Acute endotoxin tolerance downregulates superoxide anion release by the perfused liver and isolated hepatic nonparenchymal cells

This work is based on the hypothesis that low-dose lipopolysaccharide (LPS) suppresses the stimulatory and priming effects of a subsequent high-dose endotoxin on the formation of toxic oxygen-derived radicals by the perfused liver and isolated hepatic nonparenchymal cells. Such effects may in turn contribute to hyposensitivity to the lethal effect of large doses of endotoxin. Male Sprague-Dawley rats received a nonlethal (“low-dose”) intravenous injection of Escherichia coli LPS (0.5 mg/kg body weight) 12 to 120 hours before they were challenged by a “large dose” of endotoxin (10 mg/kg). Three hours after LPS challenge, the livers were perfused, and superoxide release was determined. Nonparenchymal cells were also isolated for the determination of superoxide anion formation in vitro. There was a low rate (0.14 ± 0.1 nmol/min/g liver weight) of superoxide generated by the perfused livers from rats that received the low-dose LPS 1 to 5 days previously. Control livers generated less than 0.08 nmol superoxide. A high rate (1.3 ± 0.1 nmol/min/g) of superoxide release was measured in the perfused liver 4 hours after treatment of previously untreated control rats with large-dose LPS. This was attenuated to 0.7 ± 0.04 nmol/min/g by an injection of low-dose LPS before challenge. This attenuation was time dependent; it failed to manifest at 12, 24, or 120 hours after low-dose LPS. Isolated endothelial cells, Kupffer cells, and sequestered hepatic neutrophils from rats given a high-dose LPS also generated significant amounts of superoxide both in the presence or absence of agonists, i.e., phorbol myristate acetate or opsonized zymosan. Pretreatment of rats with the low dose of LPS 48 hours before the large dose attenuated the spontaneous and primed release of superoxide by isolated nonparenchymal cells. Serum transaminase activities were also reduced in these LPS-tolerant rats. The suppressed oxygen-derived radical formation by the liver and nonparenchymal cells during acute LPS tolerance was associated with down-regulation of tumor necrosis factor (TNF) release in vivo. TNF has been shown to stimulate and prime neutrophils and Kupffer cells for increased respiratory burst. These results suggest that suppression of free radical formation may contribute to the amelioration of hepatic injury during acute endotoxin tolerance.     

Cross-Tolerance Between Acute Alcohol Intoxication and Endotoxemia

This study tests two hypotheses: (1) prior exposure to LPS induces cross-tolerance for the hepatic effects of subsequent short-term alcohol intoxication; and (2) short-term alcohol intoxication renders the liver resistant to the effects of acute endotoxemia, resulting in reduced production of superoxide and tumor necrosis factor. In the first group of experiments, male Sprague-Dawley rats were treated intravenously with E. coli lipopolysaccharide (LPS) (0.5 mg/kg) 48 hr before they were given an intravenous bolus of ethanol (1.75 g/kg), followed by 250–300 mg/kg/hr) for 3–5 hr. Superoxide release in the perfused liver was measured after the 3-hr ethanol infusion. Pre-treatment with LPS attenuated ethanol-mediated superoxide anion release by the perfused liver. The stimulatory effect of phorbol myristate acetate on hepatic release of superoxide was also decreased. In the second group of experiments, rats previously treated with ethanol for 5 hr, received an intravenous injection of LPS (1 mg/kg). At 90 min after LPS, sera were collected for tumor necrosis factor a assay. Hepatic release of superoxide anion was determined 3 hr after LPS. Acute ethanol intoxication for 5 hr significantly reduced LPS-induced serum tumor necrosis factor activity and free radical release by the perfused liver. LPS-induced mortality was also decreased. In both groups of experiments serum corticosteroid levels were reduced during cross-tolerance. These results demonstrate that cross-tolerance develops between acute alcohol intoxication and endotoxemia manifesting in reduced hepatic production of cytotoxic cytokines and superoxide anions.     

