The roleof gut microbiotain the pathogenesisof obesity...

10/23/2017

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The role of gut microbiota in the pathogenesis of obesity, insulin resistance

and type 2 diabetes mellitus

Ellen Blaak

Professor in Physiology of fat metabolism, Department of Human BiologyNUTRIM School of Nutrition and Translational Research in Metabolism

Maastricht University Medical Centre+

The Netherlands

EASO New Investigators united, Autumn School 2017, Palma de Mallorca

Department

Overview

Introduction microbiota

Microbiota composition, obesity and diabetes

Modulation of microbiota and metabolic health

• Feces transplantation studies

• antibiotics

Role of SCFA and metabolic health

– SCFA administration

Intervention with prebiotics-dietary fibers

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“Soundbites”

• Bacterial cells outnumber host cells by a factor of 10

• Adults can carry over 1 kg of gut bacteria

• We excrete our own weight in fecal bacteria per annum

• At the age of 70 we have excreted the weight of 12 elephants

• Over 50% of fecal solids is bacteria

• Each person has more colonic bacteria than the number of people that has ever been on the planet

• 1500 - >2000 species

Courtesy of Roderick Mackie and Glenn Gibson

of Human Biology

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Microbiota:fermentation

proximal colon: primarily saccharolytic

fermentation

transverse colon: combination of saccharolyticand proteolytic fermentation

distal colon: primarily proteolytic fermentation

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Qin et al. Science. 2010

Microbiota is individual specific

Is there a normal microbiota?

57 species common >90% individuals

~ 250.000 genes (at least 50% of the individuals)

Miguel Gueimonde

Adapted from:

Turnbaugh et al. Nature. 2009

Compositional vs.

functional differences

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Challenge: definition of a normal/healthy microbiota

Geographical differences

Inter-individual variation

Miguel Gueimonde

Microbiota is individual specific

Adapted from:

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Hadza tribe in Tanzania

Schnorr et al.,Nat Commun. 2014;5:3654

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Hadza tribe: different microbial genes

Rampelli et al.,Curr Biol. 2015;25(13):1682-93

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Microbiota, obesity and type 2 diabetes mellitus

• Worldwide, obesity is reaching epidemic proportions

• The obesity pandemic is not fully explained by most common gene-environment interactions

• Gut microbiota provide additional gene products that may affect host metabolism, gut physiology, body weight control and insulin sensitivity through several mechanisms

• Obesity and Type 2 diabetes mellitus are associated with differential composition of gut microbiota and/or gut microbiome

• Interventions to manipulate gut-microbiota may favorably affect metabolism and reduce diabesity risk

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Obesity – pandemic of 21st century

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Cardiovascular disease Type 2 diabetes Liver steatosis Cancer

OBESITY

of Human Biology

Obesity: an important risk factor for chronic metabolic diseases

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Gut microbiota in Human adults

• Increased Firmicutes/Bacteroidetes

(Ley et al, nature, 2006, Turnbaugh et al, 2009)

• Decreased Fimicutes/Bacteroidetes

(Collado et al,2008,Schwiertz A, Obesity, 2010, Amarugan, 2011)

Weight loss and bacteroidetes related taxa, relationship:

-positive (Nadal J, IJO, 2009)

-neutral (Duncan SH, IJO, 2008)

-negative (SantaCruz, obesity, 2009)

• Differences in other phyla and species (i.e bifidobacteria), novel diversity with each study

Outcome in human studies is

complex

Move from phyla-associated differences towards genus or species level and characterization of gene function

Gram positive anaerobic Bacteria (SCFA): lower abundance of butyrate-producing bacteria in diabetes (Qin, J et al, Nature, 2012, Karlsson et al, Nature, 2013)

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Feces transplantation from mice to mice

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From mice to mice: Gut Flora contributes to the high-fat induced metabolic diseases in mice

(Bäckhed et al, 2004, 2007)

Germ-free mice are protected from development of insulin resistance and glucose intolerance

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From man to mice: Gut microbiota from twins discordant for obesity modulate metabolism in mice

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V K Ridaura et al. Science 2013;341:1241214

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From man to man: feces transplantation and human metabolism

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Transfer of Intestinal Microbiota From Lean Donors Increases

Insulin Sensitivity in Individuals With Metabolic Syndrome

Vrieze et al, 2012of Human Biology

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Transferring microbiota between conventional and germ free rodents, humans and rodents and humans and humans have shown the role of intestinal microbiota in the development of obesity and insulin resistance, but overall effects in humans are minor.

