Acetaldehyde substoichiometrically inhibits bovine neurotubulin polymerization.

Acetaldehyde is known to form covalent adducts with tubulin and to inhibit microtubule formation. Available evidence indicates that lysine residues are prominently involved in adduct formation. Previous work has shown that lysines on tubulin can be divided into two general classes based upon their reactivity toward acetaldehyde; those of normal reactivity ("bulk" lysines) and a highly reactive lysine (HRL) located on the alpha-polypeptide subunit. We took advantage of the fact that the HRL is unreactive when tubulin is in the microtubule form to differentiate the effects of bulk from HRL adducts on tubulin polymerization. Under conditions where both bulk lysines and HRL formed adducts, 0.2 mol acetaldehyde/mol tubulin caused complete inhibition of polymerization. When we modified bulk lysines, but not HRL, tubulin polymerized essentially normally. Finally, when we first blocked bulk lysines on microtubules (HRL unreactive) using unlabeled acetaldehyde and then measured the amount of [14C]acetaldehyde adduct formed with tubulin after depolymerization (HRL reactive), 0.08 mol acetaldehyde/mol tubulin resulted in completely impaired polymerization. These data show that microtubule formation is very sensitive to even small mole fractions of acetaldehyde-modified tubulin (especially with HRL) and further suggest that small amounts of acetaldehyde adduct could be damaging to cytoskeleton function in the cell.

Gut permeability and mucosal inflammation: bad, good or context dependent.

Inflammatory bowel disease (IBD) is a multifactorial disease. A breach in the mucosal barrier, otherwise known as "leaky gut," is alleged to promote mucosal inflammation by intensifying immune activation. However, interaction between the luminal antigen and mucosal immune system is necessary to maintain mucosal homeostasis. Furthermore, manipulations leading to deregulated gut permeability have resulted in susceptibility in mice to colitis as well as to creating adaptive immunity. These findings implicate a complex but dynamic association between mucosal permeability and immune homeostasis; however, they also emphasize that compromised gut permeability alone may not be sufficient to induce colitis. Emerging evidence further supports the role(s) of proteins associated with the mucosal barrier in epithelial injury and repair: manipulations of associated proteins also modified epithelial differentiation, proliferation, and apoptosis. Taken together, the role of gut permeability and proteins associated in regulating mucosal inflammatory diseases appears to be more complex than previously thought. Herein, we review outcomes from recent mouse models where gut permeability was altered by direct and indirect effects of manipulating mucosal barrier-associated proteins, to highlight the significance of mucosal permeability and the non-barrier-related roles of these proteins in regulating chronic mucosal inflammatory conditions.

Impairment of glycoprotein secretion by phenobarbital in rat liver slices.

The effects of phenobarbital on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Phenobarbital (2 mM) decreased [14C]glucosamine and [14C]leucine incorporation into liver proteins and markedly inhibited their incorporation into medium (secretory) proteins. The inhibitory effect of phenobarbital was dose dependent and not reversible under the conditions of this study. In the presence of cycloheximide, an inhibitor of peptide synthesis, phenobarbital still inhibited the release of glycoproteins into the medium; however, the specific activity of liver glycoproteins was increased. The effects of phenobarbital on hepatic macromolecular secretion, independent of its effects on synthesis, were determined by prelabeling proteins in a liver slice system with either [14C]leucine or [14C]glucosamine. When phenobarbital was present, the secretion of these prelabeled proteins into the medium was inpaired. 12 h after intraperitoneal injections of phenobarbital, glycoprotein secretion was inhibited from liver slices prepared from the pretreated rats. This inhibition of secretion occurred even though protein synthesis was stimulated and intracellular glycosylations unaffected. The results of this study indicate that phenobarbital impairs the secretion of glycoproteins by the liver.

Effect of chronic ethanol administration on total asialoglycoprotein receptor content and intracellular processing of asialoorosomucoid in isolated rat hepatocytes.

