Role of the Gut Microbiome in the Pathogenesis of Obesity and Obesity-Related Metabolic Dysfunction

Accepted Manuscript Role of the Gut Microbiome in the Pathogenesis of Obesity and Obesity-Related Metabolic Dysfunction Kristien E. Bouter, Daniël H. van Raalte, Albert K. Groen, Max Nieuwdorp

PII: DOI: Reference:

S0016-5085(17)30141-5 10.1053/j.gastro.2016.12.048 YGAST 60968

To appear in: Gastroenterology Accepted Date: 30 December 2016 Please cite this article as: Bouter KE, van Raalte DH, Groen AK, Nieuwdorp M, Role of the Gut Microbiome in the Pathogenesis of Obesity and Obesity-Related Metabolic Dysfunction, Gastroenterology (2017), doi: 10.1053/j.gastro.2016.12.048. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Title: role of the gut microbiome in the pathogenesis of obesity and obesityrelated metabolic dysfunction

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Kristien E. Bouter1, Daniël H. van Raalte2,3, Albert K. Groen1,4 and Max Nieuwdorp1,2,3,5*

Department of Vascular Medicine, Academic Medical Center (AMC), University of

Amsterdam, The Netherlands; 2 Diabetes Center, Department of Internal Medicine, VU

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University Medical Center, Amsterdam, The Netherlands; 3 Institute for Cardiovascular Research (ICaR), VU University Medical Center, Amsterdam, The Netherlands; 4

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Department of Pediatrics, Laboratory of Metabolic Diseases, University of Groningen, UMCG, Groningen, the Netherlands; 5 Wallenberg Laboratory, University of Gothenberg, Gothenberg, Sweden.

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*Corresponding author: Max Nieuwdorp. Department of Experimental Vascular Medicine, Meibergdreef 9, Room F4-159.2, 1105 AZ Amsterdam, The Netherlands. Tel.: +

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31 20 5666612; E-mail: [email protected]

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Abstract: 146 words

Manuscript: 5813 words including references Figures: 1

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ACCEPTED MANUSCRIPT Abstract The potential role of intestinal microbiota in the etiology of various human diseases has attracted massive attention in the last decade. As such, the intestinal microbiota has been advanced as an important partaker in the development of obesity and obesity-

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related metabolic dysfunctions, amongst others. Experiments in animal models have produced evidence for a causal role of intestinal microbiota in the etiology of obesity and insulin resistance. However, with a few exceptions such causal relation is lacking

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for humans. and most publication merely report associations between intestinal

microbial composition and metabolic disorders such as obesity and type 2 diabetes

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(T2DM). Thus, the reciprocal relation between the bacteria and these metabolic disorders remains a matter of debate. The main objective of this review is to critically assess the driving role of intestinal microbe composition in the etiology, prevention and

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treatment of obesity and obesity-related metabolic dysfunction, including T2DM.

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Introduction The current and worldwide obesity pandemic is associated with lifestyle changes that are characterized by an excess energy intake and reduced physical activity. The obesity

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pandemic strongly contributes to the incidence and prevalence of type 2 diabetes

(T2DM) which through its macrovascular and microvascular complications, poses a heavy burden on the healthcare system. The mechanisms by which obesity results in

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T2DM have been partly clarified and include the development of insulin resistance at the level of adipose tissue, skeletal muscle and liver, with concurrent impaired insulin

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secretion by the pancreatic beta-cell. More recently, the influence of intestinal microbiota as an underlying mechanistic driver of obesity and its related comorbidities has come into focus. The gut microbiota, referring to bacteria as well as viruses, fungi, archaea, phages and protozoa residing in the human intestine, [1] equal the amount of

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all human cells [2] and they have the capability to interact with the host in several ways. These include, but are not limited to, functions like nutrient metabolism upon dietary intake, xenobiotic metabolism, maintenance of gut barrier function and the

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(gastrointestinal) immune system, as well as protection against translocation of

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intestinal pathogens. [3, 4]

The composition of the gutmicrobiota is considered to be influenced by many factors, starting from early life. During birth, the neonatal intestinal tract is colonized with maternal microbes originating from vaginal, fecal and skin flora. [5] Thus, the mode of birth - vaginal versus caesarian section - might influence the composition of the microbiome of the newborn’s intestinal tract. [6, 7] However, the mode of delivery is only one factor involved in the regulation of the composition of the microbial 3

ACCEPTED MANUSCRIPT community. Multiple studies indicated that composition of the microbiota varies in breast-fed infants compared to formula fed infants [8, 9] and that the introduction of solid food is another important determinant of microbial composition [10]. In humans, the microbiome changes considerably until at least year 3 when a more stable period

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sets in regarding the most predominant bacterial phyla. [11, 12] Interestingly, the

intestinal microbiota composition is only to a limited extent heritable as was observed in monozygotic twin studies [13]. Only in the last stage of life the composition changes

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again. Nevertheless, intestinal bacterial composition during life can be altered by

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changes in the diet as well as factors like medication use as will be discussed below.

