Microbially Produced Imidazole Propionate Impairs Insulin Signaling through mTORC1.

Interactions between the gut microbiota, diet, and the host potentially contribute to the development of metabolic diseases. Here, we identify imidazole propionate as a microbially produced histidine-derived metabolite that is present at higher concentrations in subjects with versus without type 2 diabetes. We show that imidazole propionate is produced from histidine in a gut simulator at higher concentrations when using fecal microbiota from subjects with versus without type 2 diabetes and that it impairs glucose tolerance when administered to mice. We further show that imidazole propionate impairs insulin signaling at the level of insulin receptor substrate through the activation of p38γ MAPK, which promotes p62 phosphorylation and, subsequently, activation of mechanistic target of rapamycin complex 1 (mTORC1). We also demonstrate increased activation of p62 and mTORC1 in liver from subjects with type 2 diabetes. Our findings indicate that the microbial metabolite imidazole propionate may contribute to the pathogenesis of type 2 diabetes.

Novel Proteome Targets Marking Insulin Resistance in Metabolic Syndrome.

CONTEXT/OBJECTIVE: In order to better understand which metabolic differences are related to insulin resistance in metabolic syndrome (MetSyn), we used hyperinsulinemic-euglycemic (HE) clamps in individuals with MetSyn and related peripheral insulin resistance to circulating biomarkers. DESIGN/METHODS: In this cross-sectional study, HE-clamps were performed in treatment-naive men (n = 97) with MetSyn. Subjects were defined as insulin-resistant based on the rate of disappearance (Rd). Machine learning models and conventional statistics were used to identify biomarkers of insulin resistance. Findings were replicated in a cohort with n = 282 obese men and women with (n = 156) and without (n = 126) MetSyn. In addition to this, the relation between biomarkers and adipose tissue was assessed by nuclear magnetic resonance imaging. RESULTS: Peripheral insulin resistance is marked by changes in proteins related to inflammatory processes such as IL-1 and TNF-receptor and superfamily members. These proteins can distinguish between insulin-resistant and insulin-sensitive individuals (AUC = 0.72 ± 0.10) with MetSyn. These proteins were also associated with IFG, liver fat (rho 0.36, p = 1.79 × 10-9) and visceral adipose tissue (rho = 0.35, p = 6.80 × 10-9). Interestingly, these proteins had the strongest association in the MetSyn subgroup compared to individuals without MetSyn. CONCLUSIONS: MetSyn associated with insulin resistance is characterized by protein changes related to body fat content, insulin signaling and pro-inflammatory processes. These findings provide novel targets for intervention studies and should be the focus of future in vitro and in vivo studies.

Intestinal permeability is associated with aggravated inflammation and myofibroblast accumulation in Graves' orbitopathy: the MicroGO study.

Background: Graves' disease (GD) and Graves' orbitopathy (GO) result from ongoing stimulation of the TSH receptor due to autoantibodies acting as persistent agonists. Orbital pre-adipocytes and fibroblasts also express the TSH receptor, resulting in expanded retro-orbital tissue and causing exophthalmos and limited eye movement. Recent studies have shown that GD/GO patients have a disturbed gut microbiome composition, which has been associated with increased intestinal permeability. This study hypothesizes that enhanced intestinal permeability may aggravate orbital inflammation and, thus, increase myofibroblast differentiation and the degree of fibrosis. Methods: Two distinct cohorts of GO patients were studied, one of which was a unique cohort consisting of blood, fecal, and retro-orbital tissue samples. Intestinal permeability was assessed by measuring serum lipopolysaccharide-binding protein (LBP), zonulin, TLR5, and TLR9 ligands. The influx of macrophages and accumulation of T-cells and myofibroblast were quantified in orbital connective tissue. The NanoString immune-oncology RNA targets panel was used to determine the transcriptional profile of active fibrotic areas within orbital sections. Results: GO patients displayed significantly higher LBP serum concentrations than healthy controls. Within the MicroGO cohort, patients with high serum LBP levels also showed higher levels of zonulin and TLR5 and TLR9 ligands in their circulation. The increased intestinal permeability was accompanied by augmented expression of genes marking immune cell infiltration and encoding key proteins for immune cell adhesion, antigen presentation, and cytokine signaling in the orbital tissue. Macrophage influx was positively linked to the extent of T cell influx and fibroblast activation within GO-affected orbital tissues. Moreover, serum LBP levels significantly correlated with the abundance of specific Gram-negative gut bacteria, linking the gut to local orbital inflammation. Conclusion: These results indicate that GO patients have enhanced intestinal permeability. The subsequent translocation of bacterial compounds to the systemic circulation may aggravate inflammatory processes within the orbital tissue and, as a consequence, augment the proportion of activated myofibroblasts, which actively secrete extracellular matrix leading to retro-orbital tissue expansion. These findings warrant further exploration to assess the correlation between specific inflammatory pathways in the orbital tissue and the gut microbiota composition and may pave the way for new microbiota-targeting therapies.

