Whey proteins inhibit food intake and tend to improve oxidative balance in obese zucker rats.

BACKGROUND/AIMS: Whey proteins (WP), obtained from milk after casein precipitation, represent a heterogeneous group of proteins. WP are reported to inhibit food intake in diet-induced experimental obesity; WP have been proposed as adjuvant therapy in oxidative stress-correlated pathologies. This work evaluates the effects of WP in comparison with casein, as a source of alimentary proteins, on food intake, weight growth and some indexes of oxidative equilibrium in Zucker Rats, genetically prone to obesity. METHODS: We monitored food intake and weight of Zucker Rats during the experiment, and some markers of oxidative equilibrium. RESULTS: WP induced significant decrease of food intake in comparison to casein (WP 80.41 ± 1.069 ml/day; CAS: 88.95 ± 1.084 ml/day; p < 0.0005). Body weight growth was slightly reduced, and the difference was just significant (WP 128.2 ± 6.56 g/day; CAS 145.2 ± 3.29 g/day; p = 0.049), while plasma HNE level was significantly lower in WP than in CAS (WP 41.2 ± 6.3 vs CAS 69.61 ± 4.69 pmol/ml, p = 0.007). Mild amelioration of oxidative equilibrium was indicated by a slight increase of total glutathione both in the liver and in the blood and a significant decrease of plasma 4-hydroxynonenal in the group receiving WP. CONCLUSIONS: The effect of WP on food intake and weight growth in Zucker Rats is particularly noteworthy since the nature of their predisposition to obesity is genetic; the possible parallel amelioration of the oxidative balance may constitute a further advantage of WP since oxidative stress is believed to be interwoven to obesity, metabolic syndrome and their complications.

Biochemical and morphologic effects after extended liver resection in rats: preliminary results.

After hepatic resection and transplantation with a partial graft, death and regeneration of the hepatocytes coexist in the liver. However, when the functional liver mass is inadequate to ensure a proper balance between regeneration vs functional and metabolic demands, small-for-size syndrome develops. We assessed the early effects of extended hepatic resection on liver function in a rat model. Six male Sprague-Dawley rats underwent 80% resection of the liver, and 6 rats served as a control group. At 6 hours after resection, blood samples were obtained from the hepatic vein for measurement of reduced glutathione (GSH), oxidized glutathione (GSSG), and hepatic venous oxygen saturation (Shvo(2)), and for standard liver function tests including determination of concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase, and total bilirubin. The remnant lobe was removed for GSH assay and histopathologic analysis. In the resection group, values were significantly higher for ALT (P = .002), AST (P = .002), and Shvo(2) (P = .01), whereas a significant decrease was observed for blood GSH (P = .009) but not liver GSH. Also in the resection group, we observed characteristic hepatocyte vacuolization with a gradient from periportal acinar zone 1 to the centrolobular area, the presence of hemorrhagic necrosis, and several leukocyte adhesions. The Shvo(2) and GSH data suggest early alteration of oxygen metabolism, as demonstrated by the reduction in oxygen uptake and decreased liver GSH secretion, with preservation of hepatic GSH. Mitochondrial dysfunction and oxidative injury seem to have a crucial role in early onset of liver damage.

Whey proteins influence hepatic glutathione after CCl4 intoxication.

Whey proteins (WP) are known to contain more cysteine than casein (CAS), so it is suggested that they should ameliorate the oxidative equilibrium in the organisms. To evaluate the influence of a WP-based diet on liver glutathione (GSH) content, male Sprague-Dawley rats were fed for 3 weeks a balanced liquid diet containing either WP or CAS as main source of protein. Liver GSH content was evaluated at the end of the treatment by high performance liquid chromatography (HPLC), both in basal conditions and after oxidative stress induced by CCl4 acute intoxication. In basal conditions, WP diet significantly increased hepatic GSH in comparison to CAS diet. After CCl4 intoxication, hepatic GSH was negligibly increased in CAS group, while its increase was much more marked in WP group, so that the difference between the two diets was significant; this suggests that WP provided rats with better ability to increase their GSH synthesis in case of need.

Impact of ischemia/reperfusion on transplanted livers procured from elderly cadaveric donors.

