Chantal Chabo1€, Jacques Amar2,3, Rémy Burcelin1
Running title: tissue and gut microbiota in type 2 diabetes
1 Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), 31432 Toulouse, France.
2 Rangueil Hospital, Department of Therapeutics, Toulouse, France.
3 Institut National de la Santé et de la Recherche Médicale (INSERM), U558, Toulouse, France.
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Abstract
The discovery of the role of gut and more recently tissue microbiota on the control of metabolic diseases led us to more precisely describe the different bacterial components present in human blood from a general population and compare with prediabetic and diabetic high-fat diet-induced mouse models. In a cohort of 311 patients, we quantified bacterial DNA (16S rRNA gene) in total blood cells DNA, and found that the concentration of 16S rRNA DNA was positively correlated with fasting glycemia, insulin, and triglycerides, as well as treated diabetes, and negatively correlated with HDL cholesterol. In a subset of diabetic patients and paired controls, we cloned and identified some bacterial groups and species and showed that blood from diabetic patients was enriched in Gram-negative bacteria, mainly from phylum Proteobacteria. Furthermore, blood concentration of genes involved in the biosynthesis of lipopolysaccharides (LPS) such as
lpxA and lpxB, as well as genes involved in nitric oxide (NO) metabolism, such as qnorB, were
increased in diabetics. We further validated these findings in a model of prediabetic and diabetic mice fed a high-fat diet, and extended these observations to mesenteric adipose tissue, suggesting that a change in tissue microbiota and its corresponding functional genes (microbiome), can constitute an early factor involved in the development of diabetes. So, we suggested a possible involvement of bacterial NO-reductases in vascular endothelial dysfunction before the onset of diabetes, leading to an increased recruitment of macrophages carrying Gram-negative bacteria inside adipose tissue, where an LPS-triggered low grade inflammation can then occur to subsequently impair insulin signaling. Furthermore, intestinal microbiota was quantitatively as well as qualitatively changed before the onset of diabetes in mice, with an important increase in Gram-negative to Gram-positive bacteria ratio, in favor of Bacteroides-Prevotella group, harboring non-inflammatory forms of LPS. But mechanisms linking gut microbiota dysbiosis to an increase bacterial translocation, before the onset of high-fat diet-induced diabetes, remain to be elucidated.
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Introduction
All mammalians house trillions of various bacteria into their gut that constitute the intestinal microbiota (1). It is inherited at birth, colonizes the intestine during the first 3-5 years of life and is specie-, age-, and sex-dependent (2). Then, throughout life the bacterial ecology tends to vary mostly according to the environment (3). The recent advent of high-throughput sequencing technologies allowed a major step towards the understanding of the molecular relationships between host and its microbiota. This second genome, the microbiome, has now been identified in a catalogue of more than 4, which is currently updated to 8 millions, non-redundant microbial putative genes and 1 to 2,000 prevalent bacterial species (4). The gut microbiome can classify humans through 3 enterotypes which are not nation- or continent- specific (5). It is more than 100-400 times larger than the human genome, can evolve dynamically according to the nutrition of individuals (4, 5). Each individual has at least 160 shared species and a number of well- balanced host-microbial molecular relationships that would define groups of individuals. An important matter is that individuals respond differently to diet (6, 7) and diseases such as obesity (8), independently from their genetic background or diabetes (9). Furthermore, we and others identified that obese and type 2 diabetic phenotypes in rodents fed a fat-enriched diet, are highly correlated with specific profiles of the intestinal microbiota (10, 11). One step further was made when the causal role of intestinal microbiota on the control of metabolic disease, was unequivocally determined through microbiota transfer in mouse (12) and evoked in humans (13). The first one was related to the bacterial genes involved in the fermentation of dietary fibers that produce short chain fatty acids which are then captured by the host as a supplementary source of energy (12, 14). A second molecular mechanism was related to the regulation by intestinal microbiota of the gut production of fast induced adipocyte factor (FIAF) that controls the circulating lipoprotein synthase and favors triglyceride storage in the liver (15). We also identified lipopolysaccharides (LPS) from Gram-negative bacteria as causal molecular links between intestinal microbiota and the well-defined metabolic inflammation (16). LPS from intestinal origin accumulates into the blood following a fat-enriched meal, leading to the establishment of a metabolic endotoxemia that triggers inflammation leading to diabetes and obesity (6, 17). Recently, in front of the highly intricated diversity of intestinal microbiota, we described the presence of a microbiota in tissues such as blood (18) and adipose tissue (19) but of much less diversity. 16S rRNA DNA pyrosequencing revealed the presence of bacteria from the phylum
Proteobacteria in large proportion in blood with predominance of the family Burkholderiaceae. In
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represented by Gram-negative LPS-containing Proteobacteria, was increased, therefore defining
16Sr RNA DNA as a predictive biomarker of the disease (18). To precise the molecular
interactions between host and microbiota, we thought here to analyze the presence of master genes involved in LPS biosynthesis (20). We also thought to analyze whether bacterial genes could regulate known molecules, involved at the interface of metabolism, vascular function, and inflammation, such as nitric oxide (NO) (21-24).
In the present study, we quantified by qPCR bacterial DNA in the blood of 311 apparently healthy patients, and found a positive correlation between bacterial DNA concentration and several clinical parameters from features of the metabolic syndrome. Diversity of this blood microbiota was further analyzed by denaturing gradient gel electrophoresis (DGGE), sequencing, and group-, specie- or gene-specific qPCR in a subset of diabetic and paired control patients. Animal preclinical studies were finally performed to validate findings observed in humans with an additional attention to the structure of intestinal microbiota.
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