Species- and strain-level assessment using rrn long-amplicons suggests donor's influence on gut microbial transference via fecal transplants in metabolic syndrome subjects.

Fecal microbiota transplantation (FMT) is currently used for treating Clostridium difficile infection and explored for other clinical applications in experimental trials. However, the effectiveness of this therapy could vary, and partly depend on the donor's bacterial species engraftment, whose evaluation is challenging because there are no cost-effective strategies for accurately tracking the microbe transference. In this regard, the precise identification of bacterial species inhabiting the human gut is essential to define their role in human health unambiguously. We used Nanopore-based device to sequence bacterial rrn operons (16S-ITS-23S) and to reveal species-level abundance changes in the human gut microbiota of a FMT trial. By assessing the donor and recipient microbiota before and after FMT, we further evaluated whether this molecular approach reveals strain-level genetic variation to demonstrate microbe transfer and engraftment. Strict control over sequencing data quality and major microbiota covariates was critical for accurately estimating the changes in gut microbial species abundance in the recipients after FMT. We detected strain-level variation via single-nucleotide variants (SNVs) at rrn regions in a species-specific manner. We showed that it was possible to explore successfully the donor-bacterial strain (e.g., Parabacteroides merdae) engraftment in recipients of the FMT by assessing the nucleotide frequencies at rrn-associated SNVs. Our findings indicate that the engraftment of donors' microbiota is to some extent correlated with the improvement of metabolic health in recipients and that parameters such as the baseline gut microbiota configuration, sex, and age of donors should be considered to ensure the success of FMT in humans. The study was prospectively registered at the Dutch Trial registry - NTR4488 (https://www.trialregister.nl/trial/4488).

Short-chain fatty acids and bile acids in human faeces are associated with the intestinal cholesterol conversion status.

BACKGROUND AND PURPOSE: The analysis of human faecal metabolites can provide an insight into metabolic interactions between gut microbiota and the host organism. The creation of metabolic profiles in faeces has received little attention until now, and reference values, especially in the context of dietary and therapeutic interventions, are missing. Exposure to xenobiotics significantly affects the physiology of the microbiome, and microbiota manipulation and short-chain fatty acid administration have been proposed as treatment targets for several diseases. The aim of the present study is to give concomitant concentration ranges of faecal sterol species, bile acids and short-chain fatty acids, based on a large cohort. EXPERIMENTAL APPROACH: Sterol species, bile acids and short-chain fatty acids in human faeces from 165 study participants were quantified by LC-MS/MS. For standardization, we refer all values to dry weight of faeces. Based on the individual intestinal sterol conversion, we classified participants into low and high converters according to their coprostanol/cholesterol ratio. KEY RESULTS: Low converters excrete more straight-chain fatty acids and bile acids than high converters; 5th and 95th percentile and median of bile acids and short-chain fatty acids were calculated for both groups. CONCLUSION AND IMPLICATIONS: We give concentration ranges for 16 faecal metabolites that can serve as reference values. Patient stratification into high or low sterol converter groups is associated with significant differences in faecal metabolites with biological activities. Such stratification should then allow better assessment of faecal metabolites before therapeutic interventions. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

Metabolite Profile of Treatment-Naive Metabolic Syndrome Subjects in Relation to Cardiovascular Disease Risk.

Metabolic syndrome (MetSyn) is an important risk factor for type 2 diabetes and cardiovascular diseases (CVD). This study aimed to find distinct plasma metabolite profiles between insulin-resistant and non-insulin resistant subjects with MetSyn and evaluate if MetSyn metabolite profiles are related to CVD risk and lipid fluxes. In a cross-sectional study, untargeted metabolomics of treatment-naive males with MetSyn (n = 132) were analyzed together with clinical parameters. In a subset of MetSyn participants, CVD risk was calculated using the Framingham score (n = 111), and lipolysis (n = 39) was measured by a two-step hyperinsulinemic euglycemic clamp using [1,1,2,3,3-2H5] glycerol to calculate lipolysis suppression rates. Peripheral insulin resistance was related to fatty acid metabolism and glycerolphosphorylcholine. Interestingly, although insulin resistance is considered to be a risk factor for CVD, we observed that there was little correspondence between metabolites associated with insulin resistance and metabolites associated with CVD risk. The latter mainly belonged to the androgenic steroid, fatty acid, phosphatidylethanolamine, and phophatidylcholine pathways. These data provide new insights into metabolic changes in mild MetSyn pathophysiology and MetSyn CVD risk related to lipid metabolism. Prospective studies may focus on the pathophysiological role of the here-identified biomarkers.

