Artificial sweeteners induce glucose intolerance by altering the gut microbiota
Authors: Zamir Halpern
Alon Harmelin
Gili Zilberman-Schapira
Yael Kuperman
Eran Segal
Ilana Kolodkin-Gal
David Zeevi
Hagit Shapiro
Tal Korem
Jotham Suez
Shlomit Gilad
Niv Zmora
Christoph A Thaiss
David Israeli
Ori Maza
Adina Weinberger
Eran Elinav
Journal: Nature (2014)
Discipline: Multidisciplinary
Keywords: Non-caloric artificial sweeteners; NAS; glucose intolerance; intestinal microbiota; antibiotic treatment
Affiliations: Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel., 1] Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot 76100, Israel [2]., 1] Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel [2]., Day Care Unit and the Laboratory of Imaging and Brain Stimulation, Kfar Shaul hospital, Jerusalem Center for Mental Health, Jerusalem 91060, Israel., 1] Internal Medicine Department, Tel Aviv Sourasky Medical Center, Tel Aviv 64239, Israel [2] Research Center for Digestive Tract and Liver Diseases, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel [3] Digestive Center, Tel Aviv Sourasky Medical Center, Tel Aviv 64239, Israel., The Nancy and Stephen Grand Israel National Center for Personalized Medicine (INCPM), Weizmann Institute of Science, Rehovot 76100, Israel., Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot 76100, Israel., Department of Veterinary Resources, Weizmann Institute of Science, Rehovot 76100, Israel., Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel., 1] Research Center for Digestive Tract and Liver Diseases, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel [2] Digestive Center, Tel Aviv Sourasky Medical Center, Tel Aviv 64239, Israel.

Abstract:  Non-caloric artificial sweeteners (NAS) are among the most widely used food additives worldwide, regularly consumed by lean and obese individuals alike. NAS consumption is considered safe and beneficial owing to their low caloric content, yet supporting scientific data remain sparse and controversial. Here we demonstrate that consumption of commonly used NAS formulations drives the development of glucose intolerance through induction of compositional and functional alterations to the intestinal microbiota. These NAS-mediated deleterious metabolic effects are abrogated by antibiotic treatment, and are fully transferrable to germ-free mice upon faecal transplantation of microbiota configurations from NAS-consuming mice, or of microbiota anaerobically incubated in the presence of NAS. We identify NAS-altered microbial metabolic pathways that are linked to host susceptibility to metabolic disease, and demonstrate similar NAS-induced dysbiosis and glucose intolerance in healthy human subjects. Collectively, our results link NAS consumption, dysbiosis and metabolic abnormalities, thereby calling for a reassessment of massive NAS usage. 