The increasing number of clinical indications for liver transplantation has forced physicians to use livers procured from elderly cadaveric donors to expand the graft pool. However, the degree of ischemia/reperfusion damage in elderly livers remains poorly investigated. In this study, the outcomes of livers procured from a group (I) of young donors (n=12; 38 +/- 12 years; range: 21-58) were compared with a group (II) from elderly donors (n=7; 68 +/- 7 years; range: 62-84) for changes in reduced glutathione, the main hepatic free radical scavenger. Reduced and oxidized glutathione were assayed by high performance liquid chromatography in liver biopsies performed just before cold ischemia and during early reperfusion. A significant decrease in reduced glutathione was observed at the time of reperfusion in both groups I (P=.0195) and II (P=.002). Before cold ischemia and during early reperfusion, no differences between young versus elderly donors were noted in the oxidized/reduced glutathione ratio, in conventional graft function markers or in liver-related hemostatic parameters. Comparable glutathione contents were measured at the time of early reperfusion in livers obtained from young and elderly cadaveric donors, suggesting that livers procured from elderly donors might be adequately protected against ischemia/reperfusion damage.

Damaging effects of advanced glycation end-products in the murine macrophage cell line J774A.1.

The interaction of reducing sugars, such as aldose, with proteins and the subsequent molecular rearrangements, produces irreversible advanced glycation end-products (AGEs), a heterogeneous class of non-enzymatic glycated proteins or lipids. AGEs form cross-links, trap macromolecules and release reactive oxygen intermediates. AGEs are linked to aging, and increase in several related diseases. The aim of this study was to assess, in a murine macrophage cell line, J774A.1, the effects of 48 h of exposure to glycated serum containing a known amount of pentosidine, a well-known AGE found in the plasma and tissues of diabetic and uremic subjects. Fetal bovine serum was incubated with ribose (50 mM) for 7 days at 37 degrees C to obtain about 10 nmol/ml of pentosidine. The cytotoxic parameters studied were cell morphology and viability by neutral red uptake, lactate dehydrogenase release and tetrazolium salt test. In the medium and in the intracellular compartment, bound and free pentosidine were evaluated by HPLC, as sensitive and specific glycative markers, and thiobarbituric acid reactive substances (TBARs), as index of the extent of lipid peroxidation. Our results confirm that macrophages are able to take up pentosidine. It is conceivable that bound pentosidine is degraded and free pentosidine is released inside the cell and then into the medium. The AGE increase in the medium was combined with an increase in TBARs, meaning that an oxidative stress occurred; marked cytotoxic effects were observed, and were followed by the release of free pentosidine and TBARs into the culture medium.

Activation of PKC-beta isoforms mediates HNE-induced MCP-1 release by macrophages.

4-Hydroxynonenal (HNE) in the concentration range detectable in many pathophysiologic conditions is able to modulate signal transduction cascades and gene expression. Here, we report the stimulating effect of 1 microM HNE on the release of the monocyte chemotactic protein-1 (MCP-1) by murine macrophages. MCP-1-increased export following 1-h cell treatment with HNE proved to be comparable to that exerted by standard amounts of bacterial lipopolysaccharide (LPS). However, the key molecular event in HNE-induced secretion of MCP-1 appeared to be the increased activity of beta-PKC isoforms, which are recognized as playing a role in the regulation of cell protein transport and secretion. On the other hand, in LPS-stimulated cells, the delta isoform was seen to be involved and was probably related to LPS-mediated effects on MCP-1 expression and synthesis. In conclusion, HNE might interact with other pro-inflammatory stimuli, like LPS, in a concerted amplification of MCP-1 production and secretion.

Orthotopic liver transplantation in a young adult (33 years old) using an 84-year-old donor.

Shortage of liver donors and the increasing number of patients on the waiting list for liver transplantation have led to a widening of the definition of liver donor suitability. Although the age limit for liver donors is controversial, current opinion is towards using liver allografts from donors older than 60 years. However, to date only a few cases that showed a good performance of liver graft by donors older than 60 years have been described. In this case report, orthotopic liver transplantation in a 33-year-old patient who received a graft from an 84-year-old donor is presented. A careful evaluation of the conventional donor-related risk factors (hemodynamics, hepatic function and histologic features) was carried out. Moreover, free radical scavenger glutathione was measured before cold ischemia and at the time of reperfusion in hepatic biopsies. After a 1-year follow-up, the recipient exhibits good general conditions and normal liver function values.

Induction of heme oxygenase 1 in liver of spontaneously diabetic rats.

