Search

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. ispartof: NATURE MEDICINE vol:28 issue:2 pages:303-+ ispartof: location:United States status: published

ObjectiveGut microbial products are involved in regulation of host metabolism. In human and experimental studies, we explored the potential role of hippurate, a hepatic phase 2 conjugation product of microbial benzoate, as a marker and mediator of metabolic health.DesignIn 271 middle-aged non-diabetic Danish individuals, who were stratified on habitual dietary intake, we applied 1H-nuclear magnetic resonance (NMR) spectroscopy of urine samples and shotgun-sequencing-based metagenomics of the gut microbiome to explore links between the urine level of hippurate, measures of the gut microbiome, dietary fat and markers of metabolic health. In mechanistic experiments with chronic subcutaneous infusion of hippurate to high-fat-diet-fed obese mice, we tested for causality between hippurate and metabolic phenotypes.ResultsIn the human study, we showed that urine hippurate positively associates with microbial gene richness and functional modules for microbial benzoate biosynthetic pathways, one of which is less prevalent in the Bacteroides 2 enterotype compared with Ruminococcaceae or Prevotella enterotypes. Through dietary stratification, we identify a subset of study participants consuming a diet rich in saturated fat in which urine hippurate concentration, independently of gene richness, accounts for links with metabolic health. In the high-fat-fed mice experiments, we demonstrate causality through chronic infusion of hippurate (20 nmol/day) resulting in improved glucose tolerance and enhanced insulin secretion.ConclusionOur human and experimental studies show that a high urine hippurate concentration is a general marker of metabolic health, and in the context of obesity induced by high-fat diets, hippurate contributes to metabolic improvements, highlighting its potential as a mediator of metabolic health.

International audience; The human gut is a home for more than 100 trillion bacteria, far more than all other microbial populations resident on the body's surface. The human gut microbiome is considered as a microbial organ symbiotically operating within the host. It is a collection of different cell lineages that are capable of communicating with each other and the host and has an ability to undergo self-replication for its repair and maintenance. As the gut microbiota is involved in many host processes including growth and development, an imbalance in its ecological composition may lead to disease and dysfunction in the human. Gut microbial degradation of nutrients produces bioactive metabolites that bind target receptors, activating signalling cascades, and modulating host metabolism. This review covers current findings on the nutritional and pharmacological roles of selective gut microbial metabolites, short-chain fatty acids, methylamines and indoles, as well as discussing nutritional interventions to modulate the microbiome.

ObjectiveGut microbial products are involved in type 2 diabetes, obesity and insulin resistance. In particular, hippurate, a hepatic phase 2 conjugation product of microbial benzoate metabolism, has been associated with a healthy phenotype. This study aims to identify metagenomic determinants and test protective effects of hippurate.DesignWe profiled the urine metabolome by 1H Nuclear Magnetic Resonance (NMR) spectroscopy to derive associations with metagenomic sequences in 271 middle-aged Danish individuals to identify dietary patterns in which urine hippurate levels were associated with health benefits. We follow up with benzoate and hippurate infusion in mice to demonstrate causality on clinical phenotypes.ResultsIn-depth analysis identifies that the urine hippurate concentration is associated with microbial gene richness, microbial functional redundancy as well as functional modules for microbial benzoate biosynthetic pathways across several enterotypes. Through dietary stratification, we identify a subset of study participants consuming a diet rich in saturated fat in which urine hippurate, independently of gene richness, accounts for links with metabolic health that we previously associated with gene richness. We then demonstrate causality in vivo through chronic subcutaneous infusions of hippurate or benzoate (20 nmol/day) resulting in improved glycemic control in mice fed a high-fat diet. Hippurate improved insulin secretion through increased β-cell mass and reduced liver inflammation and fibrosis, whereas benzoate treatment resulted in liver inflammation.ConclusionOur translational study shows that the benzoate-hippurate pathway brings a range of metabolic improvements in the context of high-fat diets, highlighting the potential of hippurate as a mediator of metabolic health.

