Probiotic-derived nanoparticles inhibit ALD through intestinal miR194 suppression and subsequent FXR activation.

BACKGROUND AND AIMS: Intestinal farnesoid X receptor (FXR) plays a critical role in alcohol-associated liver disease (ALD). We aimed to investigate whether alcohol-induced dysbiosis increased intestinal microRNA194 (miR194) that suppressed Fxr transcription and whether Lactobacillus rhamnosus GG-derived exosome-like nanoparticles (LDNPs) protected against ALD through regulation of intestinal miR194-FXR signaling in mice. APPROACH AND RESULTS: Binge-on-chronic alcohol exposure mouse model was utilized. In addition to the decreased ligand-mediated FXR activation, alcohol feeding repressed intestinal Fxr transcription and increased miR194 expression. This transcriptional suppression of Fxr by miR194 was confirmed in intestinal epithelial Caco-2 cells and mouse enteriods. The alcohol feeding-reduced intestinal FXR activation was further demonstrated by the reduced FXR reporter activity in fecal samples and by the decreased fibroblast growth factor 15 (Fgf15) messenger RNA (mRNA) in intestine and protein levels in the serum, which caused an increased hepatic bile acid synthesis and lipogeneses. We further demonstrated that alcohol feeding increased-miR194 expression was mediated by taurine-upregulated gene 1 (Tug1) through gut microbiota regulation of taurine metabolism. Importantly, 3-day oral administration of LDNPs increased bile salt hydrolase (BSH)-harboring bacteria that decreased conjugated bile acids and increased gut taurine concentration, which upregulated Tug1, leading to a suppression of intestinal miR194 expression and recovery of FXR activation. Activated FXR upregulated FGF15 signaling and subsequently reduced hepatic bile acid synthesis and lipogenesis and attenuated ALD. These protective effects of LDNPs were eliminated in intestinal FxrΔIEC and Fgf15-/- mice. We further showed that miR194 was upregulated, whereas BSH activity and taurine levels were decreased in fecal samples of patients with ALD. CONCLUSIONS: Our results demonstrated that gut microbiota-mediated miR194 regulation contributes to ALD pathogenesis and to the protective effects of LDNPs through modulating intestinal FXR signaling.

Metabolomics analysis of urine from patients with alcohol-associated liver disease reveals dysregulated caffeine metabolism.

Excess alcohol intake causes millions of deaths annually worldwide. Asymptomatic early-stage, alcohol-associated liver disease (ALD) is easily overlooked, and ALD is usually only diagnosed in more advanced stages. We explored the possibility of using polar urine metabolites as biomarkers of ALD for early-stage diagnosis and functional assessment of disease severity by quantifying the abundance of polar metabolites in the urine samples of healthy controls (n = 18), patients with mild or moderate liver injury (n = 21), and patients with severe alcohol-associated hepatitis (n = 25). The polar metabolites in human urine were first analyzed by untargeted metabolomics, showing that 209 urine metabolites are significantly changed in patients, and 17 of these are highly correlated with patients' model for end-stage liver disease (MELD) score. Pathway enrichment analysis reveals that the caffeine metabolic pathway is the most affected in ALD. We then developed a targeted metabolomics method and measured the concentration of caffeine and its metabolites in urine using internal and external standard calibration, respectively. The described method can quantify caffeine and its 14 metabolites in 35 min. The results of targeted metabolomics analysis agree with the results of untargeted metabolomics, showing that 13 caffeine metabolites are significantly decreased in patients. In particular, the concentrations of 1-methylxanthine, paraxanthine, and 5-acetylamino-6-amino-3-methyluracil are markedly decreased with increased disease severity. We suggest that these three metabolites could serve as functional biomarkers for differentiating early-stage ALD from more advanced liver injury.NEW & NOTEWORTHY Our study using both untargeted and targeted metabolomics reveals the caffeine metabolic pathway is dysregulated in ALD. Three caffeine metabolites, 1-methylxanthine, paraxanthine, and 5-acetylamino-6-amino-3-methyluracil, can differentiate the severity of early-stage ALD.

The Beneficial Effects of Lactobacillus GG Therapy on Liver and Drinking Assessments in Patients with Moderate Alcohol-Associated Hepatitis.

