Zinc and Alcoholic Liver Disease

Abstract

Zinc deficiency is one of the most consistent nutritional/biochemical observations in alcoholic liver disease (ALD). The objectives of our research are to determine how alcohol interferes with cellular zinc homeostasis and if zinc deficiency is a causal factor in the development of ALD. Metallothionein (MT) is a major protein responsible for cellular zinc homeostasis. MT-transgenic (MT-TG) mice with hepatic overexpression of MT and elevation of zinc level were resistant to ethanol-induced liver injury. MT-knockout (MT-KO) mice with a reduction of hepatic zinc were more susceptible to alcohol toxicity. However, zinc treatment also provided beneficial effects on alcohol hepatoxicity in MT-KO mice, suggesting a MT-independent action. Dietary zinc supplementation normalized hepatic zinc level and attenuated the pathological changes in the liver of mice chronically fed alcohol. Several mechanisms were involved in zinc action against alcoholic cytotoxicity. Zinc enhanced cellular antioxidant capacity and corrected alcohol metabolic switch from alcohol dehydrogenase to cytochrome P4502E1. Zinc attenuated cytokine production and TNF-α receptor- and Fas-mediated cell death pathways. Zinc restored activities of hepatocyte nuclear factor-4α (HNF-4α) and peroxisome proliferation activator-α (PPAR-α), and enhanced hepatic fatty acid β-oxidation and lipid secretion. Hepatoma cell cultures showed that zinc deprivation induces lipid accumulation via inactivating HNF-4α and PPAR-α. These results suggest that alcohol exposure interferes with hepatic zinc homeostasis, leading to cellular zinc deprivation. Inactivation of zinc proteins due to zinc release is likely an important molecular mechanism in the pathogenesis of ALD.

Introduction

Zinc deficiency has been well documented in patients with alcoholic liver disease (ALD). The zinc levels in serum and liver of patients with ALD were significantly lower than those of normal subjects. The severity of zinc deficiency tends to increase along with disease progression from alcoholic steatosis to cirrhosis [1]. Investigation of zinc metabolism in alcoholics demonstrated that alcohol consumption leads to increased zinc excretion in the urine and decreased zinc absorption from the intestine [2]. Liver is a major organ involved in zinc metabolism, however, the effects of alcohol on cellular zinc homeostasis in the liver remain largely unknown. Cellular zinc homeostasis is important in cell functions. Metallothionein (MT) is a major protein responsible for cellular zinc homeostasis [3]. Under normal physiological conditions, one MT can bind up to seven zinc ions and thus functions as a zinc reservoir. MT also functions as a zinc supplier by transferring zinc to apoproteins to form functional zinc proteins [4]. Zinc participates in cell functions mainly through binding to thousands of zinc proteins including metalloenzymes. However, zinc may be released upon oxidation of the zinc-binding cysteine residues, leading to dysfunction of zinc proteins. Alcohol metabolism in the liver generates reactive oxygen species (ROS) which may mobilize zinc from zinc proteins. This paper summarizes our research findings of the mechanistic link between zinc, zinc proteins and cell functions in the pathogenesis of ALD.

MT in Control of Zinc Homeostasis and Oxidative Stress

MTs are comprised of 61–68 amino acids with a highly conserved sequence of 20 cysteine residues [3]. MT shares an important similarity with glutathione (GSH), due to the fact that one third of their amino acids are cysteines. Importantly, the thiol groups in MTs are preferential attacking targets for free radicals compared to the sulfhydryl residues from GSH or protein fractions [5]. Thus, MTs contribute to the cellular antioxidant defense. MTs also play an important role in zinc homeostasis. It has been reported that the zinc levels in the liver correlate with MT levels [4]. MT overexpression in the livers of MT-transgenic (MT-TG) mice is associated with an increase in hepatic zinc concentration, while MT-knockout (MT-KO) KO mice showed a decrease in hepatic zinc levels [6,7]. In addition, MTs also participate in intracellular zinc trafficking. Previous studies have shown that MTs translocate from cytosol to nuclei or mitochondria inter-membrane spaces and release zinc [8]]. To determine the role of MT in alcohol-induced liver injury, both the MT-TG and MT-KO mice model were used in our studies.