MotivationIndoles have been shown to play a significant role in cardiometabolic disorders. While some individual bacterial species are known to produce indole-adducts, to our best knowledge no studies have made use of publicly available genome data to identify prokaryotes, specifically those associated with the human gut microbiota, contributing to the indole metabolic network.ResultsHere, we propose a computational strategy, comprising the integration of KEGG and BLAST, to identify prokaryote-specific metabolic reactions relevant for the production of indoles, as well as to predict new members of the human gut microbiota potentially involved in these reactions. By identifying relevant prokaryotic species for further validation studiesin vitro, this strategy represents a useful approach for those interrogating the metabolism of other gut-derived microbial metabolites relevant to human health.AvailabilityAll R scripts and files (gut microbial dataset, FASTA protein sequences, BLASTP output files) are available fromhttps://github.com/AndreaRMICL/Microbial_networks.ContactARM:andrea.rodriguez-martinez13@imperial.ac.uk; LH:lesley.hoyles@ntu.ac.uk.

International audience; The excessive endogenous glucose production (EGP) induced by glucagon participates in the development of type 2 diabetes. To further understand this hormonal control, we studied the short-term regulation by cyclic adenosine monophosphate (cAMP) of the glucose-6-phosphatase (G6Pase) enzyme, which catalyzes the last reaction of EGP. In gluconeogenic cell models, a 1-h treatment by the adenylate cyclase activator forskolin increased G6Pase activity and glucose production independently of any change in enzyme protein amount or G6P content. Using specific inhibitors or protein overexpression, we showed that the stimulation of G6Pase activity involved the protein kinase A (PKA). Results of site-directed mutagenesis, mass spectrometry analyses, and in vitro phosphorylation experiments suggested that the PKA stimulation of G6Pase activity did not depend on a direct phosphorylation of the enzyme. However, the temperature-dependent induction of both G6Pase activity and glucose release suggested a membrane-based mechanism. G6Pase is composed of a G6P transporter (G6PT) and a catalytic unit (G6PC). Surprisingly, we demonstrated that the increase in G6PT activity was required for the stimulation of G6Pase activity by forskolin. Our data demonstrate the existence of a post-translational mechanism that regulates G6Pase activity and reveal the key role of G6PT in the hormonal regulation of G6Pase activity and of EGP.Erratum inCorrection to "Post-Translational Regulation of the Glucose-6-Phosphatase Complex by Cyclic Adenosine Monophosphate Is a Crucial Determinant of Endogenous Glucose Production and Is Controlled by the Glucose-6-Phosphate Transporter". [J Proteome Res. 2016]PMID: 27111186 DOI: 10.1021/acs.jproteome.6b00284

SUMMARYGut microbiota changes are associated with increased risk of Type 2 diabetes (T2D) and obesity. Through serum metabolome profiling in patients with cardiometabolic disease (CMD) we identified significant inverse correlation between the microbial metabolite 4-cresol and T2D. Chronic administration of non toxic dose of 4-cresol in two complementary preclinical models of CMD reduced adiposity, glucose intolerance and liver triglycerides, and enhanced insulin secretion in vivo, which may be explained by markedly increased pancreas weight, augmented islet density and size, and enhanced vascularisation suggesting activated islet neogenesis. Incubation of isolated islets with 4-cresol enhanced insulin secretion, insulin content and cell proliferation. In both CMD models 4-cresol treatment in vivo was associated with altered expression of SIRT1 and the kinase DYRK1A, which may contribute to mediate its biological effects. Our findings identify 4-cresol as an effective regulator of β-cell function and T2D endophenotypes, which opens therapeutic perspectives in syndromes of insulin deficiency.

In the version of this article originally published, the received date was missing. It should have been listed as 2 January 2018. The error has been corrected in the HTML and PDF versions of this article.

