The potential of tailoring the gut microbiome to prevent and treat cardiometabolic disease.

Despite milestones in preventive measures and treatment, cardiovascular disease (CVD) remains associated with a high burden of morbidity and mortality. The protracted nature of the development and progression of CVD motivates the identification of early and complementary targets that might explain and alleviate any residual risk in treated patients. The gut microbiota has emerged as a sentinel between our inner milieu and outer environment and relays a modified risk associated with these factors to the host. Accordingly, numerous mechanistic studies in animal models support a causal role of the gut microbiome in CVD via specific microbial or shared microbiota-host metabolites and have identified converging mammalian targets for these signals. Similarly, large-scale cohort studies have repeatedly reported perturbations of the gut microbial community in CVD, supporting the translational potential of targeting this ecological niche, but the move from bench to bedside has not been smooth. In this Review, we provide an overview of the current evidence on the interconnectedness of the gut microbiome and CVD against the noisy backdrop of highly prevalent confounders in advanced CVD, such as increased metabolic burden and polypharmacy. We further aim to conceptualize the molecular mechanisms at the centre of these associations and identify actionable gut microbiome-based targets, while contextualizing the current knowledge within the clinical scenario and emphasizing the limitations of the field that need to be overcome.

Functional changes of the gastric bypass microbiota reactivate thermogenic adipose tissue and systemic glucose control via intestinal FXR-TGR5 crosstalk in diet-induced obesity.

BACKGROUND: Bariatric surgery remains the most effective therapy for adiposity reduction and remission of type 2 diabetes. Although different bariatric procedures associate with pronounced shifts in the gut microbiota, their functional role in the regulation of energetic and metabolic benefits achieved with the surgery are not clear. METHODS: To evaluate the causal as well as the inherent therapeutic character of the surgery-altered gut microbiome in improved energy and metabolic control in diet-induced obesity, an antibiotic cocktail was used to eliminate the gut microbiota in diet-induced obese rats after gastric bypass surgery, and gastric bypass-shaped gut microbiota was transplanted into obese littermates. Thorough metabolic profiling was combined with omics technologies on samples collected from cecum and plasma to identify adaptions in gut microbiota-host signaling, which control improved energy balance and metabolic profile after surgery. RESULTS: In this study, we first demonstrate that depletion of the gut microbiota largely reversed the beneficial effects of gastric bypass surgery on negative energy balance and improved glucolipid metabolism. Further, we show that the gastric bypass-shaped gut microbiota reduces adiposity in diet-induced obese recipients by re-activating energy expenditure from metabolic active brown adipose tissue. These beneficial effects were linked to improved glucose homeostasis, lipid control, and improved fatty liver disease. Mechanistically, these effects were triggered by modulation of taurine metabolism by the gastric bypass gut microbiota, fostering an increased abundance of intestinal and circulating taurine-conjugated bile acid species. In turn, these bile acids activated gut-restricted FXR and systemic TGR5 signaling to stimulate adaptive thermogenesis. CONCLUSION: Our results establish the role of the gut microbiome in the weight loss and metabolic success of gastric bypass surgery. We here identify a signaling cascade that entails altered bile acid receptor signaling resulting from a collective, hitherto undescribed change in the metabolic activity of a cluster of bacteria, thereby readjusting energy imbalance and metabolic disease in the obese host. These findings strengthen the rationale for microbiota-targeted strategies to improve and refine current therapies of obesity and metabolic syndrome. Video Abstract Bariatric Surgery (i.e. RYGB) or the repeated fecal microbiota transfer (FMT) from RYGB donors into DIO (diet-induced obesity) animals induces shifts in the intestinal microbiome, an effect that can be impaired by oral application of antibiotics (ABx). Our current study shows that RYGB-dependent alterations in the intestinal microbiome result in an increase in the luminal and systemic pool of Taurine-conjugated Bile acids (TCBAs) by various cellular mechanisms acting in the intestine and the liver. TCBAs induce signaling via two different receptors, farnesoid X receptor (FXR, specifically in the intestines) and the G-protein-coupled bile acid receptor TGR5 (systemically), finally resulting in metabolic improvement and advanced weight management. BSH, bile salt hydrolase; BAT brown adipose tissue.

