Dynamic changes of gut microbiota in mouse models of metabolic dysfunction-associated steatohepatitis and its transition to hepatocellular carcinoma.

Dysbiosis of gut microbiota may account for pathobiology in simple fatty liver (SFL), metabolic dysfunction-associated steatohepatitis (MASH), fibrotic progression, and transformation to MASH-associated hepatocellular carcinoma (MASH-HCC). The aim of the present study is to investigate gut dysbiosis in this progression. Fecal microbial rRNA-16S sequencing, absolute quantification, histopathologic, and biochemical tests were performed in mice fed high fat/calorie diet plus high fructose and glucose in drinking water (HFCD-HF/G) or control diet (CD) for 2, 16 weeks, or 14 months. Histopathologic examination verified an early stage of SFL, MASH, fibrotic, or MASH-HCC progression with disturbance of lipid metabolism, liver injury, and impaired gut mucosal barrier as indicated by loss of occludin in ileum mucosa. Gut dysbiosis occurred as early as 2 weeks with reduced α diversity, expansion of Kineothrix, Lactococcus, Akkermansia; and shrinkage in Bifidobacterium, Lactobacillus, etc., at a genus level. Dysbiosis was found as early as MAHS initiation, and was much more profound through the MASH-fibrotic and oncogenic progression. Moreover, the expansion of specific species, such as Lactobacillus johnsonii and Kineothrix alysoides, was confirmed by an optimized method for absolute quantification. Dynamic alterations of gut microbiota were characterized in three stages of early SFL, MASH, and its HCC transformation. The findings suggest that the extent of dysbiosis was accompanied with MASH progression and its transformation to HCC, and the shrinking or emerging of specific microbial species may account at least in part for pathologic, metabolic, and immunologic alterations in fibrogenic progression and malignant transition in the liver.

Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.

Helicobacter pylori infection exacerbates metabolic dysfunction-associated steatotic liver disease through lipid metabolic pathways: a transcriptomic study.

BACKGROUND: The relationship between Helicobacter pylori (H. pylori) infection and metabolic dysfunction-associated steatotic liver disease (MASLD) has attracted increased clinical attention. However, most of those current studies involve cross-sectional studies and meta-analyses, and experimental mechanistic exploration still needs to be improved. This study aimed to investigate the mechanisms by which H. pylori impacts MASLD. METHODS: We established two H. pylori-infected (Cag A positive and Cag A negative) mouse models with 16 weeks of chow diet (CD) or high-fat diet (HFD) feeding. Body weight, liver triglyceride, blood glucose, serum biochemical parameters, inflammatory factors, and insulin resistance were measured, and histological analysis of liver tissues was performed. Mouse livers were subjected to transcriptome RNA sequencing analysis. RESULTS: Although H. pylori infection could not significantly affect serum inflammatory factor levels and serum biochemical parameters in mice, serum insulin and homeostatic model assessment for insulin resistance levels increased in CD mode. In contrast, H. pylori Cag A + infection significantly aggravated hepatic pathological steatosis induced by HFD and elevated serum inflammatory factors and lipid metabolism parameters. Hepatic transcriptomic analysis in the CD groups revealed 767 differentially expressed genes (DEGs) in the H. pylori Cag A + infected group and 1473 DEGs in the H. pylori Cag A- infected group, and the "nonalcoholic fatty liver disease" pathway was significantly enriched in KEGG analysis. There were 578 DEGs in H. pylori Cag A + infection combined with the HFD feeding group and 820 DEGs in the H. pylori Cag A- infected group. DEGs in the HFD groups were significantly enriched in "fatty acid degradation" and "PPAR pathway." Exploring the effect of different Cag A statuses on mouse liver revealed that fatty acid binding protein 5 was differentially expressed in Cag A- H. pylori. DEG enrichment pathways were concentrated in the "PPAR pathway" and "fatty acid degradation." CONCLUSIONS: Clinicians are expected to comprehend the impact of H. pylori on MASLD and better understand and manage MASLD. H. pylori infection may exacerbate the development of MASLD by regulating hepatic lipid metabolism, and the H. pylori virulence factor Cag A plays a vital role in this regulation.