Hepatic steatosis is a multifactorial condition that is often observed in obese patients and is a prelude to non-alcoholic fatty liver disease. Here, we combine shotgun sequencing of fecal metagenomes with molecular phenomics (hepatic transcriptome and plasma and urine metabolomes) in two well-characterized cohorts of morbidly obese women recruited to the FLORINASH study. We reveal molecular networks linking the gut microbiome and the host phenome to hepatic steatosis. Patients with steatosis have low microbial gene richness and increased genetic potential for the processing of dietary lipids and endotoxin biosynthesis (notably from Proteobacteria), hepatic inflammation and dysregulation of aromatic and branched-chain amino acid metabolism. We demonstrated that fecal microbiota transplants and chronic treatment with phenylacetic acid, a microbial product of aromatic amino acid metabolism, successfully trigger steatosis and branched-chain amino acid metabolism. Molecular phenomic signatures were predictive (area under the curve = 87%) and consistent with the gut microbiome having an effect on the steatosis phenome (>75% shared variation) and, therefore, actionable via microbiome-based therapies. Metabolic activity of specific human gut microorganisms contributes to liver steatosis in obese women.

BACKGROUNDThe dietary methylamines choline, carnitine and phosphatidylcholine are used by the gut microbiota to produce a range of metabolites, including trimethylamine (TMA). However, little is known about the use of trimethylamine N-oxide (TMAO) by this consortium of microbes.RESULTSA feeding study using deuterated TMAO in C57BL6/J mice demonstrated microbial conversion of TMAO to TMA, with uptake of TMA into the bloodstream and its conversion to TMAO. Microbial activity necessary to convert TMAO to TMA was suppressed in antibiotic-treated mice, with deuterated TMAO being taken up directly into the bloodstream. In batch-culture fermentation systems inoculated with human faeces, growth of Enterobacteriaceae was stimulated in the presence of TMAO. Human-derived faecal and caecal bacteria (n = 66 isolates) were screened on solid and liquid media for their ability to use TMAO, with metabolites in spent media analysed by 1H-NMR. As with the in vitro fermentation experiments, TMAO stimulated the growth of Enterobacteriaceae; these bacteria produced most TMA from TMAO. Caecal/small intestinal isolates of Escherichia coli produced more TMA from TMAO than their faecal counterparts. Lactic acid bacteria produced increased amounts of lactate when grown in the presence of TMAO, but did not produce large amounts of TMA. Clostridia (sensu stricto), bifidobacteria and coriobacteria were significantly correlated with TMA production in the mixed fermentation system but did not produce notable quantities of TMA from TMAO in pure culture.CONCLUSIONSReduction of TMAO by the gut microbiota (predominantly Enterobacteriaceae) to TMA followed by host uptake of TMA into the bloodstream from the intestine and its conversion back to TMAO by host hepatic enzymes is an example of metabolic retroconversion. TMAO influences microbial metabolism depending on isolation source and taxon of gut bacterium.Correlation of metabolomic and abundance data from mixed microbiota fermentation systems did not give a true picture of which members of the gut microbiota were responsible for converting TMAO to TMA; only by supplementing the study with pure culture work and additional metabolomics was it possible to increase our understanding of TMAO bioconversions by the human gut microbiota.

One of the dieting conundrums in the age of the obesity epidemic is the cycle of weight loss and regain known as the "yo-yo effect." Thaiss et al. (2016) demonstrate that the microbiome plays a key role in this phenomenon and that simple dietary supplementations can reset the weight-rebound clock.

The microbial-mammalian symbiosis plays a critical role in metabolic health. Microbial metabolites emerge as key messengers in the complex communication between the gut microbiota and their host. These chemical signals are mainly derived from nutritional precursors, which in turn are also able to modify gut microbiota population. Recent advances in the characterization of the gut microbiome and the mechanisms involved in this symbiosis allow the development of nutritional interventions. This review covers the latest findings on the microbial-mammalian metabolic axis as a critical symbiotic relationship particularly relevant to clinical nutrition.The modulation of host metabolism by metabolites derived from the gut microbiota highlights the importance of gut microbiota in disease prevention and causation. The composition of microbial populations in our gut ecosystem is a critical pathophysiological factor, mainly regulated by diet, but also by the host's characteristics (e.g. genetics, circadian clock, immune system, age). Tailored interventions, including dietary changes, the use of antibiotics, prebiotic and probiotic supplementation and faecal transplantation are promising strategies to manipulate microbial ecology.The microbiome is now considered as an easily reachable target to prevent and treat related diseases. Recent findings in both mechanisms of its interactions with host metabolism and in strategies to modify gut microbiota will allow us to develop more effective treatments especially in metabolic diseases.

