Research ArticleMicroflora Disturbance during Progression of GlucoseIntolerance and Effect of Sitagliptin An Animal Study

Xinfeng Yan Bo Feng Peicheng Li Zhaosheng Tang and Lin Wang

Department of Endocrinology Shanghai East Hospital Tongji University School of Medicine Shanghai 200120 China

Correspondence should be addressed to Bo Feng fengbomedmailcomcn

Received 20 April 2016 Accepted 14 July 2016

Academic Editor Ruben Varela-Calvino

Copyright copy 2016 Xinfeng Yan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Emerging evidences have shown a close interplay between obesity diabetes and intestinal flora disturbanceDipeptidylpeptidase-4 inhibitor exemplified by sitagliptin is highly efficacious in treating type 2 diabetes (T2DM) yet little is known ifsitagliptin exerts beneficial effects on microbiota associated with obesity and T2DM We evaluated changes of gut microbiotafollowing the induction of obesity and T2DM in a streptozotocin treated high fathigh carbohydrate fed (HFHC-STZ) rat modeland explored the effect of sitagliptin on gut microbiota for HFHC-STZ ratsMethods Sitagliptin was administered via oral gavageto diabetic rats Fecal DNA extraction and 454 pyrosequencing based on analysis of 16S rRNA genes was utilized to determinethe overall structure of microbiota in fecal DNA samples Results Results showed that at the level of phylum there was higherabundance of Firmicutes and Tenericutes and less abundance of Bacteroidetes in obese rats compared to their lean counterpartsAt the level of genus short-chain fatty acid- (SCFA-) producing bacteria Blautia Roseburia and Clostridium and probioticsLactobacillus Bifidobacterium and so forth were identified significantly different from each other among conditions ConclusionMarked shifts of the gut microbiota structure were observed in the rats during development of glucose intolerance Intestinal florachanged in the process of glucose intolerance and treatment of sitagliptin moderately corrected the dysbiosis of microbiota inT2DM

1 Introduction

Type 2 diabetes mellitus (T2DM) is exacting a huge level ofpatient suffering and social cost worldwide Recent studiessuggested that an altered composition and diversity of gutmicrobiota could play an important role in the developmentof metabolic disorders As the second genome of humanintestinal flora has commensal relationship of mutual benefitwith the host Although not fully understood yet the gutmicrobiota is implicated in various aspects of intestinalfunction integrity including epithelial cell turnover immunemodulation and gastrointestinalmotilityThe gutmicrobiotaalso regulate energy metabolism such as breaking downdietary toxins and carcinogens manufacturing micronutri-ents fermenting indigestible food substances facilitatingthe absorption of certain electrolytes and trace mineralsand governing the growth and differentiation of enterocytesthrough the production of short-chain fatty acids (SCFA)[1ndash4] Studies have shown that obesity promotes the growth

of the Firmicutes phylum and reduces the proportion ofBacteroidetes phylum in the gut [5ndash9] Implantation of gutflora from obese mice to normal and germfree mice resultedin increased body weight and insulin resistance [9 10]supporting the notion that the bacterial species from obesegut have metabolically unfavorable properties The associa-tions between microbiota and obesity insulin resistance anddiabetes are presumably due to the impaired ability of themicrobes to extract energy from the diet [9] altered fatty acidmetabolism [11] changes in secretion of gut hormones suchas peptide YY (PYY) [12] activation of lipopolysaccharidetoll-like receptor-2 [13] and changes in the intestinal barrierintegrity [14] There seems to be an obvious growing interesttoward the relationship between gut microbes and T2DM

Sitagliptin a dipeptidyl peptidase-4 inhibitor receivedapproval from the US FDA in 2006 for treatment of type 2diabetes mellitus It prevents the enzymatic degradation ofglucagon-like peptide 1 (GLP-1) and glucagon-like peptide2 (GLP-2) GLP-1 appears to increase insulin secretion

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2016 Article ID 2093171 10 pageshttpdxdoiorg10115520162093171

2 Journal of Diabetes Research

decrease glucagon secretion decrease hepatic gluconeogen-esis improve insulin sensitivity and delay gastric emptying[15] GLP-2 appears to be an intestinal specific growthfactor promoting the growth of intestinal mucosa repairingdamaged intestinal epithelium and improving the intestinalmucosa barrier integrity [16 17] Since sitagliptin affects themetabolism of GLP-1 and GLP-2 secreted from enteroen-docrine we wonder whether sitagliptin regulates intestinalflora

Among available methodologies to study the microbialecology of complex bacterial communities the so-calledmetagenomics approach is considered to be the ldquogoldenstandardrdquo [18 19] In the current study we analyzed thestructure of intestinal flora in a HFHC progressive glucoseintolerance rat model with or without sitagliptin treatmentby 454 pyrosequencing we tried to explore whether and towhat extent sitagliptin regulated microbiota

2 Materials and Methods

21 Drug and Diets The HFHC diet (containing 198 gfat 446 g carbohydrate and 223 g protein per 100 g and40 kcal fat 40 kcal carbohydrate and 20 kcal protein byenergy) was purchased from Shanghai Laboratory AnimalCenter Chinese Academy of Science (SLACCAS) Labora-tory Animal Co Ltd (Shanghai China) The streptozotocin(STZ)was purchased fromSigmaUSA Sitagliptinwas a kindgift fromMSD China

22 Animal Experiments Fifteen four-week-old male Spra-gue-Dawley (SD) rats (pathogen-free grade average bodyweight 110 g) were purchased from SLACCAS LaboratoryAnimal (Shanghai China) All rats were acclimatized in ourlaboratory for 7 days before the initiation of the experimentAfter fasting for 12 hours fresh stool samples were collectedby stimulating the anus and immediately stored at minus80∘Cfor subsequent analysis Then the rats were fed with HFHCdiet for 4 weeks and stool samples were collected for thesecond time After fasting for 12 hours an OGTT test wasperformed to assess the insulin resistance The rats werereceiving 50 D-glucose solution by gavage at 2 gkg bodyweight and blood glucose levels were measured at 0 30 6090 and 120 minutes following the glucose challenge usingblood samples collected by tail snippingThe day after OGTTtest the HFHC fed (4 weeks on the diet) rats were injectedintraperitoneally with STZ (30mgkg body weight) to inducediabetes The animals were fed continuously on the HFHCdiet throughout the rest of the study Ten rats developeddiabetes with fasting blood glucose (FBG) gt111mmolL at2 weeks after STZ treatment [20 21] Two rats died and 3rats failed to develop diabetes Stool samples were collectedfrom the rest of the 10 rats for the third time 4 weeksafter the induction of T2DMThereafter sitagliptin (10mgkgbody weight oral gavage once a day) was administered toall 10 diabetic rats for 12 weeks followed by another stoolsample collection (Figure S1 in SupplementaryMaterial avail-able online at httpdxdoiorg10115520162093171) Bodyweight was weighed and blood glucose was checked weeklywith Bayer glucometer during the study

All animal experimental protocols were approved by theAnimal Ethics Committee of Animal Center of East HospitalTongji University and all the animal experiments were car-ried out in strict accordance with the Guidelines for Care andUse of Laboratory Animals of the Animal Ethics Committeeof Animal Center of East Hospital Tongji University Allefforts were made to minimize animal suffering

23 Fecal DNAExtraction and 454 Pyrophosphate SequencingMicrobial DNA was extracted from stool samples using theEZNA Stool DNA Kit (Omega Bio-tek Norcross GAUSA) according to manufacturerrsquos instructions The V1ndashV3regions of the bacteria 16S ribosomal DNA gene were ampli-fied by polymerase chain reaction (95∘C for 2min followedby 25 cycles at 95∘C for 30 s 55∘C for 30 s and 72∘C for 30 sand a final extension at 72∘C for 5min) using primers 27F51015840-(CCTATCCCCTGTGTGCCTTGGCAGTCGACT-3101584051015840-AGAGTTTGATCCTGGCTCAG)-31015840 and 533R 51015840-(CCA-TCTCATCCCTGCGTGTCTCCGACGACT-31015840-MID tgas-51015840-TTACCGCGGCTGCTGGCAC)-31015840 PCR reactions wereperformed in a 20120583L mixture containing 4 120583L of 5x FastPfuBuffer 2120583L of 25mM dNTPs 08 120583L of each primer (5120583M)04 120583L of FastPfu Polymerase and 10 ng of template DNAAfter purification with the AxyPrep DNA Gel Extraction Kit(Axygen Biosciences Union City CA USA) and quantifi-cation using QuantiFluor-ST (Promega US) the amplifiedmixture was used for pyrosequencing on a Roche 454 GSFLX+ Titanium platform (Roche 454 Life Sciences BranfordCT USA) according to standard protocols at Majorbio Bio-Pharm Technology Co Ltd Shanghai ChinaThe raw readswere deposited into the NCBI Sequence Read Archive (SRA)database (Accession Number SRP056522)

In total 323039 valid sequences were obtained from all40 samples with an average length of 419 bp per sequenceThe resulting sequences were processed using QIIME (ver-sion 117) After removing sequences with average qualityscore lt20 over a 50 bp sliding window sequences shorterthan 200 bp sequences with homopolymers longer than sixnucleotides and sequences with ambiguous base calls orincorrect primer sequences a total of 241879 high-qualitysequences were produced with an average length of 430 bpper sequence Operational Taxonomic Units (OTUs) wereclustered with 97 similarity cutoff using UPARSE (ver-sion 71 httpdrive5comuparse) and chimeric sequenceswere identified and removed using UCHIME The phylo-genetic affiliation of each 16S rDNA gene sequence wasanalyzed by RDP Classifier (Michigan State Universityhttprdpcmemsuedu) against the SILVA (SSU115) 16SrDNA database using confidence threshold of 70 [22]

24 Statistical Analysis Comparisons of diversity estimatorsand the metabolic indices between conditions were analyzedby the one-way repeated measures ANOVA using SPSSversion 130 for Windows OTUs that reached 97 similaritylevel were used for diversity (Shannon) rarefaction curve andShannon-Wiener curve analysis by using Mothur (version1301) [23] Heatmap figure was generated using R packagesgplots [24] at genus level A differentially abundance featurewas analyzed based on multiple hypotheses testing rare

Journal of Diabetes Research 3

Normal Obesity Diabetes Sitagliptin0

100

200

300

400

500O

TUs

Δ

ΔΔ

ΔΔ

(a)

Normal Obesity Diabetes Sitagliptin0

1

2

3

4

5

Shan

non

Δ

Δ ΔΔ

(b)

Figure 1 Pyrosequencing data summary (a) number of OTUs (b) Shannon (diversity estimator) All data were calculated at 3 distanceΔ

119875

lt 005 ΔΔ119875

lt 001 data are expressed as mean plusmn SD

Table 1 Bodyweight and blood glucose changes during progressionof glucose intolerance and after Sitagliptin treatment Data areexpressed as mean plusmn SD (compared with group obesity I

119875

lt 001compared with group diabetes 998779

119875

lt 001 998771119875

lt 005)

Conditions Body weight (g) Blood glucose (mmolL)Normal 15506 plusmn 64I 383 plusmn 025I

Obesity 39892 plusmn 2306 430 plusmn 037998779

Diabetes 41683 plusmn 3564 1881 plusmn 255I

Sitagliptin 43267 plusmn 5810 1554 plusmn 139I998771

frequency data and false discovery rate (FDR) analysis usingMetastat analysis [25] Principal coordinates analysis (PCoA)was performed based on unweighted UniFrac distance Inaddition linear discriminant analysis effect size (LEfSe) wasperformed first based on nonparametric factorial Kruskal-Wallis (KW) sum-rank test and then we performed the lineardiscriminant analysis following the Wilcoxon Signed-Ranktest to assess effect size of each differentially abundant taxonor OTU

3 Results

31 Body Weight and Blood Glucose As shown in Table 1HFHC diet resulted in significant increase of body weight(39892 plusmn 2306 g in obesity condition versus 15506 plusmn 64 gin normal condition 119875 lt 001) Furthermore STZ injectionresulted in dramatic increase of blood glucose level in dia-betes condition as compared to normal or obesity condition(119875 lt 001) As expected sitagliptin resulted in a significantreduction of blood glucose (119875 lt 005) while having nosignificant impacts on body weight (119875 = ns)

32 OGTT Test The fasting blood glucose of obese rats wassignificantly higher than that in normal control condition(430 plusmn 037mmolL versus 383 plusmn 025mmolL 119875 lt 001)and blood glucose level at 120min was 892 plusmn 123mmolLwhich should be considered IGT by definition [26] Data areexpressed as mean plusmn SD

33 Characteristics of 454 Pyrosequencing Results A total of241879 high-quality sequences of 40 samples were produced

in this study with an average of 6047 sequences per sampleA brief summary and the estimators of OTUs and diversity(Shannon) are shown in Figure 1 and detailed characteristicsof each sample are listed in Table S1 There were statisticallysignificant differences of Shannon indexes between obesecondition and sitagliptin-treated condition (432plusmn036 versus465 plusmn 020 119875 lt 005) and between diabetic conditionand sitagliptin-treated condition (399 plusmn 025 versus 465 plusmn020 119875 lt 001) suggesting significant higher diversityfound in sitagliptin condition compared to obese or diabeticcondition

The rarefaction curves of all four conditions did not leveloff at the sequencing depth of 6000 (Figure S2) Thereforethis sequencing depth was not sufficient to cover the wholebacterial diversity Thus it deserves further sequencing todetect more bacterial species in the rat feces However whenthe Shannon curves tended to be smooth the sequencing dataquantity became dramatically bigger which reflected the vastmajority of microbes in the sample (Figure S3)

34 Microbial Structures Differed Significantly among Con-ditions There were significant differences at the phylumlevel in rat fecal microbiota during progression of glucoseintolerance and after treatment with sitagliptin Figure 2demonstrated that Bacteroidetes and Firmicutes accountedfor the majority of microflora (gt90) in each conditionThe relative abundance of Firmicutes in obesity conditionwas significantly increased compared to that in the normalcondition (8628 versus 6624 119875 lt 001) while therelative abundance of Bacteroidetes decreased substantially(1216 versus 3164 119875 lt 001) There was no significantdifference between obesity and diabetes at phylum level Therelative abundance of Firmicutes in the sitagliptin conditionwas significantly less than that in the diabetic condition(6319 versus 8356 119875 lt 001) In contrast the relativeabundance of Bacteroidetes increased dramatically (3246versus 1606 119875 lt 001) It is noteworthy that the relativeabundance of Tenericutes in the obese condition significantlyincreased compared to the normal condition (115 versus061 119875 lt 005) but decreased dramatically after inducingdiabetes (115 versus 009 119875 lt 001) and then increasedlargely after sitagliptin treatment as compared to the diabetic

4 Journal of Diabetes Research

Table 2 Statistical analysis was performed by Wilcoxon Signed-Rank test Data of normal obesity diabetes and sitagliptin are relativeabundance (percentage) of all sequences in each group (119875 group obesity versus normal 119875lowast group diabetes versus obesity 119875998779 group diabetesversus sitagliptin)

Taxonomy Normal () Obesity () Diabetes () Sitagliptin () 119875

119875

lowast

119875

998779

Bacteroides (genus) 14 02 06 54 0013 0022 0005Prevotellaceae (family) 99 18 68 92 0005 0001Blautia (genus) 02 08 71 03 0012 0005 0005Roseburia (genus) 07 09 03 05 0042Ruminococcus (genus) 25 14 06 19 0025 0007Clostridium (genus) 13 46 08 07 0014 0013Lactobacillus (genus) 223 225 130 67 0028Bacillus (genus) 46 44 60 0 0005Bifidobacterium (genus) 0024 0005 0002 0085 0007Parabacteroides (genus) 02 002 01 06 0008 0024 0005

Normal Obesity Diabetes Sitagliptin0

10

20

30

40

50

60

70

80

90

100

Relat

ive a

bund

ance

()

OthersTenericutesProteobacteria

ActinobacteriaBacteroidetesFirmicutes

Figure 2 Relative abundance of different bacterial phyla in micro-biota of four conditions

condition (096 versus 009 119875 lt 001) Similar changeswere observed at the phylum of Proteobacteria

Furthermore we explored the similarities and distinc-tions of species distribution in all conditions As shown inFigure 3 we found that there were 441 species shared in thefour conditions accounting for around half of the OTUs ineach condition It is noteworthy that about one-third of thespecies only found in normal (64OTUs) or obesity (22OTUs)condition belonged to Lachnospiraceae which is capableof fermenting complex carbohydrates to SCFAs playing animportant role in maintaining intestinal homeostasis [2728] Interestingly one-third of the total species only foundin sitagliptin condition (94OTUs) belonged to Ruminococ-caceae which is a major utilizer of plant polysaccharides[29 30]

In addition Metastat analysis showed significant differ-ences of relative abundance at the level of genus or familyamong conditions (Table 2 and Table S2) And the resultat level of genus was marked in the Heatmap plot (Figure

NC

Obesity DM

Sit22

22

13

94

64

441101

19

47

94

111

33 13148

43

Unique objects all = 1283 NC = 931Obesity = 965 DM = 787 sit = 948

Figure 3 Shared OTU analysis of the different conditions Venndiagram showing the unique and shared OTUs (3 distance level)in the different conditions

S4) which showed the significant differences of BlautiaRoseburia Clostridium and so on among four conditions

There were significant variations in the composition ofintestinal bacteria species among four study conditions Acladogram representation of the microbiota structure offour conditions and their predominant bacteria performedby LEfSe and the greatest differences in taxa among fourcommunities are shown in Figure 4 and Figure S5

To compare the overall microbiota structures in all condi-tions the unweighted Unifrac distance matrix was calculatedbased on the OTUs of each sample The PCA result revealeda significant difference in gut bacterial structure among allconditions The three principal component scores accountedfor 3057 2006 and 86 of total variations respectively(Figure 5)

4 Discussion

In the study we explored the changes of rat intestinal micro-biota during progression of glucose intolerance and the effect

Journal of Diabetes Research 5

Cladogram

Candidate_division_TM7

mlk

ji

gd

ef

c

c3c0

b7b8 b9

c1 c2 Tene

ricut

es

Spiro

chae

tae

Proteobact

eria

b6b3

b5b2

b0a8

a6a3

a2

a1a0

z

yx

wv

ut

sr

pq

o n

a4a5

a7

a9 b1 b4

c4 c5

Actinobacteria

bah

NCObesityDMSit

a Bifidobacteriaceaeb Bifidobacterialesc Actinobacteriad Coriobacteriaceaee Coriobacterialesf Coriobacteriiag Bacteroidaceaeh Porphyromonadaceaei Prevotellaceaej Rikenellaceaek no rankl no rankm no rankn Bacillaceaeo Listeriaceae

p Staphylococcaceaeq Bacillalesr Carnobacteriaceaes Lacatobacillaceae

v Bacilliu Lactobacillalest Streptococcaceae

x Clostridiaceaew Christensenellaceae

z othery Family_XIII_Incertae_Sedis

a0 Peptococcaceaea1 Peptostreptococcaceaea2 Ruminococcaceaea3 Erysipelotrichaceaea4 Erysipelotrichalesa5 Erysipelotrichiaa6 Rhodospirillaceaea7 Rhodospirillales

a8 Alcaligenaceaea9 Comamonadaceaeb0 Burkholderialesb1 Rhodocyclaceaeb2 Rhodocyclalesb3 Betaproteobacteriab4 Desulfovibrionaceaeb5 Desulfovibrionalesb6 Deltaproteobacteriab7 Enterobacteriaceaeb8 Enterobacterialesb9 Gammaproteobacteriac0 Spirochaetaceaec1 Spirochaetalesc2 Spirochaetesc3 no rankc4 RF9c5 Mollicutes

Figure 4 Taxonomic representation of statistically and biologically consistent differences among condition normal obesity diabetes andsitagliptin Differences are represented by the color of the most abundant class (red indicating normal condition green obesity conditionblue diabetes condition purple sitagliptin condition and yellow nonsignificance) The diameter of each circle is proportional to the taxonrsquosabundance

6 Journal of Diabetes Research

PCoA

NC1

NC2 NC3

NC4

NC5

NC6

NC7

NC8

NC9

NC10

Obe1

Obe2Obe3

Obe4

Obe5 Obe6

Obe7

Obe8

Obe9

Obe10

DM1

DM2

DM3

DM4DM5

DM6

DM7

DM8DM9

DM10Sit1

Sit2Sit3

Sit4

Sit5

Sit6

Sit7Sit8

Sit9Sit10

minus04

minus02

00

02

04

PC3

86

00 05minus05PC2 2006

NCObe

DMSit

PCoA

NC1

NC2NC3

NC4

NC5

NC6NC7

NC8

NC9

NC10

Obe1

Obe2

Obe3

Obe4

Obe5

Obe7

Obe8Obe9

Obe10

DM1

DM2Obe2

DM3

DM4DM5

DM6

DM7

DM8DM9

DM10 Sit1

Sit2Sit3

Sit4

Sit5

Sit6

Sit7Sit8

Sit9

Sit10

00 05minus05PC1 3057

minus04

minus02

00

02

04

PC3

86

PCoA

NC1

NC2

NC3

NC4

NC5

C6NCncNC8

NC9

NC10 Obe1Obe2

Obe3Obe4

Obe5

Obe6

Obe7Obe8

Obe9

Obe10

DM1

Obe2DM3

DM4

DM5DM6

DM7

DM8DM9

DM10

Sit1

Sit2Sit3 Sit4

Sit5Sit6

Sit7Sit8

Sit9

Sit10

00 05minus05PC1 3057

minus05

00

05

PC2

20

06

NCObe

DMSit

NCObe

DMSit

Figure 5 PCA plots based on unweighted Unifrac metrics Principal components (PCs) 1 2 and 3 explained 3057 2006 and 86 Eachsymbol represents a sample

of sitagliptin onmicrobiotaWe found significantly more Fir-micutes and less Bacteroidetes in obese rats compared to theirlean counterparts similar to previous studies [31] despiteconflicting data showing no differences of Bacteroidetesand Firmicutes between obese and lean subjects [32ndash34]The increased Firmicutes in obese mice may be related tothe genes encoding enzymes that break down polysaccha-rides which cannot be digested by the host increasing the

production of monosaccharides and short-chain fatty acids(SCFA) and the conversion of these SCFA to triglyceridesin the liver The binding of SCFA to two G-protein-coupledreceptors (GPR41 and GPR43) induces peptide YY secretionwhich suppresses gut motility retards intestinal transit andsubsequently results in the increase of nutrient uptake anddeposition [35] Interestingly we also found that hydrogen-producing Prevotellaceae decreased significantly in the obese

Journal of Diabetes Research 7

condition Given the fact that hydrogen inhibits digestion theobese rats seemed likely to absorb more calories from similarenergy intake

Another interesting finding was the higher abundance ofthe phylum Tenericutes (class Mollicutes) in obese rats com-pared to their lean counterparts Certain species ofMollicutesbloom have been proved to evolve the capacity to importcertain types of carbohydrates common in westernized dietfor both mice and human being (eg glucose fructose andsucrose) and to metabolize these imported sugars to SCFAwhich could be readily absorbed by the host [36] Sitagliptindid restore the structure of gutmicrobe at the level of phylumsimilar to the lean control condition without significanteffects on body weight

Unweighted Unifrac PCA analysis confirmed aboveresults in the respect of overall microbial structures Theintestinal microbiota of the four conditions of rats werestructurally separated from each other in the three principalcomponents

