Performing Department
(N/A)
Non Technical Summary
Dietary long-chain (LC) polyunsaturated fatty acids (PUFAs) are important nutrients in the human diet obtained via consumption of foods of marine and animal origin. LC PUFA intake improves human health, in part, via stimulatory effects on skeletal muscle metabolism. The resident microbes that inhabit the gastrointestinal tract mediate some of the beneficial effects of LC PUFAs on their human host, although the chemical signals involved remain unknown. Many of the biological actions of LC PUFAs are elicited secondary to their enzymatic conversion to oxygenated lipid metabolites (oxylipins). Gut microbe-host cell interactions metabolize LC PUFA substrates to form a variety of oxylipins, but the role of these potentially bioactive lipid mediators in modulating host metabolism is poorly understood. In the proposed research project addressing the AFRI program priority area "Food and Human Health", we will investigate the role of LC PUFAs as important food components on the human gut microbiome, its metabolites, and the subsequent impact on human health via the gut-muscle axis. To accomplish this, we will utilize well-established mouse models of gut microbiota dysbiosis and oral fecal transplantation. We will first test the effect of LC PUFAs and oral antibiotics on the gut microbial community and its interrelationship with blood and muscle oxylipins. We will then test the potential role of the gut-muscle axis in mediating the protective effects of LC PUFAs in diet-induced obesity. Ultimately, this work will provide a scientific foundation regarding the nutrients in food and their impact on the gut microbiota to improve human health.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
The gut microbiome consists of ~10-100 trillion bacteria that inhabit the human gastrointestinal tract. A healthy gut microbiota functions to support nutrient digestion, energy metabolism, gut-barrier function, and the immune response. Dysregulation of gut microbiota homeostasis has been implicated in the development of obesity and type 2 diabetes. Resident gut microbes crosstalk with skeletal muscle via the gut-muscle axis. Therefore, a healthy gut microbial community is an important determinant of muscle metabolism and targeting the gut-muscle axis may be a novel strategy to promote human health and well-being.Long-chain (LC) omega-3 (n-3) polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are well-known to protect against obesity and insulin resistance. Therefore, consumption of foods rich in these nutrients such as fish, shellfish, and n-3 fortified food products (e.g., eggs, dairy, and juice) is beneficial to human health. Skeletal muscle is responsible for ~80% of whole-body glucose disposal. As such, the beneficial effects of n-3 PUFAs on whole-body insulin sensitivity are mediated in large part by their stimulatory actions on muscle glucose uptake. Recent evidence suggests that the LC omega-6 (n-6) PUFA arachidonic acid (ARA, 20:4n-6) may also benefit human health. ARA improves insulin sensitivity in rats fed a high-fat diet (26) and protects muscle cells against lipotoxicity. These data suggest that contrary to current popular belief, dietary intake of foods rich in n-6 ARA such as meat, poultry, and eggs, may also benefit human metabolic health.How LC PUFAs impact human health remains poorly understood, but much recent attention has focused on the potential role of oxygenated lipid metabolites (oxylipins). Interestingly, host-microbiome interactions have recently been suggested to confer the beneficial metabolic effects of dietary LC PUFAs upon human health. Nevertheless, how the gut microbiota transmits the benefits of LC PUFAs to host remains unknown. The overall objective of the proposed studies is to investigate the potential role of the gut-muscle axis in mediating the benefits of dietary LC PUFAs on human health. To accomplish this objective, we will focus on the following two specific aims:Aim 1: Define the role of gut microbe-host interactions in dietary LC PUFAs metabolism.1a. Determine the effect of a diet enriched in LC PUFAs on the gut microbiome diversity.1b. Establish the effect of antibiotics on the oxylipin profile of feces, blood, and muscle.Aim 2: Determine if LC PUFAs protect against metabolic disease via the gut-muscle axis. 2a. Test the effect of feeding LC PUFAs on the gut-muscle axis in diet-induced obesity.2b. Investigate the contribution of host microbiota on transfer of the protective effects of LC PUFA on muscle metabolism.These studies will investigate the potential role of the gut-muscle axis in mediating the effects of dietary LC PUFAs on human health using well-established mouse models of gut microbiota dysbiosis and transplantation. If our hypothesis is supported by the data generated, these initial proof-of-concept studies made possible by this seed grant will lay the foundation for future investigations into the role of dietary LC PUFAs and oxylipin metabolites in modulating the gut-muscle axis to benefit human health, prevent diet-related metabolic diseases, and promote healthy aging.