Obesity and type 2 diabetes are associated with low-grade inflammation and specific changes in gut microbiota composition. We previously demonstrated that administration of Akkermansia muciniphila to mice prevents the development of obesity and associated complications. However, the underlying mechanisms of this protective effect remain unclear. Moreover, the sensitivity of A. muciniphila to oxygen and the presence of animal-derived compounds in its growth medium currently limit the development of translational approaches for human medicine. We have addressed these issues here by showing that A. muciniphila retains its efficacy when grown on a synthetic medium compatible with human administration. Unexpectedly, we discovered that pasteurization of A. muciniphila enhanced its capacity to reduce fat mass development, insulin resistance and dyslipidemia in mice. These improvements were notably associated with a modulation of the host urinary metabolomics profile and intestinal energy absorption. We demonstrated that Amuc-1100, a specific protein isolated from the outer membrane of A. muciniphila, interacts with Toll-like receptor 2, is stable at temperatures used for pasteurization, improves the gut barrier and partly recapitulates the beneficial effects of the bacterium. Finally, we showed that administration of live or pasteurized A. muciniphila grown on the synthetic medium is safe in humans. These findings provide support for the use of different preparations of A. muciniphila as therapeutic options to target human obesity and associated disorders.

The human gut harbors more than 100 trillion microbial cells, which have an essential role in human metabolic regulation via their symbiotic interactions with the host. Altered gut microbial ecosystems have been associated with increased metabolic and immune disorders in animals and humans. Molecular interactions linking the gut microbiota with host energy metabolism, lipid accumulation, and immunity have also been identified. However, the exact mechanisms that link specific variations in the composition of the gut microbiota with the development of obesity and metabolic diseases in humans remain obscure owing to the complex etiology of these pathologies. In this review, we discuss current knowledge about the mechanistic interactions between the gut microbiota, host energy metabolism, and the host immune system in the context of obesity and metabolic disease, with a focus on the importance of the axis that links gut microbes and host metabolic inflammation. Finally, we discuss therapeutic approaches aimed at reshaping the gut microbial ecosystem to regulate obesity and related pathologies, as well as the challenges that remain in this area.

Consisting of trillions of non-pathogenic bacteria living in a symbiotic relationship with their mammalian host, the gut microbiota has emerged in the past decades as one of the key drivers for cardiometabolic diseases (CMD). By degrading dietary substrates, the gut microbiota produces several metabolites that bind human pharmacological targets, impact subsequent signalling networks and in fine modulate host's metabolism. In this review, we revisit the pharmacological relevance of four classes of gut microbial metabolites in CMD: short-chain fatty acids (SCFA), bile acids, methylamines and indoles. Unravelling the signalling mechanisms of the microbial-mammalian metabolic axis adds one more layer of complexity to the physiopathology of CMD and opens new avenues for the development of microbiota-based pharmacological therapies.

Introduction Le glucose est absorbe par les enterocytes par le co-transporteur SGLT1 a la membrane apicale et par le transporteur GLUT2 a la membrane basolaterale. En absence de GLUT2 le glucose pourrait etre absorbe via une voie impliquant l’activite de la glucose-6-phosphatase (G6Pase). Dans ce travail, nous avons etudie l’implication de GLUT2 et de la G6Pase dans l’absorption intestinale de glucose. Materiels et methodes L’absorption de glucose a ete mesuree in vivo par des tests oraux de tolerance au glucose realises apres 16 heures de jeune chez des souris sauvages (WT) et des souris presentant une deletion de la G6Pase (I. G6pc-/-), du transporteur GLUT2 (I. Glut2-/-) ou des deux genes (I. G6pc-/-. Glut2-/-) specifiquement au niveau de l’intestin. L’utilisation de glucose tritie nous a permis de mesurer l’absorption de glucose in vitro dans des chambres de Ussing. Resultats Quelque-soit le genotype, l’absorption intestinale de glucose est transcellulaire et met en jeu SGLT-1 car elle est supprimee par la phloridzine. L’invalidation de Glut2 dans l’intestin induit un retard d’absorption de glucose in vivo et une diminution de 50 % de la vitesse d’absorption in vitro. Les souris I. Glut2-/- compensent leur malabsorption du glucose en augmentant leur prise alimentaire (32,9 ± 0,5 g/kg versus 30,6 ± 0,7 g/kg chez les WT). L’invalidation de la G6Pase dans l’intestin n’a pas d’effet sur l’absorption intestinale de glucose mesuree in vivo mais induit une diminution de 50 % de la vitesse d’absorption in vitro. L’invalidation concomitante des deux genes n’a pas d’effet additionnel sur l’absorption de glucose. L’expression genique des autres transporteurs de glucose n’est pas modifiee dans l’intestin des souris I. G6pc-/-. Glut2-/-. Ces souris ne compensent par leur malabsorption par une augmentation de leur prise alimentaire. Conclusion Nos resultats montrent que GLUT2 et la G6Pase sont impliques, mais pas essentiels, dans l’absorption de glucose par les enterocytes. D’autres mecanismes sont induits en absence des deux proteines pour assurer l’absorption intestinale de glucose.

