The visceral adipose tissue bacterial microbiota provides a signature of obesity based on inferred metagenomic functions.

BACKGROUND: Metabolic inflammation mediated obesity requires bacterial molecules to trigger immune and adipose cells leading to inflammation and adipose depot development. In addition to the well-established gut microbiota dysbiosis, a leaky gut has been identified in patients with obesity and animal models, characterized by the presence of a tissue microbiota in the adipose fat pads. METHODS: To determine its potential role, we sequenced the bacterial 16 S rRNA genes in the visceral adipose depot of patients with obesity. Taking great care (surgical, biochemical, and bioinformatic) to avoid environmental contaminants. We performed statistical discriminant analyses to identify specific signatures and constructed network of interactions between variables. RESULTS: The data showed that a specific 16SrRNA gene signature was composed of numerous bacterial families discriminating between lean versus patients with obesity and people with severe obesity. The main discriminant families were Burkholderiaceae, Yearsiniaceae, and Xanthomonadaceae, all of which were gram-negative. Interestingly, the Morganellaceae were totally absent from people without obesity while preponderant in all in patients with obesity. To generate hypotheses regarding their potential role, we inferred metabolic pathways from the 16SrRNA gene signatures. We identified several pathways associated with adenosyl-cobalamine previously described to be linked with adipose tissue development. We further identified chorismate biosynthesis, which is involved in aromatic amino-acid metabolism and could play a role in fat pad development. This innovative approach generates novel hypotheses regarding the gut to adipose tissue axis. CONCLUSIONS: This innovative approach generates novel hypotheses regarding the gut to adipose tissue axis in obesity and notably the potential role of tissue microbiota.

Human liver microbiota modeling strategy at the early onset of fibrosis.

BACKGROUND: Gut microbiota is involved in the development of liver diseases such as fibrosis. We and others identified that selected sets of gut bacterial DNA and bacteria translocate to tissues, notably the liver, to establish a non-infectious tissue microbiota composed of microbial DNA and a low frequency live bacteria. However, the precise set of bacterial DNA, and thereby the corresponding taxa associated with the early stages of fibrosis need to be identified. Furthermore, to overcome the impact of different group size and patient origins we adapted innovative statistical approaches. Liver samples with low liver fibrosis scores (F0, F1, F2), to study the early stages of the disease, were collected from Romania(n = 36), Austria(n = 10), Italy(n = 19), and Spain(n = 17). The 16S rRNA gene was sequenced. We considered the frequency, sparsity, unbalanced sample size between cohorts to identify taxonomic profiles and statistical differences. RESULTS: Multivariate analyses, including adapted spectral clustering with L1-penalty fair-discriminant strategies, and predicted metagenomics were used to identify that 50% of liver taxa associated with the early stage fibrosis were Enterobacteriaceae, Pseudomonadaceae, Xanthobacteriaceae and Burkholderiaceae. The Flavobacteriaceae and Xanthobacteriaceae discriminated between F0 and F1. Predicted metagenomics analysis identified that the preQ0 biosynthesis and the potential pathways involving glucoryranose and glycogen degradation were negatively associated with liver fibrosis F1-F2 vs F0. CONCLUSIONS: Without demonstrating causality, our results suggest first a role of bacterial translocation to the liver in the progression of fibrosis, notably at the earliest stages. Second, our statistical approach can identify microbial signatures and overcome issues regarding sample size differences, the impact of environment, and sets of analyses. TRIAL REGISTRATION: TirguMECCH ROLIVER Prospective Cohort for the Identification of Liver Microbiota, registration 4065/2014. Registered 01 01 2014.

Liver lipopolysaccharide binding protein prevents hepatic inflammation in physiological and pathological non-obesogenic conditions.

