Performing Department
(N/A)
Non Technical Summary
Obesity is associated with an altered gut microbiota profile. Dietary fiber, food componentsbroken down by gut bacteria, beneficially shape the gut microbiota, and increasing daily fiberconsumption promotes weight loss in both obese rodents and humans. We recently analyzed theefficacy of both plant-based flours and fibers on improving metabolic homeostasis, and foundthat flours high in beta-glucan, and beta-glucan directly, along with oligofructose, are mosteffective at reducing adiposity and body weight in diet-induced obese rodents. Despite theknown interaction between dietary fiber and the gut microbiome, little is known about the exactmechanisms for how specific dietary fibers improve energy homeostasis. Our recent workhighlights a role for the small intestinal microbiota in metabolic homeostasis, an oftenoverlooked site of action, through both changes in specific bacteria, as well as gut derivedmetabolites. Based on our preliminary data, we will examine how dietary fiber can improvesmall intestinal nutrient sensing mechanisms that regulate food intake via a gut-brain axis, whichis dependent on changes in the small intestinal microbiome. We will identify specific smallintestinal bacteria that could act as potential probiotics. Additionally, we will examine how fiber-induced changes in the small intestinal microbiome alters bile acid homeostasis These aimsdirectly address the "Food and Human Health (3d)" section of the Food Safety, Nutrition, andHealth program area, as it examines the impact of food components and its metabolites on thegut microbiome to promote human health
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
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.
Project Methods
General MethodsAnimal Model and Diets: Unless indicated, all experiments involve male C57BL/6J (10 wks old) initially maintained on formulated HFD (similar to Research Diets D12451, except 10% of diet is cellulose; 47% fat, 35% carbohydrate, 4.0 kcal/g) for 6wks to obesity, and then half of mice will be switched to either HFD + 10% oligofructose (OFS; Beneo P95) or HFD + 10% β-glucan (BG; Megazyme International).Nutrient-Induced Satiation Study: After five weeks of HF feeding, mice will be surgically implanted with chronic duodenal catheters as we have much experience doing in the lab 46,67,68,72-75. We will allow for one week of recovery, with mice returning to pre-surgery baseline body weight. Mice will then be subject to small intestinal infusion tests every other day to test for small intestinal nutrient-induced satiation. For the infusion, animals will be overnight fasted and the duodenal catheter will be connected to tubing and syringe mounted in a syringe pump. A total of 0.25 mL of infusate will be given every other day at a rate of 0.025 ml min−1 (mimicking gastric emptying), after which mice will be given food and intake measured up to 3hr post-infusion. After stable baselines with saline, mice will receive intraduodenal infusions of a liquid meal (ensure), glucose, or intralipid (0.5kcal based off of previous studies), bracketed by saline infusions.Metabolic Cages and Body Composition: The Duca lab is equipped with a Sable Systems Promethion Core metabolic cage system and an EchoMRI machine. Metabolic cages can measure indirect calorimetry to determine energy expenditure and respiratory quotient, along with food intake, water intake, body weight and locomotor activity. Mice are acclimated to cages for two days before measurement of real-time recorded measurements. Additionally, lean and fat mass can be measured in unanesthetized mice with the EchoMRI machine.Statistical Analyses: All individuals performing the experimental procedures will be blinded whenever possible. As well, to avoid any time-dependent bias and variability (time of year, temperature, etc.), experiments will be conducted over a period of time. All statistics will be performed using Statistical Analysis Software. For all studies, analyses of variance (ANOVA) or repeated measures ANOVA will be followed by post-hoc Bonferroni tests, where appropriate. The mice group sizes are based power analysis calculations using our prior research and other published reports of similar experiments with rodents and gut microbiome and intestinal infusions. To calculate the sample size, we determined that ~n=10 per group is needed based on a power of 0.95 and a Type I error probability of 0.05 with an effect size calculated from our previous experiments (~0.8), which were based on η2 = 0.4 calculation from previously estimated sum of squares data.Efforts: Data will be presented at various conferences and published in peer-reviewed journalsEvaluations: Project objectives will be periodically measured to make sure that progress is being steadily made.