Faecal Microbiota transplantation affects liver DNA methylation in Non-alcoholic fatty liver disease: a multi-omics approach.

Individuals with nonalcoholic fatty liver disease (NAFLD) have an altered gut microbiota composition. Moreover, hepatic DNA methylation may be altered in the state of NAFLD. Using a fecal microbiota transplantation (FMT) intervention, we aimed to investigate whether a change in gut microbiota composition relates to altered liver DNA methylation in NAFLD. Moreover, we assessed whether plasma metabolite profiles altered by FMT relate to changes in liver DNA methylation. Twenty-one individuals with NAFLD underwent three 8-weekly vegan allogenic donor (n = 10) or autologous (n = 11) FMTs. We obtained hepatic DNA methylation profiles from paired liver biopsies of study participants before and after FMTs. We applied a multi-omics machine learning approach to identify changes in the gut microbiome, peripheral blood metabolome and liver DNA methylome, and analyzed cross-omics correlations. Vegan allogenic donor FMT compared to autologous FMT induced distinct differential changes in I) gut microbiota profiles, including increased abundance of Eubacterium siraeum and potential probiotic Blautia wexlerae; II) plasma metabolites, including altered levels of phenylacetylcarnitine (PAC) and phenylacetylglutamine (PAG) both from gut-derived phenylacetic acid, and of several choline-derived long-chain acylcholines; and III) hepatic DNA methylation profiles, most importantly in Threonyl-TRNA Synthetase 1 (TARS) and Zinc finger protein 57 (ZFP57). Multi-omics analysis showed that Gemmiger formicillis and Firmicutes bacterium_CAG_170 positively correlated with both PAC and PAG. E siraeum negatively correlated with DNA methylation of cg16885113 in ZFP57. Alterations in gut microbiota composition by FMT caused widespread changes in plasma metabolites (e.g. PAC, PAG, and choline-derived metabolites) and liver DNA methylation profiles in individuals with NAFLD. These results indicate that FMTs might induce metaorganismal pathway changes, from the gut bacteria to the liver.

Self-supervised neural network improves tri-exponential intravoxel incoherent motion model fitting compared to least-squares fitting in non-alcoholic fatty liver disease.

Recent literature suggests that tri-exponential models may provide additional information and fit liver intravoxel incoherent motion (IVIM) data more accurately than conventional bi-exponential models. However, voxel-wise fitting of IVIM results in noisy and unreliable parameter maps. For bi-exponential IVIM, neural networks (NN) were able to produce superior parameter maps than conventional least-squares (LSQ) generated images. Hence, to improve parameter map quality of tri-exponential IVIM, we developed an unsupervised physics-informed deep neural network (IVIM3-NET). We assessed its performance in simulations and in patients with non-alcoholic fatty liver disease (NAFLD) and compared outcomes with bi-exponential LSQ and NN fits and tri-exponential LSQ fits. Scanning was performed using a 3.0T free-breathing multi-slice diffusion-weighted single-shot echo-planar imaging sequence with 18 b-values. Images were analysed for visual quality, comparing the bi- and tri-exponential IVIM models for LSQ fits and NN fits using parameter-map signal-to-noise ratios (SNR) and adjusted R 2. IVIM parameters were compared to histological fibrosis, disease activity and steatosis grades. Parameter map quality improved with bi- and tri-exponential NN approaches, with a significant increase in average parameter-map SNR from 3.38 to 5.59 and 2.45 to 4.01 for bi- and tri-exponential LSQ and NN models respectively. In 33 out of 36 patients, the tri-exponential model exhibited higher adjusted R 2 values than the bi-exponential model. Correlating IVIM data to liver histology showed that the bi- and tri-exponential NN outperformed both LSQ models for the majority of IVIM parameters (10 out of 15 significant correlations). Overall, our results support the use of a tri-exponential IVIM model in NAFLD. We show that the IVIM3-NET can be used to improve image quality compared to a tri-exponential LSQ fit and provides promising correlations with histopathology similar to the bi-exponential neural network fit, while generating potentially complementary additional parameters.

Duodenal Anaerobutyricum soehngenii infusion stimulates GLP-1 production, ameliorates glycaemic control and beneficially shapes the duodenal transcriptome in metabolic syndrome subjects: a randomised double-blind placebo-controlled cross-over study.

