Gut microbiota determine insulin sensitivity · Fasting Insulin (mmol/L) MMT 80 (15-178) 85 (19-180) Cholesterol (mmol/L) 5.2 (3.5-8.8) 4.5 (3.5-7.2) TG (mmol/L) 2.0 (0.7-4.5) 1.1

Gut microbiota determine

insulin sensitivity

Dr. Max Nieuwdorp

Academic Medical Center, Amsterdam

Overview• Gutmicrobiota in health and disease

• Gutmicrobiota products in plasma associated with developing insulin resistance and CVD

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• Manipulating gutmicrobiota by fecal transplant: effect on insulin resistance

• Manipulating gutmicrobiota by broad spectrum antibiotics : effect on insulin resistance

Gutmicrobiota in health and disease

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Background IMetabolic active acterial phylae

Firmicutes (an/aerobic, gram +) • Bacillales

- listeriae-staphylococcae

• Clostridia-clostridium difficile

Bacteroidetes (anaerobic gram –)• Bacteroides

- fragilis- thetaiotaomicron

• Porphyromonas - gingivalis

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-clostridium difficile• Lactobacillales

-streptococcae-enterococcae

• Mollicutes-mycoplasma-ureaplasma urealyticum

• Porphyromonas - gingivalis

• Flavobacteriae

• Sphingobacteriales

Background IIGut microbiota

• Total bowelsurface 400m2; 6000-7000 kilogram feces in a human life produced!

• The gutmicrobiota (60% of the dry mass of feces)content consists of 10 14 (!) bacteria– 99% anaerobic

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– 99% anaerobic– 90% belong to 2 phyla:Firmicutes & Bacteroidetes– Microbiome 100 times larger than the human genome

• Foetuses in utero are sterile, develop in first 2 years, fingerprint (40-50% by parents, rest via lifestyle)

• Large fraction gut microbiota is difficult to culture, therefore only estimations of total amount of about 4000 different bacterial species could be made by microbiologists throughout the last decades

1Ley et al, Nature 2006, 444:1022-1023 2Bäckhed et al, PNAS 2004, 101:15718-15723

Background IIGut microbiota

• Total bowelsurface 400m2; 6000-7000 kilogram feces in a human life produced!

• The gutmicrobiota (60% of the dry mass of feces)content consists of 10 14 (!) bacteria– 99% anaerobic

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– 99% anaerobic– 90% belong to 2 phyla:Firmicutes & Bacteroidetes– Microbiome 100 times larger than the human genome

• Foetuses in utero are sterile, develop in first 2 years, fingerprint (40-50% by parents, rest via lifestyle)

• Large fraction gut microbiota is difficult to culture, therefore only estimations of total amount of about 400 different bacterial species could be made by microbiologists throughout the last decades

1Ley et al, Nature 2006, 444:1022-1023 2Bäckhed et al, PNAS 2004, 101:15718-15723

Background IIIHuman microbiome projects

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Background IVSymbiont role

Gutmicrobiota are involved in:• Fermenting unused energy

substrates• training the immune system• preventing growth of

harmful, pathogenic bacteria

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harmful, pathogenic bacteria (clostridium difficile)

• producing vitamins (such as biotin, B12 and vitamin K) and facilitate electrolye absorption (Magnesium, Calcium and Iron)

• hormone production(adiponectin) to direct the host to store fats

Wikoff, PNAS 2009

Background VGermfree mice and metabolism

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1Rabot, FASEB 2010

Gutmicrobiota per se affect in glucose and lipid metabolism in mice

Background VISpecific gut microbiota seem to

affect body fat composition • GF mice lower % bodyfat (assisting

nutrient uptake via firmicutes phylum)• Colonization of germ-free mice ↑ %

bodyfat by 57%

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bodyfat by 57%

Backhed et al, PNAS 2004

Background VIIGutmicrobiota habit is a

Transmissible trait• Fecal transplantation (FCT,

with obese mice feces into lean germ free mice) renders 20% increase in

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renders 20% increase in visceral fat compared to FCT of lean mice

• Just after 14 days

Turnbaugh, Nature 2006

Background VIIIGut microbiota in human disease

• Majority of phylotypes (80%) are shared between monozygous twins with large variations in the abundance of each phylotype.

