and rats in various stages of development of dietary cirrhosis was studied. A choline-deficient, low-protein and high-fat diet was used. Results were obtained on oxygen consumption and carbon dioxide production, on glucose release and uptake by the liver and on changes in the concentrations of lactate and pyruvate and of β-hydroxybutyrate and acetoacetate in the perfusion medium. 2. Oxygen consumption and carbon dioxide production were lower in fatty and cirrhotic livers than in normal livers. Ethanol had no effect on the oxygen consumption of any of the various livers. After addition of ethanol to the perfusion medium carbon dioxide production ceased almost completely in normal livers. Only a slight decrease in the carbon dioxide production occurred in fatty and cirrhotic livers. 3. With every type of liver glucose was released from the liver into the perfusion medium during the initial control period. This release continued after the addition of ethanol to the perfusion medium in experiments with normal and fatty livers, whereas with cirrhotic livers a marked uptake of glucose from the medium was found. A simultaneous release of the glycolytic end products lactate and pyruvate into the medium occurred. 4. The production of ketone bodies was equal in normal and early fatty livers (6 weeks on the fat diet). It was smaller in late fatty livers (3–4 months on the fatty diet) and in cirrhotic livers. 5. The lactate/pyruvate concentration ratio in the perfusion medium increased from 11 to 67 with normal livers, from 12 to 16 with early fatty livers, from 13 to 26 with late fatty livers and from 21 to 55 with cirrhotic livers when the livers were perfused with a medium containing ethanol. The β-hydroxybutyrate/acetoacetate concentration ratio increased from 1·2 to 8·4 in normal livers, from 2·0 to 2·8 in early fatty livers, from 1·2 to 2·4 in late fatty livers and from 2·1 to 4·0 in cirrhotic livers when ethanol was added to the medium. 6. The effects of ethanol on liver metabolism during the development of dietary cirrhosis are discussed and related to human fatty liver and cirrhosis during chronic ethanol consumption.     

Aflatoxin B1 alters protein phosphorylation in rat livers

A study was conducted on the effect of aflatoxin B1 on protein phosphorylation in rat livers by incubation of soluble and insoluble cell fractions with [gamma 32P] ATP. SDS-polyacrylamide gel electrophoresis indicated that a total of eight rat liver phosphoproteins were affected during a feeding period of 36 weeks on a carcinogenic aflatoxin B1-containing diet compared to the phosphoprotein patterns obtained from the livers of rats on a normal noncarcinogenic diet. The appearance of only two of these phosphoproteins were cAMP dependent. DEAE-cellulose chromatography employed to separate the different histone kinase activities in soluble rat liver cell fractions, showed that the specific activity of histone kinase I activity was increased but its total activity decreased while the histone kinase II activity stayed unchanged for rats submitted to the aflatoxin B1-containing diet compared to those on the normal noncarcinogenic diet.     

Autophagy is induced by ischemic preconditioning in human livers formerly treated by chemotherapy to limit necrosis

The effectiveness of ischemic preconditioning (IP) against hepatic ischemia/reperfusion injury during human liver surgery is linked to decreased apoptotic cell death as well as preservation of the ATP content in liver tissue. Overproduction of Bcl-2 is reported in preconditioned organs. In human liver biopsies exhibiting steatosis and/or vascular injuries (mainly peliosis) induced by chemotherapy, we find that the expression of Bcl-2 in centrolobular and peliotic areas colocalizes with the autophagy protein Beclin 1 in IP livers. Increased expression of phosphorylated Bcl-2 in preconditioned livers is associated with a decreased immunoprecipitation of Beclin 1 and increased expression of LC3-II. The increased number of autophagic vacuoles seen by electron microscopy confirmed that IP could trigger autophagy in chemotherapy-injured livers, probably to reduce the pro-inflammatory necrotic cell death of hepatocytes or endothelial cells and to increase ATP levels. Indeed, necrosis is less frequent (p = 0.04) in IP livers than in the others although no change in apoptosis as assessed by TUNEL assay or caspase-3, -8 and -9 expressions is observed. In conclusion, Bcl-2 and Beclin 1 could be major targets in the regulation of cell death during ischemia/reperfusion injury modulating autophagy to switch on/off necrosis and/or apoptosis.     

Theaflavin attenuates ischemia-reperfusion injury in a mouse fatty liver model

The incidence of non-alcoholic fatty liver disease (NAFLD) has been increasing, and there is a shortage of liver donors, which has led to the acceptance of steatotic livers for transplantation. However, steatotic livers are known to experience more severe acute ischemia-reperfusion (I/R) injury than normal livers upon transplantation. In the present study, we investigated the role of theaflavin, a polyphenol substance extracted from black tea, in attenuating acute I/R injury in a fatty liver model. We induced I/R in normal and steatotic livers treated with or without theaflavin. We also separated primary hepatocytes from the normal and steatotic livers, and applied RAW264.7 cells, a mouse macrophage cell line, that was pretreated with theaflavin. We observed that liver steatosis, oxidative stress, inflammation and hepatocyte apoptosis were increased in the steatotic liver compared to the normal liver, however, these changes were significantly decreased by theaflavin treatment. In addition, theaflavin significantly diminished the ROS production of steatotic hepatocytes and TNF-α production by LPS-stimulated RAW264.7 cells. We concluded that theaflavin has protective effects against I/R injury in fatty livers by anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms.     