OBJECTIVE: Although gut dysbiosis is increasingly recognised as a pathophysiological component of metabolic syndrome (MetS), the role and mode of action of specific gut microbes in metabolic health remain elusive. Previously, we identified the commensal butyrogenic Anaerobutyricum soehngenii to be associated with improved insulin sensitivity in subjects with MetS. In this proof-of-concept study, we investigated the potential therapeutic effects of A. soehngenii L2-7 on systemic metabolic responses and duodenal transcriptome profiles in individuals with MetS. DESIGN: In this randomised double-blind placebo-controlled cross-over study, 12 male subjects with MetS received duodenal infusions of A. soehngenii/ placebo and underwent duodenal biopsies, mixed meal tests (6 hours postinfusion) and 24-hour continuous glucose monitoring. RESULTS: A. soehngenii treatment provoked a markedly increased postprandial excursion of the insulinotropic hormone glucagon-like peptide 1 (GLP-1) and an elevation of plasma secondary bile acids, which were positively associated with GLP-1 levels. Moreover, A. soehngenii treatment robustly shaped the duodenal expression of 73 genes, with the highest fold induction in the expression of regenerating islet-protein 1B (REG1B)-encoding gene. Strikingly, duodenal REG1B expression positively correlated with GLP-1 levels and negatively correlated with peripheral glucose variability, which was significantly diminished in the 24 hours following A. soehngenii intake. Mechanistically, Reg1B expression is induced upon sensing butyrate or bacterial peptidoglycan. Importantly, A. soehngenii duodenal administration was safe and well tolerated. CONCLUSIONS: A single dose of A. soehngenii improves peripheral glycaemic control within 24 hours; it specifically stimulates intestinal GLP-1 production and REG1B expression. Further studies are needed to delineate the specific pathways involved in REG1B induction and function in insulin sensitivity. TRIAL REGISTRATION NUMBER: NTR-NL6630.

Effect of Fecal Microbiota Transplantation Combined With Mediterranean Diet on Insulin Sensitivity in Subjects With Metabolic Syndrome.

BACKGROUND: Recent studies demonstrate that a Mediterranean diet has beneficial metabolic effects in metabolic syndrome subjects. Since we have shown that fecal microbiota transplantation (FMT) from lean donors exerts beneficial effects on insulin sensitivity, in the present trial, we investigated the potential synergistic effects on insulin sensitivity of combining a Mediterranean diet with donor FMT in subjects with metabolic syndrome. DESIGN: Twenty-four male subjects with metabolic syndrome were put on a Mediterranean diet and after a 2-week run-in phase, the subjects were randomized to either lean donor (n = 12) or autologous (n = 12) FMT. Changes in the gut microbiota composition and bacterial strain engraftment after the 2-week dietary regimens and 6 weeks post-FMT were the primary endpoints. The secondary objectives were changes in glucose fluxes (both hepatic and peripheral insulin sensitivity), postprandial plasma incretin (GLP-1) levels, subcutaneous adipose tissue inflammation, and plasma metabolites. RESULTS: Consumption of the Mediterranean diet resulted in a reduction in body weight, HOMA-IR, and lipid levels. However, no large synergistic effects of combining the diet with lean donor FMT were seen on the gut microbiota diversity after 6 weeks. Although we did observe changes in specific bacterial species and plasma metabolites, no significant beneficial effects on glucose fluxes, postprandial incretins, or subcutaneous adipose tissue inflammation were detected. CONCLUSIONS: In this small pilot randomized controlled trial, no synergistic beneficial metabolic effects of combining a Mediterranean diet with lean donor FMT on glucose metabolism were achieved. However, we observed engraftment of specific bacterial species. Future trials are warranted to test the combination of other microbial interventions and diets in metabolic syndrome.

Assessment of Imaging Modalities Against Liver Biopsy in Nonalcoholic Fatty Liver Disease: The Amsterdam NAFLD-NASH Cohort.

