Location: Sugarbeet and Potato Research
Project Number: 3060-21650-001-034-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2021
End Date: Dec 31, 2022
(1) To determine the effects of pulse resistant starch in ameliorating diet-induced dysbiosis of the gut microbiome, intestinal permeability, inflammation, and cognitive health in a ‘humanized’ mouse model of aging. (2) To validate the causal versus casual role of gut microbiome in mediating the beneficial effects of selected pulse resistant starch on aging-associated intestinal and cognitive health.
Objective 1: C57BL/6J mice (n=144; 72M, 72F) will be purchased at 55-weeks of age and will be allowed to acclimate in the new vivarium for 2-weeks. Then, mice will be subjected to a cleansing procedure to eradicate the gut microbiota. Then, mice will be transplanted with a pool of older (age 50-60 yr) subjects’ microbiota by oral gavage and will be allowed to rest for two-weeks for microbiota stabilization. At 60-weeks of age, baseline fecal collection and bodyweight and body composition testing will be done. Then, mice will be randomly divided into five groups (n=24 per group; 12/12 M/F) and will be fed a western-style average American diet (AAD) for 20-weeks. One group will act as control and will receive AAD while four experimental groups will receive AAD supplemented (5% w/w) with purified resistant starch (RS) from dry peas (black-eyed pea), lentils (split lentil), chickpeas (Garbanzo), or dry bean (Pinto), respectively. One group will receive a widely studied fiber insulin (5% w/w) as a positive control. Fecal collection will be done again at mid-study (at 10-wk) and endpoint (at 20-wk). After 20-wk intervention, mice will undergo a series of tests to assess glucose/ insulin tolerance, leaky gut, and cognitive and motor function. The AAD diet (#D12052705) will be designed to recapitulate human diets as closely as possible, matching the ratio and contents of macronutrients, fiber, ingredients, and fatty acids, to the human diets. Bodyweight and diet intake will be measured weekly. Body composition (lean and fat mass) will be assessed at baseline, mid-study, and endpoint. The gut bacterial and fungal microbiome at the beginning (baseline), mid-study, and endpoint will be measured by employing the Earth Microbiome Project benchmark protocol. At study endpoint, mice will be deeply anesthetized and intestinal tissues, brain, liver, and white adipose tissues will be harvested. The mRNA gene expression of inflammatory genes, gut permeability genes, and senescence markers will be measured in ileum, colon, and plasma. Hormones and plasma lipids will be measured. Mouse ileum, colon, white adipose tissues, and hypothalamus will be fixed and stained for histological analyses. Objective 2: We will use 48 mice (24/24 M/F; age 72-weeks) randomized into two groups. The gut will be cleansed as described above. One group will be transplanted with the microbiota (collected at the endpoint of Objective 1) of control AAD mice. The other group will receive microbiota from a selected pulse-resistant starch group that shows greatest amelioration of gut dysbiosis, gut permeability, inflammation, and cognitive performance. After FMT procedure, the mice (now 78-weeks old) will receive AAD for 12-weeks and will be subjected to all the procedures and parameters as described above for the Objective 1 study.