Most physiological studies interested in alcohol-dependence examined ethanol as a pharmacological agent rather than a nutrient. We conducted two studies, which assessed the metabolic and endocrine ...factors involved in the regulation of alcohol and nutrient intake in alcohol-dependent (AD) subjects. We also examined the potential role of a disruption in energy balance in alcohol-dependence.
In Study-1, quantitative dietetic interviews of eating and drinking habits were conducted with 97 AD subjects. The population was split around a median alcohol intake value of 12.5 kcal/kg/day. The results showed that the "low alcohol" drinking AD subjects had high Body Mass Index (BMI) and Fat Mass (FM) and alcohol intake was compensated for by a decrease in non-alcoholic intakes. "High alcohol" drinking AD subjects, on the other hand, had low BMI and FM and the total caloric intakes were largely above norms. In Study-2, 24 AD inpatients were submitted to dietetic interviews, calorimetry and blood samplings for the measurement of biomarkers of the regulation of metabolism and satiety, on day 2, 5 and 16 of abstinence. These patients were compared with 20 controls matched for age and gender. We observed in AD patients an increase in cortisol, leptin and PYY plasma levels and a decrease in ghrelin, which might explain the observed decrease in non-alcoholic intakes. However, alcoholic and non-alcoholic intakes correlated positively with basal metabolism and negatively with leptin and leptin/BMI.
For individuals consuming below 12.5 kcal/kg/day of alcohol, alcohol intake is compensated for by a decrease in non-alcoholic nutrient intakes, probably due to changes in metabolic and satiety factors. For individuals consuming above 12.5 kcal/kg/day of alcohol, alcohol accelerates metabolism and decreases fat mass and leptin levels, and the total caloric intake largely exceeds norms. A dual model for regulation of energy intake in AD subjects is proposed.
Dysregulation of epidermal growth factor and insulin-like growth factor signaling play important roles in human hepatocellular carcinoma (HCC), leading to frequent activation of their downstream ...targets, the ras/raf/extracellular signal-regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) pathways. Salirasib is an S-prenyl-cysteine analog that has been shown to block ras and/or mTOR activation in several non hepatic tumor cell lines. We investigated in vitro the effect of salirasib on cell growth as well as its mechanism of action in human hepatoma cell lines (HepG2, Huh7, and Hep3B) and its in vivo effect in a subcutaneous xenograft model with HepG2 cells.
Salirasib induced a time and dose dependent growth inhibition in hepatocarcinoma cells through inhibition of proliferation and partially through induction of apoptosis. A 50 percent reduction in cell growth was obtained in all three cell lines at a dose of 150 μM when they were cultured with serum. By contrast, salirasib was more potent at reducing cell growth after stimulation with EGF or IGF2 under serum-free conditions, with an IC50 ranging from 60 μM to 85 μM. The drug-induced anti-proliferative effect was associated with downregulation of cyclin A and to a lesser extent of cyclin D1, and upregulation of p21 and p27. Apoptosis induction was related to a global pro-apoptotic balance with caspase 3 activation, cytochrome c release, death receptor upregulation, and a reduced mRNA expression of the apoptosis inhibitors cFLIP and survivin. These effects were associated with ras downregulation and mTOR inhibition, without reduction of ERK and Akt activation. In vivo, salirasib reduced tumour growth from day 5 onwards. After 12 days of treatment, mean tumor weight was diminished by 56 percent in the treated animals.
Our results show for the first time that salirasib inhibits the growth of human hepatoma cell lines through inhibition of proliferation and induction of apoptosis, which is associated with ras and mTOR inhibition. The therapeutic potential of salirasib in human HCC was further confirmed in a subcutaneous xenograft model.
Excessive alcohol drinking can cause pathological changes including carcinogenesis in the digestive tract from mouth to large intestine, but the underlying mechanisms are not fully understood. In ...this review, we discuss the effects of alcohol on small and large intestinal functions, such as leaky gut, dysbiosis and alterations of intestinal epithelium and gut immune dysfunctions, commonly referred to as alcohol-associated bowel disease (ABD). To date, detailed mechanistic insights into ABD are lacking. Accumulating evidence suggests a pathogenic role of ethanol metabolism in dysfunctions of the intestinal tract. Ethanol metabolism generates acetaldehyde and acetate, which could potentially promote functional disruptions of microbial and host components of the intestinal barrier along the gastrointestinal tract. The potential involvement of acetaldehyde and acetate in the pathogenesis of the underlying ABD, including cancer, is discussed. We also highlight some gaps in knowledge existing in the field of ABD. Finally, we discuss future directions in exploring the role of acetaldehyde and acetate generated during chronic alcohol intake in various pathologies affecting different sites of the intestinal tract.
