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!