INTRODUCTION: We investigated the effect of daily oral Lactobacillus rhamnosus GG (LGG) in reducing liver injury/severity and drinking in patients with alcohol use disorder and moderately severe alcohol-associated hepatitis. METHODS: Forty-six male and female individuals with alcohol use disorder and moderate alcohol-associated hepatitis (12 ≤ model for end-stage liver disease score < 20, aged 21-67 years) received either LGG (n = 24) or placebo (n = 22). Data were collected/assessed at baseline and at 1, 3, and 6 months. RESULTS: LGG treatment was associated with a significant reduction in liver injury after 1 month. Six months of LGG treatment reduced heavy drinking levels to social or abstinence levels. DISCUSSION: LGG treatment was associated with an improvement in both liver injury and drinking.

Inhibition of Sphingosine-1-Phosphate-Induced Th17 Cells Ameliorates Alcohol-Associated Steatohepatitis in Mice.

BACKGROUND AND AIMS: Chronic alcohol consumption is accompanied by intestinal inflammation. However, little is known about how alterations to the intestinal immune system and sphingolipids contribute to the pathogenesis of alcohol-associated liver disease (ALD). APPROACH AND RESULTS: We used wild-type mice, retinoid-related orphan receptor gamma t (RORγt)-deficient mice, sphingosine kinase-deficient mice, and local gut anti-inflammatory, 5-aminosalicyclic acid-treated mice in a chronic-binge ethanol feeding model. Targeted lipidomics assessed the sphingolipids in gut and liver samples. Gut immune cell populations, the amounts of sphingolipids, and the level of liver injury were examined. Alcohol intake induces a pro-inflammatory shift in immune cell populations in the gut, including an increase in Th17 cells. Using RORγt-deficient mice, we found that Th17 cells are required for alcohol-associated gut inflammation and the development of ALD. Treatment with 5-aminosalicyclic acid decreases alcohol-induced liver injury and reverses gut inflammation by the suppression of CD4+ /RORγt+ /interleukin-17A+ cells. Increased Th17 cells were due to up-regulation of sphingosine kinase 1 activity and RORγt activation. We found that S1P/S1PR1 signaling is required for the development of Th17 cell-mediated ALD. Importantly, in vivo intervention blocking of S1P/S1PR1 signaling markedly attenuated alcohol-induced liver inflammation, steatosis, and damage. CONCLUSIONS: Gut inflammation is a functional alteration of immune cells in ALD. Reducing gut Th17 cells leads to reduced liver damage. S1P signaling was crucial in the pathogenesis of ALD in a Th17 cell-dependent manner. Furthermore, our findings suggest that compounds that reduce gut inflammation locally may represent a unique targeted approach in the treatment of ALD.

Neutral Ceramidase Mediates Nonalcoholic Steatohepatitis by Regulating Monounsaturated Fatty Acids and Gut IgA+ B Cells.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is associated with obesity and an increased risk for liver cirrhosis and cancer. Neutral ceramidase (NcDase), which is highly expressed in the intestinal brush border of the small intestine, plays a critical role in digesting dietary sphingolipids (ceramide) to regulate the balance of sphingosine and free fatty acids. It remains unresolved whether obesity-associated alteration of NcDase contributes to the manifestation of NASH. Here, we revealed that NcDase deficiency in murine models of NASH prevents hepatic inflammation and fibrosis but not steatosis. APPROACH AND RESULTS: NcDase-/- mice display reduced stearoyl-CoA desaturase (SCD) 1 expression with a compositional decrease of monounsaturated fatty acids (MUFAs) under the different dietary conditions. We further found that NcDase is a functional regulator of intestinal B cells and influences the abundance and quality of the secretory IgA response toward commensal bacteria. Analysis of composition of the gut microbiota found that Clostridiales colonization was increased in NcDase-/- mice. The colonization of germ-free mice with gut microbiota from NcDase-/- mice resulted in a greater decrease in the expression of SCD1 and the level of MUFAs in the liver relative to gut microbiota from wild-type littermates, which are associated with the alternation of IgA-bound bacteria, including increase of Ruminococcaceae and reduction of Desulfovibrio. Mechanistically, NcDase is a crucial link that controls the expression of SCD1 and MUFA-mediated activation of the Wnt/ß-catenin. Very importantly, our experiments further demonstrated that Wnt3a stimulation can enhance the activity of NcDase in hepatocytes. CONCLUSIONS: Thus, the NcDase-SCD1-Wnt feedback loop promotes the diet-induced steatohepatitis and fibrosis through the regulation of intestinal IgA+ immune cells.

Theragnostic Efficacy of K18 Response in Alcohol Use Disorder with Clinically Significant Fibrosis Using Gut-Liver Axis.

