Post on 17-Apr-2018
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Supplementary Figures
Supplementary Fig. 1 Overview of workflow for the study design Integrating gut metagenomics data, human phenotypes, human serum metabolome, mouse feeding experiments and intervention data. a, Metagenomics data were aligned to the integrated gene catalog and 350524 obesity-associated genes (p<0.01) were grouped into 217 metagenomic linkage groups (MLGs) through the co-abundance patterns. b, 217 MLGs were filtered for best fitting each phenotype by random forest regression combined with spearman correlation. c, Gut microbiome functional dysbiosis were identified using reporter score. d, The difference of host circulating metabolites between control and obese samples were indicated by non-targeted metabolomics profile and validated by the targeted amino acid profile, which further associated with the MLGs and phenotypes. e, Experimental design for B. thetaiotaomicron gavage in mice. d, Characterization of the sleeve gastrectomy samples at the different levels mentioned above. CD, chow diet; HFD, high-fat diet; 0M, samples before surgery; 1M, one month after surgery; 3M, three months after surgery.
fedcba Weight loss interventionMouse experimentHost circulating metabolitesFunction level analysisAssociate with phenotypesMGWAS
MLG level analysis
Phenotype level analysisFunction level analysisAmino acids analysis
Gene level analysis
3M=23
1M=17
0M=23
Faeces and serum collecction
HFD
+B.thetaiotaomicron
HFD
+killed B.thetaiotaomicron
HFD
+PBSC
D+B.thetaiotaom
icronC
D+killed B.thetaiotaom
icronC
D+PBS
Week 6
Week 5
Week 4
Week 2
Week 3
Week 1
Case control72 OB vs 79 ControlValidation23 OB vs 26 Control *
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BMI
Hip circumference
Fasting insulin
HOMA-IR
Leptin
HbA1c
Whole body fat
FBG
HDL-C
Adiponectin
Dialister invisus (28421)
Klebsiella pneumoniae (12774)
Clostridiales bacterium (16594)
Bacteroides ovatus (11080)Haem
ophilus parainfluenzae (12511)Veillonella sp. oral taxon (12639)Bacteroides intestinalis (211)Faecalibacterium
prausnitzii (13388)Bacteroides thetaiotaom
icron (11069)
Dorea longicatena (7570)Coprococcus com
es (6940)Eubacterium
hallii (7132)Rum
inococcus obeum (7058)
Dorea longicatena (9343)
Aligned to 9.9M gene catalog
Present at least 6 samples
3M genes
p<0.01
350525 genes
Co-abundance
217 MLGs
Random Forest regress (RF)with BMI
Bacteroides thetaiotaom
icron (11069)B
acteroides intestinalis (2808)Faecalibacterium
prausnitzii (13388)E
ubacterium hallii (7132)
Rum
inococcus obeum (7058)
Dorea longicatena (9343)
Con−1216
OB−8293
... MLG
-216M
LG-217
...
HDL-C
41 phenotype
Permanova with 217MLGAdjusted p<0.05
28 phenotype
26 M
LGs
...
26 RF-selected MLGs to 28 Permanova-significanted phenotypes associations(combine RF regression and spearman correlation)
KEGG Orthology
Reporter score
87 dysbiosis module
34 dysbiosis pathway
5705 KOsnon-targeted metabolomics profiling
HPLC-MS VIP>1Fold change<0.8 or >1.2Adjusted p<0.05
148 metabolites
HMDB databaseKEGG database
Glutam
ate
Glycyl-Valine
Gam
ma-G
lutamyltyrosine
Glutam
ylphenylalanine
L-Aspartyl-L-phenylalanine
Creatine
Phenylalanine
Tyrosine
13 metabolites
...
UH
PLC-M
STargeted am
ino acid profiling
34 amino acids
GlutamatePhenylalanineTyrosineBCAA
20 amino acids
Adjusted p<0.05
SpearmanGlutamine+
217
MLG
s
Spearman
Phenotype+
weeks -3
weeks 0
FDR in this cutoff=0.047
Gene numbers>100Adjusted p<0.05
Faeces collection Chow diet
gavagegavagegavage
gavagegavagegavage
gavagegavagegavage
gavagegavagegavage
gavagegavagegavage
gavagegavagegavage
Baseline body weight
Sleeve gastrectomy
Week 7
weeks -1HFD
HFD
HFD
Chow
Chow
Chow
gavagegavagegavage
-2 0 2 4 6 8 10 -2 0 2 4
In-house database MS/MS alignment
Nature Medicine: doi:10.1038/nm.4358
2
Supplementary Fig. 2 Identification of obesity-associated gene markers in the gut metagenome a, Distribution of gene counts from all matched reads (top panel) or adjusted to 11 million matched reads per individual (bottom panel). b, Density histogram showing the p-value distribution of all genes tested in the training samples (control, n=79; obese, n=72, two-tailed Wilcoxon rank-sum test). c, Principle component analysis (PCA) for the stool samples with 350524 gene markers. Colored circles covered the individuals near the center of gravity for each cluster (<1.5σ). d, e, Density histogram showing the occurrence rate of control-enriched (d) or obesity-enriched gene markers (e) in 151 individuals.
200,000 400,000 600,000 800,000 1,000,000
0.0
e+
00
1.5e−06
3.0e−06
Gene distribution
Gene number
Density
All
Control
Obese
0.0
e+
00
1.5e−06
3.0e−06
Density
All
Control
Obese
Gene number (11 million reads)
Density
0.0 0.2 0.4 0.6 0.8 1.0
01
23
45
obese
control
PC1 13.1%
PC
2 8.1
%
Distribution of p value
Occurrence rate(n=151)
Density
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Occurrence rate(n=151)
Density
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Control-enriched gene markers OB-enriched gene markers
a b c
d e
Nature Medicine: doi:10.1038/nm.4358
3
Supplementary Fig. 3 Phyla exhibiting different abundance in lean controls (n=79) and obese patients (n=72). a, left, the five most abundant phyla are shown; right, comparison of the Bacteroidetes/Firmicutes ratio. Blue and red color represent control- and obesity- enriched phyla, respectively; ‘*’, p < 0.05. b, c, Genera (b) and species (c) that were differentially enriched in lean controls and obese patients (p < 0.01). Two-tailed Wilcoxon rank-sum test was used to determine significance. In all box plots, boxes represent the interquartile ranges (IQRs) between the first and third quartiles, and the line inside the box represents the median; whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. Circles represent data point beyond the whiskers.
