Journal Club Presentation Basis:
Nature. 2014 Oct 9;514(7521):181-6.
Feehley T, Nagler CR.
Nature. 2014 Oct 9;514(7521):176-7.
• Background:
– Non-Caloric Artificial Sweeteners (NAS)
– Microbiota
• Research article data:
– Mouse and Human studies
• Summary
• Discussion
Background: Non-Caloric Artificial Sweeteners (NAS)
• Introduced over a century ago
• Gained popularity due to:
– Reduced costs
– Low caloric intake
– Perceived health benefits for weight reduction and
normalization of blood sugar levels
• NAS consumption studies:
– Some show benefits: little induction of a glycaemic response
– Others show associations with weight gain, increased risk of
type 2 diabetes
– Interpretations complex due to NAS consumption by individuals
with existing metabolic syndrome manifestations
• FDA approved six NAS products for use in the US
Gardner et al. Diab. Care. 35(8):1798-808 (2012).
Fitch. J Acad Nutr Dietetics. 112:739–758 (2012).
Tordoff et al. Am. J. Clin. Nutr. 51:963–969 (1990).
Horwitz, et al. Diab. Care. 11:230–234 (1988).
Nettleton et al. Diab. Care. 32:688–694 (2009).
Background: Non-Caloric Artificial Sweeteners (NAS)
• Metabolism:
– Most NAS pass through GI tract without being digested by the host  directly
encounter the intestinal microbiota  central role in regulating multiple
physiological processes
Stringlike filaments of microbes grow on intestinal cells.
Credit: Weizmann Institute of Science
Background: Microbiota Development
Clemente et al. Cell 148, 1258–1270 (2012).
Background: Microbiota/host interactions
Microbiota and NAS Study
• Question: Can non-caloric artificial
sweeteners modulate the composition
and/or function of the gut microbiota
and thus affect host glucose
Credit: Weizmann Institute of Science
Experimental scheme and dosage
Commercially available NAS:
Pure saccharin:
10% solution: Sucrazit (5% saccharin,
95%glucose), Sucralite (5% Sucralose),
Sweet’n LowGold (4% Aspartame)
Well below reported toxic doses
0.1mg ml-1 solution – to meet with FDA
defined acceptable daily intake (ADI) for
saccharin in humans (5mg per kg (body
weight)), according to the following
Controls dosage:
10% solution glucose
10% solution sucrose
NAS-consuming mice developed glucose intolerance
a, b
Commercial NAS:
Controls dosage:
10% solution: Sucrazit (5% saccharin,
95%glucose), Sucralite (5% Sucralose),
Sweet’n LowGold (4% Aspartame)
Well below reported toxic doses
10% solution glucose
10% solution sucrose
Antibiotics regimens:
• Gram-negative targeting regimen A
(ciprofloxacin, metronidazole)
• Gram-positive targeting regimen B
Fig. 1
NAS-induced glucose intolerance is mediated
*** p < 0.001
through alterations to the commensal microbiota
Corroborating the findings in the obesity (HFD) setup:
NAS-consuming mice developed glucose intolerance
Pure saccharin:
0.1mg ml-1 solution – to meet the FDA defined
acceptable daily intake (ADI) for saccharin in
humans (5mg per kg (body weight))
High-Fat Diet (HFD):
60% kcal from fat
* p < 0.03
glucose intolerance
is strain
altered by microbiota
Also in Swiss-Webster
Fig. 1
Metabolic profiling of normal-chow- or HFD-fed mice
showed similar measures between NAS- and control-drinking mice
Liquids and chow consumption
Oxygen consumption
Walking distance and energy expenditure
Supp. Fig. 3, 4
Glucose intolerant NAS-drinking mice display normal
insulin levels and tolerance
Supp. Fig. 5
Causal role of the microbiota:
Faecal transplantation into normal-chow-fed germ-free mice
* p < 0.03
* p < 0.05
Metabolic derangements induced by NAS consumption
are mediated by the intestinal microbiota
Fig 1, Supp. Fig. 2
** p < 0.01
NAS mediate distinct functional alternations to the microbiota
• Saccharin consuming mice compared to controls:
– Considerable dysbiosis in the microbiota of
saccharin-consuming mice
– Alterations in > 40 operational taxonomic units
– Increases in Bacteroides
– Decreases in Clostridiales
• In germ-free recipients of stools from saccharinconsuming donors:
– Mirroring of OTUs observed
Saccharin consumption in various formulations, doses and
diets induces dysbiosis with overall similar configurations
Functional characterization of saccharin-modulated
• To compare relative species abundance:
– Shotgun metagenomic sequencing of faecal samples – genetic analysis
to examine environmental samples abundant in microscopic species
Saccharin induced the largest changes in microbial relative
species abundance
Fig. 2a, Supp. Fig. 7a, 7b
Functional characterization of saccharin-modulated
Genetic pathways abundance:
Mapped metagenomic reads to a gut microbial gene catalogue, grouping genes into KEGG
(Kyoto Encyclopedia of Genes and Genomes)
– Found changes in pathway abundance to be inversely correlated between commercial
saccharin- and glucose-consuming mice
-> Saccharin greatly affects microbiota function:
- among over-represented pathways is increased glycan degradation: glycans are fermented
to form various compounds including short chain fatty acids (SCFAs) – obesity association
Fig. 2b, c, d
Higher glycan degradation is attributed to five
bacterial taxa
• Gram-negative and positive species
• Consistent with the sharp increase of
the species in the 16S rRNA analysis
(marker of bacterial abundance)
• Consequence of higher glycan
degradation – elevated acetate and
SCFAs propionate
• Other pathways enriched:
– Starch and sucrose metabolism
– fructose and mannose metabolism
– glycerolipid and fatty acid biosynthesis
Fig. 2e,f,g
Saccharin modulates the composition and function of
the microbiome causing dysbiosis
Does saccharin directly affect the microbiota?
