Performing Department
Ag Animal Sciences
Non Technical Summary
Rationale: The prevalence of childhood obesity is steadily increasing in industrialized countries. Evidence suggests that consumption of dairy products, especially milk, might help prevent childhood obesity. The reason for this association is not yet clear. However, the milk may have the capacity to affect obesity by "programming" adipose stem cells. In a recent discovery, microRNAs (miRNAs), which are 18-25 nucleotide endogenous small RNAs involved in post-transcriptional gene silencing, have been found encapsulated in microvesicles (i.e., exosomes) within cow's milk. The miRNAs in milk exosomes appear to be quite stable and maybe horizontally transferred through intestinal absorption even after the postnatal period. If absorbed, miRNAs could affect the differentiation of stem cells, including athe ones responsible for the formation of fat cells. There is a strong potential that if miRNAs in milk exosomes are absorbed following consumption, it will affect adipogenesis via reprogramming of stem cells.Approach: To test this we will run three separate experiments Experiment 1, ten weaned pigs (5 weeks of age) will be randomly assigned to receive whole cow milk (750 mL/day) or liquid sucrose in addition to the normal diet for 3 months. At the end of 3 months, pigs will be euthanized and stem cells from the fat tissue will be harvested to see the capacity to become fat cells and thewhole transcriptome sequencing. For Experiment 2, one dairy cow will be infused intravenously with a saline solution containing an isotope tracer (13C-glycine) for 12h between two milkings. Milk from the cow will be fed to three piglets and used to isolate exosomes. Whole blood from the piglets will be collected at 1, 3, and 6h after feeding and exosomes isolated. miRNAs will be extracted from exosomes. Stem cells from the fat tissue will be isolated 12h after feeding to extract miRNAs. The horizontal transfer of miRNAs containing the isotpe from milk, blood, and stem cells will be assessed by measuring isotope enrichment in samples using liquid chromatography-tandem mass spectrometry. For Experiment 3 milk from the experiment 1 will be collected monthly to isolate exosomes and extract RNA. Whole blood from the pigs will be collected and exosomes isolated to extract RNA. The extracted RNA will be used to measure type of miRNA . To infer effects of isolated miRNAs on transcriptome of stem cells from the fat tissue, data from Experiment 3 will be combined with data from Experiment 1 using bioinformatics approaches. To determine direct effect of milk miRNAs on stem cells from the fat tissue, stem cells will be cultivated with exosomes isolated from the milk for 6h and transcriptome measuredand the formation of fat cellsassessed.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Our long-term goal is to determine the nutrigenomic effects of dairy products on human health.Our goalin this proposal is to assess if miRNAs from cow's milk play a role in controlling obesity by affecting ASC. Our central hypothesis is that miRNAs present in raw milk are horizontally transferred and prevent obesity by regulating expression of genes involved in ASC adipogenesis. Our rationale is that the demonstration of absorption and effects of miRNAs from cow's milk on pig ASC will establish a strong scientific framework for future research and, ultimately, clinical trials.Objective #1: Assess the in vivo effect of milk on the abundance, proliferation, and differentiation capacity of adipose-derived stem cells and size of mature adipocytes. Our working hypothesis is that consumption of cow's milk decreases the number and adipogenesis of porcine ASC in vivo and reduces the size (i.e. lipid accumulation) of mature adipocytes.Objective #2: Determine if miRNAs present in cow's milk exosomes are horizontally transferred into the blood stream through intestinal absorption and then transported into adipose-derived stem cells. Our working hypothesis, based on recent scientific data, is that miRNAs from cow's milk are horizontally transferred and present in ASC.Objective #3: Determine the effects of miRNAs present in exosomes of cow's milk have on the transcriptome of adipose-derived stem cells. Our working hypothesis is that miRNAs in exosomes from cow's milk have an effect on the transcriptome of porcine ASC.
