Biomedical subjects

Emanuele Albano

Publications and source records attributed to Emanuele Albano.

At least 19 recordsLinked to original sources

PubMed2006

Detection of auto-antibodies against cytochrome P4502E1 (CYP2E1) in chronic hepatitis C.

BACKGROUND/AIMS: Chronic hepatitis C (CHC) is often associated with auto-immune reactions. In the light of the role of alcohol in promoting CHC progression, we have investigated the possible presence of auto-reactivity against the ethanol-inducible cytochrome P4502E1 (CYP2E1) in CHC patients with and without alcohol consumption. METHODS: The IgG reactivity against recombinant human CYP2E1 was evaluated by solid-phase immunoassays in 102 CHC patients with different alcohol consumption and 59 HCV-free controls. RESULTS: Auto-antibodies against CYP2E1 were significantly (p<0.0001) increased in CHC patients as compared to controls. Anti-CYP2E1 IgG above the 97th percentile in the controls were evident in 41 (40%) CHC patients. Competition experiments revealed that CYP2E1 recognition was not due to the cross-reactivity with CYP2D6. The detection of anti-CYP2E1 IgG was unrelated to alcohol consumption and no difference in gender, age, aminotransferase levels and virus genotype was evident among the patients with or without anti-CYP2E1 auto-antibodies. However, anti-CYP2E1 auto-reactivity was significantly (p=0.025) associated with the severity of periportal/periseptal interface hepatitis. Moreover, confocal microscopy demonstrated that anti-CYP2E1 IgG associated with CHC recognized CYP2E1 exposed on the outer side of hepatocyte plasma membranes. CONCLUSIONS: HCV infection favours the breaking of self-tolerance against CYP2E1 that might contribute to hepatocyte injury.

Adult↗

PubMed2006

Heterozygous beta-globin gene mutations as a risk factor for iron accumulation and liver fibrosis in chronic hepatitis C.

BACKGROUND: Iron accumulation is a well-known risk factor for the progression of chronic hepatitis C (CHC) to fibrosis. However, the profibrogenic role of the genes controlling iron homeostasis is still controversial. AIM: To evaluate the relative role of haemachromatosis (HFE), ferroportin and beta-globin gene mutations in promoting iron accumulation and fibrosis in patients with CHC. METHODS: Genetic analysis was performed together with the assessment of hepatic iron content and histology in 100 consecutive HIV-antibody and hepatitis B surface antigen-negative patients with biopsy-proven CHC. RESULTS: Among the patients investigated, 12 were heterozygous for various beta-globin gene mutations (39[C-->T], IVS1.1[G-->A], 22 7 bp deletion and IVS1.6[T-->C]) and 29 carried HFE (C282Y, H63D and S65C) gene mutations. One further patient was heterozygous for both HFE (H63D) and beta-globin (39[C-->T]) variants, whereas 58 had the wild-type alleles of both the genes. Hepatic iron concentration (HIC) and hepatic stainable iron were significantly higher (p<0.05) in patients with CHC carrying beta-globin mutations than in those with HFE mutations or the wild-type alleles. Multivariate analysis confirmed that the presence of beta-globin mutations was independently associated with both HIC (p = 0.008) and hepatic-stainable iron (odds ratio (OR) 6.11; 95% CI 1.56 to 23.92; p = 0.009). Moderate/severe fibrosis or cirrhosis (Ishak's score >2) was observed in 48 of 100 patients. Logistic regression demonstrated that age (OR 1.05; 95% CI 1.02 to 1.09; p<0.005) and beta-globin mutations (OR 4.99; 95% CI 1.22 to 20.3; p = 0.025) were independent predictors of the severity of fibrosis. CONCLUSIONS: Heterozygosis for beta-globin mutations is a novel risk factor for both hepatic iron accumulation and the progression to fibrosis in patients with CHC.

Adult↗

PubMed2006

Role of p38 map kinase in glycine-induced hepatocyte resistance to hypoxic injury.

