Galactose oxidation in liver.

Rats fed a high galactose diet (40%, $\text{w}\text{w}$) for 72 h excreted more than 170 μmol of galactonic acid per day in the urine and accumulated galactonic acid in several tissues, especially liver, intestine, heart, and kidney. Rat liver microsomes incubated in the presence of 30 mm galactose produced galactonic acid. The product of the oxidation of d-galactose was identified as galactonic acid by combined gas-liquid chromatography- mass spectrometry analysis of the trimethylsilyl derivative. Optimal activity was observed at pH 8.0 and was inhibited by heavy metal ions, sulfhydryl reagents, cyanide in the absence of substrate, and various drugs. The apparent K_m for galactose was 32.9 mm and V was 160 nmol galactonic acid/4 h/mg microsomal protein. The highest galactose oxidizing specific activity was found in the microsomal fraction; specific activity in the mitochondrial fraction was one half of that in the microsomal fraction, and the soluble fraction had none. The activity was specific for d-galactose, although unidentified oxidation products of d-altrose, d-talose, and 2-deoxy-d-galactose were detected by gas chromatography. Oxidation of galactose did not require oxygen. The addition of NAD⁺ and NADP⁺ to the incubation system had little effect on the galactose oxidation; FAD and FMN inhibited the activity. Galactose-dependent formation of hydrogen peroxide was demonstrated during the incubation period. Microsomes from rats and mice contained similar galactose-oxidizing activity whereas those from bovine and chicken liver sources contained 45.3 and 5.4%, respectively. The activity was not detectable in microsomes from guinea pig liver. A liver sample, obtained at autopsy of a galactosemia patient, contained 0.18 μmol of galactonate/g suggesting that an enzyme system similar to that described herein may be responsible for the production of galactonate in human galactosemics.     

Conformational changes in rat liver chromatin after liver regeneration.

N-Pyrenemaleimide, a fluorescent probe that specifically labels histone H3 of rat liver chromatin in situ, was used to monitor the accessibility of histone H3 in chromatin isolated from rat liver at different times during degeneration. At times of maximum DNA synthesis (18--24 h after hepatectomy), the accessibility of the probe was found to be markedly (40--50%) increased. This increase is abolished, however, by treatment of the chromatin fibres with high salt (2 M-NaCl) or detergent. Tryptophan fluorescence was also enhanced at points of maximum DNA synthesis, suggesting that some non-histone tryptophan-containing protein was being synthesized. The polarization of the labelled histone H3 is not markedly altered, suggesting that fibre aggregation or dissociation does not occur. Mononucleosomes extracted from sham-operated and hepatectomized animals did not exhibit any difference in binding to the probe. Also, analysis of the chromatin protein by electrophoresis on detergent- and acid/urea/ Triton-X-100-containing polyacrylamide gels showed no detectable difference in histone H3 : 1, H3 : 2 or H3 : 3 subclasses.     

Endocannabinoids and liver disease. I. Endocannabinoids and their receptors in the liver

Cannabinoid receptors (CB1 and CB2) and their endogenous ligands (endocannabinoids) have recently emerged as novel mediators of liver diseases. Endogenous activation of CB1 receptors promotes nonalcoholic fatty liver disease (NAFLD) and progression of liver fibrosis associated with chronic liver injury; in addition, CB1 receptors contribute to the pathogenesis of portal hypertension and cirrhotic cardiomyopathy. CB2 receptor-dependent effects are also increasingly characterized, including antifibrogenic effects and regulation of liver inflammation during ischemia-reperfusion and NAFLD. It is likely that the next few years will allow us to delineate whether molecules targeting CB1 and CB2 receptors are useful therapeutic agents for the treatment of chronic liver diseases.     

Phosphatidylinositol-degrading enzymes in liver lysosomes.

