Ruth Ann Ross

Inhibitory effect of ethanol on AMPK phosphorylation is mediated in part through elevated ceramide levels

Ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMP-activated protein kinase (AMPK). This study shows that the inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of the phosphorylation of upstream kinases and the activation of protein phosphatase 2A (PP2A).Inhibition of AMPK phosphorylation by palmitate was attributed to ceramide-dependent PP2A activation. We hypothesized that the inhibitory effect of ethanol on AMPK phosphorylation was mediated partly through the generation of ceramide. The effect of ethanol and inhibitors of ceramide synthesis on AMPK phosphorylation, ceramide levels, and PP2A activity were assessed in rat hepatoma cells (H4IIEC3). The effect of ethanol on hepatic ceramide levels was also studied in C57BL/6J mice fed the Lieber-DeCarli diet. In H4IIEC3 cells, ceramide reduced AMPK phosphorylation when they were treated for between 4 and 12 h. The basal level of AMPK phosphorylation in hepatoma cells was increased with the treatment of ceramide synthase inhibitor, fumonisin B1. Ethanol treatment significantly increased cellular ceramide content and PP2A activity by approximately 18-23%, when the cells were treated with ethanol for between 4 and 12 h. These changes in intracellular ceramide concentrations and PP2A activity correlated with the time course over which ethanol inhibited AMPK phosphorylation. The activation of PP2A and inhibition of AMPK phosphorylation caused by ethanol was attenuated by fumonisin B1 and imipramine, an acid sphingomyelinase (SMase) inhibitor. There was a significant increase in the levels of ceramide and acid SMase mRNA in the livers of ethanol-fed mice compared with controls. We concluded that the effect of ethanol on AMPK appears to be mediated in part through increased cellular levels of ceramide and activation of PP2A.

Induction of alcohol dehydrogenase activity and mRNA in hepatoma cells by dexamethasone.

Liver alcohol dehydrogenase activity was present in rat H4IIE hepatoma cells. Dexamethasone increased the enzyme activity two- to fourfold in these cells, but not in HepG2 cells. Enzyme induction was observed at dexamethasone concentrations as low as 10(-9) M, and the induction was maximal at 3 days. The increase in enzyme activity was accompanied by an increase in alcohol dehydrogenase mRNA on Northern blots and a two- to fourfold increase in alcohol dehydrogenase mRNA levels as estimated from cytoplasmic dot blots. There was no effect of dexamethasone on alpha-tubulin mRNA levels. Insulin, triiodothyronine, and growth hormone had no effect on alcohol dehydrogenase activity or mRNA levels. The induction of alcohol dehydrogenase mRNA by dexamethasone could be blocked by actinomycin D, but not by protein synthesis inhibitors. Superinduction of the mRNA (approximately twofold) was observed with dexamethasone in the presence of cycloheximide. Southern blots of genomic DNA from rat liver and H4IIE cells revealed no differences in alcohol dehydrogenase gene structure. The induction of alcohol dehydrogenase activity and mRNA levels by dexamethasone may be due to an increase in the rate of transcription of the alcohol dehydrogenase gene.

Regulation of human and rat brain metabolism: inhibitory action of phenylalanine and phenylpyruvate on glycolysis, protein, lipid, DNA, and RNA metabolism.