Alcohol use is a leading cause of chronic liver disease worldwide, and changes in the microbiome associated with alcohol use contribute to patients’ risk for liver disease progression. Less is known ...about the effects of alcohol use on the intestinal viral microbiome (virome) and interactions between bacteriophages and their target bacteria. We studied changes in the intestinal virome of 62 clinically well‐characterized patients with alcohol use disorder (AUD) during active alcohol use and after 2 weeks of alcohol abstinence, by extracting virus‐like particles and performing metagenomic sequencing. We observed decreased abundance of Propionibacterium, Lactobacillus, and Leuconostoc phages in patients with active AUD when compared with controls, whereas after 2 weeks of alcohol abstinence, patients with AUD demonstrated an increase in the abundance of Propionibacterium, Lactobacillus, and Leuconostoc phages. The intestinal virome signature was also significantly different in patients with AUD with progressive liver disease, with increased abundance of phages targeting Enterobacteria and Lactococcus species phages compared with patients with AUD with nonprogressive liver disease. By performing moderation analyses, we found that progressive liver disease is associated with changes in interactions between some bacteriophages and their respective target bacteria. In summary, active alcohol use and alcohol‐associated progressive liver disease are associated with changes in the fecal virome, some of which are partially reversible after a short period of abstinence. Progression of alcohol‐associated liver disease is associated with changes in bacteriophage–bacteria interactions.
The intestinal virome of patients with alcohol use disorder (AUD) is significantly different from controls. Abstinence from alcohol in patients with AUD is also associated with significant differences in the intestinal virome, trending toward that of controls. Patients with progressive liver disease as determined by liver stiffness measurement and serum caspase‐cleaved and intact cytokeratin 18 (CK18‐M65) have a different intestinal virome signature than those with nonprogressive disease, which may be in part due to changes in bacteriophage–bacteria correlations with liver disease progression.
Chronic alcohol consumption is associated with intestinal fungal dysbiosis, yet we understand little about how alterations of intestinal fungi (mycobiota) contribute to the pathogenesis of ...alcohol-associated liver disease. By reanalyzing internal transcribed spacer 2 amplicon sequencing of fecal samples from a cohort of 66 patients with alcohol use disorder for presence (as opposed to relative abundance) of fungal species, we observed that the presence of Malassezia restricta was associated with increased markers of liver injury. M. restricta exacerbates ethanol-induced liver injury both in acute binge and chronic ethanol-feeding models in mice. Using bone marrow chimeric mice, we found that the disease exacerbating effect by M. restricta was mediated by C-type lectin domain family 4, member N on bone marrow-derived cells. M. restricta induces inflammatory cytokines and chemokines in Kupffer cells through C-type lectin domain family 4, member N signaling. Targeting fungal pathobionts might be a therapeutic strategy for alcohol-associated liver disease.
Emerging evidence highlights that targeting the gut microbiota could be an interesting approach to improve alcohol liver disease due to its important plasticity. This study aimed to evaluate the ...effects of inulin supplementation on liver parameters in alcohol use disorder (AUD) patients (whole sample) and in a subpopulation with early alcohol-associated liver disease (eALD).
Fifty AUD patients, hospitalized for a 3-week detoxification program, were enrolled in a randomized, double-blind, placebo-controlled study and assigned to prebiotic (inulin) versus placebo for 17 days. Liver damage, microbial translocation, inflammatory markers and 16S rDNA sequencing were measured at the beginning (T1) and at the end of the study (T2).
Compared to placebo, AST (β = 8.55, 95% CI 2.33:14.77), ALT (β = 6.01, 95% CI 2.02:10.00) and IL-18 (β = 113.86, 95% CI 23.02:204.71) were statistically significantly higher in the inulin group in the whole sample at T2. In the eALD subgroup, inulin supplementation leads to specific changes in the gut microbiota, including an increase in Bifidobacterium and a decrease of Bacteroides. Despite those changes, AST (β = 14.63, 95% CI 0.91:28.35) and ALT (β = 10.40, 95% CI 1.93:18.88) at T2 were higher in the inulin group compared to placebo. Treatment was well tolerated without important adverse events or side effects.
This pilot study shows that 17 days of inulin supplementation versus placebo, even though it induces specific changes in the gut microbiota, did not alleviate liver damage in AUD patients. Further studies with a larger sample size and duration of supplementation with adequate monitoring of liver parameters are needed to confirm these results. Gut2Brain study: https://clinicaltrials.gov/ct2/show/NCT03803709
Fédération Wallonie-Bruxelles, FRS-FNRS, Fondation Saint-Luc.
Alcohol-associated liver disease is one of the most prevalent diseases around the world, with 10-20% of patients developing progressive liver disease. To identify the complex and correlated nature of ...metabolic and microbial data types in progressive liver disease, we performed an integrated analysis of the fecal and serum metabolomes with the gut microbiome in a cohort of 38 subjects, including 15 patients with progressive liver disease, 16 patients with non-progressive liver disease, and 7 control subjects. We found that although patients were generally clustered in three groups according to disease status, metabolites showed better separation than microbial species. Furthermore, eight serum metabolites were correlated with two microbial species, among which seven metabolites were decreased in patients with progressive liver disease. Five fecal metabolites were correlated with three microbial species, among which four metabolites were decreased in patients with progressive liver disease. When predicting progressive liver disease from non-progressive liver disease using correlated metabolic and microbial signatures with the random forest model, correlated serum metabolites and microbial species showed great predictive power, with the area under the receiver operating characteristic curve achieving 0.91. The multi-omics signatures identified in this study are helpful for the early identification of patients with progressive alcohol-associated liver disease, which is a key step for therapeutic intervention.
