It’s been almost a decade since Jotham Suez, a microbiologist at Johns Hopkins University, first started looking into artificial sweeteners and their health effects. In 2014, as a postdoctoral researcher at the Weizmann Institute of Science in Israel, he worked on a study in mice that suggested artificial sugar alternatives present in everything from lipsticks to toothpaste could lead to obesity and related health condition like diabetes and cardiovascular diseases.

Those early findings sparked controversy, says Suez. Though the study wasn’t the first time scientists had looked at the link between artificial sweeteners and obesity, it was the first one to detail a potential mechanism for it: The sweeteners changed the intestinal bacteria of mice, which play important roles in regulating metabolism, appetite, and fat storage.

“The food industry went ballistic because obviously this is a major threat,” says Robert Lustig, a neuroendocrinologist at University of California, San Francisco, who was not involved in the work. “They came up with a zillion reasons why the study was no good, particularly that it wasn’t in humans.”

See “Sweetened Drinks Linked to Higher Mortality Risk

Now, working in the lab of Weizmann Institute of Science immunologist Eran Elinav, Suez and colleagues have taken an array of common artificial sweeteners—saccharin, sucralose, aspartame, and stevia—and filled that gap in the literature by testing them in humans. Their findings, described today (August 19) in Cell, suggest these sweeteners do indeed alter the gut microbiome, as was seen in prior mouse work. This, the researchers say, adversely impacts glucose tolerance, a measure of how readily the body moves sugar from the blood into muscle and fat, possibly leading to weight gain and diabetes.

“In the past several decades, there has been a massive increase in the prevalence of these metabolic conditions” that arise from impaired glucose tolerance, says Suez, who coauthored the new study. “This strategy [of using non-caloric sweeteners] has been around for a while as an alternative to caloric sweeteners, but our findings beg the question [of] whether they are producing the benefits or not.”

To test whether sweeteners impact gut microbes and glycemic responses, the researchers recruited volunteers with normal blood glucose levels who did not consume sweeteners in their diet to participate in a randomized controlled trial. Volunteers deemed eligible after an initial screening were split into one of six groups. Four groups consumed six commercially available sachets of either aspartame, saccharin, sucralose, or stevia every day for 14 days. As all four sweeteners contained the sugar glucose as a bulking agent, a fifth group received an equivalent amount of glucose (five grams), while the sixth received no intervention. All participants measured their blood glucose levels before, during, and after the treatment period using a glucose monitor that was attached to their upper arm. They also performed glucose tolerance tests (GTTs), which evaluate the body’s glycemic response by measuring how quickly blood glucose levels return to a baseline after glucose is consumed, and collected stool and saliva samples that were analyzed for their microbiomes.

When the researchers profiled the participants’ oral and gut microbiomes, they found that there were significant differences in the populations of bacteria before and after regular ingestion of artificial sweeteners, particularly for participants who took sucralose and saccharin. The control groups that ingested glucose or took no supplement did not experience changes in their microbiomes.

The participants who took sucralose and saccharin also exhibited larger peaks in blood glucose in GTTs conducted during the treatment period than those in the glucose control group, suggesting that those sweeteners may push the body towards glucose intolerance, where tissues struggle to absorb glucose from the blood. No significant differences in glycemic responses were observed for the glucose-only, aspartame, or stevia groups when compared with the no intervention control.

See “How the Gut Differentiates Artificial Sweeteners from Sugars

While these findings are alarming, says Lustig, the “slam dunk” came when the researchers used mouse experiments in the study to prove cause and effect between certain sweeteners, gut microbes, and glucose intolerance.

Suez and his colleagues took microbes from the feces of participants with elevated blood sugar levels and inserted the bacteria into germ-free mice by feeding them a slurry. After a few days, when the microbes colonized the animals’ gut, the researchers looked at the glycemic responses of these mice. They found that the animals’ ability to regulate blood glucose levels were also inhibited.

“This is fundamental because it proves causation, not just correlation,” says Lustig. However, he notes that this is a medium-term study that doesn’t follow the weight or glycemic responses of human participants over six months or a year. Doing so, according to him, will better answer the ultimate question about the effects of artificial sweeteners on weight gain and the development of health conditions like diabetes.

Daniel Garrido, a microbiologist at Pontifical Catholic University of Chile who was not involved in the work, also notes that the mechanism that connects changes in gut microbiome to glucose intolerance is still unknown. “But the study will get researchers thinking about it, which is an important step in the right direction,” he says.

Suez says the team is already working on this. The group wants to develop algorithms that will help identify which microbes in the gut make people susceptible to glucose intolerance after drinking or eating artificial sweeteners, information that could eventually help people make the best dietary decisions for their personal microbial makeup.

Editor’s note (August 19): This article has been updated to clarify the research teams involved in the study.

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