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Bile acids are hepatic metabolites and have many properties considered to be relevant to the pathophysiology of NAFLD. Circulating levels of the intestinal microbiome-modified bile acid deoxycholate are increased in cirrhosis.To further elucidate the role of bile acids and intestinal microbiota linked to bile acids in progressively severe NAFLD, a multiomic study of feces including 16S rRNA sequencing, microbial transcriptomics and metabolomics was performed in a cohort with varying phenotypes of NAFLD. Several bile acids of microbial origin derived from deoxycholic acid (DCA) (glycodeoxycholate, 7-ketodeoxycholic acid, dehydrocholic acid) increased with disease activity and fibrosis stage. These were linked to increased expression of microbial bile salt hydrolase, bile acid operon (BaiCD) and hydroxysteroid dehydrogenases (hdhA) required for DCA and downstream metabolite synthesis providing a mechanistic basis for altered bile acid profiles with disease progression. Bacteroidetes and several genera of Lachnospiraceae family containing DCA generating genes increased with increasing disease severity, whereas several potentially beneficial microbes sensitive to antibacterial effects of DCA e.g., Ruminococcaceae were decreased. The clinical relevance of these data was confirmed in an independent cohort enrolled in a clinical trial for NASH where at entry DCA and its conjugates were associated with advanced fibrosis. In patients treated with placebo, DCA declined in those with fibrosis regression and increased in those with fibrosis progression. DCA rose further in those with compensated cirrhosis when they experienced decompensation.These findings demonstrate a role for bile acids and the bile acid dependent microbiome in the development and progression of NAFLD and set the stage to leverage these findings for NASH biomarker development and for therapeutics.

Background and aims Alcohol use disorder (AUD) is associated with microbial alterations that worsen with cirrhosis. Fecal microbiota transplant (FMT) could be a promising approach. Approach and results In this phase 1, double-blind, randomized clinical trial, patients with AUD-related cirrhosis with problem drinking (AUDIT-10 > 8) were randomized 1:1 into receiving one placebo or FMT enema from a donor enriched in Lachnospiraceae and Ruminococcaceae. Six-month safety was the primary outcome. Alcohol craving questionnaire, alcohol consumption (urinary ethylglucuronide/creatinine), quality of life, cognition, serum IL-6 and lipopolysaccharide-binding protein, plasma/stool short-chain fatty acids (SCFAs), and stool microbiota were tested at baseline and day 15. A 6-month follow-up with serious adverse event (SAE) analysis was performed. Twenty patients with AUD-related cirrhosis (65 ± 6.4 years, all men, Model for End-Stage Liver Disease 8.9 ± 2.7) with similar demographics, cirrhosis, and AUD severity were included. Craving reduced significantly in 90% of FMT versus 30% in placebo at day 15 (P = 0.02) with lower urinary ethylglucuronide/creatinine (P = 0.03) and improved cognition and psychosocial quality of life. There was reduction in serum IL-6 and lipopolysaccharide-binding protein and increased butyrate/isobutyrate compared with baseline in FMT but not placebo. Microbial diversity increased with higher Ruminococcaceae and other SCFAs, producing taxa following FMT but not placebo, which were linked with SCFA levels. At 6 months, patients with any SAEs (8 vs. 2, P = 0.02), AUD-related SAEs (7 vs. 1, P = 0.02), and SAEs/patient (median [interquartile range], 1.5 [1.25] vs. 0 [0.25] in FMT, P = 0.02) were higher in placebo versus FMT. Conclusions This phase 1 trial shows that FMT is safe and associated with short-term reduction in alcohol craving and consumption with favorable microbial changes versus placebo in patients with alcohol-associated cirrhosis with alcohol misuse. There was also a reduction in AUD-related events over 6 months in patients assigned to FMT.

BACKGROUND AND AIMS The role of the intestinal microbiome in alcoholic hepatitis is not established. The aims of this study were to (1) characterize the fecal microbial ecology associated with alcoholic hepatitis, (2) relate microbiome changes to disease severity, and (3) infer the functional relevance of shifts in microbial ecology. APPROACH AND RESULTS The fecal microbiome in patients with moderate alcoholic hepatitis (MAH) or severe alcoholic hepatitis (SAH) was compared with healthy controls (HCs) and heavy drinking controls (HDCs). Microbial taxa were identified by 16S pyrosequencing. Functional metagenomics was performed using PICRUSt. Fecal short chain fatty acids (SCFAs) were measured using a liquid chromatography-mass spectrometry platform. A total of 78 participants (HC, n = 24; HDC, n = 20; MAH, n = 10; SAH, n = 24) were studied. HDC had a distinct signature compared with HC with depletion of Bacteroidetes (46% vs. 26%; P = 0.01). Alcoholic hepatitis was associated with a distinct microbiome signature compared with HDC (area under the curve = 0.826); differential abundance of Ruminococcaceae, Veillonellaceae, Lachnospiraceae, Porphyromonadaceae, and Rikenellaceae families were the key contributors to these differences. The beta diversity was significantly different among the groups (permutational multivariate analysis of variance [PERMANOVA] P

