This study examined the effects of artificial sweeteners on the gut microbiome. Mice given saccharin, aspartame, or sucralose showed glucose intolerance and changes to their gut bacteria including increased Bacteroides and Clostridiales. Fecal transfers from saccharin-fed mice reproduced these effects in germ-free mice. Human studies found correlations between sweetener use and increased fasting glucose as well as changes to gut bacteria. Fecal transfers between human responders and non-responders to sweeteners showed causal effects on the microbiome. The research suggests artificial sweeteners disrupt the gut microbiome in ways that decrease glucose tolerance.

1. T i f f a n y L y o n s
Artificial Sweeteners and the
Gut Microbiota

2. Overview
 Diabetes health trends and statistics
 Introduction to artificial sweeteners
 Study on diet soda
 Study on obese mice
 Primary research topic – Suez et al.
 Ingestion
 Sequencing
 Human trials
 Conclusions and future research

3. Diabetes
Statistics
Number of non-
institutionalized with
diagnosed diabetes.
1980 = 5.6 million
2011 = 20.9 million

4. Diabetes Statistics
 Diabetes in America
 2014 = 29 million
 9.3% of the population
 Pre-diabetes = 86 million
 $245 billion in medical cost
 Dietary sugar and caloric consumption are, “too
high” AHA & ADA.

5. T w o n a m i n g c o n v e n t i o n s
• n o n - n u t r i t i v e s w e e t e n e r s ( N N S )
• n o n - c a l o r i c a r t i f i c i a l s w e e t e n e r s ( N A S )
Introduction to Artificial
Sweeteners

6. Introduction to Artificial Sweeteners
 Sucralose
 Aspartame
 Saccharin
 Acesulfame-k
 Sugar Alcohols
 6000 products between 1999-2004
 40% increased use since 2000

7. 7 y e a r o b s e r v a t i o n a l s t u d y c o n d u c t e d b y
N e t t l e t o n e t a l . 2 0 0 9
Diet Soda Study

8. Diet Soda Study
 Food consumption questionnaires (2000-2002)
 Incident diabetes at 3 visits (2002-2007)
 Fasting glucose >126 mg/dl
Or
 Metabolic syndrome
 Consumption of at least 1 diet soda/per day
 36% increased risk of DM2
 67% increased risk of metabolic syndrome
 Adjusted for confounders

9. Diet Soda Study
“…observational data cannot establish causality,
consumption of diet soda at least daily was
associated with significantly greater risks of select
incident metabolic syndrome components and type 2
diabetes”
Nettleton et al. 2009

10. S t u d y c o n d u c t e d b y T u r n b a u g h e t a l . ( 2 0 0 6 )
c o m p a r i n g a n d s e q u e n c i n g t h e g u t m i c r o b i o m e
o f o b e s e , l e a n , a n d l e p t i n d e f i c i e n t m i c e
Obesity and the Gut
Microbiome

11. Obese Mice
 Experiment 1: Germ-free mice colonized with distal
gut microbiota from standard mice
 Increased body fat within 10-14 days
 Experiment 2: Leptin deficient mice
 16SrRNA sequencing in (ob/+ and +/+)
 Over represented Bacteriodes & Firmicutes

12. Obese Mice

13. Primary Research Article

14. Introductory Facts
 Cohorts of randomly chosen C57Bl/6 WT adult male
mice
 Adult male out-bred Swiss Webster germ-free
 Artificial sweeteners: 95% glucose or fructose

15. NAS Consumptions – Mice
 Supplemented drinking water (10% solution)
 Aspartame, sucralose, or saccharin
 Glucose
 Sucrose
 Plain water
 Below toxic level and ADI
 11 weeks
 Glucose tolerance tested at 0, 15, 30, 60, 90, and 120
min.

