Mechanism of the alcohol cyclic pattern: role of catecholamines

The cause of the urinary alcohol level (UAL) cycle in rats fed ethanol at a constant rate has been shown to involve the hypothalamic-pituitary thyroid axis. Because the effect of thyroid hormone on the metabolic rate is augmented by catecholamines, the role of catecholamines was investigated by using the intragastric ethanol feeding model of alcoholic liver disease in which the UAL cycles over a 6- to 10-day period. The diet was supplemented with ephedrine and caffeine to test the hypothesis that the UAL cycle involves catecholamines. The UAL was followed to see whether the cycle was ablated by catecholamine supplements. Ethanol fed alone increased the blood levels of catecholamines significantly more than did ephedrine fed alone. However, blood catecholamine levels were significantly higher when ethanol was fed with ephedrine compared with the sum of ethanol and ephedrine fed alone. This indicated that the effect of ethanol and ephedrine were synergistic. The UAL cycle was completely ablated in the ethanol + ephedrine-fed rats. These rats tolerated a much higher dose of ethanol, indicating that they metabolized alcohol faster due to an increase in metabolic rate caused by ephedrine. In the ethanol + ephedrine-fed rats the liver pathology included significantly higher alanine amino transferase (ALT) in the blood and centrilobular ischemic necrosis in the liver. Necrosis was not present in the rats fed ephedrine alone. In conclusion, catecholamine supplements prevented the UAL cycle by increasing the metabolic rate to the point at which fluctuations in the metabolic rate caused by alcohol were prevented.     

Modifications in P62 occur due to proteasome inhibition in alcoholic liver disease

P62 is capable of binding the polyubiquitin chain that targets proteins for degradation by the proteasome through its ubiquitin associated domain (UBA). Immunostaining of hepatocytes from human liver with alcoholic hepatitis showed colocalization of ubiquitin and P62 in Mallory bodies. Rats fed ethanol chronically and their controls showed that P62 is colocalized with the proteasome in hepatocytes as shown by confocal microscopy. P62 cosedimented with 26S proteasomes isolated from livers of control and alcohol fed rats. P62 was increased in the 26S proteasome fraction when the proteasome chymotrypsin-like (ChT-L) activity decreased in rats fed ethanol. PS-341, a potent proteasome inhibitor was used to compare the inhibition of the proteasome with the inhibition which occurs with ethanol feeding. P62 protein levels were also increased in the purified proteasome fraction of rats given PS-341. This data indicates that modifications in P62 occur due to proteasome inhibition in experimental alcoholic liver disease.     

Catecholamines are involved in the mechanism of the urinary alcohol level cycle in rats fed ethanol intragastrically at a constant rate

Studies have indicated that blood alcohol levels cycle exists when ethanol is fed continuously using the intragastric feeding rat model of early alcoholic liver disease. The aim of the present study was to determine the role played by catecholamines in the pathogenesis of the blood alcohol cycling observed when ethanol is fed at a constant rate. The rats were tested at the peaks and troughs of the urinary alcohol level (UAL) cycle and the results were compared with controls. Blood catecholamine levels were markedly increased at the peaks, but not at the troughs. Propranolol, a beta adrenergic blocker, attenuated the amplitude of the cycle. Phenoxybenzamine, an alpha blocker disrupted the cycle and elevated ethanol to fatal levels. The results indicate that both alpha and beta adrenergic mechanisms are required for the cycle to occur.     