Abstract In human and rat brain the activity of key glycolytic enzymes and glycolysis were examined during development. The differential inhibitory effect of l -phenylalanine and phenylpyruvate on key glycolytic enzymes, on lactate production and on the incorporation of labeled glucose into lipid, protein, RNA and DNA was investigated in the brain of differentiating human and rat. 1. (1)|In the fetal human brain hexokinase, pyruvate kinase and glycolytic activities were approximately 10% of those observed in the adult. These parameters in the average brain cell gradually rose during differentiation. A similar pattern for these three glycolytic parameters was observed in the differentiating rat brain. 2. (2)|The sensitivity to inhibition by l -phenylalanine and phenylpyruvate was the same order of magnitude for the glycolytic enzymes and lactate production during differentiation. However, since the activities were very low in the fetal and the newborn brain, these parameters may be more vulnerable to inhibition by these compounds that accumulate in the untreated phenylketonuric patients. 3. (3)|The human and fetal brain has a very high cellularity (600–700 millions per g) during gestation and continues to decrease until it reaches a level of about 100 millions of cells per gram wet weight in the adult brain. This emphasizes the importance of expressing biochemical results on a per cell basis during differentiation. 4. (4)|Studies on the mechanism of effect of l -phenylalanine and phenylpyruvate in slices from differentiating human and adult brain were carried out. The kinetic results suggest that these compounds act through a competitive mechanism and the inhibitions were reversible by increasing the substrate level. 5. (5)|The effect of phenylpyruvate on lactate production from glucose, glucose-6-phosphate and fructose-1,6-diphosphate was also explored in the fortified supernatant fluid system from brains of differentiating human and rat. Phenylpyruvate resulted in a progressive inhibition of lactate production from all three substrates. Reciprocal plots, the Dixon plot and the reversibility of the inhibition exerted by phenylpyruvate suggested a competitive type of inhibition. 6. (6)|Since the metabolic inhibitions exerted by phenylalanine and phenylpyruvate on carbohydrate metabolism were reversible, it is possible that the high carbohydrate diet might be able to protect the developing brain from the potential damage of the high levels of these compounds in the phenylketonuric patients. 7. (7)|When increasing concentrations of phenylpyruvate were added to brain slices from differentiating rat, there was a steady, dose-dependent decrease in the incorporation of labeled glucose into lipid, RNA and DNA, which in all cases was more pronounced in slices from the fetal brain than from the adult. It is striking that there was a progressive decrease in the incorporation of label into protein with increasing concentrations of phenylpyruvate in the fetal brain but the adult brain was not affected at any concentration. These data are in agreement with the postulate that phenylpyruvate might interfere with the biosynthesis of strategic macromolecules in the differentiating brain. The results of this work also agree with clinical experience that points to the early stages of brain development as especially vulnerable to toxic effects, including metabolic disturbances. 8. (8)|The enzymatic attacking points of l -phenylalanine and phenylpyruvate involve the inhibition of key enzymes of glycolysis (hexokinase and pyruvate kinase) and of the direct oxidative pathway (6-phosphogluconate dehydrogenase). The inhibition of these enzymes might explain, at least in part, the inhibition of the biosynthesis of key macromolecules in the brain.

MicroRNA-223 ameliorates alcoholic liver injury by inhibiting the IL-6–p47phox–oxidative stress pathway in neutrophils

Objectives Chronic-plus-binge ethanol feeding activates neutrophils and exacerbates liver injury in mice. This study investigates how recent excessive drinking affects peripheral neutrophils and liver injury in alcoholics, and how miR-223, one of the most abundant microRNAs (miRNAs) in neutrophils, modulates neutrophil function and liver injury in ethanol-fed mice. Designs Three hundred alcoholics with (n=140) or without (n=160) recent excessive drinking and 45 healthy controls were enrolled. Mice were fed an ethanol diet for 10 days followed by a single binge of ethanol. Results Compared with healthy controls or alcoholics without recent drinking, alcoholics with recent excessive drinking had higher levels of circulating neutrophils, which correlated with serum levels of alanine transaminase (ALT) and aspartate transaminase (AST). miRNA array analysis revealed that alcoholics had elevated serum miR-223 levels compared with healthy controls. In chronic-plus-binge ethanol feeding mouse model, the levels of miR-223 were increased in both serum and neutrophils. Genetic deletion of the miR-223 gene exacerbated ethanol-induced hepatic injury, neutrophil infiltration, reactive oxygen species (ROS) and upregulated hepatic expression of interleukin (IL)-6 and phagocytic oxidase (phox) p47phox. Mechanistic studies revealed that miR-223 directly inhibited IL-6 expression and subsequently inhibited p47phox expression in neutrophils. Deletion of the p47phox gene ameliorated ethanol-induced liver injury and ROS production by neutrophils. Finally, miR-223 expression was downregulated, while IL-6 and p47phox expression were upregulated in peripheral blood neutrophils from alcoholics compared with healthy controls. Conclusions miR-223 is an important regulator to block neutrophil infiltration in alcoholic liver disease and could be a novel therapeutic target for the treatment of this malady.

Imipramine blocks ethanol-induced ASMase activation, ceramide generation, and PP2A activation, and ameliorates hepatic steatosis in ethanol-fed mice