Impacts of liver macrophages, gut microbiota, and bile acid metabolism on the differences in iHFC diet-induced MASH progression between TSNO and TSOD mice.

BACKGROUND: Tsumura-Suzuki non-obese (TSNO) mice exhibit a severe form of metabolic dysfunction-associated steatohepatitis (MASH) with advanced liver fibrosis upon feeding a high-fat/cholesterol/cholate-based (iHFC) diet. Another ddY strain, Tsumura-Suzuki diabetes obese (TSOD) mice, are impaired in the progression of iHFC diet-induced MASH. AIM: To elucidate the underlying mechanisms contributing to the differences in MASH progression between TSNO and TSOD mice. METHODS: We analyzed differences in the immune system, gut microbiota, and bile acid metabolism in TSNO and TSOD mice fed with a normal diet (ND) or an iHFC diet. RESULTS: TSOD mice had more anti-inflammatory macrophages in the liver than TSNO mice under ND feeding, and were impaired in the iHFC diet-induced accumulation of fibrosis-associated macrophages and formation of histological hepatic crown-like structures in the liver. The gut microbiota of TSOD mice also exhibited a distinct community composition with lower diversity and higher abundance of Akkermansia muciniphila compared with that in TSNO mice. Finally, TSOD mice had lower levels of bile acids linked to intestinal barrier disruption under iHFC feeding. CONCLUSIONS: The dynamics of liver macrophage subsets, and the compositions of the gut microbiota and bile acids at steady state and post-onset of MASH, had major impacts on MASH development.

Nutritional Interventions, Probiotics, Synbiotics and Fecal Microbiota Transplantation in Steatotic Liver Disease : Pediatric Fatty Liver and Probiotics.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a major health problem worldwide, and the strongest determinant of liver disease in children. The possible influence of high-fat/low-fiber dietary patterns with microbiota (e.g., increased Firmicutes/Bacteroidetes ratio), and ultimately with MASLD occurrence and progression has been elucidated by several association studies. The possible mechanisms through which microbes exert their detrimental effects on MASLD include gut vascular barrier damage, a shift towards non-tolerogenic immunologic environment, and the detrimental metabolic changes, including a relative reduction of propionate and butyrate in favor of acetate, endogenous ethanol production, and impairment of the unconjugated bile acid-driven FXR-mediated gut-liver axis. The impact of nutritional and probiotic interventions in children with MASLD is described.

Alteration of Gut Microbiota Composition in the Progression of Liver Damage in Patients with Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD).

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a complex disorder whose prevalence is rapidly growing in South America. The disturbances in the microbiota-gut-liver axis impact the liver damaging processes toward fibrosis. Gut microbiota status is shaped by dietary and lifestyle factors, depending on geographic location. We aimed to identify microbial signatures in a group of Chilean MASLD patients. Forty subjects were recruited, including healthy controls (HCs), overweight/obese subjects (Ow/Ob), patients with MASLD without fibrosis (MASLD/F-), and MASLD with fibrosis (MASLD/F+). Both MASLD and fibrosis were detected through elastography and/or biopsy, and fecal microbiota were analyzed through deep sequencing. Despite no differences in α- and ß-diversity among all groups, a higher abundance of Bilophila and a lower presence of Defluviitaleaceae, Lachnospiraceae ND3007, and Coprobacter was found in MASLD/F- and MASLD/F+, compared to HC. Ruminococcaceae UCG-013 and Sellimonas were more abundant in MASLD/F+ than in Ow/Ob; both significantly differed between MASLD/F- and MASLD/F+, compared to HC. Significant positive correlations were observed between liver stiffness and Bifidobacterium, Prevotella, Sarcina, and Acidaminococcus abundance. Our results show that MASLD is associated with changes in bacterial taxa that are known to be involved in bile acid metabolism and SCFA production, with some of them being more specifically linked to fibrosis.