It has been suggested that diabetes induces an increase in oxidative stress; the increased expression of heme-oxygenase 1 (HO-1) in liver is believed to be a sensitive marker of the stress response. The aim of this study was to examine whether diabetes is able to induce HO-1 expression in liver. The specific mRNA was amplified by RT/PCR and calibrated with amplified beta-actin mRNA. The mRNA HO-1 levels in the liver of spontaneously diabetic rats were increased by 1.8 fold compared with non diabetics; this supports the hypothesis of weak but significant oxidative damage due to chronic hyperglycaemia. This work represents the first in vivo study exploring the semi-quantitative expression of HO-1 in the liver of spontaneously diabetic rats.

Effects of ischemia-reperfusion on hepatic glutathione and plasmatic markers of graft function during in situ split-liver transplantation in adult recipients.

In situ split-liver transplantation is a new surgical technique where the bipartition of a single liver allows procurement of a right graft (segments I, IV, V-VIII) for an adult recipient (75% of the total liver volume), and a left graft (segments II and III) for a child recipient. The present study was designed to assess the effects of ischemia-reperfusion on right grafts obtained by in situ split-liver transplantation. To this aim, hepatic glutathione and conventional plasmatic markers of allograft function (alanine and aspartate aminotransferase, total bilirubin, prothrombin time, lactate dehydrogenase, gamma-glutamyltranspeptidase, and alkaline phosphatase) were evaluated in four adult recipients. At the time of reperfusion, a marked glutathione decrease was found in the segment VI in three cases, whereas the amount of glutathione in segment IV was related to the duration of cold ischemia in all cases. Upon reperfusion, a marked increase in plasmatic alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase was found. A recovery in prothrombin time was observed from the first day in three cases. An increasing trend in total bilirubin, gamma-glutamyltranspeptidase, and alkaline phosphatase was noted from the second day after transplant. This preliminary study suggests a possible relationship between the duration of cold ischemia, amount of glutathione in segment IV of the right graft, and the trend in plasmatic markers of allograft damage during in situ split-liver transplantation in adult recipients.

Glutathione depletion induces apoptosis of rat hepatocytes through activation of protein kinase C novel isoforms and dependent increase in AP-1 nuclear binding.

Treatment of isolated rat hepatocytes with the glutathione depleting agents L-buthionine-S,R-sulfoximine or diethylmaleate reproduced various cellular conditions of glutathione depletion, from moderate to severe, similar to those occurring in a wide spectrum of human liver diseases. To evaluate molecular changes and possible cellular dysfunction and damage consequent to a pathophysiologic level of GSH depletion, the effects of this condition on protein kinase C (PKC) isoforms were investigated, since these are involved in the intracellular specific regulatory processes and are potentially sensitive to redox changes. Moreover, a moderate perturbation of cellular redox state was found to activate novel PKC isoforms, and a clear relationship was shown between novel kinase activation and nuclear binding of the redox-sensitive transcription factor, activator protein-1 (AP-1). Apoptotic death of a significant number of cells, confirmed in terms of internucleosomal DNA fragmentation was a possible effect of these molecular reactions, and was triggered by a condition of glutathione depletion usually detected in human liver diseases. Finally, the inhibition of novel PKC enzymatic activity in cells co-treated with rottlerin, a selective novel kinase inhibitor, prevented glutathione-dependent novel PKC up-regulation, markedly moderated AP-1 activation, and protected cells against apoptotic death. Taken together, these findings indicate the existence of an apoptotic pathway dependent on glutathione depletion, which occurs through the up-regulation of novel PKCs and AP-1.

Scanning force microscopy reveals structural alterations in diabetic rat collagen fibrils: role of protein glycation.

