“So, now artificial sweeteners cause glucose intolerance?”

Well, I’m not going to blow my load by telling you what I think just yet, but if you’re at all interested in nutrition, I’m guessing your feed has been overflowing with mentions of the recent Nature paper “Artificial sweeteners induce glucose intolerance by altering the gut microbiota” by Suez and coworkers (Suez et al, 2014). My feed has been bursting with mentions of this, like here, here, here, here and here. Now I’ve had the time to look through it and it is some very extensive and thorough work. I’m guessing that the Nature editors demanded loads and loads of additional documentation because they knew this would be a controversial piece. It is undoubtedly something that has taken several years to prepare and I’m guessing the group behind it has been guarding it with a lot of secrecy until now (they wouldn’t want someone to beat them to the punch). Come along and I’ll try to walk you through their work and provide you with my interpretation.

Intro

The study goes through a number of experiments to gradually build a case that non-caloric artificial sweeteners (NAS) can cause glucose intolerance through a modification of the gut microbiota. Glucose intolerance is an impaired ability to clear glucose from the bloodstream and this is a bad thing because glucose is a highly reactive compound, which reacts in a rather non-specific way with molecules it encounters (this is termed glycosylation). These reaction occurs with speeds that are dependent on the concentration of the reactants, meaning that more blood glucose equals more glycosylation. Glycosylation alters the function of the molecules (think having a massive sign implanted in your forehead) and makes them immunogenic, i.e. irritants to the immune system. Sustained elevated blood glucose slowly damages the circulation, the eyes, tactile senses, the kidneys and pretty much everything else and will slowly kill you.

And while going though a number of experiments in the text (instead of separating it into results and discussion as is more normal in clinical scientific journals)  is a good way to explain a scientific story, the extremely compact format of Nature articles and the limited space available to figures, makes the article very hard to read. Hell, I thought it was kind of heavy and that shit is my job.

I’ll try to walk the persistent readers through the experiments and some of the essential results, but if that is TL:DR, I recommend you just read the headlines of the experiments below until you reach the “what does it mean” headline further down the page ;o)

Experiment 1 – NAS impairs glucose tolerance in mice

In the first part of the experiment the investigators show that consumption of the sweeteners Saccharin, Sucralose or Aspartame impairs glucose tolerance, i.e. the ability to clear glucose from the blood stream from ingestion of 40 mg glucose, a so-called oral glucose tolerance test (OGTT). They find that glucose clearance is impaired after having ingested NAS for 11 weeks, relative to glucose or water ingestion for the same period (see figure below). The authors show this for mice that have been fed a normal chow diet as well as for mice on a high-fat diet (also known to mess up their glucose control). The investigators also went through considerable effort to show that this difference could not be caused by differences in weight, physical activity or enegy expenditure.

One thing that should be noted about this part of the experiment was that the effect of NAS on glucose tolerance were differentiated somewhat between artificial sweetener types. Saccharin had the biggest effect, with sucralose coming in second, but with aspartame having an effect comparable to glucose. That is, aspartame did not produce this effect on glucose clearance following an OGTT. But – It looks like all the NAS were grouped together in the statistical analysis (probably to obtain increased statistical power), so it was actually not tested if there were differences between the NAS types. Despite of this, they selected saccharin to continue onwards with. You and I can both see that the hyperglyemic response to saccharin was strongest, but it is not good science to just select something because it looks a certain way. Normally that requires a (statistical) reason. But that may be pedantic on my behalf…

Experiment 2 – antibiotics prevent NAS induced impairment in glucose tolerance in mice

Next, the researchers demonstrated that this NAS-induced glucose intolerance could be ablated by antibiotic administration, which indicates that it is an effected mediated by microorganisms in the gut. I’m personally guessing that an initial discovery of this phenomenon led the investigators down this very road. They even tried out different types of antibiotics, which each kill different types of bacteria (Gram positive and gram negative). this showed that the NAS-indiuced effect was mediated by more than one group of bacteria.

glucose concentration following OGTT in intervention groups

glucose concentrations in the blood of mice from different intervention groups following OGTT in intervention groups

A interesting note is that antibiotics reduced the glucose levels following an OGTT for all conditions, even in animals that had only consumed glucose or fructose. I did not realise antibiotics did that…

Glycemic responses across intervention groups

Glycemic responses across intervention groups. The AUC refers to area under the curve, which essentially means that it is the sum of all the time points for a given animal in the above figure.