Atorvastatin protects against liver and vascular damage in a model of diet induced steatohepatitis by resetting FXR and GPBAR1 signaling.

Farnesoid-x-receptor (FXR) agonists, currently trialed in patients with non-alcoholic steatosis (NAFLD), worsen the pro-atherogenic lipid profile and might require a comedication with statin. Here we report that mice feed a high fat/high cholesterol diet (HFD) are protected from developing a pro-atherogenic lipid profile because their ability to dispose cholesterol through bile acids. This protective mechanism is mediated by suppression of FXR signaling in the liver by muricholic acids (MCAs) generated in mice from chenodeoxycholic acid (CDCA). In contrast to CDCA, MCAs are FXR antagonists and promote a CYP7A1-dependent increase of bile acids synthesis. In mice feed a HFD, the treatment with obeticholic acid, a clinical stage FXR agonist, failed to improve the liver histopathology while reduced Cyp7a1 and Cyp8b1 genes expression and bile acids synthesis and excretion. In contrast, treating mice with atorvastatin mitigated liver and vascular injury caused by the HFD while increased the bile acids synthesis and excretion. Atorvastatin increased the percentage of 7α-dehydroxylase expressing bacteria in the intestine promoting the formation of deoxycholic acid and litocholic acid, two GPBAR1 agonists, along with the expression of GPBAR1-regulated genes in the white adipose tissue and colon. In conclusion, present results highlight the central role of bile acids in regulating lipid and cholesterol metabolism in response to atorvastatin and provide explanations for limited efficacy of FXR agonists in the treatment of NAFLD.

Influence of Gut Microbiota and its Metabolites on Progression of Metabolic Associated Fatty Liver Disease.

Metabolic associated fatty liver disease (MAFLD) has become a prevalent chronic liver disease worldwide because of lifestyle and dietary changes. Gut microbiota and its metabolites have been shown to play a critical role in the pathogenesis of MAFLD. Understanding of the function of gut microbiota and its metabolites in MAFLD may help to elucidate pathological mechanisms, identify diagnostic markers, and develop drugs or probiotics for the treatment of MAFLD. Here we review the pathogenesis of MAFLD by gut microbiota and its metabolites and discuss the feasibility of treating MAFLD from the perspective of gut microbes.

Microbiomics, Metabolomics, Predicted Metagenomics, and Hepatic Steatosis in a Population-Based Study of 1,355 Adults.

BACKGROUND AND AIMS: Previous small studies have appraised the gut microbiome (GM) in steatosis, but large-scale studies are lacking. We studied the association of the GM diversity and composition, plasma metabolites, predicted functional metagenomics, and steatosis. APPROACH AND RESULTS: This is a cross-sectional analysis of the prospective population-based Rotterdam Study. We used 16S ribosomal RNA gene sequencing and determined taxonomy using the SILVA reference database. Alpha diversity and beta diversity were calculated using the Shannon diversity index and Bray-Curtis dissimilarities. Differences were tested across steatosis using permutational multivariate analysis of variance. Hepatic steatosis was diagnosed by ultrasonography. We subsequently selected genera using regularized regression. The functional metagenome was predicted based on the GM using Kyoto Encyclopedia of Genes and Genomes pathways. Serum metabolomics were assessed using high-throughput proton nuclear magnetic resonance. All analyses were adjusted for age, sex, body mass index, alcohol, diet, and proton-pump inhibitors. We included 1,355 participants, of whom 472 had steatosis. Alpha diversity was lower in steatosis (P = 1.1∙10-9 ), and beta diversity varied across steatosis strata (P = 0.001). Lasso selected 37 genera of which three remained significantly associated after adjustment (Coprococcus3: ß = -65; Ruminococcus Gauvreauiigroup: ß = 62; and Ruminococcus Gnavusgroup: ß = 45, Q-value = 0.037). Predicted metagenome analyses revealed that pathways of secondary bile-acid synthesis and biotin metabolism were present, and D-alanine metabolism was absent in steatosis. Metabolic profiles showed positive associations for aromatic and branched chain amino acids and glycoprotein acetyls with steatosis and R. Gnavusgroup, whereas these metabolites were inversely associated with alpha diversity and Coprococcus3. CONCLUSIONS: We confirmed, on a large-scale, the lower microbial diversity and association of Coprococcus and Ruminococcus Gnavus with steatosis. We additionally showed that steatosis and alpha diversity share opposite metabolic profiles.

