Diabetes Care Volume 41, November 2018 2385 Gut Microbiota Differs in Isabel Leiva-Gea,1 Lidia Sanchez-Alcoholado,´ 2 Composition and Functionality Beatriz Mart´ın-Tejedor,1 Daniel Castellano-Castillo,2,3 Between Children With Type 1 Isabel Moreno-Indias,2,3 Antonio Urda-Cardona,1 Diabetes and MODY2 and Healthy Francisco J. Tinahones,2,3 Jose´ Carlos Fernandez-Garc´ ´ıa,2,3 and Control Subjects: A Case-Control Mar´ıa Isabel Queipo-Ortuno~ 2,3 Study Diabetes Care 2018;41:2385–2395 | https://doi.org/10.2337/dc18-0253 OBJECTIVE Type 1 diabetes is associated with compositional differences in gut microbiota. To date, no microbiome studies have been performed in maturity-onset diabetes of the young 2 (MODY2), a monogenic cause of diabetes. Gut microbiota of type 1 diabetes, MODY2, and healthy control subjects was compared. PATHOPHYSIOLOGY/COMPLICATIONS RESEARCH DESIGN AND METHODS This was a case-control study in 15 children with type 1 diabetes, 15 children with MODY2, and 13 healthy children. Metabolic control and potential factors mod- ifying gut microbiota were controlled. Microbiome composition was determined by 16S rRNA pyrosequencing. 1Pediatric Endocrinology, Hospital Materno- Infantil, Malaga,´ Spain RESULTS 2Clinical Management Unit of Endocrinology and Compared with healthy control subjects, type 1 diabetes was associated with a Nutrition, Laboratory of the Biomedical Research significantly lower microbiota diversity, a significantly higher relative abundance of Institute of Malaga,´ Virgen de la Victoria Uni- Bacteroides Ruminococcus Veillonella Blautia Streptococcus versityHospital,Universidad de Malaga,M´ alaga,´ , , , , and genera, and a Spain lower relative abundance of Bifidobacterium, Roseburia, Faecalibacterium, and 3Centro de Investigacion´ BiomedicaenRed(CIBER)´ Lachnospira. Children with MODY2 showed a significantly higher Prevotella abun- de Fisiopatolog´ıa de la Obesidad y Nutricion,´ In- dance and a lower Ruminococcus and Bacteroides abundance. Proinflammatory stituto Salud Carlos III, Madrid, Spain cytokines and lipopolysaccharides were increased in type 1 diabetes, and gut per- Corresponding author: Jose´ Carlos Fernandez-´ Garc´ıa, [email protected]. meability (determined by zonulin levels) was significantly increased in type 1 Received 5 February 2018 and accepted 26 diabetes and MODY2. The PICRUSt analysis found an increment of genes related to August 2018. lipid and amino acid metabolism, ABC transport, lipopolysaccharide biosynthesis, This article contains Supplementary Data online arachidonic acid metabolism, antigen processing and presentation, and chemokine at http://care.diabetesjournals.org/lookup/suppl/ signaling pathways in type 1 diabetes. doi:10.2337/dc18-0253/-/DC1. I.L.-G. and L.S.-A. contributed equally to this CONCLUSIONS work. Gut microbiota in type 1 diabetes differs at taxonomic and functional levels not only © 2018 by the American Diabetes Association. in comparison with healthy subjects but fundamentally with regard to a model of Readers may use this article as long as the work nonautoimmune diabetes. Future longitudinal studies should be aimed at eval- is properly cited, the use is educational and not for profit, and the work is not altered. More infor- uating if the modulation of gut microbiota in patients with a high risk of type 1 mation is available at http://www.diabetesjournals diabetes could modify the natural history of this autoimmune disease. .org/content/license. 2386 Gut Microbiota in Type 1 Diabetes and MODY2 Diabetes Care Volume 41, November 2018 Type 1 diabetes is a multifactorial immune- caused by a heterozygous inactivating diseases or infectious diseases or un- mediated disease characterized by the mutation in the glucokinase (GCK) gene dergoing treatment with antibiotics, pre- progressive loss of insulin-producing b- (14). To date, no studies have evaluated biotics, or probiotics or any other medical cells in the islets of Langerhans in the the gut microbiome structure in MODY2 treatment that could potentially influ- pancreas. The causes that lead to the patients. Nevertheless, MODY2 is a highly ence intestinal microbiota 3 months be- appearance of type 1 diabetes have not attractive model to assess the relation of fore inclusion. yet been fully identified, with genetic gut microbiota with type 1 diabetes, as The parents of all the participants factors playing a major role; however, MODY2 is not normally associated with completed a structured interview to ob- environmental factors are also closely obesity, a glycemic control similar to tain health status, lifestyle aspects, and linked, such as birth delivery mode (1), type 1 diabetes can be achieved, and, dietary habits. Patients with type 1 di- diet in early life (cow milk proteins or importantly, its