Researchers say consumption of artificial sweeteners such as saccharin, sucralose and aspartame led to increased risk of glucose intolerance, a condition linked to diabetes. Sweet ‘N Low contains saccharin. Equal original contains aspartame. Splenda contains sucralose. (iStockPhoto) 
 
 Artificial sweeteners might be triggering higher blood-sugar levels in some people and contributing to the problems they were designed to combat, such as diabetes and obesity, according to new findings published Wednesday in the journal Nature. 
 
 Although the precise reasons behind the blood-sugar changes remain uncertain, researchers suspect that artificial sweeteners could be disrupting the microbiome, a vast and enigmatic ecosystem of bacteria in our guts. 
 
 In a series of experiments, researchers found that several of the most widely used types of non-calorie sweeteners in food and drinks — saccharin, sucra­lose and aspartame — caused mice to experience increased risk of glucose intolerance, a condition that can lead to diabetes. 
 
 “We are talking about very dramatic increases,” said one of the study’s co-authors, Eran Segal, a computational biologist at the Weizmann Institute of Science in Israel. 
 
 The same scientists also monitored what happened to seven human volunteers who did not typically use artificial sweeteners but were given regular doses of saccharin over the course of a week. Four developed significant glucose intolerance. Separately, the researchers analyzed nearly 400 people and found that the gut bacteria of those who used artificial sweeteners were noticeably different from people who did not. 
 
 View Graphic Using some artificial sweeteners might increase blood sugar 
 
 “This huge and poorly understood microbial world, which resides in each and every one of us starting from birth, has been shown to play a fundamental role in many aspects of our physiology, as well as in [our] susceptibility to common human diseases,” said Eran Elinav, another of the study’s co-authors and an immunologist at the Weizmann Institute. 
 
 Wednesday’s findings add an intriguing new dimension to the long-running, contentious debate over the potential health benefits and risks of artificial sweeteners, which are among the most common food additives and are consumed by hundreds of millions of people around the globe. 
 
 While some past studies have found that the products pose no health risks and effectively help people cut calories and sugar intake, other research has suggested that certain artificial sweeteners might actually contribute to obesity and other problems, including cancer. 
 
 Segal and Elinav insisted that their findings are preliminary and shouldn’t be taken as a recommendation on whether people should reconsider using artificial sweeteners. 
 
 “We do not view that as our role,” Segal said. “Rather, as scientists, we simply point to the immense body of experiments that we carried out in both humans and in mice. . . . This study and these results should prompt additional debates and study into what is currently a massive use of artificial sweeteners.” 
 
 Elinav added: “This issue is far from being resolved.” 
 
 Lisa Lefferts, a senior scientist at the nonprofit Center for Science in the Public Interest, agreed. She praised the rigor behind Wednesday’s study and the “very provocative results” it produced, but she said the findings don’t negate past studies showing that people are more likely to gain weight when drinking sugar-sweetened beverages than when using artificial sweeteners. 
 
 Rather, she said, the various ways the scientists measured the effect of saccharin on the gut microbiota suggest a definite, if unresolved, link. 
 
 “We have more confidence now that there really is something going on, at least in animals,” Lefferts said. “What does that mean over the long term for the population? We still don’t have that answer.” 
 
 In addition, Lefferts and the study’s authors said people shouldn’t see the findings as a suggestion that sugar-sweetened drinks are somehow preferable to artificial sweeteners. 
 
 “We must stress, by no means are we saying that sugary drinks are healthy and that sugary drinks should be brought back as a healthy part of our nutrition,” Elinav said 
 
 The Food and Drug Administration has approved six different types of sugar substitutes, or “high-intensity” sweeteners, with the most recent coming this year. The three types included in Wednesday’s study involving mice — saccharin, sucralose and aspartame — are more commonly known by the popular brand names Sweet n’ Low, Splenda and Equal, respectively. However, the researchers relied primarily on saccharin in their mouse and human experiments. 
 
 Perhaps no sweetener has proven more controversial than saccharin, which was discovered not long after the end of the Civil War. In 1977, the FDA tried to ban saccharin because of safety concerns after studies showing that rats had developed bladder cancer after receiving high doses of the chemical sweetener. 
 
 Congress blocked that effort, instead passing legislation that required a warning label on products containing saccharin. In 2001, federal legislation removed the requirement for a warning label altogether. 
 