Modulation of F-met-leu-phe Induced Chemotactic Activity and Superoxide Production by Neutrophils during Chronic Ethanol Intoxication

Chronic alcohol consumption has been associated with increased migration of neutrophils into liver that could contribute to the development of alcoholic liver disease. Mild endotoxemia may be at least partially responsible for this condition since endotoxemia was shown to be present in virtually all chronic alcoholics. This study examines the release of superoxide anion and chemotactic activity by Kupffer cells and sequestered hepatic as well as blood neutrophils during chronic alcohol intoxication (16 weeks) alone, and following an intravenous injection of Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) 3 hr before cell isolation. Chronic ethanol consumption increased the total neutrophil yield per liver, but did not change the f-met-leu-phe induced chemotactic activity by both hepatic and blood neutrophils. However, the combined insults of ethanol and LPS increased the chemotactic activity and superoxide anion generation by these cells. Plasma from ethanol-fed rats was highly chemotactic to syngeneic normal rat neutrophils. This activity was increased 1.75-fold in the plasma obtained from chronic ethanol plus endotoxin-injected rats. The chemotactic activity of Kupffer cells was not significantly modulated during ethanol intoxication plus endotoxin treatment. The f-met-leu-phe-induced superoxide anion release by Kupffer cells was enhanced after LPS treatment. Chronic ethanol consumption did not induce any effect on this parameter. These observations suggest that functional alterations in neutrophils during chronic ethanol intoxication may contribute to hepatic injury.     

Long-Term Ethanol Feeding Enhances Susceptibility of the Liver to Orally Administered Lipopolysaccharides in Rats

Background Endotoxin has been implicated in the pathogenesis and progression of alcoholic liver disease. However, it is still unclear how long-term ethanol feeding affects absorption of endotoxin from the intestine and susceptibility of the liver to gut-derived endotoxin. The object of this study was to determine the effect of long-term ethanol feeding on hepatic susceptibility to orally administered endotoxin.
Methods Male Wistar rats that weighed approximately 150 g were pair-fed with an ethanol-containing liquid diet or a control diet for 35 days. In some experiments, 0, 10, or 20 mg/kg of lipopolysaccharides (LPS) was added to the liquid diet for 7 days beginning on day 29. On day 36, the animals were killed for blood biochemistry and histologic examination of the liver. We also determined plasma endotoxin levels after 20 mg/kg of LPS administration using a gastric tube. In another set of experiments, we determined intestinal permeability using FD4 (fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4000 D).
Results With 10 mg/kg of LPS, serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels were significantly increased in the ethanol-fed rats but not in controls. After 20 mg/kg of LPS administration, more substantial increases in serum ALT and ALP levels were observed in ethanol-fed rats as compared with control diet-fed rats. Plasma endotoxin levels in long-term ethanol-fed rats were higher than those in control rats after intragastric administration of high-dose endotoxin (20 mg/kg). Furthermore, intestinal permeability to FD4 was increased by long-term ethanol administration.
Conclusions Long-term ethanol feeding increases intestinal permeability to and absorption of endotoxin, which can sequentially enhance hepatic susceptibility to orally administered endotoxin. This model has potential as a subclinical experimental model for the study of alcoholic liver disease.     

Depressed hepatic glutathione and increased diene conjugates in alcoholic liver disease

The role of lipid peroxidation in the pathogenesis of alcoholic liver disease has been a subject of controversy. In order to study this question we measured hepatic glutathione and diene conjugates in liver biopsies from 16 alcoholics with different stages of liver injury and 8 nonalcoholics with liver disease. Patients with alcoholic liver disease were found to have decreased hepatic glutathione compared to patients with liver disease unrelated to alcohol (22.1±2.5 vs 33.5±4.6 nmol/mg protein,P<0.05). The decrease in glutathione was accompanied by an increase in diene conjugates in hepatic lipids (3.37±0.14 vs 2.26±0.21 OD at 232 nm/mg lipid,P<0.001). The changes were present in all stages of alcoholic liver damage including fatty liver but unrelated to nutritional status in these patients. They support the concept that lipid peroxidation may be an important mechanism in the pathogenesis of alcoholic liver disease.     

Hypoxia increases plasma glutathione disulfide in rats

We tested the hypothesis that hypoxia causes cellular oxidative stress by measuring plasma concentrations of glutathione disulfide (GSSG) in rats exposed to acute and subacute hypoxia. In awake, unanesthetized, catheter-implanted rats, exposure to 8% O₂ for 10 min caused pulmonary vasoconstriction and increased plasma GSSG. This increase in plasma GSSG was reversible upon re-exposure to room air. In another group of rats exposed to 48 hours of hypobaric hypoxia (P_b 450 mmHg, equivalent to about 14,500 feet altitude), plasma GSSG, but not total glutathione, was significantly increased over control values (2.83±0.24 vs 1.84±0.14 nmol/ml,p<0.05). While lung tissue GSSG in high altitude-exposed rats were somewhat higher than in controls (17.4±7.0 vs 11.9±3.6 nmol/g wet lung wt.), the difference was not statistically significant. Treatment of the rats with a radical scavenger, DMSO, before altitude exposure, blocked the increase in plasma GSSG (1.86±0.16 nmol/ml). We conclude that both acute and subacute hypoxia increase plasma GSSG in rats and speculate that hypoxia induces cellular oxidative stress in vivo.     

