Serum metabolomic analysis reveals several novel metabolites in association with excessive alcohol use - an exploratory study.

Appropriate screening tool for excessive alcohol use (EAU) is clinically important as it may help providers encourage early intervention and prevent adverse outcomes. We hypothesized that patients with excessive alcohol use will have distinct serum metabolites when compared to healthy controls. Serum metabolic profiling of 22 healthy controls and 147 patients with a history of EAU was performed. We employed seemingly unrelated regression to identify the unique metabolites and found 67 metabolites (out of 556), which were differentially expressed in patients with EAU. Sixteen metabolites belong to the sphingolipid metabolism, 13 belong to phospholipid metabolism, and the remaining 38 were metabolites of 25 different pathways. We also found 93 serum metabolites that were significantly associated with the total quantity of alcohol consumption in the last 30 days. A total of 15 metabolites belong to the sphingolipid metabolism, 11 belong to phospholipid metabolism, and 7 metabolites belong to lysolipid. Using a Venn diagram approach, we found the top 10 metabolites with differentially expressed in EAU and significantly associated with the quantity of alcohol consumption, sphingomyelin (d18:2/18:1), sphingomyelin (d18:2/21:0,d16:2/23:0), guanosine, S-methylmethionine, 10-undecenoate (11:1n1), sphingomyelin (d18:1/20:1, d18:2/20:0), sphingomyelin (d18:1/17:0, d17:1/18:0, d19:1/16:0), N-acetylasparagine, sphingomyelin (d18:1/19:0, d19:1/18:0), and 1-palmitoyl-2-palmitoleoyl-GPC (16:0/16:1). The diagnostic performance of the top 10 metabolites, using the area under the ROC curve, was significantly higher than that of commonly used markers. We have identified a unique metaboloic signature among patients with EAU. Future studies to validate and determine the kinetics of these markers as a function of alcohol consumption are needed.

Stress-Responsive Gene FK506-Binding Protein 51 Mediates Alcohol-Induced Liver Injury Through the Hippo Pathway and Chemokine (C-X-C Motif) Ligand 1 Signaling.

BACKGROUND AND AIMS: Chronic alcohol drinking is a major risk factor for alcohol-associated liver disease (ALD). FK506-binding protein 51 (FKBP5), a cochaperone protein, is involved in many key regulatory pathways. It is known to be involved in stress-related disorders, but there are no reports regarding its role in ALD. This present study aimed to examine the molecular mechanism of FKBP5 in ALD. APPROACH AND RESULTS: We found a significant increase in hepatic FKBP5 transcripts and protein expression in patients with ALD and mice fed with chronic-plus-single binge ethanol. Loss of Fkbp5 in mice protected against alcohol-induced hepatic steatosis and inflammation. Transcriptomic analysis revealed a significant reduction of Transcriptional enhancer factor TEF-1 (TEA) domain transcription factor 1 (Tead1) and chemokine (C-X-C motif) ligand 1 (Cxcl1) mRNA in ethanol-fed Fkbp5-/- mice. Ethanol-induced Fkbp5 expression was secondary to down-regulation of methylation level at its 5' untranslated promoter region. The increase in Fkbp5 expression led to induction in transcription factor TEAD1 through Hippo signaling pathway. Fkbp5 can interact with yes-associated protein (YAP) upstream kinase, mammalian Ste20-like kinase 1 (MST1), affecting its ability to phosphorylate YAP and the inhibitory effect of hepatic YAP phosphorylation by ethanol leading to YAP nuclear translocation and TEAD1 activation. Activation of TEAD1 led to increased expression of its target, CXCL1, a chemokine-mediated neutrophil recruitment, causing hepatic inflammation and neutrophil infiltration in our mouse model. CONCLUSIONS: We identified an FKBP5-YAP-TEAD1-CXCL1 axis in the pathogenesis of ALD. Loss of FKBP5 ameliorates alcohol-induced liver injury through the Hippo pathway and CXCL1 signaling, suggesting its potential role as a target for the treatment of ALD.

Levels of circulating follicular helper T cells, T helper 1 cells, and the prognostic significance of soluble form of CD40 ligand on survival in patients with alcoholic cirrhosis.

BACKGROUND: Excessive drinkers (ED) and patients with alcoholic liver disease (ALD) are several times more susceptible to bacterial and viral infections and have a decrease in antibody responses to vaccinations. Follicular helper T (TFH) cells are essential to select B cells in the germinal center and to produce antibodies. TFH cells express both a membrane-associated and a soluble form of CD40 ligand (sCD40L); in which the latter form is released to circulation upon T cell activation. The effect of alcohol on TFH cells has not been studied. OBJECTIVES: The goals of this study are to determine the levels of TFH and T helper 1 (Th1) cells in ED and those with alcoholic cirrhosis (AC) when compared to healthy controls and to determine the prognostic significance of sCD40L in a cohort of patients with AC. METHODS: Controls, ED, and those with AC were enrolled. Baseline demographic, laboratory tests, and peripheral blood mononuclear cells (PBMCs) were isolated and assessed via flow cytometry for TFH cells. In vitro study was performed to determine the ability of PBMCs to secrete interferon (IFN)-γ upon stimulation. Serum sCD40L were also determined and its prognostic significance was tested in a cohort of AC patients. RESULTS: The levels of circulating TFH (cTFH) cells were significantly lower in peripheral blood of subjects with ED and AC compared to controls (P<0.05). IFN-γ secretion from PBMCs upon stimulation was also lower in ED and those with cirrhosis. Serum sCD40L was significantly lower in ED and AC when compared to that in controls (P<0.0005). Its level was an independent predictor of mortality. CONCLUSIONS: Patients with AC had significantly lower level of cTFH and sCD40L. The level of sCD40L was an independent predictor of mortality in these patients.