BACKGROUND/AIMS: Glycine hepatoprotection is well known. However, the mechanisms involved are still poorly characterized. METHODS: Glycine protection was investigated in isolated rat hepatocytes pretreated with 2 mmol/L glycine 15 min before incubation under hypoxic conditions. RESULTS: Glycine significantly reduced Na+ overload and hepatocyte death caused by hypoxia. Glycine protection required the activation of a signal pathway involving Src, Pyk2 and p38 MAP kinases. Glycine treatment also induced a 11% increase of hepatocyte volume and transient ATP release. The prevention of cell swelling by hepatocyte incubation in a hypertonic medium as well as the degradation of extracellular ATP with apyrase or the block P2 purinergic receptors with suramin reverted glycine-induced cytoprotection and inhibited Src, Pyk2 and p38 MAPK activation. Glycine down-modulated Na+/H+ exchanger (NHE) activity, without affecting the development of intracellular acidosis during hypoxia. Such an effect was reverted by inhibiting p38 MAPK that also abolished glycine protection against Na+ overload caused by hypoxia. CONCLUSIONS: Glycine-induced ATP release in response to a moderate hepatocyte swelling led to the autocrine stimulation of P2 receptors and to the activation of Src, Pyk2 and p38 MAPK that increased hepatocyte resistance to hypoxia by preventing Na+ influx through NHE.

Adenosine Triphosphate↗

PubMed2006

Purinergic P2Y2 receptors promote hepatocyte resistance to hypoxia.

BACKGROUND/AIMS: ATP stimulation of purinergic P2 receptors (P2YR and P2XR) regulates several hepatic functions. Here we report the involvement of ATP-mediated signals in enhancing hepatocyte tolerance to lethal stress. METHODS: The protection given by purinergic agonists was investigated in rat hepatocytes exposed to hypoxia. RESULTS: ATP released after hypotonic stress (200 mOsm/L) as well as P2YR agonists prevented hepatocyte killing by hypoxia with efficiency ranking UTP > ATPgammaS > ADPbetaS, whereas the P2XR agonist, methylene-adenosine-5'-triphosphate, was ineffective. Adenosine-5'-O-3-thiotriphosphate (ATPgammaS; 100 micromol/L) also prevented Na+ -overload in hypoxic cells by inhibiting the Na+/H+ exchanger, without interfering with hypoxic acidosis. ATPgammaS activated Src and promoted a Src-dependent stimulation of both ERK1/2 and p38MAPK. Blocking p38MAPK with SB203580 reverted the protection given by ATPgammaS on both cell viability and Na+ accumulation, whereas ERK1/2 inhibition with PD98058 was ineffective. An increased phosphorylation of ERK1/2 was also evident in untreated hypoxic hepatocytes. PD98058 ameliorated Na+ accumulation and cell death caused by hypoxia. Hepatocyte pre-treatment with ATPgammaS reverted ERK1/2 activation in hypoxic cells. SB203580 blocked the effects of ATPgammaS on both ERK1/2 and Na+/H+ exchanger. CONCLUSIONS: The activation of p38MAPK by P2Y2R increases hepatocyte resistance to hypoxia by down-modulating ERK1/2-mediated signals that promote Na+ influx through the Na+/H+ exchanger.

Adenosine Triphosphate↗

PubMed2006

PI3K-dependent lysosome exocytosis in nitric oxide-preconditioned hepatocytes.