Fusobacteria
ActinobacteriaFirmicutes
ProteobacteriaBacteroidetes
−12
−10 −8 −6 −4 −2 0
ControlOB
SebaldellaIlyobacterOlsenella
SlackiaMitsuokella
GranulicatellaSolobacterium
ActinomycesGemella
LactobacillusAcidaminococcus
FusobacteriumMegasphaera
CollinsellaCoprococcus
DoreaRuminococcus
BlautiaThermincolaMannheimiaMesoplasma
GordoniaLeptospira
NitrosomonasCupriavidus
ActinoplanesHeliobacterium
SphingopyxisDesulfitobacterium
DelftiaKingella
PelotomaculumThermoanaerobacter
AcidovoraxActinobacillus
ThermoanaerobacteriumAcetivibrioVictivallisNeisseria
AggregatibacterAkkermansiaOxalobacterOscillibacter
HaemophilusHoldemania
AnaerotruncusOdoribacter
Alistipes
−20
−15
−10 −5
ControlOB
Significant different genera
Coriobacterium glomeransStreptococcus downei
Streptococcus pseudopneumoniaeFusobacterium varium
Fusobacterium ulceransunclassified Streptococcus oralis
Fusobacterium mortiferumClostridium kluyveriCollinsella stercoris
Collinsella intestinalisClostridium spiroformeCollinsella aerofaciens
Eubacterium halliiDorea longicatena
[Ruminococcus] gnavusDorea formicigenerans
Coprococcus comes[Ruminococcus] torques
unclassified Ruminococcus sp.Mannheimia haemolyticaSerratia proteamaculans
Actinobacillus pleuropneumoniaeAggregatibacter actinomycetemcomitans
Neisseria meningitidisNeisseria cinerea
Porphyromonas endodontalisNeisseria sicca/macacae
Oscillibacter valericigenesNeisseria subflavaNeisseria mucosa
Aggregatibacter aphrophilusPorphyromonas gingivalisClostridiales genomo sp.Akkermansia muciniphila
Aggregatibacter segnisPrevotella denticola
Clostridium hylemonaePorphyromonas asaccharolytica
unclassified Lachnospiraceae bacteriumunclassified Alistipes sp.
Eubacterium dolichumBacteroides helcogenes
Alistipes shahiiOdoribacter splanchnicus
unclassified Erysipelotrichaceae bacteriumBacteroides thetaiotaomicron
Bacteroides ovatusEnterococcus faecium
Bacteroides xylanisolvensFaecalibacterium prausnitzii
Prevotella orisAlistipes putredinis
Bacteroides uniformis
−20
−15
−10 −5
ControlOB
a
b
*
*
Abundance(lg)
Top five abundant phyla
Significante diferent species
Abundance(lg) Abundance(lg)
P-value=0.062
02
46
810
BFr
atio
(Bac
tero
idet
es /
Firm
icut
es)
Control OB
*
c
Nature Medicine: doi:10.1038/nm.4358
4
Supplementary Fig. 4 Validation of the enrichment of 26 BMI-associated MLGs in the test set (control, n=26; obese, n=23). Blue and red color represent control- and obesity- enriched MLGs, respectively; ‘&’ denotes MLGs with p < 0.05 in both 151 samples and 49 samples (two-tailed Wilcoxon rank-sum test). In all box plots, boxes represent the interquartile ranges (IQRs) between the first and third quartiles, and the line inside the box represents the median; whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. Circles represent data point beyond the whiskers.
−30
−25
−20
−15
−10
Dialister invisus(28421)Klebsiella pneumoniae(12774)Clostridiales bacterium(16594)
Faecalibacterium prausnitzii(13264)Bacteroides ovatus(11116)Bacteroides ovatus(11080)
Haemophilus parainfluenzae(12502)Haemophilus parainfluenzae(12511)
Veillonella sp. oral taxon(12639)Con−1216
Bacteroides intestinalis(173)Bacteroides intestinalis(243)Bacteroides intestinalis(211)
Bacteroides intestinalis(2808)Faecalibacterium prausnitzii(13388)
Bacteroides thetaiotaomicron(11069)OB−8283OB−7834
Dorea longicatena(7570)Coprococcus comes (6940)
Eubacterium hallii(7132)Ruminococcus obeum(7058)
Dorea longicatena(9343)OB−8241OB−8295OB−8293
−30
−25
−20
−15
151 samples 49 samples
MLG abundance (lg) MLG abundance (lg)
&&
&
&
&
&
&
&&
&
&
&
&&
&
Control OBControl OB
Nature Medicine: doi:10.1038/nm.4358
5
Supplementary Fig. 5 Functional characterization of the obese microbiome The relative abundances of KEGG pathways (a) and modules (b) were compared between obese patients and controls (control, n=79; obese, n=72). KEGG pathways or modules with reporter score >1.65 or < -1.65 are shown. Blue and red color represent control- and obesity- enriched pathways or modules, respectively.