Various mechanisms have been proposed to explain theinfluence of microbiota on insulin resistance and T2DMincludingmetabolic endotoxemiamodifications in the secre-tion of the incretins and butyrate production Decrease ofSCFA-producing bacteria has been commonly observed inmetabolic diseases including T2DM [37] and even colorectalcancer [38] SCFA-producing bacteria have been previouslyshown to benefit the host through protecting the mucosafrom damage induced by pathogens supplying colonocytenutrients mitigating inflammation and so forth [39 40]Recent studies in mice have shown that an increase incolonic production of short-chain fatty acids triggers intesti-nal gluconeogenesis (IGN) via complementary mechanismsButyrate activates IGN gene expression through a cAMP-dependent mechanism in enterocytes whereas propionateitself a substrate of IGN activates IGN gene expressionvia the portal nervous system and the fatty acid receptorFFAR3 In rodents the result of increased IGN is beneficialto glucose and energy homeostasis with reductions in hepaticglucose production appetite and body weight [41] Belong-ing to either Clostridium Eubacterium or Fusobacteriumthe butyrate-producing bacteria mainly exist in cecum andcolon Fusobacterium prausnitzii and Roseburia intestinalisare predominant species in butyric acid producing bacteriain human and animal intestine [42] At the level of genusour data suggested that the relative abundance of butyrate-producing Clostridium and Roseburia in the diabetic ratsdecreased significantly compared to the lean and obeseconditions Moreover we also observed that the SCFA-producing bacteria Blautia increased significantly in the dia-betic condition compared to the obese condition Becker etal demonstrated that the addition of Clostridium butyricumas a second butyrate-producing bacterium to SIHUMI (asimplified human intestinal microbiota Anaerostipes caccaeBacteroides thetaiotaomicron Bifidobacterium longum Blau-tia producta Clostridium ramosum Escherichia coli and Lac-tobacillus plantarum) led to an increase of butyrate by 56butdecrease of Bifidobacterium longum Blautia producta and Ecoli indicating that either of these organisms competes withClostridium butyricum for the same substrates or Clostridium

butyricum forms inhibitory substances [43] Thus it seemsreasonable to suppose that changes ofClostridiumRoseburiaand Blautia show an opposite trend In other words theincrease of SCFA-producing bacteria like Blautia leads todecrease of butyrate-producing bacteria like Clostridium andRoseburia In the current study the structure of SCFA-producing bacteria seemed to be abnormal in diabetic ratsAfter sitagliptin treatment Roseburia increased and Blautiadecreased while Clostridium showed no change

Probiotics such as Lactobacillus and Bifidobacteriumnormally reside in the intestinal tract and regulate gutmicroflora and mucosal immunity Recent data also suggestthat microflora especially probiotics exert beneficial effectson gastrointestinal discomforts such as diarrhea abdominalpain and bloating [44] Lactobacillus produces lactic acidCO2 acetic acid andor ethanol which may contribute to a

more acidic environment through homo- or heterofermenta-tive metabolism [45 46] Our data showed that Lactobacillusand Bifidobacterium decreased in the diabetic conditionHowever sitagliptin made Bifidobacterium increased withno significant effect on Lactobacillus which seemed to beconsistent with the notion we discussed above

Marked changes in microbiota composition after met-formin treatment were observed in high-fat diet- (HFD-) fedconditions especially Akkermansia belonging to the Verru-comicrobia phylum showed the most conspicuous changes[47] suggesting a possible interaction between HFD met-formin and intestinal microbiota Forslund et al found sig-nificant increase of Escherichia spp and decrease of Intestini-bacter spp in human gut microbiome in metformin-treatedT2DM especially the latter is resistant to oxidative stress andable to degrade fucose indicative of an indirect involvementin mucus degradation [41] Interestingly there are consistentdata from numerous studies showing that sitagliptin resultsin reduced gastrointestinal discomfort when combined withmetformin a classic antihyperglycemia agent also known forits gastrointestinal side effects For instance Reasner et alshowed that sitagliptin and metformin fixed dose combina-tion resulted in a significant reduction on abdominal pain anddiarrhea compared tometforminmonotherapy [48] In addi-tion a 104-week clinical study (Harmony) also showed thatdiarrhea incidence was reduced in sitagliptin plus metformingroup as compared to metformin monotherapy group [49]Though the underlying mechanism of effect of metforminand sitagliptin on intestinal flora remains unclear our resultsseem to provide a possible explanation arguing that thiseffect could be related to favorable changes of microflora aftersitagliptin treatment

We supposed that the effect of sitagliptin on microbiotawas related to GLP-2 which helps improve the intestinalmucosa barrier integrity Besides recent studies found thatDPP-4 may act as the protease activated receptor 2 (PAR2)agonists involved in the pathophysiology of PAR2 leading toproliferation and inflammation in smooth muscle cells ThusDPP-4 inhibitor can relieve edema of intestinal wall and alle-viate the intestinal inflammation [50 51] Integrated intestinalmucosa barrier and improved intestinal environment maybe not fit for bacteria related with obesity and diabetes oropportunistic pathogen to colonize

8 Journal of Diabetes Research

In summary our data highlights the potential beneficialeffect of sitagliptin on gut microbe of diabetic or obese ani-mals which constituted a novel exciting observation callingfor further investigation on numerous involved microbiotaspecies These data when combined with the results offuture clinical studies may facilitate the development oroptimization of novelmicrobiota based T2DM interventionalstrategies

5 Conclusions

Taken together our data suggest that the structure of gut floraespecially SCFA-producing bacteria seems to be abnormalin a progressive glucose intolerance rat model Treatmentwith sitagliptin improved gut dysbiosis moderately To thebest of our knowledge our study seems to be the firstreport showing that a DPP-4 inhibitor may exert positiveregulation on gut flora dysregulated during progression ofglucose intolerance and obesity in an animal model Futureresearch will be directed to elucidate whether the favorablechanges of microflora contribute to better metabolic controland reduced gastrointestinal side effects of DPP4 inhibitorwhen combined with metformin

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Competing Interests

The authors declare no competing financial interests

Authorsrsquo Contributions

Bo Feng designed the study Xinfeng Yan Peicheng Liand Zhaosheng Tang performed the experiments Bo FengXinfeng Yan Zhaosheng Tang and Lin Wang analyzed thedata and contributed to the discussion for interpreting thedata Bo Feng and Xinfeng Yan wrote and revised the paperXinfeng Yan prepared all the figures and tables All authorsreviewed the paper

Acknowledgments

The authors thankMajorbio company for 454 pyrophosphatesequencing and statistical analysis they also thank JayleenGrams MD PhD Zhao Bin MD PhD Zhu XiaolinMD PhD and Li Huiying MS from MSD for scien-tific and editing support This work was granted by theNational Basic Research Program of China (973 Program)no 2011CB504006 Key Specialty Construction Project ofPudong Health and Family Planning Commission of Shang-hai (no PWZz2013-04) andClinical ResearchProgramof theChinese Medical Association (no 12030510351)

References

[1] A Ouwehand E Isolauri and S Salminen ldquoThe role of theintestinalmicroflora for the development of the immune systemin early childhoodrdquo European Journal of Nutrition vol 41supplement 1 pp I32ndashI37 2002

[2] T S Stappenbeck L VHooper and J I Gordon ldquoDevelopmen-tal regulation of intestinal angiogenesis by indigenousmicrobesvia Paneth cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 24 pp 15451ndash154552002

[3] T Klaenhammer E Altermann F Arigoni et al ldquoDiscoveringlactic acid bacteria by genomicsrdquoAntonie van Leeuwenhoek vol82 no 1ndash4 pp 29ndash58 2002

[4] K Makarova A Slesarevb Y Wolf et al ldquoComparativegenomics of the lactic acid bacteriardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no42 pp 15611ndash15616 2006

[5] E EsteveW Ricart and J-M Fernandez-Real ldquoGutmicrobiotainteractions with obesity insulin resistance and type 2 diabetesdid gut microbiote co-evolve with insulin resistancerdquo CurrentOpinion in Clinical Nutrition and Metabolic Care vol 14 no 5pp 483ndash490 2011

[6] F Backhed H Ding T Wang et al ldquoThe gut microbiota as anenvironmental factor that regulates fat storagerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 101 no 44 pp 15718ndash15723 2004

[7] M Bajzer and R J Seeley ldquoPhysiology obesity and gut florardquoNature vol 444 no 7122 pp 1009ndash1010 2006

[8] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009

[9] P J Turnbaugh R E Ley M A Mahowald V Magrini ER Mardis and J I Gordon ldquoAn obesity-associated gut micro-biome with increased capacity for energy harvestrdquo Nature vol444 no 7122 pp 1027ndash1031 2006

[10] R E Ley F Backhed P Turnbaugh et al ldquoObesity alters gutmicrobial ecologyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 102 no 31 pp11070ndash11075 2005

[11] V R Velagapudi R Hezaveh C S Reigstad et al ldquoThe gutmicrobiota modulates host energy and lipid metabolism inmicerdquo Journal of Lipid Research vol 51 no 5 pp 1101ndash1112 2010

[12] M Diamant E E Blaak and W M de Vos ldquoDo nutrient-gut-microbiota interactions play a role in human obesity insulinresistance and type 2 diabetesrdquo Obesity Reviews vol 12 no 4pp 272ndash281 2011

[13] A M Caricilli P K Picardi L L de Abreu et al ldquoGutmicrobiota is a key modulator of insulin resistance in TLR 2knockout micerdquo PLoS Biology vol 9 no 12 Article ID e10012122011

[14] P D Cani S Possemiers T Van De Wiele et al ldquoChanges ingut microbiota control inflammation in obese mice througha mechanism involving GLP-2-driven improvement of gutpermeabilityrdquo Gut vol 58 no 8 pp 1091ndash1103 2009

[15] D J Drucker ldquoEnhancing incretin action for the treatment oftype 2 diabetesrdquo Diabetes Care vol 26 no 10 pp 2929ndash29402003

[16] D J Drucker and B Yusta ldquoPhysiology and pharmacology ofthe enteroendocrine hormone glucagon-like peptide-2rdquoAnnualReview of Physiology vol 76 pp 561ndash583 2014

Journal of Diabetes Research 9

[17] C S Marathe C K Rayner K L Jones and M HorowitzldquoGlucagon-like peptides 1 and 2 in health and disease a reviewrdquoPeptides vol 44 pp 75ndash86 2013

[18] P B Eckburg E M Bik C N Bernstein et al ldquoMicrobiologydiversity of the human intestinal microbial florardquo Science vol308 no 5728 pp 1635ndash1638 2005

[19] S R Gill M Pop R T DeBoy et al ldquoMetagenomic analysis ofthe human distal gut microbiomerdquo Science vol 312 no 5778pp 1355ndash1359 2006

[20] Y S Zhou B Li et al ldquoA rat model of type 2 diabetes mellitusinduced by high fat chow and low dose streptozocin injectionrdquoActa Laboratorium Animals Scientia Sinica vol 13 no 3 pp154ndash158 2005

[21] Y Jialin L Guo L Youpin et al ldquoEstablishing a rat modelsimilar to the adult patient of the general type 2 diabetes by long-term fat-enriched fed and lower dose of streptozocin-treatedratsrdquo Acta Laboratorium Animals Scientia Sinica vol 11 no 3pp 138ndash141 2003

[22] K R Amato C J Yeoman A Kent et al ldquoHabitat degradationimpacts black howler monkey (Alouatta pigra) gastrointestinalmicrobiomesrdquo ISME Journal vol 7 no 7 pp 1344ndash1353 2013

[23] P D Schloss S L Westcott T Ryabin et al ldquoIntroduc-ing mothur open-source platform-independent community-supported software for describing and comparing microbialcommunitiesrdquoApplied and EnvironmentalMicrobiology vol 75no 23 pp 7537ndash7541 2009

[24] G R Warnes B Bolker L Bonebakker et al gplots Various RProgramming Tools for Plotting Data Version 2170 2015

[25] J R White N Nagarajan and M Pop ldquoStatistical methods fordetecting differentially abundant features in clinical metage-nomic samplesrdquo PLoS Computational Biology vol 5 no 4Article ID e1000352 2009

[26] Y L Wu G X Xi et al ldquoAn experimental study of impairedglucose tolerance in Wistar ratsrdquo Chinese Remedies amp Clinicsvol 7 no 4 pp 281ndash283 2007

[27] M Cotta and R Forster ldquoThe family lachnospiraceae includingthe genera butyrivibrio lachnospira and roseburiardquo in TheProkaryotes Volume 4 Bacteria Firmicutes Cyanobacteria pp1002ndash1021 Springer Berlin Germany 3th edition 2006

[28] J M W Wong R de Souza C W C Kendall A Emam andD J A Jenkins ldquoColonic health fermentation and short chainfatty acidsrdquo Journal of Clinical Gastroenterology vol 40 no 3pp 235ndash243 2006

[29] H J Flint K P Scott S H Duncan P Louis and E ForanoldquoMicrobial degradation of complex carbohydrates in the gutrdquoGut Microbes vol 3 no 4 pp 289ndash306 2012

[30] X Ze S H Duncan P Louis and H J Flint ldquoRuminococcusbromii is a keystone species for the degradation of resistantstarch in the human colonrdquo ISME Journal vol 6 no 8 pp 1535ndash1543 2012

[31] R E Ley ldquoObesity and the human microbiomerdquo CurrentOpinion in Gastroenterology vol 26 no 1 pp 5ndash11 2010

[32] S H Duncan G E Lobley G Holtrop et al ldquoHuman colonicmicrobiota associated with diet obesity and weight lossrdquoInternational Journal of Obesity vol 32 no 11 pp 1720ndash17242008

[33] V Mai Q M McCrary R Sinha and M Glei ldquoAssociationsbetween dietary habits and body mass index with gut micro-biota composition and fecal water genotoxicity an observa-tional study in African American and Caucasian Americanvolunteersrdquo Nutrition Journal vol 8 article 49 2009

[34] R Jumpertz D S Le P J Turnbaugh et al ldquoEnergy-balancestudies reveal associations between gut microbes caloric loadand nutrient absorption in humansrdquo The American Journal ofClinical Nutrition vol 94 no 1 pp 58ndash65 2011

[35] O O Erejuwa S A Sulaiman and M S Ab Wahab ldquoMod-ulation of gut microbiota in the management of metabolicdisorders The prospects and challengesrdquo International Journalof Molecular Sciences vol 15 no 3 pp 4158ndash4188 2014

[36] P J Turnbaugh F Backhed L Fulton and J I Gordon ldquoDiet-induced obesity is linked to marked but reversible alterations inthe mouse distal gut microbiomerdquo Cell Host andMicrobe vol 3no 4 pp 213ndash223 2008

[37] J Qin J Wang Y Li et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[38] T Wang G Cai Y Qiu et al ldquoStructural segregation ofgut microbiota between colorectal cancer patients and healthyvolunteersrdquoThe ISME Journal vol 6 no 2 pp 320ndash329 2012

[39] K M Maslowski A T Vieira A Ng et al ldquoRegulation ofinflammatory responses by gutmicrobiota and chemoattractantreceptorGPR43rdquoNature vol 461 no 7268 pp 1282ndash1286 2009

[40] C De Filippo D Cavalieri M Di Paola et al ldquoImpact of dietin shaping gut microbiota revealed by a comparative studyin children from Europe and rural Africardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 33 pp 14691ndash14696 2010

[41] K Forslund FHildebrand TNielsen et al ldquoDisentangling type2 diabetes and metformin treatment signatures in the humangut microbiotardquo Nature vol 528 no 7581 pp 262ndash266 2015

[42] G L Hold A Schwiertz R I Aminov M Blaut and H J FlintldquoOligonucleotide probes that detect quantitatively significantgroups of butyrate-producing bacteria in human fecesrdquo Appliedand Environmental Microbiology vol 69 no 7 pp 4320ndash43242003

[43] N Becker J Kunath G Loh and M Blaut ldquoHuman intestinalmicrobiota characterization of a simplified and stable gnotobi-otic rat modelrdquo Gut Microbes vol 2 no 1 pp 25ndash33 2011

[44] S Nobaek M-L Johansson G Molin S Ahrne and BJeppsson ldquoAlteration of intestinal microflora is associated withreduction in abdominal bloating and pain in patients withirritable bowel syndromerdquo The American Journal of Gastroen-terology vol 95 no 5 pp 1231ndash1238 2000

[45] G T Macfarlane and S Macfarlane ldquoHuman colonic micro-biota ecology physiology and metabolic potential of intestinalbacteriardquo Scandinavian Journal of Gastroenterology vol 32supplement 222 pp 3ndash9 1997

[46] E G Zoetendal A D L Akkermans W M Akkermans-van Vliet J A G M De Visser and W M De Vos ldquoThehost genotype affects the bacterial community in the humangastrointestinal tractrdquoMicrobial Ecology in Health and Diseasevol 13 no 3 pp 129ndash134 2001

[47] N-R Shin J-C Lee H-Y Lee et al ldquoAn increase in theAkkermansia spp population induced by metformin treatmentimproves glucose homeostasis in diet-induced obesemicerdquoGutvol 63 no 5 pp 727ndash735 2014

[48] C Reasner L Olansky T L Seck et al ldquoThe effect of initialtherapy with the fixed-dose combination of sitagliptin andmetformin compared with metforminmonotherapy in patientswith type 2 diabetesmellitusrdquoDiabetes Obesity andMetabolismvol 13 no 7 pp 644ndash652 2011

[49] B Ahren S L Johnson M Stewart et al ldquoHARMONY 3 104-week randomized double-blind placebo- and active-controlled

10 Journal of Diabetes Research

trial assessing the efficacy and safety of albiglutide comparedwith placebo sitagliptin and glimepiride in patients with type2 diabetes taking metforminrdquo Diabetes Care vol 37 no 8 pp2141ndash2148 2014

[50] S Enjoji T Ohama and K Sato ldquoRegulation of epithelial celltight junctions by protease-activated receptor 2rdquoThe Journal ofVeterinary Medical Science vol 76 no 9 pp 1225ndash1229 2014

[51] N Wronkowitz S W Gorgens T Romacho et al ldquoSolu-ble DPP4 induces inflammation and proliferation of humansmoothmuscle cells via protease-activated receptor 2rdquoBiochim-ica et Biophysica Acta vol 1842 no 9 pp 1613ndash1621 2014

Journal of Diabetes Research 3

Normal Obesity Diabetes Sitagliptin0

100

200

300

400

500O

TUs

Δ

ΔΔ

ΔΔ

(a)

Normal Obesity Diabetes Sitagliptin0

1

2

3

4

5

Shan

non

Δ

Δ ΔΔ

(b)

Figure 1 Pyrosequencing data summary (a) number of OTUs (b) Shannon (diversity estimator) All data were calculated at 3 distanceΔ

119875

lt 005 ΔΔ119875

lt 001 data are expressed as mean plusmn SD

Table 1 Bodyweight and blood glucose changes during progressionof glucose intolerance and after Sitagliptin treatment Data areexpressed as mean plusmn SD (compared with group obesity I

119875

lt 001compared with group diabetes 998779

119875

lt 001 998771119875

lt 005)

Conditions Body weight (g) Blood glucose (mmolL)Normal 15506 plusmn 64I 383 plusmn 025I

Obesity 39892 plusmn 2306 430 plusmn 037998779

Diabetes 41683 plusmn 3564 1881 plusmn 255I

Sitagliptin 43267 plusmn 5810 1554 plusmn 139I998771

frequency data and false discovery rate (FDR) analysis usingMetastat analysis [25] Principal coordinates analysis (PCoA)was performed based on unweighted UniFrac distance Inaddition linear discriminant analysis effect size (LEfSe) wasperformed first based on nonparametric factorial Kruskal-Wallis (KW) sum-rank test and then we performed the lineardiscriminant analysis following the Wilcoxon Signed-Ranktest to assess effect size of each differentially abundant taxonor OTU

3 Results

31 Body Weight and Blood Glucose As shown in Table 1HFHC diet resulted in significant increase of body weight(39892 plusmn 2306 g in obesity condition versus 15506 plusmn 64 gin normal condition 119875 lt 001) Furthermore STZ injectionresulted in dramatic increase of blood glucose level in dia-betes condition as compared to normal or obesity condition(119875 lt 001) As expected sitagliptin resulted in a significantreduction of blood glucose (119875 lt 005) while having nosignificant impacts on body weight (119875 = ns)

32 OGTT Test The fasting blood glucose of obese rats wassignificantly higher than that in normal control condition(430 plusmn 037mmolL versus 383 plusmn 025mmolL 119875 lt 001)and blood glucose level at 120min was 892 plusmn 123mmolLwhich should be considered IGT by definition [26] Data areexpressed as mean plusmn SD

33 Characteristics of 454 Pyrosequencing Results A total of241879 high-quality sequences of 40 samples were produced

in this study with an average of 6047 sequences per sampleA brief summary and the estimators of OTUs and diversity(Shannon) are shown in Figure 1 and detailed characteristicsof each sample are listed in Table S1 There were statisticallysignificant differences of Shannon indexes between obesecondition and sitagliptin-treated condition (432plusmn036 versus465 plusmn 020 119875 lt 005) and between diabetic conditionand sitagliptin-treated condition (399 plusmn 025 versus 465 plusmn020 119875 lt 001) suggesting significant higher diversityfound in sitagliptin condition compared to obese or diabeticcondition

The rarefaction curves of all four conditions did not leveloff at the sequencing depth of 6000 (Figure S2) Thereforethis sequencing depth was not sufficient to cover the wholebacterial diversity Thus it deserves further sequencing todetect more bacterial species in the rat feces However whenthe Shannon curves tended to be smooth the sequencing dataquantity became dramatically bigger which reflected the vastmajority of microbes in the sample (Figure S3)

34 Microbial Structures Differed Significantly among Con-ditions There were significant differences at the phylumlevel in rat fecal microbiota during progression of glucoseintolerance and after treatment with sitagliptin Figure 2demonstrated that Bacteroidetes and Firmicutes accountedfor the majority of microflora (gt90) in each conditionThe relative abundance of Firmicutes in obesity conditionwas significantly increased compared to that in the normalcondition (8628 versus 6624 119875 lt 001) while therelative abundance of Bacteroidetes decreased substantially(1216 versus 3164 119875 lt 001) There was no significantdifference between obesity and diabetes at phylum level Therelative abundance of Firmicutes in the sitagliptin conditionwas significantly less than that in the diabetic condition(6319 versus 8356 119875 lt 001) In contrast the relativeabundance of Bacteroidetes increased dramatically (3246versus 1606 119875 lt 001) It is noteworthy that the relativeabundance of Tenericutes in the obese condition significantlyincreased compared to the normal condition (115 versus061 119875 lt 005) but decreased dramatically after inducingdiabetes (115 versus 009 119875 lt 001) and then increasedlargely after sitagliptin treatment as compared to the diabetic

4 Journal of Diabetes Research

Table 2 Statistical analysis was performed by Wilcoxon Signed-Rank test Data of normal obesity diabetes and sitagliptin are relativeabundance (percentage) of all sequences in each group (119875 group obesity versus normal 119875lowast group diabetes versus obesity 119875998779 group diabetesversus sitagliptin)