The Phosphodiesterase III Inhibitor Olprinone Decreases Sensitivity of Rat Kupffer Cells to Endotoxin

Background: Sensitivity of Kupffer cells to endotoxin [lipopolysaccharide (LPS)] and overproduction of tumor necrosis factor-α (TNF-α) are critical for progression of alcoholic liver injury. Therefore, suppression of TNF-α should prove useful for treatment of alcoholic liver injury. However, a transient increase of intracellular calcium ([Ca²⁺]_i) is required for LPS-induced TNF-α production by the macrophage cell line. The phosphodiesterase III inhibitor olprinone has been shown to suppress [Ca²⁺]_i level in vascular smooth muscle cells. Accordingly, the purpose of this study was to determine whether olprinone could prevent sensitization of Kupffer cells to endotoxin.
Methods: Kupffer cells were isolated by collagenase digestion and differential centrifugation. LPS was added to Kupffer cells 24 hr after incubation with or without olprinone (0.1 μmol/liter). After addition of LPS (10 μg/ml) to culture media, [Ca²⁺]_i was measured using a fluorescent indicator, fura-2.
Results: LPS increased [Ca²⁺]_i of Kupffer cells in control rats from basal levels (28 ± 4 nmol/liter) to 280 ± 14 nmol/liter. This increase was blunted by olprinone (91 ± 8 nmol/liter). Similarly, olprinone diminished the LPS (1 μg/ml)-induced TNF-α production by Kupffer cells by 30% (2220 ± 116 vs. 1386 ± 199 pg/ml; p < 0.05).
Conclusions: These results indicate that olprinone decreases sensitivity of Kupffer cells to endotoxin.     

Role of glucagon as a contributor to glucose intolerance in acute and chronic uremia

It has been suggested that increased sensitivity to glucagon may contribute to glucose intolerance in uremia. In order to evaluate this possibility systematically, we have assessed the effect of glucagon on hepatic glucose outflow, formation of cAMP, and activation of adenylate cyclase by livers obtained from acutely and chronically uremic rats and their respective sham operated controls. Glucagon infused at rates of 6 ng/min/kg rat resulted in minimal and equivalent increases in hepatic glucose outflow and cAMP accumulation when livers from acutely uremic and control rats were perfused for 30 min. However, at glucagon infusion rates of 18 ng/min/kg, glucose efflux from perfused livers of acutely uremic rats was significantly reduced (p < 0.001) compared to perfused livers of control rats (4.64 ± .9 vs 12.7 ± 2.4 μmol/g liver) and cAMP accumulation was also significantly lower (p < 0.01) (1352 ± 222 vs 3100 ± 348 pmol/g liver). Basal adenylate cyclase activity of hepatic membranes obtained from uremic and control rats was similar, and was stimulated by glucagon concentrations ranging from 10^?8 to 10^?6 at equivalent rates in both groups. In livers from chronically uremic rats, glucagon infused at rates of 6 ng/kg/min significantly increased hepatic glucose outflow (32.5 ± 6.9 μmolg liver). However this was not greater than that of control animals (37.6 ± 9.2). Furthermore, cAMP accumulation was significantly lower (p < .02) in chronically uremic rats than in controls, and activation of adenylate cyclase by glucagon was similar in both groups. These findings indicate that glucagon does not increase glucose efflux, cAMP accumulation or enhance activation of adenylate cyclase by isolated perfused livers from either acutely or chronically uremic rats. Thus, glucose intolerance in uremic rats does not appear to be due to increase hepatic glucose output resulting from increased sensitivity to glucagon.     

