Diabetes Care Volume 37, August 2014 2343 Gut Dysbiosis and Detection of Junko Sato,1 Akio Kanazawa,1,2 Fuki Ikeda,1 Tomoaki Yoshihara,1 “Live Gut Bacteria” in Blood of Hiromasa Goto,1 Hiroko Abe,1 Koji Komiya,1 Minako Kawaguchi,1 Japanese Patients With Type 2 Tomoaki Shimizu,1 Takeshi Ogihara,1 Yoshifumi Tamura,1,3 Yuko Sakurai,1 Diabetes Risako Yamamoto,1 Tomoya Mita,1 Yoshio Fujitani,1,4 Hiroshi Fukuda,5 6 6 Diabetes Care 2014;37:2343–2350 | DOI: 10.2337/dc13-2817 Koji Nomoto, Takuya Takahashi, Takashi Asahara,6 Takahisa Hirose,7 Satoru Nagata,8,9 Yuichiro Yamashiro,8 and Hirotaka Watada1–4,10 OBJECTIVE Mounting evidence indicates that the gut microbiota are an important modifier of obesity and diabetes. However, so far there is no information on gut microbiota and “live gut bacteria” in the systemic circulation of Japanese patients with type 2 diabetes. RESEARCH DESIGN AND METHODS 1Department of Metabolism & Endocrinology, Using a sensitive reverse transcription–quantitative PCR (RT-qPCR) method, we Juntendo University Graduate School of Medi- cine, Tokyo, Japan determined the composition of fecal gut microbiota in 50 Japanese patients with 2Center for Therapeutic Innovations in Diabetes, type 2 diabetes and 50 control subjects, and its association with various clinical Juntendo University Graduate School of Medi- parameters, including inflammatory markers. We also analyzed the presence of cine, Tokyo, Japan PATHOPHYSIOLOGY/COMPLICATIONS 3 gut bacteria in blood samples. Sportology Center, Juntendo University Gradu- ate School of Medicine, Tokyo, Japan 4 RESULTS Center for Beta Cell Biology and Regeneration, Juntendo University Graduate School of Medi- The counts of the Clostridium coccoides group, Atopobium cluster, and Prevotella cine, Tokyo, Japan (obligate anaerobes) were significantly lower (P < 0.05), while the counts of total 5Department of General Medicine, Juntendo Lactobacillus (facultative anaerobes) were significantly higher (P < 0.05) in fecal University Graduate School of Medicine, Tokyo, Japan samples of diabetic patients than in those of control subjects. Especially, the counts 6Yakult Central Institute for Microbiological of Lactobacillus reuteri and Lactobacillus plantarum subgroups were significantly Research, Tokyo, Japan higher (P < 0.05). Gut bacteria were detected in blood at a significantly higher rate 7Department of Metabolism & Endocrinology, in diabetic patients than in control subjects (28% vs. 4%, P < 0.01), and most of these Toho University School of Medicine, Tokyo, Japan 8Probiotics Research Laboratory, Juntendo Uni- bacteria were Gram-positive. versity Graduate School of Medicine, Tokyo, Japan CONCLUSIONS 9Department of Pediatrics, Tokyo Women’s This is the first report of gut dysbiosis in Japanese patients with type 2 diabetes as Medical University, Tokyo, Japan 10 assessed by RT-qPCR. The high rate of gut bacteria in the circulation suggests Center for Molecular Diabetology, Juntendo University Graduate School of Medicine, Tokyo, translocation of bacteria from the gut to the bloodstream. Japan Corresponding author: Akio Kanazawa, akana@ The gut microbiota are essential for the host immune system (1), digestion, includ- juntendo.ac.jp. ing the breakdown of complex carbohydrates such as dietary fibers, and the pro- Received 2 December 2013 and accepted 28 duction of organic acids to maintain an appropriate pH environment in the gut (2). March 2014. The study of gut microbiota is rapidly progressing, and it is no exaggeration to say This article contains Supplementary Data online that the introduction of culture-independent approaches based on 16S rRNA anal- at http://care.diabetesjournals.org/lookup/ suppl/doi:10.2337/dc13-2817/-/DC1. ysis has led to a paradigm shift in this field (2,3). In addition to its physiological © 2014 by the American Diabetes Association. importance, gut dysbiosis is associated with obesity through the increased avail- Readers may use this article as long as the work ability of energy-rich foods such as Western diet (4–6). Together with previous data, is properly cited, the use is educational and not new information on the pathophysiological roles of the gut and blood microbiota in for profit, and the work is not altered. 2344 Gut Dysbiosis in Japanese Patients With Diabetes Diabetes Care Volume 37, August 2014 the development of atherosclerosis patients with type 2 diabetes and 50 con- 5 days of submitting fecal samples. One have been reported (7–9). The impor- trol subjects by detecting 16S rRNA. We milliliter of blood was added to 2 mL of tant concept of the role of gut micro- also investigated the relation among var- RNAprotect bacterial reagent (Qiagen, biota in insulin resistance was first ious clinical parameters, food intake, and Hilden, Germany) immediately after col- described by Cani and colleagues gut microbiota to determine the clinical lection. The samples were stored at (4,10–13). In a series of studies, these significance of gut microbiota in Japanese 2808C. Both the fecal and blood samples investigators demonstrated that intesti- patients with type 2 diabetes. were transported at 