Taxonomy Normal () Obesity () Diabetes () Sitagliptin () 119875

119875

lowast

119875

998779

Bacteroides (genus) 14 02 06 54 0013 0022 0005Prevotellaceae (family) 99 18 68 92 0005 0001Blautia (genus) 02 08 71 03 0012 0005 0005Roseburia (genus) 07 09 03 05 0042Ruminococcus (genus) 25 14 06 19 0025 0007Clostridium (genus) 13 46 08 07 0014 0013Lactobacillus (genus) 223 225 130 67 0028Bacillus (genus) 46 44 60 0 0005Bifidobacterium (genus) 0024 0005 0002 0085 0007Parabacteroides (genus) 02 002 01 06 0008 0024 0005

Normal Obesity Diabetes Sitagliptin0

10

20

30

40

50

60

70

80

90

100

Relat

ive a

bund

ance

()

OthersTenericutesProteobacteria

ActinobacteriaBacteroidetesFirmicutes

Figure 2 Relative abundance of different bacterial phyla in micro-biota of four conditions

condition (096 versus 009 119875 lt 001) Similar changeswere observed at the phylum of Proteobacteria

Furthermore we explored the similarities and distinc-tions of species distribution in all conditions As shown inFigure 3 we found that there were 441 species shared in thefour conditions accounting for around half of the OTUs ineach condition It is noteworthy that about one-third of thespecies only found in normal (64OTUs) or obesity (22OTUs)condition belonged to Lachnospiraceae which is capableof fermenting complex carbohydrates to SCFAs playing animportant role in maintaining intestinal homeostasis [2728] Interestingly one-third of the total species only foundin sitagliptin condition (94OTUs) belonged to Ruminococ-caceae which is a major utilizer of plant polysaccharides[29 30]

In addition Metastat analysis showed significant differ-ences of relative abundance at the level of genus or familyamong conditions (Table 2 and Table S2) And the resultat level of genus was marked in the Heatmap plot (Figure

NC

Obesity DM

Sit22

22

13

94

64

441101

19

47

94

111

33 13148

43

Unique objects all = 1283 NC = 931Obesity = 965 DM = 787 sit = 948

Figure 3 Shared OTU analysis of the different conditions Venndiagram showing the unique and shared OTUs (3 distance level)in the different conditions

S4) which showed the significant differences of BlautiaRoseburia Clostridium and so on among four conditions

There were significant variations in the composition ofintestinal bacteria species among four study conditions Acladogram representation of the microbiota structure offour conditions and their predominant bacteria performedby LEfSe and the greatest differences in taxa among fourcommunities are shown in Figure 4 and Figure S5

To compare the overall microbiota structures in all condi-tions the unweighted Unifrac distance matrix was calculatedbased on the OTUs of each sample The PCA result revealeda significant difference in gut bacterial structure among allconditions The three principal component scores accountedfor 3057 2006 and 86 of total variations respectively(Figure 5)

4 Discussion

In the study we explored the changes of rat intestinal micro-biota during progression of glucose intolerance and the effect

Journal of Diabetes Research 5

Cladogram

Candidate_division_TM7

mlk

ji

gd

ef

c

c3c0

b7b8 b9

c1 c2 Tene

ricut

es

Spiro

chae

tae

Proteobact

eria

b6b3

b5b2

b0a8

a6a3

a2

a1a0

z

yx

wv

ut

sr

pq

o n

a4a5

a7

a9 b1 b4

c4 c5

Actinobacteria

bah

NCObesityDMSit

a Bifidobacteriaceaeb Bifidobacterialesc Actinobacteriad Coriobacteriaceaee Coriobacterialesf Coriobacteriiag Bacteroidaceaeh Porphyromonadaceaei Prevotellaceaej Rikenellaceaek no rankl no rankm no rankn Bacillaceaeo Listeriaceae

p Staphylococcaceaeq Bacillalesr Carnobacteriaceaes Lacatobacillaceae

v Bacilliu Lactobacillalest Streptococcaceae

x Clostridiaceaew Christensenellaceae

z othery Family_XIII_Incertae_Sedis

a0 Peptococcaceaea1 Peptostreptococcaceaea2 Ruminococcaceaea3 Erysipelotrichaceaea4 Erysipelotrichalesa5 Erysipelotrichiaa6 Rhodospirillaceaea7 Rhodospirillales

a8 Alcaligenaceaea9 Comamonadaceaeb0 Burkholderialesb1 Rhodocyclaceaeb2 Rhodocyclalesb3 Betaproteobacteriab4 Desulfovibrionaceaeb5 Desulfovibrionalesb6 Deltaproteobacteriab7 Enterobacteriaceaeb8 Enterobacterialesb9 Gammaproteobacteriac0 Spirochaetaceaec1 Spirochaetalesc2 Spirochaetesc3 no rankc4 RF9c5 Mollicutes

Figure 4 Taxonomic representation of statistically and biologically consistent differences among condition normal obesity diabetes andsitagliptin Differences are represented by the color of the most abundant class (red indicating normal condition green obesity conditionblue diabetes condition purple sitagliptin condition and yellow nonsignificance) The diameter of each circle is proportional to the taxonrsquosabundance

6 Journal of Diabetes Research

PCoA

NC1

NC2 NC3

NC4

NC5

NC6

NC7

NC8

NC9

NC10

Obe1

Obe2Obe3

Obe4

Obe5 Obe6

Obe7

Obe8

Obe9

Obe10

DM1

DM2

DM3

DM4DM5

DM6

DM7

DM8DM9

DM10Sit1

Sit2Sit3

Sit4

Sit5

Sit6

Sit7Sit8

Sit9Sit10

minus04

minus02

00

02

04

PC3

86

00 05minus05PC2 2006

NCObe

DMSit

PCoA

NC1

NC2NC3

NC4

NC5

NC6NC7

NC8

NC9

NC10

Obe1

Obe2

Obe3

Obe4

Obe5

Obe7

Obe8Obe9

Obe10

DM1

DM2Obe2

DM3

DM4DM5

DM6

DM7

DM8DM9

DM10 Sit1

Sit2Sit3

Sit4

Sit5

Sit6

Sit7Sit8

Sit9

Sit10

00 05minus05PC1 3057

minus04

minus02

00

02

04

PC3

86

PCoA

NC1

NC2

NC3

NC4

NC5

C6NCncNC8

NC9

NC10 Obe1Obe2

Obe3Obe4

Obe5

Obe6

Obe7Obe8

Obe9

Obe10

DM1

Obe2DM3

DM4

DM5DM6

DM7

DM8DM9

DM10

Sit1

Sit2Sit3 Sit4

Sit5Sit6

Sit7Sit8

Sit9

Sit10

00 05minus05PC1 3057

minus05

00

05

PC2

20

06

NCObe

DMSit

NCObe

DMSit

Figure 5 PCA plots based on unweighted Unifrac metrics Principal components (PCs) 1 2 and 3 explained 3057 2006 and 86 Eachsymbol represents a sample

of sitagliptin onmicrobiotaWe found significantly more Fir-micutes and less Bacteroidetes in obese rats compared to theirlean counterparts similar to previous studies [31] despiteconflicting data showing no differences of Bacteroidetesand Firmicutes between obese and lean subjects [32ndash34]The increased Firmicutes in obese mice may be related tothe genes encoding enzymes that break down polysaccha-rides which cannot be digested by the host increasing the

production of monosaccharides and short-chain fatty acids(SCFA) and the conversion of these SCFA to triglyceridesin the liver The binding of SCFA to two G-protein-coupledreceptors (GPR41 and GPR43) induces peptide YY secretionwhich suppresses gut motility retards intestinal transit andsubsequently results in the increase of nutrient uptake anddeposition [35] Interestingly we also found that hydrogen-producing Prevotellaceae decreased significantly in the obese

Journal of Diabetes Research 7

condition Given the fact that hydrogen inhibits digestion theobese rats seemed likely to absorb more calories from similarenergy intake

Another interesting finding was the higher abundance ofthe phylum Tenericutes (class Mollicutes) in obese rats com-pared to their lean counterparts Certain species ofMollicutesbloom have been proved to evolve the capacity to importcertain types of carbohydrates common in westernized dietfor both mice and human being (eg glucose fructose andsucrose) and to metabolize these imported sugars to SCFAwhich could be readily absorbed by the host [36] Sitagliptindid restore the structure of gutmicrobe at the level of phylumsimilar to the lean control condition without significanteffects on body weight

Unweighted Unifrac PCA analysis confirmed aboveresults in the respect of overall microbial structures Theintestinal microbiota of the four conditions of rats werestructurally separated from each other in the three principalcomponents

Various mechanisms have been proposed to explain theinfluence of microbiota on insulin resistance and T2DMincludingmetabolic endotoxemiamodifications in the secre-tion of the incretins and butyrate production Decrease ofSCFA-producing bacteria has been commonly observed inmetabolic diseases including T2DM [37] and even colorectalcancer [38] SCFA-producing bacteria have been previouslyshown to benefit the host through protecting the mucosafrom damage induced by pathogens supplying colonocytenutrients mitigating inflammation and so forth [39 40]Recent studies in mice have shown that an increase incolonic production of short-chain fatty acids triggers intesti-nal gluconeogenesis (IGN) via complementary mechanismsButyrate activates IGN gene expression through a cAMP-dependent mechanism in enterocytes whereas propionateitself a substrate of IGN activates IGN gene expressionvia the portal nervous system and the fatty acid receptorFFAR3 In rodents the result of increased IGN is beneficialto glucose and energy homeostasis with reductions in hepaticglucose production appetite and body weight [41] Belong-ing to either Clostridium Eubacterium or Fusobacteriumthe butyrate-producing bacteria mainly exist in cecum andcolon Fusobacterium prausnitzii and Roseburia intestinalisare predominant species in butyric acid producing bacteriain human and animal intestine [42] At the level of genusour data suggested that the relative abundance of butyrate-producing Clostridium and Roseburia in the diabetic ratsdecreased significantly compared to the lean and obeseconditions Moreover we also observed that the SCFA-producing bacteria Blautia increased significantly in the dia-betic condition compared to the obese condition Becker etal demonstrated that the addition of Clostridium butyricumas a second butyrate-producing bacterium to SIHUMI (asimplified human intestinal microbiota Anaerostipes caccaeBacteroides thetaiotaomicron Bifidobacterium longum Blau-tia producta Clostridium ramosum Escherichia coli and Lac-tobacillus plantarum) led to an increase of butyrate by 56butdecrease of Bifidobacterium longum Blautia producta and Ecoli indicating that either of these organisms competes withClostridium butyricum for the same substrates or Clostridium

butyricum forms inhibitory substances [43] Thus it seemsreasonable to suppose that changes ofClostridiumRoseburiaand Blautia show an opposite trend In other words theincrease of SCFA-producing bacteria like Blautia leads todecrease of butyrate-producing bacteria like Clostridium andRoseburia In the current study the structure of SCFA-producing bacteria seemed to be abnormal in diabetic ratsAfter sitagliptin treatment Roseburia increased and Blautiadecreased while Clostridium showed no change

Probiotics such as Lactobacillus and Bifidobacteriumnormally reside in the intestinal tract and regulate gutmicroflora and mucosal immunity Recent data also suggestthat microflora especially probiotics exert beneficial effectson gastrointestinal discomforts such as diarrhea abdominalpain and bloating [44] Lactobacillus produces lactic acidCO2 acetic acid andor ethanol which may contribute to a

more acidic environment through homo- or heterofermenta-tive metabolism [45 46] Our data showed that Lactobacillusand Bifidobacterium decreased in the diabetic conditionHowever sitagliptin made Bifidobacterium increased withno significant effect on Lactobacillus which seemed to beconsistent with the notion we discussed above

Marked changes in microbiota composition after met-formin treatment were observed in high-fat diet- (HFD-) fedconditions especially Akkermansia belonging to the Verru-comicrobia phylum showed the most conspicuous changes[47] suggesting a possible interaction between HFD met-formin and intestinal microbiota Forslund et al found sig-nificant increase of Escherichia spp and decrease of Intestini-bacter spp in human gut microbiome in metformin-treatedT2DM especially the latter is resistant to oxidative stress andable to degrade fucose indicative of an indirect involvementin mucus degradation [41] Interestingly there are consistentdata from numerous studies showing that sitagliptin resultsin reduced gastrointestinal discomfort when combined withmetformin a classic antihyperglycemia agent also known forits gastrointestinal side effects For instance Reasner et alshowed that sitagliptin and metformin fixed dose combina-tion resulted in a significant reduction on abdominal pain anddiarrhea compared tometforminmonotherapy [48] In addi-tion a 104-week clinical study (Harmony) also showed thatdiarrhea incidence was reduced in sitagliptin plus metformingroup as compared to metformin monotherapy group [49]Though the underlying mechanism of effect of metforminand sitagliptin on intestinal flora remains unclear our resultsseem to provide a possible explanation arguing that thiseffect could be related to favorable changes of microflora aftersitagliptin treatment

We supposed that the effect of sitagliptin on microbiotawas related to GLP-2 which helps improve the intestinalmucosa barrier integrity Besides recent studies found thatDPP-4 may act as the protease activated receptor 2 (PAR2)agonists involved in the pathophysiology of PAR2 leading toproliferation and inflammation in smooth muscle cells ThusDPP-4 inhibitor can relieve edema of intestinal wall and alle-viate the intestinal inflammation [50 51] Integrated intestinalmucosa barrier and improved intestinal environment maybe not fit for bacteria related with obesity and diabetes oropportunistic pathogen to colonize

8 Journal of Diabetes Research

In summary our data highlights the potential beneficialeffect of sitagliptin on gut microbe of diabetic or obese ani-mals which constituted a novel exciting observation callingfor further investigation on numerous involved microbiotaspecies These data when combined with the results offuture clinical studies may facilitate the development oroptimization of novelmicrobiota based T2DM interventionalstrategies

5 Conclusions

Taken together our data suggest that the structure of gut floraespecially SCFA-producing bacteria seems to be abnormalin a progressive glucose intolerance rat model Treatmentwith sitagliptin improved gut dysbiosis moderately To thebest of our knowledge our study seems to be the firstreport showing that a DPP-4 inhibitor may exert positiveregulation on gut flora dysregulated during progression ofglucose intolerance and obesity in an animal model Futureresearch will be directed to elucidate whether the favorablechanges of microflora contribute to better metabolic controland reduced gastrointestinal side effects of DPP4 inhibitorwhen combined with metformin

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Competing Interests

The authors declare no competing financial interests

Authorsrsquo Contributions

Bo Feng designed the study Xinfeng Yan Peicheng Liand Zhaosheng Tang performed the experiments Bo FengXinfeng Yan Zhaosheng Tang and Lin Wang analyzed thedata and contributed to the discussion for interpreting thedata Bo Feng and Xinfeng Yan wrote and revised the paperXinfeng Yan prepared all the figures and tables All authorsreviewed the paper

Acknowledgments

The authors thankMajorbio company for 454 pyrophosphatesequencing and statistical analysis they also thank JayleenGrams MD PhD Zhao Bin MD PhD Zhu XiaolinMD PhD and Li Huiying MS from MSD for scien-tific and editing support This work was granted by theNational Basic Research Program of China (973 Program)no 2011CB504006 Key Specialty Construction Project ofPudong Health and Family Planning Commission of Shang-hai (no PWZz2013-04) andClinical ResearchProgramof theChinese Medical Association (no 12030510351)

References

[1] A Ouwehand E Isolauri and S Salminen ldquoThe role of theintestinalmicroflora for the development of the immune systemin early childhoodrdquo European Journal of Nutrition vol 41supplement 1 pp I32ndashI37 2002

[2] T S Stappenbeck L VHooper and J I Gordon ldquoDevelopmen-tal regulation of intestinal angiogenesis by indigenousmicrobesvia Paneth cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 24 pp 15451ndash154552002

[3] T Klaenhammer E Altermann F Arigoni et al ldquoDiscoveringlactic acid bacteria by genomicsrdquoAntonie van Leeuwenhoek vol82 no 1ndash4 pp 29ndash58 2002

[4] K Makarova A Slesarevb Y Wolf et al ldquoComparativegenomics of the lactic acid bacteriardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no42 pp 15611ndash15616 2006

[5] E EsteveW Ricart and J-M Fernandez-Real ldquoGutmicrobiotainteractions with obesity insulin resistance and type 2 diabetesdid gut microbiote co-evolve with insulin resistancerdquo CurrentOpinion in Clinical Nutrition and Metabolic Care vol 14 no 5pp 483ndash490 2011

[6] F Backhed H Ding T Wang et al ldquoThe gut microbiota as anenvironmental factor that regulates fat storagerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 101 no 44 pp 15718ndash15723 2004

[7] M Bajzer and R J Seeley ldquoPhysiology obesity and gut florardquoNature vol 444 no 7122 pp 1009ndash1010 2006

[8] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009

[9] P J Turnbaugh R E Ley M A Mahowald V Magrini ER Mardis and J I Gordon ldquoAn obesity-associated gut micro-biome with increased capacity for energy harvestrdquo Nature vol444 no 7122 pp 1027ndash1031 2006

[10] R E Ley F Backhed P Turnbaugh et al ldquoObesity alters gutmicrobial ecologyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 102 no 31 pp11070ndash11075 2005

[11] V R Velagapudi R Hezaveh C S Reigstad et al ldquoThe gutmicrobiota modulates host energy and lipid metabolism inmicerdquo Journal of Lipid Research vol 51 no 5 pp 1101ndash1112 2010

[12] M Diamant E E Blaak and W M de Vos ldquoDo nutrient-gut-microbiota interactions play a role in human obesity insulinresistance and type 2 diabetesrdquo Obesity Reviews vol 12 no 4pp 272ndash281 2011

[13] A M Caricilli P K Picardi L L de Abreu et al ldquoGutmicrobiota is a key modulator of insulin resistance in TLR 2knockout micerdquo PLoS Biology vol 9 no 12 Article ID e10012122011

[14] P D Cani S Possemiers T Van De Wiele et al ldquoChanges ingut microbiota control inflammation in obese mice througha mechanism involving GLP-2-driven improvement of gutpermeabilityrdquo Gut vol 58 no 8 pp 1091ndash1103 2009

[15] D J Drucker ldquoEnhancing incretin action for the treatment oftype 2 diabetesrdquo Diabetes Care vol 26 no 10 pp 2929ndash29402003

[16] D J Drucker and B Yusta ldquoPhysiology and pharmacology ofthe enteroendocrine hormone glucagon-like peptide-2rdquoAnnualReview of Physiology vol 76 pp 561ndash583 2014

Journal of Diabetes Research 9

[17] C S Marathe C K Rayner K L Jones and M HorowitzldquoGlucagon-like peptides 1 and 2 in health and disease a reviewrdquoPeptides vol 44 pp 75ndash86 2013

[18] P B Eckburg E M Bik C N Bernstein et al ldquoMicrobiologydiversity of the human intestinal microbial florardquo Science vol308 no 5728 pp 1635ndash1638 2005

[19] S R Gill M Pop R T DeBoy et al ldquoMetagenomic analysis ofthe human distal gut microbiomerdquo Science vol 312 no 5778pp 1355ndash1359 2006

[20] Y S Zhou B Li et al ldquoA rat model of type 2 diabetes mellitusinduced by high fat chow and low dose streptozocin injectionrdquoActa Laboratorium Animals Scientia Sinica vol 13 no 3 pp154ndash158 2005

[21] Y Jialin L Guo L Youpin et al ldquoEstablishing a rat modelsimilar to the adult patient of the general type 2 diabetes by long-term fat-enriched fed and lower dose of streptozocin-treatedratsrdquo Acta Laboratorium Animals Scientia Sinica vol 11 no 3pp 138ndash141 2003

[22] K R Amato C J Yeoman A Kent et al ldquoHabitat degradationimpacts black howler monkey (Alouatta pigra) gastrointestinalmicrobiomesrdquo ISME Journal vol 7 no 7 pp 1344ndash1353 2013

[23] P D Schloss S L Westcott T Ryabin et al ldquoIntroduc-ing mothur open-source platform-independent community-supported software for describing and comparing microbialcommunitiesrdquoApplied and EnvironmentalMicrobiology vol 75no 23 pp 7537ndash7541 2009

[24] G R Warnes B Bolker L Bonebakker et al gplots Various RProgramming Tools for Plotting Data Version 2170 2015

[25] J R White N Nagarajan and M Pop ldquoStatistical methods fordetecting differentially abundant features in clinical metage-nomic samplesrdquo PLoS Computational Biology vol 5 no 4Article ID e1000352 2009

[26] Y L Wu G X Xi et al ldquoAn experimental study of impairedglucose tolerance in Wistar ratsrdquo Chinese Remedies amp Clinicsvol 7 no 4 pp 281ndash283 2007

[27] M Cotta and R Forster ldquoThe family lachnospiraceae includingthe genera butyrivibrio lachnospira and roseburiardquo in TheProkaryotes Volume 4 Bacteria Firmicutes Cyanobacteria pp1002ndash1021 Springer Berlin Germany 3th edition 2006

[28] J M W Wong R de Souza C W C Kendall A Emam andD J A Jenkins ldquoColonic health fermentation and short chainfatty acidsrdquo Journal of Clinical Gastroenterology vol 40 no 3pp 235ndash243 2006

[29] H J Flint K P Scott S H Duncan P Louis and E ForanoldquoMicrobial degradation of complex carbohydrates in the gutrdquoGut Microbes vol 3 no 4 pp 289ndash306 2012

[30] X Ze S H Duncan P Louis and H J Flint ldquoRuminococcusbromii is a keystone species for the degradation of resistantstarch in the human colonrdquo ISME Journal vol 6 no 8 pp 1535ndash1543 2012

[31] R E Ley ldquoObesity and the human microbiomerdquo CurrentOpinion in Gastroenterology vol 26 no 1 pp 5ndash11 2010

[32] S H Duncan G E Lobley G Holtrop et al ldquoHuman colonicmicrobiota associated with diet obesity and weight lossrdquoInternational Journal of Obesity vol 32 no 11 pp 1720ndash17242008

[33] V Mai Q M McCrary R Sinha and M Glei ldquoAssociationsbetween dietary habits and body mass index with gut micro-biota composition and fecal water genotoxicity an observa-tional study in African American and Caucasian Americanvolunteersrdquo Nutrition Journal vol 8 article 49 2009

[34] R Jumpertz D S Le P J Turnbaugh et al ldquoEnergy-balancestudies reveal associations between gut microbes caloric loadand nutrient absorption in humansrdquo The American Journal ofClinical Nutrition vol 94 no 1 pp 58ndash65 2011

[35] O O Erejuwa S A Sulaiman and M S Ab Wahab ldquoMod-ulation of gut microbiota in the management of metabolicdisorders The prospects and challengesrdquo International Journalof Molecular Sciences vol 15 no 3 pp 4158ndash4188 2014

[36] P J Turnbaugh F Backhed L Fulton and J I Gordon ldquoDiet-induced obesity is linked to marked but reversible alterations inthe mouse distal gut microbiomerdquo Cell Host andMicrobe vol 3no 4 pp 213ndash223 2008

[37] J Qin J Wang Y Li et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[38] T Wang G Cai Y Qiu et al ldquoStructural segregation ofgut microbiota between colorectal cancer patients and healthyvolunteersrdquoThe ISME Journal vol 6 no 2 pp 320ndash329 2012

[39] K M Maslowski A T Vieira A Ng et al ldquoRegulation ofinflammatory responses by gutmicrobiota and chemoattractantreceptorGPR43rdquoNature vol 461 no 7268 pp 1282ndash1286 2009

[40] C De Filippo D Cavalieri M Di Paola et al ldquoImpact of dietin shaping gut microbiota revealed by a comparative studyin children from Europe and rural Africardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 33 pp 14691ndash14696 2010

