The roleof gut microbiotain the pathogenesis of obesity, insulin resistance and type 2 diabetes mellitus

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1 The roleof gut microbiotain the pathogenesis of obesity, insulin resistance and type 2 diabetes mellitus Ellen Blaak EASO New Investigators united, Autumn School 2017, Palma de Mallorca Professor in Physiology of fat metabolism, NUTRIM School of Nutrition and Translational Research in Metabolism Maastricht University Medical Centre + The Netherlands 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 1

2 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 >2000 species Courtesy of Roderick Mackie and Glenn Gibson Microbiota:fermentation transverse colon: combination of saccharolytic and proteolytic fermentation proximal colon: primarily saccharolytic fermentation distal colon: primarily proteolytic fermentation 2

3 Microbiota is individual specific Is there a normal microbiota? 57 species common >90% individuals ~ genes (at least 50% of the individuals) Qin et al. Science Compositional vs. functional differences Adapted from: Miguel Gueimonde Turnbaugh et al. Nature Microbiota is individual specific Challenge: definition of a normal/healthy microbiota Inter-individual variation Geographical differences Adapted from: Miguel Gueimonde 3

4 Hadza tribe in Tanzania Schnorr et al., Nat Commun. 2014;5:3654 Hadza tribe: different microbial genes Rampelli et al., Curr Biol. 2015; 25(13):

5 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 Obesity pandemic of 21 st century Department 5

6 Obesity: an important risk factor for chronic metabolic diseases OBESITY Cardiovascular disease Type 2 diabetes Liver steatosis Cancer 6

7 Gut microbiota in Human adults Outcome in human studies is complex 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 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) Feces transplantation from mice to mice 7

8 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 From man to mice: Gut microbiota from twins discordant for obesity modulate metabolism in mice V K Ridaura et al. Science 2013;341:

9 From man to man: feces transplantation and human metabolism Transfer of Intestinal Microbiota From Lean Donors Increases Insulin Sensitivity in Individuals With Metabolic Syndrome Vriezeet al,

10 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. 10

11 Gut microbiota in overweight, insulin resistance and diabetes Microbiota Complex CHO Hypertrophic adipocytes Bile acids L-cell SCFA Preadipocyte recruitment GPCR ANGPTL4 GLP1 PYY Altered adipokine secretion Insulin resistance Reduced fatty acid trapping Impaired lipolysis Energy Harvest, Metabolic effects SCFA LPS Lipid spill-over Systemic inflammation Lipid accumulation Insulin resistance Inflammation Liver Impaired fat oxidation Lipid accumulation Insulin resistance Muscle Pancreas Altered insulin secr/prod Altered glucagon secr/prod Antibiotics intervention Treatment Placebo Amoxicillin Vancomycin 1500 mg/day 7 days 40-70y IFG/IGT HOMA-IR>2.2 n=56 Timing wk 1 pre wk 2 2d wash out wk 3 post wk 8 followup Reijnders et al, Cell metabolism,

12 Distinct changes in microbiota composition Vanco: Increased gram negative Proteobacteria Microbiota composition less grampositive bacteria decreased diversity After 8 weeks similarity with baseline still lower, diversity slightly lower Amox: no diversity and/or composition differences Reijnders et al, Cell metabolism, 2016 No effect on insulin sensitivity Two-step hyperinsulinemic-euglycemic clamp Endogenous Glucose Production Rate of Disappearance No effect on whole body insulin resistance Suppression of Free Fatty Acids 8 wk after stopping the active intervention % Reijnders et al, Cell metabolism,

13 Reijnders et al, Cell metabolism, 2016 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? 13

14 Function microbiota: saccharolytic en proteolytic activity Saccharolysis leads primarily to SCFA and gasses SCFA: acetate, propionate, butyrate lactate -> only accumulates when there is a fast fermentation CO 2, CH 4, H 2 Proteolysis in addition leads to toxic metabolites BCFA: iso-butyrate, iso-valerate ammonia phenolics: phenol, indol, p-cresol, skatol H 2 S, CH 3 SH, etc Site of fermentation transverse colon: combination of saccharolytic and proteolytic fermentation proximal colon: primarily saccharolytic fermentation distal colon: primarily proteolytic fermentation 14

15 Major SCFA Acetate Propionate Butyrate Functionality of microbiota: SCFA Canfora, E. E. et al. (2015) Short-chain fatty acids in control of body weight and insulin sensitivity Nature Reviews Endocrinology doi: /nrendo

16 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? Distal and proximal colonic acetate infusion and metabolic profile Aim: To investigate differential effects of proximal and distal colonic infusions with sodium acetate on human fat oxidation, energy expenditure and circulating metabolic markers Study design Double blind, placebo controlled, randomized crossover study Six healthy overweight males (BMI kg/m 2 ) Aged 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 Van der Beek et al, Clin Sci