MT-TG mice are characterized with 10-fold increase in hepatic MT level and 2-fold increase in hepatic zinc level in comparison with wild-type (WT) mice [6]. The MT-TG mice along with WT controls were subjected to alcohol intoxication by three gastric doses of alcohol at 5 g/kg at 12-hour intervals [9]. Alcohol administration in the WT mice caused prominent microvesicular steatosis along with necrosis and elevation of serum alanine aminotransferase. Ultrastructural changes of the hepatocytes include fat accumulation, organelle abnormality and focal cytoplasmic degeneration. The alcohol-induced hepatotoxicity was significantly inhibited in the MT-TG mice. Furthermore, alcohol treatment decreased hepatic-reduced GSH, but increased oxidized GSH (GSSG) along with lipid peroxidation, protein oxidation and superoxide generation in the WT mice. This hepatic oxidative stress was significantly suppressed in the MT-TG mice. However, MT overexpression in the liver did not affect ethanol-induced decrease in NAD+/NADH ratio or increase in cytochrome P4502E1 (CYP2E1) activity. These results indicate that MTs are effective agents in protection against alcohol-induced liver injury, and the hepatic protection by MTs is likely through inhibition of alcohol-induced oxidative stress.

A MT-KO mice model was used to determine if the protective action observed in MT-TG mice is dependent MT or zinc, because a 2-fold increase in hepatic zinc level is associated with MT overexpression in the MT-TG mice. MT-KO mice along with their WT controls were treated with three gastric doses of alcohol at 5 g/kg at 12-hour intervals [10]. Zinc sulfate was injected via intraperitoneal injection at 5 mg/kg/day for 3 days before alcohol treatment. Because zinc injection will induce MT expression in the liver of WT mice but not in the MT-KO mice, we were able to dissect the role of zinc from MT. The MT-KO mice demonstrated an assay background level of hepatic MT and a lower hepatic zinc level in comparison with WT mice. Zinc treatment significantly elevated hepatic MT concentrations only in WT mice and increased zinc concentrations in both MT-KO and WT mice. Alcohol treatment caused degenerative morphological changes and necrotic appearance in the liver of MT-KO mice, while microvesicular steatosis was the only alcohol-induced change in the liver of WT mice. Alcohol treatment decreased hepatic GSH concentrations and increased hepatic lipid peroxidation products in the MT-KO mice, which was more severe than that in the WT mice. All of these alcohol-induced toxic responses in the liver were significantly suppressed by zinc treatment in both MT-KO and WT mice, although the zinc effects in MT-KO mice were less than those in WT mice. These results demonstrate that zinc, independent of MT, plays an important role in protection from alcoholic liver injury. However, MT is required to maintain high levels of zinc in the liver, suggesting that the protective actions by MT in the liver are mediated, at least partially, by zinc.

Zinc in Control of Alcohol Metabolism and Oxidative Stress

Under normal physiological conditions, alcohol dehydrogenase (ADH) is the major enzyme responsible for alcohol metabolism in the liver. However, chronic alcohol consumption induces CYP2E1 rather than ADH [11,12]. Alcohol metabolism via the CYP2E1 pathway generates ROS, and previous studies have shown CYP2E1 is the major player in alcohol-induced oxidative stress [13]. Zinc is a co-factor of ADH, and removal of zinc from ADH led to a complete loss of its catalytic activity. Thus, alcohol-induced zinc depletion is most likely linked to a shift of the ethanol metabolic pathway from ADH to CYP2E1 which favors ROS generation. Zinc also plays an important role in regulation of cellular GSH that is vital to cellular antioxidant defense [14]. To determine if zinc supplementation protects against alcohol-induced oxidative stress and liver injury and if zinc action is independent of MT, MT-KO mice along with WT mice were pair-fed an alcohol liquid diet for 12 weeks [15]. Zinc sulfate was supplemented at 75 ppm in the liquid diets; this led to an average zinc intake of 33 mg/kg/day.