[41] K Forslund FHildebrand TNielsen et al ldquoDisentangling type2 diabetes and metformin treatment signatures in the humangut microbiotardquo Nature vol 528 no 7581 pp 262ndash266 2015

[42] G L Hold A Schwiertz R I Aminov M Blaut and H J FlintldquoOligonucleotide probes that detect quantitatively significantgroups of butyrate-producing bacteria in human fecesrdquo Appliedand Environmental Microbiology vol 69 no 7 pp 4320ndash43242003

[43] N Becker J Kunath G Loh and M Blaut ldquoHuman intestinalmicrobiota characterization of a simplified and stable gnotobi-otic rat modelrdquo Gut Microbes vol 2 no 1 pp 25ndash33 2011

[44] S Nobaek M-L Johansson G Molin S Ahrne and BJeppsson ldquoAlteration of intestinal microflora is associated withreduction in abdominal bloating and pain in patients withirritable bowel syndromerdquo The American Journal of Gastroen-terology vol 95 no 5 pp 1231ndash1238 2000

[45] G T Macfarlane and S Macfarlane ldquoHuman colonic micro-biota ecology physiology and metabolic potential of intestinalbacteriardquo Scandinavian Journal of Gastroenterology vol 32supplement 222 pp 3ndash9 1997

[46] E G Zoetendal A D L Akkermans W M Akkermans-van Vliet J A G M De Visser and W M De Vos ldquoThehost genotype affects the bacterial community in the humangastrointestinal tractrdquoMicrobial Ecology in Health and Diseasevol 13 no 3 pp 129ndash134 2001

[47] N-R Shin J-C Lee H-Y Lee et al ldquoAn increase in theAkkermansia spp population induced by metformin treatmentimproves glucose homeostasis in diet-induced obesemicerdquoGutvol 63 no 5 pp 727ndash735 2014

[48] C Reasner L Olansky T L Seck et al ldquoThe effect of initialtherapy with the fixed-dose combination of sitagliptin andmetformin compared with metforminmonotherapy in patientswith type 2 diabetesmellitusrdquoDiabetes Obesity andMetabolismvol 13 no 7 pp 644ndash652 2011

[49] B Ahren S L Johnson M Stewart et al ldquoHARMONY 3 104-week randomized double-blind placebo- and active-controlled

10 Journal of Diabetes Research

trial assessing the efficacy and safety of albiglutide comparedwith placebo sitagliptin and glimepiride in patients with type2 diabetes taking metforminrdquo Diabetes Care vol 37 no 8 pp2141ndash2148 2014

[50] S Enjoji T Ohama and K Sato ldquoRegulation of epithelial celltight junctions by protease-activated receptor 2rdquoThe Journal ofVeterinary Medical Science vol 76 no 9 pp 1225ndash1229 2014

[51] N Wronkowitz S W Gorgens T Romacho et al ldquoSolu-ble DPP4 induces inflammation and proliferation of humansmoothmuscle cells via protease-activated receptor 2rdquoBiochim-ica et Biophysica Acta vol 1842 no 9 pp 1613ndash1621 2014

4 Journal of Diabetes Research

Table 2 Statistical analysis was performed by Wilcoxon Signed-Rank test Data of normal obesity diabetes and sitagliptin are relativeabundance (percentage) of all sequences in each group (119875 group obesity versus normal 119875lowast group diabetes versus obesity 119875998779 group diabetesversus sitagliptin)

Taxonomy Normal () Obesity () Diabetes () Sitagliptin () 119875

119875

lowast

119875

998779

Bacteroides (genus) 14 02 06 54 0013 0022 0005Prevotellaceae (family) 99 18 68 92 0005 0001Blautia (genus) 02 08 71 03 0012 0005 0005Roseburia (genus) 07 09 03 05 0042Ruminococcus (genus) 25 14 06 19 0025 0007Clostridium (genus) 13 46 08 07 0014 0013Lactobacillus (genus) 223 225 130 67 0028Bacillus (genus) 46 44 60 0 0005Bifidobacterium (genus) 0024 0005 0002 0085 0007Parabacteroides (genus) 02 002 01 06 0008 0024 0005

Normal Obesity Diabetes Sitagliptin0

10

20

30

40

50

60

70

80

90

100

Relat

ive a

bund

ance

()

OthersTenericutesProteobacteria

ActinobacteriaBacteroidetesFirmicutes

Figure 2 Relative abundance of different bacterial phyla in micro-biota of four conditions

condition (096 versus 009 119875 lt 001) Similar changeswere observed at the phylum of Proteobacteria

Furthermore we explored the similarities and distinc-tions of species distribution in all conditions As shown inFigure 3 we found that there were 441 species shared in thefour conditions accounting for around half of the OTUs ineach condition It is noteworthy that about one-third of thespecies only found in normal (64OTUs) or obesity (22OTUs)condition belonged to Lachnospiraceae which is capableof fermenting complex carbohydrates to SCFAs playing animportant role in maintaining intestinal homeostasis [2728] Interestingly one-third of the total species only foundin sitagliptin condition (94OTUs) belonged to Ruminococ-caceae which is a major utilizer of plant polysaccharides[29 30]

In addition Metastat analysis showed significant differ-ences of relative abundance at the level of genus or familyamong conditions (Table 2 and Table S2) And the resultat level of genus was marked in the Heatmap plot (Figure

NC

Obesity DM

Sit22

22

13

94

64

441101

19

47

94

111

33 13148

43

Unique objects all = 1283 NC = 931Obesity = 965 DM = 787 sit = 948

Figure 3 Shared OTU analysis of the different conditions Venndiagram showing the unique and shared OTUs (3 distance level)in the different conditions

S4) which showed the significant differences of BlautiaRoseburia Clostridium and so on among four conditions

There were significant variations in the composition ofintestinal bacteria species among four study conditions Acladogram representation of the microbiota structure offour conditions and their predominant bacteria performedby LEfSe and the greatest differences in taxa among fourcommunities are shown in Figure 4 and Figure S5

To compare the overall microbiota structures in all condi-tions the unweighted Unifrac distance matrix was calculatedbased on the OTUs of each sample The PCA result revealeda significant difference in gut bacterial structure among allconditions The three principal component scores accountedfor 3057 2006 and 86 of total variations respectively(Figure 5)

4 Discussion

In the study we explored the changes of rat intestinal micro-biota during progression of glucose intolerance and the effect

Journal of Diabetes Research 5

Cladogram

Candidate_division_TM7

mlk

ji

gd

ef

c

c3c0

b7b8 b9

c1 c2 Tene

ricut

es

Spiro

chae

tae

Proteobact

eria

b6b3

b5b2

b0a8

a6a3

a2

a1a0

z

yx

wv

ut

sr

pq

o n

a4a5

a7

a9 b1 b4

c4 c5

Actinobacteria

bah

NCObesityDMSit

a Bifidobacteriaceaeb Bifidobacterialesc Actinobacteriad Coriobacteriaceaee Coriobacterialesf Coriobacteriiag Bacteroidaceaeh Porphyromonadaceaei Prevotellaceaej Rikenellaceaek no rankl no rankm no rankn Bacillaceaeo Listeriaceae

p Staphylococcaceaeq Bacillalesr Carnobacteriaceaes Lacatobacillaceae

v Bacilliu Lactobacillalest Streptococcaceae

x Clostridiaceaew Christensenellaceae

z othery Family_XIII_Incertae_Sedis

a0 Peptococcaceaea1 Peptostreptococcaceaea2 Ruminococcaceaea3 Erysipelotrichaceaea4 Erysipelotrichalesa5 Erysipelotrichiaa6 Rhodospirillaceaea7 Rhodospirillales

a8 Alcaligenaceaea9 Comamonadaceaeb0 Burkholderialesb1 Rhodocyclaceaeb2 Rhodocyclalesb3 Betaproteobacteriab4 Desulfovibrionaceaeb5 Desulfovibrionalesb6 Deltaproteobacteriab7 Enterobacteriaceaeb8 Enterobacterialesb9 Gammaproteobacteriac0 Spirochaetaceaec1 Spirochaetalesc2 Spirochaetesc3 no rankc4 RF9c5 Mollicutes

Figure 4 Taxonomic representation of statistically and biologically consistent differences among condition normal obesity diabetes andsitagliptin Differences are represented by the color of the most abundant class (red indicating normal condition green obesity conditionblue diabetes condition purple sitagliptin condition and yellow nonsignificance) The diameter of each circle is proportional to the taxonrsquosabundance

6 Journal of Diabetes Research

PCoA

NC1

NC2 NC3

NC4

NC5

NC6

NC7

NC8

NC9

NC10

Obe1

Obe2Obe3

Obe4

Obe5 Obe6

Obe7

Obe8

Obe9

Obe10

DM1

DM2

DM3

DM4DM5

DM6

DM7

DM8DM9

DM10Sit1

Sit2Sit3

Sit4

Sit5

Sit6

Sit7Sit8

Sit9Sit10

minus04

minus02

00

02

04

PC3

86

00 05minus05PC2 2006

NCObe

DMSit

PCoA

NC1

NC2NC3

NC4

NC5

NC6NC7

NC8

NC9

NC10

Obe1

Obe2

Obe3

Obe4

Obe5

Obe7

Obe8Obe9

Obe10

DM1

DM2Obe2

DM3

DM4DM5

DM6

DM7

DM8DM9

DM10 Sit1

Sit2Sit3

Sit4

Sit5

Sit6

Sit7Sit8

Sit9

Sit10

00 05minus05PC1 3057

minus04

minus02

00

02

04

PC3

86

PCoA

NC1

NC2

NC3

NC4

NC5

C6NCncNC8

NC9

NC10 Obe1Obe2

Obe3Obe4

Obe5

Obe6

Obe7Obe8

Obe9

Obe10

DM1

Obe2DM3

DM4

DM5DM6

DM7

DM8DM9

DM10

Sit1

Sit2Sit3 Sit4

Sit5Sit6

Sit7Sit8

Sit9

Sit10

00 05minus05PC1 3057

minus05

00

05

PC2

20

06

NCObe

DMSit

NCObe

DMSit

Figure 5 PCA plots based on unweighted Unifrac metrics Principal components (PCs) 1 2 and 3 explained 3057 2006 and 86 Eachsymbol represents a sample

of sitagliptin onmicrobiotaWe found significantly more Fir-micutes and less Bacteroidetes in obese rats compared to theirlean counterparts similar to previous studies [31] despiteconflicting data showing no differences of Bacteroidetesand Firmicutes between obese and lean subjects [32ndash34]The increased Firmicutes in obese mice may be related tothe genes encoding enzymes that break down polysaccha-rides which cannot be digested by the host increasing the

production of monosaccharides and short-chain fatty acids(SCFA) and the conversion of these SCFA to triglyceridesin the liver The binding of SCFA to two G-protein-coupledreceptors (GPR41 and GPR43) induces peptide YY secretionwhich suppresses gut motility retards intestinal transit andsubsequently results in the increase of nutrient uptake anddeposition [35] Interestingly we also found that hydrogen-producing Prevotellaceae decreased significantly in the obese

Journal of Diabetes Research 7

condition Given the fact that hydrogen inhibits digestion theobese rats seemed likely to absorb more calories from similarenergy intake

Another interesting finding was the higher abundance ofthe phylum Tenericutes (class Mollicutes) in obese rats com-pared to their lean counterparts Certain species ofMollicutesbloom have been proved to evolve the capacity to importcertain types of carbohydrates common in westernized dietfor both mice and human being (eg glucose fructose andsucrose) and to metabolize these imported sugars to SCFAwhich could be readily absorbed by the host [36] Sitagliptindid restore the structure of gutmicrobe at the level of phylumsimilar to the lean control condition without significanteffects on body weight

Unweighted Unifrac PCA analysis confirmed aboveresults in the respect of overall microbial structures Theintestinal microbiota of the four conditions of rats werestructurally separated from each other in the three principalcomponents

Various mechanisms have been proposed to explain theinfluence of microbiota on insulin resistance and T2DMincludingmetabolic endotoxemiamodifications in the secre-tion of the incretins and butyrate production Decrease ofSCFA-producing bacteria has been commonly observed inmetabolic diseases including T2DM [37] and even colorectalcancer [38] SCFA-producing bacteria have been previouslyshown to benefit the host through protecting the mucosafrom damage induced by pathogens supplying colonocytenutrients mitigating inflammation and so forth [39 40]Recent studies in mice have shown that an increase incolonic production of short-chain fatty acids triggers intesti-nal gluconeogenesis (IGN) via complementary mechanismsButyrate activates IGN gene expression through a cAMP-dependent mechanism in enterocytes whereas propionateitself a substrate of IGN activates IGN gene expressionvia the portal nervous system and the fatty acid receptorFFAR3 In rodents the result of increased IGN is beneficialto glucose and energy homeostasis with reductions in hepaticglucose production appetite and body weight [41] Belong-ing to either Clostridium Eubacterium or Fusobacteriumthe butyrate-producing bacteria mainly exist in cecum andcolon Fusobacterium prausnitzii and Roseburia intestinalisare predominant species in butyric acid producing bacteriain human and animal intestine [42] At the level of genusour data suggested that the relative abundance of butyrate-producing Clostridium and Roseburia in the diabetic ratsdecreased significantly compared to the lean and obeseconditions Moreover we also observed that the SCFA-producing bacteria Blautia increased significantly in the dia-betic condition compared to the obese condition Becker etal demonstrated that the addition of Clostridium butyricumas a second butyrate-producing bacterium to SIHUMI (asimplified human intestinal microbiota Anaerostipes caccaeBacteroides thetaiotaomicron Bifidobacterium longum Blau-tia producta Clostridium ramosum Escherichia coli and Lac-tobacillus plantarum) led to an increase of butyrate by 56butdecrease of Bifidobacterium longum Blautia producta and Ecoli indicating that either of these organisms competes withClostridium butyricum for the same substrates or Clostridium

butyricum forms inhibitory substances [43] Thus it seemsreasonable to suppose that changes ofClostridiumRoseburiaand Blautia show an opposite trend In other words theincrease of SCFA-producing bacteria like Blautia leads todecrease of butyrate-producing bacteria like Clostridium andRoseburia In the current study the structure of SCFA-producing bacteria seemed to be abnormal in diabetic ratsAfter sitagliptin treatment Roseburia increased and Blautiadecreased while Clostridium showed no change

Probiotics such as Lactobacillus and Bifidobacteriumnormally reside in the intestinal tract and regulate gutmicroflora and mucosal immunity Recent data also suggestthat microflora especially probiotics exert beneficial effectson gastrointestinal discomforts such as diarrhea abdominalpain and bloating [44] Lactobacillus produces lactic acidCO2 acetic acid andor ethanol which may contribute to a

more acidic environment through homo- or heterofermenta-tive metabolism [45 46] Our data showed that Lactobacillusand Bifidobacterium decreased in the diabetic conditionHowever sitagliptin made Bifidobacterium increased withno significant effect on Lactobacillus which seemed to beconsistent with the notion we discussed above

Marked changes in microbiota composition after met-formin treatment were observed in high-fat diet- (HFD-) fedconditions especially Akkermansia belonging to the Verru-comicrobia phylum showed the most conspicuous changes[47] suggesting a possible interaction between HFD met-formin and intestinal microbiota Forslund et al found sig-nificant increase of Escherichia spp and decrease of Intestini-bacter spp in human gut microbiome in metformin-treatedT2DM especially the latter is resistant to oxidative stress andable to degrade fucose indicative of an indirect involvementin mucus degradation [41] Interestingly there are consistentdata from numerous studies showing that sitagliptin resultsin reduced gastrointestinal discomfort when combined withmetformin a classic antihyperglycemia agent also known forits gastrointestinal side effects For instance Reasner et alshowed that sitagliptin and metformin fixed dose combina-tion resulted in a significant reduction on abdominal pain anddiarrhea compared tometforminmonotherapy [48] In addi-tion a 104-week clinical study (Harmony) also showed thatdiarrhea incidence was reduced in sitagliptin plus metformingroup as compared to metformin monotherapy group [49]Though the underlying mechanism of effect of metforminand sitagliptin on intestinal flora remains unclear our resultsseem to provide a possible explanation arguing that thiseffect could be related to favorable changes of microflora aftersitagliptin treatment

We supposed that the effect of sitagliptin on microbiotawas related to GLP-2 which helps improve the intestinalmucosa barrier integrity Besides recent studies found thatDPP-4 may act as the protease activated receptor 2 (PAR2)agonists involved in the pathophysiology of PAR2 leading toproliferation and inflammation in smooth muscle cells ThusDPP-4 inhibitor can relieve edema of intestinal wall and alle-viate the intestinal inflammation [50 51] Integrated intestinalmucosa barrier and improved intestinal environment maybe not fit for bacteria related with obesity and diabetes oropportunistic pathogen to colonize

8 Journal of Diabetes Research

In summary our data highlights the potential beneficialeffect of sitagliptin on gut microbe of diabetic or obese ani-mals which constituted a novel exciting observation callingfor further investigation on numerous involved microbiotaspecies These data when combined with the results offuture clinical studies may facilitate the development oroptimization of novelmicrobiota based T2DM interventionalstrategies

5 Conclusions

Taken together our data suggest that the structure of gut floraespecially SCFA-producing bacteria seems to be abnormalin a progressive glucose intolerance rat model Treatmentwith sitagliptin improved gut dysbiosis moderately To thebest of our knowledge our study seems to be the firstreport showing that a DPP-4 inhibitor may exert positiveregulation on gut flora dysregulated during progression ofglucose intolerance and obesity in an animal model Futureresearch will be directed to elucidate whether the favorablechanges of microflora contribute to better metabolic controland reduced gastrointestinal side effects of DPP4 inhibitorwhen combined with metformin

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Competing Interests

The authors declare no competing financial interests

Authorsrsquo Contributions

Bo Feng designed the study Xinfeng Yan Peicheng Liand Zhaosheng Tang performed the experiments Bo FengXinfeng Yan Zhaosheng Tang and Lin Wang analyzed thedata and contributed to the discussion for interpreting thedata Bo Feng and Xinfeng Yan wrote and revised the paperXinfeng Yan prepared all the figures and tables All authorsreviewed the paper

Acknowledgments

The authors thankMajorbio company for 454 pyrophosphatesequencing and statistical analysis they also thank JayleenGrams MD PhD Zhao Bin MD PhD Zhu XiaolinMD PhD and Li Huiying MS from MSD for scien-tific and editing support This work was granted by theNational Basic Research Program of China (973 Program)no 2011CB504006 Key Specialty Construction Project ofPudong Health and Family Planning Commission of Shang-hai (no PWZz2013-04) andClinical ResearchProgramof theChinese Medical Association (no 12030510351)

References

[1] A Ouwehand E Isolauri and S Salminen ldquoThe role of theintestinalmicroflora for the development of the immune systemin early childhoodrdquo European Journal of Nutrition vol 41supplement 1 pp I32ndashI37 2002

[2] T S Stappenbeck L VHooper and J I Gordon ldquoDevelopmen-tal regulation of intestinal angiogenesis by indigenousmicrobesvia Paneth cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 24 pp 15451ndash154552002

[3] T Klaenhammer E Altermann F Arigoni et al ldquoDiscoveringlactic acid bacteria by genomicsrdquoAntonie van Leeuwenhoek vol82 no 1ndash4 pp 29ndash58 2002

[4] K Makarova A Slesarevb Y Wolf et al ldquoComparativegenomics of the lactic acid bacteriardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no42 pp 15611ndash15616 2006

[5] E EsteveW Ricart and J-M Fernandez-Real ldquoGutmicrobiotainteractions with obesity insulin resistance and type 2 diabetesdid gut microbiote co-evolve with insulin resistancerdquo CurrentOpinion in Clinical Nutrition and Metabolic Care vol 14 no 5pp 483ndash490 2011

[6] F Backhed H Ding T Wang et al ldquoThe gut microbiota as anenvironmental factor that regulates fat storagerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 101 no 44 pp 15718ndash15723 2004

[7] M Bajzer and R J Seeley ldquoPhysiology obesity and gut florardquoNature vol 444 no 7122 pp 1009ndash1010 2006

[8] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009

[9] P J Turnbaugh R E Ley M A Mahowald V Magrini ER Mardis and J I Gordon ldquoAn obesity-associated gut micro-biome with increased capacity for energy harvestrdquo Nature vol444 no 7122 pp 1027ndash1031 2006

[10] R E Ley F Backhed P Turnbaugh et al ldquoObesity alters gutmicrobial ecologyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 102 no 31 pp11070ndash11075 2005

[11] V R Velagapudi R Hezaveh C S Reigstad et al ldquoThe gutmicrobiota modulates host energy and lipid metabolism inmicerdquo Journal of Lipid Research vol 51 no 5 pp 1101ndash1112 2010

[12] M Diamant E E Blaak and W M de Vos ldquoDo nutrient-gut-microbiota interactions play a role in human obesity insulinresistance and type 2 diabetesrdquo Obesity Reviews vol 12 no 4pp 272ndash281 2011

[13] A M Caricilli P K Picardi L L de Abreu et al ldquoGutmicrobiota is a key modulator of insulin resistance in TLR 2knockout micerdquo PLoS Biology vol 9 no 12 Article ID e10012122011

[14] P D Cani S Possemiers T Van De Wiele et al ldquoChanges ingut microbiota control inflammation in obese mice througha mechanism involving GLP-2-driven improvement of gutpermeabilityrdquo Gut vol 58 no 8 pp 1091ndash1103 2009

[15] D J Drucker ldquoEnhancing incretin action for the treatment oftype 2 diabetesrdquo Diabetes Care vol 26 no 10 pp 2929ndash29402003

[16] D J Drucker and B Yusta ldquoPhysiology and pharmacology ofthe enteroendocrine hormone glucagon-like peptide-2rdquoAnnualReview of Physiology vol 76 pp 561ndash583 2014

Journal of Diabetes Research 9

[17] C S Marathe C K Rayner K L Jones and M HorowitzldquoGlucagon-like peptides 1 and 2 in health and disease a reviewrdquoPeptides vol 44 pp 75ndash86 2013

[18] P B Eckburg E M Bik C N Bernstein et al ldquoMicrobiologydiversity of the human intestinal microbial florardquo Science vol308 no 5728 pp 1635ndash1638 2005

[19] S R Gill M Pop R T DeBoy et al ldquoMetagenomic analysis ofthe human distal gut microbiomerdquo Science vol 312 no 5778pp 1355ndash1359 2006

[20] Y S Zhou B Li et al ldquoA rat model of type 2 diabetes mellitusinduced by high fat chow and low dose streptozocin injectionrdquoActa Laboratorium Animals Scientia Sinica vol 13 no 3 pp154ndash158 2005

[21] Y Jialin L Guo L Youpin et al ldquoEstablishing a rat modelsimilar to the adult patient of the general type 2 diabetes by long-term fat-enriched fed and lower dose of streptozocin-treatedratsrdquo Acta Laboratorium Animals Scientia Sinica vol 11 no 3pp 138ndash141 2003

[22] K R Amato C J Yeoman A Kent et al ldquoHabitat degradationimpacts black howler monkey (Alouatta pigra) gastrointestinalmicrobiomesrdquo ISME Journal vol 7 no 7 pp 1344ndash1353 2013

[23] P D Schloss S L Westcott T Ryabin et al ldquoIntroduc-ing mothur open-source platform-independent community-supported software for describing and comparing microbialcommunitiesrdquoApplied and EnvironmentalMicrobiology vol 75no 23 pp 7537ndash7541 2009