(1) Background: Fibrosis in early-stage alcohol-associated liver disease (ALD) is commonly under-diagnosed in routine clinical practice. This study characterized the liver-injury and cell death response in alcohol use disorder (AUD) patients with ALD who also exhibited fibrosis and assessed the efficacy of standard of care (SOC) treatment in the improvement in liver injury. (2) Methods: Forty-eight heavy-drinking AUD patients aged 21−65 yrs. without clinical manifestations of liver injury were grouped by Fibrosis-4 (FIB-4) score, as negative (Gr.1 < 1.45, n = 21) or positive (Gr.2 ≥ 1.45, n = 27). Patients received 2-weeks (2 w) inpatient SOC. Data on demographics, drinking patterns, liver-injury, immune markers, and liver cell death (K18s) markers were analyzed at baseline (BL) and after 2 w SOC. (3) Results: Lifetime drinking (LTDH, yrs.) and acute heavy drinking (Heavy Drinking Days Past 90 Days [HDD90]) markers were significantly higher in Gr.2 vs. Gr.1. BL ALT, AST, AST:ALT and K18M65 were considerably higher in Gr.2. Dysregulated gut dysfunction and elevated immune activity were evident in Gr.2 characterized by TNF-α, IL-8 and LPS levels. After SOC, Gr.2 showed improvement in AST, ALT, AST/ALT ratio; and in the K18M65, K18M30 and K18M65/M30 ratio vs. Gr.1. The true positivity of BL IL-8 response to predict the improvement in K18M65 to normal levels among Gr.2 patients against those who did not have improvement after 2 w SOC was very high (AUROC = 0.830, p = 0.042). (4) Conclusions: Gut dysfunction, elevated cytokine response and necrotic liver cell death were elevated in AUD patients with early-stage ALD. K18 showed promise as a predictive theragnostic factor to differentiate among the AUD patients with early-stage ALD and baseline fibrosis who had improvement in liver injury against those who did not, by the levels of baseline IL-8.

Probiotic Lactobacillus rhamnosus GG Prevents Liver Fibrosis Through Inhibiting Hepatic Bile Acid Synthesis and Enhancing Bile Acid Excretion in Mice.

BACKGROUND AND AIMS: Cholestatic liver disease is characterized by gut dysbiosis and excessive toxic hepatic bile acids (BAs). Modification of gut microbiota and repression of BA synthesis are potential strategies for the treatment of cholestatic liver disease. The purpose of this study was to examine the effects and to understand the mechanisms of the probiotic Lactobacillus rhamnosus GG (LGG) on hepatic BA synthesis, liver injury, and fibrosis in bile duct ligation (BDL) and multidrug resistance protein 2 knockout (Mdr2-/- ) mice. APPROACH AND RESULTS: Global and intestine-specific farnesoid X receptor (FXR) inhibitors were used to dissect the role of FXR. LGG treatment significantly attenuated liver inflammation, injury, and fibrosis with a significant reduction of hepatic BAs in BDL mice. Hepatic concentration of taurine-ß-muricholic acid (T-ßMCA), an FXR antagonist, was markedly increased in BDL mice and reduced in LGG-treated mice, while chenodeoxycholic acid, an FXR agonist, was decreased in BDL mice and normalized in LGG-treated mice. LGG treatment significantly increased the expression of serum and ileum fibroblast growth factor 15 (FGF-15) and subsequently reduced hepatic cholesterol 7α-hydroxylase and BA synthesis in BDL and Mdr2-/- mice. At the molecular level, these changes were reversed by global and intestine-specific FXR inhibitors in BDL mice. In addition, LGG treatment altered gut microbiota, which was associated with increased BA deconjugation and increased fecal and urine BA excretion in both BDL and Mdr2-/- mice. In vitro studies showed that LGG suppressed the inhibitory effect of T-ßMCA on FXR and FGF-19 expression in Caco-2 cells. CONCLUSION: LGG supplementation decreases hepatic BA by increasing intestinal FXR-FGF-15 signaling pathway-mediated suppression of BA de novo synthesis and enhances BA excretion, which prevents excessive BA-induced liver injury and fibrosis in mice.

Cathelicidin-related antimicrobial peptide alleviates alcoholic liver disease through inhibiting inflammasome activation.