We investigated the signal mediators and the cellular events involved in the nitric oxide (NO)-induced hepatocyte resistance to oxygen deprivation in isolated hepatocytes treated with the NO donor (Z)-1-(N-methyl-N-[6-(N-methylammoniohexyl)amino])diazen-1-ium-1,2-diolate (NOC-9). NOC-9 greatly induced PI3K activation, as tested by phosphorylation of PKB/Akt. This effect was prevented by either 1H-(1,2,4)-oxadiazolo-(4,3)-quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (sGC), or KT5823, an inhibitor of cGMP-dependent kinase (cGK), as well as by farnesyl protein transferase inhibitor, which blocks the function of Ras GTPase. Bafilomycin A, an inhibitor of the lysosome-type vacuolar H+-ATPase, cytochalasin D, which disrupts the cytoskeleton-dependent organelle traffic, and wortmannin, which inhibits the PI3K-dependent traffic of lysosomes, all abolished the NOC-9-induced hepatocyte protection. The treatment with NOC-9 was associated with the PI3K-dependent peripheral translocation and fusion with the plasma membrane of lysosomes and the appearance at the cell surface of the vacuolar H+-ATPase. Inhibition of sGC, cGK, and Ras, as well as the inhibition of PI3K by wortmannin, prevented the exocytosis of lysosomes and concomitantly abolished the protective effect of NOC-9 on hypoxia-induced pHi and [Na+]i alterations and cell death. These data indicate that NO increases hepatocyte resistance to hypoxic injury by activating a pathway involving Ras, sGC, and cGK that determines PI3K-dependent exocytosis of lysosomes.

Animals↗

PubMed2006

A novel autoantigen to differentiate limited cutaneous systemic sclerosis from diffuse cutaneous systemic sclerosis: the interferon-inducible gene IFI16.

OBJECTIVE: To investigate the presence and clinical significance of autoantibodies against the interferon-inducible gene IFI16 in systemic sclerosis (SSc), systemic lupus erythematosus (SLE), and other autoimmune diseases. METHODS: Immunohistochemical analysis was used to evaluate the expression of IFI16 in skin biopsy specimens obtained from patients with SSc and patients with SLE. Levels of antibodies against IFI16 in sera from 82 patients with SSc and 100 patients with SLE were determined by enzyme-linked immunosorbent assay. Other autoimmune diseases such as primary Sjögren's syndrome (SS), rheumatoid arthritis (RA), chronic urticaria, and hepatitis C virus (HCV) infection were also examined. RESULTS: Expression of IFI16 was greatly increased and was ubiquitous in all layers of the epidermis and in the dermal inflammatory infiltrates of lesional skin from both patients with SLE and patients with SSc. Patients with SLE, those with primary SS, and those with SSc exhibited significantly higher anti-IFI16 IgG antibody levels compared with normal controls (for SLE, P < 0.002; for primary SS, P < 0.001; for SSc, P < 0.0005). Anti-IFI16 titers above the ninety-fifth percentile for control subjects were observed in 26% of the patients with SLE, 50% of those with primary SS, and 21% of those with SSc (28% of patients with limited cutaneous SSc [lcSSc] versus 4% of patients with diffuse cutaneous SSc [dcSSc]). In contrast, the prevalence of anti-IFI16 was 4% in patients with RA, 5% in those with chronic urticaria, and 13% in those with HCV infection. CONCLUSION: The results of this study provide evidence that an IFN-inducible gene, IFI16, may be involved in the pathophysiologic mechanisms of connective tissue disorders such as SSc. Moreover, a strict correlation with lcSSc was also demonstrated, thus providing a novel tool in the differential diagnosis of lcSSc from dcSSc.

Adult↗

PubMed2006

Alcohol, oxidative stress and free radical damage.