BACKGROUND: Noninvasive diagnostic methods are urgently required in disease stratification and monitoring in nonalcoholic fatty liver disease (NAFLD). Multiparametric magnetic resonance imaging (MRI) is a promising technique to assess hepatic steatosis, inflammation, and fibrosis, potentially enabling noninvasive identification of individuals with active and advanced stages of NAFLD. PURPOSE: To examine the diagnostic performance of multiparametric MRI for the assessment of disease severity along the NAFLD disease spectrum with comparison to histological scores. STUDY TYPE: Prospective, cohort. POPULATION: Thirty-seven patients with NAFLD. FIELD STRENGTH/SEQUENCE: Multiparametric MRI at 3.0 T consisted of magnetic resonance (MR) spectroscopy (MRS) with multi-echo stimulated-echo acquisition mode, magnitude-based and three-point Dixon using a two-dimensional multi-echo gradient echo, MR elastography (MRE) using a generalized multishot gradient-recalled echo sequence and intravoxel incoherent motion (IVIM) using a multislice diffusion weighted single-shot echo-planar sequence. ASSESSMENT: Histological steatosis grades were compared to proton density fat fraction measured by MRS (PDFFMRS ), magnitude-based MRI (PDFFMRI-M ), and three-point Dixon (PDFFDixon ), as well as FibroScan® controlled attenuation parameter (CAP). Fibrosis and disease activity were compared to IVIM and MRE. FibroScan® liver stiffness measurements were compared to fibrosis levels. Diagnostic performance of all imaging parameters was determined for distinction between simple steatosis and nonalcoholic steatohepatitis (NASH). STATISTICAL TESTS: Spearman's rank test, Kruskal-Wallis test, Dunn's post-hoc test with Holm-Bonferroni P-value adjustment, receiver operating characteristic curve analysis. A P-value <0.05 was considered statistically significant. RESULTS: Histological steatosis grade correlated significantly with PDFFMRS (rs  = 0.66, P < 0.001), PDFFMRI-M (rs  = 0.68, P < 0.001), and PDFFDixon (rs  = 0.67, P < 0.001), whereas no correlation was found with CAP. MRE and IVIM diffusion and perfusion significantly correlated with disease activity (rs  = 0.55, P < 0.001, rs  = -0.40, P = 0.016, rs  = -0.37, P = 0.027, respectively) and fibrosis (rs  = 0.55, P < 0.001, rs  = -0.46, P = 0.0051; rs  = -0.53, P < 0.001, respectively). MRE and IVIM diffusion had the highest area-under-the-curve for distinction between simple steatosis and NASH (0.79 and 0.73, respectively). DATA CONCLUSION: Multiparametric MRI is a promising method for noninvasive, accurate, and sensitive distinction between simple hepatic steatosis and NASH, as well as for the assessment of steatosis and fibrosis severity. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: 2.

Donor Fecal Microbiota Transplantation Alters Gut Microbiota and Metabolites in Obese Individuals With Steatohepatitis.

The intestinal microbiota has been linked to the development and prevalence of steatohepatitis in humans. Interestingly, steatohepatitis is significantly lower in individuals taking a plant-based, low-animal-protein diet, which is thought to be mediated by gut microbiota. However, data on causality between these observations in humans is scarce. In this regard, fecal microbiota transplantation (FMT) using healthy donors is safe and is capable of changing microbial composition in human disease. We therefore performed a double-blind randomized controlled proof-of-principle study in which individuals with hepatic steatosis on ultrasound were randomized to two study arms: lean vegan donor (allogenic n = 10) or own (autologous n = 11) FMT. Both were performed three times at 8-week intervals. A liver biopsy was performed at baseline and after 24 weeks in every subject to determine histopathology (Nonalcoholic Steatohepatitis Clinical Research Network) classification and changes in hepatic gene expression based on RNA sequencing. Secondary outcome parameters were changes in intestinal microbiota composition and fasting plasma metabolomics. We observed a trend toward improved necro-inflammatory histology, and found significant changes in expression of hepatic genes involved in inflammation and lipid metabolism following allogenic FMT. Intestinal microbial community structure changed following allogenic FMT, which was associated with changes in plasma metabolites as well as markers of . Conclusion: Allogenic FMT using lean vegan donors in individuals with hepatic steatosis shows an effect on intestinal microbiota composition, which is associated with beneficial changes in plasma metabolites and markers of steatohepatitis.

Gut microbiota and human NAFLD: disentangling microbial signatures from metabolic disorders.

Gut microbiota dysbiosis has been repeatedly observed in obesity and type 2 diabetes mellitus, two metabolic diseases strongly intertwined with non-alcoholic fatty liver disease (NAFLD). Animal studies have demonstrated a potential causal role of gut microbiota in NAFLD. Human studies have started to describe microbiota alterations in NAFLD and have found a few consistent microbiome signatures discriminating healthy individuals from those with NAFLD, non-alcoholic steatohepatitis or cirrhosis. However, patients with NAFLD often present with obesity and/or insulin resistance and type 2 diabetes mellitus, and these metabolic confounding factors for dysbiosis have not always been considered. Patients with different NAFLD severity stages often present with heterogeneous lesions and variable demographic characteristics (including age, sex and ethnicity), which are known to affect the gut microbiome and have been overlooked in most studies. Finally, multiple gut microbiome sequencing tools and NAFLD diagnostic methods have been used across studies that could account for discrepant microbiome signatures. This Review provides a broad insight into microbiome signatures for human NAFLD and explores issues with disentangling these signatures from underlying metabolic disorders. More advanced metagenomics and multi-omics studies using system biology approaches are needed to improve microbiome biomarkers.