2208C to the Yakult nal Gram-negative bacteria produced Central Institute for Microbiological lipopolysaccharide (LPS), which is a RESEARCH DESIGN AND METHODS Research. well-known proinflammatory molecule, Study Subjects To quantify the bacteria present in can translocate to the bloodstream We recruited Japanese type 2 diabetic thesamples,weextractedtotalRNA from a leaky gut, and causes metabolic patients who regularly visited the Outpa- fractions from feces and blood by the endotoxemia, which is associated with tient Clinic of Juntendo University Hospi- method previously described (18–21), obesity (4). More specifically, a high-fat tal between 2011 and 2012, and 50 and examined the gut microbiota com- diet enhances the disruption of the tight patients agreed to participate in the position and plasma levels of the gut junction proteins in the intestine, such study. We also recruited control subjects bacteria by using the 16S rRNA-targeted as zonula occludens-1 and occludin, who regularly visited the Outpatient RT–quantitative PCR (qPCR) using the that are involved in the gut barrier func- Clinic of Juntendo University Hospital Yakult Intestinal Flora-SCAN. Three tion in mouse models (13). This effect is for the management of hypertension, serial dilutions of the extracted RNA directly dependent on the gut micro- dyslipidemia, or thyroid disease, whose sample were used for bacterial rRNA- biota because antibiotic treatment abol- conditions were under good control or targeted RT-qPCR (18–21), and the ished diet-induced gut permeability (4). who visited Juntendo University Hospital threshold cycle values in the linear The above studies suggest that gut for routine medical checkups between range of the assay were applied to the dysbiosis and the related increased per- 2011 and 2012. Fifty control subjects standard curve to obtain the corre- , meability of the gut could serve as envi- with HbA1c levels 6.0% (42 mmol/mol) sponding bacterial cell count in each nu- ronmental factors for the development agreed to participate. Patients with the cleic acid sample. Those data were then of obesity before the development of following conditions were excluded used to determine the number of bacte- diabetes. While data from mice are con- from the study: 1) proliferative retinopa- ria per sample. The specificity of the RT- vincing, the functional links between hu- thy; 2)age$80 years; 3) serious liver qPCR assay using the group-, genus-, or man gut microbiota and disease are less disease (aspartate aminotransferase species-specificprimerswasdeter- well-understood because of various and/or alanine aminotransferase level mined as described previously (18–21). confounding factors, including age, sex, .100 IU/L) or serious kidney disease (se- The sequences of the primers are listed diet, genetics, and race (14,15). rum creatinine level .2.0 mg/dL; 4) acute in Supplementary Table 1. In Japan, the estimated number of pa- heart failure; 5) malignancy; 6)inflamma- tients with type 2 diabetes has increased tory bowel disease; and 7) a history of Measurement of Organic Acids to 10.7 million, the sixth largest such pop- treatment with antibiotics within 3 and pH ulation in the world (16). However, there months of study participation. The study The concentrations of organic acids in fe- were no human studies on gut microbiota protocol was approved by the Human ces and serum were determined using composition in Japanese patients with Ethics Committee of Juntendo University, methods described previously (21) with type 2 diabetes. The Japanese diet is and written informed consent was ob- slight modifications. Briefly, the frozen very different from the Western diet, tained from each patient before enroll- sample was homogenized in four volumes and gut microbiota are known to be influ- ment in the study. of 0.15 mol/L perchloric acid and allowed enced by food type (6), suggesting that to stand at 48C for 12 h. The suspension gut microbiota in Japanese patients with Determination of the Bacterial was centrifuged at 20,400 3 g at 48Cfor type 2 diabetes might be different from Count by 16S rRNA-Targeted Reverse 10 min. Then, the resulting supernatant those of Western individuals. Therefore, Transcription–Quantitative PCR was passed through a filter with a pore it is important to investigate gut micro- After study enrollment, the participants size of 0.45 mm (Millipore Japan, Tokyo, biota composition in Japanese patients were asked to submit fresh fecal samples. Japan). The sample was analyzed for with type 2 diabetes. The fecal samples were placed directly organic acids using a high-performance Intriguingly, Amar et al. (17) reported into two tubes (;1.0 g/tube) by the par- liquid chromatography system (432 Con- that a high-fat diet induces bacterial ticipants or hospital staff members; one ductivity Detector; Waters Co., Milford, translocation from the gut to the blood- tube contained 2 mL of RNAlater (an RNA MA), and pH in feces was analyzed using stream in humans by detecting bacterial stabilization solution; Ambion, Austin, the IQ 150 pH/Thermometer (IQ Scientific genomic DNA encoding 16S rRNA.