Introduction L’augmentation de la production hepatique de glucose (PHG) est associee au developpement du diabete de type 2. La PHG depend de l’activite glucose-6-phosphatase (G6Pase), qui catalyse l’hydrolyse du glucose-6-phosphate en glucose. Le flux de glucose produit par la G6Pase est dependant de la temperature suggerant l’implication de mecanismes membranaires dans la regulation de la PHG. Nous avons fait l’hypothese que la caveoline-1, composant principal des caveoles, participait a ces regulations. Materiels et methodes La PHG a ete mesuree in vitro et in vivo dans le foie de souris sauvages et invalidees pour la caveoline-1 (Cav1-/-). La localisation de la G6PC (sous-unite catalytique de la G6Pase) a ete etudiee in vivo par immuno-fluorescence sur des coupes de foie de souris. Les localisations de la G6PC et de la Cav1 ont ete etudiees in vitro via la surexpression de proteines chimeriques fluorescentes dans les cellules hepatiques HepG2. Resultats A jeun, l’absence de Cav1 induit une diminution de 50 % de la PHG parallelement a une augmentation du G6P et du glycogene, suggerant que l’absence de Cav1 inhibe l’activite G6Pase. Nous avons fait l’hypothese que la Cav1 controle l’activite G6Pase via la regulation de sa localisation. In vitro , la G6PC et la Cav1 sont colocalisees et migrent ensemble vers la membrane plasmique. In vivo , la localisation de la G6PC est ponctuee dans les hepatocytes sauvages et diffuse dans les hepatocytes Cav1-/-. L’analyse de la sequence peptidique de la G6PC suggere l’existence d’un site potentiel de liaison a la Cav1. La mutation de ce site d’interaction se traduit in vitro par une localisation diffuse de la G6PC comme observe dans les souris Cav1-/-. Conclusion Ces resultats constituent la premiere demonstration que la production de glucose est controlee par des processus cellulaires. Leur caracterisation permettrait de proposer de nouvelles cibles therapeutiques pour le traitement du diabete de type 2. Declaration d’interet Les auteurs declarent ne pas avoir d’interet direct ou indirect (financier ou en nature) avec un organisme prive, industriel ou commercial en relation avec le sujet presente.