Hyperphosphorylation of rat liver proteasome subunits: the effects of ethanol and okadaic acid are compared

In experimental alcoholic liver disease, protein degradation by the ATP-ubiquitin-proteasome pathway is inhibited. Failure of the proteasome to eliminate cytoplasmic proteins leads to the accumulation of oxidized and otherwise modified proteins. One possible explanation for the inhibition of the proteasome is hyperphosphorylation of proteasome subunits. To examine this possibility, the 26S proteasomes from the liver of rats fed ethanol and a pair-fed control were studied by isolating the proteasomes in a purified fraction. The effect of ethanol on the phosphorylation of proteasomal subunits was compared with the hyperphosphorylation of the proteasomes caused by okadaic acid given to rats in vivo. Ethanol ingestion caused an inhibition of the chymotrypsin-like activity of the purified proteasome. The 2D electrophoresis and Western blot analysis of the purified 20S and 26S proteasomes from the ethanol-fed rats indicated that hyperphosphorylation of proteasomal subunits had occured. The proteasomal alpha type subunits C9/alpha3 and C8/alpha7 were hyperphosphorylated compared to the controls. Chymotrypsin-like activity was also inhibited by okadaic acid treatment similar to ethanol feeding. The 26S proteasome fraction examined by isoelectric focusing gel revealed many hyperphosphorylated bands in the proteasomes from the okadaic acid treated and the ethanol fed rat livers compared with the controls. In conclusion hyperphosphorylation of the proteasome subunits occurs in the ethanol treated proteasomal subunits which could be one mechanism of the inhibition of the 26S proteasome caused by ethanol feeding.     

Changes in 20S proteasome activity during ageing of the LOU rat

Muscular functions decline and muscle mass decreases during ageing. In the rat, there is a 27% decrease in muscle protein between 18 and 34 months of age. We examined age-related changes in the proteasome-dependent proteolytic pathway in rats at 4, 18, 24, 29 and 34 months of age. The three best characterised activities of the proteasome (chymotrypsin-like, trypsin-like and peptidylglutamyl peptide hydrolase) increased to 29 months and then decreased in the senescent animal. These variations in activity were accompanied by an identical change in the quantity of 20S proteasome measured by Western blot, whereas the S4 subunit of the 19S regulator and the quantity of ubiquitin-linked proteins remained constant. mRNA of subunits C3, C5, C9, and S4 increased in the senescent animal, but ubiquitin mRNA levels were unchanged. These findings suggest that the 20S proteasome may be partly responsible for the muscular atrophy observed during ageing in the rat.     

Ethanol withdrawal induced CYP2E1 degradation in vivo, blocked by proteasomal inhibitor PS-341.

The aim of this study was to characterize CYP2E1 degradation in vivo using PS-341, a potent proteasome inhibitor. Previously, only in vitro evidence showed that CYP2E1 induced by ethanol is degraded by the proteasome. Male Wistar rats were given ethanol intragastrically for 30 d. Ethanol was withdrawn at the same time that PS-341 was injected, 24 h before the rats were sacrificed. The liver proteasomal chymotrypsin-like activity (ChT-L) in rats fed ethanol was inhibited. After ethanol withdrawal, the proteasomal ChT-L activity returned to control levels. In the ethanol-withdrawn rats injected with PS-341, the ChT-L activity was significantly inhibited before withdrawal (p <.001). Ethanol treatment induced a 3-fold increase in CYP2E1 levels determined by Western blot. When ethanol was withdrawn, CYP2E1 decreased to control levels. In ethanol-withdrawn rats injected with PS-341, CYP2E1 remained at the induced level. These results show, for the first time, that the proteasome is responsible for ethanol-induced CYP2E1 degradation in vivo.     

The effect of ethanol-induced cytochrome p4502E1 on the inhibition of proteasome activity by alcohol

The present investigation was undertaken to determine the effect of CYP2E1 induction by ethanol on the inhibition of proteasomal activity in wild-type and CYP2E1 knockout C57 black mice. The proteasomal chymotrypsin-like activity decreased significantly in ethanol-fed wild-type mice liver, but was not reduced in ethanol-fed knockout mice liver. The 26S proteasomal activity was decreased more by ethanol feeding than was the 20S proteasomal fraction. Individual hepatocytes lost immunostaining of the proteasomes in the centrilobular zone in the livers of ethanol-fed wild-type mice and the knockout mouse liver. There was increased product of protein oxidation in the liver in the wild type but not in the knockout mice given ethanol. Taken together, these results suggest that CYP2E1 induction was responsible for the decrease in proteasome activity seen in the wild-type mice which head to the accumulation of oxidized proteins which were increased as the result of free radicals generated by CYP2E1 metabolism of ethanol.     