Project Methods
Objective 1: Assess the in vivo effect of milk on the abundance, proliferation, and differentiation capacity of adipose-derived stem cells and size of mature adipocytes.Approach.1.1.Effect of feeding milk on abundance, proliferation, and differentiation of ASC. Studies using live animals will be supervised by the OSU Institutional Animal Care and Use Committee. The pig is a well-suited animal model for this research because it is physiologically similar to humans. Thus, we will purchase 13 weaned (5 weeks of age) male pigs of the same breed. Ten piglets will be used for this objective and 3 will be used for objective 2. The use of 5 pigs per group was determined using power analysis of transcriptomics datausing 21,630 transcripts, 80% power, 1% false positive, 2-fold-change, and a standard deviation of 0.6 as criteria. The pigs will be hosted in the OSU Hogg Animal Metabolism Barn and fed a pelleted commercial pig chow (CHS Inc., USA) at 3% of body weight daily. Pigs will be randomly assigned to receive 3 cups/day (750 mL) of whole cow's milk as for USDA guidelinesor isoenergetic liquid maltose dextrin for 3 months. Raw milk will be obtained from the OSU Dairy Center, which is equipped with Afimilk Dairy Management System (Kibbutz Afikim, Israel) that automatically measures milk components, which allows for calorie adjustment of the treatments and verifies the quality of the milk. At the end of 3 months, pigs will be humanely euthanized and ASC harvested from the back fat . Proportion and quantification of isolated ASC will be performed by flow cytometer using CD90 and CD34 as markers. Quantification of ASC will be also done by a colony-forming unit assay. The quantification of adipogenesis will be performed by inducing adipogenesis in triplicates passage 1 ASC in 24-well plates and cells harvested after 0, 7, 14, and 21 days, fixed, and stained with Oil Red O and hematoxylin. Number and size of lipid droplets in nucleated cells will be measured by ImageJ and quantification of accrued Oil Red O performed. Finally, passage 1 ASC will be cultured in 12-well plates for cell proliferation assay using Vybrant® MTT Cell Proliferation Assay Kit (Life Technologies, USA). Data will be checked for normal distribution by Univariate procedure of SAS (SAS Inst. Inc., USA) and outlier checked with studentized residuals by REG procedure of SAS. The data will be statistically analyzed by ANOVA or GLIMMIX with treatment or treatment×time as the main effect and pig as random.1.2.Effect of feeding milk on ASC transcriptome. Total RNA will be extracted from an aliquot of isolated ASC from 1.2.1 using Mag-Bind Total RNA kit (Omega, USA) associated with the KingFisher Duo (Thermo Scientific, USA). The extracted RNA will be sent to the Center for Genome Research and Biocomputing at OSU for whole transcriptome analysis by RNA sequencing (RNAseq) using an Illumina HiSeq 2000 platform. Samples will be multiplex-read with 5 samples per lane to obtain a minimum of 30 million 100 bp paired-end reads per sample. We will use the Tuxedo Suite to map the RNA-Seq reads to the pig genome, and quantify isoform expression. The statistical effect of milk with pig as random will be evaluated. The p-values will be adjusted with Benjamini-Hochberg false discovery rateand the functional analysis performed.1.3 .Effect of feeding milk and adipocytes size. Adipose tissue from back fat and mesenteric will be obtained from euthanized pigs in 1.1 fixed with formalin, sectioned at 10 μm using a Leica cryostat (Leica Biosystems, Germany), and stained with Oil Red O and hematoxylin.Size of adipocytes and content of Oil Red O will be determined using Cell Profiler software. Statistical analysis will be performed as for 1.1.Objective 2: Determine if miRNAs present in cow's milk exosomes are horizontally transferred into the blood stream through intestinal absorption and transported into adipose-derived stem cells.ApproachOne lactating dairy cow will be injected intravenously using a peristaltic pump a saline solution with Glycine-2-13C (Sigma-Aldrich, USA) as purine base precursorand infused at 547 μmol/min for 12h starting immediately after milking. 750 mL of milk from the injected cow will be fed to 3 piglets purchase as in 1.2.1 and partly used to isolate exosomes by centrifugation. Whole blood from the piglets will be collected at 1, 3, and 6h after feeding. Exosomes will be isolated from blood. ASC from euthanized pigs will be isolated from subcutaneous fat 12h after feeding as for Objective 1 and purified using antibody for CD90 and CD34 conjugated with Pierce NHS- Activated Magnetic Beads (Thermo Scientific, USA) by means of Kingfisher Duo. Total RNA with enriched miRNA will be isolated from exosomes from milk and blood and from purified ASC using mirVana™ miRNA Isolation Kit (Life Technologies, USA). Horizontal transfer of radiolabelled miRNAs from milk, blood, and ASC will be assessed by measuring isotope enrichment in treated vs. control samples (pre-treatment for milk and blood and from ASC from treated pigs in Objective 1) using liquid chromatography-tandem mass spectrometry at the OSU Mass Spectrometry Facilities.Objective 3: Determine the effects of miRNAs present in exosomes of cow's milk have on the transcriptome of adipose-derived stem cellsApproach.3.1 Effect of miRNAs from milk on ASC in vivo: a bioinformatics approach. RNA will be isolated as Objective 2 from exosomes present in 3 cow milk samples and blood from the 10 pigs used in Objective 1 collected 12h after feeding milk. The extracted RNA will be enrich for miRNAs as described in Objective 2 and then sequenced. Differentially expressed microRNAs will be compared by computing a p-value with a binomial model, and then adjusted with a Benjamini-Hochberg correction. To infer effects of isolated miRNAs on transcriptome of ASC, data from Objective 3 will be combined with data from Objective 1 using the Core and Networks analyses capability of Ingenuity Pathway Analysis software (Ingenuity Systems, USA). Due to the high conservation of bovine and pig miRNAs (only 1% difference, based on a comparison of annotated microRNAs in miRBase), only the miRNAs significantly enriched in treated vs. control pigs will be used to pair with ASC transcriptome. We will identify the regulatory impact of significantly enriched miRNAs by analyzing their target sites in the 3' UTRs of downregulated genes in ASC.3.2Direct effect of miRNA from milk on ASC in vitro. Twelve biological replicates of ASC at passage 1 from each pig from Objective 1 will be cultivated in vitro in 24 well-plates for 24hwith or without exosomes isolated from cow's milk as for Objective 2. ASC from 3 wells from each pig will be pooled and RNA extracted and sequenced for whole transcriptome analysis, plus statistical analysis, as for Objective 1. Data will be analyzed using Ingenuity Pathway Analysis and Dynamic Impact Approach. The ASC in the other wells will be tested for adipogenesis as for Objective 1 with analysis performed in triplicate at 0, 14 and 21 days. Effect of milk miRNAs on ASC will be uncovered by change in transcriptome and adipogenesis.