[24] G R Warnes B Bolker L Bonebakker et al gplots Various RProgramming Tools for Plotting Data Version 2170 2015

[25] J R White N Nagarajan and M Pop ldquoStatistical methods fordetecting differentially abundant features in clinical metage-nomic samplesrdquo PLoS Computational Biology vol 5 no 4Article ID e1000352 2009

[26] Y L Wu G X Xi et al ldquoAn experimental study of impairedglucose tolerance in Wistar ratsrdquo Chinese Remedies amp Clinicsvol 7 no 4 pp 281ndash283 2007

[27] M Cotta and R Forster ldquoThe family lachnospiraceae includingthe genera butyrivibrio lachnospira and roseburiardquo in TheProkaryotes Volume 4 Bacteria Firmicutes Cyanobacteria pp1002ndash1021 Springer Berlin Germany 3th edition 2006

[28] J M W Wong R de Souza C W C Kendall A Emam andD J A Jenkins ldquoColonic health fermentation and short chainfatty acidsrdquo Journal of Clinical Gastroenterology vol 40 no 3pp 235ndash243 2006

[29] H J Flint K P Scott S H Duncan P Louis and E ForanoldquoMicrobial degradation of complex carbohydrates in the gutrdquoGut Microbes vol 3 no 4 pp 289ndash306 2012

[30] X Ze S H Duncan P Louis and H J Flint ldquoRuminococcusbromii is a keystone species for the degradation of resistantstarch in the human colonrdquo ISME Journal vol 6 no 8 pp 1535ndash1543 2012

[31] R E Ley ldquoObesity and the human microbiomerdquo CurrentOpinion in Gastroenterology vol 26 no 1 pp 5ndash11 2010

[32] S H Duncan G E Lobley G Holtrop et al ldquoHuman colonicmicrobiota associated with diet obesity and weight lossrdquoInternational Journal of Obesity vol 32 no 11 pp 1720ndash17242008

[33] V Mai Q M McCrary R Sinha and M Glei ldquoAssociationsbetween dietary habits and body mass index with gut micro-biota composition and fecal water genotoxicity an observa-tional study in African American and Caucasian Americanvolunteersrdquo Nutrition Journal vol 8 article 49 2009

[34] R Jumpertz D S Le P J Turnbaugh et al ldquoEnergy-balancestudies reveal associations between gut microbes caloric loadand nutrient absorption in humansrdquo The American Journal ofClinical Nutrition vol 94 no 1 pp 58ndash65 2011

[35] O O Erejuwa S A Sulaiman and M S Ab Wahab ldquoMod-ulation of gut microbiota in the management of metabolicdisorders The prospects and challengesrdquo International Journalof Molecular Sciences vol 15 no 3 pp 4158ndash4188 2014

[36] P J Turnbaugh F Backhed L Fulton and J I Gordon ldquoDiet-induced obesity is linked to marked but reversible alterations inthe mouse distal gut microbiomerdquo Cell Host andMicrobe vol 3no 4 pp 213ndash223 2008

[37] J Qin J Wang Y Li et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[38] T Wang G Cai Y Qiu et al ldquoStructural segregation ofgut microbiota between colorectal cancer patients and healthyvolunteersrdquoThe ISME Journal vol 6 no 2 pp 320ndash329 2012

[39] K M Maslowski A T Vieira A Ng et al ldquoRegulation ofinflammatory responses by gutmicrobiota and chemoattractantreceptorGPR43rdquoNature vol 461 no 7268 pp 1282ndash1286 2009

[40] C De Filippo D Cavalieri M Di Paola et al ldquoImpact of dietin shaping gut microbiota revealed by a comparative studyin children from Europe and rural Africardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 33 pp 14691ndash14696 2010

[41] K Forslund FHildebrand TNielsen et al ldquoDisentangling type2 diabetes and metformin treatment signatures in the humangut microbiotardquo Nature vol 528 no 7581 pp 262ndash266 2015

[42] G L Hold A Schwiertz R I Aminov M Blaut and H J FlintldquoOligonucleotide probes that detect quantitatively significantgroups of butyrate-producing bacteria in human fecesrdquo Appliedand Environmental Microbiology vol 69 no 7 pp 4320ndash43242003

[43] N Becker J Kunath G Loh and M Blaut ldquoHuman intestinalmicrobiota characterization of a simplified and stable gnotobi-otic rat modelrdquo Gut Microbes vol 2 no 1 pp 25ndash33 2011

[44] S Nobaek M-L Johansson G Molin S Ahrne and BJeppsson ldquoAlteration of intestinal microflora is associated withreduction in abdominal bloating and pain in patients withirritable bowel syndromerdquo The American Journal of Gastroen-terology vol 95 no 5 pp 1231ndash1238 2000

[45] G T Macfarlane and S Macfarlane ldquoHuman colonic micro-biota ecology physiology and metabolic potential of intestinalbacteriardquo Scandinavian Journal of Gastroenterology vol 32supplement 222 pp 3ndash9 1997

[46] E G Zoetendal A D L Akkermans W M Akkermans-van Vliet J A G M De Visser and W M De Vos ldquoThehost genotype affects the bacterial community in the humangastrointestinal tractrdquoMicrobial Ecology in Health and Diseasevol 13 no 3 pp 129ndash134 2001

[47] N-R Shin J-C Lee H-Y Lee et al ldquoAn increase in theAkkermansia spp population induced by metformin treatmentimproves glucose homeostasis in diet-induced obesemicerdquoGutvol 63 no 5 pp 727ndash735 2014

[48] C Reasner L Olansky T L Seck et al ldquoThe effect of initialtherapy with the fixed-dose combination of sitagliptin andmetformin compared with metforminmonotherapy in patientswith type 2 diabetesmellitusrdquoDiabetes Obesity andMetabolismvol 13 no 7 pp 644ndash652 2011

[49] B Ahren S L Johnson M Stewart et al ldquoHARMONY 3 104-week randomized double-blind placebo- and active-controlled

10 Journal of Diabetes Research

trial assessing the efficacy and safety of albiglutide comparedwith placebo sitagliptin and glimepiride in patients with type2 diabetes taking metforminrdquo Diabetes Care vol 37 no 8 pp2141ndash2148 2014

[50] S Enjoji T Ohama and K Sato ldquoRegulation of epithelial celltight junctions by protease-activated receptor 2rdquoThe Journal ofVeterinary Medical Science vol 76 no 9 pp 1225ndash1229 2014

[51] N Wronkowitz S W Gorgens T Romacho et al ldquoSolu-ble DPP4 induces inflammation and proliferation of humansmoothmuscle cells via protease-activated receptor 2rdquoBiochim-ica et Biophysica Acta vol 1842 no 9 pp 1613ndash1621 2014

Journal of Diabetes Research 5

Cladogram

Candidate_division_TM7

mlk

ji

gd

ef

c

c3c0

b7b8 b9

c1 c2 Tene

ricut

es

Spiro

chae

tae

Proteobact

eria

b6b3

b5b2

b0a8

a6a3

a2

a1a0

z

yx

wv

ut

sr

pq

o n

a4a5

a7

a9 b1 b4

c4 c5

Actinobacteria

bah

NCObesityDMSit

a Bifidobacteriaceaeb Bifidobacterialesc Actinobacteriad Coriobacteriaceaee Coriobacterialesf Coriobacteriiag Bacteroidaceaeh Porphyromonadaceaei Prevotellaceaej Rikenellaceaek no rankl no rankm no rankn Bacillaceaeo Listeriaceae

p Staphylococcaceaeq Bacillalesr Carnobacteriaceaes Lacatobacillaceae

v Bacilliu Lactobacillalest Streptococcaceae

x Clostridiaceaew Christensenellaceae

z othery Family_XIII_Incertae_Sedis

a0 Peptococcaceaea1 Peptostreptococcaceaea2 Ruminococcaceaea3 Erysipelotrichaceaea4 Erysipelotrichalesa5 Erysipelotrichiaa6 Rhodospirillaceaea7 Rhodospirillales

a8 Alcaligenaceaea9 Comamonadaceaeb0 Burkholderialesb1 Rhodocyclaceaeb2 Rhodocyclalesb3 Betaproteobacteriab4 Desulfovibrionaceaeb5 Desulfovibrionalesb6 Deltaproteobacteriab7 Enterobacteriaceaeb8 Enterobacterialesb9 Gammaproteobacteriac0 Spirochaetaceaec1 Spirochaetalesc2 Spirochaetesc3 no rankc4 RF9c5 Mollicutes

Figure 4 Taxonomic representation of statistically and biologically consistent differences among condition normal obesity diabetes andsitagliptin Differences are represented by the color of the most abundant class (red indicating normal condition green obesity conditionblue diabetes condition purple sitagliptin condition and yellow nonsignificance) The diameter of each circle is proportional to the taxonrsquosabundance

6 Journal of Diabetes Research

PCoA

NC1

NC2 NC3

NC4

NC5

NC6

NC7

NC8

NC9

NC10

Obe1

Obe2Obe3

Obe4

Obe5 Obe6

Obe7

Obe8

Obe9

Obe10

DM1

DM2

DM3

DM4DM5

DM6

DM7

DM8DM9

DM10Sit1

Sit2Sit3

Sit4

Sit5

Sit6

Sit7Sit8

Sit9Sit10

minus04

minus02

00

02

04

PC3

86

00 05minus05PC2 2006

NCObe

DMSit

PCoA

NC1

NC2NC3

NC4

NC5

NC6NC7

NC8

NC9

NC10

Obe1

Obe2

Obe3

Obe4

Obe5

Obe7

Obe8Obe9

Obe10

DM1

DM2Obe2

DM3

DM4DM5

DM6

DM7

DM8DM9

DM10 Sit1

Sit2Sit3

Sit4

Sit5

Sit6

Sit7Sit8

Sit9

Sit10

00 05minus05PC1 3057

minus04

minus02

00

02

04

PC3

86

PCoA

NC1

NC2

NC3

NC4

NC5

C6NCncNC8

NC9

NC10 Obe1Obe2

Obe3Obe4

Obe5

Obe6

Obe7Obe8

Obe9

Obe10

DM1

Obe2DM3

DM4

DM5DM6

DM7

DM8DM9

DM10

Sit1

Sit2Sit3 Sit4

Sit5Sit6

Sit7Sit8

Sit9

Sit10

00 05minus05PC1 3057

minus05

00

05

PC2

20

06

NCObe

DMSit

NCObe

DMSit

Figure 5 PCA plots based on unweighted Unifrac metrics Principal components (PCs) 1 2 and 3 explained 3057 2006 and 86 Eachsymbol represents a sample

of sitagliptin onmicrobiotaWe found significantly more Fir-micutes and less Bacteroidetes in obese rats compared to theirlean counterparts similar to previous studies [31] despiteconflicting data showing no differences of Bacteroidetesand Firmicutes between obese and lean subjects [32ndash34]The increased Firmicutes in obese mice may be related tothe genes encoding enzymes that break down polysaccha-rides which cannot be digested by the host increasing the

production of monosaccharides and short-chain fatty acids(SCFA) and the conversion of these SCFA to triglyceridesin the liver The binding of SCFA to two G-protein-coupledreceptors (GPR41 and GPR43) induces peptide YY secretionwhich suppresses gut motility retards intestinal transit andsubsequently results in the increase of nutrient uptake anddeposition [35] Interestingly we also found that hydrogen-producing Prevotellaceae decreased significantly in the obese

Journal of Diabetes Research 7

condition Given the fact that hydrogen inhibits digestion theobese rats seemed likely to absorb more calories from similarenergy intake

Another interesting finding was the higher abundance ofthe phylum Tenericutes (class Mollicutes) in obese rats com-pared to their lean counterparts Certain species ofMollicutesbloom have been proved to evolve the capacity to importcertain types of carbohydrates common in westernized dietfor both mice and human being (eg glucose fructose andsucrose) and to metabolize these imported sugars to SCFAwhich could be readily absorbed by the host [36] Sitagliptindid restore the structure of gutmicrobe at the level of phylumsimilar to the lean control condition without significanteffects on body weight

Unweighted Unifrac PCA analysis confirmed aboveresults in the respect of overall microbial structures Theintestinal microbiota of the four conditions of rats werestructurally separated from each other in the three principalcomponents

Various mechanisms have been proposed to explain theinfluence of microbiota on insulin resistance and T2DMincludingmetabolic endotoxemiamodifications in the secre-tion of the incretins and butyrate production Decrease ofSCFA-producing bacteria has been commonly observed inmetabolic diseases including T2DM [37] and even colorectalcancer [38] SCFA-producing bacteria have been previouslyshown to benefit the host through protecting the mucosafrom damage induced by pathogens supplying colonocytenutrients mitigating inflammation and so forth [39 40]Recent studies in mice have shown that an increase incolonic production of short-chain fatty acids triggers intesti-nal gluconeogenesis (IGN) via complementary mechanismsButyrate activates IGN gene expression through a cAMP-dependent mechanism in enterocytes whereas propionateitself a substrate of IGN activates IGN gene expressionvia the portal nervous system and the fatty acid receptorFFAR3 In rodents the result of increased IGN is beneficialto glucose and energy homeostasis with reductions in hepaticglucose production appetite and body weight [41] Belong-ing to either Clostridium Eubacterium or Fusobacteriumthe butyrate-producing bacteria mainly exist in cecum andcolon Fusobacterium prausnitzii and Roseburia intestinalisare predominant species in butyric acid producing bacteriain human and animal intestine [42] At the level of genusour data suggested that the relative abundance of butyrate-producing Clostridium and Roseburia in the diabetic ratsdecreased significantly compared to the lean and obeseconditions Moreover we also observed that the SCFA-producing bacteria Blautia increased significantly in the dia-betic condition compared to the obese condition Becker etal demonstrated that the addition of Clostridium butyricumas a second butyrate-producing bacterium to SIHUMI (asimplified human intestinal microbiota Anaerostipes caccaeBacteroides thetaiotaomicron Bifidobacterium longum Blau-tia producta Clostridium ramosum Escherichia coli and Lac-tobacillus plantarum) led to an increase of butyrate by 56butdecrease of Bifidobacterium longum Blautia producta and Ecoli indicating that either of these organisms competes withClostridium butyricum for the same substrates or Clostridium

butyricum forms inhibitory substances [43] Thus it seemsreasonable to suppose that changes ofClostridiumRoseburiaand Blautia show an opposite trend In other words theincrease of SCFA-producing bacteria like Blautia leads todecrease of butyrate-producing bacteria like Clostridium andRoseburia In the current study the structure of SCFA-producing bacteria seemed to be abnormal in diabetic ratsAfter sitagliptin treatment Roseburia increased and Blautiadecreased while Clostridium showed no change

Probiotics such as Lactobacillus and Bifidobacteriumnormally reside in the intestinal tract and regulate gutmicroflora and mucosal immunity Recent data also suggestthat microflora especially probiotics exert beneficial effectson gastrointestinal discomforts such as diarrhea abdominalpain and bloating [44] Lactobacillus produces lactic acidCO2 acetic acid andor ethanol which may contribute to a

more acidic environment through homo- or heterofermenta-tive metabolism [45 46] Our data showed that Lactobacillusand Bifidobacterium decreased in the diabetic conditionHowever sitagliptin made Bifidobacterium increased withno significant effect on Lactobacillus which seemed to beconsistent with the notion we discussed above

Marked changes in microbiota composition after met-formin treatment were observed in high-fat diet- (HFD-) fedconditions especially Akkermansia belonging to the Verru-comicrobia phylum showed the most conspicuous changes[47] suggesting a possible interaction between HFD met-formin and intestinal microbiota Forslund et al found sig-nificant increase of Escherichia spp and decrease of Intestini-bacter spp in human gut microbiome in metformin-treatedT2DM especially the latter is resistant to oxidative stress andable to degrade fucose indicative of an indirect involvementin mucus degradation [41] Interestingly there are consistentdata from numerous studies showing that sitagliptin resultsin reduced gastrointestinal discomfort when combined withmetformin a classic antihyperglycemia agent also known forits gastrointestinal side effects For instance Reasner et alshowed that sitagliptin and metformin fixed dose combina-tion resulted in a significant reduction on abdominal pain anddiarrhea compared tometforminmonotherapy [48] In addi-tion a 104-week clinical study (Harmony) also showed thatdiarrhea incidence was reduced in sitagliptin plus metformingroup as compared to metformin monotherapy group [49]Though the underlying mechanism of effect of metforminand sitagliptin on intestinal flora remains unclear our resultsseem to provide a possible explanation arguing that thiseffect could be related to favorable changes of microflora aftersitagliptin treatment

We supposed that the effect of sitagliptin on microbiotawas related to GLP-2 which helps improve the intestinalmucosa barrier integrity Besides recent studies found thatDPP-4 may act as the protease activated receptor 2 (PAR2)agonists involved in the pathophysiology of PAR2 leading toproliferation and inflammation in smooth muscle cells ThusDPP-4 inhibitor can relieve edema of intestinal wall and alle-viate the intestinal inflammation [50 51] Integrated intestinalmucosa barrier and improved intestinal environment maybe not fit for bacteria related with obesity and diabetes oropportunistic pathogen to colonize

8 Journal of Diabetes Research

In summary our data highlights the potential beneficialeffect of sitagliptin on gut microbe of diabetic or obese ani-mals which constituted a novel exciting observation callingfor further investigation on numerous involved microbiotaspecies These data when combined with the results offuture clinical studies may facilitate the development oroptimization of novelmicrobiota based T2DM interventionalstrategies

5 Conclusions

Taken together our data suggest that the structure of gut floraespecially SCFA-producing bacteria seems to be abnormalin a progressive glucose intolerance rat model Treatmentwith sitagliptin improved gut dysbiosis moderately To thebest of our knowledge our study seems to be the firstreport showing that a DPP-4 inhibitor may exert positiveregulation on gut flora dysregulated during progression ofglucose intolerance and obesity in an animal model Futureresearch will be directed to elucidate whether the favorablechanges of microflora contribute to better metabolic controland reduced gastrointestinal side effects of DPP4 inhibitorwhen combined with metformin

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Competing Interests

The authors declare no competing financial interests

Authorsrsquo Contributions

Bo Feng designed the study Xinfeng Yan Peicheng Liand Zhaosheng Tang performed the experiments Bo FengXinfeng Yan Zhaosheng Tang and Lin Wang analyzed thedata and contributed to the discussion for interpreting thedata Bo Feng and Xinfeng Yan wrote and revised the paperXinfeng Yan prepared all the figures and tables All authorsreviewed the paper

Acknowledgments

The authors thankMajorbio company for 454 pyrophosphatesequencing and statistical analysis they also thank JayleenGrams MD PhD Zhao Bin MD PhD Zhu XiaolinMD PhD and Li Huiying MS from MSD for scien-tific and editing support This work was granted by theNational Basic Research Program of China (973 Program)no 2011CB504006 Key Specialty Construction Project ofPudong Health and Family Planning Commission of Shang-hai (no PWZz2013-04) andClinical ResearchProgramof theChinese Medical Association (no 12030510351)

References

[1] A Ouwehand E Isolauri and S Salminen ldquoThe role of theintestinalmicroflora for the development of the immune systemin early childhoodrdquo European Journal of Nutrition vol 41supplement 1 pp I32ndashI37 2002

[2] T S Stappenbeck L VHooper and J I Gordon ldquoDevelopmen-tal regulation of intestinal angiogenesis by indigenousmicrobesvia Paneth cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 24 pp 15451ndash154552002

[3] T Klaenhammer E Altermann F Arigoni et al ldquoDiscoveringlactic acid bacteria by genomicsrdquoAntonie van Leeuwenhoek vol82 no 1ndash4 pp 29ndash58 2002

[4] K Makarova A Slesarevb Y Wolf et al ldquoComparativegenomics of the lactic acid bacteriardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no42 pp 15611ndash15616 2006

[5] E EsteveW Ricart and J-M Fernandez-Real ldquoGutmicrobiotainteractions with obesity insulin resistance and type 2 diabetesdid gut microbiote co-evolve with insulin resistancerdquo CurrentOpinion in Clinical Nutrition and Metabolic Care vol 14 no 5pp 483ndash490 2011

[6] F Backhed H Ding T Wang et al ldquoThe gut microbiota as anenvironmental factor that regulates fat storagerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 101 no 44 pp 15718ndash15723 2004

[7] M Bajzer and R J Seeley ldquoPhysiology obesity and gut florardquoNature vol 444 no 7122 pp 1009ndash1010 2006

[8] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009

[9] P J Turnbaugh R E Ley M A Mahowald V Magrini ER Mardis and J I Gordon ldquoAn obesity-associated gut micro-biome with increased capacity for energy harvestrdquo Nature vol444 no 7122 pp 1027ndash1031 2006

[10] R E Ley F Backhed P Turnbaugh et al ldquoObesity alters gutmicrobial ecologyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 102 no 31 pp11070ndash11075 2005

[11] V R Velagapudi R Hezaveh C S Reigstad et al ldquoThe gutmicrobiota modulates host energy and lipid metabolism inmicerdquo Journal of Lipid Research vol 51 no 5 pp 1101ndash1112 2010

[12] M Diamant E E Blaak and W M de Vos ldquoDo nutrient-gut-microbiota interactions play a role in human obesity insulinresistance and type 2 diabetesrdquo Obesity Reviews vol 12 no 4pp 272ndash281 2011

[13] A M Caricilli P K Picardi L L de Abreu et al ldquoGutmicrobiota is a key modulator of insulin resistance in TLR 2knockout micerdquo PLoS Biology vol 9 no 12 Article ID e10012122011

[14] P D Cani S Possemiers T Van De Wiele et al ldquoChanges ingut microbiota control inflammation in obese mice througha mechanism involving GLP-2-driven improvement of gutpermeabilityrdquo Gut vol 58 no 8 pp 1091ndash1103 2009

[15] D J Drucker ldquoEnhancing incretin action for the treatment oftype 2 diabetesrdquo Diabetes Care vol 26 no 10 pp 2929ndash29402003

[16] D J Drucker and B Yusta ldquoPhysiology and pharmacology ofthe enteroendocrine hormone glucagon-like peptide-2rdquoAnnualReview of Physiology vol 76 pp 561ndash583 2014

Journal of Diabetes Research 9

[17] C S Marathe C K Rayner K L Jones and M HorowitzldquoGlucagon-like peptides 1 and 2 in health and disease a reviewrdquoPeptides vol 44 pp 75ndash86 2013

[18] P B Eckburg E M Bik C N Bernstein et al ldquoMicrobiologydiversity of the human intestinal microbial florardquo Science vol308 no 5728 pp 1635ndash1638 2005

[19] S R Gill M Pop R T DeBoy et al ldquoMetagenomic analysis ofthe human distal gut microbiomerdquo Science vol 312 no 5778pp 1355ndash1359 2006

[20] Y S Zhou B Li et al ldquoA rat model of type 2 diabetes mellitusinduced by high fat chow and low dose streptozocin injectionrdquoActa Laboratorium Animals Scientia Sinica vol 13 no 3 pp154ndash158 2005

[21] Y Jialin L Guo L Youpin et al ldquoEstablishing a rat modelsimilar to the adult patient of the general type 2 diabetes by long-term fat-enriched fed and lower dose of streptozocin-treatedratsrdquo Acta Laboratorium Animals Scientia Sinica vol 11 no 3pp 138ndash141 2003

[22] K R Amato C J Yeoman A Kent et al ldquoHabitat degradationimpacts black howler monkey (Alouatta pigra) gastrointestinalmicrobiomesrdquo ISME Journal vol 7 no 7 pp 1344ndash1353 2013