• Different gut microbiota composition in lean vs obese humans Bacteriodes

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in lean vs obese humans– Obese humans have fewer

Bacteroides and more Firmicutes

– Other human studies could not support specific changes

• Thus, relationship between obesity and bacterial groups remained a matter of debate in humans..

Turnbaugh, Nature 2010: Ley et al, Nature 2006;Duncan Obesity 2008

Bacteriodes

Firmicutes

Gutmicrobiota products in plasma associated with developing insulin resistance and CVD

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Background IXHuman gutmicrobiota and enterotypes

• 2-3 dominant enterotypes (“bloodgroups”): bacteriodes, prevotella and ruminococcus.

• enterotypes are dependent of diet, but seem

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• enterotypes are dependent of diet, but seem independent of nationality, sex, or age

Arumugam, Nature 2011; Wu, Science 2011

Background XEnterotypes and dietary composition

• Enterotype 1: increased biotin/vitamin b7 � coenzyme in the

metabolism of fatty acids and gluconeogenesis.

• Enterotype 2: increased thiamin/vitamin b1 � coenzyme in the

catabolism of sugars and amino acids.

• Bacteroides (enterotype 1)�� selected by high fat/protein diet

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• Bacteroides (enterotype 1)�� selected by high fat/protein diet

• Prevotella (enterotype 2)�� selected by carbohydrates diet

Arumugam, Nature 2011; Wu, Science 2011

Gutmicrobiota in gutphysiology

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RS Kootte, Diabetes Obesity and Metabolism 2011.

Microbiota products and insulin resistance

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Wang, Nature Medicine 2011

Microbiota products and cardiovascular disease

• Eggs, milk, liver, red meat, poultry, shell

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meat, poultry, shell fish and fish, are believed to be the major dietary sources for choline (PC), and hence TMAO production

• Wang, Nature 2011

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All human observations/conclusions are deriverd from cross sectional

studies….

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…causality remains to be proven in humans!

studies….

Manipulating gutmicrobiota by fecal transplant: effect on

insulin resistance

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Objectives FATLOSE trial

• Examine the effect of donor feces transplantation in subjects with metabolic syndrome on:

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metabolic syndrome on:

– Glucose homeostasis

– Fecal short chain fatty acids

– Gut microbiota composition (prox-distal gut)

Donors

Screening donors:

- Questionnaire (bowel habits, travel history, medication, etc)

- Faeces (parasites, Clostridium difficile, SSYC)

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- Faeces (parasites, Clostridium difficile, SSYC)

- Bloodborn viruses (Hepatitis, HIV, HTLV, CMV, EBV)

- Checklist day before treatment (been ill? different sexual contact? travelling? no antibiotics in last 3 months?)

FATLOSE trial(Faecal Administration To LOSE metabolic

syndrome)

• Study design: double blind RCT

- allogenic FT (from lean male volunteers, n=9)

- autologic FT (own faeces) n= 9

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- autologic FT (own faeces) n= 9

• Inclusion criteria:– male subjects

•BMI ≥ 30 kg/m2

•FPG ≥ 5.6 mmol/l

•Age 21-65 years

•No medication use

1: donor and patiënt go to the toilet• randomisation

2:

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3: Gastro-duodenoscopy with jejunal biopsies

Duodenal tube

Bowel lavage with cetomacrogol

Infusion faeces (either own or lean donors)

Outcome measures• Insulin sensitivity

– Hyperinsulinemic euglycemic clamp at T=0 and T=6wks– Energy intake (dietlists) and Resting Energy Expenditure (REE)