As previously indicated, alterations in the gut microbiota composition and diversity have been linked to obesity and T2DM in association studies. In addition, various mechanisms have been proposed to explain how gut microbiota drive obesity and its

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metabolic complications, including interaction with metabolic processes and the immune system of the host. This review will provide a critical overview of the current knowledge of gut microbiota and its potential pathogenic role in the development of

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human disease with a focus on obesity and T2DM. Moreover, we will also discuss the

state.

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potential role of gut microbiota in prevention and treatment of this adverse metabolic

Shaping factors of the intestinal microbial community: diet and the innate immune system The human gut lumen contains a broad variety of microorganisms, most notably bacteria. Although bacteria were generally considered as pathogens, an essential 4

ACCEPTED MANUSCRIPT symbiotic interaction between the human host and intestinal bacteria is the forging and maintenance of the immune system in the gut. The first recognition of this cross-talk came from findings in germ-free (GF) mice, where defects in the development and function of their immune system were described [14] The cross-talk between bacteria

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and host immune system is probably instrumental for the fact that the gut microbiota composition is unique for each individual. Despite the intra-individual variation, five phyla dominate the intestinal community. These phyla are the Actinobacteria,

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Bacteroidetes, Firmicutes, Proteobacteria and Verrucomicrobia. The microbial

composition changes along the gastro-intestinal tract, caused by the high-acidity and

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higher oxygen content in the stomach and duodenum. The proximal gastro-intestinal tract is thus enriched with Firmicutes, Lactobacilli and Proteobacteria, while more distally Bacteroidetes, Firmicutes and Akkermansia municiphilia are present [15]. In healthy individuals, the composition of the intestinal microbiota is diverse. This seems

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to be an important characteristic because diversity has been shown to be decreased in obese subjects as well as T2DM patients [16] . Marked differences in microbial composition have been reported in obese and T2DM patients. Qin et al [17] reported

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decreased occurrence of butyrate producing bacteria such as Clostridiales, Eubacterium

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rectale, Faecalibacterium prausnitzii and Roseburia intestinalis. Interestingly, mucin degrading bacteria such as Akkermansia muciniphila and Desulfovirbrio were enriched in samples derived from T2DM patients. Paradoxically, a membrane protein of Akkermansia mucinophilia was recently shown to improve obesity and T2D in mice [18] Changes in the Firmicutes/Bacteroidetes ratio have been repeatedly reported to associate with obesity and insulin resistance [19]. These alterations were related to measures of glycemic control including fasting glucose and HbA1c concentrations. 5

ACCEPTED MANUSCRIPT Moreover, obese subjects with low gene diversity and a less diverse composition of the microbiota are characterized by higher BMI and fat mass, lower insulin sensitivity, dyslipidemia as well as increased markers of inflammation. Nevertheless, these data should be interpreted with caution as, a recent meta-analysis of Finucane et al. [20]

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indicated that the reproducibility of these human studies is limited. When pooling four studies, differences in phylum-level taxonomic composition were confirmed in lean and obese subjects and although the authors conclude that no association between BMI and

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taxonomic composition of fecal microbes was found in the largest cohorts (Human

Microbiome Project and MetaHit) . The authors conclude that perhaps the differences

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found between studies are possibly due to differences in diet, geography or metabolic (diabetes) status. In this regard, it is important to note that glucose-lowering agents may have a large impact. While this has been documented for metformin [21, 22] , the extent to which other drugs affect the microbiome remains to be studied. Thus, larger studies

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with more power to investigate the association of the microbiome and obesity and T2DM are eagerly awaited.