Alcoholic liver disease (ALD) is associated with gut dysbiosis and hepatic inflammasome activation. While it is known that antimicrobial peptides (AMPs) play a critical role in the regulation of bacterial homeostasis in ALD, the functional role of AMPs in the alcohol-induced inflammasome activation is unclear. The aim of this study was to determine the effects of cathelicidin-related antimicrobial peptide (CRAMP) on inflammasome activation in ALD. CRAMP knockout (Camp-/-) and wild-type (WT) mice were subjected to binge-on-chronic alcohol feeding and synthetic CRAMP peptide was administered. Serum/plasma and hepatic tissue samples from human subjects with alcohol use disorder and/or alcoholic hepatitis were analyzed. CRAMP deficiency exacerbated ALD with enhanced inflammasome activation as shown by elevated serum interleukin (IL)-1ß levels. Although Camp-/- mice had comparable serum endotoxin levels compared to WT mice after alcohol feeding, hepatic lipopolysaccharide (LPS) binding protein (LBP) and cluster of differentiation (CD) 14 were increased. Serum levels of uric acid (UA), a Signal 2 molecule in inflammasome activation, were positively correlated with serum levels of IL-1ß in alcohol use disorder patients with ALD and were increased in Camp-/- mice fed alcohol. In vitro studies showed that CRAMP peptide inhibited LPS binding to macrophages and inflammasome activation stimulated by a combination of LPS and UA. Synthetic CRAMP peptide administration decreased serum UA and IL-1ß concentrations and rescued the liver from alcohol-induced damage in both WT and Camp-/- mice. In summary, CRAMP exhibited a protective role against binge-on-chronic alcohol-induced liver damage via regulation of inflammasome activation by decreasing LPS binding and UA production. CRAMP administration may represent a novel strategy for treating ALD. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Profiling of Polar Metabolites in Mouse Feces Using Four Analytical Platforms to Study the Effects Of Cathelicidin-Related Antimicrobial Peptide in Alcoholic Liver Disease.

Alterations in gut bacterial homeostasis result in changes in intestinal metabolites. To investigate the effects of alcohol on fecal metabolites and the role of cathelicidin-related antimicrobial peptide (CRAMP) in alcoholic liver disease (ALD), CRAMP knockout (KO) and their control wild type (WT) mice were fed a Lieber-DeCarli liquid diet with or without alcohol. Polar metabolites in mouse feces were analyzed by GC × GC-MS and 2DLC-MS, and the concentrations of short chain fatty acids (SCFAs) were measured by GC-MS. A total of 95 and 190 metabolites were detected by GC × GC-MS and 2DLC-MS, respectively. Among the significantly changed metabolites, taurine and nicotinic acid were decreased in WT mice fed alcohol, which were also down-regulated in KO mice fed without alcohol. Interestingly, these two metabolites were increased in KO mice fed alcohol compared to them in WT controls. Additionally, SCFAs were significantly decreased in WT mice fed alcohol and in KO mice fed without alcohol, whereas two branched-chain SCFAs were increased by alcohol treatment in KO mice. In summary, the analytical platforms employed in this study successfully dissected the alterations of polar metabolites and SCFAs in fecal samples, which helped understand the effects of alcohol consumption and CRAMP in intestinal metabolism and alcohol-induced liver injury.

Proteomic Analysis Reveals Novel Mechanisms by Which Polychlorinated Biphenyls Compromise the Liver Promoting Diet-Induced Steatohepatitis.

Environmental pollution contributes to fatty liver disease pathogenesis. Polychlorinated biphenyl (PCB) exposures have been associated with liver enzyme elevation and suspected steatohepatitis in cohort studies. Male mice treated with the commercial PCB mixture, Aroclor 1260 (20 mg/kg), and fed high fat diet (HFD) for 12 weeks developed steatohepatitis. Receptor-based modes of action including inhibition of the epidermal growth factor (EGF) receptor were previously proposed, but other mechanisms likely exist. Objectives were to identify and validate the pathways, transcription factors, and mechanisms responsible for the steatohepatitis associated with PCB and HFD coexposures. Comparative proteomics analysis was performed in archived mouse liver samples from the aforementioned chronic exposure study. Pathway and transcription factor analysis (TFA) was performed, and selected results were validated. Liver proteomics detected 1103 unique proteins. Aroclor 1260 upregulated 154 and downregulated 93 of these. Aroclor 1260 + HFD coexposures affected 55 pathways including glutathione metabolism, intermediary metabolism, and cytoskeletal remodeling. TFA of Aroclor 1260 treatment demonstrated alterations in the function of 42 transcription factors including downregulation of NRF2 and key nuclear receptors previously demonstrated to protect against steatohepatitis (e.g., HNF4α, FXR, PPARα/δ/γ, etc.). Validation studies demonstrated that Aroclor 1260 significantly reduced HNF4α protein levels, while Aroclor 1260 + HFD reduced expression of the HNF4α target gene, albumin, in vivo. Aroclor 1260 attenuated EGF-dependent HNF4α phosphorylation and target gene activation in vitro. Aroclor 1260 reduced levels of NRF2, its target genes, and glutathione in vivo. Aroclor 1260 attenuated EGF-dependent NRF2 upregulation, in vitro. Aroclor 1260 indirectly activated hepatic stellate cells in vitro via induction of hepatocyte-derived TGFß. PCB exposures adversely impacted transcription factors regulating liver protection, function, and fibrosis. PCBs, thus, compromised the liver by reducing its protective responses against nutritional stress to promote diet-induced steatohepatitis. The identified mechanisms by which environmental pollutants influence fatty liver disease pathogenesis require confirmation in humans.