 The American Medical Association and the American Diabetes Association have cautiously backed the use of non-caloric sugar substitutes as a way to fight obesity and diabetes, saying that the products can be part of a healthy diet as long as the calories saved aren’t replaced by consuming more food over the course of a day. ||||| Mice 
 
 C57Bl/6 WT adult male mice were randomly assigned (without blinding) to treatment groups and were given commercial artificial sweeteners (saccharin-, sucralose- or aspartame-based) or pure saccharin (Sigma Aldrich) in drinking water and fed a high-fat (HFD D12492, 60% kcal from fat, Research Diets) or standard polysaccharide normal-chow diet (Harlan-Teklad). Compared groups were always fed from the same batch of diet. For antibiotic treatment, mice were given a combination of ciprofloxacin (0.2 g l−1) and metronidazole (1 g l−1) or vancomycin (0.5 g l−1) in their drinking water. All antibiotics were obtained from Sigma Aldrich. Adult male outbred Swiss-Webster mice (a widely used mouse strain in germ-free experiments) served as recipients for faecal transplants and were housed in sterile isolators (Park Bioservices). For faecal transplantation experiments, 200 mg of stool (from mouse pellets or human swabs) was resuspended in 5 ml of PBS under anaerobic conditions, vortexed for 3 min and allowed to settle by gravity for 2 min. Transplant into recipient mice were achieved by gavage with 200 µl of the supernatant and maintained on standard normal-chow diet and water throughout the experiment. All animal studies were approved by the Weizmann Institute of Science Institutional Animal Care and Usage committee (IACUC), application numbers 08680114-3 and 00550113-3; all animal experiments involving transfer of human microbiota into mice were approved by the Weizmann Institute of Science Bioethics and Embrionic Stem Cell Research oversight (ESCRO) committee. 
 
 Artificial and caloric sweeteners 
 
 The following commercially available NAS were dissolved in mice drinking water to obtain a 10% solution: Sucrazit (5% saccharin, 95% glucose), Sucralite (5% Sucralose), Sweet’n Low Gold (4% Aspartame). 10% glucose (J. T. Baker) and 10% sucrose (Sigma Aldrich) solutions were used for controls. The administered doses of 10% commercial NAS dissolved in water were well below their reported toxic dose (6.3 g per kg (body weight)27, 16 g per kg (body weight)28, and 4 g per kg (body weight)29, for saccharin, sucralose and aspartame, respectively). For experiments conducted with pure saccharin (Sigma Aldrich) a 0.1 mg ml−1 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: 
 
 Glucose and insulin tolerance tests 
 
 Mice were fasted for 6 h during the light phase, with free access to water. In all groups of mice where the drinking regime was other than water, it was substituted for water for the period of the fasting and glucose or insulin tolerance test. Blood from the tail vein was used to measure glucose levels using a glucometer (Bayer) immediately before and 15, 30, 60, 90 and 120 min after oral feeding with 40 mg glucose (J. T. Baker) or intra-peritoneal injection with 0.1 U per kg (body weight) Insulin (Biological Industries). Plasma fasting insulin levels were measured in sera collected immediately before the start of GTT using ELISA (Ultra Sensitive Mouse Insulin ELISA Kit, Crystal Chem). 
 
 Metabolic studies 
 
 Food and drink intake and energy expenditure were measured using the PhenoMaster system (TSE-Systems, Bad Homburg, Germany), which consists of a combination of sensitive feeding sensors for automated measurement and a photobeam-based activity monitoring system detects and records ambulatory movements, including rearing and climbing, in each cage. All parameters were measured continuously and simultaneously. Mice were trained singly-housed in identical cages before data acquisition. To calculate total caloric intake, the following values were used: Chow 3 kcal g−1, sucrose 0.3938 kcal ml−1, glucose 0.4 kcal ml−1, saccharin 0.38 kcal ml−1, sucralose 0.392 kcal ml−1 and aspartame 0.38 kcal ml−1. 
 