Postbinge Effects of Acute Alcohol Intoxication on Hepatic Free Radical Formation

The present studies were performed to test the hypothesis that Kupffer and endothelial cells are activated after recovery from an acute alcohol binge, which is accompanied by formation of oxygen-derived radicals. These radicals have been implicated in the pathogenesis of alcohol-mediated tissue injury in a number of organs. Male Sprague-Dawley rats received an intravenous injection of 20% ethanol in saline (1.75 g/kg), followed by an intravenous infusion (250 to 300 mg/kg/hr) for 12 hr. At the end of 12-hr infusion, ethanol was replaced by saline, and the infusion was continued for a further 6 hr. This was referred to as the recovery period. The 6-hr recovery period was selected because superoxide anion generation by the perfused liver peaked at this time point. Superoxide anion formation by the perfused liver was measured by the superoxide dismutase-inhibit-able reduction of ferricytochrome c. Kupffer and endothelial cells were isolated for the determination of in vivo glucose uptake and in vitro superoxide anion release. Results show that a significant ( p < 0.05) amount of superoxide (1.54 nmol/min/g) was generated by the perfused liver at 6 hr recovery after 12 hr of ethanol infusion. Serum ALT activity was also elevated in this treatment group. Time-matched control-saline infused animals or ethanol-treated animals without a recovery period released <0.2 nmol/min/g of superoxide. The postrecovery superoxide production and an accompanying increase in the in vivo glucose uptake were also observed in isolated Kupffer and endothelial cells. Depletion of Kupffer cells by gadolinium chloride before ethanol treatment and recovery was associated with significant attenuation of free radical formation by the perfused liver and reduction of serum ALT. These studies demonstrate that recovery from an acute alcohol binge has a stimulating effect on hepatic sinusoidal superoxide production, and it may also affect liver function.     

Glucagon stimulation of hepatic gluconeogenesis in rats fed a high-protein, carbohydrate-free diet

We previously observed increased gluconeogenesis in isolated liver of rats fed a high protein, carbohydrate-free (HP) diet and postulated that this was due to enhanced secretion of glucagon. To test this hypothesis, the effects of glucagon, dibutyryl cyclic AMP (DB-CAMP), and insulin on hepatic gluconeogenesis were examined. In perfused liver of control rats, alanine conversion to carbohydrate amounted to 20.39 ± 3.15 μmole/100 g body wt/90 min. Gluconeogenesis was increased to 35.14 ± 2.66 by glucagon; to 31.40 ± 1.0 by DB-CAMP, and suppressed to 14.56 + 1.15 by insulin. When both glucagon and insulin were added to the medium, only the stimulatory action of glucagon was evident. In perfused liver of HP-fed rats, alanine incorporation into carbohydrate totalled 51.79 ± 4.36. Gluconeogenesis was elevated by glucagon to 65.23 + 1.78, by DB-CAMP to 62.27 + 2.41, and was unaffected by insulin, 49.73 + 3.11. In the presence of both glucagon and insulin, only the glucagon effect was apparent. Levels of intermediary metabolites involved in gluconeogenesis were determined in control and HP livers. Concentrations of alanine, lactate, and pyruvate were reduced in HP liver, while phosphoenol-pyruvate (PEP) was increased, findings that suggest enhanced conversion of pyruvate to PEP. Fructose diphosphate concentration was normal; however, fructose-6-phosphate was greatly elevated, suggesting that fructose diphosphatase was stimulated by HP feeding. Similar changes in hepatic metabolites have been found following exposure of normal liver to glucagon. Plasma insulin values were not altered by HP feeding (C = 36.7 ± 3.7 μU/ml; HP = 32.3 ± 2.8). Plasma-glucagon levels were higher in HP rats than in controls (C = 80 ± 7.5 pg/ml; HP = 137.8 ± 16.7; p < 0.01). When the molar ratio of insulin to glucagon in plasma was calculated, the value in HP-fed rats was reduced, thus demonstrating a relative as well as an absolute rise of glucagon concentration (C = 11.1 ± 1.1; HP = 5.76 ± 0.6; p < 0.001). Liver cyclic AMP was greater in HP-fed rats than in controls (C = 0.83 ± 0.04 nmole/g; HP = 1.17 ± 0.07; p < 0.001), a finding that indicates that the liver was exposed to an increased amount of glucagon.     

Inhibition of nuclear factor-kappaB activation by IRFI 042, protects against endotoxin-induced shock