Microbiome and metabolome features of the cardiometabolic disease spectrum.

Previous microbiome and metabolome analyses exploring non-communicable diseases have paid scant attention to major confounders of study outcomes, such as common, pre-morbid and co-morbid conditions, or polypharmacy. Here, in the context of ischemic heart disease (IHD), we used a study design that recapitulates disease initiation, escalation and response to treatment over time, mirroring a longitudinal study that would otherwise be difficult to perform given the protracted nature of IHD pathogenesis. We recruited 1,241 middle-aged Europeans, including healthy individuals, individuals with dysmetabolic morbidities (obesity and type 2 diabetes) but lacking overt IHD diagnosis and individuals with IHD at three distinct clinical stages-acute coronary syndrome, chronic IHD and IHD with heart failure-and characterized their phenome, gut metagenome and serum and urine metabolome. We found that about 75% of microbiome and metabolome features that distinguish individuals with IHD from healthy individuals after adjustment for effects of medication and lifestyle are present in individuals exhibiting dysmetabolism, suggesting that major alterations of the gut microbiome and metabolome might begin long before clinical onset of IHD. We further categorized microbiome and metabolome signatures related to prodromal dysmetabolism, specific to IHD in general or to each of its three subtypes or related to escalation or de-escalation of IHD. Discriminant analysis based on specific IHD microbiome and metabolome features could better differentiate individuals with IHD from healthy individuals or metabolically matched individuals as compared to the conventional risk markers, pointing to a pathophysiological relevance of these features.

Impairment of gut microbial biotin metabolism and host biotin status in severe obesity: effect of biotin and prebiotic supplementation on improved metabolism.

OBJECTIVES: Gut microbiota is a key component in obesity and type 2 diabetes, yet mechanisms and metabolites central to this interaction remain unclear. We examined the human gut microbiome's functional composition in healthy metabolic state and the most severe states of obesity and type 2 diabetes within the MetaCardis cohort. We focused on the role of B vitamins and B7/B8 biotin for regulation of host metabolic state, as these vitamins influence both microbial function and host metabolism and inflammation. DESIGN: We performed metagenomic analyses in 1545 subjects from the MetaCardis cohorts and different murine experiments, including germ-free and antibiotic treated animals, faecal microbiota transfer, bariatric surgery and supplementation with biotin and prebiotics in mice. RESULTS: Severe obesity is associated with an absolute deficiency in bacterial biotin producers and transporters, whose abundances correlate with host metabolic and inflammatory phenotypes. We found suboptimal circulating biotin levels in severe obesity and altered expression of biotin-associated genes in human adipose tissue. In mice, the absence or depletion of gut microbiota by antibiotics confirmed the microbial contribution to host biotin levels. Bariatric surgery, which improves metabolism and inflammation, associates with increased bacterial biotin producers and improved host systemic biotin in humans and mice. Finally, supplementing high-fat diet-fed mice with fructo-oligosaccharides and biotin improves not only the microbiome diversity, but also the potential of bacterial production of biotin and B vitamins, while limiting weight gain and glycaemic deterioration. CONCLUSION: Strategies combining biotin and prebiotic supplementation could help prevent the deterioration of metabolic states in severe obesity. TRIAL REGISTRATION NUMBER: NCT02059538.

Multiomics reveal unique signatures of human epiploic adipose tissue related to systemic insulin resistance.

OBJECTIVE: Human white adipose tissue (AT) is a metabolically active organ with distinct depot-specific functions. Despite their locations close to the gastrointestinal tract, mesenteric AT and epiploic AT (epiAT) have only scarcely been investigated. Here, we aim to characterise these ATs in-depth and estimate their contribution to alterations in whole-body metabolism. DESIGN: Mesenteric, epiploic, omental and abdominal subcutaneous ATs were collected from 70 patients with obesity undergoing Roux-en-Y gastric bypass surgery. The metabolically well-characterised cohort included nine subjects with insulin sensitive (IS) obesity, whose AT samples were analysed in a multiomics approach, including methylome, transcriptome and proteome along with samples from subjects with insulin resistance (IR) matched for age, sex and body mass index (n=9). Findings implying differences between AT depots in these subgroups were validated in the entire cohort (n=70) by quantitative real-time PCR. RESULTS: While mesenteric AT exhibited signatures similar to those found in the omental depot, epiAT was distinct from all other studied fat depots. Multiomics allowed clear discrimination between the IS and IR states in all tissues. The highest discriminatory power between IS and IR was seen in epiAT, where profound differences in the regulation of developmental, metabolic and inflammatory pathways were observed. Gene expression levels of key molecules involved in AT function, metabolic homeostasis and inflammation revealed significant depot-specific differences with epiAT showing the highest expression levels. CONCLUSION: Multi-omics epiAT signatures reflect systemic IR and obesity subphenotypes distinct from other fat depots. Our data suggest a previously unrecognised role of human epiploic fat in the context of obesity, impaired insulin sensitivity and related diseases.