The involvement of free radical mechanisms in the pathogenesis of alcoholic liver disease (ALD) is demonstrated by the detection of lipid peroxidation markers in the liver and the serum of patients with alcoholism, as well as by experiments in alcohol-feed rodents that show a relationship between alcohol-induced oxidative stress and the development of liver pathology. Ethanol-induced oxidative stress is the result of the combined impairment of antioxidant defences and the production of reactive oxygen species by the mitochondrial electron transport chain, the alcohol-inducible cytochrome P450 (CYP) 2E1 and activated phagocytes. Furthermore, hydroxyethyl free radicals (HER) are also generated during ethanol metabolism by CYP2E1. The mechanisms by which oxidative stress contributes to alcohol toxicity are still not completely understood. The available evidence indicates that, by favouring mitochondrial permeability transition, oxidative stress promotes hepatocyte necrosis and/or apoptosis and is implicated in the alcohol-induced sensitization of hepatocytes to the pro-apoptotic action of TNF-alpha. Moreover, oxidative mechanisms can contribute to liver fibrosis, by triggering the release of pro-fibrotic cytokines and collagen gene expression in hepatic stellate cells. Finally, the reactions of HER and lipid peroxidation products with hepatic proteins stimulate both humoral and cellular immune reactions and favour the breaking of self-tolerance during ALD. Thus, immune responses might represent the mechanism by which alcohol-induced oxidative stress contributes to the perpetuation of chronic hepatic inflammation. Together these observations provide a rationale for the possible clinical application of antioxidants in the therapy for ALD.

Alcohol Drinking↗

PubMed2005

Effects of N-acetylcysteine on ethanol-induced hepatotoxicity in rats fed via total enteral nutrition.

The effects of the dietary antioxidant N-acetylcysteine (NAC) on alcoholic liver damage were examined in a total enteral nutrition (TEN) model of ethanol toxicity in which liver pathology occurs in the absence of endotoxemia. Ethanol treatment resulted in steatosis, inflammatory infiltrates, occasional foci of necrosis, and elevated ALT in the absence of increased expression of the endotoxin receptor CD 14, a marker of Kupffer cell activation by LPS. In addition, ethanol treatment induced CYP 2 E1 and increased TNFalpha and TGFbeta mRNA expression accompanied by suppressed hepatic IL-4 mRNA expression. Ethanol treatment also resulted in the hepatic accumulation of malondialdehyde (MDA) and hydroxynonenal (HNE) protein adducts, decreased antioxidant capacity, and increased antibody titers toward serum hydroxyethyl radical (HER), MDA, and HNE adducts. NAC treatment increased cytosolic antioxidant capacity, abolished ethanol-induced lipid peroxidation, and inhibited the formation of antibodies toward HNE and HER adducts without interfering with CYP 2 E1 induction. NAC also decreased ethanol-induced ALT release and inflammation and prevented significant loss of hepatic GSH content. However, the improvement in necrosis score and reduction of TNFalpha mRNA elevation did not reach statistical significance. Although a direct correlation was observed among hepatic MDA and HNE adduct content and TNFalpha mRNA expression, inflammation, and necrosis scores, no correlation was observed between oxidative stress markers or TNFalpha and steatosis score. These data suggest that ethanol-induced oxidative stress can contribute to inflammation and liver injury even in the absence of Kupffer cell activation by endotoxemia.

Acetylcysteine↗

PubMed2005

Anti-phospholipid antibodies associated with alcoholic liver disease target oxidized phosphatidylserine on apoptotic cell plasma membranes.

BACKGROUND/AIMS: Circulating anti-phospholipid antibodies (aPL) are often present in patients with alcoholic liver disease (ALD). The observations that defects in the disposal of apoptotic corpses leads to the development of aPL prompted us to investigate whether ALD-associated aPL might recognize antigens in apoptotic cells. METHODS: Apoptosis was induced in HuT-78 human T-lymphoma and HepG2 hepatoma cells by, respectively, FAS ligation with CH11 monoclonal antibodies or the incubation with ethanol (400 mmol/L). RESULTS: Flow cytometry revealed that IgG from ALD patients with high aPL titers selectively bind to the surface of apoptotic, but not to viable cells. No binding was instead evident using either control or aPL-negative ALD sera. ELISA assays using different oxidized phospholipids as antigens showed that anti-phospholipid reactivity of ALD sera was mainly directed towards oxidized cardiolipin and phosphatidylserine. The pre-adsorption of aPL-positive sera with oxidized phosphatidylserine, but not with oxidized cardiolipin, lowered aPL binding to apoptotic HuT-78 cells by about 50%. No effect was instead observed by pre-adsorption with oxidation-protected phospholipids or with human serum albumin adducted with different lipid peroxidation products. CONCLUSIONS: aPL associated with ALD target apoptotic cells by specifically recognizing oxidized phosphatidylserine, suggesting a possible link between hepatocyte apoptosis and anti-phospholipid auto-reactivity in ALD.