 In microbiomes
of diabetic mice
** p < 0.01
Fig. 3, Supp. Fig 8
Human microbiome functioning
• Does the human microbiome function similarly after NAS consumption?
• Population study:
381 non-diabetic individuals: 44% males and 56% females; age 43.3 ± 13.2
High-NAS consumers (40) and non-consumers (236)
Examined the relationship between long-term NAS consumption (based on a
validated food frequency questionnaire) and various clinical parameters
• Clinical parameters found to be increased in NAS consumers compared to
Weight and waist-to-hip ratio
Haemoglobin (HbA1C%) – indicates glucose [c] over the previous 3 months
Glucose tolerance test (GTT, measures impaired glucose tolerance)
Serum alanine aminotransferase (ALT, measures hepatic damage that is likely
to be secondary, in this context, to non-alcoholic fatty liver disease)
Acute saccharin consumption impairs glycaemic
control in humans by inducing dysbiosis
• Non-consumers (236) and high-NAS
consumers (40):
Randomly characterized 16S rRNA
Found positive correlations between
multiple taxonomic entities and NAS
** p < 0.002
Fig. 4
Enterobacteriaceae family (Pearson
r=0.36, FDR corrected P<10-6)
Deltaproteobacteria class (Pearson
r=0.33, FDR corrected P<10-5)
Actinobacteria phylum (Pearson r=0.27,
FDR corrected P<0.0003)
Did not detect statistically significant
correlations between OTU abundances
and BMI  correlations are not due to
distinct BMI
Acute saccharin consumption impairs glycaemic
control in humans by inducing dysbiosis
• Initial assessment of NAS consumption/
blood glucose causation:
• 7 healthy volunteers (NAS nonconsumers):
• 5 males, 2 females, 28 – 36 y.o.
Fig. 4
• 7 day consumption of commercial
saccharin (5 mg per kg (body weight)) as 3
divided daily doses equivalent to 120 mg
• Continuous monitoring by glucose
Acute saccharin consumption impairs glycaemic
control in humans by inducing dysbiosis
• Responders developed
poorer glycemic
response 5-7 d after
• Microbiome
configuration (16s
rRNA analysis) from
responders clustered
differently from nonresponders
• Microbiome
composition changed
in NAS responders
Fig. 4
Acute saccharin consumption impairs glycaemic
control in humans by inducing dysbiosis
• Transfer of
transplanted sample
from responder
induced glucose
intolerance in
recipient germ-free
• Germ-free mice
transplanted with
replicated some of
the donor saccharininduced dysbiosis
* p < 0.004
Orders: Bacteroidales Lactobacillales, Clostridiales
Fig. 4
• NAS-consuming mice developed glucose intolerance
• NAS-regulated glucose intolerance is mediated by gut
• NAS modulate microbiota to induce glucose intolerance
• NAS-altered gut microbiota is functionally altered
• Acute NAS consumption may impair glycaemic control
in humans by inducing dysbiosis
• Several of the bacterial taxa that were altered by NAS
consumption – previously associated with type 2 diabetes:
– Increased Bacteroides, lowered Clostridiales
• Enrichment for glycan degradation pathway – link to enhanced
energy harvest and thus regulation of multiple processes in the
• Comparing current report to other reports is complex, due to
diverse ways of microbiota analysis
• Human response to NAS may be personalized
• Personalized nutrition – personalized medical outcome
Thank you!

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