Glycosaminoglycan degradationBenzoate degradation
Fluorobenzoate degradationValine, leucine and isoleucine degradation
N−Glycan biosynthesisBiosynthesis of siderophore group nonribosomal peptides
Riboflavin metabolismLipoic acid metabolism
Citrate cycle (TCA cycle)Aminobenzoate degradation
Flagellar assemblyPyruvate metabolism
Carbon fixation pathways in prokaryotesmRNA surveillance pathway
Folate biosynthesisBetalain biosynthesis
Amino sugar and nucleotide sugar metabolismPeptidoglycan biosynthesis
Fructose and mannose metabolismGlycerolipid metabolism
ABC transportersStarch and sucrose metabolism
Lipopolysaccharide biosynthesisC5−Branched dibasic acid metabolism
Chloroalkane and chloroalkene degradationPhenylalanine, tyrosine and tryptophan biosynthesis
Thiamine metabolismRibosome
Sulfur relay systemTerpenoid backbone biosynthesis
Selenocompound metabolismPorphyrin and chlorophyll metabolism
Galactose metabolismPhosphotransferase system (PTS)
−2 0 2 4 6 8 10
−2 0 2 4
Pathway reporter score
Module reporter scorea b
Methanogenesis, formate => methaneMethanogenesis, acetate => methane
Arginine/ornithine transport systemPyruvate oxidation, pyruvate => acetyl−CoA
Complex I (NADH dehydrogenase), NADH dehydrogenase IChondroitin sulfate degradation
Manganese/iron transport systemHeparan sulfate degradation
2−oxoisovalerate:ferredoxin oxidoreductaseHistidine degradation, histidine => N−formiminoglutamate => glutamate
Leucine degradation, leucine => acetoacetate + acetyl−CoAEthylmalonyl pathway
N−glycan precursor biosynthesisMenaquinone biosynthesis, chorismate => menaquinone
GPI−anchor biosynthesis, core oligosaccharideindolepyruvate:ferredoxin oxidoreductase
Dermatan sulfate degradationManganese/zinc/iron transport system
2−Aminoethylphosphonate transport systemKeratan sulfate degradation
beta−Carotene biosynthesis, GGAP => beta−caroteneReductive citric acid cycle (Arnon−Buchanan cycle)
Non−phosphorylative Entner−Doudoroff pathway, gluconate => glyceraldehyde + pyruvatePhosphonate transport system
Cysteine biosynthesis, serine => cysteineAminoacyl−tRNA biosynthesis, eukaryotes
Iron(III) transport systemalpha−Hemolysin/cyclolysin transport system
RNA polymerase, archaeaType VI secretion system
Complex IV (Cytochrome c oxidase), cytochrome c oxidase, cbb3−type2−oxoglutarate:ferredoxin oxidoreductase
Ribosome, eukaryotesCAM (Crassulacean acid metabolism), light
Citrate cycle, second carbon oxidationAscorbate biosynthesis, animals, glucose−1P => ascorbate
C4−dicarboxylic acid cycle, NADP+ −malic enzyme typeHemophore/metalloprotease transport system
Pyridoxal biosynthesis, erythrose−4P => pyridoxal−5POctopine/nopaline transport system
Phosphatidylcholine (PC) biosynthesis, PE => PCMethane oxidation, methylotroph, methane => CO2
Adhesin protein transport systemC5 isoprenoid biosynthesis, mevalonate pathway
PTS system, arbutin−, cellobiose−, and salicin−specific II componentD−Methionine transport system
Ribose transport systemGlutamine transport system
Methionine biosynthesis, apartate => homoserine => methionineMelatonin biosynthesis, tryptophan => serotonin => melatonin
PTS system, ascorbate−specific II componentMaltose/maltodextrin transport system
Putative sugar transport systemPTS system, fructose−specific II−like component
Shikimate pathway, phosphoenolpyruvate + erythrose−4P => chorismateCitrate cycle, first carbon oxidation
PTS system, mannitol−specific II componentPTS system, galactosamine−specific II component
PTS system, trehalose−specific II componentGlutathione transport systemMolybdate transport system
V−type ATPase, prokaryotesPTS system, beta−glucosides−specific II component
Thiamine biosynthesis, AIR => thiamine−P/thiamine−2PPTS system, galactitol−specific II component
PTS system, maltose and glucose−specific II componentHeme biosynthesis, glutamate => protoheme/siroheme
PTS system, fructose−specific II componentHistidine biosynthesis, PRPP => histidine
Type IV secretion systemNitrogen fixation, N2 => ammonia
Cobalt transport systemATP synthase
F−type ATPase, bacteriaLeucine biosynthesis, pyruvate => 2−oxoisovalerate => leucine
N−Acetylglucosamine transport systemPTS system, glucitol/sorbitol−specific II component
Putative ABC transport systemPTS system, lactose−specific II component
Glutamate transport systemCobalamin biosynthesis, cobinamide => cobalamin
PTS system, N−acetylgalactosamine−specific II componentPTS system, cellobiose−specific II component
PTS system, arbutin−like II componentRibosome, bacteria
PTS system, sucrose−specific II componentPTS system, mannose−specific II component
Nature Medicine: doi:10.1038/nm.4358
6
Supplementary Fig. 6 Metabolomics profiling of serum from controls and obese patients. a, PCA scores plots constructed with 3782 metabolites on 151 samples (orange points) and 67 QC samples (blue points). b, PLS-DA score plots constructed with 3782 metabolites on 72 obese samples (red points) and 79 control samples (blue points); c, Statistical validation of the PLS-DA using permutation analysis (1000 times). Comparison of explained variation (R2) between the true value (R2=0.636) and the R2
distribution (between [0.232, 0.483]) in 1000 times permutation. d, e, PCA scores plots on 151 samples (d) and another set of 35 samples (e) using 148 metabolites that differed in abundance between 72 obese samples and 79 control samples (online methods).
PC1
PC2
0−12−24−36−48−60 12 24 36 48 60
0−9
−18−27−36−45−54
9182736 Samples
QC
PC1(5.856%)
PC
2(6.
6369
%)
0−20−40−60 20 40 60 80 100 120
−20−40−60−80
−100−120
0204060 Control
OB
ControlOB
ControlOB
R2
Frequency
0.2 0.4 0.6 0.8
020
4060
80
PCA of 151 samples based on 148 metabolites
PC1(18.4701%)
PC
2(5.
9218
%)
0−10 10
0
−10
10
PCA of 35 samples based on 148 metabolites
PC1(22.1605%)
PC
2(7.