Adult↗

PubMed2004

Use of molecular simulation for mapping conformational CYP2E1 epitopes.

The identification of the epitopes recognized by autoantibodies against cytochrome P450s (CYPs) associated with drug-induced hepatotoxicity is difficult because of their conformational nature. In the present investigation, we used a novel approach based on the analysis of the whole molecule antigenic capacity following single amino acid substitutions to identify the conformational epitopes on CYP2E1. A molecular model of CYP2E1 was generated based on the CYP2C5 crystal structure, and potential motifs for amino acid exchanges were selected by computer simulation in the surface of alpha helices and beta sheets. Fourteen modified, apparently correctly folded CYP2E1 variants were produced in Escherichia coli and evaluated in immunoprecipitation experiments using sera with anti-CYP2E1 autoreactivity from 10 patients with halothane hepatitis and 12 patients with alcoholic liver disease. Ala substitution of Glu-248 and Lys-251 as well as of Lys-324, Lys-342, Lys-420, and Phe-421 severely decreased or abolished CYP2E1 recognition by the majority of both the halothane hepatitis and alcoholic liver disease sera, whereas the other substitutions had only minor effects. Based on the structural model, these substitutions identified two distinct epitopes on the CYP2E1 surface corresponding to the G-helix and an area formed by juxtaposition of the J' and K'' helices, respectively. The combined use of molecular modeling and single amino acid mutagenesis is thus a useful approach for the characterization of conformational epitopes recognized by autoantibodies.

Anesthetics, Inhalation↗

PubMed2004

Lack of sexual dimorphism in alcohol-induced liver damage (ALD) in rats treated chronically with ethanol-containing low carbohydrate diets: The role of ethanol metabolism and endotoxin.

Evidence has been presented suggesting that females are significantly more susceptible to alcohol-induced liver damage (ALD) than males. In the current study, we examined sexual dimorphism in hepatic pathology, metabolism and cytokine profiles using two different rat models of ALD. Male and female Sprague-Dawley or Wistar rats were fed ethanol-containing low-carbohydrate liquid diets using oral or intragastric methods for 42 or 60 days. In both models, ethanol treatment produced similar significant liver hyperplasia accompanied by increases in plasma ALT, steatosis, inflammation and necrosis (p < 0.05). Greater pathology scores were observed in the intragastrically infused rats. Males did not differ significantly from females in serum ALT values or pathology despite greater elevations in TNFalpha and IL-1beta mRNAs in ethanol-treated female rat livers (p < 0.05). Furthermore, there was no sexual dimorphism in blood ethanol concentrations or CYP2E1-induction even though sexually dimorphic alterations in other hepatic cytochrome P450 enzymes were observed. These data do not support previous observations that female rats have a greater susceptibility to ethanol-induced hepatotoxicity than males.

Alanine Transaminase↗

PubMed2004

Preconditioning-induced cytoprotection in hepatocytes requires Ca(2+)-dependent exocytosis of lysosomes.

A short period of hypoxia reduces the cytotoxicity produced by a subsequent prolonged hypoxia in isolated hepatocytes. This phenomenon, termed hypoxic preconditioning, is mediated by the activation of adenosine A2A-receptor and is associated with the attenuation of cellular acidosis and Na+ overload normally occurring during hypoxia. Bafilomycin, an inhibitor of the vacuolar H+/ATPase, reverts the latter effects and abrogates the preconditioning-induced cytoprotection. Here we provide evidence that the acquisition of preconditioning-induced cytoprotection requires the fusion with plasma membrane and exocytosis of endosomal-lysosomal organelles. Poisons of the vesicular traffic, such as wortmannin and 3-methyladenine, which inhibit phosphatydilinositol 3-kinase, or cytochalasin D, which disassembles the actin cytoskeleton, prevented lysosome exocytosis and also abolished the preconditioning-associated protection from acidosis and necrosis provoked by hypoxia. Preconditioning was associated with the phosphatydilinositol 3-kinase-dependent increase of cytosolic [Ca2+]. Chelation of free cytosolic Ca2+ in preconditioned cells prevented lysosome exocytosis and the acquisition of cytoprotection. We conclude that lysosome-plasma membrane fusion is the mechanism through which hypoxic preconditioning allows hepatocytes to preserve the intracellular pH and survive hypoxic stress. This process is under the control of phosphatydilinositol 3-kinase and requires the integrity of the cytoskeleton and the rise of intracellular free calcium ions.