Lipopolysaccharide binding protein (LBP) knockout mice models are protected against the deleterious effects of major acute inflammation but its possible physiological role has been less well studied. We aimed to evaluate the impact of liver LBP downregulation (using nanoparticles containing siRNA- Lbp) on liver steatosis, inflammation and fibrosis during a standard chow diet (STD), and in pathological non-obesogenic conditions, under a methionine and choline deficient diet (MCD, 5 weeks). Under STD, liver Lbp gene knockdown led to a significant increase in gene expression markers of liver inflammation (Itgax, Tlr4, Ccr2, Ccl2 and Tnf), liver injury (Krt18 and Crp), fibrosis (Col4a1, Col1a2 and Tgfb1), endoplasmic reticulum (ER) stress (Atf6, Hspa5 and Eif2ak3) and protein carbonyl levels. As expected, the MCD increased hepatocyte vacuolation, liver inflammation and fibrosis markers, also increasing liver Lbp mRNA. In this model, liver Lbp gene knockdown resulted in a pronounced worsening of the markers of liver inflammation (also including CD68 and MPO activity), fibrosis, ER stress and protein carbonyl levels, all indicative of non-alcoholic steatohepatitis (NASH) progression. At cellular level, Lbp gene knockdown also increased expression of the proinflammatory mediators (Il6, Ccl2), and markers of fibrosis (Col1a1, Tgfb1) and protein carbonyl levels. In agreement with these findings, liver LBP mRNA in humans positively correlated with markers of liver damage (circulating hsCRP, ALT activity, liver CRP and KRT18 gene expression), and with a network of genes involved in liver inflammation, innate and adaptive immune system, endoplasmic reticulum stress and neutrophil degranulation (all with q-value<0.05). In conclusion, current findings suggest that a significant downregulation in liver LBP levels promotes liver oxidative stress and inflammation, aggravating NASH progression, in physiological and pathological non-obesogenic conditions.

Obesity Is Associated with the Severity of Periodontal Inflammation Due to a Specific Signature of Subgingival Microbiota.

The aim of this study was to analyze the link between periodontal microbiota and obesity in humans. We conducted a cohort study including 45 subjects with periodontitis divided into two groups: normo-weighted subjects with a body mass index (BMI) between 20 and 25 kg/m2 (n = 34) and obese subjects with a BMI > 30 kg/m2 (n = 11). Our results showed that obesity was associated with significantly more severe gingival inflammation according to Periodontal Inflamed Surface Area (PISA index). Periodontal microbiota taxonomic analysis showed that the obese (OB) subjects with periodontitis were characterized by a specific signature of subgingival microbiota with an increase in Gram-positive bacteria in periodontal pockets, associated with a decrease in microbiota diversity compared to that of normo-weighted subjects with periodontitis. Finally, periodontal treatment response was less effective in OB subjects with persisting periodontal inflammation, reflecting a still unstable periodontal condition and a risk of recurrence. To our knowledge, this study is the first exploring both salivary and subgingival microbiota of OB subjects. Considering that OB subjects are at higher periodontal risk, this could lead to more personalized preventive or therapeutic strategies for obese patients regarding periodontitis through the specific management of oral microbiota of obese patients.

Low-Diversity Microbiota in Apical Periodontitis and High Blood Pressure Are Signatures of the Severity of Apical Lesions in Humans.

(1) Background: In developed countries, the prevalence of apical periodontitis (AP) varies from 20% to 50% for reasons that could be associated with the apical periodontitis microbiota ecology. (2) Methods: We performed a clinical study in the Odontology department of Toulouse hospital in France, to sequence the 16S rRNA gene of AP microbiota and collect clinical parameters from 94 patients. Forty-four patients were characterized with a PAI (periapical index of AP severity) score lower or equal to 3, while the others had superior scores (n = 50). (3) Results: The low diversity of granuloma microbiota is associated with the highest severity (PAI = 5) of periapical lesions (Odds Ratio 4.592, IC 95% [1.6329; 14.0728]; p = 0.001; notably, a lower relative abundance of Burkholderiaceae and a higher relative abundance of Pseudomonas and Prevotella). We also identified that high blood pressure (HBP) is associated with the increase in PAI scores. (4) Conclusions: Our data show that a low diversity of bacterial ecology of the AP is associated with severe PAI scores, suggesting a causal mechanism. Furthermore, a second risk factor was blood pressure associated with the severity of apical periodontitis.

Integrative study of diet-induced mouse models of NAFLD identifies PPARα as a sexually dimorphic drug target.