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• Altered adipokine

secretion

• Insulin resistance

•Reduced fatty acid

trapping

• Impaired lipolysis

Hypertrophic adipocytes

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Gut microbiota in overweight, insulin resistance and diabetes

Muscle•Impaired fat oxidation

• Lipid accumulation


Lipid spill-overSystemic inflammation

Liver

• Lipid accumulation


• Inflammation

Preadipocyte

recruitment

Microbiota

SCFA

Complex CHO

L-cell

Pancreas

Altered insulin secr/prodAltered glucagon secr/prod

Bile acids

GLP1

PYY

LPSEnergy

Harvest, Metabolic effects

SCFA

ANGPTL4GPCR

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Antibiotics intervention

40-70y

IFG/IGT

HOMA-IR>2.2

n=56

wk 1 pre

wk 2

2d wash out

wk 3 post

wk 8 follow-up

Treatment Timing

Placebo

Amoxicillin

Vancomycin

1500 mg/day

7 days

of Human Biology Reijnders et al, Cell metabolism, 2016

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Microbiota composition

• Vanco:

– Increased gram negative Proteobacteria

– less gram-positive bacteria

– decreased diversity

– After 8 weeks similarity with baseline still lower, diversity slightly lower

• Amox:

• no diversity and/or composition differences

Distinct changes in microbiota composition


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No effect on insulin sensitivity

� Two-step hyperinsulinemic-euglycemic clamp

� Endogenous Glucose Production

� Rate of Disappearance

� Suppression of Free Fatty Acids

%


No effect on whole body insulin resistance 8 wk after stopping the active intervention

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Department of Human Biology Reijnders et al, Cell metabolism, 2016

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Perspectives

• Modulation of adult microbiota by 7-day antibiotics does not affect host metabolism

• Host metabolism remained unchanged at 8-week

follow-up, despite deviant microbiota

• Contradicts many rodents studies!

• More frequent antibiotic use, long term diet effects?

• Does metabolic phenotype play a role?: relatively healthy vs prediabetic state

• Stable vs dynamic state?of Human Biology

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Function microbiota: saccharolytic en proteolyticactivity

• Saccharolysis leads primarily to SCFA and gasses

• SCFA: acetate, propionate, butyrate• lactate -> only accumulates when there is a fast fermentation

• CO2, CH4, H2

• Proteolysis in addition leads to toxic metabolites• BCFA: iso-butyrate, iso-valerate• ammonia• phenolics: phenol, indol, p-cresol, skatol• H2S, CH3SH, etc

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Site of fermentation

proximal colon: primarily saccharolytic

fermentation

transverse colon: combination of saccharolyticand proteolytic fermentation

distal colon: primarily proteolytic fermentation

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Acetate

Propionate

Butyrate

Major SCFA

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Canfora, E. E. et al. (2015) Short-chain fatty acids in control of body weight and insulin sensitivityNature Reviews Endocrinology doi:10.1038/nrendo.2015.128

Functionality of microbiota: SCFA

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Colonic acetate infusion and metabolic profile

Hypothesis:Colonic administration of SCFA has beneficial effects on human substrate and energy metabolism

Question:

Where to administer? Distal / Proximal?

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Distal and proximal colonic acetate infusion and metabolic profile

Aim:

To investigate differential effects of proximal and distal colonic infusions with sodiumacetate on human fat oxidation, energy expenditure and circulating metabolicmarkers

Study design

• Double blind, placebo controlled, randomized crossover study

• Six healthy overweight males (BMI 25 – 34.9 kg/m2)

Aged 20 – 50 years;

Weight stable for at least 3 months (± 2 kg)

No use of antibiotics, pre- or probiotics

Intervention

1. Sodium acetate 100mmol/L (12mmol in 120mL water)

2. Sodium acetate 180mmol/L (21.6mmol in 120mL water)

3. Placebo (0.9% NaCl) in 120mL

of Human Biology Van der Beek et al, Clin Sci 2017

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Intervention protocol


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Test day protocol


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Fat oxidation

ANOVA * p<0.05 placebo vs 180mM

Ingestion oral glucose solution

break

break

Placebo 100 mmol/L 180 mmol/L-2

-1

0

1

2

3

Fa

t oxi

da

tion

(g

fat 2

h-1

) *

p=0.055

iAUC, distal

placebo

100mmol/L

180mmol/L


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� Increasing colonic and systemic acetate beneficially affect the

metabolic profile

� Validated distal colonic infusion as a good model to study SCFA

effects on metabolism

Circulating acetate �Fat oxidation �PYY �TNF-α �Lipolysis �

No significant effects


Distal, not proximal, colonic acetate infusion improves metabolic profile

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Combinations of SCFA and metabolic profile