The histologic spectrum of nonalcoholic fatty liver disease (NAFLD) includes fatty liver (NAFL) and steatohepatitis (NASH), which can progress to cirrhosis in up to 20% NASH patients. Bile acids (BA) are linked to pathogenesis and therapy of NASH. We (a) characterized plasma BA profile in biopsy proven NAFL and NASH, and compared to controls, and (b) related plasma BA profile to liver histologic features, disease activity and fibrosis. Liquid chromatography/mass spectrometry quantified BAs. Descriptive statistics, paired and multiple group comparisons, and regression analyses were performed. Eighty six patients (24 controls, 25 NAFL and 37 NASH, mean age 51.8 years and BMI 31.9 kg/m2), 66% were women. Increased total primary BAs and decreased secondary BAs (both p

The multidrug resistance 2 knockout (Mdr2-/- ) mouse is a well-established model of cholestatic cholangiopathies. Female Mdr2-/- mice develop more severe hepatobiliary damage than male Mdr2-/- mice, which is correlated with a higher proportion of taurocholate in the bile. Although estrogen has been identified as an important player in intrahepatic cholestasis, the underlying molecular mechanisms of gender-based disparity of cholestatic injury remain unclear. The long noncoding RNA H19 is an imprinted, maternally expressed, and estrogen-targeted gene, which is significantly induced in human fibrotic/cirrhotic liver and bile duct-ligated mouse liver. However, whether aberrant expression of H19 accounts for gender-based disparity of cholestatic injury in Mdr2-/- mice remains unknown. The current study demonstrated that H19 was markedly induced (∼200-fold) in the livers of female Mdr2-/- mice at advanced stages of cholestasis (100 days old) but not in age-matched male Mdr2-/- mice. During the early stages of cholestasis, H19 expression was minimal. We further determined that hepatic H19 was mainly expressed in cholangiocytes, not hepatocytes. Both taurocholate and estrogen significantly activated the extracellular signal-regulated kinase 1/2 signaling pathway and induced H19 expression in cholangiocytes. Knocking down H19 not only significantly reduced taurocholate/estrogen-induced expression of fibrotic genes and sphingosine 1-phosphate receptor 2 in cholangiocytes but also markedly reduced cholestatic injury in female Mdr2-/- mice. Furthermore, expression of small heterodimer partner was substantially inhibited at advanced stages of liver fibrosis, which was reversed by H19 short hairpin RNA in female Mdr2-/- mice. Similar findings were obtained in human primary sclerosing cholangitis liver samples. Conclusion H19 plays a critical role in the disease progression of cholestasis and represents a key factor that causes the gender disparity of cholestatic liver injury in Mdr2-/- mice. (Hepatology 2017;66:869-884).

Specific alterations in hepatic lipid composition characterize the spectrum of nonalcoholic fatty liver disease (NAFLD), which extends from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). However, the plasma lipidome of NAFLD and whether NASH has a distinct plasma lipidomic signature are unknown. A comprehensive analysis of plasma lipids and eicosanoid metabolites quantified by mass spectrometry was performed in NAFL (n = 25) and NASH (n = 50) subjects and compared with lean normal controls (n = 50). The key findings include significantly increased total plasma monounsaturated fatty acids driven by palmitoleic (16:1 n7) and oleic (18:1 n9) acids content (P < 0.01 for both acids in both NAFL and NASH). The levels of palmitoleic acid, oleic acid, and palmitoleic acid to palmitic acid (16:0) ratio were significantly increased in NAFLD across multiple lipid classes. Linoleic acid (8:2n6) was decreased (P < 0.05), with a concomitant increase in γ-linolenic (18:3n6) and dihomo γ-linolenic (20:3n6) acids in both NAFL and NASH (P < 0.001 for most lipid classes). The docosahexanoic acid (22:6 n3) to docosapentenoic acid (22:5n3) ratio was significantly decreased within phosphatidylcholine (PC), and phosphatidylethanolamine (PE) pools, which was most marked in NASH subjects (P < 0.01 for PC and P < 0.001 for PE). The total plasmalogen levels were significantly decreased in NASH compared with controls (P < 0.05). A stepwise increase in lipoxygenase (LOX) metabolites 5(S)-hydroxyeicosatetraenoic acid (5-HETE), 8-HETE, and 15-HETE characterized progression from normal to NAFL to NASH. The level of 11-HETE, a nonenzymatic oxidation product of arachidonic (20:4) acid, was significantly increased in NASH only. Conclusions: Although increased lipogenesis, desaturases, and LOX activities characterize NAFL and NASH, impaired peroxisomal polyunsaturated fatty acid (PUFA) metabolism and nonenzymatic oxidation is associated with progression to NASH.