Our previous data showed the inhibitory effect of ethanol on AMP-activated protein kinase phosphorylation, which appears to be mediated, in part, through increased levels of hepatic ceramide and activation of protein phosphatase 2A (Liangpunsakul S, Sozio MS, Shin E, Zhao Z, Xu Y, Ross RA, Zeng Y, Crabb DW. Am J Physiol Gastrointest Liver Physiol 298: G1004-G1012, 2010). The effect of ethanol on AMP-activated protein kinase phosphorylation was reversed by imipramine, suggesting that the generation of ceramide via acid sphingomyelinase (ASMase) is stimulated by ethanol. In this study, we determined the effects of imipramine on the development of hepatic steatosis, the generation of ceramide, and downstream effects of ceramide on inflammatory, insulin, and apoptotic signaling pathways, in ethanol-fed mice. The effect of ethanol and imipramine (10 μg/g body wt ip) on ceramide levels, as well as inflammatory, insulin, and apoptotic signaling pathways, was studied in C57BL/6J mice fed the Lieber-DeCarli diet. Ethanol-fed mice developed the expected steatosis, and cotreatment with imipramine for the last 2 wk of ethanol feeding resulted in improvement in hepatic steatosis. Ethanol feeding for 4 wk induced impaired glucose tolerance compared with controls, and this was modestly improved with imipramine treatment. There was a significant decrease in total ceramide concentrations in response to imipramine in ethanol-fed mice treated with and without imipramine (287 ± 11 vs. 348 ± 12 pmol/mg tissue). The magnitude and specificity of inhibition on each ceramide species differed. A significant decrease was observed for C16 (28 ± 3 vs. 33 ± 2 pmol/mg tissue) and C24 (164 ± 9 vs. 201 ± 4 pmol/mg tissue) ceramide. Ethanol feeding increased the levels of the phosphorylated forms of ERK slightly and increased phospho-p38 and phospho-JNK substantially. The levels of phospho-p38 and phospho-JNK were reduced by treatment with imipramine. The activation of ASMase and generation of ceramide in response to ethanol feeding may underlie several effects of ethanol. ASMase inhibitors may be considered as a therapeutic target for alcohol-induced hepatic steatosis and activation of stress kinases.

Estradiol induces class I alcohol dehydrogenase activity and mRNA in kidney of female rats

Rat kidney contains alcohol dehydrogenase (ADH) activity which appears to be similar or identical to the class I ADH expressed in liver. Both tissues contain a 1.6-kb transcript which hybridizes with an ADH cDNA under stringent conditions. Kidney ADH activity is responsive to estradiol. The enzyme activity in the kidneys of sham-operated and ovariectomized animals was the same. Treatment of either group of animals by intramuscular injection of estradiol (1 mg/kg body wt/day) for 10 days induced ADH activity in kidney two- to threefold, whether the activity was expressed as U/g tissue, U/g protein, or U/mg DNA. Estradiol induced kidney ADH mRNA in both ovariectomized and sham-operated rats approximately twofold. Thus, induction of ADH mRNA accounts for the increase in ADH activity. In situ hybridization indicated that the ADH mRNA was present in the inner cortex and medulla of the kidney. Methylation patterns of the ADH gene were examined. The gene resides in a methylated region of chromatin without any of the typical features of a HpaII tiny fragment (HTF) island. Two MspI sites flanking the transcription start site are undermethylated in liver compared with kidney and spleen. This suggests that methylation of this gene may play a role in the tissue-specific expression of ADH.

Effect of ethanol on hydrogen peroxide-induced AMPK phosphorylation

AMP-activated protein kinase (AMPK) responds to oxidative stress. Previous work has shown that ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMPK and reduced the amount of AMPK protein. Ethanol generates oxidative stress in the liver. Since AMPK is activated by reactive oxygen species, it seems paradoxical that ethanol would inhibit AMPK in the hepatoma cells. In an attempt to understand the mechanism whereby ethanol inhibits AMPK, we studied the effect of ethanol on AMPK activation by exogenous hydrogen peroxide. The effects of ethanol, hydrogen peroxide, and inhibitors of protein phosphatase 2A (PP2A) [either okadaic acid or PP2A small interference RNA (siRNA)] on AMPK phosphorylation and activity were examined in rat hepatoma cells (H4IIEC3) and HeLa cells. In H4IIEC3 cells, hydrogen peroxide (H(2)O(2), 1 mM) transiently increased the level of phospho-AMPK to 1.5-fold over control (P < 0.05). Similar findings were observed in HeLa cells, which do not express the upstream AMPK kinase, LKB1. H(2)O(2) markedly increased the phosphorylation of LKB1 in H4IIEC3 cells. Ethanol significantly inhibited the phosphorylation of PKC-zeta, LKB1, and AMPK caused by exposure to H(2)O(2). This inhibitory effect of ethanol required its metabolism. More importantly, the inhibitory effects of ethanol on H(2)O(2)-induced AMPK phosphorylation were attenuated by the presence of the PP2A inhibitor, okadaic acid, or PP2A siRNA. The inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of PKC-zeta and LKB1 phosphorylation and the activation of PP2A.