Circulating bacterial signature is linked to metabolic disease and shifts with metabolic alleviation after bariatric surgery.

BACKGROUND: The microbiome has emerged as an environmental factor contributing to obesity and type 2 diabetes (T2D). Increasing evidence suggests links between circulating bacterial components (i.e., bacterial DNA), cardiometabolic disease, and blunted response to metabolic interventions. In this aspect, thorough next-generation sequencing-based and contaminant-aware approaches are lacking. To address this, we tested whether bacterial DNA could be amplified in the blood of subjects with obesity and high metabolic risk under strict experimental and analytical control and whether a putative bacterial signature is related to metabolic improvement after bariatric surgery. METHODS: Subjects undergoing bariatric surgery were recruited into sex- and BMI-matched subgroups with (n = 24) or without T2D (n = 24). Bacterial DNA in the blood was quantified and prokaryotic 16S rRNA gene amplicons were sequenced. A contaminant-aware approach was applied to derive a compositional microbial signature from bacterial sequences in all subjects at baseline and at 3 and 12 months after surgery. We modeled associations between bacterial load and composition with host metabolic and anthropometric markers. We further tested whether compositional shifts were related to weight loss response and T2D remission. Lastly, bacteria were visualized in blood samples using catalyzed reporter deposition (CARD)-fluorescence in situ hybridization (FISH). RESULTS: The contaminant-aware blood bacterial signature was associated with metabolic health. Based on bacterial phyla and genera detected in the blood samples, a metabolic syndrome classification index score was derived and shown to robustly classify subjects along their actual clinical group. T2D was characterized by decreased bacterial richness and loss of genera associated with improved metabolic health. Weight loss and metabolic improvement following bariatric surgery were associated with an early and stable increase of these genera in parallel with improvements in key cardiometabolic risk parameters. CARD-FISH allowed the detection of living bacteria in blood samples in obesity. CONCLUSIONS: We show that the circulating bacterial signature reflects metabolic disease and its improvement after bariatric surgery. Our work provides contaminant-aware evidence for the presence of living bacteria in the blood and suggests a putative crosstalk between components of the blood and metabolism in metabolic health regulation.

The Effect of FGF21 and Its Genetic Variants on Food and Drug Cravings, Adipokines and Metabolic Traits.

Fibroblast growth factor 21 (FGF21) is a regulator of addictive behavior. Increasing evidence suggests an impact of FGF21 on eating behavior, food and drug cravings and on other adipokines like insulin-like growth factor 1 (IGF-1) or adiponectin. We investigated the association of serum FGF21 and genetic variants with aspects of food and drug craving and obesity related metabolic parameters including serum adipokine levels. Standardized questionnaires, blood samples and anthropometric data of the Sorbs cohort (n = 1046) were analyzed using SPSS. For genetic analyses, the FGF21-locus ±10 kb was genotyped and analyzed using PLINK. Validation was conducted in a second independent cohort (n = 704). FGF21 was significantly associated with alcohol and coffee consumption, smoking and eating behavior (disinhibition). We confirmed correlations of FGF21 serum levels with IGF-1, adiponectin, pro-enkephalin, adipocyte fatty-acid-binding protein, chemerin and progranulin. FGF21 genetic variants were associated with anthropometric and metabolic parameters, adipokines, food and drug craving while strongest evidence was seen with low-density lipoprotein cholesterol (LDL-C). We highlight the potential role of FGF21 in food and drug cravings and provide new insights regarding the link of FGF21 with other adipokines as well as with metabolic traits, in particular those related to lipid metabolism (LDL-C).