Introduction L'obesite est associee a une inflammation chronique de faible intensite caracterisee par une augmentation des niveaux circulants de cytokines pro-inflammatoires comme l'IL-6. Cette inflammation de faible intensite peut amener au developpement du diabete de type 2 via une insulino-resistance et/ou une defaillance des cellules β. Materiels et methodes Nous avons developpe une approche originale en etudiant la reponse de 4 souches de souris a un regime gras (HFD). Cette approche permet de caracteriser leur signature metabolique par 1H-RMN et permet d'iden-tifier des biomarqueurs metaboliques de l'intolerance au glucose, du diabete et des mecanismes moleculaires sous-jacents grâce a un criblage des interactions pharmacologiques de ces biomarqueurs metaboliques. Resultats Nous avons montre que la secretion urinaire du metabolite microbien trimethylamine (TMA) est anti correlee avec la secretion d'insuline chez les souris nourries avec le regime HFD. Nous avons ensuite crible les cibles pharmacologiques potentielles de la TMA et avons identifie une inhibition specifique d'une kinase centrale dans la voie de transduction des TLRs, responsables de l'induction de l'inflammation chronique de faible intensite. Afin de mieux caracteriser le role de la TMA dans l'inhibition de l'inflammation, nous avons confirme ses proprietes immuno-modulatrices in vitro et in vivo . Nos resultats demontrent qu'un metabolite issu du metabolisme de la flore intestinale est capable d'inhiber la reponse inflammatoire au LPS dans des monocytes et dans des souris. Conclusion Dans cette etude, utilisant une approche originale, nous avons montre que la TMA urinaire inhibe l'inflammation transduite par la voie des TLRs ce qui pourrait participer a diminuer les effets de l'inflammation chronique de faible intensite. Cette etude met en evidence le role du metabolisme microbien intestinale dans le developpement du diabete de type 2. Declaration d’interet Les auteurs declarent ne pas avoir d'interet direct ou indirect (financier ou en nature) avec un organisme prive, industriel ou commercial en relation avec le sujet presente.

Introduction Les defauts structuraux et fonctionnels des cellules beta pancreatiques, elements cles dans le developpement du diabete, peuvent etre medies par des signaux extra-pancreatiques. Des etudes physiologiques chez plusieurs souches de souris ont identifie des adaptations souches-specifiques de profils de secretion d’insuline apres un regime gras. Nos objectifs sont d’identifier et de valider des biomarqueurs metaboliques associes aux variations de secretion d’insuline apres regime gras chez la souris. Materiels et methodes Les profils metabolomiques (1H RMN) plasmatiques et urinaires ont ete obtenus chez des souris C57BL6/J, DBA2, 129S6, BALB/c et C3H en regime gras (40%) ou controle. L’impact du trimethylamine-N-oxide (TMAO) sur la secretion d’insuline en reponse au glucose (GSIS) a ete etudie in vitro sur des ilots isoles de souris C57BL6/J et in vivo apres 10 jours de traitement (injection quotidienne s.c.) par un test de tolerance au glucose par voie orale (OGTT). Resultats L’analyse des profils metabolomiques a identifie des metabolites de la flore bacterienne intestinale, parmi lesquels le TMAO, caracteristiques des variations de secretion d’insuline entre souches de souris en regime gras. In vitro, le TMAO (10–3M) augmente la secretion d’insuline basale (x3,7 ; p Conclusion Par profilage metabolomique, nous avons identifie la voie du TMAO, deja impliquee dans les maladies cardiovasculaires et la steatose hepatique, comme biomarqueur de defaut insulino-secretoire. Ces resultats apportent des elements nouveaux dans notre connaissance du role de la flore bacterienne intestinale dans les regulations physiopathologiques de l’hote.

Introduction La production endogene de glucose, restreinte au foie, aux reins et a l’intestin grele, est necessaire au maintien de la glycemie. Dans le foie, le glucose produit est transporte par le transporteur Glut2. Cependant, les hepatocytes de souris invalidees pour Glut2 sont capables de produire du glucose via une voie vesiculaire, sous stimulation par l’AMPc. Dans ce travail, nous avons fait l’hypothese que cette stimulation est necessaire a la liberation de glucose par la voie vesiculaire. Materiels et methodes La liberation de glucose a partir de 14C-pyruvate a ete etudiee dans des hepatocytes primaires, issus de souris sauvages ou invalidees pour Glut2 specifiquement dans le foie (L-Glut2-/-). In vivo, la sortie de glucose neoglucogenique hepatique a ete etudiee lors d’un test de tolerance au pyruvate realise apres 16 h de jeune. Resultats Chez les souris sauvages, la liberation de glucose est inhibee de 48 ± 7 % par le dynasore (inhibiteur de la fission vesiculaire), de 37 ± 6 % par la cytochalasin B (inhibiteur de Glut2) et de 68 ± 10 % par les deux inhibiteurs. L’incorporation du 14C-pyruvate dans le glucose libere est de 9,5 ± 1,4 % dans les hepatocytes de souris sauvages, mais elle est virtuellement inexistante dans les hepatocytes de souris L-Glut2-/- (1,5 ± 1,1 %, p Conclusion La liberation de glucose d’origine neoglucogenique par les hepatocytes depend d’une voie vesiculaire et de Glut2. Nos etudes in vitro et in vivo suggerent qu’en absence de Glut2, les hepatocytes ne peuvent pas liberer de glucose « neoglucogenique » sans stimulation par l’AMPc. La liberation de glucose via la voie vesiculaire serait donc dependante d’une activation par l’AMPc.