The major pathway by which liver lysosomal enzymes degrade phosphatidylinositol is through an EDTA-insensitive formation of phosphorylinositol. This is in distinct contrast with the Ca2+-dependent production of phosphorylinositol from phosphatidylinositol, which is located in the cytosol. Lysosomal enzymes can also totally deacylate phosphatidylinositol, producing glycerophosphorylinositol.     

Cardiac abnormalities in liver cirrhosis.

Cirrhosis is associated with several circulatory abnormalities. A hyperkinetic circulation characterized by increased cardiac output and decreased arterial pressure and peripheral resistance is typical. Despite this hyperkinetic circulation, some patients with alcoholic cirrhosis have subclinical cardiomyopathy with evidence of abnormal ventricular function unmasked by physiologic or pharmacologic stress. Florid congestive alcoholic cardiomyopathy develops in a small percentage, but the concurrent presence of cirrhosis seems to retard the occurrence of overt heart failure. Even nonalcoholic cirrhosis may be associated with latent cardiomyopathy, although overt heart failure is not observed. Tense ascites is associated with some cardiac compromise, and removing or mobilizing ascitic fluid by paracentesis or peritoneovenous shunting results in short-term increases in cardiac output. Cirrhosis also appears to be associated with a decreased risk of major coronary atherosclerosis and an increased risk of bacterial endocarditis. Small hemodynamically insignificant pericardial effusions may be seen in ascitic patients. The release of atrial natriuretic peptide appears to be unimpaired in cirrhosis, although the kidney may be hyporesponsive to its natriuretic effects.     

Recent advances in alcoholic liver disease. IV. Dysregulated cytokine metabolism in alcoholic liver disease

Alcoholic liver disease (ALD) remains a leading cause of death from liver disease in the United States for which there is no FDA-approved therapy. Abnormal cytokine metabolism is a major feature of ALD. Elevated serum concentration levels of TNF-alpha and TNF-alpha-inducible cytokines/chemokines, such as IL-6, -8, and -18, have been reported in patients with alcoholic hepatitis and/or cirrhosis, and levels correlated with markers of the acute phase response, liver function, and clinical outcome. Studies in animal models support an etiologic role for cytokines in the liver injury of ALD. Cytokines, such as transforming growth factor-beta, play a critical role in the fibrosis of ALD. Multiple new strategies are under investigation to modulate cytokine metabolism as a form of therapy for ALD.     

Activating immunity in the liver. II. IFN-beta attenuates NK cell-dependent liver injury triggered by liver NKT cell activation

Dendritic cell (DC)-dependent activation of liver NKT cells triggered by a single i.v. injection of a low dose (10-100 ng/mouse) of alpha-galactosyl ceramide (alphaGalCer) into mice induces liver injury. This response is particularly evident in HBs-tg B6 mice that express a transgene-encoded hepatitis B surface Ag in the liver. Liver injury following alphaGalCer injection is suppressed in mice depleted of NK cells, indicating that NK cells play a role in NK T cell-initiated liver injury. In vitro, liver NKT cells provide a CD80/86-dependent signal to alphaGalCer-pulsed liver DC to release IL-12 p70 that stimulates the IFN-gamma response of NKT and NK cells. Adoptive transfer of NKT cell-activated liver DC into the liver of nontreated, normal (immunocompetent), or immunodeficient (RAG(-/-) or HBs-tg/RAG(-/-)) hosts via the portal vein elicited IFN-gamma responses of liver NK cells in situ. IFN-beta down-regulates the pathogenic IL-12/IFN-gamma cytokine cascade triggered by NKT cell/DC/NK cell interactions in the liver. Pretreating liver DC in vitro with IFN-beta suppressed their IL-12 (but not IL-10) release in response to CD40 ligation or specific (alphaGalCer-dependent) interaction with liver NKT cells and down-regulated the IFN-gamma response of the specifically activated liver NKT cells. In vivo, IFN-beta attenuated the NKT cell-triggered induction of liver immunopathology. This study identifies interacting subsets of the hepatic innate immune system (and cytokines that up- and down-regulate these interactions) activated early in immune-mediated liver pathology.