Chronic alcohol exposure caused a significant decrease in hepatic zinc concentrations in both WT and MT-KO mice with a lower value in the latter, which was normalized by zinc supplementation. Zinc supplementation attenuated alcohol-induced liver injury as measured by histopathological and ultrastructural changes, serum alanine transferase activity and hepatic tumor necrosis factor-α (TNF-α) in both MT-KO and WT mice. Zinc supplementation inhibited accumulation of ROS indicated by dihydroethidium fluorescence and the consequent oxidative damage as assessed by immunohistochemical detection of 4-hydroxynonenal and nitrotyrosine and quantitative analysis of malondialdehyde and protein carbonyl in the liver. Zinc supplementation suppressed alcohol-elevated CYP2E1 activity, but increased the activity of ADH in the liver. Zinc supplementation also prevented alcohol-induced decreases in GSH concentration and GSH peroxidase activity and increased GSH reductase activity in the liver. Although alcohol-induced hepatotoxicity in MT-KO mice was more severe than that in WT mice, the beneficial effects of zinc supplementation were very close in MT-KO and WT mice, suggesting that the zinc action is independent of MT.

To elucidate the question of why zinc supplementation produced a similar effect in MT-KO and WT mice, the liver MT concentrations were compared after zinc treatment for a short term (3 doses) and long term (12 weeks). Surprisingly, the hepatic MT was increased by 10-fold by 3 doses of zinc treatment, but only 2-fold after 12 weeks of zinc supplementation. Thus, the protective effects of short-term zinc treatment may be generated from the actions of both MT and zinc. However, the protective effects of dietary zinc supplementation against chronic alcohol exposure-induced liver injury are largely dependent of the actions of zinc per se rather than MT. These data indicate that zinc has inhibitory effects on alcohol-induced oxidative stress and liver injury. The suppression of alcohol-induced CYP2E1 activity and preservation of GSH concentration and GSH peroxidase activity at least partially contribute to the antioxidant effects of zinc supplementation.

Zinc in Abrogation of Alcohol-Induced Hepatic Inflammation and Apoptosis

Apoptotic cell death of hepatocytes as demonstrated in both clinical and experimental studies is a feature of ALD [16,17]. Zinc is known to participate in multiple cellular functions, including metabolism, cell signaling and gene regulation. Zinc deprivation in cell culture studies has been shown to induce apoptosis in diverse cell lines including HepG2 cells [18,19,20]. Zinc deprivation also potentiates death receptor-mediated apoptosis [21]. We have previously shown that zinc supplementation attenuates apoptotic cell death induced by D-galactosamine/TNF-α or acute alcohol intoxication [22,23]. However, neither D-galactosamine/TNF-α nor acute alcohol intoxication induced hepatitis which is characterized in alcoholic patients. Thus, a long-term (6 months) dietary alcohol exposure to mice was conducted in our laboratory to mimic alcoholic hepatitis, and the effect of zinc supplementation on alcohol-induced hepatocyte apoptosis was determined [24].

Adult male mice fed an alcohol liquid diet for 6 months developed hepatitis as indicated by neutrophil infiltration and elevation of chemokines, keratinocyte chemoattractant and monocyte chemoattractant protein-1 levels. Apoptotic cell death was detected in alcohol-exposed mice by a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and confirmed by the increased activities of caspase-3 and −8. Zinc supplementation attenuated alcoholic hepatitis and reduced the number of TUNEL-positive cells in association with inhibition of caspase activities. Alcohol exposure caused oxidative stress as indicated by ROS accumulation, mitochondrial GSH depletion and decreased MT levels in the liver, which were suppressed by zinc supplementation. The mRNA levels of TNF-α, TNF-R1, FasL, Fas, FAF-1 and caspase-3 in the liver were upregulated by alcohol exposure, which were attenuated by zinc supplementation. Zinc supplementation also prevented alcohol-elevated serum and hepatic TNF-α levels and TNF-α R1 and Fas proteins in the liver. These results suggest that zinc supplementation prevented hepatocyte apoptosis in mice subjected to a long-term alcohol exposure, and the action of zinc is likely through suppression of oxidative stress, caspase activation and death receptor signaling pathways.