Correlation of plasma metabolites with glucose and lipid fluxes in human insulin resistance.

OBJECTIVE: Insulin resistance develops prior to the onset of overt type 2 diabetes, making its early detection vital. Direct accurate evaluation is currently only possible with complex examinations like the stable isotope-based hyperinsulinemic euglycemic clamp (HIEC). Metabolomic profiling enables the detection of thousands of plasma metabolites, providing a tool to identify novel biomarkers in human obesity. DESIGN: Liquid chromatography mass spectrometry-based untargeted plasma metabolomics was applied in 60 participants with obesity with a large range of peripheral insulin sensitivity as determined via a two-step HIEC with stable isotopes [6,6-2H2]glucose and [1,1,2,3,3-2H5]glycerol. This additionally enabled measuring insulin-regulated lipolysis, which combined with metabolomics, to the knowledge of this research group, has not been reported on before. RESULTS: Several plasma metabolites were identified that significantly correlated with glucose and lipid fluxes, led by plasma (gamma-glutamyl)citrulline, followed by betaine, beta-cryptoxanthin, fructosyllysine, octanylcarnitine, sphingomyelin (d18:0/18:0, d19:0/17:0) and thyroxine. Subsequent machine learning analysis showed that a panel of these metabolites derived from a number of metabolic pathways may be used to predict insulin resistance, dominated by non-essential amino acid citrulline and its metabolite gamma-glutamylcitrulline. CONCLUSION: This approach revealed a number of plasma metabolites that correlated reasonably well with glycemic and lipolytic flux parameters, measured using gold standard techniques. These metabolites may be used to predict the rate of glucose disposal in humans with obesity to a similar extend as HOMA, thus providing potential novel biomarkers for insulin resistance.

Treatment with Anaerobutyricum soehngenii: a pilot study of safety and dose-response effects on glucose metabolism in human subjects with metabolic syndrome.

Dysbiosis of the intestinal microbiota has been implicated in insulin resistance, although evidence regarding causality in humans is scarce. We performed a phase I/II dose-finding and safety study on the effect of oral intake of the anaerobic butyrogenic strain Anaerobutyricum soehngenii on glucose metabolism in 24 subjects with metabolic syndrome. We found that treatment with A. soehngenii was safe and observed a significant correlation between the measured fecal abundance of administered A. soehngenii and improvement in peripheral insulin sensitivity after 4 weeks of treatment. This was accompanied by an altered microbiota composition and a change in bile acid metabolism. Finally, we show that metabolic response upon administration of A. soehngenii (defined as improved insulin sensitivity 4 weeks after A. soehngenii intake) is dependent on microbiota composition at baseline. These data in humans are promising, but additional studies are needed to reproduce our findings and to investigate long-term effects, as well as other modes of delivery.

Infusion of donor feces affects the gut-brain axis in humans with metabolic syndrome.

OBJECTIVE: Increasing evidence indicates that intestinal microbiota play a role in diverse metabolic processes via intestinal butyrate production. Human bariatric surgery data suggest that the gut-brain axis is also involved in this process, but the underlying mechanisms remain unknown. METHODS: We compared the effect of fecal microbiota transfer (FMT) from post-Roux-en-Y gastric bypass (RYGB) donors vs oral butyrate supplementation on (123I-FP-CIT-determined) brain dopamine transporter (DAT) and serotonin transporter (SERT) binding as well as stable isotope-determined insulin sensitivity at baseline and after 4 weeks in 24 male and female treatment-naïve metabolic syndrome subjects. Plasma metabolites and fecal microbiota were also determined at these time points. RESULTS: We observed an increase in brain DAT after donor FMT compared to oral butyrate that reduced this binding. However, no effect on body weight and insulin sensitivity was demonstrated after post-RYGB donor feces transfer in humans with metabolic syndrome. Increases in fecal levels of Bacteroides uniformis were significantly associated with an increase in DAT, whereas increases in Prevotella spp. showed an inverse association. Changes in the plasma metabolites glycine, betaine, methionine, and lysine (associated with the S-adenosylmethionine cycle) were also associated with altered striatal DAT expression. CONCLUSIONS: Although more and larger studies are needed, our data suggest a potential gut microbiota-driven modulation of brain dopamine and serotonin transporters in human subjects with obese metabolic syndrome. These data also suggest the presence of a gut-brain axis in humans that can be modulated. NTR REGISTRATION: 4488.