• Short chain fatty acids (acetate, propionate, butyrate)– T=0 and 6wks

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• Gut microbiota composition– HITChip: phylogenetic microarray analysis (HITTchip) on

faecessamples– T=0 and 6wks

Results Baseline characteristics

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A.Vrieze, Gastroenterology 2012 in revision

1) Effect donorfaeces on intake/metabolism:

1000

1500

2000

2500

RE

E (k

cal/d

)

1000

2000

3000

ener

gy

inta

ke/d

ay (k

cal)

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0wks

6wks

0wks

6wks

0

500

allogenic faeces autologous faeces

RE

E (k

cal/d

)

0 2 6 12 0 2 6 120

1000

allogenic faeces autologous faeces

ener

gy

inta

ke/d

ay (k

cal)

• No changes in weight in both groups

• No changes in whole body energy metabolism or daily dietary (caloric) intake


2) Effect donor faeces on whole body insulin sensitivity

• Increased rate of disappearance (Rd)

Median allo T0: 26.2 µmol/kg.min


80

Per

iphe

ral i

nsul

in s

ensi

tivity

min

)

P<.05

ns

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Median auto T0: 21.0 µmol/kg.min

Median auto T6: 19.5 µmol/kg.min


0 wks

6 wks

0 wks

6 wks

0

20

40

60

Leancontrols

Autologous gut microbiotainfusion

Allogenic gut microbiotainfusion

Per

iphe

ral i

nsul

in s

ensi

tivity

(Rd,

µµ µµ mol

/kg

. min

)

0 w

ks

6 wks

0

20

40

60

80P<.05

Allogenic gut microbiota infusion

Pe

rip

hera

l in

sulin

se

nsi

tivit

y(R

d, µµ µµ

mol

/kg

. min

)

0 wks

6 wks

0

10

20

30

40

Autologous gut microbiota infusion

ns

Per

iph

eral

insu

lin s

ens

itivi

ty

(Rd,

µµ µµm

ol/k

g. m

in)

3) Effect donor faeces on hepatic insulin sensitivity

• Increased suppression of endogenous glucose production (EGP)

Median allo T0: 51.5 %60

80

100P=.08

He

pat

ic in

sulin

se

nsi

tiv

ity

(% s

up

pre

ssio

n E

GP

)

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Median allo T6: 61.6 %

Median auto T0: 53.8 %

Median auto T6: 52.4 %


Basel

ine

Before

After

Before

After

0

20

40

60

Allogenic gut microbiotainfusion

Leancontrols

Autogous gut microbiotainfusion

He

pat

ic in

sulin

se

nsi

tiv

ity

(% s

up

pre

ssio

n E

GP

)

0 wks

6 wks

0

20

40

60

80

100P=.08

Allogenic gut microbiota infusion

Hepa

tic in

sulin

sen

sitiv

ity(%

sup

pres

sion

EG

P)

0 wks

6 wks

0

20

40

60

80

ns

autologous gut microbiota infusion

Hep

atic

insu

lin s

ensi

tivity

(% s

uppr

essi

on E

GP)

4) Changes in fecal butyrate and proprionate

40

60 Lean donor T0

Allogenic FT T0

Allogenic FT T6

Autologic FT T0*

Mea

n S

CF

A (

mm

ol/k

g f

eces

)

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Aceta

te

Propio

nate

Butyra

te

0

20

Autologic FT T0

Autologic FT T6*

*

Mea

n S

CF

A (

mm

ol/k

g f

eces

)


Faecalibacterium prausnitzii et rel. negatively correlated with fecal butyrate

5) Effect donorfaeces on gutmicrobiota

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Qin J, Zoetendal E, de Vos WM. Nature 2010: 464 (7285):58-65

6) Fecal samples before and after lean donor Tx

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7) Small intestinal biopsy before and after lean donor Tx

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Caution

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Eiseman, Surgery 1958

Manipulating gutmicrobiota by broad spectrum antibiotics: effect on insulin resistance

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Antibiotic use and obesity

• Retrospective studies show an association between antibiotic use and obesity

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and obesity

• BMI increase more after gram positive eradication (vancomycin)!