Zinc in Regulation of Hepatic Lipid Metabolism

Alcoholic steatosis (fatty liver) is one of the earliest pathological changes in ALD, and accumulation of lipid in the hepatocyte makes the liver susceptible to inflammatory mediators or other toxic agents, leading to further progression to hepatitis and eventually fibrosis [25]. Alcoholic steatosis is a reversible stage of liver damage, and reduction of steatosis will likely halt or slow the progression of ALD. While previous studies have shown that alterations in fatty acid synthesis, fatty acid oxidation, and triglyceride-rich very-low-density lipoprotein (VLDL) secretion are involved in the pathogenesis of alcoholic steatosis [26,27,28], the molecular mechanisms underlying the alcohol effects on lipid homeostasis have not been fully defined. Increasing evidence suggests that zinc plays a critical role in regulation of hepatic lipid metabolism. A lower hepatic zinc level was associated with steatosis in leptin receptor deficiency rats [29]. Feeding rats with a zinc-deficient diet (a single nutrient deficiency) caused hepatic lipid accumulation in association with dysregulation of a large number of genes involved in lipid metabolism [30,31,32]. Given the fact that dietary zinc deficiency facilitates hepatic lipid accumulation and alcohol causes hepatic zinc deficiency, zinc supplementation would provide beneficial effects on alcoholic steatosis.

Adult male mice were pair-fed an alcohol or isocaloric maltose dextrin liquid diet for 16 weeks with or without zinc supplementation for the last 4 weeks [33]. Oil red O staining demonstrated that alcohol exposure caused lipid droplet accumulation in the liver. Zinc supplementation to alcohol-fed mice remarkably reduced the number and the size of lipid droplets in the liver. Quantitative assay of hepatic lipid content showed alcohol exposure caused remarkable increases in hepatic triglyceride, cholesterol and free fatty acids, which was significantly reduced by zinc supplementation. To understand the mechanism of how zinc supplementation reverses alcoholic steatosis, hepatic fatty acid β-oxidation and lipid export were determined. Hepatic fatty acid β-oxidation was not affected by alcohol exposure, but accelerated by zinc supplementation. The VLDL export capacity as estimated by Triton WR1339 method was impaired by alcohol exposure, which was normalized by zinc supplementation. Hepatic genes involved in fatty acid β-oxidation (Cpt1a and Acadl) and VLDL secretion (Mttp and Apob) were further measured. The mRNA levels of Acadl, Mttp and ApoB were not affected by chronic alcohol exposure, but were significantly increased by zinc supplementation. These results suggest that zinc depletion is a causal factor in the development of alcoholic steatosis, and dietary zinc supplementation reduces hepatic lipid accumulation in mice previously exposed to alcohol by enhancing fatty acid β-oxidation and VLDL secretion.