BACKGROUND: The aim of our study was to investigate the effect of IRFI 042, a novel dual vitamin E-like antioxidant, on nuclear factor-kappaB (NF-kappaB) activation, TNF-alpha gene priming and on the release of the mature protein during endotoxin shock. METHODS: Endotoxin shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg kg(-1) of Salmonella enteritidis lipopolysaccharide (LPS). Survival rate, mean arterial blood pressure, serum TNF-alpha and plasma malondialdehyde (MAL) levels were investigated. We then evaluated in the liver TNF-alpha mRNA levels, NF-kappaB binding activity and the inhibitory protein IkappaBalpha. Moreover we studied in LPS stimulated (50 microg ml(-1)) peritoneal macrophages (Mphi), NF-kappaB activation, cytoplasmic IkappaB-alpha degradation, the message for TNF-alpha, and TNF-alpha and MAL levels. RESULTS: LPS administration reduced survival rate (0%, 72 h after LPS administration), decreased mean arterial blood pressure, augmented serum TNF-alpha (60+/-11 ng ml(-1)) and enhanced plasma malondialdehyde (MAL) levels (55+/-7.1 nmol l(-1)). LPS shocked rats also had increased TNF-alpha mRNA levels, augmented liver NF-kappaB binding activity in the nucleus and decreased levels of the inhibitory protein IkappaBalpha. In addition, in vitro LPS stimulation (50 microg ml(-1)) significantly induced NF-kappaB activation and cytoplasmic IkappaBalpha degradation in Mphi, enhanced TNF-alpha mRNA levels and increased Mphi TNF-alpha and MAL. Treatment with IRFI 042 (20 mg kg(-1), i.v., 5 min after endotoxin challenge) protected against LPS-induced lethality (90% survival rate 24 h and 80% survival rate 72 h after LPS injection, respectively), reduced hypotension, blunted plasma MAL (9.0+/-0.9 nmol l(-1)) and decreased serum TNF-alpha (15+/-3 ng ml(-1)). The antioxidant also inhibited the loss of IkappaBalpha protein from the hepatic cytoplasm, blunted the increased NF-kappaB binding activity in the liver and decreased hepatic liver mRNA for TNF-alpha. Furthermore 'in vitro' IRFI 042 (50 microM) significantly inhibited activation of NF-kappaB through inhibition of IkappaBalpha degradation, reduced the amount of TNF-alpha mRNA, decreased LPS-induced TNF-alpha release and blunted lipid peroxidation (MAL) in LPS stimulated Mphi. CONCLUSIONS: These data suggest that IRFI 042 blocks the activation of NF-kappaB, reduces TNF-alpha mRNA levels, and finally reverses endotoxic shock.     

Ethanol Suppresses Endotoxin But Not Platelet Activating Factor-Induced Hypotension and Nitric Oxide

Ethanol (ETOH) inhibits the immune response to endotoxemia. The early stage of endotoxin (LPS)-induced shock is associated with an acute phase cardiovascular depression (APCD). Release of platelet activating factor (PAF) and tumor necrosis factor alpha (TNFa) with upregulation of nitric oxide (NO) production may initiate the APCD. Since ETOH inhibits induction of NO synthase (iNOS) mNRA by LPS, we postulate that ETOH may mask the APCD associated with endotoxemia. To test this, Sprague-Dawley rats (280–320 g, n = 5–6/group) were given LPS [0.75 mg/kg, intravenously (iv)] or PAF (10 to 150 pg/kg, iv) 30 min after administration of sterile saline (PBS), BN-5073 a mixed PAF antagonist (0.50 μg/kg, iv), or ETOH t2.2-5.5 g/kg, intra-peritoneally (ip)]. Cardiovascular parameters and plasma concentrations of nitrate and nitrite (RNI), ETOH, TNFα, and neutrophil (PMN) generation of RNI were measured. LPS and PAF both produced APCD. LPS-induced APCD was associated with tachycardia, elevated plasma TNFα and RNI, and ex wivo generation of RNI by PMNs. ETOH and EN-50730 prevented LPS-induced APCD and increases in RNI and TNFα. ETOH, however, increased the mortality associated with APCD. PAF produced only hypotension, bradycardia and elevated plasma levels of TNFα. ETOH and LNMMA did not affect PAF-induced APCD. EN-50730 inhibited PAF-induced APCD and plasma TNFα. We conclude that 1) ETOH inhibits the APCD and induction of NO characteristic of endotoxemia and 2) ETOH-induced suppression of LPS-mediated APCD may be mediated in part by suppression of release of intracellular PAF. Ethanol may increase the morbidity and mortality of endotoxemia by masking the hypotension and humoral changes characteristic of early endotoxemia thereby delaying appropriate therapy and by diminution of the protective effects of endogenous NO.     