Probiotic Lactobacillus rhamnosus GG prevents progesterone metabolite epiallaopregnanolone sulfate-induced hepatic bile acid accumulation and liver injury.

Intrahepatic cholestasis of pregnancy (ICP) is gestation-specific liver disease associated with liver injury and increased serum and hepatic bile acids. Although the mechanism of ICP is still not fully understood, the reproductive hormones seem to play an important role. Recent studies show that a progesterone metabolite, epiallopregnanolone sulfate (PM5S), is supraphysiologically elevated in the serum of ICP patients, indicating it may play an etiology role in ICP. Bile acid homeostasis is controlled by multiple mechanisms including farnesoid X receptor (FXR)-mediated bile acid export and synthesis. It is known that cholic acid (CA), a primary bile acid, can activate FXR, which is inhibited by PM5S, an FXR antagonist. Here we employed a mouse model of concurrent exposure of CA and PM5S-induced liver injury and determined the effects of probiotic Lactobacillus rhamnosus GG (LGG) in the prevention of the bile acid disorders and liver injury. Mice challenged with CA + PM5S had significantly increased levels of serum and hepatic bile acids and bilirubin and liver enzyme. Pretreatment with LGG significantly reduced bile acid and bilirubin levels associated with reduced liver enzyme level and mRNA expression levels of pro-inflammatory cytokines. We also showed that the beneficial effects of LGG is likely mediated by hepatic FXR activation and bile salt export pump (BSEP) upregulation. In conclusion, our results provide a rationale for the application of probiotics in the management of ICP through gut microbiota-mediated FXR activation.

Chalcone Derivative L6H21 Reduces EtOH + LPS-Induced Liver Injury Through Inhibition of NLRP3 Inflammasome Activation.

BACKGROUND: Chronic alcohol intake increases circulating endotoxin levels causing excessive inflammation that aggravates the liver injury. (E)-2,3-dimethoxy-4'-methoxychalcone (L6H21), a derivative of chalcone, has been found to inhibit inflammation in cardiac diseases and nonalcoholic fatty liver disease. However, the use of L6H21 in alcoholic liver disease to inhibit exotoxin-associated inflammation has not been explored. In this study, we examined the effects of L6H21 on EtOH + LPS-induced hepatic inflammation, steatosis, and liver injury and investigated the underlying mechanisms. METHODS: C57BL6 mice were treated with 5% EtOH for 10 days, and LPS was given to the mice 6 hours before sacrificing. One group of mice was supplemented with L6H21 with EtOH and LPS. RAW264.7 cells were used to analyze the effects of L6H21 on macrophage activation. RESULTS: EtOH + LPS treatment significantly increased hepatic steatosis and serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), which were reduced by L6H21 treatment. EtOH + LPS treatment increased hepatic inflammation, as shown by the increased hepatic protein levels of Toll-like receptor-4, p65, and p-IκB, and increased oxidative stress, as shown by protein carbonyl levels and reactive oxygen species formation, which were reduced by L6H21 treatment. In addition, L6H21 treatment markedly inhibited EtOH + LPS-elevated hepatic protein levels of NLRP3, cleaved caspase-1, cleaved IL-1ß, and caspase-1-associated apoptosis. CONCLUSIONS: Our results demonstrate that L6H21 treatment inhibits EtOH + LPS-induced liver steatosis and injury through suppression of NLRP3 inflammasome activation. L6H21 may be used as an alternative strategy for ALD prevention/treatment.

Intestinal HIF-1α deletion exacerbates alcoholic liver disease by inducing intestinal dysbiosis and barrier dysfunction.