OBJECTIVE: We evaluated the influence of sex on the pathophysiology of non-alcoholic fatty liver disease (NAFLD). We investigated diet-induced phenotypic responses to define sex-specific regulation between healthy liver and NAFLD to identify influential pathways in different preclinical murine models and their relevance in humans. DESIGN: Different models of diet-induced NAFLD (high-fat diet, choline-deficient high-fat diet, Western diet or Western diet supplemented with fructose and glucose in drinking water) were compared with a control diet in male and female mice. We performed metabolic phenotyping, including plasma biochemistry and liver histology, untargeted large-scale approaches (liver metabolome, lipidome and transcriptome), gene expression profiling and network analysis to identify sex-specific pathways in the mouse liver. RESULTS: The different diets induced sex-specific responses that illustrated an increased susceptibility to NAFLD in male mice. The most severe lipid accumulation and inflammation/fibrosis occurred in males receiving the high-fat diet and Western diet, respectively. Sex-biased hepatic gene signatures were identified for these different dietary challenges. The peroxisome proliferator-activated receptor α (PPARα) co-expression network was identified as sexually dimorphic, and in vivo experiments in mice demonstrated that hepatocyte PPARα determines a sex-specific response to fasting and treatment with pemafibrate, a selective PPARα agonist. Liver molecular signatures in humans also provided evidence of sexually dimorphic gene expression profiles and the sex-specific co-expression network for PPARα. CONCLUSIONS: These findings underscore the sex specificity of NAFLD pathophysiology in preclinical studies and identify PPARα as a pivotal, sexually dimorphic, pharmacological target. TRIAL REGISTRATION NUMBER: NCT02390232.

Screening for anti-adipogenic, pro-lipolytic and thermogenic plant extracts by models associating intestinal epithelial cells with human adipose cells.

PURPOSE: Excessive fat mass accumulation in obesity leads to diverse metabolic disorders, increased risks of cardiovascular diseases and in some cases, mortality. The aim of this study was to screen the actions of botanical extracts intended for oral use on human adipose tissue, using an in vitro screening model combining human intestinal cells with human adipose cells. This was to find the most effective extracts on lipid accumulation, UCP1 expression and ATP production in pre-adipocytes and on adipocyte lipolysis. METHODS: In this study, 25 individual plant extracts were screened for their effects on human adipose cells. Consequently, an original in vitro model was set up using the Caco-2 cell line, to mimic the intestinal passage of the extracts and then exposing human adipose cells to them. The biological actions of extracts were thus characterized, and compared with a coffee extract standard. The most effective extracts, and their combinations, were retained for their actions on lipid accumulation, the expression of the thermogenic effector UCP1 and ATP production in pre-adipocytes as well as on lipolysis activity of mature adipocytes. RESULTS: The biphasic culture system combining human Caco-2 cells with human adipose cells was verified as functional using the green coffee extract standard. Out of the 25 plant extracts studied, only 7 and their combinations were retained due to their potent effects on adipose cells biology. The data showed that compared to the coffee extract standard, Immortelle, Catechu, Carrot and Rose hip extracts were the most effective in reducing lipid accumulation and increased UCP1 expression in human pre-adipocytes. CONCLUSION: This study reveals the potential inhibitory effects on lipid accumulation and thermogenic activity of Immortelle, Catechu, Carrot and Rose hip extracts, and for the first time synergies in their combinations, using an in vitro model mimicking as closely as possible, human intestinal passage linked to adipose cells. These findings need to be confirmed by in vivo trials.

Cytoskeletal transgelin 2 contributes to gender-dependent adipose tissue expandability and immune function.

During adipogenesis, preadipocytes' cytoskeleton reorganizes in parallel with lipid accumulation. Failure to do so may impact the ability of adipose tissue (AT) to shift between lipid storage and mobilization. Here, we identify cytoskeletal transgelin 2 (TAGLN2) as a protein expressed in AT and associated with obesity and inflammation, being normalized upon weight loss. TAGLN2 was primarily found in the adipose stromovascular cell fraction, but inflammation, TGF-ß, and estradiol also prompted increased expression in human adipocytes. Tagln2 knockdown revealed a key functional role, being required for proliferation and differentiation of fat cells, whereas transgenic mice overexpressing Tagln2 using the adipocyte protein 2 promoter disclosed remarkable sex-dependent variations, in which females displayed "healthy" obesity and hypertrophied adipocytes but preserved insulin sensitivity, and males exhibited physiologic changes suggestive of defective AT expandability, including increased number of small adipocytes, activation of immune cells, mitochondrial dysfunction, and impaired metabolism together with decreased insulin sensitivity. The metabolic relevance and sexual dimorphism of TAGLN2 was also outlined by genetic variants that may modulate its expression and are associated with obesity and the risk of ischemic heart disease in men. Collectively, current findings highlight the contribution of cytoskeletal TAGLN2 to the obese phenotype in a gender-dependent manner.-Ortega, F. J., Moreno-Navarrete, J. M., Mercader, J. M., Gómez-Serrano, M., García-Santos, E., Latorre, J., Lluch, A., Sabater, M., Caballano-Infantes, E., Guzmán, R., Macías-González, M., Buxo, M., Gironés, J., Vilallonga, R., Naon, D., Botas, P., Delgado, E., Corella, D., Burcelin, R., Frühbeck, G., Ricart, W., Simó, R., Castrillon-Rodríguez, I., Tinahones, F. J., Bosch, F., Vidal-Puig, A., Malagón, M. M., Peral, B., Zorzano, A., Fernández-Real, J. M. Cytoskeletal transgelin 2 contributes to gender-dependent adipose tissue expandability and immune function.

