Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Nutritional Sciences
Non Technical Summary
The intestine absorbs nutrients and regulates how nutrients are metabolized. Our research highlights the importance of the intestine in coordinating nutrient utilization and enhancing metabolic efficiency - Mice lacking MGAT2, an enzyme mediating intestinal fat absorption, absorb a normal amount of fat, but exhibit increased energy expenditure, resulting in resistance to obesity and associated metabolic disorders induced by excessive caloric intake, independent of macronutrient composition. However, the underlying mechanisms remain elusive. Our recent studies showed that mice lacking MGAT2 have increased plasma bile acid levels and altered composition. These changes in bile acid metabolism are associated with enhanced glycemic control and can be manipulated with two different antibiotic treatments, implicating the involvement of gut microbiota. Thus, we hypothesize that loss of MGAT2 in the intestine selects for microbial communities, altering bile acid metabolism/signaling, which in turn increases energy expenditure, reduces metabolic efficiency, and enhances glycemic control. In this project, we will determine 1) if antibiotic treatments that altered plasma bile acid levels also modulate energy expenditure, 2) if gut microbial communities selected under MGAT2 deficiency confer increases in plasma bile acids and energy expenditure, and 3) if mice without MGAT2 exclusively in the intestine, have gut microbiota similar to mice without MGAT2 in any tissues, and if these mice also exhibit increased plasma bile acids and energy expenditure. Our findings will provide insights into mechanisms by which interactions between intestinal processing of diet and gut microbiota modulate metabolism, needed to identify interventions to combat metabolic diseases and promote animal production.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Using genetically engineered mice and a comprehensive metabolic phenotyping platform for rodents, the proposed project will examine how an enzyme involved in intestinal lipid processing, gut microbiota, and diet may interact to modulate glycemic control and whole-body energy balance. It will also determine the effects of two different antibiotic treatments, one of which simulates antibiotic treatment that promotes growth/animal production, on energy expenditure. Findings from this project will have implications on both animal production and human nutrition.Aim 1-To determine if increased plasma primary bile acids mediate by MGAT2 deficiency and/or antibiotic treatments lead to increased energy expenditure.Aim 2- To determine if germ-free mice conventionalized with gut microbes from Mogat2 mice exhibit elevated energy expenditure and gain less weight upon high-fat feeding, as compared to those conventionalized with microbes from WT.Aim 3- To determine if mice lacking MGAT2 exclusively in the intestine exhibit increased energy expenditure only when they have elevated plasma bile acids.In this project, we will testour overarching hypothesis thatloss of MGAT2 in the intestineselects for certain microbial communitiesthatalter bile acid metabolism/signaling, which in turnincreases energy expenditure, reduces metabolic efficiency, and enhances glycemic control.
Project Methods
Our overarching hypothesis is thatloss of MGAT2 in the intestineselects for certain microbial communities thatalter bile acid metabolism/signaling, which in turn increases energy expenditure, reduces metabolic efficiency, and enhances glycemic control.In this proposal, we will test this hypothesis with three approaches: In Aim 1, we will compare the effects of perturbing gut microbiota on energy balance of Mogat2-/-and wildtype mice using two different antibiotic treatments: a chronic exposure to a low-dose penicillin, simulating an antibiotic growth promoter in animal production, or a regimen of broad-spectrum antibiotics that increased plasma bile acids. In Aim 2, we will examine offspring of germ-free, wildtype dams that were co-housed with either conventionally raised Mogat2-/- or wildtype mice to determine if alteration in bile acid metabolism and increased energy expenditure seen in Mogat2-/-mice are transferable through their gut microbiota. In Aim 3, we will determine if loss of MGAT2 specifically in intestinal epithelia is sufficient to alter gut microbiota and bile acid metabolism, leading to increases in energy expenditure and enhanced glycemic control.Experimental/Methodological approachAim 1: To determine if increased plasma primary bile acids mediated by MGAT2 deficiency and/or antibiotic treatments lead to increased energy expenditure. Approach: We will use an experimental design of 2 genotypes (Mogat2-/- and WT) by 3 treatments (control, broad-spectrum antibiotics, and low-dose penicillin). Mogat2-/- and WT littermates will be generated by intercrossing Mogat2+/- mice and randomly assigned into one of the three treatment groups. Plasma bile acids will be measured before and after antibiotic treatments. Food intake, energy expenditure and substrate utilization - oxygen consumption and carbon dioxide production - when mice are fed a regular mix-meal chow or a semi-purified diet containing 10% (low-fat) or 45% (high-fat) calories from fat (D12450B and D12451, Research Diets, New Brunswick, NJ) will be assessed in our metabolic phenotyping platform with indirect calorimetry, and feces will be collected to assess energy intake.Aim 2: To determine if germ-free mice conventionalized with gut microbes from Mogat2-/- mice exhibit elevated energy expenditure and gain less weight upon high-fat feeding, as compared to those conventionalized with microbes from WT.Approach: To generate mice harboring gut microbiota from Mogat2-/- or those from WT mice, we will co-housed pregnant germ-free WT mice with female conventionally raised Mogat2-/- or WT mice in the plastic flexible vinyl gnotobiotic isolators available from our collaborator Dr. F. Rey at UW-Madison. We will collect and analyze feces from their offspring to identify mice harboring respective gut microbiota and assess their plasma bile acids and metabolic phenotypes in response to different diets, as described. Mice will then be maintained on a high-fat diet to assess long-term gain in body weight and fat mass.Microbiome processing: DNA will be isolated from feces using a bead-beating protocol. The V4 region of the 16S rRNA gene will be amplified using barcoded primers. To maximize sequence depth with the most cost-effective approach, we will employ Illumina-based sequencing of all samples using a MiSeq available to our collaborator Dr. G. Suen's lab at UW-Madison.Analyses of sequencing data: A quality control step will remove all sequences that are of short length (under 250 bp) or are of poor quality (too many uncertain calls or low signal intensity) using the program MOTHUR. This will be followed by the removal of chimeras (the accidental amplification of a sequence from two disparate original sequences during PCR). Sequences that share 97% or more sequence similarity will then be clustered into groups. This corresponds to the accepted level for resolving sequence identity at the species level (so-called operational taxonomic units, OTUs).Two broad scale analyses, alpha and beta diversity, will be used to quantify and compare samples. For alpha diversity, the number of species present in a sample (richness) will be determined using the Chao 1 index and will be coupled to the abundance of the species to calculate overall alpha diversity using Shannon's index. Beta diversity analyses will be used to gain insights into the structure of the gut microbiota and will be performed on the OTUs generated from the raw sequence data. Comparisons between samples will be performed using a number of metrics, including weighted and unweighted UNIFRAC, and the Bray-Curtis dissimilarity analysis. These approaches quantify the compositional similarity or dissimilarity between microbial communities in terms of both total diversity and phylogenetic distance, allowing for visualization using PCA. We will further identify those specific taxa contributing to the differences between samples by employing correlation analyses on the total community composition via analysis of similarity (ANOSIM) in R and permutation testing of multivariate homogeneity of group dispersions (PERMDISP). Trends causing changes in dispersion will be identified by log-linear models, and those OTUs contributing to the phenotypic differences between samples will be identified by an analysis of similarity percentages (SIMPER).Aim 3: To determine if mice lacking MGAT2 exclusively in the intestine exhibit increased energy expenditure only when they have elevated plasma bile acids. Approach: We will measure plasma bile acids, assess metabolic responses to different diets, and analyze cecal microbiome, as described above, using four groups of age-and sex-matched mice: Mogat2IKO mice and their control littermates (Mogat2f/f) as well as Mogat2-/- mice and control littermates.