Chronic ethanol administration markedly impairs the process of receptor-mediated endocytosis (RME) of a representative asialoglycoprotein, asialoorosomucoid (ASOR), by the liver (Casey et al. (1987) J. Biol. Chem. 262, 2704-2710). Decreased surface binding was the major defect reported in our initial study, along with impaired internalization and degradation of 125I-ASOR in chronically-fed ethanol animals. In this study, we further characterized these impairments by examining the content of intracellular receptors and by investigating ligand processing directed by these intracellular receptors. Ethanol administration for 5-7 weeks decreased intracellular ASOR receptor content by 40%, a result which was confirmed by using both a ligand-binding assay and an antibody-binding assay. In addition to a decreased number of intracellular receptors, an impairment in intracellular processing of receptor-ligand complexes was identified. In ethanol-fed animals, dissociation of receptor-ligand complexes was decreased during steady-state conditions of endocytosis at 37 degrees C. Impaired receptor-ligand dissociation did not alter the fate of the ligand which was to undergo diacytosis (ligand recycling), but did appear to impair degradation of intracellular ligand. These results indicate that chronic ethanol administration decreases ligand binding due to a decreased number of receptors and impairs intracellular processing of ASOR in hepatocytes.

Ethanol-induced alterations of plasma membrane assembly in the liver.

The effects of acute ethanol administration on the assembly of glycoproteins into the hepatic plasma membrane were studied in the rat. When [14C]fucose and N-acetyl[3H]mannosamine, a sialic acid precursor, were injected following an acute dose of ethanol, the incorporation of these precursors into the total pool of membrane glycoproteins was minimally affected. This finding indicated that ethanol treatment did not appreciably alter the glycosylation of proteins in the Golgi apparatus. However, the assembly of labeled fucoproteins and sialoproteins into the plasma membrane was markedly inhibited in the ethanol-treated animals. This inhibition of plasmalemmal glycoprotein assembly was accompanied by a corresponding accumulation of labeled glycoproteins in the cytosolic fraction of the hepatocyte. The content of labeled glycoproteins in the Golgi complex was not significantly altered by ethanol treatment. These results indicate that ethanol administration impairs the late stages of hepatic plasma membrane assembly and further suggest that ethanol administration interferes with the flow of membrane components from the Golgi apparatus to the surface membrane.

Potentiation of ethanol fatty liver in rats by chronic administration of nicotinic acid.

Two groups of rats were fed isocalorically on alcohol and control semi-liquid diet for 28 days; two other groups had the same diets except for supplementation with nicotinic acid at 50 mg/100 ml of diet, Blood ethanol levels were unaffected by nicotinic acid administration, even though nicotinic acid was well absorbed and stored in the liver. Lipid analyses of liver and plasma after 28 days revealed that nicotinic acid, per se, stimulated fatty infiltration of the liver and this effect was potentiated when given in conjunction with ethanol.

Paradoxical urinary excretion of D-glucaric acid in acute viral hepatitis.

The urinary excretion of D-glucaric acid and the plasma clearance of antipyrine were estimated during the acute phase of viral hepatitis and again during recovery. The plasma clearance of antipyrine was impaired during the acute stage of hepatitis, while the urinary excretion of D-glucaric acid was paradoxically high. Both parameters returned to normal during recovery. These findings suggest that the use of urinary D-glucaric acid excretion as an index of microsomal enzyme induction is unreliable when there is liver injury.

Assay for free and total choline activity in biological fluids and tissues of rats and man with Torulopsis pintolopessi.

The sensitive, specific growth response to choline activity of the thermophilic enteric yeast Torulopsis pintolopessi enables estimation of free and bound choline activity in rat and human fluids and tissues- as little as 10 ng/ml of choline is measurable. Unlike other microbial assays, estimation of unbound (free) choline activity is not influenced by methionine or phospholipids. The method also distinguishes differences in choline activity of fluids and tissues from choline-deficient and choline-replete rats. Free and bound choline activity in blood, red blood cells, plasma, and liver from choline-deficient rats were almost 2-fold lower than from choline-supplemented animals. Free and bound choline activity in whole brain from choline-deficient rats were signifigantly higher (more than 2-fold). The application of the T. pintolopessi method in studying choline status in man and reasons for high choline activity in brain of choline-deficient rats are suggested.

Lipoatrophic diabetes and end-stage liver disease secondary to nonalcoholic steatohepatitis with recurrence after liver transplantation.

BACKGROUND: Lipoatrophic diabetes is an insulin resistance syndrome characterized by the complete or partial lack of adipose tissue and disturbances in lipid and glucose metabolism. Nonalcoholic steatohepatitis (NASH) is a well-described change in liver pathology consisting of steatosis, hepatitis, and fibrosis that can be associated with lipoatrophic diabetes. RESULTS: This article describes the first reported case of lipoatrophic diabetes with NASH leading to liver failure and liver transplantation. Before transplantation, the patient required 600-700 U of insulin/day. After transplantation, a dramatic decline in her insulin requirements was observed, despite corticosteroids. Eighteen months after transplantation, her glycemic control worsened, and she developed recurrent NASH on serial liver biopsies. CONCLUSIONS: NASH associated with lipoatrophic diabetes can recur after liver transplantation, and in this case, was accompanied by increased insulin requirements. These results suggest that the development of NASH itself may contribute to the insulin resistance observed in lipoatrophic diabetes.