Animals↗

PubMed2004

Oxidative stress as a trigger for cellular immune responses in patients with alcoholic liver disease.

Serum antibodies reactive with neo-antigens generated during ethanol metabolism have been identified in patients with alcoholic liver disease (ALD), although their role in the pathogenesis of disease remains unclear. In this study, we characterized peripheral blood mononuclear cell (PBMC) T-cell and antibody responses to human serum albumin (HAS) adducted with acetaldehyde under reducing conditions (AcA-HSA) or with malondialdehyde (MDA-HSA) in patients with advanced ALD (AALD, n = 28), heavy drinkers with no liver disease (NALD, n = 14), and mild/moderate drinking controls (n = 22). Peak proliferative responses of PBMC were assessed in vitro by tritiated thymidine incorporation after the addition of optimized concentrations of antigen or OKT3. Antibody titers were determined by enzyme-linked immunosorbent assay (ELISA). MDA-HSA induced PBMC T-cell proliferation was significantly higher in ALD than in NALD or control patients. Moreover, 10 of 28 (36%) of ALD patients had significant T-cell proliferative responses to MDA-HSA compared to 0 of 14 (0%, P =.02) of the NALD group and 2 of 22 (9%, P <.05) of controls. No significant difference in PBMC T-cell response to Aca-HSA was seen between subject groups. Patients with positive cellular responses to MDA had higher serum anti-MDA antibody titers than those not exhibiting a positive cellular response (P <.005). In conclusion, the pattern of cellular and humoral responses to MDA adducts suggests that the development of these responses may be a susceptibility factor for the development of advanced alcoholic liver disease. The apparent importance of T-cell responses to MDA adducts suggests that oxidative stress may represent an important stimulus for the development of cellular immune responses associated with advanced ALD.

Acetaldehyde↗

PubMed2004

Role of phosphatidylinositol 3-kinase in the development of hepatocyte preconditioning.

BACKGROUND & AIMS: Ischemic preconditioning has been proved effective in reducing ischemia/reperfusion injury during liver surgery. However, the mechanisms involved are still poorly understood. Here, we have investigated the role of phosphatidylinositol 3-kinase (PI3K) in the signal pathway leading to hepatic preconditioning. METHODS: PI3K activation was evaluated in isolated rat hepatocytes preconditioned by 10-minute hypoxia followed by 10-minute reoxygenation. RESULTS: Hypoxic preconditioning stimulated phosphatidylinositol-3,4,5-triphosphate production and the phosphorylation of PKB/Akt, a downstream target of PI3K. Conversely, PI3K inhibition by wortmannin or LY294002 abolished hepatocyte tolerance against hypoxic damage induced by preconditioning. PI3K activation in preconditioned hepatocytes required the stimulation of adenosine A 2A receptors and was mimicked by adenosine A 2A receptors agonist CGS21680. In the cells treated with CGS21680, PI3K activation was prevented either by inhibiting adenylate cyclase and PKA with, respectively, 2,5-dideoxyadenosine and H89 or by blocking Galphai-protein and Src tyrosine kinase with, respectively, pertussis toxin and PP2. H89 also abolished the phosphorylation of adenosine A 2A receptors. However, the direct PKA activation by forskolin failed to stimulate PI3K. This suggested that PKA-phosphorylated adenosine A 2A receptors may activate PI3K by coupling it with Galphai-protein through Src. We also observed that, by impairing PI3K-mediated activation of phospholypase Cgamma (PLCgamma), wortmannin and LY294002 blocked the downstream transduction of preconditioning signals via protein kinase C (PKC) delta/ isozymes. CONCLUSIONS: PI3K is activated following hepatocyte hypoxic preconditioning by the combined stimulation of adenosine A 2A receptors, PKA, Galphai protein, and Src. By regulating PKC-/delta-dependent signals, PI3K can play a key role in the development of hepatic tolerance to hypoxia/reperfusion.

