Fat-1 Transgenic Mice With Augmented n3-Polyunsaturated Fatty Acids Are Protected From Liver Injury Caused by Acute-On-Chronic Ethanol Administration.

Alcohol-associated liver disease (ALD) is the leading cause of liver disease worldwide, and alcohol-associated hepatitis (AH), a severe form of ALD, is a major contributor to the mortality and morbidity due to ALD. Many factors modulate susceptibility to ALD development and progression, including nutritional factors such as dietary fatty acids. Recent work from our group and others showed that modulation of dietary or endogenous levels of n6-and n3-polyunsaturated fatty acids (PUFAs) can exacerbate or attenuate experimental ALD, respectively. In the current study, we interrogated the effects of endogenous n3-PUFA enrichment in a mouse model which recapitulates features of early human AH using transgenic fat-1 mice which endogenously convert n6-PUFAs to n3-PUFAs. Male wild type (WT) and fat-1 littermates were provided an ethanol (EtOH, 5% v/v)-containing liquid diet for 10 days, then administered a binge of EtOH (5 g/kg) by oral gavage on the 11th day, 9 h prior to sacrifice. In WT mice, EtOH treatment resulted in liver injury as determined by significantly elevated plasma ALT levels, whereas in fat-1 mice, EtOH caused no increase in this biomarker. Compared to their pair-fed controls, a significant EtOH-mediated increase in liver neutrophil infiltration was observed also in WT, but not fat-1 mice. The hepatic expression of several cytokines and chemokines, including Pai-1, was significantly lower in fat-1 vs WT EtOH-challenged mice. Cultured bone marrow-derived macrophages isolated from fat-1 mice expressed less Pai-1 and Cxcl2 (a canonical neutrophil chemoattractant) mRNA compared to WT when stimulated with lipopolysaccharide. Further, we observed decreased pro-inflammatory M1 liver tissue-resident macrophages (Kupffer cells, KCs), as well as increased liver T regulatory cells in fat-1 vs WT EtOH-fed mice. Taken together, our data demonstrated protective effects of endogenous n3-PUFA enrichment on liver injury caused by an acute-on-chronic EtOH exposure, a paradigm which recapitulates human AH, suggesting that n3-PUFAs may be a viable nutritional adjuvant therapy for this disease.

Feeding mice a diet high in oxidized linoleic acid metabolites does not alter liver oxylipin concentrations.

The oxidation of dietary linoleic acid (LA) produces oxidized LA metabolites (OXLAMs) known to regulate multiple signaling pathways in vivo. Recently, we reported that feeding OXLAMs to mice resulted in liver inflammation and apoptosis. However, it is not known whether this is due to a direct effect of OXLAMs accumulating in the liver, or to their degradation into bioactive shorter chain molecules (e.g. aldehydes) that can provoke inflammation and related cascades. To address this question, mice were fed a low or high LA diet low in OXLAMs, or a low LA diet supplemented with OXLAMs from heated corn oil (high OXLAM diet). Unesterified oxidized fatty acids (i.e. oxylipins), including OXLAMs, were measured in liver after 8 weeks of dietary intervention using ultra-high pressure liquid chromatography coupled to tandem mass-spectrometry. The high OXLAM diet did not alter liver oxylipin concentrations compared to the low LA diet low in OXLAMs. Significant increases in several omega-6 derived oxylipins and reductions in omega-3 derived oxylipins were observed in the high LA dietary group compared to the low LA group. Our findings suggest that dietary OXLAMs do not accumulate in liver, and likely exert pro-inflammatory and pro-apoptotic effects via downstream secondary metabolites.

Ileum Gene Expression in Response to Acute Systemic Inflammation in Mice Chronically Fed Ethanol: Beneficial Effects of Elevated Tissue n-3 PUFAs.

Chronic alcohol consumption leads to disturbances in intestinal function which can be exacerbated by inflammation and modulated by different factors, e.g., polyunsaturated fatty acids (PUFAs). The mechanisms underlying these alterations are not well understood. In this study, RNA-seq analysis was performed on ileum tissue from WT and fat-1 transgenic mice (which have elevated endogenous n-3 PUFAs). Mice were chronically fed ethanol (EtOH) and challenged with a single lipopolysaccharide (LPS) dose to induce acute systemic inflammation. Both WT and fat-1 mice exhibited significant ileum transcriptome changes following EtOH + LPS treatment. Compared to WT, fat-1 mice had upregulated expression of genes associated with cell cycle and xenobiotic metabolism, while the expression of pro-inflammatory cytokines and pro-fibrotic genes was decreased. In response to EtOH + LPS, fat-1 mice had an increased expression of genes related to antibacterial B cells (APRIL and IgA), as well as an elevation in markers of pro-restorative macrophages and γδ T cells that was not observed in WT mice. Our study significantly expands the knowledge of regulatory mechanisms underlying intestinal alterations due to EtOH consumption and inflammation and identifies the beneficial transcriptional effects of n-3 PUFAs, which may serve as a viable nutritional intervention for intestinal damage resulting from excessive alcohol consumption.