Introduction La glucose-6-phosphatase (G6Pase) est l’enzyme cle de la production endogene de glucose, au carrefour de la glycogenolyse et de la neoglucogenese. Des donnees du laboratoire montrent une augmentation de l’activite G6Pase par l’AMPc, dependante de la PKA. Nous avons etudie la phosphorylation de la sous unite catalytique de la G6Pase (G6PC) par mutagenese dirigee et par spectrometrie de masse (MS). La G6PC etant faiblement exprimee, nous avons developpe au prealable une methode d’immunoprecipitation. Materiels et methodes L’activite G6Pase est mesuree apres mutation des sites de phosphorylation theoriques de la PKA sur la G6PC en alanine (empeche la phosphorylation) ou en aspartate (mime une phosphorylation constitutive). Afin d’en determiner les eventuels sites de phosphorylation, l’analyse structurale de la G6PC endogene d’hepatocytes est realisee par MS (nanoLC-MS/MS) apres immunoprecipitation et separation sur gel monodimensionnel. Resultats L’analyse de sequence de la G6PC revele deux sites potentiels de phosphorylation (T145 et T255) dont les mutations n’ont aucun effet sur l’activite G6Pase dans des cellules fibroblastiques. L’analyse nanoLC-MS/MS a permis l’identification de deux proteines dans l’immunoprecipitat dont la G6PC. Cette derniere a ete detectee dans la region de 30–40KDa, ce qui est coherent avec sa masse attendue. Quatre peptides de la G6PC ont ete identifies (un cytoplasmique, deux transmembranaires et un dans la lumiere du RE). Ces resultats doivent encore etre completes pour permettre d’identifier les sites de phosphorylation. Discussion La mutagenese dirigee n’a pas permis de mettre en evidence de site de phosphorylation. Cependant, nous montrons pour la premiere fois une technique d’immunoprecipitation enrichissant significativement la sous-unite catalytique de la G6Pase, et permettant ainsi sa detection par MS. Cette technique ouvre enfin la voie a l’etude approfondie de la G6PC et de ses regulations. Grâce a cela, nous serons capable d’en determiner par MS les eventuelles phosphorylations et, le cas echeant, de les localiser.

Introduction La production de glucose est composee de deux mecanismes : la glycogenolyse et la neoglucogenese. La voie de sortie du glucose est dependante de Glut2 mais aussi d’un mecanisme encore non identifie dependant de mouvements membranaires. Nos travaux portent sur l’identification de cette voie et se basent sur l’hypothese selon laquelle des vesicules d’exocytose pourraient jouer un role dans la regulation de la production de glucose et dans la liberation du glucose dans le milieu extracellulaire. Materiels et methodes La liberation de glucose d’hepatocytes de souris C57/Bl6 (5 semaines, n = 8) nourries et a jeun, en culture primaire, a ete etudiee en presence d’un inhibiteur chimique des dynamines (Dynasore, 80 μM, 1 h). De plus, la localisation de l’enzyme cle de la production de glucose, la Glucose-6-phosphatase, apres traitement au dynasore, a ete etudiee grâce a une proteine chimere par microscopie confocale a fluorescence dans la lignee cellulaire hepatique humaine HepG2. Resultats La liberation de glucose par des hepatocytes de souries nourries (glycogenolyse) n’est pas diminuee en presence de Dynasore alors que celle d’hepatocytes de souris a jeun (neoglucogenese) est diminuee de 45 % (p Discussion Les dynamines etant des GTPases impliquees dans la formation des vesicules, ces resultats suggerent fortement l’implication de vesicules dans le processus de sortie du glucose mais aussi dans sa regulation. Ces donnees suggerent egalement que cette voie vesiculaire jouerait un role preferentiel dans la neoglucogenese par rapport a la glycogenolyse. Ceci impliquerait une dissociation de ces deux mecanismes de production de glucose et supporterait l’hypothese de pools differents de G6P emise en 1987 par Jungermann.