The gut microbiota to the brain axis in the metabolic control.

The regulation of glycemia is under a tight neuronal detection of glucose levels performed by the gut-brain axis and an efficient efferent neuronal message sent to the peripheral organs, as the pancreas to induce insulin and inhibit glucagon secretions. The neuronal detection of glucose levels is performed by the autonomic nervous system including the enteric nervous system and the vagus nerve innervating the gastro-intestinal tractus, from the mouth to the anus. A dysregulation of this detection leads to the one of the most important current health issue around the world i.e. diabetes mellitus. Furthemore, the consequences of diabetes mellitus on neuronal homeostasis and activities participate to the aggravation of the disease establishing a viscious circle. Prokaryotic cells as bacteria, reside in our gut. The strong relationship between prokaryotic cells and our eukaryotic cells has been established long ago, and prokaryotic and eukaryotic cells in our body have evolved synbiotically. For the last decades, studies demonstrated the critical role of the gut microbiota on the metabolic control and how its shift can induce diseases such as diabetes. Despite an important increase of knowledge, few is known about 1) how the gut microbiota influences the neuronal detection of glucose and 2) how the diabetes mellitus-induced gut microbiota shift observed participates to the alterations of autonomic nervous system and the gut-brain axis activity.

The gut microbiome and heart failure: A better gut for a better heart.

Despite the development of new drugs and therapeutic strategies, mortality and morbidity related to heart failure (HF) remains high. It is also the leading cause of global mortality. Several concepts have been proposed to explore the underlying pathogenesis of HF, but there is still a strong need for more specific and complementary therapeutic options. In recent years, accumulating evidence has demonstrated that changes in the composition of gut microbiota, referred to as dysbiosis, might play a pivotal role in the development of several diseases, including HF. HF-associated decreased cardiac output, resulting in bowell wall oedema and intestine ischaemia, can alter gut structure, peamibility and function. These changes would favour bacterial translocation, exacerbating HF pathogenesis at least partly through activation of systemic inflammation. Although our knowledge of the precise molecular mechanisms by which gut dysbiosis influance HF is still limited, a growing body of evidence has recently demonstrated the impact of a series of gut microbiome-derived metabolites, such as trimetylamine N-oxide, short-chain fatty acids or secondary bile acids, which have been shown to play critical roles in cardiac health and disease. This review will summarize the role of gut microbiota and its metabolites in the pathogenesis of HF. Current and future preventive and therapeutic strategies to prevent HF by an adequate modulation of the microbiome and its derived metabolites are also discussed.

Oral microbiota-induced periodontitis: a new risk factor of metabolic diseases.

It has recently become evident that the periodontium (gingiva, desmodontal ligament, cementum and alveolar bone) and the associated microbiota play a pivotal role in regulating human health and diseases. The oral cavity is the second largest microbiota in the body with around 500 different bacterial species identified today. When disruption of oral cavity and dysbiosis occur, the proportion of strict anaerobic Gram-negative bacteria is then increased. Patients with periodontitis present 27 to 53% more risk to develop diabetes than the control population suggesting that periodontitis is an aggravating factor in the incidence of diabetes. Moreover, dysbiosis of oral microbiota is involved in both periodontal and metabolic disorders (cardiovascular diseases, dyslipidaemia …). The oral diabetic dysbiosis is characterized by a specific bacteria Porphyromonas, which is highly expressed in periodontal diseases and could exacerbate insulin resistance. In this review, we will address the nature of the oral microbiota and how it affects systemic pathologies with a bidirectional interaction. We also propose that using prebiotics like Akkermansia muciniphila may influence oral microbiota as novel therapeutic strategies. The discovery of the implication of oral microbiota for the control of metabolic diseases could be a new way for personalized medicine.