Experiment 3 – NAS-induced glucose intolerance can be transferred to NAS naive mice through faecal transplants

Then the investogators proceeded to do faecal transplants from animals that had consumed saccharin for some time, to sacchaerin-naive animals. After 6 days, the transplanted animals displayed the same impaired glucose tolerance, as the saccharin-stimulated animals.

Glucose levels followingn OGTT in animals having received faecal transplants from saccharin stimulanted animals or controls consuming glucose

Glucose levels followingn OGTT in animals having received faecal transplants from saccharin stimulanted animals or controls consuming glucose

This very strongly supports that the hyperglycemic response is related to changes in gut microbiota.

Experiment 4 – NAS modulates the gut microbiota

In order to find out what caused the changes in saccharin-ingesting animals, the investigators proceeded to do a characterisation of the microbiota through sequencing of the 16S ribosomal RNA gene. This served to identify the bacteria species present in the, well, shit of the animals.

This analysis revealed that saccharin consumption indeed did change the microbiota. About 20 bacteria mapped mostly to the Bacteroides genus or Clostridiales orders were more abundant following saccharin ingestion, whereas about 20 bacteria mapped mistly to Clostridiales orders or Lactobacillus Reuterii subtypes were less abundant.

The same analysis showed that in the animals that had faecal transplant driven glucose intolerance, some of the same patterns, but not all, were observed. Again, this supports the hypothesis of microbiota driven changes in glucose tolerance.

Experiment 5 – NAS causes changes in GENEs present in gut gene prevalence

Next, the investigators did socalled shotgum sequencing. This analysis determines the presence of genes across ALL the DNA purified from stool samples, i.e. the entire microbiota, shed cells from the intestines and possible even gut parasites. When they mapped the coding sequences (genes) found in the sequenced DNA to microbial genes, they found that the abundance of certain genes had changed relative to the abundance in saccharin-naive animals.

This is interesting because it may provide a hint of how this change in microbiota can modulate glucose tolerance. The saccharin-exposed animals had a higher abundance of genes in glycan-degradation pathways. These genes are associated with fermentation of glycans into short-chain fatty acids (SCFAs). The investigators found that the presence of propionate and acetate SCFAs were increased in saccharin-exposed animals in agreement with the these sequencing results.

When the genes found in this shotgun sequencing were mapped to specific bacteria, they found that much of the increase in the abundance could be mapped to 5 specific bacteria of which 2 matched with the data from the 16S rRNA sequencing. While 2 out of 5 may not sound of much, you have to remember that there are tens of thousands of microbial species present in the gut

Experiment 6 – changes in gut microbiota can be induced by culturing feces with saccharin in vitro (ex vivo)

Next, the investigators proceeded to culture faecal matter with or without saccharin in the media. They observed that this changed the bacterial composition of the culture, specifically towards more species from the Bacterioidetes phylum (in agreement with the findings above). Next, they transplanted these cultures into saccharin-naive animals and found that transplanted animal having received transplants from the saccharin cultures developed glucose intolerance too.

glycemic response to OGTT in mice following transplantation with microbiota stimulated with saccharin in vitro/ex vivo

glycemic response to OGTT in mice following transplantation with microbiota stimulated with saccharin in vitro/ex vivo. Note that this is an AUC figure showing the total blood glucose across all time points following and OGTT.

Experiment 7 – In a human cohort, NAS consumption was associated with a impaired glucose tolerance (and metabolic control biomarkers)

Having built a strong case for the effects of saccharin, the investigators moved into humans. From an ongoing clinical nutrition study with 381 non-diabetic participants, the investigators found a correlation between intake of of NAS and various measures of poor glucose tolerance.

So far this is by far the weakest part of the study. There is no information about how this population was selected, no information about the questionnaire used to ask for NAS consumption, no information about weight distribution between high and low-NAS consumers. Without these informations, this looks a lot like a fishing expedition. I’m particularly interested in knowing if attempts were made to characterize what artificial sweeteners the study participants were eating. This is relevant because the hyperglycemic effect seemed to be present for only saccharin and sucralose.