Disturbances in the murine hepatic circadian clock in alcohol-induced hepatic steatosis.

To investigate the role of the circadian clock in the development of alcohol-induced fatty liver disease we examined livers of mice chronically alcohol-fed over 4-weeks that resulted in steatosis. Here we show time-of-day specific changes in expression of clock genes and clock-controlled genes, including those associated with lipid and bile acid regulation. Such changes were not observed following a 1-week alcohol treatment with no hepatic lipid accumulation. Real-time bioluminescence reporting of PERIOD2 protein expression suggests that these changes occur independently of the suprachiasmatic nucleus pacemaker. Further, we find profound time-of-day specific changes to the rhythmic synthesis/accumulation of triglycerides, cholesterol and bile acid, and the NAD/NADH ratio, processes that are under clock control. These results highlight not only that the circadian timekeeping system is disturbed in the alcohol-induced hepatic steatosis state, but also that the effects of alcohol upon the clock itself may actually contribute to the development of hepatic steatosis.

Activation of carbohydrate response element-binding protein by ethanol.

Objective Carbohydrate response element–binding protein (ChREBP) is a transcription factor involved in hepatic lipogenesis. Its function is in part under the control of AMP-activated protein kinase (AMPK) and protein phosphatase 2A (PP2A). Given known effects of ethanol on AMPK and PP2A, it is plausible that ethanol might enhance fatty acid synthesis by increasing the activity of ChREBP. We hypothesized that another potential pathway of ethanol-induced hepatic steatosis is mediated by activation of ChREBP. Methods The effects of ethanol on ChREBP were assessed in hepatoma cells and in C57BL/6J mice fed with the Lieber-DeCarli diet. Results When the cells were exposed to ethanol (50 mM) for 24 hours, the activity of a liver pyruvate kinase (LPK) promoter–luciferase reporter was increased by ∼4-fold. Ethanol feeding of mice resulted in the translocation of ChREBP from cytosol to the nucleus. Protein phosphatase 2A activity was increased in the liver of ethanol-fed mice by 22%. We found no difference in the levels of hepatic Xu-5-P between ethanol-fed mice and controls. Transfection of a constitutively active AMPK expression plasmid suppressed the basal activity of the LPK luciferase reporter and abolished the effect of ethanol on the reporter activity. However, transfection of rat hepatoma cells with a dominant-negative AMPK expression plasmid induced basal LPK luciferase activity by only ∼20%. The effect of ethanol on ChREBP was attenuated in the presence of okadaic acid, an inhibitor of PP2A. Conclusions The effects of ethanol on AMPK and PP2A may result in activation of ChREBP, providing another potential mechanism for ethanol-induced hepatic steatosis. However, additional okadaic acid–insensitive effects appear to be important as well.

LncRNA AK054921 and AK128652 are potential serum biomarkers and predictors of patient survival with alcoholic cirrhosis

Alcoholic liver disease (ALD) is one of the leading causes of chronic liver disease. Recent studies have demonstrated the roles of long noncoding RNAs (lncRNAs) in the pathogenesis of several disease processes. However, the roles of lncRNAs in patients with ALD remain unexplored. Global profiling for human lncRNAs from peripheral blood RNA was performed in a well‐characterized cohort of healthy controls (HC; n = 4), excessive drinkers (ED) without liver disease (n = 4), and those with alcoholic cirrhosis (AC) with different severities (n = 12). The expression of unique lncRNA signatures were validated in a separate cohort of HC (n = 17), ED (n = 19), AC (n = 48), and human liver tissues with ALD (n = 19). A detailed analysis of plasma lncRNAs in AC subjects with different severities compared with HC identified 244 commonly up‐regulated lncRNAs and 181 commonly down‐regulated lncRNAs. We further validated top 20 most differentially up‐ and down‐regulated lncRNAs in ED and AC compared with HC and also determined the expression of selected lncRNAs in human liver tissues with or without AC. Among those lncRNAs, AK128652 and AK054921 were two of the most abundantly expressed lncRNAs in normal human plasma and liver, and their levels were significantly elevated in AC. The prognostic significance of AK128652 and AK054921 was determined in 48 subjects with AC who were followed prospectively for 520 days. The expression of AK128652 and AK054921 was inversely associated with survival in patients with AC. Conclusion: lncRNAs AK054921 and AK128652 are potential biomarkers to predict the progression to ALD in individuals with excessive alcohol consumption and are predictors of survival in patients with AC. (Hepatology Communications 2017;1:513–523)