[23] P D Schloss S L Westcott T Ryabin et al ldquoIntroduc-ing mothur open-source platform-independent community-supported software for describing and comparing microbialcommunitiesrdquoApplied and EnvironmentalMicrobiology vol 75no 23 pp 7537ndash7541 2009

[24] G R Warnes B Bolker L Bonebakker et al gplots Various RProgramming Tools for Plotting Data Version 2170 2015

[25] J R White N Nagarajan and M Pop ldquoStatistical methods fordetecting differentially abundant features in clinical metage-nomic samplesrdquo PLoS Computational Biology vol 5 no 4Article ID e1000352 2009

[26] Y L Wu G X Xi et al ldquoAn experimental study of impairedglucose tolerance in Wistar ratsrdquo Chinese Remedies amp Clinicsvol 7 no 4 pp 281ndash283 2007

[27] M Cotta and R Forster ldquoThe family lachnospiraceae includingthe genera butyrivibrio lachnospira and roseburiardquo in TheProkaryotes Volume 4 Bacteria Firmicutes Cyanobacteria pp1002ndash1021 Springer Berlin Germany 3th edition 2006

[28] J M W Wong R de Souza C W C Kendall A Emam andD J A Jenkins ldquoColonic health fermentation and short chainfatty acidsrdquo Journal of Clinical Gastroenterology vol 40 no 3pp 235ndash243 2006

[29] H J Flint K P Scott S H Duncan P Louis and E ForanoldquoMicrobial degradation of complex carbohydrates in the gutrdquoGut Microbes vol 3 no 4 pp 289ndash306 2012

[30] X Ze S H Duncan P Louis and H J Flint ldquoRuminococcusbromii is a keystone species for the degradation of resistantstarch in the human colonrdquo ISME Journal vol 6 no 8 pp 1535ndash1543 2012

[31] R E Ley ldquoObesity and the human microbiomerdquo CurrentOpinion in Gastroenterology vol 26 no 1 pp 5ndash11 2010

[32] S H Duncan G E Lobley G Holtrop et al ldquoHuman colonicmicrobiota associated with diet obesity and weight lossrdquoInternational Journal of Obesity vol 32 no 11 pp 1720ndash17242008

[33] V Mai Q M McCrary R Sinha and M Glei ldquoAssociationsbetween dietary habits and body mass index with gut micro-biota composition and fecal water genotoxicity an observa-tional study in African American and Caucasian Americanvolunteersrdquo Nutrition Journal vol 8 article 49 2009

[34] R Jumpertz D S Le P J Turnbaugh et al ldquoEnergy-balancestudies reveal associations between gut microbes caloric loadand nutrient absorption in humansrdquo The American Journal ofClinical Nutrition vol 94 no 1 pp 58ndash65 2011

[35] O O Erejuwa S A Sulaiman and M S Ab Wahab ldquoMod-ulation of gut microbiota in the management of metabolicdisorders The prospects and challengesrdquo International Journalof Molecular Sciences vol 15 no 3 pp 4158ndash4188 2014

[36] P J Turnbaugh F Backhed L Fulton and J I Gordon ldquoDiet-induced obesity is linked to marked but reversible alterations inthe mouse distal gut microbiomerdquo Cell Host andMicrobe vol 3no 4 pp 213ndash223 2008

[37] J Qin J Wang Y Li et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[38] T Wang G Cai Y Qiu et al ldquoStructural segregation ofgut microbiota between colorectal cancer patients and healthyvolunteersrdquoThe ISME Journal vol 6 no 2 pp 320ndash329 2012

[39] K M Maslowski A T Vieira A Ng et al ldquoRegulation ofinflammatory responses by gutmicrobiota and chemoattractantreceptorGPR43rdquoNature vol 461 no 7268 pp 1282ndash1286 2009

[40] C De Filippo D Cavalieri M Di Paola et al ldquoImpact of dietin shaping gut microbiota revealed by a comparative studyin children from Europe and rural Africardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 33 pp 14691ndash14696 2010

[41] K Forslund FHildebrand TNielsen et al ldquoDisentangling type2 diabetes and metformin treatment signatures in the humangut microbiotardquo Nature vol 528 no 7581 pp 262ndash266 2015

[42] G L Hold A Schwiertz R I Aminov M Blaut and H J FlintldquoOligonucleotide probes that detect quantitatively significantgroups of butyrate-producing bacteria in human fecesrdquo Appliedand Environmental Microbiology vol 69 no 7 pp 4320ndash43242003

[43] N Becker J Kunath G Loh and M Blaut ldquoHuman intestinalmicrobiota characterization of a simplified and stable gnotobi-otic rat modelrdquo Gut Microbes vol 2 no 1 pp 25ndash33 2011

[44] S Nobaek M-L Johansson G Molin S Ahrne and BJeppsson ldquoAlteration of intestinal microflora is associated withreduction in abdominal bloating and pain in patients withirritable bowel syndromerdquo The American Journal of Gastroen-terology vol 95 no 5 pp 1231ndash1238 2000

[45] G T Macfarlane and S Macfarlane ldquoHuman colonic micro-biota ecology physiology and metabolic potential of intestinalbacteriardquo Scandinavian Journal of Gastroenterology vol 32supplement 222 pp 3ndash9 1997

[46] E G Zoetendal A D L Akkermans W M Akkermans-van Vliet J A G M De Visser and W M De Vos ldquoThehost genotype affects the bacterial community in the humangastrointestinal tractrdquoMicrobial Ecology in Health and Diseasevol 13 no 3 pp 129ndash134 2001

[47] N-R Shin J-C Lee H-Y Lee et al ldquoAn increase in theAkkermansia spp population induced by metformin treatmentimproves glucose homeostasis in diet-induced obesemicerdquoGutvol 63 no 5 pp 727ndash735 2014

[48] C Reasner L Olansky T L Seck et al ldquoThe effect of initialtherapy with the fixed-dose combination of sitagliptin andmetformin compared with metforminmonotherapy in patientswith type 2 diabetesmellitusrdquoDiabetes Obesity andMetabolismvol 13 no 7 pp 644ndash652 2011

[49] B Ahren S L Johnson M Stewart et al ldquoHARMONY 3 104-week randomized double-blind placebo- and active-controlled

10 Journal of Diabetes Research

trial assessing the efficacy and safety of albiglutide comparedwith placebo sitagliptin and glimepiride in patients with type2 diabetes taking metforminrdquo Diabetes Care vol 37 no 8 pp2141ndash2148 2014

[50] S Enjoji T Ohama and K Sato ldquoRegulation of epithelial celltight junctions by protease-activated receptor 2rdquoThe Journal ofVeterinary Medical Science vol 76 no 9 pp 1225ndash1229 2014

[51] N Wronkowitz S W Gorgens T Romacho et al ldquoSolu-ble DPP4 induces inflammation and proliferation of humansmoothmuscle cells via protease-activated receptor 2rdquoBiochim-ica et Biophysica Acta vol 1842 no 9 pp 1613ndash1621 2014

6 Journal of Diabetes Research

PCoA

NC1

NC2 NC3

NC4

NC5

NC6

NC7

NC8

NC9

NC10

Obe1

Obe2Obe3

Obe4

Obe5 Obe6

Obe7

Obe8

Obe9

Obe10

DM1

DM2

DM3

DM4DM5

DM6

DM7

DM8DM9

DM10Sit1

Sit2Sit3

Sit4

Sit5

Sit6

Sit7Sit8

Sit9Sit10

minus04

minus02

00

02

04

PC3

86

00 05minus05PC2 2006

NCObe

DMSit

PCoA

NC1

NC2NC3

NC4

NC5

NC6NC7

NC8

NC9

NC10

Obe1

Obe2

Obe3

Obe4

Obe5

Obe7

Obe8Obe9

Obe10

DM1

DM2Obe2

DM3

DM4DM5

DM6

DM7

DM8DM9

DM10 Sit1

Sit2Sit3

Sit4

Sit5

Sit6

Sit7Sit8

Sit9

Sit10

00 05minus05PC1 3057

minus04

minus02

00

02

04

PC3

86

PCoA

NC1

NC2

NC3

NC4

NC5

C6NCncNC8

NC9

NC10 Obe1Obe2

Obe3Obe4

Obe5

Obe6

Obe7Obe8

Obe9

Obe10

DM1

Obe2DM3

DM4

DM5DM6

DM7

DM8DM9

DM10

Sit1

Sit2Sit3 Sit4

Sit5Sit6

Sit7Sit8

Sit9

Sit10

00 05minus05PC1 3057

minus05

00

05

PC2

20

06

NCObe

DMSit

NCObe

DMSit

Figure 5 PCA plots based on unweighted Unifrac metrics Principal components (PCs) 1 2 and 3 explained 3057 2006 and 86 Eachsymbol represents a sample

of sitagliptin onmicrobiotaWe found significantly more Fir-micutes and less Bacteroidetes in obese rats compared to theirlean counterparts similar to previous studies [31] despiteconflicting data showing no differences of Bacteroidetesand Firmicutes between obese and lean subjects [32ndash34]The increased Firmicutes in obese mice may be related tothe genes encoding enzymes that break down polysaccha-rides which cannot be digested by the host increasing the

production of monosaccharides and short-chain fatty acids(SCFA) and the conversion of these SCFA to triglyceridesin the liver The binding of SCFA to two G-protein-coupledreceptors (GPR41 and GPR43) induces peptide YY secretionwhich suppresses gut motility retards intestinal transit andsubsequently results in the increase of nutrient uptake anddeposition [35] Interestingly we also found that hydrogen-producing Prevotellaceae decreased significantly in the obese

Journal of Diabetes Research 7

condition Given the fact that hydrogen inhibits digestion theobese rats seemed likely to absorb more calories from similarenergy intake

Another interesting finding was the higher abundance ofthe phylum Tenericutes (class Mollicutes) in obese rats com-pared to their lean counterparts Certain species ofMollicutesbloom have been proved to evolve the capacity to importcertain types of carbohydrates common in westernized dietfor both mice and human being (eg glucose fructose andsucrose) and to metabolize these imported sugars to SCFAwhich could be readily absorbed by the host [36] Sitagliptindid restore the structure of gutmicrobe at the level of phylumsimilar to the lean control condition without significanteffects on body weight

Unweighted Unifrac PCA analysis confirmed aboveresults in the respect of overall microbial structures Theintestinal microbiota of the four conditions of rats werestructurally separated from each other in the three principalcomponents

Various mechanisms have been proposed to explain theinfluence of microbiota on insulin resistance and T2DMincludingmetabolic endotoxemiamodifications in the secre-tion of the incretins and butyrate production Decrease ofSCFA-producing bacteria has been commonly observed inmetabolic diseases including T2DM [37] and even colorectalcancer [38] SCFA-producing bacteria have been previouslyshown to benefit the host through protecting the mucosafrom damage induced by pathogens supplying colonocytenutrients mitigating inflammation and so forth [39 40]Recent studies in mice have shown that an increase incolonic production of short-chain fatty acids triggers intesti-nal gluconeogenesis (IGN) via complementary mechanismsButyrate activates IGN gene expression through a cAMP-dependent mechanism in enterocytes whereas propionateitself a substrate of IGN activates IGN gene expressionvia the portal nervous system and the fatty acid receptorFFAR3 In rodents the result of increased IGN is beneficialto glucose and energy homeostasis with reductions in hepaticglucose production appetite and body weight [41] Belong-ing to either Clostridium Eubacterium or Fusobacteriumthe butyrate-producing bacteria mainly exist in cecum andcolon Fusobacterium prausnitzii and Roseburia intestinalisare predominant species in butyric acid producing bacteriain human and animal intestine [42] At the level of genusour data suggested that the relative abundance of butyrate-producing Clostridium and Roseburia in the diabetic ratsdecreased significantly compared to the lean and obeseconditions Moreover we also observed that the SCFA-producing bacteria Blautia increased significantly in the dia-betic condition compared to the obese condition Becker etal demonstrated that the addition of Clostridium butyricumas a second butyrate-producing bacterium to SIHUMI (asimplified human intestinal microbiota Anaerostipes caccaeBacteroides thetaiotaomicron Bifidobacterium longum Blau-tia producta Clostridium ramosum Escherichia coli and Lac-tobacillus plantarum) led to an increase of butyrate by 56butdecrease of Bifidobacterium longum Blautia producta and Ecoli indicating that either of these organisms competes withClostridium butyricum for the same substrates or Clostridium

butyricum forms inhibitory substances [43] Thus it seemsreasonable to suppose that changes ofClostridiumRoseburiaand Blautia show an opposite trend In other words theincrease of SCFA-producing bacteria like Blautia leads todecrease of butyrate-producing bacteria like Clostridium andRoseburia In the current study the structure of SCFA-producing bacteria seemed to be abnormal in diabetic ratsAfter sitagliptin treatment Roseburia increased and Blautiadecreased while Clostridium showed no change

Probiotics such as Lactobacillus and Bifidobacteriumnormally reside in the intestinal tract and regulate gutmicroflora and mucosal immunity Recent data also suggestthat microflora especially probiotics exert beneficial effectson gastrointestinal discomforts such as diarrhea abdominalpain and bloating [44] Lactobacillus produces lactic acidCO2 acetic acid andor ethanol which may contribute to a

more acidic environment through homo- or heterofermenta-tive metabolism [45 46] Our data showed that Lactobacillusand Bifidobacterium decreased in the diabetic conditionHowever sitagliptin made Bifidobacterium increased withno significant effect on Lactobacillus which seemed to beconsistent with the notion we discussed above

Marked changes in microbiota composition after met-formin treatment were observed in high-fat diet- (HFD-) fedconditions especially Akkermansia belonging to the Verru-comicrobia phylum showed the most conspicuous changes[47] suggesting a possible interaction between HFD met-formin and intestinal microbiota Forslund et al found sig-nificant increase of Escherichia spp and decrease of Intestini-bacter spp in human gut microbiome in metformin-treatedT2DM especially the latter is resistant to oxidative stress andable to degrade fucose indicative of an indirect involvementin mucus degradation [41] Interestingly there are consistentdata from numerous studies showing that sitagliptin resultsin reduced gastrointestinal discomfort when combined withmetformin a classic antihyperglycemia agent also known forits gastrointestinal side effects For instance Reasner et alshowed that sitagliptin and metformin fixed dose combina-tion resulted in a significant reduction on abdominal pain anddiarrhea compared tometforminmonotherapy [48] In addi-tion a 104-week clinical study (Harmony) also showed thatdiarrhea incidence was reduced in sitagliptin plus metformingroup as compared to metformin monotherapy group [49]Though the underlying mechanism of effect of metforminand sitagliptin on intestinal flora remains unclear our resultsseem to provide a possible explanation arguing that thiseffect could be related to favorable changes of microflora aftersitagliptin treatment

We supposed that the effect of sitagliptin on microbiotawas related to GLP-2 which helps improve the intestinalmucosa barrier integrity Besides recent studies found thatDPP-4 may act as the protease activated receptor 2 (PAR2)agonists involved in the pathophysiology of PAR2 leading toproliferation and inflammation in smooth muscle cells ThusDPP-4 inhibitor can relieve edema of intestinal wall and alle-viate the intestinal inflammation [50 51] Integrated intestinalmucosa barrier and improved intestinal environment maybe not fit for bacteria related with obesity and diabetes oropportunistic pathogen to colonize

8 Journal of Diabetes Research

In summary our data highlights the potential beneficialeffect of sitagliptin on gut microbe of diabetic or obese ani-mals which constituted a novel exciting observation callingfor further investigation on numerous involved microbiotaspecies These data when combined with the results offuture clinical studies may facilitate the development oroptimization of novelmicrobiota based T2DM interventionalstrategies

5 Conclusions

Taken together our data suggest that the structure of gut floraespecially SCFA-producing bacteria seems to be abnormalin a progressive glucose intolerance rat model Treatmentwith sitagliptin improved gut dysbiosis moderately To thebest of our knowledge our study seems to be the firstreport showing that a DPP-4 inhibitor may exert positiveregulation on gut flora dysregulated during progression ofglucose intolerance and obesity in an animal model Futureresearch will be directed to elucidate whether the favorablechanges of microflora contribute to better metabolic controland reduced gastrointestinal side effects of DPP4 inhibitorwhen combined with metformin

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Competing Interests

The authors declare no competing financial interests

Authorsrsquo Contributions

Bo Feng designed the study Xinfeng Yan Peicheng Liand Zhaosheng Tang performed the experiments Bo FengXinfeng Yan Zhaosheng Tang and Lin Wang analyzed thedata and contributed to the discussion for interpreting thedata Bo Feng and Xinfeng Yan wrote and revised the paperXinfeng Yan prepared all the figures and tables All authorsreviewed the paper

Acknowledgments

The authors thankMajorbio company for 454 pyrophosphatesequencing and statistical analysis they also thank JayleenGrams MD PhD Zhao Bin MD PhD Zhu XiaolinMD PhD and Li Huiying MS from MSD for scien-tific and editing support This work was granted by theNational Basic Research Program of China (973 Program)no 2011CB504006 Key Specialty Construction Project ofPudong Health and Family Planning Commission of Shang-hai (no PWZz2013-04) andClinical ResearchProgramof theChinese Medical Association (no 12030510351)

References

[1] A Ouwehand E Isolauri and S Salminen ldquoThe role of theintestinalmicroflora for the development of the immune systemin early childhoodrdquo European Journal of Nutrition vol 41supplement 1 pp I32ndashI37 2002

[2] T S Stappenbeck L VHooper and J I Gordon ldquoDevelopmen-tal regulation of intestinal angiogenesis by indigenousmicrobesvia Paneth cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 24 pp 15451ndash154552002

[3] T Klaenhammer E Altermann F Arigoni et al ldquoDiscoveringlactic acid bacteria by genomicsrdquoAntonie van Leeuwenhoek vol82 no 1ndash4 pp 29ndash58 2002

[4] K Makarova A Slesarevb Y Wolf et al ldquoComparativegenomics of the lactic acid bacteriardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no42 pp 15611ndash15616 2006

[5] E EsteveW Ricart and J-M Fernandez-Real ldquoGutmicrobiotainteractions with obesity insulin resistance and type 2 diabetesdid gut microbiote co-evolve with insulin resistancerdquo CurrentOpinion in Clinical Nutrition and Metabolic Care vol 14 no 5pp 483ndash490 2011

[6] F Backhed H Ding T Wang et al ldquoThe gut microbiota as anenvironmental factor that regulates fat storagerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 101 no 44 pp 15718ndash15723 2004

[7] M Bajzer and R J Seeley ldquoPhysiology obesity and gut florardquoNature vol 444 no 7122 pp 1009ndash1010 2006

[8] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009

[9] P J Turnbaugh R E Ley M A Mahowald V Magrini ER Mardis and J I Gordon ldquoAn obesity-associated gut micro-biome with increased capacity for energy harvestrdquo Nature vol444 no 7122 pp 1027ndash1031 2006

[10] R E Ley F Backhed P Turnbaugh et al ldquoObesity alters gutmicrobial ecologyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 102 no 31 pp11070ndash11075 2005

[11] V R Velagapudi R Hezaveh C S Reigstad et al ldquoThe gutmicrobiota modulates host energy and lipid metabolism inmicerdquo Journal of Lipid Research vol 51 no 5 pp 1101ndash1112 2010

[12] M Diamant E E Blaak and W M de Vos ldquoDo nutrient-gut-microbiota interactions play a role in human obesity insulinresistance and type 2 diabetesrdquo Obesity Reviews vol 12 no 4pp 272ndash281 2011

[13] A M Caricilli P K Picardi L L de Abreu et al ldquoGutmicrobiota is a key modulator of insulin resistance in TLR 2knockout micerdquo PLoS Biology vol 9 no 12 Article ID e10012122011

[14] P D Cani S Possemiers T Van De Wiele et al ldquoChanges ingut microbiota control inflammation in obese mice througha mechanism involving GLP-2-driven improvement of gutpermeabilityrdquo Gut vol 58 no 8 pp 1091ndash1103 2009

[15] D J Drucker ldquoEnhancing incretin action for the treatment oftype 2 diabetesrdquo Diabetes Care vol 26 no 10 pp 2929ndash29402003

[16] D J Drucker and B Yusta ldquoPhysiology and pharmacology ofthe enteroendocrine hormone glucagon-like peptide-2rdquoAnnualReview of Physiology vol 76 pp 561ndash583 2014

Journal of Diabetes Research 9

[17] C S Marathe C K Rayner K L Jones and M HorowitzldquoGlucagon-like peptides 1 and 2 in health and disease a reviewrdquoPeptides vol 44 pp 75ndash86 2013

[18] P B Eckburg E M Bik C N Bernstein et al ldquoMicrobiologydiversity of the human intestinal microbial florardquo Science vol308 no 5728 pp 1635ndash1638 2005

[19] S R Gill M Pop R T DeBoy et al ldquoMetagenomic analysis ofthe human distal gut microbiomerdquo Science vol 312 no 5778pp 1355ndash1359 2006

[20] Y S Zhou B Li et al ldquoA rat model of type 2 diabetes mellitusinduced by high fat chow and low dose streptozocin injectionrdquoActa Laboratorium Animals Scientia Sinica vol 13 no 3 pp154ndash158 2005

[21] Y Jialin L Guo L Youpin et al ldquoEstablishing a rat modelsimilar to the adult patient of the general type 2 diabetes by long-term fat-enriched fed and lower dose of streptozocin-treatedratsrdquo Acta Laboratorium Animals Scientia Sinica vol 11 no 3pp 138ndash141 2003

[22] K R Amato C J Yeoman A Kent et al ldquoHabitat degradationimpacts black howler monkey (Alouatta pigra) gastrointestinalmicrobiomesrdquo ISME Journal vol 7 no 7 pp 1344ndash1353 2013

[23] P D Schloss S L Westcott T Ryabin et al ldquoIntroduc-ing mothur open-source platform-independent community-supported software for describing and comparing microbialcommunitiesrdquoApplied and EnvironmentalMicrobiology vol 75no 23 pp 7537ndash7541 2009

[24] G R Warnes B Bolker L Bonebakker et al gplots Various RProgramming Tools for Plotting Data Version 2170 2015

[25] J R White N Nagarajan and M Pop ldquoStatistical methods fordetecting differentially abundant features in clinical metage-nomic samplesrdquo PLoS Computational Biology vol 5 no 4Article ID e1000352 2009

[26] Y L Wu G X Xi et al ldquoAn experimental study of impairedglucose tolerance in Wistar ratsrdquo Chinese Remedies amp Clinicsvol 7 no 4 pp 281ndash283 2007

[27] M Cotta and R Forster ldquoThe family lachnospiraceae includingthe genera butyrivibrio lachnospira and roseburiardquo in TheProkaryotes Volume 4 Bacteria Firmicutes Cyanobacteria pp1002ndash1021 Springer Berlin Germany 3th edition 2006

[28] J M W Wong R de Souza C W C Kendall A Emam andD J A Jenkins ldquoColonic health fermentation and short chainfatty acidsrdquo Journal of Clinical Gastroenterology vol 40 no 3pp 235ndash243 2006

[29] H J Flint K P Scott S H Duncan P Louis and E ForanoldquoMicrobial degradation of complex carbohydrates in the gutrdquoGut Microbes vol 3 no 4 pp 289ndash306 2012

[30] X Ze S H Duncan P Louis and H J Flint ldquoRuminococcusbromii is a keystone species for the degradation of resistantstarch in the human colonrdquo ISME Journal vol 6 no 8 pp 1535ndash1543 2012

