Gut microbiota determine insulin sensitivity Dr. Max Nieuwdorp Academic Medical Center, Amsterdam
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
A.Vrieze, Gastroenterology 2012 in revision
2) Effect donor faeces on whole body insulin sensitivity
• Increased rate of disappearance (Rd)
Median allo T0: 26.2 µmol/kg.min
Median allo T6: 45.3 µmol/kg.min
80
Per
iphe
ral i
nsul
in s
ensi
tivity
min
)
P<.05
ns
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Median allo T6: 45.3 µmol/kg.min
Median auto T0: 21.0 µmol/kg.min
Median auto T6: 19.5 µmol/kg.min
A.Vrieze, Gastroenterology 2012 in revision
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 %
A.Vrieze, Gastroenterology 2012 in revision
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
)
A.Vrieze, Gastroenterology 2012 in revision
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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A.Vrieze, Gastroenterology 2012 in revision
7) Small intestinal biopsy before and after lean donor Tx
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A.Vrieze, Gastroenterology 2012 in revision
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
)
Median Vanco T0: 31.9 umol/kg .min
Median Vanco T1: 25.7 umol/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