Ultra-processed Food Consumption, Gut Microbiota, and Glucose Homeostasis

Advancing age is associated with gut dysbiosis, low-grade chronic inflammation, progressive insulin resistance, and increased risk of type 2 diabetes (T2D). Prediabetes is present in 45-50% of middle-aged/older adults, and declines in glucose tolerance are evident in the third or fourth decade of life. Thus, there is an urgent need to identify new approaches for the prevention of type 2 diabetes among middle-aged adults. Observational research has linked intake of ultra-processed foods (UPF), which comprise ~60% of total energy intake in US adults, with increased risk of T2D. Ex vivo and animal research suggests that components of UPF alter gut microbiota composition and initiate a cascade of events leading to intestinal inflammation and impaired glycemic control. Whether mid-life adults (aged 45-65 yrs) are susceptible to the adverse impact of UPF consumption on glucose homeostasis is unknown. The overall objective of this study is to establish proof-of-concept for an impairment in glucose homeostasis following increases in UPF consumption in mid-life adults, in order to conduct a larger, more comprehensive and mechanistic trial in the future. In addition, changes in gut microbial composition and function, intestinal inflammation and permeability, serum endotoxin concentrations, and inflammatory cytokines as potential mechanisms by which UPF consumption influences glucose homeostasis will be investigated.

Following a two- week eucaloric lead-in diet, participants will be provided and consume a diet emphasizing UPF (81% energy). Diets will be eucaloric (50% carbohydrate, 35% fat,15% protein) matched for dietary soluble and insoluble fiber, added sugar, mono- and polyunsaturated fat, saturated fat, antioxidant nutrients, sodium, pre- and probiotics, and overall diet quality, for 6 weeks.

Following a two- week eucaloric lead-in diet, participants will be provided and consume a diet without UPF (0% energy). Diets will be eucaloric (50% carbohydrate, 35% fat,15% protein) matched for dietary soluble and insoluble fiber, added sugar, mono- and polyunsaturated fat, saturated fat, antioxidant nutrients, sodium, pre- and probiotics, and overall diet quality, for 6 weeks.

Insulin sensitivity assessed using a 2-hour oral glucose tolerance test (75g glucose load). Blood will be collected at baseline (fasting), and thereafter at 30-minute intervals (5 total measurements in 2 hours) at baseline and post 6-weeks high or no UPF diet.

2 timepoints (standardized diet lead-in [baseline]), 6-weeks post high or no UPF diet, 2-hour test in laboratory

24-hour glucose control (24-hour mean glucose concentration) will be assessed using continuous glucose monitoring for a 6-day period at baseline and post high or no UPF diet.

24-hour glucose control (24-hour AUC) will be assessed using continuous glucose monitoring for a 6-day period at baseline and post high or no UPF diet.

24-hour glucose control (time in range) will be assessed using continuous glucose monitoring for a 6-day period at baseline and post high or no UPF diet.

Change in 24-hour glucose control (glycemic variability [GV]) from baseline to 6-weeks post high or no UPF diet

24-hour glucose control (GV) will be assessed using continuous glucose monitoring for a 6-day period at baseline and post high or no UPF diet.

Change in 24-hour glucose control (postprandial glucose) from baseline to 6-weeks post high or no UPF diet

Free-living postprandial glucose concentration will be assessed using continuous glucose monitoring for a 6-day period at baseline and post high or no UPF diet.

Inflammatory Cytokines, including TNF alpha, IL-6, and MCP-1 will be measured by ELISA (American Diagnostica Inc).

5-minute blood collection in the laboratory, 2 timepoints (baseline, 6 weeks post high or no UPF diet)

Serum endotoxin will be assessed using the PyroGene Recombinant Factor C endotoxin assay (Lonza, Basel, Switzerland).

5-minute blood collection in the laboratory, 2 timepoints (baseline, 6 weeks post high or no UPF diet)

Stool samples will be collected daily for 3 days before and during the final 3 days of the diet interventions. Samples will be collected daily and placed in sterile plastic containers, stored in personal freezers, and placed in coolers for transport then immediately frozen at -80°C until final processing and analysis.

Stool samples will be collected daily for 3 days before and during the final 3 days of the diet interventions. Samples will be collected daily and placed in sterile plastic containers, stored in personal freezers, and placed in coolers for transport then immediately frozen at -80°C until final processing and analysis.

Intestinal inflammation will be assessed using fecal calprotectin, lactoferrin, and lipocalin-2, measured using ELISA.

Intestinal permeability will be assessed using serum zonulin (Immunodiagnostik AG, Bensheim, Germany) concentrations, measured using ELISA.

Flow Mediated Dilation of the brachial artery will be assessed using duplex ultrasonography (GE Logiq e) with a high resolution linear array transducer. Reactive hyperemia will be produced by inflation of a pediatric BP cuff around the forearm for 5 minutes. Off line analysis of baseline and post-reactive hyperemic diameters and velocities will be performed using edge detection software (Vascular Analysis Tools, Medical Imaging Applications, Inc). Endothelium independent vasodilation (EID) will be assessed by measuring brachial arterial dilation for 10 minutes following administration of 0.4 mg of sublingual nitroglycerine. Both FMD and EID will be expressed as mm and % change from baseline diameter.

Change in arterial stiffness (Carotid femoral pulse wave velocity) from baseline to 6-weeks post high or no UPF diet

Carotid femoral (C-F) pulse wave velocity (PWV), the primary measure of arterial stiffness, will be measured. C-F waveforms will be obtained via tonometry (NIHem, Cardiovascular Engineering, Inc). Aortic PWV will be calculated from signal averaged waveforms using the ECG as the fiducial point, and body surface measurements.

Change in arterial stiffness (Beta-stiffness index) from baseline to 6-weeks post high or no UPF diet

Beta-stiffness index will be measured using high resolution ultrasonography and tonometry of the carotid artery.

Inclusion Criteria: Weight stable for previous 6 months 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 Estrogen or testosterone usage is acceptable, if on stable dose for >6 months Exclusion Criteria: BMI >35 kg/m2 Diabetes or diabetes medication Antibiotic, prebiotic or prebiotic use in prior 3 months TCHOL >6.2 mmol/L; TG >4.5 mmol/L Blood pressure (BP) > 159/99 mmHg (Stable BP on antihypertensive medications used for >6 months is acceptable) 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 Food allergies or aversions 3 or fewer stools per week or regular laxative use