BACKGROUND & AIMS: Alcoholic liver disease (ALD) is characterized by gut dysbiosis and increased gut permeability. Hypoxia inducible factor 1α (HIF-1α) has been implicated in transcriptional regulation of intestinal barrier integrity and inflammation. We aimed to test the hypothesis that HIF-1α plays a critical role in gut microbiota homeostasis and the maintenance of intestinal barrier integrity in a mouse model of ALD. METHODS: Wild-type (WT) and intestinal epithelial-specific Hif1a knockout mice (IEhif1α-/-) were pair-fed modified Lieber-DeCarli liquid diet containing 5% (w/v) alcohol or isocaloric maltose dextrin for 24 days. Serum levels of alanine aminotransferase and endotoxin were determined. Fecal microbiota were assessed. Liver steatosis and injury, and intestinal barrier integrity were evaluated. RESULTS: Alcohol feeding increased serum levels of alanine aminotransferase and lipopolysaccharide, hepatic triglyceride concentration, and liver injury in the WT mice. These deleterious effects were exaggerated in IEhif1α-/- mice. Alcohol exposure resulted in greater reduction of the expression of intestinal epithelial tight junction proteins, claudin-1 and occludin, in IEhif1α-/- mice. In addition, cathelicidin-related antimicrobial peptide and intestinal trefoil factor were further decreased by alcohol in IEhif1α-/- mice. Metagenomic analysis showed increased gut dysbiosis and significantly decreased Firmicutes/Bacteroidetes ratio in IEhif1α-/- mice compared to the WT mice exposed to alcohol. An increased abundance of Akkermansia and a decreased level of Lactobacillus in IEhif1α-/- mice were also observed. Non-absorbable antibiotic treatment reversed the liver steatosis in both WT and IEhif1α-/- mice. CONCLUSION: Intestinal HIF-1α is essential for the adaptative response to alcohol-induced changes in intestinal microbiota and barrier function associated with elevated endotoxemia and hepatic steatosis and injury. LAY SUMMARY: Alcohol consumption alters gut microbiota and multiple intestinal barrier protecting factors that are regulated by intestinal hypoxia-inducible factor 1α (HIF-1α). Absence of intestinal HIF-1α exacerbates gut leakiness leading to an increased translocation of bacteria and bacterial products to the liver, consequently causing alcoholic liver disease. Intestinal specific upregulation of HIF-1α could be developed as a novel approach for the treatment of alcoholic liver disease.

FGF 21 deficiency slows gastric emptying and reduces initial blood alcohol concentration in mice exposed to acute alcohol in fasting state.

Excess alcohol consumption can lead to alcoholic liver disease. Fibroblast growth factor 21 (FGF21) is a metabolic regulator with multiple physiologic functions. Previous study demonstrated that FGF21 deficiency exacerbated alcohol-induced liver injury and exogenous FGF21 administration protected liver from chronic alcohol-induced injury. In this study, we aimed to explore the role of FGF21 in alcohol metabolism in mice. FGF21 knockout (KO) mice and the wild type(WT) control mice were divided into two groups and fasted for 24 h followed by a bonus of alcohol treatment at a dose of 5 g/kg body weight via gavage. Serum alcohol concentration was measured after gavage at 0.5, 2, 3, 4 and 6 h, respectively. At the end, gastric and liver tissues were collected. Serum alcohol concentration of KO mice was significantly lower than that of WT at 0.5 h after alcohol expose. There were no significant differences in alcohol dehydrogenase (ADH) activity and aldehyde dehydrogenase 2 (ALDH2) activity in gastric and liver tissues between WT and the KO mice. However, gastric emptying time of KO mice was much longer than that of WT mice. In addition, the intestinal permeability and serum GLP-1 level of KO mice were significantly higher than that of WT mice. These results suggest that FGF21 deficiency slow gastric emptying rate and indirectly influence initial alcohol metabolism in mice exposed to acute alcohol. Our findings provide additional information for understanding the gastrointestinal mechanism of alcoholic liver disease and other alcohol use disorders.

Nuclear receptors and nonalcoholic fatty liver disease.

