Background
Resveratrol is a bioactive polyphenol that has shown promising results in the prevention of insulin resistance and type 2 diabetes caused by obesity. Owing to the low bioavailability of resveratrol, it has been postulated that resveratrol may impart its beneficial effects through combined effects on the host and microbiota. Importantly, changes in the gut microbiota have been associated with the progression of metabolic and obesity-related diseases, and our recent work has shown that oral administration of resveratrol to obese mice produces taxonomic changes as well as changes in the predicted function of the gut microbiome. Furthermore, recent findings from our lab have demonstrated that transplantation of fecal matter from healthy resveratrol-fed mice is sufficient to improve glucose homeostasis in obese mice.  However, the precise mechanism of how these fecal transplants improve glucose homeostasis is currently unknown.

Aims
The aims of this study are to (1) determine if oral supplementation of resveratrol is sufficient to rescue impaired glucose tolerance in obese mice; (2) determine whether live bacteria are required for the beneficial effects of fecal microbiome transplants (FMTs); and (3) elucidate the mechanisms by which FMTs improves glucose homeostasis.

Methods

Oral Supplementation of Resveratrol: 8 week-old C57Bl/6 mice were fed a high-fat, high-sucrose (HFHS) (45 kcal% fat, 17 kcal% sucrose) diet for 5 weeks and then randomly assigned to (1) continue a HFHS diet or (2) receive a HFHS diet supplemented with resveratrol (0.4% resveratrol diet ad libitum) for 2 weeks. 

Fecal Microbiome Transplants (FMTs): Fecal slurry was prepared from feces of conventional-raised donor mice maintained on a Chow ± resveratrol (0.4% resveratrol diet ad libitum) for 8 weeks. In a separate group, 8 week-old C57Bl/6 mice were subjected to a HFHS diet for 5 weeks. Following an overnight fast (day 0), fecal matter from donor mice (Chow ± resveratrol) was transplanted via oral gavage on days 1, 3, and 5. 

Heat-killed FMTs: Prepared fecal slurry was autoclaved in a sterilization pouch for 15 minutes. A separate group of 8 week-old C57Bl/6 mice was subjected to the same experimental timeline as mentioned above. Mice were maintained on a HFHS diet for 5 weeks, fasted overnight (day 0), and received 3 oral gavages of fecal slurry (resveratrol or heat-killed resveratrol) on days 1, 3, and 5. 

Intraperitoneal Glucose Tolerance Tests (IP-GTTs): Mice were fasted for 5-6 hours and then received 2 g/kg body weight glucose (dissolved in sterile 0.9% saline) injections intraperitoneally. IP-GTTs were completed in all treatment groups 3 days prior to the overnight fast and on day 11 to confirm the effects of the treatment. 

Tissue Cytokine Measurement: Frozen colon tissues were powdered using a mortar and pestle, and homogenized in ice-cold PBS (with 0.05% Tween-20) for cytokine extraction. Samples were centrifuged at 10,000 rpm for 10 minutes and then the supernatant was used to measure cytokines using an enzyme-linked immunosorbent assay (ELISA) duo set (R&D Systems, Inc., Minneapolis, MN) as indicated in the manufacturer’s protocol.

Results
In obese mice, oral supplementation of resveratrol for 2 weeks is not sufficient to rescue impaired glucose tolerance. However, FMT from resveratrol-fed donor mice to obese mice is able to improve glucose homeostasis within 11 days of the first transplant. In addition, transplantation of heat-killed resveratrol slurry was able to produce similar beneficial effects, improving the glucose homeostasis of obese mice over the same time frame. Tissue analyses of colon indicate decreased levels of inflammatory cytokines TNF-α, IL-8, and IL-1β in mice receiving resveratrol FMTs.

Discussion
Our results show that transplantation of fecal material from resveratrol-fed donor mice to obese mice is sufficient to improve glucose homeostasis during obesity-induced insulin resistance, while oral supplementation of resveratrol for two weeks has no significant impact. This finding shows that the fecal material from resveratrol-fed mice contains is more potent and efficacious than resveratrol itself. Furthermore, heat-killed resveratrol FMTs improve glucose homeostasis in obese mice, demonstrating that non-living bacterial, metabolites or other components within the feces of resveratrol-fed mice are responsible for the beneficial actions of resveratrol FMTs. Since we show that the FMT reduces inflammatory markers in the colon, anti-inflammatory effects of resveratrol-FMTs may be an integral mechanism by which resveratrol improves glucose homeostasis in obesity.

Fecal transplant from resveratrol-fed mice to obese mice improves glucose homeostasis and lowers intestinal inflammation