The role of the transjugular intrahepatic portal-systemic shunt in the management of variceal bleeding.

For patients who present with variceal bleeding refractory to endoscopic and pharmacologic methods, TIPS is a new and effective therapy. Stents are used in selected patients with decompensated liver disease and those who anticipate liver transplantation within 6 to 12 months. Surveillance of TIPS with ultrasound, with or without venography, is recommended to diagnose and treat stenosis or occlusion before variceal hemorrhage recurs. Hepatic encephalopathy may develop in a subset of patients, but it is usually well controlled with conservative measures. Child-Pugh and APACHE scores are predictive of patient survival after TIPS. Randomized controlled trials will be necessary to assess whether TIPS is useful, safe, and cost effective for the management of variceal bleeding in patients with end stage liver disease.

Substoichiometric inhibition of microtubule formation by acetaldehyde-tubulin adducts.

We have shown previously that acetaldehyde forms stable covalent adducts with tubulin, resulting in impaired microtubule formation. The present study explored the mechanism responsible for impaired microtubule formation caused by the substoichiometric stable binding of acetaldehyde to tubulin. The free tubulin dimer was much more reactive with acetaldehyde than microtubules, binding more than twice as much aldehyde. The dimer also formed nearly twice as many stable adducts on its alpha-chain as on its beta-chain, whereas microtubules exhibited an equal distribution of adducts between the two subunits. These data confirm that the alpha-chain of free tubulin, but not microtubules, has an accessible highly reactive lysine (HRL) residue that is a preferential target of acetaldehyde binding. Adduct formation with the HRL residue also correlated with impaired tubulin polymerization, and only 0.08 moles of acetaldehyde bound per mole of HRL was required for complete inhibition; however, adducts with other lysine residues (bulk adducts) did not affect assembly. Adducts to microtubule-associated proteins (MAPs) also impaired the assembly of tubulin, but were much less effective than HRL adducts. In a copolymerization assay, HRL-adducted tubulin, in addition to being itself assembly incompetent, also interfered with polymerization of normal (unadducted) tubulin. Bulk adducts did not alter assembly and were incorporated normally into the growing polymer. When tubulin was cleaved by the proteolytic enzyme, subtilisin, microtubule formation could readily take place in the absence of MAPs. In this polymerization system, HRL adducts, but not bulk adducts, still markedly inhibited assembly. When low concentrations of acetaldehyde (50 microM) were used to generate HRL adducts, an adduct on only 1 out of 20 tubulin molecules was sufficient to totally block polymerization. These findings indicate that substoichiometric amounts of acetaldehyde bound to HRL of tubulin can markedly inhibit microtubule formation via direct interference of dimer-dimer interactions, and further suggest that low concentrations of acetaldehyde could generate sufficient amounts of HRL adducts in cellular systems to alter microtubule formation and function.

Specificity of N-ethyl lysine of a monoclonal antibody to acetaldehyde-modified proteins prepared under reducing conditions.

A monoclonal antibody has been developed that recognizes only protein-acetaldehyde (AA) adducts prepared under reducing conditions: 5 mM AA with 30 mM sodium cyanoborohydride overnight at 37 degrees. This monoclonal antibody is a mouse IgG2b that has been designated RT1.1. The primary adduct formed when proteins are exposed to acetaldehyde under reducing conditions is N-ethyl lysine (NEL). To examine the epitope specificity of RT1.1, inhibition ELISAs were developed using NEL and other possible inhibitors, such as arginine, ethylamine, lysine and proteins modified with AA under non-reducing conditions. RT1.1 (at half-maximum optical density, 50 ng/mL) was inhibited only by NEL and was independent of the carrier or the pH of the buffer used in the ELISA. Further evidence indicating that NEL is the epitope recognized by RT1.1 was obtained using mouse and human epidermal growth factor (EGF). Both proteins contain one alpha amino group but only the human-EGF contains lysine residues with epsilon amino groups. In experiments where these two proteins were modified with AA under reducing conditions, RT1.1 reacted only with human-EGF. These studies demonstrate that RT1.1 is specific for NEL that is formed by the ethylation of proteins with acetaldehyde under reducing conditions. Additionally, these studies demonstrate that the procedures and methods used herein may be useful for characterizing other antibodies prepared to AA-modified proteins under a variety of defined in vitro chemical conditions.