Nuclear receptors are transcription factors which sense changing environmental or hormonal signals and effect transcriptional changes to regulate core life functions including growth, development, and reproduction. To support this function, following ligand-activation by xenobiotics, members of subfamily 1 nuclear receptors (NR1s) may heterodimerize with the retinoid X receptor (RXR) to regulate transcription of genes involved in energy and xenobiotic metabolism and inflammation. Several of these receptors including the peroxisome proliferator-activated receptors (PPARs), the pregnane and xenobiotic receptor (PXR), the constitutive androstane receptor (CAR), the liver X receptor (LXR) and the farnesoid X receptor (FXR) are key regulators of the gut:liver:adipose axis and serve to coordinate metabolic responses across organ systems between the fed and fasting states. Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and may progress to cirrhosis and even hepatocellular carcinoma. NAFLD is associated with inappropriate nuclear receptor function and perturbations along the gut:liver:adipose axis including obesity, increased intestinal permeability with systemic inflammation, abnormal hepatic lipid metabolism, and insulin resistance. Environmental chemicals may compound the problem by directly interacting with nuclear receptors leading to metabolic confusion and the inability to differentiate fed from fasting conditions. This review focuses on the impact of nuclear receptors in the pathogenesis and treatment of NAFLD. Clinical trials including PIVENS and FLINT demonstrate that nuclear receptor targeted therapies may lead to the paradoxical dissociation of steatosis, inflammation, fibrosis, insulin resistance, dyslipidemia and obesity. Novel strategies currently under development (including tissue-specific ligands and dual receptor agonists) may be required to separate the beneficial effects of nuclear receptor activation from unwanted metabolic side effects. The impact of nuclear receptor crosstalk in NAFLD is likely to be profound, but requires further elucidation. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

Saturated and Unsaturated Dietary Fats Differentially Modulate Ethanol-Induced Changes in Gut Microbiome and Metabolome in a Mouse Model of Alcoholic Liver Disease.

Alcoholic liver disease (ALD) ranks among major causes of morbidity and mortality. Diet and crosstalk between the gut and liver are important determinants of ALD. We evaluated the effects of different types of dietary fat and ethanol on the gut microbiota composition and metabolic activity and the effect of these changes on liver injury in ALD. Compared with ethanol and a saturated fat diet (medium chain triglycerides enriched), an unsaturated fat diet (corn oil enriched) exacerbated ethanol-induced endotoxemia, liver steatosis, and injury. Major alterations in gut microbiota, including a reduction in Bacteroidetes and an increase in Proteobacteria and Actinobacteria, were seen in animals fed an unsaturated fat diet and ethanol but not a saturated fat diet and ethanol. Compared with a saturated fat diet and ethanol, an unsaturated fat diet and ethanol caused major fecal metabolomic changes. Moreover, a decrease in certain fecal amino acids was noted in both alcohol-fed groups. These data support an important role of dietary lipids in ALD pathogenesis and provide insight into mechanisms of ALD development. A diet enriched in unsaturated fats enhanced alcohol-induced liver injury and caused major fecal metagenomic and metabolomic changes that may play an etiologic role in observed liver injury. Dietary lipids can potentially serve as inexpensive interventions for the prevention and treatment of ALD.

Up-regulation of Siah1 by ethanol triggers apoptosis in neural crest cells through p38 MAPK-mediated activation of p53 signaling pathway.

Seven in absentia homolog 1 (Siah1) is one of the E3 ubiquitin ligases and plays a key role in regulating target protein degradation. This study was designed to test the hypothesis that Siah1 mediates ethanol-induced apoptosis in NCCs through p38 MAPK-mediated activation of the p53 signaling pathway. We found that exposure of NCCs to ethanol resulted in the increases in the total protein levels of p53 and the phosphorylation of p53 at serine 15. Ethanol exposure also resulted in a significant increase in the phosphorylation of p38 MAPK. Knock-down of Siah1 dramatically reduced the ethanol-induced increase in the phosphorylation of p38 MAPK. Knock-down of Siah1 by siRNA or down-regulation of p38 MAPK by either siRNA or inhibitor significantly diminished ethanol-induced accumulations of p53 and the phosphorylation of p53. In addition, ethanol exposure resulted in a significant increase in the expression of p53 downstream targets and apoptosis in NCCs, which can be significantly diminished by down-regulation of Siah1 with siRNA. Knock-down of p38 MAPK by siRNA also dramatically reduced the ethanol-induced apoptosis. These results demonstrate that Siah1 plays a crucial role in ethanol-induced apoptosis in NCCs, and that the up-regulation of Siah1 by ethanol can trigger apoptosis through p38 MAPK-mediated activation of the p53 signaling pathway.

Fenofibrate increases cardiac autophagy via FGF21/SIRT1 and prevents fibrosis and inflammation in the hearts of Type 1 diabetic mice.