Animals↗

PubMed2004

Alcoholic liver disease in rats fed ethanol as part of oral or intragastric low-carbohydrate liquid diets.

The intragastric administration of ethanol as part of a low-carbohydrate diet results in alcohol hepatotoxicity. We aimed to investigate whether comparable liver injury can be achieved by oral diet intake. Male Sprague-Dawley rats were fed ethanol as part of low-carbohydrate diets for 36-42 days either intragastrically or orally. Liver pathology, blood ethanol concentration, serum alanine amino transferase (ALT), endotoxin level, hepatic CYP2E1 induction, and cytokine profiles were assessed. Both oral and intragastric low-carbohydrate ethanol diets resulted in marked steatosis with additional inflammation and necrosis accompanied by significantly increased serum ALT, high levels of CYP2E1 expression, and production of auto-antibodies against malondialdehyde and hydroxyethyl free radical protein adducts. However, cytokine profiles differed substantially between the groups, with significantly lower mRNA expression of the anti-inflammatory cytokine interleukin 4 observed in rats fed low-carbohydrate diets orally. Inflammation and necrosis were significantly greater in rats receiving low-carbohydrate alcohol diets intragastrically than orally. This was associated with a significant increase in liver tumor necrosis factor alpha and interleukin 1beta gene expression in the intragastric model. Thus, oral low-carbohydrate diets produce more ethanol-induced liver pathology than oral high-carbohydrate diets, but hepatotoxicity is more severe when a low-carbohydrate diet plus ethanol is infused intragastrically and is accompanied by significant increases in levels of proinflammatory cytokines.

Animals↗

PubMed2003

Signal pathway responsible for hepatocyte preconditioning by nitric oxide.

Nitric oxide (NO) improves liver resistance to hypoxia/reperfusion injury acting as a mediator of hepatic preconditioning. However, the mechanisms involved are still poorly understood. In this study, we have investigated the mechanisms by which short-term exposure to the NO donor (Z)-1-(N-methyl-N-[6-(N-methylammoniohexyl)amino])-diazen-1-ium-1,2-diolate (NOC-9) increases hepatocyte tolerance to hypoxic injury. Isolated rat hepatocytes preincubated 15 min with NOC-9 (0.250 mM) became resistant to the killing caused by hypoxia. NOC-9 cytoprotection did not involve the activation of protein kinase C, but was instead blocked by inhibiting soluble guanylate cyclase with 1H-(1,2,4)-oxadiazolo-(4,3) quinoxalin-1-one (ODQ) (50 microM) or cGMP-dependent kinase (cGK) with KT 5823 (5 microM). Conversely, cGMP analogue, 8Br-cGMP (50 microM) mimicked the effect of NOC-9. Western blot analysis revealed that hepatocyte treatment with NOC-9 or 8Br-cGMP significantly increased dual phosphorylation of p38 MAPK. The activation of p38 MAPK was abolished by inhibiting guanylate cyclase or cGK. Pretreatment with NO significantly reduced intracellular Na(+) accumulation in hypoxic hepatocytes. This effect was reverted by KT 5823 as well as by the p38 MAPK inhibitor SB203580. SB203580 also reverted NOC-9 protection against hypoxic injury. Altogether, these results demonstrated that NO can induce hepatic preconditioning by activating p38 MAPK through a guanylate cyclase/cGK-mediated pathway.