Obesity-associated deficits in inhibitory control are phenocopied to mice through gut microbiota changes in one-carbon and aromatic amino acids metabolic pathways.

BACKGROUND: Inhibitory control (IC) is critical to keep long-term goals in everyday life. Bidirectional relationships between IC deficits and obesity are behind unhealthy eating and physical exercise habits. METHODS: We studied gut microbiome composition and functionality, and plasma and faecal metabolomics in association with cognitive tests evaluating inhibitory control (Stroop test) and brain structure in a discovery (n=156), both cross-sectionally and longitudinally, and in an independent replication cohort (n=970). Faecal microbiota transplantation (FMT) in mice evaluated the impact on reversal learning and medial prefrontal cortex (mPFC) transcriptomics. RESULTS: An interplay among IC, brain structure (in humans) and mPFC transcriptomics (in mice), plasma/faecal metabolomics and the gut metagenome was found. Obesity-dependent alterations in one-carbon metabolism, tryptophan and histidine pathways were associated with IC in the two independent cohorts. Bacterial functions linked to one-carbon metabolism (thyX,dut, exodeoxyribonuclease V), and the anterior cingulate cortex volume were associated with IC, cross-sectionally and longitudinally. FMT from individuals with obesity led to alterations in mice reversal learning. In an independent FMT experiment, human donor's bacterial functions related to IC deficits were associated with mPFC expression of one-carbon metabolism-related genes of recipient's mice. CONCLUSION: These results highlight the importance of targeting obesity-related impulsive behaviour through the induction of gut microbiota shifts.

Minor Changes in the Composition and Function of the Gut Microbiota During a 12-Week Whole Grain Wheat or Refined Wheat Intervention Correlate with Liver Fat in Overweight and Obese Adults.

BACKGROUND: Whole grain wheat (WGW) products are advocated as a healthy choice when compared with refined wheat (RW). One proposed mechanism for these health benefits is via the microbiota, because WGW contains multiple fibers. WGW consumption has been proposed to ameliorate nonalcoholic fatty liver disease, in which microbiota might play a role. OBJECTIVES: We investigated the effect of WGW compared with RW intervention on the fecal microbiota composition and functionality, and correlated intervention-induced changes in bacteria with changes in liver health parameters in adults with overweight or obesity. METHODS: We used data of a 12-wk double-blind, randomized, controlled, parallel trial to examine the effects of a WGW (98 g/d) or RW (98 g/d) intervention on the secondary outcomes fecal microbiota composition, predicted microbiota functionality, and stool consistency in 37 women and men (aged 45-70 y, BMI 25-35 kg/m2). The changes in microbiota composition, measured using 16S ribosomal RNA gene sequencing, after a 12-wk intervention were analyzed with nonparametric tests, and correlated with changes in liver fat and circulating concentrations of liver enzymes including alanine transaminase, aspartate transaminase, γ-glutamyltransferase, and serum amyloid A. RESULTS: The WGW intervention increased the mean (± SD) relative abundances of Ruminococcaceae_UCG-014 (baseline: 2.2 ± 4.6%, differential change over time (Δ) 0.51 ± 4.2%), Ruminiclostridium_9 (baseline: 0.065 ± 0.11%, Δ 0.054 ± 0.14%), and Ruminococcaceae_NK4A214_group (baseline: 0.37 ± 0.56%, Δ 0.17 ± 0.83%), and also the predicted pathway acetyl-CoA fermentation to butyrate II (baseline: 0.23 ± 0.062%, Δ 0.035 ± 0.059%), compared with the RW intervention (P values <0.05). A change in Ruminococcaceae_NK4A214_group was positively correlated with the change in liver fat, in both the WGW (ρ = 0.54; P = 0.026) and RW (ρ = 0.67; P = 0.024) groups. CONCLUSIONS: In middle-aged overweight and obese adults, a 12-wk WGW intervention increased the relative abundance of a number of bacterial taxa from the family Ruminococcaceae and increased predicted fermentation pathways when compared with an RW intervention. Potential protective health effects of replacement of RW by WGW on metabolic organs, such as the liver, via modulation of the microbiota, deserve further investigation.This trial was registered at clinicaltrials.gov as NCT02385149.