Retracted: No

PubPeer comments:
 # This study is about saccharin, not artificial sweeteners in general. ### It shows that saccharin causes a + -20% change in ~40 gut microbiome taxa, leads to altered glucose tolerance, but leaves insulin tolerance and levels identical. Effect is similar in mice and human I am a pre-diabetic patient with a PhD in genomics and I would like to understand the effect of *Artificial sweeteners the gut microbiota*. This paper is an extremely highly cited (1400+) paper in the most prestigious journal in the scientific world. Thus I dug into this paper. ### 1. How much sweetener? Science is complicated, but ultimately you have to be clear about: - We added X amount of substance and - We observed Y. ***So how many mg of each commercial sweetener did the mice consume?*** It seems that this is basically not disclosed. ----- In the methods they only write: **They prepare a 10% solution of 3 commercial sweeteners, in drinking water**. (Of note: they are clearer in the pure saccharin experiment, see below) So: - How much drinking water did each experiment ? t - While not disclosed here, typically (in other studies) infinite water is provided to mice. (See followup) - Was the addition of the commercial sweeteners affect the water consumption? - They added 10% sweetener to water, it is crazy concentrated, see below. - I expect the some of following effects: - Small sweetness makes animals consume more water (bliss point, below) - Very strong sweetness can be repulsive u2192 less consumption - **The  liquid mice drink has ~10% sugar in all sweetener conditions** (besides the sweetener, below), mice should drink more, because it does not quell thirst as good as pure water. - This is the sugar equivalent of drinking *non-diet* soda instead of water - Also note that sweeteners have very different relative sweetness **in human**: *Saccharin~350x; Aspartame ~200x; Sucralose ~200-1000x (times table sugar)* yet they were given the same amount, meaning the the Sucralose-water was 3.3x sweeter than the Aspartame-water. They do write that average water consumption is 2ml, but unspecific in which condition, in fact it is written for another experiment (pure saccharin). **From Methods:** **Artificial and caloric sweeteners.** The following commercially available NAS were dissolved in mice drinking water to obtain a 10% solution: Sucrazit (5% sac- charin, 95% glucose), Sucralite (5% Sucralose), Sweetu2019n Low Gold (4% Aspartame). 10% glucose (J. T. Baker) and 10% sucrose (Sigma Aldrich) solutions were used for controls. [...] [commenter:] **u2192 That is 100 mg ml** For experiments conducted with pure saccharin (Sigma Aldrich) a 0.1 mg ml solution was used in order to meet with FDA defined ADI for saccharin in humans (5 mg per kg (body weight)), according to the following calculation: [...] u2192 **0,1 mg ml** ### 2. Relevance ##### For the commercial sweeteners experiment: - They prepare a 10% solution and replace drinking water with it. (No further details, so I assume they mean 10g in 100 ml water) - This is crazy sweet: One saccharin tablet (that it take) is 60mg (similar % of saccharin). So in a glass of water (250g), I would put 25g sweetener for 10%, that is 417 tablets of saccharin. - Did the mice even drink any  drinking water ? This is so sweet that definitely no human would drink it. - At least in humans low amount of sweetness would increase drinking, while high amount would decrease (known as [bliss point] ) in food industry, see also [here] , or [here] ) ##### Relevance notes on the sugar content Because they use a high amount of ready commercial sweetener (and not pure sweeter), which is a sweetener (4% - 5%) : glucose (95%) mix, - The mice are on a 190mg sugar day diet for a long time (= 2g water * 10% commercial sweetener * 95% sugar content) - As a comparison, the OGTT test they do involves 40mg sugar. - Another comparison: this would mean you as a human to consume 1 2 kg pure additional sugar a day: | Value | Explanation | | ----- | ------------------------------------------------------------ | | 0.03 | average mouse weight (methods) | | 80 | example human weight | | 2667 | times heavier (human mice) | | 190 | mg sugar per day consumed by mice (above, calculated from methods) | | 0.51 | **kg** sugar per day, ***consumption equivalent by human*** | **Interestingly, this difference in sugar consumption** (0 in water control, 190mg day in sugar controls) **have absolutely no effect on developing glucose intolerance** 11 weeks later (Fig.1.c) u2013 the 3 black lines are all the same: ![Image] This supports their claim that sugar has no effect on  diabetes , only sweeteners. It is surprising to me based on what doctors say about sugar and diabetes. ##### Relevance notes on the pure saccharin experiment: - The mice drink 5 mg kg (body weight) saccharin. - That is for 80kg human 400 mg saccharin per day. - Which is the **equivalent of 50 saccharin tablets per day for a human.** - I eat 3 tablets a day (also consume sugar). ### 3. How much effect? While figure 3 shows fold changes *upon fecal transplants into germ free mice*, [Extended Data Figure 6 ] shows **the fold change in the actual pure-saccharin feeding experiments**. - **They identify around ~40 taxa, that change + or - 1.2 fold (=+-20%)**. - (Unclear to me how much % of the total microbiome is this 40 taxa, e.g. by biomass) ![img] This is then correlated with impaired glucose tolerance in the saccharin condition, but these mice display normal **insulin levels and insulin tolerance** ([Extended Data Figure 5] ). In the fecal transplantation study they induce up to 2 log ! fold changes, which then leads to impaired glucose tolerance. But this is an artificial change in microbiome that was not caused by saccharin consumption: - 1.2 fold change in some taxa when consuming very high amount of pure saccharing - 4 fold change in some taxa after fecal transplant. (=logn(2) = 2.718^2) So fecal transplant seem to induce much bigger changes in microbiome (and possibly in different taxa) than saccharin. This change (which is not observed under saccharin treatment) then also leads to glucose intolerance, as in the saccharin treatment . ### ### 4. Limited Conclusions I would like to engage the authors or anyone in the field to clarify these, but without further comment, I find it hard to draw solid conclusions from this study. 1. **One thing is clear, that this study is about saccharin, not about artificial sweeteners.** 2. Saccharin induces a ~20% change in ~40 taxa in your gut (but not clear if this 40 taxa are 1% or 100% of your gut metabiome-biomass or all taxa present). 3. Saccharin leads to impaired glucose tolerance but insulin tolerance or levels are unaffected. 4. This effect is conserved in 4 out of 7 human participants, and this 4 already had a different microbiome before taking saccharine. (In Fig 4., not discussed here.) **These sound rather different than the title suggests.** In details: The commercial sweetener part of the paper is has a large number of confounders (Unclear how much sweetener consumed; Excess glucose; Excess water; 8x higher liquid consumption) and therefore I think its hard to conclude anything. (*E.g Saccharin has impact Y, but the saccharin condition drinks ~9x more sugary water, thus consumes 100% more additive sugar, and eats half of their normal food. Plus they have massively increased metabolism.* In later supplements you find evidence that most of this is due to sugar, not saccharin.) **Also, most findings of the study are not challenged here**, it seems like a very long reaching study. I just find it super weird that a Nature is fine with publishing a paper that essentially does not describe how much sweetener the mice consumed. Please correct me if I misunderstood something, probably this summary contains error, I spent a few hours, not month on it. Looking forward for discussion. #### Disclosure: I have no financial, scientific or other conflict of interest. --------------------------------------------------------------------------------------------------- ### 5. Followup When you go to [Extended Data Figure 3] , you find that the effects I described in (1.) are actually dramatic: 1. **Commercial Saccharin dramatically increases the  water intake 9 fold** (= 90% water, 9.5% sugar, 0.5% saccharin). In 72 hrs, 1. ~ 70 ml iquid is consumed by saccharin condition 2. ~ 8 ml iquid is consumed by pure water condition 1. That is ~9x more liquid, (that as reminder is 10% sugar vs. water) ![ExtFig.3] In **Extended Data Figure 4** it becomes clear that when you add pure saccharin (opposed to 95% sugar+ saccharin) the liquid intake equals pure water: (Same for food consumption, *however these mice are on high fat diet, not on regular diet as in the commercial sweetener test.*) ![ExtFig.4a] The relationship to sugar content is unclear (Ext. Fig 3 C), but mice consume very different amounts of food: anywhere from 4.5 to 9.5 g in 72hours. ![ExtFig.4C] , 
  the OGTT test they do involves 40mg sugar. Oral glucose tolerance tests in adults usually give 75 grams of glucose, a little over a gram per kilogram of body weight in a 70 kg person. ,