Current Molecular Pharmacology - Volume 10, Issue 3, 2017

This article reviews recent studies on CYP2E1-mediated alcoholic liver injury, the induction of CYP2A5 by alcohol and the mechanism for this upregulation, especially the permissive role of CYP2E1 in the induction of CYP2A5 by alcohol and the CYP2E1-ROS-Nrf2 pathway, and protective effects of CYP2A5 against ethanol-induced oxidative liver injury. Ethanol can induce CYP2E1, an active generator of reactive oxygen species (ROS), and CYP2E1 is a contributing factor for alcoholinduced oxidative liver injury. CYP2A5, another isoform of cytochrome P450, can also be induced by ethanol. Chronic feeding of ethanol to wild type mice increased CYP2A5 catalytic activity, protein and mRNA levels as compared to pair-fed controls. This induction was blunted in CYP2E1 knockout (cyp2e1-/-) mice but was restored when human CYP2E1 was reintroduced and expressed in cyp2e1-/- mice. Ethanol-induced CYP2E1 co-localized with CYP2A5 and preceded the elevation of CYP2A5. The antioxidants N-acetyl cysteine and vitamin C lowered the alcohol elevation of ROS and blunted the alcohol induction of CYP2A5, but not CYP2E1, suggesting ROS play a novel role in the crosstalk between CYP2E1 and CYP2A5. The antioxidants blocked the activation of Nrf2, a transcription factor known to upregulate expression of CYP2A5. When alcohol-induced liver injury was enhanced in Nrf2 knockout (Nrf2-/-) mice, alcohol elevation of CYP2A5 but not CYP2E1 was also lower in Nrf2-/- mice. CYP2A5 knockout (cyp2a5-/-) mice exhibited an enhanced alcoholic liver injury compared with WT mice as indicated by serum ALT, steatosis and necroinflammation. Alcohol-induced hyperglycemia were observed in cyp2a5-/- mice but not in WT mice.

Here, we describe research on the involvement of the transcription factor, Early Growth Response- 1 (Egr-1) in alcohol-induced liver injury, specifically, fatty liver (steatosis), one of the earliest and most frequent signs of liver injury that occurs after heavy drinking. Egr-1 is a ubiquitous transcription factor found in nearly all cell types. However, because the liver is the principal site of ethanol oxidation, it sustains the greatest damage from alcohol abuse. Thus, this review focuses on how alcohol consumption causes changes in the hepatic expression of Egr-1, which, in turn causes downstream alterations in the expression of other genes to cause liver pathology. Ironically, while such changes in Egr-1 expression clearly favor steatosis and even fibrosis development, the absence of Egr-1 expression can actually exacerbate liver injury after excessive alcohol consumption or after exposure to other hepatotoxins. The existing literature on Egr-1 is extensive. Here, we confine our initial description of Egr-1 to its principal molecular characteristics, its biological functions, and its involvement in certain pathologies that are either directly or obliquely related to alcoholic liver disease. We describe experimental data that clearly implicate Egr-1 function in alcohol-induced steatosis and fibrosis, showing that ethanol-elicited regulation of Egr-1 expression depends on the generation of acetaldehyde and that the absence of Egr-1 diminishes alcohol-induced triglyceride accumulation. Overall, the existing evidence for the involvement of Egr-1 as a key link in alcohol-induced liver disease is strong. The evidence underscores the potential role of Egr-1 and several other transcription factors as therapeutic targets in the alleviation of alcoholic liver disease, which, even after decades of treatment options, still remains difficult to manage in the clinic.