Maternal prebiotic supplementation reduces fatty liver development in offspring through altered microbial and metabolomic profiles in rats.

A maternal high-fat/sucrose diet, in the presence of maternal obesity, can program increased susceptibility to obesity and metabolic disease in offspring. In particular, nonalcoholic fatty liver disease risk is associated with poor maternal nutrition and obesity status, which may manifest via alterations in gut microbiota. Here, we report that in a preclinical model of diet-induced maternal obesity, maternal supplementation of a high-fat/sucrose diet with the prebiotic oligofructose improves glucose tolerance, insulin sensitivity, and hepatic steatosis in offspring following a long-term high-fat/sucrose dietary challenge compared with offspring of untreated dams. These improvements are associated with alterations in gut microbial composition and serum inflammatory profiles in early life and improvements in inflammatory and fatty-acid gene expression profiles in tissues. Serum metabolomics analysis highlights potential metabolic links between the gut microbiota and the degree of steatosis, including alterations in 1-carbon metabolism. Overall, our data suggest that maternal prebiotic intake protects offspring against hepatic steatosis and insulin resistance following 21 wk of high fat/sucrose diet, which is in part due to alterations in gut microbiota.-Paul, H. A., Collins, K. H., Nicolucci, A. C., Urbanski, S. J., Hart, D. A., Vogel, H. J., Reimer, R. A. Maternal prebiotic supplementation reduces fatty liver development in offspring through altered microbial and metabolomic profiles in rats.

The gut-liver-kidney axis: Novel regulator of fatty liver associated chronic kidney disease.

Increased interest in understanding the liver-kidney axis in health and disease during the last decade unveiled multiple recent evidence that suggested a strong association of fatty liver diseases with chronic kidney disease (CKD). Low-grade systemic inflammation is thought to be the major contributing factor to the pathogenesis of CKD associated with fatty liver. However, other contributing factors largely remained unclear, for example, gut microbiota and intestinal barrier integrity. Homeostasis of the gut microbiome is very crucial for the health of an individual. Imbalance in the gut microbiota leads to various diseases like fatty liver disease and CKD. On the contrary, disease conditions can also distinctly change gut microbiota. In this review, we propose the pathogenic role of the gut-liver-kidney axis in the development and progression of CKD associated with chronic fatty liver diseases, either non-alcoholic fatty liver disease or non-alcoholic steatohepatitis in experimental models and humans. Further, we discuss the therapeutic potential and highlight the future research directions for therapeutic targeting of the gut-liver-kidney axis.

Treatment with camu camu (Myrciaria dubia) prevents obesity by altering the gut microbiota and increasing energy expenditure in diet-induced obese mice.