Animals↗

PubMed2003

Recent insights on the mechanisms of liver preconditioning.

Ischemia/reperfusion is the main cause of hepatic damage consequent to temporary clamping of the hepatoduodenal ligament during liver surgery as well as graft failure after liver transplantation. In recent years, a number of animal studies have shown that pre-exposure of the liver to transient ischemia, hyperthermia, or mild oxidative stress increases the tolerance to reperfusion injury, a phenomenon known as hepatic preconditioning. The development of hepatic preconditioning can be differentiated into 2 phases. An immediate phase (early preconditioning) occurs within minutes and involves the direct modulation of energy supplies, pH regulation, Na(+) and Ca(2+) homeostasis, and caspase activation. The subsequent phase (late preconditioning) begins 12-24 hours after the stimulus and requires the synthesis of multiple stress-response proteins, including heat shock proteins HSP70, HSP27, and HSP32/heme oxygenase 1. Hepatic preconditioning is not limited to parenchymal cells but ameliorates sinusoidal perfusion, prevents postischemic neutrophil infiltration, and decreases the production of proinflammatory cytokines by Kupffer cells. This latter effect is important in improving systemic disorders associated with hepatic ischemia/reperfusion. The signals triggering hepatic preconditioning have been partially characterized, showing that adenosine, nitric oxide, and reactive oxygen species can activate multiple protein kinase cascades involving, among others, protein kinase C and p38 mitogen-activated protein kinase. These observations, along with preliminary studies in humans, give a rationale to perform clinical trials aimed at verifying the possible application of hepatic preconditioning in preventing ischemia/reperfusion injury during liver surgery.

Animals↗

PubMed2003

Mechanisms of hepatocyte protection against hypoxic injury by atrial natriuretic peptide.

Atrial natriuretic peptide (ANP) reduces ischemia and/or reperfusion damage in several organs, but the mechanisms involved are largely unknown. We used freshly isolated rat hepatocytes to investigate the mechanisms by which ANP enhances hepatocyte resistance to hypoxia. The addition of ANP (1 micromol/L) reduced the killing of hypoxic hepatocytes by interfering with intracellular Na(+) accumulation without ameliorating adenosine triphosphate (ATP) depletion and pH decrease caused by hypoxia. The effects of ANP were mimicked by 8-bromo-guanosine 3', 5'-cyclic monophosphate (cGMP) and were associated with the activation of cGMP-dependent kinase (cGK), suggesting the involvement of guanylate cyclase-coupled natriuretic peptide receptor (NPR)-A/B ANP receptors. However, stimulating NPR-C receptor with des-(Gln(18), Ser(19),Gly(20),Leu(21),Gly(22))-ANP fragment 4-23 amide (C-ANP) also increased hepatocyte tolerance to hypoxia. C-ANP protection did not involve cGK activation but was instead linked to the stimulation of protein kinase C (PKC)-delta through G(i) protein- and phospholipase C-mediated signals. PKC-delta activation was also observed in hepatocytes receiving ANP. The inhibition of phospholipase C or PKC by U73122 and chelerythrine, respectively, significantly reduced ANP cytoprotection, indicating that ANP interaction with NPR-C receptors also contributed to cytoprotection. In ANP-treated hepatocytes, the stimulation of both cGK and PKC-delta was coupled with dual phosphorylation of p38 mitogen-activated protein kinase (MAPK). The p38 MAPK inhibitor SB203580 abolished ANP protection by reverting p38 MAPK-mediated regulation of Na(+) influx by the Na(+)/H(+) exchanger. In conclusion, ANP recruits 2 independent signal pathways, one mediated by cGMP and cGK and the other associated with G(i) proteins, phospholipase C, and PKC-delta. Both cGK and PKC-delta further transduce ANP signals to p38 MAPK that, by maintaining Na(+) homeostasis, are responsible for ANP protection against hypoxic injury.

Animals↗