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It has recently become clear that gut microbiota as well as their endogenous metabolic

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function enabling the digestion of food components, is related to dietary intake. A large cohort study showed a few years ago that specific dietary intake is closely associated with development of obesity in humans [23] With every 4-year interval, weight increase was positively associated with the intake of processed foods (like potato chips), sugarsweetened beverages and red meat, while weight loss in these intervals was associated with the intake of vegetables, fibers and yogurt. In this respect, it is interesting to note many of these specific dietary compounds have been linked to altered gut microbiota 6

ACCEPTED MANUSCRIPT composition. Both food emulsifiers (like carboxymethylcellulose and polysorbate-80 often found in processed foods) [24] and artificial sweeteners [21] have been associated with altered microbial composition and the development of obesity and metabolic syndrome. Moreover, red meat intake (via carnitine and choline) has been

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linked to altered gut microbiota composition and an adverse cardiometabolic state [25]. The dietary compounds that are associated with weight reduction in the above

mentioned paper, including vegetables [26] , fibers [27] and yogurt [28] are all reported

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to alter fecal microbial composition. With regard to intake of probiotics , a double

blinded randomized placebo controlled intervention trial in overweight subjects with a

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BMI between 24,2 and 30,7 showed beneficial effects of lactobacillius Gasseri on weightreduction compared to fermented milk use only[29] suggesting that targeted (microbiota based) dietary interventions might exert beneficial metabolic effects.,In this respect the landmark paper by Zeevi et al [30]is quite interesting, since this group

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elegantly demonstrated in over 800 subjects that it is indeed possible to use microbiota composition of individual subjects to predict their metabolic (glycemic) response to a diet. Moreover, although preliminary, their data suggest that these effects are partly

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mediated via dietary-derived metabolites affecting our innate immune system [31].

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Despite these interesting findings, two large and independent Caucasian-based cohorts recently showed that the influence of endogenous factors, previous disease, medication and lifestyle (including diet) could explain only 18.7% of the inter-individual variation of fecal microbial composition. These results underscore that there are still important steps to be made towards a better understanding of environment-diet-microbe-host interactions. [32, 33].

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ACCEPTED MANUSCRIPT As mentioned above, in addition to diet oral medication can also influence the gut microbiome. Antibiotics can alter microbiome (decrease in Bifidobacterium genus) [33, 34] whereas antibiotics use at early age has been associated with higher weight gain [35]. In line, other frequently used medication in obese subjects and T2DM like proton

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pump inhibitors [36] and metformin [21, 22] been reported to affect gut microbial

composition. Thus, the composition of the gut microbiota is determined by a complex interplay of host genetic and (innate) immunologic factors, the environment and inter-

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species competition. However, a recent study [37] challenged this notion indicating that microbial cross talk is dominant over exogenous (host) factors. Applying a procedure

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called dissimilarity overlap curves (DOC), the authors looked at general relationships between the presence of bacterial species independent of the host. If bacterial cross-talk is independent of the host, presence of a bacterial species should be predictive of the relative proportion of that species in the microbial community. This was indeed

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observed, suggesting that the influence of the host on the major species distribution may be small. Although recent studies in monozygote twins provided evidence for heritability of a number of microbial species [38], it cannot be excluded that these

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observed differences might be due to the immune system than genetic background.[39]

Mechanisms by which gut microbiota may drive obesity and type 2 diabetes Microbiota-derived metabolites How gut microorganisms interact with the host is still largely enigmatic, probably due to the multiple pathways that may be involved. Bacteria may provide the host with up to 10% of energy via fermentation of dietary components that otherwise cannot be 8

ACCEPTED MANUSCRIPT digested by the intestinal digestive system of the host. Digestion of dietary fibers in the large intestine produces metabolites like short chain fatty acids (SCFA), comprising mainly acetate, propionate and butyrate. The latter has been shown to be an important energy source for colonocytes whereas propionate and acetate are rapidly absorbed and

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serve as energy substrates for the liver. However, a small amount of these SCFA is

released into the circulation, exerting a diverse array of metabolic and brain-related effects [40] . SCFAs can also activate the energy sensor AMP-activated protein kinases

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(AMPK) in the liver and muscle triggering the activation of peroxisome proliferatoractivated receptor-gamma coactivator (PGC-1alpha) and members of the peroxisome

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proliferator-activated receptor family, thereby stimulating glucose uptake and oxidation of fatty acids thus resulting in improved glycemic control at least in murine models. [41]. The role of acetate in energy metabolism is controversial. Studies in mice demonstrated that dietary supplementation of acetate ameliorates obesity and its comorbidity, insulin

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resistance. However, a recent study in rats reported the opposite showing acetate induces obesity and insulin resistance [42] and a study confirming these data in human is awaited. Intracranial perfusion experiments in this study indicated that acetate

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controls metabolism via sympathetic effects. In line, other animal studies have linked

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microbiota-generated metabolites to the brain neural circuits [43]. However, none of these relations including effect of microbiota on incretins have even been proven in obese humans. [44]. Clearly, more studies are required to solve these controversies and may delineate the complexity of the pathways that can be influenced by the SCFAs and differences between rodents and humans.