OBJECTIVE: The consumption of fruits is strongly associated with better health and higher bacterial diversity in the gut microbiota (GM). Camu camu (Myrciaria dubia) is an Amazonian fruit with a unique phytochemical profile, strong antioxidant potential and purported anti-inflammatory potential. DESIGN: By using metabolic tests coupled with 16S rRNA gene-based taxonomic profiling and faecal microbial transplantation (FMT), we have assessed the effect of a crude extract of camu camu (CC) on obesity and associated immunometabolic disorders in high fat/high sucrose (HFHS)-fed mice. RESULTS: Treatment of HFHS-fed mice with CC prevented weight gain, lowered fat accumulation and blunted metabolic inflammation and endotoxaemia. CC-treated mice displayed improved glucose tolerance and insulin sensitivity and were also fully protected against hepatic steatosis. These effects were linked to increased energy expenditure and upregulation of uncoupling protein 1 mRNA expression in the brown adipose tissue (BAT) of CC-treated mice, which strongly correlated with the mRNA expression of the membrane bile acid (BA) receptor TGR5. Moreover, CC-treated mice showed altered plasma BA pool size and composition and drastic changes in the GM (eg, bloom of Akkermansia muciniphila and a strong reduction of Lactobacillus). Germ-free (GF) mice reconstituted with the GM of CC-treated mice gained less weight and displayed higher energy expenditure than GF-mice colonised with the FM of HFHS controls. CONCLUSION: Our results show that CC prevents visceral and liver fat deposition through BAT activation and increased energy expenditure, a mechanism that is dependent on the GM and linked to major changes in the BA pool size and composition.

Amelioration of metabolic syndrome by metformin associates with reduced indices of low-grade inflammation independently of the gut microbiota.

Metformin beneficially impacts several aspects of metabolic syndrome including dysglycemia, obesity, and liver dysfunction, thus making it a widely used frontline treatment for early-stage type 2 diabetes, which is associated with these disorders. Several mechanisms of action for metformin have been proposed, including that it acts as an anti-inflammatory agent, possibly as a result of its impact on intestinal microbiota. In accord with this possibility, we observed herein that, in mice with diet-induced metabolic syndrome, metformin impacts the gut microbiota by preventing its encroachment upon the host, a feature of metabolic syndrome in mice and humans. However, the ability of metformin to beneficially impact metabolic syndrome in mice was not markedly altered by reduction or elimination of gut microbiota, achieved by the use of antibiotics or germfree mice. Although reducing or eliminating microbiota by itself suppressed diet-induced dysglycemia, other features of metabolic syndrome including obesity, hepatic steatosis, and low-grade inflammation remained suppressed by metformin in the presence or absence of gut microbiota. These results support a role for anti-inflammatory activity of metformin, irrespective of gut microbiota, in driving some of the beneficial impacts of this drug on metabolic syndrome.

Endotoxin regulates matrix genes increasing reactive oxygen species generation by intercellular communication between palmitate-treated hepatocyte and stellate cell.

Previous studies have shown that gut-derived bacterial endotoxins contribute in the progression of simple steatosis to steatohepatitis, although the mechanism(s) remains inaccurate to date. As hepatic stellate cells (HSC) play a pivotal role in the accumulation of excessive extracellular matrix (ECM), leading to collagen deposition, fibrosis, and perpetuation of inflammatory response, an in vitro model was developed to investigate the crosstalk between HSC and hepatocytes (human hepatoma cell) pretreated with palmitate. Bacterial lipopolysaccharide (LPS) stimulated HSC with phosphorylation of the p38 mitogen-activated protein kinase/NF-κB pathway, while several important pro-inflammatory cytokines were upregulated in the presence of hepatocyte-HSC. Concurrently, fibrosis-related genes were regulated by palmitate and the inflammatory effect of endotoxin where cells were more exposed or sensitive to reactive oxygen species (ROS). This interaction was accompanied by increased expression of the mitochondrial master regulator, proliferator-activated receptor gamma coactivator alpha, and a cytoprotective effect of the agent N-acetylcysteine suppressing ROS production, transforming growth factor-ß1, and tissue inhibitor of metalloproteinase-1. In summary, our results demonstrate that pro-inflammatory mediators LPS-induced promote ECM rearrangement in hepatic cells transcriptionally committed to the regulation of genes encoding enzymes for fatty acid metabolism in light of differences that might require an alternative therapeutic approach targeting ROS regulation.