Adipose tissue derived bacteria are associated with inflammation in obesity and type 2 diabetes.

OBJECTIVE: Bacterial translocation to various organs including human adipose tissue (AT) due to increased intestinal permeability remains poorly understood. We hypothesised that: (1) bacterial presence is highly tissue specific and (2) related in composition and quantity to immune inflammatory and metabolic burden. DESIGN: We quantified and sequenced the bacterial 16S rRNA gene in blood and AT samples (omental, mesenteric and subcutaneous) of 75 subjects with obesity with or without type 2 diabetes (T2D) and used catalysed reporter deposition (CARD) - fluorescence in situ hybridisation (FISH) to detect bacteria in AT. RESULTS: Under stringent experimental and bioinformatic control for contaminants, bacterial DNA was detected in blood and omental, subcutaneous and mesenteric AT samples in the range of 0.1 to 5 pg/µg DNA isolate. Moreover, CARD-FISH allowed the detection of living, AT-borne bacteria. Proteobacteria and Firmicutes were the predominant phyla, and bacterial quantity was associated with immune cell infiltration, inflammatory and metabolic parameters in a tissue-specific manner. Bacterial composition differed between subjects with and without T2D and was associated with related clinical measures, including systemic and tissues-specific inflammatory markers. Finally, treatment of adipocytes with bacterial DNA in vitro stimulated the expression of TNFA and IL6. CONCLUSIONS: Our study provides contaminant aware evidence for the presence of bacteria and bacterial DNA in several ATs in obesity and T2D and suggests an important role of bacteria in initiating and sustaining local AT subclinical inflammation and therefore impacting metabolic sequelae of obesity.

NPY1R-targeted peptide-mediated delivery of a dual PPARα/γ agonist to adipocytes enhances adipogenesis and prevents diabetes progression.

OBJECTIVE: PPARα/γ dual agonists have been in clinical development for the treatment of metabolic diseases including type 2 diabetes and dyslipidemia. However, severe adverse side effects led to complications in clinical trials. As most of the beneficial effects rely on the compound activity in adipocytes, the selective targeting of this cell type is a cutting-edge strategy to develop safe anti-diabetic drugs. The goal of this study was to strengthen the adipocyte-specific uptake of the PPARα/γ agonist tesaglitazar via NPY1R-mediated internalization. METHODS: NPY1R-preferring peptide tesaglitazar-[F7, P34]-NPY (tesa-NPY) was synthesized by a combination of automated SPPS and manual couplings. Following molecular and functional analyses for proof of concept, cell culture experiments were conducted to monitor the effects on adipogenesis. Mice treated with peptide drug conjugates or vehicle either by gavage or intraperitoneal injection were characterized phenotypically and metabolically. Histological analysis and transcriptional profiling of the adipose tissue were performed. RESULTS: In vitro studies revealed that the tesaglitazar-[F7, P34]-NPY conjugate selectively activates PPARγ in NPY1R-expressing cells and enhances adipocyte differentiation and adiponectin expression in adipocyte precursor cells. In vivo studies using db/db mice demonstrated that the anti-diabetic activity of the peptide conjugate is as efficient as that of systemically administered tesaglitazar. Additionally, tesa-NPY induces adipocyte differentiation in vivo. CONCLUSIONS: The use of the tesaglitazar-[F7, P34]-NPY conjugate is a promising strategy to apply the beneficial PPARα/γ effects in adipocytes while potentially omitting adverse effects in other tissues.

Homeostatic, reward and executive brain functions after gastric bypass surgery.

Obesity in part arises from the regular overconsumption of palatable, caloric-dense foods. This maladaptive eating behavior has been described as impulsive, compulsive and even addictive, and has its origins in molecular and cellular aberrations in the gut and brain. Mounting evidence from human and rodent studies suggests that Roux-en-Y gastric bypass (RYGB) surgery persistantly promotes lower caloric intake by modifying gut-brain communication. In this Review, we discuss how the changes in gut hormones, nutrient sensing andmicrobiota brought about by RYGB together favourably regulate homeostatic, reward and executive brain functions. We further speculate on how this lastingly establishes a negative whole-body energy balance in the face of plenty. Future studies will more completely characterize the role of modified gut-brain communication in the healthier eating behavior following RYGB, possibly facilitating the development of more effective, non-surgical weight loss treatments.