Project Methods
Aim 1: Define the role of gut microbe-host interactions in dietary LC PUFAs metabolism: Male C57BL6/J mice (n=48 total) will be housed under specific pathogen free (SPF) conditions with a semi-purified low-fat AIN-93M diet (Harlan Teklad TD.94048, 3.6 kcal/g, 10.3% kcal fat, 75.9% kcal carbohydrate, and 13.7% kcal protein) and water provided ad-libitum. At 12-weeks of age, half the mice (n=24) will be randomized to receive an oral broad-spectrum antibiotic cocktail containing ampicillin (1 g/L), neomycin (1 g/L), metronidazole (1 g/L), and vancomycin (0.5 g/L). Antibiotics will be administered continuously to mice in drinking water for a duration of 6-weeks. The remaining mice (n=24) will receive drinking water lacking antibiotics. Following a 2-week pretreatment period to induce preemptive dysbiosis of the gut microbiome, mice (n=6 with and n=6 without antibiotics) will be randomized to receive one of four different energy matched diets (3.6 kcal/g) each containing 4.1% (w/w) fat (10.3% of kcal), 68.3% (w/w) carbohydrate (75.9% kcal), and 12.4% protein (13.7% kcal). The custom experimental diets are based on AIN-93M to include 0.48% (w/w) of either ARA, EPA, or DHA ethyl esters and 3.52% soybean oil in place of the usual 4% (w/w) soybean oil. Twelve mice (n=6 with and n=6 without antibiotics) will be maintained on the AIN-93M diet containing 4% soybean oil to serve as experimental controls.1a. Determine the effect of a diet enriched in LC PUFAs on the gut microbiome: Total community DNA will be extracted from feces samples with the QIAamp PowerFecal Pro DNA kit. 16S rRNA gene sequence libraries will be constructed using the Illumina MiSeq sequencing platform. Briefly, the V4 region of the bacterial 16S rRNA gene will be amplified using 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT) primers. A mock community (ATCC MSA-1002TM) and a water control will also be amplified as control samples. The amplified DNA will be normalized using SequalPrep Normalization Plate Kit (Invitrogen) and pooled. The pooled samples will be sequenced (Illumina MiSeq 2 × 250 bp paired end). Sequence files will be imported into the QIIME2 platform for further analysis (52). Amplicon sequence variants (ASVs) will be generated from demultiplexed paired-end reads with DADA2 and taxonomy assignment will be based on the SILVA database. Alpha and beta diversities and community similarity analysis will also be conducted in the qiime2 pipeline. Alpha diversity will be evaluated by observed features and Chao1 for richness, Pielou's evenness for evenness, Faith-PD for phylogenetic diversity. Principle coordinate (PCoA) analyses based on Bray-Curtis dissimilarity and weighted UniFrac distance will be used for beta diversity analysis.1b: Establish the effect of oral antibiotics on the oxylipin profile of blood, muscle, and feces: Muscle tissue and feces samples will be homogenized in phosphate buffered saline. The fatty acid profile of plasma, muscle and feces will be determined by gas chromatography-mass spectrometry (GS-MS). Oxylipins will be isolated from blood plasma, feces, and muscle homogenates by solid phase extraction (SPE). Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based oxylipin profiling will be performed at the Wayne State Lipidomic Core in collaboration with Dr. Maddipati. This established method monitors a total of 143 oxylipins encompassing all known major PUFA metabolites of the COX, LOX, and CYP pathways as well as those of non-enzymatic origin.Aim 2: Determine if LC PUFAs protect against metabolic disease via the gut-muscle axis: Male C57BL6/J mice (n=30 total) will be housed under SPF conditions with a low fat semi-purified AIN-93M diet (Harlan Teklad, TD94048, described in full under Aim 1) and water