Low Incidence of Spontaneous Type 1 Diabetes in Non-Obese Diabetic Mice Raised on Gluten-Free Diets Is Associated with Changes in the Intestinal Microbiome

<div><p>Human and animal studies strongly suggest that dietary gluten could play a causal role in the etiopathogenesis of type 1 diabetes (T1D). However, the mechanisms have not been elucidated. Recent reports indicate that the intestinal microbiome has a major influence on the incidence of T1D. Since diet is known to shape the composition of the intestinal microbiome, we investigated using non-obese diabetic (NOD) mice whether changes in the intestinal microbiome could be attributed to the pro- and anti-diabetogenic effects of gluten-containing and gluten-free diets, respectively. NOD mice were raised on gluten-containing chows (GCC) or gluten-free chows (GFC). The incidence of diabetes was determined by monitoring blood glucose levels biweekly using a glucometer. Intestinal microbiome composition was analyzed by sequencing 16S rRNA amplicons derived from fecal samples. First of all, GCC-fed NOD mice had the expected high incidence of hyperglycemia whereas NOD mice fed with a GFC had significantly reduced incidence of hyperglycemia. Secondly, when the fecal microbiomes were compared, <i>Bifidobacterium</i>, <i>Tannerella</i>, and <i>Barnesiella</i> species were increased (p = 0.03, 0.02, and 0.02, respectively) in the microbiome of GCC mice, where as <i>Akkermansia</i> species was increased (p = 0.02) in the intestinal microbiomes of NOD mice fed GFC. Thirdly, both of the gluten-free chows that were evaluated, either egg white based (EW-GFC) or casein based (C-GFC), significantly reduced the incidence of hyperglycemia. Interestingly, the gut microbiome from EW-GFC mice was similar to C-GFC mice. Finally, adding back gluten to the gluten-free diet reversed its anti-diabetogenic effect, reduced <i>Akkermansia</i> species and increased <i>Bifidobacterium</i>, <i>Tannerella</i>, and <i>Barnesiella</i> suggesting that the presence of gluten is directly responsible for the pro-diabetogenic effects of diets and it determines the gut microflora. Our novel study thus suggests that dietary gluten could modulate the incidence of T1D by changing the gut microbiome.</p></div

Effect of Dietary Gluten Upon Specific OTU (Genus) Abundance.

<p>A. Correspondence analysis (CA) plot shows the degree of correlation between specific OTUs and diet in all of the gluten-containing chows group (GCC) and gluten-free chows group (GFC) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078687#pone-0078687-g005" target="_blank">fig 5</a>.</p

Impact of dietary gluten upon the incidence of hyperglycemia and production of anti-insulin IgG in NOD Mice.

<p>Blood glucose levels were measured once every two(gluten-containing) chow (N = 16), and the other that were weaned and maintained upon a gluten-free (casein-based) chow (N = 19). (A) Overall incidence of hyperglycemia. (B) The incidence of hyperglycemia in males and (C) females on the different diets. There were 8 Std-GCC males, 8 Std-GCC females, 8 C-GFC males, and 11 C-GFC females (D). p values are given in parenthesis. Anti-insulin IgG levels were evaluated in the standard (▪, N = 9) and casein-based (▴, N = 8) chow-fed NOD mice every 7 weeks for a total of three time points. P<0.05 for each time point.</p

Effect of dietary gluten upon the composition of the intestinal microbiomes in NOD mice.

<p>Microbiomes of mice on gluten free chows were compared with those of mice on gluten containing chows. For the gluten free chow derived microbiomes, microbiomes from five mice on an egg-white based gluten-free chow and five from a casein-based gluten-free chow were grouped together to make a total of 10 gluten-free chow mice (▾). For the microbiomes derived from mice on gluten containing chows, five were derived from mice on standard (gluten containing) chow and five were derived from mice on gluten-supplemented casein-based chow, making a total of 10 gluten associated microbiomes (•). These were evaluated using a multi-dimensional scale analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078687#pone-0078687-g005" target="_blank">Fig 5A</a>). Richness was determined by observed Operational Taxonomic Units (OTU) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078687#pone-0078687-g005" target="_blank">Fig 5B</a>).</p

Effect of Dietary Gluten Upon Specific OTU (Genus) Abundance.

<p>Mean relative abundance for each of 6 genera in all of the gluten-containing chows group (GCC) and gluten-free chows group (GFC) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078687#pone-0078687-g005" target="_blank">fig 5</a> are plotted in a box and whisker plot. The six genera plotted are A) Bacteroides, B) Akkermansia, C) Bifidobacterium, D) Tannerella, E) Barnesiella, F) Clostridiales. The respective p values are: A) 0.005843 B) 0.01783 C) 0.03615 D) 0.01761 E) 0.0234 and F) 0.0541.</p

Additional file 1: of Variable responses of human and non-human primate gut microbiomes to a Western diet

Supplementary tables and figures. (DOC 2591 KB

Additional file 1: of Xylan degradation by the human gut Bacteroides xylanisolvens XB1AT involves two distinct gene clusters that are linked at the transcriptional level

Table S1. RNA-seq mapping assessment. Table S2. Xylanase specific activity of B xylanisolvens XB1AT. Table S3. Proteins identified by MALDI-TOF MS or LC-ESI-MS/MS over-produced upon growth of B. xylanisolvens XB1AT on OSX relative to xylose. Table S4. Composition of the commercial oat-spelt xylan (SERVA, France) used in this study. Table S5. Primers used for RT-PCR (to amplify the intergenic regions between two consecutive ORFs within PUL 43). Table S6. Primers used for relative RT-qPCR. Table S7. Primers used for insertion mutagenesis into PUL 43 HTCS gene (BXY_29350). Figure S1. Growth of B. xylanisolvens XB1AT (Wt) and PUL 43 HTCS (BXY_29350) mutant on glucose, xylose, wheat arabinoxylan (WAX) and oat-spelt xylan (OSX). Figure S2. B. xylanisolvens XB1AT gene expression in response to oat-spelt xylan (OSX) and xylose relative to glucose obtained from RNA-seq analysis. Figure S3. B. xylanisolvens XB1AT PUL expression in response to xylose relative to glucose at late-log phase obtained from RNA-seq analysis. Figure S4. Schematic layout of the mutant construction and validation of pGERM:HTCS insertion into PUL 43 HTCS gene (BXY_29350) of B. xylanisolvens XB1A genome. (XLSX 454 kb