Gut microbiome and health : mechanistic insights

The gut microbiota is now considered as one of the key elements contributing to the regulation of host health. Virtually all our body sites are colonised by microbes suggesting different types of crosstalk with our organs. Because of the development of molecular tools and techniques (ie, metagenomic, metabolomic, lipidomic, metatranscriptomic), the complex interactions occurring between the host and the different microorganisms are progressively being deciphered. Nowadays, gut microbiota deviations are linked with many diseases including obesity, type 2 diabetes, hepatic steatosis, intestinal bowel diseases (IBDs) and several types of cancer. Thus, suggesting that various pathways involved in immunity, energy, lipid and glucose metabolism are affected. In this review, specific attention is given to provide a critical evaluation of the current understanding in this field. Numerous molecular mechanisms explaining how gut bacteria might be causally linked with the protection or the onset of diseases are discussed. We examine well-established metabolites (ie, short-chain fatty acids, bile acids, trimethylamine N-oxide) and extend this to more recently identified molecular actors (ie, endocannabinoids, bioactive lipids, phenolic-derived compounds, advanced glycation end products and enterosynes) and their specific receptors such as peroxisome proliferator-activated receptor alpha (PPAR alpha) and gamma (PPAR gamma), aryl hydrocarbon receptor (AhR), and G protein-coupled receptors (ie, GPR41, GPR43, GPR119, Takeda G protein-coupled receptor 5). Altogether, understanding the complexity and the molecular aspects linking gut microbes to health will help to set the basis for novel therapies that are already being developed.Peer reviewe

Pasteurized Akkermansia muciniphila increases whole-body energy expenditure and fecal energy excretion in diet-induced obese mice

Accumulating evidence points to Akkermansia muciniphila as a novel candidate to prevent or treat obesity-related metabolic disorders. We recently observed, in mice and in humans, that pasteurization of A. muciniphila increases its beneficial effects on metabolism. However, it is currently unknown if the observed beneficial effects on body weight and fat mass gain are due to specific changes in energy expenditure. Therefore, we investigated the effects of pasteurized A. muciniphila on whole-body energy metabolism during high-fat diet feeding by using metabolic chambers. We confirmed that daily oral administration of pasteurized A. muciniphila alleviated diet-induced obesity and decreased food energy efficiency. We found that this effect was associated with an increase in energy expenditure and spontaneous physical activity. Strikingly, we discovered that energy expenditure was enhanced independently from changes in markers of thermogenesis or beiging of the white adipose tissue. However, we found in brown and white adipose tissues that perilipin2, a factor associated with lipid droplet and known to be altered in obesity, was decreased in expression by pasteurized A. muciniphila. Finally, we observed that treatment with pasteurized A. muciniphila increased energy excretion in the feces. Interestingly, we demonstrated that this effect was not due to the modulation of intestinal lipid absorption or chylomicron synthesis but likely involved a reduction of carbohydrates absorption and enhanced intestinal epithelial turnover. In conclusion, this study further dissects the mechanisms by which pasteurized A. muciniphila reduces body weight and fat mass gain. These data also further support the impact of targeting the gut microbiota by using specific bacteria to control whole-body energy metabolism.Peer reviewe

Toxicological safety evaluation of pasteurizedAkkermansia muciniphila

Gut microorganisms are vital for many aspects of human health, and the commensal bacteriumAkkermansia muciniphilahas repeatedly been identified as a key component of intestinal microbiota. Reductions inA. muciniphilaabundance are associated with increased prevalence of metabolic disorders such as obesity and type 2 diabetes. It was recently discovered that administration ofA. muciniphilahas beneficial effects and that these are not diminished, but rather enhanced after pasteurization. PasteurizedA. muciniphilais proposed for use as a food ingredient, and was therefore subjected to a nonclinical safety assessment, comprising genotoxicity assays (bacterial reverse mutation and in vitro mammalian cell micronucleus tests) and a 90-day toxicity study. For the latter, Han Wistar rats were administered with the vehicle or pasteurizedA. muciniphilaat doses of 75, 375 or 1500 mg/kg body weight/day (equivalent to 4.8 x 10(9), 2.4 x 10(10), or 9.6 x 10(10)A. muciniphilacells/kg body weight/day) by oral gavage for 90 consecutive days. The study assessed potential effects on clinical observations (including detailed arena observations and a modified Irwin test), body weight, food and water consumption, clinical pathology, organ weights, and macroscopic and microscopic pathology. The results of both in vitro genotoxicity studies were negative. No test item-related adverse effects were observed in the 90-day study; therefore, 1500 mg/kg body weight/day (the highest dose tested, equivalent to 9.6 x 10(10)A. muciniphilacells/kg body weight/day) was established as the no-observed-adverse-effect-level. These results support that pasteurizedA. muciniphilais safe for use as a food ingredient.Peer reviewe