Alcohol Consumption in Rats Potentiates the Deleterious Effect of Gram-Negative Sepsis on Hepatic Hyaluronan Uptake

The role of hepatic sinusoidal endothelial cells (SECs) in the pathologic changes of the liver associated with alcohol consumption is not fully understood. The measurement of hyaluronan (HA) uptake by the SECs provides a useful means for assessing the functional state of these cells. In this study, we determined the effect of acute and chronic exposure to alcohol in rats in the absence and presence of subcutaneous Escherichia co/i-induced sepsis on plasma HA concentration and HA uptake by the isolated, perfused liver. Rats were administered ethanol (two doses of 0.2 g/100 g body weight, intraperitoneal, 24 and 15 hr before killing) or fed a liquid diet for 8–10 weeks, containing alcohol (36% of the total calories) or dextrin (in isocaloric amounts). Twenty-one hr before euthanizing for liver perfusion, animals were injected subcutaneously with live E. coli (sepsis) or sterile saline (control). Neither acute nor chronic alcohol exposure by themselves altered plasma HA levels. However, both treatments exacerbated the hyperhyaluronanemic effect of sepsis. Thus, in acutely alcohol-treated rats, sepsis induced a 187% (p &< 0.05) increase in plasma levels of HA, whereas in nonalcohol septic rats, the increase was only 54% (p &< 0.05). Likewise, sepsis resulted in a greater increase in the plasma levels of HA (871%) in alcohol-fed rats than it did in liquid diet, control-fed rats (323%, p &< 0.05). The rate of HA uptake by the isolated, perfused liver was not altered by either acute or chronic alcohol exposure. However, alcohol exposure markedly potentiated the inhibitory effect of sepsis on the capacity of the liver to take up HA. Thus, in acutely alcohol-treated rats, sepsis decreased HA uptake (60-80%, p &< 0.05), whereas in the corresponding nonalcoholic control group the decrease was evident only at the beginning of HA infusion. In chronically alcohol-fed rats, sepsis induced an 80% (p &< 0.05) inhibition of HA uptake, whereas in diet-fed control rats the inhibition was only 60% (p &< 0.05). The inhibition by sepsis of HA uptake by the isolated, perfused liver provides an explanation for the previously observed hyperhy-aluronanemia in septic humans and animals. Because alcohol alone does not alter HA metabolism, the results suggest that acute and chronic alcohol exposure influences the communication between liver cells leading to downregulation of HA clearance by SECs.     

Impaired renal mevalonate metabolism in nephrotic syndrome: A stimulus for increased hepatic cholesterogenesis independent of GFR and hypoalbuminemia

Increased hepatic lipogenesis in the nephrotic syndrome is not adequately explained by hypoalbuminemia. In this disorder an enhanced delivery of the cholesterol precursor mevalonic acid (MVA) to the liver may be an unidentified stimulus to cholesterogenesis. Since the kidneys are the major site of mevalonate excretion and metabolism by either the sterol or nonsterol shunt pathways, an impairment of any of these metabolic alternatives could result in redistribution of mevalonate to the liver. Male Sprague-Dawley rats rendered nephrotic by puromycin aminonucleoside had their kidneys perfused with Krebs-Henseleit-bicarbonate buffer containing albumin, glucose and 5-¹⁴C-MVA. The number five carbon label was utilized so that any ¹⁴CO₂ produced would represent mevalonate shunt pathway activity. The isolated perfused kidney was used to eliminate confounding variables. In eight control kidneys perfused for 2 hr 62 ± 2% of the MVA was removed from the perfusate compared to 50 ± 2% in five nephrotic kidneys (p < .006). Urinary MVA recovery was 22 ± 2% in controls, 15 ± 1% in nephrotics (p < .05). The incorportion of ¹⁴C into renal tissue lipids was not different in the two groups. Recovery of ¹⁴CO₂ was two times greater in controls than in nephrotics (p < .006). Inulin clearance per gram of kidney and sodium reabsorption were similar for the two groups. Isolated perfused kidneys from nephrotic rats metabolize MVA abnormally such that less is excreted, less is oxidized, and more is available for recirculation to the liver. This occurs independently of hypoalbuminemia, a change in glomerular filtration rate, or an overt histo-pathologic lesion. These events create an environment for increased hepatic cholesterol synthesis.     

