The objectives of this study were to lay the methodological groundwork for field studies of microRNA analysis in exosomes from small sample volumes of human milk, and assess exosome and microRNA content in infant formulas. When human milk was stored at 4°C for 4 weeks, the count of exosome-sized vesicles decreased progressively to 49% ± 13% of that in fresh milk. Exosomes were purified from 1 mL of fresh human milk and their microRNA content was assessed by microRNA-sequencing analysis and compared with that in infant formulas. We identified 221 microRNAs in exosomes from 3 samples of fresh human milk; 84 microRNAs were present in all 3 samples. MicroRNAs were not detectable in infant formulas and their exosome-sized vesicles, which appeared to be casein micelles. We conclude that large-scale studies of microRNAs in human milk exosomes are feasible, and exosomes and microRNAs are not detectable in formulas.
Exosomes are nanoparticles that transfer cargos from donor cells to recipient cells where they elicit changes in gene expression and metabolism. Evidence suggests that exosomes and their cargos are also absorbed from dietary sources such as bovine milk, and bovine exosomes promote the growth of myofibers in murine C2C12 myotube cell cultures. The aim of the current study was to determine whether the dietary intake of bovine milk exosomes alters strength, gene expression and amino acid profiles in murine skeletal muscles. Male and female C57BL/6 mice, age three weeks, were fed an AIN93G-based, exosome and RNA-depleted (ERD) diet for six weeks; controls were fed an exosome and RNA-sufficient (ERS) diet. Variables of feeding behavior, metabolism, grip strength, liver and kidney function, amino acid profiles, and gene expression patterns were analyzed by using metabolic cages, grip strength analyzers, clinical chemistry analyzers, targeted LC/MS-MS, and RNA sequencing analysis. The diets had no effect on food and water intake, respiratory exchange rate, physical activity, grip strength, markers of liver and kidney dysfunction, and amino acid profiles in muscle. Only twelve and nine mRNAs were differentially expressed in skeletal muscle from female and male mice, respectively, fed ERD and ERS diets. The modest effect of the ERD diet on gene expression and levels of free amino acids in skeletal muscle is consistent with observations that bovine milk exosomes and their cargos accumulate in tissues other than skeletal muscle.
Background Humans and mice absorb bovine milk exosomes and their RNA cargos. Objectives The objectives of this study were to determine whether milk exosome– and RNA-depleted (ERD) and exosome- and RNA-sufficient (ERS) diets alter the concentrations of purine metabolites in mouse livers, and to determine whether diets depleted of bovine milk alter the plasma concentration and urine excretion of purine metabolites in adults and infants, respectively. Methods C57BL/6 mice were fed ERD (providing 2% of the microRNA cargos compared with ERS) and ERS diets starting at age 3 wk; livers were collected at age 7 wk. Plasma and 24-h urine samples were collected from healthy adults who consumed (DCs) or avoided (DAs) dairy products. Spot urine samples were collected from healthy infants fed human milk (HM), milk formula (MF), or soy formula (SF) at age 3 mo. Purine metabolites were analyzed in liver, plasma, and urine; mRNAs and microRNAs were analyzed in the livers of female mice. Results We found that 9 hepatic purine metabolites in ERD-fed mice were 1.76 ± 0.43 times the concentrations in ERS-fed mice (P < 0.05). Plasma concentrations and urine excretion of purine metabolites in DAs was ≤1.62 ± 0.45 times the concentrations in DCs (P < 0.05). The excretion of 13 purine metabolites in urine from SF infants was ≤175 ± 39 times the excretion in HM and MF infants (P < 0.05). mRNA expression of 5′-nucleotidase, cytosolic IIIB, and adenosine deaminase in mice fed ERD was 0.64 ± 0.52 and 0.60 ± 0.28 times the expression in mice fed ERS, respectively. Conclusion Diets depleted of bovine-milk exosomes and RNA cargos caused increases in hepatic purine metabolites in mice, and in plasma and urine from human adults and infants, compared with exosome-sufficient controls. These findings are important, because purines play a role in intermediary metabolism and cell signaling.
BackgroundExosomes are small, cargo‐containing vesicles that are secreted by donor cells to elicit changes in gene expression and metabolism in recipient cells. We have discovered that dietary exosomes from bovine milk can be absorbed by non‐bovine species; studies in murine C2C12 myotube cell cultures suggest that bovine exosomes promote the growth of myofibers.HypothesisThe dietary intake of bovine milk exosomes alters amino acid metabolism, and possibly muscle protein accretion, in non‐bovine species.SignificanceThe dietary intake of exosomes might promote muscle protein accretion in patients with sarcopenia and low‐birth‐weight babies.MethodsC57BL/6 mice, age 3 weeks, were fed an exosome‐depleted (Exo−) AIN93G‐based diet for four weeks; controls were fed an exosome‐sufficient (Exo+) diet. Livers were harvested. The hepatic metabolome was assessed by non‐targeted LC/MS‐MS and by peak intensity analysis; the hepatic transcriptome was assessed by RNAseq using an Illumina HiSeq2500 platform. A second cohort of mice was fed Exo− or Exo+ diets for 4–6 weeks for subsequent analysis of grip strength, respiratory exchange ratio (RER), feeding and activity patterns; skeletal muscle samples are currently analyzed by RNAseq. Statistical significance was assessed using unpaired, two‐tailed t‐test.ResultsHepatic concentrations of several amino acids were significantly higher (up to 18 times) in mice fed the Exo− diet than in Exo+ controls (Fig. 1). The mRNA expression of branched chain amino acid (BCAA) transporters 1 (cytoplasm) and 2 (mitochondria) was greater in mice fed Exo− compared to Exo+ (n.s. for BCAT2; Fig. 2). The RER was not affected by feeding, whereas grip strength was a moderate 5% higher in Exo+ vs. Exo− females after only 4 weeks of feeding (n.s.; not shown). The trend toward a greater grip strength was not caused by differences in food and water consumption or physical activity, which were not significantly different between treatment groups in females.Conclusion & future plansDiets defined by their bovine milk exosome content alter amino acid metabolism in mice. Future studies will investigate protein metabolism by using stable isotopes, and assess muscle protein accretion in response to stimuli such as muscle wasting.Support or Funding InformationNIFA 2015‐67017‐23181, NIFA 2016‐67001‐25301/NIH DK107264, NIH 1P20GM104320, the Gerber Foundation, and USDA Hatch Act and W3002.
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