Hepatocyte MyD88 affects bile acids, gut microbiota and metabolome contributing to regulate glucose and lipid metabolism

OBJECTIVE: To examine the role of hepatocyte myeloid differentiation primary-response gene 88 (MyD88) on glucose and lipid metabolism. DESIGN: To study the impact of the innate immune system at the level of the hepatocyte and metabolism, we generated mice harbouring hepatocyte-specific deletion of MyD88. We investigated the impact of the deletion on metabolism by feeding mice with a normal control diet or a high-fat diet for 8 weeks. We evaluated body weight, fat mass gain (using time-domain nuclear magnetic resonance), glucose metabolism and energy homeostasis (using metabolic chambers). We performed microarrays and quantitative PCRs in the liver. In addition, we investigated the gut microbiota composition, bile acid profile and both liver and plasma metabolome. We analysed the expression pattern of genes in the liver of obese humans developing non-alcoholic steatohepatitis (NASH). RESULTS: Hepatocyte-specific deletion of MyD88 predisposes to glucose intolerance, inflammation and hepatic insulin resistance independently of body weight and adiposity. These phenotypic differences were partially attributed to differences in gene expression, transcriptional factor activity (ie, peroxisome proliferator activator receptor-α, farnesoid X receptor (FXR), liver X receptors and STAT3) and bile acid profiles involved in glucose, lipid metabolism and inflammation. In addition to these alterations, the genetic deletion of MyD88 in hepatocytes changes the gut microbiota composition and their metabolomes, resembling those observed during diet-induced obesity. Finally, obese humans with NASH displayed a decreased expression of different cytochromes P450 involved in bioactive lipid synthesis. CONCLUSIONS: Our study identifies a new link between innate immunity and hepatic synthesis of bile acids and bioactive lipids. This dialogue appears to be involved in the susceptibility to alterations associated with obesity such as type 2 diabetes and NASH, both in mice and humans

IGF2 mRNA Binding Protein 2 Transgenic Mice Are More Prone to Develop a Ductular Reaction and to Progress Toward Cirrhosis

The insulin-like growth factor 2 (IGF2) mRNA binding proteins (IMPs/IGF2BPs) IMP1 and 3 are regarded as oncofetal proteins, whereas the hepatic IMP2 expression in adults is controversially discussed. The splice variant IMP2-2/p62 promotes steatohepatitis and hepatocellular carcinoma. Aim of this study was to clarify whether IMP2 is expressed in the adult liver and influences progression toward cirrhosis. IMP2 was expressed at higher levels in embryonic compared to adult tissues as quantified in embryonic, newborn, and adult C57BL/6J mouse livers and suggested by analysis of publicly available human data. In an IMP2-2 transgenic mouse model microarray and qPCR analyses revealed increased expression of liver progenitor cell (LPC) markers Bex1, Prom1, Spp1, and Cdh1 indicating a de-differentiated liver cell phenotype. Induction of these LPC markers was confirmed in human cirrhotic tissue datasets. The LPC marker SPP1 has been described to play a major role in fibrogenesis. Thus, DNA methylation was investigated in order to decipher the regulatory mechanism of Spp1 induction. In IMP2-2 transgenic mouse livers single CpG sites were differentially methylated, as quantified by amplicon sequencing, whereas human HCC samples of a human publicly available dataset showed promoter hypomethylation. In order to study the impact of IMP2 on fibrogenesis in the context of steatohepatitis wild-type or IMP2-2 transgenic mice were fed either a methionine-choline deficient (MCD) or a control diet for 2-12 weeks. MCD-fed IMP2-2 transgenic mice showed a higher incidence of ductular reaction (DR), accompanied by hepatic stellate cell activation, extracellular matrix (ECM) deposition, and induction of the LPC markers Spp1, Cdh1, and Afp suggesting the occurrence of de-differentiated cells in transgenic livers. In human cirrhotic samples IMP2 overexpression correlated with LPC marker and ECM component expression. Progression of liver disease was induced by combined MCD and diethylnitrosamine (DEN) treatment. Combined MCD-DEN treatment resulted in shorter survival of IMP2-2 transgenic compared to wild-type mice. Only IMP2-2 transgenic livers progressed to cirrhosis, which was accompanied by strong DR. In conclusion, IMP2 is an oncofetal protein in the liver that promotes DR characterized by de-differentiated cells toward steatohepatitis-associated cirrhosis development with poor survival