F. Thuny, PLoS 2010

Study protocol

• Male obese subjects with metabolic syndrome were randomised to– 7 days amoxicillin 3dd 500mg (n=10)

OR

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OR

– 7 days vancomycin 3dd 500 mg (n=10)

• Before and after 7 days of AB:– Hyperinsulinemic clamp (day -1 & day 10)

– Gut microbiota composition (HITChip) follows

Baseline characteristics AV trial- Median (range)

Amoxicillin

(n=10)

Vancomycin

(n=10)

Age (y) 61.0 (37-65) 51.0 (27-61)

BMI (kg/m2) 33.0 (26.9-40.2) 34.0 (29.3-44.3)

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Weight (in kg) 112.7 (88.6-129.0) 117.8 (96.0-140.2)

Fasting glucose (mmol/l) MMT 5.6 (5.2-7.8) 5.3 (4.6-7.6)

Fasting Insulin (mmol/L) MMT 80 (15-178) 85 (19-180)

Cholesterol (mmol/L) 5.2 (3.5-8.8) 4.5 (3.5-7.2)

TG (mmol/L) 2.0 (0.7-4.5) 1.1 (0.5-4.2)

HDL (mmol/L) 1.1 (0.7-1.5) 1.0 (0.7-1.5)

LDL (mmol/L) 3.2 (1.6-5.9) 2.7 (2.0-5.3)

hsCRP 2.5 (0.6-18.1) 1.9 (0.6-20.0)

* P<0.05

Periferal insulin resistance

80

p<0.05

Per

iph

eral

insu

lin s

ensi

tivi

ty

Median Amoxi T0: 26.7 umol/kg .min

Median Amoxi T1: 25.8 umol/kg .min

Median Vanco T0: 31.9 umol/kg .min

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0 w

ks 1wk

0 wks 1w

k

0

20

40

60

amoxicillin vancomycin

Per

iph

eral

insu

lin s

ensi

tivi

ty(R

d,

µµ µµmol

/kg

. min

)



Significant reduction in periferal IR after grampositive elimination

Hepatic insulin resistance

Median Amoxi T0: 62.8 %

Median Amoxi T1: 58.9 %100

150

Hep

atic

insu

lin s

ensi

tivi

ty(%

su

pp

ress

ion

EG

P)

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Median Vanco T0: 66.7 %

Median Vanco T1: 66.5 %

No effect antibiotics on hepatic IR

0 wks 1w

k

0 wks 1w

k

0

50

amoxicillin vancomycin

Hep

atic

insu

lin s

ensi

tivi

ty(%

su

pp

ress

ion

EG

P)

Gut microbiota and human metabolism

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Vrieze/Nieuwdorp et al, Diabetologia 2010, 53(4):606-13.

Take home message• Gutmicrobiome is 100x larger than genome

• Symbiont role gutmicrobiota in humans

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• Fecaltransplantation and antibiotics (gram positive) are able to shape host gutmicrobiota in human pathophysiology

• Gutmicrobiota involved in human glucose probably act via Short Chain Fatty Acids (butyrate en proprionate)

Acknowledgments

Willem de VosWUR

Ruud KootteAMC

Anne Vrieze

AMCGeesje Dallinga

AMC

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Mireille Serlie AMC

(Obese)Volunteersand patients

Erwin ZoetendalWUR

Bert Groen UMCG

Joep Bartelsman

Stan Hazen

Cleveland ClinicErik Stroes

AMC

Documents

Gut microbiota determine insulin sensitivity · Fasting Insulin (mmol/L) MMT 80 (15-178) 85 (19-180) Cholesterol (mmol/L) 5.2 (3.5-8.8) 4.5 (3.5-7.2) TG (mmol/L) 2.0 (0.7-4.5) 1.1