Induction of hepatic 11beta-hydroxysteroid dehydrogenase type 1 in patients with alcoholic liver disease

Background and aim The alcohol‐induced pseudo‐Cushing's syndrome is an important differential diagnosis of hypercortisolism that is poorly understood. Two isozymes of 11β‐hydroxysteroid dehydrogenase (11β‐HSD) interconvert hormonally active cortisol (F) and inactive cortisone (E). Previously we have shown higher urinary F : E metabolite ratios (a reflection of global 11β‐HSD activity) in patients with alcoholic liver disease (ALD) compared to patients with chronic liver disease (CLD) of other aetiologies, suggesting that the phenotype of alcoholic pseudo‐Cushing's may relate to altered metabolism of F. Subjects and methods We performed selective venous sampling of the hepatic, renal and peripheral veins measuring F and E concentrations (using in‐house radioimmunoassay) in 20 patients with histologically confirmed ALD and 19 patients with CLD. Six patients who also had selective venous sampling for investigation of suspected hyperaldosteronism were used as ‘normal’ controls. Results There was a significant difference in the hepatic F gradient (mean ± SEM) between groups, indicating increased F production in the liver in patients with ALD (34·5 ± 21·7 nmol/l) compared to those with CLD (–21·0 ± 18·5 nmol/l) (P < 0·05) and normals (–19·7 ± 17·2 nmol/l) (P < 0·05). 11β‐HSD1 mRNA expression was increased fivefold in the ALD group compared with normal controls (P < 0·01). Conclusions These results indicate significant induction of HSD11B1 gene expression and activity in patients with ALD during short‐ and long‐term abstinence from alcohol. The mechanism is unknown but might be explained on the basis of alcohol‐induced changes in intracellular redox potential or as a protective mechanism to limit liver inflammation and injury. Selective 11β‐HSD1 inhibitors may offer a novel therapeutic approach to treat alcoholic pseudo‐Cushing's.     

Reduced hepatic glycogen stores in patients with liver cirrhosis

Abstract: Background: Patients with alcoholic liver cirrhosis have reduced hepatic glycogen stores but the mechanisms leading to this finding are not clear. Methods: We therefore determined the hepatic glycogen content in patients with alcoholic (n = 9) or biliary cirrhosis (n = 8), and in control patients undergoing liver surgery (n = 14). All patients were in the postabsorptive state. In addition, we performed a morphometric analysis of the livers, and measured activities and mRNA expression of several enzymes involved in glycogen metabolism. Cirrhotic and control patients were similar regarding age and body weight. Results: Cirrhotic patients had a reduced glycogen content per gram liver wet weight (17 ± 11 versus 45 ± 17 mg/g, P < 0.05), per milliliter hepatocytes (28 ± 16 versus 52 ± 21 mg/ml, P < 0.05) and per liver (28 ± 17 versus 64 ± 22 g, P < 0.05), the reduction being observed in both patients with alcoholic or biliary cirrhosis. Liver histology confirmed these findings and revealed that the decrease in liver glycogen in cirrhotic patients was not homogenous across cirrhotic lobules. Activities of glycogen synthase and phosphorylase (total activity and active form) were not different between cirrhotic and control patients, whereas hepatic mRNA expression was decreased in cirrhotics by approximately 50%. The activity of glucokinase was decreased in cirrhotic as compared in control patients (0.06 ± 0.30 versus 0.42 ± 0.21 U/ml hepatocytes, P < 0.05), the reduction being observed in both patients with alcoholic or biliary cirrhosis. Conclusions: We conclude that patients with alcoholic or biliary cirrhosis have decreased hepatic glycogen stores per volume of hepatocytes and per liver. Decreased activity of glucokinase may represent an important mechanism leading to this finding.     

Hyperbaric oxygen pretreatment attenuates hepatic reperfusion injury

ABSTRACT— Microcirculatory derangement, energy depletion and lipid peroxidation have been related to development of ischemia-reperfusion injury in the liver. This study investigates the effects of hyperbaric oxygen (HBO) on hepatic ischemia-reperfusion injury. Adult, male Sprague-Dawley rats were used. Three groups were evaluated: 1) sham-operated control (laparotomy only, no ischemia, no HBO), n=8; 2) ischemia control (1-h ischemia, 2-h reperfusion, no HBO), n=8; and 3) HBO pretreatment (100% oxygen, 2.5 atm absolute, 90 min) plus ischemia (1-h ischemia, 2-h reperfusion), n=8. An in vivo microscope was used to investigate hepatic microcirculation. Tissue malondialdehyde (MDA) and adenosine triphosphate (ATP) were determined. In comparison with the ischemia control group, HBO significantly improved harmful insults following ischemia-reperfusion. HBO lessened adherent leukocyte count (6.00±1.31 cells/200 μm vs 11.38±2.88 cells/200 μm), and improved flow velocity (1.72±0.26 mm/s vs 0.83±0.19 mm/s) in post-sinusoidal venules. HBO also reduced MDA (1.04±0.24 nmol/mg protein vs 2.24±0.49 μmol/g protein), and increased ATP (2.03±0.17 μmol/g wet wt vs 0.73±0.11 μmol/g wet wt) levels. This study demonstrates that HBO before ischemia may ameliorate the ischemia-reperfusion injury of the liver in the rat model.     