Impact of metformin and Dysosmobacter welbionis on diet-induced obesity and diabetes: from clinical observation to preclinical intervention.

peer reviewed[en] AIMS/HYPOTHESIS: We aimed to investigate the association between the abundance of Dysosmobacter welbionis, a commensal gut bacterium, and metabolic health in human participants with obesity and diabetes, and the influence of metformin treatment and prebiotic intervention. METHODS: Metabolic variables were assessed and faecal samples were collected from 106 participants in a randomised controlled intervention with a prebiotic stratified by metformin treatment (Food4Gut trial). The abundance of D. welbionis was measured by quantitative PCR and correlated with metabolic markers. The in vitro effect of metformin on D. welbionis growth was evaluated and an in vivo study was performed in mice to investigate the effects of metformin and D. welbionis J115T supplementation, either alone or in combination, on metabolic variables. RESULTS: D. welbionis abundance was unaffected by prebiotic treatment but was significantly higher in metformin-treated participants. Responders to prebiotic treatment had higher baseline D. welbionis levels than non-responders. D. welbionis was negatively correlated with aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and fasting blood glucose levels in humans with obesity and type 2 diabetes. In vitro, metformin had no direct effect on D. welbionis growth. In mice, D. welbionis J115T treatment reduced body weight gain and liver weight, and improved glucose tolerance to a better level than metformin, but did not have synergistic effects with metformin. CONCLUSIONS/INTERPRETATION: D. welbionis abundance is influenced by metformin treatment and associated with prebiotic response, liver health and glucose metabolism in humans with obesity and diabetes. This study suggests that D. welbionis may play a role in metabolic health and warrants further investigation. CLINICAL TRIAL: NCT03852069

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Role of Gelatinases in the Development of Adipose Tissue