[31] R E Ley ldquoObesity and the human microbiomerdquo CurrentOpinion in Gastroenterology vol 26 no 1 pp 5ndash11 2010

[32] S H Duncan G E Lobley G Holtrop et al ldquoHuman colonicmicrobiota associated with diet obesity and weight lossrdquoInternational Journal of Obesity vol 32 no 11 pp 1720ndash17242008

[33] V Mai Q M McCrary R Sinha and M Glei ldquoAssociationsbetween dietary habits and body mass index with gut micro-biota composition and fecal water genotoxicity an observa-tional study in African American and Caucasian Americanvolunteersrdquo Nutrition Journal vol 8 article 49 2009

[34] R Jumpertz D S Le P J Turnbaugh et al ldquoEnergy-balancestudies reveal associations between gut microbes caloric loadand nutrient absorption in humansrdquo The American Journal ofClinical Nutrition vol 94 no 1 pp 58ndash65 2011

[35] O O Erejuwa S A Sulaiman and M S Ab Wahab ldquoMod-ulation of gut microbiota in the management of metabolicdisorders The prospects and challengesrdquo International Journalof Molecular Sciences vol 15 no 3 pp 4158ndash4188 2014

[36] P J Turnbaugh F Backhed L Fulton and J I Gordon ldquoDiet-induced obesity is linked to marked but reversible alterations inthe mouse distal gut microbiomerdquo Cell Host andMicrobe vol 3no 4 pp 213ndash223 2008

[37] J Qin J Wang Y Li et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[38] T Wang G Cai Y Qiu et al ldquoStructural segregation ofgut microbiota between colorectal cancer patients and healthyvolunteersrdquoThe ISME Journal vol 6 no 2 pp 320ndash329 2012

[39] K M Maslowski A T Vieira A Ng et al ldquoRegulation ofinflammatory responses by gutmicrobiota and chemoattractantreceptorGPR43rdquoNature vol 461 no 7268 pp 1282ndash1286 2009

[40] C De Filippo D Cavalieri M Di Paola et al ldquoImpact of dietin shaping gut microbiota revealed by a comparative studyin children from Europe and rural Africardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 33 pp 14691ndash14696 2010

[41] K Forslund FHildebrand TNielsen et al ldquoDisentangling type2 diabetes and metformin treatment signatures in the humangut microbiotardquo Nature vol 528 no 7581 pp 262ndash266 2015

[42] G L Hold A Schwiertz R I Aminov M Blaut and H J FlintldquoOligonucleotide probes that detect quantitatively significantgroups of butyrate-producing bacteria in human fecesrdquo Appliedand Environmental Microbiology vol 69 no 7 pp 4320ndash43242003

[43] N Becker J Kunath G Loh and M Blaut ldquoHuman intestinalmicrobiota characterization of a simplified and stable gnotobi-otic rat modelrdquo Gut Microbes vol 2 no 1 pp 25ndash33 2011

[44] S Nobaek M-L Johansson G Molin S Ahrne and BJeppsson ldquoAlteration of intestinal microflora is associated withreduction in abdominal bloating and pain in patients withirritable bowel syndromerdquo The American Journal of Gastroen-terology vol 95 no 5 pp 1231ndash1238 2000

[45] G T Macfarlane and S Macfarlane ldquoHuman colonic micro-biota ecology physiology and metabolic potential of intestinalbacteriardquo Scandinavian Journal of Gastroenterology vol 32supplement 222 pp 3ndash9 1997

[46] E G Zoetendal A D L Akkermans W M Akkermans-van Vliet J A G M De Visser and W M De Vos ldquoThehost genotype affects the bacterial community in the humangastrointestinal tractrdquoMicrobial Ecology in Health and Diseasevol 13 no 3 pp 129ndash134 2001

[47] N-R Shin J-C Lee H-Y Lee et al ldquoAn increase in theAkkermansia spp population induced by metformin treatmentimproves glucose homeostasis in diet-induced obesemicerdquoGutvol 63 no 5 pp 727ndash735 2014

[48] C Reasner L Olansky T L Seck et al ldquoThe effect of initialtherapy with the fixed-dose combination of sitagliptin andmetformin compared with metforminmonotherapy in patientswith type 2 diabetesmellitusrdquoDiabetes Obesity andMetabolismvol 13 no 7 pp 644ndash652 2011

[49] B Ahren S L Johnson M Stewart et al ldquoHARMONY 3 104-week randomized double-blind placebo- and active-controlled

10 Journal of Diabetes Research

trial assessing the efficacy and safety of albiglutide comparedwith placebo sitagliptin and glimepiride in patients with type2 diabetes taking metforminrdquo Diabetes Care vol 37 no 8 pp2141ndash2148 2014

[50] S Enjoji T Ohama and K Sato ldquoRegulation of epithelial celltight junctions by protease-activated receptor 2rdquoThe Journal ofVeterinary Medical Science vol 76 no 9 pp 1225ndash1229 2014

[51] N Wronkowitz S W Gorgens T Romacho et al ldquoSolu-ble DPP4 induces inflammation and proliferation of humansmoothmuscle cells via protease-activated receptor 2rdquoBiochim-ica et Biophysica Acta vol 1842 no 9 pp 1613ndash1621 2014

Journal of Diabetes Research 7

condition Given the fact that hydrogen inhibits digestion theobese rats seemed likely to absorb more calories from similarenergy intake

Another interesting finding was the higher abundance ofthe phylum Tenericutes (class Mollicutes) in obese rats com-pared to their lean counterparts Certain species ofMollicutesbloom have been proved to evolve the capacity to importcertain types of carbohydrates common in westernized dietfor both mice and human being (eg glucose fructose andsucrose) and to metabolize these imported sugars to SCFAwhich could be readily absorbed by the host [36] Sitagliptindid restore the structure of gutmicrobe at the level of phylumsimilar to the lean control condition without significanteffects on body weight

Unweighted Unifrac PCA analysis confirmed aboveresults in the respect of overall microbial structures Theintestinal microbiota of the four conditions of rats werestructurally separated from each other in the three principalcomponents

Various mechanisms have been proposed to explain theinfluence of microbiota on insulin resistance and T2DMincludingmetabolic endotoxemiamodifications in the secre-tion of the incretins and butyrate production Decrease ofSCFA-producing bacteria has been commonly observed inmetabolic diseases including T2DM [37] and even colorectalcancer [38] SCFA-producing bacteria have been previouslyshown to benefit the host through protecting the mucosafrom damage induced by pathogens supplying colonocytenutrients mitigating inflammation and so forth [39 40]Recent studies in mice have shown that an increase incolonic production of short-chain fatty acids triggers intesti-nal gluconeogenesis (IGN) via complementary mechanismsButyrate activates IGN gene expression through a cAMP-dependent mechanism in enterocytes whereas propionateitself a substrate of IGN activates IGN gene expressionvia the portal nervous system and the fatty acid receptorFFAR3 In rodents the result of increased IGN is beneficialto glucose and energy homeostasis with reductions in hepaticglucose production appetite and body weight [41] Belong-ing to either Clostridium Eubacterium or Fusobacteriumthe butyrate-producing bacteria mainly exist in cecum andcolon Fusobacterium prausnitzii and Roseburia intestinalisare predominant species in butyric acid producing bacteriain human and animal intestine [42] At the level of genusour data suggested that the relative abundance of butyrate-producing Clostridium and Roseburia in the diabetic ratsdecreased significantly compared to the lean and obeseconditions Moreover we also observed that the SCFA-producing bacteria Blautia increased significantly in the dia-betic condition compared to the obese condition Becker etal demonstrated that the addition of Clostridium butyricumas a second butyrate-producing bacterium to SIHUMI (asimplified human intestinal microbiota Anaerostipes caccaeBacteroides thetaiotaomicron Bifidobacterium longum Blau-tia producta Clostridium ramosum Escherichia coli and Lac-tobacillus plantarum) led to an increase of butyrate by 56butdecrease of Bifidobacterium longum Blautia producta and Ecoli indicating that either of these organisms competes withClostridium butyricum for the same substrates or Clostridium

butyricum forms inhibitory substances [43] Thus it seemsreasonable to suppose that changes ofClostridiumRoseburiaand Blautia show an opposite trend In other words theincrease of SCFA-producing bacteria like Blautia leads todecrease of butyrate-producing bacteria like Clostridium andRoseburia In the current study the structure of SCFA-producing bacteria seemed to be abnormal in diabetic ratsAfter sitagliptin treatment Roseburia increased and Blautiadecreased while Clostridium showed no change

Probiotics such as Lactobacillus and Bifidobacteriumnormally reside in the intestinal tract and regulate gutmicroflora and mucosal immunity Recent data also suggestthat microflora especially probiotics exert beneficial effectson gastrointestinal discomforts such as diarrhea abdominalpain and bloating [44] Lactobacillus produces lactic acidCO2 acetic acid andor ethanol which may contribute to a

more acidic environment through homo- or heterofermenta-tive metabolism [45 46] Our data showed that Lactobacillusand Bifidobacterium decreased in the diabetic conditionHowever sitagliptin made Bifidobacterium increased withno significant effect on Lactobacillus which seemed to beconsistent with the notion we discussed above

Marked changes in microbiota composition after met-formin treatment were observed in high-fat diet- (HFD-) fedconditions especially Akkermansia belonging to the Verru-comicrobia phylum showed the most conspicuous changes[47] suggesting a possible interaction between HFD met-formin and intestinal microbiota Forslund et al found sig-nificant increase of Escherichia spp and decrease of Intestini-bacter spp in human gut microbiome in metformin-treatedT2DM especially the latter is resistant to oxidative stress andable to degrade fucose indicative of an indirect involvementin mucus degradation [41] Interestingly there are consistentdata from numerous studies showing that sitagliptin resultsin reduced gastrointestinal discomfort when combined withmetformin a classic antihyperglycemia agent also known forits gastrointestinal side effects For instance Reasner et alshowed that sitagliptin and metformin fixed dose combina-tion resulted in a significant reduction on abdominal pain anddiarrhea compared tometforminmonotherapy [48] In addi-tion a 104-week clinical study (Harmony) also showed thatdiarrhea incidence was reduced in sitagliptin plus metformingroup as compared to metformin monotherapy group [49]Though the underlying mechanism of effect of metforminand sitagliptin on intestinal flora remains unclear our resultsseem to provide a possible explanation arguing that thiseffect could be related to favorable changes of microflora aftersitagliptin treatment

We supposed that the effect of sitagliptin on microbiotawas related to GLP-2 which helps improve the intestinalmucosa barrier integrity Besides recent studies found thatDPP-4 may act as the protease activated receptor 2 (PAR2)agonists involved in the pathophysiology of PAR2 leading toproliferation and inflammation in smooth muscle cells ThusDPP-4 inhibitor can relieve edema of intestinal wall and alle-viate the intestinal inflammation [50 51] Integrated intestinalmucosa barrier and improved intestinal environment maybe not fit for bacteria related with obesity and diabetes oropportunistic pathogen to colonize

8 Journal of Diabetes Research

In summary our data highlights the potential beneficialeffect of sitagliptin on gut microbe of diabetic or obese ani-mals which constituted a novel exciting observation callingfor further investigation on numerous involved microbiotaspecies These data when combined with the results offuture clinical studies may facilitate the development oroptimization of novelmicrobiota based T2DM interventionalstrategies

5 Conclusions

Taken together our data suggest that the structure of gut floraespecially SCFA-producing bacteria seems to be abnormalin a progressive glucose intolerance rat model Treatmentwith sitagliptin improved gut dysbiosis moderately To thebest of our knowledge our study seems to be the firstreport showing that a DPP-4 inhibitor may exert positiveregulation on gut flora dysregulated during progression ofglucose intolerance and obesity in an animal model Futureresearch will be directed to elucidate whether the favorablechanges of microflora contribute to better metabolic controland reduced gastrointestinal side effects of DPP4 inhibitorwhen combined with metformin

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Competing Interests

The authors declare no competing financial interests

Authorsrsquo Contributions

Bo Feng designed the study Xinfeng Yan Peicheng Liand Zhaosheng Tang performed the experiments Bo FengXinfeng Yan Zhaosheng Tang and Lin Wang analyzed thedata and contributed to the discussion for interpreting thedata Bo Feng and Xinfeng Yan wrote and revised the paperXinfeng Yan prepared all the figures and tables All authorsreviewed the paper

Acknowledgments

The authors thankMajorbio company for 454 pyrophosphatesequencing and statistical analysis they also thank JayleenGrams MD PhD Zhao Bin MD PhD Zhu XiaolinMD PhD and Li Huiying MS from MSD for scien-tific and editing support This work was granted by theNational Basic Research Program of China (973 Program)no 2011CB504006 Key Specialty Construction Project ofPudong Health and Family Planning Commission of Shang-hai (no PWZz2013-04) andClinical ResearchProgramof theChinese Medical Association (no 12030510351)

References

[1] A Ouwehand E Isolauri and S Salminen ldquoThe role of theintestinalmicroflora for the development of the immune systemin early childhoodrdquo European Journal of Nutrition vol 41supplement 1 pp I32ndashI37 2002

[2] T S Stappenbeck L VHooper and J I Gordon ldquoDevelopmen-tal regulation of intestinal angiogenesis by indigenousmicrobesvia Paneth cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 24 pp 15451ndash154552002

[3] T Klaenhammer E Altermann F Arigoni et al ldquoDiscoveringlactic acid bacteria by genomicsrdquoAntonie van Leeuwenhoek vol82 no 1ndash4 pp 29ndash58 2002

[4] K Makarova A Slesarevb Y Wolf et al ldquoComparativegenomics of the lactic acid bacteriardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no42 pp 15611ndash15616 2006

[5] E EsteveW Ricart and J-M Fernandez-Real ldquoGutmicrobiotainteractions with obesity insulin resistance and type 2 diabetesdid gut microbiote co-evolve with insulin resistancerdquo CurrentOpinion in Clinical Nutrition and Metabolic Care vol 14 no 5pp 483ndash490 2011

[6] F Backhed H Ding T Wang et al ldquoThe gut microbiota as anenvironmental factor that regulates fat storagerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 101 no 44 pp 15718ndash15723 2004

[7] M Bajzer and R J Seeley ldquoPhysiology obesity and gut florardquoNature vol 444 no 7122 pp 1009ndash1010 2006

[8] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009

[9] P J Turnbaugh R E Ley M A Mahowald V Magrini ER Mardis and J I Gordon ldquoAn obesity-associated gut micro-biome with increased capacity for energy harvestrdquo Nature vol444 no 7122 pp 1027ndash1031 2006

[10] R E Ley F Backhed P Turnbaugh et al ldquoObesity alters gutmicrobial ecologyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 102 no 31 pp11070ndash11075 2005

[11] V R Velagapudi R Hezaveh C S Reigstad et al ldquoThe gutmicrobiota modulates host energy and lipid metabolism inmicerdquo Journal of Lipid Research vol 51 no 5 pp 1101ndash1112 2010

[12] M Diamant E E Blaak and W M de Vos ldquoDo nutrient-gut-microbiota interactions play a role in human obesity insulinresistance and type 2 diabetesrdquo Obesity Reviews vol 12 no 4pp 272ndash281 2011

[13] A M Caricilli P K Picardi L L de Abreu et al ldquoGutmicrobiota is a key modulator of insulin resistance in TLR 2knockout micerdquo PLoS Biology vol 9 no 12 Article ID e10012122011

[14] P D Cani S Possemiers T Van De Wiele et al ldquoChanges ingut microbiota control inflammation in obese mice througha mechanism involving GLP-2-driven improvement of gutpermeabilityrdquo Gut vol 58 no 8 pp 1091ndash1103 2009

[15] D J Drucker ldquoEnhancing incretin action for the treatment oftype 2 diabetesrdquo Diabetes Care vol 26 no 10 pp 2929ndash29402003

[16] D J Drucker and B Yusta ldquoPhysiology and pharmacology ofthe enteroendocrine hormone glucagon-like peptide-2rdquoAnnualReview of Physiology vol 76 pp 561ndash583 2014

Journal of Diabetes Research 9

[17] C S Marathe C K Rayner K L Jones and M HorowitzldquoGlucagon-like peptides 1 and 2 in health and disease a reviewrdquoPeptides vol 44 pp 75ndash86 2013

[18] P B Eckburg E M Bik C N Bernstein et al ldquoMicrobiologydiversity of the human intestinal microbial florardquo Science vol308 no 5728 pp 1635ndash1638 2005

[19] S R Gill M Pop R T DeBoy et al ldquoMetagenomic analysis ofthe human distal gut microbiomerdquo Science vol 312 no 5778pp 1355ndash1359 2006

[20] Y S Zhou B Li et al ldquoA rat model of type 2 diabetes mellitusinduced by high fat chow and low dose streptozocin injectionrdquoActa Laboratorium Animals Scientia Sinica vol 13 no 3 pp154ndash158 2005

[21] Y Jialin L Guo L Youpin et al ldquoEstablishing a rat modelsimilar to the adult patient of the general type 2 diabetes by long-term fat-enriched fed and lower dose of streptozocin-treatedratsrdquo Acta Laboratorium Animals Scientia Sinica vol 11 no 3pp 138ndash141 2003

[22] K R Amato C J Yeoman A Kent et al ldquoHabitat degradationimpacts black howler monkey (Alouatta pigra) gastrointestinalmicrobiomesrdquo ISME Journal vol 7 no 7 pp 1344ndash1353 2013

[23] P D Schloss S L Westcott T Ryabin et al ldquoIntroduc-ing mothur open-source platform-independent community-supported software for describing and comparing microbialcommunitiesrdquoApplied and EnvironmentalMicrobiology vol 75no 23 pp 7537ndash7541 2009

[24] G R Warnes B Bolker L Bonebakker et al gplots Various RProgramming Tools for Plotting Data Version 2170 2015

[25] J R White N Nagarajan and M Pop ldquoStatistical methods fordetecting differentially abundant features in clinical metage-nomic samplesrdquo PLoS Computational Biology vol 5 no 4Article ID e1000352 2009

[26] Y L Wu G X Xi et al ldquoAn experimental study of impairedglucose tolerance in Wistar ratsrdquo Chinese Remedies amp Clinicsvol 7 no 4 pp 281ndash283 2007

[27] M Cotta and R Forster ldquoThe family lachnospiraceae includingthe genera butyrivibrio lachnospira and roseburiardquo in TheProkaryotes Volume 4 Bacteria Firmicutes Cyanobacteria pp1002ndash1021 Springer Berlin Germany 3th edition 2006

[28] J M W Wong R de Souza C W C Kendall A Emam andD J A Jenkins ldquoColonic health fermentation and short chainfatty acidsrdquo Journal of Clinical Gastroenterology vol 40 no 3pp 235ndash243 2006

[29] H J Flint K P Scott S H Duncan P Louis and E ForanoldquoMicrobial degradation of complex carbohydrates in the gutrdquoGut Microbes vol 3 no 4 pp 289ndash306 2012

[30] X Ze S H Duncan P Louis and H J Flint ldquoRuminococcusbromii is a keystone species for the degradation of resistantstarch in the human colonrdquo ISME Journal vol 6 no 8 pp 1535ndash1543 2012

[31] R E Ley ldquoObesity and the human microbiomerdquo CurrentOpinion in Gastroenterology vol 26 no 1 pp 5ndash11 2010

[32] S H Duncan G E Lobley G Holtrop et al ldquoHuman colonicmicrobiota associated with diet obesity and weight lossrdquoInternational Journal of Obesity vol 32 no 11 pp 1720ndash17242008

[33] V Mai Q M McCrary R Sinha and M Glei ldquoAssociationsbetween dietary habits and body mass index with gut micro-biota composition and fecal water genotoxicity an observa-tional study in African American and Caucasian Americanvolunteersrdquo Nutrition Journal vol 8 article 49 2009

[34] R Jumpertz D S Le P J Turnbaugh et al ldquoEnergy-balancestudies reveal associations between gut microbes caloric loadand nutrient absorption in humansrdquo The American Journal ofClinical Nutrition vol 94 no 1 pp 58ndash65 2011

[35] O O Erejuwa S A Sulaiman and M S Ab Wahab ldquoMod-ulation of gut microbiota in the management of metabolicdisorders The prospects and challengesrdquo International Journalof Molecular Sciences vol 15 no 3 pp 4158ndash4188 2014

[36] P J Turnbaugh F Backhed L Fulton and J I Gordon ldquoDiet-induced obesity is linked to marked but reversible alterations inthe mouse distal gut microbiomerdquo Cell Host andMicrobe vol 3no 4 pp 213ndash223 2008

[37] J Qin J Wang Y Li et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[38] T Wang G Cai Y Qiu et al ldquoStructural segregation ofgut microbiota between colorectal cancer patients and healthyvolunteersrdquoThe ISME Journal vol 6 no 2 pp 320ndash329 2012

[39] K M Maslowski A T Vieira A Ng et al ldquoRegulation ofinflammatory responses by gutmicrobiota and chemoattractantreceptorGPR43rdquoNature vol 461 no 7268 pp 1282ndash1286 2009

[40] C De Filippo D Cavalieri M Di Paola et al ldquoImpact of dietin shaping gut microbiota revealed by a comparative studyin children from Europe and rural Africardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 33 pp 14691ndash14696 2010

[41] K Forslund FHildebrand TNielsen et al ldquoDisentangling type2 diabetes and metformin treatment signatures in the humangut microbiotardquo Nature vol 528 no 7581 pp 262ndash266 2015

[42] G L Hold A Schwiertz R I Aminov M Blaut and H J FlintldquoOligonucleotide probes that detect quantitatively significantgroups of butyrate-producing bacteria in human fecesrdquo Appliedand Environmental Microbiology vol 69 no 7 pp 4320ndash43242003

[43] N Becker J Kunath G Loh and M Blaut ldquoHuman intestinalmicrobiota characterization of a simplified and stable gnotobi-otic rat modelrdquo Gut Microbes vol 2 no 1 pp 25ndash33 2011

[44] S Nobaek M-L Johansson G Molin S Ahrne and BJeppsson ldquoAlteration of intestinal microflora is associated withreduction in abdominal bloating and pain in patients withirritable bowel syndromerdquo The American Journal of Gastroen-terology vol 95 no 5 pp 1231ndash1238 2000

[45] G T Macfarlane and S Macfarlane ldquoHuman colonic micro-biota ecology physiology and metabolic potential of intestinalbacteriardquo Scandinavian Journal of Gastroenterology vol 32supplement 222 pp 3ndash9 1997

[46] E G Zoetendal A D L Akkermans W M Akkermans-van Vliet J A G M De Visser and W M De Vos ldquoThehost genotype affects the bacterial community in the humangastrointestinal tractrdquoMicrobial Ecology in Health and Diseasevol 13 no 3 pp 129ndash134 2001

[47] N-R Shin J-C Lee H-Y Lee et al ldquoAn increase in theAkkermansia spp population induced by metformin treatmentimproves glucose homeostasis in diet-induced obesemicerdquoGutvol 63 no 5 pp 727ndash735 2014

[48] C Reasner L Olansky T L Seck et al ldquoThe effect of initialtherapy with the fixed-dose combination of sitagliptin andmetformin compared with metforminmonotherapy in patientswith type 2 diabetesmellitusrdquoDiabetes Obesity andMetabolismvol 13 no 7 pp 644ndash652 2011

[49] B Ahren S L Johnson M Stewart et al ldquoHARMONY 3 104-week randomized double-blind placebo- and active-controlled