BACKGROUND: The main functional property of collagen is to provide a supporting framework to almost all tissues: the effects of non-enzymatic glycation on this protein are deleterious and in diabetes mellitus contribute to the mechanism of late complications. The aim of this work is to provide evidence by scanning force microscopy of modifications in collagen structure caused by high glucose concentration, in vivo and in vitro, and to correlate the data with markers of non-enzymatic glycation. METHODS: Tendon fibrils were obtained from the tails of 8-month-old rats (BB/WOR/MOL¿BB) which developed diabetes spontaneously at least 12 weeks before they were killed, and from diabetes-resistant rats of the same strain (BB/WOR/MOL¿WB). A scanning force microscope (SFM; Nanoscope III) equipped with a Contact Mode Head was used for imaging. Band interval, diameter and depth of D-band gap were measured in non-diabetic and diabetic tail tendon fibrils and in fibrils incubated with glucose (0.5 M for 2 weeks). Fructosamine was determined in the tendon fibrils by a colorimetric method and pentosidine was evaluated in acid-hydrolyzed samples by coupled reverse phase-ionic exchange column HPLC. RESULTS: Incubated fibrils revealed modifications in radius (228+/-5 nm) and gap depth (3.65+/-0.10 nm) that closely reproduce diabetes-induced damage (236+/-3 and 3.20+/-0.04 nm respectively) and were significantly different from the pattern seen in non-diabetic fibrils (151+/-1 and 2.06+/-0.03 nm; p<0.001). Both fructosamine and pentosidine were higher in diabetic (3.82+/-1.43 nmol/mg and 2.23+/-0.24 pmol/mg collagen respectively) and in glucose-incubated fibrils (9.27+/-0.55 nmol/mg and 5.15+/-0.12 pmol/mg collagen respectively) vs non-diabetic tendons (1.29+/-0.08 nmol/mg and 0.88+/-0.11 pmol/mg collagen respectively; p<0.01); during the time course of incubation, an early increase in fructosamine was seen, whereas pentosidine increased later. The D-band parameter was similar in all three groups, indicating that axial organization is not modified by non-enzymatic glycation. CONCLUSION: This is the first description obtained with SFM of diabetes-induced ultrastructural changes in collagen fibrils. Moreover, the data presented are consistent with the concept that chronic exposure of collagen to glucose in vivo or in vitro leads to similar structural modifications in collagen fibrils, probably through crosslinks. The correlation between morphologic parameters and both markers of glycation provides strong evidence for a crucial role of this non-enzymatic modification.

Oxidative stress induces increase in intracellular amyloid beta-protein production and selective activation of betaI and betaII PKCs in NT2 cells.

Amyloid beta-protein (Abeta) aggregation produces an oxidative stress in neuronal cells that, in turn, may induce an amyloidogenic shift of neuronal metabolism. To investigate this hypothesis, we analyzed intra- and extracellular Abeta content in NT2 differentiated cells incubated with 4-hydroxy-2,3-nonenal (HNE), a major product of lipid peroxidation. In parallel, we evaluated protein kinase C (PKC) isoenzymes activity, a signaling system suspected to modulate amyloid precursor protein (APP) processing. Low HNE concentrations (0.1-1 microM) induced a 2-6 fold increase of intracellular Abeta production that was concomitant with selective activation of betaI and betaII PKC isoforms, without affecting either cell viability or APP full-length expression. Selective activation of the same PKC isoforms was observed following NT2 differentiation. Our findings suggest that PKC beta isoenzymes are part of cellular mechanisms that regulate production of the intracellular Abeta pool. Moreover, they indicate that lipid peroxidation fosters intracellular Abeta accumulation, creating a vicious neurodegenerative loop.

Effects of vitamin E on dolichol content of rats acutely treated with 1,2-dichloroethane.

Previous investigations have demonstrated that 1,2-dichloroethane (DCE) poisoning affects dolichol (Dol) concentration in rat liver. Dol, a long-chain polyprenol, is considered an important membrane component: as dolichyl phosphate, it is rate limiting for the synthesis of glycoprotein; as free or fatty acid, it is highly concentrated in the Golgi apparatus (GA) where it can increase membrane fluidity and permeability, required glycoprotein maturation and secretion. DCE biotransformation may stimulate pro-oxidant events through hepatocellular glutathione depletion. Since the molecules of Dol are susceptible to oxidative degradation, the aim of this investigation is to verify whether vitamin E (vit. E) supplementation in rats is able to prevent Dol breakdown during acute DCE treatment. Before acute DCE administration (628 mg/kg body weight), a group of male Wistar rats were pretreated with vit. E (33 mg/kg body weight) for 3 days. High-performance liquid chromatography analysis has shown that within 5-60 min after DCE administration, the Dol concentration decreased in liver homogenate, cytosol, microsomes and GA. Particularly, 60 min after the treatment, Dol levels in the trans Golgi fraction were 71% lower than in controls. Rat pre-treatment with vit. E prevented the DCE-induced decrease in Dol concentrations of all liver fractions considered, in particular the reduction of total-Dol observed in the trans Golgi fraction 60 min after treatment was only 40%. These data suggest that hepatic metabolism of DCE is able to promote peroxidative attacks which lead to the degradation of Dol molecules. The pre-treatment of rats with vit. E results in a good, although not complete, prevention of total-Dol depletion after DCE poisoning.

Lipoperoxidation in hepatic subcellular compartments of diabetic rats.