9263
%)
0−10 10
0
−10
10
a b c
d e
Nature Medicine: doi:10.1038/nm.4358
7
Supplementary Fig. 7 Metabolites that differed in abundance between lean controls and obese patients. a, Heat map and hierarchical clustering of 148 metabolites exhibiting significant differences in the abundance in 151 samples. Blue and red row annotations in the left denote metabolites enriched in lean controls and obese patients, respectively (online methods). b, Comparison of the abundance of identified metabolites between lean controls and obese patients. All indicated metabolites differed significantly in abundance in the 151 samples (control, n=79; obese, n=72, two-tailed Wilcoxon rank-sum test, adjusted p < 0.05, left panel), and ‘*’ denotes metabolites that also differed significantly in abundance in the 35 samples (control, n=25; obese, n=10, p < 0.05, right panel). c, Targeted metabolomics profiling of amino acids in the serum of 139 samples. Amino acids in blue and red denote amino acids significantly enriched in lean controls and obese patients, respectively (control, n=73; obese, n=66, two-tailed Wilcoxon rank-sum test, adjusted p<0.05). Ala-leu, Alanyl-leucine; 3-m-his, 3-Methyl-histidine; GABA, γ -aminobutyric acid; AADA, 2-aminoadipic acid; 1-m-his, 1-Methyl-histidine; AABA, α -aminoisobutyric acid. In all box plots, boxes represent the interquartile ranges (IQRs) between the first and third quartiles, and the line inside the box represents the median; whiskers
−6 −4 −2 0 2 4 6
ControlOB
−3 −2 −1 0 1 2 3 4
ControlOB
151 samples 35 samples
Abundance (lg)
Pipecolate
*1-Oleoylglycerophosphocholine
1-Palmitoylglycerophosphocholine
*17-Hydroxyprogesterone
*Urate
Creatine
*Gamma-Glutamyltyrosine
*Glutamylphenylalanine
L-Aspartyl-L-phenylalanine
Glycyl-Valine
*Phenylalanine
*Tyrosine
*Glutamate
Abundance (lg)
−10
−5
0
5
10
Heatmap of 148 metabolites
Metabolites
Sam
ples
Abun
danc
e
m/z 239.1636859m/z 520.3895842m/z 417.3358141m/z 130.0861253m/z 472.2387049m/z 547.3463815m/z 460.2812805m/z 333.2027954m/z 335.2184098m/z 540.3651944m/z 522.3530986m/z 525.3615259m/z 548.2988509m/z 539.3113102m/z 520.337815m/z 523.3465562m/z 547.2973235m/z 542.3200207m/z 543.3235097m/z 129.065683m/z 456.4038228m/z 540.3043567m/z 476.2757932m/z 518.3225725m/z 198.0847424m/z 220.0665549m/z 480.3075641m/z 481.3109385m/z 500.2735705m/z 480.2974119m/z 337.2727022m/z 478.29167m/z 290.1495844m/z 520.3715605m/z 247.0162878m/z 288.1339592m/z 464.2168213m/z 585.2693431m/z 557.3482532m/z 235.130051m/z 160.1329969m/z 248.1472669m/z 132.0764161m/z 233.1168861m/z 247.1321566m/z 151.0748623m/z 265.1427707m/z 353.2654424m/z 533.2974337m/z 333.2453843m/z 317.2504276m/z 366.2624411m/z 548.369626m/z 397.3088834m/z 445.3296172m/z 190.0895414m/z 169.0490706m/z 170.0525581m/z 346.180077m/z 359.2977436m/z 218.211018m/z 302.3046227m/z 303.30807m/z 461.3212264m/z 737.6179902m/z 212.9993731m/z 467.1772696m/z 580.2604602m/z 331.2257717m/z 169.0354551m/z 197.0805477m/z 473.1848407m/z 473.1849181m/z 505.1403354m/z 166.0861034m/z 281.112688m/z 313.1537335m/z 315.1604544m/z 94.04117103m/z 336.1393031m/z 131.0490501m/z 147.0438715m/z 182.0806541m/z 245.1491627m/z 393.1754385m/z 153.0655705m/z 189.1343547m/z 220.1177968m/z 347.2206109m/z 159.0276007m/z 137.0454626m/z 291.0695123m/z 339.1817768m/z 243.1223484m/z 352.1803337m/z 201.0907619m/z 315.1558712m/z 375.295025m/z 329.2464842m/z 346.2729756m/z 153.0405383m/z 616.1743381m/z 365.1048923m/z 329.2313835m/z 351.2132387m/z 496.3406009m/z 393.2235578m/z 499.3460994m/z 687.4872517m/z 567.8866747m/z 590.4015161m/z 520.2598131m/z 444.3676873m/z 452.2757906m/z 207.050528m/z 381.0228226m/z 188.0551862m/z 148.0602313m/z 271.9647307m/z 311.1232352m/z 261.1441747m/z 295.1283692m/z 296.1318007m/z 269.1377315m/z 181.0854463m/z 263.0884035m/z 242.1098546m/z 255.1221576m/z 256.1255147m/z 420.2630139m/z 279.0985922m/z 287.2209906m/z 360.2399973m/z 524.4477933m/z 352.2238785m/z 387.2519731m/z 388.2553945m/z 506.5063445m/z 472.3409446m/z 474.3569121m/z 175.1076445m/z 327.2308414m/z 379.2808484m/z 357.2987744m/z 377.2651074m/z 593.3325943m/z 453.2384303m/z 591.3161836
a
b
−6 −4 −2
0 2 4
Amino acid
Abundance(lg)
ControlOB
CysteineCystine
Histamine3-m-his
HomoserAla-leu
CitrullineMethionineAsparagine
ThreonineHistidine
Serine
GABAGlutamine
Glycine
AADA
AABAEthanolamine
1-m-hisβ-alanine
AspartateTryptophan
ProlineArginineLeucine
PhenylalanineOrnithine
Hydroxyproline
AlanineGlutamateIsoleucine
ValineLysine
Tyrosine
c
Nature Medicine: doi:10.1038/nm.4358
8
represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. Circles represent data point beyond the whiskers.
Supplementary Fig. 8 Co-variation of MLG markers and circulating metabolites a, Co-inertia analysis (CIA) of the relationships between 217 MLGs and 148 metabolites. Each sample is represented with an arrow. Blue and red color represent lean controls and obese patients, respectively. The sample projections in MLG and metabolite space are represented by the starting point and the end of the arrow, respectively. b, Canonical correlation analysis (CCA) revealing the association between 20 amino acids and 217 MLGs that differed significantly in abundance between cases and controls (see supplementary Table 16 and 4). Blue and red points represent control-enriched and obesity-enriched MLGs, respectively. AADA, 2-aminoadipic acid; AABA, α-aminoisobutyric acid.
−2 −1 0 1 2
−20
24
6
Axis1(53.13%)
Axi
s2(5
.39%
)
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−2 0 2 4
−20
24
68
10
CCA1 5%
CC
A2
2.7%
Asparagine
Histamine
Arginine
Glutamine
Glycine Ethanolamine
Aspartate
Citrulline
Glutamate
β-alanine
AlanineAADA
AABA
Cystine
lysine
Tyrosine
ValineLeucine
Isoleucine
Phenylalanine
CIA of MLG and metabolites (p=0.001)
a b
Nature Medicine: doi:10.1038/nm.4358
9
Supplementary Fig. 9 Associations of clinical indices with circulating concentration of amino acids Spearman’s rank correlation coefficient between 35 clinical indices and 20 amino acids that differed significantly in abundances between cases and controls (see supplementary Table 1 and 16). 139 samples were used for Spearman’s rank correlation, ‘+’ denotes adjusted p < 0.05; ‘*’denotes adjusted p < 0.01. AADA, 2-aminoadipic acid; AABA, α-aminoisobutyric acid.