The effects of betaine treatment on rats fed an acute bolus of ethanol at 3 and 12 h post bolus: a microarray analysis

Betaine, a methyl donor active in methionine metabolism, is effective in preventing and reversing experimental alcohol liver disease. The metabolic and molecular biologic mechanisms involved in this prevention are only partially known. To further investigate how betaine modifies the effects of ethanol on the liver, rats were given an acute ethanol bolus with or without betaine and the results were compared to isocaloric dextrose-fed controls. Livers were subjected to microarray analysis, and functional pathways and individual gene expression changes were analyzed. Experimental groups were compared by Venn diagrams showing that both ethanol and betaine caused a change in the expression of a large number of genes indicating that the changes were global. The bio-informatic analysis showed that all the KEGG functional pathways were affected and mainly down regulated at 3 h post bolus when ethanol plus betaine were compared with ethanol-fed rats. The most profound effect of betaine was on the metabolic pathways both at 3 and 12 h post bolus. At 3 h, the changes in gene expression were mostly down regulated, but at 12 h, the changes were regulated equally up and down. This hypothesis-driven analysis showed that the effects of betaine on the effects of ethanol were partly transient.     

Gene expression modifications in the liver caused by binge drinking and S-adenosylmethionine feeding. The role of epigenetic changes

Chronic ethanol ingestion, achieved by feeding ethanol at a constant rate using intragastric tube feeding, alters the expression of genes in the liver. This is done by epigenetic mechanisms, which depend on the blood alcohol levels at the time of killing. However, acute bolus feeding of ethanol changes gene expression without lasting epigenetic changes. This occurs with histone 3 methylation and acetylation modifications. The gene expression response to an acute bolus of ethanol might be modified by feeding S-adenosylmethionine (SAMe), a methyl donor. In the present study, rats were given a bolus of ethanol (6 g/kg body weight (bw), SAMe (1 g/kg bw), ethanol + SAMe, or isocaloric glucose. The group of rats (n = 3) were killed at 3 and 12 h post bolus, and gene microarray analysis was performed on their liver cells. SAMe reduced the 3 h blood ethanol levels and increased the ALT levels at 3 h. Venn diagrams showed that alcohol changed the expression of 646 genes at 3 h post bolus and 586 genes at 12 h. SAMe changed the expression of 1,012 genes when fed with ethanol 3 h post ethanol bolus and 554 genes at 12 h post ethanol bolus. SAMe alone changed the expression of 1,751 genes at 3 h and 1,398 at 12 h. There were more changes in gene expression at 3 h than at 12 h post ethanol when ethanol alone was compared to the dextrose control. The same was true when SAMe was compared to SAMe + ethanol. Ethanol up regulated gene expression in most functional pathways at 3 h. However, when SAMe was fed with ethanol at 3 h, most pathways were down regulated. At 12 h, however, when ethanol was fed, the pathways were half up regulated and half down regulated. The same was true when SAMe + ethanol was fed. The expression of epigenetically important genes, such as BHMT and Foxn3, was up regulated 3 h post alcohol bolus. At 3 h, SAMe down regulated the expression of genes, such as BHMT, Mat2a, Jun, Tnfrs9, Ahcy 1, Tgfbr1 and 2, and Pcaf. At 12 h, the insulin signaling pathways were half down regulated by ethanol, which was partly prevented by SAMe. The MAPK pathway was up regulated by ethanol, but SAMe did not prevent this. In conclusion, profound changes in gene expression evolved between 3 h and 12 post ethanol bolus. SAMe down regulated these changes in gene expression at 3 h, and less so at 12 h.