Impairment of the asialoglycoprotein receptor by ethanol oxidation.

It is well established that ethanol exposure impairs the process of receptor-mediated endocytosis in hepatic cells, although the molecular mechanism(s) and the physiological consequence(s) of this impairment are unclear. Because addressing these mechanistic questions is difficult in vivo, we have developed a recombinant cell line of hepatic origin capable of metabolizing ethanol. In this study, we have used these recombinant cells, designated HAD cells, to investigate the ethanol-induced impairment to the receptor-mediated endocytosis of the hepatic asialoglycoprotein receptor. Comparing the binding of the ligand asialoorosomucoid in both the parental Hep G2 cells and the recombinant HAD cells, maintained in the presence and absence of ethanol, revealed decreased ligand binding in the HAD cells. This impairment was accentuated by prolonging the ethanol exposure, reaching approximately 40% in both surface and total receptor populations by 7 days. Addition of the alcohol dehydrogenase inhibitor pyrazole to the ethanol-containing medium abolished this impairment, indicating that the decreased binding was a result of the alcohol dehydrogenase-mediated oxidation of ethanol. Furthermore, using antibody specific to the asialoglycoprotein receptor, it was demonstrated that the ethanol-induced impairment in ligand binding was a consequence of decreased ligand binding and not a result of diminished receptor numbers. These results indicated that ethanol oxidation was required for the ethanol-induced impairment in ligand binding, and that the reduced ligand binding was a result of a decrease in the ability of the ligand to bind to the receptor.

The interaction of acetaldehyde with tubulin.

Acetaldehyde covalently binds to purified tubulin in vitro to form both stable and unstable adducts. The formation of stable adducts can be greatly facilitated by the inclusion of the relatively gentle and Schiff base specific reducing agent, sodium cyanoborohydride. Although the tubulin molecule has multiple lysine resides available to react with acetaldehyde, certain key lysine residues on the alpha-chain appear to be selective targets for adduct formation. The formation of alpha-chain specific stable acetaldehyde-tubulin adducts results in functional impairment of the ability of tubulin to polymerize. Under relatively physiologic conditions where acetaldehyde-to-protein ratios are low, alpha-chain specific binding is prominent. These results, coupled with the studies presented in another report in this volume, raise the possibility that low levels of adduct formation may be detrimental to the structure or function of certain proteins (e.g. tubulin) in the liver. The alteration of this or other biologically important proteins by sustained low levels of adduct formation may contribute to the pathogenesis of alcoholic liver injury.

Acetaldehyde and microtubules.

Acetaldehyde covalently binds to tubulin to form stable and unstable adducts. Although tubulin has numerous lysine residues available to react with acetaldehyde, a key highly reactive lysine (HRL) on the alpha chain appears to be a preferential target for stable binding. The HRL residue is available for selective binding when tubulin is in the free (dimer) state but not when it is in the polymerized (microtubule) state. Stable binding of acetaldehyde to the HRL residue markedly inhibits tubulin assembly into microtubules, whereas stable binding to other residues (bulk adducts) has little influence on assembly. Substoichiometric stable binding of acetaldehyde to the HRL is sufficient to inhibit polymerization, via direct interference of tubulin dimer-dimer interactions, and an HRL adduct on only one out of 20 tubulin molecules can totally inhibit polymerization. These findings, along with our previous studies demonstrating impaired microtubule-dependent protein trafficking pathways in livers of ethanol-fed animals, indicate that low acetaldehyde concentrations, formed during ethanol oxidation in vivo, could generate sufficient amounts of HRL adducts on the alpha chain of tubulin in cellular systems to alter microtubule formation and function. In addition to alpha-tubulin, calmodulin and actin have also been found to have enhanced reactivity toward acetaldehyde. Thus, a general hypothesis to describe cellular injury induced by acetaldehyde adducts can be formulated: during ethanol oxidation, acetaldehyde forms stable adducts via binding to reactive lysine residues of preferential target proteins, resulting in selective functional impairment of these proteins and ultimately leading to cellular injury.

Results of liver transplantation in diabetic recipients.

