Journal Club Presentation Basis:
Nature. 2014 Oct 9;514(7521):181-6.
Feehley T, Nagler CR.
Nature. 2014 Oct 9;514(7521):176-7.
Overview
• 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
https://www.sciencenews.org
http://www.nature.com/nature/journal/v449/n716
4/fig_tab/nature06244_F1.html
Background: Microbiota Development
Gram
negative
Gram
positive
Clemente et al. Cell 148, 1258–1270 (2012).
Background: Microbiota/host interactions
http://www.quantumrevolution.net/wpcontent/uploads/2013/02/microbiome.jpg
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
metabolism?
Aspartame
Saccharin
Sucralose
Credit: Weizmann Institute of Science
Experimental scheme and dosage
10wk.o.
•
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
calculation:
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
(vancomycin)
Saccharin
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
•
c
Pure saccharin:
–
d
•
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
NAS-induced
glucose intolerance
is strain
altered by microbiota
Also in Swiss-Webster
mouse
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
Normal
chow
HFD
Supp. Fig. 3, 4
Glucose intolerant NAS-drinking mice display normal
insulin levels and tolerance
a,c
b
Supp. Fig. 5
Normal
chow
HFD
Causal role of the microbiota:
Faecal transplantation into normal-chow-fed germ-free mice
Normal
chow
* p < 0.03
* p < 0.05
HFD
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
(OTUs)
– 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
microbiota
• To compare relative species abundance:
– Shotgun metagenomic sequencing of faecal samples – genetic analysis
to examine environmental samples abundant in microscopic species
High
Low
Saccharin induced the largest changes in microbial relative
species abundance
Fig. 2a, Supp. Fig. 7a, 7b
Functional characterization of saccharin-modulated
microbiota
•
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
Glycan
degradation
pathways
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:
•
•
•
•
Non-randomized
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
non-consumers:
•
•
•
•
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
(172)
Found positive correlations between
multiple taxonomic entities and NAS
consumption:
–
–
–
•
** 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.
Non-responders
Fig. 4
Responders
• 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
measurements
Acute saccharin consumption impairs glycaemic
control in humans by inducing dysbiosis
• Responders developed
poorer glycemic
response 5-7 d after
treatment
• 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
mice
• Germ-free mice
transplanted with
‘responders’
microbiome
replicated some of
the donor saccharininduced dysbiosis
* p < 0.004
Orders: Bacteroidales Lactobacillales, Clostridiales
Fig. 4
Summary
• NAS-consuming mice developed glucose intolerance
• NAS-regulated glucose intolerance is mediated by gut
microbiota
• 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
Discussion
• 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
organism
• 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!