In 1999 the World Health Organization (WHO) declared obesity a worldwide epidemic. Today, it is estimated that around 500 million people worldwide are obese, and more than 1.6 billion are overweight. Diseases associated with excess body weight include among others: cardiovascular disease, hypertension, stroke, diabetes type II, atherosclerosis and certain forms of cancer. As a result, obesity recently became the number one cause of preventable death.Obesity is defined as an excessively high amount of adipose tissue in relation to lean body mass. Development of adipose tissue is a complex process in which several growth factors, cytokines, hormones and proteinases contribute to adipogenesis (formation of adipocytes), angiogenesis (formation of blood vessels) and proteolytic remodeling of the extracellular matrix (ECM). The matrix metalloproteinases (MMPs) form a family of zinc-dependent endopeptidases that play an important role in these processes by degradation of ECM and basement membrane components, by altering cell interactions and/or by activation of latent growth factors. This has led to the hypothesis that modulation of the activity of certain MMPs may have an effect on adipose tissue development. In the present study we have focused on the contribution of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). It was previously shown that they are both expressed by adipocytes and modulated during development of adipose tissue. To evaluate their potential role in adipogenesis, we set up a range of in vivo and in vitro experiments using different murine models.In vivo:- MMP-2 and MMP-9 deficient mice were kept on a high fat diet (HFD) for 15 weeks and compared to wild-type littermates. This revealed no differences for the MMP-9-deficient mice. Incontrast, MMP-2 deficient mice had a 20% reduction of body weight, associated with smaller fat pads en hypotrophy of adipocytes and preservation of collagen.- Wild-type mice on a HFD treated with a relative gelatinase specific inhibitor (Tolylsam) for 15 weeks, developed less adipose tissue, resulting in lower body weights. This was associated with adipocyte hypotrophy and reduced vascularization and higher levels of total collagen. - Leptin deficient ob/ob mice (a genetically induced obesity model) were significantly leaner when treated with Tolylsam.- Adipose tissue growth was impaired in wild obese type mice that were switched to a HFD supplemented with Tolylsam, whereas mice that were kept on the HFD only, continued to gain weight. Tolylsam treatment resulted in preservation of collagen. - De novo fat pad formation in NUDE mice following injection of 3T3-F442A preadipocytes was impaired in mice treated with Tolylsam. - MMP-2 deficient mice developed less adipose tissue when treated with Tolylsam. This was associated with adipocyte hypotrophy and reduced angiogenesis. In vitro:- Downregulation of MMP-2 expression in 3T3-F442A preadipocytes resulted in reduced differentiation into mature adipocytes. - 3T3-F442A preadipocytes cultured in the presence of Tolylsam showed increased differentiation as compared to vehicle treated cells. This may be related to the relatively low substrate specificity of Tolylsam.Taken together, these results reveal divergent functions for gelatinases in the development of adipose tissue: MMP-2 appears to enhance adipose tissue development in mice on high fat diet, at least in part by contributing to adipocyte growth, whereas MMP-9 does not seem crucial. In addition, adipose tissue development can be impaired by oral administration of the relatively gelatinase-specific inhibitor Tolylsam, which is not only a MMP inhibitor, preserving collagen in the ECM, but also impairs blood vessel formation.LIST OF ABBREVIATIONS 9 INTRODUCTION 11 1. Obesity 11 1.1 The outbreak of an epidemic 11 1.2 Definition and diagnosis of obesity 12 1.3 Medical complications of obesity 13 1.4 The burden of excess weight 14 1.5 Causes of obesity 15 1.6 Treatment of obesity 15 2. Adipose tissue 17 2.1 More than just storage 17 2.2 The adipocyte and its surroundings 18 2.3 Adipose tissue development 19 2.4 Adipogenesis 21 2.5 Murine models to study adipogenesis 23 3. Matrix Metalloproteinases (MMPs) 25 3.1 A historical perspective 25 3.2 Classification and general structure 25 3.3 Regulation of MMP activity 27 3.4 Models to study MMPs 33 3.5 Function 34 3.6 Synthetic inhibitors of MMPs 37 3.7 MMPs and adipose tissue development 38 3.8 Gelatinases 39 3.9 Genes of gelatinases 40 3.10 MT1-MMP/TIMP-2 mediated MMP-2 activation 41 AIMS 45 MATERIALS AND METHODS 47 1. Animal models 47 1.1 Mouse strains 47 1.2 Murine models 47 1.2.1 Nutritionally induced obesity in gelatinase deficient mice (model 1) 49 1.2.2 Nutritionally induced obesity in mice treated with a gelatinase inhibitor (model 2) 49 1.2.3 Treatment with a gelatinase inhibitor of mice with established obesity (model 3) 51 1.2.4 de novo adipogenesis (model 4) 52 1.3 Analysis 52 1.3.1 Histological analysis 52 1.3.2 Gelatinase determination 53 1.3.3 Analysis of mRNA expression 54 1.3.4 Metabolic parameters 55 2. Cell culture experiments 55 2.1 in vitro differentiation 55 2.2 In vitro gene silencing 55 2.3 Analysis 57 2.3.1 Assessment of cell proliferation and viability 57 2.3.2 Differentiation assay 57 2.3.3 Gelatinase determination 57 2.3.4 Analysis of mRNA expression 57 3. Statistical analysis 58 RESULTS 61 1. Effect of MMP-9 deficiency on adipose tissue development 61 1.1 Body weight and adipose tissue composition of mice kept on standard fat diet 61 2.2 Body weight and adipose tissue composition of mice kept on high fat diet 64 2. Effect of MMP-2 deficiency on adipose tissue development 70 2.1 Body weight and adipose tissue composition of mice kept on standard fat diet 70 2.2 Body weight and adipose tissue composition of mice kept on high fat diet 72 3. Effect of gelatinase inhibition on adipose tissue development in wild-type mice 77 4. Effect of gelatinase inhibition on adipose tissue development in leptin deficient (ob/ob) mice 83 5. Effect of gelatinase inhibition on adipose tissue development in wild-type obese mice 87 6. Effect of gelatinase inhibition on adipose tissue development in MMP-2 deficient mice 94 7. Effect of gelatinase inhibition on de novo adipogenesis 97 8. In vitro experiments 99 8.1 Effect of gelatinase inhibition on 3T3-F442A preadipocyte differentiation 99 8.2 Effect of MMP-2 knock-down on 3T3-F442A preadipocytes differentiation 103 DISCUSSION 107 Background 107 Lessons from MMP knock-out mice 108 Gelatinase inhibition in murine models of obesity 109 Lessons from in vitro experiments 113 General conclusion 114 MMPs as therapeutic targets 114 Limitations and suggestions for future experiments 115 LIST OF PUBLICATIONS DISCUSSED IN THIS THESIS 117 REFERENCES 118 SUMMARY 143 SAMENVATTING 145 ACKNOWLEDGMENTS 147 CURRICULUM VITAE 151 LIST OF PUBLICATIONS 153nrpages: 154status: publishe

Mediterranean diet, gut microbiota and health: when age and calories do not add up!

A healthy diet is generally recognised as a diet that supports the physiological and energetic requirements of the body and provides sufficient, though not excessive, amounts of micronutrients and macronutrients. Despite this self-evident definition, the implementation of this basic principle has proven very difficult in modern western society. [...