Multimodal Positron Emission Tomography Imaging to Quantify Uptake of 89Zr-Labeled Liposomes in the Atherosclerotic Vessel Wall.

Nanotherapy has recently emerged as an experimental treatment option for atherosclerosis. To fulfill its promise, robust noninvasive imaging approaches for subject selection and treatment evaluation are warranted. To that end, we present here a positron emission tomography (PET)-based method for quantification of liposomal nanoparticle uptake in the atherosclerotic vessel wall. We evaluated a modular procedure to label liposomal nanoparticles with the radioisotope zirconium-89 (89Zr). Their biodistribution and vessel wall targeting in a rabbit atherosclerosis model was evaluated up to 15 days after intravenous injection by PET/computed tomography (CT) and PET/magnetic resonance imaging (PET/MRI). Vascular permeability was assessed in vivo using three-dimensional dynamic contrast-enhanced MRI (3D DCE-MRI) and ex vivo using near-infrared fluorescence (NIRF) imaging. The 89Zr-radiolabeled liposomes displayed a biodistribution pattern typical of long-circulating nanoparticles. Importantly, they markedly accumulated in atherosclerotic lesions in the abdominal aorta, as evident on PET/MRI and confirmed by autoradiography, and this uptake moderately correlated with vascular permeability. The method presented herein facilitates the development of nanotherapy for atherosclerotic disease as it provides a tool to screen for nanoparticle targeting in individual subjects' plaques.

Intestinal Ralstonia pickettii augments glucose intolerance in obesity.

An altered intestinal microbiota composition has been implicated in the pathogenesis of metabolic disease including obesity and type 2 diabetes mellitus (T2DM). Low grade inflammation, potentially initiated by the intestinal microbiota, has been suggested to be a driving force in the development of insulin resistance in obesity. Here, we report that bacterial DNA is present in mesenteric adipose tissue of obese but otherwise healthy human subjects. Pyrosequencing of bacterial 16S rRNA genes revealed that DNA from the Gram-negative species Ralstonia was most prevalent. Interestingly, fecal abundance of Ralstonia pickettii was increased in obese subjects with pre-diabetes and T2DM. To assess if R. pickettii was causally involved in development of obesity and T2DM, we performed a proof-of-concept study in diet-induced obese (DIO) mice. Compared to vehicle-treated control mice, R. pickettii-treated DIO mice had reduced glucose tolerance. In addition, circulating levels of endotoxin were increased in R. pickettii-treated mice. In conclusion, this study suggests that intestinal Ralstonia is increased in obese human subjects with T2DM and reciprocally worsens glucose tolerance in DIO mice.

In Vivo PET Imaging of HDL in Multiple Atherosclerosis Models.

OBJECTIVES: The goal of this study was to develop and validate a noninvasive imaging tool to visualize the in vivo behavior of high-density lipoprotein (HDL) by using positron emission tomography (PET), with an emphasis on its plaque-targeting abilities. BACKGROUND: HDL is a natural nanoparticle that interacts with atherosclerotic plaque macrophages to facilitate reverse cholesterol transport. HDL-cholesterol concentration in blood is inversely associated with risk of coronary heart disease and remains one of the strongest independent predictors of incident cardiovascular events. METHODS: Discoidal HDL nanoparticles were prepared by reconstitution of its components apolipoprotein A-I (apo A-I) and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine. For radiolabeling with zirconium-89 ((89)Zr), the chelator deferoxamine B was introduced by conjugation to apo A-I or as a phospholipid-chelator (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-deferoxamine B). Biodistribution and plaque targeting of radiolabeled HDL were studied in established murine, rabbit, and porcine atherosclerosis models by using PET combined with computed tomography (PET/CT) imaging or PET combined with magnetic resonance imaging. Ex vivo validation was conducted by radioactivity counting, autoradiography, and near-infrared fluorescence imaging. Flow cytometric assessment of cellular specificity in different tissues was performed in the murine model. RESULTS: We observed distinct pharmacokinetic profiles for the two (89)Zr-HDL nanoparticles. Both apo A-I- and phospholipid-labeled HDL mainly accumulated in the kidneys, liver, and spleen, with some marked quantitative differences in radioactivity uptake values. Radioactivity concentrations in rabbit atherosclerotic aortas were 3- to 4-fold higher than in control animals at 5 days' post-injection for both (89)Zr-HDL nanoparticles. In the porcine model, increased accumulation of radioactivity was observed in lesions by using in vivo PET imaging. Irrespective of the radiolabel's location, HDL nanoparticles were able to preferentially target plaque macrophages and monocytes. CONCLUSIONS: (89)Zr labeling of HDL allows study of its in vivo behavior by using noninvasive PET imaging, including visualization of its accumulation in advanced atherosclerotic lesions. The different labeling strategies provide insight on the pharmacokinetics and biodistribution of HDL's main components (i.e., phospholipids, apo A-I).