Introduction Le diabete de type 2 est caracterise par une hyperglycemie chronique, qui engendre la production d’especes actives de l’oxygene (EAOs), generatrices de phenomenes de glucotoxicite. Cette hyperglycemie est associee a une induction paradoxale de l’expression de la sous-unite catalytique de la glucose-6-phosphatase (G6PC), enzyme cle de la neoglucogenese, concourant probablement a la deregulation de la production hepatique de glucose observee chez les diabetiques. Nous avons fait l’hypothese que le glucose regule l’activite transcriptionnelle du gene G6pc via les EAOs. Materiels et methodes La production d’EAOs induite par un traitement de 24 h avec 25 mM de glucose est mesuree dans des hepatocytes primaires de rat et la lignee HepG2. L’activite transcriptionnelle du promoteur G6pc (gene rapporteur) est etudiee dans la lignee HepG2. La liaison de facteurs de transcription au promoteur G6pc de rat est etudiee par immunoprecipitation de chromatine issue d’hepatocytes de rats. Resultats L’induction de l’activite transcriptionnelle du gene G6pc par le glucose est annulee lorsque la quantite d’EAOs est reduite par un traitement antioxydant ou lorsque l’activite transactivatrice du facteur HIF-1alpha (hypoxia inducible factor 1alpha) est annulee par la surexpression de son dominant negatif. Dans les hepatocytes, le glucose induit la stabilisation du facteur HIF-1alpha et sa liaison au promoteur G6pc. La production d’EAOs ou une forte concentration de glucose stimule l’interaction entre HIF-1alpha et le co-facteur CBP (CREB binding protein), permettant a CBP d’etre recrute sur le promoteur G6pc. Cette regulation est mise en evidence dans le foie de souris rendues obeses et diabetiques par un regime hypercalorique. Conclusion Cette etude caracterise le role des EAOs et de HIF-1alpha dans la regulation de l’activite transcriptionnelle du gene G6pc par le glucose. Elle met egalement en evidence un mecanisme graduel de regulation d’HIF-1alpha par le glucose, dependant des EAOs, semblable a celui decrit lors de l’hypoxie.

Introduction Les regimes hyperproteiques sont souvent recommandes par les dieteticiens car ils induisent une perte de poids chez les individus en surpoids et obeses. De plus, il a ete montre qu’ils ameliorent generalement l’equilibre glycemique des patients diabetiques de type 2. L’absorption intestinale des peptides issus de la digestion des proteines alimentaires fait principalement intervenir le transporteur PEPT1. Le but de cette etude a ete d’etudier les expressions genique et proteique de PEPT1 au cours du jeune ou apres un apport alimentaire enrichi en proteines. Materiels et methodes Des groupes de 5 rats Sprague-Dawley ont ete soumis soit a un jeune de 48 ou 96 h, soit a 5 j de regime enrichi a 55 % de proteines (regime a base de proteines de viande animale) au lieu de 17 % chez les animaux temoins. Les quantites de proteine et d’ARNm pour PEPT1 ont ete determinees par Western blot et par RT-qPCR dans les jejunums preleves sous anesthesie generale. Resultats Par rapport au groupe temoin, on observe une augmentation de 225 et de 509 % de la quantite d’ARNm pour le gene de PEPT1 dans les groupes soumis a un jeune de 48 et 96 h respectivement (p Conclusion On observe donc dans l’intestin, une augmentation des quantites d’ARNm et de proteines pour PEPT1 au cours du jeune et apres un regime hyperproteique. Ces resultats suggerent qu’il existe une induction de l’expression intestinale de PEPT1 par les proteines d’origine alimentaire. Chez les diabetiques de type2, cette induction pourrait jouer un role important dans l’amelioration des parametres glycemiques par les proteines alimentaires.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20412-9.