 In vitro anaerobic culturing 
 
 Pooled faecal matter from naive adult WT C57Bl/6 male mice was resuspended in 5 ml PBS in an anaerobic chamber (Coy Laboratory Products, 75% N 2 , 20% CO 2 , 5% H 2 ), vortexed for 3 min and allowed to settle by gravity for 2 min. 500 ml of the supernatant were added to a tube containing Chopped Meat Carbohydrate Broth, PR II (BD) and 500 ml of a 5 mg ml−1 saccharin solution or an equal volume of PBS. Every 3 days, 500 ml of culture were diluted to fresh medium containing saccharin or PBS. After 9 days, cultures were used for inoculation of germ-free mice. 
 
 Taxonomic microbiota analysis 
 
 Frozen faecal samples were processed for DNA isolation using the MoBio PowerSoil kit according to the manufacturer’s instructions. 1 ng of the purified faecal DNA was used for PCR amplification and sequencing of the bacterial 16S rRNA gene. ~365bp Amplicons spanning the variable region 2 (V2) of the 16S rRNA gene were generated by using the following barcoded primers: Fwd 5′-AGAGTTTGATCCTGGCTCAG-3′, Rev 5′-TGCTGCCTCCCGTAGGAGT-3′. The reactions were subsequently pooled in an equimolar ratio, purified (PCR clean kit, Promega), and used for Illumina MiSeq sequencing to a depth of at least 18,000 reads per sample (mean reads per sample 139,148 ± 5264 (s.e.m.)). Reads were then processed using the QIIME (quantitative insights into microbial ecology) analysis pipeline as described30, 31, version 1.8. Paired-end joined sequences were grouped into operational taxonomic units (OTUs) using the UCLUST algorithm and the GreenGenes database32. Sequences with distance-based similarity of 97% or greater over median sequence length of 353 bp were assigned to the same OTU. Samples were grouped according to the treatment. Analysis was performed at each taxonomical level (phylum, genus and OTU level) separately. For each taxon, G test was performed between the different groups. P values were FDR-corrected for multiple hypothesis testing. 
 
 Shotgun pyrosequencing and sequence mapping 
 
 This was performed as previously described33, with the following modifications: 1 μg of DNA was sheared using the Covaris 5200 system (Covaris, Inc., Woburn, MA, USA), followed by end repair, ligation to adapters, an 8-cycle PCR amplification (Kappa HiFi) and sequenced using an Illumina HiSeq to a minimal depth of 11,773,345 reads per sample (mean reads per sample 20,296,086 ± 637,379 (s.e.m.), read length 51 bp). Illumina sequence reads were mapped to the human microbiome reference genome database of the Human Microbiome Project (http://hmpdacc.org/HMREFG/, ref. 16), and to a gut microbial gene catalogue34 using GEM mapper35 with the following parameters: 
 
 Microbial species abundance was measured as the fraction of reads that mapped to a single species in the database. An expectation–maximization (EM) algorithm adapted from Pathoscope36 was employed to determine the correct assignment of reads that mapped to more than one species. We considered only species for which at least 10% of the genome was covered (each coverage bin was 10,000-bp long) in at least one of the growth conditions (saccharin, water, or glucose). Reads mapped to the gut microbial gene catalogue were assigned a KEGG ID according to the mapping available with the catalogue. Genes were subsequently mapped to KEGG pathways, and only pathways whose gene coverage was above 0.2 were included. To calculate the contribution of different bacteria to the overrepresentation of glycan degradation pathways, reads that were mapped to genes in the gut microbial gene catalogue that belong to glycan degrading pathways were extracted and re-mapped the HMP reference genome database, seeking germs that had the highest contribution. 
 
 Short chain fatty acid quantification 
 
 To determine the level of free fatty acids analytic HPLC (Agilent 1260) was performed as described previously37. In brief, standard solutions of acetate, butyrate and propionate (all from Sigma-Aldrich) were prepared at various concentrations (0.01–0.2 M). These solutions were analysed using HPLC, successive with quadrupole time-of-flight mass spectrometry with a step-gradient of solvent solution from 0% to 60% of CH 3 CN with 0.1% formic acid to obtain calibration curve for each fatty acid. Faecal media samples were dissolved with 0.1% formic acid and analysed in a similar manner to measure the total concentration of all three free fatty acids. 
 