* * + * * * * * * * * * * * *
* * * * * * * * * * * * *
+ + +* * * * * * * * + *
* * * * * * + * + *
* * * +
+ + * + +
+ * + * * * + * + + *
+ * * * * * + * *
+ + + * * * * * + + + * * * *
+ * * * * * + * * * * +
+ * + * * * * * * + + * * +
+ * * * + * * * * +
+ * * * * * * * * * *
* * * * * + * * * + * * *
* + * * * * * * + + *
* * * * * * * * * * + * + + * + *
* * * * * * * * * * + * * * + *
+ * * * * * * * * * * * * * *
* + * + * * * + + + + * + +* + + * * * * * + * * * + + +* * + * * * * * * * * + * +* * + * * * * * + + * + + * *
* * * * + * * * * * * * + + * *
* * * * + * * * * * * * + + * *
* * * * + * * * * + * * * * * + * + *
* * * * + * * * * * * * * * * * + *
* * * * * * * * * + * * * + + * * *
* * + * * * * * * * * * * * * * *
* * * * * * * * * * * * + * * *
+ * * * + * * * * * * * * * * * *
* * + * * * * * * + * * * * * * * *
* * + * * * * * + * * * + * * * *
* * * * * * * * + * * * * * * * * *
* * + * * * * * * + * * * * * * * *
−0.5 0 0.5
Spearman’s correlation
AdiponectinHDL−CTotal bilirubinFIAFALPTNFαHeart rate Fasting plasma glucoseIL−6HbA1c2−h plasma glucoseASTTotal cholesterolLDL−CWhole body fat WBCLeptinhsCRPUric acidDBPSBPHOMA−B2−h serum insulinFasting serum insulinHOMA−IRLBPHip circumferenceBMITriglyceridesGGTALTNeck circumferenceWeightWaist circumferenceWHR
Gly
cine
His
tam
ine
Glu
tam
ine
Citr
ullin
eAs
para
gine
Cys
tine
Aspa
rtate
Glu
tam
ate
Phen
ylal
anin
eAA
DA
AABA
Ar
gini
neIs
oleu
cine
Valin
eLe
ucin
eβ−
alan
ine
Etha
nola
min
eLy
sine
Alan
ine
Tyro
sine
Nature Medicine: doi:10.1038/nm.4358
10
Supplementary Fig. 10 KOs involved in glutamate metabolism that differed significantly in abundance between controls and obese patients. Distinct KOs assigned to enzymatic reactions involved in glutamate metabolism and harbored by the MLGs correlated with the levels of glutamate in Fig. 3. KOs in blue and red represent KOs significantly enriched in lean controls and obese patients, respectively (control, n=79; obese, n=72, two-tailed Wilcoxon rank-sum test, p<0.05). ‘$’ denotes the KOs that are involved in the conversion of glutamine-to-glutamate. In all box plots, boxes represent the interquartile ranges (IQRs) between the first and third quartiles, and the line inside the box represents the median; whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. Circles represent data point beyond the whiskers.
−12
−11
−10 −9 −8
Control
OB
K10206
K02500
K01956
K02501
K01657
K01658
K00821
K02232
K06215
K08681
K14260
K00832
K01951
K00811
K01580
$
$
$
$
$
$
$
$
Abundance(lg)
KOs involved in glutamate metabolism in 151 samples
Nature Medicine: doi:10.1038/nm.4358
11
Supplementary Fig. 11 Effects of B. thetaiotaomicron supplementation on metabolic parameters. a, Baseline body weight of three groups in normal chow diet are shown, n=12 for each group. b, The percentage of white adipose tissue mass relative to body weight was compared among the three groups fed the normal chow diet, n=12 for each group. c, Baseline body weight of three groups fed the high-fat diet, n=8 for each group. d, The percentage of white adipose tissue mass relative to body weight among the three groups fed the high-fat diet, n=8,8,7 for each group, respectively. e-f, plasma adiponectin (e) and leptin (f) in the three indicated groups fed the normal chow diet, n=12 for each group. g, Fasting blood glucose was measured in the three groups fed the normal chow diet, n=12 for each group. h, Fasting plasma insulin level was measured in the three groups fed the normal chow diet, n=9 for each group. i, Fasting blood glucose in the three groups fed the high-fat diet, n=8,8,7 for each group, respectively. j, Fasting plasma insulin levels of the three groups fed the high-fat diet, n=6, 5, 5 for each group, respectively. k-l, Representative western blotting of the expression of total and phosphorylated AKT in the three indicated groups with or without insulin stimulation for ten minutes of mice fed the normal chow diet (k) or the high-fat diet (l) (insulin: 1IU/kg and 1.5 IU/kg for normal chow diet mice and high-fat diet mice, respectively). Hsp90 was used as the internal control. Uncropped blots are shown in Supplementary Fig.15. n=3 western blots per condition. m-n, Food intake (m) and 24h fecal caloric content (n) were measured in the three groups fed the normal chow diet, n=6 (cages, two mice per cage) for each group. o-p, Food intake (o) and 24h fecal caloric content (p) of the three groups fed the high-fat diet, n=4 (cages,
PBS KBT LBT0
10
20
30
Base
line
body
wei
ght (
g)
CD
iWAT eWAT0.0
0.5
1.0
1.5
2.0
2.5
Tiss
ue w
eigh
t (%
of b
ody
wei
ght)
PBSKBTLBT
0.06
CD
PBS KBT LBT0
10
20
30
Base
line
body
wei
ght (
g)
HFD
iWAT eWAT0
2
4
6
Tiss
ue w
eigh
t (%
of b
ody
wei
ght)
PBSKBTLBT
**
***
HFD
PBS KBT LBT0
1
2
3
4
5Bl
ood
gluc
ose
(mm
ol/L
)
HFD
PBS KBT LBT0.0
0.1
0.2
0.3
Plas
ma
insu
lin (n
g/m
l)
HFD
PBS KBT LBT-0.2
0.0
0.2
0.4
0.6
0.8
Plas
ma
insu
lin (n
g/m
l)
CD
a b c d e f
g h i j k
m n o p
p-AKT
AKT
Hsp90
p-AKT
AKT
Hsp90
PBS KBT LBT0
2
4
6
Bloo
d gl
ucos
e (m
mol
/L)
CD
PBS KBT LBT0
1
2
3
4
5
Food
inta
ke (g
/mou
se/d
)
CD
PBS KBT LBT0
1
2
3
4
5
Food
inta
ke (g
/mou
se/d
)
HFD
l
PBS KBT LBT0
20
40
60
80
Plas
ma
adip
onec
tin (μ
g/m
l)
CD
0.09
0.06
PBS KBT LBT0
500
1000
1500
2000
Plas
ma
lept
in (p
g/m
l)
CD
PBS KBT LBT0
4
8
12
16
24h
feca
l cal
oric
cot
ent (
KJ)
CD
PBS KBT LBT0
2
4
6
8
24h
feca
l cal
oric
cot
ent (
KJ)
HFD
*
PBS KBT LBT PBS KBT LBTlnsulin - lnsulin +
lnsulin - lnsulin +
PBS KBT LBT PBS KBT LBT
Nature Medicine: doi:10.1038/nm.4358
12
two mice per cage) for each group. PBS, phosphate buffer saline, KBT, heated killed B. thetaiotaomicron, LBT, live B. thetaiotaomicron; iWAT, inguinal subcutaneous white adipose tissue; eWAT, epididymal white adipose tissue; CD, normal chow diet, HFD, high-fat diet. Significance between every two groups was calculated using two-tailed Student’s t-test. Data are shown as mean ± s.d. *, p <0.05; **, p <0.01; Error bars, s.d..