IL-1 receptor like 1 protects against alcoholic liver injury by limiting NF-κB activation in hepatic macrophages.

BACKGROUND & AIM: Alcohol consumption increases intestinal permeability and causes damage to hepatocytes, leading to the release of pathogen- and damage-associated molecular pattern molecules (PAMPs and DAMPs), stimulating hepatic macrophages and activating NF-κB. The resultant inflammation exacerbates alcoholic liver disease (ALD). However, much less is known about the mechanisms attenuating inflammation and preventing disease progression in most heavy drinkers. Interleukin (IL)-33 is a DAMP (alarmin) released from dead cells that acts through its receptor, IL-1 receptor like 1 (ST2). ST2 signaling has been reported to either stimulate or inhibit NF-κB activation. The role of IL-33/ST2 in ALD has not been studied. METHODS: Serum levels of IL-33 and its decoy receptor, soluble ST2 (sST2) were measured in ALD patients. Alcohol-induced liver injury, inflammation and hepatic macrophage activation were compared between wild-type, IL-33-/- and ST2-/- mice in several models. RESULTS: Elevation of serum IL-33 and sST2 were only observed in patients with severe decompensated ALD. Consistently, in mice with mild ALD without significant cell death and IL-33 release, IL-33 deletion did not affect alcohol-induced liver damage. However, ST2-deletion exacerbated ALD, through enhancing NF-κB activation in liver macrophages. In contrast, when extracellular IL-33 was markedly elevated, liver injury and inflammation were attenuated in both IL-33-/- and ST2-/- mice compared to wild-type mice. CONCLUSION: Our data revealed a dichotomous role of IL-33/ST2 signaling during ALD development. At early and mild stages, ST2 restrains the inflammatory activation of hepatic macrophages, through inhibiting NF-κB, and plays a protective function in an IL-33-independent fashion. During severe liver injury, significant cell death and marked IL-33 release occur, which triggers IL-33/ST2 signaling and exacerbates tissue damage. LAY SUMMARY: In mild ALD, ST2 negatively regulates the inflammatory activation of hepatic macrophages, thereby protecting against alcohol-induced liver damage, whereas in the case of severe liver injury, the release of extracellular IL-33 may exacerbate tissue inflammation by triggering the canonical IL-33/ST2L signaling in hepatic macrophages.

Chronic free-choice drinking in crossed high alcohol preferring mice leads to sustained blood ethanol levels and metabolic tolerance without evidence of liver damage.

BACKGROUND: Crossed high alcohol preferring (cHAP) mice were selectively bred from a cross of the HAP1 × HAP2 replicate lines, and we demonstrate blood ethanol concentrations (BECs) during free-choice drinking that are reminiscent of those observed in alcohol-dependent humans. Therefore, this line may provide an unprecedented opportunity to learn about the consequences of excessive voluntary ethanol (EtOH) consumption, including metabolic tolerance and liver pathology. Cytochrome p450 2E1 (CYP2E1) induction plays a prominent role in driving both metabolic tolerance and EtOH-induced liver injury. In this report, we sought to characterize cHAP drinking by assessing whether pharmacologically relevant BEC levels are sustained throughout the active portion of the light-dark cycle. Given that cHAP intakes and BECs are similar to those observed in mice given an EtOH liquid diet, we assessed whether free-choice exposure results in metabolic tolerance, hepatic enzyme induction, and hepatic steatosis. METHODS: In experiment 1, blood samples were taken across the dark portion of a 12:12 light-dark cycle to examine the pattern of EtOH accumulation in these mice. In experiments 1 and 2, mice were injected with EtOH following 3 to 4 weeks of access to water or 10% EtOH and water, and blood samples were taken to assess metabolic tolerance. In experiment 3, 24 mice had 4 weeks of access to 10% EtOH and water or water alone, followed by necropsy and hepatological assessment. RESULTS: In experiment 1, cHAP mice mean BEC values exceeded 80 mg/dl at all sampling points and approached 200 mg/dl during the middle of the dark cycle. In experiments 1 and 2, EtOH-exposed mice metabolized EtOH faster than EtOH-naïve mice, demonstrating metabolic tolerance (p < 0.05). In experiment 3, EtOH-drinking mice showed greater expression of hepatic CYP2E1 than water controls, consistent with the development of metabolic tolerance (p < 0.05). EtOH access altered neither hepatic histology nor levels of alcohol dehydrogenase and aldehyde dehydrogenase. CONCLUSIONS: These results demonstrate that excessive intake by cHAP mice results in sustained BECs throughout the active period, leading to the development of metabolic tolerance and evidence of CYP2E1 induction. Together, these results provide additional support for the cHAP mice as a highly translational rodent model of alcoholism.