 Analysis of the relationship between NAS consumption and clinical parameters in humans 
 
 All human studies were approved by the Tel Aviv Sourasky Medical Center Institutional Review Board, approval number TLV-0658-12, TLV-0050-13 and TLV-0522-10; Kfar Shaul Hospital Institutional Review Board, approval number 0-73; and the Weizmann Institute of Science Bioethics and Embrionic Stem Cell Research oversight (ESCRO) committee. The trial was reported to http://clinicaltrials.gov/, identifier NCT01892956. The study did not necessitate or involve randomization. For each individual in the clinical nutritional study, after signing an informed consent, multiple parameters were collected including BMI, body circumferences, fasting glucose levels, general questionnaire, complete blood counts and general chemistry parameters, a validated long-term food frequency questionnaire38, 39, 40. 
 
 Long-term NAS consumption was quantified directly from answers to an explicit question regarding artificial sweeteners that participants filled out in their food frequency questionnaire. We then used the Spearman correlation to examine the relationship between NAS consumption and each of the above parameters, and FDR corrected for the multiple hypotheses tests performed. 
 
 Statistics 
 
 The following statistical analyses were used: in GTT, a two-way ANOVA and Bonferroni post-hoc analysis were used to compare between groups in different time-points, and one-way ANOVA and Tukey post hoc analysis or unpaired two-sided Student t-test were used to compare between AUC of multiple or two groups, respectively. Bartlett’s or F-test for equal variance were employed and no significant difference was observed between variances of the compared groups. For comparison of taxonomic data, a G-test was used and P values were FDR-corrected for multiple hypothesis testing. In metagenomics and clinical and taxonomic data from humans, Pearson and Spearman were used for correlation tests, and Mann–Whitney U was used to compare clinical parameters between groups. P < 0.05 was considered significant in all analyses (* denotes P < 0.05, **P < 0.01, ***P < 0.001). In all relevant panels, symbols or horizontal lines represent the mean, and error bars represent s.e.m. For mouse experiments, cohort sizes match common practice of the described experiments. For human experiments, sample size was chosen to validate statistical analyses. No mice or data points were excluded from analyses. In the human studies, all humans older than 18 years of age who enrolled were included. Exclusion criteria included pregnancy. ||||| When artificial sweeteners are in the news, it’s rarely positive. In the last few years, sweeteners have been linked to everything from Type 2 diabetes to cardiovascular disease, high blood pressure, and stroke. Still, products like Splenda and Sweet‘N Low remain a cornerstone of many a weight-loss strategy, mostly because doctors don’t quite understand how sweeteners contribute to disease. That may soon change, however, as results from a study, published today in Nature, point to a possible mechanism behind these adverse health effects. 
 
 "Our results suggest that in a subset of individuals, artificial sweeteners may affect the composition and function of the gut microbiome" in a way that would lead to high blood-sugar levels, said Eran Elinav, an immunologist at the Weizmann Institute of Health in Israel and a co-author of the study, during a press conference yesterday. This, the researchers say, is bad for human health because when sugar levels are high in the blood, the body can’t break it down, so it ends up being stored as fat. 
 
 High blood sugar ends up being stored as fat. 
 
 To reach these conclusions, Elinav and his team first tested the effect of three common artificial sweeteners — aspartame, sucralose, saccharin — on rodents. They found that each of the sweeteners induced a change in blood sugar levels that surpassed that of the mice who consumed actual sugar. And later tests involving the main sweetening agent in Sweet‘N Low, saccharin, yielded similar results in both lean and obese mice. 
 
 But mammals don’t actually digest artificial sweeteners — that’s why they’re "calorie-free" — so the reasons why these mice were experiencing blood-glucose alterations was still mysterious, Elinav said. Still, the researchers had an idea: maybe the bacteria that lived in the guts of the mice were interacting with the sweeteners. 
 
 So the researchers performed several experiments to test their idea. In one, they gave antibiotics to mice who had been fed sweeteners regularly. Antibiotics kill gut bacteria, and when these mice had their microbial guests cleaned out, their blood sugar levels went back to normal. In another experiment, the scientists transplanted feces — a rich source of gut microbes — from sweetener-fed mice into rodents that had never consumed artificial sweeteners. The procedure caused the recipient mice to experience oddly high blood glucose, like the mice in the sweetener group. Finally, Elinav and his colleagues used genetic analysis to reveal that alterations in the composition of microbial colonies were also accompanied by changes in bacterial function — changes that could very well explain why the mice were experiencing such high blood sugar. 
 