Beneficial effects of an endogenous enrichment in n3-PUFAs on Wnt signaling are associated with attenuation of alcohol-mediated liver disease in mice.

Alcohol-associated liver disease (ALD) is a major human health issue for which there are limited treatment options. Experimental evidence suggests that nutrition plays an important role in ALD pathogenesis, and specific dietary fatty acids, for example, n6 or n3-PUFAs, may exacerbate or attenuate ALD, respectively. The purpose of the current study was to determine whether the beneficial effects of n3-PUFA enrichment in ALD were mediated, in part, by improvement in Wnt signaling. Wild-type (WT) and fat-1 transgenic mice (that endogenously convert n6-PUFAs to n3) were fed ethanol (EtOH) for 6 weeks followed by a single LPS challenge. fat-1 mice had less severe liver damage than WT littermates as evidenced by reduced plasma alanine aminotransferase, hepatic steatosis, liver tissue neutrophil infiltration, and pro-inflammatory cytokine expression. WT mice had a greater downregulation of Axin2, a key gene in the Wnt pathway, than fat-1 mice in response to EtOH and LPS. Further, there were significant differences between WT and fat-1 EtOH+LPS-challenged mice in the expression of five additional genes linked to the Wnt signaling pathway, including Apc, Fosl1/Fra-1, Mapk8/Jnk-1, Porcn, and Nkd1. Compared to WT, primary hepatocytes isolated from fat-1 mice exhibited more effective Wnt signaling and were more resistant to EtOH-, palmitic acid-, or TNFα-induced cell death. Further, we demonstrated that the n3-PUFA-derived lipid mediators, resolvins D1 and E1, can regulate hepatocyte expression of several Wnt-related genes that were differentially expressed between WT and fat-1 mice. These data demonstrate a novel mechanism by which n3-PUFAs can ameliorate ALD.

Mechanisms, biomarkers and targets for therapy in alcohol-associated liver injury: From Genetics to nutrition: Summary of the ISBRA 2018 symposium.

The symposium "Mechanisms, Biomarkers and Targets for Therapy in Alcohol-associated Liver Injury: From Genetics to Nutrition" was held at the 19th Congress of International Society for Biomedical Research on Alcoholism on September 13th, 2018 in Kyoto, Japan. The goal of the symposium was to discuss the importance of genetics and nutrition in alcoholic liver disease (ALD) development from mechanistic and therapeutic perspectives. The following is a summary of this session addressing the gene polymorphisms in ALD, the role of zinc in gut-liver axis perturbations associated with ALD, highlighting the importance of dietary fat in ALD pathogenesis, the hepatic n6 and n3 PUFA oxylipin pattern associated with ethanol-induced liver injury, and finally deliberating on new biomarkers for alcoholic hepatitis and their implications for diagnosis and therapy. This summary of the symposium will benefit junior and senior faculty currently investigating alcohol-induced organ pathology as well as undergraduate, graduate, and post-graduate students and fellows.

Ethanol and unsaturated dietary fat induce unique patterns of hepatic ω-6 and ω-3 PUFA oxylipins in a mouse model of alcoholic liver disease.

Alcoholic liver disease (ALD), a significant health problem, progresses through the course of several pathologies including steatosis, steatohepatitis, fibrosis, and cirrhosis. There are no effective FDA-approved medications to prevent or treat any stages of ALD, and the mechanisms involved in ALD pathogenesis are not well understood. Bioactive lipid metabolites play a crucial role in numerous pathological conditions, as well as in the induction and resolution of inflammation. Herein, a hepatic lipidomic analysis was performed on a mouse model of ALD with the objective of identifying novel metabolic pathways and lipid mediators associated with alcoholic steatohepatitis, which might be potential novel biomarkers and therapeutic targets for the disease. We found that ethanol and dietary unsaturated, but not saturated, fat caused elevated plasma ALT levels, hepatic steatosis and inflammation. These pathologies were associated with increased levels of bioactive lipid metabolites generally involved in pro-inflammatory responses, including 13-hydroxy-octadecadienoic acid, 9,10- and 12,13-dihydroxy-octadecenoic acids, 5-, 8-, 9-, 11-, 15-hydroxy-eicosatetraenoic acids, and 8,9- and 11,12-dihydroxy-eicosatrienoic acids, in parallel with an increase in pro-resolving mediators, such as lipoxin A4, 18-hydroxy-eicosapentaenoic acid, and 10S,17S-dihydroxy-docosahexaenoic acid. Elucidation of alterations in these lipid metabolites may shed new light into the molecular mechanisms underlying ALD development/progression, and be potential novel therapeutic targets.

Development, Prevention, and Treatment of Alcohol-Induced Organ Injury: The Role of Nutrition.