10 Journal of Diabetes Research

trial assessing the efficacy and safety of albiglutide comparedwith placebo sitagliptin and glimepiride in patients with type2 diabetes taking metforminrdquo Diabetes Care vol 37 no 8 pp2141ndash2148 2014

[50] S Enjoji T Ohama and K Sato ldquoRegulation of epithelial celltight junctions by protease-activated receptor 2rdquoThe Journal ofVeterinary Medical Science vol 76 no 9 pp 1225ndash1229 2014

[51] N Wronkowitz S W Gorgens T Romacho et al ldquoSolu-ble DPP4 induces inflammation and proliferation of humansmoothmuscle cells via protease-activated receptor 2rdquoBiochim-ica et Biophysica Acta vol 1842 no 9 pp 1613ndash1621 2014

8 Journal of Diabetes Research

In summary our data highlights the potential beneficialeffect of sitagliptin on gut microbe of diabetic or obese ani-mals which constituted a novel exciting observation callingfor further investigation on numerous involved microbiotaspecies These data when combined with the results offuture clinical studies may facilitate the development oroptimization of novelmicrobiota based T2DM interventionalstrategies

5 Conclusions

Taken together our data suggest that the structure of gut floraespecially SCFA-producing bacteria seems to be abnormalin a progressive glucose intolerance rat model Treatmentwith sitagliptin improved gut dysbiosis moderately To thebest of our knowledge our study seems to be the firstreport showing that a DPP-4 inhibitor may exert positiveregulation on gut flora dysregulated during progression ofglucose intolerance and obesity in an animal model Futureresearch will be directed to elucidate whether the favorablechanges of microflora contribute to better metabolic controland reduced gastrointestinal side effects of DPP4 inhibitorwhen combined with metformin

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Competing Interests

The authors declare no competing financial interests

Authorsrsquo Contributions

Bo Feng designed the study Xinfeng Yan Peicheng Liand Zhaosheng Tang performed the experiments Bo FengXinfeng Yan Zhaosheng Tang and Lin Wang analyzed thedata and contributed to the discussion for interpreting thedata Bo Feng and Xinfeng Yan wrote and revised the paperXinfeng Yan prepared all the figures and tables All authorsreviewed the paper

Acknowledgments

The authors thankMajorbio company for 454 pyrophosphatesequencing and statistical analysis they also thank JayleenGrams MD PhD Zhao Bin MD PhD Zhu XiaolinMD PhD and Li Huiying MS from MSD for scien-tific and editing support This work was granted by theNational Basic Research Program of China (973 Program)no 2011CB504006 Key Specialty Construction Project ofPudong Health and Family Planning Commission of Shang-hai (no PWZz2013-04) andClinical ResearchProgramof theChinese Medical Association (no 12030510351)

References

[1] A Ouwehand E Isolauri and S Salminen ldquoThe role of theintestinalmicroflora for the development of the immune systemin early childhoodrdquo European Journal of Nutrition vol 41supplement 1 pp I32ndashI37 2002

[2] T S Stappenbeck L VHooper and J I Gordon ldquoDevelopmen-tal regulation of intestinal angiogenesis by indigenousmicrobesvia Paneth cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 24 pp 15451ndash154552002

[3] T Klaenhammer E Altermann F Arigoni et al ldquoDiscoveringlactic acid bacteria by genomicsrdquoAntonie van Leeuwenhoek vol82 no 1ndash4 pp 29ndash58 2002

[4] K Makarova A Slesarevb Y Wolf et al ldquoComparativegenomics of the lactic acid bacteriardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no42 pp 15611ndash15616 2006

[5] E EsteveW Ricart and J-M Fernandez-Real ldquoGutmicrobiotainteractions with obesity insulin resistance and type 2 diabetesdid gut microbiote co-evolve with insulin resistancerdquo CurrentOpinion in Clinical Nutrition and Metabolic Care vol 14 no 5pp 483ndash490 2011

[6] F Backhed H Ding T Wang et al ldquoThe gut microbiota as anenvironmental factor that regulates fat storagerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 101 no 44 pp 15718ndash15723 2004

[7] M Bajzer and R J Seeley ldquoPhysiology obesity and gut florardquoNature vol 444 no 7122 pp 1009ndash1010 2006

[8] P J Turnbaugh M Hamady T Yatsunenko et al ldquoA core gutmicrobiome in obese and lean twinsrdquoNature vol 457 no 7228pp 480ndash484 2009

[9] P J Turnbaugh R E Ley M A Mahowald V Magrini ER Mardis and J I Gordon ldquoAn obesity-associated gut micro-biome with increased capacity for energy harvestrdquo Nature vol444 no 7122 pp 1027ndash1031 2006

[10] R E Ley F Backhed P Turnbaugh et al ldquoObesity alters gutmicrobial ecologyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 102 no 31 pp11070ndash11075 2005

[11] V R Velagapudi R Hezaveh C S Reigstad et al ldquoThe gutmicrobiota modulates host energy and lipid metabolism inmicerdquo Journal of Lipid Research vol 51 no 5 pp 1101ndash1112 2010

[12] M Diamant E E Blaak and W M de Vos ldquoDo nutrient-gut-microbiota interactions play a role in human obesity insulinresistance and type 2 diabetesrdquo Obesity Reviews vol 12 no 4pp 272ndash281 2011

[13] A M Caricilli P K Picardi L L de Abreu et al ldquoGutmicrobiota is a key modulator of insulin resistance in TLR 2knockout micerdquo PLoS Biology vol 9 no 12 Article ID e10012122011

[14] P D Cani S Possemiers T Van De Wiele et al ldquoChanges ingut microbiota control inflammation in obese mice througha mechanism involving GLP-2-driven improvement of gutpermeabilityrdquo Gut vol 58 no 8 pp 1091ndash1103 2009

[15] D J Drucker ldquoEnhancing incretin action for the treatment oftype 2 diabetesrdquo Diabetes Care vol 26 no 10 pp 2929ndash29402003

[16] D J Drucker and B Yusta ldquoPhysiology and pharmacology ofthe enteroendocrine hormone glucagon-like peptide-2rdquoAnnualReview of Physiology vol 76 pp 561ndash583 2014

Journal of Diabetes Research 9

[17] C S Marathe C K Rayner K L Jones and M HorowitzldquoGlucagon-like peptides 1 and 2 in health and disease a reviewrdquoPeptides vol 44 pp 75ndash86 2013

[18] P B Eckburg E M Bik C N Bernstein et al ldquoMicrobiologydiversity of the human intestinal microbial florardquo Science vol308 no 5728 pp 1635ndash1638 2005

[19] S R Gill M Pop R T DeBoy et al ldquoMetagenomic analysis ofthe human distal gut microbiomerdquo Science vol 312 no 5778pp 1355ndash1359 2006

[20] Y S Zhou B Li et al ldquoA rat model of type 2 diabetes mellitusinduced by high fat chow and low dose streptozocin injectionrdquoActa Laboratorium Animals Scientia Sinica vol 13 no 3 pp154ndash158 2005

[21] Y Jialin L Guo L Youpin et al ldquoEstablishing a rat modelsimilar to the adult patient of the general type 2 diabetes by long-term fat-enriched fed and lower dose of streptozocin-treatedratsrdquo Acta Laboratorium Animals Scientia Sinica vol 11 no 3pp 138ndash141 2003

[22] K R Amato C J Yeoman A Kent et al ldquoHabitat degradationimpacts black howler monkey (Alouatta pigra) gastrointestinalmicrobiomesrdquo ISME Journal vol 7 no 7 pp 1344ndash1353 2013

[23] P D Schloss S L Westcott T Ryabin et al ldquoIntroduc-ing mothur open-source platform-independent community-supported software for describing and comparing microbialcommunitiesrdquoApplied and EnvironmentalMicrobiology vol 75no 23 pp 7537ndash7541 2009

[24] G R Warnes B Bolker L Bonebakker et al gplots Various RProgramming Tools for Plotting Data Version 2170 2015

[25] J R White N Nagarajan and M Pop ldquoStatistical methods fordetecting differentially abundant features in clinical metage-nomic samplesrdquo PLoS Computational Biology vol 5 no 4Article ID e1000352 2009

[26] Y L Wu G X Xi et al ldquoAn experimental study of impairedglucose tolerance in Wistar ratsrdquo Chinese Remedies amp Clinicsvol 7 no 4 pp 281ndash283 2007

[27] M Cotta and R Forster ldquoThe family lachnospiraceae includingthe genera butyrivibrio lachnospira and roseburiardquo in TheProkaryotes Volume 4 Bacteria Firmicutes Cyanobacteria pp1002ndash1021 Springer Berlin Germany 3th edition 2006

[28] J M W Wong R de Souza C W C Kendall A Emam andD J A Jenkins ldquoColonic health fermentation and short chainfatty acidsrdquo Journal of Clinical Gastroenterology vol 40 no 3pp 235ndash243 2006

[29] H J Flint K P Scott S H Duncan P Louis and E ForanoldquoMicrobial degradation of complex carbohydrates in the gutrdquoGut Microbes vol 3 no 4 pp 289ndash306 2012

[30] X Ze S H Duncan P Louis and H J Flint ldquoRuminococcusbromii is a keystone species for the degradation of resistantstarch in the human colonrdquo ISME Journal vol 6 no 8 pp 1535ndash1543 2012

[31] R E Ley ldquoObesity and the human microbiomerdquo CurrentOpinion in Gastroenterology vol 26 no 1 pp 5ndash11 2010

[32] S H Duncan G E Lobley G Holtrop et al ldquoHuman colonicmicrobiota associated with diet obesity and weight lossrdquoInternational Journal of Obesity vol 32 no 11 pp 1720ndash17242008

[33] V Mai Q M McCrary R Sinha and M Glei ldquoAssociationsbetween dietary habits and body mass index with gut micro-biota composition and fecal water genotoxicity an observa-tional study in African American and Caucasian Americanvolunteersrdquo Nutrition Journal vol 8 article 49 2009

[34] R Jumpertz D S Le P J Turnbaugh et al ldquoEnergy-balancestudies reveal associations between gut microbes caloric loadand nutrient absorption in humansrdquo The American Journal ofClinical Nutrition vol 94 no 1 pp 58ndash65 2011

[35] O O Erejuwa S A Sulaiman and M S Ab Wahab ldquoMod-ulation of gut microbiota in the management of metabolicdisorders The prospects and challengesrdquo International Journalof Molecular Sciences vol 15 no 3 pp 4158ndash4188 2014

[36] P J Turnbaugh F Backhed L Fulton and J I Gordon ldquoDiet-induced obesity is linked to marked but reversible alterations inthe mouse distal gut microbiomerdquo Cell Host andMicrobe vol 3no 4 pp 213ndash223 2008

[37] J Qin J Wang Y Li et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[38] T Wang G Cai Y Qiu et al ldquoStructural segregation ofgut microbiota between colorectal cancer patients and healthyvolunteersrdquoThe ISME Journal vol 6 no 2 pp 320ndash329 2012

[39] K M Maslowski A T Vieira A Ng et al ldquoRegulation ofinflammatory responses by gutmicrobiota and chemoattractantreceptorGPR43rdquoNature vol 461 no 7268 pp 1282ndash1286 2009

[40] C De Filippo D Cavalieri M Di Paola et al ldquoImpact of dietin shaping gut microbiota revealed by a comparative studyin children from Europe and rural Africardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 33 pp 14691ndash14696 2010

[41] K Forslund FHildebrand TNielsen et al ldquoDisentangling type2 diabetes and metformin treatment signatures in the humangut microbiotardquo Nature vol 528 no 7581 pp 262ndash266 2015

[42] G L Hold A Schwiertz R I Aminov M Blaut and H J FlintldquoOligonucleotide probes that detect quantitatively significantgroups of butyrate-producing bacteria in human fecesrdquo Appliedand Environmental Microbiology vol 69 no 7 pp 4320ndash43242003

[43] N Becker J Kunath G Loh and M Blaut ldquoHuman intestinalmicrobiota characterization of a simplified and stable gnotobi-otic rat modelrdquo Gut Microbes vol 2 no 1 pp 25ndash33 2011

[44] S Nobaek M-L Johansson G Molin S Ahrne and BJeppsson ldquoAlteration of intestinal microflora is associated withreduction in abdominal bloating and pain in patients withirritable bowel syndromerdquo The American Journal of Gastroen-terology vol 95 no 5 pp 1231ndash1238 2000

[45] G T Macfarlane and S Macfarlane ldquoHuman colonic micro-biota ecology physiology and metabolic potential of intestinalbacteriardquo Scandinavian Journal of Gastroenterology vol 32supplement 222 pp 3ndash9 1997

[46] E G Zoetendal A D L Akkermans W M Akkermans-van Vliet J A G M De Visser and W M De Vos ldquoThehost genotype affects the bacterial community in the humangastrointestinal tractrdquoMicrobial Ecology in Health and Diseasevol 13 no 3 pp 129ndash134 2001

[47] N-R Shin J-C Lee H-Y Lee et al ldquoAn increase in theAkkermansia spp population induced by metformin treatmentimproves glucose homeostasis in diet-induced obesemicerdquoGutvol 63 no 5 pp 727ndash735 2014

[48] C Reasner L Olansky T L Seck et al ldquoThe effect of initialtherapy with the fixed-dose combination of sitagliptin andmetformin compared with metforminmonotherapy in patientswith type 2 diabetesmellitusrdquoDiabetes Obesity andMetabolismvol 13 no 7 pp 644ndash652 2011

[49] B Ahren S L Johnson M Stewart et al ldquoHARMONY 3 104-week randomized double-blind placebo- and active-controlled

10 Journal of Diabetes Research

trial assessing the efficacy and safety of albiglutide comparedwith placebo sitagliptin and glimepiride in patients with type2 diabetes taking metforminrdquo Diabetes Care vol 37 no 8 pp2141ndash2148 2014

[50] S Enjoji T Ohama and K Sato ldquoRegulation of epithelial celltight junctions by protease-activated receptor 2rdquoThe Journal ofVeterinary Medical Science vol 76 no 9 pp 1225ndash1229 2014

[51] N Wronkowitz S W Gorgens T Romacho et al ldquoSolu-ble DPP4 induces inflammation and proliferation of humansmoothmuscle cells via protease-activated receptor 2rdquoBiochim-ica et Biophysica Acta vol 1842 no 9 pp 1613ndash1621 2014

Journal of Diabetes Research 9

[17] C S Marathe C K Rayner K L Jones and M HorowitzldquoGlucagon-like peptides 1 and 2 in health and disease a reviewrdquoPeptides vol 44 pp 75ndash86 2013

[18] P B Eckburg E M Bik C N Bernstein et al ldquoMicrobiologydiversity of the human intestinal microbial florardquo Science vol308 no 5728 pp 1635ndash1638 2005

[19] S R Gill M Pop R T DeBoy et al ldquoMetagenomic analysis ofthe human distal gut microbiomerdquo Science vol 312 no 5778pp 1355ndash1359 2006

[20] Y S Zhou B Li et al ldquoA rat model of type 2 diabetes mellitusinduced by high fat chow and low dose streptozocin injectionrdquoActa Laboratorium Animals Scientia Sinica vol 13 no 3 pp154ndash158 2005

[21] Y Jialin L Guo L Youpin et al ldquoEstablishing a rat modelsimilar to the adult patient of the general type 2 diabetes by long-term fat-enriched fed and lower dose of streptozocin-treatedratsrdquo Acta Laboratorium Animals Scientia Sinica vol 11 no 3pp 138ndash141 2003

[22] K R Amato C J Yeoman A Kent et al ldquoHabitat degradationimpacts black howler monkey (Alouatta pigra) gastrointestinalmicrobiomesrdquo ISME Journal vol 7 no 7 pp 1344ndash1353 2013

[23] P D Schloss S L Westcott T Ryabin et al ldquoIntroduc-ing mothur open-source platform-independent community-supported software for describing and comparing microbialcommunitiesrdquoApplied and EnvironmentalMicrobiology vol 75no 23 pp 7537ndash7541 2009

[24] G R Warnes B Bolker L Bonebakker et al gplots Various RProgramming Tools for Plotting Data Version 2170 2015

[25] J R White N Nagarajan and M Pop ldquoStatistical methods fordetecting differentially abundant features in clinical metage-nomic samplesrdquo PLoS Computational Biology vol 5 no 4Article ID e1000352 2009

[26] Y L Wu G X Xi et al ldquoAn experimental study of impairedglucose tolerance in Wistar ratsrdquo Chinese Remedies amp Clinicsvol 7 no 4 pp 281ndash283 2007

[27] M Cotta and R Forster ldquoThe family lachnospiraceae includingthe genera butyrivibrio lachnospira and roseburiardquo in TheProkaryotes Volume 4 Bacteria Firmicutes Cyanobacteria pp1002ndash1021 Springer Berlin Germany 3th edition 2006

[28] J M W Wong R de Souza C W C Kendall A Emam andD J A Jenkins ldquoColonic health fermentation and short chainfatty acidsrdquo Journal of Clinical Gastroenterology vol 40 no 3pp 235ndash243 2006

[29] H J Flint K P Scott S H Duncan P Louis and E ForanoldquoMicrobial degradation of complex carbohydrates in the gutrdquoGut Microbes vol 3 no 4 pp 289ndash306 2012

[30] X Ze S H Duncan P Louis and H J Flint ldquoRuminococcusbromii is a keystone species for the degradation of resistantstarch in the human colonrdquo ISME Journal vol 6 no 8 pp 1535ndash1543 2012

[31] R E Ley ldquoObesity and the human microbiomerdquo CurrentOpinion in Gastroenterology vol 26 no 1 pp 5ndash11 2010

[32] S H Duncan G E Lobley G Holtrop et al ldquoHuman colonicmicrobiota associated with diet obesity and weight lossrdquoInternational Journal of Obesity vol 32 no 11 pp 1720ndash17242008

[33] V Mai Q M McCrary R Sinha and M Glei ldquoAssociationsbetween dietary habits and body mass index with gut micro-biota composition and fecal water genotoxicity an observa-tional study in African American and Caucasian Americanvolunteersrdquo Nutrition Journal vol 8 article 49 2009

[34] R Jumpertz D S Le P J Turnbaugh et al ldquoEnergy-balancestudies reveal associations between gut microbes caloric loadand nutrient absorption in humansrdquo The American Journal ofClinical Nutrition vol 94 no 1 pp 58ndash65 2011

[35] O O Erejuwa S A Sulaiman and M S Ab Wahab ldquoMod-ulation of gut microbiota in the management of metabolicdisorders The prospects and challengesrdquo International Journalof Molecular Sciences vol 15 no 3 pp 4158ndash4188 2014

[36] P J Turnbaugh F Backhed L Fulton and J I Gordon ldquoDiet-induced obesity is linked to marked but reversible alterations inthe mouse distal gut microbiomerdquo Cell Host andMicrobe vol 3no 4 pp 213ndash223 2008

[37] J Qin J Wang Y Li et al ldquoA metagenome-wide associationstudy of gut microbiota in type 2 diabetesrdquoNature vol 490 no7418 pp 55ndash60 2012

[38] T Wang G Cai Y Qiu et al ldquoStructural segregation ofgut microbiota between colorectal cancer patients and healthyvolunteersrdquoThe ISME Journal vol 6 no 2 pp 320ndash329 2012

[39] K M Maslowski A T Vieira A Ng et al ldquoRegulation ofinflammatory responses by gutmicrobiota and chemoattractantreceptorGPR43rdquoNature vol 461 no 7268 pp 1282ndash1286 2009

[40] C De Filippo D Cavalieri M Di Paola et al ldquoImpact of dietin shaping gut microbiota revealed by a comparative studyin children from Europe and rural Africardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 33 pp 14691ndash14696 2010

[41] K Forslund FHildebrand TNielsen et al ldquoDisentangling type2 diabetes and metformin treatment signatures in the humangut microbiotardquo Nature vol 528 no 7581 pp 262ndash266 2015

[42] G L Hold A Schwiertz R I Aminov M Blaut and H J FlintldquoOligonucleotide probes that detect quantitatively significantgroups of butyrate-producing bacteria in human fecesrdquo Appliedand Environmental Microbiology vol 69 no 7 pp 4320ndash43242003

[43] N Becker J Kunath G Loh and M Blaut ldquoHuman intestinalmicrobiota characterization of a simplified and stable gnotobi-otic rat modelrdquo Gut Microbes vol 2 no 1 pp 25ndash33 2011

[44] S Nobaek M-L Johansson G Molin S Ahrne and BJeppsson ldquoAlteration of intestinal microflora is associated withreduction in abdominal bloating and pain in patients withirritable bowel syndromerdquo The American Journal of Gastroen-terology vol 95 no 5 pp 1231ndash1238 2000

[45] G T Macfarlane and S Macfarlane ldquoHuman colonic micro-biota ecology physiology and metabolic potential of intestinalbacteriardquo Scandinavian Journal of Gastroenterology vol 32supplement 222 pp 3ndash9 1997

[46] E G Zoetendal A D L Akkermans W M Akkermans-van Vliet J A G M De Visser and W M De Vos ldquoThehost genotype affects the bacterial community in the humangastrointestinal tractrdquoMicrobial Ecology in Health and Diseasevol 13 no 3 pp 129ndash134 2001

[47] N-R Shin J-C Lee H-Y Lee et al ldquoAn increase in theAkkermansia spp population induced by metformin treatmentimproves glucose homeostasis in diet-induced obesemicerdquoGutvol 63 no 5 pp 727ndash735 2014

[48] C Reasner L Olansky T L Seck et al ldquoThe effect of initialtherapy with the fixed-dose combination of sitagliptin andmetformin compared with metforminmonotherapy in patientswith type 2 diabetesmellitusrdquoDiabetes Obesity andMetabolismvol 13 no 7 pp 644ndash652 2011

[49] B Ahren S L Johnson M Stewart et al ldquoHARMONY 3 104-week randomized double-blind placebo- and active-controlled

10 Journal of Diabetes Research

trial assessing the efficacy and safety of albiglutide comparedwith placebo sitagliptin and glimepiride in patients with type2 diabetes taking metforminrdquo Diabetes Care vol 37 no 8 pp2141ndash2148 2014

[50] S Enjoji T Ohama and K Sato ldquoRegulation of epithelial celltight junctions by protease-activated receptor 2rdquoThe Journal ofVeterinary Medical Science vol 76 no 9 pp 1225ndash1229 2014

[51] N Wronkowitz S W Gorgens T Romacho et al ldquoSolu-ble DPP4 induces inflammation and proliferation of humansmoothmuscle cells via protease-activated receptor 2rdquoBiochim-ica et Biophysica Acta vol 1842 no 9 pp 1613ndash1621 2014

10 Journal of Diabetes Research

trial assessing the efficacy and safety of albiglutide comparedwith placebo sitagliptin and glimepiride in patients with type2 diabetes taking metforminrdquo Diabetes Care vol 37 no 8 pp2141ndash2148 2014

[50] S Enjoji T Ohama and K Sato ldquoRegulation of epithelial celltight junctions by protease-activated receptor 2rdquoThe Journal ofVeterinary Medical Science vol 76 no 9 pp 1225ndash1229 2014

[51] N Wronkowitz S W Gorgens T Romacho et al ldquoSolu-ble DPP4 induces inflammation and proliferation of humansmoothmuscle cells via protease-activated receptor 2rdquoBiochim-ica et Biophysica Acta vol 1842 no 9 pp 1613ndash1621 2014