It is known that an accumulation of lipoperoxidative aldehydes malondialdehyde (MDA) and 4-hydroxynonenal (HNE) takes place in liver mitochondria during aging. The existence and role of an increased extra- and intra-cellular oxidative stress in diabetes, an aging-accelerating disease, is currently under discussion. This report offers evidence that lipoperoxidative aldehydes accumulate in liver microsomes and mitochondria at a higher rate in spontaneously diabetic BB/WOR rats than in control non-diabetic animals (HNE content, diabetes vs. control: microsomes 80.6+/-19.9 vs. 25.75+/-3.6 pmol/mg prot, p = .024; mitochondria 77.4+/-15.4 vs. 26.5+/-3.5 pmol/mg prot, p = .0103). Liver subcellular fractions from diabetic rats, when exposed to the peroxidative stimulus ADP/Fe, developed more lipoperoxidative aldehydes than those from non diabetic rats (HNE amount, diabetes vs. control: microsomes 3.60+/-0.37 vs. 2.33+/-0.22 nmol/mg prot, p = .014; mitochondria 3.62+/-0.26 vs. 2.30+/-0.17 nmol/mg prot, p = .0009). Liver subcellular fractions of diabetic rats developed more fluorescent chromolipids related to HNE-phospholipid adducts, either after in vitro peroxidation (microsomes: p = .0045; mitochondria: p = .0023) or by exposure to exogenous HNE (microsomes: p = .049; mitochondria: p = .0338). This higher susceptibility of diabetic liver membranes to the non-enzymatic attack of HNE may be due to an altered phospholipid composition. Moreover, a decreased activity of the HNE-metabolizing systems can be involved: diabetic liver mitochondria and microsomes were unable to consume exogenous HNE at the same rate as non-diabetic membranes; the difference was already significant after 5' incubation (microsomes p<.001; mitochondria p<.001). These data show an increased oxidative stress inside the hepatocytes of diabetic rats; the impairment of the HNE-metabolizing systems can play a key role in the maintenance and propagation of the damage.

Regulation of rat hepatocyte protein kinase C beta isoenzymes by the lipid peroxidation product 4-hydroxy-2,3-nonenal: A signaling pathway to modulate vesicular transport of glycoproteins.

A major aldehydic end product of the peroxidation of arachidonic acid, 4-hydroxy-2,3-nonenal (HNE), has recently been considered for its potential involvement in a variety of cell functions. Here we report on the differential regulation of rat hepatocyte protein kinase C (PKC) isoforms by concentrations of HNE actually detectable in specific biological fluids or tissues. PKC betaI and, to a much greater extent, PKC betaII activities were markedly increased by 0.1 micromol/L HNE (final concentration in cell medium) whereas they were unaffected or even inhibited by 1 to 10 micromol/L HNE. On the contrary, the calcium independent PKC delta activity was inhibited by 0.1 micromol/L and increased by 1 and 10 micromol/L. Further, we show here that HNE-induced stimulation of PKC betaI and betaII activities, both in cytosolic and in membrane fractions, is paralleled by a marked stimulation of the anterograde transport of a lysosomal enzyme within the central vacuolar system. In fact, the treatment with 0.1 micromol/L HNE accelerated the PKC-dependent transport of lysosomal procathepsin D from the trans-Golgi network to the endosomal-lysosomal compartment and, in addition, increased the exocytosis of mature cathepsin D (CD) from these compartments. On the other hand, hepatocyte cotreatment with a selective inhibitor of classic PKCs prevented the aldehyde-induced activation of CD transport. These results support the possible involvement of HNE in the PKC-dependent regulation of the traffic of secretory glycoproteins, and point to remarkable implications of this aldehyde in the pathophysiology of various exocytic processes including hepatocyte lipoprotein secretion.

Mechanisms of inactivation of hepatocyte protein kinase C isoforms following acute ethanol treatment.

Acute ethanol exposure of rat isolated hepatocytes leads to a significant decrease (-30%) in cytosolic enzymatic activity of classic protein kinase C (PKC) isoforms, while immunoreactive protein level measured by Western Blot remains unaffected. The inactivation of classic cytosolic isoforms appears dependent on the modification of the enzyme function, probably due to ethanol metabolism. In fact, pretreatment with 4-methylpyrazole (4MP), an inhibitor of alcohol dehydrogenase, fully prevented such damage. After ethanol treatment, a decrease of about 40% in both enzymatic activity and immunoreactive protein level of novel PKC isoforms was evident both in the soluble and particulate fractions. Even if 4MP cell pre-treatment afforded protection in this case too, the inhibitory action of ethanol on novel PKC hepatocyte isoforms involves a proteolytic mechanism as shown by Western Blot analysis. The reproduction of PKC inactivation by ethanol in hepatocyte lysate excluded a role of peroxisomal hydrogen peroxide in the pathogenesis of the damage investigated. This damage was not reduced by addition of catalase to the lysate model system.