provided ad-libitum. To induce obesity and insulin resistance, male 8-week-old C57BL6/J mice (n=6) will be fed for a duration of 12-weeks with a semi-purified obesogenic Western-style high-fat and sugar-rich diet lacking LC PUFA. (TD96132). This hypercaloric diet provides 4.5 kcal/g energy and is comprised of 20.9% w/w protein (18.7% kcal) from casein, 20.2% (w/w) fat (40.7% kcal) from hydrogenated vegetable oil (10% w/w) and beef tallow (10% w/w), and 45.5% (w/w) carbohydrate (40.6% kcal) from sucrose (18.26% w/w), maltodextrin (12.0% w/w), and corn starch (16% w/w). Three additional groups of mice (n=6/group) will be fed a custom semi-purified obesogenic diet based on TD96132 in which a proportion of usual 10% (w/w) hydrogenated vegetable oil will be replaced with 0.48% (w/w) of either ARA, EPA, or DHA. LC PUFA ethyl esters will be used in diet formulations to avoid stability issues of handling free fatty acids while maintaining a purity necessary to elucidate the specific metabolic fate of individual LC PUFAs. A fifth group of mice (n=6) maintained on a low-fat semi-purified AIN-93M diet will serve as healthy lean controls. Bodyweight and food intake will be measured twice weekly. During the last week of the experiment, oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT) will be performed following a 6 h fast. Total body fat and lean mass will be measured one day before sacrifice via Dual-energy X-ray absorptiometry (DEXA). At 20-weeks of age, mice will be fasted for 6 h prior to euthanasia and tissue collection.2a. Test the effect of feeding LC PUFAs on the gut-muscle axis in obesity: Contractility and fatigability of the TA will be measured in-situ and EDL and SOL contractile function measured in-vitro. Following muscle functional testing, mice will be euthanized and hindlimb muscles including GAST, PLA, SOL, TA, and EDL will be harvested and weighed. Gene and protein expression of mitochondrial enzymes, inflammatory cytokines, and components of the insulin signaling cascade (e.g., Akt phosphorylation) will be measured in the GAST and TA by quantitative reverse transcription PCR (RT-qPCR) and western blotting respectively Muscle histology including fiber type, myofiber cross-sectional area (CSA), and intramyocellular lipid content will be assessed on EDL and SOL cross-sections via immunohistochemistry/fluorescent microscopy. Oxylipins will be detected and quantified in the QUAD by LC-MS/MS. Gut microbiome diversity will be profiled by 16S rRNA sequencing.2b. Investigate the contribution of host microbiota on transfer of protective effects of LC PUFA on muscle metabolism: Feces will be collected from the same mice studied in Aim 2a. Donor mice are fed an obesogenic TD96132 diet lacking LC PUFAs or a modified TD96132 diet in which 0.48% (w/w) vegetable oil is replaced with individual LC PUFAs including either ARA, EPA, or DHA. To prepare fecal transplantations, feces will be suspended in 5 ml of PBS anaerobically. Recipient male 8-week-old C57BL6 germ-free mice (n=24 total) will be colonized by oral gavage with 200 µL of a pooled fecal microbiota suspension from each of the four groups of donor mice (n=6/group). Following oral microbiota gavage, all recipient mice will be fed the same Western-style high-fat and sugar-rich diet as the donor mice (TD96132) without any LC PUFA for a duration of 12-weeks. The germ-free mouse diet will be double irradiated for sterilization and will contain 1.5× vitamin mix to account for losses. Bodyweight and food intake will be measured twice weekly. Body composition will be measured one day before sacrifice via EchoMRI. At 20-weeks of age mice will be fasted for 6 h prior to euthanasia. Functional, molecular, and biochemical assays of muscle metabolism will be performed as in Aim 2a.