To investigate acute effects of distal colonic infusions of SCFA combinations on substrate and energy

metabolism

of Human Biology Canfora et al, Scientific reports, 2017

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Intervention protocol (1)

Double blind, placebo controlled, randomized crossover study with 4 distal

infusions:

• Placebo:

40mmol NaCl

• High sodium acetate (60:20:20):

24mmol NaAc, 8mmol NaBu, 8mmol NaPr

• High sodium butyrate (45:35:20):

18mmol NaAc , 14mmol NaBu, 8mmol NaPr

• High sodium propionate (45:20:35):

18mmol Na Ac, 8mmol NaBu, 14mmol NaPr

• All diluted in 200mL water

0

20

40

60

80

100

60:20:20 45:35:20 45:35:20 Placebo

sodium chloride

sodium propionate

sodium butyrate

sodium acetate

% S

CFA/N

aC

lin

solu

tions

45:20:35


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SCFA increase Fat oxidation

*** p<0.001, ** p<0.01 Ingestion oral glucose

***


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SCFA increase Energy Expenditure

*** p<0.001, * p<0.05, # p<0.1 Ingestion oral glucose solution

*

plac

ebo

high

ace

tate

high

pro

pion

ate

high

but

yrat

e

0

20

40

iAUC energy expenditure, fasted

****

#

iAU

C E

E k

J/t0

-t1

20


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Circulating acetate is associated with fat oxidation and energy expenditure

-10000 -5000 5000 10000

-4

-2

2

4

6

Fasting plasma acetate (µmol/L 2h-1)

Fa

stin

g f

at o

xid

atio

n (

g fa

t 2h

-1)

-10000 -5000 5000 10000

-50

50

100

150

Fasting plasma acetate (µmol/L 2h-1) Re

stin

g e

ne

rgy

exp

en

ditu

re (

kJ/

2h

-1)

Acetate vs. fat oxidation r=0.328 (P=.0228)

Acetate vs. energy expenditure r=0.349 (P=.0149)

Canfora, et al. (2017), Scientific Reports (accepted)

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Fat oxidation ��Energy Expenditure ��PYY ��IL-1β ↓lipolysis ↓

Acetate maybe a target to prevent and treat obesity-related co-morbidities


What are the underlying mechanisms?

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Why do we hypothesize that the distal colon should be targeted?

Kuwahara (2014), Frontiers in Endocrinology

Higher density of PYY-producing L-cells in the distal colon

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Increased fat oxidation and EE only after distal administration:

Acetate bypass the liver � circulating acetate� � uptake in oxidative tissues � pAMPK� � fat oxidation�

CirculatingAcetate ��

Acetate �� pAMPK�� fat oxidation��1,2,3

1Sakakibara et al., 2006; 2Yamashita et al., 2009; 3Kimura et al., 2013; 4 den Besten et al., 2015

Acetate �� pAMPK�� fat oxidation��1,2,4

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Fermentation of fibers by the gut microbiota –proximal versus distal

Which fibers are best?

��

Canfora, et al. (2017), Scientific Reports;7(1):2360

fermenteddistally

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Prebiotics: definition

a selectively fermented ingredient * that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus

conferring benefit(s) upon host health

* largely restricted to carbohydrates until now

Blatchford, Venema and others 2014, Intl J Probiotics Prebiotics 8(4)

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Beneficial effects of prebiotics – a snapshot (1)

FOS supplementation in rats:

increase in serum GLP-1 and proglucagon mRNA expression in the

proximal colon of rats

a two fold increase in GLP-1 producing L-cells in the proximal colon (Cani

et al., 2004; Cani et al., 2007).

adipose tissue mass was decreased and glucose control and leptin

sensitivity were increased: effects of SCFA

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Beneficial effects of prebiotics – a snapshot (2)

Targeted delivery of proprionate

to the colon acutely increases

PYY and GLP1 and reduces

energy intake

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Chambers ES et al, Gut, 2015

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Beneficial effects of prebiotics – a snapshot (3)Towards dietary intervention study with galacto-

oligosaccharides targeting colonic acetate

Study design

• Double blind, placebo controlled, randomized parallel study

• 46 volunteers:

– Males and postmenopausal females aged 45-70 years;

– BMI 28 – 40 kg/m2;

Impaired glucose tolerance (IGT) and/or impaired fasting glucose (IFG);

Weight stable for at least 3 months (± 2 kg);

No use of antibiotics, pre- or probiotics for 3 months

Intervention groups:

1. Vivinal GOS 3x 5 gram per day for 12 weeks

2. Maltodextrin 3x 4.4 gram per day for 12 weeks (isocaloric placebo)

IGT: 2h plasma glucose during 75g OGTT 7.8-11.1 mmol/l

IFG: plasma glucose ≥ 5.6 mmol/l

of Human Biology Canfora et al, Gastroenterology, july 2017

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Pre Post2.5

3.0

3.5

4.0

4.5

5.0

5.5

GOS

Bif

ido

ba

cte

riu

m (

log

10

sig

na

l in

ten

sit

y)

Pre Post2.5

3.0

3.5

4.0

4.5

5.0

5.5

Placebo

Bif

ido

ba

cte

riu

m (

log

10

sig

na

l in

ten

sit

y)

B

GOS increased faecal Bifidobacterium spp.

5.0 ± 0.3 fold increasedrelative abundance (P=0.009)

Canfora, et al. (2017), Gastroenterology


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GOS did not affect faecal and plasma SCFA concentrations

Acetate Propionate Butyrate

Faeces

Plasma

Canfora et al, Gastroenterology, july 2017of Human Biology

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GOS did not affect markers of insulin sensitivity

GOS Placebo0

1

2

3

4Pre

Post

M-v

alu

e (

mg

/kg

/min

)

Pre Post0

2

4

6

8

GOS

M-v

alu

e (

mg

/kg

/min

)

Pre Post0

2

4

6

8

Placebo

M-v

alu

e (

mg

/kg

/min

)

A B

Adipose tissueInsulin sensitivity

HOMA-IR

Pre

Post

GOS Placebo0

20

40

60

80

100Pre

Post

FF

A s

up

pre

ss

ion

(%

)

D

GOS Placebo0

2

4

6

8Pre

Post

HO

MA

-IR

C

Peripheral Insulin sensitivity


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Prebiotics studiesAltogether, the evidence points to the production of SCFA as the primary mechanism by which prebiotics mediate the beneficial effects on satiety and adipose tissue metabolism. Controversial findings remain and may relate to the site of fermentation of the prebiotics and the metabolic phenotype of the studied subjects.

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Does one size fits all?

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Towards personalised approaches

0 10 20 300

10

20

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50A

B/F-ratio

Rd, µm

ol/m

2/m

in

0 10 20 300

20

40

60

80

100C

B/F-ratio

suppre

ssi

on E

GP, %

0 2 4 6 80

10

20

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50B

B/F-ratio

Rd, µm

ol/m

2/m

in

0 2 4 6 80

20

40

60

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100D

B/F-ratio

suppre

ssi

on E

GP, %

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Males females

Most et al, Benef Microbes. 2017 Aug 24;8(4):557-562

0 10 20 300

10

20

30

40

50A

B/F-ratio

Rd, µm

ol/m

2/m

in

0 10 20 300

20

40

60

80

100C

B/F-ratio

suppre

ssi

on E

GP, %

0 2 4 6 80

10

20

30

40

50B

B/F-ratio

Rd, µm

ol/m

2/m

in

0 2 4 6 80

20

40

60

80

100D

B/F-ratio

suppre

ssi

on E

GP, %

males females

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In Summary

• Feces transplantation studies do show a role of microbiota in weight

gain and insulin resistance, effects in humans are relatively minor

• Modulation of microbiota by means of antibiotics did not affect

metabolic health after 7 days and in the longer term

• SCFA may be an important link between gut microbiota and metabolic

health

• Distal, but not proximal, acetate infusion may improve fat oxidation

and metabolic profile

• Rectal infusion of SCFA combinations all increase fat oxidation,

energy expenditure and metabolic profile

• We propose that acetate is the main driver of this effect

• Dietary fiber intervention focussed on targeted production of SCFA

may improve intervention outcome with respect to metabolic profile,

but this requires further confirmation

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Collaborations/Acknowledgements

• Gijs Goossens

• Johan Jocken

• Birgitta van der Kolk

• Dorien Reijnders

• Emanuel Canfora

• Rudi Stinkens

• Max Vogel

• Jasper Most

• Mattea Müller

• Kenneth Verboven

• Yvonne Essers

• Nicole Hoebers

Dept Human Biology, MUMC

And external

Collaborations

Top Institute Food and Nutrition

(project GH003, microbiota, energy balance

and metabolism)

EU consortia: EU-Lipgene, MIRdiet, EDIPS, Diogenes

Department

Future Directions…..

The roleof gut microbiotain the pathogenesisof obesity...

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