Alcohol and nutrition have the potential to interact at multiple levels. For example, heavy alcohol consumption can interfere with normal nutrition, resulting in overall malnutrition or in deficiencies of important micronutrients, such as zinc, by reducing their absorption or increasing their loss. Interactions between alcohol consumption and nutrition also can affect epigenetic regulation of gene expression by influencing multiple regulatory mechanisms, including methylation and acetylation of histone proteins and DNA. These effects may contribute to alcohol-related organ or tissue injury. The impact of alcohol-nutrition interactions has been assessed for several organs and tissues, including the intestine, where heavy alcohol use can increase intestinal permeability, and the liver, where the degree of malnutrition can be associated with the severity of liver injury and liver disease. Alcohol-nutrition interactions also play a role in alcohol-related lung injury, brain injury, and immune dysfunction. Therefore, treatment involving nutrient supplementation (e.g., with zinc or S-adenosylmethionine) may help prevent or attenuate some types of alcohol-induced organ damage.

Ethanol and dietary unsaturated fat (corn oil/linoleic acid enriched) cause intestinal inflammation and impaired intestinal barrier defense in mice chronically fed alcohol.

Alcohol and dietary fat both play an important role in alcohol-mediated multi-organ pathology, including gut and liver. In the present study we hypothesized that the combination of alcohol and dietary unsaturated fat (USF) would result in intestinal inflammatory stress and mucus layer alterations, thus contributing to disruption of intestinal barrier integrity. C57BL/6N mice were fed Lieber-DeCarli liquid diets containing EtOH and enriched in USF (corn oil/linoleic acid) or SF (medium chain triglycerides: beef tallow) for 8 weeks. Intestinal histology, morphometry, markers of inflammation, as well as levels of mucus protective factors were evaluated. Alcohol and dietary USF triggered an intestinal pro-inflammatory response, characterized by increase in Tnf-α, MCP1, and MPO activity. Further, alcohol and dietary USF, but not SF, resulted in alterations of the intestinal mucus layer, characterized by decreased expression of Muc2 in the ileum. A strong correlation was observed between down-regulation of the antimicrobial factor Cramp and increased Tnf-α mRNA. Therefore, dietary unsaturated fat (corn oil/LA enriched) is a significant contributing factor to EtOH-mediated intestinal inflammatory response and mucus layer alterations in rodents.

Probiotics restore bowel flora and improve liver enzymes in human alcohol-induced liver injury: a pilot study.

The effects of chronic alcohol consumption on the bowel flora and the potential therapeutic role of probiotics in alcohol-induced liver injury have not previously been evaluated. In this study, 66 adult Russian males admitted to a psychiatric hospital with a diagnosis of alcoholic psychosis were enrolled in a prospective, randomized, clinical trial to study the effects of alcohol and probiotics on the bowel flora and alcohol-induced liver injury. Patients were randomized to receive 5 days of Bifidobacterium bifidum and Lactobacillus plantarum 8PA3 versus standard therapy alone (abstinence plus vitamins). Stool cultures and liver enzymes were performed at baseline and again after therapy. Results were compared between groups and with 24 healthy, matched controls who did not consume alcohol. Compared to healthy controls, alcoholic patients had significantly reduced numbers of bifidobacteria (6.3 vs. 7.5 log colony-forming unit [CFU]/g), lactobacilli (3.15 vs. 4.59 log CFU/g), and enterococci (4.43 vs. 5.5 log CFU/g). The mean baseline alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transpeptidase (GGT) activities were significantly elevated in the alcoholic group compared to the healthy control group (AST: 104.1 vs. 29.15 U/L; ALT: 50.49 vs. 22.96 U/L; GGT 161.5 vs. 51.88 U/L), indicating that these patients did have mild alcohol-induced liver injury. After 5 days of probiotic therapy, alcoholic patients had significantly increased numbers of both bifidobacteria (7.9 vs. 6.81 log CFU/g) and lactobacilli (4.2 vs. 3.2 log CFU/g) compared to the standard therapy arm. Despite similar values at study initiation, patients treated with probiotics had significantly lower AST and ALT activity at the end of treatment than those treated with standard therapy alone (AST: 54.67 vs. 76.43 U/L; ALT 36.69 vs. 51.26 U/L). In a subgroup of 26 subjects with well-characterized mild alcoholic hepatitis (defined as AST and ALT greater than 30 U/L with AST-to-ALT ratio greater than one), probiotic therapy was associated with a significant end of treatment reduction in ALT, AST, GGT, lactate dehydrogenase, and total bilirubin. In this subgroup, there was a significant end of treatment mean ALT reduction in the probiotic arm versus the standard therapy arm. In conclusion, patients with alcohol-induced liver injury have altered bowel flora compared to healthy controls. Short-term oral supplementation with B. bifidum and L. plantarum 8PA3 was associated with restoration of the bowel flora and greater improvement in alcohol-induced liver injury than standard therapy alone.