PROJECT SUMMARY The prevalence of overweight and hyperglycemia has markedly increased over the past four decades, constituting a global health threat due to the association of these conditions with type 2 diabetes and cardiovascular disease. A common dietary approach for the management and prevention of weight gain and hyperglycemia is to replace sugar in the diet with non-nutritive sweeteners (NNS) that do not elicit a postprandial glycemic response and are calorie-free. Despite the immense popularity of this approach, epidemiological evidence in humans suggests that NNS may in fact counterintuitively promote the same conditions they aim to treat. For lack of insufficient evidence of causality and no clear mechanism through which these “metabolically inert” compounds can affect human metabolism, their use is still widely endorsed. In my graduate work, I demonstrated that three common NNS (saccharin, sucralose, aspartame) can promote glucose intolerance in mice, and that this effect on metabolism is mediated by the disruptive effect of NNS on the gut microbiome. The ongoing clinical trial that I am currently leading, together with my published work, demonstrate that in humans, susceptibility to this detrimental effect is person-specific and depends on the pre-exposure microbiome composition. While it is now evident that NNS can in fact cause metabolic derangements, and that the microbiome mediates the effects of NNS on the host, the mechanisms through which NNS shape the microbiome, and through which the NNS-perturbed microbiome affects host health, remain unknown. As my data show direct interaction between NNS and the microbiome, in Aim 1 we will determine if bacteria that bloom in the presence of NNS can metabolize the NNS compounds, identify the products of these reactions, and determine whether these products are sufficient to affect host health. By identifying NNS-responsive bacteria, we would be able to use the microbiome to predict which individuals will be susceptible to their detrimental effects. While some bacteria bloom in the presence of NNS, other diminish, and we will identify the mechanisms of NNS toxicity to bacteria. In Aim 2, we will identify the mechanisms through which the NNS-perturbed microbiome affects metabolic health in mice. We will follow- up on my observation that the NNS-perturbed microbiome has enhanced capacity for glycan degradation and ask whether this leads to metabolic derangements in two downstream mechanisms including the production of short chain fatty acids, and erosion of the intestinal mucus layer leading to metabolic endotoxemia. We will complement these approaches by analyzing two large datasets of host transcriptome and microbiome profile in NNS-exposed humans and mice. Collectively, these approaches would improve our understanding of the microbiome contribution to the pathogenesis of metabolic syndrome, and support evidence-based policies regarding health benefits and risks of NNS.