cause is not of autoim- abetes and MODY2 were instructed to gluten-containing cereals) (2), and wide- mune origin (15). follow a standard diabetic diet, contain- spread usage of antibiotics (3), all fac- We hypothesize that if the fecal micro- ing 40–50% of calories from carbohy- tors closely related to gut microbiota. biota in type 1 diabetes differs from that drates, 20–30% from fat, and 20% from Recent studies have associated the of MODY2, the gut microbiota profile protein. Dietary intake patterns were microbiome with the development of could constitute a novel associated risk determined from a food frequency ques- type 1 diabetes; animal studies have factor for the type 1 diabetes autoim- tionnaire. demonstrated a close link between in- mune process. On the contrary, if the The written informed consents of the testinal microbiota and type 1 diabetes fecal microbiota were similar and differ- children’s guardians or parents were in BioBreeding diabetes-prone rats (4) ent from the microbiota of healthy con- obtained. The sampling and experimen- and in nonobese diabetic mice (5). Also, trol subjects, this would indicate that tal processes were performed with the in children with type 1 diabetes, auto- differences in intestinal microbiota could approval of the local Ethics Committee immune positivity has been related to be attributed to hyperglycemia per se. of the Regional Hospital of Malaga.´ changes in microbiota composition (6,7). Therefore, the aim of this case-control Laboratory Measurements In a previous study, we found that in study is to evaluate the gut microbiota Serum glucose, cholesterol, triglycerides, comparison with healthy control sub- profile, functional capacity, low-grade and interleukin-1b (IL-1b) and IL-10 cyto- jects, children with type 1 diabetes pre- inflammation, and gut permeability be- kines were measured by ELISA as previ- sented large significant differences in tween patients with type 1 diabetes and ously described (17). Concentrations of IL-6, the relative abundance of predominant MODY2 and healthy control subjects. IL-13, and tumor necrosis factor (TNF)-a phyla, families, and genera (8). Poten- were quantified by ELISA assay kits (Thermo tially, the altered microbiota profile in RESEARCH DESIGN AND METHODS Fisher Scientific) in serum samples ac- type 1 diabetes may be associated with This study was a case-control study, in- cording to the instructions of the man- alterations in the gut immune system, cluding 15 children with type 1 diabetes, ufacturer. The detection limits were as such as increased gut permeability (9). 15 children with MODY2, and 13 healthy follows: 7.8–500 pg/mL for IL-6, 1.6–100 Recent studies have shown that com- control children, all under 18 years old, pg/mL for IL-13, and 15.6–1,000 pg/mL mensal bacteria are crucial for the ma- of Caucasian origin and with the same for TNF-a. turing and functioning of the mucosal geographical location. immune system. Moreover, an impaired Type 1 diabetes was diagnosed accord- DNA Extraction, Pyrosequencing of 16S integrity of the intestinal barrier with ing to the criteria of the American Di- rRNA Sequences, and Bioinformatic an increase in permeability has been abetes Association (16) and the positivity Analysis described in both animal models and of at least two persistent, confirmed anti- Study participants collected their fecal human type 1 diabetes studies (10). islet autoantibodies (anti-insulin autoan- samples in a sterile and hermetically Therefore, given that intestinal microbes tibodies, GAD autoantibodies, or tyrosine sealed receptacle provided by the re- may affect intestinal permeability, intes- phosphatase autoantibodies). MODY2 search team. Fecal samples were col- tinal ecology could also play a crucial role children were diagnosed by suggestive lected in the morning of the day of in the development of type 1 diabetes clinical history, negative anti-islet auto- sample receipt and were immediately (11). On the other hand, zonulin, a phys- antibodies, and positive genetic testing. refrigerated in household freezers and iological modulator of intercellular tight Healthy control subjects were children transported to the laboratory during the junctions, increases gut permeability and with negative anti-islet autoantibodies, following 4 h. Frozen fecal samples were macromolecule absorption, and previous matched to children with type 1 diabetes transported with ice to avoid important studies claim a role for zonulin as a capital andMODY2forage,sex,race,BMI,mode changes of temperature that might cause regulator of intestinal barrier function in of delivery, and duration of breastfeed- bacterial DNA degradation and were the genesis of metabolic disorders (12). ing. In addition, patients with type 1 subsequently stored