Nature Medicine: doi:10.1038/nm.4358
13
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−0.4 −0.2 0.0 0.2 0.4
−0.3
−0.2
−0.1
0.0
0.1
0.2
CD
PC1 45.3 %
PC
2 8
.6 %
●
●
●
PBS beforeKBT beforeLBT beforePBS afterKBT afterLBT after
●
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●
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−0.4 −0.3 −0.2 −0.1 0.0 0.1 0.2 0.3
−0.2
−0.1
0.0
0.1
HFD
PC1 54.4 %
PC
2 6
.9 %
Phylum CD
Rel
ativ
e ab
unda
nce
(%)
0
20
40
60
80
100
PBSKBTLBTPBSKBTLBT
PBSKBTLBTPBSKBTLBT
PBSKBTLBTPBSKBTLBT
PBSKBTLBTPBSKBTLBT
Phylum HFD
Rel
ativ
e ab
unda
nce
(%)
0
20
40
60
80
100BacteroidetesFirmicutesUnclassifiedProteobacteriaTM7CyanobacteriaActinobacteriaTenericutesVerrucomicrobiaDeferribacteres
BacteroidetesFirmicutesUnclassifiedProteobacteriaTM7CyanobacteriaActinobacteriaTenericutesVerrucomicrobiaDeferribacteres
●
●
●
PBS beforeKBT beforeLBT beforePBS afterKBT afterLBT after
a
b c
d
f
e
g
Genus CD
Rel
ativ
e ab
unda
nce
(%)
0
20
40
60
80
100
PBSKBTLBT
PBSKBTLBT
Parabacteroides Unclassified Oscillospira BlautiaTuricibacterCoprococcus AcinetobacterRuminococcusLactobacillusOthers(<0.5%)BacteroidesAllobaculumStenotrophomonasComamonasFlavobacteriumBilophila AnaeroplasmaSutterella StaphylococcusSphingobacteriumPrevotellaOdoribacterMycoplanaEscherichiaElizabethkingiaBacillusAkkermansia Achromobacter
Rel
ativ
e ab
unda
nce
(%)
0
20
40
60
80
100 ParabacteroidesUnclassifiedOscillospiraBlautiaTuricibacterCoprococcus AcinetobacterRuminococcusLactobacillusOthers(<0.5%)Bacteroides cAllobaculumStenotrophomonasComamonasFlavobacteriumBilophilaAnaeroplasmaSutterellaStaphylococcusSphingobacteriumPrevotellaOdoribacterMycoplanaEscherichia ElizabethkingiaBacillusAkkermansia Achromobacter
b;ca;c
a
a
Genus HFD
05
1015
2025
30BFr
atio
(Bac
tero
idet
es/F
irmic
utes
)
05
1015
2025
BFr
atio
(Bac
tero
idet
es/F
irmic
utes
)
aa
a
a;c
a;b
b
b;c
b;c
b;c
b
;b
P-value=0.03
Before After
Before After
Before After
Before After
After After
PBS KBT LBT0
2
4
6
8
10 CD***
***
PBS KBT LBT0
2
4
6
8
10***
***
HFD
Lg B
. the
taio
taom
icro
n/g
feca
l DN
A
Lg B
. the
taio
taom
icro
n/g
feca
l DN
A
Nature Medicine: doi:10.1038/nm.4358
14
Supplementary Fig. 12 The effects of B. thetaiotaomicron gavage on gut microbiota in mice. a, PCoA based on Unweighted UniFrac analysis on operational taxonomic units (OTUs). b-c, The relative abundance of microbial phyla (left) and the Bacteroidetes/Firmicutes ratio (right) in the indicated groups on the normal chow diet (b) and the high-fat diet (c), respectively. d-e, The relative abundance of microbial genera in the indicated groups fed the normal chow diet (d) or the high-fat diet (e), respectively. f-g, qPCR detection of B. thetaiotaomicron in fecal DNA of the three indicated groups fed the normal chow diet (f) or the high-fat diet (g), respectively. PBS, gavage with phosphate buffer saline, n=8; KBT, gavage with heated killed B. thetaiotaomicron, n=8; LBT, gavage with B. thetaiotaomicron, n=7; before, before the experiment; after, seven weeks after the experiment. CD, normal chow diet, HFD, high-fat diet. a, p<0.05 in PBS vs LBT; b, p<0.05 in KBT vs LBT; c, p<0.05 in PBS vs KBT; *** p<0.0001.
