1  

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.

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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.

* * + * * * * * * * * * * * *

* * * * * * * * * * * * *

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+ + + * * * * * + + + * * * *

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* * * * + * * * * * * * + + * *

* * * * + * * * * * * * + + * *

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* * + * * * * * * + * * * * * * * *

−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

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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

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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

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−0.1

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CD

PC1 45.3 %

PC

2 8

.6 %

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PBS beforeKBT beforeLBT beforePBS afterKBT afterLBT after

●

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−0.1

0.0

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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

●

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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

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05

1015

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(Bac

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05

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BFr

atio

(Bac

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idet

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irmic

utes

)

aa

a

a;c

a;b

b

b;c

b;c

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;b

P-value=0.03

Before After

Before After

Before After

Before After

After After

PBS KBT LBT0

2

4

6

8

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PBS KBT LBT0

2

4

6

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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

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Training samples ROC curve

False positive rate

True

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best cut−off: 0.4551FPR: 0.125TPR: 0.9494AUC: 0.9687

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ese

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Test samples ROC curve

False positive rate

True

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itive

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0 50 100 150 200

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RandomForest classfication for 42 marker MLGs for obese

Variable numbers

Cla

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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

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Gene count

Num

ber

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P-value=0.020P-value=0.007

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.20.

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PC1 11.7 %

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2 9

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●

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ConOBbefore1M3M

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+

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+

+

+

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++

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

++++++++

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Case vs control

S0 vs S

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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