About the gut microbiome as a pharmacological target in atherosclerosis.

The contribution of intestinal bacterial strains (gut microbiota) in the development of cardiometabolic disease is increasingly recognized as potential diagnostic and pharmacological target. Changes in the intestinal bacterial composition and subsequent altered diversity has been associated with presence of chronic low-grade inflammation of mesenteric visceral adipose tissue, a known feature of malign obesity which can eventually lead to insulin resistance and type 2 diabetes mellitus. However, causality still needs to be proven. In this regard, both fecal transplantation studies as well as multiethnic prospective cohorts can help to identify the causally involved driving intestinal bacterial strains in human cardiometabolism. Ultimately, it is expected that novel diagnostic markers as well as therapeutics (pharmabiotics and vaccine strategies) can be developed.

Carriers of loss-of-function mutations in EXT display impaired pancreatic beta-cell reserve due to smaller pancreas volume.

Exotosin (EXT) proteins are involved in the chain elongation step of heparan sulfate (HS) biosynthesis, which is intricately involved in organ development. Loss of function mutations (LOF) in EXT1 and EXT2 result in hereditary exostoses (HME). Interestingly, HS plays a role in pancreas development and beta-cell function, and genetic variations in EXT2 are associated with an increased risk for type 2 diabetes mellitus. We hypothesized that loss of function of EXT1 or EXT2 in subjects with hereditary multiple exostoses (HME) affects pancreatic insulin secretion capacity and development. We performed an oral glucose tolerance test (OGTT) followed by hyperglycemic clamps to investigate first-phase glucose-stimulated insulin secretion (GSIS) in HME patients and age and gender matched non-affected relatives. Pancreas volume was assessed with magnetic resonance imaging (MRI). OGTT did not reveal significant differences in glucose disposal, but there was a markedly lower GSIS in HME subjects during hyperglycemic clamp (iAUC HME: 0.72 [0.46-1.16] vs. controls 1.53 [0.69-3.36] nmol·l-1·min-1, p<0.05). Maximal insulin response following arginine challenge was also significantly attenuated (iAUC HME: 7.14 [4.22-10.5] vs. controls 10.2 [7.91-12.70] nmol·l-1·min-1 p<0.05), indicative of an impaired beta-cell reserve. MRI revealed a significantly smaller pancreatic volume in HME subjects (HME: 72.0±15.8 vs. controls 96.5±26.0 cm3 p = 0.04). In conclusion, loss of function of EXT proteins may affect beta-cell mass and insulin secretion capacity in humans, and render subjects at a higher risk of developing type 2 diabetes when exposed to environmental risk factors.

NADP+ -dependent IDH1 R132 mutation and its relevance for glioma patient survival.

The isocitrate dehydrogenase 1 (IDH1) mutation occurs in high frequency in glioma and secondary glioblastoma (GBM). Mutated IDH1 produces the oncometabolite 2-hydroxyglutarate rather than α-ketoglutarate or isocitrate. The oncometabolite is considered to be the major cause of the association between the IDH1 mutation and gliomagenesis. On the other hand, the IDH1 mutation in GBM is associated with prolonged patient survival. This association is not well understood yet but IDH1 involvement in epigenetic silencing of O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme is considered to be an important mechanism. However, it was shown recently that the IDH1 mutation and MGMT silencing are independent prognostic factors. Here, we hypothesize that the IDH1 mutation reduces the capacity to produce NADPH and thus reduces the capacity to scavenge reactive oxygen species that are generated during irradiation and chemotherapy. IDH1 activity is responsible for two-thirds of the NADPH production capacity in normal brain, whereas the IDH1 mutation reduces this capacity by almost 40%. Therefore, we hypothesize that the reduced NADPH production capacity due to the IDH1 mutation renders GBM cells more vulnerable to irradiation and chemotherapy thus prolonging survival of the patients.