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ACCEPTED MANUSCRIPT In addition to SCFAs, gut microbes produce a multitude of other metabolites that may influence host metabolism. An elegant case in point is the bacterial metabolite trimethylamine (TMA). The group of Hazen [45] has reported a number of studies showing that TMA is converted to trimethylamineoxide (TMAO) in the liver by the

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hepatic enzyme flavinemonooxygenase 3 (FMO3). TMAO induces atherosclerosis in mice and TMAO plasma levels have been shown to correlate with incidence of cardiovascular disease in humans. In a recent study of the same group [46], it was shown that TMAO

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may exert its action via influencing blood platelet hyper responsiveness and thrombosis, which for the first time provides a mechanistic link between TMAO and cardiovascular

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risk. Unravelling the metabolic pathways involved in TMAO metabolism provides a beautiful example how the interaction between microbial activity and host metabolism can be elucidated. In addition, this approach allows targeted treatment focused at

disease [47].

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specific metabolites produced by the involved microbial strains that are linked to

Finally, next to production of novel metabolites, gut bacteria may alter the

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physicochemical properties of endogenous metabolites. An example of such a process is

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the metabolism of bile acids in the ileum and large intestine. Bile acids are the major degradation product of cholesterol and serve as a metabolic soap in the proximal small intestine solubilizing lipids and fat soluble vitamins. In addition, after (re)absorption in the terminal ileum, bile acids generate a postprandial signal via activation of the nuclear hormone receptor farnesoid X receptor (FXR) in ileum and liver, and via the G-protein coupled receptor TGR5 in entero-endocrine L cells, but also in immune cells [48] . Intestinal bacteria modify the properties of the bile acids in two independent ways. On 10

ACCEPTED MANUSCRIPT the one hand, they deconjugate the molecules, splitting off taurine or glycine molecules. Subsequently, the unconjugated bile acids can be dehydroxylated by specific strains from the clostridium genus in the large intestine. Dehydroxylation renders the bile acid molecules more hydrophobic and better ligands for both FXR and TGR5 receptors.

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Further indications that bile acids play an important role in regulation of host energy metabolism stems from experiments with Roux-en-Y gastric (RYGB) bypass surgery. It is well known that RYGB surgery is the most effective intervention to induce weight loss

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[49]. Additionally, the intervention is effective in the long-term regarding weight loss, and was shown to decrease mortality rate compared to conventional treatment with diet

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and exercise. The, almost direct, effects measured in metabolic parameters are not likely to be explained by the decreased calorie intake or weight loss alone. Supporting an important role for bile acids, vertical sleeve gastrectomy (VSG) in (non bile acid responding) FXR knock-out mice failed to reproduce the beneficial effects of the surgery

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[50] and simple diversion of the biliary flow from duodenum to ileum reproduced all beneficial effects of RYGB on energy metabolism in rats. [51] Zhang et al found a new established composition of gut microbiota in subjects undergoing RYBG surgery,

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differing from obese or normal-weight individuals. [52] To test the hypothesis that the

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positive effects of RYGB on weight and other clinical parameters is driven by changes in gut microbial composition, Liou et al transplanted feces from mice after RYGB surgery or a sham surgery into germ-free mice. Indeed, this resulted in a significant weight loss and a decreased fat mass in the mice receiving feces from the RYGB surgery mice compared to the mice receiving feces from the sham surgery animals. [53] A possible next step would be to confirm the effect of this kind of fecal transplantation with post RYGB donors in humans. 11

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Bacterial translocation Next to metabolic interaction, exchange at the innate immune level may play an important role in the crosstalk between intestinal microbiome and host metabolism.