BACKGROUND: The results of orthotopic liver transplantation (OLTx) in patients with diabetes mellitus (DM) are not well defined. METHODS: Between 1985 and 1991, 45 adult patients with pretransplantation DM (5 type I, 40 type II) underwent OLTx at our center as identified by retrospective chart review. We compared this diabetic recipient group to a case-control nondiabetic group matched for age, gender, primary liver disease, weight, and timing of OLTx. A total of 30 variables were collected and analyzed with McNemar's test for categorical data, paired t tests for continuous data, and survival and repeated measures analysis for longitudinal data. RESULTS: No differences between diabetic and nondiabetic recipients were noted in patient or graft survival, the incidence or severity of rejection, blood transfusions, operative complications, readmissions, major infections, or number of hospital days after OLTx. However, the incidence of minor bacterial (p = 0.046) and minor fungal (p = 0.035) infections were higher in the DM group. Serum blood urea nitrogen (p = 0.02) and creatinine (p = 0.03) levels were also higher in patients with diabetes versus control patients during the first year after OLTx. CONCLUSIONS: In carefully selected patients with pretransplantation DM, OLTx can be accomplished with results similar to nondiabetic recipients in spite of a higher incidence of minor infections and renal dysfunction.

Orthotopic hepatic transplantation in patients with type I diabetes mellitus.

Six transplantations of the liver were performed over a period of six years in five adult patients with Type I diabetes mellitus (DM). The diabetic group included two males and three females with a mean age of 36 years and a mean duration of DM of 20 years. Primary diseases of the liver included two instances of primary biliary cirrhosis, two instances of sclerosing cholangitis and one instance of autoimmune chronic hepatitis. Three patients also received a simultaneous whole organ pancreatic transplant. All patients were managed with cyclosporine and prednisone immunosuppression with selective OKT3 induction. Patient and hepatic allograft survival rates were 80 and 67 percent, respectively, after a mean follow-up period of 4.7 years. One of the three pancreatic grafts was successful and resulted in euglycemia for two years. Three patients have subsequently undergone successful renal transplantation at one, two and one-half, and six and one-half years after hepatic transplantation. Although transplantation of the liver can be performed safely in carefully selected patients with Type I DM, these patients are still at risk for the development of progressive nephropathy. Renal transplantation is an acceptable therapeutic alternative when this occurs.

Host resistance in liver disease--its evaluation and therapeutic modification.

We have attempted to review some of the factors involved in host resistance and the variations in the individual responses to the infectious and noxious agents to which we are increasingly exposed. Few treatment modalities are available to favorably influence host resistance, but clearly, this approach to the treatment of liver disease represents an opportunity for the future.

Increased covalent binding of acetaldehyde to calmodulin in the presence of calcium.

The regulatory protein, calmodulin, undergoes major conformational changes in response to changes in intracellular calcium concentration. Furthermore, calmodulin has been reported to have lysine residues which markedly increase their reactivity toward electrophilic substances in the calcium-loaded state. We found that calmodulin formed two to three times more stable adducts with acetaldehyde in the calcium-loaded state as compared to the calcium-free state. Competition-binding studies showed that calmodulin could preferentially compete with albumin for acetaldehyde in the presence, but not in the absence, of calcium. When calmodulin was in the calcium-loaded state, trifluoperazine, an inhibitor of calmodulin activity, significantly decreased the stable binding of acetaldehyde to the protein, whereas in the calcium-free state, minimal effects on binding were observed. Since calmodulin is involved in regulation of multiple important processes in the cell, it is possible that acetaldehyde-calmodulin adducts could contribute to liver injury by perturbation of calcium-dependent homeostatic mechanisms within the hepatocyte.

Effect of serum lipoproteins of bile obstructed rats on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in perfused rat liver.

Total lipoproteins as well as fractionated VLDL + LDL and HDL from fasted control rats and bile-ligated rats were tested in liver perfusion for their effect on 3-hydroxy-3-methylglutaryl CoA reductase activity in normal rat livers. The total lipoproteins of bile-obstructed rats had 3 times greater capacity to increase 3-hydroxy-3-methylglutaryl CoA reductase activity than that of the control total lipoproteins. When the fractionated lipoproteins were tested from fasted control rats, it was found that the major stimulating activity was in the HDL fraction with minor activity in the VLDL + LDL fraction. When these plasma components isolated from fasted bile-ligated rats were tested, it was found that the major activity had shifted to the VLDL + LDL fraction with the HDL having only a minor stimulatory role. The possible mechanism of action of the abnormal lipoproteins associated with bile obstruction in regulating 3-hydroxy-3-methylglutaryl CoA reductase activity is discussed.