Supplementary Fig. 13 Random forest classifier containing 42 MLG markers identified from the case-control cohort. a, Distribution of the error rate in random forest classification of obese patients as the number of top ranking MLGs increased (online methods). The model was trained by
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Training samples ROC curve
False positive rate
True
pos
itive
rate
•
best cut−off: 0.4551FPR: 0.125TPR: 0.9494AUC: 0.9687
0.0
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0.6
0.8
1.0
RandomForest in the training samples
Pro
babi
lity
of th
e ob
ese
patie
nts
Control OB 0.0 0.2 0.4 0.6 0.8 1.0
0.0
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Test samples ROC curve
False positive rate
True
pos
itive
rate
•
best cut−off: 0.478FPR: 0.08696TPR: 0.8462AUC: 0.9214
0 50 100 150 200
0.0
0.2
0.4
0.6
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RandomForest classfication for 42 marker MLGs for obese
Variable numbers
Cla
ss−s
peci
fic p
redi
ctio
n er
ror
a b
c d
Nature Medicine: doi:10.1038/nm.4358
15
relative abundance of 217 MLGs in 151 training samples (control, n=79; obese, n=72). The red line marks the optimal set of the number of MLGs (n=42). b, Receiver operating curve (ROC) for the training set. c, The probability of obesity in the training set based on the model in a. d, ROC for the test set (control, n=26; obese, n= 23).
Nature Medicine: doi:10.1038/nm.4358
16
1010.5
11.5
12.5
11
12
Control OB 0M 1M 3M
P-value=0.061P-value=0.174
a
2e+05
4e+05
8e+05
6e+05
1e+06
Gene count
Num
ber
of g
enes
Control OB 0M 1M 3M
P-value=0.020P-value=0.007
b α-diversity
Shan
non
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.20.
00.
20.
4
PC1 11.7 %
PC
2 9
.5 %
●
●
ConOBbefore1M3M
PC1
c
++++++
+
++++
+
++
++++++++++++
+
+
+
++
++++++
++
Case vs control
S0 vs S
1
S0 vs S
3
S3 vs control
Phosphotransferase system (PTS)Flagellar assemblyRibosomeThiamine metabolismPhenylalanine, tyrosine and tryptophan biosynthesisStarch and sucrose metabolismSelenocompound metabolismSulfur relay systemBenzoate degradationValine, leucine and isoleucine degradationPyruvate metabolismCitrate cycle (TCA cycle)Glycosaminoglycan degradationLipoic acid metabolismAminobenzoate degradationFluorobenzoate degradationBiosynthesis of siderophore group nonribosomal peptidesCarbon fixation pathways in prokaryotesmRNA surveillance pathwayN−Glycan biosynthesisRiboflavin metabolismABC transportersPorphyrin and chlorophyll metabolismPeptidoglycan biosynthesisTerpenoid backbone biosynthesisChloroalkane and chloroalkene degradationBetalain biosynthesisGalactose metabolismAmino sugar and nucleotide sugar metabolismFolate biosynthesisFructose and mannose metabolismLipopolysaccharide biosynthesisGlycerolipid metabolismC5−Branched dibasic acid metabolism
++++++++
++++
+
+
++
+
++++
++
+++++++++++
+
+
++
+
+++
+++
+++++
+
++
+
+
++++++++++++
+++
++
+
+
+
+
+++
Case vs control
S0 vs S
1S
0 vs S3
S3 vs control
Aminoacyl−tRNA biosynthesis, eukaryotesRibosome, bacteriaATP synthaseF−type ATPase, bacteriaPutative ABC transport systemHistidine biosynthesis, PRPP => histidineShikimate pathway, phosphoenolpyruvate + erythrose−4P => chorismateThiamine biosynthesis, AIR => thiamine−P/thiamine−2PLeucine biosynthesis, pyruvate => 2−oxoisovalerate => leucineCitrate cycle, first carbon oxidationMethionine biosynthesis, apartate => homoserine => methioninePTS system, galactosamine−specific II componentPTS system, glucitol/sorbitol−specific II componentPTS system, galactitol−specific II componentMolybdate transport systemPTS system, fructose−specific II−like componentPTS system, mannitol−specific II componentGlutamine transport systemPTS system, N−acetylgalactosamine−specific II componentGlutathione transport systemPTS system, ascorbate−specific II componentRibose transport systemPTS system, arbutin−like II componentN−Acetylglucosamine transport systemPTS system, lactose−specific II componentCobalamin biosynthesis, cobinamide => cobalaminGlutamate transport systemPTS system, sucrose−specific II componentCobalt transport systemNitrogen fixation, N2 => ammoniaPTS system, mannose−specific II componentPTS system, cellobiose−specific II componentV−type ATPase, prokaryotesType IV secretion systemMelatonin biosynthesis, tryptophan => serotonin => melatoninPTS system, beta−glucosides−specific II componentPTS system, maltose and glucose−specific II componentPTS system, fructose−specific II componentPTS system, arbutin−, cellobiose−, and salicin−specific II componentMaltose/maltodextrin transport systemPutative sugar transport systemHeme biosynthesis, glutamate => protoheme/sirohemeD−Methionine transport systemPTS system, trehalose−specific II componentHeparan sulfate degradationbeta−Carotene biosynthesis, GGAP => beta−caroteneEthylmalonyl pathwayReductive citric acid cycle (Arnon−Buchanan cycle)indolepyruvate:ferredoxin oxidoreductaseMethanogenesis, acetate => methaneN−glycan precursor biosynthesisArginine/ornithine transport systemRNA polymerase, archaeaMenaquinone biosynthesis, chorismate => menaquinoneComplex I (NADH dehydrogenase), NADH dehydrogenase IMethanogenesis, formate => methaneChondroitin sulfate degradationCitrate cycle, second carbon oxidationPhosphonate transport systemAscorbate biosynthesis, animals, glucose−1P => ascorbateAdhesin protein transport systemComplex IV (Cytochrome c oxidase), cytochrome c oxidase, cbb3−typeCAM (Crassulacean acid metabolism), light2−oxoglutarate:ferredoxin oxidoreductaseCysteine biosynthesis, serine => cysteinealpha−Hemolysin/cyclolysin