16. NAS Consumptions – Mice

17. NAS Consumptions – Mice
 Correlation between high fat diet (HFD) and glucose
intolerance (obesity set-up)
 60% of total kcal
 Commercial saccharin vs. glucose control
 Pure saccharin vs. plain water control
 ADI ~5mg/kg (body weight)
 Out bred Swiss Webster mice

18. Commercial Saccharin Pure Saccharin
NAS Consumptions – Mice

19. NAS Consumptions – Mice
 Metabolic profiling PhenoMaster
 Liquids
 Chow
 Oxygen
 Kcal expenditure (movement)
 Serum insulin (fasting and non)
 Confounding

22. Microbiota Connection
 Cohorts
 Lean and HFD
 Commercial or pure NAS
 Glucose or water
 Antibiotics
 Ciprofloxacin and metronidazole
 Vancomycin
 Tested after 4 weeks

23. Microbiota Connection

24. Microbiota – Causal Relationship
 In vitro fecal cultures
 Anaerobic
 PBS
 Meat carbohydrate broth
 Commercial saccharin or pure saccharin
 Incubated 9 days
 Inoculated germ free mice
 Glucose tolerance tested after 6 days

25. Commercial Saccharin Pure Saccharin
Microbiota – Causal Relationship

26. Sequencing
 16SrRNA
 Week 0 vs. week 11 for all cohorts
 Donor and transplant cohorts
 40 different OTU
 Increased
 Bacteroides genus
 Clostridiales order
 Decreased
 Lactobacillus reuteri
 Clostridiales

27. Sequencing Continued
 Shotgun metagenomic sequencing
 Human genome microbiome project
 Increased Bacteroides vulgatus
 Decreased Akkermansia mucinicphila
 Gut microbial gene catalogue
 Kyoto Encyclopedia of Genes and Genomes grouped – 115
pathways
 Heat sensing

29. Sequencing Continued
 Glycan degradation
 Produce short chain fatty acids (SCFAs)
 Increased energy harvest
 Associated with human and mice obesity
 High levels of propionate and acetate in stool (sacch)
 Gram-positive and gram-negative bacteroides
 Other alterations
 Decreased glucose transport pathways
 Increased starch, sucrose, fructose, and mannose metabolism.
Increased glycerolipid and fatty acid biosynthesis

30. Sequencing Continued

31. Sequencing Continued
“Altogether, saccharin consumption results in
distinct diet-dependent functional alterations in the
microbiota, including normal-chow-related
expansion in glycan degradation contributed by
several of the increased taxa, ultimately resulting in
elevated stool SCFA levels, characteristic of
increased microbial energy harvest.”
Suez et al 2014

32. Direct Saccharin Correlation
 In Vitro cultures from germ free mice
 Cultured anaerobically in the presence of saccharin or control
 9 day incubation
 Gavage administration
 https://www.youtube.com/watch?v=oYCmKIhveFY
 Fecal samples
 16SrRNA
 Shotgun metagenomic sequencing

33. Direct Saccharin Correlation

34. Human Trials 1
 381 individuals
 44% males
 56% females
 Age 43.3 (+/- 13.2 years)
 Usage questionnaire
 BMI
 Body circumference
 Fasting glucose
 Complete blood and chemistry counts

35. Human Trials 1
 Positive correlations
 Fasting glucose
 HbA1C
 Elevated serum alanine aminotransferase (note card)
 Statistically corrected for BMI and body circumferences
 16SrRNA
 172 randomly selected
 Enterobacteriaceae, Deltaproteobacteria, and Actinobacteria
phylum
 Correlation not seen in BMI (note card?)

36. Human Trials 2
 7 healthy volunteers
 5 males
 2 females
 Age 28-36
 1 week
 Days 2-7 maximum ADI (3x40mg doses)
 4 out of 7 responded
 Decreased glucose tolerance 5-7 days after consumption
 16SrRNA sequencing on fecal samples

38. Human Trial 2 – Causal Relationship
 Fecal transplantation
 Samples from 2 responders & 2 non-responders
 Days 1 and 7
 Germ-free mice
 Results
 Responders showed considerable disbyosis between day 1 & 7
 Responders clustered differently from non-responders

39. Responder Non-responder
Human Trial 2 – Causal Relationship

40. Conclusion and Future Research
 NAS mediate deleterious changes to the gut
microbiome that decrease glucose tolerance
 Humans trials support the mediation of dysbiosis via
NNS or NAS
 Exact amount in human trials
 Non-responders
 Differences of gut microbiota
 Increased exposure time