Alcohol-related liver disease is a public health care burden globally. Only 10-20% of patients with alcohol use disorder have progressive liver disease. This study aimed to identify lipid biomarkers ...for the early identification of progressive alcohol-related liver disease, which is a key step for early intervention. We performed untargeted lipidomics analysis in serum and fecal samples for a cohort of 49 subjects, including 17 non-alcoholic controls, 16 patients with non-progressive alcohol-related liver disease, and 16 patients with progressive alcohol-related liver disease. The serum and fecal lipidome profiles in the two patient groups were different from that in the controls. Nine lipid biomarkers were identified that were significantly different between patients with progressive liver disease and patients with non-progressive liver disease in both serum and fecal samples. We further built a random forest model to predict progressive alcohol-related liver disease using nine lipid biomarkers. Fecal lipids performed better (Area Under the Curve, AUC = 0.90) than serum lipids (AUC = 0.79). The lipid biomarkers identified are promising candidates for the early identification of progressive alcohol-related liver disease.
Mechanisms underlying alcohol‐induced liver injury and its progression still remain incompletely understood. Animal models can only address some aspects of the pathophysiology that requires studies ...directly in humans, which are scarce. We assessed liver inflammatory and immune responses at early stages of alcoholic liver disease in a unique cohort of alcohol‐dependent patients undergoing a highly standardized alcohol withdrawal program. In active drinkers, quantitative real‐time polymerase chain reaction revealed alcohol‐induced activation of tumor necrosis factor alpha, interleukin (IL)‐1β, and nuclear factor kappa B in liver tissue already at early disease stages. Double immunofluorescence staining indicated that this proinflammatory response was restricted to activated, CD68‐positive macrophages. In parallel, down‐regulation of IL‐6, inhibition of the signal transducer and activator of transcription 3 (Stat3) pathway, as well as blunted cyclin D expression in hepatocytes, reduced proliferation and favored hepatocyte apoptosis. In addition, immunofluorescence and quantitative real‐time polymerase chain reaction of liver tissue showed that alcohol also activated the toll‐like receptor (TLR) 7–interferon (IFN) axis in hepatocytes, which was confirmed in alcohol‐stimulated primary human hepatocytes and precision‐cut liver slices in vitro. Activation of the TLR7–IFN axis strongly correlated with liver fibrosis markers and disease progression. Two weeks of abstinence attenuated the inflammatory response but did not allow recovery of the defective Stat3 pathway or effect on fibrosis‐associated factors. Conclusion: In humans, inflammation, activation of the TLR7–IFN axis, and inhibition of Stat3‐dependent repair mechanisms in early alcoholic liver disease pave the way for fibrosis development and ultimately disease progression.
Why some patients with alcohol dependence develop progressive liver disease remains largely unknown. Here we describe inflammatory and immune mechanisms that increase liver damage and impair liver regeneration and repair. These changes already operate at early stages of alcoholic liver disease in humans. They likely prepare an environment that favors progression of liver disease if drinking habits persist and could constitute a future target for therapy.
Alcoholic liver disease (ALD) is associated with changes in the intestinal microbiota. Functional consequences of alcohol‐associated dysbiosis are largely unknown. The aim of this study was to ...identify a mechanism of how changes in the intestinal microbiota contribute to ALD. Metagenomic sequencing of intestinal contents demonstrated that chronic ethanol feeding in mice is associated with an over‐representation of bacterial genomic DNA encoding choloylglycine hydrolase, which deconjugates bile acids in the intestine. Bile acid analysis confirmed an increased amount of unconjugated bile acids in the small intestine after ethanol administration. Mediated by a lower farnesoid X receptor (FXR) activity in enterocytes, lower fibroblast growth factor (FGF)‐15 protein secretion was associated with increased hepatic cytochrome P450 enzyme (Cyp)‐7a1 protein expression and circulating bile acid levels. Depletion of the commensal microbiota with nonabsorbable antibiotics attenuated hepatic Cyp7a1 expression and reduced ALD in mice, suggesting that increased bile acid synthesis is dependent on gut bacteria. To restore intestinal FXR activity, we used a pharmacological intervention with the intestine‐restricted FXR agonist fexaramine, which protected mice from ethanol‐induced liver injury. Whereas bile acid metabolism was only minimally altered, fexaramine treatment stabilized the gut barrier and significantly modulated hepatic genes involved in lipid metabolism. To link the beneficial metabolic effect to FGF15, a nontumorigenic FGF19 variant—a human FGF15 ortholog—was overexpressed in mice using adeno‐associated viruses. FGF19 treatment showed similarly beneficial metabolic effects and ameliorated alcoholic steatohepatitis. Conclusion: Taken together, alcohol‐associated metagenomic changes result in alterations of bile acid profiles. Targeted interventions improve bile acid–FXR–FGF15 signaling by modulation of hepatic Cyp7a1 and lipid metabolism, and reduce ethanol‐induced liver disease in mice. (Hepatology 2018;67:2150‐2166).
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