The investigators did 16S rRNA sequencing of 172 randomly selected individuals from this cohort and found what they claim is a positive correlation between NAS consumption and the presence of certain microorganisms. It is not made clear how NAS consumption was scored and more importantly, when doing this many statistical tests (they tested over 3000 bacteria), about 5% will be positive by chance alone, which is why “correction for multiple testing” is normally used. This was not done here and if it WAS done it’d have eaten pretty much all of these purported statistically sound correlations. That is something the editors at Nature should have commented on.

Also, in the paper it is not made clear how the observed changes related to the microbiotic  changes observed in the animal studies. I’m not familiar enough with microbiology to assess that.

Experiment 8 – In a human intervention study, NAS consumption caused impaired glucose tolerance in 4 of 7 subjects

In the next part of the study, 7 subjects (that did not normally consume artificial sweeteners) were fed saccharin at a level corresponding to the FDA’s maximal acceptable daily intake (ADI) for 6 days. During this period, 4 of the 7 individuals developed significantly worsened glycemic control. Important stuff!

Glycemic responses in responders and non-responders early (day 1-4) vs late (day 5-7) in the saccharin stimulation period. Subjects had daily OGTT tests.

Glycemic responses in responders and non-responders early (day 1-4) vs late (day 5-7) in the saccharin stimulation period. Subjects had daily OGTT tests.

Next, the microbiota of these subjects were characterized. It was found that the biota were different between responders and non-responders at both baseline and following saccharin consumption and that the biota changed in responders, but not in non-responders. Again, the changes in microbiota with saccharin ingestion weren’t related to the changes observed in mice earlier in the study, which sucks.

It goes without saying, that the number of subjects here is quite low and there is no negative control group. Furthermore, arbitrarily selecting some subjects to be responders to a given intervention, solely on the premise of their response in such a small study is very questionable from a statistical point of view, but I think the results in the other experiments provides the authors with enough credit to permit this.

Experiment 9 – GLUCOSE INTOLERANCE CAN BE INDUCED IN NAS-NAIVE MICE, THROUGH FAECAL TRANSPLANTS FROM HUMAN NAS-RESPONDERS, BUT NOT FROM NON-RESPONDERS

Next, faecal transplants were done from both baseline and following saccharin stimulation from both saccharin responders and non-responders into groups of mice. and oh, lo and behold, glucose intolerance could be transferred through faecal transplant from saccharin stimulated responders but not non-responders and not with faecal transplants from before the saccharin stimulation. Inter-species faecal transplants, sounds pretty japanese gameshow’ish to me, but it actually makes a strong point here.

Glycemic responses in animals having received faecal transplants from human saccharin responders and non-responders.

Glycemic responses in animals having received faecal transplants from human saccharin responders and non-responders.

But what does it mean?

Well, unless the researchers have manipulated their data wildly, which I doubt, it actually really does look like saccharin induces glucose intolerance to a significant degree, in (some) humans!

But, because there’s a “but”, it really doesn’t make a difference… – Why? Because saccharin is hardly used anymore… Saccharin was the artificial sweetener of the 80’s and consumption these days constitute less than 10% of total artificial sweetener consumption in the western world, maybe even less than 5%.

use of artificial sweeteners in non-alcoholic beverages in Denmark

use of artificial sweeteners in non-alcoholic beverages in Denmark. From Leth et al, 2007)

And this is a problem with the study. What the study shows is that Saccharin modifies gut microbiota which in turn causes glucose intolerance, NOT that artificial sweeteners as such does so. Other artificial sweeteners may do it, and the first experiments in the study indicate that that is the case for sucralose at least, but it is not a question that is pursued throughout the study. Having a paper title that is unprecise and unprecise in a manner that one could be tempted to think was so due to a desire of greater popular impact/controversy on the behalf of the authors is not in the interest of science.

But, I’m diverging… I think the study looks extremely solid and it is hard to argue against the findings of transferring glucose intolerance through faecal transplants. Again, unless, manipulated with, somehow, this should lead to some sort of reappraisal of Saccharin. There are some things in the study that could have been done better. Personally, I would have ditched the 341-person cohort study and spent a little more resources on the 7-person intervention study. This would have strengthened the paper considerably.