Oral vancomycin treatment does not alter markers of postprandial inflammation in lean and obese subjects.

Intake of a high-fat meal induces a systemic inflammatory response in the postprandial which is augmented in obese subjects. However, the underlying mechanisms of this response have not been fully elucidated. We aimed to assess the effect of gut microbiota modulation on postprandial inflammatory response in lean and obese subjects. Ten lean and ten obese subjects with metabolic syndrome received oral vancomycin 500 mg four times per day for 7 days. Oral high-fat meal tests (50 g fat/m2 body surface area) were performed before and after vancomycin intervention. Gut microbiota composition, leukocyte counts, plasma lipopolysaccharides (LPS), LPS-binding protein (LBP), IL-6 and MCP-1 concentrations and monocyte CCR2 and cytokine expression were determined before and after the high-fat meal. Oral vancomycin treatment resulted in profound changes in gut microbiota composition and significantly decreased bacterial diversity in both groups (phylogenetic diversity pre- versus post-intervention: lean, 56.9 ± 7.8 vs. 21.4 ± 6.6, P < 0.001; obese, 53.9 ± 7.8 vs. 21.0 ± 5.9, P < 0.001). After intervention, fasting plasma LPS significantly increased (lean, median [IQR] 0.81 [0.63-1.45] EU/mL vs. 2.23 [1.33-3.83] EU/mL, P = 0.017; obese, median [IQR] 0.76 [0.45-1.03] EU/mL vs. 1.44 [1.11-4.24], P = 0.014). However, postprandial increases in leukocytes and plasma LPS were unaffected by vancomycin in both groups. Moreover, we found no changes in plasma LBP, IL-6 and MCP-1 or in monocyte CCR2 expression. Despite major vancomycin-induced disruption of the gut microbiota and increased fasting plasma LPS, the postprandial inflammatory phenotype in lean and obese subjects was unaffected in this study.

Improvement of Insulin Sensitivity after Lean Donor Feces in Metabolic Syndrome Is Driven by Baseline Intestinal Microbiota Composition.

The intestinal microbiota has been implicated in insulin resistance, although evidence regarding causality in humans is scarce. We therefore studied the effect of lean donor (allogenic) versus own (autologous) fecal microbiota transplantation (FMT) to male recipients with the metabolic syndrome. Whereas we did not observe metabolic changes at 18 weeks after FMT, insulin sensitivity at 6 weeks after allogenic FMT was significantly improved, accompanied by altered microbiota composition. We also observed changes in plasma metabolites such as γ-aminobutyric acid and show that metabolic response upon allogenic FMT (defined as improved insulin sensitivity 6 weeks after FMT) is dependent on decreased fecal microbial diversity at baseline. In conclusion, the beneficial effects of lean donor FMT on glucose metabolism are associated with changes in intestinal microbiota and plasma metabolites and can be predicted based on baseline fecal microbiota composition.

Insights into the role of the microbiome in obesity and type 2 diabetes.

The worldwide prevalence of obesity and type 2 diabetes mellitus (T2DM) continues to rise at an alarming pace. Recently the potential role of the gut microbiome in these metabolic disorders has been identified. Obesity is associated with changes in the composition of the intestinal microbiota, and the obese microbiome seems to be more efficient in harvesting energy from the diet. Lean male donor fecal microbiota transplantation (FMT) in males with metabolic syndrome resulted in a significant improvement in insulin sensitivity in conjunction with an increased intestinal microbial diversity, including a distinct increase in butyrate-producing bacterial strains. Such differences in gut microbiota composition might function as early diagnostic markers for the development of T2DM in high-risk patients. Products of intestinal microbes such as butyrate may induce beneficial metabolic effects through enhancement of mitochondrial activity, prevention of metabolic endotoxemia, and activation of intestinal gluconeogenesis via different routes of gene expression and hormone regulation. Future research should focus on whether bacterial products (like butyrate) have the same effects as the intestinal bacteria that produce it, in order to ultimately pave the way for more successful interventions for obesity and T2DM. The rapid development of the currently available techniques, including use of fecal transplantations, has already shown promising results, so there is hope for novel therapies based on the microbiota in the future.