Reduced obesity, diabetes, and steatosis upon cinnamon and grape pomace are associated with changes in gut microbiota and markers of gut barrier.

Increasing evidence suggests that polyphenols have a significant potential in the prevention and treatment of risk factors associated with metabolic syndrome. The objective of this study was to assess the metabolic outcomes of two polyphenol-containing extracts from cinnamon bark (CBE) and grape pomace (GPE) on C57BL/6J mice fed a high-fat diet (HFD) for 8 wk. Both CBE and GPE were able to decrease fat mass gain and adipose tissue inflammation in mice fed a HFD without reducing food intake. This was associated with reduced liver steatosis and lower plasma nonesterified fatty acid levels. We also observed a beneficial effect on glucose homeostasis, as evidenced by an improved glucose tolerance and a lower insulin resistance index. These ameliorations of the overall metabolic profile were associated with a significant impact on the microbial composition, which was more profound for the GPE than for the CBE. At the genus level, Peptococcus were decreased in the CBE group. In the GPE-treated group, several key genera that have been previously found to be linked with HFD, metabolic effects, and gut barrier integrity were affected: we observed a decrease of Desulfovibrio, Lactococcus, whereas Allobaculum and Roseburia were increased. In addition, the expression of several antimicrobial peptides and tight junction proteins was increased in response to both CBE and GPE supplementation, indicating an improvement of the gut barrier function. Collectively, these data suggest that CBE and GPE can ameliorate the overall metabolic profile of mice on a high-fat diet, partly by acting on the gut microbiota.

Western Diet-Induced Dysbiosis in Farnesoid X Receptor Knockout Mice Causes Persistent Hepatic Inflammation after Antibiotic Treatment.

Patients who have liver cirrhosis and liver cancer also have reduced farnesoid X receptor (FXR). The current study analyzes the effect of diet through microbiota that affect hepatic inflammation in FXR knockout (KO) mice. Wild-type and FXR KO mice were on a control (CD) or Western diet (WD) for 10 months. In addition, both CD- and WD-fed FXR KO male mice, which had hepatic lymphocyte and neutrophil infiltration, were treated by vancomycin, polymyxin B, and Abx (ampicillin, neomycin, metronidazole, and vancomycin). Mice were subjected to morphological analysis as well as gut microbiota and bile acid profiling. Male WD-fed FXR KO mice had the most severe steatohepatitis. FXR KO also had reduced Firmicutes and increased Proteobacteria, which could be reversed by Abx. In addition, Abx eliminated hepatic neutrophils and lymphocytes in CD-fed, but not WD-fed, FXR KO mice. Proteobacteria and Bacteroidetes persisted in WD-fed FXR KO mice even after Abx treatment. Only polymyxin B could reduce hepatic lymphocytes in WD-fed FXR KO mice. The reduced hepatic inflammation by antibiotics was accompanied by decreased free and conjugated secondary bile acids as well as changes in gut microbiota. Our data revealed that Lactococcus, Lactobacillus, and Coprococcus protect the liver from inflammation.

Gut microbiota profiling of pediatric nonalcoholic fatty liver disease and obese patients unveiled by an integrated meta-omics-based approach.