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It is important to note that in the insulin resistant state and T2DM, a low-grade

inflammatory state exists driven by multiple factors (including altered fatty acid

metabolism) that are linked to impaired insulin sensitivity in muscle and adipose tissue,

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as well as defective pancreatic islet function [54, 55]. The primary trigger(s) underlying this state of chronic metabolic inflammation is still unclear, but intestinal microbiota

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have been linked to this chronic low grade inflammatory state. Cani et al were the first to report that a low-grade endotoxemia exists in mice fed a high- fat diet, which was linked to obesity, insulin resistance and diabetes, [56], probably via altered innate [57] and adaptive immunity [58]. Data from cohort studies have suggested similar relations

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in human T2DM [59]. It is thought that plasma levels of lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, can induce a host of proinflammatory responses via activation of Toll-like receptors 2, 4 and 5, which are crucial

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parts of the pattern recognition receptors (PRR). Moreover, increased portal vein as well

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as systemic plasma LPS concentrations, enabled by a dietary-induced increase in gut permeability and disruption of tight-junction proteins that connect epithelial cells [60] may be held accountable for this phenomenon. However, risk of exogenous contamination [61] might be a limiting factor to translate these results.

How could the gut microbiome be exploited in preventing or treating obesity and T2DM?

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ACCEPTED MANUSCRIPT As detailed, convincing evidence for a potential causal role of microbiota in diverse diseases including obesity and T2DM comes from studies in rodent models (Fig 1). In a landmark study, Turnbaugh et al showed that transplantation of feces from obese mice in lean germ free recipient mice induced a surprising high increase in weight in the

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recipient mice, who are normally resistant to high-fat diets. [62] Vice versa, fecal

microbiota transplantation (FMT) of feces from lean mice in obese animals induced weight loss without affecting food intake suggesting increased energy dissipation.

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Transfer of human feces into GF mice has also been carried out. Ridaura et al showed in a very interesting experiment that FMT of feces derived from twin pairs discordant for

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obesity to germ-free mice induced more weight gain after FMT of feces from the obese co-twin compared to feces from the lean co-twin. [38] These studies, albeit in rodents, demonstrate that transplantation of feces in feasible and is able to induce significant metabolic effects. Oral treatment with a mixture of beneficial bacterial strains may be a

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successful treatment modality in the future. However, until the right combination of strains has been found, FMT may serve as alternative, but this requires stringent

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development of standard operation procedures (SOPs).

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Interestingly, FMT has had a long history in medicine for treating a number of human diseases. It was not until the late 1950s that FMT garnered interest again when the first reports about its use to treat fulminant enterocolitis appeared in the scientific literature [63]However, FMT’s breakthrough as the method of choice for the treatment of persistent Clostridium difficile infection (CDI) came only after a double blind randomized trial [64], which demonstrated 94% efficacy of FMT compared with 31% after antibiotics treatment. FMT as a treatment modality is currently tested in a range of other 13

ACCEPTED MANUSCRIPT diseases, such as inflammatory bowel disease (IBD), irritable bowel syndrome, obesity and metabolic syndrome as well as neurological and psychological disorders, although most reports of the latter category are merely anecdotal. Yet, the results so far are not convincing. Reports about effects of FMT on remission in IBD patients show

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considerable heterogeneity in terms of clinical effect [65, 66] underscoring that the composition of the gut microbiota may play a role in the etiology of these diseases.

Similarly, peripheral insulin sensitivity in obese subjects significantly improved 6 weeks

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after lean donor FMT, but at 12 weeks, no significant effect was observed any longer on metabolic parameters nor on fecal microbiota composition [67] An interesting, although

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also anecdotal finding supporting a stable effect of feces transfer between humans is the case of FMT from an overweight daughter to her mother suffering from CDI. At baseline mother had a BMI of 26 while, without changing lifestyle in this period, after 36 months her BMI was increased to 34,5 suggesting that underweight could also be modulated by

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targeting the gutmicrobiota. [68] The reason why FMT has such variable efficacy is probably due to the stability and resilience of the gut microbiota. CDI patients who have been treated multiple times with antibiotics have most of their bacterial diversity wiped

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out; FMT effectively repopulates this vacated space with a stable and healthy bacterial

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community [69]. In contrast, FMT recipients with metabolic syndrome were obese but otherwise healthy individuals with a stable and diverse fecal microbiota. Further investigation of why FMT failed to engraft novel species or change the gut microbiota in these naïve patients confirmed that their bacterial community is largely resilient to accepting new members in their midst. This “gated community” makes exceptions only for donor strains that are closely related – as identified via single nucleotide variants – to species that are already present in the patient’s intestine [77]. Finally, in a recent 14

