DIETARY FIBER IMPROVES ENERGY HOMEOSTASIS VIA CHANGES IN SMALL INTESTINAL MICROBIOTA-HOST INTERACTIONS

Goal 1: Examine the impact of β-glucan and OFS on small intestinal nutrient-sensing mechanisms.Obj1.1 - Effect of OFS and BG treatment on energy homeostasis: Mice (n=10/group) will be maintained on HFD for 6wks and then switched to HFD-dietary fiber (10% OFS or BG) or remain on HFD-only for 8wks. During the last week of HFD-feeding, mice will be placed in metabolic cages to get baseline measurements, and then will stay in the metabolic cages throughout dietary switch, with EchoMRI measurements weekly. Following the study, mice will be fasted 5hr and small intestinal contents will be collected (for Obj 2.1 and 3.1), along with portal plasma, colon contents and liver (for Obj3.1), and distal small intestinal mucosa (for Obj 3.1).Obj1.2 - Effect of OFS and BG treatment on nutrient-induced satiation: Mice (n=10/group) will be subject to surgeries and nutrient-induced satiation studies.Following the study, mice will be fasted for 5hr and sacrificed small intestine will be collected for western blot for measurement of nutrient receptors known to induce release of gut peptides as previously done in our labObj1.3 - Role of gut-brain axis: Ex-vivo characterization of gut-brain axis: Mice (n=6/group and treatment) willbe subject to ensure infusion or saline, and sacrificed one hour later. Hindbrain and nodose ganglia will be sectioned, and IHC will be performed. C-fos staining (a marker of neuronal activity) will be measured in the NTS (where vagal afferents terminate) and nodose (cell bodies of vagal afferent neurons). GLP-1 and CCK will be measured in portal plasma via ELISA.GLP-1/CCK antagonism in-vio: Mice (n=10/group and treatment) will follow protocol for Obj1.2, except only ensure will be tested. Following switch to HFD-fiber diets, mice will follow the timeline: saline, ensure, ensure+antagonist, saline+antagonist. Antagonists will be injected intraperitoneal 15min before infusion: exendin-9 (GLP-1R; 25 nmol/kg) or MK-801 (CCK-1R:0.5 mg/kg).Goal 2: Determine the role of the small intestinal microbiota in mediating the beneficial effects of prebiotics on energy homeostasis.Obj2.1 - Small intestinal microbiota analysis of obese mice treated with dietary fiber: SI content samples collected from Obj1.1 will be analyzed, in addition to a group of mice (n=10/group) following the identical protocol to Obj1.1 except they will be sacrificed after 1wk of dietary treatment.Obj2.2 - Acute (nutrient-induced satiation) and chronic inoculations (energy homeostasis) of small intestinal microbiota: HFD mice will be implanted with SI catheter and receive transfer of SI MB frommice that after 6wks of HFD-feeding will be switch to 1wk of HFD-10% OFS or BG or stay on HFD-only.For the acute study, mice (n=10/group) will be subject to nutrient-induced satiation study where after recovery from surgery, HFD mice will be tested for sensitivity to intestinal nutrients.For the chronic study, mice (n=10/group) will be follow timeline similar to Obj1.1, with HFD mice placed in metabolic chamber at 5wks, then instead of switching to the fiber diet at wk6, will be subject to bowel cleansing and SI inoculation described above. One donor mouse will be used per recipient due to the fact that inoculation will be repeated every 2wks to minimize shift in gut microbiota from maintenance diet.Obj2.3 - Acute (nutrient-induced satiation) and chronic inoculations (energy homeostasis) of probiotics.For the acute study, mice (n=10/group) will be subject to nutrient-induced satiation study where after recovery from surgery, HFD mice will be tested for sensitivity to intestinal nutrients, as outlined in Obj1.2. Following this, mice will receive daily bacterial infusion directly via the small intestinal catheter and tested.For the chronic study, mice (n=10/group) will be follow timeline similar to Obj1.1, with HFD mice placed in metabolic chamber at 5wks, then instead of switching to the fiber diet at wk6, mice will be subject to daily bacterial infusion via the small intestinal catheter.Goal 3: Determine the role of bile acid signaling in mediating the beneficial effects of prebiotics on energy homeostasis.Obj3.1 - Enterohepatic bile analysis of obese mice treated with dietary fiber: Bile acids will be quantified in the small intestine, portal vein, liver, and colon by UPLC-MSObj3.2 - Impact of endogenous bile acid signaling on the acute (nutrient-induced satiation) and chronic (energy homeostasis) effects of dietary fiber: To determine if microbial-induced changes in the bile acids are necessary for the effects of dietary fibers on food intake and energy homeostasis, mice (n=10/group) will be treated with BSH inhibitor caffeic acid phenethyl ester (CAPE, 75 mg/kg/day)in drinking water or unadulterated drinking water at the start of dietary switch for acute or chronic studies. After metabolic assessments, small intestinal bile acids will be quantified to confirm increased bile acid conjugation with BSH inhibition. For acute study (nutrient-induced satitation), mice will follow protocol identical to Obj1.2, and for chronic study mice will follow protocol identical to Obj1.1.Obj3.3 - Acute (nutrient-induced satiation) and chronic treatments (energy homeostasis) of bile acids: Mice will follow protocol identical to Obj2.3 for both acute and chronic studies, except instead of a probiotic in the SI catheter, mice will be treated with daily bile acids identified to be beneficial in Exp 3.1.