成人脂肪肝整肝肝细胞分离及大量冻存的实验研究

目的建立成人脂肪肝整肝肝细胞的分离以及人肝细胞大量冻存技术,为生物人工肝提供稳定的人肝细胞来源。方法采用胶原酶经肝静脉逆行灌注的方法分离成人重度脂肪肝的整肝肝细胞,并比较常规冻存和程序冻存后肝细胞在细胞活性、贴壁率、LDH漏出量及白蛋白合成能力的差异。结果采用添加N-乙酰半胱氨酸(NAC)的胶原酶灌注液分离肝细胞的产量为(7.4±0.5)×10~6 cells/g肝组织,活性为(81.4±3.4)%,而未添加NAC组的肝细胞产量为(5.6±0.8)×10~6 cells/g肝组织和活性为(67.3±5.0)%,差异均有统计学意义(P0.05)。分离的人肝细胞采用程序冻存后在细胞活性、贴壁率及白蛋白合成能力方面均相应高于常规冻存组(P0.05),LDH漏出量低于常规冻存组(P0.05)。结论应用添加NAC的胶原酶灌注液经肝静脉逆行灌注可提高脂肪肝整肝肝细胞分离的活性及产量,采用程序冻存的方法可以提高冻存人肝细胞的活性,满足生物人工肝对肝细胞的需要。     

All-trans retinoic acid suppresses liver injury induced by Propionibacterium acnes and lipopolysaccharide in rats

All-trans retinoic acid (ATRA) has been reported to exert major effects on the immune system, including monocytes/macrophages. The present study was designed to determine whether ATRA would modulate macrophage-associated liver injury induced by Propionibacterium acnes and lipopolysaccharide (LPS) in rats. All-trans retinoic acid administration alleviated the liver injury and reduced the incidence of death following hepatic failure. Serum alanine aminotransferase (ALT) levels 5 h after, and survival rates within 12 h after the administration of LPS were significantly lower in the ATRA-treated group (134 ± 119 IU/L and 72.7%) compared with the control group (713 ± 411 IU/L and 18.2%; P < 0.05). Histological findings supported these results. These effects may be due to suppression of tumour necrosis factor-α (TNF-α) and superoxide anions produced by activated macrophages. Serum levels of TNF-α 1 h after LPS administration were significantly lower in the ATRA-treated group (60.5 ± 7.0 ng/mL) as compared with the control group (105.2 ± 39.3 ng/mL; P < 0.05). Formazan deposition that was generated by the perfusion of the liver with nitroblue tetrazolium, also suggested suppression of the release of superoxide anions from hepatic macrophages. These results suggest that ATRA acts as an immunomodulator in liver injury by suppressing the activation of liver macrophages.     

Uptake and catabolism of gamma-aminobutyric acid by the isolated perfused rat liver

Serum concentrations of gamma-aminobutyric acid (GABA) are increased in liver failure, possibly because of decreased hepatic GABA catabolism. To study in detail the role of the liver in GABA metabolism, uptake and catabolism of GABA by isolated perfused liver from normal rats and rats with galactosamine- or carbon tetrachloride-induced liver failure were measured. Hepatic GABA uptake was almost complete at GABA concentrations of up to 10 microM and approached saturation at a concentration of 50 microM. The apparent affinity of hepatic GABA uptake was 38 microM and the apparent maximal velocity was 158 nmol/g.min. Hepatic GABA uptake was sodium-dependent. gamma-Aminobutyric acid taken up by the liver was rapidly catabolized as measured by 14CO2 formation from [U-14C]GABA. Aminooxyacetic acid, a GABA transaminase inhibitor, completely and irreversibly inhibited hepatic GABA catabolism and thereby also inhibited hepatic GABA uptake. Although uptake of GABA by livers of carbon tetrachloride- or galactosamine-treated rats was decreased (apparent maximal velocity, 103 and 98 nmol/g.min, respectively), at physiologic GABA concentrations in the perfusate GABA uptake and catabolism was not different from that of untreated controls. The observed impairment of hepatic GABA uptake or catabolism by the diseased liver would be expected to contribute to increased GABA levels in peripheral blood plasma in liver failure. However, the magnitude of the observed impairment would be insufficient to account for a 10-fold increase in such levels.