Something that should be considered is that this hyperglycemic effect of saccharin seems to be pretty qualitative. The first experiments seemed to indicate that saccharin and sucralose had this effect on glucose tolerance, whereas aspartame did not. So even though sweetness in a molecule is associated with certain molecular shapes and structures, it doesn’t look like all sweetness is associated with the hyperglycemic effect described in this paper. Hell, saccharin and sucralose looks nothing like each other, so I wouldn’t know what parts of them were involved in this mechanism. So the findings here do not apply to artificial sweeteners in general and very likely not to aspartame at all.

One might ask: why is this effect of sweeteners not showing up in the studies elsewhere? There is actually one study from 2012 that showed that saccharin intake adversely affected glucose homeostasis in rats (Swithers et al, 2012), so it is not completely new, but the microbiota mechanism is. I’ve also been looking for it in human studies and it appears that in the previous studies on artificial sweeteners, the investigators either did not separate sweetener types or did not look into glucose tolerance. A recent meta analysis have shown that artificial sweetener use in general is both safe and that substituting sugar intake for artificial sweeteners are associated with modest weight loss (Miller et al, 2014). This is not necessarily in conflict with the present study as the use of saccharin only comprises a small fraction of the total artificial sweetener use. Therefore any potentially adverse effects from saccharin would have “drowned” in the data from the other sweeteners.

I think that was that. I’ll probably come back to take out typos and make minor edits for the next days. Post a comment and let me know what you think.

References

Leth, T., Fabricius, N., & Fagt, S. (2007). Estimated intake of intense sweeteners from non-alcoholic beverages in Denmark. Food Additives and Contaminants, 24(3), 227–235. doi:10.1080/02652030601019429

Miller, P. E., & Perez, V. (2014). Low-calorie sweeteners and body weight and composition: a meta-analysis of randomized controlled trials and prospective cohort studies. American Journal of Clinical Nutrition, 100(3), 765–777. doi:10.3945/ajcn.113.082826

Suez, J., Korem, T., Zeevi, D., Zilberman-Schapira, G., Thaiss, C. A., Maza, O., et al. (2014). Artificial sweeteners induce glucose intolerance by altering the gut microbiota. Nature, –. doi:10.1038/nature13793

9 Comments

  1. Rasmus Bagklog on 2014-09-20 at 06:40

    First!

  2. Ole Dines Olsen on 2014-09-23 at 08:44

    First, thanks for a really nice and thorough review of the article, only read the headlines myself. It does look interesting and especially the mouse part seems quite solid. But you might want to change shotgum to shotgun.

    • incognitodk on 2014-09-23 at 09:43

      hah! Yes, that'd might be a good idea ;o)

      Anders

  3. BioP.. on 2014-09-24 at 03:59

    Great article interpretation Dr. Anders… I am presenting this paper this Friday in my Senior Seminar class for biology, and you helped me understand this paper immensely. My group and I had a few questions, like how did the bacteroides and clostridiales contribute to the glucose intolerance?

    • incognitodk on 2014-09-24 at 08:11

      Actually, I don't really know, but in the paper it is found that there are more genes in the microbiota og saccharin stimulated animals, related to improved energy extraction. So, the authors hint at the hyperglycemic responses being caused by improved energy extraction. I think that's a s close as we'll get to an answer. But i think it smells fishy. I'm guessing there's also an interaction with small intestine nutrient absorption and possible even at hepatic glucose clearance. Just a hunch. I don't think gut energy extrraction is sufficient for hyperglycemic responses this strong

      Anders

  4. Mari on 2014-09-25 at 13:25

    Since cyclamate seems to be included in large quantities in majority of soft drinks, would be very interesting to know whether it has the same effect as saccharin or sucralose. I too noticed the AUC of aspartame being like glucose, although the initial rise at 15 min was more like the other two artificial sweeteners…

    • incognitodk on 2014-09-25 at 14:05

      I totally agree. Also I'd like to see the latter part of the study repeated with sucralose, as this had a response quite similar to saccharin. thank you for your comment.

      Anders

  5. compressore on 2016-09-23 at 11:24

    An outstanding share! I’ve just forwarded this onto a friend who had been doing a little homework on this.

    And he actually ordered me breakfast because I stumbled upon it for him…
    lol. So allow me to reword this…. Thank YOU for the meal!!
    But yeah, thanx for spending time to talk about this topic here on your web page.

  6. pakistan video song mp4 on 2016-11-10 at 12:14

    Great post.

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