Long-term Relapse of Type 2 Diabetes After Roux-en-Y Gastric Bypass: Prediction and Clinical Relevance.

OBJECTIVE: Roux-en-Y gastric bypass (RYGB) induces type 2 diabetes remission (DR) in 60% of patients at 1 year, yet long-term relapse occurs in half of these patients. Scoring methods to predict DR outcomes 1 year after surgery that include only baseline parameters cannot accurately predict 5-year DR (5y-DR). We aimed to develop a new score to better predict 5y-DR. RESEARCH DESIGN AND METHODS: We retrospectively included 175 RYGB patients with type 2 diabetes with 5-year follow-up. Using machine learning algorithms, we developed a scoring method, 5-year Advanced-Diabetes Remission (5y-Ad-DiaRem), predicting longer-term DR postsurgery by integrating medical history, bioclinical data, and antidiabetic treatments. The scoring method was based on odds ratios and variables significantly different between groups. This score was further validated in three independent RYGB cohorts from three European countries. RESULTS: Compared with 5y-DR patients, patients who had relapsed after 5 years exhibited more severe type 2 diabetes at baseline, lost significantly less weight during the 1st year after RYGB, and regained more weight afterward. The 5y-Ad-DiaRem includes baseline (diabetes duration, number of antidiabetic treatments, and HbA1c) and 1-year follow-up parameters (glycemia, number of antidiabetic treatments, remission status, 1st-year weight loss). The 5y-Ad-DiaRem was accurate (area under the receiver operating characteristic curve [AUROC], 90%; accuracy, 85%) at predicting 5y-DR, performed better than the Diabetes Remission score (DiaRem) and the Advanced-DiaRem (AUROC, 81% and 84%; accuracy, 79% and 78%, respectively), and correctly reclassified 13 of 39 patients misclassified with the DiaRem. The 5y-Ad-DiaRem robustness was confirmed in the independent cohorts. CONCLUSIONS: The 5y-Ad-DiaRem accurately predicts 5y-DR and appears relevant to identify patients at risk for relapse. Using this score could help personalize patient care after the 1st year post-RYGB to maximize weight loss, limit weight regains, and prevent relapse.

Dietary Assessment in the MetaCardis Study: Development and Relative Validity of an Online Food Frequency Questionnaire.

BACKGROUND: The European study MetaCardis aims to investigate the role of the gut microbiota in health and cardiometabolic diseases in France, Germany, and Denmark. To evaluate long-term diet-disease relationships, a food frequency questionnaire (FFQ) was found to be the most relevant dietary assessment method for the MetaCardis study. OBJECTIVE: The objectives of this study were to describe the development of three semiquantitative online FFQs used in the MetaCardis study-one FFQ per country-and to assess the relative validity of the French MetaCardis FFQ. DESIGN: The layout and format of the MetaCardis FFQ was based on the European Prospective Investigation of Cancer (EPIC)-Norfolk FFQ and the content was based on relevant European FFQs. Portion size and nutrient composition were derived from national food consumption surveys and food composition databases. To assess the validity of the French MetaCardis FFQ, a cross-sectional study design was utilized. PARTICIPANTS/SETTING: The validation study included 324 adults recruited between September 2013 and June 2015 from different hospitals in Paris, France. MAIN OUTCOME MEASURES: Food intakes were measured with both the French MetaCardis FFQ and 3 consecutive self-administered web-based 24-hour dietary recalls (DRs). STATISTICAL ANALYSES PERFORMED: Several measures of validity of the French MetaCardis FFQ were evaluated: estimations of food groups, energy, and nutrient intakes from the DRs and the FFQ, Spearman and Pearson correlations, cross-classification, and Bland-Altman analyses. RESULTS: The French MetaCardis FFQ tended to report higher food, energy, and nutrient intakes compared with the DRs. Mean correlation coefficient was 0.429 for food, 0.460 for energy, 0.544 for macronutrients, 0.640 for alcohol, and 0.503 for micronutrient intakes. Almost half of participants (44.4%) were correctly classified within tertiles of consumption, whereas 12.9% were misclassified in the opposite tertile. Performance of the FFQ was relatively similar after stratification by sex. CONCLUSIONS: The French MetaCardis FFQ was found to have an acceptable level of validity and may be a useful instrument to rank individuals based on their food and nutrient intakes.