The Wnt pathway is a highly conserved signal transduction pathway that plays an important role in diverse aspects of hepatic physiology. The Wnt pathway, consisting of canonical and noncanonical arms, is composed of secreted glycoproteins, cell surface receptors and co-receptors, and complex intracellular regulatory machinery that regulate a large number of cellular functions.β-Catenin is the main effector of the canonical Wnt pathway and hepatocyte-specific loss of the protein leads to increased susceptibility to alcoholic steatohepatitis. Hepatocytes with disrupted β-catenin demonstrate mitochondrial dysfunction, defective oxidative phosphorylation, and increased oxidative stress.β- Catenin knockout mice have decreased expression of alcohol metabolizing enzymes and increased blood alcohol levels that along with hypoglycemia and hyperammonemia, lead to increased mortality upon alcohol exposure. Disruption of hepatic β-catenin affects fatty acid oxidation and fasting ketogenesis and thereby profoundly affects systemic energy homeostasis. Given the combined roles of Wnt/β-catenin signaling in hepatocellular bioenergetics and regeneration, the Wnt pathway also contributes to alcohol-induced hepatic fibrogenesis and hepatocarcinogenesis. Targeting the Wnt/β-catenin pathway represents an attractive strategy for the treatment of alcohol-induced liver disease.

Chronic alcohol consumption can lead to the development of alcoholic fatty liver disease. The underlying pathogenic mechanisms however, have not been fully elucidated. Here, we review the current state of the art regarding the application of lipidomics to study alcohol's effect on hepatic lipids. It is clear that alcohol has a profound effect on the hepatic lipidome, with documented changes in the major lipid categories (i.e. fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids and prenol lipids). Alcohol';s most striking effect is the marked change in the hepatic fatty acyl pool. This effect includes increased levels of 18-carbon fatty acyl chains incorporated into multiple lipid species, as well as a general shift toward increased unsaturation of fatty acyl moieties. In addition to our literature review, we also make several recommendations to consider when designing lipidomic studies into alcohol’s effects. These recommendations include integration of lipidomic data with other measures of lipid metabolism, inclusion of multiple experimental time points, and presentation of quantitative data. We believe rigorous analysis of the hepatic lipidome can yield new insight into the pathogenesis of alcohol-induced fatty liver. While the existing literature has been largely descriptive, the field is poised to apply lipidomics to yield a new level of understanding on alcohol’s effects on hepatic lipid metabolism.

Alcoholic fatty liver disease (AFLD) and non-alcoholic fatty liver disease (NAFLD) are two pathological conditions that are spreading worldwide. Both conditions are remarkably similar with regard to the pathophysiological mechanism and progression despite different causes. Oxidative stressinduced mitochondrial dysfunction through post-translational protein modifications and/or mitochondrial DNA damage has been a major risk factor in both AFLD and NAFLD development and progression. Cytochrome P450-2E1 (CYP2E1), a known important inducer of oxidative radicals in the cells, has been reported to remarkably increase in both AFLD and NAFLD. Interestingly, CYP2E1 isoforms expressed in both endoplasmic reticulum (ER) and mitochondria, likely lead to the deleterious consequences in response to alcohol or in conditions of NAFLD after exposure to high fat diet (HFD) and in obesity and diabetes. Whether CYP2E1 in both ER and mitochondria work simultaneously or sequentially in various conditions and whether mitochondrial CYP2E1 may exert more pronounced effects on mitochondrial dysfunction in AFLD and NAFLD are unclear. The aims of this review are to briefly describe the role of CYP2E1 and resultant oxidative stress in promoting mitochondrial dysfunction and the development or progression of AFLD and NAFLD, to shed a light on the function of the mitochondrial CYP2E1 as compared with the ER-associated CYP2E1. We finally discuss translational research opportunities related to this field.