High-fat diet modifies the PPAR-γ pathway leading to disruption of microbial and physiological ecosystem in murine small intestine.

Diet is among the most important factors contributing to intestinal homeostasis, and basic functions performed by the small intestine need to be tightly preserved to maintain health. Little is known about the direct impact of high-fat (HF) diet on small-intestinal mucosal defenses and spatial distribution of the microbiota during the early phase of its administration. We observed that only 30 d after HF diet initiation, the intervillous zone of the ileum-which is usually described as free of bacteria-became occupied by a dense microbiota. In addition to affecting its spatial distribution, HF diet also drastically affected microbiota composition with a profile characterized by the expansion of Firmicutes (appearance of Erysipelotrichi), Proteobacteria (Desulfovibrionales) and Verrucomicrobia, and decrease of Bacteroidetes (family S24-7) and Candidatus arthromitus A decrease in antimicrobial peptide expression was predominantly observed in the ileum where bacterial density appeared highest. In addition, HF diet increased intestinal permeability and decreased cystic fibrosis transmembrane conductance regulator (Cftr) and the Na-K-2Cl cotransporter 1 (Nkcc1) gene and protein expressions, leading to a decrease in ileal secretion of chloride, likely responsible for massive alteration in mucus phenotype. This complex phenotype triggered by HF diet at the interface between the microbiota and the mucosal surface was reversed when the diet was switched back to standard composition or when mice were treated for 1 wk with rosiglitazone, a specific agonist of peroxisome proliferator-activated receptor-γ (PPAR-γ). Moreover, weaker expression of antimicrobial peptide-encoding genes and intervillous bacterial colonization were observed in Ppar-γ-deficient mice, highlighting the major role of lipids in modulation of mucosal immune defenses.

Lixisenatide requires a functional gut-vagus nerve-brain axis to trigger insulin secretion in controls and type 2 diabetic mice.

Endogenous glucagon-like peptide-1 (GLP-1) regulates glucose-induced insulin secretion through both direct ß-cell-dependent and indirect gut-brain axis-dependent pathways. However, little is known about the mode of action of the GLP-1 receptor agonist lixisenatide. We studied the effects of lixisenatide (intraperitoneal injection) on insulin secretion, gastric emptying, vagus nerve activity, and brain c-Fos activation in naive, chronically vagotomized, GLP-1 receptor knockout (KO), high-fat diet-fed diabetic mice, or db/db mice. Lixisenatide dose-dependently increased oral glucose-induced insulin secretion that is correlated with a decrease of glycemia. In addition, lixisenatide inhibited gastric emptying. These effects of lixisenatide were abolished in vagotomized mice, characterized by a delay of gastric emptying and in GLP-1 receptor KO mice. Intraperitoneal administration of lixisenatide also increased the vagus nerve firing rate and the number of c-Fos-labeled neurons in the nucleus tractus solitarius (NTS) of the brainstem. In diabetic mouse models, lixisenatide increased the firing rate of the vagus nerve when administrated simultaneously to an intraduodenal glucose. It increased also insulin secretion and c-Fos activation in the NTS. Altogether, our findings show that lixisenatide requires a functional vagus nerve and neuronal gut-brain-islets axis as well as the GLP-1 receptor to regulate glucose-induced insulin secretion in healthy and diabetic mice. NEW & NOTEWORTHY Lixisenatide is an agonist of the glucagon-like protein (GLP)-1 receptor, modified from exendin 4, used to treat type 2 diabetic patients. However, whereas the mode of action of endogenous GLP-1 is extensively studied, the mode of action of the GLP-1 analog lixisenatide is poorly understood. Here, we demonstrated that lixisenatide activates the vagus nerve and recruits the gut-brain axis through the GLP-1 receptor to decrease gastric emptying and stimulate insulin secretion to improve glycemia.

Transfer of dysbiotic gut microbiota has beneficial effects on host liver metabolism.