Estimating steatosis and fibrosis: Comparison of acoustic structure quantification with established techniques.

AIM: To compare ultrasound-based acoustic structure quantification (ASQ) with established non-invasive techniques for grading and staging fatty liver disease. METHODS: Type 2 diabetic patients at risk of non-alcoholic fatty liver disease (n = 50) and healthy volunteers (n = 20) were evaluated using laboratory analysis and anthropometric measurements, transient elastography (TE), controlled attenuation parameter (CAP), proton magnetic resonance spectroscopy ((1)H-MRS; only available for the diabetic cohort), and ASQ. ASQ parameters mode, average and focal disturbance (FD) ratio were compared with: (1) the extent of liver fibrosis estimated from TE and non-alcoholic fatty liver disease (NAFLD) fibrosis scores; and (2) the amount of steatosis, which was classified according to CAP values. RESULTS: Forty-seven diabetic patients (age 67.0 ± 8.6 years; body mass index 29.4 ± 4.5 kg/m²) with reliable CAP measurements and all controls (age 26.5 ± 3.2 years; body mass index 22.0 ± 2.7 kg/m²) were included in the analysis. All ASQ parameters showed differences between healthy controls and diabetic patients (P < 0.001, respectively). The ASQ FD ratio (logarithmic) correlated with the CAP (r = -0.81, P < 0.001) and (1)H-MRS (r = -0.43, P = 0.004) results. The FD ratio [CAP < 250 dB/m: 107 (102-109), CAP between 250 and 300 dB/m: 106 (102-114); CAP between 300 and 350 dB/m: 105 (100-112), CAP ≥ 350 dB/m: 102 (99-108)] as well as mode and average parameters, were reduced in cases with advanced steatosis (ANOVA P < 0.05). However, none of the ASQ parameters showed a significant difference in patients with advanced fibrosis, as determined by TE and the NAFLD fibrosis score (P > 0.08, respectively). CONCLUSION: ASQ parameters correlate with steatosis, but not with fibrosis in fatty liver disease. Steatosis estimation with ASQ should be further evaluated in biopsy-controlled studies.

Effects of weight loss and exercise on chemerin serum concentrations and adipose tissue expression in human obesity.

Chemerin is a chemoattractant adipokine that regulates adipogenesis and may induce insulin resistance. Chemerin serum concentrations are elevated in obese, insulin-resistant, and inflammatory states in vivo. Here we investigate the role of omental (OM) and subcutaneous (SC) adipose tissue chemerin and CMKLR1 messenger RNA (mRNA) expression in human obesity. In addition, we test the hypothesis that changes in chemerin serum concentrations are primarily associated with reduced body fat mass in the context of 3 weight loss intervention studies. Chemerin serum concentration was measured in 740 individuals in a cross-sectional (n = 629) study including a subgroup (n = 161) for which OM and SC chemerin mRNA expression has been analyzed as well as in 3 interventions including 12 weeks of exercise (n = 60), 6 months of calorie-restricted diet (n = 19) studies, and 12 months after bariatric surgery (n = 32). Chemerin mRNA is significantly higher expressed in adipose tissue of patients with type 2 diabetes mellitus and correlates with circulating chemerin, body mass index (BMI), percentage body fat, C-reactive protein, homeostasis model assessment of insulin resistance, and glucose infusion rate in euglycemic-hyperinsulinemic clamps. CMKLR1 mRNA expression was not significantly different between the 2 fat depots. Obesity surgery-induced weight loss causes a significant reduction on both OM and SC chemerin expression. All interventions led to significantly reduced chemerin serum concentrations. Decreased chemerin serum concentrations significantly correlate with improved glucose infusion rate and reduced C-reactive protein levels independently of changes in BMI. Insulin resistance and inflammation are BMI-independent predictors of elevated chemerin serum concentrations. Reduced chemerin expression and serum concentration may contribute to improved insulin sensitivity and subclinical inflammation beyond significant weight loss.