ACCEPTED MANUSCRIPT small case series of patients with CDI it was shown that transfer of sterile filtrates from donor stool rather than FMT was effective in eliminating symptoms suggesting that other fecal components such as metabolites and bacteriophages mediate many of the

Conclusions and future perspectives

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effects of FMT[70]

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Given the current obesity and T2DM epidemic, novel treatment options are urgently warranted. Prebiotics including fiber are an option, but might be more effective in

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combination with spiking the diet with beneficial bacterial strains. The gut microbiota has received much attention due to its association with obesity and T2DM., although causality remains to be proven. Unfortunately, contrasting data are frequently reported and many studies have been carried out in rodents that have a markedly different gut

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microbiota and (innate) immune system. underscoring that murine causation studies may, but does not necessarily equate to human causation regading metabolic microbiota in metabolism [71] Clearly, we are only beginning to understand the mechanisms by

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which gut microbiota (or their metabolites) drive host-metabolism and immunological function in humans. This is partly caused by the fact that the techniques necessary to

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address these questions are also in development, including assessment of sequencings methods, computational analysis, isolation of bacterial metabolites in both plasma and feces as well as validation as to whether bacterial translocation exists. Improvements in standardization of these techniques will provide a big step forward for this field.

Some encouraging progress has been made in terms of investigating causality using FMT in obesity humans. However, FMT procedures require further standardization. First, 15

ACCEPTED MANUSCRIPT SOP’s need to be made to guarantee feces of similar quality and purification, as it may be possible that not only bacteria from donor feces may be responsible for metabolic effects, but also other microorganisms like phages or even host proteins and metabolites. This means that differences in the healthy donors may be responsible for

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the different outcomes. Second, the protocols in terms of frequency and dose of FMT need to be standardized. Similarly, it may be beneficial to combine FMT with a specific diet or antibiotic pretreatment, to stimulate engraftment of donor bacteria. Also delivery

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in capsules could enhance the efficacy of FMT. Third, the safety of fecal transplant needs to be monitored in terms of (serious) adverse events that are encountered. Finally, as

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previously discussed, more clarity is needed whether FMT with encapsulated wellcharacterized mixtures of cultured microorganisms that have been proven to cure human disease is more beneficial. A strong effect of a single strain is unlikely to improve human health. Indeed, a combination of several novel probiotic bacterial strains has

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been shown to successfully treat patients with CDI suggesting that this may be way to go [72]. To this end, it will be important to move away from small, cross sectional studies towards larger prospective studies [73]. In this regard,, Falcony et al estimated that a

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sample size of 1700 subjects per study is probably needed to adequately asses the

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relationship between obesity and microbiota composition in a cohort study when one corrects for age, gender and other variables[74]

In conclusion the intestinal microbiota has great therapeutic potential in obesity and T2DM given the fact that multiple studies showed correlations between the microbiota composition and these conditions. Therefore, further studies are needed to establish the role of fecal microbiota in metabolism which will most likely lead to sub-classifications 16

ACCEPTED MANUSCRIPT of disease types. For example, one might envision diabetes cases that are directly driven by intestinal bacterial composition versus cases of diabetes, in which gutmicrobiota do not play a major role in the pathophysiology of this disease. Ultimately, these efforts may lead to the development of more personalized diagnostic and

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therapeutic tools by identifying specific strains or ‘tailor-made’ bacterial mixtures while improving engraftment by either diet of medication (eg short term oral antibiotics) to

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improve metabolic control. .

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CONFLICTS OF INTEREST

M.N. is in the Scientific Advisory Board of Caelus Pharmaceuticals, the Netherlands. None of these conflicts of interest is directly related to the research currently described.

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FUNDING

M. Nieuwdorp is supported by a ZONMW-VIDI grant 2013 (016.146.327) and CVON Young Talent grant 2012. DVR is supported by the Dutch Diabetes Fonds - Junior

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Fellowship (2015.81) and a Marie Curie Fellowship.

FIGURE LEGENDS.

Fig 1 | Schematic overview of functions attributed to intestinal bacterial strains.. SCFA; short chain fatty acids. GPR41; G-protein coupled receptor 41, GPR43; G-protein coupled receptor 43, GLP1;

Glucagon Like Protein 1, PYY; peptide YY, LPS;

lipopolysaccharide, TLR4; Toll like receptor 4, TMA; trimethylamine; FMO3; FlavinContaining Monooxygenase, TMAO; Trimethylamine-N-oxide, FXR; farnesoid X receptor TGR5 transmembrane G protein-coupled receptor.

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