Gut microbiota dysbiosis has been implicated in a variety of systemic disorders, notably metabolic diseases including obesity and impaired liver function, but the underlying mechanisms are uncertain. To investigate this question, we transferred caecal microbiota from either obese or lean mice to antibiotic-free, conventional wild-type mice. We found that transferring obese-mouse gut microbiota to mice on normal chow (NC) acutely reduces markers of hepatic gluconeogenesis with decreased hepatic PEPCK activity, compared to non-inoculated mice, a phenotypic trait blunted in conventional NOD2 KO mice. Furthermore, transferring of obese-mouse microbiota changes both the gut microbiota and the microbiome of recipient mice. We also found that transferring obese gut microbiota to NC-fed mice then fed with a high-fat diet (HFD) acutely impacts hepatic metabolism and prevents HFD-increased hepatic gluconeogenesis compared to non-inoculated mice. Moreover, the recipient mice exhibit reduced hepatic PEPCK and G6Pase activity, fed glycaemia and adiposity. Conversely, transfer of lean-mouse microbiota does not affect markers of hepatic gluconeogenesis. Our findings provide a new perspective on gut microbiota dysbiosis, potentially useful to better understand the aetiology of metabolic diseases.

Periodontitis induced by Porphyromonas gingivalis drives periodontal microbiota dysbiosis and insulin resistance via an impaired adaptive immune response.

OBJECTIVE: To identify a causal mechanism responsible for the enhancement of insulin resistance and hyperglycaemia following periodontitis in mice fed a fat-enriched diet. DESIGN: We set-up a unique animal model of periodontitis in C57Bl/6 female mice by infecting the periodontal tissue with specific and alive pathogens like Porphyromonas gingivalis (Pg), Fusobacterium nucleatum and Prevotella intermedia. The mice were then fed with a diabetogenic/non-obesogenic fat-enriched diet for up to 3 months. Alveolar bone loss, periodontal microbiota dysbiosis and features of glucose metabolism were quantified. Eventually, adoptive transfer of cervical (regional) and systemic immune cells was performed to demonstrate the causal role of the cervical immune system. RESULTS: Periodontitis induced a periodontal microbiota dysbiosis without mainly affecting gut microbiota. The disease concomitantly impacted on the regional and systemic immune response impairing glucose metabolism. The transfer of cervical lymph-node cells from infected mice to naive recipients guarded against periodontitis-aggravated metabolic disease. A treatment with inactivated Pg prior to the periodontal infection induced specific antibodies against Pg and protected the mouse from periodontitis-induced dysmetabolism. Finally, a 1-month subcutaneous chronic infusion of low rates of lipopolysaccharides from Pg mimicked the impact of periodontitis on immune and metabolic parameters. CONCLUSIONS: We identified that insulin resistance in the high-fat fed mouse is enhanced by pathogen-induced periodontitis. This is caused by an adaptive immune response specifically directed against pathogens and associated with a periodontal dysbiosis.

Associations between hepatic miRNA expression, liver triacylglycerols and gut microbiota during metabolic adaptation to high-fat diet in mice.

AIMS/HYPOTHESIS: Despite the current pandemic of metabolic diseases, our understanding of the diverse nature of the development of metabolic alterations in people who eat a high-fat diet (HFD) is still poor. We recently demonstrated a cardio-metabolic adaptation in mice fed an HFD, which was characterised by a specific gut and periodontal microbiota profile. Since the severity of hepatic disease is characterised by specific microRNA (miRNA) signatures and the gut microbiota is a key driver of both hepatic disease and miRNA expression, we analysed the expression of three hepatic miRNA and studied their correlation with hepatic triacylglycerol content and gut microbiota. METHODS: Two cohorts of C57BL/6 4-week-old wild-type (WT) male mice (n = 62 and n = 96) were fed an HFD for 3 months to provide a model of metabolic adaptation. Additionally 8-week-old C57BL/6 mice, either WT or of different genotypes, with diverse gut microbiota (ob/ob, Nod1, Cd14 knockout [Cd14KO] and Nod2) or without gut microbiota (axenic mice) were fed a normal chow diet. Following which, glycaemic index, body weight, blood glucose levels and hepatic triacylglycerol levels were measured. Gut (caecum) microbiota taxa were analysed by pyrosequencing. To analyse hepatic miRNA expression, real-time PCR was performed on total extracted miRNA samples. Data were analysed using two-way ANOVA followed by the Dunnett's post hoc test, or by the unpaired Student's t test. A cluster analysis and multivariate analyses were also performed. RESULTS: Our results demonstrated that the expression of miR-181a, miR-666 and miR-21 in primary murine hepatocytes is controlled by lipopolysaccharide in a dose-dependent manner. Of the gut microbiota, Firmicutes were positively correlated and Proteobacteria and Bacteroides acidifaciens were negatively correlated with liver triacylglycerol levels. Furthermore, the relative abundance of Firmicutes was negatively correlated with hepatic expression of miR-666 and miR-21. In contrast, the relative abundance of B. acidifaciens was positively correlated with miR-21. CONCLUSIONS/INTERPRETATION: We propose the involvement of hepatic miRNA, liver triacylglycerols and gut microbiota as a new triad that underlies the molecular mechanisms by which gut microbiota governs hepatic pathophysiology during metabolic adaptation to HFD.