Characterization of insulinase from mammalian and non-mammalian livers

The Michaelis constants (Km's) and maximum reaction velocities (Vmax's) for the degradation of beef insulin by livers from frogs, guinea pigs, rats, a rabbit, a dog and a pig were determined. The Km's for mammalian livers appear to be species-dependent and range from 0.25 microM to 0.65 microM. The Km for frog liver was somewhat lower, averaging 0.13 microM. The Km is independent of animal age, but the enzyme concentrations (Vmax) were greatly reduced in the fetal guinea pig and 3 day rat compared to the adult livers. There appears to be no relation between Km and the chemical dissimilarity between beef insulin and endogenous insulin of the species, since guinea pig liver insulinase had a Km (0.50 microM) intermediate between dog (0.47 microM) and pig (0.65 microM) liver insulinase although guinea pig insulin has a markedly different amino acid sequence and biologic activity.     

Response of normal and reperfused livers to glucagon stimulation: NMR detection of blood flow and high-energy phosphates

The effects of glucagon on blood flow and high-energy phosphates in control and in rat livers damaged by ischemia were studied using in vivo nuclear magnetic resonance (NMR) spectroscopy. Normal livers and livers which had been made ischemic for 20, 40, and 60 min followed by 60 min of reperfusion were studied. Ischemia led to a loss in adenosine triphosphate (ATP) within 30 min. Reperfusion after 20 min of ischemia led to complete recovery of ATP. 60 min of reperfusion after 40 or 60 min of ischemia led to only a 76% and 48% recovery of ATP, respectively. Glucagon, at doses up to 2.5 mg/kg body weight, caused no changes in the inorganic phosphate (Pi) to ATP ratio in normal livers as measured by ³¹P-NMR spectroscopy. In livers which had been made ischemic for 20, 40, or 60 min, glucagon caused an increase in the Pi/ATP ratio of 18%, 40%, and 40%, respectively. ¹⁹F-NMR detection of the washout of trifluoromethane from liver was used to measure blood flow. Glucagon-stimulated flow in the normal liver in a dose-dependent manner, with 2.5 mg glucagon/kg body weight leading to a 95% increase in flow. Ischemia for 20, 40, and 60 min followed by 60 min of reperfusion led to hepatic blood flows which were 63%, 68%, and 58% lower than control liver. In reperfused livers, blood flow after glucagon-stimulation was reduced to 56%, 43%, and 48% of control glucagon-stimulated flow after 20, 40, and 60 min of ischemia. These results indicate that ischemia followed by reperfusion leads to deceases in hepatic blood flow prior to alterations in ATP and the response of the liver to glucagon is altered in the reperfused liver.     

Amino acid release by isolated perfused cirrhotic livers

Livers of normal and cirrhotic rats were perfused in vitro with and without amino acid substrates (2.3 mM ornithine, 10 mM glutamine or 20 mM alanine) in order to assess urea formation and amino acid release. The rates of urea production were lower in the livers of cirrhotic rats when compared to those of controls only in perfusions with added substrates. The release of several amino acids by livers of cirrhotic rats was higher than that of controls although the pattern of amino acids in the perfusate was different from that reported in plasma during hepatic insufficiency.     

Deficit in nitric oxide production in cirrhotic rat livers is located in the sinusoidal and postsinusoidal areas

Intrahepatic nitric oxide (NO) production is decreased in cirrhotic livers. Our objective was to identify, in cirrhotic rat livers, intrahepatic vascular segments where the deficit of NO facilitates the effect of vasoconstrictors. By using a modified rat liver perfusion system with measurement of both the perfusion and sinusoidal (wedged hepatic vein) pressures, we studied the effect of the NO synthase blocker N(omega)-nitro-l-arginine (l-NNA) on the response to methoxamine (alpha(1)-adrenoreceptor agonist) in different segments of the intrahepatic circulation of normal and cirrhotic rat livers. l-NNA enhanced the presinusoidal, sinusoidal, and postsinusoidal responses to methoxamine in normal livers as well as the presinusoidal response in cirrhotic livers. However, l-NNA did not change the already enhanced sinusoidal/postsinusoidal response to methoxamine in cirrhotic livers. The postsinusoidal response to methoxamine was higher in cirrhotic rats with ascites than in those without ascites. We concluded that NO modulates the presinusoidal, sinusoidal, and postsinusoidal vascular tone in normal livers. NO production in cirrhotic rat livers is severely impaired in the sinusoidal and postsinusoidal areas but is preserved in the presinusoidal area, as evidenced by its normal response to l-NNA. We speculate that an increased postsinusoidal response to catecholamines may participate in the genesis of ascites in cirrhosis.     

Differential gene expression of livers from ApoE deficient mice

A genomic survey for differentially expressed genes was performed to livers of ApoE deficient mice using human cDNA microarray containing approximately 9,000 human cDNA clones. Due to the homology between mouse and human, hybridization was performed at lower stringency condition, 10 degrees below the regular hybridizing temperature. Gene expression profiles of livers corresponding to high levels of blood cholesterol were generated at genomic scale. Thirty-seven genes were randomly selected from a pool of differentially expressed genes and subjected to semi-quantitative RT-PCR, further confirmed the result from microarray hybridization. These included genes associated with atherosclerosis, and novel genes that implied novel pathways correlated to high levels of blood cholesterol. It is promising using human cDNA microarray, the most complete collection among all species, to study other mammalian systems with satisfying speed and accuracy.