Lipin-1, a mammalian phosphatidic acid phosphatase (PAP), is a bi-functional molecule involved in various signaling pathways via its function as a PAP enzyme in the triglyceride synthesis pathway and in the nucleus as a transcriptional co-regulator. In the liver, lipin-1 is known to play a vital role in controlling the lipid metabolism and inflammation process at multiple regulatory levels. Alcoholic fatty liver disease (AFLD) is one of the earliest forms of liver injury and approximately 8-20% of patients with simple steatosis can develop into more severe forms of liver injury, including steatohepatitis, fibrosis/ cirrhosis, and eventually hepatocellular carcinoma (HCC). The signal transduction mechanisms for alcohol-induced detrimental effects in liver involves alteration of complex and multiple signaling pathways largely governed by a central and upstream signaling system, namely, sirtuin 1 (SIRT1)-AMP activated kinase (AMPK) axis. Emerging evidence suggests a pivotal role of lipin-1 as a crucial downstream regulator of SIRT1-AMPK signaling system that is likely to be ultimately responsible for development and progression of AFLD. Several lines of evidence demonstrate that ethanol exposure significantly induces lipin-1 gene and protein expression levels in cultured hepatocytes and in the livers of rodents, induces lipin-1-PAP activity, impairs the functional activity of nuclear lipin-1, disrupts lipin-1 mRNA alternative splicing and induces lipin-1 nucleocytoplasmic shuttling. Such impairment in response to ethanol leads to derangement of hepatic lipid metabolism, and excessive production of inflammatory cytokines in the livers of the rodents and human alcoholics. This review summarizes current knowledge about the role of lipin-1 in the pathogenesis of AFLD and its potential signal transduction mechanisms.

For more than 30 years, lipid droplets (LDs) were considered as an inert bag of lipid for storage of energy-rich fat molecules. Following a paradigm shift almost a decade ago, LDs are presently considered an active subcellular organelle especially designed for assembling, storing and subsequently supplying lipids for generating energy and membrane synthesis (and in the case of hepatocytes for VLDL secretion). LDs also play a central role in many other cellular functions such as viral assembly and protein degradation. Here, we have explored the structural and functional changes that occur in hepatic and adipose tissue LDs following chronic ethanol consumption in relation to their role in the pathogenesis of alcoholic liver injury.

Introduction: Ethanol metabolism in the liver results in oxidative stress, altered cytokine production and fat accumulation in the liver. Thus, it is thought that the accumulation of benign fat into the liver in conjunction with a “second hit” leads to liver failure. However, we have recently developed the use of precision-cut liver slices (PCLSs) as an in vitro culture model in which to investigate the pathophysiology of alcohol-induced liver injury. In this review, these studies will be discussed and newer data presented. Methods: Original investigations into the use of PCLS were obtained from chow fed rats (200-300g). PCLSs were cultured 24-96h in media, 25 mM ethanol, or 25 mM ethanol and 0.5 mM 4- methylpyrazole (4-MP). PCLSs were examined for at different times and evaluated for glutathione (GSH) levels, extent of lipid peroxidation (TBARS assay), cytokine production (ELISA and RT-PCR) and myofibroblast activation. Age-matched rats were fed high fat diets for 13 months, PCLSs were prepared, and evaluated as outlined above. In recently, human and mouse PCLSs were cut, equilibrated, and evaluated using the methods outlined as above. Results: In these studies, it was shown that the PCLSs from rats, mice and human livers retained excellent viability over a 96 hour period of incubation. During this time period, alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P4502E1 levels were viable. After 24 hours of ethanol exposure, fatty livers and fibrogenic responses developed and could be prevented/reversed with the 4-MP. In a separate study using overly obese rats, ethanol metabolism was decreased in PCLSs as compared to age-matched controls (AMC). However, higher levels of triglycerides and lipid peroxidation were found in PCLSs from obese rats compared to AMC. Also, increased concentrations of the proinflammatory cytokines (TNF-α and IL-6) were found in the culture supernatants. In contrast, decreased levels of reduced glutathione (GSH) and heme oxygenase I (HO-1) levels were detected. Conclusion: Within 24h of incubation, ethanol metabolism by PCLSs initiates fat accumulation in the liver at which point there is an activation of myofibroblasts. Thus, fatty liver is the first response to ethanol and sensitizes the liver to other products of oxidative stress that result in inflammation and the start of liver failure ending in cirrhosis. Thus, from these studies it appears that PCLSs can be utilized to determine the mechanisms(s) by which ethanol exposure leads to the development and/or progression of alcoholic liver disease (ALD).