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes a nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). The specific types and amounts of lipids that accumulate in NAFLD are not fully defined. The free fatty acid (FFA), diacylglycerol (DAG), triacylglycerol (TAG), free cholesterol (FC), cholesterol ester, and phospholipid contents in normal livers were quantified and compared to those of NAFL and NASH, and the distribution of fatty acids within these classes was compared across these groups. Hepatic lipids were quantified by capillary gas chromatography. The mean (nmol/g of tissue) DAG (normal/NAFL/NASH: 1922 versus 4947 versus 3304) and TAG (13,609 versus 128,585 versus 104,036) increased significantly in NAFLD, but FFA remained unaltered (5533 versus 5929 versus 6115). There was a stepwise increase in the mean TAG/DAG ratio from normal livers to NAFL to NASH (7 versus 26 versus 31, P0.001). There was also a similar stepwise increment in hepatic FC (7539 versus 10,383 versus 12,863, P0.05 for NASH). The total phosphatidylcholine (PC) decreased in both NAFL and NASH. The FC/PC ratio increased progressively (0.34 versus 0.69 versus 0.71, P0.008 for both). Although the levels for linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) remained unaltered, there was a decrease in arachidonic acid (20:4n-6) in FFA, TAG, and PC (P0.05 for all) in NASH. Eicosapentanoic acid (20:5n-3) and docosahexanoic acid (22:6n-3) were decreased in TAG in NASH. The n-6:n-3 FFA ratio increased in NASH (P0.05).NAFLD is associated with numerous changes in the lipid composition of the liver. The potential implications are discussed.

The patterns of fat distribution and their relationship to severity of nonalcoholic fatty liver disease (NAFLD) are unknown. The objectives of this study were to define the fat distribution patterns and their relationship to histological severity and metabolic parameters in subjects with NAFLD. Anthropometric indices and total body fat were measured in 123 subjects. Fat distribution patterns were defined as: general, abdominal, limb, truncal, and dorsocervical lipohypertrophy (DCL) a novel finding in NAFLD. Eighty-one (66%) of the subjects were obese, and 94 (76%) had abdominal obesity. Thirty-five (28.5%) had DCL. Whereas body mass index (BMI) correlated best with the presence of diabetes (r 0.22, P < 0.05), waist circumference (WC) correlated best with hypertension (r 0.2, P < 0.05), hypertriglyceridemia (r 0.37, P < 0.001), and insulin resistance (homeostasis model of assessment for insulin resistance [r 0.68, P < 0.0001]). None of the patterns of fat distribution were significantly associated with severity of hepatic steatosis. Abdominal obesity (WC) correlated with inflammation (r 0.2, P < 0.05) only. DCL correlated significantly with the severity of all histological parameters except steatosis. Whereas DCL was the single greatest contributor to the variability in severity of histological parameters, a model combining BMI, WC, and DCL showed the greatest contribution to the variability in severity of individual histological parameters. The addition of steatosis grade to the model significantly increased its contribution to the range of lobular inflammation. Conclusion: WC predicts metabolic risk profile with the most significance. However, DCL is most strongly associated with severity of steatohepatitis. WC and BMI added modestly to the contribution of DCL to severity of nonalcoholic steatohepatitis. (HEPATOLOGY 2007;46:1091-1100.)

The fasting-activated longevity protein sirtuin 1 (SirT1, ref. 1) promotes gluconeogenesis in part, by increasing transcription of the key gluconeogenic genes pepck1 and g6pase2,3, through deacetylating PGC-1α and FOXO1 (ref. 4). In contrast, signal transducer and activator of transcription 3 (STAT3) inhibits glucose production by suppressing expression of these genes5,6. It is not known whether the inhibition of gluconeogenesis by STAT3 is controlled by metabolic regulation. Here we show that STAT3 phosphorylation and function in the liver were tightly regulated by the nutritional status of an animal, through SirT1-mediated deacetylation of key STAT3 lysine sites. The importance of the SirT1-STAT3 pathway in the regulation of gluconeogenesis was verified in STAT3-deficient mice in which the dynamic regulation of gluconeogenic genes by nutritional status was disrupted. Our results reveal a new nutrient sensing pathway through which SirT1 suppresses the inhibitory effect of STAT3, while activating the stimulatory effect of PGC-1α and FOXO1 on gluconeogenesis, thus ensuring maximal activation of gluconeogenic gene transcription. The connection between acetylation and phosphorylation of STAT3 implies that STAT3 may have an important role in other cellular processes that involve SirT1.