transport systemC4−dicarboxylic acid cycle, NADP+ −malic enzyme type2−oxoisovalerate:ferredoxin oxidoreductaseGPI−anchor biosynthesis, core oligosaccharideMethane oxidation, methylotroph, methane => CO2Octopine/nopaline transport systemType VI secretion system2−Aminoethylphosphonate transport systemHemophore/metalloprotease transport systemPhosphatidylcholine (PC) biosynthesis, PE => PCKeratan sulfate degradationRibosome, eukaryotesPyridoxal biosynthesis, erythrose−4P => pyridoxal−5PC5 isoprenoid biosynthesis, mevalonate pathwayPyruvate oxidation, pyruvate => acetyl−CoAManganese/zinc/iron transport systemManganese/iron transport systemNon−phosphorylative Entner−Doudoroff pathway, gluconate => glyceraldehyde + pyruvateIron(III) transport systemDermatan sulfate degradationHistidine degradation, histidine => N−formiminoglutamate => glutamateLeucine degradation, leucine => acetoacetate + acetyl−CoA
8
6
4
2
0
−2
−4
d e
0-1 M
0-3 M
MLG abundance (lg)
MLG abundance (lg)
1M-e
nric
hed
0M-e
nric
hed
f
g−30 −25 −20 −15 −10
3M
-enrich
ed
0M
-enrich
ed
−30 −25 −20 −15 −10
1M Samples0M Samples
Ruminococcus torques(8038)Dorea longicatena(7570)
Ruminococcus torques(7997)Collinsella aerofaciens(8613)
OB−28149Dorea longicatena(7593)Ruminococcus sp.(8908)
Collinsella aerofaciens(6710)OB−21281
Dorea longicatena(9343)Faecalibacterium cf. prausnitzii(3979)
Dorea longicatena(9349)OB−8935
Con−27927Con−2500
Con−12885OB−20906Con−3480
Bacteroides eggerthii(11877)Faecalibacterium prausnitzii(3943)
Con−2328Con−13802
OB−8196Con−2488
Streptococcus salivarius(8493)Clostridiales bacterium(16594)
Bacteroides thetaiotaomicron(11069)Lachnospiraceae bacterium(538)
Akkermansia muciniphila(74)Dialister invisus(28421)
Akkermansia muciniphila(63)Klebsiella oxytoca(12807)
Akkermansia muciniphila(69)
Dorea formicigenerans(28646)Ruminococcus gnavus(8062)Ruminococcus torques(8038)
Dorea longicatena(7570)Coprococcus comes(6940)
Ruminococcus gnavus(7680)Ruminococcus torques(7997)
OB−28149Dorea longicatena(7593)
Coprococcus comes(6834)OB−21275
Dorea longicatena(9343)OB−9891OB−9651
Dorea longicatena(9349)OB−7820
OB−20937OB−7776
OB−20906Eubacterium dolichum(7818)Bacteroides uniformis(11898)
Bacteroides thetaiotaomicron(11021)Streptococcus salivarius(8493)
Con−5231Bacteroides intestinalis(173)Bacteroides intestinalis(243)
Con−19475Roseburia hominis(14574)
Bacteroides intestinalis(211)Con−3508Con−5220
Clostridiales bacterium(16594)Bacteroides thetaiotaomicron(11069)
Bacteroides intestinalis(2808)Veillonella sp. Oral(12639)
Haemophilus parainfluenzae(12511)Con−3439
Akkermansia muciniphila(75)Akkermansia muciniphila(10938)
Con−553Akkermansia muciniphila(74)
Con−4754Con−1216Con−1400Con−5622Con−3564
Dialister invisus(28421)Con−16701
Akkermansia muciniphila(63)Fusobacterium ulcerans(7655)Oxalobacter formigenes(1300)
Anaerotruncus colihominis(14893)Klebsiella oxytoca(12807)
Akkermansia muciniphila(69)Con−4707Con−536
Bacteroides intestinalis(2744)Haemophilus parainfluenzae (12502)
Con−10882
−25 −20 −15 −10Abundance(lg)
3M0M
K00266K01776K10206K00831K00931K02501K01657K01658K06215K08681K00823K01955K01951K00603K13821K00841K00819K00681K00261K08969K03342K00830K00825
KOs involved in glutamate metabolism
86420−2−4−6
h
3M Samples0M Samples
Nature Medicine: doi:10.1038/nm.4358
17
Supplementary Fig. 14 Gut microbiome alterations following weight loss by sleeve gastrectomy a, PCoA based on Bray-Curtis similarity index on 217 MLGs. Top left panel showing the density distribution of the projection in PC1. b-c, gene count (b) and α-diversity (within-sample diversity, c) of 151 samples (control, n=79; obese, n=72) and intervention samples (0M, n=23; 1M, n=17; 3M, n=23) at the gene level. d-e, Heat map and hierarchical clustering of reporter score of KEGG pathways (d) and modules (e) from four comparisons: obese (n=72) vs. control (n=79), 0M (n=17) vs. 1M (n=17), 0M (n=23) vs. 3M (n=23), 3M vs. control. For the comparison of 3M vs. control, we randomly picked 23 samples from 79 controls to compare with 23 3M samples for 100 times and used the mean of the 100 results. KO pathways and modules with |reporter score| > 1.65 in the comparison between 79 lean controls and 72 obese patients are shown. +, |reporter score| > 1.65. f, MLGs (Supplementary Table 4) that significantly differed in abundances in 0M and 1M samples (0M, n=17, 1M, n=17). g, MLGs that significantly differed in abundances in 0M and 3M samples (0M, n=23, 3M, n=23). Control-enriched MLGs are shown in blue and obesity-enriched MLGs in red. h, Distinct KOs assigned to enzymatic reactions involved in glutamate metabolism were shown. KOs in blue and red represent KOs significantly enriched in 3M samples and 0M samples, respectively (0M, n=23, 3M, n=23). For f-h, two-tailed Wilcoxon matched-pairs signed rank test, p<0.05. 0M, baseline; 1M, one month after SG; 3M, three months after SG. In all box plots, boxes represent the interquartile ranges (IQRs) between the first and third quartiles, and the line inside the box represents the median; whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. Circles represent data point beyond the whiskers. The notches show the 95% confidence interval for the medians.
Supplementary Fig. 15 Uncropped gels for western blots in supplementary Fig. 11k (left) and supplementary Fig. 11l (right).
70100130250KD
KD7055
35
35
KD7055
70
100
KD
55
35
KD7055
KD7055
PBS KBT LBT PBS KBT LBTlnsulin - lnsulin +
lnsulin - lnsulin +
PBS KBT LBT PBS KBT LBT
p-AKT
AKT
Hsp90
p-AKT
AKT
Hsp90
Nature Medicine: doi:10.1038/nm.4358