There is evidence that nonalcoholic fatty liver disease (NAFLD) is affected by gut microbiota. Therefore, we investigated its modifications in pediatric NAFLD patients using targeted metagenomics and metabolomics. Stools were collected from 61 consecutive patients diagnosed with nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), or obesity and 54 healthy controls (CTRLs), matched in a case-control fashion. Operational taxonomic units were pyrosequenced targeting 16S ribosomal RNA and volatile organic compounds determined by solid-phase microextraction gas chromatography-mass spectrometry. The α-diversity was highest in CTRLs, followed by obese, NASH, and NAFL patients; and ß-diversity distinguished between patients and CTRLs but not NAFL and NASH. Compared to CTRLs, in NAFLD patients Actinobacteria were significantly increased and Bacteroidetes reduced. There were no significant differences among the NAFL, NASH, and obese groups. Overall NAFLD patients had increased levels of Bradyrhizobium, Anaerococcus, Peptoniphilus, Propionibacterium acnes, Dorea, and Ruminococcus and reduced proportions of Oscillospira and Rikenellaceae compared to CTRLs. After reducing metagenomics and metabolomics data dimensionality, multivariate analyses indicated a decrease of Oscillospira in NAFL and NASH groups and increases of Ruminococcus, Blautia, and Dorea in NASH patients compared to CTRLs. Of the 292 volatile organic compounds, 26 were up-regulated and 2 down-regulated in NAFLD patients. Multivariate analyses found that combination of Oscillospira, Rickenellaceae, Parabacteroides, Bacteroides fragilis, Sutterella, Lachnospiraceae, 4-methyl-2-pentanone, 1-butanol, and 2-butanone could discriminate NAFLD patients from CTRLs. Univariate analyses found significantly lower levels of Oscillospira and higher levels of 1-pentanol and 2-butanone in NAFL patients compared to CTRLs. In NASH, lower levels of Oscillospira were associated with higher abundance of Dorea and Ruminococcus and higher levels of 2-butanone and 4-methyl-2-pentanone compared to CTRLs. CONCLUSION: An Oscillospira decrease coupled to a 2-butanone up-regulation and increases in Ruminococcus and Dorea were identified as gut microbiota signatures of NAFL onset and NAFL-NASH progression, respectively. (Hepatology 2017;65:451-464).

Endotoxinemia contributes to steatosis, insulin resistance and atherosclerosis in chronic hepatitis C: the role of pro-inflammatory cytokines and oxidative stress.

PURPOSE: Endotoxin is a component of the outer membrane of gram-negative bacteria that live in the intestine. Endotoxinemia is reported in non-alcoholic fatty liver disease and in cirrhotic patients, causing various biological and clinical effects in the host. It is not known whether endotoxinemia occurs in chronic hepatitis C patients (CHC), therefore we evaluated the occurrence of endotoxinemia and its effect on inflammation, liver damage, insulin resistance (IR) and atherosclerosis. METHODS: Consecutive CHC patients assessed by liver biopsy were enrolled. Endotoxinemia was evaluated by LAL test. IR was estimated by HOMA-IR. Serum TNF-α, IL-8, adiponectin and MCP-1 were measured with ELISA tests. Oxidative stress was estimated by circulating IgG against malondialdehyde adducts with human serum albumin (MDA-HAS). Carotid atherosclerosis was assessed by ultrasonography. RESULTS: Endotoxinemia was found in 60% of the 126 patients enrolled. A serum level-dependent association between endotoxinemia, steatosis (p < 0.001) and HOMA-IR (p < 0.006) was observed. Patients with endotoxinemia showed significant increase in TNF-α and IL8 levels. TNF-α correlated with steatosis (p <  0.001) and HOMA-IR (p < 0.03), whereas IL8 correlated with steatosis (p =  <0.001), TNF-α (p < 0.04) and atherosclerosis (p < 0.01). The highest levels of endotoxinemia were associated with oxidative stress and a higher prevalence of carotid atherosclerosis. Multivariate logistic regression analysis showed that the independent factors associated with endotoxinemia were hepatic steatosis, HOMA-IR, IL8 and MDA-HAS. CONCLUSIONS: Endotoxinemia occurs with high frequency in CHC patients and contributes to the development of hepatic steatosis, IR and atherosclerosis through increased pro-inflammatory cytokines and oxidative stress. Anti-endotoxin treatment could be of clinical relevance.