Animal and observational research in humans suggest that specific types of non-nutritive sweeteners (NNS) may impair glycemic control. However, whether NNS consumption impacts glucose homeostasis in middle-aged/older adults with prediabetes is unknown, and potential mechanisms by which this could occur have yet to be identified. The overall objective of this R21 proposal is to establish proof-of-concept for alterations in glucose homeostasis following intake of saccharin, but not acesulfame potassium, in middle-aged/older adults with prediabetes compared to a eucaloric diet with no NNS.

Observational research has linked intake of non-nutritive sweeteners (NNS), which are consumed daily by ~50% of middle-aged/older U.S. adults, with increased risk of type 2 diabetes (T2D). This risk may be exacerbated by advancing age, which is associated with low-grade chronic inflammation and increased risk of T2D. Current T2D prevention recommendations related to NNS usage are unclear and confusing; use as an alternative to added sugar intake is suggested but long-term NNS use is discouraged despite minimal research to support this recommendation. Animal and observational human studies suggest detrimental effects of some NNS on glucose homeostasis. Longer-term human studies largely demonstrate null findings. Differences in study design and a lack of rigor in existing research contribute to inconclusive findings. In addition, NNS are often studied as a single entity yet types of NNS vary in their absorption and metabolism (e.g., two commonly consumed NNS, saccharin and acesulfame potassium). Whether NNS consumption impacts glucose homeostasis in middle-aged/older adults with prediabetes is unknown, and potential mechanisms by which this could occur have yet to be identified. The overall objective of this R21 proposal is to establish proof-of-concept for alterations in glucose homeostasis following intake of saccharin, but not acesulfame potassium, in middle-aged/older adults with prediabetes compared to a eucaloric diet with no NNS. We will investigate changes in inflammatory markers as potential mechanisms by which saccharin intake influences glucose homeostasis. Following a 2-week eucaloric lead-in diet, 30 middle-aged/older adults (40+ yrs) with prediabetes will be randomly assigned to 1 of 3 controlled feeding conditions for 6 weeks (10 participants per group): saccharin, acesulfame potassium, or a control group (no NNS). Standardized diets will be matched for macronutrients (50% carbohydrate, 35% fat, 15% protein) and other variables to avoid the potential confounds of weight change and dietary factors which may influence study outcomes (e.g., added sugars). All groups will receive identical diets, other than the additional NNS for the two NNS groups. 24-hr glycemic control using continuous glucose monitoring and insulin sensitivity and beta cell function via oral glucose tolerance test (OGTT), serum endotoxin, and inflammatory cytokines, including C-reactive protein, will be measured before and following the 6-week dietary treatment period. This research may have clinical practice and policy implications by informing U.S. dietary guidelines and guidelines for T2D prevention, which devote minimal attention to NNS and provide unclear guidance on NNS use due largely to a lack of rigorously-designed controlled feeding trials.

Inclusion Criteria:

• Age 40+ years
• Prediabetic (fasting glucose concentration of 100-125 mg/dL, 2-hour oral glucose tolerance test glucose concentration of 140-199 mg/dL, or a HbA1c value of 5.7% to 6.4%)
• Weight stable for previous 6 months (±2 kg)
• BMI <40 kg/m2
• Sedentary to recreationally active
• No plans to gain/lose weight or change physical activity level
• Willing to pick up food daily and consume foods provided for an 8-week period
• Verbal and written informed consent
• Approval by Medical Director
• Consume less than one serving of non-nutritive sweetener per week

Exclusion Criteria:

• BMI >40 kg/m2
• Diabetes or diabetes medication
• Antibiotic, prebiotic or prebiotic use in prior 3 months
• Uncontrolled hypertension (blood pressure (BP) > 159/99 mmHg)
• Diagnosed inflammatory bowel disease
• Past or current heart diseases, stroke, respiratory disease, endocrine or metabolic disease, or hematological-oncological disease
• Vegetarian or vegan
• Pregnant or plans to become pregnant
• Breastfeeding
• Food allergies or aversions, Phenylketonuria (PKU)
• Estrogen or testosterone usage

De-identified study data will be posted to Virginia Tech's data repository, VTechData (https:// data.lib.vt.edu/). Datasets selected for sharing will be made accessible through VTechData, managed by the University Libraries at Virginia Tech. VTechData highlights, preserves, and provides access to data generated at Virginia Tech. The system relies on item/dataset level metadata as the primary building block to data discovery, access, and reuse. Published datasets are to be made accessible for at least five years.

Data will be available following completion of the trial and will be available for at least 5 years

Access to trial IPD can be requested by qualified researchers engaging in independent scientific research, and will be provided following review and approval of a research proposal and Statistical Analysis Plan (SAP) and execution of a Data Sharing Agreement (DSA).