Changes in blood microbiota profiles associated with liver fibrosis in obese patients: A pilot analysis.

The early detection of liver fibrosis among patients with nonalcoholic fatty liver disease (NAFLD) is an important clinical need. In view of the suggested role played by bacterial translocation in liver disease and obesity, we sought to investigate the relationship between blood microbiota and liver fibrosis (LF) in European cohorts of patients with severe obesity. We carried out a cross-sectional study of obese patients, well characterized with respect to the severity of the NAFLD, in the cohort FLORINASH. This cohort has been divided into a discovery cohort comprising 50 Spanish patients and then in a validation cohort of 71 Italian patients. Blood bacterial DNA was analyzed both quantitatively by 16S ribosomal DNA (rDNA) quantitative polymerase chain reaction and qualitatively by 16S rDNA targeted metagenomic sequencing and functional metagenome prediction. Spanish plasma bile acid contents were analyzed by liquid chromatography/mass spectrometry. The 16S rDNA concentration was significantly higher in patients of the discovery cohort with LF. By 16S sequencing, we found specific differences in the proportion of several bacterial taxa in both blood and feces that correlate with the presence of LF, thus defining a specific signature of the liver disease. Several secondary/primary bile acid ratios were also decreased with LF in the discovery cohort. We confirmed, in the validation cohort, the correlation between blood 16S rDNA concentration and LF, whereas we did not confirm the specific bacterial taxa signature, despite a similar trend in patients with more-severe fibrosis. CONCLUSION: Changes in blood microbiota are associated with LF in obese patients. Blood microbiota analysis provides potential biomarkers for the detection of LF in this population. (Hepatology 2016;64:2015-2027).

Periodontal dysbiosis linked to periodontitis is associated with cardiometabolic adaptation to high-fat diet in mice.

Periodontitis and type 2 diabetes are connected pandemic diseases, and both are risk factors for cardiovascular complications. Nevertheless, the molecular factors relating these two chronic pathologies are poorly understood. We have shown that, in response to a long-term fat-enriched diet, mice present particular gut microbiota profiles related to three metabolic phenotypes: diabetic-resistant (DR), intermediate (Inter), and diabetic-sensitive (DS). Moreover, many studies suggest that a dysbiosis of periodontal microbiota could be associated with the incidence of metabolic and cardiac diseases. We investigated whether periodontitis together with the periodontal microbiota may also be associated with these different cardiometabolic phenotypes. We report that the severity of glucose intolerance is related to the severity of periodontitis and cardiac disorders. In detail, alveolar bone loss was more accentuated in DS than Inter, DR, and normal chow-fed mice. Molecular markers of periodontal inflammation, such as TNF-α and plasminogen activator inhibitor-1 mRNA levels, correlated positively with both alveolar bone loss and glycemic index. Furthermore, the periodontal microbiota of DR mice was dominated by the Streptococcaceae family of the phylum Firmicutes, whereas the periodontal microbiota of DS mice was characterized by increased Porphyromonadaceae and Prevotellaceae families. Moreover, in DS mice the periodontal microbiota was indicated by an abundance of the genera Prevotella and Tannerella, which are major periodontal pathogens. PICRUSt analysis of the periodontal microbiome highlighted that prenyltransferase pathways follow the cardiometabolic adaptation to a high-fat diet. Finally, DS mice displayed a worse cardiac phenotype, percentage of fractional shortening, heart rhythm, and left ventricle weight-to-tibia length ratio than Inter and DR mice. Together, our data show that periodontitis combined with particular periodontal microbiota and microbiome is associated with metabolic adaptation to a high-fat diet related to the severity of cardiometabolic alteration.