Ethanol-induced effects on expression level, activity, and distribution of protein kinase C isoforms in rat liver Golgi apparatus.

Acute ethanol administration induces significant modifications both in secretive and formative membranes of rat liver Golgi apparatus. The decrease in glycolipoprotein secretion and their retention into the hepatocyte contribute to the pathogenesis of alcohol-induced fatty liver. Molecular and cellular mechanisms behind the ethanol-induced injury of the liver secretory pathway are not yet completely defined. In this study on intact livers from ethanol-treated rats, the involvement of the Golgi compartment in the impairment of hepatic glycolipoprotein secretion has been correlated with changes in the expression level, subcellular distribution and enzymatic activity of protein kinase C (PKC) isoforms. Acute ethanol exposure determined a translocation of classic PKCs and delta isoform from the cytosol to cis and trans Golgi membranes, the site of glycolipoprotein retention in the hepatic cell. A marked stimulation of cytosolic epsilon PKC activity was observed throughout the period of treatment. The presence of activated PKC isozymes at the Golgi compartment of alcohol-treated rat livers may play a role in hepatic secretion and protein accumulation. Direct and indirect effects of ethanol consumption on PKC isozymes and Golgi function are discussed.

1,1,2,2-Tetrachloroethane-induced early decrease of dolichol levels in rat liver microsomes and Golgi apparatus.

Dolichols are long-chain polyprenols containing 14-22 isoprene units, present in mammalian tissues as free dolichol (Free-Dol), fatty acyl dolichyl esters (Dol-FA), and dolichyl phosphate (Dol-P). The hepatic level of Dol-P seems to be a rate-limiting factor for glycosylation processes. Previous studies from our laboratory demonstrated the susceptibility of the dolichol molecule to undergo radical attacks. Since the toxicity of 1,1,2,2-tetrachloroethane (TTCE)is dependent on the free-radical production during hepatic biotrasformation, it was of interest to determine whether this haloalkane might affect glycosylation mechanisms by changing dolichol levels and distribution in rat liver microsomes and Golgi apparatus (GA). Male Sprague-Dawley rats received a single dose of TTCE (574 mg/kg body weight) and were then sacrificed at different times (5, 15, 30, or 60 min). In the TTCE-treated rats both serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and hepatic triglycerides (TG) were significantly higher than control, while microsomal glucose 6-phosphatase (G6Pase) activity was decreased. In total microsomes Dol-P levels considered rate-limiting for the biosynthesis of the N-glycosylated proteins were significantly lower than in the control group 15 min after TTCE treatment. In normal rat liver, F1 secretory fraction of CA is 60-fold enriched in total dolichol content with respect to microsomes. In this compartment the total dolichol content, essential for the increase in membrane fluidity and permeability required for glycoprotein maturation and secretion, decreased significantly 5 min after TTCE treatment. Our results suggest that TTCE may affect dolichol functions in rat liver.

Effect of ethanol administration on the level of dolichol in rat liver microsomes and Golgi apparatus.

Data obtained in our laboratory had suggested that acute ethanol administration (6 g/kg body weight) selectively and rapidly affects the intracellular system of protein glycosylation at the level of the Golgi apparatus. Dolichols are important membrane components, and dolichyl phosphate is a glycosyl sugar carrier for N-glycosylation of proteins in endoplasmic reticulum and is considered rate-limiting for this process. In this study, modifications in the concentration and distribution of liver microsomal dolichols after acute ethanol administration were investigated. Between 3 and 24 hr after ethanol administration, the microsomal dolichyl phosphate concentration was significantly lower than in control animals. The highest reduction was observed at 12 hr (-52%). An earlier and more marked reduction of total dolichol was observed in the Golgi apparatus, and, in particular, in the secretory fraction F1 (-70% at 6 hr). Ethanol treatment of isolated hepatocytes led to a significant reduction of the de novo synthesis of both dolichyl phosphate and free dolichol. Moreover, in vitro experiments have demonstrated that pro-oxidant agents lead to a significant decrease of both free dolichol and dolichyl phosphate. Our results suggest that acute ethanol administration induces a marked decrease of dolichols, probably by increasing the degradation and impairing the biosynthetic pathway of these molecules.