 Findings in mice aren't nearly as convincing as findings in people 
 
 But findings in mice aren’t nearly as convincing as findings in people, so the researchers set out to investigate human sweetener consumption. In the first experiment, the researchers analyzed the blood-sugar levels and gut bacteria colonies of 381 participants. And, as expected, Elinav and his colleagues found that people who consumed sweeteners in large quantities also showed disturbances in several metabolic parameters — including increased weight — as well as distinct microbial changes in their guts. 
 
 The results from the second, much smaller human experiment might actually be the most illuminating. 
 
 "We followed for a single week a group of seven human volunteers who do not consume sweeteners as part of their normal diet," Elinav said. During that period, the researchers gave them a single dose of saccharin, and monitored their vitals. After just four days, half the participants showed microbial alterations and increases in blood sugar levels, he explained, "while the other subset had no meaningful effect immediately following the consumption of sweeteners." 
 
 In other words: some people are more susceptible to the effects of artificial sweetener than others. 
 
 A causal link 
 
 The handful of studies suggest that consuming non-caloric artificial sweeteners boosts the risk glucose intolerance in both humans and mice, as a result of changes in gut microbe function, the researchers wrote in their report. Yet, because of the preliminary nature of their results and the small number of human participants involved, they stopped short of suggesting that people change their eating habits. "By no means are we prepared to make recommendations as to the use and dosage of artificial sweeteners based on the results of this study," said Eran Segal, a study co-author also at the Weizmann Institute of Health. 
 
 Other researchers, however, were more forthcoming. 
 
 "People need to be much more mindful of what they are eating and drinking and make efforts to avoid products that have added sweeteners in any form" said Susan Swithers, a behavioral neuroscientist at Purdue University who wasn’t part of the Nature study, in an email to The Verge. The studies showed not only a causal link between the changes in the gut and artificial sweeteners, but that the observed changes happen quickly, she wrote. 
 
 "People need to be much more mindful of what they are eating and drinking." 
 
 Not everyone agrees with the design the researchers used to address the question about artificial sweeteners and weight gain. Christopher Gardner, a food scientist at Stanford University who didn’t participate in the study, says that the fact that the researchers gave the FDA's maximal acceptable daily intake of saccharin to the human participants — about 5 mg / kg body weight per day — isn’t ideal. In a real-life setting, that dose would be the equivalent to a 150-pound person consuming 42 12-ounce sodas per day, or 8.5 packets of pink Sweet 'n Low per day. "That may be ‘acceptable’ according to some set of guidelines," Gardner wrote in an email, "but it should be noted that realistically this is a very high dose they are using and one that wouldn't be consumed by a typical consumer." 
 
 Still, the idea that we might finally have an explanation for the adverse health effects seen in certain sweetener studies is worth paying attention to. Should the findings prove reproducible, doctors will be tasked with understanding why some people are susceptible to microbiome alterations, while others aren’t. And sweetener companies will have to address the criticism — in addition to rethinking their marketing strategies. "The work is important," Swithers said, "because it underscores the role that artificial sweeteners may play in contributing to the very problems they were designed to help."
– If you're drinking diet soda or consuming other products with artificial sweeteners to cut your risk of obesity and diabetes, a new study published in Nature has some bad news: You might be doing more harm than good. Researchers found that sweeteners such as saccharin, sucralose, and aspartame can interact in a complex way with bacteria in our gut and lead to higher blood-sugar levels, reports the Wall Street Journal. Scientists say studies on both mice and people back up the assertion, though the findings may not apply to all. In one of the human experiments, for example, researchers gave foods with such sweeteners to seven people who normally never consumed them and found that four of the subjects had markedly higher blood-sugar levels in just four days. "In other words: Some people are more susceptible to the effects of artificial sweetener than others," explains the Verge. It all depends, apparently, on your particular microbiome, which the Washington Post describes as the "vast and enigmatic ecosystem of bacteria in our guts." The sweetener industry is already disputing the research, but "the scope of our discovery is cause for a public reassessment of the massive and unsupervised use of artificial sweeteners," says a study co-author at Israel's Weizmann Institute of Science. Far more research is needed before it's definitive, but NPR grabs this quote from the director of the Human Microbiome Program at New York University: "I can just tell you ... as a middle-aged man who's concerned about his diet and his waistline—and [as] somebody who drinks diet soda—I didn't drink any yesterday." (Meanwhile, US bellies are getting bigger.)