Fenofibrate (FF), as a peroxisome-proliferator-activated receptor α (PPARα) agonist, has been used clinically for decades to lower lipid levels. In the present study, we examined whether FF can be repurposed to prevent the pathogenesi of the heart in Type 1 diabetes and to describe the underlying mechanism of its action. Streptozotocin (STZ)-induced diabetic mice and their age-matched control mice were treated with vehicle or FF by gavage every other day for 3 or 6 months. FF prevented diabetes-induced cardiac dysfunction (e.g. decreased ejection fraction and hypertrophy), inflammation and remodelling. FF also increased cardiac expression of fibroblast growth factor 21 (FGF21) and sirtuin 1 (Sirt1) in non-diabetic and diabetic conditions. Deletion of FGF21 gene (FGF21-KO) worsened diabetes-induced pathogenic effects in the heart. FF treatment prevented heart deterioration in the wild-type diabetic mice, but could not do so in the FGF21-KO diabetic mice although the systemic lipid profile was lowered in both wild-type and FGF21-KO diabetic mice. Mechanistically, FF treatment prevented diabetes-impaired autophagy, reflected by increased microtubule-associated protein 1A/1B-light chain 3, in the wild-type diabetic mice but not in the FGF21-KO diabetic mice. Studies with H9C2 cells in vitro demonstrated that exposure to high glucose (HG) significantly increased inflammatory response, oxidative stress and pro-fibrotic response and also significantly inhibited autophagy. These effects of HG were prevented by FF treatment. Inhibition of either autophagy by 3-methyladenine (3MA) or Sirt1 by sirtinol (SI) abolished FF's prevention of HG-induced effects. These results suggested that FF could prevent Type 1 diabetes-induced pathological and functional abnormalities of the heart by increasing FGF21 that may up-regulate Sirt1-mediated autophagy.

FGF21 mediates alcohol-induced adipose tissue lipolysis by activation of systemic release of catecholamine in mice.

Alcohol consumption leads to adipose tissue lipoatrophy and mobilization of FFAs, which contributes to hepatic fat accumulation in alcoholic liver disease. This study aimed to investigate the role of fibroblast growth factor (FGF)21, a metabolic regulator, in the regulation of chronic-binge alcohol-induced adipose tissue lipolysis. FGF21 KO mice were subjected to chronic-binge alcohol exposure, and epididymal white adipose tissue lipolysis and liver steatosis were investigated. Alcohol exposure caused adipose intracellular cAMP elevation and activation of lipolytic enzymes, leading to FFA mobilization in both WT and FGF21 KO mice. However, alcohol-induced systemic elevation of catecholamine, which is known to be a major player in adipose lipolysis by binding to the ß-adrenergic receptor, was markedly inhibited in KO mice. Supplementation with recombinant human FGF21 to alcohol-exposed FGF21 KO mice resulted in an increase in fat loss in parallel with an increase of circulating norepinephrine concentration. Furthermore, alcohol consumption-induced fatty liver was blunted in the KO mice, indicating an inhibition of fatty acid reverse transport from adipose to the liver in the KO mice. Taken together, our studies demonstrate that FGF21 KO mice are protected from alcohol-induced adipose tissue excess-lipolysis through a mechanism involving systemic catecholamine release.

Hepatic and fecal metabolomic analysis of the effects of Lactobacillus rhamnosus GG on alcoholic fatty liver disease in mice.

The interactions among the gut, liver, and immune system play an important role in liver disease. Probiotics have been used for the treatment and prevention of many pathological conditions, including liver diseases. Comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOF MS) was used herein, in conjunction with chemometric data analysis, to identify metabolites significantly affected by probiotics in mice fed with or without alcohol. The metabolomics analysis indicates that the levels of fatty acids increased in mouse liver and decreased in mouse feces when mice were chronically exposed to alcohol. Supplementing the alcohol-fed mice with culture supernatant from Lactobacillus rhamnosus GG (LGGs) normalized these alcohol-induced abnormalities and prevented alcoholic liver disease (ALD). These results agree well with previous studies. In addition to diet-derived long chain fatty acids (LCFAs), LGGs may positively modify the gut's bacterial population to stimulate LCFA synthesis, which has been shown to enhance intestinal barrier function, reduce endotoxemia, and prevent ALD. We also found that several amino acids, including l-isoleucine, a branched chain amino acid, were downregulated in the liver and fecal samples from animals exposed to alcohol and that the levels of these amino acids were corrected by LGGs. These results demonstrate that LGGs alleviates alcohol-induced fatty liver by mechanisms involving increasing intestinal and decreasing hepatic fatty acids and increasing amino acid concentration.