Zinc in Maintaining Activities of PPAR-α and HNF-4α

PPAR-α and HNF-4α are zinc finger transcription factors and belong to a nuclear hormone superfamily [34]. PPAR-α is one of the most well-recognized transcription factors in mechanistic studies on the pathogenesis of alcoholic steatosis and has been well studied [35,36]. Chronic alcohol exposure in mice has been reported to suppress the DNA-binding activity of PPAR-α, without affecting the protein level [37]. Treatment with the PPAR-α agonist, WY14,643, attenuated alcoholic steatosis in association with upregulating mRNA protein and activity of PPAR-α. HNF-4α is a master regulator of hepatic gene expression [38], and it has been shown to bind to the reporters of more than 1,200 genes involved in most aspects of hepatocyte function [39]. However, the role of HNF-4α in alcoholic steatosis remains unknown. Zinc is a central component in the zinc figure structure which is required for DNA-binding activity. Elimination of zinc coordination by ROS or iron from the zinc fingers will disassemble the zinc figure structure of these proteins, leading to defective DNA binding and decreased transcription of target genes [40,41]. Thus, inactivation of zinc transcription factors may account for alcohol- induced cellular disorders. To determine the link between zinc and zinc finger transcription factors in ALD, effects of alcohol and zinc on HNF-4α and PPAR-α were determined with both the chronic alcohol exposure mice model and hepatoma cell culture model [33].

In the liver of mice chronically fed alcohol for 3 months, DNA-binding activity of HNF-4α was reduced, although the mRNA and protein levels were not affected. Zinc supplementation upregulated the mRNA level of HNF-4α and attenuated alcohol-reduced DNA-binding activity of HNF-4α. Chronic alcohol exposure decreased the protein level and DNA-binding activity of PPAR-α without affecting the mRNA level. Again, zinc supplementation attenuated alcohol-diminished protein level and DNA-binding activity of PPAR-α. The direct link between zinc and zinc finger transcription factors was defined by experimental zinc depletion in HepG2 cell culture. Zinc deprivation with or without zinc supplementation did not significantly affect the protein levels of HNF-α and PPAR-α. However, the DNA-binding activity of HNF-4α and PPAR-α was significantly decreased by zinc deprivation, which was reversed by adding back zinc in the media. HNF-4α and PPAR-α regulatory proteins related to fatty acids β-oxidation (ACADM, ACADL, ACADVL) and lipid secretion (MTTP, ApoB) were then determined, and zinc deprivation reduced the protein levels of ACADL, MTP and ApoB. Oil red O staining showed that zinc deprivation causes lipid droplet accumulation in the HepG2 cells. Zinc deprivation significantly increased the cellular concentrations of triglycerides and free fatty acids. These results suggest that alcohol exposure reduces the DNA-binding activity of HNF-4α and PPAR-α at least partially through mobilizing zinc from these zinc finger transcription factors. Reactivation of HNF-4α and PPAR-α is likely the most important molecular mechanism underlying zinc action on hepatic lipid homeostasis.

Conclusion

Our research findings suggest that zinc deficiency is a causal factor in the development of ALD. Inactivation of zinc proteins due to zinc release under oxidative stress condition at least partially accounts for alcohol-induced metabolic disorders and/or cell injury. Dietary zinc supplementation provides protection against alcohol-induced liver injury through modulating multiple pathways, including oxidative stress, alcohol metabolic pathways, lipid metabolic pathways, cytokine production and cell death signaling. MTs play an important role in cellular zinc homeostasis, and the hepatic zinc status correlates well with hepatic MT concentrations. Short-term zinc treatment can induce significant MT expression in the liver, and at least partially medicates the protective effects of zinc on acute alcohol intoxication. However, long-term dietary zinc supplementation induced only limited MT expression in the liver. Therefore, the beneficial effects of long-term dietary zinc supplementation on alcohol-induced liver damage are mainly generated from the actions of zinc per se rather than MTs. Because of the importance of MTs in maintaining cellular zinc homeostasis and promoting zinc trafficking, exploring methods that reverse alcohol-induced hepatic MT depletion is one of attractive strategies in efforts to restore the activities of zinc proteins. There are thousands of zinc proteins in the cells, and thus high-throughput technologies such as metalloproteomics should be introduced to future studies in order to understand the molecular mechanisms of zinc deficiency in mediating alcohol-induced liver damage.

Disclosure Statement

The author declares that no financial or other conflict of interest exists in relation to the content of the article.