17 Intervention protocol Van der Beek et al, Clin Sci 2017 Test day protocol Van der Beek et al, Clin Sci

18 Fat oxidation break Fat oxidation (g fat 2h -1 ) iauc, distal * p=0.055 Placebo 100 mmol/l 180 mmol/l placebo 100mmol/L 180mmol/L break ANOVA * p<0.05 placebo vs 180mM Ingestion oral glucose solution Van der Beek et al, Clin Sci 2017 Distal, not proximal, colonic acetate infusion improves metabolic profile No significant effects Circulating acetate Fat oxidation PYY TNF-α Lipolysis Increasing colonic and systemic acetate beneficially affect the metabolic profile Validated distal colonic infusion as a good model to study SCFA effects on metabolism Van der Beek et al, Clin Sci

19 Combinations of SCFA and metabolic profile To investigate acute effects of distal colonic infusions of SCFA combinations on substrate and energy metabolism Canfora et al, Scientific reports, 2017 Intervention protocol (1) Double blind, placebo controlled, randomized crossover study with 4 distal infusions: 100 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 % SCFA/NaCl in solutions :20:20 45:35:20 45:35:20 45:20:35 Placebo sodium chloride sodium propionate sodium butyrate sodium acetate All diluted in 200mL water Canfora et al, Scientific reports,

20 SCFA increase Fat oxidation *** *** p<0.001, ** p<0.01 Ingestion oral glucose Canfora et al, Scientific reports, 2017 SCFA increase Energy Expenditure * iauc energy expenditure, fasted iauc EE kj/t0-t * *** # *** p<0.001, * p<0.05, # p<0.1 Ingestion oral glucose solution placebo high acetate high propionate high butyrate Canfora et al, Scientific reports,

21 Circulating acetate is associated with fat oxidation and energy expenditure Fa stin g fa t o xid a tio n (g fa t 2 h -1 ) Fasting plasma acetate (µmol/l 2h -1 ) R e stin g e ne rg y e xp e nd iture (kj/2h -1 ) Fasting plasma acetate (µmol/l 2h -1 ) Acetate vs. fat oxidation r=0.328 (P=.0228) Acetate vs. energy expenditurer=0.349 (P=.0149) Canfora, et al. (2017), Scientific Reports (accepted) Department Acetate maybe a target to prevent and treat obesityrelated co-morbidities Fat oxidation Energy Expenditure PYY IL-1β lipolysis What are the underlying mechanisms? Canfora et al, Scientific reports,

22 Why do we hypothesize that the distal colon should be targeted? Higher density of PYY-producing L-cells in the distal colon Kuwahara (2014), Frontiers in Endocrinology Department Increased fat oxidation and EE only after distal administration: Acetate bypass the liver circulating acetate uptake in oxidative tissues pampk fat oxidation Acetate pampk fat oxidation 1,2,3 Circulating Acetate Acetate pampk fat oxidation 1,2,4 1 Sakakibara et al., 2006; 2 Yamashita et al., 2009; 3 Kimura et al., 2013; 4 den Besten et al.,

23 Fermentation of fibers by the gut microbiota proximal versus distal fermented distally Which fibers are best? Canfora, et al. (2017), Scientific Reports;7(1):2360 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) 23

24 Beneficial effectsof 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 Department Beneficial effects of prebiotics a snapshot (2) Targeted delivery of proprionate to the colon acutely increases PYY and GLP1 and reduces energy intake Chambers ES et al, Gut,

25 Beneficial effects of prebiotics a snapshot (3) Towards dietary intervention study with galactooligosaccharides targeting colonic acetate Study design Double blind, placebo controlled, randomized parallel study 46 volunteers: Males and postmenopausal females aged years; BMI kg/m 2; 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 IGT: 2h plasma glucose during 75g OGTT mmol/l IFG: plasma glucose 5.6 mmol/l 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) Canfora et al, Gastroenterology, july 2017 GOS increased faecal Bifidobacterium spp. 5.0 ± 0.3 fold increased relative abundance (P=0.009) B Bifidobacterium (log 10 signal intensity) Canfora, et al. (2017), Gastroenterology Pre GOS Post Bifidobacterium (log 10 signal intensity) Pre Placebo Post Canfora et al, Gastroenterology, july

26 GOS did not affect faecal and plasma SCFA concentrations Acetate Propionate Butyrate Faeces Plasma Canfora et al, Gastroenterology, july 2017 GOS did not affect markers of insulin sensitivity Peripheral Insulin sensitivity A M -v a lue (m g/k g/m in) Pre Post B M -value (m g/kg/m in) M -value (m g/kg/m in) GOS Placebo 0 Pre Post 0 Pre Post GOS Placebo HOMA-IR C H O M A -IR Pre Post Pre Post Adipose tissue Insulin sensitivity D FFA suppression (% ) Pre Post 0 GOS Placebo 0 GOS Placebo Canfora et al, Gastroenterology, july

27 Prebiotics studies Altogether, 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. Department Does one size fits all? Department 27

28 Towards personalised approaches A A Rd, µmol/m 2 /min Rd, µmol/m 2 /min males Males B/F-ratio B/F-ratio B B Rd, µmol/m 2 /min Rd, µmol/m 2 /min females females B/F-ratio B/F-ratio C 100 C B/F-ratio B/F-ratio suppression EGP, % suppression EGP, % D 100 D B/F-ratio B/F-ratio suppression EGP